Attempted induction of reductive acetogenesis into the rumen fermentation in vitro.

Rumen and caecal contents, obtained from slaughterhouse cattle and rumen contents obtained from a fistulated wether were incubated in vitro with ground hay in the presence and absence of, respectively, casein hydrolysate and mucin. Differences in stoichiometry of rumen and caecal fermentations, indicative of reductive acetogenesis in the caecum, were confirmed, except for incubations with free amino acids. Net fermentation end product production was determined after correction for amounts formed in incubations without the substrate. These determined amounts of hay fermentation end products were compared with the amounts calculated from incubations of hay with added casein hydrolysate or mucin, corrected for amounts formed from the latter added substrates incubated alone. With casein hydrolysate, no differences between the determined and calculated amounts were observed, excluding the occurrence of reductive acetogenesis from hay in the presence of free amino acids. With mucin, the calculated amounts indicated an inhibition of methanogenesis, accompanied by increased amounts of proprionate, butyrate and valerate production. This finding was probably related to the greater availability of easily fermented carbohydrates in the presence of mucins. The absence of an increased acetate production in the incubations with added head space hydrogen gas, also indicate the absence of reductive acetogenesis from hay in the presence of mucin. Stoichiometric considerations also indicate that neither free amino acids, nor mucin, induce reductive acetogenesis in short-term in vitro incubations of rumen contents with hay.

Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora.

Washed bacterial suspensions obtained from the pig hindgut were incubated under 13CO2 in a buffer containing NaH13CO3 and carbohydrates. Incorporation of 13C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H2 added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H2 markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H2 significantly increased the incorporation of 13CO2 into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H2 partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO2 and H2 (represented mostly by double labelling). A highly significant interaction between BES and H2 concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.

NMR study of 13CO2 incorporation into short-chain fatty acids by pig large-intestinal flora.

The nuclear magnetic resonance technique was used to study carbon dioxide reduction by the pig large-intestinal flora. Washed bacterial cell suspensions were incubated for 6 and 15 h under 13CO2 and H2 as the gas phase and with a buffer containing NaH13CO3 and cellobiose and amino acids (casein hydrolysate) as substrates. Methane was produced in all incubation media. Significant amounts of single- as well as multiple-labelled acetate and butyrate were formed, demonstrating synthesis of acetate from H2 + CO2. Propionate was labelled mainly on the carboxyl group, which was attributed to an enzymatic exchange of the carboxyl group of propionate with 13CO2. These results indicate that the reduction of CO2 to acetate may be an important pathway for microbial production of acetate in the pig large intestine even in the presence of methanogenesis.