锌抗性菌株的筛选及其强化印度芥菜修复锌污染土壤的研究

发布时间：2018-04-20 04:06

本文选题：锌污染土壤 + 根际微生物　；参考：《江南大学》2017年硕士论文

【摘要】：土壤重金属污染已成为当今面临的主要环境问题之一,而重金属污染土壤的植物修复技术因其成本投入较低、对土壤环境扰动小、不引起二次污染以及可美化景观等特点成为当前学术界研究的热点。超积累植物被认为是实施重金属污染环境植物修复不可缺少的材料。然而,大多数超积累植物生长周期长、生物量小,加上土壤中重金属生物有效性低,严重限制了其应用于大规模污染土壤修复的潜力。为了提高植物修复效率,接种根际微生物被认为是提高超积累植物修复效果的重要途径。本研究从土壤中分离得到一株对重金属锌具有较强抗性且对难溶性锌有促溶作用的菌株BC109-2,经形态和分子生物学鉴定,确定其为一株微紫青霉菌,命名为Penicillium janthinellum BC109-2。锌抗性菌株BC109-2对锌的最小抑制浓度(MIC)达到2100 mg L-1,在PDB培养基(含1000 mg L-1 ZnCO3)中摇床振荡培养72 h后,培养液中可溶性锌含量增加了113%,该菌株对难溶态锌具有较强的溶解作用。在筛选得到高锌耐受性和溶解性菌株BC109-2的基础上,对锌在菌株细胞中的分布与化学形态进行分析并对细胞抗氧化系统进行毒性评价。菌株细胞中的锌含量与培养基中的锌浓度成正比;锌由于强烈的阴阳离子相互作用结合到细胞壁上,占菌株细胞总锌含量的14%~21%;液泡由于区室化作用可以作为毒性物质的另一个存储室,细胞液和液泡中的锌含量占细胞总量的45%~49%。另外,细胞中的锌大多以不溶态、草酸盐态以及残留态三种非活跃状态存在。在不同浓度的锌胁迫下的抗氧化酶活性的变化表明,锌的加入会导致在真菌细胞中的氧化应激响应,其中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)三种抗氧化酶通过协调作用,直接参与清除细胞内产生的活性氧自由基(ROS)的过程,且POD含量变化最为明显;谷胱甘肽转硫酶(GST)能催化还原型谷胱甘肽(GSH)和羟基自由基的结合而不产生氧化型谷胱甘肽(GSSG),但其活性增加并不明显;除此之外,谷胱甘肽还原酶(GR)活性因为受到高浓度锌胁迫而降低,导致GSH含量下降远远超过GSSG升高量。细胞可以通过抗氧化酶(SOD、CAT、POD、GR和GST)和非酶抗氧化剂(GSH和GSSG)的综合作用使脂质过氧化水平保持在一个相对稳定的水平,以增强细胞抵御外界非生物刺激的能力,使细胞得以正常生长。最后对菌株BC109-2的促植物生长指标进行评价,并研究供试菌株对印度芥菜修复锌污染土壤的促进作用。锌耐受菌株BC109-2能产吲哚乙酸(IAA)和铁载体,并且具有ACC脱氨酶活性和溶磷能力,且能促进植物根系伸长;盆栽实验结果表明,接种了根际促生菌的印度芥菜的地上部分对土壤中Zn的总提取量与不接种菌的对照组相比提高了42%,说明根际促生菌BC109-2可以促进印度芥菜吸收和富集土壤中的锌,具有一定修复锌污染土壤的应用潜力。
[Abstract]:Soil heavy metal pollution has become one of the main environmental problems, and the phytoremediation technology of heavy metal contaminated soil has low cost and little disturbance to soil environment. The characteristics of not causing secondary pollution and beautifying landscape have become the focus of academic research. Superaccumulative plants are regarded as indispensable materials for phytoremediation of heavy metal polluted environment. However, the long growth cycle and small biomass of most superaccumulative plants, coupled with the low bioavailability of heavy metals in soil, seriously limit their potential for remediation of contaminated soils on a large scale. In order to improve the efficiency of phytoremediation, inoculation of rhizosphere microorganisms is considered to be an important way to improve the effect of hyperaccumulative phytoremediation. In this study, a strain BC109-2 with strong resistance to heavy metal zinc and solubilization of insoluble zinc was isolated from soil. The strain BC109-2 was identified as Penicillium janthinellum BC109-2 by morphological and molecular biological identification. The minimum inhibition concentration of zinc resistance strain BC109-2 was 2100 mg L -1. After shaking culture in PDB medium (containing 1000 mg L -1 ZnCO 3) for 72 h, the soluble zinc content in the culture medium increased by 113%, and the strain had a strong dissolution effect on insoluble zinc. On the basis of screening high zinc tolerance and solubility strain BC109-2, the distribution and chemical forms of zinc in the strain cells were analyzed and the cytotoxicity of the antioxidant system was evaluated. The content of zinc in the cell of the strain is proportional to the concentration of zinc in the medium; zinc is bound to the cell wall because of the strong interaction of anion and anion. The vacuoles can be used as another storage room for toxic substances because of the compartmentalization of the vacuoles, and the zinc content in the cell fluid and vacuole accounts for 4549% of the total number of cells. In addition, most of the zinc in cells were insoluble, oxalate and residual states. The changes of antioxidant enzyme activity under different concentrations of zinc stress showed that the addition of zinc resulted in oxidative stress response in fungal cells. Superoxide dismutase (SOD), catalase catalase (cat) and peroxidase (POD) were involved in the process of scavenging the reactive oxygen free radicals (Ros) by coordinating action, and the change of POD content was the most obvious. Glutathione transferase (GST) can catalyze the binding of reduced glutathione glutathione (GSH) with hydroxyl radical without producing oxidized glutathione GSSGN, but its activity increase is not obvious. Glutathione reductase (Glutathione reductase) activity decreased due to high concentration of zinc stress, resulting in the decrease of GSH content far more than the increase of GSSG. Cells can maintain the level of lipid peroxidation at a relatively stable level through the combined effects of antioxidant enzymes such as SOD, CAT, PODN, gr and GST, and non-enzymatic antioxidants, GSH and GSSGG, in order to enhance the ability of cells to resist external abiotic stimuli and to make the cells grow normally. Finally, the plant growth index of strain BC109-2 was evaluated, and the promoting effect of the strain on the remediation of zinc contaminated soil by Indian mustard was studied. Zinc tolerant strain BC109-2 can produce indoleacetic acid (IIA) and iron carrier, and it has the activity of ACC deaminase and the ability of dissolving phosphorus, and can promote the elongation of plant root system. The total amount of Zn extracted from soil by the aboveground part of Indian mustard inoculated with rhizosphere growth promoting bacteria increased 42 ~ 2% compared with that of the control group without inoculation, which indicated that BC109-2 could promote the absorption and accumulation of zinc in soil by Indian mustard (Brassica juncea). It has the potential of remediation of zinc contaminated soil.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X53;X172

【参考文献】