968 J SCI IND RES VOL 70 NOVEMBER 2011 Journal of Scientific & Industrial Research Vol. 70, November 2011, pp. 968-975 *Author for correspondence Tel: 91-471-2515361; Fax 91-471-2495949 E-mail: binodkannur@gmail.com Media engineering for production of poly- β-hydroxybutyrate by Bacillus firmus NII 0830 Sreelatha K Deepthi, Parameswaran Binod*, Raveendran Sindhu and Ashok Pandey Biotechnology Division, National Institute for Interdisciplinary Science & Technology (NIIST), CSIR, Trivandrum 695 019, India Received 18 February 2011; revised 30 August 2011; accepted 02 September 2011 This study evaluates biodiesel industry generated crude glycerol as sole carbon source for the production of poly-β - hydroxybutyrate (PHB) using Bacillus firmus NII 0830. Pretreatment of crude glycerol from biodiesel industry was tried using strong acids. Biomass yield and PHB accumulation by B. firmus NII 0830 in submerged fermentation was compared using crude as well as pretreated glycerol as sole carbon source. It was observed that crude glycerol from biodiesel industry, without any pretreatment, can be used for PHB production. The organism when grown in mineral salt media containing crude glycerol as carbon source gave a maximum yield of biomass (3 g/l) and PHB (53% of cell dry wt). Response surface methodology (RSM) showed that presence of nitrogen and phosphate in fermentation media favored biomass production but reduced PHB production. Optimized crude glycerol media for maximum PHB accumulation contains ammonium sulphate (4 g/l) and K 2 HPO 4 (0.8 g/l). Keywords: Biodiesel industry, Biopolymer, Glycerol, Poly- β-hydroxybutyrate (PHB), Response Surface Methodology (RSM) Introduction Biopolymers originate from renewable sources and, being biodegradable, have a less negative impact on environment compared to petroleum based materials 1 . Poly- β-hydroxybutyrate (PHB) is a most common type of polyhydroxyalkanoate (PHA), synthesized by many bacterial species as intracellular carbon and energy source when grown under suboptimal cultural conditions. PHB is an eco-friendly substitute and an ideal candidate for making biodegradable plastics 2,3 . Important applications of PHB include development of tissue engineered cardiovascular products 4 , PHB based films as matrices for pesticides 5 , treatment of osteomyelitis 6 and food packaging 7 . However, PHB has not been commercialized for large scale applications due to high cost of production and less efficient recovery methods 8 . Efforts are being made to use less expensive carbon sources to reduce cost of PHA. Use of various carbon sources (malt, soya 9 , corn steep liquor 10 , crude glycerol from biodiesel industry 11,12 , jackfruit seed powder 13 , date syrup 14 , molasses 15,16 etc.) are reported for the production of PHA using various bacteria. Because of increasing demand for biodiesel, large amounts of glycerol would be accumulated as a byproduct and can serve as a promising cheap carbon source. For every 3 mol of methyl esters produced, 1 mol of glycerol is obtained, equivalent to about 10% of total product 17 . Crude glycerol (CG) contains glycerol (30-60 wt%), besides soaps, remaining catalyst, water, and esters formed during transesterification process 18 . Utilization of glycerol for PHB production offers a potentially cheap substrate for the production of biodegradable plastics and also adds value to bio-diesel production economics. Glycerol is the most energetically favorable substrate for the formation of acetyl-CoA, the precursor for PHB synthesis. A number of Bacillus species are reported to accumulate 9-67% cell dry weight PHA 16,19-22 . This study utilizes biodiesel industry generated CG as carbon source for PHB production by B. firmus NII 0830 under variations in fermentation conditions. Experimental Section Microorganism, Maintenance and Inoculum Preparation B. firmus NII 0830 was obtained from NII Microbial Culture Collection (NIICC), NIIST, Trivandrum, India. Strain was maintained on nutrient agar slants. Inoculums were prepared in LB media and incubated at 30°C for 16 h on a rotary shaker at 250 rpm. Seed cultures