are mainly supplied by degradation of intracellular carbon compounds. In microalgae, these carbon compounds such as starch
are produced through the fixation of CO 2 by using light energy
(photosynthesis).
The light-independent pathway involves anaerobic fermentation of high molecular weight organic substrates into soluble
metabolite products such as ethanol, formate, etc. In addition to
these metabolites, pyruvate oxidation by pyruvate-ferredoxin oxidoreductase (PFOR) enzyme leads to the formation of acetyl-CoA
and CO 2 . This reaction is also associated with the reduction of
ferredoxin, through which electrons are transferred to hydrogenase
for the catalytic production of H 2 [48–50]. In fact, throughout the
hydrogen production process by microalgae, hydrogenase is the key
enzyme, whose active site is highly sensitive to oxygen, and thus, a
biphasic production strategy is necessary to produce hydrogen
under photosynthesis condition [66]. It should be mentioned
that hydrogenase activity can be externally induced by adding
DCMU (a PSII electron chain uncoupler) [51, 52]. To resolve
the problem of oxygen sensitivity of hydrogenase, microalgae
should be first grown photosynthetically to accumulate biomass,
and then hydrogen production should be pursued under anoxic
conditions. To achieve anoxic conditions, inhibition of photosystem II (PSII) through the application of herbicides or sulfur deprivation has been suggested [53, 54].
A number of approaches based on genetic engineering solutions
have been offered to improve hydrogen production in microalgae.
Induction of anaerobic conditions is among such approaches, which
could result in suppression of D2 protein (an essential protein in
PSII) leading to a brief period of anaerobiosis and consequently
increased hydrogen production. A copper-responsive nuclear transgene is necessary for the expression of the D2 protein, and therefore, addition of copper could adversely affect the production rate of
this protein [55]. Other strategies include increasing hydrogenase
activity and its resistance to oxygen, preventing electron consumption by other parallel pathways to increase electron flux toward
hydrogen production pathway and finally increasing the sources of
electrons [56, 57]. For instance, Kruse et al. claimed enhanced
hydrogen production in an engineered C. reinhardtii by 5 to 13
folds (compared to the wild type) through blocking the cyclic
electron transfer around photosystem I resulting in increased diversion of electrons toward hydrogenases [58].
4 Genetic Engineering of Microalgae
Transformation in microalgae can occur in chloroplast, nuclear,
or mitochondrial genomes [59]. Although most enzymes are
encoded in the nuclear genome, it should be mentioned that
162
Mohammad Pooya Naghshbandi et al.
are produced through the fixation of CO 2 by using light energy
(photosynthesis).
The light-independent pathway involves anaerobic fermentation of high molecular weight organic substrates into soluble
metabolite products such as ethanol, formate, etc. In addition to
these metabolites, pyruvate oxidation by pyruvate-ferredoxin oxidoreductase (PFOR) enzyme leads to the formation of acetyl-CoA
and CO 2 . This reaction is also associated with the reduction of
ferredoxin, through which electrons are transferred to hydrogenase
for the catalytic production of H 2 [48–50]. In fact, throughout the
hydrogen production process by microalgae, hydrogenase is the key
enzyme, whose active site is highly sensitive to oxygen, and thus, a
biphasic production strategy is necessary to produce hydrogen
under photosynthesis condition [66]. It should be mentioned
that hydrogenase activity can be externally induced by adding
DCMU (a PSII electron chain uncoupler) [51, 52]. To resolve
the problem of oxygen sensitivity of hydrogenase, microalgae
should be first grown photosynthetically to accumulate biomass,
and then hydrogen production should be pursued under anoxic
conditions. To achieve anoxic conditions, inhibition of photosystem II (PSII) through the application of herbicides or sulfur deprivation has been suggested [53, 54].
A number of approaches based on genetic engineering solutions
have been offered to improve hydrogen production in microalgae.
Induction of anaerobic conditions is among such approaches, which
could result in suppression of D2 protein (an essential protein in
PSII) leading to a brief period of anaerobiosis and consequently
increased hydrogen production. A copper-responsive nuclear transgene is necessary for the expression of the D2 protein, and therefore, addition of copper could adversely affect the production rate of
this protein [55]. Other strategies include increasing hydrogenase
activity and its resistance to oxygen, preventing electron consumption by other parallel pathways to increase electron flux toward
hydrogen production pathway and finally increasing the sources of
electrons [56, 57]. For instance, Kruse et al. claimed enhanced
hydrogen production in an engineered C. reinhardtii by 5 to 13
folds (compared to the wild type) through blocking the cyclic
electron transfer around photosystem I resulting in increased diversion of electrons toward hydrogenases [58].
4 Genetic Engineering of Microalgae
Transformation in microalgae can occur in chloroplast, nuclear,
or mitochondrial genomes [59]. Although most enzymes are
encoded in the nuclear genome, it should be mentioned that
162
Mohammad Pooya Naghshbandi et al.