## 3 Results  
We observed, throughout three different experiments conducted between 2015 and 2016, considerable variation in the development of voltage production following the inoculation process of MFCs. The lag phase is defined here as the time until the MFC reaches 30 mV. This value was chosen arbitrarily as a value that was higher than base-line voltage, indicating that the biofilm was starting to acclimate to the anode, yet was still in an initial stage of development. Startup time was defined as the time to reach steady-state voltage production, i.e., the time to reach the maximum voltage that a particular anodic biofilm was able to produce under specific given conditions. The startup phase of MFCs from all three experiments (N = 16), therefore, could be classified into either \"efficient\" (shorter than 25 days) or \"non-efficient\" (Figure 1). This heterogeneity in startup time efficiency necessitates specific biomarkers for identifying dominant key species involved in forming an efficient anodic biofilm at early stages. Recently, we showed that while all anodic biofilms were composed of _Desulfuronondaceae_, MFCs differed in startup times [17]. Although _Desulfuronondaceae_ has been found to be an important member of the anodic biofilm, very little is currently known regarding the molecular activity within MFC systems [25]. In this study, our main goal was to investigate strain level dynamics within the _Desulfuronondaceae_ arising in the anodic biofilm. The _Geobacteraceae_ family are thought to be the typical and most predominant electroactive bacteria in MFC systems, thus, this taxonomic group has been widely studied and probes targeting it have been developed [20; 21; 22; 26; 27]. Because _Geobacteraceae_ and _Desulfuronondaceae_ are phylogenetically closely related [28], we tested several semi-specific published primer sets on different DNA samples in an effort to differentiate between them by PCR without resorting to sequencing (Table 1 and Supplementary Figure S1). Only one PCR primer set, hereby referred to as _Desulfurononas_ dichlorinator-targeted primers [23], was indeed specific to _Desulfurononas_ (Figure 2) and showed a clear differentiation between _Desulfuronondaceae_ and _Geobacteraceae_. Figure 2 shows clearly that not all samples that show the presence of the _Desulfuronondaceae_ family by the amplicon sequencing analysis, have a positive PCR product with the specific 16S-_Desulfuronondaceae_ primer set. For example, according to amplicon sequencing results, 21% of Sample 3 was composed of the _Desulfuronondaceae_ family, however, it had no product from the 16S-_Desulfuronondaceae_ primer set reaction. Notably, this was not due to the lower relative abundance of Sample 3, as other samples with a lower _Desulfuronondaceae_ relative abundance did produce a PCR  
Figure 1: Current density production growth curve of two representative microbial fuel cells (MFCs), efficient and non-efficient. \(\lambda\) is lag phase length, the time it took to reach 30 mV. product (Table 2). Interestingly, Samples 1 and 3 were anodic biofilm DNA samples taken from an efficient and non-efficient MFC, respectively. Remarkably, all the samples that did yield a PCR product with the _Desulfurononas_ dechlorinator-targeted primers originated in efficient MFCs, while samples from non-efficient MFCs \begin{table}
\begin{tabular}{c c c} \hline  & _Geobacteraceae_ & _Desulfurononadaceae_ \\
**Sample ID** & **Relative Abundance According** & **Relative Abundance According** \\  & **to Amplicon Sequencing \({}^{*}\)** & **to Amplicon Sequencing** \\ \hline  
19 & 0 & 0 \\ \hline \end{tabular} \({}^{*}\) Relative abundance of _Geobacteraceae_/_Desulfurononadaceae_ is according to 16S- rDNA amplicon sequencing, representing relative abundance of each operational taxonomic unit (OTU) from the entire community in each sample.  
\end{table}