Heterotrophic cultivation of microalgae for pigment production: A review

Biotechnology Advances 36 (2018) 54–67

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Heterotrophic cultivation of microalgae for pigment production: A review a,1

Jianjun Hu

, Dillirani Nagarajan

b,c,1

a

, Quanguo Zhang , Jo-Shu Chang

c,d,⁎⁎

T

a,b,e,⁎

, Duu-Jong Lee

a

Collaborative Innovation Center of Biomass Energy, Henan Agricultural University, Henan Province, Zhengzhou 450002, China Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan d Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan e Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Carotenoids Lutein Astaxanthin Phycocyanin Microalgae Heterotrophic metabolism

Pigments (mainly carotenoids) are important nutraceuticals known for their potent anti-oxidant activities and have been used extensively as high end health supplements. Microalgae are the most promising sources of natural carotenoids and are devoid of the toxic effects associated with synthetic derivatives. Compared to photoautotrophic cultivation, heterotrophic cultivation of microalgae in well-controlled bioreactors for pigments production has attracted much attention for commercial applications due to overcoming the difficulties associated with the supply of CO2 and light, as well as avoiding the contamination problems and land requirements in open autotrophic culture systems. In this review, the heterotrophic metabolic potential of microalgae and their uses in pigment production are comprehensively described. Strategies to enhance pigment production under heterotrophic conditions are critically discussed and the challenges faced in heterotrophic pigment production with possible alternative solutions are presented.

1. Introduction Carotenoids are the most abundant and widely distributed pigments on earth, second only to chlorophyll. Chemically, they are lipophilic isoprenoid compounds composed of a C40 backbone. The structural diversity for the various carotenoids is provided by the number and position of the conjugate double bonds, cyclization at one or both ends and oxygenation of the backbone (Britton, 1995). Carotenes are the hydrocarbon carotenoids, while xanthophylls are the oxygenated version. In photosynthetic organisms like plants and algae, carotenoids are associated with the light harvesting complex of photosynthesis, functioning as accessory pigments and are also known for their photoprotective effect of photosystems from oxidative damage (Varela et al., 2015). Animals and humans are incapable of de novo carotenoid synthesis, and rely on dietary sources for acquirement of these essential nutrients. Carotenoids are important nutraceuticals because of their known beneficial effects including anti-oxidant, anti-ageing, anti-inflammatory, anti-angiogenic, cardio protective and hepato-protective properties (Zhang et al., 2014a,b). The global carotenoid market is estimated at US$ 1.24 billion in 2016 and projected to reach US$ 1.53 billion by 2021, with a compounded annual growth rate (CAGR) of 3.78% from 2016 to 2021 (http://www.marketsandmarkets.com/

Market-Reports/carotenoid-market-158421566.html). Microalgae are being increasingly recognized as a potential source of carotenoids. Carotenoids are essential for the survival of microalgae, to protect the cells from the reactive oxygen species generated during photosynthesis and high light intensity and to dissipate excess light as heat by the xanthophyll cycle. It has been shown that disruption of the enzymes involved in carotenoid synthesis, phytoene synthase and phytoene desaturase can lead to autophagy in Chlamydomonas reinhardtii. Autophagy is triggered in these mutants when exposed to high light, as the photo protection provided by carotenoids is compromised (Pérez-Pérez et al., 2012). The major carotenoid of commercial interest from microalgae are β-carotene, lutein and astaxanthin. Lutein is known for its protective role against macular degeneration of the eye and dietary lutein is important as it cannot be synthesized by humans. Astaxanthin is a well-known anti-oxidant and has cardio protective, neuro protective, anti-cancerous and anti-diabetic properties. β-carotene, is pro-vitamin A and is also an anti-oxidant with cardio protective effects (Spolaore et al., 2006). Microalgae, including cyanobacteria, are among the oldest photosynthetic organisms on earth, and together with the protists, they are the primary producers in aquatic ecosystems. The ability of microalgae to convert atmospheric inorganic carbon to organic biomass with the help of

⁎

Correspondence to: Duu-Jong Lee, Collaborative Innovation Center of Biomass Energy, Henan Agricultural University, Henan Province, Zhengzhou 450002, China. Correspondence to: Jo-Shu Chang, Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan. E-mail addresses: [email protected] (J.-S. Chang), [email protected] (D.-J. Lee). 1 Co-first author. ⁎⁎

http://dx.doi.org/10.1016/j.biotechadv.2017.09.009 Received 4 June 2017; Received in revised form 26 August 2017; Accepted 20 September 2017 Available online 22 September 2017 0734-9750/ © 2017 Elsevier Inc. All rights reserved.

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Neochloris (Molares-Sanchez et al., 2011) are capable of both autotrophic and heterotrophic growth. The major advantages of cultivating microalgae under heterotrophic conditions can be summarized as follows:

sunlight is their hallmark feature, and they are being increasingly recognized as a potential biofuel feedstock in the recent years. The unicellular nature of microalgae simplifies large scale cultivation and the microalgal biomass with its constituent components has applications in various industries ranging from pharmaceutical, nutraceutical and health care products (Mata et al., 2010). The global algae market is expected to be worth of about US$ 1.1 billion by 2024, with a CAGR of around 7% (http://www. transparencymarketresearch.com/pressrelease/algae-market.htm). The storage components or energy reserves of microalgae are primarily polysaccharides and lipids/triglycerides. Microalgae are capable of accumulating very high levels of lipids (55–70%) and carbohydrates (as high as 70%), when cultivated under appropriate conditions (Vitova et al., 2015). These components can be used in the biofuel industry for the production of biodiesel, bioethanol, biobutanol, biohydrogen, biomethane and so on. The other bioactive compounds extracted from microalgae, like the pigments, functional polysaccharides, and polyunsaturated fatty acids are the green and natural alternatives for a wide variety of chemical based components used in health care and cosmetics (Spolaore et al., 2006). The microalgal biomass itself or the residual biomass left after extraction of any valuable product can be subjected to thermochemical conversion, yielding biochar, natural gas and fuel oils. Complete utilization of the microalgal biomass is thus possible, with the advent of various technologies. Microalgae are very versatile with regard to its adapting technologies and very robust, as they can be seen in almost every environment. Microalgae has also been used for bioremediation of wastewater, as they can grow in marginal land and utilize the nutrients present in any wastewater for biomass production and hence alleviating the problems of eutrophication (Yen et al., 2013). Even though microalgae can be a potential source of carotenoids, mass cultivation of microalgal biomass with optimal light supply is a major concern to be overcome. Based on their metabolism, microalgae can be photoautotrophic, photoheterotrophic, mixotrophic and heterotrophic. The most commonly cultivated microalgae are photoautotrophic, utilizing sunlight and atmospheric CO2 as their energy and carbon source, respectively. Open systems are the most preferred systems for the large scale cultivation of photoautotrophic microalgae as they have many advantages: (i) can utilize sunlight as energy source and atmospheric air as CO2 source, (ii) very low installation and operating costs, and (iii) lower energy consumption (Brennan and Owende, 2010). Most commercial establishments utilize open systems for cultivation of microalgae. But it is limited to certain robust species that grow under specific conditions like high salinity or alkaline pH, protecting the culture from contaminants. Spirulina, Dunaliella and Chlorella are successfully cultivated outdoors for single cell protein and pigment production (Mata et al., 2010). Closed photobioreactors (PBR) are often the cultivation method of choice for the production of high end pharmaceutical compounds that demand the maintenance of axenic cultures in a pure state. PBR requires successful design based on the organism to be cultivated and the product in question, accompanied by high installation and operating costs. In both open systems and PBR, efficient supply of optimal light intensity is still a major concern. In open systems, pond depths are limited to be around 20 cm which allows maximum penetration of light and in closed PBRs vigorous mixing is often needed to prevent a sub-population of cells being in the dark zone undergoing respiratory loss of biomass (Chang et al., 2016). Growing heterotrophic microalgae in the absence of light in conventional bioreactors seems to be a very viable option for economic cultivation of microalgae. Heterotrophic cultivation of microalgae is possible in the existing infrastructure for bacterial fermentations, and the major hurdle of light supply is overcome (Bumbak et al., 2011). A number of microalgal species are capable of growing in the dark using organic carbon sources. Obligate heterotrophs, such as the marine thraustochytrids, are cultivated solely in heterotrophic mode for the production of polyunsaturated fatty acids. Several Chlorella species, like C. protothecoides (Shi et al., 1999), C. vulgaris (Liang et al., 2009), C. zofingensis (Ip and Chen, 2005a) and C. minutissima (Bhatnagar et al., 2010) and other microalgae like Tetraselmis (Azma et al., 2011) and

a) The problem of optimal light supply for the culture is overcome. Heterotrophic microalgae can grow and metabolize in the absence of light or under dark conditions, using an organic carbon as the energy and carbon source (Chen, 1996). b) Cell densities in the order of 100 g/L can be achieved in heterotrophic cultivation, which in turn simplifies harvesting of the biomass (Morales-Sánchez et al., 2015). In contract, under photoautotrophic conditions, the maximum cell density of microalgae that can be achieved in photobioreactors is around 40 g/L, while in outdoor open-pond or raceway-pond cultures, the cell concentration is usually lower than 10 g/L. This significantly increases the energy consumption of cell harvesting and the cost of biomass production (Scaife et al., 2015). c) The heterotrophic cultivation can be carried out in conventional industrial scale fermenters, which offer a better control over the process parameters like pH, temperature, oxygen levels and carbon source (Perez-Garcia et al., 2011). Substrate inhibition with very high initial substrate concentration can be overcome by process strategies, such as fed-batch and continuous fermentations, and even at very high cell densities, the cell growth is not limited by selfshading of light supply that normally happens in photoautotrophic systems (Chen et al., 2011). d) From the economic perspective, heterotrophic cultivation could be much more beneficial than photoautotrophic cultivation. The input energy to ATP conversion ratio is higher for heterotrophic cultivation (18% of energy obtained can be converted to ATP, while only 10% was converted under photoautotrophic conditions (Yang et al., 2000)). Similar observations were made by Behrens (2005), who calculated the conversion efficiency of input energy in the form of electricity to ATP and NADPH and concluded that heterotrophic cultivation is economically more advantageous than photoautotrophic cultivation; the cost per kg of dry biomass for heterotrophic cultivation was calculated as US$ 2, while for photoautotrophic cultivation it was about US$ 11 (Behrens, 2005). Although heterotrophic cultivation possesses many advantages as mentioned above, it does not fix CO2 during growth (unlike photoautotrophic growth). Instead, it generates CO2 so does not positively contribute to the mitigation of global CO2 emissions. From economic view, the major cost for conducting heterotrophic cultivation is the installation and equipment costs which account for up to 42% of total investment costs, as well as the cost of organic carbon source (e.g., glucose or acetate) (Lowrey et al., 2015). Compared to PBRs, the microbial fermenters are not specially designed for a particular microalgal species and the universal design can be mass produced and acquired at comparable costs (Behrens, 2005). It is also possible to reduce the costs of carbon source and other nutritional requirements by the use of waste biomass resources and recycling of nutrients and media in cultures (Lowrey et al., 2016). Comparison of cost evaluation and life cycle assessment (LCA) on pigments production from autotrophic and heterotrophic microalgal cultures has been lacking, but a comparative LCA for biodiesel production from microalgae by photoautotrophic (sunlight and CO2), mixotrophic and heterotrophic cultivation (sugarcane and sugar beet as carbon source) is available (Orfield et al., 2015). The study showed that among the three cultivation modes, the net energy ratio (NER) was the highest for heterotrophic method. NER for heterotrophic cultivation was around 0.6 to 1.6, while for photoautotrophic cultivation it was 1.3 (Orfield et al., 2015). In a similar study, LCA assessment of biodiesel production from the heterotrophic cultivation of a marine Thrasutochyrid using glycerol as a carbon source revealed that the biodiesel derived from heterotrophic 55

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secondary transporters facilitating the transport of metabolites across the membrane (Fischer, 2011) and completing the metabolic network comprising of the cytosol, chloroplast and mitochondria. It has been reported that Chlamydomonas chloroplasts can export 3-phosphoglycerate, dihydroxyacetone phosphate, hexose phosphates and glycolate in the presence of light (Johnson and Alric, 2013). Once glucose has been converted to pyruvate, it can be further metabolized by aerobic or anaerobic pathways i.e., respiration or fermentation. For photoautotrophic cells, in the absence of photosynthesis in dark, anoxia develops and the sugars from storage polysaccharides undergo fermentation. C. reinhardtii is endowed with the complete set of enzymes for the fermentation of pyruvate to acetate. Anaerobic fermentation of pyruvate in C. reinhardtii under anoxic conditions is associated with the evolution of hydrogen, as the hydrogenase genes of are induced under anoxic conditions. Under aerobic conditions in heterotrophic algae, respiration occurs with the complete oxidation of glucose to CO2 via EMP, PPP and the tricarboxylic acid cycle (TCA cycle) and ATP is generated by oxidative phosphorylation. Dark respiration in photoautotrophic during alternate light/dark cycles is mainly maintenance respiration, which provides energy for macromolecular turnover and maintenance of the gradient across cell membranes in solution, and growth respiration occurring in heterotrophic cells provides all the necessary components for growth: ATP, carbon skeletons and reducing equivalents (Raven, 1976; Geider and Osborne, 1989). Since oxygen is required for respiration, aeration of the cultures is an important parameter for algal cultures grown heterotrophically on glucose. When microalgae are grown under heterotrophic conditions, the respiration efficiency is very close to the theoretically maximum for cell growth, whereas under dark conditions, respiration of photoautotrophic cells contributes only up to 30% of the biomass growth (Perez-Garcia et al., 2011). Acetate is another commonly used organic carbon source for the mixotrophic and heterotrophic cultivation of microalgae. Acetate uptake in microalgae is ATP dependent, probably involving a monocarboxylic acid/ proton transporter protein and once in the cytosol, acetate is converted to acetyl CoA. The conversion can be a single step reaction catalyzed by acetyl CoA synthase or a two-step method involving acetate kinase and phosphate acetyltransferase (Johnson and Alric, 2013). Acetyl CoA can enter TCA cycle or can be metabolized by glyoxylate cycle, an alternative cycle for the conversion of monocarboxylic acids. Glyoxylate cycle bypasses the CO2 releasing steps of TCA cycle and is more efficient in incorporating the imported carbon into biomass. Glyoxylate cycle is also important in gluconeogenesis, which results in starch accumulation in cells grown on acetate in dark. The by-products of glyoxylate cycle are succinate, fumarate, malate, and oxaloacetate, and of these the first step of gluconeogenesis catalyzed by phosphoenol pyruvate (PEP) carboxykinase converts oxaloacetate to phosphoenol pyruvate (Johnson and Alric, 2012). Glyoxylate cycle has also been reported in certain cyanobacteria (Zhang and Bryant, 2015), although it is absent in Synechocystis sp. (Knoop et al., 2013). Disruption of the key enzyme of glyoxylate cycle, isocitrate lyase in C. reinhardtii redirects the carbon flux towards lipid accumulation. There is a decrease in the enzymes of glyoxylate cycle, gluconeogenesis and the β-oxidation of fatty acids. Since oxidation by TCA cycle is enhanced, there is an elevation in response to oxidative stress, with an increase in superoxide dismutase and ascorbate peroxidase (Plancke et al., 2014). Despite the presence of a genome encoded, central carbohydrate metabolizing pathway, many microalgae are incapable of growth on organic carbon substrates under dark conditions. The main reason attributed to obligate phototrophy is the absence of specific sugar transporters in many species (Chen and Chen, 2006). Microalgae that are capable of growth on glucose possess a glucose/H + symporter that can simultaneously transport one molecule of glucose and one proton at the expense of one molecule of ATP (Tanner, 2000). In Chlorella, this transporter is induced in the presence of glucose under dark conditions

microalgae has a superior or comparable greenhouse gas footprint as that of fossil fuel derived diesel. The greenhouse gas footprint for fossil derived diesel, hydroprocessed microalgae derived biodiesel and transesterified microalgae derived biodiesel were 85.1 g CO2 emission (CO2e)/MJ, 71.5 g CO2e/MJ, 89.9 g CO2e/MJ, respectively (Chang et al., 2014). The comparatively high costs associated with such methods are offset by the high cell densities achieved and also they are perfectly suited for the axenic cultivation of microalgae for the production of high quality bioactive compounds as pharmaceutics (Morales-Sánchez et al., 2015). In this review, we discuss the metabolic aspects of microalgae in heterotrophic mode of cultivation and focus mainly on the production of photosynthetic pigments from microalgae in dark. Pigment production in microalgae is mainly associated with light intensity and the challenges faced in dark cultivation are also discussed in detail. 2. Microalgal metabolism in heterotrophy All organisms require carbon skeletons as building blocks, energy and reducing equivalents for the synthesis of proteins, nucleic acids and other biopolymers essential for the growth and survival of the cell. Energy is required for metabolism and cell division. Metabolic requirements of anabolism are generally met by catabolism. The energy and carbon requirements of microalgae vary depending on their metabolic mode. In photoautotrophic mode, sunlight is utilized as the energy source and CO2 is used as the carbon source (Chen et al., 2011). In the light reactions of photosynthesis, cells utilize sunlight as an energy source to excite the photosystems and drive photo phosphorylation, obtaining the reductant NADPH2 and energy rich ATP (Masojıdek et al., 2012). The reducing equivalents and ATP are utilized in the dark reactions and inorganic carbon is converted into carbohydrates, as represented by the following equation:

CO2 + 4H+ + 4e− → (CH2 O) + H2 O The reaction requires 2 molecules of NADPH2 and 3 molecules of ATP to fix one molecule of CO2. The enzyme RuBisCO (Ribulose-1,5bisphosphate carboxylase/oxygenase), catalyzes the addition of CO2 to ribulose bisphosphate, forming 2 molecules of phosphoglycerate, which is further processed to yield triose phosphates. Ribulose 5 phosphate is regenerated for further CO2 fixation. (Masojídek et al., 2012). In heterotrophic mode, the organic carbon acquired by microalgae are catabolized or degraded, similar to bacteria. Microalgae are genetically capable of metabolizing organic carbon for energy and carbon (Chen, 1996). The presence of carbon metabolizing enzymes in microalgae reflects their basic metabolic mode: they store the fixed carbon as polymers like starch and this is later broken down and utilized in dark conditions for biomass growth and cell division. Starch provides glucose for the central carbohydrate pathway, either the EmbdenMayerhoff-Parnas Pathway (EMP pathway or glycolysis) or the Pentose Phosphate pathway (PPP), yielding NADH and ATP (Boyle and Morgan, 2009). In C. reinhardtii, the glycolysis pathway is compartmentalized; the initial steps or the upper half including the enzymes for starch breakdown, glucose phosphorylation and conversion of glucose to glyceraldehyde-3-phosphate is present in the chloroplast, whereas the final steps or the lower half of the cycle, from glyceraldehyde-3-phosphate to pyruvate is present in the cytosol (Klein, 1986). The genes for glycolysis are not encoded in the chloroplast genome, but in the nuclear genome (Maul et al., 2002). Starch is hydrolyzed to glucose, phosphorylated and fed into glycolysis in the chloroplast. Glyceraldehyde-3phosphate is then exported into the cytosol for conversion to pyruvate. In contrast, in the other pathway for glucose utilization (i.e., the PPP pathway), the enzymes associated with this pathway are seen in both chloroplast and cytoplasm, while it was reported that a major part of the conversion occurs in the chloroplast (Klein, 1986). The plastid membrane or the chloroplast membrane is loaded with a variety of transporters of various specificities including ion channels, primary and 56

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in a specific location in the human body and function as a part of the system: together with zeaxanthin, lutein and its oxidized derivatives are accumulated in the macula of human eye. The macular pigment, as they are called, protect the eyes from oxidative damage by blue light, improve visual acuity and increased lutein intake was shown to protect from age related macular degeneration (Krinsky et al., 2003). As for all carotenoids, humans depend on dietary intake of lutein. Lutein, and its oxidized forms has been identified in human fetal eyes right from the second trimester, indicating the protective role of lutein in eye health (Panova et al., 2017). Lutein has also been positively associated with improved respiratory health, probably because of its anti-oxidative effect (Melo van Lent et al., 2016). The structure of lutein has a long polyene structure with conjugated double bonds that endows lutein with its potent anti-oxidant property. The anti-oxidant activity of lutein has been implicated in protection against cardiovascular diseases (Wang et al., 2013; Chung et al., 2017), oxidative damage caused by UV-B radiation (Aimjongjun et al., 2013) or the drug cisplatin (Serpeloni et al., 2012). Lutein is also identified as the primary ingredient in microlagal extractions that protect the eye from harmful glycosylation of retina in diabetic individuals, implicating its protective role against diabetic retinopathy (Sun et al., 2011). Lutein is used as a food additive (E161b in the European Union) and also as a feed additive to deepen the color of egg yolks and brighten the feather of poultry (Lin et al., 2015). The global lutein market was estimated as US$ 135 million in 2015 and will continue to rise until 2024 with a growth rate of 5.3% (Global Market Insights Lutein Market Report, 2016). The current commercial supply of lutein is solely dependent on marigold flower petals of the genus Tagetus (Lin et al., 2015). The petals are harvested and pure lutein or lutein esters are extracted in a multi-step process dependent on the final product of use. The major disadvantages of using terrestrial plants like marigold as a lutein source is the seasonal variation in availability of flowers for processing and the requirement of skilled labor for processing and extraction. Microalgae can be the potential source of lutein, as many algal species like Muriellopsis, Scenedesmus, Dunaliella and Chlorella are capable of producing lutein at high cellular content from 4 to 7.5 mg/g dry weight of algal biomass (FernándezSevilla et al., 2010). Heterotrophic cultures have also been used for the production of lutein, mainly from the genera Chlorella, as it can be seen from Table 1. Lutein yields as high as 5.3 mg/g has been obtained from heterotrophic cultivation (Shi, 2002) and about 7.5 mg/g in mixotrophic cultivation (Chen et al., 2017), which is comparable to photoautotrophic cultivation. Glucose is the preferable carbon source for C. protothecoides and C. pyrenoidosa for heterotrophic cultivation, while acetate seems to be the preferred carbon source in mixotrophic cultivation for lutein production (Table 1). Various carbon sources for heterotrophic growth and lutein production was tested for C. pyrenoidosa, and it was found that glucose promoted growth and cellular lutein content, while even a combination of other sugars were not utilized properly, resulting in poor cell growth and lower lutein content (Theriault, 1965). Glycerol has been used with little success in mixotrophic cultivation of Scenedesmus sp. (Yen et al., 2011). C. protothecoides CS-41 seems to be the prototype strain for heterotrophic lutein production, with higher lutein yields obtained from N-limited, fed-batch cultures (Shi, 2002). Glucose is the preferred carbon source (Shi et al., 1999) and urea is the optimal nitrogen source for higher biomass growth (Shi et al., 2000), while an increase in temperature from 25 °C to 35 °C improved cellular lutein content (Shi et al., 2006). Instead of light stress, lutein accumulation in heterotrophic cultures can be triggered by inducing oxidative stress with the addition of chemicals like hydrogen peroxide, sodium hypochlorite and ferrous ions. In C. protothecoides, the addition of these chemical induced carotenogenesis and the highest lutein content of 2 mg/g was obtained (Wei et al., 2008). Thus, heterotrophic cultivation of microalgae is a promising approach in facing the demand for natural lutein in the nutraceutical market.

(Komor and Tanner, 1974), and a set of amino acid transporters are also co-induced facilitating the transport of amino acids like glycine, L-alanine, L-proline, L-serine, L-arginine L-lysine and proline (Cho et al., 1981). In Chlamydomonas, uptake of glucose from the medium increases with increase in the concentration of glucose, and reached a plateau at about 5 mg/L, suggesting the presence of a transport system that could be saturated, but none is reported till now (Bennet and Hobbie, 1972). The low concentrations of glucose transported into the cells were mainly used for respiration but not for growth, and the authors suggested that growth alone cannot measure the heterotrophic metabolic capacity in any given microalgae. Recently, an inducible D-xylose/H + symporter was reported in D-glucose induced C. sorokiniana. Xylose transport was inhibited in the presence of hexoses like glucose, galactose and fructose, but not by arabinose or ribose (Zheng et al., 2014b). The presence of xylose also induced the expression of xylose utilizing enzymes - xylose reductase (XR) and xylitol dehydrogenase (XDH), resulting in the accumulation of xylitol (Zheng et al., 2014b). This is similar to the induction of the hexose transporters in Chlorella sp. by hexoses, but not by pentoses or other disaccharides (Komor et al., 1985). Another important reason for obligate phototrophy could be the presence of an inefficient or insufficient carbon metabolizing pathway (Wood et al., 2004). This is similar to obligate heterotrophs like Thraustochytrids, as they lost the photosynthetic power during evolution, but still possess a non-functional chloroplast (Mishra, 2015). It has been reported that for some obligate autotrophic cyanobacteria, incorporation of the imported carbon into building blocks required for biomass growth was restricted to certain groups of amino acids due to the presence of an incomplete TCA cycle, which provides the intermediates for cell growth (Smith et al., 1967). It was shown that the acetate used as carbon source contributes for only 10% of the newly synthesized carbon, whereas chemoautotrophs assimilated the imported acetate in over 40% of their building blocks. Metabolic analyses revealed certain features typical of incomplete TCA cycle: absence of αketoglutarate dehydrogenase, extremely low levels of malic and succinic dehydrogenase, and absence of NADH2 oxidase (Smith et al., 1967). As the TCA cycle is the main source of metabolic intermediates for biomass growth, an impaired TCA cycle will not support growth but only provide energy. For heterotrophic cultivation, the most important characteristics that should be seen in a microalgae are as follows: active growth and metabolism in the absence of light, growth on relatively inexpensive sterilized media, the ability to withstand hydrodynamic stresses that could be experienced in conventional fermenters, and adaptability to harsh environmental conditions (Chen and Chen, 2006). A few other desirable characteristics of a microalgae to be successfully cultivated in heterotrophic conditions include: versatile substrate utilization capacity - the ability to utilize a variety of organic carbon sources, prompting the utilization of waste lignocellulosic biomass and other materials as a carbon source, and higher biomass productivity to obtain very high cell density cultures under dark conditions. Light intensity is the major growth limiting part in photoautotrophic cultivation and for pigment production. In the following sections, pigment production by microalgae in heterotrophic conditions is discussed in detail. 3. Pigment production by heterotrophic cultivation 3.1. Lutein Lutein ((3R, 3′R, 6′R)-β, ε-carotene-3, 3′-diol) is a primary xanthophyll pigment present in plants and green algae, involved in light harvesting during photosynthesis and protection of the photosystems from photo oxidative damage. Lutein is responsible for the bright yellow color seen in many flowers, fruits and vegetables and is especially rich in green leafy vegetables (kale, spinach, broccoli, peas, parsley), corn and egg yolks. It is the only carotenoid to be accumulated 57

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Table 1 Mixotrophic and heterotrophic cultivation of microalgae for the production of lutein. Microalgae used

Carbon source

Cultivation mode

Lutein content

Lutein productivity

Reference

Chlorella pyrenoidosa 7-11-05 Chlorella pyrenoidosa 7-11-05 Chlorella pyrenoidosa 15-2070 Chlorella pyrenoidosa 15-2070 Chlorella sorokiniana 211-32 Chlorella sorokiniana MR-1 mutant Chlorella sorokiniana CCAP 211/8K Chlorella sorokiniana CCAP 211/8K Chlorella sorokiniana Mb-1 Chlorella sorokiniana Mb-1 Chlorella sorokiniana Mb-1 M-12 Chlorella sorokiniana Mb-1 M-12 Chlorella protothecoides CS-41

Glucose 30 g/L Glucose 230 g/L Glucose 40 g/L Glucose 10 g/L 100 mM glucose 40 mM acetate 100 mM glucose 40 mM acetate Acetate

Mixotrophic, fed batch, tetracycline 100 μg/mL Heterotrophic, batch, Erythromycin 20 μg/mL Heterotrophic, batch

218 mg/L

–

Theriault (1965)

378 mg/L xanthophylls, with lutein for around 70% 2.5 mg/g

–

Theriault (1965)

Heterotrophic, fed-batch with 400 g/L glucose Mixotrophic, batch

178 mg/L

Wu et al. (2007)

33 mg/L, 2.6 mg/g

Cordero et al. (2011)

Mixotrophic, batch

22 mg/L, 2.8 mg/g

Cordero et al. (2011)

Mixotrophic

1.07 mg/g

Acetate

Cyclic autotrophic/heterotrophic

1.16 mg/g

Acetate 6 g/L Acetate 6 g/L

Mixotrophic, semi batch Mixotrophic, batch

5.21 mg/g 5.86 mg/g

5.67 mg/L·d 2.39 mg/L·d

Wagenen et al. (2015) Wagenen et al. (2015) Chen et al. (2016) Chen et al. (2017)

Acetate 6 g/L

Mixotrophic, batch

7.52 mg/g

3.63 mg/L·d

Chen et al. (2017)

Acetate 6 g/L

Mixotrophic, Outdoor cultivation in tubular PBR Heterotrophic, batch, Basal medium

6.85 mg/g

1.35 mg/L·d

Chen et al. (2017)

16 mg/L, 4.6 mg/g

Shi et al. (1997)

Heterotrophic, batch, Kuhl medium Heterotrophic, batch, Basal medium, 30L fermenter Heterotrophic, batch, 3.7L fermenter

14.8 mg/L, 4.4 mg/g

Shi et al. (1997)

66.3 mg/L, 4.9 mg/g

Shi et al. (1997)

2.63 mg/g

Zhang et al. (1999)

Heterotrophic, batch, 3.7L fermenter

4.45 mg/g, 77.43 mg/L

Shi et al. (2000)

Heterotrophic, batch, 3.7L fermenter

4.29 mg/g, 68.42 mg/L

Shi et al. (2000)

Heterotrophic, batch, 3.7L fermenter

4.58 mg/g, 83.81 g/L

Shi et al. (2000)

Mixotrophic

6.48 mg/g

Shi and Chen (1999)

Heterotrophic

4.43 mg/g

Shi and Chen (1999)

Heterotrophic, 30L fermenter, fed batch, N-limited Heterotrophic, batch, NaClO, H2O2, Fe2 + as inducers Mixotrophic, Repeated fed batch Mixotrophic, batch

5.35 mg/g, 209 mg/L

49.18 mg/L.d

Shi et al. (2002)

1.98 mg/g, 31.4 mg/L

–

Wei et al. (2008)

Glucose 9 g/L Glucose 6 g/L Glucose 36 g/L,

Chlorella protothecoides CS-41 Chlorella protothecoides CS-41

Chlorella protothecoides CS-41

Chlorella protothecoides CS-41 Chlorella protothecoides UTEX 29 Scenedesmus sp. Coccomyxa acidophila

Wu et al. (2009)

Glucose 40 g/L Urea 3.6 g/L Glucose 40 g/L Nitrate 1.7 g/L Glucose 40 g/L Ammonium 1.7 g/L Glucose 40 g/L Urea 1.7 g/L Glucose 40 g/L Nitrate 10 g/L Glucose 40 g/L Nitrate 10 g/L Glucose 40 g/L Urea 3.6 g/L Glucose Glycerol 3 g/L Urea 0.67 g/L

0.2% by weight, 2.5 mg/g 3.55 mg/g

.

0.36 mg/L d

Yen et al. (2011) Casal et al. (2011)

reproductive performance and egg quality of aquatic animals due to the potent anti-oxidant activity and has been shown to enhance disease resistance and immune response in farmed fishes against infectious diseases (Lim et al., 2017). The US FDA approved astaxanthin as a nutritional supplement in 1999, owing to its beneficial effects as a potent anti-oxidant (Fassett and Coombes, 2011). The anti-oxidant activity of astaxanthin is shown to be superior to other carotenoids zeaxanthin, lutein, canthaxanthin and β-carotene and is 500 times more potent than α-tocopherol, and hence astaxanthin has been dubbed as “super vitamin E” (Lorenz and Cysewski, 2000). The molecular structure of astaxanthin is attributed for the potent anti-oxidant activity. Structurally, astaxanthin is composed of a long, unsaturated polyene chain of 40 carbon atoms, with two terminal polar ionone rings, each with an unreacted hydroxyl group. The polar end- non polar chainpolar end structure is strikingly similar to membrane structure, which is also polar-non polar-polar orientation, and astaxanthin can traverse the cell membrane. Hence, astaxanthin can be found in cell membranes and lipoproteins protecting them from oxidative damage. When positioned in the cell membrane, astaxanthin does not alter the structural integrity or the electron density of the double layered membrane (McNulty et al.,

3.2. Astaxanthin Astaxanthin (3,3′-dihydroxy-β, β-1-carotene-4,4′-dione) is a secondary xanthophyll and are the oxygenated derivatives of carotenoids. Astaxanthin can be commonly observed as the bright pink color seen in marine organisms like salmonid fishes, shrimps, krill, crabs, lobsters, crayfish, trout and other crustaceans. They are also responsible for the vibrant feather color in birds like flamingo, quails and storks. Astaxanthin is primarily produced by marine algae and bacteria. As mentioned previously, humans are incapable of carotenoid synthesis and are totally dependent on dietary intake. Seafood is the major source of astaxanthin for humans and its almost always insufficient. The key application of astaxanthin is mainly in the aquaculture industry as a feed additive, to enhance the color in farmed fish and shrimp (Yaakob et al., 2014). The United States Food and Drug administration (FDA) approved astaxanthin as a feed additive in 1987 (Fassett and Coombes, 2011). Supplementing astaxanthin is not only required for color improvement, it is also needed for producing good quality seafood for consumption. It has been reported that addition of astaxanthin has improved growth rate and survival of larvae in aquaculture, improved 58

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1997; Sarada et al., 2002). However, commercial cultivation of H. pluvialis is challenged by slow growth rate of the strain and the contamination with faster growing microalgae and bacteria in open cultivation systems (Liu et al., 2014). The heterotrophic Chlorella zofingensis is being touted as the potential alternative for astaxanthin production under dark conditions (Liu et al., 2014). C. zofingensis is capable of growth in the dark using glucose, fructose, and sucrose as carbon sources, without much compromise on the production of astaxanthin (Liu et al., 2012). Versatility in substrate utilization has been utilized by growing the algae in waste biomass resources like waste molasses as carbon sources (Liu et al., 2012; Liu et al., 2013). Carotenogenesis can be induced in C. zofingensis by the addition of chemicals that can create oxidative stress like sodium hypochlorite and hydrogen peroxide (Table 2). Moreover, commercial success with Chlorella heterotrophic cultivation has been previously reported in Japan (Lee, 1997) with biomass yields as high as 80 g/L (Iwamoto, 2004), and cultivation of astaxanthin producing Chlorella species might just be logistically possible with high yields.

2007). The structure has in total 13 double bonds, making it the most potent anti-oxidant. Astaxanthin is also known for its cardio protective properties (Régnier et al., 2015; Kishimoto et al., 2016), by protecting the endothelial cells from oxidative damage and preventing fat deposits. Astaxanthin can enhance humoral immunity in mice by increasing antibody production depending on the antigen, suppress nonspecific polyclonal B-cell activation and increase the production of high-affinity, specific IgG antibodies (Jyonouchi et al., 1994). Astaxanthin also augmented tumor immunity in mice and delays autoimmune responses, implicating the possible therapeutic role of astaxanthin in delaying auto immune response in autoimmune prone patients and reduces cancer risk by enhancing T-helper cell functions (Jyonouchi et al., 1994). The market value for astaxanthin was estimated at 280 metric tons valued at US$ 447 million in 2014, was further projected to reach 670 metric tons valued at US$ 1.1 billion by 2020 (Industry experts market report, 2015). Commercial astaxanthin market is dominated by synthetic astaxanthin derived from petrochemical derivatives (> 95%), with the companies DSM and BASF leading the production (Lim et al., 2017). However, natural astaxanthin derived from H. pluvialis proved to be superior to the synthetic derivative; the intracellular antioxidant capacity of naturally occurring esterified astaxanthin from H. pluvialis is 90 times more potent than the synthetic astaxanthin without any reported toxicity. Synthetic astaxanthin is present in free form, while naturally occurring astaxanthin derived from H. pluvialis is esterified with the presence of one or two fatty acids facilitating absorption and bioactivity (Régnier et al., 2015). Rising awareness among customers and increasing regulations for the use of synthetic derivatives has also been instrumental in increasing the demand for natural astaxanthin. Natural astaxanthin has been derived from three main sources: the yeast Xanthophyllomyces dendrorhous, the bacteria Agrobacterium aurantiacum and Paracoccus carotinifaciens (Dufosse, 2006), and green algae: mainly Haematococcus pluvialis (Li et al., 2011), and more recently Chlorella zofingensis (Liu et al., 2014) and Chlorococcum. Of these, only astaxanthin derived from H. pluvialis has received the FDA approval for use as human nutritional supplement, while others are primarily used as aquaculture feed. H. pluvialis is the most promising producer of astaxanthin, as it can grow rapidly and accumulate astaxanthin up to 5% by weight under stress conditions like high light, salinity stress, nutrient deprivation and ion stress. The actively growing green vegetative cells, upon cessation of cell division by stress conditions or nutrient deprivation turn into hematocysts or aplanospores, accumulating astaxanthin and turning red. Haematococcus astaxanthin currently accounts for about 1% of the market demand, and production is severely lagging behind demands leading to high prices. Heterotrophic cultivation of microalgae for astaxanthin production is an economically feasible option for commercially viable astaxanthin. Heterotrophic cultivation of various microalgae for the production of astaxanthin is summarized in Table 2. Heterotrophic growth of H pluvialis has been reported much earlier (Droop, 1955), but was not pursued due to the relatively low growth compared to autotrophic conditions and the belief that astaxanthin can accumulate only under high light stress. Under dark conditions, H. pluvialis can assimilate acetate, and addition of acetate in the medium enhanced carotenogenesis, with rapid transition to hematocysts which was delayed in the absence of acetate (Kobayashi et al., 1991; Choi et al., 2002). The addition of a photosynthesis inhibitor did not prevent growth with acetate under illumination, while acetate assimilation rate was doubled in the absence of light (Kobayashi et al., 1992). Ferrous ions are known to enhance astaxanthin accumulation and when added with acetate astaxanthin accumulation was higher (Kobayashi et al., 1991; Ma and Chen, 2001b). Salt stress, with increasing NaCl concentration is known to enhance astaxanthin accumulation in H. pluvialis (Kobayashi et al.,

3.3. Phycocyanin Phycocyanin (PC) is the blue colored phycobiliprotein found exclusively in cyanobacteria, cryptophytes and red algae. PC often imparts the characteristic blue color to cyanobacteria, and hence the name blue green algae. The chromophore present in PC is an open chain tetrapyrrole ring called phycocyanobilin. Structurally, PC is composed of two protein subunits: α-chain with one phycocyanobilin unit and a βchain with two phycocyanobilin units (Contreras-Martel et al., 2007). In cyanobacteria and red algae, PC is accumulated in specialized organs called phycobilisomes and take part in photosynthesis. PCs are the major light harvesting pigments in cyanobacteria with an absorption maxima of 620 nm (Eriksen, 2008). PC is known to possess anti-oxidative, anti-inflammatory, anti-carcinogenic, neuro-protective and hepato protective effects bestowed by the chromophore phycocyanobilin (Romay et al., 2003; Fernandéz-Rojas et al., 2014). The phycocyanobilin can be reduced to phycocyanorubin, which is structurally similar to the plasma anti-oxidant bilirubin. PC has the potential to be a commercial anti-oxidant since the commercial form of bilirubin called biliverdin needs to be extracted from animals and could be easily replaced by natural PC (Sørensen et al., 2013). Currently, PC is mainly used as a coloring agent because of its vibrant color and was approved for use as a coloring agent by the U.S FDA. Phycocyanin derived from Arthrospira platensis is used as a natural pigment in chewing gums, candies, jellies and dairy products (Sekar and Chandramohan, 2008). PC absorb orange and red light at 620 nm and emits fluorescence at 650 nm and hence they are widely used in clinical and immunological labelling assays (Spolaore et al., 2006). They can be conjugated to antibodies and proteins to be used in diagnostic assays. Photoautotrophic cultivation of A. platensis is the sole source of natural PC (Eriksen, 2016) and it is cultivated in open systems in the Asia-pacific regions particularly (Lee, 1997). Despite the challenges associated with the cultivation of A. platensis and extraction of intracellular PC, there are no other particular sources of natural PC. A. platensis is also capable of heterotrophic growth with glucose as a carbon source, with a PC content of 57 mg/g, about half of that obtained in mixotrophic cultivation (Marquez et al., 1993). The unicellular red alga, Galdieria sulphuraria, is a promising candidate for heterotrophic PC production. G. sulphuraria is a polyextremophilic algae, capable of growing endolithically in the dark at very low pH. It is capable of growth of 27 different sugars and sugar alcohols as a sole carbon source under heterotrophic and mixotrophic conditions (Gross and Schnarrenberger, 1995). As it can be seen from Table 3, G. sulphuraria is capable of heterotrophic growth and PC production on

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Table 2 Mixotrophic and heterotrophic cultivation of microalgae for the production of Astaxanthin. Microalga used

Carbon source

Cultivation mode

Astaxanthin content

Haematococcus pluvialis Flotow NIES-144

22.5 mM acetate

Mixotrophic, batch, 20 μM DCMU

11 mg/g cells

22.5 mM acetate

Heterotrophic, batch, 20 μM DCMU Heterotrophic, batch, NaCl stress

10.5 mg/g cell

12.5 mg/L 10.3 mg/L 12.58 mg/L 11.78 mg/L

Ip and Chen (2005c)

30 g/L glucose 30 g/L glucose

Mixotrophic, batch Heterotrophic, batch Heterotrophic, batch, 10 mM H2O2, 0.5 mM NaClO Heterotrophic, batch, 1 mM peroxynitrite Heterotrophic, batch, Nitryl chloride Heterotrophic, fed-batch,

Kobayashi et al. (1992) Kobayashi et al. (1992) Kobayashi et al. (1997) Ip et al. (2004) Ip and Chen (2005a) Ip and Chen (2005b)

10.99 mg/L 32 mg/L

Ip and Chen (2005c) Sun et al. (2008)

30 g/L glucose

Heterotrophic, batch

1 mg/g

1.6 mg/L·d

Liu et al. (2012)

30 g/L pretreated molasses 20 g/L Glucose

Heterotrophic, batch

1 mg/g

1.7 mg/L·d

Liu et al. (2012)

Heterotrophic, batch

1.21 mg/g

1.95 mg/L·d

Liu et al. (2013)

20 g/L Fructose 20 g/L Sucrose 20 g/L Sugar mixture 20 g/L Treated cane molasses 40 g/L glucose

Heterotrophic, Heterotrophic, Heterotrophic, Heterotrophic,

1.17 mg/g 1.23 mg/g 1.18 mg/g 1.11 mg/g

1.73 mg/L·d 1.93 mg/L·d 1.79 mg/L·d 1.99 mg/L·d

Liu Liu Liu Liu

44 g/L 44 g/L 44 g/L 44 g/L 44 g/L

Mixotrophic, batch, 0.1 mM H2O2 Heterotrophic, batch, 0.1 mM H2O2 Mixotrophic, batch Mixotrophic, batch, 0.1 mM H2O2 Mixotrophic, batch, 0.1 mM H2O2, 0.5 mM Fe2 + Mixotrophic, batch, 0.01 mM methyl viologen, 0.5 mM Fe2 + Heterotrophic, batch

Haematococcus pluvialis Flotow NIES-144 Chlorella zofingiensis ATCC30412 C. zofingiensis ATCC30412 C. zofingiensis ATCC 30412 C. zofingiensis ATCC 30412 C. zofingiensis ATCC 30412 C. zofingiensis ATCC 30412

C. zofingensis Mutant E17

Chlorella zofingiensis ATCC 30412 Chlorococcum sp. Chlorococcum sp.

45 mM Acetate 30 g/L glucose 50 g/L glucose 30 g/L glucose 30 g/L glucose

Glucose Glucose Glucose Glucose Glucose

44 g/L Glucose Haematococcus pluvialis

1.986 g/L Sodium acetate

batch batch batch batch

Heterotrophic, batch

30 pg/cell (9 μg/mL)

Astaxanthin productivity

–

Reference

et et et et

al. al. al. al.

(2013) (2013) (2013) (2013)

0.96 mg/g, 10.24 mg/ L 7.08 mg/g 1.8 mg/g 5.8 mg/g 6.5 mg/g 7.1 mg/g

Wang and Peng (2008) Ma and Chen (2001a) Ma and Chen (2001a) Ma and Chen (2001b) Ma and Chen (2001b) Ma and Chen (2001b)

6.3 mg/g

Ma and Chen (2001b)

160 pg/cell

Tripathi et al. (1999)

Table 3 Mixotrophic and heterotrophic cultivation of microalgae for the production of Phycocyanin. Microalga used

Carbon source

Cultivation mode

Phycocyanin content

Galdieria sulphuraria 074G

Glucose 50 g/L

Heterotrophic, batch

3.6 mg/g

Schmidt et al. (2005)

Heterotrophic, batch Heterotrophic, batch Heterotrophic, batch

3.4 mg/g 4.3 mg/g 11.2 mg/g

Schmidt et al. (2005) Schmidt et al. (2005) Schmidt et al. (2005)

Heterotrophic, fed batch

350 mg/L

Schmidt et al. (2005)

G. sulphuraria 074G

Fructose 50 g/L Sucrose 50 g/L Molasses 7.5 g/L plus Glucose 45 g/L Sugar beet molasses sucrose 50 g/ L, total sugar up to 750 g/L Glucose 500 g/L

Heterotrophic, fed batch

1.4–2.9 g/L

G. sulphuraria 074G

Glucose, fructose, glycerol 5 g/L

2–4 mg/g 8–12 mg/g

G. sulphuraria 074G

Glucose 5 g/L Restaurant waste with glucose 5 g/L Bakery waste with glucose 5 g/L Glucose 5 g/L

Heterotrophic, batch Heterotrophic, carbon limited, nitrogen replete Heterotrophic, batch Heterotrophic, batch

Graverholt and Eriksen (2007) Sloth et al. (2006) Sloth et al. (2006)

18 mg/g 20 mg/g

Sloth et al. (2017) Sloth et al. (2017)

Heterotrophic, batch Heterotrophic, batch

21.8 mg/g 25–30 mg/g

Sloth et al. (2017) Sørensen et al. (2013)

Glucose 2 g/L

Heterotrophic, Batch

57 mg/g

Marquez et al. (1993)

Glucose 2 g/L Glucose 2.5 g/L

Mixotrophic, batch Photoheterotrophic, batch

131 mg/g 322 mg/L

Marquez et al. (1993) Chen et al. (1996)

Acetate 2 g/L Glucose 2 g/L

Photoheterotrophic, batch Mixotrophic, fed-batch

246 mg/L 795 mg/L

Chen et al. (1996) Chen and Zhang (1997)

G. sulphuraria strain 074G S. platensis NIES-39 Spirulina platensis UTEX 1926 S. platensis UTEX 1926

60

Phycocyanin productivity

0.5–0.9 g/L·d

Reference

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(Hlavova et al., 2015) (b) developing molecular tools and kits for the genetic modification of the genome, and (c) design and development of expression vectors in algae for the expression of heterologous proteins and so on and so forth (Radakovits et al., 2010). The pioneering work with regards to heterotrophic cultivation of microalgae is the proposed conversion of obligate phototrophic algae to heterotrophic metabolism. As discussed in detail in Section 2, microalgae are capable of carbohydrate metabolism and the absence of sugar transporters is the obstacle to be overcome. Zaslavskaia et al. reported trophic conversion of the obligate autotroph Phaeodactylum tricornutum to heterotroph by cloning and expressing the glucose transporter gene glut1 from human erythrocytes (Zaslavskaia et al., 2001). Transformed cells achieved five times higher cell densities when grown in light with glucose, and in the absence of light cell densities equal to phototrophic growth has been achieved The glucose transporter gene of Chlorella kessleri hup1 was expressed in Chlamydomonas reinhardtii and the transformant was able to grow in dark with the utilization of glucose. The mutant line Stm6Glc4 showed enhanced photobiological hydrogen production (150%) in the presence of 1 mM glucose, compared to the wild type Stm6 (Doebbe et al., 2007). Trophic conversion of commercially important algae can pave the way for economic high cell density cultivation in conventional fermenters. Another way to improve pigment production is to use random mutagenesis and isolate high yielding mutants of an existing commercial strain of algae. This step is more convenient and direct, instead of going through the process of constructing a pathway or heterologous expression of certain genes. Random mutagenesis has been applied to improve lutein production in C. sorokiniana (Cordero et al., 2011; Chen et al., 2017), and for improving astaxanthin production in H. pluvialis (Tripathi et al., 2001; Gómez et al., 2013). Directed evolution of a gene mimics the evolution process under laboratory conditions, and it involves an iterative process of applying selective pressure to a library of variants to identify mutants with desirable properties (Pourmir and Johannes, 2012). Adaptive laboratory evolution was applied to Phaeodactylum tricornutum by growing the cells in the presence of photo oxidative stress inducing high light stress under light emitting diode (LED) lamps (Yi et al., 2015). The selected strains after the exposure were adapted to the selective process and the carotenoid content in the selected strains were increased. Random mutagenesis with ultraviolet radiation could also produce mutants with high carotenoid accumulating capacity. Astaxanthin has been the target of many genetic engineering approaches, since the commercial source of astaxanthin is chemical synthesis or the only available natural source if from microalgae. Cloning and overexpression of certain key target genes in the astaxanthin producing organisms could direct the flux towards astaxanthin production and improve astaxanthin yield and productivity. Two key genes have been identified for the enhanced production of astaxanthin and can direct the flux of carotenoid synthesis from β-carotene towards astaxanthin. Those include (i) β-carotene hydroxylase (bch) that catalyzes the conversion of β-carotene to zeaxanthin and (ii) β-carotene ketolase (bkt) that catalyzes the conversion of zeaxanthin to astaxanthin. The bkt gene of H. pluvialis has been cloned and overexpressed in Escherichia coli and along with the carotene hydroxylase gene crtZ from Erwinia, accumulation of astaxanthin was shown in E. coli (Breitenbach et al., 1996). An engineered E. coli strain was constructed with the expression of the following enzymes that could generate geranylgeranyl diphosphate and convert it into astaxanthin: isopentenyl diphosphate (IPP) isomerase (idi gene of E. coli), geranylgeranyl diphosphate (GGPP) synthase (gps gene of the archaebacterium Archaeoglobus fulgidus), and the astaxanthin biosynthesis enzymes crtWZYIB genes from the marine bacterium Agrobacterium aurantiacum (Wang et al., 1999). The resultant recombinant strain produced 50 times higher astaxanthin. Similarly, the bkt gene from H. pluvialis (SAG 34-1a) was cloned and overexpressed in the same organism, leading to 2–3 fold increased accumulation of astaxanthin. A higher accumulation of the intermediates echinenone and canthaxanthin were also reported and it

glucose, fructose, sucrose and even waste biomass resources like molasses and restaurant waste. PC yields as high as 30 mg/g can be obtained by fed-batch and high density continuous cultures, and with high cell densities and PC content, extraction and purification can be easier, proving this to be a potential candidate for heterotrophic production of PC. Other than the above mentioned pigments, the microalga Dunaliella salina is also the sole source of naturally derived β-carotene and are exclusively cultured in open ponds under high salinity conditions for the commercial scale production of β-carotene (Spolaore et al., 2006). Dunaliella species is capable of heterotrophic growth with glucose and acetate (Gladue and Maxey, 1994). It was shown that D. salina V-101 can grow mixotrophically with 10 mM glucose under low light intensity, while increasing glucose concentration above 20 mM resulted in acidification of the culture, increasing sensitivity to the antibiotic hygromycin and bleaching of the cells (Anila et al., 2013). Production of β-carotene from D. salina under photoheterotrophic cultivation of D. salina with acetate (Mojaat et al., 2008) and mixotrophic cultivation with glucose (Morowvat and Ghasemi, 2016) have also been reported. However, these were not pursued further due to the low growth potential and low β-carotene productivity of D. salina under sub optimal mixotrophic cultivation. Thus, the major carotenoids that can be extracted in an economically beneficial way from heterotrophically cultivated microalgae are lutein, astaxanthin and phycocyanin. 4. Strategies for improving pigment production under heterotrophic or mixotrophic cultivation of microalgae The most abundant light harvesting pigment in nature is chlorophyll, seen in abundance all around us, in green leafy plants. The other pigments are often masked by chlorophyll, and are often shown in autumn in senescence, where chlorophyll degrades and the synthesis of other pigments are induced (Lev-Yadun and Gould, 2007). Xanthophylls like lutein are the primary carotenoids and are essential for survival under light as they are an integral part of the photosynthetic apparatus. The secondary carotenoids like astaxanthin are mainly synthesized as a stress response and are induced by certain stress like increased reactive oxygen species, salt stress and high light intensity (Guedes et al., 2011). Having said this, it must be noted that xanthophylls can be synthesized in dark in the absence of photosynthetic requirements. Microalgae grown in dark are still genetically capable of producing carotenoids, albeit at lower levels. This is in contrast to thraustochytrids, the obligate heterotrophs, who have lost their photosynthetic ability along with pigment production capacity in evolution (Mishra, 2015). It was reported in the red alga G. sulphuraria, c-phycocyanin continues to be synthesized in the dark (Eriksen, 2008). This sounds promising for the heterotrophic production of carotenoids in the dark, where light induction cannot happen. High light stress is the most commonly employed stress factor to induce lutein accumulation in microalgae, and lutein along with zeaxanthin protects photosystem II from photooxidative damage (Jahns and Holzwarth, 2012). Mixotrophic cultivation can be another viable option for lutein production with light stress. In the case of astaxanthin, a stress factor must be applied for inducing carotenogenesis. In this section, we discuss the strategies that can be employed to enhance carotenoid production in dark conditions/heterotrophic cultivation. 4.1. Genetic engineering strategies to improve pigment production Genetic engineering is the most powerful tool available in the current era for the development of custom made organisms that can carry out a desired function under specific conditions. Genetic engineering in microalgae has seen a tremendous growth in the last two decades with (a) the nuclear genome sequencing of many model organisms, including Chlamydomonas reinhardtii, Cyanidioschyzon merolae, Nannochloropsis sp., Ostreococcus Tauri, Phaeodactylum tricornutum, Thalassiosira pseudonana, 61

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essential for growth and hence cannot be applied as inducer. Generation of oxidative stress in cultures by various methods has been used by researchers to induce carotenogenesis, as it can be seen from Tables 1 and 2. In C. zofingensis, induction by oxidative stress by reactive oxygen species or reactive nitrogen species can enhance carotenoid accumulation. Application of peroxynitrite as an inducer increased astaxanthin accumulation from 9.9 to 11.8 mg/L, whereas in the presence of nitryl chloride, astaxanthin content increased to 10.99 mg/L (Ip and Chen, 2005c). Hydroxyl radicals generated by hydrogen peroxide were also effective in inducing carotenogenesis in C. zofingensis, where astaxanthin accumulation increased from 9.9 mg/L to 12.58 mg/L (Ip and Chen, 2005b). The addition of hydroxyl radicals increased astaxanthin accumulation in Chlorococcum sp. under heterotrophic conditions, where astaxanthin content increased almost 80% (Ma and Chen, 2001a). Lutein production is thought to be primarily enhanced by high light intensity, as lutein is primarily a photosynthetic pigment. In the microalga C. protothecoides, reactive oxygen species (ROS) generated by a combination of H2O2, NaClO and Fe2 + ions stimulated lutein accumulation and the lutein yield reached up to 31.4 mg/L (Wei et al., 2008). Fe2 + ions can generate the most potent hydroxyl radical by the Fenton reaction and are known to enhance astaxanthin accumulation in H. pluvialis. Thus, oxidative stress generated by certain chemicals are effective in inducing carotenogenesis for the accumulation of both lutein and astaxanthin.

was observed that the expression of the upstream genes of the carotenoid synthesis pathway has also been upregulated (Kathiresan et al., 2015). The bkt and bch genes form H. pluvialis 34-1n was cloned and expressed in Chlamydomonas reinhardtii CC-849 by transformation and integration into the nuclear genome. The transformants could accumulate 34% more astaxanthin than the wild type and also produced 29% and 30% more carotenoids and xanthophylls respectively (Zheng et al., 2014a,b). Another target gene for enhancing astaxanthin expression is phytoene desaturase (pds) that catalyzes the conversion of phytoene to ζ-carotene. A single amino acid substitution in this gene could induce the resistance to herbicide norflurazon, and the presence of norflurazon can act as an inducer to maintain the expression levels of this gene. A C. zofingensis mutant generated by chemical mutagenesis, possessed a psd gene resistant to norflurazon and could grow in the presence of 0.25 μM norflurazon. High light induction or glucose induction increases the accumulation of astaxanthin in heterotrophic conditions by 36–44% (Liu et al., 2010). Very few studies report mutagenesis for improvement of lutein production by microalgae as mentioned earlier, as most of the commercial lutein comes from the marigold Tagetes patula or Tagetus erecta. The process has long been commercialized, but still suffers from low supply. Lutein from microalgae is still in its infancy in the market and genetic engineering strategies needs to be applied. It was observed that the major bottleneck in lutein synthesis is the low flux towards lycopene production. Once lycopene production is enhanced, lutein accumulation can be enhanced (Wu et al., 2009). Yeh et al. confirmed the same by transcriptome analysis of a high lutein accumulating strain Desmodesus sp. JSC3. The authors reported that enhancing lycopene synthesis in the initial growth phase could lead the flux towards αcarotene and lutein in the late exponential phase (Yeh et al., 2017). As for enhancing lutein accumulation in microalgae, enhancing flux towards lycopene synthesis and probably blocking the astaxanthin producing bch and bkt genes could greatly enhance lutein accumulation. Such a study has not been reported till now.

4.3. Other carotenogenesis enhancing factors in heterotrophic conditions In the absence of any stimuli to induce carotenogenesis, an increased C/ N ratio in the culture medium is known to enhance pigment production in microalgae under heterotrophic conditions. It was shown that under heterotrophic conditions, biomass production and astaxanthin content of C. zofingensis is directly proportional to glucose concentration (Ip and Chen, 2005a). Maximum astaxanthin content of 1.01 mg/g and 10.29 mg/L was obtained at 50 g/L glucose, and the astaxanthin content doubled compared to 5 g/L glucose. Similar results were seen in C. protothecoides, where an increase in glucose concentration from 10 to 40 g/L increased lutein production from 19·39 to 76·56 mg/L (Shi et al., 1999). The β-carotene oxygenase (ctrO) gene was isolated from C. zofingensis, and it was shown that the expression of the gene is up regulated in the presence of glucose, both under phototrophic and heterotrophic cultivations (Huang et al., 2006). The effect of each monosaccharide on growth and astaxanthin production varied, and it was observed that glucose and maltose supported best growth and astaxanthin levels, compared to fructose, galactose, lactose and sucrose. Highest transcription of the carotenogenesis genes was obtained with glucose and maltose (Sun et al., 2008). Thus, an increase in the carbon source, preferably glucose, can enhance carotenogenesis in pigment producing heterotrophic Chlorella. In the mixotrophic or heterotrophic cultivation of H. pluvialis, acetate is mostly used as the carbon source. The formation of cysts that accumulate astaxanthin was enhanced in the presence of acetate and other free radical inducers like Fe2 +. The addition of acetate to a 4-day old vegetative culture induces the formation of cysts within 24 h, whereas it might take about 6 days in normal culture without addition of acetate (Kobayashi et al., 1991). Pyruvate, at concentrations higher than that of acetate, are not inhibitory and promoted carotenogenesis. Acetate was better assimilated by H. pluvialis under illumination, demonstrating mixotrophic mode of cultivation (Jeon et al., 2005). It was also shown that addition of acetate increased the C/N ratio of the medium simulating nitrogen depletion that might induce cyst formation and enhance carotenogenesis (Kakizono et al., 1992). Nitrogen depletion in photoautotrophic growth results in limited cell growth and the survivors accumulate astaxanthin (Harker et al., 1996). Salt stress is also known to induce astaxanthin accumulation in H. pluvialis (Sarada et al., 2002; Kobayashi et al., 1997). The search for alternative inducers of carotenogenesis instead of high light for the production of pigments under heterotrophy is essential to achieve enhanced and economic pigment productivity.

4.2. Oxidative stress as an alternative inducer for carotenogenesis Carotenoids are very effective anti-oxidants and are the primary line of defense against photo-oxidative damage by the products of photosynthesis, singlet oxygen and they are very efficient in scavenging peroxyl radicals which are the by-products of lipid oxidation (Stahl and Sies, 2003). Singlet oxygen is primarily produced under high light condition, where the excited triplet state chlorophyll physically interacts with stable molecular oxygen, generating the highly reactive singlet oxygen, as denoted by 1O2. Singlet oxygen generated within the chloroplast can react with lipid membranes, proteins and nucleic acids, causing oxidative damage (Ledford and Niyogi, 2005). Since singlet oxygen predominantly interacts with double bonded molecules, carotenoids with a backbone of conjugated double bonds can quench their harmful effects. The reaction describing the quenching of singlet oxygen by a carotenoid is given as follows (Fiedor and Burda, 2014): 1O 2

+ Crt → 3O2 + 3Crt∗

3Crt∗

→ Crt + heat

The highly reactive singlet molecular oxygen (1O2) is converted to a relatively stable triplet oxygen (molecular oxygen in its ground state incapable of chemical interaction with other molecules, 3O2) by its direct physical interaction with a carotenoid molecule (Crt) generating an excited triplet state carotenoid (3Crt⁎). The carotenoid triplet state returns to the ground state (Crt) by dissipation of excess energy as heat, and the harmful effects of these triplet state carotenoids are negligible because of their low energy and half-life period (Fiedor and Burda, 2014). Carotenoids act as a catalyst for deactivating 1O2. High light intensity is efficient in inducing oxidative stress and induction of carotenogenesis, but under dark heterotrophic cultivation light is not 62

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5. Challenges faced and future research directions

Comparison with other red algal genomes revealed that only G. sulphuraria genome consists of up to 28 putative sugar transporters (Barbier et al., 2005). This particular adaptability of the alga to the utilization of a variety of sugars is a reflection of its natural niche; G. sulphuria is a polyextremophile, it can survive very low pH of 0.5 to 1.5, tolerance to heavy metals, and is capable of growth endolithically in dark (Schilling and Oesterhelt, 2007). A glucose induced transport system for xylose uptake was reported recently for C. sorokiniana and the genes for xylose metabolism were up-regulated in the presence of xylose (Zheng et al., 2014b). The glucose induction of these transporters indicate the presence of low affinity, nonspecific transporters that can transport any hexose or pentose in the medium. Specific transporter for glucose has been the only one characterized. So, a genome search of many commercial microalgae for putative sugar transporters and their characterization might help isolate or identify microalgae that are capable of sugar transport and hence heterotrophic growth. Glucose and acetate are the most commonly used organic carbon sources for heterotrophic cultivation of microalgae, and most other commercially important bacteria. The high cost of acquiring high quality fermentation grade glucose might reflect in the associated product price. However, with the advent of new technologies, organic carbon can be extracted from a variety of sources in a relatively inexpensive manner. Acetate is abundant in wastewater and can be used as a carbon source for microalgae (Lowrey et al., 2015). Glucose can be obtained by the hydrolysis of lignocellulosic biomass, which is the second generation feedstock for biofuels production. C. zofingiensis, and its mutant E17 were able to grow in glucose, treated and untreated molasses and accumulate astaxanthin with a productivity of up to 1.99 mg/L·d (Liu et al., 2012; Liu et al., 2013). The red alga Galdieria sulphuraria was able to grow on restaurant and bakery waste rich in glucose in dark conditions and was able to accumulate phycocyanin up to 20–22 mg/g (Sloth et al., 2017). Lohrey and Kochergin proposed colocation of sugar mills and algae production facilities, wherein the byproducts of the sugar mill can be valorized in algal culture (Lohrey and Kochergin, 2012). The bagasse that is abundant in sugar mills would be used for energy and CO2 production to support algal growth and the energy intensive harvesting and drying of algae. Supposing the algae are rich in lipids, then biodiesel can be produced with algal meal as a feed source. The authors reported that by co-locating an algal production facility in a 10,000 ton/d cane sugar mill, the following can be achieved from the waste resources: a 530 ha algal farm can be sustained, the GHG emissions of the mill can be reduced by 15% without using fossil fuels and a biodiesel production capacity of 5.8 million L of biodiesel/year can be obtained with a net energy ratio of 1.5 (Lohrey and Kochergin, 2012). This could also be possible with heterotrophic pigment producing algae, and it would be an interesting avenue to explore. Contamination of the microalgal culture with competing heterotrophic bacteria is another major problem in the maintenance of axenic cultures of microalgae for the production of pigments. In photoautotrophic culture, microalgae have the photosynthetic advantage in the absence of organic carbon and has an edge over contaminating heterotrophic bacteria. Under heterotrophic conditions, both microalgae and bacteria compete for organic carbon and bacteria with very less doubling times can easily grow and outnumber microalgae. Antibiotics can be used for the maintenance of axenic status of microalgal cultures (Jones et al., 1973) and it has been reported that periodic purification of microalgal cultures with antibiotics is essential to maintain the productivity of the strain (Han et al., 2016; Choi et al., 2008; Cho et al., 2002). The antibiotics used depends on the bacteria associated with microalgae, their concentration and nature. It is essential to check the susceptibility of microalgae before treating them with antibiotics as some microalgae are sensitive to antibiotics at higher concentrations (Han et al., 2016). The type of antibiotic and the treatment dosage can be determined by a disc diffusion test and optimized before treating the microalga. Tetraselmis suecica was treated

Heterotrophic cultivation of microalgae has many advantages for large scale culturing of microalgae. The primary concern in photoautotrophic is the continuous supply of optimal light intensity for photosynthesis and biomass production. Even though it has been shown that alternative light/dark cycle can enhance biomass and photosynthetic efficiency of certain microalgae (Xue et al., 2011), night biomass loss of up to 22% was reported in the light/dark cultivation of potential commercial strains like Nannochloropsis salina, Chlorella sorokiniana, and Picochlorum sp. (Edmundson and Huesemann, 2015). Heterotrophic cultivation eliminates the light supply problem, growing microalgal cells in dark conditions. For bioremediation of wastewater using microalgae, heterotrophic or mixotrophic cultivation is best suited, as wastewaters are rich in organic carbon source and the dark waters severely affect light penetration (Christenson and Sims, 2011). Despite the many advantages, heterotrophic cultivation of microalgae suffers from many challenges and research directed towards addressing these issues might help realize the potential of heterotrophic cultivation of microalgae. The first and the most overlooked question in case of heterotrophic cultivation of microalgae is that most of the microalgae isolated for commercial purposed were usually screened for photoautotrophic growth. Except for a few heterotrophic microalgae, the cultivation of microalgae was solely photoautotrophic. The screening and isolation of microalgae based on their ability to utilize organic carbon sources would unlock the untapped metabolic potential of microalgae to produce products of commercial interest in heterotrophic cultivation. The microalgae culture collection centers currently do not have many heterotrophic microalgae (Lee, 2004). Glaude and Maxley screened almost 120 strains for heterotrophy to be used as feed for aquaculture and it was found that 52 strains were capable of growth using glucose as the carbon source in the dark and certain strains were able to use acetate and glycerol. The heterotrophic strains were mainly of the genera Tetraselmis, Chlorella, Nannochloropsis and Dunaliella (Gladue and Maxey, 1994). This screening was to isolate heterotrophic strains with high biomass productivity for use as aquaculture feeds, but not for any special product of interest. Thirty-five strains of Spirulina were tested for their ability to grow in the dark with glucose or fructose as carbon source (Mühling et al., 2005). Of those, 34 strains were able to utilize glucose in dark and 24 strains were able to utilize fructose in the dark, indicating the rich source of heterotrophic Spirulina in laboratory collections. Recently, heterotrophic microalgae were screened for biodiesel production (Jia et al., 2014) and wastewater treatment (TianYuan et al., 2014). More studies like this are the need of the hour to realize the potential of heterotrophic microalgae. Second important issue is the sugar utilization potential of microalgae that endows them with the capacity of heterotrophic growth. As discussed previously in Section 2 and Section 4, sugar transport in microalgae are not studied widely and the Hup1 protein involved in the symport of glucose and a proton in C. kessleri is the only one that has been identified at the molecular level. A constitutive glucose transport and utilization system was reported for Scenedesmus obliquus grown in high rate oxidation ponds for wastewater treatment, and glucose utilization efficiency enhances after shifting to heterotrophic growth conditions (Abeliovich and Weisman, 1978). A similar glucose transport system was reported in Neochloris oleoabundans when grown under glucose, and glucose transport was blocked when the proton gradient was destabilized indicating symport kind of sugar transport (MoralesSánchez et al., 2011). Genome analysis of Chlorella protothecoides sp. 0710 revealed the presence of three homologous Hup1 like proteins, but they were not characterized experimentally (Gao et al., 2014). However, the red alga Galdieria sulphuraria used for the heterotrophic production of phycocyanin possess at least 14 sugar transporters that can transport a variety of sugars including deoxy sugars, polyols, both in D- and L- configurations (Schilling and Oesterhelt, 2007). 63

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proteins and the well-established culture procedures can be adapted for the production of various bioproducts. Microalgal technology is still in its infancy, and hence needs encouragement in the form of state subsidies to be developed into a powerful industrial sector.

with different methods in succession to obtain an axenic culture: extensive centrifugation and washing, mild sonication to remove adherent bacterial cells, followed by treatment with the combination of 5 mg/mL vancomycine and 10 mg/mL neomycine yielded the axenic culture (Azma et al., 2010). Then this was subjected to adaptive evolution to heterotrophy: the culture was initially grown in photoautotrophic mode, then with alternate light/dark cycle with a steady decrease of light phase and finally the microalga was able to grow heterotrophically using 10 g/L glucose in the dark. This method has potential only for microalgal species that are known to be capable of heterotrophic growth like Chlorella. Other concerns regarding heterotrophic growth is the inability to produce light induced metabolites like pigments (PerezGarcia et al., 2011). The alternative methods used to induce pigment production in microalgae has already been discussed in detail in Section 4. Thus, heterotrophic cultures have the potential to be used as a sustainable resource for the production of pigments in place of chemical synthesis. Cultivation of microalgae in conventional bacterial fermenters in the presence and absence of light and the associated production of carotenoids was reported as early as 1965 (Theriault, 1965). In the study, antibiotics were added to prevent bacterial fermentation, and with fed-batch or continuous fermentation strategy to alleviate substrate inhibition and C. pyrenoidosa biomass accumulated at concentrations of 100–300 g/L in 30 L fermenters. The author also reported that aeration of the cultures was an important parameter to be taken care of while culturing heterotrophic algae in fermenters (Theriault, 1965). More recently, C. protothecoides was cultivated successfully in an industrial scale fermenter of 11,000 L, with a biomass content of 14.2 g/L and lipid content of around 45% and was used as a feedstock for biodiesel production (Li et al., 2007). Commercial success has been achieved for heterotrophic microalgal cultivation mainly in the production of polyunsaturated fatty acids (PUFA) like docosahexaenoic acid (DHA) by Crypthecodinium cohinii and Schizochytrium sp. The commercial scale process and the accompanied downstream processes were described in detail by Cohen and Ratledge (2010). The possibility and convenience of cultivating microalgae in fermenters has also been mentioned (Bumbak et al., 2011; Barclay et al., 2013). However, still very few studies have been done with regard to pigment production by heterotrophic microalgae. All the process parameters for the cultivation of microalgae in conventional fermenters are similar to the cultivation of other microbes: the temperature, pH and the medium components are optimized based on the microbial strain used and the process requirements. Fed-batch and continuous systems with substrate addition at intervals are recommended to overcome substrate inhibition (Bumbak et al., 2011). Aeration is the single most important parameter that affect microalgal growth in fermenters, since the aerobic metabolism of organic carbon requires oxygen. Other than supplying sufficient oxygen for biomass production, oxygen levels does not influence pigment production, whereas oxygen levels are extremely important in the production of PUFAs and the fatty acid synthase is oxygen dependent (Qu et al., 2013). Air-life and bubble column type of aeration and agitation and the use of impellars without sharp edges (blunt impellars) were recommended as the aeration mode for microalgal cultivation (Behrens, 2005). Microalgae have been produced in a very large scale and are consumed as a dietary supplement, and with the adverse effects associated with the use of synthetic or chemical based products, microalgae are also being explored for a number of products ranging from health supplements, pharmaceuticals, nutraceuticals and cosmaceuticals. Microalgae based products are catching up with the developing countries. For instance, the algae based bioethanol company Algenol from the Unites states has gone into a joint venture with the Indian petroleum refinery giant Reliance industries for microalgae based bioethanol production in India. The pilot plant is functioning and several cycles of microalgal growth has been demonstrated. Taiwan, China and Japan are pioneers in the production of Chlorella and Spirulina as single cell

6. Conclusions Carotenoids are nature's way of overcoming the oxidative stress associated with respiratory metabolism in eukaryotes. Even though animals are incapable of synthesizing these protective entities, plants and microalgae rich in carotenoids are a part of their diet and hence they can benefit from carotenoids. In the current scenario, there is a large demand for natural carotenoids in place of the synthetic ones currently available in the market and microalgae are the most encouraging natural source of carotenoids. Photoautotrophic cultivation of microalgae with high light intensity is the most commonly used strategy for production of pigments from microalgae, since carotenoid synthesis and accumulation is associated with light harvesting in photosynthesis. Carotenoids are also synthesized in dark and heterotrophic cultivation seems an interesting option for the commercial production of carotenoids. The cost associated with acquiring the carbon source and equipment operation of heterotrophic cultivation can be offset by the price of carotenoids as they are high value products. From careful survey of literature, it is safe to recommend that species of the genera Chlorella are the most suitable candidates for heterotrophic production of lutein and astaxanthin. In particular, C. protothecoides is a worthy choice for lutein production and C. zofingensis is a favorable alternative for H. pluvialis regarding astaxanthin production. Phycocyanin can be obtained in higher yields from the red alga G. sulphuraria, compared to A. platensis. Very high yields comparable to photoautotrophic cultivation has been obtained for lutein, astaxanthin and PC from heterotrophic cultivation. Focus on these organisms and employment of cultivation and process engineering strategies can overcome the bottlenecks associated with heterotrophic cultivation for the production of carotenoids. Acknowledgements This work is financially supported by National Natural Science Foundation of China (Nos. U1304528, 51576060) and Innovation Scientists and Technicians Troop Construction Projects of Henan Province (16HASTIT026). References Abeliovich, A., Weisman, D., 1978. Role of heterotrophic nutrition in growth of the alga Scenedesmus obliquus in high-rate oxidation ponds. Appl. Environ. Microbiol. 35, 32–37. Aimjongjun, S., Sutheerawattananonda, M., Limpeanchob, N., 2013. Silk lutein extract and its combination with vitamin E reduce UVB-mediated oxidative damage to retinal pigment epithelial cells. J. Photochem. Photobiol. B 124, 34–41. Anila, N., Simon, D.P., Chandrashekar, A., Sarada, R., 2013. Glucose-induced activation of H +−ATPase in Dunaliella salina and its role in hygromycin resistance. J. Appl. Phycol. 25 (1), 121–128. Azma, M., Mohamad, R., Rahim, R.A., Ariff, A.B., 2010. Improved protocol for the preparation of Tetraselmis suecica axenic culture and adaptation to heterotrophic cultivation. Open. Biotechnol. J. 4, 36–46. Azma, M., Mohamed, M.S., Mohamad, R., Rahim, R.A., Ariff, A.B., 2011. Improvement of medium composition for heterotrophic cultivation of green microalgae, Tetraselmis suecica, using response surface methodology. Biochem. Eng. J. 53, 187–195. Barbier, G., Oesterhelt, C., Larson, M., Halgren, R., Wilkerson, C., Garavito, R., Benning, C., Weber, A., 2005. Comparative genomics of two closely related unicellular thermoacidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolic flexibility of G. sulphuraria and significant differences in carbohydrate metabolism of both algae. Plant Physiol. 137, 460–474. Barclay, W., Apt, K., Dong, X.D., 2013. Commercial production of microalgae via fermentation. In: Richmond, A., Hu, Q. (Eds.), Handbook of Microalgal Culture: Applied Phycology and Biotechnology, second edition. Blackwell Publishing Ltd.. Behrens, P.W., 2005. Photobioreactors and fermentors: the light and dark sides of growing algae. In: Andersen, R.A. (Ed.), Algal Culturing Techniques. Elsevier, Oxford, pp. 189–204. Bennet, M.E., Hobbie, J.E., 1972. The uptake of glucose by Chalmydomonas sp. J. Phycol.

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