Methane Production In Anaerobic Digestion Research Articles

Advancing anaerobic digestion with MnO2-modified biochar: Insights into performance and mechanisms

The use of bio-based composites to enhance the methane production in anaerobic digestion has attracted considerable attention. Nevertheless, the study of electron transfer mechanisms and the applications of biochar/MnO2 (MBC) in complex systems remains largely unexplored. Biochar composited with MnO2 at 10:1 mass ratio (MBC10) increased the content of volatile fatty acids by 9.09 % during acidogenic phase. During the methanogenic experiments using acetate, cumulative methane production (CMP) rose by 5.83 %, and in the methanogenic experiments using food waste, CMP increased by 24.32 %. Microbial community analysis indicated an enrichment of Syntrophomonas, Bacilli, and Methanosaetaceae in the MBC10 group. This enrichment occurred mainly due to the redox capability of MnO2 enhancing MBC capacitance, thereby facilitating microbial electron transfer processes. Additionally, under 2 g/L ammonia nitrogen concentration and 30 g/L organic load, the CMP of MBC10 increased by 12.74 % and 9.44 %, respectively, compared to the BC600 group. This study illuminates MBC's electron transfer mechanisms and applications, facilitating its wider practical adoption and fostering future innovations.

Enhancing methane production in anaerobic digestion of waste activated sludge by combined thermal hydrolysis and photocatalysis pretreatment

Thermal hydrolysis (TH) is promising for sludge pretreatment, but the refractory substances generated at high temperatures inhibit anaerobic digestion. In this study, a novel combined TH and photocatalytic pretreatment method was proposed to improve the anaerobic digestion performance of waste activated sludge. The results showed that the combined pretreatment (170 °C, 0.5 g/L TiO2) increased methane yield by 66 % from 111 ± 5 m L/g VS to 185 ± 5 m L/g VS. After TH pretreatment, photocatalysis further promoted sludge solubilization by destroying extracellular polymeric substances, resulting in an increase in released soluble organic matter from 292 ± 16 mg/L to 4,091 ± 85 mg/L. In addition, photocatalysis improved the biodegradability of sludge by reducing the melanoidin and humic acid contents by 26 % and 20 %, respectively. The proposed novel pretreatment method effectively overcomes the bottleneck of TH technology and provides an alternative pretreatment technology for improving sludge resource recovery.

Mechanistic exploration of COVlD-19 antiviral drug ritonavir on anaerobic digestion through experimental validation coupled with metagenomics analysis

Aggregation of antiviral drugs (ATVs) in waste activated sludge (WAS) poses considerable environmental risk, so it is crucial to understand the behavior of these agents during WAS treatment. This study investigated the effects of ritonavir (RIT), an ATV used to treat human immunodeficiency virus infection and coronavirus disease 2019, on anaerobic digestion (AD) of WAS to reveal the mechanisms by which it interferes with anaerobic flora. The dosage influence results showed that methane production in AD of WAS decreased by 46.56 % when RIT concentration was increased to 1000 μg/kg total suspended solids (TSS). The AD staging test revealed that RIT mainly stimulated microbial synthesis of the extracellular polymeric substance (EPS), limiting organic matter solubilization. At 500 μg/kg TSS, RIT decreased CHO and CHON levels in dissolved organic matter by 23.12 % and 56.68 %, respectively, significantly reducing substrate availability to microorganisms. Metagenomic analysis of microbial functional gene sets revealed that RIT had greater inhibitory effects on protein and amino acid metabolism than on carbohydrate metabolism. Under RIT stress, methanogens switched from hydrogenotrophic and acetotrophic methanogenesis to methylotrophic and acetotrophic methanogenesis.

Enhance the biomethane yield of food waste by anaerobic fermentation

This research investigates optimizing food waste (FW) concentrations for enhanced methane production in anaerobic digestion (AD) systems. Various FW concentrations (10, 20, 30, 40, 50, and 100 % v/v) were assessed for their impact on methane yield, pH stability, volatile fatty acid (VFA) levels, and microbial community composition. The study found that FW concentrations up to 20 % v/v maximized methane production, achieving a peak yield of 140.20 mL CH4/gVS within an ideal pH range of 6.00–7.00. However, higher FW concentrations (>30 % v/v) significantly reduced methane output, with 100 % v/v halting production due to excessive VFA accumulation and pH drops. Key microbial players included acetoclastic methanogens like Methanosaeta and hydrogenotrophic methanogens such as Methanospirillum. These findings emphasize the importance of managing FW concentrations to maintain AD system efficiency, providing valuable insights into sustainable waste management and renewable energy production.

A method for predicting methane production from anaerobic digestion of kitchen waste under small sample conditions.

Anaerobic digestion (AD) has the great potential to treat organic waste and achieve remarkable results effectively. However, it is very tough to establish an accurate mechanistic model for this process. Data-driven modeling technology has opened a new door to solving this problem. While when the sample set is small, traditional data-driven modeling methods are often powerless. In this paper, an effective method is proposed for data-driven high-precision modeling in small sample scenarios. A time series generative adversarial network (TimeGAN) is first utilized to augment the original high-quality small-sample data collected during the AD methane production. A novel hybrid kernel extreme learning machine (HKELM) is then designed to form a better structure of the data-driven model, whose regularization coefficient C0 is optimized by the sparrow search algorithm (SSA). Finally, this semi-finished model (SSA-HKELM) is trained by the augmented data to form the final mathematical model (TimeGAN-SSA-HKELM) for the AD methane generation process. Comparative experiments of the methane daily production prediction error haveverified the effectiveness of the method, which can be extended to other similar small sample data-driven modeling scenarios.

Estimation of some AM2 model parameters from a derived empirical logistic function of methane production

Because of its capability to convert organic wastes into renewable energy and into some components useful for agriculture, the anaerobic digestion technology can reduce greenhouse gas emissions in the atmosphere and the pollution. Thus, anaerobic digestion can contribute to achieving some of sustainable development goals. Consequently, many theoretical and empirical approaches are proposed for estimating, predicting and optimizing the methane produced by anaerobic digestion. In this context, the logistic function is a mathematical model that can be used to approximate empirical data of the temporal methane production in anaerobic digestion. In a previous paper, under some appropriate approximations, we have derived from AM2 model a single analytical expression in a form of a logistic function for describing the evolution of methane production in batch bioreactors. In the present paper, by comparing the three standard parameters associated with the classical empirical logistic function with that of the derived one from AM2 model; some relationships between them have been established. These relations are exploited for estimating some coefficients and parameters of AM2 model with respect to empiric logistic function parameters and vice-versa. Moreover, this possibility enables more qualitative insight about the evolution of the methane production and the influence of AM2 parameters and coefficients as well as their interaction over its processes.

Heterogeneous g-C3N4/polyaniline composites enhanced the conversion of organics into methane during anaerobic wastewater treatment

In this study, g-C3N4/PANI was prepared by in situ oxidative polymerization. Graphite-phase carbon nitride (g-C3N4) with surface defects was deposited onto the surface of conductive polyaniline (PANI) to form a p-n heterojunction. This construction aimed to create an efficient heterogeneous catalyst, increasing the surface defect level and active sites of the composite, and augmenting its capability to capture and transfer extracellular electrons under anaerobic conditions. This addresses the challenge of low efficiency in direct interspecies electron transfer between bacteria and archaea during anaerobic digestion for methane production. The results showed that the prepared g-C3N4/PANI increased the CH4 yield and CH4 production rate by 82% and 96%, respectively. Notably, the conductivity and XPS test results showed that the ratio of g-C3N4 to PANI was 0.15, and the composite exhibited favorable conductivity, with a uniform distribution of pyrrolic nitrogen, pyridinic nitrogen, and graphitic nitrogen, each accounting for approximately 30%. Furthermore, g-C3N4/PANI effectively enhanced the metabolic efficiency of intermediate products such as acetate and butyrate. Analysis of the microbial community structure revealed that g-C3N4/PANI led to a significant increase in the abundance of hydrogenotrophic methanogen Methanolinea (from 48% to 64%) and enriched Clostridium (a rise of 1%) with direct interspecies electron transfer capability. Microbial community function analysis demonstrated that the addition of g-C3N4/PANI boosted the activities of key enzymes involved in anaerobic digestion, including phosphate transacetylase (PTA), phospho-butyryl transferase (PTB), and NAD-independent lactate dehydrogenase (NNLD), by 47%, 135%, and 153%, respectively. This acceleration in enzymatic activity promoted the metabolism of acetyl-CoA, butyryl-CoA, and pyruvate. Additionally, the function of ABC transporters was enhanced, thereby improving the efficiency of material and energy exchange among microorganisms.

Enhancement of anaerobic digestion of dairy wastewater by addition of conductive materials with or without the combination of external voltage application

AbstractBACKGROUNDThe addition of conductive materials or the application of external voltage is a promising novel methodology to enhance methane production in anaerobic digesters. However, there is limited knowledge about their individual or combined effects on the anaerobic digestion of dairy wastewater. The aim of this study was to examine the effects of granular activated carbon (GAC) or magnetite addition and external voltage application, both individually and in combination, for the anaerobic digestion of real, undiluted dairy wastewater.RESULTSThe results showed that the estimated maximum methane production rate increased significantly (P < 0.05) from 143 ± 8 mL gVSadded−1 day−1 in the control reactors to 163 ± 11 mL gVSadded−1 day−1 and 166 ± 11 mL gVSadded−1 day−1 in the reactors containing GAC, with or without the combination of external voltage, respectively. The addition of GAC, in both cases, resulted in higher consumption rate of acetate, indicating a promotion of the methanogenesis step. By contrast, the application of external voltage or the addition of magnetite had no significant effect on either methane production rate nor methane yield. Moreover, the analysis of the microbial community showed that the addition of GAC resulted in the enrichment of Desulfobacterota, Methanosarcina, Candidatus Methanofastidiosum and Methanolinea.CONCLUSIONOverall, GAC addition seems a promising strategy to increase methane production in anaerobic digesters treating dairy wastewater. © 2024 The Author(s). Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Tris (2-chloroethyl) phosphate presence inhibits methane production from anaerobic digestion: Alterations in organic matter transformation, cell physiological status, and microbial community

Organophosphate flame retardants (OPFRs) are widely used in consumer products, leading to their unavoidable release into the environment, especially accumulation in anaerobic environments and posing potential risks. This study focused on Tris(2-chloroethyl) phosphate (TCEP), a representative OPFR, to investigate its effects on carbon transformation and methane production in anaerobic digestion. Increasing TCEP concentrations from control to 16 mg/L resulted in decreased cumulative methane yield (from 235.4 to 196.3 mL/g COD) and maximum daily methane yield (from 40.8 to 16.17 mL/(g COD·d)), along with an extended optimal anaerobic digestion time (from 15 to 20 days). Mechanistic analysis revealed TCEP binding to tyrosine-like proteins in extracellular polymeric substances, causing cell membrane integrity impairment. The TCEP-caused alteration of the physiological status of cells was demonstrated to be a significant contribution to the inhibited bioprocesses including acidogenesis, acetogenesis, and methanogenesis. Illumina Miseq sequencing showed TCEP decreasing the relative abundance of acidogens (58.8 % to 46.0 %) and acetogens (7.1 % to 5.0 %), partly shifting the methanogenesis pathway from acetoclastic to hydrogenotrophic methanogenesis. These findings enhance understanding of TCEP's impact on anaerobic digestion, emphasizing the environmental risk associated with its continued accumulation.

Impact of Polyethylene-Glycol-Induced Water Potential on Methane Yield and Microbial Consortium Dynamics in the Anaerobic Degradation of Glucose.

This study investigated the relationship between water potential (Ψ) and the cation-induced inhibition of methane production in anaerobic digesters. The Ψ around methanogens was manipulated using polyethylene glycol (PEG) in a batch anaerobic reactor, ranging from -0.92 to -5.10 MPa. The ultimate methane potential (Bu) decreased significantly from 0.293 to 0.002 Nm3 kg-1-VSadded as Ψ decreased. When Ψ lowered from -0.92 MPa to -1.48 MPa, the community distribution of acetoclastic Methanosarcina decreased from 59.62% to 40.44%, while those of hydrogenotrophic Methanoculleus and Methanobacterium increased from 17.70% and 1.30% to 36.30% and 18.07%, respectively. These results mirrored changes observed in methanogenic communities affected by cation inhibition with KCl. Our findings strongly indicate that the inhibitory effect of cations on methane production may stem more from the water stress induced by cations than from their direct toxic effects. This study highlights the importance of considering Ψ dynamics in understanding cation-mediated inhibition in anaerobic digesters, providing insights into optimizing microbial processes for enhanced methane production from organic substrates.

Metagenome reveals the possible mechanism that microbial strains promote methanogenesis during anaerobic digestion of food waste

For better understanding the mechanism of microbial strains promoting methane production, four strains Hungatella xylanolytica A5, Bacillus licheniformis B1, Paraclostridium benzoelyticum C2 and Advenella faeciporci E1 were inoculated into anaerobic digestion systems. After bioaugmentation, the cumulative methane production of A5, B1, C2 and E1 groups elevated by 11.68%, 8.20%, 18.21% and 15.67% compared to CK group, respectively. The metagenomic analysis revealed that the species diversity and uniformity of the experimental groups was improved, and hydrolytic acidifying bacteria, represented by Clostridiaceae, Anaerolineaceae and Oscillospiraceae, and methanogens, such as Methanotrichaceae and Methanobacteriaceae, were enriched. Meanwhile, the abundance of key genes in carbohydrate, pyruvate and methane metabolism was increased in the inoculated groups, providing reasonable reasons for more methane production. The strengthening mechanism of microbial strains in this study offered a theoretical foundation for selecting a suitable bioaugmentation strategy to solve the problems of slow start-up and low methane production in anaerobic digestion.

Effect of bioaugmentation on gas production and microbial community during anaerobic digestion in a low-temperature fixed-bed reactor.

Low temperature is one of the limiting factors for anaerobic digestion in cold regions. To improve the efficiency of anaerobic digestion for methane production in stationary reactors under low-temperature conditions, and to improve the structure of the microbial community for anaerobic digestion at low temperatures. We investigated the effects of different concentrations of exogenous Methanomicrobium (10, 20, 30%) and different volumes of carbon fiber carriers (0, 10, 20%) on gas production and microbial communities to improve the performance of low-temperature anaerobic digestion systems. The results show that the addition of 30% exogenous microorganisms and a 10% volume of carbon fiber carrier led to the highest daily (128.15 mL/g VS) and cumulative (576.62 mL/g VS) methane production. This treatment effectively reduced the concentrations of COD and organic acid, in addition to stabilizing the pH of the system. High-throughput sequencing analysis revealed that the dominant bacteria under these conditions were Acidobacteria and Firmicutes and the dominant archaea were Candidatus_Udaeobacter and Methanobacterium. While the abundance of microorganisms that metabolize organic acids was reduced, the functional abundance of hydrogenophilic methanogenic microorganisms was increased. Therefore, the synergistic effect of Methanomicrobium bioaugmentation with carbon fiber carriers can significantly improve the performance and efficiency of low-temperature anaerobic fermentation systems.

Combined hydrothermal pretreatment of agricultural and forestry wastes to enhance anaerobic digestion for methane production

The dense structure and significant differences in the properties of agricultural and forestry wastes make it difficult to achieve anaerobic conversion. This study employed hydrothermal pretreatment (HTP) and combined hydrothermal pretreatment (CHTP) to enhance the anaerobic digestion (AD) performance of agricultural and forestry organic wastes. At a specific hydrothermal condition of 140℃ for 30 mins, the cumulative methane production of garden waste (GW), chicken manure (CM), duck manure (DM), and pig manure (PM) increased by 14.70%, 14.18%, 27.48%, and 21.81%, respectively. Under the same conditions, CHTP of garden waste with livestock and poultry manures showed noticeable synergistic effects, resulting in the methane production potential of garden waste mixed with chicken manure, duck manure, and pig manure increased by 28.82%, 36.59%, and 37.56%, respectively, compared with the untreated group. Metabolomic analysis showed that CHTP could significantly promote the decomposition of biomass carbohydrates, proteins, and lipids, leading to increased levels of small molecular metabolites. Analysis of microbial community diversity showed that HTP and CHTP increase the abundance of specific functional microbial communities, such as hydrolysis bacteria and methanogens, thereby promoting the anaerobic production of methane from biomass waste. Energy balance analysis shows that HTP-AD and CHTP-AD have a positive net energy benefit. This study provides an efficient CHTP technology and theoretical support for the resource utilization and energy conversion of agricultural and forestry wastes.

Optimum Magnetite–Zeolite Nanoparticles Loading on the Cathode of Microbial Electrochemical–Anaerobic Digestion System for Enhancing Methane Generation

Microbial electrochemical systems (MESs) can enhance methane production in anaerobic digestion, but their performance is hindered by inadequate electron transfer between the cathode and microbes. Magnetite–zeolite (MZ)‐based catalysts accelerate electron exchange; however, the optimal loading of MZ is crucial for maximizing MES performance. In this study, different loadings of the MZ catalyst ranging from 0 to 2 mg cm−2 on the cathode were evaluated to obtain an optimum loading for maximizing methane recovery from MES. Electrochemical analyses demonstrated enhanced electrochemical reduction kinetics of the cathode with increased MZ loading from 0 to 2 mg cm−2. Among the tested loadings, the MES with 1 mg cm−2 MZ on the cathode exhibited the highest methane production rate at 548 mL L−1 d−1, surpassing other MES operations with 2 and 0.5 mg cm−2 (499 and 434 mL L−1 d−1, respectively). Additionally, the MES with 1 mg cm−2 MZ demonstrated the highest soluble chemical oxygen demand removal efficiency of 87% and total coulomb recovery of 1444.06C ± 42.60C, with the lowest amount of volatile fatty acids accumulation. Consequently, MZ is recommended as a superior catalyst for enhancing MES performance and suggested to utilize 1 mg cm−2 MZ loading on the cathode to maximize methane production.

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.

Acyl homoserine lactone-based regulation strategy for improved methane production in anaerobic digestion of agricultural wastes

The acyl homoserine lactone (AHL)-based regulation strategy presents a considerable potential in improving anaerobic digestion (AD) efficiency of complex substrates. However, the reinforcement mechanisms are still unclear. In this study, the effects of different AHL types (C4-HSL, C7-HSL, C12-HSL) and different adding times (0 d, 5 d, 15 d) on AD performance of agricultural wastes (corn straw and cattle manure) were investigated. The results indicated that all types of AHLs exerted positive effects on AD, and the adding time rather than the AHL type was the key factor affecting AD processes. The C12-5d group obtained the highest accumulated methane production (310.7 ± 2.56 mL/g VS), which increased by 47.71% than that of the Control group. The exogenous AHLs facilitated the hydrolysis and acidification of complex substrates and the utilization of released low-molecular-weight organic acids in AD systems. Furthermore, the role of methanogenic archaea was upregulated and the dominance of bacteria was relatively weakened in AD systems, which favored the establishment of their balanced and symbiotic metabolic relationship. The enriched Methanobacterium strengthened the hydrogenotrophic methanogenesis pathway. This study provides new insights into the regulation strategy of AD for agricultural waste treatment based on AHL addition.

Effects of inocula on methane production and the microbial community in a rice straw anaerobic digestion system

Field experiments have shown that the mixed anaerobic digestion of straw with livestock and poultry manure can improve the efficiency of biogas production. We hypothesize that inoculation with livestock and poultry manure increase the efficiency of methane production by improving the diversity and structure of microbial community in the field anaerobic digestion system. In this study, an experimental system was established to simulate the microbial community of the mixed anaerobic digestion system in the field. The methane production and taxonomic composition of bacterial and archaeal communities in the rice straw anaerobic digestion systems were determined. We aim to reveal the effects of the inocula on the diversity and composition of bacterial and archaeal communities and the mechanism underlying the impact on biogas production in the field. The following results were obtained. (1) Inoculation significantly improved the efficiency of methane production in anaerobic digestion, and the best performance was achieved with treatment A (cleared biogas slurry), which obtained methane production that were 39.59 %, 106.85 %, and 123.00 % higher than in treatment B (pig manure extract), C (cleared biogas slurry + pig manure extract), and the control, respectively. (2) The inocula increased the abundance and diversity of bacterial communities in the early and middle stages and archaeal communities in the early stage. (3) The inocula also had a significant impact on bacterial and archaeal community composition in the early and middle stages. The inocula increased the relative abundance of Aquamicrobium_A, Proteiniphilum, and Cryptobacteroides but reduced the relative abundance of Macellibacteroides, Acinetobacter, and Phocaeicola. The dominant genus of archaeal community was altered from Methanosarcina to Methanocorpusculum. (4) It is concluded that inoculation significantly increase methane production as the inocula improve the abundance and diversity of bacteria and archaea of anaerobic digestion systems in the early and middle stages of digestion, as well as improve the structure and metabolic pathway of archaeal communities.

Applicability of Rice Husk Residue Generated by the Silica Extraction Process to Anaerobic Digestion for Methane Production

Rice husks are a feedstock of biogenic silica because of their high silica content. After silica extraction, a solid residue comprising mostly carbohydrates is present. Solid residue valorization is important for closed-loop systems using rice husk and has minimal negative environmental impacts. In this study, we used solid rice husk that was generated by silica extractionto anaerobic digestion for producing biomethane. The rice husk residue was characterized in terms of total solids, volatile solids, pH, composition, and particle size. Changing the characteristics increased biogas production by 2.48-fold compared to that of raw rice husk. The residue produced 166.4 mL-biogas g−1 vs. and 100.4 mL CH 4 g−1 VS, much more than previously reported. Microbial community analysis, which was conducted to investigate the biological reasons for increased biogas and methane, found increased Bacteroidetes levels in the rice husk samples. Among archaeal communities, Bathyarchaeota was more abundant in all rice husk samples than in the inoculum. The rice husk residue contained more operational taxonomic units than other samples. These changes in the microbial community significantly influenced the anaerobic digestion of the rice husk residue and improved methane production. Our findings provide a basis for the cleaner utilization of rice husk residue to produce renewable energy.

Effects of cold isostatic press pretreatment of rice straw on microstructure and efficiency of anaerobic digestion for methane production

In this study, rice straw was pretreated using by cold isostatic pressure to disrupt its microstructure and improve the performance of anaerobic digestion, and the optimal process parameters were optimized using the response surface methodology. The results showed that cold isostatic pressure pretreatment under optimal conditions (pressure of 400 MPa and holding time of 9 min) was effective in disrupting the structure of rice straw and improving its biodegradability. The cumulative methane production of the cold isostatic pressure pretreatment group increased by 76% compared to the untreated group. In addition, microbial community analysis showed that the relative abundance of Firmicutes, Halobacterota, DMER64 and Methanosaeta was higher in groups pretreated with cold isostatic pressure than in untreated groups. This study demonstrated the potential of pretreatment of rice straw with cold isostatic pressure to increase methane production during anaerobic digestion.

Effects of magnetite on nitrate-dependent anaerobic oxidation of methane in an anaerobic membrane biofilm reactor: Metatranscriptomic analysis and mechanism prediction

Magnetite has been widely reported to accelerate methane production in anaerobic digestion. However, the effects of magnetite on the process of methane consumption, specifically nitrate-dependent anaerobic oxidation of methane (AOM), and its underlying mechanisms remain poorly understood. In this study, the addition of magnetite significantly improved the removal of nitrate in an anaerobic membrane biofilm reactor (AnMBfR) (25.2 ± 2.5 mg NO3−-N/L/d) compared to that in an AnMBfR without magnetite (8.7 ± 4.2 mg NO3−-N/L/d). Metatranscriptomic analysis showed that genes encoding type IV pilus assembly proteins were highly expressed in nitrate/nitrite-reducing bacteria in the biofilms of the two AnMBfRs. Specifically, the transcript abundance of these genes was observed to be higher in the presence of magnetite (gene expression levels, log2FPKM = 13.08) compared to the absence of magnetite (12.04). Biofilms with magnetite exhibited a linear increase in electron transfer resistance with increasing temperature, and the decrease in pH significantly increased their conductivity, indicating a metallic-like conductivity. Contrarily, an increase in temperature or decrease in pH did not influence the resistance or conductivity of biofilms without magnetite. Some of these cytochrome c (CYC)-like proteins were electrically active in the electrochemical Fourier transform infrared spectra and were closely associated with the outer-membrane multi-heme c-type cytochromes (OMCs). The intensities of the OMC-like bands in the surface-enhanced resonance Raman spectra with biofilms in the presence of magnetite were higher than those without magnetite. This study suggests that magnetite promotes the nitrate-dependent AOM process because of the formation of methanotrophic consortia based on direct interspecies electron transfer (DIET).