construction in a lead halide perovskite photocathode, producing a photocurrent of
−5 mA/cm
3 at 0 V versus RHE [58].
There are other oxides besides TiO 2 able to harvest photons and work as photocathodes, for instance, Cu 2 O and ZnO. The bandgap of ZnO is too high and
works only in the UV, whereas Cu 2 O has a suitable bandgap but is prone to
oxidation. However, their layer-by-layer combination allows harvesting of light
energy within the visible range and protection against corrosion, as well as hosting
hydrogenases to perform the hydrogen production [59]. The oxide layers were
deposited on the surface of a FTO electrode. First, Cu 2 O was deposited by alkaline
electrodeposition and then ZnO by the spin coating of precursors with further
annealing. On its top, a histidine-tagged hydrogenase from E. coli was drop-casted
for its physical adsorption during 15 min. The biocathode delivered 0.8 mA Â
cm
−2 upon illumination with 100 mWÁcm
−2 VIS LED light (1 Sun equivalent),
which is tenfold higher than the photocurrent provided by a hydrogenase-less
photocathode. The hydrogen produced was measured with a unisense H 2 external
sensor, delivering 0.68 nmol of H 2 after accumulation during 200 s. Such H 2
amount measured was compared to the charge measured in the circuit, rendering a
Faradaic efficiency of 1%.
Another set of photobiocathodes has recently been developed in the absence of
the classical semiconductors, by combining photosystem I (PSI) and hydrogenase
on the top of an electrode and connecting them with two redox polymers: one
Os-modified polymer for collecting the excited electrons from PSI and another
cobaltocene-derived polymer to connect the hydrogenase to the electrode [60]. The
key in this first proof-of-concept work was substituting a viologen-derived polymer
by the cobaltocene-derived polymer, which has a much lower redox potential and
therefore providing a larger driving force to push the catalytic cycle of the D. gigas
NiFe-hydrogenase towards H 2 production. A following work using layer-by-layer
controlled strategies to connect PSI and another hydrogenase, increasing the H 2
production, has been recently published [61].
3 Microbial Production of H 2
The use of microbial whole-cells as catalysts for H 2 production has obvious
advantages over the use of enzymes, which have a short lifetime and require
lengthy and expensive processes to be isolated. Microbial cells are much more
robust and are capable of growth and self-repair, and the corresponding bioprocesses are easily scaled up and provide an opportunity for the use of sustainable
substrates, such as sunlight or wastes. Several kinds of bioprocesses have been
explored to produce hydrogen biologically (biohydrogen, BioH 2 ), namely
bio-photolysis of water by algae, photo-fermentation by photosynthetic bacteria and
dark fermentation by anaerobic bacteria and archaea [62–65]. Here, we will focus
specifically on dark fermentation, which is regarded as the most promising
approach due to the simplicity of the process, ease of scale-up, high rates of BioH 2
258
M. Martins et al.
−5 mA/cm
3 at 0 V versus RHE [58].
There are other oxides besides TiO 2 able to harvest photons and work as photocathodes, for instance, Cu 2 O and ZnO. The bandgap of ZnO is too high and
works only in the UV, whereas Cu 2 O has a suitable bandgap but is prone to
oxidation. However, their layer-by-layer combination allows harvesting of light
energy within the visible range and protection against corrosion, as well as hosting
hydrogenases to perform the hydrogen production [59]. The oxide layers were
deposited on the surface of a FTO electrode. First, Cu 2 O was deposited by alkaline
electrodeposition and then ZnO by the spin coating of precursors with further
annealing. On its top, a histidine-tagged hydrogenase from E. coli was drop-casted
for its physical adsorption during 15 min. The biocathode delivered 0.8 mA Â
cm
−2 upon illumination with 100 mWÁcm
−2 VIS LED light (1 Sun equivalent),
which is tenfold higher than the photocurrent provided by a hydrogenase-less
photocathode. The hydrogen produced was measured with a unisense H 2 external
sensor, delivering 0.68 nmol of H 2 after accumulation during 200 s. Such H 2
amount measured was compared to the charge measured in the circuit, rendering a
Faradaic efficiency of 1%.
Another set of photobiocathodes has recently been developed in the absence of
the classical semiconductors, by combining photosystem I (PSI) and hydrogenase
on the top of an electrode and connecting them with two redox polymers: one
Os-modified polymer for collecting the excited electrons from PSI and another
cobaltocene-derived polymer to connect the hydrogenase to the electrode [60]. The
key in this first proof-of-concept work was substituting a viologen-derived polymer
by the cobaltocene-derived polymer, which has a much lower redox potential and
therefore providing a larger driving force to push the catalytic cycle of the D. gigas
NiFe-hydrogenase towards H 2 production. A following work using layer-by-layer
controlled strategies to connect PSI and another hydrogenase, increasing the H 2
production, has been recently published [61].
3 Microbial Production of H 2
The use of microbial whole-cells as catalysts for H 2 production has obvious
advantages over the use of enzymes, which have a short lifetime and require
lengthy and expensive processes to be isolated. Microbial cells are much more
robust and are capable of growth and self-repair, and the corresponding bioprocesses are easily scaled up and provide an opportunity for the use of sustainable
substrates, such as sunlight or wastes. Several kinds of bioprocesses have been
explored to produce hydrogen biologically (biohydrogen, BioH 2 ), namely
bio-photolysis of water by algae, photo-fermentation by photosynthetic bacteria and
dark fermentation by anaerobic bacteria and archaea [62–65]. Here, we will focus
specifically on dark fermentation, which is regarded as the most promising
approach due to the simplicity of the process, ease of scale-up, high rates of BioH 2
258
M. Martins et al.