Bioconversion of carbon dioxide to methane using hydrogen and hydrogenotrophic methanogens

Biogas produced from organic wastes contains energetically usable methane and unavoidable amount of carbon dioxide. The exploitation of whole biogas energy is locally limited and utilization of the natural gas transport system requires CO₂ removal or its conversion to methane. The biological conversion of CO₂ and hydrogen to methane is well known reaction without the demand of high pressure and temperature and is carried out by hydrogenotrophic methanogens. Reducing equivalents to the biotransformation of carbon dioxide from biogas or other resources to biomethane can be supplied by external hydrogen. Discontinuous electricity production from wind and solar energy combined with fluctuating utilization cause serious storage problems that can be solved by power-to-gas strategy representing the production of storable hydrogen via the electrolysis of water. The possibility of subsequent repowering of the energy of hydrogen to the easily utilizable and transportable form is a biological conversion with CO₂ to biomethane. Biomethanization of CO₂ can take place directly in anaerobic digesters fed with organic substrates or in separate bioreactors. The major bottleneck in the process is gas-liquid mass transfer of H₂ and the method of the effective input of hydrogen into the system. There are many studies with different bioreactors arrangements and a way of enrichment of hydrogenotrophic methanogens, but the system still has to be optimized for a higher efficiency. The aim of the paper is to gather and critically assess the state of a research and experience from laboratory, pilot and operational applications of carbon dioxide bioconversion and highlight further perspective fields of research.     

Rumen methanogens: a review

The Methanogens are a diverse group of organisms found in anaerobic environments such as anaerobic sludge digester, wet wood of trees, sewage, rumen, black mud, black sea sediments, etc which utilize carbon dioxide and hydrogen and produce methane. They are nutritionally fastidious anaerobes with the redox potential below −300 mV and usually grow at pH range of 6.0–8.0 [1]. Substrates utilized for growth and methane production include hydrogen, formate, methanol, methylamine, acetate, etc. They metabolize only restricted range of substrates and are poorly characterized with respect to other metabolic, biochemical and molecular properties.     

三硝酸丙三酯对厌氧真菌和甲烷菌代谢产物、纤维水解酶活性及菌群丰度的影响

【背景】近年来硝酸酯化合物抑制瘤胃甲烷生成的能力受到广泛关注,但鲜有研究关注其对瘤胃内主要纤维降解菌——厌氧真菌的影响。【目的】研究三硝酸丙三酯(Nitroglycerin,NG)对厌氧真菌和甲烷菌活性和代谢的影响。【方法】利用厌氧真菌(Piromyces sp.F1)纯培养及与甲烷菌共培养(Piromyces sp.F1+Methanobrevibacter sp.),比较不同剂量NG(0.0、6.6、13.2和19.8μmol/L)对厌氧真菌和甲烷菌的代谢终产物、主要纤维水解酶活性及菌群丰度的影响。【结果】厌氧真菌共培养组中,6.6μmol/L NG完全抑制了甲烷的生成,积累了氢气和甲酸(P0.05),降低了乙酸浓度以及木聚糖酶和羧甲基纤维素酶活性(P0.05),但未显著影响厌氧真菌和甲烷菌的丰度(P0.05)。厌氧真菌纯培养组中,6.6μmol/L NG显著降低了甲酸、乙酸、乙醇、木聚糖酶和羧甲基纤维素酶活性(P0.05),但总产气量和氢气产量没有显著差异(P0.05)。在2个培养体系中,随着剂量的升高,NG对厌氧真菌和甲烷菌的抑制增强。【结论】NG能够抑制甲烷菌的活性,但对厌氧真菌也有抑制作用。     

Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens

Methane-forming archaea are strictly anaerobic microbes and are essential for global carbon fluxes since they perform the terminal step in breakdown of organic matter in the absence of oxygen. Major part of methane produced in nature derives from the methyl group of acetate. Only members of the genera Methanosarcina and Methanosaeta are able to use this substrate for methane formation and growth. Since the free energy change coupled to methanogenesis from acetate is only − 36 kJ/mol CH₄, aceticlastic methanogens developed efficient energy-conserving systems to handle this thermodynamic limitation. The membrane bound electron transport system of aceticlastic methanogens is a complex branched respiratory chain that can accept electrons from hydrogen, reduced coenzyme F₄₂₀ or reduced ferredoxin. The terminal electron acceptor of this anaerobic respiration is a mixed disulfide composed of coenzyme M and coenzyme B. Reduced ferredoxin has an important function under aceticlastic growth conditions and novel and well-established membrane complexes oxidizing ferredoxin will be discussed in depth. Membrane bound electron transport is connected to energy conservation by proton or sodium ion translocating enzymes (F₄₂₀H₂ dehydrogenase, Rnf complex, Ech hydrogenase, methanophenazine-reducing hydrogenase and heterodisulfide reductase). The resulting electrochemical ion gradient constitutes the driving force for adenosine triphosphate synthesis. Methanogenesis, electron transport, and the structure of key enzymes are discussed in this review leading to a concept of how aceticlastic methanogens make a living. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Isolation of natural cultures of anaerobic fungi and indigenously associated methanogens from herbivores and their bioconversion of lignocellulosic materials to methane

This study aimed to obtain natural cultures of anaerobic fungi and their indigenously associated methanogens from herbivores and investigate their ability to degrade lignocelluloses to methane. Eight natural cultures were obtained by Hungate roll tube technique. The fungi were identified as belonging to Piromyces, Anaeromyces and Neocallimastix respectively by microscopy, and the methanogens as Methanobrevibacter spp. by 16S rRNA gene sequencing. In vitro studies with rice straw showed that these cultures degraded 33.5-48.3% substrate and produced 0.33-0.84 mmol/(100 ml culture) methane. Two cultures were further selected for their ability to degrade different lignocellulosic materials and could produce 0.38-1.27 mmol/(100 ml culture) methane. When methanogens were inhibited, the lignocellulose-degrading ability of cultures significantly reduced. In conclusion, natural cultures of anaerobic fungi with indigenously associated methanogens with high fiber degradation ability were obtained, and these cultures may have the potential in industrial use in lignocelluloses degradation and methane production.     

The anaerobic decomposition of benzoic acid during methane fermentation

Anaerobic rupture of the benzoic acid ring was investigated. Carbon 4 was converted primarily to carbon dioxide. Following ring rupture during methane fermentation, propanoic acid is an intermediate, and carbon 4 of benzoate becomes its carboxyl.Contribution No. 1285-j, Division of Biology, Kansas State University, Manhattan, KS 66506. This work was supported in part by funds from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506. Paper II of this series is Fina and Fiskin (1960)     

共存于厌氧真菌分离培养液中瘤胃甲烷菌的检测及其多样性分析

对分离自山羊瘤胃的真菌分离培养液中甲烷菌进行16SrDNA扩增、DGGE分析、RFLP及测序分析,研究共存于真菌分离培养液中甲烷菌的种类及其多样性。DGGE结果显示:从厌氧真菌分离至第45代,甲烷菌多样性指数由1·32降至0·99,相似性最低为34·7%;第45代至62代,多样性指数由0·99升至1·15,相似性最低为89·2%。RFLP多态性分析69个克隆共得到5个操作分类单元,选择其中6个具有代表性的序列进行测序。序列及系统进化分析表明,属于其中3个操作分类单元的克隆最相似菌都是UnculturedarchaealsymbiontPA202,相似性均为95%,没有与这些克隆相似性较高的已培养甲烷菌;属于另外2个操作分类单元的克隆最相似菌都是Unculturedrumenmethanogen956,相似性均为97%,最相似已知菌为Methanobrevibactersp.NT7,相似性为97%。结果表明,真菌培养液中存在目前尚未分离培养的瘤胃甲烷菌。     

On-field study of anaerobic digestion full-scale plants (Part II): new approaches in monitoring and evaluating process efficiency

Biogas plants need easy and practical tools for monitoring and evaluating their biological process efficiency. As soon as, in many cases, biomass supply present considerable costs, full-scale anaerobic digestion (AD) processes must approach, as much as possible, the potential biogas yield of the organic mixture fed to the biodigesters. In this paper, a new indicator is proposed (the bio-methane yield, BMY), for measuring the efficiency in full-scale AD processes, based on a balance between the biochemical methane potential (BMP) of the input biomass and the residual BMP of the output materials (digestate). For this purpose, a one-year survey was performed on three different full-scale biogas plants, in the Italian agro-industrial context, and the bio-chemical processes were fully described in order to calculate their efficiencies (BMY = 87-93%) and to validate the new indicator proposed, as useful and easily applicable tool for full-scale AD plants operators.     

Effects of ensiling, silage additives and storage period on methane formation of biogas crops

Effects of the ensiling process, storage periods of up to 1 year and several chemical and biological silage additives on biomethanation were assessed for maize, sorghum, forage rye and triticale with the aim to identify optimised conditions for silage production of crops used as feedstock in biogas plants. Ensiling, prolonged storage and biological silage additives showed positive effects on methane yield of up to 11%. These could be attributed to increases in organic acids and alcohols during ensiling. A regression model including acetic acid, butyric acid and ethanol accounts for 75-96% of the variation in methane yield. Storage periods of up to 1 year for properly ensiled crops could be possible without losses in methane production, considering the increase in methane yield and the losses of dry matter during this period. However, taking storage losses into account silage additives showed little effect on methane production.     

Biological conversion of methane to liquid fuels: Status and opportunities

Methane is the main component of natural gas and biogas. As an abundant energy source, methane is crucial not only to meet current energy needs but also to achieve a sustainable energy future. Conversion of methane to liquid fuels provides energy-dense products and therefore reduces costs for storage, transportation, and distribution. Compared to thermochemical processes, biological conversion has advantages such as high conversion efficiency and using environmentally friendly processes. This paper is a comprehensive review of studies on three promising groups of microorganisms (methanotrophs, ammonia-oxidizing bacteria, and acetogens) that hold potential in converting methane to liquid fuels; their habitats, biochemical conversion mechanisms, performance in liquid fuels production, and genetic modification to enhance the conversion are also discussed. To date, methane-to-methanol conversion efficiencies (moles of methanol produced per mole methane consumed) of up to 80% have been reported. A number of issues that impede scale-up of this technology, such as mass transfer limitations of methane, inhibitory effects of H₂S in biogas, usage of expensive chemicals as electron donors, and lack of native strains capable of converting methane to liquid fuels other than methanol, are discussed. Future perspectives and strategies in addressing these challenges are also discussed.     

Using enriched cultures for elevation of anaerobic syntrophic interactions between acetogens and methanogens in a high-load continuous digester

Volatile fatty acids (VFAs) are key intermediates in anaerobic digestion. Enriched acetogenic and methanogenic cultures were used for syntrophic anaerobic digestion of VFAs in a high-load continuous reactor fed with acetic (HAc), propionic (HPr) and butyric (HBu) acids at maximum concentrations of 5, 3 and 4 g/L, respectively. Interactive effects of HPr, HBu and HAc were analyzed. Furthermore, hydraulic retention time (HRT) and methanogen to acetogen population ratio (M/A) were investigated as key microbiological and operating variables of VFA anaerobic degradations. Optimum conditions were found to be HPr = 1125.0 mg/L, HBu = 1833.4 mg/L, HAc = 1727.4 mg/L, HRT = 21 h and M/A = 2.5 (corresponding to maximum VFA removal and biogas production rate (BPR)). Results of verification experiments and predicted values from fitted correlations were in close agreement at 95% confidence interval. HRT and M/A had positive effects on VFA removal and BPR. M/A was the most important factor that affected BPR. All VFAs inhibited VFA removals.     

Competition and coexistence of sulfate-reducing bacteria,acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol⁻¹, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80–85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10–15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol⁻¹, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40–45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community.     

Isolation and characterization of a motile hydrogenotrophic methanogen from rice paddy field soil in Japan

A hydrogenotrophic motile methanogen was isolated from flooded Japanese paddy field soil. Anaerobic incubation of the paddy soil on H(2)-CO(2) at 20 degrees C led to the enrichment of symmetrically curved motile autofluorescent rods. The methanogenic strain TM20-1 isolated from the culture was halotolerant and utilized H(2)-CO(2), 2-propanol-CO(2), or formate as a sole methanogenic substrate. Based on the 16S rRNA gene sequence similarity (94.8%) with Methanospirillum hungateii, and on the physiological and phenotypic characteristics, TM20-1 was suggested to be a newly identified species belonging to the genus Methanospirillum. This is the first report of isolation of the genus Methanospirillum strain from a rice paddy field.     

Stirring and biomass starter influences the anaerobic digestion of different substrates for biogas production

Here, we present the results of lab‐scale experiments conducted in a batch mode to determine the biogas yield of lipid‐rich waste and corn silage under the effect of stirring. Further semi‐continuous experiments were carried out for the lipid‐rich waste with/without stirring. Additionally, it was analyzed how the starter used for the batch experiment influences the digestion process. The results showed a significant stirring effect on the anaerobic digestion only when seed sludge from a biogas plant was used as a starter. In this case, the experiments without stirring yielded only about 50% of the expected biogas for the investigated substrates. The addition of manure slurry to the batch reactor as part of the starter improved the biogas production. The more diluted media in the reactor allowed a better contact between the bacteria and the substrates making stirring not necessary.     

水生生态系统中金属依赖型甲烷厌氧氧化过程的研究进展

甲烷是一种比CO_2更活跃的温室气体,微生物驱动的甲烷厌氧氧化(anaerobicoxidationof methane,AOM)过程对于降低全球甲烷的排放有着重要意义。参与AOM反应的最终电子受体主要分为三类,即硫酸盐、亚硝酸盐/硝酸盐以及以Fe(III)、Mn(IV)等为代表的金属离子。可溶性金属物质和不溶性金属矿物都可以被用作AOM的电子受体,这大大提高了参与金属依赖型甲烷厌氧氧化(metal-dependent anaerobic oxidation of methane,Metal-AOM)微生物的生态价值。目前研究聚焦在功能菌群、生态分布等方面。部分甲烷厌氧氧化古菌(anaerobic methanotrophic archaea,ANME)具有直接或间接参与Metal-AOM过程的能力。但由于功能菌群纯化富集和分离具有一定难度,有关其生理生化和生态学等特征的研究受到限制。同时,随着Metal-AOM被发现存在于不同水生生境中,其在污染治理领域的应用也被广泛讨论,但是河口生境尚缺乏深入研究。本文从Metal-AOM的发现入手,阐述了参与该过程的主要微生物及其在水域环境下的生态分布,并介绍了Metal-AOM的反应机制和在实际应用中的机遇与挑战。最后,根据现有研究结果,提出对功能菌群、机制及环保应用的研究展望,包括微生物分离纯化和影响因素、菌群代谢活性和作用机制的解析以及新型生产工艺的设计和发展应用,以期为今后的环境污染治理和工业应用提供借鉴意义。     

Direct conversion of cellulose to methane by anaerobic fungus Neocallimastix frontalis and defined methanogens

Co-cultures of N. frontalis with a formate-utilizing methanogen, Methanobacterium formicicum and/or an aceticlastic methanogen, Methanosaeta concilii, were performed for methane production from cellulose. In the co-culture with M. formicicum, ca. 16 mM CH₄ was produced after 7 days without accumulation of H₂ and formate. In the co-culture with M. concilii, 12 mM CH₄ was produced after 17 days with decreasing acetate production. In the tri-culture of N. frontalis with M. formicicum and M. concilii, 24 mM CH₄ was produced after 17 days where acetate still remained at 23 mM, but production of lactate and ethanol decreased. When a 4-times concentrated culture broth of M. concilii was inoculated in this tri-culture system in a bioreactor, 150 mM CH₄ was produced after 24 days by feeding of cellulose, although 57 mM acetate still accumulated.     

Production of methane and hydrogen by anaerobic ciliates containing symbiotic methanogens

Rates of methane production by three anaerobic ciliates containing symbiotic methanogens (the marine Metopus contortus and Plagiopyla frontata, and the limnic Metopus palaeformis) were quantified. Hydrogen production by normal (containing active symbionts), aposymbiotic and BES-treated cells was also measured in the case of the marine species. Methanogenesis was closely coupled to host metabolism and growth; at maximum ciliate growth rates (20°C) each methanogen produced about 1 fmol CH₄ per hour corresponding to about 7, 4 and 0.35 pmol per ciliate per hour for M. contortus, P. frontata and M. palaeformis, respectively. Normal cells produced traces of H₂. Hydrogen production by BES-treated or aposymbiotic cells accounted for 75 and 45% of the methane production of normal M. contortus and P. frontata cells, respectively. However, it is possible that hydrogen production was partly inhibited in the absence of methanogens. Theoretical considerations suggest that hydrogen transfer is significant to the metabolism of larger anaerobic ciliates. Ciliates with methanogens produced CH₄ under microaerobic conditions due to their ability to maintain an anoxic intracellular environment at low external oxygen tensions. Methanogenesis was still detectable at a pO₂ of 0.63 kPa (3 %atm sat).     

A new algorithm to characterize biodegradability of biomass during anaerobic digestion: influence of lignin concentration on methane production potential

We examined the influence of fibrous fractions of biomass on biochemical methane potential (BMP) with the objective of developing an economical and easy-to-use statistical model to predict BMP, and hence the biodegradability of organic material (BD) for biogas production. The model was developed either for energy crops (grass, maize, and straw) or for animal manures, or as a combined model for these two biomass groups. It was found that lignin concentration in volatile solids (VS) was the strongest predictor of BMP for all the biomass samples. The square of the sample correlation coefficient (R(2)) from the BMP versus lignin was 0.908 (p<0.0001), 0.763 (p<0.001) and 0.883 (p<0.001) for animal manure, energy crops and the combined model, respectively. Validation of the combined model was carried out using 65 datasets from the literature.     

Elimination of methane generated from landfills by biofiltration: a review

The production of biogas in landfills, its composition and the problems resulting from its generation are all reviewed. Biofiltration is a promising option for the control of emissions to atmosphere of the methane contained in biogas issued from the smaller and/or older landfills. A detailed review of the methane biofiltration literature is presented. The microorganisms, mainly the methanotrophs, involved in the methane biodegradation process, and their needs in terms of oxygen and carbon dioxide utilization, are described. Moreover, the influence of nutrients such as copper, nitrogen and phosphorus, and the process operating conditions such as temperature, pH and moisture content of the biofilter bed, are also presented. Finally, the performance of various filter beds, in terms of their elimination capacities, is presented for laboratory scale biofilters and landfill covers.     

Continuously-stirred anaerobic digester to convert organic wastes into biogas: system setup and basic operation

Anaerobic digestion (AD) is a bioprocess that is commonly used to convert complex organic wastes into a useful biogas with methane as the energy carrier. Increasingly, AD is being used in industrial, agricultural, and municipal waste(water) treatment applications. The use of AD technology allows plant operators to reduce waste disposal costs and offset energy utility expenses. In addition to treating organic wastes, energy crops are being converted into the energy carrier methane. As the application of AD technology broadens for the treatment of new substrates and co-substrate mixtures, so does the demand for a reliable testing methodology at the pilot- and laboratory-scale. Anaerobic digestion systems have a variety of configurations, including the continuously stirred tank reactor (CSTR), plug flow (PF), and anaerobic sequencing batch reactor (ASBR) configurations. The CSTR is frequently used in research due to its simplicity in design and operation, but also for its advantages in experimentation. Compared to other configurations, the CSTR provides greater uniformity of system parameters, such as temperature, mixing, chemical concentration, and substrate concentration. Ultimately, when designing a full-scale reactor, the optimum reactor configuration will depend on the character of a given substrate among many other nontechnical considerations. However, all configurations share fundamental design features and operating parameters that render the CSTR appropriate for most preliminary assessments. If researchers and engineers use an influent stream with relatively high concentrations of solids, then lab-scale bioreactor configurations cannot be fed continuously due to plugging problems of lab-scale pumps with solids or settling of solids in tubing. For that scenario with continuous mixing requirements, lab-scale bioreactors are fed periodically and we refer to such configurations as continuously stirred anaerobic digesters (CSADs). This article presents a general methodology for constructing, inoculating, operating, and monitoring a CSAD system for the purpose of testing the suitability of a given organic substrate for long-term anaerobic digestion. The construction section of this article will cover building the lab-scale reactor system. The inoculation section will explain how to create an anaerobic environment suitable for seeding with an active methanogenic inoculum. The operating section will cover operation, maintenance, and troubleshooting. The monitoring section will introduce testing protocols using standard analyses. The use of these measures is necessary for reliable experimental assessments of substrate suitability for AD. This protocol should provide greater protection against a common mistake made in AD studies, which is to conclude that reactor failure was caused by the substrate in use, when really it was improper user operation.