284 Marine Macro- and Microalgae: An Overview
Bioethanol production
Ethanol for biofuels is currently produced from the fermentation of starches or cellulose-derived
sugars. The predominant energy storage polysaccharide in Chlorophyta (green algae), Dinophyta
(dinoflagellates), Glaucophyta and Rhodophyta (red algae) is starch, while Phaeophyceae (brown algae)
and Bacillariophyceae (diatoms) store glucans in laminarian and chrysolaminarin, respectively. In
industry, the algal polysaccharides are firstly hydrolyzed and then fermented to ethanol by other organisms
such as yeast. However, an approach that would couple ethanol production directly to photosynthetic
carbon fixation in situ may be preferred. Numerous microalgae have fermentative metabolic pathways
to ethanol, but the coupling of ethanol production to photoautotrophic metabolism would require
changes in regulatory pathways or the insertion of new metabolic pathways (Radakovits et al. 2010a).
Increasing carbohydrate production in algae would help biofuel production. Proposed strategies to that
end include overexpression of key enzymes in starch biosynthesis (e.g., ADP-glucose pyrophosphorylase
or isoamylase), knockout of starch degrading enzymes (e.g., glucan-water dikinases and amylases), and
secretion of soluble carbohydrates (Work et al. 2012).
As brown macroalgae does not contain lignin, the sugars can be released by such simple
operations as milling or crushing. This bio-architectural feature gives macroalgae a distinct advantage
over lignocellulosic biomass, by facilitating higher yields and avoiding the need for energy-intensive
pretreatment and hydrolytic processes before fermentation (Wargacki et al. 2012). However, seaweeds
have been ignored as a source of renewable fuel because their primary sugar component is not easily
fermented. A microbe able to extract sugars from brown seaweeds and convert them into low-carbon,
renewable fuels, and chemicals was recently engineered (Wargacki et al. 2012).
Production of hydrogen
The biosynthesis of hydrogen has attracted an outstanding interest in the last decades due to its potential
application as non-polluting and renewable biofuel. Hydrogen can indeed, be converted to electricity
thus liberating high amounts of energy (122 kJ/g) and releasing only water as a collateral product (Ballat
2008). Reviews about hydrogen production, facts, and potentials, in green microalgae and cyanobacteria
include Burgess et al. (2011); Eroglu and Melis (2011); Esquivel et al. (2011); Srirangan et al. (2011);
Masukawa et al. (2012); Antal et al. (2015); Dubini and Ghirardi (2015); Khanna and Lindblad (2015);
Oey et al. (2016); Khetkorn et al. (2017); Nagarajan et al. (2017) and Martin and Frymier (2017).
Enzymes involved in hydrogen production in green microalgae and cyanobacteria
Cyanobacteria and eukaryotic microalgae have evolved different systems to produce hydrogen via specific
enzymes. Green microalgae possess [FeFe]-hydrogenases, whereas cyanobacteria may produce hydrogen
by a [NiFe]-hydrogenase or, in the case of nitrogen-fixing cyanobacteria, by a [MoFe]-nitrogenase.
Green algae [FeFe]-hydrogenases are located in the chloroplast and act in a unidirectional way
reducing free protons into hydrogen. The enzyme is ferredoxin-dependent, and the reducing power may
be supplied by the photosynthetic electron transport chain in the light, or by fermentative metabolism
through a pyruvate-ferredoxin oxidoreductase (PFOR) (Burgess et al. 2011) (Fig. 1A). The active site
of [FeFe]-hydrogenases contains a six-iron complex, termed the H-cluster, formed by a [4Fe4S] cubane
connected to a di-iron subcluster [2Fe] through a cysteine thiolate (Mulder et al. 2010). C. reinhardtii
contains two [FeFe]-hydrogenase-encoding genes, namely HydA1 and HydA2, of which the former
contributes to 75% of total hydrogen-production (Meuser et al. 2012). The formation of an active enzyme
requires expression of a maturation cassette composed of genes HydE, HydF and HydG. Transcription,
maturation, and activity of [FeFe]-hydrogenase are oxygen-sensitive (Srirangan et al. 2011). Oxygen
reacts in a stepwise manner with the di-iron subcluster, releasing reactive oxygen-species that subsequently
attack the [4Fe4S] cubane leading to complete and irreversible destruction of the H-cluster (Stripp et al.
2009; Lambertz et al. 2011). This property of the enzyme is the main bottleneck for efficient hydrogenproduction in green microalgae, as will be discussed below.
Bioethanol production
Ethanol for biofuels is currently produced from the fermentation of starches or cellulose-derived
sugars. The predominant energy storage polysaccharide in Chlorophyta (green algae), Dinophyta
(dinoflagellates), Glaucophyta and Rhodophyta (red algae) is starch, while Phaeophyceae (brown algae)
and Bacillariophyceae (diatoms) store glucans in laminarian and chrysolaminarin, respectively. In
industry, the algal polysaccharides are firstly hydrolyzed and then fermented to ethanol by other organisms
such as yeast. However, an approach that would couple ethanol production directly to photosynthetic
carbon fixation in situ may be preferred. Numerous microalgae have fermentative metabolic pathways
to ethanol, but the coupling of ethanol production to photoautotrophic metabolism would require
changes in regulatory pathways or the insertion of new metabolic pathways (Radakovits et al. 2010a).
Increasing carbohydrate production in algae would help biofuel production. Proposed strategies to that
end include overexpression of key enzymes in starch biosynthesis (e.g., ADP-glucose pyrophosphorylase
or isoamylase), knockout of starch degrading enzymes (e.g., glucan-water dikinases and amylases), and
secretion of soluble carbohydrates (Work et al. 2012).
As brown macroalgae does not contain lignin, the sugars can be released by such simple
operations as milling or crushing. This bio-architectural feature gives macroalgae a distinct advantage
over lignocellulosic biomass, by facilitating higher yields and avoiding the need for energy-intensive
pretreatment and hydrolytic processes before fermentation (Wargacki et al. 2012). However, seaweeds
have been ignored as a source of renewable fuel because their primary sugar component is not easily
fermented. A microbe able to extract sugars from brown seaweeds and convert them into low-carbon,
renewable fuels, and chemicals was recently engineered (Wargacki et al. 2012).
Production of hydrogen
The biosynthesis of hydrogen has attracted an outstanding interest in the last decades due to its potential
application as non-polluting and renewable biofuel. Hydrogen can indeed, be converted to electricity
thus liberating high amounts of energy (122 kJ/g) and releasing only water as a collateral product (Ballat
2008). Reviews about hydrogen production, facts, and potentials, in green microalgae and cyanobacteria
include Burgess et al. (2011); Eroglu and Melis (2011); Esquivel et al. (2011); Srirangan et al. (2011);
Masukawa et al. (2012); Antal et al. (2015); Dubini and Ghirardi (2015); Khanna and Lindblad (2015);
Oey et al. (2016); Khetkorn et al. (2017); Nagarajan et al. (2017) and Martin and Frymier (2017).
Enzymes involved in hydrogen production in green microalgae and cyanobacteria
Cyanobacteria and eukaryotic microalgae have evolved different systems to produce hydrogen via specific
enzymes. Green microalgae possess [FeFe]-hydrogenases, whereas cyanobacteria may produce hydrogen
by a [NiFe]-hydrogenase or, in the case of nitrogen-fixing cyanobacteria, by a [MoFe]-nitrogenase.
Green algae [FeFe]-hydrogenases are located in the chloroplast and act in a unidirectional way
reducing free protons into hydrogen. The enzyme is ferredoxin-dependent, and the reducing power may
be supplied by the photosynthetic electron transport chain in the light, or by fermentative metabolism
through a pyruvate-ferredoxin oxidoreductase (PFOR) (Burgess et al. 2011) (Fig. 1A). The active site
of [FeFe]-hydrogenases contains a six-iron complex, termed the H-cluster, formed by a [4Fe4S] cubane
connected to a di-iron subcluster [2Fe] through a cysteine thiolate (Mulder et al. 2010). C. reinhardtii
contains two [FeFe]-hydrogenase-encoding genes, namely HydA1 and HydA2, of which the former
contributes to 75% of total hydrogen-production (Meuser et al. 2012). The formation of an active enzyme
requires expression of a maturation cassette composed of genes HydE, HydF and HydG. Transcription,
maturation, and activity of [FeFe]-hydrogenase are oxygen-sensitive (Srirangan et al. 2011). Oxygen
reacts in a stepwise manner with the di-iron subcluster, releasing reactive oxygen-species that subsequently
attack the [4Fe4S] cubane leading to complete and irreversible destruction of the H-cluster (Stripp et al.
2009; Lambertz et al. 2011). This property of the enzyme is the main bottleneck for efficient hydrogenproduction in green microalgae, as will be discussed below.