68 to 0.70 at 620 nm) by centrifugation at 12,000 rpm for 10 min. The pellet was washed thrice with sodium chloride solution (0.9%, w/v) and then resuspended in sodium chloride solution (0.9%, w/v). Fe3O4 nanoparticles were prepared as previously selleck compound described . Fe3O4 powder (1.0 g) was put into 100 ml distilled water to form the Fe3O4 particle suspension. After ultrasonic disruption (25 KHz, 10 min; BUG25-06, Branson, MO, USA) of the suspension, the Fe3O4 nanoparticles were well dispersed in distilled water to form a stable suspension. Fe3O4 particle suspension (1%, w/v) and cell suspension were mixed with the ratio of cell wet weight to Fe3O4 of 1 (w/w). Microbial
cells and Fe3O4 nanoparticles were fully mixed by vortexing, then the mixture was incubated at 30°C for 2 h in a dark shaker to obtain microbial cell/Fe3O4 biocomposites. All TSA HDAC supplier biodegradation experiments were carried out in 100-ml flasks containing 10-ml MSM at 30°C on a reciprocal shaker at 180 rpm. In each experiment, 3,500 μg of carbazole was added to MSM, and the microbial cell/Fe3O4 biocomposites made by 2 ml mixture of Fe3O4 particle suspension selleck chemical and cell suspension served as biocatalysts. Additionally, the same amount of cells
was conducted in the batch biodegradation experiment. All the subsequent experiments contained the same amount of carbazole and biocatalysts as above. In the recycle experiments, after each batch of biodegradation, the microbial cell/Fe3O4 biocomposites were collected using a magnetic field, and then
were washed thrice with MSM to remove the free cells. After the MSM was drained, 10 ml of fresh MSM containing carbazole was added to repeat the cycle. All experiments were performed in triplicate. After each batch of biodegradation, the biodegradation mixture was added 20 ml ethanol, followed by centrifugation (12,000 rpm for 20 min) and filtration. Residual contents of carbazole were determined using High-performance liquid chromatography (HPLC). HPLC was performed with an Agilent 1100 series (Hewlett-Packard) instrument equipped with a reversed-phase C18 column (4.6 mm × 150 mm, Hewlett-Packard). The mobile phase was a 2-hydroxyphytanoyl-CoA lyase mixture of methanol and deionized water (90:10, v/v) at a flow rate of 0.5 ml min-1, and carbazole was monitored at 254 nm with a variable-wavelength detector. The size and morphology of magnetic nanoparticles and microbial cell/Fe3O4 biocomposite were determined by transmission electronic microscopy (TEM; JEM-100cx II, JEOL, Akishima-shi, Japan). The sample was prepared by evaporating a drop of properly diluted microbial cell/Fe3O4 biocomposite or nanoparticle suspension on a carbon copper grid. The morphology of free cells was determined using a scanning electron microscope (SEM; S-570, Hitachi, Chiyoda-ku, Japan). Magnetization curves for the magnetic immobilized cells were obtained with a vibrating sample magnetometer (MicroMag 2900/3900, Princeton Measurements Corp., Westerville, OH, USA).