Zosteric acid--an effective antifoulant for reducing fresh water bacterial attachment on coatings.Zosteric acid, a natural product present in eelgrass, has been found to prevent the attachment of some bacteria and barnacles. The results indicate that it may also be effective at reducing the early stages of biofouling bi·o·foul·ing
The impairment or degradation of something, such as a ship's hull or mechanical equipment, as a result of the growth or activity of living organisms. , such as the attachment of bacteria that lead to a biofilm Biofilm
An adhesive substance, the glycocalyx, and the bacterial community which it envelops at the interface of a liquid and a surface. When a liquid is in contact with an inert surface, any bacteria within the liquid are attracted to the surface and adhere . In this study, the ability of zosteric acid in reducing the early stages of fouling was evaluated using attachment studies of fresh water bacteria via two approaches. First, plain coatings were submersed in water containing zosteric acid and either enriched Lake Erie Lake Erie
Great Lake; once so polluted, referred to as Lake Eerie. [Am. Hist.: NCE, 887]
See : Filth bacteria or Pseudomonas putida Pseudomonas putida is a gram-negative rod-shaped saprophytic soil bacterium. Based on 16S rRNA analysis, P. putida has been placed in the P. putida group, to which it lends its name. , a model fresh water bacteria. It was found that zosteric acid with a concentration one-tenth of its E[C.sub.50] (the concentration eliminates 50% of the bacteria) was able to reduce bacterial attachment by more than 90%. The second approach incorporated zosteric acid into silicone coatings in the presence of a common solvent to achieve the slow release of zosteric acid; such coatings were then subjected to the bacterial attachment. A ~75% reduction in bacterial attachment was found for 1 wt% zosteric acid entrapped Sylgard[R] 184, a model silicone coating, but the reduction only achieved ~55% for 1 wt% zosteric acid entrapped in a commercial silicone coating, RTV RTV Room Temperature Vulcanizing (elastomer sealant)
RTV Radio Television (educational major)
RTV ReplayTV (digital video recorder brand)
RTV Real-Time Video
RTV Return To Vendor 11.
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 adsorption, adhesion of the molecules of liquids, gases, and dissolved substances to the surfaces of solids, as opposed to absorption, in which the molecules actually enter the absorbing medium (see adhesion and cohesion). of organic matter, such as macromolecules Macromolecules
A large molecule composed of thousands of atoms.
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macromolecules and protein fragments, onto a surface. (1,2) This is followed by the attachment of a complex community of bacteria, diatoms diatoms
a series of unicellular algae, microscopic in size, with cell walls containing silica. Members of the family Diatomaceae. Their remains accumulate as geological deposits and are mined. See diatomaceous earth. , protozoa, and algae algae (ăl`jē) [plural of Lat. alga=seaweed], a large and diverse group of primarily aquatic plantlike organisms. These organisms were previously classified as a primitive subkingdom of the plant kingdom, the thallophytes (plants that 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 drinking water
supply of water available to animals for drinking supplied via nipples, in troughs, dams, ponds and larger natural water sources; an insufficient supply leads to dehydration; it can be the source of infection, e.g. leptospirosis, salmonellosis, or of poisoning, e.g. 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 an·ti·foul·ing
Counteracting or preventing the building up of deposits on underwater surfaces, such as the undersides of boats: antifouling paint. (AF) substances were biocides made of organo-mercury, lead, and dichloro-diphenyl-trichloroethane (DDT DDT or 2,2-bis(p-chlorophenyl)-1,1,1,-trichloroethane, chlorinated hydrocarbon compound used as an insecticide. First introduced during the 1940s, it killed insects that spread disease and feed on crops. ). 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 TBT,
n See theta brainwave training.
TBT Transcervical balloon tuboplasty, see there ), 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 cinnamic acid
A white crystalline acid, C6H5CHCHCOOH, obtained from cinnamon or from balsams such as storax or made synthetically and used chiefly to manufacture perfumery compounds. is shown in Figure 1. The antifouling capability of zosteric acid was attributed to the sulfate sulfate, chemical compound containing the sulfate (SO4) radical. Sulfates are salts or esters of sulfuric acid, H2SO4, formed by replacing one or both of the hydrogens with a metal (e.g., sodium) or a radical (e.g., ammonium or ethyl). 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.
[FIGURE 1 OMITTED]
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 elastomer (ĭlăs`təmər), substance having to some extent the elastic properties of natural rubber. The term is sometimes used technically to distinguish synthetic rubbers and rubberlike plastics from natural rubber. kit manufactured by Dow Corning Dow Corning is a multinational corporation headquartered in Midland, Michigan, USA. Dow Corning specializes in silicon and silicone-based technology, offering more than 7,000 products and services. Dow Corning is equally owned by The Dow Chemical Company and Corning, Inc. , 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 calcium carbonate, CaCO3, white chemical compound that is the most common nonsiliceous mineral. It occurs in two crystal forms: calcite, which is hexagonal, and aragonite, which is rhombohedral. , and ethyl ethyl (ĕth`əl), CH3CH2, organic free radical or alkyl group derived from ethane by removing one hydrogen atom. silicate silicate, chemical compound containing silicon, oxygen, and one or more metals, e.g., aluminum, barium, beryllium, calcium, iron, magnesium, manganese, potassium, sodium, or zirconium. Silicates may be considered chemically as salts of the various silicic acids. (part C) and a tin-based catalyst (dibutyl tin dilaurate, part D). Microscope glass slides purchased from VWR VWR Van Waters and Rogers
VWR Viewer File 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 sodium chloride, NaCl, common salt. Properties
Sodium chloride is readily soluble in water and insoluble or only slightly soluble in most other liquids. It forms small, transparent, colorless to white cubic crystals. , 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 (Asynchronous Communications Server) See network access server. graded pyridine pyridine (pĭr`ĭdēn) or azine (ăz`ēn), C5H5N, colorless, flammable, toxic liquid with a putrid odor. It melts at −42°C; and boils at 115.5°C;. , 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 microbial
pertaining to or emanating from a microbe.
the breakdown of organic material, especially feedstuffs, by microbial organisms. 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 DNA analysis Any technique used to analyze genes and DNA. See Chromosome walking, DNA fingerprinting, Footprinting, In situ hybridization, Jeffries' probe, Jumping libraries, PCR, RFLP analysis, Southern blot hybridization. ). 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 Pseudomonas
A genus of gram-negative, nonsporeforming, rod-shaped bacteria. Motile species possess polar flagella. They are strictly aerobic, but some members do respire anaerobically in the presence of nitrate. sp. and Pseudomonas fluorescens The introduction of this article is too short.
To comply with Wikipedia's lead section guidelines, it should be expanded. . The second, Pseudomonas putida (from American Type Culture Collection American Type Culture Collection (ATCC) is a private, not-for-profit biological resource center whose mission focuses on the acquisition, authentication, production, preservation, development and distribution of standard reference microorganisms, cell lines and other materials for , #12633), was used as the model fresh water bacteria. Both cultures were maintained as described elsewhere. (24,28)
A contact angle goniometer goniometer /go·ni·om·e·ter/ (go?ne-om´e-ter)
1. an instrument for measuring angles.
2. a plank that can be tilted at one end to any height, used in testing for labyrinthine disease. (Model 100-00 from Rame-Hart, Inc.), optical microscopes (IX 70, Olympus and Infini Tube, Edmund Scientific), and an atomic force microscope atomic force microscope (AFM), device that uses a spring-mounted probe to image individual atoms on the surface of a material. Unlike the scanning tunneling microscope, which is also a scanning probe microscope, the AFM can be used on materials that do not conduct (Metrology 2000, Molecular Imaging) were used for characterization of the coatings. They were all equipped with CCD CCD
in full charge-coupled device
Semiconductor device in which the individual semiconductor components are connected so that the electrical charge at the output of one device provides the input to the next device. video cameras for spontaneously capturing the images of interest. A digital conductivity meter (Traceable[R]) and a UV-visible spectrophotometer spectrophotometer, instrument for measuring and comparing the intensities of common spectral lines in the spectra of two different sources of light. See photometry; spectroscope; spectrum. (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 ster·il·ize
tr.v. ster·il·ized, ster·il·iz·ing, ster·il·iz·es
1. To make free from live bacteria or other microorganisms.
2. in an autoclave autoclave
Vessel, usually of steel, able to withstand high temperatures and pressures. The chemical industry uses various types of autoclaves in manufacturing dyes and in other chemical reactions requiring high pressures. 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 bulk modulus
Numerical constant that describes the elastic properties of a solid or fluid under pressure from all sides. It is the ratio of the tensile strength or compressive force per unit surface area to the change in volume per unit volume of the solid or fluid and thus 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 miscibility (miˈ·s·biˑ·l of the zosteric acid solution with silicone, since silicone is immiscible immiscible /im·mis·ci·ble/ (i-mis´i-b'l) not susceptible to being mixed.
Incapable of being mixed or blended, as oil and water. 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 Bacterial growth
The processes of both the increase in number and the increase in mass of bacteria. Growth has three distinct aspects: biomass production, cell production, and cell survival. 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 factorial
For any whole number, the product of all the counting numbers up to and including itself. It is indicated with an exclamation point: 4! (read “four factorial”) is 1 × 2 × 3 × 4 = 24. 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 anova
see analysis of variance.
ANOVA Analysis of variance, see there ) via MiniTab software was applied to evaluate the effect of concentration and time of attachment. The Tukey's Honestly Significant Different Test (HSD HSD Human Services Department
HSD High Speed Data
HSD Hillsboro School District (Hillsboro, OR)
HSD Hybrid Synergy Drive (Toyota/Lexus)
HSD High School Diploma
HSD Historical Society of Delaware ) 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 non·sig·nif·i·cant
1. Not significant.
2. Having, producing, or being a value obtained from a statistical test that lies within the limits for being of random occurrence. results were those with p > 0.05.
Coating Properties Evaluation
Coating properties, mainly surface wettability and bulk elastic modulus elastic modulus
or elastic constant
In materials science and physical metallurgy, any of various numbers that quantify the response of a material to elastic or springy deflection. , 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 sessile /ses·sile/ (ses´il) attached by a broad base, as opposed to being pedunculated or stalked.
Permanently attached or fixed; not free-moving. drops that formed on the surface were captured using a Dazzle DVC (1) (Digital Video Camera) A camcorder that records in digital format. See DV.
(2) (Digital Video Cassette) An earlier term for the DV format. See DV.
(3) See desktop videoconferencing. 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 sci·on
1. A descendant or heir.
2. also ci·on A detached shoot or twig containing buds from a woody plant, used in grafting. 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.
[FIGURE 2 OMITTED]
The elastic modulus of the coatings was measured using the JKR JKR J.K. Rowling (author of the Harry Potter book series)
JKR Jabatan Kerja Raya (Ministry of Public Works, Malaysia)
JKR Joanne Kathleen Rowling (author of Harry Potter Book series) 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.
[FIGURE 3 OMITTED]
The surface morphology was evaluated with an optical microscope (OM) and an atomic force microscope (AFM (Atomic Force Microscope) A device used to image materials at the atomic level. AFMs are used to solve processing and materials problems in electronics, telecom, biology and other high-tech industries. ). 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 antibacterial /an·ti·bac·te·ri·al/ (-bak-ter´e-al) destroying or suppressing growth or reproduction of bacteria; also, an agent that does this.
adj. 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 (1) (Numbering Plan Area) The Bellcore/Telcordia telephone area code system in use in the U.S., Canada, Alaska, Hawaii and islands in the Caribbean. See NPA code.
(2) (Network Professional Association, San Diego, CA, www.npanet. , 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 e·lon·gate
tr. & intr.v. e·lon·gat·ed, e·lon·gat·ing, e·lon·gates
To make or grow longer.
adj. or elongated
1. Made longer; extended.
2. Having more length than width; slender. 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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[FIGURE 5 OMITTED]
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 In statistics, the average amount a number varies from the average number in a series of numbers.
(statistics) standard deviation - (SD) A measure of the range of values in a set of numbers. of the measurements. The elastic modulus, depending on the crosslinking density of Sylgard 184 (a highly hydrophobic hydrophobic /hy·dro·pho·bic/ (-fo´bik)
1. pertaining to hydrophobia (rabies).
2. not readily absorbing water, or being adversely affected by water.
3. 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.
[FIGURE 6 OMITTED]
[FIGURE 7 OMITTED]
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.
[FIGURE 8 OMITTED]
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.
[FIGURE 9 OMITTED]
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 The Ohio Board of Regents is the coordinating board for higher education in Ohio. The board was created in 1963 by the Ohio General Assembly to: provide higher education policy advice to the Governor of Ohio and the Ohio General Assembly; develop a strategy involving Ohio's public (R5905-OBR), and Faculty Research Grant (UA FRG 1533) of The University of Akron Enrollment in fall 2006 was 23,539 students. The school offers more than 200 undergraduate degrees  and 100 graduate degrees . The University's best-known program is its College of Polymer Science and Polymer Engineering, which is located in a 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.
(1) Clare, A.S., Rittschof, D., Gerhart, D.J., and Maki, J.S., "Molecular Approaches to Nontoxic Antifouling," Invertebrate invertebrate (ĭn'vûr`təbrət, –brāt'), any animal lacking a backbone. The invertebrates include the tunicates and lancelets of phylum Chordata, as well as all animal phyla other than Chordata. Reproduction and Development, 22(1-3), p. 67-76 (1992).
(2) Michael, T. and Smith, C.M., "Lectins Lectins
A class of proteins of nonimmune origin that bind carbohydrates reversibly and noncovalently without inducing any change in the carbohydrate. Lectins bind a variety of cells having cell-surface glycoproteins (carbohydrate bound proteins) or glycolipids Probe Molecular Films in Biofouling--Characterization of Early Films on Nonliving and Living Surfaces," Mar. Ecol.: Prog. Ser., 119(1-3), p. 229-236, 1995.
(3) Little, B.J., Wagner, P.A., and Gerchakov, S.M., "Mechanisms for Microbial Corrosion in Marine Environments--An Electrochemical electrochemical /elec·tro·chem·i·cal/ (-kem´i-k'l) pertaining to interaction or interconversion of chemical and electrical energies.
adj. Assessment," in Marine Biodeterioration: Advanced Techniques Applicable to the Indian Ocean, Thompson M., Sarojini, R., and Nagabhushanam, R., (Eds.), A.A. Balkema: Rotterdam, p. 377-384, 1988.
(4) Little, B.J., Wagner, P. Maki, J.S., Walch, M., and Mitchell, R., "Factors Influencing the Adhesion of Microorganisms to Surfaces," J. Adhes., 20(3), p. 187-210 (1986).
(5) Rittschof, D., "Natural Product Antifoulants and Coatings Development," in Mar. Chem. Ecol., McClintock, J.B. and Baker, B.J. (Eds.), CRC (Cyclical Redundancy Checking) An error checking technique used to ensure the accuracy of transmitting digital data. The transmitted messages are divided into predetermined lengths which, used as dividends, are divided by a fixed divisor. Press, Boca Raton FL, Chapt. 17, 2001.
(6) Callow, M.E., "The Status and Future of Biocides in Marine Biofouling Prevention," in Recent Advances in Marine Biotechnology, Vol. 3: Biofilms, Bioadhesion, Corrosion and Biofouling, Fingerman, M., Nagabhushanam, R., and Thompson, M.F. (Eds.), Science Publishers, Enfield NH, p. 109-126, 1999.
(7) O'Connor, N.J. and Richardson, D.L., "Effects of Bacteria Films on Attachment of Barnacle barnacle, common name of the sedentary crustacean animals constituting the subclass Cirripedia. Barnacles are exclusively marine and are quite unlike any other crustacean because of the permanently attached, or sessile, mode of existence for which they are highly (Balanus improvisus Darwin) Larvae Larvae, in Roman religion
Larvae: see lemures. : Laboratory and Field Studies," J. Exp. Mar. Biol. Ecol., 206, p. 69-81 (1996).
(8) Geesey, G.G. and Bryers, J.D., "Biofouling of Engineered Materials and Systems," in Biofilms II: Process Analysis and Applications, Bryers, J.D. (Ed.) Wiley-Liss, Inc., New York New York, state, United States
New York, Middle Atlantic state of the United States. It is bordered by Vermont, Massachusetts, Connecticut, and the Atlantic Ocean (E), New Jersey and Pennsylvania (S), Lakes Erie and Ontario and the Canadian province of , p. 237-279, 2000.
(9) Bennett, R.F., "Industrial Manufacture and Applications of Tributyltin Compounds," in Tributyltin: Case Study of an Environmental Contaminant contaminant /con·tam·i·nant/ (kon-tam´in-int) something that causes contamination.
something that causes contamination. , de Mora MORA, In civil law. This term, in mora, is used to denote that a party to a contract, who is obliged to do anything, has neglected to perform it, and is in default. Story on Bailm. Sec. 123, 259; Jones on Bailm. 70; Poth. Pret a Usage, c. 2, Sec. 2, art. 2, n. , S.J. (Ed.), Cambridge University Press Cambridge University Press (known colloquially as CUP) is a publisher given a Royal Charter by Henry VIII in 1534, and one of the two privileged presses (the other being Oxford University Press). , New York, Chapt. 2, 1996.
(10) Alzieu, C., "Tributyltin: Case Study of a Chromic chromic /chro·mic/ (kro´mik) of, pertaining to, or related to chromium.
chromic phosphate P 32 Contaminant in the Coastal Environment," Ocean and Coastal Management, 40(1), p. 23-36 (1998).
(11) Lewis J.A., "Marine Biofouling and its Prevention on Underwater Surfaces," Mater. Forum, 22, p. 41-61 (1998).
(12) Ponasik, J., Conova, S., Kinghorn, D., Kinney, W.A., Rittschof, D., and Ganem, B., "Pseudoceratidine, a Marine Natural Product with Antifouling Activity: Synthetic and Biological Studies," Tetrahedron tetrahedron: see polyhedron. , 54(25), p. 6977-6986 (1998).
(13) Brady, R.F., "Clean Hulls Without Poisons: Devising and Testing Nontoxic Marine Coatings," J. COAT. TECHNOL., 72, No. 900, 45 (2000).
(14) Brady R.F. and Singer, I.L., "Mechanical Factors Favoring Release from Fouling Release Coatings," Biofouling, 15(1-3), p. 73-81 (2000).
(15) Brady, R.F. and Aronson, C.L., "Elastomeric Fluorinated fluorinated
material to which a fluoride has been added, e.g. water for human consumption treated as a prophylaxis against tooth decay. Polyurethane Coatings for Nontoxic Fouling Control," Biofouling, 19 (Supplement), p. 59-62 (2003).
(16) Estarlich F.F., Lewey, S.A., Nevell, T.G., Thorpe, A.A., Tsibouklis, J., and Upton, A.C., "The Surface Properties of Some Silicone and Fluorosilicone Coating Materials Immersed in Seawater seawater
Water that makes up the oceans and seas. Seawater is a complex mixture of 96.5% water, 2.5% salts, and small amounts of other substances. Much of the world's magnesium is recovered from seawater, as are large quantities of bromine. ," Biofouling, 16(2-4), p. 263-275 (2000).
(17) Stein, J., Truby, K., Wood, C.D., Stein, J., Gardner, M., Swain, G., Kavanagh, C., Kovach, B., Schultz, M., Wiebe, D., Holm, E., Montemarano, J., Wendt, D., Smith, C., and Meyer, A., "Silicone Foul Release Coatings: Effect of the Interaction of Oil and Coating Functionalities on the Magnitude of Macrofouling Attachment Strengths," Biofouling, 19 (Supplement), p. 71-82 (2003).
(18) Wynne K.J., Swain, G.W., Fox, R.B., Bullock, S., and Uilk, J., "Two Silicone Nontoxic Fouling Release Coatings: Hydrosilation Cured PDMS (Product Data Management System) See PDM. and CaC[O.sub.3] Filled, Ethoxysiloxane Cured RTV11," Biofouling, 16(2-4), p. 277-288 (2000).
(19) Armstrong, E., Boyd, K.G., Pisacane, A., Peppiatt, C.J., and Burgess, J.G., "Marine Microbial Natural Products in Antifouling Coatings," Biofouling, 16(2-4), p. 215-224 (2000).
(20) Rittschof, D., "Natural Product Antifoulants: One Perspective on the Challenges Related to Coatings Development," Biofouling, 15(1-3), p. 119-127 (2000).
(21) Sundberg, D.C., Vasishtha, N., Zimmerman, R.C., and Smith, C.M., "Selection, Design and Delivery of Environmentally Benign Antifouling Agents," Naval Research Reviews, XLIX: p. 51-59 (1997).
(22) Burnell, T.B., Carpenter, J.C., Carroll, K.M., Cella, J.A., Resue, J.A., Rubinsztajn, G., Serth-Guzzo, J., Stein, J., Truby, K.E., Webb, K.K., Schultz, M., Swain, G.W., and Zimmerman, R.C., "Advances in Nontoxic Silicone Biofouling Release Coatings," Technical Information Series, GE Research & Development Center (1997).
(23) Todd, J., Zimmerman, R.C., Crews, P., and Alberte, R.S., "The Antifouling Activity of Natural and Synthetic Phenolic-Acid Sulfate Esters," Phytochemistry phytochemistry,
n the scientific study and classification of the chemical constituents of plants. , 34(2), p. 401-404 (1993).
(24) Xu, Q.W., Barrios Barrios is a name of Hispanic origin. The name may refer to: Persons
n. and Zosteric Acid and Their Potential Applications as Antifoulants." Environ. Toxicol., 20(5) (2005).
(25) Fusetani, N., Matsunaga, S., and Konosu, S., "Bioactive bi·o·ac·tive
Of or relating to a substance that has an effect on living tissue.
having an effect on or eliciting a response from living tissue. Marine Metabolites Metabolites
Substances produced by metabolism or by a metabolic process.
Mentioned in: Interactions . 2. Halistanol Sulfate, an Anti-Microbial Novel Steroid Sulfate from the Marine Sponge Halichondria CF Moorei Bergquist," Tetrahedron Letters, 22(21), p. 1985-1988 (1981).
(26) Nakatsu, T., Walker, R.P., Thompson, J.E., and Faulkner, D.J., "Biologically-active Sterol Sterol
Any of a group of naturally occurring or synthetic organic compounds with a steroid ring structure, having a hydroxyl (—OH) group, usually attached to carbon-3. Sulfates from the Marine Sponge Toxadocia-zumi," Experientia, 39(7), p. 759-761 (1983).
(27) Kanazawa, S., Fusetani, N., and Matsunaga, S., "Bioactive Marine Metabolites. 42. Halistanol Sulfates-A-E, New Steroid Sulfates from a Marine Sponge, Epipolasis-SP," Tetrahedron, 48(26), p. 5467-5472 (1992).
(28) Mendez-Sanchez, N., Cutright, T.J., and Qiao, P.Z., "Simultaneous Evaluation of Composite Biodeterioration and Changes in the Physicochemical physicochemical /phys·i·co·chem·i·cal/ (fiz?i-ko-kem´ik-il) pertaining to both physics and chemistry.
1. Relating to both physical and chemical properties. and Biological Water Characteristics," International Biodeterioration and Biodegradation, 52(3), p. 187-196 (2003).
(29) Chaudhury, M.K., and Whitesides, G.M., "Direct Measurement of Interfacial Interactions Between Semispherical sem·i·spher·i·cal
Somewhat spherical in shape. Lenses and Flat Sheets of Poly(dimethylsiloxane) and Their Chemical Derivatives," Langmuir, 7(5), p. 1013-1025 (1991).
(30) Fernandez-Alba, A.R., Hernando M.D., Piedra L., and Chisti Y., "Toxicity Evaluation of Single and Mixed Antifouling Biocides Measured with Acute Toxicity acute toxicity Pharmacology Illness caused by a single exposure to a toxic substance Bioassays," Anal. Chim. Acta, 456(2), p. 303-312 (2002).
(31) Haslbeck, E.G., Kavanagh, C.J., Shin, H.W., Banta, W.C., Song, P., and Loeb, G.I., "Minimum Effective Release Rate of Antifoulants. 2. Measurement of the Effect of TBT and Zosteric Acid on Hard Fouling," Biofouling, 10(1-3), p. 175-186 (1996).
(32) Koberstein, J.T., "Polymer Surfaces and Interfaces," MRS MRS - Modifiable Representation System.
An integration of logic programming into Lisp.
["A Modifiable Representation System", M. Genesereth et al, HPP 80-22, CS Dept Stanford U 1980]. Bull., 21(1), p. 16-17 (1996).
(33) Barrios, C.A., Xu, Q.-W., Cutright T., and Zhang Newby, B.-M., "Incorporating Zosteric Acid into Silicone Coatings to Achieve its Slow Release While Reducing Fresh Water Bacterial Attachment," Colloids Surf., B: Biointerfaces, 41, p. 83-93 (2005).
Bi-min Zhang Newby,** Teresa Cutright, ([dagger]) Carlos A. Barrios, and Qingwei Xu ([dagger]) -- The University of Akron*
Presented at the American Chemical Society The American Chemical Society (ACS) is a learned society (professional association) based in the United States that supports scientific inquiry in the field of chemistry. Founded in 1876 at New York University, the ACS currently has over 160,000 members at all degree-levels and in (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.