Abstract

   Studies on rodents indicate the daily oscillations of the gut
   microbiota have biological implications for host. However, the
   responses of fluctuating gut microbes to the dynamic nutrient
   substrates are not fully clear. In the study, we found that the feed
   intake, nutrient substrates, microbiota and metabolites in the colon
   underwent asynchronous oscillation within a day. Short-chain fatty
   acids (SCFAs) including acetate, propionate, butyrate and valerate
   peaked during T24 ~ T27 (Timepoint 24, 12:00 pm, T27, 03:00 am) whereas
   branched SCFAs isobutyrate and isovalerate peaked during T09 ~ T12.
   Further extended local similarity analysis (eLSA) revealed that the
   fluctuation of feed intake dynamically correlated with the colonic
   carbon substrates which further influenced the oscillation of sugar
   metabolites and acetate, propionate, butyrate and valerate with a
   certain time shift. The relative abundance of primary degrader
   Ruminococcaceae taxa was highly related to the dynamics of the carbon
   substrates whereas the fluctuations of secondary degraders
   Lactobacillaceae and Streptococcaceae taxa were highly correlated with
   the sugar metabolites. Meanwhile, colonic nitrogen substrates were
   correlated with branched amino acids and the branched SCFAs.
   Furthermore, we validated the evolution of gut microbes under different
   carbohydrate and protein combinations by using an in vitro fermentation
   experiment. The study pictured the dynamics of the micro-ecological
   environment within a day which highlights the implications of the
   temporal dimension in studies related to the gut microbiota. Feed
   intake, more precisely substrate intake, is highly correlated with
   microbial evolution, which makes it possible to develop chronotherapies
   targeting the gut microbiota through nutrition intervention.

   Subject terms: Microbial ecology, Microbiome

Introduction

   The mammalian digestive tract harbors trillions of microorganisms whose
   whole genome is ten times larger than its host^[32]1. Recently,
   increasing evidence shows that the microbial consortia undergo
   spatiotemporal evolution to adapt to the environmental variations in
   the gut ecosystem^[33]2–[34]6. Along the temporal dimension, the gut
   microbiota undergoes seasonal and daily variations in composition and
   function^[35]6,[36]7. It is worth noting that the oscillation of the
   gut microbiota has great importance on the host metabolism, normal
   physiological rhythm and gut homeostasis^[37]8,[38]9. However, most of
   these studies were conducted on rodents. As pigs have significant
   differences in habits, eating patterns, body size, behavior, life span
   and gut microbiota compared with rodents. Besides, considering the
   comparability between pigs and humans in terms of genetic information,
   anatomic characteristics, eating habits and physiology, observations
   from the pigs could have a great reference value for human research on
   gut microbiota^[39]10.

   Intrinsic and extrinsic factors including host genotype, physiology,
   species, age, gender, environmental factors, feeding pattern and the
   nutrient composition of feed affect the gut microbiota and its
   fluctuation^[40]11,[41]12. Among these factors, feed, more precisely
   the accessibility and composition of the nutrients, has a significant
   impact on the composition and function dynamics of the gut
   microbiota^[42]3,[43]6,[44]7,[45]13,[46]14. Under the ad libitum
   circumstances, the circadian clock, which synchronizes the behaviors
   and physiology including food taking and wake-sleep circle, is the most
   critical factor influencing the gut microbial dynamics^[47]15. As a
   result of rhythmic feed-taking behavior, the metabolites in the serum
   undergo robustly oscillating and are relevant to the gut microbiota
   fluctuation^[48]9,[49]14,[50]16–[51]18. The direct effect of dietary
   interventions functions probably through altering the substrates and
   metabolome profile in the intestine^[52]19–[53]21. However, so far, the
   fluctuation of substrates and metabolites in the colon of growing pigs
   is unclear. How nutrients shape the gut microbial community and how
   they drive the metabolic pathways remains unknown.

   Of note, light is the primary zeitgeber for the mammalian circadian
   clock and clear time shifts exist between the zeitgeber and certain
   physiological phenomena^[54]22,[55]23. Likely, the effect of nutrient
   substrate on intestinal microbes may take a specific time to
   manifest^[56]24,[57]25. Therefore, it is reasonable to consider the
   time shift between the substrates and the microbes in their crosstalk.
   It is of importance for a better understanding of the dynamic
   microbe-metabolite interactions and providing references for developing