Accelerated Methane Production Research Articles

Effects of Fe–N co-modified biochar on methanogenesis performance, microbial community, and metabolic pathway during anaerobic co-digestion of alternanthera philoxeroides and cow manure

The performance of anaerobic digestion (AD) is susceptible to disturbances in feedstock degradation, intermediates accumulation, and methanogenic archaea activity. To improve the methanogenesis performance of the AD system, Fe–N co-modified biochar was prepared under different pyrolysis temperatures (300,500, and 700 °C). Meanwhile, pristine and Fe-modified biochar were also derived from alternanthera philoxeroides (AP). The aim was to compare the effects of Fe–N co-modification, Fe modification, and pristine biochar on the methanogenic performance and explicit the responding mechanism of the microbial community in anaerobic co-digestion (coAD) of AP and cow manure (CM). The highest cumulative methane production was obtained with the addition of Fe-N-BC500 (260.38 mL/gVS), which was 42.37 % higher than the control, while the acetic acid, propionic acid, and butyric acid concentration of Fe-N-BC were increased by 147.58 %, 44.25 %, and 194.06 % compared with the control, respectively. The co-modified biochar enhanced the abundance of Chloroflexi and Methanosarcina in the AD system. Metabolic pathway analysis revealed that the increased methane production was related to the formation and metabolism of volatile fatty acids and that Fe-N-BC500 enhanced the biosynthesis of coenzyme A and the cell activity of microorganisms, accelerating the degradation of propionic acid and enhancing the hydrogenotrophic methanogenesis pathway. Overall, Fe–N co-modified biochar was proved to be an effective promoter for accelerated methane production during AD.

Effect of co-digestion and hydrodynamic disintegration on the methane potential of sewage sludge and organic fraction of municipal solid waste with consideration of the carbon footprint

The study objective was to investigate the applicability of co-digestion and/or hydrodynamic disintegration as potential methods of methane production intensification from sewage sludge (SS) and the organic fraction of municipal solid waste (OFMSW) with consideration of carbon footprint (CF) assessment. Methane production was determined by means of biochemical methane potential tests. For co-digestion, the highest increase in methane production (53.8 %) was observed for the SS:OFMSW mixing ratio 40:60. Hydrodynamic disintegration at all tested energy densities (10, 30 and 60 kJ/L) caused release of soluble organic compounds from both substrates, although accelerated methane production was only observed at 10 kJ/L. An increase in methane potential for substrates disintegrated separately did not imply analogical results for the disintegration of their mixture. Within the assumed limits, CF per 1 Nm3 of produced methane, CF per 1 t of wet feedstock for the digester, and estimated CF per 1 kWh produced were also calculated. Comparable values of all indicators were obtained for monodigestion and co-digestion. The highest CF indices were achieved for monodigestion of disintegrated SS and increased significantly with applied energy density. The obtained results can serve as valuable material for operators of wastewater treatment plants facilitating the selection of sustainable methods of increasing renewable energy production.

Revealing sustainable energy opportunities through the integrated use of Canna indica biomass and buffalo manure for biogas generation

Mature Canna indica L., contains significant percentages of hemicellulose (21.6±0.22%) and lignin (20.14±0.13%), showing its high potential as a biogas source. This study explores the potential of using C. indica biomass harvested from waterlogged clay areas for biogas production. The research focuses on optimizing the anaerobic co-digestion process with swine dung through varying calcium oxide (CaO) pretreatment concentrations during a 45-day experiment. CaO pretreatment significantly enhances biogas yield, with 2% CaO yielding the highest biogas production at 8024.10 mL. Methane concentration analysis reveals that higher CaO concentrations, notably 2% and 3%, accelerate methane production, indicating an optimal CaO concentration of around 2% for maximizing methane yield. This study outperforms others in anaerobic co-digestion, achieving a methane concentration of 64.93%. Data on C. indica at different CaO concentrations as a substrate underscores the need for precise CaO tuning for optimal methane production. The findings open avenues for sustainable waste management and renewable energy production, hinting at promising developments in energy solutions through optimized anaerobic co-digestion processes using C. indica and buffalo dung.

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).

Low-temperature torrefaction assisted with solid-state KOH/urea pretreatment for accelerated methane production in wheat straw anaerobic digestion

Low-temperature torrefaction assisted with solid-state KOH/urea applied onto wheat straw was proposed to break down the lignocellulosic material to enhance biomethane production in anaerobic digestion (AD). The optimization of key parameters applying the Box-Behnken design and response surface methodology showed that an addition of 0.1 g/gstraw KOH/urea at 180 °C while torrefying for 30 min was the optimal condition for producing biomethane. Results indicate that co-applying KOH and urea in torrefaction synergistically enhanced the biodegradability of straw by effectively removing lignin and largely retaining cellulose, giving rise to a 41 % increase in the cumulative methane production compared to untreated straw (213 mL/g-volatile solids (VSraw)) from batch AD. Additionally, the nitrogen- and potassium-rich digestates helped to improve soil fertility, thus achieving a zero-waste discharge. This study demonstrated the feasibility of using solid-state KOH/urea assisted low-temperature torrefaction as an effective pretreatment method to promote methane production during AD.

Varied promotion effects and mechanisms of biochar on anaerobic digestion (AD) under distinct food-to-microorganism (F/M) ratios and biochar dosages

Biochar (BC) promotes the performance of anaerobic digestion (AD) through different routes, such as enriching microbes, buffering pH and promoting electron transfer. However, the mechanisms and processes of AD that enhanced by BC under various food to microorganism (F/M) ratios are still unclear. The organic transformations, bioelectrochemical characteristics and microbial consortia under the different BC dosages and F/M ratios were studied to reveal the role of BC in an AD process. The electron transfer system (ETS) was proportional to BC dosage and considered to be a key for AD promotion. At the F/M ratios of 0.5 and 1.0, BC accelerated methane production mainly by promoting ETS. The most enhanced specific methanation activities (SMAs) were obtained with 10.0 g/L BC, and the promotion efficiency under the F/M ratio of 1.0 was significantly higher (P < 0.05) than that under the F/M ratio of 0.5. Under the higher F/M ratio of 2.0, BC shortened the entire AD duration for 5.0 ∼ 13.0 days and guaranteed the resilience of AD by expanding the thermodynamic window of syntrophic methanogenesis via direct interspecies electron transfer (DIET). The COD balance analysis and the ecological functional profiles of microbes demonstrated that BC promoted both the anabolism and catabolism of anaerobes, and enhanced the DIET by converting hydrotrophic methanogenesis into acetolastic methanogenesis pathway. Besides, excessive BC enhanced SMA and simultaneously triggered superfluous biomass growth and thus decreased CH4 yield. This study provided an important reference for further application of BC under various F/M ratios and dosages in AD.

Consolidation of hydrogenotrophic methanogenesis by sulfidated nanoscale zero-valent iron in the anaerobic digestion of food waste upon ammonia stress

The feasibility of adding sulfidated nanoscale zero-valent iron (S-nZVI) into anaerobic systems to improve anaerobic digestion of food waste (FW) under ammonia stress was evaluated in this study. The addition of S-nZVI improved the methane production compared to nanoscale zero-valent iron (nZVI), indicating that sulfidation significantly reinforced the enhancement effect of nZVI in consolidating the hydrogenotrophic methanogenesis. The promoted methanogenic performance was associated with chemical reaction and variances of microbial community induced by S-nZVI. With the characteristics of generation of Fe2+ and slow-release of H2, S-nZVI made the anaerobic system respond positively in facilitating extracellular polymeric substances secretion and optimizing the microbial community structure. Moreover, microbial community analysis showed that S-nZVI addition enriched the species related to biohydrogen production (e.g., Prevotella) and ammonia-tolerant hydrogenotrophic methanogenesis (e.g., Methanoculleus), possibly enhancing the hydrogenotrophic methanogenesis pathway to accelerate methane production. Therefore, adding S-nZVI into the anaerobic systems might propose a feasible engineering strategy to improve the methanogenic performance of the anaerobic digestion of FW upon ammonia stress.

Carbon-based conductive materials enhance biomethane recovery from organic wastes: A review of the impacts on anaerobic treatment

Amongst the most important sustainable waste management strategies, anaerobic biotechnology has had a central role over the past century in the management of high-pollution load sources, such as food, agricultural and municipal wastes. During anaerobic digestion (AD), valuable by-products such as digestate and biogas are produced. Biogas (mainly composed of methane) is generated through a series of reactions between bacteria and archaea. Enhancement of AD process with higher methane yield, accelerated methane production rate, and shorter start-up time is possible via tapping into a novel methanogenic pathway discovered a decade ago. This fundamentally new concept that is a substitute to interspecies hydrogen transfer is called direct interspecies electron transfer (DIET). DIET, a thermodynamically more feasible way of electron transfer, has been proven to occur between bacteria and methanogens. It is well-documented that amendment of carbon-based conductive materials (CCMs) can stimulate DIET via serving as an electrical conduit between microorganisms. Therefore, different types of CCMs such as biochar and activated carbon have been amended to a variety of AD reactors and enhancement of process performance was reported. In this review, a comparative analysis is presented for enhancement of AD performance in relation to major CCM related factors; electrical conductivity, redox properties, particle size and dosage. Additionally, the impacts of AD operational conditions such as organic loading rate and temperature on CCM amended reactors were discussed. Further, the changes in microbial communities of CCM amended reactors were reviewed and future perspectives along with challenges for CCM application in AD have been provided.

Adding activated carbon to the system with added zero-valent iron further improves anaerobic digestion performance by alleviating ammonia inhibition and promoting DIET

The addition of activated carbon (AC) may improve the efficiency of anaerobic digestion (AD) system to which zero-valent iron (ZVI) has been added. This study investigated the effects of the addition of AC on the AD system with ZVI added. Results showed that the cumulative methane production of 5 g/L ZVI + 5 g/L AC (5ZVI + 5AC) was 2.77–35.0% higher than that of 5 g/L ZVI, 10 g/L ZVI and control. The combined addition shortened the time to reach maximum daily methane yield by 18.2% compared to ZVI groups. Meanwhile, 5ZVI+5AC had the lowest free ammonia concentration (average 461 mg/L) amongst all groups, thus effectively alleviating the ammonia inhibition. Microbial community analysis showed that the combined addition of ZVI and AC enriched hydrogenotrophic methanogens (Methanoculleus) and direct interspecies electron transfer related microorganisms (Syntrophomonas and Methanosaeta), possibly enhancing electron exchange to accelerate methane production.

Revealing the role of conductive materials on facilitating direct interspecies electron transfer in syntrophic methanogenesis: A thermodynamic analysis

Facilitating the direct interspecies electron transfer with supplying versatile conductive materials to accelerate methane production in anaerobic digestion has received considerable attention, while the stimulatory mechanism of conductive materials on the direct interspecies electrons transfer still remains unclear. Herein, the thermodynamic characteristics of syntrophic methanogenesis induced by different conductive materials in various fermentation conditions were investigated to explore the stimulatory mechanism. The results showed that the supplementation of conductive materials significantly affected the Gibbs free energy changes of both propionate oxidation and electrotrophic methanogenesis. Activated carbon contributed to propionate oxidation as a thermodynamically favorable reaction with Gibbs free energy changes in the range of −310.9 and −399.4 kJ mol−1 and the available energy of propionate oxidation was 4.4 times higher than that with c-type cytochrome at a CO2 partial pressure of 0.4 bar. Conductive material with a low redox potential facilitated electrotrophic methanogenesis as a thermodynamically favorable reaction. The Gibbs free energy change of propionate oxidation increased after elevating CO2 partial pressure or electrons concentration but decreased with an increase in pH, temperature, or the propionate concentration.

Carbon-based conductive materials accelerated methane production in anaerobic digestion of waste fat, oil and grease

Little is known about the effect of carbon-based conductive material (CM) addition on the anaerobic co-digestion of fat, oil and grease (FOG) and waste activated sludge (WAS). In this study, three types of carbon-based CMs (nano-graphite (NG), granular activated carbon (GAC), and carbon cloth (CC)) and nine dosages were evaluated for their influences on co-digestion performance. The best dosage was achieved at 0.2 g/L NG, 10 g/L GAC, and 1 cm × 5 cm CC with 13–22% incremental methane production, 25–55% increased VS removal and 28–32% enhanced COD conversion efficiency compared to the control. The highest total amount of bacteria/archaea was found in CC (1 cm × 5 cm), followed by GAC at 10 g/L and NG at 0.2 g/L, which were all higher than those of the control. Microbial community analysis revealed that direct interspecies electron transfer (DIET)-mediated syntrophic acetate oxidation (SAO) enabling faster acetate conversion might be responsible for the enhancement of methane production.

Ferroferric oxide promotes metabolism in Anaerolineae other than microbial syntrophy in anaerobic methanogenesis of antibiotic fermentation residue

Antibiotic fermentation residue (AFR) is a kind of protein-rich biosolids that would be utilized as valuable substrates for biogas production. However, the effectiveness of conductive material supplementation and its effect on the microbiome in anaerobic digestion of AFR have not been fully elucidated. The objective of this study is to access the impact of Fe3O4 addition on anaerobic digestion of AFR and reveal its microbial mechanism. By adding Fe3O4 at concentration of 150 mg/gTS to two typical AFRs, the methane yield was decreased while the methane production rate was significantly enhanced by 48% and 21%, respectively. Genomic analyses revealed that the Fe3O4 addition altered the microbial community by selecting the unknown genus of Anaerolineae with the metabolic capacity of carbohydrate hydrolysis and proteolysis. Meanwhile, the hydrogenotrophic methanogenesis was the dominant pathway in the AFR digestion system, which was mostly contributed by the Methanoculleus and Methanolinea. More interestingly, Methanoculleus might possess fermentative capability involved in the versatile hydrolysis pathway. Although the genes related to electrically conductive pili were detected, the induction of direct interspecies electron transfer by Fe3O4 supplementation was not confirmed in this study. In summary, except the accelerated methane production rate, Fe3O4 addition functioned as biostimulator for Anaerolineae other than electron conduit in anaerobic methanogenesis in the AFR digestion system.

Microbial electrolysis cells (MEC) accelerated methane production from the enhanced hydrolysis and acidogenesis of raw waste activated sludge

The anaerobic digestion coupled with the microbial electrolysis cell (MEC-AD) is generally thought to accelerate methane production from the hydrolysate of biomass. This work studied the methanogenesis performance and response of functional microorganisms in MEC-AD feeding with raw waste activated sludge (rWAS) and heat pretreated waste activated sludge (hWAS), respectively. The results showed that the methane productivity of rWAS and hWAS were both substantially enhanced by applied voltage at 0.8 V, being 7.8 times and 2.1 times higher than that without voltage supply (the first open circuit, stage I). Surprisingly, when applied voltage was afterward decreased back to 0 V, the enhanced observation of hWAS almost gone, but the methane productivity of rWAS remained as high as 6.3 mL gVSSin−1d−1, which were still 6.2 times higher than that in the first open circuit. Different from the enhancement of methanogenesis on hWAS, the applied voltage in rWAS not only benefited to enrich the electricigens and methanogens, but also specially to enrich the fermentative bacteria and syntrophic acetogenic bacteria in both electrode biofilms. While this advantageous microbial structure in rWAS did not disappear with the removal of voltage supply except electricigens. Therefore, the enhanced hydrolysis-fermentation and synergy of acetogenic bacteria and hydrogenotrophic methanogens might be the main reason for keeping the high efficiency of methanogenesis with rWAS as substrate in MEC-AD. The findings reported in this study is economically and environmentally attractive, which might support high and stable methane production in traditional AD by enriching functional microorganisms with less energy input in the future.

Enhancement of syntrophic acetate oxidation pathway via single walled carbon nanotubes addition under high acetate concentration and thermophilic condition

The effect of single walled carbon nanotubes (SWCNT) on methane production under high acetate concentration and thermophilic condition was evaluated. An isotope labeling experiment verified that >85% of methane was generated from syntrophic acetate oxidation (SAO) at 50, 100 and 150 mM acetate and almost 100% at 200 mM. SWCNT addition had little effect on the methanogenesis pathway, whereas it accelerated methane production via decreasing lag phase times and increasing maximum methane production rates. Electrochemical impedance spectroscopy (EIS) results revealed the electrical resistivity of sludge in groups of SWCNT was distinctly smaller than CK groups, indicating higher sludge conductivity was achieved. Further, the results of communities described that Coprothermobacter and Thermacetogenium played the most important role in SAO under all conditions. Meanwhile, the enriched Thermacetogenium and direct interspecies electron transfer (DIET) pathway in SAO consortia contributed to the acceleration of methane production via SWCNT addition.

Enhancing biomethane production and pyrene biodegradation by addition of bio-nano FeS or magnetic carbon during sludge anaerobic digestion

ABSTRACT Pyrene exerts toxic effects on methanogens during anaerobic digestion of sludge, thus affecting the efficiency of sludge treatment. This study evaluated the facilitated direct interspecific electron transfer (DIET) between bacteria and methanogens when bio-nano FeS or magnetic carbon is added into anaerobic reactors. Results showed that adding 200 mg/L bio-nano FeS or magnetic carbon clearly reduced the accumulation of short-chain fatty acids and avoided acidification during 25 days of anaerobic digestion. The methane productions were 98.38 L/kg total solid (TS) and 73.69 L/kg TS in the bio-nano FeS and magnetic carbon systems, respectively, which accelerated methane production by 58.1% and 33.4%, respectively, compared with the control system (40.26 L/kg TS). The pyrene removal rates reached 77.5% and 72.1% in the bio-nano FeS and magnetic carbon systems, whereas it was only 40.8% in the control system. Analysis of microbial community structure revealed that methanogens (e.g. Methanosarcina and Methanosaeta) and extracellular electron-transfer bacteria (e.g. Pseudomonas, Cloastridia, and Synergistetes) were enriched in the reactors added with bio-nano FeS or magnetic carbon. This result indicates that the addition of bio-nano FeS or magnetic carbon may promote the activity and growth of microorganisms to improve the efficiency of methane production and pyrene degradation by enhancing DIET.

Anaerobic co-digestion of food waste and sewage sludge under mesophilic and thermophilic conditions: Focusing on synergistic effects on methane production

Globally, food waste (FW) and sewage sludge (SS) are among major organic wastes that needs to be properly handled. Anaerobic digestion (AD) has been a widely accepted method to treat those wastes and simultaneously recover energy and nutrients. The objective of this study was to elucidate synergistic effects in co-digestion of FW and SS by incubating at mesophilic and thermophilic conditions. The results demonstrated temperature did not significantly affect the cumulative methane yields (CMY); instead, higher temperature resulted in accelerated methane production. Although not clearly shown on CMYs (less than 10% increase), positive synergistic effects on methane production rates were characterized by a co-digestion impact factor (CIF) during the early phase of co-digestion. This early synergism (up to 24% increase) was primarily attributed to the accelerated hydrolysis due to addition of readily degradable FW, which may have practical implication for selection of retention time in order to optimize digestion process.

Comparative study of different thermal pretreatment techniques for accelerated methane production from rice straw

This study emphasizes the use of different thermal pretreatment methods (i.e., hot air oven, hot water bath, autoclave and microwave) on pretreatment of rice straw prior to anaerobic degradation. These pretreatment methods resulted in reasonably high solubilization of recalcitrant biomass. Maximum solublisation was attained by microwave (190 °C; exposure time-4 min) (66.6%) followed by autoclave, hot air oven and hot water bath methods. Accelerated cumulative methane yield of microwave pretreated sample achieved was 367.71 mL/g-VSadded, which was 160.68 mL/g-VSadded higher than the control. Combining this high temperature conditions with lesser exposure time also improved the energy balance of the process. The physicochemical characteristics of pretreated rice straw were investigated by Fourier transform infrared spectroscopy (FTIR), Field emission scanning electron microscopy (FESEM) and X-ray diffraction (XRD), FESEM analysis showed the increased porosity of the rice straw, FTIR analysis showed the omission of hemicellulose from the matrix and with XRD analysis reduced value of crystallinity index supports the destruction of the crystalline matrix.

Sludge-based biochar-assisted thermophilic anaerobic digestion of waste-activated sludge in microbial electrolysis cell for methane production

The development of microbial electrolysis cells (MECs) for methane production from waste activated sludge (WAS) is arrested due to the limited methane yield and fragile system stability. This study proposed a strategy to accelerate and stabilize MEC via 1.0 g/g DM (dry matter) sludge-based biochar (BC). The results showed that BC clearly accelerated methane production by 24.7% and enhanced VS removal efficiency by 17.9%, compared to control group. Variations of SCOD, proteins, carbohydrates and VFAs indicated biochar promoted hydrolysis and acidogenesis process. Cyclic voltammetry (CV) curves and coulombic efficiency (CE) suggested organic matters degradation and electron generation on anode were enhanced with supplement of biochar. Microbial community analyses revealed that biochar addition could both promote DIET through substituting exoelectrogen (e.g., Thermincola) on anode and enrich hydrogenotrophic methanogens (e.g., Methanothermobacter) on cathode, which is beneficial to development of MEC as to methane recovery from organic matters.

Effect and microbial reaction mechanism of rice straw biochar on pore methane production during mainstream large-scale aerobic composting in China

This study investigated the effect of rice straw biochar (RSB) on methane production during mainstream large-scale aerobic composting by using high-throughput sequencing and quantitative PCR. A 55-day large-scale aerobic composting experiment under cyclical forced-turning with aeration was conducted with control (CK) and experimental groups (BC), namely, without and with 10% RSB. The methanogen/methanotroph and microbial community of samples were analyzed by mcrA/pmoA and 16S rRNA/internal transcribed spacer region, respectively. The pore methane concentration had a positive relationship with mcrA and mcrA/pmoA. RSB accelerated methane production, which was reflected by an increase in mcrA. The diversity of bacteria and fungi increased by addition of RSB. Addition of RSB contributed to increasing the RAs of Methanobrevibacter and Methylobacterium. Methanobrevibacter was related to CH4 production, and Methylobacterium related to CH4 consumption through the metabolism of methyl material. Thus, the biochar has always increased the diversity of bacteria, including Methanobrevibacter and Methylobacterium. The optimal way to control CH4 production is to reduce the number of mcrA/pmoA. Meanwhile, RSB contributed to methane production because of its small particle size in this study. The biochar particle size could be further optimized during large-scale aerobic composting process.

Magnetite Triggering Enhanced Direct Interspecies Electron Transfer: A Scavenger for the Blockage of Electron Transfer in Anaerobic Digestion of High-Solids Sewage Sludge.

At present, high-solids anaerobic digestion of sewage sludge has drawn great attention due to the superiority of its small land area footprint and low energy consumption. However, a high organic loading rate may cause acids accumulation and ammonia inhibition, thus leading to an inhibited pseudo-steady state in which electron transfer through interspecies hydrogen transfer (IHT) between acetogens and methanogens is blocked. In this study, adding 50 mg/g TS (total solid) magnetite clearly reduced the accumulation of short-chain fatty acids and accelerated methane production by 26.6%. As demonstrated, the individual processes of anaerobic digestion could not be improved by magnetite when methanogenesis was interrupted. Analyzing stable carbon isotopes and investigating the methanogenesis pathways using acetate and H2/CO2 as substrates together proved that direct interspecies electron transfer (DIET) was enhanced by magnetite. Metatranscriptomic analysis and determination of key enzymes showed that IHT could be partially substituted by enhanced DIET, and acetate-dependent methanogenesis was improved after the blockage of electron transfer was scavenged. Additionally, the expression of both pili and c-type cytochromes was found to decrease, indicating that magnetite could replace their roles for efficient electron transfer between acetogens and methanogens; thus, a robust chain of electron transfer was established.