Effect of electron acceptors on product selectivity and carbon flux in carbon chain elongation with Megasphaera hexanoica

Megasphaera hexanoica is a bacterial strain following the reverse β-oxidation pathway to synthesize caproate (CA) using lactate (LA) as an electron donor (ED) and acetate (AA) or butyrate (BA) as electron acceptors (EA). Differences in the type and concentration of EA lead to distinctions in product distribution and energy bifurcation of carbon fluxes in ED pathways, thereby affecting CA production. In this study, the effect of various ratios of AA, BA, and AA+BA as EA on carbon flux and CA specific titer during the carbon chain elongation in M. hexanoica was explored. The results indicated that the maximum levels of CA were 18.81 mM and 31.48 mM when the molar ratios of LA/AA and LA/BA were 10:1 and 3:1, respectively. Meanwhile, when AA and BA were used as combined EA (LA, AA, and BA molar amounts of 100, 23, and 77 mM), a maximum CA production of 39.45 mM was obtained. Further analysis revealed that the combined EA exhibited a CA production carbon flux of 49 % (4.3 % and 19.5 % higher compared to AA or BA, respectively) and a CA production specific titer of 45.24 mol (80.89 % and 58.51 % higher compared to AA or BA, respectively), indicating that the effective carbon utilization rate and CA production efficiency were greatly improved. Finally, a scaled-up experiment was conducted in a 1.2 L (working volume) automated bioreactor, implying high biomass (optical density at 600 nm or OD600 = 1.809) and a slight decrease in CA production (28.45 mM). A decrease in H2 production (4.11 g/m3) and an increase in CO2 production (0.632 g/m3) demonstrated the appropriate metabolic adaptation of M. hexanoica to environmental changes such as stirring shear.