Novel approach for the development of axenic microalgal cultures from environmental samples

Abstract

We demonstrated a comprehensive approach for development of axenic cultures of microalgae from environmental samples. A combination of ultrasonication, fluorescence-activated cell sorting (FACS), and micropicking was used to isolate axenic cultures of Chlorella vulgaris Beyerinck (Beijerinck) and Chlorella sorokiniana Shihira & R.W. Krauss from swine wastewater, and Scenedesmus sp. YC001 from an open pond. Ultrasonication dispersed microorganisms attached to microalgae and reduced the bacterial population by 70%, and when followed by cell sorting yielded 99.5% pure microalgal strains. The strains were rendered axenic by the novel method of micropicking and were tested for purity in both solid and liquid media under different trophic states. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene confirmed the absence of unculturable bacteria, whereas fluorescence microscopy and scanning electron microscopy (SEM) further confirmed the axenicity. This is the most comprehensive approach developed to date for obtaining axenic microalgal strains without the use of antibiotics and repetitive subculturing.

Microalgal diversity fosters stable biomass productivity in open ponds treating wastewater

“…All dominant, cultivable, unialgal strains from HRAP were isolated using micropicking followed by continuous serial dilutions 18 . Further morphological analysis followed by phylogenetic analysis of the unialgal strains revealed that these strains were Pediastrum sp., Parachlorella sp., Dictyosphaerium sp., Desmodesmus sp., and Scenedesmus sp.…”

“…Briefly, all cultures were grown in BG11 agar plates and liquid medium in triplicates with a continuous illumination at a light intensity of 130 µmol·m −2 ·s −1 and temperature of 25 °C for seven days. Isolated prokaryotic and eukaryotic strains were identified by sequencing 16S rRNA gene and 18S rRNA gene, respectively 18 . The details of the primer sets are described in the succeeding section.…”

“…Five microalgae strains were isolated by using of a micropipette under a microscope (Nikon F, Japan) followed by dilution in liquid and solid BG11 medium 18 . Choice of microalgae was made based on dominance through biovolume calculations, and cultivability.…”

Improvement of phytoplankton culture isolation using single cell sorting by flow cytometry

“…, Cho et al. ), one reason for this being the generally low level of recovery of viable cultures after sorting.…”

“…Flow cytometry sorting has been successfully applied to recover natural populations for genetic and genomic characterization (Shi et al 2009, Woyke et al 2009, Rinke et al 2013, as well as for physiological measurements such as their capacity to fix carbon (Jardillier et al 2010). However, this technique has relatively rarely been used to bring novel microorganisms into culture (Sensen et al 1993, Sieracki et al 2005, Sinigalliano et al 2010, Cho et al 2013), one reason for this being the generally low level of recovery of viable cultures after sorting.…”