1

SPME-GC/MS Analysis of Volatile Organic Compounds Produced by Oral Cavity Streptococci

by Amanda Dell’Olio

B.S. in Chemistry, Northeastern University

A thesis submitted to

The Faculty of

the College of Science of

Northeastern University

in partial fulfillment of the requirements

for the degree of Master of Science

December 5, 2017

Thesis directed by

Dr. Adam B. Hall

Director, Core Mass Spectrometry Facility

Barnett Institute of Chemical and Biological Analysis

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Acknowledgements

I would like to thank Dr. Adam Hall, my principal investigator, for his insight, expertise,

and assistance with the project. I’d particularly like to thank him for assistance with and

troubleshooting of the GC/MS and for his reviews of my thesis.

I’d also like to thank Violetta Medik and her advisor, Dr. Slava Epstein, for partnering

with me on this project and allowing me to undertake the analytical chemistry side of the project

while they handled the microbiology side of it. I am grateful to Violetta for teaching me the

SPME-GC/MS method, for providing me with bacterial samples to test, and for providing me

with some figures to use in my thesis.

Thank you to Dr. Adam Hall, Dr. Slava Epstein, and Dr. Paul Vouros for serving on my

thesis committee and taking the time to review my thesis.

Thank you as well to my other colleagues in the Northeastern University community for

giving me the tools and education to complete this thesis.

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Abstract of Thesis

According to the Human Oral Microbiome Project, dental plaque consists of about 700

different species of bacteria.1 Dental plaque is the reason that the average American adult has

between ten and seventeen decayed, missing, or filled permanent teeth and most of the

population suffers from at least a minor case of gingivitis.1 Metagenomics research has provided

useful information about dental plaque composition in various stages of health and disease, but it

is unclear what ecological forces cause the establishment and composition of the microbiome. It

is known that dental plaque formation is affected by interactions and competitive or mutualistic

relationships between different strains of bacteria. This interaction can be driven by the

production of volatile organic compounds (VOCs). The ecological roles of these compounds in

the headspace was previously overlooked, but it is known that interkingdom interactions and

bacterial behavior are affected by bacterial VOCs.2 SPME-GC/MS was used in a series of

experiments to determine the VOC profiles of several strains of oral Streptococci to gain insight

about what role this may play in the establishment of healthy oral flora. Every strain of bacteria

appeared to have a different VOC profile associated with it, but analysis with PCA plots did not

reveal any distinct differences that clearly separated strains. It was observed that culturing on

agar versus in broth affected the VOC profiles although there were other factors that differed in

these experiments such as temperature, so it cannot be confirmed that culture medium alone

changes the VOC profile. Co-cultured samples were compared to individual strain samples, and

although unique compounds were rarely produced in co-culture compared to the individual

strains, some compounds, such as 1-butanol, were often overexpressed in the co-cultures

suggesting they may play a role in competitive or synergistic relationships. Time course

experiments revealed that the VOC profiles change over time, even just over the course of a day,

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but there isn’t always a trend in the abundances of antibacterial compounds that could affect

bacterial growth. For instance, no trend was observed when looking at acetic acid levels whereas

1-butanol levels seemed to generally increase over time. Still, it is difficult to confirm VOC

profiles and abundance levels of all compounds. Hundreds of compounds were identified using

the NIST library associated with the ChemStation software, but these compounds often appear to

be misidentified. In the VOC profiles of supposedly identical samples analyzed on different

days and of triplicate samples analyzed concurrently, differences are seen in the ChemStation

NIST library identification of a given peak at the same retention time in the replicate samples.

The data looks slightly more similar when viewed using PCA and cluster plots to visualize the

results, but there are still some inconsistencies. Using a Kruskal-Wallis test to statistically

analyze chromatograms of replicate samples gives a P value that indicates the results are not

statistically different, providing validity to the method. Still, to confirm any VOCs of interest as

being major contributors to VOC profiles or to the development of healthy oral flora, standards

must be analyzed using the same SPME-GC/MS method and the resulting mass spectra must be

compared to known mass spectra. No VOCs have been confirmed yet as being key antibacterial

compounds, but acetic acid, 1-butanol, and dimethylamine all have antibacterial properties and

show varying levels of abundance in the oral Streptococci samples that were analyzed. The

following results, while comprehensive, represent a preliminary approach for which further

studies should clarify and expand upon these findings.

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Table of Contents

Acknowledgements ......................................................................................................................... 2

Abstract of Thesis ........................................................................................................................... 3

Table of Contents ............................................................................................................................ 5

List of Figures ................................................................................................................................. 7

List of Tables .................................................................................................................................. 9

List of Symbols & Abbreviations ................................................................................................. 10

1. Introduction ........................................................................................................................... 11

2. Background ............................................................................................................................ 12

2.1. Dental Plaque Formation ................................................................................................... 12

2.2. Streptococci........................................................................................................................ 13

2.3. Bacterial VOCs .................................................................................................................. 14

2.4. Solid-Phase Microextraction (SPME)................................................................................ 15

2.5. Gas Chromatography (GC) ................................................................................................ 17

2.6. Mass Spectrometry (MS) ................................................................................................... 18

3. Materials and Methods .......................................................................................................... 19

3.1. Bacterial Samples............................................................................................................... 19

3.2. SPME Procedure ................................................................................................................ 19

3.3. GC/MS Analysis ................................................................................................................ 19

4. Results and Discussion .......................................................................................................... 20

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4.1. VOC Identification and VOCs of Interest ......................................................................... 20

4.2. Broth versus Agar .............................................................................................................. 26

4.3. Co-culturing ....................................................................................................................... 34

4.4. Time Course Experiments.................................................................................................. 39

4.4.1. 10 Day Time Course of Single Strains and Co-cultures ................................................ 39

4.4.2. 27 Hour Time Course of SA .......................................................................................... 44

4.5. Assessment of Reproducibility .......................................................................................... 47

4.5.1. Day to Day Reproducibility ........................................................................................... 49

4.5.2. 3 Day Time Course Analysis of SR4 and CM7 in Triplicate......................................... 52

4.6. Chemometrics .................................................................................................................... 58

5. Conclusions ........................................................................................................................... 62

6. Future Directions ................................................................................................................... 64

References ..................................................................................................................................... 66

Appendices .................................................................................................................................... 68

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List of Figures

Figure 1 Novel Streptococci strains identified by the Epstein lab10 ............................................ 13

Figure 2 Close-up diagram of SPME device16 ............................................................................. 15

Figure 3 SPME procedure showing the headspace extraction step and thermal desorption within

the GC injection port16 .................................................................................................................. 17

Figure 4 Identification of analytes in AC15, ACC21, and ACS2 bacterial cultures ................... 20

Figure 5 Venn diagram showing similarities and differences in the VOC profiles of SA, SR4,

OBCR6, and CM7 strains cultured on agar .................................................................................. 21

Figure 6 Unknown VOC mass spectrum (top) versus known hexanal mass spectrum (bottom) 23

Figure 7 Experimental acetic acid mass spectrum (top) versus known acetic acid mass spectrum

(bottom)......................................................................................................................................... 24

Figure 8 Experimental dimethylamine mass spectrum (top) versus known dimethylamine mass

spectrum (bottom) ......................................................................................................................... 24

Figure 9 Experimental 1-butanol mass spectrum (top) versus known 1-butanol mass spectrum

(bottom)......................................................................................................................................... 25

Figure 10 GC/MS chromatograms of CM7, CM6, and OBCR6 and the media blank cultured on

agar versus in broth10 .................................................................................................................... 26

Figure 11 Heat map showing the relative abundance of different chemical classes identified in

all samples cultured in broth10 ...................................................................................................... 27

Figure 12 Heat map showing the relative abundance of different chemical classes identified in

all samples cultured on agar10 ....................................................................................................... 27

Figure 13 VOC abundance changes when using broth or agar to culture (A) OBCR6, (B) CM6,

(C) CM7, and (D) SR4 .................................................................................................................. 30

Figure 14 Acetic acid increase from SR4 when cultured in broth versus agar ............................ 31

Figure 15 GC chromatograms of co-cultured bacteria samples ................................................... 34

Figure 16 1-butanol levels in single strains and two-strain co-cultures ....................................... 36

Figure 17 Ethanol levels in single strains and two-strain co-cultures.......................................... 37

Figure 18 3-methyl-1-butanol levels in single strains and two-strain co-cultures ....................... 38

Figure 19 PCA plot of single strain and co-culture time course data with zoomed in section

highlighting clustering based on day ............................................................................................ 39

Figure 20 Acetic acid levels in SR4 over a 10-day time course .................................................. 41

Figure 21 Acetic acid levels in SR4 and SR4 co-cultures over a ten-day time course ................ 42

Figure 22 Dimethylamine levels in CM7 over a 10-day time course .......................................... 43

Figure 23 Dimethylamine levels in CM7 and CM7 co-cultures over a ten-day time course ...... 44

Figure 24 Initial presence of 1-butanol during SA time course study ......................................... 45

Figure 25 Changing 1-butanol levels over 1-day SA time course study ..................................... 46

Figure 26 CM6 chromatograms and VOC profiles cultured on agar (top) and in broth (bottom)49

Figure 27 Venn diagrams of SR4 triplicates’ VOC profiles on A) day 1, B) day 2, and C) day 3

....................................................................................................................................................... 54

Figure 28 Venn diagrams of CM7 triplicates’ VOC profiles on A) day 1, B) day 2, and C) day 3

....................................................................................................................................................... 56

Figure 29 A) Clustering plot and B) PCA plot of CM7, OBCR6, SA, and SR4 samples ........... 59

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Figure 30 PCA plot of all agar blanks from the course of this study ........................................... 60

Figure 31 PCA and clustering plots of A) SR4 triplicates and B) CM7 triplicates over 3-day time

course10 ......................................................................................................................................... 61

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List of Tables

Table 1 Biological activity of compounds of interest10................................................................ 22

Table 2 SA x SR4 co-culture VOC profile comparison ............................................................... 35

Table 3 CM7 agar and broth duplicate VOC profiles .................................................................. 50

Table 4 OBCR6 agar and broth duplicate VOC profiles ............................................................. 50

Table 5 SR4 agar and broth duplicate VOC profiles ................................................................... 51

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List of Symbols & Abbreviations

° degrees

± plus or minus

μm micrometers

amu atomic mass units

B. subtilis Bacillus subtilis

BLIS Bacteriocin-Like Inhibitory Substances

C Celsius

CAR Carboxen

CE Capillary Electrophoresis

CV Coefficient of Variation

DVB Divinylbenzene

E. coli Escherichia coli

EI Electron Ionization

et al. et alia (“and others”)

GC Gas Chromatography

GC/MS Gas Chromatography/Mass Spectrometry

g/mol grams per mole

HPLC High-Performance Liquid Chromatography

ID Identification

LLE Liquid-Liquid Extraction

m meter

min minutes

mm millimeter

MS Mass Spectrometry

mw molecular weight

m/z mass to charge ratio

Pa Pascal

PCA Principal Component Analysis

PDMS Polydimethylsiloxane

Rep Replicate

SPME Solid-Phase Microextraction

TIC Total Ion Chromatogram

USA United States of America

VOC Volatile Organic Compound

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1. Introduction

The average American adult has from ten to seventeen decayed, missing, or filled

permanent teeth, and most of the population also has at least a minor case of gingivitis.1 Dental

plaque is comprised of approximately 700 bacterial species according to the Human Oral

Microbiome Project.1 The primary cause of dental decay is Streptococcus mutans.3 Recent

advancements in metagenomics research has provided valuable information about dental plaque

composition in various stages of health and disease; however, it is still unclear what the

ecological forces are that cause the establishment and composition conditions of the

microbiome.4 Compositional studies have been done to characterize and map the microbiome,5

but these studies simply provide information about types of organisms populating specific

niches, not information about the organisms’ functions. They do not give information about

mechanisms of interactions, such as competing or synergistic relationships, which prevents the

ecology of the biofilm from being manipulated to favor the development of a healthy oral flora

and to prevent dental problems.

As dental plaque forms, growth conditions for more bacteria become more favorable.1

Different strains of bacteria can interact with each other and establish competitive or mutualistic

relationships. One type of interaction between oral Streptococci is driven by the production of

volatile organic compounds (VOCs). VOCs are small organic molecules or compounds that are

naturally volatile at ambient temperature. In different physicochemical conditions, bacteria

produce a wide range of VOCs as primary or secondary metabolites.2,6 The ecological roles of

these compounds in the headspace were generally overlooked, but it is known that interkingdom

interactions and bacterial behavior are affected by bacterial VOCs.2 Spatially separated

Streptococci actively colonizing the surfaces of different teeth can communicate through these

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VOCs. The VOCs allow species growing in physically separated niches to affect each other if

there are air gaps present such as in the oral cavity. This would allow oral Streptococci to

potentially establish a competitive environment even where there is no means of communication

through diffusible molecules. Several strains of oral Streptococci were selected to study the

degrees of interactions of the bacteria and to analyze the VOC profiles that could be contributing

to increased bacterial growth and therefore increased levels of tooth decay and gum disease.

Solid-phase microextraction—gas chromatography/mass spectrometry (SPME-GC/MS) was

selected as the analytical tool to use to identify VOCs in different strains of oral Streptococci.

2. Background

2.1. Dental Plaque Formation

Dental plaque is constantly evolving. As dental plaque forms, growth conditions for more

bacteria become more favorable. Initially, hydrophobic molecules and macromolecules adhere to

the surface of teeth. The molecules are mainly salivary glycoproteins, specifically mucins, and

antibodies that form an acquired pellicle or conditioning film. The film is a substrate for bacterial

adhesion by primary colonizers. As the primary colonizers divide and produce extracellular

polymers, a more favorable environment for the adhesion of secondary colonizers is created.

More bacterial levels develop following this process, and an oxygen gradient is established. The

deeper layers are under completely anaerobic conditions, which allows for colonization by strict

anaerobes.1 Still, the dental conditioning film is primarily colonized by mostly facultatively

anaerobic bacteria which can grow with or without oxygen. Most Streptococci are facultative

anaerobes,7 and plaque from the oral cavity is 80 percent Streptococci.8, 9 Early colonizers

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provide the substrate for later colonizers, so the spatial and temporal composition of plaque is

affected by competitive or mutualistic interactions between early colonizers.8

2.2. Streptococci

Figure 1 Novel Streptococci strains identified by the Epstein lab10

Some Streptococci are an important part of animals’ and humans’ normal microbial flora,

but some strains can cause acute to chronic diseases.7 Oral Streptococci are a heterogeneous

group of gram-positive, non-spore forming, nonmotile, Firmicutes from the order Lactobacillales

and are divided into five groups—mutans, salivarius, anginosus, sanguinis, and mitis.8 The

Epstein lab at Northeastern University identified fourteen novel strains of oral Streptococci as

shown in Figure 1. Three separate groups were identified—mitis consisting of seven isolates,

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salivarius consisting of two isolates, and anginosus consisting of five isolates. Biological studies

were carried out by the Epstein lab to determine pairs of strains that showed growth

enhancement or suppression. Strains of interest were selected for VOC analysis. OBCR6, part of

the S. anginosus group, was selected because of its ability to cause invasive pyrogenic infections

of the central nervous system, lung, liver, and spleen. SA, CM6, SV, and SR4 induced OCBR6

growth when co-cultured so these strains were also selected for studies. SR4 is a member of the

S. salivarius group. Some strains of S. salivarius are being trialed for their use as a probiotic to

prevent oral infections. Other S. salivarius strains produce antimicrobial peptides called

Bacteriocin-Like Inhibitory Substances (BLIS), which inhibit Streptococcus pyogenes, which

causes Strep throat infections.7

2.3. Bacterial VOCs

Interactions by VOCs are a fairly new and unexplored area of research. A study by Tait et

al. evaluated the use of solid-phase microextraction—gas chromatography/mass spectrometry to

analyze VOCs produced by gram-negative and gram-positive bacteria.11 They investigated the

effect of culture medium, SPME fiber type, and GC column and determined that culture medium

and SPME fiber type significantly affected VOC profiles while GC column polarity had little

effect.11 Other studies investigated interactions between different strains of bacteria that were

influenced by VOC production. Heal et al. observed in 2002 that E. coli resistance to ampicillin

is induced when E. coli cells are cultured in spatial proximity but in physical separation from a

dense population of a different E. coli strain.12 In 2014, Gabreva et al. demonstrated that the soil

bacterium’s, Pseudomonas fluorescens’s, growth, antibiotic production, and gene expression

were affected by the VOCs emitted by different soil bacteria.13 In an in vitro study by Chai et al.,

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VOCs emitted by B. subtilis enhanced the biofilm formation of other B. subtilis cells that were

physically separated but growing in proximity.14 VOC driven interactions are more common and

widespread than initially thought. One study by Aydin et al. already demonstrated the use of

VOC analysis in a medical or dental setting by determining that VOCs can be quantified and

used to identify the source of halitosis.15 Analysis of VOCs produced by oral Streptococci could

provide insight into the factors that drive healthy plaque formation and the establishment and

maintenance of healthy dental plaque.

2.4. Solid-Phase Microextraction (SPME)

Figure 2 Close-up diagram of SPME device16

For this thesis, VOC profiles for individual bacterial strains and co-cultures were

determined using solid-phase microextraction (SPME) and gas chromatography/mass

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spectrometry (GC/MS). SPME was invented by Pawliszyn in 1989 as a simple, efficient, and

solventless sample preparation method.16 It was first applied to environmental and food analysis,

but now it is being used in environmental and food chemistry as well as in the analysis of

alcoholic beverages, biological fluids, hair, breath, and more. It is especially applicable to and

widely used for the analysis of volatile and semi-volatile compounds from biological,

environmental, and food samples.16 As opposed to liquid-liquid extraction (LLE), SPME

involves one step and one device, shown in Figure 2, for extraction, concentration, and transfer

to the GC. It reduces sample preparation time, decreases solvent-related costs, and can improve

limits of detection. SPME is used to extract analytes from gaseous samples by inserting a needle

through the septum into the headspace of a sample and exposing the thin fused-silica fiber coated

with a thin polymer film stationary phase such as polydimethylsiloxane (PDMS), which is the

most commonly used stationary phase.16 The coating is selected based on compounds of interest

so that it will interact with compounds best depending on the class of compound and its polarity.

The polymer concentrates the analytes through adsorption. SPME is routinely used with gas

chromatography, high-performance liquid chromatography (HPLC), and capillary

electrophoresis (CE). In the case of GC, the fiber and needle are retracted and removed from the

headspace, and the analytes are thermally desorbed within the hot GC injection port. It is a very

sensitive method and is ideally suited for coupling to mass spectrometry.16 Figure 3 shows the

SPME procedure when coupling with GC/MS.16

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Figure 3 SPME procedure showing the headspace extraction step and thermal desorption within

the GC injection port16

SPME is known to have some reproducibility issues, and it is largely affected by

temperature and extraction time so it is important to control these factors to improve

reproducibility.16 The SPME fiber can also become fully saturated and will only concentrate

volatiles and semi-volatiles that interact well with the fiber’s stationary phase so appropriate

fiber use is necessary to obtain optimal results.16

2.5. Gas Chromatography (GC)

Gas chromatography is used to separate compounds that can be volatized. It was invented

by A.T. James and A.J.P. Martin in 1952.17, 18 The technique involves physically separating

compounds from each other in a long column typically ranging from 15 to 60 meters in length.19

An inert carrier gas such as nitrogen, helium, or hydrogen serves as the mobile phase and flows

through a heated column. In this type of chromatography, the mobile phase is not used for

interacting with the analytes, it simply helps the analytes flow through the column. The column

is packed with a silicon oxide based material or is coated with a polymeric wax. The sample is

vaporized in the injection port, flows through the column assisted by the carrier gas, and then

analytes are detected by detectors such as flame ionization or thermal conductivity. It is a widely

18

used technique but is only applicable to gaseous or volatile compounds that are thermally stable.

Therefore, it is not commonly used for biomolecules since compounds with large molecular

weights like peptides and proteins are thermally degraded and are beyond the detection

capabilities of commonly used GC detectors.20

2.6. Mass Spectrometry (MS)

GC can be coupled to mass spectrometry to aid in identifying compounds. Mass

spectrometry is used to determine the molecular weight of ions in vacuum. It began being used

during World War II when it was largely used by the petroleum industry for the quantitative

analysis of refined products.18 Sample molecules are first ionized in the ion source. The sample

must be ionized so that it can be manipulated by magnetic and electric fields. After ionization,

the mass analyzer separates compounds according to their mass-to-charge ratios. A detector

registers the signals associated with the analytes and sends the information to a computer for

analysis. This method is very sensitive, but it can be hard to differentiate a complex sample with

compounds having the same mass. It also requires a high vacuum of about 10-5 Pa to operate.20

The sensitivity and ability to analyze volatile compounds made SPME-GC/MS ideal for

the analysis of VOCs produced by oral cavity bacteria in this study. SPME is simple, efficient,

and solventless, limiting the need for additional materials, and it allows for sampling from the

bacterial culture tubes without opening the tubes and releasing the VOCs or killing the bacteria.

SPME is also easily coupled to GC/MS. Gas chromatography was ideal for this study since the

analytes being studied were volatile. Mass spectrometry is a highly sensitive technique that has

low sample requirement and allowed for selective identification of compounds based on mass to

charge ratios.

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3. Materials and Methods

3.1. Bacterial Samples

Various bacterial samples were either plated on agar or grown in broth under anaerobic

conditions by Violetta Medik and incubated for varying lengths of time at 37°C. The following

eighteen strains were used in this study: BS35a, BS21, AS20, AS14, ACS2, ACC21, AC15,

CM6, CM7, OBCR6, SR4, SA, BS29, SO, SR1, SR5, SV, and SM. Most of the experiments

were carried out using SR4, SA, OBCR6, CM6, and CM7.

3.2. SPME Procedure

Divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 μm) fibers

were purchased from Supelco along with the SPME holder (Supelco Park Bellefonte,

Pennsylvania, USA). The SPME fiber was attached to the SPME holder and conditioned prior to

use by inserting it into the GC injection port at 270°C for 30 minutes. The SPME fiber was

exposed to the headspace of either agar or broth blanks or bacterial culture samples by injecting

the SPME needle through the septum of the culture tube and injecting the fiber into the

headspace. For broth samples, the incubation tube was placed in a water bath at 50°C ± 5°C. For

agar samples, headspace extraction was done at ambient temperature to prevent the agar from

melting. The VOCs were allowed to adsorb to the fiber for 20 minutes. The fiber was then

retracted and injected into the injection port of the GC/MS at 270°C for five minutes.

3.3. GC/MS Analysis

The analysis of VOCs produced by the bacterial samples was performed using GC/MS.

An Agilent 6890 GC/5973N MS (Santa Clara, California, USA) with helium (grade 5.0;

Middlesex Gases & Technologies, Inc.; Everett, Massachusetts, USA) as the gas type was used.

20

Analytes were separated on an RTX-5 silicon MS column (30 m x 0.32 mm x 0.33 μm; Restek,

Bellefonte, Pennsylvania, USA). The GC/MS method used split injection with an injection port

temperature of 270°C and a five-minute desorption time. The oven temperature was ramped from

40°C to 250°C. The total GC run time was 24 minutes. Electron impact ionization, positive ion

mode, and a mass range of 35-650 amu was used for MS detection and analysis.

VOCs were identified using ChemStation’s data analysis software and the NIST mass

spectral library. Chromatographic peaks were selected at their apex and the mass spectrum was

compared to the software’s existing library to determine the compound’s identification. For

compounds that were of interest, the mass spectra were compared to known mass spectra from

NIST Chemistry Webbook to confirm or refute the library identification.

4. Results and Discussion

4.1. VOC Identification and VOCs of Interest

Figure 4 Identification of analytes in AC15, ACC21, and ACS2 bacterial cultures

21

After each sample of bacteria was analyzed using SPME-GC/MS, the VOCs present in

the headspace were identified using the data from the NIST mass spectral library associated with

the ChemStation data analysis software. Each bacterial sample was compared to a media blank

analyzed in the same session to determine which analytes were unique to the bacteria and not

coming from the culture media. The compounds were identified and listed in table format along

with their retention times as shown in Figure 4.

Figure 5 Venn diagram showing similarities and differences in the VOC profiles of SA, SR4,

OBCR6, and CM7 strains cultured on agar

Most of the experiments were conducted with the SR4, SA, OBCR6, and CM7 bacterial

strains cultured on agar. Although all the strains are oral Streptococci, there was little overlap in

their VOC profiles as shown in Figure 5. Acetic acid is the only compound all four strains

produced, and no combination of three of the strains produced more than one compound in

common. Still, in most samples, acetic acid and 3-methyl-1-butanol were present in large

22

amounts and ethanol was frequently present. 3-methylbutanal and carbon dioxide were detected

in the media blanks, but the abundance increased when bacteria were present.

Some analytes had very small percent similarities to the compounds identified by the

ChemStation software, and sometimes the same compound was identified at different retention

times. This shows the software library has some limitations and was not always correctly

identifying the VOCs present in these samples. Further comparison with known MS data would

be necessary to confirm all the library identifications. Hundreds of unique VOCs were identified

so the identity of each one was not confirmed, but VOCs of interest were selected for

confirmation and further analysis based on their biological activities.

Table 1 Biological activity of compounds of interest10

The biological activity of select compounds was evaluated by Violetta Medik as shown in

Table 1. The antibacterial and antifungal properties of hexanal and the fact that it is produced by

bacteria other than those being studied here made it a compound of interest. Hexanal is an alkyl

aldehyde found in human biofluids. Among mediators of oxidative stress, the highly reactive

secondary aldehydic lipid peroxidation products can initiate the processes of spontaneous

mutagenesis and carcinogenesis. It can also act as a growth-regulating factor and signaling

23

molecule, and is associated with the development of bad flavors.21 Hexanal was initially

identified as a VOC detected in several samples, but upon comparison of the experimental mass

spectrum to the known mass spectrum, it became clear the library misidentified this VOC.

Figure 6 Unknown VOC mass spectrum (top) versus known hexanal mass spectrum (bottom)

Figure 6 shows the difference between what was initially thought to be hexanal and the

known EI mass spectrum of hexanal. The strong presence of the peaks at 70, 80, and 94 m/z

make it impossible for this VOC to be hexanal (MW = 100.16 g/mol) so other biologically active

VOCs were investigated.

Acetic acid was present in nearly every sample that was analyzed, and it has antibacterial

properties, so it could contribute to competing relationships between different bacterial strains.

Acetic acid is a synthetic carboxylic acid that is known to have antibacterial and antifungal

properties, but the mechanism of action is not known. It is believed that lipid solubility can be

improved by undissociated acetic acid, which allows for more fatty acid accumulation on the cell

membrane and in other structures in the cell wall. Carbohydrate metabolism can be inhibited by

acetic acid, which could result in organism death.22

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Figure 7 Experimental acetic acid mass spectrum (top) versus known acetic acid mass spectrum

(bottom)

The library identification of acetic acid was a stronger match to the actual data than it was

for hexanal. Figure 7 shows key ions at 43, 45, and 60 m/z allowing this compound to be

confidently identified as acetic acid and further investigated.

Figure 8 Experimental dimethylamine mass spectrum (top) versus known dimethylamine mass

spectrum (bottom)

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Dimethylamine was selected as another compound of interest since it is seen in other

non-Streptococcus bacteria and it is antibacterial. The strong peak at 44 m/z and the less intense

peak at 45 m/z in Figure 8 match the known dimethylamine spectrum. The peak at 28 m/z in the

known spectrum cannot be matched with a peak in the experimental spectrum because the mass

range truncated at 35 m/z.

Figure 9 Experimental 1-butanol mass spectrum (top) versus known 1-butanol mass spectrum

(bottom)

1-butanol, also called biobutanol when biologically produced, is a known antibacterial

and antifungal. Gut microbial fermentation produces it in small amounts through the butanoate

metabolic pathway.23 Figure 9 shows that this compound could be confirmed as a VOC produced

by various strains of bacteria. The relative intensities of the peaks at 39, 41, 42, 43, 55, and 56

m/z make it a likely match to 1-butanol. In studies of the effect of agar versus broth as a culture

medium, of co-culturing, and of incubation time, acetic acid, dimethylamine, and 1-butanol were

monitored for changes in their abundances.

26

4.2. Broth versus Agar

Figure 10 GC/MS chromatograms of CM7, CM6, and OBCR6 and the media blank cultured on

agar versus in broth10

Some strains were cultured on only agar or only in broth while others were cultured in

both types of media to investigate the effects media had on VOC profiles. Figure 10 illustrates

that the chromatograms generated for identical strains of bacteria differed based on what type of

media was used for culturing. Even the blank samples differed greatly.

27

Figure 11 Heat map showing the relative abundance of different chemical classes identified in

all samples cultured in broth10

Figure 12 Heat map showing the relative abundance of different chemical classes identified in

all samples cultured on agar10

28

In general, in samples cultured on agar, VOCs belonging to more chemical classes were detected

than in samples cultured in broth as seen in Figure 11 compared to Figure 12. Ninety different

compounds and ten different chemical classes were detected when culturing on agar versus in

broth.

A)

-500000

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

0 5 10 15 20 25

Ab

un

dan

ce

Retention time (min)

OBCR6

Agar

Broth

Compound ID Retention time (min) % difference agar/broth Abundance increase when using agar?

ethoxyethene 1.2-1.4 160.4 yes

1-butanol 1.5 23.5 no

glycerin 6.4-6.6 175.0 yes

benzeneacetaldehyde 7.1 48.4 no

29

B)

C)

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CM6

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Compound ID Retention time (min) % difference agar/broth Abundance increase when using agar?

ethylene oxide 0.99 63.7 no

butanal 1.1-1.4 44.0 no

acetic acid 1.3 34.6 no

glycerin 6.7-6.8 220.4 yes

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Agar

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Compound ID Retention time (min) % difference agar/broth Abundance increase when using agar?

3-methyl-1-butanol 2.2 18.2 no

glycerin 6.4 75.2 no

30

D)

Figure 13 VOC abundance changes when using broth or agar to culture (A) OBCR6, (B) CM6,

(C) CM7, and (D) SR4

Occasionally, samples cultured on agar also produced VOCs with greater abundance.

This is apparent in the chromatograms for OBCR6 in Figure 10. There are two abundant peaks

with the same retention times in both the agar and broth chromatograms, but the agar

chromatogram shows a greater abundance. Taking a closer look at the OBCR6 chromatograms in

Figure 13 (A), we see that four of the same compounds were identified in broth and agar

samples. A semi-quantitative approach was taken by peak area integrations to analyze the

abundance of these compounds. The integrated peak areas of the same compound in broth and

agar samples were compared and a percent difference was calculated for the integrated area of

the compound seen in agar versus in broth. More than one and a half times more ethoxyethane

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Agar

Broth

Compound ID Retention time (min) % difference agar/broth Abundance increase when using agar?

ethylene oxide 0.97-0.98 55.9 no

ethanol 1.1 88.5 no

butanal 1.3-1.4 6.0 no

methylcyclopentane 1.4-1.5 5.4 no

3-methyl-1-butanol 2.1-2.2 23.9 no

2-methyl-1-butanol 2.1-2.3 11.3 no

31

and glycerin amounts were detected when samples were cultured using agar rather than broth.

Yet, the detected amounts of 1-butanol, a previously identified antifungal and antibacterial, and

benzeneacetaldehyde decreased, and only a 23.5% and 48.4% difference in abundances was seen

respectively. Similarly, Figure 13 (B) shows that for the CM6 strain, more than twice as much

glycerin was detected in agar samples compared to broth samples, but the amounts of ethylene

oxide, butanal, and acetic acid that were detected decreased when culturing with agar. The two

common compounds identified in CM7 agar and broth samples (Figure 13 (C)) and the six

common compounds identified in SR4 agar and broth samples (Figure 13 (D)) were all more

abundant when samples were cultured using broth. SR4 agar and broth samples had the greatest

number of VOCs in common out of the four strains investigated here, and the differences

between SR4 agar and broth VOCs are least significant. Butanal, methylcyclopentane, and 2-

methyl-1-butanol levels only differed by 6.0%, 5.4%, and 11.3% respectively when culturing

with broth versus agar.

Figure 14 Acetic acid increase from SR4 when cultured in broth versus agar

In Figure 14, the peak corresponding to acetic acid is circled.

32

Different samples of SR4 cultured in broth and on agar also showed the presence of

acetic acid, another one of the previously identified antibacterial compounds of interest. Figure

14 shows the full chromatograms and a zoomed in inset showing the acetic acid peaks circled.

The abundance of acetic acid is about 1.4 times greater in broth compared to agar, suggesting

broth may enhance the antibacterial effect of acetic acid. Still, two sets of SR4 samples yielded

different acetic acid results so there is a lack of consistency even when comparing samples

cultured with the same type of media.

Apart from abundance changes, the broth samples often reveal a greater number of VOCs

detected, especially when looking at the CM7 and SR4 spectra in Figure 13. Nevertheless, out of

all the VOCs identified, 148 unique VOCs were detected in agar-cultured samples as seen in

Appendix A. Whereas, 144 unique VOCs were detected in broth-cultured samples even though

more strains were cultured in broth than on agar as seen in Appendix B. The tendency of broth-

cultured samples to show more VOCs in their profiles illustrates that several of the same

compounds were present in many different strains, while agar-cultured samples tended to

produce more compounds that were present in few or no other strains.

There is clearly a difference between cultures sampled when using broth versus agar, but

it is difficult to say what contributes to this difference. Tait et al. also saw different VOC profiles

in their studies based on what media was used, but their studies were only done using different

types of broth and no agar.11 In this study, all the broth versus agar comparative samples were

analyzed on the same day using the same fiber so that should have provided some control.

However, the SPME extraction was done at ambient temperature for samples on agar and was

done at 50°C ± 5°C for samples in broth. The broth samples were heated to 50°C ± 5°C to help

release the VOCs from the broth solution. The agar samples could not be heated to this

33

temperature because the agar melts at 40°C. The temperature difference could be a reason why

agar samples gave different results than broth samples. Precision and reproducibility with SPME

are largely affected by extraction temperature.16 Less volatile analytes are more likely to be

present in the chromatograms of broth samples since the extraction temperature was higher,

which correlates with the increased number of VOCs seen in many broth samples’ spectra.

Lower extraction temperatures tend to yield chromatograms with analytes having lower retention

times, which explains why the broth sample chromatograms showed more compounds much

later in the spectrum than the agar sample chromatograms which had the lower extraction

temperature. Furthermore, the diffusion of VOCs in a liquid versus a gas is different. VOCs

produced by samples cultured in broth are initially produced within the liquid broth and must

diffuse through the liquid into the headspace to be extracted by the SPME fiber. The bacteria

cultured on agar are already atop the solid agar, so the VOCs are produced and released directly

into the headspace. They only must diffuse through the gaseous headspace to be extracted by the

SPME fiber. It is possible that some VOCs cannot diffuse as well through liquids as opposed to

gases, which would prevent them from being detected in broth-cultured samples.

34

4.3. Co-culturing

Figure 15 GC chromatograms of co-cultured bacteria samples

Based on the biological data available, four different strains of bacteria—SA, OBCR6,

CM7, and SR4—were selected to carry out most of the experiments including experiments with

co-cultures. These four strains were analyzed individually and as co-cultures of SA with

OBCR6, SA with SR4, SR4 with CM7, SR4 with OBCR6, SA with OBCR6 and CM7, SA with

SR4 and CM7, SA with OCBR6 and SR4, and SA with OBCR6, SR4, and CM7.

Chromatograms were generated for each of the co-cultures, as seen in Figure 15, and compared

to the individual strains’ chromatograms. Tables such as Table 2 were created for each co-

culture.

35

Table 2 SA x SR4 co-culture VOC profile comparison

SA SR4 Co-culture

Retention

Time

(min)

Compound ID

Retention

Time

(min)

Compound ID

Retention

Time

(min)

Compound ID

0.81 carbon dioxide 0.817 carbon dioxide 0.977 dl-alanyl-dl-methionine

0.963 dl-alanine 0.921 dimethylamine 1.046 N-

(methylthio)carbonyloxamide

1.053 dl-allo-cystathionine 0.963 cyclobutanol 1.074 ethanol

1.116 N,N’-dimethyl-1,2-

ethanediamine 1.039 formaldehyde oxime trimer 1.13 cycloserine

1.234 cyclobutanol 1.498 1-butanol 1.248 dimethylamine

1.268 dimethylamine 2.206 3-methyl-1-butanol 1.303 ethylene oxide

1.463 8-[N-aziridylethylamino]-

2,6-dimethyloctene-2 3.219 hexamethylcyclotrisiloxane 1.463 ethylene oxide

1.49 1,3-butanediamine 6.031 glycerin 1.484 5-methyl-2-hexanamine

2.226 1,1,-

dioxidetetrahydrothiophene-

3-ol

7.031 glycerin 1.532 cyclobutanol

6.746 Glycerin

1.567 1-butanol

6.864 Xylitol

1.65 1-butanol

7.155 benzeneacetaldehyde

2.164 3-methyl-1-butanol

7.738 3-ethyl-2,5-dimethyl-

pyrazine 2.227 3-methyl-1-butanol

11.307 2-butyl-3,5-dimethyl-

pyrazine 2.275 2-methyl-1-butanol

15.042 4-(1,1,3,3-

tetramethylbutyl)-phenol 2.921 2-(methylamino)ethanol

16.333 methyl ether farnesol

8.294 2,3,4,5-tetramethyl-2-

cyclopenten-1-one

9.974

glutaric acid, dodecyl 1-

naphthyl ester

In Table 2, yellow boxes indicate a compound present in the co-culture and the two individual

strains and red boxes indicate a compound present in the co-culture and one individual strain.

White boxes indicate a compound that was only present in the co-culture or individual strains.

Overall, in all the samples there were some compounds that were present in the co-

culture and in one or more of the individual strains. There were also some compounds unique to

each co-culture that were not seen in any individual strain. Attention should ideally be paid to the

compounds that are present in the co-cultures and none of the individual strains and to the

compounds that appear to be overexpressed or have increased abundance levels in co-cultures

since these compounds may be the cause of growth promotion or inhibition when multiple strains

are cultured together. For this study, it was more beneficial to focus on known antibacterials.

Once again, a semi-quantitative approach was taken by peak area integrations. The integrated

36

peak areas of compounds detected in the single strains were compared to the integrated peak

areas of those same compounds detected in the co-cultures. One integrated area was divided by

another to determine approximately by how much the abundance increased or decreased.

Figure 16 1-butanol levels in single strains and two-strain co-cultures

When looking at 1-butanol, for example, there was a difference in abundance when

strains were cultured individually versus with one other strain as shown in Figure 16. SR4 and

CM7 produced 1-butanol on their own, while SA and OBCR6 did not produce it when cultured

alone. However, when SA and OBCR6 were co-cultured, 1-butanol became present in the

headspace. When SA and SR4 were co-cultured, the 1-butanol levels were more than three times

greater than the amount produced by SR4 alone, and when SR4 was co-cultured with OBCR6,

the amount of 1-butanol present was again more than three times greater than the amount

produced by SR4 alone. Finally, when SR4 and CM7, two 1-butanol producing strains, were co-

cultured, the amount of 1-butanol present in the co-culture was about 1.7 times greater than the

combined amounts of 1-butanol present in the individual strains. Co-culturing seems to elicit an

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increased production of 1-butanol across all samples meaning there is an increased antibacterial

presence in the samples. This would suggest a competitive environment is established when two

strains are cultured together.

Figure 17 Ethanol levels in single strains and two-strain co-cultures

This sort of overexpression was also seen with other compounds such as ethanol and 3-

methyl-1-butanol. Ethanol was not selected for biological activity analysis in this study, but it is

known to have antibacterial activity and is often used as a topical disinfectant. As Figure 17

illustrates, ethanol was not present in the headspace of SR4 or SA samples but was present in the

headspace of the co-culture of these strains. Co-culturing SA with OBCR6 produced an ethanol

level about 3.7 times greater than the level produced by OBCR6 alone. Co-culturing SR4 with

CM7 caused ethanol levels to almost double compared to the level found in CM7 alone, and co-

culturing SR4 with OBCR6 produced an amount approximately 6.4 times greater than the

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amount produced by OBCR6 alone. Once again, co-culturing elicited an increased antibacterial

presence across all samples.

Figure 18 3-methyl-1-butanol levels in single strains and two-strain co-cultures

Still, overexpression does not always mean that higher levels of antibacterials are being

produced. 3-methyl-1-butanol was determined not to have antibacterial or antifungal properties,

as shown in Table 1, yet Figure 18 shows that this compound was also detected in greater

abundance in co-cultures than in individual strains. In this case, the VOC was not present at all in

the SA sample but was present in the SR4, OBCR6, and CM7 samples. Co-culturing SA with

OBCR6 and with SR4 showed approximately a four time and an eight time increase respectively

in 3-methyl-1-butanol levels compared to the levels found in just the OBCR6 and SR4 samples

when using the same semi-quantitative approach as previously described. The SR4 and CM7 co-

culture produced levels more than four times greater than levels produced by SR4 and CM7

combined, and the SR4 and OBCR6 co-culture produced levels about 4.8 times greater than

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levels produced by SR4 and OBCR6 combined. All of the co-cultures showed an increase in 3-

methyl-1-butanol levels compared to the individual strains, but 3-methyl-1-butanol is not

antibacterial or antifungal, so overexpression of a VOC does not automatically mean two strains

are establishing a competitive environment with each other.

4.4. Time Course Experiments

4.4.1. 10 Day Time Course of Single Strains and Co-cultures

Figure 19 PCA plot of single strain and co-culture time course data with zoomed in section

highlighting clustering based on day

40

Two time course experiments were carried out to determine how the VOC profiles of the

bacterial samples change over time. In one experiment, four individual strains, SA, SR4,

OBCR6, and CM7, and five co-cultures of two strains, SAxSR4, SAxOBCR6, SAxCM7,

SR4xCM7, and SR4xOBCR6, were analyzed by SPME-GC/MS after 1, 2, 3, 4, 5, and 10 days.

The VOCs were compared each day for all the samples as Appendix C shows for SR4 and

SAxSR4 samples. This time course experiment revealed variation in the VOC profiles for all the

samples over the course of the study. Acetic acid was almost always present in every strain each

day. Ethanol was almost always present in SR4. 3-methyl-1-butanol was almost always present

in the co-culture of SR4 with SA and with CM7. N-hexylmethylamine was frequently present in

SA co-cultured with CM7. Beyond those similarities, the VOC profiles varied from day to day.

Figure 19 shows a PCA plot of the individual strains and co-cultures. There is more

clustering based on day of the time course than based on sample. This means more similarities

exist between the TICs of different strains analyzed on a given day than between the same strains

analyzed on different days. This suggests the different strains and different co-cultures don’t

produce significantly different VOC profiles. VOCs appear to be more affected by sampling time

than by the sample. Yet, this seems to contradict previous data, such as that shown in Figure 5,

which showed little overlap in the VOC profiles of four different Streptococci strains analyzed

after one day of incubation. Figure 5 displays data based on the VOC identifications, whereas

Figure 19 displays data based on the TICs. This seemingly contradictory data could be explained

by the TICs from different strains being similar while the VOC identifications were different

possibly due to misidentification of compounds by the NIST library in the ChemStation

software.

41

A greater number of VOCs were typically detected in all the samples in the first few days

of the time course. However, this does not necessarily indicate that the Streptococci strains

produce fewer VOCs as time progresses since the sampling methodology would prevent that

from being confirmed. For the time course experiments, the same culture tube for each sample

was used each day. It is possible that in the initial days of sampling, some VOCs were

completely extracted and were not present later in the study if the rate of production of those

VOCs was not constant or decreased. Fully extracting some VOCs early on would also increase

the probability of extracting VOCs which do not react as favorably with the SPME fiber later in

the time course since there would be less competition for fiber surface area. Additionally, the

culture tubes are air tight with a septum cover; however, it is possible to introduce outside

contaminants when piercing the septum with the SPME needle and when removing the SPME

needle.

Figure 20 Acetic acid levels in SR4 over a 10-day time course

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Again, attention was paid to biologically active VOCs of interest. Acetic acid levels in

SR4 and the co-cultures with SR4 after each day of sample incubation and VOC extraction were

compared. Figure 20 shows the acetic acid level in the SR4 sample during each day of the time

course. Acetic acid was present every day, but there was no trend in how the levels of it

increased or decreased throughout the study. Acetic acid levels were highest on day 4 and lowest

on day 5, but nothing can be said about the rate of acetic acid production over the course of ten

days.

Figure 21 Acetic acid levels in SR4 and SR4 co-cultures over a ten-day time course

Acetic acid levels in the co-cultures with SR4 were also determined to see how acetic

acid levels change over time in co-cultures compared to in the individual strain as shown in

Figure 21. Like with the SR4 sample, there was no trend in acetic acid production in any of the

SR4 co-cultures over the ten-day time course. There was not a particular day when acetic acid

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Acetic Acid Levels

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43

production was highest for all samples, and no sample consistently produced the greatest amount

of acetic acid. Over the whole study, acetic acid was most abundant in SR4 on day four and least

abundant in SR4xOBCR6 on day four. Time does not appear to play a role in acetic acid

abundance when samples are co-cultured or cultured individually.

Figure 22 Dimethylamine levels in CM7 over a 10-day time course

Similarly, dimethylamine was present in the CM7 sample. Figure 22 shows

dimethylamine levels over the course of the ten-day time course, and once again, there was no

trend in the abundance of this VOC. It wasn’t even detected on day five of the time course, so

nothing can be said about the rate of production of dimethylamine by CM7.

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Figure 23 Dimethylamine levels in CM7 and CM7 co-cultures over a ten-day time course

Only two co-cultures with CM7 were analyzed, and with these co-cultures, there was still

no increasing or decreasing trend in dimethylamine abundance as Figure 23 illustrates. No CM7

single strain or co-culture sample consistently had the highest or lowest levels of dimethylamine.

Overall, the highest dimethylamine level was seen in the CM7 sample on day four, but then this

same sample on day five had the lowest overall dimethylamine level. Time does not appear to

play a role in dimethylamine production either when samples are co-cultured or cultured

individually.

4.4.2. 27 Hour Time Course of SA

In the other time course experiment, SA was sampled at 1, 2, 4, 6, 8, 16, and 27 hours to

monitor the VOC profiles over the course of one day since samples were typically analyzed after

one day of incubation. The VOCs were analyzed and compared as shown in Appendix D. There

was clearly variation in the VOC profile over the course of the day. There were few VOCs

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consistently present other than carbon dioxide, ethylene oxide, semioxamazide, 2,5-dimethyl-3-

(3-methylbutyl)pyrazine, 1-butanol, and 3-methyl-1-butanol, suggesting that the bacteria produce

different VOCs over the course of one day just as they produced different VOCs over the course

of several days.

Figure 24 Initial presence of 1-butanol during SA time course study

Focusing on 1-butanol as an antibacterial of interest during the SA time course study, it

was observed that the level of this VOC was not constant throughout the day. Figure 24 shows

that initially, after one hour, no 1-butanol was present. 1-butanol was first detected after four

hours. The inset of Figure 24 highlights the differences between the chromatograms from one

and four hours. The four-hour chromatogram has a double peak circled with the left peak

corresponding to 1-butanol and the right peak being a slightly coeluting compound. The one-

46

hour chromatogram has a single peak circled, which matches the coeluting compound from the

four-hour chromatogram, but 1-butanol is not seen. There is a slight discrepancy between

retention times in the two chromatograms, but this can be attributed to normal instrumental

variations unless a retention time locking feature is utilized which was not used here.

Figure 25 Changing 1-butanol levels over 1-day SA time course study

After the fourth hour, 1-butanol was present throughout the remainder of the time course

study of SA. Figure 25 shows how the levels changed. Overall, there is an increasing trend in the

1-butanol levels. Hour four is the outlier, but the abundance of 1-butanol tended to increase over

approximately the first 16 hours, although not at a steady rate, and then remained fairly constant

for the remainder of the time course. From the initial observation of 1-butanol at four hours to

the end of the time course at 27 hours, the 1-butanol levels more than doubled. This suggests that

time does play a role in SA’s 1-butanol production.

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In general, the two time course experiments show that both co-cultures and individual

strains produce different VOCs each day and during each day. Some compounds were initially

present then were not seen in the chromatograms of later time points, and some compounds were

not initially present but appeared at several time points later in the study. There were typically

more VOCs present in the beginning of the time course experiments, but this could be the result

of sampling from the same culture tube for each sample at each time point in both time course

experiments. Time does not seem to affect all antibacterial compound levels since it did not play

a role in acetic acid or dimethylamine levels, but it did correlate to a difference in 1-butanol

levels, so it may be a factor for the abundance of some antibacterials.

4.5. Assessment of Reproducibility

After sampling several different strains and co-cultures multiple times, it became clear

that the same VOC profiles were not being generated even when the samples were the same

strain, prepared under identical conditions, and analyzed after the same length of time. Samples

were not always analyzed the same number of hours after incubation, which could affect the

VOC profile as seen in the SA time course study. However, unlike in the SA study, they were

left at room temperature until they were to be analyzed, which would prevent further bacteria

growth and should have kept the VOC profile relatively constant. Regardless, there appeared to

be differences in the presence and abundance of key compounds, as mentioned in section 4.1

regarding acetic acid in two identical sets of SR4 broth and agar samples, so assessments of

reproducibility were carried out.

SPME is known to have some reproducibility issues, largely due to limitations associated

with the SPME fiber and a lack of automation of the process.24 The SPME fiber is mechanically

weak, often has poor efficiency, and the coating can degrade. There are a limited number of

48

choices commercially available for fiber coatings, which plays a crucial role in what types of

analytes can be extracted. Polar analytes are especially difficult to recover while maintaining

reproducibility.24 For this study, a mixed mode DVB/CAR/PDMS fiber was used since DVB and

CAR are useful for extracting highly polar compounds while PDMS is mainly useful for

extracting nonpolar compounds. This should have reduced issues associated with extracting polar

compounds. Still, GC/MS is not ideally suited for polar compounds unless they are derivatized

so there is still the potential for some issues. Also, the automation limitation could not be

addressed for this study. For every sample, the SPME needle and fiber was manually injected

into the headspace, removed, and injected into the GC. In-tube SPME, also called capillary

microextraction, analysis exists which can be connected to GC and HPLC automatic sampling

devices, thereby improving precision and reproducibility, 24 but it was not used for this study.

Extraction is also largely affected by temperature so slight changes in room temperature could

have affected the extraction of VOCs from agar-plated samples. Still, the room temperature was

consistently approximately 18°C so only slight variations were expected. The known limitations

of SPME were addressed as best as possible, but some reproducibility issues were expected due

to the nature of the technique.

49

4.5.1. Day to Day Reproducibility

Figure 26 CM6 chromatograms and VOC profiles cultured on agar (top) and in broth (bottom)

In Figure 26, Rep 1 refers to the first day the sample was analyzed, and Rep 2 refers to the

second day an identical sample was analyzed. Green boxes indicate the presence of a compound

in both samples.

Four individual strains of bacteria—CM6, CM7, OBCR6, and SR4—were cultured in

broth and on agar and analyzed once on two different days. Samples were prepared and analyzed

in an identical manner each day. Figure 26 shows the chromatographic data and the

corresponding VOC profile that was generated for CM6 cultured on agar and in broth.

50

Table 3 CM7 agar and broth duplicate VOC profiles

Table 4 OBCR6 agar and broth duplicate VOC profiles

Rep 1 Rep 2 Rep 1 Rep 2

(0.595) hexamethylcyclotrisiloxane (1.032) ethanol (1.074) ethanol

(0.901) carbon dioxide (1.081) acetic acid

(0.97) carbon dioxide (1.345) 2,3-epoxybutane

(1.005) dl-alanine (1.365) glutaraldehyde

(1.067) ethanol (1.977) 2,4,4-trimethyl-1-pentene (1.99) 2,4,4-trimethyl-1-pentene

(1.345) sec-butylamine (2.206) 3-methyl-1-butanol (2.212) 3-methyl-1-butanol

(1.373) propanamide (2.247) 2-methyl-1-butanol (2.254) 2-methyl-1-butanol

(1.567) 1-butanol (4.156) p-xylene (4.115) p-xylene

(2.206) 3-methyl-1-butanol (4.233) 2-hexanol

(2.254) (S)-2-methyl-1-butanol (4.566) 1,3-dimethylbenzene (4.524) 1,3-dimethylbenzene

(5.649) octamethylcyclotetrasiloxane (6.017) diisopropyl sulfide

(6.475) glycerin (6.892) 2-ethyl-1-hexanol (6.829) glycerin

(7.121) benzeneacetaldehyde (7.121) glycerin

(7.766) 3-ethyl-2,5-dimethylpyrazine (7.697) 3-ethyl-2,5-dimethylpyrazine

(8.766) 1,1,3-trimethyl-3-(2-methyl-2-

propenyl)-cyclopentane (8.703) 1,2,3,4-butanetetrol, [S-(R",R')]-

(10.939) 4-ethyl-2-methyl-5-propylthiazole

(15.070) 4-(1,1,3,3-tetramethylbutyl)phenol (15.014) 4-(1,1,3,3-tetramethylbutyl)phenol

(17.395) N-allyloxycarbonyl-l-proline hexyl ester

(18.395) N-allyloxycarbonyl-l-proline hexyl ester

(18.548) hexahydro-3-(2-

methylpropyl)pyrrolo(1,2-a)pyrazine-1,4-dione

CM7 agar CM7 broth

It doesn't look like any VOCs from bacteria adsorbed to the fiber. The two present compounds are likely from the stationary phase.

Rep 1 Rep 2 Rep 1 Rep 2

(0.907) carbon dioxide (0.97) 1-methoxy-2-propanamine

(0.977) 2-iodohiistidine (0.977) carbon dioxide (1.012) dl-alanine

(1.067) ethanol (1.074) ethanol (1.053) nitro-L-arginine

(1.13) cyclopropyl carbinol (1.144) (S)-1,3-butanediol

(1.352) 2-butanone (1.345) ethoxyethene (1.268) acetic acid (1.171) acetic acid

(1.372) 2-methylfuran (1.373) propanamide (1.234) ethoxyethene

(1.56) 1-butanol (1.352) cyclobutanol

(2.226) 3-methyl-1-butanol (2.206) 3-methyl-1-butanol

(1.421) N-(3-methylaminopropyl)-N-

methylformamide

(6.399) glycerin (1.636) 1-butanol (1.491) 1-butanol

(7.121) benzeneacetaldehyde (1.983) 2,4,4-trimethyl-1-pentene (1.81) 2,4,4-trimethyl-1-pentene

(2.06) N-methyl-1,3-propanediamine

(6.579) glycerin

(7.162) benzeneacetaldehyde (7.058) benzeneacealdehyde

(7.732) 3-ethyl-2,5-dimethylpyrazine (7.648) 3-ethyl-2,5-dimethylpyrazine

OBCR6 agar OBCR6 broth

51

Table 5 SR4 agar and broth duplicate VOC profiles

In Figure 26, Table 3, Table 4, and Table 5, the same compound was sometimes identified at

multiple retention times. This is likely due to the actual compound, whether it be an isomer or an

entirely different compound, not being present in the NIST library associated with ChemStation,

so the software identified the closest matching compound rather than the actual compound.

Rep 1 Rep 2 Rep 1 Rep 2

(0.817) carbon dioxide (0.727) carbon dioxide (0.880) carbon dioxide

(0.921) dimethylamine (0.831) formaldehyde oxime trimer

(0.956) ethylene oxide (0.984) ethylene oxide (0.88) 2-formylhistamine

(1.039) formaldehyde oxime trimer (0.942) dl-alanine

(1.074) ethanol (0.956) ethylene oxide (0.97) ethylene oxide

(1.13) furan (1.011) cyclobutanol

(1.352) butanal

(1.032) N-

(methylthio)carbonyloxamide

(1.352) 2-methylfuran (1.379) 2-methylfuran (1.046) formaldehyde oxime trimer (1.053) ethanol

(1.498) 1-butanol (1.477) methylcyclopentane (1.102) acetic acid (1.095) acetic acid

(2.206) 3-methyl-1-butanol (2.213) 3-methyl-1-butanol (1.227) N-ethyl-N'-nitroguanidine (1.192) trimethylsilanol

(2.261) 2-methyl-1-butanol (1.269) 3-methylpentane (1.241) 3-methylpentane

(3.219) hexamethylcyclotrisiloxane (1.31) butanal

(6.031) diisopropyl sulfide (6.003) diisopropyl sulfide (1.372) cyclopropyl carbinol

(7.031) glycerin (1.407) 1-butanol (1.4) methylcyclopentane

(1.449) methylcyclopentane (1.532) 1-butanol

(1.608) tert-

butylpentamethyldisiloxane

(1.851) 2,4,4-trimethyl-1-pentene

(1.97) 2,4,4-trimethyl-1-pentene (2.046) 3-methyl-1-butanol

(2.087) (S)-2-methyl-1-butanol

(2.206) 3-methyl-1-butanol

(2.247) pentafluoropropionic acid,

heptyl ester

(3.157) hexamethylcyclotrisiloxane 3.122 hexamethylcyclotrisiloxane

(3.941) 3,3-dimethylbutanamide (3.962) p-xylene

(4.545) 1,3-dimethylbenzene (4.378) 1,3-dimethylbenzene

(5.885) glycerin

(6.322) 2-methyl-1,4-

benzenediamine

(6.753) 2-ethyl-1-hexanol

(6.864) glycerin

(7.065) benzeneacetaldehyde

(7.725) 3-ethyl-2,5-

dimethylpyrazine

(7.634) 3-ethyl-2,5-

dimethylpyrazine

(15.035) 4-(1,1,3,3-

tetramethylbutyl)phenol

(15) 4-(1,1,3,3-

tetramethylbutyl)phenol

SR4 agar SR4 broth

52

The data was generated and analyzed similarly for all the other samples. Table 3, Table 4, and

Table 5 show the VOC profiles for CM7, OBCR6, and SR4 samples respectively. Although the

samples analyzed each day were supposedly identical sets, the VOC identifications varied

greatly from day to day. In Figure 26, Table 3, Table 4, and Table 5 green shaded boxes indicate

compounds that were present in each sample. Unshaded boxes indicate a VOC that was present

in one replicate but not the other. Empty spaces indicate the lack of any compound at a retention

time when a compound was present in the replicate sample. For example, as Figure 26 shows,

CM6 on agar had only four compounds detected on both days, and ten compounds were detected

that were present in only one of the replicates. Every sample had similar results. Table 3 shows

that CM7 agar samples initially appeared to have no overlap in their VOC profiles. Replicate 1

only had two compounds present, both of which are siloxanes that can be attributed to the

stationary phase of the fiber. VOCs produced by CM7 did not adsorb to the fiber, the GC/MS

experienced an instrumental error, or the CM7 culture did not successfully grow resulting in an

invalid VOC profile so this sample set was not considered when determining the level of

reproducibility

4.5.2. 3 Day Time Course Analysis of SR4 and CM7 in Triplicate

To further assess the reproducibility of this SPME-GC/MS method, three samples of SR4

prepared identically and three samples of CM7 prepared identically were analyzed each day for

three days in a row. The same six sample tubes were resampled on each of the days. Once again,

the experiment did not yield reproducible VOC identifications from the ChemStation library.

53

A)

B)

54

C)

Figure 27 Venn diagrams of SR4 triplicates’ VOC profiles on A) day 1, B) day 2, and C) day 3

Figure 27 shows that the SR4 replicates’ VOC profiles varied greatly each day. On day

one, the only compounds the triplicates shared were carbon dioxide, methanethiol, acetic acid,

and 3-methyl-1-butanol. On day two, they only shared carbon dioxide, acetic acid, and 3-methyl-

1-butanol, and on day three, they all only shared acetic acid and 3-methyl-1-butnaol in the VOC

profiles. Two replicates each day occasionally shared one or two compounds in common, but

there was a wide variety of compounds that differed between each replicate. To address the

continuous reproducibility issue, a new SPME fiber was used to analyze CM7 since the one that

was currently being used was probably approaching the end of its life cycle.

55

A)

B)

56

C)

Figure 28 Venn diagrams of CM7 triplicates’ VOC profiles on A) day 1, B) day 2, and C) day 3

Figure 28 shows a comparison of the VOC profiles of the CM7 triplicates analyzed using

a brand new SPME fiber. Once again, the same three CM7 sample tubes were sampled each day

of the study. The new fiber was appropriately conditioned and had its surface area fully intact,

but using a new fiber did not improve reproducibility of the VOCs identified in the triplicates.

On days one and three, the only compound the triplicates shared was acetic acid, and on day two,

they only shared acetic acid and N-(methylthio)carbonyloxamide. Again, few compounds were

shared between even just two of the replicates. Replicates one and three only produced acetic

acid, and the rest of the respective profiles differed greatly.

There are often challenges associated with SPME, especially in terms of maintaining

extraction conditions to have good reproducibility, but reproducibility issues in these

57

experiments could be attributed to the ChemStation library being limited if the VOCs produced

are not present in the library. As described in section 4.1 and shown in Figure 6, the library

identification of a promising antibacterial compound, hexanal, proved to be incorrect. To

investigate if the reproducibility issue was associated with library misidentifications of the VOCs

or with differing TICs, the Kruskal-Wallis test was performed using Prism software.

Chromatographic data for the CM7 triplicates cultured on agar and analyzed by SPME-GC/MS

on day one of the time course was imported into Prism and analyzed to determine the means and

coefficients of variations (CVs). All data associated with a CV value less than 20% was omitted.

The Kruskal-Wallis test was performed on the remaining data and a P value of 0.9090 was

obtained indicating that the data is not significantly different (P > 0.05). This reveals that the

total ion chromatograms (TICs) are reproducible. The lack of reproducibility with identifying

VOCs speaks to the need for a more robust library of compounds within the ChemStation

software or to the need for the creation of a specific bacterial VOC library. This also confirms

the importance of verifying the library identifications by comparing the experimental mass

spectra to known mass spectra.

Analyzing selected antibacterial compounds to confirm their identity sometimes yields

inconsistent results in terms of abundance as well. Acetic acid’s presence was confirmed in every

sample. In SR4 on day one, there was a 9.13% difference in abundance levels between replicates

one and two and a 10.4% difference in abundance levels between replicates one and three and

two and three, which are tolerable differences. In CM7 on day one, it was expected that the

percent differences in abundance would be smaller since a new SPME fiber was used. However,

there was a 72.2% difference in abundance levels between replicates one and two, a 68.0%

difference in abundance levels between replicates one and three, and a 4.76% difference in

58

abundance levels between replicates two and three. The presence of some VOCs also varied. For

example, on day three, SR4 replicates one and three had dimethylamine present. The abundance

level of dimethylamine in each of these samples differed by 2.30%, which is in the realm of

acceptable reproducibility. A 15% difference is typically considered acceptable, and a 10%

difference is typically considered good. However, dimethylamine was absent from replicate two

even though everything else in the method, such as resampling, time, and temperature, was

consistent. Ideally, if throughput of the method was higher, many replicates could be analyzed

concurrently, and the results could be averaged to generate more standardized spectra for each

sample, yet that was not feasible for this study.

4.6. Chemometrics

Chemometrics tools were used to determine how different the data is since the hundreds

of VOC identifications were not an accurate indicator of this. Past3 software was used to create

cluster and PCA plots based on the chromatographic data generated after one day of incubation

for CM7, OBCR6, SA, and SR4. All the one-day, single strain data for these four strains from

the course of this project was used to create the plots to see how the strains differ from each

other and to further see how data from samples of the same strain differs or compares.

59

A)

B)

Figure 29 A) Clustering plot and B) PCA plot of CM7, OBCR6, SA, and SR4 samples

60

Figure 29 shows the plots created using the data for the four strains. The crimson color

represents SA samples, dark orange represents SR4 samples, blue violet represents OBCR6

samples, and black represents CM7 samples. The different colors would be expected to cluster

together if the results were similar for the samples of each strain. Instead, there is no distinct

clustering. The SA samples have the closest proximity to each other indicating the greatest

similarity among these samples, while the SR4 and CM7 samples have the farthest proximity to

each other indicating the greatest differences among these samples. Still, all the different strains’

samples overlap showing no distinct differences or separation between the individual strains.

Figure 30 PCA plot of all agar blanks from the course of this study

There is even unexpected variability throughout the VOC profiles of all the agar blanks

that were used over the course of this study. Most of the blanks fall in the third quadrant in

61

Figure 30. However, the clustering is not very tight. The variability among the supposedly

identical blanks suggests it would be best to attempt to improve the workflow by controlling

factors such as sample storage, sample temperature, and sampling time during the day.

A)

B)

Figure 31 PCA and clustering plots of A) SR4 triplicates and B) CM7 triplicates over 3-day time

course10

Looking again at the SR4 and CM7 triplicates used to assess reproducibility, the

chemometrics tools again show a slight lack of consistency. In Figure 31, each color represents a

62

different day of the time course. Overall, two of the three samples on each day typically cluster

together, indicating that these samples have similar TICs. Yet, the one differing but supposedly

identical sample shows that there is variability. It would be beneficial to analyze a larger number

of replicates to try to obtain some overall consistency and average the TIC data to generate a

typical chromatogram for each strain.

5. Conclusions

Dental plaque plagues a large portion of the population causing various tooth and gum

problems. The spatial and temporal composition of plaque is affected by competitive or

mutualistic interactions between bacteria that colonize the conditioning film early on during

plaque formation. These interactions between oral Streptococci can be driven by the production

of VOCs so analysis of oral bacteria VOCs could provide insight into factors that drive healthy

plaque formation. Analysis of VOCs also has relevance with drug sniffing dogs, explosives,

flavors and fragrances, and other biological specimens so the technique can be applied widely.

Using SPME-GC/MS, it was determined that every strain of oral Streptococci analyzed in

this study has a unique VOC profile. PCA plots suggest that the TICs of different strains are

similar, so different strains likely produce many of the same compounds. The VOC profile of a

strain differs when cultured on broth or on agar. Samples cultured on agar tend to show

detectable VOC profiles that include compounds from more chemical classes than samples

cultured in broth. However, broth-cultured samples of any given strain tend to have increased

abundance levels of compounds that are present when culturing with broth or agar. Still, it

cannot be confirmed if these results are directly attributed to using broth versus agar since the

difference in SPME extraction temperatures for the two types of samples could have played a

large role in the resulting TICs and VOC profiles. Co-culturing strains also alters VOC profiles,

63

produces some VOCs not seen in the individual strains, and often induces the bacteria to produce

larger amounts of some VOCs. Time course experiments revealed that the VOC profiles change

over time, whether it be over several days or just over the course of a single day.

No key VOCs that contribute to plaque formation and bacterial communication have been

confirmed yet, but some VOCs have been identified as being potential contributors to bacterial

growth due to their antibacterial properties such as acetic acid, dimethylamine, and 1-butanol.

Acetic acid was present in nearly every sample. When culturing with broth compared to agar,

there was an increased abundance of acetic acid. The time course experiments did not reveal any

trend in the levels of acetic acid present in samples, but the levels did consistently change. It was

also observed that co-culturing alters the acetic acid levels, but there isn’t a consistent increase or

decrease in the levels compared to those found in individual strains. 1-butanol was not detected

in every sample, but co-culturing strains either caused 1-butanol to be detected if it wasn’t

detected in the individual strains or caused the amount of 1-butanol detected to increase. As

opposed to acetic acid levels, 1-butanol levels seem to generally increase over time. For

dimethylamine, no increasing or decreasing trend in abundance during the time course studies

was observed, and co-culturing did not consistently increase levels of this compound as it did for

1-butanol.

SPME is known to have some reproducibility issues, but the lack of reproducibility in

easily identifying VOCs in these experiments made it difficult to identify any compound with

confidence without comparing each one individually to known mass spectra. Efforts were taken

to maintain sample preparation and analysis conditions throughout the course of the study yet

ChemStation library ID results varied from day to day and varied between replicates analyzed

consecutively. Using a new SPME fiber did not improve reproducibility. However, performing a

64

Kruskal-Wallis statistical analysis test showed that total ion chromatograms for replicate samples

were very similar (P = 0.9090) and not significantly different as it appeared when looking at

VOC profiles and cluster plots alone. In future experiments, sample preparation and analysis

conditions will be more normalized to try to further improve reproducibility of the VOC

identifications.

6. Future Directions

Moving forward, to determine if the differences in VOC profiles when culturing strains in

broth versus on agar were a result of the type of media, further experiments should be conducted

testing both groups of samples at the same temperature, ideally at 37°C since this is the

temperature at which the bacteria were incubated. This would eliminate the potential affect

temperature had on the SPME extraction, but other variables such as diffusion rate in liquid

versus gas would still make it challenging to accurately determine the effect of culture media

type on VOC profiles of oral Streptococci.

More time course studies should be conducted with a slightly altered method. Enough

sample tubes of each strain or co-culture should be prepared for each point in the time course.

Each tube should be sampled once with no resampling. For example, tube 1 of SR4 should be

sampled after one day, tube 2 of SR4 should be sampled after two days, and so on. This would

eliminate any sources of error that could have arisen from piercing the septum multiple times or

fully extracting certain VOCs early in the study.

Furthermore, all samples should be analyzed a precise amount of time after incubation,

even when left at room temperature. Samples were left at room temperature after being in the

incubator at 37°C prior to analysis for varying numbers of hours. Room temperature does not

65

promote bacterial growth, so the sample contents should have remained constant, but analyzing

the samples one versus three hours, for example, after they reached room temperature could have

altered the results.

Any compound identified as being potentially important in oral Streptococci

communication and the formation of dental plaque must be analytically confirmed. Three VOCs

were selected for further analysis in this study, but there were several other potentially

interesting compounds seen in at least one of the samples that were analyzed by SPME-GC/MS.

For instance, 2-fluoroacetamide disrupts the citric acid cycle, semioxamazide is a byproduct in

the synthesis of antimicrobial molecules, octodrine is an antibacterial compound, dl-threitol is a

building block in the chemical synthesis of other compounds of interest, and cyclobutanol is

potentially antimicrobial, and all were seen in at least one VOC profile. If any of these or other

compounds are selected for further analysis, the experimental mass spectra should be compared

to known mass spectra. Standards of the compounds should be spiked into broth or agar and

analyzed using the same SPME-GC/MS method to confirm retention times and mass spectra and

therefore final identifications. This could help elucidate what VOCs are contributors to

Streptococci growth and dental plaque formation. Standards were not analyzed in these

experiments because this served as a preliminary study to assess the presence of VOCs in oral

Streptococci.

66

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68

Appendix A All VOCs identified in agar

Y corresponds to yes, N corresponds to no.

Produced by Strain?

VOC OBCR6 CM6 SR4 CM7 SA SO SV SR5 SR1 S.mutans

(S)-2-methyl-1-butanol N N N Y N N N Y N N

[(aminocarbonyl)amino]oxoacetic

acid N N N Y N N N N N N

[S-(Rx,Rx)]-1,2,3,4-butanetetrol N N N Y N N N N N N

1-(trimethylsilyl)-1-propyne N N N Y N N N N N N

1-butanol N N N Y N N Y N N N

1-butanol, 3-methyl-, formate N N N N Y N Y N N N

1-methoxy-2-propanamine N N N N Y N N N N N

1-methyl-2-phenoxyethylamine N N N Y N N N N N N

1-propylaziridine Y N N N N N N N N N

1-silacyclo-3-pentene N N Y N N N N N N N

1,2,4-thiadiazole, 3,5-

bis(carbamoylmethylthio)- N N Y N N N N N N N

1,3-diazine N N Y N N N N N N N

1,3-dimethylbenzene N N Y N N N N N N N

1,3-dimethylcyclopentane N N Y N N N N N N N

12-methylaminolauric acid N N Y N N N N N N N

2-(adamantan-1-yl)-N-(1-

adamantan-1-ylethyl)acetamide N N Y N N N N N N N

2-(methylamino)ethanol N N Y Y N N N N N N

2-(vinyloxy)ethanol N N N Y N N N N N N

2-amino-1-(o-

hydroxyphenyl)propane N N Y N N N N N N N

2-amino-5-[(2-

carboxy)vinyl]imidazole N N N Y N N N N N N

2-chlorobutyramide N N N Y Y N N N N N

2-cyclopenten-1-one N N N N Y N N N N N

2-fluoroacetamide N N Y N N N N N N N

2-hexenoic acid, ethyl ester N N N Y N N N N N N

2-iodohiistidine N N Y N N N N N N N

2-methyl-1-butanol N N Y Y N N Y N N N

2-methyl-1-propene N N Y N N N N N N N

2-methyl-3-decen-5-one N N N Y N N N N N N

2-methylaminomethyl-1,3-

dioxolane Y N N Y Y N N N N N

2-methylbutanal Y Y Y Y N N N N N N

2-methylfuran Y Y N N Y N N N N N

2-methylpiperazine N N N N Y N N N N N

69

2-methylpropanamide N N N Y N N N N N N

2-octanamine N N Y Y N N N N N N

2-pentanamine N N Y Y N N N N N N

2-propanamine N N Y Y N N N N N N

2-thiopheneacetic acid, 2-

methyloct-5-yn-4-yl ester N N N Y N N N N N N

2-thiopheneacetic acid, tetradecyl

ester N N N Y N N N N N N

2-thiophenecarboxylic acid, 5-

(1,1-dimethylethocy)- N N N Y N N N N N N

2,2,2-trifluoroacetamide Y N Y N N N N N N N

2,3-epoxybutane N Y N Y N N N N N N

2,3,3-trimethyl-cyclobutanone N Y N N N N N N N N

2,4-disocyanato-1-methylbenzene N Y N N N N N N N N

2,4,4-trimethyl-1-pentene N N N Y N N N N N N

2,5,6-trimethyldecane N N N Y N N N N N N

3-(methylamino)propanenitrile N N Y N N N N N N N

3-amino-2-methylbutanoic acid N N Y N N N N N N N

3-chloro-5,5-dimethylcyclohex-2-

enone N N N Y N N N N N N

3-methyl-1-butanol Y Y Y Y Y Y N Y Y Y

3-methyl-2-pentanone N N N Y N N N N N N

3-methylbutanal Y N Y Y Y N N N N N

3-methylpentane N N N Y N N N N N N

3,3-dimethyl-4-methylamino-

butan-2-one Y N Y N N N N N N N

4-amino-1-propylpiperidine N Y N N N N N N N N

4-methyl-2-hexanamine N Y N N N N N N Y N

4-methyl-2-pentanamine N N N Y N N Y N N N

4,4-dimethyl-3-oxopentanenitrile N N N Y N N N N N N

5-(thiophen-2-yl)methyl-2H-

tetrazole N N N N Y N N N N N

5-methyl-2-heptanamine N N Y Y N N N N N N

5-methyl-2-hexanamine N N Y N N N N N N N

5-methyl-2-phenyl-1H-indole N N Y N N N N N N N

6,9-dichloro-2-methoxyacridine N N N N Y N N N N N

acetaldehyde N Y N Y N N N N N N

acetic acid Y Y Y Y N Y N N Y Y

acetic acid, 2-(N-methyl-N-

phosphonatomethyl)amino- N N N Y Y N N N N N

acetic acid, hydroxy[(1-oxo-2-

propenyl)amino]- N N Y N Y N N N N N

acetic acid, sodium salt N N Y N N N N N N N

acetone N N Y N N N N N N N

70

arsenous acid, tris(trimethylsilyl)

ester N N N N Y N N N N N

benzaldehyde N N N N Y N N N N N

benzeneacetaldehyde Y Y N Y Y N N N N N

benzeneethanamine, 2-fluoro-

beta,5-dihydroxy-N-methyl- N N Y N N N N N N N

benzeneethanamine, 2,5-difluoro-

beta,3,4-trihydroxy-N-methyl N N Y N N N N N N N

bromodichloromethane N N Y N N N N N N N

butanal N N N Y N N N N N N

butylated hydroxytoluene N N N Y Y N N N N N

carbamic acid, methyl ester Y N N N N N N N N N

carbamimidoylsulfanylacetic acid N N N Y N N N N N N

carbazic acid, 3-(1-

propylbutylidene)-, ethyl ester N N Y N N N N N N N

carbon dioxide Y Y Y Y Y N Y Y N Y

cis-1,2-dimethylcyclopentane N N Y N N N N N N N

cyclobutanol N Y N Y Y N N N N N

cyclohexan-1,4,5-triol-3-one-1-

carboxylic acid N N Y N N N N N N N

cyclohexane N N Y N N N N N N N

cyclopropyl carbinol N N Y Y Y N N N N N

dimethyl ether N N N N N Y N N N N

dimethylamine Y N Y Y Y N N Y N N

dimethyldisulfide Y N Y N Y N N N N N

dimethylsilanediol N N Y N Y N N N N N

dl-alanine Y N N Y N N N N Y N

dl-alanyl-dl-methionine N N Y N Y N N N N N

dl-allo-cystathionine Y N Y N Y N N N N N

dl-cystathionine N N Y Y N N N N N N

ethanol Y Y Y Y Y Y N N Y Y

ethoxyethene Y N Y Y N N N N N N

ethyl 2-(2-chloroacetamido)-

3,3,3-trifluorolactate N N N Y N N N N N N

ethyl aminomethylformimidate N N Y N N N N N N N

ethyl hydrogen oxalate Y N N Y N N N N N N

ethylene oxide Y Y Y Y Y N N N N Y

fluoroethyne N N N Y N N N N N N

formaldehyde oxime trimer Y N Y N N N N N N N

formyltrimethylurea N N N Y N N N N N N

furan Y Y Y Y N N N N N N

glutaraldehyde Y N N N Y N N N N N

glycerin Y Y Y Y N N N N N N

71

hexamethylcyclotrisiloxane Y N Y Y Y N N N N N

hexamethyldisiloxane N N Y N N N N N N N

hexanal N N Y N N N N N N N

hydroxy[(1-oxo-2-

propenyl)amino]acetic acid N N N Y N N N N N N

maprotiline N N N N Y N N N N N

methanethiol N N Y N N N N N N N

methional N N Y Y N N N N N N

methylcyclopentane N N Y Y N N N N N N

methylpent-4-enylamine N N Y N N N N N N N

methylsilane Y N Y Y N N N N N N

N-(1-

cyclohexylethyl)propanamide N N Y N N N N N N N

N-(1-methylpropyl)-acetamide N N Y N N N N N N N

N-(3,5-dinitropyridin-2-yl)-L-

aspartic acid N N N Y Y N N N N N

N-(methylthio)carbonyloxamide N N N Y Y N N N N N

n-ethylformamide N N Y N N N N N N N

n-hexylmethylamine Y N Y Y Y N N N N N

N-methyl-1,2-ethanediamine N N N Y N N N N N N

N-methyl-2-phenyl-1-

propylamine Y N N N N N N N N N

N,2-dimethyl-1-propanamine N N Y Y N N N N N N

N,N-

dimethylmethanesulfonamide N N Y N N N N N N N

N,N,N',N'-

tetramethylsulfonamide N N N N Y N N N N N

N,N'-bis(1-methylethyl)urea Y N N N N N N N N N

N,N'-dimethyl-1,2-ethanediamine N N N N Y N N N N N

O-butyl,O-1,2,2-trimethylpropyl

methylphosphonate N N N N Y N N N N N

o-xylene N N Y Y N N N N N N

octamethylcyclotetrasiloxane N N Y N Y N N N N N

p-xylene N N Y N N N N N N N

pentanal Y N N N N N N N N N

phenylephrine N N Y Y Y N N N N N

phthalic acid, di(2-propylpentyl)

ester N N N Y N N N N N N

propanamide Y N N Y N N N N N N

sarcosine, N-isobutyryl-,

tetradecyl ester N N Y N N N N N N N

sarcosine, N-valeryl-, hexadecyl

ester N N Y N N N N N N N

sec-butylamine N N Y Y Y N N N N N

silver acetate N N N Y N N N N N N

72

sulfurous acid, diethyl ester N N N Y N N N N N N

thiophene-3-ol, tetrahydro-, 1,1-

dioxide N N Y Y N N N N N N

toluene N N Y N N N N N N N

topotecan N N Y N N N N N N N

trichloromethane N N Y N N N N N N N

trimethylurea N N Y N N N N N N N

triphenylphosphine oxide N N Y N Y N N N N N

tuaminoheptane Y N N Y N N N N N N

73

Appendix B All VOCs identified in broth

Y corresponds to yes, N corresponds to no.

Produced by Strain?

OBCR6 CM6 CM7 SR4 SA AC15 ACC21 ACS2 AS14 AS20 BS29 BS21 BS35a SO SR1 SR5 SV

(S)-1,3-butanediol Y N N N N N N N N N N N N N N N N

(S)-2-methyl-1-butanol N N N Y N N N N N N N N Y N N N N

1-acetoxynonadecane N N N N N N N N N N N N N Y N N N

1-butanol Y N N Y Y N N N Y N N N N N N N N

1-butanol, 3-methyl- N N N N N N N N N N N Y Y N N N N

1-heptene N N N N N N N N Y N N N N N N N N

1-methoxy-2-propanamine Y N N N N N N N N N N N N N N N N

1,1,-dioxide

tetrahydrothiophene-3-ol N N N N Y N N N N N N N N N N N N

1,1,3-trimethyl-3-(2-methyl-2-

propenyl)-cyclopentane N N Y N N N N N N N Y N N N N N N

1,2-propanediamine N N N N Y N N N N N N N N N N N N

1,2,3,4-butanetetrol, [S-

(RN,RN)]- N N N N Y N N N N N N N N N N N N

1,3-butanediamine N N N N Y N N N N N N N N N N N N

1,3-dimethylbenzene N N Y Y N N N N N N N N N N N N N

2-(methylamino)ethanol N N N N N N N N N N N N N N N Y N

2-[(1-methylethyl)thio]pentane N N N N Y N N N N N N N N N N Y N

2-amino-5-[(2-

carboxy)vinyl]imidazole N N N N N N N N N N N N Y N N N N

2-butyl-3,5-dimethylpyrazine N N N N Y N N N N N N N N N N N Y

2-chloro-N-

(hydroxymethyl)acetamide N N N N N N N N N N Y N N N Y N N

2-ethyl-1-dodecanol N N N N Y N N N N N N N N N N N N

2-ethyl-1-hexanol N Y Y Y N Y Y Y N Y Y N Y Y N N N

2-ethyl-5-methylpyrazine N N N N N N N N N N N N N Y N N N

2-ethylcyclobutanol N N N N N N N N N N N N N Y N N N

2-ethylcyclobutanone N N N N N N N N Y N N N N N N N N

2-formylhistamine N N N Y N N N N N N N N N N N N N

2-heptanone N N N N N N N N Y N N N N N N N N

2-hexanamine N N N N Y N N N N N N N N N N N N

2-hexanol N N Y N N N N N N N N N N N N N N

2-hexenoic acid, ethyl ester N N N N N N N N N N N N N Y N N N

2-hydroxy-2,4,6-

cycloheptatrien-1-one N N N N Y N N N N N N N N N N N N

2-hydroxypropanamide N N N N N N N N Y N Y N N N N N Y

2-methyl-1-butanol N N Y N N N N N Y N N N N N N Y N

2-methyl-1,4-benzenediamine N N N Y N N N N N N N N N N N N N

2-methyl-butanal N Y N N N Y N N N N N N N N N N N

2-methylpropanamide N N N N N Y N N N N N N N N N N N

74

2-methylfuran N N N N N N N N Y N N Y N N N N N

2-nonanone N N N N N N N N Y N N N N N N N N

2-octanamine N N N N Y N N N N N N N N N N N N

2-propanamine N N N N Y N N Y N N N N N N N N N

2-propen-1-ol N N N N N N N N Y N N N N N N N N

2,2-dimethylglutaric anhydride N N N N N N N N N Y N N N N N N N

2,2'-thiobisbutane N N N N N N N N N N N N N Y N N N

2,3-dicyanopropionamide N N N N N N N N N N N N N N N N Y

2,3-epoxybutane N N Y N N N N N N N N Y N N N N N

2,3-pentanedione N N N N N N N N N N N N N N N N N

2,4-dimethyl-

benzo[h]quinoline N N N N Y N N N N N N N N N N N N

2,4,4-trimethyl-1-pentene Y N Y Y Y Y Y Y Y Y Y Y Y N N N N

4-ethylbenzoic acid, 2,5-

dichlorophenyl ester N N N N Y N N N N N N N N N N N N

2,5-dimethyl-3-(3-

methylbutyl)pyrazine N N N N Y N N N N N N N N Y N Y N

3-amino-2-methylbutanoic

acid N N Y N N N N N N N N N N N N N N

3-chloro-1,2-propanediol N N N N Y N N N N N N N N Y N N N

3-ethyl-2,5-dimethylpyrazine Y N Y Y Y N N N N N N N N Y Y Y Y

3-methylbutanal N Y Y N N Y Y Y N Y N Y N N N N N

3-methyl-1-butanol N N N Y Y N Y Y Y Y N N Y Y N Y N

3-methylpentane N N N Y N N N N N N N N N Y N N N

3-methylpiperidine N N N N N N N N N N N N N N N N Y

3,5-dihydroxy-4,4-dimethyl-

2,5-cyclohexadien-1-one N N N N Y N N N N N N N N N N N N

3,7,11-trimethyl-3-hydroxy-

6,10-dodecadien-1-yl acetate N N N N N N Y N N N N N N N N N N

4-(1,1,3,3-

tetramethylbutyl)phenol N N Y Y Y N N N N N N N N Y N Y Y

4-ethyl-2-methyl-5-

propylthiazole N N Y N N N N N N N N N N Y N N N

4-fluorohistamine N N Y N N N N N N N N N N N N N N

benzeneethanamine, 4-fluoro-

.beta.,3-dihydroxy-N-methyl N N N N N N N N N N N N N N N Y N

4-methyl-2-hexanamine N N N N N N N N N N N N N N N N N

8-(N-aziridylethylamino)-2,6-

dimethyloctene-2 N N N N Y N N N N N N N N N N Y Y

acetaldehyde N N Y N N N N N N N N N N N N N N

acetamide, 2-(adamantan-1-

yl)-N-(1-adamantan-1-

ylethyl)- N N N N Y N N N N N N N N N N N N

acetic acid Y Y Y Y N N N Y Y N Y Y Y N N Y N

acetic acid, sodium salt N N N Y N N N N Y Y Y Y N Y N N Y

alanine N N N N N N N N N N N Y N N N N N

amphetamine-3-methyl N N N N N Y N N N N N N N N N N Y

benzeneacetaldehyde Y N N Y Y Y Y N N N N N N N N Y Y

benzeneethanamine, 2-fluoro-

beta,5-dihydroxy-N-methyl N N N N Y N N N N N N N N N N N N

benzeneethanamine, 4-fluoro-

beta,3-dihydroxy-N-methyl N N N N Y N N N N N N N N N N N N

butanal N Y N Y Y N N Y Y N N N N N N N N

75

carbamic acid N N N N N N N N N N N N Y N N N N

carbon dioxide N N N Y Y Y Y Y Y Y N Y Y Y N N Y

cyclobutanol Y N N Y Y Y Y Y Y N Y Y N N N N Y

cyclohexan-1,4,5-triol-3-one-

1-carboxylic acid N N N N N Y Y N N N N N N N N N N

cyclopropyl carbinol N N Y Y N N N N Y N N N N N N N N

cyclotetrasiloxane,

octamethyl- N N N N N N N N N N N N Y N N N N

cyclotrisiloxane, hexamethyl- N N N N N N N N Y N N N N N N N N

cystathionine N N N N N N Y N N Y N N Y N N N N

decamethylcyclopentasiloxane N N N N N Y N Y N N N N N N N N N

dimethylamine N N N N Y N N N Y N N N N Y N Y Y

dimethyl disulfide N Y N N Y N Y Y N N N N N N N N N

dimethylamine N Y N N N N N N N N N N N N N N N

dimethylsilanediol N N N N N N N N Y N N N N Y N N Y

disulfide, dimethyl N N N N N N Y N N N N N N N N N N

dl-alanine N N N Y Y N N N N N Y N N N N N N

dl-alanyl-dl-methionine N N N N Y N N N N N N N N N N N N

dl-allo-cystathionine N N N N Y N N N N N N N N N N N N

dl-threitol N N N N Y N N N N N N N N N N N N

erythritol N N N N Y N N N N N N N N N N N N

ethanol N N Y Y Y N N N N N N Y Y Y N N N

ethoxyethene Y Y N N N Y Y Y Y Y Y Y Y N N N N

ethyldimethylsilanol N N N N N N N N N N N N Y N N N N

ethylene oxide N N Y Y Y Y Y N Y Y N N N N N N Y

fenproporex N N N N N N N N N N N N N N N Y N

fluoroethyne N N N N N N N N N N Y N Y N N N N

fluoroethyne N N N Y N N N N N N N N N N N N N

formaldehyde oxime trimer N N N N N Y N N N N N N N N N N N

formic acid, hexyl ester N N N N N N N N Y N N N N N N N N

glutaraldehyde N N Y N N N N Y N N N N N N N N N

glycerin Y Y Y Y Y N N N Y N N N N Y N Y Y

heptanal N N N N N N N N Y N Y N N N N N N

heptyl ester

pentafluoropropionic acid N N N N Y N N N N N N N N N N N N

pyrrolo(1,2-a)pyrazine-1,4-

dione, hexahydro-3-(2-

methylpropyl) N N Y N N N N N N N N N N N N N N

hexamethylcyclotrisiloxane N N N Y N N N N N N N N N Y N N N

hexanal N N N Y N N Y N N N N N N Y N N N

L-alanine, ethyl ester N N N N N N N N Y N N N N N N Y N

methyl ether farnesol N N N N Y N N N N N N N N N N N N

methylcyclopentane N N N Y N N N Y Y N N N Y N N N N

methylpent-4-enylamine N N N N Y N N N N N N N N N N N N

76

methylsilane N N N N N N N N N N N Y Y N N N N

N-

(methylthio)carbonyloxamide N N N Y Y N Y N N N N N N N N N N

l-proline, N-allyloxycarbonyl-,

hexyl ester N N Y N N N N N N N N N N N N N N

N-ethyl-N'-nitroguanidine N N N Y N N N N N N N N N N N N N

n-hexylmethylamine N N N N Y N N N N N N N N N N N N

N-methyl-1,3-propanediamine N N N N N N N N N N N N N N N N N

N,N-

dimethylmethanesulfonamide N N N N Y N N N N Y N N N N N Y N

N,N'-dimethyl-1,2-

ethanediamine N N N N Y N N N N N N N N N N N N

N1-methyl-2-methoxy-1-

propanamine N N N N N N N N N N N N N Y N N N

nitro-L-arginine Y N N N N N N N N N N N N N N N N

octamethylcyclotetrasiloxane N N N N N N N Y N N N N N N N N N

octodrine N N N N Y N N N N N N N N N N N N

p-xylene N N Y Y N N N N N N N N N Y N N N

pentafluoropropionic acid,

heptyl ester N N N Y N N N N N N N N N N N N N

pentanal N N N N N Y N N N N Y N N N N N Y

phenylethyl alcohol N N N N N N N N Y N N N N N N N N

piperazine N N N N N N N N N N N N Y N N N N

propanamide N N N N N N N N Y N N N N N N N N

pyrrolo(1,2-a)pyrazine-1,4-

dione, hexahydro-3-(2-

methylpropyl)- N N N N Y N N N Y N N N N N N N N

R-(-)-cyclohexylethylamine N N N N Y N N N N N N N N N N N N

sarcosine, n-hexanoyl-,

hexadecyl ester N N N N Y N N N N N N N N N N N N

sec-butylamine N N N N Y N Y N N Y N N Y N N N Y

semioxamazide N N N N Y N N N N N N N N N N N N

tert-

butylpentamethyldisiloxane N N N Y N N N N N N N N N N N N N

tris(trimethylsilyl) ester

arsenous acid N N N N Y N N N N N N N N N N N N

trichloromethane N N N N N N N N N N N N N Y N N Y

tricyclo[4.3.1.1(3,8)]undecane-

1-carboyxlic acid N N N N Y N N N N N N N N N N N N

trifluoroacetic acid N N N N N N Y N N N N N N N N N N

trimethylsilanol N N N Y N N N N N N N N N N N N N

triphenylphosphine oxide N N N N Y N Y N N N N N N N N N N

tuaminoheptane N N N N N N N N N N N N N N N N Y

xylitol N N N N Y N N N N N N N N N N N N

77

Appendix C 10-day time course results for SR4 and SR4xSA

Green boxes indicate a compound that was present over the course of the whole study. Yellow-

orange boxes indicate a compound that was present over the course of most of the study.

Highlighted yellow boxes indicate a compound that was only missing on one day of the study.

Retention Time (min) Compound ID Retention Time (min) Compound ID Retention Time (min) Compound ID

0.991 carbon dioxide 1.039 2-methyl-1-propene 0.963 ethylene oxide

1.032 ethanol 1.06 methylsilane 1.282 acetic acid

1.06 1-methoxy-2-propanamine 1.081 ethanol 1.338 ethoxyethene

1.095 acetic acid 1.254 2-(methylamino)ethanol 1.359 2-methylfuran

1.192 cyclobutanol 1.303 acetic acid 1.414 3-methylbutanal

1.435 3-methyl-1-butanol 2.226 3-methyl-1-butanol 1.477 3-methylbutanal

2.282 1,3-dimethylcyclopentane 1.803 2-fluoroacetamide

2.039 3-methyl-1-butanol

2.192 3-methyl-1-butanol

2.247 hexanal

SR4Day 10Day 4 Day 5

Retention Time (min) Compound ID Retention Time (min) Compound ID Retention Time (min) Compound ID

1.074 dimethylamine 1.06 ethanol 1.011 ethylene oxide

1.289 acetic acid 1.269 acetic acid 1.06 ethanol

1.345 2-pentanamine 1.338 glycidol 1.275 acetic acid

1.373 2-methylfuran 1.359 2-methylfuran 1.338 hexanal

1.428 N,2-dimethyl-1-propanamine 1.414 N-methyl-1-butanamine 1.365 2-cyclopenten-1-one

1.505 N,N'-dimethyl-1,2-ethanediamine 1.456 pentanal 1.4 acetic acid, hydroxy[(1-oxo-2-propenyl)amino]-

1.56 pentanal 2.192 3-methyl-1-butanol 1.421 acetic acid, 2-(N-methyl-N-phophonatomethyl)amino-

2.143 methylpent-4-enylamine 1.449 sec-butylamine

2.213 3-methyl-1-butanol 1.47 cyclobutanol

2.907 actinobolin 2.205 3-methyl-1-butanol

SR4xSADay 1 Day 2 Day 3

Retention Time (min) Compound ID Retention Time (min) Compound ID Retention Time (min) Compound ID

0.956 carbon dioxide 1.025 dl-alanyl-dl-methionine 0.97 ethanol

1.012 carbon dioxide 1.074 1,3-dioxan-5-ol 1.032 furan

1.06 ethanol 1.241 butanal 1.053 ethanol

1.116 N,N'-dimethyl-1,2-ehtanediamine 1.296 acetic acid 1.275 acetic acid

1.227 butanal 2.22 3-methyl-1-butanol 1.331 2-pentanamine

1.268 acetic acid 2.268 1,2,5-oxadiazol-3-carboxamide, 4,4'-azobis-, 2,2'-dioxide 2.115 3-methyl-1-butanol

1.65 2-methylbutanal 3.212 hexamethylcylcotrisiloxane 2.192 1-butanol, 3-methyl-, formate

2.372 dimethyldisulfide

3.233 hexamethylcylcotrisiloxane

3.323 hexamethylcylcotrisiloxane

SR4xSADay 10Day 4 Day 5

Retention Time (min) Compound ID Retention Time (min) Compound ID Retention Time (min) Compound ID

0.914 2,2,2-trifluoroacetamide 0.991 benzeneethanamine, 2,5-difluoro-beta,3,4-trihydroxy-N-methyl 0.991 ethylene oxide

1.067 ethanol 1.012 carbon dioxide 1.011 ethylene oxide

1.234 3-amino-2-methylbutanoic acid 1.06 ethanol 1.039 ethanol

1.282 acetic acid 1.275 acetic acid 1.206 butanal

1.345 hexanal 1.331 2,3-epoxybutane 1.254 acetic acid

1.366 2-methylfuran 1.359 2-methylfuran 1.31 2,3-epoxybutane

1.4 topotecan 1.414 bromodichloromethane 1.338 2-methylfuran

1.428 N,2-dimethyl-1-propanamine 1.456 cyclopropyl carbinol 1.393 cyclobutanol

1.463 cyclobutanol 2.192 3-methyl-1-butanol 1.442 cyclobutanol

2.206 3-methyl-1-butanol 2.233 hexanal 2.178 3-methyl-1-butanol

2.254 cis-1,2-dimethylcyclopentane 2.226 hexanal

2.733 2-fluoroacetamide

2.893 12-methylaminolauric acid

SR4Day 1 Day 2 Day 3

78

Appendix D SA 1-day time course results

The VOCs are aligned each day according to retention time and whether they were present each

day. Green boxes indicate a compound that was present for some time. Yellow-orange boxes

indicate a change in the compound present.

1 hour 2 hours 4 hours 6 hours

0.769 carbon dioxide 0.886 carbon dioxide 0.921 carbon dioxide 0.921 carbon dioxide

0.991 ethylene oxide 1.011 ethylene oxide 0.942 ethylene oxide 1.025 ethylene oxide

1.046 benzeneethanamine, 2-fluro-beta,5-dihydroxy-N-methyl

1.067 N-(methylthio)carbonyloxamide

1.463 4,4'-azobis-2,2'-dioxide-1,2,5-oxadiazol-3-carboxamide

1.539 2-octanamine

1.574 2-propanamine

1.713 1-butanol

2.192 dimethyldisulfide

2.317 3-methyl-1-butanol

2.726 benzeneethanamine, 4-fluro-beta,3-dihydroxy-N-methyl

2.942 tricyclo[4.3.1.1(3,8)]undecane-1-carboyxlic acid 2.942 decyl undec-2-en-1-yl ester phthalic acid

2.983 2,5-dichlorophenyl ester 4-ethylbenzoic acid

5.906 erythritol 5.968 3-chloro-1,2-propanediol 5.961 dl-threitol

6.058 2,4-dimethyl-benzo[h]quinoline 6.059 tri(trimethylsilyl) ester arsenous acid 5.996 dl-threitol

6.128 semioxamazide 6.121 semioxaamazide 6.128 2-[(1-methylethyl)thio]pentane

6.246 methylpent-4-enylamine

6.551 2-hydroxy-2,4,6-cycloheptatrien-1-one

6.947 2-ethyl-1-dodecanol

7.822 3-ethyl-2,5-dimethylpyrazine

11.355 2,5-dimethyl-3-(3-methylbutyl)pyrazine 11.376 2,5-dimethyl-3-(3-methylbutyl)pyrazine 11.362 2,5-dimethyl-3-(3-methylbutyl)pyrazine

12.411 2-hexanamine

17.437 l-alanine, N-(cyclohexylcarbonyl)-,tetradecyl ester 17.458 3,5-dihydroy-4,4-dimethyl-2,5-cyclohexadien-1-one

17.742 octodrine

19.27 sarcosine, n-hexanoyl-, hexadecyl ester

20.963

79

4 hours 6 hours

0.921 carbon dioxide 0.921 carbon dioxide

0.942 ethylene oxide 1.025 ethylene oxide

1.539 2-octanamine

1.574 2-propanamine

1.636 1-butanol 1.713 1-butanol

2.317 3-methyl-1-butanol

2.942 decyl undec-2-en-1-yl ester phthalic acid

5.961 dl-threitol

6.059 tri(trimethylsilyl) ester arsenous acid 5.996 dl-threitol

6.121 semioxaamazide 6.128 2-[(1-methylethyl)thio]pentane

7.822 3-ethyl-2,5-dimethylpyrazine

11.376 2,5-dimethyl-3-(3-methylbutyl)pyrazine 11.362 2,5-dimethyl-3-(3-methylbutyl)pyrazine

17.458 3,5-dihydroy-4,4-dimethyl-2,5-cyclohexadien-1-one

17.742 octodrine

20.963

80

8 hours 16 hours

0.921 ethylene oxide 1.004 ethylene oxide

1.026 carbon dioxide

1.074 dl-alanyl-dl-methionine

1.525 n-hexylmethylamine 1.504 sec-butylamine

1.546 R-(-)-cyclohexylethylamine

1.567 2-propanamine

1.608 cyclobutanol

1.72 1-butanol 1.685 1-butanol

2.233 2-octanamine

2.317 3-methyl-1-butanol 2.275 3-methyl-1-butanol

2.33 heptyl ester pentafluoropropionic acid

2.997 1,2-propanediamine

3.039 cyclobutanol

5.996 3-chloro-1,2-propanediol

6.135 semioxamazide

17.465 pyrrolo(1,2-a)pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- 17.326 acetamide, 2-(adamantan-1-yl)-N-(1-adamantan-1-ylethyl)-

20.963 triphenylphosphine oxide

81

8 hours 16 hours 27 hours

0.921 ethylene oxide 1.004 ethylene oxide 0.956 carbon dioxide

1.026 carbon dioxide 1.011 carbon dioxide

1.074 dl-alanyl-dl-methionine 1.109 ethanol

1.525 n-hexylmethylamine 1.504 sec-butylamine

1.546 R-(-)-cyclohexylethylamine

1.567 2-propanamine

1.608 cyclobutanol 1.602 cyclobutanol

1.685 1-butanol 1.685 1-butanol

2.233 2-octanamine

2.317 3-methyl-1-butanol 2.275 3-methyl-1-butanol 2.268 3-methyl-1-butanol

2.33 heptyl ester pentafluoropropionic acid 2.324 2-propanamine

2.372 dimethyl disulfide

2.997 1,2-propanediamine

3.039 cyclobutanol

5.899 1,2,3,4-butanetetrol, [S-(RN,RN)]-

5.996 3-chloro-1,2-propanediol

6.135 semioxamazide

7.176 benzeneacetaldehyde

7.773 3-ethyl-2,5-dimethylpyrazine

17.465 pyrrolo(1,2-a)pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- 17.326 acetamide, 2-(adamantan-1-yl)-N-(1-adamantan-1-ylethyl)-

20.963 triphenylphosphine oxide