INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Several microorganisms (e.g., Escherichia and Actinobacillus) grow with H₂ as the electron donor and reduce fumarate into succinate in an anaerobic respiration process (13, 16, 24). These bacteria obtain free energy and reducing power from the electron driving force generated by the E_o' difference between the coupled oxidoreduction half-reactions of [2H⁺/H₂] and [fumarate/succinate].

Methanogens are strict anaerobic bacteria that can couple H₂ or HCOOH oxidation to CO₂ reduction into methane (26). Methanogenesis produces less free energy than other anaerobic respiration processes (e.g., fumarate, nitrate, or sulfate reduction) because the E_o' difference between the oxidation-reduction half-reactions of [2H⁺/H₂] and [CO₂/CH₄] is 0.17 V, from which about 34 kJ of G°'/H₂ is produced (23).

Hydrogen oxidation by microbial hydrogenases (25) can be coupled to reduction of different biological electron carriers, including NAD⁺ (22), cytochromes, and quinones (8, 14, 29), or to certain artificial redox dyes, such as methyl-viologen and neutral red (NR) (2, 10). The influence of redox dyes, with or without electrochemical reduction systems, on altering metabolite patterns and H₂ production has been examined in several microbial processes, including the glutamate (7), butanol (4, 11), and butyrate (20) fermentations. These investigations did not determine whether electrically reduced dyes could serve as the sole electron donor for driving growth and cellular metabolism.

The energetics of living systems is driven by electron transfer processes (18). Electrons are transferred from a substrate that becomes oxidized to a final acceptor that becomes reduced. This observation implies that it may be possible to control or alter metabolism by plugging biochemical processes into an external electrochemical system. Previously, an electrochemical system (17) was used to generate reduced iron for Thiobacillus ferrooxidans growth on electrical reducing power. We have previously shown (19) that electrically reduced NR could serve as an extra electron donor and enhance growth and succinate production from glucose by Actinobacillus succinogenes. We also reported (19) that this organism contained membrane-bound hydrogenase and fumarate reductase and that electrically reduced NR could chemically reduce NAD⁺ and replace menaquinone as the electron donor for fumarate reductase.

The purpose of the present report is fourfold: first, to show the utility of this novel electrochemical reactor for replacing H₂ with electrically reduced NR as the sole electron donor for microbial growth and metabolite production; second, to show that an A. succinogenes mutant that lacks key oxidoreductases and that cannot grow on fumarate alone can grow on either H₂ plus fumarate or on electrically reduced NR plus fumarate; third, to show that in mixed methanogenic cultures electrically reduced NR can replace H₂ in CO₂ reduction to methane; and finally, to discuss the potential industrial utility of this novel electrochemical reactor system for enhancing microbial fermentation and transformation processes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

A. succinogenes growth and metabolic analysis. A. succinogenes strains 130Z and FZ-6 were obtained from our culture collection at Michigan Biotechnology Institute International (Lansing, Mich.). Bacteria were grown in 158-ml serum vials with butyl rubber stoppers containing 40 ml of medium with fumarate under a N₂ gas phase (100%; 20 lb/in²). Traces of oxygen were removed by passing the gas over heated (370°C) copper filings. Growth medium A contained the following (per liter of double-distilled water): yeast extract, 5.0 g; NaHCO₃, 10.0 g; NaH₂PO₄ · H₂O, 8.5 g; and Na₂HPO₄, 15.5 g. The pH was adjusted to 7.0 after autoclaving. Fumarate (final concentration, 50 mM) was added aseptically to the medium after autoclaving. Na₂S (final concentration, 0.02%) was added to establish strict anoxic conditions. Media were inoculated with 3.0% (by volume) samples of cultures grown in the same medium and incubated at 37°C for 24 (strain 130Z) or 48 (strain FZ-6) h. The culture samples were aseptically and anaerobically removed with 3-ml syringes. Glucose, fumarate, succinate, acetate, ethanol, and formate concentrations in the cultures were determined by high-performance liquid chromatography (5). The components were analyzed chromatographically by elution with 0.006 M H₂SO₄ from a cation-exchange resin in the hydrogen form.

Enzymatic analysis. All procedures for bacterial cultivation, cell extract preparation, and enzyme activity measurement were done under a strict anaerobic N₂ atmosphere as described previously (24). Dithiothreitol was used as a chemical reductant. A. succinogenes 130Z and FZ-6 were anaerobically cultivated on medium A (5) containing 2% glucose in a 4-liter carboy under a N₂-CO₂ (80:20) atmosphere. Cells grown for 16 h were harvested by centrifugation at 5,000 × g at 4°C for 30 min and washed three times with 100 ml of 50 mM phosphate buffer (pH 7.2). The washed cells were resuspended in the same buffer and disrupted with a French press at 2,000 lb/in² and 4°C. The cell debris was removed by centrifuging the cells twice at 40,000 × g and 4°C for 30 min. The clear, light-brown supernatant was used as an extract for enzyme assays. D-Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; NAD⁺ dependent) activity was measured as previously described (5). Fumarate reductase (EC 1.3), formate dehydrogenase (EC 1.2), hydrogenase (EC 2.12.2.1), and malate dehydrogenase (EC 1.1.1.37) (24); pyruvate dehydrogenase (EC 1.2.2.2) (21); and pyruvate formate-lyase (EC 2.3.1.54) (12) activities were measured as described previously. The oxidation and reduction reactions of pyridine nucleotide were measured spectrophotometrically at 340 nm (₃₃₄ = 6.23 mM¹ cm¹). Methyl viologen and benzyl viologen reductions were spectrophotometrically measured at 578 nm. The millimolar extinction coefficients (₅₇₈) of methyl viologen and benzyl viologen were 9.78 and 8.65 mM¹ cm¹, respectively.

Electrobiochemical reactor systems. A. succinogenes 130Z and FZ-6 were grown in an electrochemical bioreactor system containing 40 ml of medium A (24) with 50 mM fumarate, 100 µM NR, and a N₂ headspace (99.5%; 1 atm). All procedures for medium preparation, inoculation, and cultivation were the same as those for vial cultures except that Na₂S was not used because the medium was electrically reduced. The current and potential between the anode and cathode were 0.3 to 0.35 mA and 1.5 V. Anaerobic culture samples were aseptically removed with 3-ml syringes. Cells grew suspended in the liquid medium and self-immobilized on the cathode. Cell growth in the liquid was determined spectrophotometrically by measuring the optical density at 660 nm. The growth of cells adsorbed on the cathode was calculated by measuring bacterial protein concentration. The protein concentration was converted to optical density by using a predetermined calibration curve (bacterial density = protein concentration [in milligrams per milliliter] × 1.7556). Bacterial cells that were adsorbed on the cathode were washed three times with 100 ml of phosphate buffer (50 mM; pH 7.0) for 30 min to remove medium compounds. A bacterial lysate was obtained from the electrodes after alkaline treatment at 100°C for 10 min in 1 N NaOH. The protein concentration of the bacterial lysate was determined by using a calibration curve (protein concentration [in milligrams per milliliter] = A₅₉₅ × 1.3327) with the Bradford reagent (Bio-Rad, Hercules, Calif.) after cell debris was removed from the lysate by centrifugation at 10,000 × g and 4°C for 30 min.

2

1

2

2

View larger version (79K): [in this window] [in a new window]   FIG. 1.   Schematic of the electrochemical bioreactor system used (19). Cells were placed in the cathode compartment. In electricity-dependent succinogenesis, the gassing system was not used and the pH was held constant. For electricity-dependent methanogenesis, a sulfide reducing agent was added in the anode compartment. The electrical potential resulted in the following half-reactions in the anode and cathode compartments: succinogenesis, H2O  2H+ + 2e + 1/2O2 (anode reaction), 2NR+ + 2H+ + 2e  2NRH (cathode reaction), 2NRH + 2X+  2NR+ + 2XH (electron mediation), 2XH + fumarate  2X+ + succinate (succinogenesis); and methanogenesis, S2  SO42 + 8e + 8H+ (anode reaction), 8NR+ + 8e + 8H+  8NRH (cathode reaction), 8NRH + 8X+  8NR+ + 8XH (electron mediation), 8XH + CO2  8X+ + CH4 + 2H2O (methanogenesis). X, biological electron carrier(s).

Methanogenic granules growth and metabolic analysis. Methanogenic granules containing mixed cultures of fatty acid-degrading syntrophiles and methanogens were obtained from a bench scale anaerobic sludge reactor fed on a mixture of 50 mM [each] acetate, butyrate, and propionate at Michigan Biotechnology Institute International (27, 28). Methanogenic granules were cultivated in PBBM prepared without organic compounds (9). The medium was prepared without phosphate, brought to pH 7.2 with NaOH, boiled, sparged with N₂-CO₂ (80:20%) or H₂-CO₂ (80:20%), dispensed into 158-ml Wheaton serum vials, sealed with butyl rubber stoppers, and autoclaved. Phosphate, sulfide (0.01%), N₂-CO₂ (80:20%) or H₂-CO₂ (80:20%), and vitamin solution were added after autoclaving. The medium volume was 40 ml, and the initial headspace gas pressure in the serum vials was adjusted to 30 lb/in². The media were inoculated with 3.0% (by volume; protein concentration, 1.995 mg/ml) methanogenic granules and incubated at 37°C. All procedures for medium preparation, inoculation, and cultivation were the same as those used for vial cultures except that Na₂S was not added because the medium was electrically reduced. Na₂S (2%) was added to the anode compartment as a reducing agent to remove the O₂ generated. NR (100 µM) was added to the cathode compartment as an electron mediator. The current and potential between the anode and cathode were 0.4 mA and 2.0 V. CO₂ and CH₄ were analyzed with a gas chromatograph equipped with a carbosphere column and flame-ionized detector. The injector and column temperatures were 50 and 150°C, respectively, and the carrier (N₂) flow rate was 45 ml/min. Gas samples were removed with a pressure lock syringe. CO₂ consumption and CH₄ production are shown as the percentage of total gas composition in the headspace.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

We previously reported that A. succinogenes, sp. nov., wild-type strain 130Z grew and produced succinate by fermentation of H₂ and fumarate (24). Mutant FZ-6 is a succinate-overproducing mutant selected by fluoroacetate resistance that produces low levels of acetate and formate (6). Figure 2 compares the growth and succinate production of wild-type 130Z to those of mutant FZ-6 on fumarate plus or minus H₂. The wild-type strain 130Z grew equally well on fumarate plus or minus H₂, whereas strain FZ-6 grew on fumarate only in the presence of H₂ as an electron donor.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Growth and succinate production by the A. succinogenes type strain 130Z on fumarate-N2 (A) and on fumarate-H2 (B) and by mutant strain FZ-6 on fumarate-N2 (C) and on fumarate-H2 (D). , cell growth; , fumarate; , succinate; OD660, optical density at 660 nm.

To produce free energy with fumarate as the sole anaerobic energy source, fumarate reduction to succinate must be coupled to the oxidation of a reductant generated by fumarate metabolism to acetate plus formate. We compared oxidoreductase levels in A. succinogenes 130Z and FZ-6 to determine the metabolic basis for strain FZ-6's inability to grown on fumarate without H₂ (Table 1). While the strains had equivalent levels of glyceraldehyde-3P dehydrogenase, hydrogenase, fumarate reductase, and malate dehydrogenase activities, strain FZ-6 lacked pyruvate formate lyase and formate dehydrogenase activities. The absence of pyruvate-metabolizing enzymes also explains the inability of the mutant strain to grow on fumarate or pyruvate alone (data not shown).

Experiments were initiated to test if electrically reduced NR could replace H₂ for strain FZ-6 growth on fumarate. These studies were performed in an ECBS (Fig. 1) in the presence of NR, which served as an electron mediator, or electronophore. Figure 3 demonstrates that strain FZ-6 can grow by reducing fumarate to succinate with electrically reduced NR as the electron donor. The organism did not grow on fumarate alone without cathodic reduction of NR. NR serves as an electron mediator to transfer electrons from the cathode to the cellular electron transport chain. Table 2 summarizes the electrical dependence of growth and succinate production by strain FZ-6 on fumarate. At the end of growth, succinate was the only detectable fermentation product, and 92% of the starting carbon in fumarate was recovered in the succinate and cells produced. Growth and succinate production by strain FZ-6 were higher when electrically reduced NR served as the energy source (Table 2) in place of H₂ (Fig. 2).

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Electricity-dependent growth and succinate production of the A. succinogenes mutant strain FZ-6 on fumarate. The cathode compartment contained medium A, fumarate, and 100 µM NR. The potential and current between anode and cathode were 1.5 V and 0.4 to 1.5 mA. Although cell growth also takes place on the cathode, it was only measured as cells were released into the liquid medium, thus underestimating the total growth. OD600, optical density at 600 nm.

Experiments were initiated to test whether electricity could replace H₂ for microbial CO₂ reduction to methane. These studies were performed in the reactor system described above (Fig. 1). The reactor contained methanogenic granules comprised of mixed cultures of syntrophic fatty acid-degrading bacteria and methane-producing bacteria (27, 28). Figure 4 shows that CO₂ reduction to methane was dependent on either H₂ or electricity. Notably, electrically reduced NR served as a better electron donor for CO₂ reduction than H₂. Determination of total cell growth after 120 h indicated a twofold increase in cells grown on electrically reduced NR versus a 1.4-fold increase on H₂-CO₂.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Influence of electron donors on CO2 reduction to methane by methanogenic granules. (A) N2-CO2 control; (B) H2-CO2; (C) electrical reduction. For electrical reduction, the cathode compartment contained PBBM and 100 µM NR. The potential and current between the anode and cathode were 1.5 V and 0.3 to 0.35 mA, respectively. The data values represent the averages of three replicate experiments. , CO2; , CH4.

To our knowledge, this is the first report to demonstrate that H₂-utilizing microbes can utilize reducing power from the electrically reduced electron donor NR as the sole electron donor for growth and for either fumarate reduction to succinate or CO₂ reduction to methane. Previous work on electrochemically reduced redox dyes (i.e., electroenergized systems) in microbial fermentations (3, 7) focused only on altering fermentation product patterns, and the systems used were not the carefully controlled electrochemical systems used here. In these systems, bacteria were not necessarily using the reduced dye as reducing power because the high voltage supplied produced H₂, which in turn could be used as an electron donor.

Figure 5A summarizes our hypothesis explaining how electrically reduced NR can replace H₂ as an energy source for microbial growth and succinate production. In respiratory succinate metabolism linked to fumarate reduction (13), electron carriers such as quinones are reduced by an electron donor (e.g., H₂ and NADH), and the reduced quinone is subsequently oxidized. This oxidation is coupled to the production of a proton motive force (PMF) that is dependent on the electron driving force. Similarly, in respiratory methane production linked to CO₂ reduction (Fig. 5B), electron donors (e.g., H₂) may be linked to a proton-translocating electron transport chain which includes cytochrome b (8). Electrons can also be transferred from the electrode to these two different microbial electron carrier chains through electrical (i.e., cathodic) reduction of NR and its subsequent microbial oxidation. The E_o' of reduced NR (0.325) is similar to that of NADH, and its microbial oxidation generates an electron driving force coupled to the production of a PMF to account for energy conservation.

View larger version (54K): [in this window] [in a new window]   FIG. 5.   Hypothetical models for microbial energy conservation with H2 and with electrically reduced NR as sources of reducing power for fumarate reduction to succinate (A) and CO2 reduction to methane (B). The electrical reduction of NR, which serves as an electronophore, or of H2 depends on the reduction of normal cellular electron carriers (XH). Electron carrier reduction is coupled to the generation of a PMF that drives ATP synthesis and charge separation across the cell membrane. CM, cytoplasmic membrane; 2eV, electrical reducing power.

In short, NR functions as an electronophore, or artificial electron carrier, enabling electricity to indirectly supply the electron driving force needed to generate a PMF for energy conservation and the electrons needed for growth and metabolite production. Much remains to be learned about the exact biochemical mechanism(s) that accounts for the ability of NR to function in electron transfer and about how PMF and energy conservation are driven by electrically reduced NR in microbes. It will also be of interest to study further, from both fundamental and applied perspectives, the use of electrically reduced NR as a reductant to drive different metabolic processes, such as photosynthesis, fermentative production of organic alcohols or acids, biotransformation of organic compounds into higher-value drugs and specialty chemicals, and anaerobic transformations that are rate limited by reducing equivalents (e.g., aromatic-compound reductive dechlorination or oil desulfurization).

Previously, a different electrochemical reactor system was used to grow T. ferrooxidans indirectly on electrically reduced iron as the immediate electron donor (29). Our electrochemical system is quite different; it works with pure and mixed cultures, and it is more versatile, since it is not limited to metal- or H₂-oxidizing microbes because NADH is formed directly from electrically reduced NR.

In light of these results, perhaps living cells can be more easily viewed as bioelectrical devices. That is, cells can function by maintaining charge separation across their membranes and by passing currents through their electron transport chains when utilizing light, chemicals, or electricity itself in the presence of electrically reduced NR as the source of reducing power. Actually, microbial fuel cells containing Proteus vulgaris and redox dyes were placed in the anode of an electrochemical bioreactor to generate an electrical current produced from the microbial metabolism of carbohydrates (1). Thus, it appears that the utilization or production of an electric current is reversible in microbial systems as long as an appropriate electron mediator is present.