290 Marine Macro- and Microalgae: An Overview
Manipulation of carbohydrate metabolism routes is another interesting target, since these may
serve as a reducing power source for ferredoxin reduction, and ultimately for feeding electrons into
hydrogenase. The hydrogen production yield of the C. reinhardtii stm6 mutant has been improved a
further 1.5-fold by coexpression of a hexose symporter (Doebbe et al. 2007). A C. reinhardtii mutant
possessing delayed starch catabolism (sda6) showed lower initial hydrogen release rates, but an overall
increase (1.5-fold) in hydrogen production compared to a wild-type strain (Chochois et al. 2010). A
prolonged synthesis of carbohydrates upon sulfur-deprivation was possible also in the C. reinhardtii
L159I/N230Y D1 mutant, as a result of a higher amount of D1 protein that slowed-down PSII downregulation (Scoma et al. 2012). In this mutant, anaerobiosis was sustained because the higher PSII activity
was counter-balanced by a higher respiratory activity. Because of this, and probably other accompanying
phenotypes (i.e., lower chlorophyll content, accumulation of carotenoids), this mutant showed a 10-fold
increase in hydrogen production rate compared to the corresponding wild-type strain (Torzillo et al.
2009). Other strategies, undertaken also in C. reinhardtii, attempted to enhance the injection of reducing
power into ferredoxin via the anaerobic fermentative pathways. The approach in some of these cases
was to focus the electron flow up to pyruvate-ferredoxin oxidoreductase (PFOR), by knocking down
competing pathways. Knocking-out pyruvate formate lyase (PFL1) increased hydrogen production in
the dark (Philipps et al. 2011), although hydrogen evolution in the light was not significantly changed
(Burgess et al. 2012) or negatively affected (Philipps et al. 2011). Alternatively, overexpression of type
II NAD(P)H dehydrogenase (Nda2) in C. reinhardtii also enhanced hydrogen photoproduction under
nutrient-deprivation conditions by increasing the reducing state of plastoquinones (Baltz et al. 2014).
Sulfur deprivation is itself a multi-targeting strategy to induce hydrogen production in green algae
and (in some cases) non-heterocysts forming cyanobacteria (Srirangan et al. 2011), and this effect may
be mimicked by restricting sulfur uptake. Antisense technology was used in C. reinhardtii to dampen the
expression of sulphate permease (SulP) (Chen et al. 2005). The resulting strains were able to produce
hydrogen under limited amounts of sulphate during seven days after sealing the cultures, with a 5-fold
increased yield compared to a wild-type strain.
Specific strategies have been designed in the case of cyanobacteria to improve hydrogen production.
As previously mentioned, nitrogen-fixing cyanobacteria contain an uptake-hydrogenase encoded by the
hup genes, which catalyzes the unidirectional oxidation of hydrogen and therefore decreases hydrogen
yield. Elimination of the Hup activity in several Anabaena and Nostoc cyanobacteria strains has enhanced
hydrogen production compared to the corresponding wild-type strains (Happe et al. 2000; Lindbgerg et
al. 2002; Masukawa et al. 2002; Yoshino et al. 2007; Khetkorn et al. 2012b). In the case of diazotrophic
cyanobacteria, an interesting goal would be to increase heterocyst frequency upon nitrogen starvation.
However, heterocyst formation is a complex process involving activation of 600–1000 genes (Lynn
et al. 1986), and the critical regulatory steps are not yet completely understood. Candidate genes for
manipulation are hetR causing an increase in heterocyst frequency when overexpressed, patS and patN
that suppress heterocyst formation, and hglK that fortifies the glycolipid layer of the hetorocyst when
upregulated (reviewed in Srirangan et al. 2011). Indeed, patN disruption in an Anabaena strain deficient
in Hup hydrogenase, resulted in increased heterocysts formation and higher hydrogen production
compared to the parental strain (Masukawa et al. 2017). Finally, another improvement of the two-stage
process commonly used for hydrogen production with nitrogen-fixing cyanobacteria would be to avoid
the replacement of nitrogen by argon, which increases the operational cost at industrial scale (Masukawa
et al. 2012). Interestingly, a [MoFe]-nitrogenase containing citrate instead of homocitrate in the FeMo
cofactor catalyzed nitrogen reduction poorly but was still able to reduce protons effectively in a nitrogen
atmosphere (Mayer et al. 2002). In agreement with this, gene disruption of one of the genes encoding
homocitrate synthase (nifV1) in a ΔHup strain of Nostoc sp. PCC 7120 increased hydrogen production in
the presence of nitrogen, compared to the parental ΔHup strain (Masukawa et al. 2007). Another approach
has been site-directed mutagenesis of Anabaena [MoFe]-nitrogenase in residues located in the vicinity of
the active site to direct electron flow selectively to proton reduction in the presence of nitrogen. Several
Anabaena mutants increased their in vivo rates of hydrogen production in a nitrogen atmosphere. In
particular, the R284H mutant was able to accumulate up to 87% hydrogen after 1 wk under nitrogen, as
compared to the reference strain under argon (Masukawa et al. 2010).
Manipulation of carbohydrate metabolism routes is another interesting target, since these may
serve as a reducing power source for ferredoxin reduction, and ultimately for feeding electrons into
hydrogenase. The hydrogen production yield of the C. reinhardtii stm6 mutant has been improved a
further 1.5-fold by coexpression of a hexose symporter (Doebbe et al. 2007). A C. reinhardtii mutant
possessing delayed starch catabolism (sda6) showed lower initial hydrogen release rates, but an overall
increase (1.5-fold) in hydrogen production compared to a wild-type strain (Chochois et al. 2010). A
prolonged synthesis of carbohydrates upon sulfur-deprivation was possible also in the C. reinhardtii
L159I/N230Y D1 mutant, as a result of a higher amount of D1 protein that slowed-down PSII downregulation (Scoma et al. 2012). In this mutant, anaerobiosis was sustained because the higher PSII activity
was counter-balanced by a higher respiratory activity. Because of this, and probably other accompanying
phenotypes (i.e., lower chlorophyll content, accumulation of carotenoids), this mutant showed a 10-fold
increase in hydrogen production rate compared to the corresponding wild-type strain (Torzillo et al.
2009). Other strategies, undertaken also in C. reinhardtii, attempted to enhance the injection of reducing
power into ferredoxin via the anaerobic fermentative pathways. The approach in some of these cases
was to focus the electron flow up to pyruvate-ferredoxin oxidoreductase (PFOR), by knocking down
competing pathways. Knocking-out pyruvate formate lyase (PFL1) increased hydrogen production in
the dark (Philipps et al. 2011), although hydrogen evolution in the light was not significantly changed
(Burgess et al. 2012) or negatively affected (Philipps et al. 2011). Alternatively, overexpression of type
II NAD(P)H dehydrogenase (Nda2) in C. reinhardtii also enhanced hydrogen photoproduction under
nutrient-deprivation conditions by increasing the reducing state of plastoquinones (Baltz et al. 2014).
Sulfur deprivation is itself a multi-targeting strategy to induce hydrogen production in green algae
and (in some cases) non-heterocysts forming cyanobacteria (Srirangan et al. 2011), and this effect may
be mimicked by restricting sulfur uptake. Antisense technology was used in C. reinhardtii to dampen the
expression of sulphate permease (SulP) (Chen et al. 2005). The resulting strains were able to produce
hydrogen under limited amounts of sulphate during seven days after sealing the cultures, with a 5-fold
increased yield compared to a wild-type strain.
Specific strategies have been designed in the case of cyanobacteria to improve hydrogen production.
As previously mentioned, nitrogen-fixing cyanobacteria contain an uptake-hydrogenase encoded by the
hup genes, which catalyzes the unidirectional oxidation of hydrogen and therefore decreases hydrogen
yield. Elimination of the Hup activity in several Anabaena and Nostoc cyanobacteria strains has enhanced
hydrogen production compared to the corresponding wild-type strains (Happe et al. 2000; Lindbgerg et
al. 2002; Masukawa et al. 2002; Yoshino et al. 2007; Khetkorn et al. 2012b). In the case of diazotrophic
cyanobacteria, an interesting goal would be to increase heterocyst frequency upon nitrogen starvation.
However, heterocyst formation is a complex process involving activation of 600–1000 genes (Lynn
et al. 1986), and the critical regulatory steps are not yet completely understood. Candidate genes for
manipulation are hetR causing an increase in heterocyst frequency when overexpressed, patS and patN
that suppress heterocyst formation, and hglK that fortifies the glycolipid layer of the hetorocyst when
upregulated (reviewed in Srirangan et al. 2011). Indeed, patN disruption in an Anabaena strain deficient
in Hup hydrogenase, resulted in increased heterocysts formation and higher hydrogen production
compared to the parental strain (Masukawa et al. 2017). Finally, another improvement of the two-stage
process commonly used for hydrogen production with nitrogen-fixing cyanobacteria would be to avoid
the replacement of nitrogen by argon, which increases the operational cost at industrial scale (Masukawa
et al. 2012). Interestingly, a [MoFe]-nitrogenase containing citrate instead of homocitrate in the FeMo
cofactor catalyzed nitrogen reduction poorly but was still able to reduce protons effectively in a nitrogen
atmosphere (Mayer et al. 2002). In agreement with this, gene disruption of one of the genes encoding
homocitrate synthase (nifV1) in a ΔHup strain of Nostoc sp. PCC 7120 increased hydrogen production in
the presence of nitrogen, compared to the parental ΔHup strain (Masukawa et al. 2007). Another approach
has been site-directed mutagenesis of Anabaena [MoFe]-nitrogenase in residues located in the vicinity of
the active site to direct electron flow selectively to proton reduction in the presence of nitrogen. Several
Anabaena mutants increased their in vivo rates of hydrogen production in a nitrogen atmosphere. In
particular, the R284H mutant was able to accumulate up to 87% hydrogen after 1 wk under nitrogen, as
compared to the reference strain under argon (Masukawa et al. 2010).