nanocatalysts. An example of micron-size In 2 S 3 particles with an average pore
diameter of 16 nm hosting D. vulgaris Hildenborough NiFeSe-hydrogenase and
producing H 2 upon only visible light illumination, using sulphide anions as sacrificial electron donor has been reported [52]. The authors optimized the incubation
time to 6 h; in that case, the In 2 S 3 -hydrogenase complex produced about 800 lmol
H 2 Â mg (hydrogenase)
−1
 min
−1 . Compared to the activity of the system measured with MV instead of illumination, the later one kept 89% of the activity
measured chemically. The turnover frequency (TOF) of the hydrogenase under
photobioproduction was 986 s
−1 . These values match the ones obtained for another
sulphide, cadmium sulphide capped with mercaptopropionic acid (CdS-MPA), a
few years earlier [53]. In that case, the hydrogenase selected was the
FeFe-hydrogenase I from C. acetobutylicum, which provided a maximum 900 s
−1
turnover frequency for H 2 conversion using ascorbic acid as electron donor.
CdS is a very good semiconductor from this perspective, as its bandgap is
2.4 eV, and its conduction band is well aligned with the 2H
+
/H 2 redox couple. One
challenge to use sulphides to provide high-energy electrons to hydrogenases in an
aqueous solution is its lack of stability, needing capping agents to stabilize them in
the aqueous phase. The capping agents must attract the hydrogenase in an oriented
fashion to catalyze the H 2 production successfully. Mercaptoacids are suitable for
this purpose, as its thiol group can be attached to the sulphide surface via either a
M-S bond or a S–S bridge; and the carboxylic functionalities allow oriented
electrostatic orientation of C. acetobutylicum FeFe-hydrogenase with the distal
[4Fe4S] cluster facing the semiconductor surface. The influence of the mercaptocarboxylic derivative’s chain length was studied [54], finding that the shorter the
chain the more efficient was the electron transfer between the semiconductor and
the enzyme. The most efficient capping agent was 2-mercaptoacetic acid, and an
exponential decay upon longer chain was observed.
2.3.2 Heterogeneous Photobiocatalytic Production of H 2
The combination of the hydrogenases for photoproduction of H 2 with solid supports
has interesting advantages. Firstly, the sacrificial electron donor needed in homogeneous photobiocatalytic systems may be substituted and provided by the electrode or the photo-harvesting material. Secondly, a successful immobilization of the
biocatalysts provides higher stability and longer operational periods. Moreover, it
gives flexibility for the design of photobioelectrochemical cells, facilitates flow
systems and allows reutilization.
The most popular and material used for photon harvesting is TiO 2 . A very
interesting work anodizes a TiO 2 film to generate a nanosized tubular material that
can be loaded with hydrogenases [55]. After a careful selection of anodization
conditions to form the tubular TiO 2 and its modification with dithiothreitol,
hydrogenase from P. furiosus was linked via disulphide bonds. Further, the electrode protection was added by electropolymerization of polypyrrole. The resulting
electrode produced 140 lmol  cm
−2
 h
−1 of H 2 , upon illumination with 75
mW/cm
2 light power and using 3.66 units of hydrogenase.
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M. Martins et al.
diameter of 16 nm hosting D. vulgaris Hildenborough NiFeSe-hydrogenase and
producing H 2 upon only visible light illumination, using sulphide anions as sacrificial electron donor has been reported [52]. The authors optimized the incubation
time to 6 h; in that case, the In 2 S 3 -hydrogenase complex produced about 800 lmol
H 2 Â mg (hydrogenase)
−1
 min
−1 . Compared to the activity of the system measured with MV instead of illumination, the later one kept 89% of the activity
measured chemically. The turnover frequency (TOF) of the hydrogenase under
photobioproduction was 986 s
−1 . These values match the ones obtained for another
sulphide, cadmium sulphide capped with mercaptopropionic acid (CdS-MPA), a
few years earlier [53]. In that case, the hydrogenase selected was the
FeFe-hydrogenase I from C. acetobutylicum, which provided a maximum 900 s
−1
turnover frequency for H 2 conversion using ascorbic acid as electron donor.
CdS is a very good semiconductor from this perspective, as its bandgap is
2.4 eV, and its conduction band is well aligned with the 2H
+
/H 2 redox couple. One
challenge to use sulphides to provide high-energy electrons to hydrogenases in an
aqueous solution is its lack of stability, needing capping agents to stabilize them in
the aqueous phase. The capping agents must attract the hydrogenase in an oriented
fashion to catalyze the H 2 production successfully. Mercaptoacids are suitable for
this purpose, as its thiol group can be attached to the sulphide surface via either a
M-S bond or a S–S bridge; and the carboxylic functionalities allow oriented
electrostatic orientation of C. acetobutylicum FeFe-hydrogenase with the distal
[4Fe4S] cluster facing the semiconductor surface. The influence of the mercaptocarboxylic derivative’s chain length was studied [54], finding that the shorter the
chain the more efficient was the electron transfer between the semiconductor and
the enzyme. The most efficient capping agent was 2-mercaptoacetic acid, and an
exponential decay upon longer chain was observed.
2.3.2 Heterogeneous Photobiocatalytic Production of H 2
The combination of the hydrogenases for photoproduction of H 2 with solid supports
has interesting advantages. Firstly, the sacrificial electron donor needed in homogeneous photobiocatalytic systems may be substituted and provided by the electrode or the photo-harvesting material. Secondly, a successful immobilization of the
biocatalysts provides higher stability and longer operational periods. Moreover, it
gives flexibility for the design of photobioelectrochemical cells, facilitates flow
systems and allows reutilization.
The most popular and material used for photon harvesting is TiO 2 . A very
interesting work anodizes a TiO 2 film to generate a nanosized tubular material that
can be loaded with hydrogenases [55]. After a careful selection of anodization
conditions to form the tubular TiO 2 and its modification with dithiothreitol,
hydrogenase from P. furiosus was linked via disulphide bonds. Further, the electrode protection was added by electropolymerization of polypyrrole. The resulting
electrode produced 140 lmol  cm
−2
 h
−1 of H 2 , upon illumination with 75
mW/cm
2 light power and using 3.66 units of hydrogenase.
256
M. Martins et al.