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Evaluation of polymerized organo-selenium feed spacers to inhibitS. aureus and E. coli biofilm development in reverse osmosis systems
Tony Vercellino a,⁎, Phat Tran c, Ted Reid c, Abdul Hamood b, Audra Morse a
a Texas Tech University, Dept. of Civil & Environmental Engineering, USAb Texas Tech University Health Sciences Center, Dept. of Microbiology, USAc Texas Tech University Health Sciences Center, Dept. of Ophthalmology, USA
H I G H L I G H T S
• Organo-selenium is polymerized into a feed spacer polypropylene material.• Impact of organo-selenium in feed spacer was proved in synthetic wastewater stream.• Organo-selenium material reduced net effects of biofouling on RO membrane surface.• 2+ log reduction of S. aureus and E. coli biofilms on RO membrane surface
A feed spacer polymerized with organo-selenium was analyzed for biofouling inhibition to reverse osmosismembranes and feed spacers. The spacers polymerized with organo-selenium were tested for their ability toinhibit biomass formation and reduce biofilm thickness on the surface of RO membranes that the spacers werein contact with. The spacers and RO membranes were tested in a flow-cell system that exposed the samplemembranes to normal operating conditions for RO membranes over a period of 24 h. This system utilized asyntheticwastewater, selected as a highnutrient source tomodel a primarywastewater, and bacterial test strainswere chosen to represent Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria.Initial tests with spacers containing 0.55% organo-selenium in the spacer material showed inhibition levelsof 2.0 logs for total biomass, and 2.7 logs for average biofilm thickness against S. aureus only. At a spacerconcentration of 1% organo-selenium, inhibition levels of total specific biomass concentration averaged 2.9logs for each bacterial strain, and averaged a reduction of biofilm thickness of 2.9 and 3.9 logs for S. aureus andE. coli.
Membrane filtration is increasingly becoming a more commonlyused technology for water purification in situations where poor waterquality exists, and in areas that have little access to natural, near-potable water sources. Of the membrane separation techniques used,reverse osmosis (RO) is one of the most common, and is the mostselective separation process via membranes. While RO processes canperform well, the systems are prone to fouling. Fouling reduces thefunctional ability of the membrane to produce high quality water. As aresult, higher energy and cost inputs are required to meet the originaldesign demand of the RO system prior to fouling [1]. The vulnerability
ept. of Civil & Environmental1 806 742 2801x265; fax: +1
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ghts reserved.
of a membrane system to fouling is dependent on the makeup of theraw water source [2].
Biofouling has been identified as a primary culprit in the loss offunctionality in membrane filtration systems, and presents a uniqueset of challenges when attempting to address the issue. Biologicalfouling is caused by the formation of unwanted biofilms due to thenatural deposition of bacteria on a surface. The initial deposition andadhesion of bacteria onto a surface have been identified as vital stepsin the development of biofilms [3]. As a biofilm matures its attachmentto the growth surface becomes stronger, and the biofilm is able to growin size because of the stability of its attachment.
Current research has shown that the use of organo-seleniumcompounds can act as a feasible technique to reduce and inhibit theeffects of biofilm growth on a variety of surfaces [4–6]. The success oforgano-selenium compounds is linked to its ability to catalyze theformation of superoxide radicals (O2
•−) via non-enzymatic processes[7]. The superoxide radicals are produced by the catalytic reaction oforgano-selenium compounds with free oxygen and thiol compounds
2 T. Vercellino et al. / Desalination 331 (2013) 1–5
within the feed solution. Once the superoxide radicals approach abacterial cellmembrane, they can be converted to hydroperoxyl radicals(HO2
• ). The hydroperoxyl radicals can pass into the cell membrane andreact with unsaturated fatty acids, which ultimately leads to cell deathvia a chain reaction. However, while the superoxide radicals areindirectly responsible for cell death, the superoxide radical itself is notstrong enough to cause damage to the RO membrane/feed spacersurface.
Recent studies into membrane biofouling have shown that the feedspacer plays a dominant role in the formation of biofouling within spiralwound systems [8]. Current research by the author has established thatcoating the surface of a RO membrane and feed spacer with organo-selenium is effective at inhibiting biofilm formation [9,10]. Therefore,we proposed that modifying the polymer of the feed spacer withorgano-selenium rather than modifying the membrane surface wouldinhibit the formation of and reduce the negative side effects associatedwith biofilm formationon themembrane surface in a comparablemannertowhen the surfaceswere coated. This design changewould allow for thenegation of the flux loss problems associated with membrane surfacecoatings,while stillmaintaining the benefit of biofouling inhibitionwithinthe filtration system. The concern with this arrangement is that thesuperoxide radicals will lose effectiveness in the areas that are not indirect contact with the membrane surface, and biofouling will occur dueto insufficient organo-selenium concentrations in the polymerized spacerbecause of poor quality control in the manufacturing process.
The purpose of this paper is to present the results of work to evaluatethe effectiveness of feed spacers polymerized with organo-seleniumto inhibit the effects of biofouling in RO membrane systems. Thepolymerized feed spacer material was tested for effectiveness ininhibiting the growth of the Grampositive bacteria Staphylococcus aureusand the Gram negative bacteria Escherichia coli. The experimental setupfor this research utilizes a bench-scale SEPA CF II flow-cell unit thatexposes ROmembranes and feed spacers to normal operating conditionsand only studies the effect of the organo-selenium surface treatment in aprimary wastewater ersatz.
2. Experimental section
2.1. Experiment supplies
2.1.1. RO membranes and control feed spacersAll ROmembranes and control spacers used in the experiments were
purchased fromGEOsmonics (Minnetonka, MN). The SEPA CF II flow cellhas the ability to accommodate any 19cm×14cm flat sheet membrane.All RO membranes were shipped as individually packaged flat sheets.
2.2. Organo-selenium polymerization in feed spacers
The feed spacers were prepared by the thermal grafting of Seldox™,an organo-seleniummethacrylate compound, into a commercial grade,engineered polypropylene. The preliminary experiments that evaluateda concentration difference of organo-selenium at 0.55% and 1%, utilizedfeed spacers that were made in separate locations. The 0.55% spacerswere made in-house at Texas Tech University, while the 1% spacerswere manufactured at DelStar Technologies Inc. The primary focus ofthis research was on the 1% spacers manufactured through DelStarTechnologies Inc. In each case, the organo-selenium was supplied viaSelenium Ltd. for the manufacturing process. It should be noted thatthe target concentration was 0.5% for the set of spacers that ended upbeing 0.55%, but due to the cost of production, the 0.55% spacers wereused for the experiments rather than sourcing new spacers.
2.3. Synthetic wastewater feed preparation
A wastewater ersatz formulation was chosen as the feed solution tomodel a nutrient-richwastewater thatwould support biofilm formation
in the presence of microorganisms and to represent a primarywastewater influent without suspended solids. The ersatz recipe isbased on a formulation used by NASA to characterize wastewaterstreams for space-based scenarios [11]. The specific ersatz formulationrepresents a source separated waste stream made up of componentsto represent urine waste and hygiene waste.
2.4. Biofouling experiments
2.4.1. Bacterial speciesFor thefirst set of bacterial experiments, S. aureus strain AH1333was
used to test for biofouling inhibition. The primary goal of the first set ofexperimentswas to determinewhether varying the loading of seleniumin the spacer polymer resulted in higher levels of biofilm inhibition. Thesecond stage of experiments expanded to use S. aureus and E. coli strainMM294 for evaluation of biofouling inhibition. These bacteria wereselected to act as models to represent Gramnegative and Grampositivetype organisms and to assess how the organo-selenium reaction affectsthese types of organisms. The strains of S. aureus and E. coli used in theexperiments contained a GFP plasmid for fluorescent analysis. Culturesused in the experiments were grown from frozen stocks in LB brothovernight at 37 °C and supplemented with antibiotics (erythromycinfor S. aureus and carbenicillin for E. coli) prior to being inserted intothe system to hold the GFP in expression over the test periods.
2.4.2. Biofilm development on RO membranesPrior to starting the experiments, the RO system and flow cell were
thoroughly sanitized using a multi-step process to ensure that thebiofilms were growing only from the inputs from each individual test.The sterilization process included separate wash steps using sodiumhypochlorite, EDTA, SDS, and ethanol to remove any trace contaminantsremaining from prior experiments.
RO membranes used in the RO flow cell system were removed fromthe individual packaging immediately prior to use to minimize handling.Once the membrane was sterilized via UV exposure, it was thencompressed for approximately 24h at 300psi in nano-pure water withinthe system. Following the compression period, the pressure was loweredto 180psi, and the systemwas operated until the membrane maintaineda constant flux over a one hour period. The compression step ensuredthat the membrane was properly seated within the flow cell andthat there were no leaks from the filtration area. Once the compressionstep was complete, the system was operated with the syntheticwastewater media at 180 psi until the system reached a constant flux.The compression step allows for the formation of a gel layer, whichreduces the permeate flux of the system and also ensures that anyobserved permeate flux, is only due to the formation of biological foulingonce the microorganisms are injected into the system. The systemequalization in wastewater was typically allowed to run for 24 h toreach its equilibrium point. After the membrane had been properly setwithin the flow cell and reached equilibrium with the wastewatermedia, an overnight culture of the test bacteria was introduced into thesystem. Following the introduction of bacteria, the system was operatedfor another 24 h following an initial attachment period for the bacteria.During the 24hour test period, temperature measurements were closelymonitored to prevent the system's water temperature from exceeding40 °C. This monitoring ensured that the water temperature did notbecome detrimental to the growth of microorganisms. At the end of the24 hour experimental run, the RO membrane was removed from theflow-cell and sterilely separated into four sections for analysis. Eachsection of RO membrane was rinsed in a phosphate buffer solution toremove unattached biological materials and then mounted to a cultureplate for analysis. The feed spacer material was not analyzed in theseexperiments, because the focus of this work was to assess the effect oforgano-selenium polymerized spacers on inhibiting the formation ofbiofouling on the surface of RO membranes.
0.55% 1%
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ass
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3 /µm
2 )
10-6
10-5
10-4
10-3
10-2
10-1
10 0
10 1
10 2
10 3
Untreated Treated
A B
Fig. 2. Total specific S. aureusbiomass concentration (μm3biomass/μm2membrane surfacearea) on RO membrane surface due to biofouling.
A) 0.55% Organo-selenium concentration in feed spacer material.B) 1.0% Organo-selenium concentration in feed spacer material.
3T. Vercellino et al. / Desalination 331 (2013) 1–5
2.4.3. Microscopy and image analysisROmembrane sampleswere analyzedwith a confocal laser scanning
microscope (CLSM) within 15min of being removed from the RO flow-cell body to ensure minimal cell loss and maximum signal emissionfrom the GFP in the biofilm. Samples were analyzed using an OlympusIX 71 upright CLSM, and Fluoview software (Olympus America). Typicalimages produced by the CLSM can be found in Fig. 1. The data filesgenerated for each membrane sample analyzed by the CLSM wereexpanded into individual data files for every 2 μm of biofilm thicknesswithMetaMorph software (Molecular Devices). The datafiles generatedby MetaMorph were analyzed using the COMSTAT module in MatLab(MathWorks). The COMSTAT module produced values for the averagetotal specific biomass concentration (μm3/μm2) and average biofilmthickness (μm) for each membrane sample for reporting.
3. Results and discussion
3.1. Evaluation of varying organo-selenium concentrations on biofoulinginhibition
3.1.1. Inhibition of S. aureus biofilm at 0.55% organo-selenium concentrationS. aureus biofilms had an average total biomass concentration
(μm3/μm2) of 2.36×100±4.33×100 (n=12) in control experiments.When the 0.55% organo-selenium spacer was ran in place of anuntreated feed spacer, the biofilm formed by S. aureus was reduced to2.09 × 10−2 ± 1.97 × 10−2 (n= 12); yielding a 2.0 log reduction. Thesame experiments produced an average biofilm thickness (μm) of8.68 × 100± 1.35 × 101 (n= 12) in control samples and was reducedto 2.43 × 10−2± 2.02× 10−2 (n=12) in experiments with the 0.55%organo-selenium polymerized feed spacer; corresponding to a 2.7 logreduction in average biofilm thickness.
3.1.2. Inhibition of S. aureus biofilm at 1.0% organo-selenium concentrationControl experimentswere run separately for the experimentswhere
feed spacers were polymerized with a 1.0% concentration of organo-selenium because a different batch of plastic was used for extrusion.These control experiments yielded S. aureus biofilms with an averagetotal biomass concentration (μm3/μm2) of 7.55 × 101 ± 3.46 × 101
(n = 8), and an average biofilm thickness (μm) of 1.56 × 102 ±5.80×101 (n=8).When the 1.0% polymerized organo-selenium spacerwas introduced, S. aureus biofilms yielded a total average biomassconcentration (μm3/μm2) of 3.72×10−2±3.35×10−2 (n=8), and anaverage biofilm thickness (μm) of 6.06 × 10−2 ± 5.94 × 10−2 (n= 8);corresponding to a 3.6 log reduction in total average biomassconcentration and a 3.7 log reduction in average biofilm thickness.
Fig. 1. CLSM images displaying biofilms (in green) after the completion of a 24hour biofouling espacer (Left), Membrane surface when coupled with an organo-selenium polymerized spacer
3.1.3. Summary of preliminary experimentsBased on the preliminary results from evaluating the differences
between the 0.55% and 1.0% organo-seleniumpolymerized feed spacers,outlined in Figs. 2 and 3, we decided to further evaluate the potentialof the 1.0% concentration in a larger sample set as well as testingits potential against E. coli biofilms. The results of the tests usingthe 1.0% concentration showed a 1.6 log higher inhibition of totalbiomass concentration and a 1.0 log higher reduction in averagebiofilm thickness. Table 1 summarizes the results of the preliminaryexperiments.
3.2. Evaluation of 1% polymerized organo-selenium feed spacers onS. aureus and E. coli biofilm formations
3.2.1. Inhibition of S. aureus biofilm formationFor phase two testing of the 1.0% polymerized organo-selenium
spacers, new control samples were run for comparison to test samples.The control samples produced a biofilm with an average total biomassconcentration (μm3/μm2) of 1.73 × 101 ± 1.67 × 101 (n= 18), and anaverage biofilm thickness (μm) of 5.09 × 101 ± 5.48 × 101 (n = 18).When the 1.0% feed spacers were incorporated in place of thecontrol spacers, S. aureus biofilms were reduced to an average totalbiomass concentration (μm3/μm2) of 7.08 × 10−2 ± 6.71 × 10−2
xperiment using S. aureus. Pictures show:Membrane surfacewhen coupledwith a control(Right).
Fig. 3.Average S. aureus biofilm thickness (μm2) on ROmembrane surface due to biofouling,for different organo-selenium concentrations (as %) within the feed spacer polymer.
A) 0.55% Organo-selenium concentration in feed spacer material.B) 1.0% Organo-selenium concentration in feed spacer material.
Table 1Summary of results from preliminary experiments with varying spacer seleniumconcentrations.
Control spacer Polymerized spacer
Avg SD Avg SD
Average total specific biomass concentration0.55% 2.36E+00 4.33E+00 2.09E-02 1.97E-021.00% 7.55E+01 3.46E+01 3.72E-02 3.35E-02
Fig. 5. Average biofilm thickness (μm2) on RO membrane surface due to biofouling forS. aureus and E. coli. Untreated samples contain no organo-selenium; treated samplescontain 1% organo-selenium within the feed spacer polymer.
4 T. Vercellino et al. / Desalination 331 (2013) 1–5
(n = 14) as shown in Fig. 4, and an average biofilm thickness (μm)of 1.43 × 10−1 ± 2.06 × 10−1 (n = 14), as shown in Fig. 5. Overall,these results produce a net inhibition of 2.9 logs for both average totalbiomass concentration and average biofilm thickness.
3.2.2. Inhibition of E. coli biofilm formationTo quantify the effect of the treated feed spacers on a Gram negative
bacterium, tests were performed on E. coli biofilms. Control tests withE. coli yielded biofilms with an average total biomass concentration(μm3/μm2) of 2.35×101±2.00×101 (n=10), and an average thickness
S. aureus AH1333 E. coli MM29410-6
10-5
10-4
10-3
10-2
10-1
100
101
102
103
Untreated Treated
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al B
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ass
(µm
3 /µm
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Fig. 4. Total specific biomass concentration (μm3biomass/μm2membrane) onROmembranesurface due to biofouling for S. aureus and E. coli. Untreated samples contain no organo-selenium, treated samples contain 1% organo-selenium within the feed spacer polymer.
(μm) of 6.36×101±7.09×101 (n=10). In experiments where the 1.0%polymerized organo-selenium spacer was included, E. coli biofilms onthe RO membrane surface measured an average total biomassconcentration (μm3/μm2) of 4.71 × 10−2 ± 3.92 × 10−2 (n= 12), andan average thickness (μm) of 1.10 × 10−2 ± 2.54 × 10−2 (n = 10).Overall, the average total biomass concentration was inhibited by 2.9logs, and the average biofilm thickness was reduced by 3.9 logs. Thedata for total biomass reduction and inhibition of average biofilmthickness are detailed in Figs. 4 and 5, respectively.
4. Conclusions
Membrane purification of water is becoming a more prominent go-to technology for desalination in commercial, municipal, and industrialapplications. Given that membrane technologies will only keep gainingin popularity, the drawbacks of membrane treatment will need to beaddressed at an equal or greater rate. The formation of biological foulingwithin membrane filtration systems is currently one of the leadingdisadvantages to utilizing the technology.
Previous experiments by our research team have demonstrated thatorgano-selenium compounds are an effective anti-biological controltechnology [5–7], and can be successfully utilized within filtrationsystems, and produce encouraging results to further evaluate the effectof applying the organo-selenium compounds to the membrane feedspacers alone [4,9,10]. The experiments within this paper showed thatthe total average biomass concentration (μm3/μm2) was reduced byapproximately 2.9 logs for both S. aureus and E. coli biofilms, and theaverage biofilm thickness (μm) was reduced by 2.9 logs for S. aureusand 3.9 logs for E. coli. Previous experiments [9] showed that whenthe membrane feed spacers were coated with the organo-seleniummaterial, total biomass concentration was inhibited by 2.0 logs andthe average biofilm thickness was reduced by 3.3 logs in S. aureusbiofilms tested within the same wastewater ersatz solution.
While the results between surface coating and polymerization oforgano-selenium appear comparable, the polymerization process has afew distinct advantages. The primary advantage is that organo-seleniumis fully incorporated within the spacer material, which alleviates anyconcern that a surface coating may wear off over time or that damageto the spacer surface will expose an area of untreated spacer material.In addition, the incorporation of organo-selenium into the spacermaterialallows for a higher degree of control over the amount of organo-seleniumcompound present within the spacer material. Past experiments haveshown that the organo-selenium compounds contained within a coatingsolution may not attach, and the amount that attaches can vary greatly.
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However, while the incorporation of organo-selenium into the feedspacer material showed promising advantages, inconsistencies inmanufacturing showed that the effectiveness of the spacer materialwas directly tied to how well the organo-selenium compound wasmixed in the polymer batch material prior to being extruded intothe spacer material. A high degree of quality control will be requiredin the manufacturing process to ensure that there is a consistent andhomogenous dispersion of organo-selenium in the feed spacer materialprior to production. In lieu of this drawback, the incorporation oforgano-selenium into the polymer of feed spacers proved effective inreducing the formation of biofilms within an RO filtration system overshort periods of time in a primarywastewater ersatz solution. The furtherdevelopment of this technology could prove to be beneficial to the watertreatment industry, and further testing of this technology over longertime periods in different wastewater types is the next logical step foradvancement.
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