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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.

Methanol is a promising renewable energy carrier that can be used as a favourable substrate for biotechnology, due to its high energy efficiency conversion and ease of integration within existing infrastructure. Some acetogenic bacteria have the native ability to utilise methanol, along with other C1substrates such as CO2and formate, to produce valuable chemicals. Continuous cultures favour economically viable bioprocesses, however, the performance of acetogens has not been investigated at the molecular level when grown on methanol. Here we present steady-state chemostat quantification of the metabolism ofEubacterium limosum, finding maximum methanol uptake rates up to 640±22 mmol/gDCW/d, with significant fluxes to butyrate. To better understand metabolism of acetogens under methanol growth conditions, we sampled chemostats for proteomics and metabolomics. Changes in protein expression and intracellular metabolomics highlighted key aspects of methanol metabolism, and highlighted bottleneck conditions preventing formation of the more valuable product, butanol. Interestingly, a small amount of formate in methylotrophic metabolism triggered a cellular state known in other acetogens to correlate with solventogenesis. Unfortunately, this was prevented by post-translation effects including an oxidised NAD pool. There remains uncertainty around ferredoxin balance at the methylene-tetrahydrofolate reductase (MTHFR) and at the Rnf level.

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Introduction Slow, oscillatory, transcranial electrical stimulation (so-tES) applies a current over the scalp that oscillates in intensity at a frequency associated with slow wave sleep (SWS; 0.75Hz). When applied during SWS, so-tES can enhance SWS EEG power compared to sham stimulation, as well as overnight declarative memory consolidation. When applied during wake, so-tES can enhance local EEG power in the slow wave frequency range (0.5–4.5Hz) compared to sham. Therefore, this study will investigate whether so-tES can enhance the early consolidation of new learning compared to sham, when applied during wake. A preliminary analysis of data will be presented. Methods Healthy, young, right-handed adults (18–35 years) practiced a motor sequence learning task for 30 minutes, before receiving 15 minutes of active or sham so-tES (0.75Hz) during quiet wakefulness. Task performance was assessed by recording the total number of correct sequences performed in 30 seconds before practice, after practice, and after stimulation. Performance improvements will be compared between stimulation conditions. Non-invasive, electrophysiological corticospinal excitability measurements (i.e., motor-evoked potentials) were also recorded at six timepoints throughout each session, to investigate whether active so-tES can modulate corticospinal excitability differently to sham. Progress to date Data collection is ongoing, and completion is expected by late 2021. Intended outcome and impact We expect so-tES to enhance early skill consolidation during wake, and that enhanced consolidation will be associated with less variable measurements of corticospinal excitability, when compared with sham stimulation.

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