A Preliminary Study on the Potential……..1 Dayao and Egloso, 2009 INTRODUCTION Background of the Study Pollution is an inevitable problem due to population growth, urbanization and the increased demand for manufactured products in the local and export markets. Industrialization has resulted to the generation of wastes of various forms that pose serious risks to the environment and public health, thus, requiring an efficient waste regulatory management. At the advent of technology, pollution has indeed taken its toll on nature, making people harvest and manufacture products that would take eons to decay and rot at the very least. Everywhere, a plethora of biodegradable and non-biodegradable wastes can be seen. And, indeed it’s high time that people revert back to natural processes that could help solve the burgeoning problem of waste disposal since all the artificial methods that require today’s technology could contribute to the pollution that the planet is experiencing now. One such natural process that could solve the problem, or in a way even just alleviate such waste pile-up, is biodegradation. The country’s population growth rate is one of the highest in the world (Mangahas, 2006) and it places serious strains on the economy. In 2005, the population was 82.8 million, of which 51.8 million or 63% lived in urban areas. Metro Manila is the most densely populated urban area with 10.7 million (Mangahas, 2006). Over the past 3 decades, the country’s economy slid behind many Asian economies. Gross domestic product (GDP) grew at an average of only 3%, compared with 8% in the People’s Republic of China (PRC); 6% in the Republic of Korea, Singapore, Malaysia, and Thailand; and 5% in Indonesia over the last 30 years (Wallace Report 2004; Mangahas, 2006). Urbanization, decline in the economy and further population growth lead to the even higher generation of wastes that has not been managed properly and safely (DENR, 2004). A Preliminary Study on the Potential……..2 Dayao and Egloso, 2009 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 pH5.0 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 showed 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 and 118 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). A Preliminary Study on the Potential……..3 Dayao and Egloso, 2009 This study aimed 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 To determine the potential biodegrading ability of Xylaria sp. mutant strains and SDM wild type using scanning electron microscopy (SEM). Objectives The study aimed to determine the biodegrading capacity of Xylaria sp. wild type and its mutants. The specific objectives are as follows: 1. to determine if Xylaria sp. mutants and wild type can degrade natural rubber, chicken feather and polystyrene through Scanning Electron Microscopy. 2. to compare the biodegrading ability of the wild type to each mutant to find which strain is most appropriate for each type of waste 3. to observe the macroscopic/physical changes that natural rubber, chicken feather and polystyrene have undergone after incubation with the Xylaria sp. Significance of the Study The study will serve as a source of preliminary information on the potential of Xylaria sp. strains to degrade chicken feathers, polystyrene and natural rubber. Findings of this study may be utilized in the development of Xylaria sp. as a good biodegrading agent in reducing durable wastes A Preliminary Study on the Potential……..4 Dayao and Egloso, 2009 such as plastics and others, as well as in optimizing fungal technologies. This study may also provide a possible means to discover and understand 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 less expensive and 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 organism of focus used in this study, Xylaria sp., has only been identified up to the genus level. The experiment determined the potential biodegrading ability of the Xylaria sp. mutant strains and wild type on natural rubber, chicken feather and polystyrene. Other pollutants with similar biochemical structure to the aforementioned pollutants were not included in the experiment. The three pollutants were not subjected to any chemical (i.e. chemical oxidation) or physical process (i.e. compression) that would significantly alter their structure prior to the experiment. The used latext gloves to represent natural rubber were not used to handle chemicals that would affect the results of the current study. The Xylaria sp. variant strains used came from the stock culture of the Antibiotic Laboratory of the National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines – Los Baños. The strains that were used were Xylaria sp. strain mutants PNL 114, 116, 118, E26 and E35 which were all compared to the wild type SDM (sterile dark mycelia), in terms of their biodegrading capacity. The albino mutants (PNL 114, 116, 118) have been partially characterized, whereas the black mutants E26 and E35 have not been A Preliminary Study on the Potential……..5 Dayao and Egloso, 2009 characterized. Culture media and reagents were also provided by BIOTECH. Experimentation was 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 were not 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 catalysts for each pollutant degradation set-up, optimum size of inoculum for each pollutant were not within the scope of this experiment. The conditions, such as the pH of 5.0, incubation temperature of 25 ˚C and incubation period of 50 days, of the Xylaria sp. strains, used for incubation and growth were derived from the results of the study of Clutario and Cuevas (2001) on Xylaria sp.’s degradation of polyethylene. Colonization of the substrates’ surfaces was 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. The SEM was conducted after the incubation period of the set-ups. One sample per pollutant per strain was used. The non-inoculated control was also subjected to SEM. Only the initial and final weights of the pollutants were recorded. The strain treatments were in duplicates or 2 replicates per run, and 3 runs were conducted due to logistic matters and unavailability of equipment. Moreover, the set-ups were observed on the 20th, 30th and 50th day of incubation, with 50 days as the maximum incubation period. On the 20th and 30th day, only visual observation was done, 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 is only on the 50th day that the actual change in weight was determined. Aside from microscopic observations, macroscopic/physical observations were done. Yet no standard methods in determining physical change, such as tensile strength measurement for A Preliminary Study on the Potential……..6 Dayao and Egloso, 2009 natural rubber, have been conducted. In terms of data analysis, this study only focused in analyzing the biodegradation potential of Xylaria sp. strains through its colonization on natural rubber, chicken feather and polystyrene. Also, the study was concerned on whether the degradation capacity of the mutant strains is significantly different from the capacity of the wild type to degrade. Definition of Terms Anamorph – mitotic, asexual stage of a fungus that does not produce ascospores Biodegradation – material is broken down to environmentally acceptable state by microorganisms capable of utilizing it as a source of energy A Preliminary Study on the Potential……..7 Dayao and Egloso, 2009 Biodeterioration – a superficial degradation that modifies mechanical, physical and chemical properties of a given material, mainly the result of the activity of microorganisms growing on the surface or/and inside a given material Biofilm formation – a.k.a. microfouling; the process in which a complex community of microorganisms is established on a surface Bioremediation - Biotransformation – the chemical modification(s) made by an organism on a chemical compound Burrowing – the tunneling, penetrating, or infiltrating into the substrate causing holes, cracks, tearing Colonization – the establishment of an organism in a new, particular ecosystem Crack – a thin break, crevice or fissure Dent – a concavity, hollow or depression in the surface Elevation – a degree or amount of being raised Flaking – resulted into flat pieces of the substrate, particularly the surface Hole, pit – perforation or cavity in an object Holomorph – the whole fungus in all its forms, facets, and potentialities, either latent or expressed even if it reproduces by only one method Hyphae – microscopic, tubular, and threadlike or filamentous structures which compose the fungus body or mycelium or thallus and branch in all directions, spreading over or within whatever substrate a fungus utilizes as food Keratinases - A group of proteolytic enzymes which are able to hydrolyze insoluble keratins more efficiently than other proteases A Preliminary Study on the Potential……..8 Dayao and Egloso, 2009 Keratinophilic fungi – have the unique ability to degrade keratinous substrates such as hair, horns, hooves, feathers and nails Keratinolytic fungi – have the unique ability to degrade keratinous substrates completely Mycelium – the fungus body which is composed of hyphae Mycelial mat – a mass of hyphae Polymer - consists of long chains of repeated molecule units known as "mers", which intertwine to form the bulk of the plastic Plectenchyma – mycelial mats; ascomycete mycelium that are organized into fungal tissues Recalcitrance – Smooth – having a continuously even surface Spore – a minute, simple propagating unit without an embryo that serves in the production of new individuals Striation – marked with parallel grooves, ridges, or narrow bands Tearing – cause something to become splitted, divided, fragmented Teleomorph – meiotic, sexual stage of a fungus that produces ascospores Staling product – of an organism: produced by its metabolic activites A Preliminary Study on the Potential……..9 Dayao and Egloso, 2009 REVIEW OF RELATED LITERATURE In the 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. 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, risks in the excavation, handling, and transport of hazardous material (Vidali, 2001). A Preliminary Study on the Potential……..10 Dayao and Egloso, 2009 Biodegradation Figure 1: Scheme for Polymer 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 decomposition of substances by the action of microorganisms, which result to the recycling of carbon, the mineralization (CO2, H2O and salts) of organic compounds and the generation of new biomass (Dommergues & Mangenot, 1972; Lucas, et al., 2008). Moreover, biodegradation is said to take place in three stages: biodeterioration, biofragmentation and assimilation. Biodeterioration is the result of the activity of microorganisms growing on the surface and/or inside a given material (Hueck, 2001; Walsh, 2001; Lucas, et al., 2008) through mechanical, chemical and/or enzymatic means (Gu, 2003; Lucas, et al., 2008). Biofragmentation, on the other hand, is a lytic phenomenon essential for the consequent process called assimilation (Lucas, et al., 2008). Nonetheless, it is said that as all materials of organic origin, A Preliminary Study on the Potential……..11 Dayao and Egloso, 2009 both natural and synthetic polymers containing specific functional groups, are potential substrates for heterotrophic microorganisms including bacteria and fungi (Motta, et al., 2007). 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 are 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 contaminants and the environment factors (type of soil, temperature, pH, the presence of oxygen or other electron acceptors, and nutrients). Different microorganisms degrade different types of compounds and survive under different conditions (United States Environmental Protection Agency, 1996). As a prerequisite to initiate microbial growth in biodegradation, inorganic salts and sugars (e.g. glucose) are often employed. The amount must be strictly controlled to prevent the organism A Preliminary Study on the Potential……..12 Dayao and Egloso, 2009 from using this material as the sole carbon source. Growth is verified through visual inspection, but absence of growth may be due to an unsuccessful inoculation in addition to the fact that the organisms are unable to utilize the polymer as a sole carbon source. Evaluation of biodegradation also frequently involves computation of the weight loss and microscopic examination of the polymer surface. When a polymer is incubated with fungi, small circular holes are sometimes observed. The hydrophobicity of most polymers is often a major obstacle to obtaining satisfactory results from a biodegradation test. Consequently, it is customary to include a surfactant (Tween) to enhance the growth conditions. However, microorganisms themselves also often produce surfactants (Albertsson & Karlsson, 1993). Microbial growth depends on properties of polymer materials and specific environmental conditions, such as humidity, weather and atmospheric pollutants (Lugauskas et al., 2003; Lucas, et al., 2008). The ease with which polymers are degraded is entirely dependent upon their material composition (Busscher et al., 1990; Wiencek & Fletcher, 1995; Bos et al.,1999; Gu, et al., 2000b; Gu, 2003), molecular weights, crystallinity, atomic composition and conformation, the chemical bonds in the structure, the physical and chemical characteristics of the surfaces (Callow& Fletcher, 1994; Becker et al., 1994; Caldwell, et al., 1997; Gu, 2003), the indigenous microflora, and environmental conditions whether they are naturally occurring or synthetic (Albertsson & Karlsson, 1993; Gu, et al., 2000; Gu, 2003; Motta, et al., 2007). Generally, higher molecular weight results in greater resistance to degradation by microorganisms, whereas monomers, dimers and oligomers of a polymer’s repeating units are degraded and mineralized more easily. High molecular weights lead to sharply decreased solubility, making the polymer resistant to microbial attack because microorganisms need to assimilate the substrate through their cellular membrane and then degrade the substrate further by means of intracellular enzymes. At least two categories of enzymes, namely A Preliminary Study on the Potential……..13 Dayao and Egloso, 2009 extracellular and intracellular depolymerizers, are actively involved in biodegradation of polymers (Doi, 1990; Gu et al., 2000; Motta, et al., 2007). It is commonly recognized that the closer a polymer’s structure to that of a natural molecule, the more easily it is degraded and mineralized (Gu & Gu, 2005; Motta, et al., 2007). Generally, microbes cannot degrade synthetic polymers, principally the polyolefins, which are made up of only carbon and hydrogen atoms, considered to be resistant to biodegradation. This is probably due to a total lack in the polymer’s backbone of sites involving carbon-to-oxygen bonds (C=O, C–OR, C–OH), which are the real target of microbial enzymes (Motta, et al, 2007). The biodegradation of natural polymers, on the other hand, usually proceeds faster than that of their synthetic homologues because they contain traces of amino acids, vitamins, growth hormones, and enzymes (Albertsson & Karlsson, 1993). 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 & Sivan, 2008). Yet originally, Rhodococcous rubber is a known rubber- A Preliminary Study on the Potential……..14 Dayao and Egloso, 2009 degrading organism, according to the review of Rose and Steinbuchel (2005). Xanthomonas sp. strain 357, in much the same way, can degrade rubber as well. A number of fungi species are also known to biodegrade. The known fungi biodegraders are Gordonia sp., Streptomyces sp., Nectria gliocladioides, Penicillium ochrochloron and Geomyces pannorum and Trichoderma atroviride (Barraat, et al.,2003; Cheng chang, et al., 2003; Rose & Steinbuchel, 2005). Gordonia sp. and Streptomyces sp. are known rubber-degraders (Rose and Steinbuchel, 2005). Nectria gliocladioides (five strains), Penicillium ochrochloron (one strain) and Geomyces pannorum (seven strains), in a study of Barraat et al. (2003), have been observed to degrade polyurethane while simultaneously relating it to the water holding capacity of the soil. Moreover, in a study conducted by Cao et al. (2008), the fungus Trichoderma atroviride completely degraded the chicken feathers. This strain was actually isolated from a decaying feather. The list of microorganisms that could be used in biodegradation goes on for there are still more species that could degrade pollutants. And in fact, in the Philippines a fungus named Xylaria sp. has been isolated and proven to degrade polyethylene (Cuevas & Manaligod, 1997; Clutario & Cuevas, 2001). Xylaria sp. as a Potential Agent for Biodegradation Xylaria sp. tested in this study 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 A Preliminary Study on the Potential……..15 Dayao and Egloso, 2009 have designated it under Class Ascomycetes, Order Xylariales, Genus Xylaria. (Clutario & Cuevas, 2001). A previous study by Clutario and Cuevas (2001) proved that Xylaria sp. can utilize polyethylene plastic strips as an alternative carbon source, thereby degrading them into usable forms for self-sustenance. Through the use of scanning electron microscopy, the proponents of the said study observed visible damages of the surface structure of the plastic strips. There were tearing and striations caused by active burrowing of Xylaria sp. hyphae on the polyethylene material. Plastic is an extremely versatile synthetic material made of high molecular weight, semi-crystalline polymer prepared from ethylene through the cracking of crude oil, light petroleum and natural gas. For plastic bags alone, it is estimated that some 430,000 gallons of oil are needed to produce 100 million pieces of these omnipresent consumer items on the planet (Knapczyk & Simon, 1992; EcoWaste Coalition, 2008). 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. 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, A Preliminary Study on the Potential……..16 Dayao and Egloso, 2009 petioles, leaves of angiosperms and herbaceous stems, sometimes appearing terrestrial but actually attached to buried wood; growing alone or, more commonly in clusters; appearing in spring and not decaying until late summer or fall (Kuo, 2003). Some are associated with insect nests. Most decay wood and many are plant pathogens. Many are endophytes. They are commonly found throughout the temperate and tropical regions of the world. The Xylaria sp. can be distributed above, around, and beneath perithecia. It forms a unipartite stromatal layer, with a superficial or erumpent surface level. The interior of its stromata is essentially homogeneous. Conidium-bearing discs, potassium hydroxide pigments and orange granules surrounding the perithecia are absent (Rogers et al., 2002). They are mostly multiperitheciate in ascomatal number per stroma, ascomatal ostioles and ascal apical rings: are present, and the ascospore cell number is one-celled. Teleomorph and anamorph are produced on the same stromata in most species, with their anamorphs: Geniculosporium-like. Some Xylaria sp. species exist as endophytes, and have mutualistic associations with plants. The fungus secrete toxins to protect the plant from herbivory from other insects or animals, while the fungus in return feeds on the host’s tissues for nutrition, and its mycelia are scattered through seed dispersal. Endophytic Xylariaceae have been documented in conifers, monocots, dicots, ferns, and lycopsids (Brunner & Petrini, 1992; Davis, et al, 2003). According to the study of Liers et al. (2007), Xylaria polymorpha, which is said to lack peroxidase, is known to produce the enzyme laccase, a known ligninolyitc oxidoreductase. This supports the previous study of Lou and Wen (2005) wherein they discovered that Xylaria sp. along with other ascomycetes and some basidiomycetes commonly demonstrated laccase activity together with cellulolytic and xylanolytic activities. The enzymatic profiles of the aforementioned species suggests that (1) ascomycetes is potentially capable of utilizing the lignocellulosic wood components (2) laccase is apparently the main enzyme for ligninolysis unlike the white-rot basidiomycetes that A Preliminary Study on the Potential……..17 Dayao and Egloso, 2009 utilizes its ligninolytic peroxidase in the form of manganese peroxidase or lignin peroxidase in addition to lignin peroxidase. Metabolites produced by Xylaria have already been identified. In a study by Pongcharoen, et al. (2008), two xylarosides, which are glucoside derivatives, were isolated from the broth extract of a Xylaria sp. PSU-D14 strain along with two compounds, sordaricin and 2,3-dihydro-5-hydroxy-2-methyl-4H-1-benzopyran-4-one. Sordaricin showed moderate antifungal activity against Candida albicans ATCC90028 strain. Pongcharoen, W., Rukachaisirikul, V., Phongpaichit, S., Kühn, T., Pelzing, M., Sakayaroj, J. and Taylor, W. C. (2008). Metabolites from the endophytic fungus Xylaria sp. PSU-D14. Phytochemistry, 69, 1900–1902. In another study by Xiaobo, et al. (2006) they isolated a Xylaria sp. 2508 strain from the seeds of an angiosperm tree and was found to produce rich secondary metabolites. Xyloketals, xyloallenolide A, and two other allenic ethers were isolated from the spent culture medium of this fungus. One of allenic ethers is eucalyptene A which exhibited antifungal activity. Xiaobo, Z., Haiying, W., Linyu, H., Yongcheng, L., and Zhongtao, L. (2006). Medium Optimization of Carbon and Nitrogen Sources for the Production of Eucalyptene A and Xyloketal A from Xylaria sp. 2508 using Response Surface Methodology. Process Biochemistry, 41, 293–298. The Xylaria Wild Type and Mutant Strains Partial characterization of the fungus Xylaria sp. and its albino mutants, PNL 114, PNL 116 and PNL 118 was based on the study by Tavanlar and Lat (2008) in which the black fungus wild type SDM was subjected to mutagenesis, and protoplast fusion. On the other hand, the black mutants E26 and E35 have not been characterized as of yet (M. A. Tavanlar, personal communication, March 4, 2009). The aforementioned study determined morphological and biochemical characteristics or A Preliminary Study on the Potential……..18 Dayao and Egloso, 2009 markers in the wild type 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 & 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) and protects hyphae during infiltration through a substrate during colonization (M. A. Tavanlar, personal communication, March 4, 2009). This polymer of phenolic compounds provides tolerance to various environmental stresses like oxidants, microbial lysis, UV radiation, and defense responses of host plants and animals against fungal infection (Kimura & Tsuge, 1993; Tavanlar & Lat, 2008). In the study by Tavanlar and Lat (2008), after mutants were repeatedly tested on MMG (mineral medium plus 0.5% glucose) plus various supplements, Xylaria strain mutants PNL 114, 116 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 wild type, when viewed under the light microscope. These albino mutants evidently lost their melanin pigmentation and the mycelia assumed a thinner appearance than the wild type dark mycelia. This study utilized NTG in the induction of mutants from the SDM wild type. 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 A Preliminary Study on the Potential……..19 Dayao and Egloso, 2009 color (Kimura & Tsuge, 1993; Kawamura, et al., 1997; Tavanlar & Lat, 2008). Defective genes involved in the very common DHN pathway to melanin biosynthesis have been identified in some of the mutants of these fungi. Table 1 shows the three mutants which underwent further tests as presented. The study further tested the three 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 three albino mutants on mineral medium with and without supplements as compared to the wild type SDM. In summary, the results of the said study showed that the three 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 sources of carbon as compared to the wild type. These albino mutants may potentially exhibit enhanced degradation of polyethylene plastics than the wild type. Also, the proponents have speculated that the albino mutants can better survive environments with less available amounts of readily utilizable carbon sources such as the surface of plastics than the wild type. Table 1. Comparative growth of the PNL mutants and wildtype SDM on MMG and mineral medium with various supplements. Average diameter of colony (mm) Mutant Strain Medium M1 M2 M3 M4 M5 PNL 114 23.0a 14.8a 17.0a 24.2a 24.8 PNL 116 22.0a 14.5a 17.0a 24.3a 23.0 PNL 118 23.3a 12.8b 17.0a 23.0a 23.8 SDM 5b 5c 5b 12.8b 20.1 (Source: Tavanlar, M. A. T., & Lat, E. C. (2008). Partial Characterization of Mutants from a Plastic-degrading Black Fungus. Unpublished.) Measured after 4 days incubation at ART: M1 = MMG + 0.005% benomyl M2 = MM + 0.025% glucose + 1% acetamide A Preliminary Study on the Potential……..20 Dayao and Egloso, 2009 M3 = MM + 0.025% glucose + 1% PEG M4 = MM + 0.025% glucose M5 = MM + 0.5% glucose Values within the same column followed by the same letter are not significantly different at P<0.05. Table 2. Growth of the four albino mutants on mineral medium with and without supplements as compared to the wildtype SDM. Average diameter of colony (mm) Mutant Strain Medium MM MMP MMT PNL 114 41.5 36.0 38.5 PNL 116 45.0 35.0 34.8 PNL 118 39.0 36.5 32.2 SDM 16.0 16.5 14.1 (Source: Tavanlar, M. A. T., & Lat, E. C. (2008). Partial Characterization of Mutants from a Plastic-degrading Black Fungus. Unpublished.) 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 Table 3. Growth of the two black mutants on mineral medium with supplements MDPE and PEG Average diameter of Mutant Strain colony (mm) MMMDPE MMPEG E26 25.9 23.85 E35 40.15 30.65 MMMDPE = MM + medium density polyethylene MMPEG = MM + polyethylene glycol 6000 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, A Preliminary Study on the Potential……..21 Dayao and Egloso, 2009 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. Culture Methods The shaken culture method was used to provide a stock culture for fungal inocula. The medium is shaken after inoculation with spores or mycelium so that growth occurs in the body of the liquid. Nutrient uptake is very efficient, giving faster and more homogeneous growth. The technique exhibited by Xylaria growth during incubation for 50 days is the surface culture method. When a nutrient medium is inoculated with fungal spores or mycelium, the mycelium grows over the surface of the liquid to form what is variously referred to as a felt, a pad or a mat (Turner, 1971). A Preliminary Study on the Potential……..22 Dayao and Egloso, 2009 Current State of the Solid Waste in the Philippines Filipinos generate around 0.3 to 0.7 kilograms of garbage daily per person depending on income levels (World Bank, 2001). Metro Manila produces about 8,000 tons of solid waste each day and is expected to reach 13,300 tons each day in 2014 (Baroña, 2004). The National Capital Region produces the highest amount of wastes, about 23% of the country’s waste generation (Anden & Rebolledo, 2003). 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). A Preliminary Study on the Potential……..23 Dayao and Egloso, 2009 Natural Rubber Figure 2: Natural rubber is a polymer called polyisoprene, 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, 1-2% (wt/wt) carbohydrates, 0.4-1.1% (wt/wt) neutral lipids, 0.5-0.6% (wt/wt) polar lipids, 0.4-0.6% (wt/wt) inorganic components, 0.4% (wt/wt) amino acids, amides, etc.; and 50-70% (wt/wt) water. This polymer has rubber particles which are about 3- to 5- µm 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 as centrifugation to clean the latex, treatment of sodium or 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. A Preliminary Study on the Potential……..24 Dayao and Egloso, 2009 Since 1914, natural rubber has been a classic subject of biodegradation studies. (Rose & Steinbuchel, 2005). This is due to the high rate of its yearly manufacture which is several million tons, as mentioned in the study of Bereeka (2006), and its slow rate of natural degradation as reviewed by Rose and Steinbechul (2005). In fact, a number of studies abound concerning its degradation. And it has been learned that both bacteria and fungi can participate in such process. Throughout all the investigations and experimentations done, two categories of rubber- degrading microbes according mainly on growth characteristics have been established. Based on a review by Rose and Steinbuchel (2005), which recapped the aforementioned groups, the microbes that can degrade rubber can be categorized as clear zone-forming around their colonies and non-halo forming whenever isolated and cultured in latex overlay plate, which is made by overlaying a layer of latex agar medium on a basal salt medium agar. The former category was identified to mainly consist of actinomycete species. They are said to biodegrade or metabolize rubber by secreting enzymes and other substances and also they are dubbed to be slow degraders since they rarely show an abundant cell mass when grown on natural rubber directly. On the other hand, members of the second group do not form halos on latex overlay plates. They, unlike the first group, grow more when directly grown on natural rubber. In a way, their growth on rubber could be described in an adhesive manner. The second group is said to demonstrate a relatively stronger growth on rubber. Species comprising this category are the Corynebacterium-Nocardia-Mycobacterium group. They consist of the Gordonia polyisoprenivorans strains VH2 and Y2K, G. westfalica strain Kb1, and Mycobacterium fortuitum strain NF4. As demonstrated by a particular study by Linos, et al. (2000), the mechanisms that the microbes undergo when biodegrading is colonization, biofilm formation and aldehyde group formation after cultivating it on the surface of latex gloves. Such is revealed after undergoing the A Preliminary Study on the Potential……..25 Dayao and Egloso, 2009 Schiff reagent’s test. This is further examined under a scanning electron microscope. In their methodology they have indicated that the preliminary screening method to be used in finding potential rubber-degrading bacteria is by growing such bacteria or microbe on the latex overlay or by latex film on the mineral agar plates. Growth and colonization of the microbe in this medium would indicate its utilization of rubber as its sole carbon source; hence, making it as a potential rubber-degrader. Furthermore, according to a study of Bereeka (2006), the degradation of natural rubber is initiated by the oxidation of double bonds. Once this takes place, oligomeric derivatives with aldehyde and keto groups formed at their ends are assumed to be degraded by beta-oxidation. Based 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 a b b c Figure 3: (a) Gallus sp is the source of feathers for the current study, (b) bilaterally symmetric contour feather and its parts (the type chosen for the current study) A Preliminary Study on the Potential……..26 Dayao and Egloso, 2009 In the Philippines, chicken feathers aren’t a publicly recognized problem. However, the build-up of chicken feathers in the environment and landfills would only result to future pollution problems and protein wastage (Onifade, et al., 1998; Goushterova, et al.,2005; Cheng-Cheng, et al.; 2008). More so, its accumulation could serve as a breeding ground for a variety of harmful pathogens (Singh, 2004). 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 higher degree of cross-linking by disulfide bonds, hydrophobic interactions, and hydrogen bonds. Such stability renders keratin water-insoluble, extremely resistant to biodegradation and poorly susceptible to digestion by the most common peptidases like papain, trypsin and pepsin (Gradisar et al., 2005; Kim, 2007). Considering that chicken feathers have a high protein content it could 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). A Preliminary Study on the Potential……..27 Dayao and Egloso, 2009 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 & Wojciech, 2008), fungi (Gradisar, et al., 2005) and actinomycetes (Goushterova et al., 2005) have an ability to degrade the keratin in chicken feathers. A study (Burtt & Ichida, 1999) also 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. And the metabolic activity and / or antibiotic production of some bacteria may inhibit or improve the growth of other bacteria and / or fungi present. Researchers have known for decades that the plumage of birds harbors a diverse community of bacteria and fungi, including yeast (Hubilek, 1994). To our knowledge, no one has yet comprehensively characterized the microbial communities living on feathers of any species. Such information is needed to determine how microbes interact both with one another and with birds (Shawkey, et al., 2005). Outstanding keratinolytic activity among A Preliminary Study on the Potential……..28 Dayao and Egloso, 2009 keratinases produced from tested nonpathogenic filamentous fungi has been observed from Paecilomyces marquandii, Doratomyces microsporus, Aspergillus flavus (Gradisar, et al., 2005) and Aspergillus nidulans strains (Manczinger, et al., 2003; Joshi, et al., 2007). A group of proteolytic enzymes which are able to hydrolyze insoluble keratins more efficiently than other proteases are called keratinases, which could degrade feathers and make it available for its use as animal feed, fertilizer and natural gas. They are primarily classified as extracellular serine proteases, with the exception of keratinases from yeasts, which belong to the aspartic proteases. Molecular masses of these enzymes range from 20 kDa to 60 kDa. They are mostly active in alkaline environments, with optimal activity at temperatures up to 50°C. Thermostable keratinases with optimal temperatures of around 85°C and a higher molecular mass have been reported (Gradisar, et al., 2005). 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. A Preliminary Study on the Potential……..29 Dayao and Egloso, 2009 Keratinases are produced by some insects and mostly by microorganisms. They have various economic uses. Aside from their feather degrading capacity, they could be used in the leather industry as an agent in dehairing leather. Their 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). Keratinases are extensively applied also in the detergent and textiles industries, waste bioconversion, medicine, and cosmetics for drug delivery through nails and degradation of keratinized skin (Gradisar, et al., 2005). 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. 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 A Preliminary Study on the Potential……..30 Dayao and Egloso, 2009 source of carbon, nitrogen and sulfur. It was then rotated in an orbital shaker for 10 days. After 4 days, one flask showed a visual degradation of the feather. A dilution series was made afterwards so as to isolate and culture the bacteria that just degraded the feather. The strain was identified as Bacillus lichenformis strain K-508. And the confirmation of the keratinolytic activity was done by using the azokeratin as a substrate assay. A study by Kavitha (2001) studied the distribution of mycoflora inhabiting bird's feather. All the fungi were screened for keratinolytic activity. Chrysosporium spp having high keratinase activity was taken for further studies. The keratinase from Chrysosporium spp., was purified and homogeneity was confirmed by polyacrylamide gel electrophoresis. Isolation of a new microbial organism that could degrade chicken feather will help in the degradation of the chicken feathers which is now becoming a burden in the society both internationally and locally. The microbe could potentially provide the keratinase that could be used in compost technology (Maczinger, et al., 2003) or in the conversion of feather to feedstock meal additives (Tapia & Contiero, 2008). Polystyrene a b c 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 A Preliminary Study on the Potential……..31 Dayao and Egloso, 2009 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. In thermoplastics, the polymer chains are only weakly bonded (van der Waals forces). The chains are free to slide past one another when sufficient thermal energy is supplied, making the plastic formable and recyclable (eFunda, 2009). Most biodegradable polymers belong to thermoplastics (e.g. poly (lactic acid), poly(hydroxyalkanoate), poly(vinyl alcohol)) or plants, polymers (e.g. cellulose and starch), except for polyolefins which are nonbiodegradable. 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 or styrofoam 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). EPS 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, there 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 exists after polystyrene completes its "degradation process”. The basic unit of polystyrene is styrene, which is a known neurotoxin and animal carcinogen, considered very dangerous to human health and hence, strategies to avoid its discharge, eliminate it from the environment, and understand its route of degradation were the focus of much research (Mooney, Ward, & O'Connor, 2006). Studies suggest that styrene mimics estrogen in the body and can therefore disrupt normal hormone functions, possibly contributing to thyroid problems, A Preliminary Study on the Potential……..32 Dayao and Egloso, 2009 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 A Preliminary Study on the Potential……..33 Dayao and Egloso, 2009 $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 A Preliminary Study on the Potential……..34 Dayao and Egloso, 2009 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). There were studies that tested the possibility of whether copolymerizing polystyrene with other substance could make it more degradable and susceptible to microbial attack. In 1992, a study by Milstein, et al. (1992), focused on the biodegradation of a lignin-polystyrene copolymer. The white rot basidiomycete was used to degrade such complex copolymer. Such fungus released enzyme that oxidized lignin and demonstrated degradation through weight loss, UV spectrophotometric analysis and deterioration of surface of the plastic substance as seen under the SEM. A similar study by Singh and Sharma (2007) demonstrated through the process of graft copolymerization that polystyrene must be modified with natural polymers and hydrophilic monomers so as to enhance its degrading ability, thereby rendering polystyrene waste useful in 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 A Preliminary Study on the Potential……..35 Dayao and Egloso, 2009 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 Fourier Transform Infrared Spectroscopy (FTIR) spectra. In 1993, a study by Cox, et al. 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. Perlite is an amorphous volcanic glass that has a relatively high water content, typically formed by the hydration of obsidian. The biofilter with perlite completely degraded styrene and an elimination capacity of at least 70 g styrene/m3 filter bed per hour was computed. In this study, a concept in biofiltration is presented, based on the application of fungi for the degradation of waste gas compounds in biofilters containing inert support materials for the immobilization of the fungi. In principle, the application of fungi in biofilters may offer two advantages: (1) stern control of the water activity and/or pH in the filter bed is less important, since fungi are generally tolerant to low water activity and low pH, and (2) reduction of the water activity in the filter bed may improve the mass transfer of poorly water soluble waste gas compounds like styrene. Starch was shown in a study (Jasso, et al., 2004) to be useful in the degradation of polystyrene. In this study, results showed the effectiveness of concentrated activated sludge in 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 Preliminary Study on the Potential……..36 Dayao and Egloso, 2009 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 and inside 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. Further visual confirmation of deterioration of surface area is done by using the scanning electron microscope (Motta et al., 2007; Mor & Sivan, 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 permitting more of a specimen to be in focus. More so, due to its usage of electromagnets rather than A Preliminary Study on the Potential……..37 Dayao and Egloso, 2009 light, the degree of magnification could be much more controlled. (Prescott et al., 2005; Schweitzer, 2006; Chumbley, 2009) The scanning electron microscope functions by producing electrons thermoionically fired by an electron gun composed of tungsten filament cathode. Tungsten is used because of its high melting point and its lowest vapor pressure characteristics. The electron gun produces monochromatic electrons and vertically directs it to the two condensers which focus the beam to a spot of about 0.4 to 5 nm in diameter. The electron beam which has energy of a few hundred eV to 40 KeV travels in a vacuum environment and detected by detectors inside the SEM. The first condenser lens which is controlled by the coarse probe knob functions to form and restrict the amount of current in the beam. It also works with the condenser aperture, not user selectable, in discharging and constricting the high-angle electrons. The second condenser, on the other hand, is controlled by the fine probe current knob which functions to produce a thin, tight and coherent beam of electrons. Unlike in the former, the aperture in this condenser is user selectable and works also to remove high-angle electrons. After this, the beam is passed through a set of coils which scans the beam in a grid-like manner in a microsecond range to focus on points. The objective then allows the electron beam to concentrate and scan on the part of the specimen desired. Once the electron beam strikes the sample, interactions occur inside the sample; then, electrons and x-rays are emitted. The energy exchange between the electron beam and the sample results in the reflection of high-energy electrons by elastic scattering, emission of secondary electrons by inelastic scattering and the emission of electromagnetic radiation, each of which can be detected by specialized detectors. The detection of the interacting particles is detected by various instruments. These instruments count the number of interactions and show a pixel display on a CRT, the intensity then is determined by the number of reactions. The relationship of the two is said to be proportional: the more interactions, the brighter A Preliminary Study on the Potential……..38 Dayao and Egloso, 2009 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 (Prescott et al., 2005; Schweitzer, 2006; Chumbley, 2009). A Preliminary Study on the Potential……..39 Dayao and Egloso, 2009 MATERIALS AND METHODS Research Design The research design used in the study is the Randomized Complete Block Design (RCBD). The experiment consisted of three trials/runs with two replicates per strain treatment. The experimentation process was conducted at the National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines - Los Baños. Experimentation I. Preparation of Inoculum The stock cultures of Xylaria sp. which are the wild type (SDM), its three albino mutants (PNL 114, 116 and118) strains and two black strains (E25 and E36) were obtained from UPLB BIOTECH. Xylaria sp. strains were isolated by culturing them in a Potato Dextrose Agar (PDA) medium. The pH was then adjusted to pH 5.0 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. Inoculation was done using a cork borer with a diameter size of 0.5cm. The fungal pellets were bored at the margins of the colony to get actively growing and young hyphae. Two pellets were inoculated or transferred to each flask. The flasks were then subjected to shaking for enrichment and sustenance of growth. II. 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. They were subjected to surface A Preliminary Study on the Potential……..40 Dayao and Egloso, 2009 sterilization by shaking in 70% ethanol, once for 3 mins, then in sterile distilled water twice for 1min each, 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 mineral medium flask. B. Chicken feathers Fresh contour feathers from an adult, female Gallus sp. were obtained from a nearby market place where chickens are butchered and sold. The feathers were washed and 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 sterilized using an autoclave 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. They were subjected to surface sterilization by shaking in 70% ethanol, once for 3 mins, then in sterile distilled water twice for 1min each, One strip underwent SEM to check the initial surface condition of the latex glove. After which, two strips of the gloves were utilized per replicate of each treatment. This was then placed in a flask. A Preliminary Study on the Potential……..41 Dayao and Egloso, 2009 III. 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 adjusted to pH 5.0 by adding small amounts of either 0.1M NaOH or 0.1M HCl. The addition of sterilized substrate pollutant (either polystyrene, natural rubber or chicken feather) followed, then the inoculation of 2 ml of Xylaria sp. SDM wild type and it’s mutants in six flasks excluding the control. 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. Gentle scraping was applied to the samples to remove the mycelia and mucilaginous sheaths attached. Next, the pollutants were air dried. Then lastly, the final weight of the pollutants was determined using an analytical balance. Determination of Potential Degradation through Scanning Electron Microscopy 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 A Preliminary Study on the Potential……..42 Dayao and Egloso, 2009 increase, weight decrease and other physical changes 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. The wild type (SDM) micrograph was then compared with the micrographs of the five mutant species for each pollutant.
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