The extent of nitrogen isotopic fractionation in rumen bacteria is associated with changes in rumen nitrogen metabolism

by Gonzalo Cantalapiedra-Hijar, Gonzalo Martinez-Fernandez, Evelyne Forano, Stuart E. Denman, Diego Morgavi, Christopher S. McSweeney

Nitrogen use efficiency is an important index in ruminants and can be indirectly evaluated through the N isotopic discrimination between the animal and its diet (Δ¹⁵N_animal-diet). The concentration and source of N may determine both the extent of the N isotopic discrimination in bacteria and N use efficiency. We hypothesised that the uptake and release of ammonia by rumen bacteria will affect the natural ¹⁵N enrichment of the bacterial biomass over their substrates (Δ¹⁵N_{bacteria-substrate}) and thereby further impacting Δ¹⁵N_animal-diet. To test this hypothesis, two independent in vitro experiments were conducted using two contrasting N sources (organic vs inorganic) at different levels either in pure rumen bacteria culture incubations (Experiment #1) or in mixed rumen cultures (Experiment #2). In Experiment #1, tryptone casein or ammonium chloride were tested at low (1 mM N) and high (11.5 mM N) concentrations on three rumen bacterial strains (Fibrobacter succinogenes, Eubacterium limosum and Xylanibacter ruminicola) incubated in triplicate in anaerobic batch monocultures during 48h. In Experiment #2 mixed rumen cultures were incubated during 120 h with peptone or ammonium chloride at five different levels of N (1.5, 3, 4.5, 6 and 12-mM). In experiment #1, Δ¹⁵N_{bacteria-substrate} was lowest when the ammonia-consumer bacterium Fibrobacter succinogenes was grown on ammonium chloride, and highest when the proteolytic bacterial strain Xylanibacter ruminicola was grown on tryptone. In experiment #2, Δ¹⁵N_{bacteria-substrate} was lower with inorganic (ammonium chloride) vs organic (peptone) N source. A strong negative correlation between Δ¹⁵N_{bacteria-substrate} and Rikenellaceae_RC9_gut_group, a potential fibrolytic rumen bacterium, was detected. Together, our results showed that Δ¹⁵N_{bacteria-substrate} may change according to the balance between synthesis of microbial protein from ammonia versus non-ammonia N sources and confirm the key role of rumen bacteria as modulators of Δ¹⁵N_animal-diet.