Available online freely at www.isisn.org Bioscience Research Print ISSN: 1811-9506 Online ISSN: 2218-3973 Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2018 15(4): 4584-4589. OPEN ACCESS

Culture and Biorefinary of Two Freshwater Microalgae; Spirulina platensis and Chlorella vulgaris As Vitamins Sources

Abeer M.A. Mahmoud; Soaad A. Sabae and Helal A.M.

Hydrobiology Laboratory, National Institute of Oceanography and Fisheries, 101, El Kaser El Einy St., Cairo, Egypt . *Correspondence: amrhelal [email protected] Accepted: 05 Dec. 2018 Published online: 31 Dec. 2018 The bio-refinery of some microalgae can already meet the high demands of both the food and pharmaceutical industries. Spirulina platensis and Chlorella vulgaris are important microalgae for the production of high value products such as vitamins. These algae are concentrated sources of carotenoids (especially pro-vitamin A carotenoids) and other nutrients, such as vitamin B12. Their health benefits as a complementary dietary source for macro and micro nutrients have been studied and confirmed in various populations. The freshwater algae were cultured in 25 m3 open raceway pond under outdoor condition using commercial fertilizers (modified Zarrouk’s medium and BG11 medium). Therefore, the present study aimed to outdoor commercial production of these algae and utilization of their vitamins content. The analyses revealed the presence of vitamin A, C, B3, B6, B9, & B12. Vitamins content was high in Spirulina samples. Keywords: Spirulina platensis, Freshwater Microalgae, Vitamins, Chlorella vulgaris.

INTRODUCTION commonly produced by microalgae are: vitamin A In recent years, the biorefinary of microalgae (β-carotene), vitamin C, vitamin E and vitamin B has gained considerable importance. Biorefinery such as thiamine (B1), riboflavin (B2), niacin (B3), is an industrial process, where biomass is pantothenic acid (B5), pyridoxine (B6), folic acid converted into a range of biochemicals, materials (B9) and cobalamin (B12) (Jeske et al., 2011). and energy products. Some of the most Microalgae are a diverse group of microscopic biotechnologically relevant microalgae are the plants with the wide range of vitamins B1, B2, B3, green algae (Chlorophycea) Chlorella vulgaris and B6, B12, E, K, D, etc., compared with other plants the Cyanobacteria Spirulina platensis which are or animals (Avagyan, 2008). Spirulina & chlorella widely commercialized and used, mainly as are unicellular algae that are commercially nutritional supplements for humans and as animal produced world- wide. Spirulina platensis is a feed additives. Spirulina platensis, a blue-green blue-green microalga, it represents the most alga has been shown to be an excellent source of important commercial microalga for the production vitamins and phenolics (Colla et al., 2007; of biomass as health food (Vonshak and Ogbonda et al., 2007). Today the major use of Tomaselli, 2000). The nutritional value Spirulina is for the extraction of phycocyanin, a of Spirulina is well recognized with its unusual blue photosynthetic pigment. Chlorella and high protein content (60–70% by dry weight) and Spirulina microalgae are vitamin producers that its richness in vitamins, minerals, essential fatty are used in animal and human metabolisms, acids and other nutrients (Vonshak, 1997) and (Shim et al., 2008). The vitamins that are (Gershwin and Belay, 2008). Helal (2016) Mahmoud et al., Spirulina and chlorella as a source of vitamins concluded that Spirulina can be used as El-Qanater El-Khayria by using commercial unconventional source of protein. Recently modified Zarrouk’s medium (Zarrouk, 1966) in Spirulina platensis are widely being studied for its open pond raceway 25 m3 with using urea as a nutritional reasons and also for its therapeutic source of nitrogen and commercial phosphoric properties. Spirulina is the richest known natural acid as a source of phosphorus according to the source of β-carotene (pro-vitamin A). Spirulina method of Abou El-Kheir et al., (2008). contains a whole spectrum of natural mixed Microscopic examination was done to the carotene and xanthophylls phytopigments which inoculum, it was considered pure culture. The together seem to be related to its antioxidant inoculum added to culture by 5%/culture volume. activity (Pineiro et al., 2001). Spirulina & chlorella The cultures were serially expanded from a 100 algae are concentrated sources of β-carotene (pro ml Erlenmeyer flask, 5 L of cylinder, 50 L of vitamin A) and vitamin B12 (Tang and Suter, 2011). inoculum pond indoor at Hydrobiology lab. and Chlorella is unicellular freshwater microalgae. then transferred to outdoor greenhouse ponds of Chlorella, like Spirulina is mainly sold in health 1000 to 5000 L capacity. The culture of S. food stores and as a fish food. At present platensis was cultured at open air temperature in microalgal market is dominated by Chlorella and the pond. The mass production was measured by Spirulina (Becker, 2004; Pulz and Gross, 2004), the growth curve of the chlorophyll concentration mainly because of their high protein content, of algae. Biomass was harvested using 20 micron nutritive value, and moreover they are easy to mesh plankton net after 14 days. The harvested grow. Chlorella microalgae is one of the main biomass was dried outdoors 24-48 hours sun source of natural bioactive compounds used in the dried. Dried Spirulina was used as a powder. food and pharmaceutical industries (Zakaria et al., 2017). Recently, Chlorella is known and Production of Chlorella vulgaris commercialized because of its nutrients and Chlorella vulgaris used for this study was benefits. Chlorella is high in protein, vitamins and collected and cultivated from the Hydrobiology polysaccharides contents (Carballo-Cárdenas et Laboratory, National Institute of Oceanography al., (2003). and Fisheries, El-Qanater El-Khayria by using BG11 medium (Laura & Paolo, 2014). The MATERIALS AND METHODS inoculum was added by 5% of the culture medium. The cultures were serially expanded Outdoor production of Spirulina platensis& from a 100 ml Erlenmeyer flask, 5 L of cylinder, Chlorella vulgaris 100 L of inoculum container indoor at Hydrobiology lab. The concentrated culture was Production of Spirulina platensis collected and let to be participate and the let dry it Spirulina platensis used for this study was in oven at 60o C for 4 hours. The dried alga was collected from the Hydrobiology Laboratory, dried and used as powder. National Institute of Oceanography and Fisheries,

(1) (2) (3) 1-Microscopic photo 2- Outdoor greenhouse pond 3- Spirulina mat harvesting

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100 L small pond Chlorella vulgaris powder

contents of Spirulina & Chlorella were described Extraction & Isolation of Vitamins from in Table & Figure (1a-b). Spirulina microalgae Spirulina platensis& Chlorella vulgaris were richer sources of vitamin C, vitamin B3, B 9 The detection of water soluble vitamins carried out and vitamin B12, while Chlorella was richer by HPLC (Brubacher et al., 1985), operated at 30 source of vitamin B6. C°. The separation is achieved using a binary linear elution gradient with (A) 25mM NaH2PO4 Pro-Vitamin A (beta-carotene) pH = 2.5, (B) methanol. The injected volume was Spirulina was rich in ß-carotene, a principal 20µl. Detection: VWD detector set at 254 nm for provitamin A carotenoid. As shown in Figure (1-a), ascorbic acids (vitamin C) and 220nm for vitamins HPLC analysis indicates that 100 g of spirulina B3, B6, B9, & B12. contains 1100 IU, while 100g of Chlorella contains 195 IU. Vitamin A is involved in immune function, Vitamin A vision, reproduction and cellular communication After homogenization and saponification of (Johnson, 2000; Solomons, 2012). So they have a the juice sample in a solution of ethanolic fairly high concentration of ß-carotene and potassium hydroxide, (vitamin A alcohol), vitamins B3, B6, B9, B12, etc., compared with released is totally extracted with organic solvents. other plants or animals (Avagyan, 2008). Quantitation is carried out against β- carotene Table (1): Vitamins composition of Spirulina standard, the absorbance measured at 450 nm. and Chlorella.

RESULTS Vitamins(mg/kg) Spirulina chlorella Outdoor commercial production of Spirulina C 11.3 7.8 platensis& Chlorella vulgaris B3 20.05 3.4 Commercial synthetic medium for low-cost mass production of Spirulina & Chlorella in a B6 27.84 169.87 larger scale was used. The commercial culture of B9 7.5 2.1 microalgae is now over 30 years old with the main B12 21.1 5.2 microalgal species grown being Chlorella and Vitamin A Spirulina for health food, and several species for Spirulina chlorella aquaculture. Using commercial fertilizers as urea (IU/100g) 1100 195 and commercial phosphoric acid as a source of nitrogen & phosphorus in the media to decrease 1500 the cost of production. The size of Spirulina cells is larger than Chlorella cells which let it easy to 1000 harvest by using 20-micron mesh size plankton net. 500

Biorefinery of Spirulina platensis & Chlorella 0 vulgaris for utilization of vitamins Vitamin A Spirulina chlorella The HPLC investigated wide range of water (IU/100g) soluble vitamins: vitamin C (Ascorbic acid), B3 (Niacin), B6 (Pyridoxine), B9 (Inositol), B12 (Cyanocobalamin). In addition to large quantity of Figure (1-a): Vitamin A content of Spirulina β -Carotene (pro-vitamin A). The vitamins &Chlorella

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Water-soluble Vitamins Spirulina is four times as rich in B12 compounds Spirulina & Chloerlla microalgae have an as raw liver, long put forward as the best source. interesting amount of vitamin C (Table 1and However, it should be noted that a controversy Figure 1-b), this agrees with Jeske et al., (2011). surrounds the real bioavailability to humans of the Spirulina have high concentration of vitamin B3, B12 complex in spirulina (Carmichael 1994). 20.05mg/kg (Table 1and Figure 1-b). Vitamin B3 Many methods have been devised to extract is important for the metabolism of fats, cholesterol vitamin B12 from microalgae (Blanch et al., 1990). synthesis, DNA synthesis, regulation of glucose, Extracting vitamin B12 from algal material with all reduce cholesterol and cardiovascular disease the other metabolite complicates an already (Johnson & Russell, 2012). The analysis shows difficult separation process and is generally low in that Chlorella was high in vitamin B6 (Table 1and quantity (Kumudha and Sarada, 2015). The Figure 1-b), this agrees with the previous studies present analysis showed that Spirulina was high which shown that the vitamins content of Chlorella in vitamin B12 (Table 1 and Figure 1-b), which is is high. The tests have confirmed they contain necessary for red blood cell formation and DNA high levels of vitamin B6 (particularly chlorella synthesis. These agree with the previous studies algae which has six the amount of Vitamin B6 as which shown that Spirulina is a great source of Spirulina algae). Vitamin B6 (Pyridoxine) beta-carotene (provitamin A) and vitamin B12. deficiency in humans may be associated with Vitamin B12 is very useful in treatment of anemia (Harper, 1979). Spirulina and Chloerlla pernicious anemia (Vijayarani et al., 2012) and microalgae have an interesting amount of folic Belay, (2008). Chlorella contains large quantities acid; vitamin B9 (Table 1and Figure 1-b), which is of vitamin B-12 and iron (Tang and Suter, 2011; necessary for the formation of cells, preservation Jeske et al., 2011.). Beta-carotene accounts for of skin and for the normal development of bones 80% of the carotenoids present in Spirulina and and teeth (Becker, 2007; Brown et al., 1999). each kilogram of dry spirulina contains between 700 and 1700mg of beta-carotene and about Vitamin B12 100mg of cryptoxanthin; these two carotenoids It is worth stressing the exceptionally high Vitamin are convertible into vitamin A by mammals (Palla B12 (cobalamin) content, since this Vitamin is by and Busson, 1969). far the most difficult to obtain in a meatless diet because no common food plant contains it. Water soluble vitamins 180 160 140 120 100 Spirulina 80 chlorella 60

Vitamin conc.(mg/kg) 40 20 0 C B3 B6 B9 B12

Figure (1-b): Vitamins composition of Spirulina &Chlorella

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CONCLUSION Becker, W. (2004). Microalgae in human and Recently, the applications of biotechnology animal nutrition. In A. Richmond (Ed) focused on the economic microalgae such as Handbook of Microalgal Culture, Blackwell, Spirulina platensis & Chlorella vulgaris which are Oxford, pp. 312-351. commercially available. Their economic Becker EW, (2007). Micro-algae as a source of importance is related to synthesize of several proteins. Biotechnol Adv. 25(2):207–210. economic substances such as carotenoids and Belay A (2008). Spirulina (Arthrospira): Production vitamins and they can be used for the production and quality assurance. In: Gershwin ME, of functional foods. Mass production of both Belay A (eds) Spirulina in human nutrition Spirulina and chlorella in open ponds is viable and health. CRS press, Taylor and Francis under climate conditions of Egypt for a period of at group, New York, pp. 1-25. least 6 months per year. The outdoor commercial Blanche F,Thibaut D, Couder M, Muller JC (1990). cultivation of Spirulina & Chlorella has been Identification and quantitation of corrinoid carried out aiming at the production of large precursors of cobalamin from Pseudomonas quantity of biomass with low cost and utilization of denitrificans by high- performance liquid the biomass yield in vitamins production which chromatography. Anal Biochem 189: 24-29. give using of these algae as economic crops. Brown MR, Mular M, Miller I, (1999). The vitamin Spirulina and chlorella can be used as content of microalgae used in aquaculture. J unconventional source of vitamins. App Phycol. 11(3):247–255. Brubacher G, Muller-Mulot W and Southgate DAT, CONFLICT OF INTEREST (1985). Methods for the determination of The present study was performed in absence vitamins in food. Elsevier Applied Science of any conflict of interest. Publishers. London. Carballo-Cárdenas EC, Tuan PM, Janssen M and ACKNOWLEGEMENT Wijffels RH (2003). Vitamin E (α-tocopherol) The author would thank all participants and production by the marine microalgae their parents. Dunaliella tertiolecta and Tetraselmis suecica in batch cultivation Biomol. Eng. 20 139. AUTHOR CONTRIBUTIONS Carmichael W. (1994). “The Toxins of All authors contributed equally in all parts of Cyanobacteria” Sci. Am. Jan. 1994, 64-72. this study. Colla, L.M. Reinehr, C.O. Reichert, C. and Costa, J.A.V. 2007 Production of biomass and Copyrights: © 2017 @ author (s). nutraceutical compounds by Spirulina This is an open access article distributed under the platensis under different temperature and terms of the Creative Commons Attribution License nitrogen regimes. Bioresour. Technol. 98 (7): (CC BY 4.0), which permits unrestricted use, 1489–1493. distribution, and reproduction in any medium, Gershwin ME and Belay A, editors (2008). Spirulina in human nutrition and provided the original author(s) and source are health. Boca Raton: CRC Press. credited and that the original publication in this Harper H.A. (1979). Review of Physiological journal is cited, in accordance with accepted Chemistry, 17th edition. Copyright in Canada. academic practice. No use, distribution or 702 pp. reproduction is permitted which does not comply Helal A. M. (2016). Application study for producing with these terms. eco-friendly diet for fish from the blue green alga Spirulina platensis. PhD thesis, p 106. REFERENCES Jeske M, Trentini AM, Bontempo M. Clorela, O. Abou El-Kheir W. S.; Ibrahim E. A. and Helal A. (2011). alimento completo, Compêndio de M. (2008). Large Scale production of Fitoterapia. Manual da Medicina Integral. p. economically important cyanobacterium 1–2. (Spirulina platensis) by using commercial Johnson S. (2000). The possible role of gradual fertilizers. Journal of Environmental Science , accumulation of copper, cadmium, lead and 17(3), 1-19 iron and gradual depletion of zinc, Avagyan, A.B. (2008). Microalgae: Big Feed magnesium, selenium, vitamins B2, B6, D, Potential in a Small Package. Feed and E and essential fatty acids in multiple International, 16-18. sclerosis. Med Hypotheses, 55(3):239–2241.

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