氧化亚铁硫杆菌生物浸出雄黄系统研究
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摘要
雄黄为四硫化四砷(As4S4)含量超过90%的天然砷化合物。在古代中国与印度医学中,雄黄通常被作为药物治疗多种疾病。近年来,体内外实验证实雄黄可有效治疗多种形式的癌症。药用雄黄的传统制备方法包括酸提、煅烧,膜分离,以及机械研磨,然而,药用雄黄水溶性低、胃肠道吸收差、毒性高,以及生物利用度低的缺点严重限制了它的临床应用。生物浸出技术是近年来发展起来的一项湿法冶金技术,我们将这一技术与药用雄黄制备交叉融合,利用天然嗜酸微生物对雄黄进行微生物浸出。最近的研究证实：氧化亚铁硫杆菌作用雄黄的浸出液在体内外实验中均显示很强的抗癌活性。相比传统制备方法,雄黄的生物浸出能显著增强雄黄的可溶性和生物利用度,并且该方法具有效率高,环境友好度高,低成本的特性。
论文测定了亚铁离子与元素硫在单一和混合能源中生物浸出雄黄的效率。在所有设定的浸出条件下,A. ferrooxidans BY-3显著增强雄黄的溶解。而且,在只有亚铁离子存在的浸出体系中砷的浸出效率要高于其它浸出实验组。在不添加任何能源的实验组中有较高的砷浓度浓度的,这一浸出砷浓度水平高于培养基中只有硫的浸出体系,但是低于培养基中添加了亚铁离子与元素硫的浸出体系。线性和非线性模型拟合了实验数据,非线性模型显示生物浸出雄黄存在砷的溶出与移除的双效作用,而在线性模型中只存在缓慢的浸出,而没有明显砷的移除现象。
在A. ferrooxidans BY-3对雄黄的生化改性研究中,通过SEM/EDS, XRD, Raman对生物浸出后的雄黄矿物表面特征,以及A. ferrooxidans BY-3粘附于雄黄表面的行为的分析,显示雄黄改性前后均未发现副雄黄的光谱,不存在雄黄的光化现象；同时,雄黄中所含有的少量As2O3在溶解于浸出体系之后,被菌株生物转化作用转化为低毒性的As(Ⅴ)。从而证实了生物浸出技术可有效解决传统雄黄炮制与贮存所面临的两大难题：氧化与光化作用对雄黄品质与药效的不利影响。
在A. ferrooxidans BY-3生物浸出雄黄环境参数的优化研究中,通过实验数据和线性模型的分析,确定了雄黄生物浸出的最优条件为：pH1.5,35℃,20%接种量,亚铁浓度1.0g/L,矿浆密度1.5%w/v。
在抗砷菌株A. ferrooxidans BY-3细胞表面化学研究中,TEM测定了A.ferrooxidans BY-3菌细胞表面存在吸附的颗粒物质,经XRD分析证实为Fe3+复合物[Fe3O4, KFe33+(SO4)2(OH)6,等]及含砷物质[KAsO3, Na2(H2As40g)],表明A. ferrooxidans BY-3对浸出体系中可溶性砷具有极强的吸附能力；FT-IR光谱显示A. ferrooxidans BY-3的表面含有多种的化学基团如-OH或-NH、-CH、-CN、蛋白的酰胺Ⅰ类-CONH-、蛋白的酰胺Ⅱ类-CONH-、-COOH、-CN、C-O、-CH。A. ferrooxidans BY-3细胞表面存在的这些化学基团在生物吸附中发挥重要作用。
在系统生物学思想指导下,论文应用生物信息学方法设计并初步构建了A.ferrooxidans生物浸出数据整合系统(DISBAf),包括生物浸出基质数据库(DBS),生物浸出相关金属离子螯合蛋白数据库(MIRP), A.ferrooxidans生物信息数据库(Af-info),嗜酸菌菌种数据库(DAf),以及生物浸出动力学模型数据库(DDMB),为进一步实现A.ferrooxidans生物浸出雄黄体系的计算机仿真,建立了研究平台。
Realgar is a naturally occurring arsenic sulfide. It contains over 90% tetra-arsenic tetra-sulfide (As4S4). In ancient Chinese and Indian medicine, realgar was commonly applied as a drug to treat various ailments. In recent years, realgar has been confirmed to be clinically effective for treatment of several forms of cancer in vitro and in vivo. Traditional means of preparing realgar include acid extraction, calcination, membrane dialysis, and mechanical milling. However, disadvantages such as low solubility in water, inadequate absorption of gastrointestinal, high toxicity, and poor bioavailability have seriously limited clinical applications of realgar. Bioleaching, a long-standing technology in hydrometallurgy, has recently been applied to preparation of realgar using indigenous acidophilic microorganisms in the field of pharmaceuticals. Recent studies have confirmed bioleaching solution with stronger antitumor activities, both in vitro and in vivo, of realgar extracted using A. ferrooxidans. It compared to the traditional methods mentioned above, bioleaching of realgar produces extraordinary increases in solubility and bioavailability and is efficient, ecologically safe, low cost and characterized by easy storage for realgar preparation. The characteristics of the bioleaching of realgar by Acidithiobacillus ferrooxidans BY-3(A. ferrooxidans) were investigated in this work. We examined the effects of using ferrous iron and elemental sulfur as the sole and mixed energy sources on the bioleaching of realgar. Under all experimental conditions, A. ferrooxidans BY-3 significantly enhanced the dissolution of realgar. Moreover, arsenic was more efficiently leached using A. ferrooxidans BY-3 in the presence of ferrous iron than in other culture conditions. A high concentration of arsenic was observed in the absence of alternative energy sources. This concentration was higher than that in cultures with sulfur only and lower than that in cultures with ferrous iron and sulfur. Linear or nonlinear models best fit the experimental data; the nonlinear model exhibited the dual effects of dissolution and removal on the bioleaching of realgar, whereas the linear model only applied to situations of slow bioleaching rather than removal.
The characteristics of biochemical modification of realgar using A. ferrooxidans were investigated. SEM/EDS, XRD, and Raman observations were performed to elucidate the surface morphology of powder realgar and its interactions with A. ferrooxidans BY-3. The results show pararealgar was not found in after and before bioleaching. It suggested that the light-induced alteration of realgar to pararealgar that does not exist. The bioleaching of realgar can decrease toxicity by A. ferrooxidans BY-3. Moreover, modified realgar are more soluble than crude realgar in the solution. Therefore, it has been proven that the bioleaching of realgar can effectively solve the problem of partial oxidation and light-induced degradation products in traditional methods of preparing realgar. The effects of a range of operational parameters, including temperature, pH, bacteria population, ferrous iron concentration, and pulp density, on the efficiency of the bioleaching process were investigated. Experimental results demonstrated that optimum conditions in bioleaching of realgar were as follows:pH,1.5; 35℃; inoculum (v/v),20%; ferrous iron concentration,1.0 g/L; pulp density (w/v),1.5%. Surface chemical studies of A. ferrooxidans with reference to arsenic tolerance were studied. TEM observations were performed to elucidate the surface A. ferrooxidans BY-3. The results show that the existence of particulate matter on the surface of the A. ferrooxidans BY-3. The X-ray diffraction (XRD) assay indicated that the particulate matter contains Fe3+ compound [Fe3O4, KFe33+(SO4)2(OH)6,.etc] and arsenic-containing substances [KAsO3, Na2(H2As4O8)]. Fourier transform infrared spectroscopy (FTIR) analysis revealed the existence of different chemical groups (-OH or-NH,-CH,-CN,-CONH-,-CONH-,-COOH,-CN, C-O,-CH) on the surface of A. ferrooxidans BY-3. These chemical groups have a vital role to play in uptake of metals and metalloids by A. ferrooxidans BY-3. Under the guideline of systems biology, we had been preliminary built data integration system for bioleaching of A. ferrooxidans (DISBAf), which contains a series of dataset, the database of bioleaching substrates (DBS), the database of bioleaching metallic ion-related proteins (MIRP), the database of A. ferrooxidans bioinformation (Af-info), the database of Acidithiobacillus spp. (DAf) and the database for dynamics's model of bioleaching (DDMB), respectively. DISBAf will be sever as a versatile database and analysis platform for bioleaching of realgar, and achievement simulation for computer simulation.
论文测定了亚铁离子与元素硫在单一和混合能源中生物浸出雄黄的效率。在所有设定的浸出条件下,A. ferrooxidans BY-3显著增强雄黄的溶解。而且,在只有亚铁离子存在的浸出体系中砷的浸出效率要高于其它浸出实验组。在不添加任何能源的实验组中有较高的砷浓度浓度的,这一浸出砷浓度水平高于培养基中只有硫的浸出体系,但是低于培养基中添加了亚铁离子与元素硫的浸出体系。线性和非线性模型拟合了实验数据,非线性模型显示生物浸出雄黄存在砷的溶出与移除的双效作用,而在线性模型中只存在缓慢的浸出,而没有明显砷的移除现象。
在A. ferrooxidans BY-3对雄黄的生化改性研究中,通过SEM/EDS, XRD, Raman对生物浸出后的雄黄矿物表面特征,以及A. ferrooxidans BY-3粘附于雄黄表面的行为的分析,显示雄黄改性前后均未发现副雄黄的光谱,不存在雄黄的光化现象；同时,雄黄中所含有的少量As2O3在溶解于浸出体系之后,被菌株生物转化作用转化为低毒性的As(Ⅴ)。从而证实了生物浸出技术可有效解决传统雄黄炮制与贮存所面临的两大难题：氧化与光化作用对雄黄品质与药效的不利影响。
在A. ferrooxidans BY-3生物浸出雄黄环境参数的优化研究中,通过实验数据和线性模型的分析,确定了雄黄生物浸出的最优条件为：pH1.5,35℃,20%接种量,亚铁浓度1.0g/L,矿浆密度1.5%w/v。
在抗砷菌株A. ferrooxidans BY-3细胞表面化学研究中,TEM测定了A.ferrooxidans BY-3菌细胞表面存在吸附的颗粒物质,经XRD分析证实为Fe3+复合物[Fe3O4, KFe33+(SO4)2(OH)6,等]及含砷物质[KAsO3, Na2(H2As40g)],表明A. ferrooxidans BY-3对浸出体系中可溶性砷具有极强的吸附能力；FT-IR光谱显示A. ferrooxidans BY-3的表面含有多种的化学基团如-OH或-NH、-CH、-CN、蛋白的酰胺Ⅰ类-CONH-、蛋白的酰胺Ⅱ类-CONH-、-COOH、-CN、C-O、-CH。A. ferrooxidans BY-3细胞表面存在的这些化学基团在生物吸附中发挥重要作用。
在系统生物学思想指导下,论文应用生物信息学方法设计并初步构建了A.ferrooxidans生物浸出数据整合系统(DISBAf),包括生物浸出基质数据库(DBS),生物浸出相关金属离子螯合蛋白数据库(MIRP), A.ferrooxidans生物信息数据库(Af-info),嗜酸菌菌种数据库(DAf),以及生物浸出动力学模型数据库(DDMB),为进一步实现A.ferrooxidans生物浸出雄黄体系的计算机仿真,建立了研究平台。
Realgar is a naturally occurring arsenic sulfide. It contains over 90% tetra-arsenic tetra-sulfide (As4S4). In ancient Chinese and Indian medicine, realgar was commonly applied as a drug to treat various ailments. In recent years, realgar has been confirmed to be clinically effective for treatment of several forms of cancer in vitro and in vivo. Traditional means of preparing realgar include acid extraction, calcination, membrane dialysis, and mechanical milling. However, disadvantages such as low solubility in water, inadequate absorption of gastrointestinal, high toxicity, and poor bioavailability have seriously limited clinical applications of realgar. Bioleaching, a long-standing technology in hydrometallurgy, has recently been applied to preparation of realgar using indigenous acidophilic microorganisms in the field of pharmaceuticals. Recent studies have confirmed bioleaching solution with stronger antitumor activities, both in vitro and in vivo, of realgar extracted using A. ferrooxidans. It compared to the traditional methods mentioned above, bioleaching of realgar produces extraordinary increases in solubility and bioavailability and is efficient, ecologically safe, low cost and characterized by easy storage for realgar preparation. The characteristics of the bioleaching of realgar by Acidithiobacillus ferrooxidans BY-3(A. ferrooxidans) were investigated in this work. We examined the effects of using ferrous iron and elemental sulfur as the sole and mixed energy sources on the bioleaching of realgar. Under all experimental conditions, A. ferrooxidans BY-3 significantly enhanced the dissolution of realgar. Moreover, arsenic was more efficiently leached using A. ferrooxidans BY-3 in the presence of ferrous iron than in other culture conditions. A high concentration of arsenic was observed in the absence of alternative energy sources. This concentration was higher than that in cultures with sulfur only and lower than that in cultures with ferrous iron and sulfur. Linear or nonlinear models best fit the experimental data; the nonlinear model exhibited the dual effects of dissolution and removal on the bioleaching of realgar, whereas the linear model only applied to situations of slow bioleaching rather than removal.
The characteristics of biochemical modification of realgar using A. ferrooxidans were investigated. SEM/EDS, XRD, and Raman observations were performed to elucidate the surface morphology of powder realgar and its interactions with A. ferrooxidans BY-3. The results show pararealgar was not found in after and before bioleaching. It suggested that the light-induced alteration of realgar to pararealgar that does not exist. The bioleaching of realgar can decrease toxicity by A. ferrooxidans BY-3. Moreover, modified realgar are more soluble than crude realgar in the solution. Therefore, it has been proven that the bioleaching of realgar can effectively solve the problem of partial oxidation and light-induced degradation products in traditional methods of preparing realgar. The effects of a range of operational parameters, including temperature, pH, bacteria population, ferrous iron concentration, and pulp density, on the efficiency of the bioleaching process were investigated. Experimental results demonstrated that optimum conditions in bioleaching of realgar were as follows:pH,1.5; 35℃; inoculum (v/v),20%; ferrous iron concentration,1.0 g/L; pulp density (w/v),1.5%. Surface chemical studies of A. ferrooxidans with reference to arsenic tolerance were studied. TEM observations were performed to elucidate the surface A. ferrooxidans BY-3. The results show that the existence of particulate matter on the surface of the A. ferrooxidans BY-3. The X-ray diffraction (XRD) assay indicated that the particulate matter contains Fe3+ compound [Fe3O4, KFe33+(SO4)2(OH)6,.etc] and arsenic-containing substances [KAsO3, Na2(H2As4O8)]. Fourier transform infrared spectroscopy (FTIR) analysis revealed the existence of different chemical groups (-OH or-NH,-CH,-CN,-CONH-,-CONH-,-COOH,-CN, C-O,-CH) on the surface of A. ferrooxidans BY-3. These chemical groups have a vital role to play in uptake of metals and metalloids by A. ferrooxidans BY-3. Under the guideline of systems biology, we had been preliminary built data integration system for bioleaching of A. ferrooxidans (DISBAf), which contains a series of dataset, the database of bioleaching substrates (DBS), the database of bioleaching metallic ion-related proteins (MIRP), the database of A. ferrooxidans bioinformation (Af-info), the database of Acidithiobacillus spp. (DAf) and the database for dynamics's model of bioleaching (DDMB), respectively. DISBAf will be sever as a versatile database and analysis platform for bioleaching of realgar, and achievement simulation for computer simulation.
引文
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