Biomethanation processes: new insights on the effect of a high H2 partial pressure on microbial communities.
ABSTRACT: Background:Biomethanation is a promising solution to upgrade the CH4 content in biogas. This process consists in the injection of H2 into an anaerobic digester, using the capacity of indigenous hydrogenotrophic methanogens for converting the injected H2 and the CO2 generated from the anaerobic digestion process into CH4. However, the injection of H2 could cause process disturbances by impacting the microbial communities of the anaerobic digester. Better understanding on how the indigenous microbial community can adapt to high H2 partial pressures is therefore required. Results:Seven microbial inocula issued from industrial bioprocesses treating different types of waste were exposed to a high H2 partial pressure in semi-continuous reactors. After 12 days of operation, even though both CH4 and volatile fatty acids (VFA) were produced as end products, one of them was the main product. Acetate was the most abundant VFA, representing up to 94% of the total VFA production. VFA accumulation strongly anti-correlated with CH4 production according to the source of inoculum. Three clusters of inocula were distinguished: (1) inocula leading to CH4 production, (2) inocula leading to the production of methane and VFA in a low proportion, and (3) inocula leading to the accumulation of mostly VFA, mainly acetate. Interestingly, VFA accumulation was highly correlated to a low proportion of archaea in the inocula, a higher amount of homoacetogens than hydrogenotrophic methanogens and, the absence or the very low abundance in members from the Methanosarcinales order. The best methanogenic performances were obtained when hydrogenotrophic methanogens and Methanosarcina sp. co-dominated all along the operation. Conclusions:New insights on the microbial community response to high H2 partial pressure are provided in this work. H2 injection in semi-continuous reactors showed a significant impact on microbial communities and their associated metabolic patterns. Hydrogenotrophic methanogens, Methanobacterium sp. or Methanoculleus sp. were highly selected in the reactors, but the presence of co-dominant Methanosarcinales related species were required to produce higher amounts of CH4 than VFA.
Project description:Background:Commercial biogas upgrading facilities are expensive and consume energy. Biological biogas upgrading may serve as a low-cost approach because it can be easily integrated with existing facilities at biogas plants. The microbial communities found in anaerobic digesters typically contain hydrogenotrophic methanogens, which can use hydrogen (H2) as a reducing agent for conversion of carbon dioxide (CO2) into methane (CH4). Thus, biological biogas upgrading through the exogenous addition of H2 into biogas digesters for the conversion of CO2 into CH4 can increase CH4 yield and lower CO2 emission. Results:The addition of 4 mol of H2 per mol of CO2 was optimal for batch biogas reactors and increased the CH4 content of the biogas from 67 to 94%. The CO2 content of the biogas was reduced from 33 to 3% and the average residual H2 content was 3%. At molar H2:CO2 ratios > 4:1, all CO2 was converted into CH4, but the pH increased above 8 due to depletion of CO2, which negatively influenced the process stability. Additionally, high residual H2 content in these reactors was unfavourable, causing volatile fatty acid accumulation and reduced CH4 yields. The reactor microbial communities shifted in composition over time, which corresponded to changes in the reactor variables. Numerous taxa responded to the H2 inputs, and in particular the hydrogenotrophic methanogen Methanobacterium increased in abundance with addition of H2. In addition, the apparent rapid response of hydrogenotrophic methanogens to intermittent H2 feeding indicates the suitability of biological methanation for variable H2 inputs, aligning well with fluctuations in renewable electricity production that may be used to produce H2. Conclusions:Our research demonstrates that the H2:CO2 ratio has a significant effect on reactor performance during in situ biological methanation. Consequently, the H2:CO2 molar ratio should be kept at 4:1 to avoid process instability. A shift toward hydrogenotrophic methanogenesis was indicated by an increase in the abundance of the obligate hydrogenotrophic methanogen Methanobacterium.
Project description:Hydrogenotrophic methanogens can use gaseous substrates, such as H2 and CO2, in CH4 production. H2 gas is used to reduce CO2. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH4 production from CO2 and H2. CO2 and H2 were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5-5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH4 production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH4 production process. The results show that acidic operation of a CH4 production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.
Project description: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.
Project description:In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a Methanoculleus sp. and a Methanothermobacter sp.) and one species of aceticlastic methanogen (i.e., a Methanosarcina sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the Methanoculleus sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the Methanothermobacter sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the Methanosarcina sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.
Project description:The feasibility of using pre-acclimated activated carbon to start up microbial electrolysis cell assisted anaerobic digester (MEC-AD) has been testified in this study. Two identical lab-scale digesters were separately packed with granular activated carbon (GAC) and powered activated carbon (PAC), which were initially acclimated as anaerobic digester and then transferred to MEC-AD. When a voltage of 0.5 V was applied, increased methane generation and substrate removal rates were observed. Hydrogenotrophic methanogens predominated in both digesters before and after transition, indicating that the pre-cultured microbial community on carbon materials could provide necessary microbiome favorable for starting up MECs. Although a low abundance of Geobacter was detected in inoculum, a rapid propagation could be realized when reactors were subjected to the electro-stimulation. The abundance of Methanosarcina closely attached to PAC was four times than that of GAC, which might be partially contributed to the improved resilience of anaerobic digester subjected to electro-stimulation.
Project description:Anaerobic digesters become unstable when operated at a high organi c loading rate (OLR). Investigating the microbial community response to OLR disturbance is helpful for achieving efficient and stable process operation. However, previous studies have only focused on community succession during different process stages. How does community succession influence process stability? Is this kind of succession resilient? Are any key microbial indicator closely related to process stability? Such relationships between microbial communities and process stability are poorly understood.In this study, a mesophilic anaerobic digester for treating food waste (FW) was operated to study the microbial diversity and dynamicity due to OLR disturbance. Overloading resulted in proliferation of acidogenic bacteria, and the resulting high volatile fatty acid (VFA) yield triggered an abundance of acetogenic bacteria. However, the abundance and metabolic efficiency of hydrogenotrophic methanogens decreased after disturbance, and as a consequence, methanogens and acetogenic bacteria could not efficiently complete the syntrophy. This stress induced the proliferation of homoacetogens as alternative hydrogenotrophs for converting excessive H2 to acetate. However, the susceptible Methanothrix species also failed to degrade the excessive acetate. This metabolic imbalance finally led to process deterioration. After process recovery, the digester gradually returned to its original operational conditions, reached close to its original performance, and the microbial community profile achieved a new steady-state. Interestingly, the abundance of Syntrophomonas and Treponema increased during the deteriorative stage and rebounded after disturbance, suggesting they were resilient groups.Acidogenic bacteria showed high functional redundancy, rapidly adapted to the increased OLR, and shaped new microbial community profiles. The genera Syntrophomonas and Treponema were resilient groups. This observation provides insight into the key microbial indicator that are closely related to process stability. Moreover, the succession of methanogens during the disturbance phase was unsuitable for the metabolic function needed at high OLR. This contradiction resulted in process deterioration. Thus, methanogenesis is the main step that interferes with the stable operation of digesters at high OLR. Further studies on identifying and breeding high-efficiency methanogens may be helpful for breaking the technical bottleneck of process instability and achieving stable operation under high OLR.
Project description:Commercial biogas production takes place by complex microbial communities enclosed in controlled "technical ecosystems". Once established, the communities tend to be resilient towards disturbances, although the relative abundance of their members may vary. The start-up phase, during which the community establishes itself, is therefore decisive for the later performance of the reactor. In this study, we followed the first 240 days of a standard agricultural energy crop digester consisting of a 400 m(3) plug flow fermenter and a 1000 m(3) agitated post digester, operated at 40-45 °C. The feed consisted of corn and later grass silage augmented by ground wheat. Changes in both the eubacterial and methanogenic archaeal communities were followed by automated ribosomal intergenic spacer analysis (ARISA). In addition the copy number of the methyl-coenzyme reductase A (mcrA)-genes found in all known methanogens were followed by quantitative PCR, while selected samples from two phases-one early, one late-of the community structure development were subjected to high throughput sequencing. Biogas volume and composition (CH4, CO2, H2, H2S, O2), pH, ammonia-N, and volatile fatty acids (VFA), were measured as part of the routine process control. VFA/TIC values were calculated on this basis. Whereas the total gas production of the plant established itself at about 2500 m(3) biogas per day within the first months, the composition of the microbial communities showed distinct spatial and temporal differences over the investigated time period. Absolute values for DNA isolation procedures are difficult to certify, hence comparative results on community structures obtained using standardized ARISA with identical primers are of value. Moreover, ARISA patterns can be statistically analyzed to identify distinct subgroups and transitions between them as well as serial correlations. Thereby the microbial community and its structural development can be correlated with statistical relevance to changes in operational (feed) and process parameters (pH-value, biogas composition). In particular when augmented by deep sequencing data of judiciously chosen samples, this allows a hitherto unknown level of insight into the performance of technical biogas plants.
Project description:Influenced by feedstock type and microbial inoculum, different microbial groups must precisely interact for high-quality biogas yields. As a first approach for optimization, this study aimed to identify through time the biogas-producing microbial community in a 10-ton dry anaerobic digester treating cattle manure by denaturing gradient gel electrophoresis (DGGE) and metagenomics. Moreover, the associated bovine residues or feedstocks (leachate, manure, oxidation lagoon water, rumen) were also characterized to determine their contribution. A diverse and dynamic community characterized by Bacteria (82%-88%) and a considerable amount of Archaea (8%-15%) presented profiles particular to each stage of biogas production. Eukaryotes (2.6%-3.6%), mainly fungi, were a minor but stable component. Proteobacteria represented 47% of the community at the start of the run but only 18% at the end, opposite to the Bacteroidetes/Chlorobi group (8% and 20%, respectively), while Firmicutes (12%-18%) and Actinobacteria (12%-32%) remained relatively constant. Methanogens of the order Methanomicrobiales represented by several species of Methanoculleus were abundant at the end of the run (77%) contrary to Methanosarcinales (11%) and Methanobacteriales (0.7%). Therefore, methanogenesis mainly occurred by the hydrogenotrophic pathway. Manure and oxidation lagoon water seemed to contribute key microorganisms, while rumen dominated by Methanobrevibacter (72%) did not proliferate in the digester. Manure particularly possessed Methanoculleus (24%) and uncultured methanogens identified by DGGE, whereas oxidation lagoon was exclusively abundant in Methanolinea (18%) and Methanosaeta (19%). Leachate, as the microbial inoculum from a previous run, adequately preserved the biogas-producing community. These results could lead to higher biogas yields through bioaugmentation strategies by incorporating higher proportions or an enriched inoculum from the relevant feedstocks.
Project description:Anaerobic digestion is a widely used technology for waste stabilization and generation of biogas, and has recently emerged as a potentially important process for the production of high value volatile fatty acids (VFAs) and alcohols. Here, three reactors were seeded with inoculum from a stably performing methanogenic digester, and selective operating conditions (37°C and 55°C; 12 day and 4 day solids retention time) were applied to restrict methanogenesis while maintaining hydrolysis and fermentation. Replicated experiments performed at each set of operating conditions led to reproducible VFA production profiles which could be correlated with specific changes in microbial community composition. The mesophilic reactor at short solids retention time showed accumulation of propionate and acetate (42 ± 2% and 15 ± 6% of CODhydrolyzed, respectively), and dominance of Fibrobacter and Bacteroidales. Acetate accumulation (>50% of CODhydrolyzed) was also observed in the thermophilic reactors, which were dominated by Clostridium. Under all tested conditions, there was a shift from acetoclastic to hydrogenotrophic methanogenesis, and a reduction in methane production by >50% of CODhydrolyzed. Our results demonstrate that shortening the SRT and increasing the temperature are effective strategies for driving microbial communities towards controlled production of high levels of specific volatile fatty acids.
Project description:Hydrogenotrophic methanogens are an intriguing group of microorganisms from the domain Archaea. Methanogens exhibit extraordinary ecological, biochemical, and physiological characteristics and possess a huge biotechnological potential. Yet, the only possibility to assess the methane (CH4) production potential of hydrogenotrophic methanogens is to apply gas chromatographic quantification of CH4. In order to be able to effectively screen pure cultures of hydrogenotrophic methanogens regarding their CH4 production potential we developed a novel method for indirect quantification of the volumetric CH4 production rate by measuring the volumetric water production rate. This method was established in serum bottles for cultivation of methanogens in closed batch cultivation mode. Water production was estimated by determining the difference in mass increase in a quasi-isobaric setting. This novel CH4 quantification method is an accurate and precise analytical technique, which can be used to rapidly screen pure cultures of methanogens regarding their volumetric CH4 evolution rate. It is a cost effective alternative determining CH4 production of methanogens over CH4 quantification by using gas chromatography, especially if applied as a high throughput quantification method. Eventually, the method can be universally applied for quantification of CH4 production from psychrophilic, thermophilic and hyperthermophilic hydrogenotrophic methanogens.