Bioremediation of lead and manganese from wastewater using different Bacillus spp. with FTIR spectroscopic analysis

Both humans and ecosystems are seriously at risk from water contaminated with heavy metals (HMs), such as lead and manganese. This research assesses the biosorption effectiveness of different Bacillus strains, namely: Bacillus licheniformis MSRY R, Bacillus amyloliquefaciens MSRY F, and Bacillus amyloliquefaciens MSRY J, to remove Pb(II) and Mn(II) from aqueous solutions, aiming to identify the best effective strain for wastewater bioremediation in Egypt’s Nile Delta (Kitchener and No. 7 drains) in accordance with discharge standards. Results indicated that Bacillus licheniformis MSRY R eliminated 98%–99% of lead, bringing its concentration to levels close to or below the World Health Organization (WHO) safe drinking-water recommendation of 0.01 mg/L. Additionally, 77% of the manganese was removed, leaving levels that met or remained below the WHO standard of 0.4 mg/L. Notably, FTIR spectroscopic analysis revealed a distinct shift in hydroxyl, amide, carboxylate, and phosphate groups, indicating its involvement in Pb(II) and Mn(II) binding by such Bacillus strains, specifically Bacillus amyloliquefaciens MSRY J, which exhibited the greatest Extracellular Polymeric Substances (EPS)-matrix reorganization at the same time, Bacillus licheniformis MSRY R showed a strong multi-site mechanism. These findings confirm that Bacillus strains provide a viable, environmentally friendly way to remove heavy metals from contaminated water.

Extensive industrial activity and the use of agrochemicals, including fertilizers and pesticides in intensive agriculture, contribute to the accumulation of heavy metals in plants and soils. These metals present significant risks to human health through cytotoxic, mutagenic, and carcinogenic effects in consumers (Sytar et al., 2021; Scutaras and Carmen, 2023). In this context, lead (Pb(II)) is a heavy metal that occurs in wastewater from industrial activities such as battery, piping, glass, and ceramics manufacturing. Pb occurs in two forms of oxidation, that is, Pb(II) and Pb(IV). Both forms of Pb(II) are toxic to human health because they affect the brain, circulatory, and nervous systems, and can damage them. Additionally, the accumulation of Pb(II) in soil over hundreds of years may adversely affect food chains and plant photosynthesis (Diaconu et al., 2023). Manganese (Mn(II)) is a divalent, water-soluble heavy metal also participating in various metabolic reactions and activities. The presence of Mn(II) in drinking water can lead to its bioaccumulation (Rudi et al., 2020). (II) toxicity in the aquatic environment results in iron deficiency in algae, neurotoxicity in flatworms, and acute toxicity in fish (Safwat et al., 2023). The Kitchener Drain is one of the major northward-flowing channels originating from the Nile Delta and emptying into the Mediterranean Sea. It is mainly supplied by diluted treated wastewater. Approximately 75% of input water originates from agricultural drainage, 23% from domestic sewage, whereas 2% from industrial wastewater (Hendawy et al., 2025).

The removal of heavy metals from wastewater has posed a significant challenge for an extended period. A variety of techniques has been established for the extraction of hazardous metal ions from wastewater, such as chemical precipitation, ion exchange, membrane filtration, reverse osmosis, and electrodialysis. However, these conventional technologies entail substantial expenses due to the use of non-renewable materials, high costs, and the generation of toxic sludge (Khalifa et al., 2025). Bioremediation, an eco-friendly technology, is the use of living organisms to remediate contaminated water, soil, sludge, and other media. It's considered one of the most significant technologies for decontaminating water and soil from heavy metals. Among the various microbial remediation methods, biosorption has proven to be the most effective, simple, and scalable for remediation (Anusha and Natarajan, 2020). Recent research has highlighted microbial biosorption as a highly promising process, in which metal ions interact with functional groups on microbial cell surfaces, leading to their rapid removal from aqueous solutions. These interactions could encompass electrostatic attraction, ion exchange, complexation, precipitation, and other surface-related mechanisms, contingent on the microbial species and ambient conditions (Qattan, 2025).

Bacterial bioremediation is especially appealing due to bacteria’s ability to endure various environmental conditions and their possession of numerous pathways for metal sequestration and detoxification (Bisht et al., 2026). The unique character of Bacillus spp. Is the ability to form spores under adverse conditions. Due to their distinctive structure, these spores can withstand various adverse environmental conditions, including heat, dryness, humidity, and radiation. This feature gives them a competitive advantage over other bacteria and makes them commercially employed in many industrial and agricultural sectors (Pham et al., 2022; Tirloni et al., 2022).

This study aims to conduct a comparative evaluation of the biosorption capacity of various Bacillus strains for Pb(II) and Mn(II) ions from aqueous solutions. The specific objectives are the screening and selection of the most efficient strain for the biosorption of both metal ions and the assessment of its practical applicability in reducing the concentrations of these heavy metals in real wastewater samples to below the discharge limits.

Study area and samples collection

Wastewater samples were collected in September 2024 from two main drains. Wastewater samples were collected in September 2024 from two main drains. Along the first drain, samples were collected from three sampling sites at [31°21′40.75″N 30°56′44.46″E], [31°17′43.90″N 30°57′38.44″E], and [31°13′23.17″N 30°59′11.24″E]. Along the second drain, samples were collected from three sampling sites located at [31°25′37.29″N 31°10′48.40″E], [31°19′42.38″N 31°10′47.70″E], and [31°09′50.72″N 31°10′30.79″E], respectively. The locations of the two drains and all sampling sites are shown in Figure 1. 500 mL of three samples from each drain were collected at a depth of 50 cm below the surface in polythene bottles, yielding a total of six wastewater samples, which were transferred to the laboratory in an icebox for analysis.

Heavy metal analysis of wastewater

According to the WHO (World Health Organization, 2011) method, 5 mL of nitric acid was immediately added to the wastewater samples after collection to minimize adsorption of heavy metals onto the bottle walls. 50 mL of each wastewater sample was filtered through Whatman No. 42 filter paper, then through a syringe filter (0.45 μm pore size). Samples were kept in the refrigerator until analysis for heavy metals Pb(II) and Mn(II) using an Atomic Absorption Spectrophotometer (AAS) (GBC AVANTA™).

Metal solutions preparation

Aqueous solutions from [MnSO 4 and (CH 3 COO) 2 Pb [3H2O] were prepared at a concentration of 1 mg/mL for each metal ion and diluted to the required final concentration with sterile distilled water (Anusha and Natarajan, 2020). Working metal solutions at concentrations of 100, 200, and 300 mg L -1 were prepared from the stock solution.

Selection and description of Bacillus strains

Three Bacillus strains were previously isolated by Elmasry (2017), identified according to Holt et al. (1994), and matched to 16 S rRNA gene sequences in the NCBI nucleotide database. Bacillus strains and their accession numbers are presented as follows:

Selection criteria for such Bacillus species were determined based on various properties, including growth rate under harsh conditions, survivability at low pH, tolerance to bile salts, and susceptibility to antibiotics. The bacterial strains were cultured in a nutrient broth medium (g/L: tryptone 10; meat extract 5; sodium chloride 5; pH 7.2 ± 2 before autoclaving) and incubated in a shaker-incubator (180 rpm) at 37 °C for 24 h under aerobic conditions to evaluate their potential for heavy-metal bioremediation.

Experimental design

Exploration of different conditions for Pb(II) and Mn(II) bioremediation

A separate experiment was conducted to evaluate the growth responses of the three selected Bacillus strains under Pb(II) and Mn(II) stress. For each heavy metal, six pH-temperature conditions were evaluated separately: pH 6 at 32, 35, and 38 °C and pH 7 at 32, 35, and 38 °C. These conditions were designated as P1, P2, P3, P4, P5, and P6, respectively. Under each pH-temperature condition, each bacterial strain was separately exposed to three concentrations of the respective heavy metal (100, 200, and 300 mg L -1), and each treatment was performed in triplicate. The selected bacterial strains were inoculated into 50 mL conical flasks containing 20 mL of Luria-Bertani broth. The pH of the medium was adjusted to the required value using 2 N HCl or NaOH, and the cultures were incubated at the designated temperature with shaking at 180 rpm. Bacterial growth under the different heavy metal concentrations and environmental conditions was evaluated by measuring the optical density at 570 nm using a spectrophotometer (JENWAY model 6,405, United Kingdom).

Heavy metal bioremediation experiments

According to Ali et al. (2023), the wastewater bioremediation experiment was conducted using six wastewater samples collected from six different sampling sites and the three selected Bacillus strains. Each wastewater sample was independently treated with each bacterial strain. For every wastewater sample × bacterial strain combination, three independent replicates were prepared, resulting in 54 wastewater treatment pots (6 wastewater samples × 3 bacterial strains × 3 replicates). In addition, a tap-water control was prepared separately for each bacterial strain, with three replicates per strain, resulting in nine control pots. Thus, the experiment comprised a total of 63 experimental pots (54 wastewater treatment pots and 9 control pots). Each pot contained 100 mL of the corresponding water sample and was inoculated with 1 mL of the respective bacterial inoculum at a concentration of 1 × 10 8 CFU/mL. The pots were maintained under laboratory conditions for 14 days at ambient temperatures ranging from 30 to 38 °C.

Bioremoval efficiency (%)

Efficiency of bioremoval was determined by finding the percentage removal (R) from the formula R = Ci − Cf Ci × 100, where R is the percentage removal of metal, C i is the initial concentration of metal in water samples, and C f is the residual concentration of metal in water samples (Shamshad et al., 2015).

Fourier transform infrared spectroscopy (FT-IR)

FTIR data were collected in the range of 400–4,000 cm -1 using a JASCO spectrophotometer (model 6,800) at the Institute of Nanoscience and Nanotechnology, Kafrelsheikh University, Egypt. The liquid culture of bacteria was inoculated into LB broth medium containing 0 or 100 mg L -1 Pb(II) or Mn(II), incubated in a shaking incubator at 35 °C and 180 rpm min-1 for 48 h, followed by centrifugation to separate the precipitate. The sediment was washed three times with phosphate buffer solution. Then, the bacteria were dried for 24 h at 45 °C (Kenzhegulov et al., 2024).

Statistical analysis

Data are presented as mean ± standard error of mean (SEM). Differences among groups (treatments) were tested using a general linear model ANOVA in SPSS statistical software, version 16.0. Duncan’s multiple range test was used to determine significance. Statistical significance was considered at p < 0.05.

Exploration of selected strains for Pb(II) and Mn(II)

Figures 2, 3 illustrate the relationship between optical density for the growth of Bacillus strains at different concentrations (100, 200 and 300 ppm) of heavy metals under varying temperature and pH conditions, Where the symbols are from P1 to P3 represent temperatures of 32 °C, 35 °C and 38 °C, respectively at pH 6 and the symbols from P4 to P6 represent temperatures of 32 °C, 35 °C, and 38 °C, respectively at pH 7.

The data showed that different concentrations of Pb(II) significantly (P < 0.05) affected the growth of Bacillus licheniformis MSRY R, Bacillus amyloliquefaciens MSRY F, and MSRY J, with growth decreasing gradually as concentrations increased. At pH 6 (P1-P3), the OD of B. licheniformis MSRY R was high across all Pb(II) concentrations. Growth generally improved from 32 °C to 38 °C, where OD values were highest at P3, particularly at 100 ppm. At pH 7 (P4–P6), OD decreased significantly at 32 °C and 35 °C, indicating that neutral pH, along with low to intermediate temperatures, increased bacterial sensitivity to Pb(II). OD partially showed growth recovery at 38 °C (P6), suggesting that this temperature improved tolerance under pH 7 (Figure 2A).

The results in Figure 2B demonstrated that growth was high for B. amyloliquefaciens MSRY F within the pH 6 range, particularly at 35 °C and 38 °C (P2 and P3), indicating good resistance to Pb(II) ions under mildly acidic conditions. The most severe inhibition was observed at pH 7 and a temperature range of 32 °C–35 °C (P4 and P5), especially at a concentration of 300 ppm. At pH 7 °C and 38 °C (P6), the OD value increased again, but the concentration effect was weaker than in P4 and P5. Bacillus amyloliquefaciens MSRY J showed high growth activity at pH 6, with the highest level observed once again at 38 °C (P3). Nevertheless, OD values in this case were somewhat lower than those obtained for strain MSRY F under similar pH 6 conditions. Strain MSRY J proved to be the most sensitive to Pb(II) at pH 7 °C and 35 °C (P5), with a sharp decrease in OD values, particularly at 300 ppm. In turn, pH 7 °C and 38 °C (P6) allowed regaining a growth rate similar to that at pH 6 (Figure 2C).

In the case of Mn(II), the strains B. licheniformis MSRY R, B. amyloliquefaciens MSRY F, and MSRY J maintained high OD at pH 6, with the highest readings in P2 and P3. Inhibition due to increased Mn(II) concentration was observed for B. licheniformis MSRY R; however, it was lower than that observed for others. Growth was decreased during P4, specifically at 300 ppm Mn(II). However, growth was markedly enhanced in P5 and P6, indicating that 35 °C–38 °C supported Mn(II) tolerance at neutral pH. In relation to the results for the same strains compared to the Pb(II) curve, Mn(II) showed less inhibition at neutral pH and high temperatures, implying low toxicity of the metal. From the figure, the organism appears sensitive to neutral pH at high temperatures, but is more tolerant of Mn(II) than Pb(II) under similar conditions (Figure 3).

Heavy metals bioremediation

The concentration of Pb(II) (mg/L) in wastewater samples of various sources before and after treatments by three Bacillus bacterial strains was established as follows: The above-stated strains were provided in Table 1: B. licheniformis MSRY R, B. amyloliquefaciens MSRY F, and B. amyloliquefaciens MSRY J. Results show significant decreases in Pb(II) concentrations in all treated samples compared to initial concentrations, which confirm the efficiency of decontamination of Pb(II) from polluted wastewater by the bacterial strains used. The results also show minimal differences in removal efficiency among the three strains and indicate their distinct capacities for biosorption or bioaccumulation of Pb(II) from wastewater.

The concentrations of Mn(II) in wastewater samples prior to and following treatment using the same three strains of Bacillus bacteria as in Pb(II) are presented in Table 2. The data show that sample No. (1) had the highest initial concentration (0.710 mg/L), while all samples showed a decrease in concentration after treatment. It is also evident that there was variation in the strains’ efficiency at reducing manganese concentration, with B. licheniformis (R) showing the greatest ability to reduce Mn(II) across most samples.

Bioremoval efficiency (%)

The effectiveness of the removal of Pb(II) and Mn(II) from wastewater with the application of B. licheniformis MSRY R strain in six samples and the control sample. As a matter of fact, the effectiveness of Pb(II) bioremoval was very high across all samples, ranging from 98.2% to 99.3%, with the control showing the lowest percentage at 61.5%. For Mn(II), the efficiency was lower than for Pb(II), ranging from 62.8% to 73.1%, whereas the control was only 50%. The results indicate that B. licheniformis MSRY R strain has a high Pb(II) removal capability from wastewater, in contrast to Mn(II), demonstrating its high biological efficiency, especially in the removal of highly toxic heavy metals such as Pb(II) (Figure 4A). The bioremoval efficiency of Pb(II) was highly effective across all samples, ranging from 98% to 99.4%, except for the control sample, which showed the lowest efficiency at 56.4%. For Mn(II), the efficiency was lower than for Pb(II), ranging from 49.6% to 77.7%, while the control sample reached only 45.3%. The findings indicate that B. amyloliquefaciens strain MSRY F exhibits a notable capacity to remove Pb(II) from wastewater, in comparison to its capacity to remove Mn(II) (Figure 4B). The strain of B. amyloliquefaciens MSRY J in (Figure 4C) achieved high efficiency in removing Pb(II), reaching between 98% and 99.2%, and in the control achieved (51.3%), which is consistent with the general results in (Figures 4A,B). It was able to remove Mn(II) less, reaching between 47.5% and 64.2%, with only 39.1% recorded in the control sample, which indicates that this strain, despite its effectiveness in Pb(II), was less efficient in removing Mn(II) compared to strains (R) and (F).

FTIR analysis of metal-binding functional groups in Bacillus strains

In strains MSRY R, MSRY F, and MSRY J, the presence of metals affects the broad O-H/N-H region, protein amide I and II regions, carboxylate region, phosphate region, and low-wavenumber metal-O/O-P-O region. From this, it can be observed that oxygen- and nitrogen-containing functional groups in cell wall polymers and extracellular polymeric substances interact with Pb 2+ and Mn 2+ via electrostatic interactions and ion exchange/coordination (Figure 5).

For the strain Bacillus licheniformis MSRY R, it is clear that there is a multilocus effect for both metals. Pb 2+ leads to the largest effect on the phosphate group (1,073→1,060 cm -1) and the low frequency region (540→566 cm -1). There is also a small effect on the O-H/N-H, amide, and carboxylate groups. This indicates Pb 2+ interaction via coordination to O/N groups, with Pb-O coordination or phosphate immobilization. For Mn 2+, there is a large effect on the carboxylate group (1,391→1,406 cm -1) along with P-O (1,073→1,059 cm -1) and the low frequency region around 565 cm -1. Therefore, Mn 2+ interacts more with the carboxylate groups, and phosphate groups participate in binding both metals (Figure 5A).

The strain Bacillus amyloliquefaciens MSRY F in (Figure 5B) exhibits the smallest overall spectral response. Pb 2+ causes only minor changes in O–H/N–H, amide I, COO −, P–O and the low-frequency band, indicating interaction with surface groups but limited phosphate/metal–oxygen reorganization compared with R and J. Mn 2+ produces a stronger phosphate shift (1,073→1,059 cm -1) and low-frequency change (545→565 cm -1), showing that oxygen- and phosphate-containing sites respond more strongly to Mn 2+ than to Pb 2+. The −15 cm -1 C–H shift is best interpreted as a change in the surrounding membrane/protein environment rather than as direct metal coordination. Strain F therefore binds both metals, but its surface chemistry is less reorganized, especially after Pb 2+ exposure.

Strain Bacillus amyloliquefaciens MSRY J displays the greatest spectral reorganization. The broad O–H/N–H maximum shifts from 3,425 to ∼3,300 cm -1 after both metals, with increased intensity, indicating major alterations in hydrogen bonding, hydration, and the protein–polysaccharide/EPS matrix. Amide, carboxylate, and phosphate bands also intensify, supporting the extensive participation of proteins, EPS, and phosphorylated cell-wall components. The changes observed in the amide I and amide II regions (around 1,650 and 1,540 cm -1, respectively) further support the involvement of protein-associated functional groups in the spectral response to Pb 2+ and Mn 2+ exposure. Pb 2+ causes only a small P–O shift (1,073→1,070 cm -1), whereas Mn 2+ causes the larger shift (1,073→1,059 cm -1). The low-frequency control band at 664 cm -1 in control is replaced by bands at 553–567 cm -1 for pb 2+ and Mn 2+ treated samples respectively (Figure 5C).

Impact of metal concentration, pH, and temperature on heavy metal accumulation

Across all graphs, a common trend was observed: an increase in OD was associated with a decrease in concentration, with 100 ppm generally yielding maximum growth and 300 ppm yielding minimum growth. This is understandable, since heavy metals can interact with proteins, cell-wall receptors, ion balance, enzymes, and cell membranes, thereby causing oxidative stress. The general adaptability of Bacillus spp. To resist this type of stress was expected from the capabilities of this bacterial genus in bioremediation strategies, such as biosorption, EPS-sorption, bioaccumulation, bioprecipitation, and metal tolerance (Pande et al., 2022; Wróbel et al., 2023).

Moreover, it is important to note that metal toxicity was highly dependent on the pH-temperature relationship. In most cases, OD at pH 6 was higher than at pH 7, especially with Pb(II). This does not mean that pH 6 is always the best value for biosorption; it only means that these bacteria grew better under conditions where pH 6 was provided. The pH of the medium impacts not only the structure of the bacterial cell wall but also the metal species in the solution. For example, the presence of carboxyl, phosphate, hydroxyl, and amino groups in Gram-positive bacterial cell walls may lead to the formation of complexes with cations; however, their protonation/deprotonation depends on the solution pH. Also, some recent Pb(II) biosorption studies with Bacillus biomass have found that pH is an important operating parameter, especially near pH 6 (Emara et al., 2023; El-Sharkawy et al., 2024).

The comparison of Pb(II) and Mn(II) is quite crucial. In particular, Pb(II) induced greater inhibition of OD growth compared to Mn(II), especially in B. amyloliquefaciens MSRY J under pH 7 °C and 35 °C conditions. Lead is a toxic heavy metal that is not an essential element, but it can interact effectively with functional groups of proteins and cell surfaces. On the other hand, manganese is an essential element that serves as a component of various enzymes and catalysts, but it becomes toxic in excess. Such differences between the roles of Pb and Mn contribute to the fact that graphs for Mn(II) usually appear smoother and demonstrate lower inhibition of growth compared to graphs for Pb(II). Removal of manganese is associated with biological processes such as biosorption and oxidation of Mn(II), which make Mn speciation highly pH-dependent between pH 4 and 7 (Wu et al., 2022; Moeini et al., 2024).

Heavy metals bioremediation

Both Pb(II) and Mn(II) are present at relatively high levels in Kitchener Drain and Drain No. 7 raw wastewater samples, with Pb(II) levels fluctuating from about 1.5 to 3.48 mg/L and Mn(II) from 0.24 to 0.71 mg/L. These levels exceed the allowable standards for potable and irrigation water (WHO standards for Pb(II) in drinking water are 0.01 mg/L, and for Mn(II) are 0.4 mg/L), indicating a severe environmental and public health issue.

The reduction pattern exhibited by Mn(II) followed a similar trend to that of Pb(II), with B. licheniformis MSRY R once again demonstrating superior removal rates. The final concentrations of Mn(II) were reduced from initial levels of up to 0.71 mg per liter (mg/L) to 0.032–0.191 mg/L. This in Mn(II) concentrations resulted in levels exceeding the safety threshold for Mn(II) in many of the samples analyzed. These results align with the current literature, which shows that Bacillus species exhibit excellent biosorption and bioremediation capacities for heavy metals, outperforming many traditional physico-chemical methods in terms of cost-effectiveness and eco-friendliness (Hasan et al., 2024).

The near-total elimination of Pb(II) from the wastewater samples to levels below the WHO-recommended maximum, and the significant reduction in Mn(II) concentrations, attest to the strong feasibility of applying these Bacillus strains in real wastewater treatment. That this is bacterially mediated, through a process that is sustainable, energy-saving, and free of the secondary pollution frequently associated with chemical treatments, makes it even more desirable. The use of local bacterial isolates also enhances adaptation to the specific pollution environment, thereby improving remediation efficacy (Zhang et al., 2025).

Bioremoval efficiency (%)

Correlation and comparative performance across strains

The experimental graphs (Figure 4) show consistent high Pb(II) bioremoval and high Mn(II) removal by Bacillus strains: B. licheniformis MSRY R and the 2 B. amyloliquefaciens MSRY (F and J). Pb(II) removal is nearly complete under optimal conditions, whereas Mn(II) removal is also high but more strain- and time-dependent. Across the three strains, Pb(II) removal is generally similar (≈90–98%), with the maximum plateau reached in the minimum time by B. licheniformis MSRY R. Pb(II) removal by B. amyloliquefaciens MSRY F and J reaches close to >90% and exhibits partially slow kinetics and slightly greater variability, suggesting strain variation in available binding sites and extracellular metabolite production. These strains’ differences are consistent with recent comparative experiments that attribute greater Pb(II) uptake during the early phase to higher cell-wall functionalization and greater EPS production in certain B. licheniformis MSRY R strains (Gul et al., 2024). The considerably higher removal efficiency observed for Pb(II) compared with Mn(II) may be related to differences in the affinity of these metal ions for the binding sites present on the bacterial cell surface and extracellular polymeric substances (EPS). Bacillus spp. Possess various surface-associated functional groups and EPS components that can provide effective binding sites for metal ions through electrostatic interactions, complexation, ion exchange, and other surface-associated mechanisms. Recent studies have demonstrated that Pb(II) can interact strongly with bacterial EPS and may also undergo immobilization through the formation of poorly soluble Pb-containing minerals, which can contribute to its efficient removal from aqueous systems (Maumela and Serepa-Dlamini, 2025; Wang et al., 2025).

Mechanistic correlation of Pb(II) and Mn(II) removal

The concurrent high Pb(II) removal and significant Mn(II) removal indicate mechanistic overlap: (1) biosorption to cell wall functional groups (carboxyl, phosphate, hydroxyl, amine), aided by EPS enhancing effective surface area and adsorbed metal stabilization; and (2) metabolically mediated reactions (siderophore synthesis, redox enzymes) that can alter metal speciation and increase Mn(II) biomineralization (oxidation to insoluble Mn(II) oxides) (Galezowski et al., 2020). Mn(II)’s oxidation state variability renders it more vulnerable to removal based on biofilm development and metabolic activity than is Pb(II), which is mostly surface-adsorbed in near-neutral conditions (Wróbel et al., 2023).

FTIR spectral changes following metal exposure

Pb 2+ and Mn 2+ interact with a variety of ligands, as seen by the striking recurrence of O–H/N–H, amide, COO −, and P–O bands. Proteins, peptidoglycan-associated components, teichoic/phosphorylated polymers, and EPS provide ligands in the biomass of Bacillus bacteria. Similarly, new research on Bacillus reveals that the primary metal-binding functions are hydroxyl, amino/amide, carboxyl, and phosphate groups (Li et al., 2023; Mathivanan et al., 2023; El-Sharkawy et al., 2024).

Pb 2+ commonly forms strong surface complexes and may be immobilized as Pb–O, Pb–O–R, or phosphate-associated products; this interpretation is especially supported for strain R and, to a lesser extent, strain J (Han et al., 2023; El-Sharkawy et al., 2024). Mn 2+ causes the largest carboxylate perturbation in strain R and the largest phosphate/low-frequency response in strain F; in strain J, the band near 567 cm -1 is compatible with Mn–O formation. Microbial Mn removal can combine adsorption with oxidation or precipitation (Danial and Dardir, 2023; Huang et al., 2023).

Based on spectral perturbation, not adsorption capacity, train J undergoes the greatest matrix reorganization; strain R shows the clearest coordinated contribution of phosphate and carboxylate sites; strain F shows the smallest Pb-induced changes but a stronger Mn-specific phosphate/metal–oxygen response. Such differences are scientifically plausible because metal tolerance, EPS production, and surface-site accessibility vary substantially among Bacillus strains (Mathivanan et al., 2023; Moeini et al., 2024).

Bioremediation is becoming a more appealing choice for treating industrial wastewater because it is less expensive, easier to operate, and can recover resources compared with traditional physicochemical approaches. According to our research, the bioremediation of samples from Kitchener Drain and Drain No. 7 using Bacillus strains is quite successful in reducing Mn(II) and Pb(II) in wastewater. Bacillus licheniformis MSRY R outperformed the other strains tested, significantly lowering Mn(II) and Pb(II) levels within the WHO’s safety guidelines. Environmental factors had a significant impact on bioremediation efficiency. Both metal uptake and bacterial growth peaked at pH 6 and 35 °C–38 °C, underscoring the importance of managing these variables for optimal treatment outcomes. Bacillus licheniformis MSRY R once again showed the best ability under these ideal circumstances. FTIR spectroscopic analysis, a powerful method, revealed functional group differentiation and shift as binding mechanisms in Pb(II) and Mn(II) bioremediation by such Bacillus strains. Bacillus amyloliquefaciens MSRY J exhibited the greatest (EPS)-matrix reorganization. In contrast, Bacillus licheniformis MSRY R exhibited a strong multi-site mechanism, showing how bacteria change their very structure to grow and survive under heavy metal stress. This study showed that using native Bacillus isolates is a viable and sustainable approach to reducing heavy metal contamination in wastewater.