【作者】 江春玉；

【导师】 黄为一；

【作者基本信息】 南京农业大学 ， 微生物学， 2008， 博士

【摘要】 植物修复被认为是成本低廉、操作简单的土壤重金属污染修复技术。大量活跃的根际微生物可通过多种作用方式影响重金属污染环境中植物的生长和对重金属的吸收,从而影响植物对重金属污染的修复效率。本文以细菌的铅(Pb)镉(Cd)耐受性、1-氨基环丙烷-1-羧酸脱氨酶(ACCD)活性及对植物苗期的促生效应为指标,筛选出对铅镉具有较强耐受性、对玉米具有促生能力的细菌菌株。研究细菌对重金属铅、镉胁迫下植物生长和富集铅镉的影响,并对细菌提高玉米重金属耐受性的机制进行探讨,以期为植物耐受重金属机制的进一步阐明和植物—微生物联合修复途径的应用提供理论和实验依据。主要研究结果如下：1、筛选出对铅镉具有较强抗性、对玉米具有促生能力的六株菌,经生理生化特性和16S rDNA基因序列分析初步鉴定为Burkholderia cepacia J62、Rahnella sp. LR113、Microbacterium oxydans JYC17、Microbacterium foliorum G16、Enterobacter agglomerans J17-2a和Pseudomonas thivervalensis Y3-9;经安全性试验表明供试菌株对作物无致病性。2、研究六株菌与促生作用相关的生物学特性,结果表明：菌株J62、G16和Y3-9具有较高的ACCD活性；菌株J62、JYC17、J17-2a和Y3-9具有精氨酸脱羧酶活性；六株菌均能溶解磷酸钙、产生铁载体和分泌IAA。3、研究六株菌与重金属相关的生物学特性,结果表明：菌株J62、LR113、J17-2a和Y3-9能在含Pb1000 mg.L-1或Cd50 mg.L-1的培养基中生长,并对Cu、Zn和Ni具有抗性。六株菌能促进碳酸铅和碳酸镉的溶解,使培养液中Pb2+比对照(0.051 mg.L-1)增加3.45～39.3倍、Cd2+由1.03mg.L-1增加至1.23 mg.L-1～127.53 mg.L-1。菌株J62和JYC17溶解碳酸铅效果较好,菌株J62和J17-2a溶解碳酸镉效果最显著。4、以菌株J62和LR113为代表,研究细菌对铅镉的溶解机制和溶解-吸附动态,结果表明：菌株生长代谢产酸使培养液pH降低,促使培养液中Pb2+、Cd2+浓度增加,重金属的溶解改变了细菌分泌的有机酸的种类和含量。菌株J62对碳酸铅、镉的增溶能力高于LR113,菌株LR113对铅镉的吸附能力强于J62。研究菌株对土壤中不溶性铅、镉的影响,菌株J62和LR113均能在土壤中存活,菌株J62对土壤重金属铅镉的增溶能力较强,能显著提高土壤中水溶态铅和镉的浓度。4、水培条件下研究细菌J62、JYC17和Y3-9对重金属铅、镉胁迫下玉米生长和富集铅镉的影响。结果表明,三株菌对玉米生长均有促进作用。不加重金属处理时,菌株J62和JYC17显著促进玉米生长。Pb50、100 mg.L-1处理下,三株菌使玉米干重比对照增加25%～94%。Cd2、4 mg.L-1处理下,菌株使玉米根重比对照增加8%～102%,JYC17的促生作用最好。重金属胁迫程度的增加降低了菌株的促生效应。接菌处理能提高玉米根部铅、镉的浓度,比对照增加17%～182%。菌株J62、JYC17和Y3-9均提高了玉米植株的铅和镉总积累量,使铅镉富集量分别比对照增加28～126%和47～130%,菌株J62和JYC17对玉米富集重金属的促进作用显著。5、研究菌株J62、JYC17和Y3-9促进玉米生长、提高重金属耐受性的机制,结果表明,供试菌株能在玉米根际和根内定殖,接菌玉米根际和根内的细菌数量显著高于不接菌对照。接菌J62、JYC17和Y3-9降低了铅、镉胁迫下玉米的过氧化程度,使玉米根部尤其是铅处理下超氧化物歧化酶(SOD)和过氧化物酶(POD)活性降低；使玉米叶片中抗氧化剂抗坏血酸(ASA)浓度显著提高,比对照增加14%～123%；使Pb处理下还原型谷胱甘肽(GSH)含量增加；使丙二醛(MDA)含量明显降低。供试菌株使玉米叶绿素含量提高,苹果酸浓度显著增加,使玉米内源脯氨酸(Pro)浓度低于对照。采用透射电子显微镜研究菌株对玉米根部重金属铅、镉积累和分布的影响,结果表明供试菌株显著改变了玉米根部细胞内铅的富集量和分布。与对照相比,接菌J62使玉米根部吸收的Pb显著提高,接菌JYC17使铅积累颗粒变大。不接菌对照的玉米根部铅主要分布于细胞内壁和细胞膜上,接菌处理均使铅主要分布在细胞外壁和周质空间中。接菌处理改变了镉在玉米细胞中的分配,减轻了镉对玉米细胞的伤害。在镉处理的玉米根细胞内观察到供试细菌菌体的存在。6、以既能提高土壤中铅镉迁移率、又能提高玉米对铅镉抗逆性的菌株J62为试验菌株,盆栽条件下研究细菌对植物富集土壤Pb和Cd的促进作用。由盆栽1的结果可知,菌株J62能促进重金属铅镉污染土壤中玉米和番茄的生长,使地上部干重分别比对照增加54%和17%,根部干重增加75%和30%。接J62菌株促进了植物根部对铅和镉的吸收,使印度芥菜、玉米和番茄根部的Pb浓度分别比对照增加27%、59%和31%,Cd浓度分别增加174%、31%和12%。接J62菌株显著增加了玉米和番茄全株的重金属吸收量,Pb吸收量比对照增加85%和38%,Cd吸收量增加56%和61%。接J62菌株使植物根际土中抗Pb200 mg.L-1&Cd50 mg.L-1细菌和总细菌的数量远高于对照,并促进了解钾、溶磷和固氮细菌数量的增加；菌株J62能在植物根际定殖,在重金属抗性平板中检测到J62的存在。接J62菌株显著提高了玉米和番茄根际交换态铅、镉的浓度；使根际土pH值低于对照,可溶性糖含量增加6%～40%；显著提高印度芥菜和玉米根际草酸浓度和玉米、番茄根际乙酸浓度,刺激印度芥菜和玉米根际土中丁二酸和番茄根际土中柠檬酸的产生。菌株J62自身的促生特性、对植物根际细菌、pH值、重金属形态、有机酸及可溶性糖的影响可能是其促进玉米和番茄富集铅镉的原因。盆栽2的结果与盆栽1基本一致,接菌J62显著提高了玉米根部、叶部和全植株的干重；促进了玉米根部对Cd、茎部对Pb和Cd的吸收,但降低了Pb和Cd向叶片转运的能力；显著增加了玉米各器官的Pb富集量和玉米根、茎部的Cd富集量。综合而言,菌株J62是一种良好的促进玉米修复土壤重金属铅、镉污染的菌剂,具有修复受污染土壤同时进行玉米生产的潜力。更多还原

【Abstract】 Phytoremediation is considered as a cost-effective and environment-friendly technology for remediation of heavy metal-contaminated soils. A large number of active rhizosphere microorganisms affect growth and heavy metal accumulation of plant in heavy metal-polluted environment, and then change phytoremediation efficiency. In this paper, Pb&Cd-resistant and maize-growth promoting bacterial strains were screened out by their lead and cadmium resistance, ACC deaminase activity and growth promotion to corn seedlings. The effects of bacteria on plant growth and Pb and Cd accumulation under heavy metals stress and mechanism of microbes improving heavy metal tolerance of maize were studied. The results may further clarify mechanism of plant’s heavy metal tolerance and offer the theoretical and experimental basis for application of plant-microbe remediation. Major conclusions were summarized as follows:1. Bacterial strains J62, LR113, JYC17, G16, J17-2a and Y3-9 were screened out and respectively identified as Burkholderia cepacia J62, Rahnella sp. LR113, Microbacterium oxydans JYC17, Microbacterium foliorum G16, Enterobacter agglomerans J17-2a and Pseudomonas thivervalensis Y3-9. All bacteria did not cause disease to crops.2. The plant growth-promoting characteristics of isolated bacteria were studied in this paper. The results showed that test strains expected LR113 had the ACC deaminase activity. Strain J62, JYC17, J17-2a and Y3-9 had the arginine decarboxylase activity. All the strains could dissolve calcium phosphate and produce siderophores and indole acetic acid (IAA). The phosphate solubilization of strain LR113 and J62, siderophores secretion of strain J62 and LR113 and IAA production capacity of strain G16 and LR113 were better than that of other strains.3. The heavy metals characteristics of isolated bacteria were assessed. Strains J62, LR113, J17-2a and Y3-9 could grow in media with Pbl000 mg.L-1 or Cd50 mg.L-1 and tolerated Cu, Zn and Ni as well. All test bacteria could promote PbCO3 and CdCO3 solubilization. The bacteria enhanced Pb2+ contents by 3.45 to 39.3 times, compared to non-inoculated control. Cd2+contents in media were increased from 1.03 mg.L-1 to 1.23～127.53 mg.L-1. The lead carbonate activation of strain J62 and JYC17 and cadmium carbonate activation of strains J62 and J17-2a were significantly better than that of other strains. Strain J62 and LR113 were chosen to investigate release and absorption dynamic and mechanism of lead and cadmium. The data revealed that pH descent arose by strains growth increased water-soluble Pb2+or Cd2+concentration in liquid media. The insoluble Pb and Cd solubilization ability of strain J62 was higher than that of LR113. The organic acid species and concentrations produced by bacteria were determined by HPLC. In the media inoculated strain J62, Pb release had no significant effect on organic acids production and Cd release reduced organic acids contents. Carbonate lead and cadmium solubilization promoted organic acids secretion by strain LR113. The organic acids types produced by these two strains were quietly different. Lead and cadmium adsorbed by bacterial cell were measured through the desorption ability of NH4OAc, HCl and EDTA. NH4OAc extracted more Pb (6.17 mg.L-1) on J62 cells. HCl had the best desoiption capability to Cd (77.41mg.L-1) on strain J62 mycelium and Pb (33.65mg.L-1) or Cd (91.92mg.L-1) adsorbed by strain LR113. The Pb and Cd absorption capacity of strain LR113 were better than that of strain J62. The results of bacteria impact on insoluble lead and cadmium in soils demonstrated that strains J62 and LR113 could colonize in the soils; strain J62 had stronger lead and cadmium activation ability than strain LR113 and significantly increased water-soluble Pb and Cd in soils.4. Strain J62, JYC17 and Y3-9 were selected as test bacteria to research the effect of bacterial inoculation on maize growth and Pb and Cd accumulation in aqueous culture with heavy metal stress. The experiments clearly showed that all strains promoted maize growth. Strain J62 and JYC17 significantly promoted the growth of corn in nutrient solution without heavy metal ions. In nutrient solution with Pb2+50 or 100 mg.L-1, dry weights of corn inoculated with strains were higher (25%～94%) than that of non-inoculated control. In nutrient solution with Cd+2 or 4 mg.L-1, all bacteria enhanced root weight of maize by 8%～102%; the growth-promoting role of strain JYC17 was better than J62 and Y3-9. The enhancement of heavy metals stress reduced the growth-promoting capability of bacteria. Bacterial inoculation could improve lead and cadmium concentrations by 17%to 182%in com roots, whereas had little effect on lead and cadmium uptake in corn shoots. Inoculation with strain J62, JYC17 and Y3-9 all increased total lead and cadmium accumulation of maize. Compared to non-inoculated control, strain treatments added 28-126%Pb and 47-130%Cd in maize respectively. Strain J62 and JYC17 had a significant improvement on heavy metals accumulation in maize.5. The mechanism of three bacteria improving maize growth and heavy metals tolerance was further explored. The results displayed that the test strains could colonize in both root and rhizosphere of maize. The bacterial numbers of maize inoculated strains were significantly higher than that of control. Strain J62, JYC17 and Y3-9 inoculation reduced maize peroxidation level under lead or cadmium stress. In the maize inoculated with strains, SOD and POD activity reduced, especially in Pb treatments; the concentration of antioxidants ASA significantly increased by 14%-123%. GSH content increased in maize treated with Pb and slightly dropped in maize treated with Cd, which might a large number of GSH be used for PCs anabolism. The MDA contents obviously reduced in inoculated maize than that in control maize. It was dissimilar effect of different strains on endogenous Pro concentrations in maize. Strain J62 inoculation significantly declined Pro level in maize treated with Pb, but slightly increased Pro concentration in maize treated with Cd. Strain JYC17 and Y3-9 inoculation made Pro concentrations remarkable lower than that of control, in maize treated with high concentrations of Pb or Cd. The data of organic acids determined by HPLC revealed that strains incubation increased significantly malic acid concentrations in maize shoots. Bacterial effects on lead or cadmium accumulation and distribution in corn roots were studied through TEM. The results showed that bacteria significantly changed Pb concentration and distribution in maize root cells. Compared with the non-inoculated control, strain J62 Inoculation significantly increased Pb absorb in corn roots, moreover, inoculation with strain JYC17 caused larger lead particles. Pb in root of control maize mainly distributed in cellular wall and membrane, whereas Pb in maize roots inoculated bacterial strains mainly distributed in external layer of cellular wall and extracellular space. Bacterial inoculation also altered Cd distribution and alleviated Cd phytotoxicity in maize cells. Bacterial cells of test strains were observed in root cells of maize.6. The strain J62 that improved lead and cadmium bioavailability in soils and metal-tolerance of com was selected for pot experiment. The results of pot test one showed that strain J62 could promote growth of maize and tomato in lead and cadmium-contaminated soils. Dry weight of maize and tomato inoculated J62 increased by 54% and 17% in shoots and by 75% and 30% in roots. Strain J62 could improve lead and cadmium uptake in plant roots. The Pb concentrations in Indian mustard, corn and tomato inoculated strain J62 were 27%,59% and 31% respectively more than that in control. The Cd concentrations were 174%,31% and 12% higher than that in control, respectively. In conclusion, lead and cadmium total contents in maize and tomato were significantly increased by inoculation of strain J62. The Pb uptake in maize and tomato inoculated strain J62 were 85% and 38% respectively more than that in control, Cd uptake were 56% and 61% respectively more than that in control. The amount of Pb (200 mg.L-1) and Cd (50 mg.L-1)-resistant bacteria and total bacteria in rhizosphere inoculated with strain J62 were much higher than that in controls. The population of potassium-releasing, phosphorus-soluble and nitrogen-fixing bacteria in rhizosphere was also enhanced by strain inoculation. Strain J62 could colonize in test plants rhizosphere and obtain in heavy metal-resistant plates. Moreover, strain J62 inoculation also significantly improved NH4OAc-extracted Pb and Cd concentration in rhizosphere of maize and tomato. The pH values were decreased in the soils inoculated with strain and dissoluble sugar were increased by 6% to 40%, compared with the uninoculation control. Furthermore, strain J62 inoculation increased ocalic acid concentration in Indian mustard and corn rhizosphere and acetic acid concentration in maize and tomato rhizosphere, stimulated production of succinic acid in Indian mustard and maize rhizosphere and citric acid in tomato rhizosphere. Effects of strain J62 on bacterial number, pH, metal form, organic acid and soluble sugar contents might promote Pb or Cd accumulation of corn and tomato. The results of pot test two were accordant with that of test one. Burkholderia cepacia J62 is a good bioinoculant to strengthen phytoremediation in lead and cadmium contaminated soils.It is potential to apply maize inoculated with strain J62 to remedy metal-polluted soils and produce corn cobs.更多还原