1
Characterization of the Relative Importance of Human- and Infrastructure-
2
Associated Bacteria in Graywater: A Case Study
3
SUPPORTING INFORMATION
4
SCOTT P. KEELY1,4#,NICHOLE E. BRINKMAN1,4, BRIAN D. ZIMMERMAN1,3, DAVID WENDELL3,
5
KRISTEN M. EKEREN2, SUSAN K. DE LONG2, SYBIL SHARVELLE2, JAY L. GARLAND1; 1National
6
Exposure Research Laboratory, United States Environmental Protection Agency, Cincinnati, OH,
7
45268; 2Department of Civil and Environmental Engineering, Colorado State University, Fort
8
Collins, CO, 80523, 3Department of Energy, Environmental, Biological & Medical Engineering
9
and 4McMicken College of Arts and Sciences, Department of Biological Sciences, University of
10
Cincinnati, Cincinnati, OH 45221.
11
#corresponding author
12
Scott P. Keely, Ph.D.
13
National Exposure Research Laboratory
14
US Environmental Protection Agency
15
26 W. Martin Luther King Drive
16
Cincinnati, OH 45268
17
18
1
19
20
Table S1. Characteristics of Raw GW from the CSU Collection System
Parameter*
Average
Standard Deviation
TOC (mg l-1-C)
44
12.2
Turbidity (NTU)
32
4.2
NH3-N (mg l-1-N)
8.4
2.2
Total coliforms (log cfu 100 ml-1)
8.4
0.6
E. coli (log cfu 100 ml-1)
4.2
2.5
21
*TOC indicates total organic carbon; NTU indicates nephelometric turbidity units. Note that this data is
22
for GW collected post ET from the “Raw Graywater Sampling Port” in Fig. 1. Some changes to water
23
quality will have occurred due to storage (e.g., reduction in turbidity due to settling).
24
2
25
26
27
Table S2. Characteristics of GW from the UC Laundry
Parameter*
Average
Standard Deviation
Dissolved Oxygen (mg l-1)
7.4
0.6
pH
7.6
0.02
Specific Conductivity (µS)
599.0
86.1
Oxidation Reduction Potential (mV)
326.3
209.7
Salinity (ppt)
0.29
0.05
*µS indicates micro seimens; mV indicates millivolts; ppt indicates parts-per-trillion.
28
29
30
3
31
32
33
34
35
Table S3. 454 Read Statistics
Sample
Type
Number
of
Samples
Total
Number
of Raw
Reads
Total
Number
of
Unique
Reads
Percent
Chimeras
Average
Number
of HighQuality
Reads
SH
18
518,502
225,136
LA
24
914,881
440,540
ET
6
269,138
111,232
BC
3
80,994
34,007
10.39
±3.28*
2.07 ±
2.90
10.35 ±
2.39
4.45 ±
1.56
0.20†
9,789 ±
2215*
14,685 ±
2273
10,112 ±
1776
10,844 ±
1598
10,997†
Average
Number
of
Classified
Reads
(Genus)
5,063 ±
1,281*
12,234 ±
1,961
5,148 ±
1,144
6,000 ±
1433
8,415†
PW
1
38,673
11,019
*Standard deviation
†Standard deviation was not calculated because only one sample was sequenced.
36
4
Average
Number
of
Genera
Total
Number
of
Genera
86 ± 24‡
191
105 ± 21
295
53 ± 11
90
82 ± 8
107
37†
37
37
38
Table S4. OTU statistics and BLAST analysis of partially-classified 16S rRNA gene reads observed
in BC and ET
MEGAN Tree
Taxonomic group
Alphaproteobacteria
GW
BC
%
OTU
OTU
Reads length
No.
OTU-1
(bp)
1
23.55
474
%
Identity
%
Coverage
Acc. No.
Isolation
Source
97
100
JX521421
Terrestrial
Sulfidic
Springs
Betaproteobacteria
BC
2
86.24
521
99
100
AY945914
Sludgeseeded
Bioreactor
Coriobacteriaceae
BC
3
89.07
506
99
97
DQ168843
Anaerobic
Sludge
Oxalobacteraceae
BC
4
87.32
517
99
100
GU563775
Parasitic
Nematode
Propionibacteriaceae
BC
5
35.44
501
99
99
EU812976
Biogas
Reactor
Fed Corn
Silage
Xanthomonadaceae
BC
6
80.19
527
99
100
EF515229
Microbial
Fuel Cell
Anode
Bacteroidetes
ET
7
59.45
515
99
100
JX828412
IronReducing
Culture
Betaproteobacteria
ET
8
44.84
521
99
100
AY945914
Sludgeseeded
Bioreactor
Clostridiales
ET
9
42.63
515
98
100
JN368364
Pitcher
Plant
Enterobacteriaceae
ET
10
24.60
519
99
99
FJ393177
Microbial
Fuel Cell
Rhodocyclaceae
ET
11
18.86
523
99
100
JF341927
Concrete
Sewer
Biofilm
39
5
40
41
Table S5. Bonferroni-corrected p-values for GW groups
GW Group
GW Group
LA
SH
Dec. SH
SH
0.0015
Dec. SH
0.0015
0.003
ET
0.0015
0.0015
0.0045
BC
0.0015
0.0105
0.1695
ET
0.021
42
43
6
44
Figure Legends
45
Figure S1. A black-and-white screen shot of the network model in Supplemental File 2. The
46
network was constructed using the edge-weighted spring embedded model with read counts as
47
weights. The larger nodes are GW samples (i.e., SH, ET, BC and LA) and the smaller nodes
48
represent genera.
49
Figure S2. Relative abundance analysis of the classified GW genera for PW. Genera with 5 or
50
more reads are shown. The number of reads per genus is specified above each bar.
51
52
Supplemental Files
53
Data S1. Microsoft Excel file containing bacterial genera read counts for GW samples.
54
Data S2. Network model of the relationships between GWs. A network was constructed using
55
the edge-weighted spring embedded model with read counts as edge weights. The names of
56
the GW and genera are given on the nodes. Node colors and sizes represent relative number of
57
network connections. Green, yellow, brown, orange and red nodes indicated >4, 4, 3, 2 and 1
58
connections, respectively. The edge colors indicate read abundance: green specifies the
59
greatest number of reads; yellow, orange and brown are intermediate levels; and red was the
60
lowest.
61
7
62
63
Figure S1
64
65
66
67
8
68
Figure S2
69
9