1. Spirulina production using three different media: biomass, pigments and lipids production Luis G. Torres, Julio C. Roldan and Yaremi Lopez UPIBI- Bioprocesses and Food Depts. Instituto Politécnico Nacional. Acueducto s/n, La Laguna Ticoman, Gustavo A. Madero, C.P , México, D.F. MEXICO.*Corresponding author,
Background
Spirulina is a well-known microalgae that has been used for centuries as a food pigment and supplement. Recently, nutraceutic properties (antioxidant, anticancer, antibiotic, lipid control and chelating activity, among others) have been reported for this strain. The culture media for Spirulina production represents an important fraction of the total production cost. It is an alkaline and saline medium which contains various other salts. It is mandatory to develop a simpler medium which allows good cell growth, lipids, and pigments production.
Aim of this work
The aim of this work was to develop a cheap medium to growth Spirulina, which produced amounts of biomass, lipids and pigments (both soluble in water and in acetone) comparable to the media called ENERVIVA.
Methods
The strain employed in this work arises from the Lago de Texcoco, Mexico. The experiments were carried out in 500 mL baffled flasks with 300 mL of each media (al assessments by triplicate). The microalgae were cultured in a shaker with 60 mmol photons/m2s, at room temperature (23-25oC). Three different media were employed: the full commercial media called ENERVIVA, containing NaCl, NaH2CO3 and salts, a second one, consisting in the major salts of ENERVIVA medium but where the NaCl (5 g/L) was replace by sea water. The third medium was prepared using human urine, seawater and H2CO3 (See Table 1).
Figure 2. Chlorophylls a+ b in the final products of Spirulina
grew in different media.
Media
ENERVIVA
Nutrients
Urine
NaHCO (g/L) 10
NaCl (g/L) 5
Potassium nitrate (g/L) 2
Potassium Sulfate (g/L) 1
Urea (g/L)
0.02
Monobasic potassium phosphate (g/L)
0.1
Magnesium Sulfate (g/L)
0.2
Calcium Carbonate (g/L)
Iron Sulfate (g/L)
0.005
Sea Water
160 mL/L
Human Urine
10 mL/L
pH 7.2 and then sterilization
Measurements
Biomass : optical density a 700 nm, daily
pH: commercial pH-meter,
Temperature: controlled at 25+/- 2 C
Irradiance: Erlen Meyer flasks were illuminated with LEDS at
Around 60 µmol of photons/m2.s
Lipids: hot extraction with hexane, final products
Soluble in acetone-pigments : cell rupture using acetone and mortar. Acetone extracts were evaluated at different wave lengths to calculate using formula the acetone-soluble pigments chlorophylls a and b. All measurements were carried out by duplicate.
As observed in Figure 2, the medium where more chlorophyll a and b were produced was the nutrients medium. Chlorophylls a and b in the ENERIVA and urine media were very similar between them. A maximum concentration of 7.17 mg/L for chlorophyll b was found in the nutrients medium, while a maximum value of 1.45 mg/L was found for chlorophyll a in the same medium. Note that the chlorophyll b/chlorophyll a ratio were of about 4.8 for all the Spirulina products.
Regarding the lipids production (see Figure 3), it is remarkable that the urine medium promoted the higher lipids production by Spirulina (8.15 mg/L), followed by the medium ENERVIVA (5 mg/L) and the nutrients medium (with seawater) at the end, with only 0.25 mg/L. It has been reported that biomass growth and lipids production have a compromise, i.e., the production of biomass is inversely proportional to lipids production. That situation was not observed in this work, since the lowest biomass and lipids concentrations were observed for the Spirulina growing in nutrients medium. In this work could be said that pigments (chlorophyll a and b) and lipid production have a compromise.
Table 1. Composition of the three media
Results and Discussion
Figure 1. Growth of Spirulina in the three media.
Figure 1 show the Spirulina Growth in the three different media. As observed, All the time, the biomass value for the urine medium was higher tan the reference media (ENERVIVA) and the nutrients or seawater media. The ENERVIVA medium showed an intermediate value, while the seawater medium showed the lower biomass concentrations. Nevertheless, at the 15 days of culture, the biomass values for the three different medium resulted very similar. (about 3.5 g/L) . As shown at Table 2, the nutrients medium supported a little lower specific growth rate for Spirulina, if compared with ENERVIVA and urine media. Regarding the Productivity, the higher value was observed for Spirulina growing in the urine medium ( mg/L.d). Maximum biomass concentration was for the Spirulina growing in urine medium also (3.58 g/L.
Figure 3. Final lipids concentration in the final products of Spirulina grew in different media.
Conclusions
As conclusion, it can be said that two media cheaper than the ENERVIVA full medium were shown to produce good amounts of biomass, lipids and pigments, equal or higher than those reached in the ENERVIVA medium. At the 15 days of culture, the biomass values for the three different medium resulted very similar (about 3.5 g/L). Maximum biomass concentration was for the Spirulina growing in urine medium (3.58 g/L). Regarding the productivity, the higher value was observed for Spirulina growing in the urine medium ( mg/L.d). The urine medium promoted the higher lipids production by Spirulina (8.15 mg/L), followed by the medium ENERVIVA (5 mg/L) and the nutrients medium (with seawater) at the end, with only 0.25 mg/L. On the contrary, the urine medium promoted the higher lipids production by Spirulina (8.15 mg/L), followed by the medium ENERVIVA (5 mg/L) and the nutrients medium (with seawater) at the end, with only 0.25 mg/L.
Table 2. Kinetic parameters for Spirulina growing in the three different media.
Acknowledgments Authors thank Instituto Politecnico Nacional (Grant SIP and COFAA) for financial help and economical support to attend the Congress at Miami, Florida.