海洋底栖硅藻的筛选、培养和应用研究
摘要
采用海区悬挂网袋的方法对大连近海底栖硅藻进行了采集，并通过平板法分离出单一的底栖硅藻。对筛选到的底栖硅藻分别从生物学特征、营养成分、生长环境条件以及饵料应用等方面进行了研究和分析，探讨单一底栖硅藻作为海洋经济动物幼体的饵料的可行性及其潜在应用价值。
本文共分离出50余株单细胞底栖硅藻，并从其中筛选出8种有潜在开发价值的单一底栖硅藻。经鉴定它们分别为双菱缝舟藻、半裸舟形藻、双尖菱板藻细头变种、易变双眉藻眼状变种、盔状舟形藻、菱形藻、小形舟形藻和咖啡形双眉藻；其细胞大小在6.7μm×3.3μm-35.9μ×9.6μm之间，生长速率为0.44 div／d-0.73 div／d，生长过程中均能分泌胞外多聚物且细胞壁较薄，使其具有一定的附着能力和可消化性；上述特点可适宜不同生长阶段海洋经济动物幼体的摄食习性。
研究结果表明：在不同生长时期内8种底栖硅藻细胞内的蛋白质、胞内总糖、胞外总糖、粗脂肪和灰分含量分别为1.21％-9.73％、2.67％-20.76％、2.08mg／L-16.23 mg／L、26.03％-50.71％和18.18％-4810％。其中蛋白质含量较高的是半裸舟形藻、双尖菱板藻细头变种、菱形藻和咖啡双眉藻；后两种胞内、外总糖含量也均较高；粗脂肪含量最高的是易变双眉藻眼状变种；灰分含量最高的是双菱缝舟藻。脂肪酸组成因硅藻种类和不同生长时期而差异较大。盔状舟形藻脂肪酸组成最丰富，半裸舟形藻、双尖菱板藻细头变种、盔状舟形藻、小形舟形藻和咖啡双眉藻含有EPA和DHA。8种底栖硅藻矿物元素含量也很丰富，半裸舟形藻、双尖菱板藻细头变种和菱形藻部分矿物元素含量最高。
随着培养时间的延长，硅藻胞内蛋白质含量在第8-15天达到较高；胞内、外总糖含量的总体趋势升高；粗脂肪在第8天含量最高；灰分是第3天和第25天含量较高；脂肪酸在培养后期组成较丰富。
8种底栖硅藻的适宜和最适生长温度、光照、盐度和pH范围分别为15℃-30℃和20℃-25℃、500 lx-9500 lx和1500 lx-5500 lx、25-50和30-35、7-9和8-8.5。温度和pH对各营养成分含量的影响均较小，低温更利于蛋白质的积累，温度过低或过高都不利于脂肪的合成。光照和盐度对蛋白质和胞内总糖含量的影响相对较小，对胞外总糖和粗脂肪的含量影响相对较大，略低或略高于最适光照更利于营养物质的合成，低盐度条件下合成蛋白质较多，高盐度条件下更利于胞内、外总糖和粗脂肪的合成。
藻密度相同条件下，部分单种底栖硅藻饵料效果优于海区混合种。菱形藻、双菱缝舟藻和易变双眉藻眼状变种硅藻能强烈诱导虾夷马粪海胆幼体附着，附着率分别高出海区混合底栖硅藻对照组的18.33％、13.35％和6.68％。摄食盔状舟形藻、易变双眉藻眼状变种和菱形藻硅藻的海胆与海区混合硅藻对照组相比生长快、存活率高，壳径分别比对照组长55.2μm、42.82μm和39.45μm，存活率分别高出19.36％、18.77％和9.24％。皱纹盘鲍幼体在菱形藻、双尖菱板藻细头变种、半裸舟形藻和盔状舟形藻硅藻板上的附着率均高于海区混合底栖硅藻对照组，分别高出20.00％、18.33％、13.33％和6.67％。以菱形藻、双菱缝舟藻和盔状舟形藻硅藻为食的鲍幼体比海区对照组生长快，而存活率高只有菱形藻硅藻高于海区对照组，存活率为95.33％，超过海区对照组8.96％。
Benthic diatoms collected from hanging cages which were suspended beneath the surface of the coastal seawater near the seed production hatcheries were isolated by spreading plating method and their potential as food for aquaculture evaluated based on their biology characteristics, nutritional component, growth conditions and application to sea animals.
The results show that there are eight benthic diatoms which are Rhaphoneis surirella, Navicula seminulum, Hantzschia amphioxys var. leptocephala, Amphora proteus var. oculata, Navicula corymbosa, Nitzschia sp., Navicula parva and Amphora coffeaeformis. All species were within the size range ingested by juvenile sea urchins and postlarval abalone, i.e. 6.7μm×3.3μm-35.9μm×9.6μm. Cellular growth rates ranged from 0.44 divisions·day~(-1)(Rhaphoneis surirella) to 0.73 divisions-day~(-1)(Navicula seminulum).
The contents of protein, intracellular carbohydrate, extracellular carbohydrate, lipid and ash weight from eight benthic diatom species at four growth rates are 1.21%-9.73%、2.67%-20.76%、2.08mg/L-16.23mg/L、26.03%-50.71%and 18.18%-48.10%, respectively. Protein contents of N. seminulum, H. amphioxys var. leptocephala, Nitzschia sp. and A. coffeaeformis are higher than other diatoms. The contents of intracellular carbohydrate and extracellular carbohydrate from Nitzschia sp. and A. coffeaeformis are higher. Lipid content of A. proteus var. oculata is the highest in all diatoms. Ash weight of R. surirella is highest than the others. The fatty acid composition differs between diatoms at different growth phases. Among them, N. corymbosa is most abundant in fatty acid composition, and there are EPA and DHA in N. seminulum, H. amphioxys var. leptocephala, N. corymbosa, N. parva and A. coffeaeformis. Moreover, all diatoms are also rich in Mineral-element composition
Nutritional component showed significant differences between diatoms at 3, 8, 15 and 25 days. Highest contents of protein and intracellular carbohydrate from diatoms are found at 8 and 15 days. Extracellular carbohydrate contents of diatoms increase always during the experiment. Highest lipid content is found at day 8. Diatoms have the highest amounts of ash at 3 and 25 days. Fatty acid is most abundant at day 15.
The favourable and optimum temperature, light intensity, salinity and pH conditions for eight benthic diatom species are 15℃-30℃and 20℃-25℃, 500 lx-9500 lx and 1500 lx-55001x, 25-50 and 30-35, 7-9 and 8-8.5. Temperature and pH do less significantly affect all nutrient contents of diatoms. Protein contents of diatoms increase under lower temperature condition, while lower and higher temperature affect lipid contents of diatoms. Light intensity and salinity do less significantly affect protein and intracellular carbohydrate contents of diatoms, and significantly affect extracellular carbohydrate and lipid contents of diatoms. Nutritent contents are higher at lower and higher light intensity than at optimum one. Protein contents of diatoms cultured under lower salinity condition are higher, and intracellular carbohydrate, extracellular carbohydrate and lipid contents cultured under higher salinity condition are higher.
A significantly higher number of larval abalone settled on N. seminulum, H. amphioxys var. leptocephala and compared to the control and others plates. Moreover, Nitzschia sp. and N. corymbosa strongly induce larval settlement of Strongylocentrotus intermedius and Haliotis discus hannai. Growth and survival of abalone are also greatest on R. surirella, and that of sea urchin on Amphora proteus var. oculata are higher than ones on other diatoms. Nitzschia sp. and N. corymbosa also accelerate growth and survival of S. intermedius and H. discus hannai. These results indicate the effectiveness of Nitzschia sp., R. surirella, A. proteus var. oculata and N. corymbosa as single species over mixed diatoms and natural diatoms in larval settlement and juvenile growth of S. intermedius and H. discus hannai.
本文共分离出50余株单细胞底栖硅藻，并从其中筛选出8种有潜在开发价值的单一底栖硅藻。经鉴定它们分别为双菱缝舟藻、半裸舟形藻、双尖菱板藻细头变种、易变双眉藻眼状变种、盔状舟形藻、菱形藻、小形舟形藻和咖啡形双眉藻；其细胞大小在6.7μm×3.3μm-35.9μ×9.6μm之间，生长速率为0.44 div／d-0.73 div／d，生长过程中均能分泌胞外多聚物且细胞壁较薄，使其具有一定的附着能力和可消化性；上述特点可适宜不同生长阶段海洋经济动物幼体的摄食习性。
研究结果表明：在不同生长时期内8种底栖硅藻细胞内的蛋白质、胞内总糖、胞外总糖、粗脂肪和灰分含量分别为1.21％-9.73％、2.67％-20.76％、2.08mg／L-16.23 mg／L、26.03％-50.71％和18.18％-4810％。其中蛋白质含量较高的是半裸舟形藻、双尖菱板藻细头变种、菱形藻和咖啡双眉藻；后两种胞内、外总糖含量也均较高；粗脂肪含量最高的是易变双眉藻眼状变种；灰分含量最高的是双菱缝舟藻。脂肪酸组成因硅藻种类和不同生长时期而差异较大。盔状舟形藻脂肪酸组成最丰富，半裸舟形藻、双尖菱板藻细头变种、盔状舟形藻、小形舟形藻和咖啡双眉藻含有EPA和DHA。8种底栖硅藻矿物元素含量也很丰富，半裸舟形藻、双尖菱板藻细头变种和菱形藻部分矿物元素含量最高。
随着培养时间的延长，硅藻胞内蛋白质含量在第8-15天达到较高；胞内、外总糖含量的总体趋势升高；粗脂肪在第8天含量最高；灰分是第3天和第25天含量较高；脂肪酸在培养后期组成较丰富。
8种底栖硅藻的适宜和最适生长温度、光照、盐度和pH范围分别为15℃-30℃和20℃-25℃、500 lx-9500 lx和1500 lx-5500 lx、25-50和30-35、7-9和8-8.5。温度和pH对各营养成分含量的影响均较小，低温更利于蛋白质的积累，温度过低或过高都不利于脂肪的合成。光照和盐度对蛋白质和胞内总糖含量的影响相对较小，对胞外总糖和粗脂肪的含量影响相对较大，略低或略高于最适光照更利于营养物质的合成，低盐度条件下合成蛋白质较多，高盐度条件下更利于胞内、外总糖和粗脂肪的合成。
藻密度相同条件下，部分单种底栖硅藻饵料效果优于海区混合种。菱形藻、双菱缝舟藻和易变双眉藻眼状变种硅藻能强烈诱导虾夷马粪海胆幼体附着，附着率分别高出海区混合底栖硅藻对照组的18.33％、13.35％和6.68％。摄食盔状舟形藻、易变双眉藻眼状变种和菱形藻硅藻的海胆与海区混合硅藻对照组相比生长快、存活率高，壳径分别比对照组长55.2μm、42.82μm和39.45μm，存活率分别高出19.36％、18.77％和9.24％。皱纹盘鲍幼体在菱形藻、双尖菱板藻细头变种、半裸舟形藻和盔状舟形藻硅藻板上的附着率均高于海区混合底栖硅藻对照组，分别高出20.00％、18.33％、13.33％和6.67％。以菱形藻、双菱缝舟藻和盔状舟形藻硅藻为食的鲍幼体比海区对照组生长快，而存活率高只有菱形藻硅藻高于海区对照组，存活率为95.33％，超过海区对照组8.96％。
Benthic diatoms collected from hanging cages which were suspended beneath the surface of the coastal seawater near the seed production hatcheries were isolated by spreading plating method and their potential as food for aquaculture evaluated based on their biology characteristics, nutritional component, growth conditions and application to sea animals.
The results show that there are eight benthic diatoms which are Rhaphoneis surirella, Navicula seminulum, Hantzschia amphioxys var. leptocephala, Amphora proteus var. oculata, Navicula corymbosa, Nitzschia sp., Navicula parva and Amphora coffeaeformis. All species were within the size range ingested by juvenile sea urchins and postlarval abalone, i.e. 6.7μm×3.3μm-35.9μm×9.6μm. Cellular growth rates ranged from 0.44 divisions·day~(-1)(Rhaphoneis surirella) to 0.73 divisions-day~(-1)(Navicula seminulum).
The contents of protein, intracellular carbohydrate, extracellular carbohydrate, lipid and ash weight from eight benthic diatom species at four growth rates are 1.21%-9.73%、2.67%-20.76%、2.08mg/L-16.23mg/L、26.03%-50.71%and 18.18%-48.10%, respectively. Protein contents of N. seminulum, H. amphioxys var. leptocephala, Nitzschia sp. and A. coffeaeformis are higher than other diatoms. The contents of intracellular carbohydrate and extracellular carbohydrate from Nitzschia sp. and A. coffeaeformis are higher. Lipid content of A. proteus var. oculata is the highest in all diatoms. Ash weight of R. surirella is highest than the others. The fatty acid composition differs between diatoms at different growth phases. Among them, N. corymbosa is most abundant in fatty acid composition, and there are EPA and DHA in N. seminulum, H. amphioxys var. leptocephala, N. corymbosa, N. parva and A. coffeaeformis. Moreover, all diatoms are also rich in Mineral-element composition
Nutritional component showed significant differences between diatoms at 3, 8, 15 and 25 days. Highest contents of protein and intracellular carbohydrate from diatoms are found at 8 and 15 days. Extracellular carbohydrate contents of diatoms increase always during the experiment. Highest lipid content is found at day 8. Diatoms have the highest amounts of ash at 3 and 25 days. Fatty acid is most abundant at day 15.
The favourable and optimum temperature, light intensity, salinity and pH conditions for eight benthic diatom species are 15℃-30℃and 20℃-25℃, 500 lx-9500 lx and 1500 lx-55001x, 25-50 and 30-35, 7-9 and 8-8.5. Temperature and pH do less significantly affect all nutrient contents of diatoms. Protein contents of diatoms increase under lower temperature condition, while lower and higher temperature affect lipid contents of diatoms. Light intensity and salinity do less significantly affect protein and intracellular carbohydrate contents of diatoms, and significantly affect extracellular carbohydrate and lipid contents of diatoms. Nutritent contents are higher at lower and higher light intensity than at optimum one. Protein contents of diatoms cultured under lower salinity condition are higher, and intracellular carbohydrate, extracellular carbohydrate and lipid contents cultured under higher salinity condition are higher.
A significantly higher number of larval abalone settled on N. seminulum, H. amphioxys var. leptocephala and compared to the control and others plates. Moreover, Nitzschia sp. and N. corymbosa strongly induce larval settlement of Strongylocentrotus intermedius and Haliotis discus hannai. Growth and survival of abalone are also greatest on R. surirella, and that of sea urchin on Amphora proteus var. oculata are higher than ones on other diatoms. Nitzschia sp. and N. corymbosa also accelerate growth and survival of S. intermedius and H. discus hannai. These results indicate the effectiveness of Nitzschia sp., R. surirella, A. proteus var. oculata and N. corymbosa as single species over mixed diatoms and natural diatoms in larval settlement and juvenile growth of S. intermedius and H. discus hannai.
引文
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