hydrogen and oxygen when an appropriate amount of energy is applied. In order to
produce H 2 with minimal energy loss during the reduction of protons, catalysts are
a must. The most used catalyst for H 2 production from water is platinum, an
electrocatalyst that, when working at optimal conditions and absence of poisoning
agents, produces H 2 at high rates and negligible overpotential [1]. Unfortunately,
platinum is a non-abundant noble element (0.003 ppm of Pt on Earth’s crust) that
cannot become the solution for global problems, and thus alternative ways to reduce
protons to H 2 are intensely studied. As other metallic catalysts are not as efficient as
Pt for H 2 evolution, research has also focused on biocatalysts. In general, biocatalytic processes can be divided between those that employ purified enzymes and
those based on whole-cells. In this chapter, we review both types of biocatalytic
systems applied to H 2 production.
2 Enzymatic Production of H 2
Although some other redox metalloenzymes, such as nitrogenases, produce H 2 as a
by-product, hydrogenases are the only ones that specifically catalyze the reversible
reaction of H 2 production/uptake with almost no overpotential [2]. Hydrogenases
are classified according to the metal content of their active site (Fig. 1), thus they
either belong to the FeFe-hydrogenases, NiFe-hydrogenases or Fe-hydrogenases
groups [3]. Not all hydrogenases have the same ability for catalyzing H 2 -production. For instance, O 2 -tolerant membrane-bound NiFe-hydrogenases are catalytically biased towards to H 2 -oxidation and only are capable of H 2 -production at quite
acidic pH values [4]. On the other hand, FeFe-hydrogenases have very high turnover numbers for H 2 -production at neutral pH and opposite to NiFe-hydrogenases
they are hardly inhibited by product accumulation [5, 6]. However,
FeFe-hydrogenases are very sensitive to irreversible O 2 inhibition [7, 8] or
destruction of the active site by light [9], which may hamper practical applications.
2.1 Enzymatic Production of H 2 Driven by Reduced
Compounds
In an early work by Zorin and coworkers, it was reported for the first time that the
soluble NiFe-hydrogenase isolated from the H 2 -oxidizing bacterium Alcaligenes
eutrophus (currently named Ralstonia eutropha) was able to produce H 2 under air
or even under 100% O 2 atmosphere using NAD(P)H as electron donor [10]. More
recently, this activity under aerobic conditions has been explained in a combined
spectroscopic and computational study by the capability of NAD(P)H to transfer
simultaneously 2 electrons to the oxidized active site and form the active reduced
state [11]. However, the rates of H 2 production are very low even under anaerobic
conditions, up to 0.045 µmol  min
−1
 mg
−1 [10], because the standard redox
potential (E
0 ) of NAD(P)H is higher than that of H 2 . Therefore, the process is only
248
M. Martins et al.
produce H 2 with minimal energy loss during the reduction of protons, catalysts are
a must. The most used catalyst for H 2 production from water is platinum, an
electrocatalyst that, when working at optimal conditions and absence of poisoning
agents, produces H 2 at high rates and negligible overpotential [1]. Unfortunately,
platinum is a non-abundant noble element (0.003 ppm of Pt on Earth’s crust) that
cannot become the solution for global problems, and thus alternative ways to reduce
protons to H 2 are intensely studied. As other metallic catalysts are not as efficient as
Pt for H 2 evolution, research has also focused on biocatalysts. In general, biocatalytic processes can be divided between those that employ purified enzymes and
those based on whole-cells. In this chapter, we review both types of biocatalytic
systems applied to H 2 production.
2 Enzymatic Production of H 2
Although some other redox metalloenzymes, such as nitrogenases, produce H 2 as a
by-product, hydrogenases are the only ones that specifically catalyze the reversible
reaction of H 2 production/uptake with almost no overpotential [2]. Hydrogenases
are classified according to the metal content of their active site (Fig. 1), thus they
either belong to the FeFe-hydrogenases, NiFe-hydrogenases or Fe-hydrogenases
groups [3]. Not all hydrogenases have the same ability for catalyzing H 2 -production. For instance, O 2 -tolerant membrane-bound NiFe-hydrogenases are catalytically biased towards to H 2 -oxidation and only are capable of H 2 -production at quite
acidic pH values [4]. On the other hand, FeFe-hydrogenases have very high turnover numbers for H 2 -production at neutral pH and opposite to NiFe-hydrogenases
they are hardly inhibited by product accumulation [5, 6]. However,
FeFe-hydrogenases are very sensitive to irreversible O 2 inhibition [7, 8] or
destruction of the active site by light [9], which may hamper practical applications.
2.1 Enzymatic Production of H 2 Driven by Reduced
Compounds
In an early work by Zorin and coworkers, it was reported for the first time that the
soluble NiFe-hydrogenase isolated from the H 2 -oxidizing bacterium Alcaligenes
eutrophus (currently named Ralstonia eutropha) was able to produce H 2 under air
or even under 100% O 2 atmosphere using NAD(P)H as electron donor [10]. More
recently, this activity under aerobic conditions has been explained in a combined
spectroscopic and computational study by the capability of NAD(P)H to transfer
simultaneously 2 electrons to the oxidized active site and form the active reduced
state [11]. However, the rates of H 2 production are very low even under anaerobic
conditions, up to 0.045 µmol  min
−1
 mg
−1 [10], because the standard redox
potential (E
0 ) of NAD(P)H is higher than that of H 2 . Therefore, the process is only
248
M. Martins et al.