Project Proposal (Primary Protocol)

Isolation and characterisation of LDPE degrading bacteria having plant growth promoting actives
Aabeer Kumar Basu (Microbiology Department, Vijaygarh Jyotish Ray College) Anirban Dutta (Chemistry Department, Krishnagar Government College)

Key-Words: Low Density Polythene (LDPE), LDPE degradation, N-fixation, P-solubilisation, IAA-production

Objective: This project is aimed at isolation, characterisation and cultivation of Low Density Poly Ethene (LDPE) degrading bacteria and assessing their plant growth promoting activities. For assaying plant growth promoting abilities, three properties will be studied: ability to fix nitrogen, ability to solubilise inorganic phosphorous and production of Indole Acetic Acid (IAA). After being through these tasks, an attempt would be made to manufacture a bio-fertiliser using the bacterial strains isolated in this project.

Methodology:
(The tests/experiments and their procedures, as on requirement, may be changed.)

1. Making LDPE powder: LDPE films would be cut into small pieces, immersed in xylene and boiled for 15 minutes and then crushed. LDPE powder so obtained would be washed with ethanol to remove residual xylene and allowed to evaporate to remove excess ethanol. Powder would be dried in hot air oven at 60 0C overnight (refer to no. 5 and 6 in the reference list). 2. Collection of samples: Soil samples will be collected from various waste disposal vats and dustbins. If possible, with permission, soil samples will be collected from _________________________________________________________. 3. Isolation and characterisation of LDPE degrading bacterial strains: Selective enrichment techniques would be employed to isolate LDPE degrading bacteria. The media used will be minimal media with LDPE as chief carbon source (refer to no. 8 in the reference list). Characterisation will be done through various staining techniques and biochemical tests (refer to no. 8 in the reference list). 4. Assessment of LDPE degradation: LDPE degradation by the bacterial strains would be monitored using any one or a combination of the following methods:

Project Proposal (Primary Protocol)

5.

6.

7. 8.

4.1. By measuring the change in tensile strength and percentage extension of the LDPE films after being inoculated with bacteria for a time period (refer to no. 6 in the reference list). 4.2. By measuring the Biological Oxygen Demand (BOD) of the media with LDPE as sole carbon source (refer to no. 6 in the reference list). 4.3. By trubidimetric analysis of the media, and plotting the growth curve of the bacterial strains (refer to no. 6 in the reference list). 4.4. Microscopic analysis of LDPE films after being inoculated with bacteria for a time period (refer to no. 4 in the reference list). 4.5. By using modified Sturm Test, i.e., quantifying the amount of CO2 produced by the bacteria by degrading LDPE (refer to no. 4 in the reference list). Assessing N-fixing abilities of the isolated strains: N-fixing ability of isolated bacterial strains would be assessed using either or both the following methods: 5.1. By assessing their acetylene reduction activity (ARA), using Burks N-free medium. 5.2. By using N-free mannitol agar plate. Assessing P-solubilising abilities of the isolated strains: P-solubilisation test would be conducted qualitatively by plating the isolated bacterial strains in agar containing precipitated tricalcium phosphate. Bacterial culture would be streaked on the surface of the agar plates. The presence of clearing zone, if present, around the bacterial colonies after overnight incubation would be considered indication of P-solubilisation. Assessing IAA production by the isolated strains: Patten and Glick (refer to no. 2 and 7 in the reference list) method of assaying IAA production would be used. Producing bio-fertiliser: This section is provisional and completely dependent on the success of the previous sections. After isolating LDPE degrading bacteria having the three before mentioned plant growth promoting activities, a trial would be made to check their abilities to enhance plant growth in combination with each other. The chance of their success when used as bio-fertiliser will be observed.

References: 1. Husen, E., Screening of soil bacteria for plant growth promotion activities in vitro; Indonesian Journal of Agricultural Science 4(1) 2003:27-31. 2. Patten, C.L., and Glick, B.R., Role of Pseudomonas putida Indole Acetic Acid in Development of Host Plant Root System; Appl. Environ. Microbiol. 2002, 68(8):3795. 3. Karnwal, K., Production of IAA by fluorescent Pseudomonas in the Presence of LTryptophan and Rice Roots Exudates; Journal of Plant Pathology, 2009, 91 (1), 61-63. 4. R. Pramila and K. Vijay Ramesh, Biodegradation of LDPE by Fungi Isolated From Marine Water a SEM Analysis; African Journal of Microbiology Research, Vol. 5(28), pp. 5013-5018. 5. Kumar, S., et al, Isolation and Identification of LDPE Degrading Fungi from Municipal Solid Waste; JCPRC, 2013, 5(3):78-81.

Project Proposal (Primary Protocol)

6. Sharma, A., and Sharma, A., Degradation Assessment of Low Density Polythene (LDP) and Polythene (PP) by an Indigenous Isolate of Pseudomonas stutzeri; JSIR, Vol. 63, March 2004, pp 293-296. 7. Shakina J. et al, Microbial Degradation of Synthetic Polyesters form Renewable Resources; Indian Journal of Science, Volume 1, Number 1, November 2012. 8. Cappuccino, J. G., and Sherman, N., Microbiology: A Laboratory Manual; 7 th edition, Pearson Education, Inc., Pages: 53-92, 203-208, 459-466, 512.