3.2.5. Gas Phase Composition

The composition of the gas phase in the samples is shown in Table 4. In the samples containing sewage sludge and/or rumen fluid pretreated at 38.6 ◦C, significant concentrations of CH4 were observed in the gas phase. The addition of rumen fluid increases cell wall degradation and thus allows microorganisms easier access to nutrients; therefore, more CH4 is formed [56].

Besides CH4, other gases such as CO2 and H2S were also analyzed and detected in the gas phase of the samples. The mixture of grass and rumen fluid (G + R) contains the highest amount of CO2. Rumen fluid degrades the lignocellulosic fibers in the grass and consumes oxygen, which leads to CO2 increase [63]. Accordingly, CO2 production could be used as an indicator of the degradation efficiency of lignocellulosic biomass during rumen fermentation [30].

The concentrations of H2S were more significant in the samples containing rumen fluid (G + R and G + S + R). Slightly higher concentrations of this gas were found in mixtures containing grass. The production of H2S comes mostly from microbial degradation of organic matter [64].


**Table 4.** Gas phase composition.

On the other hand, when pretreatment was performed at 80 ◦C, production of the gases under analysis was negligible. The reason for such results is that bacteria in materials are highly active only in mesophilic ranges (between 25 and 42 ◦C) [65] and thermophilic ranges (between 50 and 65 ◦C) [66]. At temperatures below 15 ◦C and above 70 ◦C, methanogenic bacteria are limited in activity [67].
