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Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Published: June 1st, 2022



1Department of Molecular Biology and Biochemistry, University of California Irvine, 2Department of Ecology and Evolutionary Biology, University of California Irvine, 3Entech Instruments Inc.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host's microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.

Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

Volatile organic compounds (VOCs) have great promise for bacterial detection and identification because they are emitted from all organisms, and different microbes have unique VOC signatures. Volatile molecules have been utilized as a non-invasive measurement for detecting various respiratory infections including chronic obstructive pulmonary disease1, tuberculosis2 in urine3, and ventilator-associated pneumonia4, in addition to distinguishing subjects with cystic fibrosis (CF) from healthy control subjects5,6. Vola....

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1. Headspace Sorbent Pen (HSP) and sample analysis considerations

NOTE: The HSP containing the sorbent Tenax TA was selected to capture a broad range of volatiles. Tenax has a lower affinity for water compared to other sorbents, which enables it to trap more VOCs from higher-moisture samples. Tenax also has a low level of impurities and can be conditioned for re-use. Sorbent selection was also made in consideration with the column installed in the GC-MS (see the Table of Materials

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Mono- and co-cultures of S. aureus, P. aeruginosa, and A. baumannii
The mono- and co-cultures consisted of the bacterial species S. aureus, P. aeruginosa, and A. baumannii. These are common opportunistic pathogens found in human wounds and chronic infections. To identify the volatile molecules present in the mono- and co-cultures, a short 1-h extraction was performed at .......

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To identify volatile production in in vitro cultures and human-associated samples, volatile analysis of mono- and co-cultures of P. aeruginosa, S. aureus, and A. baumanii and stable isotope probing of different biological samples were performed. In the analysis for the mono- and co-cultures, volatiles were detected by performing a short extraction for 1 h at 70 °C. The volatile analysis of mono- and co-cultures allowed the survey of the compounds produced both by individual species and dur.......

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We thank Heather Maughan and Linda M. Kalikin for careful editing of this manuscript. This work was supported by NIH NHLBI (grant 5R01HL136647-04).


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Name Company Catalog Number Comments
13C glucose Sigma-Aldrich 389374-1G
2-Stg Diaph Pump Entech Instruments 01-10-20030
20 mL VOA vials Fisher Scientific 5719110
24 mm Black Caps with hole, no septum Entech Instruments 01-39-76044B holds lid liner in place on vial
24 mm vial liner for sorbent pens Entech Instruments SP-L024S allows pens to make a vacuum seal at top of vial
5600 Sorbent pen extraction unit (SPEU) Entech Instruments 5600-SPES 5600 Sorbent Pen Extraction Unit -120 VAC
96-well assay plates Genesee 25-224
Brain Heart Infusion (BHI) media Sigma-Aldrich 53286-500G
ChemStation Stofware Agilent
DB-624 column Agilent 122-1364E 60 m, 0.25 mm ID, 1.40 micron film thickness, in GC-MS
Deuterium oxide Sigma-Aldrich 151882-1L
Dexsi sofware Dexsi (open source)
GC-MS (7890A GC and 5975C inert XL MSD with Triple-Axis Detector) Agilent 7890A GC and 5975C inert XL MSD with triple-axis detector
Headspace Bundle HS-B01, 120VA Entech Instruments SP-HS-B01 Items for running headspace extraction included in bundle
Headspace sorbent pen (HSP) - blank Entech Instruments SP-HS-0
Headspace sorbent pen (HSP) Tenax TA (35/60 Mesh) Entech Instruments SP-HS-T3560
Microcentrifuge tubes (2 mL) VWR 53550-792
O-rings Entech Instruments SP-OR-L024
Sample Preparation Rail Entech Instruments
Sorbent pen thermal conditioner Entech Instruments 3801-SPTC
Todd Hewitt (TH) media Sigma T1438-500G

  1. Van Berkel, J. J. B. N., et al. A profile of volatile organic compounds in breath discriminates COPD patients from controls. Respiratory Medicine. 104 (4), 557-563 (2010).
  2. Nakhleh, M. K., et al. Detecting active pulmonary tuberculosis with a breath test using nanomaterial-based sensors. European Respiratory Journal. 43 (5), 1522-1525 (2014).
  3. Lim, S. H., et al. Rapid diagnosis of tuberculosis from analysis of urine volatile organic compounds. ACS Sensors. 1 (7), 852-856 (2016).
  4. Schnabel, R., et al. Analysis of volatile organic compounds in exhaled breath to diagnose ventilator-associated pneumonia. Scientific Reports. 5, 17179 (2015).
  5. Paff, T., et al. Exhaled molecular profiles in the assessment of cystic fibrosis and primary ciliary dyskinesia. Journal of Cystic Fibrosis. 12 (5), 454-460 (2013).
  6. Robroeks, C. M. H. H. T., et al. Metabolomics of volatile organic compounds in cystic fibrosis patients and controls. Pediatric Research. 68 (1), 75-80 (2010).
  7. Neerincx, A. H., et al. Hydrogen cyanide emission in the lung by Staphylococcus aureus. European Respiratory Journal. 48 (2), 577-579 (2016).
  8. Goeminne, P. C., et al. Detection of Pseudomonas aeruginosa in sputum headspace through volatile organic compound analysis. Respiratory Research. 13, 87 (2012).
  9. Joensen, O., et al. Exhaled breath analysis using Electronic Nose in cystic fibrosis and primary ciliary dyskinesia patients with chronic pulmonary infections. PLOS ONE. 9 (12), 115584 (2014).
  10. Nasir, M., et al. Volatile molecules from bronchoalveolar lavage fluid can 'rule-in' Pseudomonas aeruginosa and 'rule-out' Staphylococcus aureus infections in cystic fibrosis patients. Scientific Reports. 8 (1), 826 (2018).
  11. Tyc, O., Zweers, H., de Boer, W., Garbeva, P. Volatiles in inter-specific bacterial interactions. Frontiers in Microbiology. 6, 1412 (2015).
  12. Gao, B., et al. Tracking polymicrobial metabolism in cystic fibrosis airways: Pseudomonas aeruginosa metabolism and physiology are influenced by Rothia mucilaginosa-derived metabolites. mSphere. 3 (2), 00151 (2018).
  13. Schoenheimer, R., Rittenberg, D. Deuterium as an indicator in the study of intermediary metabolism. Science. 82 (2120), 156-157 (1935).
  14. Neubauer, C., et al. Refining the application of microbial lipids as tracers of Staphylococcus aureus growth rates in cystic fibrosis sputum. Journal of Bacteriology. 200 (24), 00365 (2018).
  15. Cordell, R. L., Pandya, H., Hubbard, M., Turner, M. A., Monks, P. S. GC-MS analysis of ethanol and other volatile compounds in micro-volume blood samples-quantifying neonatal exposure. Analytical and Bioanalytical Chemistry. 405 (12), 4139-4147 (2013).
  16. Mayor, A. S. R. Optimisation of sample preparation for direct SPME-GC-MS analysis of murine and human faecal volatile organic compounds for metabolomic studies. Journal of Analytical & Bioanalytical Techniques. 5 (2), 184 (2014).
  17. Camarasu, C. C. Headspace SPME method development for the analysis of volatile polar residual solvents by GC-MS. Journal of Pharmaceutical and Biomedical Analysis. 23 (1), 197-210 (2000).
  18. Charry-Parra, G., DeJesus-Echevarria, M., Perez, F. J. Beer volatile analysis: optimization of HS/SPME coupled to GC/MS/FID. Journal of Food Science. 76 (2), 205-211 (2011).
  19. Bicchi, C., Cordero, C., Liberto, E., Rubiolo, P., Sgorbini, B. Automated headspace solid-phase dynamic extraction to analyse the volatile fraction of food matrices. Journal of Chromatography A. 1024 (1), 217-226 (2004).
  20. Trujillo-Rodríguez, M. J., Anderson, J. L., Dunham, S. J. B., Noad, V. L., Cardin, D. B. Vacuum-assisted sorbent extraction: An analytical methodology for the determination of ultraviolet filters in environmental samples. Talanta. 208, 120390 (2020).
  21. Mollamohammada, S., Hassan, A. A., Dahab, M. Immobilized algae-based treatment of herbicide-contaminated groundwater. Water Environment Research. 93 (2), 263-273 (2021).
  22. Psillakis, E. The effect of vacuum: an emerging experimental parameter to consider during headspace microextraction sampling. Analytical and Bioanalytical Chemistry. 412 (24), 5989-5997 (2020).
  23. Carmody, L. A., et al. The daily dynamics of cystic fibrosis airway microbiota during clinical stability and at exacerbation. Microbiome. 3, 12 (2015).
  24. Carmody, L. A., et al. Fluctuations in airway bacterial communities associated with clinical states and disease stages in cystic fibrosis. PLOS ONE. 13 (3), 0194060 (2018).
  25. Mahboubi, M. A., et al. Culture-based and culture-independent bacteriologic analysis of cystic fibrosis respiratory specimens. Journal of Clinical Microbiology. 54 (3), 613-619 (2016).

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