Using Advanced Spectroscopy and Organic Matter Characterization to Evaluate the Impact of Oxidation on Cyanobacteria

Abstract

Drinking water treatment plants throughout the world are increasingly facing the presence of toxic cyanobacteria in their source waters. During treatment, the oxidation of cyanobacteria changes cell morphology and can potentially lyse cells, releasing intracellular metabolites. In this study, a combination of techniques was applied to better understand the effect of oxidation with chlorine, ozone, potassium permanganate, and hydrogen peroxide on two cell cultures (Microcystis, Dolichospermum) in Lake Champlain water. The discrepancy observed between flow cytometry cell viability and cell count numbers was more pronounced for hydrogen peroxide and potassium permanganate than ozone and chlorine. Liquid chromatography with organic carbon and nitrogen detection was applied to monitor the changes in dissolved organic matter fractions following oxidation. Increases in the biopolymer fraction after oxidation with chlorine and ozone were attributed to the release of intracellular algal organic matter and/or fragmentation of the cell membrane. A novel technique, Enhanced Darkfield Microscopy with Hyperspectral Imaging, was applied to chlorinated and ozonated samples. Significant changes in the peak maxima and number of peaks were observed for the cell walls post-oxidation, but this effect was muted for the cell-bound material, which remained relatively unaltered.

Keywords: cell morphology; cyanobacteria; enhanced darkfield microscopy/hyperspectral imaging; intracellular organic matter; oxidation.

Figure 1

Impact of chlorine, ozone, potassium permanganate, and hydrogen peroxide on the fraction of viable vs. injured/dead cells and cell numbers. The control sample contained 4 × 10⁵ cells/mL of Microcystis and Dolichospermum in Lake Champlain water. Viable vs. injured/dead cell concentrations were determined with flow cytometry.

Figure 2

Organic matter fractions before (control) and after the oxidation of Microcystis and Dolichospermum in Lake Champlain water. The three different control samples are the same matrix, conducted as separate experiments. Components were identified via LC-OCD-OND based on Huber et al. [41]: BB = building blocks; HS = humic substances; BP = Biopolymer; LMW = low molecular weight.

Figure 3

EDM image of Dolichospermum before oxidation, illustrating different pixels targeted for spectral analysis: intracellular or cell-bound material, cell links, and cell wall. The EDM image for Microcystis with labels for intracellular or cell-bound material and cell wall is shown in Figure S3.

Figure 4

Impact of chlorine (CT = 37.5 mg-min/L) and ozone (0.5 mg/L, 5 min) on Microcystis (a) cell-bound material and (b) cell wall. EDM was used to find a pixel containing only the cell-bound material or cell wall where HSI spectra were collected. Instrument responses were normalized to the maximum value of each spectra for comparison.

Figure 5

Impact of chlorine (CT = 37.5 mg-min/L) and ozone (0.5 mg/L, 5 min) on Dolichospermum (a) cell-bound material, (b) cell wall, and (c) cell links. EDM was used to find a pixel containing only the cell-bound material, cell wall, or links between cells where HSI spectra were collected. Instrument responses were normalized to the maximum value of each spectra for comparison.