Results and Discussion Spirullina production in 200 L bubble columns with different mixing intensities: pigment profiles and proximal analysis of the products Luis G. Torres, Yaremi López, Raúl. Gálvez, Julio C. Roldan and Luis J. Corzo-Ríos UPIBI- Bioprocesses and Food Depts. Instituto Politécnico Nacional. Acueducto s/n, La Laguna Ticoman, Gustavo A. Madero, C.P. 07340, México, D.F. MEXICO.*Corresponding author, Antecedents Regarding the quality of the products, it can be said that the pigments that were identified by TLC in the three bubbled columns products are very similar to each other, but different from the commercial Spirullina sample. Pigments identified in the Spirullina biomass were fucocianine, alofucocianine, chlorophyll a, b and c, diadinochrome, b-carotene and pheophytine (a chlorophyll-Mg degradation product). See Figure 3 and Table 1. Spirullina is a well-known microalgae that has been used for centuries as a food pigment and supplement. Recently, its nutracetutic properties (antioxidant, anticancer, antibiotic, lipid control and chelating activity, among others) have been reported for this strain. It is necessary to develop an efficient and inexpensive process to grow these microalgae without using large production ponds. Te relationship between mixing in the column bioreactors and the quality of the Spirullina produced (proximal analysis, pigments, lipids, etc) has not been previously reported. Aim of this work Figure 3. TCL for the acetone- soluble pigments Table 2 shows the pigments production by Spirullina in the three bioreactors. In respect to the water-soluble pigments, both fucocianine and alofucocianine were found in all samples (home-made and commercial one). Higher concentration values were found in the comercial sample (37 and 27 mg/g dry weight, respectively) in comparison with the concentration values found in bioreactor 1, 2 and 3 products: 26 and 14 mg/g dry weight; 17 and 6 mg/g dry weight and 13 and 6 mg/g dry weight, respectively. The aim of this work was to establish the effect of mixing throughout air injection over the quantity and quality of Spirullina production using bubbled columns: No aereation (3), low aereation i.e., 0.02 vvm (2) and high aereation, i.e., 0.04 vvm (1). Methods Three bubbled columns of 200 L (see Figure 1) were used for the Spirullina culture using a defined alkaline and saline medium at three different mixing intensities. Since the medium contains large amounts of inorganic carbon (10 g/L of H2CO3), it was considered that the supplied air only worked to suspend the microalgae cells and provide mixing within the column. A Spirullina comercial product (Pronat-Ultra, Mexico) was employed as comparison against the home-made products. Table 2. Pigments (water and acetone-soluble) production in the three bioreactors. Measurements Biomass : optical density a 700 nm, daily pH: comercial pH-meter, daily at 10 am Temperature: daily at 10 am Irradiance: light meter equipment, daily at 10 am Lipids: hot extraction with hexane, final products Proximal analysis: OAC, final products Soluble in water-pigments: Cell rupture freezing/de-freezing thrice. Solubilization in 0.1N Phosphates buffer, reading of absorbances at defined nm values, for calculations. (phycocianine, allophycocianine and phycorerithrine) Soluble in acetone-pigments : cell rupture using acetone and mortar. TCL (ZnO) mobil phase was acetone 20% /petroleum ether (80%). Various nm abosrbances of the acetone extract were evaluated to calculate using formula the acetone-soluble pigments (chlorophylls a, b, and c, diadinochrome, b-carotene and pheophytine. Pigments only for final produts. Comercial 1 2 3 sample Bioreactors Note: Fucoci- fucocianine, Alofucoc- alofucocianine, Chl a, b, c- Chlorophyll a, b and c, Pheo-pheophytine, Diadin-diadinochrome, bCarot- b-carotene. Figure 1. Scheme of the 200 L bioreactors Results and Discussion It was observed that the best aeration rate for the process was 0.02 vvm, as a maximum Spirullina content of 1.84 g/L (15 days) was achieved. The 0.04 vvm experiment produced less Spirulina in 15 days (1.36 g/L) and the unmixed column produced even less Spirullina in 13 days (1.14 g/L) (see Figure 2 and Table 1). Regarding the lipids in the final samples, the maximum value was found for the bioreactor 1 with high mixing (2.17 mg/L), followed by the comercial product (2.1 mg/L), the bioreactor 2 with low mixing (1.5 mg/L) and finally, the bioreactor 3 without mixing (1.4 mg/L). Figure 5. Ficocianine extracted from different Spirullina samples. E, F and G correspond to bioreactors 1, 2 and 3, respectively. Figure 4. Percentages of pigment production for the different Spirullina products. Table 1. Biomass, biomass productivity and pigments production for reactor 1, 2 and 3. The comercial Spirullina pigments profile is shown at Figure 4. It is remarkable the high amounts of fucocianine (36%), alofucocianine (27%) and chlorophyll a (20%). Regarding the sample arising from reactor 1 (high mixing), the higher concentration was found for pheophytin (41%) followed by fucocianine (26%), alofucocianine (14%) and b-carotene (11%). In the case of the sample arising from reactor 2 (low mixing), the higher pigments concentrations were for: pheophytin (51%), fucocianine (17%), and b-carotene (14%). Finally, for the product arising from reactor 3 (no mixing), the main pigments found were pheophytin (56%), followed by b-carotene (17%), fucocianin (13%), and alophycocianine (6%). All these pigments have many applications in the industry of pigments, food additives and cosmetics. Pheophytin is not reported as a pigments, but it has been reported the high oxidation capability of this pigment, which could be very interesting for pharmaceutic applications. Conclusions It is feasible to cultivate Spirullina algae in photo-reactors of 200L, using an air flow of 0.04 vvm. With this flow, a good mixing level is promoted and biomass concentrations of 1.84 g/L are achieved with a biomass productivity of 0.1415 g/L.day. The quality of the products obtained was significantly affected by the mixing. The proximal analysis and the pigment profiles were very different from each other for the three products generated. Best protein contents were observed in the reactor without any mixing (no aereation). Regarding the lipids in the final samples, the maximum value was found for the bioreactor 1 with high mixing (2.17 mg/L), followed by the comercial product (2.1 mg/L), the bioreactor 2 with low mixing (1.5 mg/L) and finally, the bioreactor 3 without mixing (1.4 mg/L). Regarding the total pigments, more product was obtained in the reactor with low mixing (0.02 vvm). Specifically, the fucocianine and allofucocianine concentrations were better in the well mixed reactor (0.04 vvm) 26 and 14 mg/g biomass, respectively). Table 2. Proximal analysis for Spirullina produced at bioreactors 1, 2 and 3. As for the proximal analysis, the three bioreactor samples differ very little between them, but they are very different from the commercial product, especially with respect to the protein content, which is about 40% for homemade products and > 60% for the sample commercial Spirullina (see Table 2). Acknowledgments Authors thank Instituto Politecnico Nacional (Grant SIP 20160635 and COFAA) for finacial help and economical support to attend the Congress at Miami, Florida. Authors thank to M. Torre y A. Urzua (UPCI/Edo de Mexico) for the help in the pigments analysis.