Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials in Soil and Water

*Xiaoniu Yu and Qiwei Zhan*

## **Abstract**

This chapter presents a new method for treatment of heavy metal ions in soil or water. Heavy metal pollution in soil and water has become one of the serious environmental problems. Heavy metal pollution can degrade soil quality and ecosystems, contaminate crops, and threaten human health. At present, there are three ways to repair heavy metals in soil or water, including physical, chemical, and biological technologies. The microbial mineralization technology can be applied to remove heavy metal pollutants which contaminated soil and water and has been paid with more attention in recent years. Heavy metal ions can be mineralized by phosphate-mineralization microbe to form stable phosphate nanomaterials compared to mineralization of carbonate-mineralization microbe in the environments. Therefore, heavy metal pollution can well be removed from soil or water by microbial mineralization method.

**Keywords:** heavy metal pollution, soil, water, phosphate-mineralization microbe, phosphate nanomaterials

### **1. Introduction**

The density of the heavy metal is bigger than 4.5 g/cm3 or more, and atomic numbers are from 23 (V) to 92 (U) of heavy metal elements, such as lead, nickel, zinc, copper, iron, cadmium, chromium, etc. [1]. They are widely used in industrial production and discharged into the environment due to the failure to process heavy metals in mining and industrial production. Soil and water are the ultimate destination of these heavy metals due to their particularity. Compared with air pollution, water pollution, and industrial solid waste pollution, heavy metals in soil and water are invisible and concealed. The pollution of heavy metals in soil and water can lead to the degradation of soil fertility, the reduction of crop yields, and the decline in quality, which seriously affects the environmental quality and sustainable economic development and threatens people's food safety [2]. Heavy metal pollution has become a global concern [1, 3–9]. Whether in the soil or water, different heavy metals enter the bottom of the human food chain and finally enter the human body [2]. Heavy metals are very difficult to be biodegraded and can easily be biomagnified in the human body. Heavy metals can interact strongly with proteins and enzymes in the human body, making them inactive, or they can accumulate in certain organs of the human body, causing chronic poisoning, which is a serious threat to people's lives, health, and safety [2, 5].

The content, migration, and damage of heavy metal ions are high in the electroplating wastewater irrigation area, the tailings accumulation area, and the surrounding cultivated soil. Since heavy metal pollution has already caused a major threat to human survival and health, countries around the world have formulated corresponding laws and regulations to strictly limit the emission of heavy metals. At the same time, areas of heavy metal pollution can well be repaired through physical, chemical, and biological technologies to reduce the harm [9]. Physical and chemical methods to repair heavy metal pollution have been widely reported, including chemical curing, soil leaching, electrokinetic repair, etc. The physicochemical methods may cause secondary pollution because of its damage to soil structure and indigenous microorganisms. Bioremediation technology is a more effective method for controlling polluted soil and water and has become a new hotspot in current environmental engineering science and technology research, including phytoremediation, microbial adsorption, and microbial mineralization technologies. The use of microorganisms (bacteria, algae, yeast, etc.) to reduce or eliminate heavy metal pollution has been reported widely at home and abroad. The mechanism of bioremediation heavy metals includes: (1) cell metabolism—a specific metabolic pathway can make heavy metal ions precipitation or into low-toxic; (2) biosorption—using living cells, inanimate organisms metal-binding proteins and polypeptides or biopolymers as biosorbents, vacuolar swallowing, precipitation and redox reactions; and (3) biomineralization—heavy metal ions are precipitated into biominerals by microbial hydrolysate.

At present, the problem of heavy metal pollution in soil and water is becoming more and more serious, and the use of carbonate-mineralization microbe to repair heavy metals pollution has attracted people's attention because of its excellent characteristics [10, 11]. Although many studies have been carried out, the stability of carbonate in the environment is lower than that of phosphate. Therefore, this chapter proposes that phosphate-mineralization microbe mineralizes heavy metal ions to form stable phosphates. Based on the principle of biomineralization, organic phosphate monoester is hydrolyzed by phosphatase produced by phosphatemineralization microbe of growth and metabolism and then obtains phosphate ions. Heavy metal ions can combine with phosphate ions to form phosphate minerals in soil or water, thereby reducing the risk of heavy metals being absorbed by plants and the harm to the natural environment.

## **2. Methodology**

*Bacillus subtilis* was purchased by China Center of Industrial Culture Collection. Pure water was made by small ultrapure water machine. All raw materials were of analytically pure grade and used without further purification.

*Bacillus subtilis* of cultivation [12, 13]: 5 g of tryptone and 3 g of beef extract were completely dissolved in 1 L of pure water, and its pH was adjusted to 7.0 using diluted NaOH solution. 1 L of above solution was added to two beaker flasks (1000 mL), and each bottle was added 500 mL of mixture solution and wrapped by paper and gauze. Two beaker flasks were placed in autoclave under 125°C and 0.1 Mpa for 30 min. Then they were cooled to room temperature under natural conditions. Next, 3 mL of *Bacillus subtilis* strains were added to each beaker flask and cultivated in the oscillation incubator (170r·min<sup>−</sup><sup>1</sup> ) at 28°C for 22–24 hours. As a result, the harvested microorganisms were kept in a refrigerator at 4°C before use. Optical density (OD) of *Bacillus subtilis* liquid was measured by UV spectrophotometer at 600 nm of wavelength with OD range of 1.23–1.36.

**115**

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials…*

was added to 100 mL of *Bacillus subtilis* under static conditions for 24 hours at room temperature. The mixture solution of *Bacillus subtilis* and organic phosphate monoester was centrifuged, and then the centrifugal solution was obtained. The pH of solution was adjusted to 7, 8, 9, 10, and 11 with diluted HCl and NaOH solution. BaCl2·2H2O powder was added to the above solution at different pH. Precipitated solution was stilled for 24 hours at room temperature after stirring for 1 min. All products were dried at 60°C for 24 hours. After that, all powder precipitates were

Microbiological reduction-precipitation of cerium phosphates [15]: Organic phosphate monoester (pH = 8.8–9.3) was added to 100 mL of *Bacillus subtilis* under static conditions for 24 hours. Next, 0.01 and 0.02 mol of Ce(SO4)2 were added to above solution. The precipitated solution was stood under static conditions for 24 hours. As a result, two powder precipitates were dried at 60°C for 24 hours. After

Syntheses of iron phosphates [16]: Organic phosphate monoester (pH = 8.8–9.3) was injected into 200 mL of *Bacillus subtilis* and was allowed to stand under static conditions for 24 hours at room temperature. After 24 hours, FeSO4 ·7H2O (12 mM) was added to the above mixture solution with 2 min of stirring. The precipitated solution was allowed to stand under static conditions for 72 hours at room temperature. The products were then dried at 60°C for 24 hours. After that, powder precipi-

Fourier-transform infrared spectroscopy (FTIR) spectra of the samples were recorded with a Nicolet 5700 spectrometer using the KBr pellet technique

by powder X-ray diffraction (XRD) with a Bruker D8-Discover diffractometer with Cu Kα radiation (λ = 1.5406 Å). Scanning electron microscopy (SEM, FEI Company, the Netherlands, operating voltage 20 kV) with an energy-dispersive X-ray spectroscopy (EDS) was used to conduct morphological studies and to measure elemental compositions of the samples. Transmission electron microscopy (TEM) images were obtained on a FEI, G2 20 equipment. The samples were dispersed in anhydrous ethanol before being tested. Thermogravimetricdifferential scanning calorimetry (DSC-TG) was carried out on STA449 F3 thermogravimetric analyzer (NETZSCH, Germany). The analyses were carried out simultaneously in a nitrogen atmosphere at a heating rate of 10°C/minute between room temperature and 700–800°C under an N2 flow of 20 mL·min<sup>−</sup><sup>1</sup>

The average crystallite size of biophosphate nanomaterials was estimated by the

where *D* is the average crystallite size of samples (nm), *k* is the shape factor (0.9), λ is the wavelength of Cu Kα radiation equal to 1.5406 Å, *β* is defined as the full width of the peak at half of the maximum intensity (FWHM), and θ is the

The FTIR spectra of barium phosphates obtained at pH = 8 and 11 were dis-

appears in the spectrum of any compounds. A shoulder peak at 2973.47 cm<sup>−</sup><sup>1</sup>

**3. Barium phosphate nanoparticles prepared by microbial** 

. The phase purity of products was examined

<sup>β</sup> cos<sup>θ</sup> (1)

was KBr mull absorption, which

.

that, two powder specimens were collected and characterized.

Syntheses of barium phosphates [14]: organic phosphate monoester (pH = 8.8–9.3)

*DOI: http://dx.doi.org/10.5772/intechopen.84296*

tates were obtained and characterized.

in the range of 399–4000 cm<sup>−</sup><sup>1</sup>

Scherrer formula [13]:

*D* = \_\_\_\_\_ *<sup>k</sup>*

diffraction angle of the selected reflection.

played in **Figure 1**. The peak at 3415.62 cm<sup>−</sup><sup>1</sup>

**mineralization method**

collected.

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials… DOI: http://dx.doi.org/10.5772/intechopen.84296*

Syntheses of barium phosphates [14]: organic phosphate monoester (pH = 8.8–9.3) was added to 100 mL of *Bacillus subtilis* under static conditions for 24 hours at room temperature. The mixture solution of *Bacillus subtilis* and organic phosphate monoester was centrifuged, and then the centrifugal solution was obtained. The pH of solution was adjusted to 7, 8, 9, 10, and 11 with diluted HCl and NaOH solution. BaCl2·2H2O powder was added to the above solution at different pH. Precipitated solution was stilled for 24 hours at room temperature after stirring for 1 min. All products were dried at 60°C for 24 hours. After that, all powder precipitates were collected.

Microbiological reduction-precipitation of cerium phosphates [15]: Organic phosphate monoester (pH = 8.8–9.3) was added to 100 mL of *Bacillus subtilis* under static conditions for 24 hours. Next, 0.01 and 0.02 mol of Ce(SO4)2 were added to above solution. The precipitated solution was stood under static conditions for 24 hours. As a result, two powder precipitates were dried at 60°C for 24 hours. After that, two powder specimens were collected and characterized.

Syntheses of iron phosphates [16]: Organic phosphate monoester (pH = 8.8–9.3) was injected into 200 mL of *Bacillus subtilis* and was allowed to stand under static conditions for 24 hours at room temperature. After 24 hours, FeSO4 ·7H2O (12 mM) was added to the above mixture solution with 2 min of stirring. The precipitated solution was allowed to stand under static conditions for 72 hours at room temperature. The products were then dried at 60°C for 24 hours. After that, powder precipitates were obtained and characterized.

Fourier-transform infrared spectroscopy (FTIR) spectra of the samples were recorded with a Nicolet 5700 spectrometer using the KBr pellet technique in the range of 399–4000 cm<sup>−</sup><sup>1</sup> . The phase purity of products was examined by powder X-ray diffraction (XRD) with a Bruker D8-Discover diffractometer with Cu Kα radiation (λ = 1.5406 Å). Scanning electron microscopy (SEM, FEI Company, the Netherlands, operating voltage 20 kV) with an energy-dispersive X-ray spectroscopy (EDS) was used to conduct morphological studies and to measure elemental compositions of the samples. Transmission electron microscopy (TEM) images were obtained on a FEI, G2 20 equipment. The samples were dispersed in anhydrous ethanol before being tested. Thermogravimetricdifferential scanning calorimetry (DSC-TG) was carried out on STA449 F3 thermogravimetric analyzer (NETZSCH, Germany). The analyses were carried out simultaneously in a nitrogen atmosphere at a heating rate of 10°C/minute between room temperature and 700–800°C under an N2 flow of 20 mL·min<sup>−</sup><sup>1</sup> . The average crystallite size of biophosphate nanomaterials was estimated by the Scherrer formula [13]:

$$D = \frac{k\lambda}{\beta \cos \theta} \tag{1}$$

where *D* is the average crystallite size of samples (nm), *k* is the shape factor (0.9), λ is the wavelength of Cu Kα radiation equal to 1.5406 Å, *β* is defined as the full width of the peak at half of the maximum intensity (FWHM), and θ is the diffraction angle of the selected reflection.

## **3. Barium phosphate nanoparticles prepared by microbial mineralization method**

The FTIR spectra of barium phosphates obtained at pH = 8 and 11 were displayed in **Figure 1**. The peak at 3415.62 cm<sup>−</sup><sup>1</sup> was KBr mull absorption, which appears in the spectrum of any compounds. A shoulder peak at 2973.47 cm<sup>−</sup><sup>1</sup>

*Nanomaterials - Toxicity, Human Health and Environment*

microbial hydrolysate.

**2. Methodology**

and the harm to the natural environment.

The content, migration, and damage of heavy metal ions are high in the electroplating wastewater irrigation area, the tailings accumulation area, and the surrounding cultivated soil. Since heavy metal pollution has already caused a major threat to human survival and health, countries around the world have formulated corresponding laws and regulations to strictly limit the emission of heavy metals. At the same time, areas of heavy metal pollution can well be repaired through physical, chemical, and biological technologies to reduce the harm [9]. Physical and chemical methods to repair heavy metal pollution have been widely reported, including chemical curing, soil leaching, electrokinetic repair, etc. The physicochemical methods may cause secondary pollution because of its damage to soil structure and indigenous microorganisms. Bioremediation technology is a more effective method for controlling polluted soil and water and has become a new hotspot in current environmental engineering science and technology research, including phytoremediation, microbial adsorption, and microbial mineralization technologies. The use of microorganisms (bacteria, algae, yeast, etc.) to reduce or eliminate heavy metal pollution has been reported widely at home and abroad. The mechanism of bioremediation heavy metals includes: (1) cell metabolism—a specific metabolic pathway can make heavy metal ions precipitation or into low-toxic; (2) biosorption—using living cells, inanimate organisms metal-binding proteins and polypeptides or biopolymers as biosorbents, vacuolar swallowing, precipitation and redox reactions; and (3) biomineralization—heavy metal ions are precipitated into biominerals by

At present, the problem of heavy metal pollution in soil and water is becoming more and more serious, and the use of carbonate-mineralization microbe to repair heavy metals pollution has attracted people's attention because of its excellent characteristics [10, 11]. Although many studies have been carried out, the stability of carbonate in the environment is lower than that of phosphate. Therefore, this chapter proposes that phosphate-mineralization microbe mineralizes heavy metal ions to form stable phosphates. Based on the principle of biomineralization, organic

phosphate monoester is hydrolyzed by phosphatase produced by phosphate-

analytically pure grade and used without further purification.

and cultivated in the oscillation incubator (170r·min<sup>−</sup><sup>1</sup>

tometer at 600 nm of wavelength with OD range of 1.23–1.36.

mineralization microbe of growth and metabolism and then obtains phosphate ions. Heavy metal ions can combine with phosphate ions to form phosphate minerals in soil or water, thereby reducing the risk of heavy metals being absorbed by plants

*Bacillus subtilis* was purchased by China Center of Industrial Culture Collection. Pure water was made by small ultrapure water machine. All raw materials were of

) at 28°C for 22–24 hours. As

*Bacillus subtilis* of cultivation [12, 13]: 5 g of tryptone and 3 g of beef extract were completely dissolved in 1 L of pure water, and its pH was adjusted to 7.0 using diluted NaOH solution. 1 L of above solution was added to two beaker flasks (1000 mL), and each bottle was added 500 mL of mixture solution and wrapped by paper and gauze. Two beaker flasks were placed in autoclave under 125°C and 0.1 Mpa for 30 min. Then they were cooled to room temperature under natural conditions. Next, 3 mL of *Bacillus subtilis* strains were added to each beaker flask

a result, the harvested microorganisms were kept in a refrigerator at 4°C before use. Optical density (OD) of *Bacillus subtilis* liquid was measured by UV spectropho-

**114**

**Figure 1.** *FTIR spectra of barium phosphates prepared at pH = 8 (a) and 11 (b) [14].*

**Figure 2.** *XRD patterns of barium phosphates precipitated at pH = 7, 8, 9, 10, and 11 [14].*

corresponded to hinder rotation. The peak at 1650.24 cm<sup>−</sup><sup>1</sup> reveals thermal vibrations of H<sup>+</sup> in an intermolecular phonon vibration associated with H<sup>+</sup> -PO4 <sup>3</sup><sup>−</sup>. The peak at 1258.78 cm<sup>−</sup><sup>1</sup> was P=O stretching vibration. The peaks at 1391.75, 1076.58, and 982.25 cm<sup>−</sup><sup>1</sup> corresponded to stretching vibration of P-O (H). The P=O of deformation vibration was found at 892.04 cm<sup>−</sup><sup>1</sup> and to be nondegenerate. The P-O(H) wagging and rocking vibration was found at 537.45 cm<sup>−</sup><sup>1</sup> . The above results are similar with literatures reported, as shown in **Figure 1a** [17–20]. The peaks are at 3411.74, 1645.18, 1403.90, 1017.95, 870.66, and 566.45 cm<sup>−</sup><sup>1</sup> in the FTIR spectrum with respect to the OH and PO4 <sup>3</sup><sup>−</sup> (**Figure 1b**), which were in accordance with the data reported in the literature [21].

**117**

**Figure 3.**

*(i, j) pH =11 [14].*

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials…*

Chemical compositions of barium phosphates were analyzed by XRD technique, as shown in **Figure 2**. Barium phosphates of patterns could be readily indexed using the reported structures of BaHPO4 (JCPDS Card No. 72–1370) at pH < 10 (**Figure 2a–c**). When pH was 10, the mixture of BaNaPO4, BaHPO4, and Ba5(PO4)3OH (JCPDS No. 70–1787, 09–0113 and 36–0272) was found, as shown in **Figure 2d**. The mixture of BaNaPO4 (JCPDS No. 70–1787) and Ba5(PO4)3OH (JCPDS No. 78–1441) was precipitated at pH = 11(**Figure 2e**). Above results

*SEM and TEM images of barium phosphates: (a, b) pH = 7, (c, d) pH = 8, (e, f) pH = 9, (g, h) pH = 10, and* 

*DOI: http://dx.doi.org/10.5772/intechopen.84296*

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials… DOI: http://dx.doi.org/10.5772/intechopen.84296*

**Figure 3.**

*Nanomaterials - Toxicity, Human Health and Environment*

corresponded to hinder rotation. The peak at 1650.24 cm<sup>−</sup><sup>1</sup>

*FTIR spectra of barium phosphates prepared at pH = 8 (a) and 11 (b) [14].*

*XRD patterns of barium phosphates precipitated at pH = 7, 8, 9, 10, and 11 [14].*

P-O(H) wagging and rocking vibration was found at 537.45 cm<sup>−</sup><sup>1</sup>

at 3411.74, 1645.18, 1403.90, 1017.95, 870.66, and 566.45 cm<sup>−</sup><sup>1</sup>

deformation vibration was found at 892.04 cm<sup>−</sup><sup>1</sup>

in an intermolecular phonon vibration associated with H<sup>+</sup>

are similar with literatures reported, as shown in **Figure 1a** [17–20]. The peaks are

was P=O stretching vibration. The peaks at 1391.75, 1076.58,

<sup>3</sup><sup>−</sup> (**Figure 1b**), which were in accordance with the

corresponded to stretching vibration of P-O (H). The P=O of

reveals thermal vibra-

and to be nondegenerate. The


. The above results

in the FTIR spectrum

<sup>3</sup><sup>−</sup>. The

**116**

tions of H<sup>+</sup>

**Figure 2.**

**Figure 1.**

peak at 1258.78 cm<sup>−</sup><sup>1</sup>

with respect to the OH and PO4

data reported in the literature [21].

and 982.25 cm<sup>−</sup><sup>1</sup>

*SEM and TEM images of barium phosphates: (a, b) pH = 7, (c, d) pH = 8, (e, f) pH = 9, (g, h) pH = 10, and (i, j) pH =11 [14].*

Chemical compositions of barium phosphates were analyzed by XRD technique, as shown in **Figure 2**. Barium phosphates of patterns could be readily indexed using the reported structures of BaHPO4 (JCPDS Card No. 72–1370) at pH < 10 (**Figure 2a–c**). When pH was 10, the mixture of BaNaPO4, BaHPO4, and Ba5(PO4)3OH (JCPDS No. 70–1787, 09–0113 and 36–0272) was found, as shown in **Figure 2d**. The mixture of BaNaPO4 (JCPDS No. 70–1787) and Ba5(PO4)3OH (JCPDS No. 78–1441) was precipitated at pH = 11(**Figure 2e**). Above results

indicated that HPO4 <sup>2</sup><sup>−</sup> and PO4 <sup>3</sup><sup>−</sup> ions were obtained by *Bacillus subtilis* hydrolyzing organic phosphate monoester. HPO4 <sup>2</sup><sup>−</sup> ions were formed at pH = 7, 8, and 9. HPO4 <sup>2</sup><sup>−</sup> and PO4 <sup>3</sup><sup>−</sup> ions were formed at pH = 10. PO4 <sup>3</sup><sup>−</sup> ions were formed at pH = 11. Therefore, the barium phosphates were mainly BaHPO4 at pH < 10 [22]. The barium phosphates were mainly the mixture of BaNaPO4, BaHPO4, and Ba5(PO4)3OH at pH = 10 [22]. The barium phosphates were mainly the mixture of BaNaPO4 and Ba5(PO4)3OH at pH = 11 [22]. Organic phosphate monoesters can well be hydrolyzed through *Bacillus subtilis*.

SEM and TEM images showed shape of barium phosphates were nanoparticles, as shown in **Figure 3**. SEM images showed that barium phosphates were nanoagglomerates, (**Figure 3a, c, e, g**, and **i**). The size of the nano-agglomerates was less than 3 μm. TEM images indicated that morphology of barium phosphate particles was mainly irregular flakes with diameter in the range of 20–100 nm, as shown in **Figure 3b, d, f, h**, and **j**. The average size of barium phosphates was calculated by the Scherrer formula (1), and the average size was 48.56, 36.60, 31.89, 33.27, and 34.79 nm when pH were 7, 8, 9, 10, and 11, respectively [13]. Above results showed that barium phosphate nanomaterials could be easily prepared by the centrifuged solution of *Bacillus subtilis* and organic phosphate monoester reacting with BaCl2·2H2O, which were different from chemical and biomineralization methods [17, 22].

## **4. Microbiological reduction-precipitation of nanostructured cerium phosphates**

Elemental compositions of cerium phosphates were analyzed by EDS spectra, as shown in **Figure 4**. The elements C, O, Na, P, S, and Ce were found in cerium phosphates when P/Ce molar ratio was 1:1 and 2:1 (**Figure 4a** and **b**). The raw files and cerium phosphates could be indexed by MDI jade 5.0 procedure. The indexed results indicated CePO4@NaCe(SO4)2(H2O) and CePO4 were found when P/Ce molar ratio was 1:1 and 2:1, respectively, as shown in **Figure 5a** and **b**. The standard cards of CePO4@NaCe(SO4)2(H2O) used were JCPDS Card No. 74–1889 and 86–0526. CePO4 was JCPDS Card No. 75–1880.

The average crystallite size of cerium phosphates was calculated via the Scherrer formula (1), as shown in **Table 1** [13]. The results showed that the average size of CePO4@NaCe(SO4)2(H2O) nanoparticles was bigger than CePO4.

**119**

**Figure 6.**

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials…*

Cerium phosphates and *Bacillus subtilis* were determined by FTIR spectra, as shown in **Figure 6**. According to literature reported, the peaks at 3438.03, 2968.49

to, respectively, OH, C-H, C=O or N-H, C-O, etc. (**Figure 3a**) [23]. Above results showed that these functional groups play important roles in the process of adsorption and reduction of Ce(IV) [23]. **Figure 6b** indicated the peak at 1013.15 cm<sup>−</sup><sup>1</sup>

corresponded

**(deg)**

**β (deg)** **D (nm)**

<sup>3</sup><sup>−</sup> groups. The peaks

was

and 1348.28, 1636.09, 1233.71, 1092.30, 1405.87, and 537.82 cm<sup>−</sup><sup>1</sup>

*FTIR spectra: (a) Bacillus subtilis, (b) CePO4@NaCe(SO4)2(H2O), and (c) CePO4 [15].*

attributed to the asymmetry stretching vibration of the PO4

*DOI: http://dx.doi.org/10.5772/intechopen.84296*

*XRD patterns: (a) CePO4@NaCe(SO4)2(H2O) and (b) CePO4 [15].*

*Average crystallite size of cerium phosphates [15].*

**Sample P/Ce (molar ratio) θ**

CePO4 2:1 15.67 0.504 15.61 CePO4@NaCe(SO4)2(H2O) 1:1 15.68 0.243 32.34

**Figure 5.**

**Table 1.**

**Figure 4.** *EDS spectra: (a) CePO4@NaCe(SO4)2(H2O) and (b) CePO4 [15].*

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials… DOI: http://dx.doi.org/10.5772/intechopen.84296*

**Figure 5.**

*XRD patterns: (a) CePO4@NaCe(SO4)2(H2O) and (b) CePO4 [15].*


#### **Table 1.**

*Nanomaterials - Toxicity, Human Health and Environment*

<sup>2</sup><sup>−</sup> and PO4

<sup>3</sup><sup>−</sup> ions were formed at pH = 10. PO4

pH = 11. Therefore, the barium phosphates were mainly BaHPO4 at pH < 10 [22]. The barium phosphates were mainly the mixture of BaNaPO4, BaHPO4, and Ba5(PO4)3OH at pH = 10 [22]. The barium phosphates were mainly the mixture of BaNaPO4 and Ba5(PO4)3OH at pH = 11 [22]. Organic phosphate monoesters can

SEM and TEM images showed shape of barium phosphates were nanoparticles,

**Figure 3b, d, f, h**, and **j**. The average size of barium phosphates was calculated by the Scherrer formula (1), and the average size was 48.56, 36.60, 31.89, 33.27, and 34.79 nm when pH were 7, 8, 9, 10, and 11, respectively [13]. Above results showed that barium phosphate nanomaterials could be easily prepared by the centrifuged solution of *Bacillus subtilis* and organic phosphate monoester reacting with BaCl2·2H2O, which

**4. Microbiological reduction-precipitation of nanostructured cerium** 

Elemental compositions of cerium phosphates were analyzed by EDS spectra, as shown in **Figure 4**. The elements C, O, Na, P, S, and Ce were found in cerium phosphates when P/Ce molar ratio was 1:1 and 2:1 (**Figure 4a** and **b**). The raw files and cerium phosphates could be indexed by MDI jade 5.0 procedure. The indexed results indicated CePO4@NaCe(SO4)2(H2O) and CePO4 were found when P/Ce molar ratio was 1:1 and 2:1, respectively, as shown in **Figure 5a** and **b**. The standard cards of CePO4@NaCe(SO4)2(H2O) used were JCPDS Card No. 74–1889 and

The average crystallite size of cerium phosphates was calculated via the Scherrer formula (1), as shown in **Table 1** [13]. The results showed that the average size of

as shown in **Figure 3**. SEM images showed that barium phosphates were nanoagglomerates, (**Figure 3a, c, e, g**, and **i**). The size of the nano-agglomerates was less than 3 μm. TEM images indicated that morphology of barium phosphate particles was

mainly irregular flakes with diameter in the range of 20–100 nm, as shown in

were different from chemical and biomineralization methods [17, 22].

CePO4@NaCe(SO4)2(H2O) nanoparticles was bigger than CePO4.

ing organic phosphate monoester. HPO4

well be hydrolyzed through *Bacillus subtilis*.

<sup>3</sup><sup>−</sup> ions were obtained by *Bacillus subtilis* hydrolyz-

<sup>2</sup><sup>−</sup> ions were formed at pH = 7, 8, and

<sup>3</sup><sup>−</sup> ions were formed at

indicated that HPO4

**phosphates**

<sup>2</sup><sup>−</sup> and PO4

9. HPO4

**118**

**Figure 4.**

*EDS spectra: (a) CePO4@NaCe(SO4)2(H2O) and (b) CePO4 [15].*

86–0526. CePO4 was JCPDS Card No. 75–1880.

*Average crystallite size of cerium phosphates [15].*

#### **Figure 6.**

*FTIR spectra: (a) Bacillus subtilis, (b) CePO4@NaCe(SO4)2(H2O), and (c) CePO4 [15].*

Cerium phosphates and *Bacillus subtilis* were determined by FTIR spectra, as shown in **Figure 6**. According to literature reported, the peaks at 3438.03, 2968.49 and 1348.28, 1636.09, 1233.71, 1092.30, 1405.87, and 537.82 cm<sup>−</sup><sup>1</sup> corresponded to, respectively, OH, C-H, C=O or N-H, C-O, etc. (**Figure 3a**) [23]. Above results showed that these functional groups play important roles in the process of adsorption and reduction of Ce(IV) [23]. **Figure 6b** indicated the peak at 1013.15 cm<sup>−</sup><sup>1</sup> was attributed to the asymmetry stretching vibration of the PO4 <sup>3</sup><sup>−</sup> groups. The peaks

#### **Figure 7.**

*SEM images of CePO4@NaCe(SO4)2(H2O) (a, c) and CePO4 (b, d), TEM images of CePO4@NaCe(SO4)2(H2O) (e, g) and CePO4 (f, h), and ED images of CePO4@NaCe(SO4)2(H2O) (i) and CePO4 (j) [15].*

at 620.65 and 540.68 cm<sup>−</sup><sup>1</sup> were assigned to the O-P-O bending vibration [24]. The peaks at 1109.67 and 654.77 cm<sup>−</sup><sup>1</sup> corresponded to the stretching vibration of SO4 2− group and the bending vibration of O-S-O, respectively. Weak transmission bands of PO4 were at 1057.28 cm<sup>−</sup><sup>1</sup> and bending at 539.12 and 615.75 cm<sup>−</sup><sup>1</sup> , as shown in **Figure 6c**, in good agreement with the investigations on CePO4 by Yang [24]. Other peaks in **Figure 6b** and **c** may be ascribed to the absorption vibration of N-H, C=O, COO–, etc., according to the FTIR of *Bacillus subtilis*.

The morphology and crystalline nature of cerium phosphates were observed by SEM, TEM, and electron diffraction (ED) images, as shown in **Figure 7**. SEM images showed shape of CePO4@NaCe(SO4)2(H2O) and CePO4 was nano-clusters and sphere-like with a narrow diameter distribution (**Figure 4a, b, c**, and **d**). TEM images further confirmed that a large number of nanoparticles were stacked

**121**

**Figure 9.**

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials…*

together and formed the big agglomerates. The size of CePO4@NaCe(SO4)2(H2O) and CePO4 was in the range of 20–50 nm and 5–25 nm, respectively. Nanosheets and nanorods in CePO4@NaCe(SO4)2(H2O) and CePO4 were observed, as shown in **Figure** 7**g** and **h**. ED patterns showed that CePO4@NaCe(SO4)2(H2O) and CePO4

**5. Microbiological reduction-precipitation of nanostructured iron** 

and the strong peak at 1644.90 cm<sup>−</sup><sup>1</sup>

FTIR spectrum of iron phosphates was observed in **Figure 8**. The broad peaks

stretching vibration in water. The iron phosphates showed the peaks at 1056.82 and

Fe elements were found in EDS spectrum, as shown in **Figure 9a**. Therefore, iron phosphates were composed by O, P, and Fe elements. XRD further confirmed iron phosphates could be readily indexed to the reported structures of Fe3(PO4)2·8H2O (JCPDS Card No.03–0070), and no peaks attributed to impurities (**Figure 9b**).

were attributed to the O-H

<sup>3</sup><sup>−</sup> groups [17–20, 21, 24–26]. O, P, and

nanomaterials were well crystallized, as shown **Figure 7i** and **j**.

were stretching vibration of PO4

*DOI: http://dx.doi.org/10.5772/intechopen.84296*

**phosphates**

at 3000–3500 cm<sup>−</sup><sup>1</sup>

553.89 cm<sup>−</sup><sup>1</sup>

**Figure 8.**

*FTIR spectrum of iron phosphates [16].*

*EDS spectrum and XRD patterns of iron phosphates [16].*

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials… DOI: http://dx.doi.org/10.5772/intechopen.84296*

together and formed the big agglomerates. The size of CePO4@NaCe(SO4)2(H2O) and CePO4 was in the range of 20–50 nm and 5–25 nm, respectively. Nanosheets and nanorods in CePO4@NaCe(SO4)2(H2O) and CePO4 were observed, as shown in **Figure** 7**g** and **h**. ED patterns showed that CePO4@NaCe(SO4)2(H2O) and CePO4 nanomaterials were well crystallized, as shown **Figure 7i** and **j**.

## **5. Microbiological reduction-precipitation of nanostructured iron phosphates**

FTIR spectrum of iron phosphates was observed in **Figure 8**. The broad peaks at 3000–3500 cm<sup>−</sup><sup>1</sup> and the strong peak at 1644.90 cm<sup>−</sup><sup>1</sup> were attributed to the O-H stretching vibration in water. The iron phosphates showed the peaks at 1056.82 and 553.89 cm<sup>−</sup><sup>1</sup> were stretching vibration of PO4 <sup>3</sup><sup>−</sup> groups [17–20, 21, 24–26]. O, P, and Fe elements were found in EDS spectrum, as shown in **Figure 9a**. Therefore, iron phosphates were composed by O, P, and Fe elements. XRD further confirmed iron phosphates could be readily indexed to the reported structures of Fe3(PO4)2·8H2O (JCPDS Card No.03–0070), and no peaks attributed to impurities (**Figure 9b**).

**Figure 8.** *FTIR spectrum of iron phosphates [16].*

**Figure 9.** *EDS spectrum and XRD patterns of iron phosphates [16].*

*Nanomaterials - Toxicity, Human Health and Environment*

**120**

**Figure 7.**

at 620.65 and 540.68 cm<sup>−</sup><sup>1</sup>

of PO4 were at 1057.28 cm<sup>−</sup><sup>1</sup>

peaks at 1109.67 and 654.77 cm<sup>−</sup><sup>1</sup>

COO–, etc., according to the FTIR of *Bacillus subtilis*.

were assigned to the O-P-O bending vibration [24]. The

group and the bending vibration of O-S-O, respectively. Weak transmission bands

*SEM images of CePO4@NaCe(SO4)2(H2O) (a, c) and CePO4 (b, d), TEM images of CePO4@NaCe(SO4)2(H2O)* 

*(e, g) and CePO4 (f, h), and ED images of CePO4@NaCe(SO4)2(H2O) (i) and CePO4 (j) [15].*

**Figure 6c**, in good agreement with the investigations on CePO4 by Yang [24]. Other peaks in **Figure 6b** and **c** may be ascribed to the absorption vibration of N-H, C=O,

The morphology and crystalline nature of cerium phosphates were observed by SEM, TEM, and electron diffraction (ED) images, as shown in **Figure 7**. SEM images showed shape of CePO4@NaCe(SO4)2(H2O) and CePO4 was nano-clusters and sphere-like with a narrow diameter distribution (**Figure 4a, b, c**, and **d**). TEM images further confirmed that a large number of nanoparticles were stacked

and bending at 539.12 and 615.75 cm<sup>−</sup><sup>1</sup>

corresponded to the stretching vibration of SO4

2−

, as shown in

**Figure 10.** *TG-DSC curves of Fe3(PO4)2·8H2O [16].*

**Figure 11.** *SEM images (a, b), TEM images (c, d, e, f), and ED patterns (g) of Fe3(PO4)2·8H2O [16].*

**123**

**Conflict of interest**

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials…*

Fe3(PO4)2·8H2O and crystallization of Fe3(PO4)2, respectively.

indicated Fe3(PO4)2·8H2O was polycrystalline in nature (**Figure 11g**).

pH were 7, 8, 9, 10, and 11, respectively.

in structure with the size of 31.89 nm.

The authors declare no conflict of interest.

**Notes/thanks/other declarations**

TG-DSC curves of iron phosphates were analyzed from room temperature to 800°C, as shown in **Figure 10**. The TG curve showed total weight loss was about 28.38%, which consisted with the theoretical value (28.69%). TG also confirmed chemical composition of iron phosphates was Fe3(PO4)2·8H2O. Crystal water in Fe3(PO4)2·8H2O could be removed at temperature range of 61–250°C. DSC curve showed endothermic peaks at 142.1°C and 536.1°C were decomposition points of

SEM, TEM, and ED images of Fe3(PO4)2·8H2O were observed in **Figure 11**. SEM images showed that the shape of Fe3(PO4)2·8H2O was honeycomb-like, as shown in **Figure 11a** and **b**. TEM images indicated that the morphology of Fe3(PO4)2·8H2O was quadrilateral, hexagonal, and irregular structure with size in the range of 20–200 nm (**Figure 11c, d, e**, and **f**). The average crystallite size was calculated through the Scherrer formula (1), which was 31.89 nm [13]. The ED patterns

1.*Bacillus subtilis* could hydrolyze organic phosphate monoester to form phosphate ions. Barium phosphate nanoparticles could well be prepared by the centrifuged solution of *Bacillus subtilis* and organic phosphate monoester reacting with barium ions under different pH. TEM images indicated that shape of barium phosphate particles was mainly irregular flakes. The average size of barium phosphates were 48.56, 36.60, 31.89, 33.27, and 34.79 nm when

2.Nanostructures of CePO4 and CePO4@NaCe(SO4)2(H2O) have been successfully synthesized through the mixture solution of *Bacillus subtilis* and organic phosphate monoester reacting with Ce(IV). The average crystalline size of CePO4 and CePO4@NaCe(SO4)2(H2O) was 15.61 and 32.34 nm, respectively. TEM images indicated that shape of CePO4@NaCe(SO4)2(H2O) and CePO4

3.FTIR spectrum, EDS, XRD, and TG-DSC confirmed the chemical composition of iron phosphates was Fe3(PO4)2·8H2O. TEM images showed that the morphology of Fe3(PO4)2·8H2O powder was quadrilateral, hexagonal, and irregular

4.The content of phosphate ions can be increased by adjusting the amount of substrate and pH in the mixture solution of *Bacillus subtilis* and organic phosphate monoester. At same time, the removal efficiency of heavy metal ions can well be increased in soil and water. The different phosphate nanomaterials can

This work was supported by the National Nature Science Foundation of China (51702238) and the Opening Funds of Jiangsu Key Laboratory of Construction Materials (CM2018–02); the financial support is gratefully acknowledged.

be prepared using centrifugation liquid or bacterial liquid.

was nanosheets and nanorods with nonuniform size, respectively.

*DOI: http://dx.doi.org/10.5772/intechopen.84296*

**6. Conclusions**

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials… DOI: http://dx.doi.org/10.5772/intechopen.84296*

TG-DSC curves of iron phosphates were analyzed from room temperature to 800°C, as shown in **Figure 10**. The TG curve showed total weight loss was about 28.38%, which consisted with the theoretical value (28.69%). TG also confirmed chemical composition of iron phosphates was Fe3(PO4)2·8H2O. Crystal water in Fe3(PO4)2·8H2O could be removed at temperature range of 61–250°C. DSC curve showed endothermic peaks at 142.1°C and 536.1°C were decomposition points of Fe3(PO4)2·8H2O and crystallization of Fe3(PO4)2, respectively.

SEM, TEM, and ED images of Fe3(PO4)2·8H2O were observed in **Figure 11**. SEM images showed that the shape of Fe3(PO4)2·8H2O was honeycomb-like, as shown in **Figure 11a** and **b**. TEM images indicated that the morphology of Fe3(PO4)2·8H2O was quadrilateral, hexagonal, and irregular structure with size in the range of 20–200 nm (**Figure 11c, d, e**, and **f**). The average crystallite size was calculated through the Scherrer formula (1), which was 31.89 nm [13]. The ED patterns indicated Fe3(PO4)2·8H2O was polycrystalline in nature (**Figure 11g**).

## **6. Conclusions**

*Nanomaterials - Toxicity, Human Health and Environment*

**122**

**Figure 11.**

*SEM images (a, b), TEM images (c, d, e, f), and ED patterns (g) of Fe3(PO4)2·8H2O [16].*

**Figure 10.**

*TG-DSC curves of Fe3(PO4)2·8H2O [16].*


### **Conflict of interest**

The authors declare no conflict of interest.

### **Notes/thanks/other declarations**

This work was supported by the National Nature Science Foundation of China (51702238) and the Opening Funds of Jiangsu Key Laboratory of Construction Materials (CM2018–02); the financial support is gratefully acknowledged.

## **Author details**

Xiaoniu Yu1,2 and Qiwei Zhan3 \*

1 College of Civil Engineering and Architecture, Wenzhou University, Wenzhou, China

2 School of Environment, Tsinghua University, Beijing, China

3 School of Materials Science and Engineering, Anhui University of Technology, Maanshan, China

\*Address all correspondence to: zhanqw@ahut.edu.cn

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**125**

ma11060945

*Phosphate-Mineralization Microbe Repairs Heavy Metal Ions That Formed Nanomaterials…*

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[10] Qian CX, Xiaoniu Yu XN, Wang X. Potential uses and cementing mechanism of bio-carbonate cement and bio-phosphate cement. AIP Advances. 2018;**8**:095224. DOI:

[11] Li L, Qian CX, Cheng L, Wang RX. A laboratory investigation of microbe-inducing CdCO3 precipitate treatment in Cd2+ contaminated soil. Journal of Soils and Sediments. 2010;**10**:248-254. DOI: 10.1007/

2017;**26**:859-863

10.1063/1.5040730

s11368-009-0089-6

[12] Yu XN, Qian CX, Wang X.

DOI: 10.1166/sam.2015.2392

Biosynthesis of magnesium phosphates and its thermal property. Science of Advanced Materials. 2015;**7**:1730-1733.

[13] Wang X, Qian CX, Yu XN. Synthesis of nano-hydroxyapatite via microbial method and its characterization. Applied Biochemistry and

Biotechnology. 2014;**173**:1003-1010. DOI: 10.1007/s12010-014-0911-5

[14] Yu XN, Qian CX, Wang X. Bioinspired synthesis of barium phosphates nanoparticles and its characterization. Digest Journal of Nanomaterials and Biostructures. 2015;**10**:199-205

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*DOI: http://dx.doi.org/10.5772/intechopen.84296*

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**Author details**

Maanshan, China

China

Xiaoniu Yu1,2 and Qiwei Zhan3

\*

2 School of Environment, Tsinghua University, Beijing, China

\*Address all correspondence to: zhanqw@ahut.edu.cn

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 College of Civil Engineering and Architecture, Wenzhou University, Wenzhou,

3 School of Materials Science and Engineering, Anhui University of Technology,

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Section 5

Applications of

Nanomaterials

Section 5
