Partial Characterization of the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,

Chicken Feathers, and Polystyrene

An Undergraduate Thesis Proposal

In Partial Fulfillment of the

Requirements in

Biology 200: Undergraduate Thesis

AY 2008-2009

Dayao, Janine Erica P.

Egloso, Mary Bernadette V.

August 15, 2008

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).

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 250C on a mineral

medium of pH5 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 shows that Xylaria sp. has indeed a

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

The Xylaria strain mutants PNL 114, 116,117 and 118 which will be 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).

This proposed study aims 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.

Objectives

The proposed study aims to determine the biodegrading capacity of Xylaria sp. wild type and

its mutants.

The specific objectives are as follows:

1. to determine if Xylaria sp. mutants and wild type can degrade natural rubber as a carbon

source

2. to determine if Xylaria sp. mutants and wild type can degrade chicken feathers as a carbon

and nitrogen source

3. to determine if Xylaria sp. mutants and wild type can degrade polystyrene as a carbon

source

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

most appropriate for each type of waste

5. to examine the treated pollutants under the scanning electron microscope (SEM) to check if

the pollutants have been biodegraded by the Xylaria sp. mutant strains and wildtype

Significance of the Study

Findings of this study might be utilized in the development of Xylaria sp. as a good

biodegrading agent in reducing durable wastes such as plastics and others, as well as in optimizing

fungal technologies. This study may also provide a way or ways in the discovery of 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 concern

about their waste disposal methods, and also to assure a good source of 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 experiment will serve as a source of preliminary information on the potential of Xylaria

sp. strains to degrade chicken feathers, polystyrene and natural rubber. Other pollutants with similar

biochemical structure to the aforementioned pollutants will not be included in the experiment. For

the methodology, the Xylaria sp. that will be used will only come from the stock culture of UP Los

Baños Biotech Institute. The strains that will be used are Xylaria strain mutants PNL 114, 116,117

and 118, which will all be compared to the wildtype SDM (sterile dark mycelia), in terms of their

biodegrading capacity. Culture media and reagents will also be provided by BIOTECH.

Experimentation will be done both in the Microbiology thesis room of the UP Manila, College of

Arts and Sciences and 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 will not be 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 a catalysts for

each pollutant degradation set-up, optimum size of inoculum for each pollutant aren’t within the

scope of this experiment. The conditions for incubation and growth such as the pH of 5, incubation

temperature of 25 ˚C and incubation period of 50 days of the Xylaria strains are followed after the

experiment of Clutario and Cuevas’s experiment on Xylaria’s degradation of polyethylene. The

observation of colonization through scanning electron microscopy (SEM) will be done in

BIOTECH. This will be done twice: before and after the incubation period. Only the crude weight

percent difference of the colonization of the wastes will be recorded. The runs will only be done

thrice and in duplicate per run due to logistic matters and unavailability of equipment. Moreover,

observations of the set-ups will be noted on the 20th, 30th and 50th day of incubation, with 50 days as

the maximum incubation period. On the 20th and 30th day, the observation will only be visual since

removing the pollutants from the flask will likely contaminate the set-up. More so, designing another

set-up for the 20th and 30th day cannot be performed due to the limitation of materials and reagents.

So it’s only on the 50th day that the actual weight loss determination will take place. In terms of data

analysis, this experiment will only focus in analyzing the degradation potential of Xylaria sp. strains

on natural rubber, chicken feathers and polystyrene. Also, it will be concerned on whether the

degradation capacity of the mutant strains is significantly different from the capacity of the wild type

to degrade.

REVIEW OF RELATED LITERATURE

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

of natural resources, thus, various activities was done with negligence and carelessness.

Contaminated lands generally result from past industrial activities when awareness of the health and

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

less well recognized than today. Currently however, the consequences of our previous actions are

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

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

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

(Vidali, 2001).

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

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

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

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

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

material. Additionally, most of them are very difficult and increasingly expensive (Vidali, 2001).

BIODEGRADATION

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

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

biodegradation. Biodegradation is the breakdown of natural substances through the action of

enzymes secreted by organisms such as microbes and fungi. Only waste materials made up of natural

polymers can be degraded by microbes and fungi. Biodegradation works in such a manner that the

organisms involved utilize, or more appropriately, metabolize these wastes as sources of nutrients

such as carbon or nitrogen. (Tortora, et al., 2005). It uses relatively low-cost, low-technology

techniques, which generally have a high public acceptance and can often be carried out on site

(Vidali, 2001). Biodegradation agents like bacteria and fungi must be healthy and active for

biodegradation to be highly efficient. Biodegradation technologies create optimum environmental

conditions to help the growth and increase the number of microbial or fungal populations for them to

detoxify the maximum amount of contaminants (United States Environmental Protection Agency,

1996).

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

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

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

concentration in the environment at which it may cause health risks to nearby inhabitants such as

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

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

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

the site conditions, the quantity and toxicity of contaminant chemicals and the environment factors

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

Different microorganisms degrade different types of compounds and survive under different

conditions (United States Environmental Protection Agency, 1996).

MICROORGANISMS USED IN BIODEGRADATION

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

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

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

the capacity to biodegrade pollutants.

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

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

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

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

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

degrade polystyrene (Mor and Sivan, 2008). Yet originally, Rhodococcus rubber is a known rubber-

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

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

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

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

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

Steinbuchel, 2005)

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

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

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

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

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

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

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

more species that could degrade pollutants. And in fact, in the Philippines a fungus has been isolated

and proven to degrade polyethylene (Cuevas and Manaligod, 1997; Clutario and Cuevas, 2001).

Xylaria sp. AS A POTENTIAL AGENT FOR BIODEGRADATION

Xylaria sp. was discovered by Cuevas and Manaligod (1997) (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 have designated it under Class

Ascomycetes, Order Xylariales, Genus Xylaria (Clutario and 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 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 and Simon, 1992; EcoWaste

Coalition, 2008).

Figure 1. Xylaria in its natural habitat.

Figure 2. Xylaria fruiting body surrounded by flask-shaped structures called peritheca.

Figure 3. Cross section of Xylaria stroma.

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,

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 and Petrini, 1992; Davis, et al, 2003).

Xylariaceae endophytes are hypothesized to be quiescent colonizers that decompose lignin

and cellulose when a plant dies. Nonetheless there are also some xylariaceous fungi that only exist as

endophytes. No obvious benefit to living host plants has been documented for Xylariaceae (Petrini et

al., 1995; Whalley, 1996; Rogers, 2000; Davis, et al., 2003)

A review of empirical studies on antagonistic interactions between endophytes and grazers,

insects and microbial pathogens summarizes five general properties of endophyte mutualism: (1) the

endophyte is ubiquitous in a given host, geographically widespread, and causes minimal disease

symptoms in the host plant; (2) vertical transmission or efficient horizontal transmission of the

fungus occurs; (3) the fungus grows throughout host tissue, or, if confined to a particular organ, a

high proportion of such organs are infected; (4) the fungus produces secondary metabolites likely to

be antibiotic or toxic; and (5) the endophyte is taxonomically related to known herbivore or

pathogen antagonists (Carroll, 1988; 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 & Wen (2005) wherein they discovered that Xylaria sp. along

with other ascomycetes and some basidiomycetes commonly demonstrated laccase activity together

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

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

(2) laccase is apparently the main enzyme for ligninolysis unlike the white-rot basidiomycetes that

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

addition to lignin peroxidase.

THE XYLARIA WILDTYPE AND MUTANT STRAINS

Partial characterization of the fungus Xylaria sp. and its mutants was based on the study by

Tavanlar and Lat (2008) in which the black fungus wildtype SDM was subjected to mutagenesis, and

protoplast fusion was performed. The aforementioned study determined morphological and

biochemical characteristics or markers in the wildtype 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

wildtype SDM that there was a high deposition of melanin. When deposited in the outer layer of the

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

studies on Magnaporthe grisea (Howard et al., 1991; Tavanlar and 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). 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 and Tsuge, 1993; Tavanlar and 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,

116,117 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 wildtype, when viewed under the light microscope.

These albino mutants evidently lost their melanin pigmentation and the mycelia assumed a thinner

appearance than the wildtype dark mycelia. This study utilized NTG in the induction of mutants

from the SDM wildtype. 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 and Tsuge, 1993; Kawamura, et al., 1997; Tavanlar and 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 four mutants which underwent further tests as

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

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

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

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

that the four 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 source of carbon as compared

to the wildtype. These albino mutants may potentially exhibit enhanced degradation of polyethylene

plastics than the wildtype. 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 wildtype.

Table 1. Comparative growth of the PNL mutants and wildtype SDM on MMG and mineral medium

with various supplements.

Code Average diameter of colony (mm)Medium

M1 M2 M3 M4 M5PNL 114 23.0a 14.8a 17.0a 24.2a 24.8

116 22.0a 14.5a 17.0a 24.3a 23.0117 17.5a 12.8b 17.0a 25.3a 23.8118 23.3a 12.8b 17.0a 23.0a 23.8

SDM 5b 5c 5b 12.8b 20.1

Measured after 4 days incubation at ART:M1 = MMG + 0.005% benomylM2 = MM + 0.025% glucose + 1% acetamideM3 = MM + 0.025% glucose + 1% PEGM4 = 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.

Code Average diameter of colony (mm)Medium

MM MMP MMTPNL 114 41.5 36.0 38.5

116 45.0 35.0 34.8117 38.5 31.0 31.5118 39.0 36.5 32.2

SDM 16.0 16.5 14.1

Measured after 3 days incubation at ART:MM = mineral mediumMMP = MM + 1% w/v polyethylene glycol 6000MMT = MM + 1%v/v Tween 80

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

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

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

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

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

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

glutamate decarboxylase (NuLiv Lifestyle, 2008).

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

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

nuclear ribosomal ITS1-5.8SITS2 sequence analysis, and was labeled F0010 strain. The fungus was

endophytic to Abies holophylla, and the study evaluated its in vivo antifungal activity against plant

pathogenic fungi. The antibiotics were determined to be griseofulvin and dechlorogriseofulvin

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

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

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

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

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

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

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

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

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).

Polystyrene

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. One of the most common forms and

uses of polystyrene is the EPS which stands for Expanded Polystyrene. The industry manufactures

such product by mixing polystyrene with blowing agents in the form of carbon dioxide and pentane

which comprises 5%-10% of its composition. The EPS is also called foamed polystyrene and it is

said to be 30 times lighter than regular polystyrene. This substance is popularly used in the form of

beverage cups and insulating materials. (Friend, 2005). The basic unit of polystyrene which is

styrene, which is a known neurotoxin and animal carcinogen, is considered very harmful to human

health. In fact, it inflicts neurological and hematological disorder especially to factory workers. EPS

food packaging is the one accountable for the leaking out of styrene. Styrene leak or leech is

triggered when acids from our juices when placed in such EPS cups and when food with Vitamin A

a b c

content is placed inside a microwave leading the styrene to accumulate in our system. (Californians

Against Waste, 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 and Sivan, 2008). According to the

Californians Against Waste (2008), it is very difficult to recycle due to its light weight property,

which accounts for why it’s expensive to recycle. Imagine just recycling a ton of polystyrene, needs

a budget of $3000. Hence, it has a negative scrap-value. More so, it’s due to this light weight

property that they find polystyrene hard to transport since polystyrene is advised to be always kept

food-free and uncontaminated when recycled. The build-up of polystyrene in landfills, as reported

by CAW (2008), will contribute to plastic marine debris, since even when it is disposed of properly

it is carried by natural agents such as wind or other forces to the ocean. As manifested, there is an

excess of it in the environment and it is a major pollutant. (Mor and Sivan, 2008). For almost three

decades ago, polystyrene was first ban due to the utilization CFC material for its generation. In fact

there was a hype heralding that it is recyclable. After some time the companies that invested for its

recycling process disappeared. This move confirms that, indeed, recycling polystyrene is not an easy

thing to do. Now, the problem is back and the attention of scientists is focused on the recycling of

disposable foamed polystyrene. But recycling it would cost much in terms of energy, waste and

management point of view. (Californians Against Waste, 2008). A way of solving such impending

problem, is through biodegradation (Mor and Sivan, 2008; Singh and Sharma, 2007).

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

will be named here. A study by Mor and Sivan (2008), dealt with the monitoring of biofilm

formation of the microbe Rhodococcus sp. strain C208 on polystyrene. Their aim was to observe the

kinetics of biofilm formation and of whether polystyrene would be degraded. They used two

methods in quantifying the biofilm biomas: modified crystal violet staining and observation of the

protein content of the biofilm. The C208 strain was cultured in a flask containing polystyrene flakes

with the addition of mineral oil (0.0055% w/v), which induced more biofilm build-up. The study

concluded that after an extension of 8th weeks of incubation, loss of 0.8% (gravimetric weight loss)

of polystyrene weight was found. From this, Mor and Sivan (2008) regarded C208 to demonstrate a

high affinity towards polystyrene through biofilm formation which lead to it’s degradation. The

C208 strain is a biofilm-producing actinomycete that has first colonized and degraded polyethylene

(Orr et al., 2004).

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

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

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

white rot basidiomycete was used to degrade such lignin-polystyrene complex copolymer. Such

fungi released enzyme that oxidized lignin and demonstrated the degradation through weight loss,

UV spectrophotometric analysis and deterioration of surface of the plastic substance as seen under

the SEM. A similar study by Singh and Sharma (2007) demonstrated through the process of graft

copolymerization that polystyrene must be modified with natural polymers and hydrophilic

monomers so as to enhance its degrading ability and so as to render polystyrene waste useful in

diminishing metal ion pollution in water. According to the mentioned study, the degrading rate of

polystyrene increased to 37% after subjecting it to soil burial method for 160 days.

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

polystyrene using the fungi Curvularia sp. After about nine weeks of incubation, microscopic

examination revealed that hyphae had grown on the polystyrene. The colonization of the fungi and

it’s adhesion to the surface of the substance, according to Motta, et al. (2007), is a crucial step

towards polymer biodegradation.

As demonstrated, 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. (Mor and Sivan,

2008; Motta et al., 2007).

Natural Rubber

Figure 5: Natural rubber is a polymer called polyisoprene, which can be 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).

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

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

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

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

degradation. And it has been learned that both bacteria and fungi can participate in such process.

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

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

Rose and Steinbuchel (2005), which recapped the aforementioned groups, the microbes that can

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

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

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

actinomycetes species. They are said to biodegrade or metabolize rubber by secreting enzymes and

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

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

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

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

The second group is said to demonstrate a relatively stronger growth on rubber. Species comprising

this category are the Corynebacterium-Nocardia-Mycobacterium group. They consist of the

Gordonia polyisoprenivorans strains VH2 and Y2K, G. westfalica strain Kb1, and Mycobacterium

fortuitum strain NF4.

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

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

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

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

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

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

latex film on the mineral agar plates. Growth and colonization of the microbe in this medium would

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

degrader.

Furthemore, according to 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

Figure 6: (a) A rooster will be the source of feathers for the current study, (b) Major types of feathers: radially symmetric downy feather, bilaterally symmetric contour feather, and bilaterally asymmetric flight feather or remiges (the type chosen for the current study) and (c) male flight feather, a sample of a caudal feather or rectrice

In the Philippines, chicken feathers aren’t a publicly recognized problem. But, experiments

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

Philippines-Los Baños, scientist Menandro Acda has ventured into recycling chicken feather into a

low-cost building material. The scientist quoted that, recycling it would be more advisable than

burning it since the incineration problem could cause environmental hazards. (Morales, 2008).

Moreover, in the US alone, 2 billion pounds of chicken feathers are produced by the poultry industry

(Comis, 2008). Chicken feathers, by nature, are made up of over 90% protein (Cheng-cheng, et al.,

2008). And this protein is none other than keratin. It’s actually the most abundant protein. It is not

easily degraded due to its tightly packed structural arrangement which is in the form of alpha keratin

or beta keratin. The key to its stability lies on the cross-linking by disulfide bonds, hydrophobic

interactions, and hydrogen bonds. Such stability renders keratin water-insolube and non-degradable

by the enzymes papain, trypsin and pepsin. (Gradisar et al., 2005). In a study conducted by Onifade,

et al. (1998) and Goushterova, et al. (2005), as cited in the journal of Cheng-Cheng, et al. (2008),

the build-up of chicken feathers in the environment and landfills would only result to future

pollution problems and protein wastage. More so, its accumulation could serve as a breeding ground

for a variety of harmful pathogens (Singh, 2004).

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

animal feed, but first its protein must be degraded (Tapia and Contiero, 2008). Yet this is said to

need so much water and energy (Frazer, 2004). Old methods of degrading the chicken feathers such

a

b

c

as alkali hydrolysis and steam pressure cooking are no longer advisable. They cause so much energy

wastage and they unfortunately destroy the configuration of proteins. (Cheng-cheng, et al., 2008).

Incineration is also a method used in degrading such waste but it causes so much energy loss

and carbon dioxide build-up in the environment. Other methods of disposal are landfilling, burning,

natural gas production and treatment for animal feed. But subjecting it to burning and land filing

costs a lot and it contributes air, soil and water contamination. (Joshi et al., 2007).

A wiser suggestion or approach would be the use of microbes in degrading these chicken

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

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

already been done. In fact, studies have already proven that keratinolytic microbes such as Bacillus

(Maczinger, et al., 2003; Rodziewicz and Wojciech, 2008; Joshi et al., 2007), fungi (Gradisar, et al.,

2005) and actinomycetes (Goushterova et al., 2005). It has also been demonstrated that Aspergillus

nidulasn, a known imperfect Ascomycete which produces the toxin aflatoxin, shows an outstanding

keratinolytic activity. The enzymes that perform keratin degradation are called keratinase, which

could degrade feathers and make it available for its use as animal feed, fertilizer and natural gas.

The enzymes are said to degrade the beta-keratin component and the main idea behind such

biodegradation is that the microbes use the feather as their carbon, nitrogen, sulfur and energy for

their nourishment. (Joshi et al.,2007 ; Manczinger, et al., 2003). According to study of Cheng-gang

et al. (2008), the keratinase enzyme is inducible whenever substrates of keratin composition are

present. Among the all the keratin-inducing substrates, feathers (made up of beta-keratin) are the

ones commonly utilized. Yet both alpha-keratin and beta-keratin substrates can be used in feather

degradation. It is reported that the mechanism behind the degradation of chicken feather is yet to be

elucidated. But according to Kunert (2000) in the study of Cheng-gang et al. (2008), the proposed

primary step in keratinolysis is the deamination which produces an alkaline environment. Such

environment is needed to induce substrate swelling, sulphitolysis and proteolytic attack. In the same

study of Cheng-gang et al. (2008), the degradation of feathers produced amino acid residues such as

threonine, valine, methionine, isoleucine, phenylalanine and lysine. It was elucidated that this could

be due to the high disulfide content of the feathers.

Keratinases isolated from microbes have various economic uses. Aside from its feather

degrading capacity, it could be used in the leather industry as an agent in dehairing leather. Its by-

product, the feather hydrolysate, could also be used as animal feed additive. (Joshi, et al., 2007).

Furthermore, potentially, the said hydolysate could be used in the generation of organic fertilizer,

edible films and amino acids which are considered rare, as cited by Brandelli in the journal of Joshi

et al. (2007).

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 10 th day of incubation, through colony

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

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

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

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

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

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

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

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

identified as Bacillus lichenformis strain K-508. And the confirmation of the keratinolytic activity

was done by using the azokeratin as a substrate assay.

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

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

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

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

additives (Tapia and Contiero, 2008).

METHODOLOGY

I. Research Design

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

(RCBD). The experiment will consist of two trials/runs with three replicates per treatment. The

experimentation process will be conducted in UP Los Baños Institute of Biotechnology and at the

Microbiology thesis room of the CAS, UP-Manila.

II. Experimentation

A. Preparation of Inoculum

The stock culture of Xylaria sp. and its four albino mutant strains will be obtained from

UPLB Biotech. Xylaria sp. will be isolated by culturing it in a Potato Dextrose Agar (PDA) medium.

The pH will be adjusted to pH 5 and it will be incubated at 25˚C. After 2-5 days, the fungi will be

transferred into a mineral medium with 5% glucose flask and subjected to a shaker for enrichment

and sustenance to further growth.

B. Preparation of Pollutants

A. Polystyrene

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

styroplates. The strips will be weighed in two’s. The strips will be surface sterilized

using 70% ethanol solution. It will then be oven dried. The weight will serve as the

initial weight. One polystyrene representative will undergo SEM to visually see the

initial status of the strips before colonization. Afterwhich, two strips per replicate of

each treatment will be placed in a flask.

B. Chicken feathers

Fresh contour feathers from an adult, male Gallus sp. will be obtained from a

nearby market place where chickens are butchered and sold. The feathers will be

washed. And it will be cut from their tips to 3 cm in length. Each cut feather will be

weighed and placed in a foil. The weight obtained will serve as the initial weight. One

representative of the feather will be obtained and will undergo SEM to check the

initial surface of the feathers. The feathers will then be autoclaved for 20 minutes at

15psi. One 3 cm feather will be used per replicate of each treatment and it will be

placed in a flask.

C. Rubber

Obtain rubber latex gloves size 5. Cut the gloves into strips of the same sizes,

approximate area to be about 2x2 cm. Weigh the gloves by two’s. The weight will

serve as the initial weight. One strip will undergo SEM to check the initial surface

condition of the latex glove. Then the strips will be autoclaved for 20mins at 15 psi.

Afterwhich 2 strips of the gloves will be utilized per replicate of each treatment. This

will then be placed in a flask.

C. Biodegradation Proper using Culture Method

Two sets of flasks will be prepared containing 15 ml Mineral Medium each, in duplicate.

0.5% glucose will be added in set A (which consists of only 1 set-up per pollutant per strain) and B.

The pH will be adjusted to pH 5 by adding small amounts of either 0.1M NaOH or 0.1M HCl. Then

2 ml of the microorganism which came from the mineral medium flask will be added. The addition

of pollutant in set B only will follow right after. The flasks will be subjected in a shaker for 4 hours

to homogenize the medium. The culture will be observed for some time then when all the glucose

has been used up and the fungi have grown into a considerable mass as examined visually, another

MM + 0.5% glucose will be added in set A only, leaving the set B flasks to utilize the solid

pollutants as the sole carbon source. The extent of colonization will be carefully examined every day

until rate of colony growth can be predicted (growth in mm/day). But if otherwise, the addition of

the MMG (mineral medium+0.5%glucose) to both sets of flasks will be continued until the fungi

have grown and thrived. Incubate for 50 days, with the flasks in a room with more or less 250C in

temperature. This is to observe if changes have already occurred even as early as 20 days or 30 days,

but the maximum incubation is 50 days which was applied in the previous studies (Clutario and

Cuevas, 2001; Tavanlar and Lat, 2008). The solid pollutants will then be removed from the culture

medium and examined under a scanning electron microscope (SEM). The pollutants will undergo

SEM before and after colonization for a better comparison of changes, if any, in their surface

structures.

Note:

* This step is intended for each mutant and for the wild type. Since we have 4 mutant strains and a

wild type, this step will be repeated five times multiplied with the number of pollutants to be used.

D. Determination of Amount of Degradation through Colonization

Incubation periods and set-up observations are done after 20, 30 and 50 days. After each

incubation period, the remaining pollutant will be weighed in grams. This measurement will be

recorded as the final weight. The percent weight loss of pollutant will be determined using the

formula:

% weight loss = (initial weight – final weight)

Initial weight

Statistical Analysis

The analysis that will be used for this study is the ANOVA for Random Complete Block

Design (RCBD). The blocks will be the Xylaria sp. and the mutants while the treatment will be the

three pollutants namely natural rubber, chicken feather and polystyrene.

Hypothesis

(Hypotheses for the blocksF for rows)

Ho1: There is no significant difference in the biodegrading ability of the different Xylaria sp. strains

on the pollutants.

Ha1: There is a significant difference in the biodegrading ability of the different Xylaria sp. strains

on the pollutants.

(Hypotheses for the pollutantsF for columns)

Ho2: There is no significant difference in the degree of biodegradation of polystyrene, natural rubber

& chicken feathers due to Xylaria sp.

Ha2: There is a significant difference in the degree of biodegradation of polystyrene, natural rubber

& chicken feathers due to Xylaria sp.

Dummy Tables

Table 1. Percent Weight Loss in two runs of the (insert pollutant name here) due to Xylaria sp.

strains

Replicate

1

Replicate 2 Replicate 3 Replicate

4

Replicate

5

Replicate 6 Mean

Wild

type

Mutant 1

Mutant 2

Mutant 3

Mutant 4

Note:

*replicate 1-2 = run 1

**replicate 3-4 = run 2

*** replicate 5-6 = run 3

***Table 1 will be used for each pollutant hence it will be repeated thrice for each of the three

incubation periods.

Table 2. Mean Values of the Percent Weight Loss of Polystyrene, Natural Rubber and Chicken

Feathers due to degradation of Xylaria sp. strains

Xylaria strain Polystyrene Natural Rubber Chicken Feathers

Wild-type

Mutant1

Mutant2

Mutant3

Mutant4

Table 3. ANOVA for Randomized Complete Block Design of the Potential Biodegrading Ability of

Xylaria sp. Strains on Chicken Feathers, Natural Rubber and Polystyrene

Source of Variation SS df MS F P-value F crit

Rows (Xylaria strain)

Columns (pollutant)

Error

α = 0.05

In the event that the F value for rows or columns shows any significant difference, a post

hoc analysis would be conducted. For the F value for rows, which are the pollutants, the Multiple

Analysis test will be used and it will be followed by the Tukey’s test. While for the F value for

columns, the Dunnet’s test will be applied.

BUDGET OUTLINE

Materials

Chicken feathers P300

Styroplates P30

Foil, tissue, cotton P200

Reagents

Potato Dextrose agar P1000

Mineral medium P500

0.5% Glucose P1200

70% Ethanol solution P800

Distilled water P500

Thesis Proposal

Printing P2000

Photocopied materials P1500

Materials such as Bond Papers etc. P2000

Scanning Electron Microscopy P 25,000

Miscellaneous

Fares P5000

Glasswares P400

Others P2000

TOTAL: Php 46,500

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