Chemicals and preparation of culture media
Phenanthrene, ZnCl2, MnCl2, HgCl2, CrO3, PbCl2 and NiCl2 were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA); Escravos crude oil (see Table S1 in Supplementary document for its characteristics) was obtained from ‘Chevron Nigeria Limited’, and all other chemicals were of analytical reagent grade. Phenanthrene (100 mg) was dissolved in hexane (100 ml), shared into 10 Erlenmeyer flasks (500 ml) and nitrogen-free mineral salts medium (N-free MSM) was added to each flasks containing 10 mg hexane-free phenanthrene to make up 100 mg l-1, final concentration, before sterilization as earlier reported (Oyetibo et al., 2013, 2017). Similarly, a 1% (v/v) final concentration of crude oil-MSM whereby crude oil formed an immiscible oily layer on aqueous MSM in Erlenmeyer flasks was prepared as earlier explained (Oyetibo et al., 2017). The N-free MSM contained (g l-1) Na2HPO4, 2.13 g; KH2PO4, 1.30 g; NaCl (instead of NH4Cl), 0.50 g and MgSO4.7H2O, 0.20 g; pH was adjusted to 6.9. Sterile trace elements solution, SL-6 (1.0 ml l-1) (Atlas, 2005) was aseptically added to the medium after sterilization. All sterilization processes were by moist heat in autoclave at 121 oC for 15 min unless otherwise stated.
Site description and sampling
Ikeja Industrial Estate, Lagos, Nigeria was established in the mid-1960s and industrial activities of the estate were peaked in the late-1980 until Nigeria’s economy began to experience downturn. Despite that, the estate still housed plethora of diverse industries that discharge their multicomponent wastewaters into the environment, forming “Odo-Iyalaro” stream, which empties into Lagos lagoon and finally into the Atlantic Ocean. Composite samples (10) of sediments were randomly collected from the site and GPS coordinates were as presented in the supplementary document (Table S2).
Geochemical and physical parameters analyses
Physico-geochemical parameters including pH, chlorides, total dissolved solids (TDS), cation-exchange capacity (CEC), acidity, alkalinity, chemical oxygen demand (COD), and biochemical oxygen demand (BOD) were determined as previously reported (Oyetibo et al., 2017, 2019). A total of 15 HMs/metalloids were quantified in the sediment via inductively coupled plasma mass spectrometry (ICP-MS) (ELAN 9000, Perkin Elmer SCIEX, Boston, MA, USA) after digesting dry sample (0.1 g) with HNO3/HCl (4:1, v/v) in a microwave oven (Multiwave 3000, Anton Paar, Graz, Austria) as described in previous report (Oyetibo et al., 2019). Extraction of total hydrocarbons was with n-hexane and quantified using standard gravimetric method coupled with GC-FID as previously reported (Obayori et al., 2009).
Isolation of heavy metals resistant bacteria with potential to fix free nitrogen
Isolation of multi-resistant bacteria was based on culture enrichment procedure where samples were inoculated into N2-free MSM amended with sterile HMs solutions (1%) and glucose (10 g l-1). The HMs solutions consisted of ZnCl2 (0.5 mmol l-1), MnCl2 (0.5 mmol l-1), HgCl2 (0.01 mmol l-1), CrO3 (0.1 mmol l-1), PbCl2 (0.5 mmol l-1), and NiCl2 (0.5 mmol l-1). After incubation (30 oC; 96 h; 50 ×g), the culture was diluted appropriately, and plated onto N2-free MSM containing 1.5% bacteriological agar (Wako Pure Chemicals Ltd., Japan) and supplemented with 2% HMs solutions. The choice of Zn2+, Mn2+, Hg2+, Cr6+, Pb2+ and Ni2+ in this study was because they were individually predominant (≥ 5% [33.4 mg kgdw-1]) in the sediment. Pure cultures were further screened for resistance to higher concentrations of the HgCl2 to select those colonies that showed high resistance.
Determination of mercury resistance and sequestration pattern of bacterial isolates
Mercury resistance and sequestration pattern was chosen for further analyses in this study because of its recent global attention towards public health. Isolates grown in Luria Bertani (LB) broth (containing per litre: 10 g tryptone, 5 g yeast extract, and 5 g NaCl) for 18 h at 30 oC were harvested by centrifugation (7,000 × g, 10 min), washed twice with sterile buffered phosphate solution, and re-suspended in the same buffer solution. The cell density of bacterial suspensions was determined by measuring the absorbance at 600 nm in relation to a calibration curve (1010 cfu l-1 = 1 OD unit). LB broth amended with HgCl2 (10 – 240 µmol l-1) in aliquots (5 ml) into test tubes were inoculated with 50 μl inoculums (OD600, 0.1). Growth media without HMs but inoculated with test organisms, and growth media amended with HgCl2 but inoculated with dead cell mass (boiled thrice at 100 oC, 10 min. at 12 h interval to totally kill sporogenous bacteria) served as positive and negative controls, respectively.
Growth of the bacteria was measured using absorbance at 600 nm (OD600 nm) with extrapolated viable counts, and plating out to confirm bacterial purity and viability. Resistance was tested as minimum tolerance concentrations (MTCs) for the isolated strains after incubation (36 h; 30 oC). The MTC was defined as the highest concentration of Hg2+ which did not affect the viability of organisms (OD600 > 0.1). The total mercury in LB broth before inoculation, culture and supernatant (100 µl) were quantified directly without any pre-treatment using a fully automated thermal vaporization mercury analysis system, Mercury/MA-3000 (Nippon Instrument Corp., Osaka, Japan) as earlier reported (Oyetibo et al., 2015, 2016a, 2016b). Nevertheless, capabilities of bacterial strains to sequester Hg2+ were determined as volatilisation, bioaccumulation/adsorption, and bio-removal efficiencies using , and respectively. Where VE is volatilisation efficiency, MQM is the quantity of Hg in medium before inoculation with bacterial strain, CQ is the quantity of Hg in culture, AE is bioaccumulation/adsorption efficiency, SQ is the quantity of Hg in cell-free supernatant of centrifuged (14,000 × g, 2 min) culture, and RE is the removal efficiency.
Molecular characterization and identification of the selected bacteria
Pure colonies of overnight culture of selected bacteria were suspended in mix-solutions of Cica Geneus DNA Extraction Reagent (Kanto Chemical Co. Inc., Tokyo, Japan) and genomic DNA was extracted using two temperature regimes (72 oC, 20 min; 93 oC, 3 min) according to manufacturer’s instructions. The 16S rRNA genes in the purified genomic DNA samples were amplified by using Ex-Taq polymerase (TaKaRa, Ohtsu, Japan) according to the manufacturer’s instructions. A domain bacteria-specific PCR primer set of 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GGHTACCTTGTTACGACTT-3') (Weisburg et al., 1991) was used to amplify 16S rRNA genes (approximately 1,500 nucleotides). The PCR condition was: initial denaturation (94 °C, 5 min); followed by 35 cycles of denaturation (94 °C, 30 s); annealing (50 °C, 30 s), and extension (72 °C, 2 min). The final extension was at 72 °C for 7 min. The PCR products were confirmed by electrophoresis after staining with ethidium bromide. Clean amplicons (after elution through SephacrylS-300 [GE Healthcare Bio-Sciences, Sweden]) was subjected to cycle (sequencing) PCR using each of the primer and a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster, CA, USA). Cycle (sequencing) PCR condition was: initial denaturation (96 °C, 1 min); followed by 25 cycles of denaturation (96 °C, 10 s); annealing (50 °C, 5 s), and extension (60 °C, 4 min). The cycle PCR products were purified (elution through Sephadex G-50 [Sigma-Aldrich, Germany]), vacuum-dried, resuspended with Formamide (⁓ 10 µl) and sequenced using the Applied Biosystems 3130xl genetic analyzer. Sequences were compared to those present in the Ribosomal Database Project (RDP) at Michigan State University (Sequence Match (msu.edu)), and aligned with the CLUSTALW program. Phylogenetic trees were obtained using the Mega Software (Mega 11; Tamura et al., 2021), for the neighbor-joining method. The confidence of the phylogenetic trees was analyzed by the bootstrap method (resampling value was 1000). All 16S rRNA gene sequences were deposited in the DDBJ/EMBL/GenBank databases under accession numbers LC681799-LC681830.
Crude oil degradation in N2-free chemically defined medium supplemented with Hg
Seven bacterial strains, identified as Bacillus sp., showing luxuriant growth in LB broth supplemented with 160 µmol l-1 HgCl2, were selected for crude oil degradation assay and Hg sequestration analysis. Triplicate 250-ml flasks containing 50 ml of N2-free MSM supplemented with sterile HgCl2 solution (40 µmol l-1) and crude oil (1%) as sole source of carbon and energy were prepared to mimic a N2-limiting ecosystem that is co-contaminated with HMs and PHs. The flasks were inoculated with washed bacterial cells (1 ml) and incubated at 30 (±2) oC with shaking (50 × g; 18 d). Growth-associated depletion of crude oil was determined by gas chromatography equipped with flame ionization detection (GC-FID) analysis of residual crude oil upon extraction with hexane (Obayori et al., 2009; Oyetibo et al., 2017). Control experiments and Hg sequestration assay were as explained in the earlier Section.
Statistical analyses
All experiments, readings and analyses were in triplicates. All values represented mean of triplicate experimental data. Column statistics, row statistics, and one-way ANOVA at 95 % confidence were determined using GraphPad Prism 5 (GraphPad Software, Inc., Avenida de la Playa La Jolla, CA., USA).