Applied Microbiology and Biotechnology
Metagenomes reveal microbial structures, functional
potentials, and biofouling-related genes in a
membrane bioreactor
Jinxing Ma1, Zhiwei Wang1*, Huan Li2, Hee-Deung Park3, Zhichao Wu1
1. State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental
Science and Engineering, Tongji University, Shanghai 200092, PR China
2. Majorbio Pharm Technology Co.,Ltd., Shanghai 201203, PR China
3. School of Civil, Environmental and Architectural Engineering, Korea University, Anam-Dong,
Seongbuk-Gu, Seoul, South Korea
Corresponding Author
* Tel./fax: +86-21-65980400; E-mail address: [email protected] (Z. Wang)
S1
Section I Metagenomic DNA extraction protocol
Initially, 500 mL of S1 was filtered through a 0.1-μm filter membrane (Supor®-100, Pall
Corporation, U.S.). 20 mL of S2, S3 and S4 were centrifuged at 6000 rpm for 10 min, and the
sediments were recovered though decantation of the supernatant. The extraction of DNA from
microbial cells collected from the filter membrane (S1) and sediments (S2, S3 and S4) was then
conducted using the E.Z.N.A.® Soil DNA kit (Omega Bio-Tek, Inc., Norcross, GA, U.S.). Briefly,
a 15-mL tube containing 1.0 g of spiked samples, 0.5 g of glass beads, and a series of buffers was
subject to vortexing and bead beating. DNA samples were isolated by using isopropanol-kit
elution buffer, treated with inhibitor removal reagents and bound to spin columns. DNA was then
washed and eluted from columns with elution buffer according to the manufacturer’s protocol.
Afterwards, the quality of DNA samples was assessed using a 1.0 % (w/v) agarose gel
electrophoresis and the concentration measured with a NanoDrop spectrophotometer.
Table S1 Operating parameters of the MBR and CAS system
MBR
CAS system
Treatment capacity, m3/d
0.3
Treatment capacity, m3/d
60,000
Membrane flux, L/(m2 h)
15
Effluent suction cycle, min
10/2
Anaerobic zone HRTa, h
1.6
Anaerobic zone HRT, h
1.5
Anoxic zone HRT, h
6.7
Anoxic zone HRT, h
1.5
Oxic zone HRT, h
6.7
Oxic zone HRT, h
4.7
SRTb, d
60
SRT, d
10
MLSSc, g/L
4~5
MLSS, g/L
2.5
R1d
3
R1d
0.5~1.0
R2e
0.5
Ref
2~3
S2
Anaerobic and anoxic zone DO, mg/L
<0.2
Oxic zone DO, mg/L
1~3
a. HRT, hydraulic retention time; b. SRT, sludge retention time; c. MLSS, concentration of mixed liquors
suspended solids; d. R1, return rate of mixed liquor from oxic zone to anoxic zone; e. R2, return rate of mixed
liquor from anoxic zone to anaerobic zone; f. Ref, return rate of sludge to anaerobic zone; g. DO, concentration
of dissolved oxygen.
Fig. S1 Pairwise comparison of COG classification of S3-S1 and S3-S4.
S3
80
80
Predicted proteins
S1
S2
60
60
40
40
20
20
0
0
0
20
40
60
80
80
0
40
60
80
40
60
80
80
S3
Predicted proteins
20
S4
60
60
40
40
20
20
0
0
0
20
40
60
80
SSU rRNA
0
20
SSU rRNA
Fig. S2 Correlation between different taxonomic results based on SSU rRNA gene tags and
predicted proteins at phylum level.
S4
500
Flavobacterium
400
300
200
100
0
0.0
.2
.4
.6
.8
1.0
.2
.4
.6
.8
1.0
.4
.6
.8
1.0
.4
.6
.8
1.0
500
Thauera
400
Number of reads
300
200
100
0
0.0
500
Dechloromonas
400
300
200
100
0
0.0
.2
200
Comamonas
150
100
50
0
0.0
.2
Identity
Fig. S3 Identities of protein-coding genes of S1 with the representative sequences of genera of
Flavobacterium, Thauera, Comamonas and Dechloromonas. Abundances of the predicted
proteins are corrected by mapping the sequencing reads to the assembly contigs.
S5
35
S3
S2
S1
30
b
20
15
10
5
Thiobacillus
Rubrivivax
Chryseobacterium
Rhodanobacter
Kangiella
Azospira
Polaromonas
Chthoniobacter
Azoarcus
Acidovorax
Thauera
Solitalea
Sulfurovum
Pseudogulbenkiania
Neisseria
Hahella
Candidatus_Accumulibacter
Sulfurimonas
Leptothrix
Dechloromonas
Chromobacterium
Pseudomonas
Flavobacterium
No_Rank
Other
0
S6
Other
0
30
25
20
15
Number of sequences
S3
S2
S1
35
Pseudovibrio
Photobacterium
Nitrobacter
Hydrogenivirga
Sulfuricella
Desulfurispirillum
Brevundimonas
Thioalkalivibrio
Campylobacter
Thiocapsa
Methylobacter
Nitrospira
Thiobacillus
Maricaulis
Methylibium
Hahella
Aromatoleum
Curvibacter
Burkholderia
Stenotrophomonas
Magnetospirillum
Sulfurovum
Sorangium
Nitratiruptor
Beggiatoa
Variovorax
Sterolibacterium
Saccharophagus
Flavobacterium
Chromobacterium
Pseudoxanthomonas
Pseudogulbenkiania
Leptothrix
Azoarcus
Aeromonas
Shewanella
Polaromonas
Hydrogenophaga
Tolumonas
Sulfurimonas
Candidatus_Accumulibacter
Arcobacter
No_Rank
Methyloversatilis
Albidiferax
Acinetobacter
Thiovulum
Rhodanobacter
Alicycliphilus
Acidovorax
Azospira
Sulfuricurvum
Dechloromonas
Thauera
Sulfurospirillum
Geobacter
Pseudomonas
25
Number of sequences
40
a
10
5
0
Other
Cecembia
Azoarcus
Cupriavidus
Pedobacter
Haliscomenobacter
Maribacter
Riemerella
Belliella
Azospira
Alicycliphilus
Rhodothermus
Solitalea
Thauera
Dyadobacter
Runella
Niastella
Thiobacillus
Niabella
Candidatus_Accumulibacter
Dechloromonas
Campylobacter
Pseudomonas
Wolinella
Acidovorax
Flavobacterium
No_Rank
Sulfurimonas
0
d
S3
S2
S1
12
10
8
6
Number of sequences
S3
S2
S1
14
Other
Zobellia
Sulfurovum
Methylomicrobium
Nitrosomonas
Cecembia
Thauera
Maribacter
Alicycliphilus
Marivirga
Dechloromonas
Leptospira
Leptonema
Indibacter
Muricauda
Cupriavidus
Polaromonas
Azoarcus
Leptothrix
Sphingomonas
Syntrophobacter
Imtechella
Flavobacterium
Sulfurimonas
Haliscomenobacter
Candidatus_Accumulibacter
Acidovorax
No_Rank
Pseudomonas
20
15
10
Number of sequences
16
c
4
2
30
25
5
Fig. S4 Taxonomy of the coding genes in denitrification. (a) coding genes of nar, (b) coding
genes of nirK (NO-forming), (c) coding genes of norB, and (d) coding genes of nosZ. Taxonomy
of the corresponding sequences was conducted using BLAST search against NCBI database
S7
(ftp://ftp.ncbi.nih.gov/pub/taxonomy). For each sequence, the blast result with the highest bit
score was selected.
(a)
(b)
0.35
4.0
S3
0.30
0.25
0.20
0.15
0.10
0.05
r(amo)/r(AOB) (E-3)
Relative abundance (%)
S2
0.00
3.0
2.0
1.0
0.0
S2
S3
Fig. S5 a) Relative abundances of AOB and NOB in S2 and S3 and b) r(amo)/r(AOB) in S2 and
S3. r(amo) represents the relative abundances of amo in predicted proteins and r(AOB) the
relative abundances of AOB based on 16S rRNA gene tags.
Table S2 Abundances of coding genes associated with nitrification, denitrification and
dissimilatory nitrate reduction to ammonium.a
Definition
Gene name
S1
S2
S3
Ammonia monooxygenase
amoA
0
0
34
amoB
0
0
39
amoC
0
4
35
Hydroxylamine oxygenase
hao
202
33
81
Nitrate reductase
napA
784
355
585
napB
364
179
137
napC
247
192
148
S8
napG
746
109
220
napH
874
381
296
narB
0
0
0
narC
0
0
0
narG
2025
1306
1013
narH
630
380
453
narI
239
321
199
nirK
1162
1936
1182
nirS
0
0
0
norB
1140
2346
1512
norC
216
441
305
Nitrous oxide reductase
nosZ
1202
2721
3628
Nitrite reductase
nrfA
1157
138
136
nrfB
22
0
0
nrfC
218
0
0
nrfD
260
0
0
nrfH
0
0
0
Nitrite reductase (NO-forming)
Nitric oxide reductase
a. Total reads of S1~S3 mapped to KEGG pathways are 5156200, 6080374 and 5624636, respectively.
S9