Metals tolerance in moderately thermophilic isolates from a spent copper sulfide heap, closely related to Acidithiobacillus caldus, Acidimicrobium ferrooxidans and Sulfobacillus thermosulfidooxidans

Selective enrichments enabled the recovery of moderately thermophilic isolates with copper bioleaching ability from a spent copper sulfide heap. Phylogenetic and physiological characterization revealed that the isolates were closely related to Sulfobacillus thermosulfidooxidans, Acidithiobacillus caldus and Acidimicrobium ferrooxidans. While isolates exhibited similar physiological characteristics to their corresponding type strains, in general they displayed similar or greater tolerance of high copper, zinc, nickel and cobalt concentrations. Considerable variation was found between species and between several strains related to S. thermosulfidooxidans. It is concluded that adaptation to metals present in the bioleaching heap from which they were isolated contributed to but did not entirely explain high metals tolerances. Higher metals tolerance did not confer stronger bioleaching performance, suggesting that a physical, mineralogical or chemical process is rate limiting for a specific ore or concentrate.     

Effects of Stimulation of Copper Bioleaching on Microbial Community in Vineyard Soil and Copper Mining Waste

Long-term copper application in vineyards and copper mining activities cause heavy metal pollution sites. Such sites need remediation to protect soil and water quality. Bioremediation of contaminated areas through bioleaching can help to remove copper ions from the contaminated soils. Thus, the aim of this work was to evaluate the effects of different treatments for copper bioleaching in two diverse copper-contaminated soils (a 40-year-old vineyard and a copper mining waste) and to evaluate the effect on microbial community by applying denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA amplicons and DNA sequence analysis. Several treatments with HCl, H₂SO₄, and FeSO₄ were evaluated by stimulation of bioleaching of copper in the soils. Treatments and extractions using FeSO₄ and H₂SO₄ mixture at 30°C displayed more copper leaching than extractions with deionized water at room temperature. Treatment with H₂SO₄ supported bioleaching of as much as 120 mg kg⁻¹ of copper from vineyard soil after 115 days of incubation. DGGE analysis of the treatments revealed that some treatments caused greater diversity of microorganisms in the vineyard soil compared to the copper mining waste. Nucleotide Blast of PCR-amplified fragments of 16S rRNA gene bands from DGGE indicated the presence of Rhodobacter sp., Silicibacter sp., Bacillus sp., Paracoccus sp., Pediococcus sp., a Myxococcales, Clostridium sp., Thiomonas sp., a firmicute, Caulobacter vibrioides, Serratia sp., and an actinomycetales in vineyard soil. Contrarily, Sphingomonas was the predominant genus in copper mining waste in most treatments. Paracoccus sp. and Enterobacter sp. were also identified from DGGE bands of the copper mining waste. Paracoccus species is involved in the copper bioleaching by sulfur oxidation system, liberating the copper bounded in the soils and hence promoting copper bioremediation. Results indicate that stimulation of bioleaching with a combination of FeSO₄ and H₂SO₄ promoted bioleaching in the soils and can be employed ex situ to remediate copper-impacted soils.     

Bioleaching review part B:

This review describes the historical development and current state of metals leaching and sulfide mineral biooxidation by the minerals industries. During the past 20 years commercial processes employing microorganisms for mineral recovery have progressed from rather uncontrolled copper dump leaching to mineral oxidation and leaching in designed bioheaps for oxidation of refractory gold ores and for copper recovery. Also during this period of time, stirred tank bioleaching has been commercialized for cobalt recovery and for biooxidation of refractory gold ores. Chalcopyrite bioleaching in stirred tanks is on the verge of commercialization. Commercial applications of biohydrometallurgy have advanced due to favorable process economics and, in some cases, reduced environmental problems compared to conventional metal recovery processes such as smelting. Process development has included recognition of the importance of aeration of bioheaps, and improvements in stirred tank reactor design and operation. Concurrently, knowledge of the key microorganisms involved in these processes has advanced, aided by advances in molecular biology to characterize microbial populations.     

块菌(松露)宿主可培养内生菌群落结构与多样性研究

探究四川凉山彝族自治州块菌主产区华山松内生菌群的结构及多样性。在会东县选取4个点(新田乡、新云乡、淌塘镇、雪山乡)的块菌宿主华山松的根、茎、叶为实验材料,通过不同培养基分离样品根、茎、叶的内生细菌、真菌、放线菌,DNA分子鉴定分离菌株的种属,最终分离得到细菌46株,其中欧文氏菌属(Erwinia)5株,沙门氏菌属(Salmonell)1株,杆菌属(Bacillus)22株,葡萄球菌属(Staphylococcus)2株,假单胞菌属(Pseudomonas)2株,类芽胞杆菌属(Paenibacillus)2株,布丘氏菌属(Buttiauxella)2株,肠杆菌属(Enterobacte)3株,爱文氏菌属(Ewingella)1株,泛菌属(Rahnella)1株,拉恩氏菌属(Rahnella Izard)2株,其他属3株;真菌19株,均为子囊菌门(Ascomycota),其中青霉属(Penicillium)4株,疱霉属(Phoma)1株,子囊菌门未知菌1株,篮状菌属(Cladosporium)1株,分枝孢子菌属(Sydowia)1株,曲霉属(Aspergillus)1株,其他属10株;放线菌33株,为链霉菌属(Streptomyces)22株,短小杆菌属(Curtobacterium)2株,短杆菌属(Brevibacterium)1株,其他属8株。研究结果表明,来自不同地点、不同植株部位、不同培养基分离得到的块菌宿主华山松内生菌有差异,证实微生物在土壤、块菌、宿主植物之间有着复杂的相互作用,为块菌的人工栽培提供参考。     

Column bioleaching of uranium embedded in granite porphyry by a mesophilic acidophilic consortium

A mesophilic acidophilic consortium was enriched from acid mine drainage samples collected from several uranium mines in China. The performance of the consortium in column bioleaching of low-grade uranium embedded in granite porphyry was investigated. The influences of several chemical parameters on uranium extraction in column reactor were also investigated. A uranium recovery of 96.82% was achieved in 97 days column leaching process including 33 days acid pre-leaching stage and 64 days bioleaching stage. It was reflected that indirect leaching mechanism took precedence over direct. Furthermore, the bacterial community structure was analyzed by using Amplified Ribosomal DNA Restriction Analysis. The results showed that microorganisms on the residual surface were more diverse than that in the solution. Acidithiobacillus ferrooxidans was the dominant species in the solution and Leptospirillum ferriphilum on the residual surface.     

海南海口温泉真菌、细菌多样性及其环境影响因素分析

【目的】海南海口含有丰富的温泉资源,对温泉微生物多样性进行研究,有助于进一步开发和利用海南温泉微生物资源。【方法】本文采用Illumina Hi Seq高通量测序技术对海口3个温泉[海甸岛荣域温泉(S1)、火山口开心农场温泉(S2)和西海岸海长流温泉(S3)]水样中微生物ITS序列和16Sr RNA基因V3-V4区进行测序及生物信息学分析,探究海口市3个不同区域的温泉真菌多样性与细菌多样性。【结果】(1)α多样性分析表明,真菌群落中,S3(29)S1(29)S2,而在细菌群落中,S2(29)S1(29)S3。β多样性分析表明,3个温泉真菌群落和细菌群落组成差异皆显著。(2)分类分析表明,温泉真菌群落优势菌门为子囊菌门(Ascomycota)和担子菌门(Basidiomycota),细菌群落优势菌门为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、Thermi、硝化螺旋菌门(Nitrospirae)、绿菌门(Chlorobi)、厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)、放线菌门(Actinobacteria)。(3) CCA (Canonical correspondence analysis)分析表明,3个温泉的真菌群落主要影响因子是温度,细菌群落主要影响因子是总磷。【结论】海南省海口市温泉中含有丰富的微生物资源,其微生物群落组成受多种环境因子影响,且影响真菌和细菌的主要环境因子不同。     

Recent advances in microbial mining

Microbial mining of copper sulphide ores, has been practiced on an industrial scale since the late 1950s. Since then, advances in microbial mining and the role of microorganisms involved in solubilization of metals have assumed commerical importance. The fact that bioleaching processes save energy, have a minimum pollution potential and are able to yield value-added by-products make these processes invaluable. The metal extraction processes using microorganisms, which are currently in active use, concern copper and uranium bioleaching. Biobeneficiation is also applied at an industrial scale for recovery of gold from arsenopyrites. The developments in these processes during the last 15 years, with particular reference to developing nations, are reviewed. Information gathered on molecular genetics of these microorganisms should lead to a better understanding and control of microbial leaching processes. Areas still needing research to sustain economic expansion of microbial mining techniques are indicated.The author is with the Agharkar Research Institute, Agarkar Road, Pune 411 004, India     

半干旱矿区排土场苜蓿恢复过程中土壤颗粒分形的演变特征

种植苜蓿恢复模式在半干旱矿区植被恢复中占重要地位。为掌握其恢复过程中土壤颗粒分形的演变特征,选择恢复年限分别为2 a、4 a、6 a和10 a(M2、M4、M6和M10)的苜蓿种植地为研究对象,通过野外分层采样与室内测定,依据颗粒体积分形理论,研究了排土场苜蓿恢复过程中土壤颗粒分形的演变特征及与土壤特性的关系。结果表明:研究区土壤各粒径含量以砂粒为主,粉粒次之,粘粒最少;随着苜蓿恢复过程,粘粒与粉粒含量整体表现为先增加后下降的趋势,且在M4阶段达到最佳,砂粒反之。土壤分形维数变化在2.09—2.57,在苜蓿恢复过程中先增大后减小,在M4阶段达到最大。土壤分形维数与粘粒、粉粒具有极显著正相关性(P0.01),与砂粒具有极显著负相关性(P0.01);土壤分形维数与电导率呈极显著负相关关系(P0.01),与pH值和速效钾含量呈显著负相关关系(P0.05),与碱解氮含量呈显著正相关关系(P0.05)。在半干旱矿区排土场采用苜蓿恢复模式,可用土壤分形维数表征土壤特性,应重视恢复年限的调控,适时进行适宜的利用与改造,确保矿区生态恢复的可持续性。     

Carotenoids from <Emphasis Type="Italic">Rhodotorula</Emphasis> and <Emphasis Type="Italic">Phaffia</Emphasis>: yeasts of biotechnological importance

Carotenoids represent a group of valuable molecules for the pharmaceutical, chemical, food and feed industries, not only because they can act as vitamin A precursors, but also for their coloring, antioxidant and possible tumor-inhibiting activity. Animals cannot synthesize carotenoids, and these pigments must therefore be added to the feeds of farmed species. The synthesis of different natural commercially important carotenoids (β-carotene, torulene, torularhodin and astaxanthin) by several yeast species belonging to the genera Rhodotorula and Phaffia has led to consider these microorganisms as a potential pigment sources. In this review, we discuss the biosynthesis, factors affecting carotenogenesis in Rhodotorula and Phaffia strains, strategies for improving the production properties of the strains and directions for potential utility of carotenoid-synthesizing yeast as a alternative source of natural carotenoid pigments.     

Isothermal DNA amplification facilitates the identification of a broad spectrum of bacteria,fungi and protozoa in <Emphasis Type="Italic">Eleutherococcus</Emphasis> sp. plant tissue cultures

In vitro cultures of Eleutherococcus sieboldianus originating from surface sterilized leaf explants were found to be associated with several microorganisms. The associations included bacteria, fungi and protozoa within the rhizosphere and inside root hairs. To determine if this phenomenon is unique to this species, plant tissue cultures of E. gracilistylus and E. senticosus were included in our studies for comparison. A methodology consisting of isothermal amplification, cloning and sequencing was established for analysing 16S ribosomal DNA of cultivated and non-cultivated bacteria from different tissue types. The same methodology was used to obtain internal transcribed spacer regions and 18S regions of fungal and protozoan rDNA. Comparative analyses of sequencing data resulted in the identification of various genera within the Firmicutes and γ-proteobacteria kingdoms and a broad spectrum of fungal genera related to several uncultured fungi. In addition, amoebal and chrysophyte species were detected. Most of the species were identified in different plant organs and in in vitro culture cell types indicating the microorganisms are systemically distributed. The presence of identical microorganisms in different plant species argues for an evolutionary long-lasting and stable association between the plant genus and the microinhabitants.     

Isolation of a strain of <Emphasis Type="Italic">Acidithiobacillus caldus</Emphasis> and its role in bioleaching of chalcopyrite

A moderately thermophilic and acidophilic sulfur-oxidizing bacterium named S₂, was isolated from coal heap drainage. The bacterium was motile, Gram-negative, rod-shaped, measured 0.4 to 0.6 by 1 to 2 μm, and grew optimally at 42–45°C and an initial pH of 2.5. The strain S₂ grew autotrophically by using elemental sulfur, sodium thiosulfate and potassium tetrathionate as energy sources. The strain did not use organic matter and inorganic minerals including ferrous sulfate, pyrite and chalcopyrite as energy sources. The morphological, biochemical, physiological characterization and analysis based on 16S rRNA gene sequence indicated that the strain S₂ is most closely related to Acidithiobacillus caldus (>99% similarity in gene sequence). The combination of the strain S₂ with Leptospirillum ferriphilum or Acidithiobacillus ferrooxidans in chalcopyrite bioleaching improved the copper-leaching efficiency. Scanning electron microscope (SEM) analysis revealed that the chalcopyrite surface in a mixed culture of Leptospirillum ferriphilum and Acidithiobacillus caldus was heavily etched. The energy dispersive X-ray (EDX) analysis indicated that Acidithiobacillus caldus has the potential role to enhance the recovery of copper from chalcopyrite by oxidizing the sulfur formed during the bioleaching progress.     

Cryptococcus haglerorum,sp. nov., an anamorphic basidiomycetous yeast isolated from nests of the leaf-cutting ant Atta sexdens

A yeast strain (CBS 8902) was isolated from the nest of a leaf-cutting ant and was shown to be related to Cryptococcus humicola. Sequencing of the D1/D2 region of the 26S ribosomal DNA and physiological characterization revealed a separate taxonomic position. A novel species named Cryptococcus haglerorum is proposed to accommodate strain CBS 8902 that assimilates n-hexadecane and several benzene compounds. Physiological characteristics distinguishing the novel species from some other members of the C. humicola complex are presented. The phylogenetic relationship of these strains to species of the genus Trichosporon Behrend is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

Four species of the unicellular green alga Chlorella, C. vulgaris, C. luteoviridis, C. minutissima, and C. zofingiensis, were characterized with respect to DNA similarities as determined by quantitative DNA hybridization procedures. In contrast to previous DNA hybridization procedures. In contrast to previous results, C. vulgaris turned out to be a homogeneous species with the exception of strain 211-11c of the Göttingen collection, which was shown to belong to C. kessleri. Similary, C. luteoviridis and C. minutissima represent well defined species in terms of phenotypic and genotypic features. Whitin C. zofingiensis on strain is clearly different with respect to DNA base composition and DNA hybridization data even though it shares phenotypic characteristics with the other strains of C. zofingiensis.     

Genomic studies on killer yeasts belonging to the genusPichia

Twenty-four species belonging to the genusPichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA.Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii andCandida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of theEcoRI andXhoI RFLP patterns withP. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis andC. albicans). However, one hybridized band, located at 3.9–4.1 Kb, seems to be peculiar to thePichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms     

Genetic variation can promote system persistence in an experimental host-parasitoid system

We tested experimentally the effects of genetic variation in host population on the host-parasitoid system persistence. The experimental systems consisted of one parasitoid wasp species (Heterospilus prosopidis), one bean weevil species (Callosobruchus chinensis), and one bean species, of which only the host species (bean weevil) was genetically manipulated. As control treatments with low genetic heterogeneity in the host population, we used two bean weevil strains (Kyoto and Niigata strains) which have several contrasting ecological traits. For the high genetic heterogeneity treatment, hybrid bean weevils which were generated by crossbreeding the strains Kyoto and Niigata were used. In the multiple-generation experiments, all three treatments had different patterns of extinction. The control treatment with the Niigata strain was very prone to system extinction, whereas the treatment with hybrids showed coexistence of constituent species in almost all replicates and also showed stabilized population dynamics. The other control treatment, using the Kyoto strain, showed intermediate proneness. To interpret the results of multiple-generation experiments, we conducted several short-term experiments. The different persistence patterns between the two control treatments were explained by the shapes of the host-finding abilities of wasps and the growth rate of the bean weevils. The mean values of many ecological traits of hybrid lines were not different from those of the Kyoto strain, but their variability increased. These outcomes corresponded well to the prediction of models by Doebeli [J Theor Biol (1997) 188:109–120]. We discuss the mechanisms in which the variability in host species population was effective for the prolonged system persistence.     

Growth promotion and cell binding ability of bovine lactoferrin to Bifidobacterium longum

Lactoferrin, a major whey protein of human milk, is considered as growth promoter for bifidobacteria, the predominant microorganisms of human intestine. In the present study, in vitro growth promotion and cell binding ability of bovine lactoferrin to several strains of Bifidobacterium longum have been demonstrated. A dose-dependent as well as strain-dependent growth promotion effect by lactoferrin was observed. Cell binding ability of lactoferrin was inspected under an inverted confocal laser scanning microscope by incubation bacterial cells with biotinylated bovine lactoferrin and FITC-conjugated avidin. Fluorescence staining showed bovine lactoferrin binding to all tested strains. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the extracted membrane and cytosolic fraction of each B. longum strain by far-Western blot technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on these results, we suggest that existence of lactoferrin-binding protein could be a common characteristic in bifidobacteria. It can also be hypothesized that lactoferrin-binding protein in bifidobacteria is not only involved in growth stimulation mechanism but also could play different roles.     

Heliorestis convoluta sp. nov., a coiled, alkaliphilic heliobacterium from the Wadi El Natroun, Egypt

A morphologically distinct heliobacterium, strain HH, was isolated from Lake El Hamra, a soda lake in the Wadi El Natroun region of northwest Egypt. Strain HH consisted of ring-shaped cells that remained attached after cell division to yield coils of various lengths. Strain HH showed several of the physiological properties of known heliobacteria and grouped in the Heliorestis clade by virtue of its phylogeny and alkaliphily. The closest relative of strain HH was the filamentous alkaliphilic heliobacterium Heliorestis daurensis. However, genomic DNA:DNA hybridization results clearly indicated that strain HH was a distinct species of Heliorestis. Based on its unique phenotypic and genetic properties we describe strain HH here as a new species of the genus Heliorestis, H. convoluta sp. nov.Dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 80th birthday.     

Characterization of the satellite DNA Msat-160 from the species <Emphasis Type="Italic">Chionomys</Emphasis><Emphasis Type="Italic">nivalis</Emphasis> (Rodentia,Arvicolinae)

The satellite DNA Msat-160 has been previously characterized in several species of the genus Microtus. Here we present the characterization of Msat-160 from Chionomys nivalis, a species with a very primitive karyotype. As in other Microtus species analyzed, C. nivalis Msat-160 is AT rich, has a monomer length of 160 bp, is undermethylated and is mainly located in all the pericentromeric heterochromatin of all autosomes and the X chromosome, but is completely absent from the Y chromosome. Hence, our results support the hypothesis that Msat-160 was initially distributed in the pericentromeric heterochromatin of all autosomes and the X chromosome. The taxonomic status of the genus Chionomys in relation to the genus Microtus is a very interesting issue, so we constructed phylogenetic dendrograms using Msat-160 sequences from several Microtus species. Although the results were not informative about this issue, the presence of Msat-160 in C. nivalis and Microtus species suggested that both genera are closely related and that this satellite DNA was present in the common ancestor. Studies of Msat-160 in different arvicoline species could help to determine the origin of this satellite and, perhaps, to establish the phylogenetic relationships of some arvicoline groups.     

Xenorhabdus japonicus sp. nov. associated with the nematode Steinernema kushidai

A new species, Xenorhabdus japonicus, is proposed as the bacterial symbiont of Steinernema kushidai isolated from field soil in Shizuoka Prefecture, Japan. Xenorhabdus japonicus could be distinguished phenotypically and genetically from other Xenorhabdus spp. The type strain of the species, SK-1, a Gram-negative, facultative anaerobe and peritrichously flagellated rod, has colonies with primary and secondary forms. The strain can be differentiated from the type strain of Xenorhabdus nematophilus by several characters, including the formation of arginine dehydrolase, phenylalanine deaminase and lysine decarboxylase, the assimilation of inosine and L-proline and acid production from inositol. The major cellular fatty acids are 16:0, cyclo 17:0 and 18:1. The ubiquinone system is Q-8. The G+C content of DNA is 45.9 mol%. The DNA of strain SK-1 has 20 to 58% homology with that of the type strains of other Xenorhabdus spp.Y. Nishimura, A. Hagiwara and T. Suzuki are with the Department of Applied Biological Science, Science University of Tokyo, Noda 278, Japan, and SDS Biotech K.K., Tsukuba Technology Centre, Tsukuba 300-26, Japan