Sep 12, 2025
Analysis of Microbial Volatile Compounds by SPME-GC/MS
- Md Sahadat Ali1,
- Jonathan D. Eisenback1,
- Fatima Tuz Zohora Mony1
- 1Virginia Tech
Protocol Citation: Md Sahadat Ali, Jonathan D. Eisenback, Fatima Tuz Zohora Mony 2025. Analysis of Microbial Volatile Compounds by SPME-GC/MS. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvodpobg4o/v1
Manuscript citation:
Ali, M. S., Mony, F. T. Z., Evans, M., Rideout, S., Haak, D., & Eisenback, J. D. (2025). Unveiling the antagonistic activity of ‘Candidatus Pseudomonas auctus’ JDE115 against Agroathelia rolfsii: A soybean nodule endophyte with biocontrol potential. (In preparation)
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 12, 2025
Last Modified: September 12, 2025
Protocol Integer ID: 227164
Keywords: Volatile organic compounds, SPME-GC/MS, Pseudomonas auctus, 1-undecene, DMDS, biocontrol, antifungal metabolites, analysis of microbial volatile compound, microbial volatile compound, antifungal activity, volatile organic compound, candidatus pseudomonas auctus, dimethyl disulfide, bacterial production
Disclaimer
This protocol is intended solely for research and educational purposes. While the procedures have been validated in the authors’ laboratory, results may vary depending on strain, media composition, and experimental conditions. Users are responsible for ensuring compliance with institutional biosafety and chemical safety regulations. The authors and Virginia Tech disclaim liability for misuse or misapplication of the protocol.
Abstract
This protocol describes the use of headspace solid-phase microextraction (SPME) and GC-MS to identify volatile organic compounds (VOCs) produced by ‘Candidatus Pseudomonas auctus’ JDE115 under different culture conditions. VOCs are adsorbed on a 50/30 µm DVB/CAR/PDMS StableFlex® fiber, thermally desorbed in a GC injector, and analyzed on a DB-5 column with an Agilent 7890 GC–5975C MSD system. Compounds are identified by comparison to Wiley and NIST spectral libraries. The assay confirms bacterial production of 1-undecene and dimethyl disulfide (DMDS), both implicated in antifungal activity
Attachments
Image Attribution
All images used in this protocol, including photographs of agar plates and assay setups, were generated by the authors at Virginia Tech. Images are original and may be used for academic and research purposes with proper citation of this protocol.
Guidelines
- Use freshly prepared PDA medium in airtight vials.
- Pre-condition SPME fibers according to manufacturer’s instructions.
- Run all samples, blanks, and controls under identical instrument conditions for reproducibility.
- Include both bacterial and fungal controls, plus medium-only blanks.
Materials
- 'Candidatus P. auctus’ JDE115 culture (LB agar, 48 h, 28 °C)
- Agroathelia rolfsii culture (optional co-culture condition)
- Potato dextrose agar (PDA) medium
- 20 mL glass vials with Teflon septa (airtight)
- 50/30 µm DVB/CAR/PDMS StableFlex® SPME fiber (Supelco, USA)
- Deionized water
- Helium (carrier gas, GC-grade)
Equipment
- Incubator (28 °C)
- Magnetic stirrer and stir bars
- Agilent® 7890 GC coupled to a 5975C mass selective detector (MSD)
- DB-5 capillary column (30 m × 250 µm I.D., 0.25 µm film thickness)
- Vortex mixer
- Analytical balance
- Pipettes and sterile tips
Troubleshooting
Safety warnings
- Handle all cultures in a biosafety cabinet when possible.
- Volatile metabolites may include hazardous compounds; avoid direct inhalation.
- GC-MS solvents and carrier gases must be handled in accordance with institutional chemical safety rules.
- Follow manufacturer instructions for safe handling of SPME fibers.
Ethics statement
This protocol involves only microbial cultures and chemical analysis, requiring no human or animal ethics approval. All procedures must be conducted in accordance with institutional biosafety and chemical safety guidelines.
Before start
- Pre-clean and bake glass vials to remove contaminants.
- Verify incubator temperature at 28 °C.
- Prepare labeled 20 mL airtight vials with Teflon septa.
- Confirm GC-MS system calibration and mass library availability.
Treatment Setup
Culture conditions:
- JDE115 (bacterial control)
- A. rolfsii (fungal control)
- JDE115 + A. rolfsii (co-culture)
- Medium-only (blank control)
Inoculate 10 mL PDA into 20 mL airtight vials, incubate at 28 °C.
Sample preparation
- Prepare airtight 20 mL vials with 10 mL PDA.
- Inoculate according to the four experimental conditions.
- Incubate at 28 °C until VOC production stage.
- Equilibrate samples at room temperature for 30 min.
- Insert pre-conditioned SPME fiber into vial headspace for 10 min exposure.
- Retract fiber and immediately inject into GC injector port.
GC-MS analysis
- Injector port: 200 °C
- Split ratio: 1:10
- Purge flow: 3 mL/min
- Carrier gas: Helium, 1.0 mL/min constant flow
- Oven program:
Initial 35 °C, hold 3 mins
Ramp 15 °C/min to 200 °C
Hold 200 °C for 5 min
- Transfer line: 220 °C
- Mass spectra: Electron ionization (EI) mode
- Library: Wiley and NIST
Data acquisition
- Collect chromatograms and spectra for each sample.
- Identify peaks by comparison to library spectra.
- Quantify target compounds by peak integration (e.g., DMDS, 1-undecene).
Protocol references
Kai M, Effmert U, Berg G, Piechulla B (2007) Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani. Arch Microbiol 187:351–360. https://doi.org/10.1007/s00203-006-0199-0
Schafer H, Myronova N, Boden R (2010) Microbial degradation of dimethylsulphide and related C1-sulphur compounds: organisms and pathways controlling fluxes of sulphur in the biosphere. J Exp Bot 61:315–334. https://doi.org/10.1093/jxb/erp355
Acknowledgements
We acknowledge the Nematode Diagnostic Lab at Virginia Tech for providing facilities and resources essential for conducting this work.