Innova 2300

SLES cleavage and conversion to biomass and CO₂ by strain S11 was compared at anoxic and oxic conditions, using 500 mg SLES L⁻¹. Anoxic batch bottles were prepared as described above. For oxic conditions, bottles were prepared with air as gas phase. Nitrate, nitrite, sulfate, anionic surfactants, and DOC were analyzed at the beginning, after 1 day and 2 weeks of incubation. All batches were gently stirred (60 rpm; Innova 2300, New Brunswick Scientific, Edison, NJ) to avoid foaming.

Extraction of FB1, FB2 and DON was based on the multi-targeted method based on the QuEChERs extraction described by López et al. [34 (link)] with minor modifications. Briefly, 1 g dry sample was spiked with 16 µl of ¹³C34-FB1 and 16 µl ¹³C34-FB2, and samples were then mixed with 3 mL ultra-pure water (Arium pro, Sartorius, Göttingen, Germany). After manual shaking, 4 mL of extraction solvent (ACN with 1% (v/v) acetic acid) were added. The extraction process consisted of 30 min shaking in a platform shaker (Innova 2300, New Brunswick Scientific, Nijmegen, The Netherlands). Afterwards, the sample was cleaned-up by vortexing with 1.6 g of anhydrous MgSO₄ for 1 min, followed by centrifugation at 1960× g for 10 min. An aliquot of 500 µL obtained from the extraction solvent supernatant was then diluted to 1 mL with 5 µL ¹³C-DON, 45 µL ACN with 0.1% (v/v) acetic acid and 450 µL water. Samples were stored at −80°C until analysis. Before analysis, samples were vortexed, and 500 µL were filtrated using a syringeless 0.45 µm PTFE filter vials (Whatman Mini-UniPrep, GE, Buckinghamshire, United Kingdom) before LC-MS/MS analysis. Two independent extractions were performed for each sample, which were also analyzed independently.

Five mL LB media (10 g/L sodium chloride, 5 g/L yeast extract and 10 g/L tryptone) were inoculated with the desired bacterial strain, followed by incubation overnight at 37 °C in a shaking incubator (Platform shaker: Innova 2300, New Brunswick Scientific Co, Enfield, USA; Incubator: Wärmeschrank für Plattformschüttler, Mytron Bio- und Solartechnik GmbH, Heilbad Heiligenstadt, Germany). Afterwards, the preparatory culture was transferred into 20 mL of LB media or a minimal media (1% LB media in depyrogenated LRW) and incubated for 18 h at room temperature. Bacterial growth was stopped by temperature reduction to 4 °C and sterile filtration (0.2 µm) of the bacterial suspension. For conservation 0.05% (v/v) sodium azide was added. Required endotoxin concentrations for endotoxin recovery studies were adjusted by dilution with depyrogenated water.

For the agitated culture initiation, 1.5 g S. chinensis microshoots (see the “Basic agar culture” section), grown on MS_Sch medium for 60 days, were placed into 125 ml Erlenmeyer flasks, filled with 50 ml liquid MS_Sch medium, and closed with silicone sponge stoppers (Carl-Roth, Karlsruhe, Germany). The cultures were maintained on the rotary shaker at 120 rpm (INNOVA 2300, Eppendorf, Enfield, US-CT), under light and temperature conditions described beforehand (see the “Basic agar culture” section).

In order to initiate liquid shoot cultures, 3.0 g of the inoculum were inserted into 250 ml Erlenmeyer flasks containing 100 ml liquid M_A medium and plugged with silicone foam stoppers (Carl Roth, Karlsruhe, Germany). The cultures were maintained on rotary shaker (120 rpm, 25.4 mm orbit, INNOVA 2300, Eppendorf, Enfield, US-CT) under photoperiod or without the presence of light. Biomass and medium samples were harvested at 2-day intervals until 52 days of experiment.
During the feeding experiments, the cultures were supplemented with LY (1.0 g l⁻¹), CH (1.0 g l⁻¹), CW (10 % v/v), or their mixture (MIX, 1.0 + 1.0 g l⁻¹ + 10 % v/v, respectively) on 34 days of the growth period and harvested on 44 days.