Determination of intermediary metabolites in Aspergillus niger

Procedures are described to recover and determine intermediary metabolites in Aspergillus niger. The methods involve rapid quenching of metabolism by direct transfer of a culture sample from a bioreactor to a buffered solution containing 60% methanol at -45 degrees C. Metabolites do not leak from the cells when this sampling technique is used. Following quenching, the mycelium is collected by filtration and washed if required. Extraction of metabolites at -20 degrees C and neutral pH using chloroform to permeabilise the cells prevents degradation of labile metabolites. An internal standard (xylitol) is used to correct for extraction recovery which was approximately 80%. To calculate actual intracellular concentrations, the intracellular volume has been determined. A value of 1.2 ml/g of dry weight was obtained for the growth conditions used. This method is suitable to determine the levels of glycolytic intermediates in Aspergillus niger mycelium. Although the measured levels were, in general, similar to values published previously and obtained by various other methods, the advantage of the procedure described here is that all metabolites can be assayed in a single extract.