Bioencapsulation of Microbial Cells For Targeted Agricultural Delivery

Critical Reviews in Biotechnology, 2011; 31(3): 211226
2011 Informa Healthcare USA, Inc.

ISSN 0738-8551 print/ISSN 1549-7801 online
DOI: 10.3109/07388551.2010.513327
REVIEW ARTICLE
Bio-encapsulation of microbial cells for targeted agricultural

delivery
Rojan P. John1, R.D. Tyagi1, S.K. Brar1, R.Y. Surampalli2, and Danielle Prvost3
INRS-ETE, Qubec, Canada, 2USEPA, Kansas City, Kansas, USA, and 3Agriculture et Agroalimentaire Canada,
Sainte-Foy, QC, Canada
Abstract
Biofertilizers, namely Rhizobium and biocontrol agents such as Pseudomonas and Trichoderma have been well
established in the field of agricultural practices for many decades. Nevertheless, research is still going on in the field
of inoculant production to find methods to improve advanced formulation and application in fields. Conventionally
used solid and liquid formulations encompass several problems with respect to the low viability of microorganisms
during storage and field application. There is also lack of knowledge regarding the best carrier in conventional
formulations. Immobilization of microorganisms however improves their shelf-life and field efficacy. In this context,
microencapsulation is an advanced technology which has the possibility to overcome the drawbacks of other
formulations, results in extended shelf-life, and controlled microbial release from formulations enhancing their
application efficacy. This review discusses different microencapsulation technologies including the production
strategies and application thereof in agricultural practices.
Keywords: Biofertilizer, encapsulation technology, microencapsulation, shelf-life, controlled release
Introduction
containing nodulating bacteria and sticking agents before

sowing (Walley etal., (2004). According to Bashan (1998),
inoculation with non-symbiotic nitrogen fixing bacteria
in large scale production was practiced in Russia during
the 1930s and 1940s. According to Bashan (1998), rhizobial inoculants were produced around the world for years
initially by small companies. In countries such as Brazil
and Argentina there is no application of chemical nitrogen fertilizer for legumes cultivation, such as soybean
and hence they are inoculated with rhizosphere bacteria
(Dobereiner etal., 1995). As Bashan (1998) reported, the
1970s witnessed major advancements in plant inoculation technologies such as the invention of nitrogen fixing
bacterium (e.g. Azospirillum) and biocontrol agents (eg.
Pseudomonas spp.). Later, various other bacterial genera
were also evaluated for plant growth promotion (Bashan,
1998). Nowadays, inoculation of seed with the desired
microorganisms and soil application of these microbes
are considered as two inoculation technologies. Direct
seed inoculation has a positive effect only with sufficient
moisture content. Moisture is critical since desiccation
Soil quality is totally affected by the application of

chemical nitrogen fertilizers and exceeding application
beyond the crop requirement creates many environmental problems in all ecosystems (Mulvaney et al., 2009).
One of the solutions for eliminating the negative effect of
chemical ammoniacal nitrogen fertilizer is crop rotation
with rhizobial symbiotic leguminous plants. Inoculation
of leguminous plants using pure cultures of Rhizobium is
a common practice (Ben Rebah etal., 2007). According to
Perret etal., (2000), the practice of rhizobial inoculation
became promising after the discovery and application of
nitrogen fixing bacteria by Hellriegel and Beijerinck in
the 1880s. According to Guthrie (1896), Beijerinck first
isolated and grew rhizobia and used them in legumes. In
earlier days, farmers transferred the fertile soil to other
sterile fields to make them suitable for agricultural use
and the transfer of the soil (after leguminous plant growth)
to other agricultural fields was considered as the primary
phase of plant inoculation technology. In the early twentieth century in Canada, seeds were treated with soil
Address for Correspondence: R.D. Tyagi, INRS-ETE, Qubec, Canada. Tel.: 418-654-2617; Fax: +1-418-654-2600; E-mail: tyagi@ete.inrs.ca
(Received 13 April 2010; revised 22 July 2010; accepted 02 August 2010)
211
212 Rojan P. John etal.

occurs at lower moisture and the germination of seeds
requires high moisture, thus formulation development is
very important.
Bioinoculant formulation
According to Xavier et al., (2004), formulation is a vital
characteristic for bioinoculant development of an effective microbial strain and it can decide the functional
success or failure of a biological agent. Formulation usually consists of the microorganism and a suitable carrier
together with additives. Additives along with carrier, aid
in the stabilization of formulation and protection of the
microbes from environmental influence during storage
and transport. It helps microorganisms to perform better
at the application site (Xavier etal., 2004). According to
Xavier etal. (2004), a formulation should be stable during
production, storage, and shipping. It should be easy to
handle and apply in order to give better performance of
inoculation. Besides, all the formulations should be costeffective (Xavier etal., 2004, Jones and Burges, 1998).
There are numerous effective solid and liquid formulations, such as peat based powders or suspensions, and
emulsions for agricultural applications. However, the lack
of availability of good carriers, environmental impacts
and factors affecting the biological agents regarding formulation lead to the search for improved formulations.
The potency of the formulation is critical for the successful commercialization of the bio-inoculants products.
Thus, formulation is a challenging and often success
limiting step in the successful commercialization of new
bio-inoculants. This necessitates the development of
stable biofertilizer or biocontrol agent formulations from
alternative raw materials which can compete with the
existing inoculants as well as the chemical alternatives.
Research is still ongoing to find an efficient formulation
for agricultural applications and this review focuses on
the bio-encapsulation aspects of microorganisms for the
development of efficacious biofertilizers and biocontrol
agent formulations.
Conventional formulation versus advanced

formulation
Common carriers in conventional formulation
According to Stephens and Rask (2000), conventional
biofertilizer or biopesticides are used in plant applications as powder, liquid or granulated forms. Powder
and liquid inoculants are generally applied to the seeds
(preinoculated seeds), while granular inoculants are
applied to the soil (to seed furrows or soil mixing) (Ben
Rebah et al., 2007). Inoculants are usually commercialized as solid and liquid inoculants (Stephens and Rask,
2000). Powder or granular inert materials may include
plant growth media or matrices, such as rockwool and
peat-based mixes, attapulgite clays, kaolinic clay, montmorillonites, saponites, mica, perlites, vermiculite, talc,
carbonates, sulfates, oxides (silicon oxides), diatomites,

phytoproducts, (ground grains, pulses flour, grain bran,

wood pulp, and lignin), synthetic silicates (precipitated
hydrated calcium silicates and silicon dioxides, organics), polysaccharides (gums, starches, seaweed extracts,
alginates, plant extracts, microbial gums), and derivatives
of polysaccharides, proteins, such as gelatin, casein, and
synthetic polymers, such as polyvinyl alcohols, polyvinyl
pyrrolidone, polyacrylates (Date and Roughley, 1977;
Dairiki and Hashimoto, 2005; Jung etal., 1982).
Need for alternative carriers

Earlier, seed coated peat-based inoculants dominated the
commercial inoculant market due to the positive impact
of peat on the storage and application processes. During
soil inoculation, peat based formulations can alter the soil
structure and can provide a new protective habitat for the
rhizobial cells (van Elsas and Heijnen, 1990). Although
peat was recognized as a very good carrier of rhizobia,
there was interest in developing alternate formulations
as its availability was not widespread and due to its negative environmental impact on peat available ecosystems
(Hynes etal., 1995). According to Albareda etal., (2008),
the liquid inoculant production process is simpler as
there is no alteration of carrier and the application of the
formulation is easier. Liquid formulations include suspensions, concentrates, and oil based products, such as
emulsions. However, bacterial survival is inferior in the
liquid type of inoculants and on liquid inoculated seed
as bacteria lack carrier protection (Albareda etal., 2008;
Singleton et al., 2002; Tittabutr et al., 2007). Alternative
formulations were developed to obtain the advantage of
a good shelf-life by additive supplementation and easy
transportation by decreasing the volume, which minimized the handling steps (John et al., 2010). Therefore,
the adoption of these formulations reduced the overall
associated cost compared to other conventional formulations. Recently, some advanced technologies have
been developed for the effective storage; transportation,
and enhanced efficiency of formulations by encapsulating rhizobial cells using polymers.
Encapsulation technology
Encapsulation is the technique of making a protective
shell or capsule around the active ingredient or cells (e.g.
microbial, macrobial cell or tissue).
Advantages of cell encapsulation

According to McLoughlin (1994), the matrix of micro
beads protects the inner cells from both mechanical
stress and the adverse conditions of the outer environment by providing a defined, constant, and protective
microenvironment. The cells can survive and metabolic
activity can be maintained for extended periods of time,
with controlled release of cells after their adaptation to
the surrounding environmental conditions (Cassidy etal.,
1996). Microencapsulation can noticeably improve the
viability of microorganisms due to its protective effects
Critical Reviews in Biotechnology
Bio-encapsulation of microbial cells 213

against detrimental environmental factors, such as pH
variation and the poisoning agents generated during
the process (Mortazavian etal., 2007). It can protect the
bacterial cells from bacteriophages, hydrogen peroxide,
short chain fatty acids, carbonyl-aromatic compounds,
and drying (Mortazavian et al., 2007). The immobilized cells showed tolerance to alcohols (Holcberg and
Margalith, 1981). This tolerance was mainly due to the
enhanced modification of the cell membrane (Groboillot
etal., 1994). Alginate contains many fatty acid impurities
and these impurities probably result in a modified fatty
acid pattern for the immobilized cells compared with
free cells (Diefenbach etal., 1992). A similar effect of tolerance against phenol in E. coli was reported (Keweloh
et al., 1990) and it was attributed to the uptake of the
saturated fatty acids present in commercial alginates and
incorporation into the cellular membrane. Later, Keweloh
etal. (1990) suggested that it was not only fatty acids but
also other ingredients of alginate that had a physiological
effect on the cell membrane (Diefenbach etal., 1992).
The release of microorganisms from micro beads is
also a simpler but controlled process. A micro-envelope
may be opened by many different means, including
fracture by heat, solvent action, diffusion, and pressure (Brannon-Peppas, 1997). Thus, the encapsulated
microorganisms possess much more efficiency than
the conventional powder and liquid formulations. Both
conventional and advanced formulations have their own
merits and demerits as illustrated in Table 1.
Macro and microencapsulation

The encapsulation of cells is of two types: macroencapsulation and microencapsulation. Macroencapsulation is the
entrapment of cells in polymeric structures of a larger size
extending from few millimeters to centimeters. Usually
animal cells or tissues are entrapped by this method to
make artificial cells or tissues. Macroencapsulation can
be defined as encapsulation by surface coating materials, such as polymeric organics (e.g., resins and plastics)
or inert inorganic materials to substantially reduce the
surface exposure to potential leaching media (http://
www.setonresourcecenter.com/cfr/40CFR/P268_030.
HTM). According to Bashan (1998), the use of macro
alginate beads in agricultural application has two major
disadvantages and these are; additional treatment during
sowing and the need for the bacteria to move through the
soil towards the plants. This problem with macro-beads
can be solved by the use of micro-beads as they can have
direct contact with the seed. According to Lin and Chen
(2002), micro beads have a size of 10100 m and macro
beads are greater than 100 m. The encapsulated material can be rounded beads, cubes or even sheaths.
Microtek laboratories has defined microencapsulation
as the process of surrounding or enveloping one substance within another substance on a very small scale,
yielding capsules ranging from less than one micron to
several hundred microns in size. The core materials will
be released either slowly through the capsule walls by
diffusion or when external conditions prompt the capsule

walls to burst, melt or dissolve (http://www.microteklabs.
com/technical_overview.pdf ). The bigger sized particles,
such as microencapsulated cells are usually released
by the latter method. The macro-encapsulation of seed
inoculants has less uniformity when mixed with seed.
From an application view point, the microencapsulation
gives uniformity in spreading, is effective and results in
an easy release of microorganism to the targeted site
due to the small size and higher surface area. The loss of
microbial cells during the preparation of microbeads is
minimal compared to large beads (Bashan 1998). It can
be easily applied to soil using sprayers or can also be
directly coated on seeds.
Microbeads and their structure

According to Park and Chang (2000), microbeads are
encapsulated microbial cells and consist of hydrogels
(capsule) coated around the microbial cell or cells. The
shape, size, and texture of the beads vary with the type
of coating material and microorganism employed. The
shape may be spherical, elliptical or irregular and contain
one or more cells (Figure 1). The filamentous core material like fungal fragments or actinobacteria can vary in
shape as they have different shapes and size. The coating
of the beads in the case of multilayered beads are durable
than other single coated gel beads. The ultrastructure of
microcapsules revealed a micro-reservoir structure in
which the wall extended well into the core and the active
agent (e.g. microbial cells), and was accommodated by
the micro-reservoirs (Figure 2). Most of the materials
used in encapsulation are polymers, either naturally
occurring (e.g. polysaccharides, proteinaceous material)
or synthetic (e.g. Polyurethane foam) (Groboillot et al.,
1994, Saucedo etal., 1989). The polymers may be homo
polymers (e.g. gelatin) or hetero or co-polymers (polylactic acid-polyglycolic acid). The polymers are selected
on the basis of the chemical composition of the monomeric units. The quantity and characters of the functional
groups contained in the monomer units define the primary structure of polymers. The chemical composition of
these monomer units gives rise to various interactions,
such as hydrogen bonding, electrostatic interactions, and
hydrophobic interactions, which are important for intraand intermolecular interactions (de Vos etal., 2009) such
as gelling or cross-linking.
Besides the chemical composition of polymer, the
molecular weight also has significant role in the encapsulation. According to Lu and Toy (2009) one of the
major drawbacks of polyethylene glycol (PEG) was its
higher molecular weight. The loading level of PEG was
inversely proportional to its molecular weight as it has
functional groups only at the terminals of the polymer
chain. The optimum molecular weight of PEG in distearoyl-N-(monomethoxy poly(ethylene glycol)succinyl)
phosphatidylethanolamine (DSPEPEG) was 2000 and
a larger molecular weight resulted in lower encapsulation (Moribe et al., 1999). Jalil and Nixon (1990) tested

Table 1. Microbial formulation: conventional versus advanced types.
Conventional formulation
Solid formulation
Liquid formulation
Shelf life is poor or average due to the
In solid formulation, the shelf life of
osmotic imbalance and lack of oxygen
microbes is poor or average due to
desiccation. This is not only in the
storage period but also in the field
application.
The cost of material and process are
The cost of raw material and production
higher compared to solid formulation.
cost is lower but unavailability of the best
material is a limiting factor.
There is the need of more cells in the

formulation development and field
application. Washing off percentage and
mortality is high.
As in the case of solid formulations the

viable biomass necessity is high. Erosion
and mortality of cells are higher due to
the lag period in adaptation.
Contamination by other microbes is

higher. Antimicrobial agent is thus
mandatory in solid formulations.
A contamination problem exists even in

the case of emulsions but less compared
to solid. Antimicrobial agent is essential
in some liquid formulation and will
negatively affect the microbial survival.
Concentrated microbial emulsion and
suspension will decrease the size of
storage vessels and transport is easier
but each step increases the production
cost.
In seed applications, there is the need of
large volumes to produce the minimum
level of cells and there is clumping many
seeds.
No or less need of sophisticated
equipment.
Transportation is costly due to the bulk

size, but some dry formulations such as
spray drying with some fillers or freeze
drying agents increase the cost of
production.
There is less bridging of seeds as the
formulation is dry.
No or less need for sophisticated

equipment.
Advanced formulation
Encapsulated cells
More compared to both conventional methods
due to the reduced influence of environment
and easy accessibility of nutrient and oxygen
through simple diffusion.
The cost of the polymeric compound is higher
than the other easily available solid and liquid
formulation components but the higher cost
types can be replaced by low cost gum or
gelling agents of biological origin.
The viability of microbes is higher in the case
of encapsulation. Controlled release to the
external environment reduces the wastage of
microbial cells. It helps the microbes to adapt
to the environment by giving protection.
The contamination of antagonistic organism is
very less due to the protective shell or sheath
outside.
Minimum storage space is required as it is

immobilized in micro or macro beads as the
low volume application and thus reduces the
initial fermentation cost.
The use of microencapsulated formulation is
low so there is no clumping of two or more
seeds.
For microcapsule preparation there is the
need of equipment relating to spray and
solidification.
Capsule
Lipophilic layer
Microbial cell
Hydrophilic layer
5
2
Active ingredient
Figure 1. Different forms of microbeads: (1) spherical, (2) oval,

(3) irregular surface, (4) multicellular, and (5) multilayered bead.
Figure 2. Diagrammatic representation of a microcapsule

showing core and shell (capsule).
poly(DL-lactic acid) having three different molecular

weights (20500, 13300 and 5200) to prepare microcapsules comprising phenobarbitone, as a reference core
using the water/oil emulsion evaporation method. Jalil
and Nixon (1990), observed a trend towards lowering
the mean microcapsule size, both by volume and population was observed with respect to a lower molecular
weight polymer. With variations in polymer molecular
weight, no effects were observed on the morphology of
the microcapsulated surface, efficiency of encapsulation,
and density (Jalil and Nixon, 1990).
Techniques of microencapsulation
Methods of encapsulation vary with the nature of applications from diverse fields for miscellaneous objectives.
Some techniques require incorporation of the supporting material along with the central core cells for stability.
The formulation can be freeze dried or made into a liquid
emulsion and a suspension to assimilate the components.
Further, these formulations can be micronized, microemulsified or coated for further modification and finally
polymerized, gellified, spray dried or coacervated to produce stabilized micro particles. For further protection,

there can be utilization of more polymer coating based
on the application and types of beads and organisms.
Alginate beads can be hardened by the post treatment
with chemicals, such as gluteraldehyde (Park and Chang,
2000). According to Park and Chang (2000) polyvinyl
alcohol could be treated with boric acid solution to make
the bead strong and long-lasting, but a prolonged cross
linking process reduces the survival of the cells.
Different methods dealing with microencapsulation
are summarized in Figure 3. The important methods
used for encapsulating microorganism are discussed in
the following text. Research has also been carried out to
increase tolerance using other additives, such as, oil and
poly-L-lysine or certain methods, such as cross linking or
coatings (Ding and Shah, 2009; Ghosh, 2006; Hyndman
etal., 1993). The first step of encapsulation is the dispersal and integration of microorganism into a liquid matrix.
This is followed by a selective process that can be used for
the production of micro-beads or a microcapsule which
depends on the organism or encapsulating materials or
application. Among the methods mentioned in Figure 3,
spray drying and extrusion are the two major commercial processes usually used in terms of product volume.
The important methods are described in the subsequent
sections.
Extrusion
Extrusion is a very old and simple method of making
capsules with hydrocolloids (King, 1995). According to
Krasaekoopt etal. (2003), the extrusion process could be
done by mixing the microorganism with hydrocolloids
followed by extrusion into a hardening solution. The size
and shape of the beads could vary based on the distance
of free-fall and the size of needle pore. Thus, the extruder
die plays an important role in the extrusion process. The
flow, deformation and temperature relationships should
also be considered while designing an extruder die
(Sokhey etal., 1997).
A pumping system can be utilized to make uniform
and small sized beads. In this case, the pressure flow (Q)
mainly depends on the viscosity of the liquid. According
to Liang (2001), viscosity is a crucial parameter for the flow
characteristics of polymer fluids. Liang (2001) reported
Coacervation
Fluidized bed
Droplet freezing
Polymerization
Spray-drying
Microencapsulation Technologies
Interfacial
polycondensation
Solvent
evaporation
Supercritical fluid
Extrusion
Gelation
Droplet gelation
Thermal gelation
Figure 3. Different processes in microencapsulation technology.

that Hirai and Eyring (1959) proposed a relationship

between viscosity and pressure as follows, based on the
Eyring hole theory of fluid,
(1)
P 0 = P 0 exp ( P P0 )

where, P and P0 are the viscosity at pressure (P) and
atmosphere pressure (P0), respectively. is the pressure coefficient which is a function of the hole volume
and absolute temperature. So, extrusion pressure must
be regulated on the basis of the viscosity of the fluid for
a better encapsulation and for stable bead formation.
Sokhey etal. (1997) tested the effect of die measurement
on the extrusion performance on food products using
maize. According to Sokhey et al. (1997) die diameter
affected the radial expansion of the extrudate but axial
and overall expansions were not affected by die diameter. Length of the die had no influence on radial expansion of bead but had a major effect on axial and overall
expansions. During extrusion, operating pressure is
inversely proportional to increasing die diameter but
directly proportional with die length. For a constant die
length, increase in operating pressure increased radial
expansion. A die with small diameter and shorter length
should be used for greater radial expansion and minimizing energy consumption. The real diameter of the
bead is again corrected by shrinkage or swelling factors.
Usually alginate beads tend to shrink during gelation
while a nylon microcapsule swells on washing (Dulieu
etal., 1999).
Emulsion technique
Water-soluble materials are widely used for applications
in agriculture, food, and pharmaceuticals (Baogou etal.,
2006) and generally a water-in-oil emulsion has been
used to make micro-beads. Krasaekoopt et al. (2003)
described emulsion technique as follows; a small volume
of the cell-water soluble polymer (e.g. alginate, gelatin)
suspension is added to a large volume of a vegetable oil.
The mixture is homogenized to form a water-in-oil emulsion and the polymer in emulsion cross-linked to form
tiny gel particles within the oil phase. The final size of the
micro-particles varies according to type of emulsification, depends on the internal phase particle size of the
emulsion. The cross-linking method depends on the type
of polymer support (e.g. gelatin can be cross-linked using
gluteraldehyde or alginate can be gelled by calcium ions).
The size of the beads is controlled by the speed of agitation. McNamee etal. (1998) used gum arabic for making
emulsions with soya oil and the mixture was spray dried
for microencapsulation. The selection of gum arabic was
due to its high solubility, low viscosity compared to other
hydrocolloids, and ability to act as an oil-in-water emulsifier. Later, McNamee etal. (2001) replaced gum Arabic
with other carbohydrates.
Spray drying
According to Picot and Lacroix (2003), microencapsulation by spray drying is a well-established process that can

produce a large quantity of material. This technology is
not considered as a good cell immobilization technique
due to a high mortality resulting from simultaneous
dehydration and high temperature inactivation of microorganisms. Picot and Lacroix (2003) micronized the
powder particles produced by spray drying and emulsification methods, using a spiral jet mill as a grinding
system. According to Picot and Lacroix (2003), the effect
of micronization on cell viability was mainly influenced
by the final particle size. Micronization was determined
to be an efficient means of reducing the particle size of
freeze-dried culture powders with a satisfactory death
rate before producing microcapsules with low diameters
for the protection of heat sensitive bacteria (Picot and
Lacroix 2003).
In accordance with Ghosh (2006), the fermented broth
can be quickly dried after mixing in a polymer solution
and sprayed into a hot chamber. The in-feed temperature
is most often kept at or close to ambient temperature for
the viability of microorganisms, such as non-sporulating
Rhizobium. Some other sporulating bacteria and spores
of fungus, such as Trichoderma sp. can withstand an even
higher drying temperature. High pressure spray nozzle
and centrifugal wheels can make very minute particles
and it can reduce the exposure time of heat-labile microorganisms. The particle size distribution of microencapsulated bacteria may be limited to a lower range than
the given particle size in conventional formulations. The
hypothetical curve of particle size distribution is shown
in Figure 4. The lower size of microbeads helps in their
better performance. The major limitation of the spray
drying process is the mortality of microorganisms as
mentioned above, along with the damage to the shell
by micro-cracks that can lead to decomposition of the
capsule. The process costs may also be high with the use
of sophisticated equipment, power consumption, and
expensive carriers.
Amiet-Charpentier et al. (1998b) reported microencapsulation of Pseudomonas fluorescens-putida using
spray-drying. No bacteria were alive at time zero, when
the inlet air temperature was at 80C for the microspheres harvested in the cone and flask. While at 60C,
there was bacterial survival and it was estimated that
nearly ~107 CFU/g of micro-particles were obtained in
the cone, after 2 months of storage. At time zero, it was
noticed that the residual moisture of the microspheres
decreased for the particles collected both in the flask
and cone, when the inlet air temperature was increased
from 60 to 80C. Thus, it is clear that desiccation due
to higher temperature destroys the microbial cells and
protective measures have to be taken for better survival.
A similar result was observed in the case of biocontrol
fungal (Beauveria brongniartii) conidia of encapsulation, when the outlet temperature was raised to nearly
53C 2C so that the viability decreased (Horaczek and
Viernstein, 2004).
Amiet-Charpentier et al. (1998b) reported that feed
rate also influence the survival of microorganisms during spray drying. A lower spray feed rate decreased the
microbial survival due to a faster desiccation rate and
vice versa. At a spray feed rate of 34mL/min, there was
no bacterial survival at time zero. By doubling the rate to
78mL/min, at time zero bacterial survival of ~104 CFU/g
micro-particles was obtained in the flask. Bacterial
survival of ~107 CFU/g was obtained in those collected
in the cone. This bacterial survival was continued to be
the same after 2 months of storage in the encapsulated
form. This increase in survival was due to the increase in
residual moisture when the spray feed rate was doubled
(Amiet-Charpentier etal., 1998b).
Coacervation
This is mainly used as a technique for controlled drug
release, and the process relies upon the decrease in
solubility of coating polymer by the addition of another
compound to the polymer solution, changing pH or temperature (Park and Chang 2000). Microencapsulation by
coacervation can be achieved by the phase separation of
the coating polymer solution. Polymer rich coacervate
wraps around the liquid core and the solvent supernatant
Frequency distribution
100
Cumulative frequency distribution
6
5
4
80
Hypothetical
microencapsulation profile
60
40
2
20
1
0
0.1
10
Particle size (m)
100
Cumulative frequency distribution
120
Frequency distribution
0
1000
Figure 4. A hypothetical curve for particle size distribution (PSD) for a normal liquid versus the microencapsulated formulation. (PSD
data derived in our laboratory as tested on a liquid formulation).


separates, finally leading to the solidification of the microparticle (Nihant etal., 1995). This method is not suitable
for producing lower sized microspheres and there is formation of harmful residues on the microspheres. Use of
supercritical gases as phase separating agents can minimize the formation of these harmful residues (Freitas
etal., 2005).
Solvent extraction/evaporation
Solvent extraction/evaporation is a simple method of
microencapsulation to achieve a controlled particle
size. This method does not require high temperatures
or inducing agents for phase separation (Freitas et al.,
2005). Vigilant choice of encapsulation materials and
conditions and a low residual solvent content in the final
preparation will give particle sizes in the nano to micrometer range (Freitas etal., 2005). According to Freitas etal.
(2005), solvent extraction/evaporation basically consists
of four major steps: (i) dispersal of the microorganism or
other active ingredients in a solution of matrix forming
material; (ii) emulsification of this phase in a successive
continuous immiscible phase; (iii) extraction or evaporation of the solvent and transforming microglobules into
solid microspheres; (iv) harvesting and drying of the
microspheres. The temperature for evaporation is not so
high in this process and is tolerable to the microorganisms. But the mortality of the microorganisms is mainly
caused by the solvent used in this process which may be
lethal to the microbe. Sometimes, higher concentration
of the solvent can desiccate the microbial cells and a longer duration of the process may affect the viability of the
organism. Thus, solvent extraction/evaporation may not
be a viable technique for formulation of the biological
control agent.
Thermal gelation
This is another promising technique in cell encapsulation
and can be achieved by cooling a warm aqueous polymer
solution. Beads are obtained by dropping the polymer
solution in cold water or oil. The cell-polymer solution
dropped into media containing a cross-linking agent
was suddenly cooled from 4045C to 25C to gelate the
micro-beads after strong vortexing in the reactor (Audet
et al., 1989). Autoclaved -carrageenan at 4045C was
mixed with cells and dropped into a cold KCl solution. To
obtain better results, the polymer can be emulsified with
warm oil and cooled by adding cold oil or by using heat
exchangers (Neufeld etal., 1991).
Pregel dissolving technique

According to Park and Chang (2000), the double step
pregel dissolving technique was developed by Lim and
Sun in 1980. The sodium alginate containing cells are
dropped into a CaCl2 solution and the calcium alginate
beads are made by a conventional method. Then the
carboxyl group of the calcium alginate bead are allowed
to react with an acid-reactive amine or imine group in a
compound such as, poly-L-lysine or polyethyleneimine.
A polyelectrolyte complex membrane is formed on the

surface of the calcium alginate bead and the bead is
retreated with sodium alginate solution to crosslink the
residual poly-L-lysine on the surface of the bead. The
solid core capsule of calcium alginate is dissolved in
sodium citrate solution and forms a liquid core with the
microbial cell (Park and Chang, 2000). This preparation
is impractical in agricultural application as there was no
microbial release for an extended time due to the high
stability of the beads. It can be used for the industrial
production of organic acids or other compounds using
anaerobic or micro-aerophilic bacteria.
Factors affecting microencapsulated

microbial cells
The selection of suitable capsule materials that are able
to remain stable in the surrounding environment is
very important. Each and every material has different
functional groups, monomer differences and an ability
to interact with other components during microencapsulation. A capsule should have sufficient mechanical
resistance to withstand the various forces during the
entire duration of production and application, especially in bioreactors. Mechanical stability does not have
much importance during agricultural applications as
the capsule has to degrade or dissolve for controlled
release into the environment. However, resistance of
microcapsules is very important during storage and
transportation.
Alginate is considered as the most common biomaterial in the encapsulation of bacterial and/or other type of
cells (Murua etal., 2008). Commercial sources of alginate
are brown algal species especially Laminaria, Macrocystis,
and others (Cheze-Lange et al., 2002). The natural alginate polymer is formed by linking D-mannuronic acid
and L-guluronic acid monomers at the C-1 and C-4 positions (Smidsrod and Skjak-Braek, 1990). Alginates create
three dimensional structures when they react with multivalent ions and it is the basis of its use as a gel matrix.
According to Murua etal. (2008), the gelling of the aqueous alginate solutions is due to the binding of divalent
cations between the G blocks of adjacent alginate chains
and creating inter-chain bridges. A natural material like
alginate is not harmful to the survival of microorganisms.
Common capsular material used in bioencapsulation is
presented in Table 2 and the advantages, disadvantages
and possible modifications of important materials are
included in Table 3.
The coating material of the capsules and type of
interstitial fluids are two other parameters that affect
the efficiency of microencapsulated cells by modifying the physicochemical characteristics of beads. As in
pregel dissolving, the hardening material of the surface
of the alginate bead helps to increase the hardness and
increase the leakage of cells. But, the coating material
can influence the material transfer in reactor applications and affect the survival of cells. The number of

entrapped cells in each bead is directly proportional to
the concentration of microbial cells in the encapsulation solution and increased concentration of the cells
in beads directly influence quantitative efficiency of
encapsulation. The quality of microbial cells, such as tolerance to pH and temperature, sporulation ability, flocculation, exopolysaccharide production, antimicrobial
activity, and additive tolerance also affect the survival of
encapsulated organisms (Sultana etal., 2000). Thermal
gelation is the best method for thermopilic organisms,
while pregel dissolving is suitable for anaerobic organisms. As mentioned earlier, the factors during microencapsulation process, such as freeze drying, spray drying,
micronization, and storage conditions are very important to avoid the mortality of contained cells and injuries
to the beads (Sultana etal., 2000). The bead size and cell
load are critical parameters for encapsulated bacteria
during storage and application.
Akin to the physical forces in the microencapsulation process, the additives used to maintain the viability of microorganisms or to maintain the properties of
the microcapsule also have great influence on the performance of microorganisms in microbeads. Additives
are the substances that aid in the stabilization and

protection of the microbial cells in a formulation during storage, transport, and at the target zone (Xavier
etal. 2004). Different additives which aid in the stability and survival rate can be applied during the formation and storage of micro-beads. A study comprising
rhizobacteria revealed that bacterial survival was
higher with the seed coating agent, Sepiret 1039G, and
rapid release of the microencapsulated bacteria were
shown using wettability measurements of agglomerated micro-particles (Amiet-Charpentier, 1999). The
physical-chemical characteristics of the coating material showed that water was the critical parameter for
the release of rhizobacteria.
As mentioned above, the formulation must be stable
from production to application site, it should enhance
the activity of the organism in the field, be cost-effective,
and be practical (Young et al., 2006). The additive is
one of the major cost factors in various formulations.
Along with carrier, additives play a crucial role in a
prolonged survival during different stages of formulation. Many commonly available materials were used
as additives according to the types of application and
Table 2. Different carrier materials used in encapsulation of microbial cells.

Carrier
Microorganism
Use
Urea-formaldehyde
Mixed culture
Crude oil, Hexadecane,
Phenanthrene degradation
Agar-alginate
Yarrowia lipolytica
Crude oil degradation
Polyurethane
Yarrowia lipolytica
Crude oil degradation
Gellan gum
Mixed culture
Gasoline degradation
Alginate
Prototheca sp.
n-Alkanes degradation
Azospirillum brasilense Cd
Chitosan, Cross-linked
with Hexamethylene
diisocyanate or glutaldehyde
Gelatin, cross-linked with
toluene-2,4-diisocyanate
Gelatinpolyphosphate
polymer
Xanthan and locust
bean gum
Eudragit, methacrylic
copolymer
Eudragit, methyl cellulose,
modified starch

Zinjarde and Pant, (2000)

Oh etal. (2000)
Moslemy etal. (2002)
Suzuki etal. (1998)
Biofertilizer
Bashan, (1986)
Pseudomonas sp.
Phenanthrene degradation
Paje etal. (1998)
Rhodococcus spp.
Benzene degradation
Sheu and Marshall, (1991)
Lactobacillus delbrueckii ssp.
Ice cream production
bulgaricus
Pseudomonas putida
Pseudomonas spp.
Benzene degradation
Naphthalene degradation
Streptococcus thermophilus
L. casei ssp. casei
L. lactis ssp. cremoris
Yoghurt production
Biomass production
Biomass production
Somerville etal. (1977)

Manohar and
Karegoudar, (1998)
Audet etal. (1988)
Arnaud etal. (1992)
Groboillotetal. (1993)
L. lactis ssp. cremoris
Biomass production
Hyndman etal. (1993)
Pseudomonas sp.
Biocontrol agent
Amiet-Charpentier etal. (1998a)
Bradyrhizobium
japonicum
Pseudomonas
fluorescens-putida
Pseudomonas
fluorescens-putida
Biofertilizer
Jung (1980)
Biofertilizer
Amiet-Charpentier etal. (1999)
Biofertilizer
Amiet-Charpentier etal. (1998b)
(ATCC 29710)
Polyacrylamide
Alginate, agar,
polyacrylamide
k-carrageenan and
locust bean gum
Reference
Mohn, (1997)
Weir etal. (1995)
The bacterial survival time of

incorporated microparticles, was up to
one year with moisture was around 25%.
Survival of bacteria was for nearly 8

months and the polymers are biological
in origin
It is a modified form of conventional
ca-alginate and provides an inner
liquid core for the microbial cells
Amiet-Charpentier etal.
(1998b)
Jung (1980)
Park and Chang (2000)
Methacrylic
copolymer,
ethylcellulose
and modified
starch
Xanthan
gum-Locust
bean gum
Alginate
polyelectrolyte
complex
Complex coacervation without any

organic solvent which is harmful to
microorganisms and no sophisticated
production procedure is needed.It is a
good method for dead or alive anaerobic
bacteria microencapsulation.
Amiet-Charpentier etal.
(1998a)
Gelatinpolyphosphate
polymer
Table 3. Advantages, disadvantages and possible modifications of different matrices.

Matrix
References
Advantage
Alginate
Bashan (1986)
Easily available and low cost compared
to others and the process is effort free.
Moisture content in the formulation is

5070 %. So, seed application will be
dehydrated soon or further moisture
addition will allow seeds to germinate
Little more expensive than the
conventional method and comprises
two steps for the preparation
The bacterial survival was affected by

the additives such as silica
Disadvantage
Alginate containing bacterial cells
is impractical due to the storage
incompatibility between bacteria and
seeds during application, seed tend to
germinate with high moisture whereas
bacteria need a higher moisture rate to
remain alive
Even in the modified form the migration
of bacteria from the oil phase to the
water phase is increased. The survival of
aerobic bacteria is very poor due to the
lack of oxygen transfer through the crosslinked capsule
Separate storage of seeds and formulation

is necessary. It can applied to the seed just
before the sowing or applied to the field after
sowing.
Currently, it is used in the medical field for
artificial cell formation or waste product
removal and possibilities in the crop field
application should be exploited.
For suspension of bacteria, it can be mixed

with paraffin oil by a w/o emulsion, whose
internal aqueous phase contains the
bacteria. Later, a double (w/o/w) emulsion
was formed in the aqueous solution of
gelatin and cross linked with gluteraldehyde.
This strategy should make it possible to keep
bacteria in an aqueous internal phase within
the polymer microparticles.
The concentration of the bacterial suspension
must be increased to prevent the dehydration
of the formulation
Possibility of modification
Incorporate bacteria and seeds as separate
or application must be at the time of sowing
to solve this problem

microorganisms. Vassilev et al. (2001a) used skimmed
milk and clay as additives to enhance the performance
of gel entrapped soil microbial inoculants during soil
application. A controversial effect was observed with the
survival of P-solubilizing bacteria on application of charcoal-soil mixed with alginate (Viveganandan and Jauhri,
2000). Young etal. (2006) showed humic acid along with
alginate was the best additive in the encapsulation of
rhizobacteria to enhance field performance. It is a challenging task to incorporate a suitable additive along
with the active ingredient without causing any negative impact on cell. Generally, the mortality of the cells
may be higher if they are directly applied to the soil, and
additives help to adapt with the environment soon after
the application. For example, materials, such as humic
acid can influence the soil dwelling organism for acclimatization to the soil environment. Materials, such as
gelatin, skimmed milk or whey protein may provide the
nutritional requirements of entrapped microorganisms.
Encapsulation of microorganisms for

agricultural applications
Soil fertility and plant growth is greatly influenced by
microbial assistance in the rhizosphere as microbial
populations in the soil are involved in fundamental
activities that ensure the stability and productivity of
agricultural and natural ecosystems (Barea etal., 2005).
The industrial sector in agriculture has mainly focussed
on the isolation and commercial cultivation of these
microorganisms for high yield and disease prevention
in an environmental friendly manner. Researchers have
developed many formulations of these microorganisms
to improve crop production and recent research focus
on advanced methods to improve formulation efficiency
during storage and application.
Bacterial application
It is well established that introduction of plant growth promoting bacteria to soil improves plant growth. Bacteria,
such as Pseudomonas fluorescens-putida, act as a plant
growth stimulator and biocontrol agent (Bashan 1998).
Some bacteria can fix nitrogen by symbiotic association
with leguminous plant root. Bacteria that are able to nodulate leguminous plants can be classified into five genera,
such as Azorhizobium, Bradyrhizobium, Mesorhizobium,
Rhizobium, and Sinorhizobium. Some plants, such as
Phaseolus vulgaris can be a host for many bacteria to nodulate the plant. The important bacteria which nodulate
P. vulgaris are R. leguminosarum bv. phaseoli (Jordan, 1984),
R. etli (Segovia etal., 1993), R. tropici (Martinez-Romero
et al., 1991), S. meliloti, R. leguminosarum bv. trifolii
(Michiels etal., 1998), and R. gallicum bv. phaseoli and
R. giardinii bv. phaseoli (Amarger etal., 1997). Meanwhile,
others are host-specific.
Rekha etal. (2007) reported that Pseudomonas putida
CC-FR2-4 and Bacillus subtilis CC-pg 104 produced a
significant effect on the shoot height of Lectuca sativa

L. seedlings. Bacteria encapsulated in beads had more

effect than free cells after 21 days of growth and thus
the authors claimed that the entrapped bacterial cells
could be a novel and feasible technique for application
in the agricultural industry. The soil application of bacterial beads at sowing time is one of the frequently used
methods for encapsulated microorganisms either as a
biocontrol or a biofertilizer. Many encapsulation studies
have been reported with bacterial beads application to
agriculture and beads were made up of polyacrylamide
(Dommergues et al., 1979), or polysaccharides, such as
alginate (Diem etal., 1988; van Elsas etal., 1992), carrageenan (Trevors etal., 1992; Bashan, 1986) and xanthan
(Jung etal., 1982; Mugnier and Jung, 1985), among others.
These bacterial beads were produced by co-extrusion of
an alginate solution to obtain spherical granules of 6mm
diameter (Digat, 1988) or by rhizobacterial macroencapsulation (Jung, 1980). Fertilizer formulation, comprising
rhizobacteria has also been encapsulated in a calcium
alginate matrix (Fages etal., 1988). These methods produced only larger encapsulated beads or material and
did not suit the seed industry, which was searching for
direct seed inoculation. Bashan (1986), described direct
bacterization of wheat seeds with beneficial rhizosphere
bacteria. The study found that the macroencapsulation of seed with alginate-containing bacterial cells was
impractical due to a storage incompatibility between
the bacteria and seeds, as the seeds need a dry environment to avoid germination during storage, but bacteria
need more moisture for their survival. As the size of the
beads is very big and almost the same size as the seeds,
there is no uniformity in the distribution of large scale
seed mixing. Direct bacterization of various seeds using
rhizobacteria (about 108 bacteria per seed) in calcium
alginate in a double inclusion technique was perfprmed
by Digat (1991) and more than 107 bacteria per seed
remained alive after 1h at 40C. Callan etal. (1991) studied the field performance of maize seeds coated with a
Pseudomonas fluorescens AB254 suspension in a methylcellulose against a disease caused by Pythium ultimum.
The treatment provided an equal effect of fungicide in
all conditions that were tested. The major drawback of
preinoculated seeds is undesired seed germination during storage, due to the high water content of the applied
formulation. In addition, drying processes after seed
coating with formulations cause water loss leading to the
mortality of the microorganism due to desiccation.
Amiet-Charpentier et al. (1998a), has recounted that
dehydration during storage prevented germination of
seed with high moisture beads by fabricating crosslinked complex coacervated gelatin-polyphosphate
beads. A suspension of bacteria in paraffin oil was used
to make water-in-oil (w/o) emulsion, where the internal
aqueous phase contained the bacteria and there was no
direct contact with seeds. Thus, a double, water-in-oilin-water (w/o/w) emulsion was formed in the aqueous
solution of gelatin and cross-linked with gluteraldehyde.
This approach should make it possible to keep bacteria

in an aqueous internal phase within the polymer microparticles. According to Amiet-Charpentier etal. (1998a),
the drawback of this system was that it was not good
for the survival of aerobic bacteria as the air-transfer
was reduced. This method can be successfully used for
the development of dead bacterial formulations and
anaerobic bacterial formulations (Amiet-Charpentier
et al., 1998a). Some reports found that polymers can
provide much longer viability of up to one year. AmietCharpentier etal. (1998b) tested three polymers, namely
methacrylic copolymer, ethylcellulose, and modified
starch for the microencapsulation and spray drying of
a plant growth promoting bacteria. Eudragit, methacrylic copolymer was found to be better for the survival
of Pseudomonas fluorescens-putida. A silica additive was
also examined for enhancing the moisture retention and
survival of bacteria. In this test there was no effect of
silica on the moisture content but it enhanced the survival of bacteria at time zero, however during storage, the
effect was negligible. Amiet-Charpentier et al. (1998b)
stated that it might be the result of the larger particle size
of silica gel than the bead as it is situated outside the gel,
and absorbed water from the bead to the outside of the
additive particle. So, the additive particle has to be compatible with the microencapsulated particle.
Amiet-Charpentier etal. (1998b) found a relationship
between bacterial survival and the residual moisture of
microspheres. The best survival of the stored bacteria
was observed at 25% of the residual moisture. In another
study, Amiet-Charpentier et al. (1999) used Eudragit
as a formulation which was the first report on bacterial
inoculant and proposed in a micro-particulate form.
The survival of the bacterial cell in micro-particles was
studied under different levels of relative humidity, such
as 0, 33, 55 and 100%. Bacterial survival was satisfactory
for the formulation at 100 % relative humidity. The presence of silica in the formulation improved the bacterial
survival when the relative humidity becomes between 55
and 33% (Amiet-Charpentier et al. 1999). Compatibility
studies were performed between film-forming agents
and the encapsulated bacteria. Compatibility was
higher with one of the two film-forming agents (in the
powder form, Sepiret and an aqueous dispersion
form, Cerprotecteur), for the seed coatings tested and
immobilized bacteria were released when in contact with
excess of water (Amiet-Charpentier etal. 1999).
Similar to biofertilizer application, bacterial agents
can also be encapsulated for their use as biocontrol
agents. A simple strategy is the application of biocontrol
agents such as bacteria, Bacillus thuringiensis. Attempts
to create autoencapsulated formulations of B. thuringiensis against European corn borer, were made by mixing
starch powder and sugar (McGuire et al., 1990; Shasha
and Dunkle, 1989). Bok etal., (1994), suggested the use
of carbohydrate rich biopolymeric gels for construction
of matrices, and they observed a 50% mortality of the
diamondback moth even after two weeks. The entrapped
biopesticide in biopolymeric gels (potato starch, rice,
rye, barley, and soybean powders) could retain its

entomocidal activity through better protection against
dessication, sunlight, heat, and the damaging effects of
UV light (Bok etal. 1993). Cote etal. (2001), reported an
extended period of mortality of Choristoneura rosaceana
with a bioencapsulated formulation of B. thuringiensis
var. kurstaki in comparison to conventional Dipel (a
registered Bacillus thuringiensis var. kurstaki (Btk)-based
formulation). Among the tested meteorological factors
such as, precipitations, temperature, and solar radiations,
precipitation was more predominant as if reduced the
persistence of the insecticidal activity (Cote etal. 2001).
Fungal application
Most fungal parts can be used as mycopesticides, but all
of them are susceptible to environmental parameters
and there is worldwide acceptance for these formulations. Fungi belonging to the genera Conidiobolus,
Entomophaga, Nomuraea, Paecilomyces, Tolypocladium,
Verticillium, Entomophthora, Erynia, Neozygites, Pandora,
Hirsutella, Metarhizium, Aschersonia, Beauveria etc. are
well known as biocontrol agents against many pests (Shah
and Pell 2003). Trichoderma sp. was used as a biocontrol
agent against many fungal pathogens. Many strains of
Trichoderma have been investigated and considered as
potential biocontrol microorganisms. Trichoderma viride
ATCC 52440, for example, is able to produce biocontrol
agents against soil borne plant pathogens. This fungus
can be used for the biocontrol of Fusarium oxysporum,
F. solani, Phytophthora capsici, Sclerotium cepivorum,
and Verticillium dahliae (Cho and Lee, 1999). Generally,
formulations of the biocontrol strains of Trichoderma
are used in the form of alginate pellets containing their
spores (Knudsen and Bin, 1990; Lewis and Papawizas,
1985), and spores and hyphal segments in gluten (Cho
and Lee, 1999).
Akin to rhizobacteria, mycorrhizal fungi play different
roles in the soil nutrient improvement and plant growth,
such as phosphate nutrition, plant water potential under
drought stress, bioprotection against various pathogens and improvement of soil structure, among others
(Vassilev etal., 2005). According to Vassilev etal. (2005),
there are at least 1350 combinations of natural, semi-synthetic, and synthetic polymers for the entrapment of biomaterials but the majority of techniques involved in situ
entrapment by using natural polysaccharide gels which
include alginates, agar, and carrageenan. According to
Vassilev etal. (2005), the first successful encapsulation of
mycorrhizal fungi was performed by Ganry etal. (1982),
with endomycorrhizal fungi. Horaczek and Viernstein
(2004), examined a spray drying mechanism for encapsulating Beauveria brogniartii for the formulation against
Melolontha melolontha. The study on fungal encapsulations in skim milk and polyvinylpyrrolidone mixture
matrix, pointed out that spray drying of the fungal aerial
conidia was highly viable after spray drying even though
there was a decline in viability during an increase in the
outlet temperature. According to Horaczek and Viernstein

(2004), the survival of fungal material was inversely
related to the storage temperatures and residual moisture levels of the spray dried powders. The inlet-outlet
temperature must be selected based on the type of aerial
material and the storage of bioencapsulated material
must be carried out at low temperature with minimum
moisture. Each and every part has a different tolerance
and usually the conidia or other thick walled spores are
used for prolonged storage and long duration during formulation production.
Mixed culture application

To attain different results together, various biological
materials belonging to several genera with synergistic
action can be applied simultaneously. Cowpea treated
with Glomus mosseae (entrapped in Ca-alginate beads)
could improve nitrogen fixation with applications along
with Bradyrhizobium (Ganry etal., 1985). Vassilev etal.
(2001a) co-entrapped G. deserticola containing root pieces
and cells of the P-solubilizing yeast Yarrowia lipolytica
in alginate gel. They pointed out that the introduction
of an entrapped inoculant formulation into soil-plant
systems enriched with insoluble inorganic phosphates
showed that plant dry weight, soluble P-acquisition, and
mycorrhizal index were higher than the treatments with
free and Ca-alginate entrapped G. deserticola included
in the presence of free cells of the yeast inoculant. A
similar study with G. deserticola and G. fasciculatum
revealed that the introduction of both inoculant formulations into soil for the growth of the tomato plant as a test
showed average levels of root colonization between 26%
(G. fasciculatum) and 32% (G. deserticola) which were
further enhanced to 39% and 49%, respectively, when
the mycorrhizal fungi were co-entrapped in the alginate beads with Y. lipolytica (Vassilev et al., 2001b).
Another experiment by Vassilev etal. (2001c) showed a
four times higher nodule number was observed when
Glomus deserticola (an arbuscular mycorrhizal fungus)
was introduced into a soil-plant system with Rhizobium
trifoli and the results were compared with the control
plant nodulated only with R. trifoli. Addition of Y. lipolytica with a soil-plant system enhanced root mycorrhizal
infection by about 14%. Co-encapsulated R. trifoli and Y.
lipolytica yielded a ten fold increase in root nodulation in
the tested plants (Vassilev etal., 2001c).
Effect of natural agents on formulation performance

The effect of sunlight is crucial in foliar application
besides eroding factors, such as rain. Sunlight, mainly
direct heat and UV rays play an important role in the
mortality of biopesticides. Even though encapsulated
bacteria or fungal material have more tolerance than
free cell formulation, the application of heat tolerant
substances in encapsulating material can drastically
reduce the effect. According to Brar et al. (2006), various UV radiation screens, such as Congo red, folic acid,
molasses, lignin, alginate, cellulose, shellac, and p-aminobenzoic acid were tested for UV protection and showed

mixed results. Protein-based polymers, such as gluten

and casein are advantageous for use as coating materials
as they are biodegradable, harmless to the environment,
cheaper, and readily available. Protein coatings prevent
the UV exposure of prepared biological materials that are
sensitive to UV light (Yu and Lee, 1997). Selection of heat
tolerant bacteria or fungal material such as spores can be
more effectively utilized in tropical areas than the easily
desiccating biomaterial.
Rainfall is another important natural agent that would
affect the persistence of biopesticides on foliage leading
to wash-off and thus a reduction of the efficiency of the
biopesticide (Behle et al., 1997). Wash-off has a negative effect not only on foliage application but also on soil
application. Encapsulation can reduce microbial washoff by rain fall or runoff water. When the bacteria were
encapsulated, the wash down rate was reduced compared
to free inoculated bacteria, as most of them remained in
the root zone (controlled release) (van Elsas etal., 1991;
1992). P. fluorescens embedded in alginate beads had the
potential to produce a biocontrol effect against bacterial wilt of tomato by its colonization on the root (Aino
etal., 1997). Trevors etal. (1992) proposed that bacterial
inoculation using alginate resulted in good rhizosphere
colonization and survival on the plant, a low wash-down
rate, and resistance to drying. Corn starch formulation
showed a higher efficiency after rainfall due to the sticking property of the starch (Brar etal., 2006). Gelatinized
or modified starch sticker addition can also enhance the
efficiency of encapsulated biopesticides by application
on leaf surfaces.
There are several other factors, such as bead size,
shape, and cell load that have to be taken into account
while applying on to foliage or other arial parts of the
plant. Papavizas etal. (1987) studied the effect of wheat
bran on the proliferation of biological material, such as
hyphae, conidia, and ascospores of Talaromyces fIavus.
Conidia and ascospores showed rapid proliferation
but hyphae did not propagate. In contrast, Lewis and
Papavizas (1985) reported some food base addititives that
can increase the hyphal proliferation of Trichoderma and
Gliocladium in alginate beads. These facts indicate that
the selection of organism, type of encapsulation material
(bacterial cell, spores, and mycelia), encapsulating agent,
additives, and others as key parameters for the encapsulated formulation with respect to their application.
Future prospects and recommendations

Due to the benefits over conventional formulations,
encapsulated formulations can be recommended for
the agricultural applications of microorganisms, both
for soil and aerial applications. Among bioencapsulated
forms, microencapsulated formulations are preferable
to efficient applications such as foliar spraying or seed
coating. Seeds of plants like alfalfa are very small and
only microencapsulated forms can be coated on such
seeds for a uniform distribution. The seed coating of

microencapsulated formulation is cost effective and uniform as a minimum volume of the formulation is used
for seed application and the bioinoculant can have an
easy access during the emergence of radicle or plumule.
Microencapsulated formulation can be applied easily on
seeds or soil-like liquid formulations after the required
dilution. In microencapsulated beads cells are highly
concentrated and the volume of these formulations is
very low; therefore these formulations can be stored in a
limited space, transported easily, and diluted many times
during applications.
The risk of a biocontrol agent in the foliar application
is higher compared to soil applications as there is the
influence of environmental parameters, such as sunlight
and rain fall. However, using an effective means of bioencapsulation along with proper screens and stickers
would enhance the effect of biocontrol activity. Besides,
the additives and the biomaterial selection are also very
important depending upon the geographical area and
mode of application. Microencapsulation of bacteria
and fungal spore is easier than the fungal mycelia due to
their microstructure. The fungal thick-walled spores possess higher tolerance than the mycelial fragments. Thus,
spores can be utilized for the effective production of
microencapsulated material than the mycelia, as it helps
produce a uniform distribution. Generally, actively growing fungal mycelia are more active in biocontrol than
applied spore formulations as they can grow faster in the
soil. The beads stored at low temperature (the survival
rate is very high under refrigerated condition) can be
brought to the optimum growth temperature to enhance
germination, and hence increase the performance on the
application site. For soil applications, actively growing
mycelia can be entrapped and these macrobeads can be
mixed with soil.
Emulsion techniques can be adopted for microencapsulation of agro-friendly microbes and other techniques
such as spray drying or solvent extraction for the superior
survival of thermo-or chemo-liable bioinoculants. In our
previous work, it was shown that emulsion formulation
is better for the concentrated bioinoculant production
and it could enhance the survival of microbes during
prolonged storage (John etal., 2010). Hence, the encapsulated beads can be kept as such in oil after production,
emulsified with suitable emulsifier, or separated from the
oil phase for further formulation developments. A bioreactor can be used for the production of microbeads with
the emulsion technique and thus agitation and feeding of
ingredients can be controlled.
Our group has proved the use of different agro-industrial waste and wastewater for efficient and economic
production of biocontrol and biofertilizing microorganisms. After harvesting and concentrating, the microbial
biomass along with the other ingredients (unutilized
polymeric substances or hydrolysed substrates) in the
medium can be used for the micro-encapsulation.
Usually the sludge contains di- or trivalent metal ions
sufficiently in its natural form and this innate character
can be explored for the self encapsulation by mixing

polymers, such as alginate. The concentrations of these
metal ions are sufficient for supporting their growth and
thus it will not affect survival. Other by-products such as
brewery sludge, powdered brewery spent grain or hydrolyzed spent brewing yeast can be supplemented to the
harvested microorganisms. The selection of additives in
polymer must be performed carefully based on the type
of microorganism and its role on the growth or survival
of microorganism. The cost effectiveness can be achieved
by replacing skimmed milk (a general additive) with
whey concentrate or whey powder. The use of these low
cost materials can also act as carbon source for microorganisms during storage and applications. Biodegradable
synthetic polymers are a good option with respect to cost
effectiveness, survival of microbes, and environmental
impact. Natural gums, such as locust bean gum, xanthan
gum and gum arabic can be applied as stickers, antidesiccant or gelling agent, and thus the formulation can
be completely environmentally friendly.
The particle size of the additive must be smaller than
the prepared beads; otherwise the microorganism has
lower accessibility to the additive. The bigger size of
the additives has a negative influence on the survival of
microorganisms due to the moisture extraction outside
of the bead. The selection of microencapsulation technologies, such as emulsion technique and spray drying and also the process parameters, such as duration
of formulation development, temperature, and curing
time must also be taken into consideration while using
the desired bacterial or fungal material. As in the case of
spray drying, the inlet air temperature, drying duration
in the column, and the storage temperature in the cone
are critical.
The disadvantages of microencapsulation technology
must not be ignored. Many of the microencapsulation
technologies require special equipment. In the desired
processes, such as spray drying, several sequential steps
are needed for proper encapsulation. Microencapsulation
duration is longer than the preparation of other formulations. The material cost used for the encapsulation
is generally higher than other formulations. Therefore,
abundant and economically feasible materials should
be adopted. Biofertilizer and biopesticides have a major
impact in agricultural applications due to their ecofriendly nature. Environmental effects must also be taken
into consideration while selecting the material and processes for microencapsulation.
The market for biocontrol agents and biofertilizer
varies with different countries as there are varied rules
and regulations for application of microbes. There has
to be a universal regulation and acceptance for bioinoculant applications and both environmentalists and
the government have to take initiatives for testing and
commercialization of these biological and eco-friendly
formulations. Consumer anxiety about live microbes is
another limiting factor for commercialization. Proper
education and awareness for solving these issues and

support from financial agencies (public and private) has
to be implemented.
Conclusions
Different solid and liquid formulations have been developed for the application of microbial biofertilizer or
biocontrol agents in agriculture, albeit with advantages
and disadvantages. The emerging field of microencapsulation is a promising step to obtain the controlled and
effective release of these microorganisms for targeted
agricultural applications. The importance of the modification and testing of these formulations is necessary for
the cost effective and environmental-friendly production
and application of this system. Most of the current studies report positive effects of utilizing these advanced formulations. Furthermore, exploitation of different types of
matrices of biological origin, effectiveness of encapsulation of each and every microorganism for agricultural
use, survival during storage and application is necessary
to overcome the drawbacks of conventional solid and
liquid formulations.
Bacterial microencapsulated formulations for soil and
seed application are superior to other formulations as it
enhances the survival of bacteria, and controlled release
with prolonged effect. Even though the cost of production
is slightly higher, microorganisms in microencapsulated
formulation are recommended due to their higher performance during storage and applications. It is expected
that in future, efficient microencapsulated formulations
will conquer the market for better agricultural production
and to protect the environment from non-degradable
chemical pollutants.
Declaration of interest
The authors would like to thank the Natural Sciences and
Engineering Research Council of Canada (Grants A4984,
Canada Research Chair), MAPAQ (807150). One of the
authors, RPJ, would like to thank the Fonds Qubcois de
la Recherche sur la Nature et les Technologies (FQRNT),
for the postdoctoral fellowship under the programme
Programme de bourses dexcellence pour tudiants
trangers.
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Critical Reviews in Biotechnology, 2011; 31(3): 211226

2011 Informa Healthcare USA, Inc.

Bio-encapsulation of microbial cells for targeted agricultural

containing nodulating bacteria and sticking agents before

Soil quality is totally affected by the application of

212 Rojan P. John etal.

Conventional formulation versus advanced

phytoproducts, (ground grains, pulses flour, grain bran,

Need for alternative carriers

Advantages of cell encapsulation

Bio-encapsulation of microbial cells 213

Macro and microencapsulation

diffusion or when external conditions prompt the capsule

Microbeads and their structure

214 Rojan P. John etal.

There is the need of more cells in the

As in the case of solid formulations the

Contamination by other microbes is

A contamination problem exists even in

Transportation is costly due to the bulk

No or less need for sophisticated

Minimum storage space is required as it is

Figure 1. Different forms of microbeads: (1) spherical, (2) oval,

Figure 2. Diagrammatic representation of a microcapsule

poly(DL-lactic acid) having three different molecular

Bio-encapsulation of microbial cells 215

Figure 3. Different processes in microencapsulation technology.

that Hirai and Eyring (1959) proposed a relationship

216 Rojan P. John etal.

Cumulative frequency distribution

Cumulative frequency distribution

Critical Reviews in Biotechnology

Bio-encapsulation of microbial cells 217

Pregel dissolving technique

A polyelectrolyte complex membrane is formed on the

Factors affecting microencapsulated

218 Rojan P. John etal.

are the substances that aid in the stabilization and

Table 2. Different carrier materials used in encapsulation of microbial cells.

Zinjarde and Pant, (2000)

Paje etal. (1998)

Sheu and Marshall, (1991)

Lactobacillus delbrueckii ssp.

Ice cream production

Somerville etal. (1977)

L. lactis ssp. cremoris

Hyndman etal. (1993)

Amiet-Charpentier etal. (1998a)

Amiet-Charpentier etal. (1999)

Amiet-Charpentier etal. (1998b)

Weir etal. (1995)

Critical Reviews in Biotechnology

2011 Informa Healthcare USA, Inc.

The bacterial survival time of

Survival of bacteria was for nearly 8

Park and Chang (2000)

Complex coacervation without any

Table 3. Advantages, disadvantages and possible modifications of different matrices.

Moisture content in the formulation is

The bacterial survival was affected by

Separate storage of seeds and formulation

For suspension of bacteria, it can be mixed

Bio-encapsulation of microbial cells 219

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