Sampling
Water samples
Water samples were collected on board the research vessel Kay-Kay II (Universidad de Concepción) quasi-monthly between November 2009 and January 2011 (13 sampling dates). Seawater was collected at depths of 0, 10, 30, 65, 70, and 80-meters using a rosette system equipped with 10-L Niskin bottles. Subsamples of 50 mL volume were transferred onboard to pre-combusted (450 ºC, 4 h) gas tight borosilicate Wheaton® bottles (darkened with aluminum foil) under a N2 atmosphere – using glove bags (Aldrich®AtmosBag) to avoid O2 contamination – then immediately poisoned with HgCl2 (0.001%) to arrest microbial activity. Bottles were crimp-sealed with ultra-pure bromobutyl stoppers (Wheaton®) and stored in the dark at 4 ºC until further analysis of toluene in the laboratory (within 12 hours). Data for temperature, salinity, O2, NO3-, NO2-, PO43-, NH4+ and chlorophyll-a were provided by the COPAS Center Time Series Oceanographic Station 18 (FONDAP COPAS) and the program Microbial Initiative in Low Oxygen off Concepción and Oregon (Moore Foundation http:// mi_loco.coas.oregonstate.edu). Volatile fatty acid (VFA) data were taken from Srain et al. (2020).
Inocula and incubation media
Inocula of seawater for laboratory experiments were obtained on January 18, 2011, from 65 m depth (10.3oC, 34.6 PSU, 4.0 µM O2) and transferred to 10 mL serum vacuum-tubes BD®. Tubes were maintained at 10°C in darkness until arrival at the laboratory. Inocula and reagents were manipulated in a laminar flow hood LABCONCO Class II Type IIA under a N2-saturated atmosphere (glove bags Aldrich© Atmosbag) achieved by bubbling with N2 (99.9% purity). O2-sensitive methylene blue (Resazurin 0.0001%, Wolfe, 2011; McDonald et al., 2012) was added to anoxic incubation vessels to detect traces of O2 contamination (> 0.7 µM). Artificial seawater for anoxic incubations was prepared according to Lovley (2006).
Experimental setup
Experimental incubations were conducted in duplicate by transferring 30 mL of artificial seawater into darkened pre-combusted (450 ºC, 4 h) borosilicate glass bottles (60 mL, Wheaton®), under a N2 saturated atmosphere. L-phenylalanine in excess (500 µM, CAS 63-91-2) and D-glucose (10 µM, CAS 50-99-7), were added as carbon and nitrogen sources, and inoculated with 10% v/v of seawater collected from the OMZ. Bottles were sealed with ultra-pure bromobutyl stoppers, keeping a headspace volume of 20 mL for sampling of volatile compounds. Incubation of bottles was carried out at 10°C in darkness, with constant orbital agitation (180 rpm). Incubations were conducted for 5 days with the headspace sampled (500µL with a Hamilton gas-tight syringe 1750) every ca. 24 hours.
Experimental treatments were (in parenthesis the initial final concentration added):
- Anoxic-1 incubation under denitrifying conditions: Phenylalanine (500 µM), glucose (10 µM) and NO3- (800 µM). This same concentration of NO3- was subsequently added daily under a N2-saturated atmosphere using a microliter syringe Hamilton® (500 µL) previously flushed with N2.
- Anoxic-2 incubation under sulfate reduction conditions: Phenylalanine (500 µM), glucose (800 µM) and SO42- (450 µM), amended with 5% v/v of reducing solution (Na2O2S x 5H2O and cysteine) to promote sulfate reduction. This same concentration of SO42- was subsequently added daily under N2-saturated atmosphere using a microliter syringe Hamilton® (500 µL) previously flushed with N2.
- Anoxic-3 incubation under fermentative conditions: Phenylalanine (500 µM) and glucose (800 µM) without external electron acceptors.
- Oxic incubation: Phenylalanine (500 µM), glucose (800 µM) and O2 (370 µM). Incubation flasks were oxygenated to saturation (8.9 ml L-1 at 10°C and 0.21 atm, Henry’s Law) with synthetic air (ca. 20% O2, 80% N2, 99.99% purity). Five mL aliquots of pure synthetic air were added daily to the incubation flasks using a 1000 µL gas-tight syringe Hamilton®.
- Experiments Anoxic-1, Anoxic-2, Anoxic-3, and Oxic were also repeated using deuterated phenylalanine (500 µM L-phenyl-d5-alanine, CAS 284664-89-7), instead of phenylalanine, to confirm that toluene synthesis came from phenylalanine.
Anoxic conditions were achieved by gently bubbling N2 into incubation bottles for 15 minutes to displace traces of O2. Abiotic controls with substrates were incubated without OMZ inocula for each treatment.
Analysis of toluene in ambient and experimental samples
Analysis of toluene was carried out through Headspace Solid Phase Micro–Extraction coupled to Gas Chromatography with MSD detection (HS–SPME–GCMS). Twenty-five mL of water was removed from each sample (n = 3), under a N2 saturated atmosphere, to generate a headspace. Environmental and experimental samples were placed on a thermostatic hotplate at 20°C, with constant stirring (using an acid-cleaned and sterilized stirring bar) for 5 minutes to reach partition equilibrium between aqueous and gas phases. Polar and non-polar volatile organic compounds were adsorbed using a fiber of 85 μm CarboxenTM / PDMS Stable Flex (SUPELCO). The fiber was inserted into the insertion septum of the gas bottle and exposed to the gas phase (headspace). After adsorption of analytes, the fiber was placed in the injection port of the gas chromatograph and exposed for 5 minutes at 250°C for desorption of the collected gaseous analytes. GC–MS analyses were conducted using a gas chromatograph Agilent 6890N series coupled to a mass spectrometer Agilent 5973 Network. The mass spectrometer was operated in electron impact mode (70 eV) and spectra of toluene standards and incubation samples were acquired in full scan mode (m/z 40-600, 2.6 s-1), with spectra for environmental samples acquired using selective ion monitoring (SIM): toluene (tropylium ion m/z 91). Chromatographic separation was achieved using a HP–Plot/Q column 30 m (0.32 mm diameter, 0.20 μm film thickness), using He as carrier gas. The oven temperature program started at 100°C (5 min), ramped to 230°C at 50°C min-1 (held 2 min), to 250ºC at 50ºC min-1 (held 7 min).
Quantification of toluene and deuterated toluene (toluene-d5) in both scan and selective ion monitoring (SIM) modes was determined using calibration curves (R2> 0.994) for which toluene standards (HPLC grade, ≥ 98.8% purity, Fisher) were added to artificial seawater in the range 1 nmol L-1 to 1 mmol L-1. Concentrations of toluene in the gas phase were calculated as CHS = Cap / (K + (VHS / VS)) with the partition coefficient K = CAP / CHS. CHS is the concentration in the headspace, CAP is the concentration in the aqueous phase, and VHS and VS are headspace and sample volumes 65, resulting in a partition coefficient of 0.9. Detection limits were calculated from the slopes and residual standard deviations derived from linear regressions of calibration curves (3 times residual error times slope)66. Limit of detection for toluene was 1 nmol L-1, whilst limit of quantification was 5 nmol L-1. Synthetic seawater was analyzed for toluene.
Analyses of dissolved free amino acids (DFAA)
Concentrations of DFAA from incubations, and from Station 18, were quantified as OPA-derivatized adducts 67 with a Shimadzu LC-10AT HPLC coupled to a Shimadzu RF-10Axl fluorescence detector (set at excitation/emission of 340/450 nm), column oven CTO 10As and autosampler (Shimadzu SIL 10 ADvp). Aliquots of 0.6 mL, mixed with 0.4 mL methanol, were derivatized in the autosampler with 60 mL ortho-phthalaldehyde/2- mercaptoethanol reagent and 100 mL sodium acetate buffer 0.1 N, pH 5, and then injected (50 mL) into the HPLC. Fifteen amino acids (asp, glu, ser, his, gly, thr, arg, ala, tyr, val, met, phe, ile, leu, lys) were separated using an Alltima C18 (5 mm, 250 x 4.6 mm) column maintained at 40°C, with a mobile phase of 5% tetrahydrofuran in 25 mM sodium acetate and methanol, and at a flow rate of 1 mL min-1. A gradient of 25–30% methanol in 35 min, 30–50% in 7 min, 50–60% in 18 min, 60–100% in 12 min was used. Initial conditions were restored within 7 min, and the column equilibrated for 10 min between injections. Amino acids were identified and quantified by comparison with chromatograms of a standard amino acid mix (Pierce 20088) run under the same conditions every 10 injections. The coefficient of variation for quantification of duplicate samples was 9.4%.
Statistical analysis
Since homogeneity of variances (Levene test) and normality of variables (Shapiro–Wilk test) were not fulfilled, we tested for significant differences between environmental data using the non-parametric Kruskal–Wallis ANOVA test. Statistical differences between experimental treatments were determined by using the Wilcoxon matched pairs test. Correlations were examined using Spearman R coefficients.
Thermodynamic Calculations
Calculated Gibbs Energy values (ΔG) for proposed oxidation reactions of toluene in the suboxic water column were calculated using activities of substrates and products, temperature and pH of the water depth of inoculum collection in austral spring or from literature. The following data and sources were used: temperature (10.2 °C), NO3- (25 µM) from the COPAS Center Oceanographic Time Series database (W. Schneider, curator), pH (7.8, this study), toluene (0.09 µM, this study), SO42- (28 mM) 68, HCO3- (2 mM)68, N2 (389 µM)69, HS- (1 µM)70,71. Activities of N2 and HS- in experimental incubations were determined stoichiometrically from the chemical equations of the proposed reactions. Thermodynamic calculations were carried out using Thermodyn© software73.