Supporting Information
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Impact of microbial diversity depletion on xenobiotic degradation
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by sewage activated sludge
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Guillermina Hernandez-Raquet, Elodie Durand, Florence Braun, Cristiana Cravo-Laureau,
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Jean-Jacques Godon
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Experimental procedures
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Activated sludge community dilution
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The activated sludge communities displaying a diversity gradient were produced by dilution
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to extinction methodology. Activated sludge was collected from the aeration tank of an urban
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sewage treatment plant (Narbonne, France) mainly handling domestic wastewater. For
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practical reasons, due to the amount of laboratory effort required to carry out the experiments,
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they were realized in three series of experiments. Then, in order to ensure a same inoculum
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for all the experiments, activated sludge was homogenised and aliquots of 50 mL were
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prepared; aliquots were simultaneously frozen and immediately kept at –20°C. Forty mL of
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thawed activated sludge were used to inoculate 360 mL of semi-synthetic sewage medium
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(SSM; dilution 10-1) and then, activated sludge was serially diluted (until to 10-8). To ensure
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the same biomass concentration of diluted cultures, a regrowth step was performed in SSM
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medium. The SSM was prepared as follows: a large volume of fresh raw sewage was
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collected at the inlet of the WWTP and transported to the lab at ambient temperature (10 min
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travel). In the lab, it was immediately centrifuged (18 500 g, 15 min, 4°C) to eliminate
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suspended particulate matter; the supernatant was then filter-sterilised (0.2 µm, Whatman)
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and collected in sterile flasks. To the sewage was added a sterile solution of peptone (670
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mg.L-1) and meat extract (700 mg.L-1) to obtain a final chemical oxygen demand (COD) of
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2 000 mgO2.L-1. The final pH was adjusted to 7.4 with filter-sterilised NaOH (10 % w/v).
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SSM was selected as a growth medium to simulate the natural environment of activated
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sludge while preventing possible interference of suspended matter on further analysis.
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Triplicate cultures inoculated with dilutions 10-1 to 10-8 were incubated in a rotary shaker
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(30°C, 150 rpm). During incubation, duplicate measurement of growth (optical density, OD)
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and residual substrate concentration [measured as COD and total organic carbon (TOC)] were
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monitored to determine the end of the growth phase (stationary phase). COD was determined
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with Spectroquant kits (Merck), based on ISO 15705 while TOC was measured with a TOC
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analyser (Shimadzu Sci. Instruments). Cultures were stopped when they reached the
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stationary phase (no more OD increase); then, they were stopped at different incubation times
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as a function of dilution. At this time, the three biological replicate cultures inoculated with
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each dilution were sampled to assess biomass concentration (dry weight, measured in
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duplicate, n=3), microbial diversity (duplicate DNA extraction for 16S rDNA-SSCP analysis;
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n=3), metabolic diversity (measured in triplicate, n=3) and phenanthrene degradation capacity
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(test realised in duplicate for two replicated dilutions; n=2).
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Community structure and diversity analysis
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Nucleic acid extractions from communities obtained after regrowth of diluted activated sludge
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were performed using 1.5 mL of each culture. Aliquots were centrifuged (18 000 g, 10 min)
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and the pellet was used for total DNA extraction using the QIAamp DNA stool (Quiagen,
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Courtaboeuf, France) following the manufacturer’s instructions. The highly variable V3
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region of 16S rDNA was PCR amplified as described previously (Peu et al., 2006). The
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expected size of the bands was around 200 bp length, and was checked using a 100 bp DNA
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ladder (Invitrogen). The 16S rDNA-PCR products were subsequently analysed by SSCP
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capillary electrophoresis following. One microlitre of each PCR product (diluted between 1
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and 10 times depending on the band intensity on the gel) was mixed with 18.925 µL of
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formamide and 0.075 µL of internal standard GeneScan ROX GS 400 (Applied Biosystems).
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Samples were heat-denatured at 95°C for 3 min and immediately placed on ice. They were
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then subjected to capillary electrophoresis for 35 min in an ABI Prism 3130 genetic analyser
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(3130 Genetic Analyser, Applied Biosystems Hitachi, Courtaboeuf, France; Wery et al.,
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2008) with four 50 cm capillary tubes. 16S rDNA-SSCP fingerprints were aligned with the
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internal standard (GeneScan ROX) and the entire area of SSCP profiles was normalized and
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the relative sub-peak background areas were calculated with StatFingerprints, software
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developed with R programme (R Development Core Team 2005, htpp://www.r-project.org)
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especially designed for 16S rDNA-SSCP analysis. From 16S rDNA-SSCP data a Simpson
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diversity index was estimated using StatFingerprints by summarizing a complex community
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represented in the SSCP profile by a single value by taking into account the number of peaks
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(number of operational taxonomic units, OTUs) and the area under each peak (relative
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abundance). The Simpson diversity index (D’) was estimated as the negative logarithm of the
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number of effective OTUs in a community, designated for each profile as 𝐷′ = −𝑙𝑛 ∑𝑆𝑖=1 𝑛𝑖2
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where ni is the relative abundance of individual OTUs (area under each SSCP peak) in the
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community (total fingerprint area) (Michelland et al., 2009; Rosenzweig, 1995). The same
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software was used to estimate Euclidian distance matrices for 16S rDNA-SSCP fingerprints
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and Biolog Ecoplates (Michelland et al., 2009). Pareto-Lorenz distribution curves were
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plotted bases on normalized SSCP-profiles. The SSCP-OTUs was ranked from high to low
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based on their relative abundance. The x axis represents the cumulative normalized proportion
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of SSCP-OTUs and the y axis represents the cumulative area under the peaks. In the Pareto-
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Lorenz curve, the perfect evenness line (45° diagonal) represents the highest evenness; as the
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curve deviates from this perfect line, the evenness of the community decreases (Wittebolle et
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al., 2008).
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Biomass production.
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Biomass concentration was determined as dry weight for the inoculum (initial biomass in
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activated sludge) and at the end of the growth phase (final biomass) of the diluted activated
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sludge. Twenty five millilitres of culture were centrifuged (18 000 g, 15 min) to recover the
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biomass. The cells were suspended in MilliQ water and dried at 105°C for 24 h.
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Metabolic diversity
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Biolog Ecoplates (Biolog, Hayward, CA, USA) were used to assess the functional metabolic
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diversity of the communities obtained from diluted activated sludge (Garland & Mills, 1991;
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Paixao et al., 2007). Biolog Ecoplates contain, in three replicate wells, 31 carbon substrates
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and a control well containing no test substrate. Each well contains a tetrazolium salt which is
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a redox indicator of substrate oxidation. To inoculate Ecoplates, the biomass of 20 mL of
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microbial communities obtained from dilutions 10-1, 10-3, 10-5 and 10-8 were harvested by
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centrifugation at the end of the growth phase (18 500 g, 15 min). The supernatant was run off
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and the biomass was resuspended in a volume of sterile physiological water to ensure a
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similar biomass concentration (30 mg.L-1). 100 µL of this suspension per well were used as
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inocula. This inoculation procedure was used to overcome possible effects of inoculum
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density and residual nutriments from SSM. The Ecoplates were incubated at 30°C and
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absorbance at 590 nm was monitored every 24 h until OD reached a stable value (no more
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than 5 days to prevent absorbance increase by liquid evaporation from the wells). For
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absorbance measurement, the Ecoplates were shaken for 10 s prior to reading and an average
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OD value of 25 measurements per well were recorded using a microplate reader (Nanoquant
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Infinite 2000, Tecan, Switzerland). Quantitative analysis of Biolog data was performed
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comparing tetrazolium dye colour development in each well after subtracting the average
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value of the three control wells containing no substrate. A threshold of 0.2 absorbance units
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was established and substrates with absorbance lower than this value were considered as non-
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degraded; for these negative wells, an OD of 0 was considered for further analysis. For OD
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values higher than 0.21 units, statistical comparisons between the raw OD data of substrate-
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containing wells and the control wells were performed using the Statigraphics Plus tool to
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identify significant differences (p=0.05) between the standard deviation (Fisher test), the
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mean (t test) and the median values (W test) of replicates. Substrate richness was defined as
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the number of substrates degraded significantly (p=0.05) in all replicates. The average well
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colour development (AWCD) was estimated adding the absorbance measured in positive
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wells divided by the total number of substrates (Garland, 1996).
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Phenanthrene Mineralisation Assay
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Phenanthrene mineralisation was assessed by respirometry based on the OECD 301F method
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for measuring the degradability of chemicals (OECD Guidelines, 1993). This respirometric
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test was carried out using the OxiTop OC110 system (WTW, Weilheim, Germany) which is
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based on a manometric principle. During microbial activity, oxygen is taken from the gas
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phase of the hermetically-sealed reaction vessels, while carbon dioxide released from
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respiration is absorbed by KOH contained in a small tube placed inside the reaction vessel.
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The ensuing reduction in pressure inside the system is continuously monitored. From the
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resulting data, the quantity of oxygen required for the biodegradation of organic compounds
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(biological oxygen demand: BOD) can be directly calculated in mg.L-1. For respirometric
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tests, a mineral solution was prepared by mixing, per litre of distilled water, 10 mL of solution
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A (8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g NaHPO4 2H2O, 0.5 g NH4Cl per litter of distilled
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water) with 1 mL of each solution B (27.5 g.L-1 CaCl2), C (22.5 g.L-1 MgSO4 x 7H2O) D (0.25
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g.L-1 FeCl3 x 6H2O) and E (15.8 g.L-1 allythioure) (OECD Guideline, 1993; Reuschenbach et
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al., 2003; Stasinakis et al., 2008). Solutions D and E were filter-sterilised (0.2µm, Whatman)
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while all others solutions were autoclaved (120°C, 20 min). Inocula for respirometric tests
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were prepared by collecting by centrifugation (18 000 g, 15 min) the biomass corresponding
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to a known dry weight (determined as above). The pellet was suspended in a precise volume
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of mineral solution to ensure an initial biomass concentration of 30 mg.L-1 (Stasinakis et al.,
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2008). In these OxiTop respirometric tests, the range of measurement was set at 0–200 mg.L-1
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and all the respirometric measurements were carried out in a final volume of 158 mL.
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Phenathrene was used as sole carbon source at an initial concentration of 50 mg.L-1,
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corresponding to a theoretical oxygen demand (ThOD) of 148 mgO2.L-1. For that, 100 µL of a
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stock phenathrene solution prepared in acetone (79 mg.mL-1) were added to the sterilized
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flasks. The acetone was allowed to evaporate until dryness then, the media was added. In
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parallel experiments, as a control of biological activity, an easy degradable substrate (acetate)
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at the same ThOD was used as sole carbon source. A second control without a substrate was
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also included for monitoring endogenous respiration. Respirometric tests were performed in
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duplicate with two duplicates (biological duplicates) of communities obtained from activated
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sludge dilutions of 10-1, 10-3, 10-5 and 10-8 (four experiments for each dilution, n=2). They
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were incubated at 30 ± 2°C for 25 days. Mineralisation data were reported as a percentage of
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the initial ThOD.
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References
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StatFingerprints: a friendly graphical interface program for processing and analysis of
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microbial fingerprint profiles. Mol Ecol Res 9: 1359-1363.
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OECD Guidelines (1993). OECD 301F: Manometric respirometric test. In OECD Guidelines
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Wery, N., Bru-Adan, V., Minervini, C., Delgenes, J.P., Garrelly, L., and Godon, J.J. (2008).
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Dynamics of Legionella spp. and bacterial populations during the proliferation of L.
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Figure S1.
A
B
10-1
10-3
10-5
10-7
10-8
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Figure S1. 16S rRNA-SSCP fingerprints of the communities obtained by dilution/regrowth
procedure of activated sludge (two replicates). Level of dilution is indicated as 10-x. The xaxis showed the elution time in SSCP profiles (unit of ABI Prism 3130, Applied Biosystem
software) and y-axis showed the relative fluorescence intensity.
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Figure S2.
1.0
of SSCP-OTUs
abundance
normalized
Cumulative
of SSCP-peaks
abundance
Cummulative
0.9
0.8
0.7
10-1
10-2
10-3
10-4
10-5
10-6
10-7
10-8
0.6
0.5
0.4
0.3
0.2
0.1
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Cummulative
proportion
of SSCP-peaks
Cumulative
normalized
proportion
of SSCP-OTUs
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Figure S2. Pareto-Lorenz distribution curves based on normalized SSCP-profiles. The dashed
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line at the 0.2 x-axis is plotted to determine the Pareto values. The level of dilution is
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indicated as 10-x.