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Abstract Background The genus Eubacterium is quite diverse and includes several acetogenic strains capable of fermenting C1-substrates into valuable products. Especially, Eubacterium limosum and closely related strains attract attention not only for their capability to ferment C1 gases and liquids, but also due to their ability to produce butyrate. Apart from its well-elucidated metabolism, E. limosum is also genetically accessible, which makes it an interesting candidate to be an industrial biocatalyst. Results In this study, we examined genomic, phylogenetic, and physiologic features of E. limosum and the closest related species E. callanderi as well as E. maltosivorans. We sequenced the genomes of the six Eubacterium strains ‘FD’ (DSM 3662T), ‘Marburg’ (DSM 3468), ‘2A’ (DSM 2593), ‘11A’ (DSM 2594), ‘G14’ (DSM 107592), and ‘32’ (DSM 20517) and subsequently compared these with previously available genomes of the E. limosum type strain (DSM 20543T) as well as the strains ‘B2’, ‘KIST612’, ‘YI’ (DSM 105863T), and ‘SA11’. This comparison revealed a close relationship between all eleven Eubacterium strains, forming three distinct clades: E. limosum, E. callanderi, and E. maltosivorans. Moreover, we identified the gene clusters responsible for methanol utilization as well as genes mediating chain elongation in all analyzed strains. Subsequent growth experiments revealed that strains of all three clades can convert methanol and produce acetate, butyrate, and hexanoate via reverse β-oxidation. Additionally, we used a harmonized electroporation protocol and successfully transformed eight of these Eubacterium strains to enable recombinant plasmid-based expression of the gene encoding the fluorescence-activating and absorption shifting tag (FAST). Engineered Eubacterium strains were verified regarding their FAST-mediated fluorescence at a single-cell level using a flow cytometry approach. Eventually, strains ‘FD’ (DSM 3662T), ‘2A’ (DSM 2593), ‘11A’ (DSM 2594), and ‘32’ (DSM 20517) were genetically engineered for the first time. Conclusion Strains of E. limosum, E. callanderi, and E. maltosivorans are outstanding candidates as biocatalysts for anaerobic C1-substrate conversion into valuable biocommodities. A large variety of strains is genetically accessible using a harmonized electroporation protocol, and FAST can serve as a reliable fluorescent reporter protein to characterize genetically engineered cells. In total eleven strains have been assigned to distinct clades, providing a clear and updated classification. Thus, the description of respective Eubacterium species has been emended, improved, aligned, and is requested to be implemented in respective databases.

Electrocatalytic CO2 reduction to CO and formate can be coupled to gas fermentation with anaerobic microorganisms. In combination with a competing hydrogen evolution reaction in the cathode in aqueous medium, the in situ, electrocatalytic produced syngas components can be converted by an acetogenic bacterium, such as Clostridium ragsdalei, into acetate, ethanol, and 2,3-butanediol. In order to study the simultaneous conversion of CO, CO2, and formate together with H2 with C. ragsdalei, fed-batch processes were conducted with continuous gassing using a fully controlled stirred tank bioreactor. Formate was added continuously, and various initial CO partial pressures (pCO0) were applied. C. ragsdalei utilized CO as the favored substrate for growth and product formation, but below a partial pressure of 30 mbar CO in the bioreactor, a simultaneous CO2/H2 conversion was observed. Formate supplementation enabled 20–50% higher growth rates independent of the partial pressure of CO and improved the acetate and 2,3-butanediol production. Finally, the reaction conditions were identified, allowing the parallel CO, CO2, formate, and H2 consumption with C. ragsdalei at a limiting CO partial pressure below 30 mbar, pH 5.5, n = 1200 min−1, and T = 32 °C. Thus, improved carbon and electron conversion is possible to establish efficient and sustainable processes with acetogenic bacteria, as shown in the example of C. ragsdalei.

The challenge of limiting global warming to below 1.5°C requires all industries to implement new technologies and change practices immediately. The aviation industry contributes 2% of human-induced CO2 emissions and 12% of all transport emissions. Decarbonising the aviation industry, which relies heavily on high-density liquid fuels, has been difficult to achieve. The problems are compounded by the continued reliance on so-called sustainable aviation fuels, which use first-generation agricultural feedstocks, creating a trade-off between biomass for food and feed and its use as a feedstock for energy generation. Decarbonising aviation is also challenging because of problems in developing electric aircraft. Alternative feedstocks already exist that provide a more feasible path towards decelerating climate change. One such alternative is to use gas fermentation to convert greenhouse gases (e.g. from food production and food waste) into fuels using microbial acetogens. Acetogens are anaerobic microorganisms capable of producing alcohols from gaseous CO, CO2 and H2. Australia offers feedstock resources for gas fermentation with abundant H2 and CO2 production in proximity to each other. In this review, we put forward the principles, approaches and opportunities offered by gas fermentation technologies to replace our dependency on fossil fuels for aviation fuel production in Australia.

Thermophily is an ancient trait among microorganisms. The molecular principles to sustain high temperatures, however, are often described as adaptations, somewhat implying that they evolved from a non-thermophilic background and that thermophiles, i.e., organisms with growth temperature optima (TOPT) above 45°C, evolved from mesophilic organisms (TOPT 25–45°C). On the contrary, it has also been argued that LUCA, the last universal common ancestor of Bacteria and Archaea, may have been a thermophile, and mesophily is the derived trait. In this study, we took an experimental approach toward the evolution of a mesophile from a thermophile. We selected the acetogenic bacterium T. kivui (TOPT 66°C) since acetogenesis is considered ancient physiology and cultivated it at suboptimal low temperatures. We found that the lowest possible growth temperature (TMIN) under the chosen conditions was 39°C. The bacterium was subsequently subjected to adaptive laboratory evolution (ALE) by serial transfer at 45°C. Interestingly, after 67 transfers (approximately 180 generations), the adapted strain Adpt45_67 did not grow better at 45°C, but a shift in the TOPT to 60°C was observed. Growth at 45°C was accompanied by a change in the morphology as shorter, thicker cells were observed that partially occurred in chains. While the proportion of short-chain fatty acids increased at 50°C vs. 66°C in both strains, Adpt45_67 also showed a significantly increased proportion of plasmalogens. The genome analysis revealed 67 SNPs compared to the type strain, among these mutations in transcriptional regulators and in the cAMP binding protein. Ultimately, the molecular basis of the adaptation of T. kivui to a lower TOPT remains to be elucidated. The observed change in phenotype is the first experimental step toward the evolution of thermophiles growing at colder temperatures and toward a better understanding of the cold adaptation of thermophiles on early Earth.

Abstract Background Ethyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO2/H2, is desirable. Acetogens present a promising microbial platform for the production of ethyl esters from these one-carbon substrates. Results We engineered the acetogen C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT), which catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 from Kluyveromyces marxianus and Atf1 from Saccharomyces cerevisiae, were expressed in C. autoethanogenum. Strains expressing Atf1 produced up to 0.2 mM ethyl acetate. Ethyl acetate production was barely detectable (

Abstract Background The replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging. Results Using a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L−1 butanol and 133 mg L−1 hexanol from fructose in complex medium, and 174 mg L−1 butanol and 15 mg L−1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L−1) at the expense of butanol (158 mg L−1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L−1 butanol and 393 mg L−1 hexanol. Conclusions We thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes.

ABSTRACT Hydrogenotrophic microbes, primarily including the three functional groups methanogens, sulfate-reducing bacteria, and reductive acetogens, use hydrogen as an energy source and play an important role in maintaining the hydrogen balance in gut ecosystems. A distorted hydrogen balance has been associated with irritable bowel syndrome (IBS). However, the role of hydrogenotrophic microbes in overall microbiota composition and function remains largely unknown. This study aims to assess the distribution and stability of hydrogenotrophic functional groups in healthy adults (HAs) and IBS patients and their association with overall microbiota composition and IBS symptoms. A two-time-point study with 4 weeks in between was performed with 27 HAs and 55 IBS patients included. Our observations revealed that methanogens showed a bimodal distribution across samples. A high-level methanogen microbiota was consistently associated with higher alpha diversity, and its composition was significantly different from that of individuals with a low-level methanogen microbiota. In general, these associations were more pronounced in IBS patients than in HAs. The differences in the copy numbers of genes indicative of total bacteria and acetogens between HAs and IBS patients and their correlations with IBS symptom severity, anxiety, depression, and quality of life (QoL) were sampling time dependent. Hydrogenotrophic functional groups did not show negative abundance correlations with each other in HAs and IBS patients. These findings suggest that methanogen levels in the gut have a pronounced association with microbiota alpha diversity and composition, and the interactions between hydrogenotrophic functional groups are complex in gut ecosystems. IMPORTANCE Hydrogenotrophic microbes play an essential role in the disposal of hydrogen and the maintenance of the hydrogen balance in gut ecosystems. Their abundances vary between individuals and have been reported to be associated with human gut disorders such as irritable bowel disease. This study confirms that methanogen levels show a bimodal distribution. Moreover, a high-level methanogen microbiota was associated with higher alpha diversity, and its composition was different from that of individuals with a low-level methanogen microbiota. These associations are more pronounced in IBS patients than in healthy subjects. In addition, associations between hydrogenotrophic microbes and IBS symptom scores vary over time, which argues for the use of longitudinal study designs. Last but not least, this study suggests that the different hydrogenotrophic microbes coexist with each other and do not necessarily compete for hydrogen in the gut. The findings in this study highlight the impact of methanogens on overall microbiota composition and function.

The depletion of fossil fuel resources and the CO2 emissions coupled with petroleum-based industrial processes present a relevant issue for the whole of society. An alternative to the fossil-based production of chemicals is microbial fermentation using acetogens. Acetogenic bacteria are able to metabolize CO or CO2 (+H2) via the Wood–Ljungdahl pathway. As isopropanol is widely used in a variety of industrial branches, it is advantageous to find a fossil-independent production process. In this study, Acetobacterium woodii was employed to produce isopropanol via plasmid-based expression of the enzymes thiolase A, CoA-transferase, acetoacetate decarboxylase and secondary alcohol dehydrogenase. An examination of the enzymes originating from different organisms led to a maximum isopropanol production of 5.64 ± 1.08 mM using CO2 + H2 as the carbon and energy source. To this end, the genes thlA (encoding thiolase A) and ctfA/ctfB (encoding CoA-transferase) of Clostridium scatologenes, adc (encoding acetoacetate decarboxylase) originating from C. acetobutylicum and sadH (encoding secondary alcohol dehydrogenase) of C. beijerinckii DSM 6423 were employed. Since bottlenecks in the isopropanol production pathway are known, optimization of the strain was investigated, resulting in a 2.5-fold increase in isopropanol concentration.

Increasing greenhouse gas emissions and decreasing fossil fuel supplies necessitate the development of alternative methods for producing petroleum-based commodities. Plastics are also primarily petroleum-based goods with rising demand, thus there is growing interest in plastic substitutes. Polyhydroxyalkanoates (PHAs) are naturally produced biopolymers that are utilized by microorganisms as a source of energy and carbon storage. Poly-3-hydroxybutyrate (PHB) is a member of the PHA family and is considered the most promising candidate to replace polyethylene (PE). PHB is naturally produced by Cupriavidus necator, but recombinant production has also been recently established. This study is the first to investigate the heterologous production of PHB with recombinant Acetobacterium woodii using CO2 + H2 as a carbon and energy source. The introduction of a synthetic PHB production pathway resulted in the production of 1.23 g/L CDW and 1.9% PHB/cell dry weight (CDW), which corresponds to a production of 23.5 mg/L PHB. PHB quantification was simplified using LipidGreen2 fluorescence measurements.

Interspecific competition for substrate and space gives rise to considerable variation in biomass distribution within the microbial community. To study microbial community in depth, we used several research methods as sampling and analytical measurements, and developed a cellular automata (CA) model that would facilitate a description of the microbial growth process based on Anaerobic Digestion Model No. 1 (ADM1) of the International Water Association (IWA). Using the CA model, we aimed to determine whether interspecific competition occurs among acidogens, acetogens and methanogens, and to examine the influence of interspecific competition on the spatial structure of microbial communities. We found that acetogens and methanogens competed for core space, resulting in a multi-layer structure. Butyrate-degrading acetogens increased in number, resulting in inhibition of propionate-degrading acetogens. Hydrogenotrophic methanogens showed stronger competitive advantage than acetotrophic methanogens. The simulation showed that the multi-layer structure of the microbial community was formed by interspecific competition. HIGHLIGHTS A two-dimensional model is developed based on cellular automata and ADM1.; New biomass-spreading rules are introduced to cellular automata.; The integrated model is evaluated by simulation of ABR start-up.; The new model can be used to simulate interspecific competition.;

Abstract Background The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited. Results In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth. Conclusions The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

Biotransformation of lignocellulose-derived synthetic gas (syngas) into acetic acid is a promising way of creating biochemicals from lignocellulosic waste materials. Acetic acid has a growing market with applications within food, plastics and for upgrading into a wide range of biofuels and bio-products. In this paper, we will review the microbial conversion of syngas to acetic acid. This will include the presentation of acetate-producing bacterial strains and their optimal fermentation conditions, such as pH, temperature, media composition, and syngas composition, to enhance acetate production. The influence of syngas impurities generated from lignocellulose gasification will further be covered along with the means to alleviate impurity problems through gas purification. The problem with mass transfer limitation of gaseous fermentation will further be discussed as well as ways to improve gas uptake during the fermentation.

Abstract Background Gas-fermenting acetogens have received a great deal of attention for their ability to grow on various syngas and waste gas containing carbon monoxide (CO), producing acetate as the primary metabolite. Among them, some Clostridium species, such as C. ljungdahlii and C. autoethanogenum, are of particular interest as they produce fuel alcohols as well. Despite recent efforts, alcohol production by these species is still unsatisfactory due to their low productivity and acetate accumulation, necessitating the isolation of strains with better phenotypes. Results In this study, a novel alcohol-producing acetogen (Clostridium sp. AWRP) was isolated, and its complete genome was sequenced. This bacterium belongs the same phylogenetic group as C. ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii based on 16S rRNA homology; however, the levels of genome-wide average nucleotide identity (gANI) for strain AWRP compared with these strains range between 95 and 96%, suggesting that this strain can be classified as a novel species. In addition, strain AWRP produced a substantial amount of ethanol (70–90 mM) from syngas in batch serum bottle cultures, which was comparable to or even exceeded the typical values obtained using its close relatives cultivated under similar conditions. In a batch bioreactor, strain AWRP produced 119 and 12 mM of ethanol and 2,3-butanediol, respectively, while yielding only 1.4 mM of residual acetate. Interestingly, the alcohologenesis of this strain was strongly affected by oxidoreduction potential (ORP), which has not been reported with other gas-fermenting clostridia. Conclusion Considering its ethanol production under low oxidoreduction potential (ORP) conditions, Clostridium sp. AWRP will be an interesting host for biochemical studies to understand the physiology of alcohol-producing acetogens, which will contribute to metabolic engineering of those strains for the production of alcohols and other value-added compounds from syngas.

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.

Diverse physiological groups congregate into environmental corrosive biofilms, yet the interspecies interactions between these corrosive physiological groups are seldom examined. We, therefore, explored Fe0-dependent cross-group interactions between acetogens and methanogens from lake sediments. On Fe0, acetogens were more corrosive and metabolically active when decoupled from methanogens, whereas methanogens were more metabolically active when coupled with acetogens. This suggests an opportunistic (win–loss) interaction on Fe0 between acetogens (loss) and methanogens (win). Clostridia and Methanobacterium were the major candidates doing acetogenesis and methanogenesis after four transfers (metagenome sequencing) and the only groups detected after 11 transfers (amplicon sequencing) on Fe0. Since abiotic H2 failed to explain the high metabolic rates on Fe0, we examined whether cell exudates (spent media filtrate) promoted the H2-evolving reaction on Fe0 above abiotic controls. Undeniably, spent media filtrate generated three- to four-fold more H2 than abiotic controls, which could be partly explained by thermolabile enzymes and partly by non-thermolabile constituents released by cells. Next, we examined the metagenome for candidate enzymes/shuttles that could catalyze H2 evolution from Fe0 and found candidate H2-evolving hydrogenases and an almost complete pathway for flavin biosynthesis in Clostridium. Clostridial ferredoxin-dependent [FeFe]-hydrogenases may be catalyzing the H2-evolving reaction on Fe0, explaining the significant H2 evolved by spent media exposed to Fe0. It is typical of Clostridia to secrete enzymes and other small molecules for lytic purposes. Here, they may secrete such molecules to enhance their own electron uptake from extracellular electron donors but indirectly make their H2-consuming neighbors—Methanobacterium—fare five times better in their presence. The particular enzymes and constituents promoting H2 evolution from Fe0 remain to be determined. However, we postulate that in a static environment like corrosive crust biofilms in lake sediments, less corrosive methanogens like Methanobacterium could extend corrosion long after acetogenesis ceased, by exploiting the constituents secreted by acetogens.

Two different isobutanol synthesis pathways were cloned into and expressed in the two model acetogenic bacteria Acetobacterium woodii and Clostridium ljungdahlii. A. woodii is specialized on using CO2 + H2 gas mixtures for growth and depends on sodium ions for ATP generation by a respective ATPase and Rnf system. On the other hand, C. ljungdahlii grows well on syngas (CO + H2 + CO2 mixture) and depends on protons for energy conservation. The first pathway consisted of ketoisovalerate ferredoxin oxidoreductase (Kor) from Clostridium thermocellum and bifunctional aldehyde/alcohol dehydrogenase (AdhE2) from C. acetobutylicum. Three different kor gene clusters are annotated in C. thermocellum and were all tested. Only in recombinant A. woodii strains, traces of isobutanol could be detected. Additional feeding of ketoisovalerate increased isobutanol production to 2.9 mM under heterotrophic conditions using kor3 and to 1.8 mM under autotrophic conditions using kor2. In C. ljungdahlii, isobutanol could only be detected upon additional ketoisovalerate feeding under autotrophic conditions. kor3 proved to be the best suited gene cluster. The second pathway consisted of ketoisovalerate decarboxylase from Lactococcus lactis and alcohol dehydrogenase from Corynebacterium glutamicum. For increasing the carbon flux to ketoisovalerate, genes encoding ketol-acid reductoisomerase, dihydroxy-acid dehydratase, and acetolactate synthase from C. ljungdahlii were subcloned downstream of adhA. Under heterotrophic conditions, A. woodii produced 0.2 mM isobutanol and 0.4 mM upon additional ketoisovalerate feeding. Under autotrophic conditions, no isobutanol formation could be detected. Only upon additional ketoisovalerate feeding, recombinant A. woodii produced 1.5 mM isobutanol. With C. ljungdahlii, no isobutanol was formed under heterotrophic conditions and only 0.1 mM under autotrophic conditions. Additional feeding of ketoisovalerate increased these values to 1.5 mM and 0.6 mM, respectively. A further increase to 2.4 mM and 1 mM, respectively, could be achieved upon inactivation of the ilvE gene in the recombinant C. ljungdahlii strain. Engineering the coenzyme specificity of IlvC of C. ljungdahlii from NADPH to NADH did not result in improved isobutanol production.

Artificially stimulated, high-yield microbial production of methane from coal is a challenging problem that continues to generate research interest. Decomposition of organic matter and production of methane from coal are the results of multiple redox reactions carried out by different communities of bacteria and archaea. Recent work by our group (Beckmann et al., 2015) demonstrated that the presence of the redox-mediating molecule neutral red, in its crystalline form on a coal surface, can increase methane production. However, hydrolysis and the acetogenesis of the coal surface are essential precursor steps for methane production by archaea. Acetogenesis is the preparation phase of methanogenesis because methanogens can only assimilate acetate, CO2 and H2 among the products formed during this process. In the present study, the surface chemical analysis of neutral red treated coal using attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS) demonstrate that the acetate production and resulting oxidation of the coal only occurred at few nanometers into the coal surface (at the nanoscale

The strictly anaerobic acetogenic bacterium Acetobacterium woodii is metabolically diverse and grows on variety of substrates which includes H2 + CO2, sugars, alcohols and diols. It is unique in producing bacterial microcompartments (BMC) during growth on different substrates such as 1,2-propanediol, 2,3-butanediol, ethanol or fructose. In this study, we analyzed the genetic organization and expression of the BMC genes within the A. woodii genome, the previously described 18 gene pdu cluster as well as four other cluster potentially encoding one or two shell proteins. Expression analysis of respective gene clusters revealed that the pdu gene cluster is highly expressed during growth on 1,2-PD, 2,3-BD, ethanol and ethylene glycol. The promoter region upstream of the pduA gene was identified and used to establish a reporter gene assay based on chloramphenicol acetyl transferase as a reporter protein. The reporter gene assay confirmed the qPCR data and demonstrated that 1,2-PD is superior over ethanol and ethylene glycol as inducer. BMCs were enriched from cells grown on 2,3- BD and 1,2-PD and shown to have typical structure in electron micrographs. Biochemical analyses revealed several of the protein encoded by the pdu cluster to be part of the isolated BMCs. These data demonstrate a very unique situation in A. woodii in which apparently one BMC gene cluster in expressed during growth on different substrates.

ABSTRACT Acetogens are anaerobic bacteria capable of fixing CO2 or CO to produce acetyl coenzyme A (acetyl-CoA) and ultimately acetate using the Wood-Ljungdahl pathway (WLP). Acetobacterium woodii is the type strain of the Acetobacterium genus and has been critical for understanding the biochemistry and energy conservation in acetogens. Members of the Acetobacterium genus have been isolated from a variety of environments or have had genomes recovered from metagenome data, but no systematic investigation has been done on the unique and various metabolisms of the genus. To gain a better appreciation for the metabolic breadth of the genus, we sequenced the genomes of 4 isolates (A. fimetarium, A. malicum, A. paludosum, and A. tundrae) and conducted a comparative genome analysis (pan-genome) of 11 different Acetobacterium genomes. A unifying feature of the Acetobacterium genus is the carbon-fixing WLP. The methyl (cluster II) and carbonyl (cluster III) branches of the Wood-Ljungdahl pathway are highly conserved across all sequenced Acetobacterium genomes, but cluster I encoding the formate dehydrogenase is not. In contrast to A. woodii, all but four strains encode two distinct Rnf clusters, Rnf being the primary respiratory enzyme complex. Metabolism of fructose, lactate, and H2:CO2 was conserved across the genus, but metabolism of ethanol, methanol, caffeate, and 2,3-butanediol varied. Additionally, clade-specific metabolic potential was observed, such as amino acid transport and metabolism in the psychrophilic species, and biofilm formation in the A. wieringae clade, which may afford these groups an advantage in low-temperature growth or attachment to solid surfaces, respectively. IMPORTANCE Acetogens are anaerobic bacteria capable of fixing CO2 or CO to produce acetyl-CoA and ultimately acetate using the Wood-Ljungdahl pathway (WLP). This autotrophic metabolism plays a major role in the global carbon cycle and, if harnessed, can help reduce greenhouse gas emissions. Overall, the data presented here provide a framework for examining the ecology and evolution of the Acetobacterium genus and highlight the potential of these species as a source for production of fuels and chemicals from CO2 feedstocks.

The formyltetrahydrofolate synthetase (FTHFS) gene is a molecular marker of choice to study the diversity of acetogenic communities. However, current analyses are limited due to lack of a high-throughput sequencing approach for FTHFS gene amplicons and a dedicated bioinformatics pipeline for data analysis, including taxonomic annotation and visualization of the sequence data. In the present study, we combined the barcode approach for multiplexed sequencing with unsupervised data analysis to visualize acetogenic community structure. We used samples from a biogas digester to develop proof-of-principle for our combined approach. We successfully generated high-throughput sequence data for the partial FTHFS gene and performed unsupervised data analysis using the novel bioinformatics pipeline “AcetoScan” presented in this study, which resulted in taxonomically annotated OTUs, phylogenetic tree, abundance plots and diversity indices. The results demonstrated that high-throughput sequencing can be used to sequence the FTHFS amplicons from a pool of samples, while the analysis pipeline AcetoScan can be reliably used to process the raw sequence data and visualize acetogenic community structure. The method and analysis pipeline described in this paper can assist in the identification and quantification of known or potentially new acetogens. The AcetoScan pipeline is freely available at https://github.com/abhijeetsingh1704/AcetoScan.

Acetogens are naturally capable of metabolizing carbon monoxide (CO), a component of synthesis gas (syngas), for autotrophic growth in order to produce biomass and metabolites such as acetyl-CoA via the Wood–Ljungdahl pathway. However, the autotrophic growth of acetogens is often inhibited by the presence of high CO concentrations because of CO toxicity, thus limiting their biosynthetic potential for industrial applications. Herein, we implemented adaptive laboratory evolution (ALE) for growth improvement of Eubacterium limosum ATCC 8486 under high CO conditions. The strain evolved under syngas conditions with 44% CO over 150 generations, resulting in a significant increased optical density (600 nm) and growth rate by 2.14 and 1.44 folds, respectively. In addition, the evolved populations were capable of proliferating under CO concentrations as high as 80%. These results suggest that cell growth is enhanced as beneficial mutations are selected and accumulated, and the metabolism is altered to facilitate the enhanced phenotype. To identify the causal mutations related to growth improvement under high CO concentrations, we performed whole genome resequencing of each population at 50-generation intervals. Interestingly, we found key mutations in CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex coding genes, acsA and cooC. To characterize the mutational effects on growth under CO, we isolated single clones and confirmed that the growth rate and CO tolerance level of the single clone were comparable to those of the evolved populations and wild type strain under CO conditions. Furthermore, the evolved strain produced 1.34 folds target metabolite acetoin when compared to the parental strain while introducing the biosynthetic pathway coding genes to the strains. Consequently, this study demonstrates that the mutations in the CODH/ACS complex affect autotrophic growth enhancement in the presence of CO as well as the CO tolerance of E. limosum ATCC 8486.

The present human population is more than three times what it was in 1950. With that, there is an increasing demand for the consumption of fossil fuels for various anthropogenic activities. This consumption is the major source of carbon dioxide emission causing greenhouse effects leading to global warming. The dependency on fossil fuels around the globe is such that it would be hard to move away from it any time soon. Hence, we must work on strategies to improve carbon dioxide fixation as we are making advancements in clean energy technology. This review explores the natural carbon dioxide fixation pathways in plants and various microorganisms and discusses their limitations and alternative strategies. It explains what necessitates the exploration of synthetic pathways and discusses strategies and matrices to consider while evaluating various pathways. This review also discusses the recent breakthroughs in the field of nanosciences that could accelerate chemical methods of carbon dioxide fixation.

Homo-acetogens are microbes that have the ability to grow on gaseous substrates such as H2/CO2/CO and produce acetic acid as the main product of their metabolism through a metabolic process called reductive acetogenesis. These acetogens are dispersed in nature and are found to grow in various biotopes on land, water and sediments. They are also commonly found in the gastro-intestinal track of herbivores that rely on a symbiotic relationship with microbes in order to breakdown lignocellulosic biomass to provide the animal with nutrients and energy. For this motive, the fermentation scheme that occurs in the rumen has been described equivalent to a consolidated bioprocessing fermentation for the production of bioproducts derived from livestock. This paper reviews current knowledge of homo-acetogenesis and its potential to improve efficiency in the rumen for production of bioproducts by replacing methanogens, the principal H2-scavengers in the rumen, thus serving as a form of carbon sink by deviating the formation of methane into bioproducts. In this review, we discuss the main strategies employed by the livestock industry to achieve methanogenesis inhibition, and also explore homo-acetogenic microorganisms and evaluate the members for potential traits and characteristics that may favor competitive advantage over methanogenesis, making them prospective candidates for competing with methanogens in ruminant animals.

Syngas is a substrate for the anaerobic bioproduction of fuels and valuable chemicals. In this study, anaerobic sludge was used for microbial enrichments with synthetic syngas and acetate as main substrates. The objectives of this study were to identify microbial networks (in enrichment cultures) for the conversion of syngas to added-value products, and to isolate robust, non-fastidious carboxydotrophs. Enrichment cultures produced methane and propionate, this last one an unusual product from syngas fermentation. A bacterium closely related to Acetobacterium wieringae was identified as most prevalent (87% relative abundance) in the enrichments. Methanospirillum sp. and propionate-producing bacteria clustering within the genera Anaerotignum and Pelobacter were also found. Further on, strain JM, was isolated and was found to be 99% identical (16S rRNA gene) to A. wieringae DSM 1911T. Digital DNA-DNA hybridization (dDDH) value between the genomes of strain JM and A. wieringae was 77.1%, indicating that strain JM is a new strain of A. wieringae. Strain JM can grow on carbon monoxide (100% CO, total pressure 170 kPa) without yeast extract or formate, producing mainly acetate. Remarkably, conversion of CO by strain JM showed shorter lag phase than in cultures of A. wieringae DSM 1911T, and about four times higher amount of CO was consumed in 7 days. Genome analysis suggests that strain JM uses the Wood-Ljungdahl pathway for the conversion of one carbon compounds (CO, formate, CO2/H2). Genes encoding bifurcational enzyme complexes with similarity to the bifurcational formate dehydrogenase (Fdh) of Clostridium autoethanogenum are present, and possibly relate to the higher tolerance to CO of strain JM compared to other Acetobacterium species. A. wieringae DSM 1911T grew on CO in medium containing 1 mM formate.

The hydrogen-dependent carbon dioxide reductase is a soluble enzyme complex that directly utilizes hydrogen (H2) for the reduction of carbon dioxide (CO2) to formate in the first step of the acetyl-coenzyme A- or Wood-Ljungdahl pathway (WLP). HDCR consists of 2 catalytic subunits, a hydrogenase and a formate dehydrogenase (FDH) and two small subunits carrying iron-sulfur clusters. The enzyme complex has been purified and characterized from two acetogenic bacteria, from the mesophile Acetobacterium woodii and, recently, from the thermophile Thermoanaerobacter kivui. Physiological studies toward the importance of the HDCR for growth and formate metabolism in acetogens have not been carried out yet, due to the lack of genetic tools. Here, we deleted the genes encoding HDCR in T. kivui taking advantage of the recently developed genetic system. As expected, the deletion mutant (strain TKV_MB013) did not grow with formate as single substrate or under autotrophic conditions with H2 + CO2. Surprisingly, the strain did also not grow on any other substrate (sugars, mannitol or pyruvate), except for when formate was added. Concentrated cell suspensions quickly consumed formate in the presence of glucose only. In conclusion, HDCR provides formate which was essential for growth of the T. kivui mutant. Alternatively, extracellularly added formate served as terminal electron acceptor in addition to CO2, complementing the growth deficiency. The results show a tight coupling of multi-carbon substrate oxidation to the WLP. The metabolism in the mutant can be viewed as a coupled formate + CO2 respiration, which may be an ancient metabolic trait.

Abstract Acetogenic bacteria are obligate anaerobes with the ability of converting carbon dioxide and other one‐carbon substrates into acetate through the Wood–Ljungdahl (WL) pathway. These substrates are becoming increasingly important feedstock in industrial microbiology. The main potential industrial application of acetogenic bacteria is the production of metabolites that constitute renewable energy sources (biofuel); such bacteria are of particular interest for this purpose thanks to their low energy requirements for large‐scale cultivation. Here, we report new genome sequences for four species, three of them are reported for the first time, namely Acetobacterium paludosum DSM 8237, Acetobacterium tundrae DSM 917, Acetobacterium bakii DSM 8239, and Alkalibaculum bacchi DSM 221123. We performed a comparative genomic analysis focused on the WL pathway's genes and their encoded proteins, using Acetobacterium woodii as a reference genome. The Average Nucleotide Identity (ANI) values ranged from 70% to 95% over an alignment length of 5.4–6.5 Mbp. The core genome consisted of 363 genes, whereas the number of unique genes in a single genome ranged from 486 in A. tundrae to 2360 in A.bacchi. No significant rearrangements were detected in the gene order for the Wood–Ljungdahl pathway however, two species showed variations in genes involved in formate metabolism: A. paludosum harbor two copies of fhs1, and A. bakii a truncated fdhF1. The analysis of protein networks highlighted the expansion of protein orthologues in A. woodii compared to A. bacchi, whereas protein networks involved in the WL pathway were more conserved. This study has increased our understanding on the evolution of the WL pathway in acetogenic bacteria.

Genomes record their own history. But if we want to look all the way back to life’s beginnings some 4 billion years ago, the record of microbial evolution that is preserved in prokaryotic genomes is not easy to read. Microbiology has a lot in common with geology in that regard. Geologists know that plate tectonics and erosion have erased much of the geological record, with ancient rocks being truly rare. The same is true of microbes. Lateral gene transfer (LGT) and sequence divergence have erased much of the evolutionary record that was once written in genomes, and it is not obvious which genes among sequenced genomes are genuinely ancient. Which genes trace to the last universal ancestor, LUCA? The classical approach has been to look for genes that are universally distributed. Another approach is to make all trees for all genes, and sift out the trees where signals have been overwritten by LGT. What is left ought to be ancient. If we do that, what do we find?

Gasification-fermentation is a thermochemical-biological platform for the production of fuels and chemicals. Biomass is gasified at high temperatures to make syngas, a gas composed of CO, CO2, H2, N2 and other minor components. Syngas is then fed to anaerobic microorganisms that convert CO, CO2 and H2 to alcohols by fermentation. This platform offers numerous advantages such as flexibility of feedstock and syngas composition and lower operating temperature and pressure compared to other catalytic syngas conversion processes. In comparison to hydrolysis-fermentation, gasification-fermentation has a major advantage of utilizing all organic components of biomass, including lignin, to yield higher fuel production. Furthermore, syngas fermentation microorganisms do not require strict CO:H2:CO2 ratios, hence gas reforming is not required. However, several issues must be addressed for successful deployment of gasification-fermentation, particularly those that involve the integration of gasification and fermentation. Most previous reviews have focused only on either biomass gasification or syngas fermentation. In this review, the critical factors that affect the integration of biomass gasification with syngas fermentation, such as carbon conversion efficiency, effect of trace gaseous species, H2 to CO ratio requirements, and microbial preference of carbon substrate, are thoroughly discussed.

Microbial electrosynthesis (MES) is a bioelectrochemical technology developed for the conversion of carbon dioxide and electric energy into multicarbon chemicals of interest. As with other biotechnologies, achieving high production rate is a prerequisite for scaling up. In this study, we report the development of a novel cathode for MES, which was fabricated by coating carbon cloth with conductive poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) polymer. Sporomusa ovata-driven MES reactors equipped with PEDOT:PSS-carbon cloth cathodes produced 252.5 ± 23.6 mmol d−1 acetate per m2 of electrode over a period of 14 days, which was 9.3 fold higher than the production rate observed with uncoated carbon cloth cathodes. Concomitantly, current density was increased to −3.2 ± 0.8 A m−2, which was 10.7-fold higher than the untreated cathode. The coulombic efficiency with the PEDOT: PSS-carbon cloth cathodes was 78.6 ± 5.6%. Confocal laser scanning microscopy and scanning electron microscopy showed denser bacterial population on the PEDOT:PSS-carbon cloth cathodes. This suggested that PEDOT:PSS is more suitable for colonization by S. ovata during the bioelectrochemical process. The results demonstrated that PEDOT: PSS is a promising cathode material for MES.

Anaerobic digestion is achieved by the combined effort of hydrolytic, acetogenic and methanogenic bacteria. Microbial dynamics and biogas production during anaerobic digestion of cow dung and rice husk were studied in this research. The experiment lasted for 30 days using a 10 L scale bio-digester. All proximate parameters reduced significantly after digestion for CD (cow dung), RH (rice husk), and CD:RH (cow dung and rice husk) except moisture content, which increased for all substrates. Ash content (1.08-1.67 mg) and crude fibre (1.27-1.96 mg) increased in CD only. The pH ranges for the substrates were CD (7.0-7.5), RH (6.1-7.6), and CD:RH (6.1-7.8). Temperature ranges were CD (27.4 oC-33.5 oC), RH (27.2 oC-33.3 oC) and CD:RH (27.3 oC-33.4 oC). The total biogas production of the substrates and components of each gas produced were, CD (4327.65 cm3 : 62.4 % CH4, 37.4 % CO2, 0.2 % H2S), RH (150 cm3 : 100 % CO2), and CD:RH (4730.55 cm3 : 73.8 % CH4, 25.8 % CO2, 0.4 % H2S). Percentage distribution of the digester’s microflora include aerobes (40.75 %), anaerobes (31.25 %), fungi (25 %) and methanogenic bacteria (3 %). Hydrolytic bacteria and fungi isolated were Bacillus spp, Enterobacter spp, Pseudomonas spp, Proteus spp, Micrococcus spp, Aspergillus spp, Penicillium spp and Streptococcus spp. Acetogens isolated were Clostridium spp, Streptococcus spp and Pseudomonas spp. Methanococcus spp and Methanobacterium spp were the only isolated methanogens. Rice husk produced the least amount of biogas.

Anaerobic digestion is achieved by the combined effort of hydrolytic, acetogenic and methanogenic bacteria. Microbial dynamics and biogas production during anaerobic digestion of cow dung and rice husk were studied in this research. The experiment lasted for 30 days using a 10 L scale bio-digester. All proximate parameters reduced significantly after digestion for CD (cow dung), RH (rice husk), and CD:RH (cow dung and rice husk) except moisture content, which increased for all substrates. Ash content (1.08-1.67 mg) and crude fibre (1.27-1.96 mg) increased in CD only. The pH ranges for the substrates were CD (7.0-7.5), RH (6.1-7.6), and CD:RH (6.1-7.8). Temperature ranges were CD (27.4 oC-33.5 oC), RH (27.2 oC-33.3 oC) and CD:RH (27.3 oC-33.4 oC). The total biogas production of the substrates and components of each gas produced were, CD (4327.65 cm3 : 62.4 % CH4, 37.4 % CO2, 0.2 % H2S), RH (150 cm3 : 100 % CO2), and CD:RH (4730.55 cm3 : 73.8 % CH4, 25.8 % CO2, 0.4 % H2S). Percentage distribution of the digester’s microflora include aerobes (40.75 %), anaerobes (31.25 %), fungi (25 %) and methanogenic bacteria (3 %). Hydrolytic bacteria and fungi isolated were Bacillus spp, Enterobacter spp, Pseudomonas spp, Proteus spp, Micrococcus spp, Aspergillus spp, Penicillium spp and Streptococcus spp. Acetogens isolated were Clostridium spp, Streptococcus spp and Pseudomonas spp. Methanococcus spp and Methanobacterium spp were the only isolated methanogens. Rice husk produced the least amount of biogas.

Anaerobic digestion is achieved by the combined effort of hydrolytic, acetogenic and methanogenic bacteria. Microbial dynamics and biogas production during anaerobic digestion of cow dung and rice husk were studied in this research. The experiment lasted for 30 days using a 10 L scale bio-digester. All proximate parameters reduced significantly after digestion for CD (cow dung), RH (rice husk), and CD:RH (cow dung and rice husk) except moisture content, which increased for all substrates. Ash content (1.08-1.67 mg) and crude fibre (1.27-1.96 mg) increased in CD only. The pH ranges for the substrates were CD (7.0-7.5), RH (6.1-7.6), and CD:RH (6.1-7.8). Temperature ranges were CD (27.4 oC-33.5 oC), RH (27.2 oC-33.3 oC) and CD:RH (27.3 oC-33.4 oC). The total biogas production of the substrates and components of each gas produced were, CD (4327.65 cm3 : 62.4 % CH4, 37.4 % CO2, 0.2 % H2S), RH (150 cm3 : 100 % CO2), and CD:RH (4730.55 cm3 : 73.8 % CH4, 25.8 % CO2, 0.4 % H2S). Percentage distribution of the digester’s microflora include aerobes (40.75 %), anaerobes (31.25 %), fungi (25 %) and methanogenic bacteria (3 %). Hydrolytic bacteria and fungi isolated were Bacillus spp, Enterobacter spp, Pseudomonas spp, Proteus spp, Micrococcus spp, Aspergillus spp, Penicillium spp and Streptococcus spp. Acetogens isolated were Clostridium spp, Streptococcus spp and Pseudomonas spp. Methanococcus spp and Methanobacterium spp were the only isolated methanogens. Rice husk produced the least amount of biogas.

Anaerobic digestion is achieved by the combined effort of hydrolytic, acetogenic and methanogenic bacteria. Microbial dynamics and biogas production during anaerobic digestion of cow dung and rice husk were studied in this research. The experiment lasted for 30 days using a 10 L scale bio-digester. All proximate parameters reduced significantly after digestion for CD (cow dung), RH (rice husk), and CD:RH (cow dung and rice husk) except moisture content, which increased for all substrates. Ash content (1.08-1.67 mg) and crude fibre (1.27-1.96 mg) increased in CD only. The pH ranges for the substrates were CD (7.0-7.5), RH (6.1-7.6), and CD:RH (6.1-7.8). Temperature ranges were CD (27.4 oC-33.5 oC), RH (27.2 oC-33.3 oC) and CD:RH (27.3 oC-33.4 oC). The total biogas production of the substrates and components of each gas produced were, CD (4327.65 cm3 : 62.4 % CH4, 37.4 % CO2, 0.2 % H2S), RH (150 cm3 : 100 % CO2), and CD:RH (4730.55 cm3 : 73.8 % CH4, 25.8 % CO2, 0.4 % H2S). Percentage distribution of the digester’s microflora include aerobes (40.75 %), anaerobes (31.25 %), fungi (25 %) and methanogenic bacteria (3 %). Hydrolytic bacteria and fungi isolated were Bacillus spp, Enterobacter spp, Pseudomonas spp, Proteus spp, Micrococcus spp, Aspergillus spp, Penicillium spp and Streptococcus spp. Acetogens isolated were Clostridium spp, Streptococcus spp and Pseudomonas spp. Methanococcus spp and Methanobacterium spp were the only isolated methanogens. Rice husk produced the least amount of biogas.

There is an immediate need to drastically reduce the emissions associated with global fossil fuel consumption in order to limit climate change. However, carbon-based materials, chemicals, and transportation fuels are predominantly made from fossil sources and currently there is no alternative source available to adequately displace them. Gas-fermenting microorganisms that fix carbon dioxide (CO2) and carbon monoxide (CO) can break this dependence as they are capable of converting gaseous carbon to fuels and chemicals. As such, the technology can utilize a wide range of feedstocks including gasified organic matter of any sort (e.g., municipal solid waste, industrial waste, biomass, and agricultural waste residues) or industrial off gasses (e.g., from steel mills or processing plants). Gas fermentation has matured to the point that large-scale production of ethanol from gas has been demonstrated by two companies. This review gives an overview of the gas fermentation process, focusing specifically on anaerobic acetogens. Applications of synthetic biology and coupling gas fermentation to additional processes are discussed in detail. Both of these strategies, demonstrated at bench-scale, have abundant potential to rapidly expand the commercial product spectrum of gas fermentation and further improve efficiencies and yields.

Microbially-influenced corrosion (MIC) contributes to the general corrosion rate (CR), which is typically measured with carbon steel coupons. Here we explore the use of carbon steel ball bearings, referred to as beads (55.0 ± 0.3 mg; Ø = 0.238 cm), for determining CRs. CRs for samples from an oil field in Oceania incubated with beads were determined by the weight loss method, using acid treatment to remove corrosion products. The release of ferrous and ferric iron was also measured and CRs based on weight loss and iron determination were in good agreement. Average CRs were 0.022 mm/yr for 8 produced waters with high numbers (105/ml) of acid-producing bacteria (APB), but no sulfate-reducing bacteria (SRB). Average CRs were 0.009 mm/yr for 5 central processing facility (CPF) waters, which had no APB or SRB due to weekly biocide treatment and 0.036 mm/yr for 2 CPF tank bottom sludges, which had high numbers of APB (106/ml) and SRB (108/ml). Hence, corrosion monitoring with carbon steel beads indicated that biocide treatment of CPF waters decreased the CR, except where biocide did not penetrate. The CR for incubations with 20 ml of a produced water decreased from 0.061 to 0.007 mm/yr when increasing the number of beads from 1 to 40. CRs determined with beads were higher than those with coupons, possibly also due to a higher weight of iron per unit volume used in incubations with coupons. Use of 1 ml syringe columns, containing carbon steel beads and injected with 10 ml/day of SRB-containing medium for 256 days gave a CR of 0.11 mm/yr under flow conditions. The standard deviation of the distribution of residual bead weights, a measure for the unevenness of the corrosion, increased with increasing CR. The most heavily corroded beads showed significant pitting. Hence the use of uniformly sized carbon steel beads offers new opportunities for screening and monitoring of corrosion including determination of the distribution of corrosion rates, which allows estimation of the probability of high rate events that may lead to failure.

Microbially influenced corrosion (MIC) in oil field pipeline systems can be attributed to many different types of hydrogenotrophic microorganisms including sulfate reducers, methanogens and acetogens. Samples from a low temperature oil reservoir in Nigeria were analyzed using DNA pyrotag sequencing. The microbial community compositions of these samples revealed an abundance of anaerobic methanogenic archaea. Activity of methanogens was demonstrated by incubating samples anaerobically in a basal salts medium, in the presence of carbon steel and carbon dioxide. Methane formation was measured in all enrichments and correlated with metal weight loss. Methanogens were prominently represented in pipeline solids samples, scraped from the inside of a pipeline, comprising over 85% of all pyrosequencing reads. Methane production was only witnessed when carbon steel beads were added to these pipeline solids samples, indicating that no methane was formed as a result of degradation of the oil organics present in these samples. These results were compared to those obtained for samples taken from a low temperature oil field in Canada, which had been incubated with oil, either in the presence or in the absence of carbon steel. Again, methanogens present in these samples catalyzed methane production only when carbon steel was present. Moreover, acetate production was also found in these enrichments only in the presence of carbon steel. From these studies it appears that carbon steel, not oil organics, was the predominant electron donor for acetate production and methane formation in these low temperature oil fields, indicating that the methanogens and acetogens found may contribute significantly to MIC.

Microbially-influenced corrosion (MIC) of iron (Fe0) by sulfate reducing bacteria (SRB) has been studied extensively. Through a mechanism, that is still poorly understood, electrons or hydrogen (H2) molecules are removed from the metal surface and used as electron donor for sulfate reduction. The resulting ferrous ions precipitate in part with the sulfide produced, forming characteristic black iron sulfide. Hydrogenotrophic methanogens can also contribute to MIC. Incubation of pipeline water samples, containing bicarbonate and some sulfate, in serum bottles with steel coupons and a headspace of 10% (vol/vol) CO2 and 90% N2, indicated formation of acetate and methane. Incubation of these samples in serum bottles, containing medium with coupons and bicarbonate but no sulfate, also indicated that formation of acetate preceded the formation of methane. Microbial community analyses of these enrichments indicated the presence of Acetobacterium, as well we of hydrogenotrophic and acetotrophic methanogens. The formation of acetate by homoacetogens, such as Acetobacterium woodii from H2 (or Fe0) and CO2, is potentially important, because acetate is a required carbon source for many SRB growing with H2 and sulfate. A consortium of the SRB Desulfovibrio vulgaris Hildenborough and A. woodii was able to grow in defined medium with H2, CO2 and sulfate, because A. woodii provides the acetate, needed by D. vulgaris under these conditions. Likewise, general corrosion rates of metal coupons incubated with D. vulgaris in the presence of acetate or in the presence of A. woodii were higher than in the absence of acetate or A. woodii, respectively. An extended MIC model capturing these results is presented.