对虾高位池水环境养殖污染和浮游微藻生态调控机制研究
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摘要
水环境的富营养化导致水华和赤潮频发,对水源和渔业生产活动带来严重危害。因此,水环境的富营养化是当今人类面临的重要生态学问题,治理水环境富营养化的有效方法和理论越来越受到人们的广泛关注,已成为当前学术界研究的热点问题。
对虾养殖业是我国海洋经济发展重要的支柱产业。我国对虾养殖面积达6.67×104hm2以上,年产量超过120×104t,约占全球对虾养殖产量的38%,是世界对虾养殖年产量最高的国家。水体富营养化是对虾养殖水环境的主要污染现象,其次是重金属污染,养殖废水排放含氮化合物是近岸海洋环境氮污染和富营养化的主要原因。虾池水体溶解态氮主要有溶解态无机氮和溶解态有机氮,溶解态无机氮主要以NH4+-N.NO2--N和N03--N、N2等形式存在,溶解态有机氮主要以Urea-N、Met-N、蛋白质等形式存在。NH4+-N和NO2--N等是养殖水体中对对虾具有毒性的含氮污染物。利用虾池生物调控水质来改善养殖环境是对虾健康养殖主要途径。浮游微藻是对虾养殖系统中重要的生物因子,具有对溶解态氮吸收和重金属离子吸附的功能特性,是虾池水体中溶解态氮转化和降低重金属离子含量的主要途径。通过虾池浮游微藻群落结构的优化,建立良性的浮游微藻群落提高其对溶解态氮吸收和重金属离子吸附效率,降低虾池水体的自身污染,从而达到改善水质的目的。关于对虾高位池浮游微藻及其对溶解态氮吸收和重金属离子吸附的研究未见报道,本论文以对虾高位池为研究对象,分别从虾池养殖污染、虾池浮游微藻、虾池浮游微藻对溶解态氮的吸收规律和调控机制、虾池浮游微藻对重金属离子吸附和虾池养殖环境的生态调控等5个方面进行研究,为虾池浮游微藻群落结构的优化和改善水质环境提供科学依据,也为环境水体富营养化的治理提供理论支撑和技术参考。主要研究内容和结果如下：
(1)通过对对虾养殖水体中各种溶解态氮变化规律的研究,发现虾池投饲量与COD呈正相关,与溶氧呈负相关,水体中的溶解态氮主要来源于投喂的人工饲料；虾池溶解态无机氮、溶解态有机氮和溶解态总氮均值分别为0.470mg·L-1、0.325mg·L-1和0.795mg·L-1,养殖后期明显高于养殖前期,均超过富营养化的阈值；养殖期间溶解态有机氮占溶解态总氮的40.88%,是虾池溶解态氮的重要组成部分。养殖过程中INT/IP平均值为5.98,IN/IP<5的时期占整个养殖过程的48%,没有出现IN/IP>30的现象；虾池水体中叶绿素a含量平均为91.6ug·L-1,与溶解态总氮之间存在显著正相关,卡尔森营养状态指数(TSI, Carlson trophic state index)平均为72.98,虾池水体处于严重的富营养化状态。水体中NH4+-N和N02--N含量均值分别为0.210mg·L-1和0.018mg·L-1,养殖后期分别为0.419mg-L-1和0.031mg·L-1,超过健康养殖的阈值。在浓度为0.040mg·L-1的NO2--N胁迫下,对虾免疫力会下降。
(2)在广东和海南等地区的养殖中期与后期的虾池中,检测出浮游微藻优势种属于5个门共计12种,其优势度在0.12-0.99之间,优势种突出和单一；在养殖前期主要以硅藻优势种群为主,养殖的中期和后期虾池水体富营养化严重,以较喜肥或者是耐污染的绿藻和蓝藻成为优势种群。养殖期间浮游微藻的细胞数平均为19.71×107cell·L-1,香农多样性指数平均为0.56,养殖水体属重度污染状态。以波吉卵囊藻(Oocystis borgei)为优势种群控制的水质环境较为稳定,其优势种群持续时间达40d～50d；硅藻优势种群相对不稳定,持续时间仅为5d～15d；小席藻(Phormidium tenue)细胞数高达2160×107cell·L-1,容易形成赤潮。以绿藻为优势种的虾池水质较稳定,形成水环境有利于对虾生长。波吉卵囊藻是虾池中分布广和适应能力强的浮游微藻,同时还具有种群稳定和持续时间长的特点。
(3)利用稳定同位素标记法,在不同生态条件下对虾池浮游微藻溶解态氮吸收速率的研究发现,虾池浮游微藻对NH4+-N.N02--N、NO3--N、Urea-N和Met-N吸收最适宜的温度范围为25℃～30℃,盐度范围为20～30,光照度范围为45.000μmol·m-2·s～126.000μmlo·m-2·s-1,藻浓度范围为3.222×108cell·L-1～4.784×108cell·L-1。当氮浓度为14.300mg·L-1时,浮游微藻对NH4+-N、N02--N和N03--N均具有最高的吸收速率,当Urea-N和Met-N浓度分别为48.400mg·L-1和20.900mg·L-1时,其吸收速率达到最高；高浓度溶解态氮对藻细胞氮吸收具有显著的抑制作用,高的NH4十-N浓度对MH4+-N吸收抑制要早于其他形式的溶解态无机氮。
这些结果表明虾池浮游微藻对溶解态氮的吸收速率与温度、盐度、光照度、藻浓度和氮浓度有密切关系,这特点在亚热带海洋沿岸水域普遍存在。在高浓度溶解态氮情况下浮游微藻对氮的吸收会出现“饱和效应”。
(4)在多种氮源共存的条件下,利用稳定同位素标记法研究了虾池水体浮游微藻在不同温度和盐度条件下对溶解态氮吸收的选择性,并通过正交实验研究影响浮游微藻溶解态氮吸收速率的生态因子。结果显示,在不同温度和盐度实验条件下,浮游微藻对NH4+-N吸收的相对优先指数(RPI)均大于对N02-_N、Urea-N和NO3--N,优先吸收NH4+-N,其次是NO2--N,最后是Urea-N和NO3--N。正交实验结果显示,在温度20℃～30℃、光照81μmol·m-2·s-1.盐度15～30、pH7.5和藻浓度4.5×108cell·L-1～5.5×108cen·L-1的条件组合下,浮游微藻对各种溶解态氮均有较高的吸收速率,影响NH4+-N和NO2--N吸收速率的主导因子是藻浓度和盐度,影响N02-N、Urea-N吸收速率的主导因子是光照。
(5)波吉卵囊藻对NH4+-N和Urea-N吸收速率模型的预测值与实测值进行样本T检验,检验结果表明模型预测值与实测值差异不显著(P>0.05),总体均值差异不显著(P>0.05),证明模拟方程拟合度较高。
(6)波吉卵囊藻细胞对Cu2+和Zn2+的吸附过程可分为三阶段：第一阶段吸附在30min内完成,吸附率达70%以上；第二阶段吸附速度减慢,在8h内完成；第三阶段为吸附平衡阶段。波吉卵囊藻对Cu+和Zn2+的吸附的最适温度范围为25～30℃,光照度为大于54.00μmol·m-2·s-1,盐度范围为10～30。波吉卵囊藻细胞浓度为28.91×107cell·L-1时对Cu2+的吸附率为52.52%,吸附量为9.469mg·g-1；藻细胞浓度为22.91×107cell·L-1时对Zn2+的吸附率为81.44%,吸附量为2.914mg·g-1。条纹小环藻细胞浓度为2.45×107cell·L-1时对Cu2+的吸附率为63.00%,吸附量为9.26mg·g-1；藻细胞浓度为1.75×107cell·L-1时对Zn2+的吸附率为60.52%；吸附量为20.06mg·g-1。在此藻细胞浓度下,藻细胞吸附的重金属离子不会影响波吉卵囊藻和条纹小环藻的生长。
(7)在对虾养殖系统中引入波吉卵囊藻,不仅能提高水体溶氧的含量、调节pH、降低COD,还能有效地吸收各种溶解态氮；水体中NH4+-N含量降低了51.7%-37.8%,N02--N含量可降低30.2%-26.4%,对虾抗病能力显著提高(P<0.05)。在虾池中培养波吉卵囊藻构建以波吉卵囊藻为主要构架的微藻群落,养殖期间其平均生物量占浮游微藻总生物量的95.35%-43.95%,优势度为0.27～0.58,成为虾池水体中的绝对优势种,作为优势种其种群持续时间长达77d,水体NH4+-N和N02--N浓度较对照池分别降低了36.98%和81.16%；波吉卵囊藻能够有效地减轻养殖密度的制约作用,对虾生长速度明显加快,养殖产量提高83.78%。在培养水温为27℃～32℃和盐度20～28的条件下,波吉卵囊藻在养殖水体中呈逻辑斯谛(Lgistic)方式增长,环境容纳量K值为6899.959x105cell·L-1,瞬时增长率r近似值为0.002,最大可持续产量(MsY)为3.45×105cell·L-1·d-1。每天从虾池水体中去除3.45×105cell·L-1·d-1藻细胞可以保持波吉卵囊藻的种群稳定。
Eutrophication of surface waters can induce algal blooms and in salt water or estuarine waters, red tide can occur frequently, which can cause impairment of both the use of the water body and the local fishery. Therefore, eutrophication is an important ecological problem, and the study of how to deal with this problem has attracted extensive attention.
Prawn aquaculture is the pillar industry of the national marine-based economy. In China, the area of prawn aquaculture exceeded6.67×104hm2, with an annual output of more than120×104t, which accounts for38%of the global output, making China the highest prawn yielding country. Eutrophication is the major pollution issue in prawn culture, the second problem is heavy metal pollution. Nitrogen compounds discharged from aquaculture waste water are the primary sources of nitrogen pollution and the resultant eutrophication in the near-shore marine environment. There are two kinds of dissolved nitrogen in prawn aquatic system. One is dissolved inorganic nitrogen in the form of ammonia nitrogen (NH4+-N), nitrite (NO2--N), nitrate (NO3--N).. The other form is dissolved organic nitrogen as Urea-N, Methyl-N and protein. Inorganic forms, NH4+-N and NO2-N, are the primary nitrogen pollutants which are harmful to prawn. The technique for maintaining healthy prawn aquaculture is to improve the culture environment by regulating the water quality through select organisms in the prawn pond. Planktonic microalgae can take up dissolved nitrogen and heavy metal ions, and can be an important biological factor in a prawn culture system. Thus, microalgae can transform dissolved nitrogen and reduce heavy metal loads. To improve prawn pond water quality, we can enhance the pollutant removal capacity of planktonic microalgae by assembling an optimum planktonic microalgae colony through enhancing the community structure of certain planktonic microalgal species. The studies on the Planktonic microalgae in the prawn high level pond and the absorption of dissolved nitrogen and heavy metal ions adsorption by them have not been reported. In this study, we chose high elevation prawn pond to study the pollution caused by prawn breeding, planktonic microalgal assemblages in prawn ponds, the ability and regulatory mechanism for planktonic microalgal uptake of dissolved nitrogen and heavy metals and the control of the prawn pond environment. This will provide a scientific basis for optimizing the community structure of planktonic microalgae and improving water quality in prawn ponds and additionally providing a theoretical and technical basis for controlling eutrophication.
The content of the major research and the results are as follows:
(1) Through the study of dissolved nitrogen from prawn culture, we found the amount of feed showed a positive correlation with chemical oxygen demand (COD) and was negative correlated with dissolved oxygen. The dissolved nitrogen came primarily from the artificial breeding diet, with the contents of dissolved inorganic-nitrogen, dissolved organic nitrogen and the total dissolved nitrogen being0.470mg·L-1,0.325mg·L-1,0.795mg·L-1respectively. In addition, the concentrations from the late breeding stage were higher than in the early breeding stage, and the threshold of eutrophication. The content of dissolved inorganic nitrogen was40.9%of the total nitrogen, which was the major source of the dissolved nitrogen in the prawn pond during the breeding period. Over the course of the breeding period, the mean ratio of inorganic nitrogen to inorganic phosphorus (IN/IP) was5.98. It was<5at48%of time and never exceeded30. The average chlorophyll-a concentration in the pond was91.6ug·L-1, which was positively correlated with the total dissolved nitrogen. The mean value of the Carlson trophic state index (TSI) was73.0, indicating that the pond was in a state of serious eutrophication. The concentrations of NH4+-N and NO2--N during the early breeding were0.21mg·L-1and0.02mg·L-1respectively, and their concentrations in the late breeding stage were0.42mg·L-1and0.03mg-L"1. Both nitrogen species were higher than the threshold for healthy prawn breeding. Prawn immunity was reduced when NO2--N achieved a concentration of0.04mg·L-1.
(2) In the Guangdong and Hainan provinces, the major species of planktonic microalgae belong to five phyla, including12species found during the middle and late stages of prawn breeding, with a the degree of dominance from0.12to0.99. During the early breeding stages, diatoms dominated, but with eutrophication during middle and late breeding stage becoming serious, green algae and blue-green algae became dominant. The average cell number of planktonic microalgae was19.71×107cell/L-1and the mean diversity index was0.56, indicating that the system was suffering from serious contamination. The period with Oocystis borgei as the dominant algae was relatively stable, remaining dominant for40d-50d. The period when the dominant population was diatoms proved to be unstable and only lasted for5d～15d. The cell number of Phormidium tenue was2160×107cell/·L-1, which potentially is a source of red tide. The additional stable period occurred when the dominant population was green algae, which was a benefit for the growth of the prawn. Oocystis borgei was widely spread, adaptable, with the characteristics of a stable species persisting for a long duration.
(3) Using a stable isotope labeling technique, the uptake rate for nitrogen of planktonic microalgae in shrimp ponds was studied under different ecological conditions. The most suitable conditions for the uptake of NH4+-N, NO2--N, NO3--N, Urea-N and Met-N by microalgae in prawn pond were a temperature of25℃～30℃, a salinity of20～30, light illumination at45μmol·m-2·s-1～126μmol·m-2·s-1, and an algal density of3.222×108cell·L-1～4.784×108cell·L-1. When the nitrogen concentration was14.3mg·L-1, the uptake rate of NH4+-N, NO2--N and NO3--N by microalgae was highest. The uptake rate of Urea-N and Met-N was the highest when the Urea-N and Met-N concentrations were48.4mg-L"1and20.9mg-L"1respectively. High concentrations of dissolved nitrogen apparently inhibit the uptake of nitrogen. The inhibition of NH4+-N uptake occurred when the concentration of NH4-N was high.
These results demonstrated the uptake rate of nitrogen by planktonic microalgae in prawn ponds was closely related to temperature, salinity, light illumination, algal concentration and nitrogen concentration, with these specific conditions commonly in found in coastal areas in subtropical regions. The "saturation effect" for nitrogen uptake by algae would exist under high dissolved nitrogen conditions.
(4) The effect of salinity and temperature on relative preference indices (RPI) for planktonic microalgae under four dissolved inorganic nitrogen concentrations was studied using a stable isotope labeling method. The dominant factor which affected the nitrogen uptake rate of planktonic microalgae was also studied using an orthogonal experiment. The results showed that the RPI of NH4+-N of planktonic microalgae was greater than that of NO2--N, Urea-N and NO3--N. Planktonic microalgae took up NH4+-N initially and NO2--N followed, the next being Urea-N and finally NO3- -N. The orthogonal experiment showed that microalgae had a higher uptake rate for the four nitrogen sources when the temperature was20℃～30℃, the light illumination was81μmol·m-2·s-1, the salinity was15～30, the pH was7.5, and the algae density was4.5×108cell·L-1～5.5×108cell·L-1. Microalgae density and salinity were the main factors that affected the uptake rate of NH4+-N and NO2--N, while light illumination was the main factor that affected the uptake rate of NO2--N and Urea-N.
(5) Predicted values and measured values of an uptake rate model for Oocystis bergei were examined using a t-test. The test results showed that there was not a statistically significant difference between the predicted values and the measured values, as well as the population mean, which indicated that the degree of model fit was high.
(6) The uptake of Cu2+and Zn2+by Oocystis borgei could be divided into three steps:the first step was to complete the uptake process within30min, with an uptake rate of more than70%; in the second step, the rate decreased and the uptake process was completed within8h; in the third step, the uptake process reached equilibrium. The optimum uptake of Cu+and Zn2+by planktonic microalgae Oocystis borgei was when the temperature was25～30℃, the light illumination was greater than54.00μmol·m-2·s-1, and the salinity was10～30. When the Oocystis borgei concentration was2.9×108cell·L-1, the adsorption rate and capacity of Cu2+were52.5%and9.5mg·g-1respectively; when the algae concentration was2.3×108cell·L-1, the uptake rate and capacity of Zn2+were81.4%and2.9mg·g-1respectively; for Cyclotella striata when its concentration was2.5×107cell·L-1, the uptake rate and capacity of Cu2+were63.0%and9.3mg·g-1respectively; when its concentration was1.8×107cell·L-1, the uptake rate and capacity of Zn2+were60.5%and20.1mg·g-1respectively. Under these algae concentrations, the growth of Oocystis borgei and Cyclotella striata would not be affected.
(7) Introducing Oocystis borgei into prawn farming systems not only improved the content of dissolved oxygen, regulated pH, reduced COD, but also effectively fixed dissolved nitrogen. Due to the introduction, NH4+-N concentration was reduced by51.7%～37.8%, NO--N concentration reduced by30.2%～26.4%, and the disease-resistance of the prawn was improved significantly (P<0.05). In the treated prawn pond, average biomass of Oocystis borgei accounted for95.86%to45.12%, The dominance is between0.27to0.58and was the dominant algae during the breeding period. As the dominant species, it persisted for about77days. Concentrations of NH4+-N and NO2- -N were reduced by37.0%and81.2%respectively. Oocystis borgei can potentially alleviate the effect of stocking-density constraints, significantly improve prawn growth, and increase aquaculture production by83.8%. With a water temperature of27℃～32℃, a salinity of20～28, Oocystis borgei showed logistic growth with a K (carrying capacity) value of6899.959×105, r (instantaneous rate of increase) approximate value of0.002, and the maximum sustainable yield (MSY) of3.45×105·L-1·d-1. Removal of3.45·105cell·L-1of algal cells every day from the shrimp pond waters can keep Oocystis borgei population stable.
对虾养殖业是我国海洋经济发展重要的支柱产业。我国对虾养殖面积达6.67×104hm2以上,年产量超过120×104t,约占全球对虾养殖产量的38%,是世界对虾养殖年产量最高的国家。水体富营养化是对虾养殖水环境的主要污染现象,其次是重金属污染,养殖废水排放含氮化合物是近岸海洋环境氮污染和富营养化的主要原因。虾池水体溶解态氮主要有溶解态无机氮和溶解态有机氮,溶解态无机氮主要以NH4+-N.NO2--N和N03--N、N2等形式存在,溶解态有机氮主要以Urea-N、Met-N、蛋白质等形式存在。NH4+-N和NO2--N等是养殖水体中对对虾具有毒性的含氮污染物。利用虾池生物调控水质来改善养殖环境是对虾健康养殖主要途径。浮游微藻是对虾养殖系统中重要的生物因子,具有对溶解态氮吸收和重金属离子吸附的功能特性,是虾池水体中溶解态氮转化和降低重金属离子含量的主要途径。通过虾池浮游微藻群落结构的优化,建立良性的浮游微藻群落提高其对溶解态氮吸收和重金属离子吸附效率,降低虾池水体的自身污染,从而达到改善水质的目的。关于对虾高位池浮游微藻及其对溶解态氮吸收和重金属离子吸附的研究未见报道,本论文以对虾高位池为研究对象,分别从虾池养殖污染、虾池浮游微藻、虾池浮游微藻对溶解态氮的吸收规律和调控机制、虾池浮游微藻对重金属离子吸附和虾池养殖环境的生态调控等5个方面进行研究,为虾池浮游微藻群落结构的优化和改善水质环境提供科学依据,也为环境水体富营养化的治理提供理论支撑和技术参考。主要研究内容和结果如下：
(1)通过对对虾养殖水体中各种溶解态氮变化规律的研究,发现虾池投饲量与COD呈正相关,与溶氧呈负相关,水体中的溶解态氮主要来源于投喂的人工饲料；虾池溶解态无机氮、溶解态有机氮和溶解态总氮均值分别为0.470mg·L-1、0.325mg·L-1和0.795mg·L-1,养殖后期明显高于养殖前期,均超过富营养化的阈值；养殖期间溶解态有机氮占溶解态总氮的40.88%,是虾池溶解态氮的重要组成部分。养殖过程中INT/IP平均值为5.98,IN/IP<5的时期占整个养殖过程的48%,没有出现IN/IP>30的现象；虾池水体中叶绿素a含量平均为91.6ug·L-1,与溶解态总氮之间存在显著正相关,卡尔森营养状态指数(TSI, Carlson trophic state index)平均为72.98,虾池水体处于严重的富营养化状态。水体中NH4+-N和N02--N含量均值分别为0.210mg·L-1和0.018mg·L-1,养殖后期分别为0.419mg-L-1和0.031mg·L-1,超过健康养殖的阈值。在浓度为0.040mg·L-1的NO2--N胁迫下,对虾免疫力会下降。
(2)在广东和海南等地区的养殖中期与后期的虾池中,检测出浮游微藻优势种属于5个门共计12种,其优势度在0.12-0.99之间,优势种突出和单一；在养殖前期主要以硅藻优势种群为主,养殖的中期和后期虾池水体富营养化严重,以较喜肥或者是耐污染的绿藻和蓝藻成为优势种群。养殖期间浮游微藻的细胞数平均为19.71×107cell·L-1,香农多样性指数平均为0.56,养殖水体属重度污染状态。以波吉卵囊藻(Oocystis borgei)为优势种群控制的水质环境较为稳定,其优势种群持续时间达40d～50d；硅藻优势种群相对不稳定,持续时间仅为5d～15d；小席藻(Phormidium tenue)细胞数高达2160×107cell·L-1,容易形成赤潮。以绿藻为优势种的虾池水质较稳定,形成水环境有利于对虾生长。波吉卵囊藻是虾池中分布广和适应能力强的浮游微藻,同时还具有种群稳定和持续时间长的特点。
(3)利用稳定同位素标记法,在不同生态条件下对虾池浮游微藻溶解态氮吸收速率的研究发现,虾池浮游微藻对NH4+-N.N02--N、NO3--N、Urea-N和Met-N吸收最适宜的温度范围为25℃～30℃,盐度范围为20～30,光照度范围为45.000μmol·m-2·s～126.000μmlo·m-2·s-1,藻浓度范围为3.222×108cell·L-1～4.784×108cell·L-1。当氮浓度为14.300mg·L-1时,浮游微藻对NH4+-N、N02--N和N03--N均具有最高的吸收速率,当Urea-N和Met-N浓度分别为48.400mg·L-1和20.900mg·L-1时,其吸收速率达到最高；高浓度溶解态氮对藻细胞氮吸收具有显著的抑制作用,高的NH4十-N浓度对MH4+-N吸收抑制要早于其他形式的溶解态无机氮。
这些结果表明虾池浮游微藻对溶解态氮的吸收速率与温度、盐度、光照度、藻浓度和氮浓度有密切关系,这特点在亚热带海洋沿岸水域普遍存在。在高浓度溶解态氮情况下浮游微藻对氮的吸收会出现“饱和效应”。
(4)在多种氮源共存的条件下,利用稳定同位素标记法研究了虾池水体浮游微藻在不同温度和盐度条件下对溶解态氮吸收的选择性,并通过正交实验研究影响浮游微藻溶解态氮吸收速率的生态因子。结果显示,在不同温度和盐度实验条件下,浮游微藻对NH4+-N吸收的相对优先指数(RPI)均大于对N02-_N、Urea-N和NO3--N,优先吸收NH4+-N,其次是NO2--N,最后是Urea-N和NO3--N。正交实验结果显示,在温度20℃～30℃、光照81μmol·m-2·s-1.盐度15～30、pH7.5和藻浓度4.5×108cell·L-1～5.5×108cen·L-1的条件组合下,浮游微藻对各种溶解态氮均有较高的吸收速率,影响NH4+-N和NO2--N吸收速率的主导因子是藻浓度和盐度,影响N02-N、Urea-N吸收速率的主导因子是光照。
(5)波吉卵囊藻对NH4+-N和Urea-N吸收速率模型的预测值与实测值进行样本T检验,检验结果表明模型预测值与实测值差异不显著(P>0.05),总体均值差异不显著(P>0.05),证明模拟方程拟合度较高。
(6)波吉卵囊藻细胞对Cu2+和Zn2+的吸附过程可分为三阶段：第一阶段吸附在30min内完成,吸附率达70%以上；第二阶段吸附速度减慢,在8h内完成；第三阶段为吸附平衡阶段。波吉卵囊藻对Cu+和Zn2+的吸附的最适温度范围为25～30℃,光照度为大于54.00μmol·m-2·s-1,盐度范围为10～30。波吉卵囊藻细胞浓度为28.91×107cell·L-1时对Cu2+的吸附率为52.52%,吸附量为9.469mg·g-1；藻细胞浓度为22.91×107cell·L-1时对Zn2+的吸附率为81.44%,吸附量为2.914mg·g-1。条纹小环藻细胞浓度为2.45×107cell·L-1时对Cu2+的吸附率为63.00%,吸附量为9.26mg·g-1；藻细胞浓度为1.75×107cell·L-1时对Zn2+的吸附率为60.52%；吸附量为20.06mg·g-1。在此藻细胞浓度下,藻细胞吸附的重金属离子不会影响波吉卵囊藻和条纹小环藻的生长。
(7)在对虾养殖系统中引入波吉卵囊藻,不仅能提高水体溶氧的含量、调节pH、降低COD,还能有效地吸收各种溶解态氮；水体中NH4+-N含量降低了51.7%-37.8%,N02--N含量可降低30.2%-26.4%,对虾抗病能力显著提高(P<0.05)。在虾池中培养波吉卵囊藻构建以波吉卵囊藻为主要构架的微藻群落,养殖期间其平均生物量占浮游微藻总生物量的95.35%-43.95%,优势度为0.27～0.58,成为虾池水体中的绝对优势种,作为优势种其种群持续时间长达77d,水体NH4+-N和N02--N浓度较对照池分别降低了36.98%和81.16%；波吉卵囊藻能够有效地减轻养殖密度的制约作用,对虾生长速度明显加快,养殖产量提高83.78%。在培养水温为27℃～32℃和盐度20～28的条件下,波吉卵囊藻在养殖水体中呈逻辑斯谛(Lgistic)方式增长,环境容纳量K值为6899.959x105cell·L-1,瞬时增长率r近似值为0.002,最大可持续产量(MsY)为3.45×105cell·L-1·d-1。每天从虾池水体中去除3.45×105cell·L-1·d-1藻细胞可以保持波吉卵囊藻的种群稳定。
Eutrophication of surface waters can induce algal blooms and in salt water or estuarine waters, red tide can occur frequently, which can cause impairment of both the use of the water body and the local fishery. Therefore, eutrophication is an important ecological problem, and the study of how to deal with this problem has attracted extensive attention.
Prawn aquaculture is the pillar industry of the national marine-based economy. In China, the area of prawn aquaculture exceeded6.67×104hm2, with an annual output of more than120×104t, which accounts for38%of the global output, making China the highest prawn yielding country. Eutrophication is the major pollution issue in prawn culture, the second problem is heavy metal pollution. Nitrogen compounds discharged from aquaculture waste water are the primary sources of nitrogen pollution and the resultant eutrophication in the near-shore marine environment. There are two kinds of dissolved nitrogen in prawn aquatic system. One is dissolved inorganic nitrogen in the form of ammonia nitrogen (NH4+-N), nitrite (NO2--N), nitrate (NO3--N).. The other form is dissolved organic nitrogen as Urea-N, Methyl-N and protein. Inorganic forms, NH4+-N and NO2-N, are the primary nitrogen pollutants which are harmful to prawn. The technique for maintaining healthy prawn aquaculture is to improve the culture environment by regulating the water quality through select organisms in the prawn pond. Planktonic microalgae can take up dissolved nitrogen and heavy metal ions, and can be an important biological factor in a prawn culture system. Thus, microalgae can transform dissolved nitrogen and reduce heavy metal loads. To improve prawn pond water quality, we can enhance the pollutant removal capacity of planktonic microalgae by assembling an optimum planktonic microalgae colony through enhancing the community structure of certain planktonic microalgal species. The studies on the Planktonic microalgae in the prawn high level pond and the absorption of dissolved nitrogen and heavy metal ions adsorption by them have not been reported. In this study, we chose high elevation prawn pond to study the pollution caused by prawn breeding, planktonic microalgal assemblages in prawn ponds, the ability and regulatory mechanism for planktonic microalgal uptake of dissolved nitrogen and heavy metals and the control of the prawn pond environment. This will provide a scientific basis for optimizing the community structure of planktonic microalgae and improving water quality in prawn ponds and additionally providing a theoretical and technical basis for controlling eutrophication.
The content of the major research and the results are as follows:
(1) Through the study of dissolved nitrogen from prawn culture, we found the amount of feed showed a positive correlation with chemical oxygen demand (COD) and was negative correlated with dissolved oxygen. The dissolved nitrogen came primarily from the artificial breeding diet, with the contents of dissolved inorganic-nitrogen, dissolved organic nitrogen and the total dissolved nitrogen being0.470mg·L-1,0.325mg·L-1,0.795mg·L-1respectively. In addition, the concentrations from the late breeding stage were higher than in the early breeding stage, and the threshold of eutrophication. The content of dissolved inorganic nitrogen was40.9%of the total nitrogen, which was the major source of the dissolved nitrogen in the prawn pond during the breeding period. Over the course of the breeding period, the mean ratio of inorganic nitrogen to inorganic phosphorus (IN/IP) was5.98. It was<5at48%of time and never exceeded30. The average chlorophyll-a concentration in the pond was91.6ug·L-1, which was positively correlated with the total dissolved nitrogen. The mean value of the Carlson trophic state index (TSI) was73.0, indicating that the pond was in a state of serious eutrophication. The concentrations of NH4+-N and NO2--N during the early breeding were0.21mg·L-1and0.02mg·L-1respectively, and their concentrations in the late breeding stage were0.42mg·L-1and0.03mg-L"1. Both nitrogen species were higher than the threshold for healthy prawn breeding. Prawn immunity was reduced when NO2--N achieved a concentration of0.04mg·L-1.
(2) In the Guangdong and Hainan provinces, the major species of planktonic microalgae belong to five phyla, including12species found during the middle and late stages of prawn breeding, with a the degree of dominance from0.12to0.99. During the early breeding stages, diatoms dominated, but with eutrophication during middle and late breeding stage becoming serious, green algae and blue-green algae became dominant. The average cell number of planktonic microalgae was19.71×107cell/L-1and the mean diversity index was0.56, indicating that the system was suffering from serious contamination. The period with Oocystis borgei as the dominant algae was relatively stable, remaining dominant for40d-50d. The period when the dominant population was diatoms proved to be unstable and only lasted for5d～15d. The cell number of Phormidium tenue was2160×107cell/·L-1, which potentially is a source of red tide. The additional stable period occurred when the dominant population was green algae, which was a benefit for the growth of the prawn. Oocystis borgei was widely spread, adaptable, with the characteristics of a stable species persisting for a long duration.
(3) Using a stable isotope labeling technique, the uptake rate for nitrogen of planktonic microalgae in shrimp ponds was studied under different ecological conditions. The most suitable conditions for the uptake of NH4+-N, NO2--N, NO3--N, Urea-N and Met-N by microalgae in prawn pond were a temperature of25℃～30℃, a salinity of20～30, light illumination at45μmol·m-2·s-1～126μmol·m-2·s-1, and an algal density of3.222×108cell·L-1～4.784×108cell·L-1. When the nitrogen concentration was14.3mg·L-1, the uptake rate of NH4+-N, NO2--N and NO3--N by microalgae was highest. The uptake rate of Urea-N and Met-N was the highest when the Urea-N and Met-N concentrations were48.4mg-L"1and20.9mg-L"1respectively. High concentrations of dissolved nitrogen apparently inhibit the uptake of nitrogen. The inhibition of NH4+-N uptake occurred when the concentration of NH4-N was high.
These results demonstrated the uptake rate of nitrogen by planktonic microalgae in prawn ponds was closely related to temperature, salinity, light illumination, algal concentration and nitrogen concentration, with these specific conditions commonly in found in coastal areas in subtropical regions. The "saturation effect" for nitrogen uptake by algae would exist under high dissolved nitrogen conditions.
(4) The effect of salinity and temperature on relative preference indices (RPI) for planktonic microalgae under four dissolved inorganic nitrogen concentrations was studied using a stable isotope labeling method. The dominant factor which affected the nitrogen uptake rate of planktonic microalgae was also studied using an orthogonal experiment. The results showed that the RPI of NH4+-N of planktonic microalgae was greater than that of NO2--N, Urea-N and NO3--N. Planktonic microalgae took up NH4+-N initially and NO2--N followed, the next being Urea-N and finally NO3- -N. The orthogonal experiment showed that microalgae had a higher uptake rate for the four nitrogen sources when the temperature was20℃～30℃, the light illumination was81μmol·m-2·s-1, the salinity was15～30, the pH was7.5, and the algae density was4.5×108cell·L-1～5.5×108cell·L-1. Microalgae density and salinity were the main factors that affected the uptake rate of NH4+-N and NO2--N, while light illumination was the main factor that affected the uptake rate of NO2--N and Urea-N.
(5) Predicted values and measured values of an uptake rate model for Oocystis bergei were examined using a t-test. The test results showed that there was not a statistically significant difference between the predicted values and the measured values, as well as the population mean, which indicated that the degree of model fit was high.
(6) The uptake of Cu2+and Zn2+by Oocystis borgei could be divided into three steps:the first step was to complete the uptake process within30min, with an uptake rate of more than70%; in the second step, the rate decreased and the uptake process was completed within8h; in the third step, the uptake process reached equilibrium. The optimum uptake of Cu+and Zn2+by planktonic microalgae Oocystis borgei was when the temperature was25～30℃, the light illumination was greater than54.00μmol·m-2·s-1, and the salinity was10～30. When the Oocystis borgei concentration was2.9×108cell·L-1, the adsorption rate and capacity of Cu2+were52.5%and9.5mg·g-1respectively; when the algae concentration was2.3×108cell·L-1, the uptake rate and capacity of Zn2+were81.4%and2.9mg·g-1respectively; for Cyclotella striata when its concentration was2.5×107cell·L-1, the uptake rate and capacity of Cu2+were63.0%and9.3mg·g-1respectively; when its concentration was1.8×107cell·L-1, the uptake rate and capacity of Zn2+were60.5%and20.1mg·g-1respectively. Under these algae concentrations, the growth of Oocystis borgei and Cyclotella striata would not be affected.
(7) Introducing Oocystis borgei into prawn farming systems not only improved the content of dissolved oxygen, regulated pH, reduced COD, but also effectively fixed dissolved nitrogen. Due to the introduction, NH4+-N concentration was reduced by51.7%～37.8%, NO--N concentration reduced by30.2%～26.4%, and the disease-resistance of the prawn was improved significantly (P<0.05). In the treated prawn pond, average biomass of Oocystis borgei accounted for95.86%to45.12%, The dominance is between0.27to0.58and was the dominant algae during the breeding period. As the dominant species, it persisted for about77days. Concentrations of NH4+-N and NO2- -N were reduced by37.0%and81.2%respectively. Oocystis borgei can potentially alleviate the effect of stocking-density constraints, significantly improve prawn growth, and increase aquaculture production by83.8%. With a water temperature of27℃～32℃, a salinity of20～28, Oocystis borgei showed logistic growth with a K (carrying capacity) value of6899.959×105, r (instantaneous rate of increase) approximate value of0.002, and the maximum sustainable yield (MSY) of3.45×105·L-1·d-1. Removal of3.45·105cell·L-1of algal cells every day from the shrimp pond waters can keep Oocystis borgei population stable.
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