Optimal mineral composition of artificial saliva for fermentation and methanogenesis in continuous culture of rumen microorganisms

In dual outflow continuous fermenters on a 75 : 25 hay : barley diet, fermentation and gas production by mixed rumen microbes were tested in relation to the concentrations of HPO42−, HCO3− and Cl− and the Na+/K+ ratio in artificial saliva, by applying a 17-run Franquart design, and by fitting second-order polynomial models. The HPO42−, HCO3−, Cl− concentrations and Na+/K+ ratio ranged from 0.1 to 4 g l−1, from 0.5 to 7 g l−1, from 0.1 to 0.5 g l−1 and from 0.5 to 15 g g−1, respectively. The major factor was the concentration of HCO3−, followed by HPO42− and Na+/K+, while the concentration of Cl− had negligible effects. The volatile fatty acid (VFA) production rate was mostly influenced by changes in pH, from 6.7 to 7.2, mediated by HCO3− and HPO42− concentrations. The analysis of the fermentation pattern confirmed the predominant action of HCO3−. Butyrate (C4) and branched chain VFA production rates, as well as C4 molar proportion in the fermentation broth were also lowered by high or low values of the Na+/K+ ratio. The action of minerals on gas production and protozoa population density was complex and involved to an equal degree several experimental factors with a non-linear behaviour. Protozoa numbers, which varied from 14 to 38 μl−1, were favoured by high Na+/K+ ratios. Using response surface analysis, the composition of a mineral base was optimised to simultaneously promote protozoa numbers and methanogenesis in vitro, at 39 μl−1 and 1.20 mmol h−1, respectively. The resulting artificial saliva contained 1.65 g l−1 HPO42−, 4.19 g l−1 HCO3−, and 0.22 g l−1 Cl−, and had a Na+/K+ ratio of 14.2 g g−1.