Papers by Author: D. Barrie Johnson

Abstract: Cueva de la Mora and Guadiana are two acidic mine pit lake in the Iberian Pyrite Belt (south-west Spain) that exhibit depth-related stratification, which is in turn reflected in the bacterial community population the different layers. Here we describe the microbial communities present in samples of sediments located close to the surface and deep within the lakes, which show interesting contrasts to planktonic communities.

Abstract: Four well-differentiated clusters have been identified among iron-oxidizing acidithiobacilli. One monophyletic group clusters with At. ferrooxidans^T and another with strains of At. ferrivorans. While At. ferrooxidans and At. ferrivorans share many physiological traits, they differ in some phenotypic characteristics such as motility, pH and temperature minima, and also in terms of genes involved in ferrous iron oxidation. The genome of a strain (CF27) of At. ferrivorans, which is characterized by its marked propensity to form macroscopic growths and biofilms, was sequenced at Genoscope (Evry, France). Comparative genomic studies were carried with all related Acidithiobacillus strains that have been sequenced to date, in order to identify genetic determinants responsible for physiological traits relating to the oxidation of iron and sulfur, and also to extracellular polymeric substances formation.

Abstract: The microbiology and geochemistry of two pit lakes at former metal mines (Cueva de la Mora and Guadiana) located in the Iberian Pyrite Belt in Spain were investigated. Both lakes are meromictic, with more acidic and oxidized mixolimnion zones overlying anoxic monimolimnion zones, and transitional chemoclines with characteristic sharp pH and redox potential gradients. Stratification in the pit lakes was reflected in the size and diversity of the microbial communities in the different zones, with the chemocline of Cueva de la Mora pit lake and the hypolimnion (the lower layer of the mixolimnion) in the Guadiana pit lake containing the most complex and abundant microbial communities. Acidophiles that oxidize and reduce both iron and sulfur co-exist within the chemocline/hypolimnion zones, implying that biogeochemical cycling of these two elements is most intense within these layers of the pit lakes. Novel species of bacteria were detected using molecular techniques and, in some cases, isolated and partially characterized. The latter included a novel acidophilic iron-reducing gammaproteobacterium (Acidibacter ferrireducens)

Abstract: An integrated bio-processing scheme was devised and tested in the laboratory for recovering copper, or other base metals, from pregnant leach solutions (PLS) using a two-step process involving both iron-reduction, and sulfate-reduction for H₂S generation and sulfide precipitation, as a potential alternative to conventional SX-EW. Reduction of ferric iron in the PLS was achieved using iron-reducing Acidithiobacillus spp. and Sulfobacillus thermosulfidooxidans in column reactors containing elemental sulfur as electron donor. Analysis of the column reactor effluents showed not only that most of the ferric iron was reduced to ferrous, but also that all of the copper (II) had been reduced to copper (I), i.e. cuprous copper. This copper (I) appeared to be complexed as it was not oxidized when exposed to ferric iron nor precipitated as a copper-sulfide when exposed to either sodium sulfide or H₂S. The data suggested that copper (II) was reduced and the resulting copper (I) complexed, with both reactions probably mediated by sulfur oxy-anions produced indirectly by the bacteria, in the anoxic sulfur column bioreactors. It was also noted that copper (I) produced chemically by reduction of copper (II) by hydroxylamine was more toxic to axenic cultures of Acidithiobacillus spp. and Sb. thermosulfidooxidans than was the copper (I) in the column effluent liquors.

Abstract: Acidithiobacillus ferrooxidans is the only acidophilic bacterium to have been reported to grow using hydrogen as sole electron donor. Hydrogen is a potentially widely available energy source in acidic environments and therefore may have more ubiquitous use among indigenous prokaryotes than currently recognized. To test this hypothesis, thirty-seven strains of acidophilic bacteria, including 24 separate designated and proposed species, were screened for their abilities to use the dissimilatory oxidation of hydrogen to support their growth. Positive results were obtained with all strains of Group I (Acidithiobacillus ferrooxidans) and Group II (At. ferridurans) iron-oxidizing acidithiobacilli, At. caldus, the three species of Sulfobacillus tested, and Acidimicrobium ferrooxidans. Growth yields approaching 10¹⁰ cells ml^-1 were obtained for aerobic growth of At. ferrooxidans and At. ferridurans on hydrogen.

Abstract: In this work we have examined the bacterial diversity from the hot spring sediment Agua del Limón (AL1) present at the geothermal Caviahue-Copahue system using a combination of molecular and cultivation techniques, with particular emphasis on indigenous anaerobic prokaryotes. Microorganisms involved in the iron (Acidithiobacillus ferrooxidans and Leptospirillum spp.) and sulphur (Acidithiobacillus spp., Thermotogales-like bacteria, Thiomonas sp., and Desulfurella sp.) cycles were identified in the clone library. Although no obvious sulfate-reducing bacteria were detected by culture-independent techniques, several isolates related to the mesophilic, spore-forming sulfate-reducer "Desulfobacillus acidavidus" strain CL4 were isolated at 30°C and 40°C. The 16S rRNA gene of another isolate showed 94% similarity to Desulfotomaculum thermobenzoicum. Sulfate-reducing enrichment cultures of the Copahue samples were also dominated by "Dsb. acidavidus" CL4.

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: 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.