288 Marine Macro- and Microalgae: An Overview
hydrogen production, as shown in Fig. 1) and begins at the onset of anaerobiosis supported by glycogen
catabolism. Efficiency of hydrogen production is limited in cyanobacteria by specific constraints of these
systems (such as the presence of an uptake [NiFe]-hydrogenase, or the low frequency of heterocyst
formation), as well as common restrictions to the green algae mechanism (such as the oxygen-sensitivity
of the hydrogenase or the competition for electrons from non-hydrogenase pathways) (Eroglu and Melis
2011; Srirangan et al. 2011).
Targets to improve hydrogen production
Based on the different pathways leading to hydrogen synthesis (Fig. 1) and the regulation mechanisms
described above, different strategies have been undertaken to obtain new strains capable of producing
hydrogen to higher yields and/or with an improved process for industrial scale up. The final goal of
some of these designs is to bypass the problem of the hydrogenase inhibition by oxygen. This may be
accomplished by engineering the enzyme to decrease its oxygen sensitivity (reviewed in Ghirardi et al.
2015). In the case of [FeFe]-hydrogenase, rational design of hydrogenase mutants may be directed to
narrow the putative oxygen diffusion channels into the active site (Ghirardi et al. 2005; Lambertz et al.
2011) or to change the redox potential of the [4Fe4S] cubane (Lambertz et al. 2011). Other strategies
include random mutagenesis (Nagy et al. 2007; Stapleton and Swartz 2010) or metagenomic analysis
to explore the natural diversity of these enzymes (Rusch et al. 2007; Warnecke et al. 2007). However,
no oxygen-insensitive [FeFe]-hydrogenase has been found so far. In contrast, oxygen-tolerant [NiFe]hydrogenases have been discovered in non-photosynthetic microorganisms, although their expression
in cyanobacteria was not successful until they were co-translated with their corresponding maturation
machinery. This strategy has permitted the expression of active oxygen-tolerant [NiFe]-hydrogenases
from Alteromonas macleodii and Thiocapsa roseopersicina in Synechococcus elongatus (Weyman et al.
2011).
As an alternative to alleviate the oxygen-sensitivity problem, another plausible approach is to decrease
the oxygen concentration in the environment of the enzyme. This has been accomplished in C. reinhardtii
by the overexpression of oxygen-sequestering proteins, such as ferrochelatase or leghemoglobin, which
caused an increase in hydrogen production up to 4.5-fold compared to the wild-type strain (Wu et al.
2010, 2011). A different strategy may be to decrease oxygen evolution through PSII. Constitutive and full
inhibition of the complex by knocking-out any of its subunits resulted in detrimental hydrogen production
(Makarova et al. 2007; Hemschemeier et al. 2008)—probably because photosynthetic activity is required
for starch accumulation. However, a regulated partial inhibition of PSII could be more effective, as
suggested by the Cy6Nac2.49 C. reinhardtii transgenic strain, where the expression of the PSII D2 protein
is down-regulated in the presence of Cu
2+
and up-regulated under copper depletion or anaerobiosis. When
this strain was grown aerobically in a medium lacking copper, anaerobiosis, and hydrogen synthesis
could be transiently induced upon copper addition (Surzycki et al. 2007). Furthermore, because in a
copper-repleted medium PSII expression is only induced under anaerobic conditions, a system based
on this strain may operate with a negative feedback mechanism. Thus, excessive oxygen evolution from
PSII (i.e., further surpassing oxygen consumption by mitochondrial respiration) would prompt downregulation of PSII expression, therefore extending the anaerobiosis period and hydrogen synthesis. Indeed,
this strain supported sustained hydrogen evolution under a light/dark regime with higher efficiency than
that achieved with the wild-type strain under the same conditions (Batyrova and Hallenbeck 2017).
However, hydrogen production yields were lower than those reported with the nutrient-deprivation
protocols. In a similar line of action, reduced oxygen concentrations may be achieved by the generation
of mutants with a decreased photosynthesis to respiration ratio. The apr1 mutant, with dramatically
reduced photosynthetic rates and a slightly increased respiration, turned into anerobiosis when placed in a
sealed container upon illumination. However, hydrogen production required the simultaneous inhibition
of the Calvin cycle by addition of glycolaldehyde (Ruhle et al. 2008), which eliminates one of the main
competitors with the hydrogenase for the electrons derived from ferredoxin. This result is indicative that
efficient hydrogen production probably requires multi-targeting approaches. Another strategy, also based
in decreasing photosynthetic oxygen evolution, employed temperature sensitive-PSII mutants (Bayro-
hydrogen production, as shown in Fig. 1) and begins at the onset of anaerobiosis supported by glycogen
catabolism. Efficiency of hydrogen production is limited in cyanobacteria by specific constraints of these
systems (such as the presence of an uptake [NiFe]-hydrogenase, or the low frequency of heterocyst
formation), as well as common restrictions to the green algae mechanism (such as the oxygen-sensitivity
of the hydrogenase or the competition for electrons from non-hydrogenase pathways) (Eroglu and Melis
2011; Srirangan et al. 2011).
Targets to improve hydrogen production
Based on the different pathways leading to hydrogen synthesis (Fig. 1) and the regulation mechanisms
described above, different strategies have been undertaken to obtain new strains capable of producing
hydrogen to higher yields and/or with an improved process for industrial scale up. The final goal of
some of these designs is to bypass the problem of the hydrogenase inhibition by oxygen. This may be
accomplished by engineering the enzyme to decrease its oxygen sensitivity (reviewed in Ghirardi et al.
2015). In the case of [FeFe]-hydrogenase, rational design of hydrogenase mutants may be directed to
narrow the putative oxygen diffusion channels into the active site (Ghirardi et al. 2005; Lambertz et al.
2011) or to change the redox potential of the [4Fe4S] cubane (Lambertz et al. 2011). Other strategies
include random mutagenesis (Nagy et al. 2007; Stapleton and Swartz 2010) or metagenomic analysis
to explore the natural diversity of these enzymes (Rusch et al. 2007; Warnecke et al. 2007). However,
no oxygen-insensitive [FeFe]-hydrogenase has been found so far. In contrast, oxygen-tolerant [NiFe]hydrogenases have been discovered in non-photosynthetic microorganisms, although their expression
in cyanobacteria was not successful until they were co-translated with their corresponding maturation
machinery. This strategy has permitted the expression of active oxygen-tolerant [NiFe]-hydrogenases
from Alteromonas macleodii and Thiocapsa roseopersicina in Synechococcus elongatus (Weyman et al.
2011).
As an alternative to alleviate the oxygen-sensitivity problem, another plausible approach is to decrease
the oxygen concentration in the environment of the enzyme. This has been accomplished in C. reinhardtii
by the overexpression of oxygen-sequestering proteins, such as ferrochelatase or leghemoglobin, which
caused an increase in hydrogen production up to 4.5-fold compared to the wild-type strain (Wu et al.
2010, 2011). A different strategy may be to decrease oxygen evolution through PSII. Constitutive and full
inhibition of the complex by knocking-out any of its subunits resulted in detrimental hydrogen production
(Makarova et al. 2007; Hemschemeier et al. 2008)—probably because photosynthetic activity is required
for starch accumulation. However, a regulated partial inhibition of PSII could be more effective, as
suggested by the Cy6Nac2.49 C. reinhardtii transgenic strain, where the expression of the PSII D2 protein
is down-regulated in the presence of Cu
2+
and up-regulated under copper depletion or anaerobiosis. When
this strain was grown aerobically in a medium lacking copper, anaerobiosis, and hydrogen synthesis
could be transiently induced upon copper addition (Surzycki et al. 2007). Furthermore, because in a
copper-repleted medium PSII expression is only induced under anaerobic conditions, a system based
on this strain may operate with a negative feedback mechanism. Thus, excessive oxygen evolution from
PSII (i.e., further surpassing oxygen consumption by mitochondrial respiration) would prompt downregulation of PSII expression, therefore extending the anaerobiosis period and hydrogen synthesis. Indeed,
this strain supported sustained hydrogen evolution under a light/dark regime with higher efficiency than
that achieved with the wild-type strain under the same conditions (Batyrova and Hallenbeck 2017).
However, hydrogen production yields were lower than those reported with the nutrient-deprivation
protocols. In a similar line of action, reduced oxygen concentrations may be achieved by the generation
of mutants with a decreased photosynthesis to respiration ratio. The apr1 mutant, with dramatically
reduced photosynthetic rates and a slightly increased respiration, turned into anerobiosis when placed in a
sealed container upon illumination. However, hydrogen production required the simultaneous inhibition
of the Calvin cycle by addition of glycolaldehyde (Ruhle et al. 2008), which eliminates one of the main
competitors with the hydrogenase for the electrons derived from ferredoxin. This result is indicative that
efficient hydrogen production probably requires multi-targeting approaches. Another strategy, also based
in decreasing photosynthetic oxygen evolution, employed temperature sensitive-PSII mutants (Bayro-