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Considering that cathode of microbial electrochemical system (MES) is a good electrons source for methane production via direct/indirect electron transfer to electroactive microorganisms, and that Fe(0) is also a confirmed electron donor for some electroactive microorganisms through metal-microbe direct electron transfer (DET), Fe(0)-cathode was equipped into an MES digester to enhance cathodic methane production. The results of this study indicated that the potential DET participator, Clostridium possibly obtained electrons directly from Fe(0)-cathode via metal-microbe electrons transfer, then transferred electrons directly to the definite DET participators, Methanosarcina/Methanothrix via microbe-microbe electrons transfer for CH4 production. In addition, Methanobacterium is another specially enriched methanogen on Fe(0)-cathode, which might obtain electrons directly from Fe(0)-cathode to produce CH4 via metal/electrode-microbe DET. The increment of conductivity of cathodic sludge in Fe(0)-cathode MES digester (R1) further confirmed the enrichment of electroactive microorganisms participating in DET process. As a consequence, a higher CH4 production (1205-1508 mL/d) and chemical oxygen demand (COD) removal (79.0%-93.8%) were achieved in R1 compared with graphite-cathode MES digester (R2, 720-1090 mL/d and 63.6%-85.6%) and the conventional anaerobic digester (R3, 384-428 mL/d and 35.2%-41.0%). In addition, energy efficiency calculated indicated that the output energy of CH4 production was 8.16 folds of electricity input in Fe(0)-cathode MES digester.

Two anaerobic membrane bioreactors (AnMBRs) equipped with different membrane pore size (0.4 or 0.05 µm) were operated at 25˚C and fed with domestic wastewater. The hydraulic retention time (HRT) of the reactors was shortened. The microbial communities of the two AnMBRs were investigated by 16S rRNA gene amplicon sequencing to see the effects of HRT. The predominant Archaea was an aceticlastic methanogen Methanosaeta. The composition of hydrogenotrophic methanogens changed with the HRTs: the population of Methanobacterium was higher for longer HRTs, whereas the population of unclassified Methanoregulaceae was higher for shorter HRTs. The Anaerolineae, Bacteroidia and Clostridia bacteria were dominant in both of the reactors, with a combined relative abundance of over 55%. The relative abundance of Anaerolineae was proportional to the biogas production performance. The change in the population of hydrogenotrophic methanogens or Anaerolineae can be used as an indicator for process monitoring. The sum of the relative abundance of Anaerolineae and Clostridia fluctuated slightly with changes in the HRT in both AnMBRs when the reactor was stably operated. The co-occurrence analysis revealed the relative abundance of the operational taxonomic units belonging to Anaerolineae and Clostridia was functionally equivalent during the treatment of real domestic sewage. A principal coordination analysis revealed that the changes in the microbial community in each reactor were consistent with the change of HRT. In addition, both the HRT and the stability of the process are important factors for maintaining microbial community structures.

Recent investigations verified CaO2 accelerated hydrolysis of lignocellulose, but its residue caused a harmful oxidizing environment. This study therefore proposed the combined CaO2 pretreatment and zero-valent iron (ZVI) addition to enhance high-solid anaerobic digestion of wheat straw, trying to eliminate negative effect of residual oxides. Effects of CaO2 pretreatment without/with ZVI addition on digestion performance were investigated. Results showed that in non-ZVI added digesters, as CaO2 dosage increased, total production of SCOD and VFAs increased linearly, while activities of coenzyme F420 and electron transport system were negatively correlated. Pretreatment using appropriate CaO2 dosage indirectly increased biogas production by promoting hydrolysis and acidification. Comparably, initial ORP in ZVI-added digesters sharply declined below −350 mV, which was suitable for methanogen growth. ZVI significantly improved activities of cellulase, hemicellulase, acetic kinase and coenzyme F420, with the highest improvement percentages of 138%, 48%, 35% and 58%, respectively. Since ZVI could provide electrons for conversion of CO2 to CH4, compared with non-ZVI added digesters (R0-R4), methane content in ZVI-added digesters (RZ0-RZ4) increased by 8.1%, 9.0%, 11.2%, 17.0% and 18.0% successively. Consequently, 0.6 g/g VS CaO2 pretreatment combined with 0.4 g/g VS ZVI addition achieved the maximum methane production, increasing by 35.6% compared to control digester.

Biogas (methane) as a source of renewable energy, was produced in the anaerobic co-digestion of blackwater (BW, municipal toilet wastewater) and organic kitchen waste (KW). The impact on methane production of various BW to KW mixing ratios, with and without the addition of granular activated carbon (GAC), were studied under thermophilic (55 °C) temperatures. GAC is reported to enhance methane production in such digestions through direct interspecies electron transfer. The results showed that the co-digestion of BW and KW under the 1:2 VS ratio significantly improved the biomethane potential (BMP). In the absence of GAC, an optimal BW:KW ratio was found to be 1:2, achieving a BMP of 0.76 g CH4-COD/g feed-COD. With GAC addition, the BMP increased to 0.81 g CH4-COD/g feed-COD, the lag phase in the digestion was significantly reduced, and the methane production rate increased. Microbial communities in the BW-KW anaerobic digestion were analyzed with and without the addition of GAC. Methanothermobacter and Methanosarcina were predominant archaea in BW-KW digests, with and without GAC amendment, while a third methanogen, Methanomassiliicoccus, was enriched with the addition of GAC to the digest. Further, through SEM image, the enrichment of pili-like stucture was observed in GAC surface.

Anaerobic digestion of cattle manure has a low efficiency due to the high hydraulic retention time (HRT) required to degrade the abundant degradation-resistant compositions, co-digestion with food waste is effective at improving the methane production. Lowering the HRT can therefore increase the methanogenic efficiency during co-digestion. This study considered the effects of different HRTs (25, 20, 15, 10, 7, 5, and 4 days) on cattle manure and food waste co-digestion. The highest methane production was achieved at 1.48 L/L/d with an HRT of 5 days. The maximum methane yields (236–257 mL/g-VS) were attained at HRT ≥15 days and decreasing the HRT to 10-5 days resulted in low methane yields and complete process failure at HRT 4 days, due to volatile fatty acids accumulated and microorganisms washed out. From a high HRT of 20 days to a low HRTs of 5 days, Bacteroidetes and Firmicutes were the dominant bacteria and the percentage of syntrophic acetate oxidizing bacteria (mainly Pelotomaculum and Pseudothermotoga) clearly increased. The dominant methanogen changed from the acetotrophic Methanosaeta to the hydrogenophilic Methanobrevibacter. These results enable biogas plants to utilize surplus amounts of cow manure and food waste in a sustainable manner with high process capacity and methane recovery.

This study aimed to isolate a novel archaeon capable of simultaneously producing methane and bioflocculants. The isolated methanogen was identified as Methanosarcina spelaei RK-23 by molecular characterization. Strain RK-23 grew with different carbon sources, but acetate was found to be the most beneficial carbon source for the production of methane and bioflocculants. Moreover, RK-23 produced methane and bioflocculants within an initial pH range of 5.0–8.0, with an optimal pH for methane production of 6.5 and flocculating activity of 7.0. In addition, strain RK-23 exhibited tolerance to high-salinity conditions and produced methane and bioflocculants within 0–1.2 M NaCl, with an optimum NaCl concentration of 0.3 M. The methane production and flocculating activity of strain RK-23 under optimal culture conditions were 17.4 mmol methane/mol acetate and 95.6%, respectively. MBF-23 mostly consisted of 83.5% carbohydrates and 14.3% proteins. Furthermore, the bridging of adsorption is believed to be the main bioflocculation mechanism in this study.

Potential CO2 leakage is a major concern for carbon capture and storage (CCS). The effects of high soil CO2 concentration on microbes is a major element of impact assessments of CO2 leakage on terrestrial ecosystems. We conducted a field experiment to investigate the responses of microbial functional groups of ammonia-oxidizers, methanogens, and methanotrophs in high soil CO2 conditions. A single-point injection gassing plot (2.5 m × 2.5 m in size), which had 52.2% CO2 in the center (radius = 0.5 m) and 5.5% in the edge (radius = 1.7 m) at 10 cm depth, was employed. N2O and CH4 emissions increased after 1 day of injection because injected CO2 was instantly utilized by nitrifiers and methanogens. This suggests that the activities of the selected microbes could be stimulated by high soil CO2 concentrations. Prolonged CO2 injection has toxic effects on aerobic nitrifiers, but may favor anaerobic methanogens. However, the early stimulatory effects of high soil CO2 on N2O and CH4 production did not last to the end of injection. These results imply that increased N2O and CH4 emissions could be the minor side effects of high soil CO2. Microbes responded faster than plants to high soil CO2, with responses observed as late as 7 days after injection. The inhibition of plant absorption of soil water and nutrients by high soil CO2 concentrations may also influence microbial responses. Moreover, high soil water content could retard underground CO2 diffusion, which would magnify CO2 impacts on plants and microbes. Our results suggest that microbial response could be used as an early indicator of the impact assessments of CO2 leakage on soil ecosystems. An understanding of the interaction among soils, plants, and microbes would be helpful in assessing the biological risks of potential CO2 leakage.

The performance and microbial community succession of a 36 L working volume plug flow reactor was evaluated for treated cattle manure at an organic loading rate of 2.67 g-TS/L/day, a temperature of 38 °C ± 1 °C, and a hydraulic retention time of 18 days. A reflux of acidizing fluid effectively enhanced anaerobic digestion performance and promoted optimization of microbial community structure. The average biogas volume production rate was 1.08 L biogas/L reactor, which was 116.5% higher than the control without reflux of acidizing fluid. The specific qmethane production yield and methane content reached 0.204 L/g VS and 70%. Moreover, methane yield achieved 0.34 m3/kg removal COD with a COD removal of about 70.56%. The bacteria genera Christensenellaceae, Bacteroidales, vadinBC27, Ruminococcaceae and Treponema_2 were further enriched. Methanosarcina became the dominant methanogen in the whole PFR operation process. This study offers new opportunities for producing renewable energy from enhanced cattle manure biodegradability.

Five mesophilic full-scale anaerobic digesters treating food waste (FW-digester), animal waste (AW-digester), and co-substrate of food waste and animal waste (CO-digesters) were monitored identify bacterial and archaeal communities and to quantify the effect of substrate characteristics on them, and to identify 'core' microorganism. The substrate characteristics and microbial communities of the FW-digester, AW-digester, and CO-digesters were statistically different. Organic concentration and [Na+] were identified as major variations that effect microbial community. Methanogen community was more diverse in AW-digester than in FW-digester. Methanogen community in CO-digester was as diverse as in AW-digester, and the most dominant species was Methanoculleus bourgensis same as in FW-digester. Twenty-one bacterial genera and four methanogen species were found in all digesters as a consequence of their metabolic versatility to degrade organic and inhibitor compounds. The results implied that these core microorganisms may contribute to maintaining a stable microbial ecosystem.

Biogas from anaerobic digestion (AD) of waste activated sludge (WAS) limited its utilization due to low value-added. In this study, an innovative addition mode for ash known as stepwise addition was developed to enhance methane production and improve CO2 scavenge from AD of sludge. Experimental results confirmed stepwise addition of ash improved methane content to 79.4%, compared to control group (69.1%). Compared to Pulse addition and Control, the cumulative CH4 production was promoted by 39.2% and 35.4%, respectively. Investigation of the mechanism indicated that stepwise addition of ash could decrease hydrolytic and acidifying enzyme activities but increase activity of coenzyme F420, compared to pulse addition group. Furthermore, stepwise addition of ash not only increased the abundance of Methanomassiliicoccus (34.48%), but also promoted amounts of CO2 capture. This method ameliorate utilization availability of sludge ash for sludge anaerobic digestion through promoting cumulative methane production and increasing CO2 storage capacity.

The interest in antimicrobial compounds as feed additives is currently increasing. Among different options, tannins seem to have several beneficial effects when employed in animals diet. The present study aimed at investigating the influence on caecal microbial communities of the supplementation of a chestnut and quebracho tannins mix in meat rabbit's diet, also considering animals live performances. Four groups of rabbits were fed with a different diet: a control diet (C); a control diet with coccidiostat (CC), and two experimental diets with 0.3% (T0.3) and 0.6% (T0.6) chestnut and quebracho tannins mix. For microbial analysis, culture-dependent and culture-independent methods were employed. Live performances were not significantly affected by tannins mix supplementations, as well as culturable microbial loads of E. coli, Enterobacteriaceae, Bacteroides spp. and Bifidobacterium spp. C. perfringens was always under the detection limit. A consistent result was obtained by qPCR. As for PCR-DGGE analysis, the Richness and evenness (Shannon-Weiner index) of bacterial communities in caecum resulted significantly higher in control samples (C and CC) than in those from rabbit fed with tannin-containing diets. Sequencing analysis revealed that the phylum Firmicutes was less represented in samples from control groups. As for the methanogen archaeal DGGE, no significant differences were found in richness and diversity among different groups, all dominated by Methanobrevibacter spp.. This work highlights the potential antimicrobial effect of chestnut and quebracho tannins mix in an in vivo system revealed by molecular analysis.

Drainage is an important mitigation strategy to reduce methane emission from rice paddies. Here, we investigated how drainage shapes structure and functioning of the paddy soil microbial community. Soil microcosms were pre-incubated for 28 days under flooded conditions followed by nine days' drainage. Upon sampling, metatranscriptome libraries were generated from flooded and drained soils. With drainage, oxygen concentration increased from suboxic (1.6 μmol/l) to near-atmospheric (240 μmol/l) levels. Concurrently, water potential decreased to −0.87 MPa (corresponding to 11% soil moisture content). Drainage did not affect the absolute (RT-qPCR) SSU rRNA abundance of Bacteria and Archaea, but changed significantly their community composition. Firmicutes (Clostridiaceae, Ruminococcaceae, and Lachnospiraceae) decreased in relative abundance, while Actinobacteria (Nocardioidaceae) and Proteobacteria (Comamonadaceae) increased. These taxon-specific dynamics were observed on rRNA and mRNA levels. Methanogen SSU rRNA was stable, but methanogen mRNA significantly decreased. This coincided with complete inhibition of the methane production potential in drained soil. Among Eukarya, protists and Amoebozoa thrived in flooded soil, while Fungi proliferated with drainage. In particular, Pezizomycotina (Ascomycota) and Agaricomycotina (Basidiomycota) were the most abundant fungal groups in dry soil. Taking community-wide mRNA expression as a proxy, the overall microbial activity was not severely affected by drainage. The proportion of mRNA in total RNA increased from 1.7% to 2%. In particular, the abundance of mRNA related to transcription and translation significantly increased. Correspondingly, the bacterial SSU rRNA transcript/gene ratio increased 12-fold with drainage, suggesting the development of a metabolically highly active microbial community well adapted to oxic and dry soil conditions. This community showed an increased transcription of genes involved in the degradation of lignin, peptidoglycan, and storage molecules such as glycogen. Under flooded conditions, the microbial community expressed a higher level of glycoside hydrolase transcripts involved in cellulose and chitin degradation.

The effect of bioaugmentation with different microorganisms on anaerobic digestion to mitigate the ammonia inhibition problem was investigated. Seven pure strains of microorganisms (including obligate aceticlastic methanogen, facultative aceticlastic methanogen, hydrogenotrophic methanogen, syntrophic acetate oxidizing bacteria (SAOB) were selected and thirteen bioaugmentation approaches were tested. Bioaugmentation with hydrogenotrophic methanogen Methanobrevibacter smithii (MBS) and SAOB Syntrophaceticu schinkii together was the optimal choice, methane production (MP) was 71.1% higher than that in Blank, the activity of hydrogenotrophic methanogenesis was greatly heightened according to specific methanogenic activity analysis. Bioaugmentation with facultative aceticlastic methanogen Methanosarcina barkeri (MSB) alone without SAOB addition was also proven efficient (MP was 59.7% higher than that in Blank), both aceticlastic and hydrogenotrophic methanogenesis were enhanced. Further evaluation with carbon isotope fractionations analysis indicated that balancing the activities of the aceticlastic and hydrogenotrophic methanogenic pathways is of great importance. 16s rRNA gene sequencing results showed that Methanobacterium spp. and Methanosaeta spp. were the dominant archaea in all 14 reactors. Nevertheless, bioaugmentation with Methanosaeta spp. did not result in a positive effect on MP. On the other hand, Methanobrevibacter spp. and Methanosarcina spp. were non-dominant archaea (even after bioaugmentation with MBS or MSB, the relative abundances were still poor (

Source diverted blackwater (i.e., toilet wastewater with or without food waste residuals) represents an ideal waste stream for bioenergy recovery through anaerobic digestion. However, due to the high free ammonia concentration contained in blackwater collected from water conserving toilets (e.g., vacuum toilets), CH4 recovery can be significantly reduced because methanogenesis is inhibited. Our current study evaluated the feasibility of enriching hydrogenotrophic methanogens (known to be more resistant than acetoclastic methanogens toward free ammonia inhibition) in anaerobic sludge with H2 supplementation for blackwater treatment. Our results demonstrated that hydrogenotrophic methanogens can be enriched by supplementing anaerobic sludge with H2 and CO2 in a volume ratio of 4:1 for 3 months. The dominance of hydrogenotrophic methanogens after 3-month enrichment was confirmed with quantitative PCR studies. It was observed that the hydrogenotrophic methanogen dominant anaerobic sludge significantly alleviated blackwater free ammonia inhibition, and led to enhanced biochemical methane production (BMP) (e.g., from 30% to 53%). Our current study offers a new treatment option for anaerobic treatment of wastewater with high free ammonia content.

The aim of this study was to evaluate and compare the treatment of petrochemical wastewater with an integrated anaerobic baffled reactor and microbial electrolysis cell (ABR-MEC) and anaerobic baffled reactor (ABR). For this purpose, the effect of hydraulic retention time (HRT) and applied voltage on the performance of ABR-MEC reactor were investigated in four phases. Regarding the results, the maximum COD removal efficiency in the ABR-MEC reactor was higher than that in ABR reactor, i.e. 96.5% versus 66.7%. Also, the maximum of yield CH4 in ABR-MEC reactor was 1.4-fold than ABR reactor. The results show that the applied voltages can increase the removal of chemical oxygen demand, methane production, accelerate the conversion of volatile fatty acids and maintain the appropriate pH range for methanogen growth.

This study evaluated how tylosin (TYL), ciprofloxacin (CIP), and sulfadimidine (SM2) affected biogas and CH4 production during anaerobic digestion (AD) via their effects on the key genes related to methane production and the methanogenic community. The results showed that TYL, CIP, and SM2 reduced the production of methane during AD by 7.5%, 21.9%, and 16.0%, respectively. After AD for five days, CIP strongly inhibited the mcrA gene, where its abundance was 49% less than that in the control. TYL and SM2 decreased the abundances of Spirochaeta and Fibrobacteres during AD. High-throughput sequencing identified 10 methanogen genera, where Methanocorpusculum, Methanobrevibacter, and Methanosarcina accounted for 99.1% of the total archaeal reads. TYL and SM2 increased the efficiency of the acetoclastic methanogen pathway (Methanosarcina) by 29.04% and 52.79%, respectively. Redundancy analysis showed that Spirochaeta, Fibrobacteres, and Methanosarcina had positive correlations with CH4 and mcrA. We found that 30 mg kg−1 CIP had a strong inhibitory effect on methane production by influencing the abundances of Methanobrevibacter and Methanosarcina during AD.

Anaerobic digestion (AD) is a viable and cost-effective method for converting organic waste into usable renewable energy. The efficiency of organic waste digestion, nonetheless, is limited due to inhibition and instability. Accordingly, biochar is an effective method for improving the efficiency of AD by adsorbing inhibitors, promoting biogas generation and methane concentration, maintaining process stability, colonizing microorganisms selectively, and mitigating the inhibition of volatile fatty acids and ammonia. This paper reviews the features of crop waste-derived biochar and its application in AD systems. Four critical roles of biochar in AD systems were identified: maintaining pH stability, promoting hydrolysis, enhancing the direct interspecies electron transfer pathway, and supporting microbial development. This work also highlights that the interaction between biochar dose, amount of organic component in the substrate, and inoculum-to-substrate ratio should be the focus of future research before deploying commercial applications.

Stimulating interspecies electron transfer (IET) by biochars has been demonstrated as an efficient means to enhance anaerobic digestion (AD). However, the enhancing effects vary significantly depending on biochar properties. The correlations between them are not fully understood. Herein, biochars with different physicochemical properties were produced from a whiskey by-product “draff” and applied in the digestion of draff. The biochar produced at 700 °C enhanced biomethane yield by 5%, whereas biochars produced at 500 and 900 °C did not increase biomethane yield. The addition of 700 °C-derived biochar in AD increased the relative abundance of methanogen Methanosarcina, which may be the electron-accepting partner in direct interspecies electron transfer. The characterisation of biochar properties suggested that moderate graphitisation and abundant active surface functional groups (such as –C=O, pyridinic-N, and graphitic-N) contribute to a more stimulating IET through both the carbon matrices and the charging – discharging cycles of surface functional groups.

International audience; A novel anaerobic methylotrophic halophilic methanogen strain SLHTYRO T was isolated from a deep hypersaline anoxic basin called "Tyro" located in the Eastern Mediterranean Sea. Cells of SLHTYRO T were motile cocci. The strain SLHTYRO T grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L −1 NaCl (optimum 135 g L −1). Strain SLHTYRO T was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYRO T was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYRO T was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYRO T should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYRO T (=DSM 108854 = JCM 32768 = UBOCC-M-3308).

The study on the factors influencing the biogenic gas production of low rank coal beds is of great significance to the search of the favorable biogenic gas enrichment areas of similar coal beds and the guidance of the microbial stimulation technologies. In this paper, the lignite samples taken from the Jiergalangtu Sag in the Erlian Basin were selected to carry out the simulation experiment of biogenic coalbed methane under different temperatures, pH values, oxidation-reduction potentials (Eh) and trace element concentrations. Then, the influences on biogenic gas production were discussed and the optimal biogenic gas production condition was determined. And the following research results were obtained. First, in the simulation experiments, the biogenic gas production of coal beds increases first and then decreases with the increase of temperature, pH and trace element concentration and the decrease of Eh. Second, in the case of temperature 30–35 °C, pH 7.0–7.5 and Eh −225 mV, the metabolism of methanogen is the most active and the most favorable for the generation of biogenic gas, so biogenic gas production can be promoted by adding the trace elements (Fe2+ and Ni2+) of proper concentration. It is concluded that a proper burial depth (300–600 m), a weak runoff hydrodynamic condition and an anaerobic environment constitute the favorable conditions for the generation of biogenic gas in the Jiergalangtu Sag, Erlian Basin. Moreover, the addition of appropriate trace elements can be an effective means of microbial stimulation. Keywords: Erlian Basin, Jiergalangtu Sag, Low-rank coalbed methane, Biogas, Bacteria detection, Methanogen, Gas production potential, Microbial stimulation

In this study, a three-stage upflow anaerobic sludge blanket (UASB) system was developed and tested for hydrogen (H2) and methane (CH4) production from cassava wastewater with an emphasis on CH4 production. The experiment was carried out at mesophilic temperature (37 °C) at different chemical oxygen demand (COD) loading rates from 5 to 18 kg/m3d (based on total liquid holding volume) with a recycle ratio of the final effluent to both the first and second bioreactors at a constant 1: 1 flow rate ratio of feed: final effluent. The first bioreactor was maintained pH at 5.5 while those of the other two bioreactors were not controlled. At an optimum COD loading rate of 15 kg/m3d, the system provided the highest COD removal level (92.5%) and the highest H2 and CH4 yields of 0.43 mL H2/g COD applied, and 328 mL CH4/g COD applied, respectively. The very high productivity of CH4 with the very low H2 productivity resulted from the recycled methanogen sludge from the third bioreactor to the first and second bioreactors. The process performance of the three-stage UASB system in terms of optimum COD loading rate and total energy yield was much higher than those of single and two-stage anaerobic processes.

A bioreactor was designed and established to simulate in situ oil reservoir conditions for investigating methanogenic conversion of crude oil by microbial community derived from production water of a high-temperature oilfield. The continuous incubation and culturing were divided into two phases (Phase I, day 0–341; and Phase II, day 341–520) to allow sampling and supplement of fresh culture medium on day 341 and 520, respectively. The average methane production rate was 4.9 and 20.8 mmol CH4·day−1 m−3 pore volume during the Phase I and Phase II, respectively. Degradation intermediates of fatty acids were detected in the culture fluids indicating the transformation of crude oil. The 16S rRNA gene sequence analysis revealed that the major bacterial community shifted from Deferribacteres to Aminicenantes and unclassified Bacteria, as well as Thermotogae and Synergistetes. The predominant methanogenic community shifted from methylotrophic methanogen (Methanolobus) to hydrogenotrophic methanogens (Methanothermobacter and Methanocalculus) and acetoclastic methanogen (Methanothrix) after 341 days of incubation, and was subsequently dominated by hydrogenotrophic methanogen (Methanothermobacter) after 520 days of incubation. This study expanded our understandings of methanogenic oil-degrading processes and the key microorganisms involved in laboratory simulation bioreactor over time of incubation. In addition, it further provides valuable insights on the microbial conversion of hydrocarbons to methane for enhancing the energy recovery from residual oil in petroleum reservoirs.

Methanobacterium formicicum (Methanobacteriaceae family) is an endosymbiotic methanogenic Archaean found in the digestive tracts of ruminants and elsewhere. It has been significantly implicated in global CH4 emission during enteric fermentation processes. In this review, we discuss current genomic and metabolic aspects of this microorganism for the purpose of the discovery of novel veterinary therapeutics. This microorganism encompasses a typical H2 scavenging system, which facilitates a metabolic symbiosis across the H2 producing cellulolytic bacteria and fumarate reducing bacteria. To date, five genome-scale metabolic models (iAF692, iMG746, iMB745, iVS941 and iMM518) have been developed. These metabolic reconstructions revealed the cellular and metabolic behaviors of methanogenic archaea. The characteristics of its symbiotic behavior and metabolic crosstalk with competitive rumen anaerobes support understanding of the physiological function and metabolic fate of shared metabolites in the rumen ecosystem. Thus, systems biological characterization of this microorganism may provide a new insight to realize its metabolic significance for the development of a healthy microbiota in ruminants. An in-depth knowledge of this microorganism may allow us to ensure a long term sustainability of ruminant-based agriculture. Keywords: Rumen ecosystem, Methanogens, Methane mitigation, Global warming, Enteric fermentation, Symbiosis, Genome-scale model, Systems biology

Sharpea and Kandleria are associated with rumen samples from low-methane-emitting sheep. Four strains of each genus were studied in culture, and the genomes of nine strains were analysed, to understand the physiology of these bacteria. All eight cultures grew equally well with d -glucose, d -fructose, d -galactose, cellobiose, and sucrose supplementation. d -Lactate was the major end product, with small amounts of the mixed acid fermentation products formate, acetate and ethanol. Genes encoding the enzymes necessary for this fermentation pattern were found in the genomes of four strains of Sharpea and five of Kandleria. Strains of Sharpea produced traces of hydrogen gas in pure culture, but strains of Kandleria did not. This was consistent with finding that Sharpea, but not Kandleria, genomes contained genes coding for hydrogenases. It was speculated that, in co-culture with a methanogen, Sharpea and Kandleria might change their fermentation pattern from a predominately homolactic to a predominately mixed acid fermentation, which would result in a decrease in lactate production and an increase in formation of acetate and perhaps ethanol. However, Sharpea and Kandleria did not change their fermentation products when co-cultured with Methanobrevibacter olleyae, a methanogen that can use both hydrogen and formate, and lactate remained the major end product. The results of this study therefore support a hypothesis that explains the link between lower methane yields and larger populations of Sharpea and Kandleria in the rumens of sheep.

Onsite septic systems use soil microbial communities to treat wastewater, in the process creating potent greenhouse gases (GHGs): methane (CH4) and nitrous oxide (N2O). Subsurface soil dispersal systems of septic tank overflow, known as leach fields, are an important part of wastewater treatment and have the potential to contribute significantly to GHG cycling. This study aimed to characterize soil microbial communities associated with leach field systems and quantify the abundance and distribution of microbial populations involved in CH4 and N2O cycling. Functional genes were used to target populations producing and consuming GHGs, specifically methyl coenzyme M reductase (mcrA) and particulate methane monooxygenase (pmoA) for CH4 and nitric oxide reductase (cnorB) and nitrous oxide reductase (nosZ) for N2O. All biomarker genes were found in all soil samples regardless of treatment (leach field, sand filter, or control) or depth (surface or subsurface). In general, biomarker genes were more abundant in surface soils than subsurface soils suggesting the majority of GHG cycling is occurring in near-surface soils. Ratios of production to consumption gene abundances showed a positive relationship with CH4 emissions (mcrA:pmoA, p 0.05). Of the three measured soil parameters (volumetric water content (VWC), temperature, and conductivity), only VWC was significantly correlated to a biomarker gene, mcrA (p = 0.0398) but not pmoA or either of the N2O cycling genes (p > 0.05 for cnorB and nosZ). 16S rRNA amplicon library sequencing results revealed soil VWC, CH4 flux and N2O flux together explained 64% of the microbial community diversity between samples. Sequencing of mcrA and pmoA amplicon libraries revealed treatment had little effect on diversity of CH4 cycling organisms. Overall, these results suggest GHG cycling occurs in all soils regardless of whether or not they are associated with a leach field system.

There is currently an urgent need for effective anti-methanogen small molecule inhibitors to help control ruminant methane emissions on a global scale. No assays system has yet been reported that would enable high-throughput screening of either rumen methanogen cultures or an assay system that utilises rumen fluid. We report here the development of a high-throughput 0.2 ml rumen fluid-based 96-well screening system that incorporates a simple substrate for fermentation with a short incubation time The fermentation parameters determined are total gas, methane and hydrogen production. The system markedly enhances the number of samples that can be processed (∼1000 incubation vials per week) compared to previously published assays. The system does not try to mimic rumen fermentation but is a means of screening compounds for their potential effect against rumen methanogens. We also assessed the system using known rumen anti-methanogen inhibitory compounds, a set of anthraquinone derivatives and 3-nitrooxypropanol. The system can differentiate between specific methane inhibition and the inhibition of overall fermentation and it discriminates between molecules that act as methanogen inhibitors and those acting as electron sinks.

Anaerobic digestion has attracted attention because it does not require power for aeration, it reduces excess sludge and it generates methane gas. However, the growth rate of anaerobic microorganisms is slow, resulting in low treatment efficiency. In this study, the impact of Fe3O4 nanoparticles (NPs) on the growth of methanogens, which is the rate-determining step in anaerobic digestion, was investigated using a pure culture of Methanosarcina barkeri as the model methanogen. M. barkeri were cultivated in iron free medium, as well as in media amended with various concentrations of Fe3O4 NPs with a mean diameter of 8.1 ± 2.4 nm. The production of methane gas was greatly increased when organisms were cultured in media containing NPs. After the methane production was saturated, methanol was newly added to the culture, which resulted in additional methane generation at a higher production rate than occurred during the initial round of cultivation in media containing 20 ppm Fe3O4 NPs. In addition, no evidence of negative impacts of Fe3O4 NPs on the growth of M. barkeri was observed. Taken together, these results strongly suggest that adding Fe3O4 NPs into the fermenter as an agent of sustained iron release can enable sustainable methane fermentation.

Coke oven gas is considered as a potential hydrogen source for biogas bio-upgrading. In this study, the effects of CO on biomethanation performance and microbial community structure of hydrogenotrophic mixed cultures were investigated under thermophilic (55 °C) and extreme-thermophilic (70 °C) conditions. 5% (v/v) CO did not inhibit hydrogenotrophic methanogenesis during semi-continuous operation, and 83–97% CO conversion to CH4 was achieved. Methanothermobacter thermoautotrophicus was the dominant methanogen at both temperatures and was the main functional archaea associated with CO biomethanation. Specific methanogenic activity test results showed that long-term 5% CO acclimation shortened the lag phase from 5 h to 1 h at 55 °C and 15 h to 3 h at 70 °C. CO2 was the preferred carbon source over CO for hydrogenotrophic methanogens and CO consumption only started when CO2 was completely depleted. M. thermoautotrophicus dominated mixed cultures showed a great potential in simultaneous hydrogenotrophic methanogenesis and CO biomethanation.

Recent findings showed that some trace elements essential for anaerobic digestion might be deficient in chicken (laying hens) manure. In this study, the long-term influence of trace element deficiency on anaerobic mono-digestion of chicken manure was investigated. Three bench-scale anaerobic reactors were operated with or without trace element supplementation. As trace element, only Se or a mix containing Co, Mo, Ni, Se, and W was added to the reactors. The results revealed that in anaerobic digestion of chicken manure at total ammonium nitrogen concentrations over 6000 mg L−1, Se supplementation was critical but not sufficient alone for long-term stable CH4 production. Addition of a mix consisting of Co, Mo, Ni, Se and W resulted in a more stable digestion performance. Daily trace element mix supplementation promoted the hydrogenotrophic Methanoculleus bourgensis, which is an ammonia tolerant methanogen. The decrease in the relative abundance of Methanoculleus detected after termination of trace element addition and resulted in accumulation of acetate and propionate that followed by a significant decrease in CH4 production.

An anaerobic membrane reactor (AnMBR) was investigated and compared with a continuously stirred tank reactor (CSTR) in order to treat thermal hydrolyzed sludge. Long term experiments were carried out by shortening the hydraulic retention time (HRT) from 30 d to 3 d under mesophilic conditions. The results obtained show that at each HRT the AnMBR had higher removal efficiencies for total solids and volatile solids, and higher specific methanogenic activities. Comparable process stability was obtained for the two reactors. A flat sheet membrane could work with the high solid system but the trans-membrane pressure appeared sensitive to total solid concentrations. Foaming occurred in the AnMBR at a HRT of 3 d, and an organic loading rate of 8.84 kg-COD/(m3·d) further deteriorated the membrane filtration. Hydrogentropic methanogens, in particular Methanospirillum, dominated the archaea community in both the AnMBR and the CSTR at a HRT of 3 d (i.e. 85% in the AnMBR and 95% in the CSTR). An obligate acetoclastic methanogen, Methanosaeta, was found at a very low level during all the HRTs. As a whole, the efficiency of methanogenesis in the AnMBR to treat thermal hydrolyzed sludge was proved to be better than in the CSTR during short HRTs.

This study compared single- versus two-phase systems for semi-continuous anaerobic digestion of food waste without pH control at varying organic loading rates (OLRs). The methanogenic reactors of both systems required trace element supplementation for stable operation at 3.0 g VS (volatile solids)/L∙d or higher OLRs. Under trace-element supplemented conditions, both systems achieved stable and efficient performance at OLRs up to 4.0 g VS/L∙d. The two-phase system outperformed the single-phase system at 1.0–4.0 g VS/L∙d OLRs, but it failed at an OLR of 5.0 g VS/L∙d. Meanwhile, the single-phase system maintained the stable performance and reached its maximum methane production at this OLR. These results suggest that a single-phase configuration is more advantageous for robust treatment of food waste without pH control at high organic and hydraulic loads. Hydrogenotrophic methanogens dominated the methanogen community throughout the experiment in both systems. Microbial community structure shifts correlated with reactor operation and performance characteristics.

The objective of this study was to evaluate the effect of biofilm and external voltage on the performance and microbial community composition of batch-fed, combined anaerobic digestion-bioelectrochemical cell (AD-BEC) systems under different operational conditions. A dextrin/peptone mixture was fed at a range of organic loading rates (0.34 to 1.37g COD/L-d). The hybrid system with both suspended biomass and biofilm without any external potential application achieved a substantially higher initial soluble COD consumption (53.7±2.3% vs. 39.7±3.7) and methane (CH4) production (331 vs. 225mL) within one day of feeding than the conventional AD system (suspended biomass only). Compared to the conventional AD system, the hybrid systems had higher resilience to shock organic loadings. A range of external potential (0.5 to 2.0V vs. Ag/AgCl) was applied to AD-BEC reactors, developed with two different start-up procedures. A potential of 2.0V resulted in water electrolysis leading to a higher CH4 production rate (105 vs. 84mL/L-d) and biogas CH4 content (88.5±1.4 vs. 64.5±1.9%) in the AD-BEC reactor (closed vs. open circuit condition, respectively). Application of external potential enriched putative exoelectrogens at the anode biofilm and hydrogenotrophic methanogens at the cathode biofilm, which may have contributed to the observed enhanced CH4 production in the AD-BEC system. A phylotype related to Methanobacterium formicicum, a hydrogenotrophic methanogen, dominated the archaeal community in the AD-BEC cathode biofilm.

Diurnal freeze-thaw cycles (FTCs) occur in the spring and autumn in boreal wetlands as soil temperatures rise above freezing during the day and fall below freezing at night. A surge in methane emissions from these systems is frequently documented during spring FTCs, accounting for a large portion of annual emissions. In boreal wetlands, methane is produced as a result of syntrophic microbial processes, mediated by a consortium of fermenting bacteria and methanogenic archaea. Further research is needed to determine whether FTCs enhance microbial metabolism related to methane production through the cryogenic decomposition of soil organic matter. Previous studies observed large methane emissions during the spring thawed period in the Sanjiang seasonal frozen marsh of Northeast China. To investigate how FTCs impact the soil microbial community and methanogen abundance and activity, we collected soil cores from the Sanjiang marsh during the FTCs of autumn 2014 and spring 2015. Methanogens were investigated based on expression level of the methyl coenzyme reductase (mcrA) gene, and soil bacterial and archaeal community structures were assessed by 16S rRNA gene sequencing. The results show that a decrease in bacteria and methanogens followed autumns FTCs, whereas an increase in bacteria and methanogens was observed following spring FTCs. The bacterial community structure, including Firmicutes and certain Deltaproteobacteria, was changed following autumn FTCs. Temperature and substrate were the primary factors regulating the abundance and composition of the microbial communities during autumn FTCs, whereas no factors significantly contributing to spring FTCs were identified. Acetoclastic methanogens from order Methanosarcinales were the dominant group at the beginning and end of both the autumn and spring FTCs. Active methanogens were significantly more abundant during the diurnal thawed period, indicating that the increasing number of FTCs predicted to occur with global climate change could potentially promote CH4 emissions in seasonal frozen marshes.

Drylot beef cow-calf systems are viable alternative management systems to traditional forage-based systems. In such confinement, adding ionophores such as monensin to the diets of beef cattle is common, and has been shown to improve feed efficiency and increase average daily gain. The addition of monensin is also commonly utilized as a strategy for methane mitigation, as this ionophore class antimicrobial acts to interfere with ion flux primarily within Gram-positive cells through its action as an ion carrier. It is widely accepted that suppression of these ruminal organisms results in the reduction of substrates for rumen methanogenic archaea, reducing methane production. However, several studies have indicated that cattle may adapt to monensin, and within weeks of feeding, may return to prior levels of methane production. Our hypothesis is that feeding monensin to bred beef heifers in confinement will temporarily decrease methane production from shifts in the methanogenic archaeal and bacterial communities. Sixteen fall-born bred heifers were randomly assigned to 2 treatment groups (n = 8 per treatment) and were fed a control diet or a diet containing monensin for 70 days using headgates. In vivo gas exchange of oxygen consumption, carbon dioxide and methane production were measured for 24-h periods throughout the trial using individual calorimeter head boxes. Rumen content sampling was conducted on day 0, 18 and 53 of the trial through oral lavage. Upon completion of sampling, DNA was isolated for ruminal bacteriome composition utilizing deep, next-generation sequencing of the V1-V3 hypervariable regions of the 16S bacterial rRNA gene. Level of methanogen 16S rRNA was quantified using qPCR. There was a significant reduction in phylum SR1 (P 0.05). Heifers fed monensin did not produce significantly less methane than the control (P > 0.05) on a liters per day basis, which was consistent throughout the study. These data suggest methane production is not reduced long-term when animals are fed monensin in confinement. Additionally, the data suggest that monensin supplementation does not suppress all classical Gram-positive populations, rather it influences finer shifts in bacterial species that may be key in ruminant function. Determining the stability of the ruminal microbiome over time in heifers fed monensin may provide further insights to long-term methane mitigation in cow-calf systems.

Paddy soil accounts for 10% of global atmospheric methane (CH4) emissions. Many types of fertilizers may enhance CH4 emissions, especially organic fertilizer. The aim of this study was to explore the effects of different fertilizers on CH4 and methanogen patterns in paddy soil. This experiment involved four treatments: chemical fertilizer (CT), organic fertilizer (OT), mixed with chemical and organic fertilizer (MT), and no fertilizer (ctrl). The three fertilization treatments were applied with total nitrogen at the same rate of 300 kg N ha−1. Paddy CH4, soil physicochemical variables and methanogen communities were quantitatively analyzed. Rhizosphere soil mcrA and pmoA gene copy numbers were determined by qPCR. Methanogenic 16S rRNA genes were identified by MiSeq sequencing. The results indicated CH4 emissions were significantly higher in OT (145.31 kg ha−1) than MT (84.62 kg ha−1), CT (77.88 kg ha−1) or ctrl (32.19 kg ha−1). Soil organic acids were also increased by organic fertilization. CH4 effluxes were significantly and negatively related to mcrA and pmoA gene copy numbers, and positively related to mcrA/pmoA. Above all, hydrogenotrophic Methanocella and acetoclastic Methanosaeta were the predominant methanogenic communities; these communities were strictly associated with soil potassium, oxalate, acetate, and succinate. Application of organic fertilizer promoted the dominant acetoclastic methanogens, but suppressed the dominant hydrogenotrophic methanogens. The transformation in methanogenic community structure and enhanced availability of C substrates may explain the increased CH4 production in OT compared to other treatments. Compared to OT, MT may partially mitigate CH4 emissions while guaranteeing a high rice yield. On this basis, we recommend the local fertilization pattern should change from 300 N kg ha−1 of organic manure to the same level of mixed fertilization. Moreover, we suggest multiple combinations of mixed fertilization merit more investigation in the future.

An anaerobic ceramic membrane bioreactor (AnCMBR) has been attracted as an alternative technology to co-manage various organic substrates. This AnCMBR study investigated process performance and microbial community structure at decreasing temperatures to evaluate the potential of AnCMBR treatment for co-managing domestic wastewater (DWW) and food waste-recycling wastewater (FRW). As a result, the water flux (≥6.9 LMH) and organic removal efficiency (≥98.0%) were maintained above 25 °C. The trend of methane production in the AnCMBR was similar except for at 15 °C. At 15 °C, the archaeal community structure did not shifted, whereas the bacterial community structure was changed. Various major archaeal species were identified as the mesophilic methanogens which unable to grow at 15 °C. Our results suggest that the AnCMBR can be applied to co-manage DWW and FRW above 20 °C. Future improvements including psychrophilic methanogen inoculation and process optimization would make co-manage DWW and FRW at lower temperature climates.

Annual monitoring of CH4 and associated microorganisms from four submerged plant species (Potamogeton crispus, Ceratophyllum demersum, Myriophyllum spicatum and Hydrilla verticillata) was conducted in a full-scale constructed wetland (CW). Plant species had a significant impact on CH4 emissions. The highest CH4 flux (5.7 mg m−2 h−1) came from P. crispus, and the lowest flux (1.6 mg m−2 h−1) was from M. spicatum. The dissolved oxygen (DO) and nitrogen of the rhizosphere was significantly affected by plant species, which were the most important factors affecting CH4 flux and methanogenic communities. Changes of DO level caused by plant species were more significant than temperature changes. Real-time quantitative PCR (q-PCR) analysis showed that the number of methanogens and methanotrophs varied with plant species and seasons, whereas the total number of eubacteria was less affected (p > 0.05). The variation of TN and NO2-N caused by plant species resulted in the differentiation of methanogen populations. Each plant species was characterized by a specific group of methanogens. Illumina sequencing showed that the dominant methanogenic populations from P. crispus and C. demersum were hydrogenotrophic archaea of the Methanoregula, while those from M. spicatum and H. verticillata were hydrogenotrophic archaea of the Methanobacterium. Both plant species and seasons affected the percentage of acetoclastic archaea of the Methanosarcina and Methanosaeta. This study indicates that the submerged plant species are important parameters in the production of CH4 emissions in CWs. The proper arrangement of submerged plant species in CWs is vital to maximize the environmental benefit.

Both elevated atmospheric carbon dioxide (CO2) and rising temperature can alter soil methane (CH4) fluxes, leading to a feedback to climate change. However, predicting this feedback needs to understand the microbial mechanisms involved in CH4 emissions driven by climate change. A 3-year field measurement of CH4 fluxes from rice paddies was taken in 2012–2014 to examine their responses to elevated CO2 (enriched up to 500 μmol mol−1) and rising canopy air temperature (above ambient 1.5–2.0 °C) using a free-air CO2 enrichment (FACE) system. Using real-time PCR and Illumina MiSeq sequencing of 16S rRNA genes, we measured the abundance and composition of methanogenic community in rhizosphere soil of rice paddies in 2014. Elevated CO2 and rising temperature showed additive effects on CH4 fluxes and methanogen abundances, where CH4 fluxes were correlated with methanogen abundances. Elevated CO2, rising temperature and their combination increased seasonal CH4 emissions by 28–120%, 38–74% and 82–143%, respectively. Either elevated CO2 or rising temperature did not significantly alter the diversity of methanogenic community, and methanogenic genera Methanosaeta, Methanosarcina, Methanobacterium, Methanocella and Methanoregula dominated in rhizosphere soils for all treatments. However, elevated CO2 induced a shift from acetoclastic to hydrogenotrophic methanogens in their relative abundances. Rising temperature stimulated CH4 emissions by increasing CH4 production per individual predominant methanogen genus. Besides the enhancement of soil C substrates and rhizosphere methanogen abundances as previously reported, an additive effect of elevated CO2 and canopy warming on CH4 emissions is also associated with elevated CO2-induced changes in the composition of methanogenic archaea and warming-stimulated the activity of methanogenic archaea in rice paddies.

Microbial production of methane is an important terminal metabolic process during organic matter degradation in marine sediments. It is generally acknowledged that hydrogenotrophic and acetoclastic methanogenesis constitute the dominant pathways of methane production; the importance of methanogenesis from methylated compounds remains poorly understood. We conducted various biogeochemical and molecular genetic analyses to characterize substrate availability, rates of methanogenesis, and methanogen community composition, and further evaluated the contribution of different substrates and pathways for methane production in deltaic surface and subsurface sediments of the Western Mediterranean Sea. Major substrates representing three methanogenic pathways, including H2, acetate, and methanol, trimethylamine (TMA), and dimethylsulfide (DMS), were detected in the pore waters and sediments, and exhibited variability over depth and between sites. In accompanying incubation experiments, methanogenesis rates from various 14C labeled substrates varied as well, suggesting that environmental factors, such as sulfate concentration and organic matter quality, could significantly influence the relative importance of individual pathway. In particular, methylotrophic and hydrogenotrophic methanogenesis contributed to the presence of micromolar methane concentrations in the sulfate reduction zone, with methanogenesis from methanol accounting for up to 98% of the total methane production in the topmost surface sediment. In the sulfate-depleted zone, hydrogenotrophic methanogenesis was the dominant methanogenic pathway (67–98%), and enhanced methane production from acetate was observed in organic-rich sediment (up to 31%). Methyl coenzyme M reductase gene (mcrA) analysis revealed that the composition of methanogenic communities was generally consistent with the distribution of methanogenic activity from different substrates. This study provides the first quantitative assessment of methylotrophic methanogenesis in marine sediments and has important implications for marine methane cycling. The occurrence of methylotrophic methanogenesis in surface sediments could fuel the anaerobic oxidation of methane (AOM) in the shallow sulfate reduction zone. Release of methane produced from methylotrophic methanogenesis could be a source of methane efflux to the water column, thus influencing the benthic methane budgets.

Ammonia inhibition easily affects the performance of anaerobic digestion (AD) for municipal sludge and the oxidization of volatile fatty acids (VFAs) is the rate-limiting step of this process. Bioaugmentation is considered to be an effective method to alleviate ammonia inhibition of AD, but most study used the hydrogenotrophic methanogens as the bioaugmentation culture. In this study, bioaugmentation of mesophilic AD (MAD) and thermophilic AD (TAD) under ammonia inhibition with syntrophic acetate and propionate oxidizing consortia was investigated. The results showed that the bioaugmented reactors recovered earlier than control reactors with 20 (MAD) and 8 (TAD) days, respectively. The high-throughput 16S rRNA gene sequencing indicated that the relative abundance of carbohydrates fermenter (Lentimicrobium), syntrophic VFAs-oxidizing bacteria (Rikenellaceae_DMER64, Smithella and Syntrophobacter) and acetoclastic and hydrogenotrophic methanogens (Methanosaeta, Methanolinea and Methanospirillum) increased in MAD after bioaugmentation. However, part of the bioaugmentation culture could not adapt to the high free ammonia (FAN) concentration in MAD and the effect was weakened. In TAD, proteolytic bacteria (Keratinibaculum and Tepidimicrobium), syntrophic VFAs-oxidizing bacteria (Syntrophomonas) and hydrogenotrophic methanogen (Methanosarcina) were strengthened. The effect of bioaugmentation in TAD was durable even at higher organic loading rate (OLR), due to its positive influence on microbial community. These results suggested that the different bioaugmentation mechanism occurred in MAD and TAD, which are derived from the synergetic effects of ammonia tolerance and microbial interactions. Our study revealed the VFAs-oxidizing consortia as bioaugmented culture could be the potential strategy to alleviate the ammonia stress of AD.

The understanding and enrichment of consortia formed by syntrophic butyrate-oxidizing bacteria and methanogens in the complex environment are crucial for effectively degrading butyrate and preventing acid inhibition. In this study, the better butyrate-tolerated and highly efficient microbial consortia were domesticated and enriched through adding butyric acid ranging from 0.2 to 4.4 g/(L·d). The volumetric biogas production continuously increased to 1.65 L/(L·d). Microbial community diversity showed that a dramatic shift of bacterial structure occurred at BAL of 1.6 g/(L·d) and the structure presented better stability at high BAL. The syntrophic consortia and the main metabolic pathways were revealed through combination of the 16S rDNA and metagenome sequencing analyses. Syntrophomonas was the major butyrate-oxidizing bacterium and oxidized butyrate mainly through β-oxidaiton. Synergistaceae and Mesotoga acted as the main acetate-oxidizing bacteria. IHT and methanogenesis pathways were strongly enhanced by DMER64 and Methanosarcina as the main H2 carrier and dominant methanogen, respectively.

International audience; The Power-to-Gas concept corresponds to the use of the electric energy surplus to produce H2 by water electrolysis, that can be further converted to methane by biomethanation. However, the fluctuant production of renewable energy sources can lead to discontinuous H2 injections into the reactors, that may interfere with the adaptation of the microbial community to high H2 partial pressures. In this study, the response of the microbial community to H2 and organic feed starvation was evaluated in in-situ and ex-situ biomethanation. The fed-batch reactors were fed with acetate or glucose and H2, and one or four weeks of starvation periods were investigated. Methane productivity was mostly affected by the four-week starvation period. However, both in-situ and ex-situ biomethanation reactors recovered their methane production rate after starvation within approximately one-week of normal operation, while the anaerobic digestion (AD) reactors did not recover their performances even after 3 weeks of normal operation. The recovery failure of the AD reactors was probably related to a slow growth of the syntrophic and methanogen microorganisms, that led to a VFA accumulation. On the contrary, the faster recovery of both biomethanation reactors was related to the replacement of Methanoculleus sp. by Methanobacterium sp., restoring the methane production in the in-situ and ex-situ biomethanation reactors. This study has shown that biomethanation processes can respond favourably to the intermittent H2 addition without compromising their CH4 production performance.

Subalpine peatlands are a critical sink of mercury (Hg) and source of methylmercury (MeHg). However, little is known about the identity of Hg methylators or the geochemical factors controlling their distribution. Here, high-throughput sequencing of 16S rRNA and hgcA genes were used to investigate the community composition of Hg methylating microbes and their relating geochemical factors in Dajiuhu, which is a subalpine Sphagnum peatland. The results showed that MeHg content in the soils varied from 0.9 to 13.3 ng·g−1, while the ratio of MeHg to total Hg varied between 0.98% and 8.13%. The majority of the hgcA sequences clustered with Geobacteraceae, followed by Methanoregulaceae. The ratio of hgcA abundance and quantitative PCR (qPCR) gene copy numbers (hgcA/qPCR) showed that iron-reducing bacteria (FeRB) have the highest Hg methylator proportion within it, followed by methanogens. Although a high abundance of sulfate-reducing bacteria (SRB) was observed based on 16S rRNA, the abundance that contained hgcA sequences was low. Notably, as the MeHg concentration increased, the community structure of Hg methylating microbes shifted from FeRB dominance to FeRB and methanogen dominance, which was predicted by the redox potential (Eh) and C/N value.

Undigested and dewatered sludge at 10% total solids was pretreated at 60 °C for 3 h and fed to a lab-scale horizontal anaerobic bioreactor for 130 days with solids retention time (SRTs) from 25 to 16 d. The low-thermal pretreatment enabled higher net energy production, improved sludge treatment efficiency, and enhanced digestion stability. The highest average biomethane yield and production rate were 138.5 mL/g VS and 0.43 L/L.d, respectively, and the economic benefit was expected to be the maximum at SRT 16 d. Pretreatment did not increase the specific methanogenic activity per unit methanogen, but resulted in higher abundance of methanogenic archaea and hydrolytic bacteria. Methanogenic population shifted from hydrogenotrophic to acetoclastic, consistent with predicted gene expression at SRT equal or below 20 d. Anaerobic digestion along with low-thermal could be a feasible management strategy for undigested dewatered sludge from small WWTPs.

The efficiency of microbial interspecies electron transfer between syntrophic bacteria and methanogens is considered a rate-limiting factor for the overall efficiency of anaerobic digestion (AD). Stimulating interspecies electron transfer by biochars has been demonstrated to be efficient to enhance AD. However, the enhancing effects vary significantly depending on biochar properties. The correlations between them are not fully under-stood. Herein, biochars with different physicochemical properties were produced from a whiskey by-product "draff" and subsequently applied in the digestion of draff. The biochar produced at 700 degrees C statistically (p less than 0.05) enhanced biomethane yield by 5%. In contrast, biochars produced at 500 and 900 degrees C did not increase biomethane yield. The addition of 700 degrees C-derived biochar in AD increased the relative abundance of the methanogen Methanosarcina, which may be the electron-accepting partner in direct interspecies electron transfer (DIET). The enrichment of Methanosarcina suggested the potential shift of the interspecies electron transfer pathway towards the DIET mode. The characterization of biochar properties suggested that moderate graphiti-zation degree and abundant active surface functional groups (such as -C = O, pyridinic-N, and graphitic-N) were correlated with a more stimulating interspecies electron transfer through both the carbon matrices and the charging - discharging cycles of surface functional groups.

International audience; Fossil-fuel overuse and global warming are calling for new techniques to provide sustainable fuels. Biomethane can be produced by anaerobic digestion of organic waste, yet microbial mechanisms involved are still debated. Traditionally, reduction of carbon dioxide (CO2) to methane (CH4) is commonly explained by interspecies electron transfer, i. e., direct interspecies electron transfer (DIET)-based CO2 reduction or mediated interspecies electron transfer (MIET)-based CO2 reduction. For DIET-based CO2 reduction, or DIET-CO2 reduction, where electrons are provided by electricigens and transferred to methanogenic archaea to complete CO 2 reduction for methane production. Methanogenesis is also executed and facilitated by acetoclastic methanogenesis in the presence of conductive materials, as evidenced recently. Here we compare DIET-CO2 reduction and acetoclastic methanogenesis mediated by conductive materials. In the past decade, DIET-CO2 reduction is considered as the backbone for methane production strategy in anaerobic engineering digestion. But increasing evidences propose the importance of acetoclastic methanogenesis strengthened by exogenous media. DIET-based CO2 reduction has been extensively reviewed. Herein, we conclude the diverse microbial mechanisms affected by conductive materials to improve potential acetoclastic methanogenesis for the first time. Increasing electron transfer in methanogenic archaea and/or between bacteria and methanogens, microbial immobilization, pH buffering capacity, providing metal ions, reducing toxicity, regulation of oxidation-reduction potential are detailed reviewed. Possible future application based on acetotrophic methanogens is suggested via conductive materials in anaerobic digestion and natural ecological environment management.

International audience; To understand the dynamics of methanogens in the human intestinal microbiota, we investigated the presence of methanogens in meconium using a polyphasic approach including microscopy and PCR-sequencing in 33 meconium samples collected from 33 pre-term neonates, in accordance with current ethics regulation. In the presence of negative controls, 90.9% samples were real-time PCR-positive for methanogens and 69.7 % were PCR-sequencing positive, identified as Methanobrevibacter (M.) smithii. Further, auto-fluorescent analysis detected methanogens in the two meconium samples analyzed, with a morphology suggesting M. smithii. Multispacer Sequence Typing found M. smithii genotypes ST1 and ST2, previously described as intestinal microbiota inhabitants. C-section delivery and non-use of peripartum antibiotics significantly correlated with PCR-detection of methanogens in meconium. These data position M. smithii among the early inhabitants of the human gut, detectable immediately after birth and suggest the contribution of methanogens to the perinatal development of intestinal microbiota and physiology.

This study evaluated the effect of temperature on methane production by CO2 reduction during microbial electrosynthesis (MES) with a mixed-culture biocathode. Reactor performance, in terms of the amount and rate of methane production, current density, and coulombic efficiency, was compared at different temperatures. The microbial properties of the biocathode at each temperature were also analyzed by 16S rRNA gene sequencing. The results showed that the optimum temperature for methane production from CO2 reduction in MES with a mixed-culture cathode was 50°C, with the highest amount and rate of methane production of 2.06±0.13mmol and 0.094±0.01mmolh-1, respectively. In the mixed-culture biocathode MES, the coulombic efficiency of methane formation was within a range of 19.15±2.31% to 73.94±2.18% due to by-product formation at the cathode, including volatile fatty acids and hydrogen. Microbial analysis demonstrated that temperature had an impact on the diversity of microbial communities in the biofilm that formed on the MES cathode. Specifically, the hydrogenotrophic methanogen Methanobacterium became the predominant archaea for methane production from CO2 reduction, while the abundance of the aceticlastic methanogen Methanosaeta decreased with increased temperature.

Direct interspecies electron transfer (DIET) is a new mechanism responsible for syntrophic methane production. Supplementation of conductive material in syntrophic communities can facilitate electron transfer and enhance methane production. Ferroferric oxide (Fe3O4) was dosed in an anaerobic sequencing batch reactor (ASBR) fed with tryptone-based synthetic wastewater to examine its effect on the anaerobic treatment process. Long-term dosage of Fe3O4 not only enhanced the maximum methane production rate by 78.3% in a reaction cycle but also improved methane production when hydrogen/carbon dioxide or acetate was used as the substrate. The conductivity of anaerobic sludge, activity of the electron transport chain, and extracellular electron transfer ability were enhanced with the addition of Fe3O4, inducing enhanced system performance for methane production. Proteiniclasticum and Prolixibacter were the enriched acidogens responsible for hydrolysis and acidification. With the addition of Fe3O4, Methanosarcina was the dominant methanogen, and metagenomic analysis further revealed the genes involved in the hydrogenotrophic pathway of methanogenesis. Methanosarcina might be involved in DIET, and Fe3O4 was responsible for its stimulation.

Methane production by ruminant husbandry, a major source of greenhouse gas emissions, has been increasingly regarded as an enormous threat to global climate warming. In the last few decades, a great number of researches have been done to mitigate methane emission in ruminant animals. More recently, as potential antimethanogenic additives, nitrocompounds have attracted extensive attention, and progressive and prospective advances have been achieved. In this review, the additive dietary effects of nitroethane, 2-nitroalcohol, 2-nitro-1-propanol, 3-nitrooxypropanol, 3-nitro-1-propanol, and 3-nitro-1-propionic acid were considered regarding their antimethanogenic role based on in vitro and in vivo studies. Along with this review, the possible inhibition action mode of these nitrocompounds in the rumen were discussed and summarized with the aim to provide scientific insights for mitigating methane emission in ruminant animals. Most rumen microbes have been reported to tolerate relatively high concentrations of nitrotoxins, and the nitrocompounds are considered to serve as alternative electron acceptors; their competitive consumption of reducing equivalents and the inhibitory effect on H2 and formate oxidation are currently well accepted regarding their inhibiting roles in ruminal methane production. Until now, the published data on the metabolic mechanism of the nitrocompounds mainly focus on their tolerance in ruminal systems that metabolized to major fraction of their corresponding amines and less fraction of nitrite. However, less research has been conducted to determine the metabolic fate of a diverse group of nitrocompounds biotransformed by rumen microbes. In addition, coenzyme M methyl-transferase (MTR) and methyl–coenzyme M reductase (MCR) have been considered as two key enzymes in the methanogenic process responsible for methyl-group transfer and hydrogen-electron transfer, respectively. However, how the nitrocompounds could affect these coenzymes’ activities during the inhibition of ruminal methanogenesis is presently unknown. Different methane emission responses to dietary nitrocompounds and their association with the methanogen community variation, coenzyme activities of MTR and MCR, and metabolic fate of the nitrogenous compounds derived from nitrocompounds by rumen microbes obviously need further investigation in future research to provide a scientific, concrete reference for the practical application of these nitrocompounds in reducing methane emission in ruminant animals.