The Study On Physiological Characteristics Of Extracellular Enzymes Of Harmful Algal Bloom Species In The Hydrolysis Of Organic Matters

Dissolved organic matter(DOM) is the largest source of dissolved nitrogen and phosphorus in the ocean. Extracellular enzymes play important roles in degrading DOM into small fractions that can be utilized by phytoplankton. Extracellular enzyme activities can not only reflect quantity and quality of organic matter in aquatic environments, but also reflect the extent of the transformation of DOM by algae and the nutritional contribution for algal growth. Difference in the regulation of extracellular enzyme activities by algaemay decide their capabilities in utilizing DOM.Three algal bloom species, Prorocentrum donghaiense, skeletonema costatum and Aureococcus anophagefferens were selected in the laboratory study to study the physiological characteristics of extracellular enzymes: the β- glucosidase activities in different carbon forms(cellobiose and oligopeptide), the leucine aminopeptidase activities in different nitrogen forms(ammonium, nitrate, urea and oligopeptide) and the alkaline phosphatase activities in different phosphorus sources(KH2PO4 and glucose-6-phosphate(G-6-P)). The objective of the thesis was to explore the difference in the physiological characteristics of extracellular enzymes among species, thus discuss the specific difference of phytoplankton in the hydrolysis of DOM. Four kinds of extracellular enzymes activities(α, β-glucosidase, leucine aminopeptidase, alkaline phosphatase) were measured in the coastal waters of Qinhuangdao in situ to explore the roles of extracellular enzymes in the hydrolysis DOM and to analyze the nutritional physiological status of phytoplankton in the seawater. The main results were as followed:1. P. donghaiense, S. costatum and A. anophagefferens all can produceβ-glucosidase. When nitrogen and phosphorus were adequate in the media, the β-glucosidase activities of three species all increased when cellobiose was added. The presence of oligopeptide can suppress the expression of β-glucosidase activities of all three species.2. P. donghaiense, S. costatum and A. anophagefferens all can produce leucineaminopeptidase no matter nitrogen were depleted or saturated. These results showed that at least part of the leucine aminopeptidase was constitutive. Leucine aminopeptidase activities of all three species were not affected by the existence of ammonia, nitrate or urea in the media; The leucine aminopeptidase activities of P. donghaiense and A. anophagefferens were promoted by oligopeptide whereas that of S. costatum was not. The leucine aminopeptidase activities of P. donghaiense and A. anophagefferens were higher than that of S. costatum. Therefore, the capacity in utilizing dissolved organic nitrogen(DON) for P. donghaiense and A. anophagefferens might be stronger than that of S. costatum. The leucine aminopeptidase activity of P. donghaiense was mainly consisted of both soluble and cell-bound states Whereas that of A.anophagefferens was mainly contributed by the cell-bound enzymes. P.donghaiense, A.anophagefferens might be competitive incompeting DON. Therefore, A. anophagefferens might be the strongest to utilize DON among the three species which mighthelp to explain the reason of the occurrence of A.anophagefferens blooms nitrogen restricted waters.3. The alkaline phosphatase of P. donghaiense and S. costatum were inducible. The expression of alkaline phosphatase was regulated by both external and internal phosphorus sources. As the phosphorus source concentrations reduced alkaline phosphatase activities of species increased. However the response of the enzyme expression of P. donghaiense to phosphorus was more sensitive than that of S. costatum. When dissolved organic phosphorus was existed, P. donghaiense can quickly produce alkaline phosphatase and hydrolyze organic matters. Under phosphorus stress status, S. costatum would produce excess alkaline phosphatase and exhauste abundant energy for growth which might be disadvantaged in the competing DOP.4. The analysis of four extracellular enzyme activities and the nutrient parameters in the coastal waters of Qinghuangdao in June, 2014 in situ demonstrated that the coastal waters of Qinhuangdao was phosphorus limited. Four kinds of extracellular enzyme activities were all mainly expressed in the size 2- 5 μm and > 5 μm which confirmed that phytoplankton play a role in organic matter decomposition in coastal ecosystems. The leucine aminopeptidase and alkaline phosphatase activities were 1 ~ 2 orders higher than α, β-glycosidase activities. Temperature, chlorophyll a concentration and DOP were probably the main factors affecting the activities of glycosidase enzymes and leucine aminopeptidase. Extracellular enzyme hydrolysis dynamics results showed that: compared with that of organic carbon, the turnover rate of organic nitrogen and phosphorus in seawater were much higher. The turnover time of alkaline phosphatase was only in a few minutes. DOP was likely to be the important source of phosphorus. DOP and DON may play important roles in the process of phytoplankton growth and might be the key factor to the outbreak of the brown tide in the coastal waters of Qinhuangdao.