Biosorption of copper, nickel, and manganese as well as the production of metal nanoparticles by Bacillus species isolated from soils contaminated with electronic wastes

Abstract

Improper electronic waste management in the world especially in developing countries such as Iran has resulted in environmental pollution. Copper, nickel, and manganese are from the most concerned soil contaminating heavy metals which found in many electronic devices that are not properly processed. The aim of this study was to investigate the biological removal of copper, nickel, and manganese by Bacillus species isolated from a landfill of electronic waste (Zainal Pass hills located in Isfahan, Iran) which is the and to produce nanoparticles from the studied metals by the isolated bacteria. The amounts of copper, nickel, and manganese in the soil was measured as 1.9 × 10⁴ mg/kg, 0.011 × 10⁴ mg/kg and 0.013 × 10⁴ mg/kg, respectively based on ICP-OES analysis, which was significantly higher than normal (0.02 mg/kg, 0.05 mg/kg, and 2 mg/kg, respectively. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of metals on the bacterial isolates was determined. The biosorption of metals by the bacteria was evaluated by inductively coupled plasma optical emission spectroscopy (ICP-OES). The metal nanoparticles were synthetized utilizing the isolates in culture media containing the heavy metals with the concentrations to which the isolates had shown resistance. X ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS) were used for the evaluation of the fabrication of the produced metal nanoparticles. Based on the findings of this study, a total of 15 bacterial isolates were obtained from the soil samples. The obtained MICs of copper, nickel, and manganese on the isolates were 40–300 mM, 4–10 mM, and 60–120 mM, respectively. The most resistant isolates to copper were FM1 and FM2 which were able to bio-remove 79.81% and 68.69% of the metal, respectively. FM4 and FM5 were respectively the most resistant isolate to nickel and manganese and were able to bio-remove 86.74% and 91.96% of the metals, respectively. FM1, FM2, FM4, and FM5 was molecularly identified as Bacillus cereus, Bacillus thuringiensis, Bacillus paramycoides, and Bacillus wiedmannii, respectively. The results of XRD, SEM and EDS showed conversion of the copper and manganese into spherical and oval nanoparticles with the approximate sizes of 20–40 nm. Due to the fact that the novel strains in this study showed high resistance to copper, nickel, and manganese and high adsorption of the metals, they can be used in the future, as suitable strains for the bio-removal of these metals from electronic and other industrial wastes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-024-01369-z.

Introduction

Mineral wastes contain toxic metals that impose severe consequences on ecosystems which can cause malfunctions in the body and even cause cancer [1, 2]. The progressive development of the production of electronic equipment and the short usable time of some of this equipment have led to the accumulation of a large number of electronic wastes [3]. These wastes contain heavy and toxic metals such as lead, arsenic, mercury, cadmium, copper, zinc, manganese, nickel, selenium, and hexavalent chromium [4, 5]. These metals can enter the soil and underground water in industrial areas [6]. Electronic waste is mainly included household equipment waste (45%), information and communications technology equipment waste (33.9%), and consumer electronics waste (13.7%) which can enter the environments without processing or following incomplete processing. The geographic distribution of these waste depends on the consumption of e-items in each population [4].

Copper is considered one of the major elements in the components of electronic products. For this reason, the copper ingredient of electrical and electronic waste may reach to more than 10%. Copper, in high concentrations, becomes toxic and leads to complications such as digestive disorders (nausea, vomiting, abdominal pain), respiratory tract and immune system disorders, damage to the liver and kidneys, and anemia [7, 8]. Nickel is mainly found in electronic devices such as nickel–cadmium and nickel metal hydride batteries and their wastes [9]. Nickel can enter the body through drinking water, food, and tobacco smoking. If nickel is absorbed in high concentrations, it can lead to an increase in free radicals which cause inflammation in the body, leading to an increase in the white blood cells count [6]. However, the most important harmful effect has been observed in people who are exposed to dust containing nickel compounds while working in nickel refining and processing factories. These effects include decreased lung function, lung cancer, irritation of the sinuses, and may also lead to loss of the smell sense [10]. Manganese is one of the few metals that is used in electronic equipment [11]. The main target of manganese (II) toxicity is the nervous system by deposition in the basal ganglia and its effect on dopaminergic neuronal enzyme activity. Other systems affected by manganese are cardiovascular and pulmonary systems [12, 13]. Symptoms including ataxia, dementia, and anxiety have been seen following manganese overexposure [13, 14].

Although ion exchange resins and activated carbons have long been known as effective adsorbents for the treatment of industrial wastewater containing metal pollutants, their high cost and low efficiency have limited their commercial use in industrial removal programs [15]. The ability of microorganisms to absorb metals from aqueous solutions has been studied since the beginning of the eighteenth century, and it has attracted more attention in recent decades [16]. Many microorganisms among bacteria, yeast, and algae can transfer dissolved metals from the surrounding environment into their cells and have been used to successfully remove heavy metal ions. The main advantages of biosorption of metals compared to industrial methods, low cost, high efficiency, minimization of chemical pollution, reuse of biosorbent and the possibility of metal recovery are highlighted [17]. Toxic compounds are used by microorganisms as energy sources for fermentation, respiration, or co-metabolism. The abilities of various microbial biomasses for bioremediation of toxic compounds under certain experimental conditions is different which is mainly because of different sorption ability of surface functional groups of microbial communities. Heavy metals may disrupt the cell membranes of various microorganisms, but many bacteria can overcome these toxic effects because of their characteristic enzymatic profiles [18].

Currently, synthesis of nanoparticles by microorganisms like bacteria has attracted a great deal of attention because of its simple synthesis, cost-effectiveness, and environmental friendship. In this green synthesis process, the cellular extract of the living organism provides a suitable nonvolatile medium for the synthesis of nanoparticles. On the other hand, the biomolecules such as proteins, carbohydrates, vitamins, polymers, and biosurfactants in the microbial cell extract can provide high stability and increased distribution for the synthesized nanoparticles [19].

Due to the high potential of microorganisms to absorb metals and producing metal nanoparticles, the aim of this study was to investigate the biosorption of copper, nickel and, manganese by Bacillus species isolated from soils containing electronic wastes and to evaluate the isolated bacteria for the production of nanoparticles from the studied metals.

Materials and methods

Sample collection

The soil samples were collected from a depth of 15 cm and transferred to clean containers. At least 3–5 samples were taken from each of 10 electronic waste burial sites located in the hills of Zainel pass, 36 km southeast of Isfahan city, at the geographical coordinates 32 ͦ 36ʹ53ʹʹN 51 ͦ 47ʹ39ʹʹE, in sterile containers. After preparing a 1:2.5 suspension of soils, their temperature and pH were measured using a calibrated mercury thermometer and a pH meter (Metrohm 827, Swiss), respectively. Also, the TOC level of the soil samples was measured based on the Walkley–Black method as a reference method using potassium dichromate (K₂Cr₂O₂) and concentrated H₂SO₄ in a TOC analyzer (Series II ANATOC SGE TM, Australia) [20, 21]. The concentration of metals was also determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). To prepare the soil samples, first 5 ml of nitric acid (0.5 M), 2 ml of hydrochloric acid (0.5 M) and a few drops of concentrated sulfuric acid were respectively added to 1 g of each soil sample at 25 °C, and the sample was dried over heat at 40 °C. Then, 100 ml of 0.01% EDTA was added to it and the solution was passed through a Whatman filter paper No. 4. The metal ions in the filtrate were then measured by an ICP-OES device (Nikon, Japan). The plasma capacity (W), plasma flow rate (L/min), Nebulizer gas flow rate (L/min), and sample flow rate (mL/min) were 1500, 15. 0.8, and 1.5, respectively. The wave length (nm) which used for the analysis of Cu, Ni, and Mn were 324.7, 231.6, and 259.3, respectively [22].

Isolation and counting of heterotrophic bacteria resistant to the studied metals in soil samples

For this purpose, PHG II agar culture medium contained 4.0 g/L peptone, 2.0 g/L glucose, 1.0 g/L yeast extract was used. After the temperature of the molten culture medium reached 55 °C, solutions containing CuSO₄, NiCl₂ and MnCl₂ (Merck, Germany), each with a concentration of 5 mM, were added separately to the culture medium and after complete mixing, the pH values were adjusted to 7. Then the culture media were distributed in sterile plates [22]. Next, 100 µl of each dilution (10^-1-10^-6) prepared from the soil sample was spread on the metal containing PHG II culture media. PHG II agar without metal was also cultured as control. The plates were incubated for 2–5 days at 30 °C in aerobic condition in an incubator (Jahl Tajhiz, Iran) and then the colonies of heterotrophic bacteria resistant to copper, nickel and manganese were counted visually in each dilution which formed lower than 300 colonies on the medium and the average colony counts were detected. Then the obtained bacterial colonies resistant to each three metals were selected and sub-cultured 2–3 times on PHG II culture medium containing the concentration of 5 mM of the same metal which the isolate was resistant to it and incubated at 30 °C for 24 h until obtaining purified colonies [22, 23].

Determining the resistance of isolated bacteria to the studied metals

First, the isolates were cultured individually on nutrient agar (Charlotte, Spain) plates containing metal salts with a concentration of 2 mM, and after observing the growth of the bacteria in the plates, the metal salt concentration was increased so much that the growth was stopped. Based on the results of these tests, all isolates were cultured on nutrient agar pates containing different concentrations of copper sulfate (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, and 300 mM), nickel chloride (2, 4, 6, 8, 10, 12, and 14 mM) and manganese chloride (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mM). By this method, the concentration limits for determining minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) for each strain were determined. In the following, the resistance of the isolates to different concentrations of heavy metals was evaluated by the agar well diffusion method. In this method, first, each isolated bacterium with a count equivalent to 0.5 McFarland standard (1.5 × 10⁸ CFU/ml) were cultured on Muller Hinton agar (Charlotte, Spain). Then, wells (with a diameter of 6 mm and a distance of 24 mm from each other) were cut in the culture and 90 µl of each concentration of each metal salt was added to each well. Sterile physiological serum was used as a negative control and tetracycline antibiotic (2 mg/ml) was used as a positive control. Then the plates were incubated at 30 °C for 24 h. After that, the diameters of growth inhibition zones (mm) were measured. The experiments were repeated three times for each concentration of the metal salts and the controls, and then the average results were recorded [22].

Determination of MIC and MBC of three metals on the isolated bacteria

The macro-dilution method was used. For this purpose, bacterial suspensions of selected isolates containing 1.5 × 10⁸ CFU/ml were inoculated separately into the tubes containing Luria Bertani (LB) broth (Charlotte, Spain) containing different concentrations of each metal (copper sulfate at the concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, and 300 mM and nickel chloride at the concentrations of 2, 4, 6, 8, 10, 12, and 14 mM). Two tubes were considered as negative control (culture medium without bacteria) and positive control (culture medium with bacteria). The tubes were incubated at 30 °C for 24 h. After that, the minimum concentration of metal that inhibited the growth of bacteria was detected by observation the optical density of the culture medium in a light spectrophotometer (Indiamart, India) at the wave length of 620 nm. The metal concentration in which no growth was seen was considered as MIC. Then a sub-culture was made by transferring the culture media to nutrient agar containing the same concentration of the metal and incubated at 30 °C for 24 h. Then the concentration of the metal in which no bacterial colonies were grown, was considered as MBC [24].

Evaluation of the growth rate of isolated bacteria resistant to the studied metals

First, a bacterial suspension with a count of 1.5 × 10⁸ CFU/ml was prepared from the 24-h culture of each isolate in LB broth (Charlotte, Spain). Then, for each isolate, one flask containing LB broth without metal salts (control group), and one flask containing two concentrations of each metal salt, lower than MIC, were prepared. To each flask, 1 ml of bacterial suspension was inoculated and incubated for 30 h at 30 °C and 150 rpm agitation. Since the addition of metal salts caused turbidity in the culture medium, the bacteria counting on LB agar was used for determination of bacterial growth instead of turbidity assessment. For this purpose, at 2-h intervals, 50 µl of each grown culture medium was transferred to LB agar, and after 24 h incubation at 30 °C, the number of colonies was counted and based on the results, the bacterial growth curve was drawn [24].

Observation of the surface morphology of the studied isolates with scanning electron microscopy (SEM)

The changes in the morphology of the bacterial isolates cell surface after the treatment with metal salts in comparison to the surface of bacteria in control groups was observed by SEM. For this purpose, first the cells were fixed with glutaraldehyde and then dehydrated with different concentrations of ethanol (30–98%, 15–20 min each). Next, the samples were dried by a freeze dryer (Labconco, USA) and examined by a SEM (ZEISS, Germany) [25, 26].

Molecular identification of resistant bacterial isolates to the studied metals

Molecular identification of selected bacteria was done using PCR and sequencing of 16S rDNA [27]. For this purpose, first DNA was extracted utilizing boiling method. The quality of the extracted DNA was determined by the measurement of A260/280 and A260/230 ratios. The extracted DNAs with the ratios ~ 1.8 were used as target DNAs in PCR. The universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-ACGGCTACCTTGTTACGACTT-3') were used for the gene amplification [28] and the obtained fragments with a size of approximately 1400 bp (Fig. 1, supplementary material) were sequenced utilizing a DNA sequencer system (Thermo Fisher Scientific Applied Biosystems 3500 Series Genetic Analyzer) based on Sanger method, following AMPure PB purification. The obtained sequences were evaluated using Chromas 2.6.6.0 software and then were aligned with other sequences in NCBI database by using BLAST server.

Fig. 1.

Resistance of the isolates FM1 and FM2 to different concentrations of copper sulfate (a and b). The resistance of the isolate FM4 to different concentrations of nickel chloride (c) and the resistance of the isolate FM5 to different concentrations of manganese chloride (d) by agar well diffusion method in Mueller Hinton agar after 24 h’ incubation at 30 °C 

Determination of the biological removal of the studied metals

In order to study the amount of biosorption of copper, nickel and manganese metals by the selected strains, first the bacteria were inoculated separately in 100 ml of LB broth and incubated for 24 h at 30 °C in a shaker incubator at 150 rpm. Then, each isolate was re-inoculated (1% v/v) to 100 ml of LB broth containing a concentration of the metal salt that the isolates had shown resistance to it and was incubated for 48 h at 30 °C in a shaker incubator at 150 rpm. After confirming the viability of the bacteria through transferring and checking their growth in LB agar containing metal salt, 1 ml of each culture was transferred to a sterile microtube and centrifuged at 10000 rpm for 8 min at 4 °C. Then, the supernatant was separated to measure the amount of metal reduction by ICP-OES, compared to the control. The remaining sediment was washed twice in sterile physiological serum and then it was dried at 60 °C for 6 h and was weighted to determine the biomass of the absorbent. To measure the amount of metal in the sediment, 1 ml nitric acid (0.5 M) was added to it and heated at 100 °C for 60 min. Then the volume of the sediment was adjusted to 5 ml by double distilled water and the amount of metal was measured by ICP-OES [29, 30]. The tests were done in three replicates. The biosorption capacity (the amount of metal absorbed per g of biosorbent), was calculated using the following Eq. (31):

Biosorption capacity = (C₀—C_e) V/m; in which C₀ is the initial concentration of the metal (mg/L), C_e is the final (end) concentration of the metal (mg/L), V is the volume of solution (L). The supernatant resulting from each centrifugation stage was collected in the washing step, and to each 1 ml of the supernatant, 1 µl nitric acid was added and the metal content was measured by ICP-OES. The biological metal removal capacity was calculated using the following Eq. (31).

Metal bioremoval percentage = 100 (C₀- C_e) /C₀; in which C₀ is the initial concentration of the metal (mg/L) and C_e is the final (end) concentration of the metal (mg/L), V is the volume of solution (L), and m is the biomass of the adsorbent (g).

Investigating the ability of strains to produce metal nanoparticles

To perform this step, first, each resistant isolate was inoculated into metal-free LB broth and incubated at 30 °C for 24 h. Then, each isolate was re-inoculated (1% v/v) to 100 ml of LB broth containing a concentration of the metal salt that the isolates had shown resistance to it and was incubated for 5 days at 30 °C in a shaker incubator at 150 rpm. Then the culture medium was centrifuged at 10000 rpm for 5 min and the supernatant was discarded. Next, to lyse the bacteria, 1 ml deionized water was added to the bacterial biomass and mixed well and then heated by boiling for 20 min. The obtained suspension was centrifuged again and the supernatant was completely discarded. Finally, the bacterial pellet was allowed to dry [31, 32]. In order to investigate the synthesis of metal nanoparticles by metal-resistant bacteria, first changes in the culture medium color were observed [33]. Then, X ray diffraction (XRD) (Asenware AW-DX300, England) with a range of angle 2θ from 10֯ to 90֯ was used to determine the crystal nature of metal nanoparticles. The spectra were obtained by changing the conditions, including (exposure time and the amount of metal) the nanoparticle production was proved. The isolates that were not able to form nanoparticles under any conditions were used as negative control in the next experiments. The crystal size of the synthesized nanoparticles was calculated using the Debye- Scherrer equation:

D = kλ/β (cosθ); in which D is the size of the nanoparticle crystals (nm), K is Scherer's constant (0.9–1), λ is the wavelength of the X-ray radiation (0.1541 nm), β is the full-width at half-maximum (FWHM) of the peak (in radians), and θ is the angle between the radiation beam and its reflection. After confirmation, the nanoparticles were selected by SEM (ZEISS, Germany) following sputter coating with 4 nm gold and then the chemical properties of them were analyzed using energy-dispersive X-ray spectroscopy (EDS) (Tescan mirror, Czech Squaki) following deposition of nanoparticles on the Si/SiO2/Pt substrate. Unbound osmium tetroxide was removed with running tap water for 1 h [34, 35].

Statistical analysis of data

Statistical package for the social sciences (SPSS) software version 16 was used for calculating the mean ± standard deviation. The Gaussian normal distribution of data was tested and one-way analysis of variance (ANOVA) was used for comparing the results at the significance level of 5%.

Results

Characteristics of the soil contaminated with electronic wastes

The soil pH was 9.4, its temperature was 27.8 °C, and its TOC was calculated as 1.8%. According to ICP-OES analysis, the amount of copper, nickel and manganese in the soil was measured as 1.9 × 10⁴ mg/kg, 0.011 × 10⁴ mg/kg, and 0.013 × 10⁴ mg/kg, respectively which was significantly higher than normal amounts (0.02 mg/kg, for copper, 0.05 mg/kg for nickel, and 2 mg/kg for manganese), according to the proposal of the World Health Organization. Considering the high concentration of the three metals in the soil sample, the presence of bacteria resistant to these metals was possible.

Isolation and counting the heterotrophic and metal-resistant bacteria

The results showed that the number of bacteria in the culture medium without metals (7967 × 10⁴ ± 530 × 10⁴ CFU/ml) was more than the number of bacteria in the medium containing nickel 5 mM (833 × 10⁴ ± 104 × 10⁴ CFU/ml), in the environment containing copper 5 mM (5804 × 10⁴ ± 356 × 10⁴ CFU/ml), and in the environment containing manganese 5 mM (1963 × 10⁴ ± 131 × 10⁴ CFU/ml) (p > 0.05, ANOVA). According to these results, the number of resistant bacteria in the culture medium containing copper was more than that in the culture medium containing manganese; and the lowest number of resistant bacteria was counted in the culture containing nickel.

The resistance of isolated bacteria to the studied heavy metals

Different bacterial isolates were obtained from the soil samples. The MICs of copper, nickel, and manganese on the isolates were determined as 40–300 mM, 4–10 mM, and 60–120 mM, respectively. The results of bacterial culture on different concentrations of metal salts showed that the isolates FM1 and FM2 were respectively the most resistant isolates to copper while FM4 and FM5 isolates were the most resistant isolates to nickel and manganese, respectively. From the resulting isolates, a 24-h culture was prepared in the presence of different concentrations of metal salts separately at a temperature of 30 °C. The results of this stage regarding copper resistant showed that the two isolates, FM1 and FM2 were able to grow in the concentrations of copper salt up to 70 mM and 150 mM, respectively while the nickel resistant isolate (FM4) was able to grow in the concentrations of nickel salt up to 10 mM and the manganese resistant isolate (FM5) was able to grow in the concentrations of manganese salt up to 100 mM. The results of investigating the resistance of the isolates to the studied metals by the agar well diffusion method showed that the isolates FM1 and FM2 were resistant to copper up to the concentration of 80 mM and 60 mM, respectively. The results also showed that the isolate FM4 was resistant to nickel up to the concentration of 10 mM and the isolate FM5 was resistant to manganese up to the concentration of 90 mM (Fig. 1).

The results of investigating the MIC and MBC of the studied metal salts on the bacterial isolates by macro-dilution method and then culturing in agar culture medium showed that the average values of MIC and MBC of copper metal on the first copper-resistant isolate (FM1) were 333.33 ± 76.4 and 383.33 ± 76.4 mM, respectively while on the second copper resistant isolate (FM2) it was 70 ± 7 and 81.67 ± 6.2 mM. These values were equal to 10.67 ± 0.9 mM and 14 ± 0.01 mM on the nickel-resistant isolate (FM4), and 130 ± 43.2 and 146.67 ± 37.7 on the manganese-resistant isolate (FM5), respectively. The results of colony counting of bacteria are shown in Fig. 2.

Fig. 2.

Colony count results of the isolates in different concentrations of metal salts in nutrient agar after 24 h’ incubation at 30 °C. a: the isolates FM1 and FM2 which were resistant to copper, c: FM4 which was resistant to nickel, and FM5 which was resistant to manganese. The results are the means of 3 biological replicates (n = 3). * and ** show the significant differences of colony count results at the levels of 0.05 and 0.01, respectively, with the bacterial counts in the absence of metal salts

Growth curve of the studied isolates

The growth curve of FM1, FM2, FM4 and FM5 isolates in LB broth culture media without metal and containing investigated metals during the incubation period of 30 h and at the temperature of 30 °C are seen in Fig. 3. As the concentration of metals increased, the growth of bacteria decreased, and these decreases in the growth rates were depended on the concentration of the metal. The observations showed that with the increase in the concentration of copper, nickel, and manganese, the time of lag phase of bacterial growth was increased and the exponential phase of the growth was progressed with a lower slope and a longer delay than the control.

Fig. 3.

The growth curve of the isolates FM1 (a), FM2 (b), FM4 (c), and FM5 (d) in LB broth culture medium at the temperature 30 °C. Ctrl (control), shows the growth curve of bacteria in metal-free medium

Changes in the cell surface morphology of metal-resistant isolates in SEM images

The results can be seen in Fig. 4. Cells had smooth and elongated cell walls before exposure to copper. The cells of isolate FM1 in the presence of copper, the bacterial cell wall lost its shape and the bacteria has a completely different appearance and irregular surface. The effect of copper on the isolate FM2 was less than on the isolate FM1. Some cells of this isolate exposed to copper had a slight change in the shape of the cell wall surface, but they kept the overall shape of the wall, but others had more changes in the cell wall, shape and size of the cells. Observation of the surface of FM4 isolate after contact with nickel chloride showed that the cell wall and cell integrity of the bacteria were completely destroyed. After being exposed to manganese chloride, the cell wall of the FM5 isolate was changed, affecting their shape and size, making them completely different than before the exposure to manganese. Bacteria are seen in the presence of manganese in a spherical form and in some places as a mass.

Fig. 4.

Scanning electron microscopy image of the cell surface of the studied isolates. The isolate FM1 in the absence (a) and presence (b) of copper sulfate; the isolate FM2 in the absence (c) and presence (d) of copper sulfate; the isolate FM4 in the absence (e) and presence (f) of nickel chloride; the isolate FM5 in the absence (g) and presence (h) of manganese chloride. Bacterial cells and metal salt aggregates are shown by yellow and green arrows, respectively

Molecular identification of the studied isolates

All of the isolated bacteria were Gram-positive bacilli with aggregate or chain arrangements. PCR product of 16S rDNA was determined in resistant isolates in agarose gel electrophoresis with a length of 1500 bp. Comparing the sequence of amplified fragments using BLAST server showed that FM1, FM2, FM4, and FM5 strains had 99.79%, 99.90%, 99.69%, 99.57%, and 99.35% genetical similarity to Bacillus cereus strain F-MOEINI 2021, Bacillus thuringiensis strain F-MOEINI 2021, Bacillus paramycoides strain F-MOEINI 2021, Bacillus wiedmannii strain F-MOEINI 2021, respectively, and their sequencies were deposited in the NCBI database with the accession numbers of MZ274432, MZ291698, MZ292904, and MZ292987, respectively.

Absorption and biological removal of copper, nickel and manganese metals by the resistant strains

The results of bacterial counting in order to check the viability and growth strength during the biosorption phase, at the initial and final time of culturing is presented in Fig. 5. The results showed that these bacteria can act as biological absorbers in laboratory conditions as the number of bacteria in the biosorption stage was increasing. Among them, the highest rate of biological removal was related to Bacillus wiedmannii by 91.96% removal and the lowest rate was related to Bacillus thuringiensis by 68.69% removal. The results are presented in Table 1.

Fig. 5.

Count of bacteria in terms of CFU/ml at the beginning and end of biosorption process. The results are the means of 3 biological replicates (n = 3). * shows the significant differences of initial and final colony counts at the levels of 0.05

Table 1.

Average biological removal of copper, nickel and manganese metals by the isolated bacteria 

Bacteria	Removed metal	The initial amount of metal (mg/l)	Amount of metal in the culture supernatant (mg/l)	Amount of metal in the Biomass (mg/l)	Metal removal percentage
Bacillus cereus	Cu	151.60 ± 11.00	30.60 ± 1.56	119.50 ± 6.84	79.81
Bacillus thuringiensis	Cu	119.80 ± 13.45	37.50 ± 2.03	81.50 ± 3.50	68.69
Bacillus paramycoides	Ni	54.32 ± 1.15	7.20 ± 0.31	46.97 ± 1.46	86.74
Bacillus wiedmannii	Mn	438.20 ± 24.06	35.20 ± 3.01	374.50 ± 15.87	91.96

Investigating the biological removal of copper, nickel and manganese metals with ICP-OES device in the studied strains showed that the isolated bacteria had a high ability to remove these metals.

Investigating the ability of the metal resistant strains for metal nanoparticles production

The obtained results indicated that among the 4 isolated strains resistant to copper, nickel and manganese; Bacillus cereus and Bacillus wiedmannii had the ability to produce copper and manganese nanoparticles, respectively. Confirming the production of nanoparticles by bacteria showed the changes in the color of LB broth from blue to green by Bacillus cereus (Fig. 6) and from light to dark by Bacillus wiedmannii (Fig. 7); which were the primary signs of copper and manganese nanoparticles production, respectively.

Fig. 6.

Color change of LB broth containing copper from blue (a) to green (b) during the synthesis of copper nanoparticles by Bacillus cereus strain FM1 along with culture medium sediments (c)

Fig. 7.

Change of LB broth containing manganese from light (a) to dark (b) during the synthesis of manganese nanoparticles by Bacillus wiedmannii strain FM5 along with culture medium sediments (c)

XRD results

To ensure the production and detecting the chemical properties of the nanoparticles, XRD analysis was performed. Figure 8-a shows the XRD pattern of the nanoparticles synthesized by Bacillus cereus isolate FM1. X-ray diffraction results confirmed the presence of the crystal structure of copper nanoparticles. This diffraction pattern had peaks at angles of 23.6º, 32.8º, 35.6º, 38.7º, 48.8º, 51.7º, 58.4º, 61.6º, 66.1º, 68.2º, 71.7º, and 74.9º can be assigned to (012),(110), (111), (111), (202), (020), (202), (113), (311), (220), (202) and (004) planes of the monoclinic (Tenorite) CuO (JCPDS card No. 48–1548) [36, 37]. The XRD analysis for the identified copper nanoparticles indicated that the most intense peak was located at 2θ = 35.6 ͦ with the half-height width equal to β = 0.398, which was determined by using the Debye Scherrer formula; the size of copper nanoparticle crystals was approximately 20 nm (Fig. 8-a). Figure 9-a shows the XRD pattern of the nanoparticles synthesized by Bacillus wiedmannii isolate FM5. X-ray diffraction results confirmed the presence of a crystalline structure of manganese nanoparticles. This diffraction pattern had main peaks at the angles of 26.5º, 28.2º, 31.6º, 34.7º, 36.1º, 45.5º, 58.2º, 60.2º, and 64.5º can be assigned to (112), (200), (103), (211), (004), (220), (321), (224), and (314) planes that compatible with JCPDS card No. 18–0803 of manganese oxide [38, 39]. XRD analysis of identified manganese nanoparticles indicated that the most intense peak was located at 2θ = 34.6 º with the width at half maximum height (FWHM) equal to β = 0.205. Using the Debye Scherrer formula, the size of manganese nanoparticles crystals was approximately 40 nm (Fig. 9-a).

Fig. 8.

A a) X ray diffraction analysis of the synthesized copper nanoparticles. b) Scanning electron microscopy image of the synthesized copper nanoparticles c) Elemental analysis of synthesized copper nanoparticles with energy-dispersive X-ray spectroscopy

Fig. 9.

A a) X ray diffraction image for synthesized manganese nanoparticles. b) Scanning electron microscopy image of synthesized manganese nanoparticles c) Elemental analysis of synthesized manganese nanoparticles by energy-dispersive X-ray spectroscopy

SEM results

Examining the SEM images confirmed the presence of copper nanoparticles in the bacterial cell mass. The obtained images showed nanoparticles of almost the same shape, which were elongated. These images showed that the particles were clustered or scattered together in some areas, and the average size of these nanoparticles was estimated to be 20–30 nm (Fig. 8-b). Examining the SEM images confirmed the presence of manganese nanoparticles in the bacterial cell mass. The obtained images showed that these particles are mostly piled together. They have oval and elongated morphology and their average diameter was estimated to be approximately 40 nm (Fig. 9-b).

EDS results

For microscopic elemental analysis, randomly selected particles were analyzed by EDS. Copper and oxygen elements were detected in the sample, which corresponded to the compounds detected in XRD. The amount of detected copper was more than other elements and equal to 22.89% by weight (Fig. 8-c). Manganese and oxygen elements were detected in the sample, which corresponded to the compounds identified in XRD. The amount of manganese detected was more than other elements and equal to 91.23% by weight (Fig. 9-c).

Discussion

Electronic wastes are among the increasing accumulated wastes in the world. The large amounts of electronic waste produced in the world can contain toxic substances [5, 36] which contaminate the environment mostly due to improper processing of these wastes in many countries [4]. Metals such as copper, nickel, and manganese are found in large amounts in electronic waste such as the wastes of rechargeable batteries and electronic boards [37, 38]. In the current study, the amounts of heavy metals copper, nickel and manganese in the soils of Zainal Pass hills located in Isfahan, Iran, containing electronic waste, was detected as 1.9 × 10⁴ mg/kg, 0.011 × 10⁴ mg/kg and 0.013 × 10⁴ mg/kg, respectively which are several times higher than the permissible limits according to the proposal of the World Health Organization [29, 39, 40].

The isolates FM1 and FM2 were resistant to copper, the isolate FM4 was resistant to nickel, and the isolate FM5 was resistant to manganese. FM1, FM2, FM4, and FM5 were identified as Bacillus cereus, Bacillus thuringiensis, Bacillus paramycoides, and Bacillus wiedmannii, respectively. Bacillus species are fast growing microorganisms that can survive in many environmental conditions. One of the unique features of this genus of bacteria is the formation of spores in adverse environmental conditions such as the presence of heavy metals [41, 42]. Pradeepa and Kavitha (2020) isolated Bacillus species, mainly Bacillus cereus, from soils contaminated with electronic wastes in the Trichy region of India and then investigated the resistance of these bacteria to heavy metals. Among 44 isolates, 31 isolates were able to grow in the concentrations of metals up to 3 mM and 4 isolates were able to grow in the concentrations of metals up to 4 mM [38]. Glibota et al. (2019) in a study isolated and molecularly identified copper-resistant bacteria from agricultural soils in olive tree farms which were contaminated due to the use of agricultural pesticides. Similar to the present study, Bacillus spp. were among the most bacteria isolated from these soils [43]. Bacillus cereus isolate FM1 and Bacillus thuringiensis isolate FM2 isolated from electronic waste soil in the present study showed high resistance to copper so that the average MIC and MBC of copper on Bacillus cereus were 333.33 ± 76.4 mM and 383.33 ± 76.4 mM, respectively and this amounts on Bacillus thuringiensis were 70 ± 7 mM and 81.67 ± 6.2 mM, respectively. Also, their increasing resistant over time especially by Bacillus cereus FM1, indicated their compatibility to high amount of copper in the studied soil.

The average values of MIC and MBC of nickel in the present study on Bacillus paramycoides isolate FM4 were 10.67 ± 0.9 and 14 ± 0.01 mM, respectively ND The average MIC and MBC of manganese on Bacillus widmannii isolate FM5 was 130 ± 43.2 and 146.67 ± 37.7 mM, respectively. Chaudhary et al. (2017) obtained two isolates of Escherichia coli (AS17b and AS21) and one isolate of Microbacterium (AS33) resistant to nickel from industrial sites contaminated with glass, which were able to tolerate nickel in an amount greater than 24 mM [44]. Kaliyaraj et al. (2019) collected printing circuit boards from electronic waste dumping sites in India and investigated the tolerance of 5 Actinobacter isolates from them to copper and nickel. The highest concentration of copper and nickel that the isolates showed resistance to it was 1500 mg/l [45]. In the study of Pormohammad and Turner (2020), the maximum concentration of nickel which tolerated by Pseudomonas aeruginosa and Staphylococcus aureus was reported to be about 4 and 8 mM, respectively [46]. Raja et al. [47] reported the MIC of 8.6 mM by nickel on the bacteria isolated from South Indian urban sewage [47] which was close to the MIC value obtained in the present study on Bacillus paramycoides isolate FM4. In the research conducted by Freitas et al. (2018) on Aeromonas hydrophila, the MIC and MBC of manganese on the isolate reported equal to 2 mM and 3 mM and the MIC and MBC of copper on the isolate reported equal to 15 mM and 16 mM [48]. Veenemans et al. (2012) obtained the MIC of manganese against Acinetobacter baumannii, Enterobacter kwaki, Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli at least 0.09 and at most 48 mM [49]. In a study, Cai et al. (2019) isolated and isolated strains of Bacillus, Schwanella, and Actinobacterium from the wastewater of electroplating industries. The maximum tolerable concentration of manganese in their study was 200 mM [50], that is higher than the MIC of manganese in the present study. The fact that the MICs in some previous studies are different from the MICs obtained in the present study is probably due to the environmental conditions of the sample collection sites and different adaptation capacity of the isolated bacteria.

The growth curve of the isolates in the present study showed that with the increase in the concentration of metals, the growth of bacteria decreased significantly and this decrease in the growth rate was dependent on the metal concentration. The observations of this study also showed that with the increase in the concentration of copper, nickel and manganese, the lag phase of the growth curve increased and the exponential phase had a lower slope and a longer delay than the control group. Although the metals examined in this research caused toxicity and decreased growth in bacteria, the growth of bacteria in the presence of the corresponding metal can be a sign of biocompatibility and toxicity tolerance of bacteria. The decrease in the number of bacteria in the presence of metals compared to the control was expected due to the toxicity of the metal and has also been observed in other studies [31, 51].

SEM images showed the morphological changes of bacteria including swelling, shortening, and irregularity of the bacterial cell wall, after being in contact with copper, nickel, and manganese. The presence of copper in the culture medium caused more drastic changes in the cell wall morphology and cell size of Bacillus cereus isolate FM1 compared to Bacillus thuringiensis isolate FM2. Bacteria become resistant to metals by producing compounds such as biosurfactant, indole acetic acid, and siderophores, and finally, functional groups present in the cell wall of bacteria such as hydroxyl groups, amines, sulfhydryl, carboxyl, phosphate and amide bonds that have a negative charge and react with metal cations such as Mn²⁺, Ni²⁺ and Cu²⁺; leading to changes in cell wall morphology [52]. Bacillus paramycoides isolate FM4 completely lost its shape after contact with nickel, and Bacillus wiedmannii isolate FM5 changed from elongated and regular to spherical and irregular shape under the influence of manganese; even in some sites clumps of bacteria were observed. These morphological changes of the bacterial surface may be due to the deposition of used metals around the cell surface and the functional groups attached to heavy metals [53].

The biological absorption of metals and its accumulation in the biomass of bacteria, as well as the removal of metals indicated the bioremediation power of the isolates [35, 54]. Investigating the biosorption potential of copper, nickel and manganese by bacteria isolated from electronic waste in the present study showed that these bacteria can continue to grow in the presence of metal salts and act as a biosorbent. The results of ICP-OES indicated that the highest biological removal rate belonged to the isolate resistant to manganese (Bacillus wiedmannii isolate FM5) with 91.96% removal, and the lowest was belonged to the isolate resistant to copper (Bacillus thuringiensis isolate FM2) with 68.69% removal. In a study, Ghaed et al. (2013) investigated the biosorption of copper by Bacillus spp. and reported that bacteria isolated from industrial wastewaters can perform biosorption with 90% efficiency [55]. Sedighi et al. (2012) isolated different strains of Bacillus thuringiensis from the acid mine effluent that contained various heavy metal ions and then investigated the absorption of copper and manganese ions in the same wastewater using the isolated bacteria. The absorption rate of copper and manganese ions in this research was equal to 99% and 98%, respectively, and with increasing contact time, the absorption rates were increased [56]. The bio-removal rate by the investigated strains in the present study was similar to the mentioned studies. Kulkarni et al. (2014) showed that Bacillus laterosporus is an effective biosorbent for nickel. The maximum absorption capacity of nickel from electronic waste soil in their study was 44.44 mg/g [57]. The biosorbent which was isolated in the present study for the biological removal of nickel was also of Bacillus genus. The biological removal of nickel was done well (86.74%) by Bacillus paramycoides strain FM4, which is similar to the results of the research by Mardiyono et al. (2019) who reported Bacillus subtilis with the ability to biologically remove nickel by 85.61% efficacy [58]. Njoku et al. (2020) investigated the biosorption of nickel by Bacillus megaterium isolated from nickel-contaminated soils and reported the amount of nickel biosorption by this bacterium as 541.50 mg [59]. The strains isolated in the study of Kashyap et al. (2021) for the biological removal of nickel belonged to Bacillus safensis and Bacillus cereus species, and the percentage of nickel biological removal was 91.3% and 57.2%, respectively [60]. Zhenggang et al. (2019) reported the biosorption ability of Bacillus cereus strain HM7 isolated from manganese ore soil samples as 98.9% [61], which is similar to the amount of metal removal by Bacillus wiedmannii isolate FM5 in the present study (91.96%). Huang et al. (2020) isolated a strain with high manganese tolerance from soil samples collected from manganese ore. Bacillus thuringiensis strain HM7 isolated in their study was able to remove manganese by 95.04% at 30 °C [52]. The amount of manganese absorption in the present study is similar to their research, which is probably due to the high level of manganese in both studied soils.

In the last decade, metal nanoparticles have gained special importance due to their application in biotechnology and having unique physicochemical properties. Synthesis of metal nanoparticles using microorganisms as an environmentally friendly method and is actually a suitable strategy as an alternative to chemical and physical methods. A better understanding of the microbial transformation pathway at the genetic level may lead to the development of new genetic tools to accelerate bioremediation strategies [62]. Tatariants et al. (2018), chemically synthetized copper nanoparticles from electronic wastes and investigated their antibacterial effects on pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The results showed that the produced copper nanoparticles had an average size of 7 nm and an efficiency of 90% while the preparation cost was 6 times lower than the commercial methods [63]. John et al. (2021) carried out the microbial synthesis of copper nanoparticles using bacterial strains isolated from Antarctica. UV–visible spectra showed the maximum absorption of nanoparticles in the range of 380–385 nm. Electron microscopy analysis showed that the synthesized nanoparticles were all dispersed, spherical in nature, and well separated without any agglomeration and of medium size. The infrared spectrum confirmed the presence of proteins on the surface of nanoparticles that acted as stabilizers [62]. In the present study, Bacillus cereus strain FM1 isolated from electronic waste soil was able to synthesize copper nanoparticles with an approximate size of 20 nm by XRD [64, 65], which in the SEM images showed an elongated morphology and were scattered without any accumulation. Studies by researchers have shown that the absorption of heavy metals during the biosorption process takes place through ion exchange, complexation and biomineralization [66]. Functional groups on the bacterial cell walls have been effective in the biosorption of metals and the production of metal nanoparticles by microorganisms [52, 67]. In the present study, it was observed that manganese nanoparticles with an efficiency of 91.23% and an approximate size of 40 nm were synthesized from Mn₃O₄ by Bacillus wiedmannii strain FM5. It was shown in Wright et al.'s study (2016) that Shewanella oneidensis PV-4 and Shewanella putrefaciens CN-32 were able to produce manganese nanoparticles [68]. In a study reported by Sinha et al. (2011) on Bacillus spp. isolated from oil-contaminated soils in India, it showed that Bacillus strain MTCC10650 was resistant to all kinds of heavy metals and had the property of manganese bioaccumulation. The formed bionanoparticles were characterized and identified using EDS, high resolution transmission electron microscope, X-ray photoelectron spectroscopy, X-ray diffraction and atomic microscope. In the presence of manganese, bacteria synthesized manganese oxide nanoparticles (MnO₂) with spherical and monodispersed shape and an average size of 4.62 ± 0.14 nm and the synthesized nanoparticles showed maximum absorption at 329 nm [69]. In the present study, it was found that Bacillus wiedmannii isolate FM5 is capable of producing manganese oxide (MnO₂) nanoparticles with an approximate size of 40 nm and an oval shape, which was in line with the study of Sinha et al. and other previous study [70].

Conclusions

The results of the present study showed the high resistance of newly identified Bacillus cereus, Bacillus thuringiensis, Bacillus paramycoides, and Bacillus wiedmannii isolated from the electronic waste landfill in Zainal hills, Isfahan, Iran to the heavy metals copper, nickel and manganese with high MBCs (up to 383.33 ± 76.4 mM for copper, up to 14 ± 0.01 mM for nickel, and up to 146.67 ± 37.7 mM for manganese). The isolates had a great ability (up to 91.96%) for removal of metals from the culture media. On the other hand, the production of metal nanoparticles by the isolated bacteria was proven in laboratory conditions. These isolates can be suitable candidates for the investigations on the biological removal of these metals from electronic wastes and other industrial wastes, and for biological production of metal nanoparticles by using these wastes.

Supplementary Information

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Authors' contributions

F. Moeini: Formal analysis, writing original draft, and investigation. F. Moeini: methodology, writing, and review and editing. F. Moeini: writing original draft, analysis of results, and review and editing. M. Doudi, and F. Moeini: formal analysis. M. Doudi, M. Foulagar, Z. Emami Karvani and F. Moeini: review and editing. M. Doudi, M. Foulagar, Z. Emami Karvani and F. Moeini.

Funding

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Declarations

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Competing interests

The authors declare no competing interests.