. Sparger Overlay N2 N2 Methods Results The views expressed are purely those of the author(s) and may not in any circumstances be regarded as stating an official position of the European Commission. This poster is part of the European Joint Programme One Health EJP. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No 773830. Effect of nitrogen gassing strategy on the in vitro cultured chicken caecal microbiota Ingrid Cardenas Rey1,2 • Teresita Bello Gonzalez1 • Kees Veldman1 • Arjan de Visser2 • Michael Brouwer1 1. Dept. of Bacteriology, Host-Pathogen Interaction and Diagnostics Development, Wageningen Bioveterinary Research, Lelystad, The Netherlands • 2. Laboratory of Genetics, Wageningen University and Research, Wageningen, The Netherlands. In vitro models are cost-efficient tools to study animal gut microbiota dynamics with controlled conditions without the ethical implications of animal studies. Nevertheless, the continuous optimisation of in vitro models is imperative to reduce experimental bias and generate reliable data. Here, we have tested the effect of two nitrogen gassing strategies (overlay vs sparger) on the chicken caecal microbiota composition over time using a continuous single-stage fermentation culture system. Conclusions Experimental conditions were set up identically in two bioreactors except for their anaerobic environment; in one nitrogen was supplied on the top of the culture (overlay) while in the other nitrogen was sparged directly into the culture (sparger). Bioreactors were inoculated with an identical cryopreserved pool of caecal content from adult chickens and the in vitro chicken caecal physiological conditions (temperature, pH, caecal movements, and reduced levels of oxygen) were real-time monitored overtime (Fig. 1). Samples were collected daily (day 0 - 8) for 16S rRNA gene sequencing and downstream analyses. Figure 2. Real-time monitored conditions (pH, temperature, dissolved oxygen (dO2)). Overlay and Sparger in light and dark colour, respectively. Figure 3. Alpha and beta diversity analyses of the in vitro chicken caecal microbiota in both bioreactors (overlay and sparger) a. Observed microbial richness over time (ASV; Amplicon sequence variant) b. Principal coordinate analysis showing changes in the microbial community composition over time c. Comparison of the top ten genera relative abundance over time. a b c In vitro simulated chicken caecal conditions remained stable over time (Fig. 2). Most variability was observed in the pH due to microbial activity. Microbiota analysis • Microbial richness fluctuated daily in both bioreactors (Fig. 3a). • Experiment time explained 63% of the compositional variation of the in vitro microbiota (p < 0,001) while the gassing strategy explained 4% (Fig. 3b). • Anaerobes as Butyricicoccus (Fig. 3c) and Lachnospiraceae were significantly higher (p < 0,001) in sparger than overlay. Contact: Ingrid.cardenasrey@wur.nl Acknowledgements Part of this work was funded by the Dutch Ministry of Agriculture, Nature and Food Quality. Our continuous single-stage in vitro model simulated the conditions necessary to closely reproduce the main taxa of the chicken caecal microbiota. However, our results also show that the choice of experimental settings, in our case gassing type, has an impact on the in vitro cultured microbial community composition over time. Thus, pre-testing optimisation and standardisation of in vitro systems should be considered as vital to reduce bias and draw meaningful conclusions. Further work: to study the effect of interventions (e.g. synbiotics) on the horizontal transmission of antimicrobial resistance genes in the in vitro chicken gut microbiota. Figure 1. Experimental set-up showing a. Origen of the initial caecal inoculum b. Two strategies of nitrogen delivery (sparger and overlay) on the in vitro chicken caecal microbiota.