Developing new approaches for monitoring and controlling the toxic cyanobacterium Microcystis through flow-cytometric analysis.

Abstract

Biological hazards principally those produced by microorganisms have been identified as a primary concern for drinking water, putting human health at great risk. One major threat to drinking water security is associated with cyanobacteria, where bloom-forming genera like Microcystis can cause anoxic environments, damage filtration systems and produce potent toxins. As future projections of climate change and anthropogenic nutrient loading continue to favour the growth of Microcystis it adds further stress to an already limited supply of clean drinking water, highlighting the need to develop new monitoring and controls of freshwater systems. Here, high through-put, real time protocols using a relatively low cost flow cytometer were developed for identifying, enumerating and analysing the single cell physiology of Microcystis. These methodologies were then adopted for monitoring environmental populations in drinking water supplies and assessed the potential of novel biological, chemical and biochemical controls. Measurements based on light scatter and fluorescence emissions from uni-algal Microcystis culture lines and fresh isolates (derived from a novel technique) were used as calibration for a flow cytometric assay to monitor Microcystis-like cells in a local reservoir. The findings for the first time reported seasonal patterns of Microcystis in a British lowland reservoir, revealing increased local densities during late summer and early autumn with temperature being the most significant factor. To assess potential Microcystis controls the mortality rates in laboratory experiments were also sampled through flow cytometry incorporating molecular probes, which enabled the analysis of single-cell physiological states after exposure to particular stressors. A grazing experiment was carried out which examined the trophic interactions of a ciliate protist, Blepharisma americanum, against a toxic and non-toxic strain of Microcystis. B. americanum died in the presence of toxic Microcystis at the same rate as a nutrient-starved control and recorded no grazing effects on cyanobacterium densities despite ingestion being observed. In contrast, non-toxic Microcystis populations were controlled when grazed by B. americanum with ciliate populations increasing, providing further insight into their ecological role within the microbial loop. The results also contradicted previous experimental organisms which were found to feed on toxic microcystin-rich cyanobacterial cells, contributing to the theory that the secondary metabolite may function as an anti-predatory molecule. A cheap naturally degrading chemical agent (acetic acid) found to control terrestrial photoautotrophs was tested on a fresh isolate of Microcystis. Applications of acetic acid were trailed in parallel with of a well-known anti-cyanobacterial compound (hydrogen peroxide) resulting in the increased formation of reactive oxygen species (ROS), membrane permeability and consequently cell mortality. For the first time in cyanobacteria acetic acid was found to induce ROS and decrease densities. Although hydrogen peroxide had a more effective dose for dose control of Microcystis the concentrations were too high for UK operational limits, which have not been set for acetic acid. The results also highlighted that freshly isolated Microcystis require increased concentrations compared to established culture lines. The production of a biochemical control derived through filtered bi-products from a nutrient depleted Microcystis culture demonstrated an auto-induced sub-lethal and lethal effect on low and exponential Microcystis densities. The cytotoxic effect was dose dependant and only induced cell mortality under light conditions, indicating a potential for anti-Microcystis compounds. Results also emphasised the importance of rigorous testing of any control measure through various cell densities / population life cycles and environmental parameters. The flow cytometric monitoring protocols developed throughout this research can be used for accurate, real time measurements of cyanobacteria like Microcystis in aquatic systems. Enhanced by flow cytometry analysis all three biological, chemical and biochemical controls of Microcystis can potentially be integrated into water treatment management, thereby increasing drinking water security.