Establishing

Using the Wood-Ljungdahl pathway, acetogens can nonphotosynthetically fix gaseous C(1) molecules, preventing them from entering the atmosphere. Many acetogens can also grow on liquid C(1) compounds such as formate and methanol, which avoid the storage and mass transfer issues associated with gaseous C(1) compounds. Substrate redox state also plays an important role in acetogen metabolism and can modulate products formed by these organisms. Butyribacterium methylotrophicum is an acetogen known for its ability to synthesize longer-chained molecules such as butyrate and butanol, which have significantly higher values than acetate or ethanol, from one-carbon (C(1)) compounds. We explored B. methylotrophicum’s C(1) metabolism by varying substrates, substrate concentrations, and substrate feeding strategies to improve four-carbon product titers. Our results showed that formate utilization by B. methylotrophicum favored acetate production and methanol utilization favored butyrate production. Cofeeding of both substrates produced a high butyrate titer of 4 g/liter when methanol was supplied in excess to formate. Testing of formate feeding strategies, in the presence of methanol, led to further increases in the butyrate to acetate ratio. Mixotrophic growth of liquid and gaseous C(1) substrates expanded the B. methylotrophicum product profile, as ethanol, butanol, and lactate were produced under these conditions. We also showed that B. methylotrophicum is capable of producing caproate, a six-carbon product, presumably through chain elongation cycles of the reverse β-oxidation pathway. Furthermore, we demonstrated butanol production via heterologous gene expression. Our results indicate that both selection of appropriate substrates and genetic engineering play important roles in determining titers of desired products. IMPORTANCE Acetogenic bacteria can fix single-carbon (C(1)) molecules. However, improvements are needed to overcome poor product titers. Butyribacterium methylotrophicum can naturally ferment C(1) compounds into longer-chained molecules such as butyrate alongside traditional acetate. Here, we show that B. methylotrophicum can effectively grow on formate and methanol to produce high titers of butyrate. We improved ratios of butyrate to acetate through adjusted formate feeding strategies and produced higher-value six-carbon molecules. We also expanded the B. methylotrophicum product profile with the addition of C(1) gases, as the organism produced ethanol, butanol, and lactate. Furthermore, we developed a transformation protocol for B. methylotrophicum to facilitate genetic engineering of this organism for the circular bioeconomy.