Microbial isolation and characterization from two flex lines from the urine processor assembly onboard the international space station.

Urine, humidity condensate, and other sources of non-potable water are processed onboard the International Space Station (ISS) by the Water Recovery System (WRS) yielding potable water. While some means of microbial control are in place, including a phosphoric acid/hexavalent chromium urine pretreatment solution, many areas within the WRS are not available for routine microbial monitoring. Due to refurbishment needs, two flex lines from the Urine Processor Assembly (UPA) within the WRS were removed and returned to Earth. The water from within these lines, as well as flush water, was microbially evaluated. Culture and culture-independent analysis revealed the presence of , , and . Fungal culture also identified and . Hybrid genome analysis of the five distinct isolates identified them as while the two isolates were identified as . Chromate-resistance gene clusters were identified through pangenomic analysis that differentiated these genomes from previously studied isolates recovered from the point-of-use potable water dispenser and/or current NCBI references, indicating that unique populations exist within distinct niches in the WRS. Beyond genomic analysis, fixed samples directly from the lines were imaged by environmental scanning electron microscopy, which detailed networks of fungal-bacterial biofilms. This is the first evidence of biofilm formation within flex lines from the UPA onboard the ISS. For all bacteria isolated, biofilm potential was further characterized, with the isolates demonstrating the most considerable biofilm formation. Moreover, the genomes of the revealed secondary metabolite gene clusters associated with quorum sensing, biofilm formation, antifungal compounds, and hemolysins. The potential production of these gene cluster metabolites was phenotypically evaluated through biofilm, bacterial-fungal interaction, and hemolytic assays. Collectively, these data identify the UPA flex lines as a unique ecological niche and novel area of biofilm growth within the WRS. Further investigation of these organisms and their resistance profiles will enable engineering controls directed toward biofilm prevention in future space station water systems.