变形假单胞菌吸附镉的机制及其吸附条件的研究
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摘要
本文概述了水体重金属污染的危害及污染现状,描述了现阶段重金属废水的主要处理方法,并介绍了生物吸附剂的种类和来源、生物吸附的影响因素、生物吸附的机理及生物吸附剂的应用等方面的研究进展。
由于长期的环境选择及适应进化,处在重金属污染环境中的微生物对重金属离子都具有一定的抗性。因此,利用这一特性,实现微生物对重金属废水的净化及对水体重金属污染的修复已成为环境保护领域的一个研究热点。本课题分离筛选到一株抗镉变形假单胞菌C-18,对该菌的抗镉机制及影响其吸附镉离子的各种理化因素,开展了系统的基础研究,主要包括以下工作：
1.采用含Cd(Ⅱ)的选择性培养基成功地从16株抗汞菌株中分离筛选出一株具有较强抗镉能力的细菌,在含Cd(Ⅱ)浓度为900mg/L的液体培养基中和Cd(Ⅱ)浓度1600mg/L的平板上能够正常生长。从形态特征、生理生化特征及分子生物学方面对该菌进行了分类鉴定,初步鉴定为变形假单菌,并构建了系统发育树。
2.通过对培养基的成分进行优化,采用糖蜜和酵母膏为主要营养,培养24h,变形假单胞菌C-18生物量OD600可达4.8左右。
3.以该菌株做为生物吸附剂,研究了理化因素对该菌吸附Cd(Ⅱ)的影响,并通过正交实验优化确定了最佳吸附条件。在吸附剂浓度1.2g/L、Cd(II)初始浓度100mg/L、菌龄24h、pH7.0、温度30℃、转速200r/min的条件下,吸附12h,对Cd(Ⅱ)的吸附率和吸附量分别达到88.8%和74mg/g。
4.吸附Cd(Ⅱ)的C-18菌体采用0.1mol/LHC1解吸30min,解吸率在80%以上
5.变形假单胞菌C-18对Cd(Ⅱ)的吸附可分为快速吸附和缓慢吸附二个阶段,在吸附过程的前30min时,吸附率可达总吸附率的90%左右,吸附12h达到平衡。该菌对Cd(Ⅱ)的吸附模型符合Langmuir和Freundlich等温吸附模型。吸附Cd(Ⅱ)的动力学模型与假二级动力学模型吻合。
6.SEM显示吸附Cd(Ⅱ)后的C-18菌体细胞表面粗糙有褶皱,可见沉积物。吸附Cd(Ⅱ)前后菌体的红外光谱(FTIR)分析表明,C-18菌体表面的活性基团参与Cd(Ⅱ)的络合反应。电感耦合等离子发射光谱仪分析C-18吸附Cd(Ⅱ)后的菌体消解液和上清液中Cd(Ⅱ)浓度,发现两者浓度之和接近于溶液初始Cd(Ⅱ)浓度。比较死菌吸附剂和活菌吸附剂对Cd(Ⅱ)的吸附效果,结果显示,二者的吸附率相当。均说明变形假单胞菌C-18对Cd(Ⅱ)的吸附机制属于被动吸附,是通过细胞壁上官能团的络合作用将Cd(Ⅱ)沉积在细胞表面。
This paper summarized the harmfulness and the present situation of water heavy metal pollution. The main technology for treatments of wastewater containing heavy metal at this stage was described. The types and sources of biosorbent, the factors of affecting on the heavy metal adsorption, the mechanism of adsorption, the biosorption mechanism and the application of biosorbent were aslo introduced.
As the long-term environmental selection and adaptive evolution, microorganisms grown in the environment of heavy metal pollution are able to tolerate heavy metals. Therefore, It has become a hot research topic of environmental protection that to purify the heavy metal wastewater and to repair the heavy metal pollution by taking use of the remarkable property of microorganisms.One strain named Pseudomonas plecoglossicida C-18 with great efficiency of cadmium resistance was successfully screened in our study. The basic research was carried out systematically on the bacteria anti-cadmium mechanism and various physical and chemical factors affecting biosorption on Cd(Ⅱ).The main content of this study are as follows:
1.A stain with strong ability to resistance cadmium was successfully isolated from 16 anti-mercury stains by the selected medium with Cd(Ⅱ).The strain could grow normally in the Cd (Ⅱ) concentration of 900mg/L of liquid medium and Cd(Ⅱ) concentration of 1600mg/L Cd(Ⅱ) of the plate respectively.Cell morphology and structure,physiology, biochemistry and molecular biology of the bacteria were classified. The strain C-18 was initially identified as Pseudomonas plecoglossicida and the phylogenetic tree was structured.
2.By optimizing the medium composition, using the molasses and yeast extract as the main nutrition, under training of 24h, the value of OD600 stands for the biomass of strain C-18 reached to about 4.8.
3.The strain C-18 served as biosorbent to study the impact of the physical and chemical factors on bacteria adsorbed Cd (Ⅱ) and verify the optimum adsorption conditions by optimizing orthogonal experiments.Nurturing by the condition of Concentration in the adsorbent 1.2g/L, Cd (Ⅱ) the initial concentration of 100mg/L, strain age 24h, pH7.0, temperature 30℃, speed 200r/min conditions, the adsorption 12h, the Cd (Ⅱ) adsorption rate and adsorption capacity achieved respectively 88.8%,and 74mg/g.
4.By Adsorption of Cd (Ⅱ) in the C-18 cell with O.lmol/LHC1 desorption 30min, The desorption rate exceeded 80%.
5.Pseudomonas C-18 with Cd (Ⅱ) adsorpted can be divided into fast adsorption and slow adsorption stage, in the first 30min of the adsorption process, the adsorption rate could reach 90%of the total absorption rate, and the adsorption achieved balance within 12h.Pseudomonas C-18 on the Cd (Ⅱ) Langmuir adsorption model and Freundlich isotherm consistent with adsorption model. The dynamic model of adsorption of Cd (Ⅱ) consistent with the pseudo-second order kinetic model.
6. Bacterial cells of Pseudomonas C-18 with adsorpted Cd (Ⅱ) appeared rough surface with folds mixed up with sediment can be showed by SEM. Adsorption of Cd (Ⅱ) bacteria before and after FTIR analysis showed that, hydroxyl, amide, carboxyl on C-18 cell surface and other active groups involved in Cd (Ⅱ) in the complex reaction. Inductively coupled plasma emission spectrometry (ICP) analysed the concentration in the digestion solution and the supernatant cell after Pseudomonas C-18 adsorbed Cd (Ⅱ), which was found the total concentration is equal to the solution concentration of Cd (Ⅱ). The results of Comparing dead cells and viable adsorbent adsorbent Cd (Ⅱ) adsorption effects shows that both the adsorption rate is equivalent. Which indicates the adsorption of Cd (Ⅱ) by Pseudomonas C-18 belongs to passive adsorption, as a result of Cd (Ⅱ) deposited on the cell surface through the complexation of functional groups on the cell surface.
由于长期的环境选择及适应进化,处在重金属污染环境中的微生物对重金属离子都具有一定的抗性。因此,利用这一特性,实现微生物对重金属废水的净化及对水体重金属污染的修复已成为环境保护领域的一个研究热点。本课题分离筛选到一株抗镉变形假单胞菌C-18,对该菌的抗镉机制及影响其吸附镉离子的各种理化因素,开展了系统的基础研究,主要包括以下工作：
1.采用含Cd(Ⅱ)的选择性培养基成功地从16株抗汞菌株中分离筛选出一株具有较强抗镉能力的细菌,在含Cd(Ⅱ)浓度为900mg/L的液体培养基中和Cd(Ⅱ)浓度1600mg/L的平板上能够正常生长。从形态特征、生理生化特征及分子生物学方面对该菌进行了分类鉴定,初步鉴定为变形假单菌,并构建了系统发育树。
2.通过对培养基的成分进行优化,采用糖蜜和酵母膏为主要营养,培养24h,变形假单胞菌C-18生物量OD600可达4.8左右。
3.以该菌株做为生物吸附剂,研究了理化因素对该菌吸附Cd(Ⅱ)的影响,并通过正交实验优化确定了最佳吸附条件。在吸附剂浓度1.2g/L、Cd(II)初始浓度100mg/L、菌龄24h、pH7.0、温度30℃、转速200r/min的条件下,吸附12h,对Cd(Ⅱ)的吸附率和吸附量分别达到88.8%和74mg/g。
4.吸附Cd(Ⅱ)的C-18菌体采用0.1mol/LHC1解吸30min,解吸率在80%以上
5.变形假单胞菌C-18对Cd(Ⅱ)的吸附可分为快速吸附和缓慢吸附二个阶段,在吸附过程的前30min时,吸附率可达总吸附率的90%左右,吸附12h达到平衡。该菌对Cd(Ⅱ)的吸附模型符合Langmuir和Freundlich等温吸附模型。吸附Cd(Ⅱ)的动力学模型与假二级动力学模型吻合。
6.SEM显示吸附Cd(Ⅱ)后的C-18菌体细胞表面粗糙有褶皱,可见沉积物。吸附Cd(Ⅱ)前后菌体的红外光谱(FTIR)分析表明,C-18菌体表面的活性基团参与Cd(Ⅱ)的络合反应。电感耦合等离子发射光谱仪分析C-18吸附Cd(Ⅱ)后的菌体消解液和上清液中Cd(Ⅱ)浓度,发现两者浓度之和接近于溶液初始Cd(Ⅱ)浓度。比较死菌吸附剂和活菌吸附剂对Cd(Ⅱ)的吸附效果,结果显示,二者的吸附率相当。均说明变形假单胞菌C-18对Cd(Ⅱ)的吸附机制属于被动吸附,是通过细胞壁上官能团的络合作用将Cd(Ⅱ)沉积在细胞表面。
This paper summarized the harmfulness and the present situation of water heavy metal pollution. The main technology for treatments of wastewater containing heavy metal at this stage was described. The types and sources of biosorbent, the factors of affecting on the heavy metal adsorption, the mechanism of adsorption, the biosorption mechanism and the application of biosorbent were aslo introduced.
As the long-term environmental selection and adaptive evolution, microorganisms grown in the environment of heavy metal pollution are able to tolerate heavy metals. Therefore, It has become a hot research topic of environmental protection that to purify the heavy metal wastewater and to repair the heavy metal pollution by taking use of the remarkable property of microorganisms.One strain named Pseudomonas plecoglossicida C-18 with great efficiency of cadmium resistance was successfully screened in our study. The basic research was carried out systematically on the bacteria anti-cadmium mechanism and various physical and chemical factors affecting biosorption on Cd(Ⅱ).The main content of this study are as follows:
1.A stain with strong ability to resistance cadmium was successfully isolated from 16 anti-mercury stains by the selected medium with Cd(Ⅱ).The strain could grow normally in the Cd (Ⅱ) concentration of 900mg/L of liquid medium and Cd(Ⅱ) concentration of 1600mg/L Cd(Ⅱ) of the plate respectively.Cell morphology and structure,physiology, biochemistry and molecular biology of the bacteria were classified. The strain C-18 was initially identified as Pseudomonas plecoglossicida and the phylogenetic tree was structured.
2.By optimizing the medium composition, using the molasses and yeast extract as the main nutrition, under training of 24h, the value of OD600 stands for the biomass of strain C-18 reached to about 4.8.
3.The strain C-18 served as biosorbent to study the impact of the physical and chemical factors on bacteria adsorbed Cd (Ⅱ) and verify the optimum adsorption conditions by optimizing orthogonal experiments.Nurturing by the condition of Concentration in the adsorbent 1.2g/L, Cd (Ⅱ) the initial concentration of 100mg/L, strain age 24h, pH7.0, temperature 30℃, speed 200r/min conditions, the adsorption 12h, the Cd (Ⅱ) adsorption rate and adsorption capacity achieved respectively 88.8%,and 74mg/g.
4.By Adsorption of Cd (Ⅱ) in the C-18 cell with O.lmol/LHC1 desorption 30min, The desorption rate exceeded 80%.
5.Pseudomonas C-18 with Cd (Ⅱ) adsorpted can be divided into fast adsorption and slow adsorption stage, in the first 30min of the adsorption process, the adsorption rate could reach 90%of the total absorption rate, and the adsorption achieved balance within 12h.Pseudomonas C-18 on the Cd (Ⅱ) Langmuir adsorption model and Freundlich isotherm consistent with adsorption model. The dynamic model of adsorption of Cd (Ⅱ) consistent with the pseudo-second order kinetic model.
6. Bacterial cells of Pseudomonas C-18 with adsorpted Cd (Ⅱ) appeared rough surface with folds mixed up with sediment can be showed by SEM. Adsorption of Cd (Ⅱ) bacteria before and after FTIR analysis showed that, hydroxyl, amide, carboxyl on C-18 cell surface and other active groups involved in Cd (Ⅱ) in the complex reaction. Inductively coupled plasma emission spectrometry (ICP) analysed the concentration in the digestion solution and the supernatant cell after Pseudomonas C-18 adsorbed Cd (Ⅱ), which was found the total concentration is equal to the solution concentration of Cd (Ⅱ). The results of Comparing dead cells and viable adsorbent adsorbent Cd (Ⅱ) adsorption effects shows that both the adsorption rate is equivalent. Which indicates the adsorption of Cd (Ⅱ) by Pseudomonas C-18 belongs to passive adsorption, as a result of Cd (Ⅱ) deposited on the cell surface through the complexation of functional groups on the cell surface.
引文
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