Enhancing hydrogen‐dependent growth of and carbon dioxide fixation by Clostridium ljungdahlii through nitrate supplementation

Abstract

Synthesis gas (syngas) fermentation via the Wood-Ljungdahl pathway is receiving growing attention as a possible platform for the fixation of CO 2 and renewable production of fuels and chemicals. However, the pathway operates near the thermodynamic limit of life, resulting in minimal adenosine triphosphate (ATP) production and long doubling times. This calls into question the feasibility of producing high-energy compounds at industrially relevant levels. In this study, we investigated the possibility of co-utilizing nitrate as an inexpensive additional electron acceptor to enhance ATP production during H 2 -dependent growth of Clostridium ljungdahlii, Moorella thermoacetica, and Acetobacterium woodii. In contrast to other acetogens tested, growth rate and final biomass titer were improved for C. ljungdahlii growing on a mixture of H 2 and CO 2 when supplemented with nitrate. Transcriptomic analysis, CO 13 2 labeling, and an electron balance were used to understand how electron flux was partitioned between CO 2 and nitrate. We further show that, with nitrate supplementation, the ATP/adenosine diphosphate (ADP) ratio and acetyl-CoA pools were increased by fivefold and threefold, respectively, suggesting that this strategy could be useful for the production of ATP-intensive heterologous products from acetyl-CoA. Finally, we propose a pathway for enhanced ATP production from nitrate and use this as a basis to calculate theoretical yields for a variety of products.This study demonstrates a viable strategy for the decoupling of ATP production from carbon dioxide fixation, which will serve to significantly improve the CO 2 fixation rate and the production metrics of other chemicals from CO 2 and H 2 in this host. K E Y W O R D Sacetogenic bacteria, anaerobic respiration, Clostridium ljungdahlii, fixation, hydrogen, nitrate

Nitrate Feed Improves Growth and Ethanol Production of Clostridium ljungdahlii With CO2 and H2, but Results in Stochastic Inhibition Events

“…We found an increasing pH due to ammonium production in preliminary bottle experiments in nitrate-containing PETC medium (Supplementary Figure S5 in Supplementary Data Sheet S2). A pH increase was also observed in the nitrate bottle experiments of Emerson et al (2019). Despite the pH-control in our experiments, all bioreactors with nitrate feed showed remarkable differences in growth, pH, acetate production, and ethanol production rates.…”

“…; and (2) does feeding nitrate influence the product spectrum under controlled pH conditions with CO 2 and H 2 ? In a recent study, nitrate was used as an alternative electron acceptor for C. ljungdahlii, while it also served as sole nitrogen source (N-source) in batch cultivations (Emerson et al, 2019). To our knowledge, this was the first study which investigated nitrate reduction by any known acetogen in detail.…”

“…At the same time, nitrate reduction led to an accumulation of ammonium. This resulted in a continuous increase of the pH from 6.0 to 8.0 during the batch cultivations in serum bottles (Emerson et al, 2019), which would intrinsically prevent ethanol production, because ethanol production is most likely triggered by a low pH (Mock et al, 2015;Richter et al, 2016). It is well-known that growth of acetogens, such as C. ljungdahlii, is highly dependent on the pH (Drake et al, 2008).…”

Effect of Cysteine, Yeast Extract, pH Regulation and Gas Flow on Acetate and Ethanol Formation and Growth Profiles ofClostridium ljungdahliiSyngas Fermentation

“…Besides in this study the amount of yeast extract did not have an impact on the final ethanol concentration in our set-up, contrary to what has been reported elsewhere (Abubackar et al, 2012;Barik et al, 1988;Phillips et al, 1993;Vega et al, 1989). One potential argument so as to why yeast extract did not have an effect in our media could be that, since the gas mixture used contained N 2 , C. ljungdahlii could potentially be fixing it, as proposed by Richter et al (2016) and Tremblay et al (2013), and thus minimizing the effect of other nitrogen sources, but this remains a controversial topic since Emerson et al (2019) could not observe any nitrogen fixation in their experiments.…”

Evaluation of Media Components and Process Parameters in a Sensitive and Robust Fed-Batch Syngas Fermentation System with Clostridium ljungdahlii

“…In addition, the studies mentioned above are not directly comparable in this case either, due to the different cultivation systems used (bottles rather than bioreactors), organisms or gas compositions. Another potential argument as to why yeast extract did not have an effect in our media could be that, since the gas mixture used contained N 2 , C. ljungdahlii could potentially be fixing it, as proposed by Richter et al [4] and Tremblay et al [25], thus minimizing the effect of other nitrogen sources, but this remains a controversial topic since Emerson et al [26] could not observe any nitrogen fixation in their experiments.…”