William Trebelcock & James Erdmann Microbe farts: detecting volatile organic compounds from fungi and bacteria using GC/MS William Trebelcock & James Erdmann

GC-MS Sampling Methods Volatile Organic Compounds (VOCs) in Microorganisms VOCs in Fungi Direct Headspace VOCs in Bacteria Solid-phase Microextraction (SPME)

GC-MS Sampling Methods Volatile Organic Compounds (VOCs) in Microorganisms VOCs in Fungi Direct Headspace VOCs in Bacteria Solid-phase Microextraction (SPME)

GC-MS Sampling Methods Direct Headspace Sampling: Quantification of VOCs using 1 mL of headspace gas. Solid Phase Microextraction (SPME): Concentration of VOCs on absorbent fiber.

Why use GC-MS? Chromatogram MS: Mass analyzer, compound ID Spectrum 100 90 80 70 60 Relative Abundance 50 40 30 20 10 5.8 6.0 6.2 6.4 6.6 6.8 Time (min) 7.0 7.2 7.4 7.6 7.8 8.0 8.2 Spectrum 100 90 80 Database spectral comparison GC: Separation 70 60 Relative Abundance 50 40 30 20 10 50 100 150 200 250 300 350 400 450 500 550 600

Selenium Tolerance - Reduction Fungus + Selenium Reduction Selenite or Selenate Selenium Crystals Volatilization

Selenium Tolerance - Volatilization Fungus + Selenium Reduction Volatilization Selenite or Selenate ? DMSe selenite selenate dimethyl selenide

Fungal Isolates Fungal Treatments: Selenate, selenite, and control Fungal Isolate Name and Abbreviated ID Isolate ID Aspergillus leporis AS117 AS2 Absidia spinosa AB134 Alternaria seleniphilia A1 Alternaria tenuissima A2 Alternaria astragali A3 Fusarium acuminatum F30 AS117 plate without selenium Fungal Treatments: Selenate, selenite, and control 30 ppm and 100 ppm concentrations 21 days growth in darkness at 22°C AS2 headspace treatment vials

Dimethyl selenide Characterization 60 80 100 120 140 160 180 200 m/z 10 20 30 40 50 70 90 Relative Abundance 109.96 92.93 107.97 90.93 106.98 79.91 105.98 89.94 111.96 96.93 77.91 76.93 112.98 73.90 58.08 47.03 131.01 175.98 145.88 159.91 196.51 187.07 RT: 0.00 - 5.52 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Time (min) 10 20 30 40 50 60 70 80 90 100 Relative Abundance 1.47 3.57 1.07 1.11 1.18 1.51 1.88 2.99 3.63 0.05 2.50 0.29 4.48 0.97 0.61 1.97 3.77 3.36 4.39 2.84 4.70 4.92 5.24

Dimethyl diselenide Characterization 60 80 100 120 140 160 180 200 m/z 10 20 30 40 50 70 90 Relative Abundance 92.94 94.94 90.95 189.91 187.90 174.88 79.93 89.95 185.91 108.96 170.89 88.94 77.94 159.86 191.91 106.97 176.88 168.88 155.87 75.93 104.98 81.94 110.95 153.90 193.91 74.02 47.29 63.86 126.87 141.18 RT: 0.00 - 5.52 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 Time (min) 10 20 30 40 50 60 70 80 90 100 Relative Abundance 1.47 3.57 1.07 1.11 1.18 1.51 1.88 2.99 3.63 0.05 2.50 0.29 4.48 0.97 0.61 1.97 3.77 3.36 4.39 2.84 4.70 4.92 5.24

Dimethyl selenide Quantification

Dimethyl diselenide Quantification

Selenium Tolerance - Volatilization Fungus + Selenium Volatilization Selenite 30 ppm DMDSe (low) DMSe 100 ppm (+) (high) Selenate

Conclusions Selenium metabolism different between plants and fungi? DMDSe analysis novel in fungi DMDSe production both concentration and species dependent Production varies between species Terry et al. 2000. ARPPPMB 15:401-432.

GC-MS Sampling Methods Volatile Organic Compounds (VOCs) in Microorganisms VOCs in Fungi Direct Headspace VOCs in Bacteria Solid-phase Microextraction (SPME)

Bacterial VOCs Produced by all species Potential for rapid ID

Infection and Lysis Brewster et al. 2012. JEMI 16:139-143.

Method Development SPME Escherichia coli K12 MS2 bacteriophage Multiplicity of infection Bacterial concentration Sampling and infection time SPME/GC-MS settings SPME

Time-step Analysis 12-hour 8-hour 4-hour 2-hour 1-hour

Conclusions Large variation in VOCs across infection time Most VOCs show up by 4 hr Other parameters optimized for SPME/GC-MS analysis

What does it all mean? Fungus Project Successful quantitation of selenocompounds Mycoremediation Novel identification of dimethyl diselenide production Ecoevolution Se biochemical pathways Phage Project Preliminary data show good potential Use in bacterial VOC fingerprint development Analysis of foodborne pathogens and other health concerns Thoughts/Conclusions Interesting patterns of volatile production/loss dihydro-2-methyl-3(2H)-thiophenone has only been previously detected in yeast Contamination or novel detection 7-decen-2-one not previously detected in microbe HS

Acknowledgements Funding Fungus Project (LCCC) Dr. Ami Wangeline Sami Haller Josh Sharpe Phage Project Nike Kabwar Jacque Black Holden Bindl (Wyoming EPSCoR SRAP) Basile Lab (UW) Mentor: Dr. Franco Basile Dr. Raj Mahat Mitch Helling Rudy Mignon Funding This project was supported in part by grants from the National Center for Research Resources (P20RR016474) and the National Institute of General Medical Sciences (P20GM103432) from the National Institutes of Health.

Thank you Questions?

SI

1-hour Infection

2-hour Infection

4-hour Infection Other peaks: t = 2.58 min. (dimethylsilanediol) t = 3.00 min. (dimethyl disulfide, 98.44%) t = 5.58 min. (dihydro-2-methyl-3(2H)-thiophenone, 91.23%) t = 6.27 min. (1-octanol, 39.85%) t = 7.09 min. (1-nonanol, 8.55%) t = 7.84 min. (1-decanol, 9.66%) t = 8.09 min. (indole, 51.02%) t = 9.21 min. (1-hexadecanol, 5.45%) t = 10.45 min. (7-decen-2-one , 18.29%)

8-hour Infection

12-hour Infection