Quorum Sensing Enhances Direct Interspecies Electron Transfer in Anaerobic Methane Production.

Direct interspecies electron transfer (DIET) provides an innovative way to achieve efficient methanogenesis, and this study proposes a new approach to upregulate the DIET pathway by enhancing quorum sensing (QS). Based on long-term reactor performance, QS enhancement achieved more vigorous methanogenesis with 98.7% COD removal efficiency. In the control system, methanogenesis failure occurred at the accumulated acetate of 7420 mg of COD/L and lowered pH of 6.04, and a much lower COD removal of 41.9% was observed. The more significant DIET in QS-enhancing system was supported by higher expression of conductive pili and the c-Cyts cytochrome secretion-related genes, resulting in 12.7- and 10.3-fold improvements. Moreover, QS enhancement also improved the energy production capability, with the increase of F-type and V/A-type ATPase expression by 6.3- and 4.2-fold, and this effect probably provided more energy for nanowires and c-Cyts cytochrome secretion. From the perspective of community structure, QS enhancement increased the abundance of Methanosaeta and Geobacter from 54.3 and 17.6% in the control to 63.0 and 33.8%, respectively. Furthermore, the expression of genes involved in carbon dioxide reduction and alcohol dehydrogenation increased by 0.6- and 7.1-fold, respectively. Taken together, this study indicates the positive effects of QS chemicals to stimulate DIET and advances the understanding of the DIET methanogenesis involved in environments such as anaerobic digesters and sediments.

Biochar derived from alkali-treated sludge residue regulates anaerobic digestion: Enhancement performance and potential mechanisms.

Biochar produced from bio-wastes has been widely used to promote the performance of anaerobic digestion. Waste activated sludge (WAS) is considered as a kind of popular precursor for biochar preparation, but the abundant resources in WAS were neglected previously. In this study, the roles of biochar prepared from raw, pretreated, and fermented sludge on anaerobic digestion were investigated. That is, parts of carbon sources and nutrients like polysaccharides, proteins, and phosphorus were firstly recovered after sludge pretreatment or fermentation, and then the sludge residuals were used as raw material to prepare biochar. The methane yield improved by 22.1% with adding the biochar (AK-BC) prepared by sludge residual obtained from alkaline pretreatment. Mechanism study suggested that the characteristics of AK-BC like specific surface area and defect levels were updated. Then, the conversion performance of intermediate metabolites and electro-activities of extracellular polymeric substances were up-regulated. As a result, the activity of electron transfer was increased with the presence of AK-BC, with increase ratio of 21.4%. In addition, the electroactive microorganisms like Anaerolineaceae and Methanosaeta were enriched with the presence of AK-BC, and the potential direct interspecies electron transfer was possibly established. Moreover, both aceticlastic and CO2-reducing methanogenesis pathways were improved by up-regulating related enzymes. Therefore, the proposed strategy can not only obtain preferred biochar but also recover abundant resources like carbon source, nutrients, and bioenergy.

Enhanced thermophilic high-solids anaerobic digestion of organic fraction of municipal solid waste with spatial separation from conductive materials in a single reactor volume.

Despite benefits such as lower water and working volume requirements, thermophilic high solids anaerobic digestion (THSAD) often fails due to the rapid build-up of volatile fatty acids (VFAs) and the associated drop in pH. Use of conductive materials (CM) can promote THSAD through stimulation of direct interspecies electron transfer (DIET), while the need for their constant dosing due to poor separation from effluent impairs economic feasibility. This study used an approach of spatially separating magnetite and granular activated carbon (GAC) from the organic fraction of municipal solid waste (OFMSW) in a single reactor for THSAD. GAC and magnetite addition could both mitigate the severe inhibition of methanogenesis after VFAs build-up to ∼28-30 g/L, while negligible methane production was observed in the control group. The highest methane yield (286 mL CH4/g volatile solids (VS)) was achieved in magnetite-added reactors, while the highest maximum CH4 production rates (26.38 mL CH4/g VS/d) and lowest lag-phase (2.83 days) were obtained in GAC-added reactors. The enrichment of GAC and magnetite biofilms with various syntrophic and potentially electroactive microbial groups (Ruminiclostridium 1, Clostridia MBA03, Defluviitoga, Lentimicrobiaceae) in different relative abundances indicates the existence of specific preferences of these groups for the nature of CM. According to predicted basic metabolic functions, CM can enhance cellular processes and signals, lipid transport and metabolism, and methane metabolism, resulting in improved methane production. Rearrangement of metabolic pathways, formation of pili-like structures, enrichment of biofilms with electroactive groups and a significant improvement in THSAD performance was attributed to the enhancement of the DIET pathway. Promising results obtained in this work due to the spatial separation of the bulk OFMSW and CM can be useful for modeling larger-scale THSAD systems with better recovery of CM and cost-effectiveness.

Metagenomic analysis reveals the mechanisms of biochar supported nano zero-valent iron in two-phase anaerobic digestion of food waste: microbial community, CAZmey, functional genes and antibiotic resistance genes.

The mechanisms of biochar supported nano zero-valent iron (BC/nZVI) on two-phase anaerobic digestion of food waste were investigated. Results indicated that the performance of both acidogenic phase and methanogenic phase was effectively facilitated. BC/nZVI with the amount of 120 mg/L increased methane production by 32.21%. In addition, BC/nZVI facilitated direct interspecies electron transfer (DIET) between Geobacter and methanogens. Further analysis showed that BC/nZVI increased the abundance of most CAZymes in acidogenic phase. The study also found that BC/nZVI had positive effects on metabolic pathways and related functional genes. The abundances of acdA and ackA in acidogenic phase were increased by 151.75% and 36.26%, respectively, and the abundances of pilA and TorZ associated with DIET were also increased. Furthermore, BC/nZVI mainly removed IMP-12, CAU-1, cmeB, ErmR, MexW, ErmG, Bla2, vgaD, MuxA, and cpxA from this system, and reduced the antibiotic resistance genes for antibiotic inactivation resistance mechanisms.

Research progress and perspective on sludge anaerobic digestion technology: A bibliometric analysis.

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.

Material and microbial perspectives on understanding the role of biochar in mitigating ammonia inhibition during anaerobic digestion.

With the increasing adoption of carbon-based strategies to enhance methanogenic processes, there is a growing concern regarding the correlation between biochar properties and its stimulating effects on anaerobic digestion (AD) under ammonia inhibition. This study delves into the relevant characteristics and potential mechanisms of biochar in the context of AD system under ammonia inhibition. The introduction of optimized biochar, distinguished by rich CO bond, abundant defect density, and high electronic capacity, resulted in a significant reduction in the lag period of anaerobic digestion system under 5.0 g/L ammonia stress, approximately by around 63 % compared to the control one. Biochar helps regulate the community structure, promotes the accumulation of acetate-consuming bacteria, in the AD system under ammonia inhibition. More examinations show that biochar promotes direct interspecies electron transfer in AD system under ammonia inhibition, as evidenced by diminished levels of bound electroactive extracellular polymeric substances, increased abundance of electroactive bacteria, and notably, the up-regulation of direct interspecies electron transfer associated genes, including the conductive pili and Cytochrome C genes, as revealed by meta-transcriptomic analysis. Additionally, gene expression related to proteins associated with ammonium detoxification were found to be up-regulated in systems supplemented with biochar. These findings provide essential evidence and insights for the selection and potential engineering of effective biochar to enhance AD performance under ammonia inhibition.

Alternating polarity as a novel strategy for building synthetic microbial communities capable of robust Electro-Methanogenesis.

Electro-methanogenic microbial communities can produce biogas with high efficiency and have attracted extensive research interest. In this study an alternating polarity strategy was developed to build electro-methanogenic communities. In two-chamber bioelectrochemical systems amended with activated carbon, the electrode potential was alternated between +0.8 V and -0.4 V vs. standard hydrogen electrode every three days. Cumulative biogas production under alternating polarity increased from 45 L/L/kg-activated carbon after start-up to 125 L/L/kg after the 4th enrichment, significantly higher than that under intermittent cathode (-0.4 V/open circuit), continuous cathode (-0.4 V), and open circuit. The communities assembled under alternating polarity were electroactive and structurally different from those assembled under other conditions. One Methanobacterium population and two Geobacter populations were consistently abundant and active in the communities. Their 16S rRNA was up-regulated by electrode potentials. Bayesian networks inferred close associations between these populations. Overall, electro-methanogenic communities have been successfully assembled with alternating polarity.

Distinctive effects of graphene oxide and reduced graphene oxide on methane production kinetics and pharmaceuticals removal in anaerobic reactors.

Graphene oxide (GO) addition to anaerobic digestion has been suggested to enhance direct electron transfer. The impact of GO (0.075 g GO g-1 VS) and biologically and hydrothermally reduced GO (bio-rGO and h-rGO, respectively) on the methane production kinetics and removal of 12 pharmaceuticals was assessed in Fed-batch reactors. A decrease of 15 % in methane production was observed in the tests with GO addition compared with the control and the h-rGO. However, bio-rGO and h-rGO substantially increased the methane production rate compared to the control tests (+40 %), in the third fed-batch test. Removal of pharmaceuticals was enhanced only during the bio-reduction of GO (1st fed-batch test), whereas once the GO was bio-reduced, it followed a similar trend in the control and h-rGO tests. The addition of GO can enhance the methane production rate and, therefore, reduce the anaerobic treatment time.

Enhancing methane production from corn straw via illumination-assisted Fe3O4/g-C3N4 nanocomposite in anaerobic digestion.

This study proposes a novel anaerobic digestion (AD) strategy combining recyclable photoactivated nanomaterials with illumination to enhance electronic transfer for anaerobic microorganisms. Results showed that 7000 Lux illumination increased methane production yield and rate. Incorporating Fe3O4 into graphite carbon nitride (g-C3N4) created a recyclable Fe3O4/g-C3N4 (FG) nanocomposite with improved light absorption, conductivity, redox properties, and methane promotion. The highest methane yield from corn straw was achieved with 7000 Lux and 1.5 g/L FG nanocomposite, 22.6% higher than the dark control. The AD system exhibited increased adenosine triphosphate content, improved redox performance, reduced electron transfer resistance, and higher photocurrent intensity. These improvements bolstered the microorganisms and key genes involved in hydrolysis and acidification, which in turn optimized the acetoclastic pathway. Furthermore, this strategy promoted microorganisms associated with direct interspecies electron transfer, fostering a favorable environment for methanogenic activities, paving the way for future anaerobic reactor developments.

Comprehensive insights into the impact of magnetic biochar on protein hydrolysis in sludge anaerobic digestion: Protein structures, microbial activities and syntrophic metabolisms.

The addition of composite conductive materials is being increasingly recognized as a promising strategy to enhance anaerobic digestion (AD) performance. However, the influence of these materials on protein hydrolysis has been poorly documented. Here, a novel magnetic biochar derived from oil sludge and straw was synthesized using different iron sources and successfully applied in sludge AD. Experimental results revealed that magnetic biochar modified by Fe2+ exhibited excellent electron transfer capacity, moderate magnetization, diverse functional groups (e.g. C=O, C-O=O-), and abundant iron distribution. These characteristics significantly enhanced the hydrolysis of tryptophan-like components, leading to increased methane production (144.44 mL gVS-1vs 79.72 mL gVS-1 in the control test). Molecular docking analysis revealed that the binding of magnetic biochar related Fe2+ and Fe3+, onto sludge proteins via hydrogen bond played a key role in promoting subsequent protein hydrolysis. Additionally, the noteworthy conservation of protein structures from α-helix and ß-sheet to random coil, along with the breakdown of the amide I-associated C=O group and amide III-related CN and NH bonds following the addition of magnetic biochar, accelerated the degradation of sludge protein. Observation of variations in protease activity, coenzyme F420, electron transfer system (ETS), and conductivity within the AD systems, particularly the enrichment of Methanospirillum and Methanosaeta archaea, as well as the Petrimonas, Comamonas, and Syntrophomonas bacteria, suggested that magnetic biochar facilitated a conducive environment by improving hydrolysis-acidification and the direct interspecies electron transfer (DIET) process for acetoclastic methanogens. Moreover, metabolic pathways further proved that tryptophan metobalism and acetoclastic methanogenesis were both facilitated by magnetic biochar. This study provides an in-depth understanding of the impact of magnetic biochar on protein hydrolysis in sewage sludge AD.

Zero-valent iron enhanced methane production of anaerobic digestion by reinforcing microbial electron bifurcation coupled with direct inter-species electron transfer.

Zero-valent iron (ZVI) can facilitate methanogens of anaerobic digestion (AD). However, the impact of ZVI on the micro-energetic strategies of AD microorganisms remains uncertain. This study aimed to elucidate the development of an energy conservation model involving direct interspecies electron transfer (DIET) and electron bifurcate (EB) by using four types of ZVI. Overall, the ZVI addition resulted in a substantial increase in methane production (1.26 to 2.18 times higher), and the effect of boron (B) doped ZVI was particularly pronounced. The underlying mechanism may be the formation of energy harvest pathway related to DIET. In detail, B-doped ZVI could enhance its interfacial binding to cytochrome c. Decreased polar solvation energy from 20.473 to 1.509 kJ/mol is beneficial for electron transfer, thereby augmenting the flavin-bounded Cytc activity and DIET process. Besides, ZVI-enhanced EB enzyme activity like HdrA2B2C2-MvhAGD could improve the EB process, which can couple with DIET for electron transfer and energy conservation. Energy analysis based on EB-coupled DIET metabolism pathways demonstrated that the ATP saved in this coupled model theoretically line in 0.25 to 0.5 mol ATP/mol substrate. Overall, this study offers valuable insights into microbial energetic strategies pertaining to the utilization of conductive materials, with the target of enhancing methane recovery efficiency from organic waste.

Biogas upgrading performance and underlying mechanism in microbial electrolysis cell and anaerobic digestion integrated system.

The productivity and efficiency of two-chamber microbial electrolysis cell and anaerobic digestion integrated system (MEC-AD) were promoted by a complex of anaerobic granular sludge and iron oxides (Fe-AnGS) as inoculum. Results showed that MEC-AD with Fe-AnGS achieved biogas upgrading with a 23%-29% increase in the energy recovery rate of external circuit current and a 26%-31% decrease in volatile fatty acids. The energy recovery rate of MEC-AD remained at 52%-57%, indicating a stable operation performance. The selectively enriched methanogens and electroactive bacteria resulted in dominant hydrogenotrophic and acetoclastic methanogenesis in the cathode and anode chambers. Mechanistic analysis revealed that MEC-AD with Fe-AnGS led to specifically upregulated enzymes related to energy metabolism and electron transfer. Fe-AnGS as inoculum could improve the long-term operation performance of MEC-AD. Consequently, this study provides an efficient strategy for biogas upgrading in MEC-AD.

Preparation of iron oxide-modified digestate biochar and effect on anaerobic digestion of kitchen waste.

Two kinds of Fe2O3-modified digestate-derived biochar (BC) were prepared and their effects on anaerobic digestion (AD) of kitchen waste (40.0 g VS/L) were investigated, with BC and Fe2O3 addition used as a comparison. The results showed that Fe2O3-modified BC (Fe2O3-BC1 prepared by co-precipitation and Fe2O3-BC2 by impregnation) significantly increased methane yield (20.8 % and 16.4 %, respectively) and reduced volatile fatty acid concentration (35.6 % and 29.6 %, respectively). Microbial high-throughput analysis revealed that Fe2O3-modified BC selectively enriched Clostridium (47.3 %) and Methanosarcina (72.2 %), suggesting that direct interspecies electron transfer contributing to improved biogas production performance was established and enhanced. Correlation analysis indicated that biogas production performance was improved by the larger specific surface area (83.4 m2/g), pore volume (0.101 cm3/g), and iron content (97.4 g/Kg) of the BC. These results offer insights for enhancing the efficacy of AD processes using Fe2O3-modified BCs as additives.

Syntrophic methane production from volatile fatty acids: Focus on interspecies electron transfer.

Methane is a renewable biomass energy source produced via anaerobic digestion (AD). Interspecies electron transfer (IET) between methanogens and syntrophic bacteria is crucial for mitigating energy barriers in this process. Understanding IET is essential for enhancing the efficiency of syntrophic methanogenesis in anaerobic digestion. Interspecies electron transfer mechanisms include interspecies H2/formate transfer, direct interspecies electron transfer (DIET), and electron-shuttle-mediated transfer. This review summarizes the mechanisms, developments, and research gaps in IET pathways. Interspecies H2/formate transfer requires strict control of low H2 partial pressure and involves complex enzymatic reactions. In contrast, DIET enhances the electron transfer efficiency and process stability. Conductive materials and key microorganisms can be modulated to stimulate the DIET. Electron shuttles (ES) allow microorganisms to interact with extracellular electron acceptors without direct contact; however, their efficiency depends on various factors. Future studies should elucidate the key functional groups, metabolic pathways, and regulatory mechanisms of IET to guide the optimization of AD processes for efficient renewable energy production.

Hydrogen-independent CO2 reduction dominates methanogenesis in five temperate lakes that differ in trophic states.

Emissions of microbially produced methane (CH4) from lake sediments are a major source of this potent greenhouse gas to the atmosphere. The rates of CH4 production and emission are believed to be influenced by electron acceptor distributions and organic carbon contents, which in turn are affected by anthropogenic inputs of nutrients leading to eutrophication. Here, we investigate how eutrophication influences the abundance and community structure of CH4 producing Archaea and methanogenesis pathways across time-resolved sedimentary records of five Swiss lakes with well-characterized trophic histories. Despite higher CH4 concentrations which suggest higher methanogenic activity in sediments of eutrophic lakes, abundances of methanogens were highest in oligotrophic lake sediments. Moreover, while the methanogenic community composition differed significantly at the lowest taxonomic levels (OTU), depending on whether sediment layers had been deposited under oligotrophic or eutrophic conditions, it showed no clear trend in relation to in situ distributions of electron acceptors. Remarkably, even though methanogenesis from CO2-reduction was the dominant pathway in all sediments based on carbon isotope fractionation values, taxonomic identities, and genomes of resident methanogens, CO2-reduction with hydrogen (H2) was thermodynamically unfavorable based on measured reactant and product concentrations. Instead, strong correlations between genomic abundances of CO2-reducing methanogens and anaerobic bacteria with potential for extracellular electron transfer suggest that methanogenic CO2-reduction in lake sediments is largely powered by direct electron transfer from syntrophic bacteria without involvement of H2 as an electron shuttle.

Different oxygen affinities of methanotrophs and Comammox Nitrospira inform an electrically induced symbiosis for nitrogen loss.

Aerobic methanotrophs establish a symbiotic association with denitrifiers to facilitate the process of aerobic methane oxidation coupled with denitrification (AME-D). However, the symbiosis has been frequently observed in hypoxic conditions continuing to pose an enigma. The present study has firstly characterized an electrically induced symbiosis primarily governed by Methylosarcina and Hyphomicrobium for the AME-D process in a hypoxic niche caused by Comammox Nitrospira. The kinetic analysis revealed that Comammox Nitrospira exhibited a higher apparent oxygen affinity compared to Methylosarcina. While the coexistence of comammox and AME-D resulted in an increase in methane oxidation and nitrogen loss rates, from 0.82 ± 0.10 to 1.72 ± 0.09 mmol CH4 d-1 and from 0.59 ± 0.04 to 1.30 ± 0.15 mmol N2 d-1, respectively. Furthermore, the constructed microbial fuel cells demonstrated a pronounced dependence of the biocurrents on AME-D due to oxygen competition, suggesting the involvement of direct interspecies electron transfer in the AME-D process under hypoxic conditions. Metagenomic and metatranscriptomic analysis revealed that Methylosarcina efficiently oxidized methane to formaldehyde, subsequently generating abundant NAD(P)H for nitrate reduction by Hyphomicrobium through the dissimilatory RuMP pathway, leading to CO2 production. This study challenges the conventional understanding of survival mechanism employed by AME-D symbionts, thereby contributing to the characterization responsible for limiting methane emissions and promoting nitrogen removal in hypoxic regions.

Riboflavin modified carbon cloth enhances anaerobic digestion treating food waste in a pilot-scale system.

Previous laboratory-scale studies have consistently shown that carbon-based conductive materials can notably improve the anaerobic digestion of food waste, typically employing reactors with regular capacity of 1-20 L. Furthermore, incorporating riboflavin-loaded conductive materials can further address the imbalance between fermentation and methanogenesis in anaerobic systems. However, there have been few reports on pilot-scale investigation. In this study, a 10 m2 of riboflavin modified carbon cloth was incorporated into a pilot-scale (2 m3) food waste anaerobic reactor to improve its treatment efficiency. The study found that the addition of riboflavin-loaded carbon cloth can increase the maximum organic loading rate (OLR) by 40% of the pilot-scale reactor, compared to the system using carbon cloth without riboflavin loading, while ensuring efficient operation of the reaction system, effectively alleviating system acidification, sustaining methanogen activity, and increasing daily methane production by 25%. Analysis of the microbial community structure revealed that riboflavin-loaded carbon cloth enriched the methanogenic archaea in the genera of Methanothrix and Methanobacterium, which are capable of extracellular direct interspecies electron transfer (DIET). And metabolic pathway analysis identified the methane production pathway, highly enriched on the reduction of acetic acid and CO2 at riboflavin-loaded carbon cloth sample. The expression levels of genes related to methane production via DIET pathway were also significantly upregulated. These results can provide important guidance for the practical application of food waste anaerobic digestion engineering.

Insight into the performance and microbial community of anaerobic digestion treating cow manure with a novel iron-functionalized activated biochar.

The main objective of this research was to evaluate the impacts of FeCl3-activated biochar (FA-BC) on anaerobic digestion (AD) treating cow manure. The study focused on improving AD performance and understanding microbial community structure with the addition of FA-BC, while comparing FA-BC with other conductive additives, such as pristine biochar (P-BC), NaOH-activated biochar (NA-BC), and magnetite. Key findings indicated that FA- BC significantly enhanced the AD performance, supported by an increase in CH4 yield of 11-16% and a reduction in the lag phase by 51%. The high surface area and electrical conductivity of FA-BC synergistically facilitated direct interspecies electron transfer (DIET), leading to these improvements. On contrast, P-BC and NA-BC were not efficient in enhancing the AD performance due to relatively low electrical conductivity. P-BC also improved the CH4 yield, but less effectively than FA-BC. The effects of NA-BC varied with its dosage, showing inhibition at higher dosages due to excessive surface area. Magnetite, despite its high conductivity, made the limited enhancement in CH4 yield owing to its low surface area. Additionally, the statistical analyses revealed that each additive differently affected specific bacterial and archaeal groups depending on their physical and chemical properties. Thus, these findings suggest that FA-BC would be a highly promising additive for enhan cing AD systems, with potential applications in waste management and renewable energy production.

Advances in microbial community, mechanisms and stimulation effects of direct interspecies electron transfer in anaerobic digestion.

Anaerobic digestion (AD) has been proven to be an effective green technology for producing biomethane while reducing environmental pollution. The interspecies electron transfer (IET) processes in AD are critical for acetogenesis and methanogenesis, and these IET processes are carried out via mediated interspecies electron transfer (MIET) and direct interspecies electron transfer (DIET). The latter has recently become a topic of significant interest, considering its potential to allow diffusion-free electron transfer during the AD process steps. To date, different multi-heme c-type cytochromes, electrically conductive pili (e-pili), and other relevant accessories during DIET between microorganisms of different natures have been reported. Additionally, several studies have been carried out on metagenomics and metatranscriptomics for better detection of DIET, the role of DIET's stimulation in alleviating stressed conditions, such as high organic loading rates (OLR) and low pH, and the stimulation mechanisms of DIET in mixed cultures and co-cultures by various conductive materials. Keeping in view this significant research progress, this study provides in-depth insights into the DIET-active microbial community, DIET mechanisms of different species, utilization of various approaches for stimulating DIET, characterization approaches for effectively detecting DIET, and potential future research directions. This study can help accelerate the field's research progress, enable a better understanding of DIET in complex microbial communities, and allow its utilization to alleviate various inhibitions in complex AD processes.

Neglected role of iron redox cycle in direct interspecies electron transfer in anaerobic methanogenesis: Inspired from biogeochemical processes.

Anaerobic digestion is an indispensable technical option towards green and low-carbon wastewater treatment, with interspecies electron transfer (IET) playing a key role in its efficiency and operational stability. The exogenous semiconductive iron oxides have been proven to effectively enhance IET, while the cognition of the physicochemical-biochemical coupling stimulatory mechanism was circumscribed and remains to be elucidated. In this study, semiconductive iron oxides, α-Fe2O3, γ-Fe2O3, α-FeOOH, and γ-FeOOH were found to significantly enhance syntrophic methanogenesis by 76.39, 72.40, 37.33, and 32.64% through redirecting the dominant IET pathway from classical interspecies hydrogen transfer to robust direct interspecies electron transfer (DIET). Their alternative roles as electron shuttles potentially substituting for c-type cytochromes were conjectured to establish an electron transport matrix associated with conductive pili. Distinguished from the conventional electron conductor mechanism of conductive Fe3O4, semiconductive iron oxides facilitated DIET intrinsically through the capacitive Fe(III/II) redox cycles coupled with secondary mineralization. The growth of Aminobacterium, Sedimentibacter, and Methanothrix was enriched and the gene copy numbers of Geobacteraceae 16S ribosomal ribonucleic acid were selectively flourished by 2.0-∼4.5- fold to establish a favorable microflora for DIET pathway. Metabolic pathways of syntrophic acetogenesis from propionate/butyrate and CO2 reduction methanogenesis were correspondingly promoted. The above findings provide new insights into the underlying mechanism of iron minerals enhancing the DIET-oriented pathway and offer paradigms for redox-mediated energy harvesting biological wastewater treatment.