Drought induces soil microbial stress responses and emissions of volatile organic compounds in an artificial tropical rainforest

VOCs and CO2

In order to capture any rapid changes in gas fluxes due to changes in microbial activity after pyruvate addition, measurement intervals were increased to high frequency (30 minutes) directly prior to labeling. After gas fluxes were expected to equilibrate, approximately 8 hr post pyruvate labeling (~ 6 PM), measurement intervals were decreased to low frequency (50 minutes) and remained at this frequency until measurements were stopped at 48 hr post labeling. For each measurement, the automatic chamber closed over the collar for a total of 10 min (pre-purge, 2.5 min; measurements, 6.5 min; post-purge, 1 min). Fluxes were measured using an automated, multiplexed Licor soil flux system (Licor 8100, Li-8150 16-port multiplexer and Lic 8100-104 Long-Term Chambers with opaque lids, Licor Inc.). The system was coupled to a Picarro G2201-i analyzer (Picarro Inc., Santa Clara, US) to measure CO2 and isotopic composition and a proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF 8000, Ionicon Inc. Innsbruck, Austria) for VOCs (including 13C-VOCs). The PTR-ToF-MS sampled the sub-flow from the soil flux system at 30 sccm via fluorinated ethylene propylene tubing heated at 60 °C. The drift voltage was 600 V, the drift temperature was 60°C and the drift pressure was 2.2 mbar, resulting in an E/N ratio of 137 Td. The time resolution was 10 ms and the mass range was up to 500 amu. The PTR-ToF was operated in the H3O+ mode, therefore only compounds having proton affinity higher than water (697 kJ/mol) underwent proton-transfer reactions and could be detected on their protonated mass to charge ratio (m/z), which includes the vast majority of VOCs . Measured ions were attributed to chemical formulas and specific chemical species based on the exact protonated m/z. PTR-TOF data were processed using the software PTRwid. To account for possible variations of the reagent ion signals, measured ion intensities were normalized to the H3O+ counts in combination with the water-cluster ion counts. At midnight, automatic calibrations were performed using standard gas cylinders containing different multi-VOC component calibration mixtures in Ultra-High Purity (UHP) nitrogen (Apel-Riemer Environmental, Inc., Colorado, USA). For a detailed description of the calibration setup see Werner et al. (2021). The concentrations of compounds included in the standard were calculated with an uncertainty of ≤23%. Concentrations of compounds not included in the calibration standard cylinders were calculated by applying the kinetic theory of proton transfer reaction with an uncertainty of ≤50%.

Select additional soil experiments were conducted with a Vocus proton transfer reaction time-of-flight mass spectrometer (PTR-TOF; TOFWERK, AG, Thun, Switzerland) coupled to a custom-built gas chromatograph (GC; Aerodyne Research, Inc., Billerica, MA, USA). The GC contains an integrated two-stage adsorbent-based thermal desorption preconcentration system for  in situ collection of VOCs prior to separation on the chromatographic column. For preconcentration, a multi-bed (Tenax TA/Graphitized Carbon/Carboxen 1000; Markes International) sorbent tube was used for the first stage of sample collection, this tube is then subjected to a post-collection water purge before the sample was thermally desorbed to a multi-bed focusing trap (Tenax, Carbopack X, Carboxen 1003; Markes International) prior to injection onto the GC column. For this study, the GC was equipped with a 30-m Rxi-624 column (Restek, 0.25 mm ID, 1.4 µm film thickness) which resolves non- to mid-polarity VOCs in the C5 - C12 volatility range prior to ionization in the PTR detector. The GC-PTR sampled from in-situ soil gas probes on an alternating timed schedule. The GC-PTR can speciate structural isomers and help identify some unknowns by matching to calibrated retention times. 

NMR 

1H-NMR bulk metabolite characterization was performed on soil water extracts. Samples (180 µL) were combined with 2,2-dimethyl-2-silapentane- 5-sulfonate-d6 (DSS-d6) in D2O (20 µL, 5 mM) and thoroughly mixed prior to transfer to 3 mm NMR tubes. Resonances corresponding to 13C labeling were identified by visual inspection, comparing labeled spectra to unlabeled spectra. Once differences were identified, the molecular location and quantification of 13C incorporation was determined by J-coupling pattern analysis and the ‘linefitting’ tool in Mestrenova, respectively. NMR spectra were acquired on a Bruker Avance III spectrometer operating at a field strength of 17.6 T (1H ν0 of 750.24 MHz) and equipped with a 5 mm Bruker TCI/CP HCN (inverse) cryoprobe with Z-gradient and at a regulated temperature of 298 K. The one-dimensional 1H spectra were acquired using a nuclear Overhauser effect spectroscopy (noesypr1d) pulse sequence. The 90° H pulse was calibrated prior to the measurement of each sample with a spectral width of 12 ppm and 1024 transients. The NOESY mixing time was 100 ms and the acquisition time was 4 s followed by a relaxation delay of 1.5 s during which presaturation of the water signal was applied. Time domain free induction decays (72114 total points) were zero filled to 131072 total points prior to Fourier transform, followed by exponential multiplication (0.3 Hz line-broadening), and semi-automatic multipoint smooth segments baseline correction. Chemical shifts were referenced to the 11H methyl or 13C signal in DSS-d6 at 0 ppm.  The 1D 11H NMR spectra of all samples were processed, assigned and analyzed using Chenomx NMR suite 9.2 (Chenomx Inc.; Edmonton, AB, Canada) with quantification of spectral intensities of compounds in the Chenomx library relative to the internal standard. Candidate metabolites present in each of the complex mixtures were determined by matching chemical shift, J-coupling and intensity information of the experimental signals against signals of the standard metabolites in the Chenomx library. Signal to noise ratios (S/N) were measured using MestReNova v 14.2.014 with the limit of quantification equal to a S/N of 10 and the limit of detection equal to a S/N of 3. Standard 2-D experiments such as 11H / 13C - heteronuclear correlation (HSQC) experiments or 2-D 11H/ 11H Total Correlation spectroscopy (TOCSY) experiments further aided corroboration of several metabolite identifications where there was sufficient S/N.

FTICR 

A 12 Tesla Bruker FTICR mass spectrometer (MS) was used to collect high-resolution mass spectra of WEOC by direct injection for secondary metabolite characterization. Approximately 100 µL of water extract was mixed with methanol (1:2) before injection onto the mass spectrometer to enhance ionization. A standard Bruker electrospray ionization (ESI) source was used to generate negatively charged molecular ions. Samples were introduced directly to the ESI source. The instrument settings were optimized by tuning on a Suwannee River fulvic acid standard, purchased from the International Humic Substances Society. Blanks (HPLC grade methanol) were analyzed at the beginning and end of the day to monitor potential carry over from one sample to another. The instrument was flushed between samples using a mixture of water and methanol. The ion accumulation time varied to account for differences in C concentration between samples. One hundred and forty-four individual scans were averaged for each sample and internally calibrated using an OM homologous series separated by 14 Da (CH2 groups). The mass measurement accuracy was <1 ppm for  singly charged ions across a broad m/z range (100–1,000 m/z). The mass resolution was ∼240 K at 341 m/z. The transient was 0.8 s. Data Analysis software (BrukerDaltonik version 4.2) was used to convert raw spectra to a list of m/z values applying FTICR–MS peak picker module with a signal-to-noise ratio (S/N) threshold set to 7 and absolute intensity threshold to the default value of 100. Putative chemical formulae were then assigned using Formularity software, as previously described. Chemical formulae were assigned based on the following criteria: S/N > 7, mass measurement error <1 ppm, and taking into consideration the presence of C, H, O, N, S, and P and excluding other elements. To ensure consistent formula assignment and eliminate mass shifts that could impact formula assignment, all sample peak lists were aligned to each other. The following rules were implemented to further ensure consistent formula assignment: (a) picking formulae with the lowest error between predicted and observed m/z, and with the lowest number of heteroatoms, and (b) the assignment of one phosphorus atom requires the presence of at least four oxygen atoms. The chemical character of thousands of peaks in each sample's ESI FTICR–MS spectrum was evaluated using van Krevelen diagrams.