Research Article: The application of rumen simulation technique (RUSITEC) for studying dynamics of the bacterial community and metabolome in rumen fluid and the effects of a challenge with Clostridium perfringens

Date Published: February 7, 2018

Publisher: Public Library of Science

Author(s): Stefanie U. Wetzels, Melanie Eger, Marion Burmester, Lothar Kreienbrock, Amir Abdulmawjood, Beate Pinior, Martin Wagner, Gerhard Breves, Evelyne Mann, Michel R. Popoff.


The rumen simulation technique (RUSITEC) is a well-established semicontinuous in vitro model for investigating ruminal fermentation; however, information on the stability of the ruminal bacterial microbiota and metabolome in the RUSITEC system is rarely available. The availability of high resolution methods, such as high-throughput sequencing and metabolomics improve our knowledge about the rumen microbial ecosystem and its fermentation processes. Thus, we used Illumina MiSeq 16S rRNA amplicon sequencing and a combination of direct injection mass spectrometry with a reverse-phase LC-MS/MS to evaluate the dynamics of the bacterial community and the concentration of several metabolites in a RUSITEC experiment as a function of time and in response to a challenge with a pathogenic Clostridium perfringens (C. perfringens) strain. After four days of equilibration, samples were collected on days 5, 6, 7, 10, 12 and 15 of the steady-state and experimental period. From a total of six fermenters, three non-infected fermenters were used for investigating time-dependent alterations; three fermenters were incubated with C. perfringens and compared with the non-infected vessels at days 10, 12 and 15. Along the time-line, there was no statistically significant change of the overall bacterial community, however, some phylotypes were enriched at certain time points. A decrease in Fibrobacter and Elusimicrobia over time was followed by an increase in Firmicutes and Actinobacteria. In contrast, classical fermentation measurements such as pH, redox potential, NH3-N, short chain fatty acids and the concentrations of metabolites determined by metabolomics (biogenic amines, hexoses and amino acids) remained stable throughout the experiment. In response to C. perfringens addition the concentrations of several amino acids increased. Although the overall bacterial community was not altered here either, some minor changes such as an enrichment of Synergistetes and Bacteroidetes were detectable over time. In conclusion, both, the bacterial community composition and the metabolome in the RUSITEC system were relatively stable during the experiment.

Partial Text

Detailed knowledge of the ruminal dynamic, anaerobic ecosystem and rumen fermentation processes are the prerequisite to understand basic rumen physiology and nutrition as well as gastrointestinal diseases such as rumen acidosis. Studying factors, which contribute to a modulation in vivo underlie timely, environmental fluctuations and host-dependent physiology [1]. The rumen simulation technique (RUSITEC) is a well-established in vitro method to simulate and to investigate rumen microbial processes, avoiding animal’s variability in a standardized environment [2]. This method is widely used for studying effects of different diets or feed additives on microbial fermentation patterns, protein synthesis and microbial growth [3, 4]. Although being a highly standardized method (e.g. in temperature, pH and buffer flow) the system is known to differ from in vivo conditions regarding absorptive processes, differences in the ratio between liquid and solid materials, lower short chain fatty acid (SCFA) concentrations and protozoal shifts compared to the donor animal [5–7]. It was proposed that the bacterial diversity decreases in the RUSITEC and that the disappearance of ciliates can be traced back to a loss of balance in the bacterial populations [7]. Using real-time PCR, Lengowski and colleagues [8] demonstrated that most changes during the adaptation of the ruminal microbial community to the RUSITEC system occur within in the first 48 h after inoculation, however, may continue for some species. The availability of high-throughput sequencing methods offers the opportunity to investigate alterations in the microbial community and microbial biochemical processes in detail. Belanche and colleagues were the first, who used next generation sequencing methods to evaluate the impact of dietary supplementation in the RUSITEC system [9, 10]. Recently, Duarte and colleagues [11] reported an effect of the sampling day on the liquid-associated microbiota in the RUSITEC using Illumina sequencing.

Since 1977, the RUSITEC system has been used and well established to evaluate certain rumen conditions in vitro [2]. Previous studies using single strand conformation polymorphism and qPCR indicated, that although the protozoa population decreases strongly in this in vitro system, the bacterial and the archaeal population are able to adapt to the RUSITEC system [8, 35]. Therefore, several fingerprint methods and qPCR have been intensively used for comparing bacterial communities within the RUSITEC system [28, 36, 37]. Belanche et al. [9, 10] were the first, who used next generation sequencing methods to evaluate the impact of dietary supplementation in the RUSITEC system and Duarte and colleagues [11] detected a shift in the microbiome between day 5 and 10 of a RUSITEC trial by using next generation sequencing. Our study investigated the stability of the bacterial community structure as well as its metabolome in the RUSITEC system along the time line in shorter intervals then in the latter studies. As a further challenge C. perfringens was added as a model organism to study the effect of a common pathogen on the stability of the bacterial community and metabolome in the RUSITEC system.