Biotechnology and Bioprocess Engineering 18: 272-279 (2013) DOI 10.1007/s12257-012-0615-z The Production of Polyhydroxyalkanoate by Bacillus licheniformis Using Sequential Mutagenesis and Optimization Kanokphorn Sangkharak and Poonsuk Prasertsan Received: 13 September 2012 / Revised: 1 December 2012 / Accepted: 6 December 2012 © The Korean Society for Biotechnology and Bioengineering and Springer 2013 Abstract The purpose of this study was to enhance the production of polyhydroxyalkanoate (PHA) by sequential mutation of Bacillus licheniformis PHAs-007, using UV and N-methyl-N'-nitro-N-nitrosoguanidine (NTG). In addition, the effect of nutrient additions and environmental conditions were optimized to increase the production of PHA. Bacillus licheniformis PHAs-007 produced high amounts of PHA (64.09 ~ 68.80% of DCW) under both synthetic and renewable substrates. After mutagenesis treatment, mutant M2-12 was selected from 380 strains, based on its high biomass and PHA concentration. The mutant M2-12 gave the highest value of specific growth rate (0.09/h), biomass (22.24 g/L) and PHA content (19.55 g/L) under optimal conditions, consisting of 3% palm oil mill effluent, with no additional trace elements, at 45 o C and pH 7. The mutant strain showed higher resistance to substrate concentrations, as well as pH and temperature, than the wild type. The accumulation of PHA was increased by 3.18- fold compared to the wild type, and the production of PHA by the mutant M2-12 was constantly retained over 12 times of cultivation. The mutation and optimization strategy appear to be suitable for producing high density PHA, reducing the medium cost and consequently lowering the production cost. Interestingly, the mutant strain could synthesize the novel PHA copolymers such as 3- hydroxyvalerate and 3-hydroxyhexanoate, which were not produced by the wild type. Keywords: palm oil mill effluent, polyhydroxyalkanoates, polyhydroxyhexanoate, polyhydroxyvalerate, sequential mutagenesis 1. Introduction Plastic garbage has become a major concern in terms of its many negative environmental impacts. A major disadvantage of synthetic plastics is that they do not degrade naturally, and many toxins are produced during their production and combustion [1,2]. Polyhydroxyalkanoate (PHA) is polyester which is naturally produced by bacteria, having similar properties to synthetic plastic, while being completely degraded by PHA depolymerases at a high rate within 3 ~ 9 months. The major drawback of PHA is their high costs [3]. Therefore, the commercial use of PHA is dependent on low production costs and the feasibility of mass production. Owing to their inherent metabolic control systems, micro- organisms usually produce PHA in very low concentrations, and although the yield may be increased by optimizing the cultural conditions, productivity is controlled ultimately by the organism’s genome [4]. Consequently, genetic alteration is an attractive route for the process development of this biotechnology. The improvement of microbial strains for the production of PHA has attracted attention in the commercial fermentation process [5]. Many groups have reported their attempts to increase the yield of PHA production by genetic improvements, using mutagenesis via mutagenic agents. The use of different mutagenic agents, such as ultraviolet (UV), X-rays and gamma radiation, as well as chemical mutagen including ethyl methane sulfonate (EMS), NTG and mustards, were demonstrated [5-9]. The effect of gamma irradiation on PHA production by Bacillus flexus was evaluated by Divyashree and Shamala [32]. Irradiation resulted in cell damage, and aided in the Kanokphorn Sangkharak * COE for Sustainable Energy and Environment, Department of Chemistry, Faculty of Science, Thaksin University, Phatthalung 93110, Thailand Tel: +66-7460-9634; Fax: +66-7460-9634 E-mail: skanokphorn@yahoo.com Poonsuk Prasertsan Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkhla 90112, Thailand RESEARCH PAPER