# Dark Fermentation of Fruit Juice Effluents: Effects of Substrate Concentration
## 2 Materials and Methods
### Experimental Procedure  
The experiments were carried out in batch reactors with an effective volume of 3 L. The reactors were equipped with various inlets and outlets, including ports for a pH probe, alkali dosing for pH control, antifoam addition, a heat exchanger for condensing water in the biogas phase, a nitrogen sparger, and a sampling port. The operational temperature was maintained at 26 \({}^{\circ}\)C using a water bath connected to the reactor jacket. Reactor content was homogenized with a magnetic stir bar rotating at 500 rpm. The pH was controlled at a stable level of 5 through automatic titration using an ADI 1030 Bio Controller (Delft, The Netherlands), which dosed a 3 M NaOH solution as needed. The operational conditions were selected based on previous studies [19; 26]. To minimize foam formation during fermentation, a 2.5% silicone antifoam solution (426R, Prolabo, Radnor, PA, USA) was continuously supplied at a rate of 2.5 \(\mu\)L/min. Anaerobic conditions were ensured by sparging the reactor with nitrogen gas at a flow rate of 40 mL/min [28]. This nitrogen stream also served as a carrier gas, transporting the produced hydrogen to an online gas analyzer (Rosemount Analytical NGA 2000 MLT, Emerson, Ferguson, MO, USA). More information about the experimental procedure can be found in the literature [19; 26]. Each batch experiment began with the addition of 1.85 L of synthetic wastewater, containing organic substrate concentrations of 0.30, 0.91, 1.52, 2.12 C mol\(\cdot\)L\({}^{-1}\), along with \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Component** & **0.30 C** & **0.91 C** & **1.52 C** & **2.12 C** \\  & **mol\(\cdot\)L\({}^{-1}\)** & **mol\(\cdot\)L\({}^{-1}\)** & **mol\(\cdot\)L\({}^{-1}\)** & **mol\(\cdot\)L\({}^{-1}\)** \\ \hline Fructose (C mol\(\cdot\)L\({}^{-1}\)) & 0.18 & 0.53 & 0.88 & 1.24 \\ Glucose (C mol\(\cdot\)L\({}^{-1}\)) & 0.06 & 0.18 & 0.30 & 0.42 \\ Sucrose (C mol\(\cdot\)L\({}^{-1}\)) & 0.06 & 0.20 & 0.33 & 0.46 \\ _Total organic substrate_ & _0.30 C mol\(\cdot\)L\({}^{-1}\)_ & _0.91 C mol\(\cdot\)L\({}^{-1}\)_ & _1.52 C mol\(\cdot\)L\({}^{-1}\)_ & _2.12 C mol\(\cdot\)L\({}^{-1}\)_ \\ (NH\({}_{4}\))Cl (mM) & 56.46 & 169.38 & 282.30 & 395.21 \\ KH\({}_{2}\)PO\({}_{4}\) (mM) & 10.10 & 30.31 & 50.52 & 70.73 \\ NaCl (mM) & 11.29 & 33.88 & 56.47 & 79.06 \\ Na\({}_{2}\)SO\({}_{4}\) (mM) & 0.92 & 2.75 & 4.58 & 6.41 \\ MgCl\({}_{2}\) 6H\({}_{2}\)O (mM) & 1.33 & 3.98 & 6.64 & 9.30 \\ EDTA (mM) & 0.38 & 1.13 & 1.88 & 2.63 \\ ZnSO\({}_{4}\cdot\)7H\({}_{2}\)O (mM) & 2.50 \(\times\) 10\({}^{-2}\) & 7.51 \(\times\) 10\({}^{-2}\) & 1.25 \(\times\) 10\({}^{-1}\) & 1.75 \(\times\) 10\({}^{-1}\) \\ FeSO\({}_{4}\cdot\)7H\({}_{2}\)O (mM) & 2.51 \(\times\) 10\({}^{-2}\) & 7.53 \(\times\) 10\({}^{-2}\) & 1.26 \(\times\) 10\({}^{-1}\) & 1.76 \(\times\) 10\({}^{-1}\) \\ MnCl\({}_{2}\cdot\)4H\({}_{2}\)O (mM) & 2.84 \(\times\) 10\({}^{-2}\) & 8.53 \(\times\) 10\({}^{-2}\) & 1.42 \(\times\) 10\({}^{-1}\) & 1.99 \(\times\) 10\({}^{-1}\) \\ CuCl\({}_{2}\)2H\({}_{2}\)O (mM) & 2.90 \(\times\) 10\({}^{-2}\) & 8.71 \(\times\) 10\({}^{-2}\) & 1.45 \(\times\) 10\({}^{-1}\) & 2.03 \(\times\) 10\({}^{-1}\) \\ CaCl\({}_{2}\) (mM) & 1.22 \(\times\) 10\({}^{-2}\) & 3.65 \(\times\) 10\({}^{-2}\) & 6.08 \(\times\) 10\({}^{-2}\) & 8.52 \(\times\) 10\({}^{-2}\) \\ CoCl\({}_{2}\cdot\)6H\({}_{2}\)O (mM) & 9.12 \(\times\) 10\({}^{-3}\) & 2.74 \(\times\) 10\({}^{-2}\) & 4.56 \(\times\) 10\({}^{-2}\) & 6.39 \(\times\) 10\({}^{-2}\) \\ NiCl\({}_{2}\cdot\)6H\({}_{2}\)O (mM) & 4.75 \(\times\) 10\({}^{-3}\) & 1.43 \(\times\) 10\({}^{-2}\) & 2.38 \(\times\) 10\({}^{-2}\) & 3.33 \(\times\) 10\({}^{-2}\) \\ Na\({}_{2}\)MoO\({}_{4}\cdot\)2H\({}_{2}\)O (mM) & 9.30 \(\times\) 10\({}^{-4}\) & 2.79 \(\times\) 10\({}^{-3}\) & 4.65 \(\times\) 10\({}^{-3}\) & 6.51 \(\times\) 10\({}^{-3}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Characteristics of the synthetic fruit juice wastewaters used in this study. 1.15 L of acclimatized mixed acidogenic bacterial culture.