# Theoretical Methane Emission Estimation from Volatile Fatty Acids in Bovine Rumen Fluid
## 3 Results and Discussion
### Confirmation Using Standard Mixtures Prepared with Purified VFAs  
It is widely reported that the main constituents of acidogenic production from rumen fluid are acetic acid, propionic acid, butyric acid, and valeric acid [31]. Considering CH\({}_{4}\) production via methanogens, the importance of precise analysis of volatile fatty acids is becoming more important. In order to assess the precision of both methods, standard VFA mixture solutions containing known concentrations of each component were prepared at various concentrations, and confirmatory analyses were performed using the conventional method and CER method. For both methods, the measurement deviation for each component was evaluated using a ratio, defined as the measured concentration over the known concentrations of the VFAs in the standard solution. The ratios are presented in Figure 3. If the ratio is closer to one (dot line), the deviation is lower; that is, the measurement is more accurate.  
Generally, the CER method resulted in lower measured deviations from known VFA concentrations. The results when measuring standard mixtures of acetic acid and propionic acid were relatively reliable for both methods, with measured-to-known concentration ratios of 1.08 and 1.03 for the conventional and CER methods, respectively. However, the deviation in results when using the conventional method was greater when measuring the less abundant VFAs; that is, the differences in measured concentrations between the two methods were especially apparent for butyric acid and other C3+ VFAs. For butyric acid, there was a factor-of-5 difference in sensitivity between the two methods, with the CER method detecting 91 percent of the known sample concentration compared with 18 percent for the conventional method. The discrepancy between the two methods and the substantially greater accuracy of the CER method was also apparent for valeric acid. However, the overall concentrations of the valeric and isovaleric acids in the rumen were the lowest observed among the measured VFAs for both methods (Figures 1 and 2). Consequently, the CER method displayed the largest improvement over the conventional method in measuring the absolute amount of butyric acid in the rumen. It has also been reported in previous research that the ratios of acetic acid and propionic acid appear to be overestimated [25; 26; 27]. However, their measured deviations from known concentrations in the CER method were consistent with the tendency of this overestimation (i.e., there was no substantial change in measurement accuracy for the acetic acid or propionic acid for the CER method vs. the conventional method). Using the evaluation of the frequency distribution, the comparison between the methods has been proven. The relative frequency of the ratios is presented in Figure 4.  
The standard deviations of the distribution were 0.456 and 0.161 for the conventional method and the CER method, respectively. It was observed that the distribution for the conventional method was specifically high at both around 0.2 and 1.9. This shape is consistent with the results of Figure 3. The evaluation of the frequency distribution obviously suggests that the analysis described above is convincing.  
To our knowledge, the results of this study provide the first quantitative proof to show the improved accuracy in the measurements of C3+ VFAs when using the CER method compared with the conventional method. Therefore, a new approach, the CER method, can be recommended when comprehensively analyzing not only the most important substances (acetic acid and propionic acid) but also others, including butyric acid. The more accurate analyses of these substances may provide a useful platform to predict the amount of methane generation from VFAs in rumen.