Topic name:

MICROBIAL BIODEGRADATION OF POLYETHYLENE BY

LAMINATION OF LIGNIN FROM BLACK LIQUOR.

Outline of the problem:

Polymers become an essential part of our daily life because of its wide
range of applications ranging from sophisticated article such as, prosthetic hips to food utensils. Among these polymers, polyethylene, expressed as CnH2n is the most widely used hydrocarbon. For this reason, large production and enormous use of this polymer lead to their accumulation in the environment which causes a severe damage in the environment because of their inertness towards microbial degradation [1, 13]. It was previously reported that polyethylene treated under UV for 26 days before buried under soil lost nearly 0.5% carbon as carbon dioxide (CO2 ) after 10 years [13]. In another work, it was observed that polyethylene sheet kept in contact with moist soil for 12 years showed no evidence of biodegradation [14]. Only partial degradation was observed in a polyethylene sheet that had been buried under soil for over 32 years [15]. From these reports, it is clear that polyethylene cannot be degraded under natural condition. On the other way, it will continue to deposit on the environment affecting terrestrial and marine eco-system. As human civilization modernized, paper became one of the most important items. So, paper industry takes an important place in the modern industry. Pulp and paper industries produce large quantities of coloured waste effluents, which mainly contain high molecular weight lignin and its derivatives. This kind of effluent, usually called black liquor for its colour, has a high level of chemical oxygen demand (COD). It is toxic for its chemical nature and its black colourisation cause a decrease in the oxygen content of the water affecting aquatic plants and animal [4]. Generally paper or pulp mill produces at least 250400 gallons of black liquor per ton of pulp or paper. Most of this black liquor is recycled for the recovery of chemicals like, soda (sodium hydroxide) and rest is used as bio fuel to produce electrical energy due to presence of high solid content in black liquor. Lignin, the main constituent of black liquor is a biopolymer and composed of phenylpropane monomeric units interconnected by a variety of carbon-carbon bonds and ether linkages [5]. Lignin is difficult to degrade either chemically or biologically due to the presence of -O-4 ether

bonds, -5 and 5-5/ carboncarbon linkages between the lignin molecules, which are not hydrolysable unlike other wood polymers (e.g., cellulose, hemicellulose) [4].

IMPORTANCE:Polyethylene and black liquor both are polluting environment in different ways and both are not usually degraded under natural condition. Polyethylene has -CH2-CH2 - repeating units and so making them hydrophobic in nature. But, for microbial degradation the surface has to be hydrophilic. Therefore, it is necessary to make the polyethylene surface hydrophilic in nature and the hydrophilic groups can be generated by means of chemical oxidation of polyethylene [1]. On the other hand black liquor contains different organic chemicals that can act as the oxidising agent. So, black liquor can be used to oxidise polyethylene. Further, lignin can be isolated from black liquor and subjected to different kinds of modification e.g esterification, epoxidation, hydroxymethylation etc [16, 17,18]. On the other hand surface of the oxidised polyethylene can be chemically modified in such a way, that it can be interlinked with chemically modified lignin from black liquor. In this way, a film of modified lignin can be deposited on the surface of the polyethylene through chemical bonding. Then, this polyethylene sheets will be subjected to microbial biodegradation using lignin eating micro-organisms. Although, it is very difficult to degrade lignin by general micro-organisms but, there are some specific fungus and bacteria that have the ability to degrade lignin. For several years, lots of researches have been carried out to find a lignin degrading microorganisms. Different species of white rot fungus, bacteria had been isolated which have the ability to degrade lignin. In some recent works black liquor effluent from paper and pulp industries were treated with white rot fungi Phanerochaete chrysosporium and Trametes versicolor [8]. On the other work, five white fungi strain Phanerochaete chrysosporium, Pleurotus ostreatus, Lentinus edodes, Trametes versicolor and S22 were grown on supporting media and their ability to degrade lignin, reducing COD was compared under different condition. It was observed that P.ostreatus showed highest efficiency for removal of lignin and reduction of COD [4].

OBJECTIVE: 1. Oxidation of the polyolefin. 2. Initiation of biodegradation of polyolefin through depositing lignin. 3. Separation, identification of microbes for lignin biodegradation. 4. In-situ biodegradation of polyolefin films. TECHNICAL PROGRAMME: Materials: 1. Black liquor: The black liquor was collected from a Kraft paper mill situated in
north-east region of India.

2. Culture media: The media used in this experiment to isolate strain is high in
nitrogenous compound and have higher pH. Content of the media 10gm Trypton, 5gm Beef extract, 10 gm Dextrose, 5 gm NaCl, and 10.6 gm Sodium carbonate (for high pH) is dissolved in 1 litre of distilled water. For solid media additional 20gm of Agar was added. Media was sterilised by autoclaving at 120o C for 20 minutes.

3. Black Liquor media: A stock of diluted black liquor is made by adding 10 ml of

concentrated black liquor with 240 ml of distilled water. For media preparation 20 ml of stock diluted black liquor is added into 80 ml of distilled black liquor and sterile by autoclaving at 120o C for 20 minutes. For solid media additional 2 gm of agar was added and autoclaved.

4. Soil from local chicken run was collected. 5. Other chemicals: potassium dichromate, concentrated sulphuric acid, polyethylene
bag.

Methods:
1. COD test: COD test is done by closed reflux method. 1.5 ml of concentrated sulphuric acid is added into previously dried test tube with screw cap. Then 2 ml of sample waste water is added. Distilled water is added as control. Then 2 ml of potassium dichromate is added and carefully shaken for the mixing of the component. Then test tube are capped and heated at 150o C for 2 hours. Test tubes are then allowed to come to room temperature and OD was measured at 645 nm.

2. Isolation of bacteria:

Bacteria are isolated from soil by serial dilution and agar

plates are kept at 30o C for 48 hours. 3. Oxidation of PE: PE sheets are cut into small pieces and taken into a two necked round bottom flask. Then 250 ml of concentrated black liquor is added. Air condenser is fitted into one neck and in another thermometer. Then it is reflux at different temperature for varying time. 4. Chemical modification of lignin: Esterification, epoxidation, hydroxymethylation [16,17,18]

5. FTIR: It was done by FTIR machine. 6. Chemical modification of Surface of Polyethylene: Different chemical
modification done on the surface of the polyethylene [19].

Work done so far:

It is already reported that chicken run soil is alkaline in nature. Kraft black liquor is alkaline is nature. So, for that reason chicken run soil was used to isolate alkaliphilic bacteria. Using nitrogenous media, two strains were isolated. One was colour less (CH1) and another white coloured water insoluble pigment producing strain (CH2W). Another strain (BH1) was isolated from diluted black liquor that grows on solid black liquor media without any carbon source or nitrogen source added. PE films were refluxed with black liquor at 80oC for 24 hours. FTIR was done of both treated and untreated film.

fig:1 comparison of FTIR between treated and untreated PE films. The peak at 1742 cm-1 is responsible for the carbonyl group that should not be present in normal PE. This peak will form due to formation of carbonyl group after oxidation of PE (11). As in this experiment commercially available PE bags are used as PE films, the presence of 1742 cm-1 may be due to minute oxidation occurred during processing of PE. Growth of the peak 1742 cm-1 was observed when PE refluxed with black liquor at 80oC for 24 hours (fig 1). From this it can be concluded that black liquor is oxidising PE.

WORK PLAN:

1st YEAR

2nd YEAR

3rd YEAR

Isolation of bacteria from different source

Incubation of bacteria with black liquor to observe reduction of COD and lignin

Comparison between isolated bacteria to obtain highest lignin degrading strain

Identification bacteria

of

Layer formed through interlinking of modified PE surface and modified lignin

Incubation of lignin layered PE film with lignin degrading bacteria.

Chemical modification of Lignin Chemical modification of the surface of the PE sheet

Oxidation of PE

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