CURRENT MICROBIOLOGY Vot. 20 (1990), pp. 251-254 Current Microbiology © Springer-Verlag New York Inc. 1990 Formate and Glucose Stimulation of Methane and Hydrogen Production in Rumen Liquor Jayne E. Ellis, 1 Alan G. Williams, 2 and David Lloyd I 1Microbiology Group (PABIO), University of Wales College of Cardiff, Cardiff, Wales, and 2Hannah Research Institute, Ayr, Scotland, UK Abstract. Membrane-inlet mass spectrometry was used to investigate the effects of increasing the concentration of the rumen metabolites, formate and glucose, upon CH4 and H2 production during fermentation by unfractionated rumen liquor. Additions of formate up to 3.6 mM stimu- lated CH4 and then excess H2 production. Each addition caused a large accumulation of H2 (>40 /xM), which returned to in situ concentrations after periods of more than 1 h. Glucose additions up to 2.0 mM gave linear increases in CH4 and H2 production. The conversion of substrate carbon into CH4 was found to decrease from 34% to 9% for formate, as concentrations were increased (1.6-3.6 mM); approximately 13.5% of the glucose carbon was converted to CH4. The rumen ecosystem represents the most econom- ically important of all natural fermentation systems [13]. Although the overall balances of inputs and outputs have been extensively studied [4, 8], the flux of nutrients through the diverse microbial niches and the interrelationships between the popu- lations is still poorly understood. The microbial ac- tivity of the rumen is in a state of dynamic equilib- rium in which intermediates are rapidly produced and utilized by the microorganisms present [9]. Thus, a short-lived nutrient pool exists in animals fed once or twice daily. Formate and H2 have been demonstrated to be important intermediates of ruminal fermentation [9]. Microorganisms producing formate generally also yield H2; these are principally the cellulolytic bacteria [7], the rumen fungi [2, t4], and the hydro- genosome-containing ciliate protozoa [19-21]. For- mate and H2 are removed from the intercellular fluid of the rumen by the methanogenic population. The production of H2 in the rumen is inextricably linked to methanogenesis [ 18]. The concentrations of these two dissolved gases have been shown to fluctuate with feeding, as revealed by membrane-inlet mass spectrometric monitoring of in situ fermentation gases from fistulated sheep [6]. The technique has also been shown to have an application in the study of laboratory fermentation systems [1, I I, 12] and is particularly useful for monitoring ruminal fermenta- tion patterns [5, 6]. It has been estimated that the production of CH4 in situ represents a loss of 6%-10% of the die- tary intake of available carbon from the host [4]. In this paper we report on the kinetics and stoichiome- tries of H2 and CH4 formation from formate or glu- cose. Our direct measurements on dissolved gases indicate figures for carbon loss to CH4 of about 13% for glucose, but as much as 34% for formate. Materials and Methods Preparation of the rumen sample. Rumen contents were obtained from fistulated cattle fed a ration of ryegrass hay (4.1 kg) and concentrates (2.8 kg) daily in two equal portions. Samples were withdrawn before the morning ration was offered and trans- ported to the laboratory in a sealed, insulated container. Prior to incubation, samples were strained through a double layer of sur- gical gauze under Nz and diluted twofold with N2-gased Simplex- type buffer (S) at 39°C [3]. All chemicals used were BDH, Ana- laR Grade (Poole, England). Mass spectrometry, A quadrupole mass spectrometer fitted with a membrane-covered inlet [10] was used to measure H2 and CH4 concentrations simultaneously and continuously. The instrument used was a type SX200 DPP16 digital peak programmer (VG Gas Analysis, Aston Way, Middlewich, Cheshire, U.K.) fitted with a turbomolecular pump (Pfieffer, Postfach 1280, D-6334, FRG) and rotary backing pump. The incubation vessel was of stainless steel with a 25-mi working volume [15], stirred at 450 rpm and Address reprint requests to: Dr. Jayne E. Ellis, Microbiology Group (PABIO), University of Wales College of Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK.