Special Features of the Model
Several features in TIM-2 are unique over other models. First of all, the peristaltic movements of the flexible membrane give a better mixing and movement of components through the entire model than would be accomplished by stirring (in a fermentor) or shaking (on a rocking-platform or otherwise). In TIM-2 there is no phase-separation of solids and liquids, which does occur in other systems. Viscous 'meals' or insoluble components can be used without problems in the system. Secondly, as eluded to above, the dialysis system is not only unique, but also required to maintain a highly active microbiota with a similar density as that found in the human large intestine. In batch incubations or less sophisticated models the microbiota is usually inoculated at a much lower density (100-fold or more) and is allowed to slowly grow to physiological densities. However, the metabolites produced by the microbiota start to inhibit further fermentation at these high densities in these systems. Since these metabolites are also taken up by the epithelial cells of the colon (colonocytes) in vivo, TIM-2 mimics much better the physiological situation in the large intestine. In fact, since all metabolites are collected and can be measured, a(n almost) complete mass-balance can be made, which is not possible in vivo, not even in animals, although in scientific studies usually animal are sacrificed to sample as much as possible, including blood samples. However, even in animals a massbalance is not possible, as e.g., butyrate—one of the SCFA—is used by the colonocytes and does not reach the blood circulation. Therefore, TIM-2 allows studying (molecular) mechanisms. This is certainly the case if labeled substrates are used, as will be discussed in other sections of this chapter.
The model is incubated by using a fecal donation from volunteers. This fecal donation can be used in two ways: (a) a fecal donation from individual one can be introduced into one of the TIM-2 systems, the donation from individual 2 in a second unit, and so on. The composition and activity of the microbiota of the individuals can then be compared on say one and the same substrate. This has for instance been done for lactulose, with ten donors (Venema et al. 2003); or (b) the fecal donations of several donors are mixed to create a standardized microbiota that can subsequently be used for ~100 experiments. This allows comparison of multiple substrates or conditions starting with the same microbiota composition. This pool of fecal donations to a certain extent mimics a (small) population. It has been argued by many that mixing different fecal samples may disturb the microbial balance within a single fecal sample, but as far as we know, no direct comparison has been done to show this. Recently, we have set out to show that by mixing a number of different fecal donations, the functionality of the microbiota is not influenced (Aguirre et al. 2014). That is, the individual microbiotas showed the same functionality as the pool and produced very similar amounts and ratios of microbial metabolites (amongst others SCFA), despite being different in microbial composition. This underscores our hypothesis that there is an enormous functional overlap between microbes in the large intestine. This is not entirely surprising, as there are only a limited number of biochemical routes (let's say 5) from e.g., glucose to acetate. Since the microbiota is composed of approximately 200 species or more, there has to be enormous functional overlap between these microorganisms.
Upon introduction of the microbiota in the system, an adaptation period of ~16 h is applied, to allow the microbes to adapt to their new environment and the feed components. After that the experimental period starts, which normally takes 72 h (see Sect. 26.2). Fecal donations can be obtained from healthy volunteers of different age-classes (baby, adults, elderly), people with a disease or disorder [e.g., inflammatory bowel disease; (van Nuenen et al. 2004)], or from lean vs. obese individuals.