Acetate-utilizing Methanogens Research Articles

Promoting the production of methane on the co-digestion of food waste and sewage sludge by aerobic pre-treatment

In this study, methane (CH4) production performance on the co-digestion of food waste (FW) and sewage sludge (SS) was compared under non-pretreatment and 6 days aerobic pre-treatment. The results showed that, compared with the non-aerobic group, the CH4 productivity increased by nearly 60% through aerobic pretreatment. More ammonium was obtained after pre-aeration, providing a suitable growth environment for methanogens. After the aerobic pretreatment, proteins and carbohydrates converted into solute protein and carbohydrate, which could be easily consumed by microorganisms, concentrations of solute protein and carbohydrate were 350 and 667 mg/L in R2 and Pre-R2, respectively. At the same time, aerobic pretreatment affected the distribution of microbial community in the system. In the stable stage of Pre-R2, Methanosarcina as acetate-utilizing methanogen, became the dominant in the reactor, whose abundance was over 93%. It meant after the aerobic pretreatment, the main pathway for methanogenesis was acetotrophy. This study revealed that pre-aeration is a simple and desirable strategy for improving the CH4 productivity on the co-digestion of FW and SS.

The effect of propionic acid accumulation on methane production in dry mesophilic anaerobic fermentation

The dry anaerobic digestion performances were investigated at the temperature of 35±1°Cwith addition of propionic acid at different concentrations in five lab-scale reactors (R1-R5) to investigate the effect of propionic acid accumulation on methane production in dry mesophilic anaerobic digestion. And the volatile fatty acids, biogas and microbial community were detected. The active of microbial community and the methane generation would be inhibited when the propionic acid exceeded 30 gL1, which affected not only syntrophic propionate-oxidizing bacteria but also acetate-utilizing methanogens. Eubacteria decreased sharply accompany with the higher concentration of propionic in R4 and R5. The relative abundance of acetoclastic methanogens was directly correlated with volatile solids removals and methane generation in dry anaerobic reactors. Otherwise, the quantity of syntrophic propionate-oxidizing bacteria was positively linear correlated with propionic acid when the concentration was below 30 gL1. The methanogenic bacteria activity in R3 was the best with the methane yield of 27.01 mLCH4 g-1 VS d-1. The results suggested that appropriate propionate could enhance methane production in the dry anaerobic fermentation.

Methanosarcina acetivorans: A Model for Mechanistic Understanding of Aceticlastic and Reverse Methanogenesis.

Acetate-utilizing methanogens are responsible for approximately two-thirds of the one billion metric tons of methane produced annually in Earth’s anaerobic environments. Methanosarcina acetivorans has emerged as a model organism for the mechanistic understanding of aceticlastic methanogenesis and reverse methanogenesis applicable to understanding the methane and carbon cycles in nature. It has the largest genome in the Archaea, supporting a metabolic complexity that enables a remarkable ability for adapting to environmental opportunities and challenges. Biochemical investigations have revealed an aceticlastic pathway capable of fermentative and respiratory energy conservation that explains how Ms. acetivorans is able to grow and compete in the environment. The mechanism of respiratory energy conservation also plays a role in overcoming endothermic reactions that are key to reversing methanogenesis.

Anaerobic degradation of 2-propanol: Laboratory and pilot-scale studies

The anaerobic degradation of 2-propanol, an important industrial solvent, was scaled-up from batch assays to a pilot expanded granular sludge bed (EGSB) reactor at 25°C. Batch studies indicated that 2-propanol followed Haldane kinetics, with a maximum rate at 10gCODL−1. Concentrations as high as 25gCODL−1 did not inhibit the degradation of ethanol, a common co-solvent. Similar specific methanogenic activities (SMA) were obtained for water-solvent and water-brewery sludges (88 and 77mlCH4g-VS−1d−1 at 5gCODL−1). Continuous degradation showed a lag-phase of three weeks with water-brewery sludge. Increases in 2-propanol load from 0.05 to 0.18kgCODkg-VS−1d−1 caused a shift from the consumption of soluble matter to methane production, indicating polyhydroxybutyrates (PHB) accumulation. Conversely, smooth increases of up to 0.29kgCODkg-VS−1d−1 allowed 2-propanol degradation without PHB accumulation. The slowdown rate of 2-propanol-oxidizer and acetate-utilizing methanogen bacteria below 20°C adversely impacted both removal and CH4 yield.

A Ferredoxin- and F420H2-Dependent, Electron-Bifurcating, Heterodisulfide Reductase with Homologs in the Domains Bacteria and Archaea.

ABSTRACTHeterodisulfide reductases (Hdr) of the HdrABC class are ancient enzymes and a component of the anaerobic core belonging to the prokaryotic common ancestor. The ancient origin is consistent with the widespread occurrence of genes encoding putative HdrABC homologs in metabolically diverse prokaryotes predicting diverse physiological functions; however, only one HdrABC has been characterized and that was from a narrow metabolic group of obligate CO2-reducing methanogenic anaerobes (methanogens) from the domain Archaea. Here we report the biochemical characterization of an HdrABC homolog (HdrA2B2C2) from the acetate-utilizing methanogen Methanosarcina acetivorans with unusual properties structurally and functionally distinct from the only other HdrABC characterized. Homologs of the HdrA2B2C2 archetype are present in phylogenetically and metabolically diverse species from the domains Bacteria and Archaea. The expression of the individual HdrA2, HdrB2, and HdrB2C2 enzymes in Escherichia coli, and reconstitution of an active HdrA2B2C2 complex, revealed an intersubunit electron transport pathway dependent on ferredoxin or coenzyme F420 (F420H2) as an electron donor. Remarkably, HdrA2B2C2 couples the previously unknown endergonic oxidation of F420H2 and reduction of ferredoxin with the exergonic oxidation of F420H2 and reduction of the heterodisulfide of coenzyme M and coenzyme B (CoMS-SCoB). The unique electron bifurcation predicts a role for HdrA2B2C2 in Fe(III)-dependent anaerobic methane oxidation (ANME) by M. acetivorans and uncultured species from ANME environments. HdrA2B2C2, ubiquitous in acetotrophic methanogens, was shown to participate in electron transfer during acetotrophic growth of M. acetivorans and proposed to be essential for growth in the environment when acetate is limiting.

Evaluation of the effect of glycerol supplementation on the anaerobic digestion of real municipal solid waste in batch mode

The effect of low glycerol supplementation (<1% v/v) in the thermophilic anaerobic digestion (AD) of real municipal solid waste (RMSW) on methane production (MP) is not known. This study explores the effect of five different glycerol supplementations (0%, 0.1%, 0.25%, 0.5% and 1%) on effluent characteristics, anaerobic consortia and MP. Specifically, by adding 0.25% v/v crude glycerol to the feed, the methane production rate increased by 48% (from 7.40±1.17 l CH4/l to 11.01±1.66 l CH4/l), in line with the increase in total volatile solids (TVS) removal (from 65±7% to 81±7) and methanogenic activity (from 110∗10−12±17∗10−12 l CH4/cell to 156∗10−12±24∗10−12 l CH4/cell). Extra glycerol added to the feed (0.5% or 1%) was shown to be non-feasible for thermophilic AD of RMSW, as inhibition of methanogenesis was observed. Fluorescent in situ hybridization (FISH) studies showed that the percentages of Eubacteria, Archaea, H2-utilising methanogens (HUM) and acetate-utilising methanogens (AUM) were stable within the 94.0–97.0%, 6.0–3.0%, 1.8–3.6% and 0.6–2.8% ranges, respectively.

Synergistic pretreatment of waste activated sludge using CaO2 in combination with microwave irradiation to enhance methane production during anaerobic digestion

To investigate the effects of combined calcium peroxide (CaO2) and microwave pretreatment on anaerobic digestion of waste activated sludge, lab-scale experiments were conducted to measure the solubilization, biodegradation, and dewaterability of the waste activated sludge. Additionally, the synergistic effects between CaO2 and microwave were studied, and the microbial activity and methanogenic archaea community structure were analyzed. Combined pretreatment considerably facilitated the solubilization and subsequent anaerobic digestion of the waste activated sludge. The optimal pretreatment condition was CaO2 (0.1g/gVSS)/microwave (480W, 2min) for methane production during the subsequent anaerobic digestion process. Under this condition, 80.2% higher CH4 accumulation yield was achieved after 16d of anaerobic digestion when compared with the control. The synergistic effects of CaO2/microwave pretreatment resulted from the different mechanisms of CaO2 and microwave treatments. Further, microwave irradiation increased OH generation from CaO2 and significantly alleviated the inhibitory effect of CaO2 on methanogens. The activities of hydrolytic enzymes and acid-forming enzymes in the waste activated sludge were improved after CaO2 (0.1g/gVSS)/microwave (480W, 2min) pretreatment. Methanogenesis enzyme activity was also higher after CaO2 treatment (0.1g/gVSS)/microwave (480W, 2min) following a lag period. Illumina MiSeq sequencing analysis indicated that acetate-utilizing methanogen (Methanosaeta sp.) and H2/CO2-utilizing methanogen (Methanospirillum sp.) abundance improved greatly following CaO2 (0.1g/gVSS)/microwave (480W, 2min) pretreatment. The percentage of CH4 in biogas with CaO2/microwave pretreatment increased by 25.4% relative to the control. The dewaterability of the waste activated sludge also improved considerably after anaerobic digestion with combined CaO2/microwave treatment. Therefore, CaO2/microwave pretreatment can be used as an effective method to recover energy from waste activated sludge.

The role of methanogens in acetic acid production under different salinity conditions

In this study, a fed-batch acidogenic reactor was operated at a 3 d hydraulic retention time (HRT) and fed with alkaline pre-treated sludge to investigate salinity effects on methanogens’ abundance, activities and their consumption of produced acetic acid (HAc) and total volatile fatty acids (VFAs). The salinity concentration was increased step-wise by adding sodium chloride. At 3‰ (parts per thousand) salinity, the average produced volatile fatty acids (VFAs) concentration was 2410.16 ± 637.62 mg COD L−1 and 2.70 ± 0.36 L methane was produced daily in the acidogenic reactor. Further batch tests indicated methanogens showed a HAc degradation rate of 3.81 mg COD g−1 VSS h−1 at initial HAc concentration of 1150 mg COD L−1, and showed tolerance up to 16‰ salinity (3.76 g Na+ L−1) as indicated by a constant HAc degradation rate. The microbiological study indicated this can be related to the predominance of acetate-utilizing Methanosarcinaceae and Methanomicrobiales in the reactor. However, with salinity increased to 20‰ and 40‰, increases in VFAs and HAc production and decreases in methane production, methanogens population, acidogenic bacteria population and acidification extent were observed. This study demonstrated presence of acetate-utilizing methanogens in an acidogenic reactor and their high tolerance to salinity, as well as their negative impacts on net VFAs production. The results would suggest the presence of methanogens in the acidogenic reactor should not be ignored and the recovery of methane from the acidogenic reactor needs to be considered to avoid carbon loss.

Changes in microbial community during hydrogen and methane production in two-stage thermophilic anaerobic co-digestion process from biowaste

In this paper, the microbial community in a two-phase thermophilic anaerobic co-digestion process was investigated for its role in hydrogen and methane production, treating waste activated sludge and treating the organic fraction of municipal solid waste. In the acidogenic phase, in which hydrogen is produced, Clostridium sp. clusters represented 76% of total Firmicutes. When feeding the acidogenic effluent into the methanogenic reactors, these acidic conditions negatively influenced methanogenic microorganisms: Methanosaeta sp., (Methanobacteriales, Methanomicrobiales, Methanococcales) decreased by 75%, 50%, 38% and 52%, respectively. At the same time, methanogenic digestion lowered the numbers of Clostridium sp. clusters due to both pH increasing and substrate reduction, and an increase in both Firmicutes genera (non Clostridium) and methanogenic microorganisms, especially Methanosaeta sp. (208%). This was in accordance with the observed decrease in acetic (98%) and butyric (100%) acid contents. To ensure the activity of the acetate-utilizing methanogens (AUM) and the acetogens, high ratios of H2-utilizing methanogens (HUM)/AUM (3.6) were required.

Anaerobic Digestion at 45°C for Sludge Treatment: A Trade-off between Performances and Capability in Producing Class a Biosolids

 Abstract—Anaerobic digestion at mesophilic and thermophilic temperatures have been widely studied and evaluated for the purpose of sludge stabilization. However, limited extensive research has been conducted on anaerobic digestion in the intermediate zone of 45°C, mainly due to the notion that limited microbial activity occurs within this zone. The objectives of this research were to evaluate the performance and the capability of anaerobic digestion at 45°C in producing class A biosolids, in comparison to a mesophilic and thermophilic anaerobic digestion system operated at 35°C and 55°C, respectively. 45°C anaerobic digestion systems were not able to achieve comparable methane yield and high quality effluent as mesophilic system, though the systems produced biogas with 66.08±2.83% methane. No ammonia inhibition was observed and the digesters were able to achieve volatile solids (VS) reduction of 47.79±1.86%. Moreover, the pathogen counts were less than 1,000 MPN/g dry solids, thus, producing Class A biosolids. However, the 45°C systems suffered from high acetate accumulation, but sufficient buffering capacity was observed. Correspondingly, the dominant methanogen existed in 45°C system was thermo-tolerant acetate-utilizing methanogen of Methanosarcinaceae species.

Investigation of the Performance and Kinetics of Anaerobic Digestion at 45&amp;amp;#176;C

The objective of this study was to evaluate the performance of anaerobic digestion (AD) within the intermediate zone, specifically at 45°C. Single-stage batch anaerobic digestion system was developed in the lab and performance was monitored for more than 2 years. The AD system was able to achieve high biogas production with about 62% - 67% methane content. The digester exhibited high acetate accumulation, but sufficient buffering capacity was observed as the pH, alkalinity and volatile fatty acids-to-alkalinity ratio were within recommended values. The system achieved 36.5% reduction of total solids (TS) and 47.8% reduction of volatile solids (VS), which exceeded the required VS destruction efficiency for Class A biosolids. The pathogen counts were less than 1000 MPN/g total solids in the effluent, which also satisfied Class A biosolids requirements. The accumulation of acetate was presumably due to the high temperature which contributed to high hydrolysis rate. Consequently, it produced large amount of toxic salts that combined with the acetate, making them not readily available to be consumed by methanogens. The slower degradation of acetate was observed by the kinetic parameters. Accumulation of acetate contributed to 52% to 71% reduction in acetate degradation process, but was not completely inhibitory. The methanogens existing in the system were mostly thermo-tolerant acetate-utilizing methanogens, and specifically from Methanosarcinaceae species.

Biomethanization from sulfate‐containing municipal solid waste: effect of molybdate on microbial consortium

AbstractBACKGROUNDThe effect of molybdate on a microbial consortium of dry thermophilic anaerobic digestion (AD) from sulfate‐containing municipal solid waste was studied.RESULTSThe study showed molybdate inhibition affecting hydrolysis, acidogenesis, and methanogenesis, but the main microbial populations in AD can sustain the molybdate load over a short period of time. Molybdate was not a bactericide for all members of the principal groups, only a few of them are affected. Although all the microbial populations decreased, the weakest groups were sulfate reducing bacteria (SRBs) and butyrate‐utilising acetogens (BUAs) and the most resistant group was propionate‐utilising acetogens (PUAs). Prior to using molybdate, the relative percentages were: Eubacteria:Archaea 80:20; acetogens 25% (BUAs 11% and PUAs 14%) and SRBs 17%. After molybdate addition the relative percentages were: Eubacteria:Archaea were 83:17, acetogens remained over 28% (BUAs 8% and PUAs 20%) and SRBs around 11%. The relation between acetate‐utilising methanogens (AUMs) and hydrogen‐utilising methanogens (HUMs) changed slightly from 55:45 to 53:47.CONCLUSIONAll biochemical reactions in AD were inhibited and an increase in food:microorganism(F:M) ratio was observed. The microorganism's removal rates and growth inhibition rates are indicators of inhibitory effect in all analysed populations by inhibitor addition. © 2013 Society of Chemical Industry

Optimisation of single-phase dry-thermophilic anaerobic digestion under high organic loading rates of industrial municipal solid waste: Population dynamics

Different high feed organic loading rates (OLRs) (from 5.7 g to 46.0 g TVS/l/d) or hydraulic retention times (HRTs) (from 15 d to 2 d) in single-phase dry-thermophilic anaerobic digestion (AD) of organic fraction municipal solid waste (OFMSW) were investigated. The specific gas production (SGP) values (0.25-0.53 m(3)/kg TVS) and the percentages of Eubacteria, Archaea, H2-utilising methanogens (HUMs) and acetate-utilising methanogens (AUMs) were stable within the ranges 80.2-91.1%, 12.4-18.5%, 4.4-9.8% and 5.5-10.9%, respectively. A HUM/AUM ratio greater than 0.7 seems to be necessary to maintain very low partial pressures of H2 required for dry AD process. Increasing OLR resulted in an increase in all the populations, except for propionate-utilising acetogens (PUAs). Optimal conditions were obtained at 3d HRT (OLR=30.7 g TVS/l/d), which is lower than the doubling time of acetogens and methanogens. The methane production (MP) was clearly higher than those reported in AD of OFMSW.

U(VI) Bioreduction with Emulsified Vegetable Oil as the Electron Donor – Microcosm Tests and Model Development

We conducted microcosm tests and biogeochemical modeling to study U(VI) reduction in contaminated sediments amended with emulsified vegetable oil (EVO). Indigenous microorganisms in the sediments degraded EVO and stimulated Fe(III), U(VI), and sulfate reduction, and methanogenesis. Acetate concentration peaked in 100-120 days in the EVO microcosms versus 10-20 days in the oleate microcosms, suggesting that triglyceride hydrolysis was a rate-limiting step in EVO degradation and subsequent reactions. Acetate persisted 50 days longer in oleate- and EVO- than in ethanol-amended microcosms, indicating that acetate-utilizing methanogenesis was slower in the oleate and EVO than ethanol microcosms. We developed a comprehensive biogeochemical model to couple EVO hydrolysis, production, and oxidation of long-chain fatty acids (LCFA), glycerol, acetate, and hydrogen, reduction of Fe(III), U(VI) and sulfate, and methanogenesis with growth and decay of multiple functional microbial groups. By estimating EVO, LCFA, and glycerol degradation rate coefficients, and introducing a 100 day lag time for acetoclastic methanogenesis for oleate and EVO microcosms, the model approximately matched observed sulfate, U(VI), and acetate concentrations. Our results confirmed that EVO could stimulate U(VI) bioreduction in sediments and the slow EVO hydrolysis and acetate-utilizing methanogens growth could contribute to longer term bioreduction than simple substrates (e.g., ethanol, acetate, etc.) in the subsurface.

Microbial community structure in a thermophilic aerobic digester used as a sludge pretreatment process for the mesophilic anaerobic digestion and the enhancement of methane production

An effective two-stage sewage sludge digestion process, consisting of thermophilic aerobic digestion (TAD) followed by mesophilic anaerobic digestion (MAD), was developed for efficient sludge reduction and methane production. Using TAD as a biological pretreatment, the total volatile suspended solid reduction (VSSR) and methane production rate (MPR) in the MAD reactor were significantly improved. According to denaturing gradient gel electrophoresis (DGGE) analysis, the results indicated that the dominant bacteria species such as Ureibacillus thermophiles and Bacterium thermus in TAD were major routes for enhancing soluble organic matter. TAD pretreatment using a relatively short SRT of 1 day showed highly increased soluble organic products and positively affected an increment of bacteria populations which performed interrelated microbial metabolisms with methanogenic species in the MAD; consequently, a quantitative real-time PCR indicated greatly increased Methanosarcinales (acetate-utilizing methanogens) in the MAD, resulting in enhanced methane production.

Evaluation of Process Performance and Sludge Properties of an up-flow staged Sludge Blanket (USSB) reactor for Treatment of Molasses Wastewater

A combination of an acidification reactor and an up-flow staged sludge bed (USSB) reactor wasapplied to treatment of molasses wastewater. The USSB, equipped with three gas solid separators, wasselected because of superior organic removal by means of good retention of granular sludge. The combinedsystem was continuously operated at mesophilic (35°C) conditions for 600 days. The USSB achieved a highorganic removal rate of 37 kgCOD/m3 day (organic loading rate of 43 kgCOD/m3 day with 86.2% CODremoval). The USSB retained high sludge concentration of approximately 58 gVSS/L based on the reactorvolume. The retained granular sludge had good settle-ability with a sludge volume index (SVI) of 4 to 20 ml/gSS. The retained sludge possessed a sufficient level of methanogenic activities for acetate (1.2 gCOD/gVSS day) and for hydrogen (1.7 gCOD/gVSS day). Analysis of microbial community revealed that genus Methanobacterium as hydrogen utilizing methanogen and order Methanosarcinales as acetate utilizingmethanogen were detected in the retained sludge of the USSB reactor. The superior performance of the USSB was attributed to good retention of a large amount of granular sludge with high methanogenic activity.

Intermittent irrigation changes production, oxidation, and emission of CH4 in paddy fields determined with stable carbon isotope technique

Intermittent irrigation is an important option for mitigating CH4 emissions from paddy fields. In order to better understand its controlling processes in CH4 emission, CH4 fluxes, CH4 production and oxidation potentials in paddy soils, and 13C-isotopic signatures of CH4 were observed in field and incubation experiments. The relative contribution of acetate to total CH4 production (fac) and fraction of CH4 oxidized (fox) in the field was also calculated using the isotopic data. At the beginning of the rice season, the theoretical ratio of acetate fermentation: H2/CO2 reduction=2:1 was reached, however, in the late season H2/CO2-dependent methanogenesis became dominant. Compared to continuous flooding, intermittent irrigation significantly reduced CH4 production potential and slightly decreased fac-value, indicating methanogens, particularly acetate-utilizing methanogens, were inhibited. CH4 oxidation was very important, especially in paddy fields under intermittent irrigation where 19–83% of the produced CH4 was oxidized. Intermittent irrigation enhanced CH4 oxidation potential slightly and raised fox-value significantly relative to continuous flooding. Intermittent irrigation significantly decreased CH4 flux creating a more positive δ13C-value of emitted CH4 by 12–22‰. A significant negative correlation was found between CH4 fluxes and values of δ13CH4 suggesting that the less the CH4 oxidation, the higher the CH4 emission, and the lower the δ13C-value of emitted CH4. Collectively, the findings show that intermittent irrigation reduced the seasonal CH4 production potential by 45% but increased the fraction of CH4 oxidized by 45–63%, thus decreasing the seasonal CH4 emission from the paddy fields by 71%, relative to continuous flooding.

Microbial community structure elucidates performance of Glyceria maxima plant microbial fuel cell.

The plant microbial fuel cell (PMFC) is a technology in which living plant roots provide electron donor, via rhizodeposition, to a mixed microbial community to generate electricity in a microbial fuel cell. Analysis and localisation of the microbial community is necessary for gaining insight into the competition for electron donor in a PMFC. This paper characterises the anode–rhizosphere bacterial community of a Glyceria maxima (reed mannagrass) PMFC. Electrochemically active bacteria (EAB) were located on the root surfaces, but they were more abundant colonising the graphite granular electrode. Anaerobic cellulolytic bacteria dominated the area where most of the EAB were found, indicating that the current was probably generated via the hydrolysis of cellulose. Due to the presence of oxygen and nitrate, short-chain fatty acid-utilising denitrifiers were the major competitors for the electron donor. Acetate-utilising methanogens played a minor role in the competition for electron donor, probably due to the availability of graphite granules as electron acceptors.

Adhesion and Coaggregation Phenomena of Acetate-utilizing Methanogens under Coexistence of Acidogens

Adhesion and Coaggregation Phenomena of Acetate-utilizing Methanogens under Coexistence of Acidogens

Estimation of Adhesion and Aggregation of Acetate-utilizing Methanogens

From the view point of fine particle technology, the effects of surface properties of methanogens, which are related to the rate-limiting step in the methane fermentation process, on their adhesion and aggregation were examined. As a result, Methanosarcina barkeri were found to be charged negatively and their isoelectric point was around pH 2. In contrast, Methanosaeta concilii was found to be non-charged and their isoelectric point was around pH 4.5. According to the ATR-FTIR analysis and acid-base titration, it was guessed that the difference of surface potential between two types of methanogens was due to the difference of the amount of carboxyl group and phosphate group on their microbial surface. Methanosarcina barkeri showed better adhesion to not the negatively-charged glass but to the positively-charged one. Percent aggregate of Methanosarcina barkeri increased with an increase in the ionic strength of the cell suspension. In contrast, percent aggregate of Methanosaeta concilii was almost constant regardless of the ionic strength. These adhesion and aggregation phenomena could be understood by the electrostatic interaction and the change of free energy of interaction between methanogen and slide glass.