Abstract
The cultivation of cyanobacteria with the addition of an organic carbon source (meaning as heterotrophic or mixotrophic cultivation) is a promising technique to increase their slow growth rate. However, most cyanobacteria cultures are infected by non-separable heterotrophic bacteria. While their contribution to the biomass is rather insignificant in a phototrophic cultivation, problems may arise in heterotrophic and mixotrophic mode. Heterotrophic bacteria can potentially utilize carbohydrates quickly, thus preventing any benefit for the cyanobacteria. In order to estimate the advantage of the supplementation of a carbon source, it is essential to quantify the proportion of cyanobacteria and heterotrophic bacteria in the resulting biomass. In this work, the use of quantitative polymerase chain reaction (qPCR) is proposed. To prepare the samples, a DNA extraction method for cyanobacteria was improved to provide reproducible and robust results for the group of terrestrial cyanobacteria. Two pairs of primers were used, which bind either to the 16S rRNA gene of all cyanobacteria or all bacteria including cyanobacteria. This allows a determination of the proportion of cyanobacteria in the biomass. The method was established with the two terrestrial cyanobacteria Trichocoleus sociatus SAG 26.92 and Nostoc muscorum SAG B-1453-12a. As proof of concept, a heterotrophic cultivation with T. sociatus with glucose was performed. After 2 days of cultivation, a reduction of the biomass partition of the cyanobacterium to 90% was detected. Afterwards, the proportion increased again.
Highlights
Cyanobacteria are an interesting source of natural bioactive compounds (Chlipala et al 2011) that possess antibacterial (Gantar et al 2011; Lamprinou et al 2015), antimycotic (Swain et al 2017) and anticancer (Kinghorn et al 2016) activity
If a carbohydrate is added to cyanobacteria cultivated under phototrophic conditions, it is possible that a heterotrophic bacterium will use most of it
The DNA yields achieved were sufficient for the quantitative polymerase chain reaction (qPCR) analyses performed
Summary
Cyanobacteria are an interesting source of natural bioactive compounds (Chlipala et al 2011) that possess antibacterial (Gantar et al 2011; Lamprinou et al 2015), antimycotic (Swain et al 2017) and anticancer (Kinghorn et al 2016) activity. Another problem during culturing cyanobacteria is adherent heterotrophic bacteria. If a carbohydrate is added to cyanobacteria cultivated under phototrophic conditions, it is possible that a heterotrophic bacterium will use most of it This falsifies the measured growth rate for the cyanobacterium. The growth of cyanobacteria is usually measured via the cell dry weight or the optical density of the cell suspension Both methods do not distinguish between cyanobacteria and heterotrophic bacteria. The simplest variant is microscopic evaluation where the cells of the different species can be distinguished and counted to determine the composition of the biomass This method is only suitable for the quantification of sized cells such as microalgae and cyanobacteria (Arias et al 2019). This method is limited to planktonically growing cyanobacteria and not suitable for filamentous- or biofilmforming species
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