Optimization of phosphate, nitrate, sucrose and incubation time on the production of ascorbic acid in suspended callus of Citrus Grandis (L.) Osbeck

Abstract

The potential of C. grandis tissue in producing high amount of ascorbic acid has been investigated in this study. In tissue culture technique, nutrients in the media have been a major factor in manipulating the final yield required. Phosphate, nitrate and sucrose with incubation time have been studied on their influence in production of ascorbic acid in C. grandis suspension cultures. The result obtained in factorial design indicates that among all four factors, only sucrose and incubation time give significant effect when present alone. However, all four factors have influence in the production when present together. This result was verified by the significant interaction among all the four factors. Then, suitable ranges of concentration for selected factors that obtained from factorial design were used in the optimization study. By using response surface methodology (RSM) through central composite design (CCD), the optimum conditions of selected factors were obtained. The result shows that when callus was supplied with 506.3mg/l phosphate, 1916.7 mg/l nitrate and 65.2 g/l sucrose and incubated in dark room for 7 days, maximum production of ascorbic acid at 34.53 ± 2.20 mg/l was obtained. The results obtained were in the range of predicted ascorbic acid and therefore the model of the study is acceptable. Then, the cultivation of callus in a bioreactor was carried out. The concentration of ascorbic acid obtained was 9 mg/l of which is very low compared to the production obtained in flask. This is due to the restriction provided by the unsuitable condition in the bioreactor for the growth of callus.