# A Lab-Made E-Nose-MOS Device for Assessing the Bacterial Growth in a Solid Culture Medium
## 3 Results and Discussion  
### Evaluation of VOCs Emitted during the Bacterial Growth by HS-SPME-GC-MS  
The VOCs emitted during the bacterium growth depend on the bacterial metabolism that is influenced by the culture media composition and nutrient sources. Thus, many bacterial VOCs have been identified and their amounts and profiles greatly depend on the culture medium, incubation time, and bacterial species and strains used, as contradictory detection/non-detection data is reported [5; 6; 9]. Background VOCs related to the growth media have also been reported [5]. Thus, as a first step, the VOCs emitted by the BHI un-inoculated medium were determined. Globally, 15 volatiles were identified (data not shown), and 10 of them were also emitted by at least one of the four bacteria evaluated, namely two alcohols (1-butanol and 1-nonanol), three pyrazines (2-ethyl-6-methyl-pyrazine, 3-ethyl-2,5-dimethylpyrazine, and trimethylpyrazine), three terpen (camphene, D-limonene, and \(\beta\)-pinene), and two other compounds (2,4-thujaadiene and indole). The volatiles were identified in both un-inoculated and inoculated (with a 10\({}^{-4}\) dilution for _E. faecalis_ and 10\({}^{-5}\) dilution for the other bacterial strains) solid culture medium (i.e., BHI agar medium), after overnight incubation. Table 1 shows the amounts (in ng of each compound as IS equivalents) of the VOCs detected during the chromatographic analysis of the volatile head-space generated during the overnight growth of the two Gram-positive (_E. faecalis_ and _S. aureus_) and the two Gram-negative (_E. coli_ and _P. aeruginosa_) bacteria, after subtracting the respective amounts quantified in the head-space of the un-inoculated culture medium. As can be seen from Table 1, VOCs belonging to 10 chemical classes were identified as the alcohols the most abundantly emitted by _E. coli_, _E. faecalis_, and _P. aeruginosa_ during their growth in BHI solid medium, while esters were the predominant volatiles produced by _S. aureus_ grown in the same culture medium. It should also be noticed that each bacterium had a specific profile of emitted VOCs, both in number of different volatiles identified as in the quantified amounts. In total, 17 VOCs were emitted during the growth of _E. faecalis_, followed by 14 VOCs for _E. coli_, 13 VOCs for _S. aureus_ and only 10 VOCs for _P. aeruginosa_. As can also be inferred, some VOCs were specifically produced by only one of the four studied bacteria (_E. coli_: 1-nonanol, 2-tridecanone, 3-ethyl-2,5-dimethylpyrazine, and D-limonene; _P. aeruginosa_: (E)-1,_A_-undecadiene; _E. faecalis_: ethanol, 2-methylbutanoic acid, acetic acid, and methyl undecyl ether; _S. aureus_: 3-chloro-2-methyl-2-pentanol, \(\alpha\)-phellandrene, trimethylpyrazine, and camphene), while others were common to two or more bacteria, although they were emitted, in general, in quite different amounts. These differences, in the emitted VOCs profiles and respective amounts generated by each bacterium growth, confirmed that VOCs can be used as bacterial identification biomarkers. However, it should be remarked that, for example, in this study, ethanol was only identified during the growth of _E. faecalis_, although according to the literature this alcohol is usually emitted by the four bacteria evaluated, being more abundant for _E. coli_ and _S. aureus_ compared to _E. faecalis_ and _P. aeruginosa_[6]. Indole is usually associated in the literature with _E. coli_[4; 5; 6; 7; 8; 9; 10; 11] but, in the present study, it was only identified for _P. aeruginosa_ and _S. aureus_. On the other hand, (E)-1,_A_-undecadiene was only identified for _P. aeruginosa_, in-line with the literature data, according to which this alkene has been associated with _Pseudomonasspecies, allowing its use as a biomarker for this species [36].