reduce ferredoxin (Fd). Under aerobic and light condition ferredoxin: NADP
+
oxidoreductase transfers the electrons from reduced ferredoxin to NADP
+ which
generates NADPH. This reducing power (NADPH) is used to fix carbon for cell
growth and for carbohydrates and/or lipids production. However, under some
conditions, the reduced ferredoxin generated by the water splitting can be directed
to reduce hydrogenase or nitrogenase enzymes for the hydrogen production. There
are two types of biophotolysis for H 2 production from microalgae: direct and
indirect biophotolysis.
2.1.1 Direct Biophotolysis
In this biological process, the reductive equivalents required for the hydrogen
production are generated directly by the photolysis of water. Photosynthetic
machinery of green algae utilizes the solar energy to split the H 2 O molecule into O 2
and H 2 . The electrons generated by the oxidation of H 2 O molecule flows to the
ferredoxin which under the optimal conditions donates the electrons directly to
hydrogenase enzyme for H 2 production. In direct biophotolysis, production of H 2 at
the reducing side of the PSI is associated with the simultaneous evolution of O 2 at
the oxidizing side of the PSII (Melis et al. 2000). Presence of O 2 limits the
hydrogen production as the hydrogenase gets deactivated at O 2 partial pressure of
<2% (Ghirardi et al. 1997). Thus, in this process H 2 evolution occurs for transient
period upon illumination, before the hydrogenase gets inactivated by the accumulated O 2 (Eroglu and Melis 2011). Several green microalgae such as
Chlamydomonas reinhardtii, Chlorella fusca, S. obliquus, Chlorococcum littorale
and Platymonas subcordiformis possess genomically encoded [FeFe]-hydrogenase
enzyme for hydrogen generation (Eroglu and Melis 2011). Among the green algae,
C. reinhardtii has been mostly investigated by many researchers for biohydrogen
production (Melis et al. 2000; Torzillo and Seibert 2013; Tsygankov et al. 2006). In
order to prevent the inactivation of H 2 -evolving enzyme by O 2 and for the sustained
production of H 2 , different methods have been investigated such as purging the
reaction mixture with inert gases (Greenbaum 1982), addition of oxygen scavenger
(Randt and Senger 1985) and depletion of sulphur in the cultivation media (Melis
et al. 2000).
The method of sulphur deprivation is a two-stage approach for the sustained
hydrogen production by green microalgae. First stage is the growth phase in which
generation of biomass takes place under suitable conditions. Second stage is the
non-growth phase in which the carbohydrate-rich algal biomass is transferred to
sulphur-deprived cultivation medium for H 2 production (Melis et al. 2000). Sulphur
is essential for the biosynthesis of PSII protein, which is made up of
sulphur-containing amino acids (cysteine or methionine). Due to the partial suppression of PSII activity, evolution of O 2 reduces and the mitochondrial respiration
further helps in the depletion of oxygen, developing the essential anaerobiosis
inside the cells. Anaerobic condition induces the [FeFe]-hydrogenase activity
(Forestier et al. 2003) which produces H 2 by utilizing 60–90% electrons contributed
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H. Singh and D. Das
+
oxidoreductase transfers the electrons from reduced ferredoxin to NADP
+ which
generates NADPH. This reducing power (NADPH) is used to fix carbon for cell
growth and for carbohydrates and/or lipids production. However, under some
conditions, the reduced ferredoxin generated by the water splitting can be directed
to reduce hydrogenase or nitrogenase enzymes for the hydrogen production. There
are two types of biophotolysis for H 2 production from microalgae: direct and
indirect biophotolysis.
2.1.1 Direct Biophotolysis
In this biological process, the reductive equivalents required for the hydrogen
production are generated directly by the photolysis of water. Photosynthetic
machinery of green algae utilizes the solar energy to split the H 2 O molecule into O 2
and H 2 . The electrons generated by the oxidation of H 2 O molecule flows to the
ferredoxin which under the optimal conditions donates the electrons directly to
hydrogenase enzyme for H 2 production. In direct biophotolysis, production of H 2 at
the reducing side of the PSI is associated with the simultaneous evolution of O 2 at
the oxidizing side of the PSII (Melis et al. 2000). Presence of O 2 limits the
hydrogen production as the hydrogenase gets deactivated at O 2 partial pressure of
<2% (Ghirardi et al. 1997). Thus, in this process H 2 evolution occurs for transient
period upon illumination, before the hydrogenase gets inactivated by the accumulated O 2 (Eroglu and Melis 2011). Several green microalgae such as
Chlamydomonas reinhardtii, Chlorella fusca, S. obliquus, Chlorococcum littorale
and Platymonas subcordiformis possess genomically encoded [FeFe]-hydrogenase
enzyme for hydrogen generation (Eroglu and Melis 2011). Among the green algae,
C. reinhardtii has been mostly investigated by many researchers for biohydrogen
production (Melis et al. 2000; Torzillo and Seibert 2013; Tsygankov et al. 2006). In
order to prevent the inactivation of H 2 -evolving enzyme by O 2 and for the sustained
production of H 2 , different methods have been investigated such as purging the
reaction mixture with inert gases (Greenbaum 1982), addition of oxygen scavenger
(Randt and Senger 1985) and depletion of sulphur in the cultivation media (Melis
et al. 2000).
The method of sulphur deprivation is a two-stage approach for the sustained
hydrogen production by green microalgae. First stage is the growth phase in which
generation of biomass takes place under suitable conditions. Second stage is the
non-growth phase in which the carbohydrate-rich algal biomass is transferred to
sulphur-deprived cultivation medium for H 2 production (Melis et al. 2000). Sulphur
is essential for the biosynthesis of PSII protein, which is made up of
sulphur-containing amino acids (cysteine or methionine). Due to the partial suppression of PSII activity, evolution of O 2 reduces and the mitochondrial respiration
further helps in the depletion of oxygen, developing the essential anaerobiosis
inside the cells. Anaerobic condition induces the [FeFe]-hydrogenase activity
(Forestier et al. 2003) which produces H 2 by utilizing 60–90% electrons contributed
204
H. Singh and D. Das