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Nutritional Microbiology, Microbiome, and Microbe Nutrition

Category: Nutritional Microbiology, Microbiome, and Microbe Nutrition (I) (Poster Session)

Nutritional Microbiology, Microbiome, and Microbe Nutrition (II) (Poster Session)

(P40-08-26) Gut Microbiota Preservation for In Vitro Fermentation Studies: Effects on Microbial Community Composition and Fermentation Outputs

Monday, July 27, 2026
9:30 AM - 10:30 AM ET
Location: Poster Board F-2
Disclosure(s):
Mariana Guzman Sanchez, B.S.: No relevant financial relationship(s) with ineligible companies to disclose.
Peter P. Jackson, PhD: No relevant financial relationship(s) with ineligible companies to disclose.


Objectives: In vitro gut fermentations are often used to assess the effects of dietary compounds on microbial metabolism and ecology. These systems are usually inoculated using fresh feces; however, logistical limitations often demand sample storage. Although fecal sample handling is known to influence fermentation outcomes, generally, there is no consensus regarding optimal preservation methods and their impacts on the reliability of results; a systematic evaluation of storage effects is needed. This study aimed to evaluate the effects of multiple short-term preservation methods of fecal samples on metabolic output and community structure for in vitro fermentations.

Methods: Fecal samples from four healthy adults were used. Each was divided into 11 aliquots, including a fresh control and five storage conditions: snap freezing in liquid nitrogen and transferring to a –80°C freezer, direct storage at –80°C and at –20°C, storage over blue ice packs (~4°C) with and without 10% w/v Oxyrase. Each was assessed for 24 and 48 h. Aliquots were subjected to a seven-day sequential batch fermentation with inulin (IQ) and sorghum arabinoxylan (SAX), separately. Gas production, pH, and short-chain fatty acids (SCFA) were measured at the end of each 24 h fermentation. Fecal input, batch, and enrichment communities (day 1 and 7) were assessed by 16S rRNA sequencing.

Results: Batch fermentations from stored samples had greater variability and deviated from fresh controls, including altered pH and SCFA production, particularly reduced propionate, in both SAX and IQ. However, sequential passaging mitigated these effects, with metabolic outputs converging toward fresh controls. Microbial communities clustered by donor, but storage significantly altered baseline and batch community composition within donors in both substrates (PERMANOVA, p < 0.01). Community structure changes were donor-specific, and decreases in Bacteroides spp. were most noticeable. Enrichment communities remained donor-driven, but reduced F and R² values implied mitigation of storage effects.

Conclusions: These findings suggest that sample storage can cause stochastic shifts in batch fermentations, compromising reliability. However, sequential passaging can be used to derive consistent microbial lineages for further analyses.

Funding Sources: This work was funded by NIH-NIDDK 1R01DK137173-01A1.