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Appl Environ Microbiol. Jan 2002; 68(1): 405–407.
PMCID: PMC126569

Electrochemical Regeneration of Fe(III) To Support Growth on Anaerobic Iron Respiration

Abstract

Here we describe artificial help for the respiratory electron flow supporting anaerobic growth of Thiobacillus ferrooxidans through exogenous electrolysis. Flux between H2 and a anode through cells was accomplished with electrochemical regeneration of iron. The electrochemical help resulted in a 12-fold increase in yield compared with the yield observed in its absence.

In 1964, Kinsel et al. discovered that the chemolithotrophic bacterium Thiobacillus ferrooxidans grew on ferrous iron electrolytically generated by an electrode (7). In this system, the electrode provided electrons to the aerobic respiratory chain of the bacterium via iron (3, 5, 12, 13). However, as a practical matter, this electrochemical help is available to only a few iron oxidizers that derive energy for growth from aerobic iron respiration. On the other hand, anaerobic iron respiration has been found in many eubacteria and archaebacteria (4, 6, 8, 15, 16, 18). These organisms are able to derive energy for growth from iron reduction mediated by various electron donors (9, 10, 14). If microorganisms could respire on ferric iron electrochemically generated by an electrode, then the electrode would be accepting electrons from a respiratory chain via iron and would support growth of bacterial cultures by regenerating a terminal electron acceptor in the total electron flow comprising anaerobic iron respiration. It is conceivable that such a novel system could facilitate culture of many iron reducers.

Apparatus for electrolytic cultivation.

Electrolytic cultivation under potentiostatic conditions was carried out with an apparatus comprised of catholyte and anolyte baths separated by a cation-exchange membrane (type A-201; Asahi Chemical, Tokyo, Japan), into which carbon and platinum mesh electrodes (40 by 70 mm), respectively, were inserted (Fig. (Fig.1).1). The anolyte bath was filled with 100 ml of Fe2(SO4)3-containing medium, while the catholyte bath was filled with medium that was identical to the anolyte bath medium except that it lacked Fe. Each medium contained (per liter of distilled water) 133 mg of (NH4)2SO4, 41 mg of K2HPO4, 490 mg of MgSO4 · 7H2O, 9 mg of CaCl2 · 2H2O, 52 mg of KCl, 1 mg of ZnSO4 · 7H2O, 2 mg of CuSO4 · 5H2O, 1 mg of MnSO4 · H2O, 0.5 mg of NaMoO4 · 2H2O, 0.5 mg of CoCl2 · 6H2O, 1 mg of Na2SeO4 · 10H2O, and 1 mg of NiCl2 · 6H2O. Forty-eight grams of ferric sulfate hydrate (60 to 80% of the ferric sulfate content) was added to 1 liter of the medium, and the pH of the medium was adjusted to 2.0 with 6 N H2SO4. An Ag-AgCl reference electrode situated in the anolyte bath between the anode and the cation-exchange membrane was used to control the anodic potential (12). The entire apparatus was placed in an airtight box (width, 30 cm; depth, 30 cm; height, 25 cm) and incubated at 30°C with an atmosphere containing 80% H2 and 20% CO2 at a pressure of 250 kPa. After the anolyte bath was inoculated with T. ferrooxidans JCM 7811 obtained from the Japan Collection of Microorganisms, the electrodes were connected to a potentiostat, and the potential driving the electrolysis was maintained at 400 mV. Culture samples were anaerobically taken from a port in the airtight box, which was connected to the electrolytic apparatus. Cell numbers were determined directly by counting with a phase-contrast microscope at a magnification of ×400. The concentrations of Fe2+ were determined by the phenanthroline method as described previously (12). The total concentration of iron was also determined by the same method after reduction of iron by NH2OH · HCl. The Fe3+ content was calculated by subtracting the Fe2+ content from the total iron content.

FIG. 1.
Schematic diagram illustrating the concept behind electrochemical regeneration of an electron acceptor for anaerobic respiration.

Growth on electrolytic respiration.

T. ferrooxidans is generally considered to be an autotrophic bacterium that can grow aerobically on soluble ferrous iron or sulfur compounds (2). This bacterium nevertheless exhibited chemolithoautotrophic growth under strictly anaerobic conditions through reduction of Fe3+ using H2 as an electron donor. Growth of the bacterium proved to be strongly related to the reduction of Fe3+, eventually yielding a cell density of 8.4 × 108 cells/ml (Fig. (Fig.2a).2a). During a 74-h incubation period, the Fe3+ added was reduced completely to Fe2+, after which growth entered a stationary phase (Fig. (Fig.2b).2b). Growth resumed, however, upon application of potential-controlled electrolysis, which regenerated Fe3+ by oxidizing Fe2+ at the anode. Throughout cultivation, the concentration of Fe3+ was kept between 30 and 50 mM by passage of 10.0 to 15.5 mA of current, and the final cell density after 142 h of electrolysis was 1010 cells/ml (Fig. 2a and b). Thus, electrolysis resulted in a 12-fold increase in cell density compared with the cell density achieved in the absence of electrolysis. On the other hand, no growth occurred in the absence of iron or H2, whether current was applied or not. In addition, no reduction occurred in the presence of iron and H2 under electrolysis conditions without cells (data not shown).

FIG. 2.
Chemolithoautotrophic growth of T. ferrooxidans strain JCM 7811 mediated by electrochemical regeneration of an electron acceptor for anaerobic iron respiration. (a) Cell density with and without electrolysis. Electrolysis was applied to cultures at different ...

To confirm that the observed increase in bacterial growth was a consequence of electrolytic respiration, in another batch of cells electrolysis was initiated 35 h after inoculation, when the cells were still growing logarithmically (Fig. 2a and d). With the assistance of electrolytic respiration, the cells grew for 130 h to a density of 7.1 × 109 cells/ml. In the absence of electrolysis, by contrast, growth stopped after 71 h, when all of the Fe3+ had been reduced to Fe2+, and the cell density was only 7.5 × 108 cells/ml (Fig. 2a and c). Apparently, the anode was able to effectively serve as a terminal electron acceptor supporting anaerobic bacterial respiration, with iron mediating the transfer of electrons from the bacterial respiratory chain to the electrode.

The potential for oxidation of Fe2+ at the anode was kept constant at 400 mV, which was sufficient to sustain oxidation of Fe3+ in the medium (12). At the same constant potential, H2 evolution was observed at the surface of the cathode during electrolysis. However, it was not possible to entirely mediate the electron transfer via the evolved gas. The small amount of evolved H2 was diluted in the airtight box containing the culture apparatus. Instead, exogenous H2 had to be supplied. Consequently, it is not clear how much the evolved H2 contributed to the bacterial growth.

The schematic diagram in Fig. Fig.11 summarizes our concept of bacterial cultivation driven by electrochemical regeneration of an electron acceptor for respiration. The electron flux begins between H2 and the bacterial cell. The bacterium oxidizes H2 anaerobically and then transfers the accepted electrons to Fe3+ in its respiratory chain. The Fe2+ generated is oxidized by the anode, completing the electron flux from H2 to the electrode through the bacterium. The oxidation of Fe2+ regenerates Fe3+ capable of accepting additional electrons. With respect to the total electron flow, the anode can support anaerobic respiration of the bacterium using iron as an electron mediator.

For the past 35 years, bacterial cultivation using electrodes has been discussed in terms of electrochemical regeneration of Fe2+ as the electron donor for aerobic growth of T. ferrooxidans (3, 5, 7, 12, 13). On the other hand, the concept of regenerating an electron acceptor for anaerobic respiration is novel and may be useful for culturing numerous as-yet-unknown organisms, since conventional isolation techniques are suitable for culturing only a small percentage of the species in an environmental sample (1, 19). Indeed, because the electrode would be able to oxidize a number of soluble iron complexes at neutral pH, anaerobic respiration and growth of a wide variety of both Bacteria and Archaea could be supported (9, 10, 14). Although iron-reducing bacteria have recently been isolated from various sediments, the deep subsurface, groundwater, and hydrothermal vents (4, 6, 8, 15, 16), electrolytic cultivation should provide another approach for isolating additional iron reducers that respire anaerobically on metals (9, 10, 14) or nitrogen compounds (17). Thus, electrochemically driven growth has the potential to be a highly productive approach for accelerating bacterial degradation of organic materials, including toxic chemicals, some of which are capable of serving as electron donors that support bacterial respiration (11).

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