An Effective Study on Rapid Degradation of Polythene Bags and Plastic waste by Microbial Species

 

V. Ratna Kumari¹, Dr. S. D. Kulkarni²

¹M.Sc II Year, Department of Environmental Sciences, New Arts, Science and Commerce College, Lal Taki Road, Ahmednagar, Maharashtra 414001.

² Head of the Department, Department of Environmental Sciences, New Arts, Science and Commerce College, Lal Taki Road, Ahmednagar, Maharashtra 414001.

 

ABSTRACT:

Human beings from the ancient era are in the path of inventing new things. Human beings have invented and developed so many things which are very much useful. One of those useful product is plastic-which constituents wide range of synthetic or semi-synthetic polymers. Polyethene is the most common plastic. The annual global production is around 80 million tonnes (Piringer and Baner 2008, p. 32.) In order to fulfil the demand of out bursting population of the world, huge quantity of polythene is produced for numerous uses such packaging (plastic bags, plastic films, geomembranes, containers including bottles). This invention of plastic by human beings has become threat to mother earth, as the plastic constitutes major share in land pollution on the earth. Plastic pollution has adverse effects on human beings, animals, birds, land, water and our total environment. We have observed that many landfills contain majorly polythene bags, water bodies in many areas are clogged due to excessive dumping of polythene bags. In this study, we study a method for rapid degradation of polythene bags and other plastic waste by some microbial species. Some of the bacterial species isolated from landfill soil, plastic industries premises, water bodies clogged with plastic waste etc are capable of degrading polythene under specific conditions. Four microbial species two bacterial (Bacillus, Pseudomonas spp.) and two fungal species (Aspergillus, Candida spp.) are capable to efficiently degrade polythene bags. Fermentation study in shake flask technique for 2 months in the laboratory scale has been conducted. Confirmation of plastic degradation was done by physical parameters and FTIR analysis. In future, this method will be devised to degrade polythene bags in household or in huge digesters in large scale level. This study will be very helpful, to minimize plastic pollution in landfills on the earth: making earth a safe place to survive for living creatures.

 

 

 

1.      INTRODUCTION:

The term plastic is a term derived from the Greek word plastikos which means able to be molded into different shape [1] and comprises of any synthetic or semi-synthetic organic polymers with high molecular mass and that are moldable. Plastics are man-made long chain polymeric molecules [2].

 

Mainly synthetically derived from petrochemicals. Plastic material such as polyethylene, polypropylene, polystyrene, polyvinyl chloride and polyethylene terephthalate have wide applications in industries and human life.

 

However, most of these conventional plastics are non-biodegradable or biodegradable at a very slow rate [3]. Hence, the accumulation of these materials has been accountable for innumerable environmental hazards like air water and soil pollution and has been a threat to the planet [4]. Plastics are traditionally composed of petroleum based resins like polythene and polypropylene which are very stable and do not get degraded in the ambient environment [5] leading to its accumulation and as a result pollution. Though plastics made of natural polymers are inherently biodegradable, the synthetic ones are the real threat to the environment. Over the last two decades research has been concentrated on microbial degradation of natural and synthetic polymers. At present, there are several general guidelines concerning the structure and biodegradability of polymers [6]. Biodegradable and natural plastics are polyesters which are produced by a range of microbes, cultured under different nutritional and environmental conditions [7]. Biodegradation of natural and synthetic plastics are carried out by microbes like bacteria, fungi and actinomycetes [8]. Biodegradation depends directly upon the molecular weight of the polymers and the rate of biodegradation declines with an increase in the molecular weight of polymers [9].

 

In this study, soils from different locations such as landfill site in Ahmednagar, Soil from the premises of Plastic recycling industry-Tejas shah industries in Ahmednagar and another water sample from stream with accumulation of plastic waste are collected. The micro-organisms isolated from those sites are verified for bio-chemical tests. The microbial strains are sent for strain identification to NCIL, Pune. The fresh cultures of microbial isolates have been prepared and subjected to mix up with polythene bags collected from garbage in shake flask. Shake flask fermentation studies with microbial strain inoculated with polythene bags pieces are studies for 2 months. Efficiency of Bacterial and fungal species to degrade polythene bags is determined by study of growth of those strains on mineral rich agar with LDPE powder, zone of clearance formed.

 

2.      MATERIALS AND METHODS:

2.1    Materials:

All the reagents required to prepare different culture media were obtained from Hi-Media Laboratories. All the glassware required for the experiment is provided by Department of Environmental Sciences, New Arts, Science and Commerce College, Ahmednagar, Maharashtra.

 

2.2 Sample Collection:

Soils enriched with plastic degrading microbial species are majorly found near plastic contaminated sites. Soils from different locations such as landfill site in Ahmednagar, Soil from the premises of Plastic recycling industry-Tejas Shah Industries in Ahmednagar are collected. The soil samples were collected from the depth of 5 cm in sterile containers and air dried in the laboratory at room temperature. A Sterile container is used to collect water sample from stream with accumulation of plastic waste near to Ahmednagar city. A thick polythene bag (40 GSM) and thin polythene (20 GSM) polythene bags were purchased from the local market.

 

2.3    Isolation of microorganisms:

Microbial isolation from soil:

A 1% (w/v) soil sample solution was prepared by adding 1gm of soil sample in 99 ml of sterile double distilled water. The soil solution was shaken properly and Serial dilution from 10^-1 to 10^-8 was carried out. For each dilution triplicate Luria Bertani (LB) agar plates were made to isolate bacteria and potato dextrose agar (PDA) plates to isolate the fungi. The LB plates were incubated at 37ºC for 2-3 days and the PDA plates were incubated at 25ºC for 4-7 days. The developed colonies were isolated and sub-cultured repeatedly to get the pure culture and preserved as slants at 4ºC.

 

Microbial isolation from water:

The 1% (v/v) water sample was prepared by adding 1 ml of collected water sample in 99 ml of sterile double distilled water. The water sample collected from the stream clogged with plastic waste is shaken properly and serial dilution from 10^-1 to 10^-8 were carried out. For each dilution triplicate Luria Bertani (LB) agar plates were made to isolate bacteria and potato dextrose agar (PDA) plates to isolate the fungi. The LB plates were incubated at 37ºC for 2-3 days and the PDA plates were incubated at 25ºC for 4-7 days. The developed colonies were isolated and sub-cultured repeatedly to get the pure culture and preserved as slants at 4ºC.

 

Screening for plastic degrading microorganisms:

To select the bio-degrading microorganism from the isolated ones, they were cultured in mineral salt agar plates containing LDPE powder at a concentration of 0.1% (w/v). The microorganisms that showed zone of clearance around their colonies were selected for further studies regarding their potency on biodegradation of plastic carry bags [10].

 

Enrichment technique was carried out to select the efficient polythene-degrading fungus. M1 broth was used to enrich fungus. 100 ml of M1 medium containing (g l⁻¹) NaNO₃ 2; KCl 0.5; MgSO₄·7H₂O 0.5; glucose 10; FeCl₃ 10; BaCl₂ 0.2 and CaCl₂ 0.5 at pH 6.8 were supplemented 0.1 % w/v of LDPE powder was taken. Enrichment for bacterial strains was carried out using mineral salt broth with pH maintained at 7.

 

2.4    Identification of plastic degrading microorganisms:

From the isolated microorganisms, two bacteria and two fungus were successfully screened for plastic biodegradation property with the help of zone of inhibition formed around the discs. The bacteria were further identified on the basis of macroscopic and microscopic examinations and biochemical analysis according to Bergey s manual (Holt et al. 1994). Fungi identification was done by lacto phenol cotton blue staining test following the keys Raper and Fennell ^[11]

 

 

Zone of inhibition around Aspergillus Spp.

 

 

Zone of inhibition around Bacillus Spp.

Fig 1: Figure showing the zone of inhibition formed on mineral salt agar plates containing LDPE powder at a concentration of 0.1% (w/v).

 

Uniform suspension of microbial strains were prepared and tested for plastic degrading activity on mineral salt agar plates with LDPE powder.

 

3.      RESULT AND DISCUSSIONS:

3.1    Fermentation study with the microbial isolates.

All the microbial isolates were screened for their potency to degrade polymer after an incubation period of 8-10 days at 25-30ºC. The efficiency of screening was based on the area of clear zone created by each microorganism around disc. Two fungal species of Aspergillus and Candida (Species identified with the help of morphological and staining technique of cotton phenol blue) and Bacterial species of Bacillus and Pseudomonas have formed the zone of clearance in the range varying in diameter from 0.5cm to 1.2cm.Based on this screening, two fungus and two bacterial species with maximum degradation activity were chosen for fermentation in M1 broth and mineral salt for fermentation study respectively. Polythene bags cutted in pieces were weighed, washed with 70 % iso-propyl alcohol then with water and introduced into the flask containing liquid broth media (Mineral salt broth for bacterial and M1 broth for fungal species).

 

The inoculums of fresh 24hr culture are taken in 0.1 % v/v in different flasks. The flasks are mounted over the shake flask assembly for 60 days and labeled properly, study was carried under fed batch culture with supply of nutrients i.e supply of fresh media during regular intervals to the flask containing microbial strains with polythene bags. Shake flasks containing mineral salt agar plates were inoculated by isolated bacteria and fungi strains RB (1), RBI (2), RFI (3) and RF (4).

 

Negative control: Flask containing media and polythene cut pieces without microbial strains is taken as negative control.

 

3.2    Degradation of thin(20 GSM) and thick plastic(40 GSM) by the screened microorganisms:

The screened microorganisms were further tested for their ability for degradation of plastics in laboratory conditions. The microorganisms were incubated in suitable broth culture under shaking condition for time period of 2 months having 20 GSM and 40 GSM of thick and thin plastic strips respectively. After a time interval of 20, 40and 60days, the plastic strips were collected from the culture, washed thoroughly with double distilled water and air dried. The strips were then weighed to study their final weight (see values in Table).

 

The two bacterial species of Bacillus and Pseudomonas revealed partial degradation of thick and thin plastic strips utilizing them as sole carbon source. On the other hand, Aspergillus species exhibited the formation of a fungal mat and plastic lumps with time, which resulted due to its degradation. Cell surface hydrophobicity of these microorganisms was the leading factor for the formation of biofilm, consequently enhancing the degradation of plastics.

 

The observations manifests that the thin plastics are more biodegradable than the thick plastics. This attributes to the fact that thin plastics are mainly composed of polyethylene as because they are 5-times thinner than the plastics. The organisms attach to the surface of the plastic strips and starts growing using the polymer as their only carbon source, degrading them into low molecular weight monomers and dimers ^[12].

 

 

a) Bacillus spp

b) Pseudomonas spp

 

c) Candida spp

d) Aspergillus spp

Fig 2: Microscopic images of Gram’s Staining of bacterial species and lacto phenol cotton blue staining of fungal species.

 

Above figure shows the morphology of bacterial and fungal species capable of degrading polythene, bacterial species seen under light microscope after Gram’s Staining and fungal species identified using lacto phenol cotton blue staining test.

 

3.3    Spectroscopic analysis:

The changes in the polymer bonds due to biodegradation of polythene by microbial strains were determined using FTIR spectrophotometer (8400 Shimadzu, Japan, with Hyper IR-1.7 software for Windows). The LDPE film exposed to the isolates was analyzed after 60 days of incubation period which was recorded from frequency of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹ at room temperature with a helium–neon laser lamp as a source of IR radiation. The media at the end of 60 days is centrifuged and supernaunt is collected from the centrifugation tube. It was observed that polythene bonds were broke down and media consists the traces of ethylene glycol which is the precursor of polythene.

 

 

Fig 3: FTIR Spectrum of polythene after degradation study.

 

Table 1: Table illustrating the variations in values after fermentation studies for 2 months.

Parameter	Thin polythene bags (20 GSM) (Results with Fungal species RI(3)
	Initial	After 20 days	After 40 days	After 60 days
Appearance	Pink color	Pink color	Light pink color	color Faded
Weight	300 mg	298 mg	296 mg	293 mg
Thickness	1.0 mm	1.00 mm	0.99 mm	0.97 mm
Appearance	Pink color	Pink color	Light pink color	color Faded
Weight	300 mg	298 mg	296 mg	293 mg
Thickness	1.0 mm	1.00 mm	0.99 mm	0.97 mm

 

Table 2: Table illustrating the variations in values after fermentation studies for 2 months for thick and thin polythene bag.

Parameter	Thick polythene bags (40 GSM) (Results with Fungal species RI(4)
	Initial	After 20 days	After 40 days	After 60 days
Appearance	Green color	Green color	Light Green color	color Faded
Weight	500 mg	499 mg	498 mg	497 mg
Thickness	2.01mm	2.01 mm	2.00 mm	2.00 mm

 

4.      CONCLUSION:

It was concluded that we have isolated, screened and used microbial species for effective degradation of polythene. So, we can use the method of microbial degradation of plastics for decomposing and treating synthetic polymers, this method if followed worldwide in large digesters and households can eliminate the problem of plastic accumulation on the earth’s surface. We can make our earth a clean, tidy and beautiful place with zero plastic accumulation.

 

 

 

5.    ACKNOWLEDGMENTS:

I am very grateful to Department of Environmental Sciences, New Arts, Science and Commerce College for supporting me in my research work. I am thankful to my guide and teachers who encouraged me to carry on this research work.

 

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Accepted on 12.07.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(10): 4421-4425.