Biocarriers improve bioaugmentation efficiency of a rapid sand filter for the treatment of 2,6-dichlorobenzamide (BAM)-contaminated drinking water

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1 Biocarriers improve bioaugmentation efficiency of a rapid sand filter for the treatment of 2,6-dichlorobenzamide (BAM)-contaminated drinking water Authors: Benjamin Horemans 1,*, Bart Raes 1, Johanna Vandermaesen 1, Yanti Simanjuntak 1, Hannelore Brocatus 1, Jeroen T Syen 1, Julie Degryse 2, Jos Boonen 2, Janneke Wittebol 3, Ales Lapanje 4,5, Sebastian R. Sørensen 6,7, Dirk Springael 1 1 Division of Soil and Water Management, Department of Earth and Environmental Sciences, Faculty of Bioscience Engineering, KULeuven, Kasteelpark Arenberg 20 bus 2459, 3001 Heverlee, Belgium 2 De Watergroep, Vooruitgangstraat 189, 1030 Brussel, Belgium 3 Bioclear, Rozenburglaan 13, 9727 DL Groningen, The Netherlands 4 Josef Stefan Institute, Jamova 49, 1000 Ljubljana, Slovenia 5 National Research Saratov State University, Astrakhanskaya 83, Saratov, Russian Federation 6 Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Øster Voldgade 10, 1350 Copenhagen K, Denmark 7 Bacterial Discovery, Novozymes A/S, Krogshoejvej 36 DK-2880 Bagsvaerd, Denmark (present address) S1

2 Table of contents qpcr protocol for bbda gene quantification..s3 Determination of total cell numbers, total cell loss, cell loss rates and AOC based on measured BAM removal rates S4 Table S1. Mean value of general characteristics of the groundwater extracted at Egenhoven- West S5 Table S2: Regressions of the total amount of calculated cells in the column and the measured cells in the effluent.s6 Figure S1. Scheme of the pilot scale...s7 Figure S2. Gene copy numbers of the catabolic gene bbda in the top layer..s8 Figure S3. Copy numbers of the catabolic gene bbda in the effluent..s9 Figure S4. Copy numbers of the catabolic gene bbda in the column at different depths at the end of the experiment....s10 Figure S5: The cell loss rate in the column expressed as bbda numbers/m³ water treated for the suspended cells and the immobilized cells for expt 1 and expt 2...S11 S2

3 qpcr protocol for bbda gene quantification qpcr was performed for each sample in duplicate in a Rotor Gene Real-Time Centrifugal DNA Amplification Apparatus (Corbett Research) using the SYBR Green detection method. The 15 μl reaction mixture contained 7.5 µl ABsolute qpcr SYBR Green mix 2x (Thermo Scientific), 0.3 µl forward primer bbda F RT (5 -ATATCACGGCCGGTACTATGCCAA-3 ) (200 nm), 0.3 µl reverse primer bbda R RT (5 -TCTTCCAAGATCGAACAACCCGGA-3 ) (200 nm) (Table 3), 3.9 µl nuclease free water and 3 µl of template DNA. The amplification reactions included 15 min at 95 C followed by 40 cycles of 20 s at 95 C, 20 s at 60 C and 20 s at 72 C. Fluorescence acquisition was performed at each cycle after the elongation step at 72 C. Used DNA standards were amplicons derived by conventional PCR from MSH1 genomic DNA and that were serially diluted (10-fold) from 10 8 copies/µl till 1 copy/µl. Conventional PCR reaction mixtures (50 µl) consisted of 0.25 µl Taq DNA polymerase (DreamTaq DNA polymerase, Thermo Scientific), 2.5 µl of 10 µm forward primer bbda F1 (5 - ATGCCCAGTGGTGCAAATCTGCCA-3 ), 2.5 µl of 10 µm reverse primer bbda R1543 (5 -CTTCTTGCGCCAATCCCCAGACTT-3 ), 1 µl of 10 mm of each dntp (Invitrogen), 5 µl 10x DreamTaq Green Buffer (Thermo Scientific), 5 µl 1% BSA (bovine serum albumin), 30 μl PCR-H2O and 1 µl (10 ng) of MSH1 genomic DNA. The amplification reaction for bbda was 5 min at 94 C followed by 35 cycles of 1 min at 94 C, 1 min at 59 C and 1 min at 72 C and ending with a final 5 min at 72 C. The amplicons were purified by agarose gel electrophoreses and subsequent gel extraction using the QIAquick Gel Extraction Kit (Qiagen) followed by purification with the Qiaquick PCR Purification Kit (Qiagen). DNA purity and concentrations were determined using Qubit (ThermoFisher). S3

4 Determination of total cell numbers, total cell loss, cell loss rates and AOC based on measured BAM removal rates The total BAM-degrading MSH1 cell numbers bcalc (cells) in the entire column for the suspended and immobilized cell strategy executed in expt 1 and expt 2 were calculated from the BAM removal rate rbam (µg BAM/min) using the following equation: Eq. 1. bcalc = rbam v with the specific degradation rate v (µg BAM/cell/min) following Monod equation (Eq. 2): Eq. 2: v = VMAX C BAM C BAM+K BAM with VMAX being the maximal specific degradation rate, CBAM the BAM concentration in the groundwater (µg BAM/L) and KBAM the Monod constant for BAM. The number of cells bcalc, batch was based on kinetics reported for fresh suspended MSH1 cells (VMAX of 5.4±0.2 x µg BAM/cell/min, KBAM of 100±14 µg/l 1 ). The number of cells bcalc, starved was based on kinetics reported for C and N starved MSH1 cells in biofilms (VMAX of 1.7±0.05 x µg BAM/cell/min and KBAM of 630±58 µg/l 1 ). To calculate the total cell loss rate Rcalc (cells/m³) in function of the treated volume VW (m³), the calculated cell numbers bcalc, starved in function of Vw was described by a logarithmic regression (Eq. 3): Eq. 3 bcalc, starved = a0 + a1 ln(vw) of which the first derivative (Eq. 4) was determined. Eq. 4 Rcalc, starved = bcalc, starve VW The measured cell loss rate or the measured bbda numbers in the effluent Reff (cells/m³) in function of VW was fitted (Eq. 5). Eq. 5 Reff = a1 VW a2 The AOC (µg AOC/L) in the treated groundwater available to MSH1 was calculated using Eq. 6 with the Rcalc, starved at near steady state conditions and the conversion factor CAOC.of 4.1 x 10 6 cells/µg AOC 2. Eq. 6 AOC = CAOC Rcalc, starved S4

5 Table S1. Mean value of general characteristics of the groundwater extracted at Egenhoven- West Characteristic Mean±SD* Unit Temperature 11.4±0.7 C Number of culturable bacteria at 22 C 12.9±14.2 CFU/mL Non-purgeable organic carbon 1.8±1.6 mg/l Ammonia 0.02±0.01 mg/l Nitrate 48.1±2.7 mg/l Nitrite 0.01±0.01 mg/l ortho-phosphate 0.1±0.0 mg/l P *Mean values 28 sampling moments before and during expt 1 and expt 2 with SD. S5

6 Table S2: Regressions of the total amount of calculated cells in the column and the measured cells in the effluent Predicted total cells in column [Cells/column] y = y0 + a. log10(v r) Predicted cells in effluent [Cells/m³] y = a. V r b Coefficient R² ANOVA P-value Coefficient R² ANOVA P-value Suspended expt 1 y0 3E a 9.25E < a -1E+12 b Embedded expt 1 y0 4E a 5.06E < a -5E+11 b Suspended expt 2 y0 4E < a 2.27E < a -1E+12 < b Embedded expt 2 y0 4E < a 6.76E < a -1E+12 < b S6

7 Figure S1. Scheme of the pilot scale column 1 and control C1 (suspended cell strategy) and column 2 and control C2 (immobilized cell strategy) (left) and picture of the complete set-up of the four sand filter columns (right) at the DWTP in Egenhoven, Belgium. S7

8 Figure S2. bbda numbers ( ) in the top layer of the column in function of the treated volume of water. For suspended cells, bbda numbers in the sand is presented, while for immobilized cells, bbda numbers in the sand and the carrier combined is presented. Pilot scale sand filters were operated for 44 days (8 m³) in case of suspended cell strategy expt 1 (top) and for 88 days (16 m³) in case of suspended cell strategy (middle) and immobilized cell strategy (bottom) in expt 2. Values are averages (n = 4) of two duplicate samples analyzed in two-fold by qpcr with standard deviation indicated by error bars. S8

9 Figure S3. bbda numbers ( ) in the effluent during groundwater treatment in function of treated water volume. Pilot scale sand filters were operated for 44 days (8 m³) in case of expt 1, and for 88 days (16 m³) in case of expt 2. Values are averages (n = 4) of two duplicate samples analyzed in two-fold by qpcr with standard deviation indicated by error bars. S9

10 Figure S4. bbda numbers in the column at different depths during groundwater treatment. For suspended cells, bbda numbers in the sand is presented, while for immobilized cells, bbda numbers in the sand and the carrier combined is presented. Pilot scale sand filters were operated for 44 days (8 m³) in case of the suspended cell strategy in expt 1 (top) and for 88 days (16 m³) in case of the suspended (middle) and immobilized cell strategy (bottom) in expt 2. Values are averages (n = 4) of two duplicate samples analyzed in two-fold by qpcr with standard deviation indicated by error bars. S10

11 Figure S5: The cell loss rate in the column expressed as bbda numbers/m³ water treated for the suspended cells (top) and the immobilized cells (bottom) for expt 1 and expt 2. The round (suspended) and square (immobilized) markers are the measured bbda numbers in the effluent. Values are averages (n = 4) of two duplicate samples analyzed in two-fold by qpcr with standard deviation indicated by error bars. The power regression (y = y0 +x a ) of the measured data (averages) is a full line. The calculated loss of bbda numbers is the first derivative of calculated bbda numbers in the column based on removal rate and the specific degradation rate of MSH1 in starvation conditions (Figure 2, SI Table S2) and is indicated by the dashed line. References 1. Sekhar, A.; Horemans, B.; Aamand, J.; Sørensen, S. R.; Vanhaecke, L.; Bussche, J. V.; Hofkens, J.; Springael, D., Surface colonization and activity of the 2,6-dichlorobenzamide (BAM) degrading Aminobacter sp. strain MSH1 at macro- and micropollutant BAM concentrations. Environ. Sci. Technol. 2016, 50, (18), van der Kooij, D.; Visser, A.; Hijnen, W. A. M., Determining the concentration of easily assimilable organic carbon in drinking water. Journal American Water Works Association 1982, 74, (10), S11