Impact of Biogenic Substrates on Sulfamethoxazole Biodegradation Kinetics by Achromobacter

Supplementary Material Section

Impact of biogenic substrates on sulfamethoxazole biodegradation kinetics by Achromobacter denitrificans strain PR1

P.Y. Nguyen1, G. Carvalho1, A.C. Reis2, O.C. Nunes2, M.A.M. Reis1, A. Oehmen1

1 UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. 2LEPABE – Chemical Engineering Department, Faculty of Engineering, University of Porto, 4200-465 Porto, Portugal

(6 pages, 3 Tables, 3 Figures)

1 S1. Direct HPLC injection of sulfamethoxazole, sulfanilic acid and 3-amino-5-methylisoxazole

For the tests of 150 mg/L and 500 µg/L, samples were taken for pH determination, and then centrifuged at

10000 rpm for 5 minutes in a VWR Microstar 17 centrifuge to remove the biomass, filtered through a 0.2

µm Spin Filter (Centrifugal filter modified nylon, VWR), and stored in 1.5 mL Eppendorf tubes at -20oC for the monitoring of SMX concentration and degradation metabolites. All selected organic compounds were analysed by HPLC using a Waters system equipped with ultraviolet (UV) and fluorescence detectors

(Waters Chromatography, Milford, MA, USA) and a Luna 5µm C18(2) 100A (150 x 3.0 mm) column

(Phenomenex Inc., Torrance, CA, USA). The mobile phase consisted of water acidified with formic acid

0.1% v/v (A): acetonitrile (B) at a flow rate of 1.2 mL/min and the eluting conditions applied consisted of

1.5 min at 5% of B; 3 min of a linear gradient up to 100% of B and finally 2.5 min at 5% B. The volume of injection was 50 µL. Samples containing the mixtures of compounds were analysed using the conditions described in Table S1.

Table S1 - HPLC methods used for the detection of SMX and biodegradation metabolites in the direct

HPLC analysis

Compound Column Flow rate Mobile Phase Composition (%) HPLC/UV temperature (mL/min) (°C) WavelengthUV (nm)

Sulfamethoxazole 5%ACN/95%H2O acidified with 35 1.2 270 formic acid 0.1% (v/v)

Sulfanilic acid 5%ACN/95%H2O acidified with 35 1.2 230 formic acid 0.1% (v/v)

3-amino-5- 5%ACN/95%H2O acidified with 35 1.2 250 methylisoxazole formic acid 0.1% (v/v)

ACN - Acetonitrile; MeOH - Methanol; H2O - Milli-Q water; UV - Ultraviolet;

S2. Direct HPLC- LC-MS/MS injection of sulfamethoxazole and 3-amino-5-methylisoxazole

For the tests of 20 µg/L and 600 ng/L, the analyses were performed using a high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) using a Dionex Ultimate 3000 system from Thermo Scientific. This equipment is equipped with a binary pump, an automatic injector

2 and a thermostatted column compartment coupled to a Mass Spectrometer TSQ Endura triple quadrupole model, from Thermo Scientific. The separation was performed on a reversed-phase column (Acquity

BEH C18 (2,1 x 50 mm, 1,7 µm), Waters)) at 40oC using an injection volume of 20 µL. The mobile phase consisted of water:formic acid 0.5% v/v supplemented with 0.01 mM ammonium acetate (A): methanol

(B) at a flow rate of 0.30 mL/min and the eluting conditions applied consisted of 2 min at 5% of B; 2 min at 20% of B, 2 min at 40% of B followed by 2 more minutes at 70% of B then a linear gradient up to 90% of B for 2 min before finally reduced to 5 % of B for the last 4 min.

Triple quadrupole operating conditions were optimized in order to work in multiple reaction monitoring mode (MRM). The optimization was based on the selection of ionization mode, optimum collision energy

(eV), ion transfer tube.

MRM transitions, the optimum collision energies and cone voltages selected for each transition are indicated in Table S2. The first transition corresponds to the most abundant and was used for quantification and the second one for confirmation purposes.

XCalibur software (version 4.1) was used for data acquisition and processing.

Ultrahigh-purity Argon (Ar) was used as collision gas. High purity nitrogen was used as sheath, aux and sweep gas.

Table S2. MS/MS parameters for the analysis of target analytes by MRM negative and positive ionization mode

Target Rt Precurso Collision MRM1 Collision MRM2 compounds (min) r ion energy (eV) Energy [M+H]+

Sulfamethoxazol 254.1 15 254.1>156 20 254.1>92 e

D4- 258.2 15 258.2>160.1 25 258.2>96.1 sulfamethoxazole

3-amino-5- 99.1 10 99>72 12 99>44 methylisoxazole

3 Table S3. Structure and physico-chemical properties of the sulfamethoxazole and biodegradation metabolites

Compound Molecular Molecular Solubility Log CAS Koc Vapor Log pKa a formula Weight Kow pressure Kd (Da) (mmHg)

2 3 3 4 Sulfamethoxazole C10H11N3O3S 253.3 610 mg/L 0.89 723-46-6 4.44 1.3E-7 2.4 1.9/5.7 (370C)1 (pH 7

a Log Kow: logarithm of the octanol-water partition coefficient 1(Yang et al. 2011); 2(Kolpin 2002); 3(Park and Choi 2008); 4(Gobel et al. 2005)

Figure S1. Relative change of SMX concentration, biomass concentration and C-sources over time by A. denitrificans strain PR1 in mineral medium B supplemented with 150 mg/L of SMX in the presence or absence of other carbon source (0.59g/L of succinate or acetate) and YE (0.2 mg/L).

Figure S2. Accumulation of 3-amino-5-methylisoxazole during degradation of SMX by A. denitrificans strain PR1. Bars represent standard deviation of two independent experiments.

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