No abstract

Summary Unlike gaseous C 1 feedstocks for acetogenic bacteria, there has been less attention on liquid C 1 feedstocks, despite benefits in terms of energy efficiency, mass transfer and integration within existing fermentation infrastructure. Here, we present growth of Eubacterium limosum ATCC8486 using methanol and formate as substrates, finding evidence for the first time of native butanol production. We varied ratios of methanol‐to‐formate in batch serum bottle fermentations, showing butyrate is the major product (maximum specific rate 220 ± 23 mmol‐C gDCW ‐1 day ‐1 ). Increasing this ratio showed methanol is the key feedstock driving the product spectrum towards more reduced products, such as butanol (maximum titre 2.0 ± 1.1 mM‐C). However, both substrates are required for a high growth rate (maximum 0.19 ± 0.011 h ‐1 ) and cell density (maximum 1.2 ± 0.043 gDCW l ‐1 ), with formate being the preferred substrate. In fact, formate and methanol are consumed in two distinct growth phases – growth phase 1, on predominately formate and growth phase 2 on methanol, which must balance. Because the second growth varied according to the first growth on formate, this suggests butanol production is due to overflow metabolism, similar to 2,3‐butanediol production in other acetogens. However, further research is required to confirm the butanol production pathway in E. limosum , particularly given, unlike other substrates, methanol likely results in mostly NADH generation, not reduced ferredoxin.

Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such asEubacterium limosum, have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in acetogens is poorly understood, and a systems-level characterization in continuous cultures is yet to be reported. Here we present the first steady-state dataset forE. limosumformatotrophic growth. At a defined dilution rate of 0.4 d-1, there was a high specific uptake rate of formate (280±56 mmol/gDCW/d), however, most carbon went to CO2(150±11 mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of pta appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of Pfl to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth.