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 V52 hours

Section 1. 16S rRNA V3 gene expression analysis

Section 4. Cell counting by flow cytometrySection 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. 2x105 cells

88 hours

S1R1 S1R2 S2R1 S2R2 S1R4 S2R3 S2R4S1R3

S1R1

S1R2

S2R1

S2R2

S1R1

S1R2

S2R1

S2R2

S1R3

S1R4

S2R3

S2R4

S1R3

S1R4

S2R3

S2R4

PT SET PT SET PT SET PT SET PT SET PT SET PT SET PT SET

S1R1

S1R2

S2R1

S2R2

S1R1

S1R2

S2R1

S2R2

S1R3

S1R4

S2R3

S2R4

S1R3

S1R4

S2R3

S2R4

Protein extraction with B-PERGel 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 0.610 V to 0.260 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 0.610 V to +0.260 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.

Sample cells/6 x 6 cm electrode

Hours to Imax Max current (Imax) EM

S1 R1 2.1E+06 35.5 -9.48 0.47S1 R2 1.6E+06 56.5 -51.3 0.5S1 R3 1.5E+06 40.5 -26.7 0.48S1 R4 9.4E+05 43 -20.4 0.510S2 R1 8.8E+05 44.2 -16.7 0.48S2 R2 4.8E+05 29.2 -4.5 0.47S2 R3 9.4E+05 37.8 -11.3 0.47S2 R4 2.8E+06 40.9 -13.3 0.510

Table S2. Peptide and protein identifications in porcine trypsin (PT) versus Streptomyces erythraeus trypsin (SET) samples.

Category Sample #Prot #IDs #Spec %IDsPT S1R1 PT 63 243 2989 8.1%SET S1R1 SET 347 1028 3418 30.1%PT S1R2 PT 148 602 4815 12.5%SET S1R2 SET 246 694 4313 16.1%PT S1R3 PT 75 362 4616 7.8%SET S1R3 SET 121 376 3642 10.3%PT S1R4 PT 217 738 6230 11.8%SET S1R4 SET 337 1180 7084 16.7%PT S2R1 PT 87 273 4891 5.6%SET S2R1 SET 226 485 6708 7.2%PT S2R2 PT 88 241 3933 6.1%SET S2R2 SET 279 669 4812 13.9%PT S2R3 PT 111 429 5109 8.4%SET S2R3 SET 308 724 6912 10.5%PT S2R4 PT 71 218 4857 4.5%SET S2R4 SET 220 493 4797 10.3%

Table S3. Spectral counts for proteins identified as associated with either the optimal or suboptimal potential using the Fisher’s exact test (FET).

    opt opt opt opt opt opt opt opt subopt subopt subopt subopt subopt subopt subopt subopt

ORF ID Fisher 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 <0.0001 0 7 4 0 1 5 2 0 0 0 0 0 0 2 0 0NODE_2320_41 <0.0001 0 1 0 0 9 13 9 8 0 0 0 0 0 6 4 1NODE_22_72 <0.0001 4 0 18 0 6 4 6 3 0 2 0 0 0 7 0 0NODE_1148_52 0.00067 0 7 4 0 0 7 8 4 0 2 0 0 0 4 2 2NODE_837_61 0.0042 0 32 8 6 24 35 30 32 5 13 12 10 6 37 23 22NODE_3683_49 0.0063 0 0 0 0 8 2 1 6 0 0 0 0 0 5 0 0NODE_277_323 0.0066 0 0 0 1 2 0 1 3 0 0 0 0 0 0 0 0NODE_277_255 0.0089 0 0 0 0 6 0 0 3 0 0 0 0 0 0 1 0NODE_837_51 0.011 0 0 0 0 4 8 3 4 0 0 0 0 0 3 2 2NODE_181_44 0.026 0 9 4 4 9 12 4 10 1 3 5 1 9 5 7 4NODE_728_49 0.028 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0NODE_2170_44 0.028 4 0 0 6 23 2 1 11 0 1 0 0 12 7 6 5NODE_16387_1 0.028 0 0 0 0 4 0 0 1 0 0 0 0 0 0 0 0NODE_893_1 0.028 5 3 2 2 51 14 9 11 1 15 6 0 8 26 12 7NODE_240_101 0.047 0 0 0 0 5 1 0 2 0 0 0 0 0 2 0 0NODE_15807_1 0.047 4 3 1 0 0 0 0 0 0 2 0 0 0 0 0 0

Suboptimal Proteins

NODE_403_5 <0.0001 9 6 5 0 52 24 20 21 16 29 18 8 29 56 42 30NODE_1573_14 0.0025 2 2 0 0 8 1 0 0 2 10 5 0 5 6 3 3NODE_2943_37 0.0026 0 5 0 0 0 2 1 2 0 0 0 0 0 7 12 10NODE_1848_121 0.0037 26 8 46 28 0 0 0 0 40 8 59 51 0 0 0 0NODE_2368_40 0.0052 1 2 0 5 3 1 1 3 2 7 6 4 2 7 2 5NODE_1173_134 0.0072 0 0 0 0 1 0 0 0 0 5 0 0 0 5 0 0NODE_1547_6 0.0081 0 1 0 0 0 1 0 0 0 0 1 0 0 0 7 4NODE_5518_1 0.0086 0 2 0 0 4 0 1 0 4 9 1 0 0 4 0 3NODE_3258_14 0.014 1 1 0 0 0 0 0 0 0 8 0 0 0 3 0 0NODE_2048_7 0.023 0 0 0 0 0 0 1 0 0 0 0 0 0 0 4 4NODE_307_76 0.023 0 0 0 0 0 0 0 1 0 0 0 0 0 2 3 3NODE_83_108 0.023 0 0 0 0 0 1 0 0 0 0 0 0 0 2 2 4NODE_1775_17 0.023 0 0 0 0 7 4 1 0 5 2 0 0 6 7 3 3NODE_6881_4 0.025 2 29 4 7 13 19 7 18 13 8 26 8 13 20 28 21NODE_508_70 0.028 0 0 0 0 2 2 3 4 0 0 0 0 6 11 6 1

NODE_3683_20 0.028 0 11 6 5 5 30 22 20 5 33 6 1 16 41 15 17NODE_2533_8 0.032 4 6 0 0 2 0 0 0 6 17 2 0 0 0 0 0NODE_2210_36 0.033 0 1 0 1 0 0 3 1 0 2 1 0 0 4 5 4NODE_476_49 0.035 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0NODE_6203_4 0.04 0 0 0 0 1 0 0 0 0 2 0 0 0 5 0 0NODE_522_69 0.042 0 2 0 0 4 4 3 4 4 5 2 0 4 9 5 2

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 opt opt opt opt opt opt opt subopt

subopt

subopt

subopt

subopt

subopt

subopt

subopt

ORF ID BB p-value NCBI annotation Bin organism 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_277_323 0.004 hypothetical protein Kordiimonas 0 0 0 1 2 0 1 3 0 0 0 0 0 0 0 0

NODE_2140_9 0.014quinoprotein alcohol dehydrogenase Marinobacter 0 7 4 0 1 5 2 0 0 0 0 0 0 2 0 0

NODE_779_155 0.016 hypothetical protein Kordiimonas 0 0 0 0 2 1 0 1 0 0 0 0 0 0 0 0NODE_7231_10 0.020 hypothetical protein Chromatiaceae 0 1 0 0 0 2 1 0 0 0 0 0 0 0 0 0NODE_22_72 0.021 sugar ABC transporter Labrenzia 4 0 18 0 6 4 6 3 0 2 0 0 0 7 0 0

NODE_5018_11 0.036acylneuraminate cytidylyltransferase Chromatiaceae 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0

NODE_1148_45 0.045 ectoine synthase Marinobacter 0 0 0 0 2 1 0 0 0 0 0 0 0 0 0 0

NODE_728_212 0.050MULTISPECIES: OmpA family protein Labrenzia 0 0 0 0 2 0 1 0 0 0 0 0 0 0 0 0

Suboptimal Proteins  NODE_403_5 0.004 flagellin Marinobacter 9 6 5 0 52 24 20 21 16 29 18 8 29 56 42 30

NODE_1573_14 0.014 membrane protein Marinobacter 2 2 0 0 8 1 0 0 2 10 5 0 5 6 3 3

NODE_2368_40 0.031hypothetical protein Plav_2552 Parvibaculum 1 2 0 5 3 1 1 3 2 7 6 4 2 7 2 5

NODE_890_9 0.041 type IV pilus secretin PilQ Chromatiaceae 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1

NODE_1573_44 0.046RND family efflux transporter MFP subunit Marinobacter 0 0 0 0 2 0 0 1 0 0 0 0 0 0 0 0

NODE_304_36 0.047DNA-directed RNA polymerase subunit alpha Chromatiaceae 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 1

NODE_2135_1 0.047 N/A N/A 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 1

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 opt opt opt opt opt opt opt subopt

subopt

subopt

subopt

subopt

subopt

subopt

subopt

ORF ID t-test p-value

NCBI annotation Bin organism 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 proteinsNODE_277_323 0.026 hypothetical protein Kordiimonas -0.30 -0.30 -0.30 0.18 0.40 -0.30 0.18 0.54 -0.30 -0.30 -0.30 -0.30 -0.30 -0.30 -0.30 -0.30

NODE_2140_9 0.036quinoprotein alcohol dehydrogenase Marinobacter -0.30 0.88 0.65 -0.30 0.18 0.74 0.40 -0.30 -0.30 -0.30 -0.30 -0.30 -0.30 0.40 -0.30 -0.30

NODE_22_72 0.036 sugar ABC transporter Labrenzia 0.65 -0.30 1.27 -0.30 0.81 0.65 0.81 0.54 -0.30 0.40 -0.30 -0.30 -0.30 0.88 -0.30 -0.30Suboptimal Proteins

NODE_2368_40 0.022hypothetical protein Plav_2552 Parvibaculum 0.18 0.32 -0.30 0.71 0.54 0.18 0.18 0.54 0.40 0.88 0.78 0.65 0.40 0.88 0.40 0.74

NODE_1573_14 0.045 membrane protein Marinobacter 0.40 0.40 -0.30 -0.30 0.93 0.18 -0.30 -0.30 0.40 1.02 0.74 -0.30 0.74 0.81 0.54 0.54

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 peptidesspecific to this or lower level

% of all matched peptides

Number of peptidesspecific to only this

level% of matched

peptidesoptimal suboptimal optimal suboptimal optimal suboptimal optimal suboptimal

Bacteria 600 626 89.55 91.65 46 52 6.87 7.61Proteobacteria 551 572 82.24 83.75 63 70 9.40 10.25Alpha 217 196 32.39 28.70 39 32 5.82 4.69Gamma 269 304 40.15 44.51 37 45 5.52 6.59Alteromonadales 223 252 33.28 36.90 0 0 0.00 0.00Rhodobacterales 167 153 24.93 22.40 0 2 0.00 0.29Chromatiales 3 3 0.45 0.44 0 0 0.00 0.00Labrenzia 134 123 20.00 18.01 58 53 8.66 7.76Marinobacter 222 251 33.13 36.75 182 202 27.16 29.58

 

V3 16S rRNA Peptide identifications from Unipept

Sample* Gamma Alpha Alteromonadaceae Ectothiorhodospiraceae Rhodobacteraceae Gamma Alpha Marinobacter Labrenzia

% % % % % % % % %S1 R1 77 23 31 45 11 38.8 36.8 32.6 23.6S1 R2 79 21 31 47 9 48.7 20.6 40.5 12.1S2 R1 75 25 23 52 10 47.8 25.7 41.2 12.8S2 R2 65 35 41 24 21 42.4 28 35 16.8

S1 R3 70 30 41 28 17 58.6 21.3 50.3 11.2S1 R4 59 41 28 28 20 45.6 30.1 39.1 19.6S2 R3 53 47 31 16 30 51.2 18.3 40.5 9.3S2 R4 75 25 23 51 10 49.7 19.3 39.8 9.9avg. opt 71 29 33 37 14 44 28 37 16ave. sub 67 33 30 36 18 51 22 42 13stdev opt 9.0 9.0 5.7 10.4 5.1 4.7 6.8 4.2 5.3stdev sub 10.5 10.5 8.5 18.5 9.7 5.4 5.4 5.3 4.8

Opt. pooled 71 29 31 38 14 44.8 36.2 37 21.2

Sub. pooled 67 33 29 37 17 48.6 31.3 40 19.6

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).

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.