Treatment of dual-flow continuous culture fermenters with an organic essential oil product minimally influenced prokaryotic microbiome

Previous research reported an essential oil (EO) product decreasing methane (CH₄) production by dual-flow continuous culture (DFCC); this product could assist organic dairy producers in decreasing emissions. Our objective was to assess the effect of this EO product on the microbial populations within DFCC. Here, we hypothesized that the EO either decreased protozoal population or induced shifts in the bacterial relative abundance to decrease CH₄ production. Metagenomic DNA was extracted from previous effluent samples taken from a DFCC system (n=2) across four experimental periods, after which samples were sequenced the 16S rRNA gene and microbial taxonomy was assigned using the SILVA v138 database. The treatments included a control (CON) diet (60:40 concentrate:orchardgrass pellet mix, 17.1% crude protein, 33.0% neutral detergent fiber, 20.1% acid detergent fiber, and 27.1% starch) fed twice daily for a total of 80 g/d dry matter, or the same CON diet with the addition of EO at 3 mg/d. Protozoa were also quantified in both fermenter contents and unpooled daily effluent samples. The statistical model included fixed effects of treatment and fermenter, and random effect of period, using either MaAsLin2 or the adonis2 function in the vegan package of R for microbial features, or SAS mixed model for protozoal counts. The results were deemed significant at <italic>Q</italic>&lt;0.05 and <italic>P</italic>&lt;0.05 for the MaAsLin2 and adonis2/SAS analyses, respectively. For the protozoal populations, the treatments had no significant effect (<italic>P</italic>&gt;0.10) on the total counts, differentiated groups, or cell outflow. The addition of EO increased the relative abundance of <italic>Methanobrevibacter</italic> and decreased that of uncultured Methanomethylophilaceae (<italic>Q</italic>&lt;0.05). In contrast, EO addition had no significant effect on archaeal α- or β-diversity (<italic>P</italic>&gt;0.05). Despite not having a significant effect on the β-diversity of archaeal and bacterial communities, EO decreased (<italic>P</italic>&lt;0.05) α-diversity indices in prokaryotic communities. Moreover, EO decreased (<italic>Q</italic>&lt;0.01) the relative abundance of Clostridia UCG-014, Rikenellaceae RC9 gut group, and Christenellaceae R7 group, and increased (<italic>Q</italic>&lt;0.01) others including <italic>Treponema</italic>, Succinivibrionaceae UCG-002, and <italic>Ruminococcus</italic>. Offsetting shifts in the relative abundance of fiber-degrading bacteria and detailed methanogen communities deserves further investigation including predicted metabolic pathways impacted by population shifts induced by this EO combination.