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

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

Bioresource Technology 284 (2019) 315–324 Contents lists available at ScienceDirect Bioresource Technology journal homepage:

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Bioresource Technology 284 (2019) 315–324

Contents lists available at ScienceDirect

Bioresource Technology journal homepage:

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


Changkai Yina, Yanwen Shena, Rongxue Yuana, Nanwen Zhua,b, , Haiping Yuana, Ziyang Loua a b

School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China Shanghai Institute of Pollution Control and Ecological Security, Shanghai 200092, PR China




Keywords: Sludge Anaerobic digestion Microbial electrolysis cells Biochar Electron transfer

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.

1. Introduction Waste activated sludge (WAS) is increasingly produced during wastewater biological treatment process (Raheem et al., 2018; Sun et al., 2018). Improper use and disposal of sewage sludge causes severe environmental impacts and health hazard to the public (Appels et al., 2008; Raheem et al., 2018). On the other hand, WAS contains high

concentration of organic matters and thus is treated as renewable bioenergy resource (Zhao et al., 2016b). The reutilization of WAS through anaerobic digestion (AD) is considered to be a preferable technology, which achieve both the reduction of sludge and the production of valuable products (e.g., biogas and short chain fatty acids) (Appels et al., 2008; Zhao et al., 2016b). With increasing energy supply concerns, strategies to enhance yield from AD of sludge are gaining

Corresponding author. E-mail address: [email protected] (N. Zhu). Received 5 March 2019; Received in revised form 28 March 2019; Accepted 29 March 2019 Available online 30 March 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.

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much attention. Microbial electrolysis cells (MECs) has been displayed to be an effective option for energy recovery in AD. The enhancement of methane production in MEC attribute anodic and cathodic reaction, as presented in Eqs. (1) and (2) (Chen et al., 2016; Lu and Ren, 2016). The supply of voltage in cathode facilitate hydrogen generation, while methanogenic archaea utilizes hydrogen to form methane. It has been proven that optimal external voltage for specific substrates in a MEC-AD system is crucial for achieving high rates of removal of pollutants, biogas production, and energy efficiency (Ding et al., 2018, 2016). Moreover, direct electrical stimulation of microbial metabolism (e.g. protein synthesis and membrane permeability) or effect on cultivation ambient for microorganisms might be another reason for the enhancement of AD efficiency in WAS treatment (Chen et al., 2016; Ding et al., 2018). A lot of studies focused on operating parameters (e.g. voltage and primary pH value) or electrode materials (e.g. graphite and carbon felt) in energy recovery from MEC-AD (Ding et al., 2018, 2016; Lusk et al., 2016; Ren et al., 2018). However, the large distance between the electrodes can hamper electron flow between the electrodes and result in ohmic losses and a difficult for proton transport, especially in disposal of sludge with high viscosity (Hassanein et al., 2017). Generally, a better sludge stabilization obtained at higher voltages, however, it might appear sensible inhibition for methanogens because to the high hydrogen partial pressure (Eq. (2)) (Hao et al., 2017; Zabranska et al., 2018). Cathode:

CO2 + 8H+ + 8e− → CH 4 + 2H2 O, E = −0.244V


H2 O + 4e− → 2H2 + 4OH−, E = −0.83V


Table 1 Characteristics of thickened sludge and inoculum.


2H2 O → O2 + 4H+, E = +1.23V




pH Total solid (TS) Volatile solid (VS) Soluble chemical oxygen demand (SCOD) Total chemical oxygen demand (TCOD) Total protein Total carbohydrates Ammonia nitrogen (NH3-N)

– g/L g/L g/L

5.94 29.2 19.2 0.48


34.50 ± 0.26

27.11 ± 0.15

g COD/L g COD/L mg N/L

14.26 ± 1.03 10.51 ± 0.14 95 ± 11

11.22 ± 0.65 6.14 ± 0.11 1330 ± 210

± ± ± ±

Inoculum 0.05 0.3 0.2 0.10

7.45 32.4 14.4 3.12

± ± ± ±

0.06 0.2 0.1 0.15

2. Materials and methods 2.1. Characteristics of WAS, inoculum and biochar Sludge was collected from Minghang municipal wastewater treatment plant (WWTP) in Shanghai, China. The sludge was sieved with a 100 mesh, thickened using a centrifuge to increase the solids concentration to 3–4% and then stored at 4 °C prior to utilization. The inoculum was obtained from a thermophilic semi-continuous reactor with long-term stable operation in our lab (Yin et al., 2018). The average characteristics of the thickened sludge and inoculum sludge are presented in Table 1. WAS, in this study, was used as substrates to prepare biochar (BC). Sludge obtained from WWTP was firstly dried in an oven (105 °C) and smashed to the diameters of 5 µm by the ball mill (Retsch-MM400, Germany). The sludge powder were then pyrolysis in the tubular furnaces. During the whole process, N2 was used as the carrier gas at the flow rate of 100 mL/min. The heating procedure started from 30 °C at a constant heating rate of 10 °C/min. The final temperature was set at 500 °C for 1.5 h. The prepared sludge-based biochar was boiled in the 1:9 hydrochloric acid solution and pickled samples were rinsed in the distilled water until pH was observed to be neutral (Li et al., 2018). The biochar was used in AD after being dried in an oven (105 °C). The main properties of BC are listed in Table 2.


CH3 COO− + 2H2 O − 8e− → 2CO2 + 7H+, E = −0.28V


Direct interspecies electron transfer (DIET) between the microbes (e.g., exoelectrogenic bacteria and methanogenic archaea) plays a significant role in improving efficiency of biomethane production from AD (Chen et al., 2014; Nagarajan et al., 2013; Shrestha et al., 2013). Conductive materials (e.g. granular activated carbon, magnetite and biochar) added to AD reactors can facilitates DIET without mediating diffusive electron carriers (e.g., hydrogen and formate) (Chen et al., 2014; Liu et al., 2012; Wang et al., 2018; Zhang et al., 2018). Adding a conductive materials in suspended sludge of MEC may accelerate DIET and improve methane production. This speculation has been supported by the results of a study conducted by Ren et al. (2018), where applying a voltage on graphite resulted in an improvement in the methane production of a wastewater MEC. However, the mechanism analysis only focused on the microbial diversity of cathodic rod. The result of methane production for sludge was not known to be influenced by anode and suspended sludge. Moreover, there is fewer research focused on the stability of MEC system. The low-cost biochar with abundant functional group was a better conductive additive for improving system stability and establishing DIET. Research on biochar-assisted MEC for sludge AD has not reported. In this work, the feasibility of whether biochar-assisted MEC promotes the production of methane from sludge was investigated, and a mechanism for efficient methane production was elucidated. To obtain comprehensive information, the effect of individual and combined treatment of WAS with biochar and MEC were first assessed. Then the details of biochar-assisted MEC how enhance methane production were explored by analyzing the sludge hydrolysis and acidogenesis process, the change of oxidation-reduction capacities in attached and suspended biomass. Additionally, the relationship between biochar and the microbial community structure was investigated. The finding acquired in this study have implications for improvements in MEC technology and energy recovery in the WAS treatment process.

2.2. Batch experiments Batch experiments were performed to assess and compare the effects of biochar on the anaerobic performance of MEC. Double-walled cylindrical vessels anaerobic reactors with a working volume of 1 L were Table 2 Characteristics of sludge-based biochar with pyrolysis temperature at 500 °C. Parameters



Yeild BET N C H S Ash contenta Si Fe Al Ca Mg K Na pH EDC EAC Conductivity

% m2/g % % % % % % % % % % % % – mmol e− g−1 mmol e− g−1 uS/cm

45.1 ± 1.3 41.8 ± 8.6 4.44 ± 0.12 24.36 ± 1.25 1.71 ± 0.20 0.28 ± 0.05 66.5 ± 1.4 16.42 ± 1.31 8.63 ± 0.38 2.41 ± 0.24 1.35 ± 0.06 0.11 ± 0.03 2.33 ± 0.15 4.75 ± 0.29 8.01 ± 0.05 0.160 ± 0.005 0.776 ± 0.011 466 ± 28



Content of metal element was calculated according to ash from biochar.

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determined by the Bradford protein method and the sulfate anthrone method with BSA and glucose as the standard substrates (Bradford et al., 1976; Zhang et al., 2017), respectively. VFAs was analyzed by a gas chromatograph (GC-2010, Shimadzu) with a chromatographic column (DB-FFAP: 30 m × 0.25 mm × 0.25 mm) and aflame ionization detector (FID). The EEM spectra of supernatant were measured using a luminescence spectrometry (F-7000 spectrophotometer, Hitachi, Japan), according to Zhou et al. (2013). To analyze differences in bacterial and archaeal communities of AD reactors, suspended biomass and electrode-attached biomass samples were taken from the reactors at end of experiment, and the DNA extraction, high-throughput 16S rDNA sequencing are detailed in Supplementary Material.

used (Supplementary Material). Two pairs of carbon felt inserted into AD reactor were used as electrode of electrolysis cell (dimensions: 8 × 8 cm; thickness: 5 mm; distance: 1 cm), and connected to a DC power (WYJ. 5 A 60 V DC. REGUL. ATED. POWER SUPPLY, Shanghai, China) through copper wires. Each reactor was fed with 800 mL WAS and 200 mL inoculum sludge. The batch experiments can be separated into 4 groups, designated as “control” (CF, sludge + carbon felt), “biochar” (BC, sludge + carbon felt + biochar), “microbial electrolysis” (ME, sludge + carbon felt + voltage) and “biochar-assisted MEC” (MEBC, sludge + carbon felt + biochar + voltage). According the literatures by Fagbohungbe et al. (2016), the dosage of sludge-based biochar was 1.0 g/g DM (day matter). The applied voltages was fixed at 0.6 V (Chen et al., 2016; Zhao et al., 2016a). Before the start-up, oxygen was removed from the head-space by injecting nitrogen gas for 5 min, and then sealed the reactor. All reactors were maintained at a thermesophilic temperature of 55 ± 1 °C by water circulation. The gas vent on each digester was hooked to a gas sampling bag for biogas collection and the volume of biogas produced was measured using a gastight syringe. The gas volume reported in this study was calibrated to standard conditions (273 K, 1 atm). The AD experiments were operated under batch mode for 22 days and terminated until the daily biogas production was lower than 1% of the total biogas production. The gas sample and liquid sample were taken from each digester periodically for analysis.

2.5. Statistical methods Data are expressed as the means ± standard deviations of the triplicate measurements, and the significances of the results were determined using analysis of variance (ANOVA). Value of p < 0.05 was considered statistically significant. The general differences in the structure of the microbial communities were evaluated by principal coordinates analysis (PCoA) based on bray-curtis distance. The differences between two samples were tested by Fisher’ exact test based on DP: Asymptotic. 3. Results and discussion

2.3. Cyclic voltammetry experiment

3.1. Effects of biochar on anaerobic performance of MEC

Cyclic voltammetry (CV) were measured for the electrocatalytic activity of electrode-attached biomass and suspended biomass from all reactors according the literature of Lee et al. (2017). A single-chamber microbial electrolysis cell with working volume of 50 mL was used for CV measurement. After the experiment, the carbon felt module was immediately removed from AD reactor and cut to 2 × 1 cm piece to be used as the anode electrode in the MEC. The MEC was equipped with a counter electrode (Pt rod) and a reference electrode (Ag/AgCl), and the distance between the anode and reference electrode was ∼1 cm. The CV for electrode-attached biomass was conducted in the presence of 0.05 M propionate-butyrate medium. For suspended sludge, at the end of experiment, 50 mL of sludge from four reactors were directly added into cell. The working electrode and counter electrode of MEC unit were a copper plate (2 × 1 cm) and a Ti/RuO2 mesh plate (2 × 2 cm), respectively. The anode potential was ramped with time (scan rate = 10 mV/s) in a cyclic manner from +1.0 V to −1.0 V vs. standard hydrogen electrode (SHE) using a multi-channel potentiostat. These tests were completed in a vacuum gloves box and to avoid influence of oxygen.

The cumulative methane yields in terms of milliliters of CH4 for different reactors were presented in Supplementary Material, and the methane production per gram VS added were calculated as shown in Fig. 1a. In comparison of CF after 22 days AD experiment, ME and BC

2.4. Analytic methods The surface area, pore size and pore volume of sludge-based biochar were determined by Quantachrome Instruments (NOVA, 2000e, USA). Carbon, hydrogen, nitrogen and oxygen contents were analyzed using elemental analyzer (Vario Macro Cube, Germany). Elements (Ca, Mg, Al, Fe, Na and K) in ash were analyzed using inductively coupled plasma devise (ICP-OES 511, USA). The electron accepting capacities (EAC) and electron donating capacities (EDC) of biochar were measured according Klupfel et al. (2014) and Chen et al. (2018). The biogas samples were analyzed for CH4, CO2 and H2 content by using a gas chromatography (GC 7890N, Agilent Technologies, USA) equipped with thermal conductive detector (TCD). Sludge samples were collected from AD reactors for TS, VS, TCOD and SCOD of sludge samples were determined according to standard methods (APHA et al., 2012), and the TS and VS content in BC and MEBC reactors were subtracted with TS and VS that biochar engendered by itself. Value of pH was read by pH meter (pHs-3C, Leici Co. Ltd., Shanghai, China). Soluble protein and soluble carbohydrate concentration were

Fig. 1. (a) Methane and carbon dioxide production, (b) VS/TS and VS removal efficiency during 22-day MEC-AD of sludge. 317

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hydrolysis rate than consumption rate of soluble organics was obtained under BC addition and voltage supply. In other studies reported, electrolysis was able to crack the microbial cells trapped in sludge gels and release the biopolymers (soluble protein and carbohydrate), especially when combined with alkaline pretreatment (Yuan et al., 2016). It can be seen that the presence of biochar help the sludge from rapid upturn in pH at day 4 (Supplementary Material), since the biochar can act as pH buffer additive due to the alkaline groups on surface (Wang et al., 2017). Meanwhile, an increase of SCOD was observed in MEBC, indicating that electrolysis in combination with biochar could effectively disrupt sludge cells provoked the leakage of more extra- and intracellular polymeric substances into the liquid of WAS. From Fig. 2a, it can also be observed that the residua concentration in each reactor followed the order: BC > CF > ME > MEBC, which was in accordance with the methane production. Soluble protein and soluble carbohydrates are two important hydrolysate during hydrolysis process of sludge. The soluble protein (Fig. 2b) in reactor with and without biochar were up to peak on day 2, and for the soluble carbohydrate (Fig. 2c), the maximum concentration were obtained on range of day 2–4. For protein and carbohydrate, the maximum accumulation in BC and MEBC were lower than CF and ME reactor, indicating the rate of degradation was higher than production rate. This might because of the protein consumption bacteria enriched in biochar surface and its pores (microhabitat). Previous study demonstrated that the porous medium enhanced the microbial population by providing abundant micropore-mesopore structure for bacteria to colonize (Xu et al., 2015). In addition, both of protein and carbohydrate in MEBC was decomposed rapidly than BC, demonstrating that the acidogenesis process was enhanced with voltage supply. It was reported that the applied with a low voltage (< 0.9 V) can expand membrane permeability and promote activity of bacteria (Chen et al., 2016; She et al., 2006), capable of enzymatic reaction during hydrolysis and acidogenesis process of organic matter. As shown in Fig. 2d, the total VFA concentration increased in server days, and the maximal concentration in MEBC and BC was obtained on day 2, prior to CF and ME (4 d). It suggested that the biochar addition could increase production rate of fatty acid regardless of imposing voltage on carbon felt. Afterwards, total VFAs concentration decline rapidly and the rate of VFA consumption were agreement with the methane production, i.e., MEBC > BC > ME > CF. From the change of DOM in terms of EEM profiles of fermentation liquid (Supplementary Material), it can be seen that amounts of humic acid-like substances and humic substances were introduced from biochar. Furthermore, humic acid-like substances have been clearly demonstrated to be capable of mediating extracellular electron transfer in many key biogechemical processes (Yuan et al., 2018). The enhancement of electron transfer accelerate organic matter degradation and methane production.

reactor had an increase of methane production by 13.6 ± 0.8% and 18.5 ± 0.9%, respectively. Interestingly, with the addition of biochar in MEC, the CH4 yield increased by 24.7 ± 0.4% compared to CF reactor, demonstrating that the biochar was positive effective in anaerobic performance in MEC system. The enhancement of methane production at 0.6 V can attribute to both the occurrence of cathode reaction (Eq. (1)) and the methanogen activity (e.g., acetate, H2/CO2) enhanced (Chen et al., 2016). In this study, the fugitive CO2 volume in ME decreased by 40.7% compared to CF, which provide a fuller explanation of the enhanced methane production when supplying voltage. However, only a 23.4% decrease of fugitive CO2 volume was observed in MEBC. It indicated that biochar is capable of facilitating anodic reaction for soluble organics degradation). On the other hand, since the large surface area and porous structure containing in biochar (Supplementary Material) provide a suitable habitat for microorganisms, the addition of biochar also resulted in an enhancement of CH4 and CO2 yield, accompanied with substrate consumption (Li et al., 2018). In addition, the high EDC (0.165 ± 0.005 mmol e− g−1) and EAC (0.776 ± 0.011 mmol e− g−1) value of biochar suggested that the sludge-based biochar possessed a high electron capacity, which is capable of electron exchange between syntrophs and methanogens to accelerate organic matter consumption and methane production (Torri and Fabbri, 2014). The fitting results of Gompertz mode presented in Table 3 showed that the lag phase was shorten by 43.4%, 9.2% and 43.4% in BC, ME and MEBC, and the maximum methane production rate increased by 74.1%, 15.0% and 66.3%, compared to CF reactor, respectively. This results suggested that the addition of biochar in BC or MEBC would achieve quickly start-up of AD experiment. Biochar derived from activated sludge were usually acted as the conductive solid conduit to quicken electron transfer in substrate consumption process, but also promoted system stability and prevented microorganisms from environmental shock (i.e., pH, salinity and VFAs). VS of raw sludge and final digestate in each reactor were presented in Fig. 1b. The VS removal efficiency in BC, ME and MEBC were increased by 3.2%, 12.1% and 17.9%, compared to CF (34.7%), respectively. It meant that combined of voltage supply and biochar addition significantly accelerates the sludge solubilization. During the AD process, the variations of organics in the solids were evaluated in terms of VS/TS ration, and it decreased from 62.9% in raw sludge to 54.1%, 54.2%, 52.3% and 51.7%, respectively. This result suggested that MEBC with a high solubilization rate converted more VS into methane, which in agreement with the methane production. The detailed process of organic matter metabolism was subsequently discussed.

3.2. Effects of biochar on hydrolysis and acidogenesis process of sludge The SCOD, soluble protein, soluble carbohydrate content and total VFAs of four reactors were presented in Fig. 2. The SCOD concentration without voltage and biochar increased with digestion time in the initial 6 day, and then decreased in the residual digestion time. The maximal of SCOD concentration was only 8300 ± 200 mg/L. However, when imposing voltage and the presence of biochar, the maximal of SCOD increased by 20% and 34% of that CF reactor, occurred at 4th day and 2nd day, respectively. The increase of SCOD indicated that a higher

3.3. Effects of biochar on oxidation-reduction capacities of MEC Cyclic voltammetry experiments were conducted to probe electroactivaty of the suspended and attached sludge caused by voltage imposition and biochar addtion. In respone to the forward scan (from −0.5 to +1.0 V), a oxidation peak were observed by the suspended sludge at +0.12 V (versus Ag/AgCl). The peaks intensity were 2.5 mA, 2.8 mA, 3.2 mA and 3.5 mA from CF, BC, ME and MEBC, respectively, which demonstrated that the imposing of voltage and dosing of biochar facilitated electron tansfer in minxed sludge. In compasion with the oxidation peaks, two reduction peaks were obsevered at −0.23 and −0.02 V (versus Ag/AgCl) and the diffrence between each reactor was insignificant. Similarly, by comparision the CV of electrode when imposing voltage, it can be found that the current of cathodic electrode (Fig. 3b) had a small diffrence. However, there was a significant enhancement on CV of anode rod (Fig. 3c). The current peak from the MEBC anode (∼0.004 A) was almost two times higher than that of ME anode (∼0.002 A), indicating that biochar could enhance oxidation

Table 3 Model fitting results of the experimental methane production data using the modified Gompertz equation. Treatment

λ (d)


3.34 1.89 3.03 1.89

± ± ± ±

0.20 0.12 0.30 0.06

M0 (mL g−1 VS-added d−1)


20.14 30.06 23.16 33.46

0.9933 0.9957 0.9892 0.9992

± ± ± ±

1.49 2.87 3.52 1.18


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Fig. 2. Time-course profiles of (a) SCOD, (b) proteins, (c) carbohydrate and (d) total VFAs concentration for batch AD experiments.

of coulombic efficiency was a result of VFAs comsuption and methane pruduction during AD process. Together with the higher CH4 production in MEBC, we can conclude that biochar-assisted MEC for sludge AD was became a alternative route to degrade organic matter and generate CH4.

reaction of oraganic matter on anode-attached biomass. A increase of current peak was also observed in BC electrode (Fig. 3d). These results demonstrated that the electro-conductive biochar can acclerate oxdation reaction on anode-attached biomass, but with a litter effect on reduction reaction on cathodic electrode. It can imply that adding biochar enhances the biological methanogenesis process, but not promote the CO2 reduction reaction (Eq. (1)). This result was mainly because of the sludge-biochar with high EAC contained several phenolic, while with few quinones and polycondensed aromatic structures (Supplementary Material) (Klupfel et al., 2014; Yuan et al., 2017). The current density formed between anode and cathode (Fig. 3e) and columbic efficiency (Fig. 3f) were further used to evaluate the change of substrate removal by anodic oxidation in the bio-electrochemical system. It can be found that the current density of MEBC reactor was higher than that of ME reacotr in 22 days experiments (including fluctuat period and stable stage). After all, sludge-biochar was usually treated as non-metallic conductive materials due to the contained massive inorganic carbon and metabolic compounds (Zhang et al., 2014). The current density in MEBC began to rise sharply on the 5th day (0.8–3.5 A/m2), while beginning on 6th day for ME reactor (0.4–2.5 A/m2) as similar as the VFA degradation rate. The increase of current density at day 5 was because of the extracellular organic matters were preferentially utilized by anodic exoelectrogenic bacteria (discussed in Section 3.4). From Fig. 3f, anodic coulombic efficiency increased from 5% to 26% in MEBC during day day 2–12, while the coulombic efficiency in ME only reached to 20%. The increase of coulombic efficiency at stable VFA removal efficiences suggested that decomposition of organic matter was acclerated by anodic oxidation in MEBC, incidcating biochar in MEC system could acclerate anodic oxidation, in aggrement with the result of hydrolysis process. The decrease

3.4. Effects of biochar on bacterial and arechal community of MEC Fig. 4a shows bacterial communitiy structures in reactors with and without biochar or voltage. Most of these bacteria belong to Fimicutes, Proteobacteria, Tenericutes, Actinobacteria and Thermotogae. Firmicutes are reported to be the key players of converting fermentable substances into simple substrates, providing more suitable substrates to methangen, such as VFAs, hydrogen or others (Ko et al., 2018). Firmicutes in attached sample are accounted for 64.5–73.9% of total bacterial 16S rDNA gene sequences, higher than that of suspended sludge, which is consisted with previous studies (Zhao et al., 2016a). Firmicutes can resist extreme environments and enhance systme stability through the generation of spores (Huang et al., 2018). At genus level, the predominant genus in Firmicutes phyla (> 5%) were Coprothermobacter, norank_o_MBA03, Anaerobaculum, Caloramator, Tepidimicrobium, Haloplasma, Defluviitoga, norank_f_SRB2 and norank_o_Run-SP154. Based on genus data from suspended and attached samples, the structural diversity of the microbial community was evaluated by principal component analysis (Fig. 4b). It can be seen that the influence of biochar on microbial diversity followed by the order: AA > S > AC (with supply of voltage), S > A (without voltage). Combing with the result of Fisher’ exact test in Supplementary Material, the bacteria with significant difference in anode were Defluviitoga, Thermincola, norank_c_A55-D21-H-B-C01 and Caloramator. 319

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Fig. 3. CV curve of (a) the suspended sludge and the bioelectrode in AD at 22 d: (b) cathodic electrode, (c) anodic electrode, (d) electrol without voltage; (e) current density and (f) CE profile obtained during the ME-AD and MEBC-AD.

6.7%, 10.2% to 11.3%, 15.1%, respectively. Reportedly, Coprothermobacter belongs to thermophilic proteolyticus-related species, and the gene C. proteolyticus degrades casein and gelatin (Sasaki et al., 2011). The enhancement of Coprothermobacter in MEBC and BC agreed with the degradation rate of protein and generation rate of VFA. Coprothermobacte, norank_o_MBA03, Haloplasma and Tepidimicrobiumthe with a great difference in cathodic elctrode. With supplement of biochar, norank_o_MBA03 and Haloplasma were enriched from 7.8%, 2.2% to 13.6%, 7.0%, respectively. It is reported that Haloplasma is strictly organotrophic halophilic anaerobe strain and capable of enriching in high salinity area (Tsavkelova et al., 2018). The enrichment of Haloplasma ascribed cations transport that leached from biochar. However, the meta-analysis of Haloplama was still not described in the AD process (Maspolim et al., 2016). Archaeal communities were also analyzed for suspended and

The species Thermincola. potens have been reported to have exoelectrogenic activity, support multiheme c-type cytochromes involvement in conducting electrons across the cell envelope of a gram-positive bacterium (Shrestha et al., 2018). The exoelectrogenic Thermincola in samples without voltage was not detected, indicated the imposing voltage enriched exoelectrogens in anodic electrode and facilitated the electrons generation and transformation to anode. However, with the supplement of biochar, the abundance of T. potens was decreased from 20.5% to 9.6%. The results suggested that electron transfer have been stimulated in MEC by the addition of biochar, which was accordance with the finding of Li et al. (2018) that DIET was enhanced by conductive materials through substituting for pili in exoelectrogenic bacteria. The bacteria with significant difference in suspended sludge were Coprothermobacter and norank_o_A55-D21-H-B-C01. Addition of biochar in BC and MEBC could increase abundance of Coprothermobacter from 320

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Fig. 4. (a) Bacterial community structure at genual level of the samples and (b) principal component analysis (PCA) based on bacterial genus. The abbreviations are as follow. S: suspended biomass, A: attached biomass, AC: attached biomass on cathodic electrode, AA: attached biomass on anodic electrode.

sequence identity) in MEC, which was known to use hydrogen as a substrate for methane production (Ishii et al., 2005). This result indicates that hydrogenotrophic methanogensis was promoted in cathode by the addition of biochar, which was in accordance with the high hydrogen partial pressure in BC and MEBC (Supplementary Material). As Sunyoto et al. (2016) stated that biochar enhanced biofilm formation and provided temporary nutrients for hydrogen-producing bacteria. PCA showed the significant shift of archaea community in AA. The presence of biochar in MEC increased the abundance of Methanosarcina

attached samples with and without an applied voltage in Fig. 5a. Unlike the bacterial communities, archaeal communities showed much simpler compositions, which include three genus: Methanothermobacter, Methanosacrina and Methanomassiliicoccus. The gene Methanothermbacter was the most dominant genus of methanogenic archaea, especially on the cathodic electrode. The addition of biochar in MEC increased the abundance of Methanothermbacter on the cathode from 86.2% to 92.3% of archaea 16S rRNA genus sequences. The Methanothermbacter sequences were affiliated with MT. thermautrophicus species (99% 321

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Fig. 5. (a) Archaeal community structure at genual level of the samples and (b) principal component analysis (PCA) based on archaea genus.

3.5. Overall understanding role of biochar in MEC

genu in AA from 9.6% to 25.2%, and several studies reported that Methanosarcina species made use of acetate to produce methane can participate in DIET (Chen et al., 2014). On the other hand, the electrically conductive biochar is reported to substitute pili of exoelectrogenic bacteria for the electron connection between Geobacter and Methanogens (Zhang et al., 2018). Conclusively, the presence of biochar in MEC provide better DIET between exoelectrogens and methanogens in anodic electrode of MEC, which was in agreement with the finding of Lee et al. (2017).

A schematic depicting the biochar-assisted MEC for sludge AD is presented in Fig. 6, which could provide us an understanding of biochar’s effects on methane production from WAS. The addition of biochar not only provide a relative large surface area for the adhesion and growth of hydrolyzing and acid-producing bacteria (Coprothermobacter), but also improve systemic buffer capacity, accelerating hydrolysis and acidogenesis of particulate organics. During the AD of sludge, the particulate organic matter is converted to soluble small molecular 322

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Fig. 6. Schematic of sludge-based biochar assisted microbial electrolysis cell.

Simultaneously, biochar as supporter on cathode is a capable of transferring electron and promoting hydrogen production. A high hydrogen production increased the proportion of hydrogenotrophic methanogenesis (Methanothermobacter). Furthermore, the hydrogenmethanogenes genus, accounting for 50–90% of all sequence, indicated the CO2-dependent methanogenesis is the dominant pathway in biochar-assisted MEC, compared to that finding of Chen et al. (2016) and Dai et al. (2016).

substance that utilized by acetotrophic methanogens to produce methane. Apart of soluble organics can be decomposed into CO2 and H+ by exoelectrogens while they flows to anode. On anodic area, the addition of biochar decreased the abundance of T. potens and increased anodic coulombic efficiency, which demonstrated the biochar could substitute exoelctrogenic T. potens to accelerate electron transfer. Exoelectrogens have the molecular machinery to transfer the electrons exogenously to the electrode surface or to soluble or insoluble electron acceptors (Kumar et al., 2016). Microbial nanowires referred to redoxactive proteins such as cytochromes present on the outer surface of bacterial cell membrane and conductive pili played an important role in facilitating extracellular electron transfer (Costa et al., 2018). Methanosarcina participated in DIET was enriched on anodic electrode, inferring that DIET had been promoted by biochar. The establishment of DIET can accelerate the consumption of VFAs by reinforcing the electron transfer between syntrophic bacteria and methanogens, enhancing acetate-dependent methanogenesis pathway. In addition, the addition of biochar enhances anodic oxidation reaction for organic matters, accelerating H+ generation and electron transfer to cathodic electrode.

4. Conclusion This study demonstrated that sludge-based biochar-assisted MEC led to more significant enhancement of methane production (24.7 ± 0.4%). The presence of biochar in MEC promoted sludge hydrolysis and acidogenesis on suspended phase and enhanced organics oxidation and proton/electron generation on anodic electrode. Simultaneously, conductive biochar could substitute exoelectrogenic Thermincola to promote DIET between exoelectrogen and methaneogen. The improvement of methane production was mainly due to the 323

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proliferation of hydrogenotrophic Methanothermobacter on the cathodic electrode. This strategy increased the methane yields and improved the system stability, which is conducive to develop MEC technology for methane recovery.

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