Fecal virome addition promotes biofilm biomass and produces distinct bacterial responses in biofilm and planktonic communities in an in vitro colon model

The gut microbiota is spatially organized, yet microorganisms at the intestinal mucus surface remain understudied in virome–microbiota research. We investigated how fecal virome addition affects biofilm biomass and bacterial composition in surface-associated and planktonic communities using a biofilm-adapted in vitro colon model, CoMiniGut. Fecal samples were collected from three healthy donors at two time points approximately one year apart. Communities from both collection time points were fermented for 48 h in reactors containing mucin-coated glass beads. Each community was exposed separately to virome fractions from all three donors, testing autologous and heterologous combinations alongside matched microbiome-only controls. Two parallel CoMiniGut systems provided reactor replicates for each donor–treatment combination. Biofilm biomass was quantified by crystal violet staining, while biofilm-associated and planktonic communities were characterized by 16S rRNA gene sequencing. Across 36 reactor-level observations, biomass was classified as increased in 18, neutral in 11, and decreased in 7. Each observation represented the mean of 5–6 beads normalized to its matched microbiome-only control. Among non-neutral observations, increases were more frequent than decreases (18 versus 7; two-sided exact binomial test, P = 0.043), whereas the magnitude of change did not differ between Increase and Decrease observations (P = 0.220). Virome addition explained a small but significant proportion of bacterial compositional variation (R² = 0.024, P = 0.029) without substantially altering alpha diversity. No interaction between virome treatment and growth mode was detected at the community level, while separate differential-abundance analyses identified different sets of virome-associated taxa in biofilm-associated and planktonic samples. Thus, fecal virome addition was associated with a directional bias towards increased biofilm biomass and distinct growth-mode-associated taxonomic patterns without extensive community restructuring.