Low Molecular Weight Organic Acids In Mangrove Wetlands And Their Impact On Heavy Metals Bioavailability

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...