Impact analysis of operating conditions on carbon dioxide reduction in microbial electrosynthesis: Insight into the substance utilization and microbial response.

Microbial electrosynthesis (MES) is facing a series of problems including low energy utilization and production efficiency of high value-added products, which seriously hinder its practical application. In this study, a more practical direct current power source was used and the anaerobic activated sludge from wastewater treatment plants was inoculated to construct the acetic acid-producing MES. The operating conditions of acetic acid production were further optimized and the specific mechanisms involving the substance utilization and microbial response were revealed. The optimum conditions were the potential of 3.0 V and pH 6.0. Under these conditions, highly electroactive biofilms formed and all kinds of substances were effectively utilized. In addition, dominant bacteria (Acetobacterium, Desulfovibrio, Sulfuricurvum, Sulfurospirillum, and Fusibacter) had high abundances. Under optimal conditions, acetic acid-forming characteristic genera (Acetobacterium) had the highest relative abundance (Biocathode-25.82 % and Suspension-17.24 %). This study provided references for the optimal operating conditions of MES and revealed the corresponding mechanisms.

Reprogramming the metabolism of an acetogenic bacterium to homoformatogenesis.

Methyl groups are abundant in anoxic environments and their utilization as carbon and energy sources by microorganisms involves oxidation of the methyl groups to CO2, followed by transfer of the electrons to an acceptor. In acetogenic bacteria, the electron acceptor is CO2 that is reduced to enzyme bound carbon monoxide, the precursor of the carboxyl group in acetate. Here, we describe the generation of a mutant of the acetogen Acetobacterium woodii in which the last step in methyl group oxidation, formate oxidation to CO2 catalyzed by the HDCR enzyme, has been genetically deleted. The mutant grew on glycine betaine as methyl group donor, and in contrast to the wild type, formed formate alongside acetate, in a 1:2 ratio, demonstrating that methyl group oxidation stopped at the level of formate and reduced electron carriers were reoxidized by CO2 reduction to acetate. In the presence of the alternative electron acceptor caffeate, CO2 was no longer reduced to acetate, formate was the only product and all the carbon went to formate. Apparently, acetogenesis was not required to sustain formatogenic growth. This is the first demonstration of a genetic reprogramming of an acetogen into a formatogen that grows by homoformatogenesis from methyl groups. Formate production from methyl groups is not only of biotechnological interest but also for the mechanism of electron transfer in syntrophic interactions in anoxic environments.

Evidence for a Putative Isoprene Reductase in Acetobacterium wieringae.

Recent discoveries of isoprene-metabolizing microorganisms suggest they might play an important role in the global isoprene budget. Under anoxic conditions, isoprene can be used as an electron acceptor and is reduced to methylbutene. This study describes the proteogenomic profiling of an isoprene-reducing bacterial culture to identify organisms and genes responsible for the isoprene hydrogenation reaction. A metagenome-assembled genome (MAG) of the most abundant (89% relative abundance) lineage in the enrichment, Acetobacterium wieringae, was obtained. Comparative proteogenomics and reverse transcription-PCR (RT-PCR) identified a putative five-gene operon from the A. wieringae MAG upregulated during isoprene reduction. The operon encodes a putative oxidoreductase, three pleiotropic nickel chaperones (2 × HypA, HypB), and one 4Fe-4S ferredoxin. The oxidoreductase is proposed as the putative isoprene reductase with a binding site for NADH, flavin adenine dinucleotide (FAD), two pairs of canonical [4Fe-4S] clusters, and a putative iron-sulfur cluster site in a Cys6-bonding environment. Well-studied Acetobacterium strains, such as A. woodii DSM 1030, A. wieringae DSM 1911, or A. malicum DSM 4132, do not encode the isoprene-regulated operon but encode, like many other bacteria, a homolog of the putative isoprene reductase (~47 to 49% amino acid sequence identity). Uncharacterized homologs of the putative isoprene reductase are observed across the Firmicutes, Spirochaetes, Tenericutes, Actinobacteria, Chloroflexi, Bacteroidetes, and Proteobacteria, suggesting the ability of biohydrogenation of unfunctionalized conjugated doubled bonds in other unsaturated hydrocarbons. IMPORTANCE Isoprene was recently shown to act as an electron acceptor for a homoacetogenic bacterium. The focus of this study is the molecular basis for isoprene reduction. By comparing a genome from our isoprene-reducing enrichment culture, dominated by Acetobacterium wieringae, with genomes of other Acetobacterium lineages that do not reduce isoprene, we shortlisted candidate genes for isoprene reduction. Using comparative proteogenomics and reverse transcription-PCR we have identified a putative five-gene operon encoding an oxidoreductase referred to as putative isoprene reductase.

Biohydrogenation of 1,3-Butadiene to 1-Butene under Acetogenic Conditions by Acetobacterium wieringae.

The environmental fate and transformation mechanism(s) of 1,3-butadiene (BD) under anoxic conditions remain largely unexplored. Anaerobic consortia that can biohydrogenate BD to stoichiometric amounts of 1-butene at a maximum rate of 205.7 ± 38.6 µM day-1 were derived from freshwater river sediment. The formation of 1-butene occurred only in the presence of both H2 and CO2 with concomitant acetate production, suggesting the dependence of BD biohydrogenation on acetogenesis. The 16S rRNA gene-targeted amplicon sequencing revealed the enrichment and dominance of a novel Acetobacterium wieringae population, designated as strain N, in the BD-biohydrogenating community. Multiple genes encoding putative ene-reductases, candidate catalysts for the hydrogenation of the C═C bond in diene compounds, were annotated on the metagenome-assembled genome of strain N, and thus attributed the BD biohydrogenation activity to strain N. Our findings emphasize an essential but overlooked role of certain Acetobacterium members (e.g., strain N) contributing to the natural attenuation of BD in contaminated subsurface environments (e.g., sediment and groundwater). Future efforts to identify and characterize the ene-reductase(s) responsible for BD biohydrogenation in strain N hold promise for the development of industrial biocatalysts capable of stereoselective conversion of BD to 1-butene.

Anaerobic Biohydrogenation of Isoprene by Acetobacterium wieringae Strain Y.

Isoprene is a ubiquitously distributed, biogenic, and climate-active organic compound. Microbial isoprene degradation in oxic environments is fairly well understood; however, studies exploring anaerobic isoprene metabolism remain scarce, with no isolates for study available. Here, we obtained an acetogenic isolate, designated Acetobacterium wieringae strain Y, which hydrogenated isoprene to a mixture of methyl-1-butenes at an overall rate of 288.8 ± 20.9 µM day-1 with concomitant acetate production at a rate of 478.4 ± 5.6 µM day-1. Physiological characterization demonstrated that isoprene was not utilized in a respiratory process; rather, isoprene promoted acetogenesis kinetically. Bioinformatic analysis and proteomics experiments revealed the expression of candidate ene-reductases responsible for isoprene biohydrogenation. Notably, the addition of isoprene to strain Y cultures stimulated the expression of proteins associated with the Wood-Ljungdahl pathway, indicating unresolved impacts of isoprene on carbon cycling and microbial ecology in anoxic environments (e.g., promoting CO2 plus H2 reductive acetogenesis while inhibiting methanogenesis). Our new findings advance understanding of microbial transformation of isoprene under anoxic conditions and suggest that anoxic environments are isoprene sinks. IMPORTANCE Isoprene is the most abundant, biologically generated, volatile organic compound on Earth, with estimated emissions in the same magnitude as methane. Nonetheless, a comprehensive knowledge of isoprene turnover in the environment is lacking, impacting global isoprene flux models and our understanding of the environmental fate and longevity of isoprene. A critical knowledge gap that has remained largely unexplored until recently is the microbiology and associated molecular mechanisms involved in the anaerobic biotransformation of isoprene. By integrating culture-dependent approaches with omics techniques, we isolated an acetogen, Acetobacterium wieringae strain Y, capable of anaerobic biohydrogenation of isoprene. We obtained the complete genome of strain Y, and proteomic experiments identified candidate ene-reductases for catalyzing the asymmetric reduction of the electronically activated carbon-carbon double bond of isoprene. We also demonstrated that isoprene biohydrogenation stimulates the expression of Wood-Ljungdahl pathway enzymes. This study emphasizes the ecological roles of specialized Acetobacterium on the natural cycling of isoprene in anoxic environments and the potential effects of isoprene biohydrogenation on acetogens and methanogens, which have implications for global climate change and bioenergy production.

Biosynthesis of butyrate from methanol and carbon monoxide by recombinant Acetobacterium woodii / Biosíntesis de butirato a partir de metanol y monóxido de carbono por Acetobacterium woodii recombinante

Methanol is one of the most widely produced organic substrates from syngas and can serve as a bio-feedstock to cultivate acetogenic bacteria which allows a major contribution to reducing greenhouse gas. Acetobacterium woodii is one of the very few acetogens that can utilize methanol to produce acetate as sole product. Since A. woodii is genetically tractable, it is an interesting candidate to introduce recombinant pathways for production of bio-commodities from methanol. In this study, we introduced the butyrate production operon from a related acetogen, Eubacterium callanderi KIST612, into A. woodii and show a stable production of butyrate from methanol. This study also reveals how butyrate production by recombinant A. woodii strains can be enhanced with addition of electrons in the form of carbon monoxide. Our results not only show a stable expression system of non-native enzymes in A. woodii but also increase in the product spectrum of A. woodii to compounds with higher economic value.(AU)

A clean in-frame knockout system for gene deletion in Acetobacterium woodii.

Acetogenic bacteria produce acetate following the fixation of CO2 via the Wood-Ljungdahl pathway. As such, they represent excellent process organisms for the production of novel chemicals and fuels from this waste greenhouse gas. Acetobacterium woodii is the model acetogen and numerous studies have been conducted investigating its biochemistry, gas consumption and use as a production chassis. However, there are a dearth of available tools for A. woodii gene modification which limits the research options available for genetic studies. Here, the previously proposed Clostridia Roadmap is implemented in A. woodii leading to the derivation of a knockout system for the generation of clean, in-frame deletions. The replicon of the Gram-positive plasmid pCD6 that originated in Clostridioides difficile was identified as being replication-defective in A. woodii, a property that was exploited to construct a pseudo-suicide knockout plasmid which was used to generate an auxotrophic, pyrE mutant. This allowed the subsequent use of a heterologous pyrE gene (from Clostridium acetobutylicum) as a counter selection marker and the deletion of a number of genes by allelic exchange. Specific mutants generated were affected in growth on glucose, fructose and ethanol as a consequence of deletion of fruA, pstG and adhE, respectively.

One substrate, many fates: different ways of methanol utilization in the acetogen Acetobacterium woodii.

Acetogenic bacteria such as Acetobacterium woodii use the Wood-Ljungdahl pathway (WLP) for fixation of CO2 and energy conservation. This pathway enables conversion of diverse substrates to the main product of acetogenesis, acetate. Methyl group containing substrates such as methanol or methylated compounds, derived from pectin, are abundant in the environment and a source for CO2 . Methyl groups enter the WLP at the level of methyltetrahydrofolic acid (methyl-THF). For methyl transfer from methanol to THF a substrate-specific methyltransferase system is required. In this study, we used genetic methods to identify mtaBC2A (Awo_c22760-Awo_c22740) as the methanol-specific methyltransferase system of A. woodii. After methyl transfer, methyl-THF serves as carbon and/or electron source and the respiratory Rnf complex is required for redox homeostasis if methanol + CO2 is the substrate. Resting cells fed with methanol + CO2 , indeed converted methanol to acetate in a 4:3 stoichiometry. When methanol was fed in combination with other electron sources such as H2  + CO2 or CO, methanol was converted Rnf-independently and the methyl group was condensed with CO to build acetate. When fed in combination with alternative electron sinks such as caffeate methanol was oxidized only and resulting electrons were used for non-acetogenic growth. These different pathways for the conversion of methyl-group containing substrates enable acetogens to adapt to various ecological niches and to syntrophic communities.

Biosynthesis of butyrate from methanol and carbon monoxide by recombinant Acetobacterium woodii.

Methanol is one of the most widely produced organic substrates from syngas and can serve as a bio-feedstock to cultivate acetogenic bacteria which allows a major contribution to reducing greenhouse gas. Acetobacterium woodii is one of the very few acetogens that can utilize methanol to produce acetate as sole product. Since A. woodii is genetically tractable, it is an interesting candidate to introduce recombinant pathways for production of bio-commodities from methanol. In this study, we introduced the butyrate production operon from a related acetogen, Eubacterium callanderi KIST612, into A. woodii and show a stable production of butyrate from methanol. This study also reveals how butyrate production by recombinant A. woodii strains can be enhanced with addition of electrons in the form of carbon monoxide. Our results not only show a stable expression system of non-native enzymes in A. woodii but also increase in the product spectrum of A. woodii to compounds with higher economic value.

Autotrophic lactate production from H2 + CO2 using recombinant and fluorescent FAST-tagged Acetobacterium woodii strains.

Lactate has various uses as industrial platform chemical, poly-lactic acid precursor or feedstock for anaerobic co-cultivations. The aim of this study was to construct and characterise Acetobacterium woodii strains capable of autotrophic lactate production. Therefore, the lctBCD genes, encoding the native Lct dehydrogenase complex, responsible for lactate consumption, were knocked out. Subsequently, a gene encoding a D-lactate dehydrogenase (LDHD) originating from Leuconostoc mesenteroides was expressed in A. woodii, either under the control of the anhydrotetracycline-inducible promoter Ptet or under the lactose-inducible promoter PbgaL. Moreover, LDHD was N-terminally fused to the oxygen-independent fluorescence-activating and absorption-shifting tag (FAST) and expressed in respective A. woodii strains. Cells that produced the LDHD fusion protein were capable of lactate production of up to 18.8 mM in autotrophic batch experiments using H2 + CO2 as energy and carbon source. Furthermore, cells showed a clear and bright fluorescence during exponential growth, as well as in the stationary phase after induction, mediated by the N-terminal FAST. Flow cytometry at the single-cell level revealed phenotypic heterogeneities for cells expressing the FAST-tagged LDHD fusion protein. This study shows that FAST provides a new reporter tool to quickly analyze gene expression over the course of growth experiments of A. woodii. Consequently, fluorescence-based reporters allow for faster and more targeted optimization of production strains.Key points •Autotrophic lactate production was achieved with A. woodii. •FAST functions as fluorescent marker protein in A. woodii. •Fluorescence measurements on single-cell level revealed population heterogeneity.

Engineering Acetobacterium woodii for the production of isopropanol and acetone from carbon dioxide and hydrogen.

The capability of four genetically modified Acetobacterium woodii strains for improved production of acetone from CO2 and hydrogen was tested. The acetone biosynthesis pathway was constructed by combining genes from Clostridium acetobutylicum and Clostridium aceticum. Expression of acetone production genes was demonstrated in all strains. In bioreactors with continuous gas supply, all produced acetic acid, acetone, and, surprisingly, isopropanol. The production of isopropanol was caused by an endogenous secondary alcohol dehydrogenase (SADH) activity at low gas-feeding rate. Although high amounts of the natural end product acetic acid of A. woodii were formed,14.5 mM isopropanol and 7.6 mM acetone were also detected, showing that this is a promising approach for the production of new solvents from C1 gases. The highest acetic acid, acetone, and isopropanol production was detected in the recombinant A. woodii [pJIR750_ac1t1] strain, with final concentrations of 438 mM acetic acid, 7.6 mM acetone, and 14.5 mM isopropanol. The engineered strain A. woodii [pJIR750_ac1t1] was found to be the most promising strain for acetone production from a gas mixture of CO2 and H2 and the formation of isopropanol in A. woodii was shown for the first time.

Comparison of Carbonic Anhydrases for CO2 Sequestration.

Strategies for depleting carbon dioxide (CO2) from flue gases are urgently needed and carbonic anhydrases (CAs) can contribute to solving this problem. They catalyze the hydration of CO2 in aqueous solutions and therefore capture the CO2. However, the harsh conditions due to varying process temperatures are limiting factors for the application of enzymes. The current study aims to examine four recombinantly produced CAs from different organisms, namely CAs from Acetobacterium woodii (AwCA or CynT), Persephonella marina (PmCA), Methanobacterium thermoautotrophicum (MtaCA or Cab) and Sulphurihydrogenibium yellowstonense (SspCA). The highest expression yields and activities were found for AwCA (1814 WAU mg-1 AwCA) and PmCA (1748 WAU mg-1 PmCA). AwCA was highly stable in a mesophilic temperature range, whereas PmCA proved to be exceptionally thermostable. Our results indicate the potential to utilize CAs from anaerobic microorganisms to develop CO2 sequestration applications.

Knowing the enemy: homoacetogens in hydrogen production reactors.

One of the bottlenecks of the hydrogen production by dark fermentation is the low yields obtained because of the homoacetogenesis persistence, a metabolic pathway where H2 and CO2 are consumed to produce acetate. The central reactions of H2 production and homoacetogenesis are catalyzed by enzyme hydrogenase and the formyltetrahydrofolate synthetase, respectively. In this work, genes encoding for the formyltetrahydrofolate synthetase (fthfs) and hydrogenase (hydA) were used to investigate the diversity of homoacetogens as well as their phylogenetic relationships through quantitative PCR (qPCR) and next-generation amplicon sequencing. A total of 70 samples from 19 different H2-producing bioreactors with different configurations and operating conditions were analyzed. Quantification through qPCR showed that the abundance of fthfs and hydA was strongly associated with the type of substrate, organic loading rate, and H2 production performance. In particular, fthfs sequencing revealed that homoacetogens diversity was low with one or two dominant homoacetogens in each sample. Clostridium carboxivorans was detected in the reactors fed with agave hydrolisates; Acetobacterium woodii dominated in systems fed with glucose; Blautia coccoides and unclassified Sporoanaerobacter species were present in reactors fed with cheese whey; finally, Eubacterium limosum and Selenomonas sp. were co-dominant in reactors fed with glycerol. Altogether, quantification and sequencing analysis revealed that the occurrence of homoacetogenesis could take place due to (1) metabolic changes of H2-producing bacteria towards homoacetogenesis or (2) the displacement of H2-producing bacteria by homoacetogens. Overall, it was demonstrated that the fthfs gene was a suitable marker to investigate homoacetogens in H2-producing reactors. KEY POINTS: • qPCR and sequencing analysis revealed two homoacetogenesis phenomena. • fthfs gene was a suitable marker to investigate homoacetogens in H2 reactors.

A quantitative metabolic analysis reveals Acetobacterium woodii as a flexible and robust host for formate-based bioproduction.

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.

Physiology and genetics of ethanologenesis in the acetogenic bacterium Acetobacterium woodii.

The acetogenic model bacterium Acetobacterium woodii is well-known to produce acetate by homoacetogenesis from sugars, but under certain conditions minor amounts of ethanol are produced in addition. Here, we have aimed to identify physiological conditions that increase electron and carbon flow towards ethanol production. Ethanol was only produced from fructose but not from H2  + CO2 , formate, pyruvate, lactate or alanine. In the absence of Na+ , the Wood-Ljungdahl pathway (WLP) of acetate formation is not functional. Therefore, the ethanol yield increased to 0.42 mol/mol (ethanol/fructose) with an ethanol/acetate ratio of 0.28 mol/mol. The presence of bicarbonate/CO2 stimulated electron and carbon flow through the WLP and led to less ethanol produced. Of the 11 potential alcohol dehydrogenase genes, the most upregulated during ethanologenesis was adh4. A deletion of adh4 led to an increase in ethanol production by 100% to a yield of 0.79 mol/mol (ethanol/fructose); this correlated with an increase in transcript abundance of adh6. In sum, our studies revealed low Na+ and bicarbonate/CO2 as factors that trigger ethanol formation and that a deletion of adh4 drastically increased ethanol formation in A. woodii.

Capture of carbon dioxide and hydrogen by engineered Escherichia coli: hydrogen-dependent CO2 reduction to formate.

In times of global climate change and the fear of dwindling resources, we are facing different considerable challenges such as the replacement of fossil fuel-based energy carriers with the coincident maintenance of the increasing energy supply of our growing world population. Therefore, CO2 capturing and H2 storing solutions are urgently needed. In this study, we demonstrate the production of a functional and biotechnological interesting enzyme complex from acetogenic bacteria, the hydrogen-dependent CO2 reductase (HDCR), in the well-known model organism Escherichia coli. We identified the metabolic bottlenecks of the host organisms for the production of the HDCR enzyme complex. Here we show that the recombinant expression of a heterologous enzyme complex transforms E. coli into a whole-cell biocatalyst for hydrogen-driven CO2 reduction to formate without the need of any external co-factors or endogenous enzymes in the reaction process. This shifts the industrial platform organism E. coli more and more into the focus as biocatalyst for CO2-capturing and H2-storage. KEY POINTS: • A functional HDCR enzyme complex was heterologously produced in E. coli. • The metabolic bottlenecks for HDCR production were identified. • HDCR enabled E. coli cell to capture and store H2 and CO2 in the form of formate.

Formate metabolism in the acetogenic bacterium Acetobacterium woodii.

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.

Propionate Production from Carbon Monoxide by Synthetic Cocultures of Acetobacterium wieringae and Propionigenic Bacteria.

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 (∼24 mM) being measured in the coculture composed of Ac. wieringae strain JM and An. neopropionicum, which also produced isovalerate (∼4 mM), butyrate (∼1 mM), and isobutyrate (0.3 mM). 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. IMPORTANCE Syngas fermentation has great potential for the sustainable production of chemicals from wastes (via prior gasification) and flue gases containing CO/CO2. Research efforts need to be directed toward expanding the product portfolio of gas fermentation, which is currently limited to mainly acetate and ethanol. This study provides the basis for a microbial process to produce propionate from CO using synthetic cocultures composed of acetogenic and propionigenic bacteria and elucidates the metabolic pathways involved. Furthermore, based on proteomics results, we hypothesize that the two bacterial species engage in an interaction that results in amino acid exchange, which subsequently promotes isovalerate and isobutyrate production. These findings provide a new understanding of gas fermentation and a coculturing strategy for expanding the product spectrum of microbial conversion of CO/CO2.

Growth of the acetogenic bacterium Acetobacterium woodii on glycerol and dihydroxyacetone.

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.

Investigation of putative genes for the production of medium-chained acids and alcohols in autotrophic acetogenic bacteria.

Gas fermentation is a technology for producing platform chemicals as well as fuels and one of the most promising alternatives to petrochemicals. Medium-chained acids and alcohols such as hexanoate and hexanol are particularly interesting due to their versatile application. This study elucidated the pathway of chain elongation in native C6 compound-producing acetogens. Essential genes of Clostridium carboxidivorans for synthesis of medium-chained acids and alcohols were identified in order to demonstrate their catalytic activity in the acetogenic model organism Acetobacterium woodii. Two such gene clusters were identified, which are responsible for conversion of acetyl-CoA to butyryl-CoA by reverse ß-oxidation. Using RT-PCR it could be demonstrated that only genes of cluster 1 are expressed constitutively with simultaneous formation of C6 compounds. Based on genes from C. carboxidivorans, a modular hexanoyl-CoA synthesis (hcs) plasmid system was constructed and transferred into A. woodii. With the recombinant A. woodii strains AWO [pPta_hcs1], AWO [pPta_hcs2], AWO [pTet_hcs1], and AWO [pTet_hcs2] butyrate and hexanoate production under heterotrophic (1.22-4.15 mM hexanoate) and autotrophic conditions (0.48-1.56 mM hexanoate) with both hcs clusters could be detected. hcs Cluster 1 from C. carboxidivorans was transferred into the ABE-fermenting strain Clostridium saccharoperbutylacetonicum as well. For further analysis, genes were also cloned into the hcs plasmid system individually. The resulting recombinant C. saccharoperbutylacetonicum strains with just individual genes neither produced hexanoate nor hexanol, but the strains containing the entire gene cluster were capable of chain elongation. A production of 0.8 mM hexanoate and 5.2 mM hexanol in the fermentation with glucose could be observed.