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Adsorption Characteristics of Cellulase and β-glucosidase on Avicel, Pretreated

Sugarcane Bagasse and Lignin

Daniele Longo Machado1, João Moreira Neto

1*, José Geraldo da Cruz Pradella

2, Antonio

Bonomi1,2

, Sarita Cândida Rabelo2, Aline Carvalho da Costa

1

1Laboratory of Fermentative and Enzymatic Process Engineering, School of Chemical

Engineering, University of Campinas, Campinas, SP, Brazil

2 Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE) – CTBE/CNPEM,

Campinas, SP, Brazil

Adsorption of Cellulase on Sugarcane Bagasse

*Address for correspondence: João Moreira Neto, Laboratory of Fermentative and Enzymatic

Process Engineering, School of Chemical Engineering, University of Campinas, Zip code:

13083-852, Campinas, SP, Brazil. Tel:+55-19-3521-3959; fax:+55-19- 3521-3965

e-mail: [email protected]


Abstract

Although adsorption is an essential step in the enzymatic hydrolysis of lignocellulosic

materials, literature reports controversial results in relation to the adsorption of the cellulolitic

enzymes on different biomasses/pretreatments, which difficults the description of this

phenomenon in hydrolysis mathematical models. In this work, the adsorption of these

enzymes on Avicel and sugarcane bagasse pretreated by the hydrothermal (HB) and

organosolv (OB) methods was evaluated. The results have shown no significant adsorption of

β-glucosidase on Avicel or HB. Increasing solids concentration from 5% (w/v) to 10% (w/v)

had no impact on the adsorption of cellulase on the different biomasses if stirring rates were

high enough (>100 rpm for Avicel and >150 rpm for BH and BO). Adsorption equilibrium

time was low for Avicel (10 minutes) when compared to the lignocellulosic materials (120

minutes). Adsorption isotherms determined at 4 and 50ºC have shown that for Avicel there

was a decrease in the maximum adsorption capacity (Emax) with the temperature increase,

while for HB increasing temperature increased Emax. Also, Emax increased with lignin content

in the material. Adsorption studies of cellulase on lignin left after enzymatic digestion of HB

shows lower but significant adsorption capacity (Emax=11.92 ± 0.76 mg/g).

Keywords: adsorption kinetics, cellulase, enzymatic hydrolysis, Langmuir isotherm, sugarcane

bagasse, β-glucosidase

Abbreviations: HB, hydrothermal bagasse; OB, organosolv bagasse; HPLC, high-performance

liquid chromatography; IL, isolated lignin; CTBE/CNPEM, Brazilian Bioethanol Science and

Technology Laboratory; FPU, Filter Paper Units; CBU, Cellobiose Units.


1. Introduction

Cellulase adsorption on the substrate and the formation of the enzyme-substrate complex are

considered critical steps in the enzymatic hydrolysis of cellulose [1]. Thus, an adequate

description of the adsorption step is indispensable to construct an accurate hydrolysis

mathematical model. Many hypotheses have been made to include the adsorption step in

mathematical models, but most of them have not been experimentally proven and there are

contradicting assumptions in different models.

Most works assume that the adsorption equilibrium time is fast (≤ 90 min) compared to the

hydrolysis time course (>24h) [2, 3]. However, many of the studies use pure cellulose as

substrate and adsorption kinetics have been shown to be influenced by the biomass

characteristics and pretreatment [4]. Zheng [5] has shown that the adsorption equilibrium

time of cellulase on sulfuric acid pretreated wild ryegrass is long (8 h), which points to the

need for experimental studies for different biomasses/pretreatments.

Among the most extensively studied aspects of the enzymatic hydrolysis of cellulose in the

last years is the decrease in yield at high solids loading. Wang et al. [6] concluded that the

decreased yield was caused by a decline in the adsorption capacity of cellulase at high

substrate concentrations. These authors have found that even when a fixed substrate/enzyme

ratio was maintained, there was a decrease in the mass of adsorbed enzymes per mass of

substrate when solids loads were changed from 1% to 5% (w/v, based on cellulose) and

determined different adsorption isotherms for different substrate concentrations. The

isotherms determination, however, must have been influenced by mass transfer limitations,

especially considering that the experiments were performed at a very low stirring rate (40

rpm).

Most of the adsorption isotherms of cellulase on biomass in the literature are determined at

4ºC to avoid the hydrolysis reaction [4, 7-9]. However, adsorption is strongly influenced by


temperature [10] and hydrolysis occurs at 50ºC, thus if the isotherm is to be used in a

mathematical model it should be determined at this temperature. Maurer et al. [11] proposed

the addition of high glucose concentrations (at least 100 times higher than the enzyme

concentration) to inhibit the hydrolysis reaction in adsorptions studies at high temperatures.

Another controversial aspect in enzymatic hydrolysis models is the unproductive adsorption

of β-glucosidase on lignin. Haven e Jorgensen [12] concluded that adsorption characteristics

are different for different β-glucosidase preparations. In the experimental conditions used in

their work, while β-glucosidase from Aspergillus niger (Novozym 188) did not adsorb on

lignin or on biomass, Cellic® CTec2 β-glucosidase adsorbed significantly, although its

catalytic activity was retained. Even so, many mathematical models in the literature consider

a reduction in the specific hydrolysis rate of cellobiose due to unproductive adsorption of β-

glucosidase on lignin [13-15]. As the adsorption of this enzyme on lignin depends on the

enzyme preparation as well as the biomass and pretreatment, experimental studies are

necessary for specific combinations of enzyme/biomass/pretreatment.

In this work the adsorption of cellulase (Celluclast 1.5 L by Novozyme, Sigma-Aldrich) and

β-glucosidase (Novozym 188, Sigma-Aldrich) was evaluated on Avicel and sugarcane

bagasse pretreated by the hydrothermal (HB) and organosolv (OB) methods. The different

materials were chosen to evaluate the difference in adsorption characteristics in

microcrystalline cellulose (Avicel) and lignocellulosic biomasses with high (Hydrothermal

bagasse) and low (Organosolv bagasse) lignin contents. The adsorption of β-glucosidase on

the biomass with higher lignin content (HB) and the influence of solids concentration and

stirring on the adsorption of cellulase on the three materials were evaluated. Experiments

were performed to calculate the adsorption equilibrium time for the three materials and

isotherms were determined at 4 and 50ºC. Also, adsorption equilibrium time and adsorption

isotherms of cellulase on lignin isolated after hydrolysis of HB were determined. Langmuir


isotherms were chosen because they represent simple mechanistic models that can be

employed to compare the kinetic properties of various enzyme-cellulose systems [18].

They are extensively used and provide a good fit to the data of cellulase adsorption on

lignocellulosic biomass in most cases presented by many authors [4, 9, 16, 17]. However,

some assumptions must be taken into consideration for the use of the Langmuir

isotherm, as the reversibility of adsorption, the interactions between non-adsorbed

species, the homogeneity of binding sites and the uniform composition between the

adsorbed cellulases [19].

2. Materials and Methods

2.1. Substrate

The substrate used in all experiments was fresh sugarcane bagasse (Saccharum officinarum)

submitted to hydrothermal (HB) and organosolv (OB) pretreatment and microcrystalline

cellulose (Avicel PH101, nominal particle size 50µm) obtained from Sigma-Aldrich. Bagasse

was provided by the sugar plant “Usina Tarumã” from Raizen group, located in Tarumã, São

Paulo, Brazil, from the same mechanical harvest (2011/12) and resulting from the last milling

after juice extraction. It was dried at room conditions for 4 days, ground in a cutting mill

(Pulverisette 19, Fritsch), subsequently sieved using a 0.5 mm sieve and stored in sealed

plastic bags.

2.2. Enzymes

The enzymes used were cellulase from Trichoderma reesei (Celluclast 1.5 L by Novozyme,

Sigma-Aldrich) and β-glucosidase from Aspergillus niger (Novozym 188, Sigma-Aldrich).

2.3. Pretreatment of lignocellulosic biomass

Two different pretreatments (hydrothermal and organosolv) were separately applied to

sugarcane bagasse. Both pretreatments were conducted at the Brazilian Bioethanol Science


and Technology Laboratory (CTBE/CNPEM) according to previously determined conditions

[20, 21].

The hydrothermal pretreatment was performed at 190 °C for 10 min in an alloy reactor C-276

(Parr, model 4554). 300 g of previously ground bagasse (particle size < 0.5 mm) were added

to the reactor with 3 L of distilled water and solid-liquid ratio of 1:10 (w/v). After

pretreatment, the biomass was washed to neutral pH for removal of the soluble compounds.

Analysis of structural carbohydrates, lignin and ash were performed according to Sluiter et al.

[22-24] and Hyman et al. [25]. The composition of the hydrothermal sugarcane bagasse (HB)

was 61.07 ± 0.97 % cellulose, 2.10 ± 0.06 % hemicelluloses, 31.97 ± 0.05 % lignin and 6.44

± 0.06 % ash.

The organosolv pretreatment was performed in the same reactor. 300 g of ground bagasse

with 3L water/ethanol solution (1:1 v/v) and solid-liquid ratio of 1:10 (w/v) were added to the

reactor. The reaction occurred in 150 minutes at a temperature of 190 °C. The pretreated

bagasse was washed with a solution of 1 % (w/v) of sodium hydroxide to solubilize the

residual lignin from the fibers. After washing with sodium hydroxide, the biomass was

washed to neutral pH for removal of the soluble compounds. The composition analysis

methodology was the same used in the hydrothermal pretreated bagasse. The composition of

the organosolv sugarcane bagasse (OB) was 86.91 ± 0.40 % cellulose, 6.63 ± 0.25 %

hemicelluloses, 4.42 ± 0.27 % lignin and 3.75 ± 0.11 % ash.

2.4. Preparation of isolated lignin samples

Lignin was obtained by hydrolyzing HB samples for 96 hours. The samples were incubated

in citrate buffer (50 mM, pH 4.8) supplemented with 0.02% sodium azide per gram of

biomass and 15 FPU of cellulase/g bagasse and 25 CBU of β-glucosidase/g bagasse at 50 °C

with stirring in an orbital shaker at 150 rpm. The enzymatic hydrolysis was conducted at 3%

bagasse (w/v) loading in a 300 mL reaction mixture in a 1 L Erlenmeyer flask. Before the


addition of cellulase and β-glucosidase, the mixture of substrate and buffer was pre-heated in

an incubator shaker to the temperature of 50°C for 60 min to allow the substrate to disperse

uniformly in the buffer. After hydrolysis, in order to separate the liquid phase from the

solid phase, the residual material (lignin residue) of the hydrolysis was filtered through

a sieve with pore size 400 mesh (<0.0015 in) and subsequently washed with water.

Desorption of the proteins bound to the residual substrate (the solid phase) after hydrolysis

was carried out according to previously described conditions [26]. After washing the residual

substrate, 300 mL of citrate buffer (50 mM, pH 5.3) containing 0.5% Tween 80 (v/v) was

added to the lignin residue. The Erlenmeyer flasks were incubated for 2 h at 44 °C in an

orbital shaker at 150 rpm. After this period, the suspension was sonicated at 40 kHz for 60

min in a USC-2800 ultrasonic water bath (Unique, Brazil). Then, the materials were washed

3 times with distilled water and dried at room temperature. The lignin content of the isolated

lignin (IL) sample was 78.12 ± 0.02 %.

2.5. Enzyme activity assays

Cellulase activity was determined as filter paper units per milliliter (FPU/mL), as

recommended by the International Union of Pure and Applied Chemistry [27, 28], using

filter paper (Whatman No. 1) as substrate. The enzymatic reaction was performed at

50ºC, for 60 min at pH 4.8. The quantification of sugars released in the medium was

performed by the DNS method described by Miller [29] and Bazán [30].

β-glucosidase activity was determined using a solution of cellobiose 15 mmol/L and

expressed in units per milliliter (CBU/mL) [31]. For quantification of glucose released, the

enzymatic Glucose GOD-PAP method was used, as described by Henry [32]. Enzyme

activity was 87.74 FPU/mL for cellulase and 617.33 CBU/mL for β-glucosidase.

2.6. Cellulase adsorption kinetics and adsorption isotherm


Cellulase adsorption kinetics was performed on Avicel, HB, OB and IL in 125 mL

Erlenmeyer flasks with 50 mM citrate buffer (pH 4.8) supplemented with 0.02% of sodium

azide per gram of biomass at different substrate concentrations (5% or 10% w/v). In order to

inhibit hydrolysis reaction in the assays performed at 50ºC, D-(+)-glucose (Sigma-Aldrich)

was added to the aqueous enzyme solution. Glucose is an inhibitor of cellulase, preventing

hydrolytic activity and complexation with the cellulose substrate. According to Maurer et al.

[11] glucose inhibition of the active site of cellulase does not affect the sorption properties,

apparently because the binding domain primarily controls adsorption. The concentration of

glucose in the reaction medium was kept 250 times greater than the protein concentration

used (4 mg/g of substrate).

The flasks were incubated in an air bath shaker (MA 832, Marconi, Brazil) at 50 °C for 1 h

before addition of the enzymes. Five stirring speeds (40, 100, 150, 200 and 250 rpm) were

tested. The flasks were removed at different time intervals during the incubation. The

supernatant from each flask was collected, centrifuged (NT 810 Novatecnica, Brazil) at 25 ºC

(2122 g, 5 min) and filtered using a hydrophilic PTFE membrane with a pore size of 0.45

µm. The protein content of the supernatant was determined using Bradford method [33].

Cellulase adsorption isotherm experiments were conducted by varying the concentration of

cellulase protein (0.1–4.5 mg/mL) added to a constant substrate concentration of 5% (w/v) at

a stirring rate of 150 rpm in 50 mM citrate buffer (pH 4.8) at 4 °C and 50 °C for the

adsorption equilibrium time determined for each substrate. The mass of protein in the

supernatant was determined as free cellulase in solution. The mass of cellulase bound to the

substrate was calculated by subtraction the mass of free protein in solution from the mass of

initial total protein. For the experiments at 50 ºC glucose was added to inhibit hydrolysis in a

cellulase-glucose ratio of 1:100 (w/w).

2.7. β-glucosidase adsorption kinetics


β-glucosidase adsorption kinetics was performed on HB in 125 mL Erlenmeyer flasks with

50 mM citrate buffer (pH 4.8) supplemented with 0.02% of sodium azide per gram of

biomass at 5% and 10% (w/v) substrate concentrations. After adding 2 mg protein/g

substrate, the flasks were incubated in an air bath shaker at 50°C and 150 rpm. The

supernatant from each flask was collected, centrifuged and filtered. The protein content of the

supernatant was determined using Bradford method [33].

2.8. HPLC Analysis

Liquid samples from composition analysis of bagasse were analysed by high performance

liquid chromatography (Model 1260 Infinity Agilent Technologies HPLC with refractive

index detector IR and UV-Vis DAD). All samples were filtered using a GS cellulose ester

membrane with a pore size of 0.22 µm (Millipore) before analysis to remove solid particles.

Analysis of sugars and acetic acid were performed using an Aminex HPX- 87H column at

35°C. The mobile phase used was a solution of H2SO4 at pH 2.6 prepared with ultra pure

water (Milli- Q) with a flow rate of 0.6 ml/min. The compounds separated in the stationary

phase were monitored with a refractive index detector at 30° C for 20 min. Quantification of

furfural and hydroxymethylfurfural were performed in a Nova-Pak C18 column (Waters Co.,

Milford, MA) at 30°C, the mobile phase was a solution of acetonitrile /water (1:8 with 1%

acetic acid) previously filtered and degassed, with a flow of 0.8 ml/min. The separated

compounds were monitored by an UV-Vis detector at 280 nm and run time of 8 min.

3. Results and discussion

3.1. Effect of stirring and solids concentration on the kinetics of cellulase adsorption

Figure 1 shows the adsorption kinetics of 4 mg cellulase/g substrate (5.5 FPU/g substrate) on

Avicel and pretreated sugarcane bagasse (HB and OB) at 10% (w/v) solids concentration for

stirring rates varying from 40 to 250 rpm. Statistical analysis using Tukey’s multiple

comparison test (p<0.05) has shown that the adsorption of cellulase on Avicel is lower at 40


rpm (2.80 0.22 mg/g Avicel, Figure 1a) and that the adsorption is similar for the assays

performed with stirring rates higher than 100 rpm (3.76 0.16 mg/g Avicel).

For the lignocellulosic substrates (HB and OB), the adsorption was statistically similar for

stirring rates higher than 150 rpm, 3.75 0.01 mg/g HB and 3.85 0.03 mg/g OB, from

Figures 1b and 1c, respectively. At 40 rpm the adsorption was of 3.33 0.02 mg/g HB and

3.25 0.05 mg/g OB and at 100 rpm 3.35 0,01 mg/g HB and 3.46 0.01 mg/g OB.

These data suggest that enzymatic hydrolysis should be performed at stirring rates higher

than 100 rpm for Avicel and higher than 150 rpm for the lignocellulosic materials (HB and

OB) in order to avoid the influence of mass transfer limitations. The lower stirring rate

required for Avicel in comparison to HB and OB may be caused by structural differences in

the materials, but also by particle size. The particle size of the Avicel used in the experiments

was approximately 50 μm, while HB and OB had particle sizes <0.5 mm.

Figures 2 shows the profiles of adsorbed cellulase/g substrate at 5% and 10% (m/v) substrate

concentrations and varying stirring rates (100, 150 and 200 rpm) for Avicel, HB and OB,

respectively.

It can be noticed from Figures 2a, b and c that the adsorption equilibrium time for cellulase

on Avicel is fast, around 10 minutes for all the conditions studied. From Figures 2d, e, f and

2g, h, i it can be seen that the adsorption of cellulases on HB and OB is fast for the first 30

min and then gets slower as it approaches equilibrium, which is reached in approximately 2 h.

The majority of the studies in the literature claim that the concentration of adsorbed cellulase

gets constant in times ≤ 90 min [2, 3, 9, 34]; other studies suggest that 30 min are enough [17,

35, 36]. In this work the adsorption equilibrium time determined for Avicel was the same as

the calculated in the work of Kumar and Wyman [4] and the value was much smaller than the

determined for the pretreated biomasses (HB and OB).


Figures 2a, b and c show that the profiles of cellulase adsorption were similar at solids

concentrations of 5 and 10% (w/v) at stirring rates of 100 rpm and higher. Wang et al. [6]

claimed that the adsorption of cellulase on Avicel (mg cellulase/g Avicel) was higher at a

solid concentration of 1% (w/v) than at 5% (w/v) and concluded that cellulase adsorption on

Avicel was influenced by solids loadings. The authors, however, worked with a low stirring

rate of 40 rpm and mass transfer limitations probably influenced their results.

Figures 2d, e and f show the profiles of cellulase adsorption on HB (5% and 10%, w/v). It

can be seen that when the stirring rate was 100 rpm, mass transfer limitations took place and

the adsorption profiles were different for the different solids loadings. The maximum

adsorption capacity was higher at 5% (w/v) solids (3.94 0.01 mg/g HB) than at 10% (w/v)

solids (3.36 0.01 mg de celulase/g HB). For stirring rates of 150 rpm and higher the

adsorption profiles were similar, which shows that once mass transfer limitations are

eliminated, the adsorption is not dependent on the solids concentration. The same behavior is

shown in Figures 2g, h and i for cellulase adsorption on OB, at stirring rates of 150 rpm up

there was no influence of solids loading on the adsorption profiles.

Ouyang et al. [37] concluded that the adsorption of cellulases on bagasse produced by

traditional sulfite pulping processing was influenced by substrate concentration and was

higher at low solids concentrations (4% w/v) than at high solids concentrations (8% w/v).

The stirring rate used in this study, however, as well as in the work of Wang et al. [6], was

low, of 100 rpm, and probably the authors also confounded an effect of mass transfer

limitation with the influence of solids loading on the adsorption stage. Therefore, it can be

concluded that stirring rates in enzymatic hydrolysis of biomass should be high enough to

avoid mass transfer limitations and in this case the mass of cellulase adsorbed by mass of

substrate is not dependent on the substrate concentration.

3.2. Adsorption of β-glucosidade on pretreated bagasse


Many works have concluded that the adsorption of β-glucosidade on pure cellulose (Avicel)

is negligible [5, 38-40]. The same was found in preliminary experiments performed in this

work (data not shown).

Assuming that β-glucosidade does not adsorb on cellulose, only the kinetics of adsorption of

this enzyme on HB, the biomass with the highest lignin content (31.97%), was evaluated.

The stirring rate used in this study was set high enough (150 rpm) to avoid mass

transfer effects as previously described in the section 3.1. Figure 3 shows the supernatant

β-glucosidade concentration after 2 mg enzyme/g substrate were added to solutions

containing 5 and 10% (w/v) of HB, corresponding to concentrations of 0.1 and 0.2 mg β-

glucosidade/mL, respectively. It can be seen that β-glucosidade concentrations were

practically constant, indicating that there was no adsorption of the enzyme on the substrate.

Some studies report significant adsorption of β-glucosidases on lignocellulosic materials [8,

39, 41, 42]. Lu et al., [43] claimed that this is an unexpected behaviour as β-glucosidades do

not have cellulose binding domain (CBD) and thus have low affinity for lignocellulosic

substrates. Berlin et al. [44] found that β-glucosidade had low affinity for isolated lignin-rich

residues. The controversy must be due to the different enzyme sources, substrates and

pretreatments used in the different works, as suggested by Haven e Jorgensen [12]. These

authors found that β-glucosidase from Aspergillus niger (Novozym 188), the same enzyme

used in the present work, did not adsorb on lignin or on biomass, corroborating the present

results.

3.3. Adsorption isotherms of cellulase on Avicel and pretreated sugarcane bagasse (HB

and OB)

The contact time between enzyme and substrate in the adsorption isotherm studies was fixed

a little higher than the previously calculated adsorption equilibrium time for each substrate in

order to make sure that equilibrium was attained. Also stirring rate was set high enough (150


rpm) to avoid mass transfer effects as previously described. The contact time for Avicel was

fixed at 30 min and for pretreated bagasse (HB and OB) at 3h. Experimental data were

adjusted to the Langmuir isotherm (Equation 1) using the software OriginPro 8.0 (OriginLab,

Northampton, MA, USA).

fp

fp

adEK

EKEE

1

max (1)

In Equation 1 Ead is the amount of adsorbed enzyme (mg enzyme/g substrate); Emax is the

maximum adsorption capacity (mg enzyme/g substrate); Ef is the free enzyme concentration

(mg enzyme/mL) and Kp is the equilibrium constant (mL/mg enzyme).

It can be seen from Figure 4, which shows the adsorption isotherms for all the substrates, that

the Lagmuir isotherm adjusted the experimental data accurately. Table 1 shows the values of

maximum adsorption capacity (Emax) and of the equilibrium constant (Kp) for all the

isotherms.

The parameters for Avicel at 4 ºC and 50 ºC (Emax= 42.09 ± 1.78 mg/g Avicel and Emax=17.46

± 0.85 mg/g Avicel, respectively) are in the same order of magnitude of results previously

reported [6, 9, 35] and these authors also reported a decrease in Emax as temperature increases.

This behavior is expected in processes described by the Langmuir model [9].

For the hydrothermal bagasse (HB), however, Emax increased from of 15.81± 0.58 mg/g BH

at 4 ºC to 36.93 ± 2.73 mg/g BH at 50 ºC. An increase of the maximum adsorption capacity

with temperature was found by Zheng et al. [9] when studying the adsorption of cellulases on

lignin isolated from lignocellulosic materials submitted to different pretreatments.

According to Zheng et al. [9] and Pareek et al. [8] the protein adsorption in lignin-rich

substrates might be due to hydrophobic interactions between lignin and cellulase. An

increasing temperature would alter protein molecular structures and expose more

hydrophobic regions to the sur7roundings, therefore leading an increase in lignin


adsorption capacity.

A comparison of the values of Emax for the three substrates (Table 1) shows that the

maximum adsorption capacity seems to be higher for biomasses with higher lignin content.

For Avicel, pure cellulose, Emax was 17.41 ± 0.85 mg/g Avicel; for OB, with 4.42% of lignin,

Emax was 29.40 ± 1.20 mg/g OB and for HB, with 31.97% of lignin, Emax was 36.93 ± 2.73

mg/g HB. The maximum enzyme concentration used in the isotherm experiments was 4.5

mg/mL, equivalent to 90 mg of protein/g of biomass.

Other authors have found that more cellulase is adsorbed on materials with higher lignin

content [16, 43]. Heiss-Blanquet et al. [34], however, found that less cellulase was adsorbed

on wheat straw submitted to organosolv pretreatment than on the same biomass submitted to

steam explosion, even though the organosolv pretreated wheat straw had the lowest lignin

content.

The parameter Kp of the Langmuir isotherm was not interpreted in this work. For

purified proteins the inverse of Kp (1/Kp) is a measure of the affinity of the protein for

the substrate. As the cellulolytic complex used in this work is composed by several

proteins, this parameter has no physical meaning [45].

3.4. Cellulase adsorption on lignin

The adsorption of cellulase on lignin has been evaluated in many works [7, 9, 17, 42, 46, 47].

These authors suggest that cellulases from Trichoderma reesei adsorb unproductively on

lignin. Studies suggest that the cellulase interactions with lignin may be through electrostatic

bonds [44, 47], hydrophobic bonds [48] and hydrogen bonds [49]. The unproductive

adsorption of the cellulolytic enzymes on lignin decreases hydrolysis yields and demands for

higher enzymes dosages to enhance the efficiency of the process, leading to higher costs [46,

50].


The adsorption kinetics of cellulase on isolated lignin (IL) (the residue of enzymatic

hydrolysis of HB, 78.12 ± 0.02 % of lignin) can be seen in Figure 5a, which shows that the

adsorption equilibrium time is around 1h for this biomass. Zheng et al. [9] evaluated the

adsorption of cellulases on lignin isolated from different biomasses and pretreatments

(diluted acid and steam explosion pretreated wheat straw and steam explosion pretreated rice

straw). They found that the adsorption equilibrium time was 1h at 4 ºC and 12h at 50 ºC, a

result very different from this work.

The maximum mass of cellulase adsorbed was 2.75 ± 0.03 mg/ g IL when 4 mg enzyme/g

biomass were added. When compared with the values obtained for HB (Figure 3 d, e and f)

and OB (Figure 3 g, h and i), around 3.75 mg/g de bagasse, it can be seen that the adsorption

on lignin is lower than on biomass, but significant.

Figure 5b shows the Langmuir isotherm for the adsorption of cellulases on lignin and the

estimated parameters are shown in Table 1. The maximum adsorption capacity (Emax=11. 92

± 0.76 mg/g LI) obtained in this work is similar to the obtained by Zheng et al. [9]. Although

the adjusted value of Emax was 22 mg/g IL in their work, this value was extrapolated, as the

maximum experimental values obtained were around 15 mg/g IL, even for the higher enzyme

concentrations considered (2 mg/mL at 1% solids, equivalent to 200 mg/g IL; a much higher

value than the 90 mg enzyme/g IL used in the present work).

Analyzing Table 1 it can be concluded that the isolated lignin presented lower adsorption

capacity when compared to the other substrates. The maximum adsorption capacity of the IL

was 1.5 times lower than of Avicel, 2 times lower than of OB and 3 times lower than HB,

which suggest that cellulases have higher affinity for cellulosic/lignocellulosic substrates than

for lignin. Palonen et al. [7] and Seo et al. [42] also have found that cellulases have higher

affinity for cellulose than for isolated lignin at 4ºC. Pareek et al. [8] and Li et al. [17],


however, concluded that isolated lignin adsorbed more cellulases than the pretreated biomass

and Avicel at 4 ºC.

4. Conclusions

The results of the present work show that the adsorption of β-glucosidase from A. niger

on Avicel and bagasse pretreated by both methods (HB and OB) was negligible, which

shows that mathematical models describing the hydrolysis of these substrates should not

consider the reduction of the specific rate of cellobiose hydrolysis due to unproductive

adsorption when β-glucosidase from A. niger is used. The adsorption equilibrium time

determined for Avicel (10 min) was much lower than that of the pretreated biomasses

(2h). In addition, the results obtained show that solids concentration up to 10% (w/v) do

not influence the adsorption kinetics at sufficiently high stirring rates (>100 rpm for

Avicel, >150 rpm for pretreated bagasse). The Langmuir isotherm adjusted well the

adsorption data at 4 and 50 ºC. The adjusted parameters at 50 ºC were very different

from the parameters at 4 °C. Therefore, if the isotherms are to be used in hydrolysis

mathematical models, they must be determined in the temperature in which hydrolysis

occurs (50 ºC). The adsorption experimental data have shown that the adsorption of

cellulase is higher for materials with high lignin contents and that this enzyme has

higher affinity for cellulose than for lignin, although the adsorption in lignin is

significant.

5. Acknowledgements

The authors acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo

(FAPESP) process number 2011/02743-5, Conselho Nacional de Desenvolvimento Científico

e Tecnológico (CNPq) for financial support, and the sugar plant “Usina Tarumã” from Raizen

group, located in Tarumã, São Paulo, Brazil for the supply of sugarcane bagasse.


6. References

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Table 1- Maximum adsorption capacity (Emax) and equilibrium constant (Kp) in the

adsorption of cellulases on Avicel and hydrothermal (HB) and organosolv (OB) pretreated

bagasse adjusted to the Langmuir isotherm (Equation 1).

Substrate Temperature (°C) Emax (mg/g) Kp (mL/mg) R2

Avicel 4 42.09 ± 1.78 4.23 ± 0.71 0.95

Avicel 50 17.41 ± 0.85 4.46 ± 0.87 0.96

HB 4 15.81 ± 0.58 9.26 ± 2.10 0.95

HB 50 36.93 ± 2.73 1.28 ± 0.25 0.96

OB 50 29.40 ± 1.204 2.68 ± 0.40 0.97

IL 50 11.92 ± 0.76 13.27 ± 6.29 0.87


Figure 1


Figure 2


Figure 3


Figure 4


Figure 5

adsorption characteristics of cellulase and β-glucosidase on avicel, pretreated sugarcane bagasse,...

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