不同价态铬对土壤生物化学活性影响的研究
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摘要
土壤酶是土壤中非常重要的一个组成部分,在土壤的生化反应中发挥着举足轻重的作用,它基本上参与了土壤中所发生的所有的生物化学反应,同时也在自然界的营养物质循环、能量代谢以及环境污染物监测与治理等等很多方面都发挥着十分重要的作用。重金属通过各种不同的途径进入土壤,可以与土壤酶之间发生作用,因此以目标物作为土壤酶,开展重金属的生态毒理研究,在理论上可以揭示土壤酶与重金属之间的作用机理,为重金属的治理与重金属的监测提供理论依据。而开展这一研究具有十分重要的理论和实践意义。
本论文通过模拟方法,对我国陕西几种主要类型土壤的脲酶、碱性磷酸酶和脱氢酶与重金属(三价铬、六价铬、三六价复合铬)的关系进行了研究,并借助了动力学的手段对土壤酶与重金属的作用机理进行分析,研究结果表明：
1.铬加入土壤中后导致土壤pH值下降。随三价铬质量浓度增加,土壤水溶液的pH值持续降低,最大降幅为3.03个pH单位；而加入六价铬,供试土壤pH值降低幅度比三价铬的要小；三、六价铬复合污染土壤后,土壤pH值降幅介于三价铬和六价铬之间。
2.供试三价铬进入土壤后,被土壤立即强烈吸附,溶液中三价铬含量不超过添加量的1%；六价铬加入土壤中,仅少数含量被土壤直接吸附沉淀,另外一部分六价铬还原为三价铬,其余六价铬主要以水溶态形式存在；三、六价铬复合污染后,部分六价铬被还原为三价铬,并最终很快被土壤吸附,因而在土壤溶液中无法检测到水溶态三价铬,但是可以检测到水溶态六价铬。
3.铬可以抑制土壤碱性磷酸酶活性,抑制作用强弱为铬复合污染>三价铬>六价铬；采用U=A／(1+B×C)模型较好地表征了土壤碱性磷酸酶活性与铬含量间的关系,并揭示出两者之间的作用机理为完全抑制作用；同一类型土样碱性磷酸酶活性的生态剂量ED10值随土壤有机质含量的降低而减小。三价铬和六价铬对土壤碱性磷酸酶活性具有交互作用。
4.铬抑制洋刀豆脲酶活性,其中三价铬的抑制作用最强,六价铬抑制作用最弱；计算获得了洋刀豆脲酶溶液受铬轻度污染时,三价铬、六价铬、三、六价铬复合污染的临界浓度分别为0.1、416.1、295.0 mg／kg。铬加入后洋刀豆脲酶Km总体处于同一个数量级,Vmax值随铬浓度增加而降低,揭示出铬对洋刀豆脲酶的作用机理为非竞争抑制。
5.不同价态铬对土壤脲酶活性影响有所差异,三价铬抑制作用,六价铬却是激活脲酶活性；其中土壤脲酶敏感性顺序为：三价铬>六价铬>三、六价铬复合。三价铬和六价铬对脲酶活性具有交互作用；土壤铬污染后,脲酶Km处于同一个数量级,Vmax随三价铬质量浓度增加而降低,随六价铬质量浓度增加而升高,三价铬对土壤脲酶作用机理为非竞争抑制。
6.铬抑制土壤脱氢酶活性,抑制作用顺序为六价铬>铬复合污染>三价铬,这一顺序和土壤脱氢酶活性是微生物活性的总体表现有关；采用U=β0-β1×C模型可较好表征酶活性与铬含量之间关系,揭示出脱氢酶在一定程度上可表征土壤铬污染的程度。三价铬和六价铬共存时存在交互效应。
7.根据剂量越小,反应越敏感的原则,通过比较采用多种土壤生化活性计算获得的生态剂量值,发现土壤受三价铬、六价铬、复合铬轻度污染时的临界浓度为37.90、10.19和33.7 mg／kg,水溶态铬临界浓度为0.08、795.88、191.63 mg·1-1。
With the development of social economy and the growth of world population, the soil, as the natural resource on which human beings live, was more and more unable to withstand the load. Nearly 1/5 of total existing arable land about 1×108hm2 in our country was polluted with different degree. The heavy metal was a major kind of pollutants. So it was one of the most important environmental researches to monitor and remediate soil polluted by heavy metal. Chromium as one of the four major of soil pollution,the biological toxicity of Cr6+ is 100～1000 times larger than Cr3+.but their ecotoxicity are different from that.
Soil enzyme was one of the principal components in soil, which took part in all of the biochemical reactions in soil and played an impotant part in nutrient circulation, energy metabolism, pollutant monitoring and remediation in nature. Heavy metal entered the soil by various ways, and then reacted with the soil enzyme.The study of ecotoxicological of heavy metals, slecting soil enzyme as the research target, could reveal the mechanism of soil enzymes and heavy metal in theory and provide the theoretical basis for removal, treatment and monitoring of heavy metal in practice. So it was of great theoretical and practical significance to work on this area. However, in the past decade most researches only focused on the relationship between some certain heavy metal and the soil enzyme activity, Rare study on different valence of heavy metals, and the conclusions were not the same, it was seldom to find report on the kinetics and mechanism of soil enzyme.
By simulative method, we systematically studied not only the relationships between urease, alkaline phosphatase and dehydrogenase and the heavy metals[Cr3+、Cr6+、Combined chromium]in several major soil types of Northern China, but also the reaction mechanism between enzymes and the metal by means of enzymatic kinetics. The results were as follows:
1. Chromium decreased soil pH remarkably. With the increase of Cr3+ concentration, the pH value of soil solution decreased continuously, the biggest drop was 3.03 units. While, when adding different concentration of Cr6+, the pH value decreased gradually, but the sample No.5, the pH value showed alkaline or alkalescent.
2. Cr3+could be adsorbed heavily by soil, and there was less than 1%remaining in the soil solution. A small number of Cr6+was adsorbed when adding Cr6+ to soil, apart from part of Cr6+ which were reduced to Cr3+and adsorbed quickly by soil, most of Cr6+ existed mainly in the form of water soluble hexavalent chromium. When adding combined chromium to the soil samples, Cr3+was quickly adsorbed by soil, Part of the Cr6+were reduced to Cr3+and quickly adsorbed by soil, the remaining Cr6+ existed in the form of water soluble. It was hard to detect water soluble Cr3+in the soil solution.
3. Chromium inhibited the soil alkaline phosphatase activity, The order of inhibition was that:Cr3++Cr6+> Cr3+> Cr6+. The model U=A/(1+B×C) (in which A, B were composite parameters) could describ the relationship better between soil enzyme activity and chromium concentration. The mechanism of different valence state of chromium on the soil alkaline phosphatase was completely inhibition. The ecological dose ED10 of alkaline phosphatase in the same type of soil decreased continuously with the decline of soil organic matter content. There was an interaction between trivalent chromium and hexavalent chromium on the soil alkaline phosphatase.
4. Chromium inhibited the activities of Jackbean urease, The order of inhibition was that: Cr3+> Cr3++Cr6+> Cr6+. The critical concentrations (ED10) of Cr3++Cr6+、Cr3+and Cr6+in Jackbean urease were 0.1,416.1,295.0 mg/kg respectively. With the increasing of chromium concentration, the kinetic parameter Km of Jackbean urease were kept in the same level, which revealed that the mechanism of chromium on Jackbean urease was noncompetitive inhibition.
5. Different valence state of chromium had the distinct influence on soil urease activity. Cr3+ inhibited the activities of soil urease, while Cr6+ activated the activities of soil urease. The order of inhibition is Cr3+> Cr6+> Cr3++Cr6*. There were an interaction between Cr3+ and Cr6+ on the soil urease activity, and it showed synergism. within the range of the test chromium concentration, the kinetic parameters Km of soil urease were keep in the same level. The Vmax value falled with the increase of Cr3+concentration, and rised with the increase of Cr6+concentration, the kinetic parameters of lou soil were different from Cinnamon soil and Sandy soil. The reason will be further studyed.. It revealed that the mechanism of Cr3+ on soil urease was noncompetitive inhibition.
6. chromium inhibited the dehydrogenase activity of soil, The order of inhibition was Crb +> Cr3++Cr6+> Cr3+, Which maybe cause that dehydrogenase activity is the overall performance of microbial activities. The model U=β0-β1×C could describ the relationship between soil dehydrogenase activity and chromium concentration. It revealed that Dehydrogenase can be characterized as an index of chromium contamination in certain extent. There were interaction between trivalent chromium and hexavalent chromium on the soil dehydrogenase activity.
7. According to the principle of that the smaller ecological dose, the more sensitiv, the critical concentrations (ED10) of Cr3+、Cr6+and Cr3++Cr6+in soils were 37.90.10.19 and 33.7 mg/kg respectively. The critical concentrations (EC10) of water soluble Cr3+、Cr6+and Cr3++ Cr6+ in soils were 0.08、795.88、191.63 mg·l-1 respectively.
本论文通过模拟方法,对我国陕西几种主要类型土壤的脲酶、碱性磷酸酶和脱氢酶与重金属(三价铬、六价铬、三六价复合铬)的关系进行了研究,并借助了动力学的手段对土壤酶与重金属的作用机理进行分析,研究结果表明：
1.铬加入土壤中后导致土壤pH值下降。随三价铬质量浓度增加,土壤水溶液的pH值持续降低,最大降幅为3.03个pH单位；而加入六价铬,供试土壤pH值降低幅度比三价铬的要小；三、六价铬复合污染土壤后,土壤pH值降幅介于三价铬和六价铬之间。
2.供试三价铬进入土壤后,被土壤立即强烈吸附,溶液中三价铬含量不超过添加量的1%；六价铬加入土壤中,仅少数含量被土壤直接吸附沉淀,另外一部分六价铬还原为三价铬,其余六价铬主要以水溶态形式存在；三、六价铬复合污染后,部分六价铬被还原为三价铬,并最终很快被土壤吸附,因而在土壤溶液中无法检测到水溶态三价铬,但是可以检测到水溶态六价铬。
3.铬可以抑制土壤碱性磷酸酶活性,抑制作用强弱为铬复合污染>三价铬>六价铬；采用U=A／(1+B×C)模型较好地表征了土壤碱性磷酸酶活性与铬含量间的关系,并揭示出两者之间的作用机理为完全抑制作用；同一类型土样碱性磷酸酶活性的生态剂量ED10值随土壤有机质含量的降低而减小。三价铬和六价铬对土壤碱性磷酸酶活性具有交互作用。
4.铬抑制洋刀豆脲酶活性,其中三价铬的抑制作用最强,六价铬抑制作用最弱；计算获得了洋刀豆脲酶溶液受铬轻度污染时,三价铬、六价铬、三、六价铬复合污染的临界浓度分别为0.1、416.1、295.0 mg／kg。铬加入后洋刀豆脲酶Km总体处于同一个数量级,Vmax值随铬浓度增加而降低,揭示出铬对洋刀豆脲酶的作用机理为非竞争抑制。
5.不同价态铬对土壤脲酶活性影响有所差异,三价铬抑制作用,六价铬却是激活脲酶活性；其中土壤脲酶敏感性顺序为：三价铬>六价铬>三、六价铬复合。三价铬和六价铬对脲酶活性具有交互作用；土壤铬污染后,脲酶Km处于同一个数量级,Vmax随三价铬质量浓度增加而降低,随六价铬质量浓度增加而升高,三价铬对土壤脲酶作用机理为非竞争抑制。
6.铬抑制土壤脱氢酶活性,抑制作用顺序为六价铬>铬复合污染>三价铬,这一顺序和土壤脱氢酶活性是微生物活性的总体表现有关；采用U=β0-β1×C模型可较好表征酶活性与铬含量之间关系,揭示出脱氢酶在一定程度上可表征土壤铬污染的程度。三价铬和六价铬共存时存在交互效应。
7.根据剂量越小,反应越敏感的原则,通过比较采用多种土壤生化活性计算获得的生态剂量值,发现土壤受三价铬、六价铬、复合铬轻度污染时的临界浓度为37.90、10.19和33.7 mg／kg,水溶态铬临界浓度为0.08、795.88、191.63 mg·1-1。
With the development of social economy and the growth of world population, the soil, as the natural resource on which human beings live, was more and more unable to withstand the load. Nearly 1/5 of total existing arable land about 1×108hm2 in our country was polluted with different degree. The heavy metal was a major kind of pollutants. So it was one of the most important environmental researches to monitor and remediate soil polluted by heavy metal. Chromium as one of the four major of soil pollution,the biological toxicity of Cr6+ is 100～1000 times larger than Cr3+.but their ecotoxicity are different from that.
Soil enzyme was one of the principal components in soil, which took part in all of the biochemical reactions in soil and played an impotant part in nutrient circulation, energy metabolism, pollutant monitoring and remediation in nature. Heavy metal entered the soil by various ways, and then reacted with the soil enzyme.The study of ecotoxicological of heavy metals, slecting soil enzyme as the research target, could reveal the mechanism of soil enzymes and heavy metal in theory and provide the theoretical basis for removal, treatment and monitoring of heavy metal in practice. So it was of great theoretical and practical significance to work on this area. However, in the past decade most researches only focused on the relationship between some certain heavy metal and the soil enzyme activity, Rare study on different valence of heavy metals, and the conclusions were not the same, it was seldom to find report on the kinetics and mechanism of soil enzyme.
By simulative method, we systematically studied not only the relationships between urease, alkaline phosphatase and dehydrogenase and the heavy metals[Cr3+、Cr6+、Combined chromium]in several major soil types of Northern China, but also the reaction mechanism between enzymes and the metal by means of enzymatic kinetics. The results were as follows:
1. Chromium decreased soil pH remarkably. With the increase of Cr3+ concentration, the pH value of soil solution decreased continuously, the biggest drop was 3.03 units. While, when adding different concentration of Cr6+, the pH value decreased gradually, but the sample No.5, the pH value showed alkaline or alkalescent.
2. Cr3+could be adsorbed heavily by soil, and there was less than 1%remaining in the soil solution. A small number of Cr6+was adsorbed when adding Cr6+ to soil, apart from part of Cr6+ which were reduced to Cr3+and adsorbed quickly by soil, most of Cr6+ existed mainly in the form of water soluble hexavalent chromium. When adding combined chromium to the soil samples, Cr3+was quickly adsorbed by soil, Part of the Cr6+were reduced to Cr3+and quickly adsorbed by soil, the remaining Cr6+ existed in the form of water soluble. It was hard to detect water soluble Cr3+in the soil solution.
3. Chromium inhibited the soil alkaline phosphatase activity, The order of inhibition was that:Cr3++Cr6+> Cr3+> Cr6+. The model U=A/(1+B×C) (in which A, B were composite parameters) could describ the relationship better between soil enzyme activity and chromium concentration. The mechanism of different valence state of chromium on the soil alkaline phosphatase was completely inhibition. The ecological dose ED10 of alkaline phosphatase in the same type of soil decreased continuously with the decline of soil organic matter content. There was an interaction between trivalent chromium and hexavalent chromium on the soil alkaline phosphatase.
4. Chromium inhibited the activities of Jackbean urease, The order of inhibition was that: Cr3+> Cr3++Cr6+> Cr6+. The critical concentrations (ED10) of Cr3++Cr6+、Cr3+and Cr6+in Jackbean urease were 0.1,416.1,295.0 mg/kg respectively. With the increasing of chromium concentration, the kinetic parameter Km of Jackbean urease were kept in the same level, which revealed that the mechanism of chromium on Jackbean urease was noncompetitive inhibition.
5. Different valence state of chromium had the distinct influence on soil urease activity. Cr3+ inhibited the activities of soil urease, while Cr6+ activated the activities of soil urease. The order of inhibition is Cr3+> Cr6+> Cr3++Cr6*. There were an interaction between Cr3+ and Cr6+ on the soil urease activity, and it showed synergism. within the range of the test chromium concentration, the kinetic parameters Km of soil urease were keep in the same level. The Vmax value falled with the increase of Cr3+concentration, and rised with the increase of Cr6+concentration, the kinetic parameters of lou soil were different from Cinnamon soil and Sandy soil. The reason will be further studyed.. It revealed that the mechanism of Cr3+ on soil urease was noncompetitive inhibition.
6. chromium inhibited the dehydrogenase activity of soil, The order of inhibition was Crb +> Cr3++Cr6+> Cr3+, Which maybe cause that dehydrogenase activity is the overall performance of microbial activities. The model U=β0-β1×C could describ the relationship between soil dehydrogenase activity and chromium concentration. It revealed that Dehydrogenase can be characterized as an index of chromium contamination in certain extent. There were interaction between trivalent chromium and hexavalent chromium on the soil dehydrogenase activity.
7. According to the principle of that the smaller ecological dose, the more sensitiv, the critical concentrations (ED10) of Cr3+、Cr6+and Cr3++Cr6+in soils were 37.90.10.19 and 33.7 mg/kg respectively. The critical concentrations (EC10) of water soluble Cr3+、Cr6+and Cr3++ Cr6+ in soils were 0.08、795.88、191.63 mg·l-1 respectively.
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