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					Polystyrene

Figure 9. SEM Micrographs of Xylaria sp. strains on polystyrene showing the changes
after 50-day incubation.




Figure 9.1 Control Non-inoculated control samples showed smooth, relatively
undamaged surface as predicted. Minor dents found on the lower right are due to
sampling handling during the experimentation.
Figure 9.2 SDM. Fungal mycelia has deeply penetrated that it resulted to the
disintegration of the surface and interior region of the polystyrene strips. White arrows
show mycelial growth, while black arrows show fungal spores.
Figure 9.3 Mutant 114 Fungal mycelia has deeply penetrated that it resulted to the
disintegration of the surface and interior region of the polystyrene strips. White arrows
showing mycelial growth.




Figure 9.4 Mutant 116 Fungal mycelia has deeply penetrated that it resulted to the
disintegration of the surface and interior region of the polystyrene strips. White arrows
show mycelial growth, while black arrow shows fungal spores.
Figure 9.5 Mutant 118. Fungal mycelia has deeply penetrated that it resulted to the
disintegration of the surface and interior region of the polystyrene strips. White arrows
show mycelial growth.




Figure 9.6 Mutant E26 Fungal mycelia has deeply penetrated that it resulted to the
disintegration of the surface of the polystyrene strips. White arrows show mycelial
growth, while black arrows show fungal spores.
Figure 9.7 E35 black strain mutant. Fungal mycelia has deeply penetrated that it
resulted to the disintegration of the surface and interior region of the polystyrene strips.
White arrows show mycelial growth, while black arrows show fungal spores.


Microscopic results

       Visually observing the polystyrene strips inoculated with mutants 114, 118, E35

and E41 revealed that the samples have small dents on the surface, and brown to black

mycelia attached to the edges and surface. There were no obvious physical damage

observed in the samples of mutant 116 but there were some reddish mycelia attached on

one of the samples. Black mycelia closely adhered to the edges and surface of SDM and

mutant E26, just as in the other mutants as well, thereby rendering their removal very

difficult by mere physical means. There were no obvious physical damage, if ever, to the

control sample.



Macroscopic results

       By visually observing the polystyrene strips inoculated with mutants 114, 118,

E35 and E41, results revealed that the samples have small dents on the surface, and

brown to black mycelia attached to the edges and surface. There were no obvious

physical damage observed in the samples of mutant 116 but there were some reddish

mycelia attached on one of the samples. Black mycelia closely adhered to the edges and

surface of SDM and mutant E26, just as in the other mutants as well, thereby rendering

their removal very difficult by mere physical means. There were no obvious physical

damage, if ever, to the control sample.
                                      DISCUSSION

Natural Rubber

       Instead of percent weight loss, the natural rubber pollutant samples were observed

under a scanning electron microscope (SEM). In general, SEM revealed that the natural

rubber samples were not pure anymore. They were not pure because samples are now

a combination of Xylaria sp. strains and natural rubber. By observing the

micrographs and comparing it to the control, all the strains demonstrated colonization on

the rubber surface. Colonization of rubber, based on the study of Linos et al. (2000) on

rubber degradation, is the first mechanism by which rubber-degrading organisms degrade

natural rubber. In the study, scanning electron microscopy revealed that the Xylaria sp.

strains grew adhesively on the natural rubber demonstrating contact and formation of

mycelial mats on the surface of the pollutant. The mycelial mats could be compared to

biofilm formation.

       Furthermore, the mechanism of colonization began with the cell directly merging

into substrate. Such attraction demonstrated the highly hydrophobic nature of the cell.

Biofilm formation observed in the surface of polymers is a mechanism by which

microorganisms secrete proteins and carbohydrates for their survival in environments

which are low in nutrients. Its formation could also indicate and suggest the solid

substrate’s utilization by the microorganisms. ( Linos et al., 2000)
       The macroscopic view of the natural rubber samples demonstrated signs of

elasticity reduction when compared to the control. Elasticity is a property of rubber that

allows it to return to its original form when subjected to stress. The loss of this property is

mostly obviously observed in areas where the fungal strain has embedded. This could be
                                      Is there a parameter in
                                      SEM that can measure
best illustrated by the black fungi strains namely SDM, E26 and E35 because the albino
                                      elasticity?
mutant’s, namely PNL 114, 116 and 118, mycelia cannot be distinguished upon physical

examination. The color of the albino mutants and the natural rubber samples are similar;

hence, it is difficult to macroscopically pinpoint and subject to stress, such as stretching,

the areas where the mycelia had embedded. By examining the micrographs, the loss of

the elasticity can be attributed to the areas where mycelia adherence is visible. These

portions of the natural rubber which are colonized suggest that the fungal strains utilized

the natural rubber as a carbon source and did not only rely on the 0.5% glucose.

       As indicated in previous studies, the proposed mechanism of natural rubber starts

with the oxidative cleavage of the double bonds in the polyisoprene chain. This is mainly

demonstrated by the Gordonia sp. In its rubber-degradation study, latex gloves were also

used, and oligomers of aldehyade and ketone groups were recovered. Another species

thoroughly investigated which could reveal the mechanism of rubber biodegradation is

the Nocardia sp. strain 835A. Results of it showed that there is also a cleavage of the

double bond at the poly(cis 1,4-isoprene). Its study demonstrated 90% weight loss after 8

weeks of incubation. (Rose and Steinbuchel, 2005; Berekaa, 2006 and Linos et al., 2000)

       By comparing the natural rubber samples of the mutant strains to the sample

incubated using the SDM strain, it could be observed that the best potential biodegrading

strain in terms of colonization is E26. The SEM revealed that the E26 colonized the entire
surface of the natural rubber sample. No trace of the natural rubber sample could be seen

as shown in figure_F. as shown, there are spores in the surface and the strain formed an

abundant mycelial mat colony. Moreover, when the E26 is stretched, there is a marked

loss of elasticity when compared to the wild type (SDM). This suggests that the natural

rubber had been utilized by the E26 strain.

        On the other hand, the least strain that demonstrated a potential biodegrading

capacity is the PNL 118 strain. There is a changed in the smoothness of the surface when

compared to the control. There were also observed elevations (what figure / label the

elevation) on the surface when viewed under SEM. This could suggest initial surface

colonization. Yet, the macroscopic view revealed nothing of note or marked difference

when compared to the control and to the wild type (SDM) except for a little decrease or

reduction in elasticity.

        The other mutant strain such as the PNL 114 demonstrated a comparable result to

the wild type. Although the micrograph Did you measure PNL 114 colonized more
                                           revealed that the
                                           colonization
efficiently than the wild type in terms of capacity?growth and presence of spores. The
                                           mycelia

macroscopic view of this strain reveals elasticity reduction similar to the SDM strain.
                         Did you compare
                         colonization/growth in colonization capacity; hence, potential
PNL 116 is almost the same with PNL 118 in theirSEM of
                           samples inoculated at different
biodegrading ability. Surface elevations and marked loss of smoothness is observed on
                           length/incubation time?
the surface. Lastly, the E35 strain showed a better colonization result than the wild type

(SDM). The growth of mycelia on the rubber surface is more efficient and established in

terms of mycelia mat formation and presence of spores. Through macroscopic

observation, there is a comparable elasticity reduction or loss towards the wild type.
       The slow colonization of Xylaria sp. strains on natural rubber could suggests that

colonization impedance due to chemicals in the latex gloves used might be taking place.

In the investigation of Berekaa et al. (2000) and Rose and Steinbuchel (2005), wherein

latex gloves were extracted using organic solvents to remove the anti-oxidants, the ones

used to prevent the ageing of the materials. The colonization efficiency of the known

biodegrading organism such as Gordonia (strains Kb2, Kd2 and VH2), Mycobacterium,

Micromonospora and Pseudomonas were enhanced compared to non-treated latex gloves.

       Moreover, the study also suggests that melanin, which is a pigment responsible

for the black pigment coloration, of the mutant strain’s E35 and E26 might be playing a

role in the degradation of the natural rubber since most of the samples that showed a

notable elasticity reduction and efficient colonization were from the black strain species

namely the SDM, E35 and E26. Further studies could be conducted to investigate such

hypothetical relationship.



Chicken Feather

       The mycelia, in general, were difficult to remove from the chicken feather

samples. Most of the chicken feathers were not pure samples anymore but a combination

of Xylaria sp. strains and chicken feathers. This was demonstrated under a Scanning

Electron microscope.

       During the preparation of the chicken feathers, they were not washed with soap,

therefore retaining their preen oil coating. Birds waterproof their feathers through the

application of preen oil to their feathers. Thus, there is limited moisture 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, 1996). The

preen oil 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, Pillai, & Hill, 2003; Shawkey, et al., 2005.)

       Furthermore, the chicken feathers were also autoclaved to destroy any

microorganism present that could compete and interfere with the determination of the

potential degrading ability of the Xylaria sp. strains. Researchers have known for decades

that the plumage of birds harbors a diverse community of bacteria and fungi, including

yeast (Hubilek, 1994). Despite recent interest in the interactions between birds and

environmental microbes, the identities and ecological roles of bacteria and other microbes

found on the feathers of wild (i.e. aerial and canopy) birds are largely unknown

(Shawkey, et al., 2005). Unfortunately, the influence of these creatures on the birds

themselves has received little attention. In a pioneering paper in this issue of The Auk, E.

H. Burtt and J. M. Ichida (1999) show that plumage microbes could influence birds in

significant ways. Their study provided evidence that many, if not most, species of birds

have bacteria in their plumage, and that some of these bacteria can rapidly degrade

feathers, at least under laboratory conditions. Extrapolating from the data of their study,

they predicted that most species of birds will have feather-degrading bacteria in their

plumage. And the metabolic activity and / or antibiotic production of some bacteria may

inhibit or improve the growth of other bacteria and / or fungi present. Thus, the growth of

Xylaria sp. in the current study may be affected (i.e. enhanced or inhibited) by microbial

communities already present in the feathers. But under SEM and through macroscopic

observation of the flasks, no growth of other organisms was detected.
       When the non-inoculated control, which showed no trace of change, was

compared with the wild type, SEM 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 as shown in figure 8.2. It is also observed that the

barbules contain more mycelia than the barbs. The PNL 114 mutant strain (in figure 8.3)

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 PNL 116 and PNL 118 albino mutant strains together with the E26 black

mutant strain (figures 8.4, 8.5 and 8.6, respectively) showed almost 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 8.6)

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 efficient colonization compared to the other the other four strains, 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.


You may identify and describe the region of colonization by color labeling it.

Polystyrene
       The results have showed 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.

       Polystyrene is a thermoplastic polymer. In thermoplastics, the polymer chains are

only weakly bonded (van der Waals forces). The chains are free to slide past one another

when sufficient thermal energy is supplied, making the plastic formable and recyclable

(eFunda, 2009).



                                    CONCLUSION
       In conclusion, some of the Xylaria sp. strains (the SDM or wild type and the black

mutant strains E35 and E26) possess potential biodegrading ability. The SDM or wild

type was observed to have the capacity to potentially biodegrade all three pollutants. The

E35 black mutant strain was seen to potentially biodegrade chicken feathers and natural

rubber. While, the E26 black mutant strain was observe to potentially biodegrade natural

rubber only.



                                RECOMMENDATIONS

       In general, the study suggests a longer incubation time to further test and confirm

the potential biodegrading ability of the Xylaria sp. strains on the three pollutants, namely

natural rubber, chicken feathers and polystyrene. It is also recommended that further

studies testing the various



Natural rubber

       Subjection of the natural rubber samples to tests that would further confirm

presence of intermediate and by-product compounds and biofilm as well such as staining

of schiff’s reagent and FTR-AITR spectroscopy is encouraged. More so, treatment of

natural rubber gloves before subjecting it to biodegradation is recommended to remove

possible chemical hindrances, such as anti-microbial chemicals and anti-oxidants.



Polystyrene

       Testing other grades of polystyrene to be biodegraded.
Feathers

       To verify the degradation of feather, more advanced test 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. If ever there are enzymes produced, they should be purified and isolated for

further studies. Testing white feathers is also suggested.
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                                    APPENDIX A

                                 BUDGET OUTLINE

Materials

       Chicken feathers                                      P300

       Styroplates                                           P30

       Foil, tissue, cotton                                  P200

Reagents

       Potato Dextrose agar                                  P1000

       Mineral medium                                        P500

       0.5% Glucose                                  P1200

       70% Ethanol solution                          P800

       Distilled water                               P500

Thesis Proposal

       Printing                                      P2000

       Photocopied materials                         P1500

       Materials such as Bond Papers etc.            P2000

Scanning Electron Microscopy                        P 25,200

Miscellaneous

       Fares                                         P5000

       Glasswares                                    P400

       Others                                        P2000

TOTAL:                                            Php 46,500
                                  APPENDIX B

Mineral Medium Formula



Malt extract                                 1g

Ammonium tartate                              5g

MgSO4.7H20                                  0.5g

CaCl2.2H20                                  0.01g

NaCl                                        0.1g

FeCl3                                       0.01g

1% w/v Thiamin                               5ml

1% w/v Trace elements                        1ml

1% m/v Tween 80                             0.2g



*Adjust to pH 5.0 by adding HCL or NaOH. Check the pH using pH paper

** For Mineral Medium Glucose (MMG), just add 0.5 % w/v glucose or 5 g/l
                                      APPENDIX C

                       Weight Loss / Gain Measurements of the Set-ups

                                    POLYSTYRENE
       Initial  Final              Initial  Final             Initial  Final
                        Difference
Strain Weight Weight               Weight Weight Difference Weight 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




                                   NATURAL RUBBER
       Initial  Final               Initial   Final            Initial  Final
                        Difference
Strain Weight Weight               Weight Weight Difference Weight Weight Difference
               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
                                   CHICKEN FEATHER
       Initial  Final               Initial  Final             Initial  Final
                        Difference
Strain Weight Weight                Weight Weight Difference Weight Weight Difference
               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

				
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