ORIGINAL ARTICLE Effects of ammonium feeding on the production of bioactive metabolites (cordycepin and exopolysaccharides) in mycelial culture of a Cordyceps sinensis fungus P.-H. Leung and J.-Y. Wu Department of Applied Biology & Chemical Technology and the State Key Laboratory of Chinese Medicine and Molecular Pharmacology, the Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Introduction Cordyceps sinensis (Berk.) Sacc., known as Dong- ChongXiaCao (winter worm-summer grass) in Chinese, is a special type of mushroom with the fruit body formed on an insect larva. It is a precious medicinal fungus, which has been used in China since ancient times, mainly as a general tonic to strengthen and improve lung and kidney functions, to restore health after prolonged sick- ness, to maintain overall body health and to boost phys- ical performance. Modern scientific studies have revealed several new bioactivities of the traditional remedy, such as antitumour, immunomodulatory anti-inflammation, antiatherosclerosis and antidiabetes (Zhu et al. 1998; Li and Tsim 2004). Wild C. sinensis is a rare and endan- gered natural species, distributed only in a few isolated areas of high plateaus at 3500–5000 m above sea level in western China. As the limited natural resource cannot meet the increasing demand for herbal medicine and health food applications, solid or liquid fermentation has been widely used for the production of Cordyceps mycelial bio- mass and components. Liquid or submerged fermentation Keywords ammonium feeding, cordycepin, Cordyceps sinensis, Cs-HK1, exopolysaccharides, mycelial culture. Correspondence Jian-Yong Wu, Department of Applied Biology & Chemical Technology and the State Key Laboratory of Chinese Medicine and Molecular Pharmacology, the Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong. E-mail: bcjywu@polyu.edu.hk 2006 ⁄ 1661: received 29 November 2006, revised 26 March 2007 and accepted 2 April 2007 doi:10.1111/j.1365-2672.2007.03451.x Abstract Aims: To examine the effects of ammonium feeding on the production of cordycepin (3¢-deoxyadenosine, a nucleoside analogue) and exopolysaccharides (EPS) in mycelial culture of a new Cordyceps sinensis fungus Cs-HK1. Methods and Results: Cs-HK1 fungus was cultivated in a liquid medium con- taining glucose, yeast extract, peptone and a few major inorganic salts. NH 4 Cl was fed to the mycelial culture at various concentrations from 5 to 40 mmol l )1 on day 3 (during exponential phase). NH 4 Cl, fed at 10 mmol l )1 , stimulated the cordycepin production most significantly, with nearly fourfold increase in the cordycepin content of mycelia (from 28Æ5 to 117 lgg )1 ), and also increased the EPS production by 40% (from 2Æ6 to 3Æ7gl )1 ). The ammo- nium feeding had a slightly positive effect at 5–10 mmol l )1 , but a negative effect at higher concentrations on the mycelium growth. Ammonium feeding also caused a sharp drop of the medium pH, owing perhaps to the uptake of NH 3 and the release of H + by the fungal cells. Conclusions: Ammonium feeding to the mycelial culture of Cs-HK1 fungus enhanced the intracellular cordycepin accumulation and the EPS production. The enhanced cordycepin production may be attributed to the uptake of ammonia for nucleoside synthesis, and the enhanced EPS to the increased uptake of glucose for EPS biosynthesis. Significance and Impact of the Study: It is useful for the production of bio- active metabolites and for understanding ammonium metabolism and its relationship to the biosynthesis of nucleosides in a precious medicinal fungus. Journal of Applied Microbiology ISSN 1364-5072 1942 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 1942–1949 ª 2007 The Authors