Methanogenic Pathway Research Articles

Simultaneous intensification of direct acetate cleavage and CO2 reduction to generate methane by bioaugmentation and increased electron transfer

Direct interspecies electron transfer coupled to CO2 reduction, DIET-CO2 reduction, to generate methane is proposed and prosperous in 2010s. It is well known that bioaugmentation and increased electron transfer benefit DIET-CO2 reduction. Herein, we studied whether other methanogenic pathways, such as H2-mediated methanogenic progress and direct acetate cleavage (acetoclastic methanogenesis), are simultaneously favorable in the presence of conductive materials (CMs). If so, contribution of DIET-CO2 reduction may be overestimated because overwhelming studies just considered this pathway. Detailed researches on whether and how Clostridium pasteurianum coupled with CMs, granular activated carbon, biochar, nano-magnetite and grapheme, influenced methanogenic progresses were conducted. Overall, C. pasteurianum enhanced methane production rate, which was further improved by some CMs. Combined with metabolism, kinetic and electrochemical analysis, experimental results showed that hydrogenotrophic methanogenesis occurred and bioaugmentation strengthened this progress, which was further motivated by CMs, such as biochar and magnetite. 16S rRNA gene analysis suggested that Methanobacteriaceae was potentially responsible for methane production. Whereafter, DIET-CO2 reduction may become prosperous according to electrochemical and thermomechanical analysis. Acetoclastic methanogenesis was also triggered by bioaugmentation based on experiments by using inhibitor, CH3F, for acetoclastic methanogenesis and carbon isotope fractionation. More importantly, magnetite and graphene, which significantly accelerated electron transfer based on electrochemical analysis, further stimulated acetoclastic methanogenesis. Methanothrix dominated and may play an important role in this stage. This work suggests that diverse methanogenic pathways may be benefited from an increase of electron transfer other than DIET-CO2 reduction. Consequently, the long-standing view that only DIET-CO2 reduction was stimulated by conductive materials may need to be reevaluated. Our research provides potential guides to increase methane production during anaerobic digestion by the enhancement of acetoclastic methanogenesis.

Chemical Pretreatments to Enrich the Acidogenic Phase in a System Coupled Packed Bed Reactor with a UASB Reactor Using Peels and Rotten Onion Waste

A large amount of onion wastes are produced by both domestically and industrially, making it necessary to search for a reuse process. These wastes causing odor and in some cases causes harm to the environment. At 2017, Mexico produced 1.6 million tons of which 10% are classified as organic waste. The aim of this study was to contribute to the reduction of onion peel residues, using chemical pretreatments (H2SO4, HCl, H3PO4 and NaOH) to enrich the acidogenic phase in the anaerobic digestion (AD) to improve the yield of methane in a system coupled packed bed reactor with an Up-flow Anaerobic Sludge Blanket reactor (UASB) using peels and rotten onion waste. In anaerobic digestion of white onion residues in a single stage (PBR1) presented inhibition due to the accumulation of VFA during the first days, which delay the onset of the methanogenic phase, which occurred 63 days after the start of the experiment. Pretreatment with H2SO4 (PBR2) decreased the start time of the methanogenic pathway, from 63 days to 35. In the same way, the coupling of the UASB reactor to the packed reactor (PBR3) allowed the decrease of the methanogenic step after 14 days, and the total time for this process was 35 days. The yield for PBR1 was 0.013 m3 CH4 kg VSrem−1, for PBR2 0.014 m3 CH4 kg VSrem−1 and PBR3 0.017 m3 CH4 kg VSrem−1. The total yield of PBR3 + UASB was 0.41 m3 CH4 kg VSrem−1.

Methanogenesis and secondary processes in a fractured claystone aquifer influenced by a former landfill site: Implications for the interpretation of dissolved gases

Similar to marine and/or freshwater sediments, dissolved methane and longer chained hydrocarbons are ubiquitous in all natural groundwater environments including highly productive porous aquifers. The isotopic and chemical characteristics of dissolved gases in groundwater related to municipal landfills commonly differ from other non-polluted environments. In the survey presented in this study we investigated the geochemical and isotopic variations of dissolved hydrocarbons in a unique study area with different well defined ecological micro-habitats influenced by a former landfill for industrial and special waste. The dissolved methane concentrations have generally remained stable over the last decade but vary by several orders of magnitude in different zones of the area. The dominant methanogenic pathways can be clearly distinguished in the different ecosystems. Methyl-type fermentation methanogenesis prevails in the shallow groundwater zone (5–15 m depth) in- and outside the landfill. Anaerobic methane oxidation coupled to sulfate reduction occurs in the shallow downstream area within a former leachate plume. At larger depths CO2 reduction is the predominant methane formation pathway except within the physically isolated landfill site. The high ethane concentrations in a small part of the study area are potentially related to the degradation of volatile organic compounds. The background ethane concentrations are higher in the deeper groundwater zones, indicating the coupling to the CO2 reduction methanogenic pathway. The propane concentrations in most wells are below the detection limit, except within the waste site.Secondary methane oxidation, which frequently occurs in the groundwater in all environments can alter the gas composition mimicing the thermogenic gas signature in the standard Bernard-diagram. The isotopic fractionation between co-existing methane and formation water and carbon dioxide, respectively, differs from that suggested in standard interpretational diagrams (e.g. Bernard-Diagram and Whiticar-plots). Thus, the standard evaluation based on methane hydrogen and carbon isotopes is also susceptible to an erroneous interpretation as non-biogenic dissolved gases in other groundwater environments.

Characterization of Anthracite-Degrading Methanogenic Microflora Enriched from Qinshui Basin in China

Microorganisms are essential for the formation of biogenic coalbed methane (CBM) and the application of microbially enhanced CBM (MECoM). Anthracite-degrading methanogenic microflora was cultivated by enrichment and subculture from produced water obtained in Qinshui Basin where CBM is explored commercially. The maximum methane yield was 255 μmol/g of coal after 23 days of cultivation at 35 °C, 1.1–1.2% of salinity, <0.15 mm of coal particle size, pH 8–9, and 5 mL of inocula/g of coal. Microflora archaea, as revealed by MiSeq, were mainly composed of Methanosaeta, an acetic acid-consuming methanogen, followed by Methanocella, a hydrogen-consuming methanogen, suggesting a diversity of methanogenic pathways with a dominance of acetoclastic methanogenesis. The bacteria mainly included Enterobacter, Acetoanaerobium, Macellibacteroides, Clostridium, and Ercella. FTIR analysis showed that the oxygen-containing groups and aliphatic groups were the main targets for microbial degradation. XRD analysis indicated that crystal nucleus structures of coal decreased after degradation. The concentrations of aliphatics were shown to increase over the incubation period based on GC–MS analysis, while the concentrations of dissolved aromatics decreased. These results show the potential for enhancing CBM production by stimulating the activity of methanogenic consortia in situ in Qinshui Basin as well as other high-rank coal reservoirs.

Biological treatment of electronic industry wastewater containing TMAH, MEA and sulfate in an UASB reactor

This study investigated the feasibility of the methanogenic treatment of electronic industry wastewater containing tetramethylammonium hydroxide (TMAH), monoethanolamine (MEA) and sulfate in a lab-scale mesophilic up-flow anaerobic sludge blanket reactor. Feeding a mixture of electronic industry wastewater and co-substrate organics to the reactor for smooth acclimatization of sludge gave complete degradation of each organics within five days. When the reactor was fed only electronic industry wastewater, total COD removal, TMAH removal and MEA removal were achieved over 80, 99 and 99%, respectively, at an organic loading rate of 11.5 kg-COD m−3 day−1. 173 mg-S L−1 of influent sulfate was almost reduced simultaneously with the COD removal. In order to evaluate performance stability, the TMAH shock load event was performed under the conditions of 11,000 mg-COD L−1 for 24 h. Inflow of high TMAH concentration inhibited TMAH degradation and sulfate reduction for more than one month, however, not MEA. The batch feeding experiment and specific activity measurement revealed degradation pathways of each organics. TMAH was degraded via methanogenic pathway without sulfate reduction, MEA was degraded via methanogenic pathway with sulfate reduction. The results indicated that methanogenic treatment was applicable to electronic industry wastewater by appropriate reactor handling.

Enhancing methane production of anaerobic sludge digestion by microaeration: Enzyme activity stimulation, semi-continuous reactor validation and microbial community analysis

Effects of microaeration pretreatment on sludge hydrolysis, biogas production and microbial community structure in anaerobic digestion (AD) were investigated by bench-scale tests and semi-continuous experiments. Bench tests showed that microaeration led to the release of dissolved organic matters, generation of volatile fatty acids and stimulation of enzyme activity. Correlation analysis showed that methane production was significantly correlated with the activity of α-glucosidase at 0.01 level, and with protease activity, released polysaccharides and VFAs at 0.05 level. Semi-continuous experiments showed that microaeration accelerated the utilization of organic matters, increased biogas production by 16.4%, enhanced methane content in biogas, and improved sludge dewaterability. Microbial community structure analysis showed that microaeration promoted enrichment of hydrolytic and fermentative bacteria in AD reactor rather than methanogenic bacteria, and aceticlastic methanogenesis was the main methanogenic pathway for methane production.

Methane formation and consumption by sediments in a cross-channel profile of a small river impoundment

Rivers are a natural source of methane (CH4) into the atmosphere and may contribute significantly to total CH4 emissions. Even though the details of sources of CH4 in rivers are not fully understood, weirs have been recognized as a hotspot of CH4 emissions. In this study, we investigated CH4 production and consumption in air-exposed river sediments along a cross-channel transect located upstream of a weir. Stable carbon isotopes were used for determination of individual methanogenic pathways. In order to understand the relationship between physicochemical and biological processes, additional parameters such as organic matter, grain median size, and carbon and nitrogen content were characterized as well. Generally, samples from the surface sediment layer (0-10 cm) had higher CH4 production than sediments from the deeper layer (10-20 cm) during the incubation experiments. Sediments near the bank zones and in the mid-channel were characterized by the highest organic carbon content (6.9 %) as well the highest methanogenic activity (2.5 mmol g-1 DW d-1). The CH4 production was predominated by H2/CO2 dependent methanogenesis in the surface sediment layer (0-10 cm), while the proportion of acetoclastic and hydrogenotrophic methanogenesis in the deeper sediment layer (10-20 cm) was balanced. The CH4 oxidation potential of sediments showed the same spatial pattern as observed for the CH4 production. Our results showed high spatial variability of sediment CH4 production and oxidation in the cross-channel profile upstream of the weir, whereas the highest CH4 dynamics were observed in the littoral zones. This variability was closely linked with the carbon and nitrogen content in the sediment samples.

Mesophilic and thermophilic anaerobic digestion of soybean curd residue for methane production: Characterizing bacterial and methanogen communities and their correlations with organic loading rate and operating temperature

To find the optimal operation parameters and provide an explanation of methanogenic pathway for methane production in mesophilic (35 °C) and thermophilic (55 °C) anaerobic digestion (MAD, TAD) of soybean curd residue (SCR), MAD and MAD were contrastively investigated for 95 days. The maximum available OLR was identified as 3.3 gVS/L for both MAD and TAD. Compared to MAD, TAD exhibited a 20% higher average methane yield (0.591 L/gVS) and a 7.5% higher volatile solids removal efficiency (74.1 ± 10.4%). Bacterial phyla Bacteroidetes, Firmicutes and Proteobacteria dominated in MAD digesters while genus Defluviitoga was selectively enriched in TAD digesters due to higher temperature and organic loading pressure. Principal coordinates analysis of methanogen community showed that both temperature and OLR were crucial environmental variables shifting the taxonomic patterns of the methanogens. The enriched methanogen genus Methanothermobacter (93%) with a hydrogenotrophic methanogenic pathway had a close correlation with the TAD performance.

Enhanced methanogenic performance and metabolic pathway of high solid anaerobic digestion of chicken manure by Fe2+ and Ni2+ supplementation

High solid anaerobic digestion (AD) is a promising technology for the treatment of organic waste in terms of efficient bioenergy production and digestate volume reduction. However, the high solid AD of chicken manure has been a challenge due to the inhibition effects caused by high ammonia levels. In this study, the addition of 280 mg/L Fe2+ and 2 mg/L Ni2+ has been proven to enhance the microbial activity and to overcome inhibition under the stressed environment. The mesophilic digesters feeding with total solid around 150 g/kg operated at an organic loading rate of 4.8 g/(L d) based on volatile solid and a 20-day hydraulic retention time. The total ammonia-nitrogen reached around 6.8 g/L in control and Fe-Ni digesters. The results from the 147-day continuously-fed experiment provided an increment of 34% methane production and a 29% reduction of volatile fatty acids against control. The acetoclastic and hydrogenotrophic methanogenic activity was increased by 89% and 40% respectively. The high throughout sequencing results showed an increased percentage of Methanosarcina sp., which may have contributed to the shifting of the methanogenic pathway towards acetoclastic methanogenesis. The positive effects of Fe2+ and Ni2+ supplementation obtained in this study lay the foundation for its use in AD of nitrogen rich materials.

CO2 conversion to methane and biomass in obligate methylotrophic methanogens in marine sediments

Methyl substrates are important compounds for methanogenesis in marine sediments but diversity and carbon utilization by methylotrophic methanogenic archaea have not been clarified. Here, we demonstrate that RNA-stable isotope probing (SIP) requires 13C-labeled bicarbonate as co-substrate for identification of methylotrophic methanogens in sediment samples of the Helgoland mud area, North Sea. Using lipid-SIP, we found that methylotrophic methanogens incorporate 60–86% of dissolved inorganic carbon (DIC) into lipids, and thus considerably more than what can be predicted from known metabolic pathways (~40% contribution). In slurry experiments amended with the marine methylotroph Methanococcoides methylutens, up to 12% of methane was produced from CO2, indicating that CO2-dependent methanogenesis is an alternative methanogenic pathway and suggesting that obligate methylotrophic methanogens grow in fact mixotrophically on methyl compounds and DIC. Although methane formation from methanol is the primary pathway of methanogenesis, the observed high DIC incorporation into lipids is likely linked to CO2-dependent methanogenesis, which was triggered when methane production rates were low. Since methylotrophic methanogenesis rates are much lower in marine sediments than under optimal conditions in pure culture, CO2 conversion to methane is an important but previously overlooked methanogenic process in sediments for methylotrophic methanogens.

Three-stage anaerobic co-digestion of food waste and waste activated sludge: Identifying bacterial and methanogenic archaeal communities and their correlations with performance parameters

A three-stage anaerobic digester setup was configured and evaluated for enhanced methane production during co-digestion of food waste and waste activated sludge and the corresponding bacterial and methanogen communities were characterized. Results showed that the average methane yield (0.496 L/gVS) in the three-stage digester was 13–52% higher than that of one- and two-stage digesters. Compared to controls, an increase of 12–47% in volatile solids reduction was achieved in the three-stage digester (69.3 ± 6.7%). Bacterial phyla Proteobacteria, Firmicutes and Bacteroidetes dominated in one-, two- and three-stage digester while genera Pseudomonas, Tissierella, and Petrimonas were selectively enriched in the three-stage digester due to functional segregation. Taxonomic analysis identified 8 dominant methanogen genera, of which Methanosarcina, Methanosaeta, Methanobacterium and Methanolinea collectively accounted for 80%. With increasing OLR and digester stage number, the dominant methanogenic pathway shifted from hydrogenotrophic pattern to acetoclastic pattern and reached a final synergy of these two.

Potential sources of dissolved methane at the Tablelands, Gros Morne National Park, NL, CAN: A terrestrial site of serpentinization

The Tablelands massif in Western Newfoundland is part of a Phanerozoic ophiolite sequence and is a terrestrial site of serpentinization. Similar to other Phanerozoic ophiolite sequences, the Tablelands possess environmental conditions that are conducive to all three established pathways of methanogenesis – abiogenic, microbial, and thermogenic – or a combination thereof. Sourcing methane from the Tablelands has thus far been limited because of the low dissolved methane concentrations. We tested dissolved gas extraction and concentration methods (vacuum extraction and gas stripping) for their effect on isotopic fractionation, and we applied these methods to sample dissolved methane for carbon and hydrogen isotopes (δ13CCH4 and δDCH4) as well as doubly substituted “clumped” isotopologue (13CH3D) analyses. Clumped isotope thermometry of methane estimated an apparent temperature of 85 ± 7 °C. The carbon isotope value of methane (δ13CCH4) for samples collected in 2017 was −27.9 ± 0.5‰, consistent with previously measured values (−27.3 ± 0.5‰) dating back to 2009. The hydrogen isotope value of methane (δDCH4) in 2017 was −175 ± 5‰. On a carbon deuterium (CD) plot, the Tablelands methane data plotted outside of the microbial field, but within an area where abiogenic and thermogenic fields overlap. A conclusive discrimination between abiogenic and thermogenic methanogenic pathways remains challenging. Nevertheless, a combination of sedimentary organic matter characterization of the underlying sedimentary units and comparison of geochemical characteristics with those described in experimental and theoretical studies suggest that the methane extracted from the Tablelands is likely a result of slow production of methane at ~85 °C from background organic sources.

Co-digestion of wastewater sludge: Choosing the optimal blend

Anaerobic co-digestion (AcoD) is a promising strategy to increase the methane production of anaerobic digestion plants treating wastewater sludge (WAS). In this work the degradability of six different mixtures of WAS with fish waste (FW) or garden-grass (GG) was evaluated and compared to the three mono-digestions. Degradation performances and methanogenic pathways, determined with the isotopic signatures of biogas, were compared across time. Fish and grass mono-digestion provided a higher final methane production than WAS mono-digestion. In co-digestion the addition of 25% of fish was enough to increase the final methane production from WAS while 50% of grass was necessary. To determine the optimal blend of WAS co-digestion two indicators were specifically designed, representing the maximum potential production (ODI) and the expected production in mono-digestion conditions (MDI). The comparison between these indicators and the experimental results showed that the most productive blend was composed of 75% of co-substrate, fish or grass, with WAS. Indeed, the final methane production was increased by 1.9 times with fish and by 1.7 times with grass associated to an increase of the methane production rate by 1.5 times. Even if the same succession of methanogenic pathways across time was observed for the different mixtures, their relative proportions were different. Sewage sludge degradation was mostly achieved through hydrogenotrophic pathway while acetoclastic pathway was dominant for fish and grass degradation. These results were confirmed by the identification of Archaea with 16S sequencing.

Hydrogenotrophic methanogenesis dominates at high pH

One potential design for a geological disposal facility (GDF) for intermediate level radioactive waste (ILW) involves the use of a cement base grout which will establish a highly alkaline environment for extended time periods [1]. Methane generation by colonising microbes could impact the long-term performance of the facility by influencing gas pressures and potentially leading to the migration of 14C to the biosphere [1]. Sediments acquired from a wide-range of anthropogenic alkaline sites in the UK were used to develop acetoclastic and hydrogenotrophic methanogen enrichment cultures over a broad range of pH values (7.0–12.0). The generation of methane from hydrogen and acetate was assessed to determine the dominant methanogenic pathways. Archaeal community analysis via Illumina MiSeq was employed to describe the populations involved and the acetoclastic inhibitor methyl fluoride was utilised to confirm the lack of acetate-dependent methane generation under alkaline conditions. High pH (pH&gt;9.0) microcosms employing alkaline sediments were dominated by hydrogen-consuming methanogens of the orders Methanobacteriales and Methanomicrobiales, with no acetate consumption detected under these conditions. In contrast, neutral pH microcosms employing control sediments were dominated by acetoclastic methanogens of the order Methanosarcinales and demonstrated high acetate consumption rates. The rate of acetate consumption and proportion of acetoclastic methanogens decreased in a linear fashion as the pH within cultures was increased, however hydrogen consumption rates remained stable up to pH 11.0. The data shown suggests hydrogenotrophic methanogenesis is the dominant methanogenic pathway at high pH which could have important consequences on gas pressures within a GDF.

Mitigation of ammonia inhibition through bioaugmentation with different microorganisms during anaerobic digestion: Selection of strains and reactor performance evaluation

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 (<2%)), but displayed pivotal roles in determining the overall microbial consortium and, in turn, improved the overall performance.

Novel insight into high solid anaerobic digestion of swine manure after thermal treatment: Kinetics and microbial community properties

Compared to traditional anaerobic digestion (AD), high solid anaerobic digestion (HSAD) had the advantages of small digester, low heating energy and less digestate. However, the methane production was poor. In our previous study, thermal treatment (70 ± 1 °C, 3 days) without any dilution could satisfactorily enhance the methane production rate of HSAD by up to 39.5%. However, effects of solid content on HSAD after thermal treatment were not yet studied. In this study, HSAD was conducted at 11.7–17.6% solid content, and the control experiment was conducted at low solid content (4.4% solid content). Results showed that HSAD's methane production rate was the highest at 11.7% solid content (158 mL CH4/g VS), and could reach up to 89.2% of that at 4.4% solid content. The utilization of organics was revealed by kinetics analysis that the readily biodegradable organics could be utilized at increasing solid content with decreasing hydrolysis rate. Furthermore, it was notable that methylotrophic methanogens predominated in HSAD with the abundance of 82.6%. This was quite unique from the general belief that AD system was usually dominated by acetoclastic or hydrogenotrophic methanogenic pathways. In this study, the microbial community structure of HSAD after thermal treatment was firstly studied, its unique specific methanogenic pathways was firstly revealed.

The origin of methane in an Eastern Ontario aquifer

Data from a comprehensive isotopic study is presented for a bedrock interface aquifer affected by Pleistocene post-glacial marine transgression in Eastern Ontario, and hosting groundwaters with anomalously high dissolved methane concentrations surpassing Province of Ontario recommended guidelines for drinking water, in an effort to constrain both the enrichment mechanisms and sources of methane. Stable isotopic signatures (δ13C & δ2H) of collected methane gas samples indicate a mixture of microbial methane gases generated via CO2 reduction and acetoclastic methanogenic pathways. Radiocarbon data for methane and dissolved inorganic/organic carbon indicate that the dominant sources of carbon in the aquifer are marine organic matter dating from both the Champlain Sea incursion (10–12 ka BP) and ancient terrestrial organic matter from an Early-Wisconsinian interstadial period. The prevalence of ancient organic matter sources is particularly evident in the methane pool. During early stages of methanogenesis, CO2 derived from the preferential decomposition of labile marine organic matter is the dominant substrate for methanogenesis, whereas a substrate transition to acetate derived from the fermentation of refractory ancient terrestrial organic matter is observed during later stages of methanogenesis. Both sources of organic matter (marine, terrestrial) are present in the glaciomarine sediment matrix associated with Champlain Sea incursion. Contributions of thermogenic methane from the underlying Billings shale are possible, and merit further investigation.

Quantification of Methanogenic Pathways Using Stable Carbon Isotopic Signatures.

In many anaerobic environments methane (CH4) is produced by methanogens, with either H2/CO2 or acetate (i.e., the methyl group) as precursors, through what are referred to as hydrogenotrophic and acetoclastic methanogenic pathways respectively. Their relative contribution to total CH4 production can be quantified by determining the stable carbon isotopic fractionation factors for both pathways as well as the isotopic signatures of CO2, CH4, and the methyl group in acetate of the sample. The procedures for measuring carbon isotopic fractionation factors of both methanogenic pathways and isotopic composition of these compounds by isotope ratio mass spectrometry are described in this chapter. The results are very helpful in evaluating the activity of the methanogens involved in each methanogenic pathway as well as those of other biological pathways with different fractionation factors.

Carbon-13 stable isotope analysis reveals the existence but insignificance of ruminal methanogenic pathway from acetate in a batch culture system

A series of in vitro batch culture incubations were conducted to investigate the extent of carboxyl group and methyl group in acetate converted to CH4 using a stable isotopic tracer method. Ground lucerne hay was incubated as a substrate with buffered ruminal fluid, and 13C labeled CH313COONa or 13CH3COONa was supplied into the incubation bottles at a dose of 0, 15, 35 or 55 mM. Increasing CH313COONa or 13CH3COONa supplementation linearly increased (P < 0.05) absolute 13CH4 and 13CO2 production (mL/bottle) at all the time points measured. Yet, the conversion rates (CR) of 13CH4 and 13CO2 produced from labeled CH313COONa or 13CH3COONa were very low. For instance, the relative 13CH4 CR (%) was ranged from 0.0028-0.14% with incubation time extended to 48 h, and the relative 13CO2 CR (%) was ranged from 0.054 to 1.3%. Moreover, acetate supplementation linearly increased (P < 0.01) the concentration of butyrate, 16S rRNA gene copies of protozoa, total methanogens, Methanosphaera stadtmanae, Methanobrevibacter smithii and Methanosarcina barkeri. In summary, our study indicates existence of methanogenesis from acetate, but such pathway of methanogenesis makes little contribution to the total CH4 production through in vitro ruminal fermentation.

Enhanced production of secondary biogenic coalbed natural gas from a subbituminous coal treated by hydrogen peroxide and its geochemical and microbiological analyses

Unmineable coal accounts for over 90% of the world’s fossil fuel resources. Fortunately, many coal seams contain indigenous microorganisms capable of utilizing the coal as a carbon source to produce secondary biogenic coalbed natural gas. However, coal bioavailability has been shown to be a significant factor that limits the extent of bioconversion. In this study, we have analyzed the rate and yield of the biogas production and assessed the gas potential, carbon balance, stable carbon isotopes, microbial communities, and microbial pathways changes resulting from the hydrogen peroxide pretreatment with the Wyoming’s Powder River Basin subbituminous coal. The results showed that coal pretreated with hydrogen peroxide can significantly enhance the bioavailability of coal for enhancing the biogas production, with a peak yield of 552.6 μmol/g coal (437.1 Scf/ton coal) at the day 184. The stable carbon isotopic analysis indicated that the δ13C values of the methane and carbon dioxide were much less negative than the published field data. This suggested that the enrichment or depletion of the precursor 13C could contribute to the shift of the carbon isotopic composition in the subsequence processes. Therefore, the data should be used with cautions for interpreting genesis of the thermogenic/biogenic methane and the methanogenic pathways. The methanogenic pathways were also investigated with re-fed experiment and microbial community analysis. The results indicated that the hydrogenotrophic pathway was not active in the original inoculum became activated after the gas production. The microbial community analysis demonstrated that the obligate hydrogenotrophic methanobacterium was the most dominant methanogens in the microcosms after the gas production. This suggested that the chemical treatment of coal has impacts on the microbial structure during the subsequent methane production phase.