The biosorption mechanism is an alternative for chemical precipitation and ultrafiltration which have been employed to treat heavy metal contamination with a limited success. In the present study, three species of
Heavy metal(s) are widespread pollutants of environmental concern as they are nondegradable and thus persistent [
Conventional methods like chemical oxidation reduction, adsorption, electrolytic recovery, and so forth are rendered futile due to either financial burden or lack of ecofriendly nature in the remedial process. Despite best human efforts, heavy metals are still increasing in their spread and concentration. This is due to indiscriminate and perilous ways of industrialization in sectors including mining, petrochemicals, and electronics. In 1990s, a new scientific area has developed which could help to recover heavy metals using biological means, that is, biosorption at less expensive manner [
Therefore, in the present study, we have assessed the biosorption ability of
For the isolation of the haloalkaliphilic bacteria, 1.0 g of soil sample was collected from solar salterns by employing standard method of soil sampling [
Genomic DNA extraction was isolated from selected three isolates by following the method described by Sambrook et al. [
To identify bacterial isolate of interest, 16S ribosomal DNA was extracted followed by amplification of 16S ribosomal DNA by PCR employing standard protocol [
The DNA sequences of the 16S rRNA gene from the isolate of interest were edited manually and BLAST searched individually to find out sequences of homology. The sequences were aligned using the programme CLUSTAL W [
Heavy metal biosorption is the ability of bacterial cells or components to adsorb, chelate, or precipitate metal ions in the solution into insoluble particles or aggregates which can be removed either by sedimentation or filtration from the solution.
Stock solutions of the heavy metals were prepared by using copper sulphate, cadmium chloride, and lead acetate of the respective metals to attain maximum solubility of the metal. The stock solutions were prepared with 1000 ppm concentration of respective metal in milli-Q grade deionised water by compensating for the salt/nonmetallic component (copper sulphate 2.5117 g, cadmium chloride 1.6308 g, and lead acetate 1.8307 g) and stored at 4°C. Standard metal solutions for the metal biosorption analysis were prepared by adding 1.0 mL stock solution to 100 mL of the media giving a final concentration of 1000 ppm.
The biosorption of the metals by the isolates was assayed in Erlenmeyer flasks containing 90 mL of metal biosorption medium (NaCl 81.0, MgCl2 7.0, MgSO4·7H2O 9.6, CaCl2 0.36, KCl 2.0, NaHCO3 0.06, NaBr 0.026, yeast extract 5.0, and glucose 3.0 g/L) [
After incubation, the biosorption of respective metal biosorption by the isolates was measured by removing the cells from the medium by centrifuging at 8000 rpm for 20 min. Standard solutions of individual metals were prepared with varying concentrations in milli-Q water. The standard’s absorption of metal solutions was measured by Atomic Absorption Spectroscopy (Shimadzu AA620, Shimadzu, Japan) at wavelengths 324.8 nm, 228.8 nm, and 283.3 nm for copper, cadmium, and lead, respectively. A standard curve was plotted from the absorption of standard metal solutions with concentration against absorption. The supernatant was analysed for residual metal concentration in the bacterial treated and culture free control media. Similarly, the residual metal was also determined by intersecting the absorption of supernatant in the standard curve [
The biosorption capability of bacterial isolates was assayed as above; the biosorption of metal ions was measured by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES optima 8300, Perkin Elmer, Massachusetts, USA). The ICP-OES was calibrated with standard working metal solutions and blank as above to set limits of detection (10–1000 ppm). The emission lines used for the analysis were 327.393 nm, 228.802 nm, and 340.458 nm for copper, cadmium, and lead, respectively, under Argon plasma with the concentric nebulizer. The residual metal concentration was deduced from internal standard curve produced from standardisation before running the samples and culture free control [
The pelleted
A total of 14 bacterial isolates were initially isolated from solar saltern soil samples on modified nutrient agar medium. These 14 bacterial isolates were selected on the basis of cultural characteristics such as colony size, colour, form, margin, and elevation and named as NSPA1, NSPA2, NSPA3, NSPA4, NSPA5, NSPA6, NSPA7, NSPA8, NSPA9, NSPA10, NSPA11, NSPA12, NSPA13, and NSPA14. The biochemical characters based on which the isolates were selected for further analysis are presented in Table
Biochemical characteristics of the selected isolates.
Biochemical characters | NSPA1 | NSPA2 | NSPA3 | NSPA4 | NSPA5 | NSPA6 | NSPA7 | NSPA8 | NSPA9 | NSPA10 | NSPA11 | NSPA12 | NSPA13 | NSPA14 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Nitrate reduction | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
Citrate utilisation | + | + | + | + | + | + | + | − | + | + | − | + | + | − |
H2S production | − | − | − | − | − | − | − | − | − | − | − | − | − | − |
Indole | − | − | − | − | − | − | − | − | − | − | − | − | − | − |
Methyl red test | + | + | − | + | − | + | − | − | + | − | + | + | − | + |
Vogesproskauer test | − | − | + | + | + | + | − | + | − | + | − | + | + | + |
Oxidase | − | − | − | − | + | − | − | + | − | − | − | − | + | − |
Catalase | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
Urease | − | + | − | + | + | + | + | − | − | − | − | + | − | − |
Starch hydrolysis | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
Cellulase hydrolysis | − | + | + | − | + | + | + | + | + | + | + | + | + | + |
Lipid hydrolysis | − | − | − | − | − | − | − | − | − | − | − | − | − | − |
Casein hydrolysis | − | − | − | − | + | − | − | + | − | − | − | − | + | − |
Gelatin liquefaction | − | − | − | − | + |
− | − | + | − | − | − | − | + |
+ |
Sucrose | − | − | − | − | + | − | − | − | − | − | − | − | + | − |
Fructose | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
Glucose | + | + | + | + | + | + | + | + | + | + | + | + | + | + |
Galactose | − | − | − | − | − | − | − | − | − | − | − | − | − | − |
Lactose | − | − | − | − | − | − | − | − | − | − | − | − | − | − |
“−” negative, “+” positive.
“
The selected potential haloalkaliphilic isolates NSPA5 were taxonomically classified using phylogenetic analysis. The amplified 16S rDNA gene using polymerase chain reaction resulted in a single discrete band of a 1.5 kb size in agarose gel. This amplified PCR product was BLAST searched against NCBI Genbank and RDP (Ribosomal Database Project) database 11.0. A distance matrix was constructed based on nucleotide sequence homology using kimura-2 parameter and phylogenetic trees were made using neighbor-joining method (Figure
Phylogenetic analysis of the isolates based on 16S rDNA sequence analysis. (a) Phylogenetic tree of isolate NSPA5, (b) phylogenetic tree of isolate NSPA8, and (c) phylogenetic tree of isolate NSPA13.
After analysing the treated samples in AAS, the isolate
Metal concentration in the medium determined by AAS after removal of the bacteria.
In this method, the emission spectrum is utilized in analysing the metal biosorption ability of the isolates unlike in Atomic Absorption Spectroscopy. Similar to the AAS results, lead was the maximum adsorbed, evident from reduced initial concentration by 89% (1000 ppm to 103.4 ppm) by
Metal concentration in the medium determined by ICP-OES after removal of the bacteria.
Scanning Electron Microscopy was used to show macro structure of the surface of dry biomass of the bacterial cells. Inca Penta FETx3 energy dispersive X-ray system gave a visible evidence of binding metal ions on the cell wall of bacterial cells. EDS spectral images clearly showed that Cd(II), Cu(II), and Pb(II) ions were adsorbed on the surface of
Energy dispersive X-ray spectroscopic (EDS) analysis for elemental composition of lead on cell surface of isolate
EDS analysis for elemental composition of lead on cell surface of isolate
EDS analysis for elemental composition of lead on cell surface of isolate
Increasing industrialization has resulted in an alarming increase in the discharge of heavy metals and other pollutants into the environment including water resources. Microorganisms have been used to remove heavy metals from the environment by various approaches like bioaccumulation and biosorption, oxidation and reduction, and methylation and demethylation [
Based on the above findings, it was concluded that the isolates,
The authors declare that there is no conflict of interests regarding the publication of this paper.
Authors are thankful to Sri Venkateswara University, Tirupati, India, for their support and encouragement.