research challenges that must be addressed to prove the technological feasibility of
the process. Understanding the molecular fundamentals of H 2 production pathway
and application of genetic and metabolic engineering approaches could enhance the
microalgal biohydrogen production. Significant advances in the development of
genetic tools have been made to overcome some of the major bottlenecks associated
with microalgal H 2 production system and to improve the product yield.
2.3.1 Oxygen Sensitivity of Hydrogen-Producing Enzymes
Biophotolysis method exploits highly active microalgal hydrogenases for H 2 production. However, the extreme O 2 sensitivity of these enzymes presents a challenge
for achieving sustained evolution of H 2 . It has been found that the presence of O 2
irreversibly inhibits the [FeFe]-hydrogenases by attacking the [4Fe–4S] domain of
the H-cluster (Stripp et al. 2009). Even O 2 not only inactivates the hydrogenases but
also imposes inhibitory effect on transcription and protein maturation (Oey et al.
2016). Therefore, several studies have been conducted to increase the O 2 tolerance
of the microalgal hydrogenases. Random and site-directed mutagenesis helped in
obtaining mutants of C. reinhardtii having tenfold high O 2 tolerance (Ghirardi et al.
2000). Xu et al. (2005) developed a recombinant cyanobacterial system by transferring the O 2 -tolerant hydrogenase genes from T. roseopersicina into
Synechococcus sp. PCC 7942. In a different approach for O 2 sequestration,
leghaemoglobin proteins (having high affinity to O 2 ) from legume plant (soybean)
were transformed into the chloroplast of C. reinhardtii. This method helped in rapid
consumption of O 2 and facilitated fourfold increase of H 2 production in transgenic
microalgal cultures (Chen et al. 2013; Wu et al. 2010).
2.4 Photon Conversion Efficiency
For the biofuel production by utilizing the photosynthetic machinery, quantum
efficiency holds paramount importance. Microalgal H 2 production system is greatly
limited by the low solar conversion efficiency. Under the controlled conditions and
low light intensities, algal cultures could achieve light-to-hydrogen energy conversion efficiency of up to 10% which is comparatively higher than obtained under
similar conditions with solar light (<4%). In bright light, the pigments of the huge
light harvesting complex (antenna system) capture more photons that can be utilized by the photosynthetic system. In such case, microalgal cells protect themselves from photodamage by dissipating (wasting) excess photons (*90%) as heat
and fluorescence via a process known as ‘energy-dependent non-photochemical
quenching’ (NPQ). This occurs at the upper layer of the algal culture; however, the
cells present at lower surface may not receive sufficient light due to the
‘self-shading effect’ imposed by dense culture. Thus, NPQ at the top layer and
the self-shading effect at lower surface result in low photon conversion efficiency.
10 Biofuels from Microalgae: Biohydrogen
219
the process. Understanding the molecular fundamentals of H 2 production pathway
and application of genetic and metabolic engineering approaches could enhance the
microalgal biohydrogen production. Significant advances in the development of
genetic tools have been made to overcome some of the major bottlenecks associated
with microalgal H 2 production system and to improve the product yield.
2.3.1 Oxygen Sensitivity of Hydrogen-Producing Enzymes
Biophotolysis method exploits highly active microalgal hydrogenases for H 2 production. However, the extreme O 2 sensitivity of these enzymes presents a challenge
for achieving sustained evolution of H 2 . It has been found that the presence of O 2
irreversibly inhibits the [FeFe]-hydrogenases by attacking the [4Fe–4S] domain of
the H-cluster (Stripp et al. 2009). Even O 2 not only inactivates the hydrogenases but
also imposes inhibitory effect on transcription and protein maturation (Oey et al.
2016). Therefore, several studies have been conducted to increase the O 2 tolerance
of the microalgal hydrogenases. Random and site-directed mutagenesis helped in
obtaining mutants of C. reinhardtii having tenfold high O 2 tolerance (Ghirardi et al.
2000). Xu et al. (2005) developed a recombinant cyanobacterial system by transferring the O 2 -tolerant hydrogenase genes from T. roseopersicina into
Synechococcus sp. PCC 7942. In a different approach for O 2 sequestration,
leghaemoglobin proteins (having high affinity to O 2 ) from legume plant (soybean)
were transformed into the chloroplast of C. reinhardtii. This method helped in rapid
consumption of O 2 and facilitated fourfold increase of H 2 production in transgenic
microalgal cultures (Chen et al. 2013; Wu et al. 2010).
2.4 Photon Conversion Efficiency
For the biofuel production by utilizing the photosynthetic machinery, quantum
efficiency holds paramount importance. Microalgal H 2 production system is greatly
limited by the low solar conversion efficiency. Under the controlled conditions and
low light intensities, algal cultures could achieve light-to-hydrogen energy conversion efficiency of up to 10% which is comparatively higher than obtained under
similar conditions with solar light (<4%). In bright light, the pigments of the huge
light harvesting complex (antenna system) capture more photons that can be utilized by the photosynthetic system. In such case, microalgal cells protect themselves from photodamage by dissipating (wasting) excess photons (*90%) as heat
and fluorescence via a process known as ‘energy-dependent non-photochemical
quenching’ (NPQ). This occurs at the upper layer of the algal culture; however, the
cells present at lower surface may not receive sufficient light due to the
‘self-shading effect’ imposed by dense culture. Thus, NPQ at the top layer and
the self-shading effect at lower surface result in low photon conversion efficiency.
10 Biofuels from Microalgae: Biohydrogen
219