The stoichiometrical relationship between gas volumes and short chain fatty acid (SCFA) production has been exemplified for an in vitro gas test based on the bicarbonate buffer. It is outlined that, even though variation in the molar proportion of acetate, propionate and butyrate will influence gas volumes, quite similar amounts of total carbon, hydrogen and oxygen are required for widely different SCFA patterns (0.748: 0.194: 0.058 or 0.459: 0.462: 0.079 for C-2: C-3: C-4), namely, 2.20-2.34 mg to produce 1 ml of gas. However, the variation in microbial biomass production per mole adenosine triphosphate (ATI)) presents a serious limitation for in vitro gas tests, since these tests reflect SCFA production only. As a consequence in vitro gas rests need to be complemented by a quantification of substrate concomitantly truly degraded to avoid selection of a substrate with proportionally higher SCFA production and lower microbial biomass yield. This intrinsic problem of in vitro gas tests was experimentally demonstrated for 61 roughages, where a gravimetric determination of microbial biomass yield showed a highly significant (r=-0.78, p < 0.0001) negative relationship between microbial biomass and gas volumes in vitro when both were related to 100 mg of substrates truly degraded. The relationship was further examined for 35 roughages, being representative of the 61 samples, using incubations with stable isotopic nitrogen (N-15). In these studies, N-15 incorporation and gas production were significantly (r=-0.78, p < 0.001) inversely related. An in vitro method is proposed which combines gas volume and substrate degradability measurements to estimate microbial yield and these estimations agreed well (r=0.82, p < 0.0001) with N-15 measurements.
