铬渣堆场铬污染特征及其铬污染土壤微生物修复研究
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摘要
我国是世界铬盐生产大国,年产量已超过16万吨,然而在其生产过程中产生大量含铬废渣。全国每年新排放铬渣约60万吨,历年累积堆存铬渣近400万吨。铬渣中含有0.3～1.5%可溶性Cr(VI),经降雨和地表水的冲刷,Cr(VI)进入周围土壤和地下水,对环境造成严重污染,目前我国受Cr(VI)严重污染的土壤达1250多万吨。铬渣堆场已经列为我国土壤污染重点治理对象。我们国家目前急需修复铬渣污染土壤的关键技术。因此,本文以湖南某工厂铬渣堆场所引起铬污染特点为基础,以修复铬污染土壤为目标,进行系统的研究,获得如下结果:
湖南某厂铬渣堆场、渣堆周围以及厂外农业用地三个区域表层土壤总铬平均含量分别是我国土壤环境质量二级标准的5.6、7.6和5.O倍;这三个区域剖面土壤中总铬分别在土壤剖面40-60cm、20-40cm、O-20cm深度累积;铬渣淋溶出的铬可迁移至底土层;铬渣堆场土壤总铬以铁锰结合态为主,而铬渣堆场周围土壤总铬以残渣态为主;铬渣堆场和其周围表层土壤水溶性六价铬平均含量分别是对照区的137.5和30.1倍;厂外农作物芹菜、白菜及莴笋分别有50%、100%及75%的样品中可食部分铬含量超过我国食品铬限量卫生标准;工厂附近居民饮用水(井水)50%的样品中六价铬含量超过我国饮用水卫生标准。
铬污染已严重抑制铬渣堆场土壤三大微生物区系(细菌、真菌和放线菌)的活性。与对照区相比,铬渣堆场土壤细菌、真菌和放线菌数量分别下降89.9%、99.8%和99.9%。污染土壤中细菌、真菌和放线菌数量均与土壤总铬和水溶性六价铬含量呈负相关关系;室内培养试验也表明,六价铬均不同程度地抑制了土壤可培养细菌、真菌和放线菌的生长和降低其多样性,其中放线菌对铬污染最敏感。铬污染对土壤多酚氧化酶和过氧化氢酶活性没有明显抑制,对土壤碱性磷酸酶的活性有轻度抑制,而土壤脱氢酶的活性严重受到抑制,可见土壤脱氢酶活性对铬污染较敏感,可用于土壤铬污染生物学预警指标。
基于微生物在极端环境下生存的胁迫机制,从铬渣堆场铬污染土壤中分离出4株六价铬耐受菌,均为嗜碱性细菌;4株Cr(VI)耐受菌中只有一株具有较强的还原Cr(VI)的能力,24h内基本还原500mg/LCr(VI)。该细菌生长曲线表明其生长与其对Cr(VI)的还原并非同步进行,细菌的铬耐受力与其铬还原能力无直接关联;用扫描电镜对该铬还原菌还原Cr(VI)前后进行形貌观察,结果显示该细菌呈杆状,尾部生有鞭毛,表面附有少量丝状物质,还原Cr(VI)后,部分菌体末端黏附着一团不定形物质,细菌介质中也有大量的不定形物质聚积;采用EDAX和XPS对该菌还原Cr(VI)后的产物成分进行鉴定,结果表明Cr是产物中主要元素,Cr(VI)还原为Cr(III),且以Cr(OH)_3形式存在;细菌生理生化特性的测试以及16S rDNA的测序及比对均显示该铬还原菌属Pannonibacter phragmitetus。
通过对培养基的优化,提出并研究了直接添加培养基激活Pannonibacter phragmitetus的活性来进行铬污染土壤的原位微生物修复新方法。在温度为30℃、土液比为1:1、碳源葡萄糖投加量为5g/kg、氮源化合物A投加量为5g/kg的情况下,该细菌能在4d内去除土壤总六价铬的效率达到92%,其中水溶性六价铬可基本上去除;5d后土壤中交换态六价铬去除率达到89%;10d后土壤碳酸盐结合态六价铬去除率达到84%。
铬渣堆场土壤的修复是由Pannonibacter phragmitetus对Cr(VI)还原作用的结果,有机质、铁氧化物和锰氧化物等土壤固相组分并未参与Cr(VI)的还原;同时细菌代谢产物及其胞外酶也未表现出对Cr(VI)的还原作用。细菌对Cr(VI)还原机理是其胞内酶的直接还原作用,且胞内酶在NADH的参与下完成对Cr(VI)的还原。铬还原酶是菌体本身的组成酶而不是诱导酶,即组成酶具有还原Cr(VI)的能力。
China is one of major countries to produce chromate and the annual output of chromate was more than 160 thousand tons. However, a large amount of chromium-containing slag was discharged from chromate industries. The accumulated amount of chromium-containing slag was more than 4 million tons and 600 thousand tons are being discharged annually. Dissolvable hexavalent chromium (Cr(VI)) accounting for 0.3～1.5% of slag can be leached into soils and groundwater by rainfall and runoff, which incurs a significant risk to the environment and human health. At present, the accumulated soils polluted by chromium-containing slag reached up to 12.5 million tons. Chromium-containing slag heap sites are concerned as an important treatment object and the key technologies for remediating the polluted soils are urgently required in our country. Therefore, the characteristics of chromium (Cr) pollution at one chromium-containing slag site and Cr(VI) bioremediation in the contaminated soils were investigated in this dissertation.
Mean concentrations of total Cr in the soils under the chromium-containing slag heap at one factory in Hunan province, in the vicinity of the slag heap and the agricultural land outside of the factory were 5.6, 7.6 and 5.0 times of the critical level of Secondary Environmental Quality Standard for Soil in China, respectively. The highest amount of total chromium in soil of these three areas was found at 40-60cm, 20-40cm and 0-20cm of soil depth respectively. Chromium was transported into the deep layer in soil profile. Fe and Mn oxides-bound chromium was the predominant fraction in the contaminated soils under the slag heap, while residual chromium was the main fraction in the soils in the vicinity of the slag heap. Mean contents of water soluble Cr(VI) in top soils under and in the vicinity of the slag heap were 137.5and 30.1 times of that in the unpolluted soils, respectively. According to the Tolerance Limit of Chromium in Foods, the occurance rates of exceeding over the critical level for the above-ground part of the celery, cabbage and lettuce on the farmland outside of the factory were 50%, 100% and 75%, respectively. Drinking water was heavily polluted and 50% of the samples exceeded the Cr(VI) Standard for Drinking Water Quality.
The populations of three soil microflora were severely affected by chromium contamination. The populations of bacteria, fungi and actinomycetes decreased by 89.9%、99.8% and 99.9% as compared with that of the control site. The populations of bacteria, fungi and actinomycetes were all negatively correlated with the contents of total Cr and water soluble Cr(VI). Incubation experiment also indicated that hexavalent chromium inhibited the growth and decreased the diversities of soil culturable bacteria, fungi and actinomycetes. Actinomycetes was the most sensitive to Cr pollution. The activities of catalase and polyphenol oxidase in soils were not obviously depressed by chromium pollution and alkaline phosphatases activity was slightly suppressed by chromium pollution. However, Cr(VI) significant inhibited dehydrogenase activity, revealing that dehydrogenase activity could be used as a biological indicator for the chromium pollution.
Four chromium-resistance strains were isolated from the contaminated soil under the chromium-containing slag heap. Only one strain exhibited a strong Cr(VI) reduction ability, which can completly reduce 500mg/L Cr (VI) within 24 h. Asynchrony of Cr(VI) reduction and cell growth was observed in this study. Moreover, Cr(VI) reduction ability of cells was independent on their resistance to Cr(VI). Scanning electron microscopy (SEM) was used to observe the morphologies of bacteria before and after Cr(VI) reduction. The results showed that the cells were rod with a flagellum at the terminal of bacteria. The discernible cluster of amorphous substances were bound to the terminal of the cells after reducing Cr(VI). Elemental composition and oxidation state of the chromium in the final product were verified by energy dispersive X-ray (EDAX) and X-ray photoelectron spectroscope (XPS) analysis, revealing that Cr was the major element that existed in trivalent state of chromium hydroxides under alkaline condition. Biochemical charasteristics and 16S rDNA sequencing showed that the chromate-reducing strain BB was a species of Pannonibacter phragmitetu.
Based on the optimization of culture medium composition and growth conditions and ability of Cr(VI) reduction, the bioremediation of Cr-contaminated soil can be achieved by adding culture medium in soils to stimulate the activity of Pannonibacter phragmitetu. The optimal condition for the Cr(VI) reduction by Pannonibacter phragmitetu was 5g glucose and 5g yeast extract per kg soil at 30℃and the ratio of soil and water was 1:1. Under the optimal condition, 92% of total Cr(VI) in soil contaminated by chromium-containing slag heap was removed and water soluble Cr(VI) was completely removed at 4 days. Meanwhile, the removal of exchangeable Cr(VI) and carbonate-bound Cr(VI) reached up to 89% at 5 days and 84% at 10 days, respectively.
The remediation of Cr-polluted soil was contributed to Cr(VI) reduction by Pannonibacter phragmitetu. Soil organic matter, iron oxides and magnesium oxides did not involve in Cr(VI) reduction in soils. The microbial metabolites and extracellular enzyme have no capability of reducing Cr(VI). The reduction of Cr(VI) by Pannonibacter phragmitetu was a direct reduction catalyzed by its intracellular enzyme. Furthermore, this intracellular enzyme was an NADH-dependent reductase. Cr(VI)-reducing enzyme was not induced by Cr(VI) but constitutively expressed in Pannonibacter phragmitetu.
湖南某厂铬渣堆场、渣堆周围以及厂外农业用地三个区域表层土壤总铬平均含量分别是我国土壤环境质量二级标准的5.6、7.6和5.O倍;这三个区域剖面土壤中总铬分别在土壤剖面40-60cm、20-40cm、O-20cm深度累积;铬渣淋溶出的铬可迁移至底土层;铬渣堆场土壤总铬以铁锰结合态为主,而铬渣堆场周围土壤总铬以残渣态为主;铬渣堆场和其周围表层土壤水溶性六价铬平均含量分别是对照区的137.5和30.1倍;厂外农作物芹菜、白菜及莴笋分别有50%、100%及75%的样品中可食部分铬含量超过我国食品铬限量卫生标准;工厂附近居民饮用水(井水)50%的样品中六价铬含量超过我国饮用水卫生标准。
铬污染已严重抑制铬渣堆场土壤三大微生物区系(细菌、真菌和放线菌)的活性。与对照区相比,铬渣堆场土壤细菌、真菌和放线菌数量分别下降89.9%、99.8%和99.9%。污染土壤中细菌、真菌和放线菌数量均与土壤总铬和水溶性六价铬含量呈负相关关系;室内培养试验也表明,六价铬均不同程度地抑制了土壤可培养细菌、真菌和放线菌的生长和降低其多样性,其中放线菌对铬污染最敏感。铬污染对土壤多酚氧化酶和过氧化氢酶活性没有明显抑制,对土壤碱性磷酸酶的活性有轻度抑制,而土壤脱氢酶的活性严重受到抑制,可见土壤脱氢酶活性对铬污染较敏感,可用于土壤铬污染生物学预警指标。
基于微生物在极端环境下生存的胁迫机制,从铬渣堆场铬污染土壤中分离出4株六价铬耐受菌,均为嗜碱性细菌;4株Cr(VI)耐受菌中只有一株具有较强的还原Cr(VI)的能力,24h内基本还原500mg/LCr(VI)。该细菌生长曲线表明其生长与其对Cr(VI)的还原并非同步进行,细菌的铬耐受力与其铬还原能力无直接关联;用扫描电镜对该铬还原菌还原Cr(VI)前后进行形貌观察,结果显示该细菌呈杆状,尾部生有鞭毛,表面附有少量丝状物质,还原Cr(VI)后,部分菌体末端黏附着一团不定形物质,细菌介质中也有大量的不定形物质聚积;采用EDAX和XPS对该菌还原Cr(VI)后的产物成分进行鉴定,结果表明Cr是产物中主要元素,Cr(VI)还原为Cr(III),且以Cr(OH)_3形式存在;细菌生理生化特性的测试以及16S rDNA的测序及比对均显示该铬还原菌属Pannonibacter phragmitetus。
通过对培养基的优化,提出并研究了直接添加培养基激活Pannonibacter phragmitetus的活性来进行铬污染土壤的原位微生物修复新方法。在温度为30℃、土液比为1:1、碳源葡萄糖投加量为5g/kg、氮源化合物A投加量为5g/kg的情况下,该细菌能在4d内去除土壤总六价铬的效率达到92%,其中水溶性六价铬可基本上去除;5d后土壤中交换态六价铬去除率达到89%;10d后土壤碳酸盐结合态六价铬去除率达到84%。
铬渣堆场土壤的修复是由Pannonibacter phragmitetus对Cr(VI)还原作用的结果,有机质、铁氧化物和锰氧化物等土壤固相组分并未参与Cr(VI)的还原;同时细菌代谢产物及其胞外酶也未表现出对Cr(VI)的还原作用。细菌对Cr(VI)还原机理是其胞内酶的直接还原作用,且胞内酶在NADH的参与下完成对Cr(VI)的还原。铬还原酶是菌体本身的组成酶而不是诱导酶,即组成酶具有还原Cr(VI)的能力。
China is one of major countries to produce chromate and the annual output of chromate was more than 160 thousand tons. However, a large amount of chromium-containing slag was discharged from chromate industries. The accumulated amount of chromium-containing slag was more than 4 million tons and 600 thousand tons are being discharged annually. Dissolvable hexavalent chromium (Cr(VI)) accounting for 0.3～1.5% of slag can be leached into soils and groundwater by rainfall and runoff, which incurs a significant risk to the environment and human health. At present, the accumulated soils polluted by chromium-containing slag reached up to 12.5 million tons. Chromium-containing slag heap sites are concerned as an important treatment object and the key technologies for remediating the polluted soils are urgently required in our country. Therefore, the characteristics of chromium (Cr) pollution at one chromium-containing slag site and Cr(VI) bioremediation in the contaminated soils were investigated in this dissertation.
Mean concentrations of total Cr in the soils under the chromium-containing slag heap at one factory in Hunan province, in the vicinity of the slag heap and the agricultural land outside of the factory were 5.6, 7.6 and 5.0 times of the critical level of Secondary Environmental Quality Standard for Soil in China, respectively. The highest amount of total chromium in soil of these three areas was found at 40-60cm, 20-40cm and 0-20cm of soil depth respectively. Chromium was transported into the deep layer in soil profile. Fe and Mn oxides-bound chromium was the predominant fraction in the contaminated soils under the slag heap, while residual chromium was the main fraction in the soils in the vicinity of the slag heap. Mean contents of water soluble Cr(VI) in top soils under and in the vicinity of the slag heap were 137.5and 30.1 times of that in the unpolluted soils, respectively. According to the Tolerance Limit of Chromium in Foods, the occurance rates of exceeding over the critical level for the above-ground part of the celery, cabbage and lettuce on the farmland outside of the factory were 50%, 100% and 75%, respectively. Drinking water was heavily polluted and 50% of the samples exceeded the Cr(VI) Standard for Drinking Water Quality.
The populations of three soil microflora were severely affected by chromium contamination. The populations of bacteria, fungi and actinomycetes decreased by 89.9%、99.8% and 99.9% as compared with that of the control site. The populations of bacteria, fungi and actinomycetes were all negatively correlated with the contents of total Cr and water soluble Cr(VI). Incubation experiment also indicated that hexavalent chromium inhibited the growth and decreased the diversities of soil culturable bacteria, fungi and actinomycetes. Actinomycetes was the most sensitive to Cr pollution. The activities of catalase and polyphenol oxidase in soils were not obviously depressed by chromium pollution and alkaline phosphatases activity was slightly suppressed by chromium pollution. However, Cr(VI) significant inhibited dehydrogenase activity, revealing that dehydrogenase activity could be used as a biological indicator for the chromium pollution.
Four chromium-resistance strains were isolated from the contaminated soil under the chromium-containing slag heap. Only one strain exhibited a strong Cr(VI) reduction ability, which can completly reduce 500mg/L Cr (VI) within 24 h. Asynchrony of Cr(VI) reduction and cell growth was observed in this study. Moreover, Cr(VI) reduction ability of cells was independent on their resistance to Cr(VI). Scanning electron microscopy (SEM) was used to observe the morphologies of bacteria before and after Cr(VI) reduction. The results showed that the cells were rod with a flagellum at the terminal of bacteria. The discernible cluster of amorphous substances were bound to the terminal of the cells after reducing Cr(VI). Elemental composition and oxidation state of the chromium in the final product were verified by energy dispersive X-ray (EDAX) and X-ray photoelectron spectroscope (XPS) analysis, revealing that Cr was the major element that existed in trivalent state of chromium hydroxides under alkaline condition. Biochemical charasteristics and 16S rDNA sequencing showed that the chromate-reducing strain BB was a species of Pannonibacter phragmitetu.
Based on the optimization of culture medium composition and growth conditions and ability of Cr(VI) reduction, the bioremediation of Cr-contaminated soil can be achieved by adding culture medium in soils to stimulate the activity of Pannonibacter phragmitetu. The optimal condition for the Cr(VI) reduction by Pannonibacter phragmitetu was 5g glucose and 5g yeast extract per kg soil at 30℃and the ratio of soil and water was 1:1. Under the optimal condition, 92% of total Cr(VI) in soil contaminated by chromium-containing slag heap was removed and water soluble Cr(VI) was completely removed at 4 days. Meanwhile, the removal of exchangeable Cr(VI) and carbonate-bound Cr(VI) reached up to 89% at 5 days and 84% at 10 days, respectively.
The remediation of Cr-polluted soil was contributed to Cr(VI) reduction by Pannonibacter phragmitetu. Soil organic matter, iron oxides and magnesium oxides did not involve in Cr(VI) reduction in soils. The microbial metabolites and extracellular enzyme have no capability of reducing Cr(VI). The reduction of Cr(VI) by Pannonibacter phragmitetu was a direct reduction catalyzed by its intracellular enzyme. Furthermore, this intracellular enzyme was an NADH-dependent reductase. Cr(VI)-reducing enzyme was not induced by Cr(VI) but constitutively expressed in Pannonibacter phragmitetu.
引文
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