Cell Fusion between Haematococcus pluvialis and Chlorella kessleri and Screening of Glucose-heterotrophic Fusants

To improve heterotrophic growth ability of Haematococcus pluvialis,improve the efficiency of large-scale cultivation,cell hybridization and fusant screening between H.pluvialis SCCAP K-0084 and heterotrophic Chlorella SAG211.11a were carried out through PEG-mediated protoplast fusion. A screening condition only permiting heterotrophic growth based on glucose along with addition of hygromycin was utilized to improve the screening efficiency,which could inhibite growth of wild-typle H.pluvialis and C.kessleri,respectively. Results showed that,Chlorella kessleri SAG211.11a had strong heterotrophic growth capacity,which could metabolite glucose,fructose,acetate,glycerol,malic acid and succinic acid in its heterotrophic growth,while three H.pluvialis strains could just utilize acetate for its growth. And H.pluvialis SCCAP K-0084 exhibited strong hygromycin-resistant ability. These characters could be applied in the process of fusant screening. Protoplast-releasing rates respectively reached 72.11%±3.94% and 42.07%±3.73% in H.pluvialis and C.kessleri. And fusants with Haematococcus form were obtained on the screening condition only permiting heterotrophic growth on glucose after effective cell fusion identified through microscopic check. These fusants had faster growth than wild type H.pluvialis in the condition with glucose as substrates. After 10 d cultivation,the cell densities respectively arrived at 6.7×10⁴,8.7×10⁴ and 6.5×10⁴ mL^-1 in three independent fusants,obviously higher than 2.45×10⁴ in wild type H.pluvialis. However,they were obviousy lower than 1.4×10⁶ mL^-1 in C.kessleri,which meant a low-efficient heterotrophic growth on glucose. It was also found that,many fusants were easy to lose their capacity of glucose utilization with extended inoculation. That might be due to inefficient recombination between two genomes.