Production of branched-chain alcohols by recombinant Ralstonia eutropha in fed-batch cultivation

Branched-chain alcohols are considered promising green energy sources due to their compatibility with existing infrastructure and their high energy density. We utilized a strain of Ralstonia eutropha capable of producing branched-chain alcohols and examined its production in flask cultures. In order to increase isobutanol and 3-methyl-1-butanol (isoamyl alcohol) productivity in the engineered strain, batch, fed-batch, and two-stage fed-batch cultures were carried out in this work. The effects of nitrogen source concentration on branched-chain alcohol production were investigated under four different initial concentrations in fermenters. A maximum 380gm^-3 of branched-chain alcohol production was observed with 2kgm^-3 initial NH₄Cl concentration in batch cultures. A pH-stat control strategy was utilized to investigate the optimum carbon source amount fed during fed-batch cultures for higher cell density. In cultures of R. eutropha strains that did not produce polyhydroxyalkanoate or branched-chain alcohols, a maximum cell dry weight of 36kgm^-3 was observed using a fed-batch strategy, when 10kgm^-3 carbon source was fed into culture medium. Finally, a total branched-chain alcohol titer of 790gm^-3, the highest branched-chain alcohol yield of 0.03gg^-1, and the maximum branched-chain alcohol productivity of 8.23gm^-3h^-1 were obtained from the engineered strain Re2410/pJL26 in a two-stage fed-batch culture system with pH-stat control. Isobutanol made up over 95% (mass fraction) of the total branched-chain alcohols titer produced in this study.