208 Effects of bacteriophages, their bacterial hosts, and combined phage-bacteria inoculations on in vitro ruminal fermentation and methane production

Abstract Bacteriophages are highly species-specific viruses that infect bacteria, offering a novel approach to microbiome manipulation by selectively targeting bacterial populations, while minimizing disruption to the overall microbial community. Phage-based modulation of the ruminal microbiota has the potential to improve feed efficiency and reduce methane production in the rumen. The objective of this study was to evaluate the effects of bacteriophages, their bacterial hosts, and combined phage-bacteria inoculations on the total biogas and methane production, and fermentation parameters from ruminal fluid culture in vitro. For this, fresh ruminal fluid was collected from four Jersey cows and pooled. Then, 12.5 mL of the pooled ruminal fluid was combined with 1.6 g of high-forage feed, and 50 mL of McDougal’s artificial saliva buffer (1:4 ratio) in 120 ml serum bottles. This combination was inoculated with one of the following treatments: phages, their respective bacterial host strains (Solibacillus spp., Rhodococcus spp., Bacillus licheniformis, and Escherichia coli), or a combination of all four phages and their bacterial hosts. Both phages and bacterial hosts were cultured and isolated from the ruminal fluid of beef cattle. The ruminal fluid culture samples were then incubated for 24 or 48 hours at 39°C. After incubation, total gas produced in the head-space of each bottle was extracted, pH of the culture measured, and methane gas concentration in the headspace gas, and volatile fatty acid (VFA) concentrations in 24h or 48 h culture media were measured using gas chromatography. Statistical analysis was conducted using Kruskal-Wallis tests with Dunnett’s correction in RStudio v4.3.3, with a significant threshold of P < 0.05. Overall, the inoculation of phages and bacterial strains resulted in minor to no changes in the total gas production, methane production, and pH of the culture either after 24 or 48 hours of incubation. However, pH was reduced (6.81 to 6.70) in the ruminal fluid culture treated with Solibacillus spp. and E. coli-inhibiting phages at 24 hours of incubation, and in the bacteria/phage cocktail treatment at 48 hours of incubation (P < 0.05). The VFA concentrations varied among different inoculation treatments, isobutyrate and valerate were higher (P < 0.05) across all treatments, except with Solibacillus spp. and E. coli, additionally, all treatments increased the concentration of isovalerate when compared to control ruminal fluid culture, without any inoculation, at 24 hours. However, this effect was not sustained at 48 hours, except for ruminal fluid culture inoculated with the bacteria/phage cocktail, which showed a distinct (P < 0.05) VFA profile from the control RF even after 48 hours of incubation. These preliminary findings indicate that phage and bacterial inoculations could influence ruminal fermentation parameters, specifically affecting pH and VFA profiles. Future studies are needed to optimize ruminal microbiota modulation using phage therapy.