Figure S1. Experimental overview. Set 1 (S1) and Set 2 (S2) reactors were inoculated on different days with separately prepared source electrode cell extracts but were otherwise treated identically. Each set contained four reactors. The electrodes of all 4 reactors were held V vs. SHE (optimal potential) for ca. 88 hours following inoculation. Cyclic voltammetry (CV) was performed on all reactors. Following CV, reactors 1 and 2 (R1 and R2) were returned to V (optimal potential), while reactors 3 and 4 (R3 and R4) electrodes were changed to V (suboptimal potential) for an additional 52 hours. At the time of sampling all electrodes were split into four sections. Section 1 was used for 16S rRNA gene expression analysis, section 2 was used for protein extraction and digestion by modified porcine trypsin (PT), section 3 was used for protein extraction and digestion by Streptomyces erythraeus trypsin (SET), and section 4 was used for cell counting. Biocathode-MCL source electrode maintained at V S1 and S2 all reactors, record CV S1 (R1/R2), S2 (R1/R2) returned to V S1 (R3/R4), S2 (R3/R4) switched to V 52 hours Section 1. 16S rRNA V3 gene expression analysis Section 4. Cell counting by flow cytometry Section 2. Protein extraction and digestion by PT Section 3. Protein extraction and digestion by SET LC-MS/MS qualitative shotgun proteomics Harvest Biocathode-MCL biofilms from electrodes and divide into 4 sections: Integrated characterization of Biocathode-MCL biofilm microbiome S1 and S2 all reactors, grow at V Prepare extract, count cells. Inoculate ca. 2x10 5 cells 88 hours
S1R1S1R2S2R1S2R2S1R4 S2R3 S2R4 S1R3 S1R 1 S1R 2 S2R 1 S2R 2 S1R 1 S1R 2 S2R 1 S2R 2 S1R 3 S1R 4 S2R 3 S2R 4 S1R 3 S1R 4 S2R 3 S2R 4 PT SET PT SET PT SET PT SET PT SET PT SET PT SET PT SET S1R 1 S1R 2 S2R 1 S2R 2 S1R 1 S1R 2 S2R 1 S2R 2 S1R 3 S1R 4 S2R 3 S2R 4 S1R 3 S1R 4 S2R 3 S2R 4 Protein extraction with B-PER Gel electrophoresis Protein digestion in gel LC-MS/MS analysis – Qstar Elite Database search – Mascot and X!Tandem Protein identification validation and quantitation - Scaffold Optimal biocathodes Suboptimal biocathodes Figure S2. Sample workflow. Electrodes from each reactor were split into four sections, two of which were used for proteomics analysis; section 2 was used for protein extraction and digestion by modified porcine trypsin (PT), section 3 was used for protein extraction and digestion by Streptomyces erythraeus trypsin (SET). Seven cut gel bands were digested for PT samples, and nine cut gel bands were digested for SET samples.
Figure S3. CV from A) Set 1 (S1) and B) Set 2 (S2) reactors: R1 (solid black line), R2 (solid gray line), R3 (dashed black line), and R4 (dashed gray line). CV was recorded at 0.2 mV/sec from V to V and back. A
B Figure S3. CV from A) Set 1 (S1) and B) Set 2 (S2) reactors: R1 (solid black line), R2 (solid gray line), R3 (dashed black line), and R4 (dashed gray line). CV was recorded at 0.2 mV/sec from V to V and back.
Figure S4. Normalized CV from both Set 1 (S1) Set 2 (S2) reactors. CV was normalized by dividing the catalytic current by the limiting current for each reactor.
Table S1. Final cell counts and electrochemical parameters from each reactor. Samplecells/6 x 6 cm electrode Hours to I max Max current (I max )EMEM S1 R12.1E S1 R21.6E S1 R31.5E S1 R49.4E S2 R18.8E S2 R24.8E S2 R39.4E S2 R42.8E
Table S2. Peptide and protein identifications in porcine trypsin (PT) versus Streptomyces erythraeus trypsin (SET) samples. CategorySample#Prot#IDs#Spec%IDs PTS1R1 PT % SETS1R1 SET % PTS1R2 PT % SETS1R2 SET % PTS1R3 PT % SETS1R3 SET % PTS1R4 PT % SETS1R4 SET % PTS2R1 PT % SETS2R1 SET % PTS2R2 PT % SETS2R2 SET % PTS2R3 PT % SETS2R3 SET % PTS2R4 PT % SETS2R4 SET %
Table S3. Spectral counts for proteins identified as associated with either the optimal or suboptimal potential using the Fisher’s exact test (FET). opt subopt ORF IDFisher p- value S1R1 PT S1R2 PT S2R1 PT S2R2 PT S1R1 SET S1R2 SET S2R1 SET S2R2 SET S1R3 PT S1R4 PT S2R3 PT S2R4 PT S1R3 SET S1R4 SET S2R3 SET S2R4 SET Optimal Proteins NODE_2140_9< NODE_2320_41< NODE_22_72< NODE_1148_ NODE_837_ NODE_3683_ NODE_277_ NODE_277_ NODE_837_ NODE_181_ NODE_728_ NODE_2170_ NODE_16387_ NODE_893_ NODE_240_ NODE_15807_ Suboptimal Proteins NODE_403_5< NODE_1573_ NODE_2943_ NODE_1848_ NODE_2368_ NODE_1173_ NODE_1547_ NODE_5518_ NODE_3258_ NODE_2048_ NODE_307_ NODE_83_ NODE_1775_ NODE_6881_ NODE_508_ NODE_3683_ NODE_2533_ NODE_2210_ NODE_476_ NODE_6203_ NODE_522_
Table S4. Spectral counts and p-values for proteins identified as associated with either the optimal or suboptimal potential using the beta binomial (BB) test (Pham et al., 2010; Underlined values were also significant using the Fisher’s exact test (FET). opt subop t ORF IDBB p-valueNCBI annotationBin organismS1R1 PT S1R2 PT S2R1 PT S2R2 PT S1R1 SET S1R2 SET S2R1 SET S2R2 SET S1R3 PT S1R4 PT S2R3 PT S2R4 PT S1R3 SET S1R4 SET S2R3 SET S2R4 SET Optimal proteins NODE_277_ hypothetical proteinKordiimonas NODE_2140_ quinoprotein alcohol dehydrogenaseMarinobacter NODE_779_ hypothetical proteinKordiimonas NODE_7231_ hypothetical proteinChromatiaceae NODE_22_ sugar ABC transporterLabrenzia NODE_5018_ acylneuraminate cytidylyltransferaseChromatiaceae NODE_1148_ ectoine synthaseMarinobacter NODE_728_ MULTISPECIES: OmpA family proteinLabrenzia Suboptimal Proteins NODE_403_50.004flagellinMarinobacter NODE_1573_ membrane proteinMarinobacter NODE_2368_ hypothetical protein Plav_2552Parvibaculum NODE_890_90.041type IV pilus secretin PilQChromatiaceae NODE_1573_ RND family efflux transporter MFP subunitMarinobacter NODE_304_ DNA-directed RNA polymerase subunit alphaChromatiaceae NODE_2135_10.047N/A Underlined values were also significant using the Fisher’s exact test (FET).
Table S5. Log-transformed (base-10) spectral counts and associated p-values for proteins identified as associated with either the optimal or suboptimal potential using the t-test (Microsoft Excel, v14.0.0). An arbitrary value of 0.5 was added to all spectral counts to eliminate zero values for the log transformation. opt subop t ORF IDt-test p- value NCBI annotationBin organismS1R 1 PT S1R2 PT S2R 1 PT S2R2 PT S1R1 SET S1R 2 SET S2R1 SET S2R2 SET S1R3 PT S1R4 PT S2R3 PT S2R4 PT S1R 3 SET S1R4 SET S2R3 SET S2R 4 SET Optimal proteins NODE_277_ hypothetical proteinKordiimonas NODE_2140_ quinoprotein alcohol dehydrogenaseMarinobacter NODE_22_ sugar ABC transporterLabrenzia Suboptimal Proteins NODE_2368_ hypothetical protein Plav_2552Parvibaculum NODE_1573_ membrane proteinMarinobacter Underlined values were also significant using the Fisher’s exact test (FET).
Table S6. Unipept analysis. Peptide counts associated with taxa from Figure 3 are listed in the first four columns. Columns five through eight list peptide counts that could not be assigned below the designated classification level. For example, 46 peptides identified from optimal potential reactors could not be assigned at a level lower than Bacteria. Number of peptides specific to this or lower level % of all matched peptides Number of peptides specific to only this level % of matched peptides optimalsuboptimaloptimalsuboptimaloptimalsuboptimaloptimalsuboptimal Bacteria Proteobacteria Alpha Gamma Alteromonadales Rhodobacterales Chromatiales Labrenzia Marinobacter
V3 16S rRNAPeptide identifications from Unipept Sample* GammaAlphaAlteromonadaceaeEctothiorhodospiraceaeRhodobacteraceaeGammaAlphaMarinobacterLabrenzia %%%%% S1 R S1 R S2 R S2 R S1 R S1 R S2 R S2 R avg. opt ave. sub stdev opt stdev sub Opt. pooled Sub. pooled Table S7. Breakdown of percentages for Alpha- and Gammaproteobacteria, as well as Alteromonadaceae (family containing Marinobacter) and Rhodobacteraceae (family containing Labrenzia) for each reactor. Peptide percentages are based on total of number of peptides identified as Bacteria in order to match the 16S rRNA gene analysis which only considers bacterial abundance. Supporting html files are available in the ProteomeXchange database under identifier PXD for 16S rRNA V3 gene expression analysis of all reactors at both potentials using the truncated mothur pipeline (RDP) or the entire mothur pipeline (mothur).
Figure S5. Unipept analysis of each individual reactor from Set 1. For each reactor, peptides were deduplicated and I and L residues were equated, advanced missed cleavage handling. Value in parentheses is electrode potential at time of sampling and number of peptides matched out of total: A) R1 (0.310 V, 454/655), B) R2 (0.310 V, 306/550), C) R3 (0.470 V, 169/312), and D) R4 (0.470 V, 598/832). Protein extracts (i.e. PT or SET extraction) from the same reactor were pooled. A. B. C. D.
Figure S6. Unipept analysis of each individual reactor from Set 2. For each reactor, peptides were deduplicated and I and L residues were equated, advanced missed cleavage handling. Value in parentheses is electrode potential at time of sampling and number of peptides matched out of total: A) R1 (0.310 V, 226/364), B) R2 (0.310 V, 297/487), C) R3 (0.470 V, 301/558), and D) R4 (0.470 V, 171/354). Protein extracts (i.e. PT or SET extraction) from the same reactor were pooled. A. B. C. D.
Figure S7. Taxonomic distribution of the 16S rRNA V3 read assignments at optimal (A) and suboptimal (B) electrode potentials using RDP classifications at the family level. A.
Figure S7. Taxonomic distribution of the 16S rRNA V3 read assignments at optimal (A) and suboptimal (B) electrode potentials using RDP classifications at the family level. B.
Figure S8. Pooled 16S rRNA V3 gene expression analysis at the A) optimal and B) suboptimal electrode potentials using the mothur workflow. A.
Figure S8. Pooled 16S rRNA V3 gene expression analysis at the A) optimal and B) suboptimal electrode potentials using the mothur workflow. B.