Grantee Research Project Results
2007 Progress Report: Molecular Approaches to Early Detection and Detoxification of Red Tides
EPA Grant Number: EM832982Title: Molecular Approaches to Early Detection and Detoxification of Red Tides
Investigators: Barreto, Jose , Volety, Aswani K. , Brown, David M.
Current Investigators: Barreto, Jose , Volety, Aswani K. , Brown, David
Institution: Florida Gulf Coast University
EPA Project Officer: Aja, Hayley
Project Period: July 1, 2006 through July 1, 2009
Project Period Covered by this Report: July 1, 2006 through June 30,2007
Project Amount: $243,750
RFA: Targeted Research Grant (2006) Recipients Lists
Research Category: Aquatic Ecosystems , Targeted Research
Objective:
- To develop and adapt assays demonstrating photocatalytic destruction of molecules, including the adaptation of a fluorescent assay for detecting hydroxyl radicals and a luminescent bacterial assay for the purpose of demonstrating biocidal killing with photocatalysts. Photocatalysis constitutes a green technology, producing reductant and oxidant species capable of inactivating toxins and killing the red tide organism without leaving behind any residual toxicity.
- As the reviewers correctly pointed out in the original submission of the proposal, titanium dioxide can only make use of the ultraviolet portion of sunlight and UV illumination is only 2-3 percent of the solar spectrum. Further, UV has the problem of poor penetration in ocean water so we proposed to adapt a new family of carbon and nitrogen doped titanium oxide photocatalysts, which would work in the visible portion of the spectrum. Initial doping experiments and their effectiveness are reported herein.
- In the process of adapting a luminescent Vibrio fischeri assay to test photocatalytic biocidal effectiveness, we have observed a susceptibility of these organisms to ammonia which is greatly enhanced by conditions that drive an alkaline flux inward (ammonia permeation), causing alkalinization and killing of the bacteria. We will explore this effect with K. brevis in year two, as part of the grant objective, dealing with red tide destruction using green technologies.
Progress Summary:
We were able to finalize the development of two photobleaching dye assays (Coates & Barreto, 2007) that reveal the effectiveness of a given photocatalyst is producing destructive reductant and oxidant species (ROS). The dyes were expected to photo-bleach because carbon-carbon pi-bonds in the conjugated dyes were broken by the destructive ROS. Both azo dyes were chosen because they are hard targets, meaning that both dyes are very resistant to being destroyed by long wave UV illumination. Tartrazine is a bright yellow, water soluble dye that was chosen because it represents a hydrophilic molecular target freely present in aqueous solution. Sudan red is a very hydrophobic dye which we encapsulated in a detergent micelle; it mimics a biological lipophilic membrane target encapsulated in the hydrophobic core ora cel1 membrane or the hydrophobic core of a lipid micelle. Notably, brevetoxin (the red tide toxin) is lipophilic so the destruction of this toxin is modeled by the Sudan experiments. Further, the biocidal ROS produced by photocatalysis would be expected to destroy hydrophobic targets encapsulated in the hydrocarbon core of a cell membrane, thus predicting biocidal activity with regard to causing damage to a cellular plasma membrane. We also adapted a preexisting nuorescent assay for detecting hydroxyl radicals to detect ROS in our photocatalytic systems (Barreto, 1995) and we further adapted the hydroxyl radical assay to a 24-well plate automated reader system.
Using the assays described above, our attempts at nitrogen doping, carbon doping and acid etching of titanium metal powders did not succeed at replacing Degussa P-25 (a known commercial photocatalyst which served as a standard reference). We determined that optimal photocatalysis occurs in the presence of ferric ion (Fe+3 we believe that the ferric ion is acting as a Fenton catalyst and driving the production of radicals and oxidants from hydrogen peroxide. In a useable photocatalytic technology, we would prefer to avoid the incorporation of iron because such solutions become very acidic and when neutralized the ferric ion precipitates leading to a loss of the Fenton catalyst.
An important finding during the project period appears to be the observation that V. fischeri (a model marine organism) can be effectively destroyed by low concentrations of ammonia (~55 millimolar at pH>9). We are interested in pursuing this finding which might be useful in destroying K. brevis. An alkaline biocide may represent a green technology which could suppress a red tide bloom in a locally contained environment (a beach, marina, or other economically sensitive location subject to a red tide infestation). We will pursue our pilot experiments in the next report period.
Development, modification and refinement of assays used in conducting the project:
- A killing assay was devised to determine the viability of the luminescent marine bacteria Vibrio fischeri after exposure to a biocidal treatment. A fluorometer was modified to function as a luminometer for this purpose. The luminescence assay constitutes a very rapid simple way to detennine biocidal effectiveness in seawater, and will be used to screen for the most effective biocides before they are tested on Karenia brevis.
- We modified a fluorescence assay (Barreto, 1995) to measure the effectiveness of photocatalytically produced ROS in water. The assay uses terephthalic acid to detect hydroxyl radicals. The signal produced by the dO$imeter correlates with the efficacy of the photocatalytic treatment in creating biocidal species.
- A novel micellar system incorporating hydrophobic Sudan red was developed to serve as a model for the photocatalytic destruction of brevetoxin (a hydrophobic toxin), assay and to measure the effectiveness of ROS in destroying membrane encapsulated targets which engender cell death.
- Modified all assays to run in an automated fashion on a TECAN well plate reader.
- We developed an assay to measure the effectiveness of ROS in destroying water soluble targets (tartrazine assay).
Development of photocatalytic dye photobleaching assays:
Figure1. Photocatalytic oxidation ofa solution of Sudan red CTAB with 0.25 mM Fe +3 and 0.1 mg/ml P25-Ti02- Control wells (left bar) were not irradiated with 365 nm UV light. After 15 minutes of irradiation of Sudan red solution, 70% of the dye was destroyed (right bar).
Figure 2. Photocatalytic oxidation ofa solution oftartrazinc with 0.25 mM Fe+3 and 0.1 mg/ml P25-Ti02- Control wells (left bar) were not irradiated with 365 nm UV light. After 15 minutes of irradiation, 22% of the dye was destroyed (right bar).
Some detailed results demonstrating the synthesis of new photocatalysts.
Nitrogen Doped Titanium Oxide Powders from Titanium (IV) Chloride
To a 100 ml volumetric flask was added 2.76 ml of titanium (IV) chloride (TiCl4) and deionized water. A white precipitate formed but dissolved after a few minutes. A 20.0 ml aliquot of the titanium (IV) chloride solution was placed into a 100 ml beaker, and to the solution was added 2.0 ml of triethylamine (NEt3). The pH of the solution was approximately 4.00. The resultant solution was stirred for 24 hours at room temperature and open to the atmosphere. The water was removed by evaporation and the resultant grey powder was heated at 350°C for 17 hours.
Evaluation of nitrogen doped Titanium Oxide Powder: A 24 well polystyrene well plate was used in this experiment. To each of three control wells were added 3.0 ml of a Sudan (IV) solution with an absorbance adjusted to 1.0 rau. To another three reaction wells were added 2.97ml of the Sudan (IV) solution, and 0.030 ml of a 0.025 M ferric nitrate (Fe(NO3)3 solution. To a third set of three reaction wells were added 2.94 ml of the Sudan (IV) solution, 0.030 ml of a 0.025 M (Fe(NO3)3 solution and 0.030 ml of a 10.0 mg/ml suspension of Degussa P25-Ti02. To a fourth set of reaction wells were added 2.97 ml of the Sudan (IV) and 0.030 ml ofa 10.0 mg/ml suspension of doped-TiO2. To a fifth set ofreaction wells were added 2.94 m) of the Sudan (IV) solulion, 0.030 ml of a 0.025 M Fe(NO3)3 solution and 0.030 ml of a 10.0 mg/ml suspension of doped-TiO2-. To a sixth set of reaction wells were added 1.94 ml of the Sudan (IV) Solulion, 0.030 ml of a 0.025 M (Fe(NO3)3 solution and 1.00 ml of a 10.0 mglml suspension of dopedTiO2-. The resultant solutions were stirred in the direct sunlight for 4.5 hours. After completion of the solar irradiation the solids were removed by centrifugation and the solutions were measured by UV-vis spectrophotometry at wavelength of 535 nm. All experiments were done in triplicate and the mean and standard deviation of three trials is reported.
Figure 3. Photocatalytic bleaching of Sudan (IV) in the presence of nitrogen doped TiO2.
Control wells contained only Sudan (IV) solution. Fe+3 shows wells containing a Sudan (IV)
solution and 0.25 mM of Fe(NO3)3. Degussa P25-TiO2 and Fe(NO3)3 was used as a reference. d-TiO2 contained doped TiO2 only. (a) shows wells containing 0.1 mg/ml doped TiO2 along with
Fe(N03)3. (b) shows wells containing 3.33 mg/ml doped TiOlalong with the Fe(NO3)3.
Figure 3 Results: The doped Ti02 powder afforded the photocatalytic destruction of the Sudan (IV) dye at concentrations of 0.10 mg/ml and 3.33 mg/ml in the presence of ferric nitrate. In this experiment; however, standard Degussa proved to be the most efficient photocatalyst in destroying the dye.
Preparation of carbon doped Titanium Oxide Powders from Titanium Carbide: 500 mg of titanium carbide (TiC) was placed in a crucible and heated at 350°C on top ora hot plate in the open atmosphere for 17 hours. The photocatalytic activity was then evaluated.
Evaluation of Doped Titanium Oxide Powder: A 24-well polystyrene plate was used in this experiment. To triplicate control wells were added 3.0 ml of a Sudan (IV) solution with an absorbance adjusted to 1.0 relative absorbance units. To a set of triplicate reaction wells were added 2.97 ml of the Sudan (IV) solution, and 0.030 ml of a 0.025 M ferric nitrate solution (Fe(NO3)3. To a second set of triplicate reaction wells were added 2.94 ml of the Sudan (IV) solution and 0.030 ml of a 10.0 mg/ml suspension of doped TiO2. To a third set of triplicate reaction wells were added 2.97 ml of the Sudan (IV) and 0.030 ml of a 10.0 mg/ml suspension of doped TiO2. To a fourth set of triplicate reaction wells were added 1.94 ml of the Sudan (IV) solution, 0.030 ml of a 0.025 M ferric nitrate solution Fe(NO3)3 and 1.00 ml of a 10.0 mg/ml suspension of doped-TiO2. The resultant solutions were stirred in the direct sunlight for 4.5 hours. After completion of the solar irradiation the solids were removed by centrifugation and the solutions were measured by UV-vis spectrophotometry at wavelength of 535 nm.
Figure 4. Photocatalytic Bleaching of Sudan (IV) in the presence of carbon dopcd-Ti02 prepared from titanium carbide (TiC). The control contained only Sudan (IV) solution. The Fe+ experiment contained a Sudan (IV) solution and 0.25 mM of Fe(N03)3 d-Ti02 shows doped Ti02 only. (a) shows the reaction well containing 0.1 mg/ml doped Ti02 along with the Fe(NO3)3. (b) shows wells containing 3.33 mg/ml doped Ti02 along with the Fc(NO3)3 Results: The doped Ti02 powder prepared from titanium carbide powder did afford the photocatalytic destruction of the Sudan (IV) dye at concentrations of 0.10 mg/ml and 3.33 mglmJ in the presence of 0.25 mM ferric nitrate.
Titanium metal powder coated with an oxide surface by acid etching:
Batch #1: Approximately 100 mg of titanium powder was etched with a proprietary acid mixture.
Batch #2: Approximately 200 mg of titanium powder was etched. The resultant suspension was heated for 1 hr directly on a hot plate with the theml0stat setting at 3. The crucible was then placed into a sand bath and heated for an additional 16.0 hrs at the same thermostat setting.
Batch #3: Approximately 500 mg of titanium powder was etched. The resultant suspension was heated and stirred for 2 hrs directly on a hot plate with the thermostat selling at 5.
Evaluation of coated Ti metal powders (THA dosimeter): Batch 1-3: To three wells of a 24well plate were added 30 μL of a 1.0 mg/mL suspension of the titanium powder (Batch # 1). To each well were added 330μL of a 30 mM THA solution and 667 ilL of de-ionized water. The wel1s were then irradiated for 5 minutes with a 365 nm ultra-violet light. Fluorescence was measured with a fluorometer at an excitation wavelength of 312 nm and an emission of 420 nm, high fiuorescence in the THA assay indicates the appearance ofa hydroxylated photoproduct and correlates with the production of hydroxyl radicals by the photocatalyst. The fluorescence was compared to that of Degussa P-25 titanium oxide powder.
Figure 5. Suspensions of coated Ti metal powder were prepared and subjected to uv-irradiation at 365 nm for 5 minutes (batch 1-3). Control wells contained Degussa P-25 Ti02 powder. Reaction wells contained the coated titanium metal powder and were subjected to uv-irradiation for 5 min. No fluorescence was observed for batch #1 while batch #2 and #3 did show minimal fluorescence.
Figure 5 Results: Degussa P-25 Ti02 showed the most photocatalytic activity compared to the coated titanium metal powder.
Significance of the photocatalytic results
Our attempts at nitrogen doping, carbon doping and acid etching of titanium metal powders did not succeed at replacing Degussa P-25 (a known commercial photocatalyst which served as a standard reference), this can be seen in Figures 3-5. The figures shown are representative, not inclusive. Several assays were used to determine photocatalytic activity: photobleaching of the aqueous dye tartrazine, photobleaching of the hydrophobic dye Sudan red, and production of hydroxyterephthalate from terephthalate by ring hydroxylation (this is a fluorescent assay which indicates the presence of hydroxyl radicals. We determined that optimal photocatalysis occurs in the presence of ferric ion (Fe+3 ), we believe that the ferric ion is acting as a Fenton catalyst and driving the production of radicals and oxidants from hydrogen peroxide. In a useable photocatalytic technology, we would prefer to avoid the incorporation of iron because such solutions become very acidic and when neutralized the ferric ion precipitates leading to a loss of the Fenton catalyst.
In ongoing experiments we have been successful at using our acid etching technique to generate photocatalytic surfaces which are very active in the long-wave UV region (365 nm) without ferric ion. These experiments are too recent to include in this report but when we obtain a very active surface, we will test these coated metals with the V.jischeri and the K. brevis killing assays described earlier in this report.
Figure 6. When treated for 1 hour with NaOH or NH40H followed by re-growth in fresh photobacterium broth for 24 hours, the luminescence of V.fischeri declines by ~60% with NaOH and 100% with NH40H treatment.
Detailed results demonstrating alkaline biocidal killing
Destruction of Vibrio fischeri with NaOH:
To each of seven tubes was added 1.2ml of photobacterium broth followed by the addition of 300μl of Vibrio fischeri from a stock culture addedjusl prior to the start of the reaction. To the control was added 300 μl of photobacterium broth. To each individual experimental tube was added photobacterium broth and 2.5% NaOH to give a total volume of 300μl with a final concentration of 17, 35, 52, 69, 86 or 104 mM NaOH. Each tube was vortexed and the luminescence read initially and at 30 second intervals. The luminescence was read on a Turner model 450 Fluorometer with the excitation filter blocked and the emission filter removed.
Figure 7. Destruction of Vibrio fischeri with varying concentrations of NaOH. The broth only control (red line) had a pH of 6.9. The 17 mM NaOH treatment (purple line) gave a stcady luminescence at approximately 55 while the 35 mM NaOH treatment (green line) showed a slight decrease in luminescence over time from approximately 21 to approximately 12. The 52 mM, 69 mM and 86 mM NaOH treatments all showed no luminescence over timc. The 104 mM NaOH, however, gave a slight, steady luminescent signal of about 3 over the reaction period.
Figure 7 Results: The experimental solutions ranged in pH from 8.5 to 12.3. As the pH rose from 8.5 to 10.4, the luminescence decreased from approximately 55 to O. As the pH rose from 10.4 to 11.9, the luminescence remained at 0. However, at pH 12.3, the luminescence was slightly above 0 at approximately 3. This reading could be explained by the noise in the instrument or slight drift of the baseline over time.
Effect of NH40H on Vibrio fischeri.
To each of seven tubes was added 1.2 ml of photobacterium broth followed by the addition of
300μl of Vibrio fischeri40H to give a total volume of 300μl with a final
concentration of 55, 111, 167,222,278 and 333 mM NH40H. Each tube was vortexed and the
luminescence read initially and at 30 second intervals. The luminescence was read on a Turner
model 450 Fluorometer with the excitation filter blocked and the emission filter removed.
Figure 8: Destruction of Vibrio fischeri with varying concentrations of NH40H. The broth only control (red line) had a pH of 6.9. The 55 mM NH40H treatment (purple line) gave a gradual decrease in luminescence from approximately 12 to approximately 4 while the 111 mM NH40H treatment (green line) showed a steady luminescence at approximately 2. The 167 mM, 222 mM, 278 mM and 333 mM NH40H treatments all showed no luminescence over time.
Figure 8 Results: The experimental solutions ranged in pH from 9.4 to 10.4. As the pH rose from 9.4 to 9.8, the luminescence decreased from approximately 13 to 2. As the pH rose from 9.8 to 10.4, the luminescence remained at 0.
Significance of the alkaline biocide results
Extreme alkalinity is lethal to all known organisms on earth, almost certainly because alkaline
environments above pH 10 are rare on this planet (and above pH 12 are non-existent).
Consequently, organisms have not adapted to such extremely basic environments and have poor
or non-existent defenses for alkalinity. Alkaline damage and injury is exacerbated by alkaline
hydroxophores (ammonia is one example) which effectively transport the hydroxide ions (responsible for creating basic conditions in water) into the cytoplasm of cells. Hydroxophores effectively cross the plasma membrane which encapsulates all living things; this transport can
raise the pH of the cytoplasm into a lethal range, far above neutrality (pH 7) and neutral pH is
generally understood to be the ideal cytoplasmic pH for all known cells. By combining mild
alkalinity (pH 8-9) with small concentrations of a hydroxophore (ammonia), we have devised a
system that is far more biocidal than alkalinity alone.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 54 publications | 3 publications in selected types | All 3 journal articles |
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Type | Citation | ||
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Barreto, JC, GS Smith, NH Strobel, PA McQuillin and TA Miller. Terephthalic acid: a dosimeter for the delection of hydroxyl radicals in vitro. Life Sciences 1995. 56(4):PL89-PL96. |
EM832982 (2007) |
not available |
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Coates CM, Caldwell W, Alberte RS, Barreto PD, Barreto JC. Beta-carotene protects sudan IV from photocatalytic degradation in a micellar model system:insights into the antioxidant properties of the "golden" Staphylococcus aureus. World Journal of Microbiology and Biotechnology 2007;23(9):1305-1310. |
EM832982 (2007) EM832982 (Final) |
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