Microbially mediated arsenic methylation, crucial for arsenic biogeochemical transformation, remains understudied in subgeothermal environments. This study aimed to investigate the activity and diversity of arsM-carrying microorganisms in sediment samples (termed YC1, YC2, and YC5) from a low-temperature hot spring. Microcosm assays revealed that only YC1 and YC2 exhibited limited As-methylating activities, generating a maximum of 2.3-3.5 mu g/L methylarsenate (MMA) and 2.2-2.8 mu g/L dimethylarsenate (DMA). The addition of lactate and arsenite significantly promoted these activities, increasing the concentrations to 26.1-184.0 mu g/L MMA and 36.5-204.0 mu g/L DMA. The arsM gene abundance also increased by 85-195%, indicating that lactate can activate arsenite methylation. YC1 and YC2, which were closer to the hot spring hole than YC5 was, had similar patterns, and shared a similar arsM community structure, dominated by Actinobacteriota, Firmicutes, and Proteobacteriota on days 7 and 21. In contrast, at YC5, the sampling site far from the spring hole, representatives of Acidobacteriota were dominant on day 7, whereas those of Actinobacteriota were prevalent on day 21. Acidobacteriota co-occurred with dimethylarsenate production, and Mycobacterium co-occurred with DMA demethylation. These findings suggested that the low-temperature arsenic hot spring possessed diverse arsenic-methylating species, whereas demethylating bacteria preferred to inhabit niches farther from the hot spring.
