The periplasmic 9.6-kilodalton c-type cytochrome of Geobacter sulfurreducens is not an electron shuttle to Fe(III).

Geobacter sulfurreducens contains a 9.6-kDa c-type cytochrome that was previously proposed to serve as an extracellular electron shuttle to insoluble Fe(III) oxides. However, when the cytochrome was added to washed-cell suspensions of G. sulfurreducens it did not enhance Fe(III) oxide reduction, whereas similar concentrations of the known electron shuttle, anthraquinone-2,6-disulfonate, greatly stimulated Fe(III) oxide reduction. Furthermore, analysis of the extracellular c-type cytochromes in cultures of G. sulfurreducens demonstrated that the dominant c-type cytochrome was not the 9.6-kDa cytochrome, but rather a 41-kDa cytochrome. These results and other considerations suggest that the 9.6-kDa cytochrome is not an important extracellular electron shuttle to Fe(III) oxides.

Role of humic-bound iron as an electron transfer agent in dissimilatory Fe(III) reduction.

The dissimilatory Fe(III) reducer Geobacter metallireducens reduced Fe(III) bound in humic substances, but the concentrations of Fe(III) in a wide range of highly purified humic substances were too low to account for a significant portion of the electron-accepting capacities of the humic substances. Furthermore, once reduced, the iron in humic substances could not transfer electrons to Fe(III) oxide. These results suggest that other electron-accepting moieties in humic substances, such as quinones, are the important electron-accepting and shuttling agents under Fe(III)-reducing conditions.

Humics as an electron donor for anaerobic respiration.

The possibility that microorganisms might use reduced humic substances (humics) as an electron donor for the reduction of electron acceptors with a more positive redox potential was investigated. All of the Fe(III)- and humics-reducing microorganisms evaluated were capable of oxidizing reduced humics and/or the reduced humics analogue anthrahydroquinone-2,6,-disulphonate (AHODS), with nitrate and/or fumarate as the electron acceptor. These included Geobacter metallireducens, Geobacter sulphurreducens, Geothrix fermentans, Shewanella alga, Wolinella succinogenes and 'S. barnesii'. Several of the humics-oxidizing microorganisms grew in medium with AHQDS as the sole electron donor and fumarate as the electron acceptor. Even though it does not reduce Fe(III) or humics, Paracoccus denitrificans could use AHQDS and reduced humics as electron donors for denitrification. However, another denitrifier, Pseudomonas denitrificans, could not. AHODS could also serve as an electron donor for selenate and arsenate reduction by W. succinogenes. Electron spin resonance studies demonstrated that humics oxidation was associated with the oxidation of hydroquinone moieties in the humics. Studies with G. metallireducens and W. succinogenes demonstrated that the anthraquinone-2,6-disulphonate (AQDS)/AHQDS redox couple mediated an interspecies electron transfer between the two organisms. These results suggest that, as microbially reduced humics enter less reduced zones of soils and sediments, the reduced humics may serve as electron donors for microbial reduction of several environmentally significant electron acceptors.

Microbiological evidence for Fe(III) reduction on early Earth.

It is generally considered that sulphur reduction was one of the earliest forms of microbial respiration, because the known microorganisms that are most closely related to the last common ancestor of modern life are primarily anaerobic, sulphur-reducing hyperthermophiles. However, geochemical evidence indicates that Fe(III) is more likely than sulphur to have been the first external electron acceptor of global significance in microbial metabolism. Here we show that Archaea and Bacteria that are most closely related to the last common ancestor can reduce Fe(III) to Fe(II) and conserve energy to support growth from this respiration. Surprisingly, even Thermotoga maritima, previously considered to have only a fermentative metabolism, could grow as a respiratory organism when Fe(III) was provided as an electron acceptor. These results provide microbiological evidence that Fe(III) reduction could have been an important process on early Earth and suggest that microorganisms might contribute to Fe(III) reduction in modern hot biospheres. Furthermore, our discovery that hyperthermophiles that had previously been thought to require sulphur for cultivation can instead be grown without the production of toxic and corrosive sulphide, should aid biochemical investigations of these poorly understood organisms.

Recovery of humic-reducing bacteria from a diversity of environments.

To evaluate which microorganisms might be responsible for microbial reduction of humic substances in sedimentary environments, humic-reducing bacteria were isolated from a variety of sediment types. These included lake sediments, pristine and contaminated wetland sediments, and marine sediments. In each of the sediment types, all of the humic reducers recovered with acetate as the electron donor and the humic substance analog, 2,6-anthraquinone disulfonate (AQDS), as the electron acceptor were members of the family Geobacteraceae. This was true whether the AQDS-reducing bacteria were enriched prior to isolation on solid media or were recovered from the highest positive dilutions of sediments in liquid media. All of the isolates tested not only conserved energy to support growth from acetate oxidation coupled to AQDS reduction but also could oxidize acetate with highly purified soil humic acids as the sole electron acceptor. All of the isolates tested were also able to grow with Fe(III) serving as the sole electron acceptor. This is consistent with previous studies that have suggested that the capacity for Fe(III) reduction is a common feature of all members of the Geobacteraceae. These studies demonstrate that the potential for microbial humic substance reduction can be found in a wide variety of sediment types and suggest that Geobacteraceae species might be important humic-reducing organisms in sediments.