Biotransformation of organic micropollutants by anaerobic sludge enzymes

1 SUPPLEMENTARY DATA Biotransformation of organic micropollutants by anaerobic sludge enzymes Lorena Gonzalez-Gil a*, Daniel Krah b, Ann-Kathrin Ghattas b, Marta Carballa a, Arne Wick b, Lissa Helmholzb, Juan M. Lema a, Thomas A. Ternes b a Department of Chemical Engineering, School of Engineering, Universidade de Santiago de Compostela, Rúa Lope Gómez de Marzoa, E-15782 Santiago de Compostela, Spain b Federal Institute of Hydrology (BfG), D-56068 Koblenz, Am Mainzer Tor 1, Germany * Corresponding author 2 Contents Section I. Chemical structure and physicochemical characteristics of OMPs ........... 3 Section II. Characterization of lysates ........................................................................ 6 Section III. Removal of OMPs in anaerobic sludge (positive control) ...................... 8 Section IV. Removal of OMPs under different lysate conditions .............................. 9 Section V. Identified TPs by LC–QToF-MS measurements .................................... 16 References ................................................................................................................ 17 3 Section I. Chemical structure and physicochemical characteristics of OMPs Table S1. Application and main physicochemical properties of the selected OMPs. OMP Application MW (g/mol) s (mg/L) H (atm m 3 /mol) pKa log Kow 10,11- DiOH-CBZ Metabolite of CBZ 270.2 290 n.f. 8.2 1.8 10,11-DiH-10-OH- CBZ (10-OH-CBZ) Metabolite of CBZ 254.3 550 n.f. 14.1 1.7 2-OH-CBZ Metabolite of CBZ 252.3 120 n.f. 9.2 2.2 3-OH-CBZ Metabolite of CBZ 252.3 110 n.f. 9.2 2.3 Acesulfame Artificial sweetener 163.2 5.8·10 5 9.0·10 -6 5.7 -1.3 Acetaminophen Analgesic 151.2 1.4·104 n.f. 9.4 0.46 N-acetyl-SMX Metabolite of SMX 295.3 1.2·103 3.1·10-15 5.7 1.2 Acyclovir Antiviral drug 225.2 2.5 3.2·10 -22 9.3 -1.6 Atenolol Beta blocker 266.3 1.3·104 1.4·10-18 9.6 0.16 Benzotriazole Corrosion inhibitor 133.2 9.7·103 3.1·10-16 9.6 1.1 Bezafibrate Lipid -regulator 361.8 0.36 6.1·10-11 3.8 4.2 Carbendazim Fungicide 191.2 29 7.5·10-10 4.2 1.5 Carbamazepine (CBZ) Anticonvulsant 236.3 112 1.1·10-10 15.9 2.5 Citalopram Antidepressant 324.4 31.1 2.7·10-11 9.8 3.7 Clarithromycin Antibiotic 748.0 0.34 1.7·10 -23 9.0 3.2 Climbazole Antimycotic 292.8 8.3 2.8·10-9 7.5 3.8 Codeine Opioid 299.4 9·103 7.6·10-14 8.2 1.2 Diatrizoate X-ray contrast medium 613.9 8.9 2.8·10 -18 2.2 1.4 Diclofenac Analgesic 296.2 2.4 4.7·10-12 4.2 4.2 Diuron Herbicide 233.1 42 5.0·10-10 nonionic 2.7 Erythromycin Antibiotic 733.9 1.4 5.4·10-29 8.9 3.1 Fluconazole Fungicide 306.3 1.0 n.f. 2.6 0.58 Iopromide X -ray contrast medium 791.1 23.7 1.0·10-28 4.2 -2.1 Iopamidol X -ray contrast medium 777.1 1.4·105 1.1·10-25 4.2 -2.4 Iomeprol X-ray contrast medium 777.1 155 n.f. 5.6 -1.8 Isoproturon Herbicide 206.3 65 1.1·10-10 nonionic 2.9 Mecoprop Herbicide 214.6 620 1.8·10-8 3.1 3.1 Metoprolol Beta blocker 267.4 1.7·10 4 1.4·10 -13 9.6 1.9 Oxazepam Anti -anxiety 286.7 20 5.5·10-10 10.9 2.2 Primidone Anticonvulsant 218.3 500 1.9·10-10 11.5 0.91 Sotalol β -blocker 257.3 1.4·105 1.0·10-10 10.1 0.24 Terbutryn Herbicide 241.4 25 2.1·10-8 4.3 3.7 Tramadol Opioid 263.4 1.2·103 1.5·10-11 9.4 3.0 Trimethoprim Antibiotic 290.3 400 2.4·10-14 7.1 0.9 Venlafaxine Antidepressant 277.4 267 2.0·10 -11 10.1 3.3 Molecular weight (MW), Henry’s law constant (H), solubility at 25 °C (s), acid dissociation constant (pKa), octanolwater coefficient (Kow). n.f. refers to not found. Data obtained from DrugBank. PhysProp, PubChem and The Human Metabolite (hmdb) databases. 4 Table S2. Chemical structures of the selected OMPs. Compound Chemical structure Compound Chemical structure 10,11-DiOH-CBZ 10-OH-CBZ 2-OH-CBZ 3-OH-CBZ Acesulfame Acetaminophen Acetyl-SMX Acyclovir Atenolol Benzotriazole Bezafibrate Carbendazim Carbamazepine (CBZ) Citalopram Clarithromycin Climbazole Codeine Diatrizoate Diclofenac Diuron 5 Erythromycin Fluconazole Iomeprol Iopamidol Iopromide Isoproturon Mecoprop Metoprolol Oxazepam Primidone Sotalol Terbutryn Tramadol Trimethoprim Venlafaxine 6 Section II. Characterization of lysates The measurement of protein concentration (Figure S1) and β-galactosidase (Figure S2), phosphatase (Figure S2) and acetate kinase activities (Figure S3) allow for selecting the most interesting lysates to perform the OMP transformation assays. Protein concentration and the enzymatic activities increased with bead beating time, although this improvement is less pronounced from 80 s onwards. In comparison with sonication, the maximum protein released through bead beating (160 s) was significantly lower, as well as β-galactosidase and acetate kinase activities, while phosphatase activity of both lysates was comparable. The use of phosphate buffer increased protein concentration by approximately 55% and 80% compared to the use of HN-buffer with sonication and bead beating, respectively. Acetate kinase activity also increased in phosphate buffer, especially upon lysis by bead beating. Nevertheless, β-galactosidase and phosphatase activities remained almost equal. Finally, the addition of detergents prior bead beating (80 s) clearly increased protein concentration, phosphatase and acetate kinase activities, but β-galactosidase activity diminished. Figure S1. Protein concentration in lysates obtained by different extraction procedures. Cell lysis by ultrasonication with HN-buffer (i.e., basic lysate, AD) or with phosphate buffer (AD PO4); cell lysis with HN-buffer by bead beating for 40 s (AD40), 80 s (AD80), 120 s (AD120), 160 s (AD160), with phosphate buffer by bead beating for 160 s (AD160PO4), with HN-buffer and detergents octylthioglucoside (AD80T), βdodecylmaltoside (AD80M) or CHAPS (AD80C) by bead beating for 80 s. Error bars depict the standard deviation of triplicated measurements (n=3). 7 Figure S2. β-galactosidase and phosphatase activities in lysates obtained by different extraction procedures. Cell lysis by ultrasonication with HN-buffer (i.e., basic lysate, AD) or with phosphate buffer (AD PO4); cell lysis with HN-buffer by bead beating for 40 s (AD40), 80 s (AD80), 120 s (AD120), 160 s (AD160), with phosphate buffer by bead beating for 160 s (AD160 PO4), with HN-buffer and detergents octylthioglucoside (AD80T), β-dodecylmaltoside (AD80M) or CHAPS (AD80C) by bead beating for 80 s. Error bars depict the standard deviation of quadruplicated measurements (n=4). Figure S3. Acetate kinase (AK) activity in lysates obtained by different extraction procedures. Cell lysis by ultrasonication with HN-buffer (i.e., basic lysate, AD) or with phosphate buffer (AD PO4); cell lysis with HN-buffer by bead beating for 80 s (AD80), 160 s (AD160), with phosphate buffer by bead beating for 160 s (AD160 PO4), with HN-buffer and detergents octylthioglucoside (AD80T), β-dodecylmaltoside (AD80M) or CHAPS (AD80C) by bead beating for 80 s. Error bars depict the standard deviation of triplicated measurements (n=3). 8 Section III. Removal of OMPs in anaerobic sludge (positive control) Figure S4. Removal of acetyl-SMX (a), clarithromycin (b), tramadol (c), trimethoprim (d), atenolol (e) and venlafaxine (f) and formation of SMX (a), O-desmethyl-tramadol (c, secondary y-axis), N,O-didesmethyl-tramadol (c, secondary y-axis) and 4- desmethyl-trimethoprim (d, secondary y-axis) in positive control experiments performed twice in triplicate with anaerobic sludge. Error bars represent maximum and minimum values of the two experiments. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 C/C0 Time (h) Acetyl-SMX Acetyl_SMX SMX 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 C/C0 Time (h) Trimethoprim Trimethoprim 4-DM-trimethoprim 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 C/C0 Time (h) Clarithromycin 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 C/C0 Time (h) Atenolol a) b) d) e) 0.00 0.05 0.10 0.15 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 C/C0 Time (h) Tramadol Tramadol O-DM-tramadol N,O-DDM-tramadol 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 20 40 60 80 C/C0 Time (h) Venlafaxine c) f) 9 Section IV. Removal of OMPs under different lysate conditions Figures S5-S7 show significant effects with respect to the basic lysate when different extraction conditions, cofactors and inhibitors were used. Tables S3-S5 summarize the maximum removal efficiencies achieved in the OMPs transformation assays. The extraction conditions and the addition of cofactors and inhibitors were tested in three independent assays, with lysates obtained from anaerobic sludge samples at different days. Hence, to avoid variability between assays not caused by the specific conditions tested, the ∆Removal values with respect the basic lysate (Tables 1-3, manuscript) were calculated considering the corresponding removal of each basic lysate, which is also specified in Tables S3-S5. Figure S5. Significant effect of extraction conditions on the transformation of acyclovir into carboxy-acyclovir (TP). Names in legends refer to basic lysate (obtained by sonication) and bead beating for 160 s (BB160). Each time point represents the molar concentration ratio (C/C0) (average of triplicates for the first and last time point and single values of composite samples for the second, third and fourth time point). Error bars depict the standard deviation (n=3) considering error propagation. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 020 40 60 80 C/C0 Time (h) Acyclovir 0.0 0.2 0.4 0.6 0.8 1.0 1.2 020 40 60 80 C/C0 Time (h) Carboxy-acyclovir Basic lysate BB160 a) b) 16 Section V. Identified TPs by LC–QToF-MS measurements Table S6. Mass accuracy, isotope ratio and retention time of the identified TPs erythromycin TP 576 and clarithromycin TP 590 (both resulting from the cleavage of cladinose, Terzic et al., 2018) as well as atenolol acid (resulting from the hydrolysis of the primary amide, Radjenović et al., 2008). Transformation product (TP) Sum formula Calculated exact mass of [M+H]+ (m/z) Measured exact mass of [M+H]+ (m/z) Mass accuracy (ppm) Calculated isotope ratio (%) Measured isotope ratio (%) RT1 (min) Erythromycin TP 576 C29H53NO10 576.3748 576.3740 -1.5 34 30 6,7 Clarithromycin TP 590 C30H55NO10 590.3904 590.3898 -0.95 35 32 7,4 Atenolol acid C14H21NO4 269.1581 268.1542 -2.8 16 17 5.0 1 Retention time Table S7. Comparison of measured MS/MS fragments present in both the MS2 spectrum of the parent compound (erythromycin and clarithromycin) as well as in the MS2 spectrum of the detected TP (erythromycin TP 576 and clarithromycin TP 590). The MS/MS fragments of the TP atenolol acid were compared with those of an authentic reference standard. Compound for comparison (parent/authentic standard) Measured masses of MS/MS fragments (m/z) Transformation product (TP) Measured masses of MS/MS fragments (m/z) Compliance of MS/MS fragments (ppm) Erythromycin (parent) 576.3749 Erythromycin TP 576 576.3710 ([M+H]+) -6.8 158.1179 158.1177 -1.3 116.1066 116.1042 -21 Clarithromycin (parent) 590.3852 Clarithromycin TP 590 590.3818 ([M+H]+) -5.8 558.3593 558.3537 -10 158.1172 158.1166 -3.8 98.0960 98.0963 3.1 Atenolol acid (authentic standard) 191.0698 Atenolol acid 191.0701 1.5 165.0542 165.0525 -10 145.0649 145.0649 0 91.0539 91.0550 12 56.0496 56.0494 -3.6 17 References Radjenović, J., Pérez, S., Petrović, M., Barceló, D., 2008. Identification and structural characterization of biodegradation products of atenolol and glibenclamide by liquid chromatography coupled to hybrid quadrupole time-of-flight and quadrupole ion trap mass spectrometry. J. Chromatogr. A 1210, 142–153. Terzic, S., Udikovic-Kolic, N., Jurina, T., Krizman-Matasic, I., Senta, I., Mihaljevic, I., Loncar, J., Smital, T., Ahel, M., 2018. Biotransformation of macrolide antibiotics using enriched activated sludge culture: Kinetics, transformation routes and ecotoxicological evaluation. J. Hazard. Mater. 349, 143–152.