Temporal and spatial variations in anaerobic microbiomes from different inoculum sources for carbon monoxide conversion in thermophilic trickle bed reactor

Trickle bed reactors (TBRs) are efficient biotechnological systems that utilize biofilm-forming microorganisms to valorize gaseous substrates, such as carbon monoxide (CO) and syngas. While CO conversion has been widely explored in batch systems, the impact of inoculum origin on continuous TBR performance is poorly understood. This study investigated the performance and microbial community dynamics of TBRs inoculated with three diverse sources: anaerobic sludge, anaerobic digestate, and cow manure. The reactors were operated under identical thermophilic conditions, using CO as the sole carbon and energy source. The reactor inoculated with anaerobic sludge demonstrated the fastest adaptation, achieving stable CO conversion within 10 days. In contrast, the other two reactors required over 70 days to reach a similar conversion level. Importantly, all reactors ultimately developed functionally stable biofilms dominated by the genera Methanothermobacter and Syntrophaceticus , showing a strong functional convergence. Spatial and temporal analysis of the microbial communities revealed that inoculum choice profoundly influenced the rate and pattern of microbial structuring. Anaerobic sludge promoted rapid and homogeneous community distribution, whereas cow manure resulted in pronounced spatial heterogeneity. These findings establish that inoculum selection is a critical determinant of early biofilm establishment and long-term process stability, providing key insights for optimizing continuous CO biomethanation in TBR systems. • Reactor startup and CO conversion were influenced by inoculum type. • The microbial community in the enriched biofilm was limited to two phyla. • All reactors formed a resilient Methanothermobacter -dominated biofilm community. • Spatial microbial dynamics varied by inoculum source. • The reactor with anaerobic sludge showed higher microbial temporal similarity.