Lysinibacillus sp.Cr-6还原六价铬特性及其在电镀铬污染土壤修复中的应用
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摘要
电镀行业在我国的工业发展中占有重要地位,同时也造成了严重的环境污染,尤其是对厂区及周边地区土壤的污染。六价铬是电镀工艺中的重要工业原材料,关于其毒性的研究已经有诸多报道,对人类健康有着不可忽视的影响。在前期电镀厂区土壤调查重了解到电镀场地土壤重金属污染较为严重,尤其是铬、铜、锌,而且有部分土壤中检出了六价铬,由于六价铬对人类健康危害大,对其进行治理迫在眉睫。本文致力于通过微生物对六价铬进行还原,并将其应用于电镀污染土壤中六价铬的修复。
本研究从上壤中分离得到一株六价铬还原菌Cr-6,根据其生理生化特性和16S rDNA序列分析,鉴定为赖氨酸芽孢杆菌Lysinibacillus sp.,对六价铬还原特性进行研究,探讨其六价铬还原机理,分析其对六价铬还原的动力学过程,在对电镀污染场地进行重金属及上壤酶活性进行分析的基础上,将该菌株应用于电镀污染上壤中六价铬污染的修复,得山主要结论如下：
(1)对六价铬还原菌进行分离纯化并鉴定种属。从土壤中驯化分离纯化得到六株能够在含有一定浓度六价铬的培养基中生长的细菌,在初始六价铬浓度为10mg/L的条件下培养,去除率从20%到100%不等,选取对六价铬处理效果最好的菌株Cr-6作为实验研究对象。对菌株Cr-6进行六价铬作用特性分析,结果表明该菌株不会对六价铬产生吸附作用,培养液中六价铬浓度的降低源于细菌对六价铬的还原作用。对菌株Cr-6分别进行革兰氏染色、透射电镜观察和平板培养,结果表明,该菌株为革兰氏阳性杆菌,在平板上生长菌落呈圆形,从中心向外出现不同层次圆圈,边缘整齐,淡乳黄色,表面粗糙、湿润,半透明,从侧面观察无明显隆起。通过16S rDNA序列分析,并结合菌株形态观察以及生理生化特征结果,判断菌株Cr-6为赖氨酸芽孢杆菌(Lysiniacillus sp.)。分别在添加六价铬和未添加六价铬条件下对细菌进行生长曲线实验,并计算世代时间,结果表明,添加一定浓度六价铬有利于促进细胞生长,延长对数期,最大光密度值分别达到3.67和2.97,对数期内世代时间分别为0.87h和1.76h。
(2)考察不同影响因子对菌株Cr-6还原六价铬的影响。菌株Cr-6在35℃条件下生长情况最好,而且温度的升高将细菌对数期提前。在40℃和45℃条件下,对六价铬的还原效果较好,在6h内还原率达到100%。培养液pH为7时,菌株Cr-6生长情况和对六价铬还原效果最好,生长pH值范围为6-9,在pH值为4和5的时候,停止生长,能够保持-定的六价铬还原效果,在pH值为10的时候,停止生长,2h后停止对六价铬的还原作用。在不同振荡速度下,振荡速度越快,菌株Cr-6生长越好,较快的振荡速度能够提高细菌最大光密度值。相对于静置培养,在振荡条件下还原效果更好。增大接种量可提高同时刻细菌浓度和六价铬还原率。在充气条件下细菌生长迅速,六价铬还原率低于厌氧条件下的六价铬还原率。在厌氧条件下,细菌停止生长,并保持了一定的六价铬还原速率。培养液中Zn2+的添加抑制了菌株Cr-6的生长,Zn2+浓度越高,抑制作用越明显,添加低浓度的Zn2+有利于促进菌株Cr-6对六价铬的还原。添加一定浓度的Cu2+,有利于促进菌株Cr-6的生长,并提高菌株Cr-6对六价铬的还原效果。
(3)对在不同六价铬初始浓度培养基(Omg/L、10mg/L、30mg/L和100mg/L)中培养12h左右细胞作透射电镜观察,结果发现六价铬浓度的增加对菌株Cr-6无明显影响,细胞形态完整。通过细胞不同组分对六价铬的还原发现,未经处理的细胞对六价铬还原效果最好,细胞培养后的上清液对六价铬不存在还原作用,细胞裂解后上清液对细胞还原效果较好,碎片对六价铬还原能力较差,48h仅还原体系中18%的六价铬。在缺乏营养物质的前提下,厌氧条件下细胞对六价铬还原效果最好,有氧条件下次之,在好氧条件下效果最差。在添加不同电子供体实验中,乳酸钠对于提高菌株还原六价铬效果作用最为明显,其次为乙酸钠、葡萄糖和甲醇,对于菌株还原六价铬均有不同程度的促进作用,NADH的添加在实验初期效果明显,然而随着时间的延长,添加NADH的体系中六价铬还原率低于未添加NADH体系中六价铬还原率。在后续选择最佳电子供体浓度实验中,当乳酸钠浓度为2mg/L时,对提高菌株还原六价铬效果促进作用最明显。
采用零级、一级和二级化学反应动力学模型对已有部分实验数据进行拟合,结果发现,在初始六价铬浓度为10mg/L、pH=7、35℃条件下,生长细胞还原六价铬动力学过程采用零级反应动力学模型拟合,相关系数可达0.9899,但对于较高初始六价铬浓度条件下延迟期以及后期高浓度六价铬对细菌还原六价铬的抑制作用的体现还有一定差距。对于在缺乏营养物质条件下,缓冲溶液体系中细胞还原六价铬过程拟合采用一级反应动力学模型较好,相关系数为0.9860,将该模型用于厌氧条件下添加乳酸钠体系中六价铬还原过程仍有较好的拟合效果,相关系数达到0.9979,但是在有氧条件下,一级反应动力学模型对于氧气影响六价铬还原效果的体现并不明显。
(4)电镀污染场地土壤剖而深度范围为0～300cm,每隔20cm取样,共采集土壤样品15个。0～220cm为壤土和粉砂质壤土,220～300cm土壤样品为粉砂质粘壤土,含水率范围为16%～25%,pH值范围为5.55～7.12,表层0～40cm土壤呈现弱酸性。相对于其他深度土壤,0-40cm和140-160cm土壤中有机质含量较高。0～20cm和140～160cm土层中总铬含量相对较高,均超出土壤环境质量标准三级(300mg/kg).0-20cm和120-140cm土层中监测到一定浓度六价铬。铁锰氧化物结合态铬和残渣态铬所占总铬比例范围在10%～80%之间,波动幅度较大。水溶态铬和可交换态铬所占总铬比例低于1％:0-20cm.40-60cm和140～160cm土层中总锌含量相对较高,均超出土壤环境质量标准三级(500mg/kg),铜含量则与背景值相当。水溶态锌和可交换态锌所占比例较小,其他四种形态均占有一定比例,而且随着采样深度的变化,呈现出一定波动。铜在土壤中以残渣态为主,所占总量比例为60%～80%。过氧化氢酶活性和磷酸酶活性在土壤剖面中随深度变化有一定的波动,主要体现在0～20cm和140～160cm土层,脲酶活性变化不明显。通过Pearson相关系数来表征土壤物理化学性质和重金属含量与士壤酶活性的关联程度,结果表明,过氧化氧酶与土壤pH值、有机质含量、总铬、六价铬、有机结合态铬、碳酸盐态锌、铁锰氧化物结合态和有机态锌有一定的相关性,磷酸酶与土壤pH值、有机质含量、总铬、有机结合态铬、碳酸盐态锌、铁锰氧化物结合态和有机态锌有一定的相关性,脲酶仅与总铬和有机结合态铬呈显著正相关(P<0.05)。
(5)根据不同含水率对菌株Cr-6还原土壤中六价铬的影响,在含水率为33%、50%和60%条件下,上壤中六价铬还原率较为接近,另外在空白对照组中未发现六价铬的还原。根据不同接种量对菌株Cr-6还原上壤中六价铬的影响,在菌液接种量为0.1mL、0.5mL和1mL条件下,当接种量为0.1mL时,在6h内还原了土壤中64%六价铬,同时刻接种量为0.5mL的条件下,六价铬还原率已经接近90%,当接种量增加至1mL,六价铬还原率仅提高了5%,而且随着时间的延长,土壤中六价铬还原率基本保持在92%左右。通过对修复后土壤中的铬、锌、铜不同形态含量进行分析,发现在修复后土壤中,水溶态铬基本末检出,铁锰氧化物结合态铬、有机结合态铬和残渣态铬所占比例有所增加,在微生物的作用下,其他形态的铬向残渣态铬转化；碳酸盐结合态锌、残渣态锌和可交换态锌所占总量比例有所降低,铁锰氧化物结合态锌和有机结合态锌所占总量比例增加,在微生物的作用下残渣态锌和有机结合态锌向碳酸盐结合态锌和铁锰氧化物结合态锌转化；残渣态铜和有机结合态铜所占总量比例有所增加,而碳酸盐结合态铜、铁锰氧化物结合态铜和可交换态铜所占总量比例则有所降低,在微生物的作用下,其他形态铜向残渣态铜和有机结合态铜转化。
The electroplating industry played an important role in China's industry, but caused serious environmental pollution, especially soil pollution in plant and surrounding areas. Cr(VI) was an important industrial raw material in electroplating process, whose toxicity had been reported and it had negligible impact on human health. According to previous survey of soil contamination in the electroplating plant, heavy metal pollution was more serious, especially chromium, copper, zinc. Cr(VI) was found at some soil site, which was harmful to human health, so it's very urgent to remediate. This study focused on bioreduction of Cr(VI) and its application in bioremediation of soil contaminated by electroplating. A Cr(VI) reducing bacteria strain Cr-6was isolated from soil. According to its physiological and biochemical characteristics and16S rDNA sequence analysis, bacteria strain Cr-6was identified as Lysinibacillus sp.. It's characteristic, mechanism and kinetic process of Cr(VI) reduction were investigated. Based on the soil analysis of heavy metal and soil enzyme activity in electroplating plant, this study used bacteria strain Cr-6on the bioremediation of Cr(VI) in soil contaminated by electroplating. The main results are as follows:
(1) Cr(VI) reducing bacteria was isolated, purified and identified. Six Cr(VI) removing bacteria strains were isolated and purified from soil with removing efficiency ranging from20%to100%at initial Cr(VI) concentration of10mg/L. This study chose bacteria strain Cr-6as objective bacteria because of its high Cr(VI) removing efficiency. The characteristic analysis of Cr(Ⅵ) reduction by bacteria strain Cr-6showed that there was no chromium absorbed by cell and decrease of Cr(Ⅵ) concentration in culture medium derived from Cr(Ⅵ) bioreduction. According to gram staining, TEM and colony observation on plate, bacreria strain Cr-6was a gram positive bacillus. Colony on plate was round with regular edge and rough surface, and showed creamy yellow color, translucent, moist and had circles at different levels from center to outside, which was no obvious bulge viewed from side. Based on sequence analysis of16S rDNA, morphological observation, physical and biochemical characteristics, bacteria strain Cr-6was identified as Lysinibacillus sp..Growth curve in culture medium with and without Cr(Ⅵ) showed that addition of Cr(Ⅵ) at a certain concentration could promote growth of bacteria and extend the logarithmic phase. Maximum optical density values with and without Cr(Ⅵ) in culture medium were3.67and2.97, and generation time during logarithmic phase were0.87h and1.76h separately.
(2) Different factors affecting Cr(Ⅵ) reduction by bacteria strain Cr-6were investigated. Bacteria strain Cr-6grew best at35℃, and increase of temperature shorted logarithmic phase. Bacteria strain Cr-6showed better Cr(Ⅵ) reducing efficiency at40℃and45℃, and could reduce all the Cr(Ⅵ) in which initial Cr(Ⅵ) concentration was10mg/L. Bacteria strain Cr-6could grow at pH value range of6-9and grew best under pH=7. When pH values were4and5, bacteria strain Cr-6stopped growing, but kept a certain Cr(Ⅵ) reducing efficiency. When pH value was10, bacteria strain Cr-6stopped growing and stopped Cr(Ⅵ) reduction after2h. Bacteria strain Cr-6grew better with quicker rotating speed, and increase of rotating speed could enhance maximum optical density value. Compared with static incubation, Cr(Ⅵ) reducing efficiency was much higher under rotating conditions. Increase of inoculation volume resulted in better bacterial growth and Cr(Ⅵ) reducing efficiency. Bacteria strain Cr-6grew more quickly under aerobic condition than anaerobic condition, but Cr(Ⅵ) efficiency under aerobic condition was lower than anaerobic condition. Bacteria strain Cr-6stopped growing under anaerobic conditions, but also showed a certain Cr(Ⅵ) reducing efficiency. Addition of Zn+at low concentration could promote bioreduction of Cr(Ⅵ), but it showed more obvious inhibition to bioreduction of Cr(Ⅵ) by bacteria strain Cr-6with higher Zn2+concentration. Addition of Cu2+at a certain concentration could promote growth of bacteria strain Cr-6and increase Cr(Ⅵ) reducing efficiency.
(3) TEM observation of cell incubating at different initial Cr(Ⅵ) concentration ranging from Omg/L to100mg/L for12h showed that increase of Cr(Ⅵ) had no obvious affection on bacteria strain Cr-6, and cell could keep integrated shape. Cell without lysate treatment had best Cr(VI) efficiency, supernatant of culture medium after cell incubation couldn't reduce Cr(VI), supernatant of lysate treatment had a certain Cr(VI) reducing efficiency, cell after lysate treatment showed lower Cr(VI) reducing efficiency and reduced18%of Cr(VI) in48h. In buffer solution without nutrient material, bacteria strain Cr-6had best Cr(VI) reducing efficiency under anaerobic condition, and the next was in initial aerobic environment, and the lowest Cr(VI) reducing efficiency was under aerobic condition. Addition of sodium lactate promoted Cr(VI) reducing efficiency most, follow by sodium acetate, glucose and methanol. At the beginning of experiment, addition of NADH promoted Cr(VI) reducing efficiency by bacteria, but Cr(VI) reducing efficiency with addition of NADH became less than Cr(VI) reducing efficiency without NADH with time. Compared with other different concentration of sodium lactate, the concentration of sodium lactate was2mg/L with highest Cr(VI) reducing efficiency.
Existing data were fitted by zero-order, first-order and second-order chemical kinetic model. When the initial Cr(VI) concentration was1Omg/L with pH=7and35℃, data of Cr(VI) reducing by growing cell could be fitted by zero-order kinetic model with correlation coefficients of0.9899, but zero-order kinetic model couldn't reflect lag phase and inhibition of Cr(VI) bioreduction by Cr(VI) of high concentration. Data of Cr(VI) bioreduction in buffer solution could be fitted by one-order kinetic model with correlation coefficients of0.9860. Data of Cr(VI) bioreduction in buffer solution under anaerobic condition with addition of sodium lactate could be fitted by one-order kinetic model with correlation coefficients of0.9979, but under aerobic condition one-order kinetic model couldn't reflect inhibition to Cr(VI) reduction by oxygen.
(4) The depth of electroplating soil profile ranged from0cm to300cm, in which fifteen soil samples were collected for every20cm. Soil samples were mainly loam and silt loam at depth of0-220cm, and were silt clay loam at depth of220-300cm. Moisture content ranged from16%to25%. Range of pH value was5.5-7.1and pH value of surface layer at depth of0-40cm was weakly acid. Organic matter of soil at depth of0-40cm and140-160cm were much higher than other soil samples. Content of total chromium at depth of0-20cm and140-160cm were relatively higher and exceeded grade three of soil environmental quality standard of300mg/kg. Cr(VI) at depth of0-20cm and120-140cm were detected. Proportion of Fe-Mn oxides bound chromium and residual chromium ranged from10%to80%, which showed a little big fluctuation. Proportion of water soluble chromium and exchangeable chromium were less than1%. Contents of zinc at depth of0-20cm,40-60cm and140-160cm were relatively high, which exceeded grade three of soil environmental quality standard of500mg/kg, and content of copper was close to background value. Proportion of water soluble zinc and exchangeable zinc were less than proportion of zinc at other four forms, which fluctuated at different depth. The main form of copper in soil was residual fraction, whose proportion ranged from60%to80%. In the soil profile, catalase activity and phosphatase activity fluctuated with depth mainly at depth of0-20cm and140-160cm, and urease activity didn't change significantly. Pearson correlation coefficient was used to characterize the degree of relevance between soil physical and chemical properties, heavy metal contents and soil enzyme activities. Results showed that soil pH, organic matter content, total chromium, Cr(VI), organic bound chromium, carbonate bound zinc, Fe-Mn oxide bound zinc and organic bound zinc had a certain relevance with catalase activity. Phosphatase activity had a certain relevance with soil pH, organic matter content, total chromium, organic bound chromium, carbonate bound zinc, Fe-Mn oxide bound zinc and organic bound zinc. Urease activity is only related to total chromium and organic bound chromium, which showed a significant positive correlation (P<0.05).
(5) According to impact of moisture content on Cr(VI) reduction in soil by bacteria strain Cr-6, Cr(VI) reducing efficiency in soil were almost the same with moisture contents of33%,50%and60%. In addition, there was no Cr(VI) reduction in blank control without adding bacteria. According to impact of inoculum volume (0.1mL,0.5mL and lmL) on Cr(VI) reduction in soil by bacteria strain Cr-6, when inoculum volume was0.1mL, bacteria strain Cr-6could reduce64%of Cr(VI) in6h, and almost90%of Cr(VI) in soil was reduced with inoculum volume of0.5mL. However when inoculum volume increased to1mL, Cr(VI) reducing efficiency only increased5%. Finally, the Cr(VI) reducing efficiency kept around92%. Different forms of chromium, zinc and copper in soil before and after bioremediation were investigated. After bioremediation, there was almost no water soluble chromium, proportion of Fe-Mn oxide bound chromium, organic bound chromium and residual chromium increased, and other forms of chromium transformed to residual chromium under the influence of bacteria strain Cr-6. Proportion of carbonate bound zinc, residual zinc and exchangeable zinc decreased, proportion of Fe-Mn oxide bound zinc and organic bound zinc increased. Residual zinc and organic bound zinc transformed to carbonate bound zinc and Fe-Mn oxide bound zinc under the influence of bacteria strain Cr-6. Proportion of residual copper and organic bound copper increased, proportion of carbonate bound copper, Fe-Mn oxide bound copper and exchangeable copper decreased. Other forms of copper transformed to residual copper and organic bound copper under the influence of bacteria strain Cr-6.
本研究从上壤中分离得到一株六价铬还原菌Cr-6,根据其生理生化特性和16S rDNA序列分析,鉴定为赖氨酸芽孢杆菌Lysinibacillus sp.,对六价铬还原特性进行研究,探讨其六价铬还原机理,分析其对六价铬还原的动力学过程,在对电镀污染场地进行重金属及上壤酶活性进行分析的基础上,将该菌株应用于电镀污染上壤中六价铬污染的修复,得山主要结论如下：
(1)对六价铬还原菌进行分离纯化并鉴定种属。从土壤中驯化分离纯化得到六株能够在含有一定浓度六价铬的培养基中生长的细菌,在初始六价铬浓度为10mg/L的条件下培养,去除率从20%到100%不等,选取对六价铬处理效果最好的菌株Cr-6作为实验研究对象。对菌株Cr-6进行六价铬作用特性分析,结果表明该菌株不会对六价铬产生吸附作用,培养液中六价铬浓度的降低源于细菌对六价铬的还原作用。对菌株Cr-6分别进行革兰氏染色、透射电镜观察和平板培养,结果表明,该菌株为革兰氏阳性杆菌,在平板上生长菌落呈圆形,从中心向外出现不同层次圆圈,边缘整齐,淡乳黄色,表面粗糙、湿润,半透明,从侧面观察无明显隆起。通过16S rDNA序列分析,并结合菌株形态观察以及生理生化特征结果,判断菌株Cr-6为赖氨酸芽孢杆菌(Lysiniacillus sp.)。分别在添加六价铬和未添加六价铬条件下对细菌进行生长曲线实验,并计算世代时间,结果表明,添加一定浓度六价铬有利于促进细胞生长,延长对数期,最大光密度值分别达到3.67和2.97,对数期内世代时间分别为0.87h和1.76h。
(2)考察不同影响因子对菌株Cr-6还原六价铬的影响。菌株Cr-6在35℃条件下生长情况最好,而且温度的升高将细菌对数期提前。在40℃和45℃条件下,对六价铬的还原效果较好,在6h内还原率达到100%。培养液pH为7时,菌株Cr-6生长情况和对六价铬还原效果最好,生长pH值范围为6-9,在pH值为4和5的时候,停止生长,能够保持-定的六价铬还原效果,在pH值为10的时候,停止生长,2h后停止对六价铬的还原作用。在不同振荡速度下,振荡速度越快,菌株Cr-6生长越好,较快的振荡速度能够提高细菌最大光密度值。相对于静置培养,在振荡条件下还原效果更好。增大接种量可提高同时刻细菌浓度和六价铬还原率。在充气条件下细菌生长迅速,六价铬还原率低于厌氧条件下的六价铬还原率。在厌氧条件下,细菌停止生长,并保持了一定的六价铬还原速率。培养液中Zn2+的添加抑制了菌株Cr-6的生长,Zn2+浓度越高,抑制作用越明显,添加低浓度的Zn2+有利于促进菌株Cr-6对六价铬的还原。添加一定浓度的Cu2+,有利于促进菌株Cr-6的生长,并提高菌株Cr-6对六价铬的还原效果。
(3)对在不同六价铬初始浓度培养基(Omg/L、10mg/L、30mg/L和100mg/L)中培养12h左右细胞作透射电镜观察,结果发现六价铬浓度的增加对菌株Cr-6无明显影响,细胞形态完整。通过细胞不同组分对六价铬的还原发现,未经处理的细胞对六价铬还原效果最好,细胞培养后的上清液对六价铬不存在还原作用,细胞裂解后上清液对细胞还原效果较好,碎片对六价铬还原能力较差,48h仅还原体系中18%的六价铬。在缺乏营养物质的前提下,厌氧条件下细胞对六价铬还原效果最好,有氧条件下次之,在好氧条件下效果最差。在添加不同电子供体实验中,乳酸钠对于提高菌株还原六价铬效果作用最为明显,其次为乙酸钠、葡萄糖和甲醇,对于菌株还原六价铬均有不同程度的促进作用,NADH的添加在实验初期效果明显,然而随着时间的延长,添加NADH的体系中六价铬还原率低于未添加NADH体系中六价铬还原率。在后续选择最佳电子供体浓度实验中,当乳酸钠浓度为2mg/L时,对提高菌株还原六价铬效果促进作用最明显。
采用零级、一级和二级化学反应动力学模型对已有部分实验数据进行拟合,结果发现,在初始六价铬浓度为10mg/L、pH=7、35℃条件下,生长细胞还原六价铬动力学过程采用零级反应动力学模型拟合,相关系数可达0.9899,但对于较高初始六价铬浓度条件下延迟期以及后期高浓度六价铬对细菌还原六价铬的抑制作用的体现还有一定差距。对于在缺乏营养物质条件下,缓冲溶液体系中细胞还原六价铬过程拟合采用一级反应动力学模型较好,相关系数为0.9860,将该模型用于厌氧条件下添加乳酸钠体系中六价铬还原过程仍有较好的拟合效果,相关系数达到0.9979,但是在有氧条件下,一级反应动力学模型对于氧气影响六价铬还原效果的体现并不明显。
(4)电镀污染场地土壤剖而深度范围为0～300cm,每隔20cm取样,共采集土壤样品15个。0～220cm为壤土和粉砂质壤土,220～300cm土壤样品为粉砂质粘壤土,含水率范围为16%～25%,pH值范围为5.55～7.12,表层0～40cm土壤呈现弱酸性。相对于其他深度土壤,0-40cm和140-160cm土壤中有机质含量较高。0～20cm和140～160cm土层中总铬含量相对较高,均超出土壤环境质量标准三级(300mg/kg).0-20cm和120-140cm土层中监测到一定浓度六价铬。铁锰氧化物结合态铬和残渣态铬所占总铬比例范围在10%～80%之间,波动幅度较大。水溶态铬和可交换态铬所占总铬比例低于1％:0-20cm.40-60cm和140～160cm土层中总锌含量相对较高,均超出土壤环境质量标准三级(500mg/kg),铜含量则与背景值相当。水溶态锌和可交换态锌所占比例较小,其他四种形态均占有一定比例,而且随着采样深度的变化,呈现出一定波动。铜在土壤中以残渣态为主,所占总量比例为60%～80%。过氧化氢酶活性和磷酸酶活性在土壤剖面中随深度变化有一定的波动,主要体现在0～20cm和140～160cm土层,脲酶活性变化不明显。通过Pearson相关系数来表征土壤物理化学性质和重金属含量与士壤酶活性的关联程度,结果表明,过氧化氧酶与土壤pH值、有机质含量、总铬、六价铬、有机结合态铬、碳酸盐态锌、铁锰氧化物结合态和有机态锌有一定的相关性,磷酸酶与土壤pH值、有机质含量、总铬、有机结合态铬、碳酸盐态锌、铁锰氧化物结合态和有机态锌有一定的相关性,脲酶仅与总铬和有机结合态铬呈显著正相关(P<0.05)。
(5)根据不同含水率对菌株Cr-6还原土壤中六价铬的影响,在含水率为33%、50%和60%条件下,上壤中六价铬还原率较为接近,另外在空白对照组中未发现六价铬的还原。根据不同接种量对菌株Cr-6还原上壤中六价铬的影响,在菌液接种量为0.1mL、0.5mL和1mL条件下,当接种量为0.1mL时,在6h内还原了土壤中64%六价铬,同时刻接种量为0.5mL的条件下,六价铬还原率已经接近90%,当接种量增加至1mL,六价铬还原率仅提高了5%,而且随着时间的延长,土壤中六价铬还原率基本保持在92%左右。通过对修复后土壤中的铬、锌、铜不同形态含量进行分析,发现在修复后土壤中,水溶态铬基本末检出,铁锰氧化物结合态铬、有机结合态铬和残渣态铬所占比例有所增加,在微生物的作用下,其他形态的铬向残渣态铬转化；碳酸盐结合态锌、残渣态锌和可交换态锌所占总量比例有所降低,铁锰氧化物结合态锌和有机结合态锌所占总量比例增加,在微生物的作用下残渣态锌和有机结合态锌向碳酸盐结合态锌和铁锰氧化物结合态锌转化；残渣态铜和有机结合态铜所占总量比例有所增加,而碳酸盐结合态铜、铁锰氧化物结合态铜和可交换态铜所占总量比例则有所降低,在微生物的作用下,其他形态铜向残渣态铜和有机结合态铜转化。
The electroplating industry played an important role in China's industry, but caused serious environmental pollution, especially soil pollution in plant and surrounding areas. Cr(VI) was an important industrial raw material in electroplating process, whose toxicity had been reported and it had negligible impact on human health. According to previous survey of soil contamination in the electroplating plant, heavy metal pollution was more serious, especially chromium, copper, zinc. Cr(VI) was found at some soil site, which was harmful to human health, so it's very urgent to remediate. This study focused on bioreduction of Cr(VI) and its application in bioremediation of soil contaminated by electroplating. A Cr(VI) reducing bacteria strain Cr-6was isolated from soil. According to its physiological and biochemical characteristics and16S rDNA sequence analysis, bacteria strain Cr-6was identified as Lysinibacillus sp.. It's characteristic, mechanism and kinetic process of Cr(VI) reduction were investigated. Based on the soil analysis of heavy metal and soil enzyme activity in electroplating plant, this study used bacteria strain Cr-6on the bioremediation of Cr(VI) in soil contaminated by electroplating. The main results are as follows:
(1) Cr(VI) reducing bacteria was isolated, purified and identified. Six Cr(VI) removing bacteria strains were isolated and purified from soil with removing efficiency ranging from20%to100%at initial Cr(VI) concentration of10mg/L. This study chose bacteria strain Cr-6as objective bacteria because of its high Cr(VI) removing efficiency. The characteristic analysis of Cr(Ⅵ) reduction by bacteria strain Cr-6showed that there was no chromium absorbed by cell and decrease of Cr(Ⅵ) concentration in culture medium derived from Cr(Ⅵ) bioreduction. According to gram staining, TEM and colony observation on plate, bacreria strain Cr-6was a gram positive bacillus. Colony on plate was round with regular edge and rough surface, and showed creamy yellow color, translucent, moist and had circles at different levels from center to outside, which was no obvious bulge viewed from side. Based on sequence analysis of16S rDNA, morphological observation, physical and biochemical characteristics, bacteria strain Cr-6was identified as Lysinibacillus sp..Growth curve in culture medium with and without Cr(Ⅵ) showed that addition of Cr(Ⅵ) at a certain concentration could promote growth of bacteria and extend the logarithmic phase. Maximum optical density values with and without Cr(Ⅵ) in culture medium were3.67and2.97, and generation time during logarithmic phase were0.87h and1.76h separately.
(2) Different factors affecting Cr(Ⅵ) reduction by bacteria strain Cr-6were investigated. Bacteria strain Cr-6grew best at35℃, and increase of temperature shorted logarithmic phase. Bacteria strain Cr-6showed better Cr(Ⅵ) reducing efficiency at40℃and45℃, and could reduce all the Cr(Ⅵ) in which initial Cr(Ⅵ) concentration was10mg/L. Bacteria strain Cr-6could grow at pH value range of6-9and grew best under pH=7. When pH values were4and5, bacteria strain Cr-6stopped growing, but kept a certain Cr(Ⅵ) reducing efficiency. When pH value was10, bacteria strain Cr-6stopped growing and stopped Cr(Ⅵ) reduction after2h. Bacteria strain Cr-6grew better with quicker rotating speed, and increase of rotating speed could enhance maximum optical density value. Compared with static incubation, Cr(Ⅵ) reducing efficiency was much higher under rotating conditions. Increase of inoculation volume resulted in better bacterial growth and Cr(Ⅵ) reducing efficiency. Bacteria strain Cr-6grew more quickly under aerobic condition than anaerobic condition, but Cr(Ⅵ) efficiency under aerobic condition was lower than anaerobic condition. Bacteria strain Cr-6stopped growing under anaerobic conditions, but also showed a certain Cr(Ⅵ) reducing efficiency. Addition of Zn+at low concentration could promote bioreduction of Cr(Ⅵ), but it showed more obvious inhibition to bioreduction of Cr(Ⅵ) by bacteria strain Cr-6with higher Zn2+concentration. Addition of Cu2+at a certain concentration could promote growth of bacteria strain Cr-6and increase Cr(Ⅵ) reducing efficiency.
(3) TEM observation of cell incubating at different initial Cr(Ⅵ) concentration ranging from Omg/L to100mg/L for12h showed that increase of Cr(Ⅵ) had no obvious affection on bacteria strain Cr-6, and cell could keep integrated shape. Cell without lysate treatment had best Cr(VI) efficiency, supernatant of culture medium after cell incubation couldn't reduce Cr(VI), supernatant of lysate treatment had a certain Cr(VI) reducing efficiency, cell after lysate treatment showed lower Cr(VI) reducing efficiency and reduced18%of Cr(VI) in48h. In buffer solution without nutrient material, bacteria strain Cr-6had best Cr(VI) reducing efficiency under anaerobic condition, and the next was in initial aerobic environment, and the lowest Cr(VI) reducing efficiency was under aerobic condition. Addition of sodium lactate promoted Cr(VI) reducing efficiency most, follow by sodium acetate, glucose and methanol. At the beginning of experiment, addition of NADH promoted Cr(VI) reducing efficiency by bacteria, but Cr(VI) reducing efficiency with addition of NADH became less than Cr(VI) reducing efficiency without NADH with time. Compared with other different concentration of sodium lactate, the concentration of sodium lactate was2mg/L with highest Cr(VI) reducing efficiency.
Existing data were fitted by zero-order, first-order and second-order chemical kinetic model. When the initial Cr(VI) concentration was1Omg/L with pH=7and35℃, data of Cr(VI) reducing by growing cell could be fitted by zero-order kinetic model with correlation coefficients of0.9899, but zero-order kinetic model couldn't reflect lag phase and inhibition of Cr(VI) bioreduction by Cr(VI) of high concentration. Data of Cr(VI) bioreduction in buffer solution could be fitted by one-order kinetic model with correlation coefficients of0.9860. Data of Cr(VI) bioreduction in buffer solution under anaerobic condition with addition of sodium lactate could be fitted by one-order kinetic model with correlation coefficients of0.9979, but under aerobic condition one-order kinetic model couldn't reflect inhibition to Cr(VI) reduction by oxygen.
(4) The depth of electroplating soil profile ranged from0cm to300cm, in which fifteen soil samples were collected for every20cm. Soil samples were mainly loam and silt loam at depth of0-220cm, and were silt clay loam at depth of220-300cm. Moisture content ranged from16%to25%. Range of pH value was5.5-7.1and pH value of surface layer at depth of0-40cm was weakly acid. Organic matter of soil at depth of0-40cm and140-160cm were much higher than other soil samples. Content of total chromium at depth of0-20cm and140-160cm were relatively higher and exceeded grade three of soil environmental quality standard of300mg/kg. Cr(VI) at depth of0-20cm and120-140cm were detected. Proportion of Fe-Mn oxides bound chromium and residual chromium ranged from10%to80%, which showed a little big fluctuation. Proportion of water soluble chromium and exchangeable chromium were less than1%. Contents of zinc at depth of0-20cm,40-60cm and140-160cm were relatively high, which exceeded grade three of soil environmental quality standard of500mg/kg, and content of copper was close to background value. Proportion of water soluble zinc and exchangeable zinc were less than proportion of zinc at other four forms, which fluctuated at different depth. The main form of copper in soil was residual fraction, whose proportion ranged from60%to80%. In the soil profile, catalase activity and phosphatase activity fluctuated with depth mainly at depth of0-20cm and140-160cm, and urease activity didn't change significantly. Pearson correlation coefficient was used to characterize the degree of relevance between soil physical and chemical properties, heavy metal contents and soil enzyme activities. Results showed that soil pH, organic matter content, total chromium, Cr(VI), organic bound chromium, carbonate bound zinc, Fe-Mn oxide bound zinc and organic bound zinc had a certain relevance with catalase activity. Phosphatase activity had a certain relevance with soil pH, organic matter content, total chromium, organic bound chromium, carbonate bound zinc, Fe-Mn oxide bound zinc and organic bound zinc. Urease activity is only related to total chromium and organic bound chromium, which showed a significant positive correlation (P<0.05).
(5) According to impact of moisture content on Cr(VI) reduction in soil by bacteria strain Cr-6, Cr(VI) reducing efficiency in soil were almost the same with moisture contents of33%,50%and60%. In addition, there was no Cr(VI) reduction in blank control without adding bacteria. According to impact of inoculum volume (0.1mL,0.5mL and lmL) on Cr(VI) reduction in soil by bacteria strain Cr-6, when inoculum volume was0.1mL, bacteria strain Cr-6could reduce64%of Cr(VI) in6h, and almost90%of Cr(VI) in soil was reduced with inoculum volume of0.5mL. However when inoculum volume increased to1mL, Cr(VI) reducing efficiency only increased5%. Finally, the Cr(VI) reducing efficiency kept around92%. Different forms of chromium, zinc and copper in soil before and after bioremediation were investigated. After bioremediation, there was almost no water soluble chromium, proportion of Fe-Mn oxide bound chromium, organic bound chromium and residual chromium increased, and other forms of chromium transformed to residual chromium under the influence of bacteria strain Cr-6. Proportion of carbonate bound zinc, residual zinc and exchangeable zinc decreased, proportion of Fe-Mn oxide bound zinc and organic bound zinc increased. Residual zinc and organic bound zinc transformed to carbonate bound zinc and Fe-Mn oxide bound zinc under the influence of bacteria strain Cr-6. Proportion of residual copper and organic bound copper increased, proportion of carbonate bound copper, Fe-Mn oxide bound copper and exchangeable copper decreased. Other forms of copper transformed to residual copper and organic bound copper under the influence of bacteria strain Cr-6.
引文
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