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

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

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

4. 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.

5. 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

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

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

8. 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

9. Dimethyl selenide Characterization60 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:
0.0
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

10. Dimethyl diselenide Characterization60 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:
0.0
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

11. Dimethyl selenide Quantification

12. Dimethyl diselenide Quantification

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

14. 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 ARPPPMB 15:

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

16. Bacterial VOCs
Produced by all species
Potential for rapid ID

17. Infection and Lysis
Brewster et al JEMI 16:

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

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

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

21. 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

22. 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.

23. Thank you
Questions?

24. SI

25. 1-hour Infection

26. 2-hour Infection

27. 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 = min. (7-decen-2-one , 18.29%)

28. 8-hour Infection

29. 12-hour Infection