Sample collection and heavy metals analysis
Wastewater samples were collected from the textile effluents. Six main drains present in and around Faisalabad, Pakistan receiving the textile effluents and surrounding different textile units were selected. From each drain, five samples were taken keeping the distance of about 1000 m between two points (Baby et al. 2014). After collection, samples were processed for determination of heavy metals i.e. cobalt (Co), chromium (Cr), nickel (Ni), lead (Pb) and zinc (Zn). Samples were digested by following the protocol as previously described by Sinha and Paul (2014) and metal analysis was done using Atomic Absorption Spectrophotometer (AAS) (Hitachi Polarized Zeeman AAS, Z-8200, Japan) following the conditions described in AOAC (1990). Based on the results of metal analysis, nickel (Ni) and cobalt (Co) were selected for further study.
Isolation of HMT bacteria
Ten fold serial dilutions of effluents were prepared in sterile distilled water up to 10−5 as described by Lucious et al. (2013). Isolation of Ni and Co tolerant bacteria was done through spread plate method as described by Samanta et al. (2012).
Determination of maximum tolerable concentration (MTC)
The highest concentration that allowed visible bacterial growth after 48 to 96 h of incubation was used to select MTC of heavy metal. The increasing concentration of both heavy metals (Ni and Co) i.e..5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10 mM were filter sterilized first and then added into nutrient agar which was sterilized through autoclaving and cooled at room temperature for testing the MTCs of bacteria (Vashishth and Khanna 2015).
Multi metal resistance (MMR)
Multi metal resistance of bacteria was determined by inoculating autoclaved and cooled nutrient agar medium incorporated with filtered sterilized solutions of Ni, Co and Cr in equal concentration i.e. 1:1:1 as described by Saini and Pant (2016).
Identification of bacteria
After 48 h of incubation, colonies were selected on the basis of morphology, shape and color. All the isolates were purified by repeated streaking on nutrient agar and stored at 4 °C for further studies. Identification up to genus level was done on the basis of cultural characteristics, microscopic examination after Gram’s staining; “shape, arrangement and staining character”, and physiological/biochemical characteristics; “motility, oxidase reaction, catalase reaction, glucose utilization and fermentation tests and starch hydrolysis”. All identification tests were performed following the protocols mentioned in Bergey’s Manual of Determinative Bacteriology (1994).
Molecular characterization
Ribotyping was done for the molecular characterization of identified HMT bacteria by amplifying 16S rRNA gene. Total genomic DNA was extracted by CTAB method (Wilson 2001). Polymerase Chain Reaction (PCR) was used for the amplification of 16S rRNA using 16S rRNA PCR primers, PA (5′-AGAGTTTGATCCTGGCTCAG-3′, and PH (5′-AAGGAGGTGATCCAGCCGCA-3′) (Zaheer et al. 2016). For ribotyping, all the isolates were grown in Luria–Bertani (LB) broth and total genomic DNA was extracted. PCR products were eluted using a gel extraction kit (Fermentas, Germany) and sent for commercial sequencing (Eurofins MWG Operon LLC, USA). After amplification, 16S rRNA sequences were compared with known sequences in the GenBank database.
Phylogenetic analysis
The 16S rRNA gene from the pure culture sequences from the NCBI database were aligned using Clustal X (Thompson et al. 1997) and the maximum likelihood (ML)-based phylogenetic tree was constructed using MEGA (version 6) (Tamura et al. 2013). Confidence in the tree topology was evaluated using bootstrap resampling methods (1000 replications), and bootstrap values of up to 90% that demonstrated good support measures were retained.
Effect of Ni and Co on bacterial growth
To observe the effect of Ni and Co on bacterial growth, growth curve experiment was conducted in nutrient broth. For this purpose, nutrient broth tubes with Ni (01 mM), Co (01 mM) and without Ni and Co (control) were prepared. For each bacterial isolate 100 ml medium was taken in one set consisting of eight test tubes for all three groups (i.e. two with metals and one control), autoclaved and then inoculated with 20 μl of freshly prepared inoculum. These tubes were incubated in shaking incubator at 37 °C at 100 rpm. Then after 0, 4, 8, 12, 16, 20, 24 and 28 h one tube out of eight in each group was removed and absorbance was measured at 600 nm. Growth curve was plotted by the readings obtained from the experiment and compared (Shakoori et al. 2010).
Evaluation of biosorption potential
Biosorption potential of isolated and characterized bacterial strain named as AMIC1was determined against two metals i.e. Ni and Co. For this purpose, one set (each containing 02 glass culture bottles) having capacity of 500 ml was prepared for each metal supplemented with 200 ml of LB broth with initial metal concentration of 50 ppm for both metals. After autoclaving each set was inoculated with 02 ml of 18-h old bacterial culture having turbidity equal to 0.5 McFarland. Culture bottles were kept under constant agitation at 37 °C for 24 and 48 h then centrifuged at 14,000 rpm for 05 min and supernatants were collected and stored at − 20 °C for heavy metal analyses. Heavy metals present in solution were measured through Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) (Ramyakrishna and Sudhamani 2016).
FT-IR analysis of bacterial biomass
Fourier transform infrared spectroscopy (FT-IR) was used to analyze the functional groups and overall nature of chemical bonds in bacterial strain. Infrared spectra of the control (bacteria grown without metal stress) and tested (bacteria grown with metal stress, Ni or Co) biomass were obtained by grinding 02 mg of freeze-dried biomass with 200 mg dry potassium bromide (KBr) powder (1:100) ratio in mortar. Obtained mixture was pressed to get translucent sample discs using pressure bench press. The FT-IR analysis was performed by using PerkinElmer Spectrum Version 10.4.3. The spectral data were collected over the range of 450–4000 cm−1 (Ramyakrishna and Sudhamani 2016).
Scanning electron microscopy (SEM)
Outer morphology of the bacterial cells before and after biosorption was examined using SEM (Carl Zeiss Supra 55 Gemin; German Technology, Jena, Germany). Prepared samples were placed on the sample holder (stub) with carbon tape. In order to increase the electron conduction and to improve the quality of micrographs, a conductive layer of gold was made with portable SC7620 ‘Mini’ sputter coater/glow discharge system (Quorum Technologies Ltd, Laughton, UK) (Michalak et al. 2014).
Statistical analysis
The data was analyzed by calculating mean ± SE, analysis of variance (ANOVA), regression, co-relation and Z test was performed by using Minitab software. P value was calculated to see the significant results. Results showing P value less than 0.05 were considered as significant (P < 0.05) and whereas P value less than 0.01 as highly significant (P < 0.01).