## 3 Results and Discussion
### Effect of MWCNT on Methane Production by the Indigenous Microorganisms in River Sediment  
Since microbial communities may be differently affected by MWCNT, experiments were performed with a river sediment. Faster conversion of the natural organic matter to methane was achieved in the assays with MWCNT-Fe and MWCNT, relatively to the incubations without MWCNT (Figure 4). Additionally, 10.2 times and 4.5 times higher cumulative methane production was achieved in the presence of MWCNT-Fe and MWCNT, relatively to the absence of MWCNT (i.e., 130 \(\pm\) 19 mL kg\({}^{-1}\), 57 \(\pm\) 26 mL kg\({}^{-1}\) and 13 \(\pm\) 7 mL kg\({}^{-1}\), respectively, after 169 days--Figure 4). Therefore, the stimulatory effects of MWCNT seem to be transversal to different microbial communities. These nanomaterials may have directly stimulated the methanogens, as referred in the previous section and also in [27], or accelerated interspecies electron transfers.  
Figure 4: Cumulative methane production (at STP conditions) in the assays performed in the absence of MWCNT (w/o MWCNT, \(\blacktriangle\)), and in the presence of MWCNT (\(\blacklozenge\)) or MWCNT-Fe (\(\bullet\)). The results presented are the averages and standard deviations for triplicate assays.  
Low organic matter content (0.8 \(\pm\) 0.1%) was quantified in the sediment, which is typical of intertidal zones [33], and the slow methane production rate in the incubations without MWCNT (Figure 4) highlight the recalcitrant nature of the sedimentary organic matter. Phosphate, iron(II) and iron(III) concentrations were traceless. Since the culture medium did not contain sulfate neither nitrate, the presence of an alternative electron acceptor other than bicarbonate was ruled out. pH values were relatively constant over the time, varying between 6.7 and 7.1 in all the assays (data not shown). ORP varied between \(-\)100 and \(-\)194 mV, and was higher (\(\sim\)45 to 50 mV difference) in the assays with MWCNT-Fe, relatively to the other conditions tested (Figure S4). A similar effect was previously reported for MWCNT in assays with pure cultures of methanogens performed without reducing agent [27]. These authors described that the initial methane production rate increased with increasing MWCNT concentrations, and the ORP also increased, up to \(-\)189 mV [27].  
Different mechanisms can support the stimulatory effects of MWCNT, as described previously by [PERSON] et al. [35]. In our assays, since MWCNT and MWCNT-Fe have similar textural characteristics (i.e., BET surface area of 283 and 266 m\({}^{2}\) g\({}^{-1}\), respectively), the higher stimulatory effect of MWCNT-Fe may be associated with the combined occurrence of different electron transfer mechanisms, including electron transfer from the Fe (Fe\({}^{2+}\)) impregnated in MWCNT to the carbon of the composites, and then to the final acceptor (i.e., CO\({}_{2}\) reduction by methanogens), as represented in Figure 5. By receiving electrons from the oxidation of the organic matter, iron regeneration will probably occur during the process and act cyclically, accelerating the reaction rates. A similar mechanism was proposed by [PERSON] et al. [35] for the reduction of the azo dye Acid Orange 10. In that work, the involvement of iron in the mechanisms of electron transfer (represented as (3) and (4) in Figure 5) was demonstrated.