Background: Human gut microbiome research has grown over the past 15 years and the scientific society has identified numerous factors that affect gut microbiota composition. Among these, diet, demographics and medication are commonly acknowledged. Yet, gut environmental factors such as transit time and pH are often overlooked despite the existing links to the human gut microbiome composition. Moreover, the current knowledge of the role of gut environmental factors on gut microbial metabolism in humans is limited.

Objective: Thus, the overall objective of this PhD thesis was to explore the possible links between gastrointestinal transit time and pH, and diet-microbiota interactions in humans.

Methods: This was achieved through 1) reviewing the current body of scientific evidence linking gastrointestinal transit time to the gut microbiota composition and metabolism (Paper I), 2) investigating the effects of whole-grain vs refined-grain diets on markers of colonic fermentation and bowel function, and how these markers associated with the faecal microbiome in a cross-over intervention trial (Paper II), and 3) by conducting a 9-day human study to explore associations between segmental transit times and pH, other gut environmental factors and diet-microbiota interactions in healthy volunteers using multi-omics approach (Paper III).

Results: Firstly, in Paper I, which is the first comprehensive review of the links between gut transit time, microbiota composition and metabolism, we found rather strong evidence that gut transit time varies between and within individuals and that these differences are linked to human gut microbiome composition in several cases. In contrast, based on the review, little evidence exists on the relation between the intra- and inter-individual variations in gut transit time and pH, and gut microbial metabolism.

Furthermore, in Paper II we found that a whole-grain diet led to an increased microbial

saccharolysis reflected by higher levels of faecal butyrate and caproate as well as to an increased stool frequency when compared to the refined-grain diet, emphasising that whole grains can modulate gut microbial metabolism and bowel habits. Moreover, faecal microbiota composition was associated with colonic transit time, faecal pH, and stool energy density indicating that differences in gut environmental factors are coupled to variations in the faecal microbiome.

Lastly in Paper III, we demonstrated that colonic transit time and pH and their respective proxy markers (stool moisture, faecal pH) significantly contributed to the intra- and inter-individual variations in the urine metabolome and/or faecal microbiome in healthy Danish adults. In particular, colonic transit time and stool moisture explained 6.2 % and 3.1 % of the inter- and intraindividual variation in the urine metabolome, respectively, while pH in the distal colon and faecal pH explained 5 % and 2.5 % of the inter- and intra-individual variations in the faecal microbiome, respectively, which has not been shown before. Importantly, we observed large day-to-day fluctuations in many microbial-derived metabolites measured in breath, urine, and faeces including microbial saccharolytic (short-chain fatty acids) and proteolytic products (branched-chain fatty acids, indoxyl-sulphate, p-cresol sulphate, phenylacetylglutamine, and other aromatic amino acidderivatives) over the 9 days of the study. While shorter transit time was associated with higher faecal concentrations of short-chain fatty acids, longer transit time was associated with increased abundance of microbial proteolytic metabolites as well as faecal dicarboxylic acids, and breath methane. Remarkably, a higher daily intake of dietary fibres was associated with lower levels of many of the proteolytic metabolites including the uremic toxin p-cresol sulphate suggesting that fibres can regulate microbial proteolysis.

Conclusion: In summary, this thesis demonstrates that transit time and pH within the

gastrointestinal tract are important determinants not only of the faecal microbiome composition but importantly of the gut microbial metabolism. Regional interactions between microbes, metabolites, and transit time and pH along the gastrointestinal tract remain to be elucidated, nonetheless, inter-individual differences in the gut environment may contribute to individualized gut microbiota compositions and microbiome-responses to foods.