## Materials and Methods  
Three of the most commonly employed conditioning mixtures in EPB-TBM tunneling activities, indicated as A, B and C, were used as mixture models for the present study. The information of each mixture reported in the safety data sheets includes the concentration of SLES and the co-presence of 1,2-Benzisothiazol-3(2H)-one in B and hexylene glycol in C, as additives, as shown in Table 1, where the Total Organic Carbon (TOC) and Chemical Oxygen Demand (COD) are also reported. For each conditioning mixture, three solutions were prepared by dissolving A, B and C in demineralized water. Three different dilution ratios were used: 1:500, 1:750 and 1:1000 (_v/v_) corresponding to 0.20, 0.13 and 0.10% vol., respectively. Biodegradability tests were performed according to the closed bottles procedure to assess the biodegradability of the investigated mixtures at different dosage (0.20 and 0.10%), and to evaluate the effect of the inoculum selected on the biodegradation rate. _Bacillus Clausii_ was compared to a mixed inoculum obtained by dissolving in water commercial soil humus (Altea Natural Humus; Composition of the dry product: Nitrogen (N) content 2 wt% and Organic (C) content 26 wt%). After a vigorous mixing, the suspension was separated by sedimentation for 30 min and the supernatant was then kept under continuous aeration [24]. For the closed bottle procedure, different mineral stock solutions were prepared according to the OECD method, in particular:  
- Solution 1 [8.50 g/L of KH\({}_{2}\)PO\({}_{4}\), 21.75 g/L of K\({}_{2}\)HPO\({}_{4}\), 33.40 g/L of Na\({}_{2}\)HPO\({}_{4}\)-2H\({}_{2}\)O and 0.50 g/L of NH\({}_{4}\)Cl];
- Solution 2 [27.50 g/L of CaCl\({}_{2}\)];
- Solution 3 [22.50 g/L of MgSO\({}_{4}\)-7H\({}_{2}\)O];
- Solution 4 [0.25 g/L of FeCl\({}_{3}\)-6H\({}_{2}\)O]. A diluting water solution was prepared by dissolving the mineral stock solutions (1 mL for each mineral) to 1.0 L of demineralized water. This solution was kept in vigorous aeration for 20 min and the foam agent was then added. The bottles were filled with 250 mL of the prepared foam agent solution and 2.0 mL of bacteria inoculum were added. Each bottle is provided by a cup with a digital display that allowed the online monitoring of the BOD value as oxygen consumption. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Conditioning Mixture** & **TOC [g/L]** & **COD [g/L]** & **SLES [wt%]** & **Additives [wt%]** \\ \hline
**A** & 154.3 & 154.9 & 10–50 & - \\
**B** & 67.1 & 86.2 & 10–20 & 0.005–0.01 \\
**C** & 74.8 & 75.6 & 5–10 & 5–10 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Characteristics of the conditioning mixtures used in the experiments. Neutral pH condition and room temperature (25 \({}^{\circ}\)C) were maintained during the closed bottle tests. TOC (determined by a TOC-L analyzer Shimadzu) and COD (determined through colorimetric method [25]) were measured to define a general description of the process by using two indexes: the BOD(t)/COD(t = 0) and COD(t)/TOC(t) ratios. The first parameter (also known as the biodegradation index [26]) defines the rate of oxidable compounds removal, while the second one could highlight the presence of reducing species different from organics. The mineralization of organic compounds into CO\({}_{2}\) and H\({}_{2}\)O was evaluated by TOC removal %, calculated by the following equation:  
\[\text{TOC (\%)}=[\text{TOC (t = 0)}-\text{TOC (t)}]/[\text{TOC (t = 0)}]\times 100 \tag{1}\]  
where TOC [mg/L] was measured at the beginning of the tests (t = 0) and at a selected time t. In the adopted conditions, the mean values of the TOC (t = 0) were 152.08, 210.77 and 316.16 mg/L (for the conditioning mixture (A); 70.03, 99.70 and 149.56 mg/L (for the conditioning mixture (B); 72.24, 115.21 and 172.67 mg/L (for the conditioning mixture (C), at 0.10, 0.13 and 0.20%, respectively.