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					                                                     A Preliminary Study on the Potential……..1
                                                                        Dayao and Egloso, 2009

                                   INTRODUCTION

       In the early times, people have always believed in the world’s abundance and

unlimited supply of natural resources; thus, various environmental activities were done

with negligence. Currently however, the consequences of our previous actions are felt

more and more as the discovery of contaminated sites over the recent years continues,

and this has posed serious risks to people's health and the environment.

       Conventional methods for remediation have been to transfer contaminated soil 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, risks in the excavation

process, handling, and transport of hazardous material (Vidali, 2001).

       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 decomposition of

substances by the action of microorganisms, which result to the recycling of carbon, the

mineralization (CO2, H2O and salts) of organic compounds and the generation of new

biomass (Dommergues & Mangenot, 1972; Lucas, et al., 2008). Moreover,

biodegradation is said to take place in three stages: biodeterioration, biofragmentation

and assimilation. Biodeterioration is the result of the activity of microorganisms growing

on the surface and/or inside a given material (Hueck, 2001; Walsh, 2001; Lucas, et al.,

2008) through mechanical, chemical and/or enzymatic means (Gu, 2003; Lucas, et al.,

2008). Biofragmentation, on the other hand, is a lytic phenomenon essential for the
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                                                                         Dayao and Egloso, 2009

consequent process called assimilation (Lucas, et al., 2008). Organisms involved

metabolize wastes as sources of nutrients such as carbon or nitrogen (Tortora, et al.,

2005). Biodegradation technologies create optimum environmental conditions to help the

growth and increase the number of microbial populations for them to detoxify the

maximum amount of contaminants (United States Environmental Protection Agency,

1996). Biodegradation wants 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 before it

may cause any health risk to nearby inhabitants (European Federation of Biotechnology,

1999).

         As a prerequisite to initiate microbial growth in biodegradation, inorganic salts

and sugars (e.g. glucose) are often employed. The amount is strictly controlled to prevent

the organism from using this material as the sole carbon source. Growth is verified

through visual inspection, but absence of growth may be due to an unsuccessful

inoculation in addition to the fact that the organisms are unable to utilize the polymer as a

sole carbon source. Evaluation of biodegradation also frequently involves calculation of

weight loss and microscopic examination of the polymer surface. When a polymer is

incubated with fungi, small circular holes are sometimes observed. Consequently, it is

customary to include a surfactant (Tween) to enhance the growth conditions. However,

microorganisms themselves also often produce surfactants (Albertsson & Karlsson,

1993).

         Microbial growth depends on the properties of polymer materials and specific

environmental conditions, such as humidity, weather and atmospheric pollutants
                                                    A Preliminary Study on the Potential……..3
                                                                       Dayao and Egloso, 2009

(Lugauskas, et al., 2003; Lucas, et al., 2008). The ease with which a polymer is degraded

is entirely dependent upon its molecular weight, crystallinity, atomic composition and

conformation, the chemical bonds in the structure, the physical and chemical

characteristics of the surfaces (Callow& Fletcher, 1994; Becker et al., 1994; Caldwell, et

al., 1997; Gu, 2003), the indigenous microflora, and whether it is naturally occurring or

synthetic (Albertsson & Karlsson, 1993; Gu, et al., 2000; Gu, 2003; Motta, et al., 2007).

Generally, higher molecular weight results in greater resistance to degradation by

microorganisms, whereas monomers, dimers and oligomers of a polymer’s repeating

units are degraded and mineralized more easily. High molecular weights lead to sharply

decreased solubility, making the polymer resistant to microbial attack because

microorganisms need to assimilate the substrate through their cellular membrane and then

degrade the substrate further by means of intracellular enzymes. At least two categories

of enzymes, namely extracellular and intracellular depolymerizers, are actively involved

in biodegradation of polymers (Doi, 1990; Gu et al., 2000; Motta, et al., 2007). It is

commonly recognized that the closer a polymer’s structure to that of a natural molecule,

the more easily it is degraded and mineralized (Gu & Gu, 2005; Motta, et al., 2007).

       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, and Rhodococcous rubber C208

have been proven to degrade plastics in the form of polyethylene. Yet originally,

Rhodococcous rubber is a known rubber-degrading organism (Rose & Steinbuchel,
                                                      A Preliminary Study on the Potential……..4
                                                                         Dayao and Egloso, 2009

2005). A number of fungal species are also known to biodegrade. Gordonia sp. and

Streptomyces sp. are known rubber-degraders (Rose & Steinbuchel, 2005). The fungus

Trichoderma atroviride completely degraded chicken feathers (Cao, et al., 2008). This

strain was isolated from a decaying feather. The list of microorganisms that could be used

in biodegradation goes on for there are still more species that are yet to be discovered to

degrade pollutants. And in fact, in the Philippines a fungus named Xylaria sp. has been

isolated and proven to degrade polyethylene (Cuevas & Manaligod, 1997; Clutario &

Cuevas, 2001).

       Xylaria sp. tested in this study was discovered by Cuevas and Manaligod (1997),

growing on a sando plastic bag, buried in forest soil and litter in the lowland secondary

forest of Mt. Makiling, Laguna (Clutario & Cuevas, 2001). The fungus comprised of

sterile melanin pigmented mycelia and was reported as an ascomycete sterile dark

mycelia (ASDM). Cultural studies have designated it under the Class Ascomycetes,

Order Xylariales, Genus Xylaria (Clutario & Cuevas, 2001). It has not been identified in

the species level.

        Xylaria sp. can utilize polyethylene plastic strips as an alternative carbon source

(Clutario & Cuevas, 2001). The fungus grew optimally at 250C on a mineral medium of

pH5.0 containing 0.5% glucose and polyethylene plastic strips as co-carbon source. A

mucilaginous sheath was produced by the fungus to help its mycelial growth adhere to

the surfaces and edges of the plastic strips. After 50 days of incubation, the strips became

embedded in the mycelial growth. Visible damage on the surface structure of the plastic

strips was observed using scanning electron microscopy (SEM). Striations and tearing

were present due to the active burrowing of Xylaria hyphae on the polyethylene material.
                                                    A Preliminary Study on the Potential……..5
                                                                       Dayao and Egloso, 2009

This showed that Xylaria sp. has indeed a potential in degrading synthetic wastes like

plastics, which are difficult to decompose.

       The fungus Xylaria sp. and its albino mutants, PNL 114, PNL 116 and PNL 118

have been partially characterized (Tavanlar & Lat, 2008) based on colony characteristics,

morphology and growth on various media. These mutants were generated by subjecting

the black wild type SDM to mutagenesis, and protoplast fusion. Furthermore,

morphological and biochemical characteristics or markers in the wild type and mutants

were determined, which can be used in the analysis of future recombinants or fusants.

The black mutants E26 and E35, on the other hand, have not been characterized as of yet

(M. A. Tavanlar, personal communication, March 4, 2009).

       It was highly apparent from the very dark (black) color of mycelium and hyphae

of the wild type SDM that there was a high deposition of melanin. When deposited in the

outer layer of the cell wall, melanin reduces the pore diameter below 1nm but remains

permeable to water (Howard et al., 1991; Tavanlar & Lat, 2008). Melanin protects

hyphae during infiltration through a substrate during colonization (M. A. Tavanlar,

personal communication, March 4, 2009). This polymer provides tolerance to various

environmental stresses like oxidants, microbial lysis, UV radiation, and defense

responses of host plants and animals against fungal infection (Kimura & Tsuge, 1993;

Tavanlar & Lat, 2008).

       After mutants were repeatedly tested on MMG (mineral medium plus 0.5%

glucose) plus various supplements, Xylaria strain mutants PNL 114, 116 and 118 were

chosen based on the retained white color of the colonies even after 7 days (Tavanlar &

Lat, 2008). The hyphae of these mutants were similar to the wild type, when viewed
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                                                                        Dayao and Egloso, 2009

under the light microscope. These albino mutants evidently lost their melanin

pigmentation and the mycelia assumed a thinner appearance than the wild type dark

mycelia. This study utilized NTG in the induction of mutants from the SDM wild type.

Exposure to NTG (N’,N”-methyl-N-nitro-N-nitrosoguanidin) induced melanin-deficient

mutants in Alternaria alternate, M. grisea, Colletotrichum lagenarium and C.

lindemuthianum The phenotypic mutations showed albino, rosy, light brown, and brown

colony color (Kimura & Tsuge, 1993; Kawamura, et al., 1997; Tavanlar & Lat, 2008).

Defective genes involved in the very common DHN pathway to melanin biosynthesis

have been identified in some of the mutants of these fungi. The three amelanotic mutants

were further selected in various media supplemented with benomyl, acetamide, PEG

6000 (polyethylene glycol), Tween 80, and glucose (Tavanlar & Lat, 2008). In summary,

the results (Tavanlar & Lat, 2008) showed that the three mutants are less dependent on

the glucose level in the medium for growth and hyphal tip extension. They exhibited the

following other characteristics: loss of melanin pigmentation, ability to utilize

polyethylene glycol (PEG), Tween 80, acetamide, and resistant to some fungicides which

contained copper hydroxide and benomyl (Tavanlar & Lat, 2008). Also, the proponents

have speculated that the albino mutants can better survive environments with less

available amounts of readily utilizable carbon sources such as the surface of plastics than

the wild type.

       The current study, on the other hand, has tested the potential use of Xylaria sp.

and its mutants as a natural biodegrading agent in biodegrading other rampant wastes

such as natural rubber, polystyrene and chicken feathers. The study determined the

capacity of Xylaria sp. wild type and its mutants to degrade natural rubber, chicken
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                                                                       Dayao and Egloso, 2009

feather and polystyrene through Scanning Electron Microscopy (SEM). The biodegrading

ability of the Xylaria variants were compared to find which strain is most appropriate for

each type of waste. After incubation, the macroscopic/physical changes that natural

rubber, chicken feather and polystyrene have undergone were observed. Moreover, the

change in weight after 50 days of incubation was noted as an indicator of biodegradation.



Natural Rubber




       Figure 2: Natural rubber is a polymer called polyisoprene, made synthetically by
                 polymerization of a small molecule called isoprene, with the help of
                 special metal compounds called Ziegler-Natta catalysts.


       Natural rubber (NR) is made from the latex of 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 produced as the

substance known as Isoprene Rubber (IR) (Linos, et. al, 2000).

       The average composition of latex glove from the Hevea brasiliensis plant is 25-

35% (wt/wt) polyisoprene; 1-1.8% (wt/wt) protein, 1-2% (wt/wt) carbohydrates, 0.4-

1.1% (wt/wt) neutral lipids, 0.5-0.6% (wt/wt) polar lipids, 0.4-0.6% (wt/wt) inorganic

components, 0.4% (wt/wt) amino acids, amides, etc.; and 50-70% (wt/wt) water (Rose &

Steinbuchel, 2005). This polymer has rubber particles which are about 3-5µm and

covered by a layer of proteins and lipids. This serves to divide the hydrophobic rubber
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                                                                        Dayao and Egloso, 2009

molecules from the hydrophilic environment. But due to some allergic potential caused

by Hevea proteins, methods to remove these proteins were applied such as centrifugation

to clean the latex, treatment of sodium or potassium hydroxide and application of

enzymatic digestion with papain or alkaline proteases.

       Since 1914, natural rubber has been a classic subject of biodegradation studies

(Rose & Steinbuchel, 2005) due to the high rate of its yearly manufacture which is

several million tons (Bereeka, 2006), and its slow rate of natural degradation (Rose &

Steinbuchel, 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 done, two categories of rubber-degrading

microbes according mainly on growth characteristics have been established (Rose &

Steinbuchel, 2005). Rubber degrading microbes 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 actinomycete

species. They are said to 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-
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                                                                        Dayao and Egloso, 2009

Nocardia-Mycobacterium group. They consist of the Gordonia polyisoprenivorans

strains VH2 and Y2K, G. westfalica strain Kb1, and Mycobacterium fortuitum strain NF4.

       The mechanisms (Linos, et al., 2000) 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. The study

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.

       Furthermore, the degradation of natural rubber is initiated by the oxidation of

double bonds (Bereeka, 2006). Once this takes place, oligomeric derivatives with

aldehyde and keto groups formed at their ends are assumed to be degraded by beta-

oxidation. The mechanism of rubber degradation of the Gordonia sp., as shown by

spectroscopy, resulted in a decrease in the number of cis-1,4 double bonds in the

polyisoprene chain, the appearance of ketone and aldehyde groups in the samples, and the

formation of two different kinds of bonding environments (Linos, et al., 2000). Such

results could be interpreted as a product of polymer chain length that had undergone

oxidative reduction thereby yielding a change in the chemical environment.


Chicken Feathers
                                                     A Preliminary Study on the Potential……..10
                                                                         Dayao and Egloso, 2009




         a                             b                                           c
Figure 3: (a) Gallus sp is the source of feathers for the current study, (b) bilaterally
        symmetric contour feather and its parts (the type chosen for the current study)
      In the Philippines, chicken feathers aren’t a publicly recognized problem.

However, the build-up of chicken feathers in the environment and landfills would only

result to future pollution problems and protein wastage (Onifade, et al., 1998;

Goushterova, et al., 2005; Cheng-Cheng, et al.; 2008). More so, its accumulation could

serve as a breeding ground for a variety of harmful pathogens (Singh, 2004). Experiments

and researches for its reuse and degradation are being explored at present. At the

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 - 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 higher degree of cross-linking

by disulfide bonds, hydrophobic interactions, and hydrogen bonds. Such stability renders

keratin water-insoluble, extremely resistant to biodegradation and poorly susceptible to
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                                                                          Dayao and Egloso, 2009

digestion by the most common peptidases like papain, trypsin and pepsin (Gradisar et al.,

2005; Kim, 2007).

       Considering that chicken feathers have a high protein content it could be used as

an animal feed, but first its protein must be degraded (Tapia & 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).

       Composting is one of the more economical and environmentally safe methods of

recycling feather wastes (Tiquia, et al., 2005). During composting, organic materials are

mixed to create a moist, aerobic environment where organic matter decomposition and

humification occur at rapid rates. 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 landfilling costs a lot and it contributes

air, soil and water contamination (Joshi, et al., 2007).

        A wiser approach would be the use of microbes in degrading chicken feathers

(Cheng-cheng, et al., 2008). Such approach is said to be an economical and environment-

friendly alternative (Joshi, et al., 2007). Experiments that tested on the degradation of

chicken feathers have already been done. In fact, studies have already proven that

keratinolytic microbes such as the bacterium Bacillus (Maczinger, et al., 2003; Joshi, et

al., 2007; Rodziewicz & Wojciech, 2008), fungi (Gradisar, et al., 2005) and

actinomycetes (Goushterova et al., 2005) have an ability to degrade the keratin in chicken
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                                                                       Dayao and Egloso, 2009

feathers. A study (Burtt & Ichida, 1999) also showed that bacteria collected from wild

birds can cause extensive damage to feathers in vitro. The damage is caused by one or

more keratin-degrading enzymes released by vegetative bacterial cells. Of course, in vitro

experiments may overestimate the potential for bacterial damage under natural

conditions. And the metabolic activity and / or antibiotic production of some bacteria

may inhibit or improve the growth of other bacteria and / or fungi present. Researchers

have known for decades that the plumage of birds harbors a diverse community of

bacteria and fungi, including yeast (Hubilek, 1994). To our knowledge, no one has yet

comprehensively characterized the microbial communities living in feathers of any

species. Such information is needed to determine how microbes interact both with one

another and with birds (Shawkey, et al., 2005). Outstanding keratinolytic activity among

keratinases produced from tested nonpathogenic filamentous fungi has been observed

from Paecilomyces marquandii, Doratomyces microsporus, Aspergillus flavus (Gradisar,

et al., 2005) and Aspergillus nidulans strains (Manczinger, et al., 2003; Joshi, et al.,

2007).

         A group of proteolytic enzymes which are able to hydrolyze insoluble keratins

more efficiently than other proteases are called keratinases. These enzymes can degrade

feathers and make them available for 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 source for nourishment (Manczinger, et al., 2003; Joshi, et al., 2007). The

keratinase enzyme is inducible whenever substrates of keratin composition are present

(Cheng-gang, et al., 2008). Among all the keratin-inducing substrates, feathers (made up
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                                                                          Dayao and Egloso, 2009

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), the

proposed primary step in keratinolysis is deamination which produces an alkaline

environment (Cheng-gang, et al., 2008). Such environment is needed to induce substrate

swelling, sulphitolysis and proteolytic attack. 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

(Cheng-gang, et al., 2008).

         Keratinases are produced by some insects and mostly by microorganisms. They

have various economic uses. Aside from their feather degrading capacity, they could be

used in the leather industry as an agent in dehairing leather. Their by-product, the feather

hydrolysate, could also be used as an animal feed additive, and potentially in the

generation of organic fertilizer, edible films and amino acids which are considered rare

(Joshi, et al., 2007). Keratinases are extensively applied also in the detergent and textiles

industries, waste bioconversion, medicine, and cosmetics for drug delivery through nails

and degradation of keratinized skin (Gradisar, et al., 2005).

Polystyrene




     a                                              b                                c
                                                     A Preliminary Study on the Potential……..14
                                                                         Dayao and Egloso, 2009

   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, an aromatic polymer and an inexpensive, hard plastic, 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. In

thermoplastics, the polymer chains are only weakly bonded (van der Waals forces). The

chains are free to slide past one another when sufficient thermal energy is supplied,

making the plastic formable and recyclable (eFunda, 2009). Most biodegradable

polymers belong to thermoplastics (e.g. poly (lactic acid), poly(hydroxyalkanoate),

poly(vinyl alcohol)) or plants, polymers (e.g. cellulose and starch), except for polyolefins

which are nonbiodegradable. 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

or styrofoam 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 (Friend, 2005).

EPS represents one of the packaging industry's toughest environmentally challenging

products, due to its enormous sustaining longevity, which consequently results in a

negative impact on our environment. Although there have been some developments using

chemical degradation of polystyrene materials, there still exists the problem of a chemical

byproduct that will remain behind, which is a noxious deposit know as a "benzene ring”.

This noxious chemical exists after polystyrene completes its "degradation process”.
                                                   A Preliminary Study on the Potential……..15
                                                                       Dayao and Egloso, 2009

       The basic unit of polystyrene is styrene, which is a known neurotoxin and animal

carcinogen, considered very dangerous to human health and hence, strategies to avoid its

discharge, eliminate it from the environment, and understand its route of degradation

were the focus of much research (Mooney, Ward, & O'Connor, 2006). Studies suggest

that styrene mimics estrogen in the body and can therefore disrupt normal hormone

functions, possibly contributing to thyroid problems, menstrual irregularities, and other

hormone-related problems, as well as breast cancer and prostate cancer. The estrogenicity

of styrene is thought to be comparable to that of bisphenol A, another potent estrogen

mimic from the world of plastics (Grinning Planet, 2008). Long-term exposure to small

quantities of styrene is also suspected of causing low platelet counts or hemoglobin

values, chromosomal and lymphatic abnormalities, and neurotoxic effects due to

accumulation of styrene in the tissues of the brain, spinal cord, and peripheral nerves,

resulting in fatigue, nervousness, difficulty sleeping, and other acute or chronic health

problems associated with the nervous system (Grinning Planet, 2008). The one

responsible for the leaking out of styrene is EPS food packaging. Styrene leak or leech is

triggered once acids from our juices are 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. (Californians Against Waste, 2008). The International Agency for Research on

Cancer lists styrene as a possible human carcinogen, though this conclusion is primarily

based on studies of workers in styrene-related chemical plants. The Vallombrosa

Consensus Statement on Environmental Contaminants and Human Fertility Compromise

includes styrene on its list of contaminants of possible concern, noting that even weak

estrogen mimics can combine with other such chemicals to have negative effects even
                                                   A Preliminary Study on the Potential……..16
                                                                       Dayao and Egloso, 2009

when the chemicals are individually present at levels that would have no impact. On the

positive side, a 2005 expert panel convened by the National Institutes of Health

concluded that there is negligible concern for developmental toxicity in embryos and

babies (Grinning Planet, 2008).

       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 & 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 is 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 is 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 Californians

Against Waste (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 & Sivan, 2008). For almost three decades ago, polystyrene was first

banned due to the utilization of 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
                                                   A Preliminary Study on the Potential……..17
                                                                       Dayao and Egloso, 2009

Waste, 2008). A way of solving such impending problem is through biodegradation

(Singh & Sharma, 2007; Mor & Sivan, 2008).

        Biodegradation has been manifested in a number of studies already. And some of

the studies will be named here. There are a large number of microbial genera capable of

metabolizing styrene as a sole source of carbon and energy and therefore, the possibility

of applying these organisms to bioremediation strategies was extensively investigated.

From the multitude of biodegradation studies, the application of styrene-degrading

organisms or single enzymes for the synthesis of value-added products such as epoxides

has emerged (Mooney, Ward, & O'Connor, 2006).

        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 biomass: 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 8 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 its 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
                                                    A Preliminary Study on the Potential……..18
                                                                        Dayao and Egloso, 2009

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 complex copolymer. Such fungus released enzyme that oxidized lignin

and demonstrated 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, thereby rendering 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. Another study (Galgali, et al., 2004) linked a series of sugar molecules such

as glucose, sucrose and lactose, to maleic anhydride functionalized polystyrene through

polymer analogous reactions to produce biodegradable polymers. Evaluation of the

biodegradability of these sugar linked polystyrene-maleic anhydride copolymers by

known fungal test organisms was done using pure culture system. After fungal treatment,

weight loss measurements confirmed the biodegradability of the carbohydrate-linked

polymers. Results revealed that the degree of susceptibility to degradation varied with the

type of test organism and the type of sugar. Then polymer degradation was confirmed

through Fourier Transform Infrared Spectroscopy (FTIR) spectra.

       In 1993, a study by Cox, et al. was conducted to enrich styrene-degrading fungi in

biofilters under conditions representative for industrial off-gas treatment. From the

support materials tested, polyurethane and perlite proved to be most suitable for

enrichment of styrene-degrading fungi. Perlite is an amorphous volcanic glass that has a
                                                      A Preliminary Study on the Potential……..19
                                                                          Dayao and Egloso, 2009

relatively high water content, typically formed by the hydration of obsidian. The biofilter

with perlite completely degraded styrene and an elimination capacity of at least 70 g

styrene/m3 filter bed per hour was computed. In this study, a concept in biofiltration is

presented, based on the application of fungi for the degradation of waste gas compounds

in biofilters containing inert support materials for the immobilization of the fungi. In

principle, the application of fungi in biofilters may offer two advantages: (1) stern control

of the water activity and/or pH in the filter bed is less important, since fungi are generally

tolerant to low water activity and low pH, and (2) reduction of the water activity in the

filter bed may improve the mass transfer of poorly water soluble waste gas compounds

like styrene.

       Starch was shown in a study (Jasso, et al., 2004) to be useful in the degradation of

polystyrene. In this study, results showed the effectiveness of concentrated activated

sludge in polymer degradation and the utility of starch inclusion as a filler to accelerate

the structural molecular changes. High impact polystyrene blended with starch was

degraded in concentrated activated sludge for 3 months. Then mechanical degradation

was determined by stress-strain tests. Examination through scanning electron microscopy

showed the presence of microorganisms in the polymer samples, and changes in polymer

morphology in areas near holes produced in samples.

       Furthermore, the study of Motta et al. (2007), explored the degradation of

oxidized polystyrene using the fungus Curvularia sp. After about nine weeks of

incubation, microscopic examination revealed that hyphae had grown on and inside the

polystyrene. The colonization of the fungus and it’s adhesion to the surface of the
                                                     A Preliminary Study on the Potential……..20
                                                                         Dayao and Egloso, 2009

substrate, such as polystyrene, according to Motta, et al. (2007), is a crucial step towards

polymer biodegradation.

       As demonstrated in several studies mentioned above, colonization is needed in

determining whether a particular microbe or organism is a potential biodegrading agent

(Motta 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 (Motta et al., 2007; Mor

& Sivan, 2008).




                            MATERIALS AND METHODS



Research Design

       The research design used in the study is the Randomized Complete Block Design

(RCBD). The experiment consisted of three trials/runs with two replicates per strain

treatment. The experimentation process was conducted at the National Institute of
                                                    A Preliminary Study on the Potential……..21
                                                                        Dayao and Egloso, 2009

Molecular Biology and Biotechnology (BIOTECH), University of the Philippines - Los

Baños.



Experimentation

I. Preparation of Inoculum

         The stock cultures of Xylaria sp. which are the wild type (SDM), its three albino

mutants (PNL 114, 116 and118) strains and two black strains (E25 and E36) were

obtained from UPLB BIOTECH. Xylaria sp. strains were isolated by culturing them in a

Potato Dextrose Agar (PDA) medium. The pH was then adjusted to pH 5.0 and it was

incubated at 25˚C. After 2-5 days, the fungi were transferred into a flask containing

mineral medium with 0.5% glucose. Inoculation was done using a cork borer with a

diameter size of 0.5cm. The fungal pellets were bored at the margins of the colony to get

actively growing and young hyphae. Two pellets were inoculated or transferred to each

flask. The flasks were then subjected to shaking for enrichment and sustenance of

growth.




II. Preparation of Pollutants

         A. Polystyrene

                    1x2 cm strips were cut from clean polystyrene food containers such as

            styroplates. The strips were weighed in two’s. They were subjected to surface

            sterilization by shaking in 70% ethanol, once for 3 mins, then in sterile

            distilled water twice for 1min each, The weight served as the initial weight.
                                             A Preliminary Study on the Potential……..22
                                                                 Dayao and Egloso, 2009

   One polystyrene representative will undergo SEM to visually see the initial

   status of the strips before colonization. After which, two strips per replicate of

   each treatment will be placed in a mineral medium flask.

B. Chicken feathers

            Fresh contour feathers from an adult, female Gallus sp. were obtained

   from a nearby market place where chickens are butchered and sold. The

   feathers were washed and cut from their tips to 3 cm in length. Each cut

   feather were weighed and placed in a foil. The weight obtained served as the

   initial weight. One representative of the feather was obtained and underwent

   SEM to visually see the initial surface status of the feathers. The feathers will

   be wrapped in a foil and then it will be sterilized using an autoclave for 20

   minutes at 15psi. One 3 cm feather was used per replicate of each treatment

   and it was placed in a flask.

C. Rubber

            Used rubber latex gloves were used. The gloves were cut into strips of

   the same sizes, and the area was approximated to be about 2x2 cm. The gloves

   were weighed by two’s. The weight served as the initial weight. They were

   subjected to surface sterilization by shaking in 70% ethanol, once for 3 mins,

   then in sterile distilled water twice for 1min each, One strip underwent SEM

   to check the initial surface condition of the latex glove. After which, two

   strips of the gloves were utilized per replicate of each treatment. This was then

   placed in a flask.
                                                     A Preliminary Study on the Potential……..23
                                                                         Dayao and Egloso, 2009

III. Biodegradation Proper using Culture Method

       Seven flasks for the wild type, five mutants and control were prepared containing

15 ml Mineral Medium each, in duplicate. Then, 0.5% glucose was added in all the

flasks. The pH was adjusted to pH 5.0 by adding small amounts of either 0.1M NaOH or

0.1M HCl. The addition of sterilized substrate pollutant (either polystyrene, natural

rubber or chicken feather) followed, then the inoculation of 2 ml of Xylaria sp. SDM wild

type and it’s mutants in six flasks excluding the control.

       The incubation period lasted for 50 days with the flasks in a room with more or

less 250C in temperature. Yet observations were made on the 20th, 30th and finally on the

50th day. The observations done on the 20th and 30th day were only visual examinations

since removing the pollutant and fungi from the flask might contaminate the culture.

Determination of the colony growth rate (growth in mm/day) had been attempted yet no

pattern had been established.

       On the 50th day, the Xylaria sp. and its mutant strains were removed from the

pollutants. The mineral medium along with some of the fungi not colonizing the

pollutants were decanted from the flasks. The emptied flasks containing the pollutants

with some remaining fungi closely adhering on its surface were rinsed once with 70%

ethyl alcohol for three minutes with shaking. And twice, for one minute each, with

distilled water to remove the remaining fungi. Gentle scraping was applied to the samples

to remove the mycelia and mucilaginous sheaths attached. Next, the pollutants were air

dried. Then lastly, the final weight of the pollutants was determined using an analytical

balance.
                                                      A Preliminary Study on the Potential……..24
                                                                          Dayao and Egloso, 2009

Determination of Potential Degradation through Scanning Electron Microscopy

          Only one sample per strain of each pollutant was used and underwent SEM. In

choosing the samples to be subjected to SEM, factors such as extent of colonization on

the surface, weight increase, weight decrease and other physical changes were

considered. The samples that best demonstrated the aforementioned properties were

picked.

          In preparing for SEM, the sample pollutants were cut into small sizes,

approximately less than a centimeter. Samples were then gold-sputtered to make it

electrically conductive. When the sputtering was done, the loading into the scanning

electron microscope (model Leica S440) followed. Images of various magnifications,

such as 100x, 200x, 500x, 1000x, 1500x and 2000x, for each sample were chosen non-

uniformly.

          After obtaining the micrographs, the Xylaria sp. strains’ images were individually

examined and contrasted with the control. The wild type (SDM) micrograph was then

compared with the micrographs of the five mutant species for each pollutant.




                                         RESULTS



          After 50 days of incubation, the final weights of the natural rubber, chicken

feather and polystyrene samples were determined using an analytical balance. Weight

gain instead of weight loss was observed for some of the samples (see appendix B). Thus,
                                                   A Preliminary Study on the Potential……..25
                                                                       Dayao and Egloso, 2009

instead of utilizing percent weight loss to determine the potential degrading ability on

natural rubber, chicken feather and polystyrene of the Xylaria sp. and its variant strains

with respect to colonization, Scanning Electron Microscopy (SEM) was used.




Natural Rubber
                                                  A Preliminary Study on the Potential……..26
                                                                      Dayao and Egloso, 2009




           a                                      b




           c                                      d




           e                                      f
Figure 5. Growth of Xylaria sp. with presence of colonies at the tip of the white arrows
(a. SDM/wild type strain, b. E26 strain, c. E35 strain, d. PNL 114 strain, e. PNL 116
strain, and f. PNL 118 strain) in mineral medium with 0.5% glucose and natural rubber;
pH of medium - 5.0 and incubation temperature of ~250C, as observed for 20 days of
incubation.
                                                  A Preliminary Study on the Potential……..27
                                                                      Dayao and Egloso, 2009




Figure 6. SEM of the non-inoculated natural rubber control without the fungus.




                                                          hy


                                          my


                                          hy
                                                                                    my




Figure 7. SEM of natural rubber surface colonized by the SDM or wild type strain after
50 days of incubation. There is presence of hyphae (hy) and mycelial mats (my).
                                                    A Preliminary Study on the Potential……..28
                                                                        Dayao and Egloso, 2009




                                     sp                     hy                    hy




                                                                 my




Figure 8. SEM of natural rubber surface colonized by the PNL 114 mutant strain after 50
days of incubation. There is presence of spore (sp), hyphae (hy) and mycelial mats (mm).




                                               my

                                                               hy



                                          sp
          sp




                                                                               my



Figure 9. SEM of natural rubber surface colonized by the PNL 116 mutant strain after 50
days of incubation. There is presence of hyphae (hy), spores (sp) and mycelial mats (my).
                                                  A Preliminary Study on the Potential……..29
                                                                      Dayao and Egloso, 2009




                                            hy


                   my




Figure 10. SEM of natural rubber surface colonized by the PNL 118 mutant strain after
50 days of incubation. There is presence of mycelial mats (my) and hypha (hy).




                                                      mc




Figure 11. SEM of natural rubber surface colonized by the E26 mutant strain after 50
days of incubation. Micrograph demonstrates profuse growth of mycelia (mc).
                                                   A Preliminary Study on the Potential……..30
                                                                       Dayao and Egloso, 2009




                                                 sp


                                                                  my




                                                                    mc




 Figure 12. SEM of natural rubber surface colonized by the E35 mutant strain after 50
days of incubation. There is presence of mycelial mats (my), spores (sp) and mycelia
(mc).


Microscopic SEM Results

       The micrographs of each mutant strain and wild type were compared to the

micrograph of the non-inoculated control to reveal the marked differences and changes

that had taken place. In figure 6, a 10um section of the non-inoculated control is shown.

Figure 7 demonstrated the growth of SDM (wild type) after 50 days which revealed the

presence of hyphae and mycelial mats in the rubber surface as indicated by the arrows. In

figure 8, micrograph of the PNL 114 mutant demonstrated mycelial mat formation. There

were also spores seen on the surface as well as hyphae. Micrograph revealed that this

mutant strain has demonstrated comparable colonization compared to the SDM. On the

hand, PNL 116 mutant (shown in figure 9) showed minimal colonization when compared

to the SDM strain since there is a minimal growth: minimal presence of hyphae, mycelial
                                                   A Preliminary Study on the Potential……..31
                                                                       Dayao and Egloso, 2009

mat and spores on the natural rubber. In figure 10, PNL 118 mutant showed no distinct

traces of mycelia or spores on the surface; hence no distinct colonization was observed

when compared to the SDM and control. But when the PNL 118 strain at 500x

magnification (see appendix C) was compared to the non-inoculated control (figure 6)

presence of mycelial mat is revealed. At 1000x magnification, the PNL 118 (figure 10)

demonstrated a clearer view of the mycelial mat formation. The E26 mutant (figure 11)

showed the greatest growth and colonization when compared to the SDM and other

mutant strains. The micrograph revealed profuse growth of mycelia as indicated by mc.

When compared to the control (see figure 6), the original rubber surface could not be

distinguished. Lastly in figure 12, E35 mutant strain also demonstrated natural rubber

surface colonization. The result is comparable to that of the SDM. Presence of mycelia

and spores were observed. Moreover, formation of mycelial mat is also present.



Macroscopic Results

       Macroscopic examination of the natural rubber samples was also done. Through

physical examination, it revealed that the control showed no change after the 50-day

incubation period. While the SDM on the other hand, even after rinsing once with 70%

ethanol and twice with water, showed signs of mycelia embedded on the surface.

However, in the case of the three albino mutants namely 114, 116 and 118, there were no

physical signs of mycelia embedded on the surface because of the color of these mutant

strains which is similar to the natural rubber color. But microscopic examination revealed

their presence. There was an observed elasticity reduction in the samples where the

mycelia have obviously adhered. The observation was obvious among the black mutant
                                                      A Preliminary Study on the Potential……..32
                                                                          Dayao and Egloso, 2009

strain samples. Yet no actual standard measurement was undertaken to measure the

percent of elasticity loss. More so, there had been an observed biofilm formation in the

form of mucilaginous sheath on the surface of the natural rubber where the Xylaria sp.

strains have grown.

Table 4. Table of the Macroscopic/Physical and Microscopic Observations of the Xylaria
    sp. Mutant Strains and Wild Type on Colonized Natural Rubber after 50 days of
                                     Incubation.

     Strain                Macroscopic/Physical              Microscopic Observations
                                Observations
 Non-inoculated        Clear surface, no trace of fungus             Clear surface
    control
     SDM               Presence of mucilaginous sheath         Presence of hyphae and
                       while in culture, mycelia adhered    mycelial mats. Colonization
                       on the surface, elasticity loss on             took place.
                      areas where mycelia has embedded
    PNL 114            Presence of mucilaginous sheath     Presence of hyphae, mycelial
                          while in culture, no distinct      mats and spore formation.
                       mycelia adherence on the rubber comparable colonization when
                                     surface                    compared to the SDM
    PNL 116            Presence of mucilaginous sheath     Presence of hyphae, mycelial
                          while in culture, no distinct      mats and spores. Minimal
                       mycelia adherence on the rubber colonization when compared to
                                     surface                           the SDM.
    PNL 118            Presence of mucilaginous sheath        Presence of mycelial mat
                          while in culture, no distinct          formation. Minimal
                       mycelia adherence on the rubber colonization when compared to
                                     surface                           the SDM.
       E26             Presence of mucilaginous sheath      Profuse growth of mycelia.
                       while in culture, mycelia adhered   Maximum colonization when
                        on the surface, elasticity loss on     compared to the SDM.
                      areas where mycelia has embedded
       E35             Presence of mucilaginous sheath       Presence of mycelia, spore
                       while in culture, mycelia adhered    formation and mycelia mat.
                        on the surface, elasticity loss on Colonization is comparable to
                      areas where mycelia has embedded             that of the SDM.
                                                   A Preliminary Study on the Potential……..33
                                                                       Dayao and Egloso, 2009

Chicken Feathers




         a                                         b




         c                                         d




         e                                         f
Figure 13. Growth of Xylaria sp. with presence of fungal colonies at the tip of the white
arrows. a. SDM/wild type strain, b. E26 strain, c. E35 strain, d. PNL 114 strain, e. PNL
116 strain, and f. PNL 118 strain) in mineral medium with 0.5% glucose and chicken
feather; pH of medium - 5.0 and incubation temperature of ~250C, as observed for 20
days of incubation.
                                                  A Preliminary Study on the Potential……..34
                                                                      Dayao and Egloso, 2009




Figure 14. SEM of non-inoculated chicken feather control without the fungus.




                                    my                                        br
                                                            br



                        bl                my


                                                               bl




Figure 15. SEM of chicken feather colonized by the SDM wild type strain after 50 days
of incubation. Profuse growth of Xylaria wild type as shown by mycelial mats (my)
closely adhering to the barbs (br) and barbules (bl).
                                                    A Preliminary Study on the Potential……..35
                                                                        Dayao and Egloso, 2009


                                                                           br




                                                                      bl
                                                    my

                                   br

                                                c

                                                                                my
                                                              bl


Figure 16. SEM of chicken feather colonized by the PNL 114 mutant strain after 50 days
of incubation. Flakes of mycelial mats (my) colonize barbs (br) and barbules (bl) of the
chicken feather. A deep crack (c) can be observed at the middle of the lower barb.



                                                     bl
                             br




                                        my

                              bl



                                                                      br
                                   my


Figure 17. SEM of chicken feather colonized by the PNL 116 mutant strain after 50 days
of incubation. Colonization demonstrated by the close adherence of mycelial mats (my)
to the barbules (bl). Parts of barbs (br) shown seem to be physically intact. No crack or
hole can observed in the barbs.
                                                    A Preliminary Study on the Potential……..36
                                                                        Dayao and Egloso, 2009




                                   br          bl

                                                     r
                                                                my




                       my




Figure 18. SEM of chicken feather colonized by the PNL 118 mutant strain after 50 days
of incubation. Smaller mycelial mats are adhering to several areas of the barbules (bl).
The rachis (r) at the middle of the SEM seems to be physically intact. No crack or hole
can be observed also.

                  my
                                                                           bl

                                                           br

              r




                                    my

                                                                                  bl

                       my
                                                                br
 Figure 19. SEM of chicken feather colonized by the E26 mutant strain after 50 days of
incubation. Presence of mycelial mats (my) adhere to the barbules (bl) while fewer
mycelia filaments or threads adhere to the barbs (br). No discernible crack or hole can be
observed.
                                                      A Preliminary Study on the Potential……..37
                                                                          Dayao and Egloso, 2009



                                                                            sp



                                                                        my

                                                 bl
                                                                                          bk
                          bk

                                            br
                  bl

                                                                 my




Figure 20. SEM of chicken feather colonized by the E35 mutant strain after 50 days of
incubation. Dense mycelial mats (my) with spores (sp) attached are shown covering
several areas of the feather sample. Weakened barbs (br) resulted to breakage (bk).
Several barbules (bl) detached from their barbs. Presence of spores (sp in white circles)
are also observed.


Microscopic SEM Results

       Micrographs of the SDM wild type, and the black and white mutant strains were

compared to the non-inoculated control (figure 14) to observe changes occurred. The

wild type strain micrograph (Fig. 15) shows colonized barbs and barbules through

prevalent mycelial attachments. No cracks or holes can be observed in the barbs. SEM of

PNL 114 mutant strain (Fig. 16) illustrates the presence of mycelial mats closely adhering

to the barbs and barbules of the chicken feather. A deep crack can be observed at the

middle of the lower barb. PNL 116 mutant strain (Fig. 17) shows adherence and

establishment of the mycelia on the barbules of the chicken feather. Any crack or hole

cannot be observed on the barbs as well. Moreover, mycelial mats are seen (Fig. 18)
                                                     A Preliminary Study on the Potential……..38
                                                                         Dayao and Egloso, 2009

attached in various areas on the feather of the SEM of PNL 118 mutant strain. Some

portions of the feathers seem to be disheveled. The barb at the middle seems to be

physically intact. The micrograph (Fig. 19) of the E26 strain demonstrates the adherence

of the mycelia on the barbs and barbules. The barbules contain more mycelia than the

barbs. No discernible crack or hole can be observed. On the other hand, dense mycelial

mats with spores attached are shown in the SEM of E35 mutant strain (Fig. 20), covering

several areas of the feather sample. At some point, the barbules are disheveled. Breakage

is also seen at some barbs.



Macroscopic Results

       Macroscopic investigation results show that in most cases, the chicken feathers

have retained their original physical appearance except for two strains namely, SDM or

the wild type and E35 black mutant strain. The two showed little signs of brittleness in

their barbules. This brittleness refers to their ability to be detached easily from the barb.

No other changes had been observed.
                                                   A Preliminary Study on the Potential……..39
                                                                       Dayao and Egloso, 2009

     Table 5. Table of the Macroscopic/Physical and Microscopic Observations of the Xylaria
        sp. Mutant Strains and Wild Type on Colonized Chicken Feather after 50 days of
                                          Incubation.
Strain        Macroscopic/Physical                      Microscopic Observations
                  Observations
Contro No physical change occurred       No observed mycelia attached and physical damage
   l
SDM Presence of mucilaginous Profuse growth as shown by mycelial mats closely
         sheath while in culture. adhering to the barbs and barbules. No discernible tearing,
         Barbules are slightly brittle cracks or holes can be observed on the barbs.
         (i.e. ability to be detached
         easily from the barb).
 PNL Presence of mucilaginous Flakes of mycelial mats colonize barbs and barbules of
 114     sheath while in culture. the chicken feather. A deep crack can be observed at the
         Retained original physical middle of the lower barb.
         characteristics
 PNL Presence of mucilaginous Colonization demonstrated by the close adherence of
 116     sheath while in culture. mycelial mats to the barbules. Parts of barbs shown seem
         Retained original physical to be physically intact. No tearing, cracks or holes can
         characteristics                 observed in the barbs.
 PNL Presence of mucilaginous Smaller mycelial mats are adhering to several areas of the
 118     sheath while in culture. barbules. The rachis at the middle of the SEM seems to be
         Retained original physical physically intact. No tearing, cracks or holes can be
         characteristics                 observed also.
 E26     Presence of mucilaginous Presence of mycelial mats adhering to the barbules while
         sheath while in culture. fewer mycelia filaments or threads adhere to the barbs. No
         Retained original physical discernible tearing, cracks or holes can be observed.
         characteristics
 E35     Presence of mucilaginous Dense mycelial mats with spores attached are shown
         sheath while in culture. covering several areas of the feather sample. Weakened
         Barbules are slightly brittle barbs resulted to breakage. Several barbules detached
         (i.e. ability to be detached from their barbs. Presence of spores are also observed.
         easily from the rachis).
                                                   A Preliminary Study on the Potential……..40
                                                                       Dayao and Egloso, 2009




Polystyrene




           a                                       b




           c                                       d




           e                                       f
Figure 21. Growth of Xylaria sp.with the presence of fungal colonies as indicated by the
tip of the white arrows (a. SDM/wild type strain, b. E26 strain, c. E35 strain, d. PNL 114
strain, e. PNL 116 strain, and f. PNL 118 strain) in mineral medium with 0.5% glucose
and polystyrene; pH of medium - 5.0 and incubation temperature of ~250C, as observed
for 20 days of incubation.
                                                   A Preliminary Study on the Potential……..41
                                                                       Dayao and Egloso, 2009




Figure 22. SEM of non-inoculated control sample.



                        cr




                                            my

                         cr                              hy
                                sp




Figure 23. SEM of polystyrene surface colonized by the SDM wild type strain after 50
days of incubation. Profuse growth resulted to dense mycelial mats (my), made of thick
hyphae (hy), which closely adhered to the surface. Residual polystyrene surfaces are
unapparent, but cracks (cr), shearing (sh) and striations can be observed. Several spores
(sp) are also shown.
                                                    A Preliminary Study on the Potential……..42
                                                                        Dayao and Egloso, 2009




                                          tr



                   my




                                                                     st




Figure 24. SEM of polystyrene surface colonized by the PNL 114 mutant strain after 50
days of incubation. Thin mycelial mats (my), which are composed of thin hyphae, closely
adhered to the surface. There is apparent tearing (tr), and striations (st) on the surface.
Several small holes or pits through the surface are also observable.




                      tr                            sp
                                                                                 st




                           my




Figure 25. SEM of polystyrene surface colonized by the PNL 116 mutant strain after 50
days of incubation. Mycelial mats (my) made of thin hyphae more closely adhered to the
surface which has completely disintegrated due to extensive tearing (tr) as shown by the
serrated edges of residual surface. Striations (st) and spores (sp) are also apparent.
                                                        A Preliminary Study on the Potential……..43
                                                                            Dayao and Egloso, 2009




                                               hy


                                       p
                                                            my

                                                                             st
                                                           tr
Figure 26. SEM of polystyrene surface colonized by the PNL 118 mutant strain after 50
days of incubation. Profuse growth closely adhered to the surface and resulted to dense
mycelial mats (my) made of thick hyphae (hy). Residual polystyrene (p) surfaces are still
apparent but are very thin. Tearing (tr) and striations (st) can be observed.




                                                   hy




                                                                my
                                              sp




Figure 27. SEM of polystyrene surface colonized by the E26 mutant strain after 50 days
of incubation. Active hyphal burrowing resulted to a deep depression (encircled) at the
middle, in which mycelial growth and spores have concentrated. Dense mycelia (my)
consisting of thick hyphae (hy) closely adhered to the surface. There is apparent tearing
(tr) on the surface, but striations are not. Several spores and holes through the surface are
also observable.
                                                      A Preliminary Study on the Potential……..44
                                                                          Dayao and Egloso, 2009




                                  hy




                                               my
                     my



                                                                                 st


Figure 28. SEM of polystyrene surface colonized by the E35 mutant strain after 50 days
of incubation. Active hyphal burrowing resulted to a deep depression (de) at the middle,
in which mycelial mat (my) has concentrated. Dense mycelia (my) made of thick hyphae
(hy) closely adhered to the surface. There is apparent tearing (tr), and striations (st) on the
surface. Several holes through the surface are also observable.



Microscopic SEM Results

       The images clearly show extensive colonization and degradation of polystyrene.

Hyphae have apparently penetrated the polymer, forming a well-defined network on its

surface. Polystyrene samples inoculated with their respective Xylaria fungal strains

illustrated remarkable difference from the non-inoculated control after incubation. The

control (fig. 22) shows a relatively smooth surface with several minor dents and shallow

depressions which may be due to handling during sample preparation, or were already

present prior to this experiment. Comparing the SDM wild type (fig. 23) to the control

revealed extensive colonization as shown by mycelial mats, or aggregations of fungal

hypha, and numerous spores found throughout the entire micrograph, including holes,

cracks and crevices due to mycelial penetration. Mycelial mats proliferated throughout
                                                    A Preliminary Study on the Potential……..45
                                                                        Dayao and Egloso, 2009

the area of interest, that traces of raw polystyrene surface is barely visible; they almost

completely covered the residual surface fragments. Mutant strains E26 (fig. 27) and E35

(fig. 28) showed equally extensive colonization as that of the wild type, and showed thick

mycelial mats, especially the E26 strain. In addition to numerous spores, both black

mutants also revealed holes or deep depressions wherein mycelial mats concentrated their

growth. Strains PNL 118 (fig. 26), 116 (fig. 25), and 114 (fig. 24), in decreasing order of

colonization extent, showed comparable results as that of the wild type. Mutant strains

PNL 118, PNL 114, E26, E35 and the wild type SDM exhibited close packing of

mycelial growth in most of the samples, whereas PNL 116 showed loosely packed

growth of its thin mycelia. PNL 114 also demonstrated thin mycelial growth, but mycelia

are closely packed. It can further be concluded from the SEM micrographs that the SDM

wild type exhibited the most extensive degradation as compared to the mutant strains, but

the degree of difference is not wide.


Macroscopic Results

       Visually observing the polystyrene strips inoculated with mutants 114, 118, E26

and E35 revealed that the samples have small dents on the surface, and brown to black

mycelia attached to the edges and surface. There were no obvious physical damage

observed in the samples of mutant 116 but there were some reddish mycelia attached on

one of the samples. Black mycelia closely adhered to the edges and surface of SDM and

mutant E26, just as in the other mutants as well, thereby rendering their removal very

difficult by mere physical means. There were no obvious physical damage, if ever, to the

control sample.
                                                                    A Preliminary Study on the Potential……..46
                                                                                        Dayao and Egloso, 2009

     Table 6. Table of the Macroscopic/Physical and Microscopic Observations of the Xylaria
     sp. Mutant Strains and Wild Type on Colonized Polystyrene after 50 days of Incubation.
Strain                   Macroscopic/Physical Observations                           Microscopic Observations
Control         Smooth, no observed mycelia attached to the surface                    Smooth, few minor dents
 SDM        Presence of mucilaginous sheath while in culture. Slightly      Profuse growth of closely packed and dense
               black mycelia attached to the edges and surface; some        mycelial mats of thick hyphae closely
              samples had no obvious physical damage; hard and not          adhered to the surface. Residual polystyrene
                brittle as the control except for colonized areas which     surfaces are unapparent, but cracks, tearing
            became softer. Presence of striations at the surface can be     and striations can be observed. Several
                                           seen.                            spores are also present.
 PNL      Presence of mucilaginous sheath while in culture. Generally,      Closely packed, thin mycelial mats
 114          samples have small dents on the surface, dark brown to        composed of thin hyphae, closely adhered
            black mycelia attached to the edges and surface; hard and       to the surface. There are noticeable, holes,
             not brittle as the control except for colonized areas which    tearing and striations.
            became softer. Presence of black strains and discoloration.
                 One replicate has a notably huge amount of mycelia
             embedded on its surface. Presence of striations and dents
                                  comparable to SDM.
 PNL      Presence of mucilaginous sheath while in culture. Generally       Mycelial mats of thin hyphae closely
 116          no obvious physical damage, hard and not brittle as the       adhered to the surface, which has
              control except for colonized areas which became softer,       completely disintegrated due to extensive
          some reddish mycelia attachments on one of the samples. No        tearing as shown by serrated edges of
               trace of strain due to its albino property although some     residual surface. Striations, holes and spores
           replicates have traces of dark/brown discolorations. Surface     are also apparent.
                 striations are minimal when compared to wild type.
 PNL          Presence of mucilaginous sheath while in culture. Small       Common surface has been almost
 118       dents on the surface (2samples), brown mycelia attached to       completely degraded. Profuse growth of
            the edges (1sample) and surface; 1 sample had no obvious        closely packed and dense mycelial mats of
            physical damage; hard and not brittle as the control except     thick hyphae closely adhered to the surface.
             for colonized areas which became softer. Others have no        Residual polystyrene surfaces are still
                visible mycelia due to the albino color of the mutant.      apparent but are very thin. Numerous holes,
                         Presence of striation and surface holes.           tearing and striations can be observed.
 E26          Presence of mucilaginous sheath while in culture. Black       Active hyphal burrowing resulted to a deep
               mycelia attached to the edges (1sample) and surface; 1       depression at the middle, in which mycelial
          sample had no obvious physical damage; hard and not brittle       growth and spores have concentrated.
              as the control except for colonized areas which became        Dense mycelia of thick hyphae closely
           softer especially the samples which were almost completely       adhered to the surface. Tearing, several
             covered with black mycelia. Minimum surface striations         spores and holes are also observable.
              compared to SDM. Minimum mycelia embedded on the
                             surface except for two samples.
 E35      Presence of mucilaginous sheath while in culture. Generally,      Active hyphal burrowing resulted to a deep
              samples have small dents on the surface, black mycelia        depressionat the middle, in which mycelial
           attached to the edges and surface; hard and not brittle as the   mat has concentrated. Dense mycelia of
              control except for colonized areas which became softer.       thick hyphae closely adhered to the surface.
            Presence of more mycelia embedded on the surface of the         There is apparent tearing, striations and
             samples compared to the wild type. There are more dents        several holes on the surface.
                   and striations on the surface compared to SDM.
                                                    A Preliminary Study on the Potential……..47
                                                                        Dayao and Egloso, 2009

                                     DISCUSSION



       Natural rubber, polystyrene and chicken feather were utilized as co-substrates of

glucose for the growth of a filamentous fungus, Xylaria sp. The fungus was grown at

25oC and at pH 5.0 in all the three runs and set-ups; the optimum conditions concluded

by the study of Clutario and Cuevas (2001). At first, the initial and final weights were

measured to detect weight change of the pollutants, but however, mycelia closely adhered

to the three substrates’ surface and grew into it, thereby rendering their removal very

difficult. Due to this, the change in weight was not used as a method. In study, the weight

gain was proven to be indicative of colonization, whereas weight loss is indicative of

biodegradation.

        The 0.5% glucose provided in the medium initiated (Cuevas & Manaligod, 1997;

Clutario & Cuevas, 2001) and sustained growth for up to 3 weeks of incubation (M. A.

Tavanlar, personal communication, March 4, 2009). Beyond this period when glucose

has been depleted, the given Xylaria sp. variant in a set-up had to utilize the provided

substrate as an alternative carbon and energy source. Moreover, it has been proven that

the mutants and SDM wild type can utilize other carbon sources aside from glucose, such

as PEG and mineral oil (M. A. Tavanlar, personal communication, March 4, 2009). The

generally good growth could only be credited to this capacity of the given fungus,

supported by the favorable environmental conditions provided, such as optimum pH and

temperature.

       At the end of the 50-day incubation period, many of the set-ups were covered by

mycelial growth. Such colonization and surface adhesion is a fundamental prerequisite to
                                                    A Preliminary Study on the Potential……..48
                                                                        Dayao and Egloso, 2009

biodegradation, although growth on a polymer surface is not adequate to deduce that

carbon from this polymer has been assimilated. Nonetheless, this colonization provides a

simple, fast and clear test to evaluate the response of a macromolecular material to

biodegradation (Clutario & Cuevas, 2001; Motta,et al., 2007).

       The samples from the three pollutants had been observed to be covered by a slimy

mucilaginous sheath or biofilm which can be easily removed (see Tables 4, 5 and 6). All

Xylaria sp. variants utilized in this study are able to form biofilm (M. A. Tavanlar,

personal communication, March 4, 2009), which is a crucial step to microbially induced

biodegradation.

       Furthermore, it should be noted that scraping had been performed gently to the

three substrate pollutants after removal of fungi from the samples so as to avoid

destruction of the surface and to further remove mycelia. Hence, the colonization

revealed in the SEM micrographs cannot be attributed to mere mycelial aggregation on

the surface since physical manipulation and removal through gentle scraping have been

performed.

       As indicated in studies, biofilm formation observed in the surface of polymers is a

mechanism by which microorganisms secrete proteins and carbohydrates for their

survival in environments which are low in nutrients. Its formation could also indicate that

there is solid substrate utilization by the microorganisms. Biofilm formation is regarded

as an initial step in biodegradation. The first step by which biofilms are formed in a

substrate is through colonization. Colonization happens when the cells of the organism

secretes extracellular polysaccharide substances (EPS). Colonization of the organism and

the eventual formation of biofilm results in the destruction of quality or performance of
                                                     A Preliminary Study on the Potential……..49
                                                                         Dayao and Egloso, 2009

the material. Active colonizers of polymer can adhere to material surfaces because they

secrete a type of glue (Capitelli et al., 2006; Lucas, et al., 2008). This substance is a

complex matrix made of polymers (e.g. nonionic and anionic polysaccharides and

proteins), which penetrate into porous structures and alters the size and the distribution of

pores and changes moisture degrees and thermal transfers. The slime matter functions to

protect microorganisms against adverse conditions such as desiccation and UV

radiations. Kaeppeli and Fiechter (1976) observed that the first step in the utilization of

hydrocarbon by microbial cells, involves a passive adsorption to lipophilic

lipopolysaccharide found in the surface of the cell to the alkane group of the polymer

(Clutario & Cuevas, 2001). Another study (Reddy, et al., 1982; Gutnick & Minas, 1987)

hypothesized that after adhesion, solubilizing agents are secreted and produced by many

microorganisms which can make use of water-immiscible compounds (Clutario &

Cuevas, 2001).

       Filamentous microorganisms develop their mycelial mats or plectenchyma, inside

the pollutant substrates. The generally more efficient performance of the black mutants,

E26 and E35, is important to note, which is due to the melanin which cover their hyphal

surfaces. The melanin protects the hyphae of the black mutants thereby making them

resistant to frictional damage during penetration into the substrate. Thus, they can more

efficiently degrade the substrate as compared to the albino mutants PNL 114, 116 and

118 (M. A. Tavanlar, personal communication, March 4, 2009). Therefore, the black

mutants also produced denser and thicker mycelial growth and more efficient degradation

of substrate. The mechanical action of hyphal apices infiltrating the substrates increases

the size of pores and provokes cracks. Hence, the substrates are weakened in resistance
                                                     A Preliminary Study on the Potential……..50
                                                                         Dayao and Egloso, 2009

and durability (Bonhomme et al., 2003; Lucas, et al., 2008). The thicker hyphae of the

wild type and black mutants (figures 22, 26 and 27) are also due to the presence of

melanin around their hypha. However, the black mutants are still undergoing tests and are

not characterized yet as of the moment (M. A. Tavanlar, personal communication, March

4, 2009).

       Prior to experimentation, the chicken feathers were autoclaved, whereas natural

rubber and polystyrene were only surface sterilized through agitation in 70% ethanol

once and in sterile distilled water twice. Polystyrene and natural rubber were initially

autoclaved, but were discarded because the samples have been greatly degraded, unlike

the chicken feather which remained intact. On the other hand, surface sterilization would

maintain the structural integrity of polystyrene and natural rubber, thus, this was the

method used. However, the chicken feathers were still autoclave to assure the elimination

of already present microbes. Scientists have known for so long that the plumage of birds

is a sanctuary for diverse populations of bacteria and fungi (Hubilek, 1994). Furthermore,

it is important to note that the samples were not subjected to weakening processes, such

as chemical oxidation of polystyrene (Motta et al.,2008) so as to simulate the natural

condition of the pollutants as it is discarded in the environment.

       Moreover, the enhanced rate of degradation in the mineral medium solution, as

compared to in situ degradation, may be due to the greater contact between the polymer

and the microorganism, and the liquid has a better buffering effect than the solid substrate

solely. Produced degradation products may also remain in the system and some may

provide nutrients for growth (Albertsson & Karlsson, 1993).
                                                      A Preliminary Study on the Potential……..51
                                                                          Dayao and Egloso, 2009

Natural Rubber

       The commercial usage of latex gloves, condoms, tires and the like has lead to the

burgeoning problem of rubber waste disposal. Several tons of rubber per year are

produced and exported by countries such as Indonesia. According to the rubber review of

biodegradation, natural rubber degradation happens slowly (Rose & Steinbuchel, 2005;

Bereeka, 2006). In response to this need, this study included the natural rubber pollutants

in the form of latex gloves, which has undergone irradiation and peroxidation processes

in its production (Linos et al., 2000). Old latex gloves were used and subjected to

biodegradation. The old latex glove is preferred to simulate the conditions of the gloves

when disposed in the environment.

       The scanning electron microscope (SEM) revealed that the natural rubber samples

were now a combination of Xylaria sp. strains and natural rubber. By observing the

micrographs (fig.7-12) and comparing it to the control (fig.6), all the strains demonstrated

colonization on the rubber surface. Growth, mycelial mat formation and presence of

spores had been observed. More so, macroscopic examination (table 4) revealed that

some of the mycelia of the Xylaria sp. remained embedded and adhered on the surface

even after gentle scraping and washing of the samples with 70% ethanol and water to

remove and rinse the mycelia. It should be noted that the scraping had been performed

gently so as to avoid destruction of the natural rubber surface. Hence, the colonization

revealed in the micrographs of the natural rubber samples cannot be attributed to mycelial

aggregation on the surface since physical manipulation and removal through gentle

scraping have been performed. Through this method, it could be said that mycelia have

adhered on the surface and colonization took place.
                                                     A Preliminary Study on the Potential……..52
                                                                         Dayao and Egloso, 2009

       Colonization of rubber, based on the study of Linos et al. (2000) on rubber

degradation, is the first mechanism by which rubber-degrading organisms degrade natural

rubber. In the study, scanning electron microscopy revealed that the Xylaria sp. strains

grew adhesively on the natural rubber by demonstrating contact through the formation of

mycelial mats, hyphae and spore formation on the surface of the pollutant. In the study of

rubber degradation by Linos et al. (2000), the mechanism of colonization was revealed. It

began with the organism directly merging into substrate. The organism demonstrated a

hydrophobic nature on the substrate upon merging.

       There was also an observed biofilm formation in the form of mucilaginous

sheaths on the surface (M. A. Tavanlar, personal communication, March 4, 2009). As

indicated in studies, biofilm formation observed in the surface of polymers is a

mechanism by which microorganisms secrete proteins and carbohydrates for their

survival in environments which are low in nutrients. Its formation could also indicate

solid substrate utilization by the microorganisms. Biofilm formation is regarded as an

initial step in biodegradation. Colonization of the organism and the eventual formation of

biofilm results in the destruction of quality or performance of the material. Moreover,

fungi and bacteria have been shown to attack and form biofilms on natural rubber

substrates (Morton & Surman, 1994; Linos et al., 2000). Hence in the study, it could be

suggested that the adherence, colonization and biofilm formation is an indication that the

Xylaria sp. strains assimilated natural rubber as a co-carbon source and it did not solely

relied on the 0.5 % glucose. Moreover, it could also be extrapolated that the initial step in

biodegradation has taken place.
                                                         A Preliminary Study on the Potential……..53
                                                                             Dayao and Egloso, 2009

       By comparing the natural rubber micrographs of the mutant strain samples (fig. 8-

12) to the sample incubated using the SDM strain (fig.7), it could be observed that E26

(fig. 12) strain demonstrated maximum colonization. The SEM revealed that the E26 (fig.

12) profusely colonized the surface of the natural rubber sample; thereby suggesting that

this strain demonstrated the greatest biodegrading potential compared to other strains.

This greatest potential is attributed to the maximum capacity of the strain, when

compared to other strains, to grow and colonize the rubber pollutant.

       On the other hand, the least strain that demonstrated a potential biodegrading

capacity is the PNL 118 (fig. 10) albino mutant strain. Minimal growth is observed in the

micrograph (see fig. 10) when compared to the other mutant strain micrographs. In PNL

118, the only visible observation at 500x (shown in the appendix C) is mycelial mat

formation. But in figure 10 at 2000x, the SEM revealed the presence of hyphae and

mycelial mats. Hence, there is still growth and colonization which could indicate that the

strain still possesses potential biodegrading ability.

       In comparing all the micrographs, SEM of all the other strains not mentioned have

revealed comparable results. All have demonstrated growth and have shown to colonize

the rubber substrates. But in general, the black Xylaria sp. mutant strains, especially the

E26 (fig. 12) strain, showed maximum growth and colonization when compared to the

albino strains namely PNL 114, 116 and 118 (fig. 8, 9 and 10). This suggests that the

melanin in the structure of the black strains could be playing a role in its biodegradation.

The melanin surrounding the hypha, thereby making it thicker than the albino strains,

could be offering resistance and protection upon hyphal tip penetration inside the natural

rubber pollutant. (M.A. Tavanlar, personal communication, March 9, 2009)
                                                     A Preliminary Study on the Potential……..54
                                                                         Dayao and Egloso, 2009

       Furthermore, the slow colonization of Xylaria sp. strains on natural rubber could

suggest that colonization impedance due to chemicals in the old latex gloves used might

be taking place. In the investigation of Bereeka et al. (2000) and Rose and Steinbuchel

(2005), the latex gloves were extracted using organic solvents to remove the anti-oxidants

before inoculation. And it has shown that the colonization efficiency on the rubber latex

gloves of the known biodegrading organism such as Gordonia (strains Kb2, Kd2 and

VH2), Mycobacterium, Micromonospora and Pseudomonas were enhanced in the treated

latex gloves as compared to the non-treated latex gloves.



Chicken Feather

       In general, all the Xylaria sp. variants efficiently colonized the feather samples

(figure 13, 14-19), although the samples did not show apparent physical damage as

compared to the undamaged control sample, except for the SDM wild type (figure 15)

and E35 (figure 20) strain which manifested slight brittleness. Efficient colonization in all

samples was revealed by the close adherence of mycelial mats to the rachis, barbs, and

barbules of the feathers. The results for SDM and E35 strains should be further noted

because despite the fact that the incubation period of 50 days is relatively short, both of

these strains already manifested signs of degradation. The SEM (figure 20) for the E35

strain even illustrated weakened barbs which resulted to breakage at some points. The

barbules of the feather samples of SDM and E35 mutant became slightly brittle, thus they

were easily detached from the barbs. Nonetheless, if given a longer incubation time, the

other strains may have revealed indications of biodegradation also, as all of them have
                                                    A Preliminary Study on the Potential……..55
                                                                        Dayao and Egloso, 2009

shown to efficiently colonize this substrate. And this is the fundamental prerequisite for

biodegradation to occur.

       In the study the chicken feathers were sterilized using an autoclave to degrade the

preen oil coating of the feathers and to destroy any microorganism present that could

compete and interfere with the determination of the potential degrading ability of the

Xylaria sp. strains. Preen oil can hamper the growth of normally occurring microbes in

birds’ plumage because it limits the presence of moisture (Shawkey, et al., 2005), which

is needed in microbial metabolic activities.

       The mycelia, in general, were difficult to remove from the chicken feather

samples. Most of them were now a combination of Xylaria sp. and chicken feathers, as

demonstrated under a scanning electron microscope.          The samples were shaken

with 70% ethanol once and in distilled water twice to remove mycelia. Gentle scraping

was also performed afterwards. However, there were few mycelia still remained closely

adhered especially in areas near the points of attachment of the rachis and barbs. In

addition during the removal of mycelia from the samples, slimy biofilm was also

observed to have surrounded all the samples. The formation of biofilm, in the form of

mucilaginous sheath (see table 5) observed, is a means by which microorganisms

establish themselves on a surface. They secrete carbohydrates and proteins to survive in

this low nutrient environment, thereby facilitating microbial activity and degradation of

polymer surface. Establishment is exemplified by the profuse growth of mycelial mats

(figure 15 and 20) in most of the samples and the presence of spores (figure 20).

       Difficulty in the degradation of chicken feather could only be attributed to their

very hard β-keratin composition. Thus, chicken feathers are highly troublesome waste
                                                   A Preliminary Study on the Potential……..56
                                                                       Dayao and Egloso, 2009

products. Therefore it is highly recommended that the incubation period be increased, an

enzyme catalyst be added, and/or the feathers be subjected to “weakening” mechanisms

prior to the addition of Xylaria sp.

       There should be a distinction made between the preliminary disintegration of

complex keratinous structures into smaller substructures, such as feathers and hairs, and

the molecular breakdown of keratin into smaller peptides. The former may be the result

of protease activities on interkeratin matrix, while the breakdown of the almost

crystalline keratin would require further degradative means. The cleavage of the cystine,

disulfide bonds may have a significant influence on keratin degradation, and this has been

described for a few microorganisms. Several keratinolytic fungi cause sulfitolysis by

excreting sulfite and by producing an acid pH at the mycelial surface (Bockle & Muller,

1997). Furthermore, keratin degradation must occur outside of the cell at the keratin

molecules through the release of a soluble reducing component into the medium, or

through a cell-bound redox system at the surface of the cells (Bockle & Muller, 1997).

       Also it might be presumed from microscopic examinations that there was no

permanent contact between mycelium and keratin particles observed, making direct

degradation of the substrate surface unlikely. However, it cannot be excluded that

degradation can occur by short contacts between mycelium and substrate (Bockle &

Muller, 1997). A high percentage of keratin represents hydrophobic and aromatic amino

acids (approximately 50%) (Gregg, et al., 1984; Gradisar, et al., 2005), it has thus been

concluded (Gradisar, et al., 2005) that keratinases are successful in hydrolysis of

keratinous materials due to the specific amino acid composition of keratins as well as to

their broad specificity.
                                                     A Preliminary Study on the Potential……..57
                                                                         Dayao and Egloso, 2009

       SEM (figure 15) of the SDM wild type sample revealed that the mycelia had

adhered to the barbules and the barbs of the pollutant. Colonization and growth is rapid in

terms of extent of coverage and adherence of mycelia. It is also observed that the

barbules contain more mycelia than the barbs. The PNL 114 mutant strain (figure 16)

showed minimal colonization when compared with the SDM or the wild type. A wider

magnification is chosen because at low magnification, mycelia adherence is not quite

obvious. The deep crack at the middle of the micrograph may have already been present

prior to this experiment. The PNL 116 and 118 albino mutants together with the E26

black mutant strains (figures 17, 18 and 19, respectively) showed almost the same and

comparable results with the PNL 114 albino mutant strain. The adherence of the strains is

minimal when compared to the wild type or the SDM. The micrographs revealed that at

various portions of the chicken feather, mycelia in minimal amount could be seen

attached on the barbules and barbs as well. Hence, it could be stated that when compared

to the wild type, they colonized poorly. On the other hand, the E35 black mutant strain

(in figure 20) showed colonization quite comparable to that of the SDM or wild type. The

micrographs justified the macroscopic physical appearances. For both the SDM and the

E35, which showed more efficient colonization as compared to the other the four strains,

they demonstrated feather samples that were quite brittle. The brittleness refers to the

ability of the barbs and barbules to be easily detached from the rachis. The results suggest

that the wild type strain and the E35 black strain are the most probable strains to

demonstrate potential biodegrading ability.
                                                     A Preliminary Study on the Potential……..58
                                                                         Dayao and Egloso, 2009

Polystyrene

       Among the three pollutants, polystyrene revealed the most extensive colonization

and degradation in the current study. Results of the albino mutants, PNL 114, 116, and

118 (fig. 24, 25 and 26) are not widely different from those of the wild type SDM (fig.

23) and black mutants, E26 and E35 (fig. 27 and 28) which showed better results in

natural rubber and chicken feather. Moreover, following incubation, it has been observed

that mucilaginous sheaths surrounded the samples, as mycelia were physically removed

through gentle scraping. Small dents have been generally found on the surface of all

samples. Brown to black mycelia were seen attached to the edges and surface, even on

some of the samples of the albino mutants PNL 114, 116 and 118 (fig. 24, 25 and 26).

According to Tavanlar (March 4, 2009), these brown or black discoloration in the albino

strains (see fig. 21) could be staling products produced by the fungus, and not melanin in

their hyphal surface. Nevertheless, the other samples of the albino mutants have no

visible mycelia due to its white color. Most samples with areas where there are noticeable

mycelial attachments were also observed to be softer or less rigid than the non-inoculated

control.    Presence of striations, dents and holes are also highly apparent on the sample

surfaces.

       In addition, the thicker hyphae of the wild type SDM (fig. 23) and black mutants

E26 and E35 (fig. 27 and 28) are due to the presence of melanin in their surface, which

give them protection and resistance in penetration during substrate colonization.

       Polystyrene, one of the most widely used synthetic polymers, has been chosen for

this study because its high recalcitrance to biodegradation is very well known. Although

considered as a recalcitrant polymer, it is nonetheless subject to biodegradation, just like
                                                     A Preliminary Study on the Potential……..59
                                                                         Dayao and Egloso, 2009

polyurethane and polyethylene (Lucas, et al., 2008). Polystyrene is also commonly

considered a plastic, as well as natural and synthetic rubber, and polyethylene. The

plasticisers and fillers used in the formulation of these plastics leave them vulnerable to

attack which usually materializes as a surface biofilm, which causes slight adverse effects

to the physical or chemical integrity of the material (Morton & Surman, 1994). Like

natural rubber and chicken feather, mucilaginous sheaths were also observed to wrap the

polystyrene samples while in culture. The microbial susceptibility of these recalcitrant

polymers is credited to the biosynthesis of lipases, esterases, ureases and proteases

(Flemming, 1998; Lugauskas et al., 2003; Lucas, et al., 2008). These enzymes require the

presence of cofactors (i.e. cations present into the material matrix and coenzymes

synthesized by microorganisms) for the breakdown of specific bonds (Pelmont, 2005;

Lucas, et al., 2008).

       The biodegradation of thermoplastic polymers such as polystyrene could proceed

via bulk and/or surface erosion (von Burkersroda et al., 2002; Pepic et al., 2008; Lucas,

et al., 2008). Bulk erosion results to fragments lost from the entire polymer mass and

changes in molecular weight due to bond cleavage. This lysis is provoked by chemicals

such as H2O, acids, bases, transition metals and radicals, or by radiations, but not by

enzymes. They are too large to penetrate throughout the matrix framework. Surface

erosion, on the other hand, result to matter being lost but no change in molecular weight

of polymers of the matrix. If the diffusion of chemicals throughout the material is faster

than the cleavage of polymer bonds, the polymer undergoes bulk erosion. If the cleavage

of bonds is faster than the diffusion of chemicals, the process occurs mainly at the surface

of the matrix (von Burkersroda et al., 2002; Pepic et al., 2008; Lucas, et al., 2008).
                                                     A Preliminary Study on the Potential……..60
                                                                         Dayao and Egloso, 2009

        It is proven from the results of this study that polystyrene does not need to be

copolymerized with other substances like lignin and sugars (i.e. glucose and sucrose) to

make it more degradable and susceptible to microbial attack, as mentioned in the

previous studies. Also, it is clearly shown in the micrograph results that not only did the

Xylaria mutant strains and wild type showed high affinity, but they actually degraded and

utilized polystyrene or EPS strips as an alternative carbon and energy source. EPS is a

closed cell, lightweight and resilient, foamed plastic composed of hydrogen and carbon

atoms. It is non-hygroscopic and does not readily absorb water vapor. Its closed-cell

structure reduces the absorption and/or migration of moisture into the insulation material

(EPS Molders Association, 2009). It is said that raw polystyrene foam will not rot or

attract fungi or mildew and has a superior R-value, thus polystyrene will insulate and

keep heating or cooling inside of any particular room or commercial space (The Foam

Factory, 2009). Because of the high level of moisture resistance and breathability of

polystyrene, fungal growth is retarded; water cannot support its growth when mycelial

growth already penetrated inside the substrate. Consequently, in the study, the set-ups

required surrounding the polystyrene strips with liquid media throughout the incubation

period. For better results, it is recommended to apply a longer incubation time than 50

days.

        Aside from keeping the experimental organism alive and free from contamination

by other organisms, the most difficult problem to overcome in providing a continuing

source of this organism for a mycological study to be successful, is sustaining the

biochemical property which is being studied, so that the experiment may be repeated with

essentially the same results several months or even years after. This is due to the fact that
                                                    A Preliminary Study on the Potential……..61
                                                                        Dayao and Egloso, 2009

fungi are extremely variable organisms – strains obtained from different sources, while

appearing morphologically identical, will not necessarily behave in the same way

biochemically. What is worse is that variation can occur within a given strain, a property

clearly exemplified in the phenomenon of “sectoring”. On a nutrient agar plate, a spore

or mycelial cell grows radially, maintaining a roughly circular boundary. If at some point

the mycelium undergoes a change, such as a mutation, the variant progeny will still grow

radially outward forming a sector. If the change is morphological (i.e. production or

absence of pigment), then the progeny sector would be visibly different from the parent,

but when the change is biochemical, then a uniform colony is produced, which in reality,

contains cells of different biochemical potentialities. Thus, if inocula are taken from

different parts of the colony, then there would be different results. This occurs during

repeated subculturing of an organism. And when a certain variant, which does not

possess the physiological or biochemical property of interest, prevails among others in

the culture, then that property will be lost altogether (Turner, 1971). This inclination to

change is universal among fungi, although some are more stable than others, hence,

according to Foster, “all investigations dealing with specific metabolic functions of a

fungus sooner or later encounter physiological degeneration manifested by progressive

loss of the function of particular interest” (Turner, 1971). This is evidently true of the

fungal biodegradation processes. It is therefore only presumed that the Xylaria variants

utilized in the present study are still the same with the ones used by Tavanlar and Lat

(2008) in their characterization study. The mentioned study (Tavanlar & Lat, 2008)

concluded that the albino or white mutants exhibited improved ability to grow on reduced

glucose levels as compared to the SDM wild type. This ability may or may not have been
                                                    A Preliminary Study on the Potential……..62
                                                                        Dayao and Egloso, 2009

lost or reduced during the course of the study, especially during subculturing, thus, it may

account as a probable reason why the albino mutants showed less ability to degrade

natural rubber, chicken feather and polystyrene. To reduce experimental errors and

chances of strain variation, i.e. of subculturing from the “wrong” part of the parent

culture, and of contamination from other organisms, transfers must be as infrequent as

possible. Due to this, and to reduce strain variation within the culture, the growth of the

stock culture should be kept to a minimum.

   Furthermore, because of the subculturing and/or mutation step (Tavanlar & Lat,

2008) done to derive the black and white mutants for this study, these could account for

the probable loss or change of the original functions of SDM and the more efficient

ability of the black mutants, E26 and E35, to degrade the given pollutants than the albino

mutants, and/or the loss or decreased ability of the albino mutants to degrade the given

pollutants as compared to the wild type when in fact, they performed better than the wild

type in the study by Tavanlar and Lat (2008). Change produced may be a block, due to

the loss of an enzyme, on a pathway to an essential metabolite, loss or reduction of a

particular function or structural modification.
                                                   A Preliminary Study on the Potential……..63
                                                                       Dayao and Egloso, 2009

                                    CONCLUSION



       Scanning Electron Microscopy (SEM) revealed colonization of Xylaria sp. strain

on natural rubber, chicken feather and polystyrene. Generally, the black strains, namely

the SDM wild type, E26 and E35, have been observed to possess a higher potential to

biodegrade natural rubber, chicken feather and polystyrene than the albino mutants PNL

114, 116 and 118. Among the black variant strains, E26 is the best colonizer for natural

rubber, whereas the albino mutants also showed potential ability to biodegrade this

substrate. On the other hand, the three black strains, SDM wild type, E26 and E35, were

best colonizers for polystyrene, but the albino mutants also showed potential ability to

biodegrade this substrate. Lastly, no significant changes occurred to the chicken feather

samples duue to the short 50-day incubation period. Nonetheless, SDM and E35 black

strains have shown to colonize chicken feather.
                                                     A Preliminary Study on the Potential……..64
                                                                         Dayao and Egloso, 2009




                                RECOMMENDATIONS



       In general, the study suggests a longer incubation time to further test and confirm

the potential biodegrading ability of the Xylaria sp. strains on the three pollutants, namely

natural rubber, chicken feathers and polystyrene. The species of Xylaria used in this

study, and the mutants derived from it, is currently unidentified. It must be further

characterized by several tests to improve nomenclature, thus reducing ambiguity, as it is

further used in other studies. It is also suggested when comparing using SEM

micrographs same magnifications will be used to make more efficient comparisons. More

so, it is recommended that non-candidate species would be used as a species of

comparison in the degradation experiment to further confirm and compare the

biodegrading capacity of Xylaria sp. strains.

       It is also recommended that further test methods and standards for biodegradable

polymers, be applied whenever possible. Further characterization may reveal other useful

properties of the wild type and mutants, applicable to biodegradation and/or

bioconversion purposes. Structural modifications can be monitored in depth through

infra-red (IR) spectroscopy. The actual utilization or “absorption” of carbon by the

mycelia can also be computed.



Natural rubber

       Subjection of the natural rubber samples to tests that would further confirm

presence of intermediate and by-product compounds and biofilm as well such as staining
                                                   A Preliminary Study on the Potential……..65
                                                                       Dayao and Egloso, 2009

of schiff’s reagent and FTR-AITR spectroscopy is encouraged. More so, treatment of

natural rubber gloves before subjecting it to biodegradation is recommended to remove

possible chemical hindrances, such as anti-microbial chemicals and anti-oxidants.

Polystyrene

       Testing other grades of polystyrene to be biodegraded.



Chicken Feather

       To verify the degradation of feather, more advanced tests should be conducted

wherein the presence of soluble proteins and amino groups concentration will be

observed. Study the possibility that Xylaria sp. can produce enzymes such as keratinase,

proteinase. If ever there are enzymes produced, they should be purified and isolated for

the determination of their biochemical properties. Optimum conditions for enzyme

activity should be determined and tested in further biodegradtion studies to attain

optimum enzymatic activities. Testing white and black feathers is also suggested.
                                                  A Preliminary Study on the Potential……..66
                                                                      Dayao and Egloso, 2009

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                                                A Preliminary Study on the Potential……..73
                                                                    Dayao and Egloso, 2009

                                  APPENDIX A

Mineral Medium Formula



Malt extract                                 1g

Ammonium tartate                              5g

MgSO4.7H20                                  0.5g

CaCl2.2H20                                  0.01g

NaCl                                        0.1g

FeCl3                                       0.01g

1% w/v Thiamin                               5ml

1% w/v Trace elements                        1ml

1% m/v Tween 80                             0.2g



*Adjust to pH 5.0 by adding HCL or NaOH. Check the pH using pH paper

** For Mineral Medium Glucose (MMG), just add 0.5 % w/v glucose or 5 g/l
                                                    A Preliminary Study on the Potential……..74
                                                                        Dayao and Egloso, 2009

                                      APPENDIX B

                       Weight Loss / Gain Measurements of the Set-ups

                                    POLYSTYRENE
       Initial  Final              Initial  Final             Initial  Final
                        Difference
Strain Weight Weight               Weight Weight Difference Weight Weight Difference
               First Run                   Second Run                 Third Run
SDM 0.0491 0.0498        -0.0007   0.0451 0.0453      -0.0002 0.0472 0.0487     -0.0015
SDM 0.0506 0.0505        0.0001    0.0444 0.0448      -0.0004 0.0507 0.0517      -0.001
 114    0.046  0.0463    -0.0003   0.0476 0.0479      -0.0003  0.052  0.0527    -0.0007
 114   0.0469 0.0473     -0.0004   0.0514   0.052     -0.0006 0.0528 0.0545     -0.0017
 116   0.0459 0.0462     -0.0003   0.0461   0.046     0.0001  0.0505 0.0553     -0.0048
 116   0.0492 0.0495     -0.0003   0.0412 0.0414      -0.0002 0.0488 0.0502     -0.0014
 118   0.0467 0.0466     0.0001    0.0481 0.0482      -0.0001  0.053  0.0527    0.0003
 118   0.0466 0.0469     -0.0003   0.0479   0.048     -0.0001 0.0478 0.0489     -0.0011
 E26   0.0468 0.0468         0     0.0472 0.0484      -0.0012 0.0515 0.0665      -0.015
 E26   0.0483 0.0468     0.0015    0.0471 0.0486      -0.0015 0.0494 0.0763     -0.0269
 E35   0.0461 0.0468     -0.0007   0.0473 0.0473         0    0.0477   0.077    -0.0293
 E35   0.0495 0.0495         0     0.0462 0.0462         0    0.0515 0.0523     -0.0008




                                   NATURAL RUBBER
       Initial  Final               Initial   Final            Initial  Final
                        Difference
Strain Weight Weight               Weight Weight Difference Weight Weight Difference
               First Run                    Second Run                 Third Run
SDM 0.1909 0.1883        0.0026     0.1976 0.1929      0.0047   0.231  0.3216    -0.0906
SDM 0.1427 0.1489        -0.0062    0.2581 0.2535      0.0046  0.3524 0.3555     -0.0031
 114   0.2041 0.2221      -0.018    0.3179 0.3117      0.0062  0.3942 0.3595     0.0347
 114   0.1183 0.1408     -0.0225    0.2741 0.2693      0.0048  0.2766 0.2472     0.0294
 116   0.1402 0.1363     0.0039     0.2797 0.2842      -0.0045  0.276  0.3283    -0.0523
 116   0.2067   0.254    -0.0473    0.3771 0.3721       0.005  0.2748 0.2864     -0.0116
 118   0.2002   0.197    0.0032     0.2853 0.2806      0.0047  0.2867 0.2202     0.0665
 118    0.149  0.2088    -0.0598    0.2915 0.2814      0.0101  0.2879 0.2709      0.017
 E26   0.1891 0.1851      0.004     0.2732 0.2698      0.0034  0.3284 0.2687     0.0597
 E26   0.2106   0.204    0.0066      0.309   0.3045    0.0045  0.2458 0.2738      -0.028
 E35   0.1183 0.1409     -0.0226    0.3229 0.3191      0.0038  0.3541 0.3877     -0.0336
 E35   0.2009 0.2098     -0.0089    0.2232 0.2162       0.007  0.3514 0.3497     0.0017
                                                 A Preliminary Study on the Potential……..75
                                                                     Dayao and Egloso, 2009

                                   CHICKEN FEATHER
       Initial  Final               Initial  Final             Initial  Final
                        Difference
Strain Weight Weight                Weight Weight Difference Weight Weight Difference
               First Run                    Second Run                 Third Run
SDM 0.0393 0.0401        -0.0008    0.0418 0.0417      0.0001  0.0413   0.041    0.0003
SDM     0.041  0.0501    -0.0091    0.0268 0.0274      -0.0006 0.0316 0.0316        0
 114   0.0367 0.0328     0.0039     0.0643 0.0635      0.0008  0.0911 0.0911        0
 114   0.1428 0.1411     0.0017     0.0263 0.0276      -0.0013 0.0328 0.0329     -0.0001
 116   0.0554 0.0542     0.0012     0.0881 0.0862      0.0019  0.0313 0.0308     0.0005
 116    0.043  0.0415    0.0015     0.0583 0.0579      0.0004  0.0718 0.0702     0.0016
 118   0.0533 0.0540     -0.0007    0.0367   0.035     0.0017  0.0954 0.0959     -0.0005
 118   0.0542 0.0543     -0.0001    0.0554 0.0538      0.0016  0.0916 0.0911     0.0005
 E26   0.0394 0.0379     0.0015     0.0484 0.0478      0.0006  0.0833 0.0839     -0.0006
 E26   0.0909 0.0989      -0.008    0.0916 0.0913      0.0003  0.0423 0.0438     -0.0015
 E35   0.0268 0.0265     0.0003     0.0299 0.0299          0    0.113  0.1131    -0.0001
 E35   0.2252 0.2254     -0.0002    0.0736 0.0746       -0.001 0.0837 0.0648     0.0189
                                            A Preliminary Study on the Potential……..76
                                                                Dayao and Egloso, 2009




                                APPENDIX C

            Natural Rubber SEM plates on Various Magnifications




   A                                           B




   C                                       D




  E                                            F
A) PNL 114 at 500x   B) PNL 116 at 500x C) PNL 118 at 500x      D) E26 at 200x E)
                         E35 at 500x F) SDM at 500x
                                             A Preliminary Study on the Potential……..77
                                                                 Dayao and Egloso, 2009




                                APPENDIX D

               Polystyrene SEM plates on Various Magnifications




   A                                     B




   C                                    D




    E                                   F


A) PNL 114 at 250x   B) PNL 116 at 200x C) PNL 118 at 200x      D) E26 at 1000x E)
                          E35 at 100x F) SDM at 200x
                                               A Preliminary Study on the Potential……..78
                                                                   Dayao and Egloso, 2009



                               APPENDIX E

           Chicken Feather SEM plates on Various Magnifications




   A                                       B




  C                                    D




  E                                    F


A) PNL 118 at 1000x   B) E35 at 500x C) E26 at 500x D) SDM at 500x E) PNL
                       114 at 100x F) PNL 116 at 100x

				
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