Algae are small photosynthetic organisms with a relatively large productivity and a small generation time. An exact definition of an alga is hard to obtain as this group covers a large variety of organisms. A rough classification is often made based on the size, separating algae into micro- and macroalgae. Microalgae are situated at the bottom of the food chain. The biochemical compounds they produce are essential for the human diet as well, although microalgae are not often found in the local grocery shops. The production of microalgae species for food supplementation has been commercialized in Asia since decades . The most popular species for this purpose are Spirulina and Chlorella. Due to their large concentration of proteins and useful lipids, they are sold as entire biomass. However, as algae are such a large group of species, a large variety of different beneficial biochemical compounds could be produced. Since only a few of the estimated 72,500 species are commercialized, this large potential remains mostly untapped [2, 3].
Figure 1. Chlorella pills
Some microalgae species are known to accumulate relatively large amounts of certain compounds. Dunaliella salina and Haematococcus pluvialis have for example been commercialized for the production of β-carotene and astaxanthin . Other microalgae species can accumulate large amounts of lipids which can be converted into biofuel . If we take the large productivity potential of these species and the fossil fuel problems into account, it doesn’t come as a surprise that the search for a microalgae-based biofuel has received a large amount of attention. Despite all the research which has been performed in this area, an economically viable algae-based biofuel has not yet been commercialized. The large costs related to the cultivation stage combined with the low fuel prices, make this a difficult business case . Combining different applications in a biorefinery concept could be a solution. These applications can be grouped according to their market volume and value, ranging from low value products with a large market volume (e.g. biofuels) to high-value products with a low market volume (e.g. food supplements). The production of both biofuels and high-value food supplements in one biorefinery concept is not feasible due to incompatible market volumes . Moreover, it doesn’t make sense to focus on a low value product, when the same feedstock could be converted and sold for higher prices as fertilizers or feed. This point of view on the commercialization of the different algae components is based on the cascading principle .
Figure 2. A microalgae biorefinery concept based on the cascading principle
A more logic strategy would therefore primarily focus on a broader commercialization of algae products, gradually increasing the scale. Learning effects and new technology developments can be implemented and the costs of microalgae cultivation can gradually decrease which can improve the commercial viability of algae-based biofuel . The algae-based biorefinery would therefore be initiated producing multiple high value products. The algae species which have been commercialized mainly focus on the production of one product. The ideal algae species for a biorefinery may therefore still remain undiscovered. The following three algae species are proposed for the introduction of a microalgae-based biorefinery: Phaeodactylum tricornutum, Nannochloropsis oceanica and Chlorella vulgaris.
Like most microalgae species, Phaeodactylum tricornutum produces several pigments to enable photosynthesis. Some of these pigments have been commercialized due to their high value and can therefore play an important role in a biorefinery.
The most abundant high-value pigment produced by Phaeodactylum tricornutum is fucoxanthin. This carotenoid, which is mainly found in brown seaweed, contributes more than 10% to the total amount of carotenoids produced in nature [7, 8]. Peng et al.  performed a review of the different beneficial characteristics of this pigment, including anti-oxidant, anti-cancer, anti-obesity, anti-diabetic and anti-photo aging activities . Figure 3 illustrates the chemical structure of fucoxanthin.
Figure 3. Fucoxanthin
Its main application at this moment is as one of the working compounds of dietary pills. Despite his abundance and beneficial effects, fucoxanthin is not yet widely known, which results in a broad range of price estimates: € 183-42,222 per kilogram. Most fucoxanthin is produced from macroalgae, because cultivation is much cheaper. However, the concentration of fucoxanthin can be much higher in microalgae .
β-carotene is another pigment produced by Phaeodactylum tricornutum. This carotenoid is the most generally known of all these pigments and is already commercialized by multiple companies, for example by using Dunaliella salina as a feedstock. It is a precursor of vitamin A and therefore also known as provitamin A. Due to its anti-oxidant features, it can inhibit or slow down the oxidation of free radicals or prevent the propagation of free radical chains . Other beneficial effects include preventive effects against cancer . β-carotene is mainly produced as a pigment or antioxidant for nutraceutical and dietary supplement applications . It is used as well as a colorant for animal feed .
Figure 4. β-carotene
More than 90 % of the total β-carotene production is chemically synthetized . However, a trans-isomer is obtained in synthetic manufacturing. Natural sources produce a mixture of trans- and cis-isomers. As the cis-isomer possesses anti-cancer properties, the price of natural β-carotene doubles the price of the synthetic form . Although this difference in isomer composition is an important factor for market differentiation, it has also been a barrier to market acceptance in the early stages of marketing of the microalgal product . An alternative natural source of β-carotene, which competes with the microalgal product, is the fungus Blakeslea trispora . Nevertheless, the microalgae Dunaliella salina is still the most important natural source for β-carotene .
Market prices for the natural β-carotene form range from € 215-2,650 per kilogram [16-19]. The total market volume of β-carotene has been estimated between € 218-272 million, with the natural β-carotene form constituting 20%-30% of the market [1, 15, 17, 20]. The compound annual growth rate has been estimated between +1.8% and +3.1%.
β-carotene has the E-number E160a in Europe. A study by the European Food Safety Authority concluded that the consumption of microalgal β-carotene is not of safety concern, given that the intake is not more than the normal consumption from natural sources (5-10 mg/day) . Current producers of β-carotene from a microalgae feedstock are EID Parry (India), Cognis/BASF (Australia/Germany), Betatene/BASF (Germany), Natural Beta Technologies (Australia), Tianjin Lantai Laboratory (China), Nature Beta Technologies/Nikken Sohonsa (Israel/Japan), Aqua Carotene Ltd (Australia), Pro Alge Biotech (India), Shaanxi Sciphar Biotechnology Co. and DSM (Netherlands) .
The third pigment which was identified as a potential biorefinery product from a Phaeodactylum tricornutum was zeaxanthin. Zeaxanthin is a less abundant isomer of lutein and its chemical structure is illustrated in Figure 5 . Lutein is a xanthophyllic pigment with antioxidant properties, which structure is illustrated in Figure 6. Lutein is accumulated in the retina of the eye, where it filtrates the blue light and protects the eye against singlet oxygen and radicals. Lutein can have a preventive function against different diseases such as age-related macular degeneration, cataract or skin diseases. However, there still exists controversy over the use of lutein and zeaxanthin as a pharmaceutical for these diseases as the exact mechanism is not yet known [23, 24]. Lutein/Zeaxanthin can be valorized in crystallized or sold in oil suspense. Like the other carotenoids, lutein is not synthesized by the human metabolism and therefore needs to be acquired to the diet . A minimum daily consumption of 6 mg is recommended. The main dietary sources of lutein are green leafy vegetables such as spinach and kale . Another dietary source is egg yolk, which contains a lower amount of lutein but with a higher bioavailability . Zeaxanthin has similar beneficial effects as lutein although it accumulates at a different location in the retina of the eye. The separation of lutein and zeaxanthin is not a straightforward process. Therefore, many researchers report a combined lutein/zeaxanthin value .
Figure 5. Lutein
Figure 6. Zeaxanthin
Other microalgae species which are known to produce high amounts of lutein are Muriellopsis sp. and Scenedesmus almeriensis . Lutein and zeaxanthin are mainly sold as a feed additive . Other applications are the use as a colorant in cosmetics and as food or pharmaceutical products . Lutein is listed in the European Union as a food additive (E616b) . The price for lutein/zeaxanthin ranges between € 910 and 15,000 per kilogram (based on commercial suppliers). The market size is estimated to be between € 123 and 165 million per year, with a CAGR of 3.6-6.1% [15, 17].
Currently, the main producers are Kemin Foods, which has a lutein-based partnership with DSM and Cognis, which was acquired by BASF. The commercial names of both lutein-based products are FloraGlo (Kemin Foods) and Xangold (Cognis). Both producers use marigold extract as a source of lutein. However, microalgae contain a much higher content of lutein compared to marigold. A relatively easy production process and a more homogenous biomass are other advantages of the use as microalgae as a lutein source. Other possible sources for commercial lutein are egg yolk, corn residues or shellfish. However, the lutein composition and/or bioavailability are lower compared to microalgae as well. A disadvantage of microalgae is the higher cost of algae biomass production compared to alternative sources .
Besides pigments, Phaeodactylum tricornutum produces another valuable product: Eicosapentaenoic acid. Eicosapentaenoic acid, or shorter EPA (see Figure 7), is a polyunsaturated fatty acid (PUFA). Together with docosahexaenoic acid (DHA), it is the main omega-3 fatty acid. A variety of health effects, such as anti-inflammatory effects, anti-cancer effects and beneficial effects for cardiovascular diseases are attributed to omega-3 fatty acids . EPA is a non-essential PUFA as it can be converted from the essential alpha-linolenic acid (ALA). However, this conversion rate is not high enough to meet the daily intake requirements .
Figure 7. EPA
The main dietary source of EPA is fish oil. However, fish do not synthesize EPA themselves, but derive it from the marine microorganisms they consume . Advantages of the production of EPA from microalgae instead of fish oils are the lack of unpleasant odor, the reduced chemical contamination risk and the higher purification potential . The current main source of EPA fish oils is the anchovery fishery. The global EPA and DHA market size is expected to rise, for example due to the rise in demand of functional foods. Therefore, microalgae-based PUFAs, which are currently expensive may have promising market perspectives as the anchovy fishery will not be able to meet these demands alone .
Besides food applications, feed applications are promising as well. However, the high production price is still a problem. Another issue is the high fat content of microalgae which can become a problem for salmon feed processing . Moreover, the salmon feed industry would require a minimum supply of 100,000 ton dry algae per year, which requires a large upscaling of current microalgae cultivation plants .
Currently, the omega-3 market for food supplements is dominated by large companies, DSM in specific. This can be both and entry barrier as an opportunity for small companies . Pharmaceutical supplements, nutritional supplements and functional food applications account for 72% of the total market volume. Of these three applications, the nutritional supplements are dominating with 59% of total market volume. Pharmaceutical applications are still limited to triglyceride reduction. However, other applications are currently under development .
Current producers of algal EPA/DHA are Ocean’s Alive (USA, Nannochloropsis), Flora Health (USA, Schizochrytium), Martek/DSM (USA/NL, Crypthecodinium), Blue Biotech(Germany, Nannochloropsis), InnovalG (France, Odontela), Photonz (New Zealand), Xiamen Huison Biotech Co. (China, Schizochrytum) and Lonza 2010 (Ulkenia) .
Prices for algal-based omega-3 oil are higher than for fish-derived omega-3 oil. For human consumption, algal-omega-3 oil has a price between € 70 and € 141 per kilogram. For feed applications a lower price, € 1.32 per kilogram, was estimated by Borowitzka . The market size for total omega-3 oil consumption ranges between 85,000 ton and 190,000 ton and is still growing .
Nannochloropsis oceanica is the second proposed feedstock for an algal-based biorefinery. It can produce several high-value products, like EPA, zeaxanthin, β-carotene, astaxanthin and canthaxanthin.
Astaxanthin is a pink-orange pigment which is used in aquaculture to provide the typical color of salmonids and crustaceans . However, as the production of microalgal-based astaxanthin is much more expensive then the synthetic form, the microalgal product cannot compete in this market. Moreover, the advantages of its natural source are not as significant in the feed market as they would be in the food market. Therefore, the microalgal astaxanthin has targeted the nutraceutical market, due to its strong antioxidant properties .
Astaxanthin has even more pronounced anti-oxidant properties than other pigments such as lutein or β-carotene . Different health effects such as protection against cardiovascular and immunological diseases and cancer have been attributed to this carotenoid . An overview of the beneficial effects of astaxanthin can be found in the study of Guerin et al. . The most common microalgae species which is used to produce astaxanthin is Haematococcus pluvialis . Figure 8 illustrates the chemical structure of astaxanthin.
Figure 8. Astaxanthin
Astaxanthin is mostly produced synthetically . However, the high costs of this synthetic process and the growing demand for natural sources have initiated research for natural alternatives. An alternative natural source for astaxanthin is the red yeast Phaffia rhodozyma . Krill and the yeast Xantophyllomyces dedrorous/Pfaffia rhodozyma are also used to produce small quantities of astaxanthin . Figure 9 gives the range of astaxanthin content over different natural sources.
Figure 9. Average content of astaxanthin in different sources 
Astaxanthin is classified as food additive with E number E161j . The price of astaxanthin ranges between € 7,150 and € 8,837 per kilogram for the natural form and between 1,762 and € 2,198 per kilogram for synthetic astaxanthin [16, 19, 34, 37]. The market size for total astaxanthin is estimated between € 176-227 million per year [15, 38]. The natural form of astaxanthin constitutes 5% of this total production .
Current producers of astaxanthin as a dietary supplement or food additive are Cyanotech (USA, Hawaii), EID Parry (India), Mera Pharma (US, Hawaii), Fuji Chemical Industry Co. (=BioReal, Sweden), US Nutra (US)/Parry Nutraceuticals (India), AlgaTech (Israel) and Blue Biotech (Germany) . Astaxanthin for coloring applications for aquaculture is produced by Blue Biotech in Germany 
The last pigment of the proposed Nannochloropsis oceanica biorefinery is canthaxanthin. Canthaxanthin is a carotenoid as well and has anti-oxidant, neuroprotective and anti-inflammatory activities . The application as chicken feed has been proven to be associated with increased hatchability . Canthaxanthin can also be related to certain anti-cancer functionalities. However, no conclusive results on this topic have been obtained yet . Figure 10 illustrates its structure.
Figure 10. Canthaxanthin
Canthaxanthin is commercially used to color egg yolk, salmons from aquaculture, and birds. It has been commercialized as a tanning pill, although it has been associated with different negative health effects . Canthaxanthin can be found in fungi, such as cantharelles, but it is produced synthetically as a bulk product . The commercial producers of canthaxanthin are DSM, Novus international (canthacol) and Versele-Laga, which produces can-tax to color birds.
Feed applications are allowed in the EU for cats, dogs, ornamental fish and birds without a maximum amount. The use of canthaxanthin as a feed additive for salmon and trout is allowed with a maximum concentration of 25 mg/kg. For poultry other than laying hens, a maximum concentration of 25 mg/kg is established, where a more strict concentration of 8 mg/kg is allowed for laying hens. Mixing canthaxanthin with other carotenoids is allowed as well, if the total concentration does not surpass 80 mg/kg in the complete feedingstuff . The maximum residual amount in the animal products has been restricted as well . These maximum concentrations are a consequence an unwanted side effect of canthaxanthin, which may form minute crystals in the retina of the eye through a reversible deposition effect at high doses [41, 43]. However, a recent tolerance study by Weber et al.  did not find any health problems related to a ten-times overdose of canthaxanthin in poultry feed. Canthaxanthin is classified as feed additive E161g .
The price for canthaxanthin ranges between € 528 and € 4,278 per kilogram (based on commercial suppliers). Its market size was estimated at approximately € 90 million per year according to a BCC report and is stagnating .
The third and last proposed biorefinery uses Chlorella vulgaris as a feedstock. Its main products are lutein/zeaxanthin, canthaxanthin, β-carotene and astaxanthin. Chlorella vulgaris is one of the most popular microalgae species and has been commercialized since decades. It has primarily been produced to be sold as entire biomass. Prices vary between € 89 per kg in the form of dry powder (Phycom, 100% purity) and € 220 per kg in the form of dry pellets of (Blue Biotech, 97% purity).
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