The indigenous microbial communities of two extremely acidic, metal-rich stratified pit lakes, located in the Iberian Pyrite Belt (Spain), were identified, and their roles in mediating transformations of carbon, iron, and sulfur were confirmed. A combined cultivation-based and culture-independent approach was used to elucidate microbial communities at different depths and to examine the physiologies of isolates, which included representatives of at least one novel genus and several species of acidophilic Bacteria. Phosphate availability correlated with redox transformations of iron, and this (rather than solar radiation) dictated where primary production was concentrated. Carbon fixed and released as organic compounds by acidophilic phototrophs acted as electron donors for acidophilic heterotrophic prokaryotes, many of which catalyzed the dissimilatory reduction in ferric iron; the ferrous iron generated was re-oxidized by chemolithotrophic acidophiles. Bacteria that catalyze redox transformations of sulfur were also identified, although these Bacteria appeared to be less abundant than the iron oxidizers/reducers. Primary production and microbial numbers were greatest, and biogeochemical transformation of carbon, iron, and sulfur, most intense, within a zone of c. 8-10 m depth, close to the chemocline, in both pit lakes. Archaea detected in sediments included two Thaumarchaeota clones, indicating that members of this recently described phylum can inhabit extremely acidic environments.
An acidophilic gammaproteobacterium, isolated from a pit lake at an abandoned metal mine in south-west Spain, was shown to be distantly related to all characterized prokaryotes, and to be the first representative of a novel genus and species. Isolate MCF85 is a Gram-negative, non-motile, rod-shaped mesophilic bacterium with a temperature growth optimum of 32-35 °C (range 8-45 °C). It was categorized as a moderate acidophile, growing optimally at pH 3.5-4.0 and between pH 2.5 and 4.5. Under optimum conditions its culture doubling time was around 75 min. Only organic electron donors were used by MCF85, and the isolate was confirmed to be an obligate heterotroph. It grew on a limited range of sugars (hexoses and disaccharides, though not pentoses) and some other small molecular weight organic compounds, and growth was partially or completely inhibited by small concentrations of some aliphatic acids. The acidophile grew in the presence of >100 mM ferrous iron or aluminium, but was more sensitive to some other metals, such as copper. It was also much more tolerant of arsenic (V) than arsenic (III). Isolate MCF85 catalysed the reductive dissolution of the ferric iron mineral schwertmannite when incubated under micro-aerobic or anaerobic conditions, causing the culture media pH to increase. There was no evidence, however, that the acidophile could grow by ferric iron respiration under strictly anoxic conditions. Isolate MCF85 is the designated type strain of the novel species Acidibacter ferrireducens (=DSM 27237(T) = NCCB 100460(T)).
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