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
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
o CONTOUR BODY, WINGS, TAIL OR FLIGHT FEATHER MADALAS TINA
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 &
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
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).
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
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
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.