Trichoderma reesei was co-cultured with Aspergillus phoenicis using dairy manure as a substrate to produce cellulase with a high level of P-glucosidase. For pure cultures of T reesei and A. phoenicis, the optimal media compositions were the same (10 g/L manure supplemented with 2 g/L KH2PO4, 2 mL/L tween-80 and 2 mg/L CoCl2) while the optimal temperature and pH were similar (25.5 degrees C and pH 5.76 for T reesei; 28.2 degrees C and pH 5.14 for A. phoenicis). The mixed culture was therefore completed at 27 degrees C and pH 5.5, which is close to the optimal values for both fungi. The mixed culture resulted in a relatively high level of total cellulase and P-glucosidase. It was also found that a high manure solid concentration (> 20 g/L) resulted in higher enzyme activities, probably due to an alleviation of the nutrients limitation at 10 g/L manure. The P-glucosidase activity and filter paper activity of the mixed fungi culture were 0.64 IU/mL and 1.54 FPU/mL, respectively, corresponding to a ratio of 0.41, which is an ideal ratio for hydrolyzing manure cellulose. To test the effectiveness of the enzymes produced by mixed culture, a crude enzyme broth was used for hydrolyzing the manure cellulose. The glucose concentration produced was significantly (p < 0.01) higher than the glucose obtained when using the commercial enzyme and the enzyme broth of the pure culture T reesei. The results indicate that the mixed culture of T reesei and A. phoenicis is an effective approach to produce a cellulolytic enzyme system for hydrolyzing manure cellulose. (c) 2005 Elsevier Ltd. All rights reserved.