# Dependence of the Staphylococcal Volatilome Composition on Microbial Nutrition
## Results and Discussion
### Influence of Medium and Nutrition on the Chemical Composition of the Volatilome  
Based on differences in glucose concentration in the four complex media, and its influence on _Staphylococcus_ spp. catabolite repression control, we posited that the volatilome of both species would strongly vary by media. Interestingly, whether we characterized the volatilome by numbers of volatiles (Figure 1a,b) or by relative abundance (Figure 1c,d) determined whether we classified this hypothesis as true or false. When viewed by the numbers of volatiles in each chemical class, the staph volatilomes in each medium were relatively uniform (Figure 1a,b). For example, in all four media, hydrocarbons were the most numerous, and hydrocarbons plus ketones made up about 50% of all volatiles detected. However, some notable variations did occur. First, the numbers of volatiles that were produced in each medium did not correlate with the availability of free glucose (Figure 1a). Second, the two high-glucose media had some interesting differences in their volatilome compositions. The TSB volatilome contained the lowest numbers of alcohols and sulfurous volatiles, while BHI produced proportionally high numbers of acids and esters but the fewest aromatics of any kind. LB, a low glucose medium, produced the greatest number of alcohols and sulfurs but the fewest halogens and no detectable ethers. Although MHB cultures had the lowest number of identified analytes, it produced a staph volatilome with the largest percentage of hydrocarbons and the lowest percentage of ketones. Comparing the staphylococcus volatilomes across the rich media not by numbers of compounds but by the relative abundance of volatiles in each chemical class told a contradictory story (Figure 1c,d). First, the relative abundance of volatiles produced in each medium did trend with the availability of glucose (TSB \(>\) BHI \(>\) LB \(>\) MHB). In addition, while acids and esters comprised 8% of the total number of volatiles detected across all four media (Figure 1b), by relative abundance, they made up approximately half (52%) of all classified volatiles, and heteroamotatics were the second most abundant at 18% (Figure 1d). Hydrocarbons, by contrast, represented only 12% of the volatilome. Additionally, differences between the four media were much more pronounced when the relative abundances of the volatiles were considered, and interesting patterns emerged related to glucose availability and catabolite repression control (Figure 1d). We observed that the two high-glucose media were the primary sources of the acids and esters in the staphylococcal volatilome, with this class representing approximately two-thirds of the volatiles produced in BHI and TSB. By contrast, staph grown in LB and MHB, the two low-glucose media, produced acids and esters at 25% and 4%, respectively. The dependency of acid production on the availability of free glucose was consistent with prior observations of the volatilome of _S. aureus_ grown in BHI, MHB, and NB [35]. More strikingly, the overall volatilome compositions of LB and MHB were very different, with the former producing a relatively even distribution of many different chemical classes, and the latter almost exclusively producing ketones, which might be caused by growth defects due to the poor uptake of amino acid monomers in MHB relative to polypeptides in LB by staphylococci [28]. The non-acid/ester portion of the volatilomes produced in high-glucose media were also unique, with TSB dominated by heteroamotatics and BHI by hydrocarbons. We quantified the chemical diversities and dissimilarities of the volatilomes in each medium using Shannon diversity and Morisita-Horn methods, respectively, and the relative abundances of volatile chemical classes (Table S5). We observed that the Shannon diversities of the staph volatilomes in BHI and TSB were relatively low (H = 0.84 and 0.73, respectively) compared to LB (H = 1.78), while their dissimilarities to each other were the lowest of any pair-wise comparison (C\({}_{MH}\) = 0.071) (Figure 2).