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

                                         INTRODUCTION



Background of the Study

       Pollution is an inevitable problem due to population growth, urbanization and the increased

demand for manufactured products in the local and export markets. Industrialization has resulted to

the generation of wastes of various forms that pose serious risks to the environment and public

health, thus, requiring an efficient waste regulatory management.

       At the advent of technology, pollution has indeed taken its toll on nature, making people

harvest and manufacture products that would take eons to decay and rot at the very least.

Everywhere, a plethora of biodegradable and non-biodegradable wastes can be seen. And, indeed it’s

high time that people revert back to natural processes that could help solve the burgeoning problem

of waste disposal since all the artificial methods that require today’s technology could contribute to

the pollution that the planet is experiencing now. One such natural process that could solve the

problem, or in a way even just alleviate such waste pile-up, is biodegradation.

       The country’s population growth rate is one of the highest in the world (Mangahas, 2006)

and it places serious strains on the economy. In 2005, the population was 82.8 million, of which 51.8

million or 63% lived in urban areas. Metro Manila is the most densely populated urban area with

10.7 million (Mangahas, 2006). Over the past 3 decades, the country’s economy slid behind many

Asian economies. Gross domestic product (GDP) grew at an average of only 3%, compared with 8%

in the People’s Republic of China (PRC); 6% in the Republic of Korea, Singapore, Malaysia, and

Thailand; and 5% in Indonesia over the last 30 years (Wallace Report 2004; Mangahas, 2006).

Urbanization, decline in the economy and further population growth lead to the even higher

generation of wastes that has not been managed properly and safely (DENR, 2004).
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                                                                                    Dayao and Egloso, 2009

       Presidential Decree (PD) 1152, or “the Philippine Environmental Code,” provides the basis

for an integrated waste management regulation starting from waste source to methods of disposal.

PD1152 has further mandated specific guidelines to manage municipal wastes (solid and liquid),

sanitary landfills and incineration, and disposal sites in the Philippines. Apart from the basic

policies of PD1152, waste management must also comply with the requirements and implementing

regulations of other specific environmental laws, such as PD984 (Pollution Control Law), PD1586

(Environmental Impact Assessment System Law), RA8749 (Clean Air Act) and RA9003 (Ecological

Solid Waste Management Act) (DENR, 2004).

       A study by Clutario and Cuevas (2001) showed that Xylaria sp. can utilize polyethylene

plastic strips as an alternative carbon source. The fungus grew optimally at 25 0C 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. This showed that Xylaria sp. has indeed

a potential in degrading synthetic wastes like plastics which are difficult to decompose.

       The Xylaria strain mutants PNL 114, 116 and 118 used in the current study, exhibited the

following 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, according to the study by Tavanlar and Lat (2008).
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                                                                                    Dayao and Egloso, 2009

        This study aimed to test 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.



Statement of the Problem

        To determine the potential biodegrading ability of Xylaria sp. mutant strains and SDM wild

type using scanning electron microscopy (SEM).



Objectives

        The study aimed to determine the biodegrading capacity of Xylaria sp. wild type and its

mutants.

        The specific objectives are as follows:

   1.      to determine if Xylaria sp. mutants and wild type can degrade natural rubber, chicken

           feather and polystyrene through Scanning Electron Microscopy.

   2.      to compare the biodegrading ability of the wild type to each mutant to find which strain is

           most appropriate for each type of waste

   3.      to observe the macroscopic/physical changes that natural rubber, chicken feather and

           polystyrene have undergone after incubation with the Xylaria sp.



Significance of the Study

        The study will serve as a source of preliminary information on the potential of Xylaria sp.

strains to degrade chicken feathers, polystyrene and natural rubber. Findings of this study may be

utilized in the development of Xylaria sp. as a good biodegrading agent in reducing durable wastes
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                                                                                  Dayao and Egloso, 2009

such as plastics and others, as well as in optimizing fungal technologies. This study may also

provide a possible means to discover and understand other important characteristics of Xylaria sp.

and its mutants, which may be used in other applications and scientific investigations. The discovery

of other sources of biodegradation agents and their potential bioactive natural products is of

paramount importance, especially nowadays that people should mostly be concerned about their

waste disposal methods, and also to assure a good source of less expensive and more accessible

ways, through research, in approaching the reduction of pollution that are safe and can possibly

boost the Philippine fungal industry in the world market.



Scope and Limitations

       The organism of focus used in this study, Xylaria sp., has only been identified up to the

genus level. The experiment determined the potential biodegrading ability of the Xylaria sp. mutant

strains and wild type on natural rubber, chicken feather and polystyrene. Other pollutants with

similar biochemical structure to the aforementioned pollutants were not included in the experiment.

The three pollutants were not subjected to any chemical (i.e. chemical oxidation) or physical process

(i.e. compression) that would significantly alter their structure prior to the experiment. The used

latext gloves to represent natural rubber were not used to handle chemicals that would affect the

results of the current study. The Xylaria sp. variant strains used came from the stock culture of the

Antibiotic Laboratory of the National Institute of Molecular Biology and Biotechnology

(BIOTECH), University of the Philippines – Los Baños. The strains that were used were Xylaria sp.

strain mutants PNL 114, 116, 118, E26 and E35 which were all compared to the wild type SDM

(sterile dark mycelia), in terms of their biodegrading capacity. The albino mutants (PNL 114, 116,

118) have been partially characterized, whereas the black mutants E26 and E35 have not been
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                                                                                    Dayao and Egloso, 2009

characterized. Culture media and reagents were also provided by BIOTECH. Experimentation was

done in the Antibiotic Laboratory in BIOTECH. Isolation and purification of active components (i.e.

enzymes) responsible for the probable degradation of chicken feathers, polystyrene and natural

rubber were not performed. The determination of new cultural optimum conditions of Xylaria sp.

strains per pollutant such as the optimum temperature for the enzyme activity, optimum pH for each

pollutant degradation activity, optimum incubation period, the need of catalysts for each pollutant

degradation set-up, optimum size of inoculum for each pollutant were not within the scope of this

experiment. The conditions, such as the pH of 5.0, incubation temperature of 25 ˚C and incubation

period of 50 days, of the Xylaria sp. strains, used for incubation and growth were derived from the

results of the study of Clutario and Cuevas (2001) on Xylaria sp.’s degradation of polyethylene.

Colonization of the substrates’ surfaces was observed through scanning electron microscopy (SEM),

done at the Electron Microscopy Laboratory of the National Institute of Engineering at the

University of the Philippines – Diliman. The SEM was conducted after the incubation period of the

set-ups. One sample per pollutant per strain was used. The non-inoculated control was also subjected

to SEM. Only the initial and final weights of the pollutants were recorded. The strain treatments

were in duplicates or 2 replicates per run, and 3 runs were conducted due to logistic matters and

unavailability of equipment. Moreover, the set-ups were observed on the 20th, 30th and 50th day of

incubation, with 50 days as the maximum incubation period. On the 20th and 30th day, only visual

observation was done, since removing the pollutants from the flask will likely contaminate the set-

up. And also, designing another set-up for the 20th and 30th day cannot be performed due to the

limitation of materials and reagents. So it is only on the 50th day that the actual change in weight was

determined. Aside from microscopic observations, macroscopic/physical observations were done.

Yet no standard methods in determining physical change, such as tensile strength measurement for
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                                                                                     Dayao and Egloso, 2009

natural rubber, have been conducted. In terms of data analysis, this study only focused in analyzing

the biodegradation potential of Xylaria sp. strains through its colonization on natural rubber, chicken

feather and polystyrene. Also, the study was concerned on whether the degradation capacity of the

mutant strains is significantly different from the capacity of the wild type to degrade.
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Definition of Terms

Anamorph – mitotic, asexual stage of a fungus that does not produce ascospores

Biodegradation – material is broken down to environmentally acceptable state by microorganisms

capable of utilizing it as a source of energy

Biodeterioration – a superficial degradation that modifies mechanical, physical and chemical

properties of a given material, mainly the result of the activity of microorganisms growing on the

surface or/and inside a given material

Biofilm formation – a.k.a. microfouling; the process in which a complex community of

microorganisms is established on a surface

Bioremediation - any process that uses microorganisms, fungi, green plants or their enzymes to

return the natural environment altered by contaminants to its original condition.

Biotransformation – the chemical modification(s) made by an organism on a chemical compound

Burrowing – the tunneling, penetrating, or infiltrating into the substrate causing holes, cracks,

tearing

Colonization – the establishment of an organism in a new, particular ecosystem

Crack – a thin break, crevice or fissure

Dent – a concavity, hollow or depression in the surface

Elevation – a degree or amount of being raised

Flaking – resulted into flat pieces of the substrate, particularly the surface

Hole, pit – perforation or cavity in an object

Holomorph – the whole fungus in all its forms, facets, and potentialities, either latent or expressed

even if it reproduces by only one method
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Hyphae – microscopic, tubular, and threadlike or filamentous structures which compose the fungus

body or mycelium or thallus and branch in all directions, spreading over or within whatever

substrate a fungus utilizes as food

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

efficiently than other proteases

Keratinophilic fungi – have the unique ability to degrade keratinous substrates such as hair, horns,

hooves, feathers and nails

Keratinolytic fungi – have the unique ability to degrade keratinous substrates completely

Mycelium – the fungus body which is composed of hyphae

Mycelial mat – a mass of hyphae

Polymer - consists of long chains of repeated molecule units known as "mers", which intertwine to

form the bulk of the plastic

Plectenchyma – mycelial mats; ascomycete mycelium that are organized into fungal tissues

Recalcitrance – the resistance of a polymer to be degraded

Smooth – having a continuously even surface

Spore – a minute, simple propagating unit without an embryo that serves in the production of new

individuals

Striation – marked with parallel grooves, ridges, or narrow bands

Tearing – cause something to become splitted, divided, fragmented

Teleomorph – meiotic, sexual stage of a fungus that produces ascospores

Staling product – of an organism: produced by its metabolic activites
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                            REVIEW OF RELATED LITERATURE



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

supply of natural resources; thus, various environmental activities were performed with negligence.

Contaminated lands are aftermaths of past industrial activities that took place when awareness of the

health and environmental effects connected with the production, use, and disposal of hazardous

substances were less well recognized. Currently however, the consequences of our previous actions

are felt more and more as the continual discovery of contaminated sites over recent years has led to

international efforts to remedy many of these sites, either as a response to the risk of adverse health

or environmental effects caused by contamination or to enable the site to be redeveloped for use

(Vidali, 2001).

       Conventional methods for remediation have been to dig up contaminated soil and remove it

to a landfill, or to cap and contain the contaminated areas of a site. Some technologies that have been

used are high-temperature incineration and various types of chemical decomposition (e.g., base-

catalyzed dechlorination, UV oxidation). These techniques have several drawbacks such as technical

complexity, high costs, risks in the excavation, handling, and transport of hazardous material

(Vidali, 2001).
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Biodegradation




                            Figure 1: Scheme for Polymer Biodegradation

       A better approach than these traditional methods is to completely destroy the pollutants if

possible, or at least to transform them to innocuous substances in a process called bioremediation or

biodegradation. Biodegradation is the 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 consequent process

called assimilation (Lucas, et al., 2008). Nonetheless, it is said that as all materials of organic origin,
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both natural and synthetic polymers containing specific functional groups, are potential substrates

for heterotrophic microorganisms including bacteria and fungi (Motta, et al., 2007). Biodegradation

works in such a manner that the organisms involved utilize, or more appropriately, metabolize these

wastes as sources of nutrients such as carbon or nitrogen (Tortora, et al., 2005). It uses relatively

low-cost, low-technology techniques, which generally have a high public acceptance and can often

be carried out on site (Vidali, 2001). Biodegradation agents like bacteria and fungi must be healthy

and active for biodegradation to be highly efficient. Biodegradation technologies create optimum

environmental conditions to help the growth and increase the number of microbial or fungal

populations for them to detoxify the maximum amount of contaminants (United States

Environmental Protection Agency, 1996).

       The general objective of biodegradation is to discern the speed (i.e. percent weight loss of

pollutant per week) of unaided biodegradation before catalysts may even be added, and then

strengthen spontaneous biodegradation only if this is not fast enough to remove the contaminant’s

concentration in the environment before it may cause any health risk to nearby inhabitants such as

people, animals and plants (European Federation of Biotechnology, 1999).

       The control and optimization of biodegradation processes are a complex system of many

factors which include: the existence of a microbial population capable of degrading the pollutants,

the site conditions, the quantity and toxicity of contaminants and the environment factors (type of

soil, temperature, pH, the presence of oxygen or other electron acceptors, and nutrients). Different

microorganisms degrade different types of compounds and survive under different conditions

(United States Environmental Protection Agency, 1996).

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

(e.g. glucose) are often employed. The amount must be strictly controlled to prevent the organism
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                                                                                  Dayao and Egloso, 2009

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 computation of the weight loss and microscopic examination of the polymer

surface. When a polymer is incubated with fungi, small circular holes are sometimes observed. The

hydrophobicity of most polymers is often a major obstacle to obtaining satisfactory results from a

biodegradation test. 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 properties of polymer materials and specific environmental

conditions, such as humidity, weather and atmospheric pollutants (Lugauskas et al., 2003; Lucas, et

al., 2008). The ease with which polymers are degraded is entirely dependent upon their material

composition (Busscher et al., 1990; Wiencek & Fletcher, 1995; Bos et al.,1999; Gu, et al., 2000b;

Gu, 2003), molecular weights, 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

environmental conditions whether they are 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
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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).

       Generally, microbes cannot degrade synthetic polymers, principally the polyolefins, which

are made up of only carbon and hydrogen atoms, considered to be resistant to biodegradation. This is

probably due to a total lack in the polymer’s backbone of sites involving carbon-to-oxygen bonds

(C=O, C–OR, C–OH), which are the real target of microbial enzymes (Motta, et al, 2007). The

biodegradation of natural polymers, on the other hand, usually proceeds faster than that of their

synthetic homologues because they contain traces of amino acids, vitamins, growth hormones, and

enzymes (Albertsson & Karlsson, 1993).



Microorganisms Used in Biodegradation

       Through time, scientific experiments have already proven the ability of some

microorganisms to biodegrade pollutants such as polyethylene, polystyrene, rubber, chicken feathers

and other types of wastes. Organisms such as bacteria and fungi have proven themselves to possess

the capacity to biodegrade pollutants.

       Bacteria such as Brevibaccillus borstelensis, Rhodococcous rubber C208, Xanthomonas sp.

strain 357 have been proven to degrade pollutants. Plastics in the form of polyethylene are known to

be degraded by the thermophilic bacterium Brevibaccillus borstelensis 707 which was isolated from

soil (Hadad et al, 2005). Another study by Orr et al. (2004) featured the Rhodococcous rubber C208

as an effective polyethylene-degrading organism. In addition to this, this strain has been proven to

degrade polystyrene (Mor & Sivan, 2008). Yet originally, Rhodococcous rubber is a known rubber-
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                                                                                    Dayao and Egloso, 2009

degrading organism, according to the review of Rose and Steinbuchel (2005). Xanthomonas sp.

strain 357, in much the same way, can degrade rubber as well.

       A number of fungi species are also known to biodegrade. The known fungi biodegraders are

Gordonia sp., Streptomyces sp., Nectria gliocladioides, Penicillium ochrochloron and Geomyces

pannorum and Trichoderma atroviride (Barraat, et al.,2003; Cheng chang, et al., 2003; Rose &

Steinbuchel, 2005).

       Gordonia sp. and Streptomyces sp. are known rubber-degraders (Rose and Steinbuchel,

2005). Nectria gliocladioides (five strains), Penicillium ochrochloron (one strain) and Geomyces

pannorum (seven strains), in a study of Barraat et al. (2003), have been observed to degrade

polyurethane while simultaneously relating it to the water holding capacity of the soil. Moreover, in

a study conducted by Cao et al. (2008), the fungus Trichoderma atroviride completely degraded the

chicken feathers. This strain was actually isolated from a decaying feather.

        The list of microorganisms that could be used in biodegradation goes on for there are still

more species that could 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. as a Potential Agent for Biodegradation

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

Clutario and Cuevas (2001), growing on a sando plastic bag, buried in forest soil and litter in the

lowland secondary forest of Mt. Makiling, Laguna. The fungus comprised of sterile melanin

pigmented mycelia and was reported as ascomycete sterile dark mycelia (ASDM). Cultural studies
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                                                                                   Dayao and Egloso, 2009

have designated it under Class Ascomycetes, Order Xylariales, Genus Xylaria. (Clutario & Cuevas,

2001).

         A previous study by Clutario and Cuevas (2001) proved that Xylaria sp. can utilize

polyethylene plastic strips as an alternative carbon source, thereby degrading them into usable forms

for self-sustenance. Through the use of scanning electron microscopy, the proponents of the said

study observed visible damages of the surface structure of the plastic strips. There were tearing and

striations caused by active burrowing of Xylaria sp. hyphae on the polyethylene material. Plastic is

an extremely versatile synthetic material made of high molecular weight, semi-crystalline polymer

prepared from ethylene through the cracking of crude oil, light petroleum and natural gas. For plastic

bags alone, it is estimated that some 430,000 gallons of oil are needed to produce 100 million pieces

of these omnipresent consumer items on the planet (Knapczyk & Simon, 1992; EcoWaste Coalition,

2008).

         According to a study by Carmen Acevedo (2007) of the University of Puerto Rico, Xylaria

biotransformed significant amounts of phenanthrene with and without surfactants. Surfactants were

tested for their ability to solubilize phenanthrene, and therefore increase the biotransformation of

phenanthrene. Results indicated that the surfactants examined can either enhance or inhibit

biotransformation depending on the fungus and concentration, which suggest that marine fungi and

particularly endophytes are potentially useful for bioremediation in marine environments.

         Xylaria is one of the most commonly encountered groups of ascomycetes with most of its

members being stromatic, peritheciate, with an iodine-positive ascus apical ring, and with one-

celled, dark ascospores on which a germination slit can be found. Xylaria species, although most

often encountered in temperate and tropical forests, saprobic on decaying hardwood stumps and

logs, also to a large extend colonize substrates such as woody legume pods and other kinds of fruit,
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                                                                                    Dayao and Egloso, 2009

petioles, leaves of angiosperms and herbaceous stems, sometimes appearing terrestrial but actually

attached to buried wood; growing alone or, more commonly in clusters; appearing in spring and not

decaying until late summer or fall (Kuo, 2003). Some are associated with insect nests. Most decay

wood and many are plant pathogens. Many are endophytes. They are commonly found throughout

the temperate and tropical regions of the world. The Xylaria sp. can be distributed above, around,

and beneath perithecia. It forms a unipartite stromatal layer, with a superficial or erumpent surface

level. The interior of its stromata is essentially homogeneous. Conidium-bearing discs, potassium

hydroxide pigments and orange granules surrounding the perithecia are absent (Rogers et al., 2002).

They are mostly multiperitheciate in ascomatal number per stroma, ascomatal ostioles and ascal

apical rings: are present, and the ascospore cell number is one-celled. Teleomorph and anamorph are

produced on the same stromata in most species, with their anamorphs: Geniculosporium-like. Some

Xylaria sp. species exist as endophytes, and have mutualistic associations with plants. The fungus

secrete toxins to protect the plant from herbivory from other insects or animals, while the fungus in

return feeds on the host’s tissues for nutrition, and its mycelia are scattered through seed dispersal.

Endophytic Xylariaceae have been documented in conifers, monocots, dicots, ferns, and lycopsids

(Brunner & Petrini, 1992; Davis, et al, 2003).

       According to the study of Liers et al. (2007), Xylaria polymorpha, which is said to lack

peroxidase, is known to produce the enzyme laccase, a known ligninolyitc oxidoreductase. This

supports the previous study of Lou and Wen (2005) wherein they discovered that Xylaria sp. along

with other ascomycetes and some basidiomycetes commonly demonstrated laccase activity together

with cellulolytic and xylanolytic activities. The enzymatic profiles of the aforementioned species

suggests that (1) ascomycetes is potentially capable of utilizing the lignocellulosic wood components

(2) laccase is apparently the main enzyme for ligninolysis unlike the white-rot basidiomycetes that
                                                               A Preliminary Study on the Potential……..17
                                                                                   Dayao and Egloso, 2009

utilizes its ligninolytic peroxidase in the form of manganese peroxidase or lignin peroxidase in

addition to lignin peroxidase.

       Metabolites produced by Xylaria have already been identified. In a study by Pongcharoen, et

al. (2008), two xylarosides, which are glucoside derivatives, were isolated from the broth extract of

a Xylaria sp. PSU-D14 strain along with two compounds, sordaricin and 2,3-dihydro-5-hydroxy-2-

methyl-4H-1-benzopyran-4-one. Sordaricin showed moderate antifungal activity against Candida

albicans ATCC90028 strain.


       In another study by Xiaobo, et al. (2006) they isolated a Xylaria sp. 2508 strain from the

seeds of an angiosperm tree and was found to produce rich secondary metabolites. Xyloketals,

xyloallenolide A, and two other allenic ethers were isolated from the spent culture medium of this

fungus. One of allenic ethers is eucalyptene A which exhibited antifungal activity.


The Xylaria Wild Type and Mutant Strains

       Partial characterization of the fungus Xylaria sp. and its albino mutants, PNL 114, PNL 116

and PNL 118 was based on the study by Tavanlar and Lat (2008) in which the black fungus wild

type SDM was subjected to mutagenesis, and protoplast fusion. On the other hand, the black mutants

E26 and E35 have not been characterized as of yet (M. A. Tavanlar, personal communication, March

4, 2009). The aforementioned study determined morphological and biochemical characteristics or

markers in the wild type and mutants that can be used in the analysis of future recombinants or

fusants. The reputed mutants were described based on colony characteristics, morphology and

growth on various media.

       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
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                                                                                  Dayao and Egloso, 2009

wall, melanin reduces the pore diameter below 1nm but remains permeable to water, based on

studies on Magnaporthe grisea (Howard et al., 1991; Tavanlar & Lat, 2008). Melanin acts in the

survival and longevity of propagules (i.e. part of a plant or fungus such as a bud or a spore that

becomes detached from the rest and forms a new organism) (Bell and Wheeler, 1986; Tavanlar and

Lat, 2008) and protects hyphae during infiltration through a substrate during colonization (M. A.

Tavanlar, personal communication, March 4, 2009). This polymer of phenolic compounds 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).

       In the study by Tavanlar and 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. The hyphae

of these mutants were similar to the wild type, when viewed 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. Table 1 shows the three mutants which underwent further tests as

presented. The study further tested the three amelanotic mutants selected in various media

supplemented with benomyl, acetamide, PEG 6000 (polyethylene glycol), Tween 80, and glucose.
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                                                                                                  Dayao and Egloso, 2009

Table 2 shows the growth of the three albino mutants on mineral medium with and without

supplements as compared to the wild type SDM. In summary, the results of the said study showed

that the three mutants are less dependent on the glucose level in the medium for growth and hyphal

tip extension. The mutants showed loss of melanin pigmentation and improved ability to grow on

reduced glucose levels, tolerate 0.1% w/v copper hydroxide and 0.005% benomyl, utilize 1% w/v

polyethylene glycol 6000, 1% v/v Tween 80 and 1% w/v acetamide as sources of carbon as

compared to the wild type. These albino mutants may potentially exhibit enhanced degradation of

polyethylene plastics than the wild type. 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.


Table 1. Comparative growth of the PNL mutants and wildtype SDM on MMG and mineral medium
with various supplements.
                                      Average diameter of colony (mm)
           Mutant Strain                          Medium
                            M1          M2          M3          M4       M5
              PNL 114      23.0a       14.8a       17.0a       24.2a     24.8
              PNL 116      22.0a       14.5a       17.0a       24.3a     23.0
              PNL 118      23.3a       12.8b       17.0a       23.0a     23.8
               SDM           5b         5c          5b         12.8b     20.1
           (Source: Tavanlar, M. A. T., & Lat, E. C. (2008). Partial Characterization of Mutants from a Plastic-degrading
           Black Fungus. Unpublished.)

       Measured after 4 days incubation at ART:

              M1 = MMG + 0.005% benomyl
              M2 = MM + 0.025% glucose + 1% acetamide
              M3 = MM + 0.025% glucose + 1% PEG
              M4 = MM + 0.025% glucose
              M5 = MM + 0.5% glucose
       Values within the same column followed by the same letter are not significantly different at

P<0.05.

Table 2. Growth of the four albino mutants on mineral medium with and without supplements as
compared to the wildtype SDM.
                                                                             A Preliminary Study on the Potential……..20
                                                                                                 Dayao and Egloso, 2009

                                                Average diameter of colony (mm)
                 Mutant Strain                             Medium
                                              MM            MMP              MMT
                    PNL 114                   41.5           36.0            38.5
                    PNL 116                   45.0           35.0            34.8
                    PNL 118                   39.0           36.5            32.2
                     SDM                      16.0           16.5            14.1
                (Source: Tavanlar, M. A. T., & Lat, E. C. (2008). Partial Characterization of Mutants from
                 a Plastic-degrading Black Fungus. Unpublished.)
       Measured after 3 days incubation at ART:

               MM = mineral medium
               MMP = MM + 1% w/v polyethylene glycol 6000
               MMT = MM + 1%v/v Tween 80

Table 3. Growth of the two black mutants on mineral medium with supplements MDPE and PEG

                                                       Average diameter of
                               Mutant Strain               colony (mm)
                                                      MMMDPE        MMPEG
                                      E26               25.9         23.85
                                      E35              40.15         30.65
               MMMDPE = MM + medium density polyethylene
               MMPEG = MM + polyethylene glycol 6000


       Another application of Xylaria aside from its biodegrading abilities is the proprietary Xylaria

nigripes extract in WulinshenPrime™ in SleepWell™ (a patented fermentation technology available

from NuLiv Science). It provides the critical, necessary and often depleted nutrients to the brain and

assists in the biochemical process in the brain to promote restful and deeper sleep so one will wake

up refreshed and energized. WulinshenPrime™ contains many essential amino acids, vitamins,

minerals, trace elements, glycoproteins, glutamic acid, γ-aminobutyric acid (GABA) and glutamate

decarboxylase (NuLiv Lifestyle, 2008).

       A study by Park (2005) showed that antifungal antibiotics for the treatment of fungal

diseases of humans and veterinary animals were produced by a fungus identified as a Xylaria sp.

according to nuclear ribosomal ITS1-5.8SITS2 sequence analysis, and was labeled F0010 strain. The
                                                                 A Preliminary Study on the Potential……..21
                                                                                     Dayao and Egloso, 2009

fungus was endophytic to Abies holophylla, and the study evaluated its in vivo antifungal activity

against plant pathogenic fungi. The antibiotics were determined to be griseofulvin and

dechlorogriseofulvin through mass and NMR spectral analyses of purified liquid cultures. Compared

to dechlorogriseofulvin, griseofulvin showed high in vivo and in vitro antifungal activity, and

effectively controlled the development of rice blast (Magnaporthe grisea), rice sheath blight

(Corticium sasaki), wheat leaf rust (Puccinia recondita), and barley powdery mildew (Blumeria

graminis f. sp. hordei), at doses of 50 to 150 μg/ml, depending on the disease. This was the first

report on the production of griseofulvin and dechlorogriseofulvin by Xylaria species.



Culture Methods

       The shaken culture method was used to provide a stock culture for fungal inocula. The

medium is shaken after inoculation with spores or mycelium so that growth occurs in the body of the

liquid. Nutrient uptake is very efficient, giving faster and more homogeneous growth. The technique

exhibited by Xylaria growth during incubation for 50 days is the surface culture method. When a

nutrient medium is inoculated with fungal spores or mycelium, the mycelium grows over the surface

of the liquid to form what is variously referred to as a felt, a pad or a mat (Turner, 1971).




Current State of the Solid Waste in the Philippines

       Filipinos generate around 0.3 to 0.7 kilograms of garbage daily per person depending on

income levels (World Bank, 2001). Metro Manila produces about 8,000 tons of solid waste each day

and is expected to reach 13,300 tons each day in 2014 (Baroña, 2004). The National Capital Region
                                                                 A Preliminary Study on the Potential……..22
                                                                                     Dayao and Egloso, 2009

produces the highest amount of wastes, about 23% of the country’s waste generation (Anden &

Rebolledo, 2003).

       Based on studies (2001) made by the National Solid Waste Management Commission

Secretariat based at the Environmental Management Bureau (EMB), it is estimated that in Metro

Manila, the per capita waste production daily is 0.5 kg. Thus, every person living in the metropolis

generates half a kilo of waste a day. With an estimated population of 10.5 million, total waste

generated in Metro Manila alone could run up to 5,250 metric tons per day or 162,750 metric tons

per month or 1.95 million metric tons per year.

       Based on another EMB study (2001) regarding the disposal of daily wastes, only about 73%

of the 5,250 metric tons of waste generated daily are collected by dump trucks hired by local

government units. The remaining 27% of daily wastes, or about 1,417.5 metric tons, end up in

canals, vacant spaces, street corners, market places, rivers and other places.

       According to a survey conducted by the EcoWaste Coalition and Greenpeace Southeast Asia

in 2006, synthetic plastics comprise 76% of the floating trash in Manila Bay, out of which 51% are

plastic bags, 19% are sachets and junk food wrappers, 5% are styrofoams and 1% is hard plastics.

The rest were rubber (10%) and biodegradable discards (13%) (EcoWaste Coalition, 2008).




Natural Rubber
                                                               A Preliminary Study on the Potential……..23
                                                                                   Dayao and Egloso, 2009

          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 the Hevea brasiliensis also known as the

rubber tree. It is mainly composed of cis-1,4 polyisoprene which has a molecular mass of about 106

Da. This could also be chemically synthesized and produce the substance known as Isoprene Rubber

(IR) (Linos, et. al, 2000).

          According to the mini review of Rose and Steinbuchel (2005), the average composition of

latex glove from the Hevea brasiliensis plant is 25 to 35% (wt/wt) polyisoprene; 1 to 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. This polymer has rubber particles which are about 3- to 5- µm and covered by a layer

of proteins and lipids. This serves to divide the hydrophobic rubber 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. Such treatments

thereby reduced the protein content of condoms and latex gloves to less than 20 µg/g of natural

rubber.

          Since 1914, natural rubber has been a classic subject of biodegradation studies. (Rose &

Steinbuchel, 2005). This is due to the high rate of its yearly manufacture which is several million

tons, as mentioned in the study of Bereeka (2006), and its slow rate of natural degradation as

reviewed by Rose and Steinbechul (2005). In fact, a number of studies abound concerning its

degradation. And it has been learned that both bacteria and fungi can participate in such process.
                                                              A Preliminary Study on the Potential……..24
                                                                                  Dayao and Egloso, 2009

       Throughout all the investigations and experimentations done, two categories of rubber-

degrading microbes according mainly on growth characteristics have been established. Based on a

review by Rose and Steinbuchel (2005), which recapped the aforementioned groups, the microbes

that can degrade rubber can be categorized as clear zone-forming around their colonies and non-halo

forming whenever isolated and cultured in latex overlay plate, which is made by overlaying a layer

of latex agar medium on a basal salt medium agar. The former category was identified to mainly

consist of actinomycete species. They are said to biodegrade or metabolize rubber by secreting

enzymes and other substances and also they are dubbed to be slow degraders since they rarely show

an abundant cell mass when grown on natural rubber directly. On the other hand, members of the

second group do not form halos on latex overlay plates. They, unlike the first group, grow more

when directly grown on natural rubber. In a way, their growth on rubber could be described in an

adhesive manner. The second group is said to demonstrate a relatively stronger growth on rubber.

Species comprising this category are the Corynebacterium-Nocardia-Mycobacterium group. They

consist of the Gordonia polyisoprenivorans strains VH2 and Y2K, G. westfalica strain Kb1, and

Mycobacterium fortuitum strain NF4.

       As demonstrated by a particular study by Linos, et al. (2000), the mechanisms that the

microbes undergo when biodegrading is colonization, biofilm formation and aldehyde group

formation after cultivating it on the surface of latex gloves. Such is revealed after undergoing the

Schiff reagent’s test. This is further examined under a scanning electron microscope. In their

methodology they have indicated that the preliminary screening method to be used in finding

potential rubber-degrading bacteria is by growing such bacteria or microbe on the latex overlay or

by latex film on the mineral agar plates. Growth and colonization of the microbe in this medium
                                                             A Preliminary Study on the Potential……..25
                                                                                 Dayao and Egloso, 2009

would indicate its utilization of rubber as its sole carbon source; hence, making it as a potential

rubber-degrader.

       Furthermore, according to a study of Bereeka (2006), the degradation of natural rubber is

initiated by the oxidation of double bonds. Once this takes place, oligomeric derivatives with

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

on the study of Linos et al. (2000), 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. 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                    b        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
                                                                A Preliminary Study on the Potential……..26
                                                                                    Dayao and Egloso, 2009

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. It’s

actually the most abundant protein. It is not easily degraded due to its tightly packed structural

arrangement which is in the form of alpha keratin or beta keratin. The key to its stability lies on the

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 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,
                                                                  A Preliminary Study on the Potential……..27
                                                                                      Dayao and Egloso, 2009

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 these 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 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 on

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, which could degrade feathers and make it
                                                                  A Preliminary Study on the Potential……..28
                                                                                      Dayao and Egloso, 2009

available for its use as animal feed, fertilizer and natural gas. They are primarily classified as

extracellular serine proteases, with the exception of keratinases from yeasts, which belong to the

aspartic proteases. Molecular masses of these enzymes range from 20 kDa to 60 kDa. They are

mostly active in alkaline environments, with optimal activity at temperatures up to 50°C.

Thermostable keratinases with optimal temperatures of around 85°C and a higher molecular mass

have been reported (Gradisar, et al., 2005). The enzymes are said to degrade the beta-keratin

component and the main idea behind such biodegradation is that the microbes use the feather as their

carbon, nitrogen, sulfur and energy for their nourishment (Manczinger, et al., 2003; Joshi, et al.,

2007). According to the study of Cheng-gang, et al. (2008), the keratinase enzyme is inducible

whenever substrates of keratin composition are present. Among all the keratin-inducing substrates,

feathers (made up of beta-keratin) are the ones commonly utilized. Yet both alpha-keratin and beta-

keratin substrates can be used in feather degradation. It is reported that the mechanism behind the

degradation of chicken feather is yet to be elucidated. But according to Kunert (2000) in the study of

Cheng-gang, et al. (2008), the proposed primary step in keratinolysis is deamination which produces

an alkaline environment. Such environment is needed to induce substrate swelling, sulphitolysis and

proteolytic attack. In the same study of Cheng-gang, et al. (2008), the degradation of feathers

produced amino acid residues such as threonine, valine, methionine, isoleucine, phenylalanine and

lysine. It was elucidated that this could be due to the high disulfide content of the feathers.

       Keratinases 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 (Joshi, et al., 2007). Furthermore, potentially, the said hydrolysate

could be used in the generation of organic fertilizer, edible films and amino acids which are
                                                                A Preliminary Study on the Potential……..29
                                                                                    Dayao and Egloso, 2009

considered rare, as cited by Brandelli in the journal of 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).

       A study by Heather Costello of the Ohio Wesleyan University collected fungal samples in

Ohio Wesleyan's Kraus Wilderness Preserve from different locations with different soil profiles.

These were analyzed and tested for fungi that can hydrolyze §-keratin. The first experiment showed

that only the quills were left in the flask. Scanning electron showed intricate details of fungal

degradation of feathers.

       In terms of experimental procedures, various methods are used in determining the

keratinolytic ability, which means it could produce keratinase and hence degrade chicken feather, of

microbes. A particular study by Tapia and Contiero (2008), used a feather meal agar, wherein the

feather served as the source of carbon, nitrogen, sulfur and energy, in cultivating the isolated

microbe Streptomyces. The growth, which occurred on the 10th day of incubation, through colony

formation of the microbe indicated that it utilized the feather as a source of its nutrients. After

which, its keratinolytic activity was tested using a modified keratin azure protocol. Another study by

Maczinger, et al. (2003) focused on the isolation of a microbe from the poultry waste that could

degrade feathers. During the preliminary elimination, they cultured the different population of

bacteria found in a partially degraded feather in a basal medium with sterilized feathers serving as its

source of carbon, nitrogen and sulfur. It was then rotated in an orbital shaker for 10 days. After 4

days, one flask showed a visual degradation of the feather. A dilution series was made afterwards so

as to isolate and culture the bacteria that just degraded the feather. The strain was identified as

Bacillus lichenformis strain K-508. And the confirmation of the keratinolytic activity was done by

using the azokeratin as a substrate assay.
                                                                     A Preliminary Study on the Potential……..30
                                                                                         Dayao and Egloso, 2009

           A study by Kavitha (2001) studied the distribution of mycoflora inhabiting bird's feather. All

the fungi were screened for keratinolytic activity. Chrysosporium spp having high keratinase activity

was taken for further studies.          The keratinase from Chrysosporium spp., was purified and

homogeneity was confirmed by polyacrylamide gel electrophoresis.

           Isolation of a new microbial organism that could degrade chicken feather will help in the

degradation of the chicken feathers which is now becoming a burden in the society both

internationally and locally. The microbe could potentially provide the keratinase that could be used

in compost technology (Maczinger, et al., 2003) or in the conversion of feather to feedstock meal

additives (Tapia & Contiero, 2008).



Polystyrene




       a                                                b                               c
   Figure 4: Polystyrene (a) Different kinds of cup made of polystyrene, (b) Styrene molecular
              formula, the repeating unit to make a large polystyrene, and (c) Model diagram of a
              styrene monomer


           Polystyrene, 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),
                                                              A Preliminary Study on the Potential……..31
                                                                                  Dayao and Egloso, 2009

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”.

       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,
                                                                A Preliminary Study on the Potential……..32
                                                                                    Dayao and Egloso, 2009

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 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
                                                               A Preliminary Study on the Potential……..33
                                                                                   Dayao and Egloso, 2009

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 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
                                                               A Preliminary Study on the Potential……..34
                                                                                   Dayao and Egloso, 2009

strain is a biofilm-producing actinomycete that has first colonized and degraded polyethylene (Orr et

al., 2004).

        There were studies that tested the possibility of whether copolymerizing polystyrene with

other substance could make it more degradable and susceptible to microbial attack. In 1992, a study

by Milstein, et al. (1992), focused on the biodegradation of a lignin-polystyrene copolymer. The

white rot basidiomycete was used to degrade such 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,
                                                                A Preliminary Study on the Potential……..35
                                                                                    Dayao and Egloso, 2009

polyurethane and perlite proved to be most suitable for enrichment of styrene-degrading fungi.

Perlite is an amorphous volcanic glass that has a 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 substrate, such as polystyrene, according to Motta, et

al. (2007), is a crucial step towards polymer biodegradation.
                                                               A Preliminary Study on the Potential……..36
                                                                                   Dayao and Egloso, 2009

       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).



Scanning Electron Microscope

       The scanning electron microscope (SEM) is a microscope that utilizes electrons rather than

light in forming images. The high energy electrons used interacts with the atoms which are present

on the surface of the specimen. During this interaction, signals such as secondary electrons,

backscattered electrons, characteristic x-rays, light, specimen current and transmitted electrons are

produced. These signals possess the details of the specimen’s surface topography, composition and

its other properties and characteristics. The SEM’s pattern follows that of a reflecting light

microscope hence it yields similar information. Yet the scanning electron microscope has a number

of advantages over the traditional light microscope. It has a resolution range of 5 nanometers and a

magnification range of about 15x to 200,000x. This means that a higher level of magnification could

be achieved by specimens which are so closely spaced. SEM also has a large depth field thereby

permitting more of a specimen to be in focus. More so, due to its usage of electromagnets rather than

light, the degree of magnification could be much more controlled. (Prescott et al., 2005; Schweitzer,

2006; Chumbley, 2009)

       The scanning electron microscope functions by producing electrons thermoionically fired by

an electron gun composed of tungsten filament cathode. Tungsten is used because of its high melting

point and its lowest vapor pressure characteristics. The electron gun produces monochromatic
                                                               A Preliminary Study on the Potential……..37
                                                                                   Dayao and Egloso, 2009

electrons and vertically directs it to the two condensers which focus the beam to a spot of about 0.4

to 5 nm in diameter. The electron beam which has energy of a few hundred eV to 40 KeV travels in

a vacuum environment and detected by detectors inside the SEM. The first condenser lens which is

controlled by the coarse probe knob functions to form and restrict the amount of current in the beam.

It also works with the condenser aperture, not user selectable, in discharging and constricting the

high-angle electrons. The second condenser, on the other hand, is controlled by the fine probe

current knob which functions to produce a thin, tight and coherent beam of electrons. Unlike in the

former, the aperture in this condenser is user selectable and works also to remove high-angle

electrons. After this, the beam is passed through a set of coils which scans the beam in a grid-like

manner in a microsecond range to focus on points. The objective then allows the electron beam to

concentrate and scan on the part of the specimen desired. Once the electron beam strikes the sample,

interactions occur inside the sample; then, electrons and x-rays are emitted. The energy exchange

between the electron beam and the sample results in the reflection of high-energy electrons by

elastic scattering, emission of secondary electrons by inelastic scattering and the emission of

electromagnetic radiation, each of which can be detected by specialized detectors. The detection of

the interacting particles is detected by various instruments. These instruments count the number of

interactions and show a pixel display on a CRT, the intensity then is determined by the number of

reactions. The relationship of the two is said to be proportional: the more interactions, the brighter

the pixel. Repetition of the process, which could be done 30 times per second, is done until the grid

scan is finished. (Schweitzer, 2006; Chumbley, 2009 and Prescott et al., 2005)

       Due to the conditions by which SEM works, such as a vacuum environment and electron

usage, specimen preparation must be done. The sample must be dry. All water must be evaporated or

remove from the system because it may vaporize in the vacuum. The size of the material should fit
                                                               A Preliminary Study on the Potential……..38
                                                                                   Dayao and Egloso, 2009

in a specimen chamber and mounted on a holder called a specimen stub. The specimens must all be

electrically conductive and grounded. All non-metals such as biological matters must be made

conductive hence it must be coated with a thin layer of conductive material by using a device called

a “sputter coater”. On the other hand, since metals are naturally conductive they no longer need to be

coated. But it must be insured that they are thoroughly cleaned and properly mounted. Through the

use of electric field and argon gas, the sputter coater works by placing the specimen in a vacuum

condition. The electric field induces the argon gas to be negatively charge by removing an electron

from the argon gas. The argon ion then exerts an attractive force to the charged gold foil by

knocking down gold atoms. The gold foil’s electrons are then sputtered or fall and settle on the

surface of the sample producing a thin gold coating. Coating is very important to prevent the

accumulation of charge during irradiation (Prescott et al., 2005; Schweitzer, 2006; Chumbley,

2009).
                                                              A Preliminary Study on the Potential……..39
                                                                                  Dayao and Egloso, 2009

                                 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 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
                                                       A Preliminary Study on the Potential……..40
                                                                           Dayao and Egloso, 2009

   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. 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……..41
                                                                                    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.



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
                                                             A Preliminary Study on the Potential……..42
                                                                                 Dayao and Egloso, 2009

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.
                                                               A Preliminary Study on the Potential……..43
                                                                                   Dayao and Egloso, 2009

                                             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, 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.
                                                              A Preliminary Study on the Potential……..44
                                                                                  Dayao and Egloso, 2009




Natural Rubber




            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……..45
                                                                                   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……..46
                                                                                   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……..47
                                                                                 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……..48
                                                                                  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

mat and spores on the natural rubber. In figure 10, PNL 118 mutant showed no distinct traces of
                                                                 A Preliminary Study on the Potential……..49
                                                                                     Dayao and Egloso, 2009

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

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.
                                                             A Preliminary Study on the Potential……..50
                                                                                 Dayao and Egloso, 2009

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……..51
                                                                                  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……..52
                                                                                 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……..53
                                                                                 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……..54
                                                                                    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……..55
                                                                                  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) attached in various areas

on the feather of the SEM of PNL 118 mutant strain. Some portions of the feathers seem to be
                                                                           A Preliminary Study on the Potential……..56
                                                                                               Dayao and Egloso, 2009

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.


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
Control     No physical change occurred            No observed mycelia attached and physical damage
 SDM        Presence of mucilaginous sheath        Profuse growth as shown by mycelial mats closely adhering to the
            while in culture. Barbules are         barbs and barbules. No discernible tearing, cracks or holes can be
            slightly brittle (i.e. ability to be   observed on the barbs.
            detached easily from the barb).
 PNL        Presence of mucilaginous sheath        Flakes of mycelial mats colonize barbs and barbules of the chicken
 114        while in culture. Retained original    feather. A deep crack can be observed at the middle of the lower barb.
            physical characteristics
 PNL        Presence of mucilaginous sheath        Colonization demonstrated by the close adherence of mycelial mats to
 116        while in culture. Retained original    the barbules. Parts of barbs shown seem to be physically intact. No
            physical characteristics               tearing, cracks or holes can observed in the barbs.
 PNL        Presence of mucilaginous sheath        Smaller mycelial mats are adhering to several areas of the barbules.
 118        while in culture. Retained original    The rachis at the middle of the SEM seems to be physically intact. No
            physical characteristics               tearing, cracks or holes can be observed also.
  E26       Presence of mucilaginous sheath        Presence of mycelial mats adhering to the barbules while fewer
            while in culture. Retained original    mycelia filaments or threads adhere to the barbs. No discernible
            physical characteristics               tearing, cracks or holes can be observed.
  E35       Presence of mucilaginous sheath        Dense mycelial mats with spores attached are shown covering several
            while in culture. Barbules are         areas of the feather sample. Weakened barbs resulted to breakage.
            slightly brittle (i.e. ability to be   Several barbules detached from their barbs. Presence of spores are
            detached easily from the rachis).      also observed.
                                                             A Preliminary Study on the Potential……..57
                                                                                 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……..58
                                                                                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……..59
                                                                                    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……..60
                                                                                   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……..61
                                                                                     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 the area of

interest, that traces of raw polystyrene surface is barely visible; they almost completely covered the
                                                               A Preliminary Study on the Potential……..62
                                                                                   Dayao and Egloso, 2009

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.
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                                                                                                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……..64
                                                                                    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 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
                                                                 A Preliminary Study on the Potential……..65
                                                                                     Dayao and Egloso, 2009

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 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
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                                                                                    Dayao and Egloso, 2009

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

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
                                                               A Preliminary Study on the Potential……..67
                                                                                   Dayao and Egloso, 2009

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).



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.
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                                                                                  Dayao and Egloso, 2009

       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.

       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
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                                                                                     Dayao and Egloso, 2009

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.

       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.
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                                                                                    Dayao and Egloso, 2009

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)

       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.
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                                                                                    Dayao and Egloso, 2009

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 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 products. Therefore it is
                                                              A Preliminary Study on the Potential……..72
                                                                                  Dayao and Egloso, 2009

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.

        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
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                                                                                     Dayao and Egloso, 2009

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.



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
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                                                                                    Dayao and Egloso, 2009

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 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
                                                                 A Preliminary Study on the Potential……..75
                                                                                     Dayao and Egloso, 2009

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).

       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
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                                                                                    Dayao and Egloso, 2009

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 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
                                                               A Preliminary Study on the Potential……..77
                                                                                   Dayao and Egloso, 2009

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 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……..78
                                                                                   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……..79
                                                                                    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 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.
                                                                A Preliminary Study on the Potential……..80
                                                                                    Dayao and Egloso, 2009

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……..81
                                                                                 Dayao and Egloso, 2009

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                                                                              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……..89
                                                                             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……..90
                                                                          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……..91
                                                                             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……..92
                                                                           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……..93
                                                                           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
A Preliminary Study on the Potential……..94
                    Dayao and Egloso, 2009

				
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