1. Daniel Sobczynski
SURE Program
Maroney Group
Chrisjoe A. Joseph, Project Leader
Julius Campecino, Graduate Student Mentor

2. Overview
 Why Hydrogen?
 Hydrogenase Specifics
 Isolation of the Ni-Fe Hydrogenase
 Hydrogen Production
 Hydrogen Utilization
 Benefits of the Project
 Future Studies

3. Why Hydrogen?
 Can be used for
electrochemical cells
 Can be formed by
biological catalysts
 Clean emissions
Image credit: www.autopten.com

4. Hydrogenase Specifics
H2 2H+
2e-+
H2 D2O+ HD + HDO
o-H2 p-H2
Image credit: Prof. Michael Maroney

5. Growing and Harvesting the cells
 Thiocapsa roseopersicina
 Phototropic
 Marine bacteria
 Withstands oxygen and
non-oxygen
environments
Centrifuge
Cell paste, stored at
-20˚C
Image credit: Julius Campecino

6. Hydrogenase Purification
supernatant
DEAE: Anion-exchange resin
filtrate DEAE with bound proteins
450mM NaCl + 20mM
Tris, pH 7.5
DEAE
clean DEAE
filtrate
filtrate butyl sepharose column*
butyl sepharose column
1mM TRIS buffer, pH 7.5
Q sepharose column
20mM TRIS buffer, pH 7.5
Q sepharose column
50mM MES buffer, pH 5.5
Q sepharose column
TRIS buffer, pH 7.5
pure hydrogenase
Dissolve 15g acetone powder in water
100 grams of ammonium sulfate,
butyl sepharose resin
300g cell paste
acetone powder
pellet
1M NaCl + 20mM Tris,
pH 7.5
native gel electrophoresis
(9% gel)

7. Electrocatalyst Design
Images credit: Prof. Dhandapani Venkataraman

8. Production of Hydrogen
O2Ti
O2Ti
O2Ti
Image credit:
Hydrogenase picture: Prof. Dhandapani Venkataraman

9. Hydrogen Utilization
Image credit:
Hydrogenase picture: Prof. Dhandapani Venkataraman

10. Additional Modification Site
Proximal Medial Distal
Images credit: Prof. Dhandapani Venkataraman

11. Benefits of the Project
 Versatility of the project
 Alternative to hydrogen
storage issue
 Water as output, and
possibly input too
Image credit:
Water drop: http://www.biofeedbackcalifornia.org

12. Future Studies
 Optimizing hydrogen
production and
utilization
 Finding a way to better
produce hydrogenase
 Integrating the project
goals into applied fields
of science

13. Butyl Seph, Tris Chromatogram
Max UV: 2200 mAU
%B concentration: 0 – 100 %
Conductivity range: 100 – 0 mS/cm
Fractions collected: All
H2ase Butyl 2 062409:10_UV H2ase Butyl 2 062409:10_Cond H2ase Butyl 2 062409:10_Conc
H2ase Butyl 2 062409:10_Fractions
-500
0
500
1000
1500
2000
mAU
50 100 150 200 250 300 350 400 ml
X1 X2 X3 X4 X5 X6 X7 X8

14. Q-Seph, Tris Chromatogram
H2ase Long QSeph A load002:10_UV H2ase Long QSeph A load002:10_Cond H2ase Long QSeph A load002:10_Conc
H2ase Long QSeph A load002:10_Fractions
500
1000
1500
2000
2500
mAU
220.0 230.0 240.0 250.0 min
X1 X2 X3 X4
Max UV: 2600 mAU
%B concentration: 60 %
Conductivity range: 25 – 50 mS/cm
Fractions collected: X2 , X3

15. Q-Seph, MES Chromatogram
H2ase MES QSephB load B 071209C001:10_UV H2ase MES QSephB load B 071209C001:10_Cond
H2ase MES QSephB load B 071209C001:10_Conc H2ase MES QSephB load B 071209C001:10_Fractions
0
50
100
150
200
250
mAU
160 180 200 220 240 ml
Waste X3 X4 Waste
Max UV: 230 mAU
%B concentration: 17.5 – 60 %
Conductivity range: 17.5 – 50 mS/cm
Fractions collected: X3 , X4

16. Pure Hydrogenase, EPR Results

17. Buffer Calculations
450 mM NaCl, 20mM Tris buffer, pH = 7.5
For 1L of buffer:
(Mol. Wt.) x (# of Mol) = 58.443g/mol x .45 mol = 26.29935 g NaCl
M1 * V1 = M2 * V2  Solve for V2
20mM * 1L = 1M * V2
V 2=20 mL of 1M Tris