Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation

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Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation Zhiqiang Zhao a, Cheng Sun a, Yang Li b, Hong Peng a, c, Yaobin Zhang a, * a Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian University of Technology), Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian, 116024, China b Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Food and Environment, Dalian University of Technology, Panjin, 124221, China c Department of Environment Science, College of Environmental Sciences, Sichuan Agricultural University-Chengdu Campus, Chengdu, Sichuan, 611130, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 April 2019 Received in revised form 7 August 2019 Accepted 10 October 2019 Available online xxx

Anaerobic co-digestion (AcoD) has been widely applied to the disposal of waste activated sludge (WAS), since it optimizes the C/N ratio and decreases the buffer capacity, which dose not solve the problem involved in the slow methanogenic metabolism that limits methanogenesis yet. In this study, a strategy of initially fermenting the polysaccharide-rich organic wastes to produce the ethanol-contained fermentation liquid (EFL) that was then mixed with WAS for further AcoD was proposed, with the aim of establishing direct interspecies electron transfer (DIET). The results showed that, the methane production rates and organic removal efficiencies in the digesters treating WAS mixed with EFL were significantly higher than that in the single-phase AcoD system about 30% and 6e7%, respectively, but the improved performances in the digesters treating WAS mixed with the neutral-pH fermentation liquid (FL) were not significant, which resulted in a low-efficiency energy recovery. The conductivity of sludge fed with WAS mixed with EFL were about 3e5 folds higher than that with the neutral-pH FL, suggesting that the DIET-based metabolism was established. Together with the special enrichment of Fe(III)reducing genus involved in Rhodoferax and high-abundance Methanospirillum capable of producing the electrical pili, a novel DIET between Rhodoferax and Methanospirillum was inferred. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Anaerobic co-digestion (AcoD) Waste activated sludge (WAS) Direct interspecies electron transfer (DIET) Ethanol-type fermentation Methanospirillum

1. Introduction Conversion of organic wastes to methane via anaerobic digestion (AD) as an effective strategy of renewable energy recovery has been widely applied to the disposal of waste activated sludge (WAS) [1,2]. Considering that WAS is characterized by the low C/N ratio and high buffer capacity due to its high-concentration ammonia nitrogen released after AD [3], a large number of studies focus on anaerobic co-digestion (AcoD) of WAS with the co-substrates that contain plenty of easily biodegradable organic matters and low alkalinity values, such as kitchen wastes [4,5], agricultural wastes [6] and animal manures [7], with the straightforward aim of achieving the optimum C/N ratio that is favorable for microbial

* Corresponding author. E-mail address: [email protected] (Y. Zhang).

metabolism and producing more methane. Schematic diagram of a current AcoD system for the disposal of WAS is shown in Fig. 1. The high-solid WAS (>20%, content of suspended solid) mixed with the co-substrates based on a defined C/N ratio is initially diluted in the sludge tank to improve the fluidity, then pumped into a sludge digester for further methanogenesis. However, this simple process improvement is insufficient to solve the major problem involved in the slow syntrophic metabolism between fermentative/syntrophic microorganisms and methanogens [8]. As the decrease of solid retention time (SRT), the balance of syntrophic metabolism may be destroyed, which can not provide the sufficient energy to support the growth of methanogens and further limit methanogenesis. Electron transfer to methanogens during syntrophic metabolism can either be direct or indirect. Hydrogen serving as the electron carriers involved in interspecies hydrogen transfer (IHT) is indirect, which depends on the hydrogen-utilizing methanogens to

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Please cite this article as: Z. Zhao et al., Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation, Renewable Energy, https://doi.org/10.1016/ j.renene.2019.10.058

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Fig. 1. Schematic diagram of a current AcoD system for the disposal of WAS.

continuously consume hydrogen and to keep the hydrogen partial pressure low enough [8]. However, it is usually observed that, the hydrogen-utilizing methanogens fail to consume hydrogen and proceed syntrophic metabolism well due to their relatively low abundance (<20%) in the methanogenic digesters [9e11]. Direct interspecies electron transfer (DIET) can proceed via electrically conductive pili [12] and outer surface c-type cytochromes OmcS [13], or a combination of biological and abiological electron transfer components, such as conductive materials [14e17]. The discovery that, acetate-utilizing methanogens, such as Methanothrix and Methanosarcina, are capable of accepting electrons via DIET for the reduction of carbon dioxide to methane is of great importance to AD [18,19], since Methanothrix and Methanosarcina are usually abundant in the methanogenic digesters but they typically grow slowly on acetate [20,21]. Therefore, it seems likely that enhancing the DIET-based syntrophic metabolism can promote the better methanogenesis during AcoD of WAS. Conductive carbon-based materials serving as the electron conduits have been widely investigated to enhance the DIET-based syntrophic metabolism in defined co-cultures [14e17] as well as in some mixed cultures [15,22e27], in which the DIET-based microorganisms such as Geobacter are abundant in the communities. However, in some methanogenic digesters treating complex organic solids, such as kitchen wastes [28] and WAS [29,30], the DIET-based microorganisms are quite rare, resulting that the improved performances are not significant even if in the presence of conductive carbon-based materials. Besides, since no study reports that the DIET-based microorganisms are capable of directly utilizing the complex organic substrates, such as carbohydrates and proteins, via DIET in defined co-cultures, the slow hydrolysis may limit their available substrates [28,31]. Some studies [32] suggested that the DIET-based syntrophic metabolism could be established between the special enrichments involved in some sulfur/Fe(III)reducing genus that might participate in the decomposition of complex organic matters and methanogens, if magnetite nanoparticles were supplemented. However, the design of magnetite nanoparticles incorporated as part of methanogenic digesters to provide a permanent conductive conduit for interspecies electron transfer will inevitably add the considerable costs and technical complexity in the practical engineering application. Previous study [24] demonstrated that, initially feeding the methanogenic digesters with ethanol, similar to the report that

metabolized ethanol via DIET in an up-flow anaerobic sludge blanket (UASB) reactor treating brewery wastes [33], could stimulating the communities to perform DIET. Although the mechanisms related to the enrichment of Geobacter with ethanol were still yet unknown, the results suggested that the energy yield from the oxidation of ethanol could support Geobacter to overcome the thermodynamical limitation of syntrophic oxidation of propionate and butyrate as well as to compete electron donors with the IHTbased microorganisms. Apart from external addition, ethanol can be internally produced from the fermentation of polysacchariderich organic wastes via adjusting the fermentation pH at 4.0e4.5 to achieve the ethanol-type fermentation [34]. A proof-of-concept study was further explored in a high organic loading rate (OLR) two-phase AD system treating dairy wastes, in which the acidogenic-phase pH was always maintained at 4.0e4.5 to produce the ethanol-abundant products that permanently stimulated the methanogenic phase to perform DIET [35]. However, due to the slow hydrolysis and high buffer capacity, simply adjusting the fermentation pH of WAS to produce ethanol is not feasible, and DIET-based syntrophic metabolism is still not established during AD of WAS. It should be pointed out that, during AcoD of WAS, the ethanol-abundant products may be produced by fermenting the co-substrates that largely contained the polysaccharide-rich organic wastes [36]. If so, it will not only improve the C/N ratio and decrease the buffer capacity that may be suitable for microbial growth and metabolism, but establish the DIET-based syntrophic metabolism. Based on these considerations, a strategy for upgrading the current method of AcoD of WAS was proposed in this study, in which the polysaccharide-rich co-substrates were initially fermented at pH 4.0e4.5 to obtain the ethanol-type fermentation liquid (EFL) in an acidogenic digester, then mixed with WAS for further AcoD in a methanogenic digester, with the aim of achieving the high-efficiency methanogenesis and energy recovery. 2. Materials and methods 2.1. Experimental setup Semi-continuous-flow experiments were simultaneously conducted in three parallel single-phase AcoD systems and twelve parallel two-phase AcoD systems. Schematic diagram of a singleand two-phase AcoD system is shown in Fig. 2. Each of the singlephase AcoD systems was operated in a cylindrical glass completelymixed digester (internal diameter of 150 mm and height of 500 mm) with a working volume of 7400 mL (referred to as sludge digester). At the top of each of the digesters, a stirring rod at a speed of 30e40 rpm connected with electric motor was placed into the sludge. The acidogenic phase of each of the two-phase AcoD systems was operated in a cylindrical glass completely-mixed digester (internal diameter of 80 mm and height of 90 mm) with a working volume of 400 mL (referred to as acidogenic digester). At the top of each of the acidogenic digesters, a stirring rod at a speed of 50e60 rpm connected with electric motor and a pH monitor were placed into the sludge. The methanogenic phase was operated in a completely-mixed digester similar to the sludge digester in a single-phase AcoD system. Each of the digesters was equipped with a gas sampling bags at the top of digester and a sludge sampling ports at the bottom of digester, and operated at a temperature of 37.0
C. At the beginning of experiments in a single-phase AcoD system, 200 mL of the polysaccharide-rich organic wastes stored in the feeding tank was initially pumped into the sludge tank with a peristaltic pump (Lange, BT100-2J, China), and then were mixed with WAS according to the volume ratio of 2:3 (Fig. 2A). Afterwards,

Please cite this article as: Z. Zhao et al., Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation, Renewable Energy, https://doi.org/10.1016/ j.renene.2019.10.058

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the sludge digester in a two-phase AcoD system was 22 d. 2.2. Seed sludge, waste activated sludge and Co-substrates Seed sludge was obtained from an anaerobic digester of a municipal sludge treatment plant in Dalian, China. Total suspended solids (TSS) and volatile suspended solids (VSS) of the seed sludge was 77300 ± 210 mg/L (mean ± standard deviation, n ¼ 3) and 28100 ± 433 mg/L, respectively. The seed sludge was stored under anaerobic conditions at a temperature of 4
C. At the beginning of experiments, each of the acidogenic digesters received a 200 mL of seed sludge, and each of the sludge digesters received a 7400 mL of seed sludge. WAS was obtained from a secondary sedimentation tank of a local municipal wastewater treatment plant in Dalian, China. The main characteristics of the WAS are shown in Table S1 (see Supporting Information). The WAS was also stored under anaerobic conditions a temperature of 4
C. Artificial dairy wastes were used as the polysaccharide-rich cosubstrates in this study. The composition (per liter) of the dairy wastes was as follows [35]: sucrose, 4.80 g; yeast extract, 0.96 g; milk powder (Songhuajiang; taobao.com; China), 4.00 g; NH4Cl, 0.92 g; KH2PO4, 0.20 g; trace element solution, 10 mL; vitamin solution, 10 ml. The composition of the trace element solution and vitamin solution was described in our previous study [27]. The chemical oxygen demand (COD) and pH of the artificial dairy wastes was about 10600 mg/L and 7.2, respectively. 2.3. Chemical analysis

Fig. 2. Schematic diagram of a single- (A) and two-phase AcoD system (B).

the mixed sludge was pumped into the sludge digester with a sludge peristaltic pump (Lange, BT300-2J, China) for further AcoD (Fig. 2A). The SRT of the sludge digester in a single-phase AcoD system was 22 d. The main characteristics of the WAS mixed with the polysaccharide-rich organic wastes (dairy wastes) are shown in Table 2. At the beginning of experiments in a two-phase AcoD system, 200 mL of the polysaccharide-rich organic wastes (dairy wastes) stored in the feeding tank was pumped into the acidogenic digester with a peristaltic pump (Lange, BT100-2J, China) for acidogenesis (Fig. 2B). The fermentation pH in the twelve parallel acidogenic digesters was maintained at 7.43, 5.96, 4.68 and 4.07 with 4M NaOH solution, respectively. The hydraulic retention time (HRT) of all the acidogenic digesters was 2 d. After acidogenesis, 200 mL of the fermentation liquid (FL) contained a small amount of suspended sludge was exhausted to the adjusting tank (Fig. 2B). The settling sludge at the bottom of the adjusting tank was returned to the acidogenic digester (sludge recirculation) with a sludge peristaltic pump (Lange, BT300-2J, China), and the supernatant liquid (clean FL) at the upside of the adjusting tank flowed into the sludge tank (Fig. 2B). The main characteristics of the FL at different fermentation pHs are shown in Table 1. The FL mixed with the WAS according to the volume ratio of 2:3 was pumped into the sludge digester with another sludge peristaltic pump (Lange, BT300-2J, China) for further AcoD. The main characteristics of the WAS mixed with FL at different pHs are shown in Table 2. The SRT of

TSS and VSS were analyzed in accordance with the Standard Methods for the Examination of Water and Wastewater. COD was determined by Hach’s method 8000 (Hach, DR/890, USA) [33]. Proteins were measured with Lowry’s method using bovine serum albumin as a standard solution [37]. Carbohydrates were measured by the phenol-sulfuric method with glucose as standard [38]. Ethanol and short-chain fatty acids (SCFAs, including acetate, propionate and butyrate) were measured by a gas chromatograph with a flame ionization detector (FID) (Tianmei, GC-7900P/FID, China) [39]. The volume of biogas in the gas sampling bag was measured by a glass syringe of 100 mL. The composition of biogas in the gas sampling was measured by another gas chromatograph (Tianmei, GC-7900, China) with a thermal conductivity detector (TCD) [40]. The equivalent relationship between COD and organic matters are as follows: 1.07 g-COD/g acetate, 1.51 g-COD/g propionate, 1.82 gCOD/g butyrate, 2.08 g-COD/g ethanol, 1.5 g-COD/g protein, 1.06 gCOD/g carbohydrate, 2.58  103 g-COD/mL methane and 0.645  103 g-COD/mL hydrogen. pH was measured by a pH analyzer (Sartorius, PB-20, Germany). 2.4. Sludge conductance After experiments, the conductivity of the sludge in the sludge digesters was measured in accordance with the method of threeprobe electrical conductance measurement [24]. The sludge was initially taken from the digesters with a 50 mL centrifuge tube from the sludge sampling port, then collected by the centrifugation at 8000 rpm for 5 min and washed three times by 0.1 M NaCl solution [41]. After washing and centrifuging, the sludge was placed on the two-gold electrodes separated by 0.5 mm non-conductive gap and crushed with a cover glass to form a confluent film that spread across the non-conductive gap [41]. An electrochemical workstation (Zhenhua, CHI1030C, China) was used to apply a voltage ramp of 0.3-0.3 V across split electrodes in steps of 0.025 V [12]. For each measurement, after allowing the exponential decay of

Please cite this article as: Z. Zhao et al., Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation, Renewable Energy, https://doi.org/10.1016/ j.renene.2019.10.058

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Table 1 Main characteristics of the fermentation liquid (FL) at different pHs. Error bars represent standard deviations (SD) of three-group parallel experiments. Parameters

FL at pH 7.43

FL at pH 5.96

FL at pH 4.68

FL at pH 4.07

pH Total COD (mg/L) Ethanol (mgCOD/L) Acetate (mgCOD/L) Propionate (mgCOD/L) Butyrate (mgCOD/L) Proteins (mgCOD/L) Carbohydrates (mgCOD/L)

7.43 ± 0.11 6943.2 ± 231.6 -a 880.3 ± 69.6 5363.4 ± 358.3 264.6 ± 9.6 7.2 ± 11.2 28.0 ± 6.6

5.96 ± 0.08 9342.1 ± 176.8 106.1 ± 1.8 2394.4 ± 243.2 4815.9 ± 136.2 1739.4 ± 166.3 10.6 ± 6.1 56.3 ± 1.0

4.68 ± 0.06 9769.9 ± 324.6 632.9 ± 21.5 5268.9 ± 1449.2 547.5 ± 153.0 2911.0 ± 488.8 5.1 ± 2.2 104.5 ± 6.8

4.07 ± 0.05 10050.1 ± 157.9 1292.5 ± 78.8 2258.1 ± 84.7 24.2 ± 4.3 443.5 ± 69.6 10.1 ± 5.9 173.6 ± 7.5

a

No detected.

Table 2 Main characteristics of the WAS mixed with the fermentation liquid (FL) at different pHs. Error bars represent standard deviations (SD) of three-group parallel experiments. Parameters

WAS with dairy wastes

WAS with FL at pH 7.43

WAS with FL at pH 5.96

WAS with FL at pH 4.68

WAS with FL at pH 4.07

pH SS (mg/L) VSS (mg/L) Total COD (mg/L) Total Proteins (mgCOD/L) Total Carbohydrates (mgCOD/L) Total SCFAs and Ethanol (mgCOD/L)

7.12 ± 0.04 50675.5 ± 2477.3 41907.2 ± 1147.3 51090.0 ± 961.7 3029.9 ± 293.5 1340.9 ± 19.1 2001.8 ± 128.4

7.06 ± 0.02 45678.3 ± 2133.6 36700.1 ± 569.2 50865.0 ± 2976.9 2680.3 ± 231.7 1224.1 ± 133.5 3916.8 ± 57.5

7.01 ± 0.03 48828.1 ± 506.7 38086.0 ± 1381.1 47850.0 ± 424.3 2964.3 ± 478.9 1242.0 ± 82.6 5279.5 ± 436.1

6.96 ± 0.03 49743.6 ± 1049.1 40352.1 ± 600.5 52215.0 ± 1704.1 2592.9 ± 139.0 1183.6 ± 38.1 3214.6 ± 122.9

7.01 ± 0.01 49850.9 ± 210.9 39880.7 ± 168.7 52820.0 ± 346.9 3292.0 ± 108.1 1804.0 ± 317.9 2951.7 ± 256.7

transient ionic current, the steady-state electronic current for each voltage was measured every second over a minimum period of 120 s [12]. The time-averaged current for each applied voltage was recorded to create the current-voltage curve. The conductivity of the sludge was calculated by the formula described in our previous study [24].

2.5. DNA extraction, PCR amplification and high-throughput sequencing After experiments, the microbial communities in the sludge digesters were analyzed by the high-throughput sequencing. The sludge was initially taken from the digesters with a 50 mL centrifuge tube from the sludge sampling port, then rinsed twice by phosphate-buffered saline (PBS; 0.13 M NaCl and 10 mM Na2HPO4 at pH 7.2) and harvested by centrifugation (110  100 g for 15 min at 4
C). The FastDNA® SPIN kit for soil (Bioteke, China) was used to extract DNA from the sludge according to the manufacturer’s protocols. The concentration and purity of the extracted DNA were determined by analyzing their absorbance at 260 and 280 nm with a Nanodrop® ND-1000 spectrophotometer (Labtech International, ND-1000, UK). 16S rRNA gene fragments were amplified via the polymerase chain reaction (PCR) with the following primer sets: Arch519F/ Arch915R and 515F/806R. The following amplification cycling scheme was used [42]: 94
C for 3 min, followed by 28 cycles of 94
C for 30 s, 53
C for 40 s and 72
C for 1 min, after which a final elongation step at 72
C for 5 min was performed. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. The pooled and purified PCR products were used to prepare DNA library by following Illumina TruSeq DNA library preparation protocol. High-throughput sequencing was performed on an Illumina Hiseq 2000 platform (Illumia, San Diego, USA) by Sangon Biotechnology Co., Ltd in Shanghai, China. Sequences were placed into various operational taxonomic units with pipeline software. Final OTUs were taxonomically classified using BLASTN against a curated database derived from GreenGenes, RDPII and NCBI (www. ncbi.nlm.nih.gov, http://rdp.cme.msu.edu) [43].

2.6. Energy evaluation After experiments, the energy recovery in the single- and twophase AcoD systems was evaluated. The energy recovery (W T) in a two-phase AcoD system was calculated by the following formula (1):

WT ¼ WA þ WM

(1)

where WA was the energy recovery in the acidogenic phase and WM was the energy recovery in the methanogenic phase. The energy recovery in the acidogenic phase (WA) was calculated by the following formula (2):

WA ¼

Dt DHS VA VM

(2)

where Dt was the time of the semi-continuous flow experiments (22 d), DHs was the energy content of methane/hydrogen based on the heat of combustion (upper heating value) (890.3/285.8 kJ/mol for methane/hydrogen), VA was the methane/hydrogen production in the acidogenic digester (L/d), and VM was molar volume of the gas at room temperature and atmosphere pressure (24.8 L/mol). The energy recovery in the methanogenic phase (WM) was calculated by the following formula (3):

WM ¼

Dt DHS VM VM

(3)

Where VM was the methane/hydrogen production in the sludge digester (L/d). The energy recovery (WS) in a single-phase AcoD system was calculated by the following formula (4):

WS ¼

Dt DHS VS VM

(4)

Where VS was the methane/hydrogen production in the sludge digester (L/d).

Please cite this article as: Z. Zhao et al., Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation, Renewable Energy, https://doi.org/10.1016/ j.renene.2019.10.058

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3. Results and discussion 3.1. Performances of acidogenic digesters in two-phase AcoD systems As the decrease of fermentation pH in the acidogenic digesters of two-phase AcoD systems, both methane production rates (Fig. 3A) and COD removal efficiencies (Table 1) significantly declined, due to the low-pH conditions that were unfavorable for methanogens. The primary acidogenic products at relatively neutral pHs (7.43 and 5.96) were propionate and acetate (Table 1), accounting for about 70e80% of total acidogenic products (Fig. 3B), suggesting that the dominant fermentation type belonged to propionate-type fermentation. Since the production of propionate can prevent the formation of Fdred and formate, both of which lead to the hydrogen production, and also consume NADH and Hþ, its formation ought to almost no hydrogen production (Fig. 3A) [44]. When the fermentation pH in the aciodgenic digesters further declined to 4.68, the primary acidogenic products were changed to butyrate and acetate (Table 1), corresponding to more than 80% of total acidogenic products (Fig. 3B), suggesting that the dominant fermentation type belonged to butyrate-type fermentation. Ethanol-type fermentation is characterized by the production of ethanol and acetate and accompanied by the significant release of hydrogen, since the balance between NAD and NADHþ is preserved [34]. The significant hydrogen and ethanol production was only observed in the acidogenic digesters operated at pH 4.07 (Fig. 3A and B). Although the ethanol and acetate production only accounted for about 35% of total acidogenic products, Megasphaera and Ethanoligenens known as the typical ethanol-type fermentation genera detected in the bacterial communities (Table S2) suggested that the dominant fermentation type in the acidogenic digesters operated at pH 4.07 belonged to the ethanol-type fermentation.

3.2. Performances of sludge digesters in single- and two-phase AcoD systems During the initial 22-day experiments, the differences in the performances of methane production (Fig. 4) and VSS removal (Table 3) in the sludge digesters of two-phase AcoD systems treating WAS mixed with the neutral-pH FL (7.43 and 5.98) were insignificant (P > 0.05). In these sludge digesters, the average

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methane production rates reached 190 mL/g-VSS/d, which were about 12% higher than that in the single-phase AcoD systems treating WAS mixed with dairy wastes (170 mL/g-VSS/d) (Table 3), and the VSS removal efficiencies were only about 1e2% higher than that in the single-phase AcoD systems (Table 3). The rates of syntrophic metabolism in the sludge digesters treating WAS mixed with the neutral-pH FL were not accelerated, since the methane conversion efficiencies were 86e87% similar to that in the singlephase AcoD systems (Table 3). Therefore, the primary reason involved in the improvement of methane production in the sludge digesters treating WAS mixed with the neutral-pH FL might be ascribed to the high-concentration SCFAs contained in the FL (Table 2). It should be pointed out that, the hydrolysis/acidification efficiencies in the acidogenic digesters at acidic pHs (4.68 and 4.07) were quite lower, since the concentration of total SCFAs and ethanol contained in the WAS mixed with the acidic-pH FL was only about 2900e3200 mgCOD/L, significantly lower than that in the WAS mixed with the neutral-pH FL (Table 2). However, the methane production rates in the sludge digesters treating WAS mixed with the acidic-pH FL further increased (Fig. 4). Especially in the sludge digesters treating WAS mixed with EFL at pH 4.07, the average methane production rates reached 213.9 ± 8.5 mL/g-VSS/ d (Table 3), which were higher than that in the single-phase AcoD systems about 25%. The methane conversion efficiencies reached 94.3%, which were higher than that in the sludge digesters treating WAS mixed with the neutral-pH FL about 8% (Table 3), suggesting that the rates of syntrophic metabolism were accelerated. It is well known that, proceeding syntrophic conversion of organic wastes to methane depends on the effective mechanisms of interspecies electron exchange between syntrophic microorganisms and methanogens [8,45]. Previous studies indicated that, initially feeding the methanogenic digesters with ethanol [24] or ethanol-abundant acidogenic products [35] could stimulate the methanogenic communities to perform DIET. Therefore, it seemed likely that, the reason involved in the high-efficiency methanogenesis in the sludge digesters treating WAS mixed with EFL might be ascribed to the establishment of DIET-based syntrophic metabolism. Although the performances in the single-phase AcoD systems during the second 22-day experiments significantly improved (P < 0.05) (Fig. 4 and Table 3), but the gap in the rates of methane production and efficiencies of VSS removal between the sludge

Fig. 3. The biogas production rates (A) and mass balance (B) in the acidogenic digesters operated at different pHs. Error bars represent standard deviations of three-group parallel experiments.

Please cite this article as: Z. Zhao et al., Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation, Renewable Energy, https://doi.org/10.1016/ j.renene.2019.10.058

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Fig. 4. The methane production rates in the sludge digesters treating WAS mixed with the fermentation liquid (FL) at different pHs. Error bars represent standard deviations of three-group parallel experiments.

Table 3 The performances of sludge digesters treating WAS mixed with fermentation liquid (FL) at different pHs. Error bars represent standard deviations (SD) of three-group parallel experiments. Stage

Parameters

Stage 1 (Day 1e22) MPRa (mL/gVSS/d) VREb (%) TREc (%) MCEd (%) Effluent SCFAs (mgCOD/ L) Stage 2 (Day 23e44) MPR (mL/gVSS/d) VRE (%) TRE (%) MCE (%) Effluent SCFAs (mgCOD/ L) a b c d

WAS with dairy wastes WAS with FL at pH 7.43 WAS with FL at pH 5.96 WAS with FL at pH 4.68 WAS with FL at pH 4.07 170.4 ± 8.3 38.8 ± 2.6 41.2 ± 1.2 86.6 ± 1.7 204.5 ± 12.1

190.8 ± 9.7 39.7 ± 1.2 40.5 ± 2.3 86.8 ± 3.1 217.4 ± 8.9

193.1 ± 8.9 39.8 ± 2.3 45.5 ± 1.3 86.4 ± 2.2 39.8 ± 2.3

41.4 ± 2.5 43.9 ± 1.7 91.6 ± 1.9 218.2 ± 11.6

40.4 ± 1.7 43.0 ± 0.9 94.3 ± 1.5 233.4 ± 17.5

197.0 ± 8.2 41.4 ± 2.1 49.1 ± 2.3 84.2 ± 2.2 427.7 ± 21.2

211.5 ± 9.3 41.1 ± 1.7 47.8 ± 1.7 81.6 ± 0.5 153.6 ± 9.6

220.4 ± 9.5 42.4 ± 2.3 51.7 ± 1.2 86.7 ± 2.5 231.9 ± 7.5

241.8 ± 8.7 46.5 ± 1.6 53.5 ± 3.1 89.3 ± 3.3 275.9 ± 17.2

256.3 ± 9.0 47.8 ± 2.2 54.9 ± 0.9 90.0 ± 1.2 109.6 ± 10.3

Methane Producion Rate (MPR). VSS Removal Efficiency (VRE). Total COD Removal Efficiency (TRE). Methane Conversion Efficiency (MCE).

digesters treating WAS mixed with the acidic-pH FL and singlephase AcoD systems further increased (Table 3). Specifically, the average methane production rates in the sludge digesters treating WAS mixed with EFL reached 256.3 ± 9.0 mL/g-VSS/d (Table 3), which were higher than that in the single-phase AcoD systems about 30%. The VSS removal efficiencies reached 47.8 ± 2.2% (Table 3), which were higher than that in the single-phase AcoD systems about 6e7%. Conversely, the gap in terms of methane production rates and VSS removal efficiencies between the sludge digesters treating WAS mixed with the neutral-pH FL (7.43 and 5.98) and the single-phase AcoD systems significantly declined (Table 3). Specifically, the average methane production rates in the sludge digesters treating WAS mixed with the neutral-pH FL increased to 210e220 mL/g-VSS/d (Table 3), but which were only higher than that in the single-phase AcoD systems about 7e12%, and the VSS removal efficiencies still only about 1e2% higher than that in the single-phase AcoD systems. These results demonstrated that, although operating the acidogenic digesters at relatively

neutral pHs could achieve the effective hydrolysis/acidification that produced more SCFAs, the performances in the two-phase AcoD systems were not significantly improved and even got closer and closer to that in the single-phase AcoD systems. 3.3. Sludge conductance The metallic-like and electrical aggregates have been considered as a potential evidence to support the DIET-based syntrophic metabolism established in the methanogenic communities [24,35,41,46], which were for the first time observed in an UASB reactor treating brewery wastes [33]. In this case, the temperature dependence of conductivity of the aggregates was consistent with the metallic-like conductivity similar to that found in Geobacter electrically conductive pili and biofilms [12,47]. Further study [13] in defined co-cultures of two Geobacter demonstrated that the mechanisms involved in the electrical aggregates were that Geobacter species facilitated DIET via their electrically conductive pili to

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form the electrical networks. Therefore, to investigate the potential establishment of the DIET-based syntrophic metabolism, the conductivity of sludge in the sludge digesters was measured (Fig. 5). The conductivity of sludge treating WAS mixed with the neutral-pH FL (7.43 and 5.96) was about 3.1e4.9 uS/cm2, similar to that in the single-phase AcoD systems (3.82 ± 0.10 uS/cm2) (Fig. 5). The conductivity of sludge treating WAS mixed with FL at pH 4.68 (5.51 ± 0.23 uS/cm2) slightly higher than that treating WAS mixed with the neutral-pH FL (Fig. 5), but the conductivity of sludge treating WAS mixed with EFL at pH 4.07 was about 3e5 folds higher than that treating WAS mixed with the neutral-pH FL (Fig. 5), suggesting that the potential DIET-based syntrophic metabolism was established. The results presented here were in accordance with that previously observed in a high-OLR two-phase AD system treating dairy wastes, which found that the sludge conductance had a nearly linear correlation with the concentration of ethanol in the acidogenic products [35].

3.4. Microbial community composition The microbial community composition was analyzed to gain insight into the microbial factors linked to the potential establishment of the DIET-based syntrophic metabolism (Table 4 and Table 5). The predominant methanogenic genus in the sludge digesters of both single- and two-phase AcoD systems belonged to Methanospirillum known as the hydrogen-utilizing methanogens typically capable of participating in IHT (Table 4). However, recent studies [48,49] revealed that, Methanospirillum hungatei JF-1 (96%, similarity), the most closest relative of Methanospirillum detected, were capable of producing the electrically conductive pili and expressing the functional genes involved in proceeding the extracellular electron transfer based on cryo electron microscopy. It should be pointed out that, the abundance of Methanospirillum in the sludge digesters treating WAS mixed with EFL was more than 10% (Table 4) higher than that in the other sludge digesters. However, the concentration of total SCFAs and ethanol that could be metabolized via IHT in the sludge digesters treating WAS mixed with EFL was the lowest (Table 1). Therefore, it seemed likely that, Methanospirillum specially enriched might directly accept electrons

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via DIET for the reduction of carbon dioxide to methane. Similar to this discovery, Lei et al. [46] using the conductive carbon cloth to enhance methanogenesis in an UASB reactor treating leachate also found that, the abundance of Methanospirillum attached to the conductive carbon cloth significantly increased, suggesting that Methanospirillum might utilize the conductive carbon cloth as the electron conduits. Methanothrix were the second dominant methanogenic genus in all the sludge digesters, accounting for about 25e30% of the communities (Table 4). Although the most closest relative of Methanothrix detected were related to Methanothrix concilii GP-6 (99%) known as the DIET-based methanogens observed in an UASB reactor treating brewery wastes [33], the differences in the abundance of Methanothrix in all the sludge digesters were very linked to the concentration of acetate (Table 1), suggesting that their most likely role was to convert acetate to methane rather than to participate in DIET. Previous study [35] demonstrated that, the ethanol-abundant acidogenic products could stimulate the methanogenic communities to enrich Geobacter with a abundance of 4e9%. In the study presented here, Geobacter were also detected in the sludge digesters treating WAS mixed with EFL, but the abundance of which was only about 0.5% (Table 5). Although the most closest relative of Geobacter detected were involved in Geobacter daltonii FRC-32 (96%) known as the dominant Geobacter in an UASB reactor treating brewery wastes (Table 5) [33], Geobacter with a abundance of 0.5% proceeding DIET with methanogens could not be sufficiently responsible for the heavy duty of maintaining the high-efficiency methanogenesis, suggesting that some other microorganisms other than Geobacter might hold a great potential of proceeding DIET. It was observed that, Rhodoferax known as the Fe(III)reducing genus were specifically enriched in the sludge digesters treating WAS mixed with the EFL (Table 5) [50]. The most closest relative of Rhodoferax detected, Rhodoferax ferrireducens T118 (98%), was capable of utilizing the various substrates including acetate, lactate, malate, propionate, pyruvate, succinate and benzoate as the electron donors in the presence of Fe(III) oxides [50]. Remarkably, the mechanisms of extracellular electron transfer to Fe(III) oxides or electrodes are very commonly linked to that of electron transfer to methanogens via DIET [51]. Therefore, it seemed likely that,

Fig. 5. The conductivity of sludge in the sludge digesters treating WAS mixed with the FL at different pHs. Error bars represent standard deviations of three-group parallel experiments.

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Table 4 The archaeal communities in the sludge digesters treating WAS mixed with the FL at different pHs. The genus level with relative abundance lower than 1.00% were classified into group ‘others’. Genus

Closest Relativea

Methanospirillum Methanothrix Methanosphaera

Methanospirillum hungatei JF-1 (96%) Methanothrix concilii GP-6 (98%) Methanothermobacter marburgensis (87%) Methanosphaerula Methanosphaerula palustris E1-9c (94%) Methanomassiliicoccus Methanomassiliicoccus luminyensis B10 (97%) Methanobrevibacter Methanobrevibacter smithii ATCC 35061 (99%) Methanoculleus Methanoculleus bourgensis MS2T (98%) Others e a

Relative Abundance (%) WAS with dairy wastes

WAS with FL at pH 7.43

WAS with FL at pH 5.96

WAS with FL at pH 4.68

WAS with FL at pH 4.07

58.6 24.2 7.1

51.1 34.8 5.0

52.3 37.1 3.9

47.1 31.5 7.2

61.7 29.4 5.9

3.0 3.1

4.6 1.0

3.4 0.2

4.4 5.1

1.4 0.2

1.8

2.1

1.9

2.6

0.9

1.1 1.1

0.5 0.9

0.4 0.8

0.8 1.3

0.2 0.3

Percentages of nucleotide similarity are shown in parentheses.

Table 5 The bacterial communities in the sludge digesters treating WAS mixed with the FL at different pHs. The genus level with relative abundance lower than 1.00% were classified into group ‘others’. Genus

Closest Relativea

Acutalibacter Anaerolineaceae Sporobacter Lutispora Williamwhitmania Tindallia Tangfeifania

Acutalibacter muris strain KB18 (86%) Anaerolineaceae bacterium CAMBI-1 (91%) Sporobacter termitidis (88%) Lutispora thermophila (89%) Williamwhitmania taraxaci strain A7P-90m (86%) Tindallia californiensis strain APO (89%) Tangfeifania diversioriginum strain DSM 2706 (87%) Diaphorobacter Diaphorobacter polyhydroxybutyrativorans strain SL-205 (98%) Dechloromonas Dechloromonas denitrificans strain ATCC BAA-841 (99%) Labilibacter Labilibacter marinus strain Y11 (86%) Pelobacter Pelobacter seleniigenes (81%) Neglecta Neglecta timonensis strain SN17 (87%) Capnocytophaga Capnocytophaga cynodegmi (86%) Wenzhouxiangella Wenzhouxiangella marina strain KCTC 42284 (80%) Syntrophomonas Syntrophomonas zehnderi OL-4 (95%) Blastopirellula Blastopirellula marina (85%) Emergencia Emergencia timonensis strain SN18 (94%) Novosphingobium Sphingopyxis baekryungensis (99%) Rhodoferax Rhodoferax ferrireducens T118 (98%) Gimesia Gimesia maris (90%) Pusillimonas Pusillimonas noertemannii (97%) Melioribacter Melioribacter roseus P3M (82%) Dethiobacter Dethiobacter alkaliphilus (87%) Geobacter Geobacter daltonii FRC-32 (96%) unclassified e Others e

a

Relative Abundance (%) WAS with dairy wastes

WAS with FL at pH WAS with FL at pH WAS with FL at pH WAS with FL at pH 7.43 5.96 4.68 4.07

12.3 6.1 4.9 3.5 3.4 3.1 3.0

9.3 5.8 6.3 2.8 2.0 2.6 2.1

12.7 3.6 5.3 4.1 1.6 1.3 1.5

9.1 5.6 5.3 3.8 2.7 3.1 2.8

11.3 4.6 6.8 4.1 1.8 3.5 1.7

2.5

2.5

2.3

2.2

2.4

2.5

3.7

4.1

3.7

3.1

2.5 1.9 1.8 1.8 1.4

2.0 1.5 2.1 1.5 1.1

1.5 1.2 2.5 1.7 1.0

2.0 1.3 2.1 1.7 1.3

2.2 1.8 1.8 2.1 1.2

1.4 1.1 1.0 1.7 0.2 0.9 0.6 0.5 0.4 0.1 16.0 25.4

0.9 1.2 1.2 1.5 0.2 1.0 1.0 1.1 1.1 0.1 18.1 27.2

0.7 1.0 1.3 1.1 0.6 0.8 1.6 1.1 0.8 0.1 22.3 24.2

1.2 1.4 0.7 1.3 0.7 1.8 0.5 1.0 0.7 0.0 16.0 28.0

1.3 1.0 1.2 1.0 4.2 0.9 1.1 0.9 0.6 0.5 15.9 23.0

Percentages of nucleotide similarity are shown in parentheses.

Rhodoferax might participate in DIET. The high-throughput sequencing also detected some anaerobic fermentation genera capable of converting complex organic matters to SCFAs and alcohols, such as Acutalibacter, Anaerolineaceae and Sporobacter, but the differences in the abundance of these genera among all the sludge digesters were not significant (Table 5). Both Capnocytophaga and Syntrophomonas belonged to the syntrophic acetate-producing genus, which metabolized the SCFAs and alcohols with the significant production of hydrogen, the differences in the abundance of which between the sludge digesters treating WAS mixed with the neutral-pH FL (7.43 and 5.96) and the acidic-pH FL (4.68 and 4.07) were also not significant (Table 5),

suggesting that the IHT-based syntrophic metabolism was not promoted. 3.5. Evaluation of energy recovery The energy recovery from the produced biogas was evaluated to gain insight into the novel strategy of AcoD of WAS linked to the establishment of the DIET-based syntrophic metabolism (Fig. 6). During the initial 22-day experiments, since no hydrogen production was detected in the sludge digesters treating WAS mixed with dairy wastes, the total energy recovery primarily from the produced methane in the single-phase AcoD systems was 2327.8 kJ

Please cite this article as: Z. Zhao et al., Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation, Renewable Energy, https://doi.org/10.1016/ j.renene.2019.10.058

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(Fig. 6A). The differences in the energy recovery between the twophase AcoD systems in which the aciodgenic digesters were operated at relatively neutral pHs (7.43 and 5.96) were not significant (Fig. 6A). Although the acidogenic digesters operated at relatively neutral pHs could achieve a certain amount of methane (Fig. 3A), the energy recovery in the acidogenic digesters only accounted for 3e10% of total energy recovery of the two-phase AcoD systems, suggesting that the primary energy production of the two-phase AcoD systems was ascribed to the methanogenic phase. During this stage, the total energy recovery of the two-phase AcoD systems was only higher than that of the single-phase AcoD systems about 6e9% (Fig. 6A). However, the total energy recovery in the two-

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phase AcoD systems in which the aciodgenic digesters were operated at acidic neutral pHs (4.68 and 4.07) significantly increased. Especially when the acidogenic digesters were operated at pH 4.07, the total energy recovery in the two-phase AcoD systems was more than 20% higher than that in the single-phase AcoD systems (Fig. 6A), even though the energy recovery from the produced hydrogen and methane in the acidogenic digesters only accounted for about 1% of the total energy recovery. During the second 22-day experiments, although the total energy recovery in both single- and two-phase AcoD systems increased (Fig. 6B), the gap in the energy recovery between the single-phase AcoD systems and two-phase AcoD systems in which the aciodgenic digesters were operated at

Fig. 6. The energy recovery in the single- and two-phase AD systems during the first 22-day semi-continuous flow experiments (A) and the second 22-day semi-continuous flow experiments (B).

Please cite this article as: Z. Zhao et al., Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation, Renewable Energy, https://doi.org/10.1016/ j.renene.2019.10.058

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Z. Zhao et al. / Renewable Energy xxx (xxxx) xxx

relatively neutral pHs (7.43 and 5.96) declined (Fig. 6B). Conversely, the gap in the energy recovery between the single-phase AcoD systems and two-phase AcoD systems in which the aciodgenic digesters were operated at the ethanol-type fermentation pH (4.07) further increased (Fig. 6B), suggesting that the DIET-based syntrophic metabolism established led to a high-efficiency energy recovery. From the view-point of engineering applications, most of acidogenic digesters are usually operated under the conditions close to the neutral pHs. In this case, lactate and propionate are the primary products after acidogenesis [44]. The syntrophic oxidation of propionate to acetate is quite unautogenous in thermodynamics, and the accumulation of propionate resulting in the failure of methanogenesis is commonly observed [10,24]. Some studies [52,53] operated the acidogenic digesters at pH 5.0e6.0 in an attempt to avoid the significant accumulation of propionate, and the fermentation type was thereby shifted to the butyrate-type fermentation, which is accompanied by the significant production of acetate. Although the syntrophic conversion of butyrate is relatively easier than that of propionate in thermodynamics [54], the metabolism of acetate via aceticlastic pathway only yields little energy that limits the growth of acetate-utilizing methanogens, such as Methanothrix or Methanosarcina. The current study presented here further demonstrated that, the acidogenic digesters operated at relatively neutral pHs had a quite small influence on the improvement of energy recovery from a long-term perspective. Although the acidogenic digesters operated at relatively neutral pHs could produce a certain amount of methane or hydrogen, the energy recovery from the biogas production in the acidogenic digesters was nearly ignored, compared with the total energy recovery in the two-phase AD systems. However, when the acidogenic digesters were operated at pH 4.0e4.5, the ethanolabundant acidogenic products did not break the balance of syntrophic metabolism, but stimulated the methanogenic communities to establish the DIET-based syntrophic metabolism that could permanently enhance the methanogenesis and increase the energy recovery. 4. Conclusions The study presented here proposed an effective strategy for upgrading the current AcoD of WAS, which not only improved the C/N ratio and decreased the buffer capacity, but held a great potential to solve the major problem involved in the slow syntrophic metabolism that limited the methanogenesis, via stimulating the methanogenic communities to establish the DIET-based syntrophic metabolism. The main conclusions of the performed analysis were: C The DIET-based syntrophic metabolism could be permanently established in the methanogenic digester treating WAS to achieve a high-efficiency methanogenesis, via ethanol-type fermentation of polysaccharide-rich co-substrates to produce ethanol. C Rhodoferax known as the Fe(III)-reducing genus were specially enriched with the stimulation of EFL, which might proceed DIET with Methanospirillum that were the dominant methanogens in the communities. C The strategy of establishing the DIET-based syntrophic metabolism during AcoD of WAS with EFL could achieve the better energy recovery, which held a great potential for upgrading the current method of AcoD of WAS. Declaration of competing interest The authors declare that the research was conducted in the

absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments The authors acknowledge the financial support from State Key Research and Development Plan (2018YFC1900901), National Natural Scientific Foundation of China (51578105, 51808097) and Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (ES201822). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.renene.2019.10.058. References
ve, R. Dewil, Principles and potential of the [1] L. Appels, J. Baeyens, J. Degre anaerobic digestion of waste-activated sludge, Prog. Energ. Combust. 34 (2008) 755e781. [2] A. Kelessidis, A.S. Stasinakis, Comparative study of the methods used for treatment and final disposal of sewage sludge in European countries, Waste Manag. 32 (2012) 1186e1195. [3] J. Mata-Alvarez, J. Dosta, M.S. Romero-Güiza, X. Fonoll, M. Peces, S. Astals, A critical review on anaerobic co-digestion achievements between 2010 and 2013, Renew. Sustain. Energy Rev. 36 (2014) 412e427. [4] J. Edwards, M. Othman, E. Crossin, S. Burn, Anaerobic co-digestion of municipal food waste and sewage sludge: a comparative life cycle assessment in the context of a waste service provision, Bioresour. Technol. 223 (2017) 237e249. [5] L.D. Nghiem, K. Koch, D. Bolzonella, J.E. Drewes, Full scale co-digestion of wastewater sludge and food waste: bottlenecks and possibilities, Renew. Sustain. Energy Rev. 72 (2017) 354e362. [6] A. Zhou, Z. Guo, C. Yang, F. Kong, W. Liu, A. Wang, Volatile fatty acids productivity by anaerobic co-digesting waste activated sludge and corn straw: effect of feedstock proportion, J. Biotechnol. 168 (2013) 234e239. n, E. Maran ~o n, Y. Ferna ndez-Nava, P. Ormaechea, Inverted [7] L. Negral, L. Castrillo phase fermentation as a pretreatment for anaerobic digestion of cattle manure and sewage sludge, J. Environ. Manag. 203 (2017) 741e744. [8] A.J.M. Stams, C.M. Plugge, Electron transfer in syntrophic communities of anaerobic bacteria and archaea, Nat. Rev. Microbiol. 7 (2009) 568e577. [9] J. Ma, L.J. Mungoni, W. Verstraete, M. Carballa, Maximum removal rate of propionic acid as a sole carbon source in UASB reactors and the importance of the macro- and micro-nutrients stimulation, Bioresour. Technol. 100 (2009) 3477e3482. [10] F.A.M. de Bok, C.M. Plugge, A.J.M. Stams, Interspecies electron transfer in methanogenic propionate degrading consortia, Water Res. 38 (2004) 1368e1375. [11] B. Demirel, P. Scherer, The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a review, Rev. Environ. Sci. Bio/Technol. 7 (2008) 173e190. [12] N.S. Malvankar, M. Vargas, K.P. Nevin, A.E. Franks, C. Leang, B. Kim, et al., Tunable metallic-like conductivity in microbial nanowire networks, Nat. Nanotechnol. 6 (2011) 573e579. [13] Z.M. Summers, H.E. Fogarty, C. Leang, A.E. Franks, N.S. Malvankar, D.R. Lovley, Direct exchange of electrons within aggregates of an evolved syntrophic coculture of anaerobic bacteria, Science 330 (2010) 1413e1415. [14] S. Chen, A. Rotaru, P.M. Shrestha, N.S. Malvankar, F. Liu, W. Fan, et al., Promoting interspecies electron transfer with biochar, Sci Rep-UK 4 (2015). [15] F. Liu, A. Rotaru, P.M. Shrestha, N.S. Malvankar, K.P. Nevin, D.R. Lovley, Promoting direct interspecies electron transfer with activated carbon, Energy Environ. Sci. 5 (2012) 8982e8989. [16] S. Chen, A. Rotaru, F. Liu, J. Philips, T.L. Woodard, K.P. Nevin, et al., Carbon cloth stimulates direct interspecies electron transfer in syntrophic co-cultures, Bioresour. Technol. 173 (2014) 82e86. [17] F. Liu, A. Rotaru, P.M. Shrestha, N.S. Malvankar, K.P. Nevin, D.R. Lovley, Magnetite compensates for the lack of a pilin-associatedc-type cytochrome in extracellular electron exchange, Environ. Microbiol. 17 (2015) 648e655. [18] A. Rotaru, P.M. Shrestha, F. Liu, B. Markovaite, S. Chen, K.P. Nevin, et al., Direct interspecies electron transfer between geobacter metallireducens and Methanosarcina barkeri, Appl. Environ. Microbiol. 80 (2014) 4599e4605. [19] A. Rotaru, P.M. Shrestha, F. Liu, M. Shrestha, D. Shrestha, M. Embree, et al., A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane, Energy Environ. Sci. 7 (2014) 408e415. [20] J. De Vrieze, T. Hennebel, N. Boon, W. Verstraete, Methanosarcina: the rediscovered methanogen for heavy duty biomethanation, Bioresour. Technol. 112 (2012) 1e9. [21] K.S. Smith, C. Ingram-Smith, Methanosaeta, the forgotten methanogen?

Please cite this article as: Z. Zhao et al., Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation, Renewable Energy, https://doi.org/10.1016/ j.renene.2019.10.058

Z. Zhao et al. / Renewable Energy xxx (xxxx) xxx Trends Microbiol. 15 (2007) 150e155. [22] H. Li, J. Chang, P. Liu, L. Fu, D. Ding, Y. Lu, Direct interspecies electron transfer accelerates syntrophic oxidation of butyrate in paddy soil enrichments, Environ. Microbiol. 17 (2015) 1533e1547. [23] Z. Zhao, Y. Zhang, Y. Li, Y. Dang, T. Zhu, X. Quan, Potentially shifting from interspecies hydrogen transfer to direct interspecies electron transfer for syntrophic metabolism to resist acidic impact with conductive carbon cloth, Chem. Eng. J. 313 (2017) 10e18. [24] Z. Zhao, Y. Zhang, Q. Yu, Y. Dang, Y. Li, X. Quan, Communities stimulated with ethanol to perform direct interspecies electron transfer for syntrophic metabolism of propionate and butyrate, Water Res. 102 (2016) 475e484. [25] Z. Zhao, Y. Zhang, D.E. Holmes, Y. Dang, T.L. Woodard, K.P. Nevin, et al., Potential enhancement of direct interspecies electron transfer for syntrophic metabolism of propionate and butyrate with biochar in up-flow anaerobic sludge blanket reactors, Bioresour. Technol. 209 (2016) 148e156. [26] J. Zhang, Y. Lu, Conductive Fe3O4 nanoparticles accelerate syntrophic methane production from butyrate oxidation in two different lake sediments, Front. Microbiol. 7 (2016). [27] Z. Zhao, Y. Zhang, T.L. Woodard, K.P. Nevin, D.R. Lovley, Enhancing syntrophic metabolism in up-flow anaerobic sludge blanket reactors with conductive carbon materials, Bioresour. Technol. 191 (2015) 140e145. [28] Y. Dang, D.E. Holmes, Z. Zhao, T.L. Woodard, Y. Zhang, D. Sun, et al., Enhancing anaerobic digestion of complex organic waste with carbon-based conductive materials, Bioresour. Technol. 220 (2016) 516e522. [29] H. Peng, Y. Zhang, D. Tan, Z. Zhao, H. Zhao, X. Quan, Roles of magnetite and granular activated carbon in improvement of anaerobic sludge digestion, Bioresour. Technol. 249 (2018) 666e672. [30] Y. Yang, Y. Zhang, Z. Li, Z. Zhao, X. Quan, Z. Zhao, Adding granular activated carbon into anaerobic sludge digestion to promote methane production and sludge decomposition, J. Clean. Prod. 149 (2017) 1101e1108. [31] Z. Zhao, Y. Li, X. Quan, Y. Zhang, Towards engineering application: potential mechanism for enhancing anaerobic digestion of complex organic waste with different types of conductive materials, Water Res. 115 (2017) 266e277. [32] Z. Yang, R. Guo, X. Shi, C. Wang, L. Wang, M. Dai, Magnetite nanoparticles enable a rapid conversion of volatile fatty acids to methane, RSC Adv. 6 (2016) 25662e25668. [33] M. Morita, N.S. Malvankar, A.E. Franks, Z.M. Summers, L. Giloteaux, A.E. Rotaru, et al., Potential for direct interspecies electron transfer in methanogenic wastewater digester aggregates, mBio 2 (2011). [34] N. Ren, B. Wang, J.C. Huang, Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor, Biotechnol. Bioeng. 54 (1997) 428e433. [35] Z. Zhao, Y. Li, X. Quan, Y. Zhang, New application of ethanol-type fermentation: stimulating methanogenic communities with ethanol to perform direct interspecies electron transfer, ACS Sustain. Chem. Eng. 5 (2017) 9441e9453. [36] F. Talebnia, D. Karakashev, I. Angelidaki, Production of bioethanol from wheat straw: an overview on pretreatment, hydrolysis and fermentation, Bioresour. Technol. 101 (2010) 4744e4753. [37] B. Frolund, T. Griebe, P.H. Nielsen, Enzymatic activity in the activated-sludge floc matrix, Appl. Microbiol. Biotechnol. 43 (1995) 755e761.

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[38] T. Masuko, A. Minami, N. Iwasaki, T. Majima, S. Nishimura, Y.C. Lee, Carbohydrate analysis by a phenolesulfuric acid method in microplate format, Anal. Biochem. 339 (2005) 69e72. [39] S. Jiang, Y. Chen, Q. Zhou, Effect of sodium dodecyl sulfate on waste activated sludge hydrolysis and acidification, Chem. Eng. J. 132 (2007) 311e317. [40] Z. Zhao, Y. Zhang, L. Wang, X. Quan, Potential for direct interspecies electron transfer in an electric-anaerobic system to increase methane production from sludge digestion, Sci. Rep-UK 5 (2015). [41] L. Li, Z. Tong, C. Fang, J. Chu, H. Yu, Response of anaerobic granular sludge to single-wall carbon nanotube exposure, Water Res. 70 (2015) 1e8. [42] J.G. Caporaso, C.L. Lauber, W.A. Walters, D. Berg-Lyons, C.A. Lozupone, P.J. Turnbaugh, et al., Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 4516e4522. [43] T.Z. DeSantis, P. Hugenholtz, N. Larsen, M. Rojas, E.L. Brodie, K. Keller, et al., Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB, Appl. Environ. Microbiol. 72 (2006) 5069e5072. [44] H. Lee, M.B. Salerno, B.E. Rittmann, Thermodynamic evaluation on H2 production in glucose fermentation, Environ. Sci. Technol. 42 (2008) 2401e2407. [45] J.R. Sieber, M.J. McInerney, R.P. Gunsalus, Genomic insights into syntrophy: the paradigm for anaerobic metabolic cooperation, Annu. Rev. Microbiol. 66 (2012) 429e452. [46] Y. Lei, D. Sun, Y. Dang, H. Chen, Z. Zhao, Y. Zhang, et al., Stimulation of methanogenesis in anaerobic digesters treating leachate from a municipal solid waste incineration plant with carbon cloth, Bioresour. Technol. 222 (2016) 270e276. [47] N.S. Malvankar, M.T. Tuominen, D.R. Lovley, Lack of cytochrome involvement in long-range electron transport through conductive biofilms and nanowires of Geobacter sulfurreducens, Energy Environ. Sci. 5 (2012) 8651e8659. [48] N. Poweleit, P. Ge, H.H. Nguyen, R.R.O. Loo, R.P. Gunsalus, Z.H. Zhou, CryoEM structure of the Methanospirillum hungatei archaellum reveals structural features distinct from the bacterial flagellum and type IV pilus, Nat. Microbiol. 2 (2017). [49] R.P. Gunsalus, L.E. Cook, B. Crable, L. Rohlin, E. McDonald, H. Mouttaki, et al., Complete genome sequence of Methanospirillum hungatei type strain JF1, Stand. Genomic Sci. 11 (2016). [50] K.T. Finneran, Rhodoferax ferrireducens sp. nov., a psychrotolerant, facultatively anaerobic bacterium that oxidizes acetate with the reduction of Fe(III), Int. J. Syst. Evol. Microbiol. 53 (2003) 669e673. [51] D.R. Lovley, T. Ueki, T. Zhang, N.S. Malvankar, P.M. Shrestha, K.A. Flanagan, et al., Geobacter: the microbe electric’s physiology, ecology, and practical applications, Adv. Microb. Physiol. 59 (2011) 1e100. [52] Y. Liu, Y. Zhang, X. Quan, Y. Li, Z. Zhao, X. Meng, et al., Optimization of anaerobic acidogenesis by adding Fe0 powder to enhance anaerobic wastewater treatment, Chem. Eng. J. 192 (2012) 179e185. [53] B. Demirel, O. Yenigün, Two-phase anaerobic digestion processes: a review, J. Chem. Technol. Biotechnol. 77 (2002) 743e755. [54] X. Dong, A.J. Stams, Evidence for H2 and formate formation during syntrophic butyrate and propionate degradation, Anaerobe 1 (1995) 35e39.

Please cite this article as: Z. Zhao et al., Upgrading current method of anaerobic co-digestion of waste activated sludge for high-efficiency methanogenesis: Establishing direct interspecies electron transfer via ethanol-type fermentation, Renewable Energy, https://doi.org/10.1016/ j.renene.2019.10.058