| In this paper, we used high performance liquid chromatography (HPLC)to study about the effect of growth offive species of marine unicellular algae (Isochrysis galbana (Tahitian strain)H29, Isochrysis galbana, Chaetoceros muelleri, Chaetoceros gracilis, Phaeodactylum tricornutum) and the content of carotenoid substances (chlorophyll-a, β-carotene, fucoxanthin, lutein) accumulated at different temperature and different light intensity to provide adequate theoretical and experimental evidence foroptimal environmental condition for culturing five species of marine unicellular algae and access to high-yielding fucoxanthin environmental condition, and providebasic data and experimental technique for screening of high-yielding algae fucoxanthin. The research results are as follows:1. The effect of growth offive species of marine unicellular algae at different temperatureIn this experiment, five kinds of marine unicellular algae were cultured at 5 temperature gradient (15,20,25,30,35 ℃) using double f/2 medium for 12 days. In the cycle of 0,3,6,9,12 daythe number of cells were counted using a hemocytometer method, and at the end of the culture algae was centrifuged and freeze-dried to obtain algae dry weight values. Experimental results show that the cell density oflsochrysis galbana (Tahitian strain)H29increased during culturing period, cells of 15,20,30,35℃ treatment were no longer growing, only 25℃ treatment’s cell density was always growing, and the maximum was 2.365× 106cell/mL, each treatment reach the final cell density in the range of 4.710×105-2.365 ×106cell/mL. The specific growth rate of 20℃ and 25℃were the maximum,The difference between the two treatments was not significant (P> 0.05). SGR of 35 ℃ was minimum of 15.67%. Isochrysis galbana’s cell density showed a gradual increase in 15,25,30,35 ℃ treatment in the first nine days.Only cells of 20℃ were always growing during twelve days whose maximum was 2.683 ×106cell/mL. Each treatment reached the last cell density in the range of 1.065-2.683×106cell/mL. The specific growth rate of 20℃ treatment reached a maximum of 39.02%, significantly higher than the other temperature treatment treatment (P<0.05). The specific growth rate of 35 "Ctreatment exhibited the lowest value of 13.10%. Chaetoceros muelleri’s cell density was increasing up to the 12th day, the cell density of 30 "Ctreatment reached a maximum of 2.294×106cell/mL. The cell density varied in the range of 4.403×105-2.294 × 106cell/mL. The specific growth rate of 15,25,30,35℃temperature treatments was not significantly different(P> 0.05).The lowest value was in 20℃ treatment, which was 3.69%. The cell density of C. gracilis increased with time. Only 20℃ treatment’seell density did not increase after9th dayand reached a maximum 2.222 X 106 cell/mL. SGR of 20℃ treatment reached the maximum of 40.22%. Phaeodactylum tricornutum’s cell density of 15,20,25,30,35℃ treatment gradually increased with time, and cell density of 30,35℃ treatment did not show significant growth, and 35℃ treatment’s cell density showed negative growth. The experimental results were thailsochrysis galbcma (Tahitian strain)H29optimum growth temperature was 20-25℃ ,Isochrysis ga/banaoptimum growth temperature was 20℃, Chaetoceros muelleri optimum temperature was 25-35 ℃,C. gracilis optimum growth temperature was 20 ℃, Phaeodactylum tricornutumshowed poor heat resistance, and optimum growth temperature was 15 ℃. Dry weight of the effects of different temperature conditions on the five species of marine microalgae were not significant (P> 0.05).600mL culture medium of five marine microalgae contained limited nutrient substances, so the amount of biomass remained stable.2. The effect of growth of five species of marine unicellular algae at different light intensityIn this experiment, five kinds of marine unicellular algae were cultured at 5 temperature gradient (50,100,150,200,250μmolm-2s-1) using double f/2 medium for 12 days. In the cycle of 0,3,6,9,12 daythe number of cells were counted using a hemocytometer method, and at the end of the culture algae was centrifuged and freeze-dried to obtain algae dry weight values.These results are that Isochrysis galbana (Tahitian strain)H29 cell density maximum was in 150μmolm-2s-1 and 200μmolm-2s-1 treatment, but there was no significant difference between the two treatments. Cell density in the range of 12 days for each treatment was in the rangeof 1.219-2.365 ×106cell/mL. The SGR of 200μmolm-2s-1 treatment was significantly higher than other light treatment. The maximum reached 24.20%. Isochrysis galbana cell density in 150μmolm-2s-1 and 200μmolm-2s-1 was higher than other treatments,but there was no significant difference between them, which were 1.867-1.881× 106cell/ mL. Cell density of each treatment in light intensity was in the range of 1.219-2.253 X 106/mL. SGR value of 200μmolm-2s-1 and 250μmolm-2s-1 treatment was no significantdifference, which were higher than the other treatment groups. Chaetoceros muelleri in 200μmolm-2s-1 treatment reached maximum value 2.202×106cell/mL. SGR value of 200μmolm-2s-1 was significantly higher than other light treatments, which was 27.73%. The cell density of C. gracilisin 250μmolm-2s-1 treatment was the highest value, the average reached 1.382×106cell/mL. The cell density of five treatment in light intensity was in the range of 7.475×105-1.731×106cell/mL, Phaeodactylum tricornutum cell density in 12thday of treatment 200μmolm-2s-1 reached the highest value of 2.202×106cell/mL. Phaeodactylum tricornutum in 50μmolm-2s-1 and 200μmolm-2s-1 treatment’s SGR value was significantly higher than other light treatment group, the difference was not significant between the three, with an average of 17.64%-19.77%. The experimental results were that Isochrysis galbana (Tahitian strain)H29 optimum light intensity was 200μmolm-2s-1, Isochrysis galbana optimal light intensity was 150-200μmolm-2s-1 Chaetoceros muellerioptimum light intensity was 200μmolm-2s-1, Phaeodactylum tricornutum optimal light intensity was 200μmolm-2s-1.Dry weight of different light intensity condition treatment on five kinds of marine microalgae cell was not significant (P> 0.05).600mL culture medium of five marine microalgae contained limited nutrient substances, so the amount of biomass remained stable.3.Effect of temperature conditions on the five marine unicellular algae in vivo accumulation of carotenoid substancesIn this study, five temperature gradient of 15,20,25,30,35℃ was seton five kinds of marine unicellular algae to determine four kinds of pigment (chlorophyll-a,β-carotene, fucoxanthin, lutein) using liquid chromatography system for qualitative and quantitative analysis. The results were that Isochrysis galbana (Tahitian strain)H29 light-harvesting pigment chlorophyll-a, fucoxanthin,β-carotene content in low temperature treatmentof 15℃was higher. And chlorophyll-a and fucoxanthin content ratio was maintained at about 3. Isochrysis galbanapigment β-carotene and fucoxanthin content with temperature change trend were consistent with the content of chlorophyll-a, reaching maximum at 15℃. Fucoxanthin content varied with the change of temperature between 2.033-4.686 mg/g. As temperature rose, four kinds of pigments’s content in Chaetoceros muellerishowed two peaks at 20℃and 35 ℃.The content of chlorophyll-a was always higher than the other pigments, lutein content was generally lower than other pigments. Chlorophyll-a,β-carotene, fucoxanthin contents reached a maximum value at 35℃,which were respectively 16.61 mg/g,4.28mg/g,5.74mg/g. C. gracilis’s four pigments contents reached two peaks, which appeared at 15℃and 30 ℃. Chlorophyll-a content was always higher than the other pigments. Lutein content was always the lowest. Chlorophyll-a, fucoxanthin, β-carotene content group had a maximum at 30℃, respectively 14.06mg/g,2.39 mg/g,1.19 mg/g. four pigment contents of Phaeodactylum tricornutum with temperature trends are also consistent, the maximum appeared at 30 ℃. Chlorophyll-a,β-carotene, fucoxanthin, lutein contents were 10.19 mg /g,2.34 mg/g,3.05 mg/g,0.70 mg/g.The experimental results were that five kinds of marine unicellular algae light-harvesting pigment (chlorophyll-a,β-carotene, fucoxanthin) under different temperature conditions were basically the same trends which were higher under optimum temperature. Light protection pigment (lutein) was affected little by temperature changes. The ratio of chlorophyll-a and fucoxanthin inlsochrysis galbana stabilized at about 3, indicating that the ratio can be used as species classification.4.Effect of light intensity conditions on the five marine unicellular algae in vivo accumulation of carotenoid substancesIn this study, five temperature gradient of 50,100,150,200,250μmoln-1 was seton five kinds of marine unicellular algae to determine four kinds of pigment (chlorophyll-a, β-carotene, fucoxanthin, lutein) using liquid chromatography system for qualitative and quantitative analysis.Light intensity conditions had close contact with pigment accumulation of marine microalgae species, lsochrysis galbana (Tahitian strain)H29 pigment content first rose then declined light intensity increased. Chlorophyll-a content changes were consistent with fucoxanthin content changes, their ratio roughly stablized at about 3, and the maximum appeared at 150μmolm-2s-1 which were 9.62 mg/g,2.98 mg/g. P-carotene content peaked at 100μmolm-2s-1 which was 4.15mg/g. Lutein in high light intensity treatments showed higher values, the highest value reached 2.49 mg/g, showing that frond at a high light intensity would synthesized more light protective pigment to prevent high light damage. Four pigment contents of Isochrysis galbana first rose then declined as light intensity gradually increased, reaching a peak at 100μmolm-2s-1 treatment. Chlorophyll-a, β-carotene, fucoxanthin, lutein contents were 11.17 mg/g,10.54 mg/g,3.86 mg/g, 1.84 mg/g. Chaetoceros muelleri’s chlorophyll-a, fucoxanthin content changes were consistent with changes in light intensity. And there were two peaks at 50 and 200μmolm-2s-1, the maximum was at 50μmolm-2s-1, their values were 4.26 mg/g, 2.23 mg/g. maximum of β-carotene appeared at 200μmolm-2s-1, which was 1.76 mg /g. Maximum Lutein content also appeared at high light treatment, which was 1.28 mg/g. C. gracilis’s chlorophyll-a, fucoxanthin contents showed gradually decreasing variation. The maximum appeared at 50μmolm-2s-1, whose values were 4.25 mg/g, 2.43 mg/g. P-carotene content was less affected by light intensity, and changed little. Lutein content was lower in low-light treatment. The peak appeared at 250μmolm-2s-1 which was 1.28 mg/g. Chlorophyll-a, fucoxanthin contents in Phaeodactylum tricornutum levels were higher in low-light group, and lower in high-light group. The maximum values were 5.35 mg/g,2.75 mg/g. β-carotene, lutein content in Phaeodactylum tricornutum was lower, and effected little by light intensity.The experiment concluded that the light intensity affected photosynthesis and pigment biosynthesis in the algal cells.and had a very close relationship with pigment. Light-harvesting pigment content of algae (chlorophyll-a,β-carotene, fucoxanthin) increased with light intensity trends consistently, which was high in low-light treatment, in order to promote photosynthesis for the growth. Photoprotective pigments (lutein) content in the high-light group increased in algal cells to protect against high light damage. |
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