A fully automated incubation system for the measurement of gas production and gas composition

• We developed a truly automatic in vitro system for gas methane and hydrogen production.
• The system is a new screening tool for methane mitigation strategies.
• We demonstrated that rumen fluid form sheep produced less methane than that of cattle.

A fully automated in vitro incubation system consisting of 32 incubation bottles (125 ml) each fitted with a pressure sensor and a solenoid valve was developed to monitor total gas production and hydrogen (H2) and methane (CH4) composition. Gas volume is calculated from the pressure readings collected by a dedicated computer and when the threshold pressure of 9 kPa is reached the fermentation gases are released into a gas chromatograph via a sampling loop. Substrates were incubated at 10 mg/ml in 60 ml of a medium with a buffer to rumen fluid ratio of 4:1. Typically, 20–25 measurements of gas volume and composition are obtained from each bottle over a 48 h incubation period. More than 60% of the measurements are obtained during the first 12 h of fermentation when rumen fermentation is most active. This allows the same model to be fitted to the data to describe the gas and CH4 production data and directly compare their kinetics for various substrates. The overall repeatability of the system has been tested over 2 years using a ‘standard ryegrass hay’ sample that was incubated as an internal control in each incubation run. Overall, repeatability of gas and CH4 production was comparable to other incubation systems with a CV of 7.2% and 12.5% for total gas and CH4 production. However, within the dataset an annual pattern was detected in the parameters of the standard hay. Differences in CH4 emissions from various substrates were largely explained by differences in short chain fatty acid production.In an initial series of test the system was used to evaluate the effect of incubation buffers or rumen fluid donor species on gas emissions and fermentation end products. While no differences were found for the different buffers used, our results indicate sheep rumen fluid leads to a slightly lower production of CH4 when compared to rumen fluid from cattle.The system presented here is the first rumen fluid batch culture system that automatically measures total gas, CH4 and H2 production and is currently used as a screening tool for substrates or additives that have a potential to lower CH4 emissions from ruminants.