Comparative study of exoelectrogenic utilization preferences and hydrogen conversion among major fermentation products in microbial electrolysis cells.

This study comparatively investigated the exoelectrogenic utilization and hydrogen conversion of major dark fermentation products (acetate, propionate, butyrate, lactate, and ethanol) from organic wastes in dual-chamber microbial electrolysis cells (MECs) alongside their mixture as a simulated dark fermentation effluent (DFE). Acetate-fed MECs showed the highest hydrogen yield (1,465 mL/g chemical oxygen demand), near the theoretical maximum yield, with the highest coulombic efficiency (105%) and maximum current density (7.9 A/m2), followed by lactate-fed, propionate-fed, butyrate-fed, mixture-fed, and ethanol-fed MECs. Meanwhile, the highest hydrogen production rate (514 mL/L anolyte∙d) was observed in ethanol-fed MECs despite their lower coulombic efficiency. Butyrate was the least favored substrate, followed by propionate, leading to significantly delayed startup and reaction. The active anodic microbial community structure varied considerably among the MECs utilizing different substrates, particularly between Geobacter and Acetobacterium dominance. The results highlight the substantial effect of the DFE composition on its utilization and current-producing bioanode development.

A comprehensive analysis of key factors influencing methane production from CO2 using microbial methanogenesis cells.

With increasing attention on carbon capture and utilization (CCU) technologies for the conversion of CO2 into chemical products, microbial methanogenesis cells (MMCs) have been extensively studied over the past few decades for biomethane production. Using rapidly accumulating data for MMCs with varying configurations and operating conditions, a comprehensive analysis was conducted here to investigate the critical factors that influence methane production rates (MPR) in these systems. A comparison of MPR and set potentials or current densities showed weak linear relationships (R2 < 0.6, p < 0.05), indicating the significant contributions of other important factors impacting methane production. A non-quantitative analysis of these additional parameters indicated the potential importance of using metal catalysts for anode materials where oxygen evolution reaction occurs, while most previous MMC research focused more on cathode materials where the biocatalytic reaction occurs. The use of undefined mixed anaerobic cultures as inocula was found to be sufficient for producing high MPRs, as the electrochemical environment at the cathode provides a strong selective pressure to converge on desirable methanogenic cultures. Other operational parameters, such as catholyte pH control and CO2 supply methods, were also important factors impacting MPR in MMCs, indicating the cumulative impact of these various factors will require careful consideration in future research.

A study of electron source preference and its impact on hydrogen production in microbial electrolysis cells fed with synthetic fermentation effluent.

Fermentation effluents from organic wastes contain simple organic acids and ethanol, which are good electron sources for exoelectrogenic bacteria, and hence are considered a promising substrate for hydrogen production in microbial electrolysis cells (MECs). These fermentation products have different mechanisms and thermodynamics for their anaerobic oxidation, and therefore the composition of fermentation effluent significantly influences MEC performance. This study examined the microbial electrolysis of a synthetic fermentation effluent (containing acetate, propionate, butyrate, lactate, and ethanol) in two-chamber MECs fitted with either a proton exchange membrane (PEM) or an anion exchange membrane (AEM), with a focus on the utilization preference between the electron sources present in the effluent. Throughout the eight cycles of repeated batch operation with an applied voltage of 0.8 V, the AEM-MECs consistently outperformed the PEM-MECs in terms of organic removal, current generation, and hydrogen production. The highest hydrogen yield achieved for AEM-MECs was 1.26 L/g chemical oxygen demand (COD) fed (approximately 90% of the theoretical maximum), which was nearly double the yield for PEM-MECs (0.68 L/g COD fed). The superior performance of AEM-MECs was attributed to the greater pH imbalance and more acidic anodic pH in PEM-MECs (5.5-6.0), disrupting anodic respiration. Although butyrate is more thermodynamically favorable than propionate for anaerobic oxidation, butyrate was the least favored electron source, followed by propionate, in both AEM- and PEM-MECs, while ethanol and lactate were completely consumed. Further research is needed to better comprehend the preferences for different electron sources in fermentation effluents and enhance their microbial electrolysis.

Potential treatment of aged cow manure using spare capacity in anaerobic digesters treating a mixture of food waste and pig manure.

With the increasing production of cow manure (CM) and the continuing decrease in the demand for manure compost, CM management has become an urgent and challenging task in Korea. In most cattle farms in Korea, CM mixed with bedding materials is left in pens exposed to the open air for several months before treatment, which makes CM an unsuitable feedstock for anaerobic digestion. This study examined the co-digestion of aged CM with a mixture of food waste and pig manure as the base substrate to assess the possibility of treating and valorizing CM using spare capacity in existing anaerobic digesters dealing with other wastes. The duplicate digesters initially fed with the base substrate were subjected to the addition of increasing amounts of CM (3-10% in the feed, w/v) over nine months. Co-feeding CM up to 5% in the feed (w/v) did not compromise the methanogenic degradation of the substrates, but adding more CM led to a significant performance deterioration likely related to the buildup of inhibitory free ammonia and H2S. Adding CM substantially influenced the digester microbial communities, especially methanogenic communities, and induced a dominance shift from aceticlastic Methanothrix to hydrogenotrophic methanogens as the CM fraction increased. The overall results suggest that the CM fraction should not exceed 5% in the feed (w/v) for its stable treatment with the base substrate in the experimental digesters. Although further studies are needed, anaerobic treatment using spare capacity in existing digesters can be a useful strategy for the management of aged CM.

High-rate microbial electrosynthesis using a zero-gap flow cell and vapor-fed anode design.

Microbial electrosynthesis (MES) cells use renewable energy to convert carbon dioxide into valuable chemical products such as methane and acetate, but chemical production rates are low and pH changes can adversely impact biocathodes. To overcome these limitations, an MES reactor was designed with a zero-gap electrode configuration with a cation exchange membrane (CEM) to achieve a low internal resistance, and a vapor-fed electrode to minimize pH changes. Liquid catholyte was pumped through a carbon felt cathode inoculated with anaerobic digester sludge, with humidified N2 gas flowing over the abiotic anode (Ti or C with a Pt catalyst) to drive water splitting. The ohmic resistance was 2.4 ± 0.5 mΩ m2, substantially lower than previous bioelectrochemical systems (20-25 mΩ m2), and the catholyte pH remained near-neutral (6.6-7.2). The MES produced a high methane production rate of 2.9 ± 1.2 L/L-d (748 mmol/m2-d, 17.4 A/m2; Ti/Pt anode) at a relatively low applied voltage of 3.1 V. In addition, acetate was produced at a rate of 940 ± 250 mmol/m2-d with 180 ± 30 mmol/m2-d for propionate. The biocathode microbial community was dominated by the methanogens of the genus Methanobrevibacter, and the acetogen of the genus Clostridium sensu stricto 1. These results demonstrate the utility of this zero-gap cell and vapor-fed anode design for increasing rates of methane and chemical production in MES.

Pilot scale microbial fuel cells using air cathodes for producing electricity while treating wastewater.

Microbial fuel cells (MFCs) can generate electrical energy from the oxidation of the organic matter, but they must be demonstrated at large scales, treat real wastewaters, and show the required performance needed at a site to provide a path forward for this technology. Previous pilot-scale studies of MFC technology have relied on systems with aerated catholytes, which limited energy recovery due to the energy consumed by pumping air into the catholyte. In the present study, we developed, deployed, and tested an 850 L (1400 L total liquid volume) air-cathode MFC treating domestic-type wastewater at a centralized wastewater treatment facility. The wastewater was processed over a hydraulic retention time (HRT) of 12 h through a sequence of 17 brush anode modules (11 m2 total projected anode area) and 16 cathode modules, each constructed using two air-cathodes (0.6 m2 each, total cathode area of 20 m2) with the air side facing each other to allow passive air flow. The MFC effluent was further treated in a biofilter (BF) to decrease the organic matter content. The field test was conducted for over six months to fully characterize the electrochemical and wastewater treatment performance. Wastewater quality as well as electrical energy production were routinely monitored. The power produced over six months by the MFC averaged 0.46 ± 0.35 W (0.043 W m-2 normalized to the cross-sectional area of an anode) at a current of 1.54 ± 0.90 A with a coulombic efficiency of 9%. Approximately 49 ± 15 % of the chemical oxygen demand (COD) was removed in the MFC alone as well as a large amount of the biochemical oxygen demand (BOD5) (70%) and total suspended solid (TSS) (48%). In the combined MFC/BF process, up to 91 ± 6 % of the COD and 91 % of the BOD5 were removed as well as certain bacteria (E. coli, 98.9%; fecal coliforms, 99.1%). The average effluent concentration of nitrate was 1.6 ± 2.4 mg L-1, nitrite was 0.17 ± 0.24 mg L-1 and ammonia was 0.4 ± 1.0 mg L-1. The pilot scale reactor presented here is the largest air-cathode MFC ever tested, generating electrical power while treating wastewater.

Vapor-Fed Cathode Microbial Electrolysis Cells with Closely Spaced Electrodes Enables Greatly Improved Performance.

Hydrogen can be electrochemically produced in microbial electrolysis cells (MECs) by current generated from bacterial anodes with a small added voltage. MECs typically use a liquid catholyte containing a buffer or salts. However, anions in these catholytes result in charge being balanced predominantly by ions other than hydroxide or protons, leading to anode acidification. To enhance only hydroxide ion transport to the anode, we developed a novel vapor-fed MEC configuration lacking a catholyte with closely spaced electrodes and an anion exchange membrane to limit the acidification. This MEC design produced a record-high sustained current density of 43.1 ± 0.6 A/m2 and a H2 production rate of 72 ± 2 LH2/L-d (cell voltage of 0.79 ± 0.00 V). There was minimal impact on MEC performance of increased acetate concentrations, solution conductivity, or anolyte buffer capacity at applied voltages up to 1.1 V, as shown by a nearly constant internal resistance of only 6.8 ± 0.3 mΩ m2. At applied external voltages >1.1 V, the buffer capacity impacted performance, with current densities increasing from 28.5 ± 0.6 A/m2 (20 mM phosphate buffer solution (PBS)) to 51 ± 1 A/m2 (100 mM PBS). These results show that a vapor-fed MEC can produce higher and more stable performance than liquid-fed cathodes by enhancing transport of hydroxide ions to the anode.

The impact of different types of high surface area brush fibers with different electrical conductivity and biocompatibility on the rates of methane generation in anaerobic digestion.

The addition of electrically conductive materials may enhance anaerobic digestion (AD) efficiency by promoting direct interspecies electron transfer (DIET) between electroactive microorganisms, but an equivalent enhancement can also be achieved using non-conductive materials. Four high surface area brush materials were added to AD reactors: non-conductive horsehair (HB) and polyester (PB), and conductive carbon fiber (CB) and stainless steel (SB) brushes. Reactors with the polyester material showed lower methane production (68 ± 5 mL/g CODfed) than the other non-conductive material (horsehair) and the conductive (graphite or stainless steel) materials (83 ± 3 mL/g CODfed) (p < 0.05). This difference was due in part to the higher biomass concentrations using horsehair or carbon (135 ± 43 mg) than polyester or stainless steel or materials (26 ± 1 mg). A microbial community analysis indicated that the relative abundance of electroactive microorganisms was not directly related to enhanced AD performance. These results show that non-conductive materials such as horsehair can produce the same AD enhancement as conductive materials (carbon or stainless steel). However, if the material, such as polyester, does not have good biomass retention, it will not enhance methane production. Thus, electrical conductivity alone was not responsible for enhancing AD performance. Polyester, which has been frequently used as a non-conductive control material in DIET studies, should not be used for this purpose due to its poor biocompatibility as shown by low biomass retention in AD tests.

Designing a marine outfall to reduce microbial risk on a recreational beach: Field experiment and modeling.

A marine outfall can be a wastewater management system that discharges sewage and stormwater into the sea; hence, it is a source of microbial pollution on recreational beaches, including antibiotic resistant genes (ARGs), which lead to an increase in untreatable diseases. In this regard, a marine outfall must be efficiently located to mitigate these risks. This study aimed to 1) investigate the spatiotemporal variability of Escherichia coli (E. coli) and ARGs on a recreational beach and 2) design marine outfalls to reduce microbial risks. For this purpose, E. coli and ARGs with influential environmental variables were intensively monitored on Gwangalli beach, South Korea in this study. Environmental fluid dynamic code (EFDC) was used and calibrated using the monitoring data, and 12 outfall extension scenarios were explored (6 locations at 2 depths). The results revealed that repositioning the marine outfall can significantly reduce the concentrations of E. coli and ARGs on the beach by 46-99%. Offshore extended outfalls at the bottom of the sea reduced concentrations of E. coli and ARGs on the beach more effectively than onshore outfalls at the sea surface. These findings could be helpful in establishing microbial pollution management plans at recreational beaches in the future.

Addition of a carbon fiber brush improves anaerobic digestion compared to external voltage application.

Two methods were examined to improve methane production efficiency in anaerobic digestion (AD) based on adding a large amount of surface area using a single electrically conductive carbon brush, or by adding electrodes as done in microbial electrolysis cells (MECs) to form a hybrid AD-MEC. To examine the impact of surface area relative to electrodes, AD reactors were fitted with a single large brush without electrodes (FB), half a large brush with two electrodes with an applied voltage (0.8 V) and operated in closed circuit (HB-CC) or open circuit (HB-OC) mode, or only two electrodes with a closed circuit and no large brush (NB-CC) (equivalent to an MEC). The three configurations with a half or full brush all had improved performance as shown by 57-82% higher methane generation rate parameters in the Gompertz model compared to NB-CC. The retained biomass was much higher in the reactors with large brush, which likely contributed to the rapid consumption of volatile fatty acids (VFAs) and therefore improved AD performance. A different microbial community structure was formed in the large-size brushes compared to the electrodes. Methanothrix was predominant in the biofilm of large-size carbon brush, while Geobacter (anode) and Methanobacterium (cathode) were highly abundant in the electrode biofilms. These results demonstrate that adding a high surface area carbon fiber brush will be a more effective method of improving AD performance than using MEC electrodes with an applied voltage.

Treatment of Cattle Manure by Anaerobic Co-Digestion with Food Waste and Pig Manure: Methane Yield and Synergistic Effect.

The management of cattle manure (CM) has become increasingly challenging because its production continues to rise, while the regulations on manure management have become increasingly stringent. In Korea, most farms produce CM as a dry mixture with lignocellulosic bedding materials (mainly sawdust), making it impractical to treat CM by anaerobic digestion. To address this problem, this study examined whether anaerobic co-digestion with food waste (FW) and pig manure (PM) could be an effective approach for the treatment of CM. The batch anaerobic digestion tests at different CM: FW: PM mixing ratios showed that more methane was produced as the FW fraction increased, and as the CM fraction decreased. The response surface models describing how the substrate mixing ratio affects the methane yield and synergistic effect (methane yield basis) were successfully generated. The models proved that the methane yield and synergistic effect respond differently to changes in the substrate mixing ratio. The maximum 30-day methane yield was predicted at 100% FW, whereas the maximum 30-day synergy index was estimated for the mixture of 47% CM, 6% FW, and 47% PM (total solids basis). The synergy index model showed that CM, FW, and PM could be co-digested without a substantial loss of their methane potential at any mixing ratio (30-day synergy index, 0.89-1.22), and that a possible antagonistic effect could be avoided by keeping the FW proportion less than 50%. The results suggest that co-digestion with PM and FW could be flexibly applied for the treatment and valorization of CM in existing anaerobic digestion plants treating FW and PM.

Individual and combined effects of magnetite addition and external voltage application on anaerobic digestion of dairy wastewater.

Direct interspecies electron transfer (DIET) between exoelectrogenic fatty acid oxidizers and electrotrophic methanogens plays an important role in keeping the overall anaerobic digestion (AD) process well-balanced. This study examined the individual and combined effects of two different DIET-promoting strategies, i.e., magnetite addition (20 mM Fe) and external voltage application (0.6 V), in continuous digesters treating dairy wastewater. Although the strategies were both effective in enhancing the process performance and stability, adding magnetite had a much greater stimulatory effect. External voltage contributed little to the methane yield, and the digester with magnetite addition alone achieved stable performance, comparable to that of the digester where both strategies were combined, at short hydraulic retention times (down to 7.5 days). Diverse (putative) electroactive microorganisms were significantly enriched under DIET-promoting conditions, particularly with magnetite addition. The overall results suggest that magnetite addition could effectively enhance AD performance and stability by promoting DIET-based electro-syntrophic microbial interactions.

Development of biocathode during repeated cycles of bioelectrochemical conversion of carbon dioxide to methane.

Functioning biocathodes are essential for electromethanogenesis. This study investigated the development of a biocathode from non-acclimated anaerobic sludge in an electromethanogenesis cell at a cathode potential of -0.7V (vs. standard hydrogen electrode) over four cycles of repeated batch operations. The CO2-to-CH4 conversion rate increased (to 97.7%) while the length of the lag phase decreased as the number of cycles increased, suggesting that a functioning biocathode developed during the repeated subculturing cycles. CO2-resupply test results suggested that the biocathode catalyzed the formation of CH4 via both direct and indirect (H2-mediated) electron transfer mechanisms. The biocathode archaeal community was dominated by the genus Methanobacterium, and most archaeal sequences (>89%) were affiliated with Methanobacterium palustre. The bacterial community was dominated by putative electroactive bacteria, with Arcobacter, which is rarely observed in biocathodes, forming the largest population. These electroactive bacteria were likely involved in electron transfer between the cathode and the methanogens.

A long-term study on the effect of magnetite supplementation in continuous anaerobic digestion of dairy effluent - Magnetic separation and recycling of magnetite.

Promotion of direct interspecies electron transfer (DIET) between exoelectrogenic bacteria and electron-utilizing methanogens has recently been discussed as a new method for enhanced biomethanation. This study evaluated the effect of magnetite-promoted DIET in continuous anaerobic digestion of dairy effluent and tested the magnetic separation and recycling of magnetite to avoid continuous magnetite addition. The applied magnetite recycling method effectively supported enhanced DIET activity and biomethanation performance over a long period (>250days) without adding extra magnetite. DIET via magnetite particles as electrical conduits was likely the main mechanism for the enhanced biomethanation. Magnetite formed complex aggregate structures with microbes, and magnetite recycling also helped retain more biomass in the process. Methanosaeta was likely the major methanogen group responsible for DIET-based methanogenesis, in association with Proteobacteria and Chloroflexi populations as syntrophic partners. The recycling approach proved robust and effective, highlighting the potential of magnetite recycling for high-rate biomethanation.

Anaerobic co-digestion of spent coffee grounds with different waste feedstocks for biogas production.

Proper management of spent coffee grounds has become a challenging problem as the production of this waste residue has increased rapidly worldwide. This study investigated the feasibility of the anaerobic co-digestion of spent coffee ground with various organic wastes, i.e., food waste, Ulva, waste activated sludge, and whey, for biomethanation. The effect of co-digestion was evaluated for each tested co-substrate in batch biochemical methane potential tests by varying the substrate mixing ratio. Co-digestion with waste activated sludge had an apparent negative effect on both the yield and production rate of methane. Meanwhile, the other co-substrates enhanced the reaction rate while maintaining methane production at a comparable or higher level to that of the mono-digestion of spent coffee ground. The reaction rate increased with the proportion of co-substrates without a significant loss in methanation potential. These results suggest the potential to reduce the reaction time and thus the reactor capacity without compromising methane production.

A long-term study on the effect of magnetite supplementation in continuous anaerobic digestion of dairy effluent - Enhancement in process performance and stability.

Interspecies electron transfer (IET) between microbial populations with different functions is critical to stable anaerobic digestion. This study, in an attempt to facilitate IET, investigated the effect of magnetite supplementation on the biomethanation of dairy effluent in continuous mode. The magnetite-added reactor (RM) was significantly more resistant and resilient to process imbalance than the reactor run without magnetite addition (RC). RC showed unstable performance with repeated process upsets, but its performance improved to be comparable to that of RM after applying magnetite supplementation. Magnetite was particularly effective in stabilizing a build-up of propionic acid and therefore improving the process robustness and reliability. The enhanced biomethanation in terms of productivity and stability was attributed to the facilitated direct IET (DIET) between exoelectrogens and methanogens via magnetite particles. Methanosaeta was the predominant methanogen group in the experimental reactors and likely played a key role in both DIET-mediated carbon dioxide-reducing and aceticlastic methanogenesis.

Mild-temperature thermochemical pretreatment of green macroalgal biomass: Effects on solubilization, methanation, and microbial community structure.

The effects of mild-temperature thermochemical pretreatments with HCl or NaOH on the solubilization and biomethanation of Ulva biomass were assessed. Within the explored region (0-0.2M HCl/NaOH, 60-90°C), both methods were effective for solubilization (about 2-fold increase in the proportion of soluble organics), particularly under high-temperature and high-chemical-dose conditions. However, increased solubilization was not translated into enhanced biogas production for both methods. Response surface analysis statistically revealed that HCl or NaOH addition enhances the solubilization degree while adversely affects the methanation. The thermal-only treatment at the upper-limit temperature (90°C) was estimated to maximize the biogas production for both methods, suggesting limited potential of HCl/NaOH treatment for enhanced Ulva biomethanation. Compared to HCl, NaOH had much stronger positive and negative effects on the solubilization and methanation, respectively. Methanosaeta was likely the dominant methanogen group in all trials. Bacterial community structure varied among the trials according primarily to HCl/NaOH addition.

Bioaugmentation of anaerobic sludge digestion with iron-reducing bacteria: process and microbial responses to variations in hydraulic retention time.

Although anaerobic digestion (AD) is a widely used option to manage waste activated sludge (WAS), there are some drawbacks related to its slow reaction rate and low energy productivity. This study examined an anaerobic WAS digester, augmented with an iron-reducing microbial consortium, relative to changes in microbial community structure and process performance at decreasing hydraulic retention times (HRTs) of 20 to 10 days. The enhanced methanation performance (approximately 40 % increase in methane yield) by the bioaugmentation was sustained until the HRT was decreased to 12.5 days, under Fe(3+)-rich conditions (ferric oxyhydroxide, 20 mM Fe). Enhanced iron-reducing activity was evidenced by the increased Fe(2+) to total Fe ratio maintained above 50 % during the stable operational phases. A further decrease in HRT to 10 days resulted in a significant performance deterioration, along with a drop in the Fe(2+) to total Fe ratio to <35 %, after four turnovers of operation. Prevailing existence of putative iron-reducing bacteria (IRBs) was identified by denaturing gradient gel electrophoresis (DGGE), with Spirochaetaceae- and Thauera-related organisms being dominant members, and clear dominance shifts among them with respect to decrease in HRT were observed. Lowering HRT led to evident shifts in bacterial community structure likely associated with washout of IRBs, leading to decreases in iron respiration activity and AD performance at a lower HRT. The bacterial community structure shifted dynamically over phases, and the community transitions correlated well with the changes in process performance. Overall, the combined biostimulation and bioaugmentation investigated in this study proved effective for enhanced methane recovery from anaerobic WAS digestion, which suggests an interesting potential for high-rate AD.

The biostimulation of anaerobic digestion with (semi)conductive ferric oxides: their potential for enhanced biomethanation.

The effect of biostimulation with ferric oxides, semiconductive ferric oxyhydroxide, and conductive magnetite on the anaerobic digestion of dairy wastewater was examined in a batch mode. The reactors supplemented with ferric oxyhydroxide (R2) and magnetite (R3) showed significantly enhanced biomethanation performance compared with the control (R1). The removal of chemical oxygen demand (COD) after 30 days was 31.9, 59.3, and 82.5% in R1, R2, and R3, respectively. The consumed COD was almost fully recovered as biogas in R2 and R3, while only 79% was recovered in R1. The total energy production as biogas was accordingly 32.2, 71.0, and 97.7 kJ in R1, R2, and R3, respectively. The reactors also differed in the acid formation profile with more propionate and butyrate found in R1 and more acetate found in R3. The enhanced biomethanation seems to be associated with variations in the bacterial community structure supposedly induced by the ferric oxides added. In contrast, no evident variation was observed in the archaeal community structure among the reactors. The potential electric syntrophy formed between Methanosaeta concilii-like methanogens and electroactive iron-reducing bacteria, particularly Trichococcus, was likely responsible for the enhanced performance. The stimulated growth of fermentative iron reducers may also have contributed by altering the metabolic characteristics of the bacterial communities to produce more favorable acidogenic products for methanogenesis. The overall results suggest the potential of biostimulation with (semi)conductive ferric oxides to enhance the rate and efficiency of the biomethanation of organic wastes. This seems to be potentially attractive, as increasing attention is being paid to the energy self-sufficiency of waste/wastewater treatment processes today.

Influence of ferric oxyhydroxide addition on biomethanation of waste activated sludge in a continuous reactor.

This study investigated the potential of enhancing the activity of iron-reducing bacteria (IRBs) to increase the biomethanation rate of waste activate sludge (WAS). The effects of biostimulation by ferric oxyhydroxide (Phase 2) and bioaugmentation with an enriched IRB consortium (Phase 3) were examined in a continuous anaerobic reactor treating WAS. Compared to the control operation (Phase 1), significant rises in methane yield (10.8-59.4%) and production rate (24.5-52.9%) were demonstrated by the biostimulation and bioaugmentation treatments. Visible structural changes were observed in bacterial community with the phases while not in archaeal community. Acinetobacter- and Spirochaetales-related populations were likely the major players driving anaerobic iron respiration and thus leading to enhanced biomethanation performance, in Phases 2 and 3, respectively. Our results suggest an interesting new potential for enhancing biomethanation of WAS.