藻菌混合固定化培养及其在海水人工育苗中的应用研究
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摘要
近年来,高密度人工海水育苗产业发展迅速,但由于育苗环境的水质恶化及投饵方式不合理,导致种苗生产的死亡率很高和一些名特优养殖品种种苗市场价格居高不下。作为一种生物反应器,藻菌混合固定化应用于海水育苗具有制作简单、去污能力强、可避免二次污染及适于长期保存等优点。对于提高高密度海水育苗种苗成活率具有很高的应用价值。本研究选取一株优质海洋饵料微藻,并从海水益生菌制剂中分离出一株芽孢杆菌,应用微生物固定化技术进行藻菌混合固定化培养,以期构建低成本、便于推广的新型固定化藻菌生物反应器并应用于海水育苗。为进一步开发适于海水育苗用的新型水质调控剂与实用供饵新技术提供实验依据和有效方法。本研究的主要内容有:
1、从形态、生理生化和分子生物学方面对选用的海洋饵料微藻进行分类鉴定;
2、从海水益生菌制剂中分离出4株益生菌,通过与藻细胞共固定后生长效果的比较,选出一株适于混合固定化的菌株,并对其进行分子鉴定。
3、对藻菌共固定培养条件进行优化,获得的优化结果为;海藻酸钠的浓度为4%,藻的接种量为21.9889×10~7个细胞,菌的接种量为58.7676×10~8个细胞,氯化钙的浓度为1.2075%。
4、研究混合固定化对藻菌生长生理的影响,发现混合固定化胶球中的藻和菌的生长速率比单独固定化分别提高了18.8%和92.6%,藻和菌干重之和也大于单独固定化;运用免疫荧光标记技术和共聚焦激光扫描显微技术分析共固定胶球中藻、菌的时空分布及荧光特性,发现共固定胶球中,藻、菌落在各个区域都生长良好,藻落荧光强度也较高,表明混合固定化有利于藻和菌的生长;
5、把固定化胶球投放于西施舌和鲍鱼幼苗养殖水体,研究固定胶球对水中氨氮、亚硝酸氮含量的去除效果、对弧菌的抑制作用以及对幼苗成活率和生长的影响。结果表明,藻菌混合固定化用于西施舌幼虫养殖,8天后氨氮去除率为95.49%,亚硝酸氮去除率为94.27%,幼虫成活率提高了16%;用于西施舌幼贝的养殖,7天后氨氮去除率为64.89%,亚硝酸氮去除率为24.18%,幼贝成活率提高了35%,弧菌去除率为90%;用于鲍鱼幼贝养殖,7天后氨氮去除率为63.99%%,亚硝酸氮去除率为68.53%,幼贝成活率提高了60%,弧菌去除率为98.8%。这说明藻菌混合固定化在水产养殖中有很好的应用前景。
In recent years, high-density synthetic seawater nursery industry is rapidly developpped. However, due to the deterioration of water quality and the unreasonable manners of nursery and feeding, it shows that mortality rates of seeding is high and the price of unique seed breeding is expensive. The application of algae-bacteria co-immobilization in marine seeding as a bioreactor are advantagous such as being easy to make, having a good decontamination capability, avoiding second pollution, being suitable for long-term preservation, and increasing the survival rate of seedings in high density nursery. In the present study, an effective method and experimental tests was implemented to develop the useful technology for baiting and new water regulation agents. Moreover, the co-immobilization culture was also built for a marine microalgae (Nannochloropsis sp. YW0980) and a bacillus {Bacillus sp.Shb-0980)isolated from marine probiotics. Our aims are that a new type of immobilized algae-bacteria bioreactor,with low-cost and easy to operate is expected to construct and use in breeding. The main contents of this study are as follows:
1. The selected baiting-microalgae was identified by morphology, physiology, biochemistry and molecular biology.
2. After four bacterial strains, isolated from a marine probiotics, were co-entrapped with the baiting -microalgae, respectively, a bacteria which was the most suitable for co-immobilized with the marine algea was selected. Then, the bacteria was identified with 16S rRNA gene.
3. The culture conditions of co-immobilization opetimized were as fellows: 4% for alginate, 1.2075% for Cacl2 , 21.9889×10~7 cells, 58.7676×10~8 cells for the inoculation cell number of algae and bacteria,respectively.
4. The growth and physiology varieties of algae and bacteria were studied when coentrapped in alginate. In comparism with the single immobilization, the growth speed of algae and bacteria co-immlbilized was inhanced 18.8% and 92.6% separately. The total dry weight of algae and bacteria coentrapped was higher than single immobilization either. With an immunofluorescent method involving immunofluorescent labeling and confocal microscopy, the space-time distribution and fluorescence characteristics of algae and bacteria in co-immobilized beads were analysised. It was found that algae and bacterea colony grow well in every area of co-immobilized beads,and the fluorescence intensity was high,either.It indicated that immobilization was propitious to the growth of algae and bacteria.
5. The immobilized beads was putted in the aquacultural wate of Mactra antiquataa and Hallotis diversicolor Reeve, researched the effect of removing the NH_3-N and NO_2-N , inhibition to vibrio,and the seeding percent. The results shew that the removal rate of NH_3-N and NO_2-N were 95.49% and 94.27%,and the larva suval rate was inhanced for 16% when the algae-bacfteria immobilization was applied to Mactra antiquataa. larvae aquaculture after 8 days;in Mactra antiquataa juveniles shellfish aquaculture, the removal rate of NH_3-N and NO_2-N were 64.89% and 24.18%,the vibrio removal rate was 90% and the juveniles shellfish suval rate was inhanced for 35% after 7 days; after 7days the removal rate of NH_3-N and NO_2-N were 63.99% and 68.53%, the vibrio removal rate was 98.8% and the juveniles shellfish suval rate was inhanced for 60% when the algae-bacfteria immobilization was applied to ebalone juveniles shellfish aquaculture. These shew a very good application prospects of the algae-bacteria co-immobilization in aquaculture.
1、从形态、生理生化和分子生物学方面对选用的海洋饵料微藻进行分类鉴定;
2、从海水益生菌制剂中分离出4株益生菌,通过与藻细胞共固定后生长效果的比较,选出一株适于混合固定化的菌株,并对其进行分子鉴定。
3、对藻菌共固定培养条件进行优化,获得的优化结果为;海藻酸钠的浓度为4%,藻的接种量为21.9889×10~7个细胞,菌的接种量为58.7676×10~8个细胞,氯化钙的浓度为1.2075%。
4、研究混合固定化对藻菌生长生理的影响,发现混合固定化胶球中的藻和菌的生长速率比单独固定化分别提高了18.8%和92.6%,藻和菌干重之和也大于单独固定化;运用免疫荧光标记技术和共聚焦激光扫描显微技术分析共固定胶球中藻、菌的时空分布及荧光特性,发现共固定胶球中,藻、菌落在各个区域都生长良好,藻落荧光强度也较高,表明混合固定化有利于藻和菌的生长;
5、把固定化胶球投放于西施舌和鲍鱼幼苗养殖水体,研究固定胶球对水中氨氮、亚硝酸氮含量的去除效果、对弧菌的抑制作用以及对幼苗成活率和生长的影响。结果表明,藻菌混合固定化用于西施舌幼虫养殖,8天后氨氮去除率为95.49%,亚硝酸氮去除率为94.27%,幼虫成活率提高了16%;用于西施舌幼贝的养殖,7天后氨氮去除率为64.89%,亚硝酸氮去除率为24.18%,幼贝成活率提高了35%,弧菌去除率为90%;用于鲍鱼幼贝养殖,7天后氨氮去除率为63.99%%,亚硝酸氮去除率为68.53%,幼贝成活率提高了60%,弧菌去除率为98.8%。这说明藻菌混合固定化在水产养殖中有很好的应用前景。
In recent years, high-density synthetic seawater nursery industry is rapidly developpped. However, due to the deterioration of water quality and the unreasonable manners of nursery and feeding, it shows that mortality rates of seeding is high and the price of unique seed breeding is expensive. The application of algae-bacteria co-immobilization in marine seeding as a bioreactor are advantagous such as being easy to make, having a good decontamination capability, avoiding second pollution, being suitable for long-term preservation, and increasing the survival rate of seedings in high density nursery. In the present study, an effective method and experimental tests was implemented to develop the useful technology for baiting and new water regulation agents. Moreover, the co-immobilization culture was also built for a marine microalgae (Nannochloropsis sp. YW0980) and a bacillus {Bacillus sp.Shb-0980)isolated from marine probiotics. Our aims are that a new type of immobilized algae-bacteria bioreactor,with low-cost and easy to operate is expected to construct and use in breeding. The main contents of this study are as follows:
1. The selected baiting-microalgae was identified by morphology, physiology, biochemistry and molecular biology.
2. After four bacterial strains, isolated from a marine probiotics, were co-entrapped with the baiting -microalgae, respectively, a bacteria which was the most suitable for co-immobilized with the marine algea was selected. Then, the bacteria was identified with 16S rRNA gene.
3. The culture conditions of co-immobilization opetimized were as fellows: 4% for alginate, 1.2075% for Cacl2 , 21.9889×10~7 cells, 58.7676×10~8 cells for the inoculation cell number of algae and bacteria,respectively.
4. The growth and physiology varieties of algae and bacteria were studied when coentrapped in alginate. In comparism with the single immobilization, the growth speed of algae and bacteria co-immlbilized was inhanced 18.8% and 92.6% separately. The total dry weight of algae and bacteria coentrapped was higher than single immobilization either. With an immunofluorescent method involving immunofluorescent labeling and confocal microscopy, the space-time distribution and fluorescence characteristics of algae and bacteria in co-immobilized beads were analysised. It was found that algae and bacterea colony grow well in every area of co-immobilized beads,and the fluorescence intensity was high,either.It indicated that immobilization was propitious to the growth of algae and bacteria.
5. The immobilized beads was putted in the aquacultural wate of Mactra antiquataa and Hallotis diversicolor Reeve, researched the effect of removing the NH_3-N and NO_2-N , inhibition to vibrio,and the seeding percent. The results shew that the removal rate of NH_3-N and NO_2-N were 95.49% and 94.27%,and the larva suval rate was inhanced for 16% when the algae-bacfteria immobilization was applied to Mactra antiquataa. larvae aquaculture after 8 days;in Mactra antiquataa juveniles shellfish aquaculture, the removal rate of NH_3-N and NO_2-N were 64.89% and 24.18%,the vibrio removal rate was 90% and the juveniles shellfish suval rate was inhanced for 35% after 7 days; after 7days the removal rate of NH_3-N and NO_2-N were 63.99% and 68.53%, the vibrio removal rate was 98.8% and the juveniles shellfish suval rate was inhanced for 60% when the algae-bacfteria immobilization was applied to ebalone juveniles shellfish aquaculture. These shew a very good application prospects of the algae-bacteria co-immobilization in aquaculture.
引文
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