**5. Hydrogen from microalgae**

*Microalgae - From Physiology to Application*

of phycocyanin that be produced.

needs to be experimentally tested.

**4. Exopolymeric substances from microalgae**

microalgae is limited as their production is strictly dependent on the maximum amount of biomass that can be generated. A suggested final concentration of higher than 10% of cell dry weight of phycocyanin is required to make a profit over the cost of pigment downstream separation [63]. With factors such as blue light and low light levels required to induce maximum cellular synthesis of phycocyanin, the growth is significantly attenuated under these low energy light conditions, thereby limiting the maximum biomass and hence the maximum quantity of phycocyanin that can be synthesized. Therefore, a dual phase production approach to maximize the biomass production under mixotrophic conditions with cheaper organic carbon source such as molasses [41] followed by the second phase of low levels of blue light to stimulate the synthesis phycocyanin can significantly maximize the total amount

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

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

**218**

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

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

equivalents derived through anaerobic catabolism of cellular carbon reserve. Therefore, all the physiological and thermodynamic limitations that apply to anaerobic catabolism of cellular would in turn limit the supply of electrons and protons to hydrogenase. (2) Hydrogenase catalyzes a reversible reaction, and Kessler [84] has shown a "photoreduction" process of uptake of hydrogen gas to reduce molecular oxygen or CO2 similar to the photosynthate derived under aerobic conditions. Whether the reduction of CO2 through uptake of hydrogen gas by hydrogenase under anaerobic conditions involves the Calvin cycle remains to be demonstrated. Regardless, the uptake of hydrogen by hydrogenase should contribute to the reduced efficiency of the hydrogenase. (3) Direct oxidation of photosysnthetically derived reducing equivalents would mean a net production of 3.1:0 ATP/NADPH per 8 photons (4 molecules of H2O) and generation of 2 H2. This would provide no anabolic advantage to the cells producing hydrogen, as this would lead to a big imbalance in the ATP/NADPH levels in the cells. (4) Often the yields of hydrogen derived under sulfur deprivation are compared to the theoretical yield of 2:1 H2/O2 per 8 photons; however, during sulfur deprivation, the anaerobiosis is created by the failure to regenerate the high sulfur photo-damaged D1 protein; therefore the hydrogen gas derived is not directly through photolysis of water but instead through catabolism of carbon reserves [84, 87]. These carbon reserve can be starch or proteins [82], and every molecule of glucose derived from the breakdown of starch, is then catabolized to pyruvate through glycolysis yielding 2 NADH and ATP, based on the observation by Melis et al. [82] of increase in acetate levels beyond 120 hour incubation in sulfur deprived anaerobic condition in the presence of light and no ethanol of formate secretion, hints towards catabolism of pyruvate to acetate possibly via phosphotransacetylase and acetate kinase, which would generate an additional molecule of NADH and ATP. Atteia et al. [88] have detected phosphotransacetylase and acetate kinase in the species *Chlamydomonas reinhardtii* that was used in Melis et al. (2000) study [82]. Overall, this process of sulfur deprivation-induced anaerobiosis leads to a total of 3 NADH and 3 ATP, and 2 molecules of acetate from 1 molecule of glucose; therefore a net loss of 8 protons and hence 4H2 in the form of 2 acetate molecules and formation of 6H2 occurs in this biochemical pathway. This, when expressed per photon cost, would result in 6H2 per 32 photons (the ideal required amount to make a molecule of glucose), which would result in 75% efficiency compared to the ideal generation of 2H2 per 8 photons directly through photolysis of water via photosynthesis. The discrepancy of the observed 20% efficiency vs. calculated 75% via fermentation can be due to the existence of other pathways competing for the same NADH. Oxidative pentose phosphate pathway has been shown to be upregulated under sulfur deprivation-induced anaerobiosis [89, 90]; however, whether reductive pentose phosphate pathway is upregulated remains unknown. This is probably due to the cellular demand for ribulose 5-phosphate to synthesize nucleic acids and NADP and NAD to maintain the integrity/protect the DNA and cellular metabolism. Nevertheless, if the glucose was catabolized only via oxidative pentose phosphate pathway, only 2 NADPH would be synthesized, which would yield just two molecules of H2, which would match the observed 20% efficiency of H2 generation. However, despite nucleic acid synthesis, further catabolism of ribulose 5-phosphate is inevitable and should lead to generation of more NADH. Furthermore, catabolism via oxidative pentose phosphate pathway solely would not explain the increase in acetate levels observed by Melis et al. [82]. On the other hand, if proteins served as carbon source during sulfur-deprived anaerobiosis, we hypothesize only amino acid with three or more carbon chains would be utilized, based on the absence of acetate consumption during sulfur-deprived anaerobiosis observed by Melis et al. [82] and the absence of H2 production and methyl viologen

**221**

H2 μg Chl<sup>−</sup><sup>1</sup>

*Physiological Limitations and Solutions to Various Applications of Microalgae*

smaller antennae size produced more hydrogen [93, 94].

ated by N2 flushing observed around 0.422 H2 μg Chl<sup>−</sup><sup>1</sup>

Hydrogen is also produced in the dark and therefore in the absence of excitation and transfer of electrons by PS I. Noth et al. [91] under anaerobic conditions gener-

lism in the dark also leads to the production of formate, acetate, and ethanol [87]. Atteia et al. [88] revealed the presence of pyruvate-formate lyase in *Chlamydomonas reinhardtii*, suggesting mixed fermentative metabolism via pyruvate-formate lyase, aldehyde-alcohol dehydrogenase, phosphotransacetylase, and acetate kinase, leading to the formation of formate, ethanol, and acetate [91]. Furthermore, Noth et al. [91] suggested pyruvate ferrodoxin oxidoreductase and not hydrogenase being responsible for H2 production in the dark. The combination of mixed acid fermentation and pyruvate ferrodoxin oxidoreductase suggests the operation of a different set of pathways in dark anaerobic conditions compared to light. More studies are needed to study to confirm this hypothesis and also to test whether these pathways are mutually exclusive. Even though the hydrogen produced were around 87% lower than that observed in the light, the absence of photosystem involvement eliminates the complication of oxygen-induced inhibition of hydrogenases and competition for NADPH by photorespiration. In addition, the complete heterotrophic nature of this production and the valuable co-products such as ethanol and acetate allows for further optimization and scaling up the fermentative reactions, leading up to hydrogen production. Overall, the process of hydrogen production from microalgae via anaerobiosis whether in the light and/or dark clearly has a strong potential, especially with the development of hydrogen fuel cell-powered cars. Given the advantage of being a cleaner source of hydrogen than thermochemical production, hydrogen production from microalgae can definitely benefit more from more research.

in *Chlamydomonas reinhardtii*. Along with the H2, fermentative metabo-

in the dark compared to 3.128

reduction when supplied with α-ketoglutarate during anaerobiosis as observed by Noth et al. [91]. Both these studies indicate that TCA cycle was not active during anaerobiosis, which explains the absence of acetate and α-ketoglutarate uptake under such conditions, therefore making catabolism of amino acid smaller than the three-carbon chain (glycine) and serving as a source for reducing equivalents for impractical H2 production. However, Noth et al. [91] did observe H2 production and methyl viologen reduction when *Chlamydomonas reinhardtii* was grown on oxaloacetate, which suggest glyoxylate pathway might be still active under anaerobiosis. It is important to note the absence of acetate uptake in sulfur deprivation-induced anaerobiosis as seen in Melis et al. [82] study and the opposite phenomena observed by Gibbs et al. [87] where acetate was readily uptaken when anaerobiosis was established via flushing with N2. This suggests that glyoxylate pathway is inhibited under sulfur deprivation-induced anaerobiosis, probably due to the iron–sulfur (Fe▬S) cluster of aconitase [92], thereby limiting the ability of cells to use external acetate as a carbon source. Therefore, sulfur deprivation-induced anaerobiosis would lead to relatively lower H2 production than N2 flushing-induced anaerobiosis, which is due to the extra one NADH and FADH produced via glyoxylate pathway that could potentially yield an additional two molecules of H2 per molecule of glucose and the two molecules of H2 per molecule of external acetate metabolized. Nevertheless, calculation of H2 per photon derived through protein catabolism is complicated by the presence of three to five carbon substrates and the various pathways through which they can be broken down to acetate. Isotope labelling studies will definitely help shed light into which amino acids are preferentially degraded during anaerobiosis and allow for a more accurate determination of H2 per photon. Studies have suggested that using genetic engineering to develop mutants lacking the ability to carry out state transitioning, cyclic electron transport, and mutants that have a

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

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

*Microalgae - From Physiology to Application*

equivalents derived through anaerobic catabolism of cellular carbon reserve. Therefore, all the physiological and thermodynamic limitations that apply to anaerobic catabolism of cellular would in turn limit the supply of electrons and protons to hydrogenase. (2) Hydrogenase catalyzes a reversible reaction, and Kessler [84] has shown a "photoreduction" process of uptake of hydrogen gas to reduce molecular oxygen or CO2 similar to the photosynthate derived under aerobic conditions. Whether the reduction of CO2 through uptake of hydrogen gas by hydrogenase under anaerobic conditions involves the Calvin cycle remains to be demonstrated. Regardless, the uptake of hydrogen by hydrogenase should contribute to the reduced efficiency of the hydrogenase. (3) Direct oxidation of photosysnthetically derived reducing equivalents would mean a net production of 3.1:0 ATP/NADPH per 8 photons (4 molecules of H2O) and generation of 2 H2. This would provide no anabolic advantage to the cells producing hydrogen, as this would lead to a big imbalance in the ATP/NADPH levels in the cells. (4) Often the yields of hydrogen derived under sulfur deprivation are compared to the theoretical yield of 2:1 H2/O2 per 8 photons; however, during sulfur deprivation, the anaerobiosis is created by the failure to regenerate the high sulfur photo-damaged D1 protein; therefore the hydrogen gas derived is not directly through photolysis of water but instead through catabolism of carbon reserves [84, 87]. These carbon reserve can be starch or proteins [82], and every molecule of glucose derived from the breakdown of starch, is then catabolized to pyruvate through glycolysis yielding 2 NADH and ATP, based on the observation by Melis et al. [82] of increase in acetate levels beyond 120 hour incubation in sulfur deprived anaerobic condition in the presence of light and no ethanol of formate secretion, hints towards catabolism of pyruvate to acetate possibly via phosphotransacetylase and acetate kinase, which would generate an additional molecule of NADH and ATP. Atteia et al. [88] have detected phosphotransacetylase and acetate kinase in the species *Chlamydomonas reinhardtii* that was used in Melis et al. (2000) study [82]. Overall, this process of sulfur deprivation-induced anaerobiosis leads to a total of 3 NADH and 3 ATP, and 2 molecules of acetate from 1 molecule of glucose; therefore a net loss of 8 protons and hence 4H2 in the form of 2 acetate molecules and formation of 6H2 occurs in this biochemical pathway. This, when expressed per photon cost, would result in 6H2 per 32 photons (the ideal required amount to make a molecule of glucose), which would result in 75% efficiency compared to the ideal generation of 2H2 per 8 photons directly through photolysis of water via photosynthesis. The discrepancy of the observed 20% efficiency vs. calculated 75% via fermentation can be due to the existence of other pathways competing for the same NADH. Oxidative pentose phosphate pathway has been shown to be upregulated under sulfur deprivation-induced anaerobiosis [89, 90]; however, whether reductive pentose phosphate pathway is upregulated remains unknown. This is probably due to the cellular demand for ribulose 5-phosphate to synthesize nucleic acids and NADP and NAD to maintain the integrity/protect the DNA and cellular metabolism. Nevertheless, if the glucose was catabolized only via oxidative pentose phosphate pathway, only 2 NADPH would be synthesized, which would yield just two molecules of H2, which would match the observed 20% efficiency of H2 generation. However, despite nucleic acid synthesis, further catabolism of ribulose 5-phosphate is inevitable and should lead to generation of more NADH. Furthermore, catabolism via oxidative pentose phosphate pathway solely would not explain the increase in acetate levels observed by Melis et al. [82]. On the other hand, if proteins served as carbon source during sulfur-deprived anaerobiosis, we hypothesize only amino acid with three or more carbon chains would be utilized, based on the absence of acetate consumption during sulfur-deprived anaerobiosis observed by Melis et al. [82] and the absence of H2 production and methyl viologen

**220**

reduction when supplied with α-ketoglutarate during anaerobiosis as observed by Noth et al. [91]. Both these studies indicate that TCA cycle was not active during anaerobiosis, which explains the absence of acetate and α-ketoglutarate uptake under such conditions, therefore making catabolism of amino acid smaller than the three-carbon chain (glycine) and serving as a source for reducing equivalents for impractical H2 production. However, Noth et al. [91] did observe H2 production and methyl viologen reduction when *Chlamydomonas reinhardtii* was grown on oxaloacetate, which suggest glyoxylate pathway might be still active under anaerobiosis. It is important to note the absence of acetate uptake in sulfur deprivation-induced anaerobiosis as seen in Melis et al. [82] study and the opposite phenomena observed by Gibbs et al. [87] where acetate was readily uptaken when anaerobiosis was established via flushing with N2. This suggests that glyoxylate pathway is inhibited under sulfur deprivation-induced anaerobiosis, probably due to the iron–sulfur (Fe▬S) cluster of aconitase [92], thereby limiting the ability of cells to use external acetate as a carbon source. Therefore, sulfur deprivation-induced anaerobiosis would lead to relatively lower H2 production than N2 flushing-induced anaerobiosis, which is due to the extra one NADH and FADH produced via glyoxylate pathway that could potentially yield an additional two molecules of H2 per molecule of glucose and the two molecules of H2 per molecule of external acetate metabolized. Nevertheless, calculation of H2 per photon derived through protein catabolism is complicated by the presence of three to five carbon substrates and the various pathways through which they can be broken down to acetate. Isotope labelling studies will definitely help shed light into which amino acids are preferentially degraded during anaerobiosis and allow for a more accurate determination of H2 per photon. Studies have suggested that using genetic engineering to develop mutants lacking the ability to carry out state transitioning, cyclic electron transport, and mutants that have a smaller antennae size produced more hydrogen [93, 94].

Hydrogen is also produced in the dark and therefore in the absence of excitation and transfer of electrons by PS I. Noth et al. [91] under anaerobic conditions generated by N2 flushing observed around 0.422 H2 μg Chl<sup>−</sup><sup>1</sup> in the dark compared to 3.128 H2 μg Chl<sup>−</sup><sup>1</sup> in *Chlamydomonas reinhardtii*. Along with the H2, fermentative metabolism in the dark also leads to the production of formate, acetate, and ethanol [87]. Atteia et al. [88] revealed the presence of pyruvate-formate lyase in *Chlamydomonas reinhardtii*, suggesting mixed fermentative metabolism via pyruvate-formate lyase, aldehyde-alcohol dehydrogenase, phosphotransacetylase, and acetate kinase, leading to the formation of formate, ethanol, and acetate [91]. Furthermore, Noth et al. [91] suggested pyruvate ferrodoxin oxidoreductase and not hydrogenase being responsible for H2 production in the dark. The combination of mixed acid fermentation and pyruvate ferrodoxin oxidoreductase suggests the operation of a different set of pathways in dark anaerobic conditions compared to light. More studies are needed to study to confirm this hypothesis and also to test whether these pathways are mutually exclusive. Even though the hydrogen produced were around 87% lower than that observed in the light, the absence of photosystem involvement eliminates the complication of oxygen-induced inhibition of hydrogenases and competition for NADPH by photorespiration. In addition, the complete heterotrophic nature of this production and the valuable co-products such as ethanol and acetate allows for further optimization and scaling up the fermentative reactions, leading up to hydrogen production. Overall, the process of hydrogen production from microalgae via anaerobiosis whether in the light and/or dark clearly has a strong potential, especially with the development of hydrogen fuel cell-powered cars. Given the advantage of being a cleaner source of hydrogen than thermochemical production, hydrogen production from microalgae can definitely benefit more from more research.
