# Dependence of the Staphylococcal Volatilome Composition on Microbial Nutrition
## 1 Introduction  
In vitro cultivation of staphylococci is fundamental to both clinical and research microbiology, and the selection of growth medium substantially influences staph growth rates, genetic integrity, pathogenicity, and metabolic capacity [1; 2; 3; 4; 5]. Depending on the species, staphylococci possess remarkable genetic plasticity, regulated by two-component systems that enable rapid responses to fluctuations in environmental conditions [1; 4; 6]. Additionally, staph is able to survive under host sequestration (also known as nutritional immunity) and microbial competition, both of which influence the availability of essential nutrients and carbon sources [1; 4; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15]. Like many heterotrophic bacteria, glucose is the preferred carbon and energy source for staphylococci, which employ catabolite repression control to regulate metabolism in glucose-rich environments. Under catabolite repression control in glucose-replete conditions, the tricarboxylic acid cycle is markedly downregulated, forcing catabolism through the Embden-Meyerhof-Parnas pathway (EMP; glycolysis) and the pentose phosphate pathway (PPP) [16; 17; 18; 19]. Once glucose and other hexose sugars are depleted, other nutrient sources, such aspeptides, will be imported and catabolized. Alternatively, under starvation conditions, intracellular biomass (amino acids, proteins, RNA) will become substrates of endogenous metabolism [17; 20; 21]. Four common media used for the enrichment of staph are brain heart infusion (BHI), lysogeny broth (LB), Mueller Hinton broth (MHB), and tryptic soy broth (TSB). All of these are complex media with high contributions of amino acids or peptides to the total organic dry weight, ranging from approximately 66% in LB to 89% in TSB and 93% in BHI and MHB. Thus, these media provide nitrogen-replete conditions for protein and nucleotide synthesis during growth. In contrast, the sugar content of these media substantially differs. TSB and BHI contain 11% and 7% dextrose (\(\alpha\)-p-glucose), respectively, by dry organic weight, while MHB provides 7% starch (amylose and amylopectin), which can be hydrolyzed by most staphylococci via \(\alpha\)-amylase to generate free sugars for glycolysis [22]. LB contains no added dextrose or starches, and the yeast extract ingredient has been shown to contribute less than 100 uM fermentable sugar equivalents (nucleotides, sugar phosphates, or oligosaccharides), which substantially slow the growth of _Escherichia coli_ and _S. aureus_ once the sugars are depleted [23; 24; 25]. A few other notable differences in the media may influence staph metabolism. For instance, MHB has low concentrations of thymine and thymidine, which will require upregulates in thymidine precursor biosynthesis during replication [26; 27]. Additionally, the proteins in BHI, LB, and TSB have been enzymatically digested, generating random length oligopeptides, while MHB contains an acid digest of casein, which generates free amino acids that are not as easily imported by staphylococci [28]. Lastly, the low lipid content of TSB, contributing <0.05% dry organic weight, may introduce a differential effect on staph metabolism compared to other media, as both _S. aureus_ and _S. epidermidis_ must activate de novo fatty acid biosynthetic pathways when exogenous fatty acids are unavailable for assimilation [29; 30; 31]. We are specifically interested in the role that growth media play on the production of the volatile metabolome (or volatilome) of staph and other microbes. In BHI, LB, MHB, and TSB, proteolysis and/or glycolysis will dominate the production of volatiles. Amino acid catabolism generates sulfur and nitrogen-containing volatiles (e.g., dimethyl disulfide, pyrazines, nitriles, and pyrroles), as well as aromatic compounds (e.g., aromatic alcohols, aldehydes, and ketones) from the catabolism of aromatic amino acids [32; 33; 34].