Lignocellulose was converted into reducing sugars by using saccharification enzymes from cocultivated Trichoderma reesei and Aspergillus niger and reducing sugars as nutrients for Zymomonas mobilis to produce bioethanol in an immobilization system. After 96 h of cultivation, cocultivated T. reesei and A. niger had enzymatical synergistic effects that enabled a reducing sugar production of 1.29 g/L and a cellulose conversion rate of 23.27%. An 18% total inoculum concentration and a 1/1 inoculation ratio of T. reesei to A. niger obtained a reducing sugar production rate and a cellulose conversion rate of 2.57 g/L and 46.27%, respectively. The co-immobilization cultivation results showed that using polyurethane as a carrier optimized total saccharification enzyme activity at an inoculum ratio of 1/1 and a total inoculum concentration of 6.5 x 10(6) spores/mL Based on the experimental results, the bioreactor design was further modified to enhance bioethanol production. The three strains (A. niger, T. reesei and Z. mobilis) were cocultivated with a co-immobilization cultivation system. The experimental results showed that, after 24 h cultivation, bioethanol production reached 0.56 g/L, and reducing sugar conversion rate reached 11.2% when using carboxymethylcellulose (CMC) substrates. The experimental results confirmed that the modified bioreactor enhances bioethanol production. However, further experiments are needed to determine how to prevent multi-stage failure of reducing medium volume. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.