Bioprocess development for high cell density cultivation of Rhizobium trifolii in semi-industrial scale stirred tank bioreactor

Rhizobium trifolii is a symbiotic N-fixing bacterium that is able to facilitate the conversion of atmospheric nitrogen into ammonia in legumes through the formation of root nodules. Although the use of Rhizobia as microbial inoculants is well-established, there is a lack of information regarding mass production of this type of bacteria in large scale. Therefore, the objective of this research is to develop an optimum cultivation condition for high cell mass production of R. trifolii in liquid culture, for 16-L and 150-L stirred-tank bioreactors. Batch cultivation under controlled and uncontrolled pH in 16-L stirred tank bioreactors yielded cell mass concentrations of 8.05 g L-1 and 16.87 g L-1, respectively. To study the limiting factors in Rhizobia cultivation, subsequent bioreactor experiments were carried out in different fed-batch mode such as constant feeding and intermittent addition method. Intermittent addition method yielded 37.95 g L-1 of cell mass which was lower compared to 69.10 g L-1 of cell mass using constant feeding strategy. Fed-batch cultivation of R. trifolli in a 150-L stirred tank bioreactor using the optimum cultivation strategy yielded a maximum cell mass of 27.32 g L-1. This was due to the decrease in oxygen transfer in a larger bioreactor. Under oxygen limited condition, exopolysaccharides (EPS) was accumulated in culture medium and reached about 9.5 g L-1.