A stepwise pretreatment of combination of bacterial treatment with NaOH/Urea (NU) treatment was conducted to enhance enzymatic hydrolysis of rice straw (RS). The results showed that the composition of RS changed significantly, the lignin and hemicellulose decreased while cellulose increased. The biological treatment with a bacterium Sphingobacterium sp. LD-1 achieved mild conditions for the sequential NU treatment, reducing the concentration of the NU solution from 7%/12% to 4%/6% and increasing the temperature from -20 degrees C to -10 degrees C. The saccharification of rice straw co-treated with bacterium Sphingobacterium sp. LD-1 and 4%/6% NU at -10 degrees C resulted in 1.396-fold and 1.372-fold increase of reducing sugar and glucose yield respectively than that of sole NU treatment. (C) 2015 Elsevier Ltd. All rights reserved.