Isolation and characterization of novel iron-oxidizing bacteria that grow at circumneutral pH

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Abstract
A gel-stabilized gradient method that employed opposing gradients of Fe2+ and O2 was used to isolate and characterize two new Fe-oxidizing bacteria from a neutral pH, Fe(2+)-containing groundwater in Michigan. Two separate enrichment cultures were obtained, and in each the cells grew in a distinct, rust-colored band in the gel at the oxic-anoxic interface. The cells were tightly associated with the ferric hydroxides. Repeated serial dilutions of both enrichments resulted in the isolation of two axenic strains, ES-1 and ES-2. The cultures were judged pure based on (i) growth from single colonies in tubes at dilutions of 10(-7) (ES-2) (ES-2) and 10(-8) (ES-1); (ii) uniform cell morphologies, i.e., ES-1 was a motile long thin, bent, or S-shaped rod and ES-2 was a shorter curved rod; and (iii) no growth on a heterotrophic medium. Strain ES-1 grew to a density of 10(8) cells/ml on FeS with a doubling time of 8 h. Strain ES-2 grew to a density of 5 x 10(7) cells/ml with a doubling time of 12.5 h. Both strains also grew on FeCO3. Neither strain grew without Fe2+, nor did they grow with glucose, pyruvate, acetate, Mn, or H2S as an electron donor. Studies with an oxygen microelectrode revealed that both strains grew at the oxic-anoxic interface of the gradients and tracked the O2 minima when subjected to higher O2 concentrations, suggesting they are microaerobes. Phylogenetically the two strains formed a novel lineage within the gamma Proteobacteria. They were very closely related to each other and were equally closely related to PVB OTU 1, a phylotype obtained from an iron-rich hydrothermal vent system at the Loihi Seamount in the Pacific Ocean, and SPB OTU 1, a phylotype obtained from permafrost soil in Siberia. Their closest cultivated relative was Stenotrophomonas maltophilia. In total, this evidence suggests ES-1 and ES-2 are members of a previously untapped group of putatively lithotrophic, unicellular iron-oxidizing bacteria.