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
Prepare extract, count cells.
Inoculate ca. 2x105 cells
S1 and S2 all reactors, grow at 0.310 V
88 hours
S1 and S2 all reactors, record CV
S1 (R3/R4), S2 (R3/R4)
S1 (R1/R2), S2 (R1/R2)
switched to 0.470 V
returned to 0.310 V
52 hours
Harvest Biocathode-MCL biofilms from electrodes and divide into 4 sections:
Section 1. 16S rRNA
V3 gene expression
analysis
Section 2. Protein
extraction and digestion
by PT
Section 3. Protein
extraction and
digestion by SET
LC-MS/MS qualitative shotgun proteomics
Integrated characterization of Biocathode-MCL biofilm microbiome
Section 4. Cell counting
by flow cytometry
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.
Suboptimal biocathodes
Optimal biocathodes
S1R1
S1R2
S2R1
S1R3
S2R2
S1R4
S2R3
S2R4
Protein extraction with B-PER
Gel electrophoresis
S1R S1R S1R S1R S2R S2R S2R S2R
1
1
2
2
1
1
2
2
PT
SET
PT
SET
PT SET
PT
S1R S1R S1R S1R S2R S2R S2R S2R
3
3
4
4
3
3
4
4
Protein digestion in gel
SET
PT
S1R S1R S1R S1R S2R S2R S2R S2R
1
1
2
2
1
1
2
2
SET PT
SET
PT
SET PT SET
S1R S1R S1R S1R S2R S2R S2R S2R
3
3
4
4
3
3
4
4
LC-MS/MS analysis – Qstar Elite
Database search – Mascot and X!Tandem
Protein identification validation and quantitation - Scaffold
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
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.
B
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
S1 R1
S1 R2
S1 R3
S1 R4
S2 R1
S2 R2
S2 R3
S2 R4
2.1E+06
1.6E+06
1.5E+06
9.4E+05
8.8E+05
4.8E+05
9.4E+05
2.8E+06
Hours to Imax Max current (Imax)
35.5
56.5
40.5
43
44.2
29.2
37.8
40.9
-9.48
-51.3
-26.7
-20.4
-16.7
-4.5
-11.3
-13.3
EM
0.47
0.5
0.48
0.510
0.48
0.47
0.47
0.510
Table S2. Peptide and protein identifications in porcine trypsin (PT) versus Streptomyces erythraeus trypsin (SET) samples.
Category
PT
SET
PT
SET
PT
SET
PT
SET
PT
SET
PT
SET
PT
SET
PT
SET
Sample
S1R1 PT
S1R1 SET
S1R2 PT
S1R2 SET
S1R3 PT
S1R3 SET
S1R4 PT
S1R4 SET
S2R1 PT
S2R1 SET
S2R2 PT
S2R2 SET
S2R3 PT
S2R3 SET
S2R4 PT
S2R4 SET
#Prot
63
347
148
246
75
121
217
337
87
226
88
279
111
308
71
220
#IDs
243
1028
602
694
362
376
738
1180
273
485
241
669
429
724
218
493
#Spec
2989
3418
4815
4313
4616
3642
6230
7084
4891
6708
3933
4812
5109
6912
4857
4797
%IDs
8.1%
30.1%
12.5%
16.1%
7.8%
10.3%
11.8%
16.7%
5.6%
7.2%
6.1%
13.9%
8.4%
10.5%
4.5%
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
ORF ID
opt
opt
opt
opt
opt
opt
opt subopt subopt subopt subopt subopt subopt subopt subopt
Fisher p- S1R1 S1R2 S2R1 S2R2 S1R1 S1R2 S2R1 S2R2 S1R3 S1R4 S2R3 S2R4 S1R3
value
PT
PT
PT
PT SET SET SET SET PT
PT
PT
PT
SET
S1R4
SET
S2R3 S2R4
SET SET
Optimal Proteins
NODE_2140_9
NODE_2320_41
NODE_22_72
NODE_1148_52
NODE_837_61
NODE_3683_49
NODE_277_323
NODE_277_255
NODE_837_51
NODE_181_44
NODE_728_49
NODE_2170_44
NODE_16387_1
NODE_893_1
NODE_240_101
NODE_15807_1
Suboptimal Proteins
<0.0001
<0.0001
<0.0001
0.00067
0.0042
0.0063
0.0066
0.0089
0.011
0.026
0.028
0.028
0.028
0.028
0.047
0.047
0
0
4
0
0
0
0
0
0
0
0
4
0
5
0
4
7
1
0
7
32
0
0
0
0
9
0
0
0
3
0
3
4
0
18
4
8
0
0
0
0
4
0
0
0
2
0
1
0
0
0
0
6
0
1
0
0
4
0
6
0
2
0
0
1
9
6
0
24
8
2
6
4
9
5
23
4
51
5
0
5
13
4
7
35
2
0
0
8
12
0
2
0
14
1
0
2
9
6
8
30
1
1
0
3
4
0
1
0
9
0
0
0
8
3
4
32
6
3
3
4
10
0
11
1
11
2
0
0
0
0
0
5
0
0
0
0
1
0
0
0
1
0
0
0
0
2
2
13
0
0
0
0
3
0
1
0
15
0
2
0
0
0
0
12
0
0
0
0
5
0
0
0
6
0
0
0
0
0
0
10
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
6
0
0
0
0
9
0
12
0
8
0
0
2
6
7
4
37
5
0
0
3
5
0
7
0
26
2
0
0
4
0
2
23
0
0
1
2
7
0
6
0
12
0
0
0
1
0
2
22
0
0
0
2
4
0
5
0
7
0
0
NODE_403_5
NODE_1573_14
NODE_2943_37
NODE_1848_121
NODE_2368_40
NODE_1173_134
NODE_1547_6
NODE_5518_1
NODE_3258_14
NODE_2048_7
NODE_307_76
NODE_83_108
NODE_1775_17
NODE_6881_4
NODE_508_70
<0.0001
0.0025
0.0026
0.0037
0.0052
0.0072
0.0081
0.0086
0.014
0.023
0.023
0.023
0.023
0.025
0.028
9
2
0
26
1
0
0
0
1
0
0
0
0
2
0
6
2
5
8
2
0
1
2
1
0
0
0
0
29
0
5
0
0
46
0
0
0
0
0
0
0
0
0
4
0
0
0
0
28
5
0
0
0
0
0
0
0
0
7
0
52
8
0
0
3
1
0
4
0
0
0
0
7
13
2
24
1
2
0
1
0
1
0
0
0
0
1
4
19
2
20
0
1
0
1
0
0
1
0
1
0
0
1
7
3
21
0
2
0
3
0
0
0
0
0
1
0
0
18
4
16
2
0
40
2
0
0
4
0
0
0
0
5
13
0
29
10
0
8
7
5
0
9
8
0
0
0
2
8
0
18
5
0
59
6
0
1
1
0
0
0
0
0
26
0
8
0
0
51
4
0
0
0
0
0
0
0
0
8
0
29
5
0
0
2
0
0
0
0
0
0
0
6
13
6
56
6
7
0
7
5
0
4
3
0
2
2
7
20
11
42
3
12
0
2
0
7
0
0
4
3
2
3
28
6
30
3
10
0
5
0
4
3
0
4
3
4
3
21
1
0.028
0.032
0.033
0.035
0.04
0.042
0
4
0
0
0
0
11
6
1
0
0
2
6
0
0
0
0
0
5
0
1
0
0
0
5
2
0
0
1
4
30
0
0
0
0
4
22
0
3
0
0
3
20
0
1
0
0
4
5
6
0
0
0
4
33
17
2
0
2
5
6
2
1
0
0
2
1
0
0
0
0
0
16
0
0
0
0
4
41
0
4
5
5
9
15
0
5
0
0
5
17
0
4
0
0
2
NODE_3683_20
NODE_2533_8
NODE_2210_36
NODE_476_49
NODE_6203_4
NODE_522_69
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).
opt
opt
opt
opt
opt
opt
opt
Kordiimonas
0
0
0
1
2
0
1
3
0
0
0
0
0
0
0
0
Marinobacter
Kordiimonas
Chromatiaceae
Labrenzia
0
0
0
4
7
0
1
0
4
0
0
18
0
0
0
0
1
2
0
6
5
1
2
4
2
0
1
6
0
1
0
3
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
7
0
0
0
0
0
0
0
0
Chromatiaceae
Marinobacter
0
0
1
0
1
0
1
0
0
2
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Labrenzia
0
0
0
0
2
0
1
0
0
0
0
0
0
0
0
0
Marinobacter
9
6
5
0
52
24
20
21
16
29
18
8
29
56
42
30
membrane protein
Marinobacter
hypothetical protein
NODE_2368_40
0.031
Plav_2552
Parvibaculum
NODE_890_9
0.041
type IV pilus secretin PilQ Chromatiaceae
RND family efflux
NODE_1573_44
0.046
transporter MFP subunit
Marinobacter
DNA-directed RNA
Underlined
values were also
significant
using the
Fisher’s
exact test
(FET).
NODE_304_36
0.047
polymerase
subunit
alpha
Chromatiaceae
NODE_2135_1
0.047
N/A
N/A
2
2
0
0
8
1
0
0
2
10
5
0
5
6
3
3
1
0
2
0
0
0
5
0
3
0
1
0
1
0
3
0
2
0
7
0
6
0
4
0
2
0
7
0
2
2
5
1
0
0
0
0
2
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
1
0
1
0
0
0
0
0
0
0
1
1
ORF ID
BB p-value
NCBI annotation
Optimal proteins
NODE_277_323
0.004
NODE_2140_9
NODE_779_155
NODE_7231_10
NODE_22_72
0.014
0.016
0.020
0.021
NODE_5018_11
NODE_1148_45
0.036
0.045
NODE_728_212
Suboptimal
Proteins
NODE_403_5
0.050
hypothetical protein
quinoprotein alcohol
dehydrogenase
hypothetical protein
hypothetical protein
sugar ABC transporter
acylneuraminate
cytidylyltransferase
ectoine synthase
MULTISPECIES: OmpA
family protein
0.004
flagellin
NODE_1573_14
0.014
Bin organism
opt subop subop subo subop subo subop subop subo
t
t
pt
t
pt
t
t
pt
S1R1 S1R2 S2R1 S2R2 S1R1 S1R2 S2R1 S2R2 S1R3 S1R4 S2R3 S2R4 S1R3 S1R4 S2R3 S2R4
PT PT PT PT SET SET SET SET PT
PT PT PT SET SET SET SET
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
ORF ID
Optimal proteins
NODE_277_323
NODE_2140_9
NODE_22_72
Suboptimal Proteins
NODE_2368_40
NODE_1573_14
NCBI annotation
Bin organism
opt subop subop subo subo subo subo subop subo
t
t
pt
pt
pt
pt
t
pt
S1R S1R2 S2R S2R2 S1R1 S1R S2R1 S2R2 S1R3 S1R4 S2R3 S2R4 S1R S1R4 S2R3 S2R
1 PT PT 1 PT PT SET 2 SET SET PT PT PT PT
3 SET SET 4
SET
SET
SET
0.026 hypothetical protein
quinoprotein alcohol
0.036 dehydrogenase
0.036 sugar ABC transporter
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
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
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 -0.30
hypothetical protein
0.022 Plav_2552
0.045 membrane protein
Parvibaculum
Marinobacter
t-test pvalue
Underlined values were also significant using the Fisher’s exact test (FET).
opt
opt
opt
opt
opt
opt
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
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
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.
Bacteria
Proteobacteria
Alpha
Gamma
Alteromonadales
Rhodobacterales
Chromatiales
Labrenzia
Marinobacter
Number of peptides
specific to this
% of all matched
or lower level
peptides
optimal suboptimal optimal suboptimal
600
626
89.55
91.65
551
572
82.24
83.75
217
196
32.39
28.70
269
304
40.15
44.51
223
252
33.28
36.90
167
153
24.93
22.40
3
3
0.45
0.44
134
123
20.00
18.01
222
251
33.13
36.75
Number of peptides
specific to only this
% of matched
level
peptides
optimal suboptimal optimal suboptimal
46
52
6.87
7.61
63
70
9.40
10.25
39
32
5.82
4.69
37
45
5.52
6.59
0
0
0.00
0.00
0
2
0.00
0.29
0
0
0.00
0.00
58
53
8.66
7.76
182
202
27.16
29.58
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).
V3 16S rRNA
Peptide identifications from Unipept
Sample*
S1 R1
S1 R2
S2 R1
S2 R2
Gamma
%
77
79
75
65
Alpha
%
23
21
25
35
Alteromonadaceae
%
31
31
23
41
Ectothiorhodospiraceae Rhodobacteraceae
%
%
45
11
47
9
52
10
24
21
Gamma
%
38.8
48.7
47.8
42.4
Alpha
%
36.8
20.6
25.7
28
Marinobacter
%
32.6
40.5
41.2
35
Labrenzia
%
23.6
12.1
12.8
16.8
S1 R3
S1 R4
S2 R3
S2 R4
avg. opt
ave. sub
stdev opt
stdev sub
70
59
53
75
71
67
9.0
10.5
30
41
47
25
29
33
9.0
10.5
41
28
31
23
33
30
5.7
8.5
28
28
16
51
37
36
10.4
18.5
17
20
30
10
14
18
5.1
9.7
58.6
45.6
51.2
49.7
44
51
4.7
5.4
21.3
30.1
18.3
19.3
28
22
6.8
5.4
50.3
39.1
40.5
39.8
37
42
4.2
5.3
11.2
19.6
9.3
9.9
16
13
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
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.
C.
B.
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.