War/d Journa/ of Microbio/ogy & Biotechno/ogy 11, 346-346 Short Communication: The effect of urea on growth of moulds and biomass yield in solid-state cultivation B. Kovac and P. Raspor* Urea, added at 2 to 20 mg/g in solid bran medium supporting growth of Aspergihs oryzue and Nk~pus digosporus, led to a higher concentration of NH4’ and pH but a decrease in biomass production. &y words: Aspergillus oryme, protein enrichment, Rhiqms oligosporm, solid-state cultivation, urea. Wheat bran is a particularly suitable material for conversion into microbial biomass because it has a good ratio of substrates and no apparent growth inhibitors. Organic and inorganic nitrogen salts have already been used to enrich wheat bran (Rhodes & Broderick 1989) and the aim of the present study was to optimize the concentration of urea needed to maximize the protein content of several filamen- tous fungi grown on bran. The method of Mitchell ef ~1. (1988) was used as a model to compare growth on agar plates with that in the bran medium. Materials and Methods Microorganisms and Growth Aspergik oryzae ATCC 16868, ATCC 22787, ATCC 12892 and Rhizopm ofigosporus ATCC 48109 were grown cm MS agar contain- ing (g/l): yeast extract, 4; glucose, 4; starch (Kemika), 0.5; agar, 20; and malt extract, 30; supplemented with urea LIP to 2.0% (w/v). The effects of urea were also studied in a solid-state, wheat-bran medium with 40% (w/w) water content. Sterile standard urea solution was added to give up to 1.0% (w/v). Media were inoculated with a spore suspension to give 105 spores/g, spread on plastic Petri dishes and incubated at 28’C for 3 days. Attalyticaf Mefhods Total N was determined by the Kjeldahl method. Non-protein-N was determined after precipitating proteins with trichloracetic acid, Chitin was digested by alkaline hydrolysis, deaminated with HN03, reacted with methylbenzothiazalonehydrozone hydrochlo- ride and FeC13 and glucosamine content determined, according to Ride & Drysdale (19711, at A650. NHa’ was determined at 660 nm using a Technicon II auto-analyser. Urea was converted to NHa’ using urease and then determined in the same way. Results and Discussion Colony growth was measured on agar over 44 to 52 h of cultivation. As Mitchell ef ul. (1988) observed, colonies of all R&opus strains grew faster than those of Aspergiks, at The authors are with the Department of Food Science and Technology, Siotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 61000 Ljubljana, Slovenia; fax: 366 61 274 092. *Corresponding author. @ 7995 Rapid Communications of Oxford Ltd 0.2’ ’ I I 1 1 1 0 0.4 0.6 1.2 1.6 2 Urea (%) Figure 1. Rate of radial growth of the colonies of Aspergi//us oryzae ATCC 16866 (W), ATCC 12892 (a), ATCC 22787 (A) and Fff~izopus o/igosporus ATCC 48109 (a) over 44 to 52 h on agar plates supplemented with various concenWaGons of urea. least in terms of colony radial growth (Figure I). However, Rhizopus colonies, though wider, were relatively sparse and colony radial growth may not be a good measure of biomass production, especially when different genera with different morphological characteristics are being compared. The concentration of chitin in the mycelium gradually increases with age (Sharma ef ul. 1977). To avoid this effect as much as possible, glucosamine content was always measured after 48 h of cultivation, a time found to be optimal, in a preliminary study, for evaluating mould re- sponse to growth medium. Urea slightly stimulated colony radial growth in Aspergiks on bran (up to 1.2% in Aspergil- 1~ ovzae ATCC 12892 and ATCC 22787, and up to 1.6% in ATCC 16868) but not by any significant amount in Rhizopus (0% to 0.4%). Measurements of glucosamine con- tent indicated that biomass production in Aspergillus and Rhizopus was maximal with 0.2% to 0.6% and 0.4% urea, respectively (Table 1). The effect of urea on pH was less pronounced in R&opus than in Aspergillus. The results indicated differences in the ability of the different Aspergiks strains to use urea and transform it to NHd’, reflected by differences in the accumulation of various N forms in the biomass (Table I). The problem of non-protein-N produc- World ~ownal of Microbiology 6 Bmtechnology, Vol 11, 1995 345