Production of potential coproducts from microalgae

CHAPTER

Production of potential coproducts from microalgae

14 I-Chen Hu,

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan

1 ­INTRODUCTION The production of biofuels from algae is not yet economically competitive with that of petroleum-based fuels. The major barriers to the commercialization of algae-derived biofuel are huge-volume cultivation and high costs for processing. Therefore, it is proposed that commercially viable biofuel production could be accomplished by simultaneously developing high-value co-products from algal biomass [1]. The high protein content of some algae is the reason for development of algae as a source of single-cell protein since the 1970s. The commercialization of Chlorella and Spirulina in Japan, Taiwan, and Mexico as food, feed, and recently health food are successful examples [2]. This was followed by the commercialization of several algal pigments, such as beta-carotene from Dunaliella salina [3] in the 1980s, astaxanthin from Haematococcus pluvialis [4] since the mid-1990s, and phycobiliproteins from cyanobacteria and algae such as rhodophytes, cryptomonads, and glaucocystophytes [5,6]. Polysaccharides derived from seaweeds such as alginate, agar, and carrageenan are widely used as gelling and thickening agents in the food industry. Finally, lipids and fatty acids from microalgae, especially long-chain polyunsaturated fatty acids (LC-PUFA), have gained particular interest due to their health benefits. Docosahexaenoic acid (DHA) from Crypthecodinium cohnii is the first commercialized algal oil product [7]. In this chapter, value-added compounds including carotenoids, phycobiliproteins, polysaccharides, and polyunsaturated fatty acids (PUFAs) are introduced. The path to commercialization is also considered. These considerations include the product quality requirements, application, market size and market characteristics of the product, and competitors (chemically synthesized products or compounds from other organisms). Compounds, microalgae sources, applications, and estimated markets are listed in Table 1 [2,8,9].

Biomass, Biofuels and Biochemicals. https://doi.org/10.1016/B978-0-444-64192-2.00014-7 © 2019 Elsevier B.V. All rights reserved.

345

Table 1  High-value compounds, sources, applications, and estimated markets [2,8,9] Price range (UD$/kg)

Estimated market (US$ million)

Compounds

Microalgae sources

Applications

Carotenoids Beta-Carotene

Dunaliella

300–1500

~200 (2019)a

3.3% (2014–2019)

Astaxanthin

Haematococcus

200–7000

~40 (2019)a

Lutein

910–15,000

<3.14 (2019)a

2.3% (2014–2019) 3.6% (2014–2019)

100–500

<1.17 (2019)a

Fucoxanthin

Scenedesmus, Muriellopsis, Chlorella Chlorella, Dunaliella (mutant) Phaeodactylum Chlorella, Dunaliella, Scenedesmus Phaeodactylum

Functional food (antioxidant, pro-vitamin A), natural pigment, and feed Functional food (antioxidant) and cosmeceuticals (antiaging) Functional food (antioxidant, eye protection) Functional food (antioxidant, eye protection) Natural pigment Functional food (antioxidant)

180–42,000

Unclear

Phycobiliproteins Phycoerythrin (PE) and Allophycocyanin (APC) C-Phycocyanin (CPC)

Bangia, Prophyridium, Spirulina Spirulina

Fluorescent reagent

500–50,000

60 (2019)

Stagnant

Natural pigment (for food and cosmetics), and pharmaceutical

500–100,000

114.8 (2022)

4.7% (2016–2022)

80–160

898.7 (2025)a

4.3% (2014–2025)

Unclear

Unclear

Unclear

Zeaxanthin Cantaxanthin

Omega-3 fatty acids Eicosapentaenoic acid (EPA) Docosahexaenoic acid (DHA) Sulfated polysaccharides

a

Nannochloropsis, Schizochytrium Crypthecodinium, Isochrysis, Schizochytrium Prophyridium

Nutritional supplement and feed Nutritional supplement and feed Functional food (antivirus, antioxidant, antiinflammatory, immune-modulator) and cosmeceuticals (antioxidant, antiinflammatory)

The estimated market is the total market value multiplied by the proportion of algal products.

CAGR

3.7% (2014–2019) 5.3% (2016–2021)

2 ­Pigments and phycobiliproteins

2 ­PIGMENTS AND PHYCOBILIPROTEINS The first image algae present is that of beautiful colors. The colorful appearance is derived from the pigments algae use to absorb visible light and initiate photosynthesis. Three major classes of photosynthetic pigments are present in algae: chlorophylls, carotenoids, and phycobilins [10]. Chlorophyll a is the primary pigment in all photosynthetic organisms. It absorbs most energy from wavelengths of violet-blue and orange-red light, and then serves as the primary electron donor in the electron transport chain [11]. However, aquatic light environments are quite different from those on land. Water both absorbs and scatters light, and this results in the intensity and color of the penetrated light changing greatly with depth. Generally, the deeper the water is, the dimmer the light is. To overcome this energy problem, marine algae develop accessory pigments that absorb the light that chlorophyll a does not. These accessory pigments include chlorophyll b (also c and d in different algae), carotenoids (such as beta-carotene in most algae), and phycobilins (in cyanobacteria and red algae) [10]. Carotenoids have been noticed and commercialized owing to their potential health benefits. Beta-carotene was the first high-value product commercially produced from microalga; the mass production of beta-carotene from Dunaliella salina was begun in the 1980s, in the USA, Australia, and Israel [3]. The second carotenoid from microalgae to be commercialized was astaxanthin, produced from Haematococcus pluvialis [4]. Since the mid-1990s, several companies have successfully produced Haematococcus pluvialis on a commercial scale and then natural astaxanthin was marketed worldwide. Compared to higher plants, microalgae usually contain considerably greater quantities of specific carotenoids. The content of beta-carotene in carrots is 0.08% of its dry weight, while in D. salina it is about 12%–14% [12–14]. Astaxanthin in H. pluvialis can reach high concentrations of >4% of dry weight [15]. The lutein content in Scenedesmus obliquus is usually over 4 mg/g, whereas the content in marigold flowers is often reported to be only 0.3 mg/g [16]. Fucoxanthin in Isochrysis galbana has been reported as 18.23 mg/g, which is up to 15 times higher than the predominant fucoxanthin producer, seaweed [17].

2.1 ­BETA-CAROTENE One molecule of beta-carotene can be cleaved by human intestinal enzymes into two molecules of vitamin A; thus, beta-carotene is the main dietary source of vitamin A [18]. For humans, beta-carotene has been developed as a dietary supplement that provides several benefits, such as immune modulation [19], antiaging effects [20], and the prevention of both cancer and cardiovascular disease [21]. Furthermore, beta-­carotene is widely used as a colorant in food and beverages, and as an animal feed additive. Beta-carotene can be derived from both natural and synthetic sources. Synthetic beta-carotene is almost 100% trans form (Fig. 1), whereas beta-carotene, found in fruits and vegetables, comprises 10% of cis isomers, and the beta-carotene derived from algae is a mixture of all-trans form and 9-cis isomer in approximately

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CHAPTER 14  Production of potential coproducts from microalgae

FIG. 1 Structures of beta-carotene stereoisomers.

equal proportions [22]. Natural beta-carotene can be obtained from microalgae (Dunaliella salina), fruits and vegetables (carrots, sweet potatoes, pumpkins, etc.), fungi, and the fermentation of microorganisms. The size of the global beta-­carotene market in 2015 was US$432.2 million, and algal beta-carotene accounted for about 35% of total revenue [23]. The market for beta-carotene in 2019 is forecast to reach US$532 million, a cumulative annual growth rate (CAGR) of 3.3% [9]. Dunaliella is the best source of commercially produced beta-carotene. The content of beta-­ carotene can reach up to 14% of biomass, whereas the average concentration of carotenoids in most algae is only 0.1%–0.2% [24]. Current major manufacturers of Dunaliella beta-carotene are BASF in Australia (40–50 tons/yr), NBT in Israel (2–3 tons/yr), and E.I.D Parry in India (1–3 tons/yr) [25]. Dunaliella is cultivated by a two-stage method for β-carotene production. In stage one, the cells are grown in small nursery ponds with a medium rich in nitrate to attain optimal biomass. In stage two, the cells are transferred to large production ponds and diluted to about one-third concentration by adding a medium deficient in nitrate [12]. A nutrient-poor medium containing a high concentration of salt (2.5 M sodium chloride) has been reported as further increasing the amount of induced beta-carotene [26].

2.2 ­ASTAXANTHIN Astaxanthin is an oxygenated carotenoid, also a kind of ketocarotenoid, known for its powerful antioxidant activity. For example, the antioxidant activity of astaxanthin is 10 times higher than that of other carotenoids [27] and over 500 times more effective than that of α-tocopherol, a kind of vitamin E [28]. Several in vitro studies also

2 ­Pigments and phycobiliproteins

show that it is a potent quencher of reactive oxygen and nitrogen species, including singlet oxygen, and single- and two-electron oxidants [29]. This powerful antioxidant activity has resulted in the extensive application of astaxanthin in foods, dietary supplements (nutraceuticals), cosmetics, and feed. In 1987, the United States Food and Drug Administration (US FDA) approved astaxanthin as a feed additive for use in the aquaculture industry; in 1999, it was further approved for use as a dietary supplement [30]. Natural sources of astaxanthin include microalgae, yeast, shrimp, krill, and plankton [31]. Haematococcus pluvialis is a freshwater green alga that can synthesize and accumulate astaxanthin (up to 4% of total cellular dry weight) under oxidative stress [32]. The crustacean (shrimp and krill) exoskeletons are in limited supply and have low astaxanthin content, whereas yeast (Phaffia rhodozyma) has a lower astaxanthin content (0.4%–2.5%) than microalgae [33]. The life cycle of Haematococcus consists of four stages of cell growth: vegetative cell growth, encystment, maturation, and germination [32,34]. The vegetative cells are motile flagellated cells with an optimum growth temperature of 22–25°C. Stress conditions, such as nutrient deprivation, high salinity, strong irradiance, high temperature, or a combination of these, trigger carotenogenesis and cell differentiation from the vegetative cell stage through to the cyst cell stage. The cyst cells are nonmotile aplanospores with thick walls. Astaxanthin is contained in the aplanospore [4], and its content comprises as much as 4% of total cellular dry weight [32]. The global market for both synthetic and natural astaxanthin in 2014 was approximately 280 metric tons, valued at US$447 million; the market has continued to grow since 2014 [35]. In 2019, the market for beta-carotene is expected to reach US$423 million, a CAGR of 2.3% [9]. However, over 95% of the astaxanthin available on the market is produced synthetically, whereas natural astaxanthin from Haematococcus comprises <1% of the commercial product [36]. Astaxanthin has three stereoisomers: (3R, 3′R), (3R, 3′S; meso), and (3S, 3′S) (Fig.  2). Synthetic astaxanthin contains an approximate 1:2:1 ratio of these, whereas Haematococcus produces only the (3S, 3′S) stereoisomer, in both free and esterified forms [37]. In singlet oxygen quenching assay, Haematococcus astaxanthin was found to be >50 times stronger than synthetic astaxanthin, and approximately 20 times stronger in free radical elimination [38]. Therefore, even though the consumption of synthetic astaxanthin dominates the market, consumer demand for natural and more effective products has been growing and thus provides a market opportunity for Haematococcus astaxanthin. Synthetic astaxanthin sells at US$2500/kg, whereas the natural product sells for over US$7000/kg. There are five major commercial producers of Haematococcus astaxanthin worldwide: Algatechnologies Ltd. (product brand name AstaPure, in Israel); AstaReal Inc. (product brand name AstaReal, in Hawaii, USA; a subsidiary of Fuji Chemical Industry, Toyama, Japan); BGG (Beijing Gingko Group) (product brand name AstaZine, production farm located in Yunnan Province, China); Cyanotech Corporation (product brand name BioAstin®, in Hawaii, USA); and Parry’s Pharmaceuticals (product brand name Zanthin, in India) [28].

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CHAPTER 14  Production of potential coproducts from microalgae

FIG. 2 Structures of astaxanthin stereoisomers.

2.3 ­OTHER CAROTENOIDS Microalgae are also potential sources of other carotenoids, such as lutein from Scenedesmus, Muriellopsis, and Chlorella; zeaxanthin from Dunaliella salina mutants; cantaxanthin from Chlorella, Dunaliella, and Scenedesmus; fucoxanthin from Phaeodactylum tricornutum; and the colorless carotenoids phytoene and phytofluene from Dunaliella [2,8]. Lutein is a xanthophyllic pigment with antioxidant properties, whereas Zeaxanthin is a less abundant isomer of lutein. Various studies have shown that lutein and zeaxanthin are accumulated in human retinas to protect them against singlet oxygen and radicals [39]. Therefore, ingesting lutein and zeaxanthin can reduce the risk of chronic eye diseases such as age-related macular degeneration (AMD) and cataracts. The principal commercial use of canthaxanthin is as a colorant for egg yolk, salmon, and birds [40]. Chemical synthesis is used to mass produce canthaxanthin, whereas the microalgal product is mainly produced by green algae [2]. Fucoxanthin has been shown to have many beneficial characteristics, which are reflected in its antioxidant, anticancer, antiobesity, antidiabetic, and antiphoto-aging properties [41]. Although the pigment content in microalgae is higher than that in macroalgae [42], most fucoxanthin is still produced from macroalgae because cultivation is much cheaper. The global market for both synthetic and natural lutein is forecast to reach US$314 million in 2019, at a CAGR of 3.6% (2014–19) [9]; the market for zeaxanthin is estimated to reach US$18 million in 2018, at a CAGR of 1.5% (2010–18) [43]; and the market for cantaxanthin is expected to be US$117 million in 2019, a CAGR of

2 ­Pigments and phycobiliproteins

3.7% (2014–19) [9]. Information regarding the market for fucoxanthin, however, is difficult to find. Although the total market for carotenoids is vast, the commercial quantity of products derived from microalgae remains very low, and the marketing of such products remains underdeveloped.

2.4 ­PHYCOBILIPROTEINS Phycobiliproteins are water-soluble proteins that are usually covalently bound with phycobilins. They are present in cyanobacteria and algae such as rhodophytes, cryptomonads, and glaucocystophytes [5,6]. The phycobilins give phycobiliproteins distinct absorption spectra ranging from 460 to 670 nm [6,44,45] in which chlorophyll a has low absorption. After capturing light energy, phycobiliproteins transfer this energy to chlorophylls during photosynthesis [5,44,45]. Phycobiliproteins are classified into three types according to level of phycobilin energy (absorption spectra): those of high energy are phycoerythrins (PEs), or phycoerythrocyanins (PECs), with main absorption at 480–580 nm; those of intermediate energy are phycocyanins (PCs), with main absorption at 600–640 nm; and those of low energy are allophycocyanins (APCs), with main absorption at 620–660 nm [6]. Isolated phycobiliproteins are highly fluorescent, because no acceptors are nearby to receive the harvested energy. Phycobiliproteins also possess several unique features, such as broad absorption in the visible light spectrum, a high extinction coefficient, high fluorescence quantum efficiency, large Stokes shift, and minimal fluorescence quenching [46–48]. These features mean that phycobiliproteins are promising fluorescent labeling reagents that can be employed in flow cytometry, fluorescence immunoassay, fluorescence microscopy, immuno-histochemistry, and other biomedical research activities [49–58]. The blue phycobiliproteins, APC, can be produced from Spirulina sp., whereas the red ones, B-PE and R-PE, are produced from red microalgae such as Porphyridium sp., Rhodella sp., and Bangia sp. at kilogram scale per year. The price of phycobiliproteins varies from US$3/mg to US$25/ mg [59]. The global market was estimated to reach approximately US$60 million in 2019; however, in 2018 the market now seems to be stagnant [9]. The leading manufacturers and suppliers of phycobiliproteins used as fluorescent labeling reagents are: Algapharma Biotech Corp. (product brand name Flogen; a subsidiary company of Far East Bio-Tec. Co. Ltd. (FEBICO) in Taiwan); Columbia Biosciences (product brand name SureLight, in Maryland, USA); and QuantaPhy, Inc. (in California, USA; its distributor is ProZyme, Inc.).

2.5 ­C-PYCOCYANIN Considered as a fluorescent labeling reagent, C-pycocyanin (C-PC) is not as effective as R-PE and APC. Nevertheless, CPC has been found to exhibit a variety of pharmacological properties, including antioxidant, antiinflammatory, neuroprotective, and hepatoprotective effects [60]. Moreover, C-PC is a natural pigment frequently used in food, cosmetics, and ink. It is sold for US$500–100,000/kg, depending on its­

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CHAPTER 14  Production of potential coproducts from microalgae

purity [2]. This is usually determined by its ratio of absorbance (A620/A280); product with a ratio of ≥0.7 is food grade, product with a ratio of approximately 3.9 is reactive grade, and product with a ratio of ≥4.0 is analytical grade [2]. The global market for C-PC in 2016 totaled US$87 million, and is expected to reach US$114.8 million by 2022, at a CAGR of 4.7% from 2016 through to 2022 [61]. The major manufacturers and suppliers of C-PC powder are Dainippon Ink and Chemicals (in Japan), Parry Nutraceuticals (in India), and Norland Biotech (in Tianjin, China).

3 ­POLYSACCHARIDES The cell walls of marine algae are rich in sulfated polysaccharides. Sulfated polysaccharides, which are derived from seaweeds such as alginate, agar, and carrageenan, are widely used as gelling and thickening agents in the food industry. Alginates are anionic polysaccharides isolated from the cell walls of brown algae, whereas carrageenan and agar are principally produced from red seaweed (Rhodophyta) [62]. The anticancer activity of fucoidans from brown algae has recently been intensively studied [63]. Regarding sulfated polysaccharides of marine microalgae, antiviral activity is the most studied characteristic [64]. In addition to antiviral activity, sulfated polysaccharides from microalgae have also been found to exhibit several biological properties, including as antioxidation, free radical scavenging, antiinflammation and immune-modulation, inhibiting tumor cell growth, hypolipidemic and hypoglycemic, and their ability to lubricate bone joints [64,65]. However, pharmaceutical products containing sulfated polysaccharides from microalgae are rare, and their market position as ingredients for health and skin care is unclear. Since the 1970s, sulfated polysaccharide from Porphyridium has probably been the most intensively researched sulfated polysaccharide [66]. Asta Technologies LTD in Israel produce and sell sulfated polysaccharides from Porphyridium to the cosmetics market in the form of powder (content >97%) and gel (content 5% or 10%). Calcium spirulan (Ca-SP), a kind of sulfated polysaccharide isolated from Spirulina platensis, is composed of two types of repeating disaccharide units, O-rhamnosylacofriose and O-hexuronosyl-rhamnose (aldobiuronic acid) [67]. Ca-SP has been evaluated to possess antiherpes simplex virus and antihuman immunodeficiency virus activities [68]. The mechanism of Ca-SP is preventing virus-cell attachment and fusion with host cells. A commercial Spirulina tablet is now available, which is alleged to contain 8% Ca-SP. Some scientists reported that under the right conditions, the yield of extracted polysaccharides was 15%–55% of the weight of the microalgae (such as Porphyridium cruentum). If the yield of polysaccharides could be optimized to 30% of algal biomass, about 5 tons of polysaccharides per 1000 m2 cultivation, and the production cost is kept low (about US$20,000 per 1000 m2 cultivation), then the break-even price of this polysaccharide product could be less than US$15/kg [69]. This price is quite a modest one for many polysaccharides, and then polysaccharides from microalgae will have good economic potential. However, the commercialization of

5  Conclusions and perspectives

polysaccharide from microalgae is still at an early stage. Target products, workable technology for extraction, and specifications remain challenges for scientists and producers to deal with.

4 ­POLYUNSATURATED FATTY ACIDS Through heterotrophic cultivation, fatty acids can comprise up to 40% of microalgae mass, and may include long-chain polyunsaturated fatty acids (LC-PUFA) such as linoleic acid (LA, 18:2 ω-6), gamma-linolenic acid (GLA, 18:3 ω-6), arachidonic acid (ARA, 20:4 ω-6), eicospentaenoic acid (EPA, 20:5 ω-3), and docosahexaenoic acid (DHA, 22:6 ω-3) [70]. DHA is the first commercialized algal oil product; the leading brand is DHASCO from the Martek Biosciences Corporation (since 2011, it has been part of Royal DSM NV). DHASCO is oil extracted from single-cell Crypthecodinium cohnii and then standardized with high oleic sunflower oil to contain 40% of DHA (w/w). Since 2001, DHASCO has also been granted GRAS status for use in infant formulas and foods (US FDA, GRAS Notice 000041, 2001). In 2012, the global market for microalgae-based DHA oil (DHA content >30%) was estimated to be nearly US$350 million, which is approximately 4614 metric tons. The infant formula application comprised 48.9% of its total market volume, followed by dietary supplements with 28.4%, and foods/beverages with 19.4% [71]. C. cohnii can readily be cultured in a medium containing sodium acetate, yeast extract, and peptone in seawater at temperatures of 15–30°C. Moreover, several methods were developed to enhance the DHA content in C. cohnii [7]. The highest DHA content reported was 65% of the total fatty acids [72]. Rapeseed meal hydrolysate and waste molasses were also used as feedstock to enhance the biomass of C. cohnii, in which DHA content was 22%–34% of the total fatty acids similar to that of algal biomass from commercial cultivation [73]. Schizochytrium, Ulkenia, and Nannochloropsis are also common sources of algal omega-3 LC-PUFA [2]. Oil from Schizochytrium sp. is rich in both DHA and EPA, whereas oil from Nannochloropsis is rich in EPA [74]. Table 2 lists commercial algal PUFA products, companies, microalgae sources, and cultivation methods [74]. The size of the global algae oil market was valued at US$1.38 billion in 2015, and is expected to reach US$2.09 billion by 2025, growing at a CAGR of 4.3% [75,76]. The fuel grade segment was the largest application by volume, accounting for >57% of the global market in 2015. Animal feed was estimated as the second largest segment, accounting for over 27%. Consequently, the application of algal oil in dietary supplements and in foods/beverages comprises only approximately 15% of the total market [75,76].

5 ­CONCLUSIONS AND PERSPECTIVES Although the demand and price for specific algal compounds (pigments, phycobiliproteins, polysaccharides, and PUFA) are increasing, the commercial quantity of products derived from microalgae remains very low. More research is needed to

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CHAPTER 14  Production of potential coproducts from microalgae

Table 2  Commercialized algal oil products, companies, microalgae sources, and cultivation methods Commercial product

Microalgae sources

Company

PUFA content

AlgaeBio Omega-3 Origins A2 EPA Pure

Algae Biosciences

20% EPA + 20% DHA

Undisclosed

Aurora Algae

Undisclosed

Life’s DHA

DSM

65% EPA (regular), 95% EPA (pharma) 40%–45% DHA

life’s DHA plus EPA EicoOil DHA-3 Sure DHAid Source Oil

Qualitas Health GCI Nutrients Lonza SourceOmega

10% EPA + 22.5% DHA 25%–30% EPA 35% DHA 35%–40% DHA 35%–40% DHA

Cultivation Oil blend from two marine strains Phototrophic, open-pond

Crypthecodinium cohnii Schizochytrium sp.

Heterotrophic fermentation

Nannochloropsis oculata Crypthecodinium cohnii Ulkenia sp.

Phototrophic, open-pond Heterotrophic fermentation Heterotrophic fermentation Heterotrophic fermentation

Schizochytrium sp.

d­ efine the workable technology for their extraction and purification to reduce the production costs. In addition, producers also have to determine the main products and establish market segmentation. The following issues remain to be worked out. To increase the production yields is the basic task, and screening for new microorganisms or genetic modifications are being assessed. Culture conditions (temperature, nutrients, light, and pH) is the other issue that must be evaluated to increase the amount of target compounds. In most cases, final recoveries are low, whereas recovery on a large scale is still challenging because not all steps, including cell disruption, extraction, and purification, are scalable. In addition, the long-term stabilities of algal products should be evaluated.

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