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					                 A Preliminary Study on the Potential
                  Biodegrading Ability of Xylaria sp.
                 on Natural Rubber, Chicken Feather
                  and Polystyrene through Scanning
                         Electron Microscopy
                   Janine Erica P. Dayao1, Mary Bernadette V. Egloso1,
                  Anna Theresa A. Santiago1 and Mary Ann T. Tavanlar2
                 1Department   of Biology, College of Arts and Sciences, University of the
                            Philippines Manila, Padre Faura St., Ermita, Manila
                 2National Institute of Molecular Biology and Biotechnology, University of the

                            Philippines Los Baños, College Laguna 4031


Dayao & Egloso
   (2009)
                          A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                                 Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Introduction


Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                        Chicken Feather




                 Medical Waste
                 including NR gloves




                 Polystyrene




Dayao & Egloso
   (2009)
                         A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                                Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                              Biodegradation
                 • breakdown of natural substances
                   through the action of enzymes
                   secreted by organisms
                 • natural and synthetic polymers
                   containing specific functional
                   groups, are potential substrates
                   for heterotrophic organisms
                 • organisms involved utilize or
                   metabolize these wastes as
                   sources of nutrients
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Xylaria sp. wild type and
                          mutants



Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • 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
                 • comprised of sterile melanin pigmented
                   mycelia
                 • reported as ascomycete sterile dark
Dayao & Egloso
   (2009)          mycelia (ASDM).
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Cultural studies: Class
                   Ascomycetes, Order Xylariales,
                   Genus Xylaria.
                 • Clutario and Cuevas (2001)
                  –Xylaria sp. can utilize polyethylene
                   plastic strips as alternative C source
                  –observed visible damages of the
                   surface structure (tearing and
Dayao & Egloso     striations caused by active burrowing)
   (2009)
                   A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                          Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • one of the most commonly encountered
                   groups of ascomycetes
                 • stromatic, peritheciate, with an iodine-
                   positive ascus apical ring, and with one-
                   celled, dark ascospores on which a
                   germination slit can be found.
                 • saprobic on decaying hardwood stumps
                   and logs
                 • quiescent colonizers that decompose
                   lignin and cellulose when a plant dies
                 • Some are associated with insect nests.
                 • many are plant pathogens and
                   endophytes.
                 • temperate and tropical regions of the
Dayao & Egloso
   (2009)
                   world.
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Xylaria sp. variant strains: SDM/Wild
                   type, PNL 114, PNL 116, PNL 118,
                   E26, E35
                 • wild type SDM mutagenesis +
                   protoplast fusion (thru NTG)
                 • determined morphological and
                   biochemical characteristics or markers
                   in the wild type and mutants
                 • High deposition of melanin in wild type
                   SDM
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                  Comparative growth of the PNL mutants and
                 wildtype SDM on MMG and mineral medium with
                             various supplements.

                                       Average diameter of colony (mm)
                    Mutant                         Medium
                    Strain           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


                  Measured after 4 days incubation at ART:
                          M1 = MMG + 0.005% benomyl
                          M2 = MM + 0.025% glucose + 1% acetamide
                          M3 = MM + 0.025% glucose + 1% PEG
                          M4 = MM + 0.025% glucose
                          M5 = MM + 0.5% glucose
Dayao & Egloso
   (2009)
                       A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                              Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Growth of the four albino mutants on mineral
                 medium with and without supplements as
                     compared to the wildtype SDM.

                                          Average diameter of colony
                       Mutant                       (mm)
                       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


                 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
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Growth of the two black mutants on mineral medium
                       with supplements MDPE and PEG


                                                           Average diameter of
                                     Mutant                   colony (mm)
                                     Strain               MMMDPE MMPEG
                                      E26                    25.9       23.85
                                      E35                   40.15       30.65


                            MMMDPE = MM + medium density polyethylene
                            MMPEG = MM + polyethylene glycol 6000




Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Results of Characterization Study (Tavanlar & Lat, 2008)
                 less dependent on glucose in the medium for growth and
                 hyphal tip extension.
                 loss of melanin pigmentation
                 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, Tween 80 and
                 acetamide as source of carbon as compared to the wild type
                 may potentially exhibit enhanced degradation of polyethylene
                 plastics than the wild type
                 may better survive environments with less available amounts
                 of readily utilizable carbon sources such as the surface of
                 plastics than the wild type

Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 POLLUTANTS




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Natural Rubber


                 • Hevea brasiliensis a.k.a rubber
                   tree
                 • cis-1,4 polyisoprene
                 • Biodegradation of rubber slow
                   process
                        Rose & Steinbuchel (2005) and
                   Bereeka (2006)
Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Chicken Feathers
                 • In the Philippines, chicken feathers
                   aren’t a publicly recognized problem
                 • build-up in the environment and
                   landfills  result to future pollution
                   problems and protein wastage
                 • accumulation could serve as a
                   breeding ground for various harmful
                   pathogens
                 • in the US, 2B lbs produced by the
                   poultry industry
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 CF = 90% keratin
                 • most abundant protein
                 • not easily degraded  tightly packed
                   structural arrangement α-keratin/β-keratin
                 • Stability higher degree of cross-linking by
                   disulfide bonds, hydrophobic interactions, and
                   hydrogen bonds
                   – water-insoluble
                   – extremely resistant to biodegradation
                   – poorly susceptible to digestion by the most
                     common peptidases like papain, trypsin and
Dayao & Egloso
                     pepsin
   (2009)
                      A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                             Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                  Keratinase
                 • A group of proteolytic enzymes
                   capable to hydrolyze insoluble
                   keratins more efficiently than other
                   proteases
                 • degrade the beta-keratin component
                 • microbes use feather as C, N, S
                   and energy for their nourishment
                 • inducible whenever substrates of
                   keratin composition are present
Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Polystyrene
                 • an aromatic polymer and an
                   inexpensive, hard plastic
                 • synthesized from the aromatic
                   monomer styrene, from petroleum
                   products.
                 • thermoplastic solid in room
                   temperature or liquid when melted
                  – polymer chains are only weakly bonded
                   (van der Waals forces)
                 • most common form  EPS/Expanded
Dayao & Egloso
   (2009)
                   Polystyrene/Styrofoam
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Polystyrene


                 • one of the packaging industry's
                   toughest environmentally
                   challenging products
                  – enormous sustaining longevity
                  –High durability but it is not
                   biodegradable
                  –very difficult to recycle light weight
Dayao & Egloso
   (2009)
                   property
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • known neurotoxin and
                     animal carcinogen
                 • mimics estrogen  disrupt normal
                   hormone functions (thyroid problems,
                   menstrual irregularities, breast and
                   prostate cancer)
                 • low platelet counts or hemoglobin values,
                   chromosomal and lymphatic abnormalities
                 • EPS food packagingresponsible for the
                   leaking/leeching out of styrene is.
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Specific Objectives



Dayao & Egloso
   (2009)
                   A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                          Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 1. to determine if Xylaria sp. mutants and
                    wild type have the ability to colonize
                    and degrade through Scanning
                    Electron Microscopy

                 2. to compare the biodegrading ability of
                    the wild type to each mutant

                 3. to observe the macroscopic/physical
                    changes of the pollutants after
                    incubation with the Xylaria sp.
Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Significance of the
                        Study



Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • serve as a source of preliminary
                   information on the potential of Xylaria
                   sp. strains

                 • findings might be utilized in the
                   development of Xylaria sp. as a good
                   biodegrading agent

                 • may provide a possible means to
                   discover and understand other
                   important characteristics of Xylaria sp.
                   and its mutants
Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Methodology


Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Xylaria sp. variant strains:
                   SDM/Wild type, PNL 114, PNL
                   116, PNL 118, E26, E35




Dayao & Egloso
   (2009)
                   A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                          Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                        Mineral
    Plate/slant
                                                        Medium
     of stock              PDA
                                                      (0.5% gluc;
      culture
                                                       pH5;25˚C)




Dayao & Egloso
   (2009)
                  A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                         Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                              15mL
                             Mineral
                             Medium




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                       0.5%
                                                       Glucose




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                              0.1M
                             NaOH/
                            0.1M HCl




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                    Substrate
                                                    pollutant




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                                                Natural rubber
                                                    Substrate
                                                                                 Chicken feather
                                                    pollutant
                                                                                 Polystyrene




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                         2 ml
                      Xylaria sp.




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                                               SDM/Wild type

                                                       2 ml                    PNL 114
                                                    Xylaria sp.
                                                                               PNL 116

                                                                               PNL 118

                                                E35                 E26




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                    PNL114




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                              PNL114               PNL114




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                  SDM/         SDM/
                  Wild         Wild
                  type         type               PNL116         PNL116                  E26            E26




                 PNL114      PNL114               PNL118         PNL118                  E35            E35




Dayao & Egloso
   (2009)
                   A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                          Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • NATURAL
                                                        SDM/        SDM/
                                                        Wild        Wild
                                                        type        type       PNL116      PNL116        E26       E26


                     RUBBER
                                                       PNL114     PNL114       PNL118      PNL118        E35       E35




                                                        SDM/        SDM/

                 • CHICKEN                              Wild
                                                        type
                                                                    Wild
                                                                    type       PNL116      PNL116        E26       E26



                      FEATHER
                                                       PNL114     PNL114       PNL118      PNL118        E35       E35




                                                        SDM/        SDM/
                                                        Wild        Wild

                 • POLYSTYRENE                          type        type       PNL116      PNL116        E26       E26




Dayao & Egloso                                         PNL114     PNL114       PNL118      PNL118        E35       E35
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
          NATURAL RUBBER CHICKEN FEATHER                                              POLYSTYRENE


RUN 1




RUN 2




RUN 3




 Dayao & Egloso
    (2009)
                   A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                          Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Substrates
                   cut to 0.5
                   x0.5 cm
                   per piece




Dayao & Egloso
   (2009)
                         A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                                Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Results and Discussion



Dayao & Egloso
   (2009)
                   A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                          Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                               Natural Rubber
                  before                                                    after

                                                                                                        E26



                                                                                                         116
                   Control


Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                           Chicken Feathers
                  before                                                    after


                                                                                                        E35



                    Control

Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                      Polystyrene
                  before                                                    after

                                                                                                         118



                                                                                                        E35

                  Control

Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                    SDM wild type                                   E26                                       E35
                                                                                                                     Staling
                                                                            Staling                                  products
                                                                            products
                 Staling
                 products




Dayao & Egloso        PNL 114                                    PNL 116                                PNL 118
   (2009)
                            A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                                   Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 SDM wild type                             E26                                       E35




                                                                                                               Staling
                                                                                                               products




Dayao & Egloso      PNL 114                                PNL 116                               PNL 118
   (2009)
                      A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                             Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 SDM wild type                        E26                                      E35

                       Staling
                       products




                    114                                   116                                     118
Dayao & Egloso
   (2009)
                      A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                             Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                           Discussion
                 • mycelia have adhered on the surface of
                   the pollutants especially for natural
                   rubber and polystyrene.

                 • Even after gentle scraping and washing
                   with 70% ethanol and water,
                        the mycelia have remained
                   embedded on the surface of the
                   substrate.
                       proof of non-aggregation on the
                   surface of the natural rubber.
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Observed biofilm formation in the form of
                   mucilaginous sheaths
                 • Xylaria strains forms biofilm
                 (M.A Tavanlar,personal communication, March 9,2008)



                 • BIOFILM
                  mechanism where microorganisms secrete
                   proteins and carbohydrates (EPS) for their survival
                   in environments which are low in nutrients
                  indicate solid substrate utilization by the
                   microorganisms.
                  1st step : COLONIZATION
                  regarded as an initial step in biodegradation.
                                   fungi and bacteria have been shown to attack and form
                                    biofilms on natural rubber substrates

                                    (Morton & Surman, 1994; Linos et al., 2000)
Dayao & Egloso
   (2009)
                           A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                                  Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Since biofilm formation was
                   observed, the general changes
                   could suggest that the fungus has
                   undergone the 1st step in
                   biodegradation.
                 • Biofilm formation = biodegradation
                   potential


Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Natural rubber                          The areas where the mycelia have
                                                         aggregated were observed to have
                                                           undergone elasticity reduction

                 Chicken Feather                       Generally, the samples have retained
                                                       original physical characteristics such
                                                           as hardness and color, when
                                                          compared to the non-inoculated
                                                                       control
                   Polystyrene                            The colonized areas were mycelia
                                                           have penetrated and cannot be
                                                          removed thru mere scraping were
                                                            observed to be softer than the
                                                                 uncolonized areas




Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Microscopic Results




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 NATURAL RUBBER




Dayao & Egloso
   (2009)
                  A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                         Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                                                             hy


                                                                                      my



                                                                                                  hy                my
CONTROL                                                   SDM




                                                                                                     my

                 sp                          hy

                          hy


                                                                                                hy

Dayao & Egloso            my
   (2009)
   PNL 114                                                  PNL 116
                      A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                             Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 hy
                                                                                                   mc




       my

                                                                  E26
   PNL 118




                      sp




                            my




Dayao & Egloso   hy
   (2009)
 E35                                                             CONTROL
                       A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                              Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Chicken Feather




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                                                                                         br

                                                                              my


                                                              bl                                            br




CONTROL                                                   SDM



                                                                                                     bl
                                                              br




                                     bl
                 my                                                           my

          br


Dayao & Egloso                                                                                                      br
   (2009)
PNL 114                                my                    PNL 116
                      A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                          bl Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                     my




                                                       r                                                       bl
            br



                 r
                                                                         my
                                                   my
 my                                bl
                                                           E26                                   br
     PNL 118




                            sp

                            my



       bk
                                  bk
bl
                      my
Dayao & Egloso
   (2009)
 E35
                                                        CONTROL
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                Polystyrene




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                                                    my

                                                                                                hy
                                                                                                                    sp


  Control                                                 SDM




                 tr     my
   my



                                                                                                                    my

                                                                   my
Dayao & Egloso
   (2009)
PNL 114                                                      PNL 116
                      A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                             Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                     hy                                                                        hy

                 p                                                   my

                          my                                                         sp
                                              st

PNL 118                                                           E26




            hy




                          my
Dayao & Egloso
   (2009)
E35                                                     st      CONTROL
                               A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                                      Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • SEM pictures:
                    fungi have adhered closely to
                   the substrate and grew on it.
                    substrate is now a combination
                   of the original pollutant which is
                   natural rubber and the Xylaria
                   strains.




Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • The adherence and growth of the
                   fungus signified colonization on the
                   substrate.
                        formation of mycelial mats and
                   through the presence of mycelia,
                   spores and hyphae.

                 • According to the studies of Linos et al.
                   (2000), colonization is the initial step in
                   the biodegradation of polymers.


Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Colonization
                 happens when the cells of the
                  organism secretes extracellular
                  polysaccharide substances (EPS).
                 results in the destruction of quality
                  or performance of the material.
                        biodeterioration is the first stage in
                             biodegradation.


                   (Morton & Surman, 1994; Linos et al., 2000;
                                               Lucas et al., 2008).
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • 0.5 % glucose + on the mineral medium
                       only initiated growth and
                   sustained the fungal growth for three
                   weeks. (Tavanlar, personal communication, march 9,2009)


                 • study of Tavanlar and Lat (2008)
                        wherein growth of Xylaria was
                   seen and measured upon its utilization
                   of mineral oil and PEG as alternative
                   carbon sources.

Dayao & Egloso
   (2009)
                       A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                              Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                  Natural Rubber
                 • In general, the black Xylaria sp.
                   mutant strains, especially the E26
                   strain, have shown the greatest
                   biodegrading potential when
                   compared to the albino strains
                   namely PNL 114, 116 and 118.
                   But among the albino  114

Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                Chicken Feather
                 • all variants efficiently colonized the
                   samples
                 • SDM and E35 showed signs of
                   degradation:
                   – weakened barbs (breaks)
                   – Slightly brittle barbules detached
                     from barbs

Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                         Polystyrene
                 • most extensive colonization
                 • Black strains slightly better
                 • Results of albino mutants not
                   widely different from black
                   mutants, and the wild type



Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • According to the study of Tavanlar and
                   Lat (2008)
                  white mutants possess thinner hyphal
                   extension tip due to the loss of melanin
                   structure around it; whereas the
                 black mutants have thicker hyphae due
                   to melanin around there structure.

                 • melanin
                 protection and resistance against the
                   pressure/friction of the substrate during
                   hyphal tip adherence and penetration
Dayao & Egloso        (M. A.Tavanlar, personal communication, March 4, 2009)
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                      Natural Rubber
                 Strain   Initial   Final       Difference   Initial   Final     Difference   Initial   Final       Difference
                          Weight    Weight                   Weight    Weight                 Weight    Weight

                                    First Run                          Second Run                       Third Run

                 SDM      0.1909    0.1883       0.0026      0.1976    0.1929       0.0047     0.231    0.3216      -0.0906
                 SDM      0.1427    0.1489      -0.0062      0.2581    0.2535       0.0046    0.3524    0.3555      -0.0031
                  114     0.2041    0.2221       -0.018      0.3179    0.3117       0.0062    0.3942    0.3595       0.0347
                  114     0.1183    0.1408      -0.0225      0.2741    0.2693       0.0048    0.2766    0.2472       0.0294
                  116     0.1402    0.1363       0.0039      0.2797    0.2842       -0.0045    0.276    0.3283      -0.0523
                  116     0.2067     0.254      -0.0473      0.3771    0.3721       0.005     0.2748    0.2864      -0.0116
                  118     0.2002     0.197       0.0032      0.2853    0.2806       0.0047    0.2867    0.2202       0.0665
                  118      0.149    0.2088      -0.0598      0.2915    0.2814       0.0101    0.2879    0.2709          0.017
                 E26      0.1891    0.1851       0.004       0.2732    0.2698       0.0034    0.3284    0.2687       0.0597
                 E26      0.2106     0.204       0.0066      0.309     0.3045       0.0045    0.2458    0.2738       -0.028
                 E35      0.1183    0.1409      -0.0226      0.3229    0.3191       0.0038    0.3541    0.3877      -0.0336
                 E35      0.2009    0.2098      -0.0089      0.2232    0.2162       0.007     0.3514    0.3497       0.0017


Dayao & Egloso
   (2009)
                          A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                                 Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                                          Polystyrene
                 Strain   Initial Weight   Final Weight   Difference   Initial Weight   Final Weight   Difference   Initial Weight   Final Weight   Difference


                                            First Run                                   Second Run                                     Third Run
                 SDM       0.0491          0.0498         -0.0007       0.0451          0.0453         -0.0002       0.0472          0.0487         -0.0015
                 SDM       0.0506          0.0505         0.0001        0.0444          0.0448         -0.0004       0.0507          0.0517         -0.001
                 114        0.046          0.0463         -0.0003       0.0476          0.0479         -0.0003        0.052          0.0527         -0.0007
                 114       0.0469          0.0473         -0.0004       0.0514           0.052         -0.0006       0.0528          0.0545         -0.0017
                 116       0.0459          0.0462         -0.0003       0.0461           0.046         0.0001        0.0505          0.0553         -0.0048

                 116       0.0492          0.0495         -0.0003       0.0412          0.0414         -0.0002       0.0488          0.0502         -0.0014
                 118       0.0467          0.0466         0.0001        0.0481          0.0482         -0.0001        0.053          0.0527         0.0003
                 118       0.0466          0.0469         -0.0003       0.0479           0.048         -0.0001       0.0478          0.0489         -0.0011
                 E26       0.0468          0.0468             0         0.0472          0.0484         -0.0012       0.0515          0.0665         -0.015
                 E26       0.0483          0.0468         0.0015        0.0471          0.0486         -0.0015       0.0494          0.0763         -0.0269
                 E35       0.0461          0.0468         -0.0007       0.0473          0.0473             0         0.0477           0.077         -0.0293
                 E35       0.0495          0.0495             0         0.0462          0.0462             0         0.0515          0.0523         -0.0008




Dayao & Egloso
   (2009)
                                  A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                                         Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                        Chicken Feather
                 Strain   Initial   Final       Difference   Initial   Final    Difference    Initial   Final       Difference
                          Weight    Weight                   Weight    Weight                 Weight    Weight

                                    First Run                          Second Run                       Third Run

                 SDM      0.0393    0.0401      -0.0008      0.0418    0.0417       0.0001    0.0413    0.041        0.0003
                 SDM      0.041     0.0501      -0.0091      0.0268    0.0274       -0.0006   0.0316    0.0316          0
                  114     0.0367    0.0328       0.0039      0.0643    0.0635       0.0008    0.0911    0.0911          0
                  114     0.1428    0.1411       0.0017      0.0263    0.0276       -0.0013   0.0328    0.0329      -0.0001
                  116     0.0554    0.0542       0.0012      0.0881    0.0862       0.0019    0.0313    0.0308       0.0005
                  116     0.043     0.0415       0.0015      0.0583    0.0579       0.0004    0.0718    0.0702       0.0016
                  118     0.0533    0.0540      -0.0007      0.0367     0.035       0.0017    0.0954    0.0959      -0.0005
                  118     0.0542    0.0543      -0.0001      0.0554    0.0538       0.0016    0.0916    0.0911       0.0005
                 E26      0.0394    0.0379       0.0015      0.0484    0.0478       0.0006    0.0833    0.0839      -0.0006
                 E26      0.0909    0.0989       -0.008      0.0916    0.0913       0.0003    0.0423    0.0438      -0.0015
                 E35      0.0268    0.0265       0.0003      0.0299    0.0299         0       0.113     0.1131      -0.0001
                 E35      0.2252    0.2254      -0.0002      0.0736    0.0746       -0.001    0.0837    0.0648       0.0189



Dayao & Egloso
   (2009)
                            A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                                   Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                     Specific Concerns




Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                         Natural rubber
                 • slow colonization
                   suggest that colonization
                   impedance due to
                   chemicals in the old latex
                   gloves used might be
                   taking place
                  (Bereeka et al.,2000 and Rose & Steinbuchel, 2005)


Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                Chicken Feather
                 • Sterilized using an autoclave
                 • very hard β-keratin composition




Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                                         Polystyrene
                 • It is proven from the results of this
                   study that polystyrene does not
                   need to be copolymerized with
                   other substances like lignin and
                   sugars (i.e. glucose and sucrose)
                   to make it more degradable and
                   susceptible to microbial attack, as
                   mentioned in the previous studies.
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Conclusion


Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Scanning Electron Microscopy (SEM)
                   revealed colonization of Xylaria sp.
                   strain on natural rubber, chicken
                   feather and polystyrene.

                 • Generally, the black strains, namely the
                   SDM wild type, E26 and E35, have
                   been observed to possess a higher
                   potential to biodegrade natural rubber,
                   chicken feather and polystyrene than
                   the albino mutants PNL 114, 116 and
                   118.
Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Among the black variant strains, E26 has
                   shown the greatest biodegrading potential for
                   natural rubber, nonetheless the albino
                   mutants also showed a potential to
                   biodegrade this substrate.

                 • Polystyrene was effectively colonized by the
                   three black strains SDM wild type, E26 and
                   E35, nonetheless the albino mutants also
                   showed potential ability to biodegrade this
                   substrate.

                 • Chicken feathers were colonized by the SDM
                   and E35 but may require a longer incubation
                   period for degradation
Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 Recommendations



Dayao & Egloso
   (2009)
                 A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                        Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • longer incubation time
                 • further taxonomic identification of
                   Xylaria strains
                 • test methods and standards for
                   biodegradable polymers
                 • Structural modifications can be
                   monitored in depth through infra-
                   red (IR) spectroscopy.
                 • The actual utilization or
                   “absorption” of carbon by the
                   mycelia can be computed
Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Internal biodegradation can be
                   evaluated by change of rheological
                   properties:

                 Tensile strength is measured with a
                  tensile tester
                 elongation at break by a mechanical
                  tester
                 elongation percentage and elasticity by
                  dynamic mechanical thermal analysis.
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Natural rubber
                 tests that would further confirm
                   presence of intermediate and by-
                   product compounds (staining of schiff’s
                   reagent and FTR-AITR spectroscopy)
                 treatment of natural rubber gloves
                   before subjecting it to biodegradation

                 • Polystyrene
                  Testing other grades of polystyrene to
                   be biodegraded.
Dayao & Egloso
   (2009)
                     A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                            Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy
                 • Feathers
                 To verify the degradation of
                   feather, more advanced tests
                   should be conducted wherein the
                   presence of soluble proteins and
                   amino groups concentration will be
                   observed.
                 Study the possibility that Xylaria
                   sp. can produce enzymes such as
                   keratinase, proteinase. Testing of
                   white feathers is also suggested
Dayao & Egloso
   (2009)
                    A Preliminary Study on the Potential Biodegrading Ability of Xylaria sp. on Natural Rubber,
                           Chicken Feathers, and Polystyrene through Scanning Electrons Microscopy