Cover sheet for SI
Authors:
Yi-Hao Luo, Ran Chen, Li-Lian Wen, Fan Meng, Yin Zhang, Chun-Yu Lai, Bruce E.
Rittmann, He-Ping Zhao*, Ping Zheng
Manuscript title:

Complete perchlorate reduction using methane as the sole electron donor

and carbon source
Number of pages:
Number of table:
Number of figures:

10
5
3

S1

Supplementary information (SI) for manuscript
Complete perchlorate reduction using methane as the sole electron
donor and carbon source
Yi-Hao Luo1, †, Ran Chen1, †, Li-Lian Wen1, Fan Meng1, Yin Zhang1, Chun-Yu Lai1, Bruce E.
Rittmann2, He-Ping Zhao1, *, Ping Zheng1

1. MOE Key Lab of Environmental Remediation and Ecosystem Health, College of
Environmental and Resource Science, Zhejiang University, Hangzhou, China.
2. Swette Center for Environmental Biotechnology, Biodesign Institute at Arizona State
University, P.O. Box 875701, Tempe, Arizona 85287-5701

* Correspondence to Dr. He-Ping Zhao. Tel: 0086-571-88982739, Fax: 0086-571-88982739, E-mail:
zhaohp@zju.edu.cn
† Contribute equally.

S2

Table S1. Equations applied for the CH4-permeation calculations:

in which α = k1KmZlfH(dm-Zm), β = k2DlfZm(dm+Zlf)
Pm-lf
P0
Phs
Q
H
Dlf
k1
k2
dm
Zm
Lm
nm

Methane pressure at the interface of
membrane and liquid film (bar)
Methane pressure in the hollow-fiber lumen
(bar)
Methane pressure in the headspace (bar)
Water flow rate in the serum bottle (7.2×10-4
m3/d)
Henry's law constant of CH4 (0.7512 m3
bar/mol)
CH4-diffusion coefficient in water (1.9×10-4
m2/d)1
Coefficient that converts CH4 from volume to
mass (1g/0.00154 m3 @ standard temperature
and pressure)
Coefficient that converts CH4 from moles to
mass (16g/mol)
Hollow-fiber out diameter (2.8×10-4 m)
Membrane thickness (5.0×10-5 m)
Hollow-fiber length (m)
Number of hollow fibers (32)

S3

Table S2.

Experimental parameters for the CH4-permeation test
Pressure
(bar)

Flow rate
(m3/d)

Fiber
Length
(m)

Fiber
Numbers

Fiber
O.D.
(µm)

Fiber
thickness
(µm)

Kma

Parameter

Temperature
(K)

Composite
fiber

298

1.00

7.2 × 10-4

0.05

32

2.8×10-4

0.5×10-4

1.03×10−7

a:

units are m3 CH4 @ standard temperature and pressure - m membrane thickness/m2

hollow fiber surface area - d – bar.

S4

Table S3. The influent and effluent concentrations of electron acceptors for each stage
NO3--N
stages
1
2
3
4
5
6
7

Influent
(mg N/L)
NA
NA
1.21±0.09
11.3±0.40
4.49±0.04
NA
NA

Effluent
(mg N/L)
0
0
0.000±0.000
3.51±1.25
0.023±0.029
0
0

NO2--N
Influent
(mg N/L)
1.69±0.006
NA
NA
NA
NA
NA
5.22±0.13

Effluent
(mg N/L)
0.027±0.021
0
0
0
0
0
0.000±0.000

S5

ClO4Influent
(mg/L)
1.32±0.09
1.01±0.008
1.02±0.017
1.04±0.052
1.0±0.003
5.43±0.017
5.07±0.096

Effluent
(mg/L)
1.01±0.14
0.17±0.11
0.011±0.006
1.02±0.069
0.006±0.007
0.057±0.097
2.54±0.40

Table S4.
PCR Program

Primers and PCR conditions for tested genes
Primers

Sequence

Target
Gene

Ref

Slope

Efficiency

320F
598R

5’-GCGCCCACCACTACATGTAYGGNCC-3’
5’-GGTGGTCGCCGTACCARTCRAA-3’

pcrA

2

-.3.106

1.10

M2f
M2r

5'-TAYGTSGGGCAGGARAAACTG-3'
5'-CGTAGA AGA AGCTGGTGCTGTT-3'

narG

3

-3.144

1.08

cd3af
R3cd

5’-GTSAACGTSAAGGARACSGG-3’
5’-GASTTCGGRTGSGTCTTGA-3’

nirS

4

-.3.419

0.995

mcrA

5

-3.221

1.04

5'-GGNGACTGGGACTTCTGG-3'
5'-CCGGMGCAACGTCYTTACC-3'

pMMO

6

-3.383

0.98

5'-GTGSTGCAYGGYTGTCGTCA-3'
5'-ACGTCRTCCMCACCTTCCTC-3'

16S
rDNA
for
bacteria

7

-3.215

1.05

5'-ATTAGATACCCSBGTAGTCC-3'
5'-GCCATGCACC WCCTCT-3'

16S
rDNA
for
archaea

8

-3.630

0.89

o

95 C 1 min
o
(95 C 5 sec
o
60 C 31 sec
o
72 C 20 sec)×40
o
72 C 1 min
o

94 C 2 min
o
(94 C 30 sec
o
58 C 20 sec
o
72 C 60 sec)×40
o
72 C 10 min
o

95 C 2 min
o
(94 C 30 sec
o
60 C 60 sec
o
72 C 60 sec)×40
o
72 C 5 min
o

94 C 10 min
o
(94 C 30 sec
o
58 C 300 sec
o
72 C 60 sec)×40
o
72 C 1 min

Mlas
rev

5'-GGTGGTGTMGGDTTCACMCARTA-3'
5'-CGTTCATBGCGTAGTTVGGRTAGT-3'

o

95 C 10 min
o
(95 C 60 sec
o
60 C 60 sec
o
72 C 60 sec)×40
o
72 C 5 min

A189F
MB661R

o

95 C 2 min
o
(95 C 10 sec
o
56 C 20 sec
o
68 C 20 sec)×40
o
72 C 1 min

16SF
16SR

o

94 C 10 min
o
(94 C 30 sec
o
58 C 20 sec
o
72 C 60 sec)×40
o
72 C 1 min

ARC787F
ARC1059R

S6

Table S5.

A16S

B16S

mcrA

pMM
O
pcrA

narG

nirS

CH4
flux
-

NO3
flux

-

NO2
flux

ClO4
flux

-

Pearson Correlation Matrix

0.919**

CH4
flux
0.304

NO3flux
-0.169

NO2flux
0.797

ClO4flux
0.439

0.689

0.010

0.558

0.748

0.058

0.384

0.137

0.903*

0.484

0.277

0.326

-0.145

-0.173

0.028
0.549

0.796
-0.325

0.014
0.834*

0.331
0.020

0.595
0.282

0.529
0.554

0.784
-0.542

0.743
-0.487

0.259
1.000

0.530
0.599

0.039
0.863*

0.970
0.843*

0.588
0.416

0.254
0.209

0.267
0.329

0.327
-0.090

0.209
1.000

0.027
0.195

0.035
0.933

0.411
0.251

0.691
-0.243

0.524
0.880*

0.865
0.339

0.195

0.711
1.000

0.007
0.508

0.632
0.624

0.642
0.625

0.021
-0.109

0.511
-0.360

0.508

0.303
1.000

0.186
0.341

0.185
-0.078

0.837
0.721

0.483
0.230

0.508
1.000

0.883
0.864*

0.106
0.075

0.661
-0.401

0.027
1.000

0.888
-0.428

0.431
-0.522

A16S

B16S

mcrA

MMO

pcrA

narG

nirS

Pearson
Correlation
Sig. 2-tailed

1.000

0.135

-0.323

0.579

0.983**

0.210

0.799

0.532

0.229

0.000

Pearson
Correlation
Sig. 2-tailed
Pearson
Correlation
Sig. 2-tailed
Pearson
Correlation
Sig. 2-tailed
Pearson
Correlation
Sig. 2-tailed
Pearson
Correlation
Sig. 2-tailed
Pearson
Correlation
Sig. 2-tailed
Pearson
Correlation
Sig. 2-tailed
Pearson
Correlation
Sig. 2-tailed
Pearson
Correlation
Sig. 2-tailed
Pearson
Correlation
Sig. 2-tailed

0.135

1.000

0.835*

0.861*

0.039
1.000

0.799
-0.323

0.835

0.532
0.579

0.039
0.861*

0.549

0.229

0.028
0.137

0.259
-0.325

0.599

0.000
0.210

0.796
0.903*

0.530
0.834*

0.209
0.863*

0.689

0.014
0.484

0.039
0.020

0.027
0.843*

0.933

0.010
0.304

0.331
0.277

0.970
0.282

0.035
0.416

0.007
0.251

0.303
0.624

0.341

0.558
-0.169

0.595
0.326

0.588
0.554

0.411
0.209

0.632
-0.243

0.186
0.625

0.508
-0.078

0.864

0.748
0.797

0.529
-0.145

0.254
-0.542

0.691
0.329

0.642
0.880*

0.185
-0.109

0.883
0.721

0.027
0.075

-0.428

1.000

0.139

0.058
0.439

0.784
-0.173

0.267
-0.487

0.524
-0.090

0.021
0.339

0.837
-0.360

0.106
0.230

0.888
-0.401

0.397
-0.522

0.139

1.000

0.384

0.743

0.327

0.865

0.511

0.483

0.661

0.431

0.288

0.793

0.983

0.919

**

**

*

0.711
**

**

*

*. Correlation is significant at the 0.05 level; ** Correlation is significant at the 0.01 level
(2-tailed).

S7

Figure S1.

The proposed AMO-D and ANMO-D pathways for CH4 oxidation coupled to

NO3-/NO2- reduction.

A:

AMO-D requires two microorganisms:

aerobic methanotrophs

oxidize methane and produce organic compounds, which are further used by denitrifiers to
reduce NO3- to N2.
microorganisms:

B: Reverse-methanogenesis-type ANMO-D requires two
Archaea reduce NO3- to NO2- and produces H2 via reverse methanogenesis,

and a denitrifier that oxidizes the H2 to drive NO2- respiration to N2.

C: Intra-aerobic-type

ANMO-D is carried out by one bacterium, which dismutates NO to form N2 and O2, with the
O2 used as a co-substrate for methane mono-oxygenation by the same bacterium.
S8

Figure S2.

Schematic of the set up for the CH4-permeation experiment (This figure is our

own work and is substantially modified from the original figure in Tang et al. (2012)).9

S9

Figure S3.

The NO3- and ClO4- concentrations in the MBfR influent and effluent in Stage 4

(A) and Stage 5 (B), when CH4 supply was stopped and recovered.

S10

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S11