Characterization of bovine ruminal and equine cecal microbial populations enriched for enhanced nitro-toxin...

The nitrotoxins 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH) are produced by a wide variety of leguminous plants, including over 150 different species and varieties of Astragalus potentially grazed by livestock. These toxins are known to be detoxified by at least one ruminal bacterium but detoxification by bacteria from other gut habitats is not known. In the present study, mixed populations of bovine ruminal and equine cecal microbes were enriched for NPA-metabolizing bacteria via consecutive 24-72 h culture in a basal minimal rumen fluid-based medium supplemented with 4.2 mM NPA and H₂ as the energy source. Rates of NPA metabolism by the respective populations increased from 58.4 ± 4.8 and 8.6 ± 11.6 nmol NPA/mL per h during initial culture to 88.9 ± 30.6 and 50.2 ± 30.9 nmol NPA/mL per h following enrichment. Results from 3-tube most probable number tests indicated that numbers of NPA-degrading microbes increased 2.1 and 1.8 log₁₀ units during enrichment from numbers measured pre-enrichment (3.9 × 10³ and 4.3 × 10¹ cells/mL for ruminal and equine cecal populations, respectively). Hydrogen, formate, and to a lesser extent, DL-lactic acid, served as electron donors to the enriched populations and CO₂ or formate were needed to maintain high rates of NPA-metabolism. The NPA-enriched populations were able to metabolize nitrate which, being a preferred electron acceptor, was antagonistic to NPA metabolism. Supplemental NPA was inhibitory to methanogenesis. Fermentation balance estimates indicated that only 47.6% of carbon available in potential substrates was recovered in headspace CO₂, volatile fatty acids or unmetabolized NPA after 72 h incubation of NPA-enriched populations that had metabolized 98% of 8.4 mM added NPA. Overall, these results reveal low level carriage of NPA-metabolizing, CO₂ or formate-requiring bacterial populations in the equine cecum yet support the concept that Denitrobacterium detoxificans-like organisms may well be the functional agents of NPA and NPOH detoxification in the populations studied here.