辐照条件下模拟放射性核素Sr~(2+)的微生物吸附研究
摘要
锶作为核爆、核事故等引起的全球性沉降中危害最大的核素之一,其污染具有隐蔽性和滞后性等特点。为寻求Sr2+的高效微生物吸附剂,探索微生物在放射性环境下对Sr2+吸附富集的可能性。实验以直接从自然环境(土壤)分离获得菌株和利用工业酵母分别作为活菌体与死菌体的研究对象,开展特定辐照条件对实验微生物吸附特性影响对比研究。
在对活菌体微生物吸附剂的Sr2+吸附研究中,通过对土壤样品中分离的47株微生物进行Sr2+的吸附效果对比分析,确定其中1株真菌和11株细菌对Sr2+的吸附效果较好。经过长期反复在含Sr2+培养液中的胁迫诱导驯化,所选12株菌在含Sr2+液体培养基中的生长能力、吸附效率及吸附速率均有增强。其中,各土壤分离菌的最大生长速率(vODmax)位置整体提前了0.5d,各菌株吸附吸附平衡基本出现在2.0d～3.0d之间,较驯化前提前了1.0d～2.5d。并通过研究确定实验条件下较佳吸附条件为初始pH=6.0～7.0,初始接种量Vi=0.5mL/25mL (1/50),初始Sr2+浓度C0=25mg/L～100mg/L。
各株菌对Sr2+动力学吸附过程为初期吸附平衡、快速吸附增长和二次吸附平衡等三个吸附阶段(10#菌只有后两个吸附过程),且三个阶段均符合准二级反应动力学模型,分阶段拟合结果也足反映了活菌体吸附过程复杂,不仅在于复杂的吸附机理,还有其吸附体系的复杂性。在对各株土壤分离菌吸附Sr2+进行等温吸附模型拟合时发现,各菌的Sr2+吸附对Langmuir均比Freundlich吸附模型要符合的好,相关系数R基本在0.98以上,但对qmax分析认为,Langmuir吸附模型对于活菌体吸附Sr2+的研究具有一定局限性。
各株菌吸附Sr2+的等温吸附类型参数KR均小于0.82,确定其对应等温吸附类型为有利于吸附行反应进行。各株菌在30℃、pH7.0等实验条件下,其△G0均为负值,表明实验条件下各株菌对Sr2+的吸附均能自发进行。
通过对各株土壤分离菌进行辐照条件下筛选及吸附特性研究过程中发现,0.5kGy和1.0kGy的辐照剂量下,观察所选12株土壤分离菌在平板上的生长情况,发现大多数实验菌株的生长受到明显抑制,但辐照情况下有无Sr2+对于各菌株的菌落形成无明显影响。其中6#菌在所有实验菌株中对辐照的耐受性最好,最后对相对耐辐照的高效土壤分离细菌进行16S rDNA序列测定并与GeneBank数据库进行比对,表明该菌株属于芽孢杆菌属(Bacillus),与短小芽孢杆菌(Bacillus pumilus)序列相似性高达100%。
辐照条件下液相吸附实验发现,就整个动力学吸附曲线而言,辐照组菌体对液相中Sr2+的吸附比无辐照对照组有迟滞现象,而接种12.0h实验组受辐照影响比接种1.0h实验组受辐照的影响要小些。先吸附Sr2+再辐照实验显示,处于吸附初期阶段(1.0d～2.0d)的实验组受辐照的影响较为显著,而吸附后期辐照前后吸附效率差值不超过1.00%。先辐照后吸附Sr2+实验认为,吸附时间对各菌株对Sr2+吸附效果的影响比辐照影响更显著。
从不同辐照条件实验下各菌株的FTIR谱图变化情况来看,来自菌体细胞上的多糖、蛋白质酰胺、脂类等成分变化,反映了实验辐照条件对各菌株产生的具体损伤作用,且辐照条件下液相中的Sr2+具有一定协同损伤作用。
在对以面包酵母菌为对象的死菌体吸附Sr2+研究过程中,通过批量实验室实验确定酵母菌对Sr2+较佳吸附条件为：pH=4.0～5.5,吸附温度T=20℃～40℃,酵母菌施加浓度Cm=2.0～6.0g/L, Sr2+初始浓度C0=0.25～1.0mmol/L。 FTIR分析认为,酵母菌吸附Sr2+的过程的确存在化学吸附过程；相对而言,酵母菌细胞上的多糖、蛋白质酰胺成分更多地参与了对Sr2+的吸附过程。
1.0kGy剂量辐照面包酵母菌,发现辐照前后其在不同温度下对Sr2+的等温吸附结果均能很好符合Langmuir和Freunlich两个吸附模型。同时,根据其对应KR取值在0.88 对辐照前后酵母菌吸附Sr2+的动力学分析发现,酵母菌对Sr2+的吸附是一个快速进行的反应过程,30℃下吸附速度要略快一些；且整个吸附动力学过程中,qt均存在30℃>20℃>10℃。另外,辐照后酵母菌对Sr2+的吸附效果比相同温度条件下辐照前实验组要略好。各实验温度下酵母菌对Sr2+的动力学二级吸附速率方程拟合结果中R均在0.999以上,拟合程度较好。在此基础上,计算获得酵母菌辐照前后吸附Sr2+的反应活化能Ea分别为9.955kJ/mol和9.162kJ/mol。这一定程度上能够表明实验条件下的辐照更有利于酵母菌对Sr2+的吸附反应顺利进行。
酵母菌对5种重金属离子与Sr2+吸附的效果综合分析发现,其对所选二价离子的吸附具有相似性特性。在相对最佳吸附条件：pH=4.5, Cm=4.0g/L, C0=1.0mmol/L下,对酵母菌对6种重金属离子的动力学吸附过程中,其单位吸附量qt和n大小顺序均为：Pb2+>Cd2+>Sr2+> Cu2+>Zn2+> Ni2+。
通过论文研究结果认为,从无放射污染土壤中分离的菌株,经过实验条件下的胁迫诱导驯化,可以提高其对Sr2+的吸附效果,且部分菌株能够在1.0kGy辐照条件下(相当于中高放条件)下对Sr2+仍具有较好吸附效果。相比之下,尽管面包酵母菌吸附效率相对实验所得土壤菌株的活菌体吸附要低,但工业废弃酵母具有量大、成本低、吸附快,干燥后死菌体方便运输且耐辐照等优点,也可成为很好的放射性Sr2+的吸附剂。
As one of most hazardous nuclide ions from global sedimentation radionuclide originated from nuclear explode and nuclear accident etc., radioisotope of Strontium has its characteristic concealment and hysteresis. For achieving efficient microbial adsorbent and exploring the feasibility of biosorption of Sr2+using microbes under radioactive environment, some living microorganism from nature (soli) and dead thalli from industry waste yeast were chose as study objects in this PhD dissertation.
47strains from soli were isolated, in which one strain of fungus and eleven strains of bacteria were confirmed have better biosorption efficiencies to Sr2+. The growth rate, biosorption efficiency and biosorption rate of these12kinds of strains were increased under domesticated intimidation stress in aqueous stable isotope of strontium. The appearance time of their highest growth rate was accelerated0.5d, while the appearance time of biosorption equilibrium was accelerated1.0～2.5d as they appeared at2.0-3.0d. The optimized biosorption condition for soli strains to Sr2+was pH=6.0～7.0, Vi=0.5mL/25mL (1/50) and C0=25mg/L～100mg/L.
The kinetic processes of different strains are composed of initial biosorption equilibrium, fast biosorption increasing and second biosorption equilibrium. These three biosorption phases were all consistent with pseudo-second order adsorption kinetics. The results showed that the biosorption process of living cells to Sr2+was extremely complex due to its complex biosorption mechanism and its complex biosorption system. The biosorption of different strains to Sr2+were all better consistent with Langmuir adsorption isotherm than Freundlich adsorption isotherm, R>0.98. However, the analysis to qmax showed that the model of Langmuir adsorption isotherm still had some limits for describing the biosorption Sr2+by living cells.
The adsorption isotherm type parameters (KR) of experimental strains to Sr2+were all less than0.82ensured the experimental condition was propitious to biosorption activity of different strains to Sr2+. Under experimental condition of303K and pH7.0, The biosorption activities of Sr2+were spontaneously due to Gibbs free energy change (ΔG0) of different stains were all below zero.
Although the growth of twelve strains soil microbes were restrained obviously under the radiant dose of0.5kGy and1.0kGy, there were no distinct evidence show the effects of Sr2+on bacteria colony formation under experimental radiation condition. The strain of6#has best radiation-resistant ability. This strain was subject to Bacillus according to analysis results of16S rDNA was100%accordance with GeneBank database of Bacillus pumilus.
As for the whole kinetic biosorption curves, under the radiation dose of1.0kGy, bacterial growth rates of test groups have retardation phenomena comparing to the control groups without radiation. The radiation effect of test groups of inoculated12.Oh in advance was weaker than the test groups of inoculated1.0h. The experimental results of radiation after adsorbed Sr2+showed that the radiation effect of different strains at initial biosorption stage (1.0～2.0d) were more obvious than anaphase biosorption stage, while the results of adsorption Sr2+after radiation showed that biosorption time has more obvious effect than radiation.
FTIR analysis results under different radiation condition showed that the characteristic peaks of amylase, protein amide and lipids on bacteria cells were slightly shifted. These indicated that bacteria cells were damaged by radiation and Sr2+has cooperation damage effects with radiation in aqueous condition.
Batch experiments about biosorption Sr2+on baker's yeast showed that the optimized adsorption condition as following:pH=4.0-5.5, T=20℃-40℃, Cm=2.0-6.0g/L, C0=0.25-].Ommol/L. FTIR results showed that its existent chemical adsorption in the biosorption process to Sr2+. The changes observed in the spectra indicated the possible involvements of functional groups on the surface of the biomass in biosorption process, and the further analysis showed that amylose and protein amide have more important roles in the biosorption of Sr2+on yeast cells.
With the radiant dose of1.0kGy, the biosorption of baker's yeast and control groups without radiation to Sr2+were all better consistent with Langmuir adsorption isotherm and Freundlich adsorption isotherm. The analysis results of adsorption isotherm type parameters (0.88 The kinetic analysis showed that the biosorption of baker's yeast to Sr2+was a fast reaction process. As the whole biosorption process, qt is being the relation at different experimental temperatures as30℃>20℃>10℃. The biosorption effect to Sr2+on baker's yeast after radiation was better than the control groups without radiation. The biosorption analysis results of baker's yeast to Sr2+were all consistent with pseudo-second order adsorption kinetics at different experimental temperatures, as R>0.999. The activation energy Ea of biosorption to Sr2+can be calculate as9.955kJ/mol and9.162kJ/mol before and after radiated to baker's yeast. These indicated that an experimental radiation condition is favor to the spontaneous biosorption of baker's yeast to Sr2+
The analysis results of biosorption five kinds of heavy metal ions and Sr2+showed that baker's yeast has similar adsorption activity to bivalent ions. At the relative optimized experimental conditions as pH=4.5, Cm=4.0g/L and Co=1.0mmol/L, the biosorption kinetics of six ions on baker's yeast showed that qt and n have the relation as following: Pb2+>Cd2+>Sr2+> Cu2+>Zn2+> Ni2+.
This study obtained some strains from soli without radiation pollution. Their biosorption abilities to Sr2+increased after domestication of intimidation stress. Some of them have satisfactory biosorption efficiencies to Sr2+under the conditions of low to moderate, even high radioactivity. As industry waste, although baker's yeast have not the similar high biosorption efficiencies to Sr2+as strains from soli, it has advantages of easily obtained, low-cost and fast adsorption, etc.. On the other hand, dry yeast cells are favor to transport and is able to bear radiation; these indicated baker's yeast may become one kind of excellent biosorbent to Sr2+in the future.
在对活菌体微生物吸附剂的Sr2+吸附研究中,通过对土壤样品中分离的47株微生物进行Sr2+的吸附效果对比分析,确定其中1株真菌和11株细菌对Sr2+的吸附效果较好。经过长期反复在含Sr2+培养液中的胁迫诱导驯化,所选12株菌在含Sr2+液体培养基中的生长能力、吸附效率及吸附速率均有增强。其中,各土壤分离菌的最大生长速率(vODmax)位置整体提前了0.5d,各菌株吸附吸附平衡基本出现在2.0d～3.0d之间,较驯化前提前了1.0d～2.5d。并通过研究确定实验条件下较佳吸附条件为初始pH=6.0～7.0,初始接种量Vi=0.5mL/25mL (1/50),初始Sr2+浓度C0=25mg/L～100mg/L。
各株菌对Sr2+动力学吸附过程为初期吸附平衡、快速吸附增长和二次吸附平衡等三个吸附阶段(10#菌只有后两个吸附过程),且三个阶段均符合准二级反应动力学模型,分阶段拟合结果也足反映了活菌体吸附过程复杂,不仅在于复杂的吸附机理,还有其吸附体系的复杂性。在对各株土壤分离菌吸附Sr2+进行等温吸附模型拟合时发现,各菌的Sr2+吸附对Langmuir均比Freundlich吸附模型要符合的好,相关系数R基本在0.98以上,但对qmax分析认为,Langmuir吸附模型对于活菌体吸附Sr2+的研究具有一定局限性。
各株菌吸附Sr2+的等温吸附类型参数KR均小于0.82,确定其对应等温吸附类型为有利于吸附行反应进行。各株菌在30℃、pH7.0等实验条件下,其△G0均为负值,表明实验条件下各株菌对Sr2+的吸附均能自发进行。
通过对各株土壤分离菌进行辐照条件下筛选及吸附特性研究过程中发现,0.5kGy和1.0kGy的辐照剂量下,观察所选12株土壤分离菌在平板上的生长情况,发现大多数实验菌株的生长受到明显抑制,但辐照情况下有无Sr2+对于各菌株的菌落形成无明显影响。其中6#菌在所有实验菌株中对辐照的耐受性最好,最后对相对耐辐照的高效土壤分离细菌进行16S rDNA序列测定并与GeneBank数据库进行比对,表明该菌株属于芽孢杆菌属(Bacillus),与短小芽孢杆菌(Bacillus pumilus)序列相似性高达100%。
辐照条件下液相吸附实验发现,就整个动力学吸附曲线而言,辐照组菌体对液相中Sr2+的吸附比无辐照对照组有迟滞现象,而接种12.0h实验组受辐照影响比接种1.0h实验组受辐照的影响要小些。先吸附Sr2+再辐照实验显示,处于吸附初期阶段(1.0d～2.0d)的实验组受辐照的影响较为显著,而吸附后期辐照前后吸附效率差值不超过1.00%。先辐照后吸附Sr2+实验认为,吸附时间对各菌株对Sr2+吸附效果的影响比辐照影响更显著。
从不同辐照条件实验下各菌株的FTIR谱图变化情况来看,来自菌体细胞上的多糖、蛋白质酰胺、脂类等成分变化,反映了实验辐照条件对各菌株产生的具体损伤作用,且辐照条件下液相中的Sr2+具有一定协同损伤作用。
在对以面包酵母菌为对象的死菌体吸附Sr2+研究过程中,通过批量实验室实验确定酵母菌对Sr2+较佳吸附条件为：pH=4.0～5.5,吸附温度T=20℃～40℃,酵母菌施加浓度Cm=2.0～6.0g/L, Sr2+初始浓度C0=0.25～1.0mmol/L。 FTIR分析认为,酵母菌吸附Sr2+的过程的确存在化学吸附过程；相对而言,酵母菌细胞上的多糖、蛋白质酰胺成分更多地参与了对Sr2+的吸附过程。
1.0kGy剂量辐照面包酵母菌,发现辐照前后其在不同温度下对Sr2+的等温吸附结果均能很好符合Langmuir和Freunlich两个吸附模型。同时,根据其对应KR取值在0.88
酵母菌对5种重金属离子与Sr2+吸附的效果综合分析发现,其对所选二价离子的吸附具有相似性特性。在相对最佳吸附条件：pH=4.5, Cm=4.0g/L, C0=1.0mmol/L下,对酵母菌对6种重金属离子的动力学吸附过程中,其单位吸附量qt和n大小顺序均为：Pb2+>Cd2+>Sr2+> Cu2+>Zn2+> Ni2+。
通过论文研究结果认为,从无放射污染土壤中分离的菌株,经过实验条件下的胁迫诱导驯化,可以提高其对Sr2+的吸附效果,且部分菌株能够在1.0kGy辐照条件下(相当于中高放条件)下对Sr2+仍具有较好吸附效果。相比之下,尽管面包酵母菌吸附效率相对实验所得土壤菌株的活菌体吸附要低,但工业废弃酵母具有量大、成本低、吸附快,干燥后死菌体方便运输且耐辐照等优点,也可成为很好的放射性Sr2+的吸附剂。
As one of most hazardous nuclide ions from global sedimentation radionuclide originated from nuclear explode and nuclear accident etc., radioisotope of Strontium has its characteristic concealment and hysteresis. For achieving efficient microbial adsorbent and exploring the feasibility of biosorption of Sr2+using microbes under radioactive environment, some living microorganism from nature (soli) and dead thalli from industry waste yeast were chose as study objects in this PhD dissertation.
47strains from soli were isolated, in which one strain of fungus and eleven strains of bacteria were confirmed have better biosorption efficiencies to Sr2+. The growth rate, biosorption efficiency and biosorption rate of these12kinds of strains were increased under domesticated intimidation stress in aqueous stable isotope of strontium. The appearance time of their highest growth rate was accelerated0.5d, while the appearance time of biosorption equilibrium was accelerated1.0～2.5d as they appeared at2.0-3.0d. The optimized biosorption condition for soli strains to Sr2+was pH=6.0～7.0, Vi=0.5mL/25mL (1/50) and C0=25mg/L～100mg/L.
The kinetic processes of different strains are composed of initial biosorption equilibrium, fast biosorption increasing and second biosorption equilibrium. These three biosorption phases were all consistent with pseudo-second order adsorption kinetics. The results showed that the biosorption process of living cells to Sr2+was extremely complex due to its complex biosorption mechanism and its complex biosorption system. The biosorption of different strains to Sr2+were all better consistent with Langmuir adsorption isotherm than Freundlich adsorption isotherm, R>0.98. However, the analysis to qmax showed that the model of Langmuir adsorption isotherm still had some limits for describing the biosorption Sr2+by living cells.
The adsorption isotherm type parameters (KR) of experimental strains to Sr2+were all less than0.82ensured the experimental condition was propitious to biosorption activity of different strains to Sr2+. Under experimental condition of303K and pH7.0, The biosorption activities of Sr2+were spontaneously due to Gibbs free energy change (ΔG0) of different stains were all below zero.
Although the growth of twelve strains soil microbes were restrained obviously under the radiant dose of0.5kGy and1.0kGy, there were no distinct evidence show the effects of Sr2+on bacteria colony formation under experimental radiation condition. The strain of6#has best radiation-resistant ability. This strain was subject to Bacillus according to analysis results of16S rDNA was100%accordance with GeneBank database of Bacillus pumilus.
As for the whole kinetic biosorption curves, under the radiation dose of1.0kGy, bacterial growth rates of test groups have retardation phenomena comparing to the control groups without radiation. The radiation effect of test groups of inoculated12.Oh in advance was weaker than the test groups of inoculated1.0h. The experimental results of radiation after adsorbed Sr2+showed that the radiation effect of different strains at initial biosorption stage (1.0～2.0d) were more obvious than anaphase biosorption stage, while the results of adsorption Sr2+after radiation showed that biosorption time has more obvious effect than radiation.
FTIR analysis results under different radiation condition showed that the characteristic peaks of amylase, protein amide and lipids on bacteria cells were slightly shifted. These indicated that bacteria cells were damaged by radiation and Sr2+has cooperation damage effects with radiation in aqueous condition.
Batch experiments about biosorption Sr2+on baker's yeast showed that the optimized adsorption condition as following:pH=4.0-5.5, T=20℃-40℃, Cm=2.0-6.0g/L, C0=0.25-].Ommol/L. FTIR results showed that its existent chemical adsorption in the biosorption process to Sr2+. The changes observed in the spectra indicated the possible involvements of functional groups on the surface of the biomass in biosorption process, and the further analysis showed that amylose and protein amide have more important roles in the biosorption of Sr2+on yeast cells.
With the radiant dose of1.0kGy, the biosorption of baker's yeast and control groups without radiation to Sr2+were all better consistent with Langmuir adsorption isotherm and Freundlich adsorption isotherm. The analysis results of adsorption isotherm type parameters (0.88
The analysis results of biosorption five kinds of heavy metal ions and Sr2+showed that baker's yeast has similar adsorption activity to bivalent ions. At the relative optimized experimental conditions as pH=4.5, Cm=4.0g/L and Co=1.0mmol/L, the biosorption kinetics of six ions on baker's yeast showed that qt and n have the relation as following: Pb2+>Cd2+>Sr2+> Cu2+>Zn2+> Ni2+.
This study obtained some strains from soli without radiation pollution. Their biosorption abilities to Sr2+increased after domestication of intimidation stress. Some of them have satisfactory biosorption efficiencies to Sr2+under the conditions of low to moderate, even high radioactivity. As industry waste, although baker's yeast have not the similar high biosorption efficiencies to Sr2+as strains from soli, it has advantages of easily obtained, low-cost and fast adsorption, etc.. On the other hand, dry yeast cells are favor to transport and is able to bear radiation; these indicated baker's yeast may become one kind of excellent biosorbent to Sr2+in the future.
引文
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