Partial Characterization of the Potential -2- I suggest you change “partial characterization.” A Study on the Potential Biodegrading Ability… The Use of SEM in the Determination of the Potential… And the like… Partial Characterization of the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber, Chicken Feathers, and Polystyrene An Undergraduate Thesis 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 -3- ABSTRACT The potential biodegrading ability of the Xylaria sp. strains on three pollutants, namely, natural rubber, chicken feather and polystyrene were determined in terms of colonization. The fungal strains namely the wild type SDM (sterile dark mycelia) and the five mutants strains (PNL 114, PNL 116, PNL 118, E26 and E35) were cultured in a pH 5.0 mineral medium flask with 0.5%, as carbon source, and the three pollutant samples, as an alternative carbon source, and stored in a 25 degrees Celsius room temperature. The initial weights of the pollutants were measured before inoculation. After 50 days of incubation period, the pollutants were removed from the medium and weights were again measured to determine percent weight loss, a method that would measure utilization of the pollutant samples. But instead of constant weight loss, weight gains which could be attributed to the adherence and embedding of mycelia of the Xylaria sp. strains on the three pollutants were also observed. The results obtained in the determination of percent weight loss were inconsistent; hence, Scanning Electron Microscopy was used to observe the surface of the pollutants and confirm whether the biodegradation happened. Micrographs and physical observations SEM results have shown that the SDM strain could potentially biodegrade the three pollutants, E35 black mutant strain could potentially biodegrade chicken feathers only and E26 could potentially biodegrade polystyrene and chicken feathers. PNL 114, 116 and 118 also demonstrated biodegrading ability in polystyrene and rubber. There was no significant PNL biodegradation in chicken feathers. Partial Characterization of the Potential -4- 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). Partial Characterization of the Potential -5- 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 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, 117 and 118 which will be used in the current study, exhibited the following characteristics: loss of melanin pigmentation, ability to utilize polyethylene glycol (PEG), Tween 80, acetamide, and resistant to some fungicides which contained copper hydroxide and benomyl, according to the study by Tavanlar and Lat (2008). Partial Characterization of the Potential -6- 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 The Xylaria sp. strains could degrade the pollutants natural rubber, chicken feather and polystyrene, as an alternative energy source, in terms of colonization to determine its potential biodegrading ability. To determine the potential biodegrading ability of… using scanning electron microscopy. Objectives The proposed study aims to determine the biodegrading capacity of Xylaria sp. wild type and Maybe these three objectivies can its mutants. be combined. You may add at the end of the statement “using Scanning The specific objectives are as follows: Electron Microscopy…” 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 Partial Characterization of the Potential -7- What is the difference between 1-3 and 5 objectives? 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 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 possible means to discover/understand (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 concerned 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. Please revise and check your tenses. / Significance 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 (please give examples). 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 Partial Characterization of the Potential -8- 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 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 Partial Characterization of the Potential -9- 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. 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). Partial Characterization of the Potential - 10 - Biodegradation A better approach than these traditional methods is to completely destroy the pollutants if possible, or at least to transform them to innocuous substances in a process called bioremediation or biodegradation. Biodegradation is the breakdown of natural substances through the action of enzymes secreted by organisms such as microbes and fungi. Only waste materials made up of natural polymers can be degraded by microbes and fungi. Biodegradation works in such a manner that the organisms involved utilize, or more appropriately, metabolize these wastes as sources of nutrients such as carbon or nitrogen. (Tortora, et al., 2005). It uses relatively low-cost, low- technology techniques, which generally have a high public acceptance and can often be carried out on site (Vidali, 2001). Biodegradation agents like bacteria and fungi must be healthy and active for biodegradation to be highly efficient. Biodegradation technologies create optimum environmental conditions to help the growth and increase the number of microbial or fungal populations for them to detoxify the maximum amount of contaminants (United States Environmental Protection Agency, 1996). The general objective of biodegradation is to discern the speed (i.e. percent weight loss of pollutant per week) of unaided biodegradation before catalysts may even be added, and then strengthen spontaneous biodegradation only if this is not fast enough to remove the contaminant’s concentration in the environment 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). Partial Characterization of the Potential - 11 - Different microorganisms degrade different types of compounds and survive under different conditions (United States Environmental Protection Agency, 1996). Microorganisms Used in Biodegradation Through time, scientific experiments have already proven the ability of some microorganisms to biodegrade pollutants such as polyethylene, polystyrene, rubber, chicken feathers and other types of wastes. Organisms such as bacteria and fungi have proven themselves to possess the capacity to biodegrade pollutants. Bacteria such as Brevibaccillus borstelensis, Rhodococcous rubber C208, Xanthomonas sp. strain 357 have been proven to degrade pollutants. Plastics in the form of polyethylene are known to be degraded by the thermophilic bacterium Brevibaccillus borstelensis 707 which was isolated from soil (Hadad et al, 2005). Another study by Orr et al. (2004) featured the Rhodococcous 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 and Steinbuchel, 2005). Gordonia sp. and Streptomyces sp. are known rubber-degraders (Rose and Steinbuchel, 2005). Nectria gliocladioides (five strains), Penicillium ochrochloron (one strain) and Geomyces pannorum (seven strains), in a study of Barraat et al. (2003), have been observed to degrade Partial Characterization of the Potential - 12 - polyurethane while simultaneously relating it to the water holding capacity of the soil. Moreover, in a study conducted by Cao et al. (2008), the fungus Trichoderma atroviride completely degraded the chicken feathers. This strain was actually isolated from a decaying feather. The list of microorganisms that could be used in biodegradation goes on for there are still more species that could degrade pollutants. And in fact, in the Philippines a fungus has been isolated and proven to degrade polyethylene (Cuevas and Manaligod, 1997; Clutario and Cuevas, 2001). Xylaria sp. as a Potential Agent for Biodegradation Xylaria sp. was discovered by Cuevas and Manaligod (1997), 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 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 Partial Characterization of the Potential - 13 - of these omnipresent consumer items on the planet (Knapczyk and 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 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. Partial Characterization of the Potential - 14 - Endophytic Xylariaceae have been documented in conifers, monocots, dicots, ferns, and lycopsids (Brunner and Petrini, 1992; Davis, et al, 2003). Xylariaceae endophytes are hypothesized to be quiescent colonizers that decompose lignin and cellulose when a plant dies. Nonetheless there are also some xylariaceous fungi that only exist as endophytes. No obvious benefit to living host plants has been documented for Xylariaceae (Petrini et al., 1995; Whalley, 1996; Rogers, 2000; Davis, et al., 2003) A review of empirical studies on antagonistic interactions between endophytes and grazers, insects and microbial pathogens summarizes five general properties of endophyte mutualism: (1) the endophyte is ubiquitous in a given host, geographically widespread, and causes minimal disease symptoms in the host plant; (2) vertical transmission or efficient horizontal transmission of the fungus occurs; (3) the fungus grows throughout host tissue, or, if confined to a particular organ, a high proportion of such organs are infected; (4) the fungus produces secondary metabolites likely to be antibiotic or toxic; and (5) the endophyte is taxonomically related to known herbivore or pathogen antagonists (Carroll, 1988; Davis, et al., 2003). According to 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 & Wen (2005) wherein they discovered that Xylaria sp. along Partial Characterization of the Potential - 15 - with other ascomycetes and some basidiomycetes commonly demonstrated laccase activity together with cellulolytic and xylanolytic activities. The enzymatic profiles of the aforementioned species suggests that (1) ascomycetes is potentially capable of utilizing the lignocellulosic wood components (2) laccase is apparently the main enzyme for ligninolysis unlike the white-rot basidiomycetes that utilizes its ligninolytic peroxidase in the form of manganese peroxidase or lignin peroxidase in addition to lignin peroxidase. The Xylaria Wildtype and Mutant Strains Partial characterization of the fungus Xylaria sp. and its mutants was based on the study by Tavanlar and Lat (2008) in which the black fungus wildtype SDM was subjected to mutagenesis, and protoplast fusion was performed. The aforementioned study determined morphological and biochemical characteristics or markers in the wildtype and mutants that can be used in the analysis of future recombinants or fusants. The reputed mutants were described based on colony characteristics, morphology and growth on various media. It was highly apparent from the very dark (black) color of mycelium and hyphae of the wild type SDM that there was a high deposition of melanin. When deposited in the outer layer of the cell wall, melanin reduces the pore diameter below 1nm but remains permeable to water, based on studies on Magnaporthe grisea (Howard et al., 1991; Tavanlar and Lat, 2008). Melanin acts in the survival and longevity of propagules (i.e. part of a plant or fungus such as a bud or a spore that becomes detached from the rest and forms a new organism) (Bell and Wheeler, 1986; Tavanlar and Lat, 2008). This polymer of phenolic compounds provides tolerance to various environmental stresses like oxidants, microbial lysis, UV radiation, and defense responses of host plants and animals against fungal infection (Kimura and Tsuge, 1993; Tavanlar and Lat, 2008). Partial Characterization of the Potential - 16 - In the study by Tavanlar and Lat (2008), after mutants were repeatedly tested on MMG (mineral medium plus 0.5% glucose) plus various supplements, Xylaria strain mutants PNL 114, 116,117 and 118 were chosen based on the retained white color of the colonies even after 7 days. The hyphae of these mutants were similar to the wildtype, when viewed under the light microscope. These albino mutants evidently lost their melanin pigmentation and the mycelia assumed a thinner appearance than the wildtype dark mycelia. This study utilized NTG in the induction of mutants from the SDM wildtype. Exposure to NTG (N’,N”-methyl-N-nitro-N-nitrosoguanidin) induced melanin-deficient mutants in Alternaria alternate, M. grisea, Colletotrichum lagenarium and C. lindemuthianum The phenotypic mutations showed albino, rosy, light brown, and brown colony color (Kimura and Tsuge, 1993; Kawamura, et al., 1997; Tavanlar and Lat, 2008). Defective genes involved in the very common DHN pathway to melanin biosynthesis have been identified in some of the mutants of these fungi. Table 1 shows the four mutants which underwent further tests as presented. The study further tested the four amelanotic mutants selected in various media supplemented with benomyl, acetamide, PEG 6000 (polyethylene glycol), Tween 80, and glucose. Table 2 shows the growth of the four albino mutants on mineral medium with and without supplements as compared to the wildtype SDM. In summary, the results of the said study showed that the four mutants are less dependent on the glucose level in the medium for growth and hyphal tip extension. The mutants showed loss of melanin pigmentation and improved ability to grow on reduced glucose levels, tolerate 0.1% w/v copper hydroxide and 0.005% benomyl, utilize 1% w/v polyethylene glycol 6000, 1% v/v Tween 80 and 1% w/v acetamide as source of carbon as compared to the wildtype. These albino mutants may potentially exhibit enhanced degradation of polyethylene plastics than the wildtype. Also, the proponents have speculated that the albino mutants can better Partial Characterization of the Potential - 17 - survive environments with less available amounts of readily utilizable carbon sources such as the surface of plastics than the wild type. Table 1. Comparative growth of the PNL mutants and wildtype SDM on MMG and mineral medium with various supplements. 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 23.8 118 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. Partial Characterization of the Potential - 18 - 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 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, Partial Characterization of the Potential - 19 - 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? Revise the title… 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). Partial Characterization of the Potential - 20 - 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 - 21 - 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 Partial Characterization of the Potential - 22 - functions, possibly contributing to thyroid problems, menstrual irregularities, and other hormone- related problems, as well as breast cancer and prostate cancer. The estrogenicity of styrene is thought to be comparable to that of Bisphenol A, another potent estrogen mimic from the world of plastics (Grinning Planet, 2008). Long-term exposure to small quantities of styrene is also suspected of causing low platelet counts or hemoglobin values, chromosomal and lymphatic abnormalities, and neurotoxic effects due to accumulation of styrene in the tissues of the brain, spinal cord, and peripheral nerves, resulting in fatigue, nervousness, 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 Partial Characterization of the Potential - 23 - $3000. Hence, it has a negative scrap-value. More so, it is due to this light weight property that they find polystyrene hard to transport since polystyrene is advised to be always kept food-free and uncontaminated when recycled. The build-up of polystyrene in landfills, as reported by Californians Against Waste (2008), will contribute to plastic marine debris, since even when it is disposed of properly it is carried by natural agents such as wind or other forces to the ocean. As manifested, there is an excess of it in the environment and it is a major pollutant (Mor & Sivan, 2008). For 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 Partial Characterization of the Potential - 24 - quantifying the biofilm biomass: modified crystal violet staining and observation of the protein content of the biofilm. The C208 strain was cultured in a flask containing polystyrene flakes with the addition of mineral oil (0.0055% w/v), which induced more biofilm build-up. The study concluded that after an extension of 8 weeks of incubation, loss of 0.8% (gravimetric weight loss) of polystyrene weight was found. From this, Mor and Sivan (2008) regarded C208 to demonstrate a high affinity towards polystyrene through biofilm formation which lead to its degradation. The C208 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 Partial Characterization of the Potential - 25 - copolymerization that polystyrene must be modified with natural polymers and hydrophilic monomers so as to enhance its degrading ability, thereby rendering polystyrene waste useful in diminishing metal ion pollution in water. According to the mentioned study, the degrading rate of polystyrene increased to 37% after subjecting it to soil burial method for 160 days. Another study (Galgali, et al., 2004) linked a series of sugar molecules such as glucose, sucrose and lactose, to maleic anhydride functionalized polystyrene through polymer analogous reactions to produce biodegradable polymers. Evaluation of the biodegradability of these sugar linked polystyrene-maleic anhydride copolymers by known fungal test organisms was done using pure culture system. After fungal treatment, weight loss measurements confirmed the biodegradability of the carbohydrate- linked polymers. Results revealed that the degree of susceptibility to degradation varied with the type of test organism and the type of sugar. Then polymer degradation was confirmed through 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. Partial Characterization of the Potential - 26 - Starch was shown in a study (Jasso, et al., 2004) to be useful in the degradation of polystyrene. In this study, results showed the effectiveness of concentrated activated sludge in polymer degradation and the utility of starch inclusion as a filler to accelerate the structural molecular changes. High impact polystyrene blended with starch was degraded in concentrated activated sludge for 3 months. Then mechanical degradation was determined by stress-strain tests. Examination through scanning electron microscopy showed the presence of microorganisms in the polymer samples, and changes in polymer morphology in areas near holes produced in samples. 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 - 27 - 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). According to the mini review of Rose and Steinbuchel (2005), the average composition of latex glove from the Hevea brasiliensis plant is 25 to 35% (wt/wt) polyisoprene; 1 to 1.8% (wt/wt) protein; 1 to 2% (wt/wt) carbohydrates; 0.4 to 1.1% (wt/wt) neutral lipids; 0.5 to 0.6% (wt/wt) polar lipids; 0.4 to 0.6% (wt/wt) inorganic components; 0.4% (wt/wt) amino acids, amides, etc.; and 50 to 70% (wt/wt) water. This polymer has rubber particles which are about 3- to 5- um and covered by a layer of proteins and lipids. This serves to divide the hydrophobic rubber molecules from the hydrophilic environment. But due to some allergic potential caused by Hevea proteins, methods to remove these proteins were applied such centrifugation to clean the latex, treatment of sodium or Partial Characterization of the Potential - 28 - potassium hydroxide and application of enzymatic digestion with papain or alkaline proteases. Such treatments thereby reduced the protein content of condoms and latex gloves to less than 20 µg/g of natural rubber. Since 1914, natural rubber has been a classic subject of biodegradation studies. (Rose 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 by Rose and Steinbuchel (2005), which recapped the aforementioned groups, the microbes that can degrade rubber can be categorized as clear zone-forming around their colonies and non-halo forming whenever isolated and cultured in latex overlay plate, which is made by overlaying a layer of latex agar medium on a basal salt medium agar. The former category was identified to mainly consist of actinomycete species. They are said to biodegrade or metabolize rubber by secreting enzymes and other substances and also they are dubbed to be slow degraders since they rarely show an abundant cell mass when grown on natural rubber directly. On the other hand, members of the second group do not form halos on latex overlay plates. They, unlike the first group, grow more when directly grown on natural rubber. In a way, their growth on rubber could be described in an adhesive manner. The second group is said to demonstrate a relatively stronger growth on rubber. Species comprising this category are the Corynebacterium-Nocardia-Mycobacterium group. They consist of the Gordonia polyisoprenivorans strains VH2 and Y2K, G. westfalica strain Kb1, and Mycobacterium fortuitum strain NF4. Partial Characterization of the Potential - 29 - 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 study of Bereeka (2006), the degradation of natural rubber is initiated by the oxidation of double bonds. Once this takes place, oligomeric derivatives with aldehyde and keto groups formed at their ends are assumed to be degraded by beta-oxidation. Based on the study of Linos et al. (2000), the mechanism of rubber degradation of the Gordonia sp., as shown by spectroscopy, resulted in a decrease in the number of cis-1,4 double bonds in the polyisoprene chain, the appearance of ketone and aldehyde groups in the samples, and the formation of two different kinds of bonding environments. Such results could be interpreted as a product of polymer chain length that had undergone oxidative reduction thereby yielding a change in the chemical environment. Chicken feathers Partial Characterization of the Potential - 30 - b a c Figure 6: (a) A rooster will be the source of feathers for the current study, (b) Major types of feathers: radially symmetric downy feather, bilaterally symmetric contour feather, and bilaterally asymmetric flight feather or remiges (the type chosen for the current study) and (c) male flight feather, a sample of a caudal feather or rectrice In the Philippines, chicken feathers aren’t a publicly recognized problem. But, experiments and researches for its reuse and degradation are being explored at present. At the University of the Philippines-Los Baños, scientist Menandro Acda has ventured into recycling chicken feather into a low-cost building material. The scientist quoted that, recycling it would be more advisable than burning it since the incineration problem could cause environmental hazards (Morales, 2008). Moreover, in the US alone, 2 billion pounds of chicken feathers are produced by the poultry industry (Comis, 2008). Chicken feathers, by nature, are made up of over 90% protein (Cheng-cheng, et al., 2008). And this protein is none other than keratin. It’s actually the most abundant protein. It is not easily degraded due to its tightly packed structural arrangement which is in the form of alpha keratin or beta keratin. The key to its stability lies on the 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), Partial Characterization of the Potential - 31 - the build-up of chicken feathers in the environment and landfills would only result to future pollution problems and protein wastage. More so, its accumulation could serve as a breeding ground for a variety of harmful pathogens (Singh, 2004). Considering that chicken feathers have a high protein content it could also be used as an animal feed, but first its protein must be degraded (Tapia and Contiero, 2008). Yet this is said to need so much water and energy (Frazer, 2004). Old methods of degrading the chicken feathers such 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). 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 and Wojciech, 2008), fungi (Gradisar, et al., 2005) and actinomycetes (Goushterova et al., 2005) have an ability to degrade the Partial Characterization of the Potential - 32 - 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. 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 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). Partial Characterization of the Potential - 33 - 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 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 Partial Characterization of the Potential - 34 - 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 and 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 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 Partial Characterization of the Potential - 35 - 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. (Schweitzer, 2006; Chumbley, 2009 and Prescott et al., 2005) 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 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 Partial Characterization of the Potential - 36 - number of reactions. The relationship of the two is said to be proportional: the more interactions, the brighter the pixel. Repetition of the process, which could be done 30 times per second, is done until the grid scan is finished. (Schweitzer, 2006; Chumbley, 2009 and Prescott et al., 2005) Due to the conditions by which SEM works, such as a vacuum environment and electron usage, specimen preparation must be done. The sample must be dry. All water must be evaporated or remove from the system because it may vaporize in the vacuum. The size of the material should fit 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. (Schweitzer, 2006; Chumbley, 2009 and Prescott et al., 2005) Partial Characterization of the Potential - 37 - 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 flask containing mineral medium with 0.5% glucose 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 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. Partial Characterization of the Potential - 38 - 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. 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. Then, 0.5% glucose was added in all the flasks. The pH was Partial Characterization of the Potential - 39 - adjusted to pH 5.0 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 mutant strains were removed from the pollutants. The mineral medium along with some of the fungi not colonizing the pollutants were decanted from the flasks. The emptied flasks containing the pollutants with some remaining fungi closely adhering on its surface were rinsed once with 70% ethyl alcohol for three minutes with shaking. And twice, for one minute each, with distilled water to remove the remaining fungi. 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 pollutant. Since we have three pollutants namely, natural rubber, chicken feather and polystyrene, this step will be repeated three times. And three times again for each pollutant in separate days to accommodate three runs. Should be in results and Determination of potential degradation through Scanning Electron Microscopy discussion Since in most flasks all the fungi cannot be completely removed from the pollutant, the data for the pollutant’s final weight showed an increase. Due to this, the percent weight loss cannot be Partial Characterization of the Potential - 40 - consistently 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. In choosing the samples to be subjected to SEM, factors such as extent of colonization on the surface, weight increase, weight decrease?, elasticity reduction (for rubber), and presence of dents ( for polystyrene), brittleness (for feathers) were considered. The samples that best demonstrated the aforementioned properties were picked. In preparing for SEM, the sample pollutants were cut into small sizes, approximately less than a centimeter. Samples were then gold-sputtered to make it electrically conductive. When the sputtering was done, the loading into the scanning electron microscope (model Leica S440) followed. Images of various magnifications, such as 100x, 200x, 500x, 1000x, 1500x and 2000x, for each sample were chosen non-uniformly. After obtaining the micrographs, the Xylaria sp. strains’ images were individually examined and contrasted with the control. After which, the wild type (SDM) micrograph was then 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 - 41 - RESULTS I weights of the natural rubber After 50 days of incubation, the final suggest that you tabulate yoursamples were determined observations both for the inoculated and inconsistent using the analytical balance. The final weight results were uninoculated since weight gain instead of samples. weight loss was observed for some of the samples (see appendix C). The weight gain could be attributed to the adherence of the fungi’s mycelia on the surface of the pollutants. Due to this, there were no more attempts to use percent weight loss as a method. Instead of utilizing percent weight loss to determine the potential degrading ability on natural rubber of the Xylaria sp. and its strains with respect to colonization, Scanning Electron Microscopy was used. Natural rubber Figure 7.1 SEM Micrograph of the Non-Inoculated Control. Smooth surface shown. Minor debris indicated by the black arrows. Find other SEM comprehensive studies on natural rubber. Partial Characterization of the Potential - 42 - Figure 7.2 shows the SDM or wild type strain. Black arrows points on the mycelia on the rubber surface. The surface of the rubber is no longer smooth. Presence of elevations could be observed which could be presumed to be mycelial mats. Color label the regions Figure 7.3 is a micrograph of the PNL 114 strain. Shown here is the colonization of the fungal strain. The red arrows points at the spores and the black arrows illustrate the presence of mycelia. Yellow arrows shows presence of what is presumed to be mycelia mats. Partial Characterization of the Potential - 43 - Figure 7.4 shows the mutant strain PNL 116. The surface of the rubber shows roughness. There are also mycelia and spores on the surface as shown by the red and black arrows, respectively. Figure 7.5 shows the PNL 118 mutant strain. The strain shows what appears to be minimal mycelia growth as pointed by the red arrows. The micrograph also shows that there are presence of presumed mycelial mats at referred to by the black arrows. Partial Characterization of the Potential - 44 - Figure 7.6 is a micrograph of the mutant strain E26. The red arrows show mycelia colony formation. No signs of the original rubber surface can be seen on the micrograph. Figure 7.7 shows the mutant strain E35. The red arrows show the mycelia and the blue arrows show the spores on the surface. There is also presence of the presumed mycelial mat as pointed by the yellow arrow. Partial Characterization of the Potential - 45 - Microscopic SEM results The micrographs of each mutant strain and wild type were compared to the micrograph of the non-inoculated control to reveal the marked differences and changes that has taken place. In figure 7.1, a 10um section of the non-inoculated control with some residues or debris is shown. Figure 7.2 demonstrates the growth of SDM (wild type) which revealed that the fungus was able to establish itself on the natural rubber as indicated by the presence of mycelia on the surface. There is also presence of what is presumed to be mycelial mats. The mycelial mats, as used here, will be defined as thin aggregations of mycelia covering the surface of natural rubber in a manner that seem to demonstrate elevated surfaces when compared to the control. In polystyrene and feather, mycelial mats refer to aggregations of entangled fungal hypha or mycelia. Figure 7.3 is a micrograph of the PNL 114 mutant. There is an observed presumed mycelial mat formation and presence of mycelia and spores on the surface. Micrograph revealed that this mutant strain has embedded itself on the rubber matrix. PNL 116 mutant (shown in figure 7.4) showed poor colonization. There is a minimal growth and thereby minimal presence of fungi and mycelia on the natural rubber. Yet the micrograph still revealed that the fungal strain has established itself on the surface through mycelial I suggest no u may mat formation. In figure 7.5, PNL 118 mutant showed thatdistinct traces of mycelia or spores on the add labels in the surface. But when compared to the non-inoculated control in figure 7.1, presence of mycelial mat is figures to indicate regions… revealed. The E26 mutant (figure 7.6) showed the strongest and maximum growth and colonization. The micrograph revealed mycelial colony and presence of spores. The strain has embedded itself on the rubber matrix and suggests loss and degradation of the rubber due to the disappearance of the rubber surface. Discussion Lastly in figure 7.7 , E35 mutant strain demonstrated natural rubber surface colonization. Presence of mycelia and spores was observed. Formation of mycelial mat is also present. Partial Characterization of the Potential - 46 - Macroscopic results Macroscopic examination of the natural rubber samples was also done. Through physical examination, it revealed that the control showed no change after the 50-day incubation period. While the SDM on the other hand, even after rinsing once with 70% ethanol and twice with water, showed signs of mycelia embedded on the surface. When compared to the control, there is an observed loss of elasticity. However, in the case of the three albino mutants namely 114, 116 and 118, there were no physical signs of mycelia embedded on the surface because of the color of these mutant strains which is similar to the natural rubber color. But microscopic examination revealed their presence. All the mutants demonstrated elasticity loss especially in areas where the mycelia have embedded. But comparable results to the wild type strain in elasticity loss are demonstrated by PNL 114, E35 and E26. Chicken Feathers Figure 8. SEM micrographs of Xylaria sp. strains on Chicken Feathers after 50-day incubation Figure 8.1 Control Partial Characterization of the Potential - 47 - Figure 8.2 SDM wild type strain is shows the whole surface, specifically the barbs (yellow arrows) and barbules ( red arrows) , is covered with mycelia as pointed by the red arrow. Figure 8.3 PNL 114 mutant strain’s micrograph shows colonization of mycelia on the barbs (red arrows) and barbules (yellow arrows) of the chicken feather. Partial Characterization of the Potential - 48 - Figure 8.4 PNL 116 mutant strain micrograph demonstrates the mycelia adhering closely to the barbules. Red arrows point on mycelia’s establishment or colonization on the surface of the feather. Figure 8.5 PNL 118 mutant strain shows mycelia are adhering to several areas of the barbules. (See the red arrows.) Partial Characterization of the Potential - 49 - Figure 8.5 E26 mutant strain micrograph shows presence of mycelia at the barbs and barbules as pointed by the blue and red arrows respectively. Figure 8.7 The E35 mutant strain shows mats of mycelia (see yellow arrows) covering several areas of the feather sample. Weakened barbs resulted to breakage (see blue arrows). Barbules detached Partial Characterization of the Potential - 50 - from their barbs. Presence of spores is shown at the upper right side of the micrograph as pointed by the red arrow. Microscopic SEM results In Figure 8.1, the micrograph shows the non-inoculated control. All the other micrographs, the SDM strain or wild type and all the mutant strains, were compared with the non-inoculated control to observe if changes took place. The wild type or SDM strain is shown at figure 8.2. The micrograph shows that the SDM strain colonized barbs and barbules by covering it with mycelia. Figure 8.3, is a 10 micron view of the PNL 114 mutant strain wherein presence of mycelia at the barbules is evident as pointed by the red arrow and at the barbules as referred to by the yellow arrow. The PNL 116 mutant strain in figure 8.4 shows adherence of the mycelia in the barbules of the chicken feather. The mycelia had established itself on the surface as shown in the micrograph. Moreover, figure 8.5 micrograph illustrates PNL 118 mutant strain. The mycelia are seen attached various areas on the feather. Some portions of the feathers seem to be disheveled. For the black strains, figure 8.6 demonstrates the chicken feather sample strain of E26.The micrograph demonstrates the adherence of the mycelia on the barbules and barbules. The barbules contain more mycelia than the barbs. Lastly, E35 strain is shown at figure 8.7. Presence of spores is seen at the upper right part. Mycelia in the form of mats (see figure 8.7) have adhered closely to the barbules which at some point are disheveled. Breakage is also seen at some barbs. Macroscopic results Macroscopic investigation results shows that, in general, the chicken feathers have retained their original physical appearance except for two strains namely, SDM or the wild type and E35 Partial Characterization of the Potential - 51 - black mutant strain. The two showed little signs of brittleness in their barbules. This brittleness refers to their ability to be detached easily from the rachis. No other changes had been observed. You may also group the figures so that it will be easy to compare.
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