Bioleaching of metal sulfides is caused by astonishingly diverse groups of bacteria. Today, at least 11 putative prokaryotic divisions can be related to this phenomenon. In contrast, the dissolution (bio)chemistry of metal sulfides follows only two pathways, which are determined by the acid-solubility of the sulfides: the thiosulfate and the polysulfide pathway. The bacterial cell can effect this sulfide dissolution by "contact" and "non-contact" mechanisms. The non-contact mechanism assumes that the bacteria oxidize only dissolved iron(II) ions to iron(III) ions. The latter can then attack metal sulfides and be reduced to iron(II) ions. The contact mechanism requires attachment of bacteria to the sulfide surface. The primary mechanism for attachment to pyrite is electrostatic in nature. In the case of Acidithiobacillus ferrooxidans, bacterial exopolymers contain iron(III) ions, each complexed by two uronic acid residues. The resulting positive charge allows attachment to the negatively charged pyrite. Thus, the first function of complexed iron(III) ions in the contact mechanism is mediation of cell attachment, while their second function is oxidative dissolution of the metal sulfide, similar to the role of free iron(III) ions in the non-contact mechanism. In both cases, the electrons extracted from the metal sulfide reduce molecular oxygen via a complex redox chain located below the outer membrane, the periplasmic space, and the cytoplasmic membrane of leaching bacteria. The dominance of either At. ferrooxidans or Leptospirillum ferrooxidans in mesophilic leaching habitats is highly likely to result from differences in their biochemical iron(II) oxidation pathways, especially the involvement of rusticyanin.

The sulfane sulfur of persulfides is the actual substrate of the sulfur-oxidizing enzymes from Acidithiobacillus and Acidiphilium spp. To identify the actual substrate of the glutathione-dependent sulfur dioxygenase (EC 1.13.11.18) elemental sulfur oxidation of the meso-acidophilic Acidithiobacillus thiooxidans strains DSM 504 and K6, Acidithiobacillus ferrooxidans strain R1 and Acidiphilium acidophilum DSM 700 was analysed. Extraordinarily high specific sulfur dioxygenase activities up to 460 nmol min "1 (mg protein) "1 were found in crude extracts. All cell-free systems oxidized elemental sulfur only via glutathione persulfide (GSSH), a non-enzymic reaction product from glutathione (GSH) and elemental sulfur. Thus, GSH plays a catalytic role in elemental sulfur activation, but is not consumed during enzymic sulfane sulfur oxidation. Sulfite is the first product of sulfur dioxygenase activity; it further reacted non-enzymically to sulfate, thiosulfate or glutathione S-sulfonate (GSSO { 3 ). Free sulfide was not oxidized by the sulfur dioxygenase. Persulfide as sulfur donor could not be replaced by other sulfane-sulfur-containing compounds (thiosulfate, polythionates, bisorganylpolysulfanes or monoarylthiosulfonates). The oxidation of H 2 S by the dioxygenase required GSSG, i.e. the disulfide of GSH, which reacted non-enzymically with sulfide to give GSSH prior to enzymic oxidation. On the basis of these results and previous findings a biochemical model for elemental sulfur and sulfide oxidation in Acidithiobacillus and Acidiphilium spp. is proposed.

Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated ␤-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.

Reliable compound-specific isotope enrichment factors are needed for a quantitative assessment of in situ biodegradation in contaminated groundwater. To obtain information on the variability on carbon and hydrogen enrichment factors (epsilonC, epsilonH) the isotope fractionation of methyl tertiary (tert-) butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) upon aerobic degradation was studied with different bacterial isolates. Methylibium sp. R8 showed a carbon and hydrogen isotope enrichment upon MTBE degradation of -2.4 +/- 0.1 and -42 +/- 4 per thousand, respectively, which is in the range of previous studies with pure cultures (Methylibium petroleiphilum PM1) as well as mixed consortia. In contrast, epsilonC of the beta/-proteobacterium L108 (-0.48 +/- 0.05 per thousand) and Rhodococcus ruber IFP 2001 (-0.28 +/- 0.06 per thousand) was much lower and hydrogen isotope fractionation was negligible (epsilonH < or = -0.2 per thousand). The varying isotope fractionation pattern indicates that MTBE is degraded by different mechanisms by the strains R8 and PM1 compared to L108 and IFP 2001. The carbon and hydrogen isotope fractionation of ETBE by L108 (epsilonC = -0.68 +/- 0.06 per thousand and epsilonH = -14 +/- 2 per thousand) and IFP 2001 (epsilonC = -0.8 +/- 0.1 per thousand and epsilonH = -11 +/- 4 per thousand) was very similar and seemed slightly higher than the fractionation observed upon MTBE degradation by the same strains. The low carbon and hydrogen enrichment factors observed during MTBE and ETBE degradation by L108 and IFP 2001 suggest a hydrolysis-like reaction type of the ether bond cleavage compared to oxidation of the alkyl group as suggested for the strains PM1 and R8. The variability of carbon and hydrogen enrichment factors should be taken into account when interpreting isotope pattern of fuel oxygenates with respect to biodegradation in contamination plumes.

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The oxidation of reduced inorganic sulfur compounds to sulfuric acid is of great importance for biohydrometallurgical technologies as well as the formation of acidic (below pH 3) and often heavy metal‐contaminated environments. The use of elemental sulfur as an electron donor is the predominant energy‐yielding process in acidic natural sulfur‐rich biotopes but also at mining sites containing sulfidic ores. Contrary to its significant role in the global sulfur cycle and its biotechnological importance, the microbial fundamentals of acidophilic sulfur oxidation are only incompletely understood. Besides giving an overview of sulfur‐oxidizing acidophiles, this review describes the so far known enzymatic reactions related to elemental sulfur oxidation in acidophilic bacteria and archaea. Although generally similar reactions are employed in both prokaryotic groups, the stoichiometry of the key enzymes is different. Bacteria oxidize elemental sulfur by a sulfur dioxygenase to sulfite whereas in archaea, a sulfur oxygenase reductase is used forming equal amounts of sulfide and sulfite. In both cases, the activation mechanism of elemental sulfur is not known but highly reactive linear sulfur forms are assumed to be the actual substrate. Inhibition as well as promotion of these biochemical steps is highly relevant in bioleaching operations. An efficient oxidation can prevent the formation of passivating sulfur layers. In other cases, a specific inhibition of sulfur biooxidation may be beneficial for reducing cooling and neutralization costs. In conclusion, the demand for a better knowledge of the biochemistry of sulfur‐oxidizing acidophiles is underlined.

Strains L10T, L108 and CIP I-2052 were originally obtained from methyl tert-butyl ether (MTBE)-contaminated groundwater and from a wastewater treatment plant, respectively. All share the ability to grow on tert-butanol, an intermediate of MTBE degradation. Cells are strictly aerobic, motile by a polar flagellum and exhibit strong pili formation. Poly β-hydroxybutyrate (PHB) granules are formed. The DNA G+C content is 69–70.5 mol% and the main ubiquinone is Q-8. The major cellular fatty acids are 16 : 1 cis-9 and 16 : 0 and the only hydroxy fatty acid is 10 : 0 3-OH. The major phospholipids are phosphatidylethanolamine (PE) 16 : 1/16 : 1 and phosphatidylglycerol 16 : 0/16 : 1. A significant amount of PE 17 : 0/16 : 1 is present. The 16S rRNA gene sequences of these strains are almost identical and form a separate line of descent in the Rubrivivax–Roseateles–Leptothrix–Ideonella–Aquabacterium branch of the Betaproteobacteria with 97 % similarity to 16S rRNA genes of the type strains of Rubrivivax gelatinosus, Leptothrix mobilis and Ideonella dechloratans. However, physiological properties, DNA–DNA relatedness values and the phospholipid and cellular fatty acid profiles distinguish the novel isolates from the three closely related genera. Therefore, it is concluded that strains L10T, L108 and CIP I-2052 represent a new genus and novel species for which the name Aquincola tertiaricarbonis gen. nov., sp. nov., is proposed. The type strain is strain L10T (=DSM 18512T=CIP 109243T).

The widely used artificial sweetener acesulfame K has long been considered recalcitrant in biological wastewater treatment. Due to its persistence and mobility in the aquatic environment, acesulfame has been used as marker substance for wastewater input in surface water and groundwater. However, recent studies indicated that the potential to remove this xenobiotic compound is emerging in wastewater treatment plants worldwide, leading to decreasing mass loads in receiving waters despite unchanged human consumption patterns. Here we show evidence that acesulfame can be mineralized in a catabolic process and used as sole carbon source by bacterial pure strains isolated from activated sludge and identified as Bosea sp. and Chelatococcus sp. The strains mineralize 1 g/L acesulfame K within 8–9 days. We discuss the potential degradation pathway and how this novel catabolic trait confirms the “principle of microbial infallibility.” Once the enzymes involved in acesulfame degradation and their genes are identified, it will be possible to survey diverse environments and trace back the evolutionary origin as well as the mechanisms of global distribution and establishment of such a new catabolic trait.