2.4. Binding of Tested Strains to B(a)P in Two Simulated Systems

The artificial inorganic system (100 μL) plus 900 μL sterilized water, containing 1.0 μg/mL B(a)P, was added to 1.0 mL bacterial suspension. After incubation at 37°C for 4 hr, the supernatant was collected by centrifugation (3000 r/min, 5 min). Chloroform (500 μL) was added to the supernatant to produce the organic phase for the detection of B(a)P. The control was designed as 1.0 μg/mL aqueous solution of B(a)P without bacterial cell addition. For each sample, B(a)P was detected by HPLC with the following conditions: UV detection of 290 nm wavelength, mobile phase of pure methanol, selection of room temperature as column temperature, flow rate of 1.0 mL/min, and injection volume of 20.0 μL [18].

We selected 100 μL PHAs plus 900 μL sterilized water which were added to 1.0 mL bacterial suspension. After incubation at 37°C for 4 hr, the level of B(a)P existing in the supernatant collected by centrifugation (3000 r/min, 5 min) was detected by HPLC. The control was 1.0 μg/mL PHAs (1.0 mL) without bacterial cell addition. B(a)P was UV-detected at 290 nm wavelength by HPLC. Acetonitrile and water were used as the mobile phase. Column temperature was room temperature with a flow rate of 1.0 mL/min as well as injection volume of 20.0 μL. For gradient elution analysis, A was acetonitrile and B was water. 60% A plus 40% B was used for isocratic elution for 1 min. After A was elevated from 60% to 100% within 20 min, eluting with 100% A was kept for at least 22 min. Then, elution A was reduced to 60% in 8 min and equilibrated with 60% A for 10 min.

The binding percentage of the tested bacteria to B(a)P was calculated according to the following equation:

where Br represents the binding rate of the tested bacterial cells to B(a)P (%); Bs indicates the level of B(a)P from the sample blank (μg/mL); and S is the level of B(a)P for each supernatant harvested from the tested bacterial cultivation (μg/mL).

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