*3.4. Microbial Community Analysis*

Once the experiment was completed, a sample was taken for the analysis of microbial communities. For this purpose, a 1 × 1 cm portion of carbon felt was cut using sterilised scissors. Genomic DNA from the electrodic sample was extracted with the PowerSoil® DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA, USA), following the manufacturer's instructions. The entire DNA extract was used for the pyrosequencing of the 16S-rRNA gene-based massive library, targeting the eubacterial region V1–V3 16S-rRNA, and performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA), utilising MiSeq equipment (Illumina, San Diego, CT, USA). The primer set used was 27Fmod (50 -AGRGTTTGATCMTGGCTCAG-30 ) /519 R modBio (50 -GTNTTACNGCGGCKGCTG-30 ). Diluted DNA extracts were used as a template for polymerase chain reaction (PCR) reactions. The obtained DNA reads were compiled in FASTq files for further bioinformatics processing. The trimming of the 16S-rRNA bar-coded sequences into libraries was carried out using QIIME software, version 1.8.018 [49]. Quality filtering of the reads was performed at Q25 quality prior to grouping into operational taxonomic units (OTUs) at a 97% sequence homology cut-off. The following steps were performed using QIIME, a denoising procedure using a denoiser algorithm [50]. Final OTUs were taxonomically classified using BLASTn against a database derived from the Ribosomal Database Project II (RDPII, http://rdp.cme.msu.edu) and the National Centre for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov).

## **4. Conclusions**

The proliferation and biocatalytic activity of *Geobacter* in air-exposed anodes depends on the anaerobic microenvironment provided by the aerobic microorganisms present in outer layers of the biofilm, which consume DO. The porous structure of the carbon felt causes the channelling of DO in internal zones. This study confirms *Geobacter*'s tolerance to sustained oxygen concentrations below 90 µM. During periods in which the biofilm is maintained without substrate, oxygen diffuses through the biofilm height. If these periods without substrate are longer than six days, the inner electroactive layers seem to be irreversibly affected, and the exoelectrogenic activity does not resume again after feeding.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4344/10/11/1341/s1, Figure S1: Dissolved oxygen profile. Rejected because the data acquisition system was accidentally interrupted at the end of the experiment; Figure S2: Another dissolved oxygen profile. Rejected because the data acquisition system was accidentally interrupted at the end of the experiment.

**Author Contributions:** R.M.A. contributed to the conceptual design of the experiment, carrying out the experimental procedures, data analysis, results interpretation and drafting the manuscript. G.P.: performed the microbiological analysis and provided assistance in the laboratory. M.I.S.-M. also provided assistance in the laboratory and contributed to results' interpretation. A.M.: contributed to results interpretation, being responsible for project administration and funding acquisition. A.E.: contributed to the conceptualization, results interpretation, project supervision and writing/reviewing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was possible thanks to the financial support by 'Consejería de Educación de la Junta de Castilla y León' (ref: LE320P18), a project co-financed by FEDER funds. R. M. Alonso thanks the University of León for his predoctoral contract.

**Conflicts of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## **References**


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