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Biofouling
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Vanillin, a potential agent to prevent biofouling of reverse osmosis membrane

Sajeesh Kappachery , Diby Paul , Jeyong Yoon & Ji Hyang Kweon
a b c a b c b

Department of Advanced Technology Fusion, Konkuk University, Seoul, Republic of Korea Department of Environmental Engineering, Konkuk University, Seoul, Republic of Korea

World Class University (WCU) Program of Chemical Convergence for Energy & Environment (C2E2), Seoul National University, Seoul, Republic of Korea Available online: 20 July 2010

To cite this article: Sajeesh Kappachery, Diby Paul, Jeyong Yoon & Ji Hyang Kweon (2010): Vanillin, a potential agent to prevent biofouling of reverse osmosis membrane, Biofouling, 26:6, 667-672 To link to this article: http://dx.doi.org/10.1080/08927014.2010.506573

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Biofouling Vol. 26, No. 6, August 2010, 667672

Vanillin, a potential agent to prevent biofouling of reverse osmosis membrane

Sajeesh Kappacherya, Diby Paulb*, Jeyong Yoonc and Ji Hyang Kweonb*
Department of Advanced Technology Fusion, Konkuk University, Seoul, Republic of Korea; bDepartment of Environmental Engineering, Konkuk University, Seoul, Republic of Korea; cWorld Class University (WCU) Program of Chemical Convergence for Energy & Environment (C2E2), Seoul National University, Seoul, Republic of Korea (Received 26 April 2010; nal version received 1 July 2010) Reverse osmosis (RO) membrane systems are widely used in water purication plants. Reduction in plant performance due to biolm formation over the membrane is an inherent problem. As quorum sensing (QS) mechanisms of microorganisms have been reported to be involved in the formation of biolm, ways are sought for quorum quenching (QQ) and thereby prevention of biolm formation. In this study using a chemostat culture run for seven days in a CDC reactor it was found that a natural QQ compound, vanillin considerably suppressed bacterial biolm formation on RO membrane. There was 97% reduction in biolm surface coverage, when grown in the presence of vanillin. Similarly, the average thickness, total biomass and the total protein content of the biolm that formed in the presence of vanillin were signicantly less than that of the control. However vanillin had no eect on 1-day old pre-formed biolm. Keywords: Aeromonas hydrophila; biofouling; reverse osmosis membrane; vanillin; COMSTAT
a

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Introduction The shortage of pure water for both domestic and industrial purpose is increasing day by day. Establishment of large scale reverse osmosis (RO) plants which can be fed with river or sea water is an option to meet this demand. One of the main problems with the RO based water purication industry is fouling of the membrane; even a small amount of fouling can cause a signicant loss of permeate ux (Pasmore et al. 2001). Several types of fouling can occur in the membrane system, eg inorganic, organic, particulate, colloidal and biofouling (Kramer and Tracey 1995). Pretreated feed water can be supplied to the RO-system to reduce fouling, but even then biofouling of the membrane is dicult to control, as some microorganism can survive the pretreatment and rapidly grow to form biolm. Thus, biofouling is recognized as most dicult to control (Baker and Dudley 1998). Other than pretreatment of feed water, modication of membrane surface properties, optimization of module arrangement, process conditions and periodic cleaning (Sheikholeslami 1999) have been tried to reduce the severity of the problem, even after long periods of such development in the eld. However, biofouling still remains as a main reason for the decline in plant performance (Saad 1992; Saeed et al. 2000). Biocides have been used to control the biofouling of the RO membranes. Chlorine is the most commonly

used biocide, but is known to deteriorate membranes (Kim et al. 2009). Chlorine based biocides have also been reported to intensify biofouling, as microorganisms subjected to low levels of biocides often exude large amounts of extracellular polysaccharides (EPS) for protection. This EPS supports biolm formation (Baker and Dudley 1998). Excessive use of biocides at high concentrations is likely to lead to environmental, ecological, and toxicological problems (Elvers et al. 2002). Hence here comes the importance of developing new and sustainable strategies for controlling biofouling of RO membranes. Several bacteria have been reported to co-ordinate community behavior and thereby biolm formation through cell to cell communication or quorum sensing mediated by, small, diusible signals (Hentzer and Givskov 2003). It is also suggested that, research into quorum sensing inhibition will provide some means for controlling the growth of biolm without the use of growth-inhibitory agents that unavoidably select for resistant organisms (Richards and Melander 2009). Compounds like salicylic acid (Rosenberg et al. 2008) urosolic acid (Ren et al. 2005), cinnamaldehyde (Brackman et al. 2008), extract from garlic (Bjarnsholt et al. 2005) and cranberries (Yamanaka et al. 2007) have all shown various degrees of antibiolm properties against a number of microorganisms in various studies. Furanones isolated from the marine red alga

*Corresponding authors. Email: jhkweon@konkuk.ac.kr; dibypaul@konkuk.ac.kr Published online 21 July 2010

ISSN 0892-7014 print/ISSN 1029-2454 online 2010 Taylor & Francis DOI: 10.1080/08927014.2010.506573 http://www.informaworld.com

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S. Kappachery et al. Company, Midland, MI, USA) with high rejection for sea water application were used for this study. Experimental design The eects of vanillin on prevention of biolm formation by an environmental strain A. hydrophila were studied using chemostat culture in a CDC reactor. The experimental variables were selected to exaggerate the biolm forming characteristics; these included low shear stress, and high nutrient concentration compared to natural waters. The reactor, medium storage tank, tank for spent medium and the tubing were autoclaved and connected aseptically. RO membrane segments (1.5 cm 6 1.5 cm) were UV sterilized before use. Vanillin stock solution (8 mg ml71) was prepared in double distilled water, and sterilized by a 0.2 mm membrane lter. The least eective concentration of vanillin (0.18 mg ml71) was used in the present experiments, based on previous work in the area (Ponnusamy et al. 2009). Chemostat inoculation and biolm formation To each of the eight removable rods in a CDC reactor, a sterile glass slide was xed in such a way that it faced the bae when placed into the reactor. Two pieces of RO- membrane were xed (feed side facing outwards) on to each glass slide by using sterile double sided cellophane tape. An overnight culture of a single colony of A. hydrophila from an LB agar plate was used to inoculate a 35 ml LB/15 medium in a conical ask (100 ml) and shaken overnight at 258C to prepare the starter culture. This culture was then used to inoculate 315 ml of LB/ 15 in the CDC reactor (1 l) to initiate biolm formation. The reactor was run with 350 ml of LB/ 15 in batch mode for 1 day and then fresh medium was continuously supplied with a ow rate of 15 ml h71. The volume of the culture medium in the reactor was maintained to 350 ml. Filter-sterilized atmospheric air was supplied at a rate of 2000 ml min71 and the speed of bae rotation was set to 125 rpm. The temperature was maintained at 25 C throughout the experiments. Another reactor was run in parallel with similar conditions except that the medium contained 0.18 mg ml71 vanillin. After the specic incubation periods of 1, 2, 3, 4 and 7 days (as separate experiments), membrane coupons were removed and the biolm was quantied by the Bradford method (Bradford 1976). Protein measurements and image analyses by confocal laser scanning microscopy (CLSM) were performed with eight samples from the designated day. Another experiment was conducted to study eects of vanillin on preformed biolm. Biolm was grown on

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Delisa pulchera, are one of most extensively studied classes of natural compounds with respect to their role in inhibiting biolm. The authors recently reported the role of 2(5-H)furanone in suppressing biolm formation by environmental strains of bacteria isolated from fouled RO membrane (Ponnusamy et al. 2010). Previous work in the authors laboratory showed that, vanillin (4-hydroxy-3-methoxy benzaldehyde) at concentrations ranging from 0.063 to 0.25 mg ml71 could reduce the biolm formation by Aeromonas hydrophila on polystyrene surface without inhibiting the growth of planktonic cells (Ponnusamy et al. 2009). In this study, even 0.18 mg ml71 vanillin reduced biolm formation by 46% which is almost equal to the reported 46.3% for 0.25 mg ml71. A possible mechanism of action of vanillin as a quorum sensing inhibition (QSI) agent is its direct interaction with acylated homoserine lactone (AHL)receptors. The QSI activity of vanillin varied with dierent AHLs (signal molecules), implying that it has also something to do with the structure of signal molecules (Ponnusamy et al. 2009). There is no possibility of organisms developing resistance to vanillin because it is both an inhibitor of quorum sensing (Choo et al. 2006; Ponnusamy et al. 2009) and prevents biolm formation at concentrations below the minimum inhibitory concentration (MIC). Considering these factors together with its nontoxic nature for human application, vanillin is a promising candidate to be tested for its ecacy in controlling biofouling of RO membrane. In this study, commercially available vanillin (Sigma-Aldrich, St Louis, USA) was tested for its potential as an agent to control biofouling of RO-membrane using a continuous culture set up in a CDC reactor (Model CBR 90-2, BioSurface Technologies Corp; Bozeman, MT-USA). Materials and methods Strain and media Aeromonas hydrophila isolated from a fouled RO membrane (supplied by a local water purication plant in Deasan, Chungbuk, Korea) was used for the study. The isolate was stored on LB agar slants at 48C for short term storage and in 20% glycerol at 7708C for long term preservation. Full strength LB medium was used to revive the culture and 1/15th strength LB (LB/ 15) was used for growing biolm. The pH of all media was set to seven. The culture medium was purchased from Difco (BD, New Jersey, USA). RO membrane Polyamide thin lm composite RO membranes (FILMTECHTM SW30HR-380, Dow Chemical

Biofouling membrane coupons as described above for specic time periods and then exposed to vanillin by adding an appropriate quantity of vanillin stock to the reactor to make the nal concentration 0.18 mg ml71. This was followed by supply of fresh LB/15 medium containing the same concentration of vanillin at a ow rate of 15 ml h71 for a period of 24 h and then biolm samples were analyzed as described above. In this case, biolm quantication was conducted on four samples and the experiments were repeated three times. Sampling and data collection At the specied time periods (as mentioned above) rods holding membrane coupons with biolm were removed and rinsed three times by dipping in 0.2 M phosphate-buered saline (PBS; pH-7). Of the two membrane coupons xed to each glass slide, one was removed using sterile forceps and kept in a conical tube containing 5 ml of 0.2 M PBS and processed for protein estimation. Then the entire glass slide with the other coupon was immersed in a Petri dish containing 2.5% glutaraldehyde (Showa Chemical-Co Ltd, Meguro-ku, Tokyo, Japan) in 0.2 M PBS and left at room temperature for 90 min for xation of the biolm. It was then rinsed with 0.2 M PBS and stained with 60 mM propidium iodide (Invitogen, Carlsdad, USA) in double distilled water for 30 min and then washed again with PBS before CLSM imaging. CLSM observation and image analysis Stained biolm samples were observed using an Olympus Fluoview FV1000 confocal microscope (Olympus, Tokyo, Japan), and image stacks (seven per sample, at random locations) were saved. Two dimensional images of the biolm were generated using the software FV10-ASW version 2 (Olympus, Tokyo, Japan), a platform associated with the confocal microscope. To determine the percentage surface coverage, the average thickness and the total-biomass of each sample, the image stacks obtained were analyzed using the image analysis software, COMSTAT (Heydorn et al. 2000). Quantication of total protein The tubes containing membrane samples with biolm were vortexed for 1 min and sonicated at 40 kHz for 5 min to bring the biolm into suspension. Then the membrane pieces were taken out and the samples centrifuged at 5000 rpm for 10 min to pellet the bacterial cells. The supernatants were decanted and total protein was extracted (B-PER II Bacterial Protein Extraction Reagent-78260, Thermo Scientic,

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Rockford, IL, USA) and quantied by using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Results and discussion Experiments conducted in the present study reinstated the potential of vanillin as an eective quorum quenching agent. There are very few known natural compounds like vanillin that could inhibit biolm formation while not aecting cell growth and it adds to the suitability of the molecule to be used for sustainable and eco-friendly control of biofouling. Brominated furanones and their derivatives have been reported to inhibit biolm formation by several bacterial species (Janssens et al. 2008). Unfortunately, the use of brominated compounds is not suitable for the water purication industry due to their potential toxicity. Previous studies reported that a higher concentration of 1 mg ml71 non-halogenated, commercially available 2(5H)-furanone could signicantly reduce A. hydrophila biolm formation on polystyrene plates (Ponnusamy et al. 2010). However, non-brominated furanone did not inhibit biolm formation by other bacteria suggesting the use of brominated furanones for broader application (Janssens et al. 2008). Vanillin is a well known food avoring agent and is cheaper and safe to use when compared to furanone. In this study biolm was grown in a CDC reactor in the presence and absence of vanillin, and after the specied incubation period, parameters like total protein, surface coverage, average thickness, and total biomass were quantied. Biolm formed in a control experiment occupied 9.3% of the membrane surface by day one, 22.4% by day two, 44.5% by day three, and 71.7% by day four and 90.9% by seventh day (Figures 1 and 2a). Similarly the average thickness of the biolm increased with days of incubation in the control experiment (Figure 2b). A similar trend was observed for total biomass (Figure 2c) and the total protein content (Figure 2d). The quantity of biolm formed in medium containing vanillin showed negligible biolm development when compared to the control experiment. The third day values of surface coverage, average thickness, total biomass and total protein for biolms grown in the presence of vanillin were 93, 97, 96 and 97% less than those of the controls, respectively (Figures 1 and 2). A similar trend was continued until the 7th day of study. In short, from day to day, the control biolm showed a signicant increase in the values of all the parameters studied, whereas in the case of biolm grown in the presence of vanillin the biolm development was signicantly suppressed. This shows that the presence of vanillin in the medium could limit biolm formation on RO membranes. Thus it is

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Figure 1. CSLM images of A. hydrophila biolm on RO membrane stained with propidium iodide (red indicates the cells/white in gray scale image). C1, C2, C3, C4, and C7 are control biolms grown for 1, 2, 3, 4, and 7 days respectively. V1, V2, V3, V4, and V7 are biolms formed in the presence of vanillin from zero hours incubation for 1, 2, 3, 4, and 7 days respectively. P1, P2, P3, P4, and P7 are biolms grown for 1, 2, 3, 4, and 7 days respectively and treated with vanillin for 24 h.

expected that in real systems, the presence of vanillin in the feed water could prolong the time period of biolm formation and reduce the frequency of membrane cleaning. Currently, the most common frequency of sanitization is every 35 days during a period of high biological activity (summer) and about every 7 days during a period of slow biological activity (winter) (http://watertechgroup.com/les/Membranes__DBNPA_-_RO_Biocide.pdf). The planktonic cell concentration in terms of optical density at 600 nm (OD600) and viable cell count per milliliter of culture broth were consistent after 24 h of batch mode, indicating the stability of the chemostat system used. Throughout the experimental period there was no signicant dierence in abundance of planktonic cells among the control and the treatment, which indicates that vanillin in the tested concentration, does not hinder the growth and proliferation of A. hydrophila. This is highly attractive as it indicates that vanillin is unlikely to pose selective pressure for the development of resistance, and thus supporting the use of vanillin for sustainable control of biofouling of RO membranes.

Figure 2. (a) Percentage of surface coverage, (b) Average thickness, (c) Total biomass, (d) Total protein content. Values are means + SD. control; treated from zero hours incubation; pre-formed biolm treated for 24 h.

Biofouling It would be advantageous if vanillin could also remove preformed biolm on a membrane as well as preventing its formation. The results in the present study showed that vanillin had no eect on a 24 h old pre-formed biolm. Introduction of vanillin could not prevent the growing biolm from further development (Figures 1 and 2). This could be explained based on the complexity of biolm structure as reported by Richards and Melander (2009). They suggested that the spider-web like nature of the biolm EPS matrix could trap inhibitory compounds before they can elicit their eect, or it can lower the rate of penetration into the biolm. The ineectiveness of vanillin on a preformed biolm could therefore be explained based on the above foresaid reasons resulting in its concentration being limited to a very low level inside the preformed complex biolm structure, rendering it ineective as a biolm inhibitor. To control biofouling in RO membrane plants operating with biologically active feed water, two dierent modes of application of antifoulant are generally used, viz. slug dosing and continuous feed. In slug dosing, a particular amount of antifoulant is used for 30 min to 3 h every 5 days (http://watertechgroup. com/les/Membranes_-_DBNPA_-_RO_Biocide.pdf.). Vanillin in the tested concentration appears to be unsuitable for slug dosing as it failed to remove preformed biolm even with 24 h treatment. Since vanillin can inhibit biolm formation when supplied from zero hours incubation, its application as continuous feed looks feasible. To do so, when the system is devoid of biolm (start of operation or just after thorough cleaning), a continuous feed maintenance program can be initiated maintaining 0.18 mg ml71 of vanillin which would delay or limit biofouling of the RO membrane. Developing a slow release system using immobilization techniques (which are underway using carbon nanotubes) can minimize the quantity of vanillin needed to maintain an appropriate concentration in the feed water. Its chemical stability in feed water; and rate of its removal from the system have to be studied in order to optimize its use on a eld scale. Conclusion Vanillin in tested concentration was found to limit the establishment of biolm on RO membrane surfaces without raising selective pressure for the growth of microorganism. Since vanillin is both naturally and commercially available it may be used in RO plants for sustainable control of biofouling. Its potential in controlling biolm formation on RO surfaces by A. hydrophila holds great promise that the growth of a whole range of bacterial biolms could be controlled by the use of similar compounds, either alone or in

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