Part2_ Draft1 _Dalet_
Document Sample
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
Shared by: Mary Bernadette Vallesfin Egloso
Mary Bernadette Vallesfin Egloso
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My friends call me Addie. I want to become a doctor someday and serve my countrymen after studying medicine in the Philippines. I also want to become a sophisticated investor and business owner someday. I truly believe in what Rob
(More...)ert Kiyosaki said in his books. It is very important to keep on improving oneself, as we live in this dynamic and competitive world. I love swimming and singing.
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