Plastics are engineered polymers comprising little natural monomer units joined into a long chain by covalent bonds shaped during a polymerization response. The term 'plastic' is gotten from the Greek word 'plastikos', which signifies 'to form
The fundamental kinds of plastic in like manner use are polyethene or polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC), polypropylene or polypropene (PP), polyethylene terephthalate (PET) and polyurethane (PUR). Polythene finds a wide scope of utilizations in people day by day use on account of its simple preparing
Cremation, reusing and land filling are a portion of the customary techniques for taking care of plastic squanders. Be that as it may, these techniques are exorbitant and regularly make new ecological troubles. When contrasted with different strategies, biodegradation is contamination agreeable.
Presence of alkB gene in the selected strains were determined to rule out its efficacy to be explored as potential microbe to be employed for biodegradation of LDPE.
Polyethylene bags(40 microns) were collected from local market. Low Density Polyethylene powder(< 400 micron) was purchased from Vijaya scientific company, Chennai, Tamil Nadu, India.
Soil samples are gathered from four distinctive plastic unloading destinations in Chennai (Pallikaranai, Perungudi, Medavakkam and Sirucheri). They were gathered at a profundity of 3 to 5cm, in a sterile polythene pack. They were firmly stuffed and deliberately moved to the lab for the examination and put away at 4°C aseptically.
Various parameters like pH, Temperature, Moisture content, Alkalinity and Organic matter were tested to study the soil morphology.
1g of soil was suspended independently in 9 ml sterile saline and serially diluted. 10-5 and 10-6 dilutions were plated on supplement agar and hatched at 37⁰C for 24-48 hours to isolate diverse bacterial strains. The colonies with various settlement morphology were chosen and sub refined in the particular media for additional utilization.
The isolated organisms were screened for its ability to degrade polyethylene using mineral salt medium. Low density polyethylene powder was added to mineral salt medium at a final concentration of 0.1% (w/v) respectively. The medium was continuously shaken at 120 rpm in shaker for 1 hour, and then sterilized.
From the isolated bacteria, totally four bacterial organisms were screened based on their ability to utilize polyethylene as sole carbon source. Bacteria were identified on the basis of microscopic examination and biochemical analysis according to Bergey’s manual of Determinative Bacteriology.
Polyethylene (LDPH) films were cut in to pieces (3x3cm), disinfected with Tween 20, distilled water and 70% ethanol for 30min, air dried for 15min in laminar air flow chamber. Polyethylene films were weighed (Initial weight). 1ml of culture was inoculated in 250ml Erlenmeyer flask containing 100ml of MSM broth (autoclaved) and polyethylene films.
To measure the accurate weight of residual polyethylene, microbial biofilms were washed off from the polyethylene surface with 2% (v/v) Sodium Dodecyl Sulfate (SDS) overnight, followed by rinsing with distilled water. This is air dried and weighed (Final weight).
The bacterial culture was grown at 37°C for 24 hours in LB broth and 10ml of culture was taken and centrifuged at 5000 rpm for 10 minutes. 1mL extraction buffer was added. 1ml of lysozyme solution was added to the above suspension and incubated at 37°C for 30 minutes with intermittent stirring. After the incubation, the lysis was completed by adding 2mL of 25% SDS solution. The preparation was heated for 10 minutes at 60°C in a water bath and finally cooled down to room temperature. 5M perchloride was sufficiently added to the lysed preparation to the final concentration of 1M. Equal volume of chloroform: isoamyl alcohol was added to the lysed preparation suspended in 1M perchloride and shaken slowly (30-60 oscillations/min) in a highly stoppered flask for 30 minutes at room temperature. The resulting emulsion was separated by centrifuging for 5 min at 10000 rpm at room temperature. After centrifugation, the top clear aqueous phase was carefully pipetted out from the coagulated protein emulsion, at the inner phase. The aqueous phase containing the nucleic acid was placed in the beaker. The nucleic acid solution was gently stirred with a sterilized glass rod and 2 volumes of 95% ethanol was added slowly down the side of the beaker. So that the ethanol was layered over the viscous aqueous phase. The stirring was continued up to the preparation to mix throughout the phase and spool all of the gelatinous thread like DNA rich precipitate on the glass rod. The excess fluid was drained off from the spooled crude DNA by pressing the rod against the wall of the beaker until no further fluid can be squeezed from the spooled preparation if the squeezing was not done sufficiently, the alcohol adhering the crude DNA will make it difficult to dissolve the DNA.
The crude DNA was dissolved in 9ml of diluted saline citrate. To the even suspension, 1mL of 3M acetate and 1M EDTA were added and transferred the preparation to 100ml beaker containing 5.4ml of isopropanol. For the storage of DNA, the crude DNA was dissolved in 9ml of saline citrate and stored at 2°c with few drops of chloroform. The isolated DNA was further analyzed by Nanovue- spectrophotometry for purity and gel electrophoresis for integrity.
Gene expression study to determine alkB gene was carried out. 38 μl of sterile triple distilled water was added to a sterile microfuge tube. Then 5 μl of 10X Taq polymerase assay buffer with MgCl2 were added. 3 μl of 2.5 mm dNTP mixed solution and 1 μl of control template DNA was added. Followed by that, 1 μl of each of forward and reverse primers were added. The reaction mixture was layered with 50 μl of mineral oil to avoid evaporation. The amplification was carried out using the following reaction conditions.
Initial denaturation at 94
Denaturation at 94
Annealing at 48
Extension at 72
Final extension at 72
Soil samples were collected from four different plastic dump sites in Chennai to isolate polyethylene (LDPH) degrading bacteria from soil. Totally 20 isolates were obtained from soil sample collected from four different sites in Chennai. First sample was from Pallikaranai dump site. Second sample was obtained from Perungudi dump site. Third one was obtained from Medavakkam dump site. The last sample was collected from Sirucheri waste disposal site.
Totally 20 isolates were obtained from soil sample collected from four different sites in Chennai (
Soil samples were serially diluted from 10-1 to 10-10. Then 10-5 and 10-6 dilutions from each sample was plated in nutrient agar medium for isolation of bacteria. After incubation, various colonies were formed. From bacterial mixed culture, colonies were sub cultured and streaked on nutrient agar medium to isolate bacterial strains.
All the isolates do not have the ability to utilize plastic as sole carbon source. Therefore, they were screened to check their ability to utilize polyethylene (LDPH) as their sole carbon source. The isolated cultures were added to the wells cut on mineral salt medium (MSM) agar and the plates were then incubated at 30-37oc for 2-4 weeks. The isolates which showed growth on MSM agar plates were able to utilize polythene (LDPH) as the sole carbon source.
Nissida suggested that clear zone technique is a convenient method for the investigation of plastic degrading bacteria. Bacteria secrete the extracellular enzymes which degrade the polymeric substances into water-soluble materials, resulting into the formation of a clear zone around the microbial culture, indicating utilization of polyethylene powder as the sole carbon source.
Absolutely four organisms were screened dependent on their capacity to use polyethylene as their sole carbon source. Among the 4 bacterial strains obtained, 2 from Perungudi, 1 from Pallikaranai and other 1 from Medavakkam. None of the isolate utilized polyethylene from Sirucheri dump site.
Soil sample |
pH |
Temperature (˚C) |
Total alkalinity (mg/l) |
Organic matter (%) |
Moisture content (%) |
PA |
6.4 |
27.9 |
50 |
0.82 |
11.85 |
PB |
7.5 |
27.6 |
40 |
1.56 |
11.26 |
PC |
6.9 |
28.5 |
60 |
0.52 |
10.38 |
PD |
8.2 |
35.2 |
30 |
0.92 |
13.50 |
Absolutely four organisms were screened dependent on their capacity to use polyethylene as their sole carbon source. The screened Bacterial isolates were identified according to Bergy’s manual of Determinative Bacteriology by performing gram staining technique, motility test and biochemical tests (
S.No |
Tests |
|
|
|
|
1 |
Gram staining |
(+) ve rods |
(-) ve rods |
(-) verods |
(+) vecocci |
2 |
Motility |
Motile |
Motile |
Non- motile |
Non- motile |
3 |
Catalase |
+ |
+ |
- |
- |
4 |
Oxidase |
Variable |
+ |
- |
- |
5 |
Indole |
- |
- |
+ |
+ |
6 |
Methyl Red |
- |
- |
+ |
+ |
7 |
Voges Proskauer |
+ |
- |
- |
- |
8 |
Citrate |
+ |
+ |
+ |
- |
9 |
Urease |
- |
- |
+ |
- |
10 |
Nitrate Reduction |
+ |
+ |
- |
- |
11 |
Glucose Utilization |
+ |
+ |
+ |
+ |
13 |
Gelatin hydrolysis |
+ |
+ |
- |
- |
14 |
Spore staining |
Spore forming |
Non- sporing |
Non- sporing |
Non- sporing |
Results of degradation of polyethylene by isolates were calculated and mentioned in
S.No |
Bacterial isolate |
Initial weight (mg) |
Final weight (mg) |
Weight loss (%) |
1 |
PAB1 |
40 |
27 |
32 |
2 |
PBB1 |
40 |
25 |
37 |
3 |
PBB3 |
40 |
24 |
40 |
4 |
PCB2 |
40 |
33 |
17 |
High degradation was determined by weight loss of LDPE. Hence, PBB1 and PBB3 shows strong degradation. Therefore, Bacterial isolates like
ALKB gene specific primer synthesized by Biosource Ltd, Bangalore was used to determine the alkane monooxygenase gene and the genomic DNA from the selected isolates were used as a template and amplified by PCR and result is depicted in
Lane 1 - 500bp ladder
Lane 2, 3 and 4 – Genomic DNA of
Lane 5, 6 and 7 –
Heiss-Blanquet S et al.
Saadoun I et al.
Two groups of the bands appeared at 316~334 bp and 460~550 bp, correspondingly, on the agarose gel, and the alkB genes showed bands between 320 and 550 bp. Smits et al.
Bacteria for LDPE biodegradation was isolated from a plastic dump wastes soil sample and were identified depending on their ability to utilize LDPE as carbon source. They were identified asb
We are thankful to our Principal, Head of the department & other faculties in our department for their support to carry out our research work.
No funding sources
None declared
The study was approved by the Institutional Ethics Committee by Akilandes and its ID number : 241/2019