Abstract

First RG validation for A. succinogenes in brewery wastewater valorization. • Traditional 16S rRNA is unsuitable due to significant expression instability. • ffh and rpsL are optimal RGs under diverse industrial wastewater conditions. • rpoD and recA are the most stable pair for temporal analysis across growth phases. • Validated RGs optimize wastewater bioconversion for sustainable water management. Converting brewery mashing wastewater (BMW) into value-added chemicals is a sustainable strategy for industrial wastewater management and water resource recovery. Actinobacillus succinogenes 130Z is a leading contender for biosynthesizing succinic acid (SA) using renewable feedstocks. Precise quantification of gene expression by reverse transcription quantitative PCR (RT-qPCR) is critical for optimizing microbial bioconversion, necessitating the use of stable reference genes (RGs) to ensure reliable normalization. Nevertheless, systematic validation of RGs for A. succinogenes under industrial-relevant conditions remains scarce. This study rigorously evaluated the stability of 12 candidate RGs across diverse culture media (including BMW), abiotic stresses (acid and product inhibition), and different growth phases using five algorithms (Comparative Δ C t, geNorm, NormFinder, BestKeeper, and RefFinder). Notably, ffh demonstrated the most robust expression stability across the entire dataset, whereas the commonly used 16S rRNA exhibited poor stability. Consequently, a condition-dependent RG selection strategy is proposed: recA and rpoD form the optimal pair for growth phase analysis, while ffh and rpsL are most reliable for diverse industrial conditions (encompassing complex media and abiotic stresses). The reliability of the identified RGs was verified through the normalization of pckA gene expression. This study constitutes the first comprehensive evaluation of RGs for A. succinogenes 130Z under industrial-relevant conditions, providing essenti