High methanol‐to‐formate ratios induce butanol production in Eubacterium limosum

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.

Characterisation of acetogen formatotrophic potential usingE. limosum

“…0.2 μM accumulation of methanol, which is below our limit of detection. In principle, it is possible, which may explain our previous observation of methanol production from formate during early growth stages, presumably when there is a highly reduced redox pool (Wood et al 2021).…”

Engineering Acetogenic Bacteria for Efficient One-Carbon Utilization

“…Recently, varied ratios of methanol-to-formate were tested in E. limosum , where a methanol-to-formate substrate ratio of 7.5:1 achieved a maximum butanol titer of 2.0 ± 1.1 mM (38 mg/L). This is the first evidence of native butanol production in E. limosum , as it has not been observed under only syngas or methanol fermentation ( Wood et al, 2021 ). Although the underlying mechanism and butanol production pathway in E. limosum remains elusive, butanol production is suspected to be due to overflow metabolism, similar to 2,3-BDO production in C. ljungdahlii or C. autoethanogenum ( Köpke et al, 2011 ).…”

Molecular understanding ofEubacterium limosumchemostat methanol metabolism

“…Yet in batch during this first growth phase, there was a maximum co-consumption uptake ratio of 1:1, which was similar to the maximum achieved in chemostat when CO 2 was removed -1.6±0.7:1 with a butyrate:acetate production ratio of 5.0±2.5 and no butanol observed (10MeOH/Formate/2D) (Figure 5). In the second phase of growth in batch, when formate was exhausted, the butyrate:acetate production ratio was 17±2.7, with a butanol titer of 0.8 mM-C. 4 In chemostat, we could not mimic this second phase of growth, highlighting this is likely an overflow metabolism.…”

“…2 Unlike utilisation of synthesis gas (syngas, a mixture of CO and H 2 ), that has been commercialised by LanzaTech from waste, 3 much less is understood about liquid C 1 feedstocks such as methanol. 4 Being liquid, methanol avoids transportation problems presented with gaseous substrates. For example, most chemical supply chains are distributed across the globe, and require long-distance transportation.…”