Process physiology study of Staphylococcus carnosus
Examensarbete för masterexamen
Biotechnology (MPBIO), MSc
Staphylococcus carnosus is a bacterium used as a starter culture in the food applica-tion industry, particularly used for fermented sausage production. In this application its specific property is to reduce nitrate to nitrite which subsequently reacts with myoglobin to form a characteristic red color. The strain has been found eÿcient in this by a mechanism suppressing nitrite from further reduction to ammonia, this is mainly under the control of the nitrogen assimilatory pathway and in particular the enzyme nitrate reductase. This process have also been found to be strongly influ-enced by oxygen. Chr. Hansen A/S is a food applications company that produces S. carnosus at an industrial scale. Recently, a patent has been been filed by the company for a production procedure in which aerobic fermentation of the cells is switched to anaerobic conditions. This has been found to increase the sought after properties of the cells. The mechanisms for increase of the nitrate reducing pro-cess in S. carnosus when grown on di˙erent carbon sources under di˙erent aerated conditions are still unknown. In this master’s thesis project the e˙ects of di˙erent oxygenated states on two di˙erent carbon sources, glucose and glycerol, have been studied. KNO3 was found to severely limit the growth, and a 2.5-fold increase of KNO3 concentration was therefor implemented. Using a continuously stirred tank reactor set-up as a chemostat cells were grown at steady states with the fixed di-lution rate 0.1 [h-1] using two levels of air flow and one level of nitrogen flow. This yielded cells at di˙erent physiological states in regards to oxygen availability. Resid-ual metabolites and media components were quantified by HPLC to gain insight to metabolic di˙erences between the physiological states. The nitrate reductase activ-ity for each of these physiological states was determined through an enzymatic assay which resulted in lower activity of the enzyme of interest under anaerobic conditions compared to aerobic conditions when grown on glucose a result contradicting theory. The results of enzymatic activity for growth on glycerol was no able to be determined for the two oxic conditions.Transcriptional levels of genes coding for the enzymes nitrate reductase and nitrite reductase were determined with qPCR. Results showed an increase in expression of reductase genes for cells grown on glycerol anaerobically in comparison to cells grown in the aerobic environment. For glucose, there were no significant di˙erences between cells grown under aerobic and anaerobic conditions.
Livsvetenskaper , Mikrobiologi , Annan industriell bioteknik , Medicinsk bioteknologi (med inriktning mot cellbiologi) , Life Science , Microbiology , Other Industrial Biotechnology , Medical Biotechnology (with a focus on Cell Biology)