极端嗜酸氧化硫硫杆菌筛选及贫黄铜矿生物浸出研究
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摘要
伴随全球高品位原矿储量减少及金属需求量日益增加，如何有效提炼低品位的贫尾矿已成为世界性难题。生物浸出工艺具有经济、环保且可有效提炼贫尾矿等优点，是解决以上问题的新型绿色冶炼技术。然而由于极低品位和复杂构成，黄铜矿的生物浸出机理尚未完善，工业化应用进展也并不理想。本文从极端嗜酸Acidithiobacillusthiooxidans(A. thiooxidans)的筛选及其极端耐酸机理研究开始，提出强化生物浸出的复合体系和组合工艺，建立了定性定量分子探针检测方法，揭示了浸出过程不同集落态的群落演变规律，并初步构建贫黄铜矿生物浸出机理模型。主要研究如下：
(1)极端嗜酸氧化硫硫杆菌筛选及耐酸机理研究。采用以异养微生物-Rhodotorulasp.作为底层培养物的双层平板法，将筛选化能自养型浸矿微生物的筛选周期缩减1/3，检出率提高3.1倍。从福建紫金矿业的工业化生物堆浸的浸出液筛得1株极端嗜酸硫氧化菌种，对无机强酸耐受能力可达pH0.2。经生理特性及双重分子特征基因鉴定为A.thioodidans ZJJN-3，保藏于中国典型微生物保藏中心-CCTCC M2012104。进而从胞内pH、细胞形态、细胞多糖、细胞膜脂肪酸成分、ATPase活性及耐酸相关蛋白转录水平等对其极端耐酸机理进行研究。伴随酸胁迫的增强，细胞荚膜层先明显增厚后逐渐褪去。细胞多糖含量也呈先上升后下降趋势。细胞膜不饱和脂肪酸比例从37.85%显著提升至49.06%，而且环丙烷脂肪酸比例维持在12.56%-18.59%。ATPase活性明显增强，相关耐酸蛋白抗逆体系GrpE/DnaK/DnaJ的基因相对转录水平提升至原有水平的2.23-3.50倍。基于以上结果初步构建A. thioodidans ZJJN-3的耐酸协同机制模型。
(2)双菌协同浸出贫黄铜矿技术研究。基于Starkey-硫化矿培养基的重复补料分批培养模式，将A. thioodidans ZJJN-3的硫转化率和生物量生产强度提高31.1%和187.9%。关键生化参数、扫描电镜(SEM)及X射线衍射(XRD)结果均表明在双菌协同效应下，增强了浸出体系中生化活跃度并减弱钝化作用。最终浸出效率为Acidithiobacillusferrooxidans CUMT-1(A. ferrooxidans CUMT-1)纯菌体系的1.81倍。分别采用恒pH、Ag+和Cl-体系抑制钝化效应和加速生化反应速率。确定最佳复合浸出体系为恒pH1.3-2.0mg·L-1Ag+-2.5g·L-1Cl-。在更高的生物量和化学反应速率基础上，有效减弱了黄钾铁矾沉淀和S0膜的钝化作用。最终Cu2+浓度和浸出率分别达55.5mg·L-1和55.0%，约为对照体系的两倍。外源能源底物、分阶段恒pH调控和补料发酵策略分别被用于缩减贫黄铜矿分批浸出过程菌矿适应期、减弱中后期黄钾铁矾钝化及降低矿物底物抑制作用。确定最优组合工艺为2g·L-1Fe2++2g·L-1S0、三阶段恒pH调控(pH1.3-1.0-0.7)和补料-分批策略(0.5+0.125×4)。在7L搅拌式发酵罐验证结果表明，组合工艺更好的平衡了各浸出阶段中生物和化学效应。最终Cu2+浓度、浸出率和平均浸出速率分别达到89.1mg·L-1、44.1%和2.23mg·L-1·d-1，比原始水平提高约53%。
(3)定量检测浸矿微生物的分子探针技术构建。结合分子探针、核酸S1酶消解功能及荧光标记等手段，建立了一种浸矿微生物专用定性定量检测方法。基于目标菌种16SrRNA保守区域设计特异性分子探针。比对结果表明分子探针与近缘菌种有2个或更多的碱基差异。进而优选了分子探针检测关键步骤的操作条件。如低非特异性吸附的封闭和洗脱条件(250μL0.1%BSA，30min，以0.5%PBST洗脱为辅助)、细胞破碎方式(300W，50%空白比，超声破碎4min，以细胞裂解液为辅助)、探针工作浓度(主探针100nM，捕获探针10nM，信号探针10nM)、杂交时间(1h，0.5h，0.5h)、杂交温度(30℃，45℃，45℃)、核酸S1酶酶切时间(20min)及荧光素抗体工作浓度(1:5000稀释比例)。最终建立分子探针检测标准曲线，线性系数达98.64%，细胞量的检测低限为103cells·mL-1左右。纯菌、混菌及浸出体系验证结果表明该检测方法特异性和准确性良好，可有效应用于后续浸出过程中群落结构演变分析。
(4)双菌集落演化规律对贫黄铜矿浸出的影响。结合分子探针检测技术，对浸出过程双菌不同集落态的演变规律及其影响进行了翔实的研究。附着细胞对浸出体系中自由细胞群落的影响显著。仅占总生物量比例3.8%-11.6%的附着细胞缺失却可导致19.1%-31.2%自由细胞损失，尤其以A. thiooxidans ZJJN-3纯菌体系最为明显。双菌体系中附着细胞的缺失使得A. thiooxidans ZJJN-3在与A. ferrooxidans CUMT-1的群落竞争力明显下降，表明A. thiooxidans ZJJN-3对附着细胞有更强的依赖性。与此同时，关键化学参数的变化也证实了附着细胞的重要性。脱附着体系中Fe2+和SO24
-浓度的最大减少量均高达40%左右。XRD及傅里叶红外变换光谱技术(FTIR)对矿渣分析均表明附着细胞与硫代谢紧密相关。约37.5%-50.5%甚至更高的浸出效率与吸附作用有直接关联。在生物和化学效应平衡的基础上，归纳总结了强化贫黄铜矿生物浸出的工艺和策略，并初步构建贫黄铜矿生物浸出机理模型。
With the global decline of rich-ore reserves and increasing metal demand, how toeffectively extract the low-grade tailings has become a worldwide problem. With theadvantages of economical, environmental and efficiently extracting of low-grade ore,bioleaching was a novel and green smelting technique for solving the above problems.However, due to the extremely low-grade and complex composition, the mechanism ofchalcopyrite bioleaching was not perfect and its industrial application was also not satisfied.In this paper, the research was started from the isolation of an extremeacidophilus-Acidithiobacillus thiooxidans (A. thiooxidans) and its extremely acid-resistancemechanism. Combination system and technology were respectively proposed for enhancingthis bioprocess. An identified and quantified method was established with the molecular probe.The community evolutions in different status were further revealed. The bioleachingmechanism model of low-grade chalcopyrite was initially established. The main studycontents were listed as follow:
(1) Isolation of the extremely acidophilus A. thiooxidans and its acid-proof mechanism.A double layers culturing method with heterotrophic strain-Rhodotorula sp. as the bottomculture was employed for efficiently isolating the chemoautotroph bioleaching microorganism.The cycle was reduced by1/3when the plating efficiency was improved by3.1times. Anextremely acidophilic sulfur-oxidizer strain was isolated from the leaching solution ofindustrial bioleaching heap in Fujian Zijin Mining. The capacity of inorganic acid-proofreached to pH0.2. The strain was identified as A. thioodidans ZJJN-3with the specialphysiological and double molecular characteristics. The strain ZJJN-3has been preserved inthe China Center for Type Culture Collection-CCTCC M2012104. Then the extremelyacid-proof mechanism was respectively investigated from the intracellular pH, cellmorphology, polysaccharide, membrane fatty acid composition, ATPase activity and thetranscription levels of acid-related proteins. With the increasing acid stress, the cell capsuleexperienced a process of significantly thickening and then gradually shedding off. The cellpolysaccharide content also increased and then dropped along with the evolution process. Theunsaturated fatty acid of cell membrane ratio significantly increased from37.85%to49.06%and cyclopropane fatty acids proportion remained at a high level about12.56%-18.59%. TheATPase activity was greatly strengthened, and the relative transcript levels of acid-proofsystem-GrpE/DnaK/DnaJ also increased to2.2-3.5times of original level. The acid-proofmechanism model in A. thioodidans ZJJN-3was further established basing on the aboveresults.
(2) Bioleaching of low-grade chalcopyrite by a synergistic two-strain system. Based onthe repeated fed-batch culture model in the Starkey-sulfide medium, the sulfur conversionpercentage and biomass productivity of A. thioodidans ZJJN-3was improved by31.1%and187.9%. The results of key biochemical parameters, Scanning electron microscope(SEM) andX ray diffraction (XRD) all implied that the biochemical activity was enhanced and thepassiavations were weakened with the synergistic effects of two strains. The bioleachingefficiency was1.81times of the pure Acidithiobacillus ferrooxidans CUMT-1(A. ferrooxidans CUMT-1) system. The pH-stat, Ag+and Cl-were respectively employed forreducing the passivation formation and accelerating the rate of biochemical reactions. Theoptimum combination system was determined as pH1.3-2.0mg·L-1Ag+-2.5g·L-1Cl-. Basedon the higher biomass and chemical reaction rate, the formation of jarosite precipitation andsulfur membrane was effectively weakened in the optimum combination system. The finalCu2+concentration and bioleaching efficiency reached55.5mg·L-1and55.0%, about twice ofthe control system. The exogenous energy substrates, multiple-phases pH-stat control andfed-batch fermentation strategies were respectively determnined to shorten strain-mineraladaptive phase, further weaken the jarosite passivation in the middle-end stage and reduce thesubstrate inhibition in batch bioleaching. The optimum integrated technology was determinedas2g·L-1Fe2++2g·L-1S0, three-stage pH-stat control (1.3-1.0-0.7) and fed-batch strategy(0.5+0.125×4). The verified results in the7L stirred tank fermenter showed that thebiological and chemical effects were better balanced in each bioleaching stage with theassistance of integration technology. The final Cu2+concentration, bioleaching efficiency andaverage production rate of Cu2+were89.1mg·L-1,44.1%and2.23mg·L-1·d-1, which wasimproved by about53%compared to the initial level.
(3) Establishment of molecular probe assay for identification and quantification ofbioleaching microorganism. With the assistances of molecular probe, eliminated function ofnuclease S1and fluorescently labeled technique, an assay was established for identifying andquantifying bioleaching microorganism. The specific probes were designed basing on theconserved16S rRNA regions of target species. The blasted result showed that there were atleast two or more bases differences with other closelty related strains. The key operatingconditions of moleacluar probe assay were futher optimized such as the sealing and elutedconditions of low non-specific adsorption (250μL0.1%BSA,30min,0.5%PBST elution),cell disruption (300W,50%duty cycle,4min, lysates secondary crushing), the workingconcentration of probes (primary probe100nM, CP10nM, SP10nM), hybridization time (1h,0.5h,0.5h), hybridization temperature (30℃,45℃,45℃), nuclease S1digestion time (20min) and working concentration of fluorescein antibody (1:5000diluted proportion). Thestandard curve of molecular probe assay was eventually established. The linear coefficientwas98.64%when the detection limitation was low to103cells·mL-1. The verification resultsof pure-strain, mixed-strains and bioleaching systems all showed a good specificity andaccuracy. It could be efficiently applied for analyzing the community structure in thesubsequent bioleaching process.
(4) The effects of community evolutions on bioleaching of low-grade chalcopyrite in thetwo-strain system. With the molecular probe technique, the evolution of microbial structure indifferent status and its effects were systematically explored. The effects of attached cell onbiomass of free cell were significant. Although with only3.8%-11.6%of total biomass, highto19.1%-31.2%loss of free cell was caused in the none-attached cell system, especially in thepure strain of A. thiooxidans ZJJN-3system. In the two-strains system, the lack of attachedcells would weaken the community competitiveness of A. thiooxidans ZJJN-3with A.ferrooxidans CUMT-1, which meant more dependence of A. thiooxidans ZJJN-3for attachedcell. The importance of attached cell was also proved by the changes chemical of key parameters. The largest reduction of Fe2+and SO24-concentrations were all high to about40%.
The results of XRD and fourier transform infrared spectroscopy (FTIR) analysis of slag allimplied that the sulfur metabolism was closely related with the attached cell.37.5%-50.5%oreven higher efficiency was closely associated with the adsorption process. On the basis of theequilibrium between biological and chemical effect, the techniques and strategies forenhancing chalcopyrite bioleaching was summarized. Futuremore, the bioleaching mechanismmodel of low-grade chalcopyrite was preliminarily established.
(1)极端嗜酸氧化硫硫杆菌筛选及耐酸机理研究。采用以异养微生物-Rhodotorulasp.作为底层培养物的双层平板法，将筛选化能自养型浸矿微生物的筛选周期缩减1/3，检出率提高3.1倍。从福建紫金矿业的工业化生物堆浸的浸出液筛得1株极端嗜酸硫氧化菌种，对无机强酸耐受能力可达pH0.2。经生理特性及双重分子特征基因鉴定为A.thioodidans ZJJN-3，保藏于中国典型微生物保藏中心-CCTCC M2012104。进而从胞内pH、细胞形态、细胞多糖、细胞膜脂肪酸成分、ATPase活性及耐酸相关蛋白转录水平等对其极端耐酸机理进行研究。伴随酸胁迫的增强，细胞荚膜层先明显增厚后逐渐褪去。细胞多糖含量也呈先上升后下降趋势。细胞膜不饱和脂肪酸比例从37.85%显著提升至49.06%，而且环丙烷脂肪酸比例维持在12.56%-18.59%。ATPase活性明显增强，相关耐酸蛋白抗逆体系GrpE/DnaK/DnaJ的基因相对转录水平提升至原有水平的2.23-3.50倍。基于以上结果初步构建A. thioodidans ZJJN-3的耐酸协同机制模型。
(2)双菌协同浸出贫黄铜矿技术研究。基于Starkey-硫化矿培养基的重复补料分批培养模式，将A. thioodidans ZJJN-3的硫转化率和生物量生产强度提高31.1%和187.9%。关键生化参数、扫描电镜(SEM)及X射线衍射(XRD)结果均表明在双菌协同效应下，增强了浸出体系中生化活跃度并减弱钝化作用。最终浸出效率为Acidithiobacillusferrooxidans CUMT-1(A. ferrooxidans CUMT-1)纯菌体系的1.81倍。分别采用恒pH、Ag+和Cl-体系抑制钝化效应和加速生化反应速率。确定最佳复合浸出体系为恒pH1.3-2.0mg·L-1Ag+-2.5g·L-1Cl-。在更高的生物量和化学反应速率基础上，有效减弱了黄钾铁矾沉淀和S0膜的钝化作用。最终Cu2+浓度和浸出率分别达55.5mg·L-1和55.0%，约为对照体系的两倍。外源能源底物、分阶段恒pH调控和补料发酵策略分别被用于缩减贫黄铜矿分批浸出过程菌矿适应期、减弱中后期黄钾铁矾钝化及降低矿物底物抑制作用。确定最优组合工艺为2g·L-1Fe2++2g·L-1S0、三阶段恒pH调控(pH1.3-1.0-0.7)和补料-分批策略(0.5+0.125×4)。在7L搅拌式发酵罐验证结果表明，组合工艺更好的平衡了各浸出阶段中生物和化学效应。最终Cu2+浓度、浸出率和平均浸出速率分别达到89.1mg·L-1、44.1%和2.23mg·L-1·d-1，比原始水平提高约53%。
(3)定量检测浸矿微生物的分子探针技术构建。结合分子探针、核酸S1酶消解功能及荧光标记等手段，建立了一种浸矿微生物专用定性定量检测方法。基于目标菌种16SrRNA保守区域设计特异性分子探针。比对结果表明分子探针与近缘菌种有2个或更多的碱基差异。进而优选了分子探针检测关键步骤的操作条件。如低非特异性吸附的封闭和洗脱条件(250μL0.1%BSA，30min，以0.5%PBST洗脱为辅助)、细胞破碎方式(300W，50%空白比，超声破碎4min，以细胞裂解液为辅助)、探针工作浓度(主探针100nM，捕获探针10nM，信号探针10nM)、杂交时间(1h，0.5h，0.5h)、杂交温度(30℃，45℃，45℃)、核酸S1酶酶切时间(20min)及荧光素抗体工作浓度(1:5000稀释比例)。最终建立分子探针检测标准曲线，线性系数达98.64%，细胞量的检测低限为103cells·mL-1左右。纯菌、混菌及浸出体系验证结果表明该检测方法特异性和准确性良好，可有效应用于后续浸出过程中群落结构演变分析。
(4)双菌集落演化规律对贫黄铜矿浸出的影响。结合分子探针检测技术，对浸出过程双菌不同集落态的演变规律及其影响进行了翔实的研究。附着细胞对浸出体系中自由细胞群落的影响显著。仅占总生物量比例3.8%-11.6%的附着细胞缺失却可导致19.1%-31.2%自由细胞损失，尤其以A. thiooxidans ZJJN-3纯菌体系最为明显。双菌体系中附着细胞的缺失使得A. thiooxidans ZJJN-3在与A. ferrooxidans CUMT-1的群落竞争力明显下降，表明A. thiooxidans ZJJN-3对附着细胞有更强的依赖性。与此同时，关键化学参数的变化也证实了附着细胞的重要性。脱附着体系中Fe2+和SO24
-浓度的最大减少量均高达40%左右。XRD及傅里叶红外变换光谱技术(FTIR)对矿渣分析均表明附着细胞与硫代谢紧密相关。约37.5%-50.5%甚至更高的浸出效率与吸附作用有直接关联。在生物和化学效应平衡的基础上，归纳总结了强化贫黄铜矿生物浸出的工艺和策略，并初步构建贫黄铜矿生物浸出机理模型。
With the global decline of rich-ore reserves and increasing metal demand, how toeffectively extract the low-grade tailings has become a worldwide problem. With theadvantages of economical, environmental and efficiently extracting of low-grade ore,bioleaching was a novel and green smelting technique for solving the above problems.However, due to the extremely low-grade and complex composition, the mechanism ofchalcopyrite bioleaching was not perfect and its industrial application was also not satisfied.In this paper, the research was started from the isolation of an extremeacidophilus-Acidithiobacillus thiooxidans (A. thiooxidans) and its extremely acid-resistancemechanism. Combination system and technology were respectively proposed for enhancingthis bioprocess. An identified and quantified method was established with the molecular probe.The community evolutions in different status were further revealed. The bioleachingmechanism model of low-grade chalcopyrite was initially established. The main studycontents were listed as follow:
(1) Isolation of the extremely acidophilus A. thiooxidans and its acid-proof mechanism.A double layers culturing method with heterotrophic strain-Rhodotorula sp. as the bottomculture was employed for efficiently isolating the chemoautotroph bioleaching microorganism.The cycle was reduced by1/3when the plating efficiency was improved by3.1times. Anextremely acidophilic sulfur-oxidizer strain was isolated from the leaching solution ofindustrial bioleaching heap in Fujian Zijin Mining. The capacity of inorganic acid-proofreached to pH0.2. The strain was identified as A. thioodidans ZJJN-3with the specialphysiological and double molecular characteristics. The strain ZJJN-3has been preserved inthe China Center for Type Culture Collection-CCTCC M2012104. Then the extremelyacid-proof mechanism was respectively investigated from the intracellular pH, cellmorphology, polysaccharide, membrane fatty acid composition, ATPase activity and thetranscription levels of acid-related proteins. With the increasing acid stress, the cell capsuleexperienced a process of significantly thickening and then gradually shedding off. The cellpolysaccharide content also increased and then dropped along with the evolution process. Theunsaturated fatty acid of cell membrane ratio significantly increased from37.85%to49.06%and cyclopropane fatty acids proportion remained at a high level about12.56%-18.59%. TheATPase activity was greatly strengthened, and the relative transcript levels of acid-proofsystem-GrpE/DnaK/DnaJ also increased to2.2-3.5times of original level. The acid-proofmechanism model in A. thioodidans ZJJN-3was further established basing on the aboveresults.
(2) Bioleaching of low-grade chalcopyrite by a synergistic two-strain system. Based onthe repeated fed-batch culture model in the Starkey-sulfide medium, the sulfur conversionpercentage and biomass productivity of A. thioodidans ZJJN-3was improved by31.1%and187.9%. The results of key biochemical parameters, Scanning electron microscope(SEM) andX ray diffraction (XRD) all implied that the biochemical activity was enhanced and thepassiavations were weakened with the synergistic effects of two strains. The bioleachingefficiency was1.81times of the pure Acidithiobacillus ferrooxidans CUMT-1(A. ferrooxidans CUMT-1) system. The pH-stat, Ag+and Cl-were respectively employed forreducing the passivation formation and accelerating the rate of biochemical reactions. Theoptimum combination system was determined as pH1.3-2.0mg·L-1Ag+-2.5g·L-1Cl-. Basedon the higher biomass and chemical reaction rate, the formation of jarosite precipitation andsulfur membrane was effectively weakened in the optimum combination system. The finalCu2+concentration and bioleaching efficiency reached55.5mg·L-1and55.0%, about twice ofthe control system. The exogenous energy substrates, multiple-phases pH-stat control andfed-batch fermentation strategies were respectively determnined to shorten strain-mineraladaptive phase, further weaken the jarosite passivation in the middle-end stage and reduce thesubstrate inhibition in batch bioleaching. The optimum integrated technology was determinedas2g·L-1Fe2++2g·L-1S0, three-stage pH-stat control (1.3-1.0-0.7) and fed-batch strategy(0.5+0.125×4). The verified results in the7L stirred tank fermenter showed that thebiological and chemical effects were better balanced in each bioleaching stage with theassistance of integration technology. The final Cu2+concentration, bioleaching efficiency andaverage production rate of Cu2+were89.1mg·L-1,44.1%and2.23mg·L-1·d-1, which wasimproved by about53%compared to the initial level.
(3) Establishment of molecular probe assay for identification and quantification ofbioleaching microorganism. With the assistances of molecular probe, eliminated function ofnuclease S1and fluorescently labeled technique, an assay was established for identifying andquantifying bioleaching microorganism. The specific probes were designed basing on theconserved16S rRNA regions of target species. The blasted result showed that there were atleast two or more bases differences with other closelty related strains. The key operatingconditions of moleacluar probe assay were futher optimized such as the sealing and elutedconditions of low non-specific adsorption (250μL0.1%BSA,30min,0.5%PBST elution),cell disruption (300W,50%duty cycle,4min, lysates secondary crushing), the workingconcentration of probes (primary probe100nM, CP10nM, SP10nM), hybridization time (1h,0.5h,0.5h), hybridization temperature (30℃,45℃,45℃), nuclease S1digestion time (20min) and working concentration of fluorescein antibody (1:5000diluted proportion). Thestandard curve of molecular probe assay was eventually established. The linear coefficientwas98.64%when the detection limitation was low to103cells·mL-1. The verification resultsof pure-strain, mixed-strains and bioleaching systems all showed a good specificity andaccuracy. It could be efficiently applied for analyzing the community structure in thesubsequent bioleaching process.
(4) The effects of community evolutions on bioleaching of low-grade chalcopyrite in thetwo-strain system. With the molecular probe technique, the evolution of microbial structure indifferent status and its effects were systematically explored. The effects of attached cell onbiomass of free cell were significant. Although with only3.8%-11.6%of total biomass, highto19.1%-31.2%loss of free cell was caused in the none-attached cell system, especially in thepure strain of A. thiooxidans ZJJN-3system. In the two-strains system, the lack of attachedcells would weaken the community competitiveness of A. thiooxidans ZJJN-3with A.ferrooxidans CUMT-1, which meant more dependence of A. thiooxidans ZJJN-3for attachedcell. The importance of attached cell was also proved by the changes chemical of key parameters. The largest reduction of Fe2+and SO24-concentrations were all high to about40%.
The results of XRD and fourier transform infrared spectroscopy (FTIR) analysis of slag allimplied that the sulfur metabolism was closely related with the attached cell.37.5%-50.5%oreven higher efficiency was closely associated with the adsorption process. On the basis of theequilibrium between biological and chemical effect, the techniques and strategies forenhancing chalcopyrite bioleaching was summarized. Futuremore, the bioleaching mechanismmodel of low-grade chalcopyrite was preliminarily established.
引文
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