凡纳滨对虾工厂化养殖水体中微小辐环藻HY01的分离鉴定及对不同氮源的响应

Isolation, identification and response of Actinocyclus exiguus HY01 to different nitrogen sources from the indoor industrial aquaculture system for Litopenaeus vannamei

中文关键词: 微小辐环藻  鉴定  氨氮  硝态氮  生长特性

英文关键词: Actinocyclus exiguous  Identification  Ammonia  Nitrate  Growth characteristics

中文摘要:

      凡纳滨对虾(Litopenaeus vannamei)工厂化养殖池中，一株硅藻在养殖中后期长期占优势，因其个体较小且细胞外壳覆盖一层硅质膜，难以用光学显微镜直接准确鉴定其分类地位。通过对该藻株进行分离纯化，利用光学显微镜和电子显微镜，结合分子生物学技术，鉴定该分离藻株为微小辐环藻HY01 (Actinocyclus exiguous HY01)。藻细胞直径约为(11.4±1.0) μm，壳面上有很多小孔，光学显微镜下不可见，且壳中央的孔密度较壳边缘稀疏，壳边缘具有眼斑结构，有3~5个唇形突。以不同浓度氨氮和硝态氮为氮源培养微小辐环藻HY01，结果显示，微小辐环藻HY01均能利用氨氮和硝态氮进行生长，最适宜生长的氨氮和硝态氮浓度分别为600和882 μmol/L，但以氨氮为氮源时微小辐环藻HY01的最大细胞密度、最高比生长速率以及蛋白含量均低于以硝态氮为氮源，表明微小辐环藻HY01可能更喜欢利用硝态氮，但对较高浓度的氨氮有一定的耐受性。

英文摘要:

      Shrimp production has expanded rapidly over the last few decades in China. With the increasing production, water pollution has become increasingly serious. An increase in nutrients in water results in eutrophication, characterized by low oxygen, high ammonia, high phosphorus, and high frequency of algal blooms, which in turn influence shrimp growth and yield. Phytoplankton is an important component of aquaculture ecosystems. It is a direct or indirect food source for cultured organisms, and can remove nitrogen and phosphorus and maintain water quality. Constructing a benign phytoplankton community can improve the nitrogen pollutant absorption efficiency, improve the environment, and reduce the environmental pollution caused by cultivation. Certain phytoplankton groups, such as diatoms and green algae, are desirable for their high nutritional value and contribution to water quality. The use of phytoplankton to purify and regulate aquaculture water quality could reduce the negative impacts of aquaculture, which is an environmental protection option with a low cost, low energy consumption, high benefit, and considerable development potential. Microalgal growth is significantly affected by factors such as temperature, light, and nutrient conditions. Therefore, the environmental adaptability of microalgae is the primary consideration for selecting and cultivating algal species. In situ isolation and screening of algal species can reduce the stress response of microalgae, which is conducive to their normal ecological function of regulating water quality. In an indoor industrial aquaculture system for Litopenaeus vannamei, one diatom species was dominant for a significant amount of time in the middle and late stages of aquaculture in 2019. This alga was small and covered with a silica membrane layer, which was difficult to identify with a light microscope. The shrimps effectively grew in the ponds where this alga was the dominant species. To identify the algae and explore its application in water quality regulation in aquaculture, the algal strain was isolated and purified from the indoor industrial aquaculture system, and identified by optical microscopy, electron microscopy, and 18S rRNA sequence analysis. The results identified this algal strain as Actinocyclus exiguous HY01. The cell diameter was approximately (11.4±1.0) μm. At present, A. exiguous is the smallest individual species in the genus Actinocyclus. There are numerous small pores on the mantle that were not visible under a light microscope. The pore density at the mantle center was less than that at the mantle margin. There is a pseudonodulus at the valve margin, and 3~5 labiate processes. Nitrogen is an indispensable element for the growth and metabolism of phytoplankton, and is the main component of nucleic acids, proteins, and chlorophyll in cells. Ammonia and nitrate are the two main forms of inorganic nitrogen in aquaculture water, which can be directly absorbed and utilized by microalgae. The addition of nitrogen nutrients can promote microalgal growth. However, the microalgae cell density did not increase as the nitrogen concentration increased. Excessive ammonia content is inconducive to algae growth, and even affects the absorption and transformation of other nitrogen sources. In this study, ammonia and nitrate of different concentrations were used as nitrogen sources to culture A. exiguous HY01. The results showed that both ammonia and nitrate could be used for A. exiguous HY01 growth. The optimum growth concentration of ammonia was 600 μmol/L. Under these conditions, the cell density, specific growth rate, and protein content of A. exiguous HY01 were the highest, at 4.54×10⁸ cells/L, 0.36±0.07, and 4.45 mg/g, respectively. When the ammonia concentration was 882 μmol/L, the cell density and specific growth rate of A. exiguous HY01 were the lowest, at 2.11×10⁸ cells/L and 0.27±0.05, respectively. When the ammonia concentration was 50 μmol/L, the A. exiguous HY01 protein content was the lowest at 1.38 mg/g. The optimum growth concentration of nitrate was 882 μmol/L. Under these conditions, the cell density, specific growth rate, and protein content of A. exiguous HY01 were the highest, at 5.92×10⁸ cells/L, 0.40±0.01, and 11.97 mg/g, respectively. When the nitrate concentration was 100 μmol/L, the cell density, specific growth rate, and protein content of A. exiguous HY01 were the lowest. In summary, the maximum cell density, maximum specific growth rate, and protein content of A. exiguous HY01 were lower in the medium containing ammonia-nitrogen than those in the medium with nitrate-nitrogen. We speculated that A. exiguous HY01 might prefer nitrate to ammonia, but has greater tolerance to high ammonia concentrations. This might be one reason why A. exiguous HY01 was dominant for a long time in the indoor industrial aquaculture system of L. vannamei in 2019. The results are expected to provide a reference for the targeted cultivation of this alga and its application in water quality regulation in aquaculture.