Citation:

Brentjens, E., E. Beall, AND R. Zucker. Analysis of Microcystis aeruginosa physiology by spectral flow cytometry: Impact of chemical and light exposure. PLOS Water. Public Library of Science, San Francisco, CA, 2(10):e0000177, (2023). https://doi.org/10.1371/journal.pwat.0000177

Impact/Purpose:

Cyanobacteria (M. aeruginosa) are an important primitive organism which generates over 50% of the oxygen in the world.  However, these photosynthetic organisms can proliferate rapidly to generate harmful algal blooms (HABs) in lakes and reservoirs which can cause detrimental environmental conditions resulting in bad quality water that is used for human consumption.  Rapid cyanobacteria growth results in blooms that are a pervasive environmental issue across the globe as these cyanobacterial blooms constitute a public health threat due to the cyanotoxins they produce. These cyanotoxins can cause acute and chronic illness in humans and even death in domestic animals.  Therefore, understanding their biology, metabolism and life cycle can help reduce the exposure of water containing cyanobacteria toxins to humans, pets, and animals. Knowledge of the existence of the cyanobacteria and their metabolic state is useful information that can be used by authorities to potentially protect our water systems from the  existence of cyanobacteria which is critical to human and ecosystem safety. We have developed a system to rapidly measure single cell cyanobacteria in suspension using a laser-based equipment called flow cytometer that can monitor the specific types of fluorescence derived from cyanobacteria. Using a violet and yellow green laser we were able to derive two different color fluorescence signals: green and red. The increase of the green fluorescence yields a signal that shows cyanobacteria have been affected by their environment or chemicals.  The decrease in the red signal suggests that the cyanobacteria have less photosynthesis and are metabolically compromised.  The change in these two parameters suggest a decrease in metabolic activity that may be used as an early sensitive marker for a change in viability status of the cyanobacteria.   The goal of this study was to examine the impact of H2O2 on M. aeruginosa fluorescence using flow cytometry. By optimizing the concentrations and timing of H2O2 interaction with cyanobacteria, we anticipate that from the toxicity data obtained, we will be able to better treat water prior to human consumption with less potential damage to the environment.

Description:

M. aeruginosa fluorescent changes were observed using a Cytek Aurora spectral flow cytometer that contains 5 lasers and 64 narrow band detectors located between 365 and 829 nm. Cyanobacteria were treated with different concentrations of H2O2 and then monitored after exposure between 1 and 8 days. The red fluorescence emission derived from the excitation of cyanobacteria with a yellow green laser (550 nm) was measured in the 652–669 nm detector while green fluorescence from excitation with a violet laser (405 nm) was measured in the 532–550 nm detector. The changes in these parameters were measured after the addition of H2O2. There was an initial increase in red fluorescence intensity at 24 hours. This was followed by a daily decrease in red fluorescence intensity. In contrast, green fluorescence increased at 24 hours and remained higher than the control for the duration of the 8-day study. A similar fluorescence intensity effect as H2O2 on M. aeruginosa fluorescence emissions was observed after exposure to acetylacetone, diuron (DCMU), peracetic acid, and tryptoline. Minimal growth was also observed in H2O2 treated cyanobacteria during exposure of H2O2 for 24 days. In another experiment, H2O2-treated cyanobacteria were exposed to high-intensity blue (14 mW) and UV (1 mW) lights to assess the effects of light stress on fluorescence emissions. The combination of blue and UV light with H2O2 had a synergistic effect on M. aeruginosa that induced greater fluorescent differences between control and treated samples than exposure to either stimulus individually. These experiments suggest that the early increase in red and green fluorescence may be due to an inhibition in the ability of photosynthesis to process photons. Further research into the mechanisms driving these increases in fluorescence is necessary.

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