Summary of macromolecular composition and growth rate of microalgae belonging to various taxa.
Despite over a century of research, the various applications of microalgae have only been realized only since the 1940s. With a repertoire of valuable products like biodiesel, astaxanthin, canthaxanthin, lutein, β-carotene, phycocyanin, chlorophyll a, polyunsaturated fatty acids, exopolymeric substance (EPS), and biohydrogen, the commercial importance of microalgae and demand for its product is gaining increasing attention. However, successful transition of the synthesis of microalgal products from laboratory to industries has yet to be realized, even after over 70 years of extensive research. This failure of commercial success of microalgal products can be attributed to the lack of understanding of the physiological role of the products and biological constraint placed by the bioenergetics and physiology, which has been largely ignored. This chapter focuses on the physiological limitations behind synthesis of microalgal products, highlights the crucial unknowns behind the role and synthesis of these products, and hints strategies to overcome the limitations to realize the commercial dream of microalgal products.
The biological reaction of photosynthesis has played a significant role in shaping the course of evolution through the great oxygenation event converting atmospheric CO2 into organic form that is being currently extracted as modern day fossil fuels and finally balancing the elemental cycles (C, N, P, and Si) on Earth. The magnitude of the impact photosynthesis reaction has on humans has encouraged scientists, engineers, and others to wonder about exploiting the potential of this reaction in different ways. Factors such as simplicity, metabolic elasticity, diversity, shorter life cycles, and the range of commercially important products synthesized make microalgae the most attractive and practical option of all the other photosynthesizing life forms. Some of the commercially important products produced by microalgae include biofuels (i.e., biodiesel, biohydrogen), high value products (i.e., astaxanthin, canthaxanthin, β-carotene), polyunsaturated fatty acids (i.e., omega-3-fatty acids), farming products (i.e., animal and fish feeds), fertilizer, single-cell proteins, and miscellaneous products such as exopolymeric substances (EPS) that have various applications. Despite the potential of microalgae in producing such a variety of commercially important products, the idea of microalgae mega factories has yet to become a reality. Several factors impede the commercial success of microalgal products. This includes existing sources (fossil fuels), technological development, and physiological limitation. In this chapter, we will describe the various known physiological limitations associated with productions of various microalgal products in a commercial scale and list possible solutions.
2. Biodiesel from microalgae
Spoehr and Milner in 1949 demonstrated that the lipid content increased in the green microalga
|Microalga||Taxa||%Lipid||%Protein||%CHO||Growth rate d−1||Source|
|Bacillariophyceae||16–50||31–35||11–17||1.96||[6, 8, 10]|
The current demand of fossil fuel is approximately 100 million barrels per day (~11,563 million liters per day), which is only expected to increase with time (105 million barrels per day by 2021) . To meet this magnitude of global demand, assuming 15 ton ha−1 year−1 lipid productivity from microalgae, around 276 million hectares of land would be required for microalgal cultivation every day. This huge demand can be reduced by increasing the lipid productivity of microalgae by minimizing energy and carbon wasting physiological processes in the cell and redirecting it towards lipid synthesis. RUBISCO, the enzyme catalyzing the dark reaction of photosynthesis, wherein the atmospheric or dissolved CO2 is converted into organic triose phosphate, has a lower affinity to CO2. In addition, oxygen is a competitive substrate for RUBISCO, catalyzing the reaction of photorespiration wherein energy and NADPH are utilized and fixed CO2 is released. Therefore, during CO2 limitation, some species of microalgae utilize a carbon concentration mechanism (CCM) that increases the concentration of CO2 at the site of RUBISCO, leading to the catalysis of the dark reaction of photosynthesis. Although the CCM is an ingenious mechanism evolved to address the lower CO2 affinity of RUBISCO and the competition by oxygen, the process consumes one to two molecules of ATP . This energy can instead be redirected towards lipid synthesis by simply bubbling CO2 into the cultures  or by selection CCM lacking microalgae species.
Finkel et al. found that the median macromolecular composition of nutrient-sufficient exponentially growing microalgae is 17.3% lipid, 32.2% protein, and 15% carbohydrates . For biofuels, lipids are the most important cellular fraction.
Light harvesting systems of microalgae are usually capable of absorbing between 350 and 700 nm; however much of the radiation extending out to 1100 nm remains unused. In addition, Raven et al. calculated a 22% loss of energy even within the spectrum of 350–700 nm . However, anoxygenic photosynthetic bacteria possess bacteriochlorophylls capable of absorbing light up to 1050 nm and perform photochemistry . These bacteriochlorophyll-based photosystem can be engineered into microalgae. By replacing photosystem I, it would extend the range of absorbed photons that can be used for photochemistry , therefore the energy available for lipid synthesis. It has been proposed that smaller cells can photosynthetically perform better larger cells [26, 27], primarily attributing to the “package effect.” Package effect refers to the inverse relationship between light harvesting ability of the cells and the cell volume. However, studies have shown that larger cells tend to decrease the antennae size, thereby counteracting the negative impacts of package effect [28, 29]. Moreover, several studies have shown that decreasing the antennae size significantly increased the photosynthetic efficiencies [30, 31]. This could be due to decreased photoinhibition and decreased allocation of resources towards light harvesting systems . However, the hypothesis that larger cells would be less photosynthetically efficient than smaller cells due to package effect was recently disproved by a recent study by Malerba et al. , which showed that larger cells developed compensatory mechanisms by reducing the antenna size, increasing the connectivity between the photosynthetic units, and decreasing the levels of photo-protective pigment β-carotene. This in turn minimized the negative significances of larger cell volume-induced package effects. Exposure to extreme conditions such as photoinhibitory light levels, UVA and UVB radiation, and nutrient and temperature stress could result in oxidative damage to cellular components especially proteins. These kinds of damage result in breakdown of the damage component and re-synthesis of these components or adaptive changes to the cellular components. These changes however have a cost in terms of energy (i.e., ATP, NADPH) and resources which could have been directed towards biomass and lipid synthesis. Protein turnover during scotophase has been shown to consume about one-third of the total respiratory ATP production . Exposure to nitrogen-limiting conditions could result in upregulation of nitrogenase in diazotrophic cyanobacteria, which in turn could result in 10% decrease in growth rates [34, 35]. These are avoidable energy and resource expenditure, which can be easily prevented by providing optimal light, nutrient, and temperature during mass cultivation.
Lipids are not secreted/excreted actively by all microalgae, with exception of
Phototrophic modes of cultivation depend on two critical factors, namely, light and carbon dioxide . Therefore, microalgae grown phototrophically can either be limited in light or carbon dioxide or both, limiting the maximum cellular density one can achieve using this mode of cultivation. In addition, various inefficiencies described above with light-harvesting abilities of microalgae, lower affinity for CO2 of RUBISCO, cost associated with protein turnover, and photoinhibition can be simply avoided by growing microalgae heterotrophically and mixotrophically. Extensive amount of research suggests enhanced biomass production under heterotrophic and mixotrophic modes of cultivation, increasing the cellular density to as much as 4 to 5 times [41, 42]. Further modification in these modes of cultivation such as using fed-batch mode has resulted in further increase in biomass as much as two- to fivefold [43, 44]. Therefore, by growing a hybrid strain of lipid-secreting
3. High valued products
Apart from lipids for biodiesel, microalgae synthesize other products of value such as astaxanthin, canthaxanthin, lutein, β-carotene, phycocyanin, chlorophyll
|Product||Use||Species (dominant)||Global market value*|
|Total US$1.2 billion in 2010|
|β-carotene||US$ 300–1500 kg−1|
|Astaxanthin||Feed additive for farmed fish; pigmenter of the fish flesh||US$ 2500–7000 kg−1|
|US$ 60 million|
|C-Phycocyanin||US$ 500 to 100,000 kg−1|
|Docosahexaenoic acid||US$140 kg−1|
|Omega-3 oils||US$1.5 billion|
|Sterols||Pharmaceutical applications or in functional foods||US$ 300 million|
|Polyhydroxyalkanoates||Production of biodegradable plastics|
The major fraction of studies on microalgal PUFA have primarily focused on screening for microalgal species and optimization of conditions that lead to enhanced PUFA products. However, we have no clear understanding on the physiological role of PUFAs in microalgae. It is hypothesized that PUFAs might play a role in homeoviscous adaptation , i.e., the enhanced fluidity provided by increased unsaturation membrane fatty acids. Additionally, the fatty acid composition and therefore the PUFA concentrations appear to have some level species specificity, suggesting varying roles . However, the physiological roles of PUFAs are not yet confirmed in microalgae, and therefore strategic optimization of their synthesis in microalgae has not been realized. Nevertheless, PUFAs make a significant portion of the neutral lipid content synthesized in microalgae; therefore optimization strategies discussed above to enhance biomass and lipid synthesis should also increase the PUFA content in microalgae.
Astaxanthin is another promising product synthesized by microalgae that has already achieved a profit of $200 million per year . Astaxanthin is a red keto-carotenoid pigment ubiquitous in nature and has antioxidative, anti-inflammatory, and anti-apoptotic properties. It is also proposed as a potential therapeutic agent for cardiovascular and neurological diseases . Apart from its pharmaceutical use, this product is also used as a pigment source in aquaculture of salmon and trout . Despite the relatively vast amount of research into its properties and applications, the mass production of astaxanthin is still unable to meet its huge market demand . This problem can be primarily attributed to the lack of knowledge on the underlying mechanisms of why these algae accumulate astaxanthin similar to PUFAs and hence the lack of better strategies to optimally produce the pigment. The most prominent hypothesis regarding its production includes a multifunctional photoprotective response to stress induced by exposure to unfavorable conditions (excess light, UV-B radiation, nutrient deprivation) leading to ROS formation [52, 53, 54]. However, this hypothesis does not explain the synthesis of astaxanthin under heterotrophic conditions [55, 56]. Therefore, more studies are needed to have a better understanding of the physiological roles of astaxanthin.
Nutrient limitation such as nitrogen, phosphorus or sulfur are widely used strategies for inducing astaxanthin accumulation in
Phycocyanin is a phycobiliprotein exclusively produced by cyanobacteria and commercially important as a high value natural blue coloring agent for food. Being a part of light-harvesting pigment-protein complex in cyanobacteria, phycocyanin is very critical in light capture and therefore is an indispensable element in the growth and survival of cyanobacteria. Phycocyanin has been shown to constitute up to 60% of the total cellular protein content . As it is involved in light capture, its synthesis is tightly regulated by the wavelength of light the cells are exposed to. Green light has been shown to stimulate the synthesis of phycocyanin, whereas red light has the opposite effect . In addition, low light levels have been also shown to induce accumulation of phycocyanin [61, 62]. Other factors such as glucose and salt have also been shown to enhance the synthesis of phycocyanin . Despite knowing their physiological roles, the commercial success of phycocyanin from 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 . 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  followed by the second phase of low levels of blue light to stimulate the synthesis phycocyanin can significantly maximize the total amount of phycocyanin that be produced.
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 . Although initially presumed as experimental artifact or a product of dead and decaying phytoplankton , 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 . 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 . 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 . Polysaccharides usually could account for 80–90% of the EPS composition even under healthy conditions . 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 , and siderophores such as ISIP2a . 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 (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-inflammatory, antivirus, antibacterial, antifungal, or anticoagulant.
Xiao et al.  in their review compared the emulsifying activity of EPS from
5. Hydrogen from microalgae
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 , 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  or using PS II lacking mutants , or selective excitation of PS I through far red light (>710 nm) , or more commonly through sulfur deprivation . 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 . However, only around 20% of this efficiency is practically achieved . 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 , 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  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 , 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.  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.  have detected phosphotransacetylase and acetate kinase in the species
Hydrogen is also produced in the dark and therefore in the absence of excitation and transfer of electrons by PS I. Noth et al.  under anaerobic conditions generated by N2 flushing observed around 0.422 H2 μg Chl−1 in the dark compared to 3.128 H2 μg Chl−1 in
Overall, the successful transition of microalgal products from laboratory to industry largely depends on addressing various physiological limitations of microalgae. Biodiesel production from microalgae, although requires further research, can achieve commercial success by simple modifications such as heterotrophic and mixotrophic cultivation of microalgae in combination with genetic engineering to gain properties such as fast-growing and high lipid-secreting ability in microalgae. High valued products from microalgae can also benefit from similar modification such as heterotrophic and mixotrophic cultivation; however, the lack of knowledge on the physiological role and the biochemical pathway regulating the synthesis of these products demands further research to strategically optimize the production to its maximal potential. EPS production by microalgae, on the other hand, has a benefit of the product being naturally secreted by microalgae; however, its commercial success is hindered by the lack of knowledge of its physiological role and the nebulous nature of its applications. Hydrogen production from microalgae is a promising candidate for being a cleaner source of energy over other alternatives; however, its failure to gain commercial attention is primarily due to the limited research invested.
The Open Access publishing fees for this article have been covered by the Texas A&M University Open Access to Knowledge Fund (OAKFund), supported by the University Libraries and the Office of the Vice President for Research.
Conflict of interest
The authors declare no conflict of interest.
Acronyms and abbreviations
Carbon dioxide hydrogen oxygen nitrogen water iron–sulfur carbon nitrogen phosphorus sulfur silica exopolymeric substances ribulose bisphosphate carboxylase oxidase nicotinamide adenine dinucleotide (reduced) nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate (reduced) nicotinamide adenine dinucleotide phosphate flavin adenine dinucleotide (reduced) flavin adenine dinucleotide carbon concentration mechanism adenosine triphosphate adenosine diphosphate ultraviolet rays A ultraviolet rays B polyunsaturated fatty acids deoxyribonucleic acid ribonucleic acid photosystem I photosystem II
ribulose bisphosphate carboxylase oxidase
nicotinamide adenine dinucleotide
(reduced) nicotinamide adenine dinucleotide
nicotinamide adenine dinucleotide phosphate
(reduced) nicotinamide adenine dinucleotide phosphate
flavin adenine dinucleotide
(reduced) flavin adenine dinucleotide
carbon concentration mechanism
ultraviolet rays A
ultraviolet rays B
polyunsaturated fatty acids