## 3 Results and Discussion  
### Effect of MWCNT on Methane Production by Anaerobic Sludge  
The cumulative methane production curves obtained in the incubations with acetate and H\({}_{2}\)/CO\({}_{2}\) are shown in Figure 2, and the calculated specific methane production rates (SMPR) are presented in Table 1. The addition of MWCNT resulted in significantly faster (\(p<0.004\)) methane production from acetate relatively to the assay without MWCNT, i.e., 1.6 and 2.1 times higher SMPR were verified in the presence of 0.5 g L\({}^{-1}\) and 1.0 g L\({}^{-1}\) MWCNT, respectively (Figure 2a, Tables 1 and 2). SMPR from acetate correlated highly with MWCNT concentration, as shown by the calculated correlation coefficient of 0.998 (Figure S2). No lag phases preceded the onset of methane production (Figure 2a). Regarding H\({}_{2}\)/CO\({}_{2}\), SMPR increased _circa_ 11% in the assays performed with 1.0 g L\({}^{-1}\) MWCNT, when compared to the assays without MWCNT (\(p<0.03\)), but no significant effect was observed with 0.5 g L\({}^{-1}\) MWCNT (Figure 2b, Tables 1 and 2). Similarly, other studies reported stimulated acetoclastic but not hydrogenotrophic methanogenesis in assays with graphene (a planar representation of CNT) [45] or MWCNT-Fe [46]. The enrichment of _Methanosaeta_ and the improvement of acetoclastic activity was also described in the presence of single-walled CNT [47] or biochar [48]. However, experiments with pure cultures of _Methanobacterium_ and MWCNT (5 g L\({}^{-1}\)) showed an increase in the initial methane production rate of approximately 17 times [27], indicating that MWCNT positively affect the activity of hydrogenotrophic methanogens growing in pure cultures. In our experiments, the beneficial effects of MWCNT toward methanogens were more noticeable in the incubation with acetate than with H\({}_{2}\)/CO\({}_{2}\). Nevertheless, faster methane production from both substrates was verified, especially at the higher MWCNT concentration tested, which shows the potential of MWCNT for increasing the efficiency of AD processes. The activity of syntrophic bacteria was indirectly assessed in this work, by monitoring the methane produced from the degradation of ethanol (Figure 3a) and butyrate (Figure 3b). The SMPR from ethanol increased 11% and 22% in the assays performed with 0.5 and 1.0 g L\({}^{-1}\) MWCNT, respectively, when compared to the assays without MWCNT (Tables 1 and 2), and a correlation coefficient of 1.000 was calculated for SMPR vs MWCNT concentration (Figure S2). Ethanol oxidation yields acetate and hydrogen/formate and is thermodynamically unfavorable at standard conditions (Equation (1)--Table S1), becoming thermodynamically feasible when these compounds are consumed by methanogens (Equations (3)-(5)--Table S1). MWCNT may have stimulated the activity of ethanol-degrading syntrophic bacteria, or/and the activity of methanogens, overall enhancing syntrophic ethanol conversion. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multicolumn{2}{c}{**MWCNT**} & \multicolumn{5}{c}{**SMPR (mL g-1 d-1)**} \\ \cline{2-7}  & **Acetate** & **H\({}_{2}\)/CO\({}_{2}\)** & **Ethanol** & **Butyrate** & **Butyrate** & **Butyrate** \\  & & & & **(1 st add.)** & **(2 nd add.)** & **(2 nd add.)** & **(2 nd add.) t \textgreater{} 20 h** \\ \hline
0 & \(38\pm 3\) a  & \(394\pm 10\) a  & \(192\pm 12\) a  & n.d. & \(18\pm 3\) a  & \(40\pm 0\) a  \\
0.5 & \(62\pm 4\) b  & \(384\pm 33\) a  & \(214\pm 4\) a  & n.d. & \(34\pm 3\) a  & \(47\pm 0\) b  \\
1.0 & \(82\pm 10\) b  & \(439\pm 16\) b  & \(235\pm 9\) b  & n.d. & \(47\pm 3\) b  & \(52\pm 3\) c  \\ \hline \hline \end{tabular}
\end{table} 
Table 1: SMPR with different MWCNT concentrations. The SMPR of butyrate after 20 h of incubation is also shown. Figure 2: Cumulative methane production (at STP conditions) in the assays amended with acetate (**a**) or H\({}_{2}\)/CO\({}_{2}\) (**b**), in the presence of different MWCNT concentrations: 0 g L\({}^{-1}\) (**a**), 0.5 g L\({}^{-1}\) (**b**) and 1.0 g L\({}^{-1}\) (**b**).