In the current scenario of the need for cost-effective remediation, our study aimed to assess the remedial potential of bacteria obtained from metal-rich wastewater. To simulate the conditions, we prepared wastewater containing five toxic metals (Cu, Cr, Ni, Fe, and Pb). Two types of metal-resistant bacteria were isolated from a prominent wastewater drain in Lahore, Pakistan. These isolated bacteria were thoroughly characterized, both phenotypically and genotypically. Subsequently, the isolated bacteria were exposed to the wastewater solution containing each of the aforementioned metals at a concentration of 250 ppm. The exposed isolates were then incubated for a duration of 15 days. After 5 days, we measured the uptake of metals by the bacterial isolates. Following the 15-day incubation period, we observed that the bacterial isolates demonstrated the maximum efficiency in removing metals, with approximately 47.5% of Fe, 77% of Ni, 75.75% of Cu, 64% of Cr, and 82.5% of Pb being removed. These findings have significant implications for the development of environmentally friendly and cost-effective strategies for metal ion remediation.

  • Assess the remedial potential of bacteria obtained from metal-rich wastewater.

  • Isolated bacteria were thoroughly characterized, both phenotypically and genotypically.

  • Bacterial isolates demonstrated maximum efficiency in removing metals, with approximately 47.5% of Fe, 77% of Ni, 75.75% of Cu, 64% of Cr, and 82.5% of Pb being removed.

Environmentalists are dealing with the alarming accumulation of persistent and non-degradable contaminants like heavy metals in the environment. Heavy metals have widespread usage in modern-day industry, i.e. fertilizer, tanning, plastic/paper manufacturing, energy/fuel production, and other metallurgical processes (Kumar et al. 2023). Metals that have an atomic density of more than 5 g per cm3 are considered heavy metals (Ali & Khan 2018). Albeit some of the heavy metals are essential for all life forms on earth, longer exposure to high concentrations of heavy metals can be toxic (Ghuge et al. 2023). Zn, Ni, Mn, Cu, and Co are required as trace minerals for the optimal growth of microbes; but at higher levels, they exert lethal effects on various organisms and human health (Isik et al. 2022; Kumar et al. 2023), whereas there are some other metals that do not have any physiological role and are deadly poisonous, even in very low quantities (Sundseth et al. 2017).

During the last three decades, efforts have been made to deal with environmental pollution caused by heavy metals. Decontamination of heavy metals from industrial and household sewage has been a major concern, and several methods, i.e. electroplating, precipitation, and ion-exchange methodology, have been devised to cope with pollutants. Although all of these methods have their own efficacy, yet at the same time, they have some cons like the production of toxic sludge/slurry. In modern-day science, biosorption provides an excellent remedial strategy having low operating costs with minimum environmental problems. In biosorption, microorganisms are utilized for the efficient uptake of metallic ions from agriculture and industrial wastewater (Ghaffar et al. 2023).

Some species of bacteria, algae, fungi, and yeast reportedly have the ability to tolerate varying levels of metals and decrease the amount of metals in aqueous solution. Microorganisms uptake heavy metal ions which are followed by the efflux of similar ions: these processes normally comprise of oxidation and reduction reactions, depending on the metal. It is also evident that several bacteria consume heavy metals for acquiring growth and energy through their extraordinary metabolic pathways, in which their enzymes specifically break down heavy metals (Ramírez Calderón et al. 2020). Different microbes having different tendencies toward heavy metals correspondingly perform significant roles in the removal of heavy metals via biogeochemical cycles (Ghaffar et al. 2023).

Microbes have become resistant to different toxic heavy metal ions by adopting various ways, such as bioaccumulation, biosorption, biomineralization, and biological transformation, to live successfully in highly contaminated environments (Priya et al. 2022). The use of microbes to treat heavy metal pollution is preferred over traditional techniques such as flocculation, chemical oxidation or reduction, ion replacement, coagulation, evaporation, reverse osmosis, etc., because of their insane operating costs (Priya et al. 2022). There are some factors that affect the sorption process, i.e. concentration of metal ions in solution, temperature of the solution, pH, etc. (Nilanjana et al. 2007). Bacteria are resistant toward heavy metals and are capable of continuing their life cycle even in high concentrations of heavy metals (Issazadeh et al. 2013; Yabalak et al. 2022). Pseudomonas, Mycobacterium, Escherichia coli, Bacillus, and Streptomyces have shown great efficiencies toward heavy metals (Sharma 2021; Ramli et al. 2022). The current study is aimed at the isolation of a metal-resistant bacterial strain from a highly-polluted wastewater channel (Hadiara Drain) and employment of the isolated strain for the efficient removal of some toxic heavy metals from artificially prepared wastewater.

Sampling and description of sampling site

The wastewater samples were collected from a pollution-rich channel of Hudiara Drain, Mohlanwal, Lahore, Pakistan (31° 24′ 35N 74° 8′ 27E). Wastewater from various industries, having a bulk variety of heavy metals, passes through the drain. This drainage channel ends in River Ravi, thus it plays a significant role in polluting the river's water. Therefore, Hudiara Drain is considered as a hotspot of pollution-resistant microbes. The wastewater samples were collected from six different localities of the drain in fresh sterile vials under sterilized conditions. Physical parameters such as pH and temperature were noted during sampling and were 7.3 and 32.4 °C, respectively. These samples were safely transported to the Applied and Environmental Microbiology Laboratory, Institute of Zoology, University of the Punjab, Lahore, Pakistan. The vials were placed in a refrigerator till further processing.

Physicochemical characterization of the collected samples

Physicochemical parameters of the samples were measured before isolating the microbes. Physical parameters include temperature, pH, electrical conductivity, total dissolved solids (TDS), total volatile solids (TVS), sulfates, phosphates, chlorides, carbonates, and bicarbonates. The parameters were measured according to the methods illustrated by Garg et al. (2001).

By using a digital pH meter (pH7110, Germany) and a digital thermometer, the pH and temperature of the samples were measured, respectively. The electrical conductivity was determined using a digital conductivity meter (UK) and readings were noted in S/m. For measuring TDS, the known volume (V) of samples was poured into Petri plates. The weight of the empty Petri plate (A) was measured before adding the desired volume of the samples. After adding known volume, the Petri plate was placed on a hot plate at 105 °C. After 2–3 h, the Petri plate was cooled down at room temperature and the final volume of the Petri plate was measured (B):
formula
where A is the weight of petri plate; B is the weight after 2–3 h of evaporation; V is the volume of the sample added in the Petri plate.
The residues of TDS were combusted at 550 °C in a muffle furnace for 1 h. After 1 h, the weight was noted according to Garg et al. (2001):
formula

For measuring the sulfate contents of the samples, the samples were filtered on a fine-quality Whatman's filter paper. Approximately 50 mL of the filtered wastewater sample was taken out and then mixed with a mixture consisting of 10 mL of NaCl–HCl solution and glycerol-ethanol solution. After that, 0.5 g of BaCl2 (Barium Chloride) was added to the mixture with continuous stirring for half an hour. After that, the reading was noted as 420 nm under a digital spectrophotometer (Garg et al. 2001).

The phosphate content of the samples was measured by digesting the wastewater sample with perchloric acid, followed by oxidation using sodium hydroxide (Garg et al. 2001). The chloride content of the samples was measured by titrating chlorides against soluble silver nitrate (AgNO3) in the presence of chromate. The titration resulted in the formation of silver nitrate (AgCl) precipitates. Following titration, free silver reacts with chromate to form sliver chromate, leaving chloride ions later on the precipitates. The end color of titration was reddish brown (Garg et al. 2001):
formula
where N is the titrant's normality.
Carbonates were estimated according to Garg et al. (2001) by titrating the selected sample with a strong acid solution (H2SO4) using phenolphthalein as an indicator:
formula
Bicarbonate contents were estimated following Garg et al. (2001) using methyl orange solution as an indicator:
formula

Isolation and pure culturing of multiple-metal-resistant bacteria

Five toxic metals (Pb, Ni, Fe, Cu, and Cr) in different concentrations were used to isolate multiple-metal-resistant bacteria (Figure 1). Stock solutions of the metals were prepared by using different required concentrations of salts as described in Table 1. Nutrient agar was used as a growth medium for maintaining bacterial growth. The medium was amended with different concentrations of toxic metals to isolate multiple-metal-resistant bacteria. The obtained metal-resistant bacterial isolates were pure cultured by streak-plate method and then characterized phenotypically as well as genotypically.
Table 1

Preparation of different concentrations of five toxic metals from their respective salts

Sr. No.Metals under analysisSalts usedConcentration of metal employed
Metal concentration (ppm)Quantity of salt used (mg/L)
Pb Pb(NO3)2 5,000 
Ni NiSO4 5,000 13.1 
Fe FeSO4 5,000 13.6 
Cu CuSO4·5H25,000 19.53 
Cr CrO3 5,000 9.61 
Sr. No.Metals under analysisSalts usedConcentration of metal employed
Metal concentration (ppm)Quantity of salt used (mg/L)
Pb Pb(NO3)2 5,000 
Ni NiSO4 5,000 13.1 
Fe FeSO4 5,000 13.6 
Cu CuSO4·5H25,000 19.53 
Cr CrO3 5,000 9.61 
Figure 1

Overview of bioremediation of heavy metals.

Figure 1

Overview of bioremediation of heavy metals.

Close modal

Phenotypic characterization of the bacterial isolates

Phenotypic characterization of the bacteria involved motility detection, Gram's, and endospore staining (Figure 2).
Figure 2

Phenotypic characteristics of the isolated strains.

Figure 2

Phenotypic characteristics of the isolated strains.

Close modal

Molecular characterization of the bacterial isolates

To identify the bacteria, we extracted the total genomic DNA from the isolates (Hussain et al. 2014). We used specific primers, namely 27f (50-AGAGTTTGATCMTGGCTCAG-30) and 1492r (50-GGTTACCTTGTTACGACTT-30), to amplify the 16S rRNA genes, which are approximately 1.5 kb in length. Polymerase chain reaction (PCR) was carried out in 50 μL vials. The PCR reaction mixture consisted of 5 μL of DNA extract, 18 μL of DNA-free water, 5 μL of 1× Taq buffer, 2 U/mL of DNA Taq polymerase, 5 μL of each primer (5 pmol), 5 μL of dNTPs (1 mM), and 5 μL of MgCl2 (25 mM). The PCR procedure involved the following steps: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 2 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 2 min.

To visualize the PCR products, we loaded them onto a 1% (w/v) agarose gel containing ethidium bromide in TAE buffer and examined them using an electrophoresis chamber. Subsequently, we purified the PCR products using a Gene Purification Kit. The amplified sequences were then subjected to sequencing using the Big Dye Terminator v3.1 cycle (Macrogen, Korea) at the DNA sequencing facility in Korea. The obtained 16S rRNA sequences were assembled using Phrap (version 0.990319). To determine their similarity with other sequences, we performed a BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/), and the resulting sequences were submitted to GenBank to obtain accession numbers.

Determination of multiple-metals-resistance of the isolate

The bacterial isolate Bacillus sp. was assessed for its resistance against different concentrations of randomly selected five toxic metals. For this purpose, a fresh inoculum of the isolated strain was prepared in nutrient broth. All wastewaters were prepared artificially using salts of the toxic metals. Five thousand ppm stock solutions of Cu, Cr, Ni, Pb, and Fe were prepared by using CuSO4·5H2O, CrO3, NiSO4, Pb(NO3)2, and FeSO4, respectively. Then, stock solutions were further diluted into 250 ppm solutions and inoculated with 5% inoculum of freshly cultured bacterial strain and placed in an incubator at 30 °C. All experiments for the remediation purpose were conducted under batches, in triplicates. Metal-polluted wastewaters, without the addition of bacteria, served as the control. The remedial potential of the bacterial species was determined in a 15-day experimental trial. Samples were withdrawn and filtered for the analysis of metal reduction after an interval of every 5 days. The filtered samples were shifted to the M2 Lab Department of Environmental Sciences, Lahore, for atomic absorption spectroscopic analysis.

Statistical analysis

The obtained data were analyzed by General Linear Model (GLM) procedures using factorial arrangements according to a Completely Randomized Design (CRD). By using Duncan's Multiple Range (DMR), the means of obtained data were separated out by using SAS 9.1. The differences between the means of obtained data were considered significant at P< 0.05.

Physicochemical analysis of the sample

The collected samples were analyzed for different physicochemical parameters by following specific procedures. By using a digital pH meter (pH7110, Germany) and a digital thermometer, the pH and temperature of the samples were measured and found to be 7.3 and 32.4°C, respectively. Organic contents were also present in quantity different from the spring water, but carbonates were absent. The value of TDS, electrical conductivity, and total volatile solids are mentioned in Table 2.

Table 2

Physicochemical characterization of the selected samples

ParametersSamples
pH 7.3 
Temperature 32.4 °C 
Electrical conductivity 4.37 S/m 
Total dissolved solids (TDS) 2,980 mg/L 
Total volatile solids (TVS) 0.07 mg/L 
Sulfates 0.402 mg/L 
Phosphates 0.078 mg/L 
Chlorides 268.38 mg/L 
Carbonates Absent 
Bicarbonates 800 mg/L 
ParametersSamples
pH 7.3 
Temperature 32.4 °C 
Electrical conductivity 4.37 S/m 
Total dissolved solids (TDS) 2,980 mg/L 
Total volatile solids (TVS) 0.07 mg/L 
Sulfates 0.402 mg/L 
Phosphates 0.078 mg/L 
Chlorides 268.38 mg/L 
Carbonates Absent 
Bicarbonates 800 mg/L 

Isolation and phenotypic characterization of the bacterial isolates

Two metal-resistant bacterial strains were isolated from the collected wastewater sample. The bacterial isolates were designated as B-1 and B-2. The phenotypic characteristics of the bacterial isolates are shown in Figure 2.

Genotypic characterization of the bacterial isolates

Sample B-1

A target sequence of 1,224 bp of 16S ribosomal DNA has been obtained using the Sanger sequencing method. When it was BLAST (basic local sequence tool) matched with the already sequenced data available in GenBank of NCBI database, the sequence showed maximum base similarity with Exiguobacterium sp. XT-14 (KR063545), Exiguobacterium profundum (KX233849) and Exiguobacterium profundum (KF928335) that were isolated from China, Bangladesh and Pakistan. All possible 25 closest matches have been mentioned in Table 3.

Table 3

The closest matches to the bacteria isolated from sample B-1

Serial No.Accession No.Species nameCountryQuery cover (%)Identity %
1. KR063545 Exiguobacterium sp. XT-14 China 99 99 
2. JF758868 Exiguobacterium arabatum India 99 99 
3. KX233849 Exiguobacterium profundum Bangladesh 99 99 
4. KF928335 Exiguobacterium profundum Pakistan 99 99 
5. KJ456599 Exiguobacterium sp. NH-Q34 China 99 99 
6. JX987048 Exiguobacterium profundum India 99 99 
7. FJ785505 Exiguobacterium sp. EB408 China 99 99 
8. JN642678 Exiguobacterium sp. YMD-1 China 99 99 
9. HM352336 Exiguobacterium sp. CmLB12 China 99 99 
10. GU815993 Exiguobacterium sp. BCH4 India 99 99 
11. EF108298 Exiguobacterium sp. R China 100 99 
12. LN846823 Exiguobacterium sp. JC358 India 99 99 
13. KX185942 Exiguobacterium profundum Pakistan 99 99 
14. KT074375 Exiguobacterium sp. RB 215 India 99 99 
15. KM873375 Exiguobacterium profundum China 99 99 
16. KM215140 Exiguobacterium profundum China 99 99 
17. KF070180 uncultured bacterium USA 99 99 
18. KF070120 uncultured bacterium USA 99 99 
19. KF269101 Exiguobacterium profundum India 99 99 
20. KC668297 Exiguobacterium sp. E4 Pakistan 99 99 
21. JX112643 Exiguobacterium profundum Pakistan 99 99 
22. FJ785504 Exiguobacterium sp. EB277 China 99 99 
23. AB681514 Exiguobacterium sp. NBRC 101652 Japan 99 99 
24. JN644510 Exiguobacterium profundum India 99 99 
25. JF241394 uncultured bacterium USA 99 99 
Serial No.Accession No.Species nameCountryQuery cover (%)Identity %
1. KR063545 Exiguobacterium sp. XT-14 China 99 99 
2. JF758868 Exiguobacterium arabatum India 99 99 
3. KX233849 Exiguobacterium profundum Bangladesh 99 99 
4. KF928335 Exiguobacterium profundum Pakistan 99 99 
5. KJ456599 Exiguobacterium sp. NH-Q34 China 99 99 
6. JX987048 Exiguobacterium profundum India 99 99 
7. FJ785505 Exiguobacterium sp. EB408 China 99 99 
8. JN642678 Exiguobacterium sp. YMD-1 China 99 99 
9. HM352336 Exiguobacterium sp. CmLB12 China 99 99 
10. GU815993 Exiguobacterium sp. BCH4 India 99 99 
11. EF108298 Exiguobacterium sp. R China 100 99 
12. LN846823 Exiguobacterium sp. JC358 India 99 99 
13. KX185942 Exiguobacterium profundum Pakistan 99 99 
14. KT074375 Exiguobacterium sp. RB 215 India 99 99 
15. KM873375 Exiguobacterium profundum China 99 99 
16. KM215140 Exiguobacterium profundum China 99 99 
17. KF070180 uncultured bacterium USA 99 99 
18. KF070120 uncultured bacterium USA 99 99 
19. KF269101 Exiguobacterium profundum India 99 99 
20. KC668297 Exiguobacterium sp. E4 Pakistan 99 99 
21. JX112643 Exiguobacterium profundum Pakistan 99 99 
22. FJ785504 Exiguobacterium sp. EB277 China 99 99 
23. AB681514 Exiguobacterium sp. NBRC 101652 Japan 99 99 
24. JN644510 Exiguobacterium profundum India 99 99 
25. JF241394 uncultured bacterium USA 99 99 

Sample B-2

Another sequence of 1,224 bp of 16S ribosomal DNA has been obtained by using the Sanger sequencing method. When it was BLAST (basic local sequence tool) matched with the already deposited data in the GenBank of NCBI database, the present sequence showed close resemblance with Bacillus sp. (MG266290), Bacillus sp. (MG266289), and Bacillus sp. (MG266286), all from China. It has been supposed as a new species because its morphological, ecological, and molecular data did not match with the already reported species of the genus Bacillus. The closest matches have been mentioned in Table 4.

Table 4

The closest matches to the bacteria isolated from sample B-2

Serial No.Accession No.Species nameCountryQuery cover (%)Identity %
1. MG266290 Bacillus sp. China 100 99 
2. MG266289 Bacillus sp. China 100 99 
3. MG266286 Bacillus sp. China 100 99 
4. MG266282 Bacillus sp. China 100 99 
5. KY082734 Bacillus firmus China 100 99 
6. KY849422 Bacillus firmus China 100 99 
7. KX783540 Bacillus sp. Argentina 100 99 
8. KY928097 Bacillus firmus India 100 99 
9. KX817957 Bacillus sp. India 100 99 
10. KY672890 Bacillus sp. India 100 99 
11. KY616401 Bacillus firmus India 100 99 
12. KX108985 Bacillus sp. Chile 100 99 
13. KX033474 Bacillus sp. China 100 99 
14. KX033473 Bacillus sp. China 100 99 
15. KX033472 Bacillus sp. China 100 99 
16. KP992901 Bacillus firmus China 100 99 
17. KX242398 Bacillus firmus India 100 99 
18. KX181398 Bacillus sp. BAB-5835 India 100 99 
19. LC094998 Bacillus sp. B17Va Japan 100 99 
20. LC094994 Bacillus sp. A46V Japan 100 99 
21. KU693281 Bacillus firmus Iran 100 99 
22. KU254653 Bacillus firmus South Korea 100 99 
23. KM979152 Bacillus sp. H2-31 China 100 99 
24. KM979080 Bacillus sp. H1-87 China 100 99 
25. KJ544043 bacterium AM0334 USA 100 99 
Serial No.Accession No.Species nameCountryQuery cover (%)Identity %
1. MG266290 Bacillus sp. China 100 99 
2. MG266289 Bacillus sp. China 100 99 
3. MG266286 Bacillus sp. China 100 99 
4. MG266282 Bacillus sp. China 100 99 
5. KY082734 Bacillus firmus China 100 99 
6. KY849422 Bacillus firmus China 100 99 
7. KX783540 Bacillus sp. Argentina 100 99 
8. KY928097 Bacillus firmus India 100 99 
9. KX817957 Bacillus sp. India 100 99 
10. KY672890 Bacillus sp. India 100 99 
11. KY616401 Bacillus firmus India 100 99 
12. KX108985 Bacillus sp. Chile 100 99 
13. KX033474 Bacillus sp. China 100 99 
14. KX033473 Bacillus sp. China 100 99 
15. KX033472 Bacillus sp. China 100 99 
16. KP992901 Bacillus firmus China 100 99 
17. KX242398 Bacillus firmus India 100 99 
18. KX181398 Bacillus sp. BAB-5835 India 100 99 
19. LC094998 Bacillus sp. B17Va Japan 100 99 
20. LC094994 Bacillus sp. A46V Japan 100 99 
21. KU693281 Bacillus firmus Iran 100 99 
22. KU254653 Bacillus firmus South Korea 100 99 
23. KM979152 Bacillus sp. H2-31 China 100 99 
24. KM979080 Bacillus sp. H1-87 China 100 99 
25. KJ544043 bacterium AM0334 USA 100 99 

Biosorption of heavy metals

The isolated bacterial strain showed different biosorption against different metals. The following results have been observed after analysis:

The isolated bacteria absorbed 47.5% of Fe on the 5th day, followed by 38% on the 10th day, and minimum absorption (33%) of Fe was observed on the 15th day of incubation. The results of the present study showed maximum biosorption of Fe (47.5%) during the initial days and the biosorption rate decreased with the passage of time (Figure 3). The bacteria isolated in this study consumed 56.5% of Ni in 5 days, followed by 77% after 10 days, and 58.5% after 15 days of interval. Ni absorption showed a bell-shaped biosorption pattern where the maximum (77%) absorption of Ni was on the 10th day of incubation in Ni-polluted water. The biosorption of Ni by bacteria was significantly (p < 0.05) high at 10-day intervals in 250 ppm metal-polluted water.
Figure 3

Periodic removal of heavy metals at 250 ppm of the added metal after 5 (blue bar), 10 (red bar), and 15 (black bar) days. Please refer to the online version of this paper to see this figure in colour: http://dx.doi.org/10.2166/wrd.2023.059.

Figure 3

Periodic removal of heavy metals at 250 ppm of the added metal after 5 (blue bar), 10 (red bar), and 15 (black bar) days. Please refer to the online version of this paper to see this figure in colour: http://dx.doi.org/10.2166/wrd.2023.059.

Close modal

The biosorption of Cu was found to be the maximum (75.75%) on the 5th day of incubation, followed by 43% after 15 days, and the lowest biosorption 30.5% was observed on the 10th day. The results suggest the maximum utilization of Cu during their earlier days of exposure to 250 ppm of artificially prepared Cu-polluted water. The bacterial isolate showed Cr reduction in a linear fashion, i.e. it did not vary significantly with the passage of time. The maximum biosorption of Cr (64.5%) was observed on the 15th day of incubation followed by 64 and 62.5% on the 5th and 10th day, respectively.

In this study, the biosorption of Pb increased with the passage of time, but it did not vary statistically significantly. The results suggest that Pb utilization remained almost similar throughout, while the maximum Pb biosorption was 85%, which was observed after 15 days, followed by 82.5 and 76.5% after the 10th and 5th days of incubation, respectively. Statistical analysis revealed non-significant differences in Pb utilization by bacteria during intervals of 5, 10, and 15 days.

Industrialization has resulted in the contamination of the environment, by the emission of heavy metals (Figure 4). Lately, bacteria have been considered as a potent source of remediating heavy metal pollution. Biosorption is the most common mechanism used by bacteria to treat metal-polluted wastewaters. In the present study, two metal-resistant bacterial strains were isolated from a heavily polluted water channel that received heavy metals from industries, as observed from previous literature (Parvin et al. 2015; Gupta et al. 2017). Isolated bacterial strains were characterized phenotypically and the results showed consistency with those of the previous work (Abbas et al. 2014).
Figure 4

Accumulation of heavy metals from natural and anthropogenic sources.

Figure 4

Accumulation of heavy metals from natural and anthropogenic sources.

Close modal

The physicochemical parameters observed in this study showed a resemblance to the previous work done by Ahmed et al. (2007). The incubation period played a role in the efficiency of biosorption bacterial strain that showed a maximum reduction on the 5th day of the incubation period for Fe and Cu, and on the 10th day for Ni. While for Cr and lead, maximum metal reduction was observed on the 15th or final days of the trial. Malik (2004) explained that during the exponential growth phase, metabolically active bacterial cells are involved in rapid biosorption of metallic ions to the negatively charged sites present on the cell wall. The rapid biosorption was further assisted by a slower energy-dependent entry into the cell (Malik 2004).

The bacterial strain under study leads to the reduction of heavy metals present in the aqueous samples by biosorption at different rates. During the present study, the maximum absorption of Pb was 23.5% and the lowest was recorded as 12%, while a Pb reduction of 35.77% was observed by Paenalcaligenes hominis, followed by Proteus mirabilis at a value of 32.75% (Sanuth & Adekanmbi 2016). However, 100% Pb removal was recorded by Ilhan et al. (2004) and Kumar et al. (2010) calculated 93% reduction by heavy metal acclimated Staphylococcus species, respectively. Recently, Zhou et al. (2023) employed phosphate solubilizing bacteria along with biochar that showed significant removal of lead. Shan et al. (2023) have reported that microbes can be efficiently used for the remediation of lead-polluted agricultural wastewaters. Peng et al. (2023) employed mixed bacteria passivation for the removal of metals such as cadmium, lead, and arsenic, and the results showed a maximum biosorption for arsenic.

Mathiyazhagan & Natarajan (2011) reported 7.01 and 6.68% Cr reduction by Thiobacillus and Pseudomonas species, respectively, which is much lower than that recorded during the present study, i.e. 36%, and results are in accordance with the findings of Sanuth & Adekanmbi (2016). Sun et al. (2023) reported that Deinococcus wulumuqiensis has tolerance against Cr up to 60 mg/L. Le et al. (2023) enhanced Cr reduction by combining Paraclostridium bifermentans G3 with cadmium sulfide nanoparticles. During the present study, the highest absorption of Ni was recorded as 43.5 and 16.5% was the minimum absorption. Öztürk et al. (2004) reported that the uptake of Ni2+ was a metabolism-independent passive binding process. An interesting result was found by Tsezos et al. (1996) that biosorption of Ni2+ was always lesser as compared to the other metals, and that it might be due to the steric hindrance by intrinsic chemical property (Tsezos et al. 1995). However, strains of Pseudomonas spp. have often been used for the biosorption of Ni2+ (Ramteke 2000; Malik 2004), where Ni2+ was biosorbed rapidly on cell surfaces. Li et al. (2022) reported that the immobilized microorganism group (that contained biochar and Citrobacter sp.) showed more reduction than the free bacteria in Ni-polluted soils. During the present study, the highest absorption rate of Cu was recorded as 24.25% and the lowest as 12%, respectively. Öztürk et al. (2004) observed that the initial metal ion absorption was directly proportional to the concentration of Cu2+. Several organisms showed sensitivity to Cu toxicity (Gordon et al. 1994) because of hydroperoxide radicals (Rodriguez-Montelongo et al. 1993) whose characteristics made them highly toxic (Nies 1999). During the current study, the maximum percentage of Fe observed was 52.5 and the lowest recorded was 24.5%, respectively. The current study has revealed that Bacillus sp. showed a varied tolerance against all five metals with a maximum reduction of lead, i.e. 85%. Hence, Bacillus sp. can be a proficient microbe for the remediation of metal-polluted wastewaters containing multiple containments.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

Abbas
S. H.
,
Ismail
I. M.
,
Mostafa
T. M.
&
Sulaymon
A. H.
2014
Biosorption of heavy metals: A review
.
J. Chem. Sci. Technol.
3
(
4
),
74
102
.
Ahmed
M.
,
Idris
A.
&
Omar
S. S. R.
2007
Physicochemical characterization of compost of the industrial tannery sludge
.
J. Eng. Sci. Technol.
2
(
1
),
81
94
.
Garg
S. K.
,
Bhatnagar
A.
,
Kalla
A.
&
Johal
M. S.
2001
Experimental Ichthyology
.
CBS
,
New Delhi
.
Ghuge
S. A.
,
Nikalje
G. C.
,
Kadam
U. S.
,
Suprasanna
P.
&
Hong
J. C.
2023
Comprehensive mechanisms of heavy metal toxicity in plants, detoxification, and remediation
.
J. Hazard. Mater.
450
,
131039
.
Gordon
A. S.
,
Howell
L. D.
&
Harwood
V.
1994
Responses of diverse heterotrophic bacteria to elevated copper concentrations
.
Can. J. Microbial.
40
(
5
),
408
411
.
Gupta
R.
,
Kumar
T.
&
Mittal
A.
2017
Isolation, identification and characterization of heavy metal resistant bacteria from soil of an iron industry, haryana (India)
.
Int. J. Pharm. Sci. Res.
7 (3),
2320
5148
.
Hussain
A.
,
Shakir
H. A.
&
Qazi
J. I.
2014
Anaerobic biodegradation of sulphate employing animal manure as a cost effective growth substrate
.
J. Anim. Plant Sci.
24
,
913
918
.
Ilhan
S.
,
Nourbakhsh
M. N.
,
Kiliçarslan
S.
&
Ozdag
H.
2004
Removal of chromium, lead and copper ions from industrial waste waters by Staphylococcus saprophyticus
.
Turk. Electron. J. Biotechnol.
2
(
2
),
50
57
.
Isik
Z.
,
Saleh
M.
,
M'barek
I.
,
Yabalak
E.
,
Dizge
N.
&
Deepanraj
B.
2022
Investigation of the adsorption performance of cationic and anionic dyes using hydrochared waste human hair
.
Biomass Convers. Biorefin.
https://doi.org/10.1007/s13399-022-02582-2
.
Issazadeh
K.
,
Jahanpour
N.
,
Pourghorbanali
F.
,
Raeisi
G.
&
Faekhondeh
J.
2013
Heavy metals resistance by bacterial strains
.
Ann. Biol. Res.
4
(
2
),
60
63
.
Kumar
A.
,
Bisht
B. S.
&
Joshi
V. D.
2010
Biosorption of heavy metals by four acclimated microbial species, Bacillus spp., Pseudomonas spp., Staphylococcus spp. and Aspergillus niger
.
J. Biol. Environ. Sci.
4
(
12
), 97–108.
Kumar
P.
,
Srivastava
S.
,
Dwivedi
K.
,
Sharma
S.
,
Chauhan
B. S.
,
Jain
S.
&
Gupta
P.
2023
Impact of heavy metal contamination on human health
.
Eur. Chem. Bull.
2
(
12
),
1366
1379
.
Mathiyazhagan
N.
&
Natarajan
D.
2011
Bioremediation on effluents from Magnesite and Bauxite mines using Thiobacillus spp. and Pseudomonas spp
.
J. Bioremed. Biodegrad.
2
(
115
),
2
.
Nies
D. H.
1999
Microbial heavy metal resistance
.
Appl. Microbiol. Biotechnol.
51
,
730
750
.
Nilanjana
D.
,
Vimala
R.
&
Karthika
P.
2007
Biosorption of heavy metals-an overview
.
Ind. J. Biotechnol.
7
,
159
169
.
Parvin
F.
,
Rahman
M. M.
,
Islam
M. M.
,
Jahan
N.
,
Shaekh
M. P. E.
,
Sarkar
I.
,
Dutta
A. K.
&
Salah Uddin
M.
2015
Isolation of mixed bacterial culture from Rajshahi Silk industrial zone and their efficiency in Azo Dye decolorization
.
Ind. J. Sci. Technol.
8
(
10
),
950
957
.
Peng
C.
,
Zhao
X.
,
Ji
X.
,
Wu
J.
,
Liang
W.
,
Song
H.
,
Zhang
W.
&
Wang
X.
2023
Mixed bacteria passivation for the remediation of arsenic, lead, and cadmium: Medium optimization and mechanisms
.
Process Saf. Environ. Prot.
170
,
720
727
.
Priya
A. K.
,
Gnanasekaran
L.
,
Dutta
K.
,
Rajendran
S.
,
Balakrishnan
D.
&
Soto-Moscoso
M.
2022
Biosorption of heavy metals by microorganisms: Evaluation of different underlying mechanisms
.
Chemosphere
307
,
135957
.
Ramírez Calderón
O. A.
,
Abdeldayem
O. M.
,
Pugazhendhi
A.
&
Rene
E. R.
2020
Current updates and perspectives of biosorption technology: An alternative for the removal of heavy metals from wastewater
.
Curr. Pollut. Rep.
6 (
1
),
8
27
.
Ramli
N. N.
,
Othman
A. R.
,
Kurniawan
S. B.
,
Abdullah
S. R. S.
&
Hasan
H. A.
2022
Metabolic pathway of Cr (VI) reduction by bacteria: A review
.
Microbiol. Res.
268
,
127288
.
Ramteke
P. W.
2000
Biosorption of nickel(II) by Pseudomonas stutzeri
.
J. Environ. Biol.
21
(
3
),
219
221
.
Rodriguez-Montelongo
L.
,
de la Cruz-Rodriguez
L. C.
,
Farías
R. N.
&
Massa
E. M.
1993
Membrane-associated redox cycling of copper mediates hydroperoxide toxicity in Escherichia coli
.
Biochim. Biophys. Acta Bioenerg.
1144
(
1
),
77
84
.
Shan
B.
,
Hao
R.
,
Zhang
J.
,
Jiani Li
J.
,
Yubo Ye
Y.
&
Anhuai Lu
A.
2023
Microbial remediation mechanisms and applications for lead-contaminated environments
.
World J. Microbiol. Biotechnol.
39
,
38
.
Sundseth
K.
,
Pacyna
J. M.
,
Pacyna
E. G.
,
Pirrone
N.
&
Thorne
R. J.
2017
Global sources and pathways of mercury in the context of human health
.
Int. J. Environ. Res. Public Health
14
(
1
),
105
.
Tsezos
M.
,
Remoudaki
E.
&
Angelatou
V.
1995
A systematic study on equilibrium and kinetics of biosorptive accumulation: The case of Ag and Ni
.
Int. Biodeterior. Biodegrad.
35
(
1–3
),
129
153
.
Tsezos
M.
,
Remoudaki
E.
&
Angelatou
V.
1996
A study of the effects of competing ions on the biosorption of metals
.
Int. Biodeterior. Biodegrad.
38
(
1
),
19
29
.
Yabalak
E.
,
Al-Nuaimy
M. N. M.
,
Saleh
M.
,
Isik
Z.
,
Dizge
N.
&
Balakrishnan
D.
2022
Catalytic efficiency of raw and hydrolyzed eggshell in the oxidation of crystal violet and dye bathing wastewater by thermally activated peroxide oxidation method
.
Environ. Res.
212
,
113210
.
https://doi.org/10.1016/j.envres.2022.113210
.
Zhou
Y.
,
Zhao
X.
,
Jiang
Y.
,
Ding
C.
,
Liu
J.
&
Zhu
C.
2023
Synergistic remediation of lead pollution by biochar combined with phosphate solubilizing bacteria
.
Sci. Total Environ.
861
,
160649
.
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