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Reverse osmosis (RO) membrane systems are widely used in water purification plants. Reduction in plant performance due to biofilm 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 biofilm, ways are sought for quorum quenching (QQ) and thereby prevention of biofilm formation. In this study using a chemostat culture, run for seven days in a CDC reactor, we found that a natural QQ compound, vanillin considerably suppressed bacterial biofilm formation on RO membrane. There was 97% reduction in biofilm surface coverage, when grown in presence of vanillin. Similarly, parameters of average thickness, total biomass and total protein content of biofilm that formed in presence of vanillin were significantly lesser than that of the control. However vanillin had no effect on one day old pre-formed biofilm.
Keywords: Aeromonas hydrophila; Biofouling; Reverse osmosis membrane; Vanillin; COMSTAT
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 RO based water purification industry is fouling of membrane; even a small amount of fouling can cause significant loss of permeate flux (Pasmore et al, 2001). Several types of fouling can occur in the membrane system, e.g. inorganic, organic, particulate, colloidal and biofouling (Kramer and Tracey et al, 1995). Pretreated feed water can be supplied to RO-system to reduce fouling, even then biofouling of membrane is difficult to control, as some microorganism can survive the pretreatment and rapidly grow to form biofilm. Thus, biofouling is recognized as the most difficult to control (Baker and Dudley, 1998). Other than pretreatment of feed water, modification 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 field; 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 micro-organisms subjected to low levels of biocides often exude large amounts of extracellular polysaccharides (EPS) for protection. This EPS supports biofilm formation (Baker and Dudley, 1998). Excessive use of biocides at high concentrations is likely to lead to environmental, ecological, and toxicological problems if the discharge containing biocides flow in to natural water bodies (Elvers et al, 2002). XXXX There arises Here comes the importance of developing new and sustainable strategies for controlling biofouling of RO membrane.
Several bacteria have been reported to co-ordinate a community behavior and thereby biofilm formation through cell to cell communication or quorum sensing mediated by, small, diffusible signals (Hentzer and Givskov, 2003). It also suggests that, research into quorum sensing inhibition will provide some means for controlling the growth of biofilm 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 antibiofilm properties against a number of microorganisms in various studies. Furanones isolated from marine red algae Delisa pulchera, are one of most extensively studied classes of natural compounds in regards to their role in inhibiting biofilm. We recently reported the role of 2(5-H)Furanone in suppressing biofilm formation by environmental strains of bacteria isolated from fouled RO membrane (Ponnusamy et al, 2010). Previous work in our laboratory showed that, vanillin (4-Hydroxy-3-methoxy benzaldehyde) at concentrations ranging from 0.063 to 0.25mg/ml could reduce the biofilm formation by A. hydrophila on polystyrene surface without inhibiting the growth of planktonic cells (Ponnusamy et al, 2009). In this study, even 0.18 mg/ml vanillin reduced the biofilm formation by 46% which is almost equal to the reported 46.3% for 0.25 mg/ml.
A possible mechanism of action of vanillin as QSI agent is its direct interaction with acylated homoserine lactone (AHL)-receptors. The QSI activity of vanillin varied with different AHLs (signal molecules); implying that it has also something to do with the structure of signal molecules (Ponnusamy et al, 2009).Vanillin, being an inhibitor of both quorum sensing (Choo et al, 2006; Ponnusamy et al, 2009) and biofilm formation below its minimum inhibitory concentration for the bacterial strains studied, there is no chance of resistance against it. Considering all these factors together with its nontoxic nature for human application, vanillin is a promising candidate to be tested for its efficacy in controlling biofouling of RO membrane. In this study commercially available vanillin (V-2375, 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
A. hydrophila isolated from a fouled RO membrane (supplied by a local water purification plant in Deasan, Chungbuk, Korea) was used for the study. The isolate was stored on LB Agar slants at 4 ËšC for short term and in 20 % glycerol at Ë‰70 ËšC for long term preservation. Full strength LB media was used to revive the culture and 1/15th strength of LB (LB/15) was used for growing biofilm; pH of all media were set to seven. Culture media was purchased from Difco (Franklin Lakes, NJ, USA).
FILMTECTM SW30HR-380, high rejection, polyamide thin film composite seawater reverse osmosis membrane manufactured (Dow Chemical Company, Midland, MI, USA) was used for this study.
Effect of vanillin on prevention of biofilm formation by environmental strain A. hydrophila was studied using chemostat culture in the CDC reactor. The experimental variables were selected to exaggerate the biofilm forming characteristics, which includes the low shear stress, and high nutrient concentration compared to natural waters. The reactor, media storage tank, tank for spent media and tubing were autoclaved and connected aseptically. RO membrane segments (1.5 cm - 1.5 cm) were UV sterilized before use. Vanillin stock solution (8 mg/mL) was prepared in double distilled water, and filter sterilized. The least effective concentration of vanillin (0.18 mg/mL) was used in the present experiments, as decided based on our previous work in the area (Ponnusamy et al, 2009).
Chemostat inoculation and biofilm formation
To each of the eight removable rods in a CDC reactor, a sterile glass slide is fixed in such a way that it faces the baffle when placed into the reactor. Two pieces of RO- membrane were fixed (feed side facing out) on to each glass slide by using a sterile double side cellophane tape.
An overnight growth of a single colony of A. hydrophila from an LB agar plate was used to inoculate a 35 mL LB/15 media in a conical flask (100mL) and shaken overnight at 25 ËšC to prepare the starter culture. This culture was then used to inoculate 315 mLof LB/15 in the CDC reactor (1L) to initiate the biofilm formation. The reactor was run with 350 ml of LB/15 in batch mode for one day and then fresh media was continuously supplied with a flow rate of 15 mL/h. Volume of the culture media in reactor was maintained to 350 mL. Filter sterilized atmospheric air was supplied at a rate of 2000 mL/min, speed of baffle rotation was set to 125 rpm. The temperature was maintained at 25ËšC throughout. Another reactor was run in parallel with similar conditions except that the medium contained 0.18mg/ml vanillin. After each specific incubation periods of 1,2,3,4 and 7 days (As separate experiments), membrane coupons were removed and biofilm quantified by total protein estimation (n= 8) and analysis of images (n= 8) taken by using confocal laser scanning microscope (CLSM).
Another experiment was conducted to study the effect of vanillin on preformed biofilm. Biofilm was grown on membrane coupons as described above for specific time periods, and was exposed to vanillin by adding appropriate quantity of vanillin stock to the reactor to make the final concentration to 0.18 mg/mL. This was followed by supply of fresh LB/15 containing same concentration of vanillin at a flow rate of 15mL/h for a period of 24h and then biofilm-samples were analyzed as described above. In this case sample size for biofilm quantification was four and the experiments were repeated three times.
Sampling and data collection
At specified time periods (as mentioned above); rods holding membrane coupons with biofilm were removed and rinsed three times by dipping in 0.2 M phosphate-buffered saline (PBS; pH-7). Out of the two membrane coupons fixed on each glass slide, one is removed using sterile forceps and kept in a conical tube containing five milliliter of 0.2 M PBS and processed for protein estimation. Then the entire glass slide with the other coupon was immersed into a Petri-dish containing 2.5% glutaraldehyde (0705-1260, Showa Chemical-Co Ltd, Meguro-ku, Tokyo, Japan) in 0.2 M PBS and left at room temperature for 90 min for fixation of biofilm; it is then rinsed with 0.2 M PBS and stained with 60µM propidium iodide in dH2O (L7012, Invitogen, Carlsdad, USA) for 30 min, washed with PBS and proceeded for CLSM imaging.
CLSM observation and image analysis
Stained biofilm samples were observed using an Olympus Fluoview FV1000 Confocal Microscope (Olympus, Tokyo, Japan), and image stacks (seven per sample, at random location) were saved. Two dimensional images of biofilm were generated using the software FV10-ASW version 2(Olympus, Tokyo, Japan), a platform associated with the confocal microscope. To determine the percentage of surface coverage, average thickness and total-biomass of each sample, image stacks obtained were analyzed using the image analysis software, COMSTAT (Heydorn et al, 2000).
Quantification of total protein
The tubes containing membrane samples with biofilm were vortexed for one minute and sonicated at 40 kHz for five minutes to bring the biofilm in to suspension. Then the membrane pieces were taken out and the samples centrifuged at 5000 rpm for 10 min to pellet the bacterial cells. Supernatant were decanted and total protein was extracted (B-PER II Bacterial Protein Extraction Reagent-78260, Thermo Scientific, Rockford, IL, USA) and were quantified by using 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 effective quorum quenching agent. In fact there are very few known natural compounds like vanillin that could inhibit biofilm formation while not affecting 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 biofilm formation by several bacterial species (Janssens et al, 2008). Unfortunately, use of brominated compounds is not suitable for the water purification industry due to their potential toxicity. Previous studies reported that, a higher concentration of 1mg/mL non-halogenated, commercially available 2(5H)-Furanone could significantly reduce A. hydrophila biofilm formation on polystyrene plates (Ponnusamy et al, 2010). However non-brominated furanone did not inhibit biofilm formation by other bacteria suggesting the use of brominated furanones for broader application (Janssens et al, 2008). Vanillin is a well known food flavoring agent and is cheaper and safe to use when compared to furanone.
In the present investigation biofilms were grown in CDC reactor in presence and absence of vanillin, and after specified time period of incubation, its structural parameters like total protein, surface coverage, average thickness, total biomass were quantified. Biofilm formed in control experiment occupied 9.3 % of 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 (Fig.1, Fig.2a). Similarly average thickness of biofilm in control increased with days of incubation (Fig.2b). Similar trend was repeated for total biomass (Fig.2c) and total protein content (Fig.2d). Quantity of biofilm formed in media containing vanillin showed negligible biofilm development when compared to the control. On third day values of surface coverage, average thickness, total biomass and total protein quantified for biofilms grown in presence of vanillin were 93, 97, 96 and 97 % less than that of control, respectively (Fig.1, 2). Similar trend was continued until the 7th day of study. In short, from day to day, control biofilm showed significant increase in values of all the parameters studied, whereas in case of biofilm grown in the presence of vanillin the biofilm development was significantly suppressed. This shows that the presence of vanillin in the medium could limit the biofilm formation on RO membrane. With this, it is expected that in real systems, the presence of vanillin in the feed water could prolong the time period of biofilm formation and would reduce the frequency of membrane cleaning as the current practices of most common frequency of sanitization is every 3-5 days during peak biological activity (summer) and about every seven days during low biological activity (winter) (http://watertechgroup.com/files/Membranes_-_DBNPA_-_RO_Biocide.pdf.).
The planktonic cell concentration in terms of optical density at 600nm (OD600 ) and viable cell count per milliliter of the culture broth were consistent after 24h of batch mode indicating the stability of the chemostat system used. Throughout the experimental period there was no significant difference in abundance of planktonic cells among control and treatment, which indicates that vanillin in tested concentration, does not hinder the growth and proliferation of A. hydrophila. This is highly attractive as it makes vanillin unlikely to pose a selective pressure for the development of resistance, supporting the use of vanillin for sustainable control of biofouling of RO membrane
It would be advantageous if vanillin could also remove preformed biofilm on membrane apart from preventing its formation. Our results in the present work showed that vanillin had no effect on a 24h old pre-formed biofilm. Upon introduction of vanillin, it could not prevent the growing biofilm from its further development (Fig.1, 2). It could be explained based on the complexity of the biofilm structure as reported by Richards and Melander (2009). They suggested that the spider-web like nature of the biofilm EPS matrix could trap inhibitory compounds before they can elicit their effect, or it can lower the rate of penetration in to the biofilm. The ineffectiveness of vanillin on a pre-formed biofilm could therefore be explained based on the above foresaid reasons that limit its concentration to a very low level inside the preformed complex biofilm structure, rendering it ineffective as a biofilm inhibitor.
To control biofouling in RO membrane plants operating with biologically active feed water, generally two different modes of application of anti-foulant are used, slug dosing and continuous feed. In slug dosing, a particular amount of anti-foulant is used for 30 min to 3h every five days (http://watertechgroup.com/files/Membranes_-_DBNPA_-_RO_Biocide.pdf.). Vanillin in concentration tested appears to be unsuitable for slug dosing as it failed to remove preformed biofilm even with 24h treatment. Use of a higher concentration of vanillin for slug dosing cannot be recommended as it can raise selective pressure to microbes which is undesirable. Since vanillin can inhibit the biofilm formation when supplied from zero hours of incubation, its application as continuous feed looks feasible. To do so, when the system is devoid of biofilm (start of operation or just after thorough cleaning), a continuous feed maintenance program can be initiated which maintains 0.18mg/ml of vanillin which would delay or limit biofouling of 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 feed water. Its chemical stability in feed water; and rate of removal from the system has to be studied to optimize its use in field scale.
Vanillin in tested concentration was found to limit establishment of biofilm on RO membrane surface 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 to control biofilm formation on RO surface by A. hydrophila holds great promise that a whole range of bacterial biofilm growth could be controlled by the use of similar compounds, either alone or in combination. These results need to be extrapolated using multiple natural compounds either singly or in combination against various biofilm forming environmental isolates either singly or in consortium in order to open vistas for enhanced biofilm control.