Pure standards were used for method development, compound identification and calibration.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS -"],"chromatography_protocol":["10 min reverse-phase dual-pump LC-method used with QTOF in negative ionization mode (Assays 1, 2, 3, 7, 10, 11)
The separation was performed using two ZORBAX RRHD Eclipse Plus column (C18, 2.1 × 100 mm, 1.8 μm; Agilent 858700-902) with the ZOBRAX Eclipse Plus (C18, 2.1 × 5 mm, 1.8 μm; Agilent 821725-901) guard columns at 40 C. The multisampler was kept at a temperature of 4 C. The injection volume was 1 μL and the flow rate was 0.4 mL/min. The mobile phases consisted of A: water + 5 mM ammonium acetate + 0.03 % acetic acid; B: methanol + 5 mM ammonium acetate + 0.03 % acetic acid. The 10 min gradient started with 35 % solvent B, which was increased to 100 % by 7 min and held for 1.7 min, before returning to 35 % solvent B at 8.8 min, which was held until 10 min. The re-equilibration gradient started with 35 % solvent B, which was then ramped up to 95 % solvent B at 0.1 min and held until 4 min before returning to the starting condition of 35 % solvent B at 4.1 min.
2 min reverse-phase LC-method used with QTOF in negative ionization mode (Assays 14)
The separation was performed using a ZORBAX RRHD Eclipse Plus column (C18, 3.0 × 50 mm, 1.8 μm; Agilent 959757-302) at 40 C. The multisampler was kept at a temperature of 4 C. The injection volume was 1 μL and the flow rate was 0.8 mL/min. The mobile phases consisted of A: water + 5 mM ammonium acetate + 0.03 % acetic acid; B: methanol + 5 mM ammonium acetate + 0.03 % acetic acid. The 2 min gradient started with 30 % solvent B, which was increased to 100 % by 0.5 min and held until 1 min, before returning to 30 % solvent B at 1.1 min until 2 min.
2 min reverse-phase LC-method used with QQQ (Assays 4, 5, 6, 8, 9)
The separation was performed using a ZORBAX RRHD Eclipse Plus (C18, 3 × 50 mm, 1.8 μm; Agilent Agilent 959757-302) or a Poroshell 120 EC-C18, 1.9um, 2.1 x 50mm (Agilent 699675-902) at 40 C. The multisampler was kept at a temperature of 4 C. The injection volume was 1 μL and the flow rate was 0.8 ml/min. The mobile phases consisted of either A: water + 5mM ammonium acetate + 0.03% acetic acid and B: methanol + 5mM ammonium acetate + 0.03% acetic acid, or A: water + 0.1 % formic acid and B: acetonitrile + 0.1 % formic acid. The 2 min gradient started with 30 % solvent B, which was increased to 100 % by 0.5 min and held until 1 min, before returning to 30 % solvent B at 1.05 min and held until 2 min.
LC-MS/MS PFAS measurements per EPA Draft Method 1633 (Assays 12, 13)
Samples were analysed according to the EPA 3rd Draft Method 1633 and the corresponding Agilent application note. In short, separation was performed using a ZORBAX Eclipse Plus column (C18, 2.1 × 100 mm, 1.8 μm; Agilent 959758-902) with a ZOBRAX Eclipse Plus (C18, 2.1 × 5 mm, 1.8 μm; Agilent 821725-901) guard column at 40 C. The multisampler was kept at a temperature of 4 C. The injection volume was 5 μl and the flow rate was 0.4 mL/min. The mobile phases consisted of A: 2 mM ammonium acetate in 95 % water + 5 % acetonitrile; B: acetonitrile. The 10 min gradient started with 2 % solvent B, which was increased to 95 % by 10 min, before returning to 2 % solvent B for a 2 min re-equilibration. LC-MS/MS analysis was performed on an Agilent 1290 Infinity II LC system coupled with an Agilent 6470 triple quadrupole (Agilent).
"],"publication":["Extensive PFAS accumulation by human gut bacteria."],"submitter_affiliation":["MRC Toxicology Unit"],"submitter_name":["Anna Elisabeth Lindell"],"organism_part":["Control Analyte","control sample role","solvent blank","Supernatant","Reference compound","Pooled Sample","Harvest of Product from Whole Cell Lysate","Cell Pellet"],"technology_type":["mass spectrometry"],"disease":[""],"extraction_protocol":["Sample extraction (Assays 1-12, 14)
50 μl sample was extracted with 200 μl ice-cold methanol:acetonitrile (1:1) containing internal standard (60 µM ibuprofen or 20 µM caffeine) and incubated at 4 C for 15 min. For Assay 12 all samples were diluted 1:1 with water and 10 µl diluted sample was extracted with 90 µl ice-cold methanol:acetonitrile (1:1) containing internal standard (final IS concentration 20 µg/l for each internal standard) and incubated at 4 C for 15 min. Sample plates were then centrifuged at 4 C and 4000 rpm for 10 min. Supernatants were transferred to 96-well plates for LC-MS analysis. Samples for concentration calibration and bacteria-free compound controls were processed in the same way.
Sample extraction per EPA Draft Method 1633 (Assay 13)
Supernatant, and pellet samples were extracted according to the EPA 3rd Draft Method 1633 for analysis of PFAS in aqueous, solid, biosolid and tissue samples47 and the corresponding Agilent application note48. In short, 500 ml supernatant or compound control samples were weighed and spiked with a known concentration of an internal standard (1 ml of 50 µg/l 13C9-PFNA). Pellet samples underwent three freeze-thaw cycles to ensure bacterial lysis. Samples were weighed, resuspended in 20 ml of 0.3 % methanolic ammonium hydroxide, spiked with a known concentration of an internal standard (1 ml of 50 µg/l 13C9-PFNA) and shaken for 30 min. Samples were then centrifuged at 2800 rpm for 10 min and the supernatant was transferred to a fresh sample tube. The remaining pellet was resuspended in 15 ml of 0.3 % methanolic ammonium hydroxide, shaken for 30 min and centrifuged again. The supernatant was added to the same collection tube and the pellet underwent the same process again using 10 ml of 0.3 % methanolic ammonium hydroxide. To the combined supernatants from each pellet circa 10 mg carbon (Agilent 5982-4482) was added. Samples were hand shaken for up to 5 min and then centrifuged for 10 min at 2800 rpm. The supernatants were collected in a fresh sample tube. Agilent solid-phase extraction (SPE) cartridges (Agilent 5610-2150) were loaded with silanized glass wool (Agilent 8500-1572) fitted with large volume adapters (Agilent 12131012 and 12131001) and conditioned with 15 ml 1% methanolic ammonium hydroxide followed by 5 ml of 0.3 M formic acid. Samples were then loaded into SPE cartridges and set to a low flow rate of circa 5 ml/min, followed by a rinse with 10 ml reagent water and 5 ml of 1:1 0.1 M aqueous formic acid:methanol, before being dried under the vacuum. Sample bottles were rinsed and eluted with 5 ml of 1% methanolic ammonium hydroxide. 25 µl acetic acid was added to each sample and vortexed. To each supernatant and compound control eluate 10 mg carbon (Agilent 5982-4482) was added, samples were handshaken for up to 5 min and then centrifuged for 10 min at 2800 rpm. All samples were filtered through a nylon syringe filter (Agilent 9301-6476, 5190-5092) into a collection tube for LCMS analysis.
"],"organism":["calibration curve","Control Analyte","Bacteroides uniformis ATCC 8492","Escherichia coli","Escherichia coli IAI1","Odoribacter splanchnicus DSM 20712","experimental blank","Quality Control"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS10622"],"author":["Anna Lindell. annalindell08@gmail.com.","Kiran Patil. kp533@mrc-tox.cam.ac.uk."],"data_transformation_protocol":["The Agilent MassHunter Qualitative Analysis 10.0 software was used to qualify the selected xenobiotic standards. TIC, EIC and EIC-fragment graphs were extracted for each compound. The Agilent MassHunter TOF Quantitative Analysis (Version 10.1) or Agilent MassHunter QQQ Quantitative Analysis software (Version 10.1) was used to quantify the xenobiotic compounds in each sample.
Calibration curves based on pure compound standards were used to estimate the concentrations of target compounds. Raw response values were used for concentration calculations for most analysis. Exceptions were Assay 7, 12 and 13, where the ratio of compound response to internal standard response was used. Data analysis was performed in RStudio Version 1.3.1093. The median of each sample group was compared to the median of the compound controls and an appropriate reduction of more than 20 % was chosen as cut-off, to ensure relevant reduction compared to the compound control distribution. In addition, statistical comparison was performed using t-test (two-sided), and p-values were FDR corrected. An adjusted p-value of less than 0.05 was considered significant.
Concentration in samples extracted according to the EPA Draft Method 1633 (Assay 13) were calculated based on the ratio with the labelled internal standard, which was spiked into the sample at a known concentration of 100 ng/l.
"],"study_factor":["Assay","PFAS concentration","Internal standard","Strain ID","Media","Compound","Unit","Sampling timepoint","Biological replicate","Optical density","Other"],"submitter_email":["annalindell08@gmail.com"],"sample_collection_protocol":["Growth assay in mGAM (Assay 1, 8)
On the day of the screen each well was inoculated 1:1 with a second passage culture to reach a starting OD600 of 0.05. For compound control wells bacteria-free mGAM was added to the respective wells. Samples were incubated at 37 C for 24 h, after which they were removed from the anaerobic chamber for sample collection. Whole culture, supernatant and compound control samples were collected and stored at -80 C until extraction.
Resting cell assay in PBS (Assay 2, 6, 7)
Each well was inoculated 1:1 with a culture in PBS to reach the desired starting OD600 of 3.75, unless otherwise specified. For compound control wells bacteria-free PBS was added to the respective wells. Samples were incubated at 37 C for 4 h, after which they were removed from the anaerobic chamber for sample collection. Whole culture, supernatant and compound control samples were collected and stored at -80 C until extraction.
PFAS time-course experiment with resting B. uniformis culture (Assay 3)
On the day of the screen each well was inoculated 1:1 with a culture in PBS to reach a starting OD600 of 3.75 and a concentration of 20 µM (9.3 mg/l) PFNA. Samples were incubated at 37 C. Bacteria free PBS was added to compound control samples. Supernatant, pellet, whole culture and compound control samples for PFNA analysis were collected at 0h, 4h, 1d, 2d, 4d, and 7d and stored at -80 C until extraction.
PFAS time-course experiment with growing B. uniformis culture (Assay 4)
On the day of the screen each tube was inoculated 1:1 with a second passage culture to reach a starting OD600 of 0.05 and a concentration of 20 µM (9.3 mg/l) PFNA. Samples were incubated at 37 C for 11 h. Bacteria free mGAM was added to compound control samples. OD600 was measured every hour and supernatant, whole culture and compound control samples for PFNA analysis were collected also every hour and stored at -80 C until extraction.
PFAS time-course experiment with stationary phase B. uniformis culture (Assay 5)
1.5 ml of stationary second passage cultures of B. unifomis or pure mGAM were spiked with 15 μl of 2 mM PFNA in DMSO (final concentration 20 µM (9.3 mg/l) PFNA, 1 % DMSO). Whole culture, supernatant and compound control samples were collected at 0, 15, 30 and 60 min and stored at -80 C until extraction.
PFHpA, PFOA, PFNA and PFDA pellet recovery (Assay 9)
To determine pellet concentration based on pellet weight across four PFAS compounds (PFHpA, PFOA, PFNA, PFDA) a resting cell assay and growth assay was conducted at an exposure concentration of 5 µM in 0.5 ml volume. On the day of the screen each well was inoculated 1:1 with a second passage culture to reach a starting OD600 of 0.05 in mGAM or a starting OD600 of 3.75 in PBS and a starting concentration of 5 µM of the respective PFAS compound (0.5 ml total volume). Samples were incubated at 37 C for 24 h (mGAM) or 37 C for 4 h (PBS), after which they were removed from the anaerobic chamber for sample collection. Supernatant was collected for LCMS analysis, pellets were weighed and also collected for LCMS analysis.
PFAS wash-out from live bacterial cells (Assay 10)
Each well was inoculated 1:1 with a culture in PBS to reach the desired starting OD600 of 3.75. For compound control wells bacteria-free PBS was added to the respective wells. Samples were incubated at 37 C for 4 h, after which they were removed from the anaerobic chamber for sample collection. Whole culture, supernatant and compound control samples were collected and stored at -80 C until extraction. The remaining cell pellet was then resuspended in fresh PBS buffer, and whole culture and supernatant samples were collected from this resuspended culture and also stored at -80 C until extraction.
PFAS accumulation in live, heat-inactivated, and lysed bacterial cultures (Assay 11)
On the day of the screen each well was inoculated 1:1 with an alive, heat-inactivated or lysed culture in PBS to reach a starting OD600 of 3.75. Second passage cultures were spun down, and the pellet was resuspended in PBS to an OD600 of 7.5. Each culture was split up into 3 aliquots: alive, heat-inactivated, lysed cultures. Live cultures were used as is. Bacteria were heat-inactivated at 70 C for 40 min and lysed cultures were additionally freeze-thawed three times and sonicated for 3 min. After adding the respective cultures or bacteria-free PBS to the respective wells the plates were sealed and incubated at 37 C. After 4 h whole culture, supernatant and compound control samples were collected and stored at -80 C until extraction.
Accumulation of a mix of 14 PFAS compounds (Assay 12)
On the day of the screen each well was inoculated 1:1 with a culture in PBS to reach a starting OD600 of 3.75 (1 mg/l for each PFAS compound: PFBA, PFBS, PFPeA, PFHxA, PFHxS, PFHpA, PFOA, PFOS, PFNA, PFDA, PFUnDA, PFDoDA, PFTrDA, PFTA). For compound control wells bacteria-free PBS was added to the respective wells. Samples were incubated at 37 C for 4 h, after which they were removed from the anaerobic chamber for sample collection. Whole culture, supernatant, pellet and compound control samples were collected and stored at -80 C until extraction.
Low concentration experiment (160 ng/l) (Assay 13)
Three biological replicates of B. uniformis were grown over the course of two days. On the day of the experiment the OD600 of the second passage cultures was measured, and the equivalent of 2x 0.5 l of OD600 of 3.75 of each replicate was centrifuged 4000 rpm for 15 min and the supernatant removed. One pellet per biological replicate was kept as negative control and stored at -80 C until extraction. The other pellet was resuspended in 0.5 l of 160 ng/l PFNA in PBS and incubated at 37 C for 1 h in HDPE bottles (Buerkle 10531712). 3x 0.5 l of 160 ng/l PFNA in PBS without bacterial pellet was used as the compound control and incubated for the same amount of time. For collection samples were removed from the anaerobic chamber and centrifuged at 4000 rpm for 15 min. Supernatant and pellet samples were stored separately at -80 C until extraction.
PFNA and PFDA accumulation by evolved populations (Assay 14)
20 µl of a second passage culture was added to 380 µl PFAS in mGAM to reach a starting concentration of 20 µM PFNA or PFDA. Bacteria free mGAM was added to compound control samples. Samples were incubated at 37 C for 24 h, after which whole culture, compound control and supernatant samples were collected for LCMS analysis.
"],"omics_type":["Metabolomics"],"study_design":["perfluoroalkyl substance","Microbiota","Liquid chromatography mass spectrometry","targeted metabolites"],"curator_keywords":["perfluoroalkyl substance","Microbiota","Liquid chromatography mass spectrometry","targeted metabolites"],"mass_spectrometry_protocol":["QTOF parameters (Assays 1, 2, 3, 7, 10, 11, 14)
In short, LC-MS analysis was performed on an Agilent 1290 Infinity II LC system coupled with an Agilent 6546 LC/Q-TOF (Agilent). The QTOF MS scan was operated in negative MS mode using four different collision energies (0V, 10V, 20V, 40V) (30-1500 m/z). The source parameters were as follows: gas temperature: 200 C, drying gas: 9 L/min, nebulizer: 20 psi, sheath gas temperature: 400 C, sheath gas flow: 12 L/min, VCap: 3000 V, nozzle voltage: 0 V, fragmentor: 110 V, skimmer 45 V, Oct RF Vpp: 750 V. The online mass calibration was performed using a reference solution (112.99 and 1033.99 m/z).The compounds were identified based on their retention time, accurate mass and fragmentation patterns. For all measured compounds pure standards were used for method development, compound identification and calibration.
Two different LC-methods were applied.
QQQ parameters (Assays 4, 5, 6, 8, 9)
In short, LC-MS/MS analysis was performed on an Agilent 1290 Infinity II LC system coupled with an Agilent 6470 triple quadrupole (Agilent). The QQQ was operated in Dynamic MRM mode. The source parameters were as follows: gas temperature: 300 C, gas flow: 10 L/min, nebulizer: 50 psi, sheath gas temperature: 300 C, sheath gas flow: 11 L/min, VCap: 3500 V (positive) or 3000 V (negative), nozzle voltage: 2000 V (positive) or 500 V (negative). Pure standards were used for method development, compound identification and calibration.
LC-MS/MS PFAS measurements per EPA Draft Method 1633 (Assays 12, 13)
Samples were analysed according to the EPA 3rd Draft Method 1633 and the corresponding Agilent application note. LC-MS/MS analysis was performed on an Agilent 1290 Infinity II LC system coupled with an Agilent 6470 triple quadrupole (Agilent). The QQQ was operated in Dynamic MRM mode. The source parameters were as follows: gas temperature: 230 C, gas flow: 6 L/min, nebulizer: 20 psi, sheath gas temperature: 355 C, sheath gas flow: 10 L/min, VCap: 3500 V (positive) or 2500 V (negative), nozzle voltage: 2000 V (positive) or 0 V (negative). Pure standards (Agilent ITA-70) and labelled standards (Wellington Laboratories MPFAC-C-ES) were used for method development, compound identification and calibration.
"],"metabolite_name":["Perfluoropentanoic acid","Perfluorooctanoic acid","Perfluoroheptanoic acid","Perfluorooctanesulfonic acid","Perfluorohexanoic acid","Perfluorohexanesulfonic acid","Perfluorododecanoic acid","Perfluorobutyric acid","Perfluorobutanesulfonic acid","Perfluorononanoic acid","Perfluoroundecanoic acid","Perfluorodecanoic acid"],"additional_accession":[]},"is_claimable":false,"name":"Extensive PFAS accumulation by human gut bacteria","description":"Per- and polyfluoroalkyl Substances (PFAS) – the so-called ‘forever chemicals’ – are a major cause of environmental and health concern due to their toxicity and long-term persistence. Yet, no efficient mechanisms for their removal have been identified. Here we report the potential of microbial strains to bioaccumulate PFAS compounds.
","dates":{"publication":"2025-06-30","submission":"2024-10-03"},"accession":"MTBLS10622","cross_references":{"MetaboLights":["MTBLC38397","MTBLC27732","MTBLC35549","MTBLC5855","MTBLC35546","MTBLC35547","MTBLC39426","MTBLC132446","MTBLC83491","MTBLC83492","MTBLC132448","MTBLC39421","MTBLC83493","MTBLC90633"],"ChEBI":["CHEBI:38397","CHEBI:27732","CHEBI:35549","CHEBI:5855","CHEBI:35546","CHEBI:35547","CHEBI:39426","CHEBI:132446","CHEBI:83491","CHEBI:83492","CHEBI:132448","CHEBI:39421","CHEBI:83493","CHEBI:90633"]}}