Continuous Supply of Non-Combustible Gas Mixture for Safe Autotrophic Culture to Produce Polyhydroxyalkanoate by Hydrogen-Oxidizing Bacteria.

Abstract

Polyhydroxyalkanoates (PHAs) are eco-friendly plastics that are thermoplastic and biodegradable in nature. The hydrogen-oxidizing bacterium Ralstonia eutropha can biosynthesize poly[(R)-3-hydroxybutyrate] [P(3HB)], the most common PHA, from carbon dioxide using hydrogen and oxygen as energy sources. In conventional autotrophic cultivation using R. eutropha, a gas mixture containing 75–80 vol% hydrogen is supplied; however, a gas mixture with such a high hydrogen content has a risk of explosion due to gas leakage. In this study, we aimed to develop an efficient cell culture system with a continuous supply of a non-combustible gas mixture (H2: O2: CO2: N2 = 3.8: 7.3: 13.0: 75.9) for safe autotrophic culture to produce P(3HB) by hydrogen-oxidizing bacteria, with a controlled hydrogen concentration under a lower explosive limit concentration. When the gas mixture was continuously supplied to the jar fermentor, the cell growth of R. eutropha H16 significantly improved compared to that in previous studies using flask cultures. Furthermore, an increased gas flow rate and agitation speed enhanced both cell growth and P(3HB) production. Nitrogen source deficiency promoted P(3HB) production, achieving up to 2.94 g/L P(3HB) and 89 wt% P(3HB) content in the cells after 144 h cultivation. R. eutropha NCIMB 11599, recombinant R. eutropha PHB-4, and Azohydromonas lata grew in a low-hydrogen-content gas mixture. R. eutropha H16 and recombinant R. eutropha PHB-4 expressing PHA synthase from Bacillus cereus YB-4 synthesized P(3HB) with a high weight-average molecular weight of 13.5–16.9 × 105. Thus, this autotrophic culture system is highly beneficial for PHA production from carbon dioxide using hydrogen-oxidizing bacteria as the risk of explosion is eliminated.