2.1. Arsenic

Arsenic (As) is a naturally occurring toxic metalloid ^[10] that is reported in soil (5–10 mg/kg), rock (1–2 mg/kg), sea water (1–3 µg/L) ^[11][12][13], as well as air and volcanic ash (0.02 µg/m

³

₃) and methylated arsine species enter into the surroundings ^[14]. Its anthropogenic sources are herbicides, pesticides ^[15], fossil fuel combustion, mining, smelting, wood preservation, sludge, manure ^[16], paint pigments, ceramic, glass industry, and food additives ^[17][18][19]. It exists as insoluble sulfides and sulfosalts ^[20]. It has four oxidation states: As

^3-

⁰

³⁺

⁵⁺

³⁺

⁵⁺

³⁺

⁵⁺, which is mostly adsorbed to the sediments in an anoxic state ^[21][22]. The histidine part of cellular proteins is a target site for the interaction of thiols of cysteine residues and/or imidazolium nitrogen with As

³⁺, which results in the inactivation of enzymes ^[23]. As

⁵⁺ interferes with protein synthesis by replacing the phosphate group during phosphorylation of the energy transfer process. The existence of either state depends on the redox state of the environmental conditions ^[24], as well as the geomicrobial population ^[25]. The US EPA and WHO has allowed 10 µg/L as the permissible concentration of As in drinking water ^[16]. Above 0.5 ppm, it is toxic for living systems resulting in symptoms of skin cancer ^[26], loss of appetite, weakness, weight loss, lethargy, chronic respiratory disorders, gastrointestinal disorders, enlargement of liver, spleen disorders, anemia ^[27], and cardiovascular disease ^[28]. Introduction of arsenic into the food chain and ground water may lead to serious concerns of arsenicosis ^[22].

Bioregulation of Arsenic by 

Bacillus

 spp.

The soluble nature of As makes its removal from the environment difficult ^[29]. Physical and chemical remediation of arsenic involves an oxidation step for converting As

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 As

³⁺

 into As

⁵⁺. 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 ^[30]. Microorganisms use As as an energy source in their metabolic processes and thus transform the toxic As

. 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 As

³⁺

 into its less toxic form, As

^5+ [31][32] by arsenic oxidase, which is present in the protoplasm of arsenic-oxidizing bacteria ^[10]. Liao et al. ^[33] reported 

 [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 ^[34][35], 

 [83,84], 

B. aryabhattai ^[36], and 

 [85], and 

B. cereus strain W2 was studied by Miyatake and Hayashi ^[37]. Anaerobic respiration of As

 strain W2 was studied by Miyatake and Hayashi [86]. Anaerobic respiration of As

⁵⁺

 by 

B. cereus was reported by Ghosh et al. ^[22]. Various other studies conducted over the years have reported the ability of 

 was reported by Ghosh et al. [71]. Various other studies conducted over the years have reported the ability of 

Bacillus spp. to uptake As ^{[38][39][40][41][42]} (

 spp. to uptake As [87,88,89,90,91] (

Table 1). As speciation, solubility, and mobilization are dependent on its methylation ^[43][44], oxidation ^[45], reduction, and respiration. The significance of oxidative phosphorylation in living organisms cannot be ignored ^[19]. Structurally, As

). 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, As

⁵⁺ has similarity with phosphate ^[16]; thus, it is a potential oxidative phosphorylation inhibitor ^[19]. As

 has similarity with phosphate [65]; thus, it is a potential oxidative phosphorylation inhibitor [68]. As

⁵⁺ enters the organism’s living system by employing two pathways; Pit and Pst ^[45], which are usually used for phosphate uptake. It interferes with phosphorylation metabolic reactions and inhibits synthesis of adenosine triphosphate ^[10]. The portal of entry for As

 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 As

³⁺ is aquaglyceroporin proteins ^[16][46][47]. After internalization, it immediately binds to the respiratory enzymes via their sulfur residue ^[17][48]. Bioremediation of As in 

 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 ^[49] (

 operon [98] (

Table 2

) by employing three genes 

arsA

, 

arsB

, 

arsC

, 

arsD

, and 

arsR

. 

arsA

 and 

arsB

 have ATPase activity, 

arsC

 transforms As

³⁺

 to As

⁵⁺

, 

arsD

 works as metallochaperone, while 

arsR acts as a repressor ^{[50][51][52][53]}. Normally, As

 acts as a repressor [99,100,101,102]. Normally, As

⁵⁺

 (less toxic form) that enters the cell is reduced to As

³⁺ (more toxic form) by ArsC and then transported out of the cell by ArsB ^[54][55] (

 (more toxic form) by ArsC and then transported out of the cell by ArsB [103,104] (

Figure 1

).

Figure 1.

 Bioregulation mechanism of arsenic in 

Bacillus

 spp.

Table 1.

 Uptake ability of 

Bacillus

 spp. for various heavy metals.

Metal	Bacterial Strains	Initial Metal Concentration (%/mg/g/mM/ppm/mg/L)	Metal Uptake Ability (%/mg/g/mM/ppm/mg/L/mol/g)	Reference

Arsenic

Bacillus

 spp. for the bioregulation of heavy metals.

Metal	Protein(s)/Gene(s)	Method(s)	Reference
Bacillus	sp. KM02	100 ppm	ars operon (arsR, arsD, arsA, arsB, arsC)	Reduction (Detoxification) Efflux Cell membrane binding, Adsorption on cell surface Complexation by exopolysaccharides51.45% (As3+)	[10][32][38][50[53][10][59]
[	59	B. licheniformis B. polimyxa	0–100 mM 0–20 mM	100 ppm (As0) 100 ppm (As0)	[39][88]
Bacillus sp. IIIJ3–1	350 smM (As5+) 10 mM (As3+)	350 mM (As5+) 10 mM (As3+)	[22][71]
B. barbaricus	-	20 mM (As5+) 0.3 mM (As3+)	[42][91]
,	161	,	162,163,164,165	B. indicus Sd/3T	0 mM 0 mM	20 mM (As5+) 30 mM (As3+)	[40][89]
]
Lead	pbr operon	Efflux	[117][118][119][166,167,168]	B. selenatiredreducens	10 mM
Copper	CueR copZA operon (CopA, CopZ, CopB)	0 mM (As5+) 0.3 mM (As3+)	YcnJ[38][87]
Efflux by chaperone		Uptake	[120][121][122	B. arsenicus con a/3	20 mM 0.5 mM	20 mM (As5+) 0.3 mM (As3+)	[41][90]
B. cereus W2	50 mg/L	1.870 mg/L (As3+)	[37][86]
B. cereus EA5 B. fusiformis EA2	15 mg/L	94.9% 99.7%	[35][84]
B. arsenicus MTCC 4380	2000 mg/L 1800 mg/L	89.462% (As5+) 83.043% (As3+)	[43][92]
Zinc	B. subtilis	178 mg/L	49.7 mg/L	[56][105]
	Bacillus sp. (KF710041) B. subtilis (KF710042)	-	73.29% 78.15%	[57][106]
	B. licheniformis	-	53%	[58][107]
	B. cereus	0–200 mg/L	66.6 mg/g	[59][108]
	B. jeotgali	75 mg/l	30%	[60][109]
	B. subtilis D215	100 mg/L	63.73%	[61][110]
	B. firmus	100 mg/L	61.8%	[62][111]
	B. altitudinis	100 mg/L	87 mg/L	[63][112]
Nickel	B. subtilis	2.14 ppm	85.61%	[64][113]
	B. subtilis BM1 B. subtilis BM2 B. subtilis BM3	2–32 mg/L	98.54% 99.2% 96.3%	[65][114]
	B. subtilis	178 mg/L	57.8 mg/g	[56][105]
	Bacillus sp. KL1	100 ppm	55.06%	[66][115]
	B. thuringiensis KUNi1	0–7.5 mM	82%	[67][116]
	B. thuringiensis OSM29	25–150 mg/L	94%	[68][117]
	B. thuringiensis	250 mg/L	15.7%	[69][118]
Cadmium	B. safensis	40 ppm 60 ppm	83.5% 98.10%	[70][119]
	B. licheniformis	-	98.34%	[71][120]
	B. catenulatus JB-022	150 mg/L	66%	[72][121]
	B. thuringiensis DM55	0.25 mM	79%	[73]
[
82
]
[
131
]
B. subtilis D215	100 mg/L	67.18%	[61][110]
,	81	]	[,8751][,9952,100],101,102][	B. sphaericus B. cereus Bacillus sp.	17.6 mg/L 44.0 mg/L 88.0 mg/L	5.6 mol/g 5.9 mol/g 6.4 mol/g	[83][132]
Bacillus sp. SG-1	-	60%	[84][133]
Chromium	B. cereus NWUAB01	100 mg/L	43%	[85][134]
	]	B. cereus	100 mg/L	81%	[86][135]
	123	]	B. salmalaya 139SI	50 ppm	20.35 mg/g	[87][136]
	B. cereus FA-3	1000 μg/ml	72%	[88][137]
	B. licheniformis	15 mg/L	95%	[89][138]
	Bacillus sp. B	500–4500 mg/L	47%	[90][139]
	B. marisflavi	200 mg/L	5.783%	[91][140]
	[	169	,	170,	B. licheniformis	300 mg/g	69.4%	[92][141]
	B. thuringiensis	250 mg/L	83.3%	[93][142]
	B. licheniformis B. laterosporus	-	62 mg/g 72.6 mg/g	[94][143]
	B. circulans B. megaterium	0.96 mg/L	34.5% 32%	[95][144]
Mercury	B. thuringiensis CASKS3	200 mg/L 400 mg/L 600 mg/L	62.4% 54% 40%	[96][145]
	B. licheniformis	50 mg/L
	B. cereus
	5 mg/L	104.1 mg/g	[	100][149]
	Bacillus sp.	1–10 mg/L	7.9 mg/g	[101][150]
Manganese	B. thuringiensis HM7	400 mg/L	95.04%	[102][151]
	B. cereus HM-5	600 mg/L	67%	[103][152]
	Bacillus sp.	13.3 mg/g	55.56 mg/g	[104][153]
Molybdenum	Bacillus
	Zinc	Zur ZosA ycdHI-yceA yciABC CadA CzcD	Physico-chemical adsorption Ion exchange Efflux Uptake	[59][108][109][108,157,158]
Nickel	CzcD CitM	Effluxsp. Zeid 14	-	200 mg/L	[105][154]
Bacillus sp. strain A.rzi	0.1 mM	Not reported	[106][155]
[	110	]	[111][159,160]
Cadmium	cad operon yvgW KinA	Efflux	[73][85][112][113][114][115][116][122,134171,172]
Chromium	ChrR	Efflux, Uptake Enzymatic reduction (Detoxification)	[124][125][126][173,174,175]
Mercury	mer operon (merR, merA, merB) MerR MerA MerB	Efflux Enzymatic reduction (Detoxification)	[127][128][129][130][131][176,177,178,179,180]
Manganese	mntABCD operon MntR MntH MneP MneS	Efflux Uptake	[132][133][134][135][181,182,183,184]
Molybdenum	modABC operon	Uptake	[99][136][137][138][139][140][141][148,185,186,187,188,189,190]
Gold	Not reported	Bioaccumulation	[142][143][191,192]	[
Silver	SilP	122	]
sil	genes	Efflux	[144][145][146][147][148][149][193,194,195,196,197,198]	Lead	B. pumilus MF472596	100–1000 ppm	96%	[74][123]
B. subtilis X3	200–1400 mg/L	590.49 mg/g	[75][124]
B. cereus	5–100 mg/L	36.71 mg/g	[76][125]
Bacillus S1 Bacillus SS19	75 and 100 mg/L 50 mg/mL	53%, 51% 57%	[77][126]
Bacillus sp. AS2	500 ppm	74.5 mg/g (99.5 %)	[78][127]
Copper	B. cereus	100 ppm	54%	[79][128]
	B. cereus	400 ppm	48%	[80][129]
	B. thuringiensis OSM29	25 mg/L	91.8%	[68][117]
	B. licheniformis	5 gm/L	32%	[81][130]
	B. thioparans	40 mg/L	27.3 mg/g70%	[97][146]
	B. cereus BW-03(pPW-05)	5–50 ppm	96.4%	[98][147]
	B. licheniformis	100 μg/mL	70%	[99][148]
	Silver	B. licheniformis R08	100 mg/L	73.6 mg/g	[107][156]

Table 2.

 The proteins, operons, and methods of removal employed by 

Arsenic

2.2. Zinc

Zinc (Zn) is one of the most profusely abundant transition elements in the Earth’s crust, and is also widely found in biological systems, coming second only to iron. Zn (atomic number 30) is a member of group XII (previously known as II-B) of the periodic table of elements, and as analogous to all members of the group, it is also characterized as a divalent metal ^[150]. At room temperature, it occurs as a brittle, lustrous metal with a blue-white hue ^[151]. In reactions concerning hydrolysis, Zn tends to act as a Lewis acid or electrophile, which catalyzes these reactions and is thereby integrated into assorted metallo-enzymes, transcription factors, and regulatory proteins ^[152]. In cells, it exhibits antioxidative properties against the formation and mitigation of free radicals and reactive oxygen species, which contributes to the perpetuation of protein stability. The significance of Zn resonates with its function as an essential nutrient in living systems, augmenting its presence in both human beings and bacteria, where more than 5% of bacterial proteins evince their dependency on Zn ^[153]. These manifold functions preponderate over its toxicity at higher concentrations in cells, which can often be promoted by the blockage of protein thiols via mis-metallation with other metals, resulting in the disruption of various biological functions ^[154].

In the environment, anthropogenic actions have shaped the presence of Zn and its compounds in industrial and agricultural wastewaters, underscoring the production (more than 12 million tons annually) and then consumption of Zn in a multitude of processes such as galvanization, metallurgy, and the pharmaceutical industry, alloy metal casting, pesticides, and production of several other consumer goods ^[155]. Moreover, mining activities and contamination of sludge in soils poses a considerable threat of Zn toxicity to the sustainability and quality of crops ^[156], which further raises concern for the purification of contaminated sites by effective methods. It has been reported that the removal of Zn in low concentrations is mediated by physical and chemical methods, but its removal by biological agents (plants, algae, microorganisms) is a method which has been gaining attention due to its many advantages that eclipse its drawbacks. The treatment of Zn-contaminated wastewaters through plants, biomass, sawdust, mollusk shells, fruit and vegetable peels, agricultural wastes, and polysaccharides such as chitosan and pectin as potentially effective biosorbents has been widely reported ^[157][158].

Bioregulation of Zinc by 

Bacillus

 spp.

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 ^[159][160]. In 

 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. ^[108] also reported a third uptake system in 

, 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 ^[108][109] (

, 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 ^{[56][57][58][59][60][61][62]} (

 was reported in recent studies [105,106,107,108,109,110,111] (

Table 1). Khan et al. ^[63] also reported the removal of Zn (87 mg/L) by 

). Khan et al. [112] also reported the removal of Zn (87 mg/L) by 

B. altitudinis

 isolated from industrial wastewater.

Figure 2.

 Uptake and efflux mechanism of 

Bacillus

 spp. for the regulation of zinc.

2.3. Nickel

Nickel (Ni) belongs to group 10 and is the 28th element in the periodic table, discovered by Swedish chemist Axel Cronstedt in its purified form for the first time in 1951. It is a hard, silvery-white transition metal which belongs to the ferromagnetic group of metals with high electrical and thermal conductivity ^[161]. It is the 24th most copious element found in the Earth’s crust, and the 5th most abundantly found in terms of weight. It is naturally found in its oxidation state (2+) which is analogous to most environmental and biological settings, though it may exhibit other valences as well (−1 to +4) ^[162]. It persists in nature in its hydroxide form at pH > 6.7, while its complexes appear to be readily soluble at pH < 6.5. It is found to exist in various forms of air- and water-resistant minerals (oxides and sulfides) ^[163], which give rise to Ni salts of strong (readily soluble in water) and weak acids (poorly soluble in water), respectively ^[164]. Natural sources of Ni in the environment are attributable to soil and rock erosion, volcanic eruptions, meteorite emissions, air-blown dust, as well as foods ^[165]. Combustion of fossil fuels and leaching from rocks and soil contribute to its presence in air and water, respectively. Moreover, anthropogenic emissions in the form of metal smelting and mining, metal refineries, Ni plating and alloy production, and effluent and sludge disposal into soil and water catalyze its presence in high concentrations in the environment ^[166]. The commercial use of Ni and its extensive applications such as production of Ni-Cd batteries, use in jewelry, orthodontic equipment, machinery, coins, food processing, clothing, and electronics promote its ubiquity in the environment, where it exists as sulfides, oxides, and less frequently, in its metallic form ^[167]. Ni toxicity has been the subject of widespread research in humans, where it is highlighted that the metal poses no considerable nutritional value in humans and poses industrial and occupational hazardous risk ^[168]. Nevertheless, it has been characterized as essential for the growth of plants, microorganisms and animals ^[169], where Ni-based enzymes and cofactors are reported to serve a key role in their function ^[170].

Bioregulation of Nickel by 

Bacillus

 spp.

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 ^[171]. Several physico-chemical methods have been employed over the years for the removal of Ni from aqueous solutions ^[172]. 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 ^[173]. In a study, corn hydrochar was treated with KOH and altered by treating polyethyleneimine (PEI) to increase adsorption of Ni ions onto the surface ^[174]. Bioremediation by Gram-positive bacteria, such as 

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 ^{[56][64][65][66]} (

 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 ^[67][68][69]. In a recent study, 

 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 ^[175]. 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. ^[110], mechanisms of Ni homeostasis and regulation have not been well characterized in 

 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 ^[176]. Members of the cation diffusion facilitator (CDF) family have long been characterized to mediate efflux of multiple metal ions, including Ni ^[111]. In 

, 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 Ni

²⁺

, Cu

⁺

, Zn

²⁺

, and Co

^2+ [110] (

 [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 ^[110] (

, consequently leading to an increase in toxicity to them [159] (

Table 2

).

Figure 3.

 Efflux mechanism of nickel in 

Bacillus

 spp.

2.4. Cadmium

Cadmium (Cd) is a member of group XII of the periodic table and is a silvery white metal in appearance, with physical and chemical properties similar to both zinc and mercury. It exists in general oxidation state (+2) and is malleable and ductile. Cd is a corrosion-resistant metal, which is not flammable and water-soluble in nature, and is generally regarded as a toxic heavy metal with wide application in the industries of batteries, plating, plastics, and pigments, contributing greatly to its toxicity ^[170]. Anthropogenic activities have led to its presence being observed in many food sources and drinks ^[177]. Cadmium oxide is often used in metal plating, catalysis, and ceramic glaze. Alongside cadmium telluride, it has been also used in the form of a thin film for use in diodes, transistors, solar cells, electrodes, and anti-reflective coatings ^[178]. Cd is also used extensively in industrial strength paints, which can pose an environmental hazard during spraying. Other sources of contamination such as Cd-containing fertilizers can pollute soils which can inadvertently enhance its absorption by humans and other living beings. Cd has no known biological function and is a great threat to all life forms ^[179], due to which its environmental exposure can be dangerous, and in some cases, fatal ^[180].

Bioregulation of Cadmium by 

Bacillus

 spp.

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 ^[181][182]. 

 [229,230]. 

cadA is transcribed into the 727-amino acid protein, which performs the function of energy-dependent Cd efflux ATPase ^[85], whereas 

 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 Cd

²⁺ operon ^[182][183]. It is well reported that 

 operon [230,231]. It is well reported that 

cadA

 gene is induced in the presence of Cd

²⁺ ions. Solovieva and Entian ^[112] documented their findings on 

 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 Cd

²⁺

 resistance. Moreover, they reported that it has similarity to 

cadA

 of 

S. aureus plasmid pI258 ^[184]. Deletion of 

 plasmid pI258 [232]. Deletion of 

yvgW

 increased the Cd

²⁺

 sensitivity in the bacterium. 

cadB

 is also plasmid-mediated and confers Cd

²⁺ resistance through a change in the binding site ^[113]. In addition to efflux mechanism, KinA and histidine kinase provide phosphate for phosphorylation which leads directly to transcription in 

 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 ^[114]. Its overexpression results in phosphate flux of the cell, thereby directly affecting the energy state of the cell wall ^[185]. It also plays a significant role in biosorption of Cd

 [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 Cd

²⁺

 ions by phosphorylating the 

Bacillus cell surface magnitude, hence it acting as a major phosphate provider ^[73]. Cd

 cell surface magnitude, hence it acting as a major phosphate provider [122]. Cd

²⁺

 enters the bacterial cell membrane via the zinc and manganese transport systems, which are chromosomally mediated. Cd

²⁺

 concentration greatly affects the whole process. If the Cd

²⁺

 ions are present in high quantity in the medium, much extracellular adsorption is observed. Otherwise, intracellular Cd

²⁺ concentration is high ^[186]. The 

 concentration is high [234]. The 

cad

 operon of 

Bacillus megaterium

 TWSL_4 contains cadC, which has Cd

²⁺

 and Zn

²⁺ metal binding motifs ^[187]. On exposure to metal ions, the first interaction is always with cell wall ^[188]. Its structure and composition play a significant role in deciding the next step of the process. Cd

 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. Cd

²⁺

 adsorption on the bacterial cell wall deals with exchanging ions like Ca

²⁺

, Mg

²⁺

, and H

⁺ ions ^[189]. Chelation is another process in which Cd

 ions [237]. Chelation is another process in which Cd

²⁺

 ions are exchanged with cell surface protons like –SO

₃H, –COOH, and –NH ^[190]. It involves sequestration via intracellular metallothionein (MT) ^[191]. Inorganic deposition of Cd

H, –COOH, and –NH [238]. It involves sequestration via intracellular metallothionein (MT) [239]. Inorganic deposition of Cd

²⁺ in the cell wall or inside cells can take place through interaction with hydroxide, carbonate, sulfate, and phosphorus ^[192]. In addition to metal concentration, biosorption is dependent on cell wall composition and cell physiology ^[193]. In Gram-positive bacterial species, resistance to Cd

 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 Cd

²⁺

 is achieved by 

cadA

 system that is plasmid-borne. Cd

²⁺ enters the bacterial cell by the MIT (metal ion transporter) system ^[115][116] (

 enters the bacterial cell by the MIT (metal ion transporter) system [164,165] (

Table 2

). The genes for Cd

²⁺ resistance are mostly plasmid-mediated. According to Chen et al. ^[194] they are found on R plasmid along with antibiotic resistance genes, e.g., in pathogens including 

 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 Cd

²⁺

 ions from the environment (

Figure 4). Basha and Rajaganesh ^[71] reported 

). Basha and Rajaganesh [120] reported 

B. licheniformis

 to be a good biosorbent for Cd

²⁺

, as it removed more than 98% of Cd

²⁺

. Other species such as 

B. catenulatus

 and 

B. safensis

 are also reported to be effective in removing Cd

^2+ [70][72] (

 [119,121] (

Table 1

).

Figure 4.

 Mechanism of cadmium bioregulation by 

Bacillus

 spp.

2.5. Lead

Lead (Pb) is a toxic heavy metal that is introduced into the environment via the weathering of rocks. Anthropogenic sources include fossil fuels, extraction and melting of metals, battery-manufacturing industries, insecticides, pigments, and fertilizers ^[195]. Tetraethyl lead (TEL) has a common application as a gasoline additive, due to which it is a source of heat and electricity ^[196]. It exists in two states: Pb

²⁺

^4+ [197]. Its toxicity determines its bioavailability as well as mobility in the soil. Its common forms are oxides, hydroxides, ionic form, metal oxyanion complexes ^[151], phosphates, and carbonates (at pH above 6). The stable and insoluble forms include oxides, sulfides, and pyromorphites ^[198]. Its exposure occurs by food, water, and inhalation, which affects the circulatory, gastrointestinal, reproductive, neurological, muscular, kidney, and genetic systems ^[74]. Dose and exposure time are prime factors ^[199]. The permissible level of Pb in drinking water is <10 µL/L ^[182].

Bioregulation of Lead by 

Bacillus

 spp.

Pb-resistant 

Bacillus spp. have been reported previously ^[197]. 

 spp. have been reported previously [245]. 

Bacillus

 uses 

pbr operon ^{[117][118][119]} and active transport ^[200] as potential strategies to combat the toxic effects of Pb (

 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, Pb

²⁺ is exchanged by Na or K cations ^[75]. 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 ^[75]. 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 ^[74][201]. Microbes resist Pb toxicity by extracellular precipitation, exclusion, volatilization, biomethylation, cell surface binding, intracellular sequestration, and enhanced siderophore production ^[74]. Much like other Gram-positive bacteria, 

 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 ^[76][77][78] (

 spp. also employ one or several of these methods to remove Pb from the contaminated environments [125,126,127] (

Table 1

).

Figure 5.

 

pbr

 operon involved in the regulation of lead resistance in 

Bacillus

 spp.

2.6. Copper

Copper (Cu) is categorized into the group I-B, and period 4 of the periodic table ^[151]. It is a soft, diamagnetic, malleable, and ductile metal with remarkable electrical and thermal conductivity. It acts as a soft and intermediate Lewis acid and tends to bind to soft bases (hydride, alkyl, thiol, phosphine) and auxiliary ligands to Cu

²⁺, such as sulfate and nitrate ^[202]. Apart from being widespread in the environment thanks to anthropogenic actions, it also exists naturally in the form of minerals such as sulfides, carbonates, and oxides. The discovery and use of Cu dates back to ancient times, with its use spanning more than five thousand years. It exists in either of its two oxidation states, which can be the oxidized, divalent cupric form (Cu

²⁺

⁺) ^[203]. The significance of Cu in biological systems is pivotal; it serves an important role as a micronutrient in several biological processes in both prokaryotic and eukaryotic organisms. However, this stands only for lower concentrations of the metal; higher concentrations tend to induce cell toxicity, resulting in intracellular damage including changes in DNA, respiration, and overall growth ^[204]. Moreover, Cu is essentially required as a co-factor in more than thirty known enzymes, due to its ability to reversibly interconvert from its less to more required forms very easily ^[205]. Elevated levels of Cu exposure are a deep-rooted cause of environmental pollution by Cu, the fundamental reason being anthropogenic processes. Industries using Cu or its compounds, Cu mining, burning of fossil fuels, inadequate treatment of wastewater, accumulation in dumps, production of phosphate-containing fertilizer, and natural processes such as erosion, volcanic eruptions, forest wildfires, and decay are all processes which greatly contribute to its presence in the environment. Furthermore, its production is also a source of direct Cu pollution, capable of harming the fragile ecosystems of soil, water, and air, respectively ^[206].

Bioregulation of Copper by 

Bacillus

 spp.

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 ^{[68][79][80][81][82][83][84][207][208]} (

 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 ^[209]. 

). 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 ^[120]. In the former, CopZ plays a key role as Cu chaperone in transferring Cu over to CopA ^[121], which contributes to uptake of Cu, while CopB is accountable for Cu efflux and detoxification ^[122] (

 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. ^[123] reported that YcnJ was associated with Cu uptake function of 

). 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

).

Figure 6.

 Uptake and efflux mechanism of copper by 

Bacillus

spp.