Escolar Documentos
Profissional Documentos
Cultura Documentos
on Natural Rubber,
Chicken Feathers, and Polystyrene
Objectives
The proposed study aims to determine the biodegrading capacity of Xylaria sp. wild type and
its mutants.
The specific objectives are as follows:
1. to determine if Xylaria sp. mutants and wild type can degrade natural rubber as a carbon
source
2. to determine if Xylaria sp. mutants and wild type can degrade chicken feathers as a carbon
and nitrogen source
3. to determine if Xylaria sp. mutants and wild type can degrade polystyrene as a carbon
source
4. to compare the biodegrading ability of the wild type to each mutant to find which strain is
most appropriate for each type of waste
5. to examine the treated pollutants under the scanning electron microscope (SEM) to check if
the pollutants have been biodegraded by the Xylaria sp. mutant strains and wildtype
In early times, people have always believed of the world’s abundance and unlimited supply
of natural resources, thus, various activities was done with negligence and carelessness.
Contaminated lands generally result from past industrial activities when awareness of the health and
environmental effects connected with the production, use, and disposal of hazardous substances were
less well recognized than today. Currently however, the consequences of our previous actions are
felt more and more as the continual discovery of contaminated sites over recent years has led to
international efforts to remedy many of these sites, either as a response to the risk of adverse health
or environmental effects caused by contamination or to enable the site to be redeveloped for use
(Vidali, 2001).
Conventional methods for remediation have been to dig up contaminated soil and remove it
to a landfill, or to cap and contain the contaminated areas of a site. Some technologies that have been
used are high-temperature incineration and various types of chemical decomposition (e.g., base-
catalyzed dechlorination, UV oxidation). These techniques have several drawbacks such as technical
complexity, high costs, involves risks in the excavation, handling, and transport of hazardous
material. Additionally, most of them are very difficult and increasingly expensive (Vidali, 2001).
BIODEGRADATION
A better approach than these traditional methods is to completely destroy the pollutants if
possible, or at least to transform them to innocuous substances in a process called bioremediation or
biodegradation. Biodegradation is the breakdown of natural substances through the action of
enzymes secreted by organisms such as microbes and fungi. Only waste materials made up of natural
polymers can be degraded by microbes and fungi. Biodegradation works in such a manner that the
organisms involved utilize, or more appropriately, metabolize these wastes as sources of nutrients
such as carbon or nitrogen. (Tortora, et al., 2005). It uses relatively low-cost, low-technology
techniques, which generally have a high public acceptance and can often be carried out on site
(Vidali, 2001). Biodegradation agents like bacteria and fungi must be healthy and active for
biodegradation to be highly efficient. Biodegradation technologies create optimum environmental
conditions to help the growth and increase the number of microbial or fungal populations for them to
detoxify the maximum amount of contaminants (United States Environmental Protection Agency,
1996).
The general objective of biodegradation is to discern the speed (i.e. percent weight loss of
pollutant per week) of unaided biodegradation before catalysts may even be added, and then
strengthen spontaneous biodegradation only if this is not fast enough to remove the contaminant’s
concentration in the environment at which it may cause health risks to nearby inhabitants such as
people, animals and plants (European Federation of Biotechnology, 1999).
The control and optimization of biodegradation processes is a complex system of many
factors which include: the existence of a microbial population capable of degrading the pollutants,
the site conditions, the quantity and toxicity of contaminant chemicals and the environment factors
(type of soil, temperature, pH, the presence of oxygen or other electron acceptors, and nutrients).
Different microorganisms degrade different types of compounds and survive under different
conditions (United States Environmental Protection Agency, 1996).
MICROORGANISMS USED IN BIODEGRADATION
Through time, scientific experiments have already proven the ability of some
microorganisms to biodegrade pollutants such as polyethylene, polystyrene, rubber, chicken feathers
and other types of wastes. Organisms such as bacteria and fungi have proven themselves to possess
the capacity to biodegrade pollutants.
Bacteria such as Brevibaccillus borstelensis, Rhodococcous rubber C208, Xanthomonas sp.
strain 357 have been proven to degrade pollutants. Plastics in the form of polyethylene are known to
be degraded by the thermophilic bacterium Brevibaccillus borstelensis 707 which was isolated from
soil. (Hadad et al, 2005). Another study by Orr et al. (2004) featured the Rhodococcous ruber C208
as an effective polyethylene-degrading organism. In addition to this, this strain has been proven to
degrade polystyrene (Mor and Sivan, 2008). Yet originally, Rhodococcus rubber is a known rubber-
degrading organism, according to the review of Rose and Steinbuchel (2005). Xanthomonas sp.
strain 357, in much the same way, can degrade rubber as well.
A number of fungi species are also known to biodegrade. The known fungi biodegraders are
Gordonia sp., Streptomyces sp., Nectria gliocladioides, Penicillium ochrochloron and Geomyces
pannorum and Trichoderma atroviride. (Barraat et al.,2003; Cheng chang et al., 2003; Rose and
Steinbuchel, 2005)
Gordonia sp. and Streptomyces sp. are known rubber-degraders (Rose and Steinbuchel,
2005). Nectria gliocladioides (five strains), Penicillium ochrochloron (one strain) and Geomyces
pannorum (seven strains), in a study of Barraat et al. (2003), have been observed to degrade
polyurethane while simultaneously relating it to the water holding capacity of the soil. Moreover, In
a study conducted by Cao et al. (2008), the fungus Trichoderma atroviride completely degraded the
chicken feathers. This strain was actually isolated from a decaying feather.
The list of microorganisms that could be used in biodegrdation goes on for there are still
more species that could degrade pollutants. And in fact, in the Philippines a fungus has been isolated
and proven to degrade polyethylene (Cuevas and Manaligod, 1997; Clutario and Cuevas, 2001).
According to the study of Liers et al. (2007), Xylaria polymorpha, which is said to lack
peroxidase, is known to produce the enzyme laccase , a known ligninolyitc oxidoreductase. This
supports the previous study of Lou & Wen (2005) wherein they discovered that Xylaria sp. along
with other ascomycetes and some basidiomycetes commonly demonstrated laccase activity together
with cellulolytic and xylanolytic activities. The enzymatic profiles of the aforementioned species
suggests that (1) ascomycetes is potentially capable of utilizing the lignocellulosic wood components
(2) laccase is apparently the main enzyme for ligninolysis unlike the white-rot basidiomycetes that
utilizes its ligninolytic peroxidase in the form of manganese peroxidase or lignin peroxidase in
addition to lignin peroxidase.
Table 2. Growth of the four albino mutants on mineral medium with and without supplements as
compared to the wildtype SDM.
Another application of Xylaria aside from its biodegrading abilities is the proprietary Xylaria
nigripes extract in WulinshenPrime™ in SleepWell™ (a patented fermentation technology available
from NuLiv Science) which provides the critical, necessary and often depleted nutrients to the brain
and assist in the biochemical process in the brain to promote restful and deeper sleep so one will
wake up fully refreshed and energized. WulinshenPrime™ contains many essential amino acids,
vitamins, minerals, trace elements, glycoproteins, glutamic acid, γ-aminobutyric acid (GABA) and
glutamate decarboxylase (NuLiv Lifestyle, 2008).
A study by Park (2005) showed that antifungal antibiotics for the treatment of fungal diseases
of humans and veterinary animals were produced by a fungus identified as a Xylaria sp. according to
nuclear ribosomal ITS1-5.8SITS2 sequence analysis, and was labeled F0010 strain. The fungus was
endophytic to Abies holophylla, and the study evaluated its in vivo antifungal activity against plant
pathogenic fungi. The antibiotics were determined to be griseofulvin and dechlorogriseofulvin
through mass and NMR spectral analyses of purified liquid cultures. Compared to
dechlorogriseofulvin, griseofulvin showed high in vivo and in vitro antifungal activity, and
effectively controlled the development of rice blast (Magnaporthe grisea), rice sheath blight
(Corticium sasaki), wheat leaf rust (Puccinia recondita), and barley powdery mildew (Blumeria
graminis f. sp. hordei), at doses of 50 to 150 μg/ml, depending on the disease. This was the first
report on the production of griseofulvin and dechlorogriseofulvin by Xylaria species.
Polystyrene
a b c
Figure 4: Polystyrene (a) Different kinds of cup made of polystyrene, (b) Styrene molecular
formula, the repeating unit to make a large polystyrene, and (c) Model diagram of
a styrene monomer
Polystyrene is in high demand. It is the most used and utilized thermoplastic in the industry
due to its durability. But it is not biodegradable. (Mor and Sivan, 2008). According to the
Californians Against Waste (2008), it is very difficult to recycle due to its light weight property,
which accounts for why it’s expensive to recycle. Imagine just recycling a ton of polystyrene, needs
a budget of $3000. Hence, it has a negative scrap-value. More so, it’s due to this light weight
property that they find polystyrene hard to transport since polystyrene is advised to be always kept
food-free and uncontaminated when recycled. The build-up of polystyrene in landfills, as reported
by CAW (2008), will contribute to plastic marine debris, since even when it is disposed of properly
it is carried by natural agents such as wind or other forces to the ocean. As manifested, there is an
excess of it in the environment and it is a major pollutant. (Mor and Sivan, 2008). For almost three
decades ago, polystyrene was first ban due to the utilization CFC material for its generation. In fact
there was a hype heralding that it is recyclable. After some time the companies that invested for its
recycling process disappeared. This move confirms that, indeed, recycling polystyrene is not an easy
thing to do. Now, the problem is back and the attention of scientists is focused on the recycling of
disposable foamed polystyrene. But recycling it would cost much in terms of energy, waste and
management point of view. (Californians Against Waste, 2008). A way of solving such impending
problem, is through biodegradation (Mor and Sivan, 2008; Singh and Sharma, 2007).
Biodegradation has been manifested in a number of studies already. And some of the studies
will be named here. A study by Mor and Sivan (2008), dealt with the monitoring of biofilm
formation of the microbe Rhodococcus sp. strain C208 on polystyrene. Their aim was to observe the
kinetics of biofilm formation and of whether polystyrene would be degraded. They used two
methods in quantifying the biofilm biomas: modified crystal violet staining and observation of the
protein content of the biofilm. The C208 strain was cultured in a flask containing polystyrene flakes
with the addition of mineral oil (0.0055% w/v), which induced more biofilm build-up. The study
concluded that after an extension of 8th weeks of incubation, loss of 0.8% (gravimetric weight loss)
of polystyrene weight was found. From this, Mor and Sivan (2008) regarded C208 to demonstrate a
high affinity towards polystyrene through biofilm formation which lead to it’s degradation. The
C208 strain is a biofilm-producing actinomycete that has first colonized and degraded polyethylene
(Orr et al., 2004).
There were studies that tested the possibility of whether copolymerizing polystyrene with
other substance could make it more degradable and susceptible to microbial attack. In 1992, a study
by Milstein, et al. (1992), focused on the biodegradation of a lignin-polystyrene copolymer. The
white rot basidiomycete was used to degrade such lignin-polystyrene complex copolymer. Such
fungi released enzyme that oxidized lignin and demonstrated the degradation through weight loss,
UV spectrophotometric analysis and deterioration of surface of the plastic substance as seen under
the SEM. A similar study by Singh and Sharma (2007) demonstrated through the process of graft
copolymerization that polystyrene must be modified with natural polymers and hydrophilic
monomers so as to enhance its degrading ability and so as to render polystyrene waste useful in
diminishing metal ion pollution in water. According to the mentioned study, the degrading rate of
polystyrene increased to 37% after subjecting it to soil burial method for 160 days.
Furthermore, the study of Motta et al. (2007), explored the degradation of oxidized
polystyrene using the fungi Curvularia sp. After about nine weeks of incubation, microscopic
examination revealed that hyphae had grown on the polystyrene. The colonization of the fungi and
it’s adhesion to the surface of the substance, according to Motta, et al. (2007), is a crucial step
towards polymer biodegradation.
Natural rubber (NR) is made from the latex of the Hevea brasiliensis also known as the
rubber tree. It is mainly composed of cis-1,4 polyisoprene which has a molecular mass of about 106
Da. This could also be chemically synthesized and produce the substance known as Isoprene Rubber
(IR). (Linos, et. al, 2000).
Since 1914, natural rubber has been a classic subject of biodegradation studies. (Rose and
Steinbuchel, 2005). This is due to the high rate of its yearly manufacture which is several million
tons, as mentioned in the study of Bereeka (2006), and its slow rate of natural degradation as
reviewed by Rose and Steinbechul (2005). In fact, a number of studies abound concerning its
degradation. And it has been learned that both bacteria and fungi can participate in such process.
Throughout all the investigations and experimentations done, two categories of rubber-degrading
microbes according mainly on growth characteristics have been established. Based on a review of
Rose and Steinbuchel (2005), which recapped the aforementioned groups, the microbes that can
degrade rubber can be categorized as clear zone-forming around their colonies and non-halo forming
whenever isolated and cultured in latex overlay plate, which is made by overlaying a layer of latex
agar medium on a basal salt medium agar. The former category was identified to mainly consist of
actinomycetes species. They are said to biodegrade or metabolize rubber by secreting enzymes and
other substances and also they are dubbed to be slow degraders since they rarely show an abundant
cell mass when grown on natural rubber directly. On the other hand, members of the second group
do not form halos on latex overlay plates. They, unlike the first group, grow more when directly
grown on natural rubber. In a way, their growth on rubber could be described in an adhesive manner.
The second group is said to demonstrate a relatively stronger growth on rubber. Species comprising
this category are the Corynebacterium-Nocardia-Mycobacterium group. They consist of the
Gordonia polyisoprenivorans strains VH2 and Y2K, G. westfalica strain Kb1, and Mycobacterium
fortuitum strain NF4.
As demonstrated by a particular study by Linos, et al. (2000), the mechanisms that the
microbes undergo when biodegrading is colonization, biofilm formation and aldehyde group
formation after cultivating it on the surface of latex gloves. Such is revealed after undergoing the
Schiff reagent’s test. This is further examined under a scanning electron microscope. In their
methodology they have indicated that the preliminary screening method to be used in finding
potential rubber-degrading bacteria is by growing such bacteria or microbe on the latex overlay or by
latex film on the mineral agar plates. Growth and colonization of the microbe in this medium would
indicate its utilization of rubber as its sole carbon source; hence, making it as a potential rubber-
degrader.
a c
Figure 6: (a) A rooster will be the source of feathers for the current study, (b) Major types of
feathers: radially symmetric downy feather, bilaterally symmetric contour feather,
and bilaterally asymmetric flight feather or remiges (the type chosen for the current
study) and (c) male flight feather, a sample of a caudal feather or rectrice
In the Philippines, chicken feathers aren’t a publicly recognized problem. But, experiments
and researches for its reuse and degradation are being explored at present. At University of the
Philippines-Los Baños, scientist Menandro Acda has ventured into recycling chicken feather into a
low-cost building material. The scientist quoted that, recycling it would be more advisable than
burning it since the incineration problem could cause environmental hazards. (Morales, 2008).
Moreover, in the US alone, 2 billion pounds of chicken feathers are produced by the poultry industry
(Comis, 2008). Chicken feathers, by nature, are made up of over 90% protein (Cheng-cheng, et al.,
2008). And this protein is none other than keratin. It’s actually the most abundant protein. It is not
easily degraded due to its tightly packed structural arrangement which is in the form of alpha keratin
or beta keratin. The key to its stability lies on the cross-linking by disulfide bonds, hydrophobic
interactions, and hydrogen bonds. Such stability renders keratin water-insolube and non-degradable
by the enzymes papain, trypsin and pepsin. (Gradisar et al., 2005). In a study conducted by Onifade,
et al. (1998) and Goushterova, et al. (2005), as cited in the journal of Cheng-Cheng, et al. (2008),
the build-up of chicken feathers in the environment and landfills would only result to future
pollution problems and protein wastage. More so, its accumulation could serve as a breeding ground
for a variety of harmful pathogens (Singh, 2004).
Considering that chicken feathers have a high protein content it could also be used as an
animal feed, but first its protein must be degraded (Tapia and Contiero, 2008). Yet this is said to
need so much water and energy (Frazer, 2004). Old methods of degrading the chicken feathers such
as alkali hydrolysis and steam pressure cooking are no longer advisable. They cause so much energy
wastage and they unfortunately destroy the configuration of proteins. (Cheng-cheng, et al., 2008).
Incineration is also a method used in degrading such waste but it causes so much energy loss
and carbon dioxide build-up in the environment. Other methods of disposal are landfilling, burning,
natural gas production and treatment for animal feed. But subjecting it to burning and land filing
costs a lot and it contributes air, soil and water contamination. (Joshi et al., 2007).
A wiser suggestion or approach would be the use of microbes in degrading these chicken
feathers. (Cheng-cheng, et al., 2008). Such approach is said to an economical and environment-
friendly alternative (Joshi, et al., 2007). Experiments that tested on the degradation of chicken have
already been done. In fact, studies have already proven that keratinolytic microbes such as Bacillus
(Maczinger, et al., 2003; Rodziewicz and Wojciech, 2008; Joshi et al., 2007), fungi (Gradisar, et al.,
2005) and actinomycetes (Goushterova et al., 2005). It has also been demonstrated that Aspergillus
nidulasn, a known imperfect Ascomycete which produces the toxin aflatoxin, shows an outstanding
keratinolytic activity. The enzymes that perform keratin degradation are called keratinase, which
could degrade feathers and make it available for its use as animal feed, fertilizer and natural gas.
The enzymes are said to degrade the beta-keratin component and the main idea behind such
biodegradation is that the microbes use the feather as their carbon, nitrogen, sulfur and energy for
their nourishment. (Joshi et al.,2007 ; Manczinger, et al., 2003). According to study of Cheng-gang
et al. (2008), the keratinase enzyme is inducible whenever substrates of keratin composition are
present. Among the all the keratin-inducing substrates, feathers (made up of beta-keratin) are the
ones commonly utilized. Yet both alpha-keratin and beta-keratin substrates can be used in feather
degradation. It is reported that the mechanism behind the degradation of chicken feather is yet to be
elucidated. But according to Kunert (2000) in the study of Cheng-gang et al. (2008), the proposed
primary step in keratinolysis is the deamination which produces an alkaline environment. Such
environment is needed to induce substrate swelling, sulphitolysis and proteolytic attack. In the same
study of Cheng-gang et al. (2008), the degradation of feathers produced amino acid residues such as
threonine, valine, methionine, isoleucine, phenylalanine and lysine. It was elucidated that this could
be due to the high disulfide content of the feathers.
Keratinases isolated from microbes have various economic uses. Aside from its feather
degrading capacity, it could be used in the leather industry as an agent in dehairing leather. Its by-
product, the feather hydrolysate, could also be used as animal feed additive. (Joshi, et al., 2007).
Furthermore, potentially, the said hydolysate could be used in the generation of organic fertilizer,
edible films and amino acids which are considered rare, as cited by Brandelli in the journal of Joshi
et al. (2007).
Isolation of a new microbial organism that could degrade chicken feather will help in the
degradation of the chicken feathers which is now becoming a burden in the society both
internationally and locally. The microbe could potentially provide the keratinase that could be used
in compost technology (Maczinger et al., 2003) or in the conversion of feather to feedstock meal
additives (Tapia and Contiero, 2008).
METHODOLOGY
I. Research Design
The research design to be used in the study is the Randomized Complete Block Design
(RCBD). The experiment will consist of two trials/runs with three replicates per treatment. The
experimentation process will be conducted in UP Los Baños Institute of Biotechnology and at the
Microbiology thesis room of the CAS, UP-Manila.
II. Experimentation
A. Preparation of Inoculum
The stock culture of Xylaria sp. and its four albino mutant strains will be obtained from
UPLB Biotech. Xylaria sp. will be isolated by culturing it in a Potato Dextrose Agar (PDA) medium.
The pH will be adjusted to pH 5 and it will be incubated at 25˚C. After 2-5 days, the fungi will be
transferred into a mineral medium with 5% glucose flask and subjected to a shaker for enrichment
and sustenance to further growth.
B. Preparation of Pollutants
A. Polystyrene
1x2 cm strips will be cut from clean polystyrene food containers such as
styroplates. The strips will be weighed in two’s. The strips will be surface sterilized
using 70% ethanol solution. It will then be oven dried. The weight will serve as the
initial weight. One polystyrene representative will undergo SEM to visually see the
initial status of the strips before colonization. Afterwhich, two strips per replicate of
each treatment will be placed in a flask.
B. Chicken feathers
Fresh contour feathers from an adult, male Gallus sp. will be obtained from a
nearby market place where chickens are butchered and sold. The feathers will be
washed. And it will be cut from their tips to 3 cm in length. Each cut feather will be
weighed and placed in a foil. The weight obtained will serve as the initial weight. One
representative of the feather will be obtained and will undergo SEM to check the
initial surface of the feathers. The feathers will then be autoclaved for 20 minutes at
15psi. One 3 cm feather will be used per replicate of each treatment and it will be
placed in a flask.
C. Rubber
Obtain rubber latex gloves size 5. Cut the gloves into strips of the same sizes,
approximate area to be about 2x2 cm. Weigh the gloves by two’s. The weight will
serve as the initial weight. One strip will undergo SEM to check the initial surface
condition of the latex glove. Then the strips will be autoclaved for 20mins at 15 psi.
Afterwhich 2 strips of the gloves will be utilized per replicate of each treatment. This
will then be placed in a flask.
Note:
* This step is intended for each mutant and for the wild type. Since we have 4 mutant strains and a
wild type, this step will be repeated five times multiplied with the number of pollutants to be used.
Hypothesis
(Hypotheses for the blocksF for rows)
Ho1: There is no significant difference in the biodegrading ability of the different Xylaria sp. strains
on the pollutants.
Ha1: There is a significant difference in the biodegrading ability of the different Xylaria sp. strains
on the pollutants.
Dummy Tables
Table 1. Percent Weight Loss in two runs of the (insert pollutant name here) due to Xylaria sp.
strains
Replicate Replicate 2 Replicate 3 Replicate Replicate Replicate 6 Mean
1 4 5
Wild
type
Mutant 1
Mutant 2
Mutant 3
Mutant 4
Note:
*replicate 1-2 = run 1
**replicate 3-4 = run 2
*** replicate 5-6 = run 3
***Table 1 will be used for each pollutant hence it will be repeated thrice for each of the three
incubation periods.
Table 2. Mean Values of the Percent Weight Loss of Polystyrene, Natural Rubber and Chicken
Feathers due to degradation of Xylaria sp. strains
Xylaria strain Polystyrene Natural Rubber Chicken Feathers
Wild-type
Mutant1
Mutant2
Mutant3
Mutant4
Table 3. ANOVA for Randomized Complete Block Design of the Potential Biodegrading Ability of
Xylaria sp. Strains on Chicken Feathers, Natural Rubber and Polystyrene
Source of Variation SS df MS F P-value F crit
Rows (Xylaria strain)
Columns (pollutant)
Error
α = 0.05
In the event that the F value for rows or columns shows any significant difference, a post
hoc analysis would be conducted. For the F value for rows, which are the pollutants, the Multiple
Analysis test will be used and it will be followed by the Tukey’s test. While for the F value for
columns, the Dunnet’s test will be applied.
BUDGET OUTLINE
Materials
Styroplates P30
Reagents
Thesis Proposal
Printing P2000
Miscellaneous
Fares P5000
Glasswares P400
Others P2000
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