# Enzymatic Activity in the Anaerobic Co-Digestion of Cavitated Coffee Waste and Sewage Sludge
## 2 Materials and Methods
### HDC and AnD Experiments  
HDC was selected as the pre-treatment technique for CW prior to its AcD with SS. These experiments were conducted using a laboratory device consisting of a cavitation reactor with replaceable cavitation inducers, circulation tank with an active volume of 30 L, pump and inverter. All mentioned elements were connected with pipelines and equipped with control valves. Moreover, this laboratory equipment had measurement devices, e.g., electromagnetic flow meter and three piezoelectric pressure gauges. In this laboratory device, the wastewater with suspended coffee circulates in a closed loop, repeatedly passing through the cavitation reactor. The operational parameters of HDC  
\begin{table}
\begin{tabular}{c c c c c c} \hline
**Parameter** & **Units** & **SS** & **Non-Cavitated CW** & **CW Cavitated for 20 min** & **CW Cavitated for 30 min** \\ \hline COD & mg/L & 47650 \(\pm\) 235 & 9750 \(\pm\) 190 & 6580 \(\pm\) 164 & 6830 \(\pm\) 125 \\ TS & g/kg & 45.3 \(\pm\) 4.1 & 9.28 \(\pm\) 2.7 & 8.21 \(\pm\) 1.5 & 7.94 \(\pm\) 0.8 \\ VS & g/kg & 33.35 \(\pm\) 2.9 & 8.21 \(\pm\) 0.5 & 7.43 \(\pm\) 0.6 & 7.08 \(\pm\) 0.5 \\ phenols & mg/L & 3.7 \(\pm\) 0.06 & 16.30 \(\pm\) 1.5 & 23.60 \(\pm\) 2.7 & 26.10 \(\pm\) 3.3 \\ pH & & 5.97 \(\pm\) 0.05 & 7.28 \(\pm\) 0.1 & 7.82 \(\pm\) 0.1 & 7.93 \(\pm\) 0.1 \\ caffeine & ppb & nd & nd & nd & 505 \(\pm\) 15 \\ \hline \end{tabular}  
* COD–chemical oxygen demand, TS—total solids, VS—volatile solids, CW—coffee waste, nd—non-detected, ppb—parts per billion.  
\end{table}
Table 1: Physico-chemical characteristics of materials used in the experiments.  
experiments are presented in Table 2. The operational conditions to conduct HDC were chosen based on optimization studies, the results of which were presented in previous work [2]. The criteria taken into account were improved biodegradability and low energy consumption for pre-treatment. Therefore, two times were chosen differing in terms of caffeine presence; importantly, caffeine release was noted only at 30 min (Table 2).  
In the second stage of the experiment, batch anaerobic digestion was conducted. Therein, our series were provided with a differing feedstock composition; the detailed operational setup is presented in Figure 1. The cavitated CW was added at an amount of 1.5 g to 400 mL of SS (R2 and R3 for CW cavitated for 20 and 30 min, respectively). Furthermore, the mono-digestion of SS (R0) and the AcD series supplied with raw CW (R1) were performed as reference tests.  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Parameter** & **Description** \\ \hline inlet pressure & 5 bar \\ HDC inducer configuration & single hole plate with total area of hole 7.0686 mm\({}^{2}\) \\ time & 20 and 30 min \\ cavitation number & 0.065 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Operational parameters of HDC pre-treatment.  
Figure 1: The main parameters of experiment (CW—coffee waste, MPW—mechanically pre-treated wastewater, HDC—hydrodynamic cavitation, SS—sewage sludge).  
The specially created BioReactor Simulator (Bioprocess Control AB, Lund, Sweden) was used to carry out the batch tests. This equipment is equipped with six reactors, each with a 2.0 L working volume. Each reactor had a mechanical stirrer that ran for a cycle of five minutes for mixing and twenty-five minutes for resting. All reactors were kept in water baths to maintain the temperature under mesophilic conditions (37 \({}^{\circ}\)C). Using a wet gas flow-measuring apparatus that worked on the basis of buoyancy and liquid displacement, along with a system for continuous data collecting, the biogas output was automatically tracked and recorded. All reactors were flushed with inert gas (N2) to provide anaerobic conditions for the experiments.