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International Journal of Ethics in Engineering & Management Education 
Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 
Microbial Degradation of Plastic (LDPE) & 
domestic waste by induced mutations in 
Pseudomanas putida 
210 
Muralidhar. S.Talkad1*, Chethan.C2, Kavya.S3, 
Qudsiya.S.S4, Shalini Maria5, Ashik Raj6, Aamir Javed7 
1-6P.G. Department of Biotechnology, R&D Centre, 
Dayananda Sagar College of Biological Sciences, 
Kumaraswamy Layout, Bangalore-560078, India 
7Jr.Embryologist.Base Fertility Medical Science Pvt, Ltd 
E-mail: aamir.javed0077@gmail.com 
Tele: 0919916389255 
1*Correspondence Author: E-mail: talkad.murali@rediffmail.com 
Abstract — The most common polymer in plastics is 
polyethylene (PE), which is made from ethylene monomers 
(CH2=CH2). In natural form it is not biodegradable. Low density 
polyethylene is a vital cause of environmental pollution. It occurs 
by choking sewer line through mishandling thus posing an 
everlasting ecological threat, the making of the genetically 
engineered microbes for bioremediation, the latter being a 
strategy to develop an accelerated evolution of pathways by DNA 
restructuring. To enhance the biodegradation of polyethylene, 
pretreatment strategies were followed. Three different 
pretreatment strategies were employed for the present study, 
three duration of Pseudomanas putida treatment to PE were 
analyzed on 7, 14, and 28th day. In the first, PE films were 
subjected for Bleach with Alkali treatment and in the second they 
were subjected to UV light (UV-C,>300nm wavelength). Third 
with the EMS induction of bacterial strains and assessed for 
polymer biodegradation by Biomass weight loss, estimation of 
total carbohydrates and total protein in the culture supernatant, 
followed by DNA isolation for Gel electrophoresis, and Mutated 
DNA Stability analysis by Capillary Gel electrophoresis were 
carried out. 
Index Terms— Microbial degradation of plastics, chemically 
treated polyethylene. Biomass, Sugar, Capillary Gel 
electrophoresis (key words) 
I. INTRODUCTION 
Biodegradation is necessary for water-soluble or 
water-immiscible polymers because they eventually enter 
streams which can neither be recycled nor incinerated. It is 
important to consider the microbial degradation of natural 
and synthetic polymers in order to understand what is 
necessary for biodegradation and the mechanisms involved. 
This requires understanding of the interactions between 
materials and microorganisms and the biochemical changes 
involved. Widespread studies on the biodegradation of 
plastics have been carried out in order to overcome the 
environmental problems associated with synthetic plastic 
waste. 
Some studies have, demonstrated partial biodegradation of 
polyethylene after shorter periods of time.it has been 
suggested that the biodegradation of polyethylene is enhanced 
by oxidative pretreatment, which increases surface 
hydrophobicity by the formation of carbonyl groups that can 
be utilized by microorganisms. (1, 2, 3) 
Some microorganisms are indeed capable of degrading the 
high molecular weight polymer (4) as was evident from a 
recent report on the biodegradation of thermooxidised 
polyethylene by P.pinophilum (5). A non ionic surfactant 
(Tween -80) to the culture medium of Pseudomonas 
aeruginosa .The surfactant apparently increased the 
hydrophobicity of the polyethylene surface and thus facilitated 
the adhesion of bacteria to the polymer (6). 
Low density polyethylene is one of the major sources of 
environmental pollution. Polyethylene is a polymer made of 
long chains of ethylene monomers. The use of polyethylene 
growing worldwide at a rate of 12% per year and about 140 
million tons of synthetic polymers are produced worldwide 
each year. With such a large amount of polyethylene gets 
accumulated in the environment, generating plastic waste 
ecological problems are needed thousands of years to 
efficiently degradation (7). 
Microorganisms can degrade plastic over 90 genera, from 
bacteria and fungi, among them; Bacillus megaterium, 
Pseudomonas sp., Azotobacter, Ralstonia eutropha, 
Halomonas sp., etc. (8). Plastic degradation by microbes due 
to the activity of certain enzymes that cause cleavage of the 
polymer chains into monomers and oligomers. Plastic that has 
been enzymatically broken down further absorbed by the 
microbial cells to be metabolized. Aerobic metabolism 
produces carbon dioxide and water. Instead of anaerobic 
metabolism produces carbon dioxide, water, and methane as 
end products (9). This study aims to isolate the bacteria from 
waste polyethylene plastics that can degrade polyethylene 
plastic.
International Journal of Ethics in Engineering & Management Education 
Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 
211 
The selected Pseudomanas putida bacterial strains were 
assessed for polymer biodegradation by Biomass weight loss, 
estimation of total carbohydrates & total protein in the culture 
supernatant. 
DNA isolation and stability studies carried out in capillary 
gel Electrophoresis. 
II. MATERIALS & METHODS 
Materials: Low density polyethylene (LDPE) which is the 
major cause of environmental pollution was used for the 
study. 
Microorganism collection 
· The bacteria Pseudomonas putida (MTCC NO: 2467) used in 
this study were procured from Microbial Type Culture 
Collection and Gene Bank (MTCC), Chandigarh. Cultures 
were maintained on LB agar plate 
· Raw materials 
Plastics is polyethylene (PE) as commercial plastic carry bags 
of LDPE were collected and cut into small strips and subjected 
for Chemical - alkali treatment. 
Chemical - alkali treated polyethylene: polyethylene bags 
were cut into small strips & transferred to fresh solution 
containing 18ml tween, 10ml bleach, and 225ml of distilled 
water & stir it to 30-60mins. Bleach consists of 5gms of 
sodium chloride, 5gms of sodium hydroxide & 10 ml of 
glacial acetic acid. Strips were transferred to beaker with 
distilled water & stir it 2 one hour. They were aseptically 
relocated to ethanol solution 70%v/v For 30 mins. Finally, the 
polyethylene strips were transferred to petridish and 
inoculated at 45°-50°c overnight. Ethanol was used as 
disinfectant to polyethylene & removes any organic matter 
adhering to its surface 
Induction of mutation by UV 
Materials required:- 
· UV germicidal light bulb (Sylvania G15T8; 254 nm 
wavelength) or Stratagene UV Cross linker 
· 230c incubator 
· Pseudomonas putida (MTCC NO: 2467) 
· LB agar plate 
Grow and mutagenize cells 
1. Grow an overnight culture of the desired pseudomonas putida 
strain in 5 ml LB agar plate at 300c. 
2. Determine the density of cell in the culture and record this 
number Adjust concentration to ~2 × 108 cells/ml if necessary. 
Transfer 1 ml of the culture to a sterile micro centrifuge tube. 
3. Pellet cells in a micro centrifuge for 5 to 10 sec at maximum 
speed, room temperature. Discard supernatant and resuspend in 
1 ml sterile water. Repeat wash. After the second wash, 
resuspend cells in 1ml of sterile water. 
Plating:- 
1. Make serial dilutions of the culture in sterile water so that each 
plate has 200 to300 viable cells. 
2. Plate 0.1 and 0.2 ml of the diluted cells on separate sets of LB 
agar plate, using ten plates in each set. Incubate all plates for 3 
to 4 days at room temperature. 
3. Irradiate all but two plates from each set with UV light using a 
dosage of 300 ergs/mm2 (there should be 40% to 70% 
survival). The nonirradiated plates will serve as controls to 
determine the degree of killing by the UV light. 
Induction of mutation by EMS 
Materials required:- Pseudomonas putida (MTCC NO: 
2467) Sterile water, LB agar plate, 0.1 M sodium phosphate 
buffer, pH 7.0, Ethyl methanesulfonate, 5% (w/v) sodium 
thiosulfate (autoclaved),13 × 100–mm culture tube, Vortex, 
300c incubator with rotating platform 
Grow and mutagenize cells 
1. Grow an overnight culture of the desired yeast strain in 5 ml 
LB agar medium at 300c. 
2. Determine the density of cell in the culture and record this 
number Adjust concentration to ~2 × 108 cells/ml if necessary. 
Transfer 1 ml of the culture to a sterile micro centrifuge tube. 
3. Pellet cells in a micro centrifuge for 5 to 10 sec at maximum 
speed, room temperature. Discard supernatant and resuspend in 
1 ml sterile water. Repeat wash. After the second wash, 
resuspend cells in 1.5 ml sterile 0.1 M sodium phosphate buffer 
pH 7.0. 
4. Add 0.7 ml cell suspension to 1 ml buffer in a 13 × 100–mm 
culture tube. Save remaining cells on ice for a control. 
5. Add 50 μl EMS to the cells and disperse by vortexing. Place on 
a rotating platform and incubate 1 hr at 300c. (EMS treatment 
should cause 40% of the cells to be killed). 
6. 
7. Transfer 0.2 ml of the treated cell suspension to a culture tube 
containing 8ml sterile 5% sodium thiosulfate, which will stop 
the mutagenesis by inactivation of EMS. If cells are to be 
stored before plating, pellet in a tabletop centrifuge 5 min at 
3000×g at 40c, resuspend in an equal volume of sterile water 
and store at 40c. 
Total Biomass: 
About 1ml of the culture was transferred into 1.5ml micro 
centrifuge tube & pelleted down at 12000rpm at 4°c for 25 
min. The pellet was dried overnight at 50°c & dry weight of 
the resulting biomass was calculated 
Total proteins: 
The total protein concentration in the supernatant was 
determined by the method reported by Lowry’s method. 
Bovine serum albumin solutions were used as standards & 
observance was measured with a spectrophotometer at 595nm. 
Total sugar: 
The total sugars were analyzed by anthrone method. Glucose 
was used as the standard & the absorbance was measured at 
495nm 
Gel electrophoresis: 
Extraction and estimation of Genomic-DNA by gel 
electrophoresis- Amnion Bioscience KIT
International Journal of Ethics in Engineering & Management Education 
Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 
212 
Capillary Gel electrophoresis analysis: 
Polyacrylamide gel-filled capillaries are usually employed, 
although new polymer formulations with greater stability to 
the applied electric field are likely to be introduced shortly. 
Agarose gels are unable to withstand the heating produced by 
the high voltages used in capillary gel electrophoresis (CGE). 
The instrument CGE Pro 9600 – CGE Lauf-Nr 
15315(Machine 3) 
Capillary Gel electrophoresis was used to analyze DNA 
fingerprinting is a useful tool for identifying the genotype of 
living organisms by determining their DNA sequence. For this 
technique, genomic DNA must be amplified by PCR. 
Capillary electrophoresis separates this amplified DNA with a 
one base pair resolution and creates specific peaks for each 
nucleotide to map the DNA sequence. 
III. RESULTS 
Characterization of the isolates: based on growth these 
cultures were identified as pseudomonas putida 
Fig: 1. Colonies of pseudomonas putida showing Plastic 
degradation 
Total Biomass 
Bacterial biomass is a direct measure of the growth of the 
culture in the medium. Chart shows the variation in biomass 
during 7, 14 & 28 days respectively 
Pseudomonas putida control 
Pseudomonas putida plastic 
Pseudomonas putida domestic waste 
Pseudomonas putida plastic + domestic waste 
Total sugars in the obtained filtrate 
The amount of total sugars produced by bacterial strain during 
7, 14 & 28 days respectively 
Sugar pseudomonas putida control
International Journal of Ethics in Engineering & Management Education 
Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 
213 
Sugar pseudomonas putida plastic 
Sugar pseudomonas putida domestic waste 
Sugar pseudomonas putida domestic waste + plastic 
Figure-2: Capillary Gel Electrophoresis - Pro 9600 Sample: 
Pseudomonas Putida/EMS/Genomic /DNA/Plastic 
Figure-3: Capillary Gel Electrophoresis - Pro 9600 Sample: 
Pseudomonas Putida/EMS/Genomic /DNA/Plastic DW 
Figure-4: Capillary Gel Electrophoresis - Pro 9600 sample: 
Pseudomonas Putida/UV/Genomic /DNA/Plastic 
Figure-5: Capillary Gel Electrophoresis - Pro 9600 sample: 
Sample: Pseudomonas Putida/UV/Genomic/DNA/Plastic+ 
DW 
Analysis of Capillary Gel electrophoresis: 
Separations of oligonucleotides and DNA sequence products 
have been accomplished in polyacrylamide gels. For 
restriction fragments and larger oligos, gels with little or no 
crosslinker seem most effective due to the larger pore size of 
the gel. Separation of deoxyoligonucleotides such as poly (dA) 
40-60 is readily accomplished in an 8% T gel with a buffer 
consisting of 100 mM Tris-borate, pH 8.3 with 2 mM EDTA 
and 7 M urea, in under 35 min with unit base resolution. 
Determining the purity of synthetic oligos is an important 
application of CGE. 
Pseudomonas Putida/EMS/Genomic /DNA/Plastic: DNA is 
Stable and the Elution happened at 67.6 Minutes, Heavy 
Molecular Weight - DNA. Lot of Pre and Post Elution of 
Genomic DNA, reason may be DNA Fragmentation. (Fig: 2).
International Journal of Ethics in Engineering & Management Education 
Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 
214 
Pseudomonas Putida/EMS/Genomic /DNA/Plastic+DW: 
Stable DNA 97.2 % Purity and Elution at 43.31, Mutation 
Stable. (Fig: 3). 
Pseudomonas Putida/UV/Genomic /DNA/Plastic: Stable 
DNA 90.8 % Purity and Elution at 41.22, Mutation Stable. 
(Fig: 4). 
Pseudomonas Putida/UV/Genomic /DNA/Plastic+ DW: 
Unstable Mutation Not Suggested to be Induced .Internal 
Folding. (Fig: 5). 
IV. DISCUSSION 
Although the bio-degradation and bio-deterioration of 
polyethylene has been demonstrated by several researchers, 
the enzymes involved and mechanisms associated with these 
phenomena are still unclear. Nevertheless, it is recognized that 
both enzymatic and abiotic factors (such UV light) can 
mediate the initial oxidation of polyethylene chains, and given 
the chemical similarity between polyethylene and olefins it 
has been suggested that the metabolic pathways for 
degradation of hydrocarbons can be used once the size of 
polyethylene molecules decrease to an acceptable range for 
enzyme action (typically from 10 to 50 carbons). The long-range 
structure and morphology of polyethylene have shown 
important roles, with amorphous regions being more prone to 
microbial attack than crystalline ones. 
The Microbial Degradation of Plastic (LDPE) polyethylene & 
domestic waste mixture with plastic when were induced with 
UV & EMS in Pseudomanas putida successfully revealed the 
beneficial response in Biomass reduction for better yield 
against growth, sugar conversion along with proteins 
utilization consistently proven in both normal and mutated 
organism as the days succeeded may be by more than a month, 
soil mixture and domestic waste with plastic : polyethylene 
bags dumping can be eco-friendly manageable to degrade and 
utilize the biomass for agricultural cultivation of crops 
May be Physico-chemically treated polyethylene films were 
found to be effectively degraded by the fungal isolates than 
untreated films. The hypothesis is that a physicochemical 
treatment of the polymer leads to its oxidation and subsequent 
breakdown assisting in the easy assimilation by the fungus 
and, hence, can be effectively used as a pretreatment strategy 
before subjecting the polymer to biodegradation (10). The 
oxidized polymer helps in adhesion of microorganisms (due to 
probable changes in the hydrophobicity of the polymer 
surface), which is a prerequisite for biodegradation (11). 
Similarly in the present study, a higher biomass was observed 
on the pretreated samples. Because carbohydrates in the 
medium constitute the main energy source for their growth 
and metabolism during the nonavailability of readily 
assimilating carbon source, microorganisms adhere to the 
polymeric surface during the formation of the biofilm, which 
is essential for bringing about degradation (12). 
It is also based on research (13) these bacteria Pseudomonas 
sp. able to degrade the plastic by 8.16% and was able to 
degrade the polythene by 20.54% within one month incubation 
anaerobically. While this type of fungi Aspergillus Glaucus 
able to degrade the plastic by 7.26% and was able to degrade 
the polythene by 28.80% within one month incubation 
anaerobically, from the results of the degradation of polythene 
faster and easier than plastic degradation. Earlier publications 
interpreted the growth of microorganisms on polyolefins, e.g. 
polyethylene as being limited to the microbial action on the 
surface of an inert support without impact on the polymers 
(14). However, it was found that polyethylene is not only 
colonized but also biodegraded by various fungi mostly 
belonging to the genera Aspergillus, Fusarium or Penicillium 
(15). Polythene and plastics are two polymers with wide 
application, both are recalcitrant and thus remain inert to 
degradation and damage that leads to accumulation in the 
environment, and create serious environmental problems. 
Therefore, further research is needed to prevent environmental 
damage caused by plastic and polythene waste contamination 
(16). 
Past research has isolated Pseudomonas putida from sludge in 
industrial waste and determined that it used o-chloronitrobenzene 
(o-CNB) as its only carbon, nitrogen, and 
energy source. Most importantly, the highest degradation of 
o-CNB (85%) by P. putida was found to be at 32°C and a pH 
of 8.0. Although o-chloronitrobenzene is not plastic, this 
research gives a general idea of ideal growing conditions for 
P. putida, and shows that it is capable of using one source as 
its only carbon, nitrogen, and energy source (17). 
Microorganisms are unable to transport the polymeric material 
directly into the cell due to the lack of its solubility in water & 
its size. They excrete extra cellular enzymes which aid in the 
degradation of polymers outside the cells (18). The superficial 
growth of hyphae on the polymer surface was a function of the 
oxidation levels of treated sample was observed (19). 
Therefore pretreated samples showed greater weight loss than 
untreated samples. 
Since the continuous introduction of recalcitrant materials, 
microorganisms are challenged to develop new pathways by 
altering their own preexisting genetic components by either 
mutation(s) in single structural and/or regulatory gene or 
perhaps recruitment of single silent gene when they encounter 
the foreign compounds (20). 
In Pseudomonas Putida, when EMS induced Genomic DNA 
isolated from Plastic+DW group showed: Stable DNA of 97.2 
% Purity and Elution at 43.31, Mutation were Stable and 
beneficial. Same as Pseudomonas Putida, when UV induced 
Genomic DNA from Plastic: showed Stable DNA with 90.8 
% Purity and Elution at 41.22, Mutation were Stable and 
beneficial.
International Journal of Ethics in Engineering & Management Education 
Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 
215 
CONCLUSION 
The Microbial Degradation of Plastic (LDPE) polyethylene & 
domestic waste mixture with plastic when were induced with 
UV & EMS in Pseudomanas putida successfully revealed the 
beneficial response better option for further utility in 
commercial or municipal dump yards at better strain 
improvement and longer duration could be an ideal organism 
for the sustainable technology 
ACKNOWLEDGMENT 
The authors are awfully thankful to Dr. Premchandra Sagar 
(Vice Chairman), Dr. Krishne Gowda, Director, Dayananda 
Sagar College of Biological Sciences, Dr. C.D. Sagar Centre 
for Life Sciences, Bangalore-560078, India, for their colossal 
guidance and support for this project. 
REFERENCES 
[1] Albertsson AC. Biodegradation of synthetic polymers. 2. 
Limited microbial conversion of C-14 in polyethylene to 
(CO-2)-C-14 by some soil fungi. J Appl Polym Sci, 22, 
3419–3433, 1978. 
[2] Albertsson AC. The shape of the biodegradation curve for 
low and high density polyethylenes in prolonged series of 
experiments. Eur Polym J, 16, 623–630, 1980. 
[3] Cornell, J.H., Kaplan, A.M. and Rogers, M.R. 
Biodegradation of photooxidized polyalkylenes. Journal of 
Applied Polymer Science, 29, 2581–2597. 1984. 
[4] Yamada-Onodera K, Mukumoto H, Katsuyaya Y, Saiganji 
A, Tani Y. Degradation of polyethylene by a fungus, 
Penicillium simplicissimum YK. Polym Degrad Stabil, 
72,323 –327, 2001. 
[5] Volke-Sepulveda, T., Saucedo-Castaneda, G., Gutierrez- 
Rojas, M., Manzur, A. and Favela-Torres, E. Thermally 
treated low density polyethylene biodegradation 
by Penicillium pinophilum and Aspergillus niger. Journal 
of Applied Polymer Science 83,305–314, 2002. 
[6] Albertsson AC, Karlsson S. The influence of biotic and 
abiotic environments on the degradation of polyethylene. 
Prog Polym Sci, 15, 177–192, 1990. 
[7] Usha R., Sangeetha T., Palaniswamy M. Screening of 
Polyethylene Degrading Microorganisms from Garbage 
Soil. Libyan Agric Res Center J Internati, 2 (4), 200-204, 
2011. 
[8] Chee J. Y., Yoga S. S., Lau N. S., Ling S. C., Abed R. M. 
M., Sudesh K. L. Bacterially Produced 
Polyhydroxyalkanoate (PHA): Converting Renewable 
Resources into Bioplastics. Appl Microbiol & Microbiol 
Biotech, a Mendez Vilas (Ed), 2010. 
[9] Glass JE, Swift G. Agricultural and Synthetic Polymers, 
Biodegradation and Utilization, ACS Symposium Series, 
433. Washington DC: American Chemical Society, p. 9-64, 
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[10] Arkatkar, A., Arutchelvi, J., Bhaduri, S., Uppara, P. V., 
Doble, M. Int. Biodeterior. Biodegrad, 63, 106 –111, 
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[11] Tribedi P, Sil AK. Cell surface hydrophobicity: a key 
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by Pseudomonas sp. AKS2. Journal of Applied 
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[12] Weibin, G.,Shimin, H., Minjiao, Y, Long, J., Dan, Y. 
Polym. Degrad.Stab, 94, 13–17, 2009. 
[13] Kathiresan K. Polythene and plastics degrading microbes 
from the mangrove soil. Rev Biol Trop (513), 629-634, 
2003. 
[14] Chandra R., Rustgi R. Biodegradable polymers. Prog. 
Polym. Sci, 23, 1273. 1998. 
[15] S. Łabużek, B. Nowak, J. Pająk. The Susceptibility of 
Polyethylene Modified with Bionolle to Biodegradation 
by Filamentous Fungi. Polish Journal of Environmental 
Studies, Vol. 13, No. 1, 59-68, 2004. 
[16] Albertsson AC, Andersson SO, Karlsson S. The 
mechanism of biodegradation of polyethylene. Polym 
Degrad Stabil, 18, 73– 87, 1987. 
[17] He, Q., Liang, S., Wang, Y., Wei, C., & Wu, H. 
Degradation of o-chloronitrobenzene as the sole carbon 
and nitrogen sources by Pseudomonas putida OCNB-1, 
Science Direct, 21, 89-95, 2009. 
[18] Brown BS, Mills J, Hulse JM. Chemical and biological 
degradation of plastics. Nature, 250, 161–163, 1974. 
[19] Potts JE. Biodegradation. In: Jelinek HHG (ed). Aspects 
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****

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Aamir Javed ArticleMicrobial Degradation of Plastic (LDPE) & domestic waste by induced mutations in Pseudomanas putida

  • 1. International Journal of Ethics in Engineering & Management Education Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) Microbial Degradation of Plastic (LDPE) & domestic waste by induced mutations in Pseudomanas putida 210 Muralidhar. S.Talkad1*, Chethan.C2, Kavya.S3, Qudsiya.S.S4, Shalini Maria5, Ashik Raj6, Aamir Javed7 1-6P.G. Department of Biotechnology, R&D Centre, Dayananda Sagar College of Biological Sciences, Kumaraswamy Layout, Bangalore-560078, India 7Jr.Embryologist.Base Fertility Medical Science Pvt, Ltd E-mail: aamir.javed0077@gmail.com Tele: 0919916389255 1*Correspondence Author: E-mail: talkad.murali@rediffmail.com Abstract — The most common polymer in plastics is polyethylene (PE), which is made from ethylene monomers (CH2=CH2). In natural form it is not biodegradable. Low density polyethylene is a vital cause of environmental pollution. It occurs by choking sewer line through mishandling thus posing an everlasting ecological threat, the making of the genetically engineered microbes for bioremediation, the latter being a strategy to develop an accelerated evolution of pathways by DNA restructuring. To enhance the biodegradation of polyethylene, pretreatment strategies were followed. Three different pretreatment strategies were employed for the present study, three duration of Pseudomanas putida treatment to PE were analyzed on 7, 14, and 28th day. In the first, PE films were subjected for Bleach with Alkali treatment and in the second they were subjected to UV light (UV-C,>300nm wavelength). Third with the EMS induction of bacterial strains and assessed for polymer biodegradation by Biomass weight loss, estimation of total carbohydrates and total protein in the culture supernatant, followed by DNA isolation for Gel electrophoresis, and Mutated DNA Stability analysis by Capillary Gel electrophoresis were carried out. Index Terms— Microbial degradation of plastics, chemically treated polyethylene. Biomass, Sugar, Capillary Gel electrophoresis (key words) I. INTRODUCTION Biodegradation is necessary for water-soluble or water-immiscible polymers because they eventually enter streams which can neither be recycled nor incinerated. It is important to consider the microbial degradation of natural and synthetic polymers in order to understand what is necessary for biodegradation and the mechanisms involved. This requires understanding of the interactions between materials and microorganisms and the biochemical changes involved. Widespread studies on the biodegradation of plastics have been carried out in order to overcome the environmental problems associated with synthetic plastic waste. Some studies have, demonstrated partial biodegradation of polyethylene after shorter periods of time.it has been suggested that the biodegradation of polyethylene is enhanced by oxidative pretreatment, which increases surface hydrophobicity by the formation of carbonyl groups that can be utilized by microorganisms. (1, 2, 3) Some microorganisms are indeed capable of degrading the high molecular weight polymer (4) as was evident from a recent report on the biodegradation of thermooxidised polyethylene by P.pinophilum (5). A non ionic surfactant (Tween -80) to the culture medium of Pseudomonas aeruginosa .The surfactant apparently increased the hydrophobicity of the polyethylene surface and thus facilitated the adhesion of bacteria to the polymer (6). Low density polyethylene is one of the major sources of environmental pollution. Polyethylene is a polymer made of long chains of ethylene monomers. The use of polyethylene growing worldwide at a rate of 12% per year and about 140 million tons of synthetic polymers are produced worldwide each year. With such a large amount of polyethylene gets accumulated in the environment, generating plastic waste ecological problems are needed thousands of years to efficiently degradation (7). Microorganisms can degrade plastic over 90 genera, from bacteria and fungi, among them; Bacillus megaterium, Pseudomonas sp., Azotobacter, Ralstonia eutropha, Halomonas sp., etc. (8). Plastic degradation by microbes due to the activity of certain enzymes that cause cleavage of the polymer chains into monomers and oligomers. Plastic that has been enzymatically broken down further absorbed by the microbial cells to be metabolized. Aerobic metabolism produces carbon dioxide and water. Instead of anaerobic metabolism produces carbon dioxide, water, and methane as end products (9). This study aims to isolate the bacteria from waste polyethylene plastics that can degrade polyethylene plastic.
  • 2. International Journal of Ethics in Engineering & Management Education Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 211 The selected Pseudomanas putida bacterial strains were assessed for polymer biodegradation by Biomass weight loss, estimation of total carbohydrates & total protein in the culture supernatant. DNA isolation and stability studies carried out in capillary gel Electrophoresis. II. MATERIALS & METHODS Materials: Low density polyethylene (LDPE) which is the major cause of environmental pollution was used for the study. Microorganism collection · The bacteria Pseudomonas putida (MTCC NO: 2467) used in this study were procured from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh. Cultures were maintained on LB agar plate · Raw materials Plastics is polyethylene (PE) as commercial plastic carry bags of LDPE were collected and cut into small strips and subjected for Chemical - alkali treatment. Chemical - alkali treated polyethylene: polyethylene bags were cut into small strips & transferred to fresh solution containing 18ml tween, 10ml bleach, and 225ml of distilled water & stir it to 30-60mins. Bleach consists of 5gms of sodium chloride, 5gms of sodium hydroxide & 10 ml of glacial acetic acid. Strips were transferred to beaker with distilled water & stir it 2 one hour. They were aseptically relocated to ethanol solution 70%v/v For 30 mins. Finally, the polyethylene strips were transferred to petridish and inoculated at 45°-50°c overnight. Ethanol was used as disinfectant to polyethylene & removes any organic matter adhering to its surface Induction of mutation by UV Materials required:- · UV germicidal light bulb (Sylvania G15T8; 254 nm wavelength) or Stratagene UV Cross linker · 230c incubator · Pseudomonas putida (MTCC NO: 2467) · LB agar plate Grow and mutagenize cells 1. Grow an overnight culture of the desired pseudomonas putida strain in 5 ml LB agar plate at 300c. 2. Determine the density of cell in the culture and record this number Adjust concentration to ~2 × 108 cells/ml if necessary. Transfer 1 ml of the culture to a sterile micro centrifuge tube. 3. Pellet cells in a micro centrifuge for 5 to 10 sec at maximum speed, room temperature. Discard supernatant and resuspend in 1 ml sterile water. Repeat wash. After the second wash, resuspend cells in 1ml of sterile water. Plating:- 1. Make serial dilutions of the culture in sterile water so that each plate has 200 to300 viable cells. 2. Plate 0.1 and 0.2 ml of the diluted cells on separate sets of LB agar plate, using ten plates in each set. Incubate all plates for 3 to 4 days at room temperature. 3. Irradiate all but two plates from each set with UV light using a dosage of 300 ergs/mm2 (there should be 40% to 70% survival). The nonirradiated plates will serve as controls to determine the degree of killing by the UV light. Induction of mutation by EMS Materials required:- Pseudomonas putida (MTCC NO: 2467) Sterile water, LB agar plate, 0.1 M sodium phosphate buffer, pH 7.0, Ethyl methanesulfonate, 5% (w/v) sodium thiosulfate (autoclaved),13 × 100–mm culture tube, Vortex, 300c incubator with rotating platform Grow and mutagenize cells 1. Grow an overnight culture of the desired yeast strain in 5 ml LB agar medium at 300c. 2. Determine the density of cell in the culture and record this number Adjust concentration to ~2 × 108 cells/ml if necessary. Transfer 1 ml of the culture to a sterile micro centrifuge tube. 3. Pellet cells in a micro centrifuge for 5 to 10 sec at maximum speed, room temperature. Discard supernatant and resuspend in 1 ml sterile water. Repeat wash. After the second wash, resuspend cells in 1.5 ml sterile 0.1 M sodium phosphate buffer pH 7.0. 4. Add 0.7 ml cell suspension to 1 ml buffer in a 13 × 100–mm culture tube. Save remaining cells on ice for a control. 5. Add 50 μl EMS to the cells and disperse by vortexing. Place on a rotating platform and incubate 1 hr at 300c. (EMS treatment should cause 40% of the cells to be killed). 6. 7. Transfer 0.2 ml of the treated cell suspension to a culture tube containing 8ml sterile 5% sodium thiosulfate, which will stop the mutagenesis by inactivation of EMS. If cells are to be stored before plating, pellet in a tabletop centrifuge 5 min at 3000×g at 40c, resuspend in an equal volume of sterile water and store at 40c. Total Biomass: About 1ml of the culture was transferred into 1.5ml micro centrifuge tube & pelleted down at 12000rpm at 4°c for 25 min. The pellet was dried overnight at 50°c & dry weight of the resulting biomass was calculated Total proteins: The total protein concentration in the supernatant was determined by the method reported by Lowry’s method. Bovine serum albumin solutions were used as standards & observance was measured with a spectrophotometer at 595nm. Total sugar: The total sugars were analyzed by anthrone method. Glucose was used as the standard & the absorbance was measured at 495nm Gel electrophoresis: Extraction and estimation of Genomic-DNA by gel electrophoresis- Amnion Bioscience KIT
  • 3. International Journal of Ethics in Engineering & Management Education Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 212 Capillary Gel electrophoresis analysis: Polyacrylamide gel-filled capillaries are usually employed, although new polymer formulations with greater stability to the applied electric field are likely to be introduced shortly. Agarose gels are unable to withstand the heating produced by the high voltages used in capillary gel electrophoresis (CGE). The instrument CGE Pro 9600 – CGE Lauf-Nr 15315(Machine 3) Capillary Gel electrophoresis was used to analyze DNA fingerprinting is a useful tool for identifying the genotype of living organisms by determining their DNA sequence. For this technique, genomic DNA must be amplified by PCR. Capillary electrophoresis separates this amplified DNA with a one base pair resolution and creates specific peaks for each nucleotide to map the DNA sequence. III. RESULTS Characterization of the isolates: based on growth these cultures were identified as pseudomonas putida Fig: 1. Colonies of pseudomonas putida showing Plastic degradation Total Biomass Bacterial biomass is a direct measure of the growth of the culture in the medium. Chart shows the variation in biomass during 7, 14 & 28 days respectively Pseudomonas putida control Pseudomonas putida plastic Pseudomonas putida domestic waste Pseudomonas putida plastic + domestic waste Total sugars in the obtained filtrate The amount of total sugars produced by bacterial strain during 7, 14 & 28 days respectively Sugar pseudomonas putida control
  • 4. International Journal of Ethics in Engineering & Management Education Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 213 Sugar pseudomonas putida plastic Sugar pseudomonas putida domestic waste Sugar pseudomonas putida domestic waste + plastic Figure-2: Capillary Gel Electrophoresis - Pro 9600 Sample: Pseudomonas Putida/EMS/Genomic /DNA/Plastic Figure-3: Capillary Gel Electrophoresis - Pro 9600 Sample: Pseudomonas Putida/EMS/Genomic /DNA/Plastic DW Figure-4: Capillary Gel Electrophoresis - Pro 9600 sample: Pseudomonas Putida/UV/Genomic /DNA/Plastic Figure-5: Capillary Gel Electrophoresis - Pro 9600 sample: Sample: Pseudomonas Putida/UV/Genomic/DNA/Plastic+ DW Analysis of Capillary Gel electrophoresis: Separations of oligonucleotides and DNA sequence products have been accomplished in polyacrylamide gels. For restriction fragments and larger oligos, gels with little or no crosslinker seem most effective due to the larger pore size of the gel. Separation of deoxyoligonucleotides such as poly (dA) 40-60 is readily accomplished in an 8% T gel with a buffer consisting of 100 mM Tris-borate, pH 8.3 with 2 mM EDTA and 7 M urea, in under 35 min with unit base resolution. Determining the purity of synthetic oligos is an important application of CGE. Pseudomonas Putida/EMS/Genomic /DNA/Plastic: DNA is Stable and the Elution happened at 67.6 Minutes, Heavy Molecular Weight - DNA. Lot of Pre and Post Elution of Genomic DNA, reason may be DNA Fragmentation. (Fig: 2).
  • 5. International Journal of Ethics in Engineering & Management Education Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 214 Pseudomonas Putida/EMS/Genomic /DNA/Plastic+DW: Stable DNA 97.2 % Purity and Elution at 43.31, Mutation Stable. (Fig: 3). Pseudomonas Putida/UV/Genomic /DNA/Plastic: Stable DNA 90.8 % Purity and Elution at 41.22, Mutation Stable. (Fig: 4). Pseudomonas Putida/UV/Genomic /DNA/Plastic+ DW: Unstable Mutation Not Suggested to be Induced .Internal Folding. (Fig: 5). IV. DISCUSSION Although the bio-degradation and bio-deterioration of polyethylene has been demonstrated by several researchers, the enzymes involved and mechanisms associated with these phenomena are still unclear. Nevertheless, it is recognized that both enzymatic and abiotic factors (such UV light) can mediate the initial oxidation of polyethylene chains, and given the chemical similarity between polyethylene and olefins it has been suggested that the metabolic pathways for degradation of hydrocarbons can be used once the size of polyethylene molecules decrease to an acceptable range for enzyme action (typically from 10 to 50 carbons). The long-range structure and morphology of polyethylene have shown important roles, with amorphous regions being more prone to microbial attack than crystalline ones. The Microbial Degradation of Plastic (LDPE) polyethylene & domestic waste mixture with plastic when were induced with UV & EMS in Pseudomanas putida successfully revealed the beneficial response in Biomass reduction for better yield against growth, sugar conversion along with proteins utilization consistently proven in both normal and mutated organism as the days succeeded may be by more than a month, soil mixture and domestic waste with plastic : polyethylene bags dumping can be eco-friendly manageable to degrade and utilize the biomass for agricultural cultivation of crops May be Physico-chemically treated polyethylene films were found to be effectively degraded by the fungal isolates than untreated films. The hypothesis is that a physicochemical treatment of the polymer leads to its oxidation and subsequent breakdown assisting in the easy assimilation by the fungus and, hence, can be effectively used as a pretreatment strategy before subjecting the polymer to biodegradation (10). The oxidized polymer helps in adhesion of microorganisms (due to probable changes in the hydrophobicity of the polymer surface), which is a prerequisite for biodegradation (11). Similarly in the present study, a higher biomass was observed on the pretreated samples. Because carbohydrates in the medium constitute the main energy source for their growth and metabolism during the nonavailability of readily assimilating carbon source, microorganisms adhere to the polymeric surface during the formation of the biofilm, which is essential for bringing about degradation (12). It is also based on research (13) these bacteria Pseudomonas sp. able to degrade the plastic by 8.16% and was able to degrade the polythene by 20.54% within one month incubation anaerobically. While this type of fungi Aspergillus Glaucus able to degrade the plastic by 7.26% and was able to degrade the polythene by 28.80% within one month incubation anaerobically, from the results of the degradation of polythene faster and easier than plastic degradation. Earlier publications interpreted the growth of microorganisms on polyolefins, e.g. polyethylene as being limited to the microbial action on the surface of an inert support without impact on the polymers (14). However, it was found that polyethylene is not only colonized but also biodegraded by various fungi mostly belonging to the genera Aspergillus, Fusarium or Penicillium (15). Polythene and plastics are two polymers with wide application, both are recalcitrant and thus remain inert to degradation and damage that leads to accumulation in the environment, and create serious environmental problems. Therefore, further research is needed to prevent environmental damage caused by plastic and polythene waste contamination (16). Past research has isolated Pseudomonas putida from sludge in industrial waste and determined that it used o-chloronitrobenzene (o-CNB) as its only carbon, nitrogen, and energy source. Most importantly, the highest degradation of o-CNB (85%) by P. putida was found to be at 32°C and a pH of 8.0. Although o-chloronitrobenzene is not plastic, this research gives a general idea of ideal growing conditions for P. putida, and shows that it is capable of using one source as its only carbon, nitrogen, and energy source (17). Microorganisms are unable to transport the polymeric material directly into the cell due to the lack of its solubility in water & its size. They excrete extra cellular enzymes which aid in the degradation of polymers outside the cells (18). The superficial growth of hyphae on the polymer surface was a function of the oxidation levels of treated sample was observed (19). Therefore pretreated samples showed greater weight loss than untreated samples. Since the continuous introduction of recalcitrant materials, microorganisms are challenged to develop new pathways by altering their own preexisting genetic components by either mutation(s) in single structural and/or regulatory gene or perhaps recruitment of single silent gene when they encounter the foreign compounds (20). In Pseudomonas Putida, when EMS induced Genomic DNA isolated from Plastic+DW group showed: Stable DNA of 97.2 % Purity and Elution at 43.31, Mutation were Stable and beneficial. Same as Pseudomonas Putida, when UV induced Genomic DNA from Plastic: showed Stable DNA with 90.8 % Purity and Elution at 41.22, Mutation were Stable and beneficial.
  • 6. International Journal of Ethics in Engineering & Management Education Website: www.ijeee.in (ISSN: 2348-4748, Volume 1, Issue 5, May 2014) 215 CONCLUSION The Microbial Degradation of Plastic (LDPE) polyethylene & domestic waste mixture with plastic when were induced with UV & EMS in Pseudomanas putida successfully revealed the beneficial response better option for further utility in commercial or municipal dump yards at better strain improvement and longer duration could be an ideal organism for the sustainable technology ACKNOWLEDGMENT The authors are awfully thankful to Dr. Premchandra Sagar (Vice Chairman), Dr. Krishne Gowda, Director, Dayananda Sagar College of Biological Sciences, Dr. C.D. Sagar Centre for Life Sciences, Bangalore-560078, India, for their colossal guidance and support for this project. REFERENCES [1] Albertsson AC. Biodegradation of synthetic polymers. 2. Limited microbial conversion of C-14 in polyethylene to (CO-2)-C-14 by some soil fungi. J Appl Polym Sci, 22, 3419–3433, 1978. [2] Albertsson AC. The shape of the biodegradation curve for low and high density polyethylenes in prolonged series of experiments. Eur Polym J, 16, 623–630, 1980. [3] Cornell, J.H., Kaplan, A.M. and Rogers, M.R. Biodegradation of photooxidized polyalkylenes. Journal of Applied Polymer Science, 29, 2581–2597. 1984. [4] Yamada-Onodera K, Mukumoto H, Katsuyaya Y, Saiganji A, Tani Y. Degradation of polyethylene by a fungus, Penicillium simplicissimum YK. Polym Degrad Stabil, 72,323 –327, 2001. [5] Volke-Sepulveda, T., Saucedo-Castaneda, G., Gutierrez- Rojas, M., Manzur, A. and Favela-Torres, E. Thermally treated low density polyethylene biodegradation by Penicillium pinophilum and Aspergillus niger. Journal of Applied Polymer Science 83,305–314, 2002. [6] Albertsson AC, Karlsson S. The influence of biotic and abiotic environments on the degradation of polyethylene. Prog Polym Sci, 15, 177–192, 1990. [7] Usha R., Sangeetha T., Palaniswamy M. Screening of Polyethylene Degrading Microorganisms from Garbage Soil. Libyan Agric Res Center J Internati, 2 (4), 200-204, 2011. [8] Chee J. Y., Yoga S. S., Lau N. S., Ling S. C., Abed R. M. M., Sudesh K. L. Bacterially Produced Polyhydroxyalkanoate (PHA): Converting Renewable Resources into Bioplastics. Appl Microbiol & Microbiol Biotech, a Mendez Vilas (Ed), 2010. [9] Glass JE, Swift G. Agricultural and Synthetic Polymers, Biodegradation and Utilization, ACS Symposium Series, 433. Washington DC: American Chemical Society, p. 9-64, 1989. [10] Arkatkar, A., Arutchelvi, J., Bhaduri, S., Uppara, P. V., Doble, M. Int. Biodeterior. Biodegrad, 63, 106 –111, 2009. [11] Tribedi P, Sil AK. Cell surface hydrophobicity: a key component in the degradation of polyethylene succinate by Pseudomonas sp. AKS2. Journal of Applied Microbiology, Volume 116, Issue 2, pages 295– 303, February 2014. [12] Weibin, G.,Shimin, H., Minjiao, Y, Long, J., Dan, Y. Polym. Degrad.Stab, 94, 13–17, 2009. [13] Kathiresan K. Polythene and plastics degrading microbes from the mangrove soil. Rev Biol Trop (513), 629-634, 2003. [14] Chandra R., Rustgi R. Biodegradable polymers. Prog. Polym. Sci, 23, 1273. 1998. [15] S. Łabużek, B. Nowak, J. Pająk. The Susceptibility of Polyethylene Modified with Bionolle to Biodegradation by Filamentous Fungi. Polish Journal of Environmental Studies, Vol. 13, No. 1, 59-68, 2004. [16] Albertsson AC, Andersson SO, Karlsson S. The mechanism of biodegradation of polyethylene. Polym Degrad Stabil, 18, 73– 87, 1987. [17] He, Q., Liang, S., Wang, Y., Wei, C., & Wu, H. Degradation of o-chloronitrobenzene as the sole carbon and nitrogen sources by Pseudomonas putida OCNB-1, Science Direct, 21, 89-95, 2009. [18] Brown BS, Mills J, Hulse JM. Chemical and biological degradation of plastics. Nature, 250, 161–163, 1974. [19] Potts JE. Biodegradation. In: Jelinek HHG (ed). Aspects of degradation and stabilization of polymers, Elsevier, New York, pp 617–658, 1978. [20] Chaudhry, G.R. & Chapalamadugu, S. Biodegradation of halogenated organic compounds. Microbiological Reviews. American Society for Microbiology, 55(1) pp, 59-79, 1991. ****