【作者】 卢豪良；

【导师】 严重玲；

【作者基本信息】 厦门大学 ， 生态学， 2007， 博士

【摘要】 当前我国城市化和工业化以及海洋生物养殖加剧了重金属的陆海迁移,导致其对沿海生态环境尤其是处于海陆交错带的红树林生态系统的污染。沿海河口区域的一些工业、生活污水,养殖污水,等通过排污管道向红树林湿地排放,使得红树林成为海岸的“污水处理场所”。城市化和工业化加剧了重金属的陆海迁移,导致其对沿海生态环境尤其是处于海陆交错带的红树林生态系统的污染。低分子量有机酸在土壤中大量存在,并在时空上影响着成土的过程,如土壤养分的供应,重金属的转移和固定,矿物的风化等起着重要的作用。在土壤溶液中,低分子量有机酸的羧基部分解离,导致其表现出弱酸性。低分子量有机酸作为配合体通过络合作用提高了土壤溶液中溶解性阳离子如铁,铝的总量。溶解性低分子量有机酸和其络合的金属离子容易从土壤孔隙中运移,并通过强烈的鳌合作用,提高的金属离子如（铜,铁）的淋溶作用。低分子量有机酸通过羧基和羟基与金属离子的调节作用而吸附在土壤矿物表面。低分子量有机酸强烈的协同作用通过解离和还原反应使矿物质与其表面的吸附作用提高了矿物质的溶解和风化作用。国内外对不同植物根系分泌的有机酸与土壤中重金属的相互作用开展了大量的研究,众多研究结论认为根系分泌的有机酸主要是通过影响重金属离子在根-土界面上的吸附-解吸、配位-解离等反应来影响金属离子在根-土界面上的化学行为。目前国内外植物根系与重金属相互作用的研究多集中于陆地森林生态系统与农田生态系统,对于陆海交错带红树林生态系统这一生态敏感区域的研究还鲜见报道。本文以福建漳江口红树林为研究对象,通过野外定位观测和室内盆栽实验,在研究红树植物根系分泌的低分子量有机酸,红树林沉积物中低分子量有机酸以及红树根际重金属形态的基础上,着重探讨红树植物在胁迫条件下根系分泌低分子量有机酸响应及机制,低分子量有机酸对重金属污染治理的作用,植物-沉积物界面重金属的形态转化,这对进一步拓展我河口湿地重金属污染研究,深入分析红树植物对河口区域重金属污染的治理有所裨益,为河口区域重金属迁移转化的生物地球化学过程提供重要基础资料,并对我国河口区域重金属污染的治理,红树林生态系统的保护有一定的指导意义。本文主要得出以下结论:1.用改进的高效液相色谱法,分析10种低分子量有机酸:草酸,酒石酸,甲酸,L-苹果酸,乳酸,乙酸,顺丁烯二酸,柠檬酸,丁酸,反丁烯二酸等低分子量有机酸,色谱条件为:Agilent 1100型高效液相色谱及其色谱工作站,AgilentZORBAX Extend-C₁₈（250 mm×4.6 mm I.D.,5μ）色谱柱。柱温为25±2℃,进样量为50μl,pH为2.5的93%的25 mM的KH₂PO₄和7%的甲醇作为流动相,DAD检测波长为210 nm时,流速为1.0 ml·min^-1,能有效的分离检测。红树植物秋茄,白骨壤,桐花树的根系分泌的低分子量有机酸主要以一元羧酸为主。植物种类的不同其分泌的低分子量有机酸种类与总量都有所不同。室内栽培红树植物秋茄、白骨壤、和桐花树幼苗根系分泌的低分子量有机酸主要为草酸,柠檬酸,乳酸,苹果酸,乙酸、丁酸,酒石酸。秋茄植物根系分泌物中柠檬酸含量最高为9.004μmol g^-1root DW,而白骨壤根系分泌物中酒石酸含量(3.760μmol g^-1rootDVV)在三者之中最高,达到3.760μmol g^-1root DW,桐花树根系分泌物中则有较多的乙酸。2.红树林不同树种组成影响了沉积物中的低分子量有机酸的组成与含量。而沉积物不同区域的低分子量有机酸分布也不相同。在秋茄,白骨壤与桐花树林地中不同区域的低分子量有机酸的含量分布均表现为:根际＞林内＞林外光滩。其中,秋茄根际、林内和光滩低分子量有机酸总含量分别为269.45μM、201.47μM、157.93μM;白骨壤根际、林内和光滩低分子量有机酸总含量分别为310.96μM、214.57μM、139.51μM;桐花树根际、林内和光滩低分子量有机酸总含量分别为251.44μM、161.20μM、103.33μM。在林地内与林外土壤溶液的低分子量有机酸中甲酸、乙酸、乳酸等一元羧酸为主要成分,而三元羧酸如柠檬酸含量并不高。而在根际土壤溶液中,苹果酸,柠檬酸,丁酸含量比林内与林外光滩高。白骨壤林内低分子量有机酸总含量为214.57μM,高于秋茄（201.47μM）和桐花树（161.20μM）林内的含量。林地表层沉积物中低分子量有机酸中一元酸的比例明显高于二元和三元酸。在秋茄林地中,根际的一元羧酸总和约为二元和三元羧酸的2倍。林外光滩与林内沉积物中都表现出相同的趋势。土壤中低分子量有机酸的含量受土壤的物理化学性质影响,如土壤粒径机械组成,水分含量,电荷分布,有机质含量,pH值等均对有机酸的含量造成影响。红树林沉积物的有机质含量比较高（5-8%）,林内有机质含量大于林外。林内沉积物略成酸性,pH值在林内介于6.51-6.76之间。沉积物的机械组成以粉粒为主,占了总体粒径组成的70%以上。在红树林林内林外表层沉积物中低分子量有机酸的含量沉积物中含水量,有机质,pH值以及可溶性盐均呈显著相关,其与有机质的相关性最大。3.红树植物大量分泌有机酸作为主动适应性反应机制适应磷胁迫,这对红树林在沉积物中获取营养有重要的意义。在缺磷状态下,红树植物秋茄,白骨壤桐花树的根系分泌的低分子量有机酸的总量显著提高。正常供磷时,秋茄根系分泌物中主要检测出的低分子量有机酸为:甲酸,乙酸,丁酸,顺丁烯二酸,乳酸,苹果酸,L-酒石酸,草酸,柠檬酸。在P胁迫7天时,乙酸,丁酸,苹果酸,草酸与柠檬酸的含量都显著高于正常供磷水平。在缺磷状态下,二元羧酸与三元羧酸的分泌量明显增大,其中以柠檬酸的分泌量最大达到35.680μmolg^-1DW root。磷胁迫时总分泌的有机酸量为正常的8倍。磷胁迫下白骨壤根系分泌物中的低分子量有机酸主要为甲酸,乙酸,丁酸,苹果酸,L-酒石酸,草酸,柠檬酸。丁酸,苹果酸与柠檬酸的含量都显著（P＜0.05）高于正常供磷水平。甲酸,乙酸分泌量与正常供磷水平相比,无显著性差异。而苹果酸为主要根系分泌的低分子量有机酸,含量为正常供磷水平的18.9倍。桐花树根系在正常供磷下分泌的低分子量有机酸主要为:甲酸,丁酸,乳酸,苹果酸,L-酒石酸,草酸,柠檬酸。磷胁迫下,丁酸,草酸,L-酒石酸,柠檬酸的含量显著（P＜0.05）高于正常的供磷水平。L-酒石酸在磷胁迫下分泌量为正常的2.3倍。磷胁迫14天时,三种红树秋茄、白骨壤和桐花树分泌的种类和总量都有所减少,总的有机酸分泌大约为缺磷7天时的70%,桐花树中乳酸未能检测到。4.采用盆栽实验,探讨了低分子量有机酸对红树植物的毒性,对沉积物溶液中重金属浓度的影响,及其对红树植物修复重金属的作用。研究结果表明低分子量有机酸对红树植物的毒性小,在沉积物中施加有机酸能有效的提高重金属的植物固定。与低分子量有机酸相比,乙二胺四乙酸（ethylenediamine tetraaceticacid,EDTA）对秋茄幼苗毒性大于低分子量有机酸。三种低分子量有机酸草酸,柠檬酸,五倍子酸在施加量浓度小于150 mmol kg^-1时对秋茄生长的生物量没有显著的影响（P＞0.5）。三种低分子量有机酸草酸,柠檬酸,五倍子酸施加量为150 mmol kg^-1,EDTA施加量为50 mmol kg^-1时,秋茄的生物量分别减少5%,36%,38%和43%。施加草酸,柠檬酸,五倍子酸和EDTA显著提高了植物体内的重金属含量。除草酸外,EDTA,柠檬酸,五倍子酸显著（P＜0.05）提高了秋茄茎中Cu,Zn和Cd的含量。EDTA与柠檬酸对茎中的Pb含量影响显著（P＜0.05）,秋茄根系表现出对重金属的富集性。EDTA与柠檬酸,五倍子酸,草酸相比,其促进了Cd由根向茎的转移。施用低分子量有机酸与EDTA提高了土壤溶液中的重金属浓度。并且随着时间逐渐减少,在施用七天时土壤溶液中Cu的浓度在EDTA,柠檬酸,五倍子酸,草酸中的浓度分别为为62.36,33.48,15.73和16.64mg l^-1。5.采用根箱法应用于室内红树植物根际研究,分离根际与全土区域,探讨在红树植物根系作用下重金属在根际微域的生态行为。实验结果表明:镉胁迫改变根系分泌的低分子量有机酸含量与种类。10 ppm Cd胁迫下秋茄根系分泌的低分子量有机酸含量达到最大为39.0457μmol g^-1DW roots。反丁烯二酸则只有在20 ppm Cd的处理中出现,在根系分泌的低分子量有机酸中,甲酸,乳酸,柠檬酸,苹果酸占主要部分,在对照处理中分别占总的低分子量有机酸的8.11%,18.52%,68.28%和2.31%。随着Cd胁迫浓度的升高,在10 ppm Cd胁迫下根系分泌的低分子量有机酸中甲酸,乳酸,柠檬酸,苹果酸比例分别为8.28%,6.57%,79.26和3.74%,而在Cd处理浓度为50 ppm时其比例分别为8.81%,2.55%,18.31%和59.65%。根系影响下,根际区域更为酸化,根际区域的土壤的pH值比非根际土壤的低0.2-0.5。对照的土壤溶液的pH值比Cd胁迫处理的高,距离根系不同距离的沉积物中其变化范围为6.43-6.54。水溶性有机碳的含量随着Cd处理浓度的增加而增大,其变化范围为17.52到27.72 mM。根系分泌的低分子量有机酸改变了根际Cd的形态。碳酸盐结合态Cd随着Cd施加浓度的升高而降低（对照的13%下降到50 ppm处理中的7%）,而交换态Cd在各个处理的中的变化趋势与此相反,对照中占6%上升到50 ppm Cd处理占39%。pH对Cd形态的改变并不显著,而红树植物秋茄根系分泌有机酸对根际重金属有效性产生显著影响（P＜0.05）,根系分泌物可能通过络合Cd,改变根际区域溶解性有机碳含量等改变了Cd生物有效性。6.野外秋茄林地沉积物中重金属含量分布规律为:根际沉积物＞非根际沉积物。野外红树林湿地中秋茄幼苗根系分泌的低分子量主要为:甲酸,丁酸,柠檬酸,苹果酸和乳酸,不同沉积物结构对秋茄根系分泌的低分子量有机酸有一定的影响,表现为在泥质滩涂与砂质滩涂中,秋茄幼苗根系分泌的柠檬酸,苹果酸和乳酸含量均存在显著性差异（P＜0.05）。沉积物中重金属的赋存形态主要铁锰氧化物结合态重金属为主,非根际沉积物中的铁锰氧化物结合态重金属含量＞根际沉积物,根际沉积物的可交换态与碳酸盐结合态重金属含量＞非根际沉积物。低分子量有机酸对重金属可溶解态的提取效率表现为:柠檬酸＞混合酸＞苹果酸＞乳酸。秋茄根系分泌低分子量有机酸,特别是柠檬酸对促进了重金属的溶解,秋茄通过根系分泌的低分子量有机酸改变重金属的形态,影响重金属的生物有效性。更多还原

【Abstract】 Advances in science and technology have enabled humans to exploit natural resources to a great extent,generating unprecedented disturbances in global elemental cycles.Mangrove ecosystems,important intertidal estuarine wetlands along the coastlines of tropical and subtropical regions,are frequently exposed to contamination of heavy metal due to high degree of industrialization and urbanization.Port development,mariculture,and rapid economic development all have resulted in a strong risk of heavy metal contamination in the coastal environment.Consequently,accumulated pollutants are of concern relative to both human and coastal ecosystem exposure and potential impact.Mangrove ecosystems,although possessing great ecological and commercial value,have in many parts of the world traditionally been considered wastelands and widely used as sites where effluents are discharged and solid wastes are dumped,including metallic anthropogenic wastes.Mangrove sediment,being anaerobic,rich organic carbon,reduced and rich in sulphide,favor the retention and accumulation of heavy metals.Sediments can therefore also act as a source of heavy metals, when triggered by changes in abiotic conditions such as pH,redox potential and salinity.Storms and human activities such as clearing and dredging can remobilize metals and facilitate transport from mangroves to coastal waters. Metals may also be transported to estuarine waters when accumulated by mangroves and concentrated in exported leaf debris,which is an important food source for higher organisms in estuarine food chains.Mangrove wetland as a buffer in the estuary therefore acts as a sink and source of heavy metal in the coastline ecosystem.Organic acids have been hypothesized to perform many functions in soil including root nutrient acquisition,mineral weathering,microbial chemotaxis and metal detoxification.However,their role in most of these processes remains unproven due to a lack of fundamental understanding about the reactions of organic acids in soil.After release from the root,however,organic acids can suffer from a number of fates such as sorption,biodegradation and metal complexation,making their behavior difficult to predict.This dissertation focused on methods for analyzing low molecular weight organic acids（LMWOAs）in root exudates and soil solutions,root responses LMWOAs to phosphorus deficiency and heavy metals stress,and variation in LMWOAs between forest types.Effect of LMWOAs on phytoextraction of heavy metals and metals species changes in rhizosphere were studied.Major conclusions were summarized as follows:1.An improved reversed-phase liquid chromatographic（RPLC）method for the analysis of LMWOAs in plant root exudates and soil solutions.Agilent 1100 reversed-phase column liquid chromatography was used for the separation and quantification of 10 LMWOAs（formic,acetic,malic,butyric,lactic,fumaric, maleic,citric,L-tartaric oxilic）in mangrove root exudates and soil solution.A mobile phase of 93%25 mM KH₂PO₄ at pH 2.5 and 7%methanol at a flow-rate of 1 ml min^-1resolved all 10 acids on a Agilent ZORBAX Extend-C₁₈（250 mm×4.6 mm I.D.,5μ）column.DAD output at 210 nm was used for the quantification of LMWOAs.Different plants have different root exudates both in quantity and quality.In mangrove Kandelia candel,Avicennia marina,and Aegiceras corniculatum root exudates,monocarboxylic acids were dominated in LMWOAs.Oxalic,citric,lactic,malic,acetic,butyric and tartaric acids was found in three mangrove species.In K.Candel,citric acid concentration(9.004μmol g^-1root DW)was highest in the root exudates LMWOAs.While In A. marina and A.corniculatum the highest concentration of LMWOAs were tartaric(3.760μmol g^-1root DW)and acetic(4.213μmol g^-1root DW)acids respectively.2.Low-molecular weight organic acids found in soils and soil solutions comprise mainly aliphatic mono-,di- and tricarboxylic acids.In soil solutions isolated from mangrove sediments we observed LMWOAs concentrations showed as following:rhizopshere＞sediments with plants＞bare beach.In K. Candel the total LMWOAs concentrations in rhizopshere,sediments with plants,bare beach soil solutions were 269.45,201.47 and 157.93μM respectively.In A.marina the total LMWOAs concentrations in rhizopshere, sediments with plants,bare beach soil solutions were 310.96,214.57 and 139.51μM respectively.In A.comiculatum the total LMWOAs concentrations in rhizopshere,sediments with plants,bare beach soil solutions were 251.44, 161.20,103.33μM respectively.In bulk soil solutions monocarboxylic acids concentrations were higher than di-and tricarboxylic acids.In rhizosphere soil solution malic,citric and butyric acids were in dominant.The total LMWOAs concentrations in three stands showed the trends A.marina（214.57μM）＞K. Candel（201.47μM）＞A.comiculatum（161.20μM）.Soil chemical and physical properties such as soil texture,water contents,electric charge distribution, organic contents,pH would influence the LMWOAs concentrations in soil solutions.The high organic maters content（5-8%）in mangrove sediments was significantly related to LMWOAs concentrations.3.Phosphorus（P）is critically needed to improve soil fertility in many parts of the world.Mangrove root can excrete organic acids to adapt P stress.Low molecular organic acids exudation from the roots of mangrove seedlings under P stress increased significantly（P＜0.05）.Low molecular weight di- and tricarboxylic acids were found dominantly in root exudates..After 7 days,in P stress treatments,acetic,butyric,malic,oxalic and citric exudation from the roots of K.Candel showed significantly higher than control treatments.Root exudated citric acid was 35.680 uMg^-1DW root,and the total concentration of LMWOAs showed 8 folds than control.Formic,acetic,butyric,tartaric,oxalic and citric were identified in root exudates under P stress treatments.Butyric, malic and citric acids were significantly higher than control,but for acetic and formic acids there were no significant differences compare with control treatments.Malic acid was 18.9 folds than that in control treatment.Butyric, oxalic,tartaric and citric acids concentrations were found significantly（P＜0.05） higher than control treatments in A.comiculatum root exudates under P stress conditions.Tartaric acid were found 2.3 folds than that in control treatments. At 14 days,three mangrove species root exudates LMWOAs concentrations were decreased under P stress.And total LMWOAs concentrations were about 70%of those at 7 days under P stress.Lactic was not identified in the root exudates of A.comiculatum.4.Pot experiments were conducted to study the low molecular weight organic acids（LMWOAs）and EDTA assistant of the mobility and phytoextraction of heavy metals by mangrove Kandelia candel.The object was to investigate whether LMWOAs（citric,oxalic and gallic acid）existed in mangrove roots exudate would have same enhancing functions as synthetic chelate EDTA on phytoextration.The results showed:citric,oxalic and gallic acid showed less toxic to K.candel seedlings as comparing to EDTA. Compare with control,the biomass production decrease 43%when supplied with EDTA in concentration of 50 mmol kg^-1.Supply oxalic,citric,galic acids and EDTA can enhance metals concentration in plants.Except for oxalic acid, citric,galic acids and EDTA significantly increased the concentration of metals in shoots of K.Candel.EDTA increased Cd to transport from root to shoot.The roots of K.Candel accumulated more heavy metals.Except for lead,citric and gallic acid significantly（P＜0.01）enhanced the Cu,Zn and Cd concentration in shoots of K.candel.LMWOAs and EDTA addition led to elevate soil solution concentrations of Cu,Pb,Zn and Cd in 7 days after chelates were addition. Copper concentration in soil solutions which supply with EDTA,citric,gallic and oxalic acids were reached 62.36,33.48,15.73 to 16.64 mgl^-1,respectively. EDTA enhanced Cd transported from roots to shoots.However,LMWOAs did not show this aspect.After 2 months grown in contaminated sediments,with or without chemical chelates addition,K.candel roots accumulated more than 6-fold total Cu,Pb,Zn and Cd concentrations than those in sediments. Mangrove may be concern as phytostabilization more than phytoextraction for remediation heavy metal pollutants in tropical and subtropical estuary wetland. 5.Changes in both LMWOAs and Cd bioavailability,directly or indirectly related to the Cd stress were studied in the laboratory.A rhizobox technique was used for Cd species changes rhizoshpere in increasing Cd concentration stress conditions（0,5,10,20,30,40 and 50 ppm）.K.candel seedlings which grown in the rhizoboxes were selected to examine their root exudates.The results showed that both quantity and quality of LMWOAs in root exudates changed after Cd supply.The total concentration of LMWOAs was 39.0457μmol g^-1DW roots in treatment with 10 ppm Cd.Monocarboxylic acids（formic, acetic,lactic,butyric and oxalic acids）,and di- and tricarbonxylic acids（maleic, fumaric,citric and L-tartaric acids）were found in root exudates.Citric,lactic and acetic acids being dominant took up 76.85%-97.87%of the total LMWOAs in root exudations.Fumaric acid was only found where mangroves were growing on 20ppm Cd.Formic,lactic,citric and malic acids being dominant in root exudates and account for 8.11%,18.52%,68.28%and 2.31%of total LMWOAs in control,respectively.With the Cd stress increasing,the ratio of these acids increased,and in 50 ppm Cd treatments reached at 8.81%,2.55%, 18.31%and 59.65%respectively.Root exudates reduced pH by 0.2-0.5 pH units in the rhizosphere compare to the bulk soil.The range of pH was 6.43 to 6.54 in the sediments.The dissolved carbon ranged from 17.52 to 27.72 mM. The proportion of exchangeable Cd and Cd bound to carbonate had a positive correlation to total LMWOAs in the rhizosphere soil.Root exudates induced changes in soil Cd species under control conditions,consisting of lower exchangeable Cd compared with increasing stress.Results indicate that the measurement of LMWOAs may be included as early biomarkers in a plant bioassay to assess the phytotoxicity of Cd-contaminated soils on mangrove plants.6.In fields,muddy sediment accumulated more heavy metal than sandy sediment.Heavy metal concentrations in rhizosediment were higher than those in sediment.Heavy metals concentrations distribution in K.Candel sediments following the order:rhizosediment＞bulk sediments.Formic,butyric, malic,citric and lactic acids were found and quantified in the root exudates, with citric and lactic acids being predominant.Total amount of LMWOAs in the sandy site was higher than that for the muddy site.Furthermore,different sediment characteristics were significant influence the amounts of malic,citric and lactic acids（P<0.05）.The percentage of exchangeable and bound to carbonates fraction heavy metal extracted by LMWOAs followed the pattern: Citric acid>Mixture acid>Malic acid>Lactic acid>Acetic acid.This indicated root exudates LMWOAs caused positive effects on metal bioavailability更多还原