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VipinChandraKalia Editor

Biodiversity, Biopolymers, Bioactive Molecules:
Volume 2
Microbial Factories
Vipin Chandra Kalia

Microbial Factories
Biodiversity, Biopolymers, Bioactive
Molecules: Volume 2
Vipin Chandra Kalia
Microbial Biotechnology and Genomics
CSIR-Institute of Genomics
and Integrative Biology
Delhi University Campus
Delhi, India

ISBN 978-81-322-2594-2 ISBN 978-81-322-2595-9 (eBook)

DOI 10.1007/978-81-322-2595-9

Library of Congress Control Number: 2015957418

Springer New Delhi Heidelberg New York Dordrecht London

Springer India 2015
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Dedicated to my family and friends

Human beings are an integral part of the environment. Biological activities

have a strong influence on physical and chemical components of the ecosys-
tem. Plants are the major contributors as producers of bioproducts, to be used
by animals and microbes. In the animal kingdom, human beings are the most
aggressive consumers, and their needs are increasing geometrically with
time. Unlike animals, human needs extend beyond food and shelter. The
innovative nature of man has led to discoveries and inventions, which appar-
ently are for the benefit of human beings. However, these developments are a
big drain on the available natural resources with a cascade effect. At the base
of this chain reaction, the most adversely affected is the energy sector. The
demand for energy is increasing rapidly because of the needs and attitudes of
humans, who are thus transforming to a society of high-end consumers. Since
fossil fuels are the major source of energy, their consumption is the root cause
of irreparable damage to the environment. Another factor which adds to the
ever-increasing environmental pollution is the unmanageable quantities of
wastes. The conventional means of disposal of wastes and waste waters,
adopted in most parts of the world, pollutes the land, atmosphere, and the
water bodies. Here, we may need to approach the most efficient organisms on
the planet Earth. These efficient organisms are the microbes, which can
metabolize organic matter content of the biowastes, especially those pro-
duced due to human activities. These bioproducts are eco-friendly, biode-
gradable, and highly energy efficient. Microbes can be exploited as factories
for producing energy (biofuels), biopolymers (bioplastics), and bioactive
molecules (antimicrobial, anticancer, antidiabetic, antioxidants, etc.). There
has been a vigorous scientific pursuit to exploit microbes for the welfare of
human beings. The most exciting are the possibilities of generating clean
fuels (biohydrogen, biodiesel, etc.) and biodegradable plastics as an alterna-
tive to nondegradable plastics. Apart from these, the most curiosity-driven
activities have been to learn about those microbes which are yet to be cul-
tured. During the last 23 decades, many scientific activities have been dem-
onstrated and published in scientific journals of repute; however, it is yet to
reach the curious young minds the graduate and postgraduate students of
our future scientists. This compilation, contributed by the experts in these
research domains, speaks a lot about the present status of microbial factories
and their future potential for the welfare of human beings. In principle,
experts exist in all domains; however, most of the times, they are too busy in
their pursuits to spare time for such activities. The young, curious, and tender

viii Preface

minds are eager to learn, but those who know what and how to say do not get
the right platform and access. I am extremely thankful to all those who read-
ily agreed to share their expertise for the Ignited Minds, to whom the book is
dedicated. Although it is impossible to acknowledge the reality and true
worth of the efforts of the contributing authors, however, I am still indebted
to their prompt responses and dedicated efforts. My inspiration to learn
well and transmit the knowledge to the next generation burgeons from the
tireless efforts and constant support of my close ones Mrs. Kanta Kalia and
Mr. R.B. Kalia (parents); Amita (wife); Sunita and Sangeeta (sisters); Ravi,
Vinod, and Satyendra (brothers); Daksh and Bhrigu (sons); and my teachers
and friends Rup, Hemant, Yogendra, Rakesh, Atya, Jyoti, Malabika, Neeru,
and Ritushree to write this book. I must also acknowledge the selfless and
dedicated support of my next-generation colleagues Prasun, Sanjay,
Subhasree, Shikha, Anjali, and Jyotsana.

Delhi, India Vipin Chandra Kalia


1 Biopolymers and Their Application

as Biodegradable Plastics ............................................................. 1
Scott Lambert
2 Approaches for the Synthesis
of Tailor-Made Polyhydroxyalkanoates ...................................... 11
Carlos F. Pea Malacara, Andrs Garca Romero,
Modesto Milln Ponce, and Tania Castillo Marenco
3 Biodegradable Polymers: Renewable
Nature, Life Cycle, and Applications .......................................... 29
Manjusha Dake
4 Phylogenetic Affiliation of Pseudomonas sp. MO2, a Novel
Polyhydroxyalkanoate-Synthesizing Bacterium ........................ 57
Parveen Kumar Sharma, Jilagamazhi Fu, Xiang Zhang,
Richard Sparling, and David B. Levin
5 Synthetic Biology Strategies
for Polyhydroxyalkanoate Synthesis ........................................... 79
Gunjan Arora, Andaleeb Sajid, Parijat Kundu,
and Mritunjay Saxena
6 Frontiers in Biomedical Engineering:
PHA-Fabricated Implants ............................................................ 91
Lalit K. Singh, Neha Dhasmana, Shashank S. Kamble,
Aditya K. Sharma, and Yogendra Singh
7 Sporulation, a Pitfall in the Path of PHB Production................ 103
Neha Dhasmana, Lalit K. Singh, Shashank S. Kamble,
Nishant Kumar, and Yogendra Singh
8 Microbial Biopolymers: The Exopolysaccharides ..................... 113
Angelina and S.V.N. Vijayendra
9 Innovations in Microalgal Harvesting
Using Biopolymer-Based Approach ............................................ 127
Chiranjib Banerjee, Rajib Bandopadhyay, Puneet Kumar Singh,
Harsh Kumar Agrawal, and Pratyoosh Shukla

x Contents

10 From Microbial Biopolymers to Bioplastics: Sustainable

Additives for PHB Processing and Stabilization ........................ 139
Stefania Angelini, Pierfrancesco Cerruti, Barbara Immirzi,
Merima Poskovic, Gabriella Santagata, Gennaro Scarinzi,
and Mario Malinconico
11 The Survivors of the Extreme: Bacterial Biofilms ..................... 161
Neha Dubey, Raja Singh, Aditya K. Sharma,
Sharmila Basu-Modak, and Yogendra Singh
12 Synthetic Biology in Aid of Bioactive Molecules ........................ 183
Shilpi Jain and Swati Shalini
13 Biotechnology Implications of Extremophiles as Life
Pioneers and Wellspring of Valuable Biomolecules ................... 193
Ilaria Finore, Licia Lama, Annarita Poli, Paola Di Donato,
and Barbara Nicolaus
14 Microbial CRISPRCas System: From Bacterial
Immunity to Next-Generation Antimicrobials ........................... 217
Alka Mehra
15 Photorhabdus: A Microbial
Factory of Insect-Killing Toxins .................................................. 235
Jyoti Kushwah and Vishal Singh Somvanshi
16 Microbial Vesicles: From Ecosystem to Diseases ....................... 241
Shashank S. Kamble, Nancy Garg, Brijendra Kumar Tiwari,
Lalit K. Singh, Neha Dhasmana, and Yogendra Singh
17 Bacteriophage Diversity in Different
Habitats and Their Role in Pathogen Control............................ 259
Nishant A. Dafale, Zubeen J. Hathi, Sarmistha Bit,
and Hemant J. Purohit
18 Metagenomics: A Systemic
Approach to Explore Microbial World ....................................... 281
Manoj Kumar, Jitendra Kumar, and Nar Singh Chauhan
19 In Silico Reconstitution of Novel
Routes for Microbial Plastic......................................................... 299
Vipin Chandra Kalia, Sadhana Lal, Rashmi, Ashwini Chauhan,
and Goutam Bhattacharyya
20 Investigating the Phylogeny of Hydrogen Metabolism
by Comparative Genomics: Horizontal Gene Transfer............. 317
Sadhana Lal, Dhananjay V. Raje, Simrita Cheema,
Atya Kapley, Hemant J. Purohit, and Vipin Chandra Kalia
21 Prokaryotic Contributions
Towards Eukaryotic Powerhouse ................................................ 347
Vipin Chandra Kalia
About the Editor

Vipin Chandra Kalia is presently working as

chief scientist at Microbial Biotechnology and
Genomics, CSIR-Institute of Genomics and
Integrative Biology, Delhi. He is a professor at the
Academy of Scientific and Innovative Research
(AcSIR), Delhi. He obtained his M.Sc. and Ph.D.
degrees in genetics from the Indian Agricultural
Research Institute, Delhi. He has been elected as
(1) Fellow of the Association of Microbiologists of India (FAMI) and (2)
Fellow of the National Academy of Sciences (FNASc). His main areas of
research are microbial biodiversity, genomics, and evolution, bioenergy, bio-
polymers, antimicrobials, quorum sensing, and quorum quenching. He has
published 85 papers in scientific journals such as (1) Nature Biotechnology,
(2) Biotechnology Advances, (3) Trends in Biotechnology, (4) Critical
Reviews in Microbiology, (5) Bioresource Technology, (6) International
Journal of Hydrogen Energy, (7) PLoS ONE, (8) BMC Genomics, and (9)
Gene. His works have been cited 2080 times with an h index of 25 and an i10
index of 39. He has edited a book: Quorum Sensing Versus Quorum
Quenching: A Battle with No End in Sight (2015, Springer India). He is pres-
ently the editor in chief of the Indian Journal of Microbiology and editor of
(1) PLoS ONE, (2) Journal of Microbiology and Biotechnology (Korea), (3)
Applied Biochemistry and Biotechnology (USA), (4) International Scholarly
Research Notices (energy), (5) Dataset Papers in Science (microbiology),
and (6) Journal of Molecular and Genetic Medicine. He is a life member of
the following scientific societies: (1) Society of Biological Chemists of India;
(2) Society for Plant Biochemistry and Biotechnology, India; (3) Association
of Microbiologists of India; (4) Indian Science Congress Association; (5)
BioEnergy Society of India, and (6) the Biotech Research Society of India
(BRSI). He is also a member of the American Society for Microbiology. He
can be contacted at vckalia@igib.res.in; vc_kalia@yahoo.co.in

Biopolymers and Their Application
as Biodegradable Plastics 1
Scott Lambert

Plastics have gained widespread use because of their plasticity in form and
function, and their benefits are wide-ranging. The drawbacks to
petrochemical plastics are that they are considered to be biologically inert.
This in turn represents a waste management problem, especially for plastic
materials with a short use phase. Presently, the development of biodegrad-
able plastics as an alternative to petrochemical plastics is seen as an
important waste management option. Polyhydroxyalkanoates (PHAs) are
polyesters produced by the bacterial fermentation of sugars and lipids.
PHAs have attracted commercial and academic interest because they are
considered to be highly biodegradable. One of the most promising areas of
application for PHAs is in the production of thin film materials for use
as packaging materials. Presently, about 40 % of the plastics produced
worldwide are utilised for packaging purposes. The disposable nature
of packaging materials makes the use of PHA plastics an attractive alter-
native. As such PHAs may help to solve some of the waste management
issues associated with single-use plastic items.

1.1 Introduction function. It is clear that plastic materials play an

important role in our everyday lives. However, the
The majority of plastic products in use today are problems associated with modern petrochemical
made from petrochemical-based polymers. These plastics from a waste management and
materials have gained widespread use in a variety environmental perspective come from the large
of industries such as packaging, transport and volumes of plastic waste materials that are gener-
agriculture, because of their plasticity in form and ated. These waste materials do not readily degrade
in landfills and composting facilities. Indeed, lit-
tered plastic materials are one of the most ubiqui-
tous and conspicuous environmental pollution
S. Lambert (*)
problems and are present in one form or another
Goethe University Frankfurt am Main,
Max-von-Laue-Str. 13, 60438 Frankfurt, Germany globally. In addition to the obvious visual impacts
e-mail: scottl210@hotmail.co.uk of littering, there are also many broader environ-

Springer India 2015 1

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_1
2 S. Lambert

mental issues as they present a persistent pollution thetic polymers to be produced on a mass scale.
problem that has hazardous consequences for The modern forms of PVC, polyurethane (PUR)
organisms and environmental degradation. and a more processable PS were created during
Presently, society is working towards the goals 19261938. The early 1950s saw the development
of sustainable development. Among others, of high-density polyethylene and polypropylene.
research and development increasingly focuses The advances in the material sciences through
on waste and pollution prevention. However, the 1960s have seen the development of plastic
both the generation of waste materials and materials produced from other natural resources,
pollution cannot always be prevented, and bio- such as PHA, PLAs, polysaccharides and aliphatic
degradation is seen as an important management polyesters (Reddy et al. 2003).
option. To this end, there is strong interest in the Today world synthetic and natural polymer
development of biodegradable materials made production is approximately 327 million t annu-
from biological resources, commonly termed ally (Fig. 1.1) and is dominated by synthetic poly-
biopolymers or bio-based polymers, as a sub- mers (315 million t), for which polyolefins (114
stitution for petrochemical-based polymers, and million t) are the most important. The polyolefins
as a potential solution to waste management and and other thermoplastics, as listed in Fig. 1.1,
environmental issues. Bio-based polymers can be are the primary commodity plastics because they
produced from various resources; these include have the desired properties and are inexpensive
carbohydrate-rich products from the agricultural to produce. World natural rubber latex production
sector (natural polymers) such as polyhydroxyal- is approximately 10.4 million t annually (NRS
kanoates, starch and cellulose (Lambert et al. 2011), and the global production of biopolymers
2014). Next are the polymers produced by is estimated to be 2.33 million t (Shen et al. 2009).
bacteria via fermentation of sugars and lipids, Packaging represents one of the most impor-
which form a class composed of polyhydroxyal- tant applications of plastics. It accounts for
kanoates (PHA), polylactides (PLA), aliphatic around 40.1 % of the total production. The other
polyesters, polysaccharides and their copolymers usages of plastics are in building and construc-
and/or blends (Lambert et al. 2014; Reddy et al. tion (20.4 %), where as automotive electrical and
2003). The two most talked about bio-based electronic equipment account for 5.67.0 %.
polymers in the scientific literature are PHAs and Apart from these major areas, markets including
PLA. This chapter focuses mainly on the micro- leisure and agriculture account for 26.9 %. The
bially formed PHAs as an alternative to tradi- benefits of plastic materials are wide ranging.
tional petrochemical plastics and their potential They are inexpensive to produces when com-
utilisation as materials for bioplastic applications. pared to alternative materials, are long lasting
and offer thermal and electrical insulation
properties, and many are resistant to chemicals
1.2 Worldwide Production and water. Plastics are flexible and very versatile
of Polymers materials, meaning that they are malleable for
design purposes. They are also strong and light-
The present-day usage of plastics can be traced weight with valuable properties for the automo-
back to the development of rubber technology tive and aviation industries.
in the nineteenth century. the discovery of
vulcanisation of natural rubber by Charles
Goodyear was a major breakthrough in this area. 1.3 Plastics and Their Waste
Subsequently, efforts were focused on the devel- Management Issues
opment of synthetic polymers. Polystyrene (PS)
and polyvinyl chloride (PVC) were discovered As outlined in the above section, the vast major-
during this era, but could not be commercialised ity of plastics are made from petrochemical poly-
due to their brittle nature. Bakelite, developed by mers. Petrochemical-based plastics are generally
Leo Baekeland, in 1909 was among the first syn- considered bioinert and do not degrade, or at
1 Biopolymers and Their Application as Biodegradable Plastics 3

Polyolefins Polyethylene (PE), polypropylene (PP)

Other thermoplastics Polyvinyl chloride (PVC), polystyrene (PS), polyethylene

terephthalate (PET), expanded PS (EPS), polymethyl
methacrylate (PMMA), polycarbonate (PC), acrylonitrile
butadiene styrene (ABS), styrene acrylonitrile (SAN), and
other high performance composites

Fibres Polyamide (PA), polyester, acrylic, and others

Elastomers (synthetic) Styrene butadiene (SBR), synthetic isoprene rubber (IR),

butyl rubber (IIR), polybutadiene rubber (BR),
acrylonitrile butadiene rubber (NBR), polychloroprene
rubber (CR), and others

Natural rubber Including latex

Biopolymers Polylactic acid (PLA), polyhydroxyalkanote (PHA),

polyhybroxybutyrate (PHB), polyhydroxybutyrate-valerate
(PHBV), cellulose, and others.

Fig. 1.1 Annual world polymer production

least degrade very slowly, in landfill and under alternative to landfill. Chinese administration
composting conditions. expects to expand its capacity for waste treatment
There are several waste streams that waste via incineration from 3 % in 2011 to 30 % by
plastic materials are likely to enter, i.e. landfill, 2020 (Cheng and Hu 2010). Japan, Denmark and
incineration, wastewater treatment plants and Sweden also have extensive incinerator infra-
recycling. The suitable treatment is a key ques- structure in place for dealing with solid waste.
tion of waste management. Different countries Incineration is seen as having a preferred waste
have a wide range of waste management prioriti- management option in terms of energy recovery
sations for solid waste materials, ranging from and reducing the need for landfill.
countries that heavily favour landfill to those that The utilisation of biodegradation processes as
favour recycling and incineration. Landfill is still a waste management option is today viewed as a
a major end-of-life disposal system employed for method to improve sustainability of managing
municipal and industrial solid waste in many societies waste. A plastic material labelled as
countries. However, waste disposal in landfill is either compostable or biodegradable should, in
becoming increasing undesirable in many coun- theory, undergo biological decomposition within
tries due to legislative pressure, rising costs and industrial composting sites. This will require the
hazards associated with landfill leachate contam- development of plastic technologies that are able
inating groundwater. Incineration is an important to fully degrade under such conditions.
4 S. Lambert

Table 1.1 Examples of PHA production from different media types

Organism Carbon source Production level [g/L] References
Azotobacter vinelandii Molasses 23 Page et al. (1992)
Pseudomonas fluorescens Molasses 22 Jiang et al. (2008)
Ralstonia eutropha Potato starch 94 Haas et al. (2008)
Cupriavidus necator Glycerol 38 Cavalheiro et al. (2009)
Cupriavidus necator Glycerol 16 Koller et al. (2005)
Escherichia coli Whey 96.2 Ahn et al. (2000)
Bacillus megaterium Sugarcane molasses 1.27 Kulpreecha et al. (2009)
Bacillus megaterium Sugarcane molasses 0.45 Kulpreecha et al. (2009)

1.4 Biopolymers: PHA and PLA group and the hydroxyl group of neighbouring
monomers form an ester bond (Madison and
Although biopolymer, at present, represents only Huisman 1999). As a general rule, PHAs are
a small portion of the current market share, their classified based on the number of carbon atoms
production costs in the future are expected to in the polymer chain. Those PHAs consisting of
decrease making them more competitive. The three to five carbon atoms in the polymer chain
following section provides an overview of the are classified as short-chain length PHAs
biopolymers PHAs and PLA. For an in-depth (scl- PHA) and can be synthesised by numerous
review of biodegradable plastics, the reader is microorganisms including Alcaligenes latus and
pointed towards recent reviews by Reddy et al. Ralstonia eutropha (Madison and Huisman
(2013) and Soroudi and Jakubowicz (2013). 1999). PHAs consisting of 614 carbon atoms
are classified as medium-chain length PHAs
(mcl-PHA) and can be synthesised by
1.4.1 Polyhydroxyalkanoates Pseudomonas oleovorans and Pseudomonas
putida (Kim et al. 2000). Some microorganisms
PHAs are polyesters produced by the bacterial such as Aeromonas hydrophila and Thiococcus
fermentation of sugars and lipids. These simple pfennigii are capable of synthesising scl-PHA
macromolecules are synthesised by a very wide and mcl-PHA copolyesters (Kim do et al. 2007).
range of microorganisms. PHAs are accumulated In addition, like all polymers, PHAs can also be
intracellularly and function as carbon and energy classified as either homopolymers or heteropoly-
reserves. Microorganisms capable of accumulating mers depending on the number of different
PHAs include the genera Alcaligenes, Bacillus hydroxyalkanoate monomer types which makes
and Pseudomonas; for a detailed list, the reader is up the monomeric unit. In terms of their material
encouraged to see Koller et al. (2010). PHA properties, the scl-PHAs display thermoplastic
molecules, extracted from the bacterial cell, have properties, while mcl-PHAs have more elastic
sufficiently high molecular mass, which exhibit properties. In addition, like all polymers, PHAs
characteristics quite similar to some common can also be classified as either homopolymers or
petrochemical plastics, for example, polyethyl- heteropolymers depending on the number of
ene and polypropylene. It is thought that there different hydroxyalkanoate monomer types which
are over 150 different types of PHAs that can be makes up the monomeric unit.
combined to provide a wide range of different The production of PHAs can be undertaken
properties. The majority of PHAs are identified using various materials including sugar-based
as primarily linear polyesters composed of media (Jiang et al. 2008; Kulpreecha et al. 2009;
3-hydroxy fatty acid monomers (Madison and Page 1992), starch-based media (Haas et al.
Huisman 1999). In these PHAs, the carboxyl 2008), whey-based media (Ahn et al. 2000), and
1 Biopolymers and Their Application as Biodegradable Plastics 5

reported to display decreased water permeability

and tensile strength (Madison and Huisman
PHAs are considered to be highly biodegrad-
Fig. 1.2 Structure of polyhydroxybutyrate able (lvarez-Chvez et al. 2012) and are
reported to be biodegradable in a range of natural
environments. The literature consists of studies
glycerol-based media (Cavalheiro et al. 2009; reporting their biodegradation potential in soils
Koller et al. 2005) (Table 1.1). The data presented (Kunioka et al. 1989; Mergaert et al. 1993;
in Table 1.1 identifies starch-based and whey-based Woolnough et al. 2010), aerobic and anaerobic
materials as easily utilised by microorganisms, as sludge and compost (Mergaert et al. 1996; Luo
shown by their high production levels. Table 1.1 and Netravali 2003) and freshwater and marine
also highlights differences in the species of water environments (Mergaert et al. 1995). PHAs
microorganism used. Azotobacter vinelandii and have been demonstrated to be degradable under
Pseudomonas fluorescens are shown to demon- laboratory conditions of soil burial, at tempera-
strate high production levels compared to Bacillus tures of 2530 C, when samples have undergone
megaterium when using sugar-based media. pre-ultraviolet exposure (Saad et al. 2010; Sadi
The most common member of this family of et al. 2010). Overall, the biodegradability of PHA
polymers and often the most talked about in the materials depends on (i) the ability of degrading
scientific literature, is polyhydroxybutyrate bacteria to secrete specific extracellular PHA
(PHB; Fig. 1.2) or more accurately poly(3- depolymerases, (ii) the structure of the resulting
hdroxybutyrate). PHB when within the cell exists material, and (iii) the polymer type as PHA copo-
in a fluid, amorphous state, but after extraction, it lymers are shown to degrade faster than homo-
becomes highly crystalline (Madison and polymers (Kunioka et al. 1989). In addition, the
Huisman 1999) and is often described as pro- major advantage of PHAs is that they are believed
ducing material films that are stiff but brittle to produce no toxic biodegradation residues.
(Woolnough et al. 2008). The brittleness of PHB The diverse ranging properties that PHAs dis-
means that it is not very stress resistant. In play make them suitable for a wide range of
addition, PHB has a melting temperature in the application. PBHV, because of its greater flexibil-
region of 170 C; this is close to the temperature ity compared to PHA, makes it a suitable mate-
where PHB thermally decomposes; therefore, rial for packaging. PHAs have also generated
PHBs ability to be processed as a homopolymer interest as biomedical devices used in tissue
is limited (Madison and Huisman 1999). To repair and as bone graft substitutes (Chen and Wu
improve the material characteristics of PHB, 2005). The continuing development of PHAs,
copolymers can be formed that contain including PHA blends and composites, promises
3-hydroxyvalerate or 3-hydroxybutyrate mono- the potential for a broader range of applications
mers. Poly(hydroxybutyrate-co-hydroxyvalerate) (Madison and Huisman 1999; Reddy et al. 2013).
(PHBV) is a copolymer and is made through the
copolymerisation of 3-hydroxybutyrate with
units of 3-hydroxyvalerate to yield PHBV, a 1.4.2 Polylactic Acid (PLA)
material with modified physicochemical
properties making it a more flexible polymer. In PLA is a thermoplastic aliphatic polyes-
addition, the prosperities of PHBV are reported ter (Fig. 1.3). It can be produced by the poly-
to be easily tailored for the required application merisation of lactic acid, a metabolic product of
by varying the valerate content of the copolymer. microbial fermentation of carbohydrates present
Increasing the hydroxyvalerate content is shown in corn or sugarcane. The PLA monomer is a chi-
to increase impact strength of PHBV copoly- ral molecule and exhibits two isomeric forms,
mers. At the same time, copolymers of PHAs are D-lactic acid and L-lactic acid. PLA is considered
6 S. Lambert

that PLA is potentially not suitable for applications

that demand a high mechanical performance unless
it is modified (Soroudi and Jakubowicz 2013).
PLA is not quite suitable for industrial sectors
Fig. 1.3 Structure of polylactic acid
such as packaging because of its limited gas
barrier properties (Singh and Sharma 2008).
There is currently considerable interest in the
to be biodegradable and microorganisms capable development of modified PLA, through the
of degrading its L-isoform have been identified. development of coploymers and composites, to
However, these microbial communities are improve material characteristics such as stiffness,
thought to not be widely distributed in soil, and permeability, crystallinity, and thermal stability
soil burial studies using L-PLA over a 6-week (Reddy et al. 2013; Soroudi and Jakubowicz 2013).
time period resulted in no weight loss (Ohkita
and Lee 2006). Therefore, the degradation of
PLAs is generally favoured under conditions that 1.5 Life Cycle Analysis
will support chemical hydrolysis of ester bonds of Biopolymers
in the first step (Garlotta 2001). This process
does not require the presence of enzymes to Life cycle analysis (LCA) is a standardised
catalyse the hydrolysis, and the rate at which this method that is used to assesses a range of impact
process happens depends on the temperature of categories (e.g. acidification, carcinogens, etc.),
the degrading environment. This process then with the intention of minimising total environ-
leads to the microbial degradation of the lower mental impacts (Askham 2012). The LCA
molecular weight compounds that are produced method has been formalised by the International
(Garlotta 2001). On the other hand, D-PLA Standards Organization (ISO) and has been
is considered hydrolysable in water, but not applied, in the literature, to evaluate the environ-
biodegradable (Shogren et al. 2003; Tokiwa and mental aspects of biopolymers derived from
Calabia 2006; Kim et al. 2006). The hydrolysis renewable resources. There are a number of stud-
of this isomeric form through the backbone ies that have compared the LCA for biopolymers
ester groups is considered slow, and a process to their petrochemical counterparts. When con-
that under ambient moisture and temperature sidering the production process only, PHAs have
conditions can take several years. This process is been identified to deliver better global warming
possible to accelerate by subjecting it to tempera- potential and energy consumption scores than
tures above 50 C (Tokiwa et al. 2009). petrochemical polymers, as well as ranking as
PLA has good mechanical properties, process- environmentally superior to petrochemical poly-
ability and good thermal properties and can be mers over almost all impact categories (Harding
moulded into bottles and containers or extruded et al. 2007). lvarez-Chvez et al. (2012) also
into fibres, films and sheets (Soroudi and showed that production consumes 3050 % less
Jakubowicz 2013). PLA has also been approved fossil energy and emits 5070 % less CO2 in
by the regulatory agencies of many countries for comparison to petrochemical polymers.
medical applications such as suture threads, Predicting the environmental impacts of plastics
implantable scaffolds and bone fixation devices. made from biological resources and ranking them
Although PLA is considered a biomaterial with alongside those predicted for their petrochemical
excellent properties comparable to conventional counterparts are not easy tasks. At present,
plastics, drawbacks have been identified when bioplastics produced from PHAs seem to be the
compared to the requirements for certain applica- preferred choice. However, one of the key
tions. PLA is a very brittle material and without questions is the use of suitable feedstocks. The
the addition of additives has been shown to have preference is to use locally available and renewable
less than 10 % elongation at break; this means carbon sources that do not compete for land with
1 Biopolymers and Their Application as Biodegradable Plastics 7

food production. Of economic interest are and sandwich bags. In the food industry, packag-
agricultural cellulose waste materials that are ing has an important role in providing protection
leftover from, for example, rice, corn and sugar- from environmental, chemical and physical
cane plants, after harvesting (Koller et al. 2010; damage (during transport). Packaging materials
lvarez-Chvez et al. 2012). In addition, the provide a barrier to block light and prevent
surplus whey from the dairy industries is avail- oxygen and moisture from deteriorating food-
able in large amounts in Europe and North stuffs. PHAs because of their unique characteris-
America and is considered as a suitable feedstock tics such as good tensile strength, printability,
(Koller et al. 2010). From a life cycle perspective, flavour and odour barriers, resistance to grease
the use of agricultural by-products as a feedstock and oil and high stability to temperatures are
for bioplastic production is expected to improve highly preferred in food packaging industry
their sustainability ranking compared to petro- (Tabone et al. 2010).
chemical plastics (lvarez-Chvez et al. 2012). PHAs can also be processed into fibres for
As always the end-of-life management will nonwoven fabrics, as well as diaper materials and
also have an important role to play. Questions other compostable personal hygiene products
that remain to be answered are as follows: (i) (Madison and Huisman 1999). The medical and
Can the recycling of bio-based plastics be as an pharmaceutical industries also offer areas for
efficient option as waste disposal through, for PHA application (Box 1.1) because of their bio-
example, composting? (ii) To what extent will degradability. Biodegradable polymers also have
combining the use of bio-based plastics with the potential to offer specific advantages in the
todays collection systems affect the recycling agriculture and horticulture industries. Plastics
of petrochemical plastics? Overall, biopolymers have various agricultural applications. These
are likely to rank favourably for environmental include irrigation piping, greenhouses, low grow
impacts during LCA, because they represent a tunnels, mulches and storage (e.g. silage bails).
decrease in fossil fuel use and global warming They are desirable because they conserve mois-
potential (Tabone et al. 2010). ture thereby reducing irrigation; reduce weed
growth and increase soil temperature which
reduces competition for soil nutrients and reduces
1.6 Applications for Biopolymers

In principle, polymers derived from bio-based Box 1.1: PHA Applications Specific to
resources could potentially find utilisation in all the Medical Industry
areas. However, the shift from traditional petro-
chemical polymers to biopolymers is mostly Sutures and suture fasteners
likely to play an important role for applications Meniscus repair and regeneration devices
that have a relatively short use phase, for example, Rivets, tacks, staples and screws
the production of thin film materials for use as Bone plates and bone plating systems
packaging materials and disposable plastic bags. Surgical mesh, repair patches and car-
Presently, about 40 % of the plastics produced diovascular patches
worldwide are utilised for packaging purposes. Vein valves and bone marrow scaffolds
The disposable nature of packaging materials Ligament and tendon grafts
makes the use of biopolymers, such as these Ocular cell implants
made from PHAs, an attractive alternative. Skin substitutes, bone graft substitutes
Packaging materials are needed to pack and and wound dressings
contain a wide variety of products, such as
liquids, powders and solids. Polyethylene is a Information adapted from the text in Chen
successful material for the use of thin films that and Wu (2005); Madison and Huisman (1999)
are used to make carrier bags, cling film, freezer
8 S. Lambert

fertiliser costs thereby improving crop yields; Askham C (2012) REACH and LCAmethodological
approaches and challenges. Int J Life Cycle Assess
and protect against adverse weather conditions.
17(1):4357. doi:10.1007/s11367-011-0329-z
The development of biodegradable mulching Cavalheiro JMBT, de Almeida MCMD, Grandfils C, da
films for use in crop production offers a practical Fonseca MMR (2009) Poly(3-hydroxybutyrate) pro-
alternative to petrochemical plastics, because duction by Cupriavidus necator using waste glycerol.
Process Biochem 44(5):509515, doi:http://dx.doi.
their collection and removal from the field are
time-consuming and costly. Chen G-Q, Wu Q (2005) The application of polyhy-
droxyalkanoates as tissue engineering materials.
Biomaterials 26(33):65656578, doi:http://dx.doi.
1.7 Concluding Thoughts Cheng H, Hu Y (2010) Municipal solid waste (MSW) as a
renewable source of energy: current and future prac-
Traditionally, plastic materials have been designed tices in China. Bioresour Technol 101(11):38163824
in the past to resist degradation. For plastic items Garlotta D (2001) A literature review of Poly(lactic acid).
that have a short use phase, such as packaging
Haas R, Jin B, Zepf FT (2008) Production of Poly(3-
materials, this has created a waste management hydroxybutyrate) from waste potato starch. Biosci
problem. The use of biological systems and the Biotechnol Biochem 72(1):253256. doi:10.1271/
creation of microbial factories for the production bbb.70503
Harding KG, Dennis JS, von Blottnitz H, Harrison STL
of monomers to produce biodegradable materials
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for uses that have a relatively short use phase processes: comparing petroleum-based polypropylene
would seem likely to be a logical strategy. The and polyethylene with biologically-based poly--
challenge, therefore, is to develop biopolymers hydroxybutyric acid using life cycle analysis.
J Biotechnol 130(1):5766, doi:http://dx.doi.
that have the necessary functionality during use,
but are able to undergo full mineralisation after Jiang Y, Song X, Gong L, Li P, Dai C, Shao W (2008)
use, and leave behind no toxic residues. At pres- High poly(-hydroxybutyrate) production by Pseudo-
ent, the biopolymer market remains small com- monas fluorescens A2a5 from inexpensive substrates.
Enzym Microb Technol 42:167172. doi:10.1016/j.
pared to their petrochemical-based counterparts.
However, this market has experienced fast growth Kim DY, Kim HW, Chung MG, Rhee YH (2007)
in the past decade and global capacity is expected Biosynthesis, modification, and biodegradation of
to reach 3.45 million metric tons by 2020 bacterial medium-chain-length polyhydroxyalkano-
ates. J Microbiol 45(2):8797
(Shen et al. 2009). For now, PLAs and PHAs are
Kim DY, Kim YB, Rhee YH (2000) Evaluation of various
expected to be the major types of biopolymers in carbon substrates for the biosynthesis of polyhydroxy-
the future (Shen et al. 2009; Peelman et al. 2013). alkanoates bearing functional groups by Pseudomonas
putida. Int J Biol Macromol 28(1):2329
Kim EY, Lee JK, Lee WK (2006) Hydrolytic kinetics of
Acknowledgements The author would like to thank
langmuir monolayers of enantiorneric poly(lactide)s.
Dr. V. C. Kalia for his critical reading of the manuscript.
Curr Appl Phys 6:735738. doi: 10.1016/j.
The author received no financial support for the research,
authorship and/or publication of this article.
Koller M, Bona R, Braunegg G, Hermann C, Horvat P,
Kroutil M, Martinz J, Neto J, Pereira L, Varila P (2005)
Production of polyhydroxyalkanoates from agricultural
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Peelman N, Ragaert P, De Meulenaer B, Adons D, Peeters Scott Lambert received
R, Cardon L, Van Impe F, Devlieghere F (2013) his Ph.D. degree from the
Application of bioplastics for food packaging. Trends University of York, UK. He
Food Sci Technol 32(2):128141, doi:http://dx.doi. is currently working as a
org/10.1016/j.tifs.2013.06.003 Research Fellow at Goethe
Reddy CSK, Ghai R, Rashmi KVC (2003) Polyhydro- University Frankfurt am
xyalkanoates: an overview. Bioresour Technol 87(2): Main. His main research
137146 interests are the biodegrad-
Reddy MM, Vivekanandhan S, Misra M, Bhatia SK, ability of plastic materials
Mohanty AK (2013) Biobased plastics and bionano- for better waste manage-
composites: current status and future opportunities. ment and the environmental effects of plastics and their
Prog Polym Sci 38(1011):16531689. doi:10.1016/j. degradation products.
Approaches for the Synthesis
of Tailor-Made 2

Carlos F. Pea Malacara, Andrs Garca Romero,

Modesto Milln Ponce, and Tania Castillo Marenco

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible
thermoplastics. These have been proposed for a wide range of biotechno-
logical applications, especially in the field of the medicine and chemistry.
PHAs are produced by more than 300 bacterial species, the most efficient
being Cupriavidus necator (formerly Ralstonia eutropha), Alcaligenes
latus, and recombinant strains of Escherichia coli. PHAs are produced
by fermentation using different culture systems, from batch culture to
exponentially fed-batch cultures, and it is known that culture conditions,
such as pH, aeration, and nutritional conditions, influence the chemical
characteristic PHAs synthesized by microorganisms; because of that, it
has been proposed that by manipulating the microbial metabolism and
culture conditions, it is possible to design biopolymers with specific
chemical properties. This paper describes four cases of PHAs production:
the copolymers of poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate
[P(3HB-co-3HV)] and poly-3-hydroxybutyrate-co-poly-3-hydroxyhex-
anoate [P(3HB-co-3HHx)], the medium-chain-length PHAs, the P3HB of
ultrahigh molecular mass, and finally, the production of other short-chain-
length PHAs, with a special emphasis on the species that have been
reported for their production as well as the molecular and fermentation
strategies evaluated in order to modify the chemical composition of PHAs.

C.F.P. Malacara (*) A.G. Romero M.M. Ponce

Institute of Biotechnology, National University of
Mexico (UNAM), 62250 Cuernavaca, Morelos, 2.1 Introduction
e-mail: carlosf@ibt.unam.mx; agromero@ibt.unam.mx; Commercial interest in bioplastics has increased
due to the possibility of replacing synthetic
T.C. Marenco materials, which have disadvantages from the
Center of Research in Biotechnology (CEIB),
environmental perspective. In this regard, polyhy-
Autonomous University of Morelos State (UAEM),
62250 Cuernavaca, Morelos, Mexico droxyalkanoates (PHAs) are a suitable option to
e-mail: tania.castillo.marenco@gmail.com substitute the plastics derived from petroleum.

Springer India 2015 11

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_2
12 C.F.P. Malacara et al.

PHA R m Name of the monomer

H 1 3-hydroxypropionate (3HP)
CH3 1 3-hydroxybutyrate (3HB)
SCL H 2 4-hydroxybutyrate (4HB)

R O C2H5 1 3-hydroxyvalerate (3HV)

H O C (CH2)m C OH H 3 5-hydroxyvalerate (5HV)
n C3H7 1 3-hydroxyhexanoate (3HHx)
Polyhydroxyalkanoate C5H11 1 3-hydroxyoctanoate (3HO)
C7H15 1 3-hydroxydecanoate (3HD)
C9H19 1 3-hydroxydodecanoate (3HDD)
C9H18 1 3-hydroxydodecenoate (3HDDe)
C11H23 1 3-hydroxytetradecanoate (3HTD)
n = 1 - 230,000 monomers.

Fig. 2.1 Chemical structure of polyhydroxyalkanoates. R alkyl group, m length of carbon chains, n number of monomers

PHAs are polyesters composed of 3-hydroxy fatty et al. 2003; Pea et al. 2014a; Leong et al. 2014).
acid monomers (Fig. 2.1) (Chen 2010; Pea et al. The subjects covered by this chapter include prop-
2014a). The main advantages of these biopolymers erties of PHAs and their applications, bacterial
are their biodegradability and biocompatibility, sources and PHAs biosynthesis, as well as the
making them suitable for a wide range of applica- influence of culture conditions (i.e., medium com-
tions, from the traditional plastic industry to their position, temperature, pH, etc.), which determine
use as materials in the biomedical field, with the composition of PHAs, including specific
emphasis on chemical composition and purity of examples regarding the production of PHAs with
the product (Pea et al. 2014a; Leong et al. 2014). different chemical compositions.
PHAs are synthesized by many microorganisms as
energy reserve material. In general, it has been
well documented that these polymers are pro- 2.2 Chemical Structure,
duced under nutrient limitation, mainly nitrogen, Physicochemical Properties,
phosphorus, or oxygen (Anderson and Dawes and Applications of PHAs
1990; Pea et al. 2011, 2014a; Ienczak et al.
2013). There is a wide variability in PHA compo- Polyhydroxyalkanoates (PHAs) are polyesters
sition that includes homopolymers, heteropoly- produced and accumulated by several bacteria as
mers, and up to 150 different types of monomers a carbon and energy reservoir. These polymers
(Steinbchel and Ltke-Eversloh 2003). The ther- protect organisms against starvation and may
momechanical properties of PHAs and therefore enable them to survive under adverse conditions.
their specific applications will depend on their The PHA accumulation occurs mainly under
chemical structure, specifically monomer composi- conditions of excess of carbon and limitation of
tion (type, ratio, and distribution) and, in the case other nutrients (Anderson and Dawes 1990;
of homopolymers, their mean molecular mass Pea et al. 2011, 2014a). These polymers are
(MMM). In this line, several attempts, that include water-insoluble and they are stored in the cytoplasm
genetic manipulation of microorganisms as well as granules (Legat et al. 2010). The monomeric
as changes on the culture conditions in which the composition of PHAs depends primarily on the
cells are grown, have been evaluated in order to microbe and the type of the carbon used for
obtain materials of specific characteristics (Reddy growth. Based on their monomeric chemical
2 Approaches for the Synthesis of Tailor-Made Polyhydroxyalkanoates 13

structure, three PHA groups can be defined: the Ienczak et al. 2013); however, some of the genera
short-chain-length PHAs (SCL-PHAs), with and species listed above could bring advantages for
monomers from 3 to 5 carbon atoms, the medium- the tailor-made production of these biopolymers,
chain-length PHAs (MCL-PHAs) composed of such as Haloferax mediterranei, which produces
units from 6 to 15 carbon atoms, and, finally, the the poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
long-chain-length PHAs (LCL-PHAs) with [P(3HB-co-3HV)] copolymer in high-cell-density
monomers of more than 15 carbon atoms (Pea cultures, reaching PHA concentrations of up to
et al.2014a; Leong et al. 2014; Fig. 2.1). On the 77.8 g L1 (Huang et al. 2006). Furthermore, pro-
other hand, PHAs could also be classified as duction of PHAs using this archaea has additional
homopolymers, such as P3HB, or copolymers advantages. For example, some archaea have the
that could be found as SCL copolymers, MCL ability to grow on hypersaline conditions, using
copolymers, and SCL-MCL-PHA copolymers. inexpensive carbon sources, and the feasibility to
The thermoelastic properties of the PHAs will be lyse cells using distilled water, which could be of
influenced by the type, ratio, and distribution of great impact for PHA recovery costs (Hezayen
the monomer units (Leong et al. 2014; Table 2.1); et al. 2000; Huang et al. 2006; Poli et al. 2011). In
homopolymers of SCL-PHAs such as the contrast, Pseudomonas species are able to produce
poly-3-hydroxybutyrate P3HB are brittle and a wide range of MCL-PHAs from cheap and
stiff materials, while copolymers of MCL-PHAs renewable substrates, such as plant oils. In addi-
have improved elastomeric properties (Reddy tion, various Bacillus spp. are able to accumulate
et al. 2003). It must be emphasized that thermo- homopolymer P3HB and copolymer P(3HB-co-
processibility, biodegradability, and biocompati- 3HV) using different carbon sources as glycerol,
bility of PHAs make them of great interest for carbohydrates, and biowaste (pea shells) and also
biomedical applications such as the emerging produce MCL-PHAs, when various carbon sources
field of tissue engineering (Hazer et al. 2012; are co-fed or when this bacterium was employed as
Pea et al. 2014a; Leong et al. 2014). host for overexpression of the biosynthetic operon
phaCAB from P. aeruginosa and C. necator (Singh
et al. 2009; Kumar et al. 2009, 2013, 2015). Another
2.3 Bacterial Sources interesting case is E. coli, a nonnatural PHA pro-
of Polyhydroxyalkanoates ducer; however, recombinant strains of this bacte-
rium harboring PHA biosynthetic genes from C.
PHAs are produced by several bacterial and archaea necator, A. lata, A. vinelandii, or Pseudomonas
species (Olivera et al. 2001; Chanprateep 2010; oleovorans are important alternatives for the pro-
Pea et al. 2014a). It is noteworthy that species able duction of a wide range of PHAs, which E. coli can
to produce and accumulate these biopolymers synthesize using a wide range of substrates. E. coli
could be found in diverse environments, from can grow in high-cell-density cultures and does not
marine sediments with genera such as Vibrio, have PHA depolymerases, unlike natural PHA
Beneckea, and Paracoccus (Lpez-Corts et al. producers (Lee 1996; Olivera et al. 2001; Reddy
2010) to soil environments, where species such as et al. 2003; Chen 2009; Centeno-Leja et al. 2014;
Azotobacter vinelandii, Bacillus spp., Cupriavidus Leong et al. 2014). In addition to the production
necator, and Pseudomonas spp. are natural produc- of PHA by bacteria and archaea in pure cultures,
ers of PHAs, or even species which can be found the use of microbial mixed cultures for the produc-
on extreme hypersaline environments, in which the tion of these polymers is an attractive alternative.
haloarchaeal genera Haloferax, Halococcus, The mixed culture of several microbial species in
Halobacterium, Halorubrum, and Haloarcula are one single process allows the use of very low-cost
an interesting group for PHA production (Legat complex substrates, or mixtures of substrates, such
et al. 2010; Poli et al. 2011; Kumar et al. 2013). as those present in waste materials (derived from
Until now only two species have been successfully agro-industry or other waste sources), with no ster-
used for PHA production at a commercial scale: C. ilization requirements and with the possibility of a
necator and Azohydromonas lata (Chen 2009; continuous process (Kleerebezem and Loosdrecht

Table 2.1 Mechanical properties and applications of PHAs

Tensile strength Elongation at modulus Crystallinity
PHA (MPa) break (%) (GPa) (%) Applications References
P3HB (1400 kDa) 161 45 2.8 71 P3HB with Mw: 1143 for scaffolds for nerve Domnguez-Daz et al.
cells (2015); Chan et al. (2014)
P3HB (230 kDa) 43 5 46 Construction of cast film for the growth of Domnguez-Daz et al.
human embryonic cells (HEK293) without any (2015)
cytotoxic effect
P(3HB-4HB) (6238 48 0.65 Biomaterials for human dermal fibroblasts and Chanprateep et al. (2010)
mol%) orthopedic support. Furthermore, P(3HB-4HB)
is a potential temporary substrate that can be
used in transplantation to replace damaged
bone or skin
P(3HB/3HB-co-3HHx) 2.0 10 0.19 53.7 Construction of matrices as cell growth Li et al. (2008)
(100/7030 mol%) supporting materials for applications in skin
engineering and in nerve regeneration
P(3HB/3HB-co-3HHx) 3.5 17 0.32 50.8
(100/6040 mol%)
P(3HO-co-3HHx) 9.0 380 0.008 Scaffolds for tissue engineering of cardiac Jana et al. (2014)
(8812 mol %) valves; suitable for engineering both soft and
P(3HB-co-3HV) 32 50 1.2 hard tissues
(8020 mol %)
C.F.P. Malacara et al.
2 Approaches for the Synthesis of Tailor-Made Polyhydroxyalkanoates 15

2007). In this type of cultures, it is very difficult to P3HB, the simplest SCL-PHA, involves three
determine the species composition; however, some enzymatic reactions; the first reaction involved
studies have started to identify some of the PHA- the condensation of two molecules of acetyl-
producing species present. Particularly interesting CoA, mainly from the tricarboxylic acid (TCA)
are those works reporting Alcaligenes, Azoarcus, cycle, into acetoacetyl-CoA by the -ketothiolase
Amaricoccus, Comamonas, Achromobacter, (encoded by phaA). Then, acetoacetyl-CoA gets
Pseudomonas, Kluyvera, Acine-tobacter, reduced to 3-hydroxybutyryl-CoA (3HB-CoA)
Paracoccus, Xanthobacter, Curto-bacterium, with the help of the enzyme acetoacetyl-CoA
Flavobacterium, and Thauera (Dionisi et al. 2005, reductase (encoded by phaB). Finally, the PHA
2006, 2007; Serafim et al. 2006; Lemos et al. synthase (encoded by phaC) polymerizes the
2008) as the dominant genera present in mixed 3-hydroxybutyryl-CoA monomers to P3HB,
cultures promoting high PHA accumulation. with the subsequent liberation of CoA (Stubbe
et al. 2005; Pea et al. 2011, 2014a). However,
biosynthesis of PHAs with different monomeric
2.4 PHA Biosynthesis Pathways compositions involved biosynthetic pathways
of hydroxyacyl-CoA thioester precursors
PHAs biosynthesis and its regulation has been (Fig. 2.2). In the case of the biosynthesis of
well documented (Anderson and Dawes 1990; SCL-copolymers such as P(3HB-co-3HV), two
Slater et al. 1992; Steinbuchel and Schlegel, pathways are involved, leading to C4 monomer
1991; Pea et al. 2011, 2014a). The synthesis of (3-hydroxybutyryl-CoA) or to C5 monomer

Fig. 2.2 Pathways involved in the biosynthesis of polyhy- are shown. Abbreviations: ACP acyl-carrier protein, 3HB
droxyalkanoates. Amino acid metabolic pathways, the tri- 3-hydroxybutyric acid, 3HA 3-hydroxyalkanoic acid, HV
carboxylic acids cycle, butyrate metabolism, fatty acid hydroxyvaleric acid, 4HB 4-hydroxybutyric acid, HHx
biosynthesis, and -oxidation pathways (from left to right) hydroxyhexanoic acid, MCL medium chain length
16 C.F.P. Malacara et al.

(3-hydroxyvaleryl-CoA) (Steinbuchel and organisms, carbon sources can vary and affect
Schlegel 1991). As it has been previously MCL-PHA production. The capability of incor-
described, the synthesis of the monomer of porating different hydroxyacyl-CoA units will be
3-hydroxybutyryl-CoA involves the condensation dependent on the PHA synthase (phaC). There
of two molecules of acetyl-CoA and its further are two types of these enzymes: the Type I which
reduction to 3-hydroxybutyryl-CoA which will is harbored by organisms such as C. necator and
be available for its incorporation into the copo- synthesizes SCL-PHAs and the Type II which is
lymer by the PHA synthase (phaC). On the present mainly in Pseudomonas and is able to
other hand, formation of the 3-hydroxyvaleryl- polymerize MCL-PHAs.
CoA involves the condensation of acetyl-CoA
and propionyl-CoA into 3-ketovaleryl-CoA, a
reaction catalyzed by the -ketothiolase (phaA). 2.5 Effect of the Culture
Afterward 3-ketovaleryl-CoA is reduced by the Conditions on the PHAs
acetoacetyl-CoA reductase (phaB) into the Synthesized by Native
monomer 3-hydroxyvaleryl-CoA which could and Recombinant Bacteria
be incorporated into the growing polymer chain
by the PHA synthase or polymerase (phaC). In It is known that the culture conditions affect the
this case, the propionyl-CoA, precursor of the chemical characteristics of PHAs synthesized by
3-ketovaleryl-CoA, could be the result of the microorganisms, and those chemical properties
amino acid metabolism, from threonine, which have an important effect on the mechanical prop-
could be converted in -ketobutyrate and then erties and therefore the final applications of PHAs.
reduced to propionyl-CoA with the help of the In this section, some cases regarding the manipu-
enzyme pyruvate dehydrogenase (Slater et al. lation of the chemical composition of PHAs
1998; Fig. 2.2), or it could be synthesized through by the manipulation of strains and the culture
-oxidation during the growth of bacteria on fatty conditions will be discussed (Tables 2.2 and 2.3).
acids, amino acids, and other substrates that
can first be converted into fatty acids (Steinbuchel Case 1: Production of the Heteropolymers
and Schlegel 1991). P(3HB-co-3HV) and P(3HB-co-3HHx)
For the biosynthesis of the MCL-PHAs, which The copolymers poly(3-hydroxybutyrate-co-3-
are composed of C6 to C15, two pathways are hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-
involved: one of these is the biosynthesis and hydroxybutyrate- co-3-hydroxyhexanoate)
degradation of fatty acids (-oxidation pathway), [P(3HB-co-3HHx)] are conformed by monomers
wherein a wide variety of substrates are available of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate
for the polymer production (Lageveen et al. 1988; (3HV) or 3-hydroxyhexanoate (3HHx), respec-
Timm and Steinbchel 1990). From the fatty acid tively. Both copolymers are interesting candidates
metabolism, precursors such as enoyl-CoA, as alternative materials for replacement of petro-
hydroxyacyl-CoA, and ketoacyl-CoA could be chemical plastics. In the case of P(3HB-co-3HV),
used as substrates for the PHA polymerase for this was manufactured and commercialized by
their further conversion into MCL-PHAs (Kraak ICI (Biopol), Zeneca BioProducts, Biomer Inc.
et al. 1997; Lageveen et al. 1988; Fig. 2.2). Fatty (Biomer), and Tianan Biologic (Enmat) (Braunegg
acid biosynthesis is built by adding two carbons et al. 1998; Chanprateep 2010).
through intermediates linked to acyl-carrier Due to the presence of 3HV or 3HHx residues,
protein (ACP), whereas in the -oxidation path- polymer crystallinity is reduced, and these
way, two carbons are reduced from the fatty acyl residues contribute to an increase of flexibility,
substrates, the whole process liberates a molecule elasticity, and melting temperature as compared
of acetyl-CoA in each cycle and their intermediates with the homopolymer P3HB (Feng et al. 2002;
are linked to CoA (Fig. 2.2). Although, both Zhuang et al. 2014). In this line, when the molar
fatty acid metabolic pathways are present in all ratio of 3HV is of only 20 mol %, the copolymer
2 Approaches for the Synthesis of Tailor-Made Polyhydroxyalkanoates 17

Table 2.2 Production of polyhydroxyalkanoates under different culture conditions

Biomass PHA content
PHA Organism Production scale Culture conditions (g L1) (%) References
P3HP Recombinant E. Fed-batch Crude glycerol Pure 5.2 5.2 Andreeen
coli fermentation 2 L glycerol For both: 300 12.0 11.8 et al.
mM, 37oC and (2010)
400 rpm for 92 h
P3HB, Recombinant E. Shake flasks Decanoate Glucose 2.53.0 1.89.3 Li et al.
P3HHx, coli and decanoate Cultures 4.35.1 5.936.4 (2011)
P3HO, kept at 37 oC and
P3HD 200 rpm
P4HB Recombinant E. Bioreactor 1 L Glycerol (20 g L1) N.S. 2.061.0 Kmpf
coli with K-4HB (4 g L1), 6.56.7 63.065.0 et al.
32 C, pH 7, 800 rpm, (2014)
30 % DO2, 1 L air/min,
for 48 h Glucose (20 g
L1) with K-4HB (4 g
L1), propionic acid,
and NZ-amines (1 g
L1), 32 C, pH 7, 800
rpm, 30 % DO2, 1 L
air/min. for 48 h
P(3HB-co- E. coli LS5218 Shake flasks M9 medium supplied 7.8 14.1 Wang et al.
3HHx) (pBBJPC) with glucose (20 g (2015)
(9010 L1), 37 C, 200 rpm,
mol %) for 48 h
P3HP E. coli Q1911 Baffled shake 100 mL of minimal 4.9 10.2 Wang et al.
(harboring both flasks 500 mL medium with glycerol (2014)
pHP302 and (20 g L1) and glucose
pHP513) (3 g L1)
P(3HB- E. coli LS5218 Shake flasks batch 50 mLof 6.5 12.1 Zhuang
3HHx-3HO- 300 mL medium supplemented et al.
3HD-3HDD- with glucose (30 g (2014)
3HTD) L1), 30 C, 250 rpm,
(21.2, 6.1, for 72 h
45.8, 11.0,
9.2, 6.8 mol
P(3HB-co- E. coli XL10 Fed-batch (first Continuous feeding of 39.8 60.5 Liu et al.
3HV) (8515 stage, glucose; glucose (20 g L1) and (2009)
mol %) second stage, propionic acid (2 g L1)
P(3HB-co- C. necator Shake flasks 250 Butyrate (0.5 %) 0.6 65 Jeon et al.
3HHx) Re2133/pCB81 mL (2014)
(6040 mol
N.S. not specified

has an excellent strength and flexibility (Luzier tissue engineering (Yang et al. 2002; Chen and
1992). Besides, in some cases, these copolymers Wu 2005; Table 2.1).
have better biocompatibility compared to either The ability to produce these copolymers is
P3HB or polylactic acid, which makes them directly attributed to the specificity of polymerase
promising materials for medical fields, for exam- synthase, which has been characterized only at
ple, in cardiovascular problems, wound-healing preliminary level in Bacillus sp. (Lee et al. 2008).
process, orthopedic issues, drug delivery, and The P(3HB-co-3HV) is synthesized by several
18 C.F.P. Malacara et al.

Table 2.3 Culture conditions for the P3HB production with different molecular masses using recombinant E. coli
strains and Azotobacter species
Mean Production Culture conditions Biomass PHB
molecular scale (g L1) content
mass (%)
Mw or Mn*
Organism (kDa) References
E. coli XL-1 Blue 20,000* Bioreactor LB medium with 7.4 48 Kusaka
(pSYL105) 2.6 L glucose 20 g L1, pH et al.
6, 37 C (1997)
E. coli JM109 1800* Shake flasks LB medium with 3.2 33 Agus et al.
(pGEM-phaCReAB) 500 mL with glucose 20 g L1, 37 (2006)
E. coli JM109 4000* 100 mL C, 14 h culture time 5.8 51
(pGEM-phaCDaAB) medium
E. coli JM109 380* 2.7 24
E. coli JM109 17048* Shake flasks LB medium with 7.99.2 5461 Agus et al.
(pGEM-phaRCBspAB) glucose 20 g L1, 37 (2010)
C at 14 and 60 h of
culture time
E. coli JM109 1800* Shake flasks LB medium with 5 24 Agus et al.
(pGEM-phaRCBspAB) glucose 20 g L1, 25 (2010)
E. coli DH5 20006200 Shake flasks LB medium with 8.011.2 3157 Hiroe
(pGETS109-pha) with glucose 20 g L1, 30 et al.
order gene phaABC, C, 130 rpm at 72 h (2012)
phaACB, phaBAC,
phaBCA, phaCAB, and
A. vinelandii UWD 4100 Shake flasks 5 % (w/v) beet N.S. N.S. Chen and
molasses at 24 h Page,
A. vinelandii OPN 3670 Shake flasks Low aeration N.S. 62 Pea et al.
(ptsIIANtr-) conditions (200 mL (2014b)
PY medium)
A. chroococcum 7B 2215 Shake flasks Microaerophilic 2.87 61.3 Myshkina
conditions et al.
N.S. not specified

bacteria such as C. necator, some species of P(3HB-co-3HV) copolymer or increasing the

Bacillus, Azotobacter, recombinant strains of 3HV fraction are based on the strategies to
E. coli, and Haloferax mediterranei. This last is improve propionate utilization. On the other
a natural P(3HB-co-3HV) producer (Don hand, the P(3HB-co-3HHx) is generally pro-
et al. 2006). The composition of P(3HB-co-3HV) duced from plant oil and fatty acids by several
produced by bacterial sources could be manipu- wild-type and recombinant bacteria. Previous
lated by the kind of carbon sources employed. reports have shown that it is possible to produce
Several studies have shown that the supply of P(3HB-co-3HHx), containing greater than 20 %
propionyl-CoA in cells is the key factor for the content of 3HHx monomer, using plant oils as
production of the 3HV fraction during the syn- carbon source (Kahara et al. 2004; Budde et al.
thesis of P(3HB-co-3HV) (Aldor et al. 2002). 2011; Riedel et al. 2012). More recently, Jeon
Therefore, most attempts aimed to produce et al. ( 2014 ) demonstrated that engineered
2 Approaches for the Synthesis of Tailor-Made Polyhydroxyalkanoates 19

C. necator can produce P(3HB-co-3HHx), when via citramalate pathway. When this strain was
this strain was grown on mixed acids or on cultured in a defined medium having 20 g L1 of
butyrate as carbon source. This strain produced a glucose as carbon source, P(3HB-co-3HV) was
polymer containing up to 40 wt % of 3HHx produced up to polymer content of 61.7 % based
monomer. It is important to point that this was the on dry weight. Furthermore, the 3HV monomer
first report for the production of P(3HB-co- fraction in P(3HB-co-3HV) increased up to 5.5
3HHx) copolymer in C. necator using butyrate. mol % by additional deletion of the genes respon-
In this section the more recent attempts to sible for the metabolism of propionyl-CoA (prpC
improve composition and production of the copo- and scpC). Another interesting case is that of
lymers P(3HB-co-3HV) and P(3HB-co-3HHx) Salmonella enterica serovar Typhimurium that by
will be discussed. expression of the E. coli (2R)-methylmalonyl-
Choi and Lee (1999) described a strategy for CoA mutase (YliK) and (2R)-methylmalonyl-
production of copolymer P(3HB-co-3HV) at CoA decarboxylase (YgfG) was able to
high concentration using a recombinant strain of biosynthesize P(3HB-co-3HV) from a single car-
E. coli with different feeding solutions contain- bon source through the generation of propionyl-
ing propionic acid and glucose. In that study, a CoA from succinyl-CoA (Aldor et al. 2002).
maximal copolymer concentration of 141.9 g L1 It is important to point out that the production
with a P(3HB-co-3HV) up to 62.1 wt % and a cost of these polymers can be significantly
3HV component of 15.3 mol % was reached. It reduced by using activated sludge instead of pure
has been reported that the copolymer composi- substrates. This step enables easy operation,
tion can be manipulated by adding propionate in since this does not require sterile conditions and
the feed (Fidler and Dennis 1992; Slater et al. uses renewable substrates as carbon sources
1992, 1998; Yim et al. 1996; Choi and Lee (Bosco and Chiampo 2010). Others have focused
1999). However, industrial production of propio- on the use of dairy waste (Pandian et al. 2010),
nate is more expensive than glucose (Poirier et al. sewage water (Hu et al. 1997; Wong et al. 2000),
1995; Aldor et al. 2002) making difficult the waste from food processing industry (Wong et al.
scale-up process for P(3HB-co-3HV) production. 2000), as well as agricultural feed stocks
In addition, propionate being toxic must be fed at (Solaiman et al. 2006). More recently, Narayanan
relatively low concentrations (Steinbchel and et al. (2014) reported that the culture of B. mycoi-
Ltke-Eversloh 2003). An alternative strategy des DFC1 in rice husk hydrolyzate in combina-
has been to design genetically modified strains in tion with gluten hydrolyzate resulted in maximum
which it is possible to induce the expression of a synthesis of P(3HB-co-3HV) in the presence of
critical gene in the polymer-producing pathway valeric acid as co-substrate at different induction
(Aldor and Keasling 2001). Some examples of intervals and concentrations.
genetic modifications that increased P(3HB-co- On the other hand, focus has been on the pro-
3HV) synthesis have been reported in different duction of P(3HB-co-3HHx), which take advan-
bacteria. For example, Yang et al. (2012), by tage of the microbial fatty acid degradation
introducing the genes of propionyl-CoA transfer- pathways (Khanna and Srivastava 2005; Budde
ase (pct), -ketothiolase (bktB), acetoacetyl-CoA et al. 2011). A recent report of P(3HB-co-3HHx)
reductase (phaB) and PHA synthase (phaC) production by C. necator has showed that the
from C. necator into E. coli strain YH090, were engineered C. necator accumulated P(3HB-co-
able to produce P(3HB-co-3HV) with an ultra- 3HHx) from fructose via the inverted -oxidation
high 3HV monomer composition reaching over pathway (Insomphun et al. 2015). Since the met-
80 wt %. More recently, Yang et al. (2014) abolic flux from acetyl-CoA to 3HB-CoA was
reported the E. coli strain (XL-1) harboring E. too high in the natural PHA producer C. necator,
coli poxB L253F V380A gene along C. necator a cellular content of 48 wt % P(3HB-co-3HHx)
prpE (propionyl-CoA synthase) and phaCAB composed of 22 mol % 3HHx was obtained. In
genes, which was able to produce propionyl-CoA this context, Wang et al. (2015) with the purpose
20 C.F.P. Malacara et al.

to produce P(3HB-co-3HHx) from glucose as and the predominant monomers are C8

carbon source designed an E. coli recombinant (3-hydroxyoctanoate; 3HO), C10, and C12
strain, where they combined the BktB-dependent (3-hydroxydodecanoate; 3HDD) (Madison and
condensation pathway with the inverted Huisman 1999; Nitschke et al. 2011). In addi-
-oxidation cycle pathway, by cloning five tion, Pseudomonas spp. produce MCL-PHAs due
exogenous genes (bktB, phaB1, phaJ, ter, and to their PHA synthases (type II), which are able
phaC). The resultant recombinant strain was able to polymerize hydroxy acids of short and medium
to produce a copolymer with a 3HHx fraction of chain length (3HASCL and 3HAMCL), covalently
10 mol %. On the other hand, the biosynthesis linked within the same polyester molecules
of P(3HB-co-3HHx) from sugars involves an (Steinbchel and Ltke-Eversloh 2003; Chen
artificial pathway that allowed to build up the et al. 2014). MCL-PHAs such as poly-3-
C6-monomer from three acetyl-CoA molecules, hydroxydodecanoate (P3HDD) and poly-3-
which is a challenge from metabolic engineering hydroxyoctanoate (P3HO) are of commercial
point of view. Based on this strategy, the recom- interest, because these polymers exhibit a consid-
binant E. coli strain PHB4 designed by Fukui erable interval of thermomechanical properties
et al. (2002) was able to accumulate P(3HB- and elastomeric behavior.
co-3HHx) up to 48 wt % of dry weight from fruc- Simon-Colin et al. (2008) showed that P. gue-
tose, although the (3HHx) monomer composition zennei was able to produce MCL-PHA copoly-
in the copolymer was lower than 1.5 mol %. mers with a great diversity in their structures and
properties. The carbon sources include saturated
Case 2: Production of Medium-Chain-Length and unsaturated monomers, from C4 to C14, but
PHAs (MCL-PHAs) preferably C8 and C10 monomers. Furthermore,
The MCL-PHAs may be used in diverse applica- this strain was able to use a broad range of carbon
tions due to their better physical and mechanical sources as carbohydrates or fatty acids. For
properties as compared with the SCL-PHAs example, when the strain was grown in glucose,
(Table 2.1). The MCL-PHAs are characterized cells accumulated 3-hydroxybutyrate (3HB: 1.3
due to their low degree of crystallinity, low melt- mol %), 3-hydroxyhexanoate (3HHx: 0.9
ing point, and glass-transition temperatures com- mol %), 3-hydroxyoctanoate (3HO: 22 mol %),
bined with their improved flexibility, elasticity, 3-hydroxydecanoate (3HD: 62.8 mol %),
and sticky properties that are required for appli- 3-hydroxydodecanoate (3HDD: 6.2 mol %),
cations in certain biomedical areas (Abe et al. 3-hydroxydodecenoate (3HDDE: 5.6 mol %),
2012; Chen et al. 2014; Table 2.1). Pseudomonas and 3-hydroxytetradecanoate (3HTD: 1.2 mol
spp. are able to produce MCL-PHAs, and their %). In contrast, when the bacterium was culti-
composition is directly related to the carbon vated using oleic acid as carbon source, the
source used as growth substrate. This is because MCL-PHAs included 3HTD (13.8 mol %) with
the former monomers, as it was previously less fraction mol of monomer 3HD (35.6 mol %).
described (Fig. 2.2), are derived from intermedi- In another study, Simon-Colin et al. (2012) using
ates of fatty acid biosynthesis or -oxidation sodium octanoate as sole carbon source observed
pathways; therefore, the nature of the PHA that P. guezennei synthesized MCL-PHAs mainly
monomers produced by Pseudomonas species composed of 3HO accounting for up to 94 mol %
will depend on the metabolism of the specific and lower amounts of 3HHx and 3HD. Recently,
carbon sources. When the carbon sources are in cultures of the P. fulva strain TY16 grown on
carbohydrates, P. aeruginosa accumulates petrochemical wastes as carbon source, the pro-
C10 (3-hydroxydecanoate; 3HD) from the bio- duction of MCL-PHAs was reported (Ni et al.
synthetic fatty acid pathway as the predominant 2010). Interestingly, when this strain was grown
monomer. However, when the carbon source in glucose, toluene, benzene, ethylbenzene, glu-
used are fatty acids, the precursors for the PHA conic acid, and acetic acid, it was able to synthe-
synthesis are produced by -oxidation pathway, size MCL-PHA copolyesters, containing saturated
2 Approaches for the Synthesis of Tailor-Made Polyhydroxyalkanoates 21

and unsaturated units of 3HDD, 3HHx, 3HO, and mer was 3HD, except for PHAs produced at 42
3HD. On the other hand, copolyesters MCL- C in which 3HO was the monomer present in
PHAs synthesized by P. fulva strain TY16 from greater proportion (43.2 %). At elevated tempera-
octanoic and decanoic acids were composed of tures, long chain monomers such as 3HD, 3HDD,
repeating units of 3HHx, 3HO, and 3HD with a and C14:1 decreased, whereas at 37 C the con-
mean molecular mass (MMM) between 42 and tent of unsaturated monomers (C12:1, C14:2,
43 kDa (Ni et al. 2010). C14:1) increased. Later on a significant decrease
Another interesting case was reported using was observed at 42 C (Haba et al. 2007). Another
the strain of P. putida KT2440, grown with interesting example is the production of MCL-
nonanoic acid and glucose as carbon sources at a PHAs by cultures of P. mediterranea using
1:11.5 (w/w) ratio for the PHA production. reagent-grade or partially refined glycerol
Under such conditions, this strain accumulated a (Pappalardo et al. 2014). The gas chromatogra-
biopolyester with the following composition: phy analysis indicated that the biopolymer struc-
3-hydroxynonanoate (66 mol %), ture was composed by six monomers: 3HHx,
3-hydroxyheptanoate (32 mol %), and 3HV (1 3HO, 3HD, 3HDD, cis 3-hydroxydodec-5-enoate
mol %). The terpolymer produced exhibited a (C12:15), and cis 3-hydroxydodec-6-enoate
MMM of 11 kDa, with a polydispersion index of (C12:16).
1.8 (Sun et al. 2009). Chan et al. (2014) evaluated Besides Pseudomonas, recombinant strains of
the PHA production employing the strain of P. E. coli are also an alternative for production of
mosselii TO7 through utilization of plant oils MCL-PHAs. For example, Li et al. (2011)
such as soybean and palm kernel oil as carbon reported an E. coli recombinant strain harboring
sources. These authors demonstrated that this phaA and phaB genes from C. necator and the
strain accumulated up to 50 % (cell dry weight) phaC2 gene from P. stutzeri (pCJY02). When
of poly-3-hydroxyoctanoate (P3HO) achieving a this strain was cultivated in medium with decano-
productivity of 2.05 g PHA L1 h1. ate, the bacterium accumulated SCL-MCL-PHAs
Hori et al. (2011) evaluated the effect of with a monomer composition of 3HB, 3HHx,
temperature (within a range from 15 to 30 C), in 3HO, and 3HD in mol ratios of 43.2:12.8:10.3:33.6.
the biosynthesis of the PHAs produced by P. However, when this strain was grown on decano-
aeruginosa IFO3924. The results indicated that ate and glucose, the recombinant strain synthe-
the MCL-PHA composition was closely depen- sized the same biopolymer, but the mol ratios
dent on the temperature and the culture time in were 3HB (83.4), 3HHx (4.0), 3HO (5.6), and
which the biopolymer accumulation is carried 3HD (7.0). These results indicated that it is pos-
out. At the beginning of the culture, 3HD and sible to modulate the monomer content and type
3-hydroxydodecenoate (C12:1) units were found of the PHA accumulated by adding different car-
in the PHA samples at all temperatures evaluated bon sources and manipulating metabolic path-
(15, 20, and 25 C). In contrast, the 3HO was ways of the host. In the same line, Zhuang et al.
detected only at 30 C. On the other hand, when (2014) designed in its E. coli host metabolic
the maximum cellular content of PHA was pathways to synthesize MCL-PHAs directly
achieved, 3HO and 3HD were the major monomer from glucose. Engineering the reversed fatty acid
units present at all the temperatures tested. -oxidation cycle, Zhuang et al. (2014) employed
Haba et al. (2007) studied the effect of tempera- this route to generate the key intermediates for
ture (in the range of 1842 C) on PHA composi- the production of MCL-PHAs in E. coli. By using
tion in cultures of P. aeruginosa 47 T2. In this a PHA synthase with broad substrate specificity
study, P. aeruginosa was grown in mineral and using glucose as carbon source, recombinant
medium supplemented with urea as nitrogen E. coli was able to produce MCL-PHA copoly-
source and 2 % of waste cooking oil. The results mers with monomer composition ranging from 4
obtained indicated that the most abundant mono- to 14 carbons. The PHA compositions in mol %
22 C.F.P. Malacara et al.

were 3HB:21.2, 3HHx:6.1, 3HO:45.8, 3HD:11.0, conditions of the culture and therefore the
3HDD:9.2, and 3HTD:6.8 (Table 2.2). oxygen availability, it is possible to modify the
MMM of the P3HB. Similar results were reported
Case 3: Production of Poly-3- by Myshkina et al. (2008); these authors evaluated
hydroxybutyrate of High Molecular Mass the P3HB production by A. chroococcum 7B,
(HMM-P3HB) under microaerophilic conditions. Under low
The third case of microbial PHAs is the poly-3- aeration condition, this strain was able to synthe-
hydroxybutyrate (P3HB), the common homopol- size P3HB with a MMM of 2215 kDa and an
ymer of SCL; it is composed by monomers of increase on aeration negatively affected the
3-hydroxybutyrate (3HB), which are linked by MMM of P3HB. They also found that the optimal
ester bonds between the hydroxyl group and the temperature for P3HB production with a high
carbonyl groups of the two adjacent monomers molecular mass was 30 C; in contrast, at low
(Fig. 2.1). This polymer has similar thermome- (20 C) or high (37 C) temperatures, the MMM
chanical properties to those found in conven- decreased.
tional petrochemical plastics (Chanprateep et al. On the other hand, an interesting example for
2010), and these properties of P3HB are highly production of P3HB with a high molecular mass
dependent on the mean molecular mass (MMM) is that reported for E. coli recombinant strains
of the polymer (Pea et al. 2014a; Domnguez- harboring the C. necator biosynthesis phbCAB
Daz et al. 2015). Previous reviews have pointed genes (Kusaka et al. 1997). These authors
out two interesting bacterial sources for the reported, for first time, the production of a
production of P3HB of high molecular mass polymer with ultrahigh molecular mass (20,000
(HMM-P3HB): one of them belongs to the genus kDa) during the stationary phase of growth by
Azotobacter, which is able to accumulate P3HB culturing E. coli XL-1 Blue (pSYL105), in a bio-
that exhibits a high molecular mass (>1000 kDa), reactor of 2.6 L, under controlled pH conditions
and the other are recombinant strains of E. coli at 6.0 with LB medium supplemented with glu-
(Pea et al. 2014a; Leong et al. 2014). Some of cose (20 g L1). Interestingly, when this E. coli
the most relevant cases are discussed here. strain was grown at pH within a range of 7.08.0,
Several authors have studied the culture the molecular mass of P3HB decreased to values
parameters that could affect the MMM of the below 5000 kDa after 12 h of culture.
P3HB synthesized by Azotobacter, finding that Another interesting case was reported by Agus
the composition of the culture medium, the et al. (2006), who demonstrated that MMM of the
oxygen availability, and temperature are some of P3HB accumulated by recombinant strains of E.
the factors that could have an important effect on coli depends on the specific PHA synthase (type
the MMM. For example, Chen and Page (1994) and organism of origin) employed. Their results
observed that the UWD strain of A. vinelandii indicated that P3HB with high number-average
accumulated P3HB with a high MMM (4100 molecular mass (Mn: 1500-4000 kDa) were syn-
kDa), when it was cultivated using beet molasses thesized by PHA synthases from C. necator (type
of 5 % (w/v) in contrast with cultures without this I), Delftia acidovorans (type I), and
substrate. These authors suggested that the nitro- Allochromatium vinosum (type III). P3HB with
gen compounds of the beet molasses such as the lowest Mn (170790 kDa) were accumulated
organic acids and salts stimulate the synthesis of by PHA synthases from Aeromonas caviae (type
P3HB of very high molecular mass (Chen and I), Pseudomonas sp. (type III), and Bacillus sp.
Page 1994). More recently, Pea et al. (2014b), (type IV). On the contrary, these authors found
in shaken flask cultivations of mutant strain OPN, out that the highest MMM were obtained using
reported a polymer with an MMM of 3670 270 the PHA synthase from D. acidovorans (4000
kDa in cultures conducted under low aeration kDa). They also observed that for the strain har-
conditions (conventional shaken flasks) as boring the PHA synthase from D. acidovorans,
compared with cultures under high aeration. an acid pH (4.8) favored the P3HB production
They proposed that by manipulating the aeration with high Mn (2100 kDa) as compared with the
2 Approaches for the Synthesis of Tailor-Made Polyhydroxyalkanoates 23

biopolymer produced under basic conditions (pH 2007). Recently, Kmpf et al. (2014) investigated
7.47.8), where the Mn was 1500 kDa, and these the production of P4HB using recombinant E.
results were similar to those observed by Kusaka coli JM109 that harbors a 4-hydroxybutyric acid
et al. 1997. When they investigated the effect of CoA transferase gene (orfZ) from Clostridium
temperature on the Mn of P3HB using E. coli kluyveri, using glycerol and propionic acid. They
recombinant strains with PHA synthase D. found that biopolymer accumulation in the cells
acidovorans, they found that at 37 C the bio- was of 80 % (dry weight) achieving 3.7 gL1
polymer exhibited a higher Mn (4300 kDa) than (Table 2.2). On the other hand, Le Meur et al.
that accumulated in the condition of 30 C (580 (2013) reported that recombinant E. coli JM109
kDa). In contrast, when Agus et al. (2006) used a was able to produce P4HB using xylose as car-
recombinant E. coli strain with PHA synthase bon source and sodium-4-hydroxybutyrate
from Bacillus sp., they found a different behavior (Na-4HB) as biopolymer precursor. The highest
to that observed with the strain containing PHA P4HB concentration achieved was 4.33 g L1
synthase from D. acidovorans. They found that with a yield (YP4HB/Na-P4HB) of 92 % g g1. Also, Le
the Mn of P3HB produced by the strain with Meur et al. (2014) using fed-batch high-density
PHA synthase from Bacillus sp. in the condition bacterial mass using glycerol as the sole carbon
of 37 C was lower (440 kDa) than the Mn of the source along with precursor 4HB for biopolymer
P3HB synthesized at 25 C (nearly 1900 kDa). synthesis achieved a concentration of 15 g L1 of
On the other hand, through rearrangement of P4HB.
gene order of phbCAB operon biosynthesis The last example is the poly-3-
(phaABC, phaACB, phaBAC, phaBCA, phaCAB, hydroxypropionate (P3HP), which combines the
and phaCBA) in recombinant E. coli DH5 properties of P3HB and poly-2-hydroxypropionate
(pGES109-pha), Hiroe et al. (2012) found that it (known as polylactic acid). Andreeen et al.
was possible to produce P3HB with different (2010) reported the conversion of glycerol to
MMM in the range between 2000 and 6200 kDa. P3HP in an E. coli recombinant strain, harboring
The results indicate that the MMM of P3HB genes encoding for glycerol dehydratase (dhaB1)
accumulated by the six strains was higher during of Clostridium butyricum, the propionaldehyde
the exponential growth phase (12 h of cultiva- dehydrogenase (pduP) of Salmonella enterica,
tion) as compared with the biopolymer produced and the PHA polymerase (phaC1) of C. necator.
at the stationary phase (72 h). They also found an After 92 h of incubation at 37 C with 300 mM of
inverse correlation between MMM and P3HB pure glycerol, 1.42 gL1 of P3HP were achieved
synthase activity, in contrast to the accumulation with a yield of 17.5 mmol P3HP mol glycerol1 con-
percentage (quantified as dry weight), which sumed, with the drawback production of ethanol
increased as the synthase activity increased (8.04 gL1), succinate (48.92 gL1), and acetate
(Hiroe et al. 2012; Table 2.3). (0.26 gL1) as by-products. Another case was
reported by Wang et al. (2014). They built a
Case 4: Production of Homopolymers of recombinant strain of E. coli (with panM, panD,
Short Chain Length: Poly-4-hydroxybutyrate pp0596, ydfG, prpE, and phaC1) for the P3HP
(P4HB) and Poly-3-hydroxypropionate production. This strain was able to produce 0.5 g
(P3HP) L1 of biopolymer when it was cultivated in
Another example of SCL-homopolymers is the shaken flasks, using glycerol and glucose as car-
poly-4-hydroxybutyrate (P4HB; Fig. 2.1), which bon sources and without any addition of precur-
is one of the most promising PHA for biomedical sors. In cultures of the same strain in stirred
applications, because of its unique properties, bioreactors in fed-batch aerobic cultures, they
which include biodegradability, biocompatibility, obtained up to 10.1 g L1 of P3HP (Wang et al.
nontoxicity, and superior mechanical properties. 2013). In the same line, Gao et al. (2014) designed
It must be emphasized that synthesis of P4HB a recombinant stable E. coli strain harboring
requires precursor like 4-hydroxybutyric acid, seven exogenous genes of P3HP synthesis
1.4-butanediol, or -butyrolactone (Valappil et al. pathway. This strain in aerobic fed-batch cultures
24 C.F.P. Malacara et al.

was able to produce 25.7 g L1 of biopolymer during cultivation of PHA-accumulating Escherichia

coli. Polym Degrad Stab 95:22502254. doi:10.1016/j.
from glycerol.
Aldor I, Keasling JD (2001) Metabolic engineering of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in
2.6 Perspectives recombinant Salmonella enterica serovar
Typhimurium. Biotechnol Bioeng 76:108114.
PHAs are biomaterials of great importance not Aldor I, Kim SW, Prather KL, Keasling JD (2002)
only due to their biodegradability and thermome- Metabolic engineering of a novel propionate-
chanical capabilities similar to those of the independent pathway for the production of poly(3-
hydroxybutyrate-co- 3-hydroxyvalerate) in
plastics derived from the petrochemical industry
recombinant Salmonella enterica serovar
but also due to their biocompatibility, which is a typhimurium. Appl Environ Microbiol 68:38483854.
characteristic required in medical and biomedical doi:10.1128/AEM.68.8
fields. In addition, the success of application of Anderson AJ, Dawes EA (1990) Occurrence, metabolism,
metabolic role, and industrial uses of bacterial polyhy-
these biopolymers will depend on their chemical
droxyalkanoates. Microbial Rev 54:450472,
nature, mainly the monomer composition and doi:0146-0749/90/040450-23
mean molecular mass, and other properties which Andreeen B, Lange AB, Robenek H, Steinbchel A
influence the mechanical properties, biodegrad- (2010) Conversion of glycerol to poly(3-
hydroxypropionate) in recombinant Escherichia coli.
ability, and biocompatibility of PHAs. Current
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advances in fermentation, purification technology, AEM.02097-09
as well as the design of mutant strains by recom- Bosco F, Chiampo F (2010) Production of polyhydroxyal-
binant DNA technology would allow the tailor- canoates (PHAs) using milk whey and dairy wastewa-
ter activated sludge production of bioplastics using
made production of new PHAs. These tailor-made
dairy residues. J Biosci Bioeng 109:418421.
PHAs can be used as materials for biomedical doi:10.1016/j.jbiosc.2009.10.012
uses, such as tissue engineering. From the eco- Braunegg G, Lefebvre G, Genser KF (1998)
nomic viewpoint, the efforts are now focusing on Polyhydroxyalkanoates, biopolyesters from renewable
resources: physiological and engineering aspects.
the design of new strains, which can use complex
J Biotechnol 65:127161. doi:10.1016/
substrates of very low cost, such as those present S0168-1656(98)00126-6
in waste materials, and having the versatility to Budde CF, Riedel SL, Hubner F, Risch S, Popovic MK,
produce PHAs with a wide chemical variety and Rha C, Sinskey AJ (2011) Growth and polyhydroxy-
butyrate production by Ralstonia eutropha in emulsi-
molecular mass.
fied plant oil medium. Appl Microbiol Biotechnol
89(5):16111619. doi:10.1007/s00253-011-3102-0
Acknowledgements The authors gratefully thank the Centeno-Leja S, Huerta-Beristain G, Giles-Gomez M,
financial support of DGAPA-UNAM (grant IT100513) Bolivar F, Gosset G, Martinez A (2014) Improving
and Conacyt (grants 131851 and 238535). poly-3-hydroxybutyrate production in Escherichia
coli by combining the increase in the NADPH pool
and acetyl-CoA availability. Antonie Van
Leeuwenhoek 105:687696. doi:10.1007/s10482-
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Rha C, Sinskey AJ (2012) Production of poly(3- Boccaccini AR, Bucke C, Roy I (2007)
hydroxybutyrate-co-3-hydroxyhexanoate) by Polyhydroxyalkanoates (PHA) biosynthesis from
Ralstonia eutropha in high cell density palm oil fer- structurally unrelated carbon sources by a newly char-
mentations. Biotechnol Bioeng 109(1):7483. acterised Bacillus spp. J Biotechnol 127:475487.
doi:10.1002/bit.23283 doi:10.1016/j.jbiotec.2006.07.015
Serafim LS, Lemos T, Rosetti S, Levantesi C, Tandoi V, Wang Q, Yang P, Liu C, Xue Y, Xian M, Zhao G (2013)
Reis MA (2006) Microbial community analysis with a Biosynthesis of poly(3-hydroxypropionate) from glyc-
high PHA storage capacity. Wat Sci Technol 54:183 erol by recombinant Escherichia coli. Bioresour Technol
188. doi:10.2166/wst.2006.386 131:548551. doi:10.1016/j.biortech.2013.01.096
28 C.F.P. Malacara et al.

Wang Q, Yang P, Xian M, Feng L, Wang J, Zhao G (2014) of Biotechnology, Mxico.

Metabolic engineering of Escherichia coli for poly(3- He is the member of the
hydroxypropionate) production from glycerol and glu- Editorial Board of the
cose. Biotechnol Lett 36:22572262. doi:10.1007/ Electronic Journal of
s10529-014-1600-8 Biotechnology and fellow
Wang Q, Luan Y, Cheng X, Zhuang Q, Qi Q (2015) of the Society of Chemical
Engineering of Escherichia coli for the biosynthesis of Industry (SCI). His inter-
poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) ests are the biopolymer
from glucose. Appl Microbiol Biotechnol. production and the biopro-
doi:10.1007/s00253-015-6380-0 cess engineering.
Wong AL, Chua H, Lo WH, Yu PHF (2000) Synthesis of
bioplastics from food industry wastes with activated Andrs Garca studied
sludge biomass. Water Sci Technol 41:5559 Biology at the UAEM,
Yang XS, Zhao K, Chen GQ (2002) Effect of surface Mexico. He later studied a Masters in Biotechnology
treatment on the biocompatibility of microbial polyhy- from the Institute of Biotechnology at UNAM. He is cur-
droxyalkanoates. Biomaterials 23:13911397. rently a Ph.D. student of
doi:10.1016/S0142-9612(01)00260-5 Biochemical Sciences at
Yang YH, Brigham CJ, Song E, Jeon JM, Rha CK, Sinskey UNAM. His work is
AJ (2012) Biosynthesis of poly(3-hydroxybutyrate-co-3- focused on understanding
hydroxyvalerate) containing a predominant amount of the metabolic pathways
3-hydroxyvalerate by engineered Escherichia coli involved in the synthesis of
expressing propionate-CoA transferase. J Appl Microbiol alginate and PHB in
113:81523. doi:10.1111/j.1365-2672.2012.05391.x. Azotobacter vinelandii.
Yang JE, Choi YJ, Lee SJ, Kang KH, Lee H, Oh YH, Lee
SH, Park SJ, Lee SY (2014) Metabolic engineering of Modesto Milln gradu-
Escherichia coli for biosynthesis of poly(3- ated in Chemical Engineer
hydroxybutyrate-co-3-hydroxyvalerate) from glucose. in 2006 from Universidad
Appl Microbiol Biotechnol 98:95104. doi:10.1007/ Autnoma del Estado de
s00253-013-5285-z Morelos in Mxico. He is currently enrolled in the program
Yim KS, Lee SY, Chang HW (1996) Synthesis of of Doctorate of Biomedical Sciences in the UNAM and
poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by works at Institute of Biotechnology. He is studying the pro-
recombinant Escherichia coli. Biotechnol Bioeng 49: cesses of synthesis and deg-
495503. doi:10.1002/(SICI)1097-0290(19960305) radation of
Zhuang Q, Wang Q, Liang Q, Qi Q (2014) Synthesis of poly-3-hydroxybutyrate
polyhydroxyalkanoates from glucose that contain (P3HB) as these processes
medium-chain-length monomers via the reversed fatty affect the molecular weight
acid -oxidation cycle in Escherichia coli. Metab Eng of the polymer in cultures of
24:7886. doi:10.1016/j.ymben.2014.05.004 A. vinelandii.

Tania Castillo received

Carlos Pea received his her M.Sc. degree in Marine
M.Sc. and Ph.D. degree in Biotechnology at CIBNOR, Mxico, and her Ph.D. degree
Biotechnology from in Biochemical Sciences at the UNAM, Mxico. Dr.
UNAM, Mxico. Dr. Pea Castillo is currently a postdoc researcher in Systems
is currently working as Biology at the CEIB/UAEM. Her main interests are the
Senior Research at Institute metabolic engineering for biopolymer production.
Biodegradable Polymers:
Renewable Nature, Life Cycle, 3
and Applications

Manjusha Dake

Biopolymers are superior to synthetic polymer due to their eco-friendly
nature. Microbial biopolymers being a good substitute for conventional
plastics causing a waste management problem. Polyhydroxyalkanoates
(PHAs) produced as microbial polyesters can provide promising prospects
for food and allied industries due to their versatile properties assisting
viscosifying, gelling, and film-forming ability. Microbial and biocatalytic
production of functionalized polyhydroxyalkanoates with novel monomer
structure and tailor-made properties can be feasible by manipulating the
metabolic network in host microbes by genetic modification enabling
them to utilize a diverse range of low-cost substrate as unsaturated fatty
acid constituents. But expensive technology associated extraction and iso-
lation of PHAs is a major hindrance for their commercial applications. A
collective knowledge about PHAs as microbial biopolymers, their produc-
tion from cheap and renewable resources, metabolic pathways involved in
their production, economics of PHA production, and decisive factors
involved could possibly assist their effective utilization as a substitute to
synthetic polymers.

3.1 Introduction

Biopolymers are natural polymers originating

from plants and microbes or synthesized chemi-
cally from biological building blocks. Production
of biopolymers from renewable resources is a
promising remedy for their sustainable develop-
M. Dake (*) ment (Reddy et al. 2003; Porwal et al. 2008;
Department of Biotechnology, Dr. D.Y. Patil Kumar et al. 2009; Patel et al. 2011; Singh et al.
Biotechnology and Bioinformatics Institute,
2015). They can be degraded using natural entity
411033 Pune, India
e-mail: manjusha.dake@dpu.edu.in; as microorganisms and their integrated enzyme
man1d_2@rediffmail.com system to simpler molecular assembly recycled in

Springer India 2015 29

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_3
30 M. Dake

the environment itself. With a low environmental environmental advantages. Microbial degrada-
footprint and eco-friendly nature, biopolymers tion of biopolymers to CO2 and water serves as a
could also prove as asset to waste processing and potential remedy to waste processing where con-
contribute to a sustainable society (Kalia et al. ventional plastics are environmentally unfriendly
2000). In nature, biopolymers often play struc- in the public perception.
tural role and other important roles in maintaining Synthesis of biopolymers and their degrada-
cell viability by conserving genetic information, tion by composting governed by microbial sys-
by storing carbon-based macromolecules for pro- tem has proved their biodegradable nature and
ducing either energy or reducing power, and by user-friendly and environmental-friendly attri-
defending an organism against attack from haz- bute. Thus, biopolymers as renewable feedstock
ardous environmental factors (Steinbuchel 2001). fit nicely within the principles underlying green
Biocompatible and biodegradable nature of bio- chemistry. Plant-derived biopolymers include
polymers makes them superior than petrochemi- starch, cellulose, oils, polyesters, or PHAs, while
cal-derived polymers. Synthetic polymers as those from animal source are proteins, fats, and
plastics derived from nonrenewable fossil (petro- hydrocarbons. Biopolymers being of natural ori-
chemical) in spite of their desirable properties as gin and derived from renewable resources are
suitability and durability. Strength, lightness, and constantly restored through natural processes in
cost are causing environmental threats due to the spite of their constant utility for human life.
short-term convenience of using and throwing Biopolymers provide energy for microbial life
these conventional plastics. Fossil fuels are con- which in turn causes the synthesis of bio-based
sidered nonrenewable where their utilization from polymeric compounds, establishing a symbiotic
earths reserves without replacing them has led to association. Biopolymers are superior to syn-
the exploitation by human society and as a result thetic polymers due to their biodegradability and
created discrepancy in the nature carbon cycle by both environmental and human compatibilities
emission of carbon dioxide. which cannot be emulated by synthetic polymers.
Synthetic plastics primarily derived from non- Biopolymers can be classified according to the
renewable fossil (petrochemical) surpassed the monomers that constitute them, as polysaccha-
market by replacing the glass, wood, and other rides, polyamides (proteins and poly -glutamic
construction material. But lack of natural acid (-PGA)), nucleic acids (DNA and RNA),
enzymes and biological processes for degrada- polyesters (polyhydroxyalkanoates, PHAs),
tion of synthetic plastic has created alarming sit- polyphosphates, and polyisoprenoids (natural
uation for environment as well as human society. rubber). Biopolymers synthesized by bacteria are
So there is a worldwide demand for biopolymers applied in food, pharma, and agriculture sector.
originating from renewable raw material (Chen Natural rubber, cellulosics, and nylon-11 are the
and Martin 2012). Persistence of such synthetic most used biopolymers, while newer biopoly-
plastics in the environment is dangerous for the mers include polylactic acid, polyhydroxyal-
human community and wildlife. Thus, nonbiode- kanoate, and bio-based thermoplastic
gradable nature makes synthetic plastic waste polyurethane (Jogdand 2014). Biopolymers from
management problems. This created an urgent plants serve as energy carrier, carbon storage
need in implementing the usage of biodegradable compounds, and protective elements against
plastics for an increasing awareness among con- pathogens. Biopolymers derived from plants
sumers regarding the use of plastic-based materi- encompass diverse group of polysaccharides as
als. Thus, deleterious effects of synthetic plastic starch, cellulose, hemicellulose, pectin, galacto-
derived from nonrenewable fossil fuel have cre- mannans (guar, locust bean gum), exudates gum
ated environmental awareness by one and all for (gum Arabic), and -glucans, while seaweed
making a transition insisting demand for use of polysaccharides include alginates and
biodegradable material. Biopolymers available carrageenans. Plant polysaccharides are
on a sustainable basis have several economic and applicable in textile, leather, pharmaceutical
3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 31

(drug formulation and drug delivery), cosmetics and aliphatic polyesters that serve as intracellular
(moisturizing ingredient; hair shampoos), agri- reservoir for carbon and energy. Such biodegrad-
culture (plant growth regulators, soil fertilizers, able polymers derived from microbial source
antifungals, and anti-nematodals), and food have the potential to help in waste management
(thickeners, gelling agent). Starch-based plant and an alternative to conventional plastics.
sources and cellulose derivatives contribute to Intrinsic properties of biopolymers assure their
almost 80 % of the bioplastic market (Rajendran promising prospects making them suitable as a
et al. 2012). Starch-based films blended with packaging material in dairy industry and flight
thermoplastic polyesters are used as packaging catering products, protecting the product from
materials, in hygiene products and agriculture moisture with increase in its shelf life.
where 50 % starch combination can be used. Biopolymers have specialized applications in
Polylactic acid, the most widely used biodegrad- fabrics, adsorbents, biosensors, and data storage
able aliphatic polyester, is derived from cane elements.
sugar. But plant-produced biopolymers present Nanoscale biopolymers produced from natural
certain limitations as limited availability, sea- polymers such as starch and chitin are useful in
sonal price fluctuations, expensive technology, therapeutics, coatings, packaging materials, and
and the absence of rheological properties for spe- bioremediation of toxic heavy metals. Microbial
cific applications (Sutherland 2005). polyhydroxyalkanoate (PHA) reserve polymers
Other limitations for plant-produced biopoly- are interesting polyesters sustainably produced
mers include lower and delayed biomass produc- from renewable material and as a result can be an
tion and adverse effect on human food chain. effective substitute for synthetic plastic material.
Compared to natural polymers originating from The present chapter reveals the biopolymers from
plant sources and those synthesized from fossil microbial origin and their environmental and bio-
fuel, microbial biopolymers are more favorable medical applications. Other members of biopoly-
and advantageous on the basis of social and eco- mers include proteins (silk, collagen, elastin, poly
nomic forecast. Compared to plant-produced amino acids, soy, wheat gluten, zein, and casein),
polysaccharides, commercial production of bio- lipids, polyphenols, and natural rubber.
active polysaccharides through the process of
microbial fermentation is always preferred. A
wide range of polysaccharides synthesized by 3.2 Microbial Biopolymers
microorganisms are xanthan, gellan, curdlan,
pullulan, chitosan, and bacterial cellulose. Microorganisms as bacteria, archaea, fungi, and
Exopolysaccharides (EPPs) from bacterial and algae produce a diverse group of intracellular
fungal source are comprised of monomeric sug- and extracellular biological macromolecules
ars such as glucose, fructose, rhamnose, gluc- such as polysaccharides, polyesters, and poly-
uronic acid, mannuronic acid, and acetyl amides termed as microbial biopolymers.
glucosamine. Microbial polysaccharides are Biopolyesters (polyhydroxyalkanoates) are
valuable and cost-effective tools as thickeners, intracellular and spherical organic inclusion syn-
bioadhesives, stabilizers, probiotics, and gelling thesized by microorganisms (Singh et al. 2015).
agents in food and cosmetics (Nicolaus et al. Microbial polysaccharides may be capsular
2010; Freitas et al. 2011) and as emulsifier, bio- polysaccharides associated with the cell surface,
sorbent, and bioflocculant in the environmental or they may be exopolysaccharides loosely con-
sector (Sam et al. 2011). In bacterial polysaccha- nected with cell surface (Cuthbertson et al.
rides applicable in food industry, clinical research 2009). Microbial polysaccharides are composed
medicines include levan, alginate, dextran, xan- of various monosaccharides such as D forms of
than, and scleroglucan. Microbial biopolymers glucose, galactose, mannose, arabinose, ribose,
also encompass biodegradable polyesters such as xylose, and uronic acids. Exopolysaccharides
polyhydroxyalkanoates (PHAs), polylactides, like pullulan, kefiran, bacterial cellulose (BC),
32 M. Dake

gellan, and levan produced by microorganisms with a molecular formula (C6H8O6) n with a
exhibit film-forming ability. The physicochemi- molar mass of 13 106 g mol1. It is a linear
cal and rheological properties of exopolysaccha- chain of copolymers of -D-mannuronate (1 4)
rides vary with the type of microbial strain. and -L-glucuronate (Wong et al. 2000). Brown
Microbial biopolymers are applicable in food algae produce 40 % by weight of alginate
and allied industries due to their viscosifying, (Matsubara et al. 2000) (Fig. 3.1). Commercial
gelling, and film-forming properties. Microbial alginates are produced. Marine microalgae pro-
biopolymers play important roles as energy ducing alginate are Ascophyllum nodosum,
reserve materials, protective agents, aid in cell Durvillea antarctica, Ecklonia maxima, Lessonia
functioning, the establishment of symbiosis, and nigrescens, etc. Bacteria including Azotobacter
osmotic adaptation in response to changing envi- sp. and Pseudomonas sp. are producing alginates
ronmental applications (Vijayendra and Shamala as extracellular polysaccharides during late expo-
2014). nential growth phase. Alginate in Azotobacter
Improvement in properties of existing poly- vinelandii prevents desiccation under adverse
saccharides for generation of novel biopolymers environmental conditions (Remminghorst and
of great commercial interest and value with Rehm 2006). Bacterial yield of alginate is
desired properties can be done by proper design- affected by different cultivation conditions as pH,
ing and creating of new property based on aeration, and oxygen supply. The batch cultiva-
requirements through controlled synthesis. The tion process is designed for commercial produc-
microbial biopolymers can be successfully pro- tion of alginate on an industrial scale. Alginate is
duced on industrial scale accompanied with the applied in biomedical science and engineering, in
use of efficient producer strain, cheaper fermen- cell and tissue immobilization (Wang et al. 2003),
tation substrates as renewable feedstock, process and as food additives (Draget et al. 2005).
design along with optimized fermentation param- Alginate is also used as a thickener in ice creams
eters like pH and temperature, and superior tech- and as a chelator for removal of metals and in dye
nology like genetic engineering. Production of printing due to its unreactive nature.
microbial polysaccharides using cheap biomass
resources like syrups and molasses, olive mill
wastewater, cheese whey, vegetable and fruit 3.2.2 Xanthan
pomace, pulp and kernels, lignocellulosic bio-
mass like rice hull and bran, as well as carbon Xanthan is an extracellular polysaccharide pro-
dioxide has been reported with a suitable pre- duced by microorganisms belonging to genus
treatment method (Oner 2013). Production of Xanthomonas like Xanthomonas campestris,
biopolymers with customized properties by Xanthomonas malvacearum, and Xanthomonas
genetic manipulation of microorganisms is car- axonopodis through the process of microbial fer-
ried out for tissue engineering and drug delivery mentation. Xanthan has molecular weight rang-
(Rehm 2009). PHAs are potential microbial poly- ing from 2 106 to 20 106 Da mol1. Xanthan, a
mers for drug delivery (Shrivastav et al. 2013). polyanionic heteropolysaccharide, is composed
Pullulan could be used as a reducing as well as a of glucose, mannose, and glucuronic acid form-
capping agent for AgNPs synthesized using pul- ing a repeated pentasaccharide unit (Fig. 3.2)
lulan act as strong antimicrobial agents (Kanmani where noncarbohydrate components as O-acetate
and Lim 2013). and pyruvate substitute on the terminal mannose.
Acetylation and pyruvylation of mannose
produce modified xanthan. It shows rigid rod-
3.2.1 Alginate like conformation. Xanthan shows desirable
properties like viscosity, pseudoplasticity,
Alginic acid is a linear, negatively charged, vis- thixotropy, and gellation applicable in food
cous or gel-like water-soluble polysaccharide industry. Bacteria producing xanthan gum are
3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 33

Fig. 3.1 Structure of alginate

Fig. 3.2 Structure of xanthan

X. campestris, X. vasculorum, X. juglandis, X. tinuous culture. Production medium with higher

fragaria, etc. Xanthan formed by Xanthomonas carbon to nitrogen ratio shows a better yield of
species serves the bacterium in acting as plant xanthan. Xanthomonas campestris is responsible
pathogen by allowing its penetration and forma- to produce xanthan by anaerobic fermentation at
tion of local lesions, soft roots, scabs, and can- a temperature of 28 C.
cers. Production of xanthan can be achieved
using cheaper carbon sources like sugarcane
molasses, whey along with yeast extract, soymeal 3.2.3 Pullulan
peptone as a complex nitrogen source, and
organic acids as a stimulator through the process Aureobasidium pullulans is a dimorphic fungi
of aerobic fermentation in batch culture or con- producing pullulan, a homopolysaccharide of
34 M. Dake

natural glucan polymer with average molecular ses, and agro-industrial waste. Pullulan is
weight ranging from 1.5 104 to 1.0 107. It is produced by various microbes including
also known as -1,4- and -1,6-glucan consisting Aureobasidium pullulans (Leathers 2003),
of 0.6 % maltotetraose and a major component of Teloschistes flavicans (Reis et al. 2002), and
maltotriose (Fig. 3.3). Structure of pullulan com- Cryphonectria parasitica (Delben et al. 2006).
posed of repeating maltotriose units linked Anionic or amphiphilic pullulan microparticles
through (16) linkage using terminal glucose play an important role in controlled drug delivery
residue of trisaccharide, while each maltotriose system (Jeong et al. 2006).
unit is made up of three -1,4-linked glucose
residues (Carolan et al. 1983). Production of pul-
lulan varies with culture conditions and type of 3.2.4 Levan
microbial strain that get decreased in the late sta-
tionary phase due to pullulanase production in Levan a neutral, biodegradable, and water-
medium (Pollock et al. 1992). Pullulan has adhe- soluble homopolysaccharide is a polyfructan
sive properties applied during formation of fibers, which consists of D-fructofuranosyl residues
moldings, and oxygen-impermeable films used joined by -(2,6) linkages forming a core along
for preservation purpose (Sakata and Otsuka with -(2,1) branching points (Fig. 3.4) (Tanaka
2009) and used for coating of food containers for et al. 1990). Levans are produced by plants
preservation of perishable fruits and vegetables. termed as phelins. Levan is produced by bacte-
It has the molecular formula (C6H10O5) n and is rial sp. like Bacillus and Pseudomonas
considered as a composite of panose or isopanose (Oliveira et al. 2007; Shih et al. 2005). Low fer-
subunits. Pullulan due to its high solubility in mentation temperature (25 C) supports opti-
water is applied during controlled drug delivery. mal production of levan, while high temperature
Consequently, pullulan can be used as food addi- (3540 C) inhibits its production. Levan has
tive, flocculant, and wound healing (Cheng et al. high solubility in oil, low viscosity, high water-
2011). Pullulan shows pH tolerance from 3 to holding capacity, good biocompatibility with
8 pH and it carbonizes at 25280 C. The produc- surfactants, and stability to heat (Sima et al.
tion of pullulan varies with fermentation param- 2011). Levans have a high molecular weight of
eters like pH and temperature, composition of 2100 million Da and the density 1.4 g/cm3.
production medium, and physiological state of Levan is a nontoxic and a soluble dietary fiber
microbial strain. Commercially, pullulan can be where the hydrolysates resulting from levan
produced using coconut by-products, beet molas- particularly help to improve gut function.

Fig. 3.3 Structure of pullulan

3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 35

Fig. 3.4 Structure of levan

Fig. 3.5 Structure of gellan

Levans can be used in foods, feeds, medicines, 3.2.5 Gellan

and cosmetics, in the chemical and biotech
industry (Rairakhwada et al. 2007; Gupta et al. Gellan gum is a linear anionic polysaccharide
2008). It is useful as encapsulating agent, thick- with a molecular mass approximately 1 105 Da
ener, emulsifier, and carrier of colors and fla- due to the presence of tetracyclic units (Gong
vors for food (Jang et al. 2001). Levan from et al. 2009). It is produced by the bacterium
Microbacterium laevaniformans, Rahnella Sphingomonas elodea using a fermentative pro-
aquatilis, and Zymomonas mobilis was found to cess through immersion method. It is a linear
exhibit in vitro antitumor activity (Yoo et al. polymer composed of a repeating tetrasaccharide
2004). It also shows cell proliferative and anti- unit consisting of glucose, glucuronic acid, and
inflammatory effects (Ki Ho Kim et al. 2005). rhamnose in 2:1:1 ratio (Fig. 3.5). The structure
Levan provides a better substitute for gum of gellan contains about 1.5 % acyl group as ace-
Arabic in a variety of foods, pharmaceuticals, tyl and glycerate molecules per unit. With respect
medicines, cosmetics, adhesives, paints, ink, to acyl groups, it is classified into high-acyl and
lithography, textile, and others. Production of low-acyl gellan where the natural form of bio-
microbial levan is generally carried out using polymer is high-acyl gellan and has shorter length
sucrose-based substrates like fructose, molas- than low-acyl gellan. Gellan with high-acyl
ses, and sugarcane juice. groups forms soft, elastic, and non-brittle gels
36 M. Dake

(Sworn 2000). Gellan inhibits sedimentation or 3.2.6 Kefiran

suspension of particles in fruit juices with pulps,
cacao, and non-soluble minerals where the sus- Kefiran is a water-soluble exopolysaccharide
pended particles are trapped in liquid gel struc- present in kefir grains. It is a glucogalactan with
tures (Young 2002). Gellan is an appropriate a molecular weight corresponding to 107 Da. The
biopolymer for coating and protecting the probi- heteropolysaccharide structure of kefiran mainly
otics by resisting acidic conditions and protecting consists of glucose and galactose in equal
cells from acidic damages. The gellan gum shows amounts (Fig. 3.6). Yeasts, bacteria producing
plastic flow behavior above 1.0 % concentration lactic and acetic acid, and mycelial fungi are
at 25 C, while formation of gel requires polysac- microbial constituents of kefir grains (Witthuhn
charide concentration over 0.8 % (Tako and et al. 2005). Kefiran is produced by Lactobacillus
Nakamura 1989). Gellan reduces oil uptake due sp. like Lactobacillus kefiranofaciens (Mitsue
to its thermo-gelling property (Bajaj and Singhal et al. 1999). Exocellular kefiran recovered from
2007). It is produced by Sphingomonas elodea culture supernatant by centrifugation and recov-
through the process of aerobic fermentation using ered by precipitation of supernatant with an equal
a carbohydrate as a substrate. Gellan gum is used amount of cold ethanol. Capsular kefiran recov-
in dairy products and sugar confectionary and to ered from cells with water at 95 C and precipi-
modify traditional gelatin dessert jellies. Gellan tated with ethanol. Kefiran has several commercial
is sold in the market with trade names Kelcogel, applications ranging from being an emulsifier,
Gelrite, Phytagel, and Gel-Gro. The thickness gelling agent, stabilizer, thickener, etc. (Cheirsilp
gellan gum can be controlled by the addition of and Radchabut 2011). Kefiran in kefir grains can
sodium and potassium salts. Plasticizers like form edible transparent films at concentrations
glycerol are added to make gellan films less brit- ranging from 5 to 10 g/kg. The addition of glyc-
tle. Gellan is applied as a novel drug delivery erol as plasticizer improved flexibility of kefiran
vehicle and in fruit products due to heat and acid film matrix compared to low density polyethyl-
stability. ene film and reduced water vapor permeability
(Piermaria et al. 2009). Kefiran films are trans-
parent showing pseudoplastic behavior, partially
soluble in water at 2537 C while completely
solubilized at 100 C.

Fig. 3.6 Structure of kefiran

3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 37

Kefiran exhibits good potential as an edible D-glucopyranose units with -1,4 linkages
film in the food and packaging industries (Fig. 3.7). It has a molecular formula (C6H10O5)
(Ghasemlou et al. 2011a, b). Coculture of L. kefi- n. Bacterial cellulose is preferred than plant cel-
ranofaciens and S. cerevisiae consumes lactic lulose due to its versatile properties like higher
acid, thereby lowering its concentration, which purity, tensile strength, water-holding capacity,
enhances cell growth and kefiran producing and crystallinity index making it more suitable
capacity of L. kefiranofaciens (Cheirsilp et al. raw material for paper and dessert foods (Shoda
2003). Production of kefiran by these bacteria and Sugano 2005). Unlike plant cellulose, bacte-
depends upon the cultural conditions like pH, rial cellulose does not require processing to
temperature, agitation as well as carbon and remove contaminating lignin and hemicelluloses
nitrogen source. The kefiran represents a very components (Nishi et al. 1990). Cellulose
important option as natural additive to the food accounts for its tensile strength and elastic modu-
and industry that can be used as thickeners and lus due to its fine network of cellulose microfi-
stabilizers in food. It also shows antimicrobial brils. Bacterial cellulose is applicable diet food,
properties. paper additive, skin tissue repair, vascular grafts,
and for regenerative medicines (Lin et al. 2013).
The processed cellulose membrane is highly
3.2.7 Cellulose applicable as in food packaging due to its hydro-
philic nature and continuous moisture removal.
Bacterial cellulose is produced extracellularly in Nutrient media with high carbon content and
the form of nanofibers by Acetobacter, limitation of nutrients favors cellulose produc-
Aerobacter, Alcaligenes, Rhizobium, tion (George et al. 2005). Various carbon sources
Agrobacterium, etc. (El-Saied et al. 2004). used during cellulose production include sucrose,
Microbial cellulose consists of bundles of cellu- mannitol, glucose, and xylose.
lose microfibrils 24 nm diameter (Nakagaito
et al. 2005). Cellulose nanofibers are arranged in
a ribbon form with 100 m length and 100 nm 3.2.8 Haloferax Exopolysaccharide
diameters. Such network of cellulose nanofibers
exhibits biocompatibility, bioadaptability, biode- Bacterial spp. of the genera such as
gradability, and chemical stability (Moreira et al. Methanosarcina, Thermococcus, Sulfolobus, and
2009). Microbial cellulose consists of microfi- Archaeoglobus represent a valuable source of
brils which are made up of hydrogen bond linked exopolysaccharides. Molecular weight of bacte-
glucan chains. Microbial cellulose is a linear and rial exopolysaccharides ranges from 10 to 30
unbranched polymer comprised of KDa (Singha 2012). EPSs have heterogeneous,

Fig. 3.7 Structure of microbial cellulose

38 M. Dake

neutral, or polyanionic nature due to the presence in oil recovery process. Haloferax denitrificans
of phosphate, sulfate, and uronic acid residues. grows with salt concentration of 1.54.5 M
The polysaccharide produced by halobacterium (Parolis et al. 1999). Production of biopolymer is
Haloferax mediterranei is well studied due to its related to nutritional composition of media, pH,
film-forming property. This exopolysaccharide temperature, physiological state, and the genetics
consists of sugars such as glucose, mannose, of organism (Finore et al. 2014). The biofilm
small amount of galactose, and ribose, along with matrix prepared with exopolysaccharides exhib-
amino sugars and uronic acids (Fig. 3.8). its interesting properties such as antimicrobial
Halophilic Archaea producing EPS are Haloferax, action, preserving activity, and biodegradability
Haloarcula, Halococcus, and Natronococcus. (Mironescu and Mironescu 2006).
EPSs are produced by extremophiles in response
to biotic and abiotic stress factors (Donot et al.
2012) and serve for protection against desicca- 3.3 Polyhydroxyalkanoates
tion and predation. Exopolysaccharide from (PHAs)
Haloferax gibbonsii (ATCC 33959) consists of
neutral sugars like D-mannose, D-glucose, Biodegradable plastics can be chemically synthe-
D-galactose, and L-rhamnose. EPS produced by sized polymer, starch-based biodegradable plas-
H. mediterranei has certain rheological proper- tics, and polyhydroxyalkanoates (PHAs) (Kalia
ties like pseudoplastic and viscoelastic behavior et al. 2003; Khanna and Srivastava 2005; Singh
and flow behavior, maintaining the structure at et al. 2009). Biodegradable plastics produced
high temperature (Mironescu and Mironescu from bacterial fermentation include
2011) and also shows higher salt resistance. Due polyhydroxyalkanoates (PHAs).
to such remarkable resistance, EPS from H. med- Polyhydroxyalkanoates are biocompatible and
iterranei is applicable as a thickening agent and branched heteropolyesters composed of 150 dif-

Fig. 3.8 Structure of

Haloferax exopolysaccharide
3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 39

ferent (R)-3-hydroxyalkanoic acid monomers PHAs are storage biopolymers in prokaryotic

containing oxoester bonds joining hydroxyl and cells that occur as granular, hydrophobic inclu-
carboxyl groups. PHAs are synthesized as stor- sion bodies (PHA granules) within the cyto-
age polymers by a wide variety of bacterial spe- plasm. PHAs granules stored as insoluble
cies, Archaea, and recombinant bacteria by inclusion bodies are coated with lipids and pro-
metabolic transformation (Table 3.1). PHAs pos- teins termed as phasins on their surface which
sess unique properties as biocompatibility, biode- prevents coalescence where lipids involved are
gradability both in aerobic and anaerobic phospholipid monolayer. All enzymes necessary
conditions including aquatic environments, bio- for synthesis of PHB from acetyl-CoA associated
based and renewable origin, and structural diver- with PHA granule as observed in R. eutropha
sity providing a remedy to reduce pollution of the (Uchino et al. 2007). Polymers stored in granules
environment (Tortajada et al. 2013). PHAs can be remain in the amorphous state in vivo, while in
also produced from CO2 (Snell and Peoples isolated state, the polymer rapidly crystallizes.
2002). The production of various types of PHA Intracellular utilization of PHA occurs by PHA
has been using several transgenic crops, includ- synthesizing organisms, while extracellular utili-
ing corn, sugarcane, and cotton (Snell and zation of PHA released from lysed cells takes
Peoples 2009). Microbial degradation of PHAs to place into the environment.
CO2 and H2O under aerobic conditions in soil, PHAs are accumulated in cell cytoplasm when
freshwater, and marine environments (Mergaert carbon is in excess and inorganic nitrogen and
et al. 1994) or to methane and water under anaer- phosphate are limiting (Koller et al. 2010; Singh
obic conditions is also reported (Volova et al. et al. 2015). Thus, the bacterial cells switch car-
2010). Their degradation occurs more rapidly bon pool to Krebs cycle for synthesis of PHA
than degradation of synthetic polyesters and lig- under such growth-limiting conditions. PHAs are
nocelluloses. PHAs consisting of various biocompatible and responsible to regulate intra-
hydroxyalkanoic acids are developed through cellular energy flow by directing the carbon pool
biotechnological routes and show thermal prop- toward metabolic pathways and protecting the
erties similar to polypropylene. cell against stress conditions (Koller et al. 2011).

Table 3.1 Microbial strains producing PHA on industrial scale

Microbial strain Polymer produced References
Cupriavidus necator (Alcaligenes eutrophus) PHB and its copolymers Volova and Kalacheva 2005
Cupriavidus necator H16 PHB Pohlmann et al. 2006
Azohydromonas latus DSM 1124 PHA Yu et al. 1999
Rhizobium meliloti, R. viciae, Bradyrhizobium japonicum PHA Mercan and Beyatli 2005
Recombinant Escherichia coli (UHMW) PHB Nikel et al. 2006
Alcaligenes latus, Staphylococcus epidermidis PHB Wong et al. 2005
Ralstonia eutropha (Cupriavidus necator) PHB, PHBV, P3HB4HB Kahar et al. 2004
Aeromonas hydrophila mcl-PHAs Lee et al. 2000
Pseudomonas aeruginosa mcl-PHAs Hoffmann and Rehm 2004
Pseudomonas putida, P. fluorescens, P. jessenii Aromatic polymers Wang et al. 2005
Bacillus sp. PHB, PHBV copolymers Full et al. 2006
Burkholderia cepacia PHB, PHBV Nakas et al. 2004
Halomonas boliviensis PHB Quillaguaman et al. 2006
Microlunatus phosphovorus PHB Akar et al. 2006
Spirulina platensis (cyanobacterium) PHB Jau et al. 2005
Halophilic archaeal species Natrialba Han et al. 2007
Haloquadratum Legault et al. 2006
40 M. Dake

They exhibit nonlinear optical activity. The PHA phase. Several halophilic archaeal species like
types are polyhydroxybutyrate (PHB), polyhy- Haloferax, Haloarcula, and Halococcus have
droxyvalerate (PHV), polyhydroxyhexanoate been also reported to produce PHAs like PHB
(PHH), and polyhydroxyoctanoate (PHO) where and PHBV.
PHB is the main biodegradable polymer (Chen Polyhydroxyalkanoates are categorized as
2009; Singh et al. 2013) (Fig. 3.9). The best- SCL-PHA (35 carbons), MCL-PHA (614 car-
known PHAs are PHB, poly-3-hydroxybutyrate- bon), and LCL-PHA (1718 carbon). Copolymers
co-3-hydroxy valerate P(HB-co-HV), of scl-PHA like hydroxybutyrate (HB) and
poly-3-hydroxoctanoate-co-polyhydroxyhexano- 3-hydroxyhexanoate improve their mechanical
ate P(HO-co-HH) (Table 3.2). property making them comparable with conven-
Pendant R group in PHAs varies from C3 to tional plastics such as polypropylene and
C14 carbon atoms (Doi and Abe 1990). PHA has polystyrene. Mcl-PHAs such as poly (hydroxyoc-
molecular weight varying from 2 105 to 3 106 tanoate-co-hydroxydecanoate)
Da. It is synthesized by 30 % soil bacteria as well [P (HO-co-HD)] behave as a complete elastic
as other bacterial sp. inhabiting activated sludge, substance. PHB is highly crystalline due to its
high seas, extreme environments, and soil. stereospecific nature with a melting temperature
Cupriavidus necator is the most commonly stud- (Tm) 170180 C (Matko et al. 2005) and Tg
ied strain for PHB production (Atlic et al. 2011). around 5 C. Unfavorable aging of PHB homo-
PHB is widely found in Bacillus; Pseudomonas; polymer can be prevented by annealing for
plant symbionts, Rhizobium; nitrogen-fixing changing its lamellar morphology and improving
Azotobacter sp.; Azohydromonas lata; and mechanical properties. Increasing porosity and
recombinant Escherichia coli (Volova 2004; surface area of PHA products enhance their deg-
Reddy et al. 2003; Kumar et al. 2013, 2014) radation rate in the environment. The control of
(Table 3.2). Microorganisms living in extreme PHA monomer composition along with new
environmental habitats accumulate endocellular processing and compounding technologies and
polyhydroxyalkanoates (PHAs) during stationary their novel properties like moisture resistance

Fig. 3.9 General structure of

polyhydroxyalkanoates (PHAs)
and their structural derivatives

Table 3.2 Polyhydroxyalkanoates (PHAs) and their structural derivatives based upon R groups
R group Polyhydroxyalkanoate Abbreviation
----CH3 Poly(3-hydroxyalkanoates) PHA
----- CH3 Poly(3-hydroxyvalerate) PHV
------(CH)2--------CH3 Poly(3-hydroxyhexanoate) PHHex
------(CH)4--------CH3 Poly(3-hydroxyoctaonate) PHO
------(CH)6--------CH3 Poly(3-hydroxydecanoate) PHD
-------- (CH2) ----- Poly(3-hydroxy-5-phenylvalerate) PHPV
3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 41

and oxygen impermeability will make PHAs Functionalized PHAs with tailor-made properties
more functional and expand the number of appli- can be generated by controlling monomer com-
cations for which these plastics can be used. position where the side chains in mcl-PHAs are
Processing PHAs using certain methods can reactive groups and a potential target for post-
alter their biodegradability and improve their biosynthetic modification. Higher fraction of
mechanical properties. Properties of polymers unsaturated side chains in PHA monomers cause
can be altered through the use of additives such inhibition of crystallization and subsequent low-
as fillers, plasticizers, stabilizers, reinforcers, and ering of melting and glass transition temperature.
pigments. The composition used in bioplastic Cross-linking of unsaturated PHAs can transform
film includes P(HB-co-HV) with acetyl tributyl them into rubbers (Bassas et al. 2008).
citrate as a plasticizer and cyclohexylphosphoric
acid/zinc stearate as a nucleating agent and ther-
mal stabilizer (Asrar and Pierre 2000). Drawing 3.3.1 PHA Production from Cheap
PHA fibers and films can change the physical and Renewable Resources:
structure of the polymer and alter its mechanical Renewable Nature
properties. Tanaka and coworkers developed and Life Cycle
melt-spinning technique that produced highly
crystalline and P (HB-co-HV) fibers with greater Biodegradable nature of PHA resulting forma-
strength (Tanaka et al. 2007). Gel-spinning tech- tion of carbon dioxide and water indicates their
niques have also been developed for preparation positive ecological impact making them an effec-
of high-strength PHA fiber (Antipov et al. 2006) tive substitute to synthetic plastic. PHAs can be
increased the toughness of P(HB-co-HV) films produced from renewable resources through the
by cross-linking the polymer and drying the films process of fermentation using agricultural feeds
under uniaxial strain. High degree of crystallinity as a source of sugars and fatty acids. Carbon
of PHB leads to a plastic that is strong but very source plays a predominant role in PHA produc-
stiff high Youngs modulus and brittle that limits tion reducing the cost by 50 %. The greatest chal-
the commercial potential of PHB. PHA copoly- lenge in PHA production is the use of waste and
mers have more favorable properties than PHB biowaste, mostly because of their substrate and
where the addition of HV units to the polymer contaminant contents (Fig. 3.10). Biowastes,
chain in case of PHB-co-HV resulted in a mate- namely, glycerol, pea shells, rice chaff, coconut
rial with a lower Youngs modulus and a greater oil cake, cottonseed cake, wafer residue, and cit-
extension to break (Sudesh et al. 2000). P rus pulp waste, have been tested as substrates to
(HB-co-HAMCL) copolymers are weaker than produce PHA (Patel et al. 2012, 2015; Kumar
PHB but are tougher and more flexible thermo- et al. 2015a,b; Singh et al. 2015). The use of
plastics. Strength of PHBV can be increased by defined media with carbohydrates (glucose,
lowering Tm and Tg based upon its variable HV sucrose, fructose), alcohols as methanol, alkanes
content. Higher HV content increases strength (C6C12), and organic acids (butyrate, valerate) is
and results in reduction in Tm and Tg values expensive for industrial scale synthesis of
(Amass et al. 1998) and crystallinity. PHB serves PHA. High-volume industrial PHA production
as a promising source of biodegradable thermo- can be possible using cheap renewable resources
plastic material useful for packaging industries to as starch, lactose, fats, and oils (Braunegg et al.
solve environmental pollution problem for waste 2002).
management strategies (Mona et al. 2001). PHB production is carried using a wide vari-
Haloferax mediterranea, a denitrifying halo- ety of carbon sources including beet molasses,
phile, might present advantages for production of ethanol, methanol, wheat and casein hydrolysate,
PHB and PBV as their culture requirements in corn steep liquor, etc. Ralstonia eutropha H16, a
terms of salinity and temperature provide little promising producer of PHA showing higher yield
opportunity for growth of contaminants. of SCL-PHA (up to 8090 %), utilize H2-CO2
42 M. Dake

Molasses: Beet molasses, Cane molasses
Polysaccharides such as starch and Cellulose

Fats & oils

Whey from dairy industry

Wastes from biodiesel production: methanol

plus glycerol exchange for heavy metal removal Organic acids

Methanol plus glycerol-a side product during

biodiesel production process

Plant and animal lipids

Sulfite liquor

Fig. 3.10 PHA production from cheap and renewable resources

mixture as a sole carbon and energy source 2007). The high cost of industrial production and
(Pohlmann et al. 2006) for production. The pro- recovery of bioplastics can be improved by opti-
duction of polyhydroxybutyrate (PHB) in mization of fermentation process, by the use of
Bacillus megaterium ATCC 6748 was carried out recombinant organisms utilizing cheap carbon
using renewable carbon and nitrogen source sources. This will facilitate the commercializa-
(Chaijamrus and Udpuay 2008). Synthesis of tion of bioplastic production. Zero-cost feed-
multicomponent PHAs is a very complicated bio- stocks like industrial vegetable wastes were used
technological task. Bacterial cultures cannot be by extremophiles for the production of biopoly-
grown in the presence of mixed carbon substrates mers like PHAs (Donato et al. 2011). Haloarcula
(CO2 + valerate, hexanoate, etc.) as monomers sp. IRU1 effectively used petrochemical waste
with different number of carbon atoms that can- water as a carbon source to produce PHB (Taran
not be incorporated into the polymer at the same 2011). Industrial production of PHB was suc-
rate during their synthesis. The fatty acid salts cessfully carried out from cardboard industry
added to the culture as co-substrates were found waste water as a sole carbon source using
to be toxic to bacteria. Production of PHB by a Enterococcus sp. NAP11 and Brevundimonas sp.
recombinant Halomonas campaniensis LS21 NAC1 isolates (Bhuwal et al. 2013). PHAs are
using energy-saving, seawater-based, continuous valuable biopolymers due to their renewable
open process, and cheaper substrates (cellulose, nature and life cycle (Fig. 3.11).
proteins, fat, and starch) was established (Yue
et al. 2014). Thus, it is essential to determine
maximal permissible concentrations of every 3.3.2 Metabolic Pathway to PHB
acid for every PHA producer. Bacterial strains of
Wautersia eutropha, B5786 and H16 grown with PHB synthase catalyzes synthesis of PHB from
CO2-heptanoic acid mixture, synthesize HV and (R)-3-hydroxybutyryl-CoA (Fig. 3.12). Under
HB. The addition of octanoate inhibited both cul- limiting conditions of nitrogen acetyl-CoA
ture growth and polymer synthesis (Volova et al. undergoes condensation with another acyl-CoA
3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 43

Molasses: Beet molasses, Cane molasses

Energy Water

Carbon dioxide

Plants Water


Oxygen Carbon Sources

(Sugars and Fatty acids)


PHAs Bioplastic products

Polyhydroxyalkanoate (Packaging, implants, etc.) Composting

Fig. 3.11 Bacterial synthesis of PHAs: renewable nature and life cycle

derivative using 3-ketothiolase resulting forma- and 4-hydroxybutyrate to the production medium
tion of acetoacetyl-CoA that is stereoselectively is required.
converted to (R)-3-hydroxybutyryl-CoA with the Functionalized PHAs are synthesized by feed-
help of NADPH-dependent acetoacetyl-CoA ing structurally related substrates like (R)-3-
reductase (Kalia et al. 2007). Polymerization of hydroxyacyl-CoAs, processed through
(R)-3-hydroxybutyryl-CoA to form the homo- -oxidation pathway by bacterial sp. like pseudo-
polyester (poly(R)-3-hydroxybutyrate) or monads (Figs. 3.13 and 3.14). Nonfatty acid pre-
copolyesters (poly-3-hydroxybutyrate-co-3- cursors such as carbohydrate like fructose,
hydroxyvalerate) is executed by PHA synthase glucose, glycerol, acetate, and ethanol are chan-
catalyzes. These polymers are stored by the bac- nelized to PHA by the de novo pathway via (R)-
terial cell as globular granules. PHAs are depoly- 3-hydroxyacyl-acyl carrier protein intermediates
merized to monomeric components that are (Fig. 3.15). A plethora of tailor-designed mcl-
metabolized to CO2 and water along with the pro- PHAs, with highly diverse structures that include
duction of ATP under growth promoting condi- acetylthioester, acetoxy, alkoxy, amino, cyano,
tions in the presence of assimilable nitrogen. cyclohexyl, epoxy, halogenated, hydroxy, or pro-
When PHA production process aims to produce pylthiol groups, can be synthesized (Tables 3.3,
copolyesters, the addition of various precursors 3.4, and 3.5). Synthesis of mcl-PHAs with
like propionate, -butyrolactone, 1,4-butanediol, desired properties can be possible by using
low-cost substrates as unsaturated fatty acid con-
44 M. Dake

gation or flocculation, and the PHA can be
recovered by the use of surfactants or hypochlo-
rite to lyse the cells for release of the intracellu-
lar product. But hypochlorite can cause some
degradation of the product so that alternative
Acetyl -CoA Krebs cycle solvent extraction method is used which removes
bacterial endotoxins and causes negligible deg-
3- Ketothiolase (PhaA) radation of PHAs. Solvent extraction is the
widely accepted method to isolate PHA from
cellular mass due to its simplicity and rapidity.
acetoacetyl- CoA
In this method, cell mass is first dried by spray
drying or by lyophilisation followed by addition
acetoaetyl-CoA reductase (PhaB) of large amounts of solvent since PHA solutions
are concentrated and highly viscous. The first
step in solvent extraction method is solubiliza-
(R)-3-hydroxybutyryl-CoA tion of PHA followed by nonsolvent precipita-
PHB synthase (PhaC) tion using methanol and ethanol. Most commonly
used solvents for extraction of PHA are chloro-
PHB form, 1,2-dichloroethane, ethylene carbonate,
1,2-propylene carbonate, and acetone. The use
Fig. 3.12 General pathway for the synthesis of PHB of solvents involves high capital and operational
cost. Alternative extraction method involves
stituents present in oils or fats providing an ideal chemical digestion method using sodium hypo-
opportunity for insertion of required functional chlorite and surfactants such as Triton X-100
groups in PHA. But a major challenge in mcl- and SDS that reduced the cost by 50 % (Yu and
PHA production is a more complex metabolic Stahl 2008; Dong and Sun 2000) and facilitates
network needed for diverting mixture of fatty rapid PHA recovery. Isolation of PHA from S.
acids in fats and oils toward PHA synthesis. meliloti using treatment with surfactant-chelate
Isolation, identification, and genetic manipula- (EDTA) showed 90 % purity (Lakshman and
tion of microbes which produce bioplastics with Shamala 2006). The action of protease secreted
different structures, properties, and applications by Microbispora sp. induced hydrolysis of S.
indicate a promising future for the industrializa- meliloti cells. In other methods, PHA is recov-
tion of bioplastics. ered by enzymatic digestion in which cell lysis is
carried out by the action of proteases (Yasotha
et al. 2006). Other methods applied for extrac-
3.3.3 Various PHA Recovery tion of PHA includes bead milling and high pres-
Methods sure (Bury et al. 2001), the use of
supercritical-carbon dioxide showing 89 %
The extraction procedure applied for the recov- recovery in C. necator (Hejazi et al. 2003), and
ery of PHAs can affect the molecular mass of the use of gamma irradiation as in B. flexus
PHB (Nuti et al. 1972; Senior and Dawes 1973). (Divyashree and Shamala 2009). Apart from the
Various different extraction methods have been solvents used, parameters applied during separa-
reported for PHAs (Table 3.6). Cells grown in tion of PHAs such as pH and temperature mini-
fermentation media were harvested by centrifu- mize degradation of PHAs.
3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 45

Step I
C16) R-CH2-CH2-CH2-C-S-CoA

O Palmitoyl- CoA

Acyl-CoA FAD

transenoyl-CoA hydratase (PhaJ)
R-CH2-C=C- C -S-CoA trans 2-Enoyl-CoA


enoyl-CoA H 2O

(R)-3-hydroxyacyl-CoA *MCL
R-CH2-C- CH2- C -S-CoA 3-hydroxyacyl-CoA epimerase

b-hydroxyacyl-CoA NAD+

R-CH2-C- CH2-C -S-CoA -Ketoacyl-CoA

(R)-ketoacyl-CoA reductase (FabG)

Step II
PHA Synthase
(R)-3-hydroxyacyl-CoA MCL MCL PHAs

Fig. 3.13 Oxidation of fatty acids with even number of carbon atoms

3.3.4 Applications (Bucci and Tavares 2005; Noda 2001; Chen

of Polyhydroxyalkanoates 2009). PHA monomers can be useful synthons in
(PHAs) the pharmaceutical industry (De Eugenio et al.
2010). Small molecular weight PHAs with ter-
PHAs are natural polymers produced by numer- tiary helical structure play an important role like
ous microorganisms like bacteria and algae ion channels (Brandl et al. 1995). Functionalized
under depletion of nitrogen and phosphorous mcl-PHAs with longer side chains are promising
with excess of carbon source. and versatile candidates for high added-value
Polyhydroxyalkanoates can be an effective sub- applications.
stitute for industrial thermoplastics and are use- Bioplastics are normally highly crystalline
ful in compostable packaging, molding, and optically active, possess piezoelectric
veterinary practice, agriculture, and biodegrad- properties, and can be used as an alternative for
able rubbers and as paint binder and resorbable conventional synthetic plastic. PHAs remain as
materials for medical applications (Fig. 3.16) a resource of chiral hydroxy acid. PHA can be
46 M. Dake

(Cn) R-CH2-CH2-CH2- CH2-C-S-CoA

O Fatty acid with odd number of carbon atoms

b-oxiation of fatty acids

CH3-C-S-CoA + CH3-CH2-C-S-CoA

Acetyl CoA Propinoyl CoA

3-Ketothiolase CoASH

3-Keto-valeryl- CoA

acetoacetoacetyl NADPH++ H+
CoA reductase

3-Hydroxyl Valeryl CoA

PHA Synthase
Polyhydroxyvalerate (PHV)

Fig. 3.14 Oxidation of fatty acids with odd number of carbon atoms

a promising candidate in microsphere- or 3.3.5 Economics of the PHA

microcapsule-based drug delivery systems due Production: Decisive Factors
to their unique physiochemical and mechanical
properties. PHA microsphere or microcapsule For successful commercialization of bioplastics,
can transport antibiotics, vaccines, and steroids production cost for PHAs should be reduced by
(Orts et al. 2008). Application of PHA as a car- optimization of fermentation parameters and
rier of drug in anticancer study is reported (Lu purification procedure, cheaper and renewable
et al. 2011). PHAs are applied in medical tools carbon source, and the use of recombinant
such as sutures, meshes, implants, repair microbes (Verlinden et al. 2007). Using sheer
patches, cardiovascular patches, orthopedic diversity of microbial world to screen the
pins, nerve guides, and tendon repair devices microbes capable of producing large amount of
(Chen and Wu 2005; Valappil et al. 2006; PHB using cheaper nutrient sources is also an
Hazer 2010). Board and paper coated with bio- important aspect. Almost 50 % cost for the pro-
based material as PHB are useful for dry foods, duction of PHA is due to the feed carbon source
as these materials have relatively high water used. Waste renewable resources like lignocellu-
vapor barrier capacity as well as confer paper losics, carbohydrates, waste lipids, or alcohols
or board mechanical strength, thereby protect- are beneficial in PHAs production (Braunegg
ing products from breakage. Composite of et al. 1998; Solaiman et al. 2006; Khardenavis
PHA and hydroxyapatite (HA) has the poten- et al. 2007). Productivity of PHAs can be
tial to be used in hard tissue replacement and enhanced by improvements in downstream pro-
regeneration. cessing where fermentation by using the continu-
3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 47

Carbohydrate (glucose, gluconate) the new one by recombinant DNA techniques can
also improve the efficiency of microbes to
produce PHA enabling them to utilize a wide
range of renewable carbon sources as substrate.
Genetically modified Aeromonas hydrophila had
acetyl-CoA enhanced ability to produce poly(3-
hydroxybutyrate- co-3-hydroxyhexanoate)
(PHBHHx) (Han et al. 2004).
malonyl-CoA Recombinant E. coli strains developed by
inserting the genes for PHA biosynthesis from R.
eutropha, yielded PHB with mass of
malonyl-ACP + acyl-ACP
311 106 Da. Genes for PHA synthesis were
reported to be cloned from Alcaligenes eutro-
3-Ketoacyl-ACP phus that were successfully expressed in E. coli
synthase CO2+ACP with 80 % PHB content (Kato et al. 1996) where
the same microbial strain is currently utilized for
producing PHA on commercial scale by the
3-Ketoacyl-ACP company Metabolix in the USA (Ren et al.
reductase 2000). Other companies producing PHA on
industrial scale are Biomatera, PolyFerm
Canada, Tianan, Bio-On, Biomer, Kaneka,
Tianzhu, etc. Thus, bioplastics will capture 30 %
3-hydroxyacyl-ACP-CoA of the total plastics market within the next decade
Transacylase (PhaG) and will decrease the reliance on nonrenewable
fossil fuel.

PHA synthase
(phaC) 3.4 Perspectives
MCL PHA Polyhydroxyalkanoates representing a renewable
Fig. 3.15 De novo synthesis pathway, acyl carrier source for bioplastics can overcome the
protein petrochemical-derived synthetic biopolymers.
Cost-effective production of PHA can be possi-
ble by bioprocess design through development of
ous production mode is beneficial for high mutant microbial strain by genetic manipulation
productivity in microbial sp. like Cupriavidus capable of utilizing low-cost renewable sub-
necator DSM 545 (Horvat et al. 2013). strates as a carbon source. The major challenge in
Application of inexpensive growth additive to PHA production is the recovery process that can
enhance rate of biomass production and genetic be optimized using low-cost eco-friendly extrac-
engineering aspects involving inactivation or tion method.
modification of enzymes causing intracellular
PHA degradation will be new criteria for enhanc- Acknowledgments The author is thankful to Springer
ing volumetric productivity of the process. and the editor Dr. V. C. Kalia for giving the opportunity to
contribute a book chapter. The support from Dr. D. Y. Patil
Alteration of metabolic pathway or introducing
Vidyapeeth, Pune, is also gratefully acknowledged.
48 M. Dake

Table 3.3 Precursors used in the literature to produce functionalized mcl-PHA (branched alkyl, cyclohexyl,
mcl-PHAs Precursor Microbial strain
Branched alkyl mcl-PHA Citronellol P. citronellolis ATCC 13674
Alkylhydroxyoctanoates P. putida GPo1
Methyloctanoates P. putida GPo1
Cyclohexyl mcl-PHA Cyclohexylbutyric acid P. cichorii YN2
Cyclohexylvaleric/butyric acid P. putida GPo1
Unsaturated mcl-PHAS Alkenes (C7C9) P. putida GPo1
Undecenoic acid P. putida KCTC 2407
Undecenoic acid P. putida GPo1
Hydroxyoctanoic acids P. putida GPo1
Dicarboxylic acids (C4C10) P. citronellolis ATCC 13.674
Undecynoic acid P. putida GPo1
Undecynoic acid P. putida KCTC 2407
Halogens mcl-PHA Bromoalkanoic acids (C6C11) P. putida GPo1
Chlorooctane P. putida GPo1
Fluorohexanoic/nonanoic acids P. putida GPo1
Fluorohexanoic/nonanoic acids P. putida KT2440
Fluorophenoxyundecanoic acid P. putida 27 N01

Table 3.4 Precursors used in the literature to produce functionalized mcl-PHA (acetoxy, ester, alkoxy, epoxy, thio,
cyano, nitro)
mcl-PHAs Precursor Microbial strain
Acetoxy mcl-PHA Octanone, octylacetate P. putida GPo1
Ester/alkoxy/epoxy mcl-PHA Alkylheptanoate P. putida GPo1
Alkxyhexanoic/octanoic/undecanoic acids P. putida GPo1
10-Epoxyundecanoic acid P. putida GPo1
C7C12 alkenes P. cichorii YN2
Soybean oil P. stutzeri 1317
Thio, sulfanyl mcl-PHA Acetylthiohexanoic acid P. putida KT2442, KT24FadB
Propylthiohexanoic acid Ralstonia eutropha DSM541
Propylthioundecanoic acid P. putida KT2440
Methylsulfanylphenoxyvaleric acid P. cichorii H45, YN2
Methylsulfanylphenoxyvaleric acid P. jessenii P161
Thiophenoxyundecanoic acid P. putida 27 N01
Cyano, nitro mcl-PHA Cyanoundecanoic acid P. putida GPo1
Cyanophenoxyhexanoic acid
Nitrophenoxyhexanoic acid
Dinitrophenylvaleric acid
Cyanophenoxyhexanoic acid P. putida KT2440
Nitrophenoxyhexanoic acid
3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 49

Table 3.5 Precursors used in the literature to produce functionalized mcl-PHA (aromatics) group at mcl-PHA side
chain: aromatics (benzoyl, methylphenoxy, phenoxy, and phenyl)
mcl-PHAs Precursor Microbial strain
Aromatics mcl-PHA Benzoylalkanoic acids (C4C8) P. cichorii YN2
Methylphenoxyalkanoic acids (C6, C8) P. putida KCTC 2407
Methylphenoxyalkanoic acids (C6, C8) P. putida GPo1
Methylphenoxyalkanoic acids (C6, C8) P. putida KCTC 2407
Phenoxyundecanoic acid P. putida GPo1
Phenoxyalkanoic acids (C6, C8, C11) P. putida GPo1
Phenoxyundecanoic acid P. putida BM01
Phenylvaleric acid P. putida BM01
Phenylvaleric acid P. putida GPo1
Phenyl, tolylvaleric/octanoic acids P. putida GPo1
Phenylalkanoic acids (C4C8) P. jessenii C8
Phenylalkanoic acids (C4C8) P. putida S12, CA-1, H4, F6, D5
Phenylalkanoic acids (C6C11) P. putida U fadA-, FadBA-PhaZ
Phenylvaleric acid P. putida GPo1
Phenylvaleric acid P. putida GPo1

Table 3.6 PHA recovery: solvent extraction methods

Extraction method Solvent used Strain used Yield
Solvent extraction Chloroform Bacillus cereus SPV 31 %
Cupriavidus necator DSM54 96 %
Acetone-water process 8085 %
Methylene chloride C. necator 98 %
Acetone, room temperature P. putida GPo1 94 %
Nonhalogenated solvents: isoamyl C. necator
propionate, propyl butyrate, isoamyl
Surfactant SDS Rec. Escherichia coli 89 %
Sodium hypochlorite Sodium hypochlorite C. necator DSM 545, Rec. coli
Surfactant-sodium SDS-sodium hypochlorite Azotobacter chroococcum G-3 87 %
Surfactant-chelate Triton X-100-EDTA Sinorhizobium meliloti
Dispersion of sodium Chloroform-sodium hypochlorite B. cereus SPV 30 %
hypochlorite and Chloroform-sodium hypochlorite C. necator, Rec. E. coli
Selective dissolution Sulfuric acid C. necator 95 %
by protons
Enzymatic digestion Microbispora sp. culture-chloroform S. meliloti
Enzyme combined with SDS-EDTA P. putida
Bromelain; pancreatin C. necator
Mechanical disruption Bead mill A. latus
High-pressure homogenization A. latus
SDS-high-pressure homogenization Methylobacterium sp. V49 98 %
Sonication Bacillus flexus 20 %
50 M. Dake

Table 3.6 (continued)

Extraction method Solvent used Strain used Yield
Supercritical fluid SC-CO2 C. necator 89 %
Cell fragility Chloroform B. flexus 43 %
Sodium hypochlorite B. flexus 50 %
Alkaline hydrolysis B. flexus 50 %
Gamma irradiation Radiation-chloroform B. flexus 4554 %
Air classification E. coli 90 %
C. necator 85 %
Spontaneous E. coli 80 % (of autolysis)


Electronic material



Sticky tape made from Cup made from
Flow pack packaging for
Calendar case made from
made from bioplastic
Spring water bottle

fresh products

Starch based carrot


Fig. 3.16 Applications of PHAs in various fields

3 Biodegradable Polymers: Renewable Nature, Life Cycle, and Applications 51

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Wang L, Shelton RM, Cooper PR, Lawson M,
Triffitt JT, Barralet JE (2003) Evaluation of Manjusha Dake
sodium alginate for bone marrow cell tissue engi- received her M.Sc. and Ph.D.
neering. Biomaterials 24:34753481. doi:10.1016/ degree in Biochemistry from
s0142-9612(03)00167-4 Shivaji University, Kolhapur.
Wang YW, Wu Q, Chen GQ (2005) Gelatin blending She is presently working as
improves the performance of poly(3-hydroxybutyrate- an Assistant Professor in
co-3-hydroxyhexanoate)films for biomedical applica- Biochemistry at Dr. D.Y. Patil
tion. Biomacromolecules 6:566571. doi:10.1021/ Biotechnology and
bm049342d Bioinformatics Institute,
Witthuhn RC, Schoeman T, Britz TJ (2005) Pune. Her current interests
Characterisation of the microbial population at differ- are biofuel and degradative
ent stages of Kefir production and Kefir grain mass enzymes. She was also a
cultivation. Int Dairy J 15:383389. doi:10.1016/j. member of the Reviewer
idairyj.2004.07.016 Board of the Journal of
Wong TY, Preston LA, Schiller NL (2000) Alginate lyase: Microbial and Biochemical Technology. Currently, she is
review of major sources and enzyme characteristics, working on applications of enzymes in bioremediation.
Phylogenetic Affiliation
of Pseudomonas sp. MO2, a Novel 4
Synthesizing Bacterium

Parveen Kumar Sharma, Jilagamazhi Fu,

Xiang Zhang, Richard Sparling,
and David B. Levin

A bacterium, isolated from wastewater after enrichment with waste canola
fryer oil, was found to synthesize 1220 % of cell dry weight (cdw) medium
chain length polyhydroxyalkanoates (mcl-PHAs) using different carbon sub-
strates. On the basis of partial 16S rDNA sequence analysis, this bacterium was
first identified as Pseudomonas putida and designated as P. putida strain MO2.
However, the full 16S rDNA gene sequence from a whole genome sequence
analysis of this strain showed 100 % identity to 16S rDNA of Pseudomonas
monteilii SB3101 and Pseudomonas monteilii SB3078. The comparison of the
cpn60 gene sequence of Pseudomonas sp. strain MO2 with strains of P. putida,
P. aeruginosa, P. fluorescens, P. stutzeri, P. syringae, and P. monteilii indicated
that this strain is more closely related to P. monteilii than P. putida type strain
KT2440. Based on gene sequence similarity index and phylogenetic analyses,
some P. putida strains, which were earlier classified as P. putida, are also more
closely related to the P. monteilii cluster. Our analyses show that P. putida is a
very diverse group with divergent strains, and many strains of P. putida that
cluster with P. monteilii species may need to be reclassified.

P.K. Sharma J. Fu D.B. Levin (*) 4.1 Introduction

Department of Biosystems Engineering, University of
Manitoba, R3T 2N2 Winnipeg, Manitoba, Canada
Polyhydroxyalkanoates (PHAs) are intracellular
e-mail: pksharmajan11@yahoo.com;
Parveen.Sharma@umanitoba.ca; polymers synthesized by bacteria under carbon
umfu23@myumanitoba.ca; excess and nitrogen or phosphorus limiting differ-
fujilagamazhi@gmail.com; david.levin@umanitoba.ca ent pathways (Steinbchel and Fchtenbusch
X. Zhang 1998; Lee et al. 1999; Steinbchel 2001; Rehm
Department of Plant Science, University of Manitoba, and Steinbchel 2001). PHAs are classified into
R3T 2N2 Winnipeg, Manitoba, Canada
two classes, short chain length (scl) and medium
e-mail: justinzhang.xl@gmail.com
chain length (mcl), depending on the carbon chain
R. Sparling
length of the repeat units. The scl-PHAs have 35
Department of Microbiology, University of Manitoba,
R3T 2N2 Winnipeg, Manitoba, Canada carbons in the polymer subunits, while mcl-PHAs
e-mail: richard.sparling@umanitoba.ca have 614 carbons in the polymer subunits. The

Springer India 2015 57

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_4
58 P.K. Sharma et al.

monomer composition of PHAs determines their rod-shaped, non-sporulating, polar-flagellated,

properties as thermoplastics, elastomers, and/or aerobic bacteria (Stanier et al. 1966; Palleroni
adhesives (Lee 1996). PHA production in P. 1984; 2005; Regenhardt et al. 2002).
putida (formerly P. oleovorans) was first report by Pseudomonas species have been differentiated on
Smet et al. (1983) when this organism was grown the basis of biochemical tests, fatty acid compo-
in n-octane. Since then, a large number of sition, siderophores, 16S rDNA sequence, and
Pseudomonas species have been identified as protein profiles (Stanier et al. 1966; Janse et al.
PHA producers and PHA production appears to 1992; Stead 1992; Vancanneyt et al.1996; Anzai
be a general feature among pseudomonads. et al. 2000; Meyer et al.2008; Santos and Ochman
Pseudomonas spp. are known to produce 2004; Goris et al. 2007; Mulet et al. 2009). The
medium chain length polyhydroxyalkanoates type I chaperonin, encoded by cpn60 (also known
(mcl-PHAs) from a variety of carbon sources, as GroEL or Hsp60), is a highly conserved pro-
such as glucose, glycerol, and fatty acids, in tein found in bacteria that has also been widely
batch and fed batch cultures (Sun et al. 2007; used for phylogenetic studies as well as for iden-
Sharma et al. 2012). Daird et al. (2002) demon- tification of bacteria (Hill et al. 2004; Lazarovits
strated mcl-PHA synthesis by different et al. 2013).
Pseudomonas species belonging to the pseudo- The gene encoding 16S rRNA has been widely
monad rRNA homology group. PHA synthesis used as a molecular sequence to reconstruct
has been demonstrated and confirmed in P. inferred phylogenies because it was assumed that
putida (Sharma et al. 2012, 2014), P. aeruginosa the intraspecific variation and horizontal transfer
(Chan et al. 2006; Pham et al. 2004), P. fluores- of this gene between organisms were low.
cens (Gamal et al. 2013), P. stutzeri (Chen et al. Pseudomonads belonging to rRNA homology
2004), P. mendocina (Daird et al. 2002; Guo group I include both fluorescent and nonfluores-
et al. 2011), P. resinovorans (Solaiman et al. cent species. Yamamoto et al. (2000) identified
2000), and P. entomophila (Chung et al. 2011, two major clusters, designated as intrageneric
2013). Recently P. mosselii TK07 was isolated cluster I (IGC I) and intrageneric cluster II (IGC
from wastewater from a vegetable oil processing II). P. aeruginosa and P. stutzeri formed two sub-
plant, and this strain was shown to synthesize clusters within the IGC I cluster, while P. putida,
mcl-PHAs from palm kernel and soybean oil P. syringae, and P. fluorescens represent three
(Chen et al. 2014). subclusters within IGC II. Pseudomonads belong-
Pseudomonas putida KT2440 (type strain of ing to these three branches are able to produce
P. putida) and P. putida Gpo1 are under develop- medium chain length PHAs from octanoate
ment for industrial PHA production. However, (Daird et al. 2002).
although several companies have commercially Phylogenies based on 16S rDNA intrageneric
produced scl-PHAs, mcl-PHAs have yet to be cluster analyses, however, lack resolution due to
produced in large scale due to their low mcl-PHA their slow rate of evolution, so other molecular
yield and the high cost of production (Elbahloul targets have been investigated to elucidate the
and Steinbchel 2009). There are only few phylogenetic relationships among Pseudomonas
reports on production of PHAs using low-cost species (Moore et al.1996; Yamamoto et al. 2000;
renewable carbon sources like animal fats and Anzai et al. 2000). Genes like atpD (ATP syn-
vegetable oils (Ashby and Foglia 1998; Cromwick thase), carA (carbamoyl phosphate synthase, sub-
et al. 1996; Fchtenbusch et al. 2000; Tan et al. unit A), recA (recombinase A), gyrB (gyrase B),
1997) and dairy waste like whey (Park et al. and rpoD (RNA polymerase sigma 70) have been
2002). However, production of PHAs from veg- employed for phylogenetic analyses (Hilario
etable oils by Pseudomonas putida has not been et al. 2004; Tayeb et al.2005; Martnez-Murcia
reported. et al. 2009). Mulet et al. (2010) demonstrated that
The genus Pseudomonas is a very heteroge- three concatenated genes (16S rRNA, gyrB, and
neous taxon containing many Gram-negative, rpoD) were successfully used for phylogenetic
4 Phylogenetic Affiliation of Pseudomonas sp. MO2, a Novel 59

analysis of pseudomonads. Konstantinidis et al. terium as a strain of P. monteilii was further con-
(2006) pointed out that concatenation of these firmed by phylogenetic analyses and gene
three genes could also be employed for the analy- similarity indices (GSI) using the cpn60 gene,
ses of other bacteria groups, including Escherichia phylogenetic analyses using the PHA synthase
coli, Salmonella, Burkholderia, and Shewanella, genes (phaC1 and phaC2), and phylogenetic
when compared with their whole genomes. analyses with several individual or concatenated
On the basis of phenotypic heterogeneity, P. housekeeping genes (dnaJ, gyrA, gyrB, rpoA,
putida has been divided into three groups: Biovar and rpoD). The pseudomonads are a diverse
A, Biovar B, and Biovar C (Palleroni 1984; group of saprophytic bacteria which are present
Stanier et al. 1966; Barrett et al. 1986; Palleroni in soil, water, plants, and animals. Pseudomonas
2005). Numerical analyses (Barrett et al.1986; putida strains are well known for degradation of
Champion et al. 1980) and 16S rRNA gene xenobiotics (Timmis 2002; Nakazawa 2003;
restriction patterns (Elomari et al. 1994) identi- Timmis and Pieper 1999; Nelson et al. 2002).
fied several subgroups within these Biovars and
further suggested that P. putida consisted of a
number of strains, some of which could consti- 4.2 Isolation and Identification
tute new species. P. putida phylogenetic studies, of a Novel PHA-Producing
based on 16S rRNA sequence comparisons, con- Bacterium
firmed that strains of Biovars A and B were dif-
ferent (Moore et al.1996; Mulet et al.2013). Pseudomonas sp. MO2 was isolated from waste-
Multilocus sequence analyses (MLSA) using water using Ramsays minimal medium with
genes like cpn60, recA, rpoA, rpoD, and dnaJ waste fryer oil as sole carbon source. The culture
have also been employed to identify intraspecific was purified by streaking on Luria Bertani plates.
diversity and to confirm the phylogenetic rela- Genomic DNA was isolated using ChargeSwitch
tionships among P. putida strains. Consequently, gDNA Mini Bacteria Kit (Life Technologies,
some strains previously described as P. putida Burlington, ON). The whole genome was
were separated into other species groups, like sequenced and assembled by Genome Quebec,
Pseudomonas mosselii (Dabboussi et al. 2002) or Montreal, using Pacific Biosciences (PacBio) RS
Pseudomonas monteilii (Elomari et al. 1997). II Sequencing Technology. The genome was
Using unique features of 16S rRNA genes, annotated using NCBI pipeline (Acc. No.
Bhushan et al. (2013) developed a strategy to JFBC00000000). Pseudomonas strains used in
identify Pseudomonas strains to the species level. present study are listed in Table 4.1. BLASTn
It was concluded that majority of P. aeruginosa, analysis of the complete 16S rDNA of
P. stutzeri, and P. syringae strains were identified Pseudomonas MO2 (1538 bp) showed 100 %
and classified properly. However, P. putida and P. identity to P. monteilii SB3078, P. monteilii
fluorescens strains may require reclassification SB3101, and Pseudomonas sp. VLB120 (Fig.
(Bossis et al. 2000). 4.2), but only 99 % identity to Pseudomonas
We isolated a mcl-PHA-synthesizing pseudo- FGI182, P. putida S16, P. putida H8234, P. putida
monad bacterium from wastewater after enrich- HB3267, and other P. putida strains. P. monteilii
ment with waste fryer oil and, on the basis of 16S SB3078 and SB3101 are benzene-, toluene-, and
rDNA sequence analysis, identified the bacte- ethylbenzene-degrading bacteria, while
rium a member of the Pseudomonas putida group Pseudomonas sp. VLB120 is a solvent-tolerant,
(99 % nucleotide homology to other P. putida styrene-degrading bacterium isolated from soil
strains). However, upon further analyses, the 16S (Dueholm et al. 2014; Otto et al. 2004). All the
rDNA sequence showed 100 % nucleotide three bacteria have recently been sequenced after
sequence homology to two newly sequenced December 2013. P. putida S16 is a solvent-tolerant
Pseudomonas monteilii strains, SB3101 and nicotine-degrading bacterium (Wang et al. 2007;
SB3078. Identification of the newly isolated bac- Tang et al. 2012). Pseudomononas putida
Table 4.1 Pseudomonas strains, genome accession numbers, and genes (locus tags) used in present study
Genome acc. no Gene locus tag
Genome name 16S rRNA cpn60 rpoA rpoD gyrA gyrB dnaJ
P. putida S16 NC_015733 PPS_r01 PPS_4332 PPS_0475 PPS_0383 PPS_1408 PPS_0012 PPS_0403
P. putida 14164 AP013070 PP4_r0010 PP4_08720 PP4_05120 PP4_04200 PP4_40040 PP4_00040 PP4_04400
P. putida SJTE-1 AKCL00000000 A210DRAFT_00860 A210DRAFT_01900 A210DRAFT_03068 A210DRAFT_02963 A210DRAFT_01435 A210DRAFT_00004 A210DRAFT_02143
P. putida CSV86 NZ_ CSV86_r20517 CSV86_04192 CSV86_08943 CSV86_22371 CSV86_27714 CSV86_19158 CSV86_22598
P. putida PC9 CP003738 B479_r26783 B479_21790 B479_02880 B479_02415 B479_06830 B479_00265 B479_02515
P. putida H8234 CP005976 L483_00455 L483_26850 L483_02465 L483_01975 L483_06360 L483_32300 L483_02075
P. putida MTCC AMZE00000000 D095DRAFT_04923 D095DRAFT_03746 D095DRAFT_02128 D095DRAFT_01341 D095DRAFT_00193 D095DRAFT_04077 D095DRAFT_02038
P. putida LS46 ALPV02000000 PPUTLS46_0r25527 PPUTLS46_013648 PPUTLS46_023893 PPUTLS46_014694 PPUTLS46_015444 PPUTLS46_016579 PPUTLS46_007106
P. putida F1 NC_009512 Pput_R0001 Pput_4363 Pput_0512 Pput_0421 Pput_3947 Pput_0004 Pput_0441
P. putida B6-2 AGCS00000000 KKKDRAFT_00141 KKKDRAFT_04728 KKKDRAFT_00530 KKKDRAFT_04519 KKKDRAFT_04318 KKKDRAFT_00025 KKKDRAFT_05822
P. putida W619 NC_010501 PputW619_R0006 PputW619_0968 PputW619_4724 PputW619_4815 PputW619_1374 PputW619_0004 PputW619_0706
P. putida B001 NZ_ PPB1DRAFT_04917 PPB1DRAFT_03185 PPB1DRAFT_02238 PPB1DRAFT_04775 PPB1DRAFT_01264 PPB1DRAFT_04934 PPB1DRAFT_03026
P. putida GB-1 NC_010322 PputGB1_R0001 PputGB1_4488 PputGB1_0508 PputGB1_0418 PputGB1_1358 PputGB1_0006 PputGB1_4727
P. putida TRO1 APBQ0100001 C206_r13460 C206_04422 C206_22169 C206_03629 C206_11184 C206_04924 C206_10802
P. putida KT2440 NC_002947 PP_16SA PP_1361 PP0479 PP_0387 PP_1767 PP_0013 PP_0407
P. putida S12 NZ_ B478DRAFT_00819 B478DRAFT_0315 B478DRAFT_0570 B478DRAFT_0025 B478DRAFT_0562 B478DRAFT_0004 B478DRAFT_05215
P. putida DOT-T1E CP003734 T1E_5788 T1E_4381 T1E_3477 T1E_2166 T1E_3016 T1E_0874 T1E_0653
P. putida Idaho AGFJ00000000 KICDRAFT_04973 KICDRAFT_05289 KICDRAFT_01684 KICDRAFT_06289 KICDRAFT_04345 KICDRAFT_04591 KICDRAFT_06260
P. putida BIRD-1 CP002290 PPUBIRD1_r0001 PPUBIRD1_4201 PPUBIRD1_0516 PPUBIRD1_0424 PPUBIRD1_3846 PPUBIRD1_0077 PPUBIRD1_0445
P. putida UW4 CP003880 PputUW4_R0030 PputUW4_04386 PputUW4_04859 PputUW4_04952 PputUW4_01358 PputUW4_00004 PputUW4_00691
P. putida ND6 CP003588 YSA_11387 YSA_02940 YSA_05974 YSA_05784 YSA_02244 YSA_05066 YSA_03420
P. putida NB2011 ASJX01000001 L321_r02742 L321_23936 L321_12624 L321_11605 L321_00737 L321_17452 L321_03911
P. monteilii SB3101 CP006979 X970_00550 X970_20960 X970_00800 X970_00290 X970_04940 X970_25675 X970_00400
P. monteilii SB3078 CP006978 X969_00560 X969_10100 X969_00810 X969_00300 X969_04965 X969_26040 X969_00410
Pseudomonas CP007012 C163_00600 C163_21760 C163_02435 C163_01930 C163_00020 C163_06545 C163_02030
Pseudomonas CP003961.1 PVLB_r25916 PVLB_04630 PVLB_22805 PVLB_23270 PVLB_02625 PVLB_00020 PVLB_03450
P. aeruginosa PA7 NC_009656 PSPA7_0811 PSPA7_4956 PSPA7_0862 PSPA7_0718 PSPA7_1961 PSPA7_0004 PSPA7_5480
P. aeruginosa PAO1 CP006831 PA0668.1 PA4385 PA4238 PA0576 PA3168 PA0004 PA4760
P. stutzeri A1501 CP000304 PST_0759 PST_3145 PST_0809 PST_0712 PST_2343 PST_0004 PST_3326
P. fluorescens Pf5 CP000076 PFL_0119 PFL_4838 PFL_5558 PFL_5663 PFL_4314 PFL_0004 PFL_0828
P. mendocina NK-01 CP002620 MDS_r003 MDS_3990 MDS_4268 MDS_4366 MDS_1954 MDS_0004 MDS_3928
P. mendocina ymp CP000680 Pmen_R0008 Pmen_3688 Pmen_3884 Pmen_4028 Pmen_1848 Pmen_0004 Pmen_3623
P. entomophila L48 CT573326 PSEEN_16s_1 PSEEN4460 PSEEN0514 PSEEN0413 PSEEN1487 PSEEN0004 PSEEN0434
P. putida H3267 CP003738 B479_r26783 B479_21790 B479_02880 B479_02415 B479_06830 B479_00265 B479_22975
Pseudomonas MO2 JFBC00000000 BC89_30710 BC89_13485 BC89_07790 BC89_22730 BC89_07010 BC89_23970 BC89_15680
P. resinovorans AP013069 PCA10_r0010 PCA10_46990 PCA10_06160 PCA10_05250 PCA10_39190 PCA10_00040 PCA10_49260
P. fulva 12-X CP002727 Psefu_R0001 Psefu_3538 Psefu_0665 Psefu_3960 Psefu_2016 Psefu_0004 Psefu_3600
P. syringae DC3000 AE016853 PSPTOimg_006280 PSPTOimg_045270 PSPTOimg_006790 PSPTOimg_005530 PSPTOimg_018060 PSPTOimg_000040 PSPTOimg_0046590
62 P.K. Sharma et al.

HB3267 was isolated from a hospital in The monomer compositions of mcl-PHAs syn-
Besanon, France, from an inpatient and is thesized (Table 4.2) in medium containing glucose
known to kill insects as well as carry antibiotic versus glycerol were very similar, and the major
resistance genes (Molina et al. 2014). component of polymers was 3-hydroxydecanoate
(5264 mol %). In medium containing free fatty
acids, 3-hydroxyoctanoate and 3-hydroxytetra-
4.3 PHA Production decanoate were the major monomers (30 mol %
by Pseudomonas sp. MO2 each), but 3-hydroxydecanoate and 3-hydroxydo-
decanoate were present in lesser amounts (17.5
Three waste substrates (waste fryer oil from 19.0 mol %). Finally, the major constituents of
McCain Food, Portage La Prairie, Manitoba, mcl-PHAs synthesized in medium containing
Canada) and two by-products of biodiesel indus- waste fryer oil and REG fatty acids were
tries (REG glycerol and REG fatty acids from 3-hydroxyoctanoate (39.5 mol %) and
REG, Danville, Illinois, USA) were used for 3-hydroxydecanoate (32.9 mol %). Not only did
PHAs production. Pseudomonas sp. MO2 was the monomer composition of the polymers vary
grown in Ramsays minimal medium (Ramsay with substrate, they also varied with the growth
et al. 1991) with REG glycerol (2 % V/V), REG state of the cells, as related to the time of incuba-
fatty acids (1 % V/V), and waste canola fryer oil tion before samples were taken. In the case of
(1 % V/V). Cell dry weight, PHA production, glucose-grown cells, 3-hydroxyhexanoate was
and PHA monomer composition were deter- more than 39 mol % at 12 h pi, but decreased
mined as described earlier (Braunegg et al. 1978; with further incubation, with a corresponding
Sharma et al. 2012). The maximum cell mass increase in 3-hydroxydecanoate, while the 3-
(measured as cell dry weight = cdw) of hydroxyoctanoate content remained more or less
Pseudomonas sp. MO2 varied with the substrate constant. In glycerol medium, a decrease in the
used. REG fatty acid-grown cultures accumu- 3-hydroxyhexanoate content of the polymer
lated maximum cdw (2.77 g/L) after 48 h post- between 12 and 96 h was accompanied by an
inoculation (h pi) followed by glucose (2.27 g/L) increase in the 3-hydroxyoctanoate and 3-
after 24 h pi (Table 4.2). The mcl-PHAs were hydroxydecanoate content. In fatty acid medium,
detected after 12 h pi; however, the production the 3-hydrohexanoate and 3-hydroxydecanoate
was very low in medium containing glucose (4.8 content remained the same from 12 to 96 h pi, but
% of cdw) and glycerol (4.13 % of cdw) com- the 3-hydroxyoctanoate and 3-hydroxytetradec-
pared with medium containing REG fatty acids anoate contents of the polymer changed.
(13.37 % of cdw) or waste fryer oil (12.37 % of Interestingly, there was no significant change in
cdw). In glucose and glycerol medium, maxi- monomer composition of the mcl-PHAs in waste
mum PHA synthesis was observed at 48 h pi, fryer oil medium from 12 to 72 h pi, but at 96 h pi,
while maximum PHA synthesis was observed the 3-hydroxydodecanoate and 3-hydroxytetra-
after 36 h pi in medium containing free fatty decanoate content increased as the mol % of
acids or waste fryer oil medium. The PHAs pro- 3-hydroxyhexanoate, 3-hydroxyoctanoate, and
duction decreased significantly after 48 h pi in 3-hydroxydodecanoate decreased.
medium containing glucose, glycerol, or waste The monomer composition of mcl-PHAs syn-
fryer oil, but not in medium containing REG free thesized by Pseudomonas sp. MO2 indicates that
fatty acids. Like other Pseudomonas strains, precursors for PHA synthesis are provided by de
Pseudomonas sp. MO2 had six pha genes (phaC- novo fatty acid synthesis pathway when the cells
1ZC2DFI) which are highly conserved (unpub- are cultured in medium containing glucose or
lished data). The pha genes of Pseudomonas sp. glycerol or by the -oxidation pathway when the
MO2 had the same size and organization as cells are grown in medium containing fatty acids
Pseudomonas putida KT2440 and Pseudomonas or waste fryer oil. The role of de novo synthesis
putida LS46 (Sharma et al. 2014). of fatty acids in the synthesis of mcl-PHAs has
Table 4.2 Cell biomass and PHA production Pseudomonas MO2 in RMM with glucose, REG glycerol, REG fatty acids, waste fryer oil, and the corresponding mcl-PHA poly-
mer monomer composition

% PHA Monomer composition (% mol)a

Substrate Incubation time (h) CDW (g/L) production HHx HO HD HDD HTD
Waste fryer oil (1 %) 12 1.48 0.04 12.78 0.69 9.25 1.22 37.66 5.17 28.05 3.83 11.52 3.27 13.55 1.71
24 2.91 0.17 15.22 0.91 8.36 1.14 40.52 0.85 33.95 1.44 10.36 1.07 6.79 0.89
36 2.77 0.07 15.05 1.39 9.04 0.76 39.50 0.48 32.92 3.28 11.47 2.33 7.05 3.29
48 2.74 0.18 12.60 0.92 9.10 0.33 39.78 0.88 33.02 1.05 12.37 1.84 5.70 0.53
72 2.64 0.09 10.43 0.78 7.80 1.13 36.73 1.23 29.11 1.68 17.59 2.82 8.75 1.06
96 2.54 0.03 8.48 1.78 3.73 2.63 39.08 2.45 26.44 3.21 18.76 1.89 11.96 2.31
Glucose (2 %) 12 0.75 0.06 4.80 2.64 15.95 2.45 2.35 3.68 1.74 3.32 55.99 3.31 23.94 4.89
24 2.27 0.38 6.29 0.34 17.62 2.12 8.58 0.81 32.31 3.76 25.56 4.18 11.90 2.18
36 1.99 0.08 16.68 2.65 12.11 3.75 13.74 1.04 52.12 8.23 18.06 2.26 3.95 1.22
48 2.05 0.05 16.94 4.37 7.53 2.10 16.39 0.28 54.89 4.52 15.76 3.68 5.40 2.78
72 1.87 0.08 16.30 3.17 8.54 1.81 19.57 1.06 44.92 6.13 19.18 2.10 7.77 1.14
96 0.92 0.13 17.21 2.87 8.28 1.88 19.25 1.78 35.76 4.23 28.76 5.12 7.93 2.26
REG glycerol (2 %) 12 0.74 0.02 4.13 0.37 15.95 2.51 2.35 0.98 1.74 0.89 55.99 0.72 23.94 5.57
24 2.01 0.06 5.37 0.53 17.72 1.87 8.58 0.58 32.31 5.56 29.56 3.29 11.90 0.93
4 Phylogenetic Affiliation of Pseudomonas sp. MO2, a Novel

36 2.14 0.07 11.32 1.24 12.11 3.04 13.74 1.71 52.12 3.04 18.06 2.22 3.95 1.22
48 1.97 0.12 12.24 0.88 7.53 1.46 16.39 1.04 54.89 2.13 15.76 1.67 5.40 3.07
72 1.92 0.09 11.71 1.32 8.54 0.43 19.57 0.71 44.92 3.15 19.18 1.89 7.77 1.59
96 1.48 0.09 9.02 1.42 8.28 1.40 19.25 2.75 35.76 4.23 28.76 4.53 7.93 1.82
REG fatty acids (1 %) 12 0.71 0.02 13.37 2.72 4.92 1.69 37.62 8.2 33.34 1.48 14.91 0.52 9.18 0.91
24 1.80 0.02 17.81 2.72 4.89 1.27 45.49 5.43 17.36 5.32 15.58 3.15 16.66 3.22
36 1.96 0.11 20.37 1.57 3.33 0.45 31.26 5.44 19.02 3.06 17.50 1.91 28.87 7.04
48 1.58 0.08 20.70 0.59 3.04 0.30 29.46 2.86 16.76 3.21 17.82 0.81 32.89 4.87
72 1.12 0.08 20.77 0.51 3.63 0.15 36.86 1.48 22.45 4.17 16.77 2.26 20.26 4.65
96 1.04 0.04 20.22 1.10 5.28 1.12 45.12 7.97 20.11 3.23 13.86 3.21 15.62 7.85
Mean of three independent replicate samples standard deviation
64 P.K. Sharma et al.

been established (Rehm et al. 2001). During fatty the branch nodes of the corresponding trees.
acid synthesis, R-3-hydroxyl fatty acid-ACP Nucleotide polymorphic sites, the average num-
intermediates are synthesized and then converted ber of nucleotide differences, nucleotide diver-
to R-3-hydroxyl fatty acid by the PhaG enzyme sity (per site), synonymous site PI (JC), and
(Matsumoto et al. 2001). In addition to this, an non-synonymous site PI (JC) were calculated
enzyme, 3-hydroxyacyl-CoA ligase, has also using DnaSP software (Rozas 2009).
been hypothesized to convert R-3-hydroxyl fatty Nucleotide sequences of the Pseudomonas sp.
acid to (R)-3-hydroxyl fatty acid-CoA (Wang MO2 cpn60 (locus tag BC89_13485), dnaJ
et al. 2012). The (R)-specific enoyl-CoA hydra- (locus tag BC89_15680), gyrA (locus tag
tase, PhaJ, is an enzyme that could potentially BC89_07010), gyrB (locus tag BC89_23970),
provide (R)-3 hydroxyacyl-CoA precursors for rpoA (locus tag BC89_07790), rpoD (locus tag
mcl-PHA synthesis derived via the fatty acid BC89_22730), phaC1 (locus tag BC89_22160),
-oxidation pathway when the bacterial cells are and phaC2 (locus tag BC89_22170) genes were
grown on fatty acids (Fiedler et al. 2002). Both retrieved from the annotated genome sequence
these enzymes are the connecting link between and compared with genes from other
fatty acid metabolism and PHA synthesis. Pseudomonas strains. Other P. putida (belonging
Changes in monomer composition of mcl-PHA to Biovar A and Biovar B), as selected by Mulet
polymers occur during the exponential growth et al. (2013), were also included in this study. For
phase, but the final polymer subunit composition comparison, other strains of Pseudomonas spe-
is determined during stationary phase when cell cies were also included in the present study,
growth is arrested by nutrient limitation (e.g., including P. aeruginosa PA7 and PAO1, P. fluore-
nitrogen) in the presence of excess carbon. Cells scens Pf0-1, P. stutzeri A1501, P. syringae pv.
under carbon excess and nutrient limitation con- tomato DC3000, P. entomophila L48, P. men-
ditions produce PHA polymers employing PHA docina NK-01 and ymp, P. monteilii SB3101 and
synthase (PhaC). Under carbon-limited (starva- SB3078, P. putida H3267, Pseudomonas sp.
tion) conditions, however, PHA depolymerase VL120, and Pseudomonas FG1182. In all, 38
(PhaZ) degrades the PHA polymers to release Pseudomonas strains were included. All of the
R-hydroxyalkanoic acids, which are then used as gene sequences mentioned above were obtained
a carbon and energy source for cell growth (de from the Integrated Microbial Genomes (IMG)
Eugenio et al. 2010). database (www.img.jgi.doe.gov). The genome
accession numbers and locus tags for all the
genes from all the Pseudomonas species included
4.4 Phylogenetic Affiliation in this study are listed in (Table 4.1).
of Pseudomonas sp. MO2 Phylogenetic analysis based on partial 16S
rDNA sequences (including sequences from Mulet
Nucleotide sequences were aligned using Bioedit et al. 2013) revealed two major clades (Fig. 4.1).
(Thompson et al. 1994) and the ends of the One clade contained P. mendocina, P. aeruginosa,
sequences were trimmed to equal lengths. and P. stutzeri, while the second clade contained
Phylogenetic analyses were conducted using several subgroups consisting of mostly P. putida
MEGA6 (Tamura et al. 2013). A series of indi- strains (Fig. 4.1), a mixture of P. monteilii strains,
vidual neighbor-joining trees based on 16S P. putida strains, and other Pseudomonas species
rDNA, cpn60, dnaJ, gyrA, gyrB, rpoA, and rpoD (group 2b), a distinct subgroup of P. putida con-
were generated. Five genes (cpn60, dnaJ, gyrA, sisting of strains W619 and NBRC 14612 (group
rpoA, and rpoD) were concatenated using DnaSP 3), and a mixture of other Pseudomonas species
software (Librado and Rozas 2009; Rozas 2009) that includes P. putida UW4, P. fluorescens, and P.
and phylogenetic trees were constructed using syringae (group 4). Pseudomonas sp. MO2 formed
the Jukes and Cantor method (Jukes and Cantor a separate subgroup (Fig. 4.1) with P. monteilii
1969; Saitou and Nei 1987). Bootstrap values SB3101 and SB3078, P. putida S16, and
greater than 50 % (from 1000) are indicated at Pseudomonas sp. VLB120. On the basis of 16S
4 Phylogenetic Affiliation of Pseudomonas sp. MO2, a Novel 65

a P.putida KT2440
P.putida LS46 b
P.putida GB-1
P.putida BIRD-1
P.putida HB3267
50 P.putida H8234
P.putida JCM 6156
P.putida SJTE-1
P.putida ND6
P.putida F1
55 P.putida PC9
P.putida TRO1
56 P.putida NB2011
P.monteilii strain SB3067
Pseudomonas sp. FGI182
P.monteilii CIP 104883
P.entomophila L48
56 P.putida MO2
P.putida S16
60 Pseudomonas sp. VLB120
P.monteilii SB 3095
P.monteilii SB3078
63 P monteilii SB3101
P.putida CSV86
P.putida W619
P.putida NBRC 14164
99 P.putida W619
P.fluorescens PfO-1
P.putida UW4
93 P.fluorescens Pf-5
79 P.syringae pv. tomato DC3000
70 P.syringae pv. tabaci ATCC 11528
P.putida MTCC 5279
69 P.mendocina ymp
P.mendocina NK-01
100 P.aeruginosa PAO1
83 P.aeruginosa PA7
79 P.stutzeri A1501
99 P.stutzeri ATCC 17588


Fig. 4.1 Molecular phylogenetic analysis of rDNA. The tree is drawn to scale, with branch lengths
Pseudomonas species by maximum likelihood method measured in the number of substitutions per site
based on Juke and Cantor model. (a) cpn60 and (b) 16S

rDNA phylogeny, it was evident that Pseudomonas of the cpn60 gene (the universal target or UT) can
sp. MO2 was more closely related to P. monteilii be used for the identification and classification of
than the P. putida type strains ATCC 12633 and P. bacteria. We used the cpn60 UT sequence to con-
putida JCM6156, from which the P. putida strain duct phylogenetic analyses for 38 Pseudomonas
KT2440 was derived. On the basis of 16S rRNA, 9 strains. Phylogenetic tree based on cpn60 gene
strains (P. putida S16, Pseudomonas MO2, P. sequence analysis separated the P. putida type
monteilii SB3101, P. monteilii 3078, P. monteilii strain KT2440 from P. fluorescens, P. aerugi-
SB3095, P. entomophila L48, P. putida CFBP5934, nosa, P. syringae, P. resinovorans, P. stutzeri, P.
and P. mosselii CFML90-83) formed a separate mendocina, and P. entomophila and confirmed
subgroup. These data suggest that Pseudomonas previous phylogenetic analyses based on 16S
MO2 is closer to P. monteilii than to P. putida. To rRNA gene sequences. The cpn60 phylogenetic
further resolve this relationship, phylogenic analy- analysis further separated P. putida strains into
ses were conducted with cpn60 sequences and five subgroups. Ten P. putida strains (B6-2, DOT-
concatenated sequences. T1E, TRO1, LS46, F1, SJTE-1, ND6, S12,
BIRD-1, and Idaho) clustered with the P. putida
type strain KT2440. Our recently isolated P.
4.4.1 Phylogenetic Analysis Based putida strain LS46 also clustered with P. putida
on cpn60 Genes KT2440. Four P. putida strains, NB2011, GB-1,
NBRC14164, and H8234, formed another sub-
Chaperonin 60 (encoded by the cpn60 gene) is a group, and six Pseudomonas strains, P. monteilii
highly conserved protein found in bacteria, and SB3101, SB3078, P. putida S16, P. putida PC2,
the analysis of this gene from wide variety of Pseudomonas sp. FGI182, and Pseudomonas
bacteria has indicated that a 549567 bp region MO2, formed a separate subgroup. Four P. putida
66 P.K. Sharma et al.

MTCC5279, P. putida CSV86, Pseudomonas 4.4.3 Phylogenetic Analysis Based

spp. VLB120, and P. entomophila L48 were clus- on phaC1 and phaC2 Genes
tered in one subgroup, while P. putida W619 did
not cluster with any of P. putida strains. The There are six genes in the polyhydroxyalkanoate
cpn60 gene of P. putida type strain KT2440 (PHA) synthesis operon: phaC1, phaZ, phaC2,
showed 99.299.6 % similarity indices relative to phaD, phaF, and phaI. The phaC1 and phaC2
ten other P. putida strains (B6-2, DOT-T1E, encode distinct PHA synthases (their gene
TRO1, LS46, F1, SJTE-1, ND6, S12, BIRD-1, sequences are divergent with only 6070 %
and Idaho), but this group had only 96.496.6 % nucleotide sequence identity). phaZ encodes the
gene similarity indices relative to P. monteilii PHA depolymerase, and the other three genes
group (SB3101, SB3078, S16, PC2, FGI182, and (phaD, phaF, and phaI) are regulatory genes
MO2). The cpn60 gene of both P. putida KT2440 (Sharma et al. 2014). All the six pha genes were
group and P. monteilii SB3101 group had low present in an operon and their organization is
(91.695.7 %) similarity indices relative to other identical to other P. putida strains (Sharma et al.
P. putida strains (W619, GB-1, NBRC14164, 2013, 2014). Phylogenetic analyses based on the
H8234, MTCC5279, CSV86, and UW4). The nucleotide sequences of phaC1 and phaC2
similarity index of cpn60 genes among 38 revealed that phaC1 and phaC2 form distinct
Pseudomonas strains supports the idea of reas- clusters (Fig. 4.3). Analyses of the phaC1 and
signing some species designated as P. putida phaC2 of Pseudomonas sp. MO2 placed these
strains into different Pseudomonas species (Fig. strains in phaC1 and phaC2 clusters with P. mon-
4.1 and Table 4.3). teilii SB3101, P. monteilii SB3078, P. putida
PC9, P. putida H3267, and P. putida S16, while
the genes from P. putida KT2440, P. putida F1, P.
4.4.2 Phylogeny Based on dnaJ putida LS46, and P. putida BIRD1 resulted in
and gyrA separate phaC1 and phaC2 clusters. Moreover,
the phylogenetic trees generated with the phaC1
Other constitutively expressed (housekeeping) and phaC2 genes contained associations that
genes, such as dnaJ, the gene encoding the were similar to those formed using cpn60 gene
molecular chaperone DnaJ, and gyrA, the gene sequences.
encoding DNA gyrase subunit A, have also been
used as phylogenetic markers. The ubiquitous
distribution of dnaJ and gyrA among bacteria and 4.4.4 Phylogenetic Analysis
their high degree of amino acid sequence conser- of Pseudomonas MO2 Based
vation are key features that make these genes on Concatenated Genes
suitable for inferring phylogenetic relationships.
dnaJ has been used to differentiate species and Phylogenetic analyses based on concatenation of
subspecies, even within genera where 16S rRNA five genes, cpn60, dnaJ, gyrA, rpoA, and rpoD,
gene sequences have low resolution, as in patho- indicated that Pseudomonas MO2 was closely
genic bacteria such as Mycobacterium (Morita associated with P. monteilii SB3101, P. monteilii
et al. 2004), Streptococcus (Itoh et al. 2006), and SB3078, P. putida S16, and P. putida PC9 (Fig.
more recently Staphylococcus (Shah et al. 2007). 4.4). This phylogenetic analysis confirmed the
Phylogenetic analyses of pseudomonads based relationships determined with 16S RNA, cpn60,
on dnaJ and gyrA genes confirmed our observa- or the phaC genes. The sequence similarity index
tions, based on 16S rRNA gene sequences and of ten P. putida strains (F1, BIRD-1, DOT-T1E,
cpn60 genes (Fig. 4.2), that Pseudomonas sp. TRO1, Idaho, LS46, ND6, SJTE-1, S12, B6-2)
MO2 always grouped with P. monteilii SB3038 with the type strain, P. putida KT2440, was 0.99.
and SB3101. However, P. monteilii group strains (P. monteilii
4 Phylogenetic Affiliation of Pseudomonas sp. MO2, a Novel 67

Table 4.3 Genome similarity indices of different Pseudomonas species based on cpn60 gene sequences
Strains KT2440 BIRD-1 LS46 MO2 H3267 3101 3078 S16
P. putida S12 0.992 0.993 0.992 0.962 0.962 0.964 0.964 0.964
P. putida Idaho 0.996 0.992 0.992 0.963 0.964 0.966 0.966 0.966
P. putida B6-2 0.991 0.991 0.997 0.961 0.962 0.964 0.964 0.964
P. putida SJTE-1 0.992 0.991 0.996 0.961 0.962 0.964 0.964 0.964
P. putida BIRD-1 0.992 1.000 0.992 0.962 0.962 0.964 0.964 0.964
P. putida TRO1 0.992 0.992 1.000 0.962 0.963 0.965 0.965 0.965
P. putida ND6 0.992 0.991 0.996 0.961 0.962 0.964 0.964 0.964
P. putida KT2440 1.000 0.992 0.992 0.965 0.966 0.968 0.968 0.968
P. putida F1 0.994 0.992 0.997 0.962 0.963 0.965 0.965 0.965
P. putida LS46 0.992 0.992 1.000 0.962 0.963 0.965 0.965 0.965
P. putida DOT-T1E 0.992 0.991 0.997 0.962 0.962 0.964 0.964 0.964
P. putida MO2 0.965 0.962 0.962 1.000 0.999 0.993 0.993 0.993
P. putida H3267 0.966 0.962 0.963 0.999 1.000 0.994 0.994 0.994
P. putida S16 0.968 0.964 0.965 0.999 0.994 1.000 1.000 1.000
P. monteilii SB3101 0.968 0.964 0.965 0.993 0.994 1.000 1.000 1.000
Pseudomonas sp. 0.971 0.968 0.968 0.978 0.979 0.982 0.982 0.982
P. putida B001 0.973 0.969 0.971 0.962 0.962 0.964 0.964 0.964
P. putida GB-1 0.957 0.957 0.957 0.95 0.951 0.953 0.953 0.953
P. putida NBRC 0.953 0.949 0.952 0.949 0.95 0.951 0.951 0.951
P. putida H8234 0.951 0.946 0.948 0.947 0.947 0.949 0.949 0.949
Pseudomonas sp. 0.942 0.939 0.94 0.947 0.947 0.949 0.949 0.949
P. putida W619 0.947 0.946 0.946 0.944 0.944 0.947 0.947 0.947
P. putida NB2011 0.947 0.942 0.944 0.949 0.95 0.951 0.951 0.951
P. entomophila L48 0.945 0.943 0.943 0.953 0.953 0.956 0.956 0.956
P. putida MTCC 0.937 0.936 0.936 0.94 0.94 0.944 0.944 0.944
P. putida CSV86 0.92 0.916 0.916 0.92 0.92 0.923 0.923 0.923
P. mendocina 0.892 0.894 0.891 0.887 0.888 0.891 0.891 0.891
P. stutzeri A1501 0.875 0.875 0.872 0.878 0.879 0.881 0.881 0.881
P. mendocina ymp 0.88 0.88 0.879 0.877 0.877 0.881 0.881 0.881
P. aeruginosa PA7 0.867 0.869 0.866 0.87 0.871 0.873 0.873 0.873
P. aeruginosa PAO1 0.869 0.869 0.866 0.87 0.871 0.875 0.875 0.875
P. fluorescens Pf0-1 0.861 0.858 0.861 0.863 0.864 0.864 0.864 0.864
P. fulva 12-X 0.854 0.856 0.855 0.855 0.856 0.858 0.858 0.858
P. resinovorans 0.877 0.878 0.875 0.877 0.877 0.881 0.881 0.881
P. syringae pv. 0.846 0.844 0.846 0.851 0.852 0.85 0.85 0.85
P. putida UW4 0.858 0.856 0.859 0.86 0.86 0.862 0.862 0.862

SB3101, P. monteilii SB3078, P. putida S16, P. sequence similarity indices with the type strain,
putida 3267, and Pseudomonas sp. MO2) had a P. putida KT2440 (Table 4.3). The association of
low sequence similarity index (0.95). A number Pseudomonas MO2 with the P. monteilii strains
of other P. putida strains also showed low was also reinforced by the very high sequence
68 P.K. Sharma et al.

a 70 P.putida TRO1 b 99
100 P.putida SJTE-1
P.putida ND6
P.putida DOT-T1E
49 P.putida F1
P.putida F1
P.putida B6-2 39 P.putida Idaho
99 P.putida LS46 P.putida DOT-T1E
P.putida B6-2
98 P.putida SJTE-1 79
100 98 P.putida TRO1
P.putida ND6
P.putida LS46
P.putida KT2440 100
80 P.putida KT2440
88 96
P.putida Idaho
45 100 P.putida S12
99 P.putida S12
94 P.putida BIRD-1 P.putida BIRD-1
P.putida GB-1 P.putida GB-1
90 100 P.putida NBRC 14164
P.putida B001
100 P.putida H8234
99 P.putida NBRC 14164
100 P.putida H8234 P.putida B001
Pseudomonas sp.FGI182 95 Pseudomonas sp.FGI182

P.monteilii SB3101 100 100 P.monteilii SB3101
88 P.monteilii SB3078
P.monteilii SB3078 52
52 P.putida S16
67 100 P.putida S16
P.putida MO2
P.putida PC9 62
85 33 100 P.putida PC9
P.putida MO2
P.putida HB3267 P.putida HB3267
99 75
Pseudomonas sp.VLB120 Pseudomonas sp.VLB120
71 P.putida W619 100 P.putida W619
P.putida NB2011 P.putida NB2011
P.putida MTCC 5279 P.putida MTCC 5279
P.putida CSV86 P.putida UW4
P.putida UW4 P.putida CSV86

0.02 0.02

Fig. 4.2 Molecular phylogenetic analysis of The tree is drawn to scale, with branch lengths measured
Pseudomonas species by maximum likelihood method in the number of substitutions per site
based on Juke and Cantor model. (a) dnaJ and (b) gyrA.

similarity indices (0.991.00) among these also in agreement with the phylogeny based on
species. cpn60 gene sequence analysis. Thus, phyloge-
The analysis of 38 sequences of six genes netic analyses using both cpn60 and five house-
(cpn60, dnaJ, gyrA, gyrB, rpoA, and rpoD) keeping genes indicated that our Pseudomonas
revealed wide variations in all these sequences. sp. MO2 isolate clustered with P. monteilii
The cpn60 (555), dnaJ (788), gyrA (2408), gyrB SB3101 rather than P. putida KT2440.
(1668), rpoA (1002), and rpoD (1220) genes con- Pseudomonas taxonomy has been revised
tained 421, 331, 1006, 700, 262, and 457 poly- from time to time using methods like DNA-rRNA
morphic sites, respectively, and the nucleotide reassociation data, as well as 16S rDNA sequence
sequence identities ranged from 26.2 % to 75.8 % analysis (De Vos and De Ley 1983). Some
(Table 4.4). The average nucleotide differences Pseudomonas species were earlier transferred to
calculated by Juke-Cantor (JC) were in the range new genera in the -, -, or -Proteobacteria.
of 54.1287.8 in the six genes. The average pair- Even within the genus Pseudomonas, new spe-
wise distance in six genes ranged from 0.0 to cies have been proposed based on inferred phylo-
0.142. It was highest in dnaJ gene sequences and genetic relationships using 16S rDNA. However,
was least in rpoA gene sequences. The nucleotide it has been observed that in the genus
diversity per site was highest in the gyrB gene, Pseudomonas, 16S rRNA-based phylogenies
while minimum nucleotide diversity was detected lack resolution at the intrageneric level due the
in the rpoA gene. Likewise, a number of synony- slow rate of evolution of 16S rDNA sequences
mous and non-synonymous sites and their diver- (Moore et al. 1996; Anzai et al. 2000).
sity rates per site varied greatly in among the six Ambiguities arising from the use of 16S rRNA-
genes. The phylogeny of 38 Pseudomonas strains based phylogenetic analyses for Pseudomonas
based on individual housekeeping genes like species have been overcome by using multilocus
rpoA, rpoD, gyrA, gyrB, and dnaJ was congruent sequence analysis (MLSA) (Yamamoto and
with other gene phylogeny trees. The results were Harayama 1998; Yamamoto et al. 2000). Mulet
100 P.putida S12
P.putida BIRD-1
P.putida B6-2
P.putida SJTE-1
P.putida ND6
100 P.putida F1
58 P.putida TRO1
P.putida DOT-T1E
100 P.putida LS46
91 P.putida Idaho
P.putida KT2440
P.putida GB-1
99 P.putida PC9
99 P.putida HB3267
100 P.putida MO2
98 50 Pseudomonas sp.FGI182
P.putida S16
99 P.monteilii SB3101
P.monteilii SB3078
70 P.putida B001
99 P.putida NBRC 14164 phaC1
P.putida H8234
P.putida W619
P.putida NB2011
P.putida MTCC 5279
83 Pseudomonas sp.VLB120
P.entomophila L48
P.putida CSV86
99 P.syringae pv.tomato DC3000
58 99 P.putida UW4
P.fluorescens Pf0-1
49 P.putida UW4
P.fulva 12-X
63 100 P.mendocina ymp
P.mendocina NK-01
90 P.resinovorans NBRC 106553
P.aeruginosa PA1
P.aeruginosa PA1
100 69 P.mendocina ymp
P.resinovorans NBRC 106553
P.fluorescens Pf0-1
P.putida CSV86
99 P.putida MTCC 5279
99 P.entomophila L48
98 P.putida NBRC 14164
100 P.putida H8234
P.putida B001
74 53 P.putida W619
P.putida NB2011
Pseudomonas sp. VLB120
P.monteilii SB3101
P.monteilii SB3078
100 P.putida S16
99 P.putida MO2
100 P.putida PC9 phaC2
100 P.putida HB3267
53 Pseudomonas sp.FGI182
P.putida GB-1
97 79 P.putida Idaho
P.putida KT2440
100 91 P.putida S12
P.putida BIRD-1
65 P.putida DOT-T1E
97 P.putida LS46
P.putida TRO1
P.putida B6-2
P.putida SJTE-1
P.putida ND6
P.putida F1


Fig. 4.3 Molecular phylogenetic analysis of phaC genes scale, with branch lengths measured in the number of sub-
of Pseudomonas species by maximum likelihood method stitutions per site
based on Juke and Cantor model. The tree is drawn to
70 P.K. Sharma et al.

100 P.putida ND6

100 P.putida SJTE-1
P.putida F1
100 P.putida DOT-T1E
100 P.putida B6-2
96 P.putida LS46
100 P.putida TRO1
P.putida KT2440
44 P.putida Idaho
56 P.putida BIRD-1
49 100 P.putida S12
P.putida GB-1
P.putida B001
Pseudomonas sp.FGI182
61 P.monteilii SB3101'
100 100 P.putida S16
P.monteilii SB3078
100 P.putida MO2
68 P.putida HB3267
100 P.putida PC9
P.putida H8234
79 100 P.putida NBRC 14164
P.putida NB2011
100 94 P.putida W619
Pseudomonas sp.VLB120
100 P.entomophila L48
63 P.putida MTCC 5279
P.putida CSV86
P.syringae pv.tomato DC3000
100 P.fluorescens Pf0-1
100 P.putida UW4
100 P.mendocina NK-01
53 P.mendocina ymp
P.fulva 12-X
100 P.stutzeri A1501
P.resinovorans NBRC 106553
83 P.aeuginosa PA7
100 P.aeruginosa PAO1


Fig. 4.4 Phylogenetic tree depicting the relationships and dnaJ), which were aligned by ClustalW and a
among Pseudomonas species. The tree is based on concat- neighbor-joining tree was generated using MEGA5 pro-
enated sequences of five genes (cpn60, rpoA, rpoD, gyrA, gram. Bootstrap values are indicated at the nodes

et al. (2010) on the basis of the comparison of (2005) and Richter and Rossell-Mra (2009),
whole genome and phylogenetic similarity of based on 9495 % ANIb similarity correspond-
Pseudomonas strains opined that DNA-DNA ing to 97 % similarity in MLSA, placed
hybridization similarity of 70 % is cut off for spe- Pseudomonas strains in separate species. They
cies cutoffs, which was corresponding to 80 % used a cutoff limit of 93 % (corresponding to
genome similarity by blast analysis and to phylo- 97 % similarity in MLSA) and this value sepa-
genetic distance of a 97 % in multigene analysis. rated P. putida from P. aeruginosa. Based on the
Other studies by Konstantinidis and Tiedje cpn60 gene similarity index, 13 P. putida strains,
Table 4.4 Analysis of Pseudomonas spp. sequences from cpn60, dnaJ, grA, gyrB, rpoA, and rpoD genes
Sequence information 16S rRNA cpn60 dnaJ gyrA gyrB rpoA rpoD
Number of sequences 38 38 38 38 38 38 38
Number of sites 794 555 788 2408 1668 1002 1220
Number of polymorphic sites 668 (84.1) 421 (75.8) 331 (42.0) 1006 (41.8) 700 (41.9) 262 (26.2) 457 (37.4)
Average number of nucleotide differences 96.82 5.83 54.1 4.37 96.32 5.81 287.8 10.5 204.1 8.46 63.05 4.70 124.58 6.61
4 Phylogenetic Affiliation of Pseudomonas sp. MO2, a Novel

Nucleotide diversity (per site) 0.487 0.007 0.097 0.009 0.136 0.005 0.133 0.003 0.136 0.037 0.067 0.003 0.112 0.004
Synonymous sites PI (JC) NA 144.36 (0.290) 203.54 (0.538) 576.20 (0.544) 401.91 (0.540) 251.88 (0.300) 326.9 (0.0499)
Non-synonymous sites PI (JC) NA 410.64 (0.122) 582.46 (0.039) 1829.8 (0.044) 1266.09 (0.047) 747.12 (0.010) 891.1 (0.137)
72 P.K. Sharma et al.

including P. putida KT2440, had an SI of 99 %. DNA-DNA hybridization, the isolate was reas-
P. monteilii SB3101 and P. monteilii SB3078 signed as a new species, P. monteilii, with type
cpn60 genes, however, had an SI of < 97 % with strain designation CFML90-60 (= CIP104883).
P. putida KT2440. Therefore, the P. monteilii Bogaerts et al. (2011) isolated additional P.
strains may be considered as species that are dis- monteilii strains from clinical specimens. The
tinct from P. putida KT2440. Our Pseudomonas clinical significance of P. monteilii is not
sp. MO2 showed > 99 % sequence identity with known, but P. monteilii is assumed to be a rare
the cpn60 sequences of P. monteilii SB3101 and opportunistic pathogen or colonizer. Recently
P. monteilii SB3078, but only 96 % sequence another strain of P. monteilii, designated QM,
identity with P. putida KT2440 cpn60 (Table was identified and shown to be capable of syn-
4.3). The comparison of similarity indices based thesizing indigoids from indoles (Qu et al.
on multilocus sequence analysis of five concate- 2012; Ma et al.2012). Another P. monteilii
nated genes also confirmed the phylogenetic strain has been identified as rhizosphere colo-
association of Pseudomonas sp. MO2 with the P. nizer (Meyer et al. 2008). P. monteilii strains
monteilii MO2 subgroup (Fig. 4.5). Thus, the have also been associated with degradation of
Pseudomonas MO2 strain should be designated aromatic and heterocyclic compounds and dye
as P. monteilii MO2. compounds (Masuda et al.2007; Lie et al. 2009;
Pseudomonas monteilii has been identified Fulekar et al. 2013). Recently, the genomes of
as versatile microorganism associated with P. monteilii SB3078 and P. monteilii SB3101,
clinical specimens, bioremediation agent, and which were identified as benzene-, toluene-,
industrial production of indigo dye (Ma and ethylbenzene-degrading bacteria, were
et al.2012). The species now designated P. mon- sequenced (Dueholm et al. 2014). P. monteilii
teilii was first isolated by Elomari et al. (1997). strains are known to express endogenous extra-
On the basis of a numerical analysis of the phe- cellular lipases, which have been purified and
notypic characteristics of 39 strains, the characterized (Wang et al. 2009). For example,
Elomari et al. (1997) isolate was considered to an extracellular lipase was isolated and purified
be related to P. putida. However, on the basis of from P. monteilii TKU009 (Wang et al. 2009).

Fig. 4.5 Similarity indices of different Pseudomonas species based on concatenated sequence of the cpn60, dnaJ,
rpoA, rpoD, and gyrB genes
4 Phylogenetic Affiliation of Pseudomonas sp. MO2, a Novel 73

These reports suggest that P. monteilii strains Barrett EL, Solanes RE, Tang JS, Palleroni NJ (1986)
Pseudomonas fluorescens biovar V: its resolution into
are versatile bacteria with multiple potential
distinct component groups and the relationship of
applications. these groups to other P. fluorescens biovars, to P.
putida, and to psychrotrophic pseudomonads associ-
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4.5 Conclusion Bhushan A, Joshi J, Shankar P, Kushwah J, Raju SC,
Purohit HJ, Kalia VC (2013) Development of genomic
A novel Pseudomonas sp. (strain MO2) was tools for the identification of certain Pseudomonas up
identified as medium chain length polyhydroxy- to species level. Indian J Microbiol 53:253263.
alkanoate producer from waste canola fryer oil.
Bogaerts P, Bouchahrouf W, Lissoir B, Denis O,
Based on phylogenetic analyses using 16S rRNA, Glupczynski Y (2011) IMP-13 producing
cpn60, six individual housekeeping genes, the Pseudomonas monteilii recovered in a hospital envi-
PHA synthase genes phaC1 and phaC2, five con- ronment. J Antimicrob Chemother 66:24342435.
catenated gene sequences, and the sequence simi-
Bossis E, Lemanceau P, Latour X, Gardan L (2000) The
larity index using the cpn60 gene, we concluded taxonomy of Pseudomonas fluorescens and
that Pseudomonas sp. strain MO2 is different Pseudomonas putida: current status and need for revi-
from P. putida KT2440 and most other P. putida sion. Agronomie 20:5163. doi:10.1051/agro:2000112
Braunegg G, Sonnleitner B, Lafferty RM (1978) A rapid
strains and therefore designated the isolate as P.
gas chromatographic method for the determination of
monteilii MO2. Pseudomonas monteilii MO2, poly--hydroxybutyric acid in microbial biomass. Eur
unlike other P. monteilii strains isolated in 1997, J Appl Microbiol Biotechnol 6:2937. doi:10.1007/
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this, P. monteilii MO2 can be developed as indus- Chan PL, Yu V, Wai L, Yu HF (2006) Production of
trial strain for mcl-PHA production or for con- medium-chain-length polyhydroxyalkanoates by
Pseudomonas aeruginosa with fatty acids and alterna-
struction of recombinant strains to produce
tive carbon sources. Appl Biochem Biotechnol
mcl-PHAs from oils. 132:933941. doi:10.1385/ABAB:132:1:933
Chen J, Liu T, Zheng Z, Chen J, Chen G (2004)
Acknowledgments This work was supported by funds Polyhydroxyalkanoate synthases PhaC1 and PhaC2
provided by the Natural Sciences and Engineering from Pseudomonas stutzeri 1317 had different sub-
Research Council of Canada (NSERC), through a strate specificities. FEMS Microbiol Lett 234:231
Strategic Programs grant (STPGP 306944-04), by 237. doi:10.1016/j.femsle.2004.03.029
Genome Canada, through the Applied Genomics Research Chen YJ, Huang YC, Lee CY (2014) Production and
in Bioproducts or Crops (ABC) program for the grant characterization of medium-chain-length polyhy-
titled Microbial Genomics for Biofuels and CoProducts droxyalkanoates by Pseudomonas mosselii TO7.
from Biorefining Processes, and by the Province of J Biosci Bioeng 118:145152. doi:10.1016/j.
Manitoba, through the Manitoba Research Innovation jbiosc.2014.01.012
Fund (MRIF). Chung AL, Jin HL, Huang LJ, Ye HM, Chen JC, Wu Q,
Chen GQ (2011) Biosynthesis and characterization of
poly (3-hydroxydodecanoate) by b-oxidation inhibited
mutant of Pseudomonas entomophila L48.
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4 Phylogenetic Affiliation of Pseudomonas sp. MO2, a Novel 77

Wang SN, Liu Z, Tang HZ, Meng J, Xu P (2007) Xiang Zhang received the
Characterization of environmentally friendly nicotine B.Eng. degree in Chemical
degradation by Pseudomonas putida biotype A strain Engineering from Tsinghua
S16. Microbiology 153:15561565. doi:10.1099/ University, Beijing, China,
mic.0.2006/005223-0 and the B.Sc. degree in
Wang SL, Lin YT, Liang TW, Chio SH, Ming LJ, Wu PC Bioinformatics from Simon
(2009) Purification and characterization of extracellu- Fraser University, Burnaby,
lar lipases from Pseudomonas monteilii TKU009 by BC Canada. He is currently
the use of soybeans as the substrate. J Ind Microbiol working as a Bioinformatics Analyst at University of
Biotechnol 36:6573. doi:10.1007/ Manitoba. His current interests are data mining, systems
s10295-008-0473-z biology, statistical modeling, genomics, and clinical trial.
Yamamoto S, Harayama S (1998) Phylogenetic relation-
ships of Pseudomonas putida strains deduced from the Richard Sparling
nucleotide sequences of gyrB, rpoD and 16 rRNA received his B.Sc. degree
genes. Int J Syst Bacteriol 48:813819. (Agr.) from McGill
doi:10.1099/00207713-48-3-813 University (Canada) and
Yamamoto S, Kasai H, Arnold DL, Jackson RW, Vivian Ph.D. degree in
A, Harayama S (2000) Phylogeny of the genus Microbiology from
Pseudomonas: intrageneric structure reconstructed University of Iowa (USA).
from the nucleotide sequences of gyrB and rpoD He is presently a Professor
genes. Microbiology 146:23852394. doi:10.1099/ in the Microbiology
mic.0.27096-0 Department at the University
of Manitoba. His focus is to
understand the metabolism
Parveen Kumar and physiology of industri-
Sharma received his ally relevant microorgan-
M.Sc. and Ph.D. degree in isms using biochemical and genomic tools, with emphasis
Microbiology from Punjab on bacteria producing bioplastics and on biofuel production
Agricultural University, from lignocellulosic materials using consolidated biopro-
Ludhiana, India. Formerly cessing strategies.
a Professor of Microbiology
at CCS Haryana David B. Levin received
Agricultural University, his B.Es. degree from
Hisar, India, he is now University of Waterloo,
working as a Research M.Sc. degree from
Scientist at University of Manitoba, Winnipeg, Canada. University of Guelph, and
His current interests are polyhydroxyalkanoate produc- Ph.D. degree from McGill
tion using industrial and agricultural by-products and con- University. He is a Professor
struction of recombinant bacteria for synthesis of novel in Department of
PHA polymers. Biosystems Engineering at the University of Manitoba.
His research focus is to understand the relationships
Jilagamazhi FU, B.Sc., between genome content, gene and gene product expres-
Inner Mongolia Agricultural sion, metabolic pathway utilization, and end-product syn-
University, Hohhot, Inner thesis to increase the efficiencies of product synthesis by
Mongolia, China, is a Ph.D. microorganisms.
candidate at University of
Manitoba, Winnipeg. He is
presently in the last year in
fulfillment of the Ph.D.
degree at Department of
Biosystems Engineering. His
current research area is syn-
thesis of medium chain length polyhydroxyalkanoates by
Pseudomonas putida.
Synthetic Biology Strategies
for Polyhydroxyalkanoate 5

Gunjan Arora, Andaleeb Sajid, Parijat Kundu,

and Mritunjay Saxena

Synthetic biologists are trying to apply engineering principles in biology
to create an artificial world with unlimited possibilities. The guiding prin-
ciple is to look beyond finding the solutions and to create new ones.
Synthetic biologists are now routinely designing synthetic genetic circuits
in bacteria and yeast. The major aim is to use microbes as cell factories for
the production of bioactive compounds with a particular focus on drugs,
biofuels, and biopolymers. In this chapter, we emphasize on understand-
ing the synthetic biology approaches and their role in creating polyhy-
droxyalkanoate (PHA)-producing microbial factories.

5.1 Introduction recent progress in systems biology and rise of

omics era intensified the application of engi-
Why it is so difficult to apply engineering prin- neering principles in biology to manipulate and
ciples in biology? There are two likely explana- concoct the cellular behavior (Cameron et al.
tions to this question. First, the complexity of 2014). The field of biology dedicated to such pur-
biological systems is still not fully understood suits is prophesied as synthetic biology.
and the second is scientific community never
tried to simplify it. The amalgamation of scien-
tific disciplines in biology always fancied scien- 5.2 Definition of Synthetic
tists. Historically, it led to creation of new Biology
branches like biochemistry, biophysics, recombi-
nant DNA technology, fermentation technology, The broad literal definition of synthetic biology
microbial technology, and systems biology. The states that it is an interdisciplinary branch of sci-
ence that combines mathematical principles to
G. Arora (*) A. Sajid P. Kundu M. Saxena
diverse biological disciplines such as molecular
CSIR-Institute of Genomics and Integrative Biology and cellular biology, systems biology and bio-
(IGIB), Delhi University Campus, Mall Road, chemistry (Gardner and Hawkins 2013; Cole
110007 Delhi, India 2014; Lienert et al. 2014). Synthetic biology
e-mail: arorag1983@gmail.com;
sajid.andaleeb@gmail.com; parijat@igib.in;
merges biology and engineering to design and
mritunjaysaxena@gmail.com construct novel biological functions and proce-

Springer India 2015 79

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_5
80 G. Arora et al.

dures that help in delivering better technologies by exerting regulatory, actuator, and/or signaling
and products (Gardner and Hawkins 2013; Eils function(s) (Brophy and Voigt 2014). This will
et al. 2015). This includes either creating new need an improvised genetic toolbox with fine pre-
biological tools or artificial construction of exist- cision. Further, there is need to pursue diverse
ing products for novel purposes. The early syn- biological applications to transform this new
thetic biology manipulations were very much field into an independent engineering discipline.
related to genetic engineering. The significant The focus would be to make engineering of biol-
difference lies in the fact that genetic engineering ogy quicker, reproducible, safer, and easy in
has its roots in molecular biology, genetics, and practice.
biochemistry, whereas synthetic biology has
foundations in systems biology and mathematics
(Hu and Dhar 2015) (Fig. 5.1). The growth in 5.3 History of Synthetic Biology
synthetic biology can be attributed more to
advancement in DNA sequencing and The classical study by Jacques and Monad
metabolomics. incepted the very first idea in synthetic biology
Integration of different modules and standard- (Monod and Jacob 1961; Cameron et al. 2014).
ization of the genetic circuits together at different The discovery of regulatory circuits like lac
levels is one big challenge in synthetic biology. operon in Escherichia coli indicated the cellular
Genetic circuit is composed of biological compo- adaptability and genetic switches. However, it
nents that permit the flow of biological message took almost four decades for biologists to under-
stand the molecular details of these switches and
how these switches can be modulated to generate
specific responses spatiotemporally.
Estimated market : 38.7 billion by 2020 The word synthetic biology was coined in the
distant past but was first described by Professor
Waclaw Szybalski in 1974 (Benner et al. 2011).
He suggested that use of advance molecular biol-
ogy tools in manipulation of genetic systems will
herald a new era of synthetic biology in the
Synthetic future. By the end of last century, advances in
automated DNA sequencing, mass spectrometry,
biology and computational biology enabled high-
throughput analysis at the genome scale. These
analyses reveal the molecular interaction net-
works in a cell involving different biomolecules:
proteins, DNA, RNA, and metabolites. To eluci-

date these molecular networks, systems biology


approaches were applied that revealed hierarchi-



cal functional modules, analogous to engineered


modules (Andrianantoandro et al. 2006). This led

to the renaissance of synthetic biology in the
beginning of this century when the first major
breakthrough was published (Elowitz and Leibler
2000; Gardner et al. 2000; Cameron et al. 2014).
In its journey of 15 years, synthetic biologists
have tried to achieve ambitious goals such as
Fig. 5.1 Pillars of synthetic biology designing novel therapeutic agents, large-scale
production of low-cost drugs, and production of
5 Synthetic Biology Strategies for Polyhydroxyalkanoate Synthesis 81

biofuels or even claimed creating artificial life the cell. Accurate operational management with
(Cameron et al. 2014; Church et al. 2014). The engineering principles is needed to regulate
advancement in genomics and metabolomics has native and heterogeneous reactions inside the
tried to remove bottlenecks and to contribute cell. These measures will modify cellular metab-
toward the success of such pursuits. The particu- olism to restore the product synthesis at amena-
lar focus on microorganisms, especially bacteria ble level. In addition to in vivo synthetic biology
and yeast, to deliver engineered technologies and approaches, the field is also trying to come up
products has helped in this immense growth with cell-free synthetic biology solutions (Smith
(Kalia et al. 2003, 2007; Anderson et al. 2006; Lu et al. 2014).
and Collins 2007; Atsumi and Liao 2008; In a cell, various genetic switches control the
Keasling 2008; Holtz and Keasling 2010; Paddon levels of diverse metabolites, and the first step is
et al. 2013; Cameron et al. 2014). The field has to understand these switches. In biology, we do
provided some vital breakthroughs in such a not have arbitrary standard units to express the
short span that one expects synthetic biology strength of biological signals, and thus, it is very
principles will be extended to answer many chal- difficult to apply engineering principles at the cel-
lenging problems. lular level (Kelwick et al. 2014). Many metabo-
lites, when accumulated, tend to be secreted
outside the cell (Valle et al. 2008; Wintermute and
5.4 The Rationale of Synthetic Silver 2010). Mass production of such compounds
Biology requires an additional control on their secretion
(Meyer and Schmidhalter 2012). Production of
For centuries, synthesis of diverse natural prod- heterogenous compounds in large quantities cre-
ucts and their derivatives such as metabolites, ates burden for the biological system (Pickens
proteins, carbohydrates, and lipids in a test tube et al. 2011; Immethun et al. 2013). In response to
remained an ardent task. Enzymes that act in a this metabolic burden, the biological systems tend
concerted manner to produce desired outcome to respond by generating spontaneous mutations.
within a cell facilitate biological reactions. These mutations often switch off the metabolic
Production of the biological compounds requires pathways required for the production of com-
a variety of intermediate components involved in pounds like polyhydroxyalkanoates (PHAs)
the metabolic pathway. The traditional approach (Zhang et al. 1994; Glick 1995; Kocharin et al.
of organic synthesis of these intermediate com- 2012). Such cells impede product synthesis and
ponents is very often difficult due to poor yields, result in lower yields (Glick 1995). Therefore, it is
instability, and hazardous nature of chemicals necessary that we use such engineered cells with
(Yeh and Lim 2007; Carothers et al. 2009; extreme caution. Further, many valuable biologi-
Wallace and Balskus 2014). A simple synthetic cal components are protected by intellectual prop-
biology solution is to manipulate metabolic path- erty rights, and application of such components
ways and eliminate the need to purify or synthe- will require cost-benefit analysis.
size the intermediate chemicals. This will help in The basic difference between synthetic biol-
the production of complex natural products in a ogy and other biological subfields is the empha-
single-step reaction inside the cell. To achieve sis on the engineering component that can be
this, metabolic engineering and genetic switches explained in mathematical terms, is amenable to
are required that can respond to cellular demands. manipulations, and can be designed to serve
Additionally, when high concentrations of meta- larger integrated systems (Heinemann and Panke
bolic products become burden for the host cell, it 2006). Synthetic biologists are in the stage where
may lead to cell death (Andersen and Krummen early engineers would be, and they believe that in
2002; Rosano and Ceccarelli 2014). To overcome the near future, they will be as competent to
these limitations, it is necessary to keep the final design and accomplish large-scale biological
product concentration below the toxic levels in engineering projects as engineers in designing
82 G. Arora et al.

the integrated circuits (Heinemann and Panke nents of synthetic biology. Synthetic biologists
2006; Schwille 2011). Research and development also came up with the Registry of Standard
of new products in engineering fields is com- Biological Parts and BioBricks, collection
pletely dependent on mathematical modeling, repository of genetic parts that can be mixed and
which is done by computer-aided simulation pro- matched to build new devices and systems (Endy
grams (Zeng and Yao 2009; Chandrasegaran 2005; Arkin 2008). Based on similar engineering
et al. 2013). These simulations or computer-aided design principles, these repositories are arguably
designs help in faster and improved analysis that the best IT infrastructures for the field at present.
aid in product testing in silico. Further, computer- However, the computational setup needs further
aided simulations can even verify the perfor- work. The first task is to integrate available chas-
mance of logical gates in integrated circuit design sis and design into powerful and intuitively
(Marchisio and Stelling 2009). This also usable workflows. The computational aspects in
decreases the cost and time in the engineering synthetic biology need more comprehensive and
product development projects. However, as bio- incorporated methods that can help even the non-
logical circuits are nonlinear, it has been very dif- specialists who want to apply synthetic biology
ficult to model such pathways by computational in diverse applications. To achieve this, Synthetic
methods till now. Computational simulation pro- Biology Open Language (SBOL; http://www.
grams that can help in protocol design and per- sbolstandard.org) has been created to advance the
formance check will be needed to validate the standards to expedite the addition of new soft-
synthetic biology components in the future ware tools (Galdzicki et al. 2014).
(Chandran et al. 2009). One of the major consid-
erations will be stochastic nature of biological
components, which is different from reliable 5.5 Ethics and Synthetic Biology
nature of engineering components. The stochas-
tic behavior creates noise and ambiguity in the Scientists of this era believe that the twenty-first
engineered biological component systems century will be the century of biology (Ventor
(Slusarczyk et al. 2012; Chen and Wu 2013; Wu and Cohen 2004). In the first decade of this cen-
et al. 2013). Therefore, the computer-aided pro- tury, a prominent technology field synthetic
gram should be able to take such noise into biology was born. It aims to create artificial life
account and define dynamic signal-to-noise ratio or reengineer biological systems with innovative
to explain the performance parameters on a real- purposes to tackle numerous challenges that
time basis. The aim is to have computer-aided mankind is facing. However, due to sensational
design software that not only can suggest the reporting, many people believe that the work of
operational workflow but also can be integral synthetic biologists will be threatening for biodi-
component in designing the framework for syn- versity and speciation, therefore demeaning and
thetic biologists (Chandran et al. 2009; Wu and disrespecting the meaning of life (Schmidt et al.
Rao 2012). Combinatorics of automatic lab 2009; Breitling et al. 2015). This may threaten
assembly in biology is a pragmatic imagination the long-standing concepts of nature. Therefore,
that is still not conceptualized well and cannot be it is important to set the strategies for synthetic
mathematically defined (Friedrich 2013). The biology to contain social, environmental, and
gap between in silico and experimental biology ethical risks (Schmidt et al. 2009; Dana et al.
will be negotiable, once matter can be expressed 2012). To ensure the progress of this field, US
in pure digital terms that will bear ontological Presidential Committee has set up the guidelines
and functional ambivalences (Wu and Rao 2012). for synthetic biology research. The report came
Till then lab automation tools may struggle to up with 18 specific recommendations that can
provide the right answers (Friedrich 2013). encourage synthetic biologist community to
Similar to electronics circuit design, modular- make specific guidelines (http://bioethics.gov/
ization and standardization are the key compo- synthetic-biology-report). The report not only
5 Synthetic Biology Strategies for Polyhydroxyalkanoate Synthesis 83

encourages the scientific pursuits in synthetic media requirements can be a bottleneck (Keasling
biology but also puts weight on the need of 2008). A robust and well-characterized organism
collaborative as well as coordinated efforts of sci- capable of growing on minimal, inexpensive car-
entists at the international level. It also discusses bon sources will be a perfect choice for many
the importance of risk assessment review before purposes including biopolymer and biofuel pro-
releasing any synthetic organisms or products. duction (Keasling 2008; Pinto et al. 2012). The
This is to monitor and safeguard any conceivable other considerations include quality control of
threat through the inadvertent environmental production process, manipulations of regulatable
release of organisms or bioactive compounds. It genetic switches, and operational control to rec-
expects that synthetic biologists will find reliable tify problems. These considerations will be easier
containment and control mechanisms so that any for engineered bacteria and other microorgan-
synthetic product or organism designed will be isms (Keasling 2008). Therefore, most synthetic
unable to multiply and sustain in the natural envi- biologists will agree on the use of bacteria, and
ronment (Kaebnick et al. 2014). The report also there are four reasons for this: (a) bacteria have
cautions synthetic biologists to refrain from simple genomes compared to eukaryotes, (b)
claiming sensational terminology, for example, genetic manipulations are simpler and faster in
creating life. It is also necessary that synthetic these organisms, (c) most genetic manipulations
biologists should get ethical training and promote can be phenotypically calculated, and (d) lower
exchange of ideas with policy makers as well as costs of culturing systems.
religious and civil society groups about useful- It is considered that most of the biological
ness of synthetic biology and related emerging hosts have the capability to inactivate or get rid
technologies. themselves altogether of the foreign genes that
create stress by metabolic burdens (Glass et al.
2006; Acevedo-Rocha et al. 2013). Thus, there is
5.6 Microbial Model Systems a need to design novel host strains that lack the
and Synthetic Biology capability to induce any mutation and can stably
maintain and propagate foreign DNA (Glass
Single-celled bacteria have been utilized for et al. 2006; Acevedo-Rocha et al. 2013). The first
many decades to perform genetic manipulations such example is provided by two independent
for the desired outcomes. Microbial factories are groups, Kolisnychenko et al. and Sharma et al.,
the key choice for most synthetic biology appli- who designed reduced-genome bacterial strains
cations (Colin et al. 2011). The fact that our for the stable expression of foreign elements
understanding of bacterial systems is far better (Kolisnychenko et al. 2002; Sharma et al. 2007).
than that of complex eukaryotic cells also indi- These strains indicate that it is much easier to
cates them being the system of choice. There are manipulate microbial genomes for synthetic biol-
key considerations for synthetic biologists to use ogy purpose that can be used for mass production
cellular systems (Keasling 2008). One of the of metabolites. The lower genome size of pro-
main concerns is genetic stability and ability of karyotic cell is easier to manipulate, and there-
the host to act on foreign elements (Keasling fore, it is easier to create minimal genome by
2008). Additionally, for the mass production of removing auxiliary genes. Besides genetic insta-
biomolecules, cost estimation is a critical factor, bility, the additional complexity is at transcrip-
and to reduce the cost, it is important to grow the tional level where different promoters regulate
cells in minimal media. Microbes can grow in the expression level of genes (Alper et al. 2005).
minimal media, while eukaryotic cell culture Such transcriptional control is very complex, and
needs additional growth supplements. Thus, it is thus, it is imperative to get tunable expression
one of the main considerations in mass produc- using a promoter system that can provide strong
tion of large number of biomolecules as complex promoter strength (Keung et al. 2015).
84 G. Arora et al.

To achieve the control on biological functions Singh et al. 2009, 2013; Kumar et al. 2013, 2014;
with predictability and reliability, synthetic biol- Liu et al. 2013). The use of microbial factories is
ogists need simple biological models to work on far better than biomass extraction from other
(Keasling 2008). Microorganisms, specifically sources and chemical synthesis of many prod-
the prokaryotic bacteria E. coli and eukaryotic ucts. Using genetic and metabolic engineering
yeast Saccharomyces cerevisiae, were always the approaches, microbial factories were reported for
favorite ones due to our vast knowledge of these products when required genes were overex-
two organisms (Keasling 2008). In their native pressed in the surrogate host such as E. coli.
form these models are also very complex. Efforts Some successful examples of chemical produc-
were made to manipulate existing organisms at tion in microbial factories include succinic acid;
genetic level by removing additional and nones- fumaric acid; malic acid; adipic acid; propanol;
sential genetic components. One of such achieve- butanol; isobutanol; diols such as 1,3-propanediol,
ments was creation of minimal E. coli strain by 1,2-propanediol, 1,4-butanediol, and
different groups for efficient biotechnological 2,3-butanediol; putrescine; cadaverine; polylac-
production (Kolisnychenko et al. 2002; Posfai tic acid; poly-gamma-glutamic acid; and bio-
et al. 2006; Trinh et al. 2008). The next step is to polymers such as polyhydroxyalkanoates
generate amplified characteristics and functions (PHAs), polyhydroxybutyrate (PHB), etc. (Liu
within these systems by further replacing or add- et al. 2010; Lee et al. 2012a, b; Seo et al. 2013)
ing specific genetic components. (Fig. 5.2).
Polyhydroxyalkanoates (PHAs) comprise a
class of biological polyesters accumulated by
5.7 The Early Advances in PHA diverse bacteria when the carbon source is in
Production by Synthetic excess and one essential growth nutrient is lim-
Biology Approaches ited. PHAs have comparable material properties
to plastics, specifically thermoplastic and elasto-
Advances in genetic engineering, metabolic engi- mers (Madison and Huisman 1999; Reddy et al.
neering, and systems biology led to the creation 2003; Philip et al. 2007). The molecular masses
of novel microbes that can offer the product of of bacterially produced PHAs are generally of
choice. Pressure on environmental resources and the order 50,0001,000,000 Da that is similar to
associated economic problems in demand and conventional plastics such as polypropylene
supply are encouraging the research to develop (Madison and Huisman 1999). Natural PHA
economical microbial factories for mass produc- being biodegradable has the environmental
tion of a variety of substances (Kumar et al. 2009; advantage over conventional plastics. Using

Fig. 5.2 Bacterial factory for Polyhydroxyalkanoates

synthetic biology (PHA) 1,2-propane-diol
Succinic acid

Poly-lactic acid
5 Synthetic Biology Strategies for Polyhydroxyalkanoate Synthesis 85

chemical hydrolysis of PHA, several commer- to more PHA production and reduced granular
cially attractive molecules can be extracted that size (Maehara et al. 1999; Tian et al. 2005).
can be used as biodegradable solvents (Madison Expression of recombinant secretory Phasin in E.
and Huisman 1999). coli paves the way for synthetic biological sys-
Despite having many advantages, PHAs, how- tem to secrete PHAs (Linton 2010; Rahman et al.
ever, are not used commercially due to high costs 2013). Secretion of PHA will be helpful in down-
(approximately $4.06.0 per kg) of mass produc- stream processing whereby it can be easily sepa-
tion (Reddy et al. 2003; Rahman et al. 2013; rated from the cell mass, which can aid in better
Kumar et al. 2015a, b). The high production costs recovery and purification (Rahman et al. 2013).
are due to lower yield, expensive feed materials, Synthetic biology tools can also be applied to
and extraction efficiency in natural environments improve the PHA production by enhancing the
(Linton 2010; Patel et al. 2012, 2015). The advent activities of PHA-synthesizing enzymes. In the
of recombinant DNA technology has made E. past, protein engineering approaches have been
coli and Bacillus subtilis the preferred organisms used for directed evolution to enhance the spe-
for the production of recombinant proteins and cific activity of PHA synthase from Aeromonas
metabolites (Verlinden et al. 2007; Singh et al. punctata and to synthesize higher amounts of
2009; Zhou et al. 2012). The early efforts of PHA in recombinant E. coli (Amara et al. 2002;
recombinant PHA production in E. coli were Foo et al. 2012). These studies indicate that syn-
based on expression of the Ralstonia eutropha thetic biology in sync with metabolic engineering
phaCAB operon (Valentin and Dennis 1997). The can help improving biocatalytic properties of
phaCAB operon expresses three proteins by PHA synthase to optimize engineered pathways
which acetyl-CoA is converted to polyhydroxy- in surrogate host like B. subtilis and E. coli.
butyrate: phaC (PHA synthase), phaA PHA synthesis is an energy-consuming mech-
(-ketothiolase), and phaB (acetoacetyl-CoA anism for the bacteria, and for commercial bio-
reductase) (Rahman et al. 2013). PHA-associated polymer production, it is imperative that we have
proteins, called Phasins, adhere to the surface of fine control over the production process (Pham
PHA polymers and help in formation of spherical et al. 2004). Synthetic genetic switches as
granules (Zhou et al. 2012). As the extraction of described earlier can help in the regulation of
polymer from the cell is one of the most impor- expression levels. However, traditional genetic
tant steps in commercial processing of PHA switches can only be modified as on or off,
polymer, development of its secretion mecha- and during production they cannot sense when
nism is of potential interest (Linton 2010). Most the cell is exhausted and stressed out. The inabil-
of the cost of PHA processing is in extraction ity of such genetic switches in responding to the
process. Synthetic modules designed to secrete dynamic needs of biological system and regulat-
polymers will eliminate the cell disruption step ing the expression will result in process impedi-
and decrease the production cost significantly. ment. Therefore, the need for smart genetic
Application of such synthetic modules will not switches arises that can sense, respond, and help
need bacterial cells lysis for the extraction of the cell to adapt to every condition. These
compounds, and therefore, it can help in develop- dynamic components that can cater to the energy
ing continuous PHA production process. There is demands of the cells and streamline the PHA pro-
a lot of focus on the development of secretion duction process will be required in a bioreactor.
mechanism by using Phasins as these proteins Synthetic biologists have come up with dynamic
are suggested to increase the surface-to-volume sensor-regulator system (DSRS) that can respond
ratio of granules for better accumulation (York to dynamic changes in a cell (Zhang et al. 2012)
et al. 2001; Rahman et al. 2013). Also, Phasin and help in biofuel production. DSRS can be
expression is shown to decrease the granule size. applied to controlled biopolymer production for
The translocation of smaller granules is much better yields, and there is similar effort with tech-
easier, and therefore, Phasin overexpression leads nology named CHIRP (Circuit for Enhanced
86 G. Arora et al.

In-vivo Regulated Bioplastics Production) (http:// Breitling R, Takano E, Gardner TS (2015) Judging
synthetic biology risks. Science 347:107
Brophy JA, Voigt CA (2014) Principles of genetic circuit
design. Nat Methods 11:508520
Cameron DE, Bashor CJ, Collins JJ (2014) A brief history
5.8 Perspective of synthetic biology. Nat Rev Microbiol 12:381390
Carothers JM, Goler JA, Keasling JD (2009) Chemical
synthesis using synthetic biology. Curr Opin
The global market of synthetic biology is Biotechnol 20:498503
expected to reach 38.7 billion by 2020 (Singh Chandran D, Bergmann FT, Sauro HM (2009) TinkerCell:
2014). This indicates synthetic biology is modular CAD tool for synthetic biology. J Biol Eng
expected to deliver important products and tech-
Chandrasegaran SK, Ramani K, Sriram RD, Horvth I,
nologies. Thus, synthetic biology can be applied Bernard A, Harik RF, Gao W (2013) The evolution,
to multiple aspects of PHA production to obtain challenges, and future of knowledge representation in
better polymer quality, enhanced production, and product design systems. Comput Aided Des
ease in extraction/processing. These methods, if
Chen BS, Wu CC (2013) Systems biology as an integrated
successful, can help decrease the production platform for bioinformatics, systems synthetic biol-
costs and large-scale commercial production of ogy, and systems metabolic engineering. Cells
PHA derivatives. 2:635688
Church GM, Elowitz MB, Smolke CD, Voigt CA, Weiss R
(2014) Realizing the potential of synthetic biology.
Acknowledgments The authors wish to thank the CSIR- Nat Rev Mol Cell Biol 15:289294
Institute of Genomics and Integrative Biology (IGIB), Cole JA (2014) Synthetic biology: old wine in new bottles
Delhi, Government of India for providing support. with an emerging language that ranges from the sub-
lime to the ridiculous? FEMS Microbiol Lett
Colin VL, Rodriguez A, Cristobal HA (2011) The role of
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5 Synthetic Biology Strategies for Polyhydroxyalkanoate Synthesis 89

Wu M, Su RQ, Li X, Ellis T, Lai YC, Wang X (2013) regulate Mycobacterium tuberculosis physiology and
Engineering of regulated stochastic cell fate determi- drug discovery modules.
nation. Proc Natl Acad Sci U S A 110:1061010615
Yeh BJ, Lim WA (2007) Synthetic biology: lessons from Parijat Kundu is a gradu-
the history of synthetic organic chemistry. Nat Chem ate in Biochemistry from
Biol 3:521525 Presidency College,
York GM, Stubbe J, Sinskey AJ (2001) New insight into Kolkata, India, and post-
the role of the PhaP phasin of Ralstonia eutropha in graduate in Biochemistry
promoting synthesis of polyhydroxybutyrate. from University of Calcutta,
J Bacteriol 183:23942397 Kolkata, India. He is cur-
Zeng Y, Yao S (2009) Understanding design activities rently working as Project
through computer simulation. Adv Eng Inform Fellow at CSIR-Institute of
23:294308 Genomics and Integrative
Zhang H, Obias V, Gonyer K, Dennis D (1994) Biology, Delhi, India. His
Production of polyhydroxyalkanoates in sucrose- current research focuses on
utilizing recombinant Escherichia coli and understanding posttransla-
Klebsiella strains. Appl Environ Microbiol tional modifications network of Mycobacterium
60:11981205 tuberculosis.
Zhang F, Carothers JM, Keasling JD (2012) Design of a
dynamic sensor-regulator system for production of Mritunjay Saxena
chemicals and fuels derived from fatty acids. Nat received his Ph.D. degree in
Biotechnol 30:354359 Life Sciences from Devi
Zhou XY, Yuan XX, Shi ZY, Meng DC, Jiang WJ, Wu LP, Ahilya Vishwavidyalaya,
Chen JC, Chen GQ (2012) Hyperproduction of poly(4- Indore, India. He is currently
hydroxybutyrate) from glucose by recombinant working as a Senior Project
Escherichia coli. Microb Cell Fact 11:54 Fellow at CSIR-Institute of
Genomics and Integrative
Biology, Delhi, India. His cur-
Gunjan Arora received rent interests are biosensors,
his Ph.D. degree in Zoology Plasmodium vivax malaria,
from University of Delhi and malaria vaccine.
and CSIR-Institute of
Genomics and Integrative
Biology (CSIR-IGIB,
Delhi, India). He has also
worked as vaccine research
innovation awardee in
Translational Health
Science and Technology
Institute and is currently
working as postdoctoral fel-
low at the National Institute of Allergy and Infectious
Diseases in the National Institutes of Health (NIH). His
current research interests include systems and synthetic
biology approaches.

Andaleeb Sajid
received her Ph.D. degree
in Biotechnology from the
CSIR-Institute of Genomics
and Integrative Biology
(CSIR-IGIB, Delhi, India).
Currently, she is working as
postdoctoral fellow at
Laboratory of Clinical
Infectious Diseases at the
National Institute of
Allergy and Infectious
Diseases in the National Institutes of Health (NIH). Her
research interests include signal transduction events that
Frontiers in Biomedical
Engineering: PHA-Fabricated 6

Lalit K. Singh, Neha Dhasmana,

Shashank S. Kamble, Aditya K. Sharma,
and Yogendra Singh

Polyhydroxyalkonoates (PHAs) are biological in origin, organic polyes-
ters comprising the industrial and biomedical interest. This chapter sum-
marizes the current advances, applications, limitations, and challenges of
biopolymers in medicine. Biopolymers not only substitute the existing
polymers, but novel combinations of diverse PHAs broaden the applicabil-
ity and utility. The PHA-based implants are new dimensions of future in
biomedical engineering.

6.1 Introduction thermoplasticity. PHAs belong to the family of

biopolymers containing polyesters of different
Polyhydroxyalkonoates (PHAs) have gained the hydroxyl-carboxylic acids. Bacteria synthesize
significant attention, with diverse applications in polyhydroxyalkanoates as energy storage com-
medicine (Kalia et al. 2000). PHA is a microbial pound normally in excess with the carbon source
biopolymer having properties like biocompat- and one component essential for growth like phos-
ibility, nontoxicity, biodegradability, and phorus, nitrogen, oxygen, and sulfur in limited
concentration (Singh et al. 2015a). Novel blends
are developed by mixing with variable amounts of
L.K. Singh (*) polymers of 3-hydroxyoctanoate, 3-hydroxyvaler-
CSIR-Institute of Genomics and Integrative Biology, ate, and 3-hydroxybutyrate (PHB) for their poten-
Allergy and Infectious Diseases, Delhi, India
tial use in various biomedical applications. These
e-mail: lalit.singh@igib.in
blends showed higher Youngs modulus and ten-
N. Dhasmana S.S. Kamble A.K. Sharma
sile strength compared to their parent molecules.
Allergy and Infectious Diseases, CSIR-Institute
of Genomics and Integrative Biology, PHAs are appealing implant material in biomate-
110007 Delhi, India rial engineering and for mechanical supports
e-mail: neha.dhasmana@igib.in; because they are natural in origin, have enhanced
shashank.kamble@igib.in; aditya.sharma@gmail.com
biocompatibility, and lack cytotoxicity and capa-
Y. Singh bility to support cell adhesion and cell growth.
Allergy and Infectious Diseases, CSIR-Institute
PHAs can be categorized as short (scl), medium
of Genomics and Integrative Biology,
Room No. 208, Mall Road, 110007 Delhi, India (mcl), and long (lcl), based on chain length
e-mail: ysingh@igib.res.in (Fig. 6.1) (Misra et al. 2006; Singh et al. 2015b).

Springer India 2015 91

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_6
92 L.K. Singh et al.

a OH O e

n = 3-5
b OH O


c OH O
n = 6-14


d O O O

n = >14

Fig. 6.1 Chemical representation of hydroxy-carboxylic acid), (d) 4HB (4-hydroxy butyric acid), (e) scl-PHA
acids, monomeric components of PHA* and category of (small chain length polyhydroxy butyric acid), (f) mcl-
PHA on the basis of carbon chain length present in the PHA (medium chain length polyhydroxy butyric acid),
monomer. (a) 3HB (3-hydroxy butyric acid), (b) 3HV (g) lcl-PHA (longer chain length polyhydroxy butyric
(3-hydroxy valeric acid), (c) 3HH (3-hydroxy hexanoic acid)

The scl-PHAs exhibit high melting temperature medical engineering. Hence, several PHA-based
and are brittle in nature, while mcl-PHAs are lesser implants have been tested in animals, while some
crystalline and having low melting temperature. PHA-based implants are in practice for human
The PHB is the most widely studied scl-PHA but applications recently. The purity of PHA always
is brittle in nature and has high crystallinity to remains an issue since the pyrogenic contami-
make PHB industrially less valuable. On the other nants gets co-purified (Singh et al. 2009). In the
hand, PHO is the extensively studied mcl-PHA future, PHAs may serve as an attractive and prom-
with higher tensile strength and low thermal stabil- ising biomaterial for diverse applications in bio-
ity and can be commercially developed. medical engineering. Although in the industrial
PHAs have been discovered and investigated sector, PHAs are widely utilized as packaging
significantly for application in medicine and material, biofuel, and drug delivery vehicle, in
healthcare in the last two decades. PHAs have this chapter, we are mainly emphasizing and
been incorporated to develop several medical describing the use of PHA as biomedical implants.
implants like sutures, orthopedic anchors, and The potential implication of PHA appears excel-
several repair devices to revolutionize the bio- lent in the tissue engineering area near future.
6 Frontiers in Biomedical Engineering: PHA-Fabricated Implants 93

6.2 PHA as In-Vivo Implants These different composites of PHAs were

in Biomedical Engineering blended to design anchors, sutures, cardiovascu-
lar chips, repair patches, nerve guiding, slings,
PHAs have been developed over the years for orthopedic anchors, guiding tissue, adhesion bar-
various applications such as medical implant, riers, and devices for regeneration, cartilage and
mechanical supportive devices, drug delivery tendon repair, bone marrow scaffolds, etc. (Fig.
matrices, chips/patches to support the cell prolif- 6.3) (Masood et al. 2014; Yang et al. 2014). We
eration, and tissue regeneration by exploiting its can achieve the desirable degradation time, bio-
multifacets properties like superior elasticity, compatibility, and favorable mechanical proper-
adaptable mechanical strength, and biocompati- ties by manipulating the PHA compositions to
bility (Chen 2009; Ihssen et al. 2009). The PHB meet the demands of specific physiological
and its copolymer production were demonstrated condition.
in Bacillus using different substrates to develop The PHA-derived implants/devices do not
the PHA-based products with sustainable evoke the immune response inside the host and
approach (Fig. 6.2) (Kumar et al. 2015a, b). are thus considered as biologically safe materials
Several investigators recommended that Bacillus (Chen and Wu 2005). Williams and colleagues
could serve as a promising candidate for large- first time demonstrated that PHB- and PHO-
scale PHA production (Kumar et al. 2013, 2014; based microsphere and tubes were non-
Patel et al. 2015; Singh et al. 2013). Numerous immunogenic in mice model. Lobler and
composites were formulated especially copoly- coworkers investigated that the PHA-based gas-
mers of HB with 3-hydroxyvalerate (P3HBV), trointestinal patches did not generate any
4-HB (P4HB), and 3-hydroxyoctanoate (P3HO). inflammatory response. Moreover, PHA-based

Fig. 6.2 Transmission electron microscope image of were observed under Tecnai G2 Spirit Transmission
PHA granules accumulation in Bacillus. Bacillus cereus Electron Microscope at 200 KV. Mainly three models
were grown on GM2 media supplemented with glycerol Micelle, Budding and Scaffold are mentioned for PHA
for 72 h. The cells were harvested at 8000 rpm and granules growth. Figure (a, b, d) shows the mixed Micelle
primarily fixed with Karnovaskys reagent for 16 h at and Budding model of PHA granule growth, while
4 C. Secondary fixation was performed with 1 % Fig. (c) represents Micelle model. Moreover interestingly
osmium tetroxide and then cells were gradually dehy- granules occupy 7080 % volume of the total cell (Scale
drated followed by infiltration. The ultrathin sections bar 100 nm)
94 L.K. Singh et al.

Oesophagus Heart valves,
Bone Marrow
patches Arteries

Periodontal Brain and
Implants Nerve Implants

Bone Implants
Skin Implants Anchors and
Liver Tissue Sutures

Fig. 6.3 Schematic representation of diverse applications implants/anchors, guiding tissue repair/regeneration
of PHA in bio-medical engineering. PHAs classified into devices like liver tissue implants, adhesion barriers,
three major classes on the basis of carbon chain length articular cartilage repair devices, nerve guiding, tendon
present in the monomeric unit: short chain length (scl- repair devices, bone marrow scaffolds, periodontal
PHA), medium chain length (mcl-PHA), and long-chain implants, and wound dressings as described in the
length (lcl-PHA). The various composites and blends summary figure. We can achieve the desirable degradation
were formulated, especially poly 3-hydroxybutyrate time, biocompatibility, and favorable mechanical
(P3HB), poly-3-hydroxyvalerate (P3HBV), poly-4- properties by manipulating the PHA compositions to meet
hydroxybutyrate (P4HB), copolymers of P3HB, and poly- the demands of specific physiological conditions. PHA
3-hydroxyoctanoate (PHO). These variants of PHAs are has opened a new window of opportunity in the field of
blended to develop anchors, sutures, cardiovascular bio-medical engineering
patches, heart valves, slings, skin implants, orthopedic

implants are capable to support cell proliferation natural extracellular matrix (Li et al. 2008). PHA-
and adhesion and shown to be biodegradable in based implant materials were approved for
nature. The degraded hydroxybutyric acid from human applications in 2007 by Food and Drug
implant elevates the cytosolic calcium that Administration USA.
enhances the attachment and cell proliferation Tissue engineering is multidisciplinary fields
(Xiao et al. 2007). An enhanced cell proliferation involving regeneration of the damaged cells or
was observed in umbilical vein and smooth mus- tissue using multiple stem cells, biomaterials,
cle cells on PHB copolymer like P3HB and poly- and signaling molecules (Bruder and Fox 1999).
3-hydroxyhexanoate (P3HBHHx) coated with Tissue engineering can be categorized into two
fibronectin (Qu et al. 2006). Poly-3- types: soft tissue engineering and hard tissue
hydroxybutyrate-co-4-hydroxybutyrate-co-3- engineering. Highly porous, biodegradable
hydroxyhexanoate (P3HB4HB3HHx) is an scaffolds with specific cell types are utilized to
improved implantation material for biomedical promote ex-vivo growth in soft tissue engineer-
applications in comparison to P3HBHHx, poly- ing. The skin, heart valves, esophagus patches,
lactic acid (PLA) (Chen 2009). These PHB, vascular patches, liver, and nerve tissue guiding
P3HBHHx, and P3HB4HB3HHx composite are the classical examples of soft tissue engineer-
exhibits increased mechanical properties when ing (Du et al. 2009; Karageorgiou and Kaplan
fabricated into nanostructure and behaves like 2005). On the other hand, scaffolds fabricated
6 Frontiers in Biomedical Engineering: PHA-Fabricated Implants 95

with biological materials are implanted inside the hydroxyhexanoate (P3HB3HHx) can serve as a
body to provide the mechanical support and successful candidate to construct the artificial
growth in the hard tissue engineering. The carti- esophagus. Such composite P3HB3HHx blended
lage and bone tissue implants belong to hard tis- artificial esophagus was implanted in the dog.
sue engineering (Yu and Fan 2008). PHA-based The experiment stimulated regeneration of the
implants/devices contain all the characteristics tissue and negligible biodegradation of the com-
that are mandatory to ideal biomaterial, i.e., posite (Chen and Wu 2005).
capable to support the cell growth and maintain
proper nutrients supply to cells and degradation
in a short period after implantations. 6.2.2 PHA-Based Heart Valves,
Artery, and Vascular Grafting/
6.2.1 Soft Tissue Implants:
Esophagus, Pericardial The bicuspid/tricuspid valve malfunctioning also
Patches results in the cardiac failure. Sodian and col-
leagues demonstrated that mcl-PHA-based
Biocompatibility of the implants is a fundamen- implants can serve an option in heart valve tissue
tal prerequisite for the biomaterial engineering engineering. The improved cell proliferation and
applications. The other features including the cell adhesion were observed in poly-4-
materials shape, surface hydrophobicity, poros- hydroxybutyric acid (P4HB) and polyglycolic
ity, mechanical strength, biochemistry of the acid (PGA) blended scaffolds (Fu et al. 2004).
material, and degradation product also contribute Generally, heart valves are implanted with sup-
to the success rate of the implant (Sun et al. plementation of fibroblast growth factor and
2005). The anchorage-dependent attachment and ascorbic acid. Wu and coworkers designed hybrid
proliferation of mammalian cell fundamentally heart valve from decellularized porcine valve
depend on the surface roughness and hydropho- coated with P3HBHHx and implanted in the
bicity of any scaffold. Myocardial infarction is sheep for 16 weeks. These coated valves exhib-
the common cause of heart failure. However, ited and promoted better proliferation of cardio-
heart transplantation is not always feasible due to myocytes and endothelial cells and less
limited number of organ donors. Tissue engineer- calcification (Wu et al. 2007). Adamus and
ing serves as an alternative option to put the peri- coworkers blended the P3HB and P3HO to
cardial patch on affected area to repair the design more elastic and tensile heart valves for
damaged heart. PHA products are fabricated into the future biomedical engineering applications
the pericardial patches, surgically placed between (Adamus et al. 2012).
the sternum and heart to prevent the adhesion The damaged or diseased blood vessels are
after the cardiac surgery. The immunogenicity of repaired through the vascular grafting. The sur-
pericardial patches was first implanted and tested geons generally implant the vascular grafts com-
in the sheep. The safety was monitored in 19 posed of Dacron TM (polyethylene terephthalate)
human patients during bypass surgery for 624 for larger diameter blood vessels. These grafts
months, which demonstrated that PHA-based are not suitable for the narrow diameter blood
implants can be used as pericardial patches vessels like in coronary artery bypass procedures.
(Malm et al. 1992; Duvernoy et al. 1995). The synthetic grafts made with P3HB4HB were
The pericardial patch serves two major functions: implanted in the dog, but degradation of the
first delivers healthy cardiomyocytes into the implant started within 2 weeks. The composite of
infarcted region and second provides the ventric- P3HHx3HO was used as graft and was found
ular resistant (Fujimoto et al. 2007). Similarly, stable till 6 months (Marois et al. 1999; Shum-
copolymer of poly-3-hydroxybutyric acid-co-3- Tim et al. 1999). Moreover, PHA blended vascu-
96 L.K. Singh et al.

lar grafts are considered as valuable alternative ricate nerve scaffolds for Schwann cell regenera-
for implication in vascular patches, atrial septal tion (Armstrong et al. 2007; Bian et al. 2009). In
defect repair devices, and valves of the vein several studies, P3HBHHx was blended ran-
(Chen et al. 2001; Chen and Wu 2005; Dai et al. domly and fabricated with poly-DL-lactide
2009; Wang et al. 2008). The P3HBHHx-based (PDLLA) in different studies. The fibronectin
blended scaffolds coated with fibronectin accumulation was higher on PDLLA biofilm
increase the cell adhesion and cellular prolifera- sheet (Gao et al. 2006). The tubular PHBV was
tion and were found suitable for vascular tissue fabricated with the poly-L-lactide-co-D,L-lac-
engineering (Zhang et al. 2007; Wu et al. 2008). tide (PLDL) and PLGA poly-lactide-co-gly-
Gaudio and colleagues designed the composites colide acid as an exterior component with
of the polycaprolactone (PCL) and P3HBV in electrospin technology. The PHBVPLGA com-
various proportions. These blended compositions posites demonstrated the healing of nerve tissues
of PHA could serve as future implants in vascular in the injured area (Yucel et al. 2010). The com-
tissue engineering (Gaudio et al. 2012; Madhavan posites/conduits made with the PHB and
et al. 2013). These vascular grafts restore the PHBHHx expand the limits of axonal regenera-
malfunctioning of the blood vessels during injury tion up to 10 mm in the sciatic nerve of rat with
and pathologic condition. However, the immune good biocompatibility that is not feasible in the
response, risk of infection during surgery, and silicone-based implants (Armstrong et al. 2007;
stability of the scaffold/implant are some serious Bian et al. 2009). Mohanna and colleagues dem-
limiting factors. onstrated artificial nerve implants supplemented
with glial growth factor repair larger nerve gap
up to 24 cm in rabbit (Mohanna et al. 2003).
6.2.3 Brain Nerve Guides Chen and colleagues demonstrated that neural
and Implants progenitor cells differentiate into neurons using
the P3HBV microspheres (Chen and Tong 2012).
Peripheral nervous system injuries can lead to Collagen is the structurally most abundant pro-
permanent disability during the surgical proce- tein of the extracellular matrix of the nerves;
dures. Nerve tissue engineering has made the Prabhakaran and colleagues designed the
neurological regeneration procedures feasible. P3HBV-collagen composite nanowires to regen-
The neurological recovery is considered less erate the nerve tissue (Prabhakaran et al. 2013).
than 2 cm in humans. Apart from extremely low They demonstrated that collagen-associated
inflammatory, cytotoxic, and immunogenic P3HBV nanofibers provided proper orientations
response, the neurological implant material and bipolar extension during nerve cell prolifer-
should mimic with the fibers scale of extracellu- ation. An enhanced cell proliferation was
lar matrix and able to prevent the infiltration observed in the composite P3HBV/collagen
(Young et al. 2002; Mohanna et al. 2003). (50:50) and P3HBV/collagen (75:25) nanofibers
Although nonbiodegradable silicone-based bio- among the randomly fabricated nanofibers.
material/implants are already in use to meet the Although P3HBV was blended with type I col-
demands in nerve tissue engineering, PHA- lagen in a random manner, still improved cell
based nerve implants can serve as a good candi- adhesion and cellular differentiation of Schwann
date for nerve regeneration. The PHB-based cells were discovered in aligned PHB/P3HBV/
implants were introduced to regenerate radial collagen fibers. Masaeli and coworkers demon-
nerves in the cats (Mohanna et al. 2003; Young strated the myelinated nerve cell regeneration
et al. 2002). The axonal regeneration was dem- and high expression of nerve growth factor on
onstrated up to 23 mm distance with normal randomly fabricated PHB/PHBV/collagen nano-
inflammatory response. Several studies indicate fibers using electrospin technology (Masaeli
that PHB and P3HBHHx can be designed to fab- et al. 2013; Merolli et al. 2014).
6 Frontiers in Biomedical Engineering: PHA-Fabricated Implants 97

6.2.4 Bone Tissue Implants: with higher content of the hydroxyapatite; conse-
Orthopedic Anchors, Sutures, quently, the scaffolds were found to have a more
and Stents porous matrix and to be a favorable environment
for osteoblast (Sultana and Wang 2012).
Bone is complex organicinorganic hybrid tissue Moreover, PHB blended with bio-glass 45S5 was
with characteristic mechanical properties such as shown to produce bone implant materials with 85
high fracture strength and flexibility. Bone tissue % porosity. Mirsa and colleagues demonstrated
engineering is an interdisciplinary field of kinesi- the scaffolds were non-immunogenic and pro-
ology and material sciences and serves as an vided better cellular attachment and proliferation
excellent tool for the treatment of damaged or of osteoblasts in rat (Misra et al. 2010). More
lost bone due to aging or traumatic shock. The recently, Wang and coworkers designed the
implant material must consist good mechanical PHBV/calcium silicate composites, which mimic
strength and properties to regulate the cellular natural extracellular matrix. The calcium silicate
proliferations, differentiation of osteoblast, and induces the expression of genes responsible for
development of bone extracellular matrix. transforming growth factor-1 and bone morpho-
Initially, scl-PHAs were considered suitable for genetic protein-7 that promotes early differentia-
the hard tissue engineering, but during the last tion of human osteoblasts (Wang et al. 2013).
decade, mcl-PHA-based blended materials were
used with enhanced mechanical properties. Wang
and colleagues evaluated PHB, P3HBHHx, and 6.2.5 Cartilage Tissue Engineering
PLA-based 3D scaffolds during cellular differen-
tiation and cell adhesion in rabbit model (Wang Cartilage is nonvascular tissue and hardly
et al. 2004). The cell adhesion, proliferation of regenerates that supports the skeletal system.
osteoblast, higher calcium deposition, and colla- However, significant investigations have been
gen synthesis in P3HBHHx-based implants were carried out in PHA-based scaffolds during the
comparative to PHB and PLA (Li et al. 2007). last decades. The PHB, P3HBV, P3HBHHx,
Cool and coworkers demonstrated that PHBV/ and various composites have been studied and
hydroxyapatite-based scaffolds exhibit low were found suitable contenders for cartilage tis-
inflammatory response and high mineralization sue engineering because of improved prolifera-
(Cool et al. 2007). Francis and colleagues tion and enhanced differentiation of
designed novel multipurpose 45S5 bio-glass chondrocytes (Sun et al. 2005). Wang and col-
based on PHB and nano-sized hydroxyapatite leagues developed the 3D-engineered
(natural mineral of the bone matrix) used for gen- P3HBHHx scaffold to repair articular cartilage
tamicin delivery during bone tissue engineering in rabbit model. The engineered P3HBHHx
(Francis et al. 2011). Hyati and colleagues dem- cartilage scaffold was incubated with chondro-
onstrated that PHB/nanohydroxyapatite-based cytes and showed effective cartilage repair with
biomaterials are favorable in the bone tissue superior dissemination of extracellular matrix
engineering. The PHB blended with 1015 % of that was observed during 16 weeks in rabbit
nanohydroxylapatite consequently yielded bone (Wang et al. 2008). Similarly, Liu and col-
like highly porous material. All scaffolds contain leagues demonstrated the neocartilage for-
the biocompatibility, mechanical properties, and mation by employing the chondrogenic
porosity like cancellous bone (Hayati et al. 2012). differentiated human adipose stem cells on
Baek and colleagues demonstrated better adhe- P3HBV scaffolds. The chondrogenic pre-dif-
sion, proliferation, and differentiation of osteo- ferentiated human adipose stem cells con-
blast cells on PHBV/hydroxyapatite scaffolds structed the neocartilage with good mechanical
immobilized with collagen I (Baek et al. 2012). strength in nude mice after 16 weeks of implan-
Sultana and Wang designed the P3HBV blended tation (Liu et al. 2010). Later, the mechanical
98 L.K. Singh et al.

strength was improved with the incorporation 6.2.7 Skin Tissue Implants
of poly-L-lactide-co-caprolactone to P3HBV
microspheres for cartilage tissue engineering The human skin serves as the largest organ of the
(Li et al. 2013). body that maintains temperature, regulates water
loss, and works as an effective barrier against
external environment. Skin tissue engineering has
6.2.6 Liver Tissue Implants revolutionized the grafting procedures for the use
of burn victims and non-healing lesions like dia-
Although PHA-based liver implants have not betic and venous ulcers. Several investigators
been tested in human till date, some reports sug- have improved the skin grafting technology for
gest that PHB-based 3D supports frameworks the betterment of human health by using various
can be used in liver tissue regeneration. The biomaterial sheets. Hyaluronic acid/gelatin/chito-
investigators suggested that hepatocytes grow on san biofilms were used to propagate the human
the PHB beads in vitro. Zhu and coworkers dem- skin fibroblast cells and then transferred to the
onstrated that composite of P3HBV and poly-3- wound (Peschel et al. 2008; Tang et al. 2008).
h y d r o x y bu t y r a t e - c o - 3 - h y d r o x y va l e r a t e PHB copolymers such as P3HB4HB and
(P3HB3HV) microspheres allows the growth of P3HB3HHx with hyaluronic acid/chitosan have
hepatocytes in HepG2 and Hep3B cell lines. The been tested in skin tissue engineering (Ji et al.
microsphere allows proliferation of the HepG2 2008; Peschel et al. 2008). Ji and coworkers dem-
and Hep3B cells to construct the multilayer struc- onstrated PHB and P3HBV copolymers provide
ture within 1 week. The HepG2 cells were unable better mechanical properties and are excellent in
to maintain the P450 activity. The cellular aggre- supporting the growth of skin cells (Ji et al. 2008).
gation enhanced 24 folds upon bovine serum Later, Kuppan and colleagues studied the human
albumin secretion after 12 days (Zhu et al. skin fibroblast cell proliferation, adhesion, and
2007a). The research group identified the vital gene expression on the PHBV-based scaffolds and
role of extracellular matrix during hepatocyte tissue culture polystyrene (Kuppan et al. 2011).
regeneration. This time PHBV microspheres P3HBV blended with chitosan scaffolds support
were conjugated with three different extracellular better growth and enhanced cell proliferation and
matrix proteins: collagen, laminin, and fibronec- adhesion in wound healing model of rat in vivo
tin. The cellular hepatic function, P450 activity, (Veleirinho et al. 2012). The chitin composite
and bovine serum albumin secretion were moni- P3HB3HV with hydrogel scaffold exhibited two-
tored for 2 weeks. The Hep3B cells demonstrated fold enhanced cell proliferation of human dermal
enhanced cellular proliferation and hepatic func- fibroblast cells. These blends showed higher
tion on matrix-coated microspheres because porosity and slow rate biodegradation (Sankar
coated microsphere mimics human physiological et al. 2012). The PHB-based hydrogels macropo-
environment (Zhu et al. 2007b). Later, investiga- rous 3D scaffolds showed the promising applica-
tors identified the effect of copolymers on cellu- tion in skin tissue engineering and grafting.
lar aggregation in Hep3B cell lines. The P3HBV
was blended with polymer PLGA serving as
good framework for liver tissue engineering with 6.3 Summary and Future
the support of hepatocyte growth factor. The Prospective
composite of P3HBV/PLGA microspheres dem-
onstrated less degradation rate and maintained PHAs have opened new window of opportunity,
surface bioactivity indicating more suitability of with the diverse applications in biomedical
the copolymer in liver tissue regeneration (Zhu engineering. The application of PHAs will be
et al. 2009). broader with the discovery of new bacterial
6 Frontiers in Biomedical Engineering: PHA-Fabricated Implants 99

strains capable to generate the homopolymers Bruder SP, Fox BS (1999) Tissue engineering of bone cell
based strategies. Clin Orthop Relat Res 367:6883,
and copolymers, especially from biowastes as
feed, in near future (Kalia et al. 2003; Porwal Chen GQ (2009) A microbial polyhydroxyalkanoates
et al. 2008; Kumar et al. 2009; Patel et al. 2012; (PHA) based bio and materials industry. Chem Soc
Kumar et al. 2015a, b). This is feasible to pro- Rev 38:24342446. doi:10.1039/b812677c
Chen W, Tong YW (2012) PHBV microspheres as neural
duce the copolymers through metabolic engi-
tissue engineering scaffold support neuronal cell
neering with manifestation of diverse microbial growth and axondendrite polarization. Acta Biomater
metabolic background (Reddy et al. 2003; Kalia 8:540548. doi:10.1016/j.actbio.2011.09.026
et al. 2007; Singh et al. 2009). Moreover, the host Chen GQ, Wu Q (2005) Polyhydroxyalkanoates as tissue
engineering materials. Biomaterials 26:65656578.
cell genome manipulations with engineered PHA
synthase enzymatic machinery can design bacte- Chen GQ, Zhang G, Park SJ, Lee SJ (2001) Industrial pro-
ria into robust microbial plastic factory. The gov- duction of poly(hydroxybutyrate-co-
ernment and institutions should take initiatives to hydroxyhexanoate). Appl Microbiol Biotechnol
57:5055, PMID: 11693933
promote the growth of biomaterial research in
Cool SM, Kenny B, Wu A, Nurcombe V, Trau M, Cassady
association with the medicine. The diverse groups AI, Grndahl L (2007) Poly(3-hydroxybutyrate-co-3-
of expertise can solve the problem of implant hydroxyvalerate) composite biomaterials for bone tis-
material limitations. sue regeneration, in vitro performance assessed by
osteoblast proliferation, osteoclast adhesion and
resorption, and macrophage pro-inflammatory
Acknowledgment The authors wish to thank the director response. J Biomed Mater Res A 82A:599610.
of CSIR-Institute of Genomics and Integrative Biology doi:10.1002/jbm.a.31174
(IGIB) and Defence Research and Development Dai ZW, Zou XH, Chen GQ (2009) Poly(3-
Establishment (DRDE), Jhansi Road, Gwalior (LSRB- hydroxybutyrate-co-3- hydroxyhexanoate) as an
268/BTB/2013 and BSC0123), Government of India, for injectable implant system for prevention of post-
providing the necessary funds and facilities. Authors are surgical tissue adhesion. Biomaterials 30:30753083.
also thankful to the Academy of Scientific and Innovative doi:10.1016/j.biomaterials.2009.02.015
Research (AcSIR), New Delhi. Lalit K Singh, Shashank S Du DJ, Furukawa KS, Ushida T (2009) 3-D culture of
Kamble, and Aakriti Gangwal are thankful to UGC, and osteoblast-like cells by unidirectional or oscillatory
Neha Dhasmana is thankful to CSIR, for granting Senior flow for bone tissue engineering. Biotechnol Bioeng
Research Fellowships. We highly acknowledge Dr. V. C. 102:16701678. doi:10.1002/bit.22214
Kalia and Mr. Prasun Kumar, from CSIR-IGIB, Delhi, Duvernoy O, Malm T, Ramstrm J, Bowald S (1995) A
and Ms. Aakriti Gangwal from UDSC New Delhi, India, biodegradable patch used as a pericardial substitute
for their critical comments in the manuscript. after cardiac surgery: 6- and 24-month evaluation with
CT. Thorac Cardiovasc Surg 43:271274. doi:10.105
Francis L, Meng D, Knowles J, Keshavarz T, Boccaccini
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102 L.K. Singh et al.

Young RC, Wiberg M, Terenghi G (2002) Poly-3- and Innovative Biology. She is studying the physiological
hydroxybutyrate (PHB): a resorbable conduit for long- significance of Ser/Thr kinases in Bacillus anthracis.
gap repair in peripheral nerves. Br J Plast Surg
55:235240. doi:10.1054/bjps.2002.3798 Shashank S. Kamble
Yu GH, Fan YB (2008) Preparation of poly(D, L-lactic received his M.Sc. degree
acid) scaffolds using alginate particles. J Biomater Sci- from University of Mumbai,
Polym Ed 19:8798. doi:10.1163/156856208783227703 India. Currently, he is pursu-
Yucel D, Kose GT, Hasirci V (2010) Polyester based nerve ing Ph.D. under the supervi-
guidance conduit design. Biomaterials 31:15961603. sion of Dr. Yogendra Singh
doi:10.1016/j.biomaterials.2009.11.013 at CSIR-IGIB, India. He is
Zhang L, Zheng Z, Xi J, Gao Y, Aob Q, Gong Y, Zhao N, interested in the host patho-
Zhang X (2007) Improved mechanical property and gen cross talk during infec-
biocompatibility of poly(3-hydroxybutyrate-co-3-hy- tion with M. tuberculosis.
droxyhexanoate) for blood vessel tissue engineering by His work focuses on the
blending with poly(propylene carbonate). Europ Polym modulation of host proteases
J 43:29752978. doi:10.1016/j.eurpolymj.2007.04.007 by M. tuberculosis.
Zhu XH, Gan SK, Wang CH, Tong YW (2007a) Proteins
combination on PHBV microsphere scaffold to regu- Aditya K. Sharma has
late Hep3B cells activity and functionality: a model of done his graduation in
liver tissue engineering system. J Biomed Mater Res Biomedical Sciences from
83A:606616. doi:10.1002/jbm.a.31257 University of Delhi and
Zhu XH, Wang CH, Tong YW (2007b) Growing tissue- Masters in Biotechnology
like constructs with Hep3B/HepG2 liver cells on from Jamia Millia Islamia.
PHBV microspheres of different sizes. J Biomed Currently, he is pursuing
Mater Res B 82B:716. doi:10.1002/jbm.b.30698 his doctoral degree from
Zhu XH, Wang CH, Tong YW (2009) In vitro character- Institute of Genomics and
ization of hepatocyte growth factor release from Integrative Biology under
PHBV/PLGA microsphere scaffold. Biomed Mater supervision of Dr. Yogendra
Res 89A:411423. doi:10.1002/jbm.a.31978 Singh. For his Ph.D., he is
working on regulation of kinase and its substrates via
phosphorylation in Mycobacterium tuberculosis.
Lalit K. Singh received
the M.Sc. degree in Yogendra Singh
Biotechnology from Rajiv received his Ph.D. degree
Gandhi Biotechnology from Vallabhbhai Patel
Centre, RTM Nagpur Chest Institute, Delhi
University, Nagpur, India. University, and postdoc-
He is currently working as toral training at National
Senior Research Fellow, Institute of Health (NIH),
CSIR-IGIB, Delhi, India. Bethesda. Currently, he is
His current research Chief Scientist at CSIR-
focuses on understanding IGIB and Professor of
the role of caseinolytic AcSIR. He is a Fellow of
proteases and Ser/Thr kinases that mediated cellular National Academy of
architecture alterations, cell wall remodeling, and sporu- Sciences, India, Fellow of
lation in Bacillus. Indian National Science Academy, and Fellow of Indian
Academy of Sciences, India. He has been honored with
Neha Dhasmana gradu- Moselio Schaechter Distinguished Service Award in 2014
ated in Microbiology in by American Society for Microbiology. He serves as an
2008 from University of Editorial Board Member of Journal of Biological
Delhi. She received her M. Chemistry. His current research interest is to understand
Sc. degree in Biochemistry the host response in pulmonary/extra-pulmonary tubercu-
in 2010 from Hamdard losis and to decipher the role of eukaryotic like Ser/Thr
University. She is currently kinases in Mycobacterium tuberculosis and Bacillus
enrolled in Ph.D. with anthracis.
Academy of Innovative and
Scientific Research
(AcSIR) and works at
CSIR-Institute of Genomics
Sporulation, a Pitfall in the Path
of PHB Production 7
Neha Dhasmana, Lalit K. Singh,
Shashank S. Kamble, Nishant Kumar,
and Yogendra Singh

The concept of bioplastic is fascinating to our world, because of its poten-
tiality to deal with one of the major global problems like plastic pollution
(Kalia et al. J Sci Ind Res 59:433445, 2000; Kalia et al. Nat Biotechnol
21:845846, 2003). Polyhydroxybutyrates (PHB) are the best example for
the polymers by plant or microorganisms from a wide range of habitats
(Reddy et al. Bioresour Technol 87:137146, 2003; Porwal et al. Bioresour
Technol 99:54445451, 2008; Singh. Environ Microbiol 17:854864,
2015). PHB refers to the polyesters of 3-hydroxybutyrate and can be
extracted from various species like Ralstonia, Bacillus, Streptomyces,
Pseudomonas, etc., which are extensively discussed in published reviews
(Singh et al. Microb Cell Fact 8:38, 2009; Jendrossek and Pfeiffer. Environ
Microbiol 16:23572373, 2014). Bioplastic has the distinct feature of
being biodegradable. Further, the use of biowaste as substratum for
bioplastic-producing organisms presents an interesting concept to deal
with another global problem of waste management (Kumar et al. J Appl
Microbiol 106:20172023, 2009; Kumar et al. Indian J Microbiol 55:17,
2015; Kumar et al. Int J Biol Macromol, 2015; Patel et al. Biomas Bioenerg
36:218225, 2012; Patel et al. Bioresour Technol 176:136141, 2015).
Both Gram-positive and Gram-negative bacteria are reported to produce
polyhydroxyalkanoates (PHA); among them Gram-negative bacteria,
Ralstonia eutropha is the most extensively studied organism (Brigham

N. Dhasmana (*) S.S. Kamble N. Kumar

Allergy and Infectious Diseases,
CSIR-Institute of Genomics and Integrative Biology,
110007 Delhi, India
e-mail: neha.dhasmana@igib.in;
shashank.kamble@igib.in; nishant.kumar@igib.in
Y. Singh (*)
L.K. Singh Allergy and Infectious Diseases, CSIR-Institute
CSIR-Institute of Genomics and Integrative Biology, of Genomics and Integrative Biology,
Allergy and Infectious Diseases, Delhi, India Room No. 208, Mall Road, 110007 Delhi, India
e-mail: lalit.singh@igib.in e-mail: ysingh@igib.res.in

Springer India 2015 103

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_7
104 N. Dhasmana et al.

et al. Appl Environ Microbiol 78:80338044, 2012). One major rationale

to investigate the Gram-positive bacteria for their ability to produce PHB
is the absence of immunogenic lipopolysaccharide which co-purifies with
the PHB when Gram-negative organisms are employed, making PHB non-
appealing for the use in medical purposes like various human tissue grafts
(Valappil et al. Antonie Van Leeuwenhoek 91:117, 2007; Singh et al.
Microb Cell Fact 8:38, 2009). Additional appeal for using Gram-positive
bacteria, specifically Bacillus spp., is its ability to produce copolymers
which are superior to their counterparts, that is, homopolymers given their
enhanced characteristics like elasticity, etc. (Patel et al. Indian J Microbiol
51:418423, 2011; Kumar et al. Indian J Microbiol 54:151157, 2014;
Kumar et al. Int J Biol Macromol, 2015). Gram-positive bacteria exist in
two alternative phases in its life cycle, that is, vegetative cells and sporula-
tion. The adverse environmental conditions drive the Bacillus vegetative
cells into their transition to spores. Sporulation is an intrinsic characteris-
tic of the Bacillus species and is mainly regulated by a master regulator of
sporulation, namely, Spo0A (Slepecky and Law. J Bacteriol 82:3742,
1961; Singh et al. Indian J Microbiol 55:234, 2015).

7.1 Introduction charide which co-purifies with the PHB when

Gram-negative organisms are employed, making
The concept of bioplastic is fascinating to our PHB non-appealing for the use in medical pur-
world, because of its potentiality to deal with one poses like various human tissue grafts (Valappil
of the major global problems like plastic pollution et al. 2007; Singh et al. 2009). Additional appeal
(Kalia et al. 2000, 2003). Polyhydroxybutyrates for using Gram-positive bacteria, specifically
(PHB) are the best example for the polymers by Bacillus spp., is its ability to produce copolymers
plant or microorganisms from a wide range of which are superior to their counterparts, that is,
habitats (Reddy et al. 2003; Porwal et al. 2008; homopolymers given their enhanced characteris-
Singh et al. 2015a). PHB refers to the polyesters of tics like elasticity, etc. (Patel et al. 2011; Kumar
3-hydroxybutyrate and can be extracted from vari- et al. 2013, 2014, 2015b). Gram-positive bacteria
ous species like Ralstonia, Bacillus, Streptomyces, exist in two alternative phases in its life cycle, that
Pseudomonas, etc., which are extensively dis- is, vegetative cells and spores. The adverse envi-
cussed in published reviews (Singh et al. 2009; ronmental conditions drive the Bacillus vegetative
Jendrossek and Pfeiffer 2014). Bioplastic has the cells into their transition to spores. Sporulation is
distinct feature of being biodegradable. Further, an intrinsic characteristic of the Bacillus species
the use of biowaste as substratum for bioplastic- and is mainly regulated by a master regulator of
producing organisms presents an interesting con- sporulation, namely, Spo0A (Slepecky and Law
cept to deal with another global problem of waste 1961; Singh et al. 2015b).
management (Kumar et al. 2009, 2015a, b;
Patel et al. 2012, 2015). Both Gram-positive
and Gram-negative bacteria are reported to pro- 7.2 Polyhydroxybutyrate
duce polyhydroxyalkanoates (PHA); among them in Sporulation
Gram-negative bacteria, Ralstonia eutropha is the
most extensively studied organism (Brigham et al. Life thrives on the energy acquired. In primitive
2012). One major rationale to investigate the forms of life, the energy requirement for the cel-
Gram-positive bacteria for their ability to produce lular processes is fulfilled by existing substrate
PHB is the absence of immunogenic lipopolysac- availability or the intrinsic storage molecules.
7 Sporulation, a Pitfall in the Path of PHB Production 105

Bacteria employ different strategies to store energy Wu et al. 2001). Moreover, the onset of PHB
in forms that can be utilized in times of need (Singh biosynthesis and sporulation is induced under the
et al. 2014). Under nutrient limiting conditions, same environmental setup which renders us to
excess carbon is stored in the form of PHB or gly- intrigue their complex relationship. One major
cogen. Under high C:N or C:P ratios or lesser dis- question still exists about the essentiality of PHB
solved oxygen, PHB biosynthesis is induced in the sporulation process of Bacillus species
(Supono et al. 2013). For example, PHB synthesis which has not been experimentally validated till
in Bacillus anthracis is catalyzed by the three now. Since both sporogenic and asporogenic
enzymes, -ketothiolase, acetoacetyl-CoA reduc- strains of Bacillus and Clostridium are reported
tase, and PHB polymerase encoded by the genes to synthesize the PHB under excess carbon in the
phaA, phaB, and phaC, respectively (Kalia et al. surrounding. Therefore, the essentiality of PHB
2007; Jendrossek and Pfeiffer 2014). The two ace- in the sporulation process remains to be eluci-
tyl-CoA units are combined together to form ace- dated. PHB has been found to be functionally rel-
toacetyl-CoA, which upon reduction gets evant in maintaining the heat resistance of the
transformed to 3-hydroxybutyrate by acetoacetyl- Bacillus cereus spores. The study asserts the role
CoA reductase. The 3-hydroxybutyrate serves as a of PHB in endotrophic sporulation process in B.
substrate for the polymerization reaction that cereus (Holmes and Christopher 1984). Weakly
causes the further extension of PHB template, buffered medium supports the PHB accumula-
catalyzed by PHB synthase PhaRC in B. anthracis. tion and the endotrophic sporulation; further, it
The size of PHB polymer ranges between 50,000 has been reported to yield heat-resistant spores as
and 1,000,000 Da. Depending on their sizes, the compared to the strongly buffered media, in
PHB polymer can be largely categorized into short which lesser PHB accumulates (Holmes and
chain length (SCL) which has C3C5 backbone Christopher 1984). However, various reports
and medium chain length (MCL) which has back- state the insignificance of PHB in sporulation
bone of aliphatic or aromatic hydroxyalkanoate, process of B. cereus and Bacillus megaterium
specifically C6C14. PHB exists in the cell as amor- (Slepecky and Law 1961). A report showed pH-
phous granules and these structures are stabilized dependent PHB accumulation in the B. cereus
as phasin proteins, PhaP (Mezzina et al. 2014). strain T with the highest accumulation at pH
A study by Emeruwa and Hawirko showed between 6.2 and 6.4, whereas the sporulation
that synthesized PHB gets catabolized during the efficiency was similar in pH 6.4 or pH 7.4 (Nakata
sporulation process in Clostridium botulinum 1963).
sporogenic strains, while PHB content is unaf-
fected in the asporogenic strains of C. botulinum.
The results indicate that synthesized PHB serves 7.3 Molecular Modulators
as an endogenous source of energy for the spore of PHB Synthesis
component synthesis during the course of sporu-
lation. Interestingly, asporogenic strains of C. 7.3.1 Spo0A
botulinum were unable to utilize the accumulated
PHB in response to nutritional stress resulting in At molecular level, the sporulation master regula-
13 % of PHB (cell dry weight) as compared to tor, Spo0A, has been shown to be crucial for the
sporogenic strains of C. botulinum (9 %) expression of PHB synthesis-related genes in
(Emeruwa and Hawirko 1973). Even in B. Bacillus thuringiensis, hence causing PHB accu-
anthracis, the sporulation-defective strain mulation in the cells. A phosphorylated form of
(clpC) has reported to accumulate higher PHB Spo0A positively regulates the cascade, which
content as compared to the wild-type strain governs the transcription of sigma factor F, whose
(Singh et al. 2015a). These results suggest that a function is specifically in the forespore compart-
major fraction of the energy acquired from PHB ment of sporulation. The study has also revealed
catabolism can be utilized to drive the sporula- the non-importance of Spo0F in the context of
tion, a high-energy-consuming process (Fig. 7.1; the PHB synthesis in B. thuringiensis cells. The
106 N. Dhasmana et al.

Fig. 7.1 PHB accumulation profile in the Bacillus sp. observed under transmission electron microscopy. Yellow
cells during vegetative growth (AC) or sporulation arrows indicate the PHB granules. Scale bar 0.5 m
phases (DF). Sections of Bacillus anthracis Sterne

spo0F deletion mutant in B. thuringiensis was 7.3.2 Phasin Proteins

unaffected in PHB accumulation, suggesting that
the molecule Spo0F and PHB synthesis are inde- Phasins are the amphiphilic proteins whose pres-
pendent of each other at molecular levels. ence determines the number, size and surface to
Broadly, Spo0A-phosphorylated form is the mas- volume ratio of the PHB granules. Phasins per-
ter regulator of many processes as it activates or fectly surround the amorphous PHB granule
represses the distinct set of genes. Among many, (Pfeiffer and Jendrossek 2012; York et al. 2001).
the AbrB protein, a global regulator of proteins, In in vitro conditions, phasin proteins are reported
is responsible for the transition of cells from to activate the PhaCA synthase in Aeromonas
exponential to stationary phase of the growth caviae, while it masks the activities of PhaCR
cycle (Strauch et al. 2005). AbrB is repressed by and PhaCD in R. eutropha and Delftia acidovo-
the phosphorylated Spo0A form. The abrB and rans, respectively. The activation of PhaCA
spo0A double mutant showed early PHB synthe- through PhaP causes the change in substrate
sis. Additionally, the phaC mutant in B. thuringi- affinity toward 3-hydroxyhexanoyl-
ensis does not seem to affect the sporulation CoA. Additionally, the overexpression of phasin
efficiency (Chen et al. 2010). proteins resulted in increased PHA production by
7 Sporulation, a Pitfall in the Path of PHB Production 107

2.3-fold (Ushimaru et al. 2014). The various used to maximize the PHB content with negligible
knockout strains of phasin genes (phaP1, phaP2, sporulation (Narayanan and Ramana 2012).
phaP3, phaP4, or in combination) in R. eutropha
were studied for their PHB accumulation ability
and molecular weight. Phasins P2, P3, and P4 7.3.5 Phosphotransacetylases
have been found to be crucial for the stability of and Phosphotransbutyrylase
PHB granules, while phasin P1 was reported to
be important for the PHB degradation in R. eutro- Phosphotransacetylases (encoded by pta) cause
pha. However, in this study, no change in the the phosphorylation of acetyl-coenzyme A result-
PHB molecular weight has been detected (Kuchta ing in the formation of acetylphosphate. The
et al. 2007). In Bradyrhizobium japonicum metabolic intermediate, acetylphosphate, is
USDA110, the PHB granules are stabilized by shown to be an activator for the PHB synthase of
three PhaP paralogs, which also contribute to the R. eutropha in heterologous system, Escherichia
organisms growth. Phasin protein in B. japonicum coli (Miyake et al. 2000). On the other hand, pta-
exhibited high affinity for PHB granules and can deficient mutant of E. coli results in increased
displace PhaR previously bound to PHB (Yoshida acetyl-CoA concentration which acts as a sub-
et al. 2013). On the other hand, phasins P2 and P4 strate for the PHB synthesis, ultimately causing
inhibit the degradation mediated by PHB depoly- enhanced PHB accumulation in pta mutant as
merases (PhaZ2, Z3, and Z7) leading to higher compared to the wild type (Miyake et al. 2000).
PHB accumulations (Eggers and Steinbchel Another study by Vazquez and colleagues
2014). revealed the positive correlation of PHB with
phosphotransbutyrylase enzyme activity in
Bacillus megaterium. Ptb activity and PHB accu-
7.3.3 Catabolite Control Protein A mulation are measured throughout the growth
phases and correlation was plotted. Various acti-
Catabolite control protein A has been known to vators (isoleucine and valine) and inhibitor (glu-
play a role in various physiological processes, for cose) of this enzyme are shown to affect the PHB
example in biofilm formation and virulence of accumulation (Vazquez et al. 2003).
bacteria like Clostridium difficile, Enterococcus
faecalis, Staphylococcus epidermidis, and
Streptococcus pneumoniae (Sadykov et al. 2011; 7.3.6 Endogenous Ethanol
Antunes et al. 2012; Gao et al. 2013). CcpA inac-
tivation in Bacillus sp. MA3.3 causes reduction Catabolism of ethanol yields acetyl-CoA and
in glucose catabolite repression and reduced NADPH which inhibits the TCA cycle and thus
PHB production on hydrolases of lignocellulose. drives the enhanced acetyl-CoA into the PHB
CcpA activates the transcription of gltAB operon biosynthetic pathway. Recently, it has been
which is responsible for ammonium metabolism reported that exogenous as well as endogenous
(Lopes et al. 2011). ethanol acts as an efficient chain transfer agent
for PHB synthesis in E. coli. The endogenous
ethanol exerts a positive effect on the molecular
7.3.4 Glucose and Peptones weight of PHB. The deletion of adhE gene
(encoding alcohol dehydrogenase) in E. coli
Narayanan and Ramana have studied the effect of results in a drastic increase in PHB molecular
glucose and peptone on PHB production and spor- weight that is 77 % (Hiroe et al. 2013). Even in
ulation of Bacillus mycoides. Since sporulation Cupriavidus necator, ethanol accumulation is
always raises an issue for maximum PHB produc- known to increase the total PHB content (Obruca
tion, the central composite rotatable design was et al. 2010).
108 N. Dhasmana et al.

7.3.7 Molecular Chaperones and has been proposed to accumulate PHB,

recently. This strain could generate the maxi-
Interestingly, apart from all the above men- mum yield of 70 % cell dry weight on substra-
tioned factors, PHB synthesis is also governed tum of sodium succinate and glycerol as carbon
by the expression of chaperones like GroEL and sources. The optimum temperature for the
GroES. The heterologous expression of PHB growth of the strain was optimized as 45C (Liu
synthase from C. necator in E. coli with chaper- et al. 2014). Similar PHA yields were obtained
one co-expression results in higher fraction with a unique strain of B. thuringiensis. It
(six fold) of soluble PHB synthase. Reduced showed higher PHA production on crude glyc-
molecular weight of polymer was observed erol to the tune of 74 % (w/w) as PHA even with-
when PHB synthase is co-expressed with out the need of N-limitation.
chaperones GroEL and GroES (Thomson
et al. 2013).
7.5 Newly Investigated Bacillus
Strains for Higher PHB
7.4 Newly Identified Bacillus Accumulation
Species as Potent PHB
Producers 7.5.1 Bacillus megaterium R11

7.4.1 Bacillus licheniformis The Bacillus megaterium strain R11 was isolated
from Singapore and has the capacity to store
Recently, a Bacillus licheniformis MSBN12 has PHB as 51 % of cell dry weight on a substratum
been isolated from marine sponge, specifically of glucose and xylose. The maximum PHB yield
Callyspongia diffusa, and maximum PHB yield was standardized up to 59 % on tryptone as a
using this strain was standardized at 6.38 nitrogen source. Oil palm empty fruit bunch is a
g/L. Comparison among different substrates by-product of Malaysia palm oil refineries and is
reveals palm jiggery as the best carbon source for rich in cellulose and hemicellulose content
B. licheniformis MSBN12 (Sathiyanarayanan (Zhang et al. 2013).
et al. 2013a).

7.5.2 Bacillus megaterium BA-019

7.4.2 Bacillus subtilis
The PHB production in the B. megaterium
Another strain of Bacillus, specifically Bacillus BA-019 has been standardized to 45.84 % of cell
subtilis MSBN17, was isolated from marine dry weight, i.e., 1.73 g/L (Kanjanachumpol et al.
sponge Callyspongia diffusa, and the PHB pro- 2013). The substrates used for the PHB produc-
duction efficiency was checked on the substrate tion with BA-019 strain were sugarcane molasses
like pulp industry waste, tamarind kernel powder, (C source) and urea (N source); further, the C/N
palm jiggery, and green gram flour. Among the ratio was standardized to 25 for maximum cell
various substrates, maximum PHB production growth (72 g/L) and PHB (1.27 g/L/h) produc-
has been standardized for pulp industry waste tion (Kulpreecha et al. 2009).
(PIW), which is 19.08 g/L (Sathiyanarayanan
et al. 2013b).
7.5.3 Bacillus megaterium H15, H16,
and H26
7.4.3 Bacillus shackletonii
Several halophilic bacterial strains were iso-
Another Gram-positive bacterium, B. shackleto- lated from solar salterns from Indian western
nii K5 has been isolated from biotrickling filter coast, specifically Goa. Three strains of B.
7 Sporulation, a Pitfall in the Path of PHB Production 109

megaterium H15, H16, and H26 were identi- 7.6 Conclusion

fied and further characterized phenotypically
and genotypically. The presence of NaCl in E2 The regulation of PHB synthesis converges from
mineral media induced extended lag phase (up multifaceted pathways in different systems like
to 24 h) but no significant difference in PHB Bacillus, Ralstonia, etc. Various endogenous as
production in the case of B. megaterium H16 well as exogenous factors are involved in govern-
strain. This strain is industrially more suitable ing the final yield of PHB. The relevance of
for its higher tolerance to salt stress (Salgaonkar Gram-positive organisms in PHB production
et al. 2013). becomes more significant due to the absence of
immunogenic LPS in purified PHB fraction. The
PHB thus purified can be applied for a large num-
7.5.4 Bacillus sphaericus NCIM 5149 ber of medical uses like skin grafts, liver grafts,
etc. The extensive research is going on for stan-
A strain of Bacillus sphaericus, specifically dardizing conditions for the maximum PHB pro-
NCIM 5149, has been reported for the first time duction from Bacillus sp. Small changes in the
to produce PHB under submerged fermentation. substratum of specifically inexpensive by-
PHB production was maximized to 49 % (2.2 products of several industries like corn steep
g/L) of cell dry weight using a central composite liquor, sugarcane molasses, etc., cause drastic
design considering three parameters, i.e., pH (6), reduction in the PHB production cost. The
inoculum age (18 h), and jackfruit hydrolysate above mentioned text specifies the newly discov-
concentration (2.5 % reducing sugar) (Ramadas ered PHB producers from Bacillus species as
et al. 2010). well as different changes in the fed-batch fermen-
tor conditions to maximize the PHB production.

7.5.5 Bacillus spp. CFR-67 Acknowledgment The authors wish to thank the Director
and CFR-256 of CSIR-Institute of Genomics and Integrative Biology
(IGIB), Government of India, for providing the necessary
funds and facilities (LSRB-268/BTB/2013 and BSC0123).
Bacillus sp. CFR 67, a well-known species for Authors are also thankful to the Academy of Scientific
the production of PHB as well as amylase, was and Innovative Research (AcSIR), New Delhi. ND is
further researched to maximize the PHB produc- Shyama Prasad Mukherjee-Senior Research Fellow sup-
ported by CSIR, India. LKS and SSK are Senior Research
tion through the addition of wheat bran hydroly-
Fellows supported by University Grant Commission,
sates, rice bran, or both. The most interesting fact India. NK is Junior Research Fellow. We highly acknowl-
is that the introduction of wheat bran or rice bran edge Dr. V. C. Kalia from CSIR-IGIB, Delhi, India, for
with ammonium acetate and corn starch results in the inspiration and critical comments in the manuscript.
significant increased polyhydroxybutyrate-
co-hydroxyvalerate copolymer production from
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112 N. Dhasmana et al.

Neha Dhasmana gradu- Nishant Kumar gradu-

ated in Microbiology in ated in Biotechnology in
2008 from University of 2009 from HNB Garhwal
Delhi. She received her M. University. He received his
Sc. degree in Biochemistry M.Sc. degree in
in 2010 from Hamdard Biosciences in 2012 from
University. She is currently Jamia Millia Islamia
enrolled in Ph.D. with University. He then com-
Academy of Innovative and pleted M.Phil. from
Scientific Research (AcSIR) Zoology Department of
and works at CSIR-Institute University of Delhi in
of Genomics and Innovative 2013. He is currently
Biology. She is studying the physiological significance of enrolled in Ph.D. with
Ser/Thr kinases in Bacillus anthracis. Academy of Innovative and Scientific Research (AcSIR)
and works at CSIR-Institute of Genomics and Innovative
Lalit K. Singh received Biology. He is studying the role of Ser/Thr kinases and
the M.Sc. degree in phosphodiesterases in Bacillus anthracis.
Biotechnology from Rajiv
Gandhi Biotechnology Yogendra Singh
Centre, RTM Nagpur received his Ph.D. degree
University, Nagpur, India. from Vallabhbhai Patel
He is currently working as Chest Institute, Delhi
Senior Research Fellow, University, and postdoc-
CSIR-IGIB, Delhi, India. toral training at National
His current research Institute of Health (NIH),
focuses on understanding Bethesda. Currently, he is
the role of caseinolytic proteases and Ser/Thr kinases that Chief Scientist at CSIR-
mediated cellular architecture alterations, cell wall remod- IGIB and Professor of
eling, and sporulation in Bacillus. AcSIR. He is a Fellow of
National Academy of
Sciences, India, Fellow of
Shashank S. Kamble Indian National Science Academy, and Fellow of Indian
received his M.Sc. degree Academy of Sciences, India. He has been honored with
from University of Moselio Schaechter Distinguished Service Award in 2014
Mumbai, India. Currently, by American Society for Microbiology. He serves as an
he is pursuing Ph.D. under Editorial Board Member of Journal of Biological
the supervision of Dr. Chemistry. His current research interest is to understand
Yogendra Singh at CSIR- the host response in pulmonary/extra-pulmonary tubercu-
IGIB, India. He is inter- losis and to decipher the role of eukaryotic like Ser/Thr
ested in the host pathogen kinases in Mycobacterium tuberculosis and Bacillus
cross talk during infection anthracis.
with M. tuberculosis. His
work focuses on the modu-
lation of host proteases by M. tuberculosis.
Microbial Biopolymers:
The Exopolysaccharides 8
Angelina and S.V.N. Vijayendra

Microorganisms produce several biopolymers. Of these, intracellularly
produced polyhydroxyalkanoates (PHAs) and extracellularly produced
exopolysaccharides (EPS) are gaining importance over the other biopoly-
mers. These naturally produced polymers can replace plant-based or petro-
leum-derived polymers. There are innumerable reports and reviews on the
production of PHA and EPS by several bacteria, fungi, actinomycetes, and
algae. This chapter briefly gives an introduction to PHA and provides
recent developments in the genetic and metabolic pathways for the synthe-
sis of microbial EPS. Different strategies used for fermentative production
and various means of downstream processing are discussed. Possible ways
to minimize the cost of production and downstream processing are covered
in this chapter. Applications of these EPS in various fields such as agricul-
ture, cosmetics, foods, medical and healthcare industry, mining, oil recov-
ery, packaging, pharmaceuticals, printing and textile industry, wastewater
treatment, etc., are presented. The potential of these polymers indicates that
these microbial cell factories can be exploited for the better of mankind.

8.1 Introduction

Biopolymers are natural polymers derived from

living organisms during their growth or from
renewable resources in the form of polysaccha-
rides and polyhydroxyalkanoates (PHAs) also
Angelina S.V.N. Vijayendra (*) known as bioplastics. Being biodegradable, these
Microbiology and Fermentation Technology, are studied for different properties from several
CSIR-Central Food Technological Research Institute, microbial sources for its various applications that
570020 Mysore, India
are of high value (Sutherland 1994). Micro-
e-mail: ange.csk@gmail.com;
svnvijayendra@cftri.res.in; organisms synthesize these biopolymers as intra-
svnvijayendra@yahoo.com cellular, structural, and extracellular polymers for

Springer India 2015 113

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_8
114 Angelina and S.V.N. Vijayendra

their function and survival that play specific roles plastic; (2) mcl-PHAs (medium chain length)
such as energy reserve materials, protective containing monomers of 614 carbon atoms, e.g.,
agents, cell functioning, symbiosis, and osmotic 3-hydroxydodecanoate (3HDD),
adaptation and support the microbes to function, 3-hydroxyoctanoate (3HO), 3-hydroxyhexanoate
adapt, multiply, and survive efficiently under (3HHx), and 3-hydroxydecanoate (3HD), have
changing environmental conditions (Vijayendra low crystallinity and tensile strength and glass
and Shamala 2014). This chapter gives an intro- transition temperatures and lower melting points
duction to PHA and mainly focuses on recent in comparison to scl-PHAs (Akaraonye et al.
developments in the area of microbial exopoly- 2010); and (3) lcl-PHAs (long chain length) con-
saccharides (EPS). taining monomers of 15 and above carbon atoms
(Luengo et al. 2003) are more ductile and easier
to mold (Kabilan et al. 2012). The scl-PHA copo-
8.1.1 Polyhydroxyalkanoates lymer produced by Rhizobium meliloti was char-
acterized (Shamala et al. 2009). The various
Polyhydroxyalkanoates (PHAs) are bioplastics monomer composition of PHAs results in a wide
produced from microorganisms that have gained range of thermoplastic to elastomeric properties,
attention as an alternative to petroleum-based viz., molecular weight, density, melting point,
plastics due to their similar properties and being crystallinity, resistance to UV and solvents, water
biodegradable and biocompatible (environment and O2 permeability, glass transition temperature,
friendly) are produced from renewable and waste and tensile strength and elongation to break (Du
resources. PHAs are accumulated as intracellular et al. 2012). Identification of PHA-producing
granules by a wide variety of microorganisms in Bacillus spp. by molecular methods was stan-
the presence of an abundant carbon source and dardized (Shamala et al. 2003). Reports are also
limited essential nutrients such as nitrogen, phos- available for PHA production by yeasts (Kocharin
phorous, or oxygen and serve as reserve carbon and Nielsen 2013; Gumel et al. 2013), algae such
and energy sources; also they do not substantially as cyanobacteria (Balaji et al. 2013), and geneti-
alter the cells osmotic state (Laycock et al. 2013). cally modified diatoms (Hempel et al. 2011). The
PHAs are polyoxoesters that are naturally use of agro-industrial waste products such as
synthesized as polymers of R-3-hydroxyalkanoic corn steep liquor, mahua flower extracts, starch,
acids and constitutes of 150 different monomers wheat, or rice bran can reduce the cost of PHA
(Steinbchel 2005). PHB (polyhydroxybutyrate) polymer (Vijayendra et al. 2007; Anil kumar
is the most common of the PHAs produced, and et al. 2007; Halami 2008; Saranya devi et al.
it was the first PHA discovered in Bacillus mega- 2012). Simultaneous production of either PHA
terium by Lemoigne in 1927. Copolymers of and amylase or PHA and EPS by Bacillus sp. and
PHB like poly(3-hydroxybutyrate-co-3-hy- Sinorhizobium sp., respectively (Saranya devi
droxyvalerate), P(3HB-co-3HV), can be synthe- et al. 2012; Shamala et al. 2012; Sreekanth et al.
sized by substituting the side chain with 2013), or PHA and -carotene by Micrococcus
functional groups, viz., hydroxyl, carboxylic, sp. was reported (Vijayendra et al. 2008a).
epoxy, phenoxy groups, halogens, etc., based on Production of multiple biopolymers using fed
the carbon sources, thereby affecting its thermal batch reactor by employing a high cell density
and mechanical properties (Zinn and Hany 2005). culture of Sinorhizobium meliloti was reported
PHAs are classified on the basis of side chain very recently (Shamala et al. 2014).
length of monomers: (1) scl-PHAs (short chain
length) containing monomers of 45 carbon
atoms, such as P(3HB), are crystalline, brittle, 8.1.2 Microbial Exopolysaccharides
and stiff, having high melting and low glass tran-
sition temperatures, whereas the copolymer Microbial exopolysaccharides (EPS) are a kind
P(3HB-co-3HV) is a strong and pliable thermo- of biopolymers synthesized by several micro-
8 Microbial Biopolymers: The Exopolysaccharides 115

organisms which include several genera of bacte- Table 8.1 Important microorganisms producing
ria, molds, and yeasts. These are gumlike
polymers synthesized by these organisms and Microorganisms Exopolysaccharides
released into the surrounding environment. EPS Acetobacter xylinum Acetan/cellulose
mainly protects the microorganisms from the sur- Agrobacterium tumefaciens Succinoglucan
rounding environment and also acts as a reserve Alcaligenes faecalis Curdlan
food material, and the producing organisms can Alcaligenes Welan
faecalis/Sphingomonas sp.
use this as a main carbon source. Based on the
Azotobacter vinelandii Alginate
sugar composition, EPS are classified as homo-
Aureobasidium pullulans Pullulan
polymers (with a single type of sugar, glucose,
Epicoccum nigrum Epiglucan
xylose, etc.) and heteropolymers (with more than
Grifola frondosa Grifolan
one type of sugar moieties, glucose, rhamnose,
Lactobacillus frumenti Fructan
mannose, etc.). Based on the presence or absence
Lactobacillus reuteri Mutan/reuteran
of uronic acid, EPS are categorized as acidic or
Lactobacillus sanfranciscensis Levan
neutral EPS, respectively. EPS have several phys- Lactic acid bacteria and yeasts Kefiran
ical and chemical properties. Some of these EPS Pseudomonas Lentinan
can form a film, some form gels, some more can aeruginosa/Lentinula edodes
increase the viscosity of solutions, etc. Besides, Leuconostoc mesenteroides Dextran
these EPS have several functional attributes; Pestalotia Pestolotan
hence, these are being used in various foods and Saccharomyces sp. Yeast glucan
pharmaceutical, medical, and other industrial Saccharomyces cerevisiae Zymosan
applications. Microbial EPS are an alternate to Schizophyllum commune Schizophyllan
plant-based or seaweed-based water-soluble Sclerotium glucanicum Scleroglucan
polymers, whose quality and production quanti- Sphingomonas elodea Gellan
ties depend on several environmental factors, Trametes versicolor Krestin
whereas the production of microbial EPS even in Weissella confusa Inulin and fructan
large-scale fermentors can be controlled accu- Xanthomonas campestris Xanthan
rately and uniformity in the quality can also be Zymomonas mobilis Levan
achieved. However, even today, the cost of micro-
bial EPS is higher than plant-based polymers.
Hence, all over the world many researchers are ages. It produces soft and hard gels when heated
exploring various ways to reduce the production at 60 and 80 C, respectively. Several lactic acid
cost by using various cheap and alternate sub- bacteria (LAB) are known to produce EPS, espe-
strates and optimizing its production and recov- cially Leuconostoc spp. and Lactobacillus spp.
ery and its applications, thus becoming a topic of (Vijayendra et al. 2008b, 2009; Badel et al. 2011;
many recent reviews (Nicolaus et al. 2010; Freitas Yadav et al. 2011). Aureobasidium pullulans, a
et al. 2011; Palaniraj and Jayaraman 2011; bimorphic fungi, produces an EPS known as pul-
Seviour et al. 2011; Donot et al. 2012; Patel et al. lulan. It is an extracellular, linear, unbranched,
2012; Singha 2012; Benny et al. 2014; Dhiya water-soluble EPS. It has maltotriose-repeating
et al. 2014; Divyasri et al. 2014; Kaur et al. 2014; units linked through -1,6-glucosidic bonds. Its
Prajapati et al. 2013; Finore et al. 2014). molecular weight varies from 4.5 104 to 6 105
Da. Sphingomonas paucimobilis, formerly EPS-Producing Microorganisms known as Pseudomonas elodea, is known to pro-
Several microorganisms produce EPS. Table 8.1 duce gellan, a gelling EPS. It can be used as a
depicts important EPS-producing microorgan- gelling and thickening agent or as a solidifying
isms. Agrobacterium sp. produces curdlan, a neu- agent in tissue cultures. It is an acidic polysac-
tral water-insoluble and alkali-soluble charide consisting of glucose, rhamnose, and
EPS. Curdlan has linear -(1,3)-glycosidic link- glucuronic acid in 3:1:1 ratio. Sinorhizobium
116 Angelina and S.V.N. Vijayendra

meliloti produces multiple biopolymers (EPS, market in view, it is considered to be economical

cellular polysaccharides, and polyhydroxyal- if the rapid growth and higher survival of the pro-
kanoates) simultaneously (Saranya Devi et al. ducing organisms are addressed (Wolfaardt et al.
2012; Shamala et al. 2014). Several cyanobacte- 1999).
ria like Anabaena, Nostoc, Spirulina, Palmella Biosynthesis of microbial EPS takes place
mucosa, etc., are also known to produce EPS using nucleotide-activated precursors. All the
(Chakraborty and Pal 2014), some of which are enzymes responsible for biosynthesis, assembly,
homopolysaccharides and some other are hetero- and transport of EPS are derived from specific
polysaccharides. Several Saccharomyces spp. gene clusters. The mechanism of biosynthesis
produce an EPS popularly known as yeast glu- varies from EPS to EPS. Extracellular glycosyl-
can. Finore et al. (2014) in their review on fer- transferases help in the polymerization of homo-
mentative production of EPS by marine bacteria polysaccharides by increasing the chain length of
indicated several bacteria, such as Aeribacillus the molecule, whereas in heteropolysaccharides
pallidus, Alteromonas sp., Halomonas alkaliant- polymerization takes place after the repeating
arctica, Pseudoalteromonas sp., Salipiger muco- units that are formed in the cytoplasm are trans-
sus, Zunongwangia profunda, etc., as EPS ferred outside the cell through a lipophilic carrier
producers. Very recently, it is reported that (Finore et al. 2014); hence, molecular weight of
Bacillus subtilis produces an EPS that has anti- the extruded polymer increases at the cell sur-
oxidant activity (Razack et al. 2014). An exhaus- face. Biosynthesis and characterization of EPS
tive review on EPS-producing fungi including from lactic acid bacteria (LAB) had been
lower filamentous fungi, higher basidiomycetes, reviewed in detail (Laws et al. 2001), and a com-
and yeasts present in different ecological niches prehension of the glycosyltransferase gene map
at laboratory scale has been made available can help in predicting the structure of their EPS
(Mahapatra and Banerjee 2013). Weissella and its repeating unit (Remminghorst and Rehm
cibaria and W. confusa produce fructan and inu- 2009). For the optimization of the EPS biosyn-
lin (Malang et al. 2015). Antarctic bacterium thesis by following a system biology approach,
Pseudoalteromonas sp. S-5 producing an EPS, knowledge of the genome structure is essential as
having anticancer activity, was reported recently EPS production in microorganisms is controlled
(Chen et al. 2015). by a specific gene cluster (Ate et al. 2013). The
biosynthetic pathway and mode of export of EPS Genetics and Metabolic in alginate production (Rehm and Valla 1997)
Pathway of EPS Synthesis and cellulose production by Acetobacter (Chawla
A majority of the microbial EPS are produced et al. 2009) and Azotobacter spp. (Gauri et al.
intracellularly and sent to the outside of the cell 2012), Rhizobium spp. (Skorupska et al. 2006),
(Rehm 2009; Ullrich 2009). However, a few of EPS from lactobacilli spp. (Badel et al. 2011;
the EPS like levan and dextran are synthesized Patten and Laws 2014), and in other microbes
and polymerized outside the producing bacterial (Madhuri and Vidya Prabhakar 2014) were
cells through the action of secreted enzymes reviewed thoroughly. Metabolic pathway of cur-
(Rehm 2009). The basics of the biosynthesis of dlan synthesis is explained in detail earlier (Donot
microbial EPS have been reviewed by Freitas et al. 2012). The biosynthesis pathway of curdlan
et al. (2011). Biosynthesis of EPS takes place in production by Agrobacterium was reviewed
three steps. These are assimilation of carbon recently (Zhan et al. 2012). The EPS biosynthesis
source, intracellular synthesis of polysaccharide, pathway exclusively in different species of bifi-
and release of the polysaccharide to the outside dobacteria was reviewed recently (Hidalgo-
environment (Vandamme et al. 2002). The EPS Cantabrana et al. 2014).
production process is known to be costlier as it Biosynthetic pathway of welan gum produc-
requires large volumes of the carbon source. tion was proposed to have two discrete systems
However, keeping the cost of the EPS in todays for the metabolism of glucose (Li et al. 2010a).
8 Microbial Biopolymers: The Exopolysaccharides 117

In the first system, glucose is converted to are also in practice for the production of micro-
glucose-1-phosphate and later to G-6-Pe and bial EPS.
from there to fructose-6-phosphate and nucleo- Various aspects of pullulan production are
tide sugar precursors. In the second system, the reviewed in the past (Seviour et al. 1992; Leathers
cell skeleton is synthesized from glucose using 2003; Singh et al. 2008; Gaur et al. 2010; Cheng
the pathways like the TCA cycle, Entner- et al. 2011; Singh and Saini 2012; Yatmaz and
Doudoroff pathway, and the PPP. A little later, Turhan 2012). Besides commonly used sugars
Wang et al. (2012) analyzed 1.4 Mb genome like sucrose, maltose, glucose, and lactose, the
sequence involved in welan production and anno- use of the jaggery, a dry concentrate of sugarcane
tated 55 coding sequences (CDSs) related to its juice, as a cheap source of carbon, for pullulan
monosaccharide metabolism and 10 CDSs production, is reported (Vijayendra et al. 2001).
responsible for its biosynthesis. Improvement in Using this carbon source at 5 % level, they could
EPS production by overexpression of NADH obtain 23 g/l of pullulan in 72 h. The other
oxidase gene in L. casei resulting in 46 % more cheaper substrates used for pullulan production
EPS was attempted (Li et al. 2015). They have such as coconut water, cashew fruit juice, fuel
overexpressed a H2O-forming NADH oxidase ethanol by-products, grape skin and pulp extract,
gene in L. casei LC2W by cloning it from hydrolyzed potato starch, molasses, peat hydro-
Streptococcus mutans under the control of consti- lysate, olive oil/wastes, carob pod/extract, hydro-
tutive promoter P23; as a result, they could notice lysates of inulin and cornmeal, corn syrup,
a 20-fold increase in the gene expression levels fermentation stillage and sucrose, beet molasses,
over the wild strain and a reduction of 22 % in spent sulfite liquor, spent grain liquor, etc., are
lactate production. reviewed elsewhere (Vijayendra and Shamala
2014). Genome shuffling through protoplast Fermentative Production of EPS fusion increased the productivity (179.7 %) of
Several physical and chemical factors affect the pullulan over the wild strain of A. pullulans
production of EPS by microorganisms. Aeration, N3387 (Kang et al. 2011). Shivakumar and
agitation, fermentation period, rate of inoculum, Vijayendra (2006) for the first time used coconut
and temperature are the important physical water as a carbon source for producing curdlan.
parameters, which can influence the production Recent developments in the fermentative pro-
of EPS. Among the chemical factors, composi- duction of microbial EPS like fermentation con-
tion of the medium, source and concentration of ditions, mode of fermentation, and optimization
carbon (Rajkumar et al. 2003; Vijayendra et al. methods of fermentative production of EPS like
2003) and nitrogen, trace elements, pH, and dis- kefiran, bacterial cellulose, levan, and gellan and
solved oxygen are considered important that can EPS of Haloferax have been reviewed recently
affect the EPS production. All over the world, (Vijayendra and Shamala 2014) and, hence, not
cheaper and alternate carbon and nitrogen sources mentioned here once again. In a fed batch fer-
are being used to reduce the production cost of mentation by Sphingomonas paucimobilis ATCC
the EPS. The use of high-yielding strains can 31461, 17.71 g/l higher gellan gum production
increase the yield of EPS. Various strategies and 57.12 % higher conversion efficiency were
involved in large-scale production of microbial achieved using the Logistic and Luedeking-Piret
EPS such as type of fermentor, bioengineering models (Wang et al. 2006). Prior to this, the use
and microbiological challenges in monitoring, of fed batch fermentation for xanthan production
and controlling conditions have been critically by Xanthomonas campestris was reported
reviewed recently (Seviour et al. 2011). To (Shamala and Prasad 2001).
increase the yield of EPS, various statistical Acetic acid stress after 24 and 26 h of fermen-
methods are being used to optimize the nutri- tation increased the yield of xanthan production
tional and physical parameters. Besides these, by Xanthomonas campestris (Shehni et al. 2011).
batch, fed batch, and continuous fermentations An increase in the yield was dependent on
118 Angelina and S.V.N. Vijayendra

increased concentration and the number of pulses degrade the polymer at later stages of recovery. A
of acetic acid addition. Later, to reduce the pro- number of chemical methods, physical methods,
duction cost, Costa et al. (2014) have used aque- and their combinations are used for the recovery
ous shrimp shell extract as a carbon and nitrogen of EPS from either broth or sludges (Sheng et al.
source for xanthan production, and the yields 2010). Comparatively, chemical methods are
were higher than that of control medium prepared more efficient than physical methods. The physi-
with sucrose. cal methods of extraction include ultrasonic, cen-
Using a statistical design (Plackett-Burman), trifugation, microwave treatment, or heating
higher yield of welan gum (22.85 g/l) was (Donot et al. 2012). DomInguez et al. (2010)
obtained with optimal medium combinations and have indicated that the cationic exchange resin
fermentation conditions such as cornstarch (43.6 method resulted in 1.6 times more yield than
g/l), cottonseed cake flour (4.1 g/l), 3 % inocu- thermal treatment. Extraction method for each
lum, initial pH 7.0, and temperature 30 C (Li EPS must be optimized individually as the prop-
et al. 2010b). Similarly, very recently, Moshaf erties of the EPS vary with each polymer. If EPS
et al. (2014) have used a second-grade date palm of higher purity is required, chemical deprotein-
for the production of xanthan and optimized its ization with trichloroacetic acid (Ayala-
production by following statistical methods like Hernndez et al. 2008), treatment with enzymes
response surface methodology (RSM) combined like proteases (Wang et al. 2007), or membrane
with a central composite design (CCD). filtration either with ultrafiltration or diafiltration
Excepting dextran, which is being produced at (Kumar et al. 2007; Bahl et al. 2010) can be used.
the commercial level, other EPS of LAB could Optimization of downstream processing can
not be commercialized due to their low yields (<1 also reduce the production cost of EPS. However,
g/L) (Badel et al. 2011). However, production of in the case of highly viscous EPS like xanthan,
18 g/L of heteropolysaccharide by a Leuconostoc the recovery costs account for over 70 %
sp. CFR 2181 using a cheap semisynthetic (Torrestiana-Sanchez et al. 2007). Recent devel-
medium in a short period of 4 h of fermentation opments in the recovery of biopolymers includ-
at 25 C was reported earlier (Vijayendra and ing microbial EPS were reviewed (Kreyenschulte
Sharat Babu 2008). Galle and Arendt (2014) et al. 2014). Before solvent precipitation, heat
recently published an exhaustive review on fer- treatment at 80 C for 30 min can precipitate
mentative production of EPS by LAB, exclu- most of the thermosensitive proteins present in
sively isolated from sourdough, like Leuconostoc the pullulan fermentation broth without affecting
mesenteroides, Lactobacillus sanfranciscensis, the recovery of pullulan, and pullulan is recov-
Lb. rossiae, Lb. brevis, Lb. spicheri, Lb. pontis, ered using cold isopropanol and subsequent dry-
Lb. frumenti, Lb. reuteri, Weissella confusa, etc. ing at 60 C for 40 min. (Mishra and Vuppu
Method of levan production and various strate- 2013). Using response surface methodology,
gies being used for its production all over the enhanced recovery of pullulan was noticed with a
world have been extensively reviewed recently combination of solvents of ethanol, acetone, iso-
(Srikanth et al. 2015). propanol, and tetrahydrofuran as compared to
ethanol alone (Choudhury et al. 2013). Downstream Processing of EPS Even for the recovery of xanthan, the most
Downstream processing consists of inactivation common strategy is precipitation using water-
and removal of microbial cells from the fer- miscible non-solvents like acetone, ethanol, or
mented broth and recovery of the EPS from the isopropyl alcohol (Garca-Ochoa et al. 2000;
broth by precipitation and subsequent drying Salah et al. 2011), or in some other cases the use
using a drum drier. Inactivation of the cells is of polyvalent cations such as aluminum, calcium,
generally carried out by heating at pasteurization or quaternary ammonium salts is also reported
temperatures (Garca-Ochoa et al. 2000). This (Pace and Righelato 1980; Palaniraj and
heating also inactivates the enzymes which may Jayaraman 2011). However, the use of electrolytes
8 Microbial Biopolymers: The Exopolysaccharides 119

such as potassium or sodium chloride can reduce the cost of the recovery process. Downstream pro-
the volume of the alcohol or isopropyl alcohol cessing of scleroglucan can be done in three dif-
from 3 to 1.4 times (Galindo and Albiter 1996; ferent ways. After, initial heating of the broth at
Garca-Ochoa et al. 2000). Thermal treatment at 80 C for 30 min and subsequent centrifugation
80 C for 20 min at a pH of 6.36.9 enhances the are common to all the three methods. Later, the
recovery of xanthan, besides reducing the viscos- polymer is precipitated either by using the solvent
ity of the polymer, which eases the separation of alone or addition of calcium chloride and by sol-
insoluble material by filtration or by centrifuga- vent extraction or addition of calcium chloride
tion (Smith and Pace 1982). and then adjusting the pH of the broth to 1012
Agrobacterium sp. produced both water- using alkali solution (Survase et al. 2007).
soluble and water-insoluble EPS, simultaneously.
However, the water-insoluble EPS is soluble in Applications of EPS
alkaline solution. Separation of both these EPS The applications of the microbial EPS have been
from the fermented broth is a tedious task. A extensively reviewed recently (Rehm and Valla
detailed method of separating these EPS was 1997; Giavasis et al. 2000; Bajaj et al. 2007;
reported elsewhere (Shivakumar and Vijayendra Survase et al. 2007; Chawla et al. 2009; Freitas
2006). Recently, the downstream processing of et al. 2011; Poli et al. 2011; Freitas et al. 2014;
curdlan was optimized by changing the quantities Gauri et al. 2012; Singh and Saini 2012; Zhan
of NaOH and HCl (Kalyanasundaram et al. 2012). et al. 2012; Kaur et al. 2014; Madhuri and Vidya
By optimization they could reduce the ratio of Prabhakar 2014; Mahapatra and Banerjee 2013;
sample to alkali from 1:7.5 to 1:1, thus reducing Vijayendra and Shamala 2014). Table 8.2 sum-

Table 8.2 Application of some important microbial exopolysaccharides

Exopolysaccharide Areas of application
Acetan/cellulose Antimicrobial packaging films, wound dressings, as a parchment paper
Alginate Ceramics, drug delivery, foods, pharmaceuticals, paper industry, textile printing, welding
Alternan Bulking agent in foods, cosmetics, probiotics
Curdlan Drug delivery, foods, as a prebiotic, water treatment
Dextran Plasma extender, drug delivery, chromatography, foods, pharmaceuticals, paper industry
Fructan Foods
Gellan Capsules, controlled drug release, foods, gelling agent in dental and personal care items,
microencapsulating agent, tissue culture media, wrapping of fruits
Inulin Substitute for fat in foods, probiotics
Kefiran Active food packaging film, gelatination
Levan Biotech industry, beverages, food preservation, medicine, wound healing, nanoparticles
of levan in protein/drug delivery
Pullulan Oxygen barrier films for food and nonfood applications, cosmetics, capsules for drug
delivery, foods, printing, textile, photography, plywood, dental care, pharmaceuticals,
plasma extender
Reuteran Foods (bakery)
Scleroglucan Agriculture, ceramics, cosmetics, food, as a medicine, oil industry, ophthalmic solutions,
tablet coating, paints, printing inks
Succinoglucan Gelling agent and immobilizing agent
Welan Cement manufacturing, cosmetics, foods, metal working compounds, oil recovery,
pharmaceuticals, sealants
Xanthan Agrichemical sprays, explosives, foods, oil drilling, ore extraction, pesticide and
insecticide, printing, paints, water clarification
Yeast glucan Foods, oil sparing agent
120 Angelina and S.V.N. Vijayendra

marizes major applications of some important ity, porosity, and efficient barrier properties.
EPS. Due to its varied functional properties, Because of its stability at higher temperatures
microbial EPS found applications in various (150 C) and varied pH (212), welan can be
fields such as agriculture, cosmetics, food, oil used in several areas like food, medicine, as an
recovery, packaging, textile, wastewater treat- additive in concrete, as a coating material, and
ment, pharmaceuticals, medicine, and in the form for enhancing the recovery of oil (Kang et al.
of membranes. Fungal EPS also have found to 1983). The EPS production by Azotobacter in
have many applications in food, cosmetics, phar- soil helps in the soil fertility by controlling the
macy, medicine, feed, and other areas (Mahapatra ecosystem through nutrient cycling and in main-
and Banerjee 2013). Very recently, the applica- taining the soil structure in different environ-
tion of microbial polymers exclusively for pack- ments (Gauri et al. 2012).
aging food and nonfood items has been reviewed Several applications of levan such as food,
(Vijayendra and Shamala 2014). Various applica- medicine, beverage, nanotechnology, biotechnol-
tions of several EPS of LAB such as dextran, ogy, purification of proteins, and other areas have
alternan, reuteran, levan, kefiran, inulin, etc., been recently reviewed (Srikanth et al. 2015).
have been reviewed (Patel et al. 2012). Several Derivatization increased functionality of levan in
health benefits like heavy metal binding, antitu- terms of increased reducing power, antiprolife-
mor activity, anti-atherosclerotic effect, immuno- rative activity, scavenging activity, antioxidant,
modulation activity, and prebiotic effect of LAB and anticancer activity (Liu et al. 2012). At 0.1 to
and other microbial EPS are reviewed recently 1.0 % concentrations, levan is an excellent immu-
(Patel and Prajapati 2013; Madhuri and Vidya nostimulant in fishes (Gupta et al. 2011).
Prabhakar 2014; Patten and Laws 2014). An Fructooligosaccharides derived from acid hydro-
exclusive review on the use of gellan in medicine lysis of levan are considered as prebiotic agents
in the form of oral, ophthalmic, and nasal formu- (Huang et al. 2013). As reviewed by Srikanth
lations, tissue engineering, and dressing material et al. (2015), levan has several medical applica-
is made available recently (Osmaek et al. 2014). tions such as an anticlotting factor in heart sur-
Benny et al. (2014) have reviewed the applica- gery, healing wounds, after angioplasty
tions of xanthan gum in drug delivery, due to its anti-AIDS agent, and in the subcutaneous dental
potential in retarding the drug release, in the form filling. Levan is an excellent stabilizer, emulsi-
of liposomes, hydrogel, niosomes, nanoparticles, fier, and flavor enhancer and, hence, used in dairy
matrix system, or microspheres. In foods micro- beverages (Srikanth et al. 2015).
bial EPS can be used as emulsifiers, gelling
agents, thickeners, stabilizers, and viscosifying
agents to improve the texture and stability, and 8.2 Future Perspectives
their properties can be further strengthened by
cross-linking, either by physical or chemical Microbial biopolymers are in great demand.
method, to prepare modified EPS (Ahmad et al. However, the current production prices are inhib-
2015). iting its practical use in many areas. Future
Recently, phosphorylated curdlan microgels research focus can be made on improving the fer-
for in vitro drug release were prepared, and these mentation strategies and use of alternative raw
microgels had excellent biocompatibility materials, mainly by-products of agro-industrial
(Popescua et al. 2013). Prior to this, Chawla et al. sources. Optimization of simultaneous synthesis
(2009) have reviewed the production and appli- of multiple polymers can also reduce the produc-
cations of microbial celluloses in food, medical, tion cost, and more research could be focused on
pharmaceutical, mining, broadcasting, textile, this aspect.
refinery, waste treatment, etc. Bacterial cellulose
is considered to be a best alternative to wound Acknowledgments The authors wish to thank the
dressing material due to its water-holding capac- Director of CSIR-CFTRI, for providing necessary funds
8 Microbial Biopolymers: The Exopolysaccharides 121

and facilities. Angelina is thankful to UGC for granting Chen G, Qiana W, Lia J, Xua Y, Chena K (2015)
Maulana Azad National Fellowship. Exopolysaccharide of Antarctic bacterium
Pseudoaltermonas sp. S-5 induces apoptosis in K562
cells. Carbohydr Polym 121:107114. doi:10.1016/j.
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tion. Biotechnol Bioinform Bioeng 1:415421 AcSIR. He is a life member
Yatmaz E, Turhan I (2012) Pullulan production by fer- of several scientific associa-
mentation and usage in food industry. GIDA J Food tions. He is the former Editor of the Journal Food Science
37:95102 and Technology (20052013) and currently Editor of
Zhan XB, Lin CC, Zhang HT (2012) Recent advances in Indian Food Industry. His current interests are microbial
curdlan biosynthesis, biotechnological production, biopolymers and fermented foods.
and applications. Appl Microbiol Biotechnol 93:525
531. doi:10.1007/s00253-011-3740-2
Zinn M, Hany R (2005) Tailored material properties of
polyhydroxyalkanoates through biosynthesis and
chemical modification. Adv Eng Mater 7:408411.

Angelina received her

M.Sc. degree in
Microbiology from
University of Mysore,
Mysore, India. She is cur-
rently enrolled for Ph.D.
with Academy of
Innovative and Scientific
Research (AcSIR), New
Delhi, India, and works as
Senior Research Fellow at
CSIR-Central Food
Technological Research
Institute, Mysore, India. Her current research work
focuses on the production of polyhydroxyalkanoates (bio-
polymer) through high cell density fermentation.
Innovations in Microalgal
Harvesting Using Biopolymer- 9
Based Approach

Chiranjib Banerjee, Rajib Bandopadhyay,

Puneet Kumar Singh, Harsh Kumar Agrawal,
and Pratyoosh Shukla

Green unicellular microalgae increase their biomass content by the capa-
bility of entrapping CO2 for photosynthesis and are crucial for important
value product. Negative zeta value is imparted the presence of COOH and
NH2 groups. This review will give a detailing toward the forces that are
responsible for making alga stable in a solution phase. Beside this, it also
explains the various possibilities toward the recent advancement of biohar-
vesting in terms of technological aspects.

9.1 Stability of Microalgae

C. Banerjee Suspension
Department of Bio-Engineering, Birla Institute of
Technology, 835215 Ranchi, Jharkhand, India Van der Waals force exists in the case of colloidal
e-mail: cbanerjee310@gmail.com
particle in suspension and is characterized
R. Bandopadhyay according to the nature of their watersolid inter-
Department of Botany, The University of Burdwan,
713109 Golapbag, Bardhaman, West Bengal, India face. Electrostatic repulsion is a key factor for
e-mail: rajibindia@gmail.com; controlling hydrophobic and hydrophilic stabil-
rajib_bandopadhyay@yahoo.com ity. In case of hydrophobicity, an excess of cat-
P.K. Singh ions and anions may accumulate at the interface,
Department of Microbiology, Enzyme Technology resulting in electrical potential which exerts
and Protein Bioinformatics Laboratory, Maharshi repulsion on the particulates of similar potential.
Dayanand University, 124001 Rohtak, Haryana, India
e-mail: puneet.micro.rs@mdurohtak.ac.in However, for hydrophilic surfaces, typical elec-
trical charges arise from dissociation of inorganic
P. Shukla (*)
Professor and Head, Department of Microbiology, groups. Suspended particle in water usually car-
Maharshi Dayanand University, 124001 Rohtak, ries a negative or a positive surface charge. The
Haryana, India neutrality is maintained by repulsion between
e-mail: pratyoosh.shukla@gmail.com two precipitate particles when they approach
H.K. Agrawal each other, i.e., prevents the colloidal particles
Microbiology Lab, Department of Bio-Engineering, from sticking together and attracts opposite ions
Birla Institute of Technology, Mesra, 835215 Ranchi,
Jharkhand, India in the environs of the particle (Deryagin and
e-mail: harsh1681.11@bitmesra.ac.in Landau 1941) and thus creates a dispersed cloud

Springer India 2015 127

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_9
128 C. Banerjee et al.

Fig. 9.1 Algal cells showing the

nature of electrical forces (Source:
Vandamme et al. 2013)

of ions around the particulate. Thus, a double their stability in the suspension. Carboxylic
layer is created between charged surfaces and (-COOH) and amine (-NH2) are responsible for
coupled counter ions in a surrounding solution the charge on the algal cell surface. Above pH
(Fig. 9.1). 45 carboxylic groups dissociate and are nega-
A diffusion layer of counterions is formed far tively charged, whereas there is no charge on
from the particle surface due to the stability of amine groups above this pH. A negative charged
electrostatic pull and thermal diffusion. Due to is observed above 45 (Vandamme et al. 2013).
this, the potential difference between the bulk
solution and particle surface declines exponen-
tially. When a particle is moving, it withdraws its 9.2 Flocculation Mechanism
counterions toward itself and leaves behind the
ions which are far away from its surface. Thus, a How flocculants can act on the small particle
plane is set which consists of shear and the poten- (microalgae and cyanobacteria) which have a
tial difference, defined as the zeta potential (). negative surface charge that repels among each
Thus, the zeta value can be measured by other. Basically, flocs are generated by adhering
the mobility of the charged particle under electric to each other; at high pH flocculants block this
field and thereby act as an indicator for the degree surface charge by binding (Henderson et al.
of repulsion in suspension. 2008a, b) and thus settle down.
Zeta potential gives a clue headed for the sta- The proposed mechanisms are charged neu-
bility of the system. If the particles in the suspen- tralization, electrostatic patch mechanism, bridg-
sion have a large positive or negative zeta ing, sweeping flocculation, and Singhs Easy
potential, they will repel and there is dispersion Approachability Model.
stability. Particles having low zeta values could In charge neutralization, charges on the col-
not be prevented from coming together, resulting loidal particle surface are neutralized by adsorp-
in dispersion instability. Particles which have tion of oppositely charged ion thus leading to
zeta potentials more positive than (+) 30 mV are canceling the colloid particle charge. This ulti-
normally considered stable. On other hand () 30 mately leads to flocculation where the electro-
mV are also considered to be stable. The surface static repulsion between the colloidal particles
charges of the microalgal cells are responsible for disappears. In electrostatic patch mechanism,
9 Innovations in Microalgal Harvesting Using Biopolymer-Based Approach 129

charged polymer binds to oppositely charged par- completely safe (nontoxic). Synthetic flocculants
ticle. Island patches are enclosed by the oppo- in contrast are effective even in small doses (<1
site charge on polymer and are created prior to ppm or mgL-1) and comprise of long shelf life.
the absorption of polyelectrolytes, in which
oppositely charged particles come in and get in
touch with other particles giving rise to the strong 9.3 Microalgae Harvesting
attraction that leads to flocculation of particles. Technologies
Mabire et al. in 1984 described the sedimentation
rate, the clarification and the height of the sedi- Biomass could be used as feedstock for fuel, but
ment bed with stirring, adsorption isotherms, and it has its own disadvantages. It will be overbur-
electrokinetic potential (Mabire et al. 1984). dened on the agriculture which also feeds human
Bridging phenomenon was first proposed by and livestock and triggers food shortage conse-
Ruehrwein and Ward in 1952. In this mechanism, quently. Microalgae could be the solution of this
a small dosage of long chain polymer when added problem. The major challenges lie in the harvest-
to colloidal suspension results to form a bridge ing process. The recovery process has been esti-
with the adsorption of two or more particles mated to account for 2030 % of the total cost of
(Ruehrwein and Ward 1952). Here it should be biomass produced (Grima et al. 2003). A descrip-
considered that adequate unoccupied space also tion of different microalgae-harvesting technolo-
must be there to form the polymer clustering. The gies is summarized in Table 9.1.
presently described process is being achieved The different processes of harvesting are as
equal to a specific dose of polymeric material and follows:
this process is termed as steric stabilization. The
dose of polymer should be meticulous to achieve Electrical
this process. Therefore, in low polymer dosage, Biological
flocculation is low because no significant bridging Autoflocculation
occurs and in higher dosage, there will be insuffi- Physical
cient surface space for connection of polymer due Chemical
to which flocs destabilize. The literature suggests Polymer based
that flocculants, their geometrical parameters, and
the KozenyCarman permeability equation
explain the disparities of rate of filtration with
concentration of flocculants (Smellie and La Mer 9.3.1 Electrical
1958). In flocculation kinetics of nanosized parti-
cle of silica, cationic amylopectin also supports Electrical based flocculation comprises of coagu-
the bridging flocculation phenomenon (Larsson lant generation by oxidation of electrode, desta-
and Wall 1998). bilization of particulate suspension followed by
According to Singhs Easy Approachability aggregation of the destabilized particles which
Model, flocculation is characterized into hydro- will form flocs. The sequential events are, first,
lyzed and unhydrolyzed polyacrylamides and the coagulant generation by oxidation of elec-
grafted and cationic polysaccharides. As per this trode; second, destabilization of particulate sus-
model the hanging branches of polyacrylamide/ pension; and third, aggregation of the destabilized
cationic moiety will be easily accessible if particles which will form flocs. It needs electric-
attached to the backbone. This leads to the for- ity for flocculation instead of any flocculants and
mation of aggregates, thus providing the finest to flocculate the microalgae from solution and
flocculation character (Singh et al. 2000; Brostow subsequently float the algal flocs with 95 %
et al. 2007). Flocculants are divided into two cat- removing efficiency (Poleman et al. 1997).
egories: natural or synthetic. Natural flocculants Continuous harvesting of microalgae has also
are low in molecular weights; they are to be been reported via electrolysis with polarity
added in larger dose, do not last long, but are exchange (Kim et al. 2012).
130 C. Banerjee et al.

Table 9.1 Different algae-harvesting techniques and their efficiency in separating algal biomass
Method Material used Yield (%) References
Flotation Saponin and chitosan with CTAB 93.7 Kurniawati et al. 2014
Tetradecyl trimethylammonium bromide Garg et al. 2012
Foam flotation CTAB Jonathan et al. 2014
Bacterial biocoagulation PEI-coated E. coli 83 Agbakpe et al. 2014
Electrocoagulation Aluminum electrodes 100 Gao et al. 2010
Physical flocculation Ultrasound chitosan 9799 Fast and Gude 2014
Auto flocculation Phaeodactylum tricornutum 85 Spilling et al. 2010
Chemical flocculation Aluminum sulfate and Boisvert et al. 1998
Polymer-based flocculation Cationic tamarind kernel polysaccharide Pal et al. 2009

Fig. 9.2 Electrical-based DC Supply

flocculation technique


Magnetic Stir

Magnetic Stirrer

Electrocoagulationflocculation method has 9.3.2 Biological

also been applied for harvesting a Chlorella vul-
garis (freshwater) and a Phaeodactylum tricor- Biological-based flocculation or bioflocculation
nutum (marine) microalgal species (Fig. 9.2). is thought to be caused by extracellular poly-
Electro-based method with aluminum anode meric substance in the suspension. Bioflocculation
was shown to be more efficient than using an is used to harvest algae where microalgae are
iron anode. Efficiency can be considerably used for wastewater treatment; additionally
improved by dropping the initial pH and by algalbacterial biomass could be an imperative
escalating the turbulence in the microalgal sus- energy source for the production of biofuel
pension (Vandamme et al. 2011). Electro- (Craggs et al. 2012). Five different infochemicals
flocculation method produces 93.6 % of algae were isolated from a flocculating and senescent
from Botryococcus braunii after 30 min of culture of Skeletonema marinoi and Dunaliella
experimentation. Around 98.9 % of the harvest salina. Water-soluble extracts of S. marinoi
was observed in 14 min when electrofloccula- induce flocculation in the Nannochloropsis ocu-
tion was combined with dispersed-air flotation lata (Taylor et al. 2012). A novel bioflocculation
(Xu et al. 2010). technology involving fungi (Aspergillus sp.)
9 Innovations in Microalgal Harvesting Using Biopolymer-Based Approach 131

Fungi Pallets
Magnetic Stir Bar

Algae Suspension rpm

Magnetic Stirrer

Filter Paper

Clear Supernatant

Fungi-Algae Pallets

Clear Supernatant

Fig. 9.3 Biological-based flocculation technique

leads to bioflocculation of Chlorella vulgaris 9.3.3 Autoflocculation

(Fig. 9.3). The novel technology developed is
wastewater treatment technologies (Zhou et al. Autoflocculation occurs due to precipitation of
2012). Microalgae cells can be co-palletized carbonate salts by algae at high pH values. This is
into fungal pellets for easier harvest; this pro- primarily because of photosynthetic CO2 con-
cess can be described as co-palletized cell culti- sumption by algae (Sukenik and Shelef 1984).
vation. Due to the large size of fungialgae pellet, Microalgae autoflocculation was studied, and
it can be easily harvested through sieve (Zhang about 8090 % removal of algal cells was
and Hu 2012). For harvesting marine microalgae, observed; it was in combination with chemical
microbial flocculation is one of the efficient flocculation with the help of alum or C-31 poly-
separation techniques for the biodiesel produc- mer (Koopman and Lincoln 1983). The process is
tion (Lee et al. 2009). Simple separation of the shown in Fig. 9.4.
algal biomass by gravity sedimentation through
algaebacteria aggregates is also described
(Gutzeit et al. 2005). Cell wall-associated poly- 9.3.4 Physical
saccharides were responsible for self-flocculating
microalga Scenedesmus obliquus (Guo et al. The removal of algal biomass with the imple-
2013). mentation of physical forces is referred to as
132 C. Banerjee et al.

Fig. 9.4 Auto flocculation

Algae Suspension Auto Flocculation

for removing microalgae (Chen et al. 1998).

Expressions for the probability of collision and
Air Flow System
adhesion have been derived from fine particle on
flotation leading to their adhesion to each other
(Yoon and Luttrell 1989). Dispersed-air flotation
entails 7001500 m bubbles, which are formed
by a high-speed agitator (Rubio et al. 2002). This
process was utilized to remove algae
(Scenedesmus quadricauda) from water. CTAB
was found to be effective to dewater S. quadri-
cauda among three types of collectors, i.e., cat-
Flocc ionic (CTAB), anionic (SDS), and the nonionic
Magnetic Stir Bar (Triton X-100) (Chen et al. 1998). Scenedesmus
obliquus has great capacity for capturing CO2.
Magnetic Stirrer The lipid so produced was harvested using dis-
persed O3 flotation (Cheng et al. 2011).
Fig. 9.5 Physical-based flocculation Cultivation of selected thermotolerant micro-
algae in the heat discharge water and subsequent
harvesting of the algal biomass with microstainer
physical-based flocculation. Magnetic nanopar- (Wilde et al. 1991) have been described.
ticle (Fe3O4/magnetite) based separation method Tangential flow filtration (TFF) system involves
is reliable and thus can be used in algal bio- 0.45 m membrane to concentrate phytoplankton
mass recovery. It depends upon high dose of for water treatment (Petrusevski et al. 1995).
nanoparticles and a pH suitable for recovery of Submerged microfiltration was also carried out
microalgae (Fig. 9.5). From time and energy sav- and energy consumption to dewater C. vulgaris
ing, this process of flocculation is inexpensive and P. tricornutum was well described (Bilad
and nontoxic as well. It is also engaged in the et al. 2012). The economic assessment revealed
elimination of detrimental algae from freshwater. that dynamic filtration is economically more effi-
Microalgae revival is performed by the phenom- cient than tangential cross-flow filtration (Rios
enon of electrostatic attraction among nanoparti- et al. 2012). Flotation under vacuum for harvest-
cle and microalgal cells (Xu et al. 2011). ing microalgae (Barrut et al. 2013) and combina-
Freshwater and marine algae are removed by tion of chemical and biological flocculation have
silica-coated magnetic particles with the help of also been used (Zamalloa et al. 2013).
high-gradient magnetic filtration and have also
been studied (Cerff et al. 2012).
In flotation gaseous bubbles are attached to 9.3.5 Chemical
the solid particles, which are thus carried to the
liquid surface. Here, gravity plays an important Chemical-based flocculation is mainly used in
role in the separation process. Flotation is having the pretreatment of algal cell to increase its size.
more advantages than sedimentation especially It is used before using another method of floccu-
9 Innovations in Microalgal Harvesting Using Biopolymer-Based Approach 133

Chemical Flocculants Polymaric Flocculants

Flocc Flocc
Magnetic Stir Bar Magnetic Stir Bar
rpm rpm
Magnetic Stirrer Magnetic Stirrer

Fig. 9.6 Chemical-based flocculation Fig. 9.7 Polymer-based flocculation

lation such as flotation. It is mainly performed Scenedesmus obliquus and Chlorella vulgaris
from the salts like alum and ferric chloride (Fig. were studied at high pH induced to improve the
9.6). The testing of different harvesting methods efficiency of harvesting. Here, the required pH
for B braunii was performed once a week. The values were achieved by using sodium hydroxide
following conditions were applied: pH adjust- and calcium hydroxide (Castrillo et al. 2013).
ment, treatment with aluminum sulfate and Poly -glutamic acid, a microbial flocculant,
Pestan, and biopolymer treatment. The efficient was used to harvest oleaginous microalgae.
harvesting method is to be adjusted at pH 11 (Lee Response Surface Methodology (RSM) was used
et al. 1998). Likewise, Phaeodactylum sp. diatom to optimize the flocculation condition for Chlorella
flocculation was done with 0.41.3 mM calcium vulgaris (marine) and Chlorella protothecoides
hydroxide (Veloso et al. 1991) and Tetraselmis (freshwater) (Zheng et al. 2012). The highest floc-
sp. (Millamena et al. 1990). A high pH was culating activity of 95 % was achieved for
needed to flocculate with the use of calcium and Scenedesmus cultures by employing coagulants
magnesium: where pH 1012 (Ayoub et al. 1986; (8.5 mM CaCl2 and 0.2 mM FeCl3) along with
Yahi et al. 1994; Blanchemain and Grizeau 1999; 1 % bioflocculant of P. polymyxa (Kim et al.
McCausland et al. 1999) initiated flocculation of 2011). Chemical coagulants [FeCl3 and Fe2(SO4)3]
algae. A nonionic polymer Magnafloc LT-25 was along with commercial polymeric flocculant like
employed for flocculation at pH 1010.6 Drewfloc 447, Flocudex CS/5000, Chemifloc
(Knuckey et al. 2006). Here, a large amount of CV/300 and chitosan are used for flocculation.
blend of base and calcium was also used (Davis Their ability was compared for the removal of
and Foust 1969). Scenedesmus and Chlorella algalbacterial biomass during piggery wastewa-
were harvested by alum by means of charge neu- ter treatment (De Godos et al. 2011).
tralization (Grima et al. 2003). With the use of
inorganic flocculants, microalgae can also be
flocculated by satisfactorily low pH (Uduman 9.3.6 Polymer Based
et al. 2010).
Flocculation can be induced by increasing the Polymer-based flocculation includes the water-
pH of the medium followed by reusing the floc- soluble high-molecular weight linear or modified
culated medium for reculturing. About 90 % of polymers that can be used as flocculants.
the microalgae was harvested by increasing the Flocculation of textile industry wastewater, coal
pH (Scenedesmus sp., Chlorella vulgaris and suspension, kaolin, and iron ore was achieved by
Chlorococcum sp., Phaeodactylum tricornutum, using synthetic cationic tamarind kernel polysac-
Nannochloropsis oculata) (Wu et al. 2012). The charide (Pal et al. 2009), cationic glycogen (Pal
flocculationsedimentation is the costliest steps et al. 2008), and amphoteric amylopectin (Singh
of production of microalgae. For that reason et al. 2013).
134 C. Banerjee et al.

Flocculation of freshwater algae, Spirulina, were used (Banerjee et al. 2013). Recently, synthe-
Oscillatoria Chlorella, and one brackish alga, sis of aminoclays with Mg2+ or Fe3+, by solgel
Synechocystis, by means of chitosan was studied reaction-based methodology with 3-aminopropyl-
in the pH range of 49. The settled algal cells are triethoxysilane as a precursor molecule, is
intact and live but will not be redispersed by exhibited, which produces (CH2)3NH2 showing
mechanical agitation (Fig. 9.7). The agitated water covalent bonding. In aqueous solution the proton-
may be reused to produce fresh cultures of algae rich amine groups validate the competent harvest-
(Divakaran and Pillai 2002). The efficiency of ing of microalgal biomass for various marine algae
cationic starch as flocculant is a not very proficient (Farooq et al. 2013). We understand that genetic
for marine microalgae (Phaeodactylum, Nanno- modification includes insertion of flocculin pro-
chloropsis), but for freshwater (Parachlorella, tein in the cell wall of Saccharomyces cerevisiae,
Scenedesmus) it is very effective. There was desta- which leads to efficient settling to enhance product
bilization at high cationic starch concentration. clarification and recovery (Govender et al. 2008).
The requirement of the cationic starch dose for the
stimulation of flocculation increased linearly with
algal biomass quantity. Among the flocculants, 9.4 Concluding Remarks
namely, Greenfloc 120 and cationic starch, cat-
ionic starch was effective (Vandamme et al. 2010). Production of microalgae is quite expensive in
The required dose of chitosan is lesser than todays scenario. Harvesting microalgae through
that of inorganic metal salts due to the presence polymer is still lower compared to centrifugation.
of higher number of functional groups in chito- Flocculation using ultrasound is still more costly.
san (Renault et al. 2009). Recovered algal bio- It is evident that cationic starch and biopolymers
mass can be used directly in industrial production are cheaper to some extent, but probably these
of food and fuels for using chitosan as a natural are too costly for their application in biofuel pro-
flocculant (Ahmad et al. 2011). Chitosan was duction (Vandamme et al. 2013). Relatively,
found to be the most effective flocculant when it autoflocculation (alkalinity induced) is cost-
was used with dose of 100 mg/L algae broth effective than other methods (Vandamme et al.
when it was compared with ferric sulfate and 2012). On other hand, bioflocuulation by
alum (Beach et al. 2012). Chitosan was used as microbes (bacteria and fungus) is not feasible
natural flocculant to explore the green microalga owing to their culturing property.
Chlorella sorokiniana. Results showed that the Chemical flocculation has a disadvantage of
clarification efficiency of the process could reach contaminating biomass; however, a physical sepa-
above 99 % below pH 7 (Xu et al. 2013). ration process does not have this problem. Primary
Microalga Nannochloropsis sp. was harvested by research into flocculation that is stimulated by
flocculant chitosan which was modified to nano- infochemicals in microalgae is extremely needed,
chitosan by cross-linking with sodium tripoly- which would lead to an extremely controlled
phosphate. After the recovery of biomass, the method for suggesting contamination-free floccu-
effects of flocculants and type, dosage, and pH of lation. Genetic modification based flocculation is
the culture were examined (Farid et al. 2013). not a cost effective process and for assessing a
Microalgae are stable in suspension culture, but innovation flocculation technique economy feasi-
they are difficult to flocculate due to their small bility should be an important factor for consider-
size and negative charge on their surface. Cationic ation (Vandamme et al. 2013). Nevertheless, we
guar gum with the introduction of quaternary must also ensure that flocculation step is to be
amine groups could be used for flocculation. For taken into account for cost evaluation and extra
the flocculation of green algae, viz., Chlorella sp. efforts are also to be made to understand its effect
CB4 and Chlamydomonas sp. CRP7, different on the entire biofuel production process using
doses of the modified synthesized biopolymer flocculation-based technologies.
9 Innovations in Microalgal Harvesting Using Biopolymer-Based Approach 135

Acknowledgements Dr. Chiranjib Banerjee highly Cheng YL, Juang YC, Liao GY, Tsai PW, Ho SH et al
acknowledges Department of Science and Technology (2011) Harvesting of Scenedesmus obliquus FSP-3
(DST), Government of India for providing financial sup- using dispersed ozone flotation. Bioresour Technol
port as well as project grant from INSPIRE Faculty award 102:8287
scheme. Craggs R, Sutherland D, Campbell H (2012) Hectare-
scale demonstration of high rate algal ponds for
enhanced wastewater treatment and biofuel produc-
tion. J Appl Phycol 24:329337
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polymeric flocculant for the treatment of textile indus- biomass production. Trends Biotechnol 31:233239
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9 Innovations in Microalgal Harvesting Using Biopolymer-Based Approach 137

Wu Z, Zhu Y, Huang W, Zhang C, Li T et al (2012) Rajib Bandopadhyay

Evaluation of flocculation induced by pH increase for received his M.Sc. and
harvesting microalgae and reuse of flocculated Ph.D. degree (Molecular
medium. Bioresour Technol 110:496502 Biology) from University
Xu L, Wang F, Li HZ, Hu ZM, Guo C et al (2010) of Calcutta. He participated
Development of an efficient electroflocculation tech- for Southern Ocean
nology integrated with dispersed-air flotation for har- Expedition in 2011. He is
vesting microalgae. J Chem Technol Biotechnol presently serving as
85:15041507 Associate Professor in the
Xu L, Guo C, Wang F, Zheng S, Liu CZ (2011) A simple University of Burdwan,
and rapid harvesting method for microalgae by in situ WB, India. He is recipient
magnetic separation. Bioresour Technol of BOYSCAST Fellowship
102:1004710051 of DST and Korean
Xu Y, Purton S, Baganz F (2013) Chitosan flocculation to Research Fund Foreign Scientist Exchange Programme.
aid the harvesting of the microalga Chlorella sorokini- His current interests are algal biotechnology and plant
ana. Bioresour Technol 129:296301 molecular biology.
Yahi H, Elmaleh S, Coma J (1994) Algal flocculation
sedimentation by pH increase in a continuous reactor. Puneet Kumar Singh
Water Sci Technol 30:259267 graduated in Bioinformatics
Yoon RH, Luttrell GH (1989) The effect of bubble size on in 2009 from Devi Ahilya
fine particle flotation. Miner Process Extr Metall Rev University, Indore. He
5:101122 received his M.Sc. degree
Zamalloa C, Boon N, Verstraete W (2013) Decentralized in Bioinformatics in 2011
two-stage sewage treatment by chemical-biological from BIT Mesra, Ranchi,
flocculation combined with microalgae biofilm for India. He is currently pur-
nutrient immobilization in a roof installed parallel suing Ph.D. in
plate reactor. Bioresour Technol 130:152160 Microbiology, Maharshi
Zhang J, Hu B (2012) A novel method to harvest microal- Dayanand University,
gae via co-culture of filamentous fungi to form cell Rohtak, India. His current
pellets. Bioresour Technol 114:529535 research interests are in
Zheng H, Gao Z, Yin J, Tang X, Ji X et al (2012) systems biology, protein
Harvesting of microalgae by flocculation with poly engineering, and algal biofuel production.
(-glutamic acid). Bioresour Technol 112:212220
Zhou WG, Cheng YL, Li Y, Wan YQ, Liu YH et al (2012) Harsh Kumar Agrawal
Novel fungal pelletization-assisted technology for is a graduate student B.E.
algae harvesting and wastewater treatment. Appl (Biotechnology), from
Biochem Biotechnol 167:214228 Birla Institute of
Technology, Mesra,
Ranchi, India. His research
Chiranjib Banerjee interests are algal harvest-
received the M.Sc. degree ing and bioinformatics. He
in Biotechnology from is currently engaged in final
Vidyasagar University, semester project work
India, and Ph.D. from related to algal harvesting.
Department of
Bioengineering, BIT
Mesra, Ranchi, India. He
has been awarded with the
DST Inspire Faculty
Scheme. His current inter-
ests are algal biofuel, algal harvesting, and molecular tax-
onomy. He is a life member of the Association of
Microbiologists of India (AMI).
138 C. Banerjee et al.

Pratyoosh Shukla presently working as Professor and Head at Department of

received his M.Sc. degree Microbiology, Maharshi Dayanand University, Rohtak,
(Applied Microbiology India. He was awarded with Indo-US Visiting Research
Biotechnology) from Dr. Professorship by American Society of Microbiology
H.S. Gour University, (ASM) in 2014. His current interests are bioenergy,
Sagar, India, and Ph.D. enzyme technology, and protein bioinformatics.
degree in Microbiology and
From Microbial Biopolymers
to Bioplastics: Sustainable 10
Additives for PHB Processing
and Stabilization

Stefania Angelini, Pierfrancesco Cerruti,

Barbara Immirzi, Merima Poskovic,
Gabriella Santagata, Gennaro Scarinzi,
and Mario Malinconico

The term biopolymers refers to a broad class of materials that derive from
naturally occurring resources. Biopolymers can be obtained through
extraction from biomasses, but also through chemical or biotechnological
methods from raw natural substrates. They are used to produce bioplastics,
which could substitute fossil fuel-derived commodities. Among them,
polyhydroxyalkanoates (PHAs) are polyesters synthesized by microor-
ganisms as energy reserve. The most important member of PHA family is
poly(3-hydroxybutyrate) (PHB). PHB is mechanically similar to polypro-
pylene, even though its thermal instability, brittleness, and stiffness hinder
its applicability. Improving PHB physical properties can be achieved by
blending it with natural additives or by-products of industrial processes.
This work takes the form of a case study about the effects of three natural,
phenol-based, and polysaccharidic compounds on PHB properties. In par-
ticular, data on blending of two PHB matrices with a grape pomace extract
(EP), a lignocellulosic biomass (LC), and tannic acid (TA) are reported.
The preparation and characterization of PHB compounds and the effects
of the additives on processing, thermal and photooxidative stability, crys-

S. Angelini (*) P. Cerruti B. Immirzi

G. Santagata G. Scarinzi M. Malinconico
Polymer Science, IPCB-Institute for Polymers,
Composites and Biomaterials, 80078
Pozzuoli (NA), Italy
e-mail: stefania.angelini@ictp.cnr.it;
cerruti@ictp.cnr.it; barbara.immirzi@ictp.cnr.it;
gabriella.santagata@ictp.cnr.it; M. Poskovic
gennaro.scarinzi@ictp.cnr.it; Polymer Science, Novamont, 28100 Novara, Italy
mario.malinconico@ictp.cnr.it e-mail: merima.poskovic@novamont.com

Springer India 2015 139

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_10
140 S. Angelini et al.

tallization rate, and microbial digestion of PHB are also shown. An overall
improvement of polymer processability and photostability, along with
changes in crystallization rates, was observed. The study provides evidence
that natural additives have the potential for promoting the transition from
biopolymers to bioplastics in a sustainable way, both from an environmen-
tal and economical point of view.

10.1 Introduction In this chapter the biopolymers will be classified

on the basis of the approach used to produce them.
The use of synthetic polymers, derived from oil- The main methods for the production of these
based resources and aimed to the production of materials can be divided into extraction and syn-
plastics, is widely spread in the fields of medi- thetic routes. The first approach regards the extrac-
cine, food packaging, agriculture, cosmetics, fur- tion of the product from biomasses and dedicated
niture, composites, etc. (Lee et al. 2011; Kasirajan crops, agro-industrial and municipal wastes, and,
and Ngouajio 2012). Features as durability, last but not least, microorganisms. The second
toughness, and elasticity have provided great approach involves the chemical/biotechnological
benefits to modern society but have also raised polymerization of bio-derived monomers. Among
significant economical and ecological issues. the more common biopolymers, we can list poly-
Indeed, most plastics exhibit a remarkable resis- saccharides (alginate, starch, and starch blends
tance to biodegradation once landfill is disposed such as Mater-Bi or Solanyl, chitin), vegetable
and, in some cases, their recycling is not econom- and animal proteins, nucleic acids, lipids, polylac-
ically sustainable (Dintcheva et al. 1997; Kim tic acids (PLAs), polyhydroxyalkanoates (PHAs),
et al. 2001; Guo and Xu 2009). Hence, due to the and bio-based polyethylene terephthalate (PET) or
rising environmental awareness, a large and polyethylene (PE), even if the latter two are not
growing body of studies has been devoted to the biodegradable. Biopolymers, with respect to their
development of novel and sustainable polymers petrochemical-based counterparts, are generally
based on renewable resources (Scott 2000). more sustainable (Mac Gregor 2001). The use of
Biopolymers are a class of materials that derive these materials is aimed to the production of bio-
from naturally occurring supplies (Niaounakis plastics. Their main advantage, besides their natu-
2013). This definition refers to a heterogeneous ral origin, is the possibility to be disposed as
group of bio-based, mostly biodegradable poly- compost. This point is particularly attractive under
mers; a first classification is reported in Fig. 10.1. the environmental perspective as it contributes to
Currently, a univocal method of classification solve one of the main problems of traditional plas-
for this category of macromolecules does not exist. tics, that is, their lack of biodegradability.

Main classes of
biodegradable biopolymers

From From
biomass chemistry/biotechnology

Proteins Lipids Polysaccharides Polyhydroxyalkanoates Polylactides

Fig. 10.1 Different classes of biodegradable biopolymers

10 From Microbial Biopolymers to Bioplastics: Sustainable Additives for PHB Processing and Stabilization 141

10.2 The Blending Technique: which cause surface stickiness as well as poor
A Strategy to Obtain mechanical and barrier properties (Thunwall
Bioplastics et al. 2008). Therefore, plenty of studies have
from Biopolymers been dealing with blending TPS (usually from
20 % to 80 %) with biopolymers as chitosan
In many cases, biopolymers are not able to fully (Pelissari et al. 2012), cellulose and proteins
replace synthetic polymers. This is due to their (Gspr et al. 2005), PHAs (Yu 2009), and PLAs
poor chemicalphysical and mechanical proper- (Ferreira et al. 2015) with the aim of widening
ties, along with difficulties in processability (Van the application window of TPS. In particular,
de Velde and Kiekens 2002; Mitra et al. 2014). much effort has been made to develop TPS/PLA
These shortcomings strongly restrict the potential blends (Wang et al. 2007). The low interfacial
technological applications of such materials. A adhesion between hydrophilic starch and hydro-
typical way developed to modify biopolymer phys- phobic PLA has been recognized as the major
ical properties consists in blending (Mohanty et al. problem to be addressed. To promote compatibil-
2002). The blending method is a cheap and simple ity of these two materials, compatibilizers such
technique, based on mixing polymers with addi- as maleic anhydride are often used (Huneault and
tives that can act as processing aids, plasticizers, Li 2007). Another example of blending is
and UV or thermo-oxidative stabilizers. The blend- reported in the recent study carried out by Ortega-
ing technique is often used to modify the properties Toro et al. (2014), who examined the effect of
of biopolymers with the aim of widening their field hydroxypropyl methylcellulose on the micro-
of applicability (Marsano et al. 2004; Sionkowska structure, thermal, and physical properties of
2011; Wang et al. 2011). Starch-based blends rep- glycerol-plasticized starch films in the presence
resent a typical example of this approach. Natural of citric acid. The authors recorded a slowdown
starch is one of the most studied storage polysac- of starch retrogradation, due to the addition of
charides (Ellis et al. 1998). It is present in the form hydroxypropyl methylcellulose and citric acid.
of crystalline beads in plants as corn, maize, potato, Blending TPS with fossil fuel-based polymers
and other crops. Structurally, it consists of amylose as PVOH or PCL has also been tested (Kalambar
and amylopectin, a linear and a branched polymer, and Rizvi 2006). This successful approach has
both constituted by D-glucose repeating units been adopted by several companies as Rodenburg
linked together by -1,4 and -1,6 linkages (Bulon (Netherlands), Biotec (Germany), Limagrain
et al. 1998; Miao et al. 2015). (France), Livan (Canada), and Novamont (Italy),
Several studies have been devoted to the con- resulting in a number of commercially available
version of starch into a thermoplastic material products. As an example, the European leading
(Kaseem et al. 2012; Rosa et al. 2009). Most of company Novamont offers four classes of biode-
the proposed methods are based on the applica- gradable materials under the Mater-Bi trade-
tion of heat and shear forces or the addition of mark (Shen et al. 2009); all of them were based
plasticizers such as glycerol. The purpose is to on starch and synthetic components. The four
demolish the crystalline structure and weaken the classes are listed below (Bastioli 1998):
intermolecular interactions existing between
starch chains (Bastioli 1998; Stepto 2006). After Class Z: biodegradable and compostable
this treatment, starch can be processed like other blends made of TPS and PCL for films and
thermoplastics in typical injection-molding sheets
machines and extruders (Mahieu et al. 2013). The Class A: biodegradable and not compostable
aim of this physical manipulation of the polymer blends made of starch and ethylene vinyl alco-
is to produce processable materials suitable for hol copolymers for molded items,
rigid/flexible packaging. Nevertheless, the appli- Class Y: biodegradable and compostable
cations of thermoplastic starch (TPS) are limited blends made of TPS and cellulose derivatives
due to humidity sensitivity and retrogradation, for rigid injection-molded items
142 S. Angelini et al.

Class V: biodegradable, compostable, and original Ecoflex grade is a fossil-based copoly-

soluble materials for rigid and expanded arti- ester that originates from the reaction between
cles (TPS > 85 %) 1,4-butanediol, adipic acid, and terephthalic acid.
Blending Ecoflex with biological polymers such
Although Mater-Bi is used in packaging, as starch or poly(lactic acid) (PLA) provides bio-
agriculture, or consumer goods, its properties are degradable bioplastics with interesting techno-
often unsuitable for commercial purposes logical properties (Pack et al. 2012). Compounds
(Briassoulis 2007). In order to improve Mater-Bi of Ecoflex and PLA are produced by BASF
properties, several approaches have been pur- under the registered trademark Ecovio. Ibrahim
sued. In particular several studies have attempted (2009) described the preparation and character-
to produce Mater-Bi-based composites using ization of new biodegradable composites made
cellulose fibers as fillers in order to improve pro- by Ecoflex with kenaf fibers as filler. The effects
cessability and chemicalphysical performances of the filler loading on the mechanical and ther-
of the material (Alvarez et al. 2006). It is worth mal performances were examined, and an
noting that the addition of the filler produces an enhancement of tensile/flexural properties was
enhancement of mechanical properties and water observed.
barrier capability. In recent years Cerruti et al.
(2011) have used a polyphenolic extract (EP), the
biowaste of winery production, as an additive for 10.3 The PHA Family: A Particular
Mater-Bi. The authors found that EP behaved as Focus on Poly(3-
a plasticizing agent and as a modifier of Mater-Bi hydroxybutyrate) (PHB)
processing, mechanical and thermal properties.
Moreover, it also produced a decrease in the PHAs are a family of microbial polyesters with a
microbial digestion rate. wide range of properties that find application as
Another class of promising biopolymers is biodegradable and biocompatible thermoplastic
represented by PLAs, a family of biodegradable, polymers (Riggi et al. 2011; Zheng et al. 2015).
biocompatible, and nontoxic polyesters based on They are produced by microorganisms and accu-
lactic acid as monomer. Lactic acid can be mulated in microbial cytoplasm as energy storage
obtained by the fermentation of various hexoses. materials. An alternative route for the production
These sugars are produced by the hydrolysis of of PHAs is based on the use of algae. This
several polysaccharides, including starch, cellu- approach is still at an experimental level but can
lose, and lignocellulosic materials (Coelho et al. contribute to solve some of PHA restrictions,
2010). Although PLAs have a wide range of such as the high cost. Generally, these production
applications, they biodegrade slowly in soil. This processes are carried out in tanks, and they con-
feature limits their use in agriculture. Therefore, sist in two stages, the first one in which the algae
PLA blending represents a useful method that growth is promoted by optimal conditions and
allows to overcome this shortcoming. It was the second step where the algae begin to accumu-
shown that the addition of useful fillers can late PHAs as secondary metabolites. A deeply
enhance the biodegradability of the matrix and studied member of PHAs is poly(3-
improve its mechanical properties as well. Model hydroxybutyrate) (PHB). PHB is a linear polyes-
examples are those reported by some authors ter, homopolymer of 3-hydroxybutyrate, which is
who studied blends of PLAs with starch (Lv et al. accumulated in the cytoplasm of a wide variety of
2015; Guzman-Sielicka et al. 2013) and chitosan gram-positive and gram-negative microorgan-
(Nugraha et al. 2004). isms under physiological stress (Dawes and
Another commercial member of the polyester Senior 1973). PHB is a thermoplastic polymer
class is Ecoflex. This is the registered trademark that shows biodegradability in compost and in
of a family of aliphaticaromatic biodegradable different environments, such as marine water
polyesters, which are produced by BASF. The (Volova et al. 2010). Due to its mechanical and
10 From Microbial Biopolymers to Bioplastics: Sustainable Additives for PHB Processing and Stabilization 143

barrier characteristics, which are similar to those blend miscibility, mechanical behavior, morphol-
of oil-based polymers such as polyethylene, ogy, and crystallization rate of the PHB matrix
polypropylene, and polyethylene terephthalate, has been largely investigated. From around the
this material can be proposed as a substitute for 1990s, a great deal of scientific papers concern-
petroleum-derived plastics. Nevertheless, the dif- ing polymer blends based on PHB and poly(3-
fusion of PHB as a commodity polymer is ham- hydroxybutyrate-hydroxyvalerate) (PHBV) has
pered by its brittleness (with elongation at break appeared. The interest was mainly focused on
below 15 %) and excessive stiffness (Mekonnen blending these natural polymers with polyolefins
et al. 2013). This last shortcoming is the result of such as poly(vinyl alcohol) (PVA) (Greco and
the recrystallization phenomenon and physical Martuscelli 1989), polyethers as
aging that the material undergoes at room tem- poly(oxyethylene) (PEO) or
perature (de Koning and Lemstra 1993). poly(methyleneoxide) (POM) (Avella and
Moreover, another serious drawback of PHB is Martuscelli 1988; Avella et al. 1997), polyesters
represented by its thermal decomposition at tem- as polycaprolactone (PCL) (Kumagai and Doi
peratures just above the melting point (~175 C) 1992; Immirzi et al. 1994), and polyacrylates as
(Chodak 2002). This phenomenon strongly poly(methyl methacrylate) (PMMA) (Lotti et al.
restricts its processing window and limits its 1993; Yoon et al. 1993) or poly(butyl acrylate)
workability. Both the recrystallization phenome- (Immirzi et al. 1994). Another field that attracted
non and polymer degradation during processing a great deal of interest was the blending of PHBV
can be prevented and reduced by the addition of with polysaccharides as cellulose and starch
nucleating agents or lubricants (Bugnicourt et al. derivatives (Lotti and Scandola 1992; Buchanan
2014). In addition to the problems described et al. 1992; Shogren 2009) or natural fibers as
above, another strong limitation to the commer- wheat straw or hemp (Paramasivan and Abdul
cial diffusion of PHB is represented by its high Kalam 1974; Felix and Gatenholm 1991). In the
production costs. One of the main cost factors, last few years, the scientific interest has been
related to PHB production, is due to high price of devoted to blending PHB with natural additives
the carbon substrates. Generally, the substrates or industrial biowastes. The latter perspective
are pure carbohydrates as glucose or sucrose with deserves particular attention, because the dis-
competing food value. Currently, in order to posal of industrial by-products represents a sig-
reduce PHB costs, the use of less expensive raw nificant environmental issue (Rossini et al. 2013).
materials has been considered. These new sub- The incorporation of these biowastes into a bio-
strates include biomasses from the maintenance polymer offers the possibility to modify the
of green spaces, wastes, and coproducts of indus- matrix properties and, at the same time, to address
trial processes such as glycerol, sugarcane an environmental concern. In this frame, several
bagasse, and lignocellulosic feedstocks from authors investigated the effects of the addition of
agricultural or forestry residues. The major natural fibers such as coconut, hemp, jute, and
advantage of this approach is the conversion of flax into PHB matrix (Gunning et al. 2013). In
these wastes into value-added products. Another particular, Macedo et al. (2010) used coir dust, an
route to reduce PHB costs and to widen its appli- abundant and cheap biowaste that derives from
cability field is represented by the blending of the fiber separation process of the coconut, as
this polyester with suitable fillers and additives. filler material for PHB matrix. The addition of
In this regard, blends of PHB with synthetic the bio-charge produced an improvement of
polymers have been reported since the early stressstrain properties and thermal stability.
industrial availability of PHB from ICI Chemical Other examples of blending PHB with natural
Industries. Since then, a considerable amount of additives are also reported in the literature, and
literature has been published on the formulation some authors investigated the effects of chitin
and characterization of PHB/polymer blends. and chitosan (Ikejima and Inoue 2000); starch
The effect of the polymeric second component on and its derivatives, in the form of starchadipate
144 S. Angelini et al.

or grafted starchurethane (Innocentini-Mei et al. 10.4.1 Chemical and Physical

2003); and soda lignin extracted from bagasse Properties of the Natural
(Mousavioun et al. 2010). Biodegradability of Additives
PHB/chitin and PHB/chitosan films was found to
be enhanced. PHB-based blends with starch and In this paragraph the characterization of three
starch derivatives were found to be more brittle materials deriving from renewable resources,
than neat PHB, but also characterized by an used as additives in two PHB matrices, will be
improved processability due to the reduction of discussed. These materials are:
the polymer melting temperature. Soda lignin
improved the polymer thermal stability, owing to A dried hydroalcoholic extract from grape
the intermolecular interactions established pomace extract (EP)
between PHB and lignin. A lignocellulosic biomass (LC)
In the next paragraph, the effects of blending Tannic acid (TA)
PHB with organic renewable fillers, some of
which are biowastes of industrial productions, Their visual appearance is showed in Fig. 10.2.
will be discussed in depth. EP is a dark purple and granular soft paste, while
LC and TA are two fine powders, brownish and
off-white, respectively.
10.4 Case Study: Use of Grape EP is obtained from a dried Cabernet pomace
Pomace Extract, through a high-pressure hydroalcoholic extrac-
Lignocellulosic Biomass, tion (Persico et al. 2012). Briefly, a mixture of
and Tannic Acid as Additives 70:30 (v/v) ethanolwater was used, adjusting
in PHB the solution pH at 3 and using a volume ratio
between pomace and extracting solvent of 1:3.
In the subsequent sections, the outcomes acquired After solvent evaporation, a soft paste (water
on the processing, thermal stability, photodegra- amount of 20 wt%) is recovered. From the analy-
dation, and microbial digestion of two grades of sis of the dried extract, a content in simple carbo-
PHB filled with a pomace extract (EP), a ligno- hydrates and polysaccharides equal to 79.0 4.2
cellulosic biomass (LC), and the natural polyphe- wt% (Dubois 1956) and a phenol amount of
nolic tannic acid (TA) are reviewed in the 4.3 1.2 wt%, mostly composed by catechin
perspective of tailoring several PHB properties derivatives, was found (Garaguso and Nardini
relevant for its technological application. Before 2015). The remaining fraction was mainly com-
discussing blend preparation and characteriza- posed of organic acids derived from tartrates and
tion, a focus on the properties of the additives is malates (Kammerer et al. 2004). Some of the

Fig. 10.2 An optical image of EP (left), LC (middle), and TA (right)

10 From Microbial Biopolymers to Bioplastics: Sustainable Additives for PHB Processing and Stabilization 145

Fig. 10.3 Typical a b O

components found in the OH
grape pomace extract: (a)
tartaric acid, (b) pectins, HO O
(c) catechins, and (d) malic OH HO
acid OH O
c R
d O OH


Fig. 10.4 SEM micrographs of LC at two different magnifications

typical components present in EP are illustrated polysaccharide component. In fact, the scanning
in Fig. 10.3. electron microscopy (SEM) observation of LC
LC is a lignocellulosic biomass obtained as a (Fig. 10.4) reveals the heterogeneous morphol-
biowaste from the second-generation bioethanol ogy of the biomass characterized by the presence
production process. The definition second gen- of irregularly shaped lignin particles along with
eration refers to the use of fermentable sub- some cellulose/hemicellulose fibrous material.
strates not interfering with food industry, such as The lignin content of LC was investigated by
agricultural residues and by-products (Wyman means of the Klason method (Angelini et al.
1996). LC is produced using Arundo donax stems 2014), and an acid-insoluble lignin content of 56
as substrates, and it is recovered as a solid waste wt% was found.
after the fermentation process. Due to mild pro- TA is the commercial form of tannin, one of
cessing conditions upon fermentation, the highly the most important polyphenolic ingredients in
crystalline cellulose fraction is not completely the bark of trees, where it provides thermal and
converted, so that LC still contains an amount of antimicrobial protection (Larif et al. 2013). TA
146 S. Angelini et al.

and other polyphenols have also antimutagenic, transition (Tg), and residue at 700 C. EP weight
anticarcinogenic, and antioxidant activities loss started right above 100 C and a steep
(Lopes et al. 1999). Tannins can be classified into decrease was noticed until 500 C. 70 % of initial
two categories: hydrolysable and nonhydrolys- weight was lost in the temperature between
able or condensed tannins. TA consists of a cen- 150 C and 500 C, and a char value of 23 wt%
tral carbohydrate (glucose) surrounded by a was recorded at 700 C. It is likely that the fast
number of galloyl groups that range from 2 up to degradation of EP is due to the large amount of
10 according to the plant source (Fig. 10.5) carbohydrate structures, which undergo massive
(Sahiner 2015). dehydration processes at temperatures lower than
As underlined before, the aim of the work is to 200 C. LC appeared to be more stable than EP,
evaluate the effect of these natural substances as as demonstrated by the weight loss onset shifted
additives in PHB. This polymer is processed at a toward significantly higher temperatures. LC
temperature, around 200 C, which can trigger degradation started at around 250 C, and at
thermal degradation phenomena of the organic 700 C, a char of 36 wt% was calculated. The
molecules. Therefore, the thermal behavior of the higher onset temperature of this material was
additives is an important parameter to be consid- related to the presence of a remarkable aromatic
ered when PHB-based blends are prepared. The fraction, which degraded more slowly due to a
thermal behavior of EP, LC, and TA was investi- low content of free hydroxyls. The major degra-
gated through two thermal characterization tech- dation step of tannic acid occurred from 230 C
niques: thermogravimetric analysis (TGA) and to 350 C and a char yield of 27 % was recorded.
differential scanning calorimetry (DSC) under At the end of the thermal analysis experiment, a
nitrogen. The TGA thermograms and the DSC sort of carbonaceous aerogel structure formed
second heating scan are shown in Fig. 10.6a, b, due to the dehydration of hydroxyl groups of
respectively. tannic acid. As shown by Xia et al. (2015) under
Table 10.1 lists temperatures of onset (Tonset), air, tannic acid burns almost completely leaving
degradation peak in the DTG curves (TDTG), glass only 1.4 % of char residue.

Fig. 10.5 Typical a b

components present in the R R OH
tannic acid: (a) hexose O
monosaccharide and (b) OH
galloyl group
O O R=

a 0.6 b 1.2 0.10

100 LC 0.8 LC 0.12
Heat Flow, W g1 EP, TA
Derivative weight, % C1

TA 0.4 TA 0.14
Heat flow, W g1 LC

0.2 0.0 0.16

Weight, %

0.0 0.4
60 0.8
0.2 0.22
40 0.4 1.6
0.6 0.28
20 2.4 0.30
100 200 300 400 500 600 700 40 60 80 100 120 140 160 180 200
Temperature, C Temperature, C

Fig. 10.6 (a) TGA (full symbols) and DTG (empty symbols) curves and (b) DSC second heating curve of EP, LC, and
10 From Microbial Biopolymers to Bioplastics: Sustainable Additives for PHB Processing and Stabilization 147

DSC curves of EP, LC, and TA were charac- respectively. EP was added in PHB-T3, while LC
terized by an irregular baseline and did not dis- and TA were added in PHB-T19, at 15 wt% for
play any signals due to melting. A very broad each additive. The vacuum oven-dried additives
endotherm step ascribed to glass transition of LC were dissolved (EP and TA) or suspended (LC) in
was visible at temperatures higher than 100 C. methanol. A proper amount of PHB was added to
Since the glass transition temperatures were not the resulting mixtures, and then the formulations
clearly appreciable, their values were assessed were held at 50 C under stirring for solvent evap-
using the first derivative of the DSC traces oration. They were then dried under vacuum until
(Gordobil et al. 2014). By means of this proce- a constant weight was achieved. The samples
dure, a Tg value of 113 C was obtained. EP dis- were then milled in a mortar to a fine powder. The
played a softening temperature at about 50 C three obtained formulations will be referred to as
and started decomposing at 180 C as also PHB/15EP, PHB/15LC, and PHB/15TA in the
indicated by TGA, while the thermogram of TA following. In the case of PHB/15EP, dumbbell-
was more regular, evidencing a relaxation phe- shaped specimens were obtained by means of an
nomenon at 171 C. injection-molding equipment, in which the mate-
rial was processed at 200 C for 5 min. Conversely,
PHB blended with LC was compression molded
10.4.2 Preparation at 180 C in order to obtain films 150 m thick.
and Characterization of PHB/ Both EP and TA were homogeneously and finely
EP and PHB/TA Blends blended in PHB, while LC particles were dis-
and PHB/LC Biocomposite persed in the matrix, acting as a filler fraction. A
proof of this statement is given by SEM micro-
Two bacterial poly(3-hydroxybutyrate) (PHB) graphs of the fracture surfaces of neat PHB,
grades, coded T3 and T19 (supplied by Biomer, PHB/15LC, and PHB/15EP, shown in Fig. 10.7. It
Germany), were used after drying under vacuum is remarkable to note that the surface of neat poly-
in oven. The polymers average molecular weights mer is smooth due to the brittle behavior of PHB,
(Mw), as determined by gel permeation chroma- while it appears to be rough and more ductile in
tography (GPC), were 840 and 890 Kg mol1, the presence of LC and EP. In particular, a bipha-
sic system consisting of a filler fraction dispersed
in a polymeric matrix was evident for the
Table 10.1 Thermal parameters of EP, LC, and TA under PHB/15LC biocomposite. Lignocellulosic fibers
nitrogen and particles as large as 50 m were embedded in
Sample Tonset (C) TDTG (C) Tg (C) Char700 C (%) the PHB matrix, suggesting the presence of weak
EP 148 182 50 23 interactions at the interface between the filler and
TA 239 277 171 27 the polymer. Conversely, PHB/15EP appeared
LC 289 354 113 36 characterized by several globular particles of size

Fig. 10.7 Fracture surfaces micrographs of (a) neat PHB, (b) PHB/15LC, and (c) PHB/15EP
148 S. Angelini et al.

Table 10.2 Mw of unprocessed PHB-T19 compared with that of processed PHB-T19, PHB/15TA, and PHB/15LC held
at 190 C for 4000 s
Unprocessed Processed
Mw (Kg mol1) 890 47 50 34

lower than 5 m well dispersed in the polymeric 100

Storage modulus change, %

matrix. Moreover, the observation of several voids PHB-T3
80 PHB/15LC
on the surface was attributed to the EP granules PHB-T19
pullout caused by the cryogenic fracture. 60

10.4.3 Effect of the Additives
on Processing and Thermal
Stability of PHB 0
0 200 400 600 800 1000 1200
It has been demonstrated that high temperatures Time, s

adopted upon PHB processing are responsible for Fig. 10.8 Percent change of storage modulus versus time
a dramatic change in molecular weight (Vogel at 10 rad s1 for the PHB-based samples at 190 C
et al. 2007), due to the thermally triggered poly-
mer chain scission. In this case, the effect of pro-
cessing was evaluated by holding PHB-T19, aspect was investigated in detail by measuring the
PHB/15TA, and PHB/15LC samples for a time rheological properties of PHB/15EP, PHB/15LC,
period of 4000 s in the chamber of a rotational and PHB/15TA melts. Actually, the rheological
rheometer at 190 C. GPC measurements were properties are strongly related to changes in
performed dissolving PHB-based samples in molecular weight, entanglement density, effect of
chloroform, and molecular weights were mea- additives, and fillers. The rheological behavior of
sured before and after the thermal treatment PHB was measured by means of parallel-plate
(Table 10.2). rotational rheometry under oscillatory conditions
Neat PHB-T19, PHB/15LC, and PHB/15TA at 190 C. As the linear viscoelastic properties in
underwent massive thermal degradation when dynamic tests are sensitive to polymer chain scis-
held at 190 C for long times. Therefore, the addi- sion, valuable insight can be gained about poly-
tion of such fillers did not improve the polymer mer stability under these severe thermal
thermal stability. GPC analysis was also per- conditions. Figure 10.8 displays the percent
formed on unprocessed and injection-molded change of storage modulus G of the PHB-based
PHB-T3 blends with EP. Mw of unprocessed samples as a function of time.
PHB-T3 (840 Kg mol1) was significantly higher From this figure, it can be noticed that, due to
than that of the injection-molded counterpart (134 thermal degradation, the G values relative to the
Kg mol1), indicating that even processing times two PHB grades dropped down dramatically.
as long as 5 min bring about thermal degradation However, T3 resulted to be more sensitive to high
(Chen et al. 2013), causing the molecular weight temperature than T19. It can also be observed
to decrease significantly. In this regard, it is worth that the presence of the lignocellulosic filler had
noticing that the addition of 15 wt% EP was able no significant impact on the course of the pro-
to efficiently hinder polymer chain scission dur- cess. A different finding was obtained for
ing processing, since a Mw value of 648 Kg mol1 PHB/15EP, as the decrease of G was signifi-
was recorded for PHB/15EP. This finding reveals cantly slowed down, confirming that EP improved
the efficiency of EP as processing stabilizer. Such the polymer thermal stability.
10 From Microbial Biopolymers to Bioplastics: Sustainable Additives for PHB Processing and Stabilization 149

According to these data, it can be supposed ysis. Figure 10.9 shows the result of the spectral
that EP can stabilize PHB thermally; thereby, the subtraction between PHB/15TA and PHB-T19
polymer can withstand the residence times usu- spectra acquired at 190 C.
ally adopted in common processing machines. A Herein, in addition to the obvious increases due
similar result was observed with the addition of to the TA absorption peaks at about 3400 cm1
tannic acid, which determined higher viscoelastic (phenolic OH), 1610 cm1 (resonance of the aro-
properties and clearly delayed the thermal degra- matic C = C of TA), and 1520 cm1 (in-plane bend-
dation process to longer times (Auriemma et al. ing of phenyl CH bonds), a decrease of the
2015). From the literature data, the mechanism 1740 cm1 ester band of PHB is evident, accompa-
for PHB decomposition consists in a statistical nied by the appearance of a very strong increase of
chain scission occurring by means of a the signal at 1705 cm1. This finding is related to
-elimination reaction, which generates crotonic the formation of intermolecular hydrogen bonds
acid and oligomers with a crotonate end group as between the carbonyl of PHB and the hydroxyl of
thermal degradation products (Ariffin et al. TA, which is responsible for the band shift toward
2008). This process is characterized by a stage in lower frequencies, analogous to what is observed
which a pseudo-six-membered structure is for catechin-PHB blends (Li et al. 2003).
formed. In this structure, the carbonyl oxygen In Fig. 10.10, the TGA thermogram of PHB-
abstracts hydrogen ion from the -methylene. based specimens under nitrogen is reported. The
Therefore, any species bearing acidic hydrogen main thermal parameters of the studied samples
ions may interfere with this autocatalytic cycle, are reported in Table 10.3. Neat PHB-T3 and
thus affecting the polymer thermal stability. EP PHB-T19 degraded through a single weight loss
and TA possess a number of phenolic OH groups step; however, the T19 grade was endowed with a
able to act as proton donors: it can be claimed higher thermal stability with respect to T3. In
that PHB C = O oxygen atoms interact with the fact, a shift of about 15 C exists between the two
hydrogen atoms of OH (Chen et al. 2002). These thermogravimetric curves. This is particularly
hydrogen bonds are likely to interfere with the remarkable, since the two polymers had compa-
synthesis of the transition structure. Such interac- rable molecular weight and crystallinity. It has
tion validates EP and TA capability of delaying been recently reported that acidic treatments can
the polymer degradation process. The physical increase thermal stability of PHB (Arza et al.
bondings between TA hydroxyls and PHB car- 2014). In this regard, it is then likely that the
bonyls are confirmed by the results of FTIR anal- extraction protocol and work-up during manufac-

Fig. 10.9 Spectral

subtraction between
PHB/15TA and PHB-T19
FTIR spectra acquired at 1616
190 C 0.02
Absorbance, a.u.




4000 3600 3200 2000 1800 1600 1400 1200 1000 800 600
Wavenumber, cm1
150 S. Angelini et al.

Fig. 10.10 TGA of neat 100

PHB-T3 and T19 and of PHB/15LC
their compounds with EP, PHB-T3
LC, and TA 80 PHB-T19

Weight, %



150 200 250 300 350 400
Temperature, C

Table 10.3 Thermal parameters of PHB and PHB com- 10.4.4 Effects of EP, LC, and TA
pounds measured by TGA on the Crystallization of PHB
Tonset (C) TDTG (C) Char 400 C (%)
PHB-T19 264 282 0 Both the processing parameters and mechanical
PHB-T3 252 266 0 properties of polymeric materials depend on the
PHB/15EP 272 280 3 crystallization behavior. Moreover, the knowledge
PHB/15LC 259 280 5 of crystallization mechanisms is important for the
PHB/15TA 283 293 3 design of materials with tailored properties. This is
particularly relevant in applications in the field of
turing operations play a role. From the figure, it is medicine and packaging. In our case, PHB shows
also clear that both the phenol-containing addi- a low nucleation density. This results in the growth
tives own the potential to increase the thermal of very large spherulites that are responsible for
stability of the polymer. An increase in degrada- the brittleness of this polymer (El-Hadi et al.
tion temperature by about 15 C was observed for 2002a).
PHB/15EP and PHB/15TA, with respect to the The crystallization kinetics of PHB and its
parent PHB-T3 and T19, respectively, indicating blends was investigated by differential scanning
a relevant stabilization effect ascribed to the addi- calorimetry (DSC). Figure 10.11 reports the DSC
tives rich in phenols. curves of the cooling step (rate 50 C min1)
On the other hand, from the figure, the effect of after fusion and the second heating scan (rate
LC on PHB-T19 reveals that this additive has no 10 C min1) of PHB-T3 and PHB-T19 and their
influence on the thermal stability of the microbial compounds with EP and LC, respectively. The
polyester. This finding accounts for the importance thermal data recorded through DSC measure-
of the physical state and dispersion of the additive. ments are listed in Table 10.4.
In fact, SEM observations (Fig. 10.7) showed that The experimental results show that the pro-
LC was not actually blended with PHB, since large cess of crystallization depends upon the presence
insoluble particles of LC were visible in the PHB and type of additive. Neat PHB-T3 (Fig. 10.11a)
matrix. The absence of fillermatrix interactions at had a melt crystallization at 59 C; on the con-
a molecular level was also confirmed by FTIR trary, PHB/15EP did not show any specific melt-
spectroscopy. In fact, notwithstanding the presence crystallization signal. This indicates that EP is
of phenolic hydroxyls in the lignin fraction of LC, responsible for delaying the process during rapid
no hydrogen bonding was detected by comparing cooling, restricting the mobility of polymer chain
the FTIR spectrum of PHB/15LC with that of (Mousavioun et al. 2010). This result confirms
PHB-T19 (Angelini et al. 2014). that EP enhances macromolecule entanglements,
10 From Microbial Biopolymers to Bioplastics: Sustainable Additives for PHB Processing and Stabilization 151

a PHB-T3
b 20
1,5 PHB15/EP

Heat flow, W g 1

Heat flow, W g 1

0,0 0

-0,5 heating heating

10 PHB-T19
-1,0 PHB/15LC

0 50 100 150 200 250 50 25 0 25 50 75 100 125 150 175 200

Temperature, C Temperature, C

Fig. 10.11 DSC of (a) neat PHB-T3 and PHB/15EP and (b) neat PHB-T19 and PHB/15LC. Only the cooling and the
second heating ramps are reported

Table 10.4 Thermal properties of PHB-based compounds measured by DSC

Hc, melt Hc, cold
Tc, melt (C) (J g1) Tg (C) Tc, cold (C) (J g1) Tm (C) Hm (J g1)
PHB-T19 67 12.0 4 47 68.7 175 87.4
PHB-T3 59 27.2 2 47 17.6 170 74.0
PHB/15EP n.d. n.d. 1 45 40.7 169 66.6
PHB/15LC 65 42.0 5 44 26.1 173 84.3
PHB/15TA n.d. n.d. 3 75 172

which allow to get enhanced viscous melts. This adding a commercial alkali lignin (AL) in
results in the delay in the chain rearrangements PHB. They recorded that AL did not promote
during the melt crystallization. The DSC second melt crystallization of PHB, while in the second
heating ramp showed for both samples a notable heating scan, the glass transition was found to be
glass transition and a cold-crystallization exo- easily detectable. This outcome was ascribed to
therm, which were followed by a single melting the pro-degrading effect of AL on PHB, which
endotherm. The introduction of additive, which provokes depolymerization and reduction of the
acted as a mild plasticizer, affected the glass tran- molecular entanglements. In DSC traces relative
sition temperature of the polymer. PHB/15EP to PHB and PHB/15EP, at temperatures above
exhibited a cold-crystallization peak in the tem- 200 C, a remarkable effect was observed. For
perature range, which varied from 50 to 45 neat PHB, a drop in the thermogram suggested
C. The slow heating process promotes crystalli- that a thermal degradation is going on. On the
zation of the amorphous part of PHB/15EP, contrary, in the blend a steady signal was
which was not able to crystallize while the rapid recorded. These features confirm that the addi-
cooling process was taking place. A very similar tives help in enhancing the thermal stability of
result was recorded in the presence of TA PHB.
(Auriemma et al. 2015), which also restricted the Figure 10.11b reports the DSC response of
crystallization of PHB from the melt, giving rise PHB-T19 and PHB/15LC. The cooling ramp
to a less-ordered crystalline form. Moreover, dur- subsequent to the first heating run and the second
ing subsequent heating, the cold-crystallization heating scan is displayed. It is worth highlighting
peak was detected at a temperature higher by that neat PHB-T19 displayed a small, broad peak
about 30 C when compared to the neat polymer. of melt crystallization, since the rapid cooling
Angelini et al. (2014) found a similar behavior, inhibited chain mobility and hindered the crystal-
152 S. Angelini et al.

lization process of the polymer. El-Hadi et al. Therefore, stabilization against the oxidative
(2002b) showed that the broadened signal of melt degradation phenomena is important to preserve
crystallization during a fast dynamic cooling PHB physical and mechanical properties. In a
scan could be ascribed to the formation of spher- bioactive environment, degradation of polymer
ulites of large size and low density, representative is initiated through material fragmentation,
of bacterially synthesized PHB (El-Hadi et al. which is then followed by mineralization. The
2002b). In the second heating step, the PHB-T19 enzymatic activity of microorganisms shortens
glass transition was clearly evident, thus confirm- and weakens the polymer chains (Mohee et al.
ing that most of the amorphous phase did not 2008). In particular, for biodegradable polyes-
crystallize upon cooling. Nevertheless, the slow ters, molecular chain breakage proceeds by
heating of the second scan induced the formation enzyme-catalyzed hydrolytic chain scission of
of crystallites, as shown by the presence of a ester bonds (Sarasa et al. 2009). In the present
sharp cold-crystallization peak, followed by a section, the effect of the natural additives on the
melting endothermic signal. It is interesting to photooxidative stability of PHB will be dis-
emphasize that the introduction of LC strongly cussed. Moreover, the behavior of PHB-T19 and
influenced the crystallization behavior of PHB. In PHB/15LC compounds from the microbial deg-
particular, it was evidenced that during cooling, radation point of view was qualitatively assessed
the melt crystallization of PHB was markedly through SEM observation of garden soil-buried
enhanced by the presence of the filler to an extent specimens.
proportional to its amount (Angelini et al. 2014). First, the effect of UV light irradiation on the
Literature data suggest that melt- and cold- properties of plain PHB-T19 and the same poly-
crystallization temperatures are indirect parame- mer modified with 2.5 wt% and 5 wt% of EP was
ters of the crystallization rate. Generally, a studied. Dumbbell-shaped films were exposed to
decrease of Tc, cold indicates faster crystallization, UV radiation in a weathering chamber contain-
whereas lower Tc,melt indicates slower crystalliza- ing a mercury vapor discharged lamp kept at 40
tion (Weihua et al. 2004). From the data recorded C. The exposure times used were 0, 1, 2, 3, 6,
for PHB/15LC, a drop of cold-crystallization and 12 days. After a visual inspection of the
enthalpy (Hc, cold) values in PHB/LC-based bio- samples exposed to UV light, the dumbbell-
composites confirms that the crystallization pro- shaped films were subjected to tensile tests, and
cess mainly occurred during the cooling scan. Young modulus (E), strength (b), and strain (b)
These results indicate that LC was a heteroge- at break were recorded and reported in
neous nucleating agent, potentially able to con- Fig. 10.12ac.
trol the physical aging of the polymer (Weihua PHB exposure to UV radiation caused several
et al. 2004). Finally, it should be observed that Tm changes in the mechanical response of the mate-
values of PHB-T19 and PHB/15LC were not sig- rial. Long residence times in the chamber (3, 6,
nificantly different, suggesting that the introduc- and 12 days) damaged the polymer to such an
tion of the filler as a nucleating agent did not extent that it broke down. Conversely, when EP
influence the overall crystallinity of PHB. was added, it was observed that tensile tests could
be also performed on the samples subjected to
longer irradiation times. It is worth to highlight
10.4.5 Photodegradation that, with increasing EP amount, both strength
and Microbial Digestion and strain at break decreased (Fig. 10.12b, c).
of PHB in the Presence of EP These results are attributed to scission reactions
and LC that become predominant upon UV exposure
(Sadi et al. 2010), which cause a reduction on the
Polymeric materials may degrade during pro- chain entanglements. The drop of molecular
cessing at high temperatures as well as during weight, followed by the chain rearrangement,
use in the presence of oxygen, light, and heat. leads also to an increase of crystallinity,
10 From Microbial Biopolymers to Bioplastics: Sustainable Additives for PHB Processing and Stabilization 153

Fig. 10.12 Young a 6000

modulus (a) stress at break PHB/5EP
(b) and strain at break (c) 5500 PHB/2.5EP

Young Modulus (E), MPa

versus time of exposure to PHB-T3
UV radiation of PHB-T3, 5000
PHB/2.5EP, and PHB/5EP




0 1 2 3 4 5 6 7 8 9 10 11 12
Exposure time, days

b 26 PHB/5EP
Strength at break (sb), MPa

24 PHB-T3






0 1 2 3 4 5 6 7 8 9 10 11 12
Exposure time, days

3 PHB/2.5EP
Strain at break (eb), %

0 1 2 3 4 5 6 7 8 9 10 11 12
Exposure time, days
154 S. Angelini et al.

responsible for a rise of the elastic modulus value additive for a sustainable approach to photooxi-
of more than 40 % (Fig. 10.12a) (Law et al. dative stabilization of PHB.
2008). The microbial biodegradation behavior of
Photooxidation of polymers occurs through a PHB-T19 and PHB/15LC compounds was quali-
mechanism involving several steps. First, peroxy tatively assessed through SEM observation of
radicals and hydroperoxides form. These species garden soil-buried specimens, in order to evalu-
are unstable to near ultraviolet (UV) exposure ate the effect of the lignocellulosic filler on the
and cleave to give radicals capable of initiating biodegradation rate of PHB. Film specimens
further oxidative reactions. As a result, new oxi- were buried in soil and taken out at time intervals,
dized products such as alcohols and primary car- and their surface was observed through electron
bonyl compounds (predominantly aldehydes and microscopy. Figure 10.13 displays SEM pictures
ketones) are produced. The primary carbonyl- of PHB-T19 and PHB/15LC after 0, 20, and 80
based oxidation products can undergo photolysis days of burial.
according to the so-called Norrish pathways, giv- From the micrographs, significant degradation
ing rise to carboxylic acids as ultimate products. due to the microbial attack caused by the micro-
The observed evidence suggests that EP primar- organisms present in the soil was evidenced by
ily acts by scavenging highly reactive oxygen the appearance of a rough surface of the film.
radicals and preventing free radical damage to Many small cavities were formed in the early
PHB by effective H-atom transfer, as already burial period, which merged together over the
reported in the case of Mater-Bi (Cerruti et al. course of the degradation process, eventually giv-
2011). According to these results, EP is proposed ing rise to large holes. The SEM analysis showed
as an efficient, renewable, and biocompatible that degradation process started in the amorphous

Fig. 10.13 SEM micrographs of PHB and PHB/15LC after 0, 20, and 80 days in soil
10 From Microbial Biopolymers to Bioplastics: Sustainable Additives for PHB Processing and Stabilization 155

regions of the polymer. This was in accordance several additives from renewable sources, some of
with data on the degradation of PHB and which are currently regarded as biowaste, were
poly(hydroxybutyrate- co -hydroxyvalerate) used to improve thermal, processing, and crystal-
reported by Boyandin and Prudnikova (2012) lization properties of this polymer, which are tar-
that observed a faster degradation of the less geted to engineer completely bio-based plastic
crystalline copolymer specimens. The soil burial materials. It has been shown experimentally that
tests also showed that the presence of LC affected the phenol-based additives, EP and TA, enhanced
the surface degradation rate of PHB. In compari- the thermal stability of biopolymer, PHB. As a
son with neat PHB, after 20 days, the surface of consequence, the polymer could maintain high
the biocomposite was still rather smooth and molecular weights after processing. This finding
compact. Degradation was more evident with was in accordance with the slower decay in stor-
increasing burial times, and bare lignin particles age modulus, which was observed through rheo-
distinctly emerged on the sample surface; how- logical tests. Physical interactions between the
ever, no large holes were visible. The observed polymer and the additive were considered as a key
retarding effect of LC can be explained taking factor to interpret the experimental data. LC, a lig-
into account different aspects. First, as shown nocellulosic biomass, influenced the melt-crystal-
before (Sect. 10.4.4), LC was observed to act as a lization kinetics and the crystallinity as a whole,
nucleating agent that promoted PHB crystalliza- acting as a heterogeneous nucleating agent, with a
tion and reduced the sensitivity of PHB to hydro- positive effect on the physical aging of PHB. It
lysis and microbial attack. Moreover, it should be was also demonstrated that EP improved the poly-
reminded that a slight antimicrobial activity can mer photooxidative stability. Finally, with increas-
be ascribed to LC (Dong et al. 2011). In fact, ing contents of LC, the biocomposites were more
after 80 days, PHB surface was cracked, while resistant to the biotic degradation due to the anti-
lignin and cellulose fibers were apparently less microbial activity of the lignin, suggesting the pos-
damaged. It is likely that during the degradation sibility to modulate the rate of degradation of the
process, the developing filler surface layer acted products made with these materials.
as a barrier and delayed water diffusion and
microbial digestion of the polymer film. These
data were in agreement with those reported in the 10.6 Opinion
study conducted by Mousavioun et al. (2012),
based on the burial of PHB/lignin films in the soil The use of natural additives is an attracting
of a plant pot for up to 12 months. Their work approach to modulate the properties of biopoly-
revealed that lignin inhibited the colonization of mers such as PHB. The described results are
the microorganisms and made the blends more remarkable with a view to developing sustainable
resistant to microbial and fungal attack. This was alternatives to synthetic polymer additives.
attributed to the oxygen-containing lignin func- Overall, these outcomes demonstrated the feasi-
tionalities (hydroxyl and carboxylic acid) which bility of using agro-food by-products and natural
affect the antimicrobial activity. biomasses as bio-resources, aimed at improving
natural biopolymer features in the transition from
biopolymers to bioplastics. Future efforts should
10.5 Conclusions address environmentally friendly approaches to
chemical modification of such additives aimed to
Poly(3-hydroxybutyrate) (PHB) is a biodegrad- get better compatibility between matrix and addi-
able polymer, whose marketing potential is limited tives/fillers. The improvement of the interactions
by some flaws such as brittle nature and melting between matrix and additives/fillers can lead to a
temperature close to degradation stage, which significant enhancement of properties of the
restricts the processing window. In this study, resulting blends and biocomposites.
156 S. Angelini et al.

Acknowledgment The authors wish to thank the Italian Bugnicourt E, Cinelli P, Lazzeri A, Alvarez V (2014)
Minister of Research for the financial support Polyhydroxyalkanoate (PHA): review of synthesis,
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Polym 71:583590. doi:10.1016/j. Stefania Angelini
carbpol.2007.07.001 received her M.Sc. degree
Van de Velde K, Kiekens P (2002) Biopolymers: overview in MolecularIndustrial
of several properties and consequences on their appli- Biotechnology from
cations. Polym Testing 21:433442. doi:10.1016/ Perugia University, Italy.
S0142-9418(01)00107-6 She is currently enrolled in
Vogel C, Morita S, Sato H, Noda I, Ozaki Y, Siesler HW a Ph.D. in Agricultural and
(2007) Thermal degradation of poly(3- Food Science with Naples
hydroxybutyrate) and poly(3-hydroxybutyrate-co-3- University, Italy, and she
hydroxyhexanoate) in nitrogen and oxygen studied by works at IPCB-Institute for
thermogravimetric-Fourier transform infrared spec- Polymers, Composites and
troscopy. Appl Spectrosc 61:755764. Biomaterials in Pozzuoli, Italy. She is working on ligno-
doi:10.1366/000370207781393370 cellulosic biomasses and ligninsulfonates and bioresidue
Volova TG, Boyandin AN, Vasiliev AD, Karpov VA, streams of biorefineries and paper industries. Her current
Prudnikova SV, Mishukova OV, Boyarskikh UA, interests are microbial polymers, polysaccharide hydro-
Filipenko ML, Rudnev VP, Xuan BB, Dung VV, gels, and solar cells.
Gitelson II (2010) Biodegradation of polyhydroxyal-
kanoates (PHAs) in tropical coastal waters and Pierfrancesco Cerruti
identification of PHA-degrading bacteria. Polym received his M.Sc. degree
Degrad Stab 95:23502359. doi:10.1016/j. in Chemistry and his Ph.D.
polymdegradstab.2010.08.023 degree in Materials
Wang N, Yu J, Ma X (2007) Preparation and characteriza- Engineering from
tion of thermoplastic starch/PLA blends by one-step University of Naples, Italy.
reactive extrusion. Polym Int 56:14401447. He is presently full-time
doi:10.1002/pi.2302 Researcher at IPCB-
Wang X, Sang L, Luo D, Li X (2011) From collagenchi- Institute for Polymers,
tosan blends to three-dimensional scaffolds: the influ- Composites and
ences of chitosan on collagen nanofibrillar structure Biomaterials in Pozzuoli,
and mechanical property. Colloids Surf B 82:233240. Italy. His current interests are natural and synthetic poly-
doi:10.1016/j.colsurfb.2010.08.047 mers, composites, and stimuli-responsive materials.
Weihua K, He Y, Asakawa N, Inoue Y (2004) Effect of
lignin particles as a nucleating agent on crystallization
of poly(3-hydroxybutyrate). J Appl Polym Sci
94:24662474. doi:10.1002/app.21204
160 S. Angelini et al.

Barbara Immirzi Gennaro Scarinzi

received her M.Sc. degree received his M.Sc. degree
in Chemistry from in Chemistry from
University of Naples, Italy. University of Naples, Italy.
She is presently a He is presently a Researcher
Researcher at IPCB- at IPCB-Institute for
Institute for Polymers, Polymers, Composites and
Composites and Biomaterials in Pozzuoli,
Biomaterials in Pozzuoli, Italy. His current interests
Italy. Her expertise is in gel are thermoset polymers and
permeation chromatogra- lignin-based materials.
phy (GPC). Her current
interests are biodegradable
polymers applied in agri-
Mario Malinconico
culture and in nursery.
received his M.Sc. degree
in Industrial Chemistry
Merima Poskovic
from University of Naples,
received her bachelors
Italy. He is presently the
degree and Master I level in
Chief Scientist at IPCB-
Engineering in Plastic
Institute for Polymers,
Material from Polytechnic
Composites and
of Turin, Italy. She is cur-
Biomaterials in Pozzuoli,
rently student grant full time
Italy. He has been appointed
at Novamont S.p.A. Her
as Italian Representative at IUPAC by the President of the
expertise is in plastic materi-
Italian CNR for 2011. His current interests are biodegrad-
als, innovative technologies,
able polymers in packaging, aeronautics, and biomedical
and laboratory characteriza-
and agricultural fields.
tion: analytical and

Gabriella Santagata
received her M.Sc. degree
in Chemistry from
University of Naples, Italy.
She is presently a
Researcher at IPCB-
Institute for Polymers,
Composites and
Biomaterials in Pozzuoli,
Italy. Her current interests
are biodegradable polymers
applied in agriculture, food packaging, and biomedical
The Survivors of the Extreme:
Bacterial Biofilms 11
Neha Dubey, Raja Singh, Aditya K. Sharma,
Sharmila Basu-Modak, and Yogendra Singh

Biofilms are bacterias way of behaving like a multi-cellular organism.
Bacteria have been constantly evolving in the face of myriad natural chal-
lenges since the first life form appeared. Over the centuries, they have
survived under extreme conditions by virtue of their ability to form bio-
films. Biofilms have proved to be an immensely strong collaborative effort
of bacteria, and this interaction is administered via their quorum-sensing
mechanism. A biofilm plays a crucial role in survival, dispersal, transfer of
resistance genes, and generation of diversity among bacteria. It can also
act as a pathogenic factor for virulent bacteria and biofilms are often listed
as the major cause of many diseases, such as endocarditis, cystic fibrosis,
etc. Bacterial biofilms can be formed on almost every surface and, thus,
have deleterious effects on many indwelling medical devices and indus-
trial equipment. Many methodsfrom antibiotics to ultraviolet (UV)
radiationare currently being used to eliminate or reduce biofilm.
However, the most effective and eco-friendly measure involves the target-
ing of the root phenomenon, quorum sensing. Biofilm formation and dis-
persal mechanisms are being studied to increase the efficiency of biofilm
elimination. Despite the many harms they can pose, these biofilms have
been efficiently manipulated to be used for various purposes such as
wastewater treatment, microbial fuel cells, drug delivery, nanobiotechnol-

A.K. Sharma
Lab 208, Allergy and Infectious Diseases,
CSIR-Institute of Genomics and Integrative Biology,
Delhi University Campus, Mall Road,
N. Dubey (*) S. Basu-Modak New Delhi 110007, India
Department of Zoology, University of Delhi,
110007 Delhi, India Academy of Scientific & Innovative Research
e-mail: nnehadubey18@gmail.com; (AcSIR), 2- Rafi Marg, Anusandhan Bhawan,
sbasumodak@zoology.du.ac.in New Delhi 110001, India

R. Singh Y. Singh
Special center for molecular Medicine (SCMM), Allergy and Infectious Diseases, CSIR-Institute
Jawaharlal Nehru University, of Genomics and Integrative Biology,
110067 New Delhi, India Room No. 208, Mall Road, 110007 Delhi, India
e-mail: t.rajasingh1786@gmail.com e-mail: ysingh@igib.res.in

Springer India 2015 161

V.C. Kalia (ed.), Microbial Factories, DOI 10.1007/978-81-322-2595-9_11
162 N. Dubey et al.

ogy, etc. Biofilms bring about a very detailed level of complexity that
helps for better persistence of the bacterial population and at the same
time, provides us a valuable tool to address several important environmental
issues. Thus, it will be appropriate to term bacterial biofilms as remarkably
proficient assemblies of the life forms.

11.1 Introduction 11.2 Biofilm Structure

and Functions
Microorganisms are an important part of our eco-
system that have evolved over the centuries. Biofilm is mainly composed of the same or other
During evolution, microbes must undergo many species of bacteria comprising around 15 % pop-
stresses to successfully survive and divide in ulation and 85 % exo-polymer material, mainly
extreme conditions. The organisms that counter- polysaccharides. Biofilms can vary in thickness
act these external stresses eventually direct the from a monolayer to a thickness of 68 cm but,
path of their own evolution. One of the major on average, are about 100 m thick. Biofilm for-
mechanisms behind their survival is the tendency mation is a complex process involving a number
to stay together (Kalia 2014a; Kalia and Kumar of steps starting from initial adhesion to the final
2015a). Bacteria tend to reside in the form of community establishment (Donlan 2002). The
adherent communities to enable them to cope first bacterial counterparts that adhere to the sur-
with environmental pressure. It is widely estab- face generally form a weak van der Waals force-
lished that bacteria in communities survive much like interaction; this step is called the initial
better than their planktonic versions. These attachment. Later, upon non-dispersal of the first
adherent, self-supporting communities are commensals, the attachment is strengthened
termed biofilms. Biofilms are the most success- using cell adhesion molecules such as bacterial
ful bacterial environment, evolved to sustain pili, fimbriae, and cell surface proteins leading to
microorganisms from stressed environments and the irreversible attachment (Hinsa et al. 2003).
fulfill the nutrient requirements of the bacteria. These bacterial pioneers facilitate the arrival and
The first report defining the existence of bacterial adherence of the bacterial population; some
communities was by Zobell (1943). Later on, attach directly to the adhesion sites while others
Costerton devoted a majority of his work to attach to the colonizing bacteria and exopolysac-
understanding the mechanisms and benefits of charide. One interesting aspect of biofilm biology
biofilm formation (Costerton et al. 1978). Within is the communication aid. The colonizing bacte-
biofilms, the bacterial cells secrete a gluey mate- ria in the biofilm tend to communicate via quo-
rial called exopolysaccharide (EPS) that helps rum sensing, which is both an intra-species as
them adhere to any living or non-living surface, well as an inter-species phenomenon that serves
such as medical implant materials, soil particles, as a simple communication network. It regulates
metals, plastics, and, most significantly, human a number of different processes involving various
or animal tissue. A vast number of pathogens chemical messengers that bring together bacteria
often stay as biofilms to combat the hosts under a highly organized societal frame. This cel-
immune system. The biofilm bacterial population lular communication marks the maturity of the
shows a reduced growth rate and an altered tran- biofilm (De Kievit et al. 2001). The most impor-
scriptional profile. Additionally, the bacterial tant aspect is the dispersal of the biofilm.
community in biofilms are up to 1000 times more Dispersal is necessary to recruit the new micro-
resistant to anti-microbial stresses (Kalia et al. bial population and for proliferation of the bio-
2014; Kalia and Kumar 2015b). film entity. This periodic dispersal of planktonic
species is mediated by some detachment signals.
11 The Survivors of the Extreme: Bacterial Biofilms 163

Biofilms are coordinated structures that accom- hydrophobicity of the suspended matter influence
plish their own metabolic requirements; hence, the bond strength between the microbes and the
they are often compared to the higher organism surface by altering the substrate characteristics.
tissues that orchestrate their own development Other characteristics of the substrate, like tem-
and survival (Hall-Stoodley et al. 2004). (Figs. perature, pH, metal ion composition, etc., add to
11.1, 11.2, 11.3, 11.4, and 11.5) the complexity of biofilm dynamics. The sea-
The substrate is an important part of the bio- sonal behavior of biofilms along water bodies has
film assembly. Biofilms can be formed on almost been studied in detail by researchers and has been
every living or non-living surface, for example, attributed to local temperature fluctuations (Else
water bodies, seashore, rocks, pipelines, stagnant et al. 2003). Tidal and wind velocity also affects
pools, hot and cold springs, sewage pipes, pros- the establishment of primary pioneers of the bio-
thetics, body tissue, enamel, gut, nails, the inner film organization (Soini et al. 2002). Fletcher and
lining of lungs, and almost every body tissue. coworkers found an increase in cationic metal
These communities harbor interstitial water net- concentrations minimized the repulsive forces
works that channel the diffusion of nutrients between the bacterial cell and glass material,
across the biofilm. Broadly speaking, it has been thereby affecting the attachment of Pseudomonas
shown that biofilms often form comfortably on a fluorescens to the surface (Fletcher 1988). An
solid rough surface by virtue of fewer non- increase in the nutrient levels enhances the sur-
tripping forces and large surface areas. The vival of the microbial community (Bowden and
moistness of a surface enhances the likelihood of Li 1997). Cellular appendages such as pili and
biofilm formation by providing a conditional fimbriae are found to play an important role in
matrix. The moistness immediately conditions minimizing electrostatic repulsion by virtue of
the surface with different polymers and organic their hydrophobicity caused by the presence of
matters that facilitate the biofilm (Love 2010). large numbers of non-polar amino acids. Several
Conditional films can range from organic poly- studies have provided evidence of the relation
mers to human secretions like mucous, blood, between the surface protein hydrophobicity and
tears, gingival fluid, etc., as in the case of dental substrate adherence (Rodrigues and Elimelech
plaques, where the bacteria rapidly form biofilms 2009). Gram-negative bacteria consist of a hydro-
on the conditioned enamel of the tooth (Rosen philic O layer (LPS); this is why it is unable to
et al. 2005). Furthermore, the surface energy and create a functional biofilm. Perhaps this is

Weak Vander
wall Forces
EPS film




Fig. 11.1 Biofilm formation. Steps of biofilm formation: of the bacterial communities within the biofilm. 4.
1. Arrival of planktonic bacteria on a conditioned sub- Maturation of a biofilm. 5. Dispersal
strate. 2. Establishment of pioneer community. 3. Growth
164 N. Dubey et al.

Initial biofilm : Quorum sensors Mature biofilm : Quorum sensors

secreted in the biofilm environment secreted and are being absorbed by
neighbouring bacteria and establish
cell communication signalling

Fig. 11.2 Population dependent initiation and stimulation of biofilm development of quorum sensor

Fig. 11.3 Most Gram-negative bacteria have a LuxI/ secreted molecules are taken up by neighboring cells.
LuxR system. LuxI encodes the quorum-sensing mole- Inside cells, these molecules bind to a protein sensor
cules called autoinducers, namely AI-1, AI-2, and Acyl receptor encoded by LuxR. This complex then binds to
homoserine lactones. These quorum-sensing molecules the promoter regions of the target genes and hence control
are secreted in the environment constitutively. As soon as the transcription of the quorum-sensing-related processes
the population density crosses a threshold level, these

responsible for the differential biofilm formation and other ketal-linked pyruvates (Nwodo et al.
capacity between Gram-positive and Gram- 2012). The EPS matrix depends on two important
negative bacteria. properties governing the architecture and the
The key organic element of the biofilm are the strength of the biofilms. (1) The composition and
EPSs, which are mainly high-molecular-weight structure of the polysaccharide components.
polymers secreted by the bacteria to maintain the Most of the Gram-positive bacterial EPSs are
structural and functional integrity of the biofilms composed of hexose residues with either a 1, 3-
(Flemming and Wingender 2010). It mainly com- or 1, 4--linked backbone that confers the
prises sugar moiety along with non-sugar ele- strength and non-deformity to the biofilm; and
ments like protein and nucleic acids other than (2) The shape and age of the matrix. Biofilms are
organics like acetate, pyruvate, succinate, and structurally non-uniform, and the amount of EPS
phosphate. It is anionic in nature, conferred by varies hugely between young and old biofilms.
mannuronic acids, D-glucuronic, D-galacturonic, Additionally, over time, the EPS accumulates
11 The Survivors of the Extreme: Bacterial Biofilms 165

Fig. 11.4 Most Gram-positive bacteria have a LuxI/ phorylation and dephosphorylation events, affecting
LuxR system. In this system, LuxI encodes the quorum- downstream transcription factors. These specifically acti-
sensing oligopeptide moieties. These oligopeptides are vated transcription factors bind to the promoter regions of
secreted in the environment and, upon proper signal stim- specific target genes and hence regulate quorum-sensing-
ulation, binds to the surface-located LuxR receptors on specific transcriptome
the other cells. This binding facilitates a series of phos-

Fig. 11.5 An overview of biofilm: characteristics, functions and applications

166 N. Dubey et al.

components such as substratum particles, metal diversity and distributes antibiotic resistance but
ions, other cell macromolecules (such as pro- also synchronizes the biofilm cycle and popula-
teins, DNA, lipids, etc.) (Sutherland 2001). EPS tion (Nguyen et al. 2010). Thus, these biofilms
secretion is affected by the nutrient status of the serve as a diverse source of species (Peyyala and
adherence medium. Hydration of EPS prevents Ebersole 2013). The ability to secrete EPSs is
the desiccation of the biota. Extracellular DNA is higher in the concerted population spheres of the
part of the outer layer of the biofilm and, in some biofilm, while the outer edges with a looser
cases, has been seen to provide strength to the population helps in dispersion and migration of
biofilm structure. A recent report proves that the population. Alginate is the major component
physical interaction of the extracellular DNA and that regulates the formation and the dissemina-
EPS component Psl is crucial for the formation tion of bacterial biofilms (Hay et al. 2013). The
and stabilization of the biofilm skeleton in dissemination of biofilm is regulated by the
Pseudomonas aeruginosa (Wang et al. 2015). expression of the gene alginate lyase. Researchers
Among the membrane components, glucosidases have proved the important role of algL and algX
and N-acetylglucosaminidase are shown to genes in alginate biosynthesis and have also
reduce the attachment of bacteria with substrates showed an increase in the expression of algL sub-
in bacteria, e.g., P. fluorescence and Desulfovibrio stantially increased the shedding of biofilm,
desulfuricans. Additionally, LapA, a secretory while down-regulation of the gene resulted in a
protein, is well elucidated to act as an anchorage firm biofilm production (Robles-Price et al. 2004;
between bacterial cells. In P. aeruginosa, SadB is Albrecht and Schiller 2005). However, another
known to mediate adhesion and establish a more consecutive report claimed the necessity of these
permanent stage of biofilm formation (Caiazza genes for Pseudomonas biofilm architecture and
and OToole 2004). tenacity yet denies its importance in the forma-
Bacteria benefits from biofilm assembly in tion or seeding of the biofilm (Stapper et al.
many ways. The mode of communication estab- 2004). The basis behind this fascinating collabo-
lished between the communities acts as a method ration between bacteria in the biofilm lies in the
to channel nutrients, water, and cellular signals more dense and compact assembly, which leads
across the biofilm (Karatan and Watnick 2009). It to a more precise and coordinated exchange of
is also an established mode of antimicrobial signals and thus enhances the efficiency com-
resistance transfer (Hannan et al. 2010). The bio- pared with planktonic forms. Cyclic diguanosine-
film population is more resistant to many antimi- 5monophosphate (cyclic di-GMP) is the
crobial stresses than its planktonic counterparts. foremost intracellular secondary messenger that
A biofilm can be composed of single as well as positively regulates initiation and formation of
multiple species. Many natural biofilms formed biofilm (Hickman et al. 2005; Borlee et al. 2010).
across the global atmosphere usually comprise Biofilm formation relies on cell-to-cell com-
unions of multiple species. The biofilms that are munication systems that are coordinated by quo-
composed of different bacterial communities are rum sensing. Planktonic bacterial populations,
often stronger and thicker as different species upon reaching a threshold number, secrete chem-
synchronize together to stabilize the biofilm. ical messengers called quorum-sensing molecules
They has also been shown to enable the transfer into the environment and thus initiate and execute
of nucleic acid across strains in the form of con- a strictly population-dependent quorum-sensing
jugation (Molin and Tolker-Nielsen 2003). The signaling system. These chemical autoinducers
act of conjugation is synergistically coordinated are transcription factors that are activated only
amongst bacterial communities in the system. when the population reaches a certain number.
The DNA released in the environment for the These inducers bind to specific receptors and
process is autocatalytic and helps in biofilm com- hence regulate the up-regulation or down-
munity stabilization. These inter- and intra- regulation of genes in a manner that can benefit
species DNA transfers not only generate the the bacterial community and prevent assets from
11 The Survivors of the Extreme: Bacterial Biofilms 167

the competing planktonic or non-planktonic bac- film (Lynch et al. 2002). In organisms like
teria (Waters and Bassler 2005). In Gram- Bacillus cereus and Helicobacter pylori, AI-2 is
negative bacteria, the chemical inducer is acyl shown to negatively regulate the biofilm forma-
homoserine lactones (AHLs) auto inducer-1 tion on glass and polystyrene surfaces, respec-
(AI-1) which is secreted by the bacterial cells tively. Listeria monocytogenes has a regulon that
and, under proper threshold stimulus, enters the has been proved to play an important role in bio-
neighboring cells and binds to the protein recep- film formation. This regulon has four genes,
tor LuxR (Whitehead et al. 2001; Laverty et al. agrB, agrD, agrC, and agrA. In L. monocyto-
2014). Gram-positive bacteria, on the other hand, genes, the deletion mutants of agrA and agrD dis-
secrete peptides that bind to the cell surface played a defect in biofilm formation (Vivant et al.
receptor on other bacterial cells (Fleuchot et al. 2014). E. coli has a LuxS/AI-2 system, but its
2011; Monnet et al. 2014). These signals are role in biofilm formation is unclear. Rather, a
detected by two component sensors, leading to LuxR homolog/ sdiA (suppressor of cell division
the phosphorylation of the rear end proteins, inhibition) deletion mutant in E. coli showed an
which eventually bind to the DNA to impact the enhanced biofilm contributed to its function to
transcription of the target genes (Lyon et al. 2002; suppress the motility and biofilm formation in
Jimenez and Federle 2014). The common wild type (Sharma et al. 2010). In P. aeruginosa,
quorum-sensing mediator of both Gram-positive disruption of the AHL-producing gene lasI dis-
and Gram-negative is AI-2. AI-2 consists of played a major defect in biofilm formation
boron in a protein assembly that is produced by (Heydorn et al. 2002). Such wide examples
catalysis of LuxI enzyme. In most organisms, the clearly establish the inevitable role of quorum
LuXI/LuxR system composes the quorum- sensing in biofilm formation. Researchers are try-
sensing machinery, LuxR being the receptor. ing to find ways to break this quorum-sensing-
This decentralization of the quorum sensing mediated rhythm to prevent the formation of
brings together the bacterial micro-colonies as a biofilms. This inhibition can be accomplished in
well-synchronized system. Quorum sensing is a many ways, mostly targeting the auto inducer and
particularly crucial phenomenon during pro- receptor genes along with interfering with the
cesses like biofilm formation, conjugation, anti- secretion of quorum-sensing molecules
biotic resistance, and pathogenicity. Some enteric (Kjelleberg et al. 2008; Kalia 2013, 2014b; Kalia
pathogens, like Escherichia coli, Shigella, et al. 2011; Kumar et al. 2015). Recently, one of
Salmonella, Yersinia, and other Gram-negative the most robust P. aeruginosa biofilms has been
species have auto inducer type 3, which is a epi- shown to be inhibited by a synthetic compound
nephrine/norepinephrine-regulated system meta-bromo-thiolactone, which is targeted
(Walters and Sperandio 2006a, b). Other known against the quorum-sensing receptors LasR and
quorum-sensing molecules are indole; 3-hydroxy RhlR (OLoughlin et al. 2013).
palmitic acid methyl ester (3OH PAME);
3,4-dihydroxy-2-heptylquinolone (PQS); butyro-
lactones; and cyclic dipeptides (Kalia and Purohit 11.3 Epidemiology Caused by
2011). Campylobacter jejuni is a food-borne Biofilms
pathogen capable of forming biofilms in food as
well as in related industrial equipment. It has Globally, much focus has been placed on epi-
been shown that the LuxS gene mutant of C. demic diseases. These epidemics are often caused
jejuni showed a significant decrease in biofilm- by certain planktonic bacteria that are able to
forming capability compared with wild-type aggravate our immune system all at once and
(Reeser et al. 2007). The same effect is seen in give rise to severe to chronic infections. In the
Aeromonas hydrophila, a facultative aerobe in past, a large number of these epidemics has been
which the mutation in Acyl homoserine lactone controlled by targeting the specific structure of
(AHL) synthesis gene resulted in a desolated bio- the bacteria, such as proteins, DNA, membranes,
168 N. Dubey et al.

etc. Over time, we have been minimally success- common bacterial population involved belongs to
ful in getting rid of a few epidemics, but it would the coccus group like Streptococci, Enterococci,
not be wrong to say that we still have organisms and Staphylococci, further inhabited by fungal
around us that can prevail and cause many life- groups such as Candida and Aspergillus (Tunkel
threatening diseases. One major problem that sci- and Mandell 1992; Verma et al. 2006). Any
entists face during their research is the wound or injury at the endothelial point results in
non-reproducibility of the data when dealing the formation of thrombotic lesions primarily
with bacteria in vitro and in vivo. The root of this made up of platelets, fibronectin, and red blood
difference lies with the adaptation and survival cells (RBC). Under these conditions, the persist-
tactics of the bacteria in the form of biofilms. ing blood bacteria adhere and colonize the site of
Biofilms are often seen succeeding in the cases of infection. Fibronectin acts as a major point of
most relapsing diseases that are acute but consis- adhesion for these bacteria. Several bacteria pos-
tent. These diseases progress through seasonal sess fibronectin receptors that strengthen the
inertness and subsequent exacerbations, for binding. S. aureus executes this adhesion via
example, diarrhea (E. coli), dental plaque (H. many receptors such as surface receptors SasC
pylori), Staphylococcus aureus infection, middle and SasG along with biofilm-associated protein
ear infections (Haemophilus influenza), and Bap, serine aspartate repeat protein SdrC, and
many more. Time and again, such incidences fibronectin/fibrinogen-binding proteins FnBPA
have been seen to be frequent in the case of bio- and FnBPB (Vazquez et al. 2011). In
medical engineering, be it life related or equip- Staphylococcus epidermidis, accumulation-
ment related. The implantation of grafts and associated protein (Aap) and extracellular matrix-
prosthetics in advanced medicine unveiled sev- binding protein (Embp) are known to mediate
eral such cases of biofilm dispersal that eventu- cellular adhesion and biofilm formation. In
ally give rise to secondary chronic infections. Staphylococcus lugdunensis, surface-localized
Therefore, it would not be wrong to consider bio- AtlL is shown to affect biofilm formation as well
films as a successful factor for pathogens (Donlan as its internalization in eukaryotic cells (Hussain
and Costerton 2002). et al. 2015). Upon embedment and stabilization
Among the pathogenic bacterial population, in the endothelial fibrin matrix, the bacteria soon
different bacteria are known to form persistent start to multiply and coat themselves in fibrin
biofilms, e.g., L. monocytogenes, Legionella capsules, which protects it from white blood
pneumophila, S. aureus, Campylobacter spp., E. cells. These biofilms are known to shed cell
coli, Salmonella typhimurium, Vibrio cholera, clumps, which eventually cause emboli. These
Pseudomonas spp., Rhizobium spp., Enterococcus bacterial- and fungal-loaded infective emboli
spp., Candida spp., etc. These, along with many then spread the infection and can cause severe
other opportunistic pathogens, are the main complications throughout the body. Depending
causes of many diseases in humans, namely cys- on the type of bacterial endocarditis, various anti-
tic fibrosis, dental plaques, native valve endocar- biotic doses are ascertained. Penicillin was used
ditis, periodontitis, prostatitis, etc. The disease is against streptococcal endocarditis, while vanco-
spread through various methods, mostly upon mycin and rifampicin have been used as antibac-
establishment of the biofilm in a vulnerable area; terials against staphylococcal endocarditis
the cells detach in a timely manner from the site (Riedel et al. 2008). Candidal endocarditis has
and are circulated across the body. The suscepti- been treated successfully with fluconazole (Lye
ble areas catch infections and therefore increase et al. 2005). Although the antibacterial dosage
the complexity of the disease. The other method proved sufficient to control the endocarditis com-
of progression is the secretion of toxins by the plication, the very basis of biofilm establishment
Gram-negative population of the biofilm. is poorly understood.
Native valve endocarditis is a disease of the Other common forms of severity caused by
vascular connective and endothelial tissue. The opportunistic bacteria are periodontitis, which
11 The Survivors of the Extreme: Bacterial Biofilms 169

can be caused by different bacterial species such influenza (Starner et al. 2006; Cardines et al.
as Fusobacterium spp., Bacteroides spp., and 2012) and S. aureus (Hirschhausen et al. 2013).
Porphyromonas gingivalis (Darveau 2010). The focus has long since shifted towards infringe-
These bacteria utilize the conditioning film of ment of the signaling mechanism responsible for
pellicle over the enamel, which comprises phos- the tenacity of biofilm (Davies et al. 1998;
phoproteins, gingival crevice fluid, albumin, Bjarnsholt et al. 2010). A recent report has shown
lysozymes, glycoproteins, and lipids. The bacte- that the mucoid phenotype and biofilm formation
ria reside in subgingival crevice and attach with by Pseudomonas is ion-regulated, favoring the
the pellicle via extracellular glucan, further fol- concept of targeting the iron homeostasis of the
lowed by characteristic polysaccharide secretion. bacterium in the lung airway (Wiens et al. 2014).
This congregation eventually turns into dental Biofilms are a major impediment to the use of
plaques of 50100um thickness. The administra- indwelling medical devices. The incidence of
tion of tetracycline and metronidazole, along biofilm-dependent complications is usually seen
with N-acyl homo serine lactone analog, is sug- in cases of biomedical devices such as urinary
gested to be preventive for periodontitis (Gamboa catheters, prosthetic valves, and central venous
et al. 2014). catheters (Trautner and Darouiche 2004; Donlan
In cystic fibrosis, P. aeruginosa usually bene- 2011). The source of initial contamination could
fits from transmembrane conductance defects. be many, per se, blood- and tissue-borne
Disease is caused by the genetic mutagenesis of microbes, ill health, administration practices, and
the cystic fibrosis transmembrane conductance external factors, etc. When a catheter is inserted
regulator (CFTR), responsible for mucus, sweat, into the body, it stays in direct contact with blood
and digestive juice production. The disturbance and tissues and eventually is covered by cells,
in the ionic conductance within the body cavities blood platelets, tissue secretions, mucous, urine,
results in uncontrolled mucous production, which and fibrin. These accumulations act as a condi-
eventually thickens the cough and thereby acts as tioning film for the circulating bacteria. The most
a suitable habitat for the bacteria (Hiby et al. common pathogenic colonized cases are S.
2010). The bacteria are highly antibiotic tolerant aureus, Klebsiella pneumonia, P. aeruginosa,
because of the hypoxic, mucus-rich collaborative Enterococcus faecalis, and Candida albicans.
biofilm environment; thus, treating the Intrauterine devices are a major source of intra-
Pseudomonas biofilm in cystic fibrosis patients is uterine infections. Researchers have shown heavy
extremely difficult. The infection caused by P. depositions of biofilm in the distal end of such
aeruginosa is resistant to immune system clear- devices, which are heavily loaded with bacteria
ance methods such as opsonization because the such as E. coli, S. epidermidis, Enterococcus
thick mucoid alginate layer prevents the antibody spp., and anaerobic Lactobacilli spp.,
coating necessary for opsonization. However, a Corynebacterium spp., Micrococcus spp., C.
few reports have shown an effective concentra- albicans, and S. aureus, etc. (Wang et al. 2010).
tion of colistin and tobramycin in combination Many methods are being tried to eliminate or
with meropenem and ciprofloxacin to kill the reduce infections originating from biofilms.
bacterium when tested on over 15 clinical iso- Antibiotics such as tobramycin, chlorhexidine,
lates (Herrmann et al. 2010). As the pathology of and ciprofloxacin have been conservatively used
P. aeruginosa largely depends on mucus produc- to treat catheter-based biofilm infections to pre-
tion, the use of hypertonic solution has been vent biofilm accumulation on medical devices. It
demonstrated to indirectly reduce the bacterial has been shown that the ultrasound treatment of
persistence as it prompts the clearance of mucus indwelling medical devices prior to administra-
obstruction when carried out on a regular basis, tion reduces the risk of bacterial biofilms such as
although the long-term effect is yet to be exam- Pseudomonas diminuta, P. aeruginosa, and L.
ined (Elkins et al. 2006). Species other than P. monocytogenes (Hol et al. 2010; Torlak and Sert
aeruginosa that aid the disease severity are H. 2013). An anti-parasitic drug, nitazoxanide, has
170 N. Dubey et al.

been reported as inhibiting staphylococcal Researchers have been trying to devise tech-
biofilms, probably by blocking biofilm accumu- niques and methods that can utilize the benefi-
lation rather than affecting the dispersal cial part of the biofilm (Kalia and Kumar 2015a;
(Tchouaffi-Nana et al. 2010). Pentasilver hexa- Clark et al. 2012) .
oxoiodate (Ag5IO6) coating on medical devices
has been shown to have strong anti-biofilm activ-
ity (Incani et al. 2014). Another well-known 11.4.1 Waste Water Treatment
approach targeting the EPS component of the
biofilm via antagonistic and hydrolyzing enzymes Water is the most essential element of nature.
could be effective for the known lab-grown Water scarcity is now a major issue being faced by
organisms. An earlier study demonstrated the up- the human race. Many national government agen-
regulation of alginate lyase with the diffusion of cies have been trying to find an effective method
the antibiotics gentamicin and tobramycin across to overcome this problem, via water conservation
P. aeruginosa biofilms because of high EPS deg- and waste water treatment. Waste water has many
radation, resulting in a loosened matrix (Hatch contaminants, including particulate matter, micro-
and Schiller 1998). However, a major setback of organisms, organic matter, heavy metals, and
these studies was the variability of the EPS being excess nutrients such as nitrogen, phosphorus,
secreted by different biofilm-composing bacteria. etc. Current industrial wastewater treatment sub-
Though EPSs include many polymorphisms, the units are high-maintenance and high-cost con-
genetic variation of the EPS-producing genes is suming processes. Thus, microorganisms have
comparatively conserved and this could possibly been utilized to produce biofilm-based bioreac-
be used to identify certain drugs or compounds tors (Wagner and Loy 2002). Biofilm process
that can block or inhibit the transcription and treatment has several advantages over conven-
translation of the participating proteins. One tional waste water methods: convenient operation,
interesting method for disrupting the biofilm is environmentally friendly, high biomass activity,
targeting the quorum-sensing mechanism. higher toxin resistance, and low cost (Tao et al.
Researchers are putting effort into discovering 2010). Biofilm-based bioreactors are established
the genetic basis of quorum sensing. Davis et al. either on a static substrate or on a conditioned
showed the significance of acyl-homoserine lac- synthetic substrate system. In fixed-type bioreac-
tones (signaling molecules) in biofilm segrega- tors, matter such as rock, sand, or plastic is used to
tion (Davies et al. 1998). Thus, continuing on the condition biofilm, e.g., trickling filters, rotating
same line, if we were able to explore the crucial biological contactors, and sand filters. Second are
signaling paradigm behind it, we might be able to the suspension type in which microorganisms are
intervene in biofilm formation. suspended in the waste water where they multiply
and eventually settle at the bottom of the reactor
as activated sludge. This bio-sludge filters the
11.4 Applications water, and the sludge can be changed as neces-
sary, e.g., activated sludge, extended aeration, and
Microbes constitute the largest biomass of the sequential batch reactor systems. Third are the
earth. We have been living amongst all sorts of lagoon systems in which waste water is treated in
microbes since the rise of human beings. Not all water bodies such as lakes by the persisting micro
microorganism are harmful; some actually flora of the water bodies (Mancl 2002). The
contribute to maintenance of the well-being organisms in a biofilm-based bioreactor are selec-
of the human system, e.g., gut colonizers. tively used to remove the organic matter and to
Microorganisms actually tend to recycle some cause denitrification and dephosphorylation of the
of the vital elements of life, e.g., minerals, waste water (Yang et al. 2010). This process is
water, and atmospheric gases, etc. Similarly, not implemented in either fixed bed or moving
all the biofilms around us are harmful. reactors. Advantages of the moving reactors
11 The Survivors of the Extreme: Bacterial Biofilms 171

include better usage of reactor volume. In 1860, and silicone coatings as inert surface coating
the first sand filter treatment methods were used material is also favorable, attributed to the mate-
for water purification where sand acts as a support rials non-reactivity, a disadvantage being their
for biofilm establishment. In industrial systems, short-term stability (Rosenhahn et al. 2010).
rotating biological contactor, bioWev, linpor Thermal methods imply the treatment of equip-
sponge, capture sponge, and ring lace are the pre- ment with hot water for a certain time period,
ferred media for the support of biofilms. The bac- causing detachment of the microbial communi-
teria that grow on biofilms start with the selective ties (Piola and Hopkins 2012). Electrical meth-
development of autotrophs. It is very important ods involve the administration of short-term
that easier and effective methods for waste water pulses to remove diatoms and algal biomass.
treatment are introduced to obtain high-quality Another recent method uses thin nanofiltration
recycled water; thus, application of biofilm tech- (NF) film membranes of electrically conductive
nology along with industrial expertise will be a polymer-nanocomposite (ECPNC), a strong and
constructive approach. consistent electric flux to prevent biofilm deposi-
tion (De Lannoy et al. 2013). The most useful
eco-friendly method for antifouling is the use of
11.4.2 Biofouling quorum-quenching systems. Quorum quenchers
are the enzymes or inhibitors produced by bacte-
Many industries across the globe rely on high- ria, including acylases, lactonases, paraoxonases,
throughput machinery with an intricate chain of and oxidoreductases. These molecules suppress
supply pipelines and filters. These pipelines and the communication-related genes and signaling
membranes in a bioreactor system are often cascades (Mitchell 2001; Huma et al. 2011; Kalia
choked to insufficiency by the accumulation of and Purohit 2011; Kumar et al. 2013). This phe-
certain growing entities such as plants, algae, and nomenon was first reported in Bacillus spp.,
microbial biofilms, etc. This phenomenon is which secretes autoinducer inactivation gene aiiA
called biofouling (Flemming 2002). This prob- lactonases, disrupting the lactone moieties of the
lem, apart from creating unnecessary financial acyl homoserine lactones and rendering the sig-
and technical burdens, also adds to probable con- naling inactive (Huma et al. 2011; Kalia and
tamination of the produce; hence, it turns out to Purohit 2011; Kumar et al. 2013). Apart from the
be a huge nightmare for the business globally. naturally occurring quorum-sensing inhibitors,
The agents used to prevent biofouling are called many synthetic compounds have been used and
antifouling agents (Yebra et al. 2004; Gupta frequently researched to be used against biofilm-
et al. 2014; Kalia and Kumar 2015a; Kalia et al. based biofouling (Kalia 2013; Brackman and
2015) and include chemical, physical, mechani- Coenye 2015). Biofouling can be prevented first
cal, thermal, electrical, and biological methods. by using quorum quenchers to specifically inhibit
Chemical methods utilize many artificially syn- different crucial building steps such as blocking
thesized biocides that are designed to kill any the transcription of quorum-sensing molecules
organism present. The most commonly used bio- like AHL, AI-2, etc.; second, by disrupting the
cide is tributyltin (TBT). Although this method is efflux pumps of quorum-sensing molecules;
effective to a certain extent, it has some serious third, by directly blocking the quorum-sensing
implications on existing environmental and molecules and transcription activators at the pro-
marine fauna. Nowadays, copper-based antifoul- tein level using analogs; and, last and most effec-
ing coating or paints are used as a self-polishing tive, by using hydrolyzing enzymes such as
copolymers that react with ion in sea water and lactones, acylases, etc. (Sio et al. 2006; Kalia
gradually leaches off. Yet again the safety of et al. 2015). All these methods have been thor-
released copper in sea water is debatable oughly researched and are constantly being mod-
(Guardiola et al. 2012; UK Marines). The use of ified to become more eco-friendly, either by
oligo and poly ethylene glycols, fluoropolymers, immobilizing the quorum-quenching enzymes on
172 N. Dubey et al.

the matrix or magnetic beads or by embedding on the bacterial cells (Toner et al. 2005). Diels
the enzymes or inhibitors on the cell-entrapping used a moving-bed biofilm sand filte