Scard, Aaron (2017) Improvement of spectral analysis sensitivity for detecting cyanobacteria in low concentrations in freshwater containing interfering NOM and other microorganisms. PhD thesis, Victoria University.

Abstract

Phycocyanin is an accessory pigment that aids photosynthesis by capturing photons of light in cyanobacteria cultures. Cyanobacteria cultures, often referred to incorrectly as “blue-green algae”, are aqueous bacteria that can produce toxins based on the strain type and gene properties. The toxins produced have reportedly killed livestock and are potentially carcinogenic. Detectable by fluorescence along with chlorophyll-a and some fractions of natural organic matter, phycocyanin is specific to cyanobacteria cell cultures. Detection of phycocyanin in water sources can specifically relate to the presence of cyanobacteria cultures. To determine the concentration of cyanobacteria at up to 2,000 cells/mL in sampled waters, a device was required to isolate cyanobacteria cells, extract the phycocyanin pigment and concentrate phycocyanin to produce a more detectable positive emission signal. This device was known as the Isolated Pigment Analyser (IPA). Cyanobacteria cultures used in this study were Microcystis aeruginosa, Anabaena circinalis and Cylindrospermopsis raciborskii. Green algae cultures used in this study were Chlorella vulgaris and Chlamydomonas reinhardtii. Cyanobacteria isolation was performed by the filtration of intact cyanobacteria cells on 0.45 μm membrane filters. Tubular membranes with a pore size of 0.45 μm were used to collect cyanobacteria cells to enable pre-concentration and separation from dissolved organic matter interferences. Cell disruption by use of a sonication probe liberated phycocyanin from the cell. Phycocyanin was evaluated to be most stable in aqueous solutions; water for a brief period of time or sodium phosphate buffers up to 100mM. The addition of organic solvents into the extraction solution precipitated phycocyanin out of solution. Temperatures should be maintained below 20 degrees Celsius and at optimum pH of 6.0. Increased temperatures above twenty degrees resulted in decreased emission sensitivity. Anionic exchange resin DEAE Sephadex A25 was utilised to separate phycocyanin from natural organic matter and chlorophyll-a. Phycocyanin was captured with DEAE-Sephadex A25 resin and eluted with 0.16-0.27 M NaCl solution. Chlorophyll-a was not retained on the resin at all. Humic acid saturated the column and was retained on the resin. The resin was not able to be regenerated using 1 to 4 M NaCl, even when 1 M NaOH was used in the NaCl regenerated solution. The IPA was able to take an emission signal and measure a phycocyanin concentration without false positives or interference from chlorophyll-a and natural organic matter, and estimated cyanobacteria concentration in cells/mL. The IPA was 99.2 % accurate at cell concentrations <350,000 cells/mL and 85.8 % at cell concentrations <2,000 cells/mL.

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