Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019.

This study demonstrated the improved polyhydroxybutyrate (PHB) production via high cell density cultivation of Bacillus megaterium BA-019 with balanced initial total sugar concentration and carbon to nitrogen (C/N) weight ratio. In the 10 L stirred fermentor operated at 30 °C, pH 7.0, 600 rpm, and 1.0 vvm air, with the initial total sugar concentration of 60 g/L and urea at the C/N weight ratio of 10:1, 32.48 g/L cell biomass with the corresponding PHB weight content of 26.94 % and volumetric productivity of 0.73 g/L h were obtained from batch cultivation. Continuing cultivation by intermittent feeding of the sugarcane molasses along with urea at the C/N weight ratio of 12.5:1 gave much improved biomass and PHB production (90.71 g/L biomass with 45.84 % PHB content and 1.73 g/L h PHB productivity). Similar biomass and PHB yields were obtained in the 90 L stirred fermentor when using the impeller tip speed as the scale-up criterion.

Direct fermentation of L (+)-lactic acid from cassava pulp by solid state culture of Rhizopus oryzae.

This study shows that Rhizopus oryzae is capable of directly utilizing cassava pulp alone to L-lactic acid in solid state fermentation (SSF). pH control at 6.0 helped prevent end product inhibition. Increasing lactate titer was observed at the higher initial moistened water due to the higher degree of substrate swelling and hydrolysis. With shaking, limited ethanol production but no change in lactate titer was observed. Rigorous shaking gave better oxygen transfer but presumably caused cell damage leading to substrate utilization through the biosynthesis route. Supplementing cassava pulp with nitrogen enhanced growth but not lactate production. Under the optimal conditions, R. oryzae converted the sole cassava pulp into lactic acid at the titer of 206.20 mg per g initial dry pulp. With the help of commercial cellulase and glucoamylase, the dramatically increasing lactate titer of 463.18 mg per g initial dry pulp was achieved via SSF.

Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate) production by a new isolate of Bacillus megaterium.

This research aimed at increasing the cell density and production of the homopolymer polyhydroxybutyrate (PHB) by Bacillus megaterium BA-019, using renewable and inexpensive bioresources as a substrate. A higher cell density and a greater PHB production level were obtained by using sugarcane molasses and urea as carbon and nitrogen sources, respectively. The limitation of nitrogen at a C/N molar ratio of 25 resulted in enhanced cell growth and PHB production in batch cultures. Fed-batch cultivation with the feeding nutrient composed of MSM with sugarcane molasses, urea and trace elements, and controlled by a pH-stat feeding control, lead to a significantly enhanced cell concentration and PHB production. The optimal feeding medium in this system required a higher total sugar concentration (400 g/l) and a C/N molar ratio of 10 mol/mol. Under these conditions the highest attained cell mass (72.6 g/l DW) and PHB content (42% of cell dry wt.) were achieved in a short cultivation time (24 h), leading to improved PHB productivity (1.27 g/l/h). However, dissolved oxygen was limiting and thus the system is likely to be suboptimal and capable of even further improvements to the PHB production rate.

Characterization of new isolated Ralstonia eutropha 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 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 and characterization of biodegradable terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) by Alcaligenes sp. A-04.

The production of the terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate), P(3HB-co-3HV-co-4HB), by Alcaligenes sp. A-04 was investigated to determine the superior biodegradable polymer properties over those of poly(3-hydroxybutyrate), P(3HB), and its copolymers. The highest terpolymer content of 68% (w/w) was produced by Alcaligenes sp. A-04 at 60 h by shake-flask cultivation. The terpolymer with 93 mol% 4HB mole fraction units was produced when the cultivation time was extended to 96 h. Moreover, it was found that Alcaligenes sp. A-04 could utilize 1,4-butanediol for the synthesis of 3HB and 4HB monomers as well as the sodium salt of 4-hydroxybutyrate. The terpolymer content was 30% (w/w) and the composition was P(33%3HB-co-16%3HV-co-51%4HB). Next, terpolymers with 4HB mole fraction units ranging from 50 to 90 mol% were produced by varying the medium composition and cultivation time. The thermal and mechanical properties of the resulting terpolymers were different from those of the copolymers with a similar mole fraction of monomer units. The terpolymer P(4%3HB-co-3%3HV-co-93%4HB) showed an elongation of 430%, a toughness of 33 MPa, and Young's modulus of 127 MPa similar to those of low-density polyethylene. The terpolymer P(11%3HB-co-34%3HV-co-55%4HB) showed Young's Modulus of 618 MPa similar to that of polypropylene.