H₂ Production from Methyl Viologen–Dependent Hydrogenase Activity Monitored by Gas Chromatography

Materials and reagents

Biological materials

1. Hydrogenase-expressing Escherichia coli carrying hydA, hydE, hydF, and hydG genes (this recombinant bacterial strain was constructed in our laboratory according to our previous report in Kosem et al., 2023).

   Note: The function of each protein expressed from specific genes in the processes of hydrogenase maturation was reported in many published articles, such as Broderick et al. (2014) and Lubitz et al. (2014).

Reagents

1. LB broth, Miller (Nacalai Tesque, catalog number: 20068-75)

2. Methyl viologen (Tokyo Chemical Industry, catalog number: D3685)

3. Sodium dithionite (Na₂S₂O₄) (Sigma-Aldrich, catalog number: 71699-250G)

4. Pierce^TM BCA Protein Assay kit (Thermo Scientific, catalog number: 23227)

5. Sodium chloride (NaCl) (Wako Chemical, catalog number: 191-01665)

6. Tris (hydroxymethyl) aminomethane (Tris) (Nacalai Tesque, catalog number: 35434-21)

7. Hydrochloric acid (HCl) (Wako Chemical, catalog number: 080-0106)

8. Trichloroacetic acid (TCA) (Nacalai Tesque, catalog number: 34637-14)

9. 3-(N-morpholino)propanesulfonic acid (MOPS) (Nacalai Tesque, catalog number: 23415-25)

10. Sodium hydroxide (NaOH) (Chameleon Reagent, catalog number: 000-75165)

11. Ampicillin sodium (Wako, catalog number: 014-23302)

12. Streptomycin sulfate (Wako, catalog number: 194-08512)

13. Glucose (Nacalai Tesque, catalog number: 16805-35)

14. Ferric ammonium citrate (Nacalai Tesque, catalog number: 19425-12)

15. L-cysteine (Nacalai Tesque, catalog number: 10309-12)

16. Sodium fumarate (TCI, catalog number: F0070)

17. Isopropyl-β-D-thiogalactopyranoside (IPTG) (FujiFilm, catalog number: 094-05144)

Solutions

1. LB (Luria-Bertani) broth (see Recipes)

2. 1 M MOPS-NaOH (see Recipes)

3. 100 mg/mL Ampicillin sodium (see Recipes)

4. 40 mg/mL Streptomycin sulfate (see Recipes)

5. 50% (w/v) Glucose (see Recipes)

6. 250 mg/mL Ferric ammonium citrate (see Recipes)

7. 1 M Sodium fumarate (see Recipes)

8. 1 M IPTG (see Recipes)

9. 0.9% (w/v) NaCl (see Recipes)

10. 50 mM MV²⁺/Na₂S₂O₄ solution (see Recipes)

11. 1 M Tris-HCl pH 7 (see Recipes)

Recipes

1. LB broth

   Note: Dissolve the medium and all reagents in a 500 mL Erlenmeyer flask and then autoclave at 121 °C for 15 min before use.

   Reagent	Final concentration	Quantity
   LB broth	2.5 % (w/v)	5 g
   1 M MOPS-NaOH (pH 7.4) (Recipe 2)	100 mM	20 mL
   Water	n/a	180 mL
   Total	n/a	200 mL
   The following (see Recipes 3–8) are added after autoclaving:
   100 mg/mL Ampicillin sodium	100 μg/mL	0.2 mL
   40 mg/mL Streptomycin sulfate	40 μg/mL	0.2 mL
   50% (w/v) Glucose	0.5% (w/v)	2 mL
   250 mg/mL Ferric ammonium citrate	250 μg/mL	0.2 mL
   L-cysteine	2 mM	50 mg
   1 M Sodium fumarate	20 mM	4 mL
   1 M IPTG	1 mM	0.2 mL




2. 1 M MOPS-NaOH pH 7.4

   Note: Dissolve in a 100 mL beaker on a magnetic stirrer.

   Reagent	Final concentration	Quantity
   MOPS	1 M	20.9 g
   Water	n/a	80 mL
   NaOH (8 M)	n/a	Slowly add until pH 7.4
   Total	n/a	Make up final volume to 100 mL




3. 100 mg/mL Ampicillin sodium

   Note: Dissolve in a 5 mL microcentrifuge tube and mix thoroughly with gentle shaking by hand. Aliquot the stock solution in 1 mL microcentrifuge tubes (500 μL per tube) and store at -20 °C until use.

   Reagent	Final concentration	Quantity
   Ampicillin sodium	100 mg/mL	0.5 g
   Sterilized water	n/a	5 mL
   Total	n/a	5 mL




4. 40 mg/mL Streptomycin sulfate

   Note: Dissolve in a 5 mL microcentrifuge tube and mix thoroughly with gentle shaking by hand. Aliquot the stock solution in 1 mL microcentrifuge tubes (500 μL per tube) and store at -20 °C until use.

   Reagent	Final concentration	Quantity
   Streptomycin sulfate	40 mg/mL	0.2 g
   Sterilized water	n/a	5 mL
   Total	n/a	5 mL




5. 50% (w/v) Glucose

   Note: Dissolve glucose powder with warm sterilized water in a 100 mL beaker on a magnetic stirrer. Once it has completely dissolved, bring the volume up to 100 mL total. Aliquot the stock solution in 15 mL tubes (10 mL per tube) and store at -20 °C until use.

   Reagent	Final concentration	Quantity
   Glucose	50% (w/v)	50 g
   Sterilized water	n/a	60 mL
   Total	n/a	Make up final volume to 100 mL




6. 250 mg/mL Ferric ammonium citrate

   Note: Dissolve in a 5 mL microcentrifuge tube and mix thoroughly with gentle shaking by hand. Aliquot the stock solution in 1 mL microcentrifuge tubes (500 μL per tube) and store at -20 °C until use.

   Reagent	Final concentration	Quantity
   Ferric ammonium citrate	250 mg/mL	1.25 g
   Sterilized water	n/a	5 mL
   Total	n/a	5 mL




7. 1 M Sodium fumarate

   Note: Dissolve in a 50 mL tube and mix thoroughly with gentle shaking by hand. Aliquot the stock solution in 15 mL tubes (10 mL per tube) and store at -20 °C until use.

   Reagent	Final concentration	Quantity
   Sodium fumarate	1 M	8.0 g
   Sterilized water	n/a	50 mL
   Total	n/a	50 mL




8. 1 M IPTG

   Note: Dissolve in a 5 mL microcentrifuge tube and mix thoroughly with gentle shaking by hand. Aliquot the stock solution in 1 mL microcentrifuge tubes (500 μL per tube) and store at -20 °C until use.

   Reagent	Final concentration	Quantity
   IPTG	1 M	1.2 g
   Sterilized water	n/a	5 mL
   Total	n/a	5 mL




9. 0.9% (w/v) NaCl solution

   Note: Dissolve in a 100 mL beaker on a magnetic stirrer.

   Reagent	Final concentration	Quantity
   NaCl	0.9% (w/v)	0.9 g
   Water	n/a	100 mL
   Total	n/a	100 mL




10. 50 mM MV²⁺/Na₂S₂O₄ solution

    Note: Prepare fresh in an anaerobic sealed vial inside a glove box and mix thoroughly with gentle shaking by hand.

    Reagent	Final concentration	Quantity
    Methyl viologen	50 mM	0.0257 g
    Na2S2O4	250 mM	0.0870 g
    Water	n/a	2 mL
    Total	n/a	2 mL




11. 1 M Tris-HCl pH 7

    Note: Dissolve in a 100 mL beaker on a magnetic stirrer. Adjust pH with 3 M HCl in a fume hood and strictly following the Kyushu University Guidelines for Safety in Education: Laboratory Activities. A safety data sheet from the chemical company as shown in Guideline 1 in the Supporting materials.

    Reagent	Final concentration	Quantity
    Tris	1 M	12.1 g
    Water	n/a	80 mL
    HCl (3 M)	n/a	Slowly add until pH 7
    Total	n/a	Make up final volume to 100 mL

Laboratory supplies

1. Glass vial No. 3, 10 mL size (ASONE, catalog number: 5-111-03)

2. Rubber stopper (ASONE, catalog number: 5-112-01)

3. Aluminum cap (ASONE, catalog number: 5-112-03)

4. 1 mL Syringe (Terumo, catalog number: SS-01T)

5. Needle No. 27 G × 3/4" (Terumo, catalog number: NN-2719S)

6. 10 μL pipette tip (Molecular BioProducts, catalog number: 3510-05)

7. 200 μL pipette tip (Violamo, catalog number: 3-6504-12)

8. 1,000 μL pipette tip (Violamo, catalog number: 3-6504-13)

9. 1 mL microcentrifuge tube (Quality Scientific Plastics, catalog number: L-510-GRD-Q)

10. 5 mL microcentrifuge tube (ASONE, model: AST0500)

11. 96-well plate (ASONE, catalog number: 2-8085-02)

12. 99.999% N₂ gas (Air Liquide, catalog number: JAGA 13038)

13. Gloves (Showaglove, catalog number: 882.L.BLUE)

14. Paper towels

15. Disposable cuvettes (Violamo, model: UVC-Z8.5)

Equipment

1. 2–20 μL autoclavable micropipette (Nichipet Ex II, Nichiryo, catalog number: J16Y01961)

2. 20–200 μL autoclavable micropipette (Nichipet Ex II, Nichiryo, catalog number: J16Z00871)

3. 100–1,000 μL autoclavable micropipette (Nichipet Ex II, Nichiryo, catalog number: J16X11751)

4. accu-jet^® pro pipette controller (BRAND^®, catalog number: Z671533)

5. High-speed micro centrifuge (Hitachi, model: CF16RN)

6. Personal centrifuge (Front Lab, model: FLD2012)

7. Gas chromatography (Shimadzu Corp., model: GC-8A)

8. Thermal conductivity detector (Shimadzu Corp., model: GC-8AIT)

9. Integrator C-R6A chromatopac (Shimadzu Corp., catalog number: 223-04500-38)

10. Molecular sieve 5A beads (GL Sciences Inc., catalog number: 1001-11503)

11. Stainless steel column, 2 m length × 3 mm diameter (Shimadzu Corp., catalog number: 201-48705-20)

12. Shaking water bath (Yamato Scientific, model: BW101)

13. Shaking incubator (EYELA, model: FYC-100)

14. Anaerobic glove box (MIWA, model: 1ADB-3)

15. Clean bench (Hitachi Appliances Inc., model: CCV-1300E)

16. Microplate reader (Corona Electric, model: SH-1000)

17. 500 mL Erlenmeyer flask with baffle (IWAKI, model: CTE33)

18. 250 mL centrifuge bottle (Nalgene, catalog number: B1033)

19. 20 mm vial crimper (Chromatography Research Supplies, catalog number: 320990)

20. 20 mm vial decapper pliers (Kebby, catalog number: D-20)

21. 1 mL gas tight syringe (ITO Corporation, catalog number: MS-GAN100)

22. Weighing balance with 0.0001 g accuracy (Metter Toledo, model: XS204)

23. Vortex mixer (Scientific Industries, model: SI-0286)

24. pH meter (Horiba Scientific, model: 9625)

25. Water distillation apparatus (Advantec, model: RFD240NA)

26. Magnetic stirrer (Pasorina Stirrer, model: CT-MINI)

27. 100 mL Beaker (AGC Iwaki, model: CTE33)

28. Fume hood (Oriental, model: TNV-STZ-1800HCS)

Software and datasets

1. Microsoft Excel

2. SF6 (version 5.6.0, Copyright^© 2005 Corona Electric) for spectrophotometer

Procedure

1. Cultivation of hydrogenase-expressing bacteria

   1. In this protocol, a recombinant E. coli encoding hydA, hydE, hydF, and hydG genes for hydrogenase expression was utilized as a H₂-producing model constructed in our laboratory as reported in Honda et al. (2016) and Kosem et al. (2023).

      Note: This protocol can be applied to determine the efficiency of other hydrogenase-producing bacteria, as reported in Benoit et al. (2020) and Kosem et al. (2024).

   2. Aerobically pre-culture the recombinant E. coli in a 500 mL Erlenmeyer flask with 200 mL of LB broth supplemented with 100 μg/mL of ampicillin sodium, 40 μg/mL of streptomycin sulfate, 0.5% (w/v) of glucose, 250 μg/mL of ferric ammonium citrate, and 100 mM MOPS/NaOH pH 7.4. Incubate the pre-culture flask at 37 °C placed on a shaking incubator at 120 rpm until the cell density reaches an optical density of 0.4 at OD₆₀₀, monitored by a spectrophotometer using a 1 mL cell suspension in a disposable cuvette. Then, transfer the 200 mL pre-cultured E. coli suspension into a 250 mL centrifuge bottle inside an anaerobic glove box supplemented with 2 mM L-cysteine (50 mg), 20 mM sodium fumarate (4 mL of 1 M stock solution), and 1 mM IPTG (0.2 mL of 1 M stock solution), and further incubate for 18 h in the glove box for [FeFe]-hydrogenase expression (Figure 2A).

      
      

      
      Figure 2. Experimental process of MV²⁺-dependent hydrogenase activity




2. Cell harvesting and preparation

   1. In the glove box, transfer 200 mL of cell suspension cultivated in LB medium from the Erlenmeyer flask to a centrifuge bottle with a silicone cap to preserve an anaerobic environment.

   2. Centrifuge the bottle at 2,000× g for 10 min, discard the supernatant, and collect cell pellet.

   3. Wash the cell pellet once with 5 mL of 0.9% NaCl solution and centrifuge at 2,000× g for 10 min.

   4. To prepare cell suspension for experiments, resuspend the washed cell pellet in 5 mL of 0.9% NaCl solution (Figure 2A).

   5. To standardize the protocol, the concentration of cells used in the reaction is based on total protein contents measured by Pierce^TM BCA Protein Assay kit in a 96-well plate.

      Note: The protocol can be modified when different bacterial strains or biological samples are used.




3. Hydrogenase activity assay

   1. Degas all chemical reagents with pure N₂ for 5 min and place them in the anaerobic glove box before use.

   2. Prepare the following reagents as shown in Table 1 and Figure 2B.

      
      

      Table 1. Reaction mixture preparation of MV^•+-hydrogenase activity assay

      Reagents	Applied volume	Final concentration
      1 M Tris-HCl pH 7	0.2 mL	100 mM
      10 mg/mL protein of cell suspension or extract	0.2 mL	1 mg/mL
      Sterile deionized water	1.4 mL
      50 mM MV2+ in 250 mM Na2S2O4 (reduced MV•+ solution)	0.2 mL	5 mM
      Total	2 mL

      
      Notes:

      1. The reaction mixture is prepared in a 10 mL glass vial and sealed with a rubber stopper and aluminum cap inside the glove box.

      2. The protein concentration can be adjusted in different samples.

      3. In case of a negative control, the same volume of sterile water is added instead of cell suspension or extracts.

      4. The reduced MV^•+ solution is prepared in a separate vial and sealed with a rubber stopper and aluminum cap inside the glove box.

   3. Take the reaction vial and the MV^•+ vial out of the glove box.

   4. Purge the contaminated gases in each vial with pure N₂ for 5 min.

   5. Initiate the reaction by adding 0.2 mL of 50 mM MV²⁺/Na₂S₂O₄ solution into the reaction vial using a 1 mL syringe with needle No. 27 G × 3/4" and further incubate in a shaking water bath at 100 rpm and 37 °C.

      Note: The reaction mixture turns dark blue after adding MV²⁺/Na₂S₂O₄ solution into the reaction vial.

   6. Every 20 min after incubation, terminate the reaction vial by adding 0.1 mL of 100% TCA (concentration of the commercial stock solution) using a 1 mL syringe with needle No. 27 G × 3/4".

   7. Sample 1 mL of H₂ produced in a headspace of the reaction vial using a 1 mL gas tight syringe and vertically inject into a gas chromatograph for analysis. Here, a GC-8A gas chromatograph equipped with a thermal conductive detector and an integrator C-R6A chromatopac was used. The produced gas goes through molecular sieve 5A beads packed in a stainless steel column (2 m length × 3 mm diameter) with a carrier gas of argon. Operating parameters are shown in Table 2.

      
      

      Table 2. Chromatographic operating parameters

      Parameters	Units
      Pressure of carrier gas, Ar	100 kPa
      Injection volume	1 mL
      Detector temperature	50 °C
      Injection temperature	50 °C
      Column temperature	50 °C
      Detector current	60 mA

Data analysis

1. Quantification of H₂:

   The accurate amount of H₂ produced from the reaction is calculated from a calibration curve between H₂ concentration vs. peak area obtained from the chromatogram of GC analysis (Figure 3).

   
   

   
   Figure 3. Calibration plot of standard H₂ vs. peak area by gas chromatography. According to the calibration curve, the amount of H₂ can be calculated from the following equation: H₂ (mol) = (Peak area – Intercept)/Slope.

   
   

2. Calculation of hydrogenase activity

   
   

          (1)

   
   

   In which the amount of H₂ (μmol) in the headspace (Figure 4A) is measured at each time point and plotted to obtain the H₂ production rate (Figure 4B).

   Time (minutes or hours) is the incubation time of the reaction.

   Note: Normally it takes 1–2 h for the incubation period. The amount of H₂ production rate is presented in μmol per min.

   C_protein (mg/mL) is the final concentration of protein in the reaction mixture.

   V_reaction (mL) is the total volume of the reaction mixture.

   
   

   
   Figure 4. Methyl viologen (MV^•+)-dependent hydrogenase activity assay. (A) Reaction vial containing the liquid phase of reaction mixture and the gas phase of H₂ produced in a headspace. (B) H₂ production rate is calculated from a kinetic plot between the amount of H₂ in the y-axis vs. time in the x-axis. Hydrogenase activity is calculated from equation (1).

Validation of protocol

This protocol was validated in Kosem et al. (2023). Applied Catalysis A: General, DOI: 10.1016/j.apcata.2022.119019.

General notes and troubleshooting

1. To preserve the biological function of O₂-sensitive enzymes, biological samples containing hydrogenase must be protected from air. Therefore, the whole process of cell preparation and experiments must be performed under anaerobic environment or in the glovebox.

2. Kinetic parameters of different hydrogenases from various biological sources can be analyzed by varying the concentrations of methyl viologen as a substrate according to the Michaelis-Menten equation: , where v is velocity, V_max is the maximum velocity, K_m is the Michaelis constant, and [S] is the concentration of the substrate (methyl viologen).

Acknowledgments

I would like to thank Prof. Tatsumi Ishihara for his supervision on my previous research, which originally validated this protocol in Kosem et al. (2023) that was funded by a Grant-in-Aid for Specially Promoted Research (No. 21K18213) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan through the Japan Society for the Promotion of Science. I would also like to acknowledge the Department of Applied Chemistry, Faculty of Engineering, Kyushu University, and the International Institute for Carbon-Neutral Energy Research (I²CNER), Kyushu University for research facilities and instruments. I am thankful to Dr. Yuki Honda from whom I adapted this protocol.

Competing interests

The authors declare no competing interests.

References

1. Benoit, S. L., Maier, R. J., Sawers, R. G. and Greening, C. (2020). Molecular Hydrogen Metabolism: a Widespread Trait of Pathogenic Bacteria and Protists. Microbiol. Mol. Biol. Rev. 84(1): e00092-19.

2. Broderick, J. B., Byer, A. S., Duschene, K. S., Duffus, B. R., Betz, J. N., Shepard, E. M. and Peters, J. W. (2014). H-Cluster assembly during maturation of the [FeFe]-hydrogenase. J. Biol. Inorg. Chem. 19(6): 747–757.
3. Honda, Y., Hagiwara, H., Ida, S. and Ishihara, T. (2016). Application to Photocatalytic H₂ Production of a Whole‐Cell Reaction by Recombinant Escherichia coli Cells Expressing [FeFe]‐Hydrogenase and Maturases Genes. Angew. Chem. Int. Ed. 55(28): 8045–8048.
4. Kosem, N., Watanabe, M., Song, J. T., Takagaki, A. and Ishihara, T. (2023). A comprehensive study on rational biocatalysts and individual components of photobiocatalytic H₂ production systems. Appl. Catal. A: Gen. 651: 119019.
5. Kosem, N., Shen, X.-F., Ohsaki, Y., Watanabe, M., Song, J. T. and Ishihara, T. (2024). Photobiocatalytic conversion of solar energy to NH₃ from N₂ and H₂O under ambient condition. Appl. Catal. B: Environ. 342: 123431.
6. Lubitz, W., Ogata, H., Rüdiger, O. and Reijerse, E. (2014). Hydrogenases. Chem. Rev. 114(8): 4081–4148.
7. Ogo, S., Kishima, T., Yatabe, T., Miyazawa, K., Yamasaki, R., Matsumoto, T., Ando, T., Kikkawa, M., Isegawa, M., Yoon, K. S., et al. (2020). [NiFe], [FeFe], and [Fe] hydrogenase models from isomers. Sci. Adv. 6(24): eaaz8181.
8. Orgill, J. J., Chen, C., Schirmer, C. R., Anderson, J. L. and Lewis, R. S. (2015). Prediction of methyl viologen redox states for biological applications. Biochem. Eng. J. 94: 15–21.
9. Tard, C. and Pickett, C. J. (2009). Structural and Functional Analogues of the Active Sites of the [Fe]-, [NiFe]-, and [FeFe]-Hydrogenases. Chem. Rev. 109(6): 2245–2274.
10. Xuan, J., He, L., Wen, W. and Feng, Y. (2023). Hydrogenase and Nitrogenase: Key Catalysts in Biohydrogen Production. Molecules 28(3): 1392.

Supplementary information

The following supporting information can be downloaded here:

1. Figure S1. Catalytic sites of different hydrogenases.
2. Guideline 1. Safety procedure in handling HCl.