Heavy metals are one of the most persistent pollutants found on Earth due to their non-biodegradability and tendency to accumulate in the environment, causing harm to our natural ecosystems in the process (Gaur et al., 2021). There are many sources of heavy metals which can take root from natural and anthropogenic origins, with the former including soil run-off, volcanic eruptions, weathering, and erosion (Soltani-Gerdefaramarzi et al., 2021), whereas the latter are anthropogenic sources that stem from several industrial processes such as ore mining, combustion of fossil fuels, and the production of alloys, steel, plastic, dyes, and pigments, respectively (Selvi et al., 2019). Like soil and surface resources, water resources too tend to get contaminated by heavy metals, due to their closeness to industries which require large amounts of water for cooling and other processes, which ultimately enhances the precipitation of heavy metal ions onto the water bed once they enter the closest source (Briffa et al., 2020).

Among many heavy metals that are specifically categorized on the basis of their essentiality and non-essentiality to living organisms, copper (Cu) is an essential metal for biological systems as it is required for the function of various significant processes in prokaryotes and eukaryotes (Oves et al., 2016). It is a soft metal which conducts both heat and electricity, and belongs to the group IB in the Periodic Table of elements, in between nickel (Ni) and zinc (Zn). It is naturally found as a mineral, bound with other elements in the form of sulfides, oxides, and carbonates (Crichton and Pierre, 2001). Though found in two oxidation states in nature, it is usually found in its monovalent form (Cu⁺), which is required by various organisms for essential biological functions, as well as a co-factor for many enzymes, albeit in low concentrations, as elevated levels of Cu tend to induce cellular toxicity and can result in cell death (Zhao et al., 2010). Increasing levels of Cu in our environment have resulted in its pollution, which has since been aggravated due to many anthropogenic and natural factors (Izydorczyk et al., 2021; Covre et al., 2022).

Extensive research for mitigating excessive levels of heavy metals in the environment has brought about several methods for combating environmental pollution, as their accumulation poses a grave danger to sensitive ecosystems of the world (Minnikova et al., 2017). Over the years, many physical and chemical methods of remediation have been developed and critically evaluated, but not all were deemed to be sufficiently efficacious (Sayqal and Ahmed, 2021). The need for cheaper, feasible, and environmentally friendly methods was imperative, due to which the focus shifted toward other forms of adsorbents to remove heavy metals from water (Sulaiman et al., 2021). Biological agents, such as plants, algae, and microorganisms were found to be better suited for heavy metal remediation (Elwakeel et al., 2012; El-Liethy et al., 2018; Elgarahy et al., 2020), but the latter appeared to be more beneficial as they improved metal solubility due to the production of organic acids and/or by modification of soil texture due to the production of polysaccharides (Wang et al., 2017; Alves et al., 2022). Microorganisms such as bacteria are considered to be efficacious agents of bioremediation due to their generally well-known resistance mechanisms, and for their tendency to survive and endure extreme environments (Jain et al., 2011; Saeed et al., 2022). Bacterial consortia of several species such as Alcaligenes faecalis, Staphylococcus aureus, Streptococcus lactis, Micrococcus luteus, and Enterobacter aerogenes was reported to remove various heavy metals such as copper, cadmium, lead, and zinc from wastewaters (Silambarasan and Abraham, 2013). Furthermore, Bacillus sp. have also been well-reported as efficient agents of bioremediation from soil and wastewaters over the years (Joo et al., 2010; Kamika and Momba, 2013; Singh et al., 2014; Wierzba, 2015; Babar et al., 2021). Bacillus sp. are considered to be potentially effective in removing heavy metals from contaminated sources of soil and water, based on prior knowledge that Gram-positive bacteria are able to resist a much higher quantity of heavy metals than Gram-negative bacteria (Nwinyi et al., 2014). Furthermore, carboxyl group in the peptidoglycan layers of the bacterial cell wall serve a key role in the uptake of heavy metals, due to which Bacillus sp. are potential candidates for the bioremediation of Cu and its compounds (Igiri et al., 2018).

This study aimed to investigate the bioremediation potential of a Cu-resistant bacterium isolated from contaminated industrial wastewaters. The bacterium was also evaluated for its growth characteristics, antioxidative, motility and chemotactic behavior, as well as for the presence of metallothioneins. Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and Energy-Dispersive X-Ray Spectroscopy (EDX) analyses were employed to study physicochemical changes to bacterial cells pre and post Cu biosorption.

The sources of heavy metal contamination in the environment have increased in the past few decades, giving rise to many grave predicaments in nature. It has been a challenge to decontaminate industrial wastewater and soil from heavy metals. Different strategies have been designed and implemented for their removal, including filtration, oxidation/reduction, reverse osmosis and electro-chemical treatment among many others, but are not the favored option due to their cost, inefficiency, and intense labor (Algieri et al., 2021). This research work was carried out to investigate the bioremediation ability of chosen bacterial isolate for removing Cu from contaminated wastewaters. In this study, the chosen strain was identified as Bacillus altitudinis MT422188, which was isolated from the industrial areas of Kala Shah Kaku and Muridke, known for their miscellaneous industries and contamination of surrounding areas. It was affirmed in a previous study that a major share of bacteria isolated from heavy metal contaminated environments are Gram positive in nature, and are particularly members of the genus Bacillus (Aka and Babalola, 2017).

Cu-resistant bacteria isolated from various sources have been previously reported (Rathi and Nandabalan, 2017; Bhardwaj et al., 2018; Irawati and Tahya, 2021). In our study, the minimum inhibitory concentration (MIC) of B. altitudinis MT422188 for Cu was observed to be 20 mm, which was in agreement with the results of Mustapha and Halimoon (2015). The cross metal resistance behavior was also investigated (Ni > Zn > Cr > Pb > Ag > Hg). Rohini and Jayalakshmi (2015) reported the resistance of Bacillus cereus against Cu and more heavy metals. B. subtilis was reported to be capable of tolerating Cr and Cu upto 20 and 80 mm, respectively (Khusro et al., 2014). Bacillus sp. was found to have a high tolerance to many metals, with the most resistance observed against Cu at 12.5 mm, in a study conducted by Esertaş et al. (2020). Similar observations for Bacillus sp. were reported in another study (Singh and Chopra, 2014; Gillard et al., 2019). Prior to conducting further experimentation, the growth parameters such as pH, temperature, and phosphate concentration of B. altitudinis MT422188 were studied, where it demonstrated favorable growth at pH 7, 37°C, and 1 mm phosphate, respectively. B. licheniformis was also reported to demonstrate optimum growth at 37°C and pH 7 (Sher et al., 2021). pH has a pronounced effect on the solubility of heavy metal ions and the charge on the cell surface, which aids greatly in removing the former from contaminated media (Jin et al., 2018), and optimal pH (4–8) is reported to be significantly associated with all biomass forms (Sánchez-Clemente et al., 2020). The growth curves of B. altitudinis MT422188 were studied in the presence and absence of Cu, where its presence extended the lag phase of the bacterial cells for a short period of time and did not stop the growth, but resulted in its overall decrease (Figure 1). Similar growth characteristics were observed in B. licheniformis in the study of Sher et al. (2021). No effect of Cu on the final growth of B. stearothermophilus cells was observed when it was added to medium in a study (Bubela, 1973). In another study, Bacillus sp. demonstrated remarkable growth pattern in the absence of Cu, but the overall growth was observed to be decreased in the presence of increasing Cu concentrations (Khan et al., 2017), which was also evident from the findings of our study. Cu ions did not affect growth of B. thuringiensis cells greatly, indicating that they survived and grew in low concentrations of the metal (Liu et al., 2020). The half maximal effective concentration (EC₅₀) is considered to be the initial concentration of the metal ions at which the activity of the bacterial cells in the presence of metal is comparable to that of 50% of the control culture in the absence of the metal. Due to this aspect, EC₅₀ is a determinative parameter that defines the toxicity of metals with respect to bacterial cells. In this study, EC₅₀ of B. altitudinis for Cu was 5.56 mm, demonstrating that this was the dose at which 50% of the bacterial cells would survive in the presence of Cu (Figure 2). Utgikar et al. (2001) reported the EC₅₀ for Cu in their study to be <0.1 mm.

The biosorption studies depicted that in the case of Cu, the amount of extracellular Cu from the medium decreased proportionally from 7th h onwards, particularly due to the increased adsorption of Cu onto the bacterial cells, as well as some of the Cu being accumulated at intracellular level by bacterial cells, indicating that some of Cu was used for the performance of their essential functions. Growth of bacterial cells also noticeably increased after the first 6 h (Figure 3A). When DNP (inhibitor 1) was added to the medium containing Cu and bacterial cells, less pronounced but still eminent uptake was observed as there was some decrease in total Cu present in the medium. Adsorption of Cu also occurred, albeit slowly, from the 3rd to 9th h, while intracellular accumulation of Cu took place in the last 3 h (Figure 3B). DCCD (inhibitor 2) reduced the uptake of Cu from the medium but did not halt it altogether. Adsorption commenced from the 3rd h which continued till 9th h, while the intracellular accumulation was the same as in the case of DNP (Figure 3C). In the presence of both inhibitors and Cu, the bacterial growth rate was reduced. The adsorption and accumulation were also decreased but still occurred nevertheless, indicating that an ATP independent efflux system was employed by B. altitudinis MT422188 (Figure 3D). The most commonly reported Cu efflux system in bacterial species is encoded by the copZA operon, which is essential for inducing resistance to excessive levels of Cu. CueR is a protein which regulates cytosolic Cu and the operon encoding both Cu chaperones and ATPases for efflux, respectively (Gaballa and Helmann, 2003). CopZ serves the key role as chaperone for delivering Cu to CopA, a CPx-type ATPase, whereas CopB in the operon is responsible for Cu efflux and detoxification (Smaldone and Helmann, 2007; Figure 10).

In many studies, live or dead biosorbents have been used to deduce their efficiency in removing heavy metals from contaminated environments. It has been reported that rendering a bacterial biosorbent inactive by heat (autoclaving) strengthens its efficacy of biosorption (Paul et al., 2012). Therefore, passive uptake seems to be the major mechanism of biosorption in killed cells, as compared to the active uptake in live cells, respectively (Shamim et al., 2014). In this study, the killed cells of B. altitudinis MT422188 were able to remove Cu ions from the environment (Figure 3E), the findings of which were consistent with the study findings of Todorova et al. (2019) and Hu et al. (2020). In the pilot scale study, B. altitudinis MT422188 was able to remove 73 mg/l and 82 mg/l at 4 and 8 day intervals, which demonstrated its biosorption ability to be efficient in removing Cu (Figure 4). B. cereus has been reported to effectively remove more than 40 and 50% of Cu from industrial effluents (Raj et al., 2018).

Environmental factors like heavy metal stress can act as key inducers of ROS production, which are directly associated with cell death due to the alteration of intracellular redox stats of the cell (Franco et al., 2009). This increased level of ROS species in the cell can induce a state of “oxidative stress” which can be triggered by toxic concentrations of heavy metal ions and metalloids anions in the cell (Kumagai and Sumi, 2007). In order to combat this, high concentrations of heavy metal ions stimulate the increase in the activity of defense antioxidative enzymes, of which SOD and CAT are the first responders (Choudhary et al., 2007). The presence of divalent or monovalent heavy metal cations, including Cu, can induce the production of ROS through Fenton and Haber-Weiss reactions (Gunther et al., 1995). In our study, the increased activities of SOD, POX, and GR in the presence of Cu was indicative that Cu acted as a stress of B. altitudinis MT422188 cells, while CAT and APOX demonstrated no activity (Table 1). High concentrations of heavy metals are reported to trigger oxidative and non-oxidative stress, suggesting increased levels of ROS in metal stressed bacterial cells (Steunou et al., 2020). Cu-Zn SODs were previously thought to be specific to eukaryotes but are now reported to be present in many bacterial species, including Bacillus (Argüello et al., 2013). It is pertinent to note that the presence of Cu may have possibly induced the enhanced activity of Cu-Zn SOD in our study. The study findings of Wiesemann et al. (2013) demonstrated elevated SOD activity for mitigating ROS-mediated stress in cells. Another study demonstrated increased CAT activity in the face of elevated H₂O₂ stress (Behera et al., 2014), while Latef et al. (2020) reported the elevated POX activity and decreased CAT activity during oxidative stress, respectively. Metallothioneins (MTs) are proteins that are low in weight and abundant in cysteine, which also bind to heavy metal ions with remarkable avidity, and aid in regulating high toxic metal concentrations as well as metal homeostasis (Gadd, 2010). It is reported to be found in many microorganisms including bacteria and fungi, as well as eukaryotes such as plants, animals, and human beings (Chaturvedi et al., 2012). The findings of our study elucidated the presence of a 70 kDa MT protein in B. altitudinis MT422188 in the presence of Cu (Figure 5). Another study also reported the role of MT in removing metal ions from the environment via sequestration (Mulik et al., 2018). Copper homeostasis involves sequestration, efflux or detoxification as shown in Figure 11. Although essential for the cellular processes, concentrations of Cu²⁺ above 10 μm becomes toxic for the microbial cell. The detoxification mechanisms (Rohini and Jayalakshmi, 2015) are mentioned in Figure 11. During efflux mechanism, CopZ acts as chaperone and converts Cu²⁺ to Cu⁺ which ultimately enters the cells via CopA protein. CopB protein is associated with ATPase which is energy dependent and helps in Cu⁺ efflux out of the cell (Alotaibi et al., 2021; Figure 10). To the best of our knowledge, copper sequestration by MTs as well as uptake and efflux mechanisms regulated by copZA operon are apparently involved in copper homeostasis in B. altitudinis, as observed in our study.

Motility, particularly in bacteria, is generated by flagellae which enable bacteria to move forward through various movements. There are six different types of movements which cause bacterial motility, such as swimming, swarming, twitching, gliding, sliding, and darting, of which the first two are caused by flagellar movements (Prabhakaran et al., 2016). In our study, swimming, swarming, and twitching patterns of B. altitudinis MT422188 were found to be positive in the presence of Cu, which resulted in enhanced motile behavior in bacterial cells (Figures 6A–C). Enhanced motility was also observed in Bacillus sp. in the presence of Cu (Singh et al., 2014). In another study, the positive motility and chemotactic behavior of B. safensis was reported in the presence of manganese (Mn) ions (Ran et al., 2020). Chemotaxis is a process which involves the regulation of movement away or toward the concentration gradient of specific stimuli or stresses, acting either as attractant and repellent factors, respectively (Vladimirov and Sourjik, 2009). Chemotaxis in bacteria is regulated via a chemo-sensory pathway which involves several key proteins which initiate the movement away or toward an external stimulus. In this study, Cu acted as a chemoattractant for the cells, meaning that bacterial cells in solution tended to move toward Cu ions, which initiated flagellar motions of movement in B. altitudinis MT422188 (Figure 7).

For the determination of suitable adsorption isotherm, the biosorption data were examined with two mathematical models (Table 2). The study findings indicated biosorption was well-fitted by the Freundlich model (R² ≥ 0.98). The Langmuir isotherm model suggests that the process of adsorption is homogenous which occurs at specific sites in the adsorbent, while the Freundlich model suggests the adsorption occurs on a heterogenous surface with varying levels of biosorption affinities. The binding sites with a remarkably stronger binding energy tend to be involved primarily, followed by the exponential decline in the adsorptive energy until the whole process is completed (Pandhare et al., 2013). The Freundlich intensity constant (n) is regarded to be independent of the metal ion concentration, where n > 1, 1/n < 1, 1/n > 1 are suggestive of adsorption in favorable conditions, normal adsorption, and co-operative adsorption, respectively. In our study, the value of n was found to be greater than 1, which indicated the favorable biosorption of Cu ions. The results were in agreement with other studies which was also reported the Freundlich isotherm model to be well-fitted for Bacillus sp. (Liu et al., 2013; Kanamarlapudi and Muddada, 2019). In another study, the same model was also observed to be opted by Lactobacillus fermentum for the biosorption of Cu ions (Kargar and Shirazi, 2020). The study of kinetic parameters is important for the examination of the binding processes which can aid in elucidating reaction pathways and their mechanisms of action for subsequent biosorption. In order to determine the kinetic parameters of Cu for B. altitudinis, the pseudo-order (first and second) kinetics were investigated (Table 3), which demonstrated pseudo-second-order kinetics to be more suitable for our results than the former. Yang et al. (2017) also described the pseudo-second-order to be well-fitted with their study of Cu biosorption. Change in free energy, enthalpy and entropy (∆G°, ∆H°, and ∆S°, respectively) were calculated in this study in order to evaluate the thermodynamic patterns of Cu biosorption onto B. altitudinis cells. ∆G° was observed to be −9.3719, −9.64062, −9.6609, and − 4.1655 kJ/mol, respectively (Table 4). The negative values of free energy change were suggestive of the thermodynamically feasible nature of the biosorption process. The enthalpy change (∆H°) was found to be 10.431 kJ/mol, which indicated that Cu biosorption by B. altitudinis MT422188 was an endothermic process. ∆S° demonstrated the reaction to be feasible due to increased randomness at solid–liquid interface, which also agreed with the works of Kaparapu and Prasad (2018) and Sonawdekar and Gupte (2020).

The interaction of Cu with bacterial cell was investigated by FTIR. Peaks after Cu biosorption demonstrated slight shift in various functional groups such as hydroxyl (‖OH) and carboxyl (―C‖O). These peaks were indicative of Cu ion adsorption onto bacterial cells, with the cell membrane and its functional groups playing a significant part in the binding of metal ions, respectively. Many studies reported similar bands of biosorption peaks (Yang et al., 2017; Palanivel et al., 2020). SEM and EDX analyses yielded the physicochemical changes in bacterial cells in the presence of Cu, which was analyzed through SEM micrographs. These changes after Cu biosorption pertained to structural changes in bacterial cells, which were noted to be irregular and more flattened in shape as compared to cells prior Cu biosorption, as shown in Figures 8A,B. Changes in the shape of bacterial cells in response to metal ions has been reported previously by Yang et al. (2017). The changes in bacterial cells in the presence of Cu were also observed by EDX analysis, where sharp, intensified peaks in the range of 0–2 keV and at 8 keV corresponded to the presence of Cu signals in the spectrum, which was less pronounced in the control sample (Figures 9A,B). Our study findings were in agreement with Matilda et al. (2021).

This research work was conducted to investigate a Cu-resistant bacterium for its potentially efficient ability to remove Cu from polluted wastewater. The selected bacterial isolate SMK-08, identified as Bacillus altitudinis, was observed to optimally grow at pH 7, 1 mm phosphate, and 37°C, while its EC₅₀ (for Cu) was observed to be 5.56 mm. Biosorption studies indicated the efflux and uptake of Cu in bacterial cells to be an ATP independent process, whereas in the pilot scale study the efficient removal of Cu by B. altitudinis MT422188 (73 mg/l and 82 mg/l of Cu at 4 and 8 day intervals) was indicative of its efficacy. Chemotaxis and motility demonstrated positive chemoattractant behavior by B. altitudinis MT422188 cells for Cu. GR, SOD, and POX activity in bacterial cells was elevated in the presence of copper, while no expression of CAT and APOX activity was observed in either the presence or absence of Cu. Isotherm study demonstrated the Freundlich model to be the closest fit to the study, while the pseudo-second-order was suited in the kinetics of the study. The thermodynamic parameters depicted the overall process of biosorption to be spontaneous and feasible. FTIR demonstrated interaction of bacterial ―OH and ―C‖O groups with Cu after biosorption, while the SEM and EDX analyses were indicative of the interactions of Cu ions with the bacterial cell wall and other components, resulting in physicochemical changes. Taking these properties presented in our study into consideration, it could be stated that B. altitudinis MT422188 is an efficient biosorbent for Cu, and can be employed for its remediation.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

MK performed all the experimentations, writing-review, and editing. MK, RK, MH, AE, and HS performed data analysis, validation, writing–review and editing, and resources. MZ helped in execution and validation of the experimentations. MA facilitated critical review on pre-publication stages. SS conceived and designed the experimental strategies and was the main supervisor of the whole project. All authors contributed to the article and approved the submitted version.

This research was funded by Taif University Researchers Supporting Project number (TURSP - 2020/59), Taif University, Taif, Saudi Arabia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.