Isolation,Sterilization And Heterotrophic Cultivation Of Marine Microalgae

Five antibiotics (kanamycin, streptomycin, neomycin, penicillin, gentamycin) and three microalgae (Chlorella sp. C95,Phaeodactylum tricornutum and Isochrysis galbana, 8701) were applied for the study, and the sensitivities of three microalgal strains under three different concentrations of the five antibiotics were detected. The result indicated that all the five antibiotics can inhibit the growth of C95(Chlorella sp).; and except streptomycin, all other four antibiotics can facilitate the growth of Phaeodactylum tricornutum; the sensitivity of 8701(Isochrysis galbana), varied among the five antibiotics. Low concentration of streptomycin can facilitate the growth of 8701 obviously (P<0.01), kanamycin almost had not effect on the 8701, and all other three antibiotics can inhibit the growth of 8701 under high concentration.Bacteria were isolated from C95,Phaeodactylum tricornutum and 8701 for further study and marked as A1-A5, B1-B5 and C1-C3, respectively. The antibacterial inhibitive ability of every antibiotic was investigated. The result indicated that neomycin can inhibit A4 in C95 under high concentration, rather than A1. All the other 4 antibiotics can inhibit all bacteria except A4. Only kanamycin can inhibit B4 efficiently, but it had no affect on B3. Neomycin can inhibit 4 bacteria of them, the other 3 antibiotics can inhibit or kill 3 bacteria. Only neomycin can inhibit C3 significantly, but not C1. Gentamycin had not affect on the three bacteria, while kanamycin can only inhibit C1, and streptomycin and penicillin can inhibit C1 and C2 efficiently.Two combinations of antibiotics were designed after determining the kinds and concentrations of antibiotics. Each combination contained three antibiotics, added three times repeatedly. The result indicated that the combinations can inhibit most bacteria but cannot kill them completely. Three following methods were employed: (1) redesign of the combinations of antibiotics, (2) superaddition once in the heterotrophic f/2 culture, (3) three superadditions of the former combination in three passages. The result indicated that the methods (1) and (2) killed all bacteria in C95; while the bacteria paradoxically increased when three superadditions were performed. C95 was domesticated in heterotrophic culture for 20 days, and the heterotrophic cultivation of C95 was realized. And the growth feature differed vastly between two different cultures, revealed that the composition of culture is one important factor for heterotrophic cultivation.Meatime studies included isolation 24 natural microalgae from Qingdao and Hainan by plate streaking for the sake of enriching microalgae resource. The dry weights of 20 strains were measured, and the ITS sequence of 13 strains were amplified and analyzed for seeking the phylogenical position of them. Result showed that these 13 microalgae belong to three genus, Desmodesmus, Blidingia and Chlorella. The former axenization methods and results were employed to axenized 4 strains of the isolated microalgae, and 1 strain of aseptic microalgae was obtained finally.