Optimization of Protein Yield from Rice Straw via Ammonia–Steam Explosion Pretreatment and Solid-State Fermentation Using Response Surface Methodology

The combination of ammonia and steam explosion pretreatment has shown significant potential to enhance protein yield during the solid-state fermentation (SSF) of rice straw. However, the complex interactions between physicochemical changes and fermentation parameters necessitate precise optimization. This study employed a three-level Box–Behnken design (BBD) within the framework of Response Surface Methodology (RSM) to evaluate the effects of steam explosion pressure, pressure retention time, and ammonia concentration on protein yield. The SSF experiments were conducted in 5 L fermentation bottles using a co-culture of Aspergillus oryzae and Neurospora sitophila. The results demonstrated that the optimal conditions were a pressure of 1.44 MPa, a pressure retention time of 139.4 s, and an ammonia concentration of 8.3%. Under these optimized conditions, the protein yield reached 42.1% after 96 h of SSF, which was validated by a confirmatory experiment (42.8% ± 2.37%). The pretreated rice straw exhibited significantly improved lignocellulose degradation and structural porosity, providing a highly conducive substrate for microbial growth. These findings suggest that the optimized ammonia–steam explosion process is an effective and quantifiable strategy for converting agricultural waste into high-quality protein feed, contributing to sustainable bio-resource utilization.