Relevance of extracellular electron uptake mechanisms for electromethanogenesis applications.

Electromethanogenesis has emerged as a biological branch of Power-to-X technologies that implements methanogenic microorganisms, as an alternative to chemical Power-to-X, to convert electrical power from renewable sources, and CO2 into methane. Unlike biomethanation processes where CO2 is converted via exogenously added hydrogen, electromethanogenesis occurs in a bioelectrochemical set-up that combines electrodes and microorganisms. Thereby, mixed, or pure methanogenic cultures catalyze the reduction of CO2 to methane via reducing equivalents supplied by a cathode. Recent advances in electromethanogenesis have been driven by interdisciplinary research at the intersection of microbiology, electrochemistry, and engineering. Integrating the knowledge acquired from these areas is essential to address the specific challenges presented by this relatively young biotechnology, which include electron transfer limitations, low energy and product efficiencies, and reactor design to enable upscaling. This review approaches electromethanogenesis from a multidisciplinary perspective, putting emphasis on the extracellular electron uptake mechanisms that methanogens use to obtain energy from cathodes, since understanding these mechanisms is key to optimize the electrochemical conditions for the development of these systems. This work summarizes the direct and indirect extracellular electron uptake mechanisms that have been elucidated to date in methanogens, along with the ones that remain unsolved. As the study of microbial corrosion, a similar bioelectrochemical process with Fe0 as electron source, has contributed to elucidate different mechanisms on how methanogens use solid electron donors, insights from both fields, biocorrosion and electromethanogenesis, are combined. Based on the repertoire of mechanisms and their potential to convert CO2 to methane, we conclude that for future applications, electromethanogenesis should focus on the indirect mechanism with H2 as intermediary. By summarizing and linking the general aspects and challenges of this process, we hope that this review serves as a guide for researchers working on electromethanogenesis in different areas of expertise to overcome the current limitations and continue with the optimization of this promising interdisciplinary technology.

Exploring increased hydraulic retention time as a cost-efficient way of valorizing residual biogas potential.

Effective substrate utilization with low residual methane yield in the digestate is crucial for the economy and sustainability of biogas plants. The composition and residual methane potential of 29 digestate samples from plants operating at hydraulic retention times of 13-130 days were determined to evaluate the economic viability of extended digestion. Considerable contents of fermentable fractions, such as cellulose (8-23%), hemicellulose (1-18%), and protein (13-22%), were present in the digestate dry matter. The ultimate residual methane yields varied between 55 and 236 ml/g of volatile solids and correlated negatively with the logarithm of the hydraulic retention time (r = -0.64, p < 0.05). Economic analysis showed that extending the retention time in 20 days would be viable for 18 systems if methane were sold for 1.00 €/m3, with gains up to 40 €/year/m3 of newly installed reactor capacity. The results show the importance of operating at sufficient hydraulic retention time.

Enhanced process control of trickle-bed reactors for biomethanation by vertical profiling directed by hydrogen microsensor monitoring.

Biomethanation is an emerging Power-to-X technology enabling CO2 valorisation to produce biomethane using renewable H2. A promising reactor for facilitating biomethanation is the trickle bed reactor (TBR), however, these bioreactors are conventionally operated with a black-box approach, where the system is solely described by the input and output characteristics. This study employed a novel approach for process surveillance of internal dynamics in TBRs by installing multiple H2 microsensors along its vertical axis. The H2 microsensor monitoring was demonstrated for 135 days in a TBR integrated into a full-scale biogas plant. Despite achieving an overall CH4 productivity of 12.6 L L-1 d-1, the vertical positioning of microsensors revealed a clear zonation with CH4 productivity zones reaching 54.8 L L-1 d-1 and enabled early warning detection of deteriorating process performance days before detecting it in the product gas. Thus, vertically positioned microsensors present a promising solution for securing process stability.

Wood-Ljungdahl pathway utilisation during in situ H2 biomethanation.

Biogas production from organic waste is a waste-to-energy technology with the potential to contribute significantly to sustainable energy production. Upgrading of biogas using in situ biomethanation with hydrogen has the potential for surplus electricity storage, and delivery of biogas with a methane content of >90%, allowing for easier integration into the natural gas grid, as well as conversion to other products. Microbial communities in biomethanation reactors undergo changes, however, these changes are largely unexplored. In the present study, metagenome-resolved protein stable isotope probing (Protein-SIP) was applied to laboratory scale batch incubations operating under anaerobic digestion, and (pre-adapted) biomethanation conditions, fed with 13C-labelled bicarbonate, in order to gain insight into the microbial activities during CO2-reduction. The strongest and most microbially diverse isotopic incorporation was observed in the pre-adapted biomethanation incubation. Furthermore, divergent incorporation of 13C-labelled bicarbonate was also observed in the Wood-Ljungdahl pathway, with the anaerobic digester incubations primarily showing labelled proteins in the peripheral pathways leading toward production of energy and biomass. The pre-adapted biomethanation incubations consumed H2 and CO2, but did not convert it to CH4, suggesting the production of acetate in these incubations, which was supported by heavy labelling of key enzymes in the Wood-Ljungdahl pathway. Twelve (ten high quality) metagenome-assembled genomes (MAGs) coding for 13C-incorporated proteins were extracted from the metagenome, eight of which contained one or more of the key genes in the Wood-Ljungdahl pathway, one of which was affiliated to Methanosarcina. Together, the findings in the present study deepen our knowledge surrounding microbial communities in biomethanation systems, and contribute to the development of better strategies for implementation of biogas upgrading and microbial management.

Cellulolytic and Xylanolytic Microbial Communities Associated With Lignocellulose-Rich Wheat Straw Degradation in Anaerobic Digestion.

The enzymatic hydrolysis of lignocellulosic polymers is generally considered the rate-limiting step to methane production in anaerobic digestion of lignocellulosic biomass. The present study aimed to investigate how the hydrolytic microbial communities of three different types of anaerobic digesters adapted to lignocellulose-rich wheat straw in continuous stirred tank reactors operated for 134 days. Cellulase and xylanase activities were monitored weekly using fluorescently-labeled model substrates and the enzymatic profiles were correlated with changes in microbial community compositions based on 16S rRNA gene amplicon sequencing to identify key species involved in lignocellulose degradation. The enzymatic activity profiles and microbial community changes revealed reactor-specific adaption of phylogenetically different hydrolytic communities. The enzymatic activities correlated significantly with changes in specific taxonomic groups, including representatives of Ruminiclostridium, Caldicoprobacter, Ruminofilibacter, Ruminococcaceae, Treponema, and Clostridia order MBA03, all of which have been linked to cellulolytic and xylanolytic activity in the literature. By identifying microorganisms with similar development as the cellulase and xylanase activities, the proposed correlation method constitutes a promising approach for deciphering essential cellulolytic and xylanolytic microbial groups for anaerobic digestion of lignocellulosic biomass.

Stick or leave - Pushing methanogens to biofilm formation for ex situ biomethanation.

Biomethanation exploits the ability of methanogenic archaea to convert CO2 and renewable H2 from electrolysis to biomethane. Biofilm reactors are promising for biomethanation scale-up due to high CH4 productivity and low energy input for H2 gas-liquid mass transfer. Effects of operational conditions on biofilm dynamics remain largely uncharacterized but may increase reactor potentials further. This study investigated the effect of hydraulic retention time (HRT) on methanogenic biofilm activity and composition. Commercial carriers floating in liquid were exposed to H2/CO2 for 87 days with the liquid phase being subject to either 18 hours, 10 days, or 20 days HRT. Methanogenic biofilms were dominated by hydrogenotrophic methanogens, but biofilm CH4 productivity was enhanced at 18 hours HRT due to wash-out of competing planktonic species, which otherwise hampered proliferation of biofilm biomass at long HRT. It is suggested that high-rate biofilm reactors can increase methanogenic biofilm activity by minimizing the liquid's H2 exposure.

How Low Can You Go: Methane Production of Methanobacterium congolense at Low CO2 Concentrations.

Autotrophic hydrogenotrophic methanogens use H2/CO2 as sole carbon and energy source. In contrast to H2, CO2 is present in high concentrations in environments dominated by methanogens e.g., anaerobic digesters (AD), and is therefore rarely considered to be a limiting factor. Nonetheless, potential CO2 limitation can be relevant in the process of biomethanation, a power-to-gas technology, where biogas is upgraded by the addition of H2 and ideally reduce the CO2 concentration in the produced biogas to 0-6%. H2 is effectively utilized by methanogens even at very low concentrations, but little is known about the impact of low CO2 concentrations on methanogenic activity. In this study, CO2 consumption and CH4 production kinetics under low CO2 concentrations were studied, using a hydrogenotrophic methanogen, Methanobacterium congolense, as model organism. We found that both cellular growth and methane production were limited at low CO2 concentrations (here expressed as Dissolved Inorganic Carbon, DIC). Maximum rates (V max) were reached at [DIC] of 100 mM (extrapolated), with a CO2 consumption rate of 69.2 fmol cell-1 d-1 and a CH4 production rate of 48.8 fmol cell-1 d-1. In our experimental setup, 80% of V max was achieved at [DIC] >9 mM. DIC half-saturation concentrations (K m) was about 2.5 mM for CO2 consumption and 2.2 mM for CH4 production. No CH4 production could be detected below 44.4 µM [DIC]. These data revealed that the limiting concentration of DIC may be much higher than that of H2 for a hydrogenotrophic methanogen. However, DIC is not a limiting factor in ADs running under standard operating conditions. For biomethanation, the results are applicable for both in situ and ex situ biomethanation reactors and show that biogas can be upgraded to concentrations of 2% CO2 (98% CH4) while still retaining 80% V max at pH 7.5 evaluated from M. congolense. Since DIC concentration can vary significantly with pH and pCO2 during biomethanation, monitoring DIC concentration through pH and pCO2 is therefore important for keeping optimal operational conditions for the biomethanation process.

Parameters affecting acetate concentrations during in-situ biological hydrogen methanation.

Surplus electricity may be supplied to anaerobic digesters as H2 gas to upgrade the CH4 content of biogas. Acetate accumulation has been observed following H2 injections, but the parameters determining the degree of acetate accumulation are not well understood. The pathways involved during H2 consumption and acetate kinetics were evaluated in continuous lab reactors and parallel batch 13C experiments. Acetate accumulation increased during initial H2 injections as organic loading rate increased and CO2 levels decreased below 7%. The share of CH4 in H2 and 13C mass balances increased after repeated H2 injections, which corresponded with the increase of Methanomicrobiales observed via qPCR. The organic loading rate, the inorganic carbon level and level of methanogen adaption hence determine acetate kinetics during biomethanation of H2. The three identified parameters may form the base of a decision tool to assess acetate accumulation during H2 injections to an anaerobic digester.

In-situ biogas upgrading with pulse H2 additions: The relevance of methanogen adaption and inorganic carbon level.

Surplus electricity from fluctuating renewable power sources may be converted to CH4 via biomethanisation in anaerobic digesters. The reactor performance and response of methanogen population of mixed-culture reactors was assessed during pulsed H2 injections. Initial H2 uptake rates increased immediately and linearly during consecutive pulse H2 injections for all tested injection rates (0.3 to 1.7LH2/Lsludge/d), while novel high throughput mcrA sequencing revealed an increased abundance of specific hydrogenotrophic methanogens. These findings illustrate the adaptability of the methanogen population to H2 injections and positively affects the implementation of biomethanisation. Acetate accumulated by a 10-fold following injections exceeding a 4:1 H2:CO2 ratio and may act as temporary storage prior to biomethanisation. Daily methane production decreased for headspace CO2 concentrations below 12% and may indicate a high sensitivity of hydrogenotrophic methanogens to CO2 limitation. This may ultimately decide the biogas upgrading potential which can be achieved by biomethanisation.

Dynamic microbial response of sulfidogenic wastewater biofilm to nitrate.

Nitrate is one of the chemicals often added to wastewater to control hydrogen sulfide production by sulfate-reducing bacteria (SRB). While the effect of nitrate in various SRB pure cultures is well documented, the effect observed in mixed microbial communities is not consistent. This study investigates the response of mixed SRB communities to nitrate, by examining the changes in activity and community composition of sulfidogenic wastewater biofilm over a 10-day period with 10 mmol L(-1) nitrate exposure. Biofilms were enriched in SRB belonging to the Desulfobacter, Desulfobulbus, Desulfomicrobium, and Desulfovibrio genera. Nitrate exposure decreased dsrB transcription within 4 h, and sulfate consumption within 10 days, but it did not fully eliminate sulfide production in the biofilms. The effect of nitrate on SRB was genus specific; Desulfobacter and Desulfobulbus disappeared while Desulfovibrio and Desulfomicrobium persisted in the biofilms. Nitrate exposure also led to the rapid proliferation of nitrate-reducing bacteria within the biofilms, and increased the biofilm thickness. Nitrate consumption began within 2 h of nitrate exposure and gradually increased in rate over time. Transcription of the nitrate reductase napA, and the diversity of nitrate reductase genes narG and napA also increased concurrently. Our results demonstrate that some SRB, presumably those able to tolerate or detoxify nitrite, will persist in sulfidogenic wastewater biofilms despite continuous exposure to high levels of nitrate. Nitrate is therefore unlikely to provide lasting hydrogen sulfide suppression in wastewater biofilms harboring Desulfovibrio or Desulfomicrobium populations.