驯化大肠杆菌对镍离子的去除及三种细菌对染料脱色行为研究
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摘要
环境中重金属离子的污染较为隐蔽,不能被降解;而印染废水由于可生化性差、成分复杂、色度大、COD高,是目前对人类威胁最大的两大污染物。传统物理化学治理方法费用高、能耗大、易造成二次污染。目前,利用死菌体的生物吸附已成功处理了多种金属,但对Ni~(2+)吸附率普遍较低,而活细菌能将吸附在表面的金属或染料运送至胞内或释放降解酶,对其进行蓄积转化和降解。
论文以环境中常见细菌埃希氏大肠杆菌为对象,研究了培养基中Ni~(2+)浓度对大肠杆菌的生长周期及吸附行为的影响。在Ni~(2+)的培养基中对大肠杆菌进行三代驯化,通过分析驯化前后大肠杆菌的质粒DNA图谱,并采用红外光谱,透射电子显微镜、X射线光电子能谱、光学显微镜等仪器,对驯化过程中大肠杆菌的形貌及表面性质进行表征,探讨了驯化的初步机理。选择驯化效果较好的菌种,对镍离子模拟水样做去除实验,并将驯化前后大肠杆菌的吸附性能进行对比,实验证明吸附性能有所改善。此外,采用三种细菌对染料进行脱色,探讨用微生物对染料进行预处理的可行性。具体研究内容如下:
(1)初始驯化镍离子浓度的选择
大肠杆菌对镍离子的应急反应是生化反应过程,它与培养基中Ni~(2+)的浓度密切相关,研究了培养基中Ni~(2+)浓度对大肠杆菌生长及吸附行为的影响,在Ni~(2+)培养基中,Ni~(2+)对大肠杆菌的抑制作用随其浓度升高而增强。大肠杆菌对Ni~(2+)的吸附量随细菌数量增加而升高,在稳定生长期达到最大。用红外光谱对大肠杆菌的表面官能团进行分析,推测大肠杆菌对Ni~(2+)的吸附以-OH,-COOH等表面的官能团为主,可能与蛋白质也有关。XPS分析了细菌表面O、N、S、P等元素的变化,推测蛋白质等生物的大分子参与了Ni~(2+)的吸附过程。TEM观察到Ni~(2+)引起大肠杆菌的形貌变化,镍离子的毒性也使细胞壁脆性增加。当培养基中Ni~(2+)浓度为30 mg·L~(-1)时,大肠杆菌对Ni~(2+)离子的吸附率较高,因此,选该浓度下培养的大肠杆菌,作后续驯化的菌种。
(2)大肠杆菌的应急驯化及其机理初探
大肠杆菌经三代驯化,发现驯化后大肠杆菌在镍离子培养基中的适应力增强,生长速度与驯化前后Ni~(2+)浓度梯度有关,梯度越小,大肠杆菌的生长越快。驯化菌的吸附性能也增强,吸附行为与驯化的Ni~(2+)浓度有关,第一代驯化的大肠杆菌对Ni~(2+)的最大吸附量出现在稳定期(30mg·L~(-1)、100mg·L~(-1)),分别将其接入Ni~(2+)浓度为20mg·L~(-1)和150mg·L~(-1)的培养基中进行第二代驯化,在前一浓度中驯化的大肠杆菌(大肠杆菌ⅡC、D)对Ni~(2+)的吸附出现两个峰,分别在对数生长期和稳定生长期,而后者(大肠杆菌ⅡE、F)只在对数生长期出现一个峰。将两者分别接入300mg·L~(-1)的Ni~(2+)培养基中继续进行第三代驯化,从20mg·L~(-1)的Ni~(2+)培养基中转接的菌株(大肠杆菌ⅢG)也出现两个峰,而在150mg·L~(-1)转接的(大肠杆菌ⅢH)却只有一个峰。FTIR分析了吸附过程中表面官能团的作用,除-OH峰外,酰胺峰也发生了较大偏移,此外,多糖的C-O峰也有偏移,这些生物大分子都可与镍离子结合。用光学显微镜,TEM分别观察驯化细胞的形貌变化,随驯化的Ni~(2+)浓度升高,细胞胞膜局部裂解,同时发生聚集现象。碱法裂解大肠杆菌提取质粒DNA,电泳结果显示,大肠杆菌细胞有质粒,但驯化前后质粒无变化。
(3)驯化大肠杆菌对镍离子的去除效果
为评价驯化菌的吸附性能,采集天然水体,不加营养物质,经过滤灭菌后加镍离子制备模拟水样,选用吸附效果最好的大肠杆菌ⅢG(0-30-20~(-1)50 mg·L~(-1)),去除模拟水样中的Ni2(+<10 mg·L~(-1))。驯化菌对镍离子的耐性增加,它们在5 mg·L~(-1)和10 mg·L~(-1)的水样中的浊度(OD)值约为未驯化菌的3倍。在处理低浓度的Ni~(2+)水样时,驯化菌的去除率有较大提高,当水样中Ni~(2+)浓度为1 mg·L~(-1)时,驯化菌对镍离子的去除率是未驯化菌的3.16倍。红外图谱表明,更多的表面官能团参与了镍离子的去除过程。
(4 )大肠杆菌、金黄色葡萄球菌及枯草芽孢杆菌对日落黄、亚甲基蓝的脱色作用研究
以阴离子染料(日落黄)和阳离子染料(亚甲基蓝)为研究对象,探讨不同细菌对染料的脱色行为。该染料在pH2.0-9.0范围内吸光度变化小,稳定性较好。脱色实验表明,细菌对染料的脱色包括吸附和降解两个过程,细菌对染料的吸附在稳定期最强,但在降解过程中,降解酶需经合成、分泌及酶解,导致了细菌对染料的最高脱色率不在稳定期,而在衰亡期。不同的细菌对两种染料的脱色效果有较大差异。大肠杆菌对亚甲基蓝的脱色率最高达60%,金黄色葡萄球菌对日落黄的脱色率最高为56%。
Heavy-metal pollutants and dye wastewater have become the most serious problem through out the world.Metal ions are invisible and can’t be degraded, while dyes usually have complex aromatic molecular structure, which make the wastewater of high COD brilliance and intensity colors and low biodegradability. They are difficult to disposal by conventional physical or chemical treatment process mainly becauce of it’s expensiveness, energy-costly and secondary pollution. In recent years, numerous metal ions have been treated by non-living biomass very well, except nickel ion. Researchers have found that the metals biosorbed on the surface can be biotransformed after enter into the cell, and dyes will be biodegraded by the enzyme that excreted by living microoganism.
Mechanism of Escherichia coli domestication was discussed in this paper. The bacteria was domesticated for three times in the medium by increasing the Ni~(2+) concentration gradullay. Morphological and surface characteristics of E.coli were anlysized by infra-red spectrum, X-ray electron spectra, transmission electron microscopy and light microscopy, meanwhile, plasmid was also anlysized in these processes. Domesticated bacteria that of higher Ni~(2+) accumulation capacity was used to deal with the simulation water, compared to common strain, the accumulation capability turn out to be better. At the same time, three kinds of bacteria were used to decolorize dyes, and to explore the feasibility of microoganism in dye wastewater pretreatment. In this paper we have done the works as follows:
1. Selecting the initial concentration of nickel ions for domestication processes When incubated in the Ni~(2+) environment, The emergency response of E.coli was a biochemical process, which was closely related to the concentration of Ni~(2+). Therefore, the effect of concentration of Ni~(2+) on the behaviors of growth and accumulation were studied. The results showed that growth of E.coli was drastically inhibited by the increasing concentration of Ni~(2+). Accmulation capacity was enhanced with the biomass increasd, the maximal Ni~(2+) accumulation capacity occurred in the stationary phase. Results of FT-IR showed that, the Ni~(2+) accumulation mainly depend on the functional groups on the surface of biomass, sunch as–OH, -COOH, maybe protein was also involved in this process. From XPS spectra, we can draw a conclusion that the protein was certainly participated in Ni~(2+) accumulation. TEM observed that the biomorph of cells were changed and the cell wall became frangible after Ni~(2+) accumulation. The maximal accumulation rate of Ni~(2+) was obtained at the concentration of 30 mg l~(-1), it is selected as the strain for the later domestication process.
2.The primary mechanism of emergency response in domestication process
After domesticated for three times, E.coli can quickly acclimatize itself to Ni~(2+) medium, and the growth rate was controlled by the Ni~(2+) concentration, the propagated speed of the bacteria increased with the decreasing of the Ni~(2+) concentration gradients. The accumulation capability of domesticated bacteria, which related to the concentration of Ni~(2+) was also advanced. The time for maximal capacity of E.coliⅠwas at the stionary phase. There are two kinds of E.coliⅡ, one was incubated at the concentration of 20 mg·L~(-1), while another at 150mg·L~(-1), for the former one(E.coliⅡC,D), there were two periods of maximal capacity at log phase and stionary phase, repectively, and the latter (E.coliⅡE,F) it was at log phase. E.coliⅡwere domesticated for the third time at the concentration of 300mg·L~(-1). For E.coliⅢ, the periods of maximal capacity were the same to their strains. FT-IR anlysis showed that, besides the peaks of hydroxyl, peaks of amide groups shifted obviously and the peaks of C-O for amylose shifted, as well. All of these biomacromolecules can biosorbe the Ni~(2+) by combining with it. Light microscope and TEM were applied to investigated the microstrueture of cells, and observed that cells were destroyed and fissioned with the Ni~(2+) increased during domestication, furthermore, the cells aggregated together. Plasmid was prepared by NaOH method, and the profiles showed that there was only one plasmid in the E.coli, and it didn’t changed in domestication process.
3.The removal efficiency of Ni~(2+) with domesticated bacteria
Domesticated bacteria that of better Ni~(2+) accumulation capacity was used to deal with the simulation water, the concentration of Ni~(2+) in the sample was in the range of 0 ~ 10 mg·L~(-1). the tolerance of bacteria to Ni~(2+) became improvement after domestication. At the concentration of 5 mg·L~(-1) and10 mg·L~(-1), the OD600 for the domesticated E.coli was about three-fold of common one. Removal rate also improved more than 3 times. FT-IR spectra showed that more functional groups had participated in Ni~(2+) accumulation in the sample.
4. Study on the decolorization of dyes with three kinds of bacteria
Decolorization behaviors of bacteria on Sunset yellow and Methylene blue were investigated. When the pH was in the range of 2.0-9.0, the absorbance varied slightly. Decolorization experiment showed that the decolorization process of bacteria includes two steps, adsorption and degradation. In the first step, the maximal adsorption rate which depended on the numerous biomass was at the stionary phase, while in the next one, due to the synthesis, excretion and dagragation of enzyme, the maximal decolorization rate was delayed to the death phase. The decolorization rate of three kinds of bacteria for the dyes were different. The E.coli can remove 60% of the colour of Methylene blue, and, decolorization rate of sunset yellow by Staphylococcus aureus was 56%.
论文以环境中常见细菌埃希氏大肠杆菌为对象,研究了培养基中Ni~(2+)浓度对大肠杆菌的生长周期及吸附行为的影响。在Ni~(2+)的培养基中对大肠杆菌进行三代驯化,通过分析驯化前后大肠杆菌的质粒DNA图谱,并采用红外光谱,透射电子显微镜、X射线光电子能谱、光学显微镜等仪器,对驯化过程中大肠杆菌的形貌及表面性质进行表征,探讨了驯化的初步机理。选择驯化效果较好的菌种,对镍离子模拟水样做去除实验,并将驯化前后大肠杆菌的吸附性能进行对比,实验证明吸附性能有所改善。此外,采用三种细菌对染料进行脱色,探讨用微生物对染料进行预处理的可行性。具体研究内容如下:
(1)初始驯化镍离子浓度的选择
大肠杆菌对镍离子的应急反应是生化反应过程,它与培养基中Ni~(2+)的浓度密切相关,研究了培养基中Ni~(2+)浓度对大肠杆菌生长及吸附行为的影响,在Ni~(2+)培养基中,Ni~(2+)对大肠杆菌的抑制作用随其浓度升高而增强。大肠杆菌对Ni~(2+)的吸附量随细菌数量增加而升高,在稳定生长期达到最大。用红外光谱对大肠杆菌的表面官能团进行分析,推测大肠杆菌对Ni~(2+)的吸附以-OH,-COOH等表面的官能团为主,可能与蛋白质也有关。XPS分析了细菌表面O、N、S、P等元素的变化,推测蛋白质等生物的大分子参与了Ni~(2+)的吸附过程。TEM观察到Ni~(2+)引起大肠杆菌的形貌变化,镍离子的毒性也使细胞壁脆性增加。当培养基中Ni~(2+)浓度为30 mg·L~(-1)时,大肠杆菌对Ni~(2+)离子的吸附率较高,因此,选该浓度下培养的大肠杆菌,作后续驯化的菌种。
(2)大肠杆菌的应急驯化及其机理初探
大肠杆菌经三代驯化,发现驯化后大肠杆菌在镍离子培养基中的适应力增强,生长速度与驯化前后Ni~(2+)浓度梯度有关,梯度越小,大肠杆菌的生长越快。驯化菌的吸附性能也增强,吸附行为与驯化的Ni~(2+)浓度有关,第一代驯化的大肠杆菌对Ni~(2+)的最大吸附量出现在稳定期(30mg·L~(-1)、100mg·L~(-1)),分别将其接入Ni~(2+)浓度为20mg·L~(-1)和150mg·L~(-1)的培养基中进行第二代驯化,在前一浓度中驯化的大肠杆菌(大肠杆菌ⅡC、D)对Ni~(2+)的吸附出现两个峰,分别在对数生长期和稳定生长期,而后者(大肠杆菌ⅡE、F)只在对数生长期出现一个峰。将两者分别接入300mg·L~(-1)的Ni~(2+)培养基中继续进行第三代驯化,从20mg·L~(-1)的Ni~(2+)培养基中转接的菌株(大肠杆菌ⅢG)也出现两个峰,而在150mg·L~(-1)转接的(大肠杆菌ⅢH)却只有一个峰。FTIR分析了吸附过程中表面官能团的作用,除-OH峰外,酰胺峰也发生了较大偏移,此外,多糖的C-O峰也有偏移,这些生物大分子都可与镍离子结合。用光学显微镜,TEM分别观察驯化细胞的形貌变化,随驯化的Ni~(2+)浓度升高,细胞胞膜局部裂解,同时发生聚集现象。碱法裂解大肠杆菌提取质粒DNA,电泳结果显示,大肠杆菌细胞有质粒,但驯化前后质粒无变化。
(3)驯化大肠杆菌对镍离子的去除效果
为评价驯化菌的吸附性能,采集天然水体,不加营养物质,经过滤灭菌后加镍离子制备模拟水样,选用吸附效果最好的大肠杆菌ⅢG(0-30-20~(-1)50 mg·L~(-1)),去除模拟水样中的Ni2(+<10 mg·L~(-1))。驯化菌对镍离子的耐性增加,它们在5 mg·L~(-1)和10 mg·L~(-1)的水样中的浊度(OD)值约为未驯化菌的3倍。在处理低浓度的Ni~(2+)水样时,驯化菌的去除率有较大提高,当水样中Ni~(2+)浓度为1 mg·L~(-1)时,驯化菌对镍离子的去除率是未驯化菌的3.16倍。红外图谱表明,更多的表面官能团参与了镍离子的去除过程。
(4 )大肠杆菌、金黄色葡萄球菌及枯草芽孢杆菌对日落黄、亚甲基蓝的脱色作用研究
以阴离子染料(日落黄)和阳离子染料(亚甲基蓝)为研究对象,探讨不同细菌对染料的脱色行为。该染料在pH2.0-9.0范围内吸光度变化小,稳定性较好。脱色实验表明,细菌对染料的脱色包括吸附和降解两个过程,细菌对染料的吸附在稳定期最强,但在降解过程中,降解酶需经合成、分泌及酶解,导致了细菌对染料的最高脱色率不在稳定期,而在衰亡期。不同的细菌对两种染料的脱色效果有较大差异。大肠杆菌对亚甲基蓝的脱色率最高达60%,金黄色葡萄球菌对日落黄的脱色率最高为56%。
Heavy-metal pollutants and dye wastewater have become the most serious problem through out the world.Metal ions are invisible and can’t be degraded, while dyes usually have complex aromatic molecular structure, which make the wastewater of high COD brilliance and intensity colors and low biodegradability. They are difficult to disposal by conventional physical or chemical treatment process mainly becauce of it’s expensiveness, energy-costly and secondary pollution. In recent years, numerous metal ions have been treated by non-living biomass very well, except nickel ion. Researchers have found that the metals biosorbed on the surface can be biotransformed after enter into the cell, and dyes will be biodegraded by the enzyme that excreted by living microoganism.
Mechanism of Escherichia coli domestication was discussed in this paper. The bacteria was domesticated for three times in the medium by increasing the Ni~(2+) concentration gradullay. Morphological and surface characteristics of E.coli were anlysized by infra-red spectrum, X-ray electron spectra, transmission electron microscopy and light microscopy, meanwhile, plasmid was also anlysized in these processes. Domesticated bacteria that of higher Ni~(2+) accumulation capacity was used to deal with the simulation water, compared to common strain, the accumulation capability turn out to be better. At the same time, three kinds of bacteria were used to decolorize dyes, and to explore the feasibility of microoganism in dye wastewater pretreatment. In this paper we have done the works as follows:
1. Selecting the initial concentration of nickel ions for domestication processes When incubated in the Ni~(2+) environment, The emergency response of E.coli was a biochemical process, which was closely related to the concentration of Ni~(2+). Therefore, the effect of concentration of Ni~(2+) on the behaviors of growth and accumulation were studied. The results showed that growth of E.coli was drastically inhibited by the increasing concentration of Ni~(2+). Accmulation capacity was enhanced with the biomass increasd, the maximal Ni~(2+) accumulation capacity occurred in the stationary phase. Results of FT-IR showed that, the Ni~(2+) accumulation mainly depend on the functional groups on the surface of biomass, sunch as–OH, -COOH, maybe protein was also involved in this process. From XPS spectra, we can draw a conclusion that the protein was certainly participated in Ni~(2+) accumulation. TEM observed that the biomorph of cells were changed and the cell wall became frangible after Ni~(2+) accumulation. The maximal accumulation rate of Ni~(2+) was obtained at the concentration of 30 mg l~(-1), it is selected as the strain for the later domestication process.
2.The primary mechanism of emergency response in domestication process
After domesticated for three times, E.coli can quickly acclimatize itself to Ni~(2+) medium, and the growth rate was controlled by the Ni~(2+) concentration, the propagated speed of the bacteria increased with the decreasing of the Ni~(2+) concentration gradients. The accumulation capability of domesticated bacteria, which related to the concentration of Ni~(2+) was also advanced. The time for maximal capacity of E.coliⅠwas at the stionary phase. There are two kinds of E.coliⅡ, one was incubated at the concentration of 20 mg·L~(-1), while another at 150mg·L~(-1), for the former one(E.coliⅡC,D), there were two periods of maximal capacity at log phase and stionary phase, repectively, and the latter (E.coliⅡE,F) it was at log phase. E.coliⅡwere domesticated for the third time at the concentration of 300mg·L~(-1). For E.coliⅢ, the periods of maximal capacity were the same to their strains. FT-IR anlysis showed that, besides the peaks of hydroxyl, peaks of amide groups shifted obviously and the peaks of C-O for amylose shifted, as well. All of these biomacromolecules can biosorbe the Ni~(2+) by combining with it. Light microscope and TEM were applied to investigated the microstrueture of cells, and observed that cells were destroyed and fissioned with the Ni~(2+) increased during domestication, furthermore, the cells aggregated together. Plasmid was prepared by NaOH method, and the profiles showed that there was only one plasmid in the E.coli, and it didn’t changed in domestication process.
3.The removal efficiency of Ni~(2+) with domesticated bacteria
Domesticated bacteria that of better Ni~(2+) accumulation capacity was used to deal with the simulation water, the concentration of Ni~(2+) in the sample was in the range of 0 ~ 10 mg·L~(-1). the tolerance of bacteria to Ni~(2+) became improvement after domestication. At the concentration of 5 mg·L~(-1) and10 mg·L~(-1), the OD600 for the domesticated E.coli was about three-fold of common one. Removal rate also improved more than 3 times. FT-IR spectra showed that more functional groups had participated in Ni~(2+) accumulation in the sample.
4. Study on the decolorization of dyes with three kinds of bacteria
Decolorization behaviors of bacteria on Sunset yellow and Methylene blue were investigated. When the pH was in the range of 2.0-9.0, the absorbance varied slightly. Decolorization experiment showed that the decolorization process of bacteria includes two steps, adsorption and degradation. In the first step, the maximal adsorption rate which depended on the numerous biomass was at the stionary phase, while in the next one, due to the synthesis, excretion and dagragation of enzyme, the maximal decolorization rate was delayed to the death phase. The decolorization rate of three kinds of bacteria for the dyes were different. The E.coli can remove 60% of the colour of Methylene blue, and, decolorization rate of sunset yellow by Staphylococcus aureus was 56%.
引文
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