1. 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 0.310 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 0.310 V (optimal potential), while reactors 3 and 4 (R3 and R4) electrodes were changed to 0.470 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 0.310 V S1 and S2 all reactors, record CV S1 (R1/R2), S2 (R1/R2) returned to 0.310 V S1 (R3/R4), S2 (R3/R4) switched to 0.470 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 0.310 V Prepare extract, count cells. Inoculate ca. 2x10 5 cells 88 hours

2. 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.

3. 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 0.610 V to 0.260 V and back. A

4. 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 0.610 V to +0.260 V and back.

5. 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.

6. 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+0635.5-9.480.47 S1 R21.6E+0656.5-51.30.5 S1 R31.5E+0640.5-26.70.48 S1 R49.4E+0543-20.40.510 S2 R18.8E+0544.2-16.70.48 S2 R24.8E+0529.2-4.50.47 S2 R39.4E+0537.8-11.30.47 S2 R42.8E+0640.9-13.30.510

7. Table S2. Peptide and protein identifications in porcine trypsin (PT) versus Streptomyces erythraeus trypsin (SET) samples. CategorySample#Prot#IDs#Spec%IDs PTS1R1 PT6324329898.1% SETS1R1 SET3471028341830.1% PTS1R2 PT148602481512.5% SETS1R2 SET246694431316.1% PTS1R3 PT7536246167.8% SETS1R3 SET121376364210.3% PTS1R4 PT217738623011.8% SETS1R4 SET3371180708416.7% PTS2R1 PT8727348915.6% SETS2R1 SET22648567087.2% PTS2R2 PT8824139336.1% SETS2R2 SET279669481213.9% PTS2R3 PT11142951098.4% SETS2R3 SET308724691210.5% PTS2R4 PT7121848574.5% SETS2R4 SET220493479710.3%

8. 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<0.00010740152000000200 NODE_2320_41<0.000101009139800000641 NODE_22_72<0.000140180646302000700 NODE_1148_520.000670740078402000422 NODE_837_610.0042032862435303251312106372322 NODE_3683_490.00630000821600000500 NODE_277_3230.00660001201300000000 NODE_277_2550.00890000600300000010 NODE_837_510.0110000483400000322 NODE_181_440.026094491241013519574 NODE_728_490.0280000500000000000 NODE_2170_440.0284006232111010012765 NODE_16387_10.0280000400100000000 NODE_893_10.0285322511491111560826127 NODE_240_1010.0470000510200000200 NODE_15807_10.0474310000002000000 Suboptimal Proteins NODE_403_5<0.0001965052242021162918829564230 NODE_1573_140.002522008100210505633 NODE_2943_370.0026050002120000071210 NODE_1848_1210.00372684628000040859510000 NODE_2368_400.00521205311327642725 NODE_1173_1340.00720000100005000500 NODE_1547_60.00810100010000100074 NODE_5518_10.00860200401049100403 NODE_3258_140.0141100000008000300 NODE_2048_70.0230000001000000044 NODE_307_760.0230000000100000233 NODE_83_1080.0230000010000000224 NODE_1775_170.0230000741052006733 NODE_6881_40.02522947131971813826813202821 NODE_508_700.02800002234000061161 NODE_3683_200.0280116553022205336116411517 NODE_2533_80.03246002000617200000 NODE_2210_360.0330101003102100454 NODE_476_490.0350000000000000500 NODE_6203_40.040000100002000500 NODE_522_690.0420200443445204952

9. 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; http://www.oncoproteomics.nl/software/BetaBinomial.html). 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_3230.004hypothetical proteinKordiimonas0001201300000000 NODE_2140_90.014 quinoprotein alcohol dehydrogenaseMarinobacter0740152000000200 NODE_779_1550.016hypothetical proteinKordiimonas0000210100000000 NODE_7231_100.020hypothetical proteinChromatiaceae0100021000000000 NODE_22_720.021sugar ABC transporterLabrenzia40180646302000700 NODE_5018_110.036 acylneuraminate cytidylyltransferaseChromatiaceae0111000000000000 NODE_1148_450.045ectoine synthaseMarinobacter0000210000000000 NODE_728_2120.050 MULTISPECIES: OmpA family proteinLabrenzia0000201000000000 Suboptimal Proteins NODE_403_50.004flagellinMarinobacter965052242021162918829564230 NODE_1573_140.014membrane proteinMarinobacter22008100210505633 NODE_2368_400.031 hypothetical protein Plav_2552Parvibaculum1205311327642725 NODE_890_90.041type IV pilus secretin PilQChromatiaceae0000000000000021 NODE_1573_440.046 RND family efflux transporter MFP subunitMarinobacter0000200100000000 NODE_304_360.047 DNA-directed RNA polymerase subunit alphaChromatiaceae0000000000110001 NODE_2135_10.047N/A 0000000011000001 Underlined values were also significant using the Fisher’s exact test (FET).

10. 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_3230.026hypothetical proteinKordiimonas-0.30 0.180.40-0.300.180.54-0.30 NODE_2140_90.036 quinoprotein alcohol dehydrogenaseMarinobacter-0.300.880.65-0.300.180.740.40-0.30 0.40-0.30 NODE_22_720.036sugar ABC transporterLabrenzia0.65-0.301.27-0.300.810.650.810.54-0.300.40-0.30 0.88-0.30 Suboptimal Proteins NODE_2368_400.022 hypothetical protein Plav_2552Parvibaculum0.180.32-0.300.710.540.18 0.540.400.880.780.650.400.880.400.74 NODE_1573_140.045membrane proteinMarinobacter0.40 -0.30 0.930.18-0.30 0.401.020.74-0.300.740.810.54 Underlined values were also significant using the Fisher’s exact test (FET).

11. 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 Bacteria60062689.5591.6546526.877.61 Proteobacteria55157282.2483.7563709.4010.25 Alpha21719632.3928.7039325.824.69 Gamma26930440.1544.5137455.526.59 Alteromonadales22325233.2836.90000.00 Rhodobacterales16715324.9322.40020.000.29 Chromatiales330.450.44000.00 Labrenzia13412320.0018.0158538.667.76 Marinobacter22225133.1336.7518220227.1629.58

12. V3 16S rRNAPeptide identifications from Unipept Sample* GammaAlphaAlteromonadaceaeEctothiorhodospiraceaeRhodobacteraceaeGammaAlphaMarinobacterLabrenzia %%%%% S1 R1772331451138.836.832.623.6 S1 R279213147948.720.640.512.1 S2 R1752523521047.825.741.212.8 S2 R2653541242142.4283516.8 S1 R3703041281758.621.350.311.2 S1 R4594128 2045.630.139.119.6 S2 R3534731163051.218.340.59.3 S2 R4752523511049.719.339.89.9 avg. opt712933371444283716 ave. sub673330361851224213 stdev opt9.0 5.710.45.14.76.84.25.3 stdev sub10.5 8.518.59.75.4 5.34.8 Opt. pooled712931381444.836.23721.2 Sub. pooled673329371748.631.34019.6 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 PXD001590 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).

13. 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.

14. 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.

15. 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.

16. 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.

17. Figure S8. Pooled 16S rRNA V3 gene expression analysis at the A) optimal and B) suboptimal electrode potentials using the mothur workflow. A.

18. Figure S8. Pooled 16S rRNA V3 gene expression analysis at the A) optimal and B) suboptimal electrode potentials using the mothur workflow. B.