【作者】 杨文浩；

【导师】 倪吾钟；

【作者基本信息】 浙江大学 ， 植物营养学， 2014， 博士

【摘要】 土壤重金属污染已成为我国严重的环境问题,污染土壤的修复已引起广泛的关注,植物提取修复是一种极具潜力的治理方法。本研究从矿区土壤和植物调查入手,采用聚类分析和临界浓度判别相结合的方法分析了古银矿区主要植物的重金属积累特性,并进行了分类；通过采集野外条件下的根际土壤,研究了代表性矿区耐性植物根际微生物数量与活性；通过预分层根箱试验,研究了植物提取修复模拟镉污染土壤和镉-锌-铅复合污染土壤过程中根际微生物生物量、生化活性和群落结构特性,取得了以下主要研究结果：1.古银矿区调查结果表明,土壤Cd、Zn和Pb的浓度范围分别是5.7-84.4,467.2-8326.7和898.8-7381.0mg kg-1,植物地上部Cd、Zn和Pb的浓度范围分别是0.39-1053.58、71.3-3508.8和11.5-1073.0mg kg-1,植物根部Cd、Zn和Pb的浓度范围分别是2.54-222.02、71.3-3508.8和122.0-3437.7mgk-1。以地上部重金属含量、富集系数和转运系数为变量,采用聚类分析和浓度判别相结合将植物分为5类：超富集植物、潜在超富集植物、富集植物、潜在富集植物和普通积累植物。植物对Cd的积累分成2类：古银矿生态型东南景天地上部Cd含量为601.87mg kg-1,是Cd超富集植物；其余20种植物是Cd普通积累植物。植物对Zn的积累分成3类：东南景天是一种潜在Zn超富集植物；小飞蓬和野艾蒿是Zn富集植物；其余18种植物是Zn普通积累植物。植物对Pb的积累分成4类：东南景天和狭叶楼梯草是Pb潜在超富集植物；湖南悬钩子、白毛夏枯草和一年蓬是Pb富集植物；小飞蓬和野艾蒿是Pb潜在富集植物；其余14种植物是Pb普通积累植物。聚类分析和临界浓度判别相结合是一种鉴定具有重金属提取潜力植物的有效方法,聚类分析结果表明,东南景天(对于Cd和Zn)、小飞蓬和野艾蒿(对于Zn和Pb)、狭叶楼梯草、湖南悬钩子、白毛夏枯草和一年蓬(对于Pb)是具有植物提取修复污染土壤潜力的超富集／富集植物。2.古银矿生境条件下,东南景天、湖南悬钩子、过路黄和细风轮菜根际土壤微生物量碳含量、呼吸强度、脲酶、磷酸酶和转化酶活性显著(P<0.05)高于相应非根际土壤。东南景天、湖南悬钩子、过路黄和细风轮菜根际土壤中检测到的磷脂脂肪酸种类数量分别为70、60、58、54种,对应的非根际土壤分别为49、49、48、39种。东南景天、湖南悬钩子和细风轮菜根际土壤微生物群落物种丰富度指数(H)显著(P<0.05)高于非根际土壤。四种耐性植物根际土壤PLFA总量及细菌、G+菌、G-菌、真菌、放线菌、AM真菌、原生动物特征PLFA含量均显著(P<0.05)高于非根际土壤含量。东南景天根际土壤微生物量碳含量、基础呼吸速率、磷酸酶活性、转化酶活性、微生物群落丰富度指数(H)、PLFA总量以及所有群落PLFA含量(细菌、G+菌、G-菌、真菌、放线菌、AM真菌、原生动物)均显著(P<0.05)高于其余3种耐性植物。东南景天根际与其它植物根际以及非根际土壤微生物生化特性和群落结构存在显著差异。3.东南景天植物提取修复模拟Cd污染土壤的预分层根箱试验结果表明,在根系分布区,生长3个月后,根际土壤基础呼吸速率、酸性磷酸酶和转化酶活性显著(P<0.01)高于近根际和未种植植物土壤；生长6个月后,根际微生物量碳、脲酶、酸性磷酸酶和转化酶活性显著(P<0.01)高于近根际和未种植植物土壤。在预分层根际区,生长3个月后,0-2mm根际土壤酸性磷酸酶活性显著(P<0.05)高于2-10mm根际土壤和非根际土壤；生长6个月后,0-2mm根际土壤微生物量碳含量、基础呼吸速率、脲酶、酸性磷酸酶和转化酶活性显著(P<0.05)高于2-10mm根际土壤和非根际土壤,2-10mm根际土壤微生物碳含量和基础呼吸速率显著高于非根际土壤。相关分析结果表明,6个月生长后的预分层区,土壤基础呼吸速率和脲酶活性与水提取态Cd呈显著(P<0.05)负相关。东南景天植物提取修复Cd污染土壤能够降低根际土壤水溶态Cd浓度,显著提高根际微生物生化活性。4.东南景天修复模拟Cd污染土壤的预分层根箱试验结果表明,在根系分布区,生长12个月后,根际土壤中检测到的PLFA数量为53种,未种植植物土壤只有32种,(近)根际微生物群落物种丰富度指数(H)显著(P<0.05)高于未种植植物土壤,(近)根际土壤PLFA总量及细菌、G+菌、G-菌、真菌、放线菌、AM真菌、原生动物特征PLFA含量均显著(P<0.05)高于未种植植物土壤；MicrorespTM分析结果表明,根际土壤对所选14种碳源利用强度均显著(P<0.05)高于未种植植物土壤。在预分层根际区,0-2mm根际土壤中检测到的PLFA种类数量为60种,未种植植物土壤只有39种,0-8mm根际土壤微生物群落物种丰富度指数(H)显著(P<0.05)高于非根际土壤,0-2mm根际土壤中PLFA总量及细菌、真菌、放线菌、AM真菌、原生动物特征PLFA含量均显著高于非根际土,0-2mm根际土壤对15种碳源利用强度显著(P<0.05)高于非根际土。不同微生物群落磷脂脂肪酸含量和碳源利用强度与pH、HOAc提取态、NH2OH·HCl提取态和全量Cd呈显著(P<0.05)负相关,与土壤活性有机碳呈显著(P<0.05)正相关。根际土壤中活性有机碳含量的升高以及有效Cd浓度的降低是根际微生物群落结构丰度增加以及碳源利用水平增强的原因。5.东南景天修复镉-锌-铅复合污染土壤的预分层根箱试验结果表明,在根系分布区,生长3个月和6个月后,根际土壤微生物量碳含量、基础呼吸速率、脲酶、酸性磷酸酶和转化酶活性均显著(P<0.05)高于未种植植物土壤；在预分层根际区,3个月生长后,0-4mm根际土壤微生物量碳和脲酶活性显著(P<0.05)高于非根际土；6个月生长后,0-2mm根际土壤微生物量碳含量、0-8mm根际土壤基础呼吸速率和0-41mm根际土壤脲酶、酸性磷酸酶和转化酶活性显著(P<0.05)高于非根际土。相关分析表明,6个月生长后的预分层区,土壤基础呼吸速率和脲酶活性与水提取态Cd呈显著(P<0.05)负相关；微生物量碳含量、酸性磷酸酶和转化酶活性与水提取态Zn呈显著(P<0.05)负相关；微生物量碳含量、基础呼吸速率、脲酶、酸性磷酸酶和转化酶活性与水提取态Pb呈显著(P<0.05)负相关。东南景天植物修复镉-锌-铅复合污染土壤能够降低土壤水提取态Cd、Zn、Pb含量,显著提高根际微生物生化活性。6.东南景天植物提取修复镉-锌-铅复合污染土壤的预分层根箱试验结果表明,在根系分布区,生长12个月后,根际、近根际和未种植植物土壤中检测到的PLFA种类数量分别为50种、42种和29种,(近)根际微生物群落物种丰富度指数(H)显著(P<0.05)高于未种植植物土壤,(近)根际土壤PLFA总量及细菌、G+菌、G-菌、放线菌、真菌、AM真菌和原生动物特征脂肪酸含量均显著(P<0.05)高于未种植植物土壤；MicrorespTM结果表明,根际土壤对所选14种碳源利用强度均显著高于未种植植物土壤。在预分层根际区,0-2mmm根际土壤中检测到的PLFA种类数量为41种,非根际土为31种,0-10mm根际土壤微生物群落物种丰富度指数(H)显著(P<0.05)高于非根际土壤,0-2mm根际土壤PLFA总量及细菌、革兰氏阳性细菌、革兰氏阴性细菌、放线菌、真菌、AM真菌和原生动物特征磷脂脂肪酸含量均显著(P<0.05)高于2-10mm根际土壤和非根际土壤相应磷脂脂肪酸含量；0-8mm根际土壤14种碳源利用强度均显著(P<0.05)高于非根际土。土壤PLFA总量、各类微生物特征脂肪酸含量和碳源利用强度均与土壤全量、HOAc提取态、NH2OH·HCl提取态和NH4OAC提取态Cd和Zn呈显著(P<0.05)负相关,与土壤活性有机碳含量呈显著(P<0.05)正相关。根际微生物活性的提高和群落结构丰度的增加与土壤活性有机碳的升高以及Cd和Zn浓度的降低有关。更多还原

【Abstract】 Soil heavy metal pollution has become a serious environmental problem in our country. Remediation of heavy metal contaminated soil has attracted extensive attention. Phytoextraction, the use of metal hyperaccumulating plants to clean up contaminated soil, is being considered as a promising, cost-effective and non-intrusive technology for the remediation of metal polluted soils. In the present study, classification of plant species for metal accumulation and identification of potential species for phytoextraction was conducted using cluster analysis based on a practical survey. The rhizosphere soils of some representative tolerant plants were collected in the field condition. Microbial biomass and activities of these soils were studied to evaluate the rhizosphere microbial effects of the tolerant plants. Pre-stratified rhizobox experiments were conducted to determine the effects of phytoextraction on microbial properties through the measurement of soil microbial biomass, activities and community structure during remediation of artificial Cd contaminated soil and multiple heavy metals contaminated soil. The main results are summarized as follows:1. The results of the survey conduected in the ancient silver mining site showed that total metal concentrations in the soils ranged5.7-84.4mg kg-1for Cd,467.2-8326.7mg kg-1for Zn and898.8-7381.0mg kg-1for Pb. The heavy metal concentrations in plant shoots ranged39-1053.58mg kg-1for Cd,71.3-3508.8mg kg-1for Zn and11.5-1073.0mg kg-1for Pb, in the roots ranged2.54-222.02mg kg-1for Cd,71.3-3508.8mg kg-1for Zn and122.0-3437.7mg kg-1for Pb.The plant species for metal accumulation were classified by cluster analysis based on shoot metal concentration, BF and TF. Combining with the results of cluster analysis and metal hyperaccumulation thresholds, five groups such as hyperaccumulator, potential hyperaccumulator, accumulator, potential accumulator and normal accumulating plant were graded. For Cd accumulation, S. alfredii was treated as a Cd-hyperaccumulator, and the others were normal Cd-accumulating plants. For Zn accumulation, S. alfredii was considered as a potential Zn-hyperaccumulator, C. canadensis and A. lavandulaefolia were Zn-accumulators, and the others were normal Zn-accumulating plants. For Pb accumulation, S. alfredii and E. lineolatum were potential Pb-hyperaccumulators, R. hunanensis, A. decumbens and E. annuus were Pb accumulators, C. Canadensis and A. lavandulaefolia were potential Pb accumulators, and the others were normal Pb-accumulating plants. Plant species with the potential for phytoextraction were identified such as S. alfredii for Cd and Zn, C. canadensis and A. lavandulaefolia for Zn and Pb, and E. lineolatum, R. hunanensis, A. decumbens and E. annuus for Pb. Cluster analysis is effective in the classification of plant species for metal accumulation and identification of potential species for phytoextraction.2. The microbial properties of the rhizosphere soil of4different metal accumulation patterns tolerant plants were studied by collecting the rhizosphere soils in the field condition. Soil microbial biomass carbon content, basal respiration rate, urease, acid phosphatase and invertase activities in the rhizosphere of S. alfredii, R. hunanensis, L. christinae, C. gracile were significantly (P<0.05) higher than that of non-rhizosphere soils, so were soil microbial biomass carbon content, basal respiration rate, acid phosphatase and invertase activities in the rhizosphere of S. alfredii than that in rhizosphere of the other3tolerant plants. The numbers of the kinds of individual PLFAs detected in the rhizosphere of S. alfredii, R. hunanensis, L. christinae and C. gracile were70,60,58and54, while that in the non-rhizosphere of the four plants were49,49,48and39. Shannon-Weiner index (H) of soil microbial community in the rhizosphere of S. alfredii, R. hunanensis and C. gracile were significantly (P<0.05) higher than that of non-rhizosphere soils, so were the Shannon-Weiner index (H) of soil microbial community in the rhizosphere of S. alfredii than that in rhizosphere of the other3tolerant plants. The PLFA concentrations of total, bacterial, Gram-positive and Gram-negative bacterial, actinomycete, fungal, AM fungal and protozoan in the rhizosphere of S. alfredii, R. hunanensis, L. christinae and C. gracile were significantly(P<0.05) higher than that of non-rhizosphere soils, so were these PLFA concentrations in the rhizosphere of S. alfredii than that in rhizosphere of the other3tolerant plants. Soil microbial activities and community structure in the rhizosphere of S. alfredii were significant different from the rhizosphere of the other3tolerant plants and all the non-rhizosphere soils.3. A pre-stratified rhizobox experiment was conducted with hyperaccumulator Sedum alfredii to determine the effects of phytoextraction an artificial Cd contaminated soil on microbial properties. The results indicated that, in the plant-grown zone, basal respiration, acid phosphatase and invertase activities of the rhizospheric soil separated by the shaking method were significantly (P<0.01) higher than that of the near-rhizospheric soil and the unplanted soil after3months growth, so were microbial biomass carbon, urease, acid phosphatase and invertase activities of the rhizospheric soil after6months growth. In the pre-stratified area, acid phosphatase activity of the0-2mm sub-layer rhizospheric soil collected by the pre-stratified method after3months growth was significantly (P<0.05) higher than that of other sub-layer rhizospheric soils and bulk soil, and so were microbial biomass carbon, basal respiration, urease, invertase and acid phosphatase activities of the0-2mm sub-layer rhizosphere soil after6months growth. It was also observed that the soil, basal respiration rate, and urease activities were significantly negatively correlated to water soluble Cd. It was concluded that phytoextraction by Sedum alfredii could decrease the water-soluble Cd and improve soil microbial properties, especially in rhizosphere.4. A pre-stratified rhizobox experiment with a Cd-contaminated soil was conducted to assess the effects of phytoextraction by S.alfredii on soil microbial community. In the plant-grown zone, after12months growth,53kinds of PLFAs were identified in rhizospheric soil, while only32in unplanted soil. Shannon-Weiner index (H) of soil microbial community in the (near-) rhizospheric soils were significantly (P<0.05) higher than that in unplanted soil. The PLFA concentrations of total, bacterial, actinomycete, fungal, AM fungal and protozoan in (near-) rhizospheric soils were significantly (P<0.05) higher than that in unplanted soil. The utilization rates of15substrates by rhizospheric soil were significantly (P<0.05) higher than that by unplanted soil. In the pre-stratified area, from41to60kinds of individual PLFAs in sub-layer rhizospheric soils were identified, while only39in unplanted soil. Shannon-Weiner index (H) of soil microbial community in the0-8mm soil were significantly (P<0.05) higher than that in bulk soil. The PLFA concentrations of total, bacterial, actinomycete, fungal, AM fungal and protozoan in0-2mm soil were significantly (P<0.05) higher than that in bulk soil, so were the utilization rates for the15substrates. PLFAs concentrations and substrate utilization rate were negatively correlated with pH, total, HOAc and NH2OH·HCl-extractable Cd concentratoions and positively correlated with labile carbon. The increase in microbial diversity and activities of soil microbial community were attributed to the raise of labile carbon and reduction of available Cd concentrations.5. To study the effects of phytoextraction by Sedum alfredii on microbial property improvement of a multiple heavy metals contaminated soil, a rhizobox experiment was conducted under greenhouse conditions. In the plant-grown zone, microbial biomass carbon content, basal respiration, urease, acid phosphatase, invertase activities of the rhizospheric soils were significantly (P<0.05) higher than that of unplanted soils after3and6months growth. In the pre-stratified area, microbial biomass C and urease of0-4mm sub-layer rhizospheric soils were significantly (P<0.05) higher than that of bulk soil after3months growth. MBC of0-2mm and BR rate of0-8mm sub-layer rhizospheric soils were significantly higher than that of bulk soil after6months growth. So were the three enzyme activities of0-4mm sub-layer rhizospheric soils. BR rate and urease were significantly (P<0.05) negatively correlated with soluble Cd, so were microbial biomass C, acid phosphatase and intervase activities with soluble Zn, microbial biomass C, basal respiration rate and three enzyme activities with soluble Pb. Phytoextraction by S. alfredii could reduce water soluble Cd, Zn, Pb concentrations in the rhizospheric soils and increase microbial biomass C, basal respiration rate and enzyme activities of the metal polluted soil.6. A rhizobox experiment with a Cd-Zn-Pb multiple contaminated soil was conducted to investigate the effects of phytoextraction by S.alfredii on soil microbial community structure during the phytoextraction process. In the plant-grown zone, after12months growth,50kinds of PLFAs were identified in rhizospheric soil, while only29kinds of PLFAs were identified in unplanted soil. Shannon-Weiner index (H) of soil microbial community in the (near-) rhizospheric soils were significantly (P<0.05) higher than that in unplanted soil. The PLFA concentrations of total, bacterial, actinomycete, fungal, AM fungal and protozoan in (near-) rhizospheric soils were significantly (P<0.05) higher than that in unplanted soil. The utilization rates of14substrates by rhizospheric soil were significantly (P<0.05) higher than that by unplanted soil. In the pre-stratified area,41kinds of individual PLFAs in0-2mm sub-layer rhizospheric soil were identified, while only31in unplanted soil. Shannon-Weiner index (H) of soil microbial community in the0-10mm sub-layer rhizospheric soil were significantly (P<0.05) higher than that in bulk soil.The PLFA concentrations of total, bacterial, actinomycete, fungal, AM fungal and protozoan in0-2mm sub-layer rhizospheric soil soil were significantly (P<0.05) higher than that in the2-10mm sub-layer rhizospheric soils and bulk soil. The utilization rates of14substrates by microorganisms of0-8mm sub-layer rhizospheric soils were significantly (P<0.05) higher than that by bulk soil. PLFAs concentrations and substrate utilization rate were negatively correlated with total, HOAc, NH2OH·HC1, and NH4OAC-extractable Cd and Zn concentrations, and positively correlated with labile carbon. These indicated that the enhencement of microbial activities and changes of microbial community structure were attributed to the raise of labile carbon and reduction of Cd and Zn concentrations.更多还原