【作者】 徐卫华；

【导师】 刘云国；

【作者基本信息】 湖南大学 ， 环境科学， 2007， 博士

【摘要】 铬及其化合物在工业中被广泛使用,导致大量含铬废水、废渣产生,对环境造成严重污染。在铬污染治理中,将毒性极强的六价铬还原为毒性较小的三价铬是减少铬污染危害的关键。自然界中许多菌种具有Cr（Ⅵ）还原能力,因而用微生物法治理铬污染已受到人们的广泛认可。本文对几株不同细菌还原解毒六价铬的特性和机理进行了详细研究与探讨。从Cr（Ⅵ）污染环境中分离筛选出一株Cr（Ⅵ）还原菌Cr-4,通过16SrRNA基因序列分析,被鉴定为炭疽芽孢杆菌（Bacillus anthracis）。该菌株对Cr（Ⅵ）具有较好的抗性,菌体能在Cr（Ⅵ）浓度高达125mg/L的液体培养基中生长,在含Cr（Ⅵ） 50mg/L的液体培养基中生长,其细胞形态仍很完整。对Cr-4菌株还原Cr（Ⅵ）的影响因素如碳源、培养基初始pH值、Cr（Ⅵ）初始浓度、菌液接种量、温度、阴离子SO₄^2-和NO₃^-、重金属离子Cu²⁺和Zn²⁺、外源菌种等进行了研究。结果表明,碳源能明显促进Cr（Ⅵ）的还原,在本文采用的三种碳源中,苹果酸的效果最好,其次是葡萄糖,再为琥珀酸。当培养基初始pH=7.2,菌体还原50mg/L Cr（Ⅵ） 48h,添加苹果酸、葡萄糖和琥珀酸的处理Cr（Ⅵ）去除率分别达到98%、89%和83%以上,而同样条件下未添加外源碳时,72h后Cr（Ⅵ）去除率才达到60%。培养基初始pH值对菌体还原Cr（Ⅵ）也有重要影响。实验发现,Cr-4菌株在初始pH为7.0-11.0时的还原效果明显比初始pH为4.0-6.0时要好。以葡萄糖为外源碳,还原50mg/L Cr（Ⅵ） 72h后,初始pH为7.0-11.0的Cr（Ⅵ）去除率可达到85%-99%以上,而初始pH为4.0-6.0的去除率仅为35%-65%。菌体在Cr（Ⅵ）初始浓度为10-80mg/L时,都能有效还原Cr（Ⅵ）,但去除率随初始浓度升高而降低。在菌液接种量为0.5%-5%范围内,接种量越多,Cr（Ⅵ）还原越快。对比菌体分别在20℃、37℃和47℃的还原效果,发现37℃的还原效果最好。共存阴离子SO42-、NO3-和25mg/L的Zn²⁺,以及外源菌种（铜绿假单胞菌和产黄纤维单胞菌）都对Cr（Ⅵ）还原没有影响,但100mg/L的Zn²⁺对Cr（Ⅵ）还原有一定的抑制作用,而Cu²⁺对Cr（Ⅵ）还原有显著的促进作用。对细胞生长与Cr（Ⅵ）还原的关系进行考察,发现二者紧密相关。随着Cr（Ⅵ）细胞生长量的增加,Cr（Ⅵ）被还原,当Cr（Ⅵ）被还原至较低浓度时,细胞生长量又显著增加。通过检测胞内粗酶液和培养代谢液对Cr（Ⅵ）的还原作用,对菌株还原Cr（Ⅵ）的机理进行分析、研究,实验发现胞内粗酶液能还原Cr（Ⅵ）,表明Cr-4菌株对Cr（Ⅵ）的还原存在酶促机理。同时,菌体以不同的碳源为电子供体还原Cr（Ⅵ）时,其机理有差异。以葡萄糖为碳源还原Cr（Ⅵ）时,有部分是代谢产物的间接还原作用,而以苹果酸或琥珀酸为碳源时,Cr（Ⅵ）的还原主要通过菌体自身的直接还原作用来完成。此外,对还原Cr（Ⅵ）后的菌体进行能谱分析,发现菌体在培养基初始pH=9.0还原Cr（Ⅵ）时,没有吸附铬,而在培养基初始pH=7.2还原Cr（Ⅵ）时,通过阳离子交换吸附了少量三价铬。对铜绿假单胞菌（Pseudomonas aeruginosa）的抗Cr（Ⅵ）性能、还原Cr（Ⅵ）的影响因素和机理进行研究。结果表明,铜绿假单胞菌对Cr（Ⅵ）的抗性比Cr-4菌株弱。40mg/L的Cr（Ⅵ）就对细胞形态产生了毒性影响,在Cr（Ⅵ）浓度100mg/L的液体培养基中,菌体24h内没有出现生长。三种碳源:葡萄糖、苹果酸和琥珀酸都能促进Cr（Ⅵ）的还原,其中苹果酸的效果最好。菌体在培养基初始pH=7.0还原40mg/L Cr（Ⅵ）,72h后,未添加外源碳的处理Cr（Ⅵ）去除率仅为34%,而添加苹果酸的处理去除率可提高到60%。菌体在培养基初始pH为8.0-11.0时的还原效果比初始pH为4.0-7.0时要好。以葡萄糖为外加碳源,还原40mg/LCr（Ⅵ）72h后,培养基初始pH为8.0-11.0时Cr（Ⅵ）去除率可达到95%以上,而初始pH为4.0-7.0时,去除率仅为10%-50%。菌体在Cr（Ⅵ）初始浓度为10-80mg/L时,均能还原Cr（Ⅵ）,但Cr（Ⅵ）去除率随初始浓度升高而降低。菌液接种量（0.5%-5%）的增加能提高Cr（Ⅵ）的去除率。铜绿假单胞菌对Cr（Ⅵ）的还原不受共存阴离子SO42-和NO3-的影响。低浓度的Zn²⁺（25mg/L）就对Cr（Ⅵ）还原有明显的抑制作用,而Cu²⁺却能明显促进Cr（Ⅵ）的还原。铜绿假单胞菌对Cr（Ⅵ）的还原机理与分离菌Cr-4相似,存在酶促机理。实验还发现,菌体以葡萄糖为碳源还原Cr（Ⅵ）时,Cr（Ⅵ）的去除有部分是代谢产物的还原作用,而以苹果酸或琥珀酸为碳源时,Cr（Ⅵ）的去除主要通过菌体自身的还原作用完成,而没有代谢产物的作用。还原Cr（Ⅵ）后菌体的能谱分析结果表明,菌体还原Cr（Ⅵ）时（培养基初始pH=7.0）通过阳离子交换吸附了三价铬。本文还研究了Fe（Ⅲ）对异化型铁还原菌——产黄纤维单胞菌（Cellulomonas flavigena）还原Cr（Ⅵ）的影响。结果表明,FeCl3和纤铁矿能提高菌体对Cr（Ⅵ）的还原效率,但赤铁矿对菌体还原Cr（Ⅵ）没有明显的促进作用,这说明Fe（Ⅲ）对Cr（Ⅵ）还原的影响与铁氧化物的性质相关。另外,实验还发现,当Fe（Ш）/Cr（Ⅵ）的初始浓度比较高时,Cr（Ⅵ）的去除率也相应较高。pH的变化在一定程度上反映了Cr（Ⅵ）的还原状况。用分离菌Cr-4和铜绿假单胞菌处理含Cr（Ⅵ）工业废水,发现分离菌Cr-4的处理效率比铜绿假单胞菌高。将分离菌Cr-4菌液接种于Cr（Ⅵ）污染土壤,能加快土壤中Cr（Ⅵ）的还原,但使土壤中铬的活性有所提高。更多还原

【Abstract】 Chromium and its compounds are widely used in many industries and a large quantity of Cr（Ⅵ）-containing wastes are released into environment. Chromium, especially hexavalent chromium has posed great threat on environmental safety and human health. The reduction of quite toxic hexavalent chromium into less toxic trivalent chromium is the key for the detoxification of chromium. Many bacterial species have been found to be able to reduce Cr（Ⅵ） and microbial remediation has been regarded as a promising approach for the treatment of chromium pollution. In this paper, the ability and mechanisms of Cr（Ⅵ） reduction by several different strains were researched in detail.A novel Cr（Ⅵ）-reducing strain Cr-4 was isolated from Cr（Ⅵ）-contaminated environment and identified as Bacillus anthracis by sequence analysis of 16SrRNA. The isolate Cr-4 could grow in the presence of 125mg/L Cr（Ⅵ） and its cellular morphology keep quite intact in liquid medium containing 50mg/L Cr（Ⅵ）.The parameters affecting Cr（Ⅵ） reduction by isolated strain, Cr-4, were thoroughly investigated, such as carbon source, initial pH value of liquid medium, initial Cr（Ⅵ） concentration, cells inoculum amount, temperature, the anions of SO₄^2- and NO)3^-, the heavy metals of Zn²⁺ and Cu²⁺ and the exogenous strains. The results demonstrated that addition of carbon source enhanced Cr（Ⅵ） reduction obviously. Among three carbon sources applied in the experiments, malate was the best, followed by glucose, succinate. With the initial Cr（Ⅵ） concentration of 50 mg/L and initial pH value of 7.2, more than 98%, 89% and 83% of Cr（Ⅵ） was removed after 48h by addition of malate, glucose and succinate respectively, but only 60% of Cr（Ⅵ） was removed after 72h if none of carbon source was added. The initial pH value of liquid medium also had important effect on Cr（Ⅵ） reduction. With the initial Cr（Ⅵ） concentration of 50 mg/L and glucose as carbon source, the isolate Cr-4 reduced more than 85%-99% of Cr（Ⅵ） after 72h when initial pH value was in the range of 7.0-11.0, but only 35%-65% of Cr（Ⅵ） was reduced when initial pH value was in the range of 4.0-6.0. The isolated strain, Cr-4 was able to reduce Cr（Ⅵ） at initial concentration from 10 to 80 mg/L, but the Cr（Ⅵ） removal rate decreased with the increase of initial Cr（Ⅵ） concentration. With the cells inoculum amount from 0.5%-5%, the increase of cells inoculum amount leaded to the increase of Cr（Ⅵ） removal rate. The isolate Cr-4 reduced Cr（Ⅵ） more efficiently at 37℃, compared with that at 20℃and 47℃. The anions of SO₄^2- and NO₃^-, exogenous strains （Pseudomonas aeruginosa and Cellulomonas flavigena） and 25mg/L Zn²⁺ did not affect the bacterial Cr（Ⅵ） reduction, but 100mg/L Zn²⁺ inhibited Cr（Ⅵ） reduction, and Cu²⁺, however, had significant enhancing effects on Cr（Ⅵ） reduction.The relation between cells growth and Cr（Ⅵ） reduction was detected. It was observed that the cells growth of Cr-4 and Cr（Ⅵ） reduction were well correlated. The growth of cells stimulated Cr（Ⅵ） reduction and efficient Cr（Ⅵ） reduction conversely promoted the cells growth. The cell-free extracts and filtrates of cultures were used to reduce Cr（Ⅵ） in order to research the mechanisms of Cr（Ⅵ） reduction by the isolate Cr-4. The results demonstrated that the cell-free extracts was able to reduce Cr（Ⅵ）, indicating that the enzyme-catalyzed mechanism was applied in Cr（Ⅵ） reduction by the isolate Cr-4. Additionally, it was found that the mechanism was different when different carbon source was applied as the electron donors. When glucose was used as carbon source, a part of Cr（Ⅵ） was reduced by the metabolites, but no Cr（Ⅵ） was reduced by the metabolites when malate or succinate was used as carbon source. The energy-dispersive X-ray microanalyzer was employed to test the adsorption of chromium by cells after Cr（Ⅵ） reduction. The results of energy spectrums showed that Cr（Ⅲ） was adsorbed by cation exchange during Cr（Ⅵ） reduction at initial pH value of 7.2, but no chromium was adsorbed when Cr-4 reduced Cr（Ⅵ） at initial pH value of 9.0.Another strain, Pseudomonas aeruginosa, was also used to reduce Cr（Ⅵ）. Its resistance to Cr（Ⅵ）, the factors influencing Cr（Ⅵ） reduction and the mechanisms of Cr（Ⅵ） reduction were researched. It was found that the resistance of Pseudomonas aeruginosa to Cr（Ⅵ） was not as good as that of the isolate Cr-4. The cellular morphology was affected by 40mg/L Cr（Ⅵ）, and the cells was not grown in 24h in the presence of 100mg/L Cr（Ⅵ）. Three carbon sources, glucose, malate and succinate applied in the experiments, were found to promote Cr（Ⅵ） reduction, and malate had the best effects. With the initial Cr（Ⅵ） concentration of 40mg/L and initial pH value of 7.0, only 34% of Cr（Ⅵ） was removed by Pseudomonas aeruginosa after 72h if there was no carbon source was applied, and the Cr（Ⅵ） removal rate increased to 60% when malate was used as carbon source. Pseudomonas aeruginosa reduced Cr（Ⅵ） more efficiently at initial pH value of 8.0-11.0 than that at 4.0-7.0. With the initial Cr（Ⅵ） concentration of 40mg/L and glucose as carbon source, more than 95% of Cr（Ⅵ） was removed after 72h at initial pH value of 8.0-11.0, but only 10%-50% of Cr（Ⅵ） was removed at initial pH value of 4.0-7.0. The Cr（Ⅵ） removal rate decreased with the increase of initial Cr（Ⅵ） concentration ranging from 10 to 80 mg/L, and the increase of cells inoculum amount （0.5%-5%） leaded to the increase of Cr（Ⅵ） removal rate. The anions of SO₄^2- and NO₃^- had no effects on Cr（Ⅵ） reduction. Zn²⁺ inhibited Cr（Ⅵ） reduction significantly even the concentration of Zn²⁺ was only 25mg/L, but Cu²⁺ was found to promote Cr（Ⅵ） reduction significantly.The mechanism of Cr（Ⅵ） reduction by Pseudomonas aeruginosa is like that of the isolate Cr-4. Pseudomonas aeruginosa reduced Cr（Ⅵ） by enzyme-catalyzed mechanism, and a part of Cr（Ⅵ） was reduced by the metabolites when glucose was applied as carbon source, but no Cr（Ⅵ） was reduced by the metabolites when malate or succinate was used as carbon source. The energy spectrum showed that Pseudomonas aeruginosa adsorbed chromium by cation exchange during Cr（Ⅵ） reduction （initial pH=7.0）.The effect of Fe（Ⅲ） on Cr（Ⅵ） reduction by dissimilatory iron-reducing strain, Cellulomonas flavigena, was also investigated. The results demonstrated that addition of FeCl3 or lepidocrocite promoted Cr（Ⅵ） reduction and increased Cr（Ⅵ） removal rate, but addition of hematite did not lead to the increase of Cr（Ⅵ） removal rate, which indicates that the effect of Fe（Ⅲ） on Cr（Ⅵ） reduction appears different due to the diversity of iron-oxides. In addition, the results demonstrated that the Cr（Ⅵ） removal rate increased with the increase of initial concentration ratio of Fe（Ⅲ）/ Cr（Ⅵ）, and the change of pH value reflected Cr（Ⅵ） reduction to some extent.The isolate Cr-4 and Pseudomonas aeruginosa were respectively applied to treat the Cr（Ⅵ）-containing industrial wastewater. The isolate Cr-4 was found to have a higher Cr（Ⅵ） removal efficiency than Pseudomonas aeruginosa. The isolate Cr-4 was also inoculated to Cr（Ⅵ）-contaminated soil. It was observed that the cells of Cr-4 promoted Cr（Ⅵ） reduction, but increased the activity of chromium in soil.更多还原