Bioremediation with Biochar

**75**

**Chapter 5**

**Abstract**

**1. Introduction**

system of biomass materials.

Enzymes

*Hidetaka Noritomi*

Application of Biochar to Enzyme

Biochar showed the high affinity to enzymes, and enzymes were sufficiently adsorbed on the surface of biochar. Enzymes were highly stabilized in water at high temperatures by adsorbing enzymes on biochar. The remaining activity of lysozyme adsorbed on adzuki bean charcoal showed around 50% after heat treatment at 90°C for 30 min though that of free lysozyme was almost lost. Likewise, the stability of enzymes was enhanced in organic solvents by adsorbing enzymes on biochar. The conformation of α-chymotrypsin adsorbed on bamboo charcoal was hardly influenced by organic solvents, while that of free α-chymotrypsin was strongly dependent of the kind of organic solvents. Moreover, the adsorption of α-chymotrypsin on bamboo charcoal improved the transesterification of *N*-acetyl-L-tyrosine ethyl ester with *n*-butanol in organic solvents. The transesterification rate of α-chymotrypsin adsorbed on bamboo charcoal was about 760 times higher

than that of free α-chymotrypsin in *n*-butyl acetate.

**Keywords:** biochar, enzyme, enzyme carrier, adsorption, stress tolerance

An enormous amount of greenhouse gas such as CO2 has recently been emitted from industries and thereby has caused serious global warming problems [1, 2]. Accordingly, the application of biomass materials, which are carbon neutral, to energies and functional materials, is crucial to reduce greenhouse gas emissions [3, 4]. However, most of biomass materials such as forestry residues have hardly been utilized in the field of functional materials. Accordingly, the development in the high value-added application of biomass materials has been desired to provide the multiple effective utilization

Enzymes are biocatalysts, which exhibit their outstanding biological activity under mild conditions, and have widely been used in pharmacy, biotechnology, and chemical industry [5–7]. Typical applications of enzymes are biotransformation, biosensor, biofuel cell, and so on. Enzymes are generally stable in a cell. However, they are gradually denatured and inactivated under various physical and chemical stresses such as heat, organic solvents, and so on [8]. In order to enhance the stability of enzymes used in vitro, enzyme immobilization, where enzyme molecules are attached to solid carriers, has widely been used [9–11]. The main required features of enzyme carriers are chemical stability, thermal stability, insolubility under reaction

Carrier for Stress Tolerance of

#### **Chapter 5**

## Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes

*Hidetaka Noritomi*

### **Abstract**

Biochar showed the high affinity to enzymes, and enzymes were sufficiently adsorbed on the surface of biochar. Enzymes were highly stabilized in water at high temperatures by adsorbing enzymes on biochar. The remaining activity of lysozyme adsorbed on adzuki bean charcoal showed around 50% after heat treatment at 90°C for 30 min though that of free lysozyme was almost lost. Likewise, the stability of enzymes was enhanced in organic solvents by adsorbing enzymes on biochar. The conformation of α-chymotrypsin adsorbed on bamboo charcoal was hardly influenced by organic solvents, while that of free α-chymotrypsin was strongly dependent of the kind of organic solvents. Moreover, the adsorption of α-chymotrypsin on bamboo charcoal improved the transesterification of *N*-acetyl-L-tyrosine ethyl ester with *n*-butanol in organic solvents. The transesterification rate of α-chymotrypsin adsorbed on bamboo charcoal was about 760 times higher than that of free α-chymotrypsin in *n*-butyl acetate.

**Keywords:** biochar, enzyme, enzyme carrier, adsorption, stress tolerance

#### **1. Introduction**

An enormous amount of greenhouse gas such as CO2 has recently been emitted from industries and thereby has caused serious global warming problems [1, 2]. Accordingly, the application of biomass materials, which are carbon neutral, to energies and functional materials, is crucial to reduce greenhouse gas emissions [3, 4]. However, most of biomass materials such as forestry residues have hardly been utilized in the field of functional materials. Accordingly, the development in the high value-added application of biomass materials has been desired to provide the multiple effective utilization system of biomass materials.

Enzymes are biocatalysts, which exhibit their outstanding biological activity under mild conditions, and have widely been used in pharmacy, biotechnology, and chemical industry [5–7]. Typical applications of enzymes are biotransformation, biosensor, biofuel cell, and so on. Enzymes are generally stable in a cell. However, they are gradually denatured and inactivated under various physical and chemical stresses such as heat, organic solvents, and so on [8]. In order to enhance the stability of enzymes used in vitro, enzyme immobilization, where enzyme molecules are attached to solid carriers, has widely been used [9–11]. The main required features of enzyme carriers are chemical stability, thermal stability, insolubility under reaction

conditions, high affinity to enzymes, biocompatibility, the presence of reactive functional groups, availability, low price, regeneration, reusability, and so on. When enzymes are immobilized on carriers through adsorption, the catalytic activity, specificity, and stability of enzymes are influenced by the nature of carriers. Accordingly, the performance of enzymes can be enhanced by selecting an appropriate carrier.

A large quantity of the world's oldest biochar was excavated from the cave of Kara Iwatani of Hijikawacho, Ozu-shi, Ehime, Japan, with a beast bone and the human bone piece 300,000 years ago. Since the ancient period, the biochar has been used not only as a fuel but also as a soil conditioner to support human life for a long time in the world [12]. Consequently, as the biocompatibility of biochar can be expected, we have examined the application of biochar to enzyme carriers. As a result, we have found that enzymes are effectively adsorbed on biochar [13, 14], and biochar-adsorbed enzymes exhibit the high thermal stability in water [15–18]. Moreover, we have reported that the adsorption of enzymes on biochar sufficiently improves the enzyme activity in organic solvents [19–21].

In the chapter, the characterization of biochar, the adsorption of enzyme on biochar, the high temperature-tolerant property of biochar-adsorbed enzymes, and the organic solvent-tolerant property of biochar-adsorbed enzymes are discussed.

#### **2. Heat tolerance of biochar-adsorbed enzyme**

#### **2.1 Preparation and characterization of biochar**

Biochar has been prepared by pyrolyzing plant biomass waste such as bamboo waste at low temperatures under nitrogen atmosphere to produce functional groups, which were used as a binding site for the adsorption of enzymes (**Figure 1**) [13, 14]. The emission of carbon dioxide was reduced since plant biomass waste was not burned through the preparation of biochar. Moreover, the energy cost of the present preparation of biochar was suppressed, compared to that of the conventional preparation of charcoal. Consequently, the present preparation of biochar was a low-cost and environmentally benign process.

**77**

*a*

**Table 1.**

*Structural characteristics of biochar.*

**Figure 2.**

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

biochar was not formed enough at low temperatures.

groups decrease with increasing carbonization temperature [22, 23].

*SEM images of (a) adzuki bean charcoal, (b) bamboo charcoal, and (c) wood charcoal.*

**[m2 /g]**

*Specific area of adzuki bean charcoal was obtained from the CO2 isotherm.*

Adzuki bean charcoal 204a — — Bamboo charcoal 294 0.041 Less than 2.6 Wood charcoal 117 0.025 Less than 2.6

**Pore volume [cm3 /g]**

**Pore diameter peak [nm]**

**Biochar Specific surface area** 

**Figure 6** shows the relationship of the ζ potential of biochar with the solution pH [15]. The ζ potential of adzuki bean charcoal drastically decreased with

In order to observe the surface of biochar, we have examined SEM images [14]. As seen in **Figure 2**, the morphology of biochar was strongly dependent upon the kind of raw materials. The roughness of biochar was remarkably low, and any pores were not observed at the magnification measured in the present work. The surface of bamboo charcoal was smoother than that of any other charcoal. Moreover, Raman

**Table 1** shows the textural parameters of biochar obtained from low-temperature (−196°C) nitrogen adsorption isotherms, which allow the calculation of specific surface area, specific pore volume, and pore diameter peak [14]. In the table, the specific area of adzuki bean charcoal depicted the value obtained from the CO2 isotherm. The specific surface area and specific pore volume of biochar showed much small, compared with that of conventional activated carbon. The carbonizing temperature affects the surface property of charcoal [22]. The specific pore volume increases with an increase in carbonizing temperature. Consequently, the pore of

spectra of biochar showed that the structure of biochar was amorphous [13].

**Figure 3** shows CP/MAS 13C-NMR spectra of biochar [13]. Aromatic carbon (140–141, 131 ppm) was mainly detected, and C=O (200 ppm), COOH, CHO (175–190 ppm), and aromatic oxygen (150–153, 145–146 ppm) were also detected. Moreover, in order to assess the chemical property of the surface of biochar, the measurement on X-ray photoelectron spectroscopy (XPS) was carried out [19]. **Figure 4** shows the elemental ratio of the surface of biochar detected by XPS. The main element was carbon atom, and oxygen and nitrogen atoms also existed on the surface of biochar to some extent. The ratios of oxygen and nitrogen atoms in adzuki bean charcoal were greater than those in any other charcoal. Narrow scan spectra of XPS showed C▬C, C▬H, C▬O, O▬C▬O, C=O, COOH, and C▬N, as seen in **Figure 5**. Many radical species due to functional groups containing oxygen atoms, which are formed by thermal decomposition of cellulose and hemicelluloses, are detected in charcoals carbonized at 500°C by the measurement of electron spin resonance, and functional

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

**Figure 1.** *Preparation process of biochar.*

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

In order to observe the surface of biochar, we have examined SEM images [14]. As seen in **Figure 2**, the morphology of biochar was strongly dependent upon the kind of raw materials. The roughness of biochar was remarkably low, and any pores were not observed at the magnification measured in the present work. The surface of bamboo charcoal was smoother than that of any other charcoal. Moreover, Raman spectra of biochar showed that the structure of biochar was amorphous [13].

**Table 1** shows the textural parameters of biochar obtained from low-temperature (−196°C) nitrogen adsorption isotherms, which allow the calculation of specific surface area, specific pore volume, and pore diameter peak [14]. In the table, the specific area of adzuki bean charcoal depicted the value obtained from the CO2 isotherm. The specific surface area and specific pore volume of biochar showed much small, compared with that of conventional activated carbon. The carbonizing temperature affects the surface property of charcoal [22]. The specific pore volume increases with an increase in carbonizing temperature. Consequently, the pore of biochar was not formed enough at low temperatures.

**Figure 3** shows CP/MAS 13C-NMR spectra of biochar [13]. Aromatic carbon (140–141, 131 ppm) was mainly detected, and C=O (200 ppm), COOH, CHO (175–190 ppm), and aromatic oxygen (150–153, 145–146 ppm) were also detected. Moreover, in order to assess the chemical property of the surface of biochar, the measurement on X-ray photoelectron spectroscopy (XPS) was carried out [19]. **Figure 4** shows the elemental ratio of the surface of biochar detected by XPS. The main element was carbon atom, and oxygen and nitrogen atoms also existed on the surface of biochar to some extent. The ratios of oxygen and nitrogen atoms in adzuki bean charcoal were greater than those in any other charcoal. Narrow scan spectra of XPS showed C▬C, C▬H, C▬O, O▬C▬O, C=O, COOH, and C▬N, as seen in **Figure 5**. Many radical species due to functional groups containing oxygen atoms, which are formed by thermal decomposition of cellulose and hemicelluloses, are detected in charcoals carbonized at 500°C by the measurement of electron spin resonance, and functional groups decrease with increasing carbonization temperature [22, 23].

**Figure 6** shows the relationship of the ζ potential of biochar with the solution pH [15]. The ζ potential of adzuki bean charcoal drastically decreased with

#### **Figure 2.**

*Biochar - An Imperative Amendment for Soil and the Environment*

improves the enzyme activity in organic solvents [19–21].

**2. Heat tolerance of biochar-adsorbed enzyme**

**2.1 Preparation and characterization of biochar**

and environmentally benign process.

conditions, high affinity to enzymes, biocompatibility, the presence of reactive functional groups, availability, low price, regeneration, reusability, and so on. When enzymes are immobilized on carriers through adsorption, the catalytic activity, specificity, and stability of enzymes are influenced by the nature of carriers. Accordingly, the performance of enzymes can be enhanced by selecting an appropriate carrier. A large quantity of the world's oldest biochar was excavated from the cave of Kara Iwatani of Hijikawacho, Ozu-shi, Ehime, Japan, with a beast bone and the human bone piece 300,000 years ago. Since the ancient period, the biochar has been used not only as a fuel but also as a soil conditioner to support human life for a long time in the world [12]. Consequently, as the biocompatibility of biochar can be expected, we have examined the application of biochar to enzyme carriers. As a result, we have found that enzymes are effectively adsorbed on biochar [13, 14], and biochar-adsorbed enzymes exhibit the high thermal stability in water [15–18]. Moreover, we have reported that the adsorption of enzymes on biochar sufficiently

In the chapter, the characterization of biochar, the adsorption of enzyme on biochar, the high temperature-tolerant property of biochar-adsorbed enzymes, and the organic solvent-tolerant property of biochar-adsorbed enzymes are discussed.

Biochar has been prepared by pyrolyzing plant biomass waste such as bamboo waste at low temperatures under nitrogen atmosphere to produce functional groups, which were used as a binding site for the adsorption of enzymes (**Figure 1**) [13, 14]. The emission of carbon dioxide was reduced since plant biomass waste was not burned through the preparation of biochar. Moreover, the energy cost of the present preparation of biochar was suppressed, compared to that of the conventional preparation of charcoal. Consequently, the present preparation of biochar was a low-cost

**76**

**Figure 1.**

*Preparation process of biochar.*

*SEM images of (a) adzuki bean charcoal, (b) bamboo charcoal, and (c) wood charcoal.*


#### **Table 1.** *Structural characteristics of biochar.*

**Figure 3.** *CP/MAS 13C-NMR spectra of biochar.*

**Figure 4.** *The elemental ratio of the surface of biochar detected by XPS.*

increasing the pH value, exhibiting a negative value above pH 4, drops till pH 7, and was almost constant in the alkaline region. The pH dependence of the ζ potential of bamboo charcoal exhibited the same tendency to the case of adzuki bean charcoal.

#### **2.2 Adsorption of enzymes on biochar**

**Figure 7** shows the time course of the amount of lysozyme adsorbed on adzuki bean charcoal at pH 7 and 25°C when hen egg white lysozyme was employed as a model enzyme [16]. The amount of lysozyme adsorbed on adzuki bean charcoal increased with an increase in adsorption time, reached a plateau around 24 h, and

**79**

**Figure 6.**

*Effect of solution pH on ζ potential of biochar.*

**Figure 5.**

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

*The chemical bond ratio of biochar obtained from narrow scan spectra of XPS.*

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

**Figure 5.**

*Biochar - An Imperative Amendment for Soil and the Environment*

**78**

**Figure 4.**

*The elemental ratio of the surface of biochar detected by XPS.*

**2.2 Adsorption of enzymes on biochar**

increasing the pH value, exhibiting a negative value above pH 4, drops till pH 7, and was almost constant in the alkaline region. The pH dependence of the ζ potential of bamboo charcoal exhibited the same tendency to the case of adzuki bean charcoal.

**Figure 7** shows the time course of the amount of lysozyme adsorbed on adzuki bean charcoal at pH 7 and 25°C when hen egg white lysozyme was employed as a model enzyme [16]. The amount of lysozyme adsorbed on adzuki bean charcoal increased with an increase in adsorption time, reached a plateau around 24 h, and

**Figure 3.**

*CP/MAS 13C-NMR spectra of biochar.*

*The chemical bond ratio of biochar obtained from narrow scan spectra of XPS.*

**Figure 6.** *Effect of solution pH on ζ potential of biochar.*

**Figure 7.** *Time dependence of amount of lysozyme adsorbed onto adzuki bean charcoal.*


#### **Table 2.**

*Amount of enzymes adsorbed on biochar.*

was 11 μmol/g (0.16 g/g). As overall concentration of adzuki bean charcoal was 3 g/L in an aqueous solution, overall lysozyme concentration in the aqueous solution corresponded to 33 μM (0.48 mg/mL). From this result, the adsorption of lysozyme on adzuki bean charcoal was carried out for 24 h.

As seen in **Table 2**, the amount of enzymes adsorbed on biochar was strongly dependent on the kinds of enzymes and/or biochars [14, 15]. The amount of lysozyme adsorbed on biochar was almost the same among three different charcoals although the specific surface area of adzuki bean charcoal or bamboo charcoal was more than twice larger than wood charcoal, as seen in **Table 1**.

**Figure 8** shows the adsorption isotherms of lysozyme on biochar. These isotherms gradually increased [13]. The amount adsorbed on adzuki bean charcoal exhibited large, compared to the amount adsorbed on bamboo charcoal. The curves in the figure were the fitting lines with Freundlich adsorption isotherm equation (Eq. 1).

$$\mathbf{W} = \mathbf{K}\_{\mathbf{F}} \mathbf{C}^{1/n} \tag{1}$$

**81**

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

adsorbed on adzuki bean charcoal had the optimum value around neutral pH, similar to the case of bamboo charcoal. The net charge of protein molecules alters with the pH of aqueous solutions. Since the isoelectric point (pI) of lysozyme is 11, the net charge of lysozyme becomes more positive below pH 11. Concerning the ζ potential of biochar, the ζ potential of adzuki bean charcoal drastically decreased with increasing the pH value, exhibited a negative value above pH 4, dropped till pH 7, and was almost constant in the alkaline region, as shown in **Figure 6**. The pH dependence of the ζ potential of bamboo charcoal exhibited the same tendency to the case of adzuki bean charcoal. When the pH value was around the pI of lysozyme or the pH where the ζ potential of biochar approached 0 volts, a dramatic decrease in the amount of lysozyme adsorbed on biochar was observed. On the other hand, in the vicinity of neutral pH where lysozyme and the surface of biochar were charged positively and negatively, respectively, the high amount of adsorption

*Effect of pH on the amount of lysozyme adsorbed onto biochar; adsorption was carried out by incubating buffer solution (appropriate pH) containing 500 μM lysozyme and 3 g/L biochar at 120 rpm and 25°C for 24 h.*

*Adsorption isotherms of lysozyme onto biochar; adsorption was carried out by incubating buffer solution (pH 7) containing a certain amount of lysozyme and 3 g/L biochar at 120 rpm and 25°C for 24 h.*

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

**Figure 8.**

**Figure 9.**

Here, *W* and *C* are the amount adsorbed and the concentration of lysozyme, respectively. *K*F and *n* are experimental constants [24]. The curves of adzuki bean charcoal and bamboo charcoal had the correlation constants (*r* 2 ) of 0.974 and 0.998, respectively. On the other hand, the data were fitted by Langmuir adsorption isotherm equation; the curves of adzuki bean charcoal and bamboo charcoal depicted the correlation constants (*r* 2 ) of 0.721 and 0.694, respectively.

**Figure 9** shows the relationship of the amount of lysozyme adsorbed on biochar with the pH value of aqueous solutions at 25°C [14]. The curve of the amount

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

#### **Figure 8.**

*Biochar - An Imperative Amendment for Soil and the Environment*

was 11 μmol/g (0.16 g/g). As overall concentration of adzuki bean charcoal was 3 g/L in an aqueous solution, overall lysozyme concentration in the aqueous solution corresponded to 33 μM (0.48 mg/mL). From this result, the adsorption of

**Biochar Amount of enzymes adsorbed (μmol/g)**

Adzuki bean charcoal 11 17 Bamboo charcoal 9 9.8 Wood charcoal 12 21

dependent on the kinds of enzymes and/or biochars [14, 15]. The amount of

As seen in **Table 2**, the amount of enzymes adsorbed on biochar was strongly

lysozyme adsorbed on biochar was almost the same among three different charcoals although the specific surface area of adzuki bean charcoal or bamboo charcoal was

**Figure 8** shows the adsorption isotherms of lysozyme on biochar. These isotherms gradually increased [13]. The amount adsorbed on adzuki bean charcoal exhibited large, compared to the amount adsorbed on bamboo charcoal. The curves in the figure were the fitting lines with Freundlich adsorption isotherm

Here, *W* and *C* are the amount adsorbed and the concentration of lysozyme, respectively. *K*F and *n* are experimental constants [24]. The curves of adzuki bean

0.998, respectively. On the other hand, the data were fitted by Langmuir adsorption isotherm equation; the curves of adzuki bean charcoal and bamboo charcoal

**Figure 9** shows the relationship of the amount of lysozyme adsorbed on biochar

2

with the pH value of aqueous solutions at 25°C [14]. The curve of the amount

*W* = *K*F*C*1/*<sup>n</sup>* (1)

**Lysozyme α-Chymotrypsin**

) of 0.721 and 0.694, respectively.

2

) of 0.974 and

lysozyme on adzuki bean charcoal was carried out for 24 h.

*Time dependence of amount of lysozyme adsorbed onto adzuki bean charcoal.*

more than twice larger than wood charcoal, as seen in **Table 1**.

charcoal and bamboo charcoal had the correlation constants (*r*

depicted the correlation constants (*r*

**80**

equation (Eq. 1).

**Figure 7.**

**Table 2.**

*Amount of enzymes adsorbed on biochar.*

*Adsorption isotherms of lysozyme onto biochar; adsorption was carried out by incubating buffer solution (pH 7) containing a certain amount of lysozyme and 3 g/L biochar at 120 rpm and 25°C for 24 h.*

#### **Figure 9.**

*Effect of pH on the amount of lysozyme adsorbed onto biochar; adsorption was carried out by incubating buffer solution (appropriate pH) containing 500 μM lysozyme and 3 g/L biochar at 120 rpm and 25°C for 24 h.*

adsorbed on adzuki bean charcoal had the optimum value around neutral pH, similar to the case of bamboo charcoal. The net charge of protein molecules alters with the pH of aqueous solutions. Since the isoelectric point (pI) of lysozyme is 11, the net charge of lysozyme becomes more positive below pH 11. Concerning the ζ potential of biochar, the ζ potential of adzuki bean charcoal drastically decreased with increasing the pH value, exhibited a negative value above pH 4, dropped till pH 7, and was almost constant in the alkaline region, as shown in **Figure 6**. The pH dependence of the ζ potential of bamboo charcoal exhibited the same tendency to the case of adzuki bean charcoal. When the pH value was around the pI of lysozyme or the pH where the ζ potential of biochar approached 0 volts, a dramatic decrease in the amount of lysozyme adsorbed on biochar was observed. On the other hand, in the vicinity of neutral pH where lysozyme and the surface of biochar were charged positively and negatively, respectively, the high amount of adsorption

tended to be obtained. Consequently, these results indicate that the electrostatic interaction between the positively charged lysozyme and the negatively charged surface of biochar mainly contributes to the adsorption.

#### **2.3 Heat stress tolerance of enzymes adsorbed on biochar**

Modest heating causes enzymes dissolved in an aqueous solution to be denatured and inactivated by unfolding of enzyme molecules due to the disruption of weak interactions such as ionic bonds, hydrogen bonds, and hydrophobic interactions, which are prime determinants of enzyme tertiary structures [25]. In order to assess the heat stress tolerance of enzymes adsorbed on biochar, an aqueous solution containing lysozyme adsorbed on adzuki bean charcoal was incubated at high temperatures [15]. **Figure 10** shows photographs of aqueous solutions containing free lysozyme, the mixture of lysozyme and adzuki bean charcoal, and lysozyme adsorbed on adzuki bean charcoal before and after heat treatment was carried out at 90°C for 30 min, while under such heat conditions, raw eggs become hard-boiled eggs. The solution of free lysozyme immediately became turbid since thermally denatured enzymes were drastically aggregated by heat, as shown in **Figure 10(d)**. The enzyme aggregation is precipitated above 10 μM lysozyme [26]. The formation of enzyme aggregation was enhanced since the present concentration of lysozyme was 33 μM. Adzuki bean charcoal was easily dispersed in an aqueous solution due to the good wettability to water as seen in **Figure 10(b)**. Likewise, the mixture of lysozyme and adzuki bean charcoal was immediately precipitated by heat treatment due to the aggregation of denatured enzymes, as shown in **Figure 10(e)**. On the other hand, lysozyme adsorbed on adzuki bean charcoal was easily dispersed in an aqueous solution, as seen in **Figure 10(c)**. After the heat treatment, lysozyme adsorbed on adzuki bean charcoal was sufficiently dispersed in the solution, and the white enzyme aggregation and the cohesion among adzuki bean charcoals adsorbing lysozyme were not observed in the solution, as shown in **Figure 10(f )**. When enzymes dissolved in an aqueous solution are placed at high temperatures, most of enzymes are instantaneously unfolded by the disruption of weak interactions consisting of ionic bonds, hydrogen bonds, and hydrophobic interactions of enzymes [25, 27]. Additionally, unfolded enzymes are aggregated with each other, and the chemical deterioration reactions occur in unfolded enzymes. In particular, enzyme aggregation easily occurs upon the exposure of the hydrophobic surfaces of an enzyme, and this phenomenon becomes the major problem because of the fast

#### **Figure 10.**

*Photographs of lysozyme solutions before and after heat treatment at 90°C for 30 min: (a) an aqueous solution containing free lysozyme before heat treatment, (b) an aqueous solution containing free lysozyme solution and adzuki bean charcoal before heat treatment, (c) an aqueous solution containing lysozyme adsorbed on adzuki bean charcoal before heat treatment, (d) an aqueous solution containing free lysozyme after heat treatment, (e) an aqueous solution containing free lysozyme solution and adzuki bean charcoal after heat treatment, and (f) an aqueous solution containing lysozyme adsorbed on adzuki bean charcoal after heat treatment.*

**83**

**Table 3.**

**Figure 11.**

*heat treatment at pH 7.0 and 90°C.*

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

irreversible inactivation. The adsorption of lysozyme on adzuki bean charcoal could

When the remaining activity is defined as the ratio of the activity of lysozyme

To extend our study, the remaining activities of lysozyme adsorbed on biochar obtained from several kinds of plant biomass wastes have been measured after heat treatment at 90°C for 30 min. Lysozyme adsorbed on bamboo charcoal or wood charcoal exhibited the high thermal stability, similar to the case of lysozymeadsorbed adzuki bean charcoal, as shown in **Figure 12**. On the other hand, the

*Time course of remaining activity of free lysozyme and lysozyme adsorbed on adzuki bean charcoal through the* 

Free lysozyme 0.168 4 Lysozyme adsorbed on adzuki bean charcoal 0.027 28

**) Half life (min)**

**Samples Rate constant (min<sup>−</sup><sup>1</sup>**

*Rate constants and half lives of inactivation of lysozyme at 90°C.*

after heat treatment to that before heat treatment, time courses of remaining activities of free lysozyme and lysozyme adsorbed on adzuki bean charcoal through the heat treatment at pH 7.0 and 90°C are shown in **Figure 11** [16]. The remaining activities of free lysozyme and lysozyme adsorbed on adzuki bean charcoal decreased with an increase in time. As shown in **Figure 11**, the remaining activities of free and adsorbed lysozyme exhibited the correlation of first-order kinetics with heat treatment time. **Table 3** shows inactivation rate constants and half-lives of free and adsorbed lysozymes obtained from the curve fitting in **Figure 11**. The half-life of adsorbed lysozyme was seven times greater than that of free lysozyme. The remaining activity of free lysozyme was almost lost after heat treatment for 30 min, and the remaining activity in the mixture of lysozyme and adzuki bean charcoal exhibited 2%, while the remaining activity of lysozyme adsorbed on adzuki bean charcoal showed around 50%. The robust thermal stability of adsorbed lysozyme may be attributable to the suitable interaction of lysozyme with the surface of

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

adzuki bean charcoal.

inhibit the formation of enzyme aggregation.

#### *Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

irreversible inactivation. The adsorption of lysozyme on adzuki bean charcoal could inhibit the formation of enzyme aggregation.

When the remaining activity is defined as the ratio of the activity of lysozyme after heat treatment to that before heat treatment, time courses of remaining activities of free lysozyme and lysozyme adsorbed on adzuki bean charcoal through the heat treatment at pH 7.0 and 90°C are shown in **Figure 11** [16]. The remaining activities of free lysozyme and lysozyme adsorbed on adzuki bean charcoal decreased with an increase in time. As shown in **Figure 11**, the remaining activities of free and adsorbed lysozyme exhibited the correlation of first-order kinetics with heat treatment time. **Table 3** shows inactivation rate constants and half-lives of free and adsorbed lysozymes obtained from the curve fitting in **Figure 11**. The half-life of adsorbed lysozyme was seven times greater than that of free lysozyme. The remaining activity of free lysozyme was almost lost after heat treatment for 30 min, and the remaining activity in the mixture of lysozyme and adzuki bean charcoal exhibited 2%, while the remaining activity of lysozyme adsorbed on adzuki bean charcoal showed around 50%. The robust thermal stability of adsorbed lysozyme may be attributable to the suitable interaction of lysozyme with the surface of adzuki bean charcoal.

To extend our study, the remaining activities of lysozyme adsorbed on biochar obtained from several kinds of plant biomass wastes have been measured after heat treatment at 90°C for 30 min. Lysozyme adsorbed on bamboo charcoal or wood charcoal exhibited the high thermal stability, similar to the case of lysozymeadsorbed adzuki bean charcoal, as shown in **Figure 12**. On the other hand, the

**Figure 11.**

*Biochar - An Imperative Amendment for Soil and the Environment*

surface of biochar mainly contributes to the adsorption.

**2.3 Heat stress tolerance of enzymes adsorbed on biochar**

tended to be obtained. Consequently, these results indicate that the electrostatic interaction between the positively charged lysozyme and the negatively charged

Modest heating causes enzymes dissolved in an aqueous solution to be denatured and inactivated by unfolding of enzyme molecules due to the disruption of weak interactions such as ionic bonds, hydrogen bonds, and hydrophobic interactions, which are prime determinants of enzyme tertiary structures [25]. In order to assess the heat stress tolerance of enzymes adsorbed on biochar, an aqueous solution containing lysozyme adsorbed on adzuki bean charcoal was incubated at high temperatures [15]. **Figure 10** shows photographs of aqueous solutions containing free lysozyme, the mixture of lysozyme and adzuki bean charcoal, and lysozyme adsorbed on adzuki bean charcoal before and after heat treatment was carried out at 90°C for 30 min, while under such heat conditions, raw eggs become hard-boiled eggs. The solution of free lysozyme immediately became turbid since thermally denatured enzymes were drastically aggregated by heat, as shown in **Figure 10(d)**. The enzyme aggregation is precipitated above 10 μM lysozyme [26]. The formation of enzyme aggregation was enhanced since the present concentration of lysozyme was 33 μM. Adzuki bean charcoal was easily dispersed in an aqueous solution due to the good wettability to water as seen in **Figure 10(b)**. Likewise, the mixture of lysozyme and adzuki bean charcoal was immediately precipitated by heat treatment due to the aggregation of denatured enzymes, as shown in **Figure 10(e)**. On the other hand, lysozyme adsorbed on adzuki bean charcoal was easily dispersed in an aqueous solution, as seen in **Figure 10(c)**. After the heat treatment, lysozyme adsorbed on adzuki bean charcoal was sufficiently dispersed in the solution, and the white enzyme aggregation and the cohesion among adzuki bean charcoals adsorbing lysozyme were not observed in the solution, as shown in **Figure 10(f )**. When enzymes dissolved in an aqueous solution are placed at high temperatures, most of enzymes are instantaneously unfolded by the disruption of weak interactions consisting of ionic bonds, hydrogen bonds, and hydrophobic interactions of enzymes [25, 27]. Additionally, unfolded enzymes are aggregated with each other, and the chemical deterioration reactions occur in unfolded enzymes. In particular, enzyme aggregation easily occurs upon the exposure of the hydrophobic surfaces of an enzyme, and this phenomenon becomes the major problem because of the fast

*Photographs of lysozyme solutions before and after heat treatment at 90°C for 30 min: (a) an aqueous solution containing free lysozyme before heat treatment, (b) an aqueous solution containing free lysozyme solution and adzuki bean charcoal before heat treatment, (c) an aqueous solution containing lysozyme adsorbed on adzuki bean charcoal before heat treatment, (d) an aqueous solution containing free lysozyme after heat treatment, (e) an aqueous solution containing free lysozyme solution and adzuki bean charcoal after heat treatment, and (f) an aqueous solution containing lysozyme adsorbed on adzuki bean charcoal after heat treatment.*

**82**

**Figure 10.**

*Time course of remaining activity of free lysozyme and lysozyme adsorbed on adzuki bean charcoal through the heat treatment at pH 7.0 and 90°C.*


#### **Table 3.**

*Rate constants and half lives of inactivation of lysozyme at 90°C.*

mixture of lysozyme and bamboo charcoal or wood charcoal showed several percent of remaining activity.

The solution pH generally affects the activity and stability of enzymes in aqueous solutions [5]. **Figure 13** shows the remaining activity of lysozyme adsorbed on bamboo charcoal against the solution pH of adsorption medium. The remaining activity of bamboo charcoal-adsorbed lysozyme was markedly influenced by the pH of adsorption medium and showed the maximum value at pH 5.

#### **Figure 12.**

*Effect of kind of biochar on remaining activity of lysozyme adsorbed on biochar after heat treatment at 90°C for 30 min.*

#### **Figure 13.**

*Effect of pH of adsorption medium on the remaining activity of lysozyme adsorbed on bamboo charcoal after the heat treatment at 90°C for 30 min.*

**85**

**Figure 14.**

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

Three-dimensional structures of enzymes consist of secondary structures such as α-helix and β-sheet [28]. To elucidate the influence of adsorption on the structure of lysozyme, bamboo charcoal-adsorbed lysozyme has been measured by Fourier-transform infrared (FTIR) spectroscopy. **Figure 14** shows the FTIR spectra of native lysozyme and lysozyme adsorbed on bamboo at different pH. The most sensitive spectral region to enzyme secondary structural components is amide I

peptide linkages [28]. The spectral pattern of bamboo charcoal-adsorbed lysozyme was influenced by the pH of adsorption medium. To evaluate the change in the secondary structure of bamboo-adsorbed lysozyme, the ratio of the absorbance at

(0.86), where the remaining activity showed the maximum value, was similar to that of native lysozyme (0.88). Likewise, the ABS1681/ABS1647 ratio at pH 4 (0.92) was near that of native lysozyme. On the other hand, the ABS1681/ABS1647 ratios at pH 7 (0.69) and 9 (0.61) were different from that of native lysozyme. The effect of solution pH of adsorption medium on the thermal stability of bamboo charcoaladsorbed lysozyme has been summarized as follows. The structure of bamboo charcoal-adsorbed lysozyme was nearly the native structure of lysozyme when lysozyme was adsorbed on bamboo charcoal at pH 4, but the electrostatic interaction between lysozyme and bamboo charcoal could not sufficiently contribute to the thermal stability. The electrostatic interaction between lysozyme and bamboo charcoal could strongly retain the structure of lysozyme at high temperatures when lysozyme was adsorbed on bamboo charcoal at pH 5, where the native structure of lysozyme was maintained. The structure of lysozyme was partially destroyed since the electrostatic interaction was too strong to maintain the native structure of lysozyme, and the thermal stability of bamboo charcoal-adsorbed lysozyme dropped when lysozyme was adsorbed on bamboo charcoal at pH 7 and 9. Therefore, these results indicate that biochar-adsorbed enzymes exhibit the excellent thermal stability when the

*(A) FTIR spectrum of native lysozyme. (B) FTIR spectrum of lysozyme adsorbed on bamboo charcoal at pH 4. (C) FTIR spectrum of lysozyme adsorbed on bamboo charcoal at pH 5. (D) FTIR spectrum of lysozyme adsorbed on bamboo charcoal at pH 7. (E) FTIR spectrum of lysozyme adsorbed on bamboo charcoal at pH 9.*

), which is due almost entirely to the C〓O stretch vibrations of

is assigned to α-helix. The ABS1681/ABS1647 ratio at pH 5

(ABS1681/ABS1647) has been assessed since

is assigned to intramolecular β-sheet and the band

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

to the absorbance at 1647 cm<sup>−</sup><sup>1</sup>

the band located at ca. 1681 cm<sup>−</sup><sup>1</sup>

located at ca. 1647 cm<sup>−</sup><sup>1</sup>

(1700–1600 cm<sup>−</sup><sup>1</sup>

1681 cm<sup>−</sup><sup>1</sup>

#### *Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

*Biochar - An Imperative Amendment for Soil and the Environment*

of adsorption medium and showed the maximum value at pH 5.

percent of remaining activity.

mixture of lysozyme and bamboo charcoal or wood charcoal showed several

The solution pH generally affects the activity and stability of enzymes in aqueous solutions [5]. **Figure 13** shows the remaining activity of lysozyme adsorbed on bamboo charcoal against the solution pH of adsorption medium. The remaining activity of bamboo charcoal-adsorbed lysozyme was markedly influenced by the pH

*Effect of kind of biochar on remaining activity of lysozyme adsorbed on biochar after heat treatment at 90°C* 

*Effect of pH of adsorption medium on the remaining activity of lysozyme adsorbed on bamboo charcoal after* 

**84**

**Figure 13.**

*the heat treatment at 90°C for 30 min.*

**Figure 12.**

*for 30 min.*

Three-dimensional structures of enzymes consist of secondary structures such as α-helix and β-sheet [28]. To elucidate the influence of adsorption on the structure of lysozyme, bamboo charcoal-adsorbed lysozyme has been measured by Fourier-transform infrared (FTIR) spectroscopy. **Figure 14** shows the FTIR spectra of native lysozyme and lysozyme adsorbed on bamboo at different pH. The most sensitive spectral region to enzyme secondary structural components is amide I (1700–1600 cm<sup>−</sup><sup>1</sup> ), which is due almost entirely to the C〓O stretch vibrations of peptide linkages [28]. The spectral pattern of bamboo charcoal-adsorbed lysozyme was influenced by the pH of adsorption medium. To evaluate the change in the secondary structure of bamboo-adsorbed lysozyme, the ratio of the absorbance at 1681 cm<sup>−</sup><sup>1</sup> to the absorbance at 1647 cm<sup>−</sup><sup>1</sup> (ABS1681/ABS1647) has been assessed since the band located at ca. 1681 cm<sup>−</sup><sup>1</sup> is assigned to intramolecular β-sheet and the band located at ca. 1647 cm<sup>−</sup><sup>1</sup> is assigned to α-helix. The ABS1681/ABS1647 ratio at pH 5 (0.86), where the remaining activity showed the maximum value, was similar to that of native lysozyme (0.88). Likewise, the ABS1681/ABS1647 ratio at pH 4 (0.92) was near that of native lysozyme. On the other hand, the ABS1681/ABS1647 ratios at pH 7 (0.69) and 9 (0.61) were different from that of native lysozyme. The effect of solution pH of adsorption medium on the thermal stability of bamboo charcoaladsorbed lysozyme has been summarized as follows. The structure of bamboo charcoal-adsorbed lysozyme was nearly the native structure of lysozyme when lysozyme was adsorbed on bamboo charcoal at pH 4, but the electrostatic interaction between lysozyme and bamboo charcoal could not sufficiently contribute to the thermal stability. The electrostatic interaction between lysozyme and bamboo charcoal could strongly retain the structure of lysozyme at high temperatures when lysozyme was adsorbed on bamboo charcoal at pH 5, where the native structure of lysozyme was maintained. The structure of lysozyme was partially destroyed since the electrostatic interaction was too strong to maintain the native structure of lysozyme, and the thermal stability of bamboo charcoal-adsorbed lysozyme dropped when lysozyme was adsorbed on bamboo charcoal at pH 7 and 9. Therefore, these results indicate that biochar-adsorbed enzymes exhibit the excellent thermal stability when the

#### **Figure 14.**

*(A) FTIR spectrum of native lysozyme. (B) FTIR spectrum of lysozyme adsorbed on bamboo charcoal at pH 4. (C) FTIR spectrum of lysozyme adsorbed on bamboo charcoal at pH 5. (D) FTIR spectrum of lysozyme adsorbed on bamboo charcoal at pH 7. (E) FTIR spectrum of lysozyme adsorbed on bamboo charcoal at pH 9.*

native structure of enzymes is kept after the adsorption and the adsorption force is strong enough to retain the structure of enzymes against the heat stress.

#### **2.4 Organic solvent stress tolerance of enzymes adsorbed on biochar**

Biotransformation catalyzed by an enzyme in nonaqueous media has been applied to numerous synthetic processes because of the following benefits [29]: (1) The solubility of nonpolar reactants and products is improved. (2) Synthetic reactions can take place by the use of a conventional hydrolase without an expensive energy substance such as adenosine triphosphate (ATP). (3) The stereoselectivity of enzymes is markedly altered. (4) The thermal stability of enzymes is highly improved. (5) Enzymes can easily be recycled by the filtration or the centrifugation. (6) The product can easily be recovered by the evaporation when the volatile organic solvent is used as a reaction medium. (7) The contamination such as the growth of microorganisms can be inhibited by using organic solvents. However, the enzyme tends to show the low activity in organic solvents, compared to that in water since an organic solvent in general works as a denaturant of enzymes [30].

**Figure 15** shows the scheme of reaction catalyzed by α-chymotrypsin (α-CT) [30, 31]. When *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) is used as a substrate, the enzymatic reaction proceeds by the formation of enzyme intermediates between the active site of enzymes and the substrate. In water α-CT catalyzes the hydrolysis reaction of *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) with water to give *N-*acetyl-L-tyrosine (*N*-Ac-Tyr-OH). On the other hand, in organic solvents α-CT mainly catalyzes the transesterification reaction of *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) with another substrate, *n*-butanol (BuOH), to produce *N-*acetyl-L-tyrosine butyl ester (*N*-Ac-Tyr-OBu). Thus α-CT, which is hydrolase, can beneficially catalyze the synthetic reaction in organic solvents although expensive energy substances such as adenosine triphosphate (ATP) are needed for conventional synthetic reactions by enzymes to take place in water [24].

In order to evaluate the catalytic activity of enzymes adsorbed on biochar in an organic solvent, the transesterification catalyzed by α-CT adsorbed on the different kind of biochar in acetonitrile has been examined [19]. **Figure 16** shows the initial rates of *N-*acetyl-L-tyrosine butyl ester (*N*-Ac-Tyr-OBu) and *N-*acetyl-L-tyrosine (*N*-Ac-Tyr-OH) catalyzed by free and biochar-adsorbed α-CT in acetonitrile containing 5% (v/v) water. Both initial rates of *N*-Ac-Tyr-OBu and *N*-Ac-Tyr-OH catalyzed by biochar-adsorbed α-CT were much higher than those catalyzed by free α-CT. α-CT adsorbed on bamboo charcoal was the most effective of all, with respect

**Figure 15.**

*α-CT-catalyzed transesterification of N-acetyl-L-tyrosine ethyl ester (N-Ac-Tyr-OEt) with n-butanol (BuOH) to N-acetyl-L-tyrosine butyl ester (N-Ac-Tyr-OBu) and competing hydrolysis (N-Ac-Tyr-OH).*

**87**

**Table 4.**

*FTIR measurement.*

*Ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup>*

**Figure 16.**

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

mass transfer of substrates and products is rapidly facilitated [32].

**Table 4** shows the ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup>

mentioned above, the band located at ca. 1650 cm<sup>−</sup><sup>1</sup>

the band located at ca. 1630 cm<sup>−</sup><sup>1</sup>

1630 cm<sup>−</sup><sup>1</sup>

to the enhancement in the initial rates of *N*-Ac-Tyr-OBu and *N*-Ac-Tyr-OH. With regard to the transesterification catalyzed by hydrolase, which is characteristic of the nonaqueous enzymology, the initial rate of *N*-Ac-Tyr-OBu catalyzed by α-CT adsorbed on bamboo charcoal was about 50 times higher than that catalyzed by free one. Enzymes are aggregated in an organic solvent, and most of them cannot directly come in contact with the bulk organic phase containing substrates, although they are soluble in an aqueous solution. On the other hand, most of the enzymes adsorbed on biochar are directly in contact with the bulk organic phase since they are located on the surface of biochar. Accordingly, biochar-adsorbed enzymes can effectively proceed with the reaction, compared to free enzymes, since

In order to elucidate the influence of a kind of biochar on the secondary structure of α-CT, the FTIR spectra of free and biochar-adsorbed α-CT were measured.

of the ABS1650/ABS1630 ratio was bamboo charcoal-adsorbed α-CT > adzuki bean charcoal-adsorbed α-CT = wood charcoal-adsorbed α-CT > free α-CT. The order of the ABS1650/ABS1630 ratio was similar to that of the initial rate of transesterification as shown in **Figure 16**. The α-helical structure of α-CT molecule is more changeable than β-sheet, since the β-sheet structure is the main backbone of α-CT molecule [33]. Thus, the results indicate that at the higher initial rate, the transesterification is catalyzed by α-CT molecules having the secondary structure kept more highly.

*Dependence of kind of biochar on biochar-adsorbed α-CT-catalyzed transesterification. Free or biocharadsorbed α-CT was placed in acetonitrile containing 5% (v/v) water, 10 mM N-Ac-Tyr-OEt, 1000 mM BuOH,* 

**Sample ABS1650/ABS1630 (−)**

Free α-CT 1.1 Adzuki bean charcoal-adsorbed α-CT 1.3 Bamboo charcoal-adsorbed α-CT 1.5 Wood charcoal-adsorbed α-CT 1.3

 *to the absorbance at 1630 cm<sup>−</sup><sup>1</sup>*

*and 1 mM acetanilide, and the resulting mixture was shaken at 120 rpm and 25°C.*

(ABS1650/ABS1630) of free α-CT and α-CT adsorbed onto biochar. As

to the absorbance at

is assignable to α-helix, and

 *(ABS1650/ABS1630) of α-CT provided by the* 

is assignable to intramolecular β-sheet. The order

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

#### *Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

to the enhancement in the initial rates of *N*-Ac-Tyr-OBu and *N*-Ac-Tyr-OH. With regard to the transesterification catalyzed by hydrolase, which is characteristic of the nonaqueous enzymology, the initial rate of *N*-Ac-Tyr-OBu catalyzed by α-CT adsorbed on bamboo charcoal was about 50 times higher than that catalyzed by free one. Enzymes are aggregated in an organic solvent, and most of them cannot directly come in contact with the bulk organic phase containing substrates, although they are soluble in an aqueous solution. On the other hand, most of the enzymes adsorbed on biochar are directly in contact with the bulk organic phase since they are located on the surface of biochar. Accordingly, biochar-adsorbed enzymes can effectively proceed with the reaction, compared to free enzymes, since mass transfer of substrates and products is rapidly facilitated [32].

In order to elucidate the influence of a kind of biochar on the secondary structure of α-CT, the FTIR spectra of free and biochar-adsorbed α-CT were measured. **Table 4** shows the ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup> to the absorbance at 1630 cm<sup>−</sup><sup>1</sup> (ABS1650/ABS1630) of free α-CT and α-CT adsorbed onto biochar. As mentioned above, the band located at ca. 1650 cm<sup>−</sup><sup>1</sup> is assignable to α-helix, and the band located at ca. 1630 cm<sup>−</sup><sup>1</sup> is assignable to intramolecular β-sheet. The order of the ABS1650/ABS1630 ratio was bamboo charcoal-adsorbed α-CT > adzuki bean charcoal-adsorbed α-CT = wood charcoal-adsorbed α-CT > free α-CT. The order of the ABS1650/ABS1630 ratio was similar to that of the initial rate of transesterification as shown in **Figure 16**. The α-helical structure of α-CT molecule is more changeable than β-sheet, since the β-sheet structure is the main backbone of α-CT molecule [33]. Thus, the results indicate that at the higher initial rate, the transesterification is catalyzed by α-CT molecules having the secondary structure kept more highly.

#### **Figure 16.**

*Biochar - An Imperative Amendment for Soil and the Environment*

native structure of enzymes is kept after the adsorption and the adsorption force is

Biotransformation catalyzed by an enzyme in nonaqueous media has been applied to numerous synthetic processes because of the following benefits [29]: (1) The solubility of nonpolar reactants and products is improved. (2) Synthetic reactions can take place by the use of a conventional hydrolase without an expensive energy substance such as adenosine triphosphate (ATP). (3) The stereoselectivity of enzymes is markedly altered. (4) The thermal stability of enzymes is highly improved. (5) Enzymes can easily be recycled by the filtration or the centrifugation. (6) The product can easily be recovered by the evaporation when the volatile organic solvent is used as a reaction medium. (7) The contamination such as the growth of microorganisms can be inhibited by using organic solvents. However, the enzyme tends to show the low activity in organic solvents, compared to that in water since an organic solvent in general works as a denaturant of enzymes [30]. **Figure 15** shows the scheme of reaction catalyzed by α-chymotrypsin (α-CT) [30, 31]. When *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) is used as a substrate, the enzymatic reaction proceeds by the formation of enzyme intermediates between the active site of enzymes and the substrate. In water α-CT catalyzes the hydrolysis reaction of *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) with water to give *N-*acetyl-L-tyrosine (*N*-Ac-Tyr-OH). On the other hand, in organic solvents α-CT mainly catalyzes the transesterification reaction of *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) with another substrate, *n*-butanol (BuOH), to produce *N-*acetyl-L-tyrosine butyl ester (*N*-Ac-Tyr-OBu). Thus α-CT, which is hydrolase, can beneficially catalyze the synthetic reaction in organic solvents although expensive energy substances such as adenosine triphosphate (ATP) are needed for

strong enough to retain the structure of enzymes against the heat stress.

**2.4 Organic solvent stress tolerance of enzymes adsorbed on biochar**

conventional synthetic reactions by enzymes to take place in water [24].

In order to evaluate the catalytic activity of enzymes adsorbed on biochar in an organic solvent, the transesterification catalyzed by α-CT adsorbed on the different kind of biochar in acetonitrile has been examined [19]. **Figure 16** shows the initial rates of *N-*acetyl-L-tyrosine butyl ester (*N*-Ac-Tyr-OBu) and *N-*acetyl-L-tyrosine (*N*-Ac-Tyr-OH) catalyzed by free and biochar-adsorbed α-CT in acetonitrile containing 5% (v/v) water. Both initial rates of *N*-Ac-Tyr-OBu and *N*-Ac-Tyr-OH catalyzed by biochar-adsorbed α-CT were much higher than those catalyzed by free α-CT. α-CT adsorbed on bamboo charcoal was the most effective of all, with respect

*α-CT-catalyzed transesterification of N-acetyl-L-tyrosine ethyl ester (N-Ac-Tyr-OEt) with n-butanol (BuOH)* 

*to N-acetyl-L-tyrosine butyl ester (N-Ac-Tyr-OBu) and competing hydrolysis (N-Ac-Tyr-OH).*

**86**

**Figure 15.**

*Dependence of kind of biochar on biochar-adsorbed α-CT-catalyzed transesterification. Free or biocharadsorbed α-CT was placed in acetonitrile containing 5% (v/v) water, 10 mM N-Ac-Tyr-OEt, 1000 mM BuOH, and 1 mM acetanilide, and the resulting mixture was shaken at 120 rpm and 25°C.*


#### **Table 4.**

*Ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup> to the absorbance at 1630 cm<sup>−</sup><sup>1</sup> (ABS1650/ABS1630) of α-CT provided by the FTIR measurement.*

Moreover, it is suggested that the content of functional groups in bamboo charcoal is suitable to keep the secondary structure of α-CT in acetonitrile since functional groups contribute to the adsorption of α-CT on biochar.

**Figure 17** shows time course of remaining activities of free α-CT and bamboo charcoal-adsorbed α-CT through the heat treatment at 50°C [20]. The state of free α-CT in acetonitrile, where α-CT was dispersed as the solid state, was unchanged during the heat treatment, although enzymes dissolved in an aqueous solution immediately form the aggregation of thermally denatured enzymes [15]. Likewise, the enzyme aggregation and the cohesion among bamboo charcoaladsorbed α-CT were not observed in acetonitrile during the heat treatment. However, the remaining activities of free α-CT and BCP-adsorbed α-CT gradually dropped with an increase in heat time. The relation between the remaining activity of free α-CT and heat time could be correlated by first-order kinetics, while the relation between bamboo charcoal-adsorbed α-CT and heat time could be correlated by second-order kinetics. When the curve fitting was carried out in the figure, the half-life of inactivation of free α-CT was 33 min, and the half-life of inactivation of bamboo charcoal-adsorbed α-CT was 125 min. Therefore, the half-life of bamboo charcoal-adsorbed α-CT showed 3.8-fold, compared with that of α-CT. On the other hand, the half-life of inactivation of bamboo charcoaladsorbed α-CT is 15 min in aqueous solutions at 45°C [17]. Consequently, the thermal stability of bamboo charcoal-adsorbed α-CT in acetonitrile was greater than that in water. As a result, the electrostatic interaction between α-CT and bamboo charcoal, which mainly contributes to the adsorption of α-CT on bamboo charcoal, is strengthened in acetonitrile as the dielectric constant of acetonitrile is much smaller than that of water [34].

In moist air, the catalytic activity of solid enzymes is markedly dependent on the thermodynamic water activity (aw), which is defined as the ratio of the water partial pressure to the vapor pressure of pure water [35]. Similarly, the catalytic activity of biochar-adsorbed enzymes might be influenced in hydrophilic organic solvents containing low water content. **Figure 18** shows the relationship of the initial transesterification rate (Ve) and the initial hydrolysis rate (Vh) with the water activity

**Figure 17.**

*Time course of remaining activities of free α-CT and bamboo charcoal-adsorbed α-CT through the heat treatment at 50°C.*

**89**

than free α-CT.

**Figure 18.**

*charcoal (BC)-adsorbed α-CT in acetonitrile.*

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

in acetonitrile at 25°C [20]. Low water activity inhibited the inherent enzymatic hydrolysis of *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) with water, whereas the enzymatic transesterification of *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) with *n*-butanol (BuOH) was enhanced. The water activity strongly affected the initial transesterification rates catalyzed by bamboo charcoal-adsorbed α-CT and free α-CT, and the correlation between these parameters showed a bell-shaped curve. The initial transesterification catalyzed by bamboo charcoal-adsorbed α-CT exhibited about 60-fold, compared to that catalyzed by free α-CT when the maximum initial transesterification rates of free α-CT and bamboo charcoal-adsorbed α-CT were shown. On the other hand, the initial hydrolysis rates catalyzed by free α-CT and bamboo charcoal-adsorbed α-CT gradually increased with increasing the water activity. The relationship of the catalytic activity of enzymes with the water activity in organic solvents tends to depict a bell-shaped curve. The balance between the kinetic rigidity of enzyme structures and their thermodynamic stability results in the optimum water activity [29, 36]. The kinetic rigidity drops with an increase in water activity, while the native structure of enzymes is gradually influenced by the thermodynamic stability. Consequently, the catalytic activity of enzymes increases with increasing the flexibility in the rigid structure of enzymes and then decreases with increasing the disturbance of enzyme structures. On the other hand, the hydrolysis is promoted with an increase in the water activity since the high water activity results in the high overall water concentration. Concerning the reaction selectivity, which was defined as the ratio of the initial transesterification rate (Ve) to the initial hydrolysis rate (Vh), bamboo charcoal-adsorbed α-CT was much better

*Effect of water activity on the transesterification rate (Ve) and hydrolysis rate (Vh) of free α-CT and bamboo* 

**Table 5** shows the absorbance ratio at 1650 and 1630 cm<sup>−</sup><sup>1</sup>

bamboo charcoal-adsorbed α-CT [20]. The higher the absorbance ratio, the higher the secondary structure. The water activity did not affect the absorbance ratio

(ABS1650/ABS1630) of

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

**Figure 18.**

*Biochar - An Imperative Amendment for Soil and the Environment*

groups contribute to the adsorption of α-CT on biochar.

much smaller than that of water [34].

Moreover, it is suggested that the content of functional groups in bamboo charcoal is suitable to keep the secondary structure of α-CT in acetonitrile since functional

**Figure 17** shows time course of remaining activities of free α-CT and bamboo charcoal-adsorbed α-CT through the heat treatment at 50°C [20]. The state of free α-CT in acetonitrile, where α-CT was dispersed as the solid state, was unchanged during the heat treatment, although enzymes dissolved in an aqueous solution immediately form the aggregation of thermally denatured enzymes [15]. Likewise, the enzyme aggregation and the cohesion among bamboo charcoaladsorbed α-CT were not observed in acetonitrile during the heat treatment. However, the remaining activities of free α-CT and BCP-adsorbed α-CT gradually dropped with an increase in heat time. The relation between the remaining activity of free α-CT and heat time could be correlated by first-order kinetics, while the relation between bamboo charcoal-adsorbed α-CT and heat time could be correlated by second-order kinetics. When the curve fitting was carried out in the figure, the half-life of inactivation of free α-CT was 33 min, and the half-life of inactivation of bamboo charcoal-adsorbed α-CT was 125 min. Therefore, the half-life of bamboo charcoal-adsorbed α-CT showed 3.8-fold, compared with that of α-CT. On the other hand, the half-life of inactivation of bamboo charcoaladsorbed α-CT is 15 min in aqueous solutions at 45°C [17]. Consequently, the thermal stability of bamboo charcoal-adsorbed α-CT in acetonitrile was greater than that in water. As a result, the electrostatic interaction between α-CT and bamboo charcoal, which mainly contributes to the adsorption of α-CT on bamboo charcoal, is strengthened in acetonitrile as the dielectric constant of acetonitrile is

In moist air, the catalytic activity of solid enzymes is markedly dependent on the thermodynamic water activity (aw), which is defined as the ratio of the water partial pressure to the vapor pressure of pure water [35]. Similarly, the catalytic activity of biochar-adsorbed enzymes might be influenced in hydrophilic organic solvents containing low water content. **Figure 18** shows the relationship of the initial transesterification rate (Ve) and the initial hydrolysis rate (Vh) with the water activity

*Time course of remaining activities of free α-CT and bamboo charcoal-adsorbed α-CT through the heat* 

**88**

**Figure 17.**

*treatment at 50°C.*

*Effect of water activity on the transesterification rate (Ve) and hydrolysis rate (Vh) of free α-CT and bamboo charcoal (BC)-adsorbed α-CT in acetonitrile.*

in acetonitrile at 25°C [20]. Low water activity inhibited the inherent enzymatic hydrolysis of *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) with water, whereas the enzymatic transesterification of *N-*acetyl-L-tyrosine ethyl ester (*N*-Ac-Tyr-OEt) with *n*-butanol (BuOH) was enhanced. The water activity strongly affected the initial transesterification rates catalyzed by bamboo charcoal-adsorbed α-CT and free α-CT, and the correlation between these parameters showed a bell-shaped curve. The initial transesterification catalyzed by bamboo charcoal-adsorbed α-CT exhibited about 60-fold, compared to that catalyzed by free α-CT when the maximum initial transesterification rates of free α-CT and bamboo charcoal-adsorbed α-CT were shown. On the other hand, the initial hydrolysis rates catalyzed by free α-CT and bamboo charcoal-adsorbed α-CT gradually increased with increasing the water activity. The relationship of the catalytic activity of enzymes with the water activity in organic solvents tends to depict a bell-shaped curve. The balance between the kinetic rigidity of enzyme structures and their thermodynamic stability results in the optimum water activity [29, 36]. The kinetic rigidity drops with an increase in water activity, while the native structure of enzymes is gradually influenced by the thermodynamic stability. Consequently, the catalytic activity of enzymes increases with increasing the flexibility in the rigid structure of enzymes and then decreases with increasing the disturbance of enzyme structures. On the other hand, the hydrolysis is promoted with an increase in the water activity since the high water activity results in the high overall water concentration. Concerning the reaction selectivity, which was defined as the ratio of the initial transesterification rate (Ve) to the initial hydrolysis rate (Vh), bamboo charcoal-adsorbed α-CT was much better than free α-CT.

**Table 5** shows the absorbance ratio at 1650 and 1630 cm<sup>−</sup><sup>1</sup> (ABS1650/ABS1630) of bamboo charcoal-adsorbed α-CT [20]. The higher the absorbance ratio, the higher the secondary structure. The water activity did not affect the absorbance ratio

#### *Biochar - An Imperative Amendment for Soil and the Environment*


**Table 5.**

*Ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup> to the absorbance at 1630 cm<sup>−</sup><sup>1</sup> (ABS1650/ABS1630) of bamboo charcoaladsorbed α-CT provided by the FTIR measurement.*

(ABS1650/ABS1630) of bamboo charcoal-adsorbed α-CT, indicating that the secondary structure of bamboo charcoal-adsorbed α-CT does not depend on the water activity. Accordingly, the adsorption firmly makes the conformation of bamboo charcoal-adsorbed α-CT maintained. The absorbance ratio (ABS1650/ABS1630) of bamboo charcoal-adsorbed α-CT is higher than that of free α-CT, as seen in **Table 4**. The results illustrate that the water activity effectively affects the catalytic activity of bamboo charcoal-adsorbed α-CT having a native structure, compared to that of free α-CT.

The catalysis of free α-CT and bamboo charcoal-adsorbed α-CT was markedly dependent upon the nature of organic solvents as shown in **Figure 19**. The catalytic activity of bamboo charcoal-adsorbed α-CT was much superior to that of free α-CT in organic solvents. The initial transesterification rate of free α-CT in *n-*octane exhibited 813-fold, compared to that in *n*-butyl acetate, while the initial transesterification rate of bamboo charcoal-adsorbed α-CT in *n*-octane was 1.3 times greater than that in *n*-butyl acetate.

There have been some reports that the native conformation of enzymes may be altered when enzymes are immersed in organic solvents [37, 38]. **Table 6** shows the ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup> to the absorbance at 1630 cm<sup>−</sup><sup>1</sup> (ABS1650/ ABS1630) of free α-CT and bamboo charcoal-adsorbed α-CT after they were immersed in organic solvents for 24 h. The absorbance ratio (ABS1650/ABS1630) of bamboo charcoal-adsorbed α-CT after the solvent immersion was similar to that before the solvent immersion. On the other hand, the absorbance ratio (ABS1650/ ABS1630) of free α-CT was altered by the immersion in octane. Those results indicate that the conformation of bamboo charcoal-adsorbed α-CT is hardly influenced by the nature of solvents, compared to the case of free α-CT.

**Figure 20** shows the relation between the catalytic activity and the hydrophobicity defined as log P where P is a partition coefficient for a given solvent between *n*-octanol and water [39]. The initial transesterification rate of free α-CT and bamboo charcoal-adsorbed α-CT increased with an increase in the hydrophobicity of organic solvents. Likewise, the initial hydrolysis rates of free α-CT and bamboo charcoal-adsorbed α-CT tended to increase with increasing the hydrophobicity of organic solvents. The hydrophobicity contributes to the partition of water between enzyme molecules and the bulk organic phase in reaction systems [29, 40]. When a certain amount of water is added into organic solvents, the amount of water associated with enzyme molecules increases in an increase in the hydrophobicity of organic solvents. Thereby, the flexibility of enzyme molecules, which is essential for catalytic activity, is enhanced by the hydration of enzyme molecules, and then the catalytic activity increases. On the other hand, hydrolysis reaction is promoted by the increase of water molecules around enzymes. As shown in **Figure 20**, the initial transesterification rate of bamboo charcoal-adsorbed α-CT was much superior to that of free α-CT at the low hydrophobicity. For instance, the initial transesterification rate of bamboo charcoal-adsorbed α-CT was about 760 times higher than that

**91**

was shown.

**Figure 20.**

*bamboo charcoal (BC)-adsorbed α-CT.*

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

*Solvent dependence o of transesterification catalyzed by free α-CT (A) and BCP-adsorbed α-CT (B).*

 *to the absorbance at 1630 cm<sup>−</sup><sup>1</sup>*

*bamboo charcoal-adsorbed α-CT provided by the FTIR measurement after the solvent immersion.*

**Free α-CT Bamboo charcoal-adsorbed α-CT**

 *(ABS1650/ABS1630) of free α-CT and* 

**Solvent ABS1650/ABS1630 (−)**

None 1.14 1.47 Acetonitrile 1.15 1.51 *n*-Octane 1.20 1.53

of free α-CT in *n*-butyl acetate (log P = 1.7). Moreover, bamboo charcoal-adsorbed α-CT depicted the high initial transesterification rate in acetone and acetonitrile, compared to the case of free α-CT. An enzymatic reaction in hydrophilic solvents has the advantage of the solubility of a variety of substrates including drug derivatives, which are poorly soluble in hydrophobic solvents [41]. As discussed above, since the native conformation of bamboo charcoal-adsorbed α-CT was maintained in hydrophilic organic solvents, bamboo charcoal-adsorbed α-CT could exhibit the high catalytic activity, compared to the case of free α-CT. On the other hand, no correlation between the catalytic activity and the other parameter (e.g., dielectric constant, hydrogen bonding parameter, solubility parameter, and viscosity)

*Relationship of log P of solvents with transesterification rate (A) or hydrolysis rate (B) of free α-CT and* 

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

**Figure 19.**

**Table 6.**

*Ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup>*

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

**Figure 19.**

*Biochar - An Imperative Amendment for Soil and the Environment*

(ABS1650/ABS1630) of bamboo charcoal-adsorbed α-CT, indicating that the secondary structure of bamboo charcoal-adsorbed α-CT does not depend on the water activity. Accordingly, the adsorption firmly makes the conformation of bamboo charcoal-adsorbed α-CT maintained. The absorbance ratio (ABS1650/ABS1630) of bamboo charcoal-adsorbed α-CT is higher than that of free α-CT, as seen in **Table 4**. The results illustrate that the water activity effectively affects the catalytic activity of bamboo charcoal-adsorbed α-CT having a native structure, compared to that of free

 *to the absorbance at 1630 cm<sup>−</sup><sup>1</sup>*

**Water activity (−) ABS1650/ABS1630 (−)**

0.03 1.3 0.28 1.3 0.55 1.3 0.73 1.3

The catalysis of free α-CT and bamboo charcoal-adsorbed α-CT was markedly dependent upon the nature of organic solvents as shown in **Figure 19**. The catalytic activity of bamboo charcoal-adsorbed α-CT was much superior to that of free α-CT in organic solvents. The initial transesterification rate of free α-CT in *n-*octane exhibited 813-fold, compared to that in *n*-butyl acetate, while the initial transesterification rate of bamboo charcoal-adsorbed α-CT in *n*-octane was 1.3 times greater

There have been some reports that the native conformation of enzymes may be altered when enzymes are immersed in organic solvents [37, 38]. **Table 6** shows

**Figure 20** shows the relation between the catalytic activity and the hydrophobicity defined as log P where P is a partition coefficient for a given solvent between *n*-octanol and water [39]. The initial transesterification rate of free α-CT and bamboo charcoal-adsorbed α-CT increased with an increase in the hydrophobicity of organic solvents. Likewise, the initial hydrolysis rates of free α-CT and bamboo charcoal-adsorbed α-CT tended to increase with increasing the hydrophobicity of organic solvents. The hydrophobicity contributes to the partition of water between enzyme molecules and the bulk organic phase in reaction systems [29, 40]. When a certain amount of water is added into organic solvents, the amount of water associated with enzyme molecules increases in an increase in the hydrophobicity of organic solvents. Thereby, the flexibility of enzyme molecules, which is essential for catalytic activity, is enhanced by the hydration of enzyme molecules, and then the catalytic activity increases. On the other hand, hydrolysis reaction is promoted by the increase of water molecules around enzymes. As shown in **Figure 20**, the initial transesterification rate of bamboo charcoal-adsorbed α-CT was much superior to that of free α-CT at the low hydrophobicity. For instance, the initial transesterification rate of bamboo charcoal-adsorbed α-CT was about 760 times higher than that

ABS1630) of free α-CT and bamboo charcoal-adsorbed α-CT after they were immersed in organic solvents for 24 h. The absorbance ratio (ABS1650/ABS1630) of bamboo charcoal-adsorbed α-CT after the solvent immersion was similar to that before the solvent immersion. On the other hand, the absorbance ratio (ABS1650/ ABS1630) of free α-CT was altered by the immersion in octane. Those results indicate that the conformation of bamboo charcoal-adsorbed α-CT is hardly influenced by

the nature of solvents, compared to the case of free α-CT.

to the absorbance at 1630 cm<sup>−</sup><sup>1</sup>

 *(ABS1650/ABS1630) of bamboo charcoal-*

(ABS1650/

**90**

α-CT.

**Table 5.**

than that in *n*-butyl acetate.

*Ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup>*

*adsorbed α-CT provided by the FTIR measurement.*

the ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup>

*Solvent dependence o of transesterification catalyzed by free α-CT (A) and BCP-adsorbed α-CT (B).*


#### **Table 6.**

*Ratio of the absorbance at 1650 cm<sup>−</sup><sup>1</sup> to the absorbance at 1630 cm<sup>−</sup><sup>1</sup> (ABS1650/ABS1630) of free α-CT and bamboo charcoal-adsorbed α-CT provided by the FTIR measurement after the solvent immersion.*

#### **Figure 20.**

*Relationship of log P of solvents with transesterification rate (A) or hydrolysis rate (B) of free α-CT and bamboo charcoal (BC)-adsorbed α-CT.*

of free α-CT in *n*-butyl acetate (log P = 1.7). Moreover, bamboo charcoal-adsorbed α-CT depicted the high initial transesterification rate in acetone and acetonitrile, compared to the case of free α-CT. An enzymatic reaction in hydrophilic solvents has the advantage of the solubility of a variety of substrates including drug derivatives, which are poorly soluble in hydrophobic solvents [41]. As discussed above, since the native conformation of bamboo charcoal-adsorbed α-CT was maintained in hydrophilic organic solvents, bamboo charcoal-adsorbed α-CT could exhibit the high catalytic activity, compared to the case of free α-CT. On the other hand, no correlation between the catalytic activity and the other parameter (e.g., dielectric constant, hydrogen bonding parameter, solubility parameter, and viscosity) was shown.

### **3. Conclusions**

This chapter has introduced the study on the application of the biochar to enzyme carriers to develop the high value-added application of biomass materials. Biochar was thermal stable, chemical stable, insoluble under reaction conditions, available, low cost, regeneration, and reusable. Moreover, as biochar had functional groups for the interaction of enzymes and a high affinity to enzymes, enzymes were firmly adsorbed on biochar. On the other hand, the original weakness of enzymes due to the heat and organic solvent stresses could be much improved by adsorbing enzymes onto the biochar. Moreover, enzymes are strictly influenced not only by heat and organic solvents but also by ultraviolet, X-ray, sound wave, shake, freeze, pressure, shearing force, extreme ionic strength, urea, surfactant metal ion, reductant, and so on. It would be expected that suitable carriers having the high tolerance against those stresses are developed for an enzyme carrier by selecting a kind of biochar as well because there are a great variety of biomass in the earth.

### **Acknowledgements**

This work was mainly supported by a Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (No. 24561013) and a Grant-in-Aid for Scientific Research from Japan Science and Technology Agency (No. AS2111014D).

#### **Author details**

Hidetaka Noritomi Department of Applied Chemistry for Environment, Tokyo Metropolitan University, Tokyo, Japan

\*Address all correspondence to: noritomi@tmu.ac.jp

© 2018 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.

**93**

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology.

[11] Elnashar MMM. Review article: Immobilized molecules using

biomaterials and nanobiotechnology.

Journal of Biomaterials and Nanobiotechnology. 2010;**1**:61-77

[12] Anderson CR, Condron LM, Clough TJ, Fiers M, Alison S, Hill RA, et al. Biochar induced soil microbial community change: Implications for biogeochemical cycling of carbon, nitrogen and phosphorus. Pedobiologia.

[13] Noritomi H, Iwai D, Kai R, Tanaka M, Kato S. Adsorption of lysozyme on biomass charcoal powder prepared from plant biomass wastes. Journal of Chemical Engineering of

[14] Noritomi H, Hishinuma K, Kurihara S, Nishigami J, Takemoto T, Endo N, et al. Adsorption of α-chymotrypsin on plant biomass charcoal. Journal of Surface Engineered Materials and Advanced Technology. 2013;**3**:269-274

[15] Noritomi H, Kai R, Iwai D, Tanaka H, Kamila R, Tanaka M, et al. Increase in thermal stability of proteins adsorbed on biomass charcoal powder prepared from plant biomass wastes. Journal of Biomedical Science and Engineering.

[16] Noritomi H, Ishiyama R, Kai R, Iwai D, Tanaka M, Kato S. Immobilization of lysozyme on biomass charcoal powder derived from plant biomass wastes. Journal of Biomaterials and Nanobiotechnology. 2012;**3**:446-451

[17] Noritomi H, Kurihara S, Endo N, Kato S. Heat-resistant properties of α-chymotrypsin adsorbed onto biomass

Japan. 2013;**46**:196-200

2011;**4**:692-698

2007;**40**:1451-1463

2011;**54**:309-320

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

[1] Olivier JGJ, Muntean M, Peters JAHW. Trends in Global CO2 Emissions. 2017

[2] IPCC. Climate Change 2014: Synthesis Report. Switzerland: Intergovernmental Panel on Climate

[3] Ho YC, Show KY. A perspective in renewable energy production from biomass pyrolysis—Challenges and prospects. Current Organic Chemistry.

[4] Straathof AJJ. Transformation of biomass into commodity chemicals using enzymes or cells. Chemical Reviews. 2014;**114**:1871-1908

[5] Buchholz K, Kasche V, Bornscheuer

Technology. 2nd ed. Weinheim: Wiley-

[6] Silwana B, Horst CVD, Iwuoha E. Aerometric determination of cadmium, lead, and mercury metal ions using a novel polymer immobilized horseradish peroxidase biosensor system. Journal of Environmental Science and Health, Part

[7] Leech D, Kavanagh P, Schuhmann W.

UT. Biocatalyst and Enzyme

Change (IPCC); 2014

2015;**19**:423-436

Blackwell; 2012

A. 2014;**49**:1501-1511

2012;**84**:223-234

Enzymatic fuel cells: Recent progress. Electrochimica Acta.

[8] Bailey JE, Ollis DF. Biochemical Engineering Fundamentals. 2nd ed. New York: McGraw-Hill; 1986

[9] Zdata J, Meyer AS, Jesionowski T, Pinelo M. A general overview of support materials for enzyme immobilization: Characteristics, properties, practical utility. Catalysts. 2018;**8**:92-118

[10] Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lorente R. Improvement of enzyme

Report; 2017

**References**

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

#### **References**

*Biochar - An Imperative Amendment for Soil and the Environment*

This chapter has introduced the study on the application of the biochar to enzyme carriers to develop the high value-added application of biomass materials. Biochar was thermal stable, chemical stable, insoluble under reaction conditions, available, low cost, regeneration, and reusable. Moreover, as biochar had functional groups for the interaction of enzymes and a high affinity to enzymes, enzymes were firmly adsorbed on biochar. On the other hand, the original weakness of enzymes due to the heat and organic solvent stresses could be much improved by adsorbing enzymes onto the biochar. Moreover, enzymes are strictly influenced not only by heat and organic solvents but also by ultraviolet, X-ray, sound wave, shake, freeze, pressure, shearing force, extreme ionic strength, urea, surfactant metal ion, reductant, and so on. It would be expected that suitable carriers having the high tolerance against those stresses are developed for an enzyme carrier by selecting a kind of

biochar as well because there are a great variety of biomass in the earth.

for Scientific Research from Japan Science and Technology Agency

This work was mainly supported by a Grant-in-Aid for Scientific Research (C) from Japan Society for the Promotion of Science (No. 24561013) and a Grant-in-Aid

**3. Conclusions**

**Acknowledgements**

(No. AS2111014D).

© 2018 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,

Department of Applied Chemistry for Environment, Tokyo Metropolitan

**92**

**Author details**

Hidetaka Noritomi

University, Tokyo, Japan

provided the original work is properly cited.

\*Address all correspondence to: noritomi@tmu.ac.jp

[1] Olivier JGJ, Muntean M, Peters JAHW. Trends in Global CO2 Emissions. 2017 Report; 2017

[2] IPCC. Climate Change 2014: Synthesis Report. Switzerland: Intergovernmental Panel on Climate Change (IPCC); 2014

[3] Ho YC, Show KY. A perspective in renewable energy production from biomass pyrolysis—Challenges and prospects. Current Organic Chemistry. 2015;**19**:423-436

[4] Straathof AJJ. Transformation of biomass into commodity chemicals using enzymes or cells. Chemical Reviews. 2014;**114**:1871-1908

[5] Buchholz K, Kasche V, Bornscheuer UT. Biocatalyst and Enzyme Technology. 2nd ed. Weinheim: Wiley-Blackwell; 2012

[6] Silwana B, Horst CVD, Iwuoha E. Aerometric determination of cadmium, lead, and mercury metal ions using a novel polymer immobilized horseradish peroxidase biosensor system. Journal of Environmental Science and Health, Part A. 2014;**49**:1501-1511

[7] Leech D, Kavanagh P, Schuhmann W. Enzymatic fuel cells: Recent progress. Electrochimica Acta. 2012;**84**:223-234

[8] Bailey JE, Ollis DF. Biochemical Engineering Fundamentals. 2nd ed. New York: McGraw-Hill; 1986

[9] Zdata J, Meyer AS, Jesionowski T, Pinelo M. A general overview of support materials for enzyme immobilization: Characteristics, properties, practical utility. Catalysts. 2018;**8**:92-118

[10] Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fernandez-Lorente R. Improvement of enzyme

activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology. 2007;**40**:1451-1463

[11] Elnashar MMM. Review article: Immobilized molecules using biomaterials and nanobiotechnology. Journal of Biomaterials and Nanobiotechnology. 2010;**1**:61-77

[12] Anderson CR, Condron LM, Clough TJ, Fiers M, Alison S, Hill RA, et al. Biochar induced soil microbial community change: Implications for biogeochemical cycling of carbon, nitrogen and phosphorus. Pedobiologia. 2011;**54**:309-320

[13] Noritomi H, Iwai D, Kai R, Tanaka M, Kato S. Adsorption of lysozyme on biomass charcoal powder prepared from plant biomass wastes. Journal of Chemical Engineering of Japan. 2013;**46**:196-200

[14] Noritomi H, Hishinuma K, Kurihara S, Nishigami J, Takemoto T, Endo N, et al. Adsorption of α-chymotrypsin on plant biomass charcoal. Journal of Surface Engineered Materials and Advanced Technology. 2013;**3**:269-274

[15] Noritomi H, Kai R, Iwai D, Tanaka H, Kamila R, Tanaka M, et al. Increase in thermal stability of proteins adsorbed on biomass charcoal powder prepared from plant biomass wastes. Journal of Biomedical Science and Engineering. 2011;**4**:692-698

[16] Noritomi H, Ishiyama R, Kai R, Iwai D, Tanaka M, Kato S. Immobilization of lysozyme on biomass charcoal powder derived from plant biomass wastes. Journal of Biomaterials and Nanobiotechnology. 2012;**3**:446-451

[17] Noritomi H, Kurihara S, Endo N, Kato S. Heat-resistant properties of α-chymotrypsin adsorbed onto biomass charcoal powder. Journal of Biomaterials and Nanobiotechnology. 2014;**5**:179-185

[18] Noritomi H, Kurihara S, Endo N, Kato S, Uchiyama K. Effect of adsorption condition on thermal stability of proteins adsorbed onto biomass charcoal powder. International Journal of GEOMATE. 2016;**11**:2123-2128

[19] Noritomi H, Nishigami J, Endo N, Kato S, Uchiyama K. Organic solventresistant properties of proteins adsorbed onto biomass charcoal powder. International Journal of GEOMATE. 2017;**12**:140-145

[20] Noritomi H, Nishigami J, Endo N, Kato S, Uchiyama K. Influence of water activity on protease adsorbed on biochar in organic solvents. Journal of Materials Science Research. 2017;**6**:96-102

[21] Noritomi H, Nishigami J, Endo N, Kato S, Takagi S. Effect of solvent on catalysis of protease adsorbed on biochar in organic media. Journal of Materials Science Research. 2018;**7**:46-52

[22] Asada T, Ishihara S, Yamane T, Toba A, Yamada A, Oikawa K. Science of bamboo charcoal: Study on carbonizing temperature of bamboo charcoal and removal capability of harmful gases. Journal of Health Science. 2002;**48**:473-479

[23] Nishimiya K, Hata T, Imamura Y, Ishihara S. Analysis of chemical structure of wood charcoal by X-ray photoelectron spectroscopy. Journal of Wood Science. 1998;**44**:56-61

[24] Adamson AW. Physical Chemistry of Surfaces. 4th ed. New York: John Wiley & Sons; 1982

[25] Creighton TE. Protein Function: Practical Approach. Oxford: IRL Press; 1989

[26] Nohara D, Mizutani A, Sakai T. Kinetic study on thermal denaturation of hen egg-white lysozyme involving precipitation. Journal of Bioscience and Bioengineering. 1999;**87**:199-205

[27] Noritomi H, Minamisawa K, Kamiya R, Kato S. Thermal stability of proteins in the presence of aprotic ionic liquids. Journal of Biomedical Science and Engineering. 2011;**4**:94-99

[28] Surewicz WK, Mantsch HH. New insight into protein secondary structure from resolution-enhanced infrared spectra. Biochimica et Biophysica Acta. 1988;**952**:115-130

[29] Klibanov AM. Improving enzymes by using them in organic solvents. Nature. 2001;**409**:241-246

[30] Lehninger AL, Nelson DL, Cox MM. Principles of Biochemistry. 2nd ed. New York: Worth; 1993

[31] Wescott CR, Noritomi H, Klibanov AM. Rational control of enzymatic enantioselectivity through solvation thermodynamics. Journal of the American Chemical Society. 1996;**118**:10365-10370

[32] Fogler HS. Elements of Chemical Reaction Engineering. 2nd ed. New Jersey: Prentice Hall PTR; 1992

[33] Kumar A, Venkatesu P. Overview of the stability of α-chymotrypsin in different solvent media. Chemical Reviews. 2012;**112**:4283-4307

[34] Reinhardt C. Solvents and Solvent Effects in Organic Chemistry. 2nd ed. Weinheim: Wiley-VCH; 1988

[35] Acker L. Enzyme reactions in foods of low moisture content. Advances in Food Research. 1962;**11**:263-330

[36] Bell G, Janssen AEM, Halling PJ. Water activity fails to predict critical hydration level for enzyme activity in

**95**

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes*

*DOI: http://dx.doi.org/10.5772/intechopen.82094*

polar organic solvents: Interconversion of water concentrations and activities. Enzyme and Microbial Technology.

[37] Mozhaev VV, Khmelnitsky YL, Sergeeva MV, Belova AB, Klyachko NL, Levashov AV, et al. Catalytic activity and denaturation of enzymes in water/organic cosolvent mixtures. α-Chymotrypsin and laccase in mixed water/alcohol, water/glycol and water/ formamide solvents. European Journal of Biochemistry. 1989;**184**:597-602

[38] Ryu K, Dordick JS. How do organic solvents affect peroxidase structure and function? Biochemistry.

Bioengineering. 1987;**30**:81-87

[39] Laane C, Boeren S, Vos K, Veeger C. Rules for optimization of biocatalysis in organic solvents. Biotechnology and

[40] Zaks A, Klibanov AM. Enzymatic catalysis in nonaqueous solvents. The Journal of Biological Chemistry.

[41] Kise H, Hayakawa H, Noritomi H. Protease-catalyzed synthetic reactions and immobilization-activation of the enzymes in hydrophilic organic solvents. Journal of Biotechnology.

1992;**31**:2588-2598

1988;**263**:3194-3201

1990;**14**:239-254

1997;**20**:471-477

*Application of Biochar to Enzyme Carrier for Stress Tolerance of Enzymes DOI: http://dx.doi.org/10.5772/intechopen.82094*

polar organic solvents: Interconversion of water concentrations and activities. Enzyme and Microbial Technology. 1997;**20**:471-477

*Biochar - An Imperative Amendment for Soil and the Environment*

[26] Nohara D, Mizutani A, Sakai T. Kinetic study on thermal denaturation of hen egg-white lysozyme involving precipitation. Journal of Bioscience and Bioengineering. 1999;**87**:199-205

[27] Noritomi H, Minamisawa K, Kamiya R, Kato S. Thermal stability of proteins in the presence of aprotic ionic liquids. Journal of Biomedical Science and Engineering. 2011;**4**:94-99

[28] Surewicz WK, Mantsch HH. New insight into protein secondary structure from resolution-enhanced infrared spectra. Biochimica et Biophysica Acta.

[29] Klibanov AM. Improving enzymes by using them in organic solvents.

[30] Lehninger AL, Nelson DL, Cox MM. Principles of Biochemistry. 2nd ed.

1988;**952**:115-130

Nature. 2001;**409**:241-246

New York: Worth; 1993

1996;**118**:10365-10370

[31] Wescott CR, Noritomi H, Klibanov AM. Rational control of enzymatic enantioselectivity through solvation thermodynamics. Journal of the American Chemical Society.

[32] Fogler HS. Elements of Chemical Reaction Engineering. 2nd ed. New Jersey: Prentice Hall PTR; 1992

[33] Kumar A, Venkatesu P. Overview of the stability of α-chymotrypsin in different solvent media. Chemical Reviews. 2012;**112**:4283-4307

[34] Reinhardt C. Solvents and Solvent Effects in Organic Chemistry. 2nd ed.

[35] Acker L. Enzyme reactions in foods of low moisture content. Advances in Food Research. 1962;**11**:263-330

[36] Bell G, Janssen AEM, Halling PJ. Water activity fails to predict critical hydration level for enzyme activity in

Weinheim: Wiley-VCH; 1988

charcoal powder. Journal of Biomaterials and Nanobiotechnology. 2014;**5**:179-185

[18] Noritomi H, Kurihara S, Endo N, Kato S, Uchiyama K. Effect of adsorption condition on thermal stability of proteins adsorbed onto biomass charcoal powder. International Journal of GEOMATE.

[19] Noritomi H, Nishigami J, Endo N, Kato S, Uchiyama K. Organic solventresistant properties of proteins

adsorbed onto biomass charcoal powder. International Journal of GEOMATE.

[20] Noritomi H, Nishigami J, Endo N, Kato S, Uchiyama K. Influence of water activity on protease adsorbed on biochar in organic solvents.

Journal of Materials Science Research.

[21] Noritomi H, Nishigami J, Endo N, Kato S, Takagi S. Effect of solvent on catalysis of protease adsorbed on biochar in organic media. Journal of Materials Science Research.

[22] Asada T, Ishihara S, Yamane T, Toba A, Yamada A, Oikawa K. Science

[23] Nishimiya K, Hata T, Imamura Y, Ishihara S. Analysis of chemical structure of wood charcoal by X-ray photoelectron spectroscopy. Journal of

[24] Adamson AW. Physical Chemistry of Surfaces. 4th ed. New York: John

[25] Creighton TE. Protein Function: Practical Approach. Oxford: IRL Press;

of bamboo charcoal: Study on carbonizing temperature of bamboo charcoal and removal capability of harmful gases. Journal of Health Science. 2002;**48**:473-479

Wood Science. 1998;**44**:56-61

Wiley & Sons; 1982

2016;**11**:2123-2128

2017;**12**:140-145

2017;**6**:96-102

2018;**7**:46-52

**94**

1989

[37] Mozhaev VV, Khmelnitsky YL, Sergeeva MV, Belova AB, Klyachko NL, Levashov AV, et al. Catalytic activity and denaturation of enzymes in water/organic cosolvent mixtures. α-Chymotrypsin and laccase in mixed water/alcohol, water/glycol and water/ formamide solvents. European Journal of Biochemistry. 1989;**184**:597-602

[38] Ryu K, Dordick JS. How do organic solvents affect peroxidase structure and function? Biochemistry. 1992;**31**:2588-2598

[39] Laane C, Boeren S, Vos K, Veeger C. Rules for optimization of biocatalysis in organic solvents. Biotechnology and Bioengineering. 1987;**30**:81-87

[40] Zaks A, Klibanov AM. Enzymatic catalysis in nonaqueous solvents. The Journal of Biological Chemistry. 1988;**263**:3194-3201

[41] Kise H, Hayakawa H, Noritomi H. Protease-catalyzed synthetic reactions and immobilization-activation of the enzymes in hydrophilic organic solvents. Journal of Biotechnology. 1990;**14**:239-254

**97**

**1. Introduction**

**Chapter 6**

Strategy

**Abstract**

Biochar Phosphorus Sorption-

*Lucy W. Ngatia, Johnny M. Grace III, Daniel Moriasi,* 

*Alejandro Bolques, George K. Osei and Robert W. Taylor*

Phosphorus (P) eutrophication in the water bodies is of global concern. The role of biochar in the mitigation of (P) eutrophication has recently received substantial attention. Agriculture is the main source of P in the water bodies, as a result of excessive fertilizer and manure application. Excessive P results in excessive primary production in the water bodies, leading to anoxic conditions, growth of toxic algae blooms, altering plant species composition and biomass. Therefore, resulting in food web disruption, fish kill, toxins production and recreation areas degradation. When biochar is applied on farm, it has potential to sorb/adsorb P, immobilizing it, slowing its translocation to the water bodies. However, biochar effectiveness in P sorption is influenced by both feedstock type and pyrolysis temperature. The interaction between feedstock type and pyrolysis temperature influences the biochar pH, surface area, aromatic carbon, cation exchange capacity, surface charge density, biochar internal porosity and polar and nonpolar surface sites that promote nutrient absorption. Hence, biochar properties have a broad spectrum that influences how biochar reacts with P sorption; therefore, it is not appropriate to extrapolate observed results to different materials. Biochar that promote P sorption rather than desorption should be considered and designed to meet specific management practices.

**Keywords:** biochar, desorption, eutrophication, phosphorus, mitigation, sorption

Phosphorus (P) eutrophication is a major problem globally. Increased aquatic primary production attributed to P over enrichment results in eutrophication [1, 2]. Aquatic systems affected by eutrophication often exhibit harmful algal blooms, which foul water intakes and waterways, disrupt food webs, fuel hypoxia and produce secondary metabolites that are toxic to water consumers and users including human, cattle, zooplankton, shellfish, domestic pets and fish [3]. Agriculture is the main pollutant of aquatic systems [4]. Overapplication of P fertilizer and manure to soil is in itself wasteful and causes P accumulation in the soil, but the transport of P to aquatic ecosystems by erosion is also causing widespread problems

Eutrophication Mitigation

Desorption: Potential Phosphorus

### **Chapter 6**

## Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation Strategy

*Lucy W. Ngatia, Johnny M. Grace III, Daniel Moriasi, Alejandro Bolques, George K. Osei and Robert W. Taylor*

#### **Abstract**

Phosphorus (P) eutrophication in the water bodies is of global concern. The role of biochar in the mitigation of (P) eutrophication has recently received substantial attention. Agriculture is the main source of P in the water bodies, as a result of excessive fertilizer and manure application. Excessive P results in excessive primary production in the water bodies, leading to anoxic conditions, growth of toxic algae blooms, altering plant species composition and biomass. Therefore, resulting in food web disruption, fish kill, toxins production and recreation areas degradation. When biochar is applied on farm, it has potential to sorb/adsorb P, immobilizing it, slowing its translocation to the water bodies. However, biochar effectiveness in P sorption is influenced by both feedstock type and pyrolysis temperature. The interaction between feedstock type and pyrolysis temperature influences the biochar pH, surface area, aromatic carbon, cation exchange capacity, surface charge density, biochar internal porosity and polar and nonpolar surface sites that promote nutrient absorption. Hence, biochar properties have a broad spectrum that influences how biochar reacts with P sorption; therefore, it is not appropriate to extrapolate observed results to different materials. Biochar that promote P sorption rather than desorption should be considered and designed to meet specific management practices.

**Keywords:** biochar, desorption, eutrophication, phosphorus, mitigation, sorption

#### **1. Introduction**

Phosphorus (P) eutrophication is a major problem globally. Increased aquatic primary production attributed to P over enrichment results in eutrophication [1, 2]. Aquatic systems affected by eutrophication often exhibit harmful algal blooms, which foul water intakes and waterways, disrupt food webs, fuel hypoxia and produce secondary metabolites that are toxic to water consumers and users including human, cattle, zooplankton, shellfish, domestic pets and fish [3]. Agriculture is the main pollutant of aquatic systems [4]. Overapplication of P fertilizer and manure to soil is in itself wasteful and causes P accumulation in the soil, but the transport of P to aquatic ecosystems by erosion is also causing widespread problems of eutrophication [5–8]. Soil P exists in both organic and inorganic forms [9]; the inorganic P is highly influenced by soil pH and is mainly coupled with amorphous and crystalline forms of Fe, Al, and Ca [10]. Biochar exhibits potential to mitigate P eutrophication when applied on farm as a result of its high capacity to sorb P [1]. Biochar is a by-product of pyrolysis process, whereby in an energy-limited world, biomass is converted to energy products through pyrolysis process [11]. Biochar nutrient sorption capacity has been reported to exceed that of natural organic matter by a factor of 10–100 in some cases [12]. However, its capacity for P sorption is influenced by both feedstock type and pyrolysis temperature [13]. The interaction between feedstock type and pyrolysis temperature influences biochar characteristics resulting in wide spectrum of biochar properties that influence P sorption [1, 14]. Therefore, extrapolation of P sorption findings from one material to another is not appropriate; as a result of diversity of biochar response to P sorption, every material needs to be studied and designed to address the set P sorption objectives.

#### **2. Forms of phosphorus**

Soil P exists as either organic or inorganic compounds that differ significantly in their biological availability in the soil environment [9]. Phosphorus goes through different geochemical processes in soil which include adsorption, solubilization, precipitation and complexation that determine its mobility and fate [15]. The inorganic P compounds mainly couple with amorphous and crystalline forms of Fe, Al, and Ca [10] and are highly influenced by soil acidity and alkalinity [1]. Soil acidity and alkalinity influence the impact of solubilization, complexation, adsorption and precipitation on P retention and release. In acidic soils P is fixed by sorption to oxides and hydroxides of Al and Fe through formation of insoluble Al and Fe phosphates by ligand exchange and precipitation reactions [16, 17]. In alkaline soils, P is made unavailable due to formation of metal complexes such as Mg-P and Ca-P [18, 19]. The organic P in most soils is dominated by a mixture of phosphate monoesters (e.g., inositol phosphates and mononucleotides) and phosphate diesters (mainly phospholipids and nucleic acids), with smaller amounts of phosphonates (compounds with a direct carbon-phosphorus bond) and organic polyphosphates (e.g., adenosine triphosphate) [20]. Plants can acquire P from organic compounds through various mechanisms; some of the mechanisms allow plants to utilize organic P as efficiently as inorganic phosphate [21, 22]. Different environmental conditions influence availability and sorption of P, for example anaerobic condition leads to the release of P resulting from reduction of ferric to ferrous iron [23]. The presence of sulfate could lead to reaction of ferric iron with sulfate and sulfide to form ferrous iron and iron sulfide resulting in the release of P [24]. Increased temperature can reduce adsorption of P by mineral complexes in the sediment [25]. Other physicochemical processes affecting the release of P from the sediment include redox, temperature, reservoir hydrology, pH potential and environmental conditions [26]. These processes are further complicated by the influence of biological processes, for example mineralization, leading to a complex system regulating the release of P across sediment water interface [26]. Understanding of P retention and release mechanism enhances effective management of P enhancing crop production and promoting sustainability of soil and water quality [11].

#### **3. Agriculture: the major source of phosphorus to water bodies**

Increasing human population calls for increased food production. Increased food production requires increased fertilizer application which includes P fertilizer

**99**

**Figure 2.**

**Figure 1.**

*rock (1800–2000). Modified from [27].*

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation…*

(**Figure 1**) [27]. Today agriculture is heavily dependent on P derived from phosphate rock. Phosphorus is a nonrenewable resource and it is expected that economically mineable P reserves will be exhausted within 50–100 years (**Figure 2**) [27]. Crop and livestock production systems are the major cause of human alteration of the global P cycles [28]. The major source of P input to water bodies is the excessive application of fertilizer or manure on farm which causes P accumulation in soils [5]. Excess P or poor-timed application could mobilize P through runoff, negatively impacting water quality of water bodies and causing eutrophication [29, 30].

*Historical sources of phosphorus for use as fertilizers, including manure, human excreta, guano and phosphate* 

*Indicative peak phosphorus curve, illustrating that, global phosphorus reserves are likely to peak after which* 

*production will be significantly reduce. Modified from [27].*

*DOI: http://dx.doi.org/10.5772/intechopen.82092*

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation… DOI: http://dx.doi.org/10.5772/intechopen.82092*

(**Figure 1**) [27]. Today agriculture is heavily dependent on P derived from phosphate rock. Phosphorus is a nonrenewable resource and it is expected that economically mineable P reserves will be exhausted within 50–100 years (**Figure 2**) [27]. Crop and livestock production systems are the major cause of human alteration of the global P cycles [28]. The major source of P input to water bodies is the excessive application of fertilizer or manure on farm which causes P accumulation in soils [5]. Excess P or poor-timed application could mobilize P through runoff, negatively impacting water quality of water bodies and causing eutrophication [29, 30].

#### **Figure 1.**

*Biochar - An Imperative Amendment for Soil and the Environment*

of eutrophication [5–8]. Soil P exists in both organic and inorganic forms [9]; the inorganic P is highly influenced by soil pH and is mainly coupled with amorphous and crystalline forms of Fe, Al, and Ca [10]. Biochar exhibits potential to mitigate P eutrophication when applied on farm as a result of its high capacity to sorb P [1]. Biochar is a by-product of pyrolysis process, whereby in an energy-limited world, biomass is converted to energy products through pyrolysis process [11]. Biochar nutrient sorption capacity has been reported to exceed that of natural organic matter by a factor of 10–100 in some cases [12]. However, its capacity for P sorption is influenced by both feedstock type and pyrolysis temperature [13]. The interaction between feedstock type and pyrolysis temperature influences biochar characteristics resulting in wide spectrum of biochar properties that influence P sorption [1, 14]. Therefore, extrapolation of P sorption findings from one material to another is not appropriate; as a result of diversity of biochar response to P sorption, every material

needs to be studied and designed to address the set P sorption objectives.

most soils is dominated by a mixture of phosphate monoesters

(e.g., inositol phosphates and mononucleotides) and phosphate diesters (mainly phospholipids and nucleic acids), with smaller amounts of phosphonates (compounds with a direct carbon-phosphorus bond) and organic polyphosphates (e.g., adenosine triphosphate) [20]. Plants can acquire P from organic compounds through various mechanisms; some of the mechanisms allow plants to utilize organic P as efficiently as inorganic phosphate [21, 22]. Different environmental conditions influence availability and sorption of P, for example anaerobic condition leads to the release of P resulting from reduction of ferric to ferrous iron [23]. The presence of sulfate could lead to reaction of ferric iron with sulfate and sulfide to form ferrous iron and iron sulfide resulting in the release of P [24]. Increased temperature can reduce adsorption of P by mineral complexes in the sediment [25]. Other physicochemical processes affecting the release of P from the sediment include redox, temperature, reservoir hydrology, pH potential and environmental conditions [26]. These processes are further complicated by the influence of biological processes, for example mineralization, leading to a complex system regulating the release of P across sediment water interface [26]. Understanding of P retention and release mechanism enhances effective management of P enhancing crop production and promoting sustainability

**3. Agriculture: the major source of phosphorus to water bodies**

Increasing human population calls for increased food production. Increased food production requires increased fertilizer application which includes P fertilizer

Soil P exists as either organic or inorganic compounds that differ significantly in their biological availability in the soil environment [9]. Phosphorus goes through different geochemical processes in soil which include adsorption, solubilization, precipitation and complexation that determine its mobility and fate [15]. The inorganic P compounds mainly couple with amorphous and crystalline forms of Fe, Al, and Ca [10] and are highly influenced by soil acidity and alkalinity [1]. Soil acidity and alkalinity influence the impact of solubilization, complexation, adsorption and precipitation on P retention and release. In acidic soils P is fixed by sorption to oxides and hydroxides of Al and Fe through formation of insoluble Al and Fe phosphates by ligand exchange and precipitation reactions [16, 17]. In alkaline soils, P is made unavailable due to formation of metal complexes such as Mg-P and Ca-P [18, 19]. The organic P in

**2. Forms of phosphorus**

**98**

of soil and water quality [11].

*Historical sources of phosphorus for use as fertilizers, including manure, human excreta, guano and phosphate rock (1800–2000). Modified from [27].*

#### **Figure 2.**

*Indicative peak phosphorus curve, illustrating that, global phosphorus reserves are likely to peak after which production will be significantly reduce. Modified from [27].*

In many cases, P enters aquatic ecosystems sorbed to soil particles that are eroded into rivers, lakes and streams; any factor elevating soil P concentration or accelerating soil erosion potentially increase P runoff to aquatic system [31, 32]. Mobilization of P involves chemical, biochemical and biological processes. The processes are grouped into detachment or solubilization mechanisms and are defined by the physical size of the P compounds that are mobilized [33]. Potential for solubilization increases with increasing concentrations for extractable P. Depending on site conditions, diffuse P transport occurs as particulate or dissolved P in overland flow, drainage, channelized surface runoff or groundwater [34].

#### **4. Phosphorus sorption is dependent on biochar properties**

Biochar exhibits variable properties which are related to its formation; biochar properties are mainly influenced by method of pyrolysis, temperature of pyrolysis, feedstock type, particle size, pyrolysis retention time and furnace oxygen level [35–38]. Biochar has been reported to increase soil pH and cation exchange capacity (**Figure 3**) [1, 39]. Biochars exhibit high surface charge density, and CEC values up to 112 cmolc kg<sup>−</sup><sup>1</sup> have been observed [40]. The high surface charge density allows biochar to retain cations by cation exchange, whereas biochar internal porosity, high surface area and presence of both polar and non-polar surfaces sites promote absorption of nutrient [41]. Biochar is also reported to contain essential nutrients [41, 42] that could be retained and slowly released over time [43, 44].

As a result of biochar variable properties, the P sorption findings by different studies are inconsistent; some previous studies indicated no increase in P sorption

#### **Figure 3.**

*Relationship between biochar phosphorus sorption and pH for all feedstocks, switchgrass, kudzu and Chinese tallow. Modified from [1].*

**101**

**Figure 4.**

*Biochar phosphorus adsorption; Modified from [1].*

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation…*

of the sandy and clayey soils after addition of biochar [45]. There was no P sorption observed with corn biochar produced at 350 and 550°C pyrolysis temperatures [46]. Biochar produced from sugarcane bagasse, peanut hull, Brazilian pepper wood and bamboo at 200, 450 and 600°C did not indicate any clear trend in phosphorus sorption [47]. However, other studies indicated reduction of P leaching after biochar application [41, 48]. Further, biochar addition reduced P leaching after manure addition and in green roof discharge water [41, 49]. Application of 2% biochar to agricultural soil amended with swine manure resulted in reduction of P leaching by 69% [41]. Addition of 2% pecan shell biochar to loamy soil reduced P leachate by 40% [50]. Orange peel pyrolyzed at between 250 and 700°C improved P sorption by 8–83% [51]. [1] indicated that biochar pyrolyzed from kudzu and Chinese tallow at 300–700°C temperature exhibited increased P sorption; when switchgrass was pyrolyzed at 300–500°C, it exhibited P desorption, but when pyrolyzed at 500–700°C temperature, it exhibited P sorption, clearly demonstrating that feedstock type and pyrolysis temperature are major determinant of P

In [1], it is also demonstrated that increasing temperature resulted in loss of O-alkyl carbon and accumulation of aromatic carbon that favored P sorption (**Figure 6**). This variability in biochar capacity to sorb P suggests that understanding each biochar type is important to ensure appropriate application to meet target

*DOI: http://dx.doi.org/10.5772/intechopen.82092*

sorption capacity (**Figures 4** and **5**).

objectives.

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation… DOI: http://dx.doi.org/10.5772/intechopen.82092*

of the sandy and clayey soils after addition of biochar [45]. There was no P sorption observed with corn biochar produced at 350 and 550°C pyrolysis temperatures [46]. Biochar produced from sugarcane bagasse, peanut hull, Brazilian pepper wood and bamboo at 200, 450 and 600°C did not indicate any clear trend in phosphorus sorption [47]. However, other studies indicated reduction of P leaching after biochar application [41, 48]. Further, biochar addition reduced P leaching after manure addition and in green roof discharge water [41, 49]. Application of 2% biochar to agricultural soil amended with swine manure resulted in reduction of P leaching by 69% [41]. Addition of 2% pecan shell biochar to loamy soil reduced P leachate by 40% [50]. Orange peel pyrolyzed at between 250 and 700°C improved P sorption by 8–83% [51]. [1] indicated that biochar pyrolyzed from kudzu and Chinese tallow at 300–700°C temperature exhibited increased P sorption; when switchgrass was pyrolyzed at 300–500°C, it exhibited P desorption, but when pyrolyzed at 500–700°C temperature, it exhibited P sorption, clearly demonstrating that feedstock type and pyrolysis temperature are major determinant of P sorption capacity (**Figures 4** and **5**).

In [1], it is also demonstrated that increasing temperature resulted in loss of O-alkyl carbon and accumulation of aromatic carbon that favored P sorption (**Figure 6**). This variability in biochar capacity to sorb P suggests that understanding each biochar type is important to ensure appropriate application to meet target objectives.

**Figure 4.** *Biochar phosphorus adsorption; Modified from [1].*

*Biochar - An Imperative Amendment for Soil and the Environment*

flow, drainage, channelized surface runoff or groundwater [34].

values up to 112 cmolc kg<sup>−</sup><sup>1</sup>

over time [43, 44].

**4. Phosphorus sorption is dependent on biochar properties**

In many cases, P enters aquatic ecosystems sorbed to soil particles that are eroded into rivers, lakes and streams; any factor elevating soil P concentration or accelerating soil erosion potentially increase P runoff to aquatic system [31, 32]. Mobilization of P involves chemical, biochemical and biological processes. The processes are grouped into detachment or solubilization mechanisms and are defined by the physical size of the P compounds that are mobilized [33]. Potential for solubilization increases with increasing concentrations for extractable P. Depending on site conditions, diffuse P transport occurs as particulate or dissolved P in overland

Biochar exhibits variable properties which are related to its formation; biochar properties are mainly influenced by method of pyrolysis, temperature of pyrolysis, feedstock type, particle size, pyrolysis retention time and furnace oxygen level [35–38]. Biochar has been reported to increase soil pH and cation exchange capacity (**Figure 3**) [1, 39]. Biochars exhibit high surface charge density, and CEC

density allows biochar to retain cations by cation exchange, whereas biochar internal porosity, high surface area and presence of both polar and non-polar surfaces sites promote absorption of nutrient [41]. Biochar is also reported to contain essential nutrients [41, 42] that could be retained and slowly released

As a result of biochar variable properties, the P sorption findings by different studies are inconsistent; some previous studies indicated no increase in P sorption

*Relationship between biochar phosphorus sorption and pH for all feedstocks, switchgrass, kudzu and Chinese* 

have been observed [40]. The high surface charge

**100**

**Figure 3.**

*tallow. Modified from [1].*

**Figure 5.**

*Phosphorus sorption by corn stover, Ponderosa pine wood residue, and switchgrass biochars. Each data point is the mean of four replications with standard error. Modified from [11].*

**Figure 6.**

*Biochar carbon functional groups as determined by Nuclear Magnetic Resonance (NMR); modified from [1].*

#### **5. Biochar pH influence phosphorus sorption–desorption**

The influence of the changes in pH after biochar application seems to vary between different studies. Biochar application reduces soil acidity altering P complexity with metals such as Al3+, Fe3+ and Ca2+ affecting P availability, sorption and desorption (**Figure 3**) [13, 52]. As a result of higher alkalinity, biochars from legume plants increase pH much compared to biochars from non-legume plants [52].

**103**

**Figure 7.**

*standard error. Modified from [11].*

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation…*

Biochar has potential to adsorb cations such as Al3+, Fe3+ and Ca2+, which can lead to delayed P adsorption or precipitation; in addition organic molecules could sorb onto biochar reducing its capacity to chelate with Al3+, Fe3+ and Ca2+ in soil (**Figure 7**) [53]. Ref. [53] reported that P sorption was increased in acidic soil but decreased in alkaline soil, and attributed increased P sorption after biochar addition to Ca induced P sorption/precipitation and also reported that P sorption was less affected by Fe and Al oxides. In contrast, incorporation of 4% biochar into acidic soil reduced the P sorption and increased availability of sorbed P, whereas application of alkaline biochars to calcareous soil increased P sorption significantly and decreased availability of sorbed P [11]. The increase in soil pH reportedly enhances the solubility of P [54] but, in contrast [1], demonstrated that addition of biochar with high pH, Ca concentration and aromatic C reduced P solubility (**Figures 3–5**). Ref. [55] indicated that colloidal and nano-sized MgO (periclase) particles on the biochar surface played the key role in providing adsorption sites for aqueous phosphate [55]. In addition, initial solution pH and coexisting anions have potential

*P sorption by acidic soil and acidic soil (Grummit series) incubated with corn stover, Ponderosa pine wood residue or switchgrass biochars at 4 g/kg soil for 30 days. Each data point is the mean of four replications with* 

**6. Feedstock/biomass type and pyrolysis temperature influence** 

There have been diverse findings on the effect of biochar on P sorption, and in some studies, biochar application promoted availability and uptake of P as a result of biochar high anion exchange capacity; reduction of availability of Al and Fe in soil resulted in less P fixation (**Figure 8**) [50, 56]. Biochar feedstock and pyrolysis temperature affect its chemical composition and surface characteristics influencing biochar P sorption and desorption capacity [1, 11, 13]. Biochar produced from different biomass often has very different chemical characteristics and as a result influences the P sorption capacity of biochar [1, 11]. In [1], it is reported that corn stover biochar (79%) had greater sorption followed by switchgrass biochar (76%),

*DOI: http://dx.doi.org/10.5772/intechopen.82092*

to affect adsorption of P on biochar.

**phosphorus sorption**

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation… DOI: http://dx.doi.org/10.5772/intechopen.82092*

#### **Figure 7.**

*Biochar - An Imperative Amendment for Soil and the Environment*

*the mean of four replications with standard error. Modified from [11].*

**5. Biochar pH influence phosphorus sorption–desorption**

The influence of the changes in pH after biochar application seems to vary between different studies. Biochar application reduces soil acidity altering P complexity with metals such as Al3+, Fe3+ and Ca2+ affecting P availability, sorption and desorption (**Figure 3**) [13, 52]. As a result of higher alkalinity, biochars from legume plants increase pH much compared to biochars from non-legume plants [52].

*Biochar carbon functional groups as determined by Nuclear Magnetic Resonance (NMR); modified from [1].*

*Phosphorus sorption by corn stover, Ponderosa pine wood residue, and switchgrass biochars. Each data point is* 

**102**

**Figure 6.**

**Figure 5.**

*P sorption by acidic soil and acidic soil (Grummit series) incubated with corn stover, Ponderosa pine wood residue or switchgrass biochars at 4 g/kg soil for 30 days. Each data point is the mean of four replications with standard error. Modified from [11].*

Biochar has potential to adsorb cations such as Al3+, Fe3+ and Ca2+, which can lead to delayed P adsorption or precipitation; in addition organic molecules could sorb onto biochar reducing its capacity to chelate with Al3+, Fe3+ and Ca2+ in soil (**Figure 7**) [53]. Ref. [53] reported that P sorption was increased in acidic soil but decreased in alkaline soil, and attributed increased P sorption after biochar addition to Ca induced P sorption/precipitation and also reported that P sorption was less affected by Fe and Al oxides. In contrast, incorporation of 4% biochar into acidic soil reduced the P sorption and increased availability of sorbed P, whereas application of alkaline biochars to calcareous soil increased P sorption significantly and decreased availability of sorbed P [11]. The increase in soil pH reportedly enhances the solubility of P [54] but, in contrast [1], demonstrated that addition of biochar with high pH, Ca concentration and aromatic C reduced P solubility (**Figures 3–5**). Ref. [55] indicated that colloidal and nano-sized MgO (periclase) particles on the biochar surface played the key role in providing adsorption sites for aqueous phosphate [55]. In addition, initial solution pH and coexisting anions have potential to affect adsorption of P on biochar.

#### **6. Feedstock/biomass type and pyrolysis temperature influence phosphorus sorption**

There have been diverse findings on the effect of biochar on P sorption, and in some studies, biochar application promoted availability and uptake of P as a result of biochar high anion exchange capacity; reduction of availability of Al and Fe in soil resulted in less P fixation (**Figure 8**) [50, 56]. Biochar feedstock and pyrolysis temperature affect its chemical composition and surface characteristics influencing biochar P sorption and desorption capacity [1, 11, 13]. Biochar produced from different biomass often has very different chemical characteristics and as a result influences the P sorption capacity of biochar [1, 11]. In [1], it is reported that corn stover biochar (79%) had greater sorption followed by switchgrass biochar (76%),

#### **Figure 8.**

*P sorption calcareous soil (Langhei series) and calcareous soil incubated with corn stover, Ponderosa pine wood residue, and switchgrass biochars at 40 g/kg soil for 30 days. Each data point is the mean of four replications with standard error. Modified from [11].*


*Data indicate mean ± SEM (standard error of mean) between temperatures. Different letters superscript along the row indicate Tukey HSD significant difference between means of biochar recovery and pH under different temperatures. Modified from [1].*

#### **Table 1.**

*Biochar recovery and pH.*

while Ponderosa pine wood residue biochar (31%) exhibited the lowest biochar sorption. Increasing biochar pyrolysis temperature leads to loss of the volatile compounds and increased aromatic carbon, elevated pH, biochar yield decreases (**Table 1**), increased surface area and decreased surface functional groups that provide exchange capacity (**Figures 4** and **5**) [1, 14]. Beet tailing biochar pyrolyzed at 600°C adsorbed P; the adsorption was suggested to have occurred due to binding sites contained in colloidal and nano-sized MGO particles on the biochar surface [55]. [57] indicated that similar to P sorption, desorption is also influenced by feedstock, for example Inga exhibited less sorption of P compared to Lacre and Embauba feedstocks but also desorbed less P (**Figures 9** and **10**) [57].

**105**

**Figure 9.** *Desorption of PO4*

**Figure 10.**

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation…*

Therefore, P sorption–desorption varies between feedstock type and pyrolysis temperature levels, providing a possibility of designing biochars for specific soil

*Phosphorus desorption curve data for the sequential desorption of P from a degraded tropical soil with biochar added. The points represent desorption of P from the soil/biochar complex after the addition of 75, 150, 200 and 300 mg P/kg. The encircled area represents a uniform desorption curve common to all treatments with a Kd between solution concentrations of 0.1 and 0.2 mg/L of approximately 1230 L/kg. Modified from [57].*

*–P from washed biochars plotted as the cumulative fraction desorbed. Modified from [58].*

Biochar has potential to mitigate P eutrophication in the water bodies through its application in the farm. However, it is clear that biochar P sorption capacity is feedstock specific and is highly influenced by pyrolysis temperature [1]. Therefore,

management objectives (**Figures 4**, **5** and **10**) [57].

**7. Biochar potential to mitigate phosphorus eutrophication**

*DOI: http://dx.doi.org/10.5772/intechopen.82092*

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation… DOI: http://dx.doi.org/10.5772/intechopen.82092*

**Figure 9.** *Desorption of PO4 –P from washed biochars plotted as the cumulative fraction desorbed. Modified from [58].*

#### **Figure 10.**

*Biochar - An Imperative Amendment for Soil and the Environment*

while Ponderosa pine wood residue biochar (31%) exhibited the lowest biochar sorption. Increasing biochar pyrolysis temperature leads to loss of the volatile compounds and increased aromatic carbon, elevated pH, biochar yield decreases (**Table 1**), increased surface area and decreased surface functional groups that provide exchange capacity (**Figures 4** and **5**) [1, 14]. Beet tailing biochar pyrolyzed at 600°C adsorbed P; the adsorption was suggested to have occurred due to binding sites contained in colloidal and nano-sized MGO particles on the biochar surface [55]. [57] indicated that similar to P sorption, desorption is also influenced by feedstock, for example Inga exhibited less sorption of P compared to Lacre and

**Temperature 200°C 300°C 400°C 500°C 550°C 650°C 750°C** *P***-value**

*P sorption calcareous soil (Langhei series) and calcareous soil incubated with corn stover, Ponderosa pine wood residue, and switchgrass biochars at 40 g/kg soil for 30 days. Each data point is the mean of four replications* 

pH 7.1±0.4b 8.0±0.3b 9.3±1.0ab 9.0±0.7ab 10.7±0.04a 11.3±0.1a 11.3±0.1a 0.0001

pH 7.8±0.03e 9.5±0.1d 10.5±0.03c 11.3±0.1b 11.1±0.2b 12.0±0.03a 12.4±0.01a <0.0001

pH 7.0±0.03c 7.0±0.9c 8.8±0.1b 8.9±0.05b 8.9±0.1b 10.2±0.1ab 11.2±0.3a <0.0001 *Data indicate mean ± SEM (standard error of mean) between temperatures. Different letters superscript along the row indicate Tukey HSD significant difference between means of biochar recovery and pH under different temperatures.* 

78.0±1.5a 46.2±1.1b 31.7±1.3c 35.4±2.0c 24.9±0.3d 24.1±0.2d 22.8±0.2d <0.0001

74.0±2.3a 38.8±0.2b 29.8±2.0c 26.0±1.1cd 26.9±0.5cd 24.6±0.1cd 23.4±0.2d <0.0001

80.0±0.4a 36.5±2.6b 23.7±0.3c 22.2±0.3cd 20.6±0.9cd 20.8±0.8cd 17.5±0.6d <0.0001

Embauba feedstocks but also desorbed less P (**Figures 9** and **10**) [57].

**104**

Switchgrass BC recovery (%)

*with standard error. Modified from [11].*

**Figure 8.**

Kudzu

Chinese Tallow

BC recovery (%)

*Modified from [1].*

*Biochar recovery and pH.*

**Table 1.**

BC recovery (%)

*Phosphorus desorption curve data for the sequential desorption of P from a degraded tropical soil with biochar added. The points represent desorption of P from the soil/biochar complex after the addition of 75, 150, 200 and 300 mg P/kg. The encircled area represents a uniform desorption curve common to all treatments with a Kd between solution concentrations of 0.1 and 0.2 mg/L of approximately 1230 L/kg. Modified from [57].*

Therefore, P sorption–desorption varies between feedstock type and pyrolysis temperature levels, providing a possibility of designing biochars for specific soil management objectives (**Figures 4**, **5** and **10**) [57].

#### **7. Biochar potential to mitigate phosphorus eutrophication**

Biochar has potential to mitigate P eutrophication in the water bodies through its application in the farm. However, it is clear that biochar P sorption capacity is feedstock specific and is highly influenced by pyrolysis temperature [1]. Therefore, it is important to understand the interaction between feedstock type and pyrolysis temperature and how they influence biochar acidity/alkalinity, surface area, aromatic carbon, cation exchange capacity, surface charge density, biochar internal porosity and presence of both polar and non-polar surfaces sites that promote nutrient absorption [1, 13, 39, 41]. Biochar that promotes P sorption rather than release of P should be considered for P eutrophication mitigation, and biochar should be designed to meet specific management practices [57]. When applying biochar on farm, it is also important to consider the erodibility of biochar to water bodies; P is easily translocated to water bodies as particulate P [32, 34]. Therefore consideration of influence of biochar on erosion is important, for example in conditions where biochar reduces soil erosion, it results in reduced particulate P losses [59].

#### **8. Conclusion**

Biochar has potential to mitigate P eutrophication in the water bodies through biochar application on farm. However, biochar exhibits broad spectrum of properties which are mainly influenced by feedstock type and pyrolysis temperature and as a result influences P sorption. Some biochars have exhibited P sorption while others exhibited P desorption. Carbon composition of biochar and pH values of biochar and soils are major drivers of P sorption. It is not appropriate to extrapolate any single study findings to any other material as a result of the wide diversity in terms of biochar influence on P sorption. Therefore every material needs to be evaluated and its potential for P sorption determined. The usefulness of biochar on P sorption is dependent on its characteristics; to mitigate P eutrophication, employment of biochar with high P sorption is important.

#### **Acknowledgements**

Special thanks to College of Agriculture and Food Sciences, Florida A&M University, for providing a conducive environment for writing this book chapter. This work was supported by USDA/NIFA (1890 Evans-Allen Research Program), USDA-Forest Service Grant Number 17-CA-11330140-027 and USDA-ARS Grant Number 58-3070-7-009.

**107**

**Author details**

Lucy W. Ngatia1

George K. Osei4

Florida, USA

provided the original work is properly cited.

\*, Johnny M. Grace III2

and Robert W. Taylor1

\*Address all correspondence to: lucy.ngatia@famu.edu

© 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,

, Daniel Moriasi3

1 College of Agriculture and Food Sciences, Florida A&M University, Tallahassee,

2 USDA-Forest Service, Southern Research Station, Tallahassee, Florida, USA

4 School of Environment, Florida A&M University, Tallahassee, Florida, USA

3 USDA-ARS Grazinglands Research Laboratory, El Reno, Oklahoma, USA

, Alejandro Bolques1

,

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation…*

*DOI: http://dx.doi.org/10.5772/intechopen.82092*

*Biochar Phosphorus Sorption-Desorption: Potential Phosphorus Eutrophication Mitigation… DOI: http://dx.doi.org/10.5772/intechopen.82092*

#### **Author details**

*Biochar - An Imperative Amendment for Soil and the Environment*

ment of biochar with high P sorption is important.

**8. Conclusion**

**Acknowledgements**

Number 58-3070-7-009.

it is important to understand the interaction between feedstock type and pyrolysis temperature and how they influence biochar acidity/alkalinity, surface area, aromatic carbon, cation exchange capacity, surface charge density, biochar internal porosity and presence of both polar and non-polar surfaces sites that promote nutrient absorption [1, 13, 39, 41]. Biochar that promotes P sorption rather than release of P should be considered for P eutrophication mitigation, and biochar should be designed to meet specific management practices [57]. When applying biochar on farm, it is also important to consider the erodibility of biochar to water bodies; P is easily translocated to water bodies as particulate P [32, 34]. Therefore consideration of influence of biochar on erosion is important, for example in conditions where biochar reduces soil erosion, it results in reduced particulate P losses [59].

Biochar has potential to mitigate P eutrophication in the water bodies through biochar application on farm. However, biochar exhibits broad spectrum of properties which are mainly influenced by feedstock type and pyrolysis temperature and as a result influences P sorption. Some biochars have exhibited P sorption while others exhibited P desorption. Carbon composition of biochar and pH values of biochar and soils are major drivers of P sorption. It is not appropriate to extrapolate any single study findings to any other material as a result of the wide diversity in terms of biochar influence on P sorption. Therefore every material needs to be evaluated and its potential for P sorption determined. The usefulness of biochar on P sorption is dependent on its characteristics; to mitigate P eutrophication, employ-

Special thanks to College of Agriculture and Food Sciences, Florida A&M University, for providing a conducive environment for writing this book chapter. This work was supported by USDA/NIFA (1890 Evans-Allen Research Program), USDA-Forest Service Grant Number 17-CA-11330140-027 and USDA-ARS Grant

**106**

Lucy W. Ngatia1 \*, Johnny M. Grace III2 , Daniel Moriasi3 , Alejandro Bolques1 , George K. Osei4 and Robert W. Taylor1

1 College of Agriculture and Food Sciences, Florida A&M University, Tallahassee, Florida, USA

2 USDA-Forest Service, Southern Research Station, Tallahassee, Florida, USA

3 USDA-ARS Grazinglands Research Laboratory, El Reno, Oklahoma, USA

4 School of Environment, Florida A&M University, Tallahassee, Florida, USA

\*Address all correspondence to: lucy.ngatia@famu.edu

© 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.

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[2] Xu H, Paerl HW, Qin B, Zhu G, Gao G. Nitrogen and phosphorus inputs control phytoplankton growth in eutrophic Lake Taihu, China. Limnology and Oceanography.

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2000;**38**:669-674

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### *Edited by Vikas Abrol and Peeyush Sharma*

This book is intended to provide basic information and an overview of emerging researchable issues related to the use of biochar for mitigating climate change, water scarcity, soil degradation, and food security in a sustainable manner. We have aimed to compile information from diverse sources into a single volume and provide comprehensive information and analysis on biochar production technology and its implications in agriculture. This book represents basic and applied knowledge and acts as a vital tool for scientists, policymakers, and students working for global sustainability.

Published in London, UK © 2019 IntechOpen © Noppharat05081977 / iStock

Biochar - An Imperative Amendment for Soil and the Environment

Biochar

An Imperative Amendment

for Soil and the Environment

*Edited by Vikas Abrol and Peeyush Sharma*