Temporal dynamics of bromoform metabolite formation and microbial responses during in vitro rumen fermentation with Asparagopsis taxiformis

This study investigated the temporal dynamics of bromoform ( CHBr 3 ) dehalogenation from Asparagopsis taxiformis and its effects on fermentation characteristics and microbial composition in an in vitro batch culture system, generating data relevant to the safety and efficacy assessment of A. taxiformis as a methane ( CH 4 ) inhibitor. Per treatment, 6 bottles were incubated with individual rumen fluid from three rumen fistulated lactating Holstein-Friesian dairy cows, with duplicate bottles per biological replicate (3 biological × 2 technical replicates). These bottles were used for continuous measurement of gas production, spot CH 4 measurements at 0, 1, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h of incubation, as well as for sampling (at 72 h) of volatile fatty acids ( VFA ), CHBr 3 metabolites, total bromine, iodine and arsenic concentration, and microbiome composition. Substrate (0.5 g DM) comprised 60% grass and 40% corn (DM basis) with or without 0.01 g DM A. taxiformis ( Asparagopsis and Control , respectively). Samples for CHBr 3 metabolites, bromine, iodine, arsenic, and microbiome analyses were taken to study changes over time (at 1, 2, 4, 8, 12, 24, 36, 48, and 60 h) from extra bottles incubated at the same starting time containing the Asparagopsis treatment. Cumulative gas production was not affected by the addition of A. taxiformis , while cumulative CH 4 production was reduced by 94% and 78% after 24 and 72 h of fermentation, respectively. Total VFA concentration and molar proportion of acetate decreased, and molar proportion of propionate increased in Asparagopsis compared to Control. After 1 h of fermentation, only 5.3% of the added CHBr 3 was detectable, and it was below the detection limit after 8 h. The concentration of dibromomethane ( CH 2 Br 2 ) increased markedly within 1 h and remained relatively stable up to 72 h of fermentation. No bromomethane was detected. The lack of stoichiometric conversion between CHBr 3 and CH 2 Br 2 suggests that additional, unidentified brominated metabolites may have formed. Total bromine, iodine, and arsenic concentrations remained relatively stable over time. Supplementation of A. taxiformis resulted in large shifts in microbial community, including a decrease in the relative abundance of archaeal and ciliate species. Longitudinal microbiome analyses confirmed a progressive microbial community restructuring over time. The shifts in bacterial community generally indicate an adaptation to elevated hydrogen concentrations or alternative fermentation pathways. Further in vivo research is needed on the absorption, metabolism, distribution, and excretion of CHBr 3 and its derivatives in ruminants, including potential metabolism in organs beyond the rumen.