"Thesis Proposal Draft1"
Partial Characteriztion of the Potential Biodegrading Ability of the Fungi Xylaria sp. and its Four Mutants, [mutant 1, mutant 2, mutant 3, mutant 4] on Natural Rubber, Chicken Feathers, and Polystyrene A Thesis Proposal In Partial Fulfillment of the Requirements For Biology 200: Undergraduate Thesis AY 2008-2009 Dayao, Janine Erica P. Egloso, Mary Bernadette V. August 1, 2008 INTRODUCTION Background of the Study Pollution nowadays seems to be inevitable due to the ever growing population, urbanization and the increased demands for manufactured products in the local and export markets. Industrialization has resulted to the generation of more and more industrial wastes such as solid, liquid and other hazardous wastes that may pose serious risks to the environment and public health, thus, requiring an efficient waste regulatory management. The Philippine economy has grown over the years leading to even higher production of wastes that unfortunately have not been managed properly and safely (DENR, 2004). One of the laws, the Presidential Decree (PD) 1152, or “the Philippine Environmental Code,” provides a basis for an integrated waste management regulation starting from waste source to methods of disposal. PD 1152 has further mandated specific guidelines to manage municipal wastes (solid and liquid), sanitary landfill and incineration, and disposal sites in the Philippines (DENR, 2004). Apart from the basic policy rules and regulations of PD 1152, waste management must also comply with the requirements of other specific environmental laws, such as PD 984 (Pollution Control Law), PD 1586 (Environmental Impact Assessment System Law), RA 8749 (Clean Air Act) and RA 9003 (Ecological Solid Waste Management Act) and their implementing rules and regulations (DENR, 2004). In a study by Clutario and Cuevas (2001), evidences showed that Xylaria sp. can utilize polyethylene plastic strips as an alternative carbon source. The fungus grew best at 250C and at pH5. Growth at these optimum conditions was vigorous on mineral medium with 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 damages of the surface structure of the plastic strips were observed using scanning electron microscopy. Striations and tearings were present due to the active burrowing of Xylaria hyphae on the polyethylene material. This study aims to test the capacity of Xylaria sp. and its mutants in biodegrading other rampant wastes such as natural rubber, polystyrene and chicken feathers. The Rhodococcus sp. C208 strain is a biofilm-producing actinomycete that has first colonized and degraded polyethylene (Orr et al., 2004). A study of this strain by Mor and Sivan (2008) further showed that it is also capable of degrading polystyrene through biofilm formation. Another fungus, Curvularia sp., has been shown (Mota et al., 2007) to be capable of degradation of oxidized polystyrene. The colonization of the fungi and it’s adhesion to the surface of the substance, according to Mota et al., is a crucial step towards polymer biodegradation. A review by Rose and Steinbechul (2005) indicate that both bacteria and fungi can both participate in the degradation of natural rubber, which has been long studied due to its high rate of yearly manufacture. Studies have already proven that keratinolytic microbes such as Bacillus (Maczinger et al., 2003; Rodziewicz and Wojciech, 2007; Joshi et al.,2007) , other fungi ( Gradisar et al., 2005) and actinomycetes (Gousterova et al., 2005) are indeed capable of degrading chicken feathers. Objectives The 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/consume/ assimilate natural rubber as a carbon source 2. to determine if Xylaria sp. mutants and wild type can degrade/consume/ assimilate chicken feathers as a carbon and nitrogen source 3. to determine if Xylaria sp. mutants and wild type can degrade/consume/ assimilate polystyrene as a carbon source 4. to compare the biodegrading ability of the wild type to each mutant Hypothesis 1. H0: Xylaria sp. mutants and wild type have no significant biodegrading capacity in assimilating natural rubber as a carbon source. H1: Xylaria sp. mutants and wild type have significant biodegrading capacity in assimilating natural rubber as a carbon source. 2. H0: Xylaria sp. mutants and wild type have no significant biodegrading capacity in assimilating chicken feathers as a nitrogen and carbon source. H1: Xylaria sp. mutants and wild type have significant biodegrading capacity in assimilating chicken feathers as a nitrogen and carbon source. 3. H0: Xylaria sp. mutants and wild type have no significant biodegrading capacity in assimilating polystyrene as a carbon source. H1: Xylaria sp. mutants and wild type have significant biodegrading capacity in assimilating polystyrene as a carbon source. 4. H0: There is no significant difference between the biodegrading capacity of Xylaria sp. mutants and its wild type in assimilating natural rubber as a carbon source. H1: There is a significant difference between the biodegrading capacity of Xylaria sp. mutants and its wild type in assimilating natural rubber as a carbon source. 5. H0: There is no significant difference between the biodegrading capacity of Xylaria sp. mutants and its wild type in assimilating chicken feathers as a nitrogen and carbon source. H1: There is a significant difference between the biodegrading capacity of Xylaria sp. mutants and its wild type in assimilating chicken feathers as a nitrogen and carbon source. 6. H0: There is no significant difference between the biodegrading capacity of Xylaria sp. mutants and its wild type in assimilating polystyrene as a carbon source. H1: There is a significant difference between the biodegrading capacity of Xylaria sp. mutants and its wild type in assimilating polystyrene as a carbon source. Significance of the Study Findings of this study might be utilized in the development of Xylaria sp. as a good biodegradation 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 would serve as a preliminary information source for the potential degrading capacity of the Xylaria sp. four mutants and wild type. The pollutants that would be degraded would only be limited to chicken feather, polystyrene and natural rubber. Other pollutants with similar biochemical structure to the aforementioned pollutants would not be covered. Furthermore, the incubation time for the degradation process of the Xylaria sp. would only be limited to 50 days due to time constraints. REVIEW OF RELATED LITERATURE At the very advent of technology, pollution has indeed taken its toll on nature, making people harvest and manufacture products that would take eons to decay and rot at the very least. Everywhere, a plethora of biodegradable and non biodegradable wastes can be seen. And, indeed it’s high time that people revert back to natural processes that could help solve the burgeoning problem of waste disposal since all the artificial methods that require today’s technology could contribute to the pollution that the planet’s experiencing now. One such natural process that could solve the problem, or in a way even just alleviate such waste pile-up, is biodegradation. BIODEGRADATION Biodegradation is the breakdown of natural substances through the action of enzymes secreted by organisms such as microbes and fungi. Only waste materials made up of natural polymers can be degraded by microbes and fungi. Biodegradation works in such a manner that the organisms involved utilize, or more appropriately, metabolize these wastes as sources of nutrients such as carbon or nitrogen. (Tortora et al., 2005). In many cases, conditions are not favorable enough to promote spontaneous biodegradation or natural attenuation. Whenever there is insufficient quantity of naturally biodegrading organisms, there is a further need to add nutrients or other suitable organisms aside from those present already, for improved biodegradation to occur. The general objective of biodegradation is to discern the speed of unaided biodegradation before catalysts may even be added, and then strengthen spontaneous biodegradation only if this is not fast enough to remove the contaminant’s concentration in the environment at which it may cause health risks to nearby inhabitants such as people, animals and plants (European Federation of Biotechnology, 1999). Some of the Microorganisms used in Biodegradation Through time, scientific experiments have already proven the ability of some microorganisms to biodegrade pollutants such polyethylene, polystyrene, rubber, chicken feathers and the list goes on. Plastics in the form of polyethylene are known to be degraded by a thermophilic bacteria Brevibaccillus borstelensis strain 707. This bacterium has been isolated in the soil. ( Hadad et al.,2005). Another study by Orr et al. (2004) featured the Rhodococcous ruber strain C208 as an effective polyethylene-degrading organism. In addition to this, this strain has been proven to degrade polystyrene ( Mor & Sivan, 20080. Yet originally, Rhodococcus rubber is a known rubber-degrading organism, according to the review of Rose and Steinbuchel (2005). Other known rubber-degrading microorganisms are Gordonia sp., Streptomyces sp., and Xanthomonas sp. strain 357. Moreover, a study of Barraat et al. (2003) investigated the relationship of the water-holding capacity of the soil and polyurethane degradation and found out that the following organisms could biodegrade polyurethane: Nectria gliocladioides (five strains), Penicillium ochrochloron (one strain) and Geomyces pannorum (seven strains). On chicken feathers, various strains had already been isolated too. 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. Another organism proven to degrade chicken feather is Bacillus subtilis mutant strain as investigated by the study of Cheng chang et al. (2003). 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 & Manaligod, 1997). Xylaria sp. AS A POTENTIAL BIODEGRADATION AGENT A Xylaria sp. was discovered by Cuevas and Manaligod (1997), and will be used in this study. It was observed growing on a sando plastic bag, buried in forest soil and litter in the lowland secondary forest of Mt. Makiling, Laguna. The fungus comprised of sterile melanin pigmented mycelia and was reported as ascomycete sterile dark mycelia (ASDM). Cultural studies have designated it in Class Ascomycete, Order Xylariales, under the genus Xylaria (unpublished data). Xylaria is one of the most commonly encountered groups of ascomycetes with most of its members being stromatic, peritheciate, with an iodine-positive ascus apical ring, and with one-celled, dark ascospores on which a germination slit can be found. Most are inhabitants of wood, seeds, fruits, or leaves of angiosperms. Some are associated with insect nests. Most decay wood and many are plant pathogens. Many are endophytes. They are commonly found throughout the temperate and tropical regions of the world. The Xylaria sp. can be distributed above, around, and beneath perithecia. It forms a unipartite stromatal layer, with a superficial or erumpent surface level. The interior of its stromata is essentially homogeneous. Conidium-bearing discs, potassium hydroxide pigments and orange granules surrounding the perithecia are absent (Rogers et al., 2002). They are mostly multiperitheciate in ascomatal number per stroma, ascomatal ostioles and ascal apical rings: are present, and the ascospore cell number is one-celled. Teleomorph and anamorph are produced on the same stromata in most species, with their anamorphs: Geniculosporium-like. Some Xylaria sp. species exist as endophytes, and have mutualistic associations with plants. The fungus secrete toxins to protect the plant from herbivory from other insects or animals, while the fungus in return feeds on the host’s tissues for nutrition, and its mycelia are scattered through seed dispersal (Davis, et al., 2003). There is a hypothesis that Xylariaceae endophytes are quiescent colonizers that will decompose lignin and cellulose later when the plant dies (adapted from Petrini et al., 1995; Whalley, 1996 as cited by Davis, et al., 2003). Nonetheless there are also some xylariaceous fungi that only exist as endophytes (adapted from Rogers, 2000; J. D. Rogers, Washington State University, personal communication as cited by Davis, et al., 2003). No obvious benefit to living host plants has been documented for Xylariaceae (Davis, et al., 2003). A review (Carroll, 1988 as cited by Davis, et al., 2003) of empirical studies on antagonistic interactions between endophytes and grazers, insects and microbial pathogens summarizes five general properties of endophyte mutualism: (1) the endophyte is ubiquitous in a given host, geographically widespread, and causes minimal disease symptoms in the host plant; (2) vertical transmission or efficient horizontal transmission of the fungus occurs; (3) the fungus grows throughout host tissue, or, if confined to a particular organ, a high proportion of such organs are infected; (4) the fungus produces secondary metabolites likely to be antibiotic or toxic; and (5) the endophyte is taxonomically related to known herbivore or pathogen antagonists. A patented extract from Xylaria nigripes, the WulinshenPrime™ in SleepWell™ can provide important nutrients usually at a low level, to the brain and thus help in its biochemical processes to promote a more restful and deeper sleep to wake up fully revitalized. This extract contains essential amino acids, vitamins, minerals, trace elements, glycoproteins, glutamic acid, γ-aminobutyric acid (GABA) and glutamate decarboxylase. It is well established that glutamic acid assists the uptake of GABA to specific brain cell receptors. GABA's main function is to inhibit excitatory neuro-activities to exert a tranquilizing effect on the central nerve system. Glutamate decarboxylase (GAD) is involved in the synthesis of GABA. COLONIZATION OF PLASTIC BY XYLARIA SP. A previous study by Clutario and Cuevas (2001) proved that Xylaria sp. can utilize polyethylene plastic strips as an alternative carbon source, thereby degrading them into usable forms for self-sustenance. Through the use of scanning electron microscopy, the proponents of the said study observed visible damages of the surface structure of the plastic strips. There were tearing and striations caused by active burrowing of Xylaria hyphae on the polyethylene material. Plastic is an extremely versatile synthetic material made of high molecular weight, semi-crystalline polymer prepared from ethylene through the cracking of crude oil, light petroleum and natural gas (Knapczyk and Simon, 1992 as cited by Clutario and Cuevas, 2001). For plastic bags alone, it is estimated that some 430,000 gallons of oil are needed to produce 100 million pieces of these omnipresent consumer items on the planet (EcoWaste Coalition, 2008). Solid Waste Generation IN THE PHILIPPINES Filipinos generate around 0.3 to 0.7 kilograms of garbage daily per person depending on income levels, according to a study by the World Bank (2001). Metro Manila produces about 8,000 tons of solid waste each day and is expected to reach 13,300 tons each day in 2014 (Baroña, 2004). The National Capital Region produces the highest amount of wastes, about 23% of the country’s waste generation (ASRIA, [(www.asria.org)- According to a survey conducted by the EcoWaste Coalition and Greenpeace Southeast Asia in 2006, synthetic plastics comprise 76% of the floating trash in Manila Bay, out of which 51% are plastic bags, 19% are sachets and junk food wrappers, 5% are styrofoams and 1% is hard plastics. The rest were rubber (10%) and biodegradable discards (13%) (EcoWaste Coalition, 2008). For this study, the potential degradation of three pollutants that has been a topic of investigations lately and for the last decades as well will be tested. These pollutants are chicken feather, natural rubber and polystyrene. Chicken Feather 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 & 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). 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). The enzymes that perform keratin degradation are called keratinase, which could degrade feathers and make it available for its use as animal feed, fertilizer and natural gas. The enzymes are said to degrade the beta-keratin component and the main idea behind such biodegradation is that the microbes use the feather as their carbon, nitrogen, sulfur and energy for their nourishment. (Joshi et al.,2007 ; Manczinger et al., 2003). Keratinases isolated from microbes have various economic uses. Aside from its feather degrading capacity, it could be used in the leather industry as an agent in dehairing leather. Its by-product, the feather hydrolysate, could also be used as animal feed additive. (Joshi et al., 2007). Furthermore, potentially, the said hydolysate could be used in the generation of organic fertilizer, edible films and amino acids which are considered rare, as cited by Brandelli in the journal of Joshi et al.( 2007). In terms of experimental procedures, various methods are used in determining the keratinolytic ability, which means it could produce keratinase and hence degrade chicken feather, of microbes. A particular study by Tapia and Contiero (2008), used a feather meal agar, wherein the feather served as the source of carbon, nitrogen, sulfur and energy, in cultivating the isolated microbe Streptomyces. The growth, which occurred on the 10th day of incubation, through colony formation of the microbe indicated that it utilized the feather as a source of its nutrients. After which, its keratinolytic activity was tested using a modified keratin azure protocol. Another study by Maczinger et al. (2003) focused on the isolation of a microbe from the poultry waste that could degrade feathers. During the preliminary elimination, they cultured the different population of bacteria found in a partially degraded feather in a basal medium with sterilized feathers serving as its source of carbon, nitrogen and sulfur. It was then rotated in an orbital shaker for 10 days. After 4 days, one flask which showed a visual degradation of the feather. A dilution series was made afterwards so as to isolate and culture the bacteria that just degraded the feather. The strain was identified as Bacillus lichenformis strain K-508. And the confirmation of the keratinolytic activity was done by using the azokeratin as a substrate assay. Isolation of a new microbial organism that could degrade chicken feather will help in the degradation of the chicken feathers which is now becoming a burden in the society both internationally and locally. The microbe could potentially provide the keratinase that could be used in compost technology (Maczinger et al., 2003) or in the conversion of feather to feedstock meal additives (Tapia and Contiero, 2008). Polystyrene Polystyrene, an aromatic polymer, is synthesized from the aromatic monomer styrene which comes from petroleum products. It is a thermoplastic substance that could be solid in room temperature or liquid when melted. One of the most common forms and uses of polystyrene is the EPS which stands for Expanded Polystyrene. The industry manufactures such product by mixing polystyrene with blowing agents in the form of carbon dioxide and pentane which comprises 5%-10% of its composition.The EPS is also called foamed polystyrene and it is said to be 30 times lighter than regular polystyrene. This substance is popularly used in the form of beverage cups and insulating materials. (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 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 buil-up. The study concluded that after an extension of 8th weeks of incubation, loss of 0.8% (gravimetric weight loss) of polystyrene weight was found. From this, Mor and Sivan (2008) regarded C208 to demonstrate a high affinity towards polystyrene through biofilm formation which lead to it’s degradation. The C208 strain is a biofilm-producing actinomycete that has first colonized and degraded polyethylene (Orr et al., 2004). There were studies that tested the possibility of whether copolymerizing polystyrene with other substance could make it more degradable and susceptible to microbial attack. In 1992, a study by Milstein at el. (1992), focused on the biodegradation of a lignin- polystyrene copolymer. The white rot basidiomycete was used to degrade such lignin- polystyrene complex copolymer. Such fungi released enzyme that oxidized lignin and demonstrated the degradation through weight loss, UV spectrophotometric analysis and deterioration of surface of the plastic substance as seen under the SEM. A similar study by Singh and Sharma (2007) demonstrated through the process of graft copolymerization that polystyrene must be modified with natural polymers and hydrophilic monomers so as to enhance its degrading ability and so as to render polystyrene waste useful in diminishing metal ion pollution in water and. According to the mentioned study, the degrading rate of polystyrene increased to 37% after subjecting it to soil burial method for 160 days. Furthermore, the study of 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 Mota et al., is a crucial step towards polymer biodegradation. As demonstrated, colonization is needed in determining whether a particular microbe or organism is a potential biodegrading agent. (Mota et al., 2007) The growth of the microbes on the surface of the polystyrene is a step that would lead to its degradation. Further visual confirmation of deterioration of surface area is done by using the scanning electron microscope. (Mor and Sivan, 2008; Mota et al., 2007 ). Natural rubber Natural rubber (NR) is made from the latex of the Hevea brasiliensis also known as the rubber tree. It is mainly composed of cis-1,4 polyisoprene which has a molecular mass of about 106 Da. This could also be chemically synthesized and produce the substance known as Isoprene Rubber (IR). (Linos et. al, 2000). Since 1914, natural rubber has been a classic subject of biodegradation studies. (Rose and Steinbuchel, 2005). This is due to the high rate of its yearly manufacture which is several million tons, as mentioned in the study of Bereeka (2006), and its slow rate of natural degradation as reviewed by Rose and Steinbechul (2005). In fact, a number of studies abound concerning its degradation. And it has been learned that both bacteria and fungi can participate in such process. Throughout all the investigations and experimentations done, two categories of rubber-degrading microbes according mainly on growth characteristics have been established. Based on a review of Rose and Steinbuchel (2005), which recapped the aforementioned groups, the microbes that can degrade rubber can be categorized as clear zone-forming around their colonies and non-halo forming whenever isolated and cultured in latex overlay plate, which is made by overlaying a layer of latex agar medium on a basal salt medium agar. The former category was identified to mainly consist of actinomycetes species. They are said to biodegrade or metabolize rubber by secreting enzymes and other substances and also they are dubbed to be slow degraders since they rarely show an abundant cell mass when grown on natural rubber directly. On the other hand, members of the second group do not form halos on latex overlay plates. They, unlike the first group, grow more when directly grown on natural rubber. In a way, their growth on rubber could be described in an adhesive manner. The second group is said to demonstrate a relatively stronger growth on rubber. Species comprising this category are the Corynebacterium-Nocardia-Mycobacterium group. They consist of the Gordonia polyisoprenivorans strains VH2 and Y2K, G. westfalica strain Kb1, and Mycobacterium fortuitum strain NF4. As demonstrated by a particular study by Linos et al. (2000), the mechanisms that the microbes undergo when biodegrading is colonization, biofilm formation and aldehyde group formation after cultivating it on the surface of latex gloves. Such is revealed after undergoing the Schiff reagent’s test. This is further examined under a scanning electron microscope. In their methodology they have indicated that the preliminary screening method to be used in finding potential rubber-degrading bacteria is by growing such bacteria or microbe on the latex overlay or by latex film on the mineral agar plates. Growth and colonization of the microbe in this medium would indicate its utilization of rubber as its sole carbon source; hence, making it as a potential rubber-degrader. METHODOLOGY Research Design The research design to be used in the study is the Randomized Block Design (RBD). The experiment will consist of three trials containing three replicates per treatment, per trial. Collection of Materials I. Preparation of Inoculum Isolate the Xylaria sp. by culturing it in a Potato Dextrose Agar (PDA) medium. Adjust to pH 5 and incubate at 25˚C. After 2-3 days, transfer the fungi into test media. Preparation of Pollutants A. Polystyrene Cut 1x1 cm strips from polystyrene food containers B. Chicken feather Obtain fresh feathers from Gallus gallus sp. Sterilize by putting inside SM plastic bag and autoclave for 20 mins. at 15psi. C. Rubber - Obtain rubber latex gloves Preparation of Test Media* D. For plates: Prepare 2 sets of plates containing 20 ml PDA in triplicate (6 plates per mutant/wild type, per pollutant tested). Add 0.5% glucose in set A and add 0.5% of the liquid pollutant in set B. Set A is to verify if the fungi inoculated to both set-ups are active. Adjust to pH 5 by adding small amounts of either 0.1M NaOH or 0.1M HCl. Inoculate the fungi using agar discs from the PDA plate described in I. Agar discs will be obtained by cutting a 2-3 day-old inoculum on the peripheral area of a colony using a 5-8 mm diameter cork borer. The agar discs will be transferred to the PDA plates of sets A and B by using a sterile toothpick. Incubate at 25˚C. Observe the colony growth in set B and compare it always with set A. Set A will be the control group and it would indicate whether the fungi transferred is active. Then measure the colony growth by its diameter. E. For flasks and test tubes: Prepare 2 sets of flasks containing 50 ml Mineral Medium each, in triplicate. Add 0.5% glucose in set A and B. Adjust to pH 5 by adding small amounts of either 0.1M NaOH or 0.1M HCl. Then add the solid pollutant in set B only. Inoculate the fungi by using ………….. When all the glucose has been used up and the fungi had grown into a considerable mass as examined visually, add another MM + 0.5% glucose in set A only, leaving the set B flasks to utilize the solid pollutants as the sole carbon source. The extent of colonization should be carefully examined every day until rate of colony growth can be predicted (growth in mm/day). But if otherwise, continue adding the MMG (mineral medium+0.5%glucose) to both sets of flask until the fungi has grown and thrived. Incubate for 50-80days, with the flasks in a room with more or less 250C in temperature, under shaking conditions, while the test tubes in an incubator with 250C in temperature as well. II. Remove solid pollutants from the culture medium and examine under a scanning electron microscope (SEM). 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. Determination of Amount of Degradation through Colonization Percent weight loss of pollutant Statistical Analysis The data that will be obtained from the percent weight loss of the pollutants: natural rubber, chicken feathers and polystyrene, due to their colonization by Xylaria sp. and its mutants will be subjected to Duncan’s Multiple Range Test (DMRT). RESULTS AND DISCUSSIONS Dummy Tables *Table would be use for each pollutant and such table would be used for three runs Replicate 1 Replicate 2 Replicate 3 Mean Wild type Mutant 1 Mutant 2 Mutant 3 Mutant 4 *table for the Duncan’s Test Analysis NATURAL CHICKEN FEATHERS RUBBER % weight loss (mg) POLYSTYRENE Treatment % weight loss (mg) % weight loss (mg) Wildtype Mutant 1 Mutant 2 Mutant 3 Mutant 4 RECOMMENDATION LITERATURE CITED Hazardous Waste Management. 2004.Procedural Manual Title III of DAO 92-29 www.denr.gov.ph [retrieved 2008 June 21]. Bereeka, M. (2006). Colonization and microbial degradation of polyisoprene rubber by nocardioform actinomycete Nocardia sp. strain-MBR. Biotechnology 5(3): 234- 239 Cao, L.; Tan, H.; Liu, Y.; Xue, X. & Zhou S. (2008). Characterization of a new keratinolytic Trichoderma atroviride strain F6 that completely degrades native chicken feather. Letters in Applied Microbiology 46 (3), 389–394 Cheng-gang, C.; Bing-gan, L. & Xiao-dong, Z. (2008). 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