## 3 Results and Discussion
### Effect of MWCNT on Methane Production by Anaerobic Sludge
Blank assays (no added substrate) are also shown (open symbols). The results presented are the averages and standard deviations for triplicate assays. The conversion of butyrate to methane took more than 10 days and no methane was produced during the first three days, showing the very low butyrate degrading activity of the community (Figure S3). Therefore, a second butyrate addition was performed, and the cumulative methane production obtained is shown in Figure 3b. In the presence of 0.5 g L\({}^{-1}\) or 1.0 g L\({}^{-1}\) MWCNT, the SMPR \begin{table}
\begin{tabular}{c c c c c c} \hline
**MWCNT (g** & **Acetate** & **H\({}_{2}\)/CO\({}_{2}\)** & **Ethanol** & **Budyrate** & **Budyrate** \\
**L\({}^{-1}\))** & **Acetate** & **H\({}_{2}\)/CO\({}_{2}\)** & **Ethanol** & **Budyrate** & **Budyrate** \\
0.5 & 1.6 & 1.0 & 1.1 & 1.9 & 1.2 \\
1 & 2.1 & 1.1 & 1.2 & 2.6 & 1.3 \\ \hline \end{tabular}
\end{table} 
Table 2: Relative increase of the SMPR in the assays with MWCNT, compared to the assays without MWCNT. For butyrate, the increase of the SMPR in the period after 20 h of incubation is also shown. Figure 3: Cumulative methane production (at STP conditions) in the assays amended with ethanol (**a**) or butyrate (second addition) (**b**), in the presence of different MWCNT concentrations: 0 g L\({}^{-1}\) (**\(\blacktriangle\)**), 0.5 g L\({}^{-1}\) (**\(\blacklozenge\)**) and 1.0 g L\({}^{-1}\) (**\(\blacklozenge\)**). Blank assays (no added substrate) are also shown (open symbols). The results presented are the averages and standard deviations for triplicate assays. increased 1.9 and 2.6 fold, respectively (Tables 1 and 2), and was highly correlated with MWCNT concentrations (R\({}^{2}\) = 0.997, Figure S2). The SMPR in the assays without MWCNT was as low as 18 \(\pm\) 3 mL g\({}^{-1}\) d\({}^{-1}\), reflecting the poor butyrate oxidation ability of the sludge (Table 1; Figure 3b). An increase of approximately 1.5 times in methane production was previously reported by [PERSON] and [PERSON] [26] in butyrate-oxidizing enrichments containing MWCNT (\(\sim\)0.25 g L\({}^{-1}\)), where _Syntrophomonas_ and _Methanoregula_ were dominant. Also, faster butyrate conversion to acetate and methane by _Syntrophomonas wolfei_ in co-culture with _Methanospirillum hungatei_, in the presence of MWCNT, was described [27]. In our experiments, the positive effect of MWCNT on the initial methane production rate reflects an enhancement of the activity of syntrophic butyrate degrading bacteria, possibly due to the direct stimulation of hydrogenotrophic methanogens (Equations (2)-(4) and (6)--Table S1). The slope of the cumulative methane production curves changed after 20 h of incubation (Figure 3b), and thus the SMPR for this period was also calculated (Table 1). These values increased with the increase of MWCNT concentrations (R\({}^{2}\) = 1.000, Figure S2), and are statistically higher (\(p\) < 0.03) than in the experiments without MWCNT (Table 2). Considering that the sludge exhibited higher hydrogenotrophic than acetoclastic methanogenic activity, this second period (t > 20 h) was probably associated with the conversion of the acetate formed from butyrate oxidation, also pointing to a positive effect of MWCNT toward the activity of acetoclastic methanogens, ultimately improving complete butyrate conversion to methane (Equations (4) and (6)--Table S1). The improvement of methane production by the presence of conductive materials was previously attributed to their capacity to mediate electron transfer between fatty acid or ethanol consuming bacteria and methanogenic archaea. In this study, both acetoclastic and hydrogenotrophic methanogenic activities were enhanced in the presence of MWCNT which might have contributed to the faster ethanol and butyrate conversion to methane. Overall, MWCNT accelerated methane production from all the tested compounds, and this increase was higher with acetate and butyrate, for which the activity of the sludge was lower.