Search Results (14)

Anaerobic digestion is a critical technology for pollution control, resource capacity enhancement, and sludge management, necessitating improvements in its efficiency. Formate serves as an electron carrier in syntrophic oxidation of volatile fatty acids (VFAs) during anaerobic digestion. The accumulation of formate can exert an inhibitory effect on the anaerobic digestion process. However, the stress concentration and the mechanism of the formate are not as simple as theoretical calculations based on thermodynamics. Thus, we investigated the response to different concentrations of formate in the syntrophic oxidation of propionate. The anaerobic sludge system and syntrophic co-culture system were applied. The propionate showed more stable degradation when formate dosage ranged from 5 to 10 mM. However, when the formate dosage reached 50 mM, the concentration of propionate was significantly higher than that of CK group, and the propionate metabolism was significantly inhibited. The reduction in functional flora and homogeneous metabolic pathways were found to be unfavorable for the stable progression of syntrophic propionate metabolism. Thus, the enhancement of homoacetogenesis can be a strategy adopted by the sludge system to alleviate formate stress. The methylmalonyl-CoA (MMC) pathway was inhibited under formate stress; the downregulation of RNA transcription of formate dehydrogenase (FDH) and hydrogenase (Hyd) related to MMC pathway may be the main reason for the inhibition of syntrophic propionate oxidation. The anaerobic sludge experiment and the co-culture experiment elucidated the mechanism of action of formate from both macroscopic rules and microscopic molecular mechanisms, respectively. Full article

The biological upgrading of biogas to simulate natural gas properties contributes to the sustainable establishment of biogas technology. It is an alternative technology to the conventional physicochemical methods applied in biomethane plants and has been studied mainly in thermophilic conditions. Developing an enriched culture for converting the CO₂ of biogas to CH₄ in mesophilic conditions was the subject of the present study, which could facilitate the biological process and establish it in the mesophilic range of temperature. The enrichment took place via successive dilutions in a bubble bioreactor operated in fed-batch mode. The methane percentage was recorded at 95.5 ± 1.2% until the end of the experiment. The methane production rate was 0.28–0.30 L L⁻¹ d⁻¹ following the low hydrogen loading rate (1.2 ± 0.1 L L⁻¹ d⁻¹) applied to avoid acetate accumulation. Hydrogenotrophic methanogens, Methanobrevibacter sp., were identified at a proportion of 97.9% among the Archaea and 60% of the total population of the enriched culture. Moreover, homoacetogens (Sporomusa sp.) and acetate oxidizers (Proteiniphilum sp.) were also detected, indicating that a possible metabolic pathway for CH₄ production from CO₂ is via homoacetogenesis and syntrophic acetate oxidation, which kept the acetate concentration at a level of 143 ± 13 mg L⁻¹. It was found that adding NaHCO₃ was adequate to sustain the pH at 8.25. Full article

The effects of the inoculum origin, temperature or operational changes on ex situ biomethanation by complex microbial communities have been investigated; however, it remains unclear how the diversity of the inoculum influences the process and its stability. We explored the effect of microbial diversity of four inocula (coded as PF, WW, S37 and Nrich) on methane production, process stability and the formation of volatile fatty acids as by-products. The highest methane amounts produced were 3.38 ± 0.37 mmol, 3.20 ± 0.07 mmol, 3.07 ± 0.27 mmol and 3.14 ± 0.06 mmol for PF, WW, S37 and Nrich, respectively. The highest acetate concentration was found in less diverse cultures (1679 mg L⁻¹ and 1397 mg L⁻¹ for S37 and Nrich, respectively), whereas the acetate concentrations remained below 30 mg L⁻¹ in the more diverse cultures. The maximum concentration of propionate was observed in less diverse cultures (240 mg L⁻¹ and 37 mg L⁻¹ for S37 and Nrich cultures, respectively). The highly diverse cultures outperformed the medium and low diversity cultures in the long-term operation. Methanogenic communities were mainly composed of hydrogenotrophic methanogens in all cultures. Aceticlastic methanogenesis was only active in the highly diverse sludge community throughout the experiment. The more diverse the inocula, the more methane was produced and the less volatile fatty acids accumulated, which could be attributed to the high number of microbial functions working together to keep a stable and balanced process. It is concluded that the inoculum origin and its diversity are very important factors to consider when the biomethanation process is performed with complex microbial communities. Full article

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. Full article

Mixed or pure cultures can be used for biomethanation of hydrogen. Sodium 2-bromoethanesulfonate (BES) is an inhibitor of methanogenesis used to investigate competing reactions like homoacetogenesis in mixed cultures. To understand the effect of BES on the hydrogenotrophic metabolism in a biomethanation process, anaerobic granules from a wastewater treatment plant, a hydrogenotrophic enrichment culture, and pure cultures of Methanococcus maripaludis and Methanobacterium formicicum were incubated under H₂/CO₂ headspace in the presence or absence of BES, and the turnover of H₂, CO₂, CH₄, formate and acetate was analyzed. Anaerobic granules produced the highest amount of formate after 24 h of incubation in the presence of BES. Treating the enrichment culture with BES led to the accumulation of formate. M. maripaludis produced more formate than M. formicicum when treated with BES. The non-inhibited methanogenic communities produced small amounts of formate whereas the pure cultures did not. The highest amount of acetate was produced by the anaerobic granules concomitantly with formate consumption. These results indicate that formate is an important intermediate of hydrogenotrophic metabolism accumulating upon methanogenesis inhibition. Full article

Bioelectrochemical systems are emerging technologies for the reduction in CO₂ in fuels and chemicals, in which anaerobic chemoautotrophic microorganisms such as methanogens and acetogens are typically used as biocatalysts. The anaerobic digestion digestate represents an abundant source of methanogens and acetogens microorganisms. In a mixed culture environment, methanogen’s inhibition is necessary to avoid acetate consumption by the presence of acetoclastic methanogens. In this study, a methanogenesis inhibition approach based on the thermal treatment of mixed cultures was adopted and evaluated in terms of acetate production under different tests consisting of hydrogenophilic and bioelectrochemical experiments. Batch experiments were carried out under hydrogenophilic and bioelectrochemical conditions, demonstrating the effectiveness of the thermal treatment and showing a 30 times higher acetate production with respect to the raw anaerobic digestate. Moreover, a continuous flow bioelectrochemical reactor equipped with an anion exchange membrane (AEM) successfully overcomes the methanogens reactivation, allowing for a continuous acetate production. The AEM membrane guaranteed the migration of the acetate from the biological compartment and its concentration in the abiotic chamber avoiding its consumption by acetoclastic methanogenesis. The system allowed an acetate concentration of 1745 ± 30 mg/L in the abiotic chamber, nearly five times the concentration measured in the cathodic chamber. Full article

The performance of a mixed microbial community was tested in lab-scale power-to-methane reactors at 55 °C. The main aim was to uncover the responses of the community to starvation and stoichiometric H₂/CO₂ supply as the sole substrate. Fed-batch reactors were inoculated with the fermentation effluent of a thermophilic biogas plant. Various volumes of pure H₂/CO₂ gas mixtures were injected into the headspace daily and the process parameters were followed. Gas volumes and composition were measured by gas-chromatography, the headspace was replaced with N₂ prior to the daily H₂/CO₂ injection. Total DNA samples, collected at the beginning and end (day 71), were analyzed by metagenome sequencing. Low levels of H₂ triggered immediate CH₄ evolution utilizing CO₂/HCO₃⁻ dissolved in the fermentation effluent. Biomethanation continued when H₂/CO₂ was supplied. On the contrary, biomethane formation was inhibited at higher initial H₂ doses and concomitant acetate formation indicated homoacetogenesis. Biomethane production started upon daily delivery of stoichiometric H₂/CO₂. The fed-batch operational mode allowed high H₂ injection and consumption rates albeit intermittent operation conditions. Methane was enriched up to 95% CH₄ content and the H₂ consumption rate attained a remarkable 1000 mL·L⁻¹·d⁻¹. The microbial community spontaneously selected the genus Methanothermobacter in the enriched cultures. Full article

In this study, the microbiomes linked with the operational parameters in seven mesophilic full-scale AD plants mainly treating food waste (four plants) and sewage sludge (three plants) were analyzed. The results obtained indicated lower diversity and evenness of the microbial population in sludge digestion (SD) plants compared to food digestion (FD) plants. Candidatus Accumulibacter dominated (up to 42.1%) in SD plants due to microbial immigration from fed secondary sludge (up to 89%). Its potential activity in SD plants was correlated to H₂ production, which was related to the dominance of hydrogenotrophic methanogens (Methanococcus). In FD plants, a balance between the hydrogenotrophic and methylotrophic pathways was found, while Flavobacterium and Levilinea played an important role during acidogenesis. Levilinea also expressed sensitivity to ammonia in FD plants. The substantial differences in hydraulic retention time (HRT), organic loading rate (OLR), and total ammonium nitrogen (TAN) among the studied FD plants did not influence the archaeal methane production pathway. In addition, the bacterial genera responsible for acetate production through syntrophy and homoacetogenesis (Smithella, Treponema) were present in all the plants studied. Full article

This study presents a series of experiments to test the integration of syngas fermentation into a single-cell microbial electrosynthesis (MES) process. Minimal gas–liquid mass transfer is the primary bottleneck in such gas-fermentation processes. Therefore, we hypothesized that MES integration could trigger the thermodynamic barrier, resulting in higher gas–liquid mass transfer and product-formation rates. The study was performed in three different phases as batch experiments. The first phase dealt with mixed-culture fermentation at 1 bar H₂ headspace pressure. During the second phase, surface electrodes were integrated into the fermentation medium, and investigations were performed in open-circuit mode. In the third phase, the electrodes were poised with a voltage, and the second phase was extended in closed-circuit mode. Phase 2 demonstrated three times the gas consumption (1021 mmol) and 63% more production of acetic acid (60 mmol/L) than Phase 1. However, Phase 3 failed; at –0.8 V, acetic acid was oxidized to yield hydrogen gas in the headspace. Full article

Energy derived from water-rock interactions such as serpentinization and radiolysis, among others, can sustain microbial ecosystems deep within the continental crust, expanding the habitable biosphere kilometers below the earth’s surface. Here, we describe a viable microbial community including sulfate-reducing microorganisms from one such subsurface lithoautotrophic ecosystem hosted in fracture waters in the Canadian Shield, 2.4 km below the surface in the Kidd Creek Observatory in Timmins, Ontario. The ancient groundwater housed in fractures in this system was previously shown to be rich in abiotically produced hydrogen, sulfate, methane, and short-chain hydrocarbons. We have further investigated this system by collecting filtered water samples and deploying sterile in situ biosampler units into boreholes to provide an attachment surface for the actively growing fraction of the microbial community. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, and DNA sequencing analyses were undertaken to classify the recovered microorganisms. Moderately halophilic taxa (e.g., Marinobacter, Idiomarina, Chromohalobacter, Thiobacillus, Hyphomonas, Seohaeicola) were recovered from all sampled boreholes, and those boreholes that had previously been sealed to equilibrate with the fracture water contained taxa consistent with sulfate reduction (e.g., Desulfotomaculum) and hydrogen-driven homoacetogenesis (e.g., Fuchsiella). In contrast to this “corked” borehole that has been isolated from the mine environment for approximately 7 years at the time of sampling, we sampled additional open boreholes. The waters flowing freely from these open boreholes differ from those of the long-sealed borehole. This work complements ongoing efforts to describe the microbial diversity in fracture waters at Kidd Creek in order to better understand the processes shaping life in the deep terrestrial subsurface. In particular, this work demonstrates that anaerobic bacteria and known halophilic taxa are present and viable in the fracture waters presently outflowing from existing boreholes. Major cations and anions found in the fracture waters at the 2.4 km level of the mine are also reported. Full article

Biomethanation is a promising solution to convert H₂ (produced from surplus electricity) and CO₂ to CH₄ by using hydrogenotrophic methanogens. In ex situ biomethanation with mixed cultures, homoacetogens and methanogens compete for H₂/CO₂. We enriched a hydrogenotrophic microbiota on CO₂ and H₂ as sole carbon and energy sources, respectively, to investigate these competing reactions. The microbial community structure and dynamics of bacteria and methanogenic archaea were evaluated through 16S rRNA and mcrA gene amplicon sequencing, respectively. Hydrogenotrophic methanogens and homoacetogens were enriched, as acetate was concomitantly produced alongside CH₄. By controlling the media composition, especially changing the reducing agent, the formation of acetate was lowered and grid quality CH₄ (≥97%) was obtained. Formate was identified as an intermediate that was produced and consumed during the bioprocess. Stirring intensities ≥ 1000 rpm were detrimental, probably due to shear force stress. The predominating methanogens belonged to the genera Methanobacterium and Methanoculleus. The bacterial community was dominated by Lutispora. The methanogenic community was stable, whereas the bacterial community was more dynamic. Our results suggest that hydrogenotrophic communities can be steered towards the selective production of CH₄ from H₂/CO₂ by adapting the media composition, the reducing agent and the stirring intensity. Full article

Global warming is expected to increase the rate of CH₄ emission from acidic peatlands leading to an increased interest on its mechanisms of formation. The main routes are through the reduction of CO₂ by molecular hydrogen and through the cleavage of acetate. A predominance of the former, a reaction which also competes with homoacetogenesis to form acetate, may enrich the media in acetate, which could potentially be incorporated in the peat molecular markers. Acetates of triterpenoid biomarkers have been identified in peats and lake sediments and related to the input of higher plants. Nevertheless, the acetyl derivatives are found in very low amounts in fresh plants and in much lower amount than other derivatives with alcohol or ketone functional groups. The dichloromethane/methanol extracts of Asturian peat bog profiles (North Spain) were analyzed using gas chromatography/mass spectrometry (GC/MS) and compound-specific-isotope-analysis (CSIA). They show abundance of acetates of compounds with oleanane, ursane, and lupane skeletons derived from higher plants and with hopane skeleton, which can be considered a characteristic of these peats. Two families of 3-oxyhopenyl acetates with -17(21)- and -22(29)- configurations were detected in the upper part of the peat profiles, having a δ¹³C isotopic composition enriched by 4‰ compared with that of higher plant triterpenoids, and similar to that of microorganism-derived regular hopanoids. Both the acetate and ketone derivatives with the oxygenated functionality at C-3 were generally present in a given extract and tended to accumulate at certain depth in the profiles and in specific levels. The widespread occurrence of acetyl-derivatives, their higher concentration in the deeper layers of the peat, the fact that the acetates correspond to different compound families of diverse source and the very low amount of acetates identified in Ericaceae-contributing to the peat compared to the alcohols suggest that they were formed in the peat under particularly favorable environmental conditions. We postulate that these conditions could have been the existence of a medium enriched in acetic acid produced by the dominance of hydrogenotrophic methanogenesis and/or homoacetogenesis over acetoclastic methanogenesis. This phenomenon that has been preferentially described in Sphagnum bogs at high latitudes, and in the deeper layers of peat, appears to be also present in the temperate peats of the Asturian coast. Full article

Anoxic aquifers suffer from energy limitations due to the unavailability of organic substrates, as dictated by hydrogen (H₂) for various electron-accepting processes. This deficiency often results in the accumulation of persistent organic pollutants, where bioremediation using organic compounds often leads to secondary contamination. This study involves the reductive dechlorination of pentachlorophenol (PCP) by dechlorinators that do not use H₂ directly, but rather through a reduced state of humin—a solid-phase humic substance—as the extracellular electron donor, which requires an organic donor such as formate, lactate, etc. This shortcoming was addressed by the development of an anaerobic mixed culture that was capable of reductively dechlorinating PCP using humin under autotrophic conditions induced by homoacetogenesis. Here, H₂ was used for carbon-dioxide fixation to acetate; the acetate produced was used for the reduction of humin; and consequently used for dechlorination through reduced humin. The 16SrRNA gene sequencing analysis showed Dehalobacter and Dehalobacterium as the possible dechlorinators, while Clostridium and Oxobacter were identified as the homoacetogens. Thus, this work contributes to the development of an anaerobic consortium that balanced H₂ dependency, where efficiency of humin reduction extends the applicability of anaerobic microbial remediation in aquifers through autotrophy, syntrophy, and reductive dechlorination. Full article

This study examined the influence of biohydrogen fermentation under the high bicarbonate alkalinity (BA) and pH to optimize these critical parameters. When sucrose was used as a substrate, hydrogen was produced over a wide range of pH values (5–9) under no BA supplementation; however, BA affected hydrogen yield significantly under different initial pHs (5–10). The actual effect of high BA using raw piggery waste (pH 8.7 and BA 8.9 g CaCO₃/L) showed no biogas production or propionate/acetate accumulation. The maximum hydrogen production rate (0.32 L H₂/g volatile suspended solids (VSS)-d) was observed at pH 8.95 and 3.18 g CaCO₃/L. BA greater than 4 g CaCO₃/L also triggered lactate-type fermentation, leading to propionate accumulation, butyrate reduction and homoacetogenesis, potentially halting the hydrogen production rate. These results highlight that the substrate with high BA need to amend adequately to maximize hydrogen production. Full article