三类生物冶金微生物菌种的选育及其与矿物作用研究
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摘要
除氧化亚铁硫杆菌能浸出金属硫化矿，其它一些微生物也具有与矿物作用的能力，报道产胞外多糖的硅酸盐细菌胶质芽孢杆菌可以溶解铝硅酸盐矿物，产有机酸的黑曲霉真菌可以浸出氧化矿中的金属元素。作者在本研究中采用不同的方法分离筛选了以上三种类型的生物冶金微生物，并对它们的培养条件、浸矿生理及其与矿物作用效果进行了研究。
首先富集筛选了江西德兴铜矿、城门山铜矿、广东大宝山铜矿等六处矿坑水中的氧化亚铁硫杆菌，获得6个富集菌株。通过研究6个菌株的Fe~(2+)和S~0氧化能力，发现不同菌株的氧化活性存在差异，但发现Fe~(2+)氧化活性高的菌株S~0氧化活性也高。同时发现S~0培养基中的细菌浓度比Fe~(2+)培养基中细菌浓度高。使用DBS菌株浸出低品位含铁闪锌矿，浸出30d，金属锌的浸出率达到100％；浸出含铁闪锌矿精矿石，浸出率也可达到50％，说明该菌株具有良好的浸矿效果。
研究了氧化亚铁硫杆菌耐干燥、耐高温的抗逆性生理特性。发现该细菌具有较强的耐干燥能力，但不耐高温，55℃下细菌完全丧失氧化能力。同时研究了多种因素对氧化亚铁硫杆菌生长活性的影响，发现在Fe~(2+)氧化体系中添加0.25％固体物浓度的硫化矿物时，细菌的Fe~(2+)氧化速度会降低，细菌生长停滞期延长，浸出液中细菌浓度减少。当矿浆浓度增大时，由于矿物颗粒的运动及液体流动对菌体的机械损伤加剧，会使细菌的氧化活性进一步下降。在9K培养基中舔加1％的S~0时，细菌的Fe~(2+)氧化活性也会受到抑制。研究显示氧化亚铁硫杆菌具有耐受较高浓度金属离子的能力，但过高的金属离子浓度以及低浓度的有机物如细菌残体、葡萄糖、蛋白胨等会抑制细菌的氧化活性，当p[Zn~(2+)]＞50 g／L，ρ[Cu~(2+)]＞15g／L，ρ[Mg~(2+)]＞15 g/L时，细菌的Fe~(2+)氧化活性会显著降低。
本研究采用茚三酮比色法测定了浸矿过程中矿样表面吸附的细菌数量。首先研究了吸附细菌生物量的测定条件，发现细菌裂解液与茚三酮显色剂的反应产物在562 nm波长下有特征吸收峰，该吸光值的大小可代表细菌生物量的多少。研究发现在含铁闪锌矿摇瓶浸出20 d的过程中，矿粒表面的细菌吸附量由少到多，后期有50％～80％
的细菌吸附在矿粒表面。同时发现矿样粒径越小，比表面积越大，可
供吸附的位点越多，细菌吸附量就越大。通过柱浸体系中细菌的空间
分布情况研究，发现浸出20d，在浸矿柱的不同部位细菌分布有差异，
上层矿样吸附的细菌量多，下层矿样吸附的细菌量少。对细菌在矿物
表面的吸附过程及吸附机理进行了分析与探讨，认为矿物表面细菌生
物膜的形成有利于矿物的生物氧化，原因是生物膜中的细菌浓度要比
浸出液中高许多倍，在这种高细菌浓度的微环境中，FeZ十和S0的氧化
速度将大大加快。
研究发现，浸矿体系中细菌浓度过低是造成生物氧化速度慢的主
要原因之一。为了提高细菌的F扩十氧化速度，采用活性炭吸附固定细
菌，进行了小型生物反应器的研究。反应器中Fe2+的氧化速度可达到
9.38叭.h，是液体培养基中Fe扮氧化速度的20倍。研究发现反应器
中Fe2+氧化速度的提高主要是由于活性炭表面细菌浓度高，每克活性
炭表面吸附的细菌数量高达 4.69又10“个。因此认为活性炭可做为Fe2+
生物反应器中的细菌载体材料。
除氧化亚铁硫杆菌，本论文还首次进行了硅酸盐细菌和产酸黑曲
霉这两种生物冶金微生物的研究。
根据硅酸盐细菌胶质芽抱杆菌具有生物固氮及分解铝硅酸盐矿
物的特性，采用铝硅酸盐矿物无氮培养基从土样、铝土矿样等多个材
料中筛选到5个菌株。对其中生长速度最快、产胞外多糖最多的
GsY‘5“菌株进行了培养条件研究，发现该菌株在添加有高岭土、伊
利石、叶蜡石、石英等铝硅酸盐矿物的培养基中生长良好。通过正交
实验，发现该菌株在250 n1L锥形瓶中振荡培养的最适条件为pH 7.0、
温度30℃、转速200 r/min、装液量100毗。使用GsY一5#菌株浸出
由一水硬铝石分别与高岭石、伊利石、叶蜡石、石英配成的人工混合
铝土矿中的5102，矿浆浓度为5%，30℃下培养7d。结果表明一水
硬铝石分别与高岭石、伊利石、叶蜡石、石英配成的人工混合铝土矿
的AjS可由作用前的6.74、6.03、5.09、2.92分别提高到8.45、8.55、
6.79、13.54。铝土矿原矿石的刀S也从4.58提高到5.88。还发现硅
酸盐细菌能从伊利石中释放出K+。结果表明硅酸盐细菌具有一定的
脱硅、释钾能力。但发现硅酸盐细菌在培养及与矿物作用过程中很少
产酸，认为产酸不是该细菌脱硅的主要原因，胞外多糖对脱硅起主要
作用。
最后进行了产酸真菌黑曲霉菌株的筛选及浸矿效果研究。发现筛
选到的Asp一1“菌株具有较强的产酸能力，在查氏培养基中发酵培养7
d，发酵液的pH值能从6.5下降到2.0。用硫酸将恕p一1弃菌株发酵液
酸化到pH 0.5，在90℃下浸出铝土矿中的杂质铁，浸出6h，铝土矿
中FeZO3含量能从浸出前的6.36%下降到0.88%;采用同样方法浸出
在650℃下处理4h的高岭土，可以浸出高岭土中69.59%的金属铝;
使用Asp一1#培养液浸出氧化锌矿，浸出7d，锌的浸出率也可达到21 .9
%。研究结果表明黑曲霉Asp一1“菌株具有良好的浸矿效果。
A variety of microorganisms can catalyze the solubilization of minerals in natural environments. Besides Thiobacillus ferrooxidans, which can extract metal from metal sulfides, several other microorganisms such as silicate bacteria Bacillus mucilaginosus and fungi Aspergillus niger have been found to be progress in mineral bioleaching. Bacillus mucilaginosus can dissolve silicate from silicate minerals, and Aspergillus niger can dissolve metal from oxidative minerals. In this study, three types of microorganisms above have been screened from different materials using different isolating methods.
In study of Thiobacillus ferrooxidans, 6 strains have been isolated from Dexin Copper mine, Dabaoshan Copper mine, Chengmenshan Copper mine, Tongling Copper mine, Xixiashan Copper mine and Liuyang Pyrite mine using method of enriching culture. Research indicates different strains of Thiobacillus ferrooxidans process different Fe2+ and element sulfur oxidation activities. It has been found that bacterium density in S?medium is higher than that of in 9K Fe2+ medium. The Zinc extracting rate was 100 % when low grade sphalerite was bioleached with DBS strain for 30 days, and 50 % of sphalerite concentrate also can be biooxidized.
Two important physiological characteristics of Thiobacillus ferrooxidans, dryness tolerance and high temperature tolerance, have been researched in this study. The results show Thiobacillus ferrooxidans has excellent physiological characteristic of dryness tolerance and bad characteristic of high temperature tolerance because Thiobacillus ferrooxidans rapidly lose its oxidation activity at temperature of 55 ℃. Effects of several factors on oxidation activity of Thiobacillus ferrooxidans have been researched. Experimental results show the lag phase of Thiobacillus ferrooxidans was longer and bacterium density was decreased when 0.25 % pulp density of metal sulfides or 1% element sulfur was added in 9K medium. The research indicates metal sulfides and element sulfur may restrain Fe2+ oxidation activity of Thiobacillus ferrooxidans. It also be found that higher pulp density may decrease bacterial Fe2+ oxidation activity because the collision of mineral particles to bacterial cells will mechanically harm bacteri
al enzyme system. Experimental results show higher concentration of metal ions, for example [Zn2+]>50 g/L, [Cu2+]>15 g/L and [Mg2+]>15 g/L, may
IV
restrain Fe2+ oxidation activity of Thiobacillus ferrooxidcms although this microorganism can grow in medium containing definite concentrations of metallic ions, and Thiobacillus ferrooxidans was showed to be sensitive to organic substance such as bacterial residue, glucose and peptone.
Growth of Thiobacillus ferrooxidans on sphalerite surface and in 9K leach media was studied through estimation of bacterial protein using ninhydrin colorimetric method in the different leach phases. The mineral samples containing adhered bacterium are digested in 0.5 M NaOH in a boiling water bath for 25 min. The digested soup then filtered and the filtrate is neutralised to pH 7 using 0.5 M HC1. Then 1 mL of reagent ninhydrin is added to 2 mL of protein extracted solution and mixed thoroughly. The mixed solution is heated in a boiling water bath for 20 min, then the boiled solution is cooled for 6 min. After development of the color, the absorbance is measured at 562 nm using UV-1100 for the protein content, which provides a measure of the attached cell mass. This study indicated that the attachment of Thiobacillus ferrooxidans to sphalerite mineral is dependent on three factors, namely, particle size, expose period of the sphalerite mineral to the bacteria and as to whether stationary or agitation conditions are used during incubation. The results showed bacterial growth, both in the liquid and on the solid substrate, was found to be increased with increasing incubation periods for two size fractions of sphalerite mineral used. In this study 20 days of incubation were needed for any significant zinc dissolution. Experimental results showed 50 % -80 % of bacterial
首先富集筛选了江西德兴铜矿、城门山铜矿、广东大宝山铜矿等六处矿坑水中的氧化亚铁硫杆菌，获得6个富集菌株。通过研究6个菌株的Fe~(2+)和S~0氧化能力，发现不同菌株的氧化活性存在差异，但发现Fe~(2+)氧化活性高的菌株S~0氧化活性也高。同时发现S~0培养基中的细菌浓度比Fe~(2+)培养基中细菌浓度高。使用DBS菌株浸出低品位含铁闪锌矿，浸出30d，金属锌的浸出率达到100％；浸出含铁闪锌矿精矿石，浸出率也可达到50％，说明该菌株具有良好的浸矿效果。
研究了氧化亚铁硫杆菌耐干燥、耐高温的抗逆性生理特性。发现该细菌具有较强的耐干燥能力，但不耐高温，55℃下细菌完全丧失氧化能力。同时研究了多种因素对氧化亚铁硫杆菌生长活性的影响，发现在Fe~(2+)氧化体系中添加0.25％固体物浓度的硫化矿物时，细菌的Fe~(2+)氧化速度会降低，细菌生长停滞期延长，浸出液中细菌浓度减少。当矿浆浓度增大时，由于矿物颗粒的运动及液体流动对菌体的机械损伤加剧，会使细菌的氧化活性进一步下降。在9K培养基中舔加1％的S~0时，细菌的Fe~(2+)氧化活性也会受到抑制。研究显示氧化亚铁硫杆菌具有耐受较高浓度金属离子的能力，但过高的金属离子浓度以及低浓度的有机物如细菌残体、葡萄糖、蛋白胨等会抑制细菌的氧化活性，当p[Zn~(2+)]＞50 g／L，ρ[Cu~(2+)]＞15g／L，ρ[Mg~(2+)]＞15 g/L时，细菌的Fe~(2+)氧化活性会显著降低。
本研究采用茚三酮比色法测定了浸矿过程中矿样表面吸附的细菌数量。首先研究了吸附细菌生物量的测定条件，发现细菌裂解液与茚三酮显色剂的反应产物在562 nm波长下有特征吸收峰，该吸光值的大小可代表细菌生物量的多少。研究发现在含铁闪锌矿摇瓶浸出20 d的过程中，矿粒表面的细菌吸附量由少到多，后期有50％～80％
的细菌吸附在矿粒表面。同时发现矿样粒径越小，比表面积越大，可
供吸附的位点越多，细菌吸附量就越大。通过柱浸体系中细菌的空间
分布情况研究，发现浸出20d，在浸矿柱的不同部位细菌分布有差异，
上层矿样吸附的细菌量多，下层矿样吸附的细菌量少。对细菌在矿物
表面的吸附过程及吸附机理进行了分析与探讨，认为矿物表面细菌生
物膜的形成有利于矿物的生物氧化，原因是生物膜中的细菌浓度要比
浸出液中高许多倍，在这种高细菌浓度的微环境中，FeZ十和S0的氧化
速度将大大加快。
研究发现，浸矿体系中细菌浓度过低是造成生物氧化速度慢的主
要原因之一。为了提高细菌的F扩十氧化速度，采用活性炭吸附固定细
菌，进行了小型生物反应器的研究。反应器中Fe2+的氧化速度可达到
9.38叭.h，是液体培养基中Fe扮氧化速度的20倍。研究发现反应器
中Fe2+氧化速度的提高主要是由于活性炭表面细菌浓度高，每克活性
炭表面吸附的细菌数量高达 4.69又10“个。因此认为活性炭可做为Fe2+
生物反应器中的细菌载体材料。
除氧化亚铁硫杆菌，本论文还首次进行了硅酸盐细菌和产酸黑曲
霉这两种生物冶金微生物的研究。
根据硅酸盐细菌胶质芽抱杆菌具有生物固氮及分解铝硅酸盐矿
物的特性，采用铝硅酸盐矿物无氮培养基从土样、铝土矿样等多个材
料中筛选到5个菌株。对其中生长速度最快、产胞外多糖最多的
GsY‘5“菌株进行了培养条件研究，发现该菌株在添加有高岭土、伊
利石、叶蜡石、石英等铝硅酸盐矿物的培养基中生长良好。通过正交
实验，发现该菌株在250 n1L锥形瓶中振荡培养的最适条件为pH 7.0、
温度30℃、转速200 r/min、装液量100毗。使用GsY一5#菌株浸出
由一水硬铝石分别与高岭石、伊利石、叶蜡石、石英配成的人工混合
铝土矿中的5102，矿浆浓度为5%，30℃下培养7d。结果表明一水
硬铝石分别与高岭石、伊利石、叶蜡石、石英配成的人工混合铝土矿
的AjS可由作用前的6.74、6.03、5.09、2.92分别提高到8.45、8.55、
6.79、13.54。铝土矿原矿石的刀S也从4.58提高到5.88。还发现硅
酸盐细菌能从伊利石中释放出K+。结果表明硅酸盐细菌具有一定的
脱硅、释钾能力。但发现硅酸盐细菌在培养及与矿物作用过程中很少
产酸，认为产酸不是该细菌脱硅的主要原因，胞外多糖对脱硅起主要
作用。
最后进行了产酸真菌黑曲霉菌株的筛选及浸矿效果研究。发现筛
选到的Asp一1“菌株具有较强的产酸能力，在查氏培养基中发酵培养7
d，发酵液的pH值能从6.5下降到2.0。用硫酸将恕p一1弃菌株发酵液
酸化到pH 0.5，在90℃下浸出铝土矿中的杂质铁，浸出6h，铝土矿
中FeZO3含量能从浸出前的6.36%下降到0.88%;采用同样方法浸出
在650℃下处理4h的高岭土，可以浸出高岭土中69.59%的金属铝;
使用Asp一1#培养液浸出氧化锌矿，浸出7d，锌的浸出率也可达到21 .9
%。研究结果表明黑曲霉Asp一1“菌株具有良好的浸矿效果。
A variety of microorganisms can catalyze the solubilization of minerals in natural environments. Besides Thiobacillus ferrooxidans, which can extract metal from metal sulfides, several other microorganisms such as silicate bacteria Bacillus mucilaginosus and fungi Aspergillus niger have been found to be progress in mineral bioleaching. Bacillus mucilaginosus can dissolve silicate from silicate minerals, and Aspergillus niger can dissolve metal from oxidative minerals. In this study, three types of microorganisms above have been screened from different materials using different isolating methods.
In study of Thiobacillus ferrooxidans, 6 strains have been isolated from Dexin Copper mine, Dabaoshan Copper mine, Chengmenshan Copper mine, Tongling Copper mine, Xixiashan Copper mine and Liuyang Pyrite mine using method of enriching culture. Research indicates different strains of Thiobacillus ferrooxidans process different Fe2+ and element sulfur oxidation activities. It has been found that bacterium density in S?medium is higher than that of in 9K Fe2+ medium. The Zinc extracting rate was 100 % when low grade sphalerite was bioleached with DBS strain for 30 days, and 50 % of sphalerite concentrate also can be biooxidized.
Two important physiological characteristics of Thiobacillus ferrooxidans, dryness tolerance and high temperature tolerance, have been researched in this study. The results show Thiobacillus ferrooxidans has excellent physiological characteristic of dryness tolerance and bad characteristic of high temperature tolerance because Thiobacillus ferrooxidans rapidly lose its oxidation activity at temperature of 55 ℃. Effects of several factors on oxidation activity of Thiobacillus ferrooxidans have been researched. Experimental results show the lag phase of Thiobacillus ferrooxidans was longer and bacterium density was decreased when 0.25 % pulp density of metal sulfides or 1% element sulfur was added in 9K medium. The research indicates metal sulfides and element sulfur may restrain Fe2+ oxidation activity of Thiobacillus ferrooxidans. It also be found that higher pulp density may decrease bacterial Fe2+ oxidation activity because the collision of mineral particles to bacterial cells will mechanically harm bacteri
al enzyme system. Experimental results show higher concentration of metal ions, for example [Zn2+]>50 g/L, [Cu2+]>15 g/L and [Mg2+]>15 g/L, may
IV
restrain Fe2+ oxidation activity of Thiobacillus ferrooxidcms although this microorganism can grow in medium containing definite concentrations of metallic ions, and Thiobacillus ferrooxidans was showed to be sensitive to organic substance such as bacterial residue, glucose and peptone.
Growth of Thiobacillus ferrooxidans on sphalerite surface and in 9K leach media was studied through estimation of bacterial protein using ninhydrin colorimetric method in the different leach phases. The mineral samples containing adhered bacterium are digested in 0.5 M NaOH in a boiling water bath for 25 min. The digested soup then filtered and the filtrate is neutralised to pH 7 using 0.5 M HC1. Then 1 mL of reagent ninhydrin is added to 2 mL of protein extracted solution and mixed thoroughly. The mixed solution is heated in a boiling water bath for 20 min, then the boiled solution is cooled for 6 min. After development of the color, the absorbance is measured at 562 nm using UV-1100 for the protein content, which provides a measure of the attached cell mass. This study indicated that the attachment of Thiobacillus ferrooxidans to sphalerite mineral is dependent on three factors, namely, particle size, expose period of the sphalerite mineral to the bacteria and as to whether stationary or agitation conditions are used during incubation. The results showed bacterial growth, both in the liquid and on the solid substrate, was found to be increased with increasing incubation periods for two size fractions of sphalerite mineral used. In this study 20 days of incubation were needed for any significant zinc dissolution. Experimental results showed 50 % -80 % of bacterial
引文
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