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Anaerobic digestion (AD) is an effective biological treatment for stabilizing organic compounds in waste/wastewater and in simultaneously producing biogas. However, it is often limited by the slow reaction rates of different microorganisms’ syntrophic biological metabolisms. Stable and fast interspecies electron transfer (IET) between volatile fatty acid-oxidizing bacteria and hydrogenotrophic methanogens is crucial for efficient methanogenesis. In this syntrophic interaction, electrons are exchanged via redox mediators such as hydrogen and formate. Recently, direct IET (DIET) has been revealed as an important IET route for AD. Microorganisms undergoing DIET form interspecies electrical connections via membrane-associated cytochromes and conductive pili; thus, redox mediators are not required for electron exchange. This indicates that DIET is more thermodynamically favorable than indirect IET. Recent studies have shown that conductive materials (e.g., iron oxides, activated carbon, biochar, and carbon fibers) can mediate direct electrical connections for DIET. Microorganisms attach to conductive materials’ surfaces or vice versa according to particle size, and form conductive biofilms or aggregates. Different conductive materials promote DIET and improve AD performance in digesters treating different feedstocks, potentially suggesting a new approach to enhancing AD performance. This review discusses the role and potential of DIET in methanogenic systems, especially with conductive materials for promoting DIET.

Methanothrix is widely distributed in natural and artificial anoxic environments and plays a major role in global methane emissions. It is one of only two genera that can form methane from acetate dismutation and through participation in direct interspecies electron transfer (DIET) with exoelectrogens. Although Methanothrix is a significant member of many methanogenic communities, little is known about its physiology. In this study, transcriptomics helped to identify potential routes of electron transfer during DIET between Geobacter metallireducens and Methanothrix thermoacetophila . Additions of magnetite to cultures significantly enhanced growth by acetoclastic methanogenesis and by DIET, while granular activated carbon (GAC) amendments impaired growth. Transcriptomics suggested that the OmaF-OmbF-OmcF porin complex and the octaheme outer membrane c -type cytochrome encoded by Gmet_0930, were important for electron transport across the outer membrane of G. metallireducens during DIET with Mx. thermoacetophila . Clear differences in the metabolism of Mx. thermoacetophila when grown via DIET or acetate dismutation were not apparent. However, genes coding for proteins involved in carbon fixation, the sheath fiber protein MspA, and a surface-associated quinoprotein, SqpA, were highly expressed in all conditions. Expression of gas vesicle genes was significantly lower in DIET- than acetate-grown cells, possibly to facilitate better contact between membrane-associated redox proteins during DIET. These studies reveal potential electron transfer mechanisms utilized by both Geobacter and Methanothrix during DIET and provide important insights into the physiology of Methanothrix in anoxic environments. Methanothrix is a significant methane producer in a variety of methanogenic environments including soils and sediments as well as anaerobic digesters. Its abundance in these anoxic environments has mostly been attributed to its high affinity for acetate and its ability to grow by acetoclastic methanogenesis. However, Methanothrix species can also generate methane by directly accepting electrons from exoelectrogenic bacteria through direct interspecies electron transfer (DIET). Methane production through DIET is likely to further increase their contribution to methane production in natural and artificial environments. Therefore, acquiring a better understanding of DIET with Methanothrix will help shed light on ways to (i) minimize microbial methane production in natural terrestrial environments and (ii) maximize biogas formation by anaerobic digesters treating waste.

The conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters. Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here, we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in coculture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in M. acetivorans grown in coculture versus pure-culture growth on acetate revealed that transcripts for the outer-surface multiheme c- type cytochrome MmcA were higher during DIET-based growth. Deletion of mmcA inhibited DIET. The high aromatic amino acid content of M. acetivorans archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based coculture as well as the archaellum-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based cocultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen. IMPORTANCE The conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters. Potential electron donors for carbon dioxide reduction are H 2 or electrons derived from direct interspecies electron transfer (DIET) between bacteria and methanogens. Elucidating the relative importance of these electron donors has been difficult due to a lack of information on the electrical connections on the outer surfaces of methanogens and how they process the electrons received from DIET. Transcriptomic patterns and gene deletion phenotypes reported here provide insight into how a group of Methanosarcina organisms that play an important role in methane production in soils and sediments participate in DIET.

Coastal sediments are rich in conductive particles, possibly affecting microbial processes for which acetate is a central intermediate. In the methanogenic zone, acetate is consumed by methanogens and/or syntrophic acetate-oxidizing (SAO) consortia. SAO consortia live under extreme thermodynamic pressure, and their survival depends on successful partnership. Here, we demonstrate that conductive particles enable the partnership between SAO bacteria (i.e., Geobacter spp.) and methanogens ( Methanosarcina spp.) from the coastal sediments of the Bothnian Bay of the Baltic Sea. Baltic methanogenic sediments were rich in conductive minerals, had an apparent isotopic fractionation characteristic of CO 2 -reductive methanogenesis, and were inhabited by Geobacter and Methanosarcina . As long as conductive particles were delivered, Geobacter and Methanosarcina persisted, whereas exclusion of conductive particles led to the extinction of Geobacter . Baltic Geobacter did not establish a direct electric contact with Methanosarcina , necessitating conductive particles as electrical conduits. Within SAO consortia, Geobacter was an efficient [ 13 C]acetate utilizer, accounting for 82% of the assimilation and 27% of the breakdown of acetate. Geobacter benefits from the association with the methanogen, because in the absence of an electron acceptor it can use Methanosarcina as a terminal electron sink. Consequently, inhibition of methanogenesis constrained the SAO activity of Geobacter as well. A potential benefit for Methanosarcina partnering with Geobacter is that together they competitively exclude acetoclastic methanogens like Methanothrix from an environment rich in conductive particles. Conductive particle-mediated SAO could explain the abundance of acetate oxidizers like Geobacter in the methanogenic zone of sediments where no electron acceptors other than CO 2 are available. IMPORTANCE Acetate-oxidizing bacteria are known to thrive in mutualistic consortia in which H 2 or formate is shuttled to a methane-producing Archaea partner. Here, we discovered that such bacteria could instead transfer electrons via conductive minerals. Mineral SAO (syntrophic acetate oxidation) could be a vital pathway for CO 2 -reductive methanogenesis in the environment, especially in sediments rich in conductive minerals. Mineral-facilitated SAO is therefore of potential importance for both iron and methane cycles in sediments and soils. Additionally, our observations imply that agricultural runoff or amendments with conductive chars could trigger a significant increase in methane emissions. Acetate-oxidizing bacteria are known to thrive in mutualistic consortia in which H 2 or formate is shuttled to a methane-producing Archaea partner. Here, we discovered that such bacteria could instead transfer electrons via conductive minerals. Mineral SAO (syntrophic acetate oxidation) could be a vital pathway for CO 2 -reductive methanogenesis in the environment, especially in sediments rich in conductive minerals. Mineral-facilitated SAO is therefore of potential importance for both iron and methane cycles in sediments and soils. Additionally, our observations imply that agricultural runoff or amendments with conductive chars could trigger a significant increase in methane emissions.

Direct interspecies electron transfer (DIET) is a syntrophic metabolism wherein free electrons are directly transferred between microorganisms without the mediation of intermediates such as molecular hydrogen or formate. Previous research has demonstrated that Methanothrix harundinacea 6Ac is capable of reducing carbon dioxide through DIET. However, the mechanisms underlying electron uptake in M. harundinacea 6Ac during DIET remain poorly understood. This study aims to elucidate the electron and proton flux in M. harundinacea 6Ac during DIET and to propose a model for electron uptake in this organism, primarily based on the analysis of gene transcript levels, genomic characteristics of M. harundinacea 6Ac, and the pathways generating fully reduced ferridoxin (Fdred2−), reduced coenzyme F420 (F420H2), coenzyme M (CoM-SH), and coenzyme B (CoB-SH) during DIET. The findings suggest that membrane-bound heterodisulfide reductase (HdrED), F420H2-dehydrogenase lacking subunit F (Fpo−), and cytoplasmic heterodisulfide reductase (HdrABC)-subunit B of F420-reducing hydrogenase (FrhB) complex play critical roles in electron uptake in M. harundinacea 6Ac during DIET. Specifically, Fpo− is responsible for generating Fdred2− with reduced methanophenazine (MPH2), driven by a proton motive force, while HdrED facilitates the reduction of heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) to CoM-SH and CoB-SH using MPH2. Additionally, cytoplasmic heterodisulfide reductase HdrABC and subunit B of coenzyme F420-hydrogenase complex (HdrABC-FrhB complex) catalyzes the reduction of oxidized coenzyme F420 (F420) to F420H2, utilizing CoM-SH, CoB-SH, and Fdred2−. This study represents the first genetics-based functional characterization of electron and proton flux in M. harundinacea 6Ac during DIET, providing a model for further investigation of electron uptake in Methanosaeta species. Furthermore, it deepens our understanding of the mechanisms underlying electron uptake in methanogens during DIET.

Conductive iron oxides (CIO) have been proved recently to facilitate the anaerobic microbial syntrophy based on the direct interspecies electron transfer (DIET) in batch experiments. However, the effect of CIO was always insignificant in anaerobic digestion (AD) reactor especially when the DIET-based syntrophic partners were absent. In this study, the effect of magnetite on performance of AD system with sucrose as a sole carbon source was investigated, but limited enhancement was achieved during the first 36-day operation. The short-term effect of ethanol addition was further studied in the magnetite-amended AD reactor, and results showed that the AD reactor with 10gFe/L micro-sized magnetite (R3) achieved higher performance of COD removal and methane proportion compared with the other reactors (R1 without magnetite; R2 with 2gFe/L micro-sized magnetite; R4 with 2gFe/L nano-sized magnetite). Meanwhile, the pyridoxine in extracellular polymeric substances (EPS) and conductivity of anaerobic sludge from R3 increased more significantly than those of the others. Analysis of high-throughput sequencing indicated that the abundance of archaea increased in sludge from R3 and Methanosarcina responsible for DIET was dominant (63.64%). Additionally, the abundance of potential electroactive bacteria Chloroflexi in R3 was 7.57-fold, 3.61-fold and 7.37-fold as that of R1, R2 and R4, respectively. These results demonstrated that the electroactive microbes and methanogens could be enriched efficiently in anaerobic sludge via synergetic effect of magnetite addition and ethanol short-term stimulation.

Rational disposal of sludge is an ongoing concern. This work is the first attempt for in-depth statistical analysis of anaerobic digestion (AD) research in recent three decades (1986–2022) using both quantitative and qualitative approaches in bibliometrics to investigate the research progress, trends and hot spots. All publications in the Web of Science Core Collection database from 1986 to April 4, 2022 were analyzed. Results showed that the research on AD started in 1999 and the number of papers significantly increased since 2012. The research about the disposal of sewage sludge mainly focuses on energy recovery (e.g. methane and short chain volatile organic acids) by AD. Besides, different pretreatment technologies were studied in this study to eliminate the negative effects on the disposal of sludge caused by hydrolysis (rate-limiting step of AD), water content (increasing the costs) and heavy metal (toxic to the environment) of sludge. Of those, the treatment technologies related to direct interspecies electron transfer were worth further studied in the future. Towards that end, iron conductive material, iron-based advanced oxidation and biological treatment were concluded as the prospective technologies and worth to further study.

Conductive materials can facilitate syntrophic methane (CH 4 ) production by improving direct interspecies electron transfer. The effect of a conductive material, ferroferric oxide (Fe 3 O 4 ), on the anaerobic treatment of tryptone-based high-strength wastewater was investigated in two anaerobic sequencing batch reactors (ASBRs). One was the control reactor without amendment of Fe 3 O 4 , and the other was the Fe 3 O 4 -amended reactor. In an ASBR reaction cycle, the dosage of Fe 3 O 4 increased the CH 4 production rate by 12.2%, shortened the methanogenic lag phase by 32.3%, and improved the COD removal rate by 8.1%. During anaerobic treatment, the dosage of Fe 3 O 4 not only enhanced the CH 4 production from acetate but also facilitated the hydrolysis/acidification process fed by tryptone. Maximum electron transport activities were increased by 40.4, 137.3, and 64.6% in the processes of the ASBR reaction cycle, the CH 4 production from acetate, and the hydrolysis/acidification of tryptone, respectively. Among all tested quorum sensing signal substances, N -octanoyl- l -homoserine lactone was dominant in both ASBRs, with the highest concentration in the biomass and the lowest concentration in the water phase. In addition, the concentration of signal substance was high in the ASBR cycle and low in the CH 4 production from acetate and the hydrolysis/acidification of tryptone. Proteiniclasticum and vadinCA02 were dominant acidogens, and their relative abundances increased during the long-term operation with Fe 3 O 4 dosing. Methanosarcina was the predominant methanogen, contributing to interspecies electron transfer.

This study investigated the impact of stimulating direct interspecies electron transfer (DIET), by supplementing nano-sized magnetite (nFe3O4, 0.5 g Fe/g VSS) and carbon nanotubes (CNT, 1 g/L), in anaerobic digestion of oleic acid (OA) at various concentrations (0.10–4.00 g chemical oxygen demand(COD)/L). Both supplementations could enhance CH4 production, and its beneficial impact increased with increased OA concentration. The biggest improvements of 114% and 165% compared to the control were achieved by nFe3O4 and CNT, respectively, at OA of 4 g COD/L. The enhancement can be attributed to the increased sludge conductivity: 7.1 ± 0.5 (control), 12.5 ± 0.8 (nFe3O4-added), and 15.7 ± 1.1 µS/cm (CNT-supplemented). Dissolved iron concentration, released from nFe3O4, seemed to have a negligible role in improving CH4 production. The excretion of electron shuttles, i.e., humic-like substances and protein-like substances, were found to be stimulated by supplementing nFe3O4 and CNT. Microbial diversity was found to be simplified under DIET-stimulating conditions, whereby five genera accounted for 88% of the total sequences in the control, while more than 82% were represented by only two genera (Methanotrix concilli and Methanosarcina flavescens) by supplementing nFe3O4 and CNT. In addition, the abudance of electro-active bacteria such as Syntrophomonas zehnderi was significantly increased from 17% to around 45%.

The Gram-negative bacterium Geobacter metallireducens is of environmental and biotechnological significance. Crucial to the unique physiology of G. metallireducens is its extracellular electron transfer (EET) capability. This investigation sheds new light on the robust roles of the three porin-cytochrome ( pcc ) gene clusters, which are directly involved in EET across the bacterial outer membrane, in the EET of G. metallireducens . In addition to their essential roles, these gene clusters also play distinct, overlapping, and compensatory roles in the EET of G. metallireducens . The distinct roles of the pcc gene clusters enable G. metallireducens to mediate EET to a diverse group of electron acceptors for anaerobic respirations. The overlapping and compensatory roles of the pcc gene clusters enable G. metallireducens to maintain and restore its EET capability for anaerobic growth when one or two of its three pcc gene clusters are deleted from the genome.