The soluble nature of As makes its removal from the environment difficult [78]. Physical and chemical remediation of arsenic involves an oxidation step for converting As³⁺ into As⁵⁺. It may occur under atmospheric oxygen, which is usually very slow, or by using chemical oxidants like hydrogen peroxide, chlorine, or ozone. This method is very expensive and produces harmful byproducts [79]. Microorganisms use As as an energy source in their metabolic processes and thus transform the toxic As³⁺ into its less toxic form, As⁵⁺ [80,81] by arsenic oxidase, which is present in the protoplasm of arsenic-oxidizing bacteria [59]. Liao et al. [82] reported Bacillus as one the important arsenic-reducing bacteria. Arsenic removal by Bacillus spp. like B. megaterium [83,84], B. aryabhattai [85], and B. cereus strain W2 was studied by Miyatake and Hayashi [86]. Anaerobic respiration of As⁵⁺ by B. cereus was reported by Ghosh et al. [71]. Various other studies conducted over the years have reported the ability of Bacillus spp. to uptake As [87,88,89,90,91] (Table 1). As speciation, solubility, and mobilization are dependent on its methylation [92,93], oxidation [94], reduction, and respiration. The significance of oxidative phosphorylation in living organisms cannot be ignored [68]. Structurally, As⁵⁺ has similarity with phosphate [65]; thus, it is a potential oxidative phosphorylation inhibitor [68]. As⁵⁺ enters the organism’s living system by employing two pathways; Pit and Pst [94], which are usually used for phosphate uptake. It interferes with phosphorylation metabolic reactions and inhibits synthesis of adenosine triphosphate [59]. The portal of entry for As³⁺ is aquaglyceroporin proteins [65,95,96]. After internalization, it immediately binds to the respiratory enzymes via their sulfur residue [66,97]. Bioremediation of As in Bacillus spp. is done by ars operon [98] (Table 2) by employing three genes arsA, arsB, arsC, arsD, and arsR. arsA and arsB have ATPase activity, arsC transforms As³⁺ to As⁵⁺, arsD works as metallochaperone, while arsR acts as a repressor [99,100,101,102]. Normally, As⁵⁺ (less toxic form) that enters the cell is reduced to As³⁺ (more toxic form) by ArsC and then transported out of the cell by ArsB [103,104] (Figure 1).

The action of Bacillus spp. in removing Zn from contaminated environments has been highlighted in many bioremediation studies. This ability of Bacillus spp. is regulated either by acquiring resistance through plasmids or by evolving mechanisms of resistance [208,209]. In B. subtilis, Zn uptake is regulated by the Zur family, which enables the transport of Zn ions via two transporter proteins. Gaballa et al. [157] also reported a third uptake system in B. subtilis for Zn ions called ZosA (P-type ATPase), which was expressed in conditions of oxidative stress. Efflux of Zn in high concentrations is facilitated by a CPx-type ATPase efflux pump in B. subtilis, known as CadA [157,158] (Table 2, Figure 2). Moreover, the removal of Zn by Bacillus sp. such as B. subtilis, B. licheniformis, B. cereus, B. jeotgali, and B. firmus was reported in recent studies [105,106,107,108,109,110,111] (Table 1). Khan et al. [112] also reported the removal of Zn (87 mg/L) by B. altitudinis isolated from industrial wastewater.

There have been many methods of Ni removal from solid matrices, but the most effective are those which are capable of removing/treating Ni before it emanates into the environment [219]. Several physico-chemical methods have been employed over the years for the removal of Ni from aqueous solutions [220]. Regardless of which of these methods have been used in the past, newer, cheaper, and more efficient methods of adsorption have been used for Ni removal, such as the use of biomass, where sugarcane, corn cobs, citrus peels, and bark have been used [221]. In a study, corn hydrochar was treated with KOH and altered by treating polyethyleneimine (PEI) to increase adsorption of Ni ions onto the surface [222]. Bioremediation by Gram-positive bacteria, such as Bacillus spp., has been the better method for the removal of Ni ions from Ni-contaminated media. There have been many studies that demonstrate the uptake and/or removal of Ni ions from contaminated environments such as soils, wastewater, and rivers [105,113,114,115] (Table 1). B. thuringiensis has also been frequently reported to uptake and remove Ni from contaminated environments [116,117,118]. In a recent study, B. megaterium was isolated from Ni-contaminated soils and was able to uptake more than 500 mg Ni, where more than 3000 mg/L Ni salt was previously found [223]. The removal of Ni by bacteria is contingent on their inherent mechanisms of resistance, which ultimately facilitate uptake, transportation, and efflux of the metal ions in and out of the cell. According to Moore et al. [159], mechanisms of Ni homeostasis and regulation have not been well characterized in B. subtilis when compared to Gram-negative bacteria such as Escherichia coli and Helicobacter pylori, though some evidence suggests their presence [224]. Members of the cation diffusion facilitator (CDF) family have long been characterized to mediate efflux of multiple metal ions, including Ni [160]. In B. subtilis, the cation diffusion transporter CzcD is reported to provide protection to the cell amid high concentrations of Ni²⁺, Cu⁺, Zn²⁺, and Co²⁺ [159] (Figure 3). When these ions happen to be bound with citrate, these complexes, during favorable conditions, are taken up by the metal-dicitrate uptake system known as CitM in B. subtilis, consequently leading to an increase in toxicity to them [159] (Table 2).

Nature has gifted microorganisms with cadA operon to combat Cd toxicity. It is a 3.5 kb operon located on plasmid pI258. It has two genes; cadA and cadC [229,230]. cadA is transcribed into the 727-amino acid protein, which performs the function of energy-dependent Cd efflux ATPase [134], whereas cadC encodes relatively a small protein of 122 amino acids, and is a positive transcription regulator of Cd²⁺ operon [230,231]. It is well reported that cadA gene is induced in the presence of Cd²⁺ ions. Solovieva and Entian [161] documented their findings on cadA as a chromosomal determinant as well as a new gene yvgW of B. subtilis involved in Cd²⁺ resistance. Moreover, they reported that it has similarity to cadA of S. aureus plasmid pI258 [232]. Deletion of yvgW increased the Cd²⁺ sensitivity in the bacterium. cadB is also plasmid-mediated and confers Cd²⁺ resistance through a change in the binding site [162]. In addition to efflux mechanism, KinA and histidine kinase provide phosphate for phosphorylation which leads directly to transcription in B. subtilis [163]. Its overexpression results in phosphate flux of the cell, thereby directly affecting the energy state of the cell wall [233]. It also plays a significant role in biosorption of Cd²⁺ ions by phosphorylating the Bacillus cell surface magnitude, hence it acting as a major phosphate provider [122]. Cd²⁺ enters the bacterial cell membrane via the zinc and manganese transport systems, which are chromosomally mediated. Cd²⁺ concentration greatly affects the whole process. If the Cd²⁺ ions are present in high quantity in the medium, much extracellular adsorption is observed. Otherwise, intracellular Cd²⁺ concentration is high [234]. The cad operon of Bacillus megaterium TWSL_4 contains cadC, which has Cd²⁺ and Zn²⁺ metal binding motifs [235]. On exposure to metal ions, the first interaction is always with cell wall [236]. Its structure and composition play a significant role in deciding the next step of the process. Cd²⁺ adsorption on the bacterial cell wall deals with exchanging ions like Ca²⁺, Mg²⁺, and H⁺ ions [237]. Chelation is another process in which Cd²⁺ ions are exchanged with cell surface protons like –SO₃H, –COOH, and –NH [238]. It involves sequestration via intracellular metallothionein (MT) [239]. Inorganic deposition of Cd²⁺ in the cell wall or inside cells can take place through interaction with hydroxide, carbonate, sulfate, and phosphorus [240]. In addition to metal concentration, biosorption is dependent on cell wall composition and cell physiology [241]. In Gram-positive bacterial species, resistance to Cd²⁺ is achieved by cadA system that is plasmid-borne. Cd²⁺ enters the bacterial cell by the MIT (metal ion transporter) system [164,165] (Table 2). The genes for Cd²⁺ resistance are mostly plasmid-mediated. According to Chen et al. [242] they are found on R plasmid along with antibiotic resistance genes, e.g., in pathogens including K. pneumoniae, P. aeruginosa, and S. aureus. These genes are reported to be directly involved in the uptake of Cd²⁺ ions from the environment (Figure 4). Basha and Rajaganesh [120] reported B. licheniformis to be a good biosorbent for Cd²⁺, as it removed more than 98% of Cd²⁺. Other species such as B. catenulatus and B. safensis are also reported to be effective in removing Cd²⁺ [119,121] (Table 1).

Pb-resistant Bacillus spp. have been reported previously [245]. Bacillus uses pbr operon [166,167,168] and active transport [248] as potential strategies to combat the toxic effects of Pb (Table 2, Figure 5). Microorganisms immobilize it by adsorption, chelation, inorganic precipitation, complexation, and biosorption. These processes involve bacterial cell wall functional groups including phosphate, carboxyl, carbonyl, sulfhydryl, and hydroxyl groups, which confer a negative charge to the cell wall. Binding of Pb to any of them results in insoluble substance. On the outside environment, Pb²⁺ is exchanged by Na or K cations [124]. Another method is adsorption through the cell wall, as it is comprised of organic macromolecules including polypeptides, polysaccharides, and proteins, which have the ability to adsorb Pb via electrostatic forces including Van der Waal’s forces, covalent or ionic bonds [124]. Pb interferes with microbial growth, morphology, and biochemical activities by damaging the DNA, protein, and lipids and even replacing the essential ions within the enzymes [123,249]. Microbes resist Pb toxicity by extracellular precipitation, exclusion, volatilization, biomethylation, cell surface binding, intracellular sequestration, and enhanced siderophore production [123]. Much like other Gram-positive bacteria, Bacillus spp. also employ one or several of these methods to remove Pb from the contaminated environments [125,126,127] (Table 1).

Like many other heavy metal ions, Cu is considered to be essential for Bacillus subtilis, while concentrations exceeding normal amounts can be toxic for the cells. Species like B. thuringiensis, B. cereus, B. licheniformis, and B. sphaericus are also involved in the removal of Cu, when their concentrations exceed the required limit [117,128,129,130,131,132,133,255,256] (Table 1). In correlation with other bacterial species, Cu in the cytosol is regulated by CueR [257]. B. subtilis CueR is responsible for regulating the copZA operon which encodes both Cu chaperone and a P-type ATPase for Cu efflux, the latter of which is a member of the integral family which exports metal out of the cell [169]. In the former, CopZ plays a key role as Cu chaperone in transferring Cu over to CopA [170], which contributes to uptake of Cu, while CopB is accountable for Cu efflux and detoxification [171] (Table 2, Figure 1). Chillappagari et al. [172] reported that YcnJ was associated with Cu uptake function of copZA operon in B. subtilis, where their model proposed that the protein works in conjunction with other Cop proteins to facilitate Cu transport in and out of the cell (Figure 6).