Zosteric acid--an effective antifoulant for reducing fresh water bacterial attachment on coatings.
Keywords: Antifoulants, atomic force microscopy, silicones, silicates, biofouling/antifouling, zostera marina, natural product, eelgrass, biofilm
The attachment and growth of living organisms on surfaces exposed to an aqueous environment, defined as fouling, has always posed a serious problem. The majority of fouling begins with the adsorption of organic matter, such as macromolecules and protein fragments, onto a surface. (1,2) This is followed by the attachment of a complex community of bacteria, diatoms, protozoa, and algae spores to form biofilms. (3-5) The final stage encompasses the attachment of higher ordered organisms, such as barnacles, algae, tubeworms, mollusks, and sponges. (5-8) Fouling not only leads to increased fuel and maintenance costs, damage of ship-hulls and platforms, but also results in harmful contamination of drinking water systems and corrosion of mechanical equipment. (5) In order to minimize fouling, substances that can prevent the attachment and subsequent growth of organisms on solid surfaces have been widely utilized.
Many early antifouling (AF) substances were biocides made of organo-mercury, lead, and dichloro-diphenyl-trichloroethane (DDT). These early AFs posed severe environmental and human health risks, and were withdrawn voluntarily by the paint industry. (9) Antifouling paints containing tin (e.g., tributyltin, TBT), copper, zinc, cadmium, and chromium have been restricted from use due to serious environmental problems posed at even subparts per billion concentrations. (6,10-12) There is an urgent need to ascertain suitable non- or less toxic alternatives, such as foul-released coatings (13-18) or coatings containing nontoxic or less toxic compounds, such as natural product antifoulants (NPAs). (5,12,19,20)
Zosteric acid, a natural compound present in zostera marina, or eelgrass, has been found to prevent the attachment of some bacteria, algae, barnacles, and tubeworms at nontoxic concentrations. (21-24) The formula structure of zosteric acid or p- (sulfo-oxy) cinnamic acid is shown in Figure 1. The antifouling capability of zosteric acid was attributed to the sulfate ester group presented in the compound. (23,25-27) The AF effectiveness of zosteric acid has been demonstrated both in preliminary static laboratory assays (23) and with field tests. (21,22) In laboratory assays, glass slides coated with zosteric acid were used. As compared to slides without zosteric acid, the attachment of Acinetobacter sp. dropped from 90% to 30% when the concentration of zosteric acid on the slide increased from 1 [micro]g/[cm.sup.2] to 200 [micro]g/[cm.sup.2]. (23) In one field study, ceramic tiles were coated with crude zosteric acid and then placed into a marine environment for one week. No attachment of barnacles was found. In another field study, zosteric acid was simply blended into a silicone foul release coating, and then applied to panels. The panels were immersed in marine water for 60 days, with no hard fouling and much less slime fouling being observed, as compared to panels without zosteric acid. Although effective, these tests did not investigate the impact of zosteric acid on the preliminary stages of biofilm formation.
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In this study, the AF effectiveness of zosteric acid against fresh water bacterial attachment was evaluated. In particular, plain silicone coatings were first subjected to bacterial attachment with one of two fresh water bacteria (Lake Erie and P. putida) with and without zosteric acid dissolved in water. The zosteric acid concentration was varied from 5 mg/L to 500 mg/L to obtain the optimum concentration that could reduce 90% of bacterial attachment for the two fresh water bacteria. Then, zosteric acid was entrapped into silicone coatings by using a common solvent for both zosteric acid and silicone. The resulting coatings would attain the foul-release properties of silicone while controlling the release of entrapped zosteric acid to enhance the antifouling capability of the coating. The coatings were subjected to bacterial attachment studies to evaluate if such coatings would be effective in deterring bacterial attachment and the subsequent biofilm formation.
Materials and Equipment
Experiments were performed with two types of silicone: Sylgard[R] 184, an elastomer kit manufactured by Dow Corning, and RTV11, produced by GE. Sylgard 184 consists of a base elastomer (part A) and a curing agent of methyl hydrosiloxanes and a platinum catalyst (part B). RTV11 was also supplied as two parts, a base compound containing polydimethylsiloxane, calcium carbonate, and ethyl silicate (part C) and a tin-based catalyst (dibutyl tin dilaurate, part D). Microscope glass slides purchased from VWR Scientific were used as substrates. Zosteric acid (p-(sulfo-oxy) cinnamic acid, ~95% zosteric acid and its sodium salt, ~5% impurities consisting of residual sodium chloride, diester of zosteric acid/coumaric acid and diester of zosteric acid) was synthesized in our own laboratory from p-coumaric acid (98% pure) and chlorosulphonic acid (99% pure). P-coumaric and chlorosulphonic acid, along with certified ACS graded pyridine, were purchased from Sigma-Aldrich and used as received.
Two fresh water bacteria cultures were employed in the study. The first was an enriched microbial consortium isolated from Lake Erie, the specification of the bacteria was not critically defined (i.e., the specific microbes present were not identified by DNA analysis). Staining procedures were used to semiquantify the different populations present in terms of size, shape, and color. In addition to other bacteria present, two of the dominant species tended to be Pseudomonas sp. and Pseudomonas fluorescens. The second, Pseudomonas putida (from American Type Culture Collection, #12633), was used as the model fresh water bacteria. Both cultures were maintained as described elsewhere. (24,28)
A contact angle goniometer (Model 100-00 from Rame-Hart, Inc.), optical microscopes (IX 70, Olympus and Infini Tube, Edmund Scientific), and an atomic force microscope (Metrology 2000, Molecular Imaging) were used for characterization of the coatings. They were all equipped with CCD video cameras for spontaneously capturing the images of interest. A digital conductivity meter (Traceable[R]) and a UV-visible spectrophotometer (UV-1601, Shimadzu) were used for monitoring the amount of zosteric acid leached from its entrapped coating during the bacterial attachment studies.
Microscope glass slides of 7.5 cm x 2.5 cm were cut into 7.5 cm x 1.25 cm pieces. Each was treated with a stream of industrial grade nitrogen to remove dust particles, and then coated with a particular type of silicone mixture. For plain Sylgard 184 silicone, the mixture contained 10:1 by mass of A:B; whereas for plain RTV11, the mixture consisted of 99.5:0.5, by mass, of C:D. One drop (~0.05 g) of the mixture, after rigorous mixing to ensure the two parts had been uniformly mixed, was spread on the slides' surface using a doctor blade to form a coating with a thickness of ~200 [micro]m over an area of 2 cm x 1.25 cm. The mixture was allowed to flow and rearrange within this coated area while it was cured inside closed drawers (15 cm x 15 cm x 5 cm) under ambient conditions (20[degrees]C and 1 atm) for 48 hr. After curing, they were sterilized in an autoclave at ~120[degrees]C for 60 min prior to initiating the attachment study. The autoclaved coatings were also subjected to surface wettability characterizations and bulk modulus measurements.
The zosteric acid bulk-entrapped silicones were prepared by blending a solution of zosteric acid with the silicone mixture. The solvents used for making the zosteric acid solution included de-ionized (DI) water and pyridine. The purpose of pyridine was to increase the miscibility of the zosteric acid solution with silicone, since silicone is immiscible with water while zosteric acid is generally insoluble in organic solvents. The zosteric acid was first dissolved in water and then pyridine was added to obtain a final solution containing ~10 wt% zosteric acid. The zosteric acid solution and the base silicone elastomer were first thoroughly mixed to form a homogeneous dispersion. Then they received four repeated cycles of heating at 150[degrees]C for one hour with stirring (using a glass rod) followed with vacuuming at ~50 mm Hg for 10 min to totally remove the solvent. After removing the solvent, the mixture was cooled to room temperature and then the curing agent was added. The proper ratio of zosteric acid solution and the silicone mixture was adjusted to obtain silicone coatings with a particular amount of zosteric acid. For Sylgard 184, mixtures containing 0.3 wt%, 0.6 wt%, 1 wt%, and 2 wt% of zosteric acid were prepared, whereas for RTV11, only a mixture containing 1 wt% of zosteric acid was prepared. The mixtures were used to prepare zosteric acid-entrapped coatings on glass slides using the same procedures as those of plain silicone coatings, as described in the previous paragraph.
Bacterial Attachment Study
Each of the coatings was placed inside an amber bottle (60 ml) containing the specific bacterial culture in 30 ml aqueous solution with or without zosteric acid. Care was taken to place the coatings face down at a 40[degrees] angle to ensure that the attachment was not simply the result of settlement of species and organic matter. The initial population of bacteria and environmental conditions in each bottle was controlled to be as identical as possible. Bacterial growth and subsequent attachment was allowed to occur for two weeks. The coatings were then removed from the bottle, and observations were made after rinsing the coatings with fresh DI water to remove the loosely attached matter. Biofilm morphology was taken using a transmitted and a reflected light optical microscope, respectively, for Sylgard 184 and RTV11 coatings. The CCD video system attached to the microscope was used to capture the images of interest. The variations of the biofilm morphology on the surface of the silicone coatings due to the bacterial biofilm growth were examined. Various degrees of magnification were used to identify the differences in shapes and quantity of bacteria attached to the coating surface. The morphology image was enlarged to estimate the percent bacterial coverage for each coating by counting the pixels occupied by the biofilm divided by the total pixels of the image.
To determine the optimum effective zosteric acid concentration in solution, the bacterial attachment was conducted by dissolving zosteric acid in the solution and using a 2 x 2 x 7 factorial design. In this design, two different times of immersion (7 and 14 days), two types of bacteria (enriched Lake Erie and P. putida), and seven different concentrations (0, 5, 10, 20, 50, 100, and 500 mg/L) were used. Three replicates for each combination were utilized.
A two-way analysis of variance (ANOVA) via MiniTab software was applied to evaluate the effect of concentration and time of attachment. The Tukey's Honestly Significant Different Test (HSD) was used to complete a pair-wise comparison to determine the significance of the data. With this approach, statistically significant results were depicted by p-values < 0.05; nonsignificant results were those with p > 0.05.
Coating Properties Evaluation
Coating properties, mainly surface wettability and bulk elastic modulus, and surface topography were evaluated before and after water immersion. The surface wettability of the coating was evaluated via water contact angle measurements. For coatings subjected to bacterial attachment, a portion of the biofilm was removed immediately prior to measuring the contact angle. Due to the low adhesion between the biofilm and the silicone coatings, the biofilm could be completely removed using a piece of Scotch[R] tape, leaving behind a clean coating surface. Images of the sessile drops that formed on the surface were captured using a Dazzle DVC system. During contact angle measurement, several drops were randomly placed at different locations on the surface for each of the three replicates. Contact angle values were estimated using the Scion Image Software. The average of the angles measured on a particular surface was reported. The two-way ANOVA was conducted to determine the statistical significance of the data.
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The elastic modulus of the coatings was measured using the JKR method, (29) where a convex elastic lens made of Sylgard 184 was brought down into contact with the coating of interest. The force, determined using an electronic balance with an accuracy of 0.1 mg, acting between the two surfaces and the diameter of the circular contact, which was enlarged with an optical microscope, were measured to obtain the elastic modulus (E*) of the system. The modulus of the coating (EC) was deduced from the known value modulus of the elastic lens (EL). An ANOVA similar to that of the contact angle analysis was conducted to determine any significant difference in the data.
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The surface morphology was evaluated with an optical microscope (OM) and an atomic force microscope (AFM). AFM scans of the coating surface were obtained using the noncontact mode with a silicon cantilever having a spring constant of ~42 nN/nm. The scan size was 80 [micro]m x 80 [micro]m and the scan rate was 0.2 Hz. The surface roughness of the coatings on an 80 [micro]m x 80 [micro]m area was attained from the AFM scans using the NanoScope III software version 4.42r4.
RESULTS AND DISCUSSION
Antibacterial Attachment Ability of Zosteric Acid When Present in Solution
Antifoulants may prevent biofilm formation by posing a nonselective lethal toxicity toward the aqueous microorganisms. In order for zosteric acid to be an effective NPA, it must prevent attachment without posing an unacceptable toxicity level. Therefore, the toxicity of zosteric acid was evaluated (10) using both the standard Microtox test and the quantitative toxicity assessment. The E[C.sub.50] (the concentration that causes 50% of the original microbial population to die) of zosteric acid with the Microtox test was found to be 440 mg/L. Using the quantitative toxicity assessment, the values were 400 mg/L and 166 mg/L for the enriched Lake Erie consortium and P. putida, respectively. These values indicated that zosteric acid is approximately five to six orders of magnitude less toxic (30) as compared to currently used antifoulant compounds, such as TBT and SeaNine 211.
Plain Sylgard 184 silicone coatings were used to evaluate bacterial attachment when zosteric acid was simply dissolved in the water containing either enriched Lake Erie bacteria or Pseudomonas putida. It is important to note that almost all of the concentrations investigated were substantially less than its E[C.sub.50] value. Figure 2 contains representative biofilm morphologies of enriched Lake Erie bacteria (a, b, c, and d) and Pseudomonas putida (e, f, g, and h) on silicone coatings after 14 days. Bacterial attachment on the plain silicone coatings in solution containing no zosteric acid was used as the control for comparison. The controls depicted approximately 45% of surface coverage with a branch-like biofilm by the enriched Lake Erie bacteria and 36% surface coverage with a biofilm composed of an elongated shaped Pseudomonas putida, respectively (Figure 3). When the enriched Lake Erie bacteria was subjected to 5 mg/L of zosteric acid, the attachment was found to be slightly less than that of the control, showing a bacterial coverage of 33% (or a reduction of 25% of that depicted by the control). As the concentration increased to 10 mg/L, the bacterial surface coverage was reduced to 13%, and it was 11% for coatings immersed in water containing 20 mg/L zosteric acid. A clear reduction (i.e., 92%) in biofilm formation occurred when 50 mg/L zosteric acid was used, depicted by only a 3% coverage. When the zosteric acid concentration increased to 100 mg/L, the coverage was even less. With 500 mg/L of zosteric acid, even after 14 days of immersion, almost total inhibition of bacterial attachment (0.8% surface coverage) was observed. A similar trend was found for P. putida. The surface coverage by attached P. putida was 13%, 1%, and 0.4%, respectively, for coatings immersed in 20, 50, and 500 mg/L zosteric acid solutions. The attachment study showed that the concentration necessary to reduce the bacterial attachment by more than 90% was about 50 mg/L. This concentration is substantially lower than the E[C.sub.50] values (~400 mg/L for Lake Erie bacteria and ~170 mg/L for P. putida) of zosteric acid for the two bacteria used in this study. This indicated that zosteric acid is effective in deterring certain fresh water bacterial attachment at a much lower toxic level as compared to most of the currently used antifoulants. (31)
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The difference in bacterial attachment could also have resulted from the difference in coating properties, such as surface wettability and bulk modulus. In order to confirm that the difference in attachment is solely due to the presence of zosteric acid in the water, the variations of silicone coating properties after immersion in water were evaluated. Static contact angles (i.e., wettability) of the coatings only decreased significantly (P < 0.05) for the first day of immersion in the aqueous solution containing bacteria, with the values remaining constant as immersion time was extended. A very similar trend was observed when the coating was immersed in DI water without bacteria. The increase in wettability for the first day could likely have resulted from the slight reorganization of the side chain and backbone components of silicone as the system attempted to minimize the energy in the highly polar aqueous environment. (32) In all cases, bulk elastic modulus only fluctuated slightly (P > 0.05) and remained well within the standard deviation of the measurements. The elastic modulus, depending on the crosslinking density of Sylgard 184 (a highly hydrophobic elastomer), was expected to be unchanged by immersion in water. In addition, because the aqueous zosteric acid concentration was relatively low ([less than or equal to] 500 mg/L), the effects of zosteric acid on the bulk properties were anticipated to be negligible. Therefore, the difference in bacterial attachments was not the result of changes in the surface wettability and bulk modulus of the coatings.
Antibacterial Capability of Zosteric Acid-Entrapped Silicone Coatings
In earlier studies, zosteric acid had been directly blended into silicone coatings in the form of a powder, and the coatings were applied to panels to perform attachment studies (22) in a marine environment. Although the effectiveness of zosteric acid in deterring organism attachment was observed, zosteric acid leached out too fast (at a rate of ~1 mg/day-[cm.sup.2]) to make the coating useful for long-term service. For example, zosteric acid would completely leach out of a coating with a thickness of ~1 mm and a zosteric acid concentration of 10 wt% in about 100 days. In addition, the methods required to incorporate 10 wt% zosteric acid into a coating could likely alter the coating properties, as well as be economically unfeasible due to the cost of zosteric acid.
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In order to slow down the leaching of zosteric acid, we utilized the common solvent approach of zosteric acid and silicone (33) to incorporate zosteric acid, uniformly, into a model silicone (Sylgard 184) coating. The purpose of using transparent Sylgard 184 was to ensure that the distribution of zosteric acid inside the coating could be examined. To verify our approach was adequate, the direct blending of grounded zosteric acid powder with silicone under vigorous mixing was used as a control. As can be seen in Figure 4a, this approach resulted in large zosteric acid aggregates (average ~80 microns). Some of these aggregates spanned the entire thickness of the coating and created large pathways for water to enter and dissolve, and newly dissolved zosteric acid could also leach out quickly through these pathways.
When the common solvent was used, the distribution of zosteric acid became more uniform and the aggregate size decreased as the miscibility of the solvent/zosteric acid/silicone increased (Figure 4b); consequently, the leaching of zosteric acid from the coating slowed down. The details on choosing the solvent and leaching of zosteric acid from the resulting coatings have been reported elsewhere. (33) Briefly we were able to reduce the leaching rate to the range of 0.01 to 0.1 [micro]g/day-[cm.sup.2], thus increasing the possible service life of the coating to more than 10 years for coatings containing only 1 wt% of zosteric acid. On the other hand, in order to provide sufficient leaching of zosteric acid to deter the attachment of bacteria, a leaching rate in the range of ~0.1 [micro]g/day-[cm.sup.2] appeared to be optimum. Such coatings, as well as ones containing different amounts of zosteric acid, were prepared using the same incorporation solvent (50/50 water/pyridine) for use in the attachment studies with enriched Lake Erie bacteria.
For zosteric acid-entrapped coatings, the effects of zosteric acid inside the coating were evaluated first. Figure 5 contains representative images of the bacterial attachment on these coatings after one week. As the amount of zosteric acid inside the coating increased from 0 wt% to 0.3 wt%, 0.6 wt%, and 1 wt% (Figures 5a to 5d, respectively), the bacterial coverage on the coating surfaces decreased from 21% to 13%, 10%, and 5.8%. The coating properties, both surface energy (i.e., wettability) and bulk modulus, were not affected significantly (p-value < 0.05) by the amount of zosteric acid entrapped inside the coating (Figure 6), indicating that their contribution to the difference in bacterial attachment was negligible. No significant reduction in bacterial coverage was observed for the 2 wt% coating (depicted as a 5.8 [+ or -] 0.5% surface coverage) as compared to that of the 1 wt% coating (with a 6.2 [+ or -] 0.5% coverage). This suggests that 1 wt% zosteric acid entrapped into the coating could be the optimum concentration in minimizing the effects of zosteric acid incorporation on the coating properties as well as the costs.
The representative images of bacterial attachment as a function of immersion time on the pure silicone coatings and coatings containing 1 wt% zosteric acid are presented in Figure 7. After one week of immersion, the difference in biofilm formation on the silicone coatings with and without zosteric acid entrapped was clearly visible. Substantially fewer bacteria (i.e., 75-80%) were attached to the coating with entrapped zosteric acid. The enriched Lake Erie bacteria covered -25% of the surface for those samples without zosteric acid. Once attached, the biofilm continued to grow, and the coverage increased to 33%, 49%, and 52%, respectively, for two, three, and four weeks of immersion. One might notice that the bacterial coverage on the pure silicone coating after two weeks of immersion (performed in winter) differed from that obtained in the first part (performed in summer) of this study. This was attributed to the sensitivity of bacteria to the ambient conditions and the variation in initial concentrations of bacteria. For silicone coatings containing 1 wt% of zosteric acid, the bacterial coverage was 6%, 8%, 9%, and 10% for one, two, three, and four weeks of immersion, respectively. On average, the biofilm coverage for coatings containing zosteric acid was approximately 20-25% (i.e., 75-80% reduction) of those that contained no zosteric acid at each particular immersion time. The analysis of variance for the coverage against concentration and time of immersion yielded a p-value < 0.05 (0.012), showing a significant difference between the coatings with and without 1 wt% of zosteric acid.
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Morphology of the attached enriched Lake Erie bacteria on pure RTV11 silicone coatings as well as on RTV11 coatings containing 1 wt% of zosteric acid is presented in Figure 8. The presence of zosteric acid reduced the attachment to about 50-60%, a slightly less reduction as compared to that of zosteric acid-incorporated Sylgard 184. This was surprising since zosteric acid leached about 10 times faster when RTV11 was used as the coating carrier. Other factors could also affect the attachment of bacteria, such as the roughness of the coating and the interaction between zosteric acid and the additives in RTV11. The surface roughness of zosteric acid-entrapped silicone coatings before and after bacterial attachment was evaluated via AFM scanning (Figure 9). Before immersion, the surface roughness of the zosteric acid-incorporated RTV11 ([R.sub.q] ~ 25 nm) was about twice that of the zosteric acid-incorporated Sylgard 184 coatings ([R.sub.q] ~ 11 nm). As the coatings immersed in water with zosteric acid were leaching out, the surface roughness of RTV11 coatings ([R.sub.q] ~ 200 nm for two weeks of immersion) increased to a higher extent than that of the Sylgard 184 coatings ([R.sub.q] ~ 50 nm for two weeks of immersion). Increased bacterial attachment on the zosteric acid-entrapped RTV11 coatings could likely be the result of the rougher surface that led to additional surfaces to which the bacteria could adhere. Also, for RTV11 coatings immersed for two weeks in solution containing bacteria, large holes (10-20 microns), likely caused by the depletion of zosteric acid, were observed. Similar holes were also observed in the zosteric acid-entrapped Sylgard 184 coatings after two weeks, but they were smaller (< 10 microns). Two possible causes could lead to the difference in hole size. First, smaller zosteric acid aggregates were distributed more homogenously inside Sylgard 184 as compared to that inside RTV11. With this possibility, the spacing between two zosteric acid aggregates in RTV11 could be large enough for some bacteria to attach and grow into a small biofilm colony. Second, the calcium carbonate fillers inside RTV11 might interact with zosteric acid to accelerate leaching of zosteric acid and result in the large holes. This may also weaken zosteric acid's ability in deterring bacterial attachment. Therefore, the zosteric acid entrapped inside RTV11 slightly reduced its effectiveness in inhibiting bacterial attachment and growth. Nevertheless, the entrapped RTV11 coatings were still capable of reducing Lake Erie bacterial attachment by more than 50% as compared to pure RTV11 coatings.
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The effectiveness of zosteric acid as a less toxic antifoulant was evaluated by conducting bacterial attachment studies for plain silicone coatings with zosteric acid dispersed in the solution containing bacteria, as well as for coatings with entrapped zosteric acid. The surface wettability and bulk modulus of the coating were found to have little or no effect on the bacterial attachment behaviors. The bacterial attachment onto plain silicone coating surfaces was found to decrease as the concentration of zosteric acid in the solution increased. With 50 mg/L zosteric acid in the solution, the bacterial coverage was reduced by more than 90% for both fresh water bacteria: enriched Lake Erie bacteria and Pseudomonas putida. More importantly, this concentration was significantly lower than the E[C.sub.50] of the compound for each of the two types of bacteria tested.
When zosteric acid was incorporated into silicone coatings, the reduction on bacterial coverage and biofilm formation was also observed. The reduction of bacterial attachment increased as the amount of zosteric acid entrapped inside the coating increased from 0 to 1 wt%, and no difference in bacterial coverage was observed as the amount of zosteric acid further increased from 1 wt% to 2 wt%. Using 1 wt% zosteric acid as the bulk entrapped concentration, with Sylgard 184 as the coating carrier, a reduction of 75-80% in bacterial coverage was achieved; while the reduction was ~ 55% when RTV11 was used as the carrier. The smaller reduction in bacterial attachment of zosteric acid-incorporated RTV11 coatings could be attributed to the substantial increase in surface roughness as compared to those of zosteric acid-entrapped Sylgard 184. The results from this study indicated that zosteric acid could be a much less toxic but effective antifoulant compound. The hybrid of less toxic zosteric acid and excellent foul-release silicone coatings could commence a versatile approach in combating biofouling.
The financial support of the Ohio Sea Grant (Project: R/BM-2), Ohio Board of Regents (R5905-OBR), and Faculty Research Grant (UA FRG 1533) of The University of Akron is highly acknowledged. The help of Mr. Feng Song for synthesizing the zosteric acid and Mr. Sung-Hwan Choi for scanning the AFM images is greatly appreciated.
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Bi-min Zhang Newby,** Teresa Cutright, ([dagger]) Carlos A. Barrios, and Qingwei Xu ([dagger]) -- The University of Akron*
Presented at the American Chemical Society (ACS) Fall Meeting, August 2004 in Philadelphia, PA.
* Department of Chemical Engineering, Akron, OH 44325-3906.
([dagger]) Department of Civil Engineering, Akron, OH 44325-3905.
** Author to whom correspondence should be addressed. Email: firstname.lastname@example.org.
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|Date:||Jan 1, 2006|
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