Article pubs.acs.org/est
Biofouling Control with Bead-Entrapped Quorum Quenching Bacteria in Membrane Bioreactors: Physical and Biological Effects Sang-Ryoung Kim,† Hyun-Suk Oh,† Sung-Jun Jo,† Kyung-Min Yeon,† Chung-Hak Lee,*,† Dong-Joon Lim,‡ Chi-Ho Lee,§ and Jung-Kee Lee§ †
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea School of Chemical Engineering, Yeungnam University, Gyeongsan 712-749, Republic of Korea § Department of Life Science and Genetic Engineering, Paichai University, Daejeon 302-735, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Recently, interspecies quorum quenching by bacterial cells encapsulated in a vessel was described and shown to be efficient and economically feasible for biofouling control in membrane bioreactors (MBRs). In this study, free-moving beads entrapped with quorum quenching bacteria were applied to the inhibition of biofouling in a MBR. Cell entrapping beads (CEBs) with a porous microstructure were prepared by entrapping quorum quenching bacteria (Rhodococcus sp. BH4) into alginate beads. In MBRs provided with CEBs, the time to reach a transmembrane pressure (TMP) of 70 kPa was 10 times longer than without CEBs. The mitigation of biofouling was attributed to both physical (friction) and biological (quorum quenching) effects of CEBs, the latter being much more important. Because of the quorum quenching effect of CEBs, microbial cells in the biofilm generated fewer extracellular polymeric substances and thus formed a loosely bound biofilm, which enabled it to slough off from the membrane surface more easily. Furthermore, collisions between the moving CEBs and membranes gave rise to frictional forces that facilitated detachment of the biofilm from the membrane surface. CEBs bring bacterial quorum quenching closer to being a practical solution to the problem of biofouling in MBRs.
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INTRODUCTION Although membrane bioreactors (MBRs) have been in commercial use for more than two decades, membrane biofouling caused by the formation of biocakes (deposited microbial flocs plus biofilm) on the membrane surface still remains a bottleneck that limits their widespread use.1,2 Many researchers have attempted to mitigate biofouling in various ways, such as by using additives or specific media,3,4 by changes in the design of the MBR system,5 or modification of the membrane surface.6 Recently, novel biological approaches have been attempted to control biofouling using quorum quenching,7−10 that is, via disruption of quorum sensing. Quorum sensing is the cell−cell communication between microorganisms, which determines phenotypes such as biofilm formation, secretion of extracellular polymeric substances (EPS) and virulence.11,12 Yeon et al.7,8 prepared a magnetic enzyme carrier by immobilizing a quorum quenching enzyme, acylase, onto magnetic particles and demonstrated its potential as a novel approach to control biofouling in MBR. Kim et al. 9 immobilized acylase onto the membrane surface and showed mitigation of membrane biofouling. These applications, however, have drawbacks, such as the high cost of enzyme extraction and purification as well as enzyme instability. As an alternative to enzymatic quenching, Oh et al. 10 isolated bacteria that produce © 2012 American Chemical Society
quorum quenching enzymes and also developed a microbial vessel in which quorum quenching bacteria (Rhodococcus sp. BH4) were encapsulated. They observed that a submerged MBR equipped with the microvessel has a much lower biofouling tendency compared with a conventional MBR. In their study, however, quorum quenching bacteria were confined within a small vessel that was submerged in a fixed place in the MBR so that they could degrade only soluble signal molecules that were able to diffuse into the vessel. As such, the mass transfer of signal molecules from the mixed liquor to the inside of the microbial vessel was limited. The aim of this study was to find an alternative method of bacterial quorum quenching that is both more efficient than using a microbial vessel and more feasible than enzymatic quorum quenching from the viewpoint of practical application. We prepared free-moving beads using alginate and entrapped Rhodococcus sp. BH4 into highly interconnected microstructural pores of the beads, which will be called cell entrapping beads (CEBs) throughout this article. We placed CEBs directly into a Received: Revised: Accepted: Published: 836
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submerged MBR and allowed them to move freely together with other microorganisms in the mixed liquor as well as to contact the biofilm on the filtration membrane to catch up signal molecules in the biofilm more easily. It is thought that CEBs inhibit biofilm formation through interspecies quorum quenching as well as by physical washing through their collisions with the membrane surface.
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EXPERIMENTAL SECTION Quorum Quenching Bacteria. Rhodococcus sp. BH4 was isolated from a working MBR plant for wastewater treatment (capacity 18 000 m3/d, Okcheon, Korea), using an enrichment culture method described by Oh et al.10 Preparation of CEBs. Sodium alginate (Sigma-Aldrich), a nontoxic substance to bacteria,13 was used as a cell immobilization material. The isolated Rhodococcus sp. BH4 were inoculated in Luria−Bertani (Miller, US) broth at 30 °C for 24 h. The BH4 culture was centrifuged (12 000g, 15 min), washed with water, and resuspended in 3 mL of water. The BH4 suspension (200 mg BH4/mL of water) was gently mixed with 97 mL of the sterile sodium alginate suspension to make a 4% (w/v) BH4−alginate suspension. The BH4−alginate suspension was dripped into 3% (w/v) CaCl2 solution through a nozzle at a rate of 1.6 mL/min. As depicted in Supporting Information Figure S1, the dripping device consisted of a nozzle, fluid line, and pump with a velocity controller. The CEBs were formed and left in CaCl2 solution for 3 h before being washed three times with distilled water and dried at room temperature. The average size and density of CEBs were approximately 3.5 mm and 1.5 g/mL, respectively. The BH4 content of the CEBs was 6.0 mg BH4/g sodium alginate. Because the size and mechanical properties of the CEBs can be easily controlled by changing the diameter of the nozzle or the concentration of CaCl2, this method offers advantages for the preparation of diverse CEBs suitable for various types of MBRs. Measurement of Activity and Stability of CEBs. The quorum quenching activity and stability of CEBs were evaluated by the degradation rate of standard C8-HSL (N-octanoyl-DLhomoserine lactone) (Sigma-Aldrich, USA), which is one of the dominant signal molecules (autoinducers) in the MBR for wastewater treatment.7 The degradation rate of C8-HSL was measured according to the method described by previous studies.7−10 C8-HSL was added to 50 mM Tris−HCl buffer (pH 7.0, 30 mL) to a final concentration of 200 nM. Twenty individual CEBs were then added to the Tris−HCl buffer containing C8-HSL. The activity of the CEBs was measured via the decrease in the C8-HSL concentration with time. The stability of the CEBs was measured from the decrease in the C8HSL concentration for 30 min and was monitored 13 times during continuous MBR operation over 30 days. The CEBs were periodically removed from the mixed liquor, but were returned to the MBR following activity measurement. MBR Operation. Two laboratory-scale MBRs, each with a 1.6 L working volume, were operated in parallel. Three sets of operating schemes were designed for two MBRs, depending on the presence of either vacant beads or CEBs in each MBR (Figure 1): set 1, control and CEBs (with BH4 cells); set 2, control and vacant beads (without BH4 cells); set 3, vacant beads and CEBs. The number of vacant beads or CEBs inserted into each MBR was 40, and each MBR was always operated under a constant flux of 28.7 L/(m2 h). Hydraulic retention times and sludge retention times were set to 5.3 h and 25 d, respectively. The submerged hollow fiber membrane was made of polyvinylidene fluoride (ZeeWeed 500, GE-Zenon, USA) with an effective area of 13.4
Figure 1. Schematic diagram for three sets of operations of MBRs.
cm2. Activated sludge was taken from a wastewater treatment plant (Tancheon, Korea). Mixed liquor suspended solid in the MBR was maintained at 12.5−13.0 g/L. The detailed composition of the synthetic wastewater is given in Supporting Information Table S1. Measurement of Loosely and Tightly Bound Biofilms. For the qualitative and quantitative analysis of biofilm formed on the surface of membranes, biofilms were detached from the used membranes and were classified into two types: loosely bound biofilm (LB biofilm) or tightly bound biofilm (TB biofilm). The former was defined as biofilm that can be detached only by airscouring at a fixed aeration time and rate, whereas the latter, by sonication and subsequent air-scouring (Supporting Information Figure S2). The used membranes covered with biofilm were submerged in an aeration tank filled with 1 L of water. The LB biofilm was obtained after 80 min of aeration at a rate of 3 L/min. The remaining biofilm on the same used membrane was further sonicated for 10 min, followed by an additional 20 min of aeration to obtain the TB biofilm. The dry weight of each biofilm in suspension was measured. The total weight of LB and TB biofilms was regarded as the total attached biomass (TAB). Extraction of EPS from the biofilm in suspension was carried out using an ion-exchange resin method.14 A cation exchange resin in sodium form (CER, Dowex Marathon, Sigma-Aldrich) was washed for 1 h in phosphate buffer and was added to the suspension of each biofilm (10 g CER/g detached biofilm). The mixed suspension was stirred at 300 rpm for 2 h and then centrifuged at 4000g for 20 min. A pellet composed of cells and CER formed in the bottom of the tube, and the supernatant contained EPS. The weight of cells was determined by subtracting the weight of CER from the weight of the pellet. The amount of EPS was determined by subtracting the cell weight from the TAB. Extraction and Analysis of Acyl Homoserine Lacotones (AHLs). Standard AHLs and AHL extracts were analyzed by HPLC (Waters, USA). AHL was extracted from the biofilm on the used membrane as follows: The used membrane was placed in 400 mL of deionized water, and the biofilm was detached by backwashing and sonication. After the membrane was removed, 837
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Figure 2. SEM microphotographs of the beads: cross section of a vacant bead (a) ×25, (b) ×1000, and (c) ×6000. Cross section of a CEB (d) ×25, (e) ×1000, and (f) ×6000.
two (Figure 2). The vacant beads and CEBs possess a porous microstructure, with a high degree of interconnectivity (Figure 2a and d). Since there are many pores and the pore diameter in CEBs is around 300 μm, CEBs appear to provide enough space for BH4 colonization as well as low mass transfer resistance. Inside the pore, no BH4 were observed in vacant beads (Figure 2b), whereas BH4 were spread on the alginate matrix surface (Figure 2e) in CEBs. The BH4 appear as short rods with an approximate size of 1.2−2.0 μm in length and 0.5 μm in width (Figure 2e). To investigate the viability of BH4 during entrapment, CLSM images of CEBs were taken after viability staining. Before entrapment, the proportion of free live BH4 was ∼80% (±3%), on the basis of the ISA image. After entrapment, the BH4 appeared densely packed and evenly dispersed in the microstructure of the CEBs (Supporting Information Figure S4). From the images, the mean percentage of living cells entrapped in CEBs was calculated to be 65% (±5%). The damage to live cells during entrapment indicates that cell immobilization had a negative effect on cell viability. It is possible that BH4 near the surface of CEBs were killed by contact with CaCl2 solution, but the decrease in the amount of live BH4 was not substantial (Supporting Information Figure S4b). The SEM and CLSM analysis confirmed that CEBs were successfully constructed by the combination of alginate, Ca+, and BH4. Quorum Quenching Activity of Free BH4 and CEBs. The quorum quenching activity of isolated free BH4 was tested using a bioassay with C8-HSL, which was most abundant in the biofilm-formed membranes in MBRs.7 Live BH4 readily degraded C8-HSL, whereas dead BH4 hardly reduced C8-HSL levels, despite its potential physicochemical adsorption (Supporting Information Figure S5). The quorum quenching activity of CEBs was tested using the same method as for free BH4. As shown in Figure 3, CEBs degraded 91% of C8-HSL, whereas the vacant beads removed less than 10% of the C8-HSL in 60 min. The removal by vacant beads was attributed to its physicochem-
the biofilm in suspension was shaken with 100 mL of ethyl acetate for 2 h. After the organic layer was separated from the water layer using a separating funnel, it was dried with a vacuum evaporator. The residue was redissolved with 200 μL of methanol for HPLC analysis. Gradient elution was performed with a mixture of water/methanol as a mobile phase, and the UV detector was set at 210 nm. Standard AHLs were dissolved in methanol to obtain 1 mg/mL solutions. Aliquots (20 μL) of each of these solutions were added to 980 μL of methanol−water (35:65, v/v) with 0.1% formic acid. The column used in the HPLC was a Gemini C18, 50 mm × 2 mm, 5 μm particle size. AHL standard mixtures (25 μL), blanks, or AHL extracts were injected at a flow rate of 0.25 mL/min. The HPLC was connected to a fraction collector. Fractions were collected every 9 min into a test tube, reduced in volume, then loaded for the bioassay of AHL molecules using an indicating agar plate. The detailed analysis procedure for HPLC is described in the Supporting Information.15 Analytical Methods. The internal and external morphologies of the vacant beads and CEBs were identified by SEM. The live (green) or dead (red) cells inside the CEBs were stained by a Viability kit according to the manufacturer’s instructions and were observed using CLSM. The detailed analytical procedures for SEM and CLSM are described in the Supporting Information.
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RESULTS AND DISCUSSION Characterization of CEBs. The vacant beads were almost spherical, with a smooth surface and uniform size. Entrapment of quorum quenching bacteria (BH4) into the beads did not result in any significant change in either the shape or the size of beads (Supporting Information Figure S3a). The size of CEBs was ∼3.5 mm, and their density was roughly 1.5g/mL. The CEBs were able to circulate in the mixed liquor under aeration (Supporting Information Figure S3b). The cross-sectional SEM image showed the morphologies of the vacant beads and CEBs as well as the differences between the 838
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pressure (TMP) of the control and CEB MBRs were compared to evaluate the inhibition of biofouling by CEBs. As shown in Figure 4a, it took 1.8 d for the TMP to reach 70 kPa in the first cycle of the control MBR, whereas it took 18.8 d for the first cycle of the CEB-treated MBR. Thus, CEBs mitigated the formation of biofilm and extended the time required to reach the TMP of 70 kPa by ∼10-fold, compared with the control MBR. From a practical point of view, this is important because the delay in the rise of TMP is closely associated with a saving of energy in the operation of the MBR. The remarkable effect of CEBs to reduce biofouling is better than that reported by Oh et al.10 They encapsulated the same quorum quenching bacteria, BH4, into a microbial vessel, applied it to a submerged MBR, and observed that the microbial vessel resulted in much lower biofouling compared with a conventional MBR. Although a direct comparison between results obtained from CEBs and the microbial vessel is difficult, CEBs appear to be superior to the microbial vessel in terms of reducing membrane fouling. The excellent performance of CEBs could be attributed either to the inhibition of biofilm formation by quorum quenching or to the sloughing of biofilm from the membrane surface by collision between moving CEBs and the submerged membrane in the MBR. To verify this, two consecutive MBR operations were carried, out as depicted in sets 2 and 3 in Figure 1. Physical Washing Effect by Free-Moving CEBs. To confirm a physical washing effect, the control MBR and the MBR with the vacant beads were run in parallel under the same operating conditions (set 2 in Figure 1). As shown in Figure 4b, it
Figure 3. Quantitative quorum quenching activity of control, vacant beads, and CEBs (n = 4).
ical adsorption because vacant beads have neither quorum quenching bacteria nor quorum quenching enzyme. A control was also conducted to check the potential removal of C8-HSL by its adsorption onto the surface of a glass beaker, but the adsorption was negligible. Application of CEBs to the MBR. CEBs were applied to submerged MBRs to test their potential to inhibit biofouling in MBRs (set 1 in Figure 1). Two lab-scale MBRs in continuous mode were operated in parallel under identical operating conditions except for the addition of CEBs to one MBR at the start of the operation. The rise of the profiles transmembrane
Figure 4. Comparison of TMP between (a) control and CEBs MBRs, (b) control and vacant beads MBRs, and (c) vacant beads and CEBs MBRs under the same operating conditions. 839
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took 1.8 d to reach the TMP of 70 kPa in the control MBR, whereas it took 3.1 d to reach the same TMP in the MBR with vacant beads. Although the vacant beads with porous microstructures contain no BH4, they continuously circulate in the mixed liquor and collide with the surface of submerged membranes in the MBR. This collision could facilitate the detachment of biocake already deposited or formed on the membrane surface. A physical washing effect by moving media in MBRs has been previously reported.4,16 To quantitatively evaluate the physical washing effect, a separate experiment was designed. Two control MBRs with neither vacant beads nor CEBs were run until the TMP reached 70 kPa. The used hollow fiber membrane module was then removed from each control MBR, and each used membrane module was immersed in two separated beakers containing 1 L of water. Forty vacant beads were added to only one beaker, and each beaker was then aerated for 80 min (3 L/min) to assess how much biomass would be detached from each used module and to compare each experiment. Further sonication following aeration also made it possible to determine the TAB of each used membrane module. For the five repeating measurements, 0.64 g (87% of TAB) of biomass was detached in the beaker with the vacant beads, whereas 0.52 g (72% of TAB) of biomass was detached in the beaker without the vacant beads (Figure S6). On the other hand, the average TAB of both membrane modules was similar: 0.74 (±0.05) g in the beaker with the beads and 0.73 (±0.04) g in the beaker without beads, with a 5% relative standard deviation of each. Consequently, it can be concluded that the vacant beads facilitated the detachment and, thus, increased the amount of detached biomass by ∼15% through their collision with the membrane. Quorum Quenching Effect by Moving CEB. To confirm the quorum quenching effect, one MBR with vacant beads and the other MBR with CEBs were run under the same operating conditions (set 3 in Figure 1). During the operation of each MBR, a used membrane module was replaced by a new one when the TMP exceeded 70 kPa, and TMP monitoring was reperformed with the new membrane. As shown in Figure 4c, it took 17 d to reach the TMP of 70 kPa for the MBR with CEBs, whereas it took only 2 or 3 d to reach the same TMP for the MBR with the vacant beads. Eight cycles were thus repeated in the MBR with the vacant beads during only cycle with the one with CEBs. Expressed differently, CEBs extended the time required for the MBR to reach the TMP of 70 kPa by about 7-fold. It is worth noting that assuming that the physical washing effects of the vacant beads and CEBs were identical because the same number of vacant beads or CEBs were added to each MBR, the large difference in the rate of the TMP rise (set 3 in Figure 1) is attributable only to the BH4 in the porous microstructural CEBs. Mechanisms of Quorum Quenching. To investigate the mechanisms of quorum quenching, biofilms were detached from the used membrane modules after 75 h of operation in both the MBR with vacant beads and the MBR with CEBs. Biofilms from both used modules were analyzed in terms of EPS, TAB, and adhesiveness and then compared with each other. After 75 h of operation, the TMP reached 70 kPa in the MBR with vacant beads, whereas only 7.9 kPa was reached in the MBR with CEBs. This coincided well with the greater TAB in the former (0.77 g) compared with the latter (0.24 g), as shown in Table 1: less than one-third of the biomass developed on the membrane surface in the MBR with quorum quenching bacteria during the same operation period.
Table 1. The Amount of TAB, EPS and Loosely and Tightly Bound Biofilms in the Used Membrane Modules for the MBR with Vacant Beads and MBR with CEBs MBR with vacant beads a
TMP at the operating time of 75 h TABb TAB EPS cell TAB loosely bound biofilm tightly bound biofilm
MBR with CEBs
70.3 kPa
7.9 kPa
0.77 g 0.36 g (47% of TAB)c 0.41 g 0.53 g (68% of TAB)d 0.24 g (32% of TAB)d
0.24 g 0.07 g (29% of TAB)c 0.17 g 0.22 g (89% of TAB)d 0.02 g (11% of TAB)d
a
TMP: transmembrane pressure. bTAB: total attached biomass. cEPS percentage = (EPS/TAB) × 100. dBound biofilm percentage = (bound biofilm/TAB) × 100.
To characterize the detached biomass, the TAB was further divided into two components: EPS and cells. Not only the total amount, but also the proportion of EPS was much lower in the MBR with CEBs (0.07 g, 29%) than in the MBR with vacant beads (0.36 g, 47%). It is known that quorum sensing regulates the production of EPS through the transcription of target genes and determines the physiology of the microbial community.11 Previous studies confirmed that enzymatic quorum quenching decreases EPS production in the biofilm.7−9,17 Rhodococcus sp. have been reported to generate an enzyme (lactonase) that can degrade AHLs.18 Consequently, the application of CEBs (i.e., BH4) to MBRs inhibits quorum sensing between cells by reducing the concentration of AHLs and thus decreasing EPS production in the biofilm. The attached biomass was also classified into two types: LB biofilm or TB biofilm. As illustrated in Supporting Information Figure S2, one portion of the TAB biofilm (TB biofilm) requires more vigorous conditions for detachment than the other (LB biofilm), indicating that TB biofilm has stronger cohesive or adhesive forces (or both) than LB biofilm. In Table 1, both the total amount and the portion of the TB biofilm was substantially lower in the MBR with CEBs (0.02 g, 11%) than in the MBR with vacant beads (0.24 g, 32%). This could be attributed to the lower production of EPS, the key element for the construction of biofilm due to quorum quenching by CEBs.19 Identification of Signal Molecules for Quorum Sensing in MBRs. An important point concerns the demonstration of the destruction of signal molecules by CEBs. For this purpose, both MBRs in this study were run for 48 h, and the extracts from biofilm formed on the membrane surfaces from MBRs with or without CEBs were then analyzed by HPLC and a bioassay. The extract from the MBR with vacant beads showed four peaks (blue line in Figure 5a). Two of the four peaks appeared with a retention time of 3.2 and 11.8 min and were identified as C8-HSL and 3oxoC8-HSL, respectively, by comparison with the peaks of two standard signal molecules (green and yellow lines in Figure 5a). In the extract from the MBR with CEBs (red line in Figure 5a), however, no AHL was detected. To ensure that the destruction of AHLs occurred by CEBs, we collected two HPLC fractions, each after 9 min: fraction 1 was eluent collected for the first 9 min, and fraction 2 was eluent collected for the second 9 min. Both fractions were analyzed by bioassay with A136 as a reporter strain.7 The two fractions from the MBR with vacant beads showed blue colors, indicating the presence of AHLs 840
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Figure 5. Identification of AHLs extracted from the biofilm formed on the used membrane by HPLC. (a) Chromatogram of standard and extracted AHLs. (b) Bioassay of fractions (1 and 2) collected every 9 min for the MBR with vacant beads. (C) Bioassay of fractions (1 and 2) collected every 9 min for the MBR with CEBs.
Figure 6. Reconstructed CLSM images of biofilm formed on the membrane surface in (a) control MBR, (b) MBR with vacant beads, and (c) MBR with CEBs after 48 h operation, stained with SYTO9 (cell; green). Magnification: ×100. Image size: 1212 μm × 1212 μm.
(Figure 5b), whereas those from the MBR with CEBs showed no blue color, indicating the absence of AHLs (Figure 5c). Visual Confirmation of the Quorum Quenching Effect by Mobile CEBs. The quorum quenching effect of CEBs was confirmed visually using CLSM. Figure 6 represents the reconstructed CLSM images of biofilm formed on the membrane surfaces which were removed from the MBRs operated for 48 h. The amount of biofilm formed in the MBR with CEBs was the least, whereas that in the control MBR was the greatest, and that in the MBR with vacant beads was intermediate. In summary, CEBs induced a physiological change in microorganisms, including a decrease in EPS production through the disruption
of AHLs. Consequently, the cohesion between cells or the adhesion between cells and membrane was weakened, and thus, less biomass was attached to the membrane with the CEBs. In short, CEBs can inhibit biofilm formation by quorum quenching. Effect of CEBs on MBR Performance. In addition to variation in TMP, the quality of the permeated water is another important factor in MBR performance. We monitored the removal efficiencies of COD in three MBRs on the basis of their feed and permeate concentrations. Although the influent COD to three reactors was around 500 mg/L, three reactors exhibited similar COD concentrations in the permeate with more than 96% of COD removal: 6.2−19.1 mg/L for the control, 6.3−16.4 841
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(6) Liu, C. X.; Zhang, D. R.; He, Y.; Zhao, X. S.; Bai, R. B. Modification of membrane surface for anti-biofouling performance: Effect of antiadhesion and anti-bacteria approaches. J. Membr. Sci. 2010, 346, 121− 130. (7) Yeon, K. M.; Cheong, W. S.; Oh, H. S.; Lee, W. N.; Hwang, B. K.; Lee, C. H.; Beyenal, H.; Lewandowski, Z. Quorum sensing: A new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment. Environ. Sci. Technol. 2009, 43, 380−385. (8) Yeon, K. M.; Kim, J.; Lee, C. H. Magnetic enzyme carrier for effective biofouling control in the membrane bioreactor based on enzymatic quorum quenching. Environ. Sci. Technol. 2009, 43, 7403− 7409. (9) Kim, J. H.; Choi, D. C.; Yeon, K. M.; Kim, S. R.; Lee, C. H. Enzymeimmobilized nanofiltration membrane to mitigate biofouling based on quorum quenching. Environ. Sci. Technol. 2011, 45, 1601−1607. (10) Oh, H. S.; Yeon, K. M.; Yang, C. S.; Kim, S. R.; Lee, C. H.; Park, S. Y.; Han, J. Y.; Lee, J. K. Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane. Environ. Sci. Technol. 2012, 46, 4877−4884. (11) Fuqua, C.; Winans, S. C.; Greenberg, E. P. Census and consensus in bacterial ecosystems: The LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev. Microbiol. 1996, 50, 727−751. (12) Dobretsov, S.; Teplitski, M.; Paul, V. Mini-review: Quorum sensing in the marine environment and its relationship to biofouling. Biofouling 2009, 25, 413−427. (13) Leenen, E. J. T. M.; DosSantos, V. A.P.; Grolle, K. C. F.; Tramper, J.; Wijffels, R. Characteristics of and selection criteria for support materials for cell immobilization in wastewater treatment. Water Res. 1996, 30, 2985−2996. (14) Frolund, B.; Palmgren, R.; Keiding, K.; Nielsen, P. H. Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res. 1996, 30, 1749−1758. (15) Anjali, K.; Patrizia, P.; Sylvia, D. Detection of bacterial quorum sensing N-acyl homoserine lactones in clinical samples. Anal. Bioanal. Chem. 2008, 391, 1619−1627. (16) Rosenberger, S.; Helmus, F. P.; Krause, S.; Bareth, A. Principles of an enhanced MBR-process with mechanical cleaning. Water Sci. Technol. 2011, 64, 1951−1958. (17) Kim, H. W.; Oh, H. S.; Kim, S. R.; Lee, K. B.; Yeon, K. M.; Lee, C. H.; Kim, S.; Lee, J. K. Microbial population dynamics and proteomics in membrane bioreactors with enzymatic quorum quenching. Appl. Environ. Microbiol. 2012, DOI: 10.1007/s00253-012-4272-0. (18) Uroz, S.; Oger, P. M.; Chapelle, E.; Adeline, M. T.; Faure, D.; Dessaux, Y. A Rhodococcus qsdA-encoded enzyme defines a novel class of large-spectrum quorum-quenching lactonases. Appl. Environ. Microbiol. 2008, 74, 1357−1366. (19) Ahimou, F.; Semmens, M. J.; Haugstad, G.; Novak, P. J. Effect of protein, polysaccharide, and oxygen concentration profiles on biofilm cohesiveness. Appl. Environ. Microbiol. 2007, 73, 2905−2910.
mg/L with vacant beads, and 6.9−14.2 mg/L for CEBs. There was no significant difference in the operational parameters among three MBRs throughout the entire experimental period. Moreover, taking into account the volume of CEBs added to the MBR was less than 0.63% of the working reactor volume, the adsorption of COD or TN on CEBs would be negligible. Therefore, it has been concluded that quorum quenching with CEBs mitigates membrane biofouling but does not decrease microbial activity, at least for the degradation of organic matter in the MBRs. Stability of CEBs. The structural integrity and quorum quenching activity of CEBs were monitored during the operation of continuous MBR for 30 d. Surprisingly, the quorum quenching activity of CEBs increased by ∼3% after 30 d of operation, which is highly favorable for practical applications (Supporting Information Figure S7a). We also monitored the wet weight of CEBs and vacant beads during the MBR operation (Supporting Information Figure S7b). The wet weight of the CEBs increased by ∼4% after 25 d of operation, whereas that of the vacant beads did not change substantially. Thus, it can be concluded that the increase in the quorum quenching activity of CEBs is caused by the growth of BH4 within the beads. Furthermore, the sizes of the CEBs and vacant beads were nearly constant and maintained their structural integrity at least for the test period of 25 d (Supporting Information Figure S8). In summary, CEBs prepared in this study demonstrated their potential for efficient biofouling control in MBRs via both physical washing and quorum quenching effects. The application of quorum quenching bacteria entrapped in beads could be expanded to the plant-scale of MBRs with economic feasibility.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +82-2-880-7075; fax: +82-2-874-0896; e-mail: leech@ snu.ac.kr. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research is supported by the Korea Ministry of Environment as ″Converging Technology Project″ (2012001440001). REFERENCES
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dx.doi.org/10.1021/es303995s | Environ. Sci. Technol. 2013, 47, 836−842
