Mutation Breeding Of Heamatococcus Pluvialis And Isolation Of Mutants

Astaxanthin(3,3′-dihydroxy-β,β-carotene-4,4′-dione), the major pigment of crustaceans andtrout, has important applications in nutraceutical, cosmetics, food and feed industries as it is astrong colorant and has a lot of biological functions including antioxidation, anticancer,enhancing immunity, UV-light protection and so on. Nowadays, the limited output ofastaxanthin can′t meet the increasing demand. A unicellular green alga Haematococcuspluvialis has the ability to accumulate astaxanthin when was exposed to environmental ornutritional stresses, and is the richest source of natural astaxanthin. Commercial production ofastaxanthin from H. pluvialis has been paid more attention to in recent years and is a growingbusiness worldwide. The rapid growth of H. pluvialis and its high astaxanthin content are thekey issues to boosting the business.The effects of ethyl methanesulphonate (EMS), ultraviolet radiation (UV) and Sodiumajide (NaN3) on cell morphology, movement and division abilities of green alga H. pluvialiswere observed. The lethality rates, survival cell growth rates, astaxanthin contents andaccumulation rates were calculated and some strains were separated.The lethality rates of the mutagens were diffrent. The death rate of H. pluvialis 2 was7%-35% after treated with 0.2%-1.0% EMS for 4h. The death rate of H. pluvialis 0 was 10%-98%after irradiated with UV for 2min-10min. The death rate of H. pluvialis 0 was 20%-100%after treated with 30mg/L-270 mg/L NaN3. The morphology of dead cells was similar while thesurvival cells were different. The figure was blurry and anomalous, the space between the cellwall and membrane disappeared, the color was light green and some white vesicles appeared indead cells. The survial cells after EMS treatment lost the pronounced papilla and flagella on theanterior end and they swam slowly and some non-motile cells appeared, however, most of survivalcells resumed their swimming and dividing on the second day after EMS treatment. The morphology ofsurvival cells after UV irradiation and NaN3 treatment were similar except the cell wall becomethicker, the color of the cell was olive-drab. UV irradiation and NaN3 treatment heavelyrestrained the movments and reproductions of the survivors, they resumed swiming anddividing several days after the treatment. The asexual reproduction in survivors after exposed toUV and NaN3 were not synchronous, and was different from the normal asexual reproductiondue to the mother cells are motile cells instead of non-motile cells. Interesting, some survivorsafter UV irradiation and NaN3 treatment produced many mini-spores (or gamete?), they escapedfrom the mother cell gradually four or five days later. This is quite similar to early describedsexual reproduction. However, we are unable to confirm this was sexual reproduction due to nomating process have been observed afterward.The cell growths and astaxanthin contents had changed after the treatment. The growthrates in most groups were lower than the control group except the group treated with 0.4% EMSin which the growth rate increased 24%. The astaxanthin contents and accumulation rates of thetreated cells increased in most groups after the EMS treatment. In the UV irradiationexperiment, the growth rates in most groups were higher than the control group except thegroup irradiated for 8min. The cell astaxanthin content increased only in one group irradiatedfor 10min;During the NaN3 experiment, the cell growth rates increased 13%,20%and 9%ingroup trated with 60mg/L,120mg/L and 180mg/L while they decreased in groups treated with30mg/L and 240mg/L. The astaxanthin content and accumulation rate increased in treated groupcompared with the control group.Some strains were obtained. About 30 strains were selected out from the survivors after the0.4% EMS treatment. Strains H2-410 and H2-419 showed a fast cell growth behavior with 13%and 20% increase in biomass compared to the wild type, respectively. Then H2-419-4, a fast cellgrowth and high astaxanthin accumulation strain, was obtained by exposed the strain H2-419 toultraviolet radiation (UV) further. The total biomass, the astaxanthin content per cell,astaxanthin production of H2-419-4 showed 68%, 28% and 120% increase compared to itsoriginal wild type H2, respectively. HPLC data also showed an obvious proportional variationof different carotenoid compositions in the extracts of H2-419-4 and the wild type althoughwithout any peak of carotenoids was appeared or disappeared. Therefore, the mainlycompositions in strain H2-419-4 like its original wild type were free astaxanthin, monoester anddiester of astaxanthin.Other strains such as: E-L-22, UV-5-16, UV-5-14, N-2-42, N-2-43 were also determinedthe growth rate or astaxanthin contents, however they were unstable or with higher astaxanthincontent and lower growth rate.