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Applied and Environmental Microbiology, June 2005, p. 2870-2874, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2870-2874.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Energy Generation from the CO Oxidation-Hydrogen Production Pathway in Rubrivivax gelatinosus

Pin-Ching Maness,^* Jie Huang, Sharon Smolinski, Vekalet Tek, and Gary Vanzin

National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, Colorado 80401-3393

Received 3 May 2004/ Accepted 20 December 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H₂ production pathway according to the stoichiometry CO + H₂O CO₂ + H₂. Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H₂ activity was determined in the presence of the electron transport uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), the rate of H₂ production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H₂ production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H₂ reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H₂ production from gas streams containing CO.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Hydrogen is a clean fuel that addresses the issues of energy security and energy independence while preserving a pristine environment. Biomass gasification generates a gas stream enriched in CO and H₂ (synthesis gas). Many microbes have been reported to metabolize CO according to the equation CO + H₂O H₂ + CO₂ (4, 7, 9, 25). The biological CO-to-H₂ pathway is therefore ideal if it is used following biomass gasification to convert the CO component in the synthesis gas into additional H₂. One such candidate is the purple nonsulfur photosynthetic bacterium Rubrivivax gelatinosus CBS, which was isolated from its natural environment with the ability to metabolize CO, yielding H₂ (16). The CO oxidation pathway in R. gelatinosus CBS consists of at least two enzymatic steps: CO dehydrogenase (CODH) catalyzes the oxidation of CO, and hydrogenase mediates the reduction of protons, yielding H₂ (17), similar to their counterparts in Rhodospirillum rubrum (8, 11). Earlier findings documented that both R. gelatinosus strain 1 and R. rubrum can grow in darkness by using CO as their carbon substrate (14, 24). However, the growth media used in the above studies were often supplemented with complex carbon-containing nutrients such as Trypticase, yeast extract, and sodium acetate, which complicates the conclusion that CO could serve as the sole carbon and energy source. Nonetheless, new ATP synthesis was indeed detected in R. gelatinosus strain 1 in darkness when CO was added as the carbon substrate along with Trypticase (6). This finding provided the first direct evidence in a photosynthetic bacterium that the CO-to-H₂ pathway is linked to ATP production. CO metabolism in R. gelatinosus strain 1 is slightly different from that in R. gelatinosus CBS, as documented in this report, in that the latter utilizes CO equally well in both light and darkness (16), while the former only metabolizes CO in darkness and cells grown in the light do not oxidize CO (26).

The purpose of this report is to demonstrate that the CO oxidation pathway in R. gelatinosus CBS can indeed generate energy by using CO as the sole carbon substrate. We provide three lines of evidence to substantiate our findings. We observed CO-supported cell growth in darkness without supplementing the medium with other carbon-containing nutrients. We detected new ATP synthesis in darkness as soon as CO was added to the gas phase of the culture. Lastly, based on the effects of the proton ionophore carbonyl-cyanide m-chlorophenylhydrazone (CCCP) and the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), we demonstrate that energy generation during the CO-to-H₂ reaction is likely driven by a transmembrane proton gradient generated during the electron transport process.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organism and growth conditions.
Rubrivivax gelatinosus CBS was cultivated in RCVBN minimal medium in CO (20% [vol/vol]), with or without sodium malate (37 mM). Medium preparation, growth conditions, and cell dry weight determinations were done as described previously (17). The culture bottles were placed on their sides with shaking at 150 rpm to facilitate the mass transfer of CO to the aqueous phase. Illumination was provided by incandescent lamps, with a light intensity of approximately 45 µEinstein s^–1 cm^–2 reaching the culture surface.

CO-to-H₂ rate determination.
Rubrivivax gelatinosus CBS was cultured photosynthetically in CO and sodium malate to its logarithmic phase of growth (optical density [OD] at 660 nm between 0.6 and 0.8). An aliquot of the culture was then withdrawn and diluted anaerobically in 50 ml of RCVBN medium to reach a final OD of about 0.1 inside a 150-ml bottle capped with a bromobutyl septum. Dithiothreitol (1 mM) was added to ensure reducing conditions. The bottles were then purged with 20% CO, followed by shaking at 250 rpm at 30°C. After 2 hours of preincubation in darkness, the gas phase was replenished with 20% CO. Aliquots of the gas phase were sampled periodically, in darkness, for up to a 2-hour period to determine the CO and H₂ concentrations in the gas sample by gas chromatography (Agilent 5890 Series II) with an instrument equipped with a Molecular Sieve 5A column (60/80 mesh). The initial 2 hours of preincubation in CO were necessary to obtain a linear production of H₂ from CO during the second 2 hours without a lag phase. The protonophore CCCP and the ATP synthase inhibitor DCCD were added as ethanol solutions as indicated in the figure legends, while controls received only the solvent.

CODH and hydrogenase assays.
The CODH activity was determined spectrophotometrically by measuring the reduction of methyl viologen from CO at 578 nm, and hydrogenase activity was determined by the evolution of H₂ from reduced sodium dithionite mediated by methyl viologen (17). The chromatophore membranes used for these studies were prepared anaerobically via sonication as described previously (17), and the membrane-enriched supernatant was centrifuged at 200,000 x g for 90 min to obtain chromatophore membrane vesicles. Protein concentrations were determined by the method of Lowry et al. (15).

Intracellular ATP determination.
Rubrivivax gelatinosus CBS was cultured photosynthetically in RCVBN medium with CO as the sole source of carbon. When the culture OD was between 0.3 and 0.4, the gas phase of the culture was purged with argon gas and incubated in darkness for 16 h to exhaust endogenous reductants and intracellular ATP. The culture was then fed with either CO (20% [vol/vol]) or argon gas and placed in darkness with shaking to initiate the reaction. At various intervals, an aliquot of 0.5 ml of cell suspension was spun down and suspended in 0.5 ml of 25 mM glycylglycine (pH 8.0) containing 10 mM MgCl₂ (buffer). Twenty microliters of the suspended cells was withdrawn and mixed with 180 µl of dimethyl sulfoxide, followed by the addition of 0.8 ml of the buffer described above, according to the method of Tran and Unden (23). The cell extract was then placed on ice for ATP determinations. The intracellular ATP content was determined by pipetting 0.2 ml of the buffer into a Lumicuvette along with 25 µl of cell extract, followed by the pumping in of 0.1 ml of luciferase-luciferin stock (10 mg/ml; Sigma) to initiate the luminescence reaction. Light emission was quantified for 20 seconds in a luminometer (Analytical Laboratory, San Diego, Calif.). Calibration was performed with various known quantities of ATP.

Top Abstract Introduction Materials and Methods Results Discussion References

 
CO-dependent growth in light and in darkness.
To demonstrate that R. gelatinosus CBS can assimilate CO as the sole source of carbon during photosynthetic growth, we inoculated cells into 20 ml of RCVBN minimal medium inside a 300-ml Erlenmeyer flask supplemented with CO (20% [vol/vol]). Figure 1A shows that growth proceeded at a nearly linear rate, with a cell doubling time of approximately 10 h, in light. A high ratio of gas volume to liquid volume and vigorous stirring of the cell suspension were necessary to enhance gas-liquid mass transfer so that sufficient amounts of CO were dissolved in the liquid phase throughout the growth period. Although CO alone supported photosynthetic growth, the inclusion of sodium malate along with CO accelerated cell growth significantly, with a cell doubling time near 90 min, while displaying a similar CO-to-H₂ reaction rate to that of cells cultured in CO alone. Under these conditions, R. gelatinosus consumed both sodium malate and CO simultaneously (data not shown). Sodium malate was therefore included in most cultures for the experiments requiring CO-to-H₂ rate measurements described below.

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FIG. 1. Growth of Rubrivivax gelatinosus CBS with CO as the sole carbon substrate in light (A) and in darkness (B). R. gelatinosus was inoculated into RCVBN minimal medium, and CO feedings and cell growth measurements were performed as described in the text. The arrow in panel A indicates a batch feeding of CO (20% [vol/vol]). Panel B displays growth in CO () and nitrogen ().

Similarly, to demonstrate that CO alone can also support cell growth in darkness, we performed growth measurements in the dark with RCVBN minimal medium and with CO as the sole carbon and energy substrate. An Erlenmeyer flask (300 ml) containing 50 ml of bacterial suspension was bubbled twice daily for 10 min with either 20% CO or an inert N₂ gas control to ensure the removal of end products such as H₂ and CO₂. The CO and N₂ gas streams were first passed through a saturated Na₂S₂O₃ solution to remove any residual O₂ to prevent cell growth via the aerobic respiratory pathway. Although growth was slow, the data from Fig. 1B show an increase in cell turbidity in CO over a 4-day period, with a cell doubling time of nearly 2 days. The cell turbidity increase was also reflected by an increase in CFU, from 1.4 x 10⁸ to 6 x 10⁸ ml^–1, during the same period. The parallel control culture supplemented with N₂ instead of CO exhibited no growth and no increase in CFU over the same period.

Effect of CO addition on ATP formation in darkness.
The findings from Fig. 1B support the hypothesis that in addition to CO serving as a carbon substrate, the CO-to-H₂ reaction itself must yield energy to support cell growth in darkness. To determine if CO metabolism generates ATP, we prepared two identical cultures grown photosynthetically using CO as the sole carbon substrate and then determined the subsequent kinetic ATP formation in darkness upon the addition of CO. The data in Fig. 2 reveal that almost immediately after the CO addition, R. gelatinosus CBS began to produce ATP at a linear rate, reaching a steady state after 10 min in CO. Hydrogen was detected along with ATP production upon the addition of CO (data not shown). No net ATP production was detected in a control receiving only argon gas. The detection of new ATP in response to the addition of CO clearly establishes that the CO-to-H₂ pathway could conserve energy.

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FIG. 2. Kinetics of new ATP formation in darkness in response to the addition of gas. R. gelatinosus grown in the light in CO was first depleted of most of its endogenous ATP contents prior to feeding with either CO () or argon gas () at time zero. Quantities of net ATP were determined by subtracting the initial amount of ATP (4.7 nmol mg cell dry weight–1 at zero time) from the total amounts of ATP determined at various intervals after the addition of gas. The argon gas control samples were tested in duplicate, with a standard deviation of <10%.

Effect of electron transport uncoupler on rate of CO-to-H₂ reaction in darkness.
The chemiosmotic theory proposes a mechanism for energy conservation whereby an electron transport reaction generates an electrochemical gradient across the cell membrane, the dissipation of which subsequently drives ATP synthesis (13). Such an electrochemical gradient is known to exert a thermodynamic back pressure, which in turn slows down electron transport; this phenomenon is termed respiratory control and was first described for mitochondria (2). However, if this back pressure could be relieved by a protonophore causing an increase in the electron transport rate, it would provide indirect evidence that a transmembrane proton gradient is generated during electron transport. To verify this possibility, we measured the effect of various concentrations of the protonophore CCCP on the rate of H₂ production linked to CO oxidation in darkness, using cells that were previously cultured photosynthetically in CO and sodium malate as described in Materials and Methods. The data in Fig. 3A indicate that upon the addition of increasing concentrations of CCCP, the rate of H₂ production was also elevated incrementally, by more than 30%, and leveled off at approximately 20 µM CCCP. To demonstrate the effect of CCCP during kinetic measurements, we added 20 µM CCCP 14 min after the start of the reaction (Fig. 3B). An immediate increase in the H₂ production rate was observed, to a level comparable to that when CCCP was added at the beginning of the reaction. The control experiments received comparable amounts of ethanol solution to offset any solvent effect. CCCP is known to dissipate the proton gradient across an energized membrane. The enhancement in electron transport rate upon the addition of CCCP indicates that a transmembrane proton gradient is coupled to electron transport during the CO oxidation and H₂ production process. The dissipation of the proton gradient by CCCP should abolish ATP formation upon the addition of CO. Indeed, when 20 µM of CCCP was added along with CO to an R. gelatinosus culture under similar conditions to those described for Fig. 2, no new ATP was synthesized during a 25-min kinetic assay (data not shown).

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FIG. 3. (A) Effect of various CCCP concentrations on stimulation of the CO-dependent H2 production rate in darkness. The control data point (100%) receiving only ethanol solution had a rate of 0.89 mmol H2 min–1 g cell dry weight–1. (B) Kinetic effect of CCCP on H2 production when 20 µM CCCP was added either at the beginning of the reaction () or at 14 min (), as indicated by an arrow. The control () received an ethanol solution at time zero. The samples were tested in triplicate, with a standard deviation of <10%.

Effect of the ATP synthase inhibitor DCCD on CO-to-H₂ reaction in darkness.
Based on the stimulatory effect of CCCP on the CO-to-H₂ reaction rate and the potential buildup of a transmembrane proton gradient, it is reasonable to assume that an ATP synthase inhibitor such as DCCD should inhibit the overall reaction by blocking the reentry of protons (21). This assumption was indeed verified, as shown in Fig. 4A, for which various amounts of DCCD were preincubated with whole cells of R. gelatinosus in CO for 30 min prior to purging of the culture gas phase with 20% CO to initiate H₂ production rate measurements. The overall CO-to-H₂ production rate was very sensitive to DCCD, with a 25 µM concentration completely abolishing the reaction. Since the inhibition of ATP synthase by DCCD would cause a buildup of the thermodynamic back pressure, which would slow down the H₂ production rate from CO oxidation, dissipation of this proton gradient via the protonophore CCCP should relieve the back pressure and thus restore the reaction rate. The data in Fig. 4B show that after 13 min of reaction time in the presence of 5 µM DCCD, the addition of CCCP (30 µM) did indeed restore the reaction rate, from nearly 63% that of the control to approximately 87% that of the control. These findings further support the involvement of a proton gradient during CO metabolism.

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FIG. 4. Effect of DCCD on CO-dependent H2 production rate in darkness. (A) Various concentrations of DCCD were preincubated with an R. gelatinosus suspension for 30 min prior to the addition of CO to initiate reaction rate measurements as described in Materials and Methods. The control received only an ethanol solution. (B) Kinetics of DCCD inhibition and its reversal by CCCP. Three identical R. gelatinosus suspensions were incubated either in ethanol () or in 5 µM DCCD ( and ) for 30 min prior to initiation of the reaction by the addition of CO at time zero. No addition was made to one culture (), and 30 µM CCCP was added to the second culture after 14 min of reaction time (), as indicated by the arrow. The results are averages from three independent experiments.

The overall CO-to-H₂ production pathway is comprised of at least two enzymatic steps, with one catalyzed by the enzyme CODH, oxidizing CO to release protons and reducing equivalents, and the other being the proton-reducing hydrogenase reaction. We determined the effects of DCCD on the two separate reactions by assaying them independently from each other, using chromatophore membranes which we prepared anaerobically. The results in Fig. 5 show that after a preincubation of DCCD with the chromatophore for 30 min in darkness, DCCD up to approximately 3 µM had no effect on the CODH activity compared to a solvent control receiving only ethanol. On the other hand, the hydrogenase reaction displayed a high sensitivity to DCCD, with a loss of activity of nearly 40% observed in the presence of 5 µM DCCD (Fig. 5). This finding strongly implies that hydrogenase is involved not only in proton reduction yielding H₂ but also in proton translocation during the electron transport pathway resulting from CO oxidation.

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FIG. 5. Effects of DCCD on CO dehydrogenase and hydrogenase activities. Chromatophore membranes (9.8 mg protein ml–1) were preincubated with either DCCD or comparable volumes of ethanol for 30 min prior to initiating CODH measurements () by the addition of CO or hydrogenase determinations () by the addition of sodium dithionite. The chromatophore displayed a CODH activity of 1.8 µmol reduced methyl viologen/min/mg protein (100%) and a hydrogenase activity of 2.7 µmol H2 min–1 mg protein–1 (100%). Both activities were lowered by as much as 45% due to the presence of ethanol in the assays. The results are averages from two independent experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In earlier studies of R. gelatinosus strain 1, CO was reported to support cell growth in darkness as the sole carbon substrate (24). However, the oxidation of CO and its subsequent assimilation into new cell mass occurred only in darkness, as a culture grown in the light did not oxidize CO or assimilate CO into new cell materials (26). The CO oxidation system of R. gelatinosus CBS described here differs from that of strain 1 in that the CBS strain can oxidize and assimilate CO into new cell mass both in light and in darkness (Fig. 1). Once the CO oxidation pathway is induced, light has no effect on the CO-to-H₂ activity in the CBS strain (16). In another photosynthetic bacterium, R. rubrum, CO was also found to support cell growth in darkness (14). However, in addition to CO, carbon-containing yeast extract (0.1% [wt/vol]) and sodium acetate (0.82 g/liter) were also included in the medium, both of which were partially consumed during the CO-supported growth of R. rubrum in the dark, which complicates growth measurements in CO. Our data thus provide the first evidence that CO alone can support cell growth in darkness without other carbon supplementation.

Our observation that CO alone supported cell growth in darkness prompted us to examine if new ATP is produced in response to the addition of CO. The results shown in Fig. 2 verified that CO-dependent H₂ production is indeed coupled to ATP formation. This observation also confirmed the earlier findings by Champine and Uffen (6). Similarly, in the methanogenic bacterium Methanosarcina barkeri, CO oxidation to CO₂ and H₂ is coupled to the phosphorylation of ADP to ATP, even though the physiological function of its CODH is to mediate CO₂ formation from the carboxyl group of acetate during methanogenesis (5), a reaction that is not detected in photosynthetic bacteria (14).

In this report, we also document several lines of evidence demonstrating that CO-dependent H₂ production is coupled to proton translocation (Fig. 3 and 4). The stimulatory effects of CCCP on the CO-to-H₂ rate, the inhibitory action of DCCD, and the reversal of the latter by a subsequent CCCP addition are all consistent with the chemiosmotic mechanism of energy generation by proton translocation via an ATP synthase channel (13). The overall CO-to-H₂ reaction involves multiple steps catalyzed by several enzymes; it is not clear based on the above findings which step may be responsible for the generation of the proton gradient. The data shown in Fig. 5 indicate that DCCD inhibits the hydrogenase reaction, but not CODH activity, implying that the hydrogenase reaction may be the coupling site. Similar DCCD effects were also reported for R. rubrum (10). Both the CODH and hydrogenase involved in this pathway are membrane-bound proteins in R. rubrum (8) and R. gelatinosus (17). Molecular biology studies of R. rubrum revealed that the large subunit of the CO-induced hydrogenase CooH belongs to the [Ni-Fe] hydrogenase class, with homology to a subunit of the energy-conserving NADH:quinone oxidoreductase complex of various organisms (11). In addition, Fox and his coworkers reported that both CooK and CooU, putative subunits of the CO-induced hydrogenase complex, show similarity to the subunits of the NADH:quinone oxidoreductases of various organisms (10). We recently sequenced the cooH, cooK, and cooU genes of R. gelatinosus CBS, all of which displayed high degrees of similarity to their counterparts in R. rubrum (unpublished data). In addition to energy generation from the CO-dependent H₂ production pathway in both R. rubrum and R. gelatinosus, there is now growing evidence that the membrane-bound, multisubunit [Ni-Fe] type of hydrogenase is involved in energy generation in diverse microbes. The membrane-bound hydrogenases from the hyperthermophile Pyrococcus furiosus, the Ech hydrogenase from Methanosarcina barkeri, hydrogenases A and B from Methanobacterium thermoautotrophicum, and hydrogenases 3 and 4 of the formate hydrogenlyase system from Escherichia coli all display high levels of homology to the energy-conserving complex I, with a proposed role of generating a proton motive force coupled to energy generation (1, 3, 12, 18, 19, 20, 22). Nonetheless, the mechanism accounting for such proton pumping is still unknown. Presumably, by using protons as a substrate, hydrogenase and its accessory proteins play a dual role in both proton reduction and proton pumping. The energetics of hydrogen production mediated by hydrogenase therefore have important ramifications, serving not only to dissipate excess reductant but also to provide cells with energy to support growth in an anaerobic environment.

 
This work was supported by the U.S. Department of Energy Hydrogen, Fuel Cells, and Infrastructure Technologies Program.

 
* Corresponding author. Mailing address: National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, CO 80401-3393. Phone: (303) 384-6114. Fax: (303) 384-6150. E-mail: pinching_maness{at}nrel.gov.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, June 2005, p. 2870-2874, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2870-2874.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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