Genus Acidithiobacillus Research Articles

Monitoring bacterial community shifts in bioleaching of Ni–Cu sulfide

The microbial ecology of the bioleaching of Ni–Cu sulfide is poorly understood and little effort has been made to handle the microbiological components of these processes. In this study, denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes fragments from bacteria was used to evaluate the changes of the bacterial community in the process of Ni–Cu sulfide bioleaching in a shaken flask system. The results revealed that the bacterial community was disturbed after the addition of Ni–Cu sulfide. Phylogenetic analyses of 16S rRNA fragments revealed that the retrieved sequences clustered together with the genera Acidithiobacillus and Leptospirillum. Multidimensional scaling (MDS) and cluster analysis of DGGE-banding patterns revealed that the process of Ni–Cu sulfide bioleaching in 46 days was divided into three stages. During the bioleaching process of Ni–Cu sulfide, Leptospirillum was always dominant. The genera Acidithiobacillus was only detected at early and later stages of the bioleaching process. These results extend our knowledge on microbial dynamics in Ni–Cu sulfide bioleaching, a key issue required to improve commercial applications.

Leptospirillum forms a minor portion of the population in Zijinshan commercial non-aeration copper bioleaching heap identified by 16S rRNA clone libraries and real-time PCR

The distribution and diversity of acidophilic microbe at the ore surface of Zijinshan commercial non-aeration low-grade copper bioleaching heap operated at pH 0.8 were investigated. Samples taken from − 1 m, − 2 m, − 3 m from the top of heap surface were investigated by 16S rRNA gene clone library. Phylogenetic analyses of 16S rRNA fragments revealed that the retrieved bacterial sequences mainly related to genus Acidithiobacillus (66.78%), genus Leptospirillum (28.29%), genus Sulfobacillus (3.29%) and genus Ferrimicrobium (1.64%). For archaea, only Ferroplasma acidiphilum was detected. Bacterial diversity in the heap was increased from surface layer to underground. The proportion of genus Leptospirillum was sharply reduced (from 48.5% to 5%) from higher depth to lower depth and reverse correlation of increased A. ferrooxidans (from 0.95% to 15.00%) and S. thermotolerans (from undetectable to 7.00%) were found in the heap. Sulfur oxidizers including A. albertensis and A. caldus also vertically increased from higher depth to lower depth (from 50.47% to 70.00%). Samples taken from − 2 m, − 3 m from the top of heap surface were further investigated by real-time PCR, the results were consistent with clone libraries that the growth of genus Leptospirillum was decreased from higher depth to lower depth. Our finding indicated that growth inhibition of genus Leptospirillum could be achieved in the non-aeration heap and genus Acidithiobacillus especially sulfur oxidizers A. albertensis and A. caldus were dominant at the ore surface inside the non-aeration heap.

The bacterial community structure during bioleaching of a low-grade nickel sulphide ore in stirred-tank reactors at different combinations of temperature and pH

The focus of this study is to characterize the bacterial community structure present during stirred-tank bioleaching of a low-grade nickel sulphide ore at different temperatures (5 to 45 °C) and pH levels (3 and 5). This is a continuation of previous work, which was designed to assess the technical feasibility of applying elevated-pH bioleaching to this same ore from Manitoba, Canada. A combination of classical microbiological and molecular biological techniques has been used to identify and enumerate the members of the bacterial consortia over the course of the five-week experiments. DGGE analysis revealed the presence of at least 16 distinct 16S rRNA gene sequences, 14 of which are closely related to existing GenBank sequences. Two sequences were detected that are not closely related to existing GenBank sequences and may possibly be from novel species. Thirteen sequences are related to gene sequences of genera that have previously been detected in bioleaching environments ( Acidithiobacillus, Leptospirillum, Sulfobacillus, Acidiphilium, Ferrimicrobium, and Acidimicrobium). Members from the genus Acidithiobacillus were dominant at all temperatures except 45 °C, at which Sulfobacillus spp. were dominant. Many of the acidithiobacilli are most closely related to strains of Acidithiobacillus ferrooxidans, and different strains were dominant under different experimental conditions, indicating considerable phenotypic heterogeneity within the species.

Protection of chemolithoautotrophic bacteria exposed to simulated Mars environmental conditions

Current surface conditions (strong oxidative atmosphere, UV radiation, low temperatures and xeric conditions) on Mars are considered extremely challenging for life. The question is whether there are any features on Mars that could exert a protective effect against the sterilizing conditions detected on its surface. Potential habitability in the subsurface would increase if the overlaying material played a protective role. With the aim of evaluating this possibility we studied the viability of two microorganisms under different conditions in a Mars simulation chamber. An acidophilic chemolithotroph isolated from Río Tinto belonging to the Acidithiobacillus genus and Deinococcus radiodurans, a radiation resistant microorganism, were exposed to simulated Mars conditions under the protection of a layer of ferric oxides and hydroxides, a Mars regolith analogue. Samples of these microorganisms were exposed to UV radiation in Mars atmospheric conditions at different time intervals under the protection of 2 and 5 mm layers of oxidized iron minerals. Viability was evaluated by inoculation on fresh media and characterization of their growth cultures. Here we report the survival capability of both bacteria to simulated Mars environmental conditions.

Acidithiobacillus ferrivorans, sp. nov.; facultatively anaerobic, psychrotolerant iron-, and sulfur-oxidizing acidophiles isolated from metal mine-impacted environments

Phenotypic and genotypic analysis was carried out on four iron- and sulfur-oxidizing acidophilic bacteria (the "NO-37 group") isolated from different parts of the world. 16S rRNA phylogeny showed that they are highly related to each other, but are less related to the type strain of Acidithiobacillus ferrooxidans. The NO-37 group isolates are obligate chemolithoautotrophs, facultative anaerobes, diazotrophic, and psychrotolerant. They are less tolerant of extremely low pH, and in contrast to At. ferrooxidans (T), all of the NO-37 group isolates are motile. The GC contents of genomic DNA of the NO-37 group isolates were around 56 mol% and the DNA-DNA hybridization value between genomic DNA of isolate NO-37 and At. ferrooxidans (T) was 37%. It also appears that the bacteria of the NO-37 group have a different biochemical mechanism for oxidizing ferrous iron than At. ferrooxidans (T); the gene coding for the archetypal rusticyanin (RusA) was not detected in any of the NO-37 group isolates, rather a gene coding for a homologous protein (RusB) was amplified from three of the four novel isolates. Isolates of the NO-37 group clearly belong to a species that is different to those already recognized in the genus Acidithiobacillus, for which the name Acidithiobacillus ferrivorans is proposed.

<i>Acidithiobacillus</i> Played Important Role in Zijinshan Commercial Low-Grade Copper Bioleaching Heap

The distribution and diversity of acidophilic microbe at the ore surface of Zijinshan commercial low-grade copper bioleaching heap operated at pH 0.8 were investigated. Samples taken from -1m, -2 m, -3m from the top of heap surface were investigated by 16S rRNA gene clone library. Phylogenetic analyses of 16S rRNA fragments revealed that the retrieved bacterial sequences mainly related to genus Acidithiobacillus (64.6%), genus Leptospirillum (27.3%), genus Sulfobacillus (3.1%) and genus Ferrimicrobium(1.6%). For archaea, only Ferroplasma acidiphilum was detected. Bacterial diversity in the heap was increased from surface layer to underground. The proportion of genus Leptospirillum was sharply reduced (from 48.5% to 5%) from higher depth to lower depth and reverse correlation of increased A.ferrooxidans (from 0.9% to 15%) and S. thermotolerans (from undetectable to 7%) were found in the heap. Sulfur oxidizers including A.albertensis, A.caldus and A.thiooxidans also vertically increased from higher depth to lower depth (from 50.5% to 80%). These results indicated that genus Acidithiobacillus especially sulfur oxidizers A.albertensis, A.caldus and A.thiooxidans may play very important roles in the commercial low-grade copper bioleaching heap.

Genomic Lessons from Biomining Organisms: Case Study of the <i>Acidithiobacillus</i> Genus

Advances in DNA sequencing technologies have promoted the use of genome information as a key component in most of biological studies. In the case of biomining microorganisms, partial and complete genome information has provided critical clues for unraveling their physiology. This information has also provided genetic material for the generation of functional and biodiversity directed markers. In this work, we present a compilation of the most relevant findings based on genomic analysis of the model organism Acidithiobacillus ferrooxidans ATCC23270 that were extended and compared to the recently sequenced genomes of Acithiobacillus thiooxidans and Acidithiobacillus caldus. The phylogenetic relatedness of these three microorganisms has permitted the identification of a shared genomic core that encodes the common metabolic and regulatory functions critical for survival and proliferation in extremely acidic environments. We also identified microorganism-specific genomic components that are predicted to be responsible for the metabolic speciation of these microorganisms. Finally, we evaluated the impact of lateral gene transfer on these genomes in order to determine the functional contribution of this phenomenon to the fitness of these microbial representatives. The information gathered by genomic analyses in the Acidithiobacillus genus will be presented in conjunction with other biomining genomic and metagenomic information in order to generate a more comprehensive picture of the biodiversity, metabolism and ecophysiology of the bioleaching niche.

Recovery of Nickel and Zinc Using Biogenerated Sulphuric Acid

Sludge generated in automotive and related industries often contains heavy metals. Bioleaching is an attractive alternative for the treatment of metal containing solids. Bacteria of the genus Acidithiobacillus are the most important microorganisms applied to metal solubilisation. These microorganisms are able to produce sulphuric acid from the aerobic oxidation of elemental sulphur. The biogenerated sulphuric acid can be applied to the solubilisation of metals from a solid matrix. In this paper we present the results of our experiments aimed at the removal of nickel and zinc from sludge generated in the water treatment plant of an automotive industry. Acidithiobacillus thiooxidans cells were immobilised on sulphur pearls in a column reactor. The effects of sulphur pulp density and the dilution rate on the production of sulphuric acid were studied. In a second stage, sulphuric acid was used to solubilise the nickel and zinc from the sludge. The effects of different sludge pulp densities and initial acid pH were studied. High recoveries of zinc and nickel were obtained when the pH value of the sulphuric acid solution was lower than 2.0 for 1 and 2 % of pulp density.

Diversity of Iron Oxidizing Bacteria from Various Sulfidic Mine Waste Dumps

More than 100 cultures of acidophilic Fe(II)- and/or sulfur-oxidizing microorganisms from mine waste dumps in 10 different countries all over the world have been maintained in liquid media in the BGR-strain collection for many years. Our 16S rDNA analysis showed that most of the cultivated Fe(II)-oxidizers belong to four genera: Acidithiobacillus, Acidimicrobium, “Ferrimicrobium” and Leptospirillum. All analyzed Acidithiobacillus strains were identified as At. ferrooxidans. The Leptospirillum strains were affiliated with L. ferriphilum or L. ferrooxidans. The Gram-positive strains related to Acidimicrobium or ”Ferrimicrobium” were phylogenetically more diverse than the strains of the genera Acidithiobacillus and Leptospirillum and fell into three separate clusters. While several strains could be identified as syngeneic (16S rDNA) with “Ferrimicrobium acidiphilum”, two other 16S rDNA clusters were distantly related and might represent new species or even new genera. In addition, one new Sulfobacillus strain and one new Alicyclobacillus strain could be identified. Furthermore several strains related to Acidiphilium acidophilum have been detected and form one 16S rDNA cluster.

Attachment Behavior of Leaching Bacteria to Metal Sulfides Elucidated by Combined Atomic Force and Epifluorescence Microscopy

The aim of the study was to quantify and to visualize colonization of metal sulfides by pure and mixed cultures. Strains of the genera Acidithiobacillus and Leptospirillum were tested. Sessile and planktonic cells were visualized by fluorescence microscopy using 4',6-diamidino-2-phenylindole (DAPI) and FISH. Additionally, atomic force microscopy was used for the investigations on cell morphology, spatial arrangement of cells on metal sulfides and mineral surface topography. It was shown that the morphology of sessile cells was totally different as compared with planktonic ones. Interactions of different species resulted in increased production of extracellular polymeric substances (EPS) or caused negligible-attaching bacteria to be incorporated into a biofilm by the good attaching ones. Consequently, biofilm formation was furthered.

Concrete specimens biodeterioration by bacteria ofAcidithiobacillus thiooxidansandDesulfovibriogenera

Biodeterioration can be seen as a process, which decreases value of materials and constructions and has been defined as the deterioration of materials of economic importance by organisms. Since the discovery bacterial genus Acidithiobacillus caused rapid corrosion of concrete especially Acidithiobacillus thiooxidans. This paper presents the results of concrete specimens' biodeterioration study by bacteria Acidithiobacillus thiooxidans and Desulfovibrio genera under model conditions. Biodeterioration was carried out in laboratory reactor, where simultaneous effect of Acidithiobacillus thiooxidans and Desulfovibrio genera happened. The pH values of leachate, mass of concrete specimens change, Ca and Fe content in leachate were evaluated. After experiment the morphology of corroded surface were observed by scanning electron microscopy.

Enhanced Biofiltration Using Cell Attachment Promotors

H2S polluted airstreams were treated in two biotrickling filter columns packed with polyurethane (PU) foam cubes, one with cubes coated with a solution of 25 mg/L of polyethyleneimine (PEI, coated reactor) and the other containing just plain PU cubes (uncoated reactor) at empty bed residence times (EBRT) ranging from 6 to 60 s. and inlet H2S concentrations ranging from 30 to 235 ppm, (overall loads of up to 44 gH2S/m3bed/h), with overall removal efficiencies (RE) in the range of 90-100% over 125 days. The acclimatization characteristics of the coated reactor outperformed those of the uncoated one, and both the observed elimination capacity (EC) of 77 gH2S/m3bed/h and retention of volatile solids (VS) of 42 mgVS/cube were maxima in the coated reactor. Insights into the controlling removal mechanisms were also provided by means of dimensionless analysis of the experimental data. Denaturing gradient gel electrophoresis (DGGE) showed that the dominant surviving species in both units belonged to the genus Acidithiobacillus.

Dominance of Acidithiobacillus at ore surface of Zijinshan commercial low-grade copper bioleaching heap

The microbial community structure in the ore surface of Zijinshan commercial low-grade copper bioleaching heap was investigated by 16S rRNA gene clone library. For both bacteria and Archaea, 105 clones were sequenced. The dominant bacteria species present in the ore surface were Acidithiobacillus and Leptospirillum, accounting for 51.42% and 48.57%, respectively. However, for the Archaea, only one operational taxonomic unit (OUT) belonged to Ferroplasma acidiphilum. These results indicate that function of genus Acidithiobacillus in the commercial low-grade copper bioleaching heap may be underestimated. More detailed and quantitative information on microbial community structure over time are now under investigation.

SOLID2: An Antibody Array-Based Life-Detector Instrument in a Mars Drilling Simulation Experiment (MARTE)

A field prototype of an antibody array-based life-detector instrument, Signs Of LIfe Detector (SOLID2), has been tested in a Mars drilling mission simulation called MARTE (Mars Astrobiology Research and Technology Experiment). As one of the analytical instruments on the MARTE robotic drilling rig, SOLID2 performed automatic sample processing and analysis of ground core samples (0.5 g) with protein microarrays that contained 157 different antibodies. Core samples from different depths (down to 5.5 m) were analyzed, and positive reactions were obtained in antibodies raised against the Gram-negative bacterium Leptospirillum ferrooxidans, a species of the genus Acidithiobacillus (both common microorganisms in the Río Tinto area), and extracts from biofilms and other natural samples from the Río Tinto area. These positive reactions were absent when the samples were previously subjected to a high-temperature treatment, which indicates the biological origin and structural dependency of the antibody-antigen reactions. We conclude that an antibody array-based life-detector instrument like SOLID2 can detect complex biological material, and it should be considered as a potential analytical instrument for future planetary missions that search for life.

16S rDNA and 16S–23S internal transcribed spacer sequence analyses reveal inter- and intraspecific Acidithiobacillus phylogeny

In order to contribute to our understanding of Acidithiobacillus taxonomy, we determined 16S rDNA sequences and the 16S-23S internally transcribed spacer (ITS) sequence of 35 Chinese Acidithiobacillus isolates and three reference strains representing three validly described species and used them to construct phylogenetic trees. The two phylogenetic trees were roughly similar topologically, and Acidithiobacillus strains were assigned to eight phylogenetic groups. In addition, the results of phylogenetic analysis were consistent with those obtained by randomly amplified polymorphic DNA (RAPD) cluster analysis. Compared with a phylogenetic tree based on the 16S rRNA sequences, the ITS tree showed more clearly the inter- and intraspecific genealogical relationships of the genus Acidithiobacillus. Similarity values of the ITSs varied from 60.5 % to 84.7 % between representative strains of different species, and the maximum level of ITS divergence between strains belonging to the same species was 13 %. Coupling phylogenetic analysis and phenotypic characteristics, we concluded that at least each of the three Acidithiobacillus ferrooxidans phylogenetic groups should be considered a separate subspecies, and that five sulfur-oxidizing Chinese Acidithiobacillus-like isolates represent one or two new species of the genus Acidithiobacillus. The ITS may be a potential target for the development of fluorescent in situ hybridization probes for more accurately detecting distinct ecotypes of Acidithiobacillus strains and other closely related sulfur-oxidizing bacteria.

Bacterial community structure change during pyrite bioleaching process: Effect of pH and aeration

A culture-independent approach based on 16S rRNA gene clone library was used to analyze the bacterial community change during the bioleaching of pyrite. In this paper we summarize results from monitoring aerated and periodically aerated pyrite bioleaching conducted in two 3 L stirred reactors at 30 °C. Phylogenetic analyses of 16S rRNA fragments revealed that the retrieved sequences mainly related to genus Leptospirillum, genus Sulfobacillus and genus Acidithiobacillus. The remaining sequences were clustered together with genus Afipia and genus Sporacetigenium. The dynamics of phylogenetic microbial groups' change of the bioleaching processes were investigated and correlated with chemical and physical parameters in the reactors. In both aerated and periodically aerated bioleaching tests, bacteria diversity was decreased at the end. Genus Leptospirillum accounted for a high proportion when the pH was decreased from 1.6 to 1.3 and from 1.0 to 0.9, while the proportion of sequences belonging to genus Sulfobacillus increased when the pH was decreased from 1.3 to 1.0 and below pH 0.9. Under non-aerated condition, Sulfobacillus may have better growth. These results may be used to facilitate the selective depression of pyrite during copper bioleaching and improve commercial plant operation.

Genomic and phenotypic heterogeneity of Acidithiobacillus spp. strains isolated from diverse habitats in China

The genetic variability among 32 Chinese Acidithiobacillus spp. environmental isolates and four reference strains representing three recognized species of the genus Acidithiobacillus was characterized by using a combination of molecular methods, namely restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers, repetitive element PCR, arbitrarily primed PCR and 16S rRNA gene sequence analyses. 16S rRNA gene sequences revealed that all Acidithiobacillus spp. strains could be assigned to seven groups, three of which encompassed the Acidithiobacillus ferrooxidans strains from various parts of the world. A comparative analysis of the phylogenetic Group 1 and 2 was undertaken. Restriction fragment length polymorphism results allowed us to separate the 35 Acidithiobacillus strains into 15 different genotypes. An integrated phenotypic and genotypic analysis indicated that the distribution of A. ferrooxidans strains among the physiological groups were in agreement with their distribution among the genomic groups, and that no clear correlation was found between the genetic polymorphism of the Acidithiobacillus spp. strains and either the geographic location or type of habitats from which the strains were isolated. In addition, five unidentified sulfur-oxidizing isolates may represent one or two novel species of the genus Acidithiobacillus. The results showed that the Chinese Acidithiobacillus spp. isolates exhibited a high degree of genomic and phenotypic heterogeneity.

An oligonucleotide prokaryotic acidophile microarray: its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA

An oligonucleotide microarray that monitors prokaryotic diversity in extremely acidic environments has been developed. The oligonucleotide probes target most known acidophilic microorganisms, including members of the Nitrospira phylum, Acidithiobacillus genus, acidobacteria, sulfur reducing bacteria, Actinobacteria and Archaea of the Ferroplasma and Thermoplasma genera. The probes were tested for their specificity against the corresponding type strain by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. The microarray was tested and validated against well-established molecular ecology techniques such as molecular cloning and sequencing and FISH by using samples obtained from a natural extremely acidic environment, the Río Tinto (SW Spain). Also, fluorescent labelled total environmental RNA from Río Tinto samples were used as targets for microarray hybridizations. This approach allowed the detection of the most metabolically active prokaryotes of the ecosystem by simultaneously checking probes against 16S and 23S rRNAs as well as other functional genes. Seasonal and spatial variations in the relative expression of specific rRNA genes have been detected between two sampling sites that differ in several physicochemical parameters, mainly iron and sulfur content.

Bioleaching of spent Ni-Cd batteries and phylogenetic analysis of an acidophilic strain in acidified sludge

Biohydrometallurgy is a novel method to recycle discarded batteries, in which sewage sludge is used as microorganisms and culture due to the presence of indigenous Thiobacilli. A two-step continuous flow leaching system consisting of an acidifying reactor and a leaching reactor was introduced to achieve the bioleaching of spent nickel-cadmium (Ni-Cd) batteries. The acid supernatant produced in the acidifying reactor by the microorganisms with ferrous ions as the substrate was conducted into the leaching reactor to dissolve electrode materials. The efficiency of a batch treatment of batteries was examined. The results showed that the complete dissolution of two AA-sized Ni-Cd batteries with 0.6 L/d acid supernatant took about 30, 20, and 35 days for Ni, Cd, and Co, respectively. But the dissolution ability of the three metals was different. Cd and Co can be leached mostly for pH below 4.0 while the complete dissolution of Ni can be achieved for pH below 2.5. Meanwhile, a strain (named Thiooxidans. WL) accounting for the reduction of pH in the acidified sludge was isolated and sequenced. It was identified to be 100% similar to Acidithiobacillus ferrooxidans strain Tf-49 based on 16S rDNA sequence analysis. The relevant phylogenetic tree constructed indicates that the strain should be classified into genus Acidithiobacillus ferrooxidans.

Study of the Attachment Behaviour of Different Strains of <i>Acidithiobacillus</i> spp. to Pyrite

Attachment to metal sulfide surfaces is a prerequisite of bioleaching activity via the contact mode [Rohwerder et al.: Appl. Microbiol. Biotechnol. Vol. 63 (2003), p. 239]. Until now, the mechanisms and possible variations of this attachment process among leaching bacteria is not well understood. Therefore, we are studying the diversity in the attachment behaviour of different strains of the genus Acidithiobacillus, which is one of the main bacterial group involved in the bioleaching of metal sulfides. Among the species At. ferrooxidans, cells of the type strain (ATCC 23270) were found to attach the least. Whereas bacterial cells of strains A2 and R1 attached the most. Other strains that were investigated in this study included At. ferrooxidans D-26 and R7. Highly likely, these strains exhibit different attachment behaviour due to specific variations in their extracellular polymeric substance (EPS) composition as loss of part of the EPS has previously been observed to have a negative effect on attachment and bioleaching activities. Differences in several EPS parameters such as the amount of complexed Fe(III) ions and the sugar composition are possible reasons for the observed strain-specific attachment behaviour. In addition to attachment assays and microscopic studies (AFM and epifluorescence), leaching activities of the strains were comparatively investigated by short-term as well as growth experiments.