**Comparison of the Performance of the Laccase Bioconversion of Sodium Lignosulfonates in Batch, Continuous and Fed Batch Reactors**

Nidal Madad, Latifa Chebil, Hugues Canteri, Céline Charbonnel and Mohamed Ghoul

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53103

### **1. Introduction**

Wood and food processes generate high quantities of by-products such as lignin, lignosulfo‐ nates and free phenols(Rodrigues et al., 2008). These compounds are natural molecules and renewable resources, but they constitute an important source of pollution. However, they can undergo several transformations and processes (hydrolysis, bioconversion and fractio‐ nation) to provide fractions with useful properties such as antioxidants, dispersing agent and plasticizer (Benavente-Garcia et al., 2000; Madad et al., 2011; Ouyang et al., 2006; Yang et al., 2008; Zhou et al., 2006). The recovery and development of these by-products are main‐ ly carried out by chemical or physical processes such as thermal decomposition (Jiang et al., 2003), liquid (Correia et al., 2007) or membrane fractionation (Bhattacharya et al., 2005; Fer‐ reira et al., 2005; Venkateswaran and Palanivelu, 2006). The chemical process is often not en‐ vironmentally friendly and may be expensive. To overcome some drawbacks of the above mentioned processes, enzyme hydrolysis or bioconversion of these raw materials is present‐ ed as a promising way (Kobayashi et al., 2001). In fact, the enzymatic processes can be con‐ ducted under mild reaction conditions and without using toxic reagents. Moreover, in some cases, they lead to a homogeneous molecular distribution of obtained products and en‐ hanced properties (Gross et al., 1998; Joo et al., 1998; Kobayashi, 1999; Kobayashi et al., 1995; Kobayashi and Uyama, 1998; Kobayashi et al., 2001).

The use of enzymes is firstly applied to the delignification and the removal of free phe‐ nols from wastewaters (Dasgupta et al., 2007; Husain, 2010; Nazari et al., 2007; Riva, 2006; Widsten and Kandelbauer, 2008). Recently, the ability of some oxidoreductases and laccas‐

es to polymerize phenols have received great attention and applied with success in the field of wood by-products (Ikeda et al., 2001; Jeon et al., 2010; Mita et al., 2003; Reihmann and Ritter, 2006).

**2. Materials and methods**

Da ± 400, and 6.2 ± 0.3, respectively.

Sodium lignosulfonates (SLS) from (Aldrich, Sweden) : 90 wt. % of SLS, 4 wt. % of reducing sugars and 6 wt% of total impurities. The average molecular weight (Mw), the number mo‐ lecular weight (Mn) and the polydispersity (Pdi) values are equal to 17800 Da ± 1500, 2900

Comparison of the Performance of the Laccase Bioconversion of Sodium Lignosulfonates in Batch, Continuous...

http://dx.doi.org/10.5772/53103

27

The activity of laccase was determined spectrophotometrically by monitoring the oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline)-6-sulfonate (ABTS) to its cation radical as substrate at 436 nm in 50 mM sodium succinate buffer at pH 4.5 and 30 °C using quartz cuvette of path length 10 mm. Enzyme activity was expressed in units (1 U = 1 μmol ABTS oxidized

Batch operations were performed in a bioreactor with a working volume of 1 L equipped with dissolved oxygen, pH and temperature sensors. The reactor was stirred vigorously at 500 rpm to solubilise SLS at 20°C and throughout the reactions. The lignosulfonates were solubilised in phosphate buffer solution at pH 4.5 and laccase was added to initiate reac‐ tions. For the analyses, samples were drawn out from the reactor at different intervals of

Fed batch reactions were carried out by progressive adding, at different time intervals (ev‐ ery 30 minutes during the first 5 hours), of enzyme alone, substrate alone or both enzyme and substrate. The total amounts of enzyme and substrate for the three fed batch operations were 10 g/L and 30 U/mL of SLS and laccase, respectively. Samples were taken at different

The continuous stirred tank reactor was similar to the one used in batch step. Lignosulfo‐ nates (32 g/L) and laccase (63 U/mL) were prepared in two flasks separately and 500 mL of each solution were added progressively at a constant flow-rate into the reactor initially filled with buffered solution (1 L). The reactor was aerated and stirred vigorously at 500 rpm. Samples were taken at different time intervals and the enzyme activity was stopped by heat‐

time intervals and enzyme activity was stopped by heating to 90°C for five minutes.

time and laccase activity was stopped by heating at 90°C for five minutes.

Laccase from *Trametes versicolor* (21.4 U/mg) was purchased from Fluka (Sweden).

**2.1. Enzyme and chemicals**

**2.2. Laccase activity assay**

per min at room temperature).

**2.3. Batch operation**

**2.4. Fed batch operation**

ing to 90°C for five minutes.

**2.5. Continuous stirred tank reactor operation**

Depending on enzyme nature, enzymatic bioconversion of phenols requires either oxygen or hydrogen peroxide. The availability and the concentration of these substrates are essen‐ tial to these reactions. Ghosh et al. (Ghosh et al., 2008) studied the effect of dissolved oxygen concentration on laccase efficiency during the removal of 2,4-dimethylphenol. These authors experimented several techniques such as dissolution by stirring or bubbling or a high initial saturation of the medium by oxygen. They reported that, whatever the technique used, as long as dissolved oxygen inside the reactor remains high, initial rates of reactions were simi‐ lar and high compared to a reaction control with a low concentration of oxygen.

The main investigations in the field of enzymatic bioconversion were carried out in batch mode (Ghosh et al., 2008; Kim et al., 2009; Nugroho Prasetyo et al., 2010). However, in this mode, the degree of polydispersity remains high and hydroxyl phenolic groups are often only partially oxidized. This behavior, according to Areskogh et al.(Areskogh et al., 2010a) would be due to the ability of the lignosulfonates to form spherical microgels makes the phenolic groups buried in the core of the gel inaccessible. It could also be ex‐ plained the inhibition of laccase by formed polymers (Kurniawati and Nicell, 2009). An‐ other explanation is that the bioconversion by laccase is carried out in two ways leading either to C-O-C or to C-C linkages. The last way generates phenolic groups by ionic tauto‐ merisation (Areskogh et al., 2010b). The concentration of the lignosulfonates also seems to influence the conversion rate of the phenolic groups, the polydispersity and the average molecular weight of polymers formed. High Mw were reached with high lignosulfonate concentrations (Areskogh et al., 2010a).

Fed batch and continuous modes are used in chemical bioconversion to control average Mw evolution and polydispersity and could also overcome some drawbacks of batch reactions; because fed batch allows controlling the enzyme and the substrate concentrations in the me‐ dium while the continuous system avoids the accumulation of the formed polymers in the medium. In spite of the potential of these two modes of reaction few data are available on their performance in the field of laccase bioconversion of phenols. Wu et al. (Wu et al., 1999) compared phenols removal efficiency by horseradish peroxidase in batch, continuous stirred tank, fed batch and a plug flow reactors. They reported that the plug flow reactor was the most appropriate for this reaction. Areskogh et al. (Areskogh et al., 2010a) compared also the effect of a successive addition of laccase during the lignosulfonates (SLS) bioconversion. They observed only minor differences in the average molecular weight increase which is de‐ pendent on the amount of enzyme.

The aim of this paper is to compare the efficiency of lignosulfonate bioconversion by laccase in terms of phenolic OH group consumption, average molecular weight and degree of poly‐ dispersity evolution under three modes of reaction conductions: batch with different en‐ zyme/substrate ratio, continuous feed of laccase and lignosulfonates and three alternatives of fed batch feeding. The oxygen consumption was also monitored.

### **2. Materials and methods**

### **2.1. Enzyme and chemicals**

es to polymerize phenols have received great attention and applied with success in the field of wood by-products (Ikeda et al., 2001; Jeon et al., 2010; Mita et al., 2003; Reihmann

26 Environmental Biotechnology - New Approaches and Prospective Applications

Depending on enzyme nature, enzymatic bioconversion of phenols requires either oxygen or hydrogen peroxide. The availability and the concentration of these substrates are essen‐ tial to these reactions. Ghosh et al. (Ghosh et al., 2008) studied the effect of dissolved oxygen concentration on laccase efficiency during the removal of 2,4-dimethylphenol. These authors experimented several techniques such as dissolution by stirring or bubbling or a high initial saturation of the medium by oxygen. They reported that, whatever the technique used, as long as dissolved oxygen inside the reactor remains high, initial rates of reactions were simi‐

The main investigations in the field of enzymatic bioconversion were carried out in batch mode (Ghosh et al., 2008; Kim et al., 2009; Nugroho Prasetyo et al., 2010). However, in this mode, the degree of polydispersity remains high and hydroxyl phenolic groups are often only partially oxidized. This behavior, according to Areskogh et al.(Areskogh et al., 2010a) would be due to the ability of the lignosulfonates to form spherical microgels makes the phenolic groups buried in the core of the gel inaccessible. It could also be ex‐ plained the inhibition of laccase by formed polymers (Kurniawati and Nicell, 2009). An‐ other explanation is that the bioconversion by laccase is carried out in two ways leading either to C-O-C or to C-C linkages. The last way generates phenolic groups by ionic tauto‐ merisation (Areskogh et al., 2010b). The concentration of the lignosulfonates also seems to influence the conversion rate of the phenolic groups, the polydispersity and the average molecular weight of polymers formed. High Mw were reached with high lignosulfonate

Fed batch and continuous modes are used in chemical bioconversion to control average Mw evolution and polydispersity and could also overcome some drawbacks of batch reactions; because fed batch allows controlling the enzyme and the substrate concentrations in the me‐ dium while the continuous system avoids the accumulation of the formed polymers in the medium. In spite of the potential of these two modes of reaction few data are available on their performance in the field of laccase bioconversion of phenols. Wu et al. (Wu et al., 1999) compared phenols removal efficiency by horseradish peroxidase in batch, continuous stirred tank, fed batch and a plug flow reactors. They reported that the plug flow reactor was the most appropriate for this reaction. Areskogh et al. (Areskogh et al., 2010a) compared also the effect of a successive addition of laccase during the lignosulfonates (SLS) bioconversion. They observed only minor differences in the average molecular weight increase which is de‐

The aim of this paper is to compare the efficiency of lignosulfonate bioconversion by laccase in terms of phenolic OH group consumption, average molecular weight and degree of poly‐ dispersity evolution under three modes of reaction conductions: batch with different en‐ zyme/substrate ratio, continuous feed of laccase and lignosulfonates and three alternatives

of fed batch feeding. The oxygen consumption was also monitored.

lar and high compared to a reaction control with a low concentration of oxygen.

and Ritter, 2006).

concentrations (Areskogh et al., 2010a).

pendent on the amount of enzyme.

Sodium lignosulfonates (SLS) from (Aldrich, Sweden) : 90 wt. % of SLS, 4 wt. % of reducing sugars and 6 wt% of total impurities. The average molecular weight (Mw), the number mo‐ lecular weight (Mn) and the polydispersity (Pdi) values are equal to 17800 Da ± 1500, 2900 Da ± 400, and 6.2 ± 0.3, respectively.

Laccase from *Trametes versicolor* (21.4 U/mg) was purchased from Fluka (Sweden).

### **2.2. Laccase activity assay**

The activity of laccase was determined spectrophotometrically by monitoring the oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline)-6-sulfonate (ABTS) to its cation radical as substrate at 436 nm in 50 mM sodium succinate buffer at pH 4.5 and 30 °C using quartz cuvette of path length 10 mm. Enzyme activity was expressed in units (1 U = 1 μmol ABTS oxidized per min at room temperature).

### **2.3. Batch operation**

Batch operations were performed in a bioreactor with a working volume of 1 L equipped with dissolved oxygen, pH and temperature sensors. The reactor was stirred vigorously at 500 rpm to solubilise SLS at 20°C and throughout the reactions. The lignosulfonates were solubilised in phosphate buffer solution at pH 4.5 and laccase was added to initiate reac‐ tions. For the analyses, samples were drawn out from the reactor at different intervals of time and laccase activity was stopped by heating at 90°C for five minutes.

### **2.4. Fed batch operation**

Fed batch reactions were carried out by progressive adding, at different time intervals (ev‐ ery 30 minutes during the first 5 hours), of enzyme alone, substrate alone or both enzyme and substrate. The total amounts of enzyme and substrate for the three fed batch operations were 10 g/L and 30 U/mL of SLS and laccase, respectively. Samples were taken at different time intervals and enzyme activity was stopped by heating to 90°C for five minutes.

### **2.5. Continuous stirred tank reactor operation**

The continuous stirred tank reactor was similar to the one used in batch step. Lignosulfo‐ nates (32 g/L) and laccase (63 U/mL) were prepared in two flasks separately and 500 mL of each solution were added progressively at a constant flow-rate into the reactor initially filled with buffered solution (1 L). The reactor was aerated and stirred vigorously at 500 rpm. Samples were taken at different time intervals and the enzyme activity was stopped by heat‐ ing to 90°C for five minutes.

### **2.6. Size exclusion chromatography analysis (SEC)**

Samples were analysed by Size exclusion chromatography (SEC) (HPLC LaChrom Merck, Germany). The system consists of a pump L-2130, an autosampler L-2200, and a Superdex 200HR 10/30 column (24 mL, 13 μm, dextran/cross linked agarose matrix). Detection was performed using UV detector diode L-2455 at 280 nm. Before analysis, the samples were fil‐ tered using regenerated cellulose membrane (0.22 μm) and aliquots of 50 μl were injected into the SEC system. A Buffer Phosphate pH 7, 0.15 M NaCl solution was used as an eluent. The flow rate was 0.4 mL at 25°C and the pressure is maintained at 11 bars. The calibration was performed by using polystyrenes sulfonate (PSS) as a standard to define molecular weight distribution.

Chromatographs were integrated in segments of thirteen second intervals. The numberaverage molecular weight (Mn), the weight-average molecular weight (Mw), and the polydis‐ persity (Pdi) were calculated as follows (Faix, 1981):

Number average molecular weight

$$\mathcal{M}\_n = \frac{\sum\_{i=1}^n Area\_i}{\sum\_{i=1}^n \frac{Area\_i}{\mathcal{M}\_i}} \tag{1}$$

**(b)** 

7

4

5

Pdi

6


Time (min)


Comparison of the Performance of the Laccase Bioconversion of Sodium Lignosulfonates in Batch, Continuous...

**Time (h)**


Time (h)

**Figure 1.** OH phenolic residual (a), dissolved O2 (a), Mw (b) and Pdi (b) variations in batchwise operation of reaction

The performance of the bioconversion reaction of lignosulfonates by laccase can be affected by the ratio of SLS/laccase. To verify this assumption, the reaction of bioconversion was car‐ ried out with different ratios SLS/laccase; (1 g/L)/ (3 U/mL), (1 g/L)/ (30 U/mL), (10 g/L)/ (3

carried out with 10 g/L and 30 U/mL of SLS and laccase over time. ( ■ ) Pdi and ( □ ) Mw.

**3.1. Kinetic study of enzymatic bioconversion in batch mode**

Dissolved O2(%)

0.05

0.10

0.15

0.20

OH phenolic (g/L)

0.25

0.30

0.35

0.40

Mw (Da)

**3. Results and discussion**

**(a)** 

http://dx.doi.org/10.5772/53103

29

Weight average molecular weight

$$\mathcal{M}\_w = \frac{\sum\_{i=1}^n Area\_i \mathbf{x} \mathcal{M}\_i}{\sum\_{i=1}^n Area\_i} \tag{2}$$

Polydispersity

$$D = \frac{M\_w}{M\_n} \tag{3}$$

where Mi is the molecular weight and Areai the area of each segment i.

### **2.7. Determination of phenolic content**

Phenolic content was determined using the method described by Areskogh et al. (Areskogh et al., 2010a).

Comparison of the Performance of the Laccase Bioconversion of Sodium Lignosulfonates in Batch, Continuous... http://dx.doi.org/10.5772/53103 29

**Figure 1.** OH phenolic residual (a), dissolved O2 (a), Mw (b) and Pdi (b) variations in batchwise operation of reaction carried out with 10 g/L and 30 U/mL of SLS and laccase over time. ( ■ ) Pdi and ( □ ) Mw.

### **3. Results and discussion**

**2.6. Size exclusion chromatography analysis (SEC)**

28 Environmental Biotechnology - New Approaches and Prospective Applications

persity (Pdi) were calculated as follows (Faix, 1981):

Number average molecular weight

Weight average molecular weight

Polydispersity

where Mi

et al., 2010a).

weight distribution.

Samples were analysed by Size exclusion chromatography (SEC) (HPLC LaChrom Merck, Germany). The system consists of a pump L-2130, an autosampler L-2200, and a Superdex 200HR 10/30 column (24 mL, 13 μm, dextran/cross linked agarose matrix). Detection was performed using UV detector diode L-2455 at 280 nm. Before analysis, the samples were fil‐ tered using regenerated cellulose membrane (0.22 μm) and aliquots of 50 μl were injected into the SEC system. A Buffer Phosphate pH 7, 0.15 M NaCl solution was used as an eluent. The flow rate was 0.4 mL at 25°C and the pressure is maintained at 11 bars. The calibration was performed by using polystyrenes sulfonate (PSS) as a standard to define molecular

Chromatographs were integrated in segments of thirteen second intervals. The numberaverage molecular weight (Mn), the weight-average molecular weight (Mw), and the polydis‐

1

*i <sup>n</sup> <sup>n</sup> <sup>i</sup> i i*

= å

=

*M*

*i*

(1)

(2)

*Area*

*Area M*

x

*Area M*

*Area*

*w n*

Phenolic content was determined using the method described by Areskogh et al. (Areskogh

*<sup>M</sup>*<sup>=</sup> (3)

the area of each segment i.

*i i*

*i*

*n*

1

=

1

*i w n*

= å

=

*M*

is the molecular weight and Areai

**2.7. Determination of phenolic content**

*n*

1

*i*

*<sup>M</sup> <sup>D</sup>*

=

å

å

### **3.1. Kinetic study of enzymatic bioconversion in batch mode**

The performance of the bioconversion reaction of lignosulfonates by laccase can be affected by the ratio of SLS/laccase. To verify this assumption, the reaction of bioconversion was car‐ ried out with different ratios SLS/laccase; (1 g/L)/ (3 U/mL), (1 g/L)/ (30 U/mL), (10 g/L)/ (3 U/mL) and (10 g/L)/ (30 U/mL); in a stirred and aerated reactor. For the different assays Mw average, Pdi, phenol OH group content, and oxygen consumption were determined throughout the reaction. The results obtained with the four studied ratios, indicated similar profiles for the consumption of hydroxyl phenolic groups and oxygen. As an illustration, Figure 1 represents the variation of Mw average, Pdi, hydroxyl phenolic groups and oxygen evolution for the reaction with a SLS/laccase ratio equal to (10 g/L) / (30 U/mL). It appears that this reaction is made up of a two distinct steps. The first one is characterized by a rapid decrease of phenol OH group amount, dissolved oxygen, and Pdi value and a high increase of Mw average. The second one shows an increase of the dissolution of the oxygen to reach a plateau near the saturation of the medium, a progressive deceleration in the decrease of Pdi, and in the increase of Mw average and, a stabilisation of phenol OH group content around 0.1 g/L. These profiles could be explained by the fact that the first step consists of the initia‐ tion and the propagation of the enzymatic bioconversion. The rapid consumption of the oxygen ensures the formation of the SLS phenoxy radicals via laccase reduction. Thus, the role of the oxygen is important and can become a limiting step. The rapid decrease of dis‐ solved oxygen has already been reported by Ghosh et al. (Ghosh et al., 2008) during the 2,4 dimethylphenol bioconversion by laccase. The second step is rather a combination stage where the need for oxygen is negligible.

R1

OH

O

CH3

C O O CH3

Laccase

O2

R1

C O O CH3

R1 R1

O

R1

O

R1 R1

OH

O <sup>3</sup> CH

**Rearomatisation Rearomatisation** 

O <sup>3</sup>

CH CH3

CH3 <sup>C</sup>

CH3

Comparison of the Performance of the Laccase Bioconversion of Sodium Lignosulfonates in Batch, Continuous...

O

O

OH

R1

O

**Scheme 1.** Proposed reaction mechanism for the formation of C-O-C and C-C bonds when a lignosulfonates model is

1 g/L of S and 3 U/mL of E 47 % 25700 4.6 1 g/L of S and 30 U/mL of E 52 % 26400 4.1 10 g/L of S and 3 U/mL of E 73 % 30600 4.4 10 g/L of S and 30 U/mL of E 75 % 31400 4.6

**Table 1.** The conversion rate, the specific conversion rate, the final Mw and the final Pdi of reactions carried out in

**Reaction Conversion rate (%) Final Mw Final Pdi**

O

O

O

R1

O

O

3 CH

R1

oxidized by laccase. (R1) lignin fragment

batchwise operation. (S) Substrate; (E) Enzyme

3 CH

R1

R1

O

R1

O CH3

http://dx.doi.org/10.5772/53103

O

OH

CH3

31

CH3

O

O

The observed increase of the dissolved oxygen while Mw is still growing confirms theses as‐ sumption. After 24 h of reaction the hydroxyl phenolic groups are not totally oxidized; this is due to the fact that when the reaction of bioconversion is finished, the final obtained struc‐ ture of polymers contains hydroxyl groups (schema 1) (Areskogh et al., 2010b).

Table 1 reports the conversion rate of phenolic OH groups and the final Mw and Pdi values of batch reactions. These results showed also that regardless of the enzyme concentration, either 3 or 30 U/mL, the highest conversion rate of phenolic groups (73 % and 75 %) is ob‐ served at the highest SLS concentration (10g/L). For a given concentration of lignosulfonates, the enzyme concentration slighly affects the conversion rate; this means that a concentration of 3 U/mL of laccase is sufficient to polymerize the concentrations of the lignosulfonates test‐ ed in this work. It also appears that whatever the concentration of the enzyme Mw average is significantly improved at high concentrations of lignosulfonates (10 g/L). It increases from 17800 Da to 30600 Da and 31400 Da respectively for 3 U/mL and 30 U/mL of laccase. Pdi decrease approximately to a value of 4, independently of the enzyme and lignosulfonate concentrations. The high conversion yield of phenolic OH groups obtained at 10 g/L of lignosulfonates suggests that higher is generated phenoxy radicals in the reaction media, higher is the consumption of phenolic OH groups and Mw values. This may be due to the fact that the probability of establishing a contact between two phenoxy radicals is increased when their concentration in the medium is high and the C-O-C coupling is also favoured. So, this reaction is under a "kinetic control". The low Mw (26400 Da) observed with 1 g/L suggests that in the presence of a diluted solution and acid pH (4.5), the reaction is under a "thermodynamic control" which promotes C-C linkage.

Comparison of the Performance of the Laccase Bioconversion of Sodium Lignosulfonates in Batch, Continuous... http://dx.doi.org/10.5772/53103 31

U/mL) and (10 g/L)/ (30 U/mL); in a stirred and aerated reactor. For the different assays Mw average, Pdi, phenol OH group content, and oxygen consumption were determined throughout the reaction. The results obtained with the four studied ratios, indicated similar profiles for the consumption of hydroxyl phenolic groups and oxygen. As an illustration, Figure 1 represents the variation of Mw average, Pdi, hydroxyl phenolic groups and oxygen evolution for the reaction with a SLS/laccase ratio equal to (10 g/L) / (30 U/mL). It appears that this reaction is made up of a two distinct steps. The first one is characterized by a rapid decrease of phenol OH group amount, dissolved oxygen, and Pdi value and a high increase of Mw average. The second one shows an increase of the dissolution of the oxygen to reach a plateau near the saturation of the medium, a progressive deceleration in the decrease of Pdi, and in the increase of Mw average and, a stabilisation of phenol OH group content around 0.1 g/L. These profiles could be explained by the fact that the first step consists of the initia‐ tion and the propagation of the enzymatic bioconversion. The rapid consumption of the oxygen ensures the formation of the SLS phenoxy radicals via laccase reduction. Thus, the role of the oxygen is important and can become a limiting step. The rapid decrease of dis‐ solved oxygen has already been reported by Ghosh et al. (Ghosh et al., 2008) during the 2,4 dimethylphenol bioconversion by laccase. The second step is rather a combination stage

30 Environmental Biotechnology - New Approaches and Prospective Applications

The observed increase of the dissolved oxygen while Mw is still growing confirms theses as‐ sumption. After 24 h of reaction the hydroxyl phenolic groups are not totally oxidized; this is due to the fact that when the reaction of bioconversion is finished, the final obtained struc‐

Table 1 reports the conversion rate of phenolic OH groups and the final Mw and Pdi values of batch reactions. These results showed also that regardless of the enzyme concentration, either 3 or 30 U/mL, the highest conversion rate of phenolic groups (73 % and 75 %) is ob‐ served at the highest SLS concentration (10g/L). For a given concentration of lignosulfonates, the enzyme concentration slighly affects the conversion rate; this means that a concentration of 3 U/mL of laccase is sufficient to polymerize the concentrations of the lignosulfonates test‐ ed in this work. It also appears that whatever the concentration of the enzyme Mw average is significantly improved at high concentrations of lignosulfonates (10 g/L). It increases from 17800 Da to 30600 Da and 31400 Da respectively for 3 U/mL and 30 U/mL of laccase. Pdi decrease approximately to a value of 4, independently of the enzyme and lignosulfonate concentrations. The high conversion yield of phenolic OH groups obtained at 10 g/L of lignosulfonates suggests that higher is generated phenoxy radicals in the reaction media, higher is the consumption of phenolic OH groups and Mw values. This may be due to the fact that the probability of establishing a contact between two phenoxy radicals is increased when their concentration in the medium is high and the C-O-C coupling is also favoured. So, this reaction is under a "kinetic control". The low Mw (26400 Da) observed with 1 g/L suggests that in the presence of a diluted solution and acid pH (4.5), the reaction is under a

ture of polymers contains hydroxyl groups (schema 1) (Areskogh et al., 2010b).

where the need for oxygen is negligible.

"thermodynamic control" which promotes C-C linkage.

**Scheme 1.** Proposed reaction mechanism for the formation of C-O-C and C-C bonds when a lignosulfonates model is oxidized by laccase. (R1) lignin fragment


**Table 1.** The conversion rate, the specific conversion rate, the final Mw and the final Pdi of reactions carried out in batchwise operation. (S) Substrate; (E) Enzyme

### **3.2. Kinetic study of enzymatic bioconversion in continuous reactor**

The operating conditions for the continuous feeding of the enzyme and lignosulfonates were chosen to add 16 g/L and 32 U/mL of lignosulfonates and laccase respectively and to have the same residence time (24 h) as that used in the batch mode.

Although 16 g/L of SLS were added during the 24h of the reaction, the final Mw average is of the same order of magnitude as the batch with 10 g/L of SLS. These results indicate that the in‐ crease of Mw average is rather favoured by the conditions allowing a high amount and instanta‐ neous generation of free radicals rather than a progressive feeding of a high quantity of SLS. However, continuous adding of substrate and enzyme allows a low degree of polydispersity to be reached (3.7) compared to the batch (4.6). The low residual phenolic OH groups in the media and their high conversion rate suppose that the continuous mode promotes the C-O-C linkage.

Comparison of the Performance of the Laccase Bioconversion of Sodium Lignosulfonates in Batch, Continuous...

In fed batch mode three alternatives of feeding were tested i) with substrate alone, ii) with enzyme iii) or with both enzyme and substrate. For each assay the addition of substrate and enzyme was carried out in stepwise mode 10 times at a rate of 1g or 3000 U or both every 30 minutes during the five first hours of the reaction. Results are shown in figure 3, figure 4


Time (min)

0 4 8 12 16 20 24 28

Time (h)


Time (h)



Time (min)

Time (min)


Time (h)

**Figure 3.** OH phenolic residual and dissolved O2 variations in fed-batch operations over time. (a) Adding enzyme; (b)

Dissolved O2(%)

Dissolved O2(%)

0.00 0.04 0.08 0.12 0.16 0.20 0.24

OH-Ph residual (g/L)

Adding substrate; (c) Adding both enzyme and substrate.

**(c)** 

**(b)** 

**(a)** 

http://dx.doi.org/10.5772/53103

33

**3.3. Kinetic study of enzymatic bioconversion in fed batch operation**

Dissolved O2(%)

0.10 0.15 0.20 0.25 0.30 0.35 0.40

0.00 0.05 0.10 0.15 0.20 0.25

OH-Ph residual (g/L)

OH-Ph residual (g/L)

and table 2.

The obtained results are summarized in Figure 2 a and 2 b. It appears that phenolic OH group content increases slightly in the medium to reach the same level as that observed at the end of the batch reaction (~0.1 g/L) ; while the conversion rate of phenolic OH groups remains con‐ stant near 85 % throughout the duration of the reaction. This conversion is higher than that ob‐ tained in the batch. Molecular weight average (Mw) of formed polymers increases gradually to 28400 Da during the first four hours and then, as in batch mode, this increase becomes less pro‐ nounced. Pdi values decrease quickly to reach a low value (3.7) and remain more or less con‐ stant along the time incubation (Figure 2b). The dissolved oxygen (Figure 2a) also decreases over time due to its continuous consumption by the added laccase.

**Figure 2.** OH phenolic residual (a), dissolved O2 (a), instantaneous conversion rate (a), Mw (b) and Pdi (b) variations in continuous operation over time.(a) (■) Instantaneous conversion rate (□) OH-Ph residual (b) ( ■ ) Pdi and ( □ ) M<sup>w</sup>

Although 16 g/L of SLS were added during the 24h of the reaction, the final Mw average is of the same order of magnitude as the batch with 10 g/L of SLS. These results indicate that the in‐ crease of Mw average is rather favoured by the conditions allowing a high amount and instanta‐ neous generation of free radicals rather than a progressive feeding of a high quantity of SLS. However, continuous adding of substrate and enzyme allows a low degree of polydispersity to be reached (3.7) compared to the batch (4.6). The low residual phenolic OH groups in the media and their high conversion rate suppose that the continuous mode promotes the C-O-C linkage.

### **3.3. Kinetic study of enzymatic bioconversion in fed batch operation**

**3.2. Kinetic study of enzymatic bioconversion in continuous reactor**

the same residence time (24 h) as that used in the batch mode.

32 Environmental Biotechnology - New Approaches and Prospective Applications

over time due to its continuous consumption by the added laccase.

0.00

0.02

0.04

OH-Ph residual (g/L)

0.06

0.08

0.10

Mw (Da)

The operating conditions for the continuous feeding of the enzyme and lignosulfonates were chosen to add 16 g/L and 32 U/mL of lignosulfonates and laccase respectively and to have

The obtained results are summarized in Figure 2 a and 2 b. It appears that phenolic OH group content increases slightly in the medium to reach the same level as that observed at the end of the batch reaction (~0.1 g/L) ; while the conversion rate of phenolic OH groups remains con‐ stant near 85 % throughout the duration of the reaction. This conversion is higher than that ob‐ tained in the batch. Molecular weight average (Mw) of formed polymers increases gradually to 28400 Da during the first four hours and then, as in batch mode, this increase becomes less pro‐ nounced. Pdi values decrease quickly to reach a low value (3.7) and remain more or less con‐ stant along the time incubation (Figure 2b). The dissolved oxygen (Figure 2a) also decreases



Dissolved O2(%)


Time (min)

Time (h)

Time (h)

**Figure 2.** OH phenolic residual (a), dissolved O2 (a), instantaneous conversion rate (a), Mw (b) and Pdi (b) variations in continuous operation over time.(a) (■) Instantaneous conversion rate (□) OH-Ph residual (b) ( ■ ) Pdi and ( □ ) M<sup>w</sup>

**(b)** 

3

4

5

Pdi

6

7

50

60

70

Instantaneous conversion rate (%)

80

90

100

**(a)** 

In fed batch mode three alternatives of feeding were tested i) with substrate alone, ii) with enzyme iii) or with both enzyme and substrate. For each assay the addition of substrate and enzyme was carried out in stepwise mode 10 times at a rate of 1g or 3000 U or both every 30 minutes during the five first hours of the reaction. Results are shown in figure 3, figure 4 and table 2.

**Figure 3.** OH phenolic residual and dissolved O2 variations in fed-batch operations over time. (a) Adding enzyme; (b) Adding substrate; (c) Adding both enzyme and substrate.

Concerning the reaction carried out with enzyme feeding of the reactor (figure 3 a), similar results as batch mode operation were observed for both residual phenolic OH groups and oxygen consumption. In a first step a rapid oxidation of phenolic OH groups and oxygen uptake rate were observed, followed by an increase of the dissolved oxygen in the medium and a low oxidation of phenolic OH groups was observed during a second step. This behav‐ iour confirms that only a low amount of enzyme is needed to oxidise the 10 g/L of SLS and

Comparison of the Performance of the Laccase Bioconversion of Sodium Lignosulfonates in Batch, Continuous...

http://dx.doi.org/10.5772/53103

35

After 24h of reaction, the conversion rate of phenolic OH groups (73 %) was in the same magnitude as that obtained for the batch mode (table 2). As it is indicated in figure 4 and table 2, the Mw average rose gradually during the period of enzyme addition (5h) and then stabilizes around 29000 Da along the remaining time of the reaction. Pdi value decreased

For reactions carried out with the addition of substrate or enzyme and substrate, a progres‐ sive increase of phenolic OH group content during the first 5 hours (0.2 g/L) then a slight decrease were observed. Moreover, dissolved oxygen decreases and then increases quickly after each addition, in a repetitive way (figure 3b and 3c). The conversion rate of phenolic OH groups after 24 hours is 61 % for enzyme and substrate addition and 44 % for substrate

As for continuous mode, a progressive increase of Mw was observed to reach 28200 Da and 26400 Da respectively for enzyme and substrate addition and substrate feeding. The Pdi dropped quickly to 4.2 and remained constant throughout the duration of the reaction (Fig‐

The relatively low final Mw, the conversion rate and the accumulation of phenolic OH groups indicated that similar mechanisms, such as the one observed in batch mode with 1 g/L, occurre. This means that these two modes of reaction promote a "thermodynamic con‐

The obtained results in this work indicated that the increase of Mw average and the decrease of the polydispersity depend on the operating conditions. Batch mode with high concentra‐ tion of SLS (10 g/L), promotes the increase of the Mw and probably the C-O-C coupling route. This seemesto be due to the high and instantaneous generation of free radicals, fa‐ vouring the "kinetic control" of the reaction. The continuous mode also favours the forma‐ tion of C-O-C bounds and indicates that the increase of Mw is strongly affected by the high amount of phenoxyl radicals generated than the quantity of added substrate. However, con‐ tinuous feeding of enzyme and substrate leads to a low Pdi. Results for fed batch, carried out with enzyme feeding, is comparable to those obtained for batch with 10 g/L; the enzyme plays a minor role and a low amount is enough to oxidise the tested concentration of SLS.

trol" and then lead to C-C linkages instead of C-O-C coupling.

the oxygen consumption occurs only during this first step of free radical generation.

rapidly and stabilised more or less at 4.7 until the end of the reaction (24 hours).

feeding.

ure 4 and Table 2).

**4. Conclusions**

**Figure 4.** Mw (a) and Pdi (b) variations in fed-bach operations over time.(▲) Adding enzyme; (∆) Adding both enzyme and substrate; ( ) Adding substrate.


**Table 2.** The conversion rate after 5 h and 24 h of reaction, the final Mw and the final Pdi of reactions carried out in fed-bach operations.

Concerning the reaction carried out with enzyme feeding of the reactor (figure 3 a), similar results as batch mode operation were observed for both residual phenolic OH groups and oxygen consumption. In a first step a rapid oxidation of phenolic OH groups and oxygen uptake rate were observed, followed by an increase of the dissolved oxygen in the medium and a low oxidation of phenolic OH groups was observed during a second step. This behav‐ iour confirms that only a low amount of enzyme is needed to oxidise the 10 g/L of SLS and the oxygen consumption occurs only during this first step of free radical generation.

After 24h of reaction, the conversion rate of phenolic OH groups (73 %) was in the same magnitude as that obtained for the batch mode (table 2). As it is indicated in figure 4 and table 2, the Mw average rose gradually during the period of enzyme addition (5h) and then stabilizes around 29000 Da along the remaining time of the reaction. Pdi value decreased rapidly and stabilised more or less at 4.7 until the end of the reaction (24 hours).

For reactions carried out with the addition of substrate or enzyme and substrate, a progres‐ sive increase of phenolic OH group content during the first 5 hours (0.2 g/L) then a slight decrease were observed. Moreover, dissolved oxygen decreases and then increases quickly after each addition, in a repetitive way (figure 3b and 3c). The conversion rate of phenolic OH groups after 24 hours is 61 % for enzyme and substrate addition and 44 % for substrate feeding.

As for continuous mode, a progressive increase of Mw was observed to reach 28200 Da and 26400 Da respectively for enzyme and substrate addition and substrate feeding. The Pdi dropped quickly to 4.2 and remained constant throughout the duration of the reaction (Fig‐ ure 4 and Table 2).

The relatively low final Mw, the conversion rate and the accumulation of phenolic OH groups indicated that similar mechanisms, such as the one observed in batch mode with 1 g/L, occurre. This means that these two modes of reaction promote a "thermodynamic con‐ trol" and then lead to C-C linkages instead of C-O-C coupling.

### **4. Conclusions**

**3**

and substrate; ( ) Adding substrate.

**Reaction**

Fed-batch by adding both enzyme and substrate

fed-bach operations.

**0 4 8 12 16 20 24 Time (h)**

**Figure 4.** Mw (a) and Pdi (b) variations in fed-bach operations over time.(▲) Adding enzyme; (∆) Adding both enzyme

Fed-batch by adding enzyme 72 % 73 % 29400 4.7

Fed-batch by adding substrate 39 % 44 % 26500 4.2

**Table 2.** The conversion rate after 5 h and 24 h of reaction, the final Mw and the final Pdi of reactions carried out in

**Conversion rate 24 h (%)**

48 % 61 % 28200 4.2

**Final Mw (Da) Final Pdi**

**Conversion rate 5h (%)**

**0 4 8 12 16 20 24 Time (h)**

**(b)** 

**(a)** 

**4**

**5**

**Pdi**

**6**

**7**

**18000**

**22000**

**Mw (Da)**

**26000**

**30000**

34 Environmental Biotechnology - New Approaches and Prospective Applications

The obtained results in this work indicated that the increase of Mw average and the decrease of the polydispersity depend on the operating conditions. Batch mode with high concentra‐ tion of SLS (10 g/L), promotes the increase of the Mw and probably the C-O-C coupling route. This seemesto be due to the high and instantaneous generation of free radicals, fa‐ vouring the "kinetic control" of the reaction. The continuous mode also favours the forma‐ tion of C-O-C bounds and indicates that the increase of Mw is strongly affected by the high amount of phenoxyl radicals generated than the quantity of added substrate. However, con‐ tinuous feeding of enzyme and substrate leads to a low Pdi. Results for fed batch, carried out with enzyme feeding, is comparable to those obtained for batch with 10 g/L; the enzyme plays a minor role and a low amount is enough to oxidise the tested concentration of SLS.

Substrate adding and enzyme and substrate adding, as a dilute batch system, promotes C-C coupling ("thermodynamic control") and thus a low Mw increase. These results are likely to open new ways to control the enzymatic bioconversion of lignosulfonates. However these assumptions need to be verified by spectroscopy analyses of the formed polymers in order to have a better understanding of the mechanisms of allowing C-C or C-O-C coupling.

[8] Ferreira F. C, Peeva L, Boam A, Zhang S, Livingston A. 2005. Pilot scale application of the Membrane Aromatic Recovery System (MARS) for recovery of phenol from

Comparison of the Performance of the Laccase Bioconversion of Sodium Lignosulfonates in Batch, Continuous...

http://dx.doi.org/10.5772/53103

37

[9] Ghosh J. P, Taylor K. E, Bewtra J. K, and Biswas N. 2008. Laccase-catalyzed removal of 2,4-dimethylphenol from synthetic wastewater: Effect of polyethylene glycol and

[10] Gross S. M, Givens R. D, Jikei M, Royer J. R, Khan S, DeSimone J. M, Odell P. G, and Hamer G. K. 1998. Synthesis and swelling of poly(bisphenol a carbonate) using su‐

[11] Husain Q. 2010. Peroxidase mediated decolorization and remediation of wastewater containing industrial dyes: A review. Reviews Environ Sci Biotechnol 9: 117-140. [12] Ikeda R, Uyama H, Kobayashi S. 2001. Laccase-catalyzed curing of vinyl polymers

[13] Jeon J. R, Kim E. J, Murugesan K, Park H. K, Kim Y. M, Kwon J. H, Kim W. G, Lee J. Y, Chang Y. S. 2010. Laccase-catalysed polymeric dye synthesis from plant-derived phenols for potential application in hair dyeing: Enzymatic colourations driven by

[14] Jiang H, Fang Y, Fu Y, Guo Q. X. 2003. Studies on the extraction of phenol in waste‐

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[18] Kobayashi S, Shoda S. I, Uyama H. 1995. Enzymatic polymerization and oligomeriza‐

[19] Kobayashi S, Uyama H. 1998. Enzymatic Polymerization for Synthesis of Polyesters

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### **Author details**

Nidal Madad, Latifa Chebil, Hugues Canteri, Céline Charbonnel and Mohamed Ghoul

\*Address all correspondence to: latifa.chebil@ensaia.inpl-nancy.fr

Laboratoire d'Ingénierie des Biomolécules, ENSAIA-INPL, Vandœuvre-lès-Nancy, France

### **References**


[8] Ferreira F. C, Peeva L, Boam A, Zhang S, Livingston A. 2005. Pilot scale application of the Membrane Aromatic Recovery System (MARS) for recovery of phenol from resin production condensates. J Memb Sci 257: 120-133.

Substrate adding and enzyme and substrate adding, as a dilute batch system, promotes C-C coupling ("thermodynamic control") and thus a low Mw increase. These results are likely to open new ways to control the enzymatic bioconversion of lignosulfonates. However these assumptions need to be verified by spectroscopy analyses of the formed polymers in order to have a better understanding of the mechanisms of allowing C-C or C-O-C coupling.

Nidal Madad, Latifa Chebil, Hugues Canteri, Céline Charbonnel and Mohamed Ghoul

Laboratoire d'Ingénierie des Biomolécules, ENSAIA-INPL, Vandœuvre-lès-Nancy, France

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\*Address all correspondence to: latifa.chebil@ensaia.inpl-nancy.fr

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**Author details**

**References**

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[23] Mita N, Tawaki S. I, Hiroshi U, and Kobayashi S. 2003. Laccase-catalyzed oxidative polymerization of phenols. Macromolecular Bioscience 3: 253-257.

**Chapter 3**

**Biochemical Processes for Generating Fuels and**

Amy Philbrook, Apostolos Alissandratos and

Additional information is available at the end of the chapter

Christopher J. Easton

http://dx.doi.org/10.5772/55309

**1. Introduction**

**Commodity Chemicals from Lignocellulosic Biomass**

Fuels and chemicals derived from biomass are regarded as an environmentally friendly alternative to petroleum based products. The concept of using plant material as a source for fuels and commodity chemicals has been embraced by governments to alleviate dependence on the volatile petroleum market. This trend is driven not only by economics but also by social and political factors. Global warming has been associated with CO2 emissions largely originating from the combustion of fossil fuels.[1] This, together with depleting and finite carbon fossil fuel resources, and insecurity of petroleum supplies has prompted a shift towards biofuels and biomaterials.[1] The use of biomass as an economically competitive source of transport fuel was initiated by the fuel crisis in 1970 and its commercialization was led by the USA and Brazil.[2] In 2010, the USA and Brazil processing corn and sugarcane, respectively, produced 90% of the world's bioethanol. In 2008, the "food for fuel" debate emerged sparked by concerns that the use of arable land for bioethanol and biodiesel crops was placing pressure on food demand for a growing world population.[3] In June 2011, the World Bank and nine other international agencies produced a report advising governments to cease biofuel subsidies as the use of food stock for fuel production was linked to increasing food prices.[4] Subsidies were thus ended in the USA when their Senate voted overwhelmingly to end billions of dollars in bioethanol subsidies.[5] This reform resulted in USA bioethanol plants recording losses in the first quarter of 2012[6] and

is foreseen as the end of bioethanol production from corn at least in the USA.

Emerging from the "food for fuel" debate, the concept of commercializing second generation biofuels was embraced by governments as a route to produce biofuels without diminishing global food supplies.[7]. Second generation biofuels address concerns over designating arable land to grow food crops for fuel production as lignocellulosic biomass may consist of waste

> © 2013 Philbrook et al.; licensee InTech. This is an open access article 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.

© 2013 Philbrook et al.; licensee InTech. This is a paper 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.

