Statistical media optimization studies for growth and PHB production by Ralstonia eutropha

Poly-β-hydroxybutyric acid (PHB) is a natural, biodegradable polymer, which is accumulated as an energy reserve material by a large number of bacteria when, nutrients such as nitrogen or phosphorous sources are available in limiting concentrations in the presence of excess carbon source. The major problem associated with the industrial production of PHB is its high production cost. In the present study, efforts were made to optimize the growth of Ralstonia eutropha NRRL B14690 in the presence of nutrients, which would not only decrease the production cost of PHB but also help in increasing the productivity. Using fructose and ammonium sulphate as carbon and nitrogen source (with the media reported in literature), R. eutropha exhibited a maximum biomass of 3.25 g/L with a PHB concentration of 1.4 g/L in 48 h. To determine the possibility of growth potential of R. eutropha, it was grown in different carbon sources of which fructose, lactic acid, sucrose and glucose yielded good growth and PHB production. In order to incorporate cheaper nitrogen source and growth factors in media, ammonium sulphate was substituted by ammonium nitrate, urea and ammonium chloride. Urea featured highest PHB accumulation of 3.84 g/L after 60 h of growth. In place of yeast extract as a source of minerals and vitamins, corn steep liquor was used which yielded a PHB concentration of 2.66 g/L. Statistical media optimization design was then used to optimize the composition of culture medium for maximizing the productivity of PHB. A maximum of 6.65 g/L residual biomass and 6.75 g/L PHB was obtained using optimized concentrations, representing 94 and 83% validity of the predicted models for residual biomass and PHB production, respectively. A significantly higher maximum biomass of 20.73 g/L with a PHB content of 9.35 g/L was obtained in a 7 L lab scale bioreactor thus giving a yield of 0.24 g PHB/g fructose consumed. Batch kinetics can be used for model development, which will facilitate simulation of nutrient limited cultivation(s) for over accumulation of PHB.