Vanillin, a potential agent to prevent biofouling Essay

Rearward osmosis ( RO ) membrane systems are widely used in H2O purification workss. Decrease in works public presentation due to biofilm formation over the membrane is an built-in job. As quorum detection ( QS ) mechanisms of micro-organisms have been reported to be involved in the formation of biofilm, ways are sought for quorum extinction ( QQ ) and thereby bar of biofilm formation. In this survey utilizing a chemostat civilization, run for seven yearss in a CDC reactor, we found that a natural QQ compound, vanillin well suppressed bacterial biofilm formation on RO membrane. There was 97 % decrease in biofilm surface coverage, when grown in presence of vanillin. Similarly, parametric quantities of mean thickness, entire biomass and entire protein content of biofilm that formed in presence of vanillin were significantly lesser than that of the control. However vanillin had no consequence on one twenty-four hours old pre-formed biofilm.

Keywords: Aeromonas hydrophila ; Biofouling ; Reverse osmosis membrane ; Vanillin ; COMSTAT


Deficit of pure H2O for both domestic and industrial intent is increasing twenty-four hours by twenty-four hours. Constitution of big scale contrary osmosis ( RO ) workss which can be fed with river or sea H2O is an option to run into this demand. One of the chief jobs with RO based H2O purification industry is fouling of membrane ; even a little sum of fouling can do important loss of permeate flux ( Pasmore et Al, 2001 ) . Several types of fouling can happen in the membrane system, e.g. inorganic, organic, particulate, colloidal and biofouling ( Kramer and Tracey et Al, 1995 ) . Pretreated feed H2O can be supplied to RO-system to cut down fouling, even so biofouling of membrane is hard to command, as some micro-organism can last the pretreatment and quickly turn to organize biofilm. Therefore, biofouling is recognized as the most hard to command ( Baker and Dudley, 1998 ) . Other than pretreatment of provender H2O, alteration of membrane surface belongingss, optimisation of faculty agreement, procedure conditions, and periodic cleansing ( Sheikholeslami, 1999 ) have been tried to cut down the badness of the job, even after long periods of such development in the field ; biofouling still remains as a chief ground for the diminution in works public presentation ( Saad, 1992 ; Saeed et Al, 2000 ) .

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Biocides have been used to command the biofouling of the RO membranes. Chlorine is the most normally used biocide, but is known to deteriorate membranes ( Kim et al, 2009 ) . Chlorine based biocides have besides been reported to escalate biofouling, as micro-organisms subjected to low degrees of biocides frequently exude big sums of extracellular polyoses ( EPS ) for protection. This EPS supports biofilm formation ( Baker and Dudley, 1998 ) . Excessive usage of biocides at high concentrations is likely to take to environmental, ecological, and toxicological jobs if the discharge incorporating biocides flow in to natural H2O organic structures ( Elvers et al, 2002 ) . XXXX There arises Here comes the importance of developing new and sustainable schemes for commanding biofouling of RO membrane.

Several bacteriums have been reported to organize a community behaviour and thereby biofilm formation through cell to cell communicating or quorum detection mediated by, little, diffusible signals ( Hentzer and Givskov, 2003 ) . It besides suggests that, research into quorum feeling suppression will supply some agencies for commanding the growing of biofilm without the usage of growth-inhibitory agents that inescapably select for immune beings ( 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 Allium sativum ( Bjarnsholt et al, 2005 ) and cranberries ( Yamanaka et al, 2007 ) have all shown assorted grades of antibiofilm belongingss against a figure of micro-organisms in assorted surveies. Furanones isolated from marine ruddy algae Delisa pulchera, are one of most extensively studied categories of natural compounds in respects to their function in suppressing biofilm. We late reported the function of 2 ( 5-H ) Furanone in stamp downing biofilm formation by environmental strains of bacteriums isolated from fouled RO membrane ( Ponnusamy et al, 2010 ) . Previous work in our research lab showed that, vanillin ( 4-Hydroxy-3-methoxy benzaldehyde ) at concentrations runing from 0.063 to 0.25mg/ml could cut down the biofilm formation by A. hydrophila on polystyrene surface without suppressing the growing of planktonic cells ( Ponnusamy et al, 2009 ) . In this survey, even 0.18 mg/ml vanillin reduced the biofilm formation by 46 % which is about 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 ) ; connoting that it has besides something to make with the construction of signal molecules ( Ponnusamy et al, 2009 ) .Vanillin, being an inhibitor of both quorum detection ( Choo et al, 2006 ; Ponnusamy et Al, 2009 ) and biofilm formation below its minimal repressive concentration for the bacterial strains studied, there is no opportunity of opposition against it. Sing all these factors together with its atoxic nature for human application, vanillin is a promising campaigner to be tested for its efficaciousness in commanding biofouling of RO membrane. In this survey commercially available vanillin ( V-2375, Sigma-Aldrich, St Louis, USA ) was tested for its possible as an agent to command biofouling of RO-membrane utilizing a uninterrupted civilization 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 H2O purification works in Deasan, Chungbuk, Korea ) was used for the survey. The isolate was stored on LB Agar angles at 4 EsC for short term and in 20 % glycerin at E‰70 EsC for long term saving. Full strength LB media was used to resuscitate the civilization and 1/15th strength of LB ( LB/15 ) was used for turning biofilm ; pH of all media were set to seven. Culture media was purchased from Difco ( Franklin Lakes, NJ, USA ) .

RO membrane

FILMTECTM SW30HR-380, high rejection, polyamide thin movie composite saltwater contrary osmosis membrane manufactured ( Dow Chemical Company, Midland, MI, USA ) was used for this survey.

Experimental design

Consequence of vanillin on bar of biofilm formation by environmental strain A. hydrophila was studied utilizing chemostat civilization in the CDC reactor. The experimental variables were selected to overstate the biofilm organizing features, which includes the low shear emphasis, and high food concentration compared to natural Waterss. The reactor, media storage armored combat vehicle, armored combat vehicle for exhausted media and tubing were autoclaved and connected aseptically. RO membrane sections ( 1.5 centimeter – 1.5 centimeter ) were UV sterilized before usage. Vanillin stock solution ( 8 mg/mL ) was prepared in dual distilled H2O, and filter sterilized. The least effectual concentration of vanillin ( 0.18 mg/mL ) was used in the present experiments, as decided based on our old work in the country ( Ponnusamy et al, 2009 ) .

Chemostat vaccination and biofilm formation

To each of the eight removable rods in a CDC reactor, a unfertile glass slide is fixed in such a manner that it faces the baffle when placed into the reactor. Two pieces of RO- membrane were fixed ( feed side confronting out ) on to each glass slide by utilizing a unfertile dual side cellophane tape.

An nightlong growing of a individual settlement of A. hydrophila from an LB agar home base was used to inoculate a 35 milliliter LB/15 media in a conelike flask ( 100mL ) and shaken overnight at 25 EsC to fix the starter civilization. This civilization was so used to inoculate 315 mLof LB/15 in the CDC reactor ( 1L ) to originate the biofilm formation. The reactor was run with 350 milliliters of LB/15 in batch manner for one twenty-four hours and so fresh media was continuously supplied with a flow rate of 15 mL/h. Volume of the civilization media in reactor was maintained to 350 milliliter. Filter sterilized atmospheric air was supplied at a rate of 2000 mL/min, velocity of baffle rotary motion was set to 125 revolutions per minute. The temperature was maintained at 25EsC throughout. Another reactor was run in analogue with similar conditions except that the medium contained 0.18mg/ml vanillin. After each specific incubation periods of 1,2,3,4 and 7 yearss ( As separate experiments ) , membrane vouchers were removed and biofilm quantified by entire protein appraisal ( n= 8 ) and analysis of images ( n= 8 ) taken by utilizing confocal optical maser scanning microscope ( CLSM ) .

Another experiment was conducted to analyze the consequence of vanillin on preformed biofilm. Biofilm was grown on membrane vouchers as described above for specific clip periods, and was exposed to vanillin by adding appropriate measure of vanillin stock to the reactor to do the concluding concentration to 0.18 mg/mL. This was followed by supply of fresh LB/15 incorporating same concentration of vanillin at a flow rate of 15mL/h for a period of 24h and so biofilm-samples were analyzed as described above. In this instance sample size for biofilm quantification was four and the experiments were repeated three times.

Sampling and informations aggregation

At specified clip periods ( as mentioned above ) ; rods keeping membrane vouchers with biofilm were removed and rinsed three times by dunking in 0.2 M phosphate-buffered saline ( PBS ; pH-7 ) . Out of the two membrane vouchers fixed on each glass slide, one is removed utilizing unfertile forceps and maintain in a conelike tubing incorporating five millilitre of 0.2 M PBS and processed for protein appraisal. Then the full glass slide with the other voucher was immersed into a Petri-dish incorporating 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 arrested development of biofilm ; it is so 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 imagination.

CLSM observation and image analysis

Stained biofilm samples were observed utilizing an Olympus Fluoview FV1000 Confocal Microscope ( Olympus, Tokyo, Japan ) , and image tonss ( seven per sample, at random location ) were saved. Two dimensional images of biofilm were generated utilizing the package FV10-ASW version 2 ( Olympus, Tokyo, Japan ) , a platform associated with the confocal microscope. To find the per centum of surface coverage, mean thickness and total-biomass of each sample, image tonss obtained were analyzed utilizing the image analysis package, COMSTAT ( Heydorn et al, 2000 ) .

Quantification of entire protein

The tubings incorporating membrane samples with biofilm were vortexed for one minute and sonicated at 40 kilohertz for five proceedingss to convey the biofilm in to suspension. Then the membrane pieces were taken out and the samples centrifuged at 5000 revolutions per minute for 10 min to pellet the bacterial cells. Supernatant were decanted and entire protein was extracted ( B-PER II Bacterial Protein Extraction Reagent-78260, Thermo Scientific, Rockford, IL, USA ) and were quantified by utilizing protein assay kit ( Bio-Rad Laboratories, Hercules, Ca, USA ) .

Consequences and treatment

Experiments conducted in the present survey reinstated the potency of vanillin as an effectual quorum slaking agent. In fact there are really few known natural compounds like vanillin that could suppress biofilm formation while non impacting cell growing and it adds to the suitableness of the molecule to be used for sustainable and eco-friendly control of biofouling. Brominated furanones and their derived functions have been reported to suppress biofilm formation by several bacterial species ( Janssens et al, 2008 ) . Unfortunately, usage of brominated compounds is non suited for the H2O purification industry due to their possible toxicity. Previous surveies reported that, a higher concentration of 1mg/mL non-halogenated, commercially available 2 ( 5H ) -Furanone could significantly cut down A. hydrophila biofilm formation on polystyrene home bases ( Ponnusamy et al, 2010 ) . However non-brominated furanone did non suppress biofilm formation by other bacteriums proposing the usage of brominated furanones for broader application ( Janssens et al, 2008 ) . Vanillin is a good known nutrient seasoning agent and is cheaper and safe to utilize when compared to furanone.

In the present probe biofilms were grown in CDC reactor in presence and absence of vanillin, and after specified clip period of incubation, its structural parametric quantities like entire protein, surface coverage, mean thickness, entire biomass were quantified. Biofilm formed in control experiment occupied 9.3 % of membrane surface by twenty-four hours one, 22.4 % by twenty-four hours two, 44.5 % by twenty-four hours three, and 71.7 % by twenty-four hours four and 90.9 % by 7th twenty-four hours ( Fig.1, Fig.2a ) . Similarly mean thickness of biofilm in control increased with yearss of incubation ( Fig.2b ) . Similar tendency was repeated for entire biomass ( Fig.2c ) and entire protein content ( Fig.2d ) . Measure of biofilm formed in media incorporating vanillin showed negligible biofilm development when compared to the control. On 3rd twenty-four hours values of surface coverage, mean thickness, entire biomass and entire protein quantified for biofilms grown in presence of vanillin were 93, 97, 96 and 97 % less than that of control, severally ( Fig.1, 2 ) . Similar tendency was continued until the seventh twenty-four hours of survey. In short, from twenty-four hours to twenty-four hours, control biofilm showed important addition in values of all the parametric quantities studied, whereas in instance 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 restrict the biofilm formation on RO membrane. With this, it is expected that in existent systems, the presence of vanillin in the provender H2O could protract the clip period of biofilm formation and would cut down the frequence of membrane cleansing as the current patterns of most common frequence of sanitation is every 3-5 yearss during peak biological activity ( summer ) and about every seven yearss during low biological activity ( winter ) ( hypertext transfer protocol: // ) .

The planktonic cell concentration in footings of optical denseness at 600nm ( OD600 ) and feasible cell count per millilitre of the civilization stock were consistent after 24h of batch manner bespeaking the stableness of the chemostat system used. Throughout the experimental period there was no important difference in copiousness of planktonic cells among control and intervention, which indicates that vanillin in tried concentration, does non impede the growing and proliferation of A. hydrophila. This is extremely attractive as it makes vanillin improbable to present a selective force per unit area for the development of opposition, back uping the usage of vanillin for sustainable control of biofouling of RO membrane

It would be advantageous if vanillin could besides take preformed biofilm on membrane apart from forestalling its formation. Our consequences in the present work showed that vanillin had no consequence on a 24h old pre-formed biofilm. Upon debut of vanillin, it could non forestall the turning biofilm from its farther development ( Fig.1, 2 ) . It could be explained based on the complexness of the biofilm construction as reported by Richards and Melander ( 2009 ) . They suggested that the spider-web like nature of the biofilm EPS matrix could pin down repressive compounds before they can arouse their consequence, or it can take down the rate of incursion in to the biofilm. The ineffectualness of vanillin on a pre-formed biofilm could hence be explained based on the above foresaid grounds that limit its concentration to a really low degree inside the preformed complex biofilm construction, rendering it uneffective as a biofilm inhibitor.

To command biofouling in RO membrane workss runing with biologically active provender H2O, by and large two different manners of application of anti-foulant are used, slug dosing and uninterrupted provender. In bullet dosing, a peculiar sum of anti-foulant is used for 30 min to 3h every five yearss ( hypertext transfer protocol: // ) . Vanillin in concentration tested appears to be unsuitable for bullet dosing as it failed to take preformed biofilm even with 24h intervention. Use of a higher concentration of vanillin for bullet dosing can non be recommended as it can raise selective force per unit area to microbes which is unwanted. Since vanillin can suppress the biofilm formation when supplied from zero hours of incubation, its application as uninterrupted provender looks executable. To make so, when the system is barren of biofilm ( start of operation or merely after thorough cleansing ) , a uninterrupted provender care plan can be initiated which maintains 0.18mg/ml of vanillin which would detain or restrict biofouling of RO membrane. Developing a slow release system utilizing immobilisation techniques ( which are afoot utilizing C nanotubes ) can minimise the measure of vanillin needed to keep an appropriate concentration in feed H2O. Its chemical stableness in feed H2O ; and rate of remotion from the system has to be studied to optimise its usage in field graduated table.


Vanillin in tried concentration was found to restrict constitution of biofilm on RO membrane surface without raising selective force per unit area for the growing of micro-organism. Since vanillin is both of course and commercially available it may be used in RO workss for sustainable control of biofouling. Its possible to command biofilm formation on RO surface by A. hydrophila holds great promise that a whole scope of bacterial biofilm growing could be controlled by the usage of similar compounds, either entirely or in combination. These consequences need to be extrapolated utilizing multiple natural compounds either singly or in combination against assorted biofilm organizing environmental isolates either singly or in pool in order to open views for enhanced biofilm control.


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