*2.2. Experimental Setup*

Two pretreatment methods were tested on selected waste: biological pretreatment with the addition of an enzyme mixture (i.e., cattle rumen fluid) and lower and higher temperature thermal pretreatment (at 38.6 and 80 ◦C). For biological pretreatment, the temperature was set at a ruminal temperature of about 38.6 ◦C [25]. The temperature of 38.6 ◦C was chosen, since it is within the mesophilic temperature range that is optimal for microorganisms' growth and metabolism, and it is an optimal temperature for microorganisms present in the rumen fluid.

For lower temperature thermal pretreatment (mesophilic temperature range, 38.6 ◦C) and biological pretreatment with rumen fluid, batch assays were maintained by using a Thermo Scientific™ SC100 immersion circulator in a heated bath filled with deionized water. Samples placed in 1 L glass containers were exposed to pretreatment conditions for 8 days. Assays in containers were hand-mixed two to three times during pretreatments. If necessary, to reduce the pressure in containers, the gases were released to the atmosphere and containers were closed shortly afterwards. Figure 1 shows schematic representation of the experimental set-up for lower temperature thermal and biological pretreatment.

Higher temperature thermal pretreatment at 80 ◦C was performed in a laboratory drying chamber (ED 115, Binder), where the pretreatment time was 5 days.

All samples were prepared in triplicate, containing 6 wt.% of solids based on average dry matter (DM) content. The samples were prepared on a 500 g wet basis (30 g dry basis) and are shown in Table 1. Reaction mixture G contains only grass substrate, while reaction mixture S contains only sludge (both were diluted by distilled water to 6 wt.%). Other samples represent mixtures of the chosen substrates. For example, sample G + S was a mixture of grass and sewage sludge in a ratio of 1:1. For biological pretreatment, in addition to 500 g of material (G, S and G+S) 50 mL of rumen fluid was added. The addition of rumen fluid in reaction mixtures is denoted as R.
