The Selenoproteome as a Dynamic Response Mechanism to Oxidative Stress in Hydrogenotrophic Methanogenic Communities.

Methanogenesis is a critical process in the carbon cycle that is applied industrially in anaerobic digestion and biogas production. While naturally occurring in diverse environments, methanogenesis requires anaerobic and reduced conditions, although varying degrees of oxygen tolerance have been described. Microaeration is suggested as the next step to increase methane production and improve hydrolysis in digestion processes; therefore, a deeper understanding of the methanogenic response to oxygen stress is needed. To explore the drivers of oxygen tolerance in methanogenesis, two parallel enrichments were performed under the addition of H2/CO2 in an environment without reducing agents and in a redox-buffered environment by adding redox mediator 9,10-anthraquinone-2,7-disulfonate disodium. The cellular response to oxidative conditions is mapped using proteomic analysis. The resulting community showed remarkable tolerance to high-redox environments and was unperturbed in its methane production. Next to the expression of pathways to mitigate reactive oxygen species, the higher redox potential environment showed an increased presence of selenocysteine and selenium-associated pathways. By including sulfur-to-selenium mass shifts in a proteomic database search, we provide the first evidence of the dynamic and large-scale incorporation of selenocysteine as a response to oxidative stress in hydrogenotrophic methanogenesis and the presence of a dynamic selenoproteome.

Continuous biomethanation of flue gas-carbon dioxide using bio-integrated carbon capture and utilization.

Biomethanation of carbon dioxide (CO2) from flue gas is a potential enabler of the green transition, particularly when integrated with the power-to-gas chain. However, challenges arise in achieving synthetic natural gas quality when utilizing CO2 from diluted carbon sources, and the high costs of CO2 separation using amine-based solutions make large-scale implementation unfeasible. We propose an innovative continuous biomethanation system that integrates carbon capture and CO2 stripping through microbial utilization, eliminating expenses with the stripper. Stable continuous biomethane production (83-92 % methane purity) was achieved from flue gas-CO2 using a biocompatible aqueous n-methyldiethanolamine (MDEA) solution (50 mmol/L) under mesophilic and hydrogen-limiting conditions. MDEA was found to be recalcitrant to biodegradation and could be reused after regeneration. Demonstrating the microbial ability to simultaneously strip and convert the captured CO2 and regenerate MDEA provides a new pathway for valorization of flue gas CO2.

Reducing greenhouse gas emissions from pig slurry by acidification with organic and inorganic acids.

Methane (CH4) emission from pig slurry is a large contributor to the climate footprint of livestock production. Acidification of excreta from livestock animals with sulfuric acid, reduce CH4 emission and is practiced at many Danish farms. Possible interaction effects with other acidic agents or management practices (e.g. frequent slurry removal and residual slurry acidification) have not been fully investigated. Here we assessed the effect of pig slurry acidification with a range of organic and inorganic acids with respect to their CH4 inhibitor potential in several batch experiments (BS). After careful selection of promising CH4 inhibitors, three continuous headspace experiments (CHS) were carried out to simulate management of manure in pig houses. In BS experiments, more than <99% CH4 reduction was observed with HNO3 treatment to pH 5.5. Treatments with HNO3, H2SO4, and H3PO4 reduced CH4 production more than acetic acid and other organic acids when acidified to the same initial pH of 5.5. Synergistic effects were not observed when mixing inorganic and organic acids as otherwise proposed in the literature, which was attributed to the high amount of acetic acid in the slurry to start with. In the CHS experiments, HNO3 treatment reduced CH4 more than H2SO4, but increased nitrous oxide (N2O) emission, particularly when the acidification target pH was above 6, suggesting considerable denitrification activity. Due to increased N2O emission from HNO3 treatments, HNO3 reduced total CO2-eq by 67%, whereas H2SO4 reduced CO2-eq by 91.5% compared to untreated slurry. In experiments with daily slurry addition, weekly slurry removal, and residual acidification, HNO3 and H2SO4 treatments reduced CO2-eq by 27% and 48%, respectively (not significant). More cycles of residual acidification are recommended in future research. The study provides solid evidence that HNO3 treatment is not suitable for reducing CO2-eq and H2SO4 should be the preferred acidic agent for slurry acidification.

Hydrodynamic analysis of full-scale in-situ biogas upgrading in manure digesters.

The addition of hydrogen to anaerobic digesters is an emerging technique for the sustainable upgrading of biogas to biomethane with renewable electricity. However, it is critically dependent on the effective gas-liquid transfer of hydrogen, which is a sparingly soluble gas. Very little is known about the impact of liquid and gas flow and bubble size on gas-liquid transfer during H2 injection in full-scale anaerobic digesters. A computational fluid dynamic model was developed using a two-fluid approach for non-Newtonian liquid in the open-source computational fluid dynamics (CFD) platform, OpenFOAM. The newly developed model was validated against published experimental data-sets of a gas-mixed, laboratory-scale anaerobic digester, with good agreement between the numerical and experimental velocity fields. The hydrodynamics of the full-scale in-situ biomethanation system using venturi ejectors for H2 injection was then simulated to investigate gas-liquid dynamics, including gas-liquid mass transfer, at different operational conditions. Gas-liquid mixing is mainly controlled by the gas-plumes interaction, which promotes turbulence at the interaction zone, resulting in increasing gas bubbles mixing with the liquid and the gas-liquid interfacial area. However, beyond the plume interaction zone, the digester had flow short-circuiting and inactive zones. It was found that, due to this short-circuiting behaviour, an increase in gas flow-rate may not be an effective option in reducing inactive zones, although it can increase the gas-liquid interfacial area. Comparative analysis of the impact of gas flow and bubble size indicated that gas flow had a linear effect on both kLa and gas holdup, but that bubble size had a non-linear impact, with higher kLa values achieved at bubble sizes less than 2 mm. Comparison against measured data in the same system indicated the predicted kLa values were at the same level as measured kLa, at a bubble size of 2 mm.

Biogas upgrading with hydrogenotrophic methanogenic biofilms.

Hydrogen produced from periodic excess of electrical energy may be added to biogas reactors where it is converted to CH4 that can be utilized in the existing energy grid. The major challenge with this technology is gas-to-liquid mass transfer limitation. The microbial conversions in reactors designed for hydrogenotrophic methanogenesis were studied with microsensors for H2, pH, and CO2. The H2 consumption potential was dependent on the CO2 concentration, but could partially recover after CO2 depletion. Reactors with 3-dimensional biofilm carrier material and a large gas headspace allowed for a methanogenic biofilm in direct contact with the gas phase. A high density of Methanoculleus sp. in the biofilm mediated a high rate of CH4 production, and it was calculated that a reactor filled with 75% carrier material could mediate a biogas upgrading from 50 to 95% CH4 within 24 h when an equivalent amount of H2 was added.

Sporofaciens musculi gen. nov., sp. nov., a novel bacterium isolated from the caecum of an obese mouse.

A bacterial strain, designated WCA-9-b2T, was isolated from the caecal content of an 18-week-old obese C57BL/6NTac male mouse. According to phenotypic analyses, the isolate was rod-shaped, strictly anaerobic, spore-forming, non-motile and Gram-stain-positive, under the conditions tested. Colonies were irregular and non-pigmented. Analysis of the 16S rRNA gene sequence indicated that the isolate belonged to the order Clostridiales with Dorea longicatena ATCC 27755T (94.9 % sequence identity), Ruminococcus gnavus ATCC 29149T (94.8%) and Clostridium scindens ATCC 35704T (94.3%) being the closest relatives. Whole genome sequencing showed an average nucleotide identity <74.23 %, average amino acid identity <64.52-74.67 % and percentage of conserved proteins values <50 % against the nine closest relatives (D. longicatena, Ruminococcus gnavus, C. scindens, Dorea formicigenerans, Ruminococcus lactaris, Clostridium hylemonae, Merdimonas faecis, Faecalicatena contorta and Faecalicatena fissicatena). The genome-based G+C content of genomic DNA was 44.4 mol%. The major cellular fatty acids were C16 : 0 (24.5%), C18 : 1 cis9 (19.8 %), C16 : 0 DMA (11.7%), C18 : 0 (8.4%) and C14 : 0 (6.6%). Respiratory quinones were not detected. The predominant metabolic end products of glucose fermentation were acetate and succinate. Production of CO2 and H2 were detected. Based on these data, we propose that strain WCA-9-b2T represents a novel species within a novel genus, for which the name Sporofaciens musculi gen. nov., sp. nov. is proposed. The type strain is WCA-9-b2T (=DSM 106039T=CECT 30156T).

Chironomus plumosus larvae increase fluxes of denitrification products and diversity of nitrate-reducing bacteria in freshwater sediment.

Benthic invertebrates affect microbial processes and communities in freshwater sediment by enhancing sediment-water solute fluxes and by grazing on bacteria. Using microcosms, the effects of larvae of the widespread midge Chironomus plumosus on the efflux of denitrification products (N2O and N2+N2O) and the diversity and abundance of nitrate- and nitrous-oxide-reducing bacteria were investigated. Additionally, the diversity of actively nitrate- and nitrous-oxide-reducing bacteria was analyzed in the larval gut. The presence of larvae increased the total effluxes of N2O and N2+N2O up to 8.6- and 4.2-fold, respectively, which was mostly due to stimulation of sedimentary denitrification; incomplete denitrification in the guts accounted for up to 20% of the N2O efflux. Phylotype richness of the nitrate reductase gene narG was significantly higher in sediment with than without larvae. In the gut, 47 narG phylotypes were found expressed, which may contribute to higher phylotype richness in colonized sediment. In contrast, phylotype richness of the nitrous oxide reductase gene nosZ was unaffected by the presence of larvae and very few nosZ phylotypes were expressed in the gut. Gene abundance of neither narG, nor nosZ was different in sediments with and without larvae. Hence, C. plumosus increases activity and diversity, but not overall abundance of nitrate-reducing bacteria, probably by providing additional ecological niches in its burrow and gut.

Higher nitrate-reducer diversity in macrophyte-colonized compared to unvegetated freshwater sediment.

Freshwater macrophytes stimulate rhizosphere-associated coupled nitrification-denitrification and are therefore likely to influence the community composition and abundance of rhizosphere-associated denitrifiers and nitrate reducers. Using the narG gene, which encodes the catalytic subunit of the membrane-bound nitrate reductase, as a molecular marker, the community composition and relative abundance of nitrate-reducing bacteria were compared in the rhizosphere of the freshwater macrophyte species Littorella uniflora and Myriophyllum alterniflorum to nitrate-reducing communities in unvegetated sediment. Microsensor analysis indicated a higher availability of oxygen in the rhizosphere compared to unvegetated sediment, with a stronger release of oxygen from the roots of L. uniflora compared to M. alterniflorum. Comparison of narG clone libraries between samples revealed a higher diversity of narG phylotypes in association with the macrophyte rhizospheres compared to unvegetated sediment. Quantitative PCR targeting narG- and 16S rRNA-encoding genes pointed to a selective enrichment of narG gene copies in the rhizosphere. The results suggested that the microenvironment of macrophyte rhizospheres, characterized by the release of oxygen and labile organic carbon from the root system, had a stimulating effect on the diversity and relative abundance of rhizosphere-associated nitrate reducers.

Succession of Deferribacteres and Epsilonproteobacteria through a nitrate-treated high-temperature oil production facility.

Members of Epsilonproteobacteria and Deferribacteres have been implied in nitrate-induced souring control in high-temperature oil production facilities. Here we report on their diversity and abundance in the injection and production part of a nitrate-treated, off-shore oil facility (Halfdan, Denmark) and aimed to assess their potential in souring control. Nitrate addition to deoxygenated seawater shifted the low-biomass seawater community dominated by Gammaproteobacteria closely affiliated with the genus Colwellia to a high-biomass community with significantly higher species richness. Epsilonproteobacteria accounted for less than 1% of the total bacterial community in the nitrate-amended injection water and were most likely outcompeted by putative nitrate-reducing, methylotrophic Gammaproteobacteria of the genus Methylophaga. Reservoir passage and recovery of the oil resulted in a significant change in the bacterial community. Members of the thermophilic Deferribacteres were the second major fraction of the bacterial community in the production water (~30% of the total bacterial community). They were not found in the injection water and were therefore assumed to be indigenous to the reservoir. Additional diversity analysis and targeted quantification of periplasmic nitrate reductase (napA) genes indicated that most resident Deferribacteres possessed the functional potential to contribute to nitrate reduction in the system. In sum, the dominance of nitrate-reducing Deferribacteres and the low relative abundance of Epsilonproteobacteria throughout the production facility suggested that the Deferribacteres play a major role in nitrate-induced souring control at high temperatures.

Fluorescence in situ hybridization (FISH) detection of nitrite reductase transcripts (nirS mRNA) in Pseudomonas stutzeri biofilms relative to a microscale oxygen gradient.

Microsensor measurements of oxygen were combined with mRNA-targeted fluorescence in situ hybridization (FISH) to relate the expression of nitrite reductase (nirS) to oxygen concentrations in artificial biofilms of the denitrifier Pseudomonas stutzeri. A distinct zone of nirS transcript-containing cells was detected at the oxic-anoxic transition zone, below an oxygen threshold concentration of 0.7-2.5µM, depending on incubation conditions. Although not a routine technique yet, the possibility of coupling microsensor and mRNA-targeted FISH analyses described here opens for studies addressing microenvironment, identity, and actual activity of microbes in stratified environments at single cell resolution.