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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).
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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 20 th 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
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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
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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).
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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 a nd 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
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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
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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,
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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).
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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).
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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.
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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
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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)
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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).
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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
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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.
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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 10 th day of incubation, through colony
formation of the microbe indicated that it utilized the feather as a source of its nutrients. After
which, its keratinolytic activity was tested using a modified keratin azure protocol. Another study by
Maczinger, et al. (2003) focused on the isolation of a microbe from the poultry waste that could
degrade feathers. During the preliminary elimination, they cultured the different population of
bacteria found in a partially degraded feather in a basal medium with sterilized feathers serving as its
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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
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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,
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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
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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
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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
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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/m 3 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.
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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
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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 bea m 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
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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).
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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
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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.
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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 25 0C in
temperature. Yet observations were made on the 20 th, 30th and finally on the 50 th day. The
observations done on the 20 th 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
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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.
