Citric Acid Fermentation and Ageing

129. A spontaneous rise of intracellular cAMP levels in Aspergillus niger.

Majda Gradisnik-Grapulin and Matic Legisa, National Institute of Chemistry Hajdrihova 19, SI-1001 Ljubljana, Slovenia.

A spontaneous rise in intracellular cAMP levels was observed in the early stages of Aspegillus niger growth under high citric acid yielding conditions. The amount of cAMP formed was found to be dependent on initial sucrose concentration in the medium. Under higher sucrose conditions the peaks appeared earlier and were higher, while in lower sucrose media flattened peaks were observed later in fermentation. Since in media with a higher sucrose concentrations intracellular citric acid has started to accumulate earlier and more rapidly, it might be that cAMP synthesis is triggered by intracellular acidification, caused by dissociation of citric acid. Stimulation of cAMP synthesis by acidification was supported by the fact that addition of azide to the fermentation broth caused an imediate rise in cAMP levels. However, no spontaneous rise in cAMP concentration could be detected, if the cells were grown in continuously illuminated cultures, suggesting that A. niger phosphodiesterase might be photoregulated. More evidence for the light activated PDE was obtained by morphological studies under the light and dark conditions in the presence of cAMP or dbcAMP, and it was additionally supported by experiments where specific phosphodiesterase inhibitors were tested.

130. Citric acid production by solid state fermentation.

Y.D. Hang and E.E. Woodams. Cornell University, Geneva, New York 14456.

Citric acid is a commercially important product that has been obtained by submerged fermentation of glucose or sucrose by Aspergillus niger. This work was undertaken to determine the potential of food processing solid residues as a substrate for citric acid production by solid state fermentation using Aspergillus niger. Yields of citric acid varied considerably and were found to depend significantly on the strain of Aspergillus niger used and the following factors: the type of raw material fermented, the initial moisture content of the substrate, the amount of methyl alcohol present, and the fermentation time and temperature. Under favorable conditions, yields from more than 50 to nearly 90% were obtained on the basis of the amount of carbohydrate consumed. The results of this study indicate that food processing solid residues can serve as a low-cost substrate for citric acid production by solid state fermentation using Aspergillus niger.

131. Genetic improvement of industrial microorganisms: induction of further citric acid fermentation mutants of Aspergillus niger with the help of Gamma-rays.

M. S. Islam and Rehana Begum, Division of Genetics.Institute of Food & Radiation Biology, AEREP, GPO Box 3787, Savar, Dhaka, Bangladesh.

Citric acid is an organic acid of diverse economic use. The food and beverage industries use this acid extensively as a food additive. The present annual global requirement is nearly 4 lakh MTons which is produced almost entirely by fermentation of molasses sugar with selected strains of Aspergillus niger. Mutagenic techniques have been successfully employed for the improvement of Aspergillus niger strains. With the help of gamma radiations several high citric acid producing strains of Aspergillus niger were induced at our laboratory. These strains were found to yield 3-5 times more of citric acid from cane molasses than their parent strain (CA16). The induction of further mutation for higher yield of citric acid has been attempted using mutant strain, 14/20 as the parent. Three of the new isolates were selected for further test under semi-pilot scale studies, Of these, mutant strain no. 318 seems to be the most promising one which yields 88.5% citric acid in relation to sugar supplied.

132. Cloning and sequencing of cDNAs encoding citrate synthase and NADP⁺ -specific isocitrate dehydrogenase from Aspergilus niger.

Kohtaro Kirimura, Hiroyuki Nakagawa, and Shoji Usami. Department of Science and Engineering, Waseda University, Tokyo 169, Japan.

The cDNAs encoding citrate synthase (CS) and NADP⁺ specific isocitrate dehydrogenase (ICDH) from Aspergillus niger WU-2223L, a citric acid-producing strains were cloned by using designed oligonucleotide primers. The CS cDNA encodes 475 amino acid residues, and shows high homology with those of Neurospora crassa and Saccharomyces cerevisiae. The ICDH cDNA encodes 497 amino acid residues, and shows high homology with mitochondrial type ICDH of S. cerevisiae. The chromosomal gene encoding ICDH containing seven introns was also cloned and sequenced.

133. Metabolic engineering of the glycolytic pathway in Aspergillus niger.

Jaap Visser Wageningen Agricultural University, Wagningen, The Netherlands.

Genetic engineering of A. niger primary metabolism is employed to improve citric acid production by this fungus. The aim in such an approach would be to increase the metabolic flux through the pathway leading to citric acid formation. One aspect of this strategy is to decrease the fluxes through branches of the main pathway resulting in decreased formation of by-products, such as gluconic acid and oxalic acid. We have isolated a mutant that lacks an active glucose oxidase and hence does not produce gluconic acid from glucose. Another aspect is direct increase of the flux through the main pathway, for example, by overproduction of the enzymes involved. For genetic engineering of glycolysis we have isolated the genes encoding the glycolytic enzymes hexokinase, glucokinase, phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and pyruvate kinase (PKI). To date hexose phosphorylation has not been studied in detail in A. niger. We have recently found that A. niger possesses a hexokinase and a glucokinase. Moderate overexpression of PFK and PKI, either individually or simultaneously, did not increase citric acid production by the fungus significantly. Product yields (g citric acid formed per g glucose consumed) of the transformants were comparable to a wild-type strain making changes in by-product formation unlikely. Furthermore, no significant changes in the activities of other enzymes in the pathway or in the concentrations of intermediary metabolites were found. However, in strains overexpressing PFK the level of fructose-2,6-bisphosphate (F2,6BP), a positive allosteric effector of PFK, was reduced almost two-fold compared to the wild-type strain. Simulation experiments with purified PFK showed that such a reduction in the F2,6BP level could decrease the in vivo activity of PFK significantly. Thus, by decreasing the F2,6BP level A. niger seems to adapt to overexpression of PFK.