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					Definition of Terms

       At the very advent of technology, pollution has indeed taken its toll on nature,
making people harvest and manufacture products that would take eons to decay and rot
at the very least. Everywhere, a plethora of biodegradable and non biodegradable
wastes can be seen. And, indeed it’s high time that people revert back to natural
processes that could help solve the burgeoning problem of waste disposal since all the
artificial methods that require today’s technology could contribute to the pollution that
we are experiencing now. One such natural process that could solve the problem, or in
a way even just alleviate such waste pile-up, is 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).
       In many cases, conditions are not favorable enough to promote spontaneous
biodegradation or natural attenuation. Whenever there is insufficient quantity of naturally
biodegrading organisms, there is a further need to add nutrients or other suitable
organisms aside from those present already, for improved biodegradation to occur. The
general objective of biodegradation is to discern the speed 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).
Include   literature   on   other   microorganisms       that   have    been   used    for

       A Xylaria sp. was discovered by Cuevas and Manaligod (1997), and will be used
in this study. It was observed growing on a sando plastic bag, buried in forest soil and
litter in the lowland secondary forest of Mt. Makiling, Laguna. The fungus comprised of
sterile melanin pigmented mycelia and was reported as ascomycete sterile dark mycelia
(ASDM). Cultural studies have designated it in Class Ascomycota, Order Xylariales,
under the genus Xylaria (unpublished data).
       Xylaria is one of the most commonly encountered groups of ascomycetes with
most of its members being stromatic, peritheciate, with an iodine-positive ascus apical
ring, and with one-celled, dark ascospores on which a germination slit can be found.
Most are inhabitants of wood, seeds, fruits, or leaves of angiosperms. Some are
associated with insect nests. Most decay wood and many are plant pathogens. Many
are endophytes. They are commonly found throughout the temperate and tropical
regions of the world. The Xylaria sp. can be distributed above, around, and beneath
perithecia. It forms a unipartite stromatal layer, with a superficial or erumpent surface
level. The interior of its stromata is essentially homogeneous. Conidium-bearing discs,
potassium hydroxide pigments and orange granules surrounding the perithecia are
absent (Rogers et al., 2002). They are mostly multiperitheciate in ascomatal number per
stroma, ascomatal ostioles and ascal apical rings: are present, and the ascospore cell
number is one-celled. Teleomorph and anamorph are produced on the same stromata
in most species, with their anamorphs: Geniculosporium-like.
       Some Xylaria sp. species exist as endophytes, and have mutualistic associations
with plants. The fungus secrete toxins to protect the plant from herbivory from other
insects or animals, while the fungus in return feeds on the host’s tissues for nutrition,
and its mycelia are scattered through seed dispersal (Davis, et al., 2003). There is a
hypothesis that Xylariaceae endophytes are quiescent colonizers that will decompose
lignin and cellulose later when the plant dies (adapted from Petrini et al., 1995; Whalley,
1996 as cited by Davis, et al., 2003). Nonetheless there are also some xylariaceous
fungi that only exist as endophytes (adapted from Rogers, 2000; J. D. Rogers,
Washington State University, personal communication as cited by Davis, et al., 2003).
No obvious benefit to living host plants has been documented for Xylariaceae (Davis, et
al., 2003).
        A review (Carroll, 1988 as cited by Davis, et al., 2003) of empirical studies on
antagonistic interactions between endophytes and grazers, insects and microbial
pathogens summarizes five general properties of endophyte mutualism: (1) the
endophyte is ubiquitous in a given host, geographically widespread, and causes
minimal disease symptoms in the host plant; (2) vertical transmission or efficient
horizontal transmission of the fungus occurs; (3) the fungus grows throughout host
tissue, or, if confined to a particular organ, a high proportion of such organs are
infected; (4) the fungus produces secondary metabolites likely to be antibiotic or toxic;
and (5) the endophyte is taxonomically related to known herbivore or pathogen

        A patented extract from Xylaria nigripes, the WulinshenPrime™ in SleepWell™
can provide important nutrients usually at a low level, to the brain and thus help in its
biochemical processes to promote a more restful and deeper sleep to wake up fully
revitalized. This extract contains essential amino acids, vitamins, minerals, trace
elements, glycoproteins, glutamic acid, γ-aminobutyric acid (GABA) and glutamate
decarboxylase. It is well established that glutamic acid assists the uptake of GABA to
specific brain cell receptors. GABA's main function is to inhibit excitatory neuro-activities
to exert a tranquilizing effect on the central nerve system. Glutamate decarboxylase
(GAD)          is      involved        in       the        synthesis        of       GABA.

        A previous study by Clutario and Cuevas (2001) proved that Xylaria sp. can
utilize polyethylene plastic strips as an alternative carbon source, thereby degrading
them into usable forms for self-sustenance. Through the use of scanning electron
microscopy, the proponents of the said study observed visible damages of the surface
structure of the plastic strips. There were tearing and striations caused by active
burrowing of Xylaria hyphae on the polyethylene material. Plastic is an extremely
versatile synthetic material made of high molecular weight, semi-crystalline polymer
prepared from ethylene through the cracking of crude oil, light petroleum and natural
gas (Knapczyk and Simon, 1992 as cited by Clutario and Cuevas, 2001). For plastic
bags       alone,        it      is        estimated      that       some        430,000
gallons of oil are needed to produce 100 million pieces of these omnipresent consumer
items on the planet (EcoWaste Coalition, 2008).

Solid Waste Generation IN THE PHILIPPINES
       Filipinos generate around 0.3 to 0.7 kilograms of garbage daily per person
depending on income levels, according to a study by the World Bank (2001). Metro
Manila produces about 8,000 tons of solid waste each day and is expected to reach
13,300 tons each day in 2014 (Baroña, 2004). The National Capital Region produces
the highest amount of wastes, about 23% of the country’s waste generation (ASRIA,
[( According to a survey conducted by the EcoWaste Coalition and
Greenpeace Southeast Asia in 2006, synthetic plastics comprise 76% of the floating
trash in Manila Bay, out of which 51% are plastic bags, 19% are sachets and junk food
wrappers, 5% are styrofoams and 1% is hard plastics. The rest were rubber (10%) and
biodegradable discards (13%) (EcoWaste Coalition, 2008).
       For this study, the potential degradation of three pollutants that has been a topic
of investigations lately and for the last decades as well. These pollutants are chicken
feather, natural rubber and polystyrene.

Chicken Feather
       Chicken feathers comprise _ % of our waste. 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., 2007). 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 Gousterova
et al. (2005) as cited in the journal of Cheng-Cheng et al. (2007) 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 (Chandra, 2002).

       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., 2007).
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., 2007). 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, 2007; Joshi et al.,2007) , fungi ( Gradisar et al., 2005) and actinomycetes
(Gousterova et al., 2005) are indeed capable of degrading chicken feathers. 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).

       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.

       In terms of experimental procedures, various methods are used in determining
the keratinolytic ability, which means it could produce keratinase and hence degrade
chicken feather, of microbes. A particular study by Tapia and Contiero 2008, used a
feather meal agar, wherein the feather served as the source of carbon, nitrogen, sulfur
and energy, in cultivating the isolated microbe Streptomyces. The growth, which
occurred on the 10th day of incubation, through colony formation of the microbe
indicated that it utilized the feather as a source of its nutrients. After which, its
keratinolytic activity was tested using a modified keratin azure protocol. Another study
by Maczinger et al. 2003 focused on the isolation of a microbe from the poultry waste
that could degrade feathers. During the preliminary elimination, they cultured the
different population of bacteria found in a partially degraded feather in a basal medium
with sterilized feathers serving as its source of carbon, nitrogen and sulfur. It was then
rotated in an orbital shaker for 10 days. After 4 days, one flask which showed a visual
degradation of the feather. A dilution series was made afterwards so as to isolate and
culture the bacteria that just degraded the feather. The strain was identified as Bacillus
lichenformis strain K-508. And the confirmation of the keratinolytic activity was done by
using the azokeratin as a substrate assay.

       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).

          Polystyrene, an aromatic polymer, is synthesized from the aromatic monomer
styrene which comes from petroleum products. It is a thermoplastic substance that
could be solid in room temperature or liquid when melted. One of the most common
forms and uses of polystyrene is the EPS which stands for Expanded Polystyrene. The
industry manufactures such product by mixing polystyrene with blowing agents in the
form of carbon dioxide and pentane which comprises 5%-10% of its composition.
( The EPS is also called foamed polystyrene
and it is said to be 30 times lighter than regular polystyrene. This substance is popularly
used     in   the     form         of    beverage        cups   and     insulating    materials.
( The basic unit of polystyrene
which is styrene, which is a known neurotoxin and animal carcinogen, is considered
very harmful to human health. In fact, it inflicts neurological and hematological disorder
especially to factory workers. EPS food packaging is the one accountable for the
leaking out of styrene. Styrene leak or leech is triggered when acids from our juices
when placed in such EPS cups and when food with Vitamin A content is placed inside a
microwave      leading       the        styrene     to     accumulate     in    our     system.

       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 (CAW), 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, 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. ( 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 buil-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 Rhodococcus sp. 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 at el. (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 and.
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 Mota 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
Mota et al., is a crucial step towards polymer biodegradation.

       As demonstrated, colonization is needed in determining whether a particular
microbe or organism is a potential biodegrading agent. (Mota et al., 2007) The growth of
the microbes on the surface of the polystyrene is a step that would lead to its
degradation. Further visual confirmation of deterioration of surface area is done by
using the scanning electron microscope. (Mor and Sivan, 2008; Mota et al., 2007 ).

Natural rubber
       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

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