An Investigation of Stability and Species and Strain-Level Specificity in Bacterial Volatilomes

Microbial volatilomics is a rapidly growing field of study and has shown great potential for applications in food, farming, and clinical sectors in the future. Due to the varying experimental methods and growth conditions employed in microbial volatilomic studies as well as strain-dependent volatilomic differences, there is limited knowledge regarding the stability of microbial volatilomes. Consequently, cross-study comparisons and validation of results and data can be challenging. In this study, we investigated the stability of the volatilomes of multiple strains of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli across three frequently used nutrient-rich growth media. Volatilomic stability was assessed based on media-, time- and strain-dependent variation across the examined bacterial volatilomes. Strain-level specificity of the observed volatilomes of E. coli and P. aeruginosa strains was further investigated by comparing the emission of selected compounds at varying stages of cell growth. Headspace solid phase microextraction (HS-SPME) sampling coupled with gas chromatography mass spectrometry (GC-MS) was used to analyze the volatilome of each strain. The whole volatilomes of the examined strains demonstrate a high degree of stability across the three examined growth media. At the compound-level, media dependent differences were observed particularly when comparing the volatilomes obtained in glucose-containing brain heart infusion (BHI) and tryptone soy broth (TSB) growth media with the volatilomes obtained in glucose-free Lysogeny broth (LB) media. These glucose-dependent volatilomic differences were primarily seen in the emission of primary metabolites such as alcohols, ketones, and acids. Strain-level differences in the emission of specific compounds in E. coli and P. aeruginosa samples were also observed across the media. These strain-level volatilomic differences were also observed across varying phases of growth of each strain, therefore confirming that these strains had varying core and accessory volatilomes. Our results demonstrate that, at the species-level, the examined bacteria have a core volatilome that exhibits a high-degree of stability across frequently-used growth media. Media-dependent differences in microbial volatilomes offer valuable insights into identifying the cellular origin of individual metabolites. The observed differences in the core and accessory volatilomes of the examined strains illustrate the complexity of microbial volatilomics as a study while also highlighting the need for more strain-level investigations to ultimately elucidate the whole volatilomic capabilities of microbial species in the future.