D. baculatum NiFeSe-hydrogenase was used together with Eosin Y, an organic
dye, for the photocatalytic evolution of H 2 using triethanol amine as sacrificial
electron donor [48]. After optimizing the concentration of hydrogenase (4.4 nM),
Eosin Y (0.44 mM) and the pH = 7, the system generated 0.5 lmol of H 2 per hour
under anaerobic conditions using 100 mWÁcm
−2 power light at AM 1.5 visible
radiation. The oxygen tolerance of the homogeneous photobiocatalytic system was
evaluated, keeping 10% of the initial H 2 production at 21% O 2 concentration, and
the natural one is in air. These results using Eosin Y were compared with the ones
using [Ru(pby) 3 ]
2+ . The comparison in strict anaerobic conditions was favourable
to the ruthenium complex, as it produced almost double H 2 in the same conditions.
However, the former was much more sensitive to O 2 presence: at 5% O 2 the
Eosin Y system kept 80% of H 2 production, whereas the ruthenium complex only
kept 12% of H 2 production [48]. Another light harvester consisting of a heptazine
carbon nitride polymer (CN x ) was tested in combination with either the D. baculatum NiFeSe-hydrogenase or a biomimetic Ni–P photocatalyst in solution, comparing the activity of the biological and the inorganic catalysts [49]. In these cases,
the sacrificial electron donor used was EDTA 0.1 M. Under the optimized conditions (pH 6, anaerobic, 1 sun irradiation including UV radiation k > 300 nm) the
hydrogenase (16.7 nM) with CN x (5 mg) yielded 55 ± 5 lmol H 2 Â (g CN x Â
h)
−1 . The production was linear during at least 4 h before showing a rate decrease.
On the other hand, the biomimetic compound was able to produce H 2 , although in
rates tenfold lower than the hydrogenase system. This work was extended by
immobilizing the hydrogenase and the CN x on the surface of anatase TiO 2
nanoparticles, smaller than 10 nm [50]. Addition of TiO 2 to the system yielded
1.46 lmol H 2 using pH 6, EDTA 0.1 M, 5 mg of TiO 2 and 50 pmol of
NiFeSe-hydrogenase. The TiO 2 -based system showed a lower initial activity than
the direct combination of CN x with the hydrogenase, although it may be stable
under working conditions a longer time, as the experiments were run up to 72 h
instead of cutting after 50 h.
Genetic engineering has also been tested to improve the assembly of hybrid
biologic-inorganic catalysts for H 2 production. One example is the re-engineering
of an E. coli NiFe-hydrogenase for its selective surface through its distal [4Fe4S]
cluster attachment to silver nanoclusters stabilized in aqueous solution with
approximately m5kDa polymethacrylic acid (PMAA) [51]. Whereas silver managed to harvest some energy from light, the system performed much better when
including TiO 2 P-25 particles in the mixture. The best mutant (Y’222C) with Ag
and PMAA yielded 7.2 lmol H 2 Â h
−1 , the same mutant mixed with Ag, PMAA
and TiO 2 particles yielded 134 lmol H 2 Â h
−1 . The Y’222C mutation consisted on
exchanging tyrosine 222 for a cysteine. Other mutants showed lower or no activity
at all.
Some sulphides are interesting materials as semiconductors, mainly those on
which the bandgap is close to 2–2.5 eV and therefore absorb visible light irradiation. One example of suitable sulphide is indium sulphide, In 2 S 3 . This n-type
semiconductor material has a 2.2–2.3 eV bandgap and can be prepared solvothermally in such a way that provides a highly porous surface suitable to host
Biological Production of Hydrogen
255
dye, for the photocatalytic evolution of H 2 using triethanol amine as sacrificial
electron donor [48]. After optimizing the concentration of hydrogenase (4.4 nM),
Eosin Y (0.44 mM) and the pH = 7, the system generated 0.5 lmol of H 2 per hour
under anaerobic conditions using 100 mWÁcm
−2 power light at AM 1.5 visible
radiation. The oxygen tolerance of the homogeneous photobiocatalytic system was
evaluated, keeping 10% of the initial H 2 production at 21% O 2 concentration, and
the natural one is in air. These results using Eosin Y were compared with the ones
using [Ru(pby) 3 ]
2+ . The comparison in strict anaerobic conditions was favourable
to the ruthenium complex, as it produced almost double H 2 in the same conditions.
However, the former was much more sensitive to O 2 presence: at 5% O 2 the
Eosin Y system kept 80% of H 2 production, whereas the ruthenium complex only
kept 12% of H 2 production [48]. Another light harvester consisting of a heptazine
carbon nitride polymer (CN x ) was tested in combination with either the D. baculatum NiFeSe-hydrogenase or a biomimetic Ni–P photocatalyst in solution, comparing the activity of the biological and the inorganic catalysts [49]. In these cases,
the sacrificial electron donor used was EDTA 0.1 M. Under the optimized conditions (pH 6, anaerobic, 1 sun irradiation including UV radiation k > 300 nm) the
hydrogenase (16.7 nM) with CN x (5 mg) yielded 55 ± 5 lmol H 2 Â (g CN x Â
h)
−1 . The production was linear during at least 4 h before showing a rate decrease.
On the other hand, the biomimetic compound was able to produce H 2 , although in
rates tenfold lower than the hydrogenase system. This work was extended by
immobilizing the hydrogenase and the CN x on the surface of anatase TiO 2
nanoparticles, smaller than 10 nm [50]. Addition of TiO 2 to the system yielded
1.46 lmol H 2 using pH 6, EDTA 0.1 M, 5 mg of TiO 2 and 50 pmol of
NiFeSe-hydrogenase. The TiO 2 -based system showed a lower initial activity than
the direct combination of CN x with the hydrogenase, although it may be stable
under working conditions a longer time, as the experiments were run up to 72 h
instead of cutting after 50 h.
Genetic engineering has also been tested to improve the assembly of hybrid
biologic-inorganic catalysts for H 2 production. One example is the re-engineering
of an E. coli NiFe-hydrogenase for its selective surface through its distal [4Fe4S]
cluster attachment to silver nanoclusters stabilized in aqueous solution with
approximately m5kDa polymethacrylic acid (PMAA) [51]. Whereas silver managed to harvest some energy from light, the system performed much better when
including TiO 2 P-25 particles in the mixture. The best mutant (Y’222C) with Ag
and PMAA yielded 7.2 lmol H 2 Â h
−1 , the same mutant mixed with Ag, PMAA
and TiO 2 particles yielded 134 lmol H 2 Â h
−1 . The Y’222C mutation consisted on
exchanging tyrosine 222 for a cysteine. Other mutants showed lower or no activity
at all.
Some sulphides are interesting materials as semiconductors, mainly those on
which the bandgap is close to 2–2.5 eV and therefore absorb visible light irradiation. One example of suitable sulphide is indium sulphide, In 2 S 3 . This n-type
semiconductor material has a 2.2–2.3 eV bandgap and can be prepared solvothermally in such a way that provides a highly porous surface suitable to host
Biological Production of Hydrogen
255