Abstract
BACKGROUND: The China Space Station (CSS) modules feature many areas that are difficult to clean and thus susceptible to microbial outbreaks. A new sampling method utilizing an equivalent material sheet was applied to characterize the diversity of microbes that accumulated in inaccessible areas in orbit on the CSS. Equivalent material sheet is a membrane made of the same material as the wall of the module. RESULTS: Fifty samples were collected from interior surfaces (work, sleeping, and sanitary areas) of the Tianhe core module and the Wentian and Mengtian experimental modules, covering three flights by the Shenzhou (SZ)-12 to SZ-14 astronaut crews from 2021 to 2022. The numbers of culturable bacteria and fungi that accumulated during the on-orbit periods of each flight ranged from 0 to 2.83 × 10(9) colony-forming units/100 cm(2). The number of bacteria detected by quantitative PCR (qPCR) ranged from 1.24 × 10(5) to 2.59 × 10(9) rRNA gene copies/100 cm(2), with an average viability of 65.08%. A total of 103 bacterial strains and 27 fungal strains were cultured and isolated. The dominant culturable microorganisms were mainly from the genera Bacillus, Staphylococcus, Aspergillus, Cladosporium, and Penicillium. High-throughput sequencing results showed that the predominant bacteria were Pseudomonas, Stenotrophomonas, Methylobacterium-Methylorubrum, Sphingomonas, Bacillus, Staphylococcus, and Nocardiopsis. The microbial diversity in each module varied significantly with sampling time and sampling area. In the early stage of CSS construction with the SZ-12 crew, the microbial species evenness in the modules was high; later, with the SZ-13 crew, Pseudomonas began to appear as the dominant microorganism. More than half (58.80%) of the bacteria on module surfaces originated from the human skin and oral environments. Lactobacillus was present in all areas of the three modules at all sampling times. The biomarker bacteria Stenotrophomonas sp., isolated from the work area in the Tianhe core module, are typically derived from plants. SourceTracker analysis indicated that most of the microbes in the orbiting CSS came from human bodies, and that microbial diversity was significantly altered with each crew change. CONCLUSION: Future efforts at microbial prevention and control on orbit should emphasize the human and plant origins of microbes. Information on the microbial diversity in the condensate zone could be useful to guide the development of new strategies to prevent and control microbes during space flight. Video Abstract.