**3. Protein hydrolysis and amino acid degradation: low temperature catalytic hydrothermal cracking (LT-CHTC) for liquid fuel production**

### **3.1 How to convert protein-rich biomass into liquid fuels**

Based on the concept of "sustainable development goals (SDGs)" by UN and "circular economy" by EU, waste materials (by-product of main activity) must be used in effective and useful application and ideally must be back to primary resource production. In our daily life, huge amount of food waste generates and its utilization/valorization is still insufficient. This situation is not only for food waste (including house waste, municipal waste, sewage sludge, which are by-product of our daily life) but also agricultural by-product. One of the big problems of these wastes is to contain high amount of water, which is hard to exclude from the wastes. Water inside the waste (namely wet biomass) assists to cultivate fungi, and some of the wet biomass wastes are easy to rot with a bad odor and toxic component formation. Even with huge amount of energy loss, the large part of the wet wastes is forcedly burned to eliminate without effective utilization as energy or thermal purposes.

Microalgae, which has fast grow rate with CO2 stabilization through photosynthesis and attracts much attention as renewable resource, is also wet biomass as the same as daily-life wastes with containing a large amount of water mentioned above. The total difference of microalgae from the waste wet biomass is that cultivation and harvesting of microalgae can be controlled to level not only for yield but also composition. Thus, microalgae is a primary and industrial renewable resource. However, problem related to water is to be solved as well as wet biomass waste.

Sub and supercritical water technologies enable water (also in the wet biomass) to eliminate from the structure of the biomass and to use for hydrolysis of the natural polymer in the biomass as reactant. As mentioned above, supercritical water plays the important role in the CHTC for producing olefinic hydrocarbon from lipid and free fatty acid, as solvent and reactant. But, for feeding lipid into the process, extraction of the lipid from microalgae must be done. Typical lipid extraction processes from microalgae are solvent extraction and mechanical press, both of which require dry to improve lipid yield before the extraction. Also, in isoconversion concept, the residue of the extraction of lipid (protein and carbohydrate are main components) will be treated by anaerobic fermentation to form small molecules such as organic acids. Advantage of the biochemical process is high selectivity, but disadvantage is low reaction rate. For the purpose of liquid fuels production, broad product distribution in the range of small molecular weight is acceptable. Or rather, rapid transformation process has to be necessary for the residue conversion of the residual biomass containing protein and carbohydrate.

*Ro* ¼ *exp*

gas constant, *R* = 8.314 J mol�<sup>1</sup> K�<sup>1</sup>

*Advanced Supercritical Fluids Technologies*

calculated by the next equation.

described in the following equation:

where *k*<sup>o</sup> [min�<sup>1</sup>

was estimated to be high.

**Figure 3.**

**94**

*T* � *Tr ω* 

, and *T*r, as shown in the following equation.

where *T* [K] is reaction temperature,*T*<sup>r</sup> [K] is the reference temperature (here is 373 K), *t* [min] is reaction time, and*ω*is the function of activation energy,*Ea* [kJ mol�<sup>1</sup>

> *<sup>ω</sup>* <sup>¼</sup> *<sup>R</sup>* <sup>∙</sup> *<sup>T</sup>*<sup>2</sup> *r Ea*

According to the derivation of the severity model, lignin recovery (*LR*) can be

where *k*<sup>r</sup> is the rate constants in min�<sup>1</sup> at reference temperature *T*<sup>r</sup> (373 K), as

By fitting the equations to the experimental data (as shown in **Figure 3**), all the kinetic parameters were decided as to be 135 kJ mol�<sup>1</sup> for *<sup>E</sup>*<sup>a</sup> and 6.8 � <sup>10</sup>�<sup>5</sup> min�<sup>1</sup> for *k*r. The experimental data at the most severe condition (*k*r�*R*<sup>o</sup> value is over 1, and these data were obtained at 573 K for 10 min) were far from the calculated value, and the reason why the data are not fitted with the calculated value is because the cellulose in the bark was carbonized and the remained Klason lignin in the residue

As shown in **Figure 3**, lignin solubilization (lignin recovery) in hydrothermal water drastically changed between 0.1 < *k*r�*R*<sup>o</sup> value <10. The similar trend was seen in the previous reports [2, 32, 33]. From Eqs. (1), (2), and (4), the dimension-

*Dimensionless severity number for lignin recovery from bark of Japanese cedar (473–573 K for 3–40 min).*

less severity number (*k*r�*R*o) is written by the below equation:

*kr* ∙ *Ro* ¼ *ko* ∙*t*∙*exp*

*kr* <sup>¼</sup> *koexp* � *Ea*

*Lignin recovery LR* ð Þ½ �¼ *wt*% f1 � *exp*ð Þ �*kr* ∙ *Ro* g � 100 (3)

*RTr* 

] is pre-exponential factor of the reference rate constant.

*Ea*ð Þ *T* � 2*Tr R* ∙ *Tr* 2 

*t* (1)

],

(2)

(4)

(5)

Thus, the whole biomass including lipid, protein, and carbohydrate should be treated by sub and supercritical water technologies in the presence of an appropriate catalyst (so it should be advanced supercritical technology). Here, we focus on effective conversion of carbohydrates and protein into small fragments, and low temperature catalytic hydrothermal cracking (LT-CHTC) must be designed with the concept of inhibition of Maillard reaction, which is undesired reaction because complex nitrogen-containing components are produced and these will be repolymerized into non-decomposable materials like char or coke.

as a standard amino acid. In a literature [48], it was found that the rate of thermal decomposition of amino acids were correlated with Taft rule, which allows the prediction of the rate of different amino acid based on a reaction rate of standard

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst…*

that the same relation of degradation rate of amino acid in subcritical water can be found (it will be revealed in near future by our research group) and alanine is

Here, pH dependence of the reaction rate of alanine was investigated because an appropriate additive for improvement of alanine conversion will be looked for. For the objective, an overall rate constant for 1st order alanine degradation at a wide

> *<sup>k</sup>*Ala <sup>¼</sup> *<sup>k</sup>*Ala<sup>þ</sup> <sup>H</sup><sup>þ</sup> ½ �<sup>2</sup> <sup>þ</sup> *<sup>k</sup>*Ala� *Ka*<sup>1</sup> <sup>H</sup><sup>þ</sup> ½ �þ *<sup>k</sup>*Ala� <sup>∙</sup>*Ka*1*Ka*<sup>2</sup> <sup>H</sup><sup>þ</sup> ½ �<sup>2</sup> <sup>þ</sup> *Ka*<sup>1</sup> <sup>H</sup><sup>þ</sup> ½ �þ *Ka*1*Ka*<sup>2</sup>

constant of 1st order overall zwitterion alanine (Ala�) degradation, and *k*ala� is the

concentration of proton in the system. *Ka*<sup>1</sup> is the dissociation constant between

between zwitterion (Ala�) and anionic alanine (Ala�), as shown in the below

rate constant of 1st order overall anionic alanine (Ala�) degradation. [H<sup>+</sup>

Ala<sup>þ</sup> \$ *Ka*<sup>1</sup>

was almost the same as the rate of overall alanine degradation (*k*ala). The

degradation in subcritical water (260�360°C at 20 MPa) not only neutral

the revised HKF (Helgeson-Kirkham-Flowers) model [52].

where *k*ala is the rate constant of 1st order overall alanine degradation, *k*ala+ is the

Ala� \$ *Ka*<sup>2</sup>

In the literature [49], the rate of decarboxylation (which is main reaction at sub and supercritical water over 573 K [15]) was measured with monitoring CO2 formation by in situ FTIR. Here, in Eq. (6), it is considered that pH dependence of the 1st order rate constant of alanine evaluated from the rate of decarboxylation

dissociation constant of *Ka*<sup>1</sup> and *Ka*<sup>2</sup> in subcritical condition can be calculated by revised HKF model with the reported parameters [50]. We performed the alanine

condition but also acid (5 mmol dm�<sup>3</sup> of H2SO4 solution) and alkali (20 mmol dm�<sup>3</sup> of NaOH solution) conditions to know the intrinsic first-order rate constant of degradation for all kinds of alanine species (*k*ala+ for cationic, *k*ala� for zwitterion and *k*ala� for anion). **Figure 4** shows the alanine species distribution in the reaction

Here, the dissociation constant of H2SO4 was correlated by the equation proposed by Oscarson et al. [51]. For NaOH, the dissociation constant was calculated by

In the alkali condition (**Figure 4C**), only the anionic alanine (Ala�) is present. At the neutral condition (**Figure 4B**), the composition of zwitterion alanine (Ala�) is major. With increasing temperature, the composition of anionic alanine (Ala�) is drastically increased and the composition of anionic alanine (Ala�) is higher than that of zwitterion alanine (Ala�). In the acidic condition (**Figure 4A**), the compo-

) is major species and its composition becomes higher

) and zwitterion alanine (Ala�), and *Ka*<sup>2</sup> is the dissociation constant

, and reaction parameter. It is expected

(6)

] is the

) degradation, *k*ala� is the rate

Ala� (7)

amino acid with substituent parameter, *σ*\*

*DOI: http://dx.doi.org/10.5772/intechopen.89793*

range of pH condition [49] was considered:

*3.4.1 pH dependence of rate constant of alanine degradation*

rate constant of 1st order overall cationic alanine (Ala<sup>+</sup>

appropriate as the standard.

cationic (Ala<sup>+</sup>

equation:

conditions.

**97**

sition of cationic alanine (Ala<sup>+</sup>

with increasing temperatures.

### **3.2 Carbohydrate conversion into precursors of liquid fuels**

Our research group has studied protein and carbohydrate conversion in hydrothermal water (subcritical water) condition with some catalysts.

It has been revealed that hydrolysis of cellulose, which is main component of carbohydrate in biomass, is rapid in sub and supercritical water without additive [34, 35]. The hydrolysis product of cellulose, glucose, is converted into furan ring compounds [8–13] and further small molecules (including hydrogen gas) in hydrothermal and supercritical water in the presence and absence of catalyst/additive [34, 36–43].

### **3.3 Protein conversion into small fragments**

Protein-rich biomass, defatted soybean meal, was converted into oligopeptides with the advanced supercritical technology (catalytic hydrothermal conversion) [14]. Protein is easily denatured to aggregate into brownish compound, which is hard to be hydrolyzed, with heat. To alter the reactivity of peptide under the heat, rapid heating technique is helpful [14, 44]. In addition, denaturation agent is also useful to improve and accelerate hydrolysis rate of protein into small fragments [14]. The effectiveness of the denaturation agent is known as Hofmeister series, which tells that alkaline earth metal ion such as Ca2+ and Mg2+ is a strong denaturation agent [45, 46]. Further, pH of the protein solution is important factor for keeping solubility in water, and alkali condition is favor for soy protein solubilization in water [47]. In our previous study, the multiple techniques (microwave technique for rapid heating, Mg2+ addition for Hofmeister series and alkali condition) for keeping reactivity and solubility of soy protein were employed for improving the yield of oligomer (over 70% of oligopeptide yield based on the soy protein in the defatted soy bean meal) from defatted soy bean meal at 190°C under saturated pressure of water for 1 h [14]. The oligopeptide obtained from the advanced supercritical technology for protein conversion (rapid heating with high Hofmeister additive in subcritical water) shows high foaming ability, antioxidant activity, and high inhibitory activity on angiotensin I converting enzyme (ACE) [14]. We also confirmed that the oligopeptide can be converted into free amino acid with over 80% yield at an advanced supercritical fluid technology (patent application and data are not shown), and it will be reported in near future elsewhere.

### **3.4 Alanine degradation in subcritical water**

In the concept of the total protein-rich biomass conversion into liquid fuel with advanced supercritical fluid technology (new "iso-conversion" for total protein-rich biomass), free amino acid produced through hydrolysis of protein and oligopeptides is further degraded into small molecular weight components such as organic acids and aldehydes (**Figure 1**). To know the optimum condition of amino acid degradation in subcritical water at a wide range of reaction temperature, alanine is selected

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst… DOI: http://dx.doi.org/10.5772/intechopen.89793*

as a standard amino acid. In a literature [48], it was found that the rate of thermal decomposition of amino acids were correlated with Taft rule, which allows the prediction of the rate of different amino acid based on a reaction rate of standard amino acid with substituent parameter, *σ*\* , and reaction parameter. It is expected that the same relation of degradation rate of amino acid in subcritical water can be found (it will be revealed in near future by our research group) and alanine is appropriate as the standard.

### *3.4.1 pH dependence of rate constant of alanine degradation*

Here, pH dependence of the reaction rate of alanine was investigated because an appropriate additive for improvement of alanine conversion will be looked for. For the objective, an overall rate constant for 1st order alanine degradation at a wide range of pH condition [49] was considered:

$$k\_{\rm Ala} = \frac{k\_{\rm Ala^{+}}[\rm H^{+}]^{2} + k\_{\rm Ala^{+}}Ka\_{1}[\rm H^{+}] + k\_{\rm Ala^{-}} \cdot Ka\_{1}Ka\_{2}}{[\rm H^{+}]^{2} + Ka\_{1}[\rm H^{+}] + Ka\_{1}Ka\_{2}} \tag{6}$$

where *k*ala is the rate constant of 1st order overall alanine degradation, *k*ala+ is the rate constant of 1st order overall cationic alanine (Ala<sup>+</sup> ) degradation, *k*ala� is the rate constant of 1st order overall zwitterion alanine (Ala�) degradation, and *k*ala� is the rate constant of 1st order overall anionic alanine (Ala�) degradation. [H<sup>+</sup> ] is the concentration of proton in the system. *Ka*<sup>1</sup> is the dissociation constant between cationic (Ala<sup>+</sup> ) and zwitterion alanine (Ala�), and *Ka*<sup>2</sup> is the dissociation constant between zwitterion (Ala�) and anionic alanine (Ala�), as shown in the below equation:

$$\mathbf{A} \mathbf{l} \mathbf{a}^+ \overset{\text{Ka}\_1}{\leftrightarrow} \mathbf{A} \mathbf{l} \mathbf{a}^\pm \overset{\text{Ka}\_2}{\leftrightarrow} \mathbf{A} \mathbf{l} \mathbf{a}^- \tag{7}$$

In the literature [49], the rate of decarboxylation (which is main reaction at sub and supercritical water over 573 K [15]) was measured with monitoring CO2 formation by in situ FTIR. Here, in Eq. (6), it is considered that pH dependence of the 1st order rate constant of alanine evaluated from the rate of decarboxylation was almost the same as the rate of overall alanine degradation (*k*ala). The dissociation constant of *Ka*<sup>1</sup> and *Ka*<sup>2</sup> in subcritical condition can be calculated by revised HKF model with the reported parameters [50]. We performed the alanine degradation in subcritical water (260�360°C at 20 MPa) not only neutral condition but also acid (5 mmol dm�<sup>3</sup> of H2SO4 solution) and alkali (20 mmol dm�<sup>3</sup> of NaOH solution) conditions to know the intrinsic first-order rate constant of degradation for all kinds of alanine species (*k*ala+ for cationic, *k*ala� for zwitterion and *k*ala� for anion). **Figure 4** shows the alanine species distribution in the reaction conditions.

Here, the dissociation constant of H2SO4 was correlated by the equation proposed by Oscarson et al. [51]. For NaOH, the dissociation constant was calculated by the revised HKF (Helgeson-Kirkham-Flowers) model [52].

In the alkali condition (**Figure 4C**), only the anionic alanine (Ala�) is present. At the neutral condition (**Figure 4B**), the composition of zwitterion alanine (Ala�) is major. With increasing temperature, the composition of anionic alanine (Ala�) is drastically increased and the composition of anionic alanine (Ala�) is higher than that of zwitterion alanine (Ala�). In the acidic condition (**Figure 4A**), the composition of cationic alanine (Ala<sup>+</sup> ) is major species and its composition becomes higher with increasing temperatures.

Thus, the whole biomass including lipid, protein, and carbohydrate should be treated by sub and supercritical water technologies in the presence of an appropriate catalyst (so it should be advanced supercritical technology). Here, we focus on effective conversion of carbohydrates and protein into small fragments, and low temperature catalytic hydrothermal cracking (LT-CHTC) must be designed with the concept of inhibition of Maillard reaction, which is undesired reaction because

complex nitrogen-containing components are produced and these will be repolymerized into non-decomposable materials like char or coke.

Our research group has studied protein and carbohydrate conversion in hydro-

It has been revealed that hydrolysis of cellulose, which is main component of carbohydrate in biomass, is rapid in sub and supercritical water without additive [34, 35]. The hydrolysis product of cellulose, glucose, is converted into furan ring compounds [8–13] and further small molecules (including hydrogen gas) in hydrothermal and supercritical water in the presence and absence of catalyst/additive

Protein-rich biomass, defatted soybean meal, was converted into oligopeptides with the advanced supercritical technology (catalytic hydrothermal conversion) [14]. Protein is easily denatured to aggregate into brownish compound, which is hard to be hydrolyzed, with heat. To alter the reactivity of peptide under the heat, rapid heating technique is helpful [14, 44]. In addition, denaturation agent is also useful to improve and accelerate hydrolysis rate of protein into small fragments [14]. The effectiveness of the denaturation agent is known as Hofmeister series, which tells that alkaline earth metal ion such as Ca2+ and Mg2+ is a strong denaturation agent [45, 46]. Further, pH of the protein solution is important factor for keeping solubility in water, and alkali condition is favor for soy protein solubilization in water [47]. In our previous study, the multiple techniques (microwave technique for rapid heating, Mg2+ addition for Hofmeister series and alkali condition) for keeping reactivity and solubility of soy protein were employed for improving the yield of oligomer (over 70% of oligopeptide yield based on the soy protein in the defatted soy bean meal) from defatted soy bean meal at 190°C under saturated pressure of water for 1 h [14]. The oligopeptide obtained from the advanced supercritical technology for protein conversion (rapid heating with high Hofmeister additive in subcritical water) shows high foaming ability, antioxidant activity, and high inhibitory activity on angiotensin I converting enzyme (ACE) [14]. We also confirmed that the oligopeptide can be converted into free amino acid with over 80% yield at an advanced supercritical fluid technology (patent application and data are not shown), and it will be reported in near future elsewhere.

In the concept of the total protein-rich biomass conversion into liquid fuel with advanced supercritical fluid technology (new "iso-conversion" for total protein-rich biomass), free amino acid produced through hydrolysis of protein and oligopeptides is further degraded into small molecular weight components such as organic acids and aldehydes (**Figure 1**). To know the optimum condition of amino acid degradation in subcritical water at a wide range of reaction temperature, alanine is selected

**3.2 Carbohydrate conversion into precursors of liquid fuels**

thermal water (subcritical water) condition with some catalysts.

**3.3 Protein conversion into small fragments**

*Advanced Supercritical Fluids Technologies*

**3.4 Alanine degradation in subcritical water**

[34, 36–43].

**96**

**Figure 4.**

*Alanine species distribution in subcritical water at (A) acid, (B) neutral, and (C) alkali conditions at 20 MPa.*

In this part, pH dependence of the overall rate constant of alanine degradation at a wide range of reaction temperature and pH condition is explained. To correlate the rate constant, a correlation was performed:


$$-\frac{d[\text{Ala}]}{dt} = k\_{\text{Ala}}[\text{Ala}]\_0 = k\_{\text{Ala}^-}[\text{Ala}^-] + k\_{\text{Ala}^\pm}[\text{Ala}^\pm] \tag{8}$$

By using *k*ala� measured in alkali condition and the concentration of each species (Ala� and Ala�) calculated as shown in **Figure 4B**, *k*Ala� was obtained by the below equation.

$$k\_{\rm Ala^{\pm}} = \frac{k\_{\rm Ala}[\rm Ala]\_0 - k\_{\rm Ala^{-}}[\rm Ala^{-}]}{[\rm Ala^{\pm}]} \tag{9}$$

The effluent was cooled down in the cooler and collected. Reaction time was varied in the range of 60–300 s by changing flow rates of the reaction fluids and calculated based on the water density at reaction temperature (260–360°C) and

*Schematic diagram of continuous-flow system (1: alanine solution, 2: water, NaOH, or H2SO4, 3: pump, 4: relief valve, 5: valve, 6: preheating unit, 7: reaction unit, 8: cooling unit, 9: filter, 10: back-pressure regulator).*

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst…*

Alanine in the outlet solution was measured by HPLC with an ODS column (Shimpack VP-ODS, Shimazu) and PDA detector (L-7455, Hitachi) at wavelength of 200 nm. The column oven was 60°C. The carrier were sodium 50 mM of sodium phosphoric acid with 7.2 mM of hexane sulphonate set to pH 2.5 (solution A) and HPLC grade acetonitrile (solution B) with flow ratio of 96:4 (A:B) and the flow rate was 1.0 mL/min. Conversion of alanine was calculated from the concentration of alanine before and after the reaction and the rate constants (*k*Ala, *k*Ala and *k*Ala+) by already mentioned protocol: (i) *k*Ala was measured at alkali condition, (ii) *k*Ala was correlated from *k*Ala measured in neutral condition, and (iii) *k*Ala+ was obtained at acidic condition by correlation with the measured *k*Ala measured and *k*Ala.

**Figure 7** shows the Arrhenius plots of the intrinsic rate constant of each alanine species degradation in subcritical water at 20 MPa. The data for *k*Ala+ and *k*Ala were

*3.4.3 Rate constant of alanine at wide temperature and pH conditions*

pressure (20 MPa).

**Figure 5.**

**Figure 6.**

**99**

*Photograph of continuous flow reactor.*

*DOI: http://dx.doi.org/10.5772/intechopen.89793*

where [Ala]0 was the initial concentration of alanine. All brackets, [X], are correspondent to concentration of species X in mole m�<sup>3</sup> .

iii. For *k*Ala+ was obtained as the same manner of *k*Ala� mentioned above.

### *3.4.2 Experimental for alanine conversion in subcritical water*

**Figures 5** and **6** show a photograph and schematic diagram of a flow apparatus that used for subcritical water decomposition of alanine, respectively. The reactor consisted of two pumps, preheater, reactor, cooler, and back-pressure regulator. The preheater was made of SUS316 stainless steel (length 40 m., id: 1 mm, od: 1/16 inch) and placed in a GC oven (HP6890, Agilent Technologies). The reactor was also made of SUS316 stainless steel (length 100 m, id: 1 mm, od: 1/16 inch, volume of reactor: 81.2 cm3 ) placed in another GC oven (6890N, Agilent Technologies). The shell tube heat exchanger for cooling made of SUS316 stainless steel (length 30 m., id: 1 mm, od: 1/16 inch) was located in a chiller (LE-600, Advantec). The pressure was controlled at 20 MPa by the back-pressure regulator (TESCOM). The sample solution and water were supplied by pumps (PU-2100S, Jasco and NP-KX-500, Nihon Seimitsu Co., Ltd.). For rapid heating, water was fed to mixing part with flow ratio of 1:9 or 1:3 (sample solution:water) and preheated. The preheater temperature was decided by consideration of enthalpy balance to reach the mixing temperature to the targeted temperature, which was in the range of 260–360°C.

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst… DOI: http://dx.doi.org/10.5772/intechopen.89793*

**Figure 5.** *Photograph of continuous flow reactor.*

**Figure 6.**

In this part, pH dependence of the overall rate constant of alanine degradation at a wide range of reaction temperature and pH condition is explained. To correlate

*Alanine species distribution in subcritical water at (A) acid, (B) neutral, and (C) alkali conditions at*

i. At alkali condition, *k*ala� was measured and it was the same as the overall rate

ii. The apparent rate constant was measured at neutral condition as *k*ala, which includes both contribution of anionic and zwitterion alanine as below.

By using *k*ala� measured in alkali condition and the concentration of each species (Ala� and Ala�) calculated as shown in **Figure 4B**, *k*Ala� was obtained by the below

*<sup>k</sup>*Ala� <sup>¼</sup> *<sup>k</sup>*Ala½ � Ala <sup>0</sup> � *<sup>k</sup>*Ala� Ala� ½ �

where [Ala]0 was the initial concentration of alanine. All brackets, [X], are

iii. For *k*Ala+ was obtained as the same manner of *k*Ala� mentioned above.

**Figures 5** and **6** show a photograph and schematic diagram of a flow apparatus that used for subcritical water decomposition of alanine, respectively. The reactor consisted of two pumps, preheater, reactor, cooler, and back-pressure regulator. The preheater was made of SUS316 stainless steel (length 40 m., id: 1 mm, od: 1/16 inch) and placed in a GC oven (HP6890, Agilent Technologies). The reactor was also made of SUS316 stainless steel (length 100 m, id: 1 mm, od: 1/16 inch, volume

shell tube heat exchanger for cooling made of SUS316 stainless steel (length 30 m., id: 1 mm, od: 1/16 inch) was located in a chiller (LE-600, Advantec). The pressure was controlled at 20 MPa by the back-pressure regulator (TESCOM). The sample solution and water were supplied by pumps (PU-2100S, Jasco and NP-KX-500, Nihon Seimitsu Co., Ltd.). For rapid heating, water was fed to mixing part with flow ratio of 1:9 or 1:3 (sample solution:water) and preheated. The preheater temperature was decided by consideration of enthalpy balance to reach the mixing temperature to the targeted temperature, which was in the range of 260–360°C.

) placed in another GC oven (6890N, Agilent Technologies). The

*dt* <sup>¼</sup> *<sup>k</sup>*Ala½ � Ala <sup>0</sup> <sup>¼</sup> *<sup>k</sup>*Ala� Ala� ½ �þ *<sup>k</sup>*Ala� Ala� (8)

Ala� (9)

.

the rate constant, a correlation was performed:

*Advanced Supercritical Fluids Technologies*

� *<sup>d</sup>*½ � Ala

correspondent to concentration of species X in mole m�<sup>3</sup>

*3.4.2 Experimental for alanine conversion in subcritical water*

constant, *k*ala.

equation.

**Figure 4.**

*20 MPa.*

of reactor: 81.2 cm3

**98**

*Schematic diagram of continuous-flow system (1: alanine solution, 2: water, NaOH, or H2SO4, 3: pump, 4: relief valve, 5: valve, 6: preheating unit, 7: reaction unit, 8: cooling unit, 9: filter, 10: back-pressure regulator).*

The effluent was cooled down in the cooler and collected. Reaction time was varied in the range of 60–300 s by changing flow rates of the reaction fluids and calculated based on the water density at reaction temperature (260–360°C) and pressure (20 MPa).

Alanine in the outlet solution was measured by HPLC with an ODS column (Shimpack VP-ODS, Shimazu) and PDA detector (L-7455, Hitachi) at wavelength of 200 nm. The column oven was 60°C. The carrier were sodium 50 mM of sodium phosphoric acid with 7.2 mM of hexane sulphonate set to pH 2.5 (solution A) and HPLC grade acetonitrile (solution B) with flow ratio of 96:4 (A:B) and the flow rate was 1.0 mL/min.

Conversion of alanine was calculated from the concentration of alanine before and after the reaction and the rate constants (*k*Ala, *k*Ala and *k*Ala+) by already mentioned protocol: (i) *k*Ala was measured at alkali condition, (ii) *k*Ala was correlated from *k*Ala measured in neutral condition, and (iii) *k*Ala+ was obtained at acidic condition by correlation with the measured *k*Ala measured and *k*Ala.

### *3.4.3 Rate constant of alanine at wide temperature and pH conditions*

**Figure 7** shows the Arrhenius plots of the intrinsic rate constant of each alanine species degradation in subcritical water at 20 MPa. The data for *k*Ala+ and *k*Ala were largely scattered, and the correctness of the data must further be investigated. Here, we analyzed the trend of the rate constant for each species as the order and the temperature dependence (namely activation energy) should be correct. The activation energy of the rate constant of zwitterion, *k*Ala, was the highest among all the rate constants shown in **Figure 7**. Thus the reactivity of zwitterion alanine becomes higher than the others. **Figure 8** shows the simulation of pH dependence of the overall rate constant of alanine degradation in subcritical water at 20 MPa and various temperatures.

At the range of reaction temperatures from 340 (**Figure 8A**) to 280°C (**Figure 8D**), the simulation of pH dependence of the overall rate constant of alanine degradation, *k*Ala, (drawn with the bold line) well explained the experimental data (plots in the figures). As shown in **Figure 8**, over 300°C, zwitterion alanine is the most active species and surprisingly an acid or alkali catalyst is not useful for enhancing degradation rate. Below 280°C, alanine degradation should be enhanced by an acid or alkali catalyst. But lower temperature, the reaction rate is so low and decomposition of alanine requires long time. For the pretreatment of protein-rich biomass to produce the precursor of hydrocarbons such as organic acids, the enhancement of the reaction rate must be necessary. To seek an appropriate additive, some types of solid acid additives were screened.

### **3.5 Amino acid deamination with solid acid additives in hydrothermal water**

### *3.5.1 Previous studies on solid catalytic reaction of amino acid in hydrothermal (subcritical) water*

Since amino acid is monomer unit of protein and key compound in life, amino acid reaction in hydrothermal system is highly motivated to know the origin of life because protein is basic molecule for life and polypeptide formation, namely protein synthesis, should be the first step. Hydrothermal vent under deep sea is seemed to be one of the spaces where is the original place of life. To form protein, survivability of amino acid at such severe condition (hydrothermal water is partially higher than 200°C) is crucial for the origin of life, and the stability of amino acid at hydrothermal water condition is important. In the origin of life point of view, amino acid touches on the surface of inorganic materials at the deep sea near the hydrothermal vent and the effect of the inorganics on the stability of amino acid has been studied. It was reported that iron compounds enhance dimerization to form alanyl-alanine and diketopiperazine at hydrothermal condition [53]. Glycine and alanine are reacted at 100°C for 35 days with metal ferrites such as NiFeO4 to obtain

*pH dependence of the overall rate constant of alanine degradation, kAla, at 20 MPa: (A) 340°C, (B) 320°C,*

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst…*

As amino acids to be key compounds in biomass refinery, there have also been some reports concerning conversion of amino acids into chemicals with solid additives. It was reported that Pt/TiO2 promotes the reductive deamination of glutamic acid derivatives to form organic acids at 225°C for 24 h [54]. By the same research group of [54], decarbonylation of amino acids was attempted by Ru-based catalyst [55]. Reductive (hydrogenation) decarbonylation of valine to isobutylamine was developed in the presence of Ru-catalyst with isobutylamine yield of 87% in hydro-

*3.5.2 Sulfonyl solid acid catalytic reactions of alanine in hydrothermal (subcritical)*

As described above, there have been several papers concerning some solid catalytic reactions of amino acids in hydrothermal water; however, solid acid or base reactions on hydrothermal (subcritical water) reaction of amino acids was not

Based on the analysis of the pH dependence of degradation of alanine, solid acid or base catalyst must be effective. The batch experiments were performed with microwave hydrothermal small reactor (**Figure 9**). The detail of the apparatus and procedures are found elsewhere [13]. Here, conversion of alanine was investigated in hydrothermal water with and without sulfonyl solid catalyst at 150°C for 1– 60 min. Basically, 5.0 g of 110 mmol dm<sup>3</sup> of amino acid solution was loaded in a glass reactor with 0.5 g of solid sulfonyl additive such as sulfonyl carbon (detail is not shown because of the patent application) and cationic (having sulfonyl group) ionic exchange (Amberlyst 45) resin. As shown in **Figure 10**, alanine is decomposed

30% of yield of dimerization products [53].

*(C) 300°C, (D) 280°C, (E) 150°C, and (F) 100°C.*

*DOI: http://dx.doi.org/10.5772/intechopen.89793*

thermal water at 150°C for 2 h.

*water*

**Figure 8**

investigated so far.

**101**

**Figure 7.** *Intrinsic rate constant of each alanine species at 20 MPa.*

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst… DOI: http://dx.doi.org/10.5772/intechopen.89793*

**Figure 8**

largely scattered, and the correctness of the data must further be investigated. Here, we analyzed the trend of the rate constant for each species as the order and the temperature dependence (namely activation energy) should be correct. The activation energy of the rate constant of zwitterion, *k*Ala, was the highest among all the rate constants shown in **Figure 7**. Thus the reactivity of zwitterion alanine becomes higher than the others. **Figure 8** shows the simulation of pH dependence of the overall rate constant of alanine degradation in subcritical water at 20 MPa and

At the range of reaction temperatures from 340 (**Figure 8A**) to 280°C (**Figure 8D**), the simulation of pH dependence of the overall rate constant of alanine degradation, *k*Ala, (drawn with the bold line) well explained the experimental data (plots in the figures). As shown in **Figure 8**, over 300°C, zwitterion alanine is the most active species and surprisingly an acid or alkali catalyst is not useful for enhancing degradation rate. Below 280°C, alanine degradation should be enhanced by an acid or alkali catalyst. But lower temperature, the reaction rate is so low and decomposition of alanine requires long time. For the pretreatment of protein-rich biomass to produce the precursor of hydrocarbons such as organic acids, the enhancement of the reaction rate must be necessary. To seek an appro-

**3.5 Amino acid deamination with solid acid additives in hydrothermal water**

Since amino acid is monomer unit of protein and key compound in life, amino acid reaction in hydrothermal system is highly motivated to know the origin of life because protein is basic molecule for life and polypeptide formation, namely protein synthesis, should be the first step. Hydrothermal vent under deep sea is seemed to be one of the spaces where is the original place of life. To form protein, survivability of amino acid at such severe condition (hydrothermal water is partially higher than 200°C) is crucial for the origin of life, and the stability of amino acid at hydrothermal water condition is important. In the origin of life point of view, amino acid touches on the surface of inorganic materials at the deep sea near the hydrothermal vent and the effect of the inorganics on the stability of amino acid has

*3.5.1 Previous studies on solid catalytic reaction of amino acid in hydrothermal*

priate additive, some types of solid acid additives were screened.

various temperatures.

*Advanced Supercritical Fluids Technologies*

*(subcritical) water*

**Figure 7.**

**100**

*Intrinsic rate constant of each alanine species at 20 MPa.*

*pH dependence of the overall rate constant of alanine degradation, kAla, at 20 MPa: (A) 340°C, (B) 320°C, (C) 300°C, (D) 280°C, (E) 150°C, and (F) 100°C.*

been studied. It was reported that iron compounds enhance dimerization to form alanyl-alanine and diketopiperazine at hydrothermal condition [53]. Glycine and alanine are reacted at 100°C for 35 days with metal ferrites such as NiFeO4 to obtain 30% of yield of dimerization products [53].

As amino acids to be key compounds in biomass refinery, there have also been some reports concerning conversion of amino acids into chemicals with solid additives. It was reported that Pt/TiO2 promotes the reductive deamination of glutamic acid derivatives to form organic acids at 225°C for 24 h [54]. By the same research group of [54], decarbonylation of amino acids was attempted by Ru-based catalyst [55]. Reductive (hydrogenation) decarbonylation of valine to isobutylamine was developed in the presence of Ru-catalyst with isobutylamine yield of 87% in hydrothermal water at 150°C for 2 h.

### *3.5.2 Sulfonyl solid acid catalytic reactions of alanine in hydrothermal (subcritical) water*

As described above, there have been several papers concerning some solid catalytic reactions of amino acids in hydrothermal water; however, solid acid or base reactions on hydrothermal (subcritical water) reaction of amino acids was not investigated so far.

Based on the analysis of the pH dependence of degradation of alanine, solid acid or base catalyst must be effective. The batch experiments were performed with microwave hydrothermal small reactor (**Figure 9**). The detail of the apparatus and procedures are found elsewhere [13]. Here, conversion of alanine was investigated in hydrothermal water with and without sulfonyl solid catalyst at 150°C for 1– 60 min. Basically, 5.0 g of 110 mmol dm<sup>3</sup> of amino acid solution was loaded in a glass reactor with 0.5 g of solid sulfonyl additive such as sulfonyl carbon (detail is not shown because of the patent application) and cationic (having sulfonyl group) ionic exchange (Amberlyst 45) resin. As shown in **Figure 10**, alanine is decomposed

On Amberlyst 45, 2.95 eq kg<sup>1</sup> of sulfonyl group is bearded, while 1.90 eq kg<sup>1</sup> of sulfonyl group is attached on the surface of sulfonyl carbon. The catalytic activity for deamination of alanine is probably related to oxygenated function group (OFGs) such as hydroxyl and carboxyl because the sulfonyl carbon has 2.40 mmol g<sup>1</sup> of OFGs (Amberlyst 45 has no OFG except for sulfonyl group). More detail study for amino acid deamination on the sulfonyl carbon catalyst is now on-going

*Yield of deamination product, NH3, of alanine in hydrothermal water with and without sulfonyl solid additive*

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst…*

*3.5.3 Sulfonyl carbon reactions of okara in hydrothermal (subcritical) water*

acid condition (not only by sulfonyl group but also by OFGs) as predicted (**Figure 8**). The LT-CHTC, mainly for deamination, is favored as liquid fuel pretreatment as proposed in **Figure 1**. Here, to know the usefulness of the reaction for a real biomass, "okara," which is a residue of soy bean cake ("Tofu") production, was deaminated in the presence of the sulfonyl carbon. The elemental composition of okara used in this study is C:H:N:S:O (wt%) = 47.2:7.29:5.85:0.43:39.2 (the weight percentage of oxygen atom was subtraction of total weight % of C, H, N. and S from 100 and it should include ash, which will be analyzed by TGA). As a result, a large portion of amino group in okara was detached as ammonia, NH3, (detail is not shown at this moment because of the reason for patent application). Thus, the sulfonyl carbon catalytically assisted NH2-group abstraction as NH3. It means that Maillard reaction during okara conversion was less happened and much organic acids were formed. For this study, now the detail is being studied and it will be also

Through the experiments, it was revealed that sulfonyl carbon was active for deamination of amino acids. It must be resulted in the activation of alanine at strong

**4. Up-conversion of carbohydrate in ionic liquid and supercritical fluids**

Cellulose and chitin are abundant biomass and these up-conversion (valorization) into useful compounds are aggressively being investigated. Our research group has studied cellulose and chitin conversion with advanced supercritical technologies. As one of advanced supercritical technologies, ionic liquid utilization

together with supercritical fluid technologies (including subcritical and

and it will be reported in near future.

*at 150°C for 1 h (under saturated pressure of water, 0.48 MPa).*

*DOI: http://dx.doi.org/10.5772/intechopen.89793*

**Figure 11.**

reported in near future.

**mixtures**

**103**

**Figure 9.** *Microwave hydrothermal small reactor.*

### **Figure 10.**

*Main pathways of alanine degradation in hydrothermal water.*

through decarboxylation, deamination, and dimerization. To know which pathway is developed, decarboxylation product, CO2, was detected by CO2 meter and fingerprint of deamination, NH3, was analyzed by an ion chromatography.

**Figure 11** shows the results. Amberlyst 45 was inert for deamination of amino acids at hydrothermal water at 150°C. In the presence of the sulfonyl carbon, deamination of alanine was progressed. Not shown here, the other pathways of alanine degradation were not found for all the cases in these experiments.

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst… DOI: http://dx.doi.org/10.5772/intechopen.89793*

**Figure 11.**

*Yield of deamination product, NH3, of alanine in hydrothermal water with and without sulfonyl solid additive at 150°C for 1 h (under saturated pressure of water, 0.48 MPa).*

On Amberlyst 45, 2.95 eq kg<sup>1</sup> of sulfonyl group is bearded, while 1.90 eq kg<sup>1</sup> of sulfonyl group is attached on the surface of sulfonyl carbon. The catalytic activity for deamination of alanine is probably related to oxygenated function group (OFGs) such as hydroxyl and carboxyl because the sulfonyl carbon has 2.40 mmol g<sup>1</sup> of OFGs (Amberlyst 45 has no OFG except for sulfonyl group). More detail study for amino acid deamination on the sulfonyl carbon catalyst is now on-going and it will be reported in near future.

### *3.5.3 Sulfonyl carbon reactions of okara in hydrothermal (subcritical) water*

Through the experiments, it was revealed that sulfonyl carbon was active for deamination of amino acids. It must be resulted in the activation of alanine at strong acid condition (not only by sulfonyl group but also by OFGs) as predicted (**Figure 8**). The LT-CHTC, mainly for deamination, is favored as liquid fuel pretreatment as proposed in **Figure 1**. Here, to know the usefulness of the reaction for a real biomass, "okara," which is a residue of soy bean cake ("Tofu") production, was deaminated in the presence of the sulfonyl carbon. The elemental composition of okara used in this study is C:H:N:S:O (wt%) = 47.2:7.29:5.85:0.43:39.2 (the weight percentage of oxygen atom was subtraction of total weight % of C, H, N. and S from 100 and it should include ash, which will be analyzed by TGA). As a result, a large portion of amino group in okara was detached as ammonia, NH3, (detail is not shown at this moment because of the reason for patent application). Thus, the sulfonyl carbon catalytically assisted NH2-group abstraction as NH3. It means that Maillard reaction during okara conversion was less happened and much organic acids were formed. For this study, now the detail is being studied and it will be also reported in near future.

## **4. Up-conversion of carbohydrate in ionic liquid and supercritical fluids mixtures**

Cellulose and chitin are abundant biomass and these up-conversion (valorization) into useful compounds are aggressively being investigated. Our research group has studied cellulose and chitin conversion with advanced supercritical technologies. As one of advanced supercritical technologies, ionic liquid utilization together with supercritical fluid technologies (including subcritical and

through decarboxylation, deamination, and dimerization. To know which pathway is developed, decarboxylation product, CO2, was detected by CO2 meter and fingerprint of deamination, NH3, was analyzed by an ion chromatography.

**Figure 11** shows the results. Amberlyst 45 was inert for deamination of amino

acids at hydrothermal water at 150°C. In the presence of the sulfonyl carbon, deamination of alanine was progressed. Not shown here, the other pathways of alanine degradation were not found for all the cases in these experiments.

*Main pathways of alanine degradation in hydrothermal water.*

**Figure 9.**

**Figure 10.**

**102**

*Microwave hydrothermal small reactor.*

*Advanced Supercritical Fluids Technologies*

hydrothermal water) has been studied. Here, two biomass conversion process are focused on: furan ring compound formation from cellulose and hydrogenation.

hydrogenation), the synergetic effect of hydrogen and supercritical CO2 coexis-

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst…*

Hydrogen solubility was experimental measured and a simple correlation for hydrogen solubility in ionic liquids in the presence of CO2 was developed from available binary/ternary data. The correlation could provide reliable estimation of hydrogen solubility enhancement by CO2 for six ionic liquids at 313–453 K [56]. In the study [56], a definition for enhancement ratio (ER) based on molality and applied it to available hydrogen-CO2-ionic liquid systems were proposed. It was found that the ER was convenient for examining the trends of solubility change of hydrogen in an ionic liquid with CO2 concentration or temperature. Also in the study [56], hydrogen solubility in the presence of CO2 was estimated for biomass

In this chapter, the concept of liquid fuels from biomass in advanced supercritical fluid is firstly explained. To know the optimum condition for various kind of biomass, dimensionless severity number should be useful and the application for lignin recovery in hydrothermal and subcritical water was shown. Then, as the application of one of the advanced supercritical fluid technologies, hydrothermal water with catalyst process for carbohydrate and protein performed by our research group was briefly reviewed. For total biomass utilization as liquid fuel production, protein fragmentation is a key process and some of experimental research results were shown. Particularly, alanine conversion with and without additive was deeply considered. To inhibit Maillard reactions, the deamination of amino acid at low temperature was quite important, and it was found that a sulfonyl carbon was quite active for deamination of alanine at 150°C in hydrothermal water. Cellulose conversion in ionic liquid with supercritical fluid (hydrothermal water) to furan ring compound (HMF) was introduced. For hydrogenation of biomass, hydrogen solubility was controlled in the presence of supercritical carbon dioxide and it shows that ionic liquid with supercritical CO2 is favored hydrogenolysis and hydrogena-

tence must be revealed.

*DOI: http://dx.doi.org/10.5772/intechopen.89793*

soluble ionic liquids.

**Conflict of interest**

**Nomenclature**

[H<sup>+</sup>

**105**

tion reaction field for up-conversion of biomass.

The authors declare no conflict of interest.

[Ala] concentration of alanine, mol m<sup>3</sup> [Ala]0 initial concentration of alanine, mol m<sup>3</sup> [Ala] concentration of anionic alanine, mol m<sup>3</sup> [Ala] concentration of zwitterion alanine, mol m<sup>3</sup>

] concentration of proton, mol m<sup>3</sup> *K*a1 dissociation constant between cationic and

zwitterion alanine *K*a2 dissociation constant between zwitterion and anionic alanine

*E*<sup>a</sup> Activation energy, kJ mol<sup>1</sup>

*k* rate constant, min<sup>1</sup>

**5. Conclusions**

### **4.1 HMF formation from cellulose**

HMF is a platform compound that can be derived from cellulose in coming biomass society [23, 24]. It is known that the formation of HMF from cellulose is three-step reaction: (1) hydrolysis of cellulose into glucose, (2) glucose isomerization to fructose, and (3) fructose dehydration to HMF, as shown in **Figure 12**. Step 1 and step 3 are acid-catalyzed reactions, and step 2 is a base-catalyzed reaction. To increase the yield of HMF, optimization of each reaction step is necessary.

For step 1, selective glucose production by cellulose hydrolysis with sequential water addition in the presence of solid acid catalysts in 1-butyl-3-methyl imidazolium chloride ([bmIm][Cl]) under microwave irradiation was investigated at 120°C, where water is hydrothermal condition [23]. It revealed that the large amount of water added in the initial stage was inhibited to form homogeneous phase among three components (cellulose-water-[bmIm][Cl]) and some amounts of cellulose was precipitated. The precipitation of cellulose was resulted in the low yield of glucose because hydrolysis of cellulose was stopped. To keep the cellulose dissolution and improve hydrolysis of cellulose into glucose, sequential water addition where water is added in steps as the reaction proceeds was investigated. Glucose yields in the presence of Amberlyst-15 in [bmIm][Cl] were 75.0 mol% by three step of water addition [23].

Step 2 (glucose isomerization, which requires basic catalyst) was also optimized in the presence of hydrothermal water with [bmIm][Cl] at 120°C, and the effect of water on step 3 (glucose into HMF) was investigated [24]. For isomerization of glucose in the presence of 35 wt% of hydrothermal water in [bmIm][Cl], MgCO3 was the most effective base additives (23.1 mol% of fructose yield with 85.3 mol% selectivity was obtained at 120°C for 30 min). For dehydration of fructose into HMF with Amberlyst 15 (Step 3), the rate of dehydration of fructose was slightly reduced by adding water but not so large effect was confirmed. Also in the paper [24], continuous HMF production process via three-step reaction is proposed with separation of HMF from [bmIm][Cl] and water mixture. It was found that dimethyl ether was a good separation solvent and almost 100% of HMF was recovered. Finally, virtual integration of the three-step process tells us that the yield of HMF from cellulose is estimated to be 32 mol%.

## **4.2 Hydrogen solubility in ionic liquid with supercritical CO2**

The other important reaction routes from biomass into useful material are hydrogenolysis and hydrogenation of biomass [56]. Ionic liquid media has advantage of biomass conversion because solubilization of cellulose and chitin in some types of ionic liquid is high. One of drawbacks of the ionic liquid process is high viscosity. Supercritical CO2 assists reduction of viscosity of ionic liquid-cellulose mixture [26]. For the process of hydrogen addition (such as hydrogenolysis and

**Figure 12.** *HMF formation from cellulose.*

*Resource Upgrading in Advanced Supercritical Fluid (Supercritical Fluid with Catalyst… DOI: http://dx.doi.org/10.5772/intechopen.89793*

hydrogenation), the synergetic effect of hydrogen and supercritical CO2 coexistence must be revealed.

Hydrogen solubility was experimental measured and a simple correlation for hydrogen solubility in ionic liquids in the presence of CO2 was developed from available binary/ternary data. The correlation could provide reliable estimation of hydrogen solubility enhancement by CO2 for six ionic liquids at 313–453 K [56]. In the study [56], a definition for enhancement ratio (ER) based on molality and applied it to available hydrogen-CO2-ionic liquid systems were proposed. It was found that the ER was convenient for examining the trends of solubility change of hydrogen in an ionic liquid with CO2 concentration or temperature. Also in the study [56], hydrogen solubility in the presence of CO2 was estimated for biomass soluble ionic liquids.

### **5. Conclusions**

hydrothermal water) has been studied. Here, two biomass conversion process are focused on: furan ring compound formation from cellulose and hydrogenation.

HMF is a platform compound that can be derived from cellulose in coming biomass society [23, 24]. It is known that the formation of HMF from cellulose is three-step reaction: (1) hydrolysis of cellulose into glucose, (2) glucose isomerization to fructose, and (3) fructose dehydration to HMF, as shown in **Figure 12**. Step 1 and step 3 are acid-catalyzed reactions, and step 2 is a base-catalyzed reaction. To increase the yield of HMF, optimization of each reaction step is necessary.

For step 1, selective glucose production by cellulose hydrolysis with sequential

Step 2 (glucose isomerization, which requires basic catalyst) was also optimized in the presence of hydrothermal water with [bmIm][Cl] at 120°C, and the effect of water on step 3 (glucose into HMF) was investigated [24]. For isomerization of glucose in the presence of 35 wt% of hydrothermal water in [bmIm][Cl], MgCO3 was the most effective base additives (23.1 mol% of fructose yield with 85.3 mol% selectivity was obtained at 120°C for 30 min). For dehydration of fructose into HMF with Amberlyst 15 (Step 3), the rate of dehydration of fructose was slightly reduced by adding water but not so large effect was confirmed. Also in the paper [24], continuous HMF production process via three-step reaction is proposed with separation of HMF from [bmIm][Cl] and water mixture. It was found that dimethyl ether was a good separation solvent and almost 100% of HMF was recovered. Finally, virtual integration of the three-step process tells us that the yield of HMF

imidazolium chloride ([bmIm][Cl]) under microwave irradiation was investigated at 120°C, where water is hydrothermal condition [23]. It revealed that the large amount of water added in the initial stage was inhibited to form homogeneous phase among three components (cellulose-water-[bmIm][Cl]) and some amounts of cellulose was precipitated. The precipitation of cellulose was resulted in the low yield of glucose because hydrolysis of cellulose was stopped. To keep the cellulose dissolution and improve hydrolysis of cellulose into glucose, sequential water addition where water is added in steps as the reaction proceeds was investigated. Glucose yields in the presence of Amberlyst-15 in [bmIm][Cl] were 75.0 mol% by three

water addition in the presence of solid acid catalysts in 1-butyl-3-methyl

**4.1 HMF formation from cellulose**

*Advanced Supercritical Fluids Technologies*

step of water addition [23].

**Figure 12.**

**104**

*HMF formation from cellulose.*

from cellulose is estimated to be 32 mol%.

**4.2 Hydrogen solubility in ionic liquid with supercritical CO2**

The other important reaction routes from biomass into useful material are hydrogenolysis and hydrogenation of biomass [56]. Ionic liquid media has advantage of biomass conversion because solubilization of cellulose and chitin in some types of ionic liquid is high. One of drawbacks of the ionic liquid process is high viscosity. Supercritical CO2 assists reduction of viscosity of ionic liquid-cellulose mixture [26]. For the process of hydrogen addition (such as hydrogenolysis and

In this chapter, the concept of liquid fuels from biomass in advanced supercritical fluid is firstly explained. To know the optimum condition for various kind of biomass, dimensionless severity number should be useful and the application for lignin recovery in hydrothermal and subcritical water was shown. Then, as the application of one of the advanced supercritical fluid technologies, hydrothermal water with catalyst process for carbohydrate and protein performed by our research group was briefly reviewed. For total biomass utilization as liquid fuel production, protein fragmentation is a key process and some of experimental research results were shown. Particularly, alanine conversion with and without additive was deeply considered. To inhibit Maillard reactions, the deamination of amino acid at low temperature was quite important, and it was found that a sulfonyl carbon was quite active for deamination of alanine at 150°C in hydrothermal water. Cellulose conversion in ionic liquid with supercritical fluid (hydrothermal water) to furan ring compound (HMF) was introduced. For hydrogenation of biomass, hydrogen solubility was controlled in the presence of supercritical carbon dioxide and it shows that ionic liquid with supercritical CO2 is favored hydrogenolysis and hydrogenation reaction field for up-conversion of biomass.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Nomenclature**



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