Grantee Research Project Results

2008 Progress Report: Molecular Approaches to Early Detection and Detoxification of Red Tides

Objective:

An overall objective in the original proposal was the development of a photocatalytic biocidal treatment for a red tide bloom in an enclosed area such as a marina basin, bathing beach or other relatively small protected body of water which might suffer severe adverse economic impact. It is important to note that we are not sanguine about the prospects of treating very large blooms in open water due to the massive volumes involved. In smaller areas a biocidal treatment might be effective, but it needs to be inexpensive and environmentally benign; leaving no residual toxicity in the water and creating no profound adverse effects on other marine creatures.

In developing a photocatalytic biocidal assay, we used Vibrio fischeri as a model target organism because the abolition of luminescence in this organism is widely utilized as a killing assay. In the course of our experiments, we noted that alkalinity (pH >10) might be useful as a red tide biocide. We established a frame of reference for complete killing of V. Fischeri that abolished the luminescence of this microbe. We then devised a series of experiments to investigate the effects of alkaline biocides on K. brevis. 

Progress Summary:

1. The adaptation of a technique that demonstrates biocidal killing, (using the luminescent marine organism V. fischeri was developed to create a frame of reference for alkaline killing of K. brevis.
2. K. Brevis killing assays were developed using cell counting and microscopy; these techniques were time consuming so an automated well plate assay based upon chlorophyll fluorescence was developed using a well plate reader.
3. K. brevis killing by alkalinity and ammonia was investigated.

1. We developed an automated  technique (that  is simple and quantitative) to demonstrate biocidal killing of the red tide organism, K. brevis, using a TECAN well plate reader; this avoids the tedious and time consuming alternative of counting cells on a Nannoplankton grid.
2. While validating the V. fischeri killing methodology, we discovered an alkaline biocide technology that works in the lab, and may prove useful in killing red tide blooms in the field. We now report that K. brevis appears to be very sensitive to alkaline killing, even at relatively low ammonia concentrations (50 micro molar); at pH 9 K. brevis is much more susceptible to ammonia than V. fischeri. Presumably, an electroneutral ammonia flux is driving the alkalinization of the cell cytoplasm in all cases.
3. We also noted that K. brevis growth can be put into stasis at very low concentrations of ammonia.  Perhaps this observation can be used as a chemical trigger for stasis to explore the molecular biology of gene expression under bloom and stasis conditions.

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