## 2 Materials and Methods  
Coffee samples were obtained from three coffee shops (retail shops in Brno, Czech Republic: Three samples from each coffee shop). The samples differ in Arabica/Robusta ratios: Sample No. 1, Arabica 50%/Robusta 50%; sample No. 2, Arabica 80%/Robusta 20%; and sample No. 3, Arabica 100%. The samples of spent coffee grounds were dried at 105 \({}^{\circ}\)C to constant weight. Activated sewage sludge used in the experiments was taken from an activation tank at WWTP Moravany 5000 PE, Czech Republic and WWTP Modrice, 480,000 PE, Czech Republic. The cultivation medium was prepared as follows: 1 g of malt extract and 0.5 g of peptone (HiMedia Deutschland AG, Dusseldorf, Germany) dissolved in 1 dm\({}^{3}\) of tapped water, and sterilized in an autoclave. Biological oxygen demand (BOD) values were determined by aerobic system OxiTop(r) (WTW, Weilheim, Germany). The samples were divided into three groups: Unprocessed (before sludge addition), processed (after sludge addition), and filtrate (the filtrate obtained after sludge addition). In total, eight bottles (510 mL) were filled up with 15 mL of sewage sludge and 28.5 mL of cultivation media. Consequently, SCG samples (0.7 g) were added. During the experiment, different amounts of SCG were used but the most effectively processed was 0.7 g and the BOD was highest. The amount of CSG first ranged from 0.1 g to 0.5 g by 0.1 g. As the activated sludge was not inhibited and BOD was rising with the amount of SCG, the concentration was risen to 0.5 g, 0.7 g, 1 g, 3 g, 5 g, and 7 g, in order to cover wider range. The 0.7 g provided the highest BOD. We chose this amount and maintained the concentration for our next experiments and analyses to discover what is happening with SCG. The respiratory activity of microorganisms can be an important factor for wastewater treatment, since its increase allows us to achieve the maximum decomposition of complex organic compounds, their involvement in microbial metabolism and, thus, detoxification of the water environment from toxic pollutants. Two bottles without SCG addition were used as the control. Two NaOH pearls designed to absorb CO\({}_{2}\) were added to the rubber plugs in the bottle opening. Closed bottles were incubated in the dark for 5 days at 20 \({}^{\circ}\)C with constant stirring by a magnetic stirrer. Respiratory activity values were recorded with OxiTop(r) measuring heads. For analyses, the filtration of the mixture through filter paper 2R/60 (Papirna Persteinj.s.o., Persteinj, Czech Republic) was carried out after five days of incubation; the solid was dried at 60 \({}^{\circ}\)C for 24 h. Microbial respiration rates were calculated using the formula described in the paper by [PERSON] et al. (2018) [27]. The total content of phenols was determined using a UV/VIS spectrophotometer CE7/210 (Cecil Instruments, Cambridge, UK) at 765 nm according to the method described by [PERSON] et al. (2006) [28]. The caffeine content in SCG unprocessed and processed samples was determined by the modified method described by [PERSON] et al. (2013) [29]. The samples were treated with hot water (75-80 \({}^{\circ}\)C) and filtrated. Then, 1 mL of filtrate was mixed with 100 mL of distilled water, and absorbance at 272.5 nm was determined. The quantification of the caffeine was done by the usage of calibration curve. Chlorogenic acid was quantitatively determined according to the method described by [PERSON] (2016) [30]. The extraction of a coffee sample (2 g) was done with 150 mL of methanol/water (50/50, v/v). The samples were left in methanol/water solution for 3 days. The extract was filtered by a syringe filter (LUT Syringe Filter, 0.45 \(\upmu\)m, 13 mm), and the filtrate was used directly for injection. The mobile phase consisted of A) water + 1% phosphoric acid and B) acetonitrile. The gradient was adjusted as follows: 0 min--10% B, 20 min--20% B, 25 min--30% B, 35 min--40% B, 40 min--40% B. The duration of analysis was 10 min, and the flow rate was 1 mL/min.