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

Abstract

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 gamma-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 gamma-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.

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Burkholderia sacchari using wheat straw hydrolysates and gamma-butyrolactone

“…The 1 H NMR spectra showed that the polymers are composed of two monomer units, 3HB and 4HB, and the spectrum signals ( Fig. 1) appear identical to those obtained for P(3HB-co-4HB) by other authors [19,25]. The fraction of each monomer was calculated from the areas of the 4.11 ppm signal from 4HB and the 5.26 ppm signal from 3HB (C and B in Fig.1 ), bearing in mind that the 4HB and the 3HB signals correspond, respectively to two and one hydrogen atoms.…”

Challenges and Opportunities for Customizing Polyhydroxyalkanoates

“…In addition to this, nutrient level in media as compared to C affects the quality of PHA. The study of kinetics of P(3HB:4HB) production by Cupriavidus necator strain A-04 has shown that production of 4HB monomers was high under nitrogen (N) rich medium whereas, 3HB incorporation increases in N-limiting media [73]. Under N-limiting conditions 4HB precursors flux goes to 3HB-CoA, thus more 3HB is incorporated into polymer P(3HB:4HB) by C. necator.…”

Isolation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) producer from Malaysian environment using γ-butyrolactone as carbon source

“…In 1994, they described another bacteria, Comamonas acidovarans which is a wild type microorganism with suitable metabolic pathways for the production of P(3HB-co-4HB) (Saito and Doi 1994). To date, five wild type bacteria, which can produce P(3HB-co-4HB), i.e., R. eutropha, (Nakamura et al 1992;Kim et al 2005;Chanprateep et al 2008), A. latus (Hiramitsu et al 1993;Kang et al 1995), C. acidovorans (Saito and Doi 1994;Lee et al 2004), Comamonas testosteronii (Renner et al 1996) and Hydrogenophaga pseudoflava (Choi et al 1999), have been reported.…”