M. Legiša*, S. Golič Grdadolnik
National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia
 

ABSTRACT

 Until now, it was difficult to accurately determine the intracellular pH of filamentous fungi growing in large bioreactors. In order not to change the pHi values during the preparation of samples and 31P-NMR measurements, a new technique was developed that quenched the metabolism immediately after taking the cells out of the growing environment. It has been done by immersing the mycelium in cold buffered methanol at  -40 oC. The temperature of the samples was maintained low even during the accumulation of scans in the NMR magnet. Since frozen cells were surrounded by liquid buffered methanol, sufficient motion of molecules containing phosphorous was possible to enable the formation of high-resolution NMR spectra. The method has proven to be reliable enough for monitoring the changes of apparent intracellular pH that occur during hypoxia in a fermenter.