高矿浆浓度下黄铜矿浸出过程中微生物群落结构变化规律

目的：为阐明微生物群落演替及功能与浸出效率之间关系奠定基础,以及如何提高黄铜矿生物浸出效率和铜回收率提供理 论依据。方法：通过连续传代培养进行驯化,使得复合菌群的矿浆浓度耐受能力达到25 %（w/v）。采用该复合菌群在25 %矿浆浓 度下浸出黄铜矿,同时利用变性梯度凝胶电泳和克隆文库技术分析浸出过程中的微生物多样性。最后,采用实时荧光定量PCR 对 浸出过程中微生物群落结构进行定量解析。结果：28天内黄铜矿浸出率能够达到95.1 %,而驯化前的浸出率只有51.5%。该复合 菌群主要由Acidithiobacillus caldus, Sulfobacillus acidophilus,和Fereoplasma theroplasma thermophilum组成,其中Acidithbacillus caldus是浸出前期和后期的优势种群,而Sulfobacillus acidophilus在浸出中期均有竞争优势, Ferroplasma thermophilum在整个浸出过程中占 据整个群落的比例均较低。结论：本研究获得的复合菌群具有较强的浸出黄铜矿能力, Acidithiobacillus caldus和Sulfobacillus acidophilus在浸出过程中起着重要的作用,pH 值和铜浸出率与群落结构相关性较高。     

柱式反应器处理黄铜矿过程中等嗜热微生物群落变化

从中国的多个铜矿取样,在45°C条件下富集获得了一种高效的中等嗜热浸矿富集物,探讨了该富集物在柱式反应器中浸出低品位黄铜矿的pH变化以及与Cu2+浸出的关系,并采用限制性片段长度多态性(RFLP)技术分析了微生物的群落结构和种群动态变化规律。结果表明在整个浸出过程中pH变化较为明显,且一直在1.8以上,60 d内回收了13.6%的铜。RFLP结果表明:在初期,嗜铁钩端螺旋菌(Leptospirillum ferriphilum)在浸出前期占有很高比例(81%),随后逐渐降低,至后期只有13%,而耐温氧化硫化杆菌(Sulfobacillus thermotolerans)和喜温硫杆菌(Acidithiobacillus caldus)的比例逐渐升高,在中期分别达到32%和23%;至末期,耐温氧化硫化杆菌达到了79%,成为优势种群。研究加深了对中等嗜热微生物浸矿特性的了解,也为中等嗜热菌处理低品位黄铜矿的工业应用提供了可供借鉴的数据。     

铜耐受定向驯化强化嗜酸喜温硫杆菌浸出贫黄铜矿

生物浸出贫黄铜矿后期,累积的Cu2+会对微生物产生胁迫效应从而影响浸出效率,因此该类微生物的铜耐受性定向驯化变得非常关键。对嗜酸喜温硫杆菌(Acidithiobacillus caldus)进行6个月铜耐受定向驯化,将出发菌株与驯化菌株在不同铜胁迫浓度下(0、1和3 g/L Cu2+)纯培养及浸出贫黄铜矿,并比较分析关键参数变化。在纯培养体系中,3 g/L Cu2+胁迫下最高比生长速率(μmax)由驯化前0.21 d-1(13 d)提升至0.54 d-1(10 d)。在0、1和3 g/L Cu2+浸出体系中,菌株驯化后铜浸出率分别较出发菌株提高17.64%、70.93%及306.09%。扫描电镜(SEM)分析矿渣形貌差异表明伴随胁迫作用增强菌体对矿物腐蚀程度变弱,在相同铜胁迫水平下,驯化菌株浸出体系矿渣表面呈现更多潜在的吸附位点及明显腐蚀痕迹。采用傅里叶红外光谱(FTIR)分析矿渣表面关键官能团变化,驯化菌株浸出体系的矿渣呈现更多含硫基团。X射线衍射(XRD)分析矿渣成分表明驯化菌株浸出体系中呈现较为丰富的铁硫衍生物如Fe3O4、FeS。综上,驯化后A. caldus具备较强抵御铜胁迫能力,在浸出体系中保持了更活跃的生化浸出效应,有望在类似的工业生物浸出过程中发挥其潜在优势。     

兴安落叶松林不同强度火烧迹地土壤微生物群落特性研究

通过选取大兴安岭岭北部的兴安落叶松林重度、轻度火烧迹地以及为过火样地,运用磷脂脂肪酸分析方法（PLFAs）,研究了火烧对0~5和5~10 cm土层的土壤理化性质和土壤微生物群落的结构特征的影响,并探讨了火烧当年土壤微生物群落生物量和群落结构的变化规律与影响因素。研究结果表明：0~5和5~10 cm土层的土壤pH、全钾、有效磷、黏砂比等土壤理化指标受到了火烧的显著性影响;不同火烧程度对微生物类群的生物量有影响,但不显著;重度火烧迹地的土壤微生物的群落结构指标革兰氏阳性菌/革兰氏阴性菌（G+/G-）以及真菌/细菌（F/B）与轻度和未过火样地具有显著差异。RDA分析指出,G+/G-受土壤含水量影响最大,F/B受pH影响最大。说明在火烧迹地的当年,土壤水分和pH是影响土壤微生物群落结构的最重要因素。     

BP神经网络优化微生物浸矿工艺

以低品位黄铜矿溶液为原料,浸矿制备Cu2＋能有效提高低品位黄铜矿的利用价值。基于浸矿过程中存在多因素影响的现象,通过正交试验与神经网络分析方法,对浸矿条件（接种量、矿石品位、Fe2＋添加量及浸矿溶液pH）实行优化。结果表明：在正交试验组中最佳试验结果为浸矿产128．753mg／LCu^2＋;BP神经网络优化后的最佳实验组合为微生物接种量12％、矿石品位0．3％、添加Fe^2＋24g/L及浸矿溶液pH1．7,该条件下验证试验产Cu^2＋ 141．352mg／L,通过正交试验及神经网络优化提高了微生物浸出低品位黄铜矿酸性溶液Cu^2＋的产量。     

生物浸矿反应器中的微生物种群结构及其中可培养微生物的特征

【目的】本文旨在了解生物浸矿反应器中的微生物种群结构及其中可培养微生物的特征。【方法】通过构建微生物冶金反应器中矿浆原样的16S rRNA基因文库,测定16S rRNA基因序列,分析矿浆中种群结构。同时在不同培养条件下,对样品进行富集培养,分离获得纯菌株;并对各个菌株的16S rRNA基因序列,生理生化特征及对不同矿物的氧化能力进行了分析。【结果】研究中所选生物浸矿反应器中主要的微生物物种有细菌:Leptospirillum sp.,Sulfobacillus sp.,Acidithiobacillus sp.,Spingomonas sp.及古菌Sulfolobus sp.,Ferroplasma sp.等菌属。同时分离出5株纯菌株,这些菌分别与Acidithiobacillus thiooxidans,Acidithiobacillus caldus,Acidithiobacillus ferrooxidans,Leptospirillum ferriphilum,Sulfobacillus thermosul fidooxidans相似。分离获得的菌株具有氧化硫或二价铁和不同硫化矿的能力。【结论】生物浸矿反应器是个微生物种类相对简单的生境,利用非培养和培养技术全面地了解生物浸矿体系中的微生物群落及其生理、浸矿特性,有利于洞察生物浸矿过程中微生物种群结构,强化控制种群组成及浸矿活性,从而提高生物湿法冶金的效率。     

川西亚高山三种天然次生林土壤微生物多样性变化特征

开展川西亚高山相似土壤母质背景下天然次生林土壤微生物群落结构及其多样性探究,可加深次生林更新过程中土壤微生物群落结构变化的认知。选取川西米亚罗林区20世纪60年代采伐后经自然更新恢复形成的3种天然次生林（槭-桦阔叶林,ABB;桦-槭-冷杉针阔混交林,BAA;岷江冷杉林,AFF）,分析林下表层（0-20 cm）土壤微生物群落结构变化及其影响因素,结果显示：（1）3种林型土壤细菌Chao1和Shannon指数均极显著高于真菌,但仅真菌群落的Shannon指数差异显著,表现为BAA > ABB > AFF;（2）细菌群落优势门主要为变形杆菌门、酸杆菌门、疣微菌门、拟杆菌门、绿弯菌门,相对丰度占比超过82%;真菌群落则为子囊菌门和担子菌门,占比超过85%,AFF担子菌门相对丰度最高而子囊菌门最低。（3） RDA分析显示,土壤pH和乔木物种多样性（Shannon指数）是影响微生物群落结构变化的主导因子;土壤养分元素对细菌群落影响不显著,真菌群落主要受TN、TP含量显著影响。总体上,林型间乔木层物种多样性、土壤酸碱度及其氮磷含量是导致微生物群落结构变化的关键因素。     

功能菌群耦合黄铁矿浸出软锰矿的研究

【目的】将3种不同来源的环境样品混合后接种至含1%黄铁矿和1%软锰矿的培养基中进行富集培养,初步得到有一定浸矿功能的混合微生物菌群。【方法】菌群继续用于黄铁矿和低品位软锰矿共同浸出,设置未接种的体系作为对照。【结果】对浸出过程中菌群结构的变化、pH、锰浸出率和浸出残渣的成分进行分析,结果发现接种过微生物菌群的浸出体系在反应15 d后,锰浸出率达到92.48%,远高于未接菌对照组的40.34%;菌群中Thiomonas sp.所占比例从最初的2%上升到浸出结束时的93%。实验组的pH从最初的4.0下降到2.5;X射线衍射(XRD)分析发现,通过生物作用浸出的残渣中含有黄钾铁矾,说明生物代谢产生了大量的硫酸。【结论】证明微生物在两矿浸出过程中通过促进黄铁矿解离,维持体系低pH等作用加速反应的进行。结果为进一步研究微生物浸矿的作用机制和开发低品位锰矿的生物浸出工艺打下了基础。     

温度、pH对非光合微生物菌群固碳效率的影响及其成因分析

CO2是主要的温室气体,也是地球上最丰富的碳源和可再生资源,本实验通过研究温度和pH对非光合固碳微生物固碳效率的影响,以期获得非光合固碳微生物适宜的固碳条件,并通过16SrDNA序列分析,研究了不同条件对固碳微生物群落结构的影响及其与固碳效率的相关性。结果表明,好氧／厌氧固碳微生物菌群生长的最适pH均在7左右。好氧固碳微生物菌群可在较大的温度范围（10℃-40℃）内良好生长,但其固碳速率在10℃左右时最高,之后微生物固碳速率随温度上升而下降,在40℃时,微生物固碳速率开始回升。厌氧时微生物的固碳效率随温度的变化情况与好氧时相似,但总体效果要比其在好氧时低20％～25％。由DGGE图谱及戴斯系数分析可得培养温度和pH对群落结构有显著影响,不同pH和温度条件下优势菌差异显著,这可能是固碳效率变化的重要原因之一。     

嗜酸异养菌对自养菌Acidithiobacillus ferrooxidans金属离子抗性和生物浸出的影响

【目的】了解嗜酸异养菌在诸如酸性矿坑水(AMD)和生物浸出体系等极端酸性环境中对浸矿微生物产生的影响。【方法】研究由嗜酸异养菌Acidiphilium acidophilum和自养菌Acidithiobacillus ferrooxidans经长期驯化后形成的共培养体系分别在Cd2+、Cu2+、Ni2+和Mg2+胁迫下的稳定性;并将此共培养体系应用于黄铁矿和低品位黄铜矿的生物浸出实验。【结果】在上述4种金属离子分别存在的条件下,异养菌Aph.acidophilum均能促进At.ferrooxidans对亚铁的氧化,提高其对能源利用的效率。共培养体系中的异养菌Aph.acidophilum使At.ferrooxidans对Cu2+的最大耐受浓度(MTC)由2.0 g/L提高到5.0 g/L,而且共培养的细胞数量与2.0 g/L Cu2+条件下生长的At.ferrooxidans纯培养相似。另外,共培养中的At.ferrooxidans对Mg2+的MTC也由12.0 g/L提高到17.0 g/L。生物浸出实验中嗜酸异养菌Aph.acidophilum促进了At.ferrooxidans对黄铁矿样品的浸出,浸出率较其纯培养提高了22.7%;但在含铁量较低的低品位黄铜矿浸出体系中共培养和At.ferrooxidans纯培养的浸出率均低于33%。在加入2.0 g/L Fe2+的低品位黄铜矿浸出体系中,共培养和At.ferrooxidans纯培养的浸出率均得到提高,分别达到52.22%和41.27%。【结论】以上结果表明,Aph.acidophilum与At.ferrooxidans共培养在一定的环境胁迫下仍能保持其稳定性并完成各自的生态功能,并且嗜酸异养菌Aph.acidophilum适合在含铁量较高的浸出体系中与铁氧化细菌共同作用来提高生物浸出的效率。     

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°C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16SrRNA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16SrRNA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(Leptospirillumferriphilum)以及unculturedforest soil bacterium、uncultured proteobacterium。其中Acidithiobacillus caldus,Sulfobacillus thermotolerans,Leptospirillumferriphilum3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%)。关键词:中度嗜热细菌;生物多样性;生物浸矿;喜温硫杆菌(Acidithiobacillus caldus);耐温氧化硫化杆菌(Sulfobacillus thermotolerans);嗜铁钩端螺旋菌(Leptospirillumferriphilum)     

Isolation and characterization of Ferroplasma thermophilum sp. nov., a novel extremely acidophilic, moderately thermophilic archaeon and its role in bioleaching of chalcopyrite

Aims:  To isolate Ferroplasma thermophilum L1^T from a low pH environment and to understand its role in bioleaching of chalcopyrite.
Methods and Results:  Using serial dilution method, a moderately thermophilic and acidophilic ferrous iron-oxidizing archaeon, named L1^T, was isolated from a chalcopyrite-leaching bioreactor. The morphological, biochemical and physiological characteristics of strain L1^T and its role in bioleaching of chalcopyrite were studied. Strain L1^T was a nonmotile coccus that lacked cell wall. Strain L1^T had a temperature optimum of 45°C and the optimum pH for growth was 1·0. Strain L1^T was capable of chemomixotrophic growth on ferrous iron and yeast extract. Results of fatty acid analysis, DNA–DNA hybridization, G+C content, and analysis based on 16S rRNA gene sequence indicated that strain L1^T should be grouped in the genus Ferroplasma , and represented a new species, Ferroplasma thermophilum . Ferroplasma thermophilum in combination with Acidithiobacillus caldus and Leptospirillum ferriphilum could improve the copper dissolution in bioleaching of chalcopyrite.
Conclusions:  A novel extremely acidophilic, moderately thermophilic archaeon isolated from a bioleaching reactor has been identified as F. thermophilum that played an important role in bioleaching of chalcopyrite at low pH.
Significance and Impact of the Study:  This study contributes to understand the characteristics of F. thermophilum L1^T and its role in bioleaching of sulfide ores.     

Comparison of bioleaching behaviors of different compositional sphalerite using <Emphasis Type="Italic">Leptospirillum ferriphilum</Emphasis>, <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> and <Emphasis Type="Italic">Acidithiobacillus caldus</Emphasis>

Two sphalerite samples with different iron/sulphur (Fe/S) ratios, Shuikousan ore (Fe/S 0.2) and Dachang ore (Fe/S 0.52), were processed using three microbial species, Leptospirillum ferriphilum, Acidithiobacillus ferrooxidans and Acidithiobacillus caldus. Following 20 days of bioleaching in shake flask cultures, a higher zinc (Zn) extraction (96%) was achieved with Shuikousan ore than with Dachange ore (72%). The extraction efficiency increased when elemental S was added to Dachang ore to attain the same Fe/S ratio as that for Shuikousan ore. Following the addition of S, the redox potential, pH and total dissolved Fe for Dachang ore demonstrated similar behaviors to those of Shuikousan ore. Acidithiobacillus caldus and L. ferriphilum became the dominant species during the bioleaching of sphalerite with a high Fe/S ratio. In contrast, the dominant species were A. ferrooxidans and A. caldus during the bioleaching of sphalerite with a low Fe/S ratio. These results show that the Fe/S ratio has a significant influence on the bioleaching behavior of sphalerite and the composition of the microbial community.     

Microbiological and geochemical dynamics in simulated-heap leaching of a polymetallic sulfide ore

The evolution of microbial populations involved in simulated-heap leaching of a polymetallic black schist sulfide ore (from the recently-commissioned Talvivaara mine, Finland) was monitored in aerated packed bed column reactors over a period of 40 weeks. The influence of ore particle size (2-6.5 mm and 6.5-12 mm) on changes in composition of the bioleaching microflora and mineral leaching dynamics in columns was investigated and compared to fine-grain (<2 microm) ore that was bioprocessed in shake flask cultures. Both column reactors and shake flasks were inoculated with 24 different species and strains of mineral-oxidizing and other acidophilic micro-organisms, and maintained at 37 degrees C. Mineral oxidation was most rapid in shake flask cultures, with about 80% of both manganese and nickel and 68% of zinc being leached within 6 weeks, though relatively little of the copper present in the ore was solubilised. The microbial consortium that emerged from the original inoculum was relatively simple in shake flasks, and was dominated by the iron-oxidizing autotroph Leptospirillum ferriphilum, with smaller numbers of Acidimicrobium ferrooxidans, Acidithiobacillus caldus and Leptospirillum ferrooxidans. Both metal recovery and (for the most part) total numbers of prokaryotes were greater in the column reactor containing the medium-grain than that containing the coarse-grain ore. The bioleaching communities in the columns displayed temporal changes in composition and differed radically from those in shake flask cultures. While iron-oxidizing chemoautotrophic bacteria were always the most numerically dominant bacteria in the medium-grain column bioreactor, there were major shifts in the most abundant species present, with the type strain of Acidithiobacillus ferrooxidans dominating in the early phase of the experiment and other bacteria (At. ferrooxidans NO37 and L. ferriphilum) dominating from week 4 to week 40. With the coarse-grain column bioreactor, similar transitions in populations of iron-oxidizing chemoautotrophs were observed, though heterotrophic acidophiles were often the most abundant bacteria found in mineral leach liquors. Four bacteria not included in the mixed culture used to inoculate the columns were detected by biomolecular techniques and three of these (all Alicyclobacillus-like Firmicutes) were isolated as pure cultures. The fourth bacterium, identified from a clone library, was related to the Gram-positive sulfate reducer Desulfotomaculum salinum. All four were considered to have been present as endospores on the dried ore, which was not sterilized in the column bioreactors. Two of the Alicyclobacillus-like isolates were found, transiently, in large numbers in mineral leachates. The data support the hypothesis that temporal and spatial heterogeneity in mineral heaps create conditions that favour different mineral-oxidizing microflora, and that it is therefore important that sufficient microbial diversity is present in heaps to optimize metal extraction.     

The Community Dynamics of Major Bioleaching Microorganisms During Chalcopyrite Leaching Under the Effect of Organics

To determine the effect of organics (yeast extract) on microbial community during chalcopyrite bioleaching at different temperature, real-time polymerase chain reaction (PCR) was employed to analyze community dynamics of major bacteria applied in bioleaching. The results showed that yeast extract exerted great impact on microbial community, and therefore influencing bioleaching rate. To be specific, yeast extract was adverse to this bioleaching process at 30°C due to decreased proportion of important chemolithotrophs such as Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. However, yeast extract could promote bioleaching rate at 40°C on account of the increased number and enhanced work of Ferroplasma thermophilum, a kind of facultative bacteria. Similarly, bioleaching rate was enhanced under the effect of yeast extract at 50°C owing to the work of Acidianus brierleyi. At 60°C, bioleaching rate was close to 100% and temperature was the dominant factor determining bioleaching rate. Interestingly, the existence of yeast extract greatly enhanced the relative competitiveness of Ferroplasma thermophilum in this complex bioleaching microbial community.     

Chromosomally encoded arsenical resistance of the moderately thermophilic acidophile Acidithiobacillus caldus

Arsenical resistance is important to bioleaching microorganisms because these organisms release arsenic from minerals such as arsenopyrite during bioleaching. The acidophile Acidithiobacillus caldus KU was found to be resistant to the arsenical ions arsenate, arsenite, and antimony via an inducible, chromosomally encoded resistance mechanism. Because no apparent alteration of the toxic ions was observed, Acidithiobacillus (At.) caldus was tested to determine if it was resistant as a result of decreased accumulation of toxic ions. Reduced accumulation of arsenate and arsenite by induced At. caldus cells supported this hypothesis. It was also found that, with the addition of an energy source, induced At. caldus could transport arsenate and arsenite out of the cell against a concentration gradient. The lack of efflux in the absence of an added energy source and in the presence of inhibitors suggested that efflux was energy dependent. Induced At. caldus also expressed arsenate reductase activity, indicating that At. caldus has an arsenical resistance mechanism that is analogous to previously described systems from other Bacteria. Southern hybridization analysis showed that At. caldus and other gram-negative acidophiles carry an Escherichia coli arsB homologue on the chromosome.     

Bioleaching of chalcopyrite concentrate by a moderately thermophilic culture in a stirred tank reactor

A mixed culture of moderately thermophilic microorganisms was enriched from acid mine drainage samples collected from several chalcopyrite mines in China. Such mixed culture can be used to effectively extract copper from chalcopyrite. Furthermore, after being adapted to gradually increased concentration of chalcopyrite concentrate, the tolerance of the mixed culture to chalcopyrite concentrate was brought up to 80 g/L. The effects of several leaching parameters on copper recovery in stirred tank reactor also had been investigated. The results of the investigation show that it was possible to achieve a copper extraction rate of 75% in 44 days at a pulp density of 8%. The leaching rate of chalcopyrite concentrate tended to increase with dissolved total iron concentration. At low pH ranges, more microscopic counts of microorganisms were found in the solution. Furthermore, the analysis of leached residues indicates that the passivation of chalcopyrite concentrate was mainly due to a mass of jarosite and PbSO(4) on the mineral surface, other than the elemental sulphur layer. The bacterial community composition was analyzed by using Amplified Ribosomal DNA Restriction Analysis. Two moderately thermophilic bacteria species were identified as Leptospirillum ferriphilum and Acidithiobacillus caldus with abundance of 67% and 33% in the bio-pulp, respectively.     

Resistance determinants of a highly arsenic-resistant strain of Leptospirillum ferriphilum isolated from a commercial biooxidation tank

Two sets of arsenic resistance genes were isolated from the highly arsenic-resistant Leptospirillum ferriphilum Fairview strain. One set is located on a transposon, TnLfArs, and is related to the previously identified TnAtcArs from Acidithiobacillus caldus isolated from the same arsenopyrite biooxidation tank as L. ferriphilum. TnLfArs conferred resistance to arsenite and arsenate and was transpositionally active in Escherichia coli. TnLfArs and TnAtcArs were sufficiently different for them not to have been transferred from one type of bacterium to the other in the biooxidation tank. The second set of arsenic resistance genes conferred very low levels of resistance in E. coli and appeared to be poorly expressed in both L. ferriphilum and E. coli.