Papers by Author: Kevin B. Hallberg

Abstract: Biosulfidogenesis (the generation of hydrogen sulfide by microorganisms) in acidic liquors was investigated using two metabolically-distinct bacteria. One was a novel acidophilic sulfate-reducing bacterium (isolate CL4) that grew at pH 3.0 and above using glycerol as electron donor, and the other was the type strain of Acidithiobacillus ferrooxidans which was grown at pH 2.5 using hydrogen (derived from dissolution of metallic iron) as electron donor and elemental sulfur as electron acceptor. Both bacteria were grown in pH-controlled bioreactors. Isolate CL4 mediated the selective precipitation of zinc in situ, while the At. ferrooxidans bioreactor operated as an off-line system, generating hydrogen sulfide that precipitated copper in a separate reaction vessel. The potential of using acidophilic sulfidogens for the selective recovery of metals from acidic waste streams is discussed.

Abstract: It has long been recognized that isolates of iron- and sulfur-oxidizing acidophiles referred to as “Acidithiobacillus ferrooxidans” probably include more than one species, on the basis of differences in chromosomal GC contents and 16S rRNA gene sequences. Phylogenetic heterogeneity among these isolates was confirmed by phylogenetic analysis using the sequences of the 16S-23S intergenic spacers (ITS). Two main groups have been identified: the first includes the type strain of Acidithiobacillus ferrooxidans and a second comprises a cluster of newly isolated strains that have 98.5% 16S rRNA gene sequence identity with the type strain. Given that the new group of isolates have GC contents of 56 mol% as opposed to 58.8 mol% for At. ferrooxidansT, and that they share only 37% homologous DNA, these were given the new species name Acidithiobacillus ferrivorans. Further studies showed that, while strains of At. ferrivorans have many physiological traits in common with At. ferrooxidans, they also differ in some key characteristics. These include the ability to grow at temperatures as low as 4°C (as opposed to the lower limit of between 10 and 12°C for At. ferrooxidans) and the greater sensitivity of At. ferrivorans to low pH (minimum of 1.9 for growth as opposed to 1.3 for At. ferrooxidansT). Important genotypic differences include the fact that all strains of At. ferrivorans do not contain the archetypal rusticyanin gene (rusA), rather most contain a rusA homologue (rusB). Furthermore, the high potential iron-sulfur protein-encoding gene of all At. ferrivorans strains analyzed is more similar to the iro than to the hip gene characterized in At. ferrooxidansT. These results suggest that the iron oxidation pathways are different in At. ferrivorans and At. ferrooxidans.

Abstract: Bioleaching test work was performed in continuously operated multi-stage reactor systems at 70°C using a thermophilic culture treating an Aguablanca Ni-Cu concentrate from Spain and a blend of Cu concentrates from Bor, Serbia. The copper in both these concentrates occurs as chalcopyrite and therefore the use of thermophiles was applied, which resulted in copper recoveries of over 95%. Qualitative assessment of the microbial community in the bioreactors was performed by terminal restriction enzyme fragment length polymorphism (T-RFLP) and clone library analysis of the 16S rRNA genes amplified by PCR. T-RFLP analysis revealed that only archaea were present, and that the communities in both the Aguablanca and Bor systems consisted of two different microorganisms. A 16S rRNA gene clone library using DNA from the Aguablanca system was constructed and screened. Again, two archaea were detected in similar relative abundance in the population as found by T-RFLP analyses. The sequences of these two cloned genes revealed that the dominant archaeon (up to 98% of the total archaea detected) was Acidianus brierleyi, and the other was Metallosphaera sedula. Quantitative assessment of the microbial community was performed by Q-PCR and confirmed the dominance of archaea in the system with Acidianus being the dominant strain (98-99% of the total population) and a minor part of the population (1-2%) consisted of Metallosphaera. Additionally, very small amounts of Sulfolobus spp. were detected. This study, along with other recent studies on the diversity of thermoacidophiles involved in the solubilization of copper from chalcopyrite concentrates, revealed that a wider variety of thermoacidophiles are involved in bioprocessing of metal sulfides, and showed that A. brierleyi should be considered an important biomining acidophile.

Abstract: The Escondida mine, located in northern Chile, is the largest copper producing mine in the world. It has an abundant low-grade (ca. 0.5% Cu) sulfide copper ore reservoir, which is processed in large heap bioreactors at the mine. To understand better how microorganisms adapt to heap leaching environments, we have isolated and identified acidophiles from pregnant leach solution (PLS) from the heaps. Six bacteria and one archaeon were isolated directly on solid overlay media, and identified by phylogenetic analyses of their 16S rRNA genes as strains of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum ferriphilum, Acidiphilium cryptum and Ferroplasma acidiphilum. The sequences of the 16S rRNA genes from isolated strains showed high similarity with those detected previously by culture-independent analyses performed on samples from a pilot plant for this process. Of the three known species of Leptospirillum, only L. ferriphilum has been detected in Escondida PLS. Tolerance of the Escondida isolate (coded IESL-25) to copper and some other transition metals such as zinc, nickel and silver was compared with several other strains of both L. ferriphilum and Leptospirillum ferrooxidans. It was noted that all L. ferriphilum strains (including IESL-25) displayed far greater tolerance to both copper and silver than strains of L. ferrooxidans, though tolerance to zinc and nickel was similar among isolates of both species. Micro-representational-difference analysis (MRDA) was used to study the genetic response of L. ferriphilum IESL-25 to high copper concentration. Gene sequences obtained by MRDA were analyzed using available genomic information for L. ferriphilum and one copper-induced gene identified appears to be involved in lipopolysaccharide biosynthesis.

Abstract: In the Lusatia area a pilot plant for the treatment of acidic mine waters by microbial iron oxidation and a concomitant iron hydroxysulfate precipitation is operated. Molecular based studies of acidic waters from this iron hydroxysulfate producing pilot plant revealed the presence of 16S rRNA gene sequences from undescribed iron-oxidizing bacteria. Most of these were related to autotrophic Betaproteobacteria (see Heinzel et al. IBS 2007, poster number 98). For cultivating different iron-oxidizing bacteria water samples from this pilot plant were directly plated on various solid media. Double-layer plates were used, with a heterotrophic Acidiphiliumstrain in the underlayer, because of the high sensitivity of autotrophic bacteria towards organic substances. The media contained different iron-substrates with and without organic carbon sources. Colonies appeared at the latest after three weeks and they were encrusted with ferric iron. The phylogeny of the isolated strains was determined and the physiological requirements, like temperature, pH optima, preferred carbon source and iron concentrations, were analyzed. Many isolates which were related to Acidithiobacillus ferrooxidans strains could be cultivated, as well as an isolate related to the genus Thiomonas and one isolate related to a Ferribacter polymyxa species. The Thiomonas-like isolate showed best growth in media containing tryptone soya broth, sodium thiosulfate and ferrous sulfate at pH 2.5 and 30°C. The other Betaproteobacterium grew on ferrous sulfate medium at pH 2.5 between 16 and 37°C. In liquid culture experiments the cells of both isolates were attached to the iron minerals built in the medium. These physiological characteristics of the isolates helped to vary parameters in the pilot plant to optimize the process of iron oxidation and improved waste water remediation.

Abstract: Draft genome sequences of Acidithiobacillus thiooxidans ATCC 19377 and A. caldus ATCC 51756 have been annotated. Bioinformatic analysis of these two new genomes, together with that of A. ferrooxidans ATCC 23270, allows the prediction of metabolic and regulatory models for each species and has provided a unique opportunity to undertake comparative genomic studies of this group of bioleaching bacteria. In this paper, we report preliminary information on metabolic and electron transfer pathways for ten characteristics of the three acidithiobacilli: CO2 fixation, the TCA cycle, sulfur oxidation, sulfur reduction, iron oxidation, iron assimilation, hydrogen oxidation, flagella formation, Che signaling (chemotaxis) and nitrogen fixation. Predicted transcriptional and metabolic interplay between pathways pinpoints potential coordinated responses to environmental signals such as energy source, oxygen and nutrient limitations. The predicted pathway for nitrogen fixation in A. ferrooxidans will be described as an example of such an integrated response. Several responses appear to be especially characteristic of autotrophic microorganisms and may have direct implications for metabolic processes of critical relevance to the understanding of how these microorganisms survive and proliferate in extreme environments, including industrial bioleaching operations.

Abstract: Soluble manganese (Mn(II)) can cause nuisance and occasionally toxicity problems, both in surface and ground waters and in domestic water supplies. Many mine drainage waters contain highly elevated concentrations (often >100 mg l-1). Current systems for remediating mine waters, although effective for other metals, are often inefficient at removing manganese. This paper describes the development of self-sustaining, low-cost bioreactors that can be used in situ for passive removal of manganese from mine waters and other contaminated water courses of pH >4. A prototype bioreactor, set-up using Mn(IV)-coated pebbles from a fresh-water stream, was tested over one year and shown to be effective in removing Mn(II) from 10 to <0.25 mg l-1. Two species of fungi and one bacterial species were isolated from this system as Mn(II)-oxidizers. The fungi were identified as belonging to the order Pleosporales (Ascomycetes), and one was related (98 % 18S rRNA gene sequence identity) to a known Mn(II)-oxidising fungus. The bacterial isolate was closely related to the α-proteobacterium, Bosea thiooxidans. T-RFLP analysis showed that one or both of the fungal isolates were the dominant eukaryotes in the bioreactor community.