Supplementary materials:
Effects of aeration and internal recycle flow on nitrous oxide emission
from a Modified Ludzak–Ettinger process fed with glycerol
Kang Song1,2, Toshikazu Suenaga1, Willie F. Harper Jr.3, Tomoyuki Hori4, Shohei Riya1,
Masaaki Hosomi1, Akihiko Terada1*
1. Department of Chemical Engineering, Tokyo University of Agriculture & Technology,
Tokyo 184-8588, Japan
2. Research Fellow of Japan Society for the Promotion of Science, 5-3-1 Kojimachi,
Chiyoda-ku, Tokyo 102-0083, Japan
3. Department of Systems Engineering and Management, Air Force Institute of Technology,
Wright-Patterson Air Force Base, OH 45433, United States
4. National Institute of Advanced Industrial Science and Technology, Tokyo 305-8568, Japan
* Corresponding author: akte@cc.tuat.ac.jp
Tel: +81-42-388-7069 / Fax: +81-42-388-7731
I
Figure legends for supplementary materials
Fig. S1 Time courses of dissolved N2O concentration in the anoxic and aerobic tanks on day
146.
Fig. S2 Representative organic carbon and nitrogen concentrations in the MLE under
different IRFs conditions. The samples were taken at the final date of the IRF
condition (prior to changeover).
Fig. S3 Time courses of the quantities of functional genes encoding (a) amoA, (b) nirK, (c)
nirS, (d) nosZ clade I, and (e) nosZ clade II.
Fig. S4 Correlation of N2O emission rate from the MLE system versus the relative gene
abundance nosZ/(nirK + nirS).
Fig. S5 Correlation of N2O emission rate from the MLE system versus relative abundance of
the genus Rhodobacter.
Fig. S6 Relative abundances of AOB and NOB during the MLE operation.
II
70
60
50
40
30
20
10
0
13:18
13:32
13:46
14:01
14:15
14:29
14:44
14:58
15:12
15:27
15:41
15:55
16:10
16:24
16:38
16:53
17:07
17:21
17:36
17:50
18:04
18:19
18:33
18:47
19:02
19:16
19:30
19:45
19:59
20:13
20:28
20:42
20:57
21:11
21:25
21:40
21:54
22:08
22:23
22:37
22:51
Dissolved N2O concentration
(μgN/L)
80
Time of operation (Hour:minute) on day 146
Anoxic tank
Aerobic tank
350
Nitrogen concentration (mgN/L)
70
IRF = 3.6
IRF = 7.2
IRF = 1.0
60
300
50
250
40
200
30
150
20
100
10
50
0
0
NH4+
NO2-
NO3-
Total organic carbon concentration (mgN/L)
Fig. S1 Time courses of dissolved N2O concentration in the anoxic and aerobic tanks on day
146.
TOC
Fig. S2 Representative organic carbon and nitrogen concentrations in the MLE under
different IRF conditions. The samples were taken at the final date of each IRF condition
(prior to changeover).
III
Quantity (copy/ng-DNA)
(a).
amoA
1.E+04
Low aeration
High aeration
1.E+03
1.E+02
1.E+01
1.E+00
106
113
131
148
194
203
228
(b).
Quantity (copy/ng-DNA)
Day of operation
1.E+07
1.E+06
nirK
Low aeration
High aeration
1.E+05
1.E+04
1.E+03
1.E+02
1.E+01
1.E+00
106
113
131
148
194
203
228
(c).
Quantity (copy/ng-DNA)
Day of operation
1.E+07
1.E+06
nirS
High aeration
Low aeration
1.E+05
1.E+04
1.E+03
1.E+02
1.E+01
1.E+00
106
113
131
148
194
Day of operation
IV
203
228
Quantity (copy/ng-DNA)
(d).
nosZ clade I
1.E+07
High aeration
Low aeration
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02
1.E+01
1.E+00
106
113
131
148
194
203
228
Day of operation
(e).
Quantity (copy/ng-DNA)
nosZ clade II
1.E+05
High aeration
Low aeration
1.E+04
1.E+03
1.E+02
1.E+01
1.E+00
106
113
131
148
194
203
228
Day of operation
Fig. S3 Time courses of the quantities of functional genes encoding (a) amoA, (b) nirK, (c)
N2O emission rate
(μgN/min)
nirS, (d) nosZ clade I, and (e) nosZ clade II.
4
3
R² = 0.7499
P=0.012<0.05
2
1
0
0
1
2
nosZ/(nirS+nirK)
3
Fig. S4 Correlation of N2O emission rate from the MLE system versus the relative gene
abundance nosZ/(nirK + nirS).
V
Percentage of
Rhodobacter (%)
20
R² = 0.5848
P = 0.045 < 0.05
15
10
5
0
0
1
2
3
N2O emission rate (μgN/min)
4
Fig. S5 Correlation of N2O emission rate from the MLE system versus relative abundance of
the genus Rhodobacter.
High aeration
Low aeration
Fig. S6 Relative abundances of AOB and NOB during the MLE operation
VI
Table S1 Primers used in real-time quantitative PCR
Primer
Purpose
Target gene
Sequence (5’-3’)
Reference
amoA1F
qPCR
amoA
GGGGTTTCTACTGGTGGT
(Rotthauwe et al. 1997)
amoA 2R
qPCR
amoA
CCCCTCKGSAAAGCCTTCTTC
nirk 876-F
qPCR
nirK
ATYGGCGGVCAYGGCGA
(Henry et al. 2004)
nirK 1040-R
qPCR
nirK
GCCTCGATCAGRTTRTGGTT
nirS 4QF
qPCR
nirS
GTSAACGYSAAGGARACSGG
(Throback et al. 2004)
nirS 6QR
qPCR
nirS
GASTTCGGRTGSGTCTTSAYGAA
nosZ1F
qPCR
nosZ
WCSYTGTTCMTCGACAGCCAG
(Henry et al. 2006)
nosZ1R
qPCR
nosZ
ATGTCGATCARCTGVKCRTTYTC
nosZ Clade II-F
qPCR
nosZ
CTI GGICCI YTK CAY AC
(Jones et al. 2013)
nosZ Clade II-R
qPCR
nosZ
GCI GARCAR AAI TCB GTR C
Table S2 Diversity indices of microbial community structure
Glycerol
Read number
0
98
106
113
131
148
154
167
194
32196 31336 35856 25540 38560 37108 46564 35033 36846
OTUs number (<97%)
1123
1002
1128
1197
1136
1345
1350
993
1122
Shannon–Wiener’s H’
41.06
35.74
18.84
25.20
20.03
26.11
19.55
30.43
22.99
Evenness J
5.85
5.17
2.68
3.56
2.85
3.62
2.71
4.41
3.27
VII
Additional References
Henry S, Baudoin E, López-Gutiérrez JC, Martin-Laurent F, Brauman A, Philippot L (2004) Quantification of
denitrifying bacteria in soils by nirK gene targeted real-time PCR. Journal of microbiological methods
59:327-335
Henry S, Bru D, Stres B, Hallet S, Philippot L (2006) Quantitative detection of the nosZ gene, encoding nitrous
oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ genes in soils.
Appl Environ Microbiol 72:5181-9
Jones CM, Graf DR, Bru D, Philippot L, Hallin S (2013) The unaccounted yet abundant nitrous oxide-reducing
microbial community: a potential nitrous oxide sink. The ISME journal 7:417-26
Rotthauwe J-H, Witzel K-P, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a
functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied
and environmental microbiology 63:4704-4712
Throback IN, Enwall K, Jarvis A, Hallin S (2004) Reassessing PCR primers targeting nirS, nirK and nosZ genes
for community surveys of denitrifying bacteria with DGGE. FEMS microbiology ecology 49:401-17
VIII