High-resolution assessment of fecal microbiome integrity across practical storage conditions and extreme treatments

The human gut microbiome plays a critical role in host health, yet large-scale clinical trials and remote fieldwork are frequently bottlenecked by the stringent requirement for immediate sample preservation at ultra-low temperatures. While departures from this standard are assumed to introduce technical artifacts, the precise thresholds at which handling deviations distort ecological interpretations remain poorly defined. This study evaluates the structural resilience of the fecal microbial community across a spectrum of realistic storage delays (varying from standard freezing to room temperature for up to 11 days) and deliberate stress conditions, including prolonged open-air exposure, heating at 75°C, autoclaving, and ultraviolet irradiation. Using amplicon sequence variant (ASV) level analysis, we demonstrate that standard storage variations preserve overall microbial diversity and community architecture. Conversely, aggressive treatments like autoclaving and post-extraction ultraviolet exposure triggered catastrophic molecular degradation. This breakdown was characterized by massive template DNA loss, a severe collapse in bacterial diversity, and an artifactual dominance of stress-resilient Pseudomonadota and environmental contaminants. Together, these findings clarify the molecular signature of true sample degradation and provide insight into validating flexible sample-handling protocols, thereby expanding the logistical feasibility of microbiome research without major distortions in microbial community structure.