Anthraquinones and Phenols as Intermediates in the Formation of Dark‐Colored, Humic Acid‐Like Pigments by Eurotium echinulatum

Abstract Eurotium echinulatum , a fungus isolated from a vertisol in southern Spain, formed humic acid‐like pigments during growth in glucose‐asparagine or glucose‐NaNO 3 media. After 6 to 7 days the phenols, orsellinic, p ‐hydroxycinnamic, and p ‐hydroxybenzoic acids, and the anthraquinones, endocrocin, emodin, and physicion were detected in the culture media. Upon further development of the mycelial mats additional aromatic compounds were produced and after 4 to 6 weeks more than 50 different ether‐extractable phenols and anthraquinones were detected. With further incubation the amounts and number of these compounds decreased and pigment formation in the media increased. After 2 to 3 months the culture media were dark brown to black and about 1 to 3.5 g/liter of polymer could be recovered upon acidification of the culture medium. Sodium amalgam reduction of this polymer yielded numerous phenols, anthraquinones, anthrones, and possibly anthracene derivatives. Treatment with sodium dithionite yielded largely anthraquinones. The polymer contained 1 to 4.5% N depending upon the N source. From 50 to 60% of this N was released in the form of amino acids upon acid hydrolysis. With both asparagine and NaNO 3 as N sources the same amino acids were isolated from 6 N HCl hydrolysates. Quantitatively, however, there were differences in the percentage distribution of the amino acids.