1
Supporting information
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3
Fig. S1
4
The ratios of Nitrospira to the total microbial types in two samples were compared; 1)
5
enriched culture sample treated by weak sonication as shown as “original sample”, and
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2) the sample collected from cell sorter after application of the original sample in order
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to separate micro-colony from other populations, as shown as “sorted sample”. The
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ratios were estimated by FISH direct counting method. Two types of counting methods
9
were performed; 1) simply counting all the cells and calculating the ratio as “the ratio
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based on microbial cell counting”, and 2) counting free living cells and micro-colonies
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individually as shown as “the ratio based on initial growth unit counting”. Both “a
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single cell” and “a single micro-colony” were regarded as the equivalent unit and so
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were called “initial growth unit”.
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15
Fig. S2
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Representative FISH images showing the growth of (A) Nitrospira, (B) Nitrospira with
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contaminants, and (C) other microbial type in each well of 96 micro-titer plate after the
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one month of incubation. Ntspa662- and SYTOX green-doubly-stained cells
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(Nitrospira) are colored in yellow, where as other types are colored in green stained
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only by SYTOX green. All scale bars are 10 μm.
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22
23
Fig. S3
24
PCR-DGGE band profile of enrichment sample (lane 1) and pure strain ND1 (lane 2).
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26
Fig. S4
27
Nitrite consumption by strain ND1 grown at 23°C in Erlenmeyer flasks, plotted against
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nitrate production. The filled circle and filled square represent nitrite and nitrate
29
concentrations respectively. Error bars indicate the standard deviation of the mean of
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triplicate measurements.
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32
Fig. S5
33
Optimal nitrite concentration for nitrite consumption by strain ND1. The inoculum was
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extracted from a pre-culture grown at 25°C. Optimal nitrite concentration was
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determined as that yielding the highest nitrite oxidation rate during three days of
36
incubation. Error bars indicate the standard deviation of the mean of triplicate
37
measurements.
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39
Fig. S6
40
Optimal temperature for nitrite consumption by strain ND1. The inoculum was
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extracted from a pre-culture grown at 25°C. Initial nitrite concentration was 20 mg-N
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L-1. Optimal temperature was determined as that yielding the highest nitrite oxidation
43
rate during three days of incubation. Error bars indicate the standard deviation of the
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mean of triplicate measurements.
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46
47
Fig. S1
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49
50
52
53
54
55
56
57
58
The ratio of Nitrospira to the total
microbial types [%]
51
100
80
60
The ratio based on microbial
cell counting
40
The ratio based on initial
growth unit counting
20
0
Original samples
Sorted samples
59
60
61
Fig. S2
A
62
63
64
65
66
67
B
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
C
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Fig. S3
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85
86
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90
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92
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95
96
97
98
99
100
1
2
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Fig. S4
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103
2
105
106
107
108
109
110
111
Concentration (NO2- or NO3-) [mM]
104
1.5
1
0.5
112
113
114
115
116
117
118
0
0
2
4
6
Time [days]
8
10
12
119
Fig. S5
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60
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123
124
125
126
127
128
129
130
131
132
133
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136
137
138
139
140
141
142
50
Nitrite oxidizing rate [μM day-1]
122
40
30
20
10
0
0
1
2
3
4
Nitrite concentration [mM]
5
6
143
Fig. S6
144
145
0.3
146
148
149
150
151
152
153
Nitrite oxidation rate [mM d-1]
147
0.2
0.1
154
155
156
157
158
159
160
161
162
163
164
165
166
0
0
10
20
30
Temperature [˚C]
40
50
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Table S1
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169
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Sequence analysis of 5 pure cultures obtained in this study
Strain
Closest species (Accesion Number)
Similarity (%)
Closest match (Accesion Number)
Similarity (%)
ND1
Candidatus Nitrospira defluvii (DQ059545)
99.8
Uncultured Nitrospirae bacterium clone: 406 (AB252944)
100
ND7
Candidatus Nitrospira defluvii (DQ059546)
100
Uncultured Nitrospira sp. clone as2-4 (GU257604)
100
ND10
Candidatus Nitrospira defluvii (DQ059547)
99.9
Uncultured bacterium clone ar1e1116 (HM921144)
100
ND20
Candidatus Nitrospira defluvii (DQ059548)
100
Uncultured Nitrospira sp. clone as2-4 (GU257604)
100
ND33
Candidatus Nitrospira defluvii (DQ059549)
99.9
Uncultured bacterium clone ar1e1116 (HM921144)
100
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
Table S2
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193
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FISH probes used in this study.
Full name
Short name
Sequence 5'-3'
S-G-Ntspa-0662-a-A-18
Ntspa662
GGA ATT CCG CGC TCC TCT
FA%
Target group
Reference
genus Nitrospira
Daims et al ., 2001
-
Daims et al ., 2001
35%
cS-G-Ntspa-0662-a-A-18d
cNtspa662
GGA ATT CCG CTC TCC TCT
S-*-Ntspa-1431-a-A-18
Ntspa1431
TTG GCT TGG GCG ACT TCA
35%
Sublineage I of the genus Nitrospira
Maixner et al ., 2006
S-*-Ntspa-1151-a-A-20
Ntspa1151
TTC TCC TGG GCA GTC TCT CC
35 - 40%
Sublineage II of the genus Nitrospira
Maixner et al ., 2006
S-G-Nbac-1035-a-A-18 Nit3
Nit3
CCT GTG CTC CAT GCT CCG
genus Nitrobacter
Wagner et al ., 1996
-
Wagner et al ., 1996
most Bacteria
Amann et al ., 1990
40%
198
199
cS-G-Nbac-1035-a-A-18d
cNit3
CCT GTG CTC CAG GCT CCG
S-D-Bact-0338-a-A-18
EUB338
GCT GCC TCC CGT AGG AGT
S-*-BactP-0338-a-A-18
S-*-BactV-0338-a-A-18
EUB338 II
EUB338 III
GCA GCC ACC CGT AGG TGT
GCT GCC ACC CGT AGG TGT
0-50%
Planctomycetes and other bacteria
not targeted by EUB338
Verrucomicrobia and other bacteria
not targeted by EUB338
Daims et al ., 1999
Daims et al ., 1999
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201
202
References
203
Amann, R., Krumholz, L., and Stahl, D. (1990) Fluorescent-oligonucleotide probing of
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whole cells for determinative, phylogenetic, and environmental-studies in microbiology.
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206
207
Daims, H., Bruhl, A., Amann, R., Schleifer, K., and Wagner, M. (1999) The
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domain-specific probe EUB338 is insufficient for the detection of all Bacteria:
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Development and evaluation of a more comprehensive probe set. Syst Appl Microbiol
210
22: 434-444.
211
212
Daims, H., Nielsen, J., Nielsen, P., Schleifer, K., and Wagner, M. (2001) In situ
213
characterization of Nitrospira-like nitrite oxidizing bacteria active in wastewater
214
treatment plants. Appl Environ Microbiol 67: 5273-5284.
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Maixner, F., Noguera, D., Anneser, B., Stoecker, K., Wegl, G., Wagner, M., and Daims,
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H. (2006) Nitrite concentration influences the population structure of Nitrospira-like
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bacteria. Environ Microbiol 8: 1487-1495.
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220
Wagner, M., Rath, G., Koops, H., Flood, J., and Amann, R. (1996) In situ analysis of
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nitrifying bacteria in sewage treatment plants. Water Sci Technol 34: 237-244.
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