Crossing the Border between Laboratory and Field: Bacterial Quorum

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Crossing the Border between Laboratory and Field: Bacterial Quorum Quenching for Anti-Biofouling Strategy in an MBR Seonki Lee, Seung-Kook Park, Hyeokpil Kwon, Sang Hyun Lee, Kibaek Lee, Chang Hyun Nam, Sung Jun Jo, Hyun-Suk Oh, Pyung-Kyu Park, Kwang-Ho Choo, Chung-Hak Lee, and Taewoo Yi Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04795 • Publication Date (Web): 15 Jan 2016 Downloaded from http://pubs.acs.org on January 19, 2016

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Environmental Science & Technology

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Crossing the Border between Laboratory and Field:

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Bacterial Quorum Quenching for Anti-Biofouling

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Strategy in an MBR

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Seonki Lee†, Seung-Kook Park‡, Hyeokpil Kwon†, Sang Hyun Lee†, Kibaek Lee†,

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Chang Hyun Nahm†, Sung Jun Jo†, Hyun-Suk Oh†, Pyung-Kyu Park§, Kwang-Ho Choo∥,

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Chung-Hak Lee*,†, and Taewoo Yi,**,‡

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†

School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744,

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Republic of Korea

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‡

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Republic of Korea

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§

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Korea

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Hanwha Engineering and Construction, 76, Gajeong-Ro, Yuseong-Gu, Daejeon 305-804,

Department of Environmental Engineering, Yonsei University, Wonju, 220-710, Republic of

∥

Department of Environmental Engineering, Kyungpook National University, Daegu, 702-

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701, Republic of Korea

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*Corresponding author: Phone: +82-2-880-7075. Fax: +82-2-874-0896. E-mail: [email protected] **Corresponding author: Phone: +82-41-950-5874. Fax: +82-41-950-5934. E-mail: [email protected]

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ABSTRACT

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Quorum quenching (QQ) has recently been acknowledged to be a sustainable antifouling

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strategy and has been investigated widely using lab-scale membrane bioreactor (MBR)

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systems. This study attempted to bring this QQ-MBR closer to potential practical application.

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Two types of pilot-scale QQ-MBRs with QQ bacteria entrapping beads (QQ-beads) were

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installed and run at a wastewater treatment plant, feeding real municipal wastewater to test

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the systems’ effectiveness for membrane fouling control and thus the amount of energy

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savings, even under harsh environmental conditions. The rate of transmembrane pressure

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(TMP) build-up was significantly mitigated in QQ-MBR compared to that in a conventional-

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MBR. Consequently, QQ-MBR can substantially reduce energy consumption by reducing

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coarse bubble aeration without compromising the effluent water quality. The addition of QQ1

ACS Paragon Plus Environment


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beads to a conventional MBR substantially affected the EPS concentrations, as well as

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microbial floc size in the mixed liquor. Furthermore, the QQ activity and mechanical stability

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of QQ-beads were well maintained for at least four months, indicating QQ-MBR has good

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potential for practical applications.

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INTRODUCTION

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One of the pressing needs of people throughout the world is adequate supply of clean water.

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Increasing water reuse through advanced wastewater treatment has become more urgent than

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ever because of the water shortage accelerated by global population growth and climate

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change.1, 2 For over thirty years, the membrane bioreactor (MBR) has attracted considerable

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attention in the field of wastewater treatment and reuse. However, a critical issue in MBR

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that awaits a technological advancement to solve is membrane biofouling, which leads to

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high energy consumption and thus high operating costs.

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Many studies utilizing diverse approaches have been conducted and reported to mitigate

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this chronic issue of biofouling in MBRs, such as by modification of the membrane surface3,

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by changes in physical cleaning strategies (such as back flushing, ultra-sonication,

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mechanical cleaning, and vibration)4,5,6,7 or by adding specific media.7,8 Note that Yeon et al.9

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revealed that quorum sensing plays a key role in biofilm formation on the membrane surface

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in an MBR. Furthermore, they proved that the biofilm formation (biofouling) could be

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mitigated substantially by disrupting signal molecules such as N-acyl homoserine lactones

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(AHLs)10 using a quorum quenching enzyme.11,12,13,14 To overcome the drawbacks of

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enzymatic quorum quenching, such as high cost of enzyme, bacterial quorum quenching was

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developed.15,16,17,18,19,20,21,22 In detail, bacteria which produce quorum quenching enzymes

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were isolated from a real MBR plant. Subsequently, those quorum quenching bacteria were

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encapsulated in a vessel15,16,17,20 or entrapped in beads.18,21,22 They proved bacterial quorum

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quenching is very efficient in both physical and biological effects on biofouling control in

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MBR for wastewater treatment.

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To date, however, most studies examining quorum quenching (QQ) MBR have been

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performed in lab-scale reactors using synthetic wastewater. Although substantial mitigation

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of biofouling by quorum quenching has been demonstrated, such an approach has not been

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fully investigated in a larger pilot scale MBR using real wastewater. More often than not, the

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microbial environment in lab-scale reactors is notably different from that in real full-scale 2


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reactors due to the different scales of the technological equipment and the different raw

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wastewater characteristics.

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The purpose of this study was to bring the QQ-MBR closer to potential practical

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application by demonstrating the QQ effects in pilot-scale MBRs installed at a municipal

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wastewater treatment plant and fed with real wastewater. Two MBR systems, one using a

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one-stage (only one aerobic tank) process, and the other using a three-stage

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(anaerobic/aerobic/membrane tanks) process, were operated for an extended period of more

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than four months. To examine the life-span of encapsulated beads containing quorum

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quenching bacteria under real wastewater condition, their QQ activity was monitored

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regularly for that period. Considering that QQ may affect microbial floc size and extracellular

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polymeric substance (EPS), the changes of protein, polysaccharide concentrations and floc

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size distribution were monitored during the operation of conventional and QQ-MBRs. The

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energy savings through the QQ-MBR was also evaluated relative to the energy consumption

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of a conventional MBR.

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MATERIALS AND METHODS

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Preparation of the QQ-beads

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QQ-beads were prepared using dripping method. However, the material and solidification

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methods were modified to reinforce their stability by mixing polyvinyl alcohol and sodium

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alginate solutions because QQ-beads made of alginate matrix are easily decomposed in real

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wastewater. The mixture of polyvinyl alcohol, alginate and Rhodococcus sp. BH4 was

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dripped into CaCl2-boric acid solution to form beads. Then, the beads were immersed in

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sodium sulfate solution and stored in water. Rhodococcus sp. BH4, which was isolated from

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a real MBR plant was used as a QQ bacterium in this study because its biofouling control

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effect in MBR has been confirmed in a number of previous studies using synthetic

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wastewater15,16,19,22 as well as it was reported to be capable of degrading a wide range of AHL

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molecules by AHL-lactonase.24 The concentration of QQ bacteria (Rhodococcus sp. BH4) in

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beads was approximately 4~5 mg per g of beads. The average size and density of QQ-beads

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were approximately 4.3 mm and 1.05 g/mL, respectively.

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MBR configurations and operation conditions 3



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The two types of pilot-scale submerged MBRs, one- and three-stage MBRs, were installed at

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a municipal wastewater treatment plant (Daejeon, Korea), as shown in Figure 1. The one-

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stage MBR consisted of only an aerobic membrane tank which is a simple MBR system

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mostly used in previous studies. In the one-stage MBR, two types of aeration devices were

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equipped: one for oxygen supply to activated sludge, and the other for membrane scouring

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(Figure 1a). The air supply for the microorganisms was constantly maintained at 10 L/min

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during the entire operation period, whereas the air scrubbing volumes for membrane cleaning

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were varied 0.3 ~ 0.9 SADm (specific aeration demand per membrane area, m3/m2/h). Two

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one-stage MBRs with a working volume of 80 L of each were operated in parallel. In phase 1,

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one reactor was used for a vacant-MBR (with vacant-beads) and the other was used for a QQ-

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MBR (with QQ-beads). Same intensity of membrane aeration was maintained at 0.3 SADm

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for both MBRs to confirm the QQ effect in real wastewater. In phase 2, one reactor was used

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for a conventional-MBR (without bead) and the other was used for a QQ-MBR. Different

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membrane aeration intensity was applied to investigate the potential of energy savings for

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QQ-MBR, i.e., 0.9 SADm for the conventional-MBR whereas 0.3 SADm for QQ-MBR. For

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each one-stage MBR, three C-PVC membranes with a pore size of 0.4 µm (Pure-envitech,

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Korea) and an effective total surface area of 0.9 m2 were used. The flux was maintained at a

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constant 20 L/m2/h (LMH) in a continuous cycle of filtration (10 minutes), followed by

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relaxation (two minutes).

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A real conventional MBR system is composed of biological nutrient removal processes,

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such as anoxic-aerobic (A/O) or anaerobic–anoxic–aerobic (A2O) processes. To bring the

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QQ-MBR closer to potential practical application, the one-stage MBR was expanded to a

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three-stage MBR with an additional anoxic process for nutrient removal. The three-stage

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MBR consisted of an anoxic, an aerobic and a membrane tank. An air diffuser was equipped

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in each of the aerobic and membrane tanks (Figure 1b). Two three-stage MBRs were

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operated in parallel: one was a conventional-MBR (without beads) and the other was a QQ-

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MBR (with QQ-beads). Polyethersulfone membranes with a pore size of 0.04 µm (Microdyn-

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Nadir, Germany) were applied, giving an effective total surface area of 3.5 m3 to each MBR.

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Identical diffusers were equipped in each aerobic tank for oxygen supply and in each

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membrane tank for membrane cleaning. The aeration rate for oxygen supply and membrane

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cleaning were constantly maintained at 8 L/min in an aerobic tank and 0.34 SADm in a

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membrane tank, respectively. The flux was maintained at 15 L/m2/h (LMH) in a continuous

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cycle of filtration (10 minutes) followed by relaxation (2 minutes). Influent was fed into the 4


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anoxic tank, while activated sludge in the membrane tank was continuously recirculated into

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anoxic tank with a recirculation rate of 2.5 Q.

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In both types of MBRs, activated sludge was directly taken from the wastewater treatment

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plant and real wastewater, which passed sedimentation tank and screen, was fed as the

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influent. A mesh screen (size: about 2 mm) was placed in the membrane tank so that vacant-

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or QQ-beads (size: about 4.3 mm) were constantly retained in the membrane tank in each

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MBR. TMP is defined as the pressure difference between broth and permeate side of a

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membrane in a submerged MBR. The extent of biofouling in each MBR was quantitatively

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evaluated by monitoring the TMP which increases to compensate for the membrane-fouling

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under constant flux operation. When the TMP reached above 25 kPa during the MBR

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operation, used membrane was taken out and chemical washing with 1,000 ppm of NaOCl

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solution was conducted, which resulted in more than 96% of permeability recovery. The

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operating conditions of each MBR and other important specifications are provided in Table 1.

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Bioassay for the detection of QS bacteria producing AHLs

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LB agar covered with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) was used

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for the bioassay. These assays consisted of streaking the AHL reporter strain, A. tumefaciens

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A13623, on the plate, and then placing each sample to be tested. If the sample produces or

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contains AHLs, then they diffuse through the agar, developing a blue color in the reporter

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strain as a result. Positive and negative controls consisted of standard C8-HSL and distilled

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water, respectively.

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Bioluminescence assay for measuring the AHLs

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The AHL molecules were measured via a bioluminescence assay24. First, the reporter strains

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of A. tumefaciens A136 and the AHL sample were mixed and loaded into 96-well-plate. The

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plate was placed on an incubator to maintain the temperature at 30 °C for 90 min, and then

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Beta-Glo® Assay System (Promega, USA) solution was added into each well. After 40 min,

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the luminescence intensity was measured by using a luminometer (Synergy 2, Bio-Tek, USA).

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The concentration of AHL samples were determined by plotting the calibration curve of

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standard AHLs.

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Measurement of the biological and mechanical stability of the QQ-beads 5



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The activity of QQ-beads was determined by the degradation rate of C8-HSL (N-octanoyl-DL-

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homoserine lactone). C8-HSL was chosen as a representative AHL molecule because it is one

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of the dominant AHL molecules in MBRs for wastewater treatment.9,15 Standard C8-HSL

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(Sigma Aldrich, USA) was dissolved in Tris-HCl buffer (50 mM, pH 7) to a final

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concentration of 200 nM. Next, 50 QQ-beads were added to 20 mL of the C8-HSL solution

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and then reacted using orbital shaker at 50 rpm at 25 °C. The residual concentration of C8-

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HSL was measured using a bioluminescence assay. To determine the biological stability of

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QQ bead in a continuous MBR, 50 QQ-beads were withdrawn and strongly washed for

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measurement of the residual QQ activity. The relative QQ activity at each time point was

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calculated by the ratio of residual activity to initial activity. Furthermore, the viability of the

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entrapped BH4 into QQ-beads was visually observed using a confocal laser scanning

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microscope (CLSM, C1 plus, Nikon, Japan) after staining with live/dead kit (Molecular

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Probes, USA).

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To investigate the mechanical stability of QQ-beads during MBR operation, compression

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tests were performed using a texture analyzer (CT3 4500, Brookfield, USA) at a mobile

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probe (TA44) speed of 0.5 mm/s up to 50% deformation of beads. The hardness work, a

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measure of energy required to compress the sample, was calculated as the area under the

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curve of the compression plot.21

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Analytical methods

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EPS (extracellular polymeric substances) concentrations in the mixed liquor and the biofilm

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were analyzed in both vacant- and QQ-MBRs. Soluble EPS were obtained by centrifuging the

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mixed liquor (6000 rpm, 10 min) and then filtering the supernatant through a 0.45-µm syringe

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filter. Bound EPS was extracted from the pellet (activated sludge floc). EPS in the biofilm

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was obtained as follows. First, a used membrane was taken from a membrane tank and briefly

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washed with tap water. Next, the biofilm was detached from the used membrane via

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sonication (10 min) and then extracted using the modified thermal extraction method.25 The

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proteins and polysaccharides in the EPS were quantitatively determined using the Bradford

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assay and the phenol-sulfuric acid method, respectively.26

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Mixed liquor suspended solid (MLSS), chemical oxygen demand (COD), total nitrogen

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(TN) and ammonia-nitrogen (NH4-N) were measured according to standard methods. The

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average floc size was measured using a particle size analyzer (Microtrac S3500).

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RESULTS AND DISCUSSION

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QQ effects on the performance of the one-stage MBR fed with real wastewater

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The pilot-scale MBR experiments were conducted to investigate the influence of real

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wastewater on QQ effect instead of the synthetic wastewater because the characteristics of

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influent wastewater should have a large impact on MBR performance.27 Prior to applying

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QQ-beads into the MBRs, a bioassay test was conducted to confirm the presence of QS

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bacteria producing AHLs in the real wastewater. As shown in Figure S1, it was revealed that

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QS bacteria existed in the feed wastewater as well as the activated sludge used for inoculum

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in this study. In phase 1, vacant-beads with no QQ-bacterium and QQ-beads entrapped with

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QQ-bacteria were placed into the first one-stage MBR (Vacant-MBR) and the second one-

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stage MBR (QQ-MBR), respectively. The loading rate of the beads in each one-stage MBR

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was 1% in v/v (volume of beads/volume of reactor), while the same intensity of membrane

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aeration was maintained at 0.3 m3/m2/h (SADm) for both MBRs.

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As shown in Figure 2a, the rate of TMP build-up, which reflects the extent of membrane

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biofouling, was delayed by approximately 500% in the QQ-MBR compared with that in the

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vacant-MBR. Biofouling reduction in QQ-MBR could be attributed to both a biological effect

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due to quorum quenching of the QQ-beads and a physical effect due to friction between the

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beads and the membrane. Assuming that the physical cleaning effect by beads would be

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identical in both vacant- and QQ-MBRs because of the equal bead loading, the delay of the

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TMP build-up rate in QQ-MBR can be attributed solely to the QQ activity of the QQ-beads.

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The characteristics of real wastewater are more variable and unsteady, unlike that of synthetic

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wastewater, as shown in Figure S2. Note that biofouling control through bacterial QQ can

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also be achieved in an MBR, which suggests the QQ-MBR is approaching closer and closer

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to practical application.

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QQ effects on EPS relevant to biofouling

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Quorum sensing is known to be the cell-to-cell communication between microorganisms,

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which determines phenotypes, such as secretion of EPS, biofilm formation, and virulence.28,29

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Many previous studies on QQ-MBR using synthetic wastewater reported that the

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concentration of EPS was decreased in a QQ-MBR.12,18,19,20,22 To confirm the change in EPS

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concentration in the QQ-MBR fed with real wastewater, EPS analysis of the mixed liquor and

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the biocake were conducted for both vacant- and QQ-MBRs. 7



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After the same operating period of 14 days, the used membrane modules were taken out

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from both vacant- and QQ-MBRs to measure the amount of total attached biomass (TAB)

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and EPS in the biocake on the membrane surface. Mixed liquor in each MBR was also

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sampled to measure the total (i.e., polysaccharides plus protein) soluble and bound EPS (EPS

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in activated sludge floc). The total soluble and the total bound EPS in the mixed liquor in the

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QQ-MBR were reduced by approximately 60% (Figure 3a) and 15% (Figure 3b), respectively,

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compared to those in the vacant-MBR. It has been reported that the soluble EPS may have

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great influence on the membrane-biofouling in MBR.30,31,32 Especially, Wisniewski et al.32

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revealed the significant role of the soluble fraction of EPS on membrane permeability. Thus,

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even though our results show that the amount of soluble EPS was much smaller than that of

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bound EPS in the mixed liquor, the 60% reduction of soluble EPS through QQ-beads could

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substantially mitigate the biofouling in QQ-MBR. The TAB per unit area of membrane in the

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vacant-MBR (420 mg/m2) was much greater than that in the QQ-MBR (60 mg/m2), indicating

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much lower biomass was accumulated on the membrane surface in QQ-MBR after the same

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operation period. Moreover, the concentration of polysaccharide per unit membrane surface

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was significantly reduced in the QQ-MBR (Figure 3c). It has been reported that EPS plays a

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great role in biofilm formation and is also a key foulant of membrane-biofouling in an

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MBR.30,33,34,35 Thus, one of the main reasons why QQ-beads mitigate biofouling in MBR fed

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with real wastewater could be a reduction of soluble EPS in the mixed liquor as well as the

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reduction of EPS in the biocake.

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Although TMP rise-up rate was delayed, membrane-fouling still occurred in QQ-MBR. It

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may be related to other membrane-fouling mechanisms such as pore blocking, sludge

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accumulation, and other types of quorum sensing.

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Comparison of the energy consumption between conventional- and QQ-MBRs

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In submerged MBR systems, coarse bubble aeration for membrane scouring is a major energy

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consumer, often exceeding 50% share of the total energy consumption (Figure S3).

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Consequently, many efforts36,37 have been made and reported to reduce the energy consumed

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for membrane scouring. As we confirmed that the rate of TMP rise-up was substantially

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delayed in the QQ-MBR, we attempted to quantitatively evaluate how much energy savings

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would be achieved by the addition of QQ-beads. For that purpose, the conventional- and QQ-

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MBRs were run in parallel, applying the same aeration intensity for the oxygen supply, but 8


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with different coarse bubble aeration conditions for membrane cleaning. The specific aeration

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demand per membrane surface area (SADm) was 0.9 m3/m2/h for the conventional-MBR,

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whereas it was 0.3 m3/m2/h for the QQ-MBR. In other words, the membrane aeration

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intensity in the QQ-MBR was reduced to one third of that in the conventional-MBR. As

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shown in Figure 2b, even though the conventional-MBR was operated at threefold greater air

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scrubbing intensity, the TMP rise-up rate in the conventional-MBR was faster than that in the

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QQ-MBR.

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Based on Figure 2b, the specific filtration energy required for permeate suction and specific

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aeration energy for membrane cleaning in one-stage MBRs were estimated using equations (1)

268

& (2), which were derived with some assumptions described in the SI. Figure 4 shows a

269

comparison of the specific energy consumed for filtration (permeate suction) and air

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scrubbing between the conventional- and QQ-MBRs. The difference in the filtration energy

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consumption was not as high as that in air scrubbing for membrane cleaning. Note that the

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specific energy consumption for membrane scouring in the conventional-MBR was

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approximately three-times greater than that in the QQ-MBR. The specific energy

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consumption for both items was approximately 0.46 kWh/m3 in the conventional-MBR,

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whereas the amount was 0.20 kWh/m3 in the QQ-MBR. In short, QQ-MBR could reduce the

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biofouling related energy consumption by approximately 60%. In addition, considering the

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labor and chemical costs required for chemical cleaning of the used membranes, the operating

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cost could further be reduced in the QQ-MBR.

279 280

QQ effect in the three-stage MBR fed with real wastewater

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Figure 5 shows the profiles of TMP rise-up in the conventional- and QQ-MBRs operated at

282

the same aeration intensity (0.34 m3/m2/h). It took approximately 18 days in the

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conventional-MBR, whereas it took 35 days in the QQ-MBR, to reach the TMP of 20 kPa. In

284

other words, the rate of TMP rise-up was delayed by approximately 100% in the QQ-MBR

285

through the addition of QQ-beads to the membrane tank. This result indicates that the QQ

286

effect on the membrane fouling is also valid in the three-stage MBR system with an anoxic

287

process. However, the extent of the delay of TMP rise-up decreased from 500% (Figure 2a)

288

for the one-stage MBR to 100% (Figure 5) in the three-stage MBR. This reduced

289

performance might be attributed to the activated sludge recycle throughout the three tanks

290

and/or the considerably smaller loading volume of the QQ-beads (v/v) in the three-stage

291

MBR [0.27% (v/v)], which was approximately one-third of that in the one-stage MBR [1% 9



292

(v/v)]. Further research should be performed to determine the best operating conditions that

293

are able to optimize the use of the QQ-beads in the QQ-MBR. In addition, the difference of

294

MBR process between the one- and three-stage MBRs could give rise to the change of

295

microbial community, which might be another cause of reduced performance.

296 297

QQ effect on the floc size of the activated sludge

298

Microbial floc size is one of the important factors of the floc characteristics and is related to

299

membrane-biofouling.38,39 However, some reports on the QS/QQ effects on floc size are

300

contradictory to each other. It was reported that the granule formation and size is closely

301

related to AHL-based QS.40, 41 Jiang et al.14 found that the size of the activated sludge floc

302

was decreased by quorum quenching. However, Weerasekara et al.19 reported the change of

303

microbial floc size could be attributed to microbial adaptation when seeded into a new reactor,

304

instead of the effect of QQ.

305

In this work, in the one-stage MBR, the average floc size in the conventional-MBR was

306

106 µm, but that in the QQ-MBR was 88 µm (approximately 20% reduction), as shown in

307

Figure S4 and Figure 6. In the three-stage MBR, however, the floc size in all three tanks

308

became much smaller than that in one-stage MBRs. Moreover, no significant difference of

309

the average floc size was observed, not only between three tanks but also between the

310

conventional- and QQ-MBRs in each tank. Kim et al.33 and McMillan et al.42 mentioned that

311

activated sludge can be mechanically broken by the pumping device. In the three-stage MBR,

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microbial flocs are continuously recirculated via the high flow rate of the recirculation pump

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(2.5Q, 1.7 L/min), so that they could be broken mechanically by pumping shear, which may

314

be more powerful than the QQ effect. Consequently, the microbial floc size would be

315

equalized, regardless of the tank types.

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QQ effect on the effluent quality

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QQ-MBR inhibited biofouling and thus could substantially reduce the energy consumption of

319

the MBR. However, we cannot go further without verifying the effluent water quality in the

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QQ-MBR because it must be one of the main factors in the evaluation of MBR performance.

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It has been reported that certain nitrifying, denitrifying and nitrogen fixation bacteria were

322

involved in quorum sensing.43,44,45,46 In addition, QQ could affect the metabolism of QS 10


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bacteria in the activated sludge.

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We monitored the removal efficiencies of some pollutants, such as COD, T-N, NH4-N and

325

NO3-N for one-stage and three-stage MBRs. However, there was no significant difference

326

between conventional- and QQ-MBRs in both one-stage and three-stage MBRs (Figure 7 and

327

Table S2). This result indicates that quorum quenching has no adverse effect on the effluent

328

water quality in QQ-MBR fed with real wastewater.

329 330

Stability of the QQ-beads in the long-term operation of the QQ-MBR

331

On the path toward the practical application of a QQ-MBR, long-term stability of the QQ-

332

beads is required because a short life span of QQ-beads would require their frequent

333

replacement, which would increase the operational cost of the QQ-MBR. Thus, the stability

334

of QQ-beads in terms of the QQ activity, viability and mechanical strength were tested during

335

the long-term (100 ~ 140 days) operation of both QQ-MBRs.

336

The used QQ-beads were intermittently removed from the aerobic tank (one-stage MBR)

337

or the membrane tank (three-stage MBR). Next, the relative QQ-activity, defined by the

338

degradation ratio of standard C8-HSL for 120 min in the presence of used QQ-beads, was

339

monitored and plotted against the operating time (Figure 8). In the one-stage QQ-MBR, the

340

relative activity of QQ-beads decreased by 40% of their initial activity during 25 days, but

341

increased slowly up to the initial QQ activity level after approximately 60 days. In the three-

342

stage MBR, the relative activity of QQ-beads decreased by approximately 10% to 20% of the

343

initial activity during 100 days of operation. A possible explanation for the initial decrease of

344

the QQ activities might be that the QQ bacteria were adapting themselves to the new

345

environment when they were exposed to the real wastewater.

346

In parallel with the relative QQ activity of the QQ-beads, live/dead cells in the QQ-beads

347

were also observed intermittently in the three-stage QQ-MBR to monitor the survival of the

348

QQ bacteria. Because it is nearly impossible for bacteria outside the beads to migrate into the

349

beads as shown in Figure S6, it had been concluded that cells shown in Figure S5 were

350

Rhodococucs Sp. BH4 which were initially entrapped into the QQ-beads. The population of

351

live cells (Figure S5a) appeared to be much higher than that of the dead cells (Figure S5b),

352

i.e., the viability of QQ bacteria were well maintained at least for 100 days. Consequently, it

353

is believed that QQ bacteria could live on the substrates and nutrients in real wastewater. In

354

addition, the relative mechanical strength of the QQ-beads, defined by the ratio of residual

355

mechanical strength to its initial value, was measured intermittently during 100 days 11



356

operation. As shown in Figure S7, the change of the mechanical strength appeared to be

357

negligible.

358

All these results showed that the biological and mechanical stabilities of the QQ-beads were

359

well maintained, even in the harsh environment of real wastewater during the relatively long-

360

term operation of the QQ-MBR.

361

In summary, we demonstrated the effectiveness of quorum quenching for biofouling

362

control in two types of pilot-scale MBRs fed with real municipal wastewater. A QQ-MBR

363

with QQ bacteria entrapping beads could alleviate membrane fouling and thus could

364

significantly reduce energy consumption by reducing the aeration intensity required for

365

membrane cleaning compared with that in a conventional-MBR. Moreover, bacterial QQ was

366

determined to have no influence on the effluent water quality of the MBR systems. The QQ

367

activity, as well as mechanical stability of QQ-beads, was found to be well-maintained, even

368

under harsh real wastewater conditions, during the four months of operation. This study is

369

expected to bring QQ-MBR closer to possible practical applications.

370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396

Supporting Information Additional figures and table. This material is available free of charge via the Internet at http://pubs.acs.org. ACKNOWLEDGEMENTS This research was supported by the Commercializations Promotion Agency for R&D Outcomes (COMPA) (2014K000240) funded by the Ministry of Science, ICT and Future Planning (MISP) and Convergence Technology Program (2015001640001) funded by Korea Ministry of Environment (MOE). REFERENCES (1) Green, T. R.; Taniguchi, M.; Kooi, H.; Gurdak, J. J.; Allen, D. M.; Hiscock, K. M.; Treidel, H.; Aureli, A., Beneath the surface of global change: Impacts of climate change on groundwater. Journal of Hydrology 2011, 405, (3–4), 532-560. (2) Oki, T.; Kanae, S., Global hydrological cycles and world water resources. Science 2006, 313, (5790), 1068-72. (3) Liu, C. X.; Zhang, D. R.; He, Y.; Zhao, X. S.; Bai, R., Modification of membrane surface for anti-biofouling performance: Effect of anti-adhesion and anti-bacteria approaches. Journal of Membrane Science 2010, 346, (1), 121-130. (4) Judd, S., The MBR Book: Principles and Applications of Membrane Bioreactors for water and Wastewater Treatment, 2nd ed.; Elsevier: Amsterdam; Boston, 2011. (5) Kobayashi, T.; Kobayashi, T.; Hosaka, Y.; Fujii, N., Ultrasound-enhanced membranecleaning processes applied water treatments: influence of sonic frequency on filtration treatments. Ultrasonics 2003, 41, (3), 185-190. (6) van den Brink, P.; Vergeldt, F.; Van As, H.; Zwijnenburg, A.; Temmink, H.; van Loosdrecht, M. C. M., Potential of mechanical cleaning of membranes from a membrane bioreactor. Journal of 12


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and Environmental Microbiology 2005, 71, (8), 4906-4909. (46) Batchelor, S. E.; Cooper, M.; Chhabra, S. R.; Glover, L. A.; Stewart, G. S.; Williams, P.; Prosser, J. I., Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria. Appl Environ Microbiol 1997, 63, (6), 2281-6.

Table 1. MBR operating conditions One-stage MBR

Reactor type

510

Three-stage MBR

(Aerobic tank)

(Anoxic/aerobic/membrane tanks)

Total working volume (L)

80

450 (150 / 150 / 150)

Total HRT (h)

5.2

10.7

SRT (day)

25 days

Nearly infinite

Membrane type

C-PVC, Flat sheet

PES, Flat sheet

Membrane area (m2) / pore size (µm)

0.9 / 0.4

3.5 / 0.04

pH

6.5 ~ 7

6.5 ~ 7

MLSS (mg/L)

10,000 ~ 13,000

5,800 ~ 8,000

Flux (LMH)

20

15

Filtration mode (filtration/relaxation, min/min)

10 / 2

8/2

Aeration for oxygen supply (L/min)

10

8

Aeration for membrane scouring (SADm, m3/m2/h)

0.3 - 0.9

0.34

Concentration of beads (%, v(beads) / v(membrane tank))

1

0.8

SADm: specific aeration demand per membrane surface (m3/m2/h)

511

15



512 513 514 515 516 517 518 519 520

Figure 1. MBR configurations. (a) Two one-stage MBRs in parallel, each of which consists of only a membrane tank where two types of aerations are equipped: one for oxygen supply to activated sludge, and the other for membrane scouring. (b) Two three-stage MBRs in parallel, each of which consists of an anoxic, an aerobic and a membrane tank.

16


Page 16 of 22

Page 17 of 22

521 522 523 524 525 526 527 528 529 530


Figure 2. TMP profiles during the operation of two one-stage MBRs in two phases. (a) phase 1; TMP rise-up in the vacant- and QQ-MBRs at the same aeration intensity (SADm= 0.3 m3/m2/h). (b) phase 2; TMP rise-up in the conventional-MBR (SADm= 0.9 m3/m2/h) and QQMBR (SADm= 0.3 m3/m2/h) at the different aeration intensity.

531 532

(a)

17



533 534

535 536 537 538 539 540 541 542

(b)

(c)

Figure 3. EPS analysis in one-stage vacant- and QQ-MBRs. (a) Soluble EPS. (b) Bound EPS (EPS in activated sludge floc). (c) EPS in biocake. *Concentration of polysaccharides in biocakes in QQ-MBR was below the detection limit. Each bar represents the average value of duplicate measurements. Error bar: standard deviation (n=2).

18


Page 18 of 22

Page 19 of 22

543 544 545 546 547 548 549


Figure 4. Comparison of energy consumption related to biofouling between one-stage conventional- and QQ-MBRs. It was assumed that only two parameters, membrane aeration and filtration energy were affected by biofouling control of QQ.

19



550 551 552 553

Figure 5. TMP profile of three-stage conventional- and QQ-MBRs at the same membrane aeration intensity (SADm= 0.34 m3/m2/h). 160

Average microbial floc size (um)

One-stage MBR (n=1) 140

Three-stage MBR (n=4)

Vacant-MBR QQ-MBR

Conventional-MBR QQ-MBR

120 100 80 60 40 20 0 Aerobic Aerobic tank

554

Anoxic tank

Aerobic tank Membrane tank

555

Figure 6. Average microbial floc size in one-stage and three-stage MBRs. Error bar: standard

556

deviation (n=4). 20


Page 20 of 22

Page 21 of 22


120 One-stage MBR (n=4)

Removal efficiency (%)

100

Three-stage MBR (n=3)

Conventional-MBR QQ-MBR

80

60

40

20

0

COD 35 557

T-N 36

NH 374-N

COD 38

T-N 39

NH 404-N

558

Figure 7. Effluent water quality of one-stage and three-stage MBRs. (COD: chemical oxygen

559

demand, T-N: total nitrogen, NH4-N: ammonia-nitrogen). Error bar: standard deviation. (n=4

560

for one-stage MBR, n=3 for three-stage MBR).

561

21



Page 22 of 22

1.2 One-stage MBR Three-stage MBR

Relative QQ activity

1.0

0.8

0.6

0.4

0.2

0.0 0

562 563 564 565 566 567

20

40

60

80

100

120

140

Time (day)

Figure 8. Variation of the relative QQ activity of the QQ-beads during the operation of onestage and three-stage MBRs. QQ activity: degradation ratio of standard C8-HSL for 120 min in the presence of QQ-beads. TOC/Abstract Art

568

22


Crossing the Border between Laboratory and Field: Bacterial Quorum

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