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					Partial Characteriztion of the Potential Biodegrading Ability of the Fungi Xylaria sp.
 and its Four Mutants, [mutant 1, mutant 2, mutant 3, mutant 4] 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


Objectives
       The 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 degrade/consume/ assimilate
         natural rubber as a carbon source
   2.    to determine if Xylaria sp. mutants and wild type can degrade/consume/ assimilate
         chicken feathers as a carbon and nitrogen source
   3.    to determine if Xylaria sp. mutants and wild type can degrade/consume/ assimilate
         polystyrene as a carbon source
   4.    to compare the biodegrading ability of the wild type to each mutant


Hypothesis

Significance of the Study

Scope and Limitations



                           REVIEW OF RELATED LITERATURE




                                     METHODOLOGY

Research Design

Collection of Materials

   I.       Preparation of Inoculum
               Isolate the Xylaria sp. by culturing it in a Potato Dextrose Agar (PDA)
        medium. Adjust to pH 5 and incubate at 25˚C. After 2-3 days, transfer the fungi into
        test media.
Preparation of Pollutants
         A. Petroleum

          B. Polystyrene
                    Cut 1x1 cm strips from polystyrene food containers
          C. Chicken feather
                    Obtain fresh feathers from Gallus gallus sp. Sterilize by putting inside
             SM plastic bag and autoclave for 20 mins. at 15psi.
          D. Rubber
                  - Obtain rubber latex gloves

Preparation of Test Media*
         E. For plates:
                   Prepare 2 sets of plates containing 20 ml PDA in triplicate (6 plates per
            mutant/wild type, per pollutant tested). Add 0.5% glucose in set A and add
            0.5% of the liquid pollutant in set B. Set A is to verify if the fungi inoculated to
            both set-ups are active. Adjust to pH 5 by adding small amounts of either
            0.1M NaOH or 0.1M HCl. Inoculate the fungi using agar discs from the PDA
            plate described in I. Agar discs will be obtained by cutting a 2-3 day-old
            inoculum on the peripheral area of a colony using a 5-8 mm diameter cork
            borer. The agar discs will be transferred to the PDA plates of sets A and B by
            using a sterile toothpick. Incubate at 25˚C.
                   Observe the colony growth in set B and compare it always with set A.
            Set A will be the control group and it would indicate whether the fungi
            transferred is active. Then measure the colony growth by its diameter.

          F. For flasks and test tubes:
                     Prepare 2 sets of flasks containing 50 ml Mineral Medium each, in
             triplicate. Add 0.5% glucose in set A and B. Adjust to pH 5 by adding small
             amounts of either 0.1M NaOH or 0.1M HCl. Then add the solid pollutant in set
             B only. Inoculate the fungi by using ………….. When all the glucose has been
             used up and the fungi had grown into a considerable mass as examined
             visually, add another MM + 0.5% glucose in set A only, leaving the set B
             flasks to utilize the solid pollutants as the sole carbon source. The extent of
             colonization should be carefully examined every day until rate of colony
             growth can be predicted (growth in mm/day). But if otherwise, continue
             adding the MMG (mineral medium+0.5%glucose) to both sets of flask until the
             fungi has grown and thrived. Incubate for 50-80days, with the flasks in a room
             with more or less 250C in temperature, under shaking conditions, while the
             test tubes in an incubator with 250C in temperature as well.

   II.    Remove solid pollutants from the culture medium and examine 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.


For Set B:

*NATURAL RUBBER
      Natural latex gloves will be cut into pieces with masses of 0.25 g Mineral medium
then autoclave subsequently. Inoculate the fungi after the medium has cooled. Then
incubate at 25 ˚C at pH 5. Or cultivate the fungi on a latex overlay agar plate technique
wherein a layer of concentrated natural rubber is added on top of the mineral medium or
Potato dextrose agar at a concentration of 0.02% (wt/vol).


*CHICKEN FEATHERS

The basic medium used for isolation and fermentation of the feather-degrading
microorganisms contained the following constituents (g/L): NaCl (0.5), KH2PO4 (0.7),
K2HPO4 (1.4), MgSO4 (0.1) and feathers (10), pH 7.2. Cultivation was done using 500 ml
Erlenmeyer flasks containing 100 ml medium. Feather agar medium containing the basic
medium and 20 g/L of agar was used for screening the microorganisms in plates. For the
medium used for screening mutants, 10 g casein was used instead of feathers. Luria-
Bertani (LB) medium (peptone 1% (w/v), yeast extract 0.3% (w/v), NaCl 0.5% (w/v), pH
7.2) was used for inoculum preparation and isolate maintenance.
       The fungi will be inoculated on the feather medium-containing flasks and will be
shaken at 25 ˚C.


*POLYSTYRENE
Prepare 2 sets of flasks containing 50 ml Mineral Medium each, in triplicate. Add 0.5%
glucose in set A and B. Adjust to pH 5 by adding small amounts of either 0.1M NaOH or
0.1M HCl. Ten pieces of polystyrene strips cut into 1x1 cm from polystyrene food
containers are added to the set A flasks only, and were then inoculated with a loopful of
Xylaria sp. mycelium. Set B without polystyrene strips were used as control. All the sets are
incubated for 50-80days, in a room with more or less 250C in temperature.

Determination of Amount of Degradation through Colonization
Percent weight loss of pollutant

Statistical Analysis

      The data that will be obtained from the percent weight loss of the pollutants: natural
rubber, chicken feathers and polystyrene, due to their colonization by Xylaria sp. and its
mutants will be subjected to ________________ followed by Duncan’s Multiple Range Test
(DMRT).
    RESULTS

LITERATURE CITED

				
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