Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula

In vitro fermentation strategies with fecal inocula are considered cost-effective methods to gain mechanistic insights into fecal microbiota community dynamics. However, all in vitro approaches have their limitations due to inherent differences with respect to the in vivo situation mimicked, introducing possible biases into the results obtained. Here, we aimed to systematically optimize in vitro fermentation conditions to minimize drift from the initial inoculum, limit growth of opportunistic colonizers, and maximize the effect of added fiber products (here pectin) when compared to basal medium fermentations. We evaluated the impact of varying starting cell density and medium nutrient concentration on these three outcomes, as well as the effect of inoculation with fresh vs. stored fecal samples. By combining GC-MS metabolite profiling and 16s rRNA gene-based amplicon sequencing, we established that starting cell densities below 1010 cells/mL opened up growth opportunities for members the Enterobacteriaceae family. This effect was exacerbated when using fecal samples that were stored frozen at -80°C. Overgrowth of Enterobacteriaceae resulted in lowered alpha-diversity and larger community drift, possibly confounding results obtained from fermentations in such conditions. Higher medium nutrient concentrations were identified as an additional factor contributing to inoculum community preservation, although the use of a less nutrient dense medium increased the impact of fiber product addition on the obtained metabolite profiles. Overall, our microbiome observations indicated that starting cell densities of 1010 cells/mL limited opportunities for exponential growth, suppressing in vitro community biases, whilst metabolome incubations should preferably be carried out in a diluted medium to maximize the impact of fermentable substrates.

以粪便接种物（fecal inocula）开展体外发酵（in vitro fermentation）的策略，被视作获取粪便微生物群群落动态机制解析信息的高性价比方法。然而，所有体外发酵方法均存在局限性：因模拟体内环境（in vivo situation）时存在固有差异，会对所得实验结果引入潜在偏倚。本研究旨在系统性优化体外发酵条件，以期相较于基础培养基（basal medium）发酵体系，最大程度缩小与初始接种物的群落偏差、抑制机会性定植菌（opportunistic colonizers）的增殖，并强化添加的纤维类产物（本研究中为果胶）的作用效果。我们评估了不同起始细胞密度（starting cell density）与培养基营养浓度（medium nutrient concentration）对上述三项实验目标的影响，同时对比了新鲜粪便样本与冻存粪便样本接种的实验效果。通过结合气相色谱-质谱联用代谢物谱分析（GC-MS metabolite profiling）与基于16S rRNA基因的扩增子测序（16s rRNA gene-based amplicon sequencing），本研究证实：当起始细胞密度低于10^10 细胞/mL时，会为肠杆菌科（Enterobacteriaceae）的菌群成员提供增殖机会；当使用在-80℃冻存的粪便样本时，该效应会被进一步加剧。肠杆菌科的过度增殖会导致α多样性（alpha-diversity）降低与群落漂移（community drift）加剧，可能会混淆该条件下发酵实验的所得结果。更高的培养基营养浓度被确定为另一项有助于维持接种物群落稳定性的因素，尽管使用营养密度更低的培养基可提升纤维类产物添加对所得代谢物谱的影响效果。总体而言，我们的微生物组观测结果表明：将起始细胞密度设置为10^10 细胞/mL可限制指数生长（exponential growth）的增殖机会，抑制体外群落偏倚；而代谢组孵育实验优选在稀释培养基中进行，以最大化可发酵底物（fermentable substrates）的作用效果。