Partial Characterization of the Potential Biodegrading Ability of Xylaria sp. 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 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’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. The country’s population growth rate, which is one of the highest in the world (Mangahas, 2006), 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 as cited by Mangahas, 2006). Urbanization, decline in the economy and further population growth lead to the even higher generation of wastes that has not been managed properly and safely (DENR, 2004). Presidential Decree (PD) 1152, or “the Philippine Environmental Code,” provides the basis for an integrated waste management regulation starting from waste source to methods of disposal. PD1152 has further mandated specific guidelines to manage municipal wastes (solid and liquid), sanitary landfills and incineration, and disposal sites in the Philippines. Apart from the basic policies of PD1152, waste management must also comply with the requirements and implementing regulations of other specific environmental laws, such as PD984 (Pollution Control Law), PD1586 (Environmental Impact Assessment System Law), RA8749 (Clean Air Act) and RA9003 (Ecological Solid Waste Management Act) (DENR, 2004). A study by Clutario and Cuevas (2001) showed that Xylaria sp. can utilize polyethylene plastic strips as an alternative carbon source. The fungus grew optimally at 250C on a mineral medium of pH5 containing 0.5% glucose and polyethylene plastic strips as co-carbon source. A mucilaginous sheath was produced by the fungus to help its mycelial growth adhere to the surfaces and edges of the plastic strips. After 50 days of incubation, the strips became embedded in the mycelial growth. Visible damage on the surface structure of the plastic strips was observed using scanning electron microscopy (SEM). Striations and tearing were present due to the active burrowing of Xylaria hyphae on the polyethylene material. This shows that Xylaria sp. has indeed a potential in degrading synthetic wastes like plastics which are difficult to decompose. This proposed study aims to test the potential use of Xylaria sp. and its mutants as a natural biodegrading agent in biodegrading other rampant wastes such as natural rubber, polystyrene and chicken feathers. Objectives The proposed study aims to determine the biodegrading capacity of Xylaria sp. wild type and its mutants. The specific objectives are as follows: 1. to determine if Xylaria sp. mutants and wild type can assimilate natural rubber as a carbon source 2. to determine if Xylaria sp. mutants and wild type can assimilate chicken feathers as a carbon and nitrogen source 3. to determine if Xylaria sp. mutants and wild type can assimilate 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 Significance of the Study Findings of this study might be utilized in the development of Xylaria sp. as a good biodegrading agent in reducing durable wastes such as plastics and others, as well as in optimizing fungal technologies. This study may also provide a way or ways in the discovery of other important characteristics of Xylaria sp. and its mutants, which may be used in other applications and scientific investigations. The discovery of other sources of biodegradation agents and their potential bioactive natural products is of paramount importance, especially nowadays that people should mostly concern about their waste disposal methods, and also to assure a good source of more accessible ways, through research, in approaching the reduction of pollution that are safe and can possibly boost the Philippine fungal industry in the world market. Scope and Limitations The experiment will serve as a source of preliminary information on the potential of Xylaria sp. to degrade chicken feathers, polystyrene and natural rubber. Other pollutants with similar biochemical structure to the aforementioned pollutants will not be included in the experiment. For the methodology, the Xylaria sp. that will be used will only come from the stock culture of UP Los Baños Biotech Institute. Culture media and reagents will be provided by BIOTECH. Experimentation will be done both in the Microbiology thesis room of the UP Manila, College of Arts and Sciences and in the Antibiotic Laboratory in BIOTECH. Isolation and purification of active components (i.e. enzymes) responsible for the probable degradation of chicken feathers, polystyrene and natural rubber will not be performed. Observation of colonization through scanning electron microscopy (SEM) will be carried out in BIOTECH. Only the crude weight percent difference of the colonization of the wastes will be recorded. The runs will only be done twice due to logistic matters and unavailability of equipments. Moreover, the incubation time for the degradation process of Xylaria sp. strains will only be limited to 30 days. In terms of data analysis, this experiment will only focus in analyzing the degradation potential of Xylaria sp. strains on Natural rubber, Chicken Feathers and Polystyrene. And also, it will be concerned whether the degradation potential of the Mutants is significantly different from the Wild type and so as to determine the more appropriate strain to use. REVIEW OF RELATED LITERATURE 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). 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 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 is one. Plastics in the form of polyethylene are known to be degraded by the thermophilic bacterium Brevibaccillus borstelensis 707 which was isolated from soil. (Hadad et al,2005). Another study by Orr et al. (2004) featured the Rhodococcous ruber C208 as an effective polyethylene-degrading organism. In addition to this, this strain has been proven to degrade polystyrene ( Mor & 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 AGENT FOR BIODEGRADATION Xylaria sp. was discovered by Cuevas and Manaligod (1997), growing on a sando plastic bag, buried in forest soil and litter in the lowland secondary forest of Mt. Makiling, Laguna. The fungus comprised of sterile melanin pigmented mycelia and was reported as ascomycete sterile dark mycelia (ASDM). Cultural studies have designated it under Class Ascomycetes, Order Xylariales, Genus Xylaria (Clutario and Cuevas, 2001). A previous study by Clutario and Cuevas (2001) proved that Xylaria sp. can utilize polyethylene plastic strips as an alternative carbon source, thereby degrading them into usable forms for self-sustenance. Through the use of scanning electron microscopy, the proponents of the said study observed visible damages of the surface structure of the plastic strips. There were tearing and striations caused by active burrowing of Xylaria hyphae on the polyethylene material. Plastic is an extremely versatile synthetic material made of high molecular weight, semi-crystalline polymer prepared from ethylene through the cracking of crude oil, light petroleum and natural gas. For plastic bags alone, it is estimated that some 430,000 gallons of oil are needed to produce 100 million pieces of these omnipresent consumer items on the planet (Knapczyk and Simon, 1992; EcoWaste Coalition, 2008). Figure 1. Xylaria in its natural habitat. Figure 2. Xylaria fruiting body surrounded by flask-shaped structures called peritheca. Figure 3. Cross section of Xylaria stroma. Xylaria is one of the most commonly encountered groups of ascomycetes with most of its members being stromatic, peritheciate, with an iodine-positive ascus apical ring, and with one-celled, dark ascospores on which agermination 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. Xylariaceae endophytes are hypothesized to be quiescent colonizers that decompose lignin and cellulose when a plant dies. Nonetheless there are also some xylariaceous fungi that only exist as endophytes. No obvious benefit to living host plants has been documented for Xylariaceae (Petrini et al., 1995; Whalley, 1996; Rogers, 2000; Davis, et al., 2003) A review of empirical studies on antagonistic interactions between endophytes and grazers, insects and microbial pathogens summarizes five general properties of endophyte mutualism: (1) the endophyte is ubiquitous in a given host, geographically widespread, and causes minimal disease symptoms in the host plant; (2) vertical transmission or efficient horizontal transmission of the fungus occurs; (3) the fungus grows throughout host tissue, or, if confined to a particular organ, a high proportion of such organs are infected; (4) the fungus produces secondary metabolites likely to be antibiotic or toxic; and (5) the endophyte is taxonomically related to known herbivore or pathogen antagonists (Carroll, 1988; Davis, et al., 2003). SOLID WASTE IN THE PHILIPPINES Filipinos generate around 0.3 to 0.7 kilograms of garbage daily per person depending on income levels (World Bank, 2001). Metro Manila produces about 8,000 tons of solid waste each day and is expected to reach 13,300 tons each day in 2014 (Baroña, 2004). The National Capital Region produces the highest amount of wastes, about 23% of the country’s waste generation (Anden and Rebolledo, 2003). Based on studies (2001) made by the National Solid Waste Management Commission Secretariat based at the Environmental Management Bureau (EMB), it is estimated that in Metro Manila, the per capita waste production daily is 0.5 kg. Thus, every person living in the metropolis generates half a kilo of waste a day. With an estimated population of 10.5 million, total waste generated in Metro Manila alone could run up to 5,250 metric tons per day or 162,750 metric tons per month or 1.95 million metric tons per year. Based on another EMB study (2001) regarding the disposal of daily wastes, only about 73% of the 5,250 metric tons of waste generated daily are collected by dump trucks hired by local government units. The remaining 27% of daily wastes, or about 1,417.5 metric tons, end up in canals, vacant spaces, street corners, market places, rivers and other places. According to a survey conducted by the EcoWaste Coalition and Greenpeace Southeast Asia in 2006, synthetic plastics comprise 76% of the floating trash in Manila Bay, out of which 51% are plastic bags, 19% are sachets and junk food wrappers, 5% are styrofoams and 1% is hard plastics. The rest were rubber (10%) and biodegradable discards (13%) (EcoWaste Coalition, 2008). Polystyrene 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. Chicken Feathers 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 10 th day of incubation, through colony formation of the microbe indicated that it utilized the feather as a source of its nutrients. After which, its keratinolytic activity was tested using a modified keratin azure protocol. Another study by Maczinger et al. (2003) focused on the isolation of a microbe from the poultry waste that could degrade feathers. During the preliminary elimination, they cultured the different population of bacteria found in a partially degraded feather in a basal medium with sterilized feathers serving as its source of carbon, nitrogen and sulfur. It was then rotated in an orbital shaker for 10 days. After 4 days, one flask which showed a visual degradation of the feather. A dilution series was made afterwards so as to isolate and culture the bacteria that just degraded the feather. The strain was identified as Bacillus lichenformis strain K-508. And the confirmation of the keratinolytic activity was done by using the azokeratin as a substrate assay. Isolation of a new microbial organism that could degrade chicken feather will help in the degradation of the chicken feathers which is now becoming a burden in the society both internationally and locally. The microbe could potentially provide the keratinase that could be used in compost technology (Maczinger et al., 2003) or in the conversion of feather to feedstock meal additives (Tapia and Contiero, 2008). METHODOLOGY I. Research Design The research design to be used in the study is the Randomized Complete Block Design (RCBD). The experiment will consist of two trials with three replicates per treatment. The experimentation process will be conducted in UP Los Banos Institute of Biotechnology. II. Experimentation A. Preparation of Inoculum The stock culture of Xylaria sp. and its four albino mutant strains will be obtained from UPLB Biotech. Xylaria sp. will be isolated by culturing it in a Potato Dextrose Agar (PDA) medium. The pH will be adjusted to pH 5 and it will be incubated at 25˚C. After 2-3 days, the fungi will be transferred into the test media. B. Preparation of Pollutants A. Polystyrene 1x1 cm strips will be cut from clean polystyrene food containers such as coffee foam cups. And four strips per replicate of each treatment will be used. B. Chicken feathers Fresh feathers from Gallus sp. will be obtained from a nearby market place where chickens are butchered and sold. The feathers will be sterilized by placing it inside a SM plastic bag and autoclaved for 20 minutes at 15psi. One feather per replicate of treatment will be used. C. Rubber Obtain rubber latex gloves size 5 from Mercury Drugstore. Cut the gloves into strips of the same sizes, approximate area to be about 2x2. Use 2 strips per replicate of each treatment. C. Biodegradation Proper using Culture Method Two sets of flasks will be prepared containing 50 ml Mineral Medium each, in triplicate. 0.5% glucose will be added in set A and B. The pH will be adjusted to pH 5 by adding small amounts of either 0.1M NaOH or 0.1M HCl. Then the microorganism will be added using the wire loop inoculation method. The addition of pollutant in set B only will follow right after. The flasks will be subjected in a shaker for four hours to homogenize the medium. The culture will be observed for some time then when all the glucose has been used up and the fungi have grown into a considerable mass as examined visually, another MM + 0.5% glucose will be added in set A only, leaving the set B flasks to utilize the solid pollutants as the sole carbon source. The extent of colonization will be carefully examined every day until rate of colony growth can be predicted (growth in mm/day). But if otherwise, the addition the MMG (mineral medium+0.5%glucose) to both sets of flasks will be continued until the fungi have grown and thrived. Incubate for30 days, with the flasks in a room with more or less 250C in temperature.The solid pollutants will then be removed from the culture medium and examined 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. D. Determination of Amount of Degradation through Colonization After 30 days, the remaining pollutant will be weighed in grams. This measurement will be recorded as the final weight. The percent weight loss of pollutant will be determined using the formula: % weight loss = (initial weight – final weight) Initial weight Statistical Analysis The designed that will be used for this study is Random Complete Block Design (RCBD). The blocks will be the Xylaria sp. and the mutants while the treatment will be the three pollutants namely natural rubber, chicken feather and polystyrene. Your study design is RCBD so your analysis should therefore be ANOVA for RCBD (Which you can easily analyze using Microsoft excel….and no need to put the software to be used in your proposal). Then you should put an ANOVA table without data after your dummy tables. Xylaria sp and its mutants will serve as the blocks since you will be randomly distributing the pollutants to them. Your DATA/DUMMY table #2 (since okay na yung first dummy table ninyo na kasama yung replicates) will then look like this (please write an appropriate title above it): Xylaria Polystyrene Natural Chicken strain Rubber Feathers Wild- type Mutant1 Mutant2 Mutant3 Mutant4 Hypothesis (Hypotheses for the blocksF for rows) Ho1: There is no significant difference in the biodegrading ability of the different Xylaria sp. strains on the pollutants. Ha1: There is a significant difference in the biodegrading ability of the different Xylaria sp. strains on the pollutants. (Hypotheses for the pollutantsF for columns) Ho2: There is no significant difference in the degree of biodegradation of polystyrene, natural rubber & chicken feathers due to Xylaria sp. Ha2: There is a significant difference in the degree of biodegradation of polystyrene, natural rubber & chicken feathers due to Xylaria sp. Dummy Tables **Table 1 will be use for each pollutant and such table would be used for the two runs that will be conducted. Table 1. Percent Weight Loss of the (insert pollutant name here) due to Xylaria sp. strains Replicate 1 Replicate 2 Replicate 3 Mean Wild type Mutant 1 Mutant 2 Mutant 3 Mutant 4 Table 2. Mean Values of the Percent Weight Loss of Polystyrene, Natural Rubber and Chicken Feathers due to degradation of Xylaria sp. strains Xylaria strain Polystyrene Natural Chicken Rubber Feathers Wild-type Mutant1 Mutant2 Mutant3 Mutant4 Table 3. ANOVA for Randomized Complete Block Design of the Potential Biodegrading Ability of Xylaria sp. Strains on Chicken Feathers, Natural Rubber and Polystyrene Source of Variation SS df MS F P-value F crit Rows (Xylaria strain) Columns (pollutant) Error α = 0.05 LITERATURE CITED Anden, C. M. N. and Rebolledo, A. B. J. (2003) ASrIA Reports - SRI In Asian Emerging Markets: Philippines.www.asria.org/publications/lib/country/philippines_appendix.pdf [retrieved 2008, June 13] Baroña, M. L. J., 2004. Fungi: A Solution to Manila's Plastic Woes. Department of Agriculture. Bureau of Agricultural Research. Vol. 5 No. 8 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. and 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. and Xiao-dong, Z. (2008). Keratinase Production and Keratin Degradation by a Mutant Strain of Bacillus subtilis. Journal of Zhejiang University Science Vol. 9(1): 60–67. Clutario, M. T. P. and Cuevas, V. C. (2001). Colonization of Plastic by Xylaria sp. Philippine Journal of Science 130 (2): 89 – 95. Davis, E. C., Franklin, J. B., Shaw, A. J. and Vilgalys, R. (2003). Endophytic Xylaria (Xylariaceae) Among Liverworts and Angiosperms: Phylogenetics, Distribution, and Symbiosis. American Journal of Botany 90(11): 1661–1667. DENR. 2004. Hazardous Waste Management Procedural Manual Title III of DAO 92-29 http://www.env.go.jp/en/recycle/asian_net/reports/secondyearwork/philippines.pdf [retrieved 2008 June 21]. Environmental Management Bureau-DENR. Managing our Solid Waste: An Overview of the Ecological Solid Waste Management Act. http://www.emb.gov.ph/eeid/ESWM.htm [retrieved 2008 June 21] Duane, Friend (2005). The Pros and Cons of Styrofoam. Land and Water Conserving Natural Resources in Illinois no.5 Environmental Biotechnology: Bioremediation, Prevention, Detection and Monitoring, Genetic Engineering. 1999. Briefing Paper 4. 2nd ed. EFB Task Group on Public Perceptions of Biotechnology Environmental_biotechnology_English.pdf [retrieved 2008, June 4]. Fast Food Waste Threatens our Marine Environment, Drags Down Diversion Rates. Californians Against Waste Organization. [retrieved: 2008, July 1]. http://www.cawrecycles.org/issues/fast_food Gradišar, H.; Friedrich, J.;Križaj, I.; and Jerala, R. (2005). Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to Some Known Proteases. Application of Environmental Biology Vol 71(7): 3420-3422 Gushterova, A.; Vasileva-Tonkova, E.; Dimova, E.; Nedkov, P. and Thomas Haertlé (2005). Keratinase Production by Newly Isolated Antarctic Actinomycete Strains. World Journal of Microbiology and Biotechnology Vol. 21 (6-7):831-834 Joshi, S.; Tejashwini, M.; Revati, N.; Sridevi, R. and Roma, D. (2007). Isolation, Identification and Characterization of a Feather Degrading Bacterium. International Journal of Poultry Science 6 (9): 689-693 Linos, A.; Berekaa, M.; Reichelt, R.; Keller, U.; Schmitt, J.; Flemming, H.; Kroppenstedt, R. and Steinbüchel, A. (2000). Biodegradation of cis-1,4-Polyisoprene Rubbers by Distinct Actinomycetes: Microbial Strategies and Detailed Surface Analysis. Applied Environmental Microbiology 66(4):1639-1645 Mangahas, J. Y. (2006). Urbanization and Sustainability in Asia: Case Studies of Good Practice: The Philippines. http://www.adb.org/Documents/Books/Urbanization- Sustainability/summary-booklet.pdf [retrieved 2008, June 21]. Manczinger L.; Rozs, M.; Va´gvo¨ lgyi, Cs.; and Kevei, F. (2003). Isolation and Characterization of a New Keratinolytic Bacillus licheniformis strain. World Journal of Microbiology and Biotechnology Vol. 19: 35–39 Milstein, O.; Gersonde, R.; Huttermann, A.; Chen, M. & Meister, J. (1992). Fungal Biodegradation of Lignopolystyrene Graft Copolymers. Applied Environmental Microbiology Vol 58(10):3225-3232 Mor, A. and Sivan, A. (2008). Biofilm Formation and Partial Biodegradation of Polystyrene by the Actinomycete Rhodococcus rubber :Biodegradation of Polystyrene. Biodegradation Journal Motta, O.; Proto, A.; De Carlo, F.; De Caro, F.; Santoro, E.; Brunetti, L. and Capunzo, M. (2007). Utilization of Chemically Oxidized Polystyrene as Co-Substrate by Filamentous Fungi. International Journal of Hygiene and Environmental Health Orr, I.; Hadar, Y. and Sivan, A. (2004). Colonization, biofilm formation and biodegradation of polyethylene by a strain of Rhodococcus rubber. Applied Microbial Technology Vol. 65(1):97-104 Polystyrene and Food Packging Waste (2008). Californians Against WasteOrganization.[retrieved:2008,July1].http://www.cawrecycles.org/issues/polysty rene_main Rodziewicz, A. and Wojciech, A.( 2008). Biodegradation of Feather Keratin by Bacillus cereus in Pure Cultue and Compost. Electronic Journal of Polish Agricultural Universities Vol. 11(2): 20. Rogers, J. D., Ju, Y. M., and Adams, M. J. (2002). The Genus Xylaria. http://mycology.sinica.edu.tw/Xylariaceae/information.asp?qrySectionName=Xylaria &qryIDString=x040. [retrieved: 2008,July 1]. Rose, K. and Steinbüchel, A. (2005). Biodegradation of Natural Rubber and Related Compounds: Recent Insights into a Hardly Understood Catabolic Capability of Microorganisms. Applied and Environmental Microbiology Vol. 71(6):2803-2812. Singh, C. (2004). Exocellular Proteases of Malbranchea gypsea and their Role in Keratin Deterioration. Mycopathologia Vol. 143(3):147-150. Singh, B. and Sharma, N. (2007). Optimized Synthesis and Characterization of Polystyrene Graft Copolymers and Preliminary Assessment of Their Biodegradability and Application In Water Pollution Alleviation Technologies. Polymer Degradation and Stability Vol. 92(5):876-885. Tapia, D. and Contiero, J. (2008). Production and Partial Characterization of Keratinase Produced by a Microorganism Isolated from Poultry Processing Plant Wastewater. African Journal of Biotechnology Vol. 7 (3): 296-300. The Myth of Polystyrene Takeout (2008). Californians Against Waste Organization. http://www.cawrecycles.org/issues/eps_recycling.html [retrieved: 2008,July 1]. The Story of Polystyrene Bans. Californians Against Waste Organization. http://www.cawrecycles.org/issues/epsban_summary [retrieved: 2008, July 1]. Tortora, G. J., Funke, B. R., and Case, C. L. (2005) Microbiology: An Introduction. Pearson Education South Asia Pte. Ltd. 23-25 First Lok Yang Road, Jurong, Singapore 629733. 8th ed. pp. 772 – 774. Tavanlar, M. A. T. and Lat, E. C. (2008). Partial Characterization of Mutants from a Plastic- degrading Black Fungus.
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