Profiling Active Low-Abundance Microbes in As/Sb-Contaminated Soils via d-Amino Acid-Based In Situ Labeling

Soil microbial communities play a pivotal ecological role in contaminated environments. However, conventional metagenomic approaches struggle to distinguish between "potential function holders" and "in situ metabolically active executors". Here, we employed a method combining fluorescent d-amino acid labeling, fluorescence-activated cell sorting, and metagenomics (FDAA-FACS-Metagenomics) to capture and profile active microbes in complex soils. The secondary addition of As(V) and Sb(V) enhanced the community's reductive activity toward these metalloids, reshaping the active assemblages. Clostridium was markedly enriched, and several low-abundance members were activated as true executors of the reduction process. MAGs recovered via FDAA-FACS revealed an active core community with functional partitioning: some taxa participated directly in As(V)/Sb(V) reduction, while others contributed to community stability through tolerance and metabolic support. Notably, a Desulfitobacteriaceae genome (MAG29) harbored both arrAB and anrAB gene clusters, a complete Wood-Ljungdahl carbon fixation pathway, and nitrogen fixation genes. These genomic features suggest the potential for a multifunctional metabolic lifestyle involving metalloid reduction, carbon fixation, and nitrogen transformation. Such metabolic versatility may enable MAG29 to contribute to coupled carbon-nitrogen cycling and metalloid transformation under contaminated environmental conditions. These findings emphasize the important ecological roles of rare, metabolically active microbes in metalloid transformation and soil ecosystem functioning.