activated sludge membrane bioreactor system: Biodegradation and interception

Accepted Manuscript Title: Advanced treatment for actual hydrolyzed polyacrylamide-containing wastewater in a biofilm/activated sludge membrane bioreactor system: Biodegradation and interception Authors: Lanmei Zhao, Congcong Zhang, Mutai Bao, Jinren Lu PII: DOI: Reference:

S1369-703X(18)30385-1 https://doi.org/10.1016/j.bej.2018.10.020 BEJ 7071

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

9-9-2018 14-10-2018 17-10-2018

Please cite this article as: Zhao L, Zhang C, Bao M, Lu J, Advanced treatment for actual hydrolyzed polyacrylamide-containing wastewater in a biofilm/activated sludge membrane bioreactor system: Biodegradation and interception, Biochemical Engineering Journal (2018), https://doi.org/10.1016/j.bej.2018.10.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Advanced treatment for actual hydrolyzed polyacrylamide-containing wastewater in a biofilm/activated sludge

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membrane bioreactor system: Biodegradation and interception☆

Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China,

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a

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Lanmei Zhaoa,b, Congcong Zhang a,b, Mutai Baoa,b,* , Jinren Lub

College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, China

Corresponding authors: [email protected] (M. Bao), Tel/Fax: +86-532-66782509.

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

This is MCTL Contribution No. 187.

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☆

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b

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Qingdao 266100, China

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Graphical abstrcat

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1.0 The first cleaning

2

Permeability (L/hm kPa)

0.9

The second cleaning

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

60 65 70 75 80 85 90 95 10 0 10 5 11 0 11 5 12 0 12 5 13 0 13 5 14 0 14 5 15 0

0.0

Time/d

Influent Supernatant Effluent

575.0

380

. ..

479.2

360

. . .. . . .. . .. . . . . ... . . . . . .. .. . . . . .

Peak E

340

383.3

EX(nm)

320 300

287.5

280 191.7

260

Peak F

240

95.83

60

140 142 14 1446 148 150

50

40

30

20

220 10

0

HPAM concentration (mg/L)

400 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0

Biofilm/activated sludge membrane biosystem

0 250

300

350 400 EM(nm)

450

500

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Time/d

200 200

Produced

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After Treatment

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Water Reuse

Highlights

The removal efficiencies of HPAM, COD, TOC and TN achieved above 96%

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



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via BF-AS-MBR.

Membrane permeability was recovered to over 85% after the first or second

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cleaning.



LB-EPS and TB-EPS of biofilm, activated sludge and cake sludge were

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different.

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Key microorganisms were related to HPAM biodegradation, EPS and membrane fouling.

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Effluent was close to clean water and it could be used to re-prepare HPAM solution. 2

ABSTRACT This study deeply investigated the overall performance, nitrogen transformation, membrane fouling and cleaning, extracelluar polymeric substances (EPS) and microbial function in a biofilm/activated sludge membrane bioreactor (BF-AS-MBR)

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for treating actual hydrolyzed polyacrylamide (HPAM)-containing oilfield wastewater. The removal efficiencies of HPAM, COD, viscosity, TOC and TN achieved 97.5%,

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98.5%, 74.8%, 98.5% and 96.2%, respectively. Nitrogen transformation revealed

nitratation became the main reaction in the process of aerobic amide group

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bioconversion and surpassed denitrification, nitritation and ammonification in

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BF-AS-MBR system. Scanning electron microscopy (SEM) showed the morphology

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of new, contaminated and washed membranes. The membrane permeability was

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recovered to 92% and 87% after the first and second cleaning, respectively.

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Tryptophan protein-like, aromatic protein-like, simple aromatic protein-like and fulvic acid-like organics presented in loosely bound EPS (LB-EPS) of sludge.

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Polysaccharide-like and tyrosine protein-like organics presented in tightly bound EPS

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(TB-EPS) of sludge. Tryptophan protein-like organics existed solely in TB-EPS of cake sludge, and played an essential role in membrane fouling. HPAM-degrading microorganisms and nitrifiers were deeply discussed to explore its correlations with

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HPAM removal, EPS and membrane fouling. The viscosities of HPAM solution re-prepared with different effluent were compared, and the order was: supernatant after biodegradation < effluent after membrane filtration ≈ clean water. This study offered a technical support and theoretical foundation for treating actual

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HPAM-containing oilfield wastewater, and improved water reuse.

Keywords:

biofilm/activated

sludge

membrane

bioreactor,

hydrolyzed

polyacrylamide, biodegradation, membrane fouling and cleaning, extracelluar

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polymeric substances, microbial function

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1. Introduction

Hydrolyzed polyacrylamide (HPAM) solution, because of its high viscosity, was

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widely used as oil-displacing agent to improve oil recovery [1]. During the oil

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recovery process, a large amount of wastewater containing HPAM and oil is produced.

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The actual HPAM-containing oilfield wastewater has the characteristics of high

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viscosity, high salinity and multiphase coexistence of polymer/oil/water, which not

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only reduces oil recovery efficiency but also causes a series of hazards to the ecological environment [2]. Moreover, due to the population growth and industrial

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activities expansion around the world, the scarcity of fresh and sustainable water has

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become a major concern. As a consequence, there is a growing impetus to look for an eco-friendly and efficient method to reduce the impact of actual HPAM-containing

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wastewater on environment and improve water utilization efficiency [3,4]. Most previous research has concentrated on the removal methods and mechanisms

of HPAM (as the sole source of pollution). TOC and HPAM removal ratios reached 70.1% and 54.7% in a sequencing batch biofilm reactor [5]. 64.4% removal of HPAM was achieved through anaerobic-aerobic biological treatment systems [6]. HPAM and

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COD removal ratios reached 91.1% and 94.6% by anaerobic degradation combined Fenton oxidation [7]. The carbon backbone and amide group of HPAM served as energy sources for microorganisms [8,9]. Biodegradation pathways of HPAM were put forward by detecting intermediate metabolites or microbial enzymes [9-11].

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However, it is difficult to produce reusable effluent with biological methods treating actual HPAM-containing oilfield wastewater. Chemical methods may cause secondary

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pollution problems and increase costs despite it have the high removal efficiency of HPAM. In addition, other researchers used the dead-end/cross-flow system with a flat

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membrane to treat polymer-flooding oilfield wastewater and found that the leading

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substance on the total membrane resistance was HPAM (MW 100 kDa) [12,13]. But

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there exists a problem that single membrane interception for HPAM-containing

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oilfield wastewater can easily pollute membrane surfaces and quickly jam membrane

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holes. In order to improve water reuse and slow down membrane fouling, the combination technology of biodegradation and membrane interception was put

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forward in this study.

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The previous researchers linked biodegradation with membrane filtration for treating wastewater and the combined equipment was called membrane bioreactor (MBR) [14]. Friha et al. [15] used the aerobic submerged MBR to treat cosmetic

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wastewater and found that the removal ratios of anionic surfactant and COD were 98.1% and 83.7%, respectively. The removal ratios of BOD, COD and suspended solids of antibiotic wastewater in a MBR were 92.5%, 96% and 81.5%, respectively [16]. Sludge is the aggregation of microorganisms and different types of sludge have

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different performance in MBR, including suspended sludge, granular sludge and biofilm [17,18]. In a sludge system, extracellular polymeric substances (EPS) are the comprehensive concept for some high-molecular-weight mixture of polymers, such as proteins, polysaccharides and nucleic acids [19]. In addition, EPS can not only

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accelerate the aggregation of sludge flocs and the formation of biofilm, but also keep the stability of microbial community [20,21]. Meanwhile, biofilm provides favorable

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environment for the growth and metabolism of microorganisms [22]. The differences

of microbial communities and EPS were evaluated in the biofilm and suspended

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sludge [23]. Moreover, Li et al. [24] analyzed the relationship among HPAM

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biodegradation, EPS and microbial communities containing archaeal communities in

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the biofilm of the injection pipeline of Daqing oilfield. Activated sludge membrane

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bioreactor (AS-MBR) showed less membrane fouling and high permeability

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compared with biofilm membrane bioreactor (BF-MBR) system [25]. Based on these previous studies, a new type of biofilm/activated sludge membrane bioreactor

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(BF-AS-MBR) was put forward to further treat actual HPAM-containing oilfield

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wastewater in this study.

In addition, the viscosity of HPAM solution is an important factor affecting oil

recovery. Whether it’s feasible to use the effluent (after treatment) for re-preparation

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of fresh HPAM solution and make it further used in polymer flooding of oilfield is still unclear. Therefore, the objectives of this research were to (i) evaluate the overall performance of BF-AS-MBR system treating actual HPAM-containing oilfield

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wastewater, (ii) study the performance of membrane cleaning, (iii) investigate the composition and property of EPS in different sludge samples including activated sludge, biofilm and cake sludge, (iv) analyze the microbial community structure and function in whole system (v) explore the correlation among microbial function,

assess the reuse ability of effluent after different treatments.

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2. Materials and methods

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HPAM biodegradation, EPS and membrane fouling in BF-AS-MBR system, and (vi)

2.1 Experimental system

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The design of the system in this study was developed by adding biofilm based on

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the AS-MBR system (Fig. 1). The configuration of BF-AS-MBR system was

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consisted of a rectangular organic glass container of 600 mm×300 mm×400 mm with

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an effective volume of 68 L. Two flat-sheet hollow fiber membranes (Kay Membrane

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Technology Co., Ltd., Hangzhou, China), made of polyvinylidene fluoride, were immersed into the membrane compartment located in the middle-upper part of the

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bioreactor. Pore size and membrane area are 0.1 μm and 0.1 m2, respectively. 36

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polyester silk fillers (1809 cm2) with a diameter of 80mm (Jinguo Environmental Protection Equipment Co., Ltd., Jiangsu, China) provide the carrier for microorganisms and form biofilm. In addition, The air sparger was located at the

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lower part of BF-AS-MBR, which not only maintained a certain amount dissolved oxygen (DO) concentration for microbial growth and metabolism, but also achieved effective scouring for the membrane surface. The transmembrane pressure (TMP) and constant temperature in the bioreactor were obtained by a pressure gauge and a heater,

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respectively. 2.2 Wastewater Characteristics The actual HPAM-containing oilfield wastewater was obtained from Gusi Wastewater Treatment Station of Shengli Oilfield, located in Dongying, China. The

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Characteristics of actual HPAM-containing oilfield wastewater were presented in Table 1. In addition, a variety of microelements was added as nutriments for

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microorganism growth and metabolism, and the compositions (mg/L) included: AlCl3, 2.5; CoCl2·6H2O, 5; MnCl2·4H2O, 5; NiCl2·6H2O, 5; CuCl2·5H2O, 5; CaCl2, 25 [26].

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2.3 Bioreactor inocula and operation

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2.3.1 Inoculation sludge and functional microorganisms

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The oilfield sludge as the inoculated sludge from Zhan San oil production plant was

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added to the reactor. The sludge contained dominant bacteria for HPAM

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biodegradation and it could adapt to the wastewater environment better. Two separated functional strains were Bacillus cereus strain FM-4 (95% similarity,

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EU794727) and Bacillus sp. M7-23 (95% similarity, EU706321), respectively [9]. 3.6

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L of mixed functional bacteria (6.45×108 cell·mL-1) were added to BF-AS-MBR system for sludge acclimation. MLSS was about 4100 mg·L-1 in the initial stage and maintained approximately 5000 mg·L-1 after 60 days of acclimatization.

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2.3.2 Operation conditions of bioreactor HPAM-containing wastewater pumped into BF-AS-MBR was completely in touch with activated sludge and biofilm. The water after biodegradation was filtered through the two membrane modules by a suction pump. DO was maintained to 3-5 mg/L and

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aeration rate was set at 2 m3/h by an air compressor. The operation mode of suction pump was kept on 7 minutes, off 3 minutes automatically by a time controller. The parameters were set at pH of 7.2, temperature of 35 ℃, sludge retention time of 30 d and hydraulic retention time of 36 h.

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2.3.3 Sludge acclimation and sampling Glucose was used as the co-metabolic substrate to stimulate microbial growth in the

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process of sludge acclimation, which led to high enzyme activities to accelerate the

aerobic degradation of HPAM [5]. Sludge was domesticated by the way of stepwise

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increasing HPAM concentration and decreasing glucose concentration. Actual

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HPAM-containing oilfield wastewater was diluted to 50, 100, 150, 200, 250 and 300

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mg/L, respectively. The corresponding COD values of wastewater were 109, 219, 328,

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437, 547 and 656 mg/L, respectively. In order to remain COD values (656 ± 9 mg/L)

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of influent unchanged, the concentrations of glucose added were 547, 437, 328, 219, 109 and 0 mg/L, respectively. Each acclimation gradient maintained 10 d. The system

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reached a steady state after 60 days of acclimatization, and the removal efficiencies of

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HPAM and COD maintained approximately 59% and 76%, respectively. The whole operation lasted for 150 days. The water samples of influent, supernatant and effluent were collected from feed tank, discharge valve in the upper bioreactor and

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effluent pipe after membrane filtration, respectively. Three different kinds of sludge samples named biofilm sludge, activated sludge and cake sludge were collected from integrated fixed-film, aerobic activated sludge and contaminated membrane surface, respectively.

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2.4 Analytical methods The concentration of HPAM was conducted by starch-cadmium iodide method [5,27]. The specific measurement procedure was shown in Supplementary Material. The crude oil of actual oilfield wastewater was extracted by petroleum ether. Organic

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phase was determined at 225 nm by an UV spectrophotometer (Model UV-2102, PCS, China). MLSS was determined by using the standard methods [28]. TOC and TN

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were determined using a TOC analyzer (Multi N/C, Analytikjena, Gemany). COD was conducted by a COD digester (DRB200, HACH, America). The BOD5 was measured

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using a BOD analyzer (OxiDirect, Lovibond, Germany). The DO and pH were

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determined by a dissolved oxygen analyzer (Sension 6, HACH, America) and digital

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pH meter (pB-10, Sartorius Group, Germany), respectively. Viscosity was conducted

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by a rheometer (Mars III, Haake, Germany) at 25 ℃, and shear rate was set at 50 s-1.

c0  c 100% c0

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HBR 

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The biodegradation rate of HPAM (HBR, %) is obtained by

(1)

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where, c0 and c are the concentration value of influent and biodegraded sample, respectively.

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The water flux is calculated by

Jp 

V Am  t

(2) where, ΔV, Am and Δt are the volume change of effluent, effective membrane area and

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measuring time interval, respectively. The permeability is calculated by

Lp 

Jp P

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(3) where, ΔP is the transmembrane pressure.

I

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The ionic strength (mol·kg-1) is calculated by

1  mB Z B2 2

(4)

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where, mB and ZB are the molality and charge number of ions, respectively.

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2.5 Membrane cleaning

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When the transmembrane pressure (TMP) approached to 100 kPa, the membrane module cleaning procedure was performed by water cleaning and chemical cleaning.

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The water cleaning was as follows: module was washed by deionized water with

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aeration for 6 h and back washed by deionized water for 20 min. The chemical cleaning steps were as follows: the module immersed in NaOH (1.0%, w/v) and HCl

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(1.0%, w/v) solution with aeration for 6 h, respectively. Then, the module was back washed for 20 min with deionized water.

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2.6 SEM observation of the new, contaminated and washed membranes Scanning electron microscopy (SEM) provides morphology of the new,

contaminated and washed membranes. Membrane samples were dried and sputtered by thin gold layer [29] and observed by a SEM (Hitachi S-3400N, Tokyo, Japan). 2.7 Extraction and analysis of EPS 11

Microorganisms in the activated sludge and floc carriers have a dynamic EPS structure of double layers including Loosely bound EPS (LB-EPS) and Tightly bound EPS (TB-EPS) surrounding the cells. LB-EPS as the primary surface for cell flocculation and attachment are diffused from TB-EPS. EPS were acquired by the

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method of Li and Yang [30]. 50 mL of sludge mixture was centrifuged at 4,000 g for 5 min. The sludge particles left in the centrifuge tube were suspended into a solution of

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0.85% (w/v) NaCl with a temperature of 70 ℃, and formed a 50 mL volume of sludge suspension. The mixture solution was mixed for 1 min, and then centrifuged at

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4,000 g for 10 min. LB-EPS existed in the supernatant. The sludge particles were

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re-suspended into a solution of 0.85% (w/v) NaCl and the total volume was kept at 50

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mL. The sludge mixture was heated at 60 ℃ for 30 min, followed by centrifugation

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for 15 min. TB-EPS were acquired in the supernatant. The structure and intensity of

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EPS were determined by a fluorescence spectrophotometer (F-4500, Hitachi, Japan). 2.8 Methods for microbiological analysis

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The sludge samples were centrifuged at 8,000 g to collect cells. The centrifuged

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samples were cleaned three times with phosphate buffer solution (pH=7.4, 0.2 mol·L-1). Total DNA was extracted by TIANamp Bacterial Genomic DNA Maxiprep Kit and stored at -20 ℃ before further testing [31]. MiSeq platform of Novogene

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(Genome Sequencing Company) [32] was used for analyzing microbial function in BF-AS-MBR. Good’s coverage, Simpson index, Shannon index, ACE index and Chao 1 index were generated [33]. 3. Results and Discussion

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3.1 Performance of BF-AS-MBR system for treating actual HPAM-containing oilfield wastewater HPAM concentration, shear viscosity, COD and TOC in influent/supernate/effluent were presented in Fig. 2. At each acclimation gradient, HPAM concentration of

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supernatant gradually decreased with time and finally tended to stabilize. In the stable phase of actual wastewater treatment, the differences in HPAM concentration between

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supernatant (125 mg/L) and effluent (7.52 mg/L) indicated that viscous HPAM accumulated in the membrane compartment and membrane surfaces of the reactor.

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The removal efficiencies of HPAM after biodegradation and interception achieved

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58.9% and 97.5%, respectively. As shown in Table 1, oil concentration in wastewater

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was 50 ± 4 mg/L. Oil removal reached 62.7% and 95.2% after biodegradation and

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interception, respectively. BOD5 of influent and supernate reached 228 and 73.8 mg/L,

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respectively. BOD5/COD in feed water and supernate reached 0.35 and 0.47 respectively, indicating that the biodegradability of actual HPAM-containing

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wastewater was good. In addition, COD removal efficiencies after biodegradation and

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interception achieved 76.1% and 98.5%, respectively. With respect to shear viscosity of wastewater, it declined from 5.24 to 3.26 mPa·s with a shearing rate of 50 s-1 at 25 ℃ owing to HPAM biodegradation. As it was expected, the viscosity of supernatant

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was significantly reduced after membrane filtration. The shear viscosity of effluent was about 1.32 mPa·s, and it tended to viscosity of clean water. TOC removal efficiencies reached 75.0% and 98.5% after biodegradation and membrane filtration respectively, which were improved greatly, compared with our previous work that

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TOC removal efficiency reached 32.9% [4] and 70.1% [5]. The reduction of TOC in the process of biodegradation illustrated that microorganisms could utilize HPAM as an carbon source [5,34]. The carbon chain skeletons of HPAM were fractured from different positions and formed organic compounds of short chains [9].

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3.2 Nitrogen transformation in BF-AS-MBR system The removal efficiency of TN reached 38.1% and 96.2% after biodegradation and

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membrane filtration, respectively. The amount of assimilated nitrogen based on the

amount of sludge production reached 5.57 μg/(mg MLSS). It indicated that HPAM

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could be utilized as a nitrogen source by microorganisms. Amide groups of HPAM

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were hydrolyzed to ammonium ions in the early stage of HPAM biodegradation, and

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then the terminal methyl groups of the side chains were oxidized to acids by a single

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oxygenase [8]. As illustrated in Fig. 2e, nitrate nitrogen and nitrite nitrogen

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concentrations rose, and ammonium nitrogen concentration declined after aerobic biodegradation. Furthermore, nitrate concentration is 2.58 times higher than that of

group

bioconversion

and

surpassed

denitrification,

nitritation

and

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amide

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nitrite. It clarified that nitratation became the main reaction in the process of aerobic

ammonification in BF-AS-MBR system. Waki et al. [35] revealed that ammonium nitrogen could be thoroughly converted to nitrate nitrogen within the range of 3.0-8.0

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mg/L DO. Zhang et al. [36] found that HPAM biodegradation rose from 17% to 31% and nitrate nitrogen concentration increased from 9 to 18 mg/L with DO (3-4 mg/L). In addition, retention rate of TN reached 58.1% through membrane filtration. It indicated that organic compounds of short chains containing nitrogen could attach to

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membrane surfaces and be utilized as nitrogen source by the microorganisms from the cake sludge. 3.3 Membrane fouling and cleaning The membrane permeability in BF-AS-MBR system was presented in Fig. 2e.

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From the 60th day to the 90th day, membrane permeability dropped from 0.91 to 0.20 L/(h·m2·kPa) and was stabilized at 0.20 L/(h·m2·kPa). Water cleaning and chemical

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cleaning were carried out on the 90th day and the membrane permeability was

recovered to 0.84 L/(h·m2·kPa). The permeability recovery ratio reached 92% after

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the first cleaning. Membrane permeability declined with a relatively rapid speed from

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0.84 to 0.14 L/(h·m2·kPa) and was stabilized at 0.14 L/(h·m2·kPa). Water cleaning

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and chemical cleaning was implemented on the 120th day and the membrane

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permeability reached to 0.79 L/(h·m2·kPa). The permeability recovery ratio reached

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87% after the second cleaning. The membrane permeability declined with a rapid speed from 0.79 to 0.90 L/(h·m2·kPa). 36% of accumulated HPAM in the membrane

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compartment was degraded by cake sludge. 59% of accumulated HPAM was washed

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away after water and chemical cleaning. Additional 5% of HPAM accumulated in the membrane compartment. Long-term accumulation of high-viscous HPAM in the membrane surface could block membrane pores. Water cleaning and chemical

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cleaning could recover a part of the membrane performance to some extend. Combined with Fig. 3, new membrane surface was distributed with clear membrane hole and free of particles (Fig. 3a&g), and the surface of fouled membrane covered with contaminant which seemed to be dense and nonporous (Fig. 3b,d,f&h). The

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gel-like cake layer seemed to be composed by particles, EPS floc and some microorganisms which were rod-shaped (Fig. 3h). Research showed that the membrane could intercept macromolecular substances, microbial flocs and inorganic colloid substances [37]. Properties and concentrations of EPS and major components

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of flocs played an essential role in the membrane filtration in turn [38,39]. As a result, the adhesion of these bacteria clusters may have an important effect on the formation

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of small sludge particles, the degradation of organic substances and membrane fouling. The washed membrane was barely absent of microbes and macromolecular materials,

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and the membrane holes were clear to see (Fig. 3c&e). The degree of membrane

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contamination aggravated with the number of pollutions, and the recovered membrane

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membrane permeability.

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holes decreased with the number of cleanings. It was consistent with the changes of

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3.4 LB-EPS and TB-EPS in activated sludge, biofilm and cake sludge Peak A (275–280/340–350 nm), B (220/335–350 nm), C (220/295–305 nm) and D

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(270/300 nm) in the LB-EPS (Fig. 4a,c&e) represented tryptophan protein-like,

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aromatic protein-like, simple aromatic protein-like and fulvic acid-like organics, respectively [40-43]. Peak E (310–320/375–380 nm) and F (220–230/370–380 nm) of the TB-EPS (Fig. 4b,d&f）were associated with polysaccharide-like organics and

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tyrosine protein-like organics [44]. The LB-EPS of biofilm contained tryptophan protein-like, aromatic protein-like, simple aromatic protein-like and fulvic acid-like organics (Fig. 4a). The locations and profiles of peaks in activated sludge and cake sludge were similar to those of biofilm

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(Fig. 4c&e), which indicated that the three kinds of sludge shared the same LB-EPS. In biofilm, the intensity of protein-like and fulvic acid-like organics was the strongest compared to that of cake sludge and activated sludge (Table 2). The TB-EPS shared the same substances: polysaccharide-like organics and tyrosine protein-like

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substances (Fig. 4b,d&f). The intensity of the other two kinds of substances in biofilm and activated sludge was stronger than that of cake sludge (Table 2). Moreover,

4f).

These

results

indicated

that

tyrosine

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tryptophan protein-like substances existed solely in the TB-EPS of cake sludge (Fig. protein-like

substances

and

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polysaccharide-like organics facilitated formation of biofilm, sludge particles and

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cake layer. Moreover, tryptophan protein-like substances blocked membrane pore and

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played an essential role in membrane fouling [45].

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3.5 Microbial community structure and function in the whole system

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To estimate different bacterial communities of different sample, data were listed in Table 3. The effective sequence number of OTUs was 79156, 85767 and 74163,

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respectively, and the number of OTUs defined by 97% identity threshold according to

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the ribosomal database project was 1371, 1330 and 1041, respectively. The Good’s coverage of three sludge samples was always over 0.997, suggesting that obtained sequence library covered microbial community diversity of BR-AS-MBR system. Liu

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et al. [46] illustrated that Shannon and Simpson were interrelated to community diversity, and Chao 1 and Ace were bound up with community abundance. OTUs, Chao 1 and ACE index in the biofilm were higher than those of activated sludge and cake sludge. Microbial community was the most abundant in biofilm. While Simpson

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and Shannon index were the highest in activated sludge, which showed that the activated sludge had the highest microbial diversity. The Venn diagrams were presented in Fig. 5. The total OTU number was 1489 among the three sludge samples, of which the common OTU number (807) accounted

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for 54.2% (Fig. 5d). Additionally, the shared OTUs between the rest of any two sludge samples were 1093, 870 and 865 respectively, and only a few unique OTUs (13)

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appeared in cake sludge, indicating microorganisms appeared in cake sludge came from biofilm and activated sludge. Abundant shared OTUs indicated many bacteria

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simultaneously existed among three sludge samples.

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Microbial community structure and function in BF-AS-MBR system were further

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analyzed. The dominant bacteria at phyla level (Fig. 6a) were Proteobacteria (35.3%,

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46.2%, 48.9%), Firmicutes (13.3%, 5.49%, 22.3%), Bacteroidetes (13.2%, 15.2%,

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19.6%), Chloroflexi (14.1%, 9.09%, 1.96%) and Planctomycetes (8.74%, 12.2%, 2.55%), which accounted for 84.6%, 88.2% and 95.4% of the total number,

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respectively. The predonmiant phylum was Proteobacteria in biofilm and activated

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sludge [5]. Moreover, Proteobacteria were proved to play a significant role in biological hydrolysis of PAM [10]. Firmicutes were the most abundant in cake sludge than those of biofilm and activated sludge, which indicated that bacteria in cake

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sludge might induce microbial composition and accumulation in membrane surface. Chloroflexi had a function of degrading carbohydrates and promoted the formation of flocs, which were indispensable in MBR treatment process [5,47]. As a result, Chloroflexi made up a relatively large portion (14.1%) in biofilm, more than those of

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activated sludge (9.09%) and cake sludge (1.96%). The results also demonstrated that biofilm could provide a favorable condition for microbial cell clusters. At class level (Fig. 6b), the predominant bacteria of three sludge samples were affiliated

with

five

classes:

Betaproteobacteria,

Bacilli,

Sphingobacteriia,

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Alphaproteobacteria and Anaerolineae. Alphaproteobacteria and Betaproteobacteria, which belonged to Proteobacteria, accounted for 26.5%, 33.2% and 40.2% in biofilm,

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activated sludge and cake sludge, respectively. Additionally, cake sludge possessed

the most Alphaproteobacteria. Alphaproteobacteria has been reported as a major

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community for membrane biofouling [48].

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At genus level (Fig. 6c&Fig. 7), dominant bacteria were different in biofilm,

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activated sludge and cake sludge. The main dominant bacteria in biofilm include

Ignavibacterium,

Hyphomicrobium,

Woodsholea,

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unidentified_Nitrospiraceae,

M

Bacillus, Phaeodactylibacter, Azohydromonas, Nannocystis, Candidatus_Captivus,

Planctomyces, Pirellula and Pir4_lineage. The main dominant bacteria in activated

PT

sludge contain Aquimonas, Acidovorax, Zoogloea, Shinella, Rhodobacter, Rhizobium,

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Ignavibacterium, Hyphomicrobium, Cloacibacterium, Thauera, Flavobacterium, Shewanella,

Acinetobacter,

Haliangium,

Nitrosomonas,

Pseudomonas

and

unidentified_Nitrosomonadaceae. The main dominant bacteria in cake sludge contain

A

Gemmobacter, Caldimonas, Bacillus, Phaeodactylibacter, Aquimonas, Acidovorax, Zoogloea, Shinella, Rhodobacter, Rhizobium, Azohydromonsa and Nannocystis. Bacillus is a type of functional bacteria for degrading HPAM. The relative abundance of Bacillus in biofilm, activated sludge and cake sludge was 13.1%, 4.78% and 22.2%,

19

respectively. Wen et al. [49] isolated two strains for degrading PAM named Bacillus flexu and Bacillus cereus, and found more than 70% of PAM was consumed by the microorganisms. The amide group and the carbon backbone of HPAM could serve as energy source for Bacillus sp. and Bacillus cereus, which were isolated from the

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oilfield wastewater [9]. Furthermore, the relative abundance of Bacillus (22.2%), Hydrogenophaga (15.6%) and Phaeodactylibacter (14.7%) in cake sludge was more

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abundant than that of biofilm (13.1%, 4.03%, 8.17%) and activated sludge (4.78%,

6.68%, 4.74%). It indicated that Bacillus, Hydrogenophaga and Phaeodactylibacter

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had a stronger adhesion force in membrane surface and they could survive and

N

perform better than those of biofilm and activated sludge. More interestingly, the

A

appearance of the sole tryptophan protein-like substances in TB-EPS of cake sludge

M

had correlation with Bacillus, Hydrogenophaga and Phaeodactylibacter. In addition,

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nitrifiers are also functional bacteria in aerobic HPAM biodegradation, including unidentified_Nitrospiraceae, unidentified_Nitrosomonadaceae and Nitrosomonas

PT

[50,51]. The relative abundance of unidentified_Nitrospiraceae in biofilm is 5.25 and

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9.12 times higher than that of unidentified_Nitrosomonadaceae and Nitrosomonas, respectively. Combined with Section 3.2, nitrate concentration is 2.58 times higher than that of nitrite after aerobic biodegradation. It revealed that nitratation was the

A

main reaction of nitrogen conversion in aerobic HPAM biodegradation. 3.6 HPAM solution re-preparation by using the different effluent HPAM solution, because of its high viscosity, was used to enhance oil recovery. The viscosity of HPAM solution is an important factor affecting oil recovery. In order

20

to improve water reuse and save water resources, the effluent after different treatments can be used to re-prepare fresh HPAM solution. The supernatant, effluent of BF-AS-MBR and the clean water were used to prepare HPAM solution in this study. The viscosities of HPAM solution re-prepared with different effluent were compared,

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and the order was: supernatant after biodegradation < effluent after membrane filtration ≈ clean water (Fig. 8b). In Fig. 8a, the order of ionic strength in

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BF-AS-MBR system is effluent < supernatant < influent. The ionic strength (2.47 mol/kg) of supernatant after biodegradation is higher than that (0.06 mol/kg) of

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effluent after membrane filtration. Although biodegradation technology plays a vital

N

role in the removal of HPAM in wastewater, it is difficult to remove inorganic ions

A

from wastewater. Inorganic ions have a great effect on the viscosity of HPAM solution

M

and can reduce the viscosity. Xin et al. [52] revealed the viscosity of HPAM declined

ED

with inorganic salts concentrations and the order of ions in declining the viscosity was 0.5Mn2+ < 0.5Ca2+ < K+ < Na+. The viscosity of HPAM solution declined from 14.4

PT

mPa·s to 6.80 mPa·s with SO42- concentration (0-160 mg·L-1) and reduced to 5.81

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mPa·s with Fe3+ concentration (0-100 mg·L-1) [53]. Combined with the previous studies, the reason for the reduction in viscosity of HPAM solution re-prepared with supernatant after biodegradation is that supernatant after biodegradation contains

A

many inorganic ions (2.47 mol/kg). In addition, effluent after membrane filtration is close to clean water, and it can be used to re-prepare fresh HPAM solution for oil recovery. 4. Conclusion

21

The removal efficiencies of HPAM, COD, viscosity, TOC and TN achieved 97.5%, 98.5%, 74.8%, 98.5% and 96.2%, respectively. Nitrogen transformation revealed nitratation became the main reaction in the process of aerobic amide group bioconversion and surpassed denitrification, nitritation and ammonification in

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BF-AS-MBR system. The membrane permeability was recovered to 92% and 87% after the first and second cleaning, respectively. Tryptophan protein-like, aromatic

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protein-like, simple aromatic protein-like and fulvic acid-like organics presented in LB-EPS of sludge. Polysaccharide-like and tyrosine protein-like organics presented in

U

TB-EPS of sludge, and tryptophan protein-like organics existed solely in TB-EPS of

N

cake sludge. HPAM-degrading microorganisms and nitrifiers were closely associated

A

with HPAM removal, EPS and membrane fouling. The viscosities of HPAM solution

M

re-prepared with different effluent were compared, and the order was: supernatant

ED

after biodegradation < effluent after membrane filtration ≈ clean water. This study offered a technical support and theoretical foundation for treating actual

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HPAM-containing oilfield wastewater, and improved water reuse.

Acknowledgments This study was financially supported by the Key Research and Development

A

Program of Shandong Province (public welfare special project) (2017GSF217012), the National Natural Science Foundation of China (51174181) and the Major Projects of

the

National

High

Technology

Research

863(2013AA064401).

22

and

Development

Program

Appendix A. Supplementary data

A

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PT

ED

M

A

N

U

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

23

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6

4

P

Effluent

5 7

3

Supernatant 1-Peristaltic pump 2-Air compressor 3-Heater 4-Pressure gauge 5-Suction pump 6-Time controller 7-Membrane module 8-Biomass carrier 9-Cake sludge 10-Activated sludge

8

9

Influent

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10 2

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1

A

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M

A

N

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Fig. 1. Configuration of BF-AS-MBR system.

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340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0

b

Influent Supernatant Effluent

5.5

Influent Supernatant Effluent

5.0

Viscosity (mPas)

4.5 4.0 3.5 3.0 2.5

700

d 250 Influent Supernatant Effluent

300

150 100

200

50

N

100

50

ED

NO3 -N

30 20

Influent

Supernatant Different water samples

60

50

40

30

140 142 144 146 148 150 The second cleaning

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

Effluent

Time/d

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The first cleaning

60 65 70 75 80 85 90 95 10 0 10 5 11 0 11 5 12 0 12 5 13 0 13 5 14 0 14 5 15 0

40

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-

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2

M -

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f

Permeability (L/hm kPa)

TN + NH4 -N

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140 142 144 146 148 150

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30

20

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140 142 144 146 148 150

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COD (mg/L)

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600

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30

20

10

0

140 142 144 146 148 150

60

50

40

30

20

10

1.5

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0

HPAM concentration (mg/L)

a

A

Fig. 2. (a-d) HPAM concentration, shear viscosity, COD and TOC of influent, supernatant and effluent in BF-AS-MBR system; (e) TN, NH4+-N, NO2--N and NO3--N concentrations of influent, supernatant and effluent in BF-AS-MBR system; (f) Membrane permeability in the process of BF-AS-MBR operation.

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c

d

e

f

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b

h

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g

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A

N

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a

Fig. 3. (a-h) SEM observation of membrane surface. (a) and (g) The new membrane; (b) The membrane after the first contamination; (c) The membrane after the first cleaning; (d) The membrane after the second contamination; (e) The membrane after the second cleaning; (f) and (h) The membrane after the third contamination.

33

400

240.0

400

b

380 200.0

360 340

280 80.00 Peak C Peak B

0 300

350 400 EM(nm)

450

200 200

500 270.0

360

280

EX(nm)

90.00

260 Peak C Peak B

240

260 240

45.00

220

338.3 270.7 203.0 135.3

Peak F

67.67

220

0 300

350 400 EM(nm)

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393.0

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402.0 335.0

360 Peak E

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196.5 131.0 65.50

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340

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327.5

360

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500

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Peak E

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180.0

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300

400

d

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200 200

95.83

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380

220

191.7 Peak F

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40.00

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287.5

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Peak A

EX(nm)

EX(nm)

Peak D

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e

383.3

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200 200

Peak E

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479.2

360

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260

134.0

Peak F

240

67.00

220

0 450

200 200

500

0 250

300

350 400 EM(nm)

450

500

A

Fig. 4. EEM fluorescence spectra of the LB-EPS and TB-EPS from biofilm, activated sludge and cake sludge in a steady state. (a) LB-EPS from biofilm; (b) TB-EPS from biofilm; (c) LB-EPS from activated sludge; (d) TB-EPS from activated sludge; (e) LB-EPS from cake sludge; (f) TB-EPS from cake sludge.

34

c

d

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b

N

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a

A

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M

A

Fig. 5. Venn diagram of OTUs in different sludge samples. (a) biofilm and activated sludge; (b) activated sludge and cake sludge; (c) biofilm and cake sludge; (d) activated sludge, biofilm and cake sludge.

35

A ED

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CC E Relative abundance(%) 2

1

3 Betaproteobacteria

Activated sludge

Biofilm

0

36

Cake sludge

b

Activated sludge Cake sludge

c

Bacilli Sphingobacteriia

4 Alphaproteobacteria

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Biofilm

Biofilm Activated sludge Cake sludge

Planctomycetacia

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U

N

50 45 40 35 30 25

A

M

Hy dr og un Az eno id oh T ph en yd ha ag tif ro ue a ied _N Den Zomonra itr og as itr os Ac ati loe om id so a Ni on ovoma t a r M Crosodac ax et a m ea Dehyloldimonae c v o s Pr hlo ersa nas op ro ti io mo lis n n Azivib as oa rio Ph T In rcu Haaeo ric Bachells lis dac hoc ill a c t o u Feomeylib ccus rru no ac s gi ba ter n GeTer ibacter m rim cte m o r o n R S bac as Hy hod hin ter ob ell p B ho D ac a Ca revmic evoter nd un rob sia id W dim iu atu oo o m s_ dsh na s R Cap ole Pe hizo tivua do H bi s m irs um Vaicro chi rii biu a b m C ac un aul Bo ter ob se id en ac a Amtifie Steter in d_I lla R ob -1 A ey ac 0 Pl naeran ter an ro el cto lin la Pi Pimycea r4 re es l Bl G_lin lula Rhasto em eage od pir ma o e t A pi llu a En qui rellula ter mo la S o n Ps Aci hewbactas eu Ps ne an er do eu tob el xa do ac la n m te Arthomon r en o as un A id er imonas om na Ca ent P un nd ifie seu Sil Co onas id ida d_ do an xie s en tu L fu im ll tif s_ eg lv on a ied Co ion im as _N m ell ona itr pet ace s os iba ae pi ct ra er Otceae he rs

a Proteobacteria Firmicutes Bacteroidetes Chloroflexi Planctomycetes Acidobacteria Fusobacteria Nitrospirae Gemmatimonadetes Chlorobi Others

Betaproteobacteria Bacilli Sphingobacteriia Alphaproteobacteria Anaerolineae Planctomycetacia Gammaproteobacteria Cytophagia Deltaproteobacteria unidentified_Acidobacteria Others

Nitrospira

Gammaproteobacteria

Anaerolineae

Fig. 6. The taxonomic classification of the bacterial communities from different sludge samples during HPAM biodegradation under steady states. (a) Phylum level; (b) Class level; (c) Genus level.

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Activated Biofilm sludge

Cake sludge

A

Fig. 7. Heatmap of bacterial communities from different sludge samples at Genus level.

37

3.5

b

40

3.0

35 2.5

Viscosity (mPas)

-2

Ionic strength (10 mol/kg)

45

2.0 1.5 1.0

Clean water Influent after membrane filtration Supernatant after biodegradation

30 25 20 15 10

0.5

5

IP T

a

0

0.0 Influent

Supernatant Different water samples

200

Effluent

400 600 800 HPAM concentration (mg/L)

1000

A

CC E

PT

ED

M

A

N

U

SC R

Fig. 8. (a) Ionic strength of influent, supernatant and effluent in BF-AS-MBR system; (b) Viscosity of HPAM solution prepared with supernatant after biodegradation, effluent after membrane filtration and clean water.

38

Table 1. Characteristics of actual HPAM-containing oilfield wastewater in Gusi Wastewater Treatment Station of Shengli Oilfield. COD (mg/L) BOD (mg/L) Viscosity (mPa·s) HPAM (mg/L) Oil

Value

Parameter

656 ± 9

Na (mg/L)

228 ± 4

K (mg/L)

38.3 ± 1.9

2+

5.24 ± 0.07

Ca (mg/L)

304 ± 7

45.6 ± 2.4

2+

9.7 ± 1.8

2-

89 ± 3.3

Mg (mg/L) SO4 (mg/L)

16.3 ± 1.5

-

Cl (mg/L)

995 ± 10

NO3 (mg/L)

2.1 ± 0.16

pH

7.2 ± 0.1

Total phosphorus

0.14 ± 0.01

Temperature (℃)

35

A

CC E

PT

ED

M

A

N

U

NH4 (mg/L)

SC R

-

564 ± 6

+

50 ± 4 +

Value

+

IP T

Parameter

39

Table 2. Fluorescence spectral parameters of LB-EPS and TB-EPS from different sludge samples in BF-AS-ABR system.

Ex/ Em

280 /35 0

98. 7

220 /35 0

Inte nsit y

Ex/ Em

178

220 /31 0

Peak D

Inte nsit y

Ex/ Em

Inte nsit y

Ex/ Em

Inte nsit y

187

270 /30 0

105

-

-

TB -E PS

-

-

-

-

-

-

-

LB -E PS

280 /34 0

47. 5

220 /35 0

90. 8

220 /31 0

82. 0

270 /30 0

TB -E PS

-

-

-

-

LB -E PS

280 /35 0

183

320 /38 0

87. 8

101

220 /34 0

-

168

-

-

220 /30 0

-

59. 6

-

270 /30 0

A

40

Inte nsit y

-

-

211

-

-

-

-

-

320 /38 0

325

230 /38 0

180

98. 9

-

-

-

-

-

320 /38 0

120

230 /37 0

115

U

-

Ex/ Em

312

-

N

-

Peak F

230 /38 0

A 280 /36 0

-

CC E

TB -E PS

Peak E

IP T

Inte nsit y

PT

Cake sludge

Ex/ Em

Peak C

M

Activate d sludge

LB -E PS

Peak B

ED

Biofilm

Peak A

SC R

Samples

Table 3. Diversity and richness indices of microorganisms from different sludge samples in BF-AS-ABR system. Raw Sequen ces

Effectiv e Sequen ces

Biofilm

86253

83207

79156

Activated sludge

94019

90479

85767

Cake sludge

81251

78573

74163

OTUs

Chao1 index

1376

1371

1319

1330

1056

1041

ACE index

Shanno n index

Simpso n index

Good’s coverag e

1379

6.96

0.962

0.998

1322

7.47

1054

5.12

IP T

Raw Reads

0.983

0.998

0.905

0.997

A

CC E

PT

ED

M

A

N

U

SC R

Samples

41