Porapak

Acetoin (200 µg dissolved in 1 ml dichloromethane) and 2-phenylethylamine (400 µg dissolved in 1 ml dichloromethane) were separately dropped in a glass Petri dish (Ø30 × 10 mm). These Petri dishes were placed without a lid in another glass Petri dish (Ø130 × 30 mm), which had an in- and outlet (analysis chamber). After 24 h of incubation (30 °C and 1.5 µEm⁻²s⁻¹), charcoal-purified, sterile air was sucked through the inlet into the analysis chamber with a constant flow of 0.6 l min⁻¹ provided by a membrane pump (1410 Büh 12 VDC “D”, Gardner Denver Thomas GmbH, Memmingen, Germany). The volatile-enriched air was directed to a trap containing 30 mg of an adsorbent matrix (Porapak; Sigma–Aldrich, Munich, Germany). After 24 h of incubation, the volatiles were consecutively eluted with 200 and 100 µl dichloromethane. N-nonyl acetate solution was added as an internal standard (final concentration of 5 ng µl⁻¹).

Bacteria were cultivated overnight in 6 ml liquid medium (170 rpm, 30°C) and subsequently transferred into the VOC collection system (Kai et al., 2010 (link)) containing 100 ml medium. Volatiles were collected and trapped on the adsorbent material Porapak (Sigma–Aldrich, Munich, Germany) and eluted in 24 h time intervals using dichloromethane (Carl Roth GmbH+Co., KG, Karlsruhe, Germany) and an internal standard (nonylacetate, final concentration 5 ng/μl). Finally, eluates were analyzed by gas chromatography/mass spectrometry according to the procedure published in Domik et al. (2016a) (link).

The pre-cultivation of the bacterial strains and inoculation into the Erlenmeyer flasks was performed as described above. The VOC-collection system was modified as followed. Charcoal-purified and sterile air was sucked with a constant flow of 0.6 l min⁻¹ (1410 Büh 12 VDC “D”, Gardner Denver Thomas GmbH, Memmingen, Germany). After passing the cotton filter, the air stream was split, humidified and directed through two Erlenmeyer flasks (Fig. 1a). While one Erlenmeyer flask was filled with a liquid culture of S. plymuthica 4Rx13 (24 h post inoculation), the second flask contained a S. delphini 20771 culture (120 h post inoculation). Three additional setups served as control. Flask 1 and 2 contained NBII + glucose and TSB, respectively (control 1). Furthermore, a mono-culture of S. plymuthica 4Rx13 in flask 1 was combined with TSB in flask 2 (control 2) and the mono-culture of S. delphini 20771 in flask 2 was combined with NBII + glucose in flask 1 (control 3). Cultures were magnetically stirred. Volatile-enriched air streams were reunited and sucked into a trap containing 30 mg of an adsorbent matrix (Porapak; Sigma–Aldrich, Munich, Germany). VOCs were consecutively eluted after 24 h and 48 h of cultivation with 200 and 100 µl dichloromethane. N-nonyl acetate was added as an internal standard (final concentration of 5 ng µl⁻¹). The experiments were conducted in triplicate.