Effects of lipopolysaccharide dosing on bacterial community composition and fermentation in a dual-flow continuous culture system

Abstract

The objectives of this study were to evaluate the effects of lipopolysaccharide (LPS) dosing on bacterial fermentation and bacterial community composition (BCC), to set up a subacute ruminal acidosis (SARA) nutritional model in vitro, and to determine the best sampling time for LPS dosing in a dual-flow continuous culture system. Diets were randomly assigned to 6 fermentors in a replicated 3 × 3 Latin square with three 11-d experimental periods that consisted of 7 d for diet adaptation and 4 d for sample collection. Treatments were control diet (CON), wheat and barley diet (WBD) to induce SARA, and control diet + LPS (LPSD). Fermenters were fed 72 g of dry matter/d. The forage:concentrate ratio of CON was 65:35. The WBD diet was achieved by replacing 40% of dry matter of the CON diet with 50% ground wheat and 50% ground barley. The LPS concentration in LPSD was 200,000 endotoxin units, which was similar to that observed in cows with SARA. The SARA inducing and LPS dosing started at d 8. The BCC was determined by sequencing the V4 region of the 16S rRNA gene using the Illumina MiSeq platform (Illumina Inc., San Diego, CA). The LPSD and CON maintained pH above 6 for the entire experimental period, and the WBD kept pH between 5.2 and 5.6 for 4 h/d, successfully inducing SARA. Digestibility of neutral detergent fiber and crude protein in LPSD were not different from WBD but tended to be lower than CON. Lipopolysaccharide dosing had no effect on pool of VFA concentrations and profiles but decreased bacterial N; the pattern changes of VFA and LPS in LPSD started to increase and be similar to WBD 6 h after LPS dosing. Pool of LPS concentration was around 11-fold higher in WBD and 4-fold higher in LPSD than CON. In the solid fraction, the BCC of LPSD was different from WBD and tended to be different from CON. In the liquid fraction, the BCC was different among treatments. The LPS dosing increased the relative abundance of Succinimonas, Anaeroplasma, Succinivibrio, Succiniclasticum, and Ruminobacter, which are main gram-negative bacteria related to starch digestion. Our results suggest that LPS dosing does not affect pH alone. However, LPS could drive the development of SARA by affecting bacteria and bacterial fermentation. For future studies, samples are suggested to be taken 6 h after LPS dosing in a dual-flow continuous culture system.

Keywords: 16S rRNA; gram-negative bacteria; in vitro; starch digesting bacteria.