铅锌冶炼废渣堆场土壤产黄青霉菌F1浸出修复研究
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摘要
摘要：铅锌冶炼过程产生大量含重金属的废渣,并弃置于环境,导致渣场及附近土壤遭受重金属的严重污染,治理废渣堆场重金属污染土壤是国内外研究的焦点,我国尤为迫切。目前常采用的化学方法或植物修复法存在见效慢、成本高或具有二次污染潜在危害等不足。本论文对铅锌冶炼废渣堆场土壤重污染的特征、微生物的分离筛选、浸出修复工艺及其机理等开展了较系统的研究,取得了如下成果：
研究阐明了典型铅锌冶炼废渣堆场土壤的污染特征。铅锌冶炼废渣堆场土壤遭受重金属的重度污染：表层土壤中Pb、Zn、Cd和Cu的含量分别为1889.6mg·kg-1,5682mg·kg-1,48.4mg·kg-1和1848.6mg·kg-1,其中Pb超过《土壤环境质量标准(GB15618-1995)》二级标准限值的5.4倍,Zn超标18.94倍,Cd超标80.7倍,Cu超标18.5倍。且废渣堆场土壤Pb、Zn、Cd和Cu含量呈现随土层深度的增加而降低,随距污染源距离的增加而减少的空间分布规律。
基于微生物在极端环境下生存的胁迫机制,从铅锌冶炼废渣堆场重污染土壤中分离筛选出一株具产酸特性、能使浸出液pH值从6.7降到2.7的重金属耐受菌,经18S rDNA和ITS鉴定为产黄青霉菌(Penicillium chrysogenum),命名为F1。产黄青霉菌F1生长受pH值和重金属浓度的影响较大,能正常生长的pH值范围为5.0-9.0,当土液比在0-1：13.3范围内时,产黄青霉菌F1的生长符合SGompertz和Logistic模型。
通过对铅锌冶炼废渣堆场污染土壤微生物浸出条件的优化,提出了“菌株培养产酸-重金属浸出”的二步法浸出修复新方法。产黄青霉菌F1浸出修复的最佳条件为温度28℃、土液比1：20、pH7.0,碳源葡萄糖投加量为90g.L-1,氮源A投加量为3g-L-’。在最佳条件下,产黄青霉菌F1对重金属含量高达12.62g.kg-1重污染土壤中Cd、Cu、Pb、 Zn、Mn和Cr的浸出率分别为74%、59%、24%、55%、57%和25%,远远高于0.5%的柠檬酸、草酸、苹果酸和琥珀酸的浸出率。
阐述了铅锌冶炼废渣堆场土壤产黄青霉菌F1浸出的作用机制。在浸出过程中,产黄青霉菌F1通过葡萄糖代谢,产生了葡萄糖酸、丙酮酸和草酸等有机酸。产黄青霉菌F1浸出修复土壤后,菌丝细胞壁外分泌大量胞外聚合物,且周围吸附大量的金属离子,表明产黄青霉菌F1对重金属的抗性机理为重金属通过胞外聚合物的截留。
揭示了产黄青霉菌F1对土壤中重金属的形态转化规律。在浸出过程中,土壤中的Cd、,Zn和Mn主要以离子形态被浸出；Pb和Cr主要体现为形态的转换,以铁锰氧化物结合态和强有机态赋存的Pb转化成了残渣态,铁锰氧化物结合态的Cr转化成碳酸盐结合态；Cu既以离子形态被浸出,又有形态间的转换,被产黄青霉菌F1浸出后,Cu的铁锰氧化物结合态转化为水溶态和离子交换态。发现了重金属和pH值对葡萄糖氧化酶活性的调控机理,为浸出修复时土液比的确定提供了科学依据。单一重金属中,Pb、Cu、Cd、Mn和Cr对产黄青霉菌F1葡萄糖氧化酶活性具有抑制作用,Zn对葡萄糖氧化酶活性具有激活作用,以上六种重金属复合对葡萄糖氧化酶活性的影响大于单一重金属。酸性条件下产黄青霉菌F1胞外葡萄糖氧化酶的活性大于偏中性条件下的活性。
从铅锌冶炼废渣堆场土壤筛选到的产黄青霉菌F1在治理重金属污染土壤过程中具有较好的应用前景。
Abstract:A large amount of slags rich in heavy metals had been produced during nonferrous smelting process. These smelting slags were directly piled and no recycling technology was used on them, thus the soil was polluted severely by heavy metals at Pb/Zn smelting slag site. Remediation on the heavy metal polluted soil is a focus in the world, especially in China. At present, chemical remediation and phytoremediation are often used to harness heavy metal polluted soil, but they own several shortcomings such as high cost, low efficiency and the possibility of secondary pollution. The characteristics of the heavy metal polluted soil, the strain's isolation and screening, and the technology and mechanism of bioleaching were studied in this paper. Some conclusions were drawn as follows:
The characteristics of the soil at Pb/Zn smelting slag site were studied in this paper. It showed that the soil at Pb/Zn smelting slag site was severely polluted by heavy metals, the content of the heavy metals were as follows:1889.6mg·kg-1Pb,5682mg·kg-1Zn,48.4mg·kg-1Cd and1848.6mg·kg-1Cu, individually. According Chinese GB15618-1995, the content of Pb, Zn, Cd and Cu were5.4,18.94,80.7and18.5times as much as the secondary standard limit value. The heavy metals'spatial distribution pattern at the slag site was decreased with the increase of soil depth, and it was also decreased with the increase of the distance from the pollution sources.
Based on the stress mechanism of microbes to survive in extreme environment, a fungi with the ability of heavy metal tolerance and producing organic acids was selected, it was identified as Penicillium chrysogenum (P. chrysogenum) using18S rDNA and ITS, and named as F1. P. chrysogenum F1can tolerate568mg·L-1Zn,188.9mg-L"1Pb,185mg·L-1Cu,4.8mg·L-1Cd and10.4mg-L"1Cr at the same time. The growth of P. chrysogenum F1was influenced by heavy metals and pH value. During the pH value range of5.0-9.0, P. chrysogenum F1grew normally. The growth of P. chrysogenum F1was fitted for SGompertz and Logistic model during the ratio range of0-1:13.3.
A new method of two-step bioleaching was proposed. The optimization conditions of bioleaching using P. chrysogenum F1were as follows:the ratio soil to liquid of1:20, the temperature of28℃, pH value of7.0,9·L-1glucose as carbon source and3g`L-1A as nitrogen source. Under the best condition, the removal percentage of Cd, Cu, Pb, Zn, Mn and Cr was74%,59%,24%,55%,57%and25%. The removal percentage by P. chrysogenum F1was better than by nitric acid, malic acid, humic acid and oxalic acid under the concentration of0.5%.
The mechanism of bioleaching using P. chrysogenum F1was elaborated. The bioleaching remediation on heavy metal polluted soil at the slag site was the extraction of heavy metals by organic acids which produced by P. chrysogenum F1. During bioleaching process, glucose acid, pyruvic acid, citric acid, oxalic acid, malic acid and succinic acid were produced through the metabolism of glucose. After bioleaching process, there were many untouched extracellular polymeric substances and particles around the cell wall so that the heavy metal resistant mechanism of P. chrysogenum F1might be the interception of extracelluar polymeric substances.
The regularity of the fractions'transformation of heavy metals was revealed. During bioleaching, all the fractions of Cd, Zn and Mn were extracted in ionic form, Fe-Mn oxide-bonded and organic fraction of Pb were transformed into residual fraction, Fe-Mn oxide-bonded of Cr was transformed into carbonate-bonded fraction, water-soluble fraction and ion-exchangeable fraction of Cu were transformed from Fe-Mn oxide-bonded.
The regulation mechanism of glucose oxidase activity by heavy metal and pH value was found. For single metal, the inhibitory impact of Pb on extracellular glucose oxidase activity was the biggest than Cu, Cd, Mn and Cr, the activity of glucose oxidase was activated by Zn. The inhibitory impact of multi-metal was bigger than single metal. The activity of extracellular glucose oxidase was larger under acid condition than under neutral condition.
P. chrysogenum F1selected from the soil at the slag site has the great potential for the remediation on heavy metal polluted soil.
研究阐明了典型铅锌冶炼废渣堆场土壤的污染特征。铅锌冶炼废渣堆场土壤遭受重金属的重度污染：表层土壤中Pb、Zn、Cd和Cu的含量分别为1889.6mg·kg-1,5682mg·kg-1,48.4mg·kg-1和1848.6mg·kg-1,其中Pb超过《土壤环境质量标准(GB15618-1995)》二级标准限值的5.4倍,Zn超标18.94倍,Cd超标80.7倍,Cu超标18.5倍。且废渣堆场土壤Pb、Zn、Cd和Cu含量呈现随土层深度的增加而降低,随距污染源距离的增加而减少的空间分布规律。
基于微生物在极端环境下生存的胁迫机制,从铅锌冶炼废渣堆场重污染土壤中分离筛选出一株具产酸特性、能使浸出液pH值从6.7降到2.7的重金属耐受菌,经18S rDNA和ITS鉴定为产黄青霉菌(Penicillium chrysogenum),命名为F1。产黄青霉菌F1生长受pH值和重金属浓度的影响较大,能正常生长的pH值范围为5.0-9.0,当土液比在0-1：13.3范围内时,产黄青霉菌F1的生长符合SGompertz和Logistic模型。
通过对铅锌冶炼废渣堆场污染土壤微生物浸出条件的优化,提出了“菌株培养产酸-重金属浸出”的二步法浸出修复新方法。产黄青霉菌F1浸出修复的最佳条件为温度28℃、土液比1：20、pH7.0,碳源葡萄糖投加量为90g.L-1,氮源A投加量为3g-L-’。在最佳条件下,产黄青霉菌F1对重金属含量高达12.62g.kg-1重污染土壤中Cd、Cu、Pb、 Zn、Mn和Cr的浸出率分别为74%、59%、24%、55%、57%和25%,远远高于0.5%的柠檬酸、草酸、苹果酸和琥珀酸的浸出率。
阐述了铅锌冶炼废渣堆场土壤产黄青霉菌F1浸出的作用机制。在浸出过程中,产黄青霉菌F1通过葡萄糖代谢,产生了葡萄糖酸、丙酮酸和草酸等有机酸。产黄青霉菌F1浸出修复土壤后,菌丝细胞壁外分泌大量胞外聚合物,且周围吸附大量的金属离子,表明产黄青霉菌F1对重金属的抗性机理为重金属通过胞外聚合物的截留。
揭示了产黄青霉菌F1对土壤中重金属的形态转化规律。在浸出过程中,土壤中的Cd、,Zn和Mn主要以离子形态被浸出；Pb和Cr主要体现为形态的转换,以铁锰氧化物结合态和强有机态赋存的Pb转化成了残渣态,铁锰氧化物结合态的Cr转化成碳酸盐结合态；Cu既以离子形态被浸出,又有形态间的转换,被产黄青霉菌F1浸出后,Cu的铁锰氧化物结合态转化为水溶态和离子交换态。发现了重金属和pH值对葡萄糖氧化酶活性的调控机理,为浸出修复时土液比的确定提供了科学依据。单一重金属中,Pb、Cu、Cd、Mn和Cr对产黄青霉菌F1葡萄糖氧化酶活性具有抑制作用,Zn对葡萄糖氧化酶活性具有激活作用,以上六种重金属复合对葡萄糖氧化酶活性的影响大于单一重金属。酸性条件下产黄青霉菌F1胞外葡萄糖氧化酶的活性大于偏中性条件下的活性。
从铅锌冶炼废渣堆场土壤筛选到的产黄青霉菌F1在治理重金属污染土壤过程中具有较好的应用前景。
Abstract:A large amount of slags rich in heavy metals had been produced during nonferrous smelting process. These smelting slags were directly piled and no recycling technology was used on them, thus the soil was polluted severely by heavy metals at Pb/Zn smelting slag site. Remediation on the heavy metal polluted soil is a focus in the world, especially in China. At present, chemical remediation and phytoremediation are often used to harness heavy metal polluted soil, but they own several shortcomings such as high cost, low efficiency and the possibility of secondary pollution. The characteristics of the heavy metal polluted soil, the strain's isolation and screening, and the technology and mechanism of bioleaching were studied in this paper. Some conclusions were drawn as follows:
The characteristics of the soil at Pb/Zn smelting slag site were studied in this paper. It showed that the soil at Pb/Zn smelting slag site was severely polluted by heavy metals, the content of the heavy metals were as follows:1889.6mg·kg-1Pb,5682mg·kg-1Zn,48.4mg·kg-1Cd and1848.6mg·kg-1Cu, individually. According Chinese GB15618-1995, the content of Pb, Zn, Cd and Cu were5.4,18.94,80.7and18.5times as much as the secondary standard limit value. The heavy metals'spatial distribution pattern at the slag site was decreased with the increase of soil depth, and it was also decreased with the increase of the distance from the pollution sources.
Based on the stress mechanism of microbes to survive in extreme environment, a fungi with the ability of heavy metal tolerance and producing organic acids was selected, it was identified as Penicillium chrysogenum (P. chrysogenum) using18S rDNA and ITS, and named as F1. P. chrysogenum F1can tolerate568mg·L-1Zn,188.9mg-L"1Pb,185mg·L-1Cu,4.8mg·L-1Cd and10.4mg-L"1Cr at the same time. The growth of P. chrysogenum F1was influenced by heavy metals and pH value. During the pH value range of5.0-9.0, P. chrysogenum F1grew normally. The growth of P. chrysogenum F1was fitted for SGompertz and Logistic model during the ratio range of0-1:13.3.
A new method of two-step bioleaching was proposed. The optimization conditions of bioleaching using P. chrysogenum F1were as follows:the ratio soil to liquid of1:20, the temperature of28℃, pH value of7.0,9·L-1glucose as carbon source and3g`L-1A as nitrogen source. Under the best condition, the removal percentage of Cd, Cu, Pb, Zn, Mn and Cr was74%,59%,24%,55%,57%and25%. The removal percentage by P. chrysogenum F1was better than by nitric acid, malic acid, humic acid and oxalic acid under the concentration of0.5%.
The mechanism of bioleaching using P. chrysogenum F1was elaborated. The bioleaching remediation on heavy metal polluted soil at the slag site was the extraction of heavy metals by organic acids which produced by P. chrysogenum F1. During bioleaching process, glucose acid, pyruvic acid, citric acid, oxalic acid, malic acid and succinic acid were produced through the metabolism of glucose. After bioleaching process, there were many untouched extracellular polymeric substances and particles around the cell wall so that the heavy metal resistant mechanism of P. chrysogenum F1might be the interception of extracelluar polymeric substances.
The regularity of the fractions'transformation of heavy metals was revealed. During bioleaching, all the fractions of Cd, Zn and Mn were extracted in ionic form, Fe-Mn oxide-bonded and organic fraction of Pb were transformed into residual fraction, Fe-Mn oxide-bonded of Cr was transformed into carbonate-bonded fraction, water-soluble fraction and ion-exchangeable fraction of Cu were transformed from Fe-Mn oxide-bonded.
The regulation mechanism of glucose oxidase activity by heavy metal and pH value was found. For single metal, the inhibitory impact of Pb on extracellular glucose oxidase activity was the biggest than Cu, Cd, Mn and Cr, the activity of glucose oxidase was activated by Zn. The inhibitory impact of multi-metal was bigger than single metal. The activity of extracellular glucose oxidase was larger under acid condition than under neutral condition.
P. chrysogenum F1selected from the soil at the slag site has the great potential for the remediation on heavy metal polluted soil.
引文
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