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					                                                                        Partial Characterization of the Potential




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

                              Chicken Feathers, and Polystyrene




                               An Undergraduate Thesis Study




                                  In Partial Fulfillment of the

                                       Requirements in

                              Biology 200: Undergraduate Thesis

                                        AY 2008-2009



                                    Dayao, Janine Erica P.

                                 Egloso, Mary Bernadette V.




                                      February 20, 2009
                    Partial Characterization of the Potential




 ANNOUNCEMENT



  ENDORSEMENT



BIOGRAPHICAL DATA



ACKNOWLEDGMENT



TABLE OF CONTENTS



  LIST OF TABLES



 LIST OF FIGURES
           Partial Characterization of the Potential



ABSTRACT
                                                                           Partial Characterization of the Potential



                                         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).
                                                                            Partial Characterization of the Potential



       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 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, 118, E26 and E35 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).
                                                                           Partial Characterization of the Potential



        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.



Statement of the problem




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 before and after the experiment under the scanning

         electron microscope (SEM) to check if the pollutants have been biodegraded by the Xylaria

         sp. mutant strains and wildtype
                                                                           Partial Characterization of the Potential



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 be

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 the

National Institute of Molecular Biology and Biotechnology (BioTech) of the University of the

Philippines – Los Baños. The strains that will be used are Xylaria strain mutants PNL 114, 116, 118,

E26 and E35 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 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
                                                                           Partial Characterization of the Potential



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 are not

within the scope of this experiment. The conditions, such as the pH of 5, incubation temperature of

25 ˚C and incubation period of 50 days, of the Xylaria strains that will be used for incubation and

growth are followed after the experiment of Clutario and Cuevas‟ experiment on Xylaria sp.‟s

degradation of polyethylene. Colonization of the substrates‟ surfaces will be 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. This will be done once: after

the incubation period, wherein the positive control and sample per mutant per pollutant substrate

will be scanned. Only the crude weight percent difference as a measurement of Xylaria sp. strains‟

colonization on the wastes will be recorded. The runs will 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. 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‟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 biodegradation potential of Xylaria sp. strains through

its colonization 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.
                                                                             Partial Characterization of the Potential



                             REVIEW OF RELATED LITERATURE



       In 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 and

carelessness. 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, 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
                                                                           Partial Characterization of the Potential



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 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 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).
                                                                           Partial Characterization of the Potential



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 and Sivan, 2008). Yet originally, Rhodococcous 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 &

Steinbuchel, 2005).

       Gordonia sp. and Streptomyces sp. are known rubber-degraders (Rose & 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.
                                                                           Partial Characterization of the Potential



         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 has been isolated

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



Xylaria sp. as a potential agent for biodegradation

         Xylaria sp. 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 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 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).

         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
                                                                            Partial Characterization of the Potential



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 & 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)
                                                                          Partial Characterization of the Potential



       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 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.

       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

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

addition to lignin peroxidase.
                                                                         Partial Characterization of the Potential



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 & 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 & Wheeler, 1986; Tavanlar & 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 & 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,

116,117 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
                                                                         Partial Characterization of the Potential



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 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                       M5
   PNL 114            23.0a              14.8a          17.0a            24.2a                     24.8
     116              22.0a              14.5a          17.0a            24.3a                     23.0
     117              17.5a              12.8b          17.0a            25.3a                     23.8
                                                                           Partial Characterization of the Potential



     118             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% 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.

                     Code                Average diameter of colony (mm)
                                                    Medium
                                      MM             MMP              MMT
                  PNL 114             41.5            36.0            38.5
                     116              45.0            35.0            34.8
                     117              38.5            31.0            31.5
                     118              39.0            36.5            32.2
                    SDM               16.0            16.5            14.1
       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



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

nigripes extract in WulinshenPrime™ in SleepWell™ (a patented fermentation technology available
                                                                           Partial Characterization of the Potential



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

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 &

Rebolledo, 2003).
                                                                             Partial Characterization of the Potential



         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




     a                                              b                                 c
                                                                          Partial Characterization of the Potential



       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

       Many foods items utilize Foamed polystyrene (Styrofoam) packaged products. Styrofoam

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

(Styrofoam trays), there is 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 still exists after the

polystyrene (Styrofoam trays) completes its "degradation process”.


       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 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
                                                                            Partial Characterization of the Potential



thought to be comparable to that of Bisphenol A, another potent estrogen mimic from the world of

plastics (Grinning Planet, 2008). Long-term exposure to small quantities of styrene is also suspected

of causing low platelet counts or hemoglobin values, chromosomal and lymphatic abnormalities, and

neurotoxic effects due to accumulation of styrene in the tissues of the brain, spinal cord, and

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

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

for the leaking out of styrene is EPS food packaging. Styrene leak or leech is triggered once acids

from our juices are placed in such EPS cups and when food with Vitamin A content is placed inside

a microwave leading the styrene to accumulate in our system. (Californians Against Waste, 2008).

       The International Agency for Research on Cancer lists styrene as a possible human

carcinogen, though this conclusion is primarily based on studies of workers in styrene-related

chemical plants. The Vallombrosa Consensus Statement on Environmental Contaminants and

Human Fertility Compromise includes styrene on its list of contaminants of possible concern, noting

that even weak estrogen mimics can combine with other such chemicals to have negative effects

even 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
                                                                           Partial Characterization of the Potential



uncontaminated when recycled. The build-up of polystyrene in landfills, as reported by Californians

Against Waste (2008), will contribute to plastic marine debris, since even when it is disposed of

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

there is an excess of it in the environment and it is a major pollutant (Mor & Sivan, 2008). For

almost three decades ago, polystyrene was first banned due to the utilization of CFC material for its

generation. In fact there was a hype heralding that it is recyclable. After some time the companies

that invested for its recycling process disappeared. This move confirms that, indeed, recycling

polystyrene is not an easy thing to do. Now, the problem is back and the attention of scientists is

focused on the recycling of disposable foamed polystyrene. But recycling it would cost much in

terms of energy, waste and management point of view (Californians Against 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
                                                                           Partial Characterization of the Potential



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

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

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

high affinity towards polystyrene through biofilm formation which lead to its degradation. The C208

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

al., 2004).

        Fluorescent light was used to degrade polystyrene plastic (PS) in a study by Shang, Chai and

Zhu (2003). The study demonstrated that joining polymer plastic and dye-sensitized TiO2 catalyst to

form a thin film is a convenient and functional method to photodegrade plastic contaminants in the

sunlight. Results revealed that higher PS weight loss rate, lower PS average molecular weight, less

amount of volatile organic compounds, and more CO2 can be obtained in the system of PS-

(TiO2/CuPc), as compared to the PS-TiO2 system. Thus, PS photodegradation over TiO2/CuPc

composite is more absolute and efficient than over pure TiO2. This implies the prospective use of

dye-sensitized TiO2 catalyst in the systematic photodegradation of PS plastic under fluorescent light.

        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
                                                                            Partial Characterization of the Potential



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 FTIR

spectra.

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

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

tested, polyurethane and perlite proved to be most suitable for enrichment of styrene-degrading

fungi. 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
                                                                               Partial Characterization of the Potential



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.



               Ultrasonic degradation of polystyrene

       A study (2004) on the thermal and thermo-oxidative degradation of polystyrene and

polystyrene-clay nanocomposites, in which the latter showed a fairly higher glass transition

temperatures, decompose at significantly greater temperatures, and demonstrate a substantial

decrease in the maximum heat release rate on combustion. Introduction of the clay phase increases

the activation energy and affects the total heat of degradation, which suggests a change in the

reaction mechanism. Implanting layered silicates into polymers is known to dramatically modify

various physical properties including thermal stability and fire resistance.

       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 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.

       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.
                                                                         Partial Characterization of the Potential



Further visual confirmation of deterioration of surface area is done by using the scanning electron

microscope (Motta et al., 2007; Mor & Sivan, 2008).



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 &

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 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
                                                                         Partial Characterization of the Potential



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

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
                                                                          Partial Characterization of the Potential



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




                                                           c

       a                            b                                             c

       Figure 6: (a) A rooster will be the source of feathers for the current study, (b) A contour
              feather, one of the major types of feather (Bartels, 2003; Kock, 2006) (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 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).
                                                                              Partial Characterization of the Potential



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-insoluble 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 & Contiero, 2008). Yet this is said to need

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

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

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

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

feather wastes (Tiquia, et al., 2005). During composting, organic materials are mixed to create a

moist, aerobic environment where organic matter decomposition and humification occur at rapid

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

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

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

costs a lot and it contributes air, soil and water contamination (Joshi et al., 2007).
                                                                           Partial Characterization of the Potential



       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. It has also been demonstrated that Aspergillus nidulans, 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

(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.
                                                                            Partial Characterization of the Potential



        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. It‟s 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 considered rare, as cited by Brandelli in the journal of Joshi

et al. (2007).

        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.

        The results of a study by E. H. Burtt and J. M. Ichida (1999) 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.

        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
                                                                            Partial Characterization of the Potential



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 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).



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
                                                                            Partial Characterization of the Potential



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 it‟s 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 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
                                                                            Partial Characterization of the Potential



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. (Prescott et al., 2005; Schweitzer, 2006; Chumbley, 2009)

        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

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)
                                                                         Partial Characterization of the Potential




                                MATERIALS AND METHODS



Research design

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

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

experimentation process will be conducted in UP Los Baños Institute of Biotechnology.



Experimentation



A. Preparation of Inoculum

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

114, 116 and118) strains and two black strains (E25 and E36) were obtained from UPLB Biotech.

Xylaria sp. strains were isolated by culturing it in a Potato Dextrose Agar (PDA) medium. The pH

was then adjusted to pH 5 and it was incubated at 25˚C. After 2-5 days, the fungi were 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 were cut from clean polystyrene food containers such as

               styroplates. The strips were weighed in two‟s. The strips‟ surfaces were sterilized

               using 70% ethanol solution. The weight served as the initial weight. One polystyrene
                                                                 Partial Characterization of the Potential



   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 flask.



B. Chicken feathers

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

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

   washed. And it was 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 autoclaved 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. One strip will underwent

   SEM to check the initial surface condition of the latex glove. Then the strips were be

   wrapped in a foil and autoclaved for 20mins at 15 psi. After which, two strips of the

   gloves were utilized per replicate of each treatment. This was then be placed in a

   flask.
                                                                            Partial Characterization of the Potential




C. 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 and test run thrice. Then, 0.5% glucose was added in all the

flasks. The pH was adjusted to pH 5 by adding small amounts of either 0.1M NaOH or 0.1M HCl.

The inoculation of 2 ml of Xylaria sp. and it‟s mutants in six flasks excluding the control, and the

addition of pollutant (either polystyrene, natural rubber or chicken feather) in all the flasks followed

respectively.

        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 wild types 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. Next, the pollutants were air

dried. Then lastly, the final weight of the pollutants was determined using a balance.



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.
                                                                            Partial Characterization of the Potential




Determination of potential degradation through Scanning Electron Microscopy

       Since in most flasks all the fungi cannot be removed from the pollutant, the data for the

pollutant‟s final weight showed an increase. Due to this, the percent weight loss cannot and was not

utilized as a tool/method for the partial characterization of Xylaria sp. and its mutants‟ potential

biodegrading ability. Instead, a Scanning Electron Microscope, which was applied in the previous

study of Clutario and Cuevas (2001), was used to observe and image the microscopic surface of the

pollutants.

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

which demonstrated a notable colonization on the surface, weight increase or weight decrease, was

chosen.

       After obtaining the micrographs, the Xylaria sp. wild type (SDM) were then individually

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



Hypothesis

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

on the pollutants as observed under a scanning electron microscope

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

on the pollutants as observed under a scanning electron microscope.
                                                                           Partial Characterization of the Potential




                                            RESULTS

Natural Rubber

   Strain                                  General Observations
    SDM             In general, the fungi embedded its mycelia inside the latex gloves.
                 There is a remarkable elasticity reduction especially among the samples
                                        which have high fungi content.
     114         Since this strain is an albino mutant, the adherence of the fungus is not
                  visible. There is not significant loss of elasticity compared to the wild
                                                      type.
     116            No visible signs of colonization. There is no observed reduction of
                                                   elasticity.
     118          Some samples were observed to have a pink discoloration. This strain
                  is supposed to be an albino strain but black mycelia for some samples
                    have been observed to colonize. There is no observed reduction in
                                                   elasticity.
    E26          Colonization of fungus is visible and it is comparable to the wild type.
                     The mycelia embedded on the surface of the sample. Formation/
                   presence of pink discoloration were observed on the surface of some
                                                   samples.
    E35          There is an observed pinkish discoloration in a sample. This is believed
                                      to be a secretion from the fungus.
                  The fungus has embedded its mycelia inside the latex gloves. And the
                      extent of its colonization is comparable to that of the wild type.


Chicken Feathers

       Strain                                      Observations
                                                                 Partial Characterization of the Potential



       SDM    Still sturdy rachis, the barbules are still firmly attached to the
                                           rachis
        114   Still sturdy rachis, the barbules are still firmly attached to the
                                           rachis
        116   Still sturdy rachis, the barbules are still firmly attached to the
                                           rachis
        118   Still sturdy rachis, the barbules are still firmly attached to the
                                           rachis
        E26   Still sturdy rachis, the barbules are still firmly attached to the
                                           rachis
        E35   Still sturdy rachis, the barbules are still firmly attached to the
                                           rachis


Polystyrene
                                                                            Partial Characterization of the Potential



                                           DISCUSSION



       In all runs and set-ups, the fungus was grown at 25oC and at pH 5. It was very difficult to

remove the mycelia in the samples, because they closely adhered into the substrate surface and grew

into it. At the end of the 50-day incubation, it was observed that many of the set-ups were covered

by mycelial growth.



       According to Clutario and Cuevas (2001) in their study, other researchers (Sietsma, et al.,

1981; Whitekettle, 1991; Milstein, et al., 1992) mentioned that active colonizers of polymer were

able to adhere to the substrate‟s surface because they produce exocellular polymers made primarily

of nonionic and anionic polysaccharides. Such adhesion to surfaces of substrates is a decisive step in

microbially induced corrosion. 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, as cited in the

study by Clutario and 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).



Natural rubber



Chicken feather
                                                                               Partial Characterization of the Potential



       Mycelia were difficult to remove from the feathers. Some of the feather samples became

brittle and produced white powdery residues. However, most of the samples maintained their sturdy

rachis and the barbs were still firmly attached as compared to the control. Feather, as it is composed

of keratin, a protein that is very difficult to degrade due to its rigid structural arrangement.

       During the preparation of the chicken feathers, they were not washed with soap, therefore

retaining their preen oil coating. Birds waterproof their feathers through the application of preen oil

to their feathers. Thus, there is limited moisture present. Fungi need water, as a medium for diffusion

of soluble nutrients back into the cells. Without some free water, fungi cannot carry out normal

metabolism (Alexopoulos, 1996). The preen oil inhibits the growth of some bacteria, although it

appears to enhance the growth of other microbes, which may include fungi as well as yeasts

(Bandyopadhyay & Bhattacharyya, 1996; Shawkey, Pillai, & Hill, 2003; Shawkey, et al., 2005).

       It cannot be concluded yet from the results that Xylaria sp. exhibits keratinolytic activity,

which refers to the enzymatic ability to attack and utilize keratin, due to lack of further tests such as

biochemical tests which involve the isolation and purification of enzymes like keratinase and

proteinase from Xylaria. However, relatively little consideration has been given to the distinction

between keratin utilization and simple occurrence on keratinaceous material nourished by

constituents other than keratin. In acknowledgement of this distinction, fungi merely inhabiting

keratinaceous substrates but lacking manifest keratinolytic activity have sometimes been termed

„keratinophilic‟ (Scott & Untereiner, 2004). Most of these keratinophilic fungi belong to families

Arthrodermataceae and Onygenaceae, order Onygenales in Ascomycetes. This ascomycete relative

of Xylaria showed that it is able to degrade keratin through the keratin azure tube assay method,

whereby fungi are grown on an azure-dye impregnated substrate and their degradative abilities

assessed via dye released during the degradation process. According to Scott and Untereiner (2004),
                                                                            Partial Characterization of the Potential



fungi capable of the enzymatic degradation of these polymers are restricted largely to a single

lineage of filamentous fungi, the ascomycete Order Onygenales, which also contains a

disproportionate number of pathogens.

       Another keratinophilic dermatophyte was proven to degrade keratin in a study by Kunert

(1976). The fungus excretes sulfite which cleaves the disulfide bonds of keratin to cysteine and S-

sulfocysteine. The substrate, denatured by sulfitolysis, is then more easily digestible by fungal

proteases.

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

of bacteria and fungi, including yeast (Hubilek, 1994). Despite recent interest in the interactions

between birds and environmental microbes, the identities and ecological roles of bacteria and other

microbes found on the feathers of wild (i.e. aerial and canopy) birds are largely unknown (Shawkey,

et al., 2005). Unfortunately, the influence of these creatures on the birds themselves has received

little attention. In a pioneering paper in this issue of The Auk, E. H. Burtt and J. M. Ichida (1999)

show that plumage microbes could influence birds in significant ways. Their study provided

evidence that many, if not most, species of birds have bacteria in their plumage, and that some of

these bacteria can rapidly degrade feathers, at least under laboratory conditions. Extrapolating from

the data of their study, they predicted that most species of birds will have feather-degrading bacteria

in their plumage. And the metabolic activity and / or antibiotic production of some bacteria may

inhibit or improve the growth of other bacteria and / or fungi present. Thus, the growth of Xylaria

sp. in the current study may be affected (i.e. enhanced or inhibited) by microbial communities

already present in the feathers.

       It may also be suggested, according to Burtt and Ichida (1999) in an article by Dale Clayton

of The Auk, that ground and water birds have more microbes present, which include bacteria, yeast
                                                                          Partial Characterization of the Potential



and fungi, than aerial or canopy birds because their transmission is enhanced near the ground or

around water.

       According to Tiquia, et al. (2004) in their study of chicken feathers during composting, there

is a diversity of microbial communities on these feathers which has not been explored at a broader

phylogenetic range in the past. Moreover, very little is known about the microbial community

structure on feathers at different stages of composting, which may be important in the degradation

process.

       There are various bacteria in these microbial communities, which may or may not produce

common proteolytic enzymes which cannot degrade the ß-keratin sheets that constitute 90% of

feather mass. Thus, to utilize feathers as a nutrient source, bacteria, fungi and other microbes must

produce keratinolytic enzymes that convert feather keratin to peptides (Williams, et al., 1990;

Shawkey, et al., 2005). Such enzymes appear to be produced by fairly diverse groups of bacteria in

the environment (Lucas, Broennimann, Febbraro, & Heeb, 2003), but whether these bacteria are also

present and active on wild bird feather is unknown (Shawkey, et al., 2005). To our knowledge,

however, 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. Also, there is only a very small proportion (<1%) of microbes which can

be cultured by traditional methods (Amann, Ludwig, & Schleifer, 1995; Shawkey, et al., 2005) so

culture-based studies may not provide an adequate sampling of diversity (Shawkey, et al., 2005).

       Gerald Goldstein and colleagues from Ohio Wesleyan University reported from their study

of bird feathers that the microbes degrade white feathers much more readily than black feathers,

which contain melanin pigment. Melanin seems to strengthen feathers and make them more resistant
                                                                          Partial Characterization of the Potential



to degradation. Goldstein reported his findings in the July 2004 issue of the Auk, the journal of the

American Ornithologists' Union.



Polystyrene

   1. Properties of polystyrene that enables it to be degraded
   2. Any studies producing “enzyme” capable of degrading polystyrene
   3.
               Macroscopically, sample degradation is not obvious, but if given a longer time
duration for incubation, 2 – 6 months perhaps, then surely a more extensive degradation will be
observed.



Polystyrene http://www.genpak.com/cfm/polystyrene.cfm

PHYSICAL                      PROPERTIES                       OF                      POLYSTYRENE

http://www.epsmolders.org/PDF_FILES/C578%20Chart.pdf

Standard      for   Thermal       Insulation,   Polystyrene,    Boards     and        Pipe         Covering

http://www.epsmolders.org/4-cval.html

Expanded Polystyrene & R-Values http://www.epsmolders.org/4-rval.html#qa



Polystyrene                        and                     styrene                             copolymers

http://www.efunda.com/materials/polymers/properties/polymer_datasheet.cfm?MajorID=PS&

MinorID=1

Molecular                                   Structures,                                       POLYMERS

http://www.efunda.com/materials/polymers/science/polymer_science_intro.cfm

PROP                      DEPENDENCIES                          OF                            POLYMERS

http://www.efunda.com/materials/polymers/science/polymer_science_prop.cfm
                                                                   Partial Characterization of the Potential



POLYSTY PROPERTIES http://www.closedcellfoams.com/tech/StyroTech.html

POLYSTY PACKAGING COUNCIL OF THE PHIL http://www.ppcp.org.ph/poly.htm#look

NEWS http://www.ppcp.org.ph/index.htm

FAQS http://www.ppcp.org.ph/faq.htm

About http://www.ppcp.org.ph/about.htm



ED LAPIZ


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                                                                            Partial Characterization of the Potential




                                       RECOMMENDATIONS



       To verify the degradation of feather, more advanced test should be conducted wherein the

presence of soluble proteins and amino groups concentration will be observed. Study the possibility

that Xylaria can produce enzymes such as keratinase, proteinase.

       If ever there are enzymes produced, they should be purified and isolated for further studies.

       Testing white feathers also.

       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.

       Increase incubation period especially for chicken feather studies.
Partial Characterization of the Potential
                                                                             Partial Characterization of the Potential



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                                                                         Partial Characterization of the Potential



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The Foam Factory, 2009 thefoamfactory.com



                                            APPENDICES




                                      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
                                                                            Partial Characterization of the Potential



Scanning Electron Microscopy                                 P 25,000

Miscellaneous

       Fares                                                 P5000

       Glasswares                                            P400

       Others                                                P2000

TOTAL:                                                              Php 46,500




Author, A. A., & Author, B. B. (Date of publication). Title of article. Title of Journal, volume
number. doi:0000000/000000000000

Brownlie, D. Toward effective poster presentations: an annotated bibliography. European Journal of
Marketing, 41(11/12), 1245-1283. doi:10.1108/03090560710821161




Kinetics of the Thermal and Thermo-Oxidative

Degradation of a Polystyrene–Clay Nanocomposite

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GLOGER'S RULE, FEATHER-DEGRADING BACTERIA, AND COLOR VARIATION

AMONG SONG SPARROWS

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Starch-based plastic polymer degradation by the white rot fungus Phanerochaete chrysosporium

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ENRICHMENT OF FUNGI AND DEGRADATION OF STYRENE IN BIOFILTERS

Huub H.J. Cox*, Jos~ H.M. Houtman, Hans J. Doddema, and Wim Harder


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Enrichment of fungi and degradation of styrene in biofilters


Journal           Biotechnology Letters

Publisher         Springer Netherlands

ISSN              0141-5492 (Print) 1573-6776 (Online)

Issue             Volume 15, Number 7 / July, 1993

DOI

Pages             737-742

Subject Collection Biomedical and Life Sciences

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The degradation of high impact polystyrene with and without starch in concentrated activated sludge


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