The production of polyhydroxyalkanoate by Bacillus licheniformis using sequential mutagenesis and optimization

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 45oC 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.