SUPPORTING INFORMATION

Low temperature acclimation with electrical stimulation enhance the

biocathode functioning stability for antibiotics detoxification

Bin Liang1,2,3, Deyong Kong4,2, Jincai Ma5, Chongqing Wen3, Tong Yuan3, Duu-Jong Lee2,6,

Jizhong Zhou3, Aijie Wang1,2,*

1Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental

Sciences, Chinese Academy of Sciences, Beijing 100085, China2State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of

Technology, Harbin, 150090, China3Institute for Environmental Genomics and Department of Microbiology and Plant Biology,

University of Oklahoma, Norman, OK 73019, USA4Shenyang Academy of Environmental Sciences, Shenyang, 110167, China5College of Environment and Resources, Jilin University, Changchun 130021, China6Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan

*Corresponding author: E-mail: ajwang@rcees.ac.cn (A. Wang). Phone/Fax: +86 10

62915515.

Supporting Information (SI)

Detailed methods, Figures S1-S10 and Tables S1-S3.

MATERIALS AND METHODS

16S rRNA gene Illumina MiSeq sequencing

Six 10-biocathode and S25-biocathode biofilms were collected at the end of each test,

respectively. In order to collect the biomass of cathode biofilms, each biocathode carbon brush in

50 mL centrifuge tube containing 20 mL sterile 50 mM phosphate buffer solution (PBS, pH 7.0)

was vibrated twice using a vortex mixer and then the pooled suspending liquid was centrifuged

at 13,000 g for 20 min. The total genomic DNA was extracted according to a previous

method.1 DNA purity and quantity was determined by a Nano-Drop ND-1000

Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, U.S.) and by a PicoGreen

using FLUOstar Optima (BMG Labtech, Jena, Germany), respectively. For high throughput

sequencing, a primer pair consisting forward primer 515F (5’-GTGCCAGCMGCCGCGG-3’)

and reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’) targeting V4 hypervariable

regions of bacterial 16S rRNA gene was selected.2 The primers were tagged with adapter, pad

and linker sequences. Each barcode sequence was added to the reverse primer for pooling

multiple samples in one run of MiSeq sequencing. All primers were synthesized by Invitrogen

(Carlsbad, CA, U.S.). PCR amplification was performed in triplicate using a Gene Amp PCR-

System® 9700 (Applied Biosystems, Foster City, CA, USA) in a total volume of 25 μl containing

2.5 μl 10×PCR bufferⅡand 0.5 unit of AccuPrime™ Taq DNA Polymerase High Fidelity

(Invitrogen, Carlsbad, CA, USA), 0.4 μM of each primer, and 10 ng template DNA. Thermal

cycling conditions were as follows: an initial denaturation at 94°C for 1 min, and 30 cycles at

94°C for 20 s, 53°C for 25 s, and 68°C for 45 s, with a final extension at 68°C for 10 min.

Following amplification, 2 µl of PCR product was used for agarose gel (1%) detection. The

triplicate PCR reactions for each sample preparation were combined and quantified with

PicoGreen using a FLUOstar Optima (BMG Labtech, Jena, Germany). 200 ng of PCR product

from each sample was taken out and pooled with other samples’ for one sequencing run. The

pooled mixture was purified through QIAquick Gel Extraction Kit (QIAGEN Sciences,

Germantown, MD, USA) and was re-quantified with PicoGreen (BMG Labtech, Jena, Germany).

According to the MiSeqTM Reagent Kit Preparation Guide (Illumina, San Diego, CA, USA),

the purified mixture was diluted and denatured to obtain 8 pM sample DNA library, and mixed

with equal volume of 8 pM PhiX (Illumina, San Diego, CA, USA). Finally, 600 µl of mixture

library was loaded with read 1, read 2 and index sequencing primers 2 on a 500-cycle (2x250

paired ends) kit, and run on a MiSeq at Institute for Environmental Genomics of the University

of Oklahoma.

GeoChip hybridization

A total ten cathode biofilms were selected for the GeoChip analysis (v4.6, the Roche

NimbleGen, Madison, WI, U.S.). Extracted DNA (1.0 μg) from each sample was labeled with

Cy-3 dye using random primers and the Klenow fragment of DNA polymerase I.3 The labeled

DNA products were hybridized to GeoChip v4.6 in a MAUI hybridization station (BioMicro,

Salt Lake City, UT, U.S.) and scanned by a NimbleGen MS200 scanner (Roche, Madison, WI,

U.S.) as described recently.4 The scanned images were processed using a Imagene software (6.1

premium version, Biodiscovery, El Segundo, CA, U.S.). Signal intensities were measured based

on the scanned images, and spots with signal-to-noise ratios (SNR) lower than 2 were removed

before statistical analysis.5

Data analysis

Sequencing data of 16S rRNA gene amplicons was analyzed mainly by removing PhiX

sequences, using the Flash program to join the paired-end reads, trimming ambiguous reads (N)

and sequence length (shorter than 240 bp were removed) and using UCHIME to screen Chimeras

sequentially.6 Afterwards, the 16S rRNA gene sequences were classified into operational

taxonomic units (OTUs) at a 97% sequence similarity threshold.7 After the normalization

process, each sample contained approximately 39000 valid sequences. 16S rRNA gene

sequences were assigned to a taxonomy by the RDP classifier with 50% confidence.8

FIGURES S1-S10 AND TABLES S1-S3

0

5

10

15

20

25

30

35

40

0 12 24 36 48

Conc

entr

ation

of C

AP (m

g/L)

abiotic cathode (25℃)

abiotic cathode (10℃)

(a)

0

20000

40000

60000

80000

100000

120000

0 12 24 36 48

Peak

aera

of C

AP-N

O

(b)

0

20000

40000

60000

80000

100000

120000

140000

0 12 24 36 48

Peak

aera

of A

MCl

2

Time (hour)

(c)

0

20000

40000

60000

80000

100000

120000

140000

0 12 24 36 48

Peak

aera

of A

MCl

Time (hour)

(d)

Figure S1. The CAP reduction (A), the products CAP-NO (B), AMCl2 (C) and AMCl (D)

formation efficiencies with the abiotic cathodes under 10°C and 25°C were compared.

a b c d

-1.2 -0.8 -0.4 0.0 0.4

Cur

rent

(mA

)

-15

-12

-9

-6

-3

0

3

S25-biocathode10-biocathode

Cathode potential vs. SHE (V)

e

Figure S2. The CSLM micrographs of the biocathode communities enriched at 10°C (ab) and

switched 25°C (cd). Cathode biofilm communities were stained with SYTO 9 (green indicates

high growth activity cathodophilic microbes) and propidium iodide (red notes low growth

activity cathodophilic microbes). The cyclic voltammograms of the 10-biocathode and S25-

biocathode for the CAP reduction (e). The concentrations of CAP and glucose was 30 and 600

mg/L, respectively. The SCE was the reference electrode. Cyclic voltammograms were recorded

with the scan rate of 5 mV/s at 25°C.

10→25℃

10℃Figure S3. Hierarchical clustering analysis of the identified OTUs from the 10-biocathode and

S25-biocathode communities.

Carbon cycling 28.30%

Metal resistance 17.62%

Stress 13.78%

Organic remediation

12.46%

Nitrogen cycling 8.81%

Sulfur cycling 5.44%

Antibiotic resistance 2.44%

Phosphorus utiliza tion 1.59%

Electrons transfer 1.03% Others 8.53%

A

Carbon cycling 30.05%

Metal resistance 16.90%

Stress 13.06%

Organic remediation

14.64%

Nitrogen cycling 7.05%

Sulfur cycling 4.16%

Antibiotic resistance 2.12%

Phosphorus utilization 3.30%

Electrons transfer 0.50% Others 8.22%

B

Figure S4. The proportion of unique functional genes from the 10-biocathode (1067 genes) and S25-biocathode (2113 genes) communities.

Gammaproteobacteria Bacilli

Clostridia

Deltaproteobacteria

Bacteroidia

Methanomicrobia Others

Cla

ss (%

)

0

25

50

75

100Proteobacteria

Firmicutes

Bacteroidetes

Euryarchaeota Others

Phy

lum

(%)

0

5

20406080

100S25-biocathode10-biocathode

**

a

b

**

Figure S5. The microbial community structure differences between the 10-biocathode and S25-

biocathode communities at phylum (A) and class (B) level. ***, ** and * denotes significance at

0.01, 0.05, and 0.10 test levels, respectively.

EPS synthesis type IV pilin

Nor

mal

ized

ave

rage

sig

nal i

nten

sity

0

30

60

90

120

150

180

S25-biocathode10-biocathode

Figure S6. Comparison of the relative abundance of the EPS synthesis and type IV pilin related

genes from the 10-biocathode and S25-biocathode communities.

25oC10oC

Figure S7. Hierarchical clustering of cytochrome c and hydrogenase genes that with significant

intensity differences between the two biocathode groups. The genes annotation information are

from NCBI website (http://www.ncbi.nlm.nih.gov/).

4Fe_4S_ferredoxin

Fe_S_cluster_binding_protein

NADH_quinone_oxidoreductase

NADH_ubiquinone_oxidoreductase

cytochrome_b

ATP_synthase

ferredoxin_oxidoreductase

Nor

mal

ized

ave

rage

sig

nal i

nten

sity

0

4

5

10

15

20S25-biocathode10-biocathode

*

Figure S8. Comparison of the relative abundance of the electrons transport respiratory chain

related genes from the 10-biocathode and S25-biocathode communities.

** *

**

***

*

**

**

dsrA dsrB 16S rRNA

dsrA

and

dsr

B a

bund

ance

s

0

7

14

21

16S

rRN

A g

ene

(%)

0.0

0.8

1.6

2.4

S25-biocathode10-biocathode

Figure S9. Heatmap showing the abundance and diversity of Desulfovibrio sulfite reductase

genes (dsrA and dsrB) in the S25-biocathode (5 samples in the left) and 10-biocathode

communities (5 samples in the right). The inset histogram indicated the total abundances for

dsrA, dsrB and Desulfovibrio sp. 16S rRNA genes. The significant difference of each gene

intensity between the two biocathode groups were also shown.

sigma_24sigma_32

sigma_38sigma_70N

orm

aliz

ed a

vera

ge s

igna

l int

ensi

ty

0

50

100

250300350400450500

dnaKgroEL

grpE hrcAgroES totalN

orm

aliz

ed a

vera

ge s

igna

l int

ensi

ty

01020304050

90

120

150

Tota

l nor

mal

ized

sig

nal i

nten

sity

0

50

100

150

200

250

300

350S25-biocathode10-biocathode

** **

**

*

**

*

**a b

Figure S10. Comparison of the relative abundance of the heat shock responses related genes

from the 10-biocathode and S25-biocathode communities.

Table S1. Significance tests of the overall microbial community structure between the 10-

biocathode and S25-biocathode with three different statistical approaches. Any P value ≤ 0.05

was bolded.

Functionalgene categories

Jaccard Dissimilarity Bray Curtis DissimilarityAdonis

F PAnosim

R PMRPP

δ PAdonis

F PAnosim

R PMRPP

δ P

All functional genes 3.02 0.001 0.47 0.021 0.19

0.014 3.09 0.009 0.44 0.033 0.11 0.020

Antibiotic resistance 3.05 0.001 0.43 0.017 0.22

0.013 3.26 0.010 0.42 0.035 0.13 0.022

Carbon cycling 2.95 0.011 0.42 0.019 0.17

0.012 2.97 0.014 0.39 0.048 0.10 0.019

Metal resistance 3.26 0.002 0.52 0.014 0.19

0.012 3.34 0.012 0.50 0.030 0.11 0.005

Nitrogen cycling 2.77 0.011 0.44 0.017 0.17

0.015 2.74 0.013 0.40 0.050 0.10 0.020

Organic remediation 3.10 0.001 0.48 0.020 0.22

0.015 3.23 0.014 0.47 0.018 0.13 0.015

Phosphorus utilization 3.88 0.001 0.66 0.014 0.14

0.012 3.76 0.005 0.63 0.012 0.08 0.013

Sulfur cycling 2.68 0.010 0.45 0.022 0.19

0.015 2.67 0.006 0.39 0.018 0.11 0.009

Stress 2.92 0.001 0.47 0.013 0.25

0.018 3.13 0.013 0.45 0.023 0.15 0.019

e- transfer related genes# 2.84 0.034 0.36 0.027 0.20

0.025 2.89 0.061 0.34 0.060 0.12 0.064

Cytochrome c 2.79 0.054 0.35 0.054 0.19

0.036 2.75 0.050 0.32 0.063 0.11 0.060

Hydrogenase 3.24 0.009 0.23 0.035 0.25

0.020 3.73 0.007 0.23 0.044 0.15 0.028

gyrB 2.83 0.010 0.42 0.027 0.24

0.012 2.97 0.009 0.41 0.032 0.14 0.023

16S rRNA 4.02 0.001 0.99 0.002 0.60

0.002 7.71 0.005 0.69 0.009 0.50 0.009

#Electron transfer related genes included cytochrome (b, c and bd) genes, ferredoxin

oxidoreductase, 4Fe_4S ferredoxin, Fe_S cluster binding protein and NADH quinone/ubiquinone

oxidoreductase in electron transport respiratory chain and hydrogenase genes as well as

cytochrome p450 genes.

Table S2. Comparison of the microbial α diversity between the 10-biocathode and S25-

biocathode communities. Any P value <0.1 is bolded.

Indices16S rRNA gene Functional genes

25oC 10oC P 25oC 10oC PH 2.16±0.91 2.67±0.43 0.25 10.60±0.04 10.54±0.04 0.071/D 3.45±2.60 5.71±2.05 0.13 39904.0±1433.8 37789.2±1673.5 0.06Richness 688.2±196.0 571.2±114.

40.24 40178.8±1441.6 38055.6±1680.5 0.07

Table S3. Relatively abundances of nitroaromatics reducers in the S25-biocathode and 10-

biocathode communities based on the phylogenetic classification of the 16S rRNA gene

sequences. The significant difference of nitroaromatics reducers abundances between the S25-

biocathode and 10-biocathode communities were analyzed by the two-tailed unpaired t-test

(n=6). Any P value <0.1 was bolded.

Phylum Class Genus (%) S25-biocathode 10-biocathode P value ReferencesProteobacteria

γ-proteobacteria Raoultella 62.06 ± 21.72 15.39 ± 23.26 0.0005 9

Firmicutes Bacilli Enterococcus 9.09 ± 10.16 2.15 ± 1.12 0.160 10

Proteobacteria

γ-proteobacteria Aeromonas 0.41 ± 0.08 33.16 ± 19.82 0.010 11

Proteobacteria

δ-proteobacteria Desulfovibrio 0.99 ± 0.72 1.26 ± 0.99 0.611 12

Proteobacteria

γ-proteobacteria Citrobacter 1.33 ± 1.41 3.13 ± 2.18 0.201 13

Firmicutes Clostridia Clostridium 0.100 ± 0.089 0.735 ± 0.607 0.050 12

Proteobacteria

γ-proteobacteria Escherichia 0.169 ± 0.106 0.015 ± 0.006 0.016 14

Proteobacteria

γ-proteobacteria Pseudomonas 0.149 ± 0.246 0.363 ± 0.368 0.266 12

Proteobacteria

γ-proteobacteria Enterbacter 0.059 ± 0.053 0.309 ± 0.418 0.205 14

Proteobacteria

β-proteobacteria Comamonas 0.028 ± 0.027 0.193 ± 0.191 0.089 15

Proteobacteria

γ-proteobacteria Klebsiella 0.015 ± 0.017 0.004 ± 0.006 0.184 14

Firmicutes Bacilli Lactococcus 0.991 ± 1.477 0.129 ± 0.157 0.213 16

Proteobacteria

γ-proteobacteria Shewanella 0.0008 ± 0.0021 0.0024 ± 0.0047

0.485 17

Euryarchaeota Methanococci Methanococcus 0.0008 ± 0.0013 0.0004 ± 0.0010

0.548 12

Actinobacteria Actinobacteria Mycobacterium 0.015 ± 0.019 0.002 ± 0.004 0.173 15

Actinobacteria Actinobacteria Rhodococcus 0.181 ± 0.187 0.075 ± 0.077 0.243 15

Bacteroidetes Bacteroidia Bacteroides 0.039 ± 0.037 0.012 ± 0.010 0.141 12

Euryarchaeota Methanobacteria Methanobacterium

0.372 ± 0.643 0.00 ± 0.00 0.216 12

Bacteroidetes Flavobacteria Flavobacterium 0.0046 ± 0.0040 0.0018 ± 0.0014

0.153 15

Firmicutes Clostridia Eubacterium 0.00 ± 0.00 0.011 ± 0.020 0.230 15

Firmicutes Bacilli Bacillus 0.00 ± 0.00 0.0013 ± 0.0031

0.363 14

Proteobacteria

γ-proteobacteria Acinetobacter 0.043 ± 0.035 0.034 ± 0.021 0.589 18

Proteobacteria

δ-proteobacteria Geobacter 0.0029 ± 0.0025 0.0044± 0.0028 0.353 17

Total abundances 75.99 ± 11.75 60.51 ± 21.74 0.165

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