氧化亚铁硫杆菌固定化技术研究

在生物脱硫过程中 ,以H - 2软性填料作为氧化亚铁硫杆菌 (Thiobacillusferrooxidans)的固定化载体 ,构建了固定床生化反应器。考察了不同稀释率固定下床生化反应器氧化Fe2 + 的情况 ,在通气量为 330L/h ,稀释率为 0 6h-1条件下 ,Fe2 + 最大氧化速率达 7 6 7g[Fe2 + ]/L·h。该反应器连续运行 10 0d,固定化细胞稳定性良好     

酸性工业气体的细菌脱硫

以软性纤维和玻璃钢蜂窝填料作为氧化亚铁硫杆菌(Thiobacillus ferrooxidans)P3—20菌株载体,制备了细菌生物膜反应器。该反应器在启动后连续运转30天左右,Fe2+氧化达到平衡状态,软性填料反应器的Fe2+氧化速率是蜂窝填料反应器的三倍。在通气量为250l／h,稀释率为0.165h-1。条件下,以溢流液中Fe2+氧化率≥95％为标准,软性填料反应器中lye2+平均氧化速率的最大值为1170.87mg L-1.h-1。利用细菌9K氧化液,通过穿流栅孔板塔对石油催化干气和沼气进行脱硫,在塔板数仅为三块的条件下,H2S的去除率分别为71.45％和46.91％。在化学吸收过程中所形成的硫磺,易于沉淀分离,纯度达95％以上。将分离液的pH值调至2.0后,即可进入牛物膜反应器中重新氧化,循环使用。该法不需高温、高压和催化剂,H2S的选择吸收性高,无废料排放,整个工艺呈闭路循环。     

紫外线和60Co辐照诱变选育高效嗜酸氧化亚铁硫杆菌

依次利用紫外线和60Co-γ射线辐照诱变的方法对嗜酸氧化亚铁硫杆菌进行诱变,选育高效嗜酸氧化亚铁硫杆菌菌株.结果表明,紫外诱变可以有效提高嗜酸氧化亚铁硫杆菌的Fe2+氧化速率,最佳紫外线诱变时间为240 s.诱变后菌株的Fe2+氧化速率从0.273 g/L/h提高到了0.312 g/L/h.继续利用60Co-γ射线进行...     

氧化亚铁硫杆菌培养过程中沉淀的研究

为了减少氧化亚铁硫杆菌培养过程中产生的沉淀,对氧化亚铁硫杆菌培养过程中产生的沉淀物进行了研究,确定了在pH为1．5,K2HPO4用量为0．25g/l,KH2PO4为0．195g/l时菌体可以保持其最高氧化活性,同时产生最少量沉淀物的培养条件,并发现沉淀物对菌体的生长和氧化Fe^2 没有影响。利用饥饿状态的氧化亚铁硫杆菌证明了菌体在一定条件下可以利用黄铁钒沉淀中的部分离子进行生长繁殖。     

生物脱硫中氧化亚铁硫杆菌固定化技术的研究

在生物脱硫过程中,以焦碳为填料作为固定化载体,进行了氧化亚硫杆菌的固定化技术研究。在初始pH2、温度为30℃左右、通气量0.5m3/h、喷淋量1.0L/h条件下,挂膜后只需12h,Fe2 氧化率可达95.28%,其Fe2 平均氧化速率是游离细胞时的8倍。氧化亚铁硫杆菌固定化细胞经长期低pH值驯化后,仍能保持对Fe2 具有较高的氧化活性;只需20hFe2 氧化率就达95.05%,Fe2 平均氧化速率达0.38g/(L/h)。     

巨大芽孢杆菌D01吸附金(Au3+)的研究

巨大芽孢杆菌(Bacillus megaterium)D01菌体吸附Au3+的最适pH值为30,其生物吸附作用是一种快速的过程,最初5min的吸附量可达到最大吸附量的95%,温度不影响该吸附作用。在pH3.0和30℃、起始金离子浓度与菌体浓度之比为305mg/g的条件下,吸附30min,吸附率达99.1%,吸附量为302.0mg/g干菌体。D01菌体能将溶液中的Au3+还原成Au0,在细胞表面和溶液中的Au0能形成不同形状的金晶体。浸渍在SiO2和αFe2O.3的Au3+能被D01菌体还原成Au0。从电化学反应表明,D01菌体对Au3+的还原具有较好的选择性。     

氧化亚铁硫杆菌铁氧化系统分子生物学研究进展

氧化亚铁硫杆菌（Thiobacillus ferrooxidans,简称T.f）是目前研究最多、最具经济价值的浸矿微生物。由于该菌的能量代谢对于生物浸矿起决定作用,因此其机制的研究必然能促进对该菌生理特性的认识及其遗传改造。氧化亚铁硫杆菌生长方式代表了迄今所知的能够进行生长的热力学极限,可供该菌生长利用的△Eh仅有340mV,氧化Fe2+所能得到的能量很少。在 Fe2+氧化过程中,电子通过电子传递链最终传递给氧,     

枯草芽孢杆菌中性内切β-甘露聚糖酶的纯化及性质

枯草芽孢杆菌(Bacillus subtilis)BM9602产生的中性内切β甘露聚糖酶(endoβ1,4Dmannan mannanohydrolase,EC,3.2.1.78)经硫酸铵分级沉淀、DEAE纤维素(DE22)离子交换柱层析,得到电泳纯的样品,提纯了455倍,收率为59%。用SDSPAGE测得该酶的分子量为35kD。用PAGEIEF测得其等电点pI为45。酶反应的最适pH为5.8,最适温度为50℃。该酶在pH60～80,50℃以下稳定。金属离子Hg2+和Ag+对酶活性强烈抑制。酶对槐豆胶、羟丙基瓜胶、田菁胶和魔芋粉的Km值分别为38、149、113和24mg/mL,Vmax值分别为245、865、384和198μmol.min-1mg-1。酶水解甘露聚糖为甘露寡糖(不含单糖)。     

产气肠杆菌几丁质酶的分离纯化及性质研究

从自然罹病死亡的草原毛虫(Gynephorap ruoergnesis)体内分离到一株产气肠杆菌(Enterobacter aerogenes),它在几丁质的诱导下能产生较高活性的几丁质酶。发酵液经硫酸铵盐析、DEAE纤维素柱层析和Sephadex G-100柱层析分离出几丁质酶。用SDSPAGE测得该酶的分子量为425kD。水解几丁质的Km值为2.88mg/mL-1。酶反应的最适温度为55℃,最适pH值为60,金属离子对几丁质酶活性影响较大,其中Zn2+、Ba2+、Ca2+和Mn2+对酶有较强的激活作用,而Hg2+、Co2+和Mg2+则有较强的抑制作用。     

利用绿色荧光蛋白基因gfp研究芽胞杆菌的启动子活性

利用绿色荧光蛋白基因gfpmut3,分别标记苏云金芽胞杆菌(Bacillus thuringiensis)的cry3A启动子Pcry3A、BtI_BtII启动子PBtI_BtII和来自蜡状芽胞杆菌特异启动子P44-12以研究其表达差异。其中,Pcry3A和PBtI_BtII分别与gfpmut3构成融合基因,以调控gfpmut3在苏云金芽胞杆菌中的表达。将重组质粒pGFP_304(含P44-12)、pGFPExpA(含Pcry3A_ gfpmut3融合基因)和pGFPExpB(含PBtI_BtII_ gfpmut3融合基因)分别导入大肠杆菌(Escherichia coli)和苏云金芽胞杆菌后发现,P44-12和PBtI_BtII在大肠杆菌与苏云金芽胞杆菌中均可表达gfpmut3,其中PBtI_BtII在大肠杆菌中具有极强的启动基因表达的能力。而Pcry3A不能启动gfpmut3在大肠杆菌中表达,在苏云金芽胞杆菌中启动的gfpmut3表达的荧光强度也较弱。进一步通过荧光显微镜和生物活性检测器对含重组质粒pGFP_304、pGFPExpA和pGFPExpB的转化子分别进行荧光检测及微量热检测。结果表明,3种启动子驱动下的gfpmut3基因均可在苏云金芽胞杆菌无晶体突变株BMB171中表达并检测得到不同的发光类型。微量热法检测发现P44_12和PBtI_BtII启动gfpmut3表达的代谢热低于Pcry3A驱动gfpmut3表达的代谢热。     

Ferrous sulphate oxidation using Thiobacillus ferrooxidans cells immobilised on sand for the purpose of treating acid mine-drainage

Thiobacillus ferrooxidans was immobilised on sand (size 0.85 mm to 1.18 mm) for use in a repeated batch and continuously operated packed-bed bioreactor which has not been previously reported in the literature. Repeated batch operation resulted in the complete oxidation of ferrous to ferric iron. The bacteria were active immediately after 3-4 weeks in a non-aqueous medium; i.e. the sand was allowed to dry out, demonstrating the stability of the system. A lag phase of 28 days was recorded when the sand was stored dried in a sealed container for 16 weeks compared with a lag phase of 13 days for a sample frozen for 18 weeks. After a period of 10 days, continuous operation of the reactor at a dilution rate of 0.64 h(-1) resulted in 95-99% oxidation of ferrous iron or 0.31-0.33 kg m(-3) h(-1). With the use of a scanning electron microscope, images were recorded of Thiobacillus ferrooxidans on sand.     

Mathematical model of the oxidation of ferrous iron by a biofilm of Thiobacillus ferrooxidans

Microbial oxidation of ferrous iron may be a viable alternative method of producing ferric sulfate, which is a reagent used for removal of H(2)S from biogas. The paper introduces a kinetic study of the biological oxidation of ferrous iron by Thiobacillus ferrooxidans immobilized on biomass support particles (BSP) composed of polyurethane foam. On the basis of the data obtained, a mathematical model for the bioreactor was subsequently developed. In the model described here, the microorganisms adhere by reversible physical adsorption to the ferric precipitates that are formed on the BSP. The model can also be considered as an expression for the erosion of microorganisms immobilized due to the agitation of the medium by aeration.     

Sulfite oxidation by iron-grown cells of Thiobacillus ferrooxidans at pH 3 possibly involves free radicals, iron, and cytochrome oxidase

Thiobacillus ferrooxidans cells grown on ferrous iron oxidized sulfite to sulfate at pH 3, possibly by a free radical mechanism involving iron and cytochrome oxidase. A purely chemical system with low concentrations of Fe3+ simulated the T. ferrooxidans system. Metal chelators, ethylenediamine tetraacetic acid (EDTA), 4,5-dihydroxy-1-3-benzene disulfonic acid (Tiron), o-phenanthroline, and 2,2'-dipyridyl, inhibited both sulfite oxidation systems, but the T. ferrooxidans system was inhibited only after the initial brief oxygen consumption. EDTA and Tiron, strong chelators of Fe3+, inhibited the oxidation at lower concentrations than o-phenanthroline and 2,2'-dipyridyl, strong chelators of Fe2+. Inhibition of Fe3+-catalyzed sulfite oxidation by EDTA and Tiron was instant, but the inhibition by o-phenanthroline and dipyridyl was briefly delayed, presumably for the reduction of Fe3+ to Fe2+. Mannitol, a free radical scavenger, inhibited both systems to the same extent. Cyanide and azide inhibited only the T. ferrooxidans system, suggesting a role of cytochrome oxidase. It is proposed that sulfite is oxidized by a free radical mechanism initiated by Fe3+ on the cell surface of T. ferrooxidans. Cytochrome oxidase is possibly involved in the regeneration of Fe3+ from Fe2+ by the normal Fe2+-oxidizing system of T. ferrooxidans.     

Fast kinetics of fe oxidation in packed-bed reactors

Thiobacillus ferrooxidans was used in fixed-film bioreactors to oxidize ferrous sulfate to ferric sulfate. Glass beads, ion-exchange resin, and activated-carbon particles were tested as support matrix materials. Activated carbon was tested in both a packed-bed bioreactor and a fluidized-bed bioreactor; the other matrix materials were used in packed-bed reactors. Activated carbon displayed the most suitable characteristics for use as a support matrix of T. ferrooxidans fixed-film formation. The reactors were operated within a pH range of 1.35 to 1.5, which effectively reduced the amount of ferric iron precipitation and eliminated diffusion control of mass transfer due to precipitation. The activated-carbon packed-bed reactor displayed the most favorable biomass holdup and kinetic performance related to ferrous sulfate oxidation. The fastest kinetic performance achieved with the activated-carbon packed-bed bioreactor was 78 g of Fe oxidized per liter per h (1,400 mmol of Fe oxidized per liter per h) at a true dilution rate of 40/h, which represents a hydraulic retention time of 1.5 min.     

High-rate acidophilic ferrous iron oxidation in a biofilm airlift reactor and the role of the carrier material

In this study, the feasibility and engineering aspects of acidophilic ferrous iron oxidation in a continuous biofilm airlift reactor inoculated with a mixed culture of Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans bacteria were investigated. Specific attention was paid to biofilm formation, competition between both types of bacteria, ferrous iron oxidation rate, and gas liquid mass transfer limitations. The reactor was operated at a constant temperature of 30 degrees C and at pH values of 0-1.8. Startup of the reactor was performed with basalt carrier material. During the experiments the basalt was slowly removed and the ferric iron precipitates formed served as a biofilm carrier. These precipitates have highly suitable characteristics as a carrier material for the immobilization of ferrous iron-oxidizing bacteria and dense conglomerates were observed. Lowering the pH (0.6-1) resulted in dissolution of the ferric precipitates and induced granular sludge formation. The maximum ferrous iron oxidation rate achieved in this study was about 145 molFe(2+)/m(3).h at a hydraulic residence time of 0.25 h. Optimal treatment performance was obtained at a loading rate of 100 mol/m(3).h at a conversion efficiency as high as 98%. Fluorescent in situ hybridization (FISH) studies showed that when the reactor was operated at high ferrous iron conversion (>85%) for 1 month, the desirable L. ferrooxidans species could out-compete A. ferrooxidans due to the low Fe(2+) and high Fe(3+) concentrations.     

Does immobilization of Thiobacillus ferrooxidans really decrease the effect of temperature on its activity?

It has been suggested that the influence of temperature on the activity of Thiobacillus ferrooxidans is decreased when the cells are immobilized. This is contrary to normal expectations and the work presented here indicates that it is not, in fact, the case. Experimental results are presented which show that the kinetics of Fe(II) oxidation by biofilms at 30°C are significantly faster than those at 20°C. Temperature, therefore, plays an important role in the activity of T. ferrooxidans, even in the immobilized form, and results indicate that it can also be used to control the level of biomass within the immobilized cell bioreactor.     

Preliminary proteomic analysis of Thiobacillus ferrooxidans growing on elemental sulphur and Fe2+ separately

Thiobacillus ferrooxidans is one of the most important bacterium used in bioleaching, and can utilize Fe2+ or sulphide as energy source. Growth curves for Thiobacillus ferrooxidans have been tested, which show lag, logarithmic, stationary and aging phases as seen in other bacteria. The logarithmic phases were from 10 to 32 hours for Thiobacillus ferrooxidans cultivated with Fe2+ and from 4 to 12 days for Thiobacillus ferrooxidans cultivated with elemental sulphur. Differences of protein patterns of Thiobacillus ferrooxidans growing on elemental sulphur and Fe2+ separately were investigated after cultivation at 30 degrees C by the analysis of two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ ionization (MALDI)-Mass spectrometry and ESI-MS/MS. From the 17 identified protein spots, 11 spots were found more abundant when growing on elemental sulphur. By contrast 6 protein spots were found decreased at elemental cultivation condition. Among the proteins identified, cytochrome C have been previously identified as necessary elements of electron-transferring pathway for Thiobacillus ferrooxidans to oxidize Fe2+; ATP synthase alpha chain and beta are expressed increased when Thiobacillus ferrooxidans cultivated with Fe2+ as energy source. ATP synthase Beta chain is the catalytic subunit, and ATP synthase alpha chain is a regulatory subunit. The function of ATPase produces ATP from ADP in the presence of a proton gradient across the membrane.     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.     

Existence of a new type of sulfite oxidase which utilizes ferric ions as an electron acceptor in Thiobacillus ferrooxidans.

A new type of sulfite oxidase which utilizes ferric ion (Fe3+) as an electron acceptor was found in iron-grown Thiobacillus ferrooxidans. It was localized in the plasma membrane of the bacterium and had a pH optimum at 6.0. Under aerobic conditions, 1 mol of sulfite was oxidized by the enzyme to produce 1 mol of sulfate. Under anaerobic conditions in the presence of Fe3+, sulfite was oxidized by the enzyme as rapidly as it was under aerobic conditions. In the presence of o-phenanthroline or a chelator for Fe2+, the production of Fe2+ was observed during sulfite oxidation by this enzyme under not only anaerobic conditions but also aerobic conditions. No Fe2+ production was observed in the absence of o-phenanthroline, suggesting that the Fe2+ produced was rapidly reoxidized by molecular oxygen. Neither cytochrome c nor ferricyanide, both of which are electron acceptors for other sulfite oxidases, served as an electron acceptor for the sulfite oxidase of T. ferrooxidans. The enzyme was strongly inhibited by chelating agents for Fe3+. The physiological role of sulfite oxidase in sulfur oxidation of T. ferrooxidans is discussed.     

Ferrous iron-dependent volatilization of mercury by the plasma membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe(2+) medium (pH 2.5) supplemented with 6 microM Hg(2+). In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 microM Hg(2+). When incubated for 3 h in a salt solution (pH 2.5) with 0.7 microM Hg(2+), resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe(2+) was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30 degrees C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe(2+)-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 microM Hg(2+) and 1 mM Fe(2+), plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe(2+)-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe(2+)-dependent mercury volatilization activity of the plasma membrane.