Biochar alleviates inhibition effects of humic acid on anaerobic digestion: Insights to performances and mechanisms.

To recover methane from waste activated sludge through anaerobic digestion (AD) is one promising alternative to achieve carbon neutrality for wastewater treatment plants. However, humic acids (HAs) are one of the major compositions in waste activated sludge, and their accumulation performs inhibition effects on AD. This study investigated the potentials of biochar (BC) in alleviating inhibition effects of HAs on AD. Results showed that although the accumulated HAs reduced methane yield by 9.37% compared to control, the highest methane yield, 132.6 mL CH4/g VSS, was obtained after adding BC, which was 45.9% higher than that in HA group. Mechanism analysis showed that BC promoted the activities of hydrolase such as protease and α-glucosidase, which were 69.7% and 29.7% higher than those in HA group, respectively. The conversion of short-chain fatty acids was accelerated. In addition, the evolution of electroactive microorganisms like Clostridium_sensu_stricto_13 and Methanosaeta were consistent with the activitiy of electron transfer and the content of cytochrome c. Furthermore, parts of HAs rather than all of them were adsorbed by BC, and the remaining free HAs and BC formed synergistic effects on methanogenesis, then both CO2 reduction and acetoclastic methanogenesis pathways were improved. The findings may provide some solutions to alleviate inhibition effects of HAs on AD.

Role of phosphate in microalgal-bacterial symbiosis system treating wastewater containing heavy metals.

Phosphorus is one of the important factors to successfully establish the microalgal-bacterial symbiosis (MABS) system. The migration and transformation of phosphorus can occur in various ways, and the effects of phosphate on the MABS system facing environmental impacts like heavy metal stress are often ignored. This study investigated the roles of phosphate on the response of the MABS system to zinc ion (Zn2+). The results showed that the pollutant removal effect in the MABS system was significantly reduced, and microbial growth and activity were inhibited with the presence of Zn2+. When phosphate and Zn2+ coexisted, the inhibition effects of pollutants removal and microbial growth rate were mitigated compared to that of only with the presence of Zn2+, with the increasing rates of 28.3% for total nitrogen removal, 48.9% for chemical oxygen demand removal, 78.3% for chlorophyll-a concentration, and 13.3% for volatile suspended solids concentration. When phosphate was subsequently supplemented in the MABS system after adding Zn2+, both pollutants removal efficiency and microbial growth and activity were not recovered. Thus, the inhibition effect of Zn2+ on the MABS system was irreversible. Further analysis showed that Zn2+ preferentially combined with phosphate could form chemical precipitate, which reduced the fixation of MABS system for Zn2+ through extracellular adsorption and intracellular uptake. Under Zn2+ stress, the succession of microbial communities occurred, and Parachlorella was more tolerant to Zn2+. This study revealed the comprehensive response mechanism of the co-effects of phosphate and Zn2+ on the MABS system, and provided some insights for the MABS system treating wastewater containing heavy metals, as well as migration and transformation of heavy metals in aquatic ecosystems.

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

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

Advances, challenges, and prospects in microalgal-bacterial symbiosis system treating heavy metal wastewater.

Heavy metal (HM) pollution, particularly in its ionic form in water bodies, is a chronic issue threatening environmental security and human health. The microalgal-bacterial symbiosis (MABS) system, as the basis of water ecosystems, has the potential to treat HM wastewater in a sustainable manner, with the advantages of environmental friendliness and carbon sequestration. However, the differences between laboratory studies and engineering practices, including the complexity of pollutant compositions and extreme environmental conditions, limit the applications of the MABS system. Additionally, the biomass from the MABS system containing HMs requires further disposal or recycling. This review summarized the recent advances of the MABS system treating HM wastewater, including key mechanisms, influence factors related to HM removal, and the tolerance threshold values of the MABS system to HM toxicity. Furthermore, the challenges and prospects of the MABS system in treating actual HM wastewater are analyzed and discussed, and suggestions for biochar preparation from the MABS biomass containing HMs are provided. This review provides a reference point for the MABS system treating HM wastewater and the corresponding challenges faced by future engineering practices.

Contribution identification of hydrolyzed products of potassium ferrate on promoting short-chain fatty acids production from waste activated sludge.

Potassium ferrate (K2FeO4) has been extensively employed to promote short-chain fatty acids (SCFAs) production from anaerobic fermentation of waste activated sludge (WAS) because of its potent oxidizing property and formation of alkaline hydrolyzed products (potassium hydroxide, KOH and ferric hydroxide, Fe(OH)3). However, whether K2FeO4 actually works as dual functions of both an oxidizing agent and an alkalinity enhancer during the anaerobic fermentation process remains uncertain. This study aims to identify the contributions of hydrolyzed products of K2FeO4 on SCFAs production. The results showed that K2FeO4 did not execute dual functions of oxidization and alkalinity in promoting SCFAs production. The accumulation of SCFAs using K2FeO4 treatment (183 mg COD/g volatile suspended solids, VSS) was less than that using either KOH (192 mg COD/g VSS) or KOH & Fe(OH)3 (210 mg COD/g VSS). The mechanism analysis indicated that the synergistic effects caused by oxidization and alkalinity properties of K2FeO4 did not happen on solubilization, hydrolysis, and acidogenesis stages, and the inhibition effect caused by K2FeO4 on methanogenesis stage at the initial phase was more severe than that of its hydrolyzed products. It was also noted that the inhibition effects of K2FeO4 and its hydrolyzed products on the methanogenesis stage could be relieved during a longer sludge retention time, and the final methane yields using KOH or KOH & Fe(OH)3 treatment were higher than that using K2FeO4, further confirming that dual functions of K2FeO4 were not obtained. Therefore, K2FeO4 may not be an alternative strategy for enhancing the production of SCFAs from WAS compared to its alkaline hydrolyzed products. Regarding the strong oxidization property of K2FeO4, more attention could be turned to the fates of refractory organics in the anaerobic fermentation of WAS.

Revealing mechanisms of triclosan on the removal and distribution of nitrogen and phosphorus in microalgal-bacterial symbiosis system.

Microalgal-bacterial symbiosis (MABS) system performs synergistic effect on the reduction of nutrients and carbon emissions in the water treatment process. However, antimicrobial agents are frequently detected in water, which influence the performance of MABS system. In this study, triclosan (TCS) was selected to reveal the effects and mechanisms of antimicrobial agents on MABS system. Results showed that the removal efficiencies of chemical oxygen demand, NH4+-N and total phosphorus decreased by 3.0%, 24.0% and 14.3% under TCS stress. In contrast, there were no significant decrease on the removal effect of total nitrogen. Mechanism analysis showed that both the growth rate of microorganisms and the nutrients retention capacity of extracellular polymeric substances were decreased. The intracellular accumulation for nitrogen and phosphorus was promoted due to the increased cytomembrane permeability caused by lipid peroxidation. Moreover, microalgae were dominant in MABS system with ratio between microalgae and bacteria of more than 5.49. The main genus was Parachlorella, with abundance of more than 90%. Parachlorella was highly tolerant to TCS, which might be conductive to maintain its survival. This study revealed the nutrients pathways of MABS system under TCS stress, and helped to optimize the operation of MABS system.

Natural zeolite enhances anaerobic digestion of waste activated sludge: Insights into the performance and the role of biofilm.

Anaerobic digestion is widely employed for the treatment of waste activated sludge (WAS) due to its advantages like simultaneous energy recovery and sludge stabilization, promoting carbon-neutral operation of wastewater treatment plants. Natural zeolite, a low-cost and eco-friendly additive, has the potential to improve methane production from anaerobic digestion. This study investigated the effects of natural zeolite on anaerobic digestion when the substrate was WAS. It was found that methane production potential in response to natural zeolite was dosage-dependent. The optimal dosage was 0.1 g zeolite/g volatile suspended solids (VSS), with a methane yield of 181.89 ± 6.75 mL/g VSS, which increased by 20.1% compared to that of the control. Although the methane yields with other dosages of natural zeolite were higher than that of control, they were lesser than that with 0.1 g zeolite/g VSS. Natural zeolite affected transfer and conversion of proteins much more than polysaccharides in liquid phase and extracellular polymeric substances. In anaerobic digestion, natural zeolite had with little effects on WAS solubilization, while it improved hydrolysis, acidification, and methanogenesis. The dosages of natural zeolite did have significant effects on bacterial communities in biofilm rather than suspension, while the archaeal communities in biofilm and suspension were all greatly related to natural zeolite dosages. The developed biofilms promoted richness and functionality of microbial communities. The syntrophic metabolism relationships between methanogens and bacteria were improved, which was proved by selective enrichment of Methanosarcina, Syntrophomonas, and Petrimonas. The findings of this work provided some new solutions for promoting methane production from WAS, and the roles of natural zeolite in anaerobic digestion.

Insights into the roles and mechanisms of a green-prepared magnetic biochar in anaerobic digestion of waste activated sludge.

Methane is one of the most promising renewable energies to alleviate energy crisis or replace fossil fuels, which can be recovered from anaerobic digestion of bio-wastes. However, the engineering application of anaerobic digestion is always hindered by low methane yield and production rate. This study revealed the roles and mechanisms of a green-prepared magnetic biochar (MBC) in promoting methane production performance from waste activated sludge. Results showed that the methane yield reached 208.7 mL/g volatile suspended solids with MBC additive dosage of 1 g/L, increasing by 22.1 % compared to that in control. Mechanism analysis demonstrated that MBC could promote hydrolysis, acidification, and methanogenesis stages. This was because the properties of biochar were upgraded by loading nano-magnetite, such as specific surface area, surface active sites, and surface functional groups, which made MBC have greater potential to mediate electron transfer. Correspondingly, the activity of α-glucosidase and protease respectively increased by 41.7 % and 50.0 %, and then the hydrolysis performances of polysaccharides and proteins were improved. Also, MBC improved the secretion of electroactive substances like humic substances and cytochrome C, which could promote extracellular electron transfer. Furthermore, Clostridium and Methanosarcina, as well-known electroactive microbes, were selectively enriched. The direct interspecies electron transfer between them was established via MBC. This study provided some scientific evidences to comprehensively understand the roles of MBC in anaerobic digestion, with important implications for achieving resource recovery and sludge stabilization.

Insights into response mechanism of anaerobic digestion of waste activated sludge to particle sizes of zeolite.

Anaerobic digestion has been proved as one promising strategy to simultaneously achieve resource recovery and environmental pollution control for biosolid treatment, and adding exogenous materials is a potential alternative to promote the above process. This study investigated response mechanisms of anaerobic digestion of waste activated sludge (WAS) to particle sizes of zeolite. Results showed that the methane production reached 186.75 ± 7.62 mL/g volatile suspended solids (VSS) with zeolite of the particle size of 0.2-0.5 mm and the additive dosage of 0.1 g/g VSS, which increased by 22.08% compared to that in control. Mechanism study revealed that zeolite could improve hydrolysis, acidification, and methanogenesis stages. Rapid consumption rates of soluble polysaccharides and proteins were observed, correspondingly, the accumulations of short-chain fatty acids (SCFAs) were enhanced, and the compositions of SCFAs were optimized. Moreover, the activities of F420 increased by 28% with zeolite, and the syntrophic metabolism between bacteria and methanogens were promoted.

Roles of high/low nucleic acid bacteria in flocs and probing their dynamic migrations with respirogram.

Bacterial migration is crucial for the stability of activated sludge but rarely reported. The static distribution was explored by changes in bacteria concentration with extracellular polymeric substances (EPS) extractions. Next, denitrification and aeration were conducted as normal running conditions for examining the bacterial migration between floc-attached and dispersed growth. Above observations were further explored by conducting copper ion (Cu2+) shock as an extreme running condition. After extracting EPS, low nucleic acid (LNA) bacteria migrated from the sludge to the supernatant primarily, and high nucleic acid (HNA) bacteria remained in the residual sludge, suggesting that HNA bacteria mainly distributed inside the sludge while LNA bacteria outside the sludge. During the denitrification process, LNA bacteria migrated out of flocs, which increased by 6.94 × 106 events/mL in the supernatant. During the feast phase of aeration, LNA bacteria grew attached to flocs, causing the increased flocs diameter from 45.60 to 47.40 µm. During the following aerobic famine phase, LNA bacteria grew dispersedly, but HNA bacteria remained unchanged. However, a further severe famine phase drove HNA bacteria to be dispersed, breaking flocs with the decreased diameter from 48.10 to 46.50 µm. When the Cu2+ shock was employed, LNA and HNA bacteria increased but the LNA/HNA ratio decreased in the supernatant, indicating more HNA bacteria migrating to the dispersed phase. From a structural perspective, HNA bacteria distributed inside the sludge and functioned as the backbone of flocs, undertaking the maintenance of flocs stability primarily; while LNA bacteria distributed outside the sludge and functioned as filling materials, having a secondary influence on flocs stability. These processes were also probed by respirogram exactly, correlating the system-scale measurement and microscale migrations and providing an early warning signal under abnormal circumstances. The processed HNA-backbone theory is promising for regulating the stability of activated sludge based on bacterial migrations.

Insights into roles of triclosan in microalgal-bacterial symbiosis system treating wastewater.

Triclosan (TCS) is an antimicrobial agent and frequently detected in wastewater or water body. This study investigated the role of TCS in microalgal-bacterial symbiosis (MABS) system treating wastewater. The results showed that the removal efficiencies of NH4+-N, total nitrogen, and total phosphorus decreased under increased TCS stress, with decrease ratios of 26.5%, 16.9%, and 34.7%. The activities of microalgae were more affected than that of bacteria. The secretion of extracellular polymeric substances (EPSs) and activity of superoxide dismutase firstly increased and then decreased with aggravated TCS stress, while the accumulation of malondialdehyde increased, leading to increased permeability of cytomembrane and bioaccumulation of TCS. In addition, the aggregation properties of microalgae and bacteria were enhanced with TCS loading increasing, and the migration of TCS was affected by enhanced EPSs secretions and MABS aggregates. This work may provide some new insights into the roles of TCS in MABS system.

Reducing the inhibitive effect of fluorine and heavy metals on nitrate reduction by hydroxyapatite substrate in constructed wetlands.

Bio-toxic inorganic pollutants, e.g., fluorine (F) and heavy metals (HMs), in wastewaters are the potential threats to nitrate (NO3--N) reduction by microorganisms in constructed wetlands (CWs). Selection of suitable substrate with high F and HMs adsorption efficiency and capacity is a potential alternative for simultaneous removal of these pollutants in CWs. Herein, this study investigated the feasibility of applying hydroxyapatite (HA)-gravel media for F and HMs adsorption and its effect on NO3--N reduction in CWs (HA CWs) by comparing the CWs filled with gravel substrate (CK CWs). The results indicated that the removal efficiency of F, Cr, As, and NO3--N in HA CWs increased by 113.6-, 3.3-, 2.7-, and 0.6-folds, respectively, compared to CK CWs. The NO3--N reduction rate decreased by 11-46% in CK CWs after the presence of F and HMs in influent, while for HA CWs, it was only 13-22%. Excellent F and HMs adsorption capacity of HA substrate availed for wetland plants resisting F/HMs toxicity and making catalase activity lower. The HA substrate in CWs resulted in the certain succession of nitrogen-transforming bacteria, e.g., nitrifiers (Nitrospira) and denitrifiers (Thiobacillus and Desulfobacterium). More importantly, key functional genes, including nirK/nirS, korA/korB, ChrA/ChrD, arsA/arsB, catalyzing the processes of nitrogen biotransformation, energy metabolism, NO3--N and metal ions reduction were also enriched in HA CWs. This study highlights HA substrate reduce the inhibitive effect of F and HMs on NO3--N reduction, and provides new insights into how microbiota structurally and functionally respond to different substrates in CWs.

Roles of zero-valent iron in anaerobic digestion: Mechanisms, advances and perspectives.

With the rapid growth of population and urbanization, more and more bio-wastes have been produced. Considering organics contained in bio-wastes, to recover resource from bio-wastes is of great significance, which can not only achieve the resource recycle, but also protect the environment. Anaerobic digestion (AD) has been proved as one of the most promising strategies to recover bio-energy from bio-wastes, as well as to realize the reduction of bio-wastes. However, the conventional interspecies electron transfer is sensitive to environmental shocks, such as high ammonia, organic pollutants, metal ions, etc., which lead to instability or failure of AD. The recent findings have proved that the introduction of zero-valent iron (ZVI) in AD system can significantly enhance methane production from bio-wastes. This review systematically highlighted the recent advances on the roles of ZVI in AD, including underlying mechanisms of ZVI on AD, performance enhancement of AD contributed by ZVI, and impact factors of AD regulated by ZVI. Furthermore, current limitations and outlooks have been analyzed and concluded. The roles of ZVI on underlying mechanisms in AD include regulating reaction conditions, electron transfer mode and function of microbial communities. The addition of ZVI in AD can not only enhance bio-energy recovery and toxic contaminants removal from bio-wastes, but also have the potential to buffer adverse effect caused by inhibitors. Moreover, the electron transfer modes induced by ZVI include both interspecies hydrogen transfer and direct interspecies electron transfer pathways. How to comprehensively evaluate the effects of ZVI on AD and further improve the roles of ZVI in AD is urgently needed for practical application of ZVI in AD. This review aims to provide some references for the introduction of ZVI in AD for enhancing bio-energy recovery from bio-wastes.

Response of anaerobic digestion of waste activated sludge to types of alkalis: Contribution identification of metal ions.

Alkaline pretreatment is one promising strategy for promoting anaerobic digestion of waste activated sludge (WAS). This study selected three types of alkalis with monovalent (NaOH and KOH), divalent (Ca(OH)2 and Mg(OH)2), and trivalent (Fe(OH)3 and Al(OH)3) cations to reveal the roles of metal ions on short chain fatty acids (SCFAs) production. The enhanced production potentials of SCFAs were reduced by order of alkalis with monovalent, divalent, and trivalent cations. Na+, K+, Ca2+, and Mg2+ did no contributions on SCFAs production, while Fe3+ and Al3+ performed better than control, especially the latter. The mechanism analysis proved that Na+, K+, Ca2+, and Mg2+ did no significant effects on solubilization, hydrolysis, acidification and methanogenesis stages, while the first three stages were improved by Fe3+ and Al3+ and the methanogenesis stage was inhibited. The findings may provide some new insights when using alkalis or residual metal ions to improve anaerobic digestion of WAS.

Role of extracellular polymeric substances in methane production from waste activated sludge induced by conductive materials.

Conductive materials have been widely used to establish direct interspecies electron transfer (DIET) for enhancing methane production potential from anaerobic digestion of waste activated sludge (WAS). However, the roles of extracellular polymeric substances (EPSs) affected by conductive materials on anaerobic digestion have been rarely reported. This study selected four widely used conductive materials, i.e., granular active carbon (GAC), biochar (BC), zero-valent iron (ZVI), and magnetite (Mag), to reveal the roles of EPSs. Results showed that methane production potentials were increased by BC, ZVI and Mag compared to that of control, with increase ratios of 13.4 %, 22.2 %, and 12.2 %, while a decrease was observed by GAC. The contents, components and characteristics of EPSs were all affected by conductive materials. The contents of EPSs were increased by ZVI and Mag, while they were decreased by BC and GAC. The ratios between proteins and polysaccharides (PN/PS) in loosely bound EPSs (LB-EPSs) were reduced in all groups, while they were similar in tightly bound EPSs (TB-EPSs) of ZVI and Mag groups. In addition, the cytochrome C and redox properties were remarkably promoted in suspension rather than in LB- and TB-EPSs. It was found that the correlation relationships between the maximal methane production potential (Pmax) and PN/PS in EPSs were positive, as well as fluorescent substances, especially tyrosine-like and tryptophan-like substances, with R2 of 0.96 and 0.98. Furthermore, the correlation relationships also existed between EPSs and microbial communities. Clostridium and Methanobacterium, potential DIET partners, presented significant positive correlation relationships (P < 0.05) with Pmax, PN/PS and fluorescent substances in EPSs. The findings may provide some new insights for mechanism investigation of anaerobic digestion induced by conductive materials.

Role of types and dosages of cations with low valance states on microalgal-bacterial symbiosis system treating wastewater.

This study investigated the roles of cations with low valance states, including Mg2+, K+ and Li+, on microalgal-bacterial symbiosis (MABS) system treating wastewater. Results showed that Mg2+ and K+ improved pollutants removal at dosages of less than 1 mM, and a further increase led to poorer performances. Conversely, Li+ inhibited pollutants removal. Mechanism study indicated Mg2+ and K+ with dosages of 10 mM and Li + inhibited the activities of MABS biomass (especially Chlorella), with bad absorption efficiencies of 20.64 %, 13.65 % and lower than 10 %, leading to more extracellular polymeric substances production. Larger ions' charge density resulted in larger attraction of water molecules, contributing to the decreased distance between microalgae cells and increased biomass aggregation. Both these two impacts led to the order of impact degree on MABS aggregates: Mg2+ > Li+ > K+. The findings can present some new perspectives on assessing effects of cations on MABS system.

Chronic effects of benzalkonium chlorides on short chain fatty acids and methane production in semi-continuous anaerobic digestion of waste activated sludge.

As an emerging pollutant, benzalkonium chlorides (BACs) potentially enriched in waste activated sludge (WAS). However, the microbial response mechanism under chronic effects of BACs on acidogenesis and methanogenesis in anaerobic digestion (AD) has not been clearly disclosed. This study investigated the AD (by-)products and microbial evolution under low to high BACs concentrations from bioreactor startup to steady running. It was found that BACs can lead to an increase of WAS hydrolysis and fermentation, but a disturbance to acidogenic bacteria also occurred at low BACs concentration. A noticeable inhibition to methanogenesis occurred when BAC concentration was up to 15 mg/g TSS. Metagenomic analysis revealed the key genes involved in acetic acid (HAc) biosynthesis (i.e. phosphate acetyltransferase, PTA), ß-oxidation pathway (acetyl-CoA C-acetyltransferase) and propionic acid (HPr) conversion was slightly promoted compared with control. Furthermore, BACs inhibited the acetotrophic methanogenesis (i.e. acetyl-CoA synthetase), especially BAC concentration was up to 15 mg/g TSS, thereby enhanced short chain fatty acids (SCFAs) accumulation. Overall, chronic stimulation of functional microorganisms with increasing concentrations of BACs impact WAS fermentation.

Potassium ferrate followed by alkali-stripping treatment to achieve short-chain fatty acids and nitrogen recovery from waste activated sludge.

Waste activated sludge (WAS) is a potential resource to achieve carbon-neutral goal of wastewater treatment plant. However, the solubilization is always the rate-limiting step for resource recovery from anaerobic digestion of WAS. This study reported a novel strategy, i.e., potassium ferrate (PF) followed by alkali-stripping treatment, to achieve short-chain fatty acids (SCFAs) and nitrogen recovery from WAS. Results showed that whether the stripping process was conducted under alkaline condition or not, the SCFAs production potential was increased rather than reduced. The promoted SCFAs production was due to the accelerated solubilization and hydrolysis stages but the inhibited methanogenesis stage. The SCFAs yield reached 258 mg chemical oxygen demand (COD)/g volatile suspended solids (VSS), and the carbon source, including SCFAs, soluble polysaccharides and proteins, reached 384 mg COD/g VSS. The potentially recovered nitrogen was about 8.71 mg NH4+-N/g VSS. This work may provide some new solutions for enhancing resource recovery from WAS.

Role and significance of water and acid washing on biochar for regulating methane production from waste activated sludge.

Methane recovered from anaerobic digestion of waste activated sludge (WAS) can be used as the energy supplement of the wastewater treatment plant, benefiting to its carbon-neutral operation. In order to enhance methane production, biochar (BC) has been widely selected as conductive material to build direct interspecies electron transfer (DIET) in anaerobic digestion of WAS. However, the role and significance of washing strategies, including water and acid washing, on BCs for regulating methane production have not been reported. This study selected the frequently used woody- (W) and straw (S)-BCs as mode. Compared to raw W-BC, water and acid washing W-BC increased the methane yields by 19.1% and 15.7%, respectively. Differently, the methane yields among raw, water and acid washing S-BCs were similar. Mechanism study showed that both the two washing strategies optimized the properties of raw W-BC for promoting methane production. Water and acid washing W-BCs increased the electron transfer functional groups, such as ketones and quinones, which were not observed in S-BCs. Moreover, the electron-active microorganisms were enriched with the presence of water and acid washing W-BCs, and the predominant pathway for methane production shifted from hydrogentrophic to acetotrophic and DIET methanogenesis, while the microbial communities, including bacteria and archaea, were similar with the presence of raw, water and acid washing S-BCs. These findings of this work provide some new insights for production improvement regulation of methane from anaerobic digestion of wastes induced by BCs.