Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C₁-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 M, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol^-1 at a dilution rate of 0.05 h^-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C₁-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.     

Characterization of iron- and sulphide mineral-oxidizing moderately thermophilic acidophilic bacteria from an Indonesian auto-heating copper mine waste heap and a deep South African gold mine

Iron- and chalcopyrite-oxidizing enrichment cultures were obtained at 50°C from acidic, high-temperature, copper/gold mine environments in Indonesia and South Africa. Over 90% copper yield was obtained from chalcopyrite concentrate with the Indonesian enrichment in 3 months with 2% solids concentration, when pH was maintained at around 2. Neither addition of silver cations nor an enhanced nutrient concentration influenced chalcopyrite leaching. Excision and sequencing of bands from denaturing gradient gel electrophoresis of the amplified partial 16S rRNA gene showed that the enrichment cultures from different environments in South Africa and Indonesia were very simple, and similar. Chalcopyrite concentrate supported a simpler and different community than Fe²⁺. The members of the enrichment cultures were closely related to Sulfobacillus yellowstonensis and Sulfobacillus acidophilus.     

Comparison of ferric iron generation by different species of acidophilic bacteria immobilized in packed-bed reactors

Flooded packed-bed bioreactors, prepared by immobilizing four different species of acidophilic iron-oxidizing bacteria on porous glass beads, were compared for their ferric iron-generating capacities when operated in batch and continuous flow modes over a period of up to 9 months, using a ferrous iron-rich synthetic liquor and acid mine drainage (AMD) water. The bacteria used were strains of Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, a Ferrimicrobium-like isolate (TSTR) and a novel Betaproteobacterium (isolate PSTR), which were all isolated from relatively low-temperature mine waters. Three of the bacteria used were chemoautotrophs, while the Ferrimicrobium isolate was an obligate heterotroph. Greater biomass yields achievable with the Ferrimicrobium isolate resulted in greater iron oxidation efficiency in the newly commissioned bioreactor containing this bacterium, though long-term batch testing with organic carbon-free solution resulted in similar maximum iron oxidation rates in all four bioreactors. Two of the bioreactors (those containing immobilized L. ferrooxidans and Ferrimicrobium TSTR) were able to generate significantly lower concentrations of ferrous iron than the others when operated in batch mode. In contrast, when operated as continuous flow systems, the bioreactor containing immobilized PSTR was superior to the other three when challenged with either synthetic or actual AMD at high flow rates. The least effective bacterium overall was At. ferrooxidans, which has previously been the only iron-oxidizer used in the majority of reports describing ferric iron-generating bioreactors. The results of these experiments showed that different species of iron-oxidizing acidophiles have varying capacities to oxidize ferrous iron when immobilized in packed-bed bioreactors, and that novel isolates may be superior to well-known species.     

Cultivated anaerobic acidophilic/acidotolerant thermophiles from terrestrial and deep-sea hydrothermal habitats

Metabolic and phylogenetic diversity of cultivated anaerobic microorganisms from acidic continental hot springs and deep-sea hydrothermal vents was studied by molecular and microbiological methods. Anaerobic organotrophic enrichment cultures growing at pH 3.5–4.0 and 60 or 85°C with organic energy sources were obtained from samples of acidic hot springs of Kamchatka Peninsula (Pauzhetka, Moutnovski Volcano, Uzon Caldera) and Kunashir Island (South Kurils) as well as from the samples of chimneys of East Pacific Rise (13°N). The analyses of clone libraries obtained from terrestrial enrichment cultures growing at 60°C revealed the presence of archaea of genus Thermoplasma and bacteria of genus Thermoanaerobacter. Bacterial isolates from these enrichments were shown to belong to genera Thermoanaerobacter and Thermoanaerobacterium, being acidotolerant with the pH optimum for growth at 5.5–6.0 and the pH minimum at 3.0. At 85°C, domination of thermoacidophilic archaea of genus Acidilobus in terrestrial enrichments was found by both molecular and microbiological methods. Five isolates belonging to this genus possessed some phenotypic features that were new for this genus, such as flagellation or the ability to grow on monosaccharides or disaccharides. Analyses of clone libraries from the deep-sea thermoacidophilic enrichment cultures showed that the representatives of the genus Thermococcus were present at both 60 and 85°C. From the 60°C deep-sea enrichment, a strain belonging to Thermoanaerobacter siderophilus was isolated. It grew optimally at pH 6.0 with the minimum pH for growth at 3.0 and with salinity optimum at 0–2.5% NaCl and the maximum at 7%, thus differing significantly from the type strain. These data show that fermentative degradation of organic matter may occur at low pH and wide temperature range in both terrestrial and deep-sea habitats and can be performed by acidophilic or acidotolerant thermophilic prokaryotes.     

Ecology and habitats of extremophiles

This review describes the main natural extreme environments, characterized by high temperature, high and low pH and high salinity, that can be colonized by microorganisms. The environments covered are: freshwater alkaline hot springs; acidic solfatara fields; anaerobic geothermal mud and soils; acidic sulphur and pyrite areas; carbonate springs and alkaline soil; and soda and highly saline lakes. The community structure, in terms of available energy sources and representative autotrophic and heterotrophic microorganisms, is discussed for each type of habitat.The authors are with the Department of Biotechnology. Technological Institute of Iceland, Keldnaholt, 112 Reykjavik, Iceland and with the Institute of Biology, University of Iceland, Reykjavik, Iceland     

Colony formation of free-living and particle-associated sulfate-reducing bacteria

Abstract Some of the components that are likely to be involved in the respiratory chains that transfer electrons from the ferrous iron substrate of various acidophilic bacteria have been revealed by spectroscopic analysis. An apparently unique, soluble and acid-stable cytochrome was found in Leptospirillum ferrooxidans which appeared to lack any of the major cytochromes or the rusticyanin of the better-studied iron-oxidizing mesophile, Thiobacillus ferrooxidans . A specific absorption peak, only found in iron-grown cells, was revealed in a whole cell spectra of thermoacidophilic archaebacteria of different genera.     

Ferroplasma cupricumulans sp. nov., a novel moderately thermophilic, acidophilic archaeon isolated from an industrial-scale chalcocite bioleach heap

A new species of Archaea was isolated from an industrial mineral sulphide bioleach heap. Strain BH2, a non-motile pleomorphic coccus, was capable of chemomixotrophic growth on ferrous sulphate and yeast extract. Growth was not supported in the absence of yeast extract. Phylogenetic analysis based on the 16S rRNA gene showed that strain BH2 was most closely related to the species Ferroplasma acidiphilum; however, it showed only 95% sequence similarity with this species. Strain BH2 had a temperature optimum of 53.6°C and a temperature range for growth between 22 and 63°C. Thus, it is the first moderately thermophilic member of the genus Ferroplasma. The optimum pH for the growth of the strain occurred between pH 1.0 and 1.2 and the lowest pH at which growth was observed was 0.4. Based on 16S rRNA gene sequence analysis and other physiological characteristics, strain BH2 constitutes a new species within the genus Ferroplasma. The name Ferroplasma cupricumulans is proposed for the new species and strain BH2 (DSM 16651) is proposed as the type strain.     

Characterization of a β-glycosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius

In cell free extracts of the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius ATCC27009, we have identified β-gluco- and galactosidase activities showing a specific activity of 0.1 and 12 U/mg, respectively. The two enzymatic activities are associated with different polypeptides and we show here the functional cloning, the expression in Escherichia coli and the characterisation of the β-glucosidase (Aaβ-gly). The enzyme, which is optimally active and stable at temperatures above 65°C, belongs to glycoside hydrolase family 1 (GH1) and shows wide substrate specificity on different aryl-glycosides and cello-oligosaccharides with k _cat/K _M for 4-nitrophenyl-β-D-glucoside and cellobiose of 2,976 and 185 s⁻¹mM⁻¹, respectively. Interestingly, upstream to the β-glycosidase gene, we identified a second ORF homologous to the ATPase subunit of the bacterial ABC transporters (abc1) that is co-transcribed with the β-glycosidase gene glyB and that could be involved in the carbohydrate import. The activity of the enzyme on cello-oligosaccharides of up to five glucose units strongly indicates that the enzyme could be involved in vivo in the degradation of glucans together with endoglucanase enzymes previously described. This, together with the co-expression of the two genes, suggests a role for the glyB-abc1 cluster in A. acidocaldarius in the degradation of cellulose and hemicelluloses. Enzymes: EC 3.2.1.21; EC 3.2.1.23; EC 3.2.1.25; EC 3.2.1.38; EC 3.2.1.37 An erratum to this article can be found at     

Sulfobacillus benefaciens sp. nov., an acidophilic facultative anaerobic Firmicute isolated from mineral bioleaching operations

Gram-positive bacteria found as the sole Firmicutes present in two mineral bioleaching stirred tanks, and a third bacterium isolated from a heap leaching operation, were shown to be closely related to each other but distinct from characterized acidophilic iron- and sulfur-oxidizing bacteria of the genus Sulfobacillus, to which they were affiliated. One of the isolates (BRGM2) was shown to be a thermo-tolerant (temperature optimum 38.5°C, and maximum 47°C) obligate acidophile (pH optimum 1.5, and minimum 0.8), and also noted to be a facultative anaerobe, growing via ferric iron respiration in the absence of oxygen. Although isolates BRGM2 and TVK8 were able to metabolize many monomeric organic substrates, their propensity for autotrophic growth was found to be greater than that of Sulfobacillus thermosulfidooxidans and the related acidophile, Sb. acidophilus. Faster growth rates of the novel isolates in the absence of organic carbon was considered to be a major reason why they, rather than Sb. thermosulfidooxidans (which shared many physiological characteristics) more successfully exploited conditions in the stirred tanks. Based on their phylogenetic and phenotypic characteristics, the isolates are designated strains of the proposed novel species, Sulfobacillus benefaciens, with isolate BRGM2 nominated as the type strain.     

Culturable and molecular phylogenetic diversity of microorganisms in an open-dumped,extremely acidic Pb/Zn mine tailings

A combination of cultivation-based and molecular-based approaches was used to reveal the culturable and molecular diversity of the microbes inhabiting an open-dumped Pb/Zn mine tailings that was undergoing intensive acid generation (pH 1.9). Culturable bacteria found in the extremely acidic mine tailings were Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum, Sulfobacillus thermotolerans and Acidiphilium cryptum, where the number of acidophilic heterotrophs was ten times higher than that of the iron- and sulfur-oxidizing bacteria. Cloning and phylogenetic analysis revealed that, in contrast to the adjacent AMD, the mine tailings possessed a low microbial diversity with archaeal sequence types dominating the 16S rRNA gene library. Of the 141 clones examined, 132 were represented by two sequence types phylogenetically affiliated with the iron-oxidizing archaea Ferroplasma acidiphilum and three belonged to two tentative groups within the Thermoplasma lineage so far represented by only a few environmental sequences. Six clones in the library were represented by the only bacterial sequence type and were closely related to the well-described iron-oxidizer L. ferriphilum. The significant differences in the prokaryotic community structures of the extremely acidic mine tailings and the AMD associated with it highlights the importance of studying the microbial communities that are more directly involved in the iron and sulfur cycles of mine tailings.