摘要
底栖硅藻是鲍、海参等名贵海洋经济动物重要的饵料来源,从自然海域分离培养适于当地水产养殖的优良底栖硅藻饵料对海洋经济动物的苗种生产具有十分重要的现实意义。本文采用淘沙过滤的方法对福建连江海域的底栖硅藻进行了采集,并对其中一株分离得到的底栖硅藻生物学特性、培养条件、部分代谢产物的影响因子进行了详细研究,探讨单一底栖硅藻作为海洋经济动物幼体饵料的可行性和潜在应用价值,为今后大规模高密度培养提供理论依据。另外,通过固定化包埋技术,对底栖硅藻固定化培养、种质保存以及藻菌共固定化培养进行了初步研究。具体研究内容和结果如下：
1.通过平板分离法分离得到底栖硅藻3株、浮游性硅藻2株。通过形态观察对一株底栖硅藻初步鉴定为缢缩菱形藻Nitzschia constricta。营养成分分析表明,此株硅藻粗蛋白含量为7.26%,其中必需氨基酸含量达到占总氨基酸含量43.12%,谷氨酸14.56%；总不饱和脂肪酸含量大约占整个脂肪酸组成的48.3%。与其它种常见饵料硅藻相比,完全符合鲍鱼等名贵水产经济动物的饵料要求,具有较高的饵料经济价值。
2.通过测定色素、比增长速率、干重等生长指标得出,菱形藻的适宜生长温度为18·30℃,盐度10-45‰,pH适应范围6-10,光照500-1000 1x,水体适应范围较广。氮、磷、铁、硅营养盐浓度实验表明,在N03-N 14.13mg/L、NaH2PO3-P 0.87mg/L条件下生长良好；柠檬酸铁-铁浓度适应范围0.07-1.3 mg/l,硅酸钠-硅浓度适应范围0.23-23 mg/L。
3.比较不同培养条件下菱形藻胞外多糖和蛋白积累情况发现：温度对菱形藻蛋白积累无显著影响,对胞外多糖分泌影响显著,24℃条件下菱形藻胞外多糖分泌最高达6.93μg/ml;当盐度>10‰,不同培养时间内的菱形藻蛋白积累无明显差异,当盐度为30‰时,胞外多糖分泌最高为8.40μg/ml;培养液中pH变化对胞内多糖无明显规律影响,当pH≤8时,菱形藻蛋白和细胞总糖含量积累较好；硝酸氮浓度为75mg/l,菱形藻第10天蛋白含量最高为28.9μg/ml,胞外多糖分泌随培养时间的延长而增加；磷浓度的增加提高了菱形藻蛋白的积累,但在磷营养盐限制的条件下更有利于培养前期藻细胞外多糖的分泌；当柠檬酸铁浓度为3.9 mg/l时,菱形藻第15天蛋白积累最高,而铁浓度的变化对硅藻胞外多糖的分泌无明显影响；培养液中硅浓度越低,菱形藻胞外多糖分泌越高最高达10.64μg/ml,而硅浓度的变化对硅藻蛋白积累无显著影响。
4.通过海藻酸钠包埋法对底栖硅藻进行固定化研究。固定化条件正交试验结果表明,接种密度107 cell/ml、海藻酸钠浓度2%,氯化钙溶液浓度3%最有利于固定化菱形藻的生长。菱形藻固定化90天后复活培养,依然保持良好的生长速率,与对照组相比蛋白和碳水化合物含量没有明显区别。通过细胞计数和激光共聚焦扫描显微镜荧光测定发现低温和常温条件均适合菱形藻的固定化保存。
5.将缢缩菱形藻N. constricta、舟形藻Navicula sp.和三角褐指藻Phaeodactylum tricornutum分别与海洋酵母菌进行混合固定化培养尝试,发现藻菌共包埋均能不同程度的促进硅藻的生长。
The benthic diatoms are essential food source for precious marine economic animals such as abalone and sea cucumber etc. It has very important significance to isolate the good benthic diatoms food from nature for the local aquacultμre which produce marine economic animal seedlings.In the paper, benthic diatoms were collected by scraping sand and filtering in Lianjiang maritime space in Fujian. The biological characteristics, culture conditions and the impact factors which affect the metabolic products of the one isolated benthic diatom were studied to investigate the potential application value as food for aquaculture and to afford the theoretical basis for a large scale and high density cultivation. Besides, the immobilization culture of benthic diatom, germplasm conservation and algal-bacterial co-immobilization with the embedding techniques were researched. The results in detail were stated as follows:
1. Three benthic and two planktonic diatoms were acquired by spreading plating method.One of the benthic diatoms was identified as Nitzschia constricta by morphology observation.The nutritional ingredient analysis indicate that the crude protein level was 7.26%, and the essential amino acid was 43.12% of the total amino acids, and glutamic acid was 14.56%; the unsaturated fatty acid was 48.3% of the total fatty acid. Compare to other common food diatoms, it's very fit the food requests of marine economic animals such as abalone and has the good economic food value.
2. By evaluating the pigments, growth rate and dry weight, the favourable and optimum culture conditions of N. constricta are temperature 18-30℃,salinity 10-45‰, pH 6-10 and light intensity 500-1000 lx,which has the wide subject range in the water.The nitrogen,phosphorous,iron and silicon concentrations indicate that N. Constricta grow well at NO3-N 14.13 mg/L,NaH2PO3-P 0.87 mg/L and the optimum ferric citrate-Fe and silicon concentrations was 0.07-1.3 g/1,0.23-23 mg/L.
3. The protein and exopolysaccharide yield of N. Constricta under the different culture conditionds demonstrate:the temperatμre has no positive effect on protein produce, but is positive to exopolysaccharide yield which has the most content 6.93μg/ml at 24℃. There is no evident difference of protein yield in days when the salinity exceeds 10‰,but the exopolysaccharide yield the most of 8.40μg/ml at salinity 30‰.The pH changes in the cultμre solution has no regular effects to exopolysaccharide yield.It has good protein and exopolysaccharide accumμlation at pH≤8.When Sodium Nitrate is 75 mg/l,the highest protein content 28.9μg/ml was obtained at the 10th day, and the exopolysaccharide yield was increased with the cultμre time prolonged.The protein was increased at the higher phosphoroμs concentrations,bμt the limited phosphoroμs was good for exopolysaccharide yield at the early days.When the ferric citrate was 3.9 mg/l,the most protein was obtained at the 15th day,but the iron change has no evident effect to exopolysaccharide yield.The lower silicon concentration the more exopolysaccharide yield of N. Constricta, and the most is 10.64μg/ml,but the silicon changes has no evident effect to protein yield.
4. The benthic diatoms immobilization was researched by alginic acid embedding techniqμe. The Orthogonal test design showed that the inoculation density 107 cell/ml, alginate 4%, Calcium Chloride 3% are the optimized conditions for immobilizated N. Constricta growth. The reculture N. Constricta immobilizated for 90 days still has the good viability; compared to control, the protein and exopolysaccharide yield have no dofferent.Tt also found that the low and room temperature are both appropriate for N. Constricta conservation by cell counting and fluoremetry of confocal laser scanning microscope.
5. The attempt of N. Constricta, Navicula sp., Phaeodactylum Tricornutum co-immobilization with marine yeast were carried.lt found algal-bacterial co-immobilization can promote the diatoms growth.
引文
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