In R3 (Figure ), higher temporal dissimilarity was observed between samples taken from the same reactor position over experimental time, with values ranging from 0.64 to 0.89. The reactor also showed substantial spatial variability, with high dissimilarity values between different reactor positions and the sump ranging from 0.27 to 0.92 on day 85 and from 0.13 to 0.26 on day 115, indicating significant heterogeneity over time. The pronounced dissimilarity between the top and sump (reaching up to 0.92) highlights different microbial community structures. This spatial heterogeneity likely results from nutrient and gas concentration gradients, particularly CO, which may be higher at the top due to the gas flow direction. Additionally, the high organic content in the CM inoculum may have influenced biofilm formation, as solid particles from the manure could occupy spaces within the biofilm carriers. These conditions contributed to diverse microbial communities across positions, with different community compositions and potentially different functional roles at the bottom and sump positions. This spatial heterogeneity suggests that inoculum composition plays a crucial role in determining biofilm structure and impacts microbial distribution and activity over time. The observed spatial dynamics suggest that when the inoculum source is highly diverse, such as in CM, the reproducibility of established biofilm communities may be limited.