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Cheap and renewable feedstocks such as the one-carbon substrate formate are emerging for sustainable production in a growing chemical industry. We investigated the acetogen Acetobacterium woodii as a potential host for bioproduction from formate alone and together with autotrophic and heterotrophic co-substrates by quantitatively analyzing physiology, transcriptome, and proteome in chemostat cultivations in combination with computational analyses. Continuous cultivations with a specific growth rate of 0.05 h−1 on formate showed high specific substrate uptake rates (47 mmol g−1 h−1). Co-utilization of formate with H2, CO, CO2 or fructose was achieved without catabolite repression and with acetate as the sole metabolic product. A transcriptomic comparison of all growth conditions revealed a distinct adaptation of A. woodii to growth on formate as 570 genes were changed in their transcript level. Transcriptome and proteome showed higher expression of the Wood-Ljungdahl pathway during growth on formate and gaseous substrates, underlining its function during utilization of one-carbon substrates. Flux balance analysis showed varying flux levels for the WLP (0.7–16.4 mmol g−1 h−1) and major differences in redox and energy metabolism. Growth on formate, H2/CO2, and formate + H2/CO2 resulted in low energy availability (0.20–0.22 ATP/acetate) which was increased during co-utilization with CO or fructose (0.31 ATP/acetate for formate + H2/CO/CO2, 0.75 ATP/acetate for formate + fructose). Unitrophic and mixotrophic conversion of all substrates was further characterized by high energetic efficiencies. In silico analysis of bioproduction of ethanol and lactate from formate and autotrophic and heterotrophic co-substrates showed promising energetic efficiencies (70–92%). Collectively, our findings reveal A. woodii as a promising host for flexible and simultaneous bioconversion of multiple substrates, underline the potential of substrate co-utilization to improve the energy availability of acetogens and encourage metabolic engineering of acetogenic bacteria for the efficient synthesis of bulk chemicals and fuels from sustainable one carbon substrates.

The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.

Aims While gas-fermenting acetogens have been engineered to secrete non-native metabolites such as butyrate, acetate remains the most thermodynamically favourable product. An alternative to metabolic engineering is to exploit native capabilities for CO-to-acetate conversion by coculturing an acetogen with a second bacterium that provides efficient acetate-butyrate conversion. Methods and results We used dynamic metabolic modelling to computationally evaluate the CO-to-butyrate conversion capabilities of candidate coculture systems by exploiting the diversity of human gut bacteria for anaerobic synthesis of butyrate from acetate and ethanol. A preliminary screening procedure based on flux balance analysis was developed to identify 48 gut bacteria which satisfied minimal growth rate and acetate-to-butyrate conversion requirements when cultured on minimal medium containing acetate and a simple sugar not consumed by the paired acetogen. A total of 170 acetogen/gut bacterium/sugar combinations were dynamically simulated for continuous growth using a 70/30 CO/CO2 feed gas mixture and minimal medium computationally determined for each combination. Conclusions While coculture systems involving the acetogens Eubacterium limosum or Blautia producta yielded low butyrate productivities and CO-to-ethanol conversion had minimal impact on system performance, dynamic simulations predicted a large number of promising coculture designs with Clostridium ljungdahlii or C. autoethanogenum as the CO-to-acetate converter. Pairings with the gut bacterium Clostridium hylemonae or Roseburia hominis were particularly promising due to their ability to generate high butyrate productivities over a range of dilution rates with a variety of sugars. The higher specific acetate secretion rate of C. ljungdahlii proved more beneficial than the elevated growth rate of C. autoethanogenum for coculture butyrate productivity. Significance and impact of the study Our study demonstrated that metabolic modelling could provide useful insights into coculture design that can guide future experimental studies. More specifically, our predictions generated several favourable designs, which could serve as the first coculture systems realized experimentally.

Gas fermentation is one of the promising bioprocesses to convert CO2 or syngas to important chemicals. Thermophilic gas fermentation of volatile chemicals has the potential for the development of consolidated bioprocesses that can simultaneously separate products during fermentation. This study reports the production of acetone from CO2 and H2, CO, or syngas by introducing the acetone production pathway using acetyl–coenzyme A (Ac-CoA) and acetate produced via the Wood–Ljungdahl pathway in Moorella thermoacetica. Reducing the carbon flux from Ac-CoA to acetate through genetic engineering successfully enhanced acetone productivity, which varied on the basis of the gas composition. The highest acetone productivity was obtained with CO–H2, while autotrophic growth collapsed with CO2–H2. By adding H2 to CO, the acetone productivity from the same amount of carbon source increased compared to CO gas only, and the maximum specific acetone production rate also increased from 0.04 to 0.09 g-acetone/g-dry cell/h. Our development of the engineered thermophilic acetogen M. thermoacetica, which grows at a temperature higher than the boiling point of acetone (58°C), would pave the way for developing a consolidated process with simplified and cost-effective recovery via condensation following gas fermentation.

Syngas fermentation is a potential player for future emission reduction. The first demonstration and commercial plants have been successfully established. However, due to its novelty, development of syngas fermentation processes is still in its infancy, and the need to systematically unravel and understand further phenomena, such as substrate toxicity as well as gas transfer and uptake rates, still persists. This study describes a new online monitoring device based on the respiration activity monitoring system for cultivation of syngas fermenting microorganisms with gaseous substrates. The new device is designed to online monitor the carbon dioxide transfer rate (CO2 TR) and the gross gas transfer rate during cultivation. Online measured data are used for the calculation of the carbon monoxide transfer rate (COTR) and hydrogen transfer rate (H2 TR). In cultivation on pure CO and CO + H2 , CO was continuously limiting, whereas hydrogen, when present, was sufficiently available. The maximum COTR measured was approximately 5 mmol/L/h for pure CO cultivation, and approximately 6 mmol/L/h for cultivation with additional H2 in the gas supply. Additionally, calculation of the ratio of evolved carbon dioxide to consumed monoxide, similar to the respiratory quotient for aerobic fermentation, allows the prediction of whether acetate or ethanol is predominantly produced. Clostridium ljungdahlii, a model acetogen for syngas fermentation, was cultivated using only CO, and CO in combination with H2 . Online monitoring of the mentioned parameters revealed a metabolic shift in fermentation with sole CO, depending on COTR. The device presented herein allows fast process development, because crucial parameters for scale-up can be measured online in small-scale gas fermentation.

The massive consumption of fossil energy forces people to find new sources of energy. Syngas fermentation has become a hot research field as its high potential in renewable energy production and sustainable development. In this study, trophic anaerobic acetogen Morella thermoacetica was successfully immobilized by calcium alginate embedding method. The ability of the immobilized cells on production of acetic acid through syngas fermentation was compared in both airlift and bubble column bioreactors. The bubble column bioreactor was selected as the better type of bioreactor. The production of acetic acid reached 32.3 g·L−1 in bubble column bioreactor with a space–time yield of 2.13 g·L−1·d−1. The immobilized acetogen could be efficiently reused without significant lag period, even if exposed to air for a short time. A semi-continuous syngas fermentation was performed using immobilized cells, with an average space–time acetic acid yield of 3.20 g·L−1·d−1. After 30 days of fermentation, no significant decrease of the acetic acid production rate was observed.

Biohybrids composed of microorganisms and nanoparticles have emerged as potential systems for bioenergy and high-value compound production from CO2 and light energy, yet the cellular and metabolic processes within the biological component of this system are still elusive. Here we dissect the biohybrid composed of the anaerobic acetogenic bacterium Moorella thermoacetica and cadmium sulphide nanoparticles (CdS) in terms of physiology, metabolism, enzymatics and transcriptomic profiling. Our analyses show that while the organism does not grow on l-cysteine, it is metabolized to acetate in the biohybrid system and this metabolism is independent of CdS or light. CdS cells have higher metabolic activity, despite an inhibitory effect of Cd2+ on key enzymes, because of an intracellular storage compound linked to arginine metabolism. We identify different routes how cysteine and its oxidized form can be innately metabolized by the model acetogen and what intracellular mechanisms are triggered by cysteine, cadmium or blue light.

Clostridium ljungdahlii is a representative autotrophic gas-fermenting acetogen capable of converting CO2 and CO into biomass and multiple metabolites. The carbon fixation and conversion based on C. ljungdahlii have great potential for the sustainable production of bulk biochemicals and biofuels using industrial syngas and waste gases. With substantial recent advances in genetic manipulation tools, it has become possible to study and improve the metabolic capability of C. ljungdahlii in gas fermentation. The product scope of C. ljungdahlii has been expanded through the introduction of heterologous production pathways followed by the modification of native metabolic networks. In addition, progress has been made in understanding the physiological and metabolic mechanisms of this anaerobe, contributing to strain designs for expected phenotypes. In this review, we highlight the latest research progresses regarding C. ljungdahlii and discuss the next steps to comprehensively understand and engineer this bacterium for an improved bacterial gas bioconversion platform.

H2 is an important fermentation intermediate in anaerobic environments. Although H2 occurs at very low partial pressures in the environments, the culture and isolation of H2-utilizing microorganisms is usually carried out under very high H2 pressures, which might have hampered the discovery and understanding of microorganisms adapting to low H2 environments. Here we constructed a culture system designated the “iron corrosion-assisted H2-supplying (iCH) system” by connecting the gas phases of two vials (one for the iron corrosion reaction and the other for culturing microorganisms) to achieve cultures of microorganisms under low H2 pressures. We conducted enrichment cultures for methanogens and acetogens using rice paddy field soil as the microbial source. In the enrichment culture of methanogens under canonical high H2 pressures, only Methanobacterium spp. were enriched. By contrast, Methanocella spp. and Methanoculleus spp., methanogens adapting to low H2 pressures, were specifically enriched in the iCH cultures. We also observed selective enrichment of acetogen species by the iCH system (Acetobacterium spp. and Sporomusa spp.), whereas Clostridium spp. predominated in the high H2 cultures. These results demonstrate that the iCH system facilitates culture of anaerobic microorganisms under low H2 pressures, which will enable the selective culture of microorganisms adapting to low H2 environments.

A novel anaerobic, endospore-forming bacterium (strain M08 DMBT) was isolated from a terrestrial mud volcano (Taman Peninsula, Russia). Cells of the strain were motile rods 1.3–2.0 µm long and 0.4 µm in diameter. The temperature range for growth was 5–42 °C, with an optimum at 30 °C. The pH range for growth was H 6.5–11.0, with an optimum at pH 8.0. Growth of strain M08 DMBTwas observed at NaCl concentrations of 0–5.0 % (w/v) with an optimum at 1.0 %. Strain M08 DMBTutilized 3,4-dimethoxybenzoic acid, 2-methoxyphenol, carbon monoxide, glucose, fructose, mannose, xylose and yeast extract. The end product of glucose fermentation was acetate. The DNA G+C content of strain M08 DMBTwas 32.3 mol% (obtained via whole genome sequencing). The closest phylogenetic relative of strain M08 DMBTwasAlkalibaculum bacchi(familyEubacteriaceae, classClostridia) with 95.17 % 16S rRNA gene sequence similarity. Based on the phenotypic, genotypic and phylogenetic characteristics of the isolate, strain M08 DMBTis considered to represent a novel species of the genusAlkalibaculum, for which the nameAlkalibaculum sporogenessp. nov. is proposed. The type strain ofAlkalibaculum sporogenesis M08 DMBT(=KCTC 15840T=VKM B-3387T).

The trimethylamine methyltransferase MttB is the first described member of a superfamily comprising thousands of microbial proteins. Most members of the MttB superfamily are encoded by genes that lack the codon for pyrrolysine characteristic of trimethylamine methyltransferases, raising questions about the activities of these proteins. The superfamily member MtcB is found in the human intestinal isolate Eubacterium limosum ATCC 8486, an acetogen that can grow by demethylation of l-carnitine. Here, we demonstrate that MtcB catalyzes l-carnitine demethylation. When growing on l-carnitine, E. limosum excreted the unusual biological product norcarnitine as well as acetate, butyrate, and caproate. Cellular extracts of E. limosum grown on l-carnitine, but not lactate, methylated cob-(I)alamin or tetrahydrofolate using l-carnitine as methyl donor. MtcB, along with the corrinoid protein MtqC and the methylcorrinoid:tetrahydrofolate methyltransferase MtqA, were much more abundant in E. limosum cells grown on l-carnitine than on lactate. Recombinant MtcB methylates either cob(I)alamin or Co(I)-MtqC in the presence of l-carnitine and, to a much lesser extent, γ-butyrobetaine. Other quaternary amines were not substrates. Recombinant MtcB, MtqC, and MtqA methylated tetrahydrofolate via l-carnitine, forming a key intermediate in the acetogenic Wood–Ljungdahl pathway. To our knowledge, MtcB methylation of cobalamin or Co(I)-MtqC represents the first described mechanism of biological l-carnitine demethylation. The conversion of l-carnitine and its derivative γ-butyrobetaine to trimethylamine by the gut microbiome has been linked to cardiovascular disease. The activities of MtcB and related proteins in E. limosum might demethylate proatherogenic quaternary amines and contribute to the perceived health benefits of this human gut symbiont.

Significance Despite sharing the first four reactions, coutilization of the Wood–Ljungdahl pathway (WLP) with the glycine synthase-reductase pathway (GSRP) and reductive glycine pathway (RGP) to fix C1 compounds has remained unknown. In this study, using Clostridium drakei, we elucidated the role of the GSRP and RGP in the presence of the WLP, via a genome-scale metabolic model, RNA-seq, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression. Overall, the data suggested the pathways are functional under autotrophic conditions. Along with the WLP, GSRP and RGP convert CO2 to glycine and then to acetyl-phosphate and serine, which then obtain ATP by producing acetate and operate with limited reducing power. This is a unique coutilization of the pathways under autotrophic conditions in acetogens., Among CO2-fixing metabolic pathways in nature, the linear Wood–Ljungdahl pathway (WLP) in phylogenetically diverse acetate-forming acetogens comprises the most energetically efficient pathway, requires the least number of reactions, and converts CO2 to formate and then into acetyl-CoA. Despite two genes encoding glycine synthase being well-conserved in WLP gene clusters, the functional role of glycine synthase under autotrophic growth conditions has remained uncertain. Here, using the reconstructed genome-scale metabolic model iSL771 based on the completed genome sequence, transcriptomics, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression of the pathway in another acetogen, we discovered that the WLP and the glycine synthase pathway are functionally interconnected to fix CO2, subsequently converting CO2 into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO2 consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens.

The bacterial production of acetate via reductive acetogenesis along the Wood–Ljungdahl metabolic pathway is an important source of this molecule in several environments, ranging from industrial bioreactors to the human gastrointestinal tract. Here, we contributed to the study of reductive acetogens by considering mathematical modelling techniques for the prediction of bacterial growth and acetate production. We found that the incorporation of a hydrogen uptake concentration threshold into the models improves their predictions and we calculated this threshold as 86.2 mM (95% confidence interval 6.1–132.6 mM). Monod kinetics and first-order kinetics models, with the inclusion of two candidate threshold terms or reversible Michaelis–Menten kinetics, were compared to experimental data and the optimal formulation for predicting both growth and metabolism was found. The models were then used to compare the efficacy of two growth media for acetogens. We found that the recently described general acetogen medium was superior to the DSMZ medium in terms of unbiased estimation of acetogen growth and investigated the contribution of yeast extract concentration to acetate production and bacterial growth in culture. The models and their predictions will be useful to those studying both industrially and environmentally relevant reductive acetogenesis and allow for straightforward adaptation to similar cases with different organisms.

This study was designed to evaluate the in vitro and in vivo effects of reductive acetogens isolated from ruminants on methane mitigation, and milk performance, respectively. Four acetogens, Proteiniphilum acetatigenes DA02, P. acetatigenes GA01, Alkaliphilus crotonatoxidans GA02, and P. acetatigenes GA03 strains were isolated from ruminants and used in in vitro experiment. A control (without acetogen) and a positive group (with Eubacterium limosum ATCC 8486) were also included in in vitro experiment. Based on higher acetate as well as lower methane producing ability in in vitro trial, P. acetatigenes GA03 was used as inoculum for in vivo experiment. Holstein dairy cows (n = 14) were divided into two groups viz. control (without) and GA03 group (diet supplied with P. acetatigenes GA03 at a feed rate of 1% supplementation). Milk performance and blood parameters were checked for both groups. In in vitro, the total volatile fatty acids and acetate production were higher (p < 0.05) in all 4 isolated acetogens than the control and positive treatment. Also, all acetogens significantly lowered (p < 0.05) methane production in comparison to positive and control groups however, GA03 had the lowest (p < 0.05) methane production among 4 isolates. In in vivo, the rate of milk yield reduction was higher (p < 0.05) in the control than GA03 treated group (5.07 vs 2.4 kg). Similarly, the decrease in milk fat was also higher in control (0.14% vs 0.09%) than treatment. The somatic cell counts (SCC; ×103/mL) was decreased from 128.43 to 107.00 in acetogen treated group however, increased in control from 138.14 to 395.71. In addition, GA03 increased blood glucose and decreased non-esterified fatty acids. Our results suggest that the isolated acetogens have the potential for in vitro methane reduction and P. acetatigenes GA03 strain could be a candidate probiotic strain for improving milk yield and milk fat in lactating cows with lowering SCCs.

Fuelled by an ever-increasing demand for sustainable biofuels, a new generation of advanced technologies is needed. Fermentation of C1 gases (syngas, CO, CO2) offers numerous advantages for producing biofuels and chemicals from waste streams. Acetogens use the Wood Ljungdhal pathway (WLP) to fix CO and CO2 into the central metabolite acetyl-CoA to produce a wide variety of products. Recently, acetogens have received much attention as cell factories to produce various chemicals from waste resources. These include the production of ethanol, 2,3-butanediol, butyrate and butanol from syngas.Clostridium autoethanogenum is a model acetogen capable of growing on syngas. In addition to acetate, it produces significant amounts of ethanol compared to acetate. To expand the natural product spectrum of this acetogen, it is crucial to understand the metabolism and its mechanisms of regulation. Although some efforts were made towards understanding the metabolism of this acetogen using modelling, they are limited to stochiometric models (Constraint-based models). Constraint-based models (CBM) often lack the ability to provide insight into regulatory mechanisms, rate-limiting steps, or the effects of change in enzyme concentrations which are crucial for guided strain design. Recently reported oscillations in high-density continuous cultures is detrimental to productivity. Also, oscillatory behaviour provides a unique opportunity for understanding the robustness of metabolism, as cells respond to changes in environmental conditions by inherently compromising metabolic efficiency. The main aim of this thesis is to understand the advance the understanding of C. autoethanogenum metabolism and to investigate the biochemical reason behind the oscillations.Earlier studies indicate that metabolism of acetogens operates at the limits of thermodynamic equilibrium. To understand the reaction dynamics at a network scale we use a method called thermodynamic metabolic flux analysis (tMFA). tMFA is an extension of flux balance analysis that imposes thermodynamic constraints on genome-scale models to assign reaction directionalities. A primary challenge in tMFA is accounting for uncertainty in the thermodynamic parameters. The parameters are reaction Gibbs energy variables, but they cannot be independently varied without creating thermodynamic inconsistencies because reactions share metabolites. Handling compound formation energies as variables avoid this issue to an extent, but not entirely as the error in formation energies can be correlated. We developed a more elegant way of sampling parameter sets given parameter uncertainty that improves the state-of-the-art tMFA method generated from individual 95% confidence intervals. Using this framework, we reduce the volume of the multi-dimensional solution space and reduce the uncertainty of reaction Gibbs energies. We made this available as a Python package named multiTFA.Metabolic regulation is a complex process and is often multi-layered. Depending on the surrounding environment and the energetic availability, cells choose an array of regulatory mechanisms to control metabolic flux. Earlier studies attempted to decipher the key regulatory points in metabolism of acetogens. However, there is no comprehensive study available till date linking multiple omics datasets to understand the complex interplay. We performed a thermo-kinetic analysis on the autotrophic metabolism of C. autoethanogenum using the steady-state datasets of different growth conditions. A comprehensive analysis using various omics data revealed that the metabolism is not transcription controlled and rather thermodynamically controlled through the change in metabolite concentrations. This analysis also emphasizes the importance of high-quality omics datasets and the need of a kinetic model to understand the metabolic regulation.Oscillations are a primary challenge in C. autoethanogenum continuous cultures. These oscillations hinder us from reaching higher biomass concentrations and thus reaching higher ethanol productivity. Here, we quantify the limits of metabolic robustness in self-oscillating autotrophic continuous cultures of the gas-fermenting acetogen C. autoethanogenum. Gas analysis and high-resolution temporal metabolomics showed oscillations in gas uptake rates and extracellular by-products synchronised with biomass levels. We found that the intrinsic nature of gas fermentation allows for an initial growth phase on CO, followed by growth on CO and H2, after which a down cycle is observed in synchrony with a loss in H2 uptake. Intriguingly, we have not observed any correlation between oscillations and protein expression during oscillations. Intracellular metabolomics analysis revealed decreasing levels of redox ratios in synchrony with the cycles. We used a thermodynamic metabolic flux analysis (tMFA) model to investigate if regulation in acetogens is controlled at the thermodynamic level. By incorporating endo and exo-metabolomics data with the model we show that the thermodynamic driving force of critical reactions collapsed as H2 uptake is lost. The oscillations are coordinated with redox. The data indicate that metabolic oscillations in gas fermentation acetogens are controlled at the thermodynamic level. The work presented will show that thermodynamic control of metabolism, potentially contributing to metabolic efficiency in gas fermentation.

Gas fermentation by acetogens of the genus Clostridium is an attractive technology since it affords the production of biochemicals and biofuels from industrial waste gases while contributing to mitigate the carbon cycle alterations. The acetogenic model organisms C. ljungdahlii and C. autoethanogenum have already been used in large scale industrial fermentations. Among the natural products, ethanol production has already attained industrial scale. However, some acetogens are also natural producers of 2,3-butanediol (2,3-BDO), a platform chemical of relevant industrial interest. Here, we have developed a lab-scale screening campaign with the aim of enhancing 2,3-BDO production. Our study generated comparable data on growth and 2,3-BDO production of several batch gas fermentations using C. ljungdahlii and C. autoethanogenum grown on different gas substrates of primary applicative interest (CO2 · H2, CO · CO2, syngas) and on different media featuring different compositions as regards trace metals, mineral elements and vitamins. CO · CO2 fermentation was found to be preferable for the production of 2,3-BDO, and a fair comparison of the strains cultivated in comparable conditions revealed that C. ljungdahlii produced 3.43-fold higher titer of 2,3-BDO compared to C. autoethanogenum. Screening of different medium compositions revealed that mineral elements, Zinc and Iron exert a major positive influence on 2,3-BDO titer and productivity. Moreover, the CO2 influence on CO fermentation was explored by characterizing C. ljungdahlii response with respect to different gas ratios in the CO · CO2 gas mixtures. The screening strategies undertaken in this study led to the production of 2.03 ± 0.05 g/L of 2,3-BDO, which is unprecedented in serum bottle experiments.

Acetogens synthesize acetyl-CoA via the CO2-fixing Wood-Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon and energy metabolisms in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. Data integration of genome-scale transcriptomic and translatomic analyses revealed that the acetogenesis genes, including those of the Wood-Ljungdahl pathway and energy metabolism, showed changes in translational efficiency under autotrophic growth conditions. In particular, genes encoding the Wood-Ljungdahl pathway are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas genes encoding the carbonyl branch present increased translation levels in comparison to those for the methyl branch under heterotrophic growth conditions. The translation efficiency of genes in the pathways is differentially regulated by 5′ untranslated regions and ribosome-binding sequences under different growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas-fermenting acetogenic bacteria for better productivity. IMPORTANCE Acetogens are capable of reducing CO2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Here, we dissect the translational regulation of carbon and energy metabolisms in the model acetogen Acetobacterium woodii under heterotrophic and autotrophic growth conditions. The latter may be experienced when acetogen is used as a cell factory that synthesizes products from CO2 during the gas fermentation process. We found that the methyl and carbonyl branches of the Wood-Ljungdahl pathway are activated at similar translation levels during autotrophic growth. Translation is mainly regulated by the 5′-untranslated-region structure and ribosome-binding-site sequence. This work reveals novel translational regulation for coping with autotrophic growth conditions and provides the systematic data set, including the transcriptome, translatome, and promoter/5′-untranslated-region bioparts.

The production of trimethylamine (TMA) from quaternary amines such as l-carnitine or γ-butyrobetaine (4-(trimethylammonio)butanoate) by gut microbial enzymes has been linked to heart disease. This has led to interest in enzymes of the gut microbiome that might ameliorate net TMA production, such as members of the MttB superfamily of proteins, which can demethylate TMA (e.g., MttB) or l-carnitine (e.g., MtcB). Here, we show that the human gut acetogen Eubacterium limosum demethylates γ-butyrobetaine and produces MtyB, a previously uncharacterized MttB superfamily member catalyzing the demethylation of γ-butyrobetaine. Proteomic analyses of E. limosum grown on either γ-butyrobetaine or dl-lactate were employed to identify candidate proteins underlying catabolic demethylation of the growth substrate. Three proteins were significantly elevated in abundance in γ-butyrobetaine-grown cells: MtyB, MtqC (a corrinoid-binding protein), and MtqA (a corrinoid:tetrahydrofolate methyltransferase). Together, these proteins act as a γ-butyrobetaine:tetrahydrofolate methyltransferase system, forming a key intermediate of acetogenesis. Recombinant MtyB acts as a γ-butyrobetaine:MtqC methyltransferase but cannot methylate free cobalamin cofactor. MtyB is very similar to MtcB, the carnitine methyltransferase, but neither was detectable in cells grown on carnitine nor was detectable in cells grown with γ-butyrobetaine. Both quaternary amines are substrates for either enzyme, but kinetic analysis revealed that, in comparison to MtcB, MtyB has a lower apparent Km for γ-butyrobetaine and higher apparent Vmax, providing a rationale for MtyB abundance in γ-butyrobetaine-grown cells. As TMA is readily produced from γ-butyrobetaine, organisms with MtyB-like proteins may provide a means to lower levels of TMA and proatherogenic TMA-N-oxide via precursor competition.

Microbial conversion of carbon monoxide (CO) to acetate is a promising upcycling strategy for carbon sequestration. Herein, we demonstrate that CO conversion and acetate production rates of Eubacterium limosum KIST612 strain can be improved by in silico prediction and in vivo assessment. The mimicked CO metabolic model of KIST612 predicted that overexpressing the CO dehydrogenase (CODH) increases CO conversion and acetate production rates. To validate the prediction, we constructed mutant strains overexpressing CODH gene cluster and measured their CO conversion and acetate production rates. A mutant strain (ELM031) co-overexpressing CODH, coenzyme CooC2 and ACS showed a 3.1 × increased specific CO oxidation rate as well as 1.4 × increased specific acetate production rate, compared to the wild type strain. The transcriptional and translational data with redox balance analysis showed that ELM031 has enhanced reducing potential from up-regulation of ferredoxin and related metabolism directly linked to energy conservation.

Gas fermentation is a promising way to convert CO-rich gases to chemicals. We studied the use of synthetic cocultures composed of carboxydotrophic and propionigenic bacteria to convert CO to propionate. So far, isolated carboxydotrophs cannot directly ferment CO to propionate, and therefore, this cocultivation approach was investigated. Four distinct synthetic cocultures were constructed, consisting of Acetobacterium wieringae (DSM 1911T) and Pelobacter propionicus (DSM 2379T), Ac. wieringae (DSM 1911T) and Anaerotignum neopropionicum (DSM 3847T), Ac. wieringae strain JM and P. propionicus (DSM 2379T), and Ac. wieringae strain JM and An. neopropionicum (DSM 3847T). Propionate was produced by all the cocultures, with the highest titer (;24mM) being measured in the coculture composed of Ac. wieringae strain JM and An. neopropionicum, which also produced isovalerate (;4mM), butyrate (;1mM), and isobutyrate (0.3mM). This coculture was further studied using proteogenomics. As expected, enzymes involved in the Wood-Ljungdahl pathway in Ac. wieringae strain JM, which are responsible for the conversion of CO to ethanol and acetate, were detected; the proteome of An. neopropionicum confirmed the conversion of ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during cocultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, accompanied by isovalerate and isobutyrate production. This highlights the importance of explicitly looking at fortuitous microbial interactions during cocultivation to fully understand coculture behavior., This research was financially supported by Project NWO-GK-07 from the Netherlands Science Foundation (NWO), the Perspectief Programma P16-10 from NWO Applied and Engineering Sciences (AGS), by a Gravitation Grant (Project 024.002.002) of the Netherlands Ministry of Education, Culture and Science, and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020— Programa Operacional Regional do Norte. The financial support from FCT and European Social Fund (POPH-QREN) through the grant PD/BD/128030/2016 (given to Ana Luísa Arantes) and PD/BD/150583/2020 (given to João P. C. Moreira) and through the project INNOVsyn—Innovative strategies for syngas fermentation (POCI-01-0145-FEDER-031377) are gratefully acknowledged., info:eu-repo/semantics/publishedVersion

The trimethylamine methyltransferase MttB is the founding member of a widely distributed superfamily of microbial proteins. Genes encoding most members of the MttB superfamily lack the codon for pyrrolysine that distinguishes previously characterized trimethylamine methyltransferases, leaving the function(s) of most of the enzymes in this superfamily unknown. Here, investigating the MttB family member MtpB from the human intestinal isolate Eubacterium limosum ATCC 8486, an acetogen that excretes N-methyl proline during growth on proline betaine, we demonstrate that MtpB catalyzes anoxic demethylation of proline betaine. MtpB along with MtqC (a corrinoid protein) and MtqA (a methylcorrinoid:tetrahydrofolate methyltransferase) was much more abundant in E. limosum cells grown on proline betaine than on lactate. We observed that recombinant MtpB methylates Co(I)-MtqC in the presence of proline betaine and that other quaternary amines are much less preferred substrates. MtpB, MtqC, and MtqA catalyze tetrahydrofolate methylation with proline betaine, thereby forming a key intermediate in the Wood–Ljungdahl acetogenesis pathway. To our knowledge, MtpB methylation of Co(I)-MtqC for the subsequent methylation of tetrahydrofolate represents the first described anoxic mechanism of proline betaine demethylation. The activities of MtpB and associated proteins in acetogens or other anaerobes provide a possible mechanism for the production of N-methyl proline by the gut microbiome. MtpB's activity characterized here strengthens the hypothesis that much of the MttB superfamily comprises quaternary amine-dependent methyltransferases.

The aim of this study was to optimize the culture medium to enhance the growth of Clostridium autoethanogenum, an acetogenic bacterium that converts synthesis gas to ethanol and acetic acid. In order to optimize the culture medium, four components, yeast extract, trace metal solution, vitamin solution, and Na2S·9H2O, were investigated. Yeast extract was found to have the biggest influence on growth, and a concentration of 5 g/L, 10 times higher than that in basal medium, leading to 2.3 times greater growth. At 10 times the concentration in basal medium, trace metal solution inhibited the growth of the acetogen. A change in the concentration of vitamin solution did not yield any significant difference in cell growth. Lastly, growth with Na2S·9H2O showed a tendency to decrease after an initial cell mass increase for concentrations of 0.5 g/L (basal medium condition) and 0.05 g/L. However, at 5 g/L, C. autoethanogenum grew steadily.

Combining inorganic catalysts with CO2-fixing microorganisms has displayed a high efficiency for electricity-driven CO2 reduction. However, the maximum throughput can be limited by the low solubility of mediators, such as H2, that deliver reducing equivalents from the electrodes to the microbes. Here we report that the introduction of a biocompatible perfluorocarbon nanoemulsion as a H2 carrier increases the throughput of CO2 reduction into acetic acid by 190%. With the acetogen Sporomusa ovata as a model system, an average acetate titre of 6.4 ± 1.1 g l−1 (107 mM) was achieved in four days with close to 100% Faradaic efficiency. This is equivalent to a productivity of 1.1 mM h−1, among the highest in bioelectrochemical systems. A mechanistic investigation shows that the non-specific binding of perfluorocarbon nanoemulsions promotes the kinetics of H2 transfer and subsequent oxidation by more than threefold. Introducing nanoscale gas carriers is viable to alleviate throughput bottlenecks in the electricity-driven microbial CO2 reduction into commodity chemicals. H2 is a promising mediator of electrons from electrodes to microbes for chemicals production from CO2—but its low solubility limits the productivity. This work reports nanoemulsions as H2 carriers that improve the solubility and transfer kinetics of H2, increasing the productivity of the system.

In microbial fermentations at least 33% of the sugar-substrate carbon is lost as CO2 during pyruvate decarboxylation to acetyl-CoA, with the corresponding electrons lost in the form of H2. Previous attempts to reduce this carbon and electron loss focused on engineering of a single organism. In nature, most microorganisms live in complex communities where syntrophic interactions result in superior resource utilization. Here, we show that a synthetic syntrophy consisting of the solventogen Clostridium acetobutylicum, which converts simple and complex carbohydrates into a variety of chemicals, and the acetogen C. ljungdahlii which fixes CO2, achieved carbon recoveries into C2-C4 alcohols almost to the limit of substrate-electron availability, with minimal H2 and CO2 release. The syntrophic co-culture produced robust metabolic outcomes over a broad range of starting population ratios of the two organisms. We show that direct cell-to-cell interactions and material exchange among the two microbes enabled unforeseen rearrangements in the metabolism of the individual species that resulted in the production of non-native metabolites, namely isopropanol and 2,3-butanediol. This was accomplished by pathway-specific alterations of gene expression brought about by one organism on the other, and vice versa. While some of these gene-expression alterations can be explained by the exchange of metabolites that induce specific gene expression patterns, others, as demonstrated by co-culture setup in a transwell system, cannot. The latter, for now, would be attributed to complex direct physical interactions among the two organisms, thus providing a glimpse of the potential microbial complexity of simple or multicomponent microbiomes. Such direct material-transfer phenomena have not been documented in the literature. Furthermore, our study shows that syntrophic cultures offer a flexible platform for metabolite production with superior carbon recovery that can also be applied to electron-enhanced fermentations enabling even higher carbon recoveries.

Objective Homoacetogens play important roles in the production of acetate in the large intestine of monogastric mammals. However, their diversity in the porcine large intestine is still unknown. Marker gene analysis was performed to assess the effects of energy level on the diversity and population densities of homoacetogens in porcine feces. Methods Crossbred pigs were fed high or low energy-level diets. The high-intake (HI) diet was sufficient to allow a daily gain of 1.2 kg. The low-intake (LI) diet provided 0.6 times the amount of energy as the HI diet. Genetic diversity was analyzed using formyltetrahydrofolate synthetase gene (FHS) clone libraries derived from fecal DNA samples. FHS DNA copy numbers were quantified using real-time polymerase chain reaction. Results A wide variety of FHS sequences was recovered from animals in both treatments. No differences in FHS clone libraries between the HI and LI groups were found. During the experimental period, no significant differences in the proportion of FHS copy numbers were observed between the two treatment groups. Conclusion This is the first reported molecular diversity analysis using specific homoacetogen marker genes from the large intestines of pigs. There was no observable effect of feed intake on acetogen diversity.

The majority of the genes present in bacterial genomes remain poorly characterised with up to one third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to easily transformable species leaving those with low DNA-transfer rates out of reach. Here we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as a sole carbon and energy source.IMPORTANCEAlthough microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterised, leaving the function of many as either hypothetical or entirely unknown. High-throughput, transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA-transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here we developed approaches to facilitate transposon-insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases, CO and CO2, into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth providing insights into the utilisation of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon-insertion sequencing to other microbes where DNA-transfer has until now represented a barrier to progress.

Gas fermentation is a promising biological process for the conversion of CO2 or syngas into valuable chemicals. Homoacetogens are microorganisms growing autotrophically using CO2 and H2 or CO and metabolizing them to form acetate coupled with energy conservation. The challenge in the metabolic engineering of the homoacetogens is divergence of the acetate formation, whose intermediate is acetyl-CoA, to a targeted chemical with sufficient production of adenosine triphosphate (ATP). In this study, we report that an engineered strain of the thermophilic homoacetogen Moorella thermoacetica, in which a pool of acetyl-CoA is diverted to ethanol without ATP production, can maintain autotrophic growth on syngas. We estimated the ATP production in the engineered strains under different gaseous compositions by considering redox-balanced metabolism for ethanol and acetate formation. The culture test showed that the combination of retaining a level of acetate production and supplying the energy-rich CO allowed maintenance of the autotrophic growth during ethanol production. In contrast, autotrophy was collapsed by complete elimination of the acetate pathway or supplementation of H2-CO2. We showed that the intracellular level of ATP was significantly lowered on H2-CO2 in consistent with the incompetence. In the meantime, the complete disruption of the acetate pathway resulted in the redox imbalance to produce ethanol from CO, albeit a small loss in the ATP production. Thus, preservation of a fraction of acetate formation is required to maintain sufficient ATP and balanced redox in CO-containing gases for ethanol production.

Methanol is the simplest of all alcohols, is universally distributed in anoxic sediments as a result of plant material decomposition and is constantly attracting attention as an interesting substrate for anaerobes like acetogens that can convert bio-renewable methanol into value-added chemicals. A major drawback in the development of environmentally friendly but economically attractive biotechnological processes is the present lack of information on biochemistry and bioenergetics during methanol conversion in these bacteria. The mesophilic acetogen Eubacterium callanderi KIST612 is naturally able to consume methanol and produce acetate as well as butyrate. To grasp the full potential of methanol-based production of chemicals, we analysed the genes and enzymes involved in methanol conversion to acetate and identified the redox carriers involved. We will display a complete model for methanol-derived acetogenesis and butyrogenesis in Eubacterium callanderi KIST612, tracing the electron transfer routes and shed light on the bioenergetics during the process.

More than 2 million tons of glycerol are produced during industrial processes each year and, therefore, glycerol is an inexpensive feedstock to produce biocommodities by bacterial fermentation. Acetogenic bacteria are interesting production platforms and there have been few reports in the literature on glycerol utilization by this ecophysiologically important group of strictly anaerobic bacteria. Here, we show that the model acetogen Acetobacterium woodii DSM1030 is able to grow on glycerol, but contrary to expectations, only for 2-3 transfers. Transcriptome analysis revealed the expression of the pdu operon encoding a propanediol dehydratase along with genes encoding bacterial microcompartments. Deletion of pduAB led to a stable growth of A. woodii on glycerol, consistent with the hypothesis that the propanediol dehydratase also acts on glycerol leading to a toxic end-product. Glycerol is oxidized to acetate and the reducing equivalents are reoxidized by reducing CO2 in the Wood-Ljungdahl pathway, leading to an additional acetate. The possible oxidation product of glycerol, dihydroxyacetone (DHA), also served as carbon and energy source for A. woodii and growth was stably maintained on that compound. DHA oxidation was also coupled to CO2 reduction. Based on transcriptome data and enzymatic analysis we present the first metabolic and bioenergetic schemes for glycerol and DHA utilization in A. woodii.

Here, we report the genome sequence of Clostridium sp. strain P21, isolated from old hay from Stillwater, Oklahoma. This announcement describes the generation and annotation of the 5.6-Mb genomic sequence of strain P21, which will aid in studies targeting genes involved in the enhancement of acid-alcohol production.

Microbiological conversion of CO2 into biofuels and/or organic industrial feedstock is an excellent carbon-cycling strategy. Here, autotrophic anaerobic bacteria in the membrane biofilm reactor (MBfR) transferred electrons from hydrogen gas (H2 ) to inorganic carbon (IC) and produced organic acids and alcohols. We systematically varied the H2 -delivery, the IC concentration, and the hydraulic retention time in the MBfR. The relative availability of H2 versus IC was the determining factor for enabling microbial chain elongation (MCE). When the H2 :IC mole ratio was high (>2.0 mol H2 /mol C), MCE was an important process, generating medium-chain carboxylates up to octanoate (C8, 9.1 ± 1.3 mM C and 28.1 ± 4.1 mmol C m-2 d-1 ). Conversely, products with two carbons were the only ones present when the H2 :IC ratio was low (

Significance To develop an efficient artificial photosynthesis system using acetogen-nanoparticle hybrids, the efficiency of the electron–hole pair generation of nanoparticles must be enhanced to demonstrate extracellular electron utilization by the acetogen. Here we verified that Clostridium autoethanogenum, an industrially relevant acetogen, could use electrons generated from size- and structure-controlled chemically synthesized cadmium sulfide nanoparticles displayed on the cell surface under light-exposure conditions. In addition, transcriptomic analysis showed that the electrons generated from nanoparticles were largely transported to the intracellular matrix via the metal ion or flavin-binding proteins. These results illustrate the potential to increase the CO2-fixing efficiency of nanoparticle-based artificial photosynthesis by engineering cellular processes related to electron transfer generated from the cathode., Acetogenic bacteria use cellular redox energy to convert CO2 to acetate using the Wood–Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H2 as well as from inorganic materials, such as photoresponsive semiconductors. We have developed a nanoparticle-microbe hybrid system in which chemically synthesized cadmium sulfide nanoparticles (CdS-NPs) are displayed on the cell surface of the industrial acetogen Clostridium autoethanogenum. The hybrid system converts CO2 into acetate without the need for additional energy sources, such as H2, and uses only light-induced electrons from CdS-NPs. To elucidate the underlying mechanism by which C. autoethanogenum uses electrons generated from external energy sources to reduce CO2, we performed transcriptional analysis. Our results indicate that genes encoding the metal ion or flavin-binding proteins were highly up-regulated under CdS-driven autotrophic conditions along with the activation of genes associated with the WL pathway and energy conservation system. Furthermore, the addition of these cofactors increased the CO2 fixation rate under light-exposure conditions. Our results demonstrate the potential to improve the efficiency of artificial photosynthesis systems based on acetogenic bacteria integrated with photoresponsive nanoparticles.

Unabated mining and utilisation of petroleum and petroleum resources and their conversion to essential fuels and chemicals have drastic environmental consequences, contributing to global warming and climate change. In addition, fossil fuels are finite resources, with a fast-approaching shortage. Accordingly, research efforts are increasingly focusing on developing sustainable alternatives for chemicals and fuels production. In this context, bioprocesses, relying on microorganisms, have gained particular interest. For example, acetogens use the Wood-Ljungdahl pathway to grow on single carbon C1-gases (CO2 and CO) as their sole carbon source and produce valuable products such as acetate or ethanol. These autotrophs can, therefore, be exploited for large-scale fermentation processes to produce industrially relevant chemicals from abundant greenhouse gases. In addition, genetic tools have recently been developed to improve these chassis organisms through synthetic biology approaches. This review will focus on the challenges of genetically and metabolically modifying acetogens. It will first discuss the physical and biochemical obstacles complicating successful DNA transfer in these organisms. Current genetic tools developed for several acetogens, crucial for strain engineering to consolidate and expand their catalogue of products, will then be described. Recent tool applications for metabolic engineering purposes to allow redirection of metabolic fluxes or production of non-native compounds will lastly be covered., This review systematically discusses the challenges of genetically modifying acetogenic chassis, and the recent development of several genetic tools applied to engineer these industrially important microbes for sustainable production of fuels and chemicals from greenhouse gases using C1-gas fermentation.

Acetogenic bacteria are already established as biocatalysts for production of high-value compounds from C1 substrates such as H2 + CO2 or CO. However, little is known about the physiology, biochemistry and bioenergetics of acetogenesis from formate, an interesting feedstock for biorefineries. Here, we analysed formate metabolism in the model acetogen Acetobacterium woodii. Cells grew optimally on 200 mM formate to an optical density of 0.6. Formate was exclusively converted to acetate (and CO2 ) with a ratio of 4.4:1. Transcriptome analyses revealed genes/enzymes involved in formate metabolism. Strikingly, A. woodii has two genes potentially encoding a formyl-THF synthetase, fhs1 and fhs2. fhs2 forms an operon with a gene encoding a potential formate transporter, fdhC. Deletion of fhs2/fdhC led to a reduced growth rate, formate consumption and optical densities. Acetogenesis from H2 + CO2 was accompanied by transient formate production; strikingly, formate reutilization was completely abolished in the Δfhs2/fdhC mutant. Take together, our studies gave the first detailed insights into the formatotrophic lifestyle of A. woodii.

Acetogens play a very important role in anaerobic digestion and are essential in ensuring process stability. Despite this, targeted studies of the acetogenic community in biogas processes remain limited. Some efforts have been made to identify and understand this community, but the lack of a reliable molecular analysis strategy makes the detection of acetogenic bacteria tedious. Recent studies suggest that screening of bacterial genetic material for formyltetrahydrofolate synthetase (FTHFS), a key marker enzyme in the Wood-Ljungdahl pathway, can give a strong indication of the presence of putative acetogens in biogas environments. In this study, we applied an acetogen-targeted analyses strategy developed previously by our research group for microbiological surveillance of commercial biogas plants. The surveillance comprised high-throughput sequencing of FTHFS gene amplicons and unsupervised data analysis with the AcetoScan pipeline. The results showed differences in the acetogenic community structure related to feed substrate and operating parameters. They also indicated that our surveillance method can be helpful in the detection of community changes before observed changes in physico-chemical profiles, and that frequent high-throughput surveillance can assist in management towards stable process operation, thus improving the economic viability of biogas plants. To our knowledge, this is the first study to apply a high-throughput microbiological surveillance approach to visualise the potential acetogenic population in commercial biogas digesters.

Industrially applied bioprocesses for the reduction of C1 gases (CO2 and/or CO) are based in particular on (syn)gas fermentation with acetogenic bacteria and on photobioprocesses with microalgae. In each case, process engineering characteristics of the autotrophic microorganisms are specified and process engineering aspects for improving gas and electron supply are summarized before suitable bioreactor configurations are discussed for the production of organic products under given economic constraints. Additionally, requirements for the purity of C1 gases are summarized briefly. Finally, similarities and differences in microbial CO2 valorization are depicted comparing gas fermentations with acetogenic bacteria and photobioprocesses with microalgae.

An anaerobic enrichment with CO from sediments of hypersaline soda lakes resulted in a methane-forming binary culture, whereby CO was utilized by a bacterium and not the methanogenic partner. The bacterial isolate ANCO1 forms a deep-branching phylogenetic lineage at the level of a new family within the class ‘Natranaerobiia’. It is an extreme haloalkaliphilic and moderate thermophilic acetogen utilizing CO, formate, pyruvate and lactate as electron donors and thiosulfate, nitrate (reduced to ammonia) and fumarate as electron acceptors. The genome of ANCO1 encodes a full Wood–Ljungdahl pathway allowing for CO oxidation and acetogenic conversion of pyruvate. A locus encoding Nap nitrate reductase/NrfA ammonifying nitrite reductase is also present. Thiosulfate respiration is encoded by a Phs/Psr-like operon. The organism obviously relies on Na-based bioenergetics, since the genome encodes for the Na+-Rnf complex, Na+-F1F0 ATPase and Na+-translocating decarboxylase. Glycine betaine serves as a compatible solute. ANCO1 has an unusual membrane polar lipid composition dominated by diethers, more common among archaea, probably a result of adaptation to multiple extremophilic conditions. Overall, ANCO1 represents a unique example of a triple extremophilic CO-oxidizing anaerobe and is classified as a novel genus and species Natranaerofaba carboxydovora in a novel family Natranaerofabacea.

Gas fermentation by Clostridium autoethanogenum is a commercial process for the sustainable biomanufacturing of fuels and valuable chemicals using abundant, low-cost C1 feedstocks (CO and CO2) from sources such as inedible biomass, unsorted and nonrecyclable municipal solid waste, and industrial emissions. Efforts toward pathway engineering and elucidation of gene function in this microbe have been limited by a lack of genetic tools to control gene expression and arduous genome engineering methods. To increase the pace of progress, here we developed an inducible CRISPR interference (CRISPRi) system for C. autoethanogenum and applied that system toward transcriptional repression of genes with ostensibly crucial functions in metabolism., Graphical Abstract Graphical Abstract

Acetobacterium woodii utilizes the Wood-Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non-acetogenic growth on 1,2-propanediol (1,2-PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2-PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC-encapsulated propanol-generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH-dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2-PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.

Acetogenic bacteria are a diverse group of anaerobes that use the reductive acetyl coenzyme A (acetyl-CoA) (Wood-Ljungdahl) pathway for CO2 fixation and energy conservation. The conversion of 2 mol CO2 into acetyl-CoA by using the Wood-Ljungdahl pathway as the terminal electron accepting process is the most prominent metabolic feature for these microorganisms. However, here, we describe that the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. Genome analysis of Clostridium bovifaecis revealed that it contains almost the complete genes of the Wood-Ljungdahl pathway but lacks the gene encoding formate dehydrogenase, which catalyzes the reduction of CO2 to formate as the first step of the methyl branch of the Wood-Ljungdahl pathway. The lack of a gene encoding formate dehydrogenase was verified by PCR, reverse transcription-PCR analysis, enzyme activity assay, and its formate-dependent acetogenic utilization of glucose on DNA, RNA, protein, and phenotype level, respectively. The lack of a formate dehydrogenase gene may be associated with the adaption to a formate-rich intestinal environment, considering the isolating source of strain BXX. The formate-dependent acetogenic growth of Clostridium bovifaecis provides insight into a unique metabolic feature of fecal acetogens.IMPORTANCE The acetyl-CoA pathway is an ancient pathway of CO2 fixation, which converts 2 mol of CO2 into acetyl-CoA. Autotrophic growth with H2 and CO2 via the acetyl-CoA pathway as the terminal electron accepting process is the most unique feature of acetogenic bacteria. However, the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. The formate-dependent acetogenic growth of Clostridium bovifaecis was associated with its lack of a gene encoding formate dehydrogenase, which may result from adaption to a formate-rich intestinal environment. This study gave insight into a unique metabolic feature of fecal acetogens. Because of the requirement of formate for the acetogenic growth of certain acetogens, the ecological impact of acetogens could be more complex and important in the formate-rich environment due to their trophic interactions with other microbes.

Contribution to the International Chain Elongation Conference 2020 | ICEC 2020. An abstract can be found in the right column.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.

The production of bulk chemicals mostly depends on exhausting petroleum sources and leads to emission of greenhouse gases. Within the last decades the urgent need for alternative sources has increased and the development of bio-based processes received new attention. To avoid the competition between the use of sugars as food or fuel, other feedstocks with high availability and low cost are needed, which brought acetogenic bacteria into focus. This group of anaerobic organisms uses mixtures of CO2, CO and H2 for the production of mostly acetate and ethanol. Also methanol, a cheap and abundant bulk chemical produced from methane, is a suitable substrate for acetogenic bacteria. In methylotrophic acetogens the methyl group is transferred to the Wood-Ljungdahl pathway, a pathway to reduce CO2 to acetate via a series of C1-intermediates bound to tetrahydrofolic acid. Here we describe the biochemistry and bioenergetics of methanol conversion in the biotechnologically interesting group of anaerobic, acetogenic bacteria. Further, the bioenergetics of biochemical production from methanol is discussed.

Higher alcohols such as butanol (C4 alcohol) and hexanol (C6 alcohol) are superior biofuels compared to ethanol. Clostridium carboxidivorans P7 is a typical acetogen capable of producing C4 and C6 alcohols natively. In this study, the composition of trace metals in culture medium was adjusted, and the effects of these adjustments on artificial syngas fermentation by C. carboxidivorans P7 were investigated. Nickel and ferrous ions were essential for growth and metabolite synthesis during syngas fermentation by P7. However, a decreased dose of molybdate improved alcohol fermentation performance by stimulating carbon fixation and solventogenesis. In response to the modified trace metal composition, cells grew to a maximum OD600nm of 1.6 and accumulated ethanol and butanol to maximum concentrations of 2.0 and 1.0 g/L, respectively, in serum bottles. These yields were ten-fold higher than the yields generated using the original composition of trace metals. Furthermore, 0.5 g/L of hexanol was detected at the end of fermentation. The results from gene expression experiments examining genes related to carbon fixation and organic acid and solvent synthesis pathways revealed a dramatic up-regulation of the Wood–Ljungdahl pathway (WLP) gene cluster, the bcs gene cluster, and a putative CoA transferase and butanol dehydrogenase, thereby indicating that both de novo synthesis and acid re-assimilation contributed to the significantly elevated accumulation of higher alcohols. The bdh35 gene was speculated to be the key target for butanol synthesis during solventogenesis.

Understanding the metabolic dynamics of the human gastrointestinal tract (GIT) microbiota is of growing importance as research continues to link the microbiome to host health status. Microbial strains that metabolize hydrogen have been associated with a variety of both positive and negative host nutritional and health outcomes, but limited data exists for their competition in the GIT. To enable greater insight into the behaviour of these microbes, a mathematical model was developed for the metabolism and growth of the three major hydrogenotrophic groups: sulphate-reducing bacteria (SRB), methanogens and reductive acetogens. In batch culture simulations with abundant sulphate and hydrogen, the SRB outcompeted the methanogen for hydrogen due to having a half-saturation constant 106 times lower than that of the methanogen. The acetogen, with a high model threshold for hydrogen uptake of around 70 mM, was the least competitive. Under high lactate and zero sulphate conditions, hydrogen exchange between the SRB and the methanogen was the dominant interaction. The methanogen grew at 70% the rate of the SRB, with negligible acetogen growth. In continuous culture simulations, both the SRB and the methanogen were washed out at dilution rates above 0.15 h−1 regardless of substrate availability, whereas the acetogen could survive under abundant hydrogen conditions. Specific combinations of conditions were required for survival of more than one hydrogenotroph in continuous culture, and survival of all three was not possible. The stringency of these requirements and the inability of the model to simulate survival of all three hydrogenotrophs in continuous culture demonstrates that factors outside of those modelled are vital to allow hydrogenotroph coexistence in the GIT.

The biohydrogen production efficiency and performance of hydrogen-producing acetogen in a four-compartment anaerobic baffled reactor (ABR) were studied by gradually increasing the influent normal molasses wastewater (NMWW) proportion. When the influent NMWW proportion increased to 55%, ABR could develop microbial community with methanogenic function in 63 days and reach a stable operation. When the influent NMWW proportion increased to 80% and reached a stable state, ethanol fermentation was established from butyric acid fermentation in the first three compartments, whereas butyric acid fermentation in the fourth compartment was strengthened. The average biohydrogen production yield and biohydrogen production capacity by COD removal increased to as high as 12.85 L/day and 360.22 L/kg COD when the influent NMWW proportion increased from 55% to 80%, respectively. Although the biogas yield and the specific biogas production rate reached 61.54 L/day and 232 L/kg MLVSS·day, the biohydrogen production yield and specific biohydrogen production rate were only 12.85 L/day and 48 L/kg MLVSS·day, which results in hydrogen consumption by homoacetogenesis and methanogenesis.

rnf genes are widespread in anaerobic bacteria and hypothesized to encode a respiratory enzyme that couples exergonic reduction of NAD with reduced ferredoxin as a reductant to vectorial ion (Na+, H+) translocation across the cytoplasmic membrane. However, despite its importance for the physiology of these bacteria, little is known about the subunit composition and the function of subunits. Here, we have purified the entire Rnf complex from the acetogen Acetobacterium woodii or after its production in Escherichia coli. These studies revealed covalently bound flavin in RnfB and RnfD. Unfortunately, the complex did not catalyze electron transfer from reduced ferredoxin to NAD. We, therefore, concentrated on the two cytosolic subunits RnfC and RnfB. RnfC was produced in E. coli, purified and shown to have 8.3 mol iron and 8.6 mol sulfur per mol of the subunit, consistent with the presence of two [4Fe-4S] centers, which were verified by EPR analysis. Flavins could not be detected, but RnfC catalyzed NADH-dependent FMN reduction. These data confirm RnfC as NADH-binding subunit and FMN as an intermediate in the electron transport chain. RnfB could only be produced as a fusion to the maltose-binding protein. It contained 25 mol iron and 26 mol sulfur, consistent with the predicted six [4Fe 4S] centers. The FeS centers in RnfB were reduced with reduced ferredoxin as reductant. These data are consistent with RnfB as the ferredoxin-binding subunit of the complex.

Both CO and H2 can be utilized as energy sources during the autotrophic growth of Clostridium ljungdahlii. In principle, CO is a more energetically and thermodynamically favorable energy source for gas fermentation in comparison to H2. Therefore, metabolism may vary during growth under different energy sources. In this study, C. ljungdahlii was fed with CO and/or CO2/H2 at pH 6.0 with a gas pressure of 0.1 MPa. C. ljungdahlii primarily produced acetate in the presence of H2 as an energy source, but produced alcohols with CO as an energy source under the same fermentation conditions. A key enzyme activity assay, metabolic flux analysis, and comparative transcriptomics were performed for investigating the response mechanism of C. ljungdahlii under different energy sources. A CO dehydrogenase and an aldehyde:ferredoxin oxidoreductase were found to play important roles in CO utilization and alcohol production. Based on these findings, novel metabolic schemes are proposed for C. ljungdahlii growing on CO and/or CO2/H2. These schemes indicate that more ATP is produced during CO-fermentation than during H2-fermentation, leading to increased alcohol production.