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Sudden substrate dilution induces a higher rate of citric acid production by Aspergillus niger.
M Legisa and M Gradisnik-Grapulin Appl. Environ. Microbiol. 1995, 61(7):2732.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1995, p. 2732–2737 0099-2240/95/$04.00 0 Copyright 1995, American Society for Microbiology

Vol. 61, No. 7Sudden Substrate Dilution Induces a Higher Rate of Citric Acid Production by Aspergillus niger
ˇ MATIC LEGISA*
AND

ˇ MAJDA GRADISNIK-GRAPULIN

National Institute of Chemistry, SLO-61115 Ljubljana, Slovenia
Received 19 October 1994/Accepted 22 April 1995

On the basis of the present knowledge of Aspergillus niger metabolism during citric acid fermentation, an idea on how to improve theprocess was formed. Initially, a higher sucrose concentration was used for the germination of spores, which caused a higher intracellular level of the osmoregulator, glycerol, to be present. When citric acid started to be excreted into the medium, the substrate was suddenly diluted. Optimization of this procedure resulted in a nearly tripled volumetric rate (grams per liter per hour) of acidproduction, while the overall fermentation time was halved compared with the usual batch process. Yet, a characteristic delay was observed at the start of the acid excretion after the dilution. Hypo-osmotic shock caused a prominent elevation of intracellular cyclic AMP levels. Simultaneously, the specific activity of 6-phosphofructo-1-kinase increased significantly, probably due to phosphorylation ofthe protein molecule by cyclic AMP-dependent protein kinase. Specific 6-phosphofructo-1-kinase activity was much higher in the treated than in the normally growing mycelium. The metabolic flow through glycolysis was expected to be higher, which should contribute to a higher volumetric rate of acid production. Citric acid is one of the most important bulk-produced organic acids. Some 400,000 tons areproduced per year, largely by processes involving Aspergillus niger (15). Citric acid is a primary product of A. niger metabolism. In order to force a microorganism to excrete a primary metabolite, the microorganism must be grown under suboptimal conditions for biomass production. For citric acid accumulation, the fungus is grown in a medium lacking manganese ions but with a high initial sucroseconcentration and high dissolved oxygen tension (19). Since it is difficult to dissolve much oxygen in solutions with a high sucrose concentration, a compromise is necessary and has been empirically determined. Routinely, 14 to 22% sucrose is used to achieve high yields (21). The rate of citric acid excretion is determined in the initial phase of growth during spore germination. We have shown thatglycerol plays an important role at that stage. It inhibits NADP -specific isocitrate dehydrogenase (10), the enzyme localized predominantly in the mitochondrial compartment (3). Since mitochondrial NADP -specific enzyme has been found to be the most active enzyme converting isocitrate to 2-oxoglutarate (3, 14), its inhibition results in a diminished metabolic flux through the tricarboxylic acid (TCA)cycle and causes intracellular accumulation of citric acid (9). But first glycerol synthesized in the cytosol (11) must enter the mitochondrial matrix by passing through two membranes which are believed to be partially permeable (9). Glycerol acts primarily as an osmoregulator at the early stages of growth; later, it is metabolized. A high initial level of intracellular glycerol was followed by a...