Method for storing gaseous hydrogen through producing methanoate (formate)

Abstract

The present invention relates to a method for storing gaseous hydrogen, comprising the steps of producing methanoate (formate) through contacting gaseous hydrogen with carbon dioxide in the presence of a hydrogen dependent carbondioxide reductase (HDCR), and thereby storing of said gaseous hydrogen. The HDCR and/or its complex is preferably derived from Acetobacterium woodii.

Claims

1. A method for storing gaseous hydrogen, comprising the steps of producing methanoate (formate) through contacting gaseous hydrogen with carbon dioxide in the presence of a hydrogen dependent carbon dioxide reductase (HDCR) which catalyzes the direct conversion of H.sub.2 and CO.sub.2 into HCOOH, and thereby storing of said gaseous hydrogen, wherein said HDCR is part of an enzyme complex with a formate dehydrogenase accessory protein, an electron transfer protein, and a subunit harboring the active site characteristic of an [FeFe]-hydrogenase.

2. The method according to claim 1, wherein said HDCR is selected from FdhF1 or FdhF2 of Acetobacterium woodii.

3. The method according to claim 1, wherein said method is performed under standard ambient temperature and pressure or at between about 20? C. to about 40? C. and normal pressure.

4. The method according to claim 1, wherein said method further comprises inhibiting the metabolism of formate.

5. The method according to claim 1, further comprising the step of converting carbon monoxide into carbon dioxide using a CO dehydrogenase and a ferredoxin.

6. The method according to claim 1, wherein said method is performed in vitro, in vivo and/or in culture.

7. The method according to claim 1, further comprising the release of hydrogen from the methanoate as produced.

8. A method for storing gaseous hydrogen, comprising the steps of producing methanoate (formate) through contacting gaseous hydrogen with carbon dioxide in the presence of a hydrogen dependent carbon dioxide reductase (HDCR), which catalyzes the direct conversion of H.sub.2 and CO.sub.2 into HCOOH, wherein said HDCR is part of an enzyme complex with a formate dehydrogenase accessory protein, an electron transfer protein, and a subunit harboring the active site characteristic of an [FeFe]-hydrogenase, and thereby storing of said gaseous hydrogen wherein said method comprises the use of a recombinant bacterial organism comprising a genetic modification, wherein said genetic modification comprises transformation of said microorganism with exogenous bacterial nucleic acid molecules encoding the proteins FdhF1 and/or FdhF2, FdhD, HycB1 and/or HycB2, and HydA2, HycB3, and optionally AcsA of Acetobacterium woodii, or homologs thereof, whereby expression of said proteins increases the efficiency of producing formate from CO.sub.2 and H.sub.2.

9. The method according to claim 1, wherein said formate dehydrogenase accessory protein is FdhD of Acetobacterium woodii or a homolog thereof.

10. The method according to claim 1, wherein said complex further comprises a CO dehydrogenasand a ferredoxin, or a homolog thereof.

11. The method according to claim 10, wherein said CO dehydrogenase is AcsA of Acetobacterium woodii, or a homolog thereof.

12. The method according to claim 1, wherein said electron transfer protein is HycB1 or HycB2 of Acetobacterium woodii, or a homolog thereof.

13. The method according to claim 1, wherein said subunit harboring the active site characteristic of an [FeFe]-hydrogenase is HydA2 of Acetobacterium woodii, or a homolog thereof.

14. The method according to claim 4, wherein said inhibition of metabolism is accomplished by Na depletion using sodium ionopheres.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the productions of formate using whole cell catalysis. Cell suspensions of A. woodii (1 mg/ml) were incubated using a gas phase of 0.8?10.sup.5 Pa H.sub.2 and 0.2?10.sup.5 Pa CO.sub.2 (A). Adding the Na.sup.+ ionophore ETH2120 (30 ?M) gives rise to the production of up to 8 mM formate and the production of acetate stopped (B).

(2) FIG. 2 shows the production of formate using HCO.sub.3.sup.? or CO.sub.2 as substrate. Cell suspensions of A. woodii (1 mg/ml) were incubated using a gas phase of 0.8?10.sup.5 Pa H.sub.2 and 0.2?10.sup.5 Pa CO.sub.2 or 1?10.sup.5 Pa H.sub.2 with 300 mM KHCO.sub.3.

(3) FIG. 3 shows the relationship of final formate concentration to initial HCO.sub.3.sup.?. Cell suspensions of A. woodii (1 mg/ml) were incubated with increasing amounts of initial HCO.sub.3.sup.? and a gas phase of 1?10.sup.5 Pa H.sub.2.

BRIEF DESCRIPTION OF THE SEQUENCES

(4) Sequence ID NOs. 1 to 8 show the amino acid sequences of the enzymes FdhF1, HycB1, FdhF2, HycB2, FdhD, HycB3, HydA2, and AcsA of A. woodii, respectively.

EXAMPLES

(5) Measurements with the Isolated HCDR

(6) For the purification of HCDR A. woodii (DSM 1030) was grown at 30? C. under anaerobic conditions in 20-1-liter flasks using 20 mM fructose to an OD.sub.600 of ?2.5. All buffers used for preparation of cell extracts and purification contained 2 mM DTE and 4 ?M resazurin. All purification steps were performed under strictly anaerobic conditions at room temperature in an anaerobic chamber filled with 100% N.sub.2 and 2-5% H.sub.2. The cell free extract was prepared as described previously (Schuchmann et al., J Biol Chem. 2012 Sep. 7; 287(37):31165-71). Membranes were removed by centrifugation at 130000 g for 40 minutes. Part of the supernatant containing the cytoplasmic fraction with approximately 1600 mg protein was used for the further purification. Ammonium sulfate (0.4 M) was added to the cytoplasmic fraction. Half of this sample was loaded onto a Phenyl-Sepharose high performance column (1.6 cm?10 cm) equilibrated with buffer A (25 mM Tris/HCl, 20 mM MgSO.sub.4, 0.4 M (NH.sub.4).sub.2SO.sub.4, 20% glycerol, pH 7.5). Methylviologen-dependent formate dehydrogenase activity elutet around 0.33 M (NH.sub.4).sub.2SO.sub.4 in a linear gradient of 120 ml from 0.4 M to 0 M (NH.sub.4).sub.2SO.sub.4. This step was repeated with the second half of the sample in a separate run to gain more protein since otherwise large amounts of the activity eluted in the flowthrough. The pooled fractions of both runs were diluted to a conductivity of below 10 mS/cm with buffer C (25 mM Tris/HCl, 20 mM MgSO.sub.4, 20% glycerol, pH 7.5) and applied to a Q-Sepharose high performance column (2.6 cm?5 cm) equilibrated with buffer C. Protein was eluted with a linear gradient of 160 ml from 150 mM to 500 mM NaCl. Formate dehydrogenase eluted at around 360 mM NaCl. Pooled fractions were concentrated using ultrafiltration in 100-kDa VIASPIN tubes and applied to a Superose 6 10/300 GL prepacked column equilibrated with buffer C and eluted at a flow rate of 0.5 ml/min. Formate dehydrogenase activity eluted as a single peak. Pooled fractions were stored at 4? C.

(7) Measurements of HCDR activity were performed at 30? C. with in buffer 1 (100 mM HEPES/NaOH, 2 mM DTE, pH 7.0) in 1.8 ml anaerobic cuvettes sealed by rubber stoppers, containing 1 ml buffer and a gas phase of 0.8?10.sup.5 Pa H.sub.2 and 0.2?10.sup.5 CO.sub.2. Production of formate was measured using Formate dehydrogenase of Candida boidinii with 2 mM NAD in the assay and production of NADH was followed.

(8) For measurements with ferredoxin as electron carrier Ferredoxin was purified from Clostridium pasteurianum. For reduction of ferredoxin CO dehydrogenase of A. woodii was purified and in these experiments the gas phase of the cuvettes was changed to 100% CO (1.1?10.sup.5 Pa).

(9) Whole Cells

(10) For experiments with whole cells, the inventors used cell suspensions of A. woodii for the conversion of H.sub.2 and CO.sub.2 to formate. The energy metabolism of A. woodii is strictly sodium ion dependent, and the ATP synthase uses Na.sup.+ as the coupling ion. Thus, by omitting sodium ions in the buffer or by adding sodium ionophores (the inventors used the ionophore ETH2120 in this study), it is possible to switch off the energy metabolism specifically. Cells suspended in imidazole buffer (50 mM imidazole, 20 mM MgSO.sub.4, 20 mM KCl, 4 mM DTE, pH 7.0) containing 20 mM NaCl converted H.sub.2+CO.sub.2 to acetate, and only small amounts of formate were produced from a gas phase of 0.8?10.sup.5 Pa H.sub.2 and 0.2?10.sup.5 CO.sub.2. By adding ETH2120 (30 ?M), acetate production ceased almost completely and formate was produced with an initial rate of 2 ?mol/min?mg cell protein (FIG. 1). In agreement with the results obtained from the purified enzyme, formate production was also observed when hydrogen was absent and the electron donor was CO but with lower rates compared to hydrogen as electron donor.

(11) The results in FIG. 1 demonstrate that a maximal amount of around 8 mM of formate was produced in this experiment. The inventors next tested, if the final formate concentration is proportional to the initial gas pressure. From 0.5 to 2?10.sup.5 Pa H.sub.2+CO.sub.2 the final formate concentration did not increase. Since the inventors observed a pH drop during the experiment, the inventors examined if the lower pH is the limiting factor. Increasing the buffer concentration from 50 to 200 mM resulted in a final formate concentration of around 14?3 mM. If CO.sub.2 was exchanged with KHCO.sub.3 the effect was even more dramatic. By using the base HCO.sub.3.sup.? the overall process is almost pH neutral compared to the production of formic acid from CO.sub.2. The genome of A. woodii encodes for a carboanhydrase that allows the rapid interconversion of CO.sub.2 and HCO.sub.3.sup.?. FIG. 2 shows the production of formate from initially 300 mM KHCO.sub.3 (with 1?10.sup.5 Pa H.sub.2) compared to CO.sub.2 as substrate. Finally up to 184?5 mM formate were produced with KHCO.sub.3.

(12) The relationship of the final formate concentration to the initial concentration of HCO.sub.3.sup.? is shown in FIG. 3. Up to 300 mM HCO.sub.3.sup.?, the final formate concentration increases with increasing substrate concentration. Furthermore the final formate concentration fits well to the theoretic thermodynamic limit of the reaction underlining the independence of the carboxylation of CO.sub.2/HCO.sub.3.sup.? from other cellular processes. At 1?10.sup.5 Pa H.sub.2, the thermodynamic equilibrium is approximately [HCO.sub.3.sup.?]=[HCOOH], so equimolar concentrations of substrate and product. At concentrations of HCO.sub.3.sup.? above 300 mM, this relationship does not exist anymore and the final amount of formate produced ceased around 300 mM.