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, † CHUNG-HAK LEE,* ,† HALUK BEYENAL, ‡ 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, thin- layer 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. 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 ad- vanced 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. Although various biofouling control techniques have been developed through engineering (3), material (4), and chem- istry 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 auto- inducers 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) demon- strating the presence of an autoinducer in the MBR, (2) showing a correlation between QS activity and membrane biofouling, and (3) developing QS-based membrane bio- fouling 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 * Corresponding author tel.: +82-2-880-7075; fax: +82-2-874-0896; e-mail: leech@snu.ac.kr. † Seoul National University. ‡ Washington State University. § Montana State University. FIGURE 1. Concept of QS-based biofouling control in MBR. Environ. Sci. Technol. 2009, 43, 380–385 380 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 2, 2009 10.1021/es8019275 CCC: $40.75  2009 American Chemical Society Published on Web 12/18/2008