Chemicals and soil samples
Pyrene (purity 99%) was purchased from Sigma-Aldrich Co. HPLC grade acetonitrile was purchased from Honeywell, Inc. Chemicals for mineral basal media (MBM) were purchased from different suppliers. Pyrene was dissolved in acetone at 10 mg/ml and the final concentration of both PAHs used in liquid broth media was 0.1 mg/ml.
The oil-contaminated soil samples were collected in sterile 50 ml falcon tubes from three different sites located on Banimalek road in Abha, Saudi Arabia. Sample A was collected from the site where gasoline is spilled from trucks, sample B was from a gasoline station, while sample C was collected from an oil exchange station. All the samples were stored at 4oC until further use.
Growth media and culturing conditions
Minimal base medium (MBM) [1g (NH4)2SO4, 0.8 g K2HPO4, 0.2 g KH2PO4, 0.2 g MgSO4.7H2O, 0.1 g CaCl2.2H2O, and 5 mg FeSO4.7H2O in 1L of distilled water, pH 7.2] supplemented with pyrene was used for the suspension of oil-contaminated soil samples. In brief, one gram of each contaminated soil sample was suspended in 100 ml of MBM supplemented with 0.1 mg/ml of pyrene in 250 ml flasks [20]. The samples were incubated at 30oC for 7 days at 150 rpm [21]. Subsequently, 10 ml from each culture were sub-cultured into 90 ml of the same media under similar growth conditions. After two more sub-culturing under similar conditions, 100 µl from each culture were plated on MBM-agar plates coated with 1 ml solution of pyrene (0.1 mg/ml) dissolved in acetone. All the plates were incubated at 27oC for 2-3 days. The markedly bigger size colonies showing distinct morphology and color were re-streaked on pyrene-coated MBM agar plates. Isolates, which consistently produced a bigger size colony with distinct morphology and color, were taken for pyrene degradation study.
Growth kinetics using BD MGIT 960 BACTEC
For growth kinetics, the Becton Dickinson MGIT 960 BACTEC Automated Mycobacterial Detection system in BSL3 mycobacterial laboratory at Southern Region Military Hospital, Khamies Mushayet, Saudi Arabia, was used. The cells were inoculated into 7 ml of MBM (control) or MBM supplemented with 0.01% pyrene at a very low density (106 cfu/ml) in a test tube. The test tube contains a fluorophore at its bottom, which fluoresces in the presence of CO2 produced by the growing bacterial cells. The tubes were incubated at 37oC in the BACTEC system. Fluorescent readings were recorded regularly at intervals of 24 hours. The fluorescence signal is displayed in the form of growth units, which were plotted against the time.
Bacterial isolate identification methods
VITEK® MS and VITEK® 2 COMPACT from bioMérieux were used for the identification of bacterial isolates. VITEK® MS uses Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) technology to obtains the protein fingerprinting from whole bacterial cells, which it later uses to searches the protein fingerprinting database of bacterial isolates. VITEK® 2 COMPACT works on biochemical tests measuring carbon source utilization, enzymatic activities, and antibiotic resistance. Both VITEK® MS and VITEK® 2 COMPACT in BSL3 mycobacterial laboratory at Southern Region Military Hospital, Khamies Mushayet, Saudi Arabia, were used as per the manufacturer’s instructions.
Gram staining and scanning electron microscopy
Gram staining was performed on bacterial cells grown in tryptic soy broth to an OD600 of 0.4. Cells were heat-fixed on a glass slide and flooded with crystal violet for one min. After rinsing the slide with water, the cells were treated with iodine for one min. Subsequently, the slide was rinsed with water, and cells were flooded with counter stain safranine for one min. The slide was rinsed with water and observed under 100x objective of Nikon microscope, fitted with 10MP USB 2.0 color CMOS C-mount camera.
For scanning electron microscopy (SEM), the bacterial broth was centrifuged and fixated in 2.5% glutaraldehyde buffered to pH 7.2 with 0.1 M Na-phosphate buffer for 24h at room temperature, and then washed thoroughly with Na-phosphate buffer three times. Then post-fixed with 1% osmium in Na–phosphate buffer for one hour and dehydrated successively with different concentrations of ethanol of 30%, 50%, 70%, 95%, and 100%. For each ethanol volume, the sample was incubated for 10 minutes except 100% ethanol, which was for one hour. Subsequently, the sample was immersed in a critical point dryer and mounted onto aluminum metal stub. The sample was coated with a thin layer of gold by Edward’s sputtering coater and examined with a JEOL JSM 6360-LV (Tokyo, Japan) microscope in the Electron Microscope Unit of KKU, Pathology department, College of medicine.
DNA extraction and 16s rRNA sequencing
The genomic DNA of the bacterial isolates was extracted using the DNeasy® Plant Mini Kit (QIAGEN). After checking the purity and intactness of the genomic DNA on 1% agarose gel, it was sent to Macrogen, Korea (https://dna.macrogen.com/eng/) for 16s rRNA sequencing. In brief, PCR product of ~ 1.5 Kb amplified from genomic DNA using universal flanking primers 27F (5’AGAGTTTGATCC TGGCTCAG3’), 1492R (5’TACGGYTACCTTGTTACGACTT3’) were purified and sequenced by Internal primers 785F (5’GGATTAGATACCCTGGTA3’), 907R (5’CCGTCAA TTCMTTTRAGTTT3’) in both the directions using ABI 3730 automated sequencer (Macrogen, Seoul, Korea). The compiled sequence obtained from both the internal primers was blasted using BLASTN against the NCBI database.
Sequence alignment, phylogenetic tree analysis and gene search
The 16S rRNA sequences of each bacterial isolate obtained were aligned and compared with the known 16S rRNA sequences in the GenBank database using the basic local alignment search tool (BLASTN) available at the National Center for Biotechnology Information website (NCBI) (http://www.ncbi.nlm.nih.gov/blast). The nearest neighboring sequences in the database with high homology percentage scores were aligned using MEGA-11 (Molecular Evolutionary Genetic Analysis, version 11). To determine the taxonomic position of the isolate, a phylogenetic tree was constructed with MEGA-11 using a maximum likelihood algorithm. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to identify and locate the genes involved in the catabolism of PAHs in the genome of a reference strain K. oxytoca CAV1374.
High performance liquid chromatography
Pyrene degraded by the bacterial isolate K. oxytoca was estimated by high-performance liquid chromatography (HPLC) of the supernatant of the bacterial culture grown in MBM supplemented with pyrene as a sole source of carbon. In brief, 1 ml each of an overnight nutrient-broth-grown bacterial culture was inoculated into 100 ml of MBM supplemented with 0.1mg/ml pyrene in triplicates. For the spontaneous degradation of pyrene, 100 ml of MBM supplemented with 0.1mg/ml pyrene in a separate flask was included as a control. All the triplicate cultures of each isolate including control were incubated at 30oC for 18 days at 150 rpm. 2 ml samples (in triplicates) for each bacterial isolate were withdrawn at the regular time intervals of 3, 6 9, and 12 days. After spinning down the cells, the concentration of the pyrene in the culture supernatant of each sample was determined using HPLC coupled with Shimadzu UV-VIS Detector SPD-10A, wherein 10µl sample volume was subjected to 254 nm ultraviolet detection at 30oC. Average values calculated at each time point were normalized with the control and presented as percentages.