Isolation, Characterisation and Evaluation of Plastic Degrading Properties of Soil Biofilms Collected from Chennai District

Vijayakrishnan S R, Deepa Parvathi V, R Sumitha*

Department of Biomedical Sciences, Sri Ramachandra Institute of Higher Education and Research,

Porur, Chennai - 116.

*Corresponding Author E-mail: sumithamadhu79@gmail.com

ABSTRACT:

Environmental pollution due to accumulation of synthetic polymers namely plastics is a growing concern which threatens the terrestrial marine flora and fauna. Traditional methods of plastic disposal include incineration and disposal into landfills or water bodies. Incineration of polyethylene, polystyrene leads to emission of a large amount of carbon monoxide which is toxic if inhaled and also a potent greenhouse gas. Degradation of plastic by microorganisms is an efficient and eco-friendly method employed for rapid rate of disintegration. The biofilm present in the contaminated soil survives by adapting to harsh environment by secreting hydrolysing enzymes which are potent in degradation of the accumulated plastics. The present study deals with the isolation, characterisation and evaluation of plastic degrading properties of microorganisms isolated from various soil samples collected from Chennai district. Soil samples were collected aseptically from various locations & isolated by standard plate count method. The isolated organisms were identified by staining methods and characterized by phylogenetic analysis. The organism Pseudomonas aeruginosa and Streptomyces fulvissimus were further subjected to plastic degradation testing. The present study demonstrates the ability of Pseudomonas aeruginosa and Streptomyces fulvissimus to degrade polyethylene sheets.

KEYWORDS: Plastic degradation, Pseudomonas aeruginosa, Streptomyces fulvissimus, Phylogenetic analysis, Biofilm.

INTRODUCTION:

Plastics are synthetic polymers that are resilient to degradation, inexpensive, lightweight, and thus have a very wide variety of applications in day-to-day life. Plastic pollution is a very serious issue. Plastic, being non-biodegradable, accumulates wherever disposed. This leads to environmental pollution and the accumulation of plastics¹. Smaller plastic particles (<5mm) called microplastics tend to leach into the surrounding soil. Leaching refers to the process by which small plastic molecules are incorporated into the soil.

Plastics have also been shown to degrade soil fertility by reducing water and nutrient retention capacity. Animals who accidentally ingest plastics also suffer from toxic effects like organ damage, intestinal blockage, and weakened immune systems. Indirect consumption of the degraded plastic metabolites from the plant and animal source in humans leads to severe damage in humans causing ovarian chromosomal damage, decreased sperm production, rapid puberty, rapid changes in the immune system, type-2 diabetes, cardiovascular disorder, obesity, etc². Plastic degradation is the utmost concern in the future to reduce environmental toxicity. Studies have shown that traditional methods like incineration of polyethylene lead to the emission of a large amount of carbon monoxide which is toxic if inhaled and also a potent greenhouse gas.

A novel eco-friendly method is necessary for the degradation of plastic without causing adverse effects. Secondary metabolites include enzymes, antibiotics are non-growth linked products produced by the organism in its lifetime. These are produced during the late growth phase or stationary phase of the organism’s life. These metabolites do not serve any role in the growth or development of the organism but rather is a metabolite that gives it a better chance of survival in a harsh environment. Biofilms are an association of bacteria that are adhered to each other in a matrix. A biofilm generally has properties and metabolites which are normally not found in the constituent organisms. Microbial biofilms growing in polluted soils have a higher chance of producing secondary metabolites like plastic degrading enzymes to adapt to their environment. This will allow them to utilise the plastics present in the soil as sources of energy and biosynthesis. These biofilms can colonise the surface of the polymer. They secrete their enzymes on the colonised surface, degrading them into simpler units. All known plastic degrading enzymes produced by bacteria belong to the class of hydrolases which hydrolyse the linkages between the monomeric units of the plastics in the presence of water. This class of enzymes includes lipases, esterase, PETases, peroxidases, and cutinases. This converts the polymer into simpler and smaller units. The final products of enzymatic degradation by bacteria are Carbon dioxide, Methane, Water, and Nitrogen gas. These products can be utilised by the microbes for the biosynthesis of carbon compounds, amino acids, and proteins required for their growth and survival³. This enzymatic degradation of plastic is a 2-step process. First, the enzyme attaches to the hydrophobic clefts which are found in the polymer structure. Once it has attached to the clefts, it can catalyse the degradation of the polymer in the presence of a water molecule. This occurs by breaking the bonds which connect the monomers. For example, esterase act on the ester bonds in polyesters and break the bond to give a carboxylic acid and alcohol group. The increase in the accumulation of used plastics in the environment due to improper disposal methods and careless littering has become a serious concern. It is becoming important to find an eco-friendly way to degrade plastic to protect the environment from further damage due to plastics⁴. The present study aims to isolate, characterise and observe the plastic degrading properties of microorganisms isolated from various soil samples collected from Chennai districts

MATERIALS AND METHODOLOGY:

Collection of Soil Sample:

Soil samples were collected aseptically from the following locations. Sample A was collected from 13.14°N,80.28°E, Sample B from 13.00°N,80.10°E, Sample C from 13.00°N,80.10°E, Sample D from 13.027°N, 80.251°E and Sample E from 13.024°N,80.263°E respectively. Standard plate count method for isolation of soil samples.

Processing of Soil Sample:

Plate Count Method:

1g of each soil sample was added to 50ml of distilled water and mixed well to prepare the stock sample. 1ml of stock sample was taken in a test tube using a sterile pipette and made up to 10 ml using distilled water to prepare 10^-1 dilution of soil sample. Subsequent dilutions of the soil sample were done to obtain dilutions from 10^-2 to 10^-6. 1000mL of plate count agar was prepared and sterilised. 1ml from each dilution of Sample A was added to the appropriate plate count agar plate using a sterile pipette. A L-Rod was used to ensure an even distribution of the sample across the plate. Similarly, plates were prepared for Samples B, C, D and E. All the plates were incubated in a bacteriological incubator at 37℃ for 48 hours. Colonies of interest were identified and isolated from the culture plate ⁵.

Morphological Characterisation by Gram Staining:

A smear was made from the single colony of interest and after the drying crystal violet stain was flooded to a heat fixed smear of the organism of interest. The slide was washed with distilled water and iodine was added. The iodine was washed off and Gram’s decolouriser was added followed by Safranin stain. The safranin was then washed off and the slide was allowed to dry. The stained smear was then viewed under the microscope to identify morphology and were differentiated and classified based on the cell wall. Based on the differential staining the organisms were isolated and grown on the selective medium.

Phylogenetic Analysis of Organism:

Isolation of DNA from Organism:

A small amount of culture was added to 10ml of Cetrimonium bromide buffer. 0.5ml of β-mercaptoethanol and 20μl of proteinase K were added to the mixture; after transferring them into centrifuge tubes. It was incubated for 1hour at 37°C followed by incubation for 30 minutes at 65°C. Equal volume of phenol: chloroform was added to the mixture. It was then centrifuged at 10000rpm for 10 minutes at 4°C. The aqueous phase was transferred into a new tube to which equal volume of chloroform was added and centrifuged at 10000rpm for 10 minutes at 4°C. After centrifugation the aqueous phase was again transferred into a new tube and 2.5 volume of ethanol was added. This was centrifuged at 10000rpm for 15 minutes at 4°C. The supernatant was discarded and 70% ethanol was added to the pellet. This was followed by centrifugation at 10000 rpm for 10minutes at 4°C. The supernatant was discarded and the pellet was air dried. The pellet was dissolved in a 1x TE buffer and stored at -20°C for further use ⁶.

Agarose Gel Electrophoresis:

The DNA sample was run on 1% Agarose. The 16s rRNA gene forms a distinct band on the gel in an electric field. 1 kb base pair ladder was used to calculate the number of base pairs in the 16s rRNA gene. The bands were visualised under a UV transilluminator.

Polymerase Chain Reaction:

The PCR was carried out by following the below specifications. Initial denaturation at 94°C for 5 minutes. Denaturation at 94⁰C for 45 seconds. Primer annealing at 47⁰C for 1 minute. Extension at 72⁰C for 1minute 20 seconds. The PCR cycle was repeated 35 times. A Final extension was done at 72⁰C for 5 minutes. The PCR product was stored at 4⁰C for further use. Sequencing of 16s rRNA gene was done using Sanger sequencing. The obtained sequence was BLASTed to determine the organism species and strain.

Plastic Degrading Activity of Isolated Microorganism:

To each nutrient agar plate, lawn culture of the organism of interest was streaked. Four 2.5cm x 2.5cm strips of sterilised polyethylene were added aseptically. The initial weight of the strips with the inoculum was noted. The strips with the inoculum were incubated for 10 days. The final weight of the strips was noted. The results were tabulated.

RESULTS:

Collection of Soil Sample:

Soil samples were collected from 5 different locations where soil was contaminated due to environmental pollutants. The plate count agar method was performed and the plastic degrading organisms were isolated from all the soil samples. The isolated organisms were further characterised by gram's staining. Strain A was identified as gram-positive bacilli and strain B was identified as gram-negative bacilli respectively. These strains were isolated further by using the selective media.

Phylogenetic Analysis of Organism:

The isolated strains were sent for phylogenetic analysis. The 16s rRNA genome sequence was performed for both species. BLAST sequencing was performed on the obtained sequences from the PCR as shown in Fig 2 respectively. Strain A was identified as Streptomyces fulvissimus as shown in Fig 3. Species B was identified as Pseudomonas aeruginosa as shown in Fig 4 respectively.

Plastic Degrading Assay:

The plastic degradation assay was performed with the isolated and identified organisms as shown in Fig 4 and Fig 5 respectively. The amount of degradation was tabulated as shown in the Table 1 and Fig 6.

Fig 6: Rate of plastic degradation by isolated organisms Pseudomonas and Streptomyces sp

DISCUSSION:

The present study deals with the degradation of polyethylene which was exposed to the solid culture medium of the isolated and identified organism. The isolated organisms were allowed to grow on the plastic sheets as shown in Fig 5 and Fig 6. The growth of Pseudomonas aeruginosa on the plastic sheet over the period of 10 days was studied as shown in Fig 14. The sheets were observed for growth and degradation every 3 days. Streaks and wrinkles were noted on the surface of the plastic sheets after the 4^th day of incubation. It was also noted that the bacteria had grown and formed colonies on the surface of the plastic sheet. No significant degradation by Streptomyces fulvissimus was observed on the plastic sheet even after the 4th day in comparison with the Pseudomonas aeruginosa plates. Previous study on the degradation of Low-Density Polyethylene by soil bacteria showed 9% in Pseudomonas putida, and 11.3% in Pseudomonas syringae strain. The present study shows 5.1% degradation by Pseudomonas aeruginosa and 1.7% by Streptomyces fulvissimus strains ⁷.

The % weight loss of the plastic sheets (degradation) over a period of 10 days by the isolated bacterial species were calculated as shown in Table 2. This indicates the degradation properties of isolated and identified species.

The difference in degradation between the Pseudomonas aeruginosa and Streptomyces fulvissimus was as depicted in Fig 7. The present study indicates that the degradation of the polyethylene sheet with the isolated colonies of Pseudomonas aeruginosa was comparatively faster than the degradation of the Streptomyces fulvissimus.

The present study indicated that the contaminated soil samples were collected since the microbial biofilm growing in the contaminated soil shows an adaptation to environmental stress and tolerance due to the contamination in the soil. Hence the microbial biofilms growing on the polluted soil are able to produce various secondary metabolites by utilising the organic and inorganic sources like Polyethylene, Polyesters, Polyaromatic Hydrocarbons, Polyamides which cause them to adapt and be able to digest and utilise the pollutants present in the soil as an energy source. Studies have shown that Pseudomonas aeruginosa possesses the ability to secrete various secondary metabolites which have plastic degrading properties. These compounds belong to the class of hydrolase enzymes. The main enzymes secreted by Pseudomonas aeruginosa are Alkane Hydroxylases, Lipases and Esterase^8-10.

Previous studies show that enzymes Hydrolase and Alkane hydroxylase secreted by Pseudomonas aeruginosa show the capability of degrading polythene ^11-12. It was also seen that the expression of the gene coding for alkane hydroxylase was greater in Pseudomonas aeruginosa strains isolated from soils contaminated with petroleum and petroleum by-products¹³.

CONCLUSION:

Degradation of plastic by microorganisms is an exciting field and has a lot of scope for future developments. Several topics of particular interest are genetically modified microorganisms to enhance biodegrading property. This is achieved by recombination of the bacterial genome using plasmids containing various plastic degrading genes. The present study shows the ability of Pseudomonas aeruginosa and Streptomyces fulvissimus to degrade polyethylene sheets. This insight on microbial degradation has opened a new avenue for safe disposal of plastics.

ACKNOWLEDEMENT:

We take this opportunity to thank the Sri Ramachandra Institute of Higher Education and Technology (DU), Porur, Chennai for providing us the fund as summer research fellowship. We would like to thank the Department of Biomedical Sciences, Sri Ramachandra Institute of Higher Education and Research for the necessities and facilities provided for the conduct of the study

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Received on 30.05.2023 Modified on 18.07.2023

Asian J. Pharm. Res. 2023; 13(4):237-243.

DOI: 10.52711/2231-5691.2023.00044

Organism	Initial Weight of Plastic Sheet	Final Weight of Plastic Sheet	Total Weight Loss	% Weight Loss
Pseudomonas aeruginosa	0.0058g	0.0045g	0.0013g	5.1%
Streptomyces fulvissimus	0.0058g	0.0050g	0.0008g	1.7%