Reduction of Soluble Iron and Reductive Dissolution of Ferric Iron-Containing Minerals by Moderately Thermophilic Iron-Oxidizing Bacteria

Five moderately thermophilic iron-oxidizing bacteria, including representative strains of the three classified species (Sulfobacillus thermosulfidooxidans, Sulfobacillus acidophilus, and Acidimicrobium ferrooxidans), were shown to be capable of reducing ferric iron to ferrous iron when they were grown under oxygen limitation conditions. Iron reduction was most readily observed when the isolates were grown as mixotrophs or heterotrophs with glycerol as an electron donor; in addition, some strains were able to couple the oxidation of tetrathionate to the reduction of ferric iron. Cycling of iron between the ferrous and ferric states was observed during batch culture growth in unshaken flasks incubated under aerobic conditions, although the patterns of oxidoreduction of iron varied in different species of iron-oxidizing moderate thermophiles and in strains of a single species (S. acidophilus). All three bacterial species were able to grow anaerobically with ferric iron as a sole electron acceptor; the growth yields correlated with the amount of ferric iron reduced when the isolates were grown in the absence of oxygen. One of the moderate thermophiles (identified as a strain of S. acidophilus) was able to bring about the reductive dissolution of three ferric iron-containing minerals (ferric hydroxide, jarosite, and goethite) when it was grown under restricted aeration conditions with glycerol as a carbon and energy source. The significance of iron reduction by moderately thermophilic iron oxidizers in both environmental and applied contexts is discussed.Moderately thermophilic acidophilic bacteria that catalyze the dissimilatory oxidation of ferrous iron are distinct both phylogenetically and in aspects of their physiology. They differ from the known acidophilic mesophilic iron oxidizers (gram-negative, nonsporulating chemolithotrophic bacteria) and the extremely thermophilic iron oxidizers (certain archaea) in several fundamental ways, including cellular morphology (they are gram-positive rods that often form endospores) and growth temperature optima, which are typically 45 to 55°C (15). In addition, the moderately thermophilic iron-oxidizing acidophiles characteristically have a highly versatile metabolism (18) and may grow as autotrophs (e.g., in media containing ferrous iron or reduced sulfur), heterotrophs (e.g., on yeast extract), mixotrophs (e.g., in media containing both ferrous iron and glucose, in which both CO₂ and glucose are used as carbon sources), or chemolithoheterotrophs (e.g., in ferrous iron-yeast extract medium, in which iron acts as the energy source and yeast extract is the carbon source). Isolates have been obtained from a range of thermal acidic environments, such as geothermal areas, self-heating mine waste spoils, and commercial mineral-processing operations (2a, 5, 14). There are currently two recognized genera of these bacteria. All but one Sulfobacillus species are iron- and sulfur-oxidizing, gram-positive, sporulating rods. Two such species have been described, Sulfobacillus thermosulfidooxidans and Sulfobacillus acidophilus, which may be distinguished by their different chromosomal DNA base compositions and by their abilities to grow autotrophically on reduced sulfur (16). The genus Acidimicrobium currently contains a single species, Acidimicrobium ferrooxidans. This organism differs from Sulfobacillus spp. by its greater capacity to fix CO₂, by its lower tolerance of ferric iron, by its apparent lack of spore formation (although it is also gram positive), and by its chromosomal DNA base composition (4). Analysis of 16S rRNA sequences has also differentiated this moderate thermophile from Sulfobacillus spp. (9).The small amount of energy associated with the oxidation of ferrous iron (−30 kJ mol⁻¹ at pH 2) can serve as the exclusive source of energy for moderately thermophilic iron-oxidizing acidophiles when they are growing autotrophically with oxygen as the terminal electron acceptor. Under limited aeration conditions, ferric iron, which is often abundant and present in a soluble form in extremely acidic environments, is a thermodynamically attractive alternative electron sink (electrode potential [E′], +780 mV). Ferric iron reduction by mesophilic chemolithotrophic and heterotrophic acidophiles has been observed previously (5, 7, 17). Some moderately thermophilic, acidophilic, heterotrophic bacteria (Alicyclobacillus-like isolates) (5a) and the extremely thermophilic archaeon Sulfolobus acidocaldarius (3) can also reduce iron. While many neutrophilic microorganisms are also able to reduce ferric iron, the ability to conserve energy to support growth by coupling organic matter oxidation exclusively to ferric iron reduction appears to be more restricted among neutrophilic bacteria (11).In this paper, we describe the dissimilatory reduction of ferric iron by representative isolates of different species of iron-oxidizing moderate thermophiles with both an organic electron donor (glycerol) and an inorganic electron donor (tetrathionate), and we also describe the reductive dissolution of ferric iron-containing minerals by a Sulfobacillus isolate.