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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: [email protected] (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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