Identification of Volatiles From N1-4

Volatiles from N1-4 were enriched with solid-phase micro-extraction (SPME) fiber (divinylbenzene/carboxen/ polydimethlsiloxane), and identified through GC-MS/MS system (5975B-7890N, Agilent Technologies Inc.). N1-4 cell were spread on the surface of NA medium in a 150 mL flask. The flask containing NA medium without N1-4 bodies was used as control. Each flask was sealed with two layer of membrane, and placed at 37°C, and darkness for 24 h to produce volatiles. Volatiles in airspace of each flask were enriched with SPME system for 30 min, then analyzed with GC-MS/MS system equipped with an Agilent HP-5MS fused-C18 capillary column (length 30 m, internal diameter 0.25 mm, 0.25 μm thickness film). The samples were analyzed with splitless injection mode. The inlet temperature was 250°C, carrier gas was helium. The compounds were analyzed using the method as follows: initial temperature at 40°C for 3 min, following a linear heating rate of 3°C ⋅ min^-1 to 160°C, and contained for 2 min, 8°C ⋅ min^-1 to 220°C, and contained for 3 min. Each sample was conducted for two times. The compounds in N1-4 samples that were not present in the control samples were considered to be the final analyzes.