*2.3. Chemical Analyses*

Before and after pretreatment studies, various parameters were measured in the liquid phase of the samples. Chemical oxygen demand (COD), total organic carbon (TOC), and the amounts of nitrogen, phosphorus and potassium (NPK) content were analyzed by cuvette tests. The following NANOCOLOR®(Macherey-Nagel) cuvette tests were used: CSB 160 and 1500,

TOC 300, TNb220, ortho-Phosphate 15 and Potassium 50. Digestion of samples was performed with a NANOCOLOR®VARIO C2 (Macherey-Nagel) heating block. Analyses of COD, TOC and NPK in filtered samples were determined photometrically using a compact photometer PF-12Plus (Macherey-Nagel). The conductivity and pH were measured by using wireless pH and conductivity sensors (Pasco), which were connected through a tablet computer and recorded via the SPARKvue app.

Untreated samples were analyzed on the first day of the experiments (day zero) and are denoted in the next section as "untreated". Samples from lower temperature thermal pretreatment at 38.6 ◦C and biological pretreatment (at the same temperature) were analyzed on the 8th day of the experiments, while samples from higher temperature thermal pretreatment at 80 ◦C were analyzed on the 5th day. The concentration of gases (CH4, CO2 and H2S) formed during pretreatment in the gas phase was monitored by an Optima 7 Biogas analyzer. Measurements in the gas phase were performed on the last day of each experiment (on the 5th or 8th day of the experiments).

**Figure 1.** Schematic representation of set-up for thermal and biological pretreatment (illustration made by authors).

**Table 1.** Composition of samples used in pretreatment tests.


The results of chemical analyses for treated and untreated samples were statistically tested (in Excel) for significant differences using the *t*-test (90% confidence level, two tailed, homoscedastic type). The deviations of the results between parallel samples were expressed with standard deviation and error bars (error bar represents one standard deviation).
