Sulfur Oxidation by New and Non-Canonical Bacteria in a Subsurface Flow Constructed Wetland Treating Domestic Wastewater

Constructed wetlands (CW) are a low-cost alternative for wastewater treatment, where microbial communities play a key role in the biotransformation of pollutants, including sulfur compounds. This study reports the identification of cultivable bacteria involved in the sulfur cycle (SC) in a subsurface-flow CW located in Tetipac, Mexico. Water, sediment, and rhizosphere samples were collected during four sampling events and plated on a mineral medium with thiosulfate. Colony-forming units were quantified, and 15 isolates were genetically identified by partial 16S rRNA gene sequencing. Bacterial abundance was higher in the rhizosphere, and the cultivable fraction was dominated by Pseudomonadota, particularly Gammaproteobacteria, accompanied by Bacteroidota and several previously uncultured lineages; genera such as <i>Achromobacter</i>, <i>Chitinophaga</i>, <i>Enterobacter</i>, <i>Pseudomonas</i>, <i>Raoultella</i> and <i>Stenotrophomonas</i> were recovered. Biochemical assays revealed heterogeneous metabolic profiles, with several isolates showing activities comparable to canonical sulfur-oxidizing bacteria (SOB). Most isolates oxidized thiosulfate and a substantial proportion oxidized elemental sulfur, with higher metabolic performance in rhizosphere isolates and a positive association with BOD₅ removal. Overall, these results indicate that the Tetipac wetland harbors a cultivable community of largely non-canonical SOB whose mixotrophic versatility and spatial differentiation suggest a key contribution to the SC and organic matter degradation in CW.