other extraction method

vault/open-notebook.commons.rouchene.2023.10.11.md

@@ -2,7 +2,7 @@
 id: ehmhzkuc7v5v02me3bng7ek
 title: '2023-10-11'
 desc: ''
-updated: 1697488367323
+updated: 1697541010476
 created: 1697023998899
 traitIds:
   - open-notebook-commons-rouchene
@@ -184,14 +184,21 @@ the five methods are not coonslusive - the search for other methods is essential
 
 1. The 45 collected soil samples, and 5 blank samples containing no
 soil, were lyophilized on an Edwards Super Modulyo freeze-drier (SciQuip Ltd., Shropshire, UK) for 7 d. Subsequently, these were ground in a Retsch MM200 stainless steel ball mill (Retsch GmbH, Haan, Germany)
-at a frequency of 20 Hz to aid recovery of metabolites from the microbial biomass (Fiehn et al., 2002; Wang et al., 2015). The samples were then stored in individual sterile glass vials at  80 �C to minimize changes in metabolites (Wellerdiek et al., 2009). The soils were extracted using 3:3:2 (v/v/v) acetonitrile-isopropanol-water, vortexed for 15 s, shakenat 4° C for 5 min, centrifuged at 1400 rpm for 2 min, and dried using a CentriVap Benchtop Centrifugal Concentrator (Labconco Corp., Kansas City, MO) (Barupal et al., 2019; Fu et al., 2019). Non-targeted primary metabolism analysis was performed using a Gerstel Automated Linear Exchange-Cold Injection System (ALEX-CIS) with Agilent gas chromatograph (GC) and Leco Pegasus IV Time Of Flight (TOF) MS at the UC Davis West Coast Metabolomics Facility using the method of Fiehn (2016). Briefly, 0.5 μl of each sample was injected onto a Rtx-5Sil MS capillary column (30 m length ° 0.25 m i.d with 10 m integrated guard column; 0.25 μm 95% dimethylsiloxane/5% diphenylpolysiloxane coating; Restek Corp., Bellefonte, PA). Using a He mobile phase, the GC thermal programme was 50 °C for 1 min, ramped to 330°C at 20° C min 1 and finally held at 330 °C for 5 min. Upon elution, samples were injected into a Pegasus IV GC-time of flight mass spectrometer (Leco Corp., St Joseph, MI), using mass resolution of 17 spectra s1 , from 80 to 500 Da, at  70 eV ionization energy and 1800 V detector voltage with a 230 °C transfer line and 250°C ion source [[https://doi.org/10.1016/j.soilbio.2020.107758]].
+at a frequency of 20 Hz to aid recovery of metabolites from the microbial biomass (Fiehn et al., 2002; Wang et al., 2015). The samples were then stored in individual sterile glass vials at  -80°C to minimize changes in metabolites (Wellerdiek et al., 2009). The soils were extracted using 3:3:2 (v/v/v) acetonitrile-isopropanol-water, vortexed for 15 s, shakenat 4° C for 5 min, centrifuged at 1400 rpm for 2 min, and dried using a CentriVap Benchtop Centrifugal Concentrator (Labconco Corp., Kansas City, MO) (Barupal et al., 2019; Fu et al., 2019). Non-targeted primary metabolism analysis was performed using a Gerstel Automated Linear Exchange-Cold Injection System (ALEX-CIS) with Agilent gas chromatograph (GC) and Leco Pegasus IV Time Of Flight (TOF) MS at the UC Davis West Coast Metabolomics Facility using the method of Fiehn (2016). Briefly, 0.5 μl of each sample was injected onto a Rtx-5Sil MS capillary column (30 m length ° 0.25 m i.d with 10 m integrated guard column; 0.25 μm 95% dimethylsiloxane/5% diphenylpolysiloxane coating; Restek Corp., Bellefonte, PA). Using a He mobile phase, the GC thermal programme was 50 °C for 1 min, ramped to 330°C at 20° C min 1 and finally held at 330 °C for 5 min. Upon elution, samples were injected into a Pegasus IV GC-time of flight mass spectrometer (Leco Corp., St Joseph, MI), using mass resolution of 17 spectra s1 , from 80 to 500 Da, at  70 eV ionization energy and 1800 V detector voltage with a 230 °C transfer line and 250°C ion source [[https://doi.org/10.1016/j.soilbio.2020.107758]].
 
     -- _Protocol steps_
      - Lyophilization of the samples for 7 days.
      -Grinding in a stainless steel ball mill Retsch at a frequency of 20 Hz.
      -The samples were then stored in individual sterile glass vials at -80°C.
      -The soils were extracted using a 3:3:2 (v/v/v) acetonitrile-isopropanol-water solution. The mixture was vortexed for 15 seconds, shaken at 4°C for 5 minutes, and then centrifuged at 1400 rpm for 2 minutes. Finally, the samples were dried using a CentriVap Benchtop CentriVap.
-     -The soils were extracted according to the following protocol: a solution of acetonitrile-isopropanol-water in proportions of 3:3:2 (v/v/v) was used. Then, the sample was vortexed for 15 seconds, shaken at 4°C for 5 minutes, and finally centrifuged at 1400 rpm for 2 minutes.
+2. Prior to use, all glassware was acid-washed (5% HCl, > 24 h) to remove chemical residues, rinsed in HPLC-grade water three times, and subsequently dry heat sterilised (150 °C, 2.5 h) (Jain et al., 2020). All equipment used to process the soil (e.g., tweezers and spatulas) were thoroughly cleaned both before and between samples with deionised water and 70% industrial methylated spirit (IMS) to prevent cross-contamination. Nutrients in the concentrations described in section 2.2 were added by pipette in 1 ml of HPLC-plus grade water (Sigma-Aldrich, Munich, Germany) evenly distributed across the surface of **10 g fresh weight of soil** in 240 ml polypropylene sample containers (Snap-Seal®; Corning, NY, USA). This type of container was used to ensure even coverage of the soil surface with the nutrient solution (soil depth ca. 10 mm). Five independent soil replicates (n = 5) were set up per treatment. Samples were subsequently incubated as described in Section 2.2. At the end of the incubation period, the **soil samples were immediately transferred to 20 ml glass vials and fumigated with ethanol-free chloroform for 24 h** to lyse microbial cells, to increase metabolite yield (Swenson et al., 2015) and limit microbial processing of metabolites, using the method of Vance et al. (1987). After fumigation, samples **were immediately frozen (−80 °C) to quench any residual metabolic and enzymatic activity** (Wellerdiek et al., 2009). From this point onwards, all samples were stored at −80 °C, unless otherwise stated and while being processed (i.e. out of the freezer), samples were kept on ice (4 °C). L**yophilisation of samples (>24 h) was then performed** using a Modulyo Freeze Dryer (ThermoFisher Corp, Waltham, MA) equipped with an RV vacuum pump (Edwards Ltd., Crawley, UK). S**amples were then mechanically ground using a Retsch MM200** stainless steel ball mill (Retsch GmbH, Haan, Germany) **for 60 s at a frequency of 20 Hz** to aid in cell lysis and metabolite recovery and to increase sample homogeneity (Wang et al., 2015; Withers et al., 2020). The ball mill was cleaned thoroughly between samples with HPLC-grade water followed by 70% IMS, to avoid contamination.
+The following extraction method is based on the hybridised methods of Swenson et al. (2015) and Fiehn et al. (2008). Briefly, we used the extraction method from Swenson et al. (2015) and the solvent makeup (3:3:2) from Fiehn et al. (2008), to ensure broad metabolite coverage, with the recovery of both polar and non-polar metabolite classes. Further, the weight of soil extracted was increased in order to maximise the volume of supernatant available for lyophilisation and subsequent analysis. A pre-experiment was used to compare the efficiency of the metabolite extraction method proposed here to that of the Swenson et al. (2015) method (the results are summarised in supplementary information Section S1).
+
+Upon soil extraction, **6 g of each fumigated, lyophilised**, **ground soil sample was weighed into a 35 ml glass centrifuge tub**e (Kimax®; DWK Life Sciences, Stoke-on-Trent, UK), **to which 24 ml of pre-cooled (−20 °C) acetonitrile (MeCN)/isopropyl alcohol (IPA)/HPLC-plus grade water (H2O) (3:3:2 v/v/v) extractant was added**, using a glass pipette. Samples were then **horizontally shaken on ice (4 °C) at a frequency of 200 Hz for 1 h**, and then centrifuged at 3320×g for 15 min (Swenson et al., 2015). Supernatants were then transferred, using glass pipettes, into 20 ml glass vials and stored at −20 °C (to ensure metabolic activity was quenched but the supernatant was not frozen). Samples were left unfiltered due to the potential of contamination from dissolution of contaminants from the filter paper and plastic housing. The supernatant was lyophilised in 2 ml glass vials using a Modulyo Freeze Dryer with RV pump attached to a SpeedVac vacuum concentrator (Savant; ThermoFisher Corp.). The vials were periodically topped up with the supernatant, taking note of the quantity added (∼15 ml total) and lyophilised to complete dryness. Samples were then shipped on dry ice (−78.5 °C) to Metabolon Inc. (Morrisville, North Carolina, USA) for untargeted LC-MS metabolomic analysis. Upon analysis, samples were dissolved in methanol:water (4:1 v/v) and subjected to the standard Metabolon sample preparation procedure. Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) analysis parameters, bioinformatics, compound ID and data curation are summarised in Supplementary information Section S2.
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 ### Composition of culture media, prepared solution supplemented, and chemicals used in the experimental section