IVM biodegradation experiments

Unless otherwise mentioned, the biodegradation of IVM by strain ZJB-18,044 was carried out in 180 mm × 18 mm test tubes with silicone caps. To evaluate the effect of growing medium on IVM degradation, strain ZJB-18,044 was grown in 5 mL LB medium or 5 mL MSM containing 20 mg/L IVM for 12 h at 30 °C and 150 rpm. Then 2 mL of the resulting LB culture and MSM culture were introduced into 3 mL MSM and 3 mL LB medium (all supplemented with IVM to final concentration of 50 mg/L) respectively, and then cultivatetd for 72 h in the dark at 30 °C and 150 rpm. The culture broth was ultrasonicated and extracted as described above, then analyzed with HPLC.

Resting cells were prepared from the LB culture by centrifugation at 10,000 × g and 4 °C for 10 min. The resulting cells were washed once with an equal volume of sterile 0.2 M phosphate buffer (pH 7.5), and then resuspended in the same buffer. IVM tolerance and utilization of growing cells was carried out by introducing 2 mL LB culture into 3 mL MSM supplemented with IVM to final concentration of 10 mg/L, 30 mg/L, 50 mg/L, 80 mg/L, 100 mg/L, 150 mg/L and 200 mg/L IVM. As for IVM tolerance and utilization of the resting cells, 2 mL resting cells suspension was used.

Degradation of IVM by strain ZJB-18,044 was further examined in 5 L fermenter (BIOTECH, China). LB culture of strain ZJB-18,044 cultivated in 500 mL flasks containing 100 mL LB at 30 °C and 150 rpm for 12 h were pooled and introduced into the 5 L fermenter (with 2.7 L MSM supplemented with IVM to final concentration of 50 mg/L or 100 mg/L) at a 10% (V/V) inoculation size. The initial incubation conditions were 30 °C, 200 rpm, aeration rate 2.0 L/(L min), and pH 7.5. The dissolved oxygen (DO) was maintained above 30% throughout the fermentation and samples were withdrawn every 12 h.

The utilization and tolerance of strain ZJB-18,044 towards avermectin (16-membered-ring macrolide), doramectin (16-membered-ring macrolide), emamectin(16-membered-ring macrolide), erythromycin (14-membered-ring macrolide), azithromycin (15-membered-ring macrolide), spiramycin (16-membered-ring macrolide) or rifampicin (ansa macrolide) were investigated by incubating the strain in MSM or MH medium separately containing the macrolides in the dark at 30 °C and 150 rpm for 72 h. Then the biomass and macrolides concentrations were determined.

To determine whether the aforementioned macrolides can induce the IVM degradation, IVM (final concentration 50 mg/L) was introduced into 48 h cultures of strain ZJB-18,044 in LB or LB separately supplemented with the macrolides, and then further incubated for 72 h.

The IVM degradation metabolites of strain ZJB-18,044 was determined by HPLC-MS analysis of the IVM degradation products of growing cells. The 4 h to 32 h culture broth of strain ZJB-18,044 in MSM with 50 mg/L IVM was ultrasonicated and centrifuged at 10,000×g and 4 °C for 10 min. Then the resulting supernatants were extracted with dichloromethane for IVM and corresponding metabolites. The HPLC-MS was performed with a Thermo Scientific LTQ XL mass analyzer attached to a Dionex UltiMate 3000 system equipped with a Hypersil gold C18 column (100 mm × 2.1 mm × 3 µm). Sample (20 µL) was loaded. The elution method is 8 min 2%–100% acetonitrile, 5 min 100%–2% acetonitrile, 2 min 2% acetonitrile in total 15 min. The flow rate of the mobile phase was 0.6 mL/min.

The IVM-tolerant mechanism of strain ZJB-18,044 was investigated in 3 L MSM medium supplemented with 50 mg/L, 100 mg/L and 200 mg/L IVM within 5 L fermenter and incubated as described above. Samples were withdrawn at 0, 6, 12, 24, 36, 48, 60, 72, 96 and 120 h, and then centrifuged at 10,000×g and 4 °C for 10 min. The harvested cells were resuspended and disrupted for determination of intracellular IVM as previous research (Wang et al. 2015).