Characterization of new isolated Ralstoniaeutropha strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments. The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession number EF988626.