Abstract
Filamentous cyanobacteria, such as Anabaena 7120 have great industrial potential due to their capability to be genetically engineered to produce next-generation biofuels while using minimal nutrients. One challenge of working with these microbes is that classical methods of quantifying cell viability are not effective due to their filamentous morphology. Therefore, fluorescent assays were evaluated to determine if they could be used as a reliable indicator of cell viability. Initially, a dual-stain assay using SYTO® 9 and SYTOX® Blue was investigated. Fluorescence from SYTO® 9 was accurately correlated with viable cells; however, SYTOX® Blue did not work as a non-viable cell indicator due to non-specific binding in both viable and non-viable cells. Autofluorescence from light harvesting pigments was also evaluated as a viable cell indicator, but unfortunately these pigments resulted in several emission peaks that couldn’t be captured by a single emission filter. Moreover, certain light harvesting pigments continued to fluoresce after the cell became non-viable. SYTO® 9 was then compared to absorbance and chlorophyll content to quantify viable Anabaena 7120 in a chemical inhibition testing protocol. This protocol requires a low initial biomass concentration to prevent binding of the chemicals to cell biomass, and at low cell densities SYTO® 9 was superior to absorbance and chlorophyll content in quantifying viability. It was also determined that SYTO® 9 allows for the evaluation of different cultivation media on the growth of cyanobacteria in photobioreactors. SYTO® 9 is a reliable, accurate indicator of viability of filamentous cyanobacteria and can be used in a high-throughput manner via a microplate reader.
Highlights
Developing processes to produce renewable fuels and chemicals remains important, as fossil fuel reserves are finite and the adverse effects of fossil fuel generated greenhouse gases are well documented (Chen et al, 2011; Von Blottnitz and Curran, 2007)
To determine if a dual-stain assay consisting of SYTO® 9 and SYTOX® Blue is capable of accurately quantifying the viability of filamentous cyanobacteria, calibration curves were generated to correlate the ratio of green (SYTO® 9, viable cells) to blue (SYTOX® Blue, non-viable cells) fluorescence against known mixtures of viable and non-viable cells
The study described showed that SYTO® 9 is a reliable and accurate indicator of filamentous cyanobacteria viability
Summary
Developing processes to produce renewable fuels and chemicals remains important, as fossil fuel reserves are finite and the adverse effects of fossil fuel generated greenhouse gases are well documented (Chen et al, 2011; Von Blottnitz and Curran, 2007). Many strains of filamentous cyanobacteria are diazotrophic, using specialized cells called heterocysts to fix atmospheric nitrogen. These attributes have led to cyanobacteria emerging as a promising platform organism for production of fuels and chemicals (Schoepp et al, 2014). SYTOX® Blue is one of several SYTOX® Dead Cell Stains that should not be able to cross intact cell membranes This fluorochrome has been successfully used to quantify non-viable cells in bioaggregates (Chen et al, 2007) and in aerobic granules (Adav et al, 2007a). SYTOX® Blue was tested as a method to quantify non-viable filamentous cyanobacteria. SYTO® 9 was evaluated for monitoring the effect of different cultivation media on cyanobacterial growth in 40 L PBRs
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