Interactive effects of temperature and pH on multispecies biofilm development in a controlled system

Environmental temperature and pH drive biofilm growth, yet their interactions remain underexplored. We investigated their joint influence on biofilm communities using a spatially explicit bead-array system, where a bacterial polyculture formed biofilms on 12 glass beads under factorial combinations of pH (5, 7, 9) and temperature (15 and 25°C). Biofilms were analyzed using quantitative PCR and MiSeq sequencing. ANOVA revealed main and interactive effects of pH and temperature on abundance, richness, and diversity. Community-level metrics and phylotypes increased with rising pH, with stronger effects at 25°C (abundance, 9.7–11.0 log10 copies; richness, 5.8–9.0; diversity, 1.8–2.2). While pH shaped community structure, assembly trajectories differed between temperatures, indicating temperature-dependent modulation of pH responses. The temperature-pH interaction also shaped spatial distribution: at 15°C, heterogeneity followed a hump-shaped pattern along the pH gradient, whereas at 25°C, it declined with increasing pH (0.58–0.17 for abundance; 0.27–0.05 for community). Elevated temperature and pH promoted larger, complex interaction networks (average number of neighbors, 4.0–5.0 at 15°C; 4.0–7.6 at 25°C) and supported the establishment of rare species. Collectively, biofilm development is shaped by a strong, non-linear temperature-pH interaction, suggesting that jointly adjusting pH and temperature may help control biofilms in engineered systems.