The role of ethanol oxidation during carboxydotrophic growth of Clostridium autoethanogenum.

The Wood-Ljungdahl pathway is an ancient metabolic route used by acetogenic carboxydotrophs to convert CO into acetate, and some cases ethanol. When produced, ethanol is generally seen as an end product of acetogenic metabolism, but here we show that it acts as an important intermediate and co-substrate during carboxydotrophic growth of Clostridium autoethanogenum. Depending on CO availability, C. autoethanogenum is able to rapidly switch between ethanol production and utilization, hereby optimizing its carboxydotrophic growth. The importance of the aldehyde ferredoxin:oxidoreductase (AOR) route for ethanol production in carboxydotrophic acetogens is known; however, the role of the bifunctional alcohol dehydrogenase AdhE (Ald-Adh) route in ethanol metabolism remains largely unclear. We show that the mutant strain C. autoethanogenum ∆adhE1a, lacking the Ald subunit of the main bifunctional aldehyde/alcohol dehydrogenase (AdhE, CAETHG_3747), has poor ethanol oxidation capabilities, with a negative impact on biomass yield. This indicates that the Adh-Ald route plays a major role in ethanol oxidation during carboxydotrophic growth, enabling subsequent energy conservation via substrate-level phosphorylation using acetate kinase. Subsequent chemostat experiments with C. autoethanogenum show that the wild type, in contrast to ∆adhE1a, is more resilient to sudden changes in CO supply and utilizes ethanol as a temporary storage for reduction equivalents and energy during CO-abundant conditions, reserving these 'stored assets' for more CO-limited conditions. This shows that the direction of the ethanol metabolism is very dynamic during carboxydotrophic acetogenesis and opens new insights in the central metabolism of C. autoethanogenum and similar acetogens.

Overflow metabolism at the thermodynamic limit of life: How carboxydotrophic acetogens mitigate carbon monoxide toxicity.

Carboxydotrophic metabolism is gaining interest due to its applications in gas fermentation technology, enabling the conversion of carbon monoxide to fuels and commodities. Acetogenic carboxydotrophs play a central role in current gas fermentation processes. In contrast to other energy-rich microbial substrates, CO is highly toxic, which makes it a challenging substrate to utilize. Instantaneous scavenging of CO upon entering the cell is required to mitigate its toxicity. Experiments conducted with Clostridium autoethanogenum at different biomass-specific growth rates show that elevated ethanol production occurs at increasing growth rates. The increased allocation of electrons towards ethanol at higher growth rates strongly suggests that C. autoethanogenum employs a form of overflow metabolism to cope with high dissolved CO concentrations. We argue that this overflow branch enables acetogens to efficiently use CO at highly variable substrate influxes by increasing the conversion rate almost instantaneously when required to remove toxic substrate and promote growth. In this perspective, we will address the case study of C. autoethanogenum grown solely on CO and syngas mixtures to assess how it employs acetate reduction to ethanol as a form of overflow metabolism.

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.

Synthetic co-cultures: novel avenues for bio-based processes.

In nature, microorganisms live in multi-species communities allowing microbial interactions. These interactions are lost upon establishing a pure culture, increasing the metabolic burden and limiting the metabolic potential of the isolated microbe. In the past years, synthetic microbial co-cultivation, using well-defined consortia of two or more microbes, was increasingly explored for innovative applications in biotechnology. As such, interspecies interactions take place without the complexity of an open mixed culture, minimizing undesired side reactions. Ultimately, synthetic co-cultivation allows to take well-characterized microbes 'off-the-shelf' to create ecosystems with improved process capabilities. This review highlights some of the recent developments on co-cultivation, focusing on waste-to-chemicals conversions. It also addresses fundamental knowledge on microbial interactions deriving from these studies, which is important to further develop our ability to engineer functional co-cultures for bioproduction.

Natranaerofaba carboxydovora gen. nov., sp. nov., an extremely haloalkaliphilic CO-utilizing acetogen from a hypersaline soda lake representing a novel deep phylogenetic lineage in the class 'Natranaerobiia'.

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

Modeling a co-culture of Clostridium autoethanogenum and Clostridium kluyveri to increase syngas conversion to medium-chain fatty-acids.

Microbial fermentation of synthesis gas (syngas) is becoming more attractive for sustainable production of commodity chemicals. To date, syngas fermentation focuses mainly on the use of Clostridium species for the production of small organic molecules such as ethanol and acetate. The co-cultivation of syngas-fermenting microorganisms with chain-elongating bacteria can expand the range of possible products, allowing, for instance, the production of medium-chain fatty acids (MCFA) and alcohols from syngas. To explore these possibilities, we report herein a genome-scale, constraint-based metabolic model to describe growth of a co-culture of Clostridium autoethanogenum and Clostridium kluyveri on syngas for the production of valuable compounds. Community flux balance analysis was used to gain insight into the metabolism of the two strains and their interactions, and to reveal potential strategies enabling production of butyrate and hexanoate. The model suggests that one strategy to optimize the production of medium-chain fatty-acids from syngas would be the addition of succinate. According to the prediction, addition of succinate would increase the pool of crotonyl-CoA and the ethanol/acetate uptake ratio in C. kluyveri, resulting in a flux of up to 60 % of electrons into hexanoate. Another potential way to further optimize butyrate and hexanoate production would be an increase of C. autoethanogenum ethanol production. Blocking either acetaldehyde dehydrogenase or formate dehydrogenase (ferredoxin) activity or formate transport, in the C. autoethanogenum metabolic model could potentially lead to an up to 150 % increase in ethanol production.

Syngas as Electron Donor for Sulfate and Thiosulfate Reducing Haloalkaliphilic Microorganisms in a Gas-Lift Bioreactor.

Biodesulfurization processes remove toxic and corrosive hydrogen sulfide from gas streams (e.g., natural gas, biogas, or syngas). To improve the efficiency of these processes under haloalkaline conditions, a sulfate and thiosulfate reduction step can be included. The use of H2/CO mixtures (as in syngas) instead of pure H2 was tested to investigate the potential cost reduction of the electron donor required. Syngas is produced in the gas-reforming process and consists mainly of H2, carbon monoxide (CO), and carbon dioxide (CO2). Purification of syngas to obtain pure H2 implies higher costs because of additional post-treatment. Therefore, the use of syngas has merit in the biodesulfurization process. Initially, CO inhibited hydrogen-dependent sulfate reduction. However, after 30 days the biomass was adapted and both H2 and CO were used as electron donors. First, formate was produced, followed by sulfate and thiosulfate reduction, and later in the reactor run acetate and methane were detected. Sulfide production rates with sulfate and thiosulfate after adaptation were comparable with previously described rates with only hydrogen. The addition of CO marginally affected the microbial community in which Tindallia sp. was dominant. Over time, acetate production increased and acetogenesis became the dominant process in the bioreactor. Around 50% of H2/CO was converted to acetate. Acetate supported biomass growth and higher biomass concentrations were reached compared to bioreactors without CO feed. Finally, CO addition resulted in the formation of small, compact microbial aggregates. This suggests that CO or syngas can be used to stimulate aggregation in haloalkaline biodesulfurization systems.

Enrichment of Anaerobic Syngas-Converting Communities and Isolation of a Novel Carboxydotrophic Acetobacterium wieringae Strain JM.

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.

"Candidatus Galacturonibacter soehngenii" Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex.

Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of "Candidatus Galacturonibacter soehngenii," from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of "Ca. G. soehngenii" was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the "Ca. G. soehngenii" MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of "Ca. G. soehngenii" enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of "Ca. G. soehngenii."

Metabolic shift induced by synthetic co-cultivation promotes high yield of chain elongated acids from syngas.

Bio-catalytic processes for sustainable production of chemicals and fuels receive increased attention within the concept of circular economy. Strategies to improve these production processes include genetic engineering of bio-catalysts or process technological optimization. Alternatively, synthetic microbial co-cultures can be used to enhance production of chemicals of interest. It remains often unclear however how microbe to microbe interactions affect the overall production process and how this can be further exploited for application. In the present study we explored the microbial interaction in a synthetic co-culture of Clostridium autoethanogenum and Clostridium kluyveri, producing chain elongated products from carbon monoxide. Monocultures of C. autoethanogenum converted CO to acetate and traces of ethanol, while during co-cultivation with C. kluyveri, it shifted its metabolism significantly towards solventogenesis. In C. autoethanogenum, expression of the genes involved in the central carbon- and energy-metabolism remained unchanged during co-cultivation compared to monoculture condition. Therefore the shift in the metabolic flux of C. autoethanogenum appears to be regulated by thermodynamics, and results from the continuous removal of ethanol by C. kluyveri. This trait could be further exploited, driving the metabolism of C. autoethanogenum to solely ethanol formation during co-cultivation, resulting in a high yield of chain elongated products from CO-derived electrons. This research highlights the important role of thermodynamic interactions in (synthetic) mixed microbial communities and shows that this can be exploited to promote desired conversions.

High Rate Biomethanation of Carbon Monoxide-Rich Gases via a Thermophilic Synthetic Coculture.

Carbon monoxide-fermenting microorganisms can be used for the production of a wide range of commodity chemicals and fuels from syngas (generated by gasification of, e.g., wastes or biomass) or industrial off-gases (e.g., from steel industry). Microorganisms are normally more resistant to contaminants in the gas (e.g., hydrogen sulfide) than chemical catalysts, less expensive and self-regenerating. However, some carboxydotrophs are sensitive to high concentrations of CO, resulting in low growth rates and productivities. We hypothesize that cultivation of synthetic cocultures can be used to improve overall rates of CO bioconversion. As a case study, a thermophilic microbial coculture, consisting of Carboxydothermus hydrogenoformans and Methanothermobacter thermoautotrophicus was constructed to study the effect of cocultivation on conversion of CO-rich gases to methane. In contrast to the methanogenic monoculture, the coculture was able to efficiently utilize CO or mixtures of H2/CO/CO2 to produce methane at high efficiency and high rates. In CSTR-bioreactors operated in continuous mode, the coculture converted artificial syngas (66.6% H2:33.3% CO) to an outflow gas with a methane content of 72%, approaching the 75% theoretical maximum. CO conversion efficiencies of 93% and volumetric production rates of 4 m3methane/m3liquid/day were achieved. This case shows that microbial cocultivation can result in a significant improvement of gas-fermentation of CO-rich gases.

A Narrow pH Range Supports Butanol, Hexanol, and Octanol Production from Syngas in a Continuous Co-culture of Clostridium ljungdahlii and Clostridium kluyveri with In-Line Product Extraction.

Carboxydotrophic bacteria (CTB) have received attention due to their ability to synthesize commodity chemicals from producer gas and synthesis gas (syngas). CTB have an important advantage of a high product selectivity compared to chemical catalysts. However, the product spectrum of wild-type CTB is narrow. Our objective was to investigate whether a strategy of combining two wild-type bacterial strains into a single, continuously fed bioprocessing step would be promising to broaden the product spectrum. Here, we have operated a syngas-fermentation process with Clostridium ljungdahlii and Clostridium kluyveri with in-line product extraction through gas stripping and product condensing within the syngas recirculation line. The main products from C. ljungdahlii fermentation at a pH of 6.0 were ethanol and acetate at net volumetric production rates of 65.5 and 431 mmol C·L-1·d-1, respectively. An estimated 2/3 of total ethanol produced was utilized by C. kluyveri to chain elongate with the reverse ß-oxidation pathway, resulting in n-butyrate and n-caproate at net rates of 129 and 70 mmol C·L-1·d-1, respectively. C. ljungdahlii likely reduced the produced carboxylates to their corresponding alcohols with the reductive power from syngas. This resulted in the longer-chain alcohols n-butanol, n-hexanol, and n-octanol at net volumetric production rates of 39.2, 31.7, and 0.045 mmol C·L-1·d-1, respectively. The continuous production of the longer-chain alcohols occurred only within a narrow pH spectrum of 5.7-6.4 due to the pH discrepancy between the two strains. Regardless whether other wild-type strains could overcome this pH discrepancy, the specificity (mol carbon in product per mol carbon in all other liquid products) for each longer-chain alcohol may never be high in a single bioprocessing step. This, because two bioprocesses compete for intermediates (i.e., carboxylates): (1) chain elongation; and (2) biological reduction. This innate competition resulted in a mixture of n-butanol and n-hexanol with traces of n-octanol.

Proteomic Analysis of the Hydrogen and Carbon Monoxide Metabolism of Methanothermobacter marburgensis.

Hydrogenotrophic methanogenic archaea are efficient H2 utilizers, but only a few are known to be able to utilize CO. Methanothermobacter thermoautotrophicus is one of the hydrogenotrophic methanogens able to grow on CO, albeit about 100 times slower than on H2 + CO2. In this study, we show that the hydrogenotrophic methanogen Methanothermobacter marburgensis, is able to perform methanogenic growth on H2/CO2/CO and on CO as a sole substrate. To gain further insight in its carboxydotrophic metabolism, the proteome of M. marburgensis, grown on H2/CO2 and H2/CO2/CO, was analyzed. Cultures grown with H2/CO2/CO showed relative higher abundance of enzymes involved in the reductive acetyl-CoA pathway and proteins involved in redox metabolism. The data suggest that the strong reducing capacity of CO negatively affects hydrogenotrophic methanogenesis, making growth on CO as a sole substrate difficult for this type of methanogens. M. marburgensis appears to partly deal with this by up-regulating co-factor regenerating reactions and activating additional pathways allowing for formation of other products, like acetate.

Production of medium-chain fatty acids and higher alcohols by a synthetic co-culture grown on carbon monoxide or syngas.

BACKGROUND: Synthesis gas, a mixture of CO, H2, and CO2, is a promising renewable feedstock for bio-based production of organic chemicals. Production of medium-chain fatty acids can be performed via chain elongation, utilizing acetate and ethanol as main substrates. Acetate and ethanol are main products of syngas fermentation by acetogens. Therefore, syngas can be indirectly used as a substrate for the chain elongation process. RESULTS: Here, we report the establishment of a synthetic co-culture consisting of Clostridium autoethanogenum and Clostridium kluyveri. Together, these bacteria are capable of converting CO and syngas to a mixture of C4 and C6 fatty acids and their respective alcohols. The co-culture is able to grow using solely CO or syngas as a substrate, and presence of acetate significantly stimulated production rates. The co-culture produced butyrate and caproate at a rate of 8.5 ± 1.1 and 2.5 ± 0.63 mmol/l/day, respectively. Butanol and hexanol were produced at a rate of 3.5 ± 0.69 and 2.0 ± 0.46 mmol/l/day, respectively. The pH was found to be a major factor during cultivation, influencing the growth performance of the separate strains and caproate toxicity. CONCLUSION: This co-culture poses an alternative way to produce medium-chain fatty acids and higher alcohols from carbon monoxide or syngas and the process can be regarded as an integration of syngas fermentation and chain elongation in one growth vessel.

Anaerobic oxidation of methane associated with sulfate reduction in a natural freshwater gas source.

The occurrence of anaerobic oxidation of methane (AOM) and trace methane oxidation (TMO) was investigated in a freshwater natural gas source. Sediment samples were taken and analyzed for potential electron acceptors coupled to AOM. Long-term incubations with (13)C-labeled CH4 ((13)CH4) and different electron acceptors showed that both AOM and TMO occurred. In most conditions, (13)C-labeled CO2 ((13)CO2) simultaneously increased with methane formation, which is typical for TMO. In the presence of nitrate, neither methane formation nor methane oxidation occurred. Net AOM was measured only with sulfate as electron acceptor. Here, sulfide production occurred simultaneously with (13)CO2 production and no methanogenesis occurred, excluding TMO as a possible source for (13)CO2 production from (13)CH4. Archaeal 16S rRNA gene analysis showed the highest presence of ANME-2a/b (ANaerobic MEthane oxidizing archaea) and AAA (AOM Associated Archaea) sequences in the incubations with methane and sulfate as compared with only methane addition. Higher abundance of ANME-2a/b in incubations with methane and sulfate as compared with only sulfate addition was shown by qPCR analysis. Bacterial 16S rRNA gene analysis showed the presence of sulfate-reducing bacteria belonging to SEEP-SRB1. This is the first report that explicitly shows that AOM is associated with sulfate reduction in an enrichment culture of ANME-2a/b and AAA methanotrophs and SEEP-SRB1 sulfate reducers from a low-saline environment.

Pathways and Bioenergetics of Anaerobic Carbon Monoxide Fermentation.

Carbon monoxide can act as a substrate for different modes of fermentative anaerobic metabolism. The trait of utilizing CO is spread among a diverse group of microorganisms, including members of bacteria as well as archaea. Over the last decade this metabolism has gained interest due to the potential of converting CO-rich gas, such as synthesis gas, into bio-based products. Three main types of fermentative CO metabolism can be distinguished: hydrogenogenesis, methanogenesis, and acetogenesis, generating hydrogen, methane and acetate, respectively. Here, we review the current knowledge on these three variants of microbial CO metabolism with an emphasis on the potential enzymatic routes and bio-energetics involved.