OPTIMATION OF ASTAXANTHIN YIELD FROM Spirogyra sp. THROUGH OXIDATIVE STRESS INDUCTION BY ADDING FE2+ AND HYDROGEN PEROXIDE

• Main Author: Ambya Fahira, Dini
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/43623
• Institution: Institut Teknologi Bandung

Astaxanthin is an antioxidant compound that belongs to the carotenoid group. Macroalgae Spirogyra sp. is known as a potential raw material for natural astaxanthin production. Astaxanthin accumulation in green algae can be increased due to the presence of oxidative stres. This research is aimed at increasing astaxanthin accumulation in Spirogyra sp. through the casting of oxidative stres by adding Fe2+ and hydrogen peroxide (H2O2). The initial stage of cultivation was carried out in two different types of medium, namely 0.5% manure medium (w/v) and 2.5% BBM medium as an optimization stage of macroalgae’s culture to determine the best type of culture medium. The addition of Fe2+ and H2O2 was carried out on the 6th day of cultivation with a ratio of 1:1, 5:1, 10:1, and 50:1. Cultivation was carried out in a 16 x 16 x 7.5 cm square container with a total volume of 1,5 liters. Lighting is provided with a light intensity of 2000-2500 lux in conditions of 12 hours of bright and 12 hours of dark. Aeration is supplied at a flow rate of 1 vvm. Cultivation temperature is maintained at 25 ± 1 ? and pH of the medium is maintained at 7 ± 0.3. Harvesting of Spirogyra sp. biomass is performed using a vacuum pump and buchner funnel. Biomass of Spirogyra sp. then weighed to get the fresh weight data then dried using freeze drying method. Dry biomass of Spirogyra sp. then taken as much as 30 mg and mashed mechanically using mortar and pestle. Spirogyra sp. is extracted using methanol: dichloromethane = 1: 3 (v/v) with a ratio of 3: 1 (w/v). Spirogyra sp. is then shaken using a shaker at 170 rpm and left for 2 days. The sample was then centrifuged at 10,000 g for 10 minutes at 4?. The supernatant is then collected and evaporated at room temperature. The supernatant that has evaporated is dissolved again by adding 2 ml of methanol (HPLC grade). The solution is then filtered using a syringe filter and put in a 2 ml vial specifically for autosampler. The measurement of astaxanthin content was done by reversed phase HPLC C18 column. The isocratic mobile phase of methanol: H2O at a ratio of 95: 5 (v / v) is carried out at a flow rate of 1 ml/min. The peak is read at a wavelength of 474 nm to quantify astaxanthin. Determination of nitrate concentration (NO3-) in the medium was carried out based on Cataldo et al. (1975) with the principle of UV spectrophotometry. The absorbance is then measured at a wavelength of 410 nm using a spectrophotometer. Determination of the model and kinetics of Spirogyra sp.’s growth is done using Matlab® software. The highest accumulation was achieved on the 10th day that give astaxanthin accumulation up to 0.3482 mg/g by the 10:1 ratio. The 10:1 ratio gave the highest astaxanthin accumulation with a sixfold increase compared to none addition of oxidative stres. However, oxidative stress due to Fe2+ and H2O2 addition causes a decrease in the growth of Spirogyra sp. biomass up to 60%. However, further analysis on productivity gave that the addition of Fe2+ and H2O2 do not increase astaxanthin productivity despite increasing the accumulation of astaxanthin in Spirogyra sp.