## 4 Discussion  
During the course of our experiments, we observed that replicate MFCs often vary greatly in their startup times, probably due to stochastic effects, for example, the rare electroactive organisms in the inoculum may be strongly affected by random events [31; 32]. This variability allowed us to investigate microbial factors affecting startup times. Although _Geobacteraceae_ species are well known as the prime and most dominant anode respiring bacteria in MFCs [33], in our system _Desulfuronadaceae_ were much more abundant in the anodic biofilm. A specific primer set was initially designed by [PERSON] et al. [23] to detect the acetate-oxidizing, PCE-dechlorinating _Desulfurononas_ group in contaminated soils. This primer set was tested on MFC samples in our study, taken during the startup phase. Interestingly, an amplification product could be associated with samples from efficient startup MFCs only. Nevertheless, this _Desulfurononas_ taxon was eventually detected in all MFCs (also non-efficient), once a stable electroactive biofilm had evolved (Figure 4). Thus, it appears to be an important member of electroactive biofilms. Of note, this specific _Desulfurononas_ taxon could also be identified in planktonic samples (Figure 4) in a simple PCR assay, thereby showing potential for MFC monitoring in real-world situations. The _Desulfurononalaceae_ taxa, observed in our study, were found to be closely related to _Desulfurononas actexigens_, an acetate-oxidizing, sulfur-reducing bacterium isolated from freshwater habitats [34]. The _Desulfurononas_ decblorinator primer set has previously been shown to be specific towards _D. actexigens_, _D. chloroethenica_, and _Desulfurononas sp. strain BB1_ but other _Desulfurononas_ strains tested, including _D. thiophila_, _D. palmitatis_, _D. actexidans_, _D. succinoxidians_, _Pelobacter acetylenicus_, and _Geobacter metallietuchens_, did not result in amplification [23]. Additionally, the _Geobacteraceae_  
Figure 5: Summary of (**a**) lag time and (**b**) startup time for all experiments in this work, i.e., 2015, 2016, 2016-S, 2016-2, and 2016-E.  
taxa, observed in our study, were closely related to _Geobacter lovleyi_ SZ, a novel _Geobacter_ species, capable of coupling the oxidation of acetate and H\({}_{2}\) to the reduction of a variety of electron acceptors, including tetrachloroethene, trichloroethene, nitrate, and uranium, a property that has previously been demonstrated mostly for members of the _Desulfuronomas_ cluster [35]. In spite of this similarity in metabolic capabilities of _Geobacter lovleyi_ SZ and the _Desulfuronomas_ strains in our reactors, and the fact both were abundant in the anodic biofilm, the former was not associated with an amplification product (Supplementary Figure S1), showing that the primer set was specific only for known _Desulfuronomas_ PCE decholrinators, as shown by [23]. Application of an anode enrichment consortium reduces startup times significantly. While several studies have reported _Desulfuronomas_ species to be abundant in microbial communities colonizing the anode [24; 36; 37], inoculation of MFCs with pure cultures of these bacteria has not been tested to date [38]. As proof-of-concept for such an approach, we have isolated a consortium of _Desulfuronomas_-enriched bacteria (Supplementary Figure S3) from an active anodic biofilm of an efficient operating MFC. The direct application of such an enriched consortium to a new sterile anode dramatically reduced lag time, from an average of five days to several hours (Figure 5). Nevertheless, stable voltage outputs that were obtained by the enrichment-treated MFCs were similar to the other treatment group. These results are contradictory with those previously reported by [PERSON] et al. [39], where directly applying a biofilm scraped from an active anode to a new one, yielded no significant difference in the lag phase as compared with unacclimated MFCs inoculated with sludge alone.