## 3 Results and Discussion
### Bacillus subtilis Profiling
However, these minimal differences could be relevant quantities to life science applications and therefore, without high precision in the sampling technique, it may not be possible to detect subtle yet important differences in VOC amount. In this study, the reproducibility of SPME Arrow was verified to be adequate for usage in microbiological sampling. Finally, optimal and precise recovery is needed for a quantitative approach to VOC production in order to compare absolute concentrations of VOCs across studies. While absolute quantification was not performed in this study, the analytical parameters established for the SPME Arrow and GC-MS approach indicate that absolute quantification of VOCs is possible and a priority for future research studies. This has recently been highlighted as being critical for advancement of life sciences applications such as monitoring of breath VOCs for medical diagnostics [44]. However, the sentiments in this call for action can also be extended to other areas of importance such as the monitoring of VOCs from decomposing remains for forensic search and recovery [45]. In these application areas, microbial communities play a significant role in VOC production and, therefore, linkage of VOC trends through robust quantification is an important consideration for future advancement in these fields. This preliminary study on _B. subtilis_ identified 25 compounds in the course of the trial. Some of these compounds exhibited significant trends over time, while some did not differ significantly from levels in the uninoculated controls. It is important to note that this was a preliminary investigation using a new sampling technique, and that changing the conditions used in this study will likely  
Figure 4: Example volatile organic compounds (VOCs) detected from _Bacillus subtilis_ in each of the three categories of compound production. Category 1 compounds (‘produced VOCs’) were higher in bacterial samples than in control samples. Category 2 compounds (‘consumed VOCs’) were higher in control samples than in bacterial samples. Category 3 compounds (‘equilibrated VOCs’) in bacterial samples eventually equilibrated with control samples early in the longitudinal data and did not exhibit a significant difference to the controls over time. Compound trends for all 25 compounds of interest are shown in the Supplementary Material, Figures S1–S3. impact the identified VOCs. For example, if more advanced separation technology was used, such as comprehensive two-dimensional gas chromatography-mass spectrometry (GC\(\times\)GC-MS), it is possible that further deconvolution of low-level co-eluting peaks may uncover additional compounds or allow more robust compound identifications. One particular example of this was the lack of a significant difference over time being detected for the compound 3-methyl-2-pentanone, despite the substantial increase in amount of this compound on days 2-4 of _B. subtilis_ monitoring. Since this particular compound has a very close retention time to toluene, but a very different chemical structure, a two-dimensional separation would benefit the chromatographic separation of these peaks and potentially improve detection of a significant trend. In addition, other experimental parameters may also contribute to the specificity of detected compounds. In this study, bacteria were cultured on standard nutrient agar; however, the use of different growth media (e.g., tryptic soy agar, blood agar), would have provided the bacteria with different sources of nutrients for their metabolism, likely impacting detected VOCs. Future studies will be aimed at identifying the variation in VOC profile with different growth media. Despite these parameters that could change the resulting VOC profile detected, this study provides preliminary data on SPME Arrow sampling dynamics from microbiological sources which will provide a foundation for future studies. Considering the multivariate nature of the data obtained from these 25 compounds of interest, visualizing the changes in the overall VOC profile of _B.