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Contaminations are challenging for monocultures, as they impact the culture conditions and thus influence the growth of the target organism and the overall biomass composition. In phycology, axenic cultures comprising a single living species are commonly strived for both basic research and industrial applications, because contaminants reduce significance for analytic purposes and interfere with the safety and quality of commercial products. We aimed to establish axenic cultures of Limnospira fusiformis, known as the food additive “Spirulina”. Axenicity is strived because it ensures that pathogens or harmful microorganisms are absent and that the harvested biomass is consistent in terms of quality and composition. For the axenic treatment, we applied sterile filtration, ultrasonication, pH treatment, repeated centrifugation, and administration of antibiotics. For testing axenicity, we considered the most common verification method plate tests with Lysogeny Broth (LB) medium, which indicated axenicity after treatments were performed. In addition, we included plate tests with Reasoner’s 2A (R2A) agar and modified Zarrouk+ medium, the latter comparable to the biochemical properties of L. fusiformis’ cultivation medium. In contrast to LB plates, the other media, particularly Zarrouk+, indicated bacterial contamination. We conclude that LB-agar plates are inappropriate for contamination screening of extremophiles. Contamination was also verified by cultivation-independent methods like flow cytometry and 16S rRNA genome amplicon sequencing. We detected taxa of the phyla Proteobacteria, Bacteriodota, Firmicutes and to a lesser extent Verrucomicrobiota. Contaminants are robust taxa, as they survived aggressive treatments. Sequencing data suggest that some of them are promising candidates for in-depth studies to commercially exploit them. Full article

Limnospira fusiformis (also known as Spirulina) is a cyanobacterium that is widely cultivated due to its economic importance. It has specific pigments such as phycocyanin that allow it to grow at different light wavelengths compared to other cultivated algae. Our study investigated the effect of yellow (590 nm) and blue (460 nm) light fields on various biochemical features, including the pigment concentration, protein content, dry weight, and cell ultrastructure of L. fusiformis. Our findings revealed that biomass growth was faster in yellow light compared to blue light, with a higher relative amount of proteins even after one day of exposure. However, after eight days, the relative protein content in yellow versus blue light was not statistically different. Furthermore, in yellow light, we observed a decrease in chlorophyll a, an increase in cyanophycin granules, and an increase in the amount of dilated thylakoids. On the other hand, in blue light, there was an increase in phycocyanin after one day, along with an increase in electron-dense bodies, which are attributable to carboxysomes. However, after eight days, the differences in pigment content compared to the control were not statistically significant. Our study showed that using specific wavelengths during the harvesting phase of spirulina growth can enhance phycocyanin content with blue light (after one day) and biomass, growth rates, and protein content with yellow light after six days. This highlights the biotechnological potential of this approach. Full article

Many phycological applications require the growth and maintenance of pure algae cultures. In some research areas, such as biochemistry and physiology, axenic growth is essential to avoid misinterpretations caused by contaminants. Nonetheless, axenicity—defined as the state of only a single strain being present, free of any other organism—needs to be verified. We compare the available methods to assess axenicity. We first purified unialgal Limnospira fusiformis cultures with an established series of axenicity treatments, and by including two additional treatment steps. The presumable axenic cultures were then tested for their axenic state by applying conventional tests on LB (lysogeny broth) agar-plates, 16S rRNA gene amplicon sequencing, flow-cytometry and epifluorescence microscopy. Only the plate tests indicated axenic conditions. We found a linear relationship between total cell counts of contaminants achieved by flow cytometry and epifluorescence microscopy, with flow cytometry counts being consistently higher. In addition, 16S rRNA gene amplicon sequencing demonstrated its superiority by not only being an efficient tool for axenicity testing, but also for identification of persistent contaminants. Although classic plate tests are still commonly used to verify axenicity, we found the LB-agar-plate technique to be inappropriate. Cultivation-independent methods are highly recommended to test for axenic conditions. A combination of flow-cytometry and 16S rRNA gene amplicon sequencing complement each other and will yield the most reliable result. Full article