Acidithiobacillus Caldus Research Articles

Sulfur Oxygenase Reductase (Sor) in the Moderately Thermoacidophilic Leaching Bacteria: Studies in Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus.

The sulfur oxygenase reductase (Sor) catalyzes the oxygen dependent disproportionation of elemental sulfur, producing sulfite, thiosulfate and sulfide. Being considered an “archaeal like” enzyme, it is also encoded in the genomes of some acidophilic leaching bacteria such as Acidithiobacillus caldus, Acidithiobacillus thiooxidans, Acidithiobacillus ferrivorans and Sulfobacillus thermosulfidooxidans, among others. We measured Sor activity in crude extracts from Sb. thermosulfidooxidans DSM 9293T. The optimum temperature for its oxygenase activity was achieved at 75 °C, confirming the “thermophilic” nature of this enzyme. Additionally, a search for genes probably involved in sulfur metabolism in the genome sequence of Sb. thermosulfidooxidans DSM 9293T was done. Interestingly, no sox genes were found. Two sor genes, a complete heterodisulfidereductase (hdr) gene cluster, three tetrathionate hydrolase (tth) genes, three sulfide quinonereductase (sqr), as well as the doxD component of a thiosulfate quinonereductase (tqo) were found. Seven At. caldus strains were tested for Sor activity, which was not detected in any of them. We provide evidence that an earlier reported Sor activity from At. caldus S1 and S2 strains most likely was due to the presence of a Sulfobacillus contaminant.

Bioleaching of chalcopyrite and bornite by moderately thermophilic bacteria: an emphasis on their interactions

Interactions between chalcopyrite and bornite during bioleaching by moderately thermophilic bacteria were investigated mainly by X-ray diffraction, scanning electron microscopy, and electrochemical measurements performed in conjunction with bioleaching experiments. The results showed that a synergistic effect existed between chalcopyrite and bornite during bioleaching by both Acidithiobacillus caldus and Leptospirillum ferriphilum and that extremely high copper extraction could be achieved when chalcopyrite and bornite coexisted in a bioleaching system. Bornite dissolved preferentially because of its lower corrosion potential, and its dissolution was accelerated by the galvanic current during the initial stage of bioleaching. The galvanic current and optimum redox potential of 390−480 mV vs. Ag/AgCl promoted the reduction of chalcopyrite to chalcocite (Cu2S), thus accelerating its dissolution.

Enhancement in copper extraction from chalcopyrite by re-inoculation of different acidophilic, moderately thermophilic microorganisms

The feasibility and optimization of strategies of microbial re-inoculation were investigated to enhance chalcopyrite bioleaching. The application of microbial re-inoculation consisted of the re-addition of cultures of Acidithiobacillus caldus, Ferroplasma thermophilum or Leptospirillum ferriphilum (with different inoculum concentrations) into the two defined different bioleaching systems that separately represented the early and middle stages of chalcopyrite bioleaching. Changes in the bioleaching performance and microbial community structure after re-inoculation were compared to that in the control bioleaching experiment without re-inoculation. Results of re-addition of pre-grown microbial cultures into the early stage indicated that the re-inoculated strain survived and/or grew in the leaching environments and meanwhile synergistically promoted the growth of other microorganisms, then accelerated the total iron/sulfur oxidation compared to the unamended control. Finally, all these factors resulted in a significant enhancement of copper extraction. Moreover, a higher re-inoculation concentration exerted a more significant improvement in copper leaching from chalcopyrite. In all experiments, re-inoculation with L. ferriphilum into the early stage showed the best enhancement in copper leaching, which significantly shortened the incubation time and improved the maximum copper extraction. Re-inoculation with iron or sulphur oxidisers into the middle stage could not or slightly improve the final leaching level of copper. These results demonstrate that re-inoculation can be a useful step to improve the bioleaching kinetics and level of chalcopyrite; however its efficacy is influenced by the functional strain selection and procedures of culture re-inoculation (including re-addition time and inoculum concentration).

Feasibility of Bioaugmentation with Iron/Sulfur Oxidizing Acidophiles to Enhance Copper Bioleaching from a Flotation Copper Ore

The feasibility of one strategy of bioaugmentation was assessed to enhance copper extraction from chalcopyrite. Bioaugmentation consisted of the re-addition of one iron/sulfur oxidizing acidophile (Acidithiobacillus caldus, Ferroplasma thermophilumorLeptospirillum ferriphilum) into the early stage (on the 5thday) of the bioleaching system. The strain selection and inoculum concentration of bioaugmentation were separately investigated by comparing changes in the bioleaching performance and leached solid residues. Results indicated that bioaugmentation with three augmented strains synergistically promoted the total microbial growth and increased the cell numbers, and then accelerated the iron/sulfur oxidation, thereby catalytically regenerated the copper leaching agents of Fe3+and H+compared to the unamended control. Finally, an enhancement in copper extraction was detected and moreover positively correlated with the introducing cell numbers. Particularly, re-addition ofL.ferriphilumon the 5thday showed the best improvement in copper leaching, which remarkably shortened the incubation time (12 days) of almost full copper extraction while only 85.8% of copper was leached after 24 days in the control. Therefore, bioaugmentation could be a useful bio-remedy to improve the bioleaching kinetics and level of copper ore.

Expression of Critical Sulfur- and Iron-Oxidation Genes and the Community Dynamics During Bioleaching of Chalcopyrite Concentrate by Moderate Thermophiles.

Sulfate adenylyltransferase gene and 4Fe-4S ferredoxin gene are the key genes related to sulfur and iron oxidations during bioleaching system, respectively. In order to better understand the bioleaching and microorganism synergistic mechanism in chalcopyrite bioleaching by mixed culture of moderate thermophiles, expressions of the two energy metabolism genes and community dynamics of free and attached microorganisms were investigated. Specific primers were designed for real-time quantitative PCR to study the expression of these genes. Real-time PCR results showed that sulfate adenylyltransferase gene was more highly expressed in Sulfobacillus thermosulfidooxidans than that in Acidithiobacillus caldus, and expression of 4Fe-4S ferredoxin gene was higher in Ferroplasma thermophilum than that in S. thermosulfidooxidans and Leptospirillum ferriphilum. The results indicated that in the bioleaching system of chalcopyrite concentrate, sulfur and iron oxidations were mainly performed by S. thermosulfidooxidans and F. thermophilum, respectively. The community dynamics results revealed that S. thermosulfidooxidans took up the largest proportion during the whole period, followed by F. thermophilum, A. caldus, and L. ferriphilum. The CCA analysis showed that 4Fe-4S ferredoxin gene expression was mainly affected (positively correlated) by high pH and elevated concentration of ferrous ion, while no factor was observed to prominently influence the expression of sulfate adenylyltransferase gene.

Identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant enzymes.

Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into fructose and nucleotide (NDP)-glucose. To date, only SuSy's from plants and cyanobacteria, both photosynthetic organisms, have been characterized. Here, four prokaryotic SuSy enzymes from the nonphotosynthetic organisms Nitrosomonas Europaea (SuSyNe), Acidithiobacillus caldus (SuSyAc), Denitrovibrio acetiphilus (SusyDa), and Melioribacter roseus (SuSyMr) were recombinantly expressed in Escherichia coli and thoroughly characterized. The purified enzymes were found to display high-temperature optima (up to 80 °C), high activities (up to 125 U/mg), and high thermostability (up to 15 min at 60 °C). Furthermore, SuSyAc, SuSyNe, and SuSyDa showed a clear preference for ADP as nucleotide, as opposed to plant SuSy's which prefer UDP. A structural and mutational analysis was performed to elucidate the difference in NDP preference between eukaryotic and prokaryotic SuSy's. Finally, the physiological relevance of this enzyme specificity is discussed in the context of metabolic pathways and genomic organization.

Diguanylate cyclase null mutant reveals that C-Di-GMP pathway regulates the motility and adherence of the extremophile bacterium Acidithiobacillus caldus.

An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.

The influence of physicochemical parameters on the bioleaching of zinc sulfide concentrates using a mixed culture of moderately thermophilic microorganisms

This work investigates the extraction of zinc from Bama zinc sulfide concentrate (Isfahan, Iran) using a mixed culture of moderately thermophilic iron- and sulfur-oxidizing microorganisms including Leptospirillum ferriphilum, Acidithiobacillus caldus, Sulfobacillus sp. and Ferroplasma sp. The effects of some physicochemical parameters, namely pH, nutrient medium type, temperature, and the external addition of ferrous iron on the zinc extraction, redox potential and iron mobilization were investigated in shake flasks. Experiments were carried out at the pulp density of 5% (w/v) and a stirring rate of 150rpm. The highest zinc extraction (91%) was achieved at 45°C, leaching time of 18days, initial pH of 1.8 and Norris nutrient medium. The zinc extraction was 3.7-fold higher in the bioleaching compared to chemical leaching. The leaching rate of the sphalerite concentrate tended to decrease with decreasing pH from 1.8 to 1.2. It was also found that the increasing of ferrous iron addition from 0.02 to 0.05M led to a decrease both in the zinc extraction and bacterial activity indicating the inhibitory effect of increased iron on the microorganisms. SEM-EDX analyses revealed both poorly crystallized iron hydroxyl sulfates and well-crystallized jarosites on the surface of the solid residues.

Biooxidation of Gold-, Silver, and Antimony-Bearing Highly Refractory Polymetallic Sulfide Concentrates, and its Comparison with Abiotic Pretreatment Techniques

Effectiveness of different pure and mixed cultures of three moderately thermophilic, extremely acidophilic bacterial strains (Acidimicrobium ferrooxidans ICP, Sulfobacillus sibiricus N1, Acidithiobacillus caldus KU) were investigated for biooxidation of highly refractory polymetallic gold ore concentrates. Despite of its complex mineralogy and the presence of a mixture of potentially inhibitory metals and metalloids, the concentrate was readily dissolved in defined mixed cultures including both iron and sulfur oxidizers, releasing as much as 80% of soluble Fe and 61% of soluble As. Factors to affect microbial mineral dissolution efficiencies (i.e. microbial As(III) oxidation ability, formation of secondary mineral precipitation (e.g. jarosite, elemental sulfur, scorodite, anglesite), and microbial population dynamics during biooxidation) were studied, based on which roles of individual microbes and their synergistic interactions during biooxidation were discussed. Applying the biooxidation pretreatment using the most efficient mixed cultures containing all three strains significantly improved the recovery of both Au (from 1.1% to 86%) and Ag (from 3.2% to 87%). Finally, this study provides one of the very few available comparisons of the effectiveness of different pretreatment techniques for refractory gold ore concentrates: Compared with other abiotic pretreatment approaches (roasting, pressure oxidation, and alkali dissolution), biooxidation was shown to be one of the most effective options in terms of the recovery of Au and Ag.

Effect of thiocyanate on BIOX® organisms: Inhibition and adaptation

Stringent environmental legislation and the desire to become zero discharge have motivated mining operations to treat and recycle process water. Cyanidation tailings effluent contains elevated concentrations of cyanide and thiocyanate (SCN−), precluding recycling to the BIOX® process without prior treatment to reduce SCN− to below 1mg/l. The current study investigated the effect of SCN− on individual microbial species. Iron oxidation by Leptospirillum ferriphilum was not affected by SCN− concentrations below 0.5mg/l, with concentration dependent inhibition observed between 0.75 and 1.25mg/l and complete inhibition of iron oxidation above 1.25mg/l. Sulphur oxidation by Acidithiobacillus caldus showed a similar trend, with limited inhibition below 1.25mg/l and almost complete inhibition above 1.25mg/l. Repeated sub-culturing at low concentrations induced adaptation, with adapted cultures currently growing at SCN− concentrations of 7mg/l. The phenomenon of inhibition at low concentration, with subsequent adaptation was repeated in stirred tank reactors, leaching a pyrite/arsenopyrite concentrate.

Synergetic effects of Ferroplasma thermophilum in enhancement of copper concentrate bioleaching by Acidithiobacillus caldus and Leptospirillum ferriphilum

Assistance of introduction of Ferroplasma thermophilum in improving copper concentrate bio-dissolution was investigated. Results in pH-controlled stirred tank reactors showed that addition of F. thermophilum into the defined microbial consortium (Acidithiobacillus caldus and Leptospirillum ferriphilum) improved the dissolution and conversion of iron and meanwhile caused significant difference in the planktonic and attached population dynamics. Thereby, copper extraction was accelerated and increased by 22.7% after 24 days in comparison to the control without F. thermophilum. The intensive study of relationships between the modified bioleaching performance and augmentation of F. thermophilum conducted in shake flasks indicated that (1) F. thermophilum was capable of bio-catalytically regenerating the sulfide-attacking agent—ferric iron in bioleaching; (2) more importantly, mixed cultures involving F. thermophilum collaboratively resisted the inhibitory effects of environmental stress conditions on cells, such as 0.04% yeast extract, 5gL−1 Cu2+ and low initial pH 1.2 demonstrated in the simple growth medium, and furthermore increased the total cell numbers and accelerated the iron or sulfur oxidation compared to pure cultures. In conclusion, these findings revealed that there were synergetic acts between F. thermophilum and At. caldus or L. ferriphilum, which contributed to an improvement in copper leaching rate and level.

Thermoacidophilic microbial community oxidizing the gold-bearing flotation concentrate of a pyrite-arsenopyrite ore

An aboriginal community of thermophilic acidophilic chemolithotrophic microorganisms (ACM) was isolated from a sample of pyrite gold-bearing flotation concentrateat 45-47 degrees C and pH 1.8-2.0. Compared to an experimental thermoacidophilic microbial consortium formed in the course of cultivation in parallel bioreactors, it had lower rates of iron leaching and oxidation, while its rate of sulfur oxidation was higher. A new thermophilic acidophilic microbial community was obtained by mutual enrichment with the micioorganisms from thie experimental and aboriginal communities during oxidation of sulfide ore flotation concentrate at 47 degrees C. The dominant bacteria of this new ACM community were Acidithiobacillus caldus strains (the most active sulfur oxidizers) and Sulfobacillus thermotolerans strains (active oxidizers of both iron and sulfur), while iron-oxidizing archaea of the family Ferroplasmaceae and heterotrophic bacteria Alicyclobacillus tolerans were the minor components. The new ACM community showed promise for leaching/oxidation of sulfides from flotation concentrates at high pulp density (S:L = 1:4).

Effect of the aeration mode and yeast extract on the oxidation of high-pyrrhotite sulfide ore flotation concentrate and on the composition of the acidophilic chemolithotrophic microbial community

Optimal aeration conditions were determined and the effect of yeast extract on biooxidation of high-pyrrhotite sulfide ore flotation concentrate in the course of continuous cultivation of an acidophilic chemolithotrophic microbial community was studied in a line of four sequential laboratory reactors; the aeration rate was 3 L/(L min), yeast extract concentration was 0.02%. The gold recovery level was 96.45% at 2.23% elemental sulfur content in the solid residue. The dominant strains identified in the community responsible for biooxidation were Acidithiobacillus caldus OL13-1, At. caldus OL13-3 = At. caldus OL12-3, and an ‘Acidiferrobacter’ strain. Strains Sulfobacillus thermosulfidooxidans OL13-2 = S. thermosulfidooxidans OL12-1 and Ferroplasma acidiphilum OL13-4 = F. acidiphilum OL12-4 were isolated in pure culture and identified.

Multi Locus Sequence Typing scheme for Acidithiobacillus caldus strain evaluation and differentiation

Phenotypic, metabolic and genetic properties of several Acidithiobacillus caldus strains indicate the existence of as yet undefined levels of variation within the species. Inspite of this, intraspecies genetic diversity has not yet been explored in detail.In this study, the design and implementation of a Multi Locus Sequence Typing (MLST) scheme for At. caldus is described. This represents the first MLST-based study applied to industrial isolates of the species. Seven informative and discriminant MLST markers were selected using a sequence-driven approach and a custom-designed bioinformatic pipeline. The allelic profiles of thirteen At. caldus strains from diverse geographical origins and industrial settings were derived using this scheme.MLST-based population structure analysis indicated only moderate amounts of genetic diversity within the set of strains, further supporting their current assignment to a single species. Also, no clear evidence for geographical isolation could be derived from this study. However, the prevalence of sequence type 1 in heap leaching industrial settings support the view that bioprocess conditions and dynamics may have a strong influence on At. caldus (microbial) microdiversity patterns.The MLST scheme presented herein is a valuable tool for the identification and classification of strains of At. caldus for either ecological or evolutionary studies and possibly also for industrial monitoring purposes.

Microbial community shifts during the process of marmatite bioleaching

Predefined mixed cultures of Acidithiobacillus ferrooxidans, Acidithiobacillus caldus and Acidithiobacillus thiooxidans strains were used to stimulate marmatite bioleaching under different temperatures. Changes in this simple bacterial community over time during the leaching process were evaluated using denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments. A. ferrooxidans and A. caldus were detected in all samples taken at 5-d intervals during a 41–46days bioleaching process. The presence of A. thiooxidans in the initial inoculum stimulated bioleaching, however the relative population of A. thiooxidans gradually declined, and this species was not detected after 30day (at 30°C) or 15days (at 35°C). Scanning electron microscopy (SEM), X-ray diffraction (XRD) and energy dispersive X-ray analysis (EDX) analyses revealed the production of S0 and jarosite during bioleaching. Bacteria were observed to be attached to mineral surfaces. Results suggested that A. ferrooxidans and A. caldus together could perform marmatite bioleaching, and that A. thiooxidans could promote the bioleaching of marmatite by A. ferrooxidans and A. caldus. This study improves the understanding of marmatite bioleaching at different temperatures.

Analysis of the microbial community in moderately acidic drainage from the Yanahara pyrite mine in Japan.

Acid rock drainage (ARD) originating from the Yasumi-ishi tunnel near the main tunnel of the Yanahara mine in Japan was characterized to be moderately acidic (pH 4.1) and contained iron at a low concentration (51 mg/L). The composition of the microbial community was determined by sequence analysis of 16S rRNA genes using PCR and denaturing gradient gel electrophoresis. The analysis of the obtained sequences showed their similarity to clones recently detected in other moderately acidic mine drainages. Uncultured bacteria related to Ferrovum- and Gallionella-like clones were dominant in the microbial community. Analyses using specific primers for acidophilic iron- or sulfur-oxidizing bacteria, Acidithiobacillus ferrooxidans, Leptospirillum spp., Acidithiobacillus caldus, Acidithiobacillus thiooxidans, and Sulfobacillus spp. revealed the absence of these bacteria in the microbial community in ARD from the Yasumi-ishi tunnel. Clones affiliated with a member of the order Thermoplasmatales were detected as the dominant archaea in the ARD microbial population.

Identification and characterization of an ETHE1-like sulfur dioxygenase in extremely acidophilic Acidithiobacillus spp.

Elemental sulfur (S(0)) oxidation in Acidithiobacillus spp. is an important process in metal sulfide bioleaching. However, the gene that encodes the sulfur dioxygenase (SDO) for S(0) oxidation has remained unclarified in Acidithiobacillus spp. By BLASTP with the eukaryotic mitochondrial sulfur dioxygenases (ETHE1s), the putative sdo genes (AFE_0269 and ACAL_0790) were recovered from the genomes of Acidithiobacillus ferrooxidans ATCC 23270 and Acidithiobacillus caldus MTH-04. The purified recombinant proteins of AFE_0269 and ACAL_0790 exhibited remarkable SDO activity at optimal mildly alkaline pH by using the GSH-dependent in vitro assay. Then, a sdo knockout mutant and a sdo overexpression strain of A. ferrooxidans ATCC 23270 were constructed and characterized. By overexpressing sdo in A. ferrooxidans ATCC 23270, a significantly increased transcriptional level of sdo (91-fold) and a 2.5-fold increase in SDO activity were observed when S(0) was used as sole energy source. The sdo knockout mutant of A. ferrooxidans displayed a slightly reduced growth capacity in S(0)-medium compared with the wild type but still maintained high S(0)-oxidizing activity, suggesting that there is at least one other S(0)-oxidizing enzyme besides SDO in A. ferrooxidans ATCC 23270 cells. In addition, no obvious changes in transcriptional levels of selected genes related to sulfur oxidation was observed in response to the sdo overexpression or knockout in A. ferrooxidans when cultivated in S(0)-medium. All the results might suggest that SDO is involved in sulfide detoxification rather than bioenergetic S(0) oxidation in chemolithotrophic bacteria.

Effect of pulp density on planktonic and attached community dynamics during bioleaching of chalcopyrite by a moderately thermophilic microbial culture under uncontrolled conditions

An enriched and adapted moderately thermophilic culture showed good performance during bioleaching of chalcopyrite under uncontrolled conditions. The copper extractions were up to 85.0%, 77.3% and 56.3% at pulp densities of 10%, 15% and 20% (w/v) within 22days, respectively. The real-time quantitative PCR was employed to monitor planktonic and attached community dynamics during bioleaching of chalcopyrite by the moderately thermophilic microbial culture. Only three species, including Acidithiobacillus caldus, Sulfobacillus acidophilus and Ferroplasma thermophilum, were detected during the whole bioleaching process. The results show that pulp density had significant effects on planktonic and attached microbial community dynamics. The succession of attached cells was different from community dynamics of their planktonic counterparts. F. thermophilum and A. caldus preferred to attach to mineral surface, especially at pulp densities of 15% and 20%.

The shift of microbial community under the adjustment of initial and processing pH during bioleaching of chalcopyrite concentrate by moderate thermophiles.

The shift of microbial community under the adjustment of different pH was analyzed by denaturing gradient gel electrophoresis (DGGE). The results indicated, at initial pH 1.0, 2.0 and 3.0, the copper extraction in 22 days amounted to 84.6%, 88.2% and 77.5%, respectively; however, when the initial pH was 2.0, processing pH was adjusted to 1.0 and 3.0 on day 16, the copper extraction in 32 days was 85% and 62.6%, respectively. DGGE analysis showed Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans and Ferroplasma thermophilum existed in bioleaching systems. At initial pH 1.0 and 3.0, S. thermosulfidooxidans and A. caldus were main microorganisms. While at initial pH 2.0, L. ferriphilum, A. caldus and S. thermosulfidooxidans were always detected. At processing pH 1.0 and 3.0, the adjustment of pH greatly inhibited the growth of L. ferriphilum; it was also found microbial community would recover gradually only if pH stimulation did not fatally affect microorganisms.

Influence of bioaugmentation with Ferroplasma thermophilum on chalcopyrite bioleaching and microbial community structure

Ferroplasma spp. are cell wall-deficient, extremely acidophilic and iron-oxidising archaea that have considerable biotechnological potential for bioleaching processes. The current study focuses on investigating the ecological importance of mixotrophic Ferroplasma thermophilum L1 during chalcopyrite bioleaching and the feasibility of bioaugmentation with this archaeon to improve the extraction of copper from chalcopyrite. Bioaugmentation consisted of the addition of highly concentrated F. thermophilum (with different inoculum concentrations) into the two defined bioleaching systems that represented the early and advanced chalcopyrite bioleaching stages. The evolution of bioleaching performance, microbial community structure and leached solid residues after bioaugmentation were compared with those in the conventional (control) bioleaching experiment. The results of bioaugmentation undertaken at the early stage showed that the growth of Leptospirillum ferriphilum and Acidithiobacillus caldus were promoted after bioaugmentation, which, in turn, assisted F. thermophilum in surviving under the same conditions. Concurrently, the mineral dissolving and copper extraction rates were significantly accelerated, especially in the ‘Fer-1/1’ test that was bioaugmented with the highest addition amount. After 24days, 90.2% of the total copper was leached out, which was higher than the value (83.4%) obtained in the control. The untimely formation of a ‘Jarosite’ passivation layer decreased the rate of copper release at the later stage of the ‘Fer-1/1’ test. The bioaugmentation of the advanced leaching stage was detrimental to the chalcopyrite dissolution; the number of added F. thermophilum cells negatively correlated with the copper recovery. The poor bioleaching was also primarily associated with the formation of iron precipitation due to the increased cell numbers of iron oxidisers. These findings indicate that different bioaugmented treatments with F. thermophilum clearly influenced chalcopyrite leaching and the microbial community. Bioaugmentation could be a useful remedy to accelerate chalcopyrite dissolution.