SUPPLEMENTARY MATERIAL for
Metagenomic analysis on seasonal microbial variations of activated sludge from
a full-scale wastewater treatment plant over four years
Feng Ju， Feng Guo, Lin Ye, Yu Xia, Tong Zhang*
Environmental Biotechnology Laboratory, Department of Civil Engineering, The University of Hong
Kong, Hong Kong SAR
Submitted to Environmental Microbiology Report
*Corresponding author phone: +852-28578551; fax: +852-25595337; e-mail: zhangt@hkucc.hku.hk
Supporting Information S1: Variation of environmental and operational parameters
Tables
Table S1
Characteristics of the environmental and operational parameters in Shatin WWTP from
July 2007 to January 2011.
Table S2
Illumina sequencing metadata of the eight activated sludge samples in this study.
Table S3
The percentage abundances (%) of the major phyla (top 15 in each sample) in the 8 AS
samples.
Table S4
Abundances of the top 20 genera in each AS sample.
Table S5
Percentages of the shared genera and their corresponding sequences.
Table S6
The relative abundances of the 28 functional categories in four ecosystems based on
SEED Subsystems implemented in MG-RAST.
Figures
Figure S1
Reproducibility of Illumina high-throughput sequencing at different sequencing depths.
Figure S2
Heat map of major genera (top 20 in each sample) of the 8 AS samples.
1
Supporting
Information
S1:
Variation
of
environmental
and
operational parameters
During the sampling period, the water temperature of aerobic tank ranged from 13.7°C to 29.6°C,
with seasonal lows (15.4±1.3 0C) in January (winter) and highs (29.1±0.5 0C) in July (summer)
(Table S1). On the contrary, salinity (Cl-1/(mg.L-1) (from the seawater toilet-flushing practice in
Hong Kong) fluctuated over the 4 years with peak concentrations in January and valley values in
July. However, variation in concentrations of MLSS and nutrients like COD, NH4-N and total
phosphorus (TP) followed mixed patterns in which their averaged concentrations in the last 2 years
(Jul-07, Jan-08, Jul-08 and Jan-09) were higher than in the first 2 years (Jul-09, Jan-10, Jul-10 and
Jan-11), while their concentrations in winter (January) were always higher than in summer (July).
2
Tables
12.7
3.3
8.9
231
1637
404
31
3.7
Jan-2008
AS08-1
15.9
5687
6.1
13.6
2.7
8.0
222
1963
389
37
4.2
Jul-2008
AS08-7
28.4
4115
4.7
11.2
3.3
6.6
281
1811
334
31
3.5
Jan-2009
AS09-1
15.3
5712
4.4
9.4
3.3
7.6
243
2061
347
33
4.6
Jul-2009
AS09-7
29.1
4584
5.2
10.9
3.9
7.7
232
1859
333
37
4.6
Jan-2010
AS10-1
16.8
6020
4.7
10.5
4.3
8.1
220
2216
478
42
5.2
Jul-2010
AS10-7
29.2
5229
11.3
25.2
3.9
7.8
233
2410
410
32
3
Jan-2011
AS11-1
13.7
5951
5.3
11.9
2.5
8.2
220
3281
648
50
8.2
TP /(mg.L-1)
COD /(mg.L-1)
5.7
/(mg.L-1)
MLSS /(g.L-1)
5038
TKN-N
HRT / (hrs)
29.6
/(m3.d-1)
DO / (mg/L)
AS07-7
Flow rate
MCRT / (d-1)
Jul-2007
( mg Cl-.L-1)
name
Salinity /
time
/(0C)
Sample
Temperature
Sampling
SRT / (d-1)
Table S1 Characteristics of the environmental and operational parameters in Shatin WWTP from July
2007 to January 2011
DO: dissolved oxygen; SRT: sludge retention time; MCRT: mean cell retention time; HRT: hydraulic retention time; COD:
chemical oxygen demand; MLSS: mixed liquor suspended solids; TKN: total Kjeldahl nitrogen. The monthly averaged
values for each item were used for statistics analysis in the present study.
3
Table S2 Illumina sequencing metadata of the 8 activated sludge samples in this study
PE1 reads after
Sample
ids
normalization
number
length
(bps)
Tags2 obtained
Number of
de-replicated PE
reads
number
Number of
yield ratio
mean length
(%)
(bps)
16/18S rRNA
gene tags3
AS07-7
25,426,000
100
24,608,407
18,608,516
75.6
169
12,624
AS08-1
25,426,000
100
24,722,092
17,800,479
72.0
170
13,529
AS08-7
25,426,000
100
24,318,551
18,428,094
75.8
168
14,599
AS09-1
25,426,000
100
24,426,682
17,687,533
72.4
166
14,596
AS09-7
25,426,000
100
24,909,848
18,345,157
73.6
172
14,786
AS10-1
25,426,000
100
24,748,187
18,318,508
74.0
163
15,473
AS10-7
25,426,000
100
25,082,442
18,601,386
74.2
163
13,689
AS11-1
25,426,000
100
24,021,724
17,001,280
70.8
165
12,977
1) PE: paired-end; 2) Tags: the sequences obtained from overlapping the metagenomic paired-end reads, allowing a minimum
overlap length of 10 bps; 3) 16/18S rRNA gene tags were identified by running BLAST against SSU-Ref database at an
e-value cutoff of 1e-5.
4
Table S3 The relative abundances (%) of the major phyla (top 15 in each sample) in the 8 AS
samples. The abundance is presented in terms of percentages of the total sequences (in a sample) that
were assigned to phylum level, sorted firstly by domain and then abundance of each phylum. P1-ratio
represents the ratio of average abundances in summer to winter samples. P2-ratio represents to ratio of
AS08-1
AS08-7
AS09-1
AS09-7
AS10-1
AS10-7
AS11-1
Average
P1-ratio
P2-ratio
Proteobacteria
40.38
39.76
38.06
38.10
41.02
41.85
44.11
42.93
40.78
2.2
1.0
0.9
Actinobacteria
21.14
29.35
19.89
33.54
15.03
23.29
15.36
17.73
21.92
6.6
0.7
1.5
Chloroflexi
7.84
6.04
9.79
7.91
12.52
11.99
16.42
12.26
10.60
3.3
1.2
0.6
Bacteroidetes
10.12
6.55
7.90
7.46
9.93
7.01
6.81
13.53
8.66
2.4
1.0
0.9
Firmicutes
4.28
6.03
6.98
4.84
3.52
4.56
2.99
5.29
4.81
1.3
0.9
1.4
Planctomycetes
6.18
5.60
4.38
4.26
4.06
3.20
3.46
2.76
4.24
1.2
1.1
1.5
Nitrospirae
4.95
1.55
4.34
0.05
2.85
1.35
1.52
1.17
2.22
1.7
3.3
1.6
Verrucomicrobia
1.13
1.91
0.96
1.70
0.87
1.67
1.04
0.96
1.28
0.4
0.6
1.3
Chlorobi
0.74
0.81
0.42
0.34
1.07
0.89
0.93
0.24
0.68
0.3
1.4
0.7
Cyanobacteria
0.63
0.27
0.14
0.14
0.48
0.15
0.48
0.15
0.31
0.2
2.4
0.9
Thermotogae
0.02
0.00
0.01
0.08
0.04
0.33
0.03
0.59
0.14
0.2
0.1
0.1
Acidobacteria
0.22
0.04
0.14
0.09
0.37
0.14
0.27
0.24
0.19
0.1
2.0
0.5
Chlamydiae
0.20
0.82
0.65
0.14
0.46
0.31
0.85
0.20
0.45
0.3
1.5
1.0
Spirochaetes
0.22
0.19
0.47
0.10
0.34
0.17
0.22
0.17
0.23
0.1
2.0
1.1
Lentisphaerae
0.03
0.07
0.13
0.19
0.14
0.14
0.25
0.18
0.14
0.1
0.9
0.6
Armatimonadetes
0.08
0.15
0.01
0.01
0.08
0.00
0.03
0.02
0.05
0.1
1.1
1.9
Rotifera
0.57
0.16
2.39
0.15
1.20
0.11
0.76
0.24
0.70
0.8
7.5
1.4
Nematoda
0.57
0.05
2.40
0.18
4.65
1.44
1.00
0.08
1.30
1.6
4.9
0.4
Arthropoda
0.08
0.12
0.06
0.16
0.03
0.06
0.01
0.05
0.07
0.0
0.5
2.8
Gastrotricha
0.03
0.00
0.01
0.06
0.07
0.20
0.03
0.00
0.05
0.1
0.5
0.3
Streptophyta
0.00
0.19
0.25
0.15
0.46
0.32
2.60
0.27
0.53
0.8
3.6
0.2
0.14
0.08
0.25
0.15
0.11
0.50
0.15
0.44
0.23
0.2
0.6
0.5
0.46
0.23
0.36
0.19
0.71
0.33
0.67
0.49
0.43
0.2
1.8
0.6
Eukaryota
Bacteria
Phyla
Archaea Euryarchaeota
Others
Other assigned
STD
AS07-7
average abundances in the first-2-years to last-2-years samples.
5
Table S4 The relative abundances of the major genera (top 20 in each AS sample) in the 8 AS
samples. The abundance is presented in terms of percentages of the total sequences in a sample that
were assigned to genus level, sorted alphabetically by phylum and then genus. Average represents the
averaged abundances of each genus in eight samples. P1-ratio represents the ratio of average abundances
in summer to winter samples. P2-ratio represents to ratio of average abundances in the first-2-years to
AS07-7
AS08-1
AS08-7
AS09-1
AS09-7
AS10-1
AS10-7
AS11-1
Average
STD
P1-ratio
P2-ratio
last-2-years samples.
Mycobacteriu
10.6
13.3
10.3
8.28
8.82
8.00
6.00
6.16
8.94
2.4
1.0
1.5
m
Candidatus
4.767
6.350
2.332
4.95
1.83
4.74
3.88
2.93
3.97
1.5
0.7
1.4
Microthrix
Iamia
2.16
2.82
2.59
2.68
2.26
1.99
1.15
1.61
2.16
0.6
0.9
1.5
Nocardioides
1.08
1.52
1.74
1.80
0.72
1.99
0.58
2.88
1.54
0.7
0.5
1.0
Bifidobacteriu
0.88
1.69
0.79
1.17
0.72
1.17
0.53
1.46
1.05
0.4
0.5
1.2
m
Tetrasphaera
0.64
4.98
0.92
5.57
0.83
3.34
0.43
2.57
2.41
2.0
0.2
1.7
Gordonia
0.08
0.46
0.13
6.96
0.48
2.63
4.60
0.56
1.99
2.6
0.5
0.9
Lewinella
2.40
1.20
2.29
0.88
1.75
1.39
2.73
0.96
1.70
0.7
2.1
1.0
Flexibacter
0.08
0.14
0.49
0.73
1.59
1.32
0.82
1.77
0.87
0.6
0.8
0.3
Chlamydiae
Waddlia
0.04
0.04
0.07
0.07
0.00
0.08
1.15
0.10
0.19
0.4
4.4
0.2
Chloroflexi
Caldilinea
5.72
4.73
5.90
3.08
4.49
3.87
3.84
3.03
4.33
1.1
1.4
1.3
Deferribacteres
Caldithrix
1.60
0.74
0.82
0.40
1.79
1.50
4.51
1.41
1.60
1.3
2.2
0.4
Firmicutes
Streptococcus
1.44
1.62
2.03
0.95
1.67
0.53
0.96
0.91
1.26
0.5
1.5
1.5
Clostridium
0.84
1.02
1.08
0.77
0.60
0.60
0.58
0.56
0.76
0.2
1.1
1.6
Blautia
0.68
1.09
1.97
1.06
0.52
1.17
0.58
0.86
0.99
0.5
0.9
1.5
Enterococcus
0.04
0.64
0.13
0.95
0.04
0.75
0.10
3.08
0.72
1.0
0.1
0.4
Nitrospirae
Nitrospira
12.7
3.99
10.9
0.15
8.34
4.13
4.89
3.89
6.14
4.2
3.0
1.3
Opisthokonta
Diplolaimella
0.009
0.00
3.607
0.00
9.73
2.78
2.40
0.00
2.31
3.3
5.7
0.2
Planctomycetes
Planctomyces
8.95
8.12
4.26
6.30
4.93
4.77
3.45
2.78
5.44
2.2
1.0
1.7
Pirellula
1.56
1.48
2.03
1.39
0.75
0.83
0.91
0.86
1.23
0.5
1.2
1.9
Rhodopirellula
0.52
0.64
0.43
0.59
1.11
0.45
0.82
0.86
0.68
0.2
1.1
0.7
Amaricoccus
2.12
3.03
2.36
2.05
1.15
1.39
1.06
1.01
1.77
0.7
0.9
2.1
Rhodobacter
2.00
2.05
2.29
3.22
2.07
1.77
1.87
1.61
2.11
0.5
1.0
1.3
Nordella
1.64
2.36
0.82
0.77
0.32
0.23
0.14
0.40
0.84
0.8
0.8
5.1
Rhodobium
1.40
1.41
1.31
0.81
1.15
0.41
0.67
1.06
1.03
0.4
1.2
1.5
Paracoccus
1.32
1.41
1.18
2.38
0.79
1.88
0.72
1.51
1.40
0.5
0.6
1.3
Hyphomicrobi
1.04
0.88
0.33
0.70
0.32
0.45
0.48
0.30
0.56
0.3
0.9
1.9
um
Methylosinus
0.84
1.02
0.62
0.84
0.56
0.71
0.34
0.81
0.72
0.2
0.7
1.4
Rhodovulum
0.76
0.67
0.79
1.32
0.75
0.83
0.77
0.45
0.79
0.2
0.9
1.3
Denitromonas
0.76
0.21
0.43
0.11
0.52
0.30
1.01
1.67
0.62
0.5
1.2
0.4
Haliea
0.76
0.14
0.33
0.33
0.72
1.62
1.53
3.18
1.08
1.0
0.6
0.2
Mesorhizobium
0.72
1.06
0.62
0.44
0.72
0.64
0.82
0.61
0.70
0.2
1.1
1.0
Haliangium
0.64
0.39
0.69
0.37
0.87
0.98
0.34
0.76
0.63
0.2
1.0
0.7
Phylum
Actinobacteria
Bacteroidetes
Proteobacteria
Genus
6
Thermotogae
Azoarcus
0.64
0.18
0.49
0.22
1.47
0.26
2.30
1.56
0.89
0.8
2.2
0.3
Zoogloea
0.44
0.18
0.20
0.15
0.28
0.38
1.15
1.11
0.48
0.4
1.1
0.3
Nannocystis
0.36
0.14
1.11
0.18
0.68
0.94
1.01
0.76
0.65
0.4
1.6
0.5
Candidatus
0.36
0.35
0.26
1.39
0.72
1.73
0.19
0.56
0.69
0.6
0.4
0.7
Alysiosphaera
Acidovorax
0.32
0.39
0.85
0.62
0.20
0.38
0.10
0.20
0.38
0.2
0.9
2.5
Nitrosomonas
0.16
0.00
0.23
0.07
1.11
0.41
1.15
0.35
0.44
0.4
3.2
0.2
Thauera
0.16
0.18
0.13
0.07
1.71
0.41
1.10
1.01
0.60
0.6
1.9
0.1
Hyphomonas
0.12
0.00
0.20
0.62
0.60
0.38
1.06
0.45
0.43
0.3
1.4
0.4
Candidatus
0.04
0.04
0.10
0.07
0.52
0.15
1.10
1.72
0.47
0.6
0.9
0.1
Thiobios
Kosmotoga
0.00
0.00
0.03
0.22
0.12
1.01
0.10
1.97
0.43
0.7
0.1
0.1
7
Table S5 Percentages of the shared genera and their corresponding sequences.
a
Number of
Number of shared
sample
b
c
Percentage in
Number of shared
Percentage in classified
genera
classified genera
sequences
sequences
8
100*
15.6
16169
79.4
7
144
22.4
17457
85.7
6
188
29.2
18234
89.5
5
233
36.2
18755
92.1
4
285#
44.3
19602
96.3
3
346
53.8
19865
97.6
2
441
68.6
20120
98.8
1
643
100.0
20363
100.0
*
100 (accounting for 79.4% of the classified sequences) out of the 643 assigned genera were shared by all 8 samples.
#
A total of 285 genera were commonly shared by more than 4 AS samples, accounting for 96.3% of all classified sequences at genus
level. There were 202 rare genera that only appeared in one sample, accounting for only 1.2% of total sequences.
a
Number of AS samples, which share the genera in the second column.
b
Percentage of the number of shared genera in the number of total classified genera.
c
Percentage of sequences of the shared genera in the total classified sequences at genus level.
8
Table S6 The relative abundances of the 28 functional categories in four ecosystems based on SEED
Subsystems implemented in MG-RAST. The results are presented using the mean value of all datasets in each
ecosystem. RSD means relative standard deviation.
AS
Level 1
Soil
Human faeces
Ocean
Total
Mean
RSD
Mean
RSD
Mean
RSD
Mean
RSD
Mean
RSD
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
(%)
Clustering-based subsystems
15.63
0.54
15.48
1.89
15.43
6.93
15.39
3.25
15.48
3.85
Carbohydrates
10.16
1.58
10.95
4.16
12.9
11.18
7.94
7.98
10.49
18.71
Protein Metabolism
7.86
0.8
8.69
3.25
8.16
4.02
9.75
4.09
8.62
8.79
Amino Acids and Derivatives
8.57
1.64
8.23
7.3
7
6.51
7.54
9.89
7.84
10.24
Miscellaneous
8.83
1.72
7.53
7.07
7.81
11.31
9.42
2.33
8.40
11.41
Cofactors, Vitamins, Prosthetic Groups, Pigments
6.3
0.84
6.89
7.21
5.8
5.24
7.81
5.07
6.70
12.39
DNA Metabolism
3.41
3.00
3.55
13.18
4.48
5.94
3.58
7.32
3.76
13.91
RNA Metabolism
4.6
1.79
4.23
10.62
5.21
15.09
3.89
5.15
4.48
15.10
Membrane Transport
1.38
1.85
3.96
6.54
4.62
9.99
4.65
4.14
3.65
33.90
Respiration
3.65
2.2
3.39
7.28
3.69
12.6
2.59
6.75
3.33
15.74
Cell Wall and Capsule
4.02
3.31
3.13
13.93
2.03
21.18
2.9
13.44
3.02
25.88
Nucleosides and Nucleotides
2.87
4.18
2.75
24.57
2.72
9.08
1.58
5.75
2.48
26.25
Virulence, Disease and Defense
2.94
2.3
3.05
9.23
2.07
5.64
2.68
3.79
2.69
15.74
Fatty Acids, Lipids, and Isoprenoids
2.55
2.71
2.7
10.53
2.04
27.08
2.37
11.04
2.42
17.30
Stress Response
3.01
0.91
2.95
6.4
3.18
8.07
3.42
2.55
3.14
7.91
Phages, Prophages, Transposable elements, Plasmids
3.6
1.71
1.9
51.35
1.55
21.21
1.24
7.27
2.07
51.02
Metabolism of Aromatic Compounds
1.64
4.45
1.74
18.25
0.62
31.17
0.84
10.23
1.21
44.28
Regulation and Cell signaling
1.65
4.29
1.67
15.99
2.59
18.23
5.38
46.43
2.82
69.92
Cell Division and Cell Cycle
1.39
2.04
1.34
7.83
1.58
16.45
1.68
4.79
1.50
13.39
Nitrogen Metabolism
1.12
2.72
1.17
10.72
1.42
11.73
0.96
8.08
1.17
17.35
Sulfur Metabolism
1.38
5.16
1.04
14.74
0.83
29.33
0.78
17.15
1.01
27.60
Phosphorus Metabolism
0.79
9.66
0.87
19.08
0.59
30.24
0.53
10.23
0.70
27.98
Motility and Chemotaxis
0.9
2.02
0.84
8.92
0.82
7.31
0.67
7.57
0.81
12.42
Iron acquisition and metabolism
0.55
8.97
0.66
45.45
1.35
28.49
0.57
16.27
0.78
52.58
Secondary Metabolism
0.38
4.51
0.38
32.39
0.5
19.89
0.18
12.06
0.36
39.30
Potassium metabolism
0.47
2.67
0.41
12.2
0.34
29.88
0.41
7.93
0.41
18.09
Dormancy and Sporulation
0.22
2.76
0.26
17.36
0.64
25.73
0.22
27.04
0.34
59.09
Photosynthesis
0.12
8.05
0.23
24.52
0.05
39.3
1.02
43.08
0.36
124.9
9
.
Figures
a•
b
600
100
400
AS08-7-1G/5G
AS08-7-1G/5G
500
300
200
Linear fitting equation:
Y=1.025X; R2=0.995
100
0
100
200
300
400
AS08-7-1G
c
500
60
40
Linear fitting equation:
2
Y=1.029X; R =0.949
0
600
0
d
20
40
60
AS08-7-1G
80
100
400
AS08-7-5G/30G
AS08-7-5G/30G
2000
80
20
0
350
300
AS08-7-5G/30G
1600
AS08-7-5G/30G
AS08-7-1G/5G
120
AS08-7-1G/5G
1200
800
Linear fitting equation:
2
Y=X; R =0.985
400
250
200
150
100
Linear fitting equation:
2
Y=1.018X; R =0.989
50
0
0
0
400
800
1200
1600
AS08-7-5G
2000
0
50
100
150
200
250
300
350
AS08-7-5G
Figure S1 Reproducibility of Illumina high-throughput sequencing at different sequencing depths. The results
are based on taxonomic analysis of four data sets derived from three replicates of sample AS08-7.
AS08-7-1G/5G and AS08-7-5G/30G represents random extraction of 1G and 5G data sets from original 5G and 30
G data sets of sample AS08-7, respectively. a and b stand for linear fitting of classes (a) and genera (b) abundances
(with abundance >5) in the two 1G data sets; c and d stand for linear fitting of classes (c) and genera (d) abundances
(with abundance >5) in the two 5G data sets.
10
Figure S2 Heat map of major genera (top 20 in each sample) of the 8 AS samples. The top 20 abundant genera
in each sample were selected (a total of 43 genera for all 8 samples) and compared with their abundances
(percentages) in other samples. The genus names in bold blue, purple and green represent those major genera with
significantly-changed abundances across P1, P2 and P1P2 (both P1 and P2), respectively, based on two-way
ANOVA analysis. The color intensity in each column displays the percentage of a genus in a sample, referring to
color key on the left.
11