12 BIOCHEMISTRY OF FUNGI 



he established that Penicillium luteum has the same fermentative 

 ability. Subsequently P. expansum, P. divaricatiim, P. citrimmi, 

 and P. spimilosum and several species of Aspergillus, including 

 A. niger, A. clavatns, and A. parasiticus, were employed under 

 similar conditions to produce citric acid. Wehmer separated 

 Citromvces from Penicillium because of this ability to produce 

 citric acid, but it soon became apparent that this physiological 

 characteristic constituted an untenable generic basis. The species 

 of Citromvces, totalling about twenty, have therefore come to be 

 included in the Genus Penicillium. 



A number of studies have been concerned with the conditions 

 required for the production of citric acid. Molliard (1922) re- 

 ported that an insufficient quantity of nitrogenous material in the 

 substratum supplied to Aspergillus niger was correlated with the 

 accumulation of citric acid; this finding was not substantiated, 

 however, in the experiments of Bernhauer (1926). 



Butkewitsch (1923) reported that both Penicillium glaucmn 

 and A. niger must be grown in an acid medium to stimulate the 

 formation of citric acid. If the medium was neutral, oxalic and 

 nitric acid were produced; if it was alkaline, oxalic acid alone was 

 formed. 



In his studies on citric acid production by A. niger Porges 

 (1932) used an inorganic mineral nutrient to which sucrose was 

 added as a source of carbon. He found that it was necessary first 

 of all to secure a heavy mycelial mat over the surface of the 

 nutrient solution. As a source of nitrogen NaN0 3 proved far 

 superior to (NH 4 ) 2 S0 4 . Both Fe and Zn were essential. Sugar 

 concentrations of 15 to 20% gave best yields. As a final condition, 

 it was requisite that the mat be undisturbed in order to provide a 

 partially anaerobic environment. 



In 1917 Currie (1917) observed that in sugar solutions fer- 

 mented by the A. niger group the lag in acidity can be accounted 

 for by citric acid, which made up the difference between total 

 acidity and oxalic acid. 



Kostytchew and Tschesnokow (1927) noted, that so long as no 

 nitrogen is being absorbed, citric acid is not accumulated. At the 

 end of approximately 48 hours the mycelial mat of A. niger will 

 have covered over the nutrient solution. This solution must then 

 be replaced by a sugar solution that lacks mineral elements. After 

 3 days' growth on such a medium A. niger will have produced a 



