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

Here we describe artificial help for the respiratory electronflow supporting anaerobic growth of Thiobacillus ferrooxidansthrough exogenous electrolysis. Flux between H₂ and a anodethrough cells was accomplished with electrochemical regenerationof iron. The electrochemical help resulted in a 12-fold increasein yield compared with the yield observed in its absence.

INTRODUCTION

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Introduction

In 1964, Kinsel et al. discovered that the chemolithotrophicbacterium Thiobacillus ferrooxidans grew on ferrous iron electrolyticallygenerated by an electrode (7). In this system, the electrodeprovided electrons to the aerobic respiratory chain of the bacteriumvia iron (3, 5, 12, 13). However, as a practical matter, thiselectrochemical help is available to only a few iron oxidizersthat derive energy for growth from aerobic iron respiration.On the other hand, anaerobic iron respiration has been foundin many eubacteria and archaebacteria (4, 6, 8, 15, 16, 18).These organisms are able to derive energy for growth from ironreduction mediated by various electron donors (9, 10, 14). Ifmicroorganisms could respire on ferric iron electrochemicallygenerated by an electrode, then the electrode would be acceptingelectrons from a respiratory chain via iron and would supportgrowth of bacterial cultures by regenerating a terminal electronacceptor in the total electron flow comprising anaerobic ironrespiration. It is conceivable that such a novel system couldfacilitate culture of many iron reducers.

Apparatus for electrolytic cultivation.

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Apparatus for electrolytic...

Electrolytic cultivation under potentiostatic conditions wascarried out with an apparatus comprised of catholyte and anolytebaths separated by a cation-exchange membrane (type A-201; AsahiChemical, Tokyo, Japan), into which carbon and platinum meshelectrodes (40 by 70 mm), respectively, were inserted (Fig.1). The anolyte bath was filled with 100 ml of Fe₂(SO₄)₃-containingmedium, while the catholyte bath was filled with medium thatwas identical to the anolyte bath medium except that it lackedFe. Each medium contained (per liter of distilled water) 133mg 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 theferric 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 bathbetween the anode and the cation-exchange membrane was usedto control the anodic potential (12). The entire apparatus wasplaced in an airtight box (width, 30 cm; depth, 30 cm; height,25 cm) and incubated at 30°C with an atmosphere containing80% H₂ and 20% CO₂ at a pressure of 250 kPa. After the anolytebath was inoculated with T. ferrooxidans JCM 7811 obtained fromthe Japan Collection of Microorganisms, the electrodes wereconnected to a potentiostat, and the potential driving the electrolysiswas maintained at 400 mV. Culture samples were anaerobicallytaken from a port in the airtight box, which was connected tothe electrolytic apparatus. Cell numbers were determined directlyby counting with a phase-contrast microscope at a magnificationof x400. The concentrations of Fe²⁺ were determined by the phenanthrolinemethod as described previously (12). The total concentrationof iron was also determined by the same method after reductionof iron by NH₂OH · HCl. The Fe³⁺ content was calculatedby subtracting the Fe²⁺ content from the total iron content.

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

Growth on electrolytic respiration.

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Growth on electrolytic...

T. ferrooxidans is generally considered to be an autotrophicbacterium that can grow aerobically on soluble ferrous ironor sulfur compounds (2). This bacterium nevertheless exhibitedchemolithoautotrophic growth under strictly anaerobic conditionsthrough reduction of Fe³⁺ using H₂ as an electron donor. Growthof the bacterium proved to be strongly related to the reductionof Fe³⁺, eventually yielding a cell density of 8.4 x 10⁸ cells/ml(Fig. 2a). During a 74-h incubation period, the Fe³⁺ added wasreduced completely to Fe²⁺, after which growth entered a stationaryphase (Fig. 2b). Growth resumed, however, upon application ofpotential-controlled electrolysis, which regenerated Fe³⁺ byoxidizing Fe²⁺ at the anode. Throughout cultivation, the concentrationof Fe³⁺ was kept between 30 and 50 mM by passage of 10.0 to15.5 mA of current, and the final cell density after 142 h ofelectrolysis was 10¹⁰ cells/ml (Fig. 2a and b). Thus, electrolysisresulted in a 12-fold increase in cell density compared withthe cell density achieved in the absence of electrolysis. Onthe other hand, no growth occurred in the absence of iron orH₂, whether current was applied or not. In addition, no reductionoccurred in the presence of iron and H₂ under electrolysis conditionswithout cells (data not shown).

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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 times (35 to 178 h [ ${blacktriangleup}$ ] and 97 to 239 h [ ${blacksquare}$ ]). The arrows indicate when electrolysis was started. No electrolysis was applied to a control culture (•). (b, c, and d) Iron concentrations and electric currents in the cultures described above. (b) Culture electrolysed at 97 to 239 h; (c) control culture; (d) culture electrolysed at 35 to 178 h. Symbols: ${circ}$ and ${triangleup}$ , time-dependent changes in Fe³⁺ and total iron concentrations, respectively; ${square}$ , current driving the electrolysis. All values are averages based on duplicate determinations with independent cultures.

To confirm that the observed increase in bacterial growth wasa consequence of electrolytic respiration, in another batchof 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 cellsgrew for 130 h to a density of 7.1 x 10⁹ cells/ml. In the absenceof electrolysis, by contrast, growth stopped after 71 h, whenall of the Fe³⁺ had been reduced to Fe²⁺, and the cell densitywas only 7.5 x 10⁸ cells/ml (Fig. 2a and c). Apparently, theanode was able to effectively serve as a terminal electron acceptorsupporting anaerobic bacterial respiration, with iron mediatingthe transfer of electrons from the bacterial respiratory chainto the electrode.

The potential for oxidation of Fe²⁺ at the anode was kept constantat 400 mV, which was sufficient to sustain oxidation of Fe³⁺in the medium (12). At the same constant potential, H₂ evolutionwas observed at the surface of the cathode during electrolysis.However, it was not possible to entirely mediate the electrontransfer via the evolved gas. The small amount of evolved H₂was diluted in the airtight box containing the culture apparatus.Instead, exogenous H₂ had to be supplied. Consequently, it isnot clear how much the evolved H₂ contributed to the bacterialgrowth.

The schematic diagram in Fig. 1 summarizes our concept of bacterialcultivation driven by electrochemical regeneration of an electronacceptor for respiration. The electron flux begins between H₂and the bacterial cell. The bacterium oxidizes H₂ anaerobicallyand then transfers the accepted electrons to Fe³⁺ in its respiratorychain. The Fe²⁺ generated is oxidized by the anode, completingthe electron flux from H₂ to the electrode through the bacterium.The oxidation of Fe²⁺ regenerates Fe³⁺ capable of acceptingadditional electrons. With respect to the total electron flow,the anode can support anaerobic respiration of the bacteriumusing iron as an electron mediator.

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

FOOTNOTES

* Corresponding author. Mailing address: Department of Bio-Science, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko-city, Chiba 270-1194, Japan. Phone: 81-471-82-1181. Fax: 81-471-83-3347. E-mail: ohmura{at}criepi.denken.or.jp.

REFERENCES

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