3.1. Sample collection

Water released from the wastewater treatment plant (effluent) was obtained from Notwane Sewage Treatment Plant situated in Gaborone, Botswana. It was collected from the sampling site in sterile 250 ml Duran bottles and immediately placed on ice in a cooler box with ice. The samples were taken to the Department of Biological Sciences, North-West University, Mafikeng for analysis. Samples were analyzed within 24 h of sampling. The treatment plant treats 40,000 m3 per day of sewage.

#### 3.2. Biodegradation of PCBs in wastewater by isolate Pseudomonas aeruginosa

In the study carried out by the author, out of the many bacteria stated in literature, only Pseudomonas aeruginosa isolated from the wastewater sampled during the study was used. The water samples were divided into two parts, one part was sterilized by autoclaving at 121C for 15 min and the other half was left unsterilized. The wastewater samples were treated with Aroclors of polychlorinated biphenyls (PCBs) obtained from SUPELCO Solutions Within™, USA, through Lehlabile Scientific, South Africa. The PCBs were supplied as Aroclors. Aroclor 1242 (Lot No. LB8851), 1248 (Lot No. LB88969), and 1260 (Lot No. LB92109) in 1 ml ampoules at concentration 1000 μg/ml dissolved in isooctane were used in this study. The purity for each Aroclor was not stated.

To each 100 ml wastewater sample in a 250 ml flask, 10 μl of polychlorinated biphenyls Aroclors mixture, herein referred to as PCBs, was added. The sterilized wastewater samples were inoculated with a colony of the 18 h old culture of the test organism, which was identified as Pseudomonas aeruginosa (with accession number from the gene bank of CP 006832 in a study carried out in 2014). Non-sterilized wastewater without bacterial inoculation (Control 1) and sterilized wastewater without inoculation (Control 2) were both treated with PCBs and were the controls. The flasks were wrapped with aluminum foil to exclude light and were incubated at 30C in the dark in a rotary shaker at 150 rpm [46]. A 5 ml was aseptically taken at 24 h intervals from each setup/flask for detection of PCBs using HPLC and spectral changes were checked at 200–800 nm using Cary 300 UV-visible spectrophotometer, for a period of 96 h.

Analysis for PCB using HPLC was carried out as described by Roy et al. with some modifications [46]. A 1 ml was sampled from each setup to check for residual PCB at 24 h interval. The compounds were extracted by adding 10 ml each of dichloromethane and acetone. The mixture was incubated in a rotary shaker for 24 h at 30C. After incubation, the mixture was centrifuged for 10 min at 12,000 rpm at 4C using a Hermle Z326k high speed microcentrifuge, Labortechnik GmbH (LASEC, South Africa). The extra water was pipetted and 4 g of anhydrous sodium sulfate mixed with a PCB-containing solvent to remove residual water. The extract was concentrated to 1.5 ml using a rotary evaporator Stuart RE300DB, LASEC, South Africa and filtered with 0.45 μm PTFE syringe filters. Extracts were analyzed by high performance liquid chromatography (HPLC) UFLC Shimadzu using a florescence detector pump RF-20A and system gold column C18 (CTO-20A). The excitation level was set at 254 nm, emission level at 390 nm. The mobile phase used was a mixture of acetonitrile and water (80:20) as described by Roy et al. [46]. Data analysis was computed using real-time analysis. All chemicals used were of HPLC grade supplied by Sigma Aldrich through Lehlabile Scientific, South Africa.
