Control and Cleaning of Membrane Biofouling by Energy Uncoupling

Dec 13, 2010 - Control and Cleaning of Membrane Biofouling by Energy Uncoupling and Cellular Communication. Huijuan Xu and Yu Liu*. Division of ...
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Environ. Sci. Technol. 2011, 45, 595–601

Control and Cleaning of Membrane Biofouling by Energy Uncoupling and Cellular Communication HUIJUAN XU AND YU LIU* Division of Environmental and Water Resources Engineering, School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798

Received August 25, 2010. Revised manuscript received November 13, 2010. Accepted November 23, 2010.

This study investigated possible biological control of membrane biofouling and membrane cleaning by disrupting energy metabolism of microorganisms. Results showed that 2,4dinitrophenol(DNP),atypicaluncoupler,couldnotonlysignificantly inhibit membrane biofouling but also enhance biofilm detachment from nylon membrane. Inhibited ATP synthesis by a chemical uncoupler resulted in lowered production of autoinducer-2 (AI-2). The standard dead-end microfiltration tests further confirmed that the reduced AI-2 was positively correlated to the reduced fouling resistance of nylon membranes. It appears that inhibition of energy metabolism would be a promising alternative for control and cleaning of membrane biofouling.

1. Introduction Membrane bioreactor has emerged as a promising technology for wastewater reclamation and has advantages of highquality product water, modular design with less space requirement. However, membrane biofouling due to microbial attachment onto membrane surface would result in lowered water flux (1). So far, several attempts have been made to mitigate membrane biofouling by coating membrane surface with antimicrobial products (2) or by modifying surface physical-chemical properties (3), whereas more and more attention has been given to identify biological triggers for control of microbial attachment on membrane surfaces (4). Physiological behaviors of microorganisms are closely related to energy metabolism including interrelated catabolic and anabolic reactions. It has been reported that granular sludge biofilm could not form when ATP synthesis was inhibited (5), i.e. energy metabolism may be one of the biological triggers of microbial attachment. This implies that membrane biofouling could be controlled through inhibition of ATP synthesis, which is achievable by chemical uncoupling of energy metabolism. Chemical uncouplers are weak acids with the ability to carry a proton through cellular membrane and thus can dissipate proton gradient and subsequently inhibit ATP synthesis (6). In addition, it has been demonstrated that microorganisms regulate their group behaviors, such as biofilm formation using signal molecules, called autoinducers among which autoinducer-2 (AI-2) has been believed to coordinate interspecies communication during biofilm formation (7, 8). * Corresponding author phone: +65-67-905-254; fax: +65-67-910676; e-mail: [email protected]. 10.1021/es102911m

 2011 American Chemical Society

Published on Web 12/13/2010

Previous study showed that AI-2 was required for formation of a mixed-species biofilm between Porphyromonas gingivalis and Streptococcus gordonii, and production of AI-2 by either species was found to be sufficient for interspecies communication and biofilm formation (8). It appears that control of cellular communication would be an alternative toward prevention of microbial attachment including membrane biofouling (4, 9). Besides control of biofilm formation, removing biofilm or enhanced biofilm detachment is another way to reduce membrane biofouling (10, 11). Many physical-chemical ways have been investigated to remove biofilm, such as backwashing, sonication, chemical cleaning, and a combination of various cleaning methods etc. (12). For chemical cleaning, the chemicals are normally alkaline, acids, metal chelating agents, surfactants, and enzymes (13, 14). However, little information is available for the effect of energy metabolism on microbial attachment and detachment. Therefore, this study attempted to investigate the role of energy metabolism in both microbial attachment onto and detachment from membrane surfaces. For this purpose, chemical uncoupler was employed to disrupt energy metabolism. Although the role of cellular communication in microbial attachment and detachment has been reported in the literature, the present study aimed to establish a link between ATP and AI-2 and further to show their combined effect on membrane biofouling. Bioenergy in the form of ATP is essentially required for all microbial activities, including synthesis of universal signaling molecules, AI-2, thus it is expected that disruption of energy metabolism and subsequent AI-2 production would help to effectively control membrane biofouling. This study would offer good insight into biological control of membrane biofouling from the aspects of energy metabolism and cellular communication.

2. Materials and Methods 2.1. Biofouling Development on Nylon Membrane. Activated sludge used in this study was taken from a local wastewater treatment plant and acclimated with a synthetic wastewater for two months. The synthetic wastewater consisted of 690 mg L-1 of sodium acetate and 240 mg L-1 ethanol as carbon source, 200 mg L-1 NH4Cl, 60 mg L-1 K2HPO4, 15 mg L-1 CaCl2 · 2H2O, 12.5 mg L-1 MgSO4 · 7H2O, and 20 mg L-1 FeSO4 · 7H2O (15). Two 2-L beakers were used as batch reactors fed with the above synthetic wastewater at an initial total organic carbon (TOC) concentration of 300 mg L-1, equivalent to 960 mg L-1 of theoretical oxygen demand (ThOD), and initial acclimated biomass concentration was fixed at 300 mg L-1 in each reactor. The dissolved oxygen concentrations in the two reactors were maintained at quasi-saturation level through air aeration, and all the experiments were carried out at 25 °C. The only difference between the two reactors is that one served as control free of 2,4-dinitrophenol (DNP) which is a typical chemical uncoupler (6) and another was supplemented with 10 mg L-1 DNP. At different exposure times, suspended microorganisms were collected from two reactors for microbial attachment experiments as well as for determination of cellular ATP and AI-2 contents. In order to investigate biofouling potential of microorganisms with and without exposure to DNP, a series of static attachment assays were conducted with hydrophilic flat sheet nylon membrane (Osmonics, Minnetonka, USA) with a pore size of 0.2 µm at 25 °C. For each attachment assay, suspended microorganisms harvested from the batch reactors at different culture times were resuspended in 30 mL of 10 mM phosphate VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Water flux through nylon membrane (a), microbial attachment on membrane (b), and CLSM images (c) of attached microorganisms with (9) and without (0) exposure to DNP. Each point represents the mean of triplicate experiments, and the error bar is 1 SD from the mean. buffered saline (PBS) solution with 100 mg dry biomass L-1 and were made contact with membranes for one hour in a Petri dish. At the end of 1 h, fouled membranes were gently rinsed three times with distilled water to remove loosely attached microorganisms. To quantify attached biomass, microorganisms on membranes were collected by the method modified from Liu (16), and TOC of the harvested biomass was quantified using a TOC analyzer (ASI-V, TOC-Vcsh, Shimadzu, Japan). Each sample was analyzed in triplicate otherwise stated. Student t-tests were employed to assess the significance of results with a 95% confidence. It should be noted that the long-term effect of DNP needs further investigation. 2.2. Microfiltration Test. In order to evaluate the biofouling potential of microorganisms with and without exposure to DNP, standard dead-end microfiltration experiments (Figure S1, Supporting Information) were carried out at 25 °C with fouled membranes collected from the microbial attachment tests as described above. Sixty milliliters of Milli-Q water (Milipore, Singapore) was filtered through the fouled membranes at 5 kPa. The filtrate was collected in a container placed on an electronic balance, and its weight was recorded at the time interval of 5 s during microfiltration. In order to analyze the resistance of fouled nylon membrane, a resistance-in-series model (17) was used for determination of intrinsic membrane resistance, pore blocking resistance, and cake layer resistance 596

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J)

∆P µRt

(1)

where J is permeate flux (m3 m-2 s-1); Rt is total filtration resistance (m-1); ∆P is applied pressure (Pa); and µ is solution viscosity (Pa · s). Rt represents the sum of the intrinsic membrane resistance (Rm), the resistance due to pore blocking (Rp), and the resistance caused by the cake layer on membrane surface (Rc) Rt ) Rm + Rp + Rc

(2)

The Rm was estimated from eq 1 by measuring water flux of virgin membrane, while Rt was determined from water flux through fouled membrane. After fouled membranes were gently wiped with a sponge and rinsed with Milli-Q water to remove the cake layer, Rm+ Rp was calculated from water flux through wiped membrane, thus Rp can be obtained. After knowing Rt, Rm and Rp, Rc can be calculated from eq 2. 2.3. Determination of ATP and AI-2. Cellular ATP was extracted from freshly collected microorganism samples according to trichloroacetic acid (TCA) method (18) and was quantified by firefly luciferin-luciferase bioluminescence method with the FLAA Adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit (Sigma-Aldrich) and a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA). The amount of autoinducer-2 (AI-2) was determined by modified Vibrio harveyi bioluminescence assay (19). Details can be found in the Supporting Information.

TABLE 1. Filtration Resistances of Nylon Membrane Fouled by Microorganisms with (DNP-Treated) and without (Control) Exposure to DNPa

control

DNP-treated a

1 2 3 4 1 2 3 4

h h h h h h h h

Rt (1011 m-1)

Rm (1011 m-1)

Rm/Rt (%)

Rp (1011 m-1)

Rp/Rt (%)

Rc (1011 m-1)

Rc/Rt (%)

5.89 5.50 5.32 5.45 4.67 4.42 5.19 4.71

1.37 1.37 1.37 1.37 1.37 1.37 1.37 1.37

23.26 24.91 25.75 25.14 29.34 31.00 26.40 29.09

0.26 0.11 0.41 0.49 0.25 0.25 0.54 0.43

4.41 2.00 7.71 8.99 5.35 5.66 10.40 9.13

4.26 4.02 3.54 3.59 3.05 2.80 3.28 2.91

72.33 73.09 66.54 65.87 65.31 63.35 63.20 61.78

Rt: total resistance; Rm: membrane resistance; Rp: pore blocking resistance; Rc: cake layer resistance.

FIGURE 2. Cellular ATP (a) and AI-2 (b) contents in suspended microorganisms with (•) and without (O) exposure to DNP. Each point represents the mean of triplicate experiments, and the error bar is 1 SD from the mean. 2.4. Staining and Visualization of Attached Microorganisms. Attached microorganisms on membrane surfaces were stained with LIVE/DEAD BacLight Bacterial Viability kits (Molecular Probes, Eugene, OR, USA). The kit contains SYTO 9 and propidium iodide (PI). Membrane permeant SYTO 9 can label intact bacteria with green fluorescence, while membrane impermeant PI only labels membranedamaged bacteria with red fluorescence. The sample staining was done according to the protocol provided by the kit suppliers. The stained sample was covered with coverslip and observed using an Olympus Fluoview FV300 confocal laser scanning microscopy (CLSM) (Olympus Optical, Tokyo, Japan) with a 100× objective. 2.5. Biofilm Development on and Detachment from Membrane. Experiments were designed to investigate the effect of DNP on microbial detachment from membrane surfaces. Biofilms were precultivated on membrane surfaces for 3, 12, and 24 h, respectively. The membranes with biofilms were soaked in PBS solutions with and without 10 mg L-1 DNP. One milligram of acclimated activated sludge was filtered on 0.2 µm nylon membrane overlaying solid media and cultured on Petri dish at 30 °C (20, 21). Solid media for biofilm culturing were prepared from Luria-Bertani (LB) agar (BD Difco, Franklin Lakes, NJ). In parallel, several membranes with 1 mg biomass were prepared for biofilm cultivation. Three membranes with fixed biomass were harvested at different culture times. One was used to determine the quantity of fixed biomass on membrane according to the standard method (22); the other two were used to estimate water flux before biofilm detachment. Afterward, one of these two membranes was soaked in PBS solution for 2 h; the other was soaked in PBS solution supplemented with 10 mg L-1 DNP for 2 h. The microfiltration tests were then conducted for determination of water flux

through membranes after biofilm detachment. Applied pressure for microfiltration was kept at 40 KPa.

3. Results 3.1. Biofouling on Nylon Membrane. The dead-end microfiltration experiments were carried out with fouled nylon membrane, and the intensity of membrane fouling was characterized by the ratio of water flux (J) of fouled membrane to water flux (Jo) of virgin membrane. Figure 1a shows that the water fluxes of nylon membranes fouled by microorganisms exposed to DNP were higher than those obtained from the controls free of DNP. The calculated filtration resistances for the fouled membranes with and without exposure to DNP are shown in Table 1. It appears that total resistance of nylon membrane fouled by microorganisms free of DNP was about 20% higher than that membrane fouled by microorganisms exposed to DNP. In addition, the fraction of cake layer resistance accounted for about 61.8% to 73.1% of the total resistance (Table 1), i.e. the main fouling mechanism for the microfiltration of fouled nylon membrane can be attributed to cake layer resistance. These suggest that DNP, a typical chemical uncoupler, could help to improve the membrane performance. Figure 1b shows microbial attachment onto membrane surfaces with and without exposure to DNP. The respective attachment of microorganisms exposed to DNP onto the nylon membrane surfaces was reduced substantially as compared to the control assay free of DNP (Student’s t-test, P < 0.05). Figure 1b shows that attachment of microorganisms exposed to 10 mg L-1 DNP for 1 h was reduced by 46% on nylon membrane compared to the control without DNP. These results were further supported by microscopic observations (Figure 1c). VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Water flux through nylon membrane with biofilms (a); biomass of biofilms on membrane (b) before treatment (0), after treatment by PBS solution (gray 0) and DNP-PBS solution (9); CLSM images (c) of 24-old biofilms on nylon membrane. Each point represents the mean of triplicate experiments, and the error bar is 1 SD from the mean. 3.2. ATP and AI-2 Contents in Suspended Biomass with and without Exposure to DNP. Figure 2a shows that the cellular ATP content of microorganisms without exposure to DNP was significantly higher than that obtained from the culture supplemented with DNP at the same culture times (Student’s t-test, P < 0.05). It should be noted that the inverse V-shape curves for ATP can be explained by consumption of energy sources over culture time. For suspended microorganisms without exposure to DNP, the ATP content increased from 5.4 × 10-7 mol g-1 biomass to 10.3 × 10-7 mol g-1 biomass, indicating a net ATP synthesis of 4.9 × 10-7 mol g-1 biomass. On the contrast, for suspended microorganisms exposed to DNP, the net synthesis of cellular ATP was only about 1.72 × 10-7 mol g-1 biomass, i.e. a 65% reduction was achieved compared to that of control free of DNP. These results suggest that DNP could effectively suppress cellular ATP synthesis. 598

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Figure 2b further shows changes in the AI-2 content in suspended microorganisms with and without exposure to DNP. It is obvious that the AI-2 content in microorganisms exposed to DNP was reduced substantially as compared to that of the control free of DNP, i.e. the AI-2 synthesis was impaired markedly by chemical uncoupler (Student’s t-test, P < 0.05). 3.3. Biofilm Detachment from Membrane by DNP. Figure 3a shows flux recovery after treatment with and without DNP. A more significant flux recovery was observed after soaking in DNP-PBS solution than in PBS solution alone. This in turn implies that DNP is capable of disrupting attached biomass from membrane surface, leading to an improved permeate flux. For example, the J/Jo for nylon membrane with 24 h-old biofilm was only about 2.6%. Nevertheless, after soaking in PBS solution for 2 h, the J/Jo was increased to 6.2% and further to 24.3% after soaking in DNP-PBS

FIGURE 4. Effects of ATP content on microbial attachment (a) and AI-2 production (b) in suspended microorganisms with (•) and without (O) exposure to DNP. Each point represents the mean of triplicate experiments, and the error bar is 1 SD from the mean. solution under the same condition. The significant difference in J/Jo between the treatments by the PBS and DNP-PBS solutions was also observed for 12 h-old biofilm (Student’s t-test, P < 0.05); however, no significant improvement in J/Jo was found for 3 h-old biofilm (Student’s t-test, P > 0.05). As shown in Figure 3b, the amount of fixed biomass on nylon membrane was reduced significantly after the treatment with the PBS and DNP-PBS solutions, respectively (Student’s t-test, P < 0.05). For example, the amount of the 12 h-old biofilm on nylon membrane was reduced by 65% after the treatment with the DNP-PBS solution compared to that by the PBS treatment. Such observation is further confirmed by the CLSM cross section images (Figure 3c), showing that after the DNP treatment, the biofilm structure on nylon membrane became loose. These all indicate that DNP would be effective in biofilm detachment from nylon membrane surface.

4. Discussion The results show that DNP can not only mitigate biofouling but also enhance biofilm detachment from membrane surface. It is well-known that biofilm formation on membrane surface is the major cause of membrane biofouling. As shown in Table 1, the main fouling mechanism for nylon membrane was cake layer resistance, where the more attached biomass on nylon membrane surface resulted in the higher cake layer resistance. Miura et al. (1) also found that biofilm formation resulted in membrane biofouling in a pilot scale membrane bioreactor treating municipal wastewater, and biofilm developed on membrane surfaces had a positive correlation with the increase in trans-membrane pressure. ATP is synthesized through coupling electron transport and oxidative phosphorylation by a proton gradient (6). DNP is a typical uncoupler which can shuttle protons through membrane bypassing the enzyme matrix (6), therefore dissipating the proton gradient and subsequently inhibiting ATP synthesis (Figure 2a). Figure 4a further shows a positive correlation between the amount of attached biomass and the ATP content of suspended microorganisms with and without exposure to DNP. These suggest that a high ATP content would favor microbial attachment onto nylon membrane. As the result, inhibited ATP synthesis by DNP would lead to the low fouling potential on nylon membrane (Figure 1a). In study of attachment of Mycoplasma pneumonia to glass surface, Feldner et al. (23) also reported that microbial attachment on glass surface was reduced significantly by carbonyl cyanide chlorophenylhydrazone (CCCP), a typical chemical uncoupler. Moreover, in the study of the mechanism of aerobic granulation, Jiang and Liu (5) found that in the presence of 3,3′,4′,5-tetrachlorosalicylanilide (TCS), a chemical uncoupler, the net synthesis of cellular ATP was reduced

FIGURE 5. Effect of AI-2 content in suspended microorganisms on cake layer resistance of nylon membrane fouled by microorganisms with (•) and without (O) exposure to DNP. Each value represents the mean of triplicate experiments, and the error bar is 1 SD from the mean. by 75% compared to the control free of TCS, leading to failure of aerobic granulation. It has been hypothesized that when microorganisms approach a substratum surface, a localized proton motive force (pmf) would be established, and microorganisms can use localized pmf and ATP as the driving force to enable cells to attach to a solid surface (24). A positive correlation between the AI-2 and ATP contents of biomass was established in Figure 4b, i.e. the lower AI-2 production was observed at the lower ATP content of biomass in cases where microorganisms were exposed to DNP. Such observation can be explained by the fact that 4,5-dihydroxy2,3-pentanedione (DPD), an AI-2 precursor, is biosynthesized from S-adenosylmethionine which is made from ATP and methionine by methionine adenosyltransferase (25, 26), meaning that the AI-2 synthesis is energy-dependent. In the study of quorum-sensing systems associated with biofilm formation by Bacteroides fragilis, Pumbwe et al. (27) also reported the similar observation when CCCP was used as chemical uncoupler. Therefore, the inhibited ATP synthesis by DNP would be responsible for the observed reduction in the AI-2 content (Figure 2b). Figure 5 shows that the cake layer resistance on nylon membrane was positively correlated to the AI-2 content of suspended microorganisms regardless of DNP treatment. This suggests that AI-2 may play a coordinating role during biofouling development on membrane. It has been known that microorganisms can use AI-2 for interspecies comVOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Effect of fixed biomass on water flux through nylon membrane before (O) and after treatment by PBS solution (gray 0) and DNP-PBS solution (2). Each point represents the mean of triplicate experiments, and the error bar is 1 SD from the mean. munication for both gram-negative and gram-positive bacteria (8). Furthermore, AI-2 has been shown to control E. coli biofilm formation through a motility regulatory mechanism, e.g. addition of synthetic AI-2 would lead to 30-fold biofilm biomass increase through improving cell motility, on the contrast, for isogenic mutant without AI-2 induced motility gene, biofilm biomass was reduced by 8-fold (28). As can be seen in Figure 5, AI-2-mediated membrane biofouling is mainly responsible for the observed decline in water flux. These suggest that control of quorum sensing would be an effective way to mitigate membrane biofouling. In addition to AI-2, N-acylhomoserine lactones (AHL) are another common autoinducers which regulate the quorum sensing system of Gram-negative bacteria, and they have been studied as the targets for controlling membrane biofouling (29). Paul et al. (30) used Acylase I, an AHLdegrading enzyme, to hydrolyze AHL and found that biofilm formation of Aeromonas hydrophila and Pseudomonas putida on reverse osmosis (RO) membrane was reduced by 20% to 24%. Yeon et al. (31) also reported that the presence of AHL in membrane bioreactor (MBR) would be responsible for membrane biofouling. This and previous studies clearly show that inhibition of cellular communication would be a promising strategy for control of membrane biofouling. Cleaning of biologically fouled membrane remains a big challenge in application of membrane technology for water reclamation. It is reasonable to consider that energy dissipation induced by a chemical uncoupler would result in less ATP synthesis that might not enough to maintain the biofilm stability and subsequently trigger cell detachment from membrane surfaces. Figure 3 shows reduced attached biomass and improved water flux after treatment by PBS and DNP-PBS solutions, respectively. The observed reduction in microbial attachment after PBS treatment would be a response of attached microorganisms to the absence of carbon source in PBS solution (32). It is observed in Figure 3 that DNP can help to disrupt biofilms developed on nylon membrane, and higher water flux was obtained after DNP treatment. This is directly supported by Figure 6 showing the relationship of water flux to attached biomass on membrane. A substantial detachment of biofilms from nylon membrane was achieved after soaking in the DNP-PBS solution due to loosened biofilm structure (Figure 3c). In addition, DNP appears to be more effective for 24 h-old biofilm detachment than 3 h-old biofilm detachment, i.e. DNP would be more effective for disruption of old biofilms than young ones. In the study of dependence of microbial attachment on sludge age, Fletcher (33) observed that microorganisms at an exponential stage would more easily attach to a solid surface than those at a stationary stage. 600

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Jiang and Liu (5) also found that the mature aerobic granular biofilm began to disintegrate when exposed to 4 mg L-1 of TCS, indicating that maintaining the stability and integrity of biofilm was metabolic energy-dependent, while similar phenomenon was reported when CCCP was used as chemical uncoupler (34). According to Spormann (34), microorganisms can response to reduced metabolic energy as a signal input to a series of signaling transduction cascade, which in turn triggers biofilm detachment. The results presented in this study demonstrate that microorganisms treated with chemical uncoupler, DNP, would have less fouling potential on nylon membrane. It was revealed that chemical uncoupler suppressed ATP synthesis and subsequent AI-2 production, which further resulted in reduced microbial attachments on membrane surface and improved membrane performance in terms of water flux. In addition, it was shown that chemical uncoupler would be a potential cleaning chemical for removing biofilm from fouled membrane surface. Consequently, this study provides direct experimental evidence that inhibition of ATP and ATP-mediated AI-2 would be a promising alternative for control of membrane biofouling.

Supporting Information Available Figure S1 and text. This material is available free of charge via the Internet at http://pubs.acs.org.

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