OPTIMIZATION OF BACILLUS PARAMYCOIDES FERMENTATION MEDIUM TO INCREASE THE ALA (5-AMINOLEVULINIC ACID) YIELD AND ITS PRODUCTION IN A 10 L FERMENTER

• Main Author: Adihayyu Monconegoro, Dicky
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/66780
• Institution: Institut Teknologi Bandung

5-Aminolevulinic acid (ALA) is an essential precursor for the biosynthesis of tetrapyrrole compounds such as heme, cytochromes, chlorophyll, porphyrins, and cobalamin, which are needed by the organism. The potential of ALA in agriculture can be used as a plant growth promoter by improving plants' photosynthetic ability and their tolerance to stress. ALA can increase the biosynthesis of chlorophyll and the content of antioxidant compounds. ALA can be produced by Bacillus paramycoides through the C5 pathway (Beale pathway). The addition of a precursor for ALA synthesis and an inhibitor of ALA converting enzyme into tetrapyrrole compound in the metabolism pathway can increase the concentration of ALA. In a previous study, the composition of the fermentation media was replaced. Optimizing carbon and nitrogen sources was done to make production costs more efficient in the industrial scale ALA production. Production of ALA by using a 200 L fermenter had been carried out, but the concentration of ALA produced was still low (179 ?M) and the precursors-inhibitor used were not cost-effective yet. Thus, media optimization was needed by using design of experiment that will give the best prediction to get high ALA yield. Furthermore, the replacement of precursor and inhibitor was needed by using low cost matarials so that the production cost are more efficient. This research aimed to optimize B. paramycoides fermentation media in producing ALA using RSM, replacing precursors and inhibitors, and conducting a trial of ALA production using a 10 L fermenter. Fermentation medium optimization used RSM - Box Behnken Design experimental design. The continuous factor that needs to be optimized was the concentration of the fermentation media composition used, such as molasses, urea, NaCl, and glucose. The upper and lower limit values that must be entered into the Minitab® software were determined based on the previous optimization medium (technical media). A total of 54 variations of the media were tested by observing cell count and ALA concentration responses. The optimization was carried out in a 250 mL Erlenmeyer flask containing 50 mL culture with initial pH 6.8 and 10% inoculum culture with 0.8 optical density (OD) at 625 nm. Then, the culture was incubated at room temperature (25-29°C), 130 RPM for 24 hours. The validation test was carried out to determine the best media composition due to RSM optimization based on the predicted maximum response results. Replacement trials of the precursors and inhibitors were conducted by replacing the precursor sodium L-glutamate monohydrate with monosodium glutamate (MSG) and the inhibitor levulinic acid with wood vinegar. The trial of ALA production in a 10 L fermenter was conducted based on the best RSM optimization result and the used of a replacement precursor-inhibitor. The fermenter experiment was carried out for 48 hours at 30°C, initial pH of 7,1 and 130 RPM of mixer speed. DO and aeration were controlled at 40-60% and 1,5-2,5 vvm. After the first 24 hours of incubation, 0,1% v?v glucose 30% and 0,5% v?v wood vinegar were added and the pH was adjusted to 6,8. The fermentation was then continued for the next 24 hours at 30°C, 110 RPM. DO and aeration were controlled at 35-50% and 1.25-1.75 vvm. The results showed that the best RSM media composition was 27.78 g/L molasses; 9.145 g/L urea; 8.838 g/L NaCl; and 32.07 g/L glucose, which resulted in 10.749 log CFU/mL and 255.30 ?M ALA. Replacement of precursor-inhibitors with MSG and wood vinegar produced 298.94 ?M ALA, significantly different from the previous medium composition. ALA production trial in a 10 L fermenter produced 581.82 ?M ALA. This study showed that the production of ALA using the best media composition of RSM optimization result and replacement of precursor-inhibitors could produce ALA (581.82 ?M) 3.2 times higher than the previous study only produced 179 ?M ALA.