**4. Exopolymeric substances from microalgae**

Studies show that microalgae actively release from 3 to 40% of the fixed carbon into the surrounding environment as exopolymeric substances, mostly polysaccharides and proteins but also nucleic acids, DNA, RNA, and other macromolecules [64]. Although initially presumed as experimental artifact or a product of dead and decaying phytoplankton [65], EPS is now universally accepted as a product that is actively secreted by microalgae. The relatively higher percentage of fixed carbon released extracellularly has led the physiologist to question the reasons behind this phenomena. Several hypotheses have been put forward, including carbon overflow, photoprotection of the over-reduced photosystems, motility, self-defense mechanisms, active selection of phycosphere residents, and passive excretion due to osmosis and permeability. The hypothesis of carbon overflow and photoprotection has been discredited due to the presence of proteins, amino acids, and vitamins in the released substances and due to the secretion of EPS during the night [66]. The hypothesis of EPS secretion as a self-defense mechanism, motility, active selection of phycosphere residents, and passive excretion due to osmosis and permeability needs to be experimentally tested.

Experimental studies have shown contrasting results in the secretion of EPS in response to environmental factors such as temperature, nutrient (N, P, and S) limitation, salinity, and heterotrophy/mixotrophy [67]. The results vary depending on the species of microalgae being tested. In addition, EPS secretion during various phases of growth was species dependent, with some showing an increased secretion during stationary phase and others in exponential phase. Overall, with no universal explanation behind the mechanisms of EPS secretion by phytoplankton, and multiple hypotheses explaining the phenomena, strategic means to regulate the production and composition of EPS release by microalgae is clearly lacking. Although more research is needed, EPS are usually composed of carbohydrates, nitrogenous compounds, lipids, and organic acids [68]. Polysaccharides usually could account for 80–90% of the EPS composition even under healthy conditions [69]. Nitrogenous compounds, such as amino acids and proteins, on the other hand, only make up to 4–7% of the total EPS secreted [70, 71]. These protein fractions can include exoenzymes like phosphatase, β-glucosidase [72], and siderophores such as ISIP2a [73]. Characterization of EPS involves quantification of organic matter released as polysaccharides, proteins, lipids, neutral sugars, and/or uronic acids. EPS characterization of these macromolecules is often performed under the assumption these are the dominant molecules, however, possibilities of the same molecules possessing both sugar chain and a protein moiety are quite certain. Moreover, rarely are the monomers that make up these polymers investigated

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*Physiological Limitations and Solutions to Various Applications of Microalgae*

inflammatory, antivirus, antibacterial, antifungal, or anticoagulant.

(e.g., sugars that make up these polysaccharides), and therefore the polymeric composition and the structure and physical properties remain unknown. Therefore, more in-depth characterization of EPS from microalgal species of potential and under different growth conditions and growth phase are needed to integrate EPS from microalgae into the algal biotechnology market. Needless to say, these characterization studies of EPS have to be performed with appropriate controls within the context of its application, whether as a surfactant, lubricant, antioxidant, anti-

Xiao et al. [74] in their review compared the emulsifying activity of EPS from *Dunaliella salina* (88% retention) reported by Mishra et al. [75] to commercially available surfactants Tween 20 (65% retention), Tween 80 (60% retention), and Triton X-100 (65% retention). Furthermore, being extracellularly secreted, EPS is not constrained by physiological feedback inhibition mechanisms, or retention capacity dictated by the maximum cell volume, or the need of expensive procedures to lyse the cell to release the products from the cell, unlike other microalgal products (such as lipid for biodiesel, PUFAs, and high valued pigments). Therefore, with better-focused research, EPS has a relatively greater potential of becoming a

With the raising awareness of cleaner and sustainable fuel, development of hydrogen fuel cell-powered cars, and the high cost and greenhouse gas emission associated with thermochemical hydrogen production, microalgae is increasingly becoming an attractive source for the fuel. First observed by Hans Gaffron in 1939 [76], this phenomenon of hydrogen production has been extensively studied since then. The production of hydrogen by microalgae only occurs during anaerobic conditions, due to the sensitivity of hydrogenase (the enzyme catalyzing the reversible reaction of hydrogen production) to oxygen. Three major enzymes that lead to the production of hydrogen in microalgae include (1) reversible/classical hydrogenases, (2) membrane-bound uptake hydrogenases, and (3) nitrogenase enzyme [77, 78]. Of all three, the reversible/classical hydrogenase is the most studied enzyme. Located in the chloroplast, the primary electron donor for this enzyme is photosystem I (PS I). However, the generation of molecular oxygen through photolysis by photosystem II (PS II) inhibits the activity of hydrogenase. Therefore, this enzyme only functions when the rate of photosynthesis is below the compensation point (rate of photosynthesis = rate of respiration). Past studies have achieved this condition by either flushing the system with argon or nitrogen [79] or using PS II lacking mutants [80], or selective excitation of PS I through far red light (>710 nm) [81], or more commonly through sulfur deprivation [82]. During a combination of anaerobiosis and below compensation point conditions, supply of electron from PSI to hydrogenase has been shown to either come from the excitation of PS I, and/ or through the photolysis of water, and/or through non-photochemical reduction of the plastoquinone pool through type II NAD(P)H dehydrogenase that mediates the transfer of electron derived from anaerobic catabolism of cellular carbon reserve,

It is proposed that under ideal conditions, one should expect a generation of 2:1 H2/O2 per 8 photons [85]. However, only around 20% of this efficiency is practically achieved [86]. The discrepancy between theory and practical estimations could be due to several physiological reasons unaccounted for in the theoretical estimation. (1) The physiological role of hydrogenase was although a mystery for the most part [86], discuss it's to act as an electron sink and hence oxidation of reducing

*DOI: http://dx.doi.org/10.5772/intechopen.90206*

commercial reality as a microalgal product.

primarily thought to be proteins at this point [83, 84].

**5. Hydrogen from microalgae**

*Physiological Limitations and Solutions to Various Applications of Microalgae DOI: http://dx.doi.org/10.5772/intechopen.90206*

(e.g., sugars that make up these polysaccharides), and therefore the polymeric composition and the structure and physical properties remain unknown. Therefore, more in-depth characterization of EPS from microalgal species of potential and under different growth conditions and growth phase are needed to integrate EPS from microalgae into the algal biotechnology market. Needless to say, these characterization studies of EPS have to be performed with appropriate controls within the context of its application, whether as a surfactant, lubricant, antioxidant, antiinflammatory, antivirus, antibacterial, antifungal, or anticoagulant.

Xiao et al. [74] in their review compared the emulsifying activity of EPS from *Dunaliella salina* (88% retention) reported by Mishra et al. [75] to commercially available surfactants Tween 20 (65% retention), Tween 80 (60% retention), and Triton X-100 (65% retention). Furthermore, being extracellularly secreted, EPS is not constrained by physiological feedback inhibition mechanisms, or retention capacity dictated by the maximum cell volume, or the need of expensive procedures to lyse the cell to release the products from the cell, unlike other microalgal products (such as lipid for biodiesel, PUFAs, and high valued pigments). Therefore, with better-focused research, EPS has a relatively greater potential of becoming a commercial reality as a microalgal product.
