Establishing Direct Interspecies Electron Transfer during Laboratory

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Establishing Direct Interspecies Electron Transfer during Laboratory-scale Anaerobic Digestion of Waste Activated Sludge via Biological Ethanol-type Fermentation Pretreatment Zhiqiang Zhao, Yang Li, Jingyi He, and Yaobin Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b02618 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

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ACS Sustainable Chemistry & Engineering

Establishing Direct Interspecies Electron Transfer during Laboratory-scale Anaerobic Digestion of Waste Activated Sludge via Biological Ethanol-type Fermentation Pretreatment Author list Dr. Zhiqiang Zhao E-mail address: [email protected] Dr. Yang Li E-mail address: [email protected] Miss. Jingyi He E-mail address: [email protected] Prof. Yaobin Zhang* E-mail address: [email protected]

Affiliations: 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. Address: No.2 Linggong Road, Ganjingzi District, Dalian City, Liaoning Province * Correspondence: Tel: +86 411 8470 6460, Fax: +86 411 8470 6263; E-mail address: [email protected]. 1

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Abstract Direct interspecies electron transfer (DIET) has been considered as an effective working mode to proceed syntrophic metabolism, which is not yet established in the anaerobic digesters treating waste activated sludge (WAS), due to the lack of DIET-based syntrophs capable of producing the biological electrical connection and slow hydrolysis/acidification limiting the available substrates for DIET-based syntrophic partners. A strategy of initially pretreating WAS at pH 4.0-4.5 with ethanol-type

fermentation

enrichments

(referred

to Biological

Ethanol-type

Fermentation Pretreatment, BEFP) for stimulating the methanogenic communities to perform DIET was proposed in this study. Under the SRT of 24 d, methane production and sludge reduction in the digesters treating WAS with BEFP were about 29.8% and 12.3% higher than that without BEFP, respectively. The digested sludge with BEFP presented a high conductance (0.9722 ± 0.0085 uS/cm/g/L-VSS), even more conductive than the aggregates in the digester initially fed with ethanol previously reported. Together with the special enrichment of Geobacter species and high abundance of Methanothrix species, it was suggested that the DIET-based methanogenic communities were established. Further investigations to compare the pretreatment at pH 10 and BEFP revealed that, although the pretreatment at pH 10 promoted the better hydrolysis and increased the content of acetate, the slow methanogenic metabolism via aceticlastic pathway could not yet provide the sufficient energy to support the growth of Methanothrix species and further metabolize acetate. However, the analysis of stable carbon isotope demonstrated that the DIET-based 2


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syntrophic metabolism established with BEFP could potentially participate in acetate oxidation that supported the better growth of Methanothrix species.

Keywords: Anaerobic Digestion (AD); Waste Activated Sludge (WAS); Direct Interspecies Electron Transfer (DIET); Biological Ethanol-type Fermentation Pretreatment (BEFP); Methanogenesis

3



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Introduction The disposal of waste activated sludge (WAS) is a problem of growing importance, representing up to 50% of the current operating costs of a wastewater treatment plant 1. Anaerobic digestion (AD) has been considered as an effective method for the disposal of WAS due to its abilities to transform organic matters into methane, as thereby it also reduces the amount of final solids 2. Commonly, the major factor limiting the effectiveness of AD of WAS is the relatively slow hydrolysis/acidification including the disintegration of sludge flocs and conversion of proteins and carbohydrates into the short-chain fatty acids (SCFAs) and alcohols 3. In that case, a large number of studies

3

have focused on the improvement of hydrolysis/acidification by the

strategies of thermal, ultrasonic, or alkaline pretreatment with the aim to simultaneously enhance the decomposition of complex organics and increase the content of acetate that are favorable for acetate-utilizing methanogens, such as Methanothrix or Methanosarcina species, which are usually the predominant methanogenic genera in the digesters

4, 5

. However, the conversion of acetate into

methane via aceticlasitic pathway only yields little energy, and Methanothrix and Methanosarcina species typically grow slowly on acetate

4, 5

. The slow syntrophic

metabolism with acetate as substrate has gradually become a problem limiting the effectiveness of AD of WAS.

The discovery that Methanothrix 6 and Methanosarcina 7 species can accept electrons via direct interspecies electron transfer (DIET) for the reduction of carbon dioxide 4


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into methane in defined co-cultures provides some novel methods to accelerate and stabilize the syntrophic metabolism. Compared with traditional syntrophic metabolism mode of interspecies hydrogen transfer (IHT), the advantages of DIET are obvious. On the one hand, DIET can proceed via electrically conductive pili or outer surface c-type cytochromes 8, or a combination of biological and abiological electron transfer components, such as conductive materials 9-12, no longer requiring the hydrogen as the essential electron carrier as well as overcomes the thermodynamic limitation of hydrogen production 13. On the other hand, DIET can provide rapider electron transfer than IHT (44.9 × 103 e−cp−1s−1 vs 5.24 × 103 e−cp−1s−1) 14, supporting Methanothrix and Methanosarcina species to g row fast. Therefore, establishing the DIET-based syntrophic metabolism during the AD process is expected to achieve the better methanogenesis. However, up to now, no evidence indicates that the DIET-based methanogenic communities are established in the digesters treating WAS. This is probably because that, the DIET-based syntrophs, such as Geobacter species, are usually not abundant in these digesters

15, 16

. Besides, since no study reports that

Geobacter species can directly utilize the complex organic matters, such as carbohydrates and proteins that are largely contained in the WAS, via DIET in defined co-cultures, the slow hydrolysis/acidification may limit the available substrates for the DIET-based syntrophic partners 17.

However, it was suggested that the DIET-based syntrophic metabolism could be established during AD of WAS if conductive materials were provided as the electrical 5



conduit for interspecies electron exchange

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15, 18

. In both studies, DIET was primarily

inferred from the faster conversion of organics into methane in the presence of conductive materials. In the study with granular active carbon (GAC)

15

, the

abundance of Methanothrix species tightly attaching to the GAC significantly declined by about 40%, but that of the hydrogen-utilizing methanogens known as the microbial evidence related to IHT increased by about 10%. In the study collectively with GAC and magnetite

18

, the Fe(III)-reducing microorganisms related to

Ruminococcaceae and Synergistaceae capable of extracellular electron transfer to Fe(III) oxides or electrodes were enriched, which might participate in the decomposition of complex organics. However, the presence of Fe(III)-reducing microorganisms is not necessarily evidence for DIET, because not all Fe(III)-reducing microorganisms are capable of DIET 10, 19.

A promising strategy for establishing the DIET-based methanogenic communities during the AD process is to initially feed the digester with ethanol, similar to starting up the digester treating brewery wastes to rapidly produce the biological electrical connection

20

. The digester initially fed with ethanol forms the highly conductive

aggregates, improves the abundance of DIET-based syntrophic partners as well as effectively maintains the syntrophic metabolism stable

20

. In order to reduce the

considerable costs and permanently provide the DIET-based syntrophic partners with ethanol, a strategy of stimulating the methanogenic communities via ethanol-type fermentation was further proposed in previous study 21. Similar to that case, it was 6


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reported that the performances of the digester treating kitchen wastes could be improved when kitchen wastes were initially fermented by yeast under the acidogenic stage via ethanol-type fermentation

22

, which however ignored the potential

establishment of the DIET-based methanogenic communities with ethanol.

The purpose of this study was to investigate whether the DIET-based syntrophic metabolism could be established during AD of WAS via improving the pretreatment method to achieve ethanol-type fermentation. If so, it would not only enhance the hydrolysis/acidification of WAS, but accelerate the conversion of SCFAs and alcohols into methane and remit the accumulation of acetate. Considering that the ethanol-producing microorganisms were usually rare in the WAS, the pretreatment was initially inoculated with the ethanol-type fermentation enrichments previously cultured, then continuously operated at pH 4.0-4.5, named after biological ethanol-type fermentation pretreatment (BEFP). The long-term performances of AD of WAS in response to the change of solid retention times (SRTs), sludge conductance and microbial communities were evaluated. Besides, the effects of pretreatment at pH 10 and BEFP on methanogenesis were particularly compared, with aim to gain the new insight into the methanogenic metabolism with acetate as substrate via DIET.

Materials and Methods WAS and Anaerobic Inoculum sludge. The initial WAS was collected from a secondary sedimentation tank of a local municipal wastewater treatment plant that 7



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used the activated sludge process in Dalian, China. The main characteristics of the initial WAS are displayed in Table 1. The anaerobic inoculum sludge used for methanogenesis was collected from an anaerobic digester of a municipal sludge treatment plant in Dalian, China. The concentration of total suspended solids (TSS) and volatile suspended solids (VSS) of the inoculum sludge was 77300 ± 210 mg/L (mean ± standard deviation, n = 3) and 28100 ± 433 mg/L, respectively. The initial WAS and inoculum sludge collected were stored under anaerobic conditions at 4 oC prior to use.

Culturing Ethanol-type Fermentation Enrichments. The ethanol-type fermentation enrichments were cultured in a completely-mixed acidogenic digester treating dairy wastes with a working volume of 1000 mL (internal diameter of 70 mm and height of 300 mm) for 120-day experiments 21. At the top of the digester, a stirring rod at a speed of 30-60 rpm connected with electric motor was placed into the suspended sludge. The composition (per liter) of the artificial dairy wastes was as follows: glucose, 9.60 g; yeast extract, 1.92 g; milk powder (Songhuajiang; taobao.com; China), 8.00 g; NH4Cl, 1.84 g; KH2PO4, 0.40 g; NaHCO3, 10.00 g; trace element solution, 10 ml; vitamin solution, 10 ml. The composition of the trace element solution and vitamin solution was described in previous study

23

. The chemical

oxygen demand (COD) and pH of this artificial wastewater was about 21200 mg/L and 7.2, respectively. The digester was always operated at pH 4.0-4.5 known as the optimum pHs of ethanol-type fermentation

24

, and a temperature of 37.0 oC. The

8


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hydraulic retention time (HRT) of the digester was 9.6 h. The initial seed sludge was obtained from an anaerobic digester of a municipal sludge treatment plant in Dalian, China. After 120-day experiments, the concentration of TSS and VSS of the enrichments was 11400 ± 170 mg/L mg/L (mean ± standard deviation, n = 3) and 8400 ± 323 mg/L, respectively.

Pretreating WAS. Pretreating the initial WAS was operated in the four cylindrical glass completely-mixed digester (internal diameter of 130 mm and height of 150 mm) with a working volume of 2000 mL. Each of the digesters was sealed air-tightly, mechanically stirred at 80 rpm, and anaerobically operated at a temperature of 30.0 oC for 8-day experiments. The flow diagram of the experimental operations are shown in Fig. 1. Specifically, 2000 mL of the initial WAS mixed with the mentioned ethanol-type fermentation enrichments (95%/5%, v/v) was added into the digester (Fig. 1B), and the fermentation pH was maintained at 4.0-4.5 with the continuous addition of 4 mol/L hydrochloric acid (HCl) solution to keep the pH stable. After pretreatment, the pH of pretreated WAS was adjusted to 7.0 ± 0.2 with 4 mol/L sodium hydroxide (NaOH) solution. Besides, the initial WAS mixed without ethanol-type fermentation enrichments were pretreated at pH 7.0 ± 0.2, which was used as the control (Fig. 1A). To investigate the potential reason leading to the improved performances of AD of WAS with BEFP, the initial WAS mixed with ethanol was pretreated at pH 7.0 ± 0.2 (Fig. 1C). The dosage of ethanol (content > 99%) was about 1.1 mL/L-WAS, and the concentration of ethanol contained in the 9



pretreated WAS was 1812.4 ± 28.2 mgCOD/L (Table 2), similar to that with BEFP (1747.4 ± 79.7 mgCOD/L). Pretreating the initial WAS at pH 10.0 ± 0.2 with continuous addition of 4 mol/L NaOH solution to keep the pH stable was conducted (Fig. 1D), and the performances of AD of WAS with pretreatment at pH 10 and BEFP were compared. After pretreatment, the pH of the pretreated WAS was adjusted to 7.0 with 4 mol/L HCl solution. The pretreated WAS was stored under anaerobic conditions at 4 oC prior to use, and the main characteristics of the pretreated WAS are shown in Table 2.

AD of WAS. The semi-continuous experiments of AD of WAS were conducted in twelve cylindrical glass completely-mixed digesters (internal diameter of 60 mm and height of 88 mm), each of which had a working volume of 240 mL. Each of the digesters was equipped with a gas sampling bag and a sludge sampling port, placed in a shaker at a speed of 30-60 rpm and operated at a temperature of 37.0 oC. Before experiments, each of the digesters received a 240 mL of the inoculum sludge. The initial SRT of AD was 34 d. Namely, during each day, 7 mL of the treated WAS was extracted out of the digester, and then 7 mL of the fresh pretreated WAS was injected into the digester by a 10 mL of syringe. After 34-day experiments, the SRT was gradually decreased from 34 d to 28 d, and finally to 24 d.

Batch Experiments with Stable Carbon Isotope. In order to investigate the potential methanogenic metabolism with acetate as substrate by DIET-based methanogenic 10


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communities established via BEFP, batch experiments with stable carbon isotope were conducted in eight 26 mL anaerobic pressure tubes that contained 10 ml of media in the dark at 37.0 oC. The initial composition of the media (per liter) was as follows: sodium acetate tagged with stable carbon isotope (CH313COONa, Cambrudge Isotope Laboratories, UAS), 1.66 g (20 mM); NH4Cl, 0.64 g; K2HPO4, 0.15 g; MgCl2·6H2O, 0.1 g; CaCl2·2H2O, 0.052 g; trace element solution, 10 ml; vitamin solution, 10 ml, respectively. The media was flushed with nitrogen and carbon dioxide (80%/20%, v/v) for 0.5 h. The sludge was collected from the digesters treating WAS with pretreatment at pH 10 and BEFP, respectively, with a 2 mL centrifuge tube from the sludge sampling port at the end of experiments. Upon preparation, all the anaerobic pressure tubes were sealed with Teflon-faced butyl rubber stoppers and then flushed with nitrogen and carbon dioxide (80%/20%, v/v) for 15 min in the headspace. Before experiments, each tube was inoculated with 1 mL sludge. The content of

13

CH4 and

13

CO2 in the headspace of the tubes was analyzed by a mass spectrometry (Agilent,

5975, USA) linked to gas chromatograph (Agilent, 6980 N, USA), and the detailed analytical method was in accordance with the descriptions by Hao et al 25.

Chemical Analysis. TSS and VSS were analyzed in accordance with the Standard Methods for the Examination of Water and Wastewater. COD was determined with Hach’s method 8000 (Hach, DR/890, USA) 26. Proteins were measured with Lowry’s method using bovine serum albumin as a standard solution 27. Carbohydrates were measured by the phenol-sulfuric method with glucose as standard 28. The SCFAs and 11



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ethanol were measured by a gas chromatograph (Tianmei, GC-7900, China) with a flame ionization detector (FID)

16

. The equivalent relationship between COD and

organics are as follows: 1.07 g-COD/g acetate, 1.51 g-COD/g propionate, 1.82 g-COD/g butyrate, 2.08 g-COD/g ethanol, 1.5 g-COD/g protein, 1.06 g-COD/g carbohydrate and 2.58 × 10-3 g-COD/mL methane (based on the molar volume of gas at 25 oC, 24.8 L/mol). The volume of biogas in the gas sampling bag was measured by a glass syringe of 100 mL. The content of biogas was measured by another gas chromatograph (Tianmei, GC-7900, China) with a thermal conductivity fetector (TCD) 16

. pH was measured by a pH analyzer (Sartorius, PB-20, Germany).

Sludge Conductance. The sludge conductance was analyzed in accordance with the method of three-probe electrical conductance measurement

20

. The sludge samples

were taken from the digesters treating control WAS, WAS with BEFP and WAS with ethanol, respectively, with a 50 mL centrifuge tube from the sludge sampling port at the end of experiments, collected by the centrifugation at 8000 rpm for 5 min and washed three times by 0.1 M NaCl solution. 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 29. 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 30. For each measurement, after allowing the exponential decay of transient ionic current, the steady-state electronic current for each voltage was measured every 12


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second over a minimum period of 120 s 30. The time-averaged current for each applied voltage was recorded to create the current-voltage curve. The conductivity was calculated by the formula previously described 20.

DNA Extraction, PCR Amplification and High-throughput Sequencing. The microbial communities of sludge in the digesters treating WAS, WAS with BEFP and WAS with ethanol were analyzed at the end of the experiments via high-throughput sequencing. The sludge samples taken from the digesters with a 50 mL centrifuge tube from the sludge sampling port were firstly rinsed twice by PBS (0.13 M NaCl and 10 mM Na2HPO4 at pH 7.2), and then 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 of the sludge according to the manufacturer’s protocols. The concentration and purity of DNA extracted were determined by analyzing its absorbance at 260 and 280 nm with a 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: 94°C for 3 minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, after which a final elongation step at 72°C for 5 minutes was performed 32. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Then the pooled and purified PCR products were used to prepare DNA library by following Illumina TruSeq DNA library preparation protocol. 13



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High-throughput sequencing was performed on an Illumina Hiseq 2000 platform (Illumia, San Diego, USA) by Sangon Biotechnology Co., Ltd. (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 33.

Calculation. Methane conversion efficiency (MCE) was used to calculate the proportion of produced methane accounting for total organic removal, representing the efficiency of syntrophic conversion of organics into methane

17

, which was

calculated based on the following formula:

MCE =

CODMethane × 100% CODInfluent − CODEffluent

Where CODInfluent is the concentration of influent TCOD (mg/L), CODEffluent is the concentration of effluent TCOD (mgCOD/L) and CODMethane is the production of methane (mgCOD/L).

Results and Discussion General Characteristics of Ethanol-type Fermentation Enrichments. The ethanol-type fermentation enrichments were offwhite and cotton-shaped, comprised primarily of tightly packed spindle and rod-shaped microorganisms. The predominant genus in the enrichments belonged to the typical fermentative bacteria, such as Mitsuokella and Prevotella species, capable of fermenting the carbohydrates and proteins with the production of acetate and succinate 14


34, 35

. Megasphaera species

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accounted for about 20% of the communities, and their fermentative products were mainly involved in ethanol and butyrate, accompanied by the production of a small amount of hydrogen

36

. Ethanoligenens species, the well-known ethanol-type

fermentation genus, accounted for 6.8% of the communities, and were primarily responsible for the significant production of ethanol and hydrogen 36.

Effects of BEFP on AD of WAS. The concentration of ethanol in the WAS with BEFP was increased to 1747.4 ± 56.3 mgCOD/L (mean ± standard deviation, n = 3) (Table 2), which was not yet detected in the control WAS. Apart from the produced ethanol, the performances of WAS hydrolysis were significantly improved via BEFP. Specifically, in the control WAS, the total proteins and carbohydrates only declined to 3501.8 ± 328.0 and 2155.7 ± 93.4 mgCOD/L, respectively, accompanied by a small increase of SCFAs and ethanol from 833.7 ± 82.9 to 1966.7 ± 123.4 mgCOD/L (Table 2). In contrast, in the WAS with BEFP, the total proteins and carbohydrates declined to 2310.8 ± 450.1 and 688.0 ± 70.2 mgCOD/L, respectively, accompanied by a significant increase of SCFAs and ethanol from 833.7 ± 82.9 to 4544.7 ± 145.6 mgCOD/L (Table 2).

Then the pretreated WAS was used for further methanogenesis (Fig. 1). Under the initial SRT of 34 d, all of the digesters performed in a similar manner (P > 0.05) with fluctuant methane production (Fig. 2A), low VSS removal efficiency (Fig. 2B) and TCOD removal efficiency (Fig. 2C). The average methane production rates (Table 3) 15



and methane conversion efficiencies (Fig. 2D) in the digesters treating both control WAS and WAS with BEFP were about 180 mL/g-VSS/d and 95%, respectively. As the SRT gradually decreased to 28 d, the differences in terms of methane production and sludge reduction between the digesters treating control WAS and WAS with BEFP were gradually significant (P < 0.05) (Fig. 2A and B). The average methane production rates in the digesters treating WAS with BEFP were 237.0 ± 17.2 mL/g-VSS/d higher than that treating control WAS about 10% (Table 3). The VSS and TCOD removal efficiencies in the digesters treating WAS with BEFP reached to 43.9 ± 2.6% and 48.2 ± 2.1% higher than that treating control WAS about 5% and 10% (Table 3), respectively. A further decrease of SRT to 24 d immediately disrupted the methanogenesis with control WAS, and the performances of methane production rates (Fig. 2A), TCOD removal efficiencies (Fig. 2C) and methane conversion efficiencies (Fig. 2D) declined soon thereafter. This might be because that, the lower SRTs resulted in the great loss of biomass that contained a large number of syntrophs and methanogens, destroying the balance of syntrophic metabolism. Conversely, the digesters treating WAS with BEFP still presented an effective and stable methanogenesis, and the gap of methane production rates (Fig. 2A) and VSS removal efficiencies (Fig. 2B) between the digesters treating control WAS and WAS with BEFP further increased. Specifically, the average methane production rates and VSS removal efficiencies in the digesters treating WAS with BEFP were about 30% and 12.3%, respectively, higher than that treating control WAS (Table 3). Despite the lower SRTs, the methane conversion efficiencies with BEFP remained higher than 16


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85% (Fig. 2D), suggesting an effective working mode of syntrophic metabolism established in the methanogenic communities.

Additional experiments that the initial WAS with ethanol was pretreated at pH 7.0 were conducted to investigate the potential reason for the improved performances via BEFP (Fig. 1). Apart from the additional ethanol leading to the significant increase of total SCFAs and ethanol (Table 2), the performances of WAS hydrolysis were similar to that without ethanol (P > 0.05) (Table 2). However, the methanogenesis and organic removal in the digesters treating WAS with ethanol were significantly higher than that treating control WAS, and slightly lower than that treating WAS with BEFP (Fig. 2 and Table 3). These results demonstrated that, enhancing the WAS hydrolysis to produce the more SCFAs and ethanol that might be favorable for methanogenesis via BEFP was not the primary reason resulting in the improvement of methanogenesis. It was suggested that, the ethanol produced via BEFP might stimulate the methanogenic communities to establish the DIET-based syntrophic metabolism as described in previous studies

20, 21

to maintain the high-effficiency methanogenesis in

response to the low-SRT impacts.

Sludge Conductance. The DIET-based syntrophs proceed the long-range electron exchange via their metallic-like and electrical pili or outer surface c-type cytochromes distributed along the pili

37

, which forms the highly electrical aggregates as an

important evidence to demonstrate the DIET-based electrical connection established 17



in the methanogenic communities

20, 21, 26, 29, 38

. The possibility that the sludge in the

digesters treating WAS with BEFP might be electrically conductive, in a manner similar to that previously described for the aggregates formed in the UASB reactor initially fed with ethanol 20, was investigated. The conductivity of the sludge treating WAS with BEFP (0.9722 ± 0.0085 uS/cm/g/L-VSS) and with ethanol (1.0003 ± 0.0254 uS/cm/g/L-VSS) were similar (Fig. 3). They were more conductive than the aggregates (0.6-0.8 uS/cm/g/L-VSS) previously described, in which DIET was the primary working mode of interspecies electron exchange, in the UASB reactor treating brewery wastes 26, suggesting a great potential of DIET-based methanogenic communities established. In contrast, the conductivity of the sludge treating control WAS was only 0.1298 ± 0.0120 uS/cm/g/L-VSS, about 1/8 of that in the digesters treating WAS with BEFP (Fig. 3). Due to the lack of DIET-based electrical connection, electron transfer to methanogens only depended on the traditional IHT that was not capable of sufficiently maintaining the syntrophic metabolism stable especially under the low-SRT conditions.

Microbial Community Composition. After 86-day experiments, the microbial communities of the sludge were evaluated to gain further insight into the DIET-based methanogenic communities established. The most abundant methanogenic genera in the inoculum sludge belonged to Methanothrix species (51.9%, relative abundance) (Fig. 4A). Although Methanothrix species were confirmed to be capable of directly accepting electron via DIET 6, the relatively lower conductivity (0.0445 ± 0.0032 18


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uS/cm/g/L-VSS) did not yet support the DIET-based methanogenic communities established. Therefore, their most likely role was to convert acetate into methane via aceticlastic pathway 5. However, in the digesters treating control WAS, the abundance of Methanothrix species significantly declined by about 20%, but that of the hydrogen-utilizing methanogens, Methanospirillum species, increased by about 13%. This might be because that, pretreating the initial WAS at pH 7.0 resulting in the production of propionate and butyrate (Table 2) that were usually degraded by the microorganisms known to metabolize these organic acids to acetate with the production of hydrogen. However, the abundance of Methanothrix species in the digesters treating WAS with ethanol was still higher than 40%, similar to that treating WAS with BEFP, suggesting ethanol might stimulate the methanogenic communities to support Methanothrix species to grow well. Another methanogenic genera capable of accepting electron via DIET, Methanosarcina species, were not yet detected (Fig. 4A). Considering that Methanosarcina species are capable of utilizing the higher concentration of acetate to produce methane than Methanothrix species, the relatively lower concentration of acetate in the digesters might be an important reason limiting the enrichment of Methanosarcina species (Table 3). Methanococcus species were specifically enriched in the digesters treating WAS both with BEFP and ethanol, but which were almost not detected in the digesters treating control WAS as well as inoculum sludge (Fig. 4A). Although their ability to directly accept electron via DIET in

defined co-cultures was not yet evaluated, it was reported

that a

hydrogenase-deletion mutant of Methanococcus species could accept electron from 19



electrode via ‘electromethanogenesis’

39

, and the potential of directly accepting

electrons from electrode is very commonly linked to the ability of directly accepting electrons from Geobacter species via DIET in the presence of conductive carbon-based materials

37

. Therefore, it seemed like that Methanococcus species

enriched with ethanol might participate in DIET to maintain the high-efficiency methanogenesis.

Additional evidence to support the DIET-based syntrophic metabolism established with BEFP was the finding that Geobacter species were specifically enriched in the methanogenic communities with a abundance of 3-4% (Fig. 4B), which were not yet detected in the communities treating control WAS as well as inoculum sludge. Shrestha et al 40 reported that, there was a moderate correlation (r = 0.67) between the abundance of Geobacter species in the aggregates of UASB reactors treating brewery wastes and aggregates conductivity, but the abundance of the fatty-acid-oxidizing bacteria, fermentative bacteria and methanogens had almost no correlation with the aggregates conductivity, suggesting that Geobacter species were a major contributor to the aggregates conductivity. In accordance with that case, the enriched Geobacter species via BEFP might proceed DIET via their electrical pili, which resulted in the sludge presenting a higher conductivity (Fig. 3) and provided the energy to support the growth of Methanothrix species (Fig. 4A).

Apart from Geobacter species, the differences in the abundance of some 20


Page 20 of 48

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fatty-acid-oxidizing and fermentative bacteria between the digesters treating control WAS and WAS with BEFP were also significant (Fig. 4B). For example, the abundance of Syntrophomonas species capable of metabolizing propionate and butyrate into acetate with the hydrogen-utilizing metahnogens in the digesters treating control WAS was about 4-5% higher than that treating WAS with BEFP 41. It was in consistence with the significant increase in the abundance of Methanospirillum species in the digesters treating control WAS. Furthermore, Aminobacterium, Proteiniphilum, Oblitimonas species were specially enriched with BEFP. Their most likely role was to convert the fermentative intermediates into SCFAs

42, 43

, such as

amino acids or long-chain fatty acids, which however were almost not detected in the digesters treating control WAS. Apart from the great loss of biomass destroying the balance of syntrophic metabolism, this might be another potential reason resulting in the low-efficiency methanogenesis in the digesters treating control WAS (Fig. 2 and Table 3). The special enrichment of Aminobacterium, Proteiniphilum, Oblitimonas species with BEFP might be attributed to that the faster syntrophic conversion of SCFAs and ethanol into methane by the DIET-based syntrophic metabolism drove the decomposition of fermentative intermediates more smoothly.

Potential of DIET-based Syntrophic Metabolism with Acetate as Substrate. Pretreating the WAS at pH 10 has been reported as the more effective method tto enhance AD of WAS than some other methods, such as ultrasonic, thermal, or thermal-alkaline pretreatment 44. This was because that, pretreating the WAS at pH 10 21



could cause the greatest soluble C:N and C:P ratios, the significant release of Fe3+, and a suitable SCFA composition that was considered as the major factor for the improved performances 45. In accordance with that case, the concentration of SCFAs in the WAS with pretreatment at pH 10 reached to 8782.1 ± 123.8 mg COD/L (Table 2), about 2 folds higher than that with BEFP. More importantly, the concentration of acetate was 5845.8 ± 145.6 mgCOD/L, accounting for about 67% of total SCFAs, similar to that previously reported 44, suggesting a huge potential of methanogenesis with acetate. However, the performances in the digesters treating WAS with pretreatment at pH 10 dropped sharply as the decrease of SRT to 24 d (Fig. 5A). For example, although the average methane production rates were still 173.6 ± 38.6 mL/g-VSS/d (Table 4), similar to that previously reported (171.2 mL/g-VSS/d) 44, the methane conversion efficiencies were only 59%, about 30% lower than that with BEFP (Fig. 5D and Table 4). The change of SCFAs and ethanol during 86-day experiments is shown in Fig. 6. It was observed that, acetate rather than propionate or butyrate significantly accumulated during the whole experiments with pretreatment at pH 10, and the concentration of acetate gradually increased to 3638.4 ± 384.3 mgCOD/L under the SRT of 24 d, only yielding an acetate removal efficiency of 37.8%. This might be because that, the conversion of acetate into methane via aceticlasitic pathway (reaction 1) only yields little energy, and Methanothrix species typically grow slowly on aceticlastic methanogenesis. Especially under the semi-continuous operations, the great loss of biomass further limited the growth of Methanothrix species, which could not obtain the sufficient energy to support their 22


Page 22 of 48

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growth as well as further metabolize acetate. In contrast, the concentration of acetate was always maintained at lower than 500 mgCOD/L in the digesters treating WAS with BEFP (Fig. 6). It seemed like that, DIET-based syntrophic metabolism established via BEFP might proceed the conversion of acetate into methane, since the direct acetate oxidation could provide the sufficient energy and Methanothrix species might metabolize acetate better when they could also obtain additional energy from DIET-derived electrons (reaction 2 and 3) 46. Besides, acetate can be also oxidized syntrophically in some methanogenic environments with hydrogen as the electron carrier by syntrophic acetate-oxidizing bacteria (reaction 4 and 5)

25, 47, 48

. However,

the syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis is often reported to occur under some stressed conditions, such as the high-concentration salts or ammonium, due to the unautogenous process in thermodynamics (reaction 4).

CH3COO- + H+ → CH4 + CO2 ∆G0´ = - 36.1 kJ/mol (based on the report by Thauer et al. 31) CH3COO- + H+ + 2H2O → 2CO2 + 8H+ + 8e8H+ + 8e- + CO2 → CH4 + 2H2O

4H2 + CO2 → CH4 + 2H2O

∆G0´ = - 221.4 kJ/mol

∆G0´ = + 185.3 kJ/mol

CH3COO- + H+ + 2H2O → 2CO2 + 4H2

(1)

(3)

∆G0´ = + 94.9 kJ/mol

∆G0´ = - 131.0 kJ/mol

(2)

(4)

(5)

Further experiments to demonstrate the potential of acetate oxidation via DIET with BEFP were conducted with stable carbon isotope (Fig. 7). Commonly, the methylic 23



Page 24 of 48

and carboxylic carbon of acetate during aceticlastic methanogenesis move to methane and carbon dioxide, respectively, while both of which during acetate oxidation both move to carbon dioxide. It was observed that, almost no

13

CH4 (Fig. 7A) and

13

CO2

(Fig. 7B) was detected in the sludge taken from the digesters treating control WAS, suggesting that acetate oxidation did not occur, and the primary working mode of acetate metabolism should be aceticlastic methanogenesis. However, there was a significant 13CH4 production in the sludge taken from the digesters treating WAS with BEFP, and the content of which was even higher than that of

12

CH4 (Fig. 7A),

suggesting that acetate oxidation was highly occurred and DIET-based methanogenic communities might participate in acetate metabolism.

The study presented here for the first time demonstrated that, the DIET-based methanogenic communities could participate in the direct oxidation of acetate with the release of electrons, similar to the observation in the anodic biofilm of microbial electrolysis cells (MECs) with acetate as substrate 49. Actually, Geobacter species are expected to have higher affinity acetate transporters than methanogens because of their ability to maintain lower acetate levels than methanogens when Fe(III) is available 50. The potential acetate oxidation by Geobacter species via DIET has been also demonstrated in defined co-cultures of G. sulfurreducens and Thiobacillus denitrificans with nitrate as the electron acceptor in the presence of magnetite

51

as

well as in defined co-cultures of G. metallireducens and G. sulfurreducens with fumarate as the electron acceptor

52

. The possibility that Geobacter species may 24


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directly oxidize acetate with DIET to Methanothrix species warrants further investigation in defined co-cultures.

Conclusions The study presented here proposed a novel method of WAS pretreatment named after BEFP. The results demonstrated that, initially pretreating the WAS with BEFP to achieve the ethanol production could stimulate the methanogenic communities to establish the DIET-based syntrophic metabolism. The primary reason was that, On the one hand, the effective hydrolysis/acidification with BEFP provided the more available substrates for the DIET-based syntrophic partners; On the other hand, the ethanol produced via BEFP continuously stimulated the methanogenic communities to specifically enrich the DIET-based syntrophs, such as Geobacter species. As a result, the methane production and sludge reduction with BEFP were significantly higher than that without BEFP. Further investigations revealed that, the DIET-based syntrophic metabolism established with BEFP could potentially participate in acetate oxidation, which was of great importance to AD of WAS, since the relatively slow methanogenic metabolism via aceticlastc pathway could not provide the sufficient energy to support the growth of Methanothrix species.

Conflict of Interest Statement 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. 25



Page 26 of 48

Acknowledgments The authors acknowledge the financial support from the National Natural Scientific Foundation of China (51578105) and the Fundamental Research Funds for the Central Universities (DUT17RC(3)081).

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Table 1. Main characteristics of the initial WAS. Error bars represent standard deviations of three-group parallel measurements.

Parameters pH TSS (total suspended solids) VSS (volatile suspended solids) TCOD (total chemical oxygen demand) SCOD (solute chemical oxygen demand) Total proteins Soluble proteins Total carbohydrates Soluble carbohydrates Total SCFAs (total short-chain fatty acids) and ethanol Ethanol Acetate Propionate Butyrate - meant not detected

35


Value ± SD 6.70 ± 0.02 62259.3 ± 871.0 mg/L 42909.3 ± 615.3 mg/L 66810 ± 1982.1 mg/L 2841.3 ± 90.5 mg/L 4402.7 ± 224.2 mgCOD/L 347.0 ± 34.0 mgCOD/L 2490.3 ± 127.3 mgCOD/L 381.8 ± 150.3 mgCOD/L 833.7 ± 82.9 mgCOD/L 511.8 ± 51.4 mgCOD/L 169.0 ± 18.2 mgCOD/L 152.9 ± 13.3 mgCOD/L

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 36 of 48

Table 2. Main characteristics of the pretreated WAS after 8-day experiments (The pH of the pretreated WAS was adjusted to 7.0 ± 0.2). Error bars represent standard deviations of three-group parallel experiments.

Items

Control WAS

WAS with BEFP

WAS with Ethanol

TSS (mg/L) VSS (mg/L) TCOD (mg/L) Total Proteins (mgCOD/L) Total Carbohydrates (mgCOD/L) Total SCFAs and ethanol (mgCOD/L) Ethanol (mgCOD/L) Acetate (mgCOD/L) Propionate (mgCOD/L) Butyrate (mgCOD/L) - meant not detected

58877.5 ± 652.7 40230.0 ± 885.3 65155.0 ± 1916.3 3501.8 ± 328.0 2155.7 ± 93.4 1966.7 ± 123.4 556.5 ± 13.4 778.2 ± 33.9 632.2 ± 24.9

62126.5 ± 266.6 42245.0 ± 405.9 61415.0 ± 530.3 2310.8 ± 450.1 688.0 ± 70.2 4544.7 ± 145.6 1747.4 ± 56.3 2064.7 ± 123.8 275.9 ± 12.3 456.8 ± 7.8

60854.5 ± 2386.5 42282.0 ± 2556.9 63295.0 ± 970.1 3446.1 ± 379.9 2072.3 ± 25.9 4322.5 ± 78.9 1812.4 ± 28.2 689.4 ± 17.9 812.3 ± 24.3 1008.4 ± 117.8

36


WAS with Pretreatment at pH 10 62275.0 ± 659.0 35874.5 ± 24.7 65160.0 ± 212.1 1555.1 ± 113.5 991.8 ± 46.1 8782.1 ± 123.8 5845.8 ± 145.6 1240.8 ± 78.1 1629.3 ± 56.3

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Table 3. Performances of AD of control WAS, WAS with BEFP and WAS with ethanol, respectively. Error bars represent standard deviations of three-group parallel experiments.

HRT (Days)

34

28

24

Items Average Methane Production Rate (mL/g-VSS/d) Effluent VSS (mg/L) Effluent Total COD (mg/L) Effluent Total Proteins (mgCOD/L) Effluent Total Carbohydrates (mgCOD/L) Effluent Total SCFAs and ethanol (mgCOD/L) Average Methane Production Rate (mL/g-VSS/d) Effluent VSS (mg/L) Effluent Total COD (mg/L) Effluent Total Proteins (mgCOD/L) Effluent Total Carbohydrates (mgCOD/L) Effluent Total SCFAs and ethanol (mgCOD/L) Average Methane Production Rate (mL/g-VSS/d) Effluent VSS (mg/L) Effluent Total COD (mg/L) Effluent Total Proteins (mgCOD/L) Effluent Total Carbohydrates (mgCOD/L) Effluent Total SCFAs and ethanol (mgCOD/L)

Control WAS 181.2 ± 21.6 27845.7 ± 3394.3 45543.3 ± 298.4 1657.0 ± 103.3 1572.4 ± 235.4 497.4 ± 53.9 216.1 ± 14.1 24507.0 ± 2928.8 37695.0 ± 1004.8 1455.7 ± 93.4 1497.3 ± 133.5 218.8 ± 25.2 179.9 ± 10.2 21726.5 ± 700.7 39876.7 ± 106.8 999.6 ± 121.7 937.8 ± 113.1 264.2 ± 59.4

37


WAS with BEFP 179.5 ± 19.9 27715.0 ± 271.5 40930.0 ± 556.9 1589.3 ± 25.9 432.3 ± 89.7 413.1 ± 111.2 237.0 ± 17.2 23708.0 ± 1282.7 31805 ± 331.4 972.4 ± 209.9 318.3 ± 164.8 189.4 ± 18.6 233.5 ± 16.5 17630.5 ± 1750.1 31653.3 ± 272.4 790.3 ± 284.0 388.0 ± 70.2 552.1 ± 29.7

WAS with Ethanol 179.3 ± 15.3 29287.0 ± 1912.7 41303.0 ± 436.7 1489.5 ± 340.6 1354.4 ± 76.3 630.1 ± 195.5 230.3 ± 11.3 24730.0 ± 162.6 32496.5 ± 287.0 906.9 ± 76.7 884.5 ± 123.4 217.2 ± 33.7 224.9 ± 15.6 16914.3 ± 399.1 33489 ± 124.5 1008.8 ± 109.2 770.0 ± 76.8 300.3 ± 41.7

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 38 of 48

Table 4. Performances of AD of WAS with pretreatment at pH 10 and BEFP, respectively. Error bars represent standard deviations of three-group parallel experiments.

HRT (Days)

34

28

24

Items Average Methane Production Rate (mL/g-VSS/d) Effluent VSS (mg/L) Effluent Total COD (mg/L) Effluent Total Proteins (mgCOD/L) Effluent Total Carbohydrates (mgCOD/L) Effluent Total SCFAs and ethanol (mgCOD/L) Average Methane Production Rate (mL/g-VSS/d) Effluent VSS (mg/L) Effluent Total COD (mg/L) Effluent Total Proteins (mgCOD/L) Effluent Total Carbohydrates (mgCOD/L) Effluent Total SCFAs and ethanol (mgCOD/L) Average Methane Production Rate (mL/g-VSS/d) Effluent VSS (mg/L) Effluent Total COD (mg/L) Effluent Total Proteins (mgCOD/L) Effluent Total Carbohydrates (mgCOD/L) Effluent Total SCFAs and ethanol (mgCOD/L)

WAS with preatment at pH 10 186.6 ± 20.3 27999.7 ± 1959.3 48353.3 ± 759.6 735.7 ± 324.4 572.4 ± 35.4 740.3 ± 144.0 215.4 ± 31.1 25103.3 ± 3247.8 40380.0 ± 2652.1 550.0 ± 569.3 602.2 ± 119.4 1330.8 ± 281.2 173.6 ± 38.6 19319.3 ± 1986.8 38970.0 ± 2652.1 1052.5 ± 37.8 886.9 ± 71.9 3754.8 ± 536.3

38


WAS with BEFP 179.5 ± 19.9 27715.0 ± 271.5 40930.0 ± 556.9 1589.3 ± 25.9 432.3 ± 89.7 413.7 ± 146.8 237.0 ± 17.2 23708.0 ± 1282.7 31805 ± 331.4 972.4 ± 209.9 318.3 ± 164.8 189.4 ± 18.6 233.5 ± 16.5 17630.5 ± 1750.1 31653.3 ± 272.4 790.3 ± 284.0 388.0 ± 70.2 552.1 ± 29.7

Page 39 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure.1. Flow diagram of the experimental operations.

39



Methane Production Rate (mL/g-VSS/d)

(A) 350

HRT = 34 d

HRT = 24 d

HRT = 28 d

300

250

200

150

WAS WAS with BEFP WAS with Ethanol

100

0

20

40

60

80

Time (Days)

(B) 100 90

VSS Removal Efficiency (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 70 60 50 40 30 20 10 0

HRT = 34 d

HRT = 28 d

40


HRT = 24 d

Page 41 of 48

(C)100 TCOD Removal Efficiency (%)

90 80


70 60 50 40 30 20 10 0

HRT = 34 d

(D) 100

WAS

HRT = 24 d

HRT = 28 d

WAS with BEFP

WAS with Ethanol

90

Methane Conversion Efficiency (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 70 60 50 40 30 20 10 0

HRT = 34 d

HRT = 28 d

HRT = 24 d

Figure.2. Methane production rates (A), VSS removal efficiencies (B), TCOD removal efficiencies (C) and methane conversion efficiencies (D) in the digesters treating WAS, WAS with BEFP and WAS with ethanol, respectively, during the 86-day experiments. Error bars represent standard deviations of three-group parallel experiments

41



1.2

1.0

0.8

2

Conductivity (uS/cm /g/L-VSS)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.6

0.4

0.2

0.0

Inoculum

WAS with BEFP

WAS

WAS with Ethanol

Figure.3. Conductivity of the sludge in the digesters treating WAS, WAS with BEFP and WAS with ethanol, respectively, after 86-day experiments. Error bars represent standard deviations of three-group parallel experiments.

42


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Page 43 of 48 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(A) WAS with Ethanol

Others Methanobrevibacter Methanosphaerula Methanosphaera Methanomassiliicoccus Methanococcus Methanothrix Methanospirillum

WAS with BEFP

WAS

Inoculum Sludge

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance (%)

(B) WAS with Ethanol

WAS with BEFP

WAS

Inoculum sludge

0

20

40

60

80

100

Relative Abundance (%)

Others Unclassified Neglecta Desulfobulbus Petrimonas Sphaerochaeta Geobacter Thermovirga Mariniphaga Oblitimonas Proteiniphilum Aminobacterium Proteiniborus Tissierella Dechloromonas Mageeibacillus Labilibacter Tangfeifania Ornatilinea Bellilinea Syntrophomonas Emergencia Desulfotomaculum Lutispora Tindallia Acutalibacter Sporobacter Levilinea

Figure.4. Archaeal (A) and bacterial (B) community structure of the sludge in the digesters treating WAS, WAS with BEFP and ethanol, respectively, as well as inoculum sludge after 86-day experiments. The genus level with relative abundance lower than 2.00% was classified into group ‘others’.

43



WAS with pretreatment at pH 10 for 8 d WAS with BEFP Methane Production Rate (mL/g-VSS/d)

(A) 350

HRT = 34 d

HRT = 28 d

HRT = 24 d

300

250

200

150

100

0

20

40

60

80

Time (Days)

(B)100 90

VSS Removal Efficiency (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 48

WAS with pretreatment at pH 10 for 8 d WAS with BEFP

80 70 60 50 40 30 20 10 0

HRT = 34 d

HRT = 28 d

44


HRT = 24 d

Page 45 of 48

(C) 100 90


TCOD Removal Efficiency (%)

80 70 60 50 40 30 20 10 0

HRT = 34 d

(D)100 Methane Conversion Efficiency (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


HRT = 28 d

HRT = 24 d


80

60

40

20

0

HRT = 34 d

HRT = 28 d

HRT = 24 d

Figure.5. Methane production rates (A), VSS removal efficiencies (B), TCOD removal efficiencies (C) and methane conversion efficiencies (D) in the digesters treating WAS with pretreatment at pH 10 and BEFP, respectively, during the 86-day experiments. Error bars represent standard deviations of three-group parallel experiments

45



Figure.6. Change of ethanol (A), acetate (B), propionate (C), butyrate (D) and total SCFAs and ethanol (E) in the digesters treating WAS with AP and BEFP, respectively, during the 86-day experiments. Error bars represent standard deviations of three-group parallel experiments.

46


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Page 47 of 48

(A) 12

12

WAS with pretreatment at pH 10 for 8 d CH4

Content of Methane (%-headspace)

12

WAS with BEFP CH4

10

13

WAS with pretreatment at pH 10 for 8 d CH4 13

WAS with BEFP CH4

8

6

4

2

0 0

2

4

6

8

10

Time (h)

(B) 12 Content of Carbon Dioxide (%-headspace)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


12

WAS with pretreatment at pH 10 for 8 d CO2 12

WAS with BEFP CO2

10

13

WAS with pretreatment at pH 10 for 8 d CO2 13

WAS with BEFP CO2

8

6

4

2

0 0

2

4

6

8

10

Time (h)

Figure.7. Methane (A) and carbon dioxide (B) production in the headspace of the tubes during the batch experiments with stable carbon isotope. Error bars represent standard deviations of four-group parallel experiments.

47



TOC Graphic:

For Table of Contents Use Only

A Brief of TOC Graphic: Establishing DIET-based syntrophic metabolism during AD of WAS via biological ethanol-type fermentation pretreatment (BEFP) to achieve the better methanogenesis.

48


Page 48 of 48

Establishing Direct Interspecies Electron Transfer during Laboratory

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