【作者】 黄鹤忠；

【导师】 梁建生；

【作者基本信息】 扬州大学 ， 植物学， 2013， 博士

【摘要】 江蓠属(Gracilaria)海藻具有分布广、适应性强、生长快、产量高、易栽培等特点,具有重要的营养、药用和环保价值,并且在海洋环境污染的生物修复和监测中具有重要的前景。针对当今近海水域以及水产养殖等水体所面临的富营养化和重金属污染问题,本文在实验生态条件下,以菊花江蓠(Gracilaria lichenoides)为主要材料,研究了其对水体氮(N)、磷(P)和4种重金属离子(Cd2+、Cr6+、Cu2+、Ni2+)的去除效应及其生理生化响应和耐受机制；同时,比较了菊花江蓠与龙须菜(Gracilaria lemaneiformis)对环境中Cu2+的生理生化响应机制。以期为江蓠作为环境污染的生物修复器或生物检测器的实际应用提供理论依据和实践指导。研究的主要结果如下：1.菊花江蓠对N、P短期(0-4h)和长期(第14d)吸收的最佳N：P比为16：1,最大N和P短期吸收速率(12.85和0.79μmol g-1DW h-1)的环境N/P浓度分别为1200/75和960/60(μmol.L-1),而最大N和P长期吸收速率(4.23和0.31μmol g-1DW h-1)的N/P浓度均为480/30μmol.L-12.混合N源条件下,江蓠对NH4+-N的吸收速率显著高于N03--N,长期吸收更为明显；光照条件有利于江蓠对NH4+-N和N03--N的吸收,尤其以N03--N更为明显。硝酸还原酶(NR)和谷氨酰胺合成酶(GS)的活性分别受环境中N03--N和NH4+-N的诱导并促进江蓠对NO3--N和NH4+-N的吸收。3.饥饿状态下的菊花江蓠在N、P营养盐缺乏条件下,生长减慢、各光合色素含量、蛋白含量、脯氨酸含量以及抗氧化能力均处于低水平,藻体颜色变浅,细胞内叶绿体、线粒体萎缩但淀粉粒增加,出现负生长；而在N/P为960/60和1200/75μmo1.L-1浓度中5-7d,江蓠的抗氧化防御系统受到显著抑制,引发大量自由基和脂质过氧化产物积累,叶绿体增多但类囊体肿胀、排列杂乱,线粒体数量减少,生长率、各光合色素和蛋白质含量显著下降。4.菊花江蓠在480/30-750/45μmol.L-1N/P浓度下的生长率(SGR)最高,光合色素、生化、抗氧化防御系统和细胞超微结构指标均显示,N/P750/45μmol.L-1和N/P480/30μmol.L-1分别是菊花江蓠短期(<7d)和长期(15d)内适宜N/P浓度的上限。在N/P480/30μmol.L-1条件下,江蓠获得最佳的生化和生长状态的N：P和ρ(NH4+-N):ρ(NO3--N)比值分别为16：1-32：1和6：0-1：1。5.在单一重金属离子(0.05mg.L-1或0.5mg.L-1)或各重金属离子质量浓度(0.5mg.L-1或1.0mg.L-1)相同的复合条件下,江蓠对重金属离子的去除率、去除效率和去除速率以及在江蓠体内外的积累量均依次为Cu2+>Ni2+>Cr6+>Cd2+。6.随着Cu2+、Ni2+、Cr6+、Cd2+胁迫浓度的提高,江蓠细胞活性氧自由基和脂质过氧化产物的积累逐渐增加,抗氧化防御体系中的SOD,CAT,POD, APX, GST, GR, T-AOC活性先被激活后被抑制,各光合色素和可溶性蛋白质含量逐渐降低；并且,细胞超微结构逐渐受到损伤或破坏(如：类囊体有肿胀,原生质穿壁转移,淀粉粒大量堆积,嗜饿小体增多,甚至叶绿体双层膜结构和核糖体消失等),并在细胞壁及叶绿体被膜上有大量Cu2+颗粒沉淀,叶绿体的元素成分发生改变。综合分析结果显示,江蓠对Cu2+、Ni2+、Cr6+、Cd2+胁迫的浓度生理耐受阈值依次为0.5、2.5、2.5、5mg.L-1。7.在过低或过高P浓度(1.5μmol.L-1或60μmol.L-1)下,江蓠对Cu2+、Ni2+、Cr6+、Cd2+胁迫的耐受性减低,表现为生长率下降和细胞超微结构损伤加剧；而其在适宜P浓度(15μmol.L-1)下,对Cu2+、Ni2+、Cr6+、Cd2+胁迫的耐受性均有所提高。8.与菊花江蓠相比,龙须菜在其生长速度、色素(叶绿素a,叶绿素b,藻胆蛋白,p-胡萝卜素)含量及光合活性方面受到Cu2+胁迫抑制的效应更显著,反应更敏感,生理耐受阈值为0.05mg.L-1。因而更适合作为Cu2+污染的潜在指示生物。更多还原

【Abstract】 With high economical, medical and environmental protection benefits, Gracilaria is the type of strong adaptability and fast growth, high yield, simple cultivation method and promotion easily. Under the experimental ecology, this article mainly investigated the bioremoval of nitrogen, phosphorus and four heavy metals iron including cadmium, chromium, copper and nickel of Gracilaria lichenoides, as well as its physiological and biochemical responses on it. Meanwhile, the mechanism of physiological and biochemical response to Cu stress in Gracilaria lichenoides and Gracilaria lemaneiformis were also compared to provide theoretical basis and practical guidance for the Gracilaria as a bioremediation or bioindicator of the environment pollution. The results showed that:1. The uptake rates (SNUR and LNUR) and uptake efficiency (SNUE and LNUE) of N (TIN) and P (PO43--P) by Gracilaria lichenoides for short-term (0-4h) and long-term (over a24h period at14th day) were studied,showing that the optimal N:P ratio in culture media which SNUR and LNUR for N and P by G. lichenoides reached the highest value among different N/P concentrations (P<0.05) was16:1. The reached maximum SNUR of N and P by G. lichenoides was12.85and0.79μmol/(gDW·h), respectively, when the N/P concentration in medium was1200/75and960/60μmol/L; and the reached maximum LNUR of N and P by G. lichenoides were4.23and0.31μmol/(gDW·h), respectively, with the N/P concentration in medium of480/30μmol/L.2. On treatment with mixed nitrogen sources, the uptake rates of different state compounds of nitrogen for Gracilaria were compared, showing the uptake rates of NH4+-N of Gracilaria was significantly higher than NO3--N, especially for long-term absorption; light treatment had significant effects on the uptake of NH4+-N and NO3--N, especially for the NO3--N absorption. What’s more, the activities of NR and GS were induced by the NH4+-N and NO3--N respectively, which in turn promoted the uptake of this two states of nitrogen.3. During the nitrogen and phosphorus starvation, the growth dropped, the color of algae turned gradually from red to yellow/white,the pigment, proline, protein content as well as the antioxidant capability of G. lichenoides were all affected and weakened; meanwhile, the atrophy of chloroplast and mitochondrial、increase of the grain starch in cell also occurred; while under the condition of the N/P concentration in medium of1200/75and960/60μmol/L for5-7days, the antioxidative defense system of G. lichenoides was obviously inhibited,leading to the accumulation of great amount of harmful free radicals and lipid peroxidation products;some damaged ultrastructure changes occurred in the chloroplasts including the thylakiod swollen and lined irregularly, number increase;volume increase, and significant accumulation of starch grains in the chloroplasts.Besides,the growth rate declined,and the pigment, protein content decreased dramatically.4. On the condition of480/30-750/45μmol.L-1N/P, the SGR of Gracilaria lichenoides reached the highest value,and the photosynthetic pigments、physiological and biochemical、antioxidative defense system and the ultrastructure indexes all indicated that the best N/P concentration of short-term (<7d) and long-term (15d) for Gracilaria lichenoides were N/P750/45μmol.L and N/P480/30μmol.L-1respectively. While treatment with N/P concentration of480/30μmol.L-1,the Gracilaria lichenoides could achieve optimum status with the ratio of N:P and ρ(NH4+-N):ρ(NO3--N) of16:1-32:1,6:0-1:1, respectively.5. In this trial, whatever the single heavy metal mass concentration (0.05mg.L-1or0.5mg.L-1)condition or composite conditions with equal heavy metal concentration (0.5mg.L-1or1.0mg.L-1) condition, the removal efficiency, removal rate, and accumulation of four heavy metals extra-and intra-cellularly of Gracilaria were all shown with the following sequence: Cu>Ni>Cr>Cd.6. With increasing Cu, Ni, Cr, Cd concentrations, the accumulation of harmful reactive oxygen species(ROS) and lipid peroxidation products(MDA) increase gradually; SOD, CAT, POD, APX, GST, GR, and T-AOC activities of antioxidative defense system were all first activated and then inhibited, both the soluble protein and photosynthetic pigments were dropped; moreover the damage of the ultrastructure of cells clearly occurred in the chloroplasts (thylakoid of chloroplast swollen, evident plasmolysis phenomena, significant accumulation of starch grains, increase of osmiophilic granule number, bilayer membrane structure of the chloroplast was ruptured, disappearing of the nucleoli, acumination of deposits of Cu on the surface of the cell wall and the chloroplast envelope),the composition of the chloroplasts elements changed gradually. The analysis results indicated that the physiological tolerant threshold concentration of Cu, Ni, Cr, Cd was0.5,2.5,2.5,5mg.L-1respectively for Gracilaria.7. Both the relative higher and lower P concentrations (1.5μmol.L-1or60pmol.L-1) have adverse effects on the stress tolerance of Gracilaria to Cu, Ni, Cr, Cd, including the decline of the growth and the aggravation of the cells’ultrastructure injury. Whereas the tolerance of Cu、Ni、Cr、Cd stress increased under the appropriate concentration of15μmol.L-1P.8. In G. lemaneiformis, decrease in growth, pigment (chlorophyll a, chlorophyll b, phycobiliprotein, β-carotene) content and photosynthetic activity was more prominent, and the threshold concentration that caused a notable inhibition in SGR was50μgL-1Cu2+, making it a suitable candidate of potential bioindicator to Cu2+pollution.更多还原