Development Of Transgenic Flocculating Scenedesmus Obliquus And Characterization Of The Flocculation Of S. Obliquus

Microalgae have attracted worldwide attention for their potentials in addressing the problems of environmental pollution and energy shortage, due to their ability in CO2mitigation, degradation of nutrients in water systems, and capture energy from sunlight. However, high cost in biomass recovery is one of the bottlenecks for their applications at large scales, and engineering microalgae with flocculating phenotype for cost-effective biomass recovery by sedimentation is one of the solutions.Scenedesmus obliquus is widely used as feed additives, which has also been considered as a potential feedstock for biodiesel production. In this thesis, the genetic manipulation platform of S. obliquus was developed, and the freely-suspended S. obliquus FSP-3was engineered with the flocculating property by expressing the gene FLO1from Saccharomyces cerevisiae for cost-effective biomass recovery. On the other hand, the flocculating property of spontaneously flocculating S. obliquus AS-6-1was studied.Axenation of S. obliquus FSP-3and AS-6-1was established first, which was the key for investigating their physiological, biochemical and genetic properties. Washing and series agar culture were employed to remove mould contaminations, and antibiotics combined with lysozyme/SDS treatments were used to control bacteria. As a result, pure cultures were obtained, and culture conditions were optimized. For S. obliquus FSP-3and AS-6-1, inoculation density, light intensity and initial pH were1×106cells/mL,6000lx and6.5, but temperatures were controlled at30℃and28℃, respectively. Consequently, the growth of S. obliquus FSP-3and AS-6-1was facilitated, and more intracellular components such as polysaccarides, proteins and lipids were accumulated.S. obliquus FSP-3was then transformed by electroporation with the plasmid pCAMBIA1302-CAT containing CaMV35S as a promoter, green fluorescent protein gene (gfp) as a reporter and chloramphenicol resistance gene (cat) as a selective marker. Parameters for the electroporation were studied, and a high transformation efficiency of494+48positive transgenic clones per106recipient cells was obtained under the conditions:50ug/mL plasmid,0.2mol/L osmotic solution,2ras pulse duration and2kV pulse voltage. The transformants were subcultured for ten months without significant degeneration. These results are leading microalgal transformation studies. Furthermore, green fluorescence signals were detected with the transformants by fluorescence microscopy and flow cytometry, indicating that the cassette was functional, and the genes between T-border were integrated into the genome of&obliquus FSP-3, which was validated by PCR, Southern blot and RT-PCR analysis. It is the first time that the efficient and stable transformation system for S. obliquus has been developed, which lays foundation for its genetic modifications.Furthermore, the plasmid containing FLO1was transformed into the freely-suspended S. obliquus FSP-3cells using the transformation system, and transformants with the flocculating phenotype were screened. The FLO1-specific products were observed on their cell surface by fluorescence microscopy and SEM. PCR and RT-PCR were performed to validate the expressing of FLO1. Therefore, the yeast FLO1gene was successfully expressed in S. obliquus FSP-3and endowed it with the flocculating phenotype. No significant difference was observed in growth between the transformants and their wild type strain.The flocculation of the transgenic S. obliquus FSP-3-FLO1was compared with that of S. obliquus AS-6-1. While large floes developed with S. obliquus FSP-3-FLO1were more suitable for biomass recovery by sedimentation, spontaneously flocculating S. obliquus AS-6-1exhibited ability to flocculate the freely-suspended S. obliquus FSP-3and C vulgaris CNW-11. Moreover, the flocculation of S. obliquus AS-6-1was thermal tolerant, and not sensitive to pH shift, EDTA and sugars, which might relate to the stability of its flocculating substances, while the flocculation of FSP-3-FLO1was sensitive to high temperature and pH shift, and inhibited by mannose and galactose, because of the expression of FLO1encoding the protein for the flocculating phenotype.At the end, the flocculating substance from S. obliquus AS-6-1was studied, which was identified to be polysaccharides with an average molecular weight of1.279×105Da, composing of neutral sugars, uronic acid and amino sugars at the ratio of16:9:1. Sugar analysis indicated that the monomers consist of glucose, mannose, galatose, rhamnose and fructose with the molar ratio of8:5:3:2:1. These flocculating substances exhibited ability to flocculate S. obliquus FSP-3and C. vulgaris CNW-11at a dosage of0.6mg/L. Meanwhile, the flocculating substance was thermo-stable, and more than65%of the initial flocculating activity was retained in the temperature range of20-60℃. In addition, it was not sensitive to pH shift.In conclusion, the transformation platform established in this work provides basis for the genetic engineering of S. obliquus. Engineering S. obliquus FSP-3with FLO1for its flocculation and study of the spontaneous flocculation of S. obliquus AS-6-1benefit their further applications at large scales, particularly for cost-effective biomass recovery.