Quorum Sensing: A New Biofouling Control

concentration of quorum quenching enzyme throughout the operation. It was constructed by combining the mixing reservoir flask (150 mL) and a vertical ...
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Environ. Sci. Technol. 2009, 43, 380–385

Quorum Sensing: A New Biofouling Control Paradigm in a Membrane Bioreactor for Advanced Wastewater Treatment KYUNG-MIN YEON,† WON-SEOK CHEONG,† HYUN-SUK OH,† WOO-NYOUNG LEE,† BYUNG-KOOK HWANG,† C H U N G - H A K L E E , * ,† H A L U K B E Y E N A L , ‡ AND ZBIGNIEW LEWANDOWSKI§ School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Korea, School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington 99164-2710, and Center for Biofilm Engineering, Montana State University, Bozeman, Montana 59717-3980

Received July 17, 2008. Revised manuscript received October 18, 2008. Accepted November 5, 2008.

Bacteria regulate specific group behaviors such as biofilm formation in response to population density using small signal molecules called autoinducers (quorum sensing, QS). In this study, the concept of bacterial QS was applied to membrane bioreactors (MBRs) for advanced wastewater treatment as a new biofouling control paradigm. The research was conducted in three phases: (1) demonstrate the presence of the autoinducer signal in MBRs, (2) correlate QS activity and membrane biofouling, (3) apply QS-based membrane biofouling control. A bioassay with Agrobacterium tumefaciens reporter strain proved that N-acyl homoserine lactone (AHL) autoinducers were produced in the MBR. Furthermore, thinlayer chromatographic analysis identified at least three different AHLs in the biocake, of which N-octanoyl-homoserine lactone was the most abundant. During continuous MBR operation, the biocake showed strong AHL activity simultaneously with abrupt increase in the transmembrane pressure, which implies that QS is in close association with membrane biofouling. Porcine kidney acylase I, which can inactivate the AHL molecule by amide bond cleavage, was confirmed to prevent membrane biofouling by quenching AHL autoinducers. From these results, it was concluded that QS could be a novel target for biofouling control in MBRs.

Although various biofouling control techniques have been developed through engineering (3), material (4), and chemistry approaches (5), all these attempts have the limitation that they are essentially not able to prevent the biofilm formation because it is intrinsically a natural biological process. In this study, the concept of bacterial quorum sensing (QS) was applied to an MBR for the control of membrane biofouling. Bacteria produce small molecules called autoinducers for the purpose of intercellular communication. When the autoinducer concentration reaches the threshold level in proportion to the cell density, it combines with the receptor protein and activates the transcription of specific genes to induce group behaviors such as bioluminescence (6), antibiotic production (7), virulence (8), biofilm formation (9) and sporulation (10). Since Davies and colleagues (9) first demonstrated that a QS mechanism was involved in the differentiation of Pseudomonas aeruginosa biofilm formation, many studies have reported that QS control can successfully reduce or prevent biofilm formation on surfaces such as medical devices (11) and plant tissue (12). These results enabled us to hypothesize that, in principle, membrane biofouling originating from biofilm formation could also be alleviated through QS control, e.g., the blocking of intercellular communication to obtain MBRs of high performance (Figure 1). No information, however, is available on the application of QS to biofouling control in MBRs. In this study, we developed a novel biofouling control paradigm in an MBR based on the concept of QS. To achieve this goal, the overall experiments were conducted in three phases: (1) demonstrating the presence of an autoinducer in the MBR, (2) showing a correlation between QS activity and membrane biofouling, and (3) developing QS-based membrane biofouling control techniques. It is worth noting that the biofouling layer on the membrane surface in an MBR consists of both deposited microbial flocs (e.g., MLSS) and growing microorganisms on the membrane surface (e.g., biofilm). To avoid confusion, the deposited MLSS is differentiated from the biofilm and the term “biocake” is used to represent the whole biofouling layer, e.g., the combination of deposited MLSS and biofilm (13).

Experimental Section

Microorganisms. Agrobacterium tumefaciens A136(Ti-)(pCF218)(pCF372) was used as a reporter strain for N-acyl homoserine lactone (AHL) autoinducers of Gram-negative bacteria (14). The AHL detection mechanism of A. tumefaciens A136 is described in the Supporting Information. Briefly, this

Introduction Over the past two decades, the membrane bioreactor (MBR) has emerged as one of the innovative technologies in advanced wastewater treatment. Recent studies with advanced molecular biological techniques have revealed that the characteristics of the biocake formed on the membrane, such as porosity (1) and biovolume (2), are closely associated with the permeability loss (membrane biofouling) in MBRs. * Corresponding author tel.: +82-2-880-7075; fax: +82-2-874-0896; e-mail: [email protected]. † Seoul National University. ‡ Washington State University. § Montana State University. 380

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FIGURE 1. Concept of QS-based biofouling control in MBR. 10.1021/es8019275 CCC: $40.75

 2009 American Chemical Society

Published on Web 12/18/2008

FIGURE 2. Schematic diagram of (a) continuous MBR and (b) batch MBR. reporter strain develops a blue color on an agar plate covered with X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) in response to exogenous AHL molecules. A. tumefaciens A136 was cultured on Luria-Bertani (LB) broth supplemented with spectinomycin and tetracycline to maintain two plasmids that provide the AHL response system (15). Activated sludge from the Si-Hwa wastewater treatment plant (Korea) was inoculated into an MBR after being acclimated to synthetic wastewater by subculturing. The composition of the synthetic wastewater is shown in the Supporting Information. The bacterial community structure in the MBR was analyzed by polymerase chain reaction (PCR)denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene according to the procedure described by Park and Lee (16). Bioassay for in Situ Detection of Total AHLs. AHLs were detected using a bioassay, which consisted of an indicating agar plate, a sterilized white filter and the sample to be tested (Figure S1). The indicating agar plate was made by mixing an overnight culture of A. tumefaciens A136 and LB agar in the ratio of 1:9. The indicating agar was also supplemented by spectinomycin and tetracycline and covered with X-gal. The sterilized white filter was used to prevent the possible microbial pollution of the indicating agar. The sample, such as biocake, was placed upon the filter. If the sample produced or contained AHLs, they diffused vertically into the indicating agar, developing blue color as a result. Thin-Layer Chromatography (TLC) for AHL Identification. AHLs were extracted from the biocake as follows: the membrane with the biocake was placed in ethyl acetate and biomass was detached from the membrane by means of sonication (150 W, 15 min). After the membrane was removed, the mixture of biomass and ethyl acetate was vortexed for 2 h. The cells were then removed by centrifugation at 9000g for 10 min. The supernatant was dried in a rotary evaporator at 30 °C and was resuspended in 250 µL of methanol. This crude AHL preparation was spotted on a C18 reverse-phase TLC plate and a chromatogram was developed in 70% methanol/30% water. After being completely dried at room temperature, the TLC plate was overlaid with 1.5% LB agar containing A. tumefaciens A136, antibiotics, and X-gal for

the imaging of the AHLs. The TLC chromatogram and the Rf value, defined by the ratio of the distance moved by the spot to that moved by the solvent front, of each AHL standard are shown in the Supporting Information (Figure S2). Reactor Operation. A laboratory-scale MBR with a working volume of 1 L was constructed similar to those described by other MBR researchers (1-3) and was in continuous operation (Figure 2a). The membrane was a hydrophilic polyvinylidene fluoride (PVDF) hollow fiber (GE-Zenon, US) with a pore size of 0.04 µm. The other operating parameters of this continuous MBR are summarized in Table S1. A batch type of MBR (Figure 2b) was also designed and operated under a total recycle mode to maintain the effective concentration of quorum quenching enzyme throughout the operation. It was constructed by combining the mixing reservoir flask (150 mL) and a vertical glass tube (3 cm diameter and 35 cm long) in which a PVDF hollow fiber membrane module with an effective area of 0.002 m2 was inserted through a T-shaped fitting. An acylase or an autoinducer, if necessary, were added to the flask containing wastewater and activated sludge and then circulated within the system by the peristaltic pump. The cell concentration was around 2500 mg MLSS/L. The membrane permeate was continuously suctioned by a peristaltic pump at a constant flux of 15 L/m2/h and then returned to the mixing reservoir (total recycle mode). Analytical Methods. Gram staining was carried out according to the procedure described by Holm and Jespersen (17). Mixed-liquor suspended solids and chemical oxygen demand were determined according to standard methods (18). Glucose, the main carbon source in synthetic wastewater, was determined using the reflectometric method with a test strip (Merck test 1.16720). Extracellular polymeric substances (EPSs) were extracted from the biocake using the cationic ion-exchange resin method (19). Protein and polysaccharides were measured using the modified Lowry method and phenol-sulfuric acid method, respectively.

Results and Discussion Evidence of the Autoinducer Signal in the MBR. PCR-DGGE analysis of the microbial community in the MBR (Figure S3a) shows that more than 10 different bacterial species were VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Characterization of the autoinducer signal in biocake: (a) A. tumefaciens A 136 bioassay result for the detection of total AHL; (b) TLC chromatogram of AHL extract from biocake for identification. present in the biocake and the mixed liquor, respectively. Among various QS autoinducers, such as AHLs of Gramnegative bacteria, modified oligopeptides of Gram-positive bacteria and autoinducr-2 (AI-2) for interspecies communication (20), we focused on the AHLs based on the Gramstaining results that Gram-negative bacteria were more dominant than Gram-positive bacteria in the biocake (Figure S3b).

When the transmembrane pressure (TMP) reached 30 kPa, the submerged module was taken out of the bioreactor and disintegrated by aseptically cutting each fiber into lengths of 3-4 cm. Then, each fiber with biocake was bioassayed for the detection of AHLs. All the tested fibers showed blue color developments, proving the presence of AHL autoinducers in the biocake formed on each fiber (Figure 3a). It has been reported that AHL autoinducers have a variety of acyl chains (Figure S4) and each bacterium uses its own AHL to regulate QS mechanism (20); we therefore anticipated that the mixed-cultured bacterial community in the MBR would produce various AHLs. To identify the various AHLs which might be involved in the MBR, 5, 10, and 15 µL of AHL extracts obtained from the biocake were developed by TLC and imaged by means of A. tumefaciens A136 bioassay. The TLC chromatogram in Figure 3b indicates the presence of at least three different AHLs. Comparison of their Rf values with those of the AHL standard showed that spots A and B were N-hexanoyl-DLhomoserine (C6-HSL) and N-octanoyl-DL-homoserine lactone (C8-HSL), respectively. The strongest blue color developed on spot B suggests that C8-HSL is the most abundant AHL in this biocake, whereas spot C has not yet been identified.

FIGURE 4. Occurrence of AHL signals in biocake during continuous MBR operation: (a) 22 h, (b) 46 h, (c) 58 h, and (d) 72 h.

FIGURE 5. Comparison of the AHL level in deposited MLSS and biofilm on membrane: (a) A. tumefaceins A136 bioassay; (b) AHL level per biomass. 382

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FIGURE 6. Correlation between QS activity and filtration resistance of biofilm: (a) experiment scheme; (b) variation of TMP and AHL levels as a function of biofilm growth time. Correlation between QS Activity and Biofouling in an MBR. The AHL signal in biocake at various operating points was monitored using bioassay. As shown in Figure 4, the blue color indicating the presence of AHLs was not detected for the early stage (22 h) but started its partial development as the operation proceeded (46 h). It is worth noting that the blue color intensified after a transition point (58 h, 72 h) simultaneously with the abrupt rise of TMP. In the operation of the MBR at constant flux, a two-stage TMP increase, i.e., an initial slow and gradual increase followed by an abrupt rise in the TMP, is often observed as shown in Figure 4d, but its mechanism remains to be clarified (21). In this context, this close correlation between TMP rise and the development of an AHL signal could provide a reasonable clue to such a TMP pattern because the correlation might imply a potential role of QS in the two-stage TMP increase. To investigate this correlation in more depth, we measured the AHL activities in the biofilm and in the deposited microbial flocs (e.g., MLSS), respectively. Different volumes of MBR mixed liquor were filtered on disk-type PVDF membranes to obtain three membranes with different MLSS depositions (0.1-0.5 mg of MLSS). These three samples were directly bioassayed to measure AHL activity (Figure 5a). In parallel, five membranes with 0.1 mg of MLSS were prepared by MLSS deposition and then they were incubated on a wastewater agar plate to let the biofilm grow on each membrane (22). However, the growth of biofilm reached the stationary phase after a certain incubation time due to the limit of the nutrient supply from the agar. At the incubation times of 0, 6, 9, 16, and 24 h, AHL activity in the biocake on each corresponding membrane was measured using bioassay (Figure 5a). Moreover, the AHL level (ng C8-HSL equivalent) per unit weight of biocake (mg) was estimated quantitatively on the basis of the blue color length on the bioassay agar plate according to Yang et al. (23) and Dong et al. (24). Detailed procedure is shown in the Supporting Information. As shown in Figure 5a and b, the AHL signal in the deposited MLSS was negligible regardless of the mass of the deposited MLSS. This means that any microbial flocs deposited on the membrane surface from the mixed liquor during the filtration do not contribute substantially to the total AHL activity, but the microorganisms grown in the biocake do. On the contrary, all four samples except the incubation time of zero show strong AHL activities per unit mass of biocake (Figure 5b). Although the samples at the

incubation time of 0-6 h hardly showed the AHL activity (data not shown), the AHL activity was suddenly demonstrated from 6 h of incubation and continuously increased thereafter. These results coincide with the previous one shown in Figure 4 a-d and reasonably support that QS would take place in the biofilm grown on the membrane surface from a certain moment during the membrane filtration to give rise to maturations of biofilm (25), which might be the cause of the “two-stage TMP increase” (21) mentioned above. To make a more quantitative correlation between AHL level and TMP variation, we measured both the filtration resistance (i.e., TMP) and the AHL level of the biofilm and correlated these two parameters. As depicted in Figure 6a, 0.1 mg of MLSS from the mixed liquor in MBR was deposited on each of 12 PVDF membranes using a filtration device and then they were incubated on wastewater agar plates to make biofilm grow on each membrane. Two membrane-biofilms were prepared at each incubation time of 0, 3, 6, 9, 12, and 24 h. One was used to measure TMP when filtering distilled water at a constant flux of 45 L/m2/h. The other was bioassayed to determine the relative AHL level. As shown in Figure 6b, the TMP and AHL levels of the biofilm increased under very similar tendencies, which makes us expect the potential of QS-based membrane biofouling control. Membrane Biofouling Control Based on QS. Three basic ways of controlling AHL QS have been reported (26): (i) blockage of AHL production, (ii) interference with the signal receptor, and (iii) inactivation of AHL signal molecules (Figure S5). Of these three QS control strategies, we adopted the third one: a quorum quenching method in which AHLs are inactivated by hydrolysis either at the lactone ring with lactonase or at the acyl-amide linkage with acylase (Figure S6). Among various quorum quenching enzymes (27), we selected porcine kidney acylase I (EC 3.5.1.14) in this study. An acylase stock solution of 1000 mg/L was added exogenously to the batch reactor (Figure 2b) to make the final concentration of 10 mg/L. If the enzyme acylase is applied directly to the MBR under a continuous operation like Figure 2a, enzyme loss could take place owing to (i) passage through the membrane pore into the permeate and (ii) removal during the excess sludge disposal. Therefore, a batch type of MBR was designed and operated under a total recycle mode to maintain the effective enzyme concentration throughout the operation. As shown clearly in Figure 7a, the addition of acylase retarded the TMP rise compared with that of the VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 7. Evidence for membrane biofouling prevention by quorum quenching: (a) TMP profile; (b) A. tumefaciens bioassay results. Bioassay was carried out after 1 day of operation.

FIGURE 8. Change of glucose concentration during the batch reactor operation in a total recycle mode. control reactor, which implies that acylase can alleviate membrane biofouling. However, the biochemical reaction catalyzed with acylase is the general cleavage of amide bonds for any substrate. The reduction of biofouling by acylase would, therefore, result from either the interference of QS by the destruction of AHL autoinducers or the degradation of any organic foulants with amide bonds. Consequently, we conducted the following experiments to clarify the role of acylase in the release of biofouling. First, C8-HSL, which had been identified as a major autoinducer in this study, was deliberately and exogenously added to the batch reactor to a final concentration of 1 mg/ L, a much higher concentration than that of the control. As shown in Figure 7a, with the addition of C8-HSL, the increase rate of TMP became more rapid than that of the control. However, when C8-HSL and acylase were added simultaneously to the reactor, the biofouling aggravated by AHL alone was counterbalanced by acylase, which proves the inactivation of AHL by acylase. This quorum quenching activity of acylase was also confirmed by A. tumefaciens A136 bioassay of biocake. As shown in Figure 7b, biocake in the acylase-added reactor showed much weaker autoinducer activity than that in the control reactor. In view of these results, it has been concluded that acylase reduced the membrane biofouling by quenching AHL autoinducers. Considering that QS regulates gene transcription and determines microbial physiology, the wastewater treatment efficiency should be checked to see possible side effects of acylase or autoinducer. Consequently, the glucose concentrations were monitored during the batch reactor operations under the four different conditions, respectively. As shown in Figure 8, the differences in the glucose concentration profiles among the four cases are negligible, which indicates that the addition of acylase and/or AHL autoinducer did not 384

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FIGURE 9. Quantitative analysis of EPSs in biocakes in the MBR under various operating conditions. affect the microbial activity. It has been concluded that QS control has nothing to do with the microbial activity required for the biodegradation of organics. In the final step, we investigated how quorum quenching prevents membrane biofouling. As it is known that EPSs play a great role in the biofilm formation and are also key foulants in MBRs (28), we measured the amount of EPSs per unit mass of biocake under the various operating conditions (Figure 9). The addition of acylase reduced the EPS concentration per unit mass of biocake, whereas the addition of an autoinducer (C8-HSL) increased it. These results not only lead to the conclusion that quorum quenching can be a control method for membrane biofouling by regulating EPS concentration, but also that they provide additional evidence for the interrelation between QS activity and biofouling.

Acknowledgments This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the government (MOST) (R0A-2007-000-20073-0).

Supporting Information Available Additional data, tables, text, and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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