Bacillus Volatile Organic Compound Analysis

Bacillus VOC collection was performed as described previously by Sarenqimuge et al. [50 (link)]. As an internal standard, 200 ng of tetralin (1,2,3,4 tetrahydronaphthalene, Sigma-Aldrich, Munich, Germany) was added to each sample before GC–MS analysis. An aliquot of 30 µL sample was transferred to another GC vial with a glass insert and placed into the tray of the GC–MS autosampler. A 2 µL sample was injected in pulsed splitless mode for analysis. The oven temperature was retained at 40 °C for 3 min and gradually increased (8 °C/min) to a final temperature of 220 °C for 10 min. Helium was used as a carrier gas (flow rate was 1.5 mL/min). A homogenous series of n-Alkenes (C_7–20) was used to determine retention indices. The MassHunter instrument (Agilent Technologies: GC 7890B, MS 5977B, Santa Clara, CA, USA) was used for data processing; MSD ChemStation software with the NIST17 and Willey11 mass spectral libraries was used to tentatively identify bacterial VOCs by their mass spectra and retention indices. The identities of the ten bioactive compounds tested in Section 2.6 were confirmed by GC–MS analysis of commercially available standards. The VOC quantification was performed by comparing the peak areas of individual compounds to the peak area of the internal standard (tetralin). From each treatment, five replicates were analyzed, and LB without bacteria was used as a control.