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Polyhydroxyalkanoate from Bacillus sp.: Its production, isolation and characterization

Divyashree, M. S. (2008) Polyhydroxyalkanoate from Bacillus sp.: Its production, isolation and characterization. PhD thesis, University of Mysore.

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Abstract

Synthetic plastics are widely used for the manufacture of packaging materials, various household and industrially important articles. However they are recalcitrant to microbial degradation and they persist in the soil leading to environment pollution. To overcome the problem of plastic pollution, attempts are being made to replace synthetic plastics by various biopolymers and bacterial polyhydroxyalkanoate (PHA) is one amongst them. PHA is composed of hydroxy fatty acids and it represents a rather complex class of storage polymers synthesized by a various bacteria and are deposited as unique water insoluble cytoplasmic nano sized inclusions. Bacillus spp. represent industrially important group of organisms that are used for various metabolite production and hence attempt was made to isolate this bacterium for polymer synthesis. Based on 16SrRNA, isolated Bacillus sp. was identified as B. flexus and it was found to posses 97% homology with B. megaterium it shared the same cluster with B. simplex. This study has led to a finding of a new unexplored B. flexus strain, which has the ability to produce PHA at 50- 60% level in the biomass. B. flexus grew optimally in media containing different salts of nitrogen, amino acids, carbon sources, plant oils, free fatty acids, economic media components such as palm oil effluent, molasses, corn starch, whey, rice and wheat bran extracts as sources of carbon/nitrogen/nutrients. PHA isolated from the cells was characterized by FTIR/GC/GC-MS/NMR spectroscopy/DSC. Homopolymer of polyhydroxybutyrate was synthesized in B. flexus cells fed with only sucrose as main carbon source and PHA copolymer of polyhydroxy(butyrateco- valerate-co-octanoate) was produced in palm oil effluent containing medium and polyhydroxy(butyrate-co-octanoate-co-decanoate) with rice bran oil as co carbon substrate. This study has shown that based on the fatty acid supplemented in the medium, medium chain length PHA such as octanoate and decanoate can be synthesized by Bacillus spp also. Response surface methodology was used for optimization of nutritional parameters for optimum growth and polymer yield. Maximum production of biomass (7 g/l) and PHA (2.2 g/l) were obtained with 11.6 g/l of KH2PO4, 4.7 g/l of ammonium phosphate and 31 g/l of sucrose. The growth kinetics of B. flexus under nutrient limitation was studied by using a simple model vi ii giving mathematical description of kinetics of microbial growth, substrate consumption and product formation. PHAs are accumulated intracellularly and hence their extraction from the biomass is a critical step for economic production. Numerous separation processes are employed for the recovery of PHA. These involve extraction by organic solvents, which is hazardous and explosive. In the present study, novel physical, chemical and biological methods were assessed for isolation of the intracellular PHA. Analysis of cell wall of B. flexus indicated that the type of nutrients used for cultivation significantly influences its composition. Cells grown in inorganic medium contained lower quantities of amino acids and lacked diaminopimilic acid in the cell wall and cells lysed easily and this can be further exploited for easier recovery of the intracellular PHA. Amongst different alkalis tested for cell hydrolysis, NH4OH efficiently digested non-PHA cellular material at pH 11, to give 50% of PHA with 98% purity. The enzyme produced by Microbispora sp. hydrolysed the cells of B. flexus to release the intracellular PHA. The enzyme was identified as a protease of 40kDa and it was purified to homogeneity. Aqueous two-phase system was successfully employed as a non-organic solvent method for the isolation of PHA from other cellular materials. Exposure of cells to gamma irradiation indicated that the irradiation resulted in cell lysis leading to easy PHA extractability, low degree of cross-linking, and improvement in molecular weight as well as tensile strength of the polymer. Lambda lytic gene was integrated into the amylase gene (amyE) locus of the chromosomal DNA of B. flexus. This caused autolysis of the recombinant cells when xylose was supplemented into the medium. The study has focused on economic production of PHA by a newly isolated strain of bacterium, which is so for not reported as PHA producer, and newer aspects of PHA isolation methods have been worked out.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Plastic pollution; Biopolymers; Bacterial polyhydroxyalkanoate (PHA)-; Isolation methods; Bacillus
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 27 Polymer Chemistry
500 Natural Sciences and Mathematics > 07 Life Sciences > 04 Microbiology
Divisions: Food Microbiology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 18 Mar 2010 07:16
Last Modified: 18 Mar 2010 07:16
URI: http://ir.cftri.com/id/eprint/9402

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