Synergetic promotion of syntrophic methane production from anaerobic digestion of complex organic wastes by biochar: Performance and associated mechanisms

Accepted Manuscript Synergetic promotion of syntrophic methane production from anaerobic digestion of complex organic wastes by biochar: performance and associated mechanisms Gaojun Wang, Qian Li, Xin Gao, Xiaochang C. Wang PII: DOI: Reference:

S0960-8524(17)32113-2 https://doi.org/10.1016/j.biortech.2017.12.004 BITE 19259

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

11 November 2017 29 November 2017 2 December 2017

Please cite this article as: Wang, G., Li, Q., Gao, X., Wang, X.C., Synergetic promotion of syntrophic methane production from anaerobic digestion of complex organic wastes by biochar: performance and associated mechanisms, Bioresource Technology (2017), doi: https://doi.org/10.1016/j.biortech.2017.12.004

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Synergetic promotion of syntrophic methane production from anaerobic digestion of complex organic wastes by biochar: performance and associated mechanisms Gaojun Wang, Qian Li, Xin Gao, Xiaochang C. Wang * International Science and Technology Cooperation Center for Urban Alternative Water Resources Development; Key Laboratory of Northwest Water Resource, Environment and Ecology, MOE; Engineering Technology Research Center for Wastewater Treatment and Reuse, Shaanxi; Key Laboratory of Environmental Engineering, Shaanxi; Xi’an University of Architecture and Technology, No. 13 Yanta Road, Xi’an 710055, China *E-mail: [email protected]

Abstract: Biochar was added to a mesophilic anaerobic digester to promote syntrophic volatile fatty acids (VFAs) oxidation and methane production from complex organic wastes. Compared with conventional operation, biochar addition effectively shortened the lag time by 27.5-64.4% and increased the maximum methane production rate by 22.4% to 40.3%. With a biochar dosage of 15g/L, the system performed well under an organic loading rate as high as 3g substrate/g inoculums. Biochar showed a remarkable buffering capacity to alleviate pH decrease caused by VFAs accumulation. In order to gain knowledge on associated mechanisms, a specific experiment was conducted using butyrate as substrate. It was identified that syntrophic degradation of butyrate to acetate occurred under high H2 partial pressure. By microbial community analysis, it was further revealed that biochar addition brought about the enrichment of 1

Anaerolineaceae and Methanosaeta, typical microorganisms for direct interspecies electron transfer. Key words: Anaerobic digestion, Biochar, Volatile fatty acids, Syntrophic metabolism, Interspecies electron transfer.

1. Introduction Anaerobic digestion (AD) is a promising sustainable technology for waste management and energy production (Mao et al., 2015; McCarty, 2001), during which many kinds of wastes, such as sewage sludge, food waste, organic municipal solid waste and industrial waste, can be treated efficiently. Additionally, the chemical energy stored in these wastes can be used to generate cleaner energy in the form of biogas by anaerobic microbes (Nishio & Nakashimada, 2007). To achieve high-efficiency energy production, stable and effective methane production during AD is expected, especially under a high organic loading rate (OLR) (Appels et al., 2011). However, high OLR increases the risk of AD system failure due to the imbalance between acidification and methanation, which may result in severe accumulation of volatile fatty acids (VFAs) and a sharp decrease in pH. In general, the degradation of VFAs, such as propionate and butyrate, are slow due to the positive Gibbs free energy, which is thought to be the rate-limiting step of AD (Müller et al., 2010). During the syntrophic metabolism of propionate and butyrate, interspecies electron transfer (IET) is a crucial process between the syntrophic VFA oxidizing bacteria and methanogenic archaea, which is achieved when H2 or formate acts as the carrier (Stams et al., 2006). However, VFAs accumulation is often accompanied with 2

a rising H2 partial pressure, which can inhibit the syntrophic degradation of VFAs (Stams & Plugge, 2009). Recently, direct interspecies electron transfer (DIET) has been identified as an alternative pathway for syntrophic electron exchange in defined co-culture of two Geobacter species. These exoelectrogenic bacteria can establish an electrical connection and achieve DIET between the syntrophic partner with the help of electrical conductive pili (e-pili) or membrane cytochromes (Lovley, 2011; Shrestha et al., 2013). Additionally, DIET has also been confirmed in complex microbial environments. In an upflow anaerobic sludge blanket (UASB) digester, DIET between Methanosaeta and Geobacter species aggregates promoted syntrophic ethanol degradation and methane production (Morita et al., 2011). In order to enhance electron transfer, some (semi)conductive materials, such as minerals and abiotic carbon, were assessed for their ability to promote DIET. Conductive mineral magnetite particles were considered to act as the electron conduits between the propionate-oxidizing bacteria and methanogenic archaea to enhance propionate degradation (Viggi et al., 2014). Furthermore, the addition of conductive carbon cloth was beneficial in the aspects of methane production, volatile solids removal and COD removal efficiencies for treating the leachate or organic fraction of municipal solids wastes (Dang et al., 2017; Lei et al., 2016), and helpful to resist heavy acidic impacts condition via DIET (Zhao et al., 2017). Granular activated carbon (GAC) was shown to increase methane production potential and waste activated sludge reduction rates by enriching species like hydrogen-utilizing methanogens and Geobacter, which are 3

capable of DIET (Liu et al., 2012). Moreover, GAC addition stimulated efficient VFA degradation under high H2 partial pressure (Zhao et al., 2016). It was reported that single-walled carbon nanotubes could induce faster substrate utilization and methane production rates, which suggested that DIET among anaerobic fermentative bacteria and methanogens was enhanced (Li et al., 2015). The addition of 1.0g/L graphene also resulted in a 25.0% enhancement in methane yield and a 19.5% increase in production rate compared with the control (Lin et al., 2017). Multiple lines of evidence suggest that electrical conductive materials stimulate DIET in anaerobic syntrophic metabolism. However, the production costs of some commercial materials, such as carbon felt, carbon nanotubes, are prohibitive, and may not be suitable for field-scale application. Moreover, the environmental risk of graphene should be assessed in regard to the post disposal of anaerobic digestate (Clemente et al., 2017; Hansen, 2016). Thus, from the viewpoint of waste management and sustainable development, adding some conductive material produced with waste, such as biochar (a carbon rich material produced from waste pyrolysis) (Lehmann & Joseph, 2015) might be a practical strategy for achieving DIET in AD systems. Recently, the effect of biochar addition on AD was studied. It has been reported that biochar addition can promote IET in co-culture systems (Chen et al., 2014). Moreover, biochar addition also has some other benefits for AD, such as mitigating mild ammonia inhibition, supporting the formation of archaea microorganism (Lu et al., 2016; Mumme et al., 2014), shortening the lag time for methane production (Luo 4

et al., 2015) and increasing the biogas production rate (Sunyoto et al., 2016). However, to the best of the authors’ knowledge, the effects of biochar addition on different organic loading rate conditions and the results of biochar dosage on complex organic wastes have not been reported. In this study, the effect of biochar dosage on AD was investigated, and the performance of biochar addition to AD systems was explored under different OLR conditions. Additionally, butyrate, which was the main VFA that accumulated in the complex organic wastes AD process, was used as a substrate for degradation in a biochar amended environment to determine if biochar could promote the syntrophic degradation of VFAs via DIET.

2. Materials and methods 2.1 Biochar preparation and characterization The raw feedstock for biochar production was sawdust, which was collected from a local timber mill. Before pyrolysis, the sawdust was air-dried at 80℃ for 24h in an oven. The dried feedstock was placed into ceramic crucibles covered with fitted lids and pyrolyzed under oxygen-limited conditions in a muffle furnace (Shanghai Jing Hong Laboratory Instrument Co., Shanghai, China). After heating at a rate of 10℃min-1, the pyrolysis temperature reached 500℃, and this was maintained for 1.5h. After the pyrolysis process, the biochar sample was cooled to room temperature and then

ground

and

sieved

to

uniform

size

fractions

of

0.25-1mm.

The

Brunauer-Emmett-Teller (BET) surface area of the biochar was measured using a V-Sorb X800 surface area analyzer (Gold APP Instrument Co., Beijing, China). 5

Organic functional groups of the biochar were measured with Fourier transform infrared spectroscopy (FT-IR, ThermoFisher, USA) under an attenuated total reflectance (ATR) model (supplementary materials).

2.2 Substrates and seed sludge The complex organic wastes used in this study were a mixture of dewatered activated sludge (DAS) and food waste (FW). The DAS was collected from dewatered sludge units at Xi’an No.5 wastewater treatment plant, Shaanxi Province, China. The FW was synthetic and based on the characteristics of FW in China (Li et al., 2017). The mixture ratio of FW to DAS was 4:1based on wet weights according to the previous study (Li et al., 2017). The substrate was preserved in a 10L tank at a temperature of 4℃ before use. The properties of the complex organic wastes were shown in supplementary materials. The seed sludge used in this study was collected from an AD plant of a brewery factory, located in Xi’an, Shaanxi province, China. The AD plant was operated under steady mesophilic condition. Seed sludge was preserved in anaerobic conditions before use. The total solids (TS) and volatile solids (VS) of the seed sludge were 6.82% and 4.65%, respectively, and the pH of the seed sludge was 7.62.

2.3 Batch experiments 2.3.1 Complex organic wastes AD experiments To evaluate the effects of different dosages of biochar on AD, addition ratios of 0, 2, 6, 10 and 15g/L biochar were established. A total of 5mL seed sludge and 4mL substrate were added into a serum bottle to maintain the VS ratio of substrate to 6

inoculums (S/I) at 1.5. The different experimental groups were named BC0, BC2, B6, BC10 and BC15 according to the biochar dosage. In the experiment that aimed to evaluate the effects of biochar addition on different OLR conditions, four groups were designed to simulate the OLR from low to extremely high conditions. Specifically, 5mL of seed sludge with different substrate doses (2, 4, 6 and 8mL) was added into the bottles to maintain the S/I at 0.75, 1.5, 2.25 and 3 (based on VS), respectively. For each group, a biochar amended bottle was set up at 15g/L biochar addition and another without biochar addition was operated as control. The bottles were referred to as BCL, CTL, BCM, CTM, BCH, CTH, BCEH and CTEH, with “BC” and “CT” representing the biochar amended or control bottle, respectively, and “L”, “M”, “H”, and “EM” representing the low, medium, high and extremely high OLR groups, respectively. Considering the abundant nutrient content of the synthetic FW (supplementary materials), tap water was added into the bottles to keep the working volume at 90mL. All of the bottles were purged with high purity nitrogen gas for 5min to maintain the anaerobic conditions in the reactors and then sealed with rubber stoppers and pressed using aluminum caps. The bottles were then placed in a water bath and stirred at 150 rpm under mesophilic (35℃) conditions. All of these experiments were conducted in duplicate.

2.3.2 Specific experiment of butyrate degradation To explore the mechanism underlying the effects to AD, butyrate, which was the main VFA accumulated in the degradation process of complex organic wastes, was 7

selected as the substrate for further study. Three batch experiment groups, namely BC (biochar amended), Control A and Control B were designed. The experiments lasted for two periods. For the 1st period, 5mL seed sludge, 5mL n-butyrate sodium solution, and 50mL nutrient medium were added into each serum bottle with a working volume of 90mL to maintain the concentration of 1300mg/L n-butyrate ions. The pH of the mixture was adjusted with 6M NaOH and 6M HCl to approximately 7.2. The nutrient solution (0.5g/L NH4Cl, 0.1g/L MgCl2·6H2O, 0.4g/L K2HPO4 and 0.05g/L CaCl2·2H2O) was added to the bottles to keep the total working volume at 90mL. To identify the pH buffering capacity of the biochar, chemical pH buffering reagent (NH4HCO3) was added to the Control A group to keep the pH stable during reaction process, while the BC and Control B groups did not receive pH adjustment. Once the bottles had completely converted the butyrate to methane, the BC and Control A groups were selected to conduct the 2nd period. Butyrate sodium was added to these two groups to keep a final butyrate concentration of 700mg/L and a total working volume of 90mL. All bottles were operated the same as described above to maintain the anaerobic conditions for the reaction. To explore the effects of high H2 partial pressure on butyrate degradation, 20mL H2 (corresponding to 0.66atm) was spiked into headspace of each serum bottle. All of these experiments were conducted in duplicate.

2.4 Analytical methods The pH values were monitored using a potable pH meter (Horiba, Kyoto, Japan). 8

Hydrogen, methane, and carbon dioxide concentrations of the biogas were measured using a gas chromatograph (GC) (GC7900, Tianmei, China) equipped with a molecular sieve packed stainless-steel column (TDX-01, length × diameter of 2.0m × 3.0mm, Shanghai Xingyi Chrome, China) and a thermal conductivity detector (TCD). The VFAs were detected with a GC (PANNO, China) equipped with a DB-FFAP column (φ 0.32mm × 50m; Agilent, USA) and a flame ionization detector (FID).

2.5 Statistical analysis The experimental data of the batch experiments was simulated using the modified Gompertz equation:

where P is methane production (mL), P0 is methane production potential (mL), Rmax is the maximum methane production rate (mL/d), t0 is the lag time (days), and e=2.718281828. Origin 8.0 software (OriginLab Corporation, USA) was used to simulate the methane production curve and to coverage the results.

2.6 Microbial community analysis To identify the effects of biochar addition on the microbial community, samples of SD0 and SD15 were collected as the methane production ended. The sludge samples were first centrifuged at 13,000 rpm for 10min, and then the pellets were rinsed with phosphate-buffered saline twice via resuspension and centrifugation at room temperature. The supernatant was then discarded, and the samples were dried in an oven at 55℃ for 10min. A Mag-Bind Soil DNA Kit (Omega Bio-Tek, USA) was used to extract DNA according to the manufacturer’s protocol. The microbial 9

communities of the sludge were analyzed via high-throughput sequencing on an Illumina platform (Illumina Miseq PE250, Sangon Biotech, Shanghai, China). For the bacteria,

primers

515F

(5’-GTGCCAGCMGCCGCGGTAA-3’)

and

806R

(5’-GGACTACHVGGGTWTCTAAT-3’) were used to amplify the V4 region of the 16S rRNA gene by PCR. For the archaea, the V3-V4 region of 16S rRNA was amplified by nested PCR. 340F (5’-CCCTAYGGGGYGCASCAG-3’) and 1000R (5’-GGCCATGCACYWCYTCTC-3’) were the primers for the first cycle, and 349F (5’-GYGCASCAGKCGMGAAW-3’)

and

806R

(5’-GGACTACHVGGGTWTCTAAT-3’) were the primers for the second cycle. After amplification, the PCR products were checked via agarose gel electrophoresis to determine the quality of the amplification. After purification with Agencourt AMPure XP magnetic beads (Backman Coulter, USA) according to the manufacture’s protocol, the PCR products were quantified using a Qubit 2.0 DNA detection kit (Sangon Biotech, Shanghai, China).

3. Results and discussion 3.1 Effects of biochar dosage on AD of complex organic wastes Cumulative methane production under different biochar dosage conditions was shown in Figure 1(a). The fitted parameters of the modified Gompertz equation were listed in Table 1. The lag time of the BC0 group was 21.2 days, while that of the BC2, BC6, BC10 and BC15 groups were 15.3, 12.1, 10.2, and 7.8 days, respectively. It was clear that biochar addition significantly shortened the lag time by 27.5% to 64.4%. 10

Moreover, Rmax of the BC0 group was 6.7mL/d, and this increased to 8.2-9.4mL/d after addition of biochar. Thus, significant positive effects of biochar on both shortening the lag time and increasing Rmax were confirmed. The variation in pH was monitored in each group until it recovered to a suitable range for methane production (Figure 1(b)). Apparently, as the anaerobic process started, the pH of each group went through a rapid decline over one day due to the rapid acidification of the carbohydrates contained in the FW (Kawai et al., 2014). In BC0 group, the pH dropped from 6.7 to 5.0, while in biochar added groups, the drop in pH were diminished (above 5.1) due to the buffering capacity derived from the ash-inorganic alkalis and organic alkalis functional groups in biochar (Yuan et al., 2011). A previous study showed that the vermicompost derived biochar had a significant effect on improving acidification during AD (Wang et al., 2017). In this current study, the pH buffering capacity introduced by biochar was confirmed but not sufficient to completely eliminate the acidification. Considering a slight pH decline in the AD system would decrease the biogas production efficiency, the addition of biochar could be an ideal strategy, in practice, to stabilize the pH. The variation in VFAs during this process was shown in Figure 2. For all groups, the VFAs rapidly accumulated over the 2nd day after addition of the substrate. The VFA concentration remained at a high level until the lag time ended. For the BC0 group, the total maximum accumulated VFA concentration was 2296.3mg/L on Day 13. For the BC2, BC6, and BC10 groups, the total maximum accumulated VFA concentrations were decreased from 2192.6 to 1947.9mg/L on day 11 and that of the 11

BC15 group was 1830.3mg/L on day 9. It appeared that biochar addition was beneficial for alleviating VFA accumulation and for shortening the duration of accumulation. The main accumulated VFAs during this process were n-butyrate and acetate. For every group, these two VFAs accounted for more than 90% of the total VFAs. Although biochar addition had no obvious effect on the accumulated VFAs types, it was interesting that the ratio of acetate to n-butyrate was significantly altered after biochar addition. The syntrophic degradation of butyrate to acetate (Equation (1)) was energetically unfavorable and only occurred together with the hydrogen-consuming methanogenic process (Equation (2)). In this study, due to the low pH value, almost no methane was produced during the lag time. However, as the biochar dosage was increased, the concentration (mg/L) ratio of acetate to n-butyrate demonstrated a significant increasing trend (Figure 3). The average ratio of acetate to n-butyrate for the BC0 group was 2.22, while that of the BC2, BC6, BC10 and BC15 groups were 3.19, 3.88, 5.35 and 10.18, respectively, which suggested that biochar addition stimulated the conversion from butyrate to acetate by promoting DIET in the absence of metabolism by hydrogen-consuming methanogen. In a previous study, it was reported that biochar could act as an ideal additive to promote IET (Chen et al., 2014). CH3CH2CH2COO- +2H2O + 2CO2 2CH3COO- + H+ + 2H2

(1)

G0’=+48.3kJ/mol 4H2 + CO2 CH4 + 2H2O

(2)

G0’= -131.7kJ/mol 12

The authors attributed this phenomenon to the electrical conductivity of biochar, though its conductivity was 1000-fold less than that of granular activated carbon (Huggins et al., 2014). Noticeably, one important property of biochar for environmental management was the redox active property, due to the richness of organic functional groups of biochar (supplementary materials)(Klüpfel et al., 2014; Yuan et al., 2017). Thus, another explanation for the occurrence of DIET was likely that the butyrate oxidizing bacteria oxidized butyrate to acetate using biochar as the temporary electron acceptor in the inactive metabolism of methanogenic archaea. However, in the control group, syntrophic butyrate degradation could not occur due to the lack of electron acceptors, which resulted in butyrate accumulation. The experimental results of this section revealed that biochar alleviated VFA accumulation and stimulated methane production in AD. From the aspects of relieving VFA accumulation and avoiding acidification, which were two main complications often responsible for the failure of AD systems in engineering applications, addition of 15g/L biochar was demonstrated as the optimal dosage for promoting methane production.

3.2 Effects of biochar addition on AD of complex organic wastes under different OLRs To explore whether biochar addition could promote methane production under high OLR, four OLR conditions were selected and the results of cumulative methane production was shown in Figure 4. For the L and M groups, methane production was achieved independent of whether biochar was added or not. However, biochar 13

addition both shortened the lag time and increased Rmax. The lag time of the BCL and BCM groups was 6.5 days and 9.1 days, respectively, which were reduced by 39.2% and 52.8% compared with that of the BCL and CTM groups. Correspondingly, compared with the Rmax of the CTL and CTM groups, those of the BCL and SDM groups were also increased. For the H and EH groups, the lag times of the BCH and BCEH groups were 14.9 days and 19.5 days, respectively, which were prolonged compared to those of the SDL and SDM groups due to the high VFA accumulation. The Rmax of the BCH and BCEH groups were 13.5mL/d and 14.3 mL/d, respectively, which were quite higher than that of the SDL and SDM groups. It was noticeable that no methane production was detected in the CTH and CTEH groups until the time point at which the methane production process had ended in the BCH and BCEH groups. In the CTH and CTEH groups, the activity of methanogenic archaea was completely inhibited due to the heavy VFA accumulation (data not shown). The pH of the CTH and CTEH groups was also maintained at lower than 5.5 over this duration. These results revealed the significance of adding biochar to the AD system, especially under high OLR conditions. Additionally, the results of this study illustrated that the metabolic activity of methanogenic archaea could be recovered after biochar addition even in the presence of high VFA accumulation. Thus, biochar addition could also be an efficient and practical strategy for the rapid metabolic recovery of acidified AD reactors. Considering the batch operation mode of this study, the continuous run mode should be studied in the future to explore the long-term response of the AD system performance to biochar addition. 14

3.3 Effect of biochar addition on butyrate degradation As the above results showed, n-butyrate and acetate were the main accumulated VFAs in the AD of complex organic wastes. Considering the fact that acetate could immediately be utilized by methanogenic archaea, n-butyrate was used as a substrate to study the effects of biochar addition on the VFAs degradation further. During the 1st period, all three groups started to produce methane accompanied by butyrate degradation after a 6-day lag time (Figure 5(a)). However, the control B group ceased methane production at day 12. The cumulative methane production of the Control B group was just less than 20% that of the other two groups. The main reason for this was the decrease in pH, which was caused by the accumulation of acetate (Figure 5(c), supplementary materials). Apparently, the pH buffering capacity was important for the AD system to maintain stable and efficient methane production. For the Control A group, to avoiding a decreasing pH, a chemical buffering reagent (1g/L NH4HCO3) was added twice (at Day 0 and Day 10). Although the pH was maintained within a suitable range during the methane production process, a stagnant period occurred between Day 10 and Day 15. In the BC group, no chemical reagent was added for controlling the pH, as an appropriate pH value was maintained during the whole process due to the buffering capacity introduced by the organic functional groups of biochar (Yuan et al., 2011). The potential of biochar as an ideal substitution for chemical reagents for controlling pH was highlighted in this experiment. During the 2nd period, to study syntrophic butyrate degradation under the conditions of a high H2 partial pressure, 20mL H2 was added to the BC and Control A 15

groups. In the BC group, methane production and butyrate degradation began without a lag time, indicating that a high H2 partial pressure did not inhibit syntrophic butyrate degradation in this group. However, for the Control A group, although 4.61 mL cumulative methane was produced over the first two days, butyrate degradation was completely inhibited. Analysis of the biogas components revealed that H2 was consumed by the hydrogenophilic methanogens over first two days. After this, the butyrate began to degrade at Day 3. This result suggested that different metabolism types of syntrophic butyrate degradation likely existed between the Control A and BC groups. Due to the positive Gibbs free energy (Equation (1)), one critical requirement of this process is that the H2 concentration should be keep extremely low (typically below 10-5 atm). This was consistent with the results of the Control A group, which started butyrate degradation after the H2 was completely consumed, and was also consistent with a previous study (Viggi et al., 2014). Nevertheless, in the BC group, butyrate degradation began under a high H2 partial pressure condition. It was suggested that DIET could be occurring in the BC group. The biochar added here was considered as a temporary electron acceptor for promoting butyrate oxidation, and methanogens accepted these electrons from the reductive biochar to reduce the carbon dioxide to methane. In this manner, the high H2 partial pressure could not inhibited butyrate oxidation.

3.4 Response of the microbial community to biochar addition To identify the response of the microbial community structure to biochar 16

addition, the microbial mixtures of BC15 and BC0 were used for microbial community analysis. As shown in Table 2, for the bacteria community after biochar addition, the Shannon index decreased from 4.73 to 4.01 and the Simpson index increased from 0.03 to 0.06. Meanwhile, for the archaea community, the Shannon index decreased from 1.90 to 1.58 and the Simpson index increased from 0.25 to 0.38. These results revealed that biochar addition decreased the diversity of both the bacteria and archaea communities. However, as presented above, VFA degradation and methane production of the BC15 group were significantly promoted compared with that of the BC0 group. Thus, it was suggested that biochar addition stimulated the growth of some predominant microbes, which were crucial for promoting VFA degradation and methane production. Analysis of 16S rRNA gene sequences showed that biochar addition caused changes in the bacteria community structure (Figure 6(a, b)). For example, the relative abundance of Anaerolineaceae in the BC15 group was 30.08%, while in the BC0 group it was only 5.47%. It appeared that biochar addition stimulated the growth of Anaerolineaceae. Multiple lines of evidence suggested that Anaerolineaceae (phylum Chloroflexi) had the capacity of DIET. First, high transcription of the pilA gene was detected in some genus of Anaerolineaceae, which was prerequisite for the formation of the electrical conductive nanowires for DIET (Yu et al., 2016). Previous reports also showed that Anaerolineaceae played key roles in the syntrophy metabolism of AD and could transfer electrons to ferric iron (Kawaichi et al., 2013; Narihiro et al., 2015). In an anaerobic system with added fulvic acids, Anaerolineaceae became the 17

dominant microbe as the dosage of fulvic acids was increased, which suggested that Anaerolineaceae were able to transfer electrons to fulvic acids for syntrophic metabolism (Dang et al., 2016). Moreover, Anaerolineaceae were considered as one of predominant population in the anode of plant-based sediments Microbial Fuel Cell (MFC) system, and their relative abundance (17%) were comparable with that of Geobacter (19%), the well-known exoelectrogenic bacteria (Cabezas et al., 2015). Figure 6(a) illustrated the similar microbial community shifts occurring at the phylum level. In the BC0 Group, Firmicutes (43.81%) were the predominant bacteria, while Chloroflexi increased from 5.52% to 30.19% and Firmicutes decreased to 12.21% after addition of biochar. These results were also consistent with (Dang et al., 2016). Together, these previous studies suggested that Anaerolineaceae were capable of extracellular electron transfer to electron acceptors. For the archaea community, as shown in Figure 6(c), Methanosaeta (Methanothrix), Methanobacterium and Methanolinea were the three predominant genera in the methane production system regardless of the addition of biochar. These three genera accounted for more than 85% of the total archaea OTUs. Noticeably, with the addition of biochar, Methanosaeta was further enriched by 43.8% (from 40.77% in the BC0 Group to 58.64% in the BC15 Group). Traditionally, Methanosaeta were considered as the methanogens producing methane by using acetate. However, in the presence of some electron donors, the Methanosaeta genus could also reduce carbon dioxide to methane via DIET (Morita et al., 2011; Rotaru et al., 2014). Furthermore, it was also found that Anaerolineaceae and Methanosaeta 18

were enriched in the conductive carbon felt particles used for enhancing the degradation of propionate, which suggested syntrophic metabolism between these two microbes was achieved via DIET (Xu et al., 2016). All the evidence suggested that the addition of biochar

could

achieve

the

enrichment

of the

electro-active

Anaerolineaceae and Methanosaeta, and further indicated the advantage of biochar assisted AD over conventional process.

4. Conclusions Syntrophic VFAs degradation and methane production was promoted by biochar addition in this study. Biochar showed a remarkable buffering capacity to alleviate the pH decrease caused by VFAs accumulation. By using butyrate as a substrate in a specific experiment, it was identified that syntrophic degradation of butyrate to acetate occurred under high H2 partial pressure, evidently supporting the suggestion that biochar would have assisted in DIET in AD process. Biochar addition also resulted in enrichment of electro-active Anaerolineaceae and Methanosaeta, which further indicated the advantage of biochar assisted AD over conventional process.

E-supplementary data for this work can be found in e-version of this paper online.

Acknowledgements: This work was supported by National Natural Science Foundation of China (Grant No. 51608430), the Natural Science Foundation for Young Scientists of Xi’an University of Architecture and Technology, China (Grant No. QN1615), and the Scientific Research Program Funded by Shaanxi Provincial Education Department (Grant No. 19

17JS077).

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Figure and Table captions Figure 1. Cumulative methane production (a) and pH variation (b) under different biochar dosages condition. Figure 2. Volatile fatty acids variation under different biochar dosages conditions. Figure 3.Acetate to n-butyrate mass concentration ratio variation under different biochar dosage conditions. Figure 4. Cumulative methane production and pH variation under different organic loading rates condition Figure 5. Cumulative methane production and VFAs concentration variation in 1st (a, c) and 2nd periods (b, d) Figure 6. Relative abundance of bacterial communities under phylum (a) and genus (b) level and archaeal communities under genus level (c) at the end of complex organic wastes anaerobic digestion. Table 1. The fitting results of methane production on different biochar dosage addition by modified Gompertz equation. Table 2. Microbial community diversity analysis of bacteria and archaea of biochar amended (BC) and Control (CT) groups.

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Cumulative methane production (mL)

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Figure 1. Cumulative methane production (a) and pH variation (b) under different biochar dosages condition.

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Figure 3. Acetate to n-butyrate mass concentration ratio variation under different biochar dosage conditions.

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Figure 5. Cumulative methane production and VFAs concentration variation in 1 st (a, c) and 2nd periods (b, d) 31

(a)

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(c) Figure 6. Relative abundance of bacterial communities under phylum (a) and genus (b) level and archaeal communities under genus level (c) at the end of complex organic wastes anaerobic digestion. 32

Table 1. The fitting results of methane production on different biochar dosage addition by modified Gompertz equation. Group t0 (day) Rmax (mL/day) P0 (mL) R2 BC0 BC2 BC6 BC10 BC15

21.2±0.2 15.3±0.1 12.1±0.1 10.2±0.2 7.5±0.2

6.7±0.1 8.7±0.2 9.4±0.2 8.2±0.2 7.8±0.2

111.7±1.5 114.6±2.1 116.2±1.7 112.1±2.1 109.5±1.8

0.997 0.996 0.996 0.993 0.993

Table 2. Microbial community diversity analysis of bacteria and archaea of biochar amended (BC) and Control (CT) groups. Sample OTU number Shannon index Simpson index Bacteria-BC 1300 4.01 0.06 Bacteria-CT 1618 4.73 0.03 Archaea-BC 506 1.58 0.38 Archaea-CT 537 1.90 0.25

33

Highlights: Biochar (BC) was added for enhancing anaerobic digestion of complex organic wastes

BC addition shortened lag time and raised maximum CH4 production rate

BC showed good buffering capacity to mitigate pH decrease caused by VFAs accumulation

BC addition promoted syntrophic oxidation of butyrate under high H 2 partial pressure

Electro-active Anaerolineaceae and Methanosaeta were enriched after BC addition

34