微生物互营产甲烷过程中的种间电子传递

甲烷作为全球第二大温室气体,是典型的可再生清洁能源,也是碳循环中的重要物质组成。大气中约74%的甲烷由产甲烷古菌和其他微生物的互营产生,种间电子传递(interspecies electron transfer, IET)是微生物菌群降低热力学能垒、实现互营产甲烷的核心过程。IET可分为间接种间电子传递(mediated interspecies electron transfer,MIET)和直接种间电子传递(direct interspecies electron transfer, DIET)两种类型,其中MIET依赖氢气、甲酸等载体完成电子的远距离传输,而DIET则依赖导电菌毛、细胞色素c等膜蛋白,通过微生物的直接接触实现电子传递。本文将从IET的研究历程出发,从电子传递机制、微生物种类、生态多样性等方面对微生物互营产甲烷过程中的两种IET类型进行比较,最后对未来待探索的方向进行展望。本综述有助于加深对微生物互营产甲烷过程中IET的理解,为解决由甲烷引发的全球气候变暖等生态问题提供理论支撑。     

Evidence for the coexistence of direct and riboflavin-mediated interspecies electron transfer in Geobacter co-culture

Geobacter species can secrete free redox-active flavins, but the role of these flavins in the interspecies electron transfer (IET) of Geobacter direct interspecies electron transfer (DIET) co-culture is unknown. Here, we report the presence of a new riboflavin-mediated interspecies electron transfer (RMIET) process in a traditional Geobacter DIET co-culture; in this process, riboflavin contributes to IET by acting as a free-form electron shuttle between free Geobacter species and serving as a bound cofactor of some cytochromes in Geobacter co-culture aggregates. Multiple lines of evidence indicate that RMIET facilitates the primary initiation of syntrophic growth between Geobacter species before establishing the DIET co-culture and provides additional ways alongside the DIET to transfer electrons to achieve electric syntrophy between Geobacter species. Redox kinetic analysis of riboflavin on either Geobacter species demonstrated that the Gmet_2896 cytochrome acts as the key riboflavin reduction site, while riboflavin oxidation by Geobacter sulfurreducens is the rate-limiting step in RMIET, and the RMIET makes only a minor contribution to IET in Geobacter DIET co-culture. The discovery of a new RMIET process in Geobacter DIET co-culture suggests the complexity of IET in syntrophic bacterial communities and provides suggestions for the careful examination of the IET of other syntrophic co-cultures.     

Influence of pH on the balance between methanogenesis and iron reduction

Methanogenesis and iron reduction play major roles in determining global fluxes of greenhouse gases. Despite their importance, environmental factors that influence their interactions are poorly known. Here, we present evidence that pH significantly influences the balance between each reaction in anoxic environments that contain ferric (oxyhydr)oxide minerals. In sediment bioreactors that contained goethite as a source of ferric iron, both iron reduction and methanogenesis occurred but the balance between them varied significantly with pH. Compared to bioreactors receiving acidic media (pH 6), electron donor oxidation was 85% lower for iron reduction and 61% higher for methanogenesis in bioreactors receiving alkaline media (pH 7.5). Thus, methanogenesis displaced iron reduction considerably at alkaline pH. Geochemistry data collected from U.S. aquifers demonstrate that a similar pattern also exists on a broad spatial scale in natural settings. In contrast, in bioreactors that were not augmented with goethite, clay minerals served as the source of ferric iron and the balance between each reaction did not vary significantly with pH. We therefore conclude that pH can regulate the relative contributions of microbial iron reduction and methanogenesis to carbon fluxes from terrestrial environments. We further propose that the availability of ferric (oxyhydr)oxide minerals influences the extent to which the balance between each reaction is sensitive to pH. The results of this study advance our understanding of environmental controls on microbial methane generation and provide a basis for using pH and the occurrence of ferric minerals to refine predictions of greenhouse gas fluxes.     

产甲烷分离物中Clostridium spp.与Methanosarcina barkeri潜在的种间直接电子传递

【目的】革兰氏阴性菌Geobacter metallireducens可以与乙酸型产甲烷菌Methanosaeta harundinacea或Methanosarcina barkeri通过种间直接电子传递(DIET)还原CO2产甲烷。本实验室前期的研究发现Methanosarcina mazei和Geobacteraceae在铁还原富集培养中形成团聚体,可能存在直接电子传递。然而,革兰氏阳性菌(如Clostridium spp.)与产甲烷菌是否存在种间直接电子传递尚不明确。【方法】采用Hungate厌氧滚管法,以乙醇为唯一电子供体从铁还原富集培养体系中获得产甲烷分离物(S6)。通过T-RFLP及克隆文库分析群落多样性,结合循环伏安法等电化学方法研究产甲烷分离物的电活性。【结果】Clostridium spp.(与C.tunisiense相似性最高)和M.barkeri分别在S6细菌和古菌群落中占优势。S6与G.metallireducens共培养后铁还原和产甲烷能力未明显增加,Clostridium spp.可能与G.metallireducens类似,将电子直接传递给产甲烷菌M.barkeri产甲烷。此外,电化学检测发现,在用透析袋包裹电极阻碍微生物与电极表面通过直接接触形成生物膜的条件下,电流密度显著降低,并且循环伏安扫描无明显氧化还原峰。【结论】产甲烷分离物S6中存在直接电子传递途径。本工作提出在产甲烷分离物中占优势的革兰氏阳性菌Clostridium spp.和M.barkeri之间可能存在种间直接电子传递。     

Advances in direct interspecies electron transfer and conductive materials: Electron flux,organic degradation and microbial interaction

Direct interspecies electron transfer (DIET) via electrically conductive pili (e-pili) and c-type cytochrome between acetogens and methanogens has been proposed as an essential pathway for methane production. Supplements of conductive materials have been extensively found to promote methane production in microbial anaerobic treatment systems. This review comprehensively presents recent findings of DIET and the addition of conductive materials for methanogenesis and summarizes important results through aspects of electron flux, organic degradation, and microbial interaction. Conductive materials improve DIET and methanogenesis by acting as either substitute of e-pili or electron conduit between e-pili and electron acceptors. Other effects of conductive materials such as the change of redox potential may also be important factors for the stimulation. The type and organic loading rate of substrates affect the occurrence of DIET and stimulating effects of conductive materials. Geobacter, which can participate in DIET, were less enriched in anaerobic systems cultivated with non-ethanol substrates, suggesting the existence of other syntrophs with the capability of DIET. The coupling of communication systems such as quorum sensing may be a good strategy to achieve the formation of biofilm or granule enriched with syntrophic partners capable of DIET.     

Respiratory interactions of soil bacteria with (semi)conductive iron‐oxide minerals

Pure‐culture studies have shown that dissimilatory metal‐reducing bacteria are able to utilize iron‐oxide nanoparticles as electron conduits for reducing distant terminal acceptors; however, the ecological relevance of such energy metabolism is poorly understood. Here, soil microbial communities were grown in electrochemical cells with acetate as the electron donor and electrodes (poised at 0.2 V versus Ag/AgCl) as the electron acceptors in the presence and absence of iron‐oxide nanoparticles, and respiratory current generation and community structures were analysed. Irrespective of the iron‐oxide species (hematite, magnetite or ferrihydrite), the supplementation with iron‐oxide minerals resulted in large increases (over 30‐fold) in current, while only a moderate increase (～10‐fold) was observed in the presence of soluble ferric/ferrous irons. During the current generation, insulative ferrihydrite was transformed into semiconductive goethite. Clone‐library analyses of 16S rRNA gene fragments PCR‐amplified from the soil microbial communities revealed that iron‐oxide supplementation facilitated the occurrence of Geobacter species affiliated with subsurface clades 1 and 2. We suggest that subsurface‐clade Geobacter species preferentially thrive in soil by utilizing (semi)conductive iron oxides for their respiration.     

Syntrophic oxidation of propionate in rice field soil at 15 and 30°C under methanogenic conditions

Propionate is one of the major intermediary products in the anaerobic decomposition of organic matter in wetlands and paddy fields. Under methanogenic conditions, propionate is decomposed through syntrophic interaction between proton-reducing and propionate-oxidizing bacteria and H(2)-consuming methanogens. Temperature is an important environmental regulator; yet its effect on syntrophic propionate oxidation has been poorly understood. In the present study, we investigated the syntrophic oxidation of propionate in a rice field soil at 15°C and 30°C. [U-(13)C]propionate (99 atom%) was applied to anoxic soil slurries, and the bacteria and archaea assimilating (13)C were traced by DNA-based stable isotope probing. Syntrophobacter spp., Pelotomaculum spp., and Smithella spp. were found significantly incorporating (13)C into their nucleic acids after [(13)C]propionate incubation at 30°C. The activity of Smithella spp. increased in the later stage, and concurrently that of Syntrophomonas spp. increased. Aceticlastic Methanosaetaceae and hydrogenotrophic Methanomicrobiales and Methanocellales acted as methanogenic partners at 30°C. Syntrophic oxidation of propionate also occurred actively at 15°C. Syntrophobacter spp. were significantly labeled with (13)C, whereas Pelotomaculum spp. were less active at this temperature. In addition, Methanomicrobiales, Methanocellales, and Methanosarcinaceae dominated the methanogenic community, while Methanosaetaceae decreased. Collectively, temperature markedly influenced the activity and community structure of syntrophic guilds degrading propionate in the rice field soil. Interestingly, Geobacter spp. and some other anaerobic organisms like Rhodocyclaceae, Acidobacteria, Actinobacteria, and Thermomicrobia probably also assimilated propionate-derived (13)C. The mechanisms for the involvement of these organisms remain unclear.     

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO₂ and either ethanol or lactate but not H₂ gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO₂ reduction, with a formate production rate and methanogenic turnover constant of 660 μM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 μM) than digestor contents, and the free flora was the primary site for formate cleavage to CO₂ and H₂ (90 μM formate per h). Inhibition of methanogenesis by CHCl₃ resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H₂ gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H₂ transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.     

互营氧化产甲烷微生物种间电子传递研究进展

甲烷是重要的温室气体,也是典型的可再生性生物质能源。目前约70%的大气甲烷排放来源于产甲烷微生物过程。在产甲烷环境中,产甲烷菌与互营细菌形成互营关系,从而克服有机质厌氧分解反应的热力学能垒,实现短链脂肪酸和醇类物质的互营氧化产甲烷过程。该过程中,种间电子传递是关键步骤。本文首先概述了甲烷的研究意义及微生物互营降解有机质产甲烷的过程,然后分别综述了种间H2转移、种间甲酸转移和种间直接电子传递这3种种间电子传递机制的起源、发展、研究现状和未来所需要解决的研究问题。     

Methanogens rapidly transition from methane production to iron reduction

Methanogenesis, the microbial methane (CH₄) production, is traditionally thought to anchor the mineralization of organic matter as the ultimate respiratory process in deep sediments, despite the presence of oxidized mineral phases, such as iron oxides. This process is carried out by archaea that have also been shown to be capable of reducing iron in high levels of electron donors such as hydrogen. The current pure culture study demonstrates that methanogenic archaea (Methanosarcina barkeri) rapidly switch from methanogenesis to iron‐oxide reduction close to natural conditions, with nitrogen atmosphere, even when faced with substrate limitations. Intensive, biotic iron reduction was observed following the addition of poorly crystalline ferrihydrite and complex organic matter and was accompanied by inhibition of methane production. The reaction rate of this process was of the first order and was dependent only on the initial iron concentrations. Ferrous iron production did not accelerate significantly with the addition of 9,10‐anthraquinone‐2,6‐disulfonate (AQDS) but increased by 11–28% with the addition of phenazine‐1‐carboxylate (PCA), suggesting the possible role of methanophenazines in the electron transport. The coupling between ferrous iron and methane production has important global implications. The rapid transition from methanogenesis to reduction of iron–oxides close to the natural conditions in sediments may help to explain the globally‐distributed phenomena of increasing ferrous concentrations below the traditional iron reduction zone in the deep ‘methanogenic’ sediment horizon, with implications for metabolic networking in these subsurface ecosystems and in past geological settings.     

Enhanced system performance by dosing ferroferric oxide during the anaerobic treatment of tryptone-based high-strength wastewater

Applied Microbiology and Biotechnology - Conductive materials can facilitate syntrophic methane (CH4) production by improving direct interspecies electron transfer. The effect of a conductive...     

Control of interspecies electron transfer flow during anaerobic digestion: dynamic diffusion reaction models for hydrogen gas transfer in microbial flocs

Dynamic reaction diffusion models were used to analyze the consequences of aggregation for syntrophic reactions in methanogenic ecosystems. Flocs from a whey digestor were used to measure all model parameters under the in situ conditions of a particular defined biological system. Fermentation simulations without adjustable parameters could precisely predict the kinetics of H(2) gas production of digestor flocs during syntrophic methanogenesis from ethanol. The results demonstrated a kinetic compartmentalization of H(2) metabolism inside the flocs. The interspecies electron transfer reaction was mildly diffusion controlled. The H(2) gas profiles across the flocs showed high H (2) concentrations inside the flocs at any time. Simulations of the syntrophic metabolism at low substrate concentrations such as in digestors or sediments showed that it is impossible to achieve high H(2) gas turnovers at simultaneously low steady-state H(2) concentrations. This showed a mechanistic contradiction in the concept of postulated low H(2) microenvironments for the anaerobic digestion process. The results of the computer experiments support the conclusion that syntrophic H(2) production may only be a side reaction of H(2) independent interspecies electron transfer in methanogenic ecosystems.     

Magnetite accelerates syntrophic acetate oxidation in methanogenic systems with high ammonia concentrations

Ammonia accumulation is a major inhibitory substance causing anaerobic digestion upset and failure in CH₄ production. At high ammonia levels, CH₄ production through syntrophic acetate oxidization (SAO) pathways is more tolerant to ammonia toxicity than the acetoclastic methanogenesis pathway, but the low CH₄ production rate through SAO constitutes the main reason for the low efficiency of energy recovery in anaerobic digesters treating ammonia‐rich substrates. In this study, we showed that acetate fermentation to CH₄ and CO₂ occurred through SAO pathway in the anaerobic reactors containing a high ammonia concentration (5.0 g l^?1 NH₄⁺–N), and the magnetite nanoparticles supplementation increased the CH₄ production rates from acetate by 36–58%, compared with the anaerobic reactors without magnetite under the same ammonia level. The mechanism of facilitated methanogenesis was proposed to be the establishment of direct interspecies electron transfer (DIET) for SAO, in which magnetite facilitated DIET between syntrophic acetate oxidizing bacteria and methanogens. High‐throughput 16S rRNA gene sequencing analysis revealed that the bacterial Geobacteraceae and the archaeal Methanosarcinaceae and Methanobacteriaceae might be involved in magnetite‐mediated DIET for SAO and CH₄ production. This study demonstrated that magnetite supplementation might provide an effective approach to accelerate CH₄ production rates in the anaerobic reactors treating wastewater containing high ammonia.     

Ethanol-to-methane activity of Geobacter-deprived anaerobic granules enhanced by conductive microparticles

Direct interspecies electron transfer (DIET) has been typically proposed as mechanism of electron transfer among methanogenic populations in granules during anaerobic digestion where Geobacter species play a key role. Using anaerobic granules where Geobacteraceae members were not prevalent − representing only 0.3% of total bacteria −, tests incubated with two co-substrates showed that the rate of methanogenesis from formate and hydrogen diminished in the presence of a non-methanogenic co-substrate such as ethanol. This could indicate that biological DIET occurs and competes with hydrogen and formate during methanogenesis. Moreover, the addition of conductive microparticles, such as stainless steel and granular activated carbon, was found to increase methanogenic activity in disintegrated granules by 190 ± 18% and 175 ± 22% respectively as compared to disintegrated granules devoid of microparticles. The addition of non-conductive microparticles such as porcelain however decreased methanogenic activity by 65 ± 3% of the disrupted granules without microparticle activity. These results indicate that syntrophic bacteria from anaerobic sludge excluding Geobacter species can also carry out conductive mineral mediated DIET.     

Intertwined interspecies relationships: approaches to untangle the microbial network

In nature, microorganisms live by interacting with each other. Microbiological studies that only consider pure cultures are not sufficient to adequately describe the natural behaviour of microbes. Several microbial interactions have been recognized to affect the growth or metabolism of others; e.g. syntrophic cometabolism, competition, production of inhibitors or activators, and predation. It is believed that third‐party organisms easily affect the two‐species relationships and these relationships form the basis of interspecies networks within microbial communities. A microbial network contributes to ‘functional redundancy’ or ‘structural diversity’ and the microbial communities effectively act as a multicellular organism. It is necessary to understand not only the physiological activity of members within microbial communities but also their roles to regulate the activity or population of others. To access the microbial network, we require (i) comprehensive determination of all possible interspecies relationships among microbes, (ii) knock‐out experiments by which certain members can be removed or suppressed, and (iii) supplemental addition of microbes or activation of certain members. Microbial network studies have started using defined microbial communities, i.e. a mixed culture that is composed of three or four species. In order to expand these studies to microflora in nature, microbial ecology requires the help of mathematical biology.     

Typical methanogenic inhibitors can considerably alter bacterial populations and affect the interaction between fatty acid degraders and homoacetogens

The effects of two typical methanogenic inhibitors [2-bromoethanesulfonate (BES) and chloroform (CHCl₃)] on the bacterial populations were investigated using molecular ecological techniques. Terminal restriction fragment length polymorphism analyses (T-RFLP) in combination with clone library showed that both the toxicants not only inhibited methanogenic activity but also considerably altered the bacterial community structure. Species of low % G + C Gram-positive bacteria (Clostridiales), high % G + C Actinomycetes, and uncultured Chloroflexi showed relatively greater tolerance of CHCl₃, whereas the BES T-RFLP patterns were characterized by prevalence of Geobacter hydrogenophilus and homoacetogenic Moorella sp. In addition, due to indirect thermodynamic inhibition caused by high hydrogen partial pressures, the growth of obligately syntrophic acetogenic Syntrophomonas and Syntrophobacter was also affected by selective inhibition of methanogenesis. Interestingly, by comparing the fermentative intermediates detected in BES- and CHCl₃-treated experiments, it was furthermore found that when methanogenesis is specifically inhibited, the syntrophic interaction between hydrogen-producing fatty acid degraders and hydrogen-utilizating homoacetogens seemed to be strengthened.     

纳米铁氧化物对Pelotomaculum schinkii培养系丙酸厌氧降解产甲烷的影响

微生物能利用导电材料进行电子传递,提高种间电子传递效率。铁基纳米导电物质可以加速土壤及厌氧消化系统中微生物间的种间电子传递,促进有机废弃物的产甲烷过程。前期获得了厌氧丙酸富集培养系,互营丙酸氧化菌（Pelotomaculum schinkii）在培养系中占优势,本研究考察了10~4 000 mg/L 纳米铁氧化物对丙酸降解产甲烷过程的作用及微生物的影响。结果表明,低浓度的铁基纳米材料对丙酸降解有一定的促进作用,而高浓度会抑制产甲烷：10~1 000 mg/L纳米Fe₃O₄对产甲烷无明显影响,1 500~4 000 mg/L最大产甲烷速率抑制了26%~80%,延滞期增加了174%~222%;10~200 mg/L纳米Fe₂O₃使最大产甲烷速率提高了21%~29%,1 500~4 000 mg/L最大产甲烷速率抑制了48%~58%,延滞期增加了29%~85%。微生物群落解析结果表明,与对照相比,10~1 000 mg/L纳米Fe₂O₃使P. schinkii相对丰度略有增加,而4 000 mg/L纳米₃O₄/Fe₂O₃使P. schinkii的相对丰度下降了70.7%和55.9%,说明高浓度纳米铁氧化物会抑制P. schinkii的活性,导致丙酸降解及产甲烷速率降低。     

Oxidation of glucose by syntrophic association between <Emphasis Type="Italic">Geobacter</Emphasis> and hydrogenotrophic methanogens in microbial fuel cell

Objective

To investigate a syntrophic interaction between Geobacter sulfurreducens and hydrogenotrophic methanogens in sludge-inoculated microbial fuel cell (MFC) systems running on glucose with an improved electron recovery at the anode.

Results

The presence of archaea in MFC reduces Coulombic efficiency (CE) due to their electron scavenging capability but, here, we demonstrate that a syntrophic interaction can occur between G. sulfurreducens and hydrogenotrophic methanogens via interspecies H₂ transfer with improvement in CE and power density. The addition of the methanogenesis inhibitor, 2-bromoethanesulfonate (BES), resulted in the reduction in power density from 5.29 to 2 W/m³, and then gradually increased to the peak value of 5.5 W/m³ when BES addition was stopped.

Conclusion

Reduction of H₂ partial pressure by archaea is an efficient approach in improving power output in a glucose-fed MFC system using Geobacter sp. as an inoculum.

     

Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Abstract Dilution of anoxic slurries of paddy soil resulted in a proportional decrease of the rates of total methanogenesis and the rate constants of H₂ turnover per gram soil. Dilution did not affect the fraction of H₂/CO₂-dependent methanogenesis which made up 22% of total CH₄ production. However, dilution resulted in a ten fold decrease of the H₂ steady state partial pressure from approximately 4 to 0.4 Pa indicating that H₂/CO₂-dependent methanogenesis was more or less independent of the H₂ pool. The rates of H₂ production calculated from the H₂ turnover rate constants and the H₂ steady state partial pressures accounted for only < 5% of H₂/CO₂-dependent methanogenesis in undiluted soil slurries and for even less after dilution. Upon dilution, the Gibbs free energy available for H₂/CO₂-dependent methanogenesis decreased from −28.4 to only −5.6 kJ per mol. The results indicate that methane was mainly produced from interspecies H₂ transfer within syntrophic bacterial associations and was not significantly affected by the outside H₂ pool.