尾矿硫氧化微生物的分离鉴定与功能验证

尾矿污染是全球备受关注的环境问题之一,其产量大、污染风险高,对环境造成了严重的威胁。尾矿条件下的硫氧化过程会产生氢离子,导致尾矿酸化,进而使得金属污染物活化和迁移,对周边环境造成危害。硫氧化微生物作为硫元素生物地球化学循环的重要参与者,其硫氧化代谢途径是尾矿中硫氧化的主要组成部分。因此,加强对尾矿原位条件下微生物驱动的硫氧化过程的理解尤为重要。然而,目前对尾矿中硫氧化微生物的分离鉴定及其对原位环境的影响仍需进一步探讨和解析。针对这一问题,该文以“世界锑都”锡矿山的尾矿为研究对象,采用平板划线法,在硫氧化条件下对尾矿原位微生物种群进行分离纯化,并结合16S rRNA和硫氧化基因(soxB)序列分析对其进行物种鉴定。同时,构建硫氧化条件微宇宙实验体系,对两株具有硫氧化基因的纯菌进行功能验证。除此以外,通过构建尾矿原位条件模拟实验体系,探究尾矿酸化与硫氧化微生物之间的联系。结果显示,该研究成功得到了两株具有硫氧化基因的纯菌,分别为Methyloversatilis sp.LJY-1和Limnobacter sp.LJY-2。硫氧化实验进一步表明,LJY-1是首次被发现的具有硫氧化功能的Methyloversatilis属类,并且LJY-1的硫氧化能力明显强于LJY-2。基于尾矿原位条件模拟实验,证明在尾矿原位条件下,微生物驱动的硫氧化过程显著加剧了尾矿酸化(P<0.05)。针对尾矿不同阶段中含水率的差异,该研究进一步揭示了淹水条件下尾矿酸化过程显著快于湿润条件(P<0.05)。研究结果对尾矿场地的管理和修复具有重要意义。

气体生物脱硫及硫回收研究进展

硫化氢脱除是石油天然气开采、炼化、煤化工、水处理等行业清洁安全生产所面临的重大任务之一。气体生物脱硫及硫回收是基于生物硫氧化原理建立起来的以硫磺回收为目标的湿法硫化氢脱除方法,具有生物催化剂可再生、生物硫磺不易堵塞等优点。围绕硫化氢脱硫的方法及应用研究,重点介绍了气体生物脱硫及硫回收过程机理的研究进展,分析总结其优势和特点,并根据现有研究不足展望其未来发展。

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.

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.

Characterization of apparent sulfur oxidation activity of thermophilic archaea in bioleaching of chalcopyrite

The apparent sulfur oxidation activities of four pure thermophilic archaea, Acidianus brierleyi （JCM 8954）, Metallosphaera sedula （YN 23）, Acidianus manzaensis （YN 25） and Sulfolobus metallicus （YN 24） and their mixture in bioleaching chalcopyrite were compared, which were characterized indirectly by the evolution of the cells concentration, pH value and sulfate ions concentration in solution. The results show that the mixed culture contributed significantly to the raising of leaching rate, which suggests that the mixed culture had a higher sulfur oxidation activity than the pure culture. Meanwhile, the results also indicate that the changes of parameters characterizing the sulfur oxidation activity of thermophilic archaea are often influenced by many factors, so it is hard to reflect accurately the specific sulfur oxidation activities among the different sulfur-oxidizing microbes when bioleaching chalcopyrite at different conditions. Accordingly, an efficient method to characterize microbial sulfur oxidation activity appears to be desirable.

微生物硫循环网络的研究进展

硫元素是所有生物的基本组成成分,是生物体必需的营养元素之一。硫氧化还原微生物的数量多、分布广、代谢途径多样化,硫化合物之间的平衡依赖于微生物代谢网络中的各种硫转化反应与代谢过程。此外,硫循环与碳、氮循环紧密相关,对地球生态循环起到了至关重要的作用。本文综述了近期微生物硫循环网络的研究进展,包括所涉及的主要微生物、硫循环的生物化学途径、硫循环的环境意义和工业应用潜能等,深入了解自然和人工生态系统中存在的硫循环过程,可为控制工农业生产中硫元素的增减与利用提供理论基础与应用方案。

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.

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.

Relationships among bioleaching performance, additional elemental sulfur, microbial population dynamics and its energy metabolism in bioleaching of chalcopyrite

To estimate the relationships among bioleaching performance, additional elemental sulfur （S0）, microbial population dynamics and its energy metabolism, bioleaching of chalcopyrite by three typical sulfur- and/or iron-oxidizing bacteria, Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum and Acidithiobacillus thiooxidans with different levels of sulfur were studied in batch shake flask cultures incubated at 30 °C. Copper dissolution capability （71%） was increased with the addition of 3.193 g/L S0, compared to that （67%） without S0. However, lower copper extraction was obtained in bioleaching with excessive sulfur. Microbial population dynamics during chalcopyrite bioleaching process was monitored by using PCR-restriction fragment length polymorphism （PCR-RFLP）. Additional S0 accelerated the growth of sulfur-oxidizing bacteria, inhibited the iron-oxidizing metabolism and led to the decrease of iron-oxidizing microorganisms, finally affected iron concentration, redox potential and bioleaching performance. It is suggested that mixed iron and sulfur-oxidizing microorganisms with further optimized additional S0 concentration could improve copper recovery from chalcopyrite.

Well-controlled stirring tank leaching to improve bio-oxidation efficiency of a high sulfur refractory gold concentrate

For the high sulfur refractory gold concentrate with 41.82%sulfur and 15.12 g/t gold,of which 82.11%was wrapped in sulfide,a well-controlled stirring tank leaching was carried out to improve the bio-oxidation efficiency.Results show that bio-oxidation pretreatment can greatly improve the gold recovery rate of high-sulfur refractory gold concentrate,and at the optimum pH 1.3 in this study,compared with the process without pH control,the oxidation rate of sulfur increased from 79.31%to 83.29%,while the recovery rate of gold increased from 76.54%to 83.23%;under this condition the activity of mixed culture could be sustained and the formation of jarosite could diminish.The results also displayed that for the high sulfur refractory gold concentrate,the recovery of gold is positively correlated with the oxidation rate of sulfur,and the recovery rate of gold increases with the increase of sulfur oxidation rate within a certain range.

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.

Formation of jarosite and its effect on important ions for Acidithiobacillus ferrooxidans bacteria

The precipitation of jarosite adversely affects the bio-leaching of copper sulfides in the Sarcheshmeh heap bio-leaching process. The variables of the initial concentration of ferrous iron in the growth medium, pH, and temperature were examined in the laboratory to determine how they affect the precipitation of jarosite in the presence of Acidithiobacillus ferrooxidans bacteria. It was found that the maximum ferric precipitate occurred at a ferrous sulfate concentration of 50 g/L, a temperature of 32 ℃, and an initial pH value of 2.2. The effects of the precipitation of ferric iron on the quantities of ions that are important for A. ferrooxidans bacteria in aqueous phase, i.e., ferric, sulfate, potassium, phosphate, and magnesium ions, also were assessed. The results showed relatively similar patterns for the ferric and potassium ions, and then reason might have been the co-precipitation of these ions as constituent elements of jarosite mineral. At pH values greater than 1.6, the solubility of phosphate ions decreased dramatically due to the co-precipitation of phosphate ions with the jarosite precipitate and due to the significant growth rate of A. ferrooxidans bacteria in this pH range. Due to the dissolution of a gangue constituent in the ore, the magnesium levels increased in the first few days of the bio-leaching process;thereafter, it decreased slightly.

Bio-decomposition of rock phosphate containing pyrites by Acidithiobacillus ferrooxidans

Leaching soluble phosphorus from rock phosphate containing pyrites by Acidithiobacillus ferrooxidans (A.f.) is feasible, and the reaction mechanism is as follows. Pyrites are oxidized by A.f. to produce H_2SO_4 and FeSO_4; the rock phosphate is decomposed by H_2SO_4, forming soluble phosphorus compounds; and Fe2+ from FeSO_4 is oxidized to Fe^3+, providing energy for the growth of A. f.. In this process, as H_2SO_4 is produced in the reaction, an acidic condition in the culture medium is formed, which benefits the growth of A. f. and aids both continuous oxidation of pyrites and leaching of soluble phosphorus from rock phosphate. The fraction of phosphorous leached can reach the largest in the presence of 1.0 g/L Fe^3+, 200 mg/L Mg^2+ and 400 mg/L NH_4^+. The optimal technological parameters on the fraction of phosphorous leached are as follows: the volume fraction of inocula of A. f., the mass ratio of pyrites to rock phosphate and the pH value are in ranges of 5%-25%, 3:1-5:1 and 1.8-2.2, respectively.

Bioleaching of chalcocite by mixed microorganisms subjected to mutation

Mixed microorganisms with elevated activity of chalcocite-leaching were screened by mutation methods. The original microorganisms collected from acid mine drainage of different sites were mixed and then treated with mutagens NO2^- , diethyl sulfate （DES）, UV and their combinations, respectively. Five groups of mixed microorganisms with much stronger ore-leaching ability were obtained by screening on the leaching media. Among them, group E of mixed microorganisms （treated with 1% DES for 60 min） with the best perfonnance on chalcocite-leaching, increases the content of Cu^2＋ by 101.4% in 20 d of leaching compared with the control culture. In addition, group E is more tolerant to Cu^2＋ in media than the control without mutation treatment. Analysis for the diversity of microbial clones indicates that half of operational taxonomic units （OTUs） in group E are Acidithiobacillus ferrooxidans. These observations suggest that group E might have potentials for industrial application.

Pilot Scale Test to Treat High Concentration Gasification Wastewater Using Catalytic Oxidation and Aerobic Biological Fluid-Bed Combination Process

The gasification wastewater is a kind of typical organic industrial wastewater with high chemical oxygen demand （COD） and ammonia nitrogen, which could not be completely degraded by the traditional physical, chemical and biological method. So it is very important to find an effective treatment process. A combination process of catalytic oxidation with noble metal catalysts and aerobic biological fluid-bed packed with the new ultrastructure biological carriers, which was developed by ourselves, was investigated to treat the gasification wastewater. The pilot scale test with 0.5 m^3/h influent flow was carried out to investigate the performance of this new combination process. The results showed that the effluent COD was 84.02 mg/L, ammonia nitrogen was 14.15 mg/L, and total phenol was 0.20 mg/L, which could completely meet the Grade I of Wastewater Discharge Standard （GB8978-1996）, when the influent average COD was 5 564 mg/L, ammonia nitrogen was 237 mg/L, and total phenol was 1 100 mg/L. The two catalytic reactors could evidently improve the wastewater biodegradability, and the value of BOD5/COD （B/C） increased from 0.23 to 0.413 in the one-stage catalytic reactor and from 0. 273 to 0.421 in two-stage catalytic reactor. The further experiment results showed that the effluent quality of this new combination process could still meet the discharge standard, when the COD loading was 8.65 kg / （m^3· d）. Most of aromatic and heterocyclic compounds were degraded effectively in this combination process.

Bioleaching of chalcopyrite by UV-induced mutagenized Acidiphilium cryptum and Acidithiobacillus ferrooxidans

The original strains Acidithiobacillusferrooxidans GF and Acidiphilium cryptum DXI-1 were isolated from the drainage of some caves riched in chalcopyrite in Dexing Mine in Jiangxi Province of China. The optimum temperature and pH for growth were 30 ℃ and 3.5 for Ac. cryptum DXI-1, and 30 ℃ and 2.0 for At. ferrooxidans GF, respectively. For Ac. cryptum DXI-1, the optimum UV radiating time was 60 s and the positive mutation rate was 22.5%. The growth curves show that Ac. cryptum after mutagenesis reached stationary phase within 60 h, which was 20 h earlier than the original strain. For At. ferrooxidans GF, the optimum mutation time was 60 s and the positive mutation rate was 35%. The most active UV-mutated strain At. ferrooxidans GF oxidized all the ferrous after 48 h. The bioleaching experiments showed that bioleaching with the mixture of UV-mutated strains of At. ferrooxidans GF and A c. cryptum DX1-1 （1：1） could extract 3.01 g/L of copper after 30 d, while the extracted copper was 2.63 g/L with the mixture of the original strains before UV-mutation. At the end of the bioleaching experiments, the proportion of the cell density in the cultures ofAc. cryptum DXI-1 andAt.ferrooxidans GF was approximately 1：5.

Enhanced Performance of Denitrifying Sulfide Removal Process by 1,2-Naphthoquinone-4-Sulphonate

The denitrifying sulfide removal(DSR) process with bio-granules comprising both heterotrophic and autotrophic denitrifiers can simultaneously convert nitrate, sulfide and acetate species into di-nitrogen gas, elemental sulfur and carbon dioxide, respectively, at high loading rates. This study has determined that the reaction rate of sulfide oxidized into sulfur could be enhanced in the presence of 1,2-naphthoquinone-4-sulphonate(NQS). The presence of NQS mitigated the inhibition effects of sulfide species on denitrification. Furthermore, the reaction rates of nitrate and acetate to nitrogen gas and CO_2, respectively, were also promoted in the presence of NQS, thereby enhancing the performance of DSR granules. The advantages and disadvantages of applying the NQS-DSR process are discussed.

Succession of Bacterial Community Inhabited Acid Mine Drainage under High Fe（Ⅱ） Concentration

To reveal the effects of Fe2＋ on bacterial communities in the early stages of minerals dissolution, two different acid mine drainage （AMD） samples were collected at Dabaoshan Mine and Shenbu Mine. Community successions of AMD niches were analyzed by Amplified Ribosomal DNA Restriction Analysis （ARDRA）, sequencing, and phylogenetic analysis in original AMD samples and their subculture under Fe2＋ concentrations. Although geochemical properties and community structures were greatly different between the two original AMD samples, bacterial community successions were still very similar under high Fe2＋ concentrations. The results showed that Acidithiobacillus ferrooxidans have competitive relationship with other bacterial species living in the AMD, including species that were also capable of oxidizing ferrous ion. A competitive relationship among different At. ferrooxidans strains likewise existed. Some of At. ferrooxidans can grow first under conditions of high ferrous ion concentration, and other At. ferrooxidans species decreased gradually and disappeared. This suggested that these species of At. ferrooxidans are most acidophilic bacteria and afford Fe3＋ to leach other metallic ion in the early stages of minerals dissolution.

高通量测序分析云南腾冲热海热泉微生物多样性

【背景】云南腾冲热海热泉中蕴含着丰富的极端微生物资源。【目的】揭示云南腾冲热海热泉中微生物物种多样性及群落结构差异,发掘酸性热泉中铁、硫氧化功能微生物。【方法】采用Illumina HiSeq高通量测序技术对3处热泉15个水体样品中微生物16SrRNA基因V4-V5区进行测序及生物信息学分析。【结果】3处热泉中共获得578061条有效序列,聚类为141个可操作分类单元(Operational taxonomic unit,OTU),包括19个门66个属。鼓鸣泉(GMQ)、蛤蟆嘴(HMZ)、黄瓜箐(HGQ)3处热泉均以泉古菌门(Crenarchaeota)和厚壁菌门(Firmicute)为主。从属水平分析,碱性热泉鼓鸣泉(GMQ)和中性热泉蛤蟆嘴(HMZ)分别注释到37、32个属,优势属均为芽孢杆菌属(Bacillus)和热棒菌属(Pyrobaculum)。酸性热泉黄瓜箐(HGQ)共注释到20个属,优势属为酸杆菌属(Acidibacillus)和酸硫杆状菌属(Acidithiobacillus),此外,具有铁、硫氧化潜力的菌属有喜酸菌属(Acidicaldus)、硫化芽孢杆菌属(Sulfobacillus)、硫化叶菌属(Sulfolobus)及生金球菌属(Metallosphaera)等,进一步通过硫氧化培养基分离获得了这些菌属中的纯菌株。【结论】云南腾冲热海热泉水体中蕴含丰富的微生物资源,热泉间微生物物种组成差异明显;酸性热泉中存在多种具有潜在铁、硫代谢功能的菌种;未分类类群、非培养类群丰度很高,尤其是蕴藏着可观的古菌资源。