嗜热硫氧化硫化杆菌一新菌株的分离与鉴定

从云南腾冲酸性热泉富集物中分离到一株适度嗜热喜酸菌YN22．该菌革兰氏染色阳性，直杆状或微弯，长约1．6～2．8μm，直径0．4～0．7μm．该菌能在25～60℃下生长，最适生长温度为53℃；生长pH为1．0～5．0，最适pH为1．5；化能自养型，0．025％（w／V）的酵母提取物对其生长有明显的促进作用，在酵母提取物存在的情况下能快速氧化Fe^2＋，但对S0和还原型硫化物的氧化能力较低．16s rDNA系统发育分析表明，该菌与嗜热硫氧化硫化杆菌（Sulfobacillus thermosulfidooxidans）的16sr DNA序列相似性达99％．YN22基因组DNA的G＋C含量为47．3m01％，与嗜热硫氧化硫化杆菌模式菌株VKMB-1 269非常接近，后者基因组DNA的G＋C含量为47.5mol％．基于形态特征、生理生化特性、系统发育学和G＋C含量的分析结果，YN22应归于硫化杆菌属（Sulfobacillus），为嗜热硫氧化硫化杆菌（Sb．thermosulfidooxidans）的一新菌株．这是国内首次分离，并经多种方法鉴定、确认的嗜热硫氧化硫化杆菌，从而为我国浸矿微生物的基础和应用研究提供了最典型的适度嗜热菌种．

嗜热硫氧化硫化杆菌与喜温嗜酸硫杆菌混合浸出铁闪锌矿研究

采用正交试验,通过考察温度、pH值、喜温嗜酸硫杆菌接种时间和喜温嗜酸硫杆菌接种浓度4个因素,研究硫氧化硫化杆菌与喜温嗜酸硫杆菌混合菌对铁闪锌矿浸出的影响。试验结果表明:氧化硫细菌的加入,有助于消除铁闪锌矿浸出过程中生成的、覆盖在矿物表面的元素硫,使得硫氧化硫化杆菌和喜温嗜酸硫杆菌混合菌浸出铁闪锌矿的效果比单一硫氧化硫化杆菌浸出效果好;混合菌浸出铁闪锌矿时浸出率达到54.2%,而单一硫氧化硫化杆菌浸出时浸出率为46.8%。正交试验结果统计分析表明混合菌浸出铁闪锌矿的最优条件为:pH=1.8、第3 d接种喜温嗜酸硫杆菌和喜温嗜酸硫杆菌接种浓度2.5×106个/mL;其中pH值是影响混合菌浸出铁闪锌矿的主要因素,其次是喜温嗜酸硫杆菌接种浓度及喜温嗜酸硫杆菌接种时间。

嗜酸硫化杆菌(Sulfobacillus sp.)的分离鉴定及其在黄铁矿浸取中的应用

【目的】分离、培养获得中高温硫化矿浸取菌,为利用其进行生物冶金研究及应用奠定基础。【方法】利用二价铁或单质硫为底物,对酸性热泉泥水样品进行富集培养,以获得能够氧化二价铁或还原性硫的菌株;根据形态学、生理生化特点及系统发育分析对分离菌株进行分类鉴定;通过分析黄铁矿的铁氧化速率评估菌种在生物冶金中应用的潜力。【结果】从哥斯达黎加酸性热泉泥水样品中分离得到两株好氧嗜酸兼性自养细菌Costa C和Costa E。两株菌革兰氏染色反应为阳性,细胞大小相近,分别为(0.4-0.6)μm×(2.5-4.0)μm和(0.4-0.7)μm×(2.4-4.9)μm,端生芽胞,生长温度范围均为30℃-55℃,最适生长温度分别为50℃和40℃,生长pH范围分别为1.2-5.0和1.4-5.0,最适生长pH均为2.8。可以利用Fe(Ⅱ)、S、K2S4O6等为能源进行自养生长,也可利用酵母浸粉等有机物生长。两株菌的16S rRNA基因与硫化杆菌属(Sulfobacillus)其他菌种的最高相似性大于99%,(G+C)%含量分别为56.1 mol%和56.7 mol%。【结论】形态学、生理生化特点及系统发育分析表明,研究中筛选获得的两株菌均属于Sulfobacillus属,分别定名为Sulfobacillus sp.strain Costa C和Sulfobacillus sp.strain Costa E。浸矿结果显示两株菌在45℃时可以氧化黄铁矿,氧化速率分别为63.0 mg/L·d与56.8 mg/L·d,表明两株菌具有在中高温条件下浸取硫化矿的能力。

不同能源条件下中度嗜热嗜酸细菌多样性分析

采用黄铁矿、黄铜矿、硫酸亚铁和硫粉混合物作为主要能源物质在50℃条件下分别培养中度嗜热细菌混合物,研究其细菌多样性.提取细菌基因组总DNA,采用PCR结合限制性酶切片段多态性分析（RFLP）方法进行细菌16S rRNA基因的系统发育分析,比较不同能源条件下富集培养的混合细菌群落构成的差异.从3个培养物中共获得阳性克隆303个并进行RFLP分析,对29种不同酶切谱型的克隆插入序列进行测定和系统发育分析.大部分序列与已报道的浸矿微生物16S rRNA序列相似性较高（89.1%～99.7%）,归属于硫化叶菌属的耐温氧化硫化杆菌（Sulfobacillus thermotolerans）和热氧化硫化杆菌（Sulfobacillus thermosulfidooxidans）,嗜酸硫杆菌属的喜温硫杆菌（Acidithiobacillus caldus）,钩端螺旋菌属的嗜铁钩端螺旋菌（Leptospirillum ferriphilum）以及uncultured forest soil bacterium、uncultured proteobacterium.其中Acidithiobacillus caldus,Sulfobacillus thermotolerans,Leptospirillum ferriphilum 3种细菌为三类能源物质培养物中的优势细菌类群.L.ferriphilum在黄铁矿培养体系（53.8%）和硫酸亚铁和硫粉为能源的培养体系中（45.9%）中丰度最高;在以黄铜矿为能源物质的培养体系中,S.thermotolerans的比例大幅上升（70.1%）.

淋溶微生物菌株的筛选及其重金属溶出效率的研究

从汕头市12个蔬菜地监测点收集到多种微生物,采用含单质硫的Waksman固体培养基进行分离纯化,从中筛选出4株菌株。通过16S rRNA测序和NCBI数据库比对,其中两株为硫氧化硫化杆菌(Sulfobacillus disulfidooxidans),另两株为嗜酸性氧化硫杆菌(Acidithiobacillus thiooxidans),分别将其编号为ST-A、ST-B、ST-C和ST-D。通过研究这4株菌对Cu、Pb、Zn和Cr的溶出效率评估了它们对重金属的淋溶和转化潜力。结果表明:用这两种菌处理土样,土壤溶液的pH值显著降低,说明反应过程中产生了酸性物质;两种菌株均对Cu、Zn、Pb、Cr有明显的溶出效果;从修复过程中Cu、Pb五态质量比分布来看,经过浸提后,大部分Cu、Pb被去除,Cu去除率大于Pb去除率,硫化杆菌属的去除效果比嗜酸硫杆菌属明显,硫化杆菌能将不溶态的Cu和Pb转换为可溶态。

基于光谱分析的砷黄铁矿生物浸出过程中铁/砷/硫形态转化研究

基于同步辐射装置的As/S的K边及Fe的L边X射线吸收近边结构光谱(XANES)和X射线衍射(SR-XRD),结合扫描电镜(SEM)、傅里叶红外光谱(FTIR)、电感耦合等离子体发射光谱(ICP-AES)及各项浸出参数的测定,系统研究了(中度嗜热菌、嗜热硫化杆菌)浸出砷黄铁矿过程中铁、砷、硫的形态转化。结果表明,在生物作用下,砷黄铁矿的溶解速率明显高于化学浸出体系,伴随矿物溶解释放到溶液中的砷和铁在生物浸出体系中主要为As(Ⅴ)和Fe3+,而在无菌化学浸出体系则主要为As(Ⅲ)和Fe2+;细菌胞外多聚物(EPS)在细菌与硫化矿物的相互作用过程中起着至关重要的作用, FTIR的结果表明,生物浸出体系中吸附在矿物表面的吸附菌的EPS中蛋白质和多糖的含量均高于游离菌EPS;SEM的结果表明,砷黄铁矿表面在生物浸出过程中逐渐被腐蚀,且有浸出产物覆盖,而化学浸出10 d后,矿物表面依旧比较光滑;SR-XRD的结果表明,元素硫(S0)、黄钾铁矾和砷酸铁在生物浸出第4 d生成,并随时间延长逐渐累积,最终成为浸出渣中的主要成分。Fe的L边XANES结果表明,在细菌作用下矿物表面逐渐被Fe(Ⅲ)浸出产物覆盖;As的K边XANES结果表明,浸出渣中砷的价态包括As(-Ⅰ), As(Ⅲ)和As(Ⅴ),拟合结果表明,经过10 d的生物浸出,砷黄铁矿、雌黄(As2S3)和砷酸铁在矿渣中所占的比例分别为18.6%, 23.5%和57.9%,化学浸出10 d后,矿渣中除未溶解的砷黄铁矿外,仅有少量砷酸铁(6.2%)形成;S的K边XANES拟合结果表明,经过10 d的生物浸出,砷黄铁矿、 S0、硫代硫酸盐、施氏矿物和黄钾铁矾在矿渣中所占的比例分别为15.3%, 23.7%, 3.5%, 11.3%和46.2%,而在化学浸出10 d后的矿渣中,仅拟合到少量S0(7.8%)。基于上述结果可以得出,铁、砷、硫在砷黄铁矿生物作用下的形态转化过程分别为:Fe(Ⅱ)-Fe(Ⅲ), As(-Ⅰ)-As(Ⅲ)-As(Ⅴ), S-→S0→S2O■→SO■。结合溶液中的浸出参数发现,随着S0、黄钾铁矾、砷酸铁和雌黄的大量累积,砷黄铁矿的生物浸出严重受阻。硫代硫酸盐的生成表明砷黄铁矿的溶解途径与黄铁矿相似。

中温及嗜热菌对不同成因黄铜矿浸出行为的影响

以斑岩型黄铜矿和矽卡岩型黄铜矿为研究对象,考察了嗜酸氧化亚铁微螺菌(L f)和嗜热硫氧化硫化杆菌(S t)对不同成因黄铜矿浸出行为的影响。结果表明,在2种不同细菌浸出体系中矽卡岩型黄铜矿均表现出比斑岩型黄铜矿浸出率高;S t浸出2种不同成因黄铜矿的效率均比L f的好。通过对不同浸矿时间黄铜矿浸出渣的XRD检测并结合黄铜矿浸出过程反应步骤的分析表明,2种细菌浸出不同成因黄铜矿的机制相同,细菌的代谢途径及反应温度是影响同类成因黄铜矿浸出率和代谢产物差异的主要原因。S t作用下浸出后期黄铜矿表面有黄钾铁矾生成,而L f浸出体系黄铜矿表面主要是S的不断积累。同种浸矿菌种浸出不同成因黄铜矿时,矽卡岩型黄铜矿在浸出第15 d有S生成,斑岩型黄铜矿在S t浸出体系S生成的时间更晚,在L f浸出体系S的生成量则更少,推测矿物性质是引起其差异的主要原因。

Electrochemical oxidation behavior of pyrite bioleaching by Acidthiobacillus ferrooxidans

The electrochemical oxidation behavior of pyrite in bioleaching system of Acidthiobacillusferrooxidans was investigated by cyclic voltammetry （CV）, polarization curve and electrochemical impedance spectroscopy （EIS）. The results show that in the presence or absence of A. ferrooxidans, the oxidation reaction of pyrite is divided into two steps： the first reaction step involves the oxidation of pyrite to S, and the second reaction step is the oxidation of S to SO4^2-. The oxidation mechanism of pyrite is not changed in the presence of A. ferrooxidans, but the oxidation rate of pyrite is accelerated. With the extension of reaction time of A. ferrooxidan with pyrite, the polarization current density of pyrite increases and the breakdown potential at which the passive film dissolves decreases. The impedance in the presence ofA. ferrooxidans is obviously lower than that in the absence of A. ferrooxidans, further indicating that microorganism accelerates the corrosion process of pyrite.

Enhancement of bio-oxidation of refractory arsenopyritic gold ore by adding pyrolusite in bioleaching system

Pyrolusite was added in the bioleaching process to enhance the bio-oxidation process. Bioleaching tests at different dosages of pyrolusite ore, pH and inoculation amounts of Acidithiobacillus ferrooxidans were studied. The results showed that the time of the bio-oxidation process was decreased obviously and the arsenic leaching rate reached 94.4% after the bioleaching. The bio-oxidation of arsenopyrite and the effective extraction of manganese from pyrolusite were achieved by the bioleaching process. After bioleaching, the leaching rate of gold from the reaction residues reached 95.8% by cyanide leaching. In the bio-oxidation process, pyrolusite increased the redox potential of the solution to accelerate the bioleaching rate. The experiment showed that there were two reaction modes in the bioleaching process.

Decomposition mechanism of pentlandite during electrochemical bio-oxidation process

Electrochemical measurements were carried out to elucidate decomposition mechanism of pentlandite using modified powder microelectrode with Acidithiobacillus ferrooxidans attached or without on the mineral powder surface.Cyclic voltammetry（CV） results show that at a low potential of about-0.2 V（vs SCE）,the pentlandite was transformed to an intermediated phase like Fe4.5-yNi4.5-xS8-z when Fe and Ni ions were evacuated from mineral lattice;when the potential was changed from-0.2 V to 0.2 V,the unstable violarite（Fe3Ni3S4） and FeNi2S4 were formed which was accompanied by element sulfur formed on the mineral surface;when the potential increased over 0.2 V,the unstable intermediated phase decomposed entirely;at a higher potential of 0.7 V,the evacuated ferrous ion was oxidized to ferric ion.The presence of Acidithiobacillus ferrooxidans made the oxidation peak current increase with initial peak potential negatively moving,and the bacteria also contributed to the sulfur removing from mineral surface,which was demonstrated by the reduction characteristic at potential ranging from-0.75 to-0.5 V.Leaching experiments and electrochemical results show that the solution acidity increasing when pH2 may impede the oxidation process slightly.

Acidthiobacillus ferrooxidans中磁小体的提取(英文)

At.f和趋磁细菌在生理特性和生长环境有一定的相似性,而且镜检发现At.f具有趋磁性,所以本文采用了趋磁细菌中磁小体的提取方法尝试提取At.f中的磁小体,用超声波破碎At.f后,以磁铁吸取其体内的磁性颗粒,经过检测,发现其体内确实存在含铁元素的磁性颗粒。提取粗样品经过电镜分析,证实其体内存在着少量由脂质包裹的磁小体。磁小体悬浮液经过蔗糖密度梯度离心纯化后,对其作透射电镜,可以清晰的看到磁小体。实验结果表明,At.f体内存在少量的磁小体,正是由于磁小体的存在,才使得At.f在外加磁场作用下发生磁生物效应。这是首次发现从酸性矿坑水分离的At.f具有趋磁性,并从中提取到了磁小体,可以利用At.f的趋磁性将其按照不同磁性进行分离,从而获得活性高的、对不同磁性矿物有特异性的高效浸矿菌种。

Preservation efficiency of new cryoprotectant used for Acidithiobacillus ferrooxidans in liquid nitrogen

The efficiency of a new cryoprotectant,GP,for the preservation of Acidithiobacillus ferrooxidans（A.ferrooxidans） strain DC in liquid nitrogen was investigated.The optimal concentration of this new cryoprotectant for the maximal viable cell recovery and the highest ferrous ion oxidation activity was determined.The results show that 30%（volume fraction） GP is optimal for the cryopreservation with 84.4% of cells surviving,completely oxidizing ferrous ions within 120 h,and growing to a final density of 5.8×107 cell/mL after 6 d in the culture.Furthermore,the optimal residual GP concentration for viable cell recovery after culture of thawed cells in 9K medium for 6 d is 0.6%（volume fraction）.At this concentration,strain DC completely oxidizes ferrous ions within 108 h and grows to a final cell density of 6.8×107 mL-1.Thus,GP is a simple,effective cryoprotectant for the preservation of A.ferrooxidans strain DC in liquid nitrogen.

一株深海中等嗜热嗜酸菌的分离及鉴定

从太平洋热液区样品中分离纯化到一株中等嗜热嗜酸菌，命名为TPY。文中对该菌株的形态、生理生化特征、16S rDNA序列以及亚铁和单质硫氧化活性进行了研究。TPY菌株为短杆状，革兰氏阳性菌，大小为（0．3～0．5）μm×（1～3）μm；最适生长温度为50℃，最适生长pH值为1．8；该菌既能利用亚铁盐、单质硫自养生长，也能利用酵母粉、葡萄糖、蛋白胨和甘油等有机物异养生长；TPY菌与Sulfobacillus acidophilus（AB089842）的16S rDNA序列高度相似，其同源性为99％。这些结果表明，TPY菌是一株来自深海的嗜酸硫化芽孢杆菌（Sulfobacillus acidophilus），该菌的成功分离将有助于对太平洋热液区微生物种群结构的全面了解。同时，TPY菌对亚铁和单质硫的良好氧化能力显示出其在生物浸矿中的应用潜力。

Comparison of Fe^(2+) oxidation by Acidithiobacillus ferrooxidans in rotating-drum and stirred-tank reactors

Fe2＋ oxidation by Acidithiobacillus ferrooxidans（At.ferrooxidans） under different solid contents by adding inert Al2O3 powder was examined in rotating-drum and stirred-tank reactors.The results show that the bioactivity of At.ferrooxidans in the stirred-tank is higher than that in the rotating-drum in the absence of Al2O3 powder,but the biooxidation rate of Fe2＋ decreases markedly from 0.23 g/（L·h） to 0.025 g/（L·h） with increasing the content of Al2O3 powder from 0 to 50%（mass fraction） in the stirred-tank probably due to the deactivation of At.ferrooxidans resulting from the collision and friction of solid particles.The increase in Al2O3 content has a little adverse effect on the bioactivity of At.ferrooxidans in the rotating-drum due to different mixing mechanisms of the two reactors.The biooxidation rate of Fe2＋ in the rotating-drum is higher than that in the stirred-tank at the same content of Al2O3 powder,especially at high solid content.The higher bioactivity of At.ferrooxidans can be maintained for allowing high solid content in the rotating-drum reactor,but its application potential still needs to be verified further by the sulfide bioleaching for the property differences of Al2O3 powder and sulfide minerals.

编码结构域Tt APuX25基因片段的分离与克隆

[目的]将Tt apux25基因片段克隆到表达载体pET21a(t)中。[方法]以热产硫化氢高温厌氧杆菌菌液为模板,根据编码结构域Tt APuX25的基因片段Tt apux25设计1对特异引物进行PCR扩增。将Tt apux25克隆到表达载体pET21a(+)中,筛选阳性克隆并对其进行测序鉴定。[结果]通过PCR扩增获得一个250～500bp的扩增片段。对获得的阳性克隆进行筛选和测序鉴定,发现所得阳性克隆为表达载体pET21a(+)中插入有序列正确的Tt apux25片段。阳性克隆中的重组质粒被命名为pEX25,并将其转化进表达宿主菌大肠杆菌Tuner感受态细胞中。[结论]该方法直接将Tt apux25基因片段克隆到表达载体pET21a(+)中,操作简便并取得了较好的效果。

Effect of pH values on extracellular protein and polysaccharide secretions of Acidithiobacillus ferrooxidans during chalcopyrite bioleaching

The effect of pH values on the extracellular protein and polysaccharide secretions of Acidithiobacillus ferrooxidans was comparatively investigated in different phases of bacterial growth during chalcopyrite bioleaching. The results indicate that the extracellular protein is always more than the extracellular polysaccharide secreted by attached cells on the chalcopyrite, on the contrary, and is always less than the extracellular polysaccharide secreted by free cells in the solution at bacterial adaptive phase, logarithmic phase and stationary phase whenever pH value is at 1.0, 1.5, 2.0 or 2.5; free cells are mainly through the secretion of extracellular polysaccharide rather than the extracellular protein to fight against disadvantageous solution environment, such as high concentration of metal ions and unsuitable pH solution; both amounts of polysaccharide and protein secreted by attached cells are mainly positively related to the solution acidity rather than the total concentration of soluble metal ions. The experimental results imply that bacteria are mainly through secreting more extracellular polysaccharide to fight against disadvantageous environment and the extracellular protein perhaps plays an important role in oxidation？reduction reactions in the bioleaching system.

Bioleaching of low-grade copper sulfide ores by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans

The grown conditions of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans were investigated,and then experiments were conducted to research the bioleaching behaviors of crude ore of copper sulfide and hand-picked concentrates of chalcopyrite by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans.The experimental results show that the bacteria grow best when the temperature is(30±1) °C and the pH value is 2.0.The bacteria concentration is 2.24×107 mL-1 in this condition.It is found that the copper extraction yield is affected by the inoculum size and the pulp density and the extraction yield increases as the inoculum size grows.The bioleaching rates reach their highest point in sulfide copper and chalcopyrite with a pulp density of 5% and 10%,respectively.Column flotation experiments of low-grade copper sulfide ores show that the bioleaching recovery reaches nearly 45% after 75 days.

Bioleaching and electrochemical properties of chalcopyrite by pure and mixed culture of Leptospirillum ferriphilum and Acidthiobacillus thiooxidans

The bioleaching of chalcopyrite was investigated using a pure and mixed culture consisting of iron-oxidizing Leptospirillum ferriphilum （L. ferriphilum） and sulfur-oxidizing Acidthiobacillus thiooxidans （.4. thiooxidans）. The electrochemical tests were conducted to investigate the bioleaching behavior of chalcopyrite by various bacteria. Bioleaching efficiency of chalcopyrite in mixed culture is higher than that in the pure culture of L.ferriphilum alone. The iron-oxidizing L.ferriphilum plays a dominant role during bioleaching of chalcopyrite in the mixed culture of L. ferriphilum and A. thiooxidans. During bioleaching, certain values of redox potential are beneficial to the decomposition of chalcopyrite. Jarosite and sulfur are observed as products of bioleaching. The addition of A. thiooxidans during leaching by L. ferriphilum can change the electrochemical control steps of leaching. The corrosion current density is substantially promoted in the culture involving bacteria, especially in the mixed culture.

Elemental Sulfur Oxidation by Thiobacillus spp. and Aerobic Heterotrophic Sulfur-Oxidizing Bacteria

The prediction of the oxidation rate of elemental sulfur （S^0） is a critical step in sulfur （S） fertilizer strategy to supply plant-available sulfur. An incubation experiment was conducted to determine the rate and amount of S^0 oxidation in relation to the contribution of Thiobacillus spp. and aerobic heterotrophic S-oxidizing bacteria. After 84 days, 16.3% and 22.4% of the total S^0 applied to the soil were oxidized at 20 and 30 ℃, respectively. The oxidation of So proved to be a two-step process with a rapid oxidation during the first 28 days and a slow oxidation from then on. The highest oxidation rate of 12.8 μg S cm ^-2 d^-1 was measured during the first two weeks at 30 ℃. At 20 ℃ the highest oxidation rate of 10.2 μg S cm^-2 d^-1 was obtained from two to four weeks after start of the experiment. On an average the soil pH declined by 3.6 and 4.0 units after two weeks of experiment. At the same time the electric conductivity increased nine times. With the oxidation of S^0 the population of Thiobacillus spp. and aerobic heterotrophic S-oxidizing bacteria increased. The corresponding values for Thiobacillus spp. and aerobic heterotrophic S-oxidizing bacteria increased from 2.9 × 10^5 and 1.4 × 10^5 g^-1 soil at the start of the experiment to 4 × 10^8 and 5.6 × 10^8 g^-1 soil 14 days after S^0 application, respectively. No Thiobacillus spp. was present eight weeks after S^0 application. The results suggested that oxidation of residual S^0 completely relied oll aerobic heterotrophic S-oxidizing bacteria.

Really active form of fluorine toxicity affecting Acidithiobacillus ferrooxidans activity in bioleaching uranium

In order to determine the mechanism of bacterial tolerance to fluorine,Acidithiobacillus ferrooxidans ATCC 23270 was domesticated and studied under the conditions of different fluorine concentrations and pH values with or without treatment by Proteinase K.The bacterial activities were observed through measuring the changes of solution potentials by platinum electrode with Ag/AgCl reference electrode and the intracellular fluorine was determined by-uorine ion-selective electrode.The results indicated that the tolerance of Acidithiobacillus ferrooxidans ATCC 23270 to fluorine could be obviously improved by domestication,HF was the effective form of fluorine to affect the bacterial activity,and pH increase or concentration change of ions of strong complex ability with fluorine ions in solution could result in false appearance of high fluorine-resistant strain.Some proteins located in cell wall or cell membrane were intimately relative with the bacterial fluorine tolerance.