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Supplementary information
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The role of quorum sensing signalling in EPS production and the assembly of a sludge
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community into aerobic granules
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Chuan Hao Tan, Kai Shyang Koh, Chao Xie, Martin Tay, Yan Zhou, Rohan Williams, Wun
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Jern Ng, Scott A. Rice, Staffan Kjelleberg*
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Corresponding author: LASKJELLEBERG@ntu.edu.sg
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Supplementary materials and methods
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Bioreactor operation
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A sequencing batch reactor was seeded with 3 000 mg L-1 MLVSS (mixed liquor volatile
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suspended solids) of floccular sludge from a bioreactor undergoing stable simultaneous
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nitrification, denitrification and phosphorus removal (SNDPR) performance (Zhou et al.
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2010), with a final working volume of 4 L at 22 ºC. The operation of the bioreactor involved
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a 6 h cycle comprised of two different phases: Phase I - feeding (8 min), anaerobic (60 min),
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aerobic (80 min at day 0 and gradually increased to 95 min by the end of week 5) and anoxic
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(40 min at day 0 and gradually increased to 50 min by the end of week 5); Phase II - feeding
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(2 min), anaerobic (30 min), aerobic (40 min at day 0 and gradually increased to 70 min by
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the end of week 5) and anoxic (30 min). Each cycle was completed with a settling stage (60
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min at day 0 and gradually decreased to 5 min by the end of week 5) and a 10 min decanting
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stage. The settling time was maintained at 5 min per cycle from week 6 onwards. A total
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volume of 2 L synthetic wastewater (SWW) (Zhou et al. 2010) was fed to the bioreactor
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during the feeding stages of each cycle and 2 L of treated effluent was discharged at the end
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of the cycle, giving a hydraulic retention time of 12 h. The pH was monitored online and
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maintained at a range of pH 6.8 to pH 8.2 under the control of programmable logic controller
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(PLC) via dosing of either 9.12 g L-1 HCl or 10.0 g L-1 NaOH. Nitrogen gas was sparged
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intermittently at a flow rate of 3.0 L min-1 into the bioreactor throughout the operation except
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for the settling and decanting stages to provide complete mixing and hydrodynamic shear
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force for granulation. Air was supplied at a flow rate of 3.0 L min-1 during the aerobic stage
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to achieve dissolved oxygen (DO) concentration of 3.0-4.0 mg L-1, through the control of
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PLC.
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Synthetic wastewater
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Synthetic wastewater (SWW), simulating typical domestic wastewater composition, was
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prepared as described (Smolders et al. 1994, Zhou et al. 2010) with the following
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components: NaCH3COOH·3H2O, 320 mg L-1; propionic acid, 33 mg L-1; NH4Cl, 77 mg L-1;
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K2HPO4, 28 mg L-1; KH2PO4, 22 mg L-1; MgCl2·7H2O, 206 mg L-1 and CaCl2·2H2O, 22 mg
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L-1, as well as trace elements comprised of FeCl3·6H2O, 0.1 mg L-1; H3BO3, 0.01 mg L-1;
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CuSO4·5H2O, 0.002 mg L-1; KI, 0.014 mg L-1; MnCl2·4H2O, 0.009 mg L-1; Na2MoO4·2H2O,
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0.0045 mg L-1; ZnSO4·7H2O, 0.009 mg L-1; CoCl2·6H2O, 0.01 mg L-1 and EDTA, 0.75 mg L-
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1
.
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Detection of AHLs in situ by Escherichia coli JBA357 bioassay
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For detection of AHLs produced in situ, a 96-well plate bioassay was adopted using the
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bioreporter E. coli JBA357 which expresses green fluorescent proteins (GFP) upon detecting
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AHLs (Andersen et al. 2001). The overnight culture of E. coli JBA357 was first diluted 1:5
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(v/v) with fresh LB5 medium and 100 µL of the diluted bioreporter culture were added to 100
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µL of sludge samples in a 96 well plate. The microtitire plate was subsequently incubated at
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22 °C, with constant shaking at 200 rpm for 4 h prior to the visualization of green fluorescent
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cells
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excitation/emission wavelength of 488 nm/522-535 nm. Heat inactivated sludge samples
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were included as negative controls. SWW spiked with synthetic AHL, 3OC6-HSL, at 10 nM
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was used as a positive control for the in situ AHL detection assay.
using
confocal
laser
scanning
microscope
(LSM710,
Carl
Zeiss)
at
an
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AHL inactivation bioassay
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Synthetic AHLs were spiked into the overnight culture of isolates at a ratio of 1:9 to achieve
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a final concentration of 5 µM. The mixtures were incubated at 22 °C, with constant shaking
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at 200 rpm for 2 h. After centrifugation and UV sterilization, residual AHLs were quantified
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using the Agrobacterium tumefaciens A136 (Fuqua and Winans 1996) agar-based spot
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bioassay (Zhang et al. 2007). pH of the cultures was recorded at the end of the experiment.
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Escherichia coli JM109 (Yanisch-Perron et al. 1985) was included as the negative control.
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Supplementary references
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Andersen JB, Heydorn A, Hentzer M, Eberl L, Geisenberger O, Christensen BB et al. (2001).
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gfp-based
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communication. Appl Environ Microbiol 67: 575-585.
N-acyl
homoserine-lactone
sensor
systems
for
detection
of
bacterial
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Fuqua C, Winans SC. (1996). Conserved cis-acting promoter elements are required for
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density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes. J
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Bacteriol 178: 435-440.
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Smolders GJ, van der Meij J, van Loosdrecht MC, Heijnen JJ. (1994). Model of the anaerobic
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metabolism of the biological phosphorus removal process: stoichiometry and pH influence.
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Biotechnol Bioeng 43: 461-470.
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Yanisch-Perron C, Vieira J, Messing J. (1985). Improved M13 phage cloning vectors and
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host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103-119.
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Zhang HB, Wang LH, Zhang LH (2007). Detection and analysis of quorum-quenching
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enzymes against acyl homoserine lactone quorum-sensing signals. Curr Protoc Microbiol.
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John Wiley and Sons, Inc.
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Zhou Y, Ganda L, Lim M, Yuan Z, Kjelleberg S, Ng WJ. (2010). Free nitrous acid (FNA)
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inhibition on denitrifying poly-phosphate accumulating organisms (DPAOs). Appl Microbiol
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Biotechnol 88: 359-369.
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Titles and legends to supplementary figures
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Supplementary Figure S1. Total ion chromatogram of a mixture of 13 standard AHLs added
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to the blank sludge supernatant sample matrix (a) and an extract from the sludge supernatant
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sample (b), as analysed using LC-MS. Isolated peaks eluted at different times from the
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sample, e.g. peak eluted at 3.72 min, were subjected to fragmentation to yield an MS/MS
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spectrum (c). The example given here (c) shows that the 3.72 min peak contains a parent ion
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[M+H]+ at m/z 182, which is composed of two key product ions corresponding to the lactone
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ring at (m/z 102) and the acyl side chain (m/z 99), a fragmentation pattern matching with that
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of the synthetic C6-HSL (c, insert).
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Supplementary Figure S2. HPLC-MS/MS profiling of AHLs extracted from the sludge
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supernatant during granulation. The identity and quantity of individual AHLs present in each
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sample were compared with the multiple reaction monitoring profiles of 13 standard AHLs.
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A total of 6 AHLs are shown (the remaining 7 AHLs were below the detection limit). Error
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bars are defined as s.e.m. (n = 3, technical replicates). The dotted line separates the different
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developmental phases of granulation, i.e. Phases I to V, while n.d. represents ‘not
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determined’.
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Supplementary Figure S3. Successional changes in microbial diversity during granulation as
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assessed by the Simpson (circle) and the Shannon-Weaver (square) diversity indices. The
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dotted line separates the different developmental phases of granulation, i.e. Phases I to V.
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Supplementary Figure S4. The correlation between AHLs accumulated in the bulk liquid of
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the bioreactor and the abundance of specific community members:
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Xanthomonadaceae bacterium (Tag 6) and (b) the Comamonadaceae bacterium (Tag 17).
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Expression of 3OC8-HSL (grey bar) correlates positively with the abundance of Tag 6 (upper
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panel, circle) and negatively with Tag 17 (bottom panel, circle). n.d. represents ‘not
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determined’. (c) HPLC-MS/MS profile of AHLs released by the overnight cultures of
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Lysobacter brunescens R037 (open bar) and Stenotrophomonas sp. P088 (filled bar) isolated
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from the bioreactor. The 16S rRNA sequences of the R037 (NCBI accession no. KC252866)
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and P088 (NCBI accession no. KC252826) match 100% to Xanthomonadaceae bacteria, Tag
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6 and Tag 14, respectively. The identity and quantity of individual AHLs present in each
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sample were compared with the multiple reaction monitoring profiles of 13 standard AHLs.
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Error bars are defined as s.e.m. (n = 3, biological replicates). (d) Inactivation of AHL by
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Diaphorobacter nitroreducens R042 isolated from the bioreactor. 3OC12-HSL was added to
(a) the
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the overnight culture of R042 to a final concentration of 5 µM for 2 h and the remaining
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3OC12-HSL was assessed by the A. tumefaciens A136 spot bioassay. E. coli JM109 was
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included as the negative control. The 16S rRNA sequence of R042 matches 100% with the
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Comamonadaceae bacterium (Tag 17).
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Supplementary Figure S5. In situ detection of AHLs produced by the granular sludge
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community using the E. coli JBA357 bioreporter. Co-incubation of the sludge sample (a and
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b), or the heat-inactivated sludge sample (c) with the bioreporter at room temperature for 4 h
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with a constant shaking at 200 rpm prior to inspection by confocal laser scanning microscope.
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The heat-inactivated sludge sample serves as a background fluorescent control for the sludge
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biomass (auto-fluorescence). Additional controls for the bioassay include addition of
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exogenous 3OC6-HSL at 10 nM to the bioreporter, a positive control (d), and the presence of
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the bioreporter alone as a negative control (e). Detection of AHLs by the bioreporter is
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indicated by the appearance of green fluorescent cells. Images of green fluorescent cells were
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captured at 488 nm (i), the total unstained cells were visualized by differential interference
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contrast (ii) and the superimposed image of the two is shown in the right-hand panels (iii).
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The black arrows indicate the sludge biomass. Magnification: a, x160; b, x400; c, x120; d-e,
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x200; Scale bar, 100 μm.
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Title to supplementary tables
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Supplementary Table S1. Multi MRMs and optimized MS/MS parameters.
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Supplementary Table S2. Total RNA sequencing studies: Statistics of microbiota data.