In this study, corn stover-based sugar served as the initial carbon source to investigate the impact of different co-culture strategies on the synthesis of short- and medium-chain fatty acids (SMCFAs) through carbon chain elongation (CCE). The results indicated that the sequential inoculation outperformed simultaneous inoculation in the M. elsdenii and Lactobacillus groups. The inclusion of Lactobacillus significantly enhanced substrate utilization, generating lactate that facilitated the integration of odd carbon carboxylates (such as propionate and valerate) into the CCE products. The caproate concentration in the M. elsdenii+L. pentosus group reached 3.4 g/L, which was 1.3 times that in the control group. Furthermore, the introduction of C. kluyveri effectively boosted the synthesis of butyrate and caproate through CCE using ethanol as an electron donor within the co-culture setup of the M. elsdenii+C. kluyveri group. Moreover, the carbon efficiency of the co-culture group increased by 10–21 % compared to the control group, and the cell protein titer was 1.5–2 times higher than that of the control group. This study has established a method for co-producing SMCFAs and cell proteins utilizing bio-sugars through a co-culture approach, offering implications for lignocellulose utilization and potential high-value applications of SMCFAs in the future.
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