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Hydrogen recovery from waste activated sludge: Role of free nitrous acid (FNA) in a pre-fermentation-microbial electrolysis cells system Zhihong Liu, Aijuan Zhou, Jiaguang Zhang, Sufang Wang, Yunbo Luan, Wenzong Liu, Aijie Wang, and Xiuping Yue ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04201 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Hydrogen recovery from waste activated sludge: Role of free nitrous

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acid (FNA) in a pre-fermentation-microbial electrolysis cells system

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Zhihong Liu 1, Aijuan Zhou 1, 2, *, Jiaguang Zhang 3, Sufang Wang 1, Yunbo Luan 4,

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Wenzong Liu 5, Aijie Wang 5, 6 and Xiuping Yue 1, 2, *

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Technology, 79 Yingzexi Road, Taiyuan 030024, P.R. China

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Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, Haidian District, Beijing 10085, P.R. China

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College of Mechanics, Taiyuan University of Technology, 79 Yingzexi Road, Taiyuan 030024, P.R. China

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College of Architecture and Civil Engineering, Taiyuan University of Technology, 79 Yingzexi Road, Taiyuan 030024, P.R. China

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Shanxi Engineer Research Institute of Sludge Disposition and Resources, Taiyuan University of Technology, 79 Yingzexi Road, Taiyuan 030024, P.R. China

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College of Environmental Science and Engineering, Taiyuan University of

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State Key Laboratory of Urban Water Resource and Environment, Harbin Institute

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of Technology (SKLUWRE, HIT), 73 Huanghe Road, Nangang District, Harbin

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150090, P.R. China

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*Corresponding author:

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College of Environmental Science and Engineering, Taiyuan University of

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Technology, 79 Yingzexi Road, Taiyuan 030024, Shanxi Province, P.R. China

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E-mail: [email protected] (A. Zhou); [email protected] (X. Yue);

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Tel./fax: +86 0351-3176581.

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Abstract

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Due to the limited hydrolysis rate of particulate organics and suitable substrates for

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hydrogen-producing bacteria in raw waste activated sludge (WAS), traditional

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fermentative hydrogen production has low hydrogen yield and energy recovery

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efficiency. The role of free nitrous acid (FNA) pretreatment on WAS and hydrogen

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recovery was investigated in a pre-fermentation-microbial electrolysis cells (MECs)

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system. The results demonstrated that WAS hydrolysis and acidification were

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enhanced by FNA pretreatment. Notably, the accumulation of acetic acid and

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propionic acid eventually reached to 55% and 22% during pre-fermentation. During

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MECs cascade utilization, volatile fatty acids (VFAs) were exhausted and the

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utilization efficiencies of soluble carbohydrates and proteins reached 62% and 41.5%,

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respectively. The hydrogen yield from FNA-pretreated sludge was 1.44 mL/g VSS,

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which was approximately 3 times than that of the control. High-throughput

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sequencing and canonical correspondence analysis revealed that FNA pretreatment

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promoted the hydrolysis and acidification of particulate organics through

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accumulating anaerobic fermentation bacteria (AFB) in pre-fermentation, furthermore,

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stimulated the increase of electrochemically active bacteria (EAB) and therefore

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enhanced the current and hydrogen production. This study may provide a sound basis

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for the potential implementation of FNA pretreatment to accomplish organics

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cascading utilization and the synchronous recovery of energy from WAS.

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Keywords

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Waste activated sludge (WAS); Free nitrous acid (FNA); Anaerobic pre-fermentation;

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Microbial electrolysis cells (MECs); Hydrogen production

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Introduction Waste activated sludge (WAS) is the most voluminous by-product of biological

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wastewater treatment, which requires proper handling, treatment and disposal.1-4 In

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China, 30~40 million tons of WAS (80% moisture content) are produced annually, and

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the amount is expected to reach 60~90 million tons by 2020. WAS is considered to be

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a potential source of organic compounds, containing large amounts of carbohydrates

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and proteins, which account for 35-61% and 7-11% of the total chemical oxygen

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demand (TCOD) of WAS.5 Relevant research has concentrated on transforming these

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considerable organics into biogas, fertilizer, or easily-biodegradable carbon sources.6

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Recently, greater attention has been paid to bio-hydrogen production from WAS due

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to its high energy yield of 142.9 KJ/g and zero pollution in utilization.7

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Unfortunately, most biodegradable organics in WAS is either enclosed inside the

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microbial cell wall or enmeshed in extracellular polymeric matrix, which further

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contributes to limit the biodegradability of these organics into hydrogen. In order to

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accelerate the hydrolysis of organic matters, WAS is often pretreated by various

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physical and chemical methods prior to test.8 Lately, free nitrous acid (FNA),

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protonated form of nitrite, was demonstrated to be an effective and promising method

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for WAS pretreatment.9-11 It was found that FNA increased biodegradability by

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generating multiple inhibitory effects on microbial metabolism of extensive

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microorganisms,12 being extraordinarily biocidal to microorganisms at parts per

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million (ppm) or even sub-ppm levels.13-14 FNA pretreatment was reported to be

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effective on enhancing hydrolysis rate, volatile fatty acids (VFAs) and methane

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production of WAS. The biodegradability of FNA-pretreated sludge could be

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promoted by 20%-50% based on biochemical methane potential (BMP) testing.10, 15-16

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Methane production could be improved by 10-30% at FNA concentrations in the 3

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range of 0.4-2.1 mg N/L for 24 h.10 Additionally, Li et al. (2016) proved the shorter

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fermentation time and highest VFAs production could achieve at 1.8 mg N/L of

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FNA.17 However, an improvement of the biogas production rate is further considered

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by upgrading conventional AD systems.18-19

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MECs is a prospective technology for hydrogen production via the utilization of

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substrates from WAS. Recently, a number of studies have suggested that combine

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MECs with anaerobic digestion (AD), could not only increase the positive synergisms

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established in the digesters, but also provide an utilization route for the existing

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digestate, which further accomplish the bio-hydrogen recovery from WAS.7 Wang et

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al. (2014) obtained the yield of hydrogen at 8.5 mg H2 g-1 VSS feeding with sodium

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dodecylsulphate (SDS)-treated WAS fermentation liquid (SFL).5 Zhou et al. (2016)

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harvest 12.90 mg H2 g-1 VSS from Rhamnolipid bio-surfactant (RL)-treated SFL.20

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In this study, the feasibility of hydrogen recovery and cascade utilization of

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organics from WAS were investigated in a pre-fermentation-microbial electrolysis

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system. The performance of FNA pretreatment on WAS solubilization and

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acidification during the pre-fermentation process was studied. The hydrogen

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production and the degradation rules of the substrates of the fermented

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FNA-pretreated WAS were analyzed during the subsequent MECs process.

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Furthermore, high-throughput sequencing analysis was used to evaluate the

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mechanism of FNA pretreatment for enhancing hydrogen recovery from WAS on the

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microorganism level. In addition, we used canonical correspondence analysis (CCA)

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to assess the correlations between major environmental variables and functional

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microbial populations.

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Material and methods

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FNA pretreatment and pre-fermentation of WAS

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WAS was collected from a Zhengyang municipal wastewater treatment plant

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(WWTP) in Jinzhong City, China. Prior to use, WAS was concentrated by settling for

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24 h at 4 °C, and the supernatant was then removed. To prevent clogging problems,

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concentrated WAS was screened with a 40-mesh sieve. The main characteristics

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(average value, plus standard deviation of triplicates) were as follows: total suspended

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solids (TSS): 26.4 ± 0.4 g/L; volatile suspended solids (VSS): 12.1 ± 0.2 g/L; total

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chemical oxygen demand (TCOD): 26.2 ± 0.2 g/L; soluble chemical oxygen demand

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(SCOD): 0.41 ± 0.05 g/L; VFAs: 255 ± 12 mg COD/L; soluble carbohydrates: 44.56 ±

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8 mg COD/L; and soluble proteins: 37.5 ± 2 mg/L, pH 7.03 ± 0.02.

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Three serum bottles with a working volume of 400 mL each were set-up for

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WAS pretreatment by FNA. This process was termed “Phase 1”. A nitrite stock

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solution was first added to reactors to achieve a NO2--N concentration of 300 mg N/L.

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The pH was adjusted to 5.5 ± 0.1 by adding 3.0 M HCl. Twenty milliliters of

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phosphate buffer solution (PBS) was added in each reactor to maintain moderately

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acidic conditions. The pretreatment lasted for 24 h, during which the temperature was

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maintained at 22 ± 1 °C. Under these conditions, the FNA concentration was titrated

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to 2.13 mg N/L based on a calculation applying the formulae SNO2--N/(Ka×10pH) and

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Ka=e-2300/(273+T) 21, which proved to be an optimal FNA dosage for WAS

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solubilization.10 To evaluate the effect of FNA pretreatment, NO2--N, NO3--N, NH4+-N,

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soluble proteins and soluble carbohydrates were measured every 4 hours.

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Anaerobic pre-fermentation experiments were carried out in six batch reactors, a

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process that was termed “Phase 2”. These reactors, with a 400-mL working volume

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each, were divided into two groups (every three reactors were replicates for each 5

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group). One group was fed concentrated WAS (hereafter referred to as the

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AD_Control test). The feedstock for the other group was FNA-pretreated WAS

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(hereafter referred to as the AD_FNA test). Some of the fresh sludge was maintained

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under aeration at room temperature overnight and then used as inoculum with an

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addition ratio of 1:9 (v/v). Nitrogen gas was pumped into reactors for 10~15 min in

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order to remove oxygen; then, all reactors were capped, sealed, and stirred in an

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air-bath shaker (120 rpm) at 35 ± 1 °C.

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MECs reactors setup and operation

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Ten single-chamber MECs reactors were constructed simultaneously and

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inoculated using aeration tank effluent from the same WWTP in Jinzhong. Details of

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volume, anode and cathode were provided previously.7, 22 The reactors were operated

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at room temperature (22 ± 1 °C), a voltage of 0.80 ± 0.01 V was applied (Switching

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Power Supply, FDPS-150, Fudantianxin Inc. China), and 100 mM PBS (pH 7.0) and

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1,500 mg/L acetate were prepared for the feedstock of the MECs set-up. The setup

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time lasted for 10 days in approximately 24-h batches until the currents reached 2 mA,

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and the coulombic efficiencies were greater than 90%. Then, six parallel reactors were

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selected for the subsequent experiment. Reactors were divided into two groups in a

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process termed “Phase 3”. The pre-fermented WAS was added to each group, mixed

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with 100 mM PBS (VWAS:VPBS=1:1) and incubated for 3 days as a cycle to make full

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use of the organics.

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DNA extraction and Illumina MiSeq sequencing

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Illumina MiSeq sequencing was used to analyze the diversity of the microbial

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community. Before DNA extraction, sludge samples of pre-fermentation experiments

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and carbon brushes (bio-anode) were centrifuged at 8000 × g to remove the

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supernatant. DNA was extracted in triplicate from sludge sediments using an EZNA® 6

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Soil DNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA) and subsequently pooled

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together. Based on the amplification of V3-V4 region of the 16S rRNA gene, the

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commonly used PCR primers 341F (5′-CCCTACACGACGCTCTTCCGATCTG-3′)

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and 805R (5′-GACTGGAGTTCCTTGGCACCCGAGA ATTCCA-3′) were selected.

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During sequencing, sample multiplexing was achieved by means of the incorporation

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of barcodes between the forward primer and 454 adaptor. Polymerase chain reactions

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(PCRs) were performed as described previously.23-24 Then, the PCR amplicon was

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purified and quantified and was further applied to sequencing on an Illumina MiSeq.

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For taxonomic analysis, the 16S rRNA gene sequences read was performed

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individually using Ribosomal Database Project (RDP) classifier. Raw sequence data

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of the study were deposited to the NCBI Short Read Archive database with the

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accession no. SRR5482157. Notably, we trimmed the adapters, barcodes, and primers

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in all raw sequences to avoid the effects of random sequencing errors. The sequence

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was removed if it was shorter than 350 bp or contained any ambiguous base cells.

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The remaining sequences were clustered into operational taxonomic units

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(OTUs) using a 97% identity threshold (3% dissimilarity level). Rarefaction curves

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were generated and alpha diversity measurements, including the Shannon index

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(http://www.mothur.org/wiki/Shannon), Chao1 index

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(http://www.mothur.org/wiki/Chao) and ACE index

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(http://www.mothur.org/wiki/Ace), were calculated for each sample. BLAST of

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taxonomic classification was conducted using MOTHUR program via SILVA database

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with a set confidence threshold of 80%. Hierarchical clustering analysis (HCA) was

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used to display the interrelationship among the four samples intuitively on the basis of

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the depths of colors, which represented the abundance of the bacteria communities. In

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this study, we performed HCA analysis using GraPhlAn 0.9.7 and iTOL 3.2.1. 7

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Cytoscape v3.2.1 was used to generate OTUs networks, which could further delineate

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the similarity and diversity between the different sludge samples. The central portion

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was shared OTU, while the species and numbers of the other bacterium showed the

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diversity among the four samples.25 Principal coordinates analysis (PCoA) visually

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reflected beta diversity, which was calculated using the distance matrices based on the

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UniFrac phylogenetic method. Canonical correspondence analyses (CCA) were

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generated by Canoco 4.5 to evaluate the specific correlations between characteristic

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genera and environmental factors measured in the study, including methane, VFAs,

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NO2--N, current, hydrogen, soluble proteins and carbohydrate concentrations. The

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relative abundance of 16 characteristic bacteria was used in the CCA analysis.

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Analysis and calculation methods

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WAS samples were centrifuged at 10,000 × g for 10 min, filtered through a

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0.45-µm cellulose nitrate membrane filter and stored at 4 °C prior to analysis. TCOD,

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SCOD, TSS, VSS, NH4-N, NO3--N and NO2--N were measured according to standard

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methods (APHA, 1998). Soluble proteins were measured using a bicinchoninic acid

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(BCA) protein quantitation kit, whereas soluble carbohydrates was determined using

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the phenol–sulfuric acid method with glucose as the standard.26 VFAs were analyzed

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using gas chromatography, and the total concentration comprised acetic acid (HAc),

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propionic acid (HPr), n-/iso- butyric acid (n-HBu/iso-HBu) and n-/iso- valeric acid

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(n-HVa/iso-HVa), expressed as the COD concentration (mg COD/L). The equivalent

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relationships between COD and various VFAs were as follows: 1.07 g-COD/g-HAc,

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1.51 g-COD/g-HPr, 1.82 g-COD/g- (n-/iso-) HBu, and 2.04 g-COD/g- (n-/iso-) HVa.

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The gases produced by anaerobic reactors and MECs were collected in gas bags, and

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the total volumes were measured by glass syringe. Gas composition was analyzed by

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gas chromatography. The voltage and current were recorded via a data recorder 8

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(model 2700, Keithley Instrument, USA) every 10 minutes. Crucial indices to judge

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the performance of MEC reactors included coulombic efficiency (CE), current density

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(A/m2, normalized to the projected cathode area), hydrogen production rate Q

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(m3-H2/m3/day), hydrogen yield YH2 (mL H2/g VSS) and energy recovery efficiency.27

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Energy efficiency (ηE, %) was calculated on the basis of electricity input as ηE = WH2 /

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Win, where WH2 is the energy content of hydrogen based on the heat of combustion

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(upper heating value of 285.83 kJ per mole of H2) and Win is the electricity input

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determined as ܹ௜௡ = ‫ܧܫ(׬‬௔௣ − ‫ܫ‬ଶ ܴ) ∙ ݀‫ݐ‬, where Eap is the applied voltage (0.8 V in

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this study) and R is the external resistance (10 Ω) 28.

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Results and discussion

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Effect of FNA pretreatment on WAS solubilization and acidification

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The degree of WAS solubilization can be expressed by the change of soluble

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protein and carbohydrate levels.29-30 As shown in Fig. 2A and B, soluble carbohydrate

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and protein levels increased with FNA pretreatment and peaked at 298.7 ± 6.5 mg

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COD/L and 665.3 ± 30.1 mg COD/L (12 h), which was 6.7 and 18.4 times higher than

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that of the control, respectively. The eventual values were still much higher than that

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of un-pretreated WAS. Remarkably, NO2--N concentration increased with treatment

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time and peaked at 226.9 ± 7.5 mg/L during the initial 12 h. NO3--N content also

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achieved its maximum (40.4 ± 3.9 mg/L), which demonstrated that the nitration

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process occurred during Phase 1 (Fig. 1A). No methane was produced but 140 mL of

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gas was collected at the end of the pretreatment, consisted with N2 and N2O. The

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results could in accordance with Wang et al. (2013), who detected only N2 on the first

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day and proved that denitrification occurred during FNA pretreatment.10 In short, the

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results indicated that FNA pretreatment was effective in enhancing WAS

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solubilization. 9

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In 96-h pre-fermentation, the effect of FNA pretreatment on soluble

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carbohydrates and proteins is shown in Fig. 2A and B. Both soluble carbohydrates and

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proteins were clearly elevated from 48 h onward and decreased slightly with the time

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extension. By comparison, soluble proteins fluctuated slightly. The final

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concentrations of soluble carbohydrates and proteins in the AD_FNA test (120 h)

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were 2.46 and 4.14 times higher than that of the AD_Control test. Analogously, as a

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by-product of protein degradation, the trend of ammonia was similar with that of

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proteins (Fig. 1B). The concentration of VFAs in the AD_FNA test reached its

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maximum (1761 mg COD/L) on the fourth day, which was 5.0-fold of that obtained in

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the AD_Control test (Fig. 2C, Phase 2). Obviously, the components of VFAs reflected

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significant difference. HAc and HPr, the two top individual VFAs, reached 55% and

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22% in the AD_FNA test, respectively (Fig. 2D). However, the levels in the

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AD_Control test were 10% and 61%, respectively (Fig. 2D). The n-HVa was the

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lowest VFAs in any of the measured reactors, which was less than 3%. Thus, FNA

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pretreatment could expedite the production of VFAs, particularly HAc.

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Hydrogen recovery and organics utilization during MECs cascade utilization

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Fig. S1A depicted the current variation during the MECs startup. The average

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current tended to be stable and eventually decreased from ~7 mA to 2 mA (> 2 mA).

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Moreover, the coulombic efficiency and hydrogen yield rate were elevated from 85 ±

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6% to 101 ± 5% and 79 ± 6% to 154 ± 12%, respectively. On basis of the energy of

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electricity input, the average energy efficiency of hydrogen yield was up to 140 ± 11%

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at 0.8 V applied voltage. These phenomena implied that the MECs were successfully

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constructed.

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During MECs operation, the variation of electron transport (current) was

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connected with soluble substrates, especially VFAs degradation.5 As shown in Fig. 10

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S1B, FNA pretreatment contributed to abundant soluble substrates for exoelectrogens,

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accordingly, the maximal current density was 11 A/m2; while the limited releases of

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soluble substrates in the control group resulted in the lower current density (only 4

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A/m2). The currents both stepped to decreases along with the consumption of soluble

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substrates (Fig. 2C). The hydrogen production yield was 1.44 mL/g VSS in the FNA

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test, which was 2.7-fold higher than that of the control test (Fig. 2E). FNA

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pretreatment promoted accumulation of HAc and HPr in pre-fermentation (account

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for 77% of VFAs), which benefited hydrogen production and further shortened

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operational time.5

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As seen from Fig. 2A, the soluble carbohydrates began to decline sharply at 126

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h and the final removal efficiency increased to 62% in the FNA test, whereas in the

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control test, it was only 43%. The change of ammonia was consistent with soluble

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proteins (Fig. 1A), which markedly increased at 126 h and then appeared to integrally

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decrease. However, the eventual removal efficiencies (34.7% and 41.5% in control

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test and FNA test, respectively) were still less than that of soluble carbohydrates (Fig.

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2B). These results implied that soluble carbohydrates were much easier to utilize by

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exoelectrogens than large-molecule proteins.

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VFAs, one of the main compositions of fermented WAS, could be easily

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consumed by exoelectrogens,28 as shown in Fig. 2C, reduced acutely in both FNA and

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control tests; the respective utilization efficiencies were 99% and 83%. It is reported

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that HAc and HPr are preferred as main electron donors, which provide significant

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current generation, while the other VFAs (HBu and HVa) mainly contribute to

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exoelectrogens metabolism.31 Accordingly, all components of VFAs were also

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consumed maximally (Fig. 2E). The results demonstrated that on the basis of FNA

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pretreatment, the soluble substrates released in pre-fermentation accomplished the 11

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cascade utilization in MECs and further achieved higher hydrogen production.

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Microbial community analysis

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The bacterial communities of AD_Control and AD_FNA, as well as the biofilm

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developed on the anodes of MECs, fed with fermented AD_Control sludge

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(AD_MEC_Control) and AD_FNA sludge (AD_MEC_FNA) were analyzed using

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Illumina MiSeq sequencing. In total, as shown in Table S1, the number of OTUs in

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the four bacteria samples was 13,697, of which 542 OTUs (4% of the total OTUs)

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were shared by all samples. The shared OTUs were mainly grouped into three phyla:

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Proteobacteria (41.9%), Bacteroidetes (12.7%), and Firmicutes (10.9%) (Fig. 4).

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Rarefaction curves for all libraries not only illustrated that the sequences of all

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samples were reasonable but also displayed shapes indicative of effective sampling of

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the community diversity (Fig. S2A). The microbial diversities of the involved

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communities in all samples were assessed based on α-diversity. Under FNA

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pretreatment, the Shannon index of samples decreased from 6.08 (AD_Control) to

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5.47 (AD_FNA), and further declined to 5.45 (AD_MEC_FNA) in the MECs system

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(Table S1). Based on Chao1 and ACE indices, which indicate richness, the

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AD_MEC_FNA sample also had relatively lower diversity (13,493 and 23,228,

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respectively). β-diversity was used to calculate and examine the similarity of the

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microbiome. PCoA exhibited the dissimilarity of bacterial community composition

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among the samples, based on unweighted UniFrac (Fig. S2B). The principal

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components 1 and 2 were 36.53% and 34.19%, respectively. In addition, the four

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samples were clearly separated from each other, which illustrated the significant

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differences in the bacterial community composition (Fig. S2B). This could be further

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demonstrated by the hierarchical clustering analysis results (Fig. 3D).

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Phylogenetic differences in 16S rRNA gene sequences were characterized at 12

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three different level classifications, including that of phylum, class and genus levels,

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to further investigate the diversity of the microbial community. The three dominant

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phyla, Proteobacteria, Bacteroidetes and Firmicutes, which have been identified in

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anaerobic32-34 or bioelectrochemical systems,35-37 were identical but with different

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relative abundance for all samples (Fig. 3A). At the class level, the majority of

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sequences belonged to 10 classes, among which Bacteroidia, Clostridia,

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Betaproteobacteria and Gammaproteobacteria were the shared predominant groups

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(Fig. 3B). Bacteroidia, Gammaproteobacteria and Clostiridia are capable of

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producing VFAs by utilizing soluble organics,38 which were all enhanced by FNA

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pretreatment no matter what subsequent treatments were. Bacteroidia and Clostiridia

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clearly increased in the AD-MEC cascading system, which may improve the

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generation of amino acids during protein hydrolysis39 and hydrogen production,40

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respectively.

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To obtain more detailed information on microbial communities, the genus level

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of different samples was examined (Fig. 3C). Macellibacteroides (class Bacteroidia),

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which can produce lactate, HAc, HBu and iso-HBu from glucose metabolism and

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utilize many kinds of carbohydrates,41 enriched to 6.6% in AD_FNA and further

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accumulated to 15.6% in AD_MEC_FNA, whereas it was only 0.4% and 1.7% in

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AD_Control and AD_MEC_Control, respectively. Petrimonas (class Bacteroidia),

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which are closely related to the utilization of various sugars and produce HPr and

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HAc as their primary products,42 peaked at 4.8% in AD_MEC_FNA.

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Proteiniclasticum (class Clostridia), which cannot utilize carbohydrate but can utilize

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soya peptone, tryptone and amino acids,43 accumulated to 4.2% in AD_FNA versus

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0.4% in AD_Control. Acetoanaerobium (class Bacilli), which can produce HAc from

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H2 and CO2,44 was enriched to 3.2% in the AD_MEC system. The detailed 13

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distribution information of these four predominant anaerobic fermentation bacteria

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(AFB) is illustrated in Fig. 4. The total relative abundance of AFB,41-49 as shown in

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Fig. 3C, illustrated significant differences among the four samples. AFB were

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enriched from 4.2% (AD_Control) to 13.6% (AD_FNA) and were further enhanced to

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19.5% (AD_MEC_Control) and 34.4% (AD_MEC_FNA) after MECs cascading

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treatment. The results demonstrated the intense solubilization and acidification of

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FNA-pretreated WAS occurred during AD_MEC cascading system. Electrochemically

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active bacteria (EAB), classified in a previous study,40 and which mainly attach to

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anodes, improved the decomposition of organic matter by consuming VFAs produced

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during pre-fermentation and converting e- and H+ (produced in the process) to H2 (Fig.

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3C). Their distributions intuitively demonstrated that EAB emerged only in MECs

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rather than in pre-fermentation (Fig. 4). The total relative abundances increased to 5.5%

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of AD_MEC_Control versus 8.7% of AD_MEC_FNA, which explained the

340

phenomenon of the higher hydrogen and current generated in the latter than that in the

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former. In addition, Ottowia, Thermomonas (class Proteobacteria) and

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Phaeodactylibacter (class Sphingobacteria), classified as nitrate-reducing bacteria

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(NRB), peaked at 13.7% in AD_FNA. Notably, Ottowia, which could contribute to

344

denitrification and produce N2O in the end,50 was enriched to 7.0% in AD_FNA

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versus 3.4% in AD_Control. Its distribution was displayed in Fig. 4, which explained

346

the denitrification that occurred in the AD_FNA test.

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Correlation between environmental variables and microbial populations

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To further explore the plausible relationship among characteristic genera, various

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environmental and performance measurements in WAS pre-fermentation and MECs

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systems, including soluble carbohydrates, VFAs, NO2--N, soluble proteins, hydrogen

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and current, we performed CCA using 16 characteristic bacteria (Fig. 5). The soluble 14

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protein and hydrogen contents as well as the current were found to be positively

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associated with the first canonical axis (accounting for 61.9% of the variance of

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genera distribution), whereas the other endpoints showed a negative correlation. For

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axis 2 (explaining 31.9% of the variance), only VFAs, soluble carbohydrates and

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NO2--N showed good positive correlations. Detailed information was shown in Table

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S2.

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The strength of the relationship between environmental variables and the

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microbial community could be indicated by the length of a vector that exists in an

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arrow-line form. As implied, soluble carbohydrates, current and hydrogen were

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significantly relevant to the microbial community based on the length of the vector,

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followed by VFAs and soluble proteins. Moreover, the intersection angle between

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VFAs and soluble carbohydrates implied VFAs and soluble carbohydrates were

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prominently correlated, and VFAs would be influenced when the soluble carbohydrate

365

content changed. Additionally, all factors were related to NO2--N. In other words, free

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nitrite increased as VFAs and soluble carbohydrates accumulated at the beginning of

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FNA pretreatment, whereas it decreased because of subsequent denitrification. During

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this process, characteristic bacterial genera, such as Ottowia, Thermomonas,

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Comamonas and Phaeodactylibacter, were enriched. As stated previously, we also

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found that current and hydrogen had a very high positive interaction with partial

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bacterial genera, including Olsenella, Cloacibacillus, Geobacter, Acetoanaerobium

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and Pseudomonas, which were the common genera in AD_MEC_Control and

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AD_MEC_FNA. Additionally, Macellibacteroides, Proteiniphilum and Petrimonas

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were the dominant genera in AD_MEC_FNA, which further contributed to hydrogen

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production. This is the reason why the hydrogen yield of AD_MEC_FNA was higher

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than that of AD_MEC_Control. In this sense, the correlation between environmental 15

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variables and characteristic genera further specified the function of the genera and

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was consistent with the conclusion of process assessment. The CCA results confirmed

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that the relationship between community structure and the measured variables may

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reveal precious insight into the WAS pre-fermentation and MECs system.

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Significance and potential implementation for practice

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Most sludge pretreatment technologies require substantial energy input, the

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external addition of chemical reagents and high costs, which hinder its large-scale

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application. In this study, FNA pretreatment has been proven to be a low-cost and

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eco-friendly technology for WAS, which can serve as an ideal approach for energy

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recovery from a WAS pre-fermentation-MECs system. Moreover, the MECs system

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would serve as the essential energy recovery processing unit for WWTPs due to its

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short operation time, high energy efficiency and excellent performance.

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Fig. 6 illustrates the enhanced energy recovery concept with the combined

390

system applied in a WWTP. In practice, FNA could be obtained in situ as a byproduct

391

from anaerobic digestion liquor during the nitritation process of wastewater treatment

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in a WWTP.10, 51-52 The produced FNA could evaluate WAS hydrolysis and

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acidification in anaerobic pre-fermentation and release adequate soluble substrates for

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MECs. MECs system would accomplish complete cascade utilization of the

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aforementioned organics by converting them to hydrogen and electric energy, which

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is beneficial for energy recovery and cost savings. It was reported that the net benefit

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($132600 per annum) can be achieved at 2.13 mg HNO2−N/L condition during

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20-days digestion from the heat and energy converted by methane.10 The benefit from

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hydrogen production was expected to be much higher than the ones from methane

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because of its higher calorific value. Additionally, the degradation of WAS lead to

401

lower disposal and transport costs. Certainly, the potential challenges should be 16

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further assessed in WWTPs practical implementation and related analysis should be

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focused on the life cycle, economic and ecological evaluations in whole processes for

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ensuring sustainability.

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Conclusion

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This study examined the feasibility of FNA pretreatment on hydrogen recovery

407

from WAS in a pre-fermentation-MEC system by process assessment associated with

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microbial community network analysis. FNA pretreatment was effective in enhancing

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WAS solubilization and acidification, promoting soluble substrates cascade utilization

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in MECs and accelerating hydrogen and energy recovery. The highest hydrogen yield

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from FNA pretreated WAS was 1.44 mL/g VSS, which was 2.7-fold higher than that

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obtained from un-pretreated WAS, meanwhile, the current density peaked to 11 A/m2

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of the former while it was only 4 A/m2 of the latter. It is worth noting that FNA

414

pretreatment stimulated accumulation of functional microorganism (AFB in

415

pre-fermentation and EAB in MECs), produced abundant soluble substrates and

416

further enhanced hydrogen production. In other words, FNA pretreatment played a

417

crucial role in the construction of the WAS innate microbial community by

418

sequencing, CCA and microbial network analysis. Zero-pollution and the in situ

419

synthesis during wastewater treatment would promote the crucial implementation of

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FNA for WAS treatment in WWTPs.

421

Supporting Information

422

Alpha diversity of four WAS samples; The eigenvalues of first two canonical

423

axes and their relationships with each environmental factor; Current generation of

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MECs; Rarefaction curves of the four bacterial communities based on sequencing of

425

16S rRNA gene; PCoA analysis based on the unweighted UniFrac analyses.

426

Acknowledgements 17

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This research was supported by the National Natural Science Foundation of

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China (NSFC, Nos. 51608345, 51708386 and 21501129), by the China Postdoctoral

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Science Foundation (Nos. 2015M570241, 2016M591416 and 2017T100170), by the

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Open Project of Key Laboratory of Environmental Biotechnology, CAS (No.

431

kf2016004), by the Key Research and Development (R&D) Project of Shanxi

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Province (No. 201603D321012) and the Scientific and Technological Project of

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Shanxi Province (No. 201701D221230 and 201601D021130).

434

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J. Syst. Evol. Microbio. 2013, 63 (6), DOI 10.1099/ijs.0.044719-0 (49) Floyd E. Dewhirst; B. J. P.; Nia Tzellas; Brittney Coleman; Julia Downes; David A. Spratt; William G. Wade. Characterization of novel human oral isolates and cloned 16S rDNA sequences that fall in the family Coriobacteriaceae: description of Olsenella gen. nov., reclassification of Lactobacillus uli as Olsenella uli comb. nov. and description of Olsenella profusa sp. nov. Int. J. Syst. Evol. Microbiol. 2001, 51, DOI 10.1099/00207713-51-5-1797. (50) Spring, S.; Jackel, U.; Wagner, M.; Kampfer, P. Ottowia thiooxydans gen. nov., sp. nov., a novel facultatively anaerobic, N2O-producing bacterium isolated from activated sludge, and transfer of Aquaspirillum gracile to Hylemonella gracilis gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 2004, 54 (Pt 1), DOI 10.1099/ijs.0.02727-0. (51) Wu, Q. L.; Guo, W. Q.; Bao, X.; Zheng, H. S.; Yin, R. L.; Feng, X. C.; Luo, H. C.; Ren, N. Q. Enhanced volatile fatty acid production from excess sludge by combined free nitrous acid and rhamnolipid treatment. Bioresour. Technol. 2017, 224, DOI 10.1016/j.biortech.2016.10.086. (52) Law, Y.; Ye, L.; Wang, Q.; Hu, S.; Pijuan, M.; Yuan, Z. Producing free nitrous acid – A green and renewable biocidal agent – From anaerobic digester liquor. Chem. Eng. J. 2015, 259, DOI 10.1016/j.cej.2014.07.138.

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Figure 1 Changes of NO3--N and NO2--N concentrations under FNA pretreatment

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during 24 h (A). Time-course profiles of NH4+-N concentration in the

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whole process (B).

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Figure 2 The cascade utilization of soluble carbohydrate (A) and protein (B) during

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the FNA pretreatment, pre-fermentation and MEC operation, respectively.

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Accumulation and consumption of VFAs in pre-fermentation and MEC

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operation (C), each component content of VFAs and corresponding

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production of methane and hydrogen yield at the end of pre-fermentation

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and MEC operation, respectively (D and E).

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Figure 3 Relative abundance was defined as the number of sequences per sample.

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Taxonomic classification of sequences from the four WAS bacterial

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communities at the phylum (A), class (B) and genus (C) levels.

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Hierarchical clustering analysis of bacterial communities of four samples

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(D).

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Figure 4 Microbial network analysis of the four WAS bacterial communities; overlap

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of the four bacterial communities based on OTU (3% distance).

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Figure 5 Canonical correspondence analysis (CCA) between enriched genera and

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environmental variables [soluble carbohydrates, VFAs, NO2--N, soluble

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proteins, hydrogen and current].

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Figure 6 Implement of FNA-based pre-fermentation-microbial electrolysis cells system in practice.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Abstract Graphic

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Synopsis

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This study displayed the crucial role of FNA pretreatment on hydrogen and energy recovery from WAS in pre-fermentation-microbial electrolysis cells system and its potential implement in WWTP.

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