Introduction

Industrial revolution has greatly increased the heavy metals, which is a big hazard for human health. Anthropogenic activities convert such metals into various forms that are highly toxic and even persist in the environment for longer duration (Banerjee et al. 2015). Major ecosystem source of such metals is industrial effluents that are being discharged by steel manufacturing, electroplating, chemical processing, and leather tanning units into the surrounding land and water resources (Tian et al. 2012). Although small concentrations of some metals such as iron, zinc, copper, and manganese are required as essential micronutrients for rapid growth, maintenance of protein structure, and act as enzyme co-factor by various life forms, others are considered most toxic and persistent environmental pollutants causing multiple cellular alterations (Deeb and Altalhi 2009; Reis et al. 2014).

Immense release of heavy metals during the last few decades has increased the world concerns about their removal (Ren et al. 2009). Industries discharge their polluted waste rich in heavy metals such as Pb, Cr, Ni, and Cd into the nearby streams. Such harmful wastewater is being supplied to agricultural land, causing long-term effects on plants, animals, and humans health (Takahashi et al. 2012; Olaniran et al. 2013). For instance, elevated concentrations of lead and cadmium intake in human have resulted in many kidney disorders and disturbance in endocrine system (Jiménez-Ortega et al. 2012). Moreover, increased nickel amount has been linked to various respiratory and skin problems (Thyssen et al. 2013). To resolve these problems, several methods have been introduced to remove these contaminants from environmental compartments (Bibi et al. 2014; Oyewo et al. 2018).

Although various physicochemical techniques such as chemical precipitation, ion exchange, electrochemical treatment, membrane technologies, and adsorption on activated sludge (Shim et al. 2014; Eiband et al. 2014; Gupta et al. 2015) have been developed for eradicating excess amount of heavy metals (Beveridge et al. 1997; Wang and Chen 2009), these methodologies are highly expensive, less environment friendly, and sometimes ineffective at low concentrations. Moreover, these processes require many reagents but result in incomplete metal removal and generate highly toxic sludge (Sibi 2016), whereas bioremediation processes involve the use of bacteria, fungi, or plants for the removal of pollutants in a natural and economical way (Marc et al. 2012). Research reports addressing bioremediation processes reflect the potential application of living or dead biomass to remove considerable amounts of these toxic metals from different compartments of an ecosystem (OK et al. 2007; El-Gendy and El-Bondkly 2016). Similarly, metal sorption could be considered as it involves rapid surface binding of metal ions to either bacteria or any other life form (Bayramoglu and Arıca 2008; Huang et al. 2013). While bioaccumulation is an active mode of metal uptake by living cells, this process generally depends upon metal speciation and its bioavailability. The cell’s ability to bioaccumulate largely depends upon their metabolic activities and various physiological, biochemical, genetic, and structural adaptations depending upon the surrounding ecosystem (Algarra et al. 2014). Aditionally, environmental conditions such as temperature, pH, and biomass concentration always have a significant impact upon the metal ions uptake capabilities of living cells (Uslu and Tanyol 2006; Ojuederie and Babalola 2017).

Industrial effluents served as a potential source for isolating microbes capable of tolerating and proficiently remediation contaminants. Bacterial cells have multiple surface binding sites for metal cations and possess metal-resistant phenotypes which support their use for environmental protection. For instance, Escherichia hermannii and Enterobacter cloacae uptake Cd and Ni, Bacillus thuringiensis can accumulate copper, Bacillus cereus sorbed lead proficiently, and Pseudomonas aeruginosa act as metallophore to eradicate metals Cd and Pb from the effluent (Selvi et al. 2012; Poornima et al. 2014: McFarlane et al. 2018).

In the present study, bacterial isolates were used to check their metal remediation potential. Many strains showed varied abilities to resist metals. Among these, Klebsiella pneumoniae MB361, Stenotrophomonas sp. MB339, and Staphylococcus sp. MB371 showed resistance towards higher concentration of metals which were selected for evaluating their bioaccumulation abilities.

Materials and methods

Isolation and screening of isolates

Industrial effluent samples were collected from Hattar Industrial State, Pakistan. The physicochemical analysis of samples was done using standard techniques and stored at 4 °C for isolation of bacterial strains. Isolation of bacteria was done by performing standard spread plate method of different industrial effluent samples. Initially 50 μl of each effluent sample was plated with mixed metals (Pb, Cr, and Ni) supplemented nutrient agar plates, incubated at 37 °C for 24 h. Further screening was done by plating the selected colonies on M9 minimal medium plates with different concentrations of selected metal salts, i.e., PbCl2, K2CrO4, and NiCl2. The minimal agar medium M9 of composition Na2HPO4 (6 g), KH2PO4 (3 g), NH4Cl (1 g), and NaCl (0.5 g) per liter of distilled water was prepared and the pH was adjusted to 7.04. After autoclaving, CaCl2 (0.1 M), MgSO4 (1 M), and casein hydrolysate (5 g) were added to the medium under sterilized conditions. The freshly grown culture was inoculated in M9 broth with 50, 100, 150, 200, and 250 μg/ml of each metal and incubated in a rotary shaker (150 rpm) at 30 °C for 24 h. The growth was determined by checking optical density of the medium at 600 nm using spectrophotometer. The growth of each strain declined with increasing concentration of metal salts. However, the isolated MB339, MB361, and MB371 showed maximum relative growth of more than 80% at 200 μg/ml. Thus, these three most promising strains were purified by three rounds of single colony streaking.

Morphological, physiological, and biochemical characterization

The cell and colony morphology of purified colonies grown on nutrient agar plates was studied. Furthermore, investigation of culture growth pattern at 25, 30, 37, and 45 °C and in the medium of pHs 5 to 11 was made in nutrient broth. Biochemical tests, including catalase, oxidase, and nitrate reductase, were performed using standard methods. In addition, isolates were tested for enzymes tryptophan deaminase, lysine decarboxylase, arginine dihydrolase production, and utilization of sugars glucose, sucrose, maltose etc. The biochemical tests were performed following Bergey’s manual of Descriptive Bacteriology (Holt et al. 1994).

DNA extraction and identification of bacterial strains

These isolates were then identified through 16S rDNA sequencing. The bacteria were cultured in LB broth at 30 °C in shaking incubator for 24 h. After extraction of genomic DNA, 16S rRNA gene was amplified by PCR using universal primers: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR product was then purified with PureLink™ Quick PCR Purification Kit (Invitrogen) and run on 0.8% agarose gel as described by Aslam et al. (2018). The 16S rRNA gene sequences obtained were compared with known sequences from the NCBI database with BLASTN algorithm. These sequences were aligned with ClustalW and phylogenetic tree was constructed by the neighbor-joining method using Molecular Evolutionary Genetic Analysis MEGA 6 software (Tamura et al. 2013).

Heavy metal accumulation studies

The selected isolates MB339, MB361, and MB371 were grown in 200 μg/ml of each metal-supplemented broth at 37 °C with 150 rpm. After every 24-h incubation, bacterial cells were harvested by centrifugation and suspended in 1 ml of distilled water. The metal content was determined after acid dissolution of bacterial cell as described by Ganje and Page (1974). Concentrations of selected heavy metals accumulated were measured in μg/ml (1 ppm) units at specific wavelengths 217 nm for Pb, 232 nm for Ni, and 357.9 nm for Cr for each metal by flame atomic absorption spectrophotometer (Spectra AA 220, Australia).

Percentage bioaccumulation was then calculated as follows:

$$ \%\mathrm{Bioaccumulation}=\frac{\left(\mathrm{Initial}\ \mathrm{metal}\ \mathrm{concentration}\hbox{-} \mathrm{Final}\ \mathrm{metal}\ \mathrm{concentration}\right)}{\left(\mathrm{Initial}\ \mathrm{metal}\ \mathrm{concentration}\right)}\times 100 $$

Effect of various factors on metal (Cr, Ni, and Pb) accumulation

Bioaccumulation potential of three isolates was assessed at five different temperatures 25, 30, 37, 40, and 45 °C and pHs 6, 7, and 8. Nutrient broth (10 ml) was prepared in sterilized conditions supplemented with 200 μg/ml of Cr, Ni, and Pb and inoculated with 50 μl of fresh bacterial cultures.

Effects of six different inoculum sizes (50, 100, 200, 300, 400, and 500 μl) and five concentrations between 100 and 500 μg/ml of each metal were also used. Furthermore, impact of incubation time (24, 48, and 72 h) in shaking incubator at 150 rpm at 37 °C was also studied. The percentage of metal accumulated was analyzed through AAS.

Statistical analysis

All experiments were performed in triplicate. The results are expressed as standard error of mean (±SEM).

Results and discussion

The industrial effluent samples collected were apparently and chemically different from one another. The color observed was blank and yellow with pH value 7.1 and 9.4 having electrical conductivity 653 and 1311 μS/s. Whereas the total hardness, dissolved, and suspended solid concentrations were a little above the permissible limits.

Isolation and characterization of metal-resistant bacteria

In the present study, bacterial isolates that were obtained using selected mixed metals (Pb, Cr, and Cd) supplemented nutrient agar medium were further tested for tolerance against twelve different metals. The bacterial cultures were purified by several rounds of single colony streaking and maintained on nutrient agar medium. Strains MB339 and MB361 were gram-negative, while MB371 was gram-positive. Isolates showed optimal growth between 30 and 37 °C. Isolates MB361 and MB371 were neutrophilic whereas MB339 exhibited slightly alkalophilic nature (Fig. 1). After the screening, isolates MB339, MB361, and MB371 tolerating more than 1000 μg/ml of Cr, Ni, and Pb were finally selected for carrying out bioaccumulation studies. The lethal concentration LC50 values for each metal (Pb, Cr, Ni) were assessed using eight different concentrations from 100 to 800 μg/ml. With increasing concentration of each metal, bacterial growth was gradually decreasing. In the case of strain MB339, the LC50 calculated for metal (Pb, Cr) was 400 μg/ml and 300 μg/ml for Ni. For strain MB361, 300 μg/ml for Cr and Ni, while 400 μg/ml for Pb was found lethal concentration. While LC50 value for bacterial strain MB371 was observed at 400 μg/ml for Pb and 300 μg/ml for Ni and Cr, research reports suggested that gram-negative bacteria always had higher metal uptake capacity than gram-positive because active metal-binding groups such as carboxy, sulphuryl, phosphorous, and amino are present in the cell wall (Thomas and Rice 2014). A previous study revealed that gram-negative Escherichia coli K-12 and Pseudomonas aeruginosa more efficiently biosorbed Cu, Cr, and Ni as compared with gram-positive strains Micrococcus luteus (Churchill et al. 1995). However, Kaewehai and Prasertson (2002) demonstrated that Enterobacter agglomerrans SM 38, Bacillus subtilis SM 29, and Bacillus subtilis WD 90 proficiently accumulate cadmium and nickel.

Fig. 1
figure 1

Growth of bacterial strains at different temperatures and pHs

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On the basis of biochemical characterization, bacterial strains were distinctly different from one another. Both MB361 and MB371 showed positive Voges-Proskauer test, nitrate reduction, catalase, and oxidase activity. These two strains were able to utilize citrate with the ability to produce urease and tryptophan deaminase. These could not hydrolyze gelatin or produce indole, but could utilize all types of sugars including glucose, mannitol, inositol, sorbitol, rhamnose, saccharose, and melibiose.

On the basis of 16S rRNA, strains were identified as Stenotrophomonas sp. MB339 (KP723528), Klebsiella sp. MB361 (KP723532), and Staphylococcus sp. MB371 (KP723530). Phylogenetically, neighbor-joining algorithm showed high similarity of strain MB339 with Stenotrophomonas sp. as described in Aslam et al. (2018). Similarly, in the case of Klebsiella pneumoniae MB361, the strain showed 99% similarity with other Klebsiella pneumoniae MB369, Klebsiella pneumoniae nucleomorph CEES16, and others given in Fig. 2. The strain Staphylococcus sp. MB371 was found to be highly similar (99%) to Staphylococcus sp. SQ7-5-9, A9, BAB-4167 and different strains of Staphylococcus sciuri.

Fig. 2
figure 2

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of Stenotrophomonas sp. MB339, Klebsiella pneumoniae MB361, and Staphylococcus sp. MB371. The sequences were aligned with ClustalW to construct tree with bootstrap value of 1000 replicates in MEGA6.0

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Effect of incubation temperature on metal bioaccumulation

The results obtained from experiments performed at different incubation temperatures showed that all the three strains accumulated Cr maximally, ranging from 37 to 40 °C (Fig. 3). Most effectively Stenotrophomonas sp. MB339 accumulated 65.85% Cr at 37 °C. In the case of Ni, all three isolates achieved optimum metal uptake at 30 °C which gradually decreased with increasing temperature up to 45 °C. Same trend of percentage Pb accumulation was recorded by MB339, MB361, and MB371 strains. Temperature is known to affect the stability of the cell wall and its configuration. Bacterial strains normally remove maximum metal at 30 °C like Acromobacter sp. and Pseudomonas stutzeri could uptake 82% copper effectively (Olmezoglu et al. 2012). On contrary to this, higher temperatures generally lead to increase in metabolic activity along with an increase in energy of the system that would result in active uptake of metals. For instance, the rate of Cr (VI) removal by S.cerevisiae was very high at 45 °C (Goyal et al. 2003; Banerjee et al. 2015).

Fig. 3
figure 3

Effect of different temperatures on percentage bioaccumulation of Cr, Ni, and Pb. Horizontal bars represent standard error of mean

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Effect of pH on metal bioaccumulation

Under natural conditions, the presence of heavy metals in wastewater greatly affects its pH (Abourached et al. 2014). Therefore, experiments were performed in order to evaluate the effect of varying pH on metal uptake. The outcomes of this study demonstrated that percentage bioaccumulation of Cr and Ni was increased with increasing pH (Fig. 4). Strain MB339 showed maximum 85.3% of Pb being sorbed at pH 7, while acidic pH significantly reduced Ni accumulation. Potential of cells to uptake metal ions varies with the type of adsorbent (biomass) and adsorbate (metal ions). At low pH (below 7), bioaccumulation percentage decrease due to hydronium ions occupying the binding sites offering repulsion in contrast to higher pH values where more ligands carrying negative charge are exposed providing space for metal ions to biosorb on cell surfaces (Dexilin et al. 2007). The decrease in removal of Pb(II) in alkaline medium is owing to the formation of Pb(OH)2 precipitates (Colak et al. 2011; Sahu et al. 2015). Bacillus thuringiensis OSM29 had greater sorption of Cd and Cu at low pH values (Oves et al. 2013). On contrary to this, Masood and Malik (2011) observed more Cr removal by Bacillus sp. FM1 because of increasing metal anions as in the case of Cr(VI) in the form of chromate ion (CrO4−2).

Fig. 4
figure 4

Effect of pH on percentage bioaccumulation of Cr, Ni, and Pb. Error bars represent standard error of mean

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Effect of initial metal concentration on bioaccumulation

Pb, Ni, and Cr accumulation of bacterial isolates was observed at five different metal concentrations, i.e., 100, 200, 300, 400, and 500 μg/ml. From the results, it can be inferred that percentage of metal accumulation of selected bacterial isolates decreased with increasing concentration of respective metals (Fig. 5). Staphylococcus sp. MB371 could efficiently accumulate 71.45% of Cr; however, a little less than that (68.54% and 65.98%) was observed in Stenotrophomonas sp. MB339 and Klebsiella pneumoniae MB361 respectively, when 100 μg/ml concentration of Cr was provided in the medium. This percentage decreased in a regular manner with increasing amount of Cr. More than 50% of 100 μg/ml Ni and Pb were accumulated by three strains which then reduced to less than 10% when the provided concentration was raised to 500 μg/ml. The decrease in bioaccumulation with increasing metal concentration may be attributed to saturation of adsorption sites (Huang et al. 2014). At a lower concentration, all the metal ions in solution could interact with the available binding sites resulting in higher bioaccumulation (Pandiyan and Mahendradas 2011; Ergul-Ulger et al. 2014).

Fig. 5
figure 5

Effect of different concentration on percentage bioaccumulation of Cr, Ni, and Pb and error bars represent standard error of mean

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Effect of inoculum size on metal bioaccumulation

All bacterial strains exhibited lowest chromium accumulation at 50 μl, whereas highest chromium accumulation was observed at 500 μl. Klebsiella pneumoniae MB361 exhibited 18.24 and 83.51% accumulation in 50 and 500 μl inoculum size, respectively (Fig. 6). Ni uptake was almost doubled when inoculum was increased from 50 (13.97%) to 500 μl (26.97%) by Stenotrophomonas sp. MB339 after 24-h incubation. A maximum of 56.7% Pb was removed by Stenotrophomonas whereas 48.17% and 38.46% were accumulated by Klebsiella pneumoniae MB361 and Staphylococcus sp. MB371 respectively, with 500 μl of inoculum. Effect of biomass on the biosorption process was investigated by other researchers for both B. cereus and B. pumilus and it was found that Pb(II) uptake increased as the inoculum size increased (Colak et al. 2011). Previous studies revealed that augmented biomass resulted in an increase in biosorption due to enlarged surface area of the biosorbent, which in turn enhances the number of binding sites (Devika et al. 2014).

Fig. 6
figure 6

Effect of inoculum size on percentage bioaccumulation of Cr, Ni, and Pb with error bars representing standard error of mean

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Effect of incubation time on bioaccumulation

Contact time is of immense significance in biosorption process (Arivalagan et al. 2014). Results depicted that as the contact time increased, there was a drastic increase in capability of isolates to bioaccumulate metals (Cr, Ni, and Pb). Highest potential to remove 200 μg/ml Cr from the medium was shown by Stenotrophomonas MB339 after 72 h. More than 30% of Cr removal took place by other two strains Klebsiella pneumoniae MB361 and Staphyloccocus sp. MB371 after 24 h and 72 h that rose to 64.52 and 65.24%, respectively (Fig. 7). A similar pattern was observed in all three strains for Ni with maximum accumulation 63.19% by Stenotrophomonas sp. MB339. Strain MB339 accumulated 27% of initial concentration of Pb after 24 h which increased to 72% with an increase in incubation time, i.e., up to 72 h.

Fig. 7
figure 7

Effect of incubation time on percentage bioaccumulation of Cr, Ni, and Pb and error bars represent standard error of mean

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The high rate of bioaccumulation was due to the greater affinity of free metal ions to the binding sites available at adsorbent and after attaining equilibrium, the percentage biosorption started to decrease. In this context, Gabr et al. (2008) concluded that initial short contact time is relatively more important to attain a higher rate of biosorption as in the case of Ni and Cd. Odokuma and Series 2012) reported a rise in metal uptake with increase in contact time for Bacillus sp., Pseudomonas sp., and Aeromonas sp. Initially the cells are not dividing rapidly or in lag phase, hence exhibit less binding sites that is why metal uptake by isolates was of no consequence (Hossain and Aditya 2013). Following this period, metal uptake as well as cell growth is amplified depending upon the availability of active binding sites. Similarly, slower rate of biosorption at short incubation time become doubled with increase in contact time up to certain limit (Abd-Alla et al. 2012; Barka et al. 2013).

Conclusion

Use of microorganisms for removal of toxic contaminants from environment is a method of potential application. In this study, the ability of bacteria to remediate heavy metals from aqueous solution was determined. Klebsiella pneumoniae MB361 exhibited the highest accumulation (83.51%) with initial concentration of 200 μg/ml Cr at 37 °C for 24 h, while 85.30% of Pb and 48.78% of Ni were accumulated by Stenotrophomonas sp. MB339 at neutral pH, 37 °C and with maximum inoculum size. Present investigation also highlighted the significance of various factors in the process of bioaccumulation. Bacteria could be efficient, promising, and prospective microorganisms for the removal of Ni, Cr, and Pb from aqueous solution, owing to their significant biosorption capacity and environmental friendly nature. These findings serve as a foundation for further exploitation of bacterial ability to remediate metals.