Microbial diversity and metabolic predictions of high-temperature streamer biofilms using metagenome-assembled genomes.

High-temperature streamer biofilm communities (SBCs) are often dominated by Aquificota, which can comprise over 90% of the microbial population in shallow water channels, such as those found at Mammoth hot springs of Yellowstone National Park and the Rehai hot springs in China. This study examines SBCs from the Dusun Tua (DT) hot spring in Malaysia (75 °C, pH 7.6), where Aquificota accounted for only ~ 35% of the total amplicon sequence variants. Amplicon and hybrid metagenomic sequencing revealed a more balanced microbial community, co-dominated by Aquificota, Chloroflexota, Desulfobacterota, Bacteroidota, Deinococcota, and Candidatus Hydrothermae, along with Thermoproteota and Micrarchaeota. To our knowledge, the co-dominance of Aquificota and Chloroflexota in SBCs has not been previously reported. The unexpected abundance of Chloroflexota may stem from dispersal from upstream Cyanobacteriota-Chloroflexota biofilms, contributing to community diversification. Genome-resolved analyses identified more than 60 medium- to high-quality metagenome-assembled genomes (MAGs), suggesting that biofilm formation was initially driven by chemoautotrophic sulfur oxidation and CO₂ fixation, followed by the gradual integration of heterotrophic taxa. Nitrogen cycling and hydrogen oxidation are likely to contribute additional sources of energy. The presence of diverse CAZymes suggests that plant litter may serve as an additional carbon source. Genome-centric analyses across multiple phyla indicated that extracellular polymeric substances (EPS), curli fibers, and other matrix components contribute to the biofilm matrix, enhancing structural resilience and supporting persistence under harsh conditions. Overall, this study highlights the distinct microbial ecology of the DT SBC and broader metabolic roles beyond Aquificota dominance. The genes identified in this study may hold biotechnological potential and serve as a valuable resource for future enzyme discovery and functional screening.