High-rate anaerobic decolorization of methyl orange from synthetic azo dye wastewater in a methane-based hollow fiber membrane bioreactor

Journal Pre-proof High-rate anaerobic decolorization of methyl orange from synthetic azo dye wastewater in a methane-based hollow fiber membrane bioreactor Ya-Nan Bai, Xiu-Ning Wang, Fang Zhang, Jun Wu, Wei Zhang, Yong-Ze Lu, Ling Fu, Tai-Chu Lau, Raymond J. Zeng

PII:

S0304-3894(19)31707-8

DOI:

https://doi.org/10.1016/j.jhazmat.2019.121753

Reference:

HAZMAT 121753

To appear in:

Journal of Hazardous Materials

Received Date:

22 October 2019

Revised Date:

23 November 2019

Accepted Date:

23 November 2019

Please cite this article as: Bai Y-Nan, Wang X-Ning, Zhang F, Wu J, Zhang W, Lu Y-Ze, Fu L, Lau T-Chu, Zeng RJ, High-rate anaerobic decolorization of methyl orange from synthetic azo dye wastewater in a methane-based hollow fiber membrane bioreactor, Journal of Hazardous Materials (2019), doi: https://doi.org/10.1016/j.jhazmat.2019.121753

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High-rate anaerobic decolorization of methyl orange from synthetic azo dye wastewater in a methane-based hollow fiber membrane bioreactor

Ya-Nan Bai1,2, Xiu-Ning Wang3, Fang Zhang1,*, Jun Wu3, Wei Zhang1,3, Yong-Ze Lu3, Ling Fu3, Tai-Chu Lau2,4, and Raymond J. Zeng1,2,3*

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Fujian Provincial Key Laboratory of Soil Environmental Health and Regulation,

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College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China 2

Advanced Laboratory for Environmental Research and Technology, USTC-CityU,

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Suzhou, P. R. China

CAS Key Laboratory of Urban Pollutant Conversion, Department of Applied

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Chemistry, University of Science and Technology of China, Hefei 230026, P. R.

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China

State Key Laboratory in Marine Pollution, Department of Biology and Chemistry,

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City University of Hong Kong, Kowloon, Hong Kong

Corresponding author:

Prof. Fang Zhang at [email protected]. Tel/Fax: +86 591 83850781

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Prof. Raymond J. Zeng at [email protected]. Tel/Fax: +86 591 83303682

Graphical abstract

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Highlights

MO decolorization achieves a high level of decolorization efficiency (~

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100%)

The maximum decolorization rate is 883 mg/L/day

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Microbial community is changed significantly after MO decolorization

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Methanomethylovorans are dominant archaea and Moranbacteria are

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dominant bacteria

Archaea and bacteria play a synergistic role in MO decolorization in the

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HfMBR

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Abstract

Anaerobic biological techniques are widely used in the reductive decolorization of textile wastewater. However, the decolorization efficiency of textile wastewater by conventional anaerobic biological techniques is generally limited due to the low biomass retention capacity and short hydraulic retention time (HRT). In this study, a methane-based hollow fiber membrane bioreactor (HfMBR) was initially inoculated 2

with an enriched anaerobic methane oxidation (AOM) culture to rapidly form an anaerobic biofilm. Then, synthetic azo dye wastewater containing methyl orange (MO) was fed into the HfMBR. MO decolorization efficiency of ~ 100% (HRT=2 to 1.5 days) and maximum decolorization rate of 883 mg/L/day (HRT= 0.5 day) were obtained by the stepwise increase of the MO loading rate into the methane-based HfMBR. Scanning electron microscopy (SEM) and fluorescence in situ hybridization

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(FISH) analysis visually revealed that archaea clusters formed synergistic consortia

with adjacent bacteria. Quantitative PCR (qPCR), phylogenetic and high-throughput

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sequencing analysis results further confirmed the biological consortia formation of

methane-related archaea and partner bacteria, which played a synergistic role in MO

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decolorization. The high removal efficiency and stable microbial structure in HfMBR

textile wastewater.

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suggest it is a potentially effective technique for high-toxic azo dyes removal from

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Keywords: Methyl orange; Decolorization; Anaerobic methane oxidation; Hollow fiber membrane bioreactor; Archaea and bacteria;

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Introduction

Azo dyes containing aromatic groups and one or more –N=N− group, represent

the largest class of dyes applied in the textile industry [1, 2]. However, approximately 8-20% of the azo dyes are eventually discharged to the textile effluent due to their incomplete utilization [3, 4], which causes high toxicity and mutagenicity in aquatic

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life and humans [5, 6]. Although a series of physiochemical methods for treating textile wastewater are technically feasible, they are costly [7]. In contrast, due to its low cost and environmental friendliness, the biological treatment approach, especially anaerobic biological techniques, is a promising strategy for decolorization of textile wastewater [8, 9]. Conventional anaerobic biological techniques, such as upflow anaerobic sludge blanket (UASB) [5], fluidized-bed loop reactor (FBLR) [8] and

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sequencing batch reactor (SBR) [10, 11], have been successfully used for the

reductive decolorization of azo dyes. However, all these biological approaches

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consume large amounts of external organic carbon, such as glucose and acetate. The achievement of complete dyes decolorization and simultaneous minimization of

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treatment of textile wastewater.

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energy and organic carbon consumption is the key breakthrough for biological

Methane is a significant greenhouse gas, as well as a renewable energy and

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available carbon source if appropriately-managed [12, 13]. Anaerobic oxidation of methane (AOM) is an important methane sink [14, 15]. AOM coupled to the microbial reduction of various electron acceptors, such as sulfate [16], nitrate [17],

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nitrite [18], Fe(III) and Mn(IV) [19, 20], plays a crucial role in mitigating methane emissions to the atmosphere. A recent study has shown that methane can also serve as electron donors for microbial decolorization of methyl orange (MO), a typical azo dye, but the decolorization was seriously inhibited when the concentration of MO was above 100 mg/L due to the high toxicity of azo dyes and its products to

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microorganisms in SBR [21]. Therefore, it is necessary to use the appropriate biological technique, in which bacterial aggregates or biofilms are formed to improve the tolerance and adaptation of anaerobic bacteria to xenobiotic compounds, such as aromatic compounds [22, 23]. In addition, methane is poorly soluble (3.5 mg per 100 mL water) [24],

thus improving methane transfer for microbe utilization is also very

important in biological techniques involving methane-utilizing microbes.

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A hollow fiber membrane bioreactor (HfMBR) is a novel and efficient

technology to deliver a gaseous substrate to microorganisms [25]. Among its

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advantages, the membrane surface is used as a carrier for microorganism attachment, and can efficiently prevent the microorganisms from being washed out from the

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system [26, 27]. In addition, the continuous-flow mode used in the HfMBR can

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accelerate liquid discharge from the system, which avoids the accumulation of toxic products. These two features are particularly important for the removal of toxic

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pollutants by slow-growing anaerobes [28]. To date, the methane-based HfMBR has been successfully used for efficient removal of highly toxic inorganics, such as perchlorate [29] and selenate [30, 31], and heavy metals, such as chromium [25, 32]

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and vanadate [33]. Meanwhile, methanotrophic archaea usually forms syntrophic consortia with partner bacteria in HfMBR, which ultimately enhance the stability and removal rates of this system to practical application level [27, 34]. Therefore, the aims of this study were to (1) investigate the feasibility of applying methane-based HfMBR as a technology for MO decolorization from

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synthetic wastewater; (2) evaluate the MO decolorization rate and efficiency by stepwise changing the influent MO concentration and HRT in HfMBR; (3) explore the microbial morphology and community performing MO decolorization in HfMBR. The performance of the HfMBR was determined by measuring the concentration of MO. Microbial morphology in the HfMBR was analyzed by fluorescence in situ hybridization (FISH) and scanning electron microscopy (SEM) analysis. In addition,

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the key microorganisms performing MO decolorization were identified using a

combination of quantitative real-time PCR (qPCR), high-throughput sequencing of

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the 16S rRNA gene and cloning library construction. Materials and methods

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HfMBR set-up

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The decolorization of MO was conducted in a laboratory-scale HfMBR. The schematic diagram of the HfMBR system is shown in Fig. 1, the HfMBR contains

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100 hollow fibers (0.70 mm inner diameter and 1.03 mm outer diameter) made of polyvinylidene fluoride (PVDF). The total volume of the membrane module is 237 mL, which includes 10 mL of hollow fiber materials, 9 mL of space inside the fibers

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for gas supply, and 218 mL of space outside the fibers for liquid. The total surface area of the membrane is 0.07 m2. A gas mixture of CH4:CO2 (95:5, v/v) was fed from the lumen of the fiber membrane through a gas cylinder and the gas pressure was controlled using a gas regulator. The liquid was recirculated through an overflow bottle (450 mL in liquid volume) using a peristaltic pump (BT100-2J; Longer pump

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Factory, Baoding, China) to provide mixing. The pH value in the reactor was maintained at 7.3~7.6 by manual injection of 1 M HCl or 1 M NaOH solutions, as required. Operation of HfMBR The HfMBR was operated for a 142-day period, which was divided into three stages, namely, the start-up stage fed with nitrate for the formation of the biofilm

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(Stage 1), MO-fed batch stage (Stage 2), and MO-fed continuous-flow stage (Stage 3). For Stage 1, the HfMBR was inoculated with 100 mL of denitrifying anaerobic

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methane oxidation (DAMO) culture that was taken from a suspended parent

bioreactor fed with methane and nitrate. The anaerobic mineral medium components

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were identical to those reported in a previous study [35]. A nitrate stock solution (80

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g/L NO3--N) was manually administered twice a week to maintain the NO3--N concentration at 200-350 mg/L after each injection. Liquid samples were taken at

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least twice per day from the reactor to determine the concentrations of nitrate and nitrite. For Stage 2, MO was used to replace the nitrate and added three or four times per day by injection of a concentrated MO stock solution (5 g/L) to reach a final

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concentration of 25-35 mg/L; at least two liquid samples were taken for each injection to determine the MO concentration. On Day 93, the HfMBR was turned into the continuous-flow stage (Stage 3). The

initial influent contained 400 mg/L MO and the HRT was set at 2 days with a MO loading rate of 200 mg/L/d. During the continuous operation, the influent MO

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concentration was stepwise increased from 400 to 800 mg/L when the MO was almost completely decolorized at each condition. From Day 154, the HRT was gradually decreased from 2 days to 0.5 day after the reactor reached steady-state each time, with the MO loading rate continually increase to 1,000 mg/L. In Stage 3, effluent samples were taken at suitable intervals to determine the MO concentration from the effluent. To verify the relation between MO decolorization and methane oxidation in the

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HfMBR, stoichiometry equilibrium tests were conducted in the batch mode at the end of Stage 3. To measure the consumption of methane, the methane supply to the

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HfMBR was stopped by disconnecting it from the gas cylinder. Freshly prepared medium was sparged with the mixed methane gas for 30 min to ensure that the

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medium was saturated with methane. The auto-overflow point was locked so that the

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headspace in the overflow bottle became the only gas phase of the entire system and the consumption of methane could be measured. Batch test was operated after a 12 h

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equilibrating period. Chemical analysis

The concentrations of nitrate and nitrite were analyzed with a water quality

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autoanalyzer (Aquakem 200 Photometric Analyzer; Thermo Fisher Scientific, Vantaa, Finland) according to the standard method. For the quantitative analysis of MO, liquid samples were first centrifuged at 10,000 rpm and the supernatant was immediately analyzed with an ultraviolet-visible spectrophotometer (UV-2401PC; Shimadzu, Kyoto, Japan) at a wavelength of 465 nm. The degradation products of MO in

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stoichiometric experiments were quantified by high-performance liquid chromatography (HPLC) on an Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA, USA) using a previously reported method [36]. Methane (200 μL) in the headspace was measured by gas chromatography (GC) on a Fuli Gas Chromatograph 9790 (Zhejiang Fuli Analytical Instrument Co., Ltd., Wenling City, China) with hydrogen as the carrier gas [35].

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Microscopic observation

The spacial distribution of the microbes at the end of each stage was

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characterized by FISH analysis. FISH was performed using a Zeiss LSM710 confocal laser scanning microscope (CLSM) (Carl Zeiss Microscopy GmbH, Jena, Germany)

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according to a previously reported protocol [37]. The morphology of microorganisms

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on the membrane surface on Day 143 was also observed by a SIRION 200 field emission scanning electron microscope (SEM) (FEI Company, Hillsboro, OR, USA)

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after pretreatment with glutaraldehyde for fixation and dehydration with a gradient of ethanol concentrations.

DNA extraction, 16S rRNA gene sequencing, qPCR and phylogenetic analysis

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DNA was extracted from the harvested detached biofilm at the end of each stage using a PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) following the instructions of the manufacturer. The 16S rRNA gene was amplified using the primer pair 341b4_F (5ʹ-CTAYGGRRBGCWGCAG-3ʹ)/806_R (5ʹGGACTACNNGGGTATCTAAT-3ʹ) reported by Lu et al. [38]. The 20 mL reaction

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solution consisted of 10 ng of Template DNA, 4 μL of 5×FastPfu Buffer, 2 μL of dNTPs (2.5mM), 0.4 μL of FastPfu Polymerase, 0.2 μL of Bovine serum albumin (BSA), and 0.8 μL of forward and reverse primers (5 μM). PCR was conducted under the following conditions: 95 oC for 3 min; 35 cycles of 95 oC for 30 s, 55 oC for 30 s and 72 oC for 45 s; followed by a final extension at 72 oC for 10 min. Sequencing of the 16S rRNA (V3-V4 region) amplicon was performed on the Illumina HiSeq

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platform at Majorbio Biopharm Technology Co., Ltd. (Shanghai, China).

The abundance of the methyl-coenzyme reductase A (mcrA) gene, 16S rRNA

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gene from archaea and bacteria at the end of each stage were determined by qPCR

analysis. The primers and qPCR conditions referred those previously described by Lai

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et al. [39] and were shown in Table S1. Phylogenetic analysis was further conducted

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on the harvested detached biofilm sample from the end of stage 3. The general primer 20F (5ʹ-TTCCGGTTGATCCYGCCRG-3ʹ) /958R (5ʹ-

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YCCGGCGTTGAMTCCAATT-3ʹ) targeting the archaeal 16S rRNA gene was used for constructing clone libraries. A total of 25 positive clones were randomly selected and sequenced by Majorbio Biopharm Technology Co., Ltd. (Shanghai, China). The

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operational taxonomic units (OTUs) were determined using the Mothur software with default parameters. The phylogenetic trees analysis was performed using Mega 7.0.26, and the representative sequences and some related homologous sequences were obtained from the NCBI Web site using the Basic Local Alignment Search Tool (BLAST). The sequences were aligned with the muscle algorithm, and the

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phylogenetic trees were constructed by the neighbor-joining statistical method using the Kimura 2-parameter as the substitution model. Support for branches was determined from 1,000 bootstrap iterations. The sequences obtained in this study have been submitted to the GenBank database and the accession numbers are MK192062MK192086. Results and discussion

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Start-up of the HfMBR

In the start-up stage (Stage 1), the HfMBR was operated to establish the biofilm

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colonization and nitrate was supplied as the sole electron acceptor. After inoculation,

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a relatively high NO3--N removal rate of 226 mg/L/d on 0.5 day was observed, and there was evident nitrite accumulation due to the high nitrate removal rate (Fig. 2a).

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However, subsequently, the removal rate of nitrate gradually decreased and there was no longer nitrite accumulation after 25 days. The NO3--N removal rate was stable at

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about 75 mg/L/d within 60 days (Fig. 2b), a mature biofilm was formed on the surface of the hollow fiber membrane and no biomass loss was detected in the effluent, which mean the start-up of the HfMBR was completed. The acclimation time of 60 d

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obtained in this study was shorter than the usual 220-300 days for the start-up of similar systems [28, 31, 34], which might be attributed to different membrane materials used. In this study, PVDF was chosen as the membrane material for the membrane module, PVDF membrane fibers with hydrophobicity and scaly structure were favorable for microbial adhesion and reduced the start-up time [40, 41]. 11

MO decolorization performance of the HfMBR On Day 61, the electron acceptor supplied to the HfMBR was changed from nitrate to MO. MO decolorization immediately occurred in Stage 2 (i.e., Day 61-92) (Fig. 3a). The decolorization rate of MO was increased from 27 mg/L/day on Day 61 to 206 mg/L/day on Day 92 with the increase of the manual feeding frequency (Fig. 3a). From Day 93, the operation of the HfMBR was shifted to the continuous stage

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(Stage 3, Day 93-142) at an initial HRT of 2 days and 400 mg/L of influent MO (Fig.

3b). The effluent only had 0.78 mg/L MO on Day 103, which suggested that MO was

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almost completely decolorized. Then the influent MO increased to 600 and 800 mg/L on Day 104 and 114, respectively, and the corresponding decolorization rate was

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increased to 298 and 398 mg/L/day, respectively (Fig. 3c). These results indicated that

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the decolorization rate continued increasing with the increase of influent MO and the potential activity of the culture was very high.

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On Day 124, the HRT was decreased to 1.5 days, and the effluent MO concentration rose to 22 mg/L on Day 128, but dropped to 3.62 mg/L on Day 131. Subsequently, the HRT was further shortened to 1 day on Day 131 and the effluent

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concentration of MO increased to 8.8 mg/L. Ultimately, the effluent MO concentration increased to around 58 mg/L and remained steady as the HRT was decreased to 0.5 day and influent MO concentration was increased to 500 mg/L on Day 138 (Fig. 3b). With the stepwise increase of the influent MO concentration from 400 to 800 mg/L (Day 93-123) and the gradual decrease of the HRT from 2 days to

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0.5 day (Day 94-142), the MO loading rate increased from 200 to 1,000 mg/L/d. Correspondingly, the MO decolorization rate increased from 199 to 883 mg/L/d (Fig. 3c). Almost complete MO decolorization (>99%) was achieved by the HfMBR with an influent MO concentration of 400-800 mg/L and an HRT between 2 days to 1.5 days. However, MO decolorization efficiency dropped to 88% at the HRT of 0.5 day (Table 1), which indicated that 0.5 day was too short to achieve complete MO

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decolorization in this HfMBR system.

To verify the electron balance between MO decolorization and methane

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oxidation, a batch test was conducted in the HfMBR at the end of Stage 3 (Day 142).

As shown in Fig. S1, 0.12 mmol (60 mg/L) of MO was completely decolorized within

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3 h, and 0.11 mmol (34 mg/L) of the metabolite 4-aminobenzenesulfonic acid (4-

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ABA) was produced (Fig. S1). Meanwhile, 0.07 mmol of methane was consumed (Fig. S1), resulting in a mole ratio of 1.71 between MO decolorization and methane

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oxidation. This measured ratio was comparable to the stoichiometric ratio of 2 (2C14H14N3SO3Na + CH4 + 2H2O → 2C6H6NSO3Na + 2C8H12N2 + CO2), which suggested that MO underwent reductive decolorization coupled to methane oxidation

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rather than adsorption.

FISH and SEM images of biofilm microorganisms The HfMBR before use was white (Fig. S2a); after MO decolorization, a black

biofilm formed on the surface of the membranes (Fig. S2b-d). The distribution of archaea and bacteria on the biofilm at the end of each stage was characterized by

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FISH analysis (Fig. 4). In inoculum, archaea appeared in small clusters (1-4 μm), bacteria were dispersed and relatively distant from archaea (Fig. 4a). In the HfMBR, archaea still occurred in clusters, but most of the observed archaeal clusters were surrounded by bacterial cells and formed synergistic consortia with them. The spatial distribution of these microbes implies the existence of a synergistic relationship. The shape and size of the consortia were also different across the stages (Fig. 4b-d). In

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Stage1, the synergistic consortia were smaller than 5 μm and formed a relatively loose rectangle (Fig. 4b). The diameter of the synergistic consortia increased visibly to ~15

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μm in Stage 2 (Fig. 4c), and finally developed into a circular shape with a diameter of ~40 um as the HRT became shorter at the continuous flow mode in Stage 3 (Fig. 4d).

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The synergistic consortia became larger and more compact over time, it is

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hypothesized that it was more stable to resist the dual challenge from the inside methane supply from the hollow fibers and the high liquid recirculating rate outside.

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In addition, in this study FISH analysis was performed for the first time to characterize the microbial community spatial distribution in the biofilm. A 3-D image of the biofilm from Stage 3 (Fig. 4e) revealed the coexistence of archaea and bacteria

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in three dimensions and that there was no preferential positioning or lamination between archaea and bacteria, despite the special substrate delivery mode of the HfMBR (methane supplied in the membrane side and MO supplied in the liquid side). The results from this study were distinct from the modeling results reported in the literature suggesting that microorganisms that utilize liquid substrates mainly grow in

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the biofilm layer close to the bulk liquid and methane-using microorganisms attach close to the membrane surface [42-44], which indicates that the microbial community distribution in the biofilm of membrane biofilm reactor is more complex and further investigation is still needed. The microbial morphology in the biofilm from demolished membrane module was observed in situ by SEM at the end of Stage 3 (Fig. 5a). The SEM images showed

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that there were two different cellular morphology on the biofilm (Fig. 5b). One

cellular morphology consisted of cocci and appeared in clusters with a diameter about

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1 m (Fig. 5c). The morphology of this aggregates was very similar to that of

methane-related spherical archaea [45]. The other cellular morphology consisted of

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bacillus and formed a tight thin layer nearby the cocci clusters (Fig. 5d), which was

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consistent with the distribution of archaea and bacteria in the FISH images of Stage 3 (Fig. 4d). These results demonstrated once again that archaea clusters formed

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synergistic consortia with adjacent bacteria for MO decolorization in the HfMBR. Microbial community structure

Microbial community in the inoculum and biofilm from Stage 1-3 was evaluated

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by high-throughput sequencing analysis (Fig.6a and 6b). In the inoculum, archaea and bacteria accounted for 37.4% and 62.6% of the microbes, respectively. All the archaea were affiliated with the Euryarchaeota phylum. The dominant bacterial phyla included NC10, Parcubacteria, Proteobacteria and Saccharibacteria with relative abundances of 17.8%, 12.6%, 8.8% and 6.2%, respectively (Fig. 6a). Compared with the inoculum,

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the abundance of bacterial rose to 95.1% in Stage 1 and eventually fell to 49.2% in Stage 3. In contrast, the archaea were sharply decreased to 4.9% in Stage 1, but gradually increased at the later stage and eventually reached to 50.8% in Stage 3. The variation of archaea and bacteria populations and related genes were also evaluated by qPCR analysis (Fig. 7). The copy numbers of 16S rRNA genes of archaea was 5.11×104 copies/ng DNA in the inoculum. It decreased to 1.72×104

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copies/ng DNA in Stage 1 and gradually increased to 1.48×105 copies/ng DNA in

Stage 3, which is consistent with the change in abundance of archaea shown above

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(Fig. 6a), indicating that both the absolute and relative amount of archaea declined in Stage 1. The mcrA gene showed a similar tendency with the 16S rRNA genes of

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archaea from Stage 1 to Stage 3. Although there was an apparent decrease of the

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archaeal 16S rRNA genes and mcrA gene in Stage 1, their copy numbers increased in Stage 2 and remained steady in Stage 3, which supported the notion that archaea play

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an important role in the high decolorization performance of MO in this HfMBR. The copy numbers of bacterial 16S rRNA gene decreased somewhat in Stage 1, indicating that bacterial death had also occurred, but the amount of bacterial cell death was

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definitely less than that of archaeal cell death, as the relative abundance of bacteria dramatically increased in Stage 1 (Fig. 6a). Both the copy numbers of archaeal and bacterial 16S rRNA genes decreased in Stage 1, which was likely due to microbial adaptation from parent suspended reactor to HfMBR. The abundance of bacteria continuously declined in Stage 2 and Stage 3, but the copy numbers of bacterial 16S

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rRNA slightly increased from stage 2 (7.15×105 copies/ng DNA) to Stage 3 (1.42× 106 copies/ng DNA), which was likely due to the inherently faster specific growth rate of bacteria compared to archaea [39]. Further analysis of the community composition at the genus level indicated that Candidatus Methanoperedens (37.2%) and Candidatus Methylomirabilis (17.8%) dominated the microbes in the inoculum. Candidatus Methanoperedens was well

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known as the anaerobic methanotrophs (ANME) archaea and belonged to the ANME-

2d lineage, which could reduce nitrate to nitrite [17]. Candidatus Methylomirabilis are

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affiliated with the NC10 phylum and reduce nitrite to N2 [18]. The abundance of Candidatus Methylomirabilis (17.4%) remained relatively stable in Stage 1, but

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Candidatus Methanoperedens became only a small fraction (4.9%) in Stage 1 (Fig.

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6b). A possible explanation is that it might be due to the accumulation of nitrite (25100 mg/L NO2--N) at the start of Stage 1 (Fig. 2a), which induced irreversible

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inhibition on Candidatus Methanoperedens. As it has been reported that high concentration of NO2--N (i.e. 30-50 mg/L) could cause serious inhibition of microbial activity [46].

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After nitrate was replaced with MO in Stage 2, the new genera of Methanomethylovorans, Candidatus_Moranbacteria and Chlorobium emerged in the biofilm, which accounted for 15.4%, 22.9% and 10.6%, respectively. Among them, Methanomethylovorans, within the family of Methanosarcinaceae, is a reported methylotrophic methanogen archaea [45], while the two bacteria, Candidatus_Moranbacteria and Chlorobium, have been reported to be capable of

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degrading ethyl tert-butyl ether [47] and sulfuric acid [48], respectively, which may play an synergistic role with methane-related archaea in MO degradation. In Stage 3, the abundance of bacteria Candidatus_Moranbacteria (17.58%) was still high. A new bacteria genus, namely Geobacter (2.26%) arose, which has been reported to be a representative redox microorganism [49, 50]. In addition, the methane-related archaea Methanosaeta appeared with an abundance of 5.1% (Fig. 6b), while Methanomethylovorans were absolutely the dominant archaea, accounting

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for 43.36% of the archaeal population in the MO decolorization biofilm. A phylogenetic analysis of archaea in Stage 3 was also conducted (Fig. 8). Sequences of the archaea clone library were grouped into two OTUs, which were 98 % similar to

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each other. We compared the representative sequences in the two OTUs with that of the known ANME and other methane oxidation related archaea from the NCBI

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database. The gene sequences from the MO decolorization biofilm were affiliated to

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Methanomethylovorans, which is consistent with the high-throughput sequencing analysis results. The sequences in these two OTUs were found to be phylogenetically related to ANME-3 and Methanosarcina. Although most Methanomethylovorans are

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reported to produce methane [45, 51, 52], they have the possibility of performing methane oxidation and releasing intermediates, which could serve as electron donor for respiration of symbiotic bacteria [53]. For instance, the methanogenic archaea

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Methanosarcina, affiliated with Methanosarcinales, has been found in manganese-, iron-, bromate- and antimonite-dependent AOM [20, 39, 54, 55]. Implications

Although the nitrate/nitrite removal rate of methane-based HfMBR has been reported to achieve a practical application level [27, 34], the organic pollutant

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removal from wastewater in HfMBR system had not yet been demonstrated until now. In this study, a high MO decolorization rate of 883 mg/L/d was achieved in a methane-based HfMBR, which was approximately twenty times higher than that in a methane-based SBR [21]. The decolorization of different azo dyes in various bioreactors fed with real or synthetic wastewater, along with the operational parameters applied are summarized (Table 2). The substrates used in these reactors

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varied from simple substrates (e.g., acetate and glucose) to complex ones like starch, peptone, molasses and sago wastewater [4, 56-59], while primary substrates may be

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better suitable for delivering reducing equivalents to azo dyes with relatively high decolorization rates [5, 10].

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The MO decolorization rate (883 mg/L/d) in this study ranks in second place

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compared with others (Table 2), which indicated that the HfMBR can achieve a preponderant decolorization rate relative to those achievable in traditional granular

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sludge and biofilm systems. Furthermore, the HfMBR provided a satisfactory extent of decolorization which was superior to most of those reported in the literature. Indeed, the decolorization efficiency of dyes in the HfMBR is not only higher than

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that of reactors fed with real textile wastewater, but also the reactors fed with synthetic wastewater. With higher HRT (1.5-2 days), the decolorization efficiency was nearly 100%, which is the key for the practical biodegradation process of azo dyes. However, although the decolorization efficiency can also achieve 97 and 100% with starch and molasses as carbon sources, the decolorization rates were relative low

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(maximum 49 mg/L/day and 113 mg/L/day) [4, 58]. The high decolorization rate and efficiency in the HfMBR could be attributed to various aspects. First, the formation of synergistic consortia between archaea clusters and adjacent bacteria, which can prevent the microorganisms from being washed out, thus leading to the rapid generation of the biofilm in the surface of the hollow fiber membrane. Second, methane was supplied from the fibers lumen and MO came from

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the aqueous phase of the HfMBR, the opposite direction of the gas and liquid mass

transfer led to the achievement of the maximum extent of the utilization of gaseous

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methane and liquid MO. Third, the continuous-flow mode accelerated the liquid

discharge from the system, which prevented the accumulation of toxic products. In

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addition, the gradually elevated MO loading rate might stimulate the growth of

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functional microbes. Overall, the good decolorization performance in this study indicated that the HfMBR might be an appropriate anaerobic reactor configuration for

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the removal of toxic organic pollutant (e.g., azo dyes) from industrial wastewater. Conclusions

A methane-based HfMBR inoculated with an AOM culture was developed and

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operated to study its MO decolorization capacity with synthetic wastewater. The main findings of this study were: 

MO decolorization achieved a high level with ~ 100% decolorization efficiency (HRT=2 to 1.5 days) and a maximum decolorization rate of 883 mg/L/day (HRT= 0.5 day)

20



Microbial community changed significantly after MO decolorization. Methanerelated archaea Methanomethylovorans were absolutely the dominant archaea, accounting for 43.36% of the archaeal population in the MO decolorization biofilm and the possible MO decolorization bacteria Candidatus_Moranbacteria accounted for 17.58% of the bacterial population.



Methane-related archaea formed consortia with adjacent bacteria, which played

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a synergistic role in the biofilm of the HfMBR for the high MO decolorization rate and efficiency.

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Declaration of interests

The authors declare that they have no known competing financial interests

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reported in this paper.

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or personal relationships that could have appeared to influence the work

AUTHOR CONTRIBUTIONS

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Study conception and design: Raymond Jianxiong Zeng, Fang Zhang

Acquisition of data:

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Ya-Nan Bai, Xiu-Ning Wang, Wei Zhang, Jun Wu, Yong-Ze Lu, Liang Fu

Analysis and interpretation of data: Ya-Nan Bai, Raymond Jianxiong Zeng

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Drafting of manuscript: Ya-Nan Bai, Fang Zhang

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Critical revision:

Acknowledgments

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Raymond Jianxiong Zeng, Tai-Chu Lau

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This work was supported by the National Natural Science Foundation of China (51178444, 51878175) and the Program for Innovative Research Team in Science and

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Technology in Fujian Province University (IRTSTFJ)

22

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Table Legends Table 1. MO decolorization profile in the HfMBR with different HRTs and influent MO concentrations in Stage 3. Table 2. Operational parameters and performance in the decolorization of different

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azo dyes under anaerobic conditions.

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Table 1. MO decolorization profile in the HfMBR with different HRTs and influent MO concentrations in Stage 3. Time (day)

HRT (day)

Influent (mg/L)

Decolorization efficiency (%)

Effluent (mg/L)

2

400

0.78

99.80

104-113

2

600

3.48

99.42

114-123

2

800

4.52

99.43

124-131

1.5

800

3.62

99.55

131-138

1

800

8.80

98.90

138-142

0.5

500

58.67

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93-103

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88.27

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Dyes

Concentration (mg/L)

Inoculum

Substrates

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Reactor

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Table 2. Operational parameters and performance in the decolorization of different azo dyes under anaerobic conditions.

Acid Orange 7 Reactive Black 8

100

Sewage sludge

APCR

Reactive Red 195

50-400

Activated sludge

UASB

Reactive Black 5 Congo Red

100-3000

Methanogenic sludge

UASB

Acid Orange 7 Direct Red 254

60-300

FBLR

HRT

Decoloriza Maximum Decol- References orization rates tion (%) (mg/L/day)

24-28

10 day

99

10

[56]

Molasses

19-22

12-72 h

60-100

113

[58]

Glucose

35

0.49-3.54 day

73-79 92-95

283

[57]

Methanogenic sludge

Acetate

37 + 2

8 & 24 h

85-92

372

[5]

Synthetic wastewater 20 Real textile wastewater -

Methanogenic sludge

Acetate

37 + 1

6-24 h

75-92 90

N/A

[8]

UASB

Real textile wastewater -

Methanogenic granular Sago 28 + 5 sludge wastewater

6 & 24 h

83-92

N/A

[59]

ASBR

Reactive Red 2

100

Secondary sludge

Starch & peptone

35

48 h

97

49

[4]

ASBR

Methyl orange

25-500

Anaerobic sludge

Glucose

35

8h

75-95

1439

[10]

400-800

DAMO culture

Methane

35

12-48 h

88-100

883

（this study）

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HfMBR

Methyl orange

Acetate

re

SCR

T (℃)

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Figure Captions Fig.1. Schematic diagram of the methane-based hollow fiber membrane reactor (HfMBR). Fig.2. Performance of the HfMBR in the start-up stage (Stage 1). (a) Nitrate and nitrite concentrations; (b) Nitrate (rNO3--N) and nitrite (rNO2--N) removal rates. Fig.3. (a) MO concentration in the batch stage (Stage 2); (b) The influent and effluent MO concentration in the continuous-flow stage with different HRT (Stage 3); (c) MO decolorization rate and loading rate in Stage 2 and Stage 3. Fig.4. FISH images of the (a) inoculum, (b) microbial distribution on the biofilm from

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Stage 1 (Day 61), (c) microbial distribution on the biofilm from Stage 2 (Day 93), (d) microbial distribution on the biofilm from Stage 3 (Day 142), and (e) 3-D image of

the biofilm from Stage 3 (Day 142). The probes used for hybridization: Cy3 EUBmix probe for general bacteria (red), FITC Arch915 probe for general archaea (green).

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Fig.5. Microscopic observations of the MO decolorization biofilm on the surface of the membranes (at the end of Stage3, Day 142) by SEM.

Fig. 6. Relative abundance of microorganisms in the inoculum and Stages 1-3, (a)

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Phylum; (b) Genus.

Fig. 7. Gene copy number of mcrA and 16S rRNA genes of archaea and bacteria in

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the inoculum and Stages 1-3 by qPCR analysis. Results are presented as the mean value plus standard deviation from three replicate qPCR reactions. Fig. 8. Phylogenetic tree of the archaeal 16S rRNA gene sequences retrieved from the

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biofilm in Stage 3 and the methane oxidation related archaeal sequences derived from

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the NCBI database. The scale bar indicates a 5% divergence.

31

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Fig.1. Schematic diagram of the methane-based hollow fiber membrane reactor

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(HfMBR).

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Fig.2. Performance of the HfMBR in the start-up stage (Stage 1). (a) Nitrate and

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nitrite concentrations; (b) Nitrate (rNO3--N) and nitrite (rNO2--N) removal rates.

33

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Fig.3. (a) MO concentration in the batch stage (Stage 2); (b) The influent and effluent MO concentration in the continuous-flow stage with different HRT (Stage 3); (c) MO decolorization rate and loading rate in Stage 2 and Stage 3.

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Fig.4. FISH images of the (a) inoculum, (b) microbial distribution on the biofilm from Stage 1 (Day 61), (c) microbial distribution on the biofilm from Stage 2 (Day 93), (d) microbial distribution on the biofilm from Stage 3 (Day 142), and (e) 3-D image of the biofilm from Stage 3 (Day 142). The probes used for

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hybridization: Cy3 EUBmix probe for general bacteria (red), FITC Arch915 probe for general archaea (green).

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Fig.5. Microscopic observations of the MO decolorization biofilm on the surface

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of the membranes (at the end of Stage 3, Day 142) by SEM.

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Fig. 6. Relative abundance of microorganisms in the inoculum and Stages 1-3, (a)

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Phylum; (b) Genus.

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Fig. 7. Gene copy number of mcrA and 16S rRNA genes of archaea and bacteria

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in the inoculum and Stages 1-3 by qPCR analysis. Results are presented as the

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mean value plus standard deviation from three replicate qPCR reactions.

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Fig. 8. Phylogenetic tree of the archaeal 16S rRNA gene sequences retrieved from

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the biofilm in Stage 3 and the methane oxidation related archaeal sequences

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derived from the NCBI database. The scale bar indicates a 5% divergence.

39