**2.1 Nanostructured layered double hydroxides**

Layered double hydroxides (LDHs) with the hydrotalcite-like structure are materials attracting interest to many research groups due to their use in many different fields, such as anion scavengers, catalysts, catalyst precursors, hosts for drugs controlled delivery, contaminant and radionuclide removal from acidic mine pit water etc. [14–16]. Layered double hydroxides (LDHs) are a class of anionic

#### **Figure 1.**

*(a) SEM micrograph of Mg-Al(OH)2PO4, 1; (b) SEM micrograph of Mg-Al(OH)2PO4PF6, 2; (c) SEM micrograph of Ca-Al(OH)2SO4, 3; (d) SEM micrograph of Ca-Al(OH)2PO4PF6, 4. Source: Khansi et al. [7].*

**11**

**Figure 3.**

**Figure 2.**

*Nano/Micro-Structured Materials: Synthesis, Morphology and Applications*

structure of LDHs with the general formula [M2+1–xM3+x(OH)2]

clays with the structure based on brucite (Mg(OH)2)-like layers [17–21]. The lattice

have a positively charged brucite-shaped layers, consisting of a divalent metal ion M2+ (e.g., Ca2+, Zn2+, Mg2+, and Ni2+) octahedrally surrounded by six OH<sup>−</sup> hydroxyl groups [22–24]. The substitution of the M2+ metal with a trivalent M3+ cation gives rise to the periodic repetition of positively charged sheets (lamellas) alternating

thetic layered double hydroxides (LDHs) have been used as adsorbents and catalyst supports. The surface area and pore volume of LDHs can be increased by pillaring the interlayer with large organic and inorganic anions [19, 26, 27]. The combination of LDHs with negatively charged polymers can result in the formation of a hybrid layer component for the preparation of nano-hybrid thin films [28–30].

*Results for the removal of Fe2+ and Ca2+ ions from aqueous solutions by compounds 1–4. Source: Khansi et al. [7].*

*(a) SEM micrograph of UCIM-1; (b) SEM micrograph of UCIM-2; (c) SEM micrograph of UCIM-3;* 

*(d) SEM micrograph of UCIM-4; (e) SEM micrograph of UCIM-5. Source: Ogar [37].*

x+(An

<sup>−</sup> ions. According to Kovačević et al. [25], syn-

<sup>−</sup>)x/n·yH2O,

*DOI: http://dx.doi.org/10.5772/intechopen.85698*

with charge-counter balancing A

## *Nano/Micro-Structured Materials: Synthesis, Morphology and Applications DOI: http://dx.doi.org/10.5772/intechopen.85698*

*Nanostructures*

processing [13].

**2. Nano/micro-structured materials**

**2.1 Nanostructured layered double hydroxides**

**10**

**Figure 1.**

*(a) SEM micrograph of Mg-Al(OH)2PO4, 1; (b) SEM micrograph of Mg-Al(OH)2PO4PF6, 2; (c) SEM micrograph of Ca-Al(OH)2SO4, 3; (d) SEM micrograph of Ca-Al(OH)2PO4PF6, 4. Source: Khansi et al. [7].*

portions, until nanometer size is reached. The bottom-up method of nanoparticle synthesis involves nucleation of atoms followed by their growth in a self-assembly fashion to form the nanomaterial. Here we will look at the different nanostructured materials prepared by bottom-up methods such as hydrothermal, [8, 9] combustion synthesis [10], gas-phase methods [11, 12], microwave synthesis and sol-gel

Layered double hydroxides (LDHs) with the hydrotalcite-like structure are materials attracting interest to many research groups due to their use in many different fields, such as anion scavengers, catalysts, catalyst precursors, hosts for drugs controlled delivery, contaminant and radionuclide removal from acidic mine pit water etc. [14–16]. Layered double hydroxides (LDHs) are a class of anionic

clays with the structure based on brucite (Mg(OH)2)-like layers [17–21]. The lattice structure of LDHs with the general formula [M2+1–xM3+x(OH)2] x+(An <sup>−</sup>)x/n·yH2O, have a positively charged brucite-shaped layers, consisting of a divalent metal ion M2+ (e.g., Ca2+, Zn2+, Mg2+, and Ni2+) octahedrally surrounded by six OH<sup>−</sup> hydroxyl groups [22–24]. The substitution of the M2+ metal with a trivalent M3+ cation gives rise to the periodic repetition of positively charged sheets (lamellas) alternating with charge-counter balancing A <sup>−</sup> ions. According to Kovačević et al. [25], synthetic layered double hydroxides (LDHs) have been used as adsorbents and catalyst supports. The surface area and pore volume of LDHs can be increased by pillaring the interlayer with large organic and inorganic anions [19, 26, 27]. The combination of LDHs with negatively charged polymers can result in the formation of a hybrid layer component for the preparation of nano-hybrid thin films [28–30].

#### **Figure 3.**

*(a) SEM micrograph of UCIM-1; (b) SEM micrograph of UCIM-2; (c) SEM micrograph of UCIM-3; (d) SEM micrograph of UCIM-4; (e) SEM micrograph of UCIM-5. Source: Ogar [37].*

When LDHs which are formed by incorporating both divalent and trivalent metal ions into the octahedral lattice, are calcined at higher temperature, mixed metal oxide nanomaterials are formed with improved catalytic activity [31–33]. LDHs are also used as supports in bio-nanocomposites for medical applications [34].

Khansi et al. [7] synthesized four metal-aluminum layered double hydroxides (LDHs): Mg-Al(OH)2PO4, **1**, Mg-Al(OH)2 PO4 PF6, **2**, Ca-Al(OH)2SO4, **3** and Ca-Al(OH)2PO4PF6, **4**, by co-precipitation method followed by mild hydrothermal processing at 60°C. In **Figure 1**, we present the SEM micrographs of the as-synthesized LDHs. The synthesized nano-/microstructured LDHs of Mg-Al(OH)2PO4, **1** (**Figure 1a**), [Mg-Al(OH)2PO4PF6], **2** (**Figure 1b**) and [Ca-Al(OH)2PO4PF6] **4** (**Figure 1d**) have their Mg—O—Al—OH and Ca—O—Al—OH layers intercalated with PO4 <sup>3</sup><sup>−</sup> and/or [PO4PF6] <sup>4</sup><sup>−</sup> anions. Their micrographs consists of uniformed nano/microspheres of M—Al layered double hydroxides, while the Ca—O—Al—OH layer intercalated with SO4 <sup>2</sup><sup>−</sup> anions (**Figure 1c**) consisted of hexagonal nano-/ microplates.

The results of the column adsorption studies showed that there is significant potential for using the synthesized nanostructured LDHs as nano filters in removing ions responsible for scale formation in oil wells. According to their report, compounds **1** and **2** removed Fe2+ with greater efficiency, while all the synthesized LDHs nanostructures effectively removed Ca2+ from the oil wells as shown in **Figure 2**.

#### **2.2 Nanostructured metal phosphonates**

A surfactant-assisted synthesis route to prepare nanometer-sized metal phosphonate particles for use as an inhibitor in porous media for scale control has been reported [35, 36]. In their method, aqueous solution of calcium chloride and zinc chloride were mixed with basic solution containing phosphonate scale inhibitors such as diethylenetriamine-penta(methylene phosphonic acid) (DTPMP) in the presence of sodium dodecyl sulfate as surfactant. Following similar procedure, Ogar [37] used hydrothermal technique in the preparation of five nanostructured metal phosphonates. In a typical synthesis, 1.5 g of sodium dodecyl sulfate (SDS) was added to 40 cm3 of 0.25 M solution of the CaCl2. This was accompanied by constant stirring for 20 min (pH = 6). Then 25 cm3 of 1.0 M solution of phenylphosphonic acid (pH = 2) was added dropwise to the above solution with constant stirring. The mixture (pH = 3) was allowed to stir for 1 h after which it was transferred into ACE glass tubes and heated for 8 h at a temperature of 120°C. This was followed by centrifugation at 2000 RPM for 20 min, filtration and drying of the nanomaterials in an electric incubator at 80°C. The product obtained was designated UCIM-1 (University of Calabar Inorganic Material). Similar procedure was employed in the synthesis of UCIM-2, UCIM-3, UCIM-4 and UCIM-5 with different complexing agent as aminomethylphosphonic acid (AmMePhA), 4,4′-biphenyl-bis-phosphonic acid (BBPhA) and different metal salt such as zinc acetate.

The SEM micrographs of UCIM-1 and UCIM-2 showed clusters of micro-rods (**Figure 3a** and **b**). The zinc phosphonates prepared in the presence of surfactant, UCIM-3 consisted of particles in the size range of 30–100 nm (**Figure 3c**), whereas UCIM-4 and UCIM-5 prepared without the surfactant consists of monodispersed nanoballs (**Figure 3d** and **e**). The synthesized nanostructured zinc and calcium phosphonates were applied in the removal of scale-forming ions from aqueous solution. A similar procedure reported for LDHs [7] was used. Small amount of glass wool was first introduced into the syringe followed by 0.2 g of the nanoparticles. Two molar solution of CaCl2 was then introduced. The set-up was left for 12 h

**13**

**Table 2.**

**Table 1.**

*Nano/Micro-Structured Materials: Synthesis, Morphology and Applications*

concentration of the ions after passing through the packed column).

during which the solution eluted was collected in sample bottles and analyzed using AAS. This was repeated for FeSO4.7H2O. The removal efficiency of Fe2+ and Ca2+ by metal phosphonate nanoparticles (UCIM-n) in percentage was calculated using the formula: efficiency (%) = Ce/Co (Co = initial concentration of the ions, Ce = eluded

The removal efficiency of scale forming ions in oil wells by the synthesized metal phosphonate nanoparticles was studied with the help of atomic absorption spectroscopic technique. The removal efficiency of Ca2+ and Fe2+ ions are presented in **Tables 1** and **2** respectively. The results showed that the surfactant-assisted zinc nanostructured material (UCIM-4) removed up to 92.04% of Fe2+ compared to UCIM-5 prepared without surfactant, which showed only 75.88% removal. The compound UCIM-4 adsorbed Ca2+ the most, followed by surfactant-assisted UCIM-1 Ca-nanoparticles with a very high percentage adsorption of 94.29 as

There are a number of interesting reports on the use of ionic liquids as a solvent and/or stabilizing agent in the synthesis of metal/metal-oxide nanoparticles. The interest is not only due to the fact that ILs can form extended hydrogen-bond networks at the liquid state, but also for their nanostructural organization which is being used as a driver for spontaneous formation of nanostructured materials [38] Taubert and co-workers [39] reported on the effects of a set of ILs based on the 1-ethyl-3-methylimidazolium cation and different anions on the formation of gold nanoparticles. They demonstrated that the nature of the ionic liquid anion was crucial in the synthesis of the gold nanoparticles. For instance, anions such as methanesulfonate (MS) and trifluoromethanesulfonate (TfO) stabilized the formation of particles in the size range of 5–7 nm at low temperature, whereas with ethyl

> **Amount eluted (ppm)**

UCIM-1 7.7780 0.4438 7.3342 94.29 UCIM-2 7.7780 1.1512 6.6268 85.20 UCIM-3 7.7780 1.0251 6.7529 86.82 UCIM-4 7.7780 0.5729 7.2051 92.63 UCIM-5 7.7780 0.2399 7.5381 96.92

*Removal efficiency of Ca2+ by the synthesized metal phosphonate nanoparticles (source: Ogar [37]).*

*Removal efficiency of Fe2+ by the synthesized metal phosphonate nanoparticles (source: Ogar [37]).*

**Amount eluted (ppm)**

UCIM-1 9.8526 4.1779 5.6747 57.60 UCIM-2 9.8526 5.1040 4.7486 45.46 UCIM-3 9.8526 5.3979 4.4547 45.21 UCIM-4 9.8526 0.7843 9.0683 92.04 UCIM-5 9.8526 0.2399 7.5381 96.92

**Amount adsorbed (ppm)**

**Amount adsorbed (ppm)**

**Percentage adsorption**

**Percentage adsorption**

*DOI: http://dx.doi.org/10.5772/intechopen.85698*

**2.3 Metal/bimetallic/metal-oxide nanoparticles**

presented in **Figure 4**.

**Nanoparticles Initial amount** 

**Nanoparticles Initial amount** 

**(ppm)**

**(ppm)**

*Nano/Micro-Structured Materials: Synthesis, Morphology and Applications DOI: http://dx.doi.org/10.5772/intechopen.85698*

during which the solution eluted was collected in sample bottles and analyzed using AAS. This was repeated for FeSO4.7H2O. The removal efficiency of Fe2+ and Ca2+ by metal phosphonate nanoparticles (UCIM-n) in percentage was calculated using the formula: efficiency (%) = Ce/Co (Co = initial concentration of the ions, Ce = eluded concentration of the ions after passing through the packed column).

The removal efficiency of scale forming ions in oil wells by the synthesized metal phosphonate nanoparticles was studied with the help of atomic absorption spectroscopic technique. The removal efficiency of Ca2+ and Fe2+ ions are presented in **Tables 1** and **2** respectively. The results showed that the surfactant-assisted zinc nanostructured material (UCIM-4) removed up to 92.04% of Fe2+ compared to UCIM-5 prepared without surfactant, which showed only 75.88% removal. The compound UCIM-4 adsorbed Ca2+ the most, followed by surfactant-assisted UCIM-1 Ca-nanoparticles with a very high percentage adsorption of 94.29 as presented in **Figure 4**.

### **2.3 Metal/bimetallic/metal-oxide nanoparticles**

There are a number of interesting reports on the use of ionic liquids as a solvent and/or stabilizing agent in the synthesis of metal/metal-oxide nanoparticles. The interest is not only due to the fact that ILs can form extended hydrogen-bond networks at the liquid state, but also for their nanostructural organization which is being used as a driver for spontaneous formation of nanostructured materials [38] Taubert and co-workers [39] reported on the effects of a set of ILs based on the 1-ethyl-3-methylimidazolium cation and different anions on the formation of gold nanoparticles. They demonstrated that the nature of the ionic liquid anion was crucial in the synthesis of the gold nanoparticles. For instance, anions such as methanesulfonate (MS) and trifluoromethanesulfonate (TfO) stabilized the formation of particles in the size range of 5–7 nm at low temperature, whereas with ethyl


**Table 1.**

*Nanostructures*

with PO4

microplates.

**Figure 2**.

was added to 40 cm3

salt such as zinc acetate.

<sup>3</sup><sup>−</sup> and/or [PO4PF6]

**2.2 Nanostructured metal phosphonates**

by constant stirring for 20 min (pH = 6). Then 25 cm3

layer intercalated with SO4

When LDHs which are formed by incorporating both divalent and trivalent metal ions into the octahedral lattice, are calcined at higher temperature, mixed metal oxide nanomaterials are formed with improved catalytic activity [31–33]. LDHs are

Khansi et al. [7] synthesized four metal-aluminum layered double hydroxides

nano/microspheres of M—Al layered double hydroxides, while the Ca—O—Al—OH

The results of the column adsorption studies showed that there is significant potential for using the synthesized nanostructured LDHs as nano filters in removing ions responsible for scale formation in oil wells. According to their report, compounds **1** and **2** removed Fe2+ with greater efficiency, while all the synthesized LDHs nanostructures effectively removed Ca2+ from the oil wells as shown in

A surfactant-assisted synthesis route to prepare nanometer-sized metal phosphonate particles for use as an inhibitor in porous media for scale control has been reported [35, 36]. In their method, aqueous solution of calcium chloride and zinc chloride were mixed with basic solution containing phosphonate scale inhibitors such as diethylenetriamine-penta(methylene phosphonic acid) (DTPMP) in the presence of sodium dodecyl sulfate as surfactant. Following similar procedure, Ogar [37] used hydrothermal technique in the preparation of five nanostructured metal phosphonates. In a typical synthesis, 1.5 g of sodium dodecyl sulfate (SDS)

phenylphosphonic acid (pH = 2) was added dropwise to the above solution with constant stirring. The mixture (pH = 3) was allowed to stir for 1 h after which it was transferred into ACE glass tubes and heated for 8 h at a temperature of 120°C. This was followed by centrifugation at 2000 RPM for 20 min, filtration and drying of the nanomaterials in an electric incubator at 80°C. The product obtained was designated UCIM-1 (University of Calabar Inorganic Material). Similar procedure was employed in the synthesis of UCIM-2, UCIM-3, UCIM-4 and UCIM-5 with different complexing agent as aminomethylphosphonic acid (AmMePhA), 4,4′-biphenyl-bis-phosphonic acid (BBPhA) and different metal

The SEM micrographs of UCIM-1 and UCIM-2 showed clusters of micro-rods (**Figure 3a** and **b**). The zinc phosphonates prepared in the presence of surfactant, UCIM-3 consisted of particles in the size range of 30–100 nm (**Figure 3c**), whereas UCIM-4 and UCIM-5 prepared without the surfactant consists of monodispersed nanoballs (**Figure 3d** and **e**). The synthesized nanostructured zinc and calcium phosphonates were applied in the removal of scale-forming ions from aqueous solution. A similar procedure reported for LDHs [7] was used. Small amount of glass wool was first introduced into the syringe followed by 0.2 g of the nanoparticles. Two molar solution of CaCl2 was then introduced. The set-up was left for 12 h

of 0.25 M solution of the CaCl2. This was accompanied

of 1.0 M solution of

<sup>4</sup><sup>−</sup> anions. Their micrographs consists of uniformed

<sup>2</sup><sup>−</sup> anions (**Figure 1c**) consisted of hexagonal nano-/

also used as supports in bio-nanocomposites for medical applications [34].

(LDHs): Mg-Al(OH)2PO4, **1**, Mg-Al(OH)2 PO4 PF6, **2**, Ca-Al(OH)2SO4, **3** and Ca-Al(OH)2PO4PF6, **4**, by co-precipitation method followed by mild hydrothermal processing at 60°C. In **Figure 1**, we present the SEM micrographs of the as-synthesized LDHs. The synthesized nano-/microstructured LDHs of Mg-Al(OH)2PO4, **1** (**Figure 1a**), [Mg-Al(OH)2PO4PF6], **2** (**Figure 1b**) and [Ca-Al(OH)2PO4PF6] **4** (**Figure 1d**) have their Mg—O—Al—OH and Ca—O—Al—OH layers intercalated

**12**

*Removal efficiency of Ca2+ by the synthesized metal phosphonate nanoparticles (source: Ogar [37]).*


**Table 2.**

*Removal efficiency of Fe2+ by the synthesized metal phosphonate nanoparticles (source: Ogar [37]).*

#### **Figure 4.**

*Bar chart showing percentage adsorption of Ca2+ and Fe2+ by the different metal phosphonate nanoparticles. Source: Ogar [37].*

sulfate (ES), polydispersed particles in the size range 15–20 nm were formed at all reaction temperature. In their report, they concluded that specific IL-gold interactions were responsible for the formation of gold particles with an IL-specific shape, size, and aggregation behavior.

Ayi et al. reported on a wet chemical approach in synthesizing titanium nanoparticles in imidazolium-based ionic liquids (ILs) under reducing conditions [40]. It was established that nanosize particles were formed with some ILs being adsorbed on the nanoparticles, thus providing the needed stabilization.

In continuation of the work on anion effects on nanoparticles formation, stable Mo and molybdenum oxide nano- and microparticles were synthesized in ILs with a variety of anions under reducing conditions by Ayi and co-workers [41]. XRD and TEM revealed a strong influence of the IL anion on the particle sizes, shapes, and crystal structures. The influence of the IL cation and the reaction temperature was found to be much less pronounced.

Very recently, Ayi's research group has used plant extracts (*Kigelia africana* fruits) to prepare silver nanoparticles and copper-silver bimetallic nanostructures and their antimicrobial potentials evaluated [42]. The aqueous extract of *Kigelia africana* was utilized in the synthesis of both metallic and bimetallic nanoparticles without additional stabilizing agent. The investigation revealed that average particle size of 10 nm were formed and stabilized by the active components of the plant extract, which were adsorbed on the surface of the particles. The synthesized particles were found to inhibit the growth of both Gram-negative and Gram-positive bacteria more than any of antibiotics tested in the study. The bimetallic nanoparticles demonstrated effectiveness against *S. aureus* with maximum ZOI of 27 mm. Walter et al., made use of 1-ethyl-3-methylimidazolium acetate (EMIMOAC) and 1-buthyl-3-methylimidazolium acetate (BMIMOAC) ionic liquids to prepare cobalt nanoparticles (CoEMIMOAC or CoBMIMOAC) via chemical reduction method [43]. The SEM and UV-Vis techniques confirmed the formation of nanoparticles (**Figure 5**). There was no difference between the nanocrystals formed in EMIMOAC or BMIMOAC indicating that the cationic part of the ionic liquid did not play significant role during nucleation and growth of the particles. The inhibition zone diameter (IZD) showed moderate susceptibility of the *Staphylococcus aureus* to CoBMIMOAC (15 mm) but high susceptibility to

**15**

**Figure 6.**

*Source: John et al. [43].*

**Figure 5.**

*Nano/Micro-Structured Materials: Synthesis, Morphology and Applications*

CoEMIMOAC with a larger inhibition zone diameter of 23 mm. Gram-negative microorganism *E. coli* is moderately susceptible to both CoEMIMOAC (14 mm),

*(a) SEM micrograph of cobalt nanocrystals prepared in EMIMOAC. Source: John et al. [43]; (b) SEM* 

*Bar chart showing nanocrystals inhibition zone diameter for Staphylococcus aureus and Escherichia coli.* 

*micrograph of cobalt nanocrystals prepared in BMIMOAC. Source: John et al. [43].*

*DOI: http://dx.doi.org/10.5772/intechopen.85698*

and CoBMIMOAC (15 mm) (**Figure 6**).

*Nano/Micro-Structured Materials: Synthesis, Morphology and Applications DOI: http://dx.doi.org/10.5772/intechopen.85698*

CoEMIMOAC with a larger inhibition zone diameter of 23 mm. Gram-negative microorganism *E. coli* is moderately susceptible to both CoEMIMOAC (14 mm), and CoBMIMOAC (15 mm) (**Figure 6**).

#### **Figure 5.**

*Nanostructures*

**Figure 4.**

*Source: Ogar [37].*

size, and aggregation behavior.

found to be much less pronounced.

sulfate (ES), polydispersed particles in the size range 15–20 nm were formed at all reaction temperature. In their report, they concluded that specific IL-gold interactions were responsible for the formation of gold particles with an IL-specific shape,

*Bar chart showing percentage adsorption of Ca2+ and Fe2+ by the different metal phosphonate nanoparticles.* 

Ayi et al. reported on a wet chemical approach in synthesizing titanium nanoparticles in imidazolium-based ionic liquids (ILs) under reducing conditions [40]. It was established that nanosize particles were formed with some ILs being

In continuation of the work on anion effects on nanoparticles formation, stable Mo and molybdenum oxide nano- and microparticles were synthesized in ILs with a variety of anions under reducing conditions by Ayi and co-workers [41]. XRD and TEM revealed a strong influence of the IL anion on the particle sizes, shapes, and crystal structures. The influence of the IL cation and the reaction temperature was

Very recently, Ayi's research group has used plant extracts (*Kigelia africana* fruits) to prepare silver nanoparticles and copper-silver bimetallic nanostructures and their antimicrobial potentials evaluated [42]. The aqueous extract of *Kigelia africana* was utilized in the synthesis of both metallic and bimetallic nanoparticles without additional stabilizing agent. The investigation revealed that average particle size of 10 nm were formed and stabilized by the active components of the plant extract, which were adsorbed on the surface of the particles. The synthesized particles were found to inhibit the growth of both Gram-negative and Gram-positive bacteria more than any of antibiotics tested in the study. The bimetallic nanoparticles demonstrated effectiveness against *S. aureus* with maximum ZOI of 27 mm. Walter et al., made use of 1-ethyl-3-methylimidazolium acetate (EMIMOAC) and 1-buthyl-3-methylimidazolium acetate (BMIMOAC) ionic liquids to prepare cobalt nanoparticles (CoEMIMOAC or CoBMIMOAC) via chemical reduction method [43]. The SEM and UV-Vis techniques confirmed the formation of nanoparticles (**Figure 5**). There was no difference between the nanocrystals formed in EMIMOAC or BMIMOAC indicating that the cationic part of the ionic liquid did not play significant role during nucleation and growth of the particles. The inhibition zone diameter (IZD) showed moderate susceptibility of the *Staphylococcus aureus* to CoBMIMOAC (15 mm) but high susceptibility to

adsorbed on the nanoparticles, thus providing the needed stabilization.

**14**

*(a) SEM micrograph of cobalt nanocrystals prepared in EMIMOAC. Source: John et al. [43]; (b) SEM micrograph of cobalt nanocrystals prepared in BMIMOAC. Source: John et al. [43].*

#### **Figure 6.**

*Bar chart showing nanocrystals inhibition zone diameter for Staphylococcus aureus and Escherichia coli. Source: John et al. [43].*
