Data Sheet 1_Molecular imaging uncovers growth media influence on biofilms’ EPS production.docx

Biofilm growth in a laboratory typically requires media rich with amino acids and other nutrients for bacterial metabolism. Microbial biofilms consist of clusters of planktonic cells grouping and secrete extracellular polymeric substance (EPS). The EPS is a complex mixture consisting of polysaccharides, fatty acids, and lipids as well as primary and secondary metabolites among other biomolecules. Choice of growth medium is important to culturing microbes, as it should allow the bacteria to replicate at rapid rates. Herein, we study the effect of media selection on biofilm culture. We investigated three growth media, including two common complex growth media, namely, Luria Broth (LB) and Tryptic Soy Broth (TSB), and a minimal growth medium, hydrogen oxidizing de-nitrifier (HOD). The latter was supplemented with glucose as the carbon energy source for aerobic growth, and nitrate was not incorporated into the media. HOD was developed to cultivate hydrogenotrophic groundwater bacteria isolated from the Hanford Site in Richland, WA. A Paenibacillus strain originating in the Hanford Site subsurface was selected as the model biofilm system. We used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to investigate the cultures over a 7-day period. Three time points were chosen based on the bacterial growth curve, corresponding to the log phase, stationary phase, and death phase, respectively. The SIMS spectral and two-dimensional (2D) imaging results show that the fatty acid peaks in HOD-grown biofilms are different from those cultured in the complex media. In the HOD-grown biofilms, biomarkers indicative of bacterial stress are localized as evidenced in ToF-SIMS 2D images. Our SIMS 2D image findings also show that distributions of prominent fatty acids and lipids, as components of the EPS and possibly bacterial plasma membrane, are influenced by the growth medium. HOD, among the three media studied, seems to offer the most distinctive metabolic behavior of the selected biofilm strain. Minimal media, such as HOD, are suggested as a suitable choice to study microbial effects on materials corrosion due to the nature of the minimal medium effects, which offer good insights into the metabolic process of biofilms.