280 PHYSIOLOGY OF THE FUNGI 



ture indicates that submerged culture may be the preferred process in 

 the future. Average yields of 72 g. of anhydrous citric acid per 100 g. 

 of sucrose in the medium have been obtained in the laboratory (Shu 

 and Johnson, 1948). 



The formula for citric acid is given below: 



CH2— COOH 



I 

 HO— CH— COOH 



CH2— COOH 



Citric acid 



Any theory of citric acid formation must take into account the following 

 facts: Citric acid, a branched-chain compound, is synthesized from carbon 

 sources containing from two to seven carbon atoms. Yields of citric 

 acid may approach 90 per cent of the sugar used (Wells et al., 1936). 

 The amount of carbon dioxide evolved is low, which suggests either 

 reutilization or a mechanism of producing the necessary intermediates 

 without the production of carbon dioxide. Reutilization of carbon 

 dioxide seems the more probable, for Foster et al. (1941) showed Asper- 

 giUus niger to utilize radioactive carbon dioxide in the synthesis of citric 

 acid. The more probable pathway of synthesis is via the Krebs cycle 

 (Chap. 7) and the supplementary formation of oxalacetic from pyruvic 

 acid and carbon dioxide (Wood-Werkman reaction). 



Gluconic acid. A considerable number of fungi produce gluconic acid. 

 These include Aspergillus niger (various isolates), A. fuscus, A. cinna- 

 momeus, A. oryzae, Penicillium glabrum, P. glaucum, P. purpurogenum 

 var. ruhrisclerotium, P. chrysogenum, P. crustaceum, and Fumago vagans. 

 Most investigators have used selected isolates of A. niger for the produc- 

 tion of gluconic acid. Details of laboratory and semi-pilot-plant investi- 

 gations may be found in the papers of Wells et al. (1937), Gastrock et al. 

 (1938), and Forges et al. (1941). 



Many factors influence the formation of gluconic acid. Isolates of 

 A. niger differ in ability to synthesize this acid. Not all isolates produce 

 the maximum amount of acid under identical conditions. Adequate 

 aeration is necessary for the enzymatic conversion of glucose to gluconic 

 acid. Gluconic acid is produced most abundantly when the pH of the 

 medium is kept near 5. Calcium carbonate is used for neutralizing the 

 gluconic acid formed. This is advantageous, for calcium gluconate is 

 used in medicine as a source of readily assimilable calcium. Frecipitation 

 of calcium gluconate during formation may be prevented by the addition 

 of boric acid or borax to the culture medium in amounts vaiying up to 

 2,000 p. p.m. (Moyer et al., 1940). Boron compounds are added after 

 the growth of mycelium is essentially complete. The mycelium may be 

 used as many as thirteen times by removing the spent medium and adding 



