DETERMINATION OF INTERMEDIARY METABOLITES IN ASPERGILLUS-NIGER

Procedures are described to recover and determine intermediary metabol ites in Aspergillus niger. The methods involve rapid quenching of meta bolism by direct transfer of a culture sample from a bioreactor to a b uffered solution containing 60% methanol at -45 degrees C. Metabolites do not leak from the cells when this sampling technique is used. Foll owing quenching, the mycelium is collected by filtration and washed if required. Extraction of metabolites at -20 degrees C and neutral pH u sing chloroform to permeabilise the cells prevents degradation of labi le metabolites. An internal standard (xylitol) is used to correct for extraction recovery which was approximately 80%. To calculate actual i ntracellular concentrations, the intracellular volume has been determi ned. A value of 1.2 ml/g of dry weight was obtained for the growth con ditions used. This method is suitable to determine the levels of glyco lytic intermediates in Aspergillus niger mycelium. Although the measur ed levels were, in general, similar to values published previously and obtained by various other methods, the advantage of the procedure des cribed here is that all metabolites can be assayed in a single extract .