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					Partial Characterization of the Potential Biodegrading Ability of Xylaria sp. on Natural
                     Rubber, Chicken Feathers, and Polystyrene




                                   A Thesis Proposal




                               In Partial Fulfillment of the
                                   Requirements For
                          Biology 200: Undergraduate Thesis
                                     AY 2008-2009




                                Dayao, Janine Erica P.
                              Egloso, Mary Bernadette V.




                                     August 1, 2008
                                      INTRODUCTION


Background of the Study
       Pollution is an inevitable problem due to population growth, urbanization and the
increased demand for manufactured products in the local and export markets.
Industrialization has resulted to the generation of wastes of various forms that pose serious
risks to the environment and public health, thus, requiring an efficient waste regulatory
management.
       At the advent of technology, pollution has indeed taken its toll on nature, making
people harvest and manufacture products that would take eons to decay and rot at the very
least. Everywhere, a plethora of biodegradable and non biodegradable wastes can be
seen. And, indeed it’s high time that people revert back to natural processes that could help
solve the burgeoning problem of waste disposal since all the artificial methods that require
today’s technology could contribute to the pollution that the planet’s experiencing now. One
such natural process that could solve the problem, or in a way even just alleviate such
waste pile-up, is biodegradation.
       The country’s population growth rate, which is one of the highest in the world
(Mangahas, 2006), places serious strains on the economy. In 2005, the population was
82.8 million, of which 51.8 million or 63% lived in urban areas. Metro Manila is the most
densely populated urban area with 10.7 million (Mangahas, 2006). Over the past 3
decades, the country’s economy slid behind many Asian economies. Gross domestic
product (GDP) grew at an average of only 3%, compared with 8% in the People’s Republic
of China (PRC); 6% in the Republic of Korea, Singapore, Malaysia, and Thailand; and 5%
in Indonesia over the last 30 years (Wallace Report 2004 as cited by Mangahas, 2006).
Urbanization, decline in the economy and further population growth lead to the even higher
generation of wastes that has not been managed properly and safely (DENR, 2004).
       Presidential Decree (PD) 1152, or “the Philippine Environmental Code,” provides the
basis for an integrated waste management regulation starting from waste source to
methods of disposal. PD1152 has further mandated specific guidelines to manage
municipal wastes (solid and liquid), sanitary landfills and incineration, and disposal sites in
the Philippines. Apart from the basic policies of PD1152, waste management must also
comply with the requirements and implementing regulations of other specific environmental
laws, such as PD984 (Pollution Control Law), PD1586 (Environmental Impact Assessment
System Law), RA8749 (Clean Air Act) and RA9003 (Ecological Solid Waste Management
Act) (DENR, 2004).
        A study by Clutario and Cuevas (2001) showed that Xylaria sp. can utilize
polyethylene plastic strips as an alternative carbon source. The fungus grew optimally at
250C on a mineral medium of pH5 containing 0.5% glucose and polyethylene plastic strips
as co-carbon source. A mucilaginous sheath was produced by the fungus to help its
mycelial growth adhere to the surfaces and edges of the plastic strips. After 50 days of
incubation, the strips became embedded in the mycelial growth. Visible damage on the
surface structure of the plastic strips was observed using scanning electron microscopy
(SEM). Striations and tearing were present due to the active burrowing of Xylaria hyphae
on the polyethylene material. This shows that Xylaria sp. has indeed a potential in
degrading synthetic wastes like plastics which are difficult to decompose.
        This proposed study aims to test the potential use of Xylaria sp. and its mutants as a
natural biodegrading agent in biodegrading other rampant wastes such as natural rubber,
polystyrene and chicken feathers.


Objectives

        The proposed study aims to determine the biodegrading capacity of Xylaria sp. wild
type and its mutants.
        The specific objectives are as follows:
   1.    to determine if Xylaria sp. mutants and wild type can assimilate natural rubber as a
         carbon source
   2.    to determine if Xylaria sp. mutants and wild type can assimilate chicken feathers as
         a carbon and nitrogen source
   3.    to determine if Xylaria sp. mutants and wild type can assimilate polystyrene as a
         carbon source
   4.    to compare the biodegrading ability of the wild type to each mutant to find which
         strain is most appropriate for each type of waste
Significance of the Study
       Findings of this study might be utilized in the development of Xylaria sp. as a good
biodegrading agent in reducing durable wastes such as plastics and others, as well as in
optimizing fungal technologies. This study may also provide a way or ways in the discovery
of other important characteristics of Xylaria sp. and its mutants, which may be used in other
applications and scientific investigations. The discovery of other sources of biodegradation
agents and their potential bioactive natural products is of paramount importance, especially
nowadays that people should mostly concern about their waste disposal methods, and also
to assure a good source of more accessible ways, through research, in approaching the
reduction of pollution that are safe and can possibly boost the Philippine fungal industry in
the world market.


Scope and Limitations
       The experiment will serve as a source of preliminary information on the potential of
Xylaria sp. to degrade chicken feathers, polystyrene and natural rubber. Other pollutants
with similar biochemical structure to the aforementioned pollutants will not be included in
the experiment. For the methodology, the Xylaria sp. that will be used will only come from
the stock culture of UP Los Baños Biotech Institute. Culture media and reagents will be
provided by BIOTECH. Experimentation will be done both in the Microbiology thesis room
of the UP Manila, College of Arts and Sciences and in the Antibiotic Laboratory in
BIOTECH. Isolation and purification of active components (i.e. enzymes) responsible for
the probable degradation of chicken feathers, polystyrene and natural rubber will not be
performed. Observation of colonization through scanning electron microscopy (SEM) will be
carried out in BIOTECH. Only the crude weight percent difference of the colonization of the
wastes will be recorded. The runs will only be done twice due to logistic matters and
unavailability of equipments. Moreover, the incubation time for the degradation process of
Xylaria sp. strains will only be limited to 30 days. In terms of data analysis, this experiment
will only focus in analyzing the degradation potential of Xylaria sp. strains on Natural
rubber, Chicken Feathers and Polystyrene. And also, it will be concerned
whether the degradation potential of the Mutants is significantly different from the Wild type
and so as to determine the more appropriate strain to use.




                           REVIEW OF RELATED LITERATURE


BIODEGRADATION
      Biodegradation is the breakdown of natural substances through the action of
enzymes secreted by organisms such as microbes and fungi. Only waste materials made
up of natural polymers can be degraded by microbes and fungi. Biodegradation works in
such a manner that the organisms involved utilize, or more appropriately, metabolize thes e
wastes as sources of nutrients such as carbon or nitrogen. (Tortora et al., 2005).
      In many cases, conditions are not favorable enough to promote spontaneous
biodegradation or natural attenuation. Whenever there is insufficient quantity of naturally
biodegrading organisms, there is a further need to add nutrients or other suitable
organisms aside from those present already, for improved biodegradation to occur. The
general objective of biodegradation is to discern the speed of unaided biodegradation
before catalysts may even be added, and then strengthen spontaneous biodegradation
only if this is not fast enough to remove the contaminant’s concentration in the environment
at which it may cause health risks to nearby inhabitants such as people, animals and plants
(European Federation of Biotechnology, 1999).


MICROORGANISMS USED IN BIODEGRADATION
      Through time, scientific experiments have already proven the ability of some
microorganisms to biodegrade pollutants such polyethylene, polystyrene, rubber, chicken
feathers and other types of wastes. Organisms such as bacteria and fungi have proven
themselves to possess the capacity to biodegrade pollutants.
      Bacteria such as Brevibaccillus borstelensis is one. Plastics in the form of
polyethylene are known to be degraded by the thermophilic bacterium Brevibaccillus
borstelensis 707 which was isolated from soil. (Hadad et al,2005). Another study by Orr et
al. (2004) featured the Rhodococcous ruber C208 as an effective polyethylene-degrading
organism. In addition to this, this strain has been proven to degrade polystyrene ( Mor &
Sivan, 20080. Yet originally, Rhodococcus rubber is a known rubber-degrading organism,
according to the review of Rose and Steinbuchel (2005). Other known rubber-degrading
microorganisms are Gordonia sp., Streptomyces sp., and Xanthomonas sp. strain 357.
Moreover, a study of Barraat et al. (2003) investigated the relationship of the water-holding
capacity of the soil and polyurethane degradation and found out that the following
organisms could biodegrade polyurethane: Nectria gliocladioides (five strains), Penicillium
ochrochloron (one strain) and Geomyces pannorum (seven strains).
      On chicken feathers, various strains had already been isolated too. In a study
conducted by Cao et al. (2008), the fungus Trichoderma atroviride completely degraded the
chicken feathers. This strain was actually isolated from a decaying feather. Another
organism proven to degrade chicken feather is Bacillus subtilis mutant strain as
investigated by the study of Cheng chang et al. (2003).
       The list of microorganisms that could be used in biodegrdation goes on for there are
still more species that could degrade pollutants. And in fact, in the Philippines a fungus has
been isolated and proven to degrade polyethylene (Cuevas & Manaligod, 1997).


Xylaria sp. AS A POTENTIAL AGENT FOR BIODEGRADATION
      Xylaria sp. was discovered by Cuevas and Manaligod (1997), growing on a sando
plastic bag, buried in forest soil and litter in the lowland secondary forest of Mt. Makiling,
Laguna. The fungus comprised of sterile melanin pigmented mycelia and was reported as
ascomycete sterile dark mycelia (ASDM). Cultural studies have designated it under Class
Ascomycetes, Order Xylariales, Genus Xylaria (Clutario and Cuevas, 2001).
      A previous study by Clutario and Cuevas (2001) proved that Xylaria sp. can utilize
polyethylene plastic strips as an alternative carbon source, thereby degrading them into
usable forms for self-sustenance. Through the use of scanning electron microscopy, the
proponents of the said study observed visible damages of the surface structure of the
plastic strips. There were tearing and striations caused by active burrowing of Xylaria
hyphae on the polyethylene material. Plastic is an extremely versatile synthetic material
made of high molecular weight, semi-crystalline polymer prepared from ethylene through
the cracking of crude oil, light petroleum and natural gas. For plastic bags alone, it is
estimated that some 430,000 gallons of oil are needed to produce 100 million pieces of
these omnipresent consumer items on the planet (Knapczyk and Simon, 1992; EcoWaste
Coalition, 2008).




                           Figure 1. Xylaria in its natural habitat.




   Figure 2. Xylaria fruiting body surrounded by flask-shaped structures called peritheca.




                         Figure 3. Cross section of Xylaria stroma.
       Xylaria is one of the most commonly encountered groups of ascomycetes with most
of its members being stromatic, peritheciate, with an iodine-positive ascus apical ring, and
with one-celled, dark ascospores on which agermination slit can be found. Most are
inhabitants of wood, seeds, fruits, or leaves of angiosperms. Some are associated with
insect nests. Most decay wood and many are plant pathogens. Many are endophytes. They
are commonly found throughout the temperate and tropical regions of the world. The
Xylaria sp. can be distributed above, around, and beneath perithecia. It forms a unipartite
stromatal layer, with a superficial or erumpent surface level. The interior of its stromata is
essentially homogeneous. Conidium-bearing discs, potassium hydroxide pigments and
orange granules surrounding the perithecia are absent (Rogers et al., 2002). They are
mostly multiperitheciate in ascomatal number per stroma, ascomatal ostioles and ascal
apical rings: are present, and the ascospore cell number is one-celled. Teleomorph and
anamorph are produced on the same stromata in most species, with their anamorphs:
Geniculosporium-like.
       Some Xylaria sp. species exist as endophytes, and have mutualistic associations
with plants. The fungus secrete toxins to protect the plant from herbivory from other insects
or animals, while the fungus in return feeds on the host’s tissues for nutrition, and its
mycelia are scattered through seed dispersal. Xylariaceae endophytes are hypothesized to
be quiescent colonizers that decompose lignin and cellulose when a plant dies .
Nonetheless there are also some xylariaceous fungi that only exist as endophytes. No
obvious benefit to living host plants has been documented for Xylariaceae (Petrini et al.,
1995; Whalley, 1996; Rogers, 2000; Davis, et al., 2003)
       A review of empirical studies on antagonistic interactions between endophytes and
grazers, insects and microbial pathogens summarizes five general properties of endophyte
mutualism: (1) the endophyte is ubiquitous in a given host, geographically widespread, and
causes minimal disease symptoms in the host plant; (2) vertical transmission or efficient
horizontal transmission of the fungus occurs; (3) the fungus grows throughout host tissue,
or, if confined to a particular organ, a high proportion of such organs are infected; (4) the
fungus produces secondary metabolites likely to be antibiotic or toxic; and (5) the
endophyte is taxonomically related to known herbivore or pathogen antagonists (Carroll,
1988; Davis, et al., 2003).
SOLID WASTE IN THE PHILIPPINES
        Filipinos generate around 0.3 to 0.7 kilograms of garbage daily per person
depending on income levels (World Bank, 2001). Metro Manila produces about 8,000 tons
of solid waste each day and is expected to reach 13,300 tons each day in 2014 (Baroña,
2004). The National Capital Region produces the highest amount of wastes, about 23% of
the country’s waste generation (Anden and Rebolledo, 2003).
        Based on studies (2001) made by the National Solid Waste Management
Commission Secretariat based at the Environmental Management Bureau (EMB), it is
estimated that in Metro Manila, the per capita waste production daily is 0.5 kg. Thus, every
person living in the metropolis generates half a kilo of waste a day. With an estimated
population of 10.5 million, total waste generated in Metro Manila alone could run up to
5,250 metric tons per day or 162,750 metric tons per month or 1.95 million metric tons per
year.
        Based on another EMB study (2001) regarding the disposal of daily wastes, only
about 73% of the 5,250 metric tons of waste generated daily are collected by dump trucks
hired by local government units. The remaining 27% of daily wastes, or about 1,417.5
metric tons, end up in canals, vacant spaces, street corners, market places, rivers and
other places.
        According to a survey conducted by the EcoWaste Coalition and Greenpeace
Southeast Asia in 2006, synthetic plastics comprise 76% of the floating trash in Manila Bay,
out of which 51% are plastic bags, 19% are sachets and junk food wrappers, 5% are
styrofoams and 1% is hard plastics. The rest were rubber (10%) and biodegradable
discards (13%) (EcoWaste Coalition, 2008).


Polystyrene
           Polystyrene, an aromatic polymer, is synthesized from the aromatic monomer
styrene which comes from petroleum products. It is a thermoplastic substance that could be
solid in room temperature or liquid when melted. One of the most common forms and uses
of polystyrene is the EPS which stands for Expanded Polystyrene. The industry
manufactures such product by mixing polystyrene with blowing agents in the form of carbon
dioxide and pentane which comprises 5%-10% of its composition. The EPS is also called
foamed polystyrene and it is said to be 30 times lighter than regular polystyrene. This
substance is popularly used in the form of beverage cups and insulating materials. (Friend,
2005). The basic unit of polystyrene which is styrene, which is a known neurotoxin and
animal carcinogen, is considered very harmful to human health. In fact, it inflicts
neurological and hematological disorder especially to factory workers. EPS food packaging
is the one accountable for the leaking out of styrene. Styrene leak or leech is triggered
when acids from our juices when placed in such EPS cups and when food with Vitamin A
content is placed inside a microwave leading the styrene to accumulate in our system.
(Californians Against Waste, 2008).
       Polystyrene is in high demand. It is the most used and utilized thermoplastic in the
industry due to its durability. But it is not biodegradable. (Mor and Sivan, 2008). According
to the Californians Against Waste (2008), it is very difficult to recycle due to its light weight
property, which accounts for why it’s expensive to recycle. Imagine just recycling a ton of
polystyrene, needs a budget of $3000. Hence, it has a negative scrap-value. More so, it’s
due to this light weight property that they find polystyrene hard to transport since
polystyrene is advised to be always kept food-free and uncontaminated when recycled. The
build-up of polystyrene in landfills, as reported by CAW (2008), will contribute to plastic
marine debris, since even when it is disposed of properly it is carried by natural agents
such as wind or other forces to the ocean. As manifested, there is an excess of it in the
environment and it is a major pollutant. (Mor and Sivan, 2008). For almost three decades
ago, polystyrene was first ban due to the utilization CFC material for its generation. In fact
there was a hype heralding that it is recyclable. After some time the companies that
invested for its recycling process disappeared. This move confirms that, indeed, recycling
polystyrene is not an easy thing to do. Now, the problem is back and the attention of
scientists is focused on the recycling of disposable foamed polystyrene. But recycling it
would cost much in terms of energy, waste and management point of view. (Californians
Against Waste, 2008). A way of solving such impending problem, is through biodegradation
(Mor and Sivan, 2008; Singh and Sharma, 2007).


       Biodegradation has been manifested in a number of studies already. And some of
the studies will be named here. A study by Mor and Sivan (2008), dealt with the monitoring
of biofilm formation of the microbe Rhodococcus sp. strain C208 on polystyrene. Their aim
was to observe the kinetics of biofilm formation and of whether polystyrene would be
degraded. They used two methods in quantifying the biofilm biomas: modified crystal violet
staining and observation of the protein content of the biofilm. The C208 strain was cultured
in a flask containing polystyrene flakes with the addition of mineral oil (0.0055% w/v), which
induced more biofilm buil-up. The study concluded that after an extension of 8 th weeks of
incubation, loss of 0.8% (gravimetric weight loss) of polystyrene weight was found. From
this, Mor and Sivan (2008) regarded C208 to demonstrate a high affinity towards
polystyrene through biofilm formation which lead to it’s degradation. The C208 strain is a
biofilm-producing actinomycete that has first colonized and degraded polyethylene (Orr et
al., 2004).
       There were studies that tested the possibility of whether copolymerizing polystyrene
with other substance could make it more degradable and susceptible to microbial attack. In
1992, a study by Milstein at el. (1992), focused on the biodegradation of a lignin-
polystyrene copolymer. The white rot basidiomycete was used to degrade such lignin-
polystyrene complex copolymer. Such fungi released enzyme that oxidized lignin and
demonstrated the degradation through weight loss, UV spectrophotometric analysis and
deterioration of surface of the plastic substance as seen under the SEM. A similar study by
Singh and Sharma (2007) demonstrated through the process of graft copolymerization that
polystyrene must be modified with natural polymers and hydrophilic monomers so as to
enhance its degrading ability and so as to render polystyrene waste useful in diminishing
metal ion pollution in water and. According to the mentioned study, the degrading rate of
polystyrene increased to 37% after subjecting it to soil burial method for 160 days.
       Furthermore, the study of Motta et al. (2007), explored the degradation of oxidized
polystyrene using the fungi Curvularia sp. After about nine weeks of incubation,
microscopic examination revealed that hyphae had grown on the polystyrene. The
colonization of the fungi and it’s adhesion to the surface of the substance, according to
Mota et al., is a crucial step towards polymer biodegradation.


       As demonstrated, colonization is needed in determining whether a particular microbe
or organism is a potential biodegrading agent. (Mota et al., 2007) The growth of the
microbes on the surface of the polystyrene is a step that would lead to its degradation.
Further visual confirmation of deterioration of surface area is done by using the scanning
electron microscope. (Mor and Sivan, 2008; Mota et al., 2007).


Natural Rubber
        Natural rubber (NR) is made from the latex of the Hevea brasiliensis also known as
the rubber tree. It is mainly composed of cis-1,4 polyisoprene which has a molecular mass
of about 106 Da. This could also be chemically synthesized and produce the substance
known as Isoprene Rubber (IR). (Linos et. al, 2000).
        Since 1914, natural rubber has been a classic subject of biodegradation studies.
(Rose and Steinbuchel, 2005). This is due to the high rate of its yearly manufacture which
is several million tons, as mentioned in the study of Bereeka (2006), and its slow rate of
natural degradation as reviewed by Rose and Steinbechul (2005). In fact, a number of
studies abound concerning its degradation. And it has been learned that both bacteria and
fungi   can   participate   in   such   process.   Throughout   all   the   investigations   and
experimentations done, two categories of rubber-degrading microbes according mainly on
growth characteristics have been established. Based on a review of Rose and Steinbuchel
(2005), which recapped the aforementioned groups, the microbes that can degrade rubber
can be categorized as clear zone-forming around their colonies and non-halo forming
whenever isolated and cultured in latex overlay plate, which is made by overlaying a layer
of latex agar medium on a basal salt medium agar. The former category was identified to
mainly consist of actinomycetes species. They are said to biodegrade or metabolize rubber
by secreting enzymes and other substances and also they are dubbed to be slow
degraders since they rarely show an abundant cell mass when grown on natural rubber
directly. On the other hand, members of the second group do not form halos on latex
overlay plates. They, unlike the first group, grow more when directly grown on natural
rubber. In a way, their growth on rubber could be described in an adhesive manner. The
second group is said to demonstrate a relatively stronger growth on rubber. Species
comprising this category are the Corynebacterium-Nocardia-Mycobacterium group. They
consist of the Gordonia polyisoprenivorans strains VH2 and Y2K, G. westfalica strain Kb1,
and Mycobacterium fortuitum strain NF4.
       As demonstrated by a particular study by Linos et al. (2000), the mechanisms that
the microbes undergo when biodegrading is colonization, biofilm formation and aldehyde
group formation after cultivating it on the surface of latex gloves. Such is revealed after
undergoing the Schiff reagent’s test. This is further examined under a scanning electron
microscope.    In their methodology they have indicated that the preliminary screening
method to be used in finding potential rubber-degrading bacteria is by growing such
bacteria or microbe on the latex overlay or by latex film on the mineral agar plates. Growth
and colonization of the microbe in this medium would indicate its utilization of rubber as its
sole carbon source; hence, making it as a potential rubber-degrader.


Chicken Feathers
       In the US alone, 2 billion pounds of chicken feathers are produced by the poultry
industry (Comis, 2008). Chicken feathers, by nature, are made up of over 90% protein
(Cheng-cheng et al., 2008). And this protein is none other than keratin. It’s actually the
most abundant protein. It is not easily degraded due to its tightly packed structural
arrangement which is in the form of alpha keratin or beta keratin. The key to its stability lies
on the cross-linking by disulfide bonds, hydrophobic interactions, and hydrogen bonds.
Such stability renders keratin water-insolube and non-degradable by the enzymes papain,
trypsin and pepsin. (Gradisar et al., 2005). In a study conducted by Onifade et al., 1998 and
Goushterova et al., 2005, as cited in the journal of Cheng-Cheng et al., 2008, the build-up
of chicken feathers in the environment and landfills would only result to future pollution
problems and protein wastage. More so, its accumulation could serve as a breeding ground
for a variety of harmful pathogens (Singh, 2004).
       Considering that chicken feathers have a high protein content it could also be used
as an animal feed, but first its protein must be degraded (Tapia & Contiero, 2008). Yet this
is said to need so much water and energy (Frazer, 2004). Old methods of degrading the
chicken feathers such as alkali hydrolysis and steam pressure cooking are no longer
advisable. They cause so much energy wastage and they unfortunately destroy the
configuration of proteins. (Cheng-cheng et al., 2008).
         Incineration is also a method used in degrading such waste but it causes so much
energy loss and carbon dioxide build-up in the environment. Other methods of disposal are
landfilling, burning, natural gas production and treatment for animal feed. But subjecting it
to burning and land filing costs a lot and it contributes air, soil and water contamination.
(Joshi et al., 2007).
         A wiser suggestion or approach would be the use of microbes in degrading these
chicken feathers. (Cheng-cheng et al., 2008). Such approach is said to an economical and
environment-friendly alternative (Joshi et al., 2007). Experiments that tested on the
degradation of chicken have already been done. In fact, studies have already proven that
keratinolytic microbes such as Bacillus (Maczinger et al., 2003; Rodziewicz and Wojciech,
2008; Joshi et al.,2007) , fungi ( Gradisar et al., 2005) and actinomycetes (Goushterova et
al., 2005). The enzymes that perform keratin degradation are called keratinase, which
could degrade feathers and make it available for its use as animal feed, fertilizer and
natural gas. The enzymes are said to degrade the beta-keratin component and the main
idea behind such biodegradation is that the microbes use the feather as their carbon,
nitrogen, sulfur and energy for their nourishment. (Joshi et al.,2007 ; Manczinger et al.,
2003).
         Keratinases isolated from microbes have various economic uses. Aside from its
feather degrading capacity, it could be used in the leather industry as an agent in dehairing
leather. Its by-product, the feather hydrolysate, could also be used as animal feed additive.
(Joshi et al., 2007). Furthermore, potentially, the said hydolysate could be used in the
generation of organic fertilizer, edible films and amino acids which are considered rare, as
cited by Brandelli in the journal of Joshi et al.( 2007).
         In terms of experimental procedures, various methods are used in determining the
keratinolytic ability, which means it could produce keratinase and hence degrade chicken
feather, of microbes. A particular study by Tapia and Contiero (2008), used a feather meal
agar, wherein the feather served as the source of carbon, nitrogen, sulfur and energy, in
cultivating the isolated microbe Streptomyces. The growth, which occurred on the 10 th day
of incubation, through colony formation of the microbe indicated that it utilized the feather
as a source of its nutrients. After which, its keratinolytic activity was tested using a modified
keratin azure protocol. Another study by Maczinger et al. (2003) focused on the isolation of
a microbe from the poultry waste that could degrade feathers. During the preliminary
elimination, they cultured the different population of bacteria found in a partially degraded
feather in a basal medium with sterilized feathers serving as its source of carbon, nitrogen
and sulfur. It was then rotated in an orbital shaker for 10 days. After 4 days, one flask which
showed a visual degradation of the feather. A dilution series was made afterwards so as to
isolate and culture the bacteria that just degraded the feather. The strain was identified as
Bacillus lichenformis strain K-508. And the confirmation of the keratinolytic activity was
done by using the azokeratin as a substrate assay.
       Isolation of a new microbial organism that could degrade chicken feather will help in
the degradation of the chicken feathers which is now becoming a burden in the society both
internationally and locally. The microbe could potentially provide the keratinase that could
be used in compost technology (Maczinger et al., 2003) or in the conversion of feather to
feedstock meal additives (Tapia and Contiero, 2008).


                                      METHODOLOGY


I. Research Design
       The research design to be used in the study is the Randomized Complete Block
Design (RCBD). The experiment will consist of two trials with three replicates per treatment.
The experimentation process will be conducted in UP Los Banos Institute of Biotechnology.


II. Experimentation


A. Preparation of Inoculum
       The stock culture of Xylaria sp. and its four albino mutant strains will be obtained
from UPLB Biotech. Xylaria sp. will be isolated by culturing it in a Potato Dextrose Agar
(PDA) medium. The pH will be adjusted to pH 5 and it will be incubated at 25˚C. After 2-3
days, the fungi will be transferred into the test media.


B. Preparation of Pollutants
          A. Polystyrene
                     1x1 cm strips will be cut from clean polystyrene food containers such
             as coffee foam cups. And four strips per replicate of each treatment will be
             used.


          B. Chicken feathers
                     Fresh feathers from Gallus sp. will be obtained from a nearby market
             place where chickens are butchered and sold. The feathers will be sterilized
             by placing it inside a SM plastic bag and autoclaved for 20 minutes at 15psi.
             One feather per replicate of treatment will be used.


          C. Rubber
                     Obtain rubber latex gloves size 5 from Mercury Drugstore. Cut the
             gloves into strips of the same sizes, approximate area to be about 2x2. Use 2
             strips per replicate of each treatment.




C. Biodegradation Proper using Culture Method
      Two sets of flasks will be prepared containing 50 ml Mineral Medium each, in
triplicate. 0.5% glucose will be added in set A and B. The pH will be adjusted to pH 5 by
adding small amounts of either 0.1M NaOH or 0.1M HCl. Then the microorganism will be
added using the wire loop inoculation method. The addition of pollutant in set B only will
follow right after. The flasks will be subjected in a shaker for four hours to homogenize the
medium. The culture will be observed for some time then when all the glucose has been
used up and the fungi have grown into a considerable mass as examined visually, another
MM + 0.5% glucose will be added in set A only, leaving the set B flasks to utilize the solid
pollutants as the sole carbon source. The extent of colonization will be carefully examined
every day until rate of colony growth can be predicted (growth in mm/day). But if otherwise,
the addition the MMG (mineral medium+0.5%glucose) to both sets of flasks will be
continued until the fungi have grown and thrived. Incubate for30 days, with the flasks in a
room with more or less 250C in temperature.The solid pollutants will then be removed from
the culture medium and examined under a scanning electron microscope (SEM).
Note:
* this step is intended for each mutant and for the wild type. Since we have 4 mutant strains
and a wild type, this step will be repeated five times multiplied with the number of pollutants
to be used.


D. Determination of Amount of Degradation through Colonization
        After 30 days, the remaining pollutant will be weighed in grams. This measurement
will be recorded as the final weight. The percent weight loss of pollutant will be determined
using the formula:
                      % weight loss = (initial weight – final weight)
                                               Initial weight


Statistical Analysis
        The designed that will be used for this study is Random Complete Block Design
(RCBD). The blocks will be the Xylaria sp. and the mutants while the treatment will be the
three pollutants namely natural rubber, chicken feather and polystyrene.


Your study design is RCBD so your analysis should therefore be ANOVA for RCBD (Which
you can easily analyze using Microsoft excel….and no need to put the software to be used
in your proposal). Then you should put an ANOVA table without data after your dummy
tables. Xylaria sp and its mutants will serve as the blocks since you will be randomly
distributing the pollutants to them. Your DATA/DUMMY table #2 (since okay na yung first
dummy table ninyo na kasama yung replicates) will then look like this (please write an
appropriate title above it):
                               Xylaria   Polystyrene Natural    Chicken
                               strain                Rubber     Feathers
                               Wild-
                               type
                               Mutant1
                               Mutant2
                               Mutant3
                             Mutant4




Hypothesis
(Hypotheses for the blocksF for rows)
Ho1: There is no significant difference in the biodegrading ability of the different Xylaria sp.
strains on the pollutants.
Ha1: There is a significant difference in the biodegrading ability of the different Xylaria sp.
strains on the pollutants.


(Hypotheses for the pollutantsF for columns)
Ho2: There is no significant difference in the degree of biodegradation of polystyrene,
natural rubber & chicken feathers due to Xylaria sp.
Ha2: There is a significant difference in the degree of biodegradation of polystyrene, natural
rubber & chicken feathers due to Xylaria sp.




Dummy Tables


**Table 1 will be use for each pollutant and such table would be used for the two runs that
will be conducted.


Table 1. Percent Weight Loss of the (insert pollutant name here) due to Xylaria sp. strains
                     Replicate 1       Replicate 2        Replicate 3         Mean
Wild type
Mutant 1
Mutant 2
Mutant 3
Mutant 4
Table 2. Mean Values of the Percent Weight Loss of Polystyrene, Natural Rubber and
Chicken Feathers due to degradation of Xylaria sp. strains


             Xylaria strain        Polystyrene Natural       Chicken
                                                Rubber       Feathers
             Wild-type
             Mutant1
             Mutant2
             Mutant3
             Mutant4




Table 3. ANOVA for Randomized Complete Block Design of the Potential Biodegrading
Ability of Xylaria sp. Strains on Chicken Feathers, Natural Rubber and Polystyrene
Source of Variation      SS      df       MS        F         P-value   F crit
Rows         (Xylaria
strain)
Columns (pollutant)
Error
α = 0.05




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