Microbial propionate production from carbon monoxide a novel bioprocess

Introduction: The fermentation of CO-rich gases by carboxidotrophic microbes is a promising way to produce valuable organic compounds. Propionate is a value-added compound with numerous industrial applications, e.g. as an antifungal agent in food and feed, and as a building block to produce plastics and herbicides. Propionate is currently produced by petrochemical processes, but it can be produced from ethanol and acetate by some propionogenic bacteria. Ethanol and acetate are usually formed by acetogenic bacteria from CO-rich gases. Accordingly, propionate can be indirectly produced from CO-rich gases, representing a new approach on the realm of microbial CO conversion. Methodology: Four distinct synthetic co-cultures were constructed, consisting of: Acetobacterium wieringae (DSM 1911T) and Pelobacter propionicus (DSM 2379T); A. wieringae (DSM 1911T) and Anaerotignum neopropionicum (DSM 3847T); A. wieringae strain JM and P. propionicus (DSM 2379T); A. wieringae strain JM and A. neopropionicum (DSM 3847T). The physiology of CO conversion to propionate was accessed and a proteogenomic analysis was performed in the best performing co-culture to get insight into the involved biochemical pathways and microbial interactions within the synthetic consortium. Results: Propionate was produced by all the co-cultures, with the highest titer (~24 mM) measured in the co-culture composed of A. wieringae strain JM + A. neopropionicum, which also produced isovalerate (~4 mM), butyrate (~1 mM), and isobutyrate (~0.3 mM). In this synthetic consortium, A. wieringae strain JM converts CO to a acetate and ethanol via the Wood-Ljungdahl pathway; acetate can also be converted to ethanol through the action of aldehyde oxidoreductase (AOR); A. neopropionicum converts ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during co-cultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, and this results in isovalerate and isobutyrate production. Conclusions: This synthetic co-culture represents a new bioprocess for the microbial production of propionate from carbon monoxide, that couples the Wood-Ljungdahl and acrylate pathways. Furthermore, this symbiosis engages an interesting perspective on how C1-fixing and C3-producing microorganisms can be used to expand the product scope of gas fermentation.Portuguese Foundation for Science and Technology (FCT): POCI-01-0145-FEDER-031377; strategic funding of UIDB/04469/2020 unit; BioTecNorte operation (NORTE-01-0145-FEDER-000004); FCT doctoral grants PD/BD/128030/2016 and PD/BD/150583/2020. Netherlands Science Foundation (NWO): Project NWO-GK-07; Perspectief Programma P16-10; Gravitation Grant, Project 024.002.002.info:eu-repo/semantics/publishedVersio

The role of ethanol oxidation during carboxydotrophic growth of clostridium autoethanogenum

The WoodLjungdahl 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 (AldAdh) 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 AdhAld 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.info:eu-repo/semantics/publishedVersio

Production of propionate from carbon monoxide by a synthetic co-culture

info:eu-repo/semantics/publishedVersio

Enrichment of anaerobic syngas-converting communities and isolation of a novel carboxydotrophic Acetobacterium wieringae strain jm

The datasets generated for this study can be found in the 16S rRNA gene sequences submitted to the European Nucleotide Database (ENA) accession numbers LR655884, LR657299 to LR657303, PRJEB33623. The Whole Genome Shotgun project of Acetobacterium wieringae strain JM has been deposited at DDBJ/ENA/GenBank under the accession VSLA00000000.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 involved research was financially supported by Project NWO-GK-07 from the Netherlands Science Foundation (NWO), a Gravitation Grant (Project 024.002.002) of the Netherlands Ministry of Education, Culture and Science, and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020–Programa Operacional Regional do Norte. The financial support from FCT and European Social Fund (POPH-QREN) through the grant PD/BD/128030/2016 given to AA, and through the project INNOVsyn – Innovative strategies for syngas fermentation (POCI-01-0145-FEDER-031377), are gratefully acknowledged.info:eu-repo/semantics/publishedVersio

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 (;24mM) being measured in the coculture composed of Ac. wieringae strain JM and An. neopropionicum, which also produced isovalerate (;4mM), butyrate (;1mM), and isobutyrate (0.3mM). This coculture was further studied using proteogenomics. As expected, enzymes involved in the Wood-Ljungdahl pathway in Ac. wieringae strain JM, which are responsible for the conversion of CO to ethanol and acetate, were detected; the proteome of An. neopropionicum confirmed the conversion of ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during cocultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, accompanied by isovalerate and isobutyrate production. This highlights the importance of explicitly looking at fortuitous microbial interactions during cocultivation to fully understand coculture behavior.This research was financially supported by Project NWO-GK-07 from the Netherlands Science Foundation (NWO), the Perspectief Programma P16-10 from NWO Applied and Engineering Sciences (AGS), by a Gravitation Grant (Project 024.002.002) of the Netherlands Ministry of Education, Culture and Science, and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020— Programa Operacional Regional do Norte. The financial support from FCT and European Social Fund (POPH-QREN) through the grant PD/BD/128030/2016 (given to Ana Luísa Arantes) and PD/BD/150583/2020 (given to João P. C. Moreira) and through the project INNOVsyn—Innovative strategies for syngas fermentation (POCI-01-0145-FEDER-031377) are gratefully acknowledged.info:eu-repo/semantics/publishedVersio

Restraining of glycoprotein VI- and integrin α2β1-dependent thrombus formation y platelet PECAM1

The platelet receptors, glycoprotein VI (GPVI) and integrin α2β1 jointly control collagen-dependent thrombus formation via protein tyrosine kinases. It is unresolved to which extent the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptor PECAM1 and its downstream acting protein tyrosine phosphatase PTPN11 interfere in this process. Here, we hypothesized that integrin α2β1 has a co-regulatory role in the PECAM1- and PTPN11-dependent restraint of thrombus formation. We investigated platelet activation under flow on collagens with a different GPVI dependency and using integrin α2β1 blockage. Blood was obtained from healthy subjects and from patients with Noonan syndrome with a gain-of-function mutation of PTPN11 and variable bleeding phenotype. On collagens with decreasing GPVI activity (types I, III, IV), the surface-dependent inhibition of PECAM1 did not alter thrombus parameters using control blood. Blockage of α2β1 generally reduced thrombus parameters, most effectively on collagen IV. Strikingly, simultaneous inhibition of PECAM1 and α2β1 led to a restoration of thrombus formation, indicating that the suppressing signaling effect of PECAM1 is masked by the platelet-adhesive receptor α2β1. Blood from 4 out of 6 Noonan patients showed subnormal thrombus formation on collagen IV. In these patients, effects of α2β1 blockage were counterbalanced by PECAM1 inhibition to a normal phenotype. In summary, we conclude that the suppression of GPVI-dependent thrombus formation by either PECAM1 or a gain-of-function of PTPN11 can be overruled by α2β1 engagement

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

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.ERC Grant (Project 323009) and the Gravitation Grant (Project 024.002.002) of the Netherlands Ministry of Education, Culture and Science, and the Netherlands Science Foundation (NWO

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 13C-labeled CH4 (13CH4) and different electron acceptors showed that both AOM and TMO occurred. In most conditions, 13C-labeled CO2 (13CO2) 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 13CO2 production and no methanogenesis occurred, excluding TMO as a possible source for 13CO2 production from 13CH4. 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.We thank Douwe Bartstra (Vereniging tot Behoud van de Gasbronnen in Noord-Holland, The Netherlands), Carla Frijters (Paques BV, The Netherlands) and Teun Veuskens (Laboratory of Microbiology, WUR, The Netherlands) for sampling; Martin Meirink (Hoogheemraadschap Hollands Noorderkwartier, The Netherlands) for physicochemical data; Freek van Sambeek for providing Figure 1; Lennart Kleinjans (Laboratory of Microbiology, WUR, The Netherlands) for help with pyrosequencing analysis, Irene Sánchez-Andrea (Laboratory of Microbiology, WUR, The Netherlands) for proof-reading and Katharina Ettwig (Department of Microbiology, Radboud University Nijmegen, The Netherlands) for providing M. oxyfera DNA. We want to thank all anonymous reviewers for valuable contributions. This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009) and the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)