Grantee Research Project Results
Final 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
Institution: Florida Gulf Coast University
EPA Project Officer: Aja, Hayley
Project Period: July 1, 2006 through July 1, 2009
Project Amount: $243,750
RFA: Targeted Research Grant (2006) Recipients Lists
Research Category: Aquatic Ecosystems , Targeted Research
Objective:
Our overall project goals were directed towards remediating the toxicity of a red tide bloom. We proposed experiments that utilized high performance photocatalysts that could produce a family of decontaminating, reductants, oxidants and radicals (ROR). Such ROR, are naturally occurring, and are very short-lived (superoxide, hydroxyl radicals), or generate peroxides. These ROR have well known bio-degradative pathways, and unlike xenobiotic disinfectants (chlorine is one example), they pose no chronic threat to the environment. We were not sanguine about remediating large stretches of ocean water containing a red tide bloom, (with its attendant release of brevetoxin). Photocatalytic decontamination is overwhelmed by vast volumes of water, since it requires UV illumination and has a finite surface area for activity, which requires residence time with the ROR present but the preceding observation is generally correct concerning attempts at remediating red tide blooms in the ocean. Immense volumes of water would need treatment by chemical or physical means to destroy K. brevis or brevetoxin in open water. Such a treatment would be too expensive, generate residual toxicity, or be too slow. If the water to be treated is decontaminated in small sequential volumes until total detoxification is achieved, small bodies of water could be effectively treated. Nevertheless, remediating toxicity from a red tide bloom in the context of decontaminating incoming water that is to be used in aquaculture, or in decontaminating a small enclosed body of water, is a worthwhile goal. Such detoxification technologies are likely to be 'multi-use' and may find application in treatment of other decontamination contexts involving defense, biomedicine and environmental remediation.
Given sufficient time (and a favorable surface to volume ratio) any photocatalyst can both kill red tide organisms and inactivate brevetoxin. Indeed, photocatalytic success is assured in such experiments (with regard to all toxin and pathogen destruction) because the generic mechanism of photocatalytic destruction will leave no carbon based molecule intact. A large photocatalytic literature exists which demonstrates the destruction of many specific toxins. To us, a more important question was the practical feasibility of photocatalytic decontamination, which will require a high performance, inexpensive, large surface area photocatalyst. Preferably a catalyst firmly adhered onto an inert substrate, so that recovery of powdered photocatalysts, by filtration or centrifugation is not needed. During this project we proposed to develop assays and to synthesize and screen high performance photocatalysts suitable for decontaminating water with an ultimate aim of remediating red tide toxicity, within the limitations described above.
We also investigated the sensitivity of K. brevis to alkaline killing. We hypothesized that by increasing the pH to at least 9.0, K brevis would be killed, and that neutralization with HCI would immediately abolish lethality, producing only sodium chloride as a byproduct and creating no chronic environmental effects. We discovered that K. brevis is hardy to alkalinity alone, but is extremely sensitive to alkaline environments in the presence of ammonia. This seemed reasonable since at high pH ammonia exists in a neutral permeable form and can create an alkaline flux into the cytoplasm, alkalinizing the cell interior and causing cell death. After noting several interesting effects regarding alkaline killing, we added several components to a biocide which enhance alkaline fluxes into the cytoplasm of any living cell and created a 'green biocide' of great effectiveness which can be 'switched off' when lethality is not required.
Summary/Accomplishments (Outputs/Outcomes):
- We developed and used two sensitive and rapid dye assays in order to determine the relative production of destructive reductants, oxidants and radicals (ROR) that can are produced during the UV illumination of Ti02 semiconductor coatings, or powders. These dye assays were used to determine the performance of new TiO2 photocatalysts and are based upon the photocatalytic destruction of carbon-carbon pi bonds (C=C) in the aromatic dye structure. We used hydrophobic micellar Sudan Red to model an encapsulated hydrophobic toxin. Hydrophobic brevetoxin probably predominates in the micellar or bound form in natural waters in the presence of surfactants, but exists in equilibrium with aqueous toxin. Given the nature of our ultimate red tide toxin target we also utilized an aqueous dye, tartrazine yellow, as a model for the destruction of a water soluble toxin. Lastly, we adapted a previously developed hydroxyl radical dosimeter, (which was originally developed for the purpose of studying hydroxyl radical damage in biological systems; Barreto, 1995) to investigate new photocatalytic materials. A publication resulted from the use of the Sudan red ye model (Coates & Barreto, 2007). Lastly, using the project data as a springboard, we later modified the assays described above to run in an automated fashion on a TECAN well plate reader.
- Using our dye and THA dosimeter assay (see outcome #1 above) we tested many photocatalytic formulations, in order to create more effective photocatalysts, and/or create a photocatalyst which operated in the visible region of the solar spectrum. Our Ti02 doping results have now shown some improvement (but with modest performance increases) but no substantial visible activity was observed. We did discover, develop, and extend the synthesis of a very effective 'titanium foil coated' photocatalyst which produces a tightly adhered coating on the face of a titanium metal; we have continued the development of this coating, which now has a very high performance, favorable physical characteristics, and is very inexpensive and simple to produce, even on very large surface areas of foil. The technology is currently on the pathway to commercialization by the PI and FGCU.
- We developed an automated f1uorometric well plate technique, (using intrinsic red tide algal fluorescence) that is simple and quantitative, to demonstrate the biocidal killing of K. brevis. Our technique replaced the tedious and time consuming alternative of counting cells on a Nannoplankton grid.
- We report the interesting finding that K. brevis appears to be hardy to alkaline killing when using NaOH at pH 9.0 but becomes very sensitive to alkalinity in the presence of ammonia, even at surprisingly low ammonia concentrations (50-100 micromolar, at pH 9). K. brevis is much more susceptible to ammonia killing at alkaline pH than our reference organism, Vibrio fischeri. As an offshoot of validating the V. fischeri killing methodology, we discovered an alkaline biocide technology that can kill all pathogenic microbes, and may prove useful in killing red tide blooms in the field (assuming that a limited and defined volume is being treated). Steps towards commercialization are also being implemented for our germicide as of this writing.
- As part of a dose response experiment to determine the non-lethal range of concentration for ammonia, we noted that K. brevis growth will go into stasis at very low concentration of ammonia. We note that this observation can be used as a chemical trigger for stasis, to explore the molecular biology of gene expression under bloom and stasis conditions.
- We did not handle or collect field samples, so those usual aspects of EPA quality control were not applicable to our project. In accordance with our QNQC plan (filed with EPA for this project), instrument calibrations were routinely and regularly performed, (mostly for fluorescence and absorbance spectrophotometric measurements). Appropriate controls were run for all experiments and weekly meetings of lab personnel were used to troubleshoot and repeat any data points of questionable validity and we ensured that reasonable standard errors bars were obtained for replicate measurements.
Our assays will be of general interest to anyone engaged in determining the efficacy of molecular destruction, with any ROR generating system, not just photocatalysis. In particular, the Sudan red bleaching system is a predictor of membrane damage, because our dye target is encapsulated within the hydrocarbon core of a micelle and this circumstance mimics a natural target within the core of a cell plasma membrane.
Our work may also have a direct application in mitigating the health and economic impacts of toxic red tides. Previously several technologies such as ozone, UV radiation, filtration, and chemical biocides have been proposed to destroy red tide organisms and brevetoxin; thus far, these systems seem impractical in terms of expense, safety and environmental consequences. Most notably, our technological approach may provide protection in an aquaculture situation when natural water is being pumped into a culture pond during a 'red tide' outbreak and must be decontaminated before use. Note that there is an inherent paradox in a system that must be absolutely lethal to pathogens and destructive to toxins, yet pose no chronic harm to the pond organisms being cultured. Our systems are 'switchable'. In a 'treatment canister system', they present a high degree of destructive power to pathogens and toxins yet they can be switched off and pumped into an aquaculture facility. The lifetimes of the killing agents are very short or they can be neutralized, thus solving the paradox described above.
Conclusions:
Based upon our work during this project, we laid the foundation for the development of useful green germicides and decontaminating agents. Our formulations are now in the final stages of development, and will be commercialized in partnership with Florida Gulf Coast University. Unlike other chemical germicides, our formula is composed from substances that are individually benign.
Shipping the components, while keeping them separate, creates no hazard in the event of a spill. When combined, our materials immediately act together synergistically, to form a germicide of great lethality, no known virus, bacterium, or fungus is likely to survive treatment; spores should also be susceptible. Nevertheless, when some of the components evaporate, our germicide loses its lethality, or it can be neutralized at any time by lowering the alkalinity of the solution with a weak acid, (which is also a benign Substance). No residual toxicity remains in either case. The mechanism of killing is generic. In theory, no organism is immune to a lethal process which involves alkalinization of the cell interior; nor is it likely that complete resistance will develop upon prolonged use.
We also developed, tested, and enhanced a new photocatalytic material which produces destructive chemicals upon UV illumination, suitable for decontaminating toxins and destroying pathogens in water. Despite their lethality, these destructive chemicals are naturally occurring, they bio-degrade and/or have very short lifetimes so they do not leave behind any residual toxicity after use. The titanium oxide semiconductor that generates these destructive chemicals is a very benign substance, Ti02 is a filler and whitener in toothpaste, and has replaced lead oxide as the white pigment in most paints.
To summarize, during the term of this project we were able to lay a foundation for the development of two separate 'green' germicidal technologies, an alkaline biocide formulation and a chemical process which generates a new type of high performance photocatalyst. Both technologies were subsequently patented, and the patents are owned by Florida Gulf Coast University. Both patents are pending as of the writing of this report. One manuscript was published, and several presentations were made, based wholly or partly on the data described herein.
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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Barreto JC , Dubetz TA, Brown DW, Barreto PD, Coates CM, Cobb A. Determining the enthalpy, free energy, and entropy for the solubility of salicylic acid with the Van’t Hoff Equation: a spectrophotometric determination of Keq. The Chemical Educator 2007;12(1):18-21. |
EM832982 (Final) |
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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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Supplemental Keywords:
Photocatalysis, detoxification, decontamination, sanitizing, germicide, biocide, marine, estuary, water, exposure, risk, health effects, ecological effects, human health, toxic, toxins, aquatic ecosystem;green chemistry, clean technology, innovative technology, treatment, cleanup, disinfection, cost-benefit, decision making, chemistry, biology, ecology, analytical, chemical modeling, southeast, service industry,algal bloom detection, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Oceanography, algal blooms, Ecology and Ecosystems, brevetoxins, HAB ecology, red tide bloom, titanium oxide treatment, algal bloom detection, algal toxins, K. brevis, Karenia brevisProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.