# Efficiency of Chemical Toilet Sewage (CTS) Co-Fermentation with Typical Energy Crops
## 2 Materials and Methods
### Analytical Methods  
COD (chemical oxygen demand), all phosphorus species, all nitrogen species, and chlorides and sulfates in the CTS were determined using a DR 5000 spectrophotometer with an HT200S mineralizer (Hach-Lange, Dusseldorf, Germany). Determination of biochemical oxygen demand (BOD\({}_{5}\)) was carried out according to PN-EN ISO 5815-1:2019-12 [23]. The pH was determined using a VWR 1000 L pH meter (Gdansk, Poland). TS, VS and MS in the maize silage, in the anaerobic sludge inoculum, and in the digestate were determined gravimetrically. TS levels in the biomass were determined by drying to a constant weight at 105 \({}^{\circ}\)C, then burning it at 550 \({}^{\circ}\)C. The loss on ignition was the VS, as per PN-EN 15935: 23022-01. Biomass samples desiccated at 105 \({}^{\circ}\)C were assayed for TC, TOC, and Ntotal using a Flash 2000 elemental particle analyzer (Thermo Scientific, USA). Ptotal was determined colorimetrically in ammonium metavanadate (V) and ammonium molybdate after prior mineralization in a mixture of sulfuric (VI) and chloride (VII) acids at 390 nm using a DR 2800 spectrophotometer (HACH Lange, Weilheim, Germany). Total protein was calculated by multiplying the value of Ntotal by the protein conversion factor of 6.25. Reducing sugars were determined colorimetrically with an anthrone reagent at 600 nm using a DR 2800 spectrophotometer (HACH Lange, Weilheim, Germany). Lipids were quantified using the Soxhlet method with a Buchi extraction apparatus (Flawil, Switzerland). The pH in H2O was determined potentiometrically with an 867 pH module (Metrohm, Herisau, Switzerland). The FOS/TAC (the ratio of the buffer capacity of the sample to the VFA levels in the sample) was determined using a TitraLab AT1000 titrator (HACH Lange, Weilheim, Germany). Total alkalinity was determined through titration testing. Magnesium was determined by atomic absorption spectrometry. Calcium and potassium were determined by flame photometry. The experiment was conducted at an accredited test laboratory of the Chemical and Agricultural Research Station (Olsztyn branch, Poland). Acute toxicity of the sludge (aqueous extracts) was measured using _Vibrio fischeri_ bacteria in an M 500 Analyzer (Azur Environmental, Carlsbad, DE, USA), following Polish \begin{table}
\begin{tabular}{c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Variant**} & **Reactor** & \multirow{2}{*}{\begin{tabular}{c} **OLR** \\ **Volume** \\ **(dm\({}^{3}\))** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **OLR** \\ **VS Load** \\ **(gVS/dm\({}^{3}\))** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **VLS Load** \\ **(gVS/dm\({}^{3}\))** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Maize** \\ **Slige** \\ **Slige** \\ **Weight** \\ **(gDay)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Maze** \\ **Slige** \\ **Weight** \\ **(gDay)** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Maze** \\ **Slige** \\ **Volume** \\ **Weight** \\ **Content** \\ **(cm\({}^{3}\))** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **Water** \\ **Volume** \\ **(cm\({}^{3}\))** \\ \end{tabular} } & \multirow{2}{*}{\begin{tabular}{c} **CTS** \\ **Volume** \\ **(cm\({}^{3}\))** \\ \end{tabular} } & \multirow{2}{*}{\
\begin{tabular}{c} **HRT** \\ **(doba)** \\ \end{tabular} } & \multirow{2}{*}{
\begin{tabular}{c} **Temperature** \\ **(cC)** \\ \end{tabular} } \\ \hline
1 & & & & & & & 67.0 & 0.0 & & & \\
2 & & & & & & 60.3 & 6.7 & & & \\
3 & & & & & & 53.6 & 13.4 & 20 & 38 \\
4 & & & & & & 46.9 & 20.1 & & & \\
5 & & & & & & 40.2 & 26.8 & & & \\
6 & & & & & & & 33.5 & 33.5 & & \\
7 & & & & & & & 26.8 & 40.2 & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Feedstock volumes and key process parameters. Standard PN-ISO 11348-2:2008. The aqueous extract was prepared by adding 4 volumes of distilled water on top of 1 volume of sludge and agitating mechanically for 24 h [24].