286 Marine Macro- and Microalgae: An Overview
Cyanobacterial [NiFe]-hydrogenases contain a nickel atom at their active site, linked to a Fe(CN) 2 CO
molecule (Eroglu and Melis 2011). Two types of [NiFe]-hydrogenases are present in cyanobacteria: an
uptake [NiFe]-hydrogenase, encoded by the hup genes, which recycles hydrogen recovering energy-rich
electrons and is present in almost all nitrogen-fixing cyanobacteria (Khetkorn et al. 2017), and a multisubunit bidirectional [NiFe]-hydrogenase, encoded by the hoxFUYH genes (Fig. 1B). The bidirectional
enzyme was thought to be peripherally associated with the cytoplasmatic membrane and to accept electrons
from NAD(P)H to produce hydrogen (Eroglu and Melis 2011; Srirangan et al. 2011). However, further
research indicated that Hox subunits are associated to thylakoids and both ferredoxin and flavodoxin have
been suggested as the main direct electron donors for this hydrogenase (Khanna et al. 2015). Hydrogen
synthesis by this enzyme seems to be sustained in the dark, under fermentative conditions, when pyruvate
may be used as electron source through pyruvate-ferredoxin oxidoreductase (PFOR). In contrast, only a
short bust of hydrogen is detected upon illumination, followed by hydrogen consumption. These enzymes
are also inhibited by oxygen, but in a reversible manner (McIntosh et al. 2011).
Nitrogen-fixing, filamentous cyanobacteria are able to synthesize hydrogen through a [MoFe]nitrogenase, localized in specialized cells named heterocysts. This enzyme contains two metal clusters,
the [8Fe-7S] P-cluster and the [1Mo-7Fe-9S-1X-homocitrate] FeMo cofactor, where the homocitrate
molecule is required for efficient nitrogen fixation (Mayer et al. 2002). The nitrogenase catalyzes the
following reaction in a unidirectional way: N 2 + 8e
–
+ 8H
+
+ 16ATP à 2NH 3 + H 2 + 16ADP + 16P i .
In the absence of molecular nitrogen, the enzyme exclusively catalyzes hydrogen production with a
higher yield. Although the nitrogenase is also oxygen-sensitive, this is not a problem for hydrogen
production since heterocysts keep an internal microoxic environment. These cells do not produce oxygen
because they lack PSII and are surrounded by a thick cell wall that restricts oxygen diffusion (Eroglu and
Melis 2011; Masukawa et al. 2012). The reaction catalyzed by the nitrogenase requires a high energy
expenditure in the form of ATP, which is achieved via cyclic photophosphorylation. The electrons are
provided by ferredoxin, which in turn requires PSI illumination. The source of both reducing power
and energy depends ultimately on the photosynthetic activity of neighbouring vegetative cells, which
synthesize carbohydrates via the photosynthetic electron transport chain and the Calvin cycle. These
carbohydrates are exported to heteterocysts and used as a source of NAD(P)H and ATP. NAD(P)H feeds
electrons into the plastoquinone pool via NADH dehydrogenase (Ndh1), the reducing equivalents being
subsequently directed to PSI, and eventually to nitrogenase (Skizim et al. 2012) (Fig. 1C).
Hydrogen production mechanisms
Green algae and cyanobacteria from different genus have shown diverse hydrogen production yields
(Meuser et al. 2009; Eroglu and Melis 2011; Khetkorn et al. 2012a; Kothari et al. 2012). Hydrogen
bioproduction by C. reinhardtii has been the most studied example among all photosynthetic organisms.
This unicellular alga has a fairly high hydrogen synthesis yield compared to other green algae, in its wildtype version (Timmins et al. 2009a); it exhibits also clear advantages for genetic-engineering studies,
as previously mentioned. When C. reinhardtii cultures are sealed to induce anaerobiosis and exposed
to light, only a transient hydrogen pulse (60–90 s) is detected, since the oxygen release concomitant to
water oxidation at PSII immediately poisons the [FeFe]-hydrogenase (Ghirardi et al. 1997). A sustained
(four days) hydrogen production was achieved by Melis et al. (2000) with C. reinhardtii under sulfur
deprivation conditions. The changes occurring after the onset of the sulfur-starvation phase have been
thoroughly studied from a transcriptomic and metabolomic perspective (Timmins et al. 2009b; Doebbe
et al. 2010; Nguyen et al. 2011). The sequence of events begins with an aerobic phase, characterized
by the accumulation of starch and triacylglycerides (Zhang et al. 2002; Timmins et al. 2009b). This
is followed by a rapid degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) that
impairs Calvin-cycle function, and thus, eliminates one of the main electron sinks of the photosynthetic
electron-transport chain. In parallel, the nutrient deficiency stress hampers new protein synthesis,
which is crucial for repair of the PSII protein D1, so, the number of active PSII centers decreases to
5–10% of normal levels (Zhang et al. 2002). Since the mitochondria activity is essentially unaffected,
the photosynthesis/respiration rate drops dramatically, the cells turn to anaerobiosis and switch on the
Cyanobacterial [NiFe]-hydrogenases contain a nickel atom at their active site, linked to a Fe(CN) 2 CO
molecule (Eroglu and Melis 2011). Two types of [NiFe]-hydrogenases are present in cyanobacteria: an
uptake [NiFe]-hydrogenase, encoded by the hup genes, which recycles hydrogen recovering energy-rich
electrons and is present in almost all nitrogen-fixing cyanobacteria (Khetkorn et al. 2017), and a multisubunit bidirectional [NiFe]-hydrogenase, encoded by the hoxFUYH genes (Fig. 1B). The bidirectional
enzyme was thought to be peripherally associated with the cytoplasmatic membrane and to accept electrons
from NAD(P)H to produce hydrogen (Eroglu and Melis 2011; Srirangan et al. 2011). However, further
research indicated that Hox subunits are associated to thylakoids and both ferredoxin and flavodoxin have
been suggested as the main direct electron donors for this hydrogenase (Khanna et al. 2015). Hydrogen
synthesis by this enzyme seems to be sustained in the dark, under fermentative conditions, when pyruvate
may be used as electron source through pyruvate-ferredoxin oxidoreductase (PFOR). In contrast, only a
short bust of hydrogen is detected upon illumination, followed by hydrogen consumption. These enzymes
are also inhibited by oxygen, but in a reversible manner (McIntosh et al. 2011).
Nitrogen-fixing, filamentous cyanobacteria are able to synthesize hydrogen through a [MoFe]nitrogenase, localized in specialized cells named heterocysts. This enzyme contains two metal clusters,
the [8Fe-7S] P-cluster and the [1Mo-7Fe-9S-1X-homocitrate] FeMo cofactor, where the homocitrate
molecule is required for efficient nitrogen fixation (Mayer et al. 2002). The nitrogenase catalyzes the
following reaction in a unidirectional way: N 2 + 8e
–
+ 8H
+
+ 16ATP à 2NH 3 + H 2 + 16ADP + 16P i .
In the absence of molecular nitrogen, the enzyme exclusively catalyzes hydrogen production with a
higher yield. Although the nitrogenase is also oxygen-sensitive, this is not a problem for hydrogen
production since heterocysts keep an internal microoxic environment. These cells do not produce oxygen
because they lack PSII and are surrounded by a thick cell wall that restricts oxygen diffusion (Eroglu and
Melis 2011; Masukawa et al. 2012). The reaction catalyzed by the nitrogenase requires a high energy
expenditure in the form of ATP, which is achieved via cyclic photophosphorylation. The electrons are
provided by ferredoxin, which in turn requires PSI illumination. The source of both reducing power
and energy depends ultimately on the photosynthetic activity of neighbouring vegetative cells, which
synthesize carbohydrates via the photosynthetic electron transport chain and the Calvin cycle. These
carbohydrates are exported to heteterocysts and used as a source of NAD(P)H and ATP. NAD(P)H feeds
electrons into the plastoquinone pool via NADH dehydrogenase (Ndh1), the reducing equivalents being
subsequently directed to PSI, and eventually to nitrogenase (Skizim et al. 2012) (Fig. 1C).
Hydrogen production mechanisms
Green algae and cyanobacteria from different genus have shown diverse hydrogen production yields
(Meuser et al. 2009; Eroglu and Melis 2011; Khetkorn et al. 2012a; Kothari et al. 2012). Hydrogen
bioproduction by C. reinhardtii has been the most studied example among all photosynthetic organisms.
This unicellular alga has a fairly high hydrogen synthesis yield compared to other green algae, in its wildtype version (Timmins et al. 2009a); it exhibits also clear advantages for genetic-engineering studies,
as previously mentioned. When C. reinhardtii cultures are sealed to induce anaerobiosis and exposed
to light, only a transient hydrogen pulse (60–90 s) is detected, since the oxygen release concomitant to
water oxidation at PSII immediately poisons the [FeFe]-hydrogenase (Ghirardi et al. 1997). A sustained
(four days) hydrogen production was achieved by Melis et al. (2000) with C. reinhardtii under sulfur
deprivation conditions. The changes occurring after the onset of the sulfur-starvation phase have been
thoroughly studied from a transcriptomic and metabolomic perspective (Timmins et al. 2009b; Doebbe
et al. 2010; Nguyen et al. 2011). The sequence of events begins with an aerobic phase, characterized
by the accumulation of starch and triacylglycerides (Zhang et al. 2002; Timmins et al. 2009b). This
is followed by a rapid degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) that
impairs Calvin-cycle function, and thus, eliminates one of the main electron sinks of the photosynthetic
electron-transport chain. In parallel, the nutrient deficiency stress hampers new protein synthesis,
which is crucial for repair of the PSII protein D1, so, the number of active PSII centers decreases to
5–10% of normal levels (Zhang et al. 2002). Since the mitochondria activity is essentially unaffected,
the photosynthesis/respiration rate drops dramatically, the cells turn to anaerobiosis and switch on the