Microbial community structure of wastewater treatment subjected
to high mortality rate due to ozonation of return activated sludge
Siavash Isazadeh, Pinar Ozdural Ozcer, and Dominic Frigon*
Department of Civil Engineering and Applied Mechanics, McGill University, 817 Sherbrooke
Street West, Montreal, QC, H3A 0C3, Canada
*
Corresponding author
Email: dominic.frigon@mcgill.ca
Tel: +1-514-398, 2475
FAX: +1-514-398, 7361
SUPPLEMENTARY MATERIAL
Number of tables=2
1
Table S1. Physical and operational characteristic of two
pilot-scale reactor.
Parameters
Control
RAS-Ozonated
Influent Flow (m3/day)
1.81±0.09 1.89±0.11
Volume of aeration tank (m3)
1.045
1.015
Volume of clarifier (m3)
0.705
0.697
Aerated SRT (d)
6.06
7.17
RAS Flow/Influent Flow
1.5
1.3
RAS suspended solids (mg/L)
3,040
3,040
Ozone contact time (min)
-
45
Ozone flow rate (L/h)
-
2-6
2
Table S2. PCR primers and FISH probes used in this study.
Probes and primers
Label Sequence (5’–3’)
Binding position b
Target Group
FA %c
Annealing
temperature
References
FISH
EUB338a
FITC GCTGCCTCCCGTAG 16S (338–355)
Most Bacteria
0 – 50
NAd
(Amann 1990)
a
GAGT
Other Bacteria not detected by
EUB338-II
FITC GCAGCCACCCGTA 16S (338–355)
0 – 50
NA
(Daims 1999)
GGTGT
EUB338
Other
Bacteria not detected by
EUB338-IIIa
FITC GCTGCCACCCGTAG
16S (338–355)
0 – 50
NA
(Daims 1999)
GTGT
EUB338
ALF968
Cy3 GGTAAGGTTCTGCG 16S (968 - 985)
Alphaproteobacteria
35
NA
(Neef 1997)
CGTT
BET42a
Cy5 GCCTTCCCACTTCG
23S (1027–1043) Betaroteobacteria
35
NA
(Manz 1992)
TTT
GCCTTCCCACATCG
Competitor for BET42a
35
NA
TTT
GAM42a
Cy3 GCCTTCCCACATCG 23S (1027–1043) Gamaproteobacteria
35
NA
(Manz 1992)
TTT
GCCTTCCCACTTCG
Competitor for GAM42a
35
NA
AGTTAGCCGGTGCT 16S (495–512)
(Lucker et al.
DELTA495a
Cy5 TTT
Deltaproteobacteria
35
NA
TCCT
2007)
AGTTAGCCGGTGCT
Competitor for DELTA495a
35
NA
TCTT
CFB563
Cy5 GGACCCTTTAAACC 16S (563–580)
Flavobacteria
20
NA
CAAT
LGC354a
Cy5 TGGAAGATTCCCTA
16S (354–371)
Firmicutes
NA
(Meier et al. 1999)
CTGC
HGC69a
Cy3 TATAGTTACCACCG 23S (1901–1918) Actinobacteria
20
NA
(Roller 1994)
CCGT
Pyrosequencing
CCTACGGGAGGCA 16S (341-357)
(Muyzer et al.
341F
NA
Most Bacteria
NA
NA
GCAG
CCGTCAATTCCTTT 16S (891-907)
907R
NA
Most Bacteria
NA
NA
(Lane 1993)
et al. 1985)
GAGTTT
Cloning
ACTCCTACGGGAG 16S (338-354)
Non-EUB338F
NA
Most Bacteria
NA
56
(Amann 1990)
GCAGCAG
TACGTGTGAAGCCC
Meth1215Re
NA
16S (-1199-1215) Methylophilaceae
NA
56
this study
TGGC
AATACGACTCACTA
T7 Universal Primer
NA
TAG
GTTTTCCCAGTCAC
M13 Universal primer NA
16s (20-41)
GAC
a EUB338, EUB338-II, EUB338-III were used in the mixture called EUB mix
b rRNA target site (Escherichia coli numbering)
c FA, formamide concentration in the hybridization buffer. For salt concentration in wash buffer see(Nielsen and Daims 2009)
d NA: Not Applicable
e Meth1215R coverage (91% of Methylophilaceae) and specificity(88%Methylophilaceae) based on ribosomal database project ( RDP Release 10, accessed in June
2012)
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References
Amann, R.I., 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow
cytometry for analyzing mixed microbial populations. Applied and Environmental
Microbiology 56(6), 1919.
Daims, H., 1999. The domain-specific probe EUB338 is insufficient for the detection of all
bacteria: Development and evaluation of a more comprehensive probe set. Systematic
and Applied Microbiology 22(3), 434.
Lane, D.J., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L., Pace, N.R., 1985. Rapid
determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proceedings
of the National Academy of Sciences 82(20), 6955-6959.
Lucker, S., Steger, D., Kjeldsen, K.U., MacGregor, B.J., Wagner, M., Loy, A., 2007. Improved
16S rRNA-targeted probe set for analysis of sulfate-reducing bacteria by fluorescence in
situ hybridization. Journal of Microbiological Methods 69(3), 523-528.
Manz, W., 1992. Phylogenetic oligodeoxynucleotide probes for the major subclasses of
proteobacteria: problems and solutions. Systematic and Applied Microbiology 15(4), 593.
Meier, H., Amann, R., Ludwig, W., Schleifer, K.H., 1999. Specific Oligonucleotide Probes for in
situ Detection of a Major Group of Gram-positive Bacteria with low DNA G+C Content.
Systematic and Applied Microbiology 22(2), 186-196.
Muyzer, G., de Waal, E.C., Uitterlinden, A.G., 1993. Profiling of complex microbial populations
by denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59(3),
695-700.
Neef, A., 1997. Anwendung der in situ Einzelzell-Identifizierung von Bakterien zur
Populationsanalyse in komplexen mikrobiellen Biozönosen, Technische Universität
München Munich, Germany.
Nielsen, P.H., Daims, H., 2009. FISH handbook for biological wastewater treatment:
identification and quantification of microorganisms in activated sludge and biofilms by
FISH, IWA Publishing, London, UK.
Roller, C., 1994. In situ probing of Gram-positive bacteria with high DNA G+ C content using
23S rRNA-targeted oligonucleotides. Microbiology 140(10), 2849.
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