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DEFENSE KEY POINTS_____ Powered By Docstoc
					                                    XYLARIA SP.



A. Mycelial adhesion

B. Biofilm formation

C. co-substrates of glucose for the growth of a filamentous fungus, Xylaria sp.

D. grown at 25oC and pH 5.0  optimum conditions concluded by the study of Clutario

   and Cuevas (2001).

E. Mycelia closely adhered + grew into it, removal very difficult  Due to this, change

   in weight was not measured.

       o In fact, the measure of the weight loss of samples even from buried materials is

           not really representative of a material’s biodegradability, since this loss of

           weight can be due to the vanishing of volatile and soluble impurities (Lucas, et

           al., 2008).

       o 0.5% glucose initiated sustained growth for up to 3 weeks of incubation (M. A.

           Tavanlar, personal communication, March 4, 2009). Beyond this period when

           glucose has been depleted, Xylaria sp. had to utilize the provided substrate as an

           alternative carbon and energy source.

o mutants + SDM wild type can utilize other carbon sources; PEG and mineral oil (M. A.

   Tavanlar, personal communication, March 4, 2009).

o The generally good growth could only be credited to this capacity of the given fungus,

   supported by the favorable environmental conditions provided, such as optimum pH and

o colonization + surface adhesion fundamental prerequisite to biodegradation

o growth on a polymer surface is not adequate to deduce that carbon from this polymer has

    been assimilated.


o Nonetheless, this colonization provides a simple, fast and clear test to evaluate the

    response of a macromolecular material to biodegradation (Clutario & Cuevas, 2001;

    Motta,et al., 2007).

o covered by a slimy mucilaginous sheath or biofilm easily removed

o All variants utilized able to form biofilm, which is a crucial step to microbially induced

    corrosion and biodegradation.

o Gentle scraping  to remove fungi; avoid destruction of the surface and to further

    detach removable mycelia.

o   colonization cannot be attributed to mere mycelial aggregation on the surface since

    physical manipulation and removal through gentle scraping have been performed.

o Biofilms are extremely intricate microbial ecosystems. They compose of complex

    consortia of bacteria, algae, fungi and grazing protozoa which may exhibit morphological

    attributes not typically correlated with the organisms when grown in pure culture (Morton

    & Surman, 1994). Active colonizers of polymer can adhere to material surfaces because

    they secrete a type of glue (Capitelli et al., 2006; Lucas, et al., 2008). This substance is a

    complex matrix made of polymers (e.g. nonionic and anionic polysaccharides and

    proteins), which penetrate into porous structures and alters the size and the distribution of

    pores and changes moisture degrees and thermal transfers. The slime matter functions to

    protect microorganisms against adverse conditions such as desiccation and UV
       radiations. Kaeppeli and Fiechter (1976) observed that the first step in the utilization of

       hydrocarbon by microbial cells, involves a passive adsorption to lipophilic

       lipopolysaccharide found in the surface of the cell to the alkane group of the polymer

       (Clutario & Cuevas, 2001). Another study (Reddy, et al., 1982; Gutnick & Minas, 1987)

       hypothesized that after adhesion, solubilizing agents are secreted and produced by many

       microorganisms which can make use of water-immiscible compounds (Clutario &

       Cuevas, 2001).

       Filamentous microorganisms develop their mycelial mats or plectenchyma, inside the

pollutant substrates. The generally more efficient performance of the black mutants, E26 and

E35, is important to note, which is due to the melanin which cover their hyphal surfaces. The

melanin protects the hyphae of the black mutants thereby making them resistant to

frictional damage during penetration into the substrate. Thus, they can more efficiently

degrade the substrate as compared to the albino mutants PNL 114, 116 and 118 (M. A.

Tavanlar, personal communication, March 4, 2009). Therefore, the black mutants also produced

denser and thicker mycelial growth and more efficient degradation of substrate. The mechanical

action of hyphal apices infiltrating the substrates increases the size of pores and provokes cracks.

Hence, the substrates are weakened in resistance and durability (Bonhomme et al., 2003; Lucas,

et al., 2008). The thicker hyphae of the wild type and black mutants (figures 22, 26 and 27) are

also due to the presence of melanin in their surface. However, the black mutants are still

undergoing tests and are not characterized yet as of the moment (M. A. Tavanlar, personal

communication, March 4, 2009).

       Prior to experimentation, the chicken feathers were autoclaved, whereas natural rubber

and polystyrene were only surface sterilized through agitation in 70% ethanol once and in sterile
distilled water twice. Polystyrene and natural rubber were initially autoclaved, but were

discarded because the samples have been greatly degraded, unlike the chicken feather which

remained intact. On the other hand, surface sterilization would maintain the structural integrity of

polystyrene and natural rubber, thus, this was the method used. However, the chicken feathers

were still autoclave to assure the elimination of already present microbes. Scientists have known

for so long that the plumage of birds are sanctuaries for diverse populations of bacteria and fungi

(Hubilek, 1994).

       Moreover, the enhanced rate of degradation in the mineral medium solution, as compared

to in situ degradation, may be due to the greater contact between the polymer and the

microorganism, and the liquid has a better buffering effect than the solid substrate solely.

Produced degradation products may also remain in the system and some may provide nutrients

for growth (Albertsson & Karlsson, 1993).

Chicken Feather

       The chicken feathers were autoclaved to destroy any microorganism present that could

compete and interfere with the determination of the potential degrading ability of the Xylaria sp.

strains. The mycelia, in general, were difficult to remove from the chicken feather samples. Most

of them were now a combination of Xylaria sp. and chicken feathers, as demonstrated under a

scanning electron microscope.         The samples were shaken with 70% ethanol once and in

distilled water twice to remove mycelia. Gentle scraping was also performed afterwards.

However, there were few mycelia still remained closely adhered especially in areas near the

points of attachment of the rachis and barbs. In addition during the removal of mycelia from the

samples, slimy biofilm was also observed to have surrounded all the samples. The formation of
biofilm, in the form of mucilaginous sheath (see table 5) observed, is a means by which

microorganisms establish themselves on a surface. They secrete carbohydrates and proteins to

survive in this low nutrient environment, thereby facilitating microbial activity and degradation

of polymer surface. Establishment is exemplified by the profuse growth of mycelial mats (figure

14 and 19) in most of the samples and the presence of spores (figure 19).

       In general, all the Xylaria sp. variants efficiently colonized the feather samples (figure 12,

14-19), although the samples did not show apparent physical damage as compared to the

undamaged control sample, except for the SDM wild type (figure 14) and E35 (figure 19) strain

which manifested slight brittleness. Efficient colonization in all samples was revealed by the

close adherence of mycelial mats to the rachis, barbs, and barbules of the feathers. The results for

SDM and E35 strains should be further noted because despite the fact that the incubation period

of 50 days is relatively short, both of these strains already manifested signs of degradation. The

SEM (figure 19) for the E35 strain even illustrated weakened barbs which resulted to breakage at

some points. The barbules of the feather samples of SDM and E35 mutant became slightly

brittle, thus they were easily detached from the rachis. Nonetheless, if given a longer incubation

time, the other strains may have revealed indications of biodegradation also, as all of them have

shown to efficiently colonize this substrate. And this is the fundamental prerequisite for

biodegradation to occur.

       Difficulty in the degradation of chicken feather could only be attributed to their very hard

β-keratin composition. Thus, chicken feathers are highly troublesome waste products. Therefore

it is highly recommended that the incubation period be increased, an enzyme catalyst be added,

and/or the feathers be subjected to “weakening” mechanisms prior to the addition of Xylaria sp.
         There should be a distinction made between the preliminary disintegration of complex

keratinous structures into smaller substructures, such as feathers and hairs, and the molecular

breakdown of keratin into smaller peptides. The former may be the result of protease activities

on interkeratin matrix, while the breakdown of the almost crystalline keratin would require

further degradative means. The cleavage of the cystine, disulfide bonds may have a significant

influence on keratin degradation, and this has been described for a few microorganisms. Several

keratinolytic fungi cause sulfitolysis by excreting sulfite and by producing an acid pH at the

mycelial surface (Bockle & Muller, 1997). Furthermore, keratin degradation must occur outside

of the cell at the keratin molecules through the release of a soluble reducing component into the

medium, or through a cell-bound redox system at the surface of the cells (Bockle & Muller,


         Also it might be presumed from microscopic examinations that there was no

permanent contact between mycelium and keratin particles observed, making direct

degradation of the substrate surface unlikely. However, it cannot be excluded that degradation

can occur by short contacts between mycelium and substrate (Bockle & Muller, 1997). A high

percentage of keratin represents hydrophobic and aromatic amino acids (approximately 50%)

(Gregg, et al., 1984; Gradisar, et al., 2005), it has thus been concluded (Gradisar, et al., 2005)

that keratinases are successful in hydrolysis of keratinous materials due to the specific amino

acid composition of keratins as well as to their broad specificity.

         SEM (figure 14) of the SDM wild type sample revealed that the mycelia had adhered to

the barbules and the barbs of the pollutant. Colonization and growth is rapid in terms of extent of

coverage and adherence of mycelia. It is also observed that the barbules contain more mycelia

than the barbs. The PNL 114 mutant strain (figure 15) showed minimal colonization when
compared with the SDM or the wild type. A wider magnification is chosen because at low

magnification, mycelia adherence is not quite obvious. The deep crack at the middle of the

micrograph may have already been present prior to this experiment. The PNL 116 and 118 albino

mutants together with the E26 black mutant strains (figures 16, 17 and 18, respectively) showed

almost the same and comparable results with the PNL 114 albino mutant strain. The adherence of

the strains is minimal when compared to the wild type or the SDM. The micrographs revealed

that at various portions of the chicken feather, mycelia in minimal amount could be seen

attached on the barbules and barbs as well. Hence, it could be stated that when compared

to the wild type, they colonized poorly. On the other hand, the E35 black mutant strain (in

figure 19) showed colonization quite comparable to that of the SDM or wild type. The

micrographs justified the macroscopic physical appearances. For both the SDM and the E35,

which showed more efficient colonization as compared to the other the four strains, they

demonstrated feather samples that were quite brittle. The brittleness refers to the ability of the

barbs and barbules to be easily detached from the rachis. The results suggest that the wild type

strain and the E35 black strain are the most probable strains to demonstrate potential

biodegrading ability.

       Additionally, because chicken feathers were not washed with soap during preparation,

their preen oil coating was retained. Birds waterproof their feathers through the application of

preen oil to their feathers. Therefore, limited moisture is present. Fungi need water, as a medium

for diffusion of soluble nutrients back into the cells. Without some free water, fungi cannot carry

out normal metabolism (Alexopoulos, et al., 1996). The preen oil also inhibits the growth of

some bacteria, although it appears to enhance the growth of other microbes, which may include
fungi as well as yeasts (Bandyopadhyay & Bhattacharyya, 1996; Shawkey, et al.,                      2003;

Shawkey, et al., 2005).


        Among the three pollutants, polystyrene revealed the most extensive colonization and degradation

in the current study. Results of the albino mutants, PNL 114, 116, and 118 are not widely different from

those of the wild type SDM and black mutants, E26 and E35 which showed better results in natural

rubber and chicken feather. Moreover, following incubation, it has been observed that mucilaginous

sheaths surrounded the samples, as mycelia were physically removed through gentle scraping. Small

dents have been generally found on the surface of all samples. Brown to black mycelia were seen attached

to the edges and surface, even on some of the samples of the albino mutants PNL 114, 116 and 118.

According to Tavanlar (March 4, 2009), these brown or black discoloration could be staling products

produced by the fungus, and not melanin in their hyphal surface. Nevertheless, the other samples of the

albino mutants have no visible mycelia due to its white color. Most samples with areas where there are

noticeable mycelial attachments were also observed to be softer or less rigid than the non-inoculated

control. Presence of striations, dents and holes are also highly apparent on the sample surfaces.

        In addition, the thicker hyphae of the wild type SDM and black mutants E26 and E35 are due to

the presence of melanin in their surface, which give them protection and resistance in penetration during

substrate colonization.

        Polystyrene, one of the most widely used synthetic polymers, has been chosen for this

study because its high recalcitrance to biodegradation is very well known. Although considered

as a recalcitrant polymer, it is nonetheless subject to biodegradation, just like polyurethane and

polyethylene (Lucas, et al., 2008). Polystyrene is also commonly considered a plastic, as well as

natural and synthetic rubber, and polyethylene. The plasticisers and fillers used in the

formulation of these plastics leave them vulnerable to attack which usually materializes as a
surface biofilm, which causes slight adverse effects to the physical or chemical integrity of the

material (Morton & Surman, 1994). Like natural rubber and chicken feather, mucilaginous

sheaths were also observed to wrap the polystyrene samples while in culture. The microbial

susceptibility of these recalcitrant polymers is credited to the biosynthesis of lipases, esterases,

ureases and proteases (Flemming, 1998; Lugauskas et al., 2003; Lucas, et al., 2008). These

enzymes require the presence of cofactors (i.e. cations present into the material matrix and

coenzymes synthesized by microorganisms) for the breakdown of specific bonds (Pelmont, 2005;

Lucas, et al., 2008).

       The biodegradation of thermoplastic polymers such as polystyrene could proceed via

bulk and/or surface erosion (von Burkersroda et al., 2002; Pepic et al., 2008; Lucas, et al., 2008).

Bulk erosion results to fragments lost from the entire polymer mass and changes in molecular

weight due to bond cleavage. This lysis is provoked by chemicals such as H2O, acids, bases,

transition metals and radicals, or by radiations, but not by enzymes. They are too large to

penetrate throughout the matrix framework. Surface erosion, on the other hand, result to matter

being lost but no change in molecular weight of polymers of the matrix. If the diffusion of

chemicals throughout the material is faster than the cleavage of polymer bonds, the polymer

undergoes bulk erosion. If the cleavage of bonds is faster than the diffusion of chemicals, the

process occurs mainly at the surface of the matrix (von Burkersroda et al., 2002; Pepic et al.,

2008; Lucas, et al., 2008).

       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. Also,

it is clearly shown in the micrograph results that not only did the Xylaria mutant strains and
wildtype showed high affinity, but they actually degraded and utilized polystyrene or EPS strips

as an alternative carbon and energy source. EPS is a closed cell, lightweight and resilient,

foamed plastic composed of hydrogen and carbon atoms. It is non-hygroscopic and does not

readily absorb water vapor. Its closed-cell structure reduces the absorption and/or migration of

moisture into the insulation material (EPS Molders Association, 2009). It is said that raw

polystyrene foam will not rot or attract fungi or mildew and has a superior R-value, thus

polystyrene will insulate and keep heating or cooling inside of any particular room or

commercial space (The Foam Factory, 2009). Because of the high level of moisture resistance

and breathability of polystyrene, fungal growth is retarded; water cannot support its growth when

mycelial growth already penetrated inside the substrate. Consequently, the set-ups required

surrounding the polystyrene strips with liquid media throughout the incubation period. For better

results, it is recommended to apply a longer incubation time than 50 days.

       Aside from keeping the experimental organism alive and free from contamination by

other organisms, the most difficult problem to overcome in providing a continuing source of this

organism for a mycological study to be successful, is sustaining the biochemical property which

is being studied, so that the experiment may be repeated with essentially the same results several

months or even years after. This is due to the fact that fungi are extremely variable organisms –

strains obtained from different sources, while appearing morphologically identical, will not

necessarily behave in the same way biochemically. What is worse is that variation can occur

within a given strain, a property clearly exemplified in the phenomenon of “sectoring”. On a

nutrient agar plate, a spore or mycelial cell grows radially, maintaining a roughly circular

boundary. If at some point the mycelium undergoes a change, such as a mutation, the variant
progeny will still grow radially outward forming a sector. If the change is morphological (i.e.

production or absence of pigment), then the progeny sector would be visibly different from the

parent, but when the change is biochemical, then a uniform colony is produced, which in reality,

contains cells of different biochemical potentialities. Thus, if inocula are taken from different

parts of the colony, then there would be different results. This occurs during repeated

subculturing of an organism. And when a certain variant, which does not possess the

physiological or biochemical property of interest, prevails among others in the culture, then that

property will be lost altogether (Turner, 1971). This inclination to change is universal among

fungi, although some are more stable than others, hence, according to Foster, “all investigations

dealing with specific metabolic functions of a fungus sooner or later encounter physiological

degeneration manifested by progressive loss of the function of particular interest” (Turner,

1971).   This is evidently true of the fungal biodegradation processes. It is therefore only

presumed that the Xylaria variants utilized in the present study are still the same with the ones

used by Tavanlar and Lat (2008) in their characterization study. The mentioned study (Tavanlar

& Lat, 2008) concluded that the albino or white mutants exhibited improved ability to grow on

reduced glucose levels as compared to the SDM wild type. This ability may or may not have

been lost or reduced during the course of the study, especially during subculturing, thus, it may

account as a probable reason why the albino mutants showed less ability to degrade natural

rubber, chicken feather and polystyrene. To reduce experimental errors and chances of strain

variation, i.e. of subculturing from the “wrong” part of the parent culture, and of contamination

from other organisms, transfers must be as infrequent as possible. Due to this, and to reduce

strain variation within the culture, the growth of the stock culture should be kept to a minimum.
   Furthermore, because of the subculturing and/or mutation step (Tavanlar & Lat, 2008) done

to derive the black and white mutants for this study, these could account for the probable loss or

change of the original functions of SDM and the more efficient ability of the black mutants, E26

and E35, to degrade the given pollutants than the albino mutants, and/or the loss or decreased

ability of the albino mutants to degrade the given pollutants as compared to the wild type when

in fact, they performed better than the wild type in the study by Tavanlar and Lat (2008). Change

produced may be a block, due to the loss of an enzyme, on a pathway to an essential metabolite,

loss or reduction of a particular function or structural modification.