A method of batch-purifying microalgae with multiple antibiotics at extremely high concentrations

A bs tract Axenic microalgal strains are highly valued in diverse microalgal studies and applications. Antibiotics,alone or in combination,are often used to avoid bacterial contamination during microalgal isolation and culture. In our preliminary trials,we found that many microalgae ceased growing in antibiotics at extremely high concentrations but could resume growth quickly when returned to an antibiotics-free liquid medium and formed colonies when spread on a solid medium. We developed a simple and highly efficient method of obtaining axenic microalgal cultures based on this observation. First,microalgal strains of different species or strains were treated with a mixture of ampicillin,gentamycin sulfate,kanamycin,neomycin and streptomycin(each at a concentration of 600 mg/L) for 3 days; they were then transferred to antibiotics-free medium for 5 days; and finally they were spread on solid f/2 media to allow algal colonies to form. With this method,five strains of Nannochloropsis sp.(Eustigmatophyceae),two strains of Cylindrotheca sp.(Bacillariophyceae),two strains of T etraselmis sp.(Chlorodendrophyceae) and one strain of Amphikrikos sp.(Trebouxiophyceae) were purified successfully. The method shows promise for batchpurifying microalgal cultures.

Purification of a Diatom and Its Identification to Cylindrotheca closterium

A diatom was purified with colony selection and continuous dilution methods. It was identified to Cylindrotheca closterium according to its morphological characteristics and rbc L and 18 s r RNA gene sequences. The alga was not sensitive to ampicillin and neomycin, but sensitive to chloramphenicol which inhibited its growth at concentrations ranging from 50 to 150 μg m L-1. The purified alga was easy to culture and its specific growth rate was 0.207 ± 0.002(d-1). It was resistant to pollution and could be harvested in an easy way. It was relatively high in lipid content(20.08% ± 0.67% of dry weight) and the combined amount of its 16:0 and 16:1(n-7), the most suitable resource of biodiesel, was as high as 64% of the total fatty acids, while the amount of polyunsaturated fatty acids reached 19.96%–20% of the total fatty acids. Thus the purified C. closterium can be cultured as a biodiesel producer or a nutrition supplement producer.

Association of triacylglyceride content and transcript abundance of genes involving in lipid synthesis of nitrogen deficient Phaeodactylum tricornutum

Phaeodactylum tricornutum is a diatom that is rich in lipids.Recently,it has received much attention as a feedstock for biodiesel production.Nitrogen defi ciency is widely known to increase the content of neutral lipids(mainly triacylglycerides,or TAGs)of microalgae,including P.tricornutum,but the mechanism is unclear.In this study,we deciphered the correlations between TAG content and nine key enzymatic genes involved in lipid synthesis in P.tricornutum.After being cultured under nitrogen-free conditions for 0,4,24,48,72,120,and 168 h,the TAG contents of P.tricornutum cells were assayed and the transcript abundances of the target genes were monitored by quantitative real-time PCR.The results show that the abundances of four target gene transcripts(LACS3,G3PDH2,G3PDH3,and G3PDH5)were positively correlated with TAG content,indicating that these genes may be involved in TAG synthesis in P.tricornutum.The fi ndings improve our understanding of the metabolic network and regulation of lipid synthesis and will guide the future genetic improvement of the TAG content of P.tricornutum.

Genetic Transformation of Nannochloropsis oculata with a Bacterial Phleomycin Resistance Gene as Dominant Selective Marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker.It can control the phleomycin resistance,and significantly increase the tolerance of hosts to zeocin.The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin.We selected ble as the selective marker for the genetic transformation of N.oculata.After the algal cells at a density of 2×10~7 cells mL^(-1) was digested with 4% hemicellulase and 2% driselase for 1 h,the protoplasts accounted for 90% of the total.The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii,yielding a recombinant expression construct p MS188.The construct was transferred into the protoplasts through electroporation(1kV,15 μS).The transformed protoplasts were cultured in fresh f/2 liquid medium,and selected on solid f/2 medium supplemented with 500 ngmL^(-1) zeocin.The PCR result proved that ble existed in the transformants.Three transformants had been cultured for at least 5 generations without losing ble.Southern blotting analysis showed that the ble has been integrated into the genome of N.oculata.The ble will serve as a new dominant selective marker in genetic engineering N.oculata.