Process Biochemistry 67 (2018) 88–91

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Short communication

Inhibition of enzymatic hydrolysis of pretreated corn stover and sugar cane T

straw by laccases
⁎
Javier Rocha-Martín ,1, Claudio Martínez-Bernal, Laura S. Zamorano,
⁎
Francisco Manuel Reyes-Sosa, Bruno Díez García
Department of Biotechnology, Abengoa Research, Campus Palmas Altas, C/Energía Solar N° 1, 41014, Seville, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Laccase detoxification has been proposed to enhance the enzymatic hydrolysis of lignocellulosics by reducing the
Bioethanol effect of inhibitors formed during the pretreatment step. Herein, we investigated the effect of the laccase
Detoxification treatment on the enzymatic hydrolysis of two lignocellulosic materials pretreated by diluted acid/steam ex-
Laccase plosion through two strategies. Simultaneous laccase treatment and enzymatic hydrolysis of pretreated corn
Enzymatic hydrolysis
stover (PCS) and sugar cane straw (PSCS) led to a significant reduction of the concentration of glucose released
Lignocellulose
from 67.7 down to 39.2 g/Kg and from 69 down to 53.5 g/Kg. Laccase treatment prior to hydrolysis of both
substrates showed the same inhibitory effect. After hydrolysis of PCS and PSCS, the glucose concentration de-
creased by approximately 44 and 21% compared to the control assays, respectively. Therefore, these results do
not support the use of laccases to detoxify pretreated lignocellulosic materials for improving the bioethanol
production.

1. Introduction [4]. For bioethanol production, steam explosion is one of the most es-
tablished methods to alter the structure of lignocellulosic biomass by
The use of lignocellulosic biomass as renewable feedstock for the transforming lignin and solubilizing hemicellulose. Nevertheless,
sustainable production of biofuels, chemicals, and materials is at- during the pretreatment of biomass by steam explosion, inhibitory
tracting increasing interest from researchers over the last decade. compounds like phenols, weak acids and furan derivatives, are gener-
Nowadays, biofuel production from agricultural and municipal solid ated from the partial degradation of sugars and lignin [4]. These by-
wastes (second-generation biofuels, 2G) is not fully deployed at com- products formed during the pretreatment process can inhibit fermen-
mercial level due to the operational costs and investment requirements. tative microorganisms and the activity of the hydrolases enzymes,
Lignocellulose is principally composed of cellulose, hemicellulose and leading to a reduction of the ethanol performance [5,6]. For this reason,
lignin, with different compositions depending on the plant species [1]. a detoxification process that could remove the inhibitors formed in the
These polymers are structured forming a complex recalcitrant structure pretreatment would help to improve the enzymatic hydrolysis and in-
that hinders the enzymatic hydrolysis into monomeric and fermentable crease ethanol yield in the 2G bioethanol production process.
sugars to produce 2G biofuels [2]. Lignin forms a matrix that wraps the In this way, the detoxification of the lignocellulosic slurries before
microfibers of crystalline cellulose closely intertwined with hemi- hydrolysis and fermentation stages could be an eco-friendly alternative
celluloses in the plant cell wall. Thus, the ease or difficulty of the hy- compared to the expensive physical/chemical methods, with higher
drolysis of these substrates by cellulases is strongly dependent on the energy requirements. Laccase detoxification has been successfully ac-
composition and content of lignin [3]. complished in slurries from steam-exploded wheat straw [6–8], spruce
Therefore, an effective biomass pretreatment is needed to disrupt [9] and poplar wood [10] for the selective removal of phenolic com-
these complex substrates and remove lignin, which would increase the pounds formed during the pretreatment. In contrast, studies in which
efficiency of the enzymatic hydrolysis. Different chemical, biological laccases are supplemented to the enzymatic hydrolysis have resulted in
and physical pretreatments, including ammonia fiber explosion, che- contradictory results depending on the pretreatment and substrate
mical treatment and steam explosion at high temperatures, have been used. For example, the addition of laccase on the hydrolysis of softwood
investigated to remove lignin and deconstruct lignocellulosic materials increased the glucose conversion [9] while an inhibitory effect was

⁎
Corresponding authors.
E-mail addresses: javier.rocha@csic.es (J. Rocha-Martín), bdiez321@yahoo.es (B. Díez García).
1
Current address: Institute of Catalysis and Petrochemistry, Consejo Superior de Investigaciones Científicas (CSIC), Department of Biocatalysis, 28049, Madrid, Spain.

https://doi.org/10.1016/j.procbio.2018.01.021
Received 22 December 2017; Received in revised form 22 January 2018; Accepted 26 January 2018
Available online 02 February 2018
1359-5113/ © 2018 Elsevier Ltd. All rights reserved.
J. Rocha-Martín et al. Process Biochemistry 67 (2018) 88–91

observed during the hydrolysis of hardwood or agricultural residues 2.3. Strains and growth conditions
like wheat straw [11,12].
In the present work, we explored the effect of the laccase treatment M. thermophila industrial strain derived from C1 UV18-25 [17,18]
on the enzymatic hydrolysis of two different lignocellulosic materials was obtained under license from Dyadic International Inc. (Jupiter,
which have been pretreated by diluted acid/steam explosion. Two Florida). Strains were grown according to Emalfarb et al. [19]. After
strategies have been developed and evaluated: simultaneous laccase five days of growth, the culture was harvested and centrifuged at
treatment and enzymatic hydrolysis of pretreated sugar cane straw 12,000 rpm for 40 min at 4 °C. The extracellular enzyme fraction from
(PSCS) and corn stover (PCS) (strategy 1); and a previous laccase the supernatant was obtained by filtration with 0.45 μm nylon filters,
treatment and a subsequent hydrolysis step of both pretreated sub- and the addition of 50 mM sodium acetate buffer at pH 5.0. This en-
strates (strategy 2). For this purpose, the hydrolysis was performed by zyme fraction, termed the complete cocktail (WC), was used to perform
using an industrial enzymatic cocktail produced by Myceliophthora the hydrolysis experiments. Protein concentration was determined by
thermophila [13] at high solid loadings and limited mixing. Significant the method of BCA with serum albumin as the standard.
reduction of the glucose concentration after enzymatic hydrolysis was
found when any of these two strategies were used. 2.4. Enzyme activity assays

Assays for laccase activity were carried out using 5 mM ABTS as the
2. Materials and methods
reducing substrate in 0.1 M sodium acetate pH 5.0 at 25 °C. The laccase
activity was measured using ABTS (εABTS = 36,000 M−1 × cm−1) as
2.1. Enzymatic reagents
substrate. One activity unit was defined as the amount of enzyme that
oxidized 1 μmol of ABTS per min.
A commercial bacterial laccase (BLc) [14] and a commercial (re-
combinant) fungal laccase from the ascomycete M. thermophila
2.5. Enzymatic hydrolysis and laccase treatments
(NS50003) (FLc) (Novozymes, Bagsvaerd, Denmark) were used for
evaluation of laccase treatments. 2,2′-azino-bis3-ethylbenzothiazoline-
Enzymatic hydrolysis experiments were carried out in 100 mL ISO
6-sulphonic acid (ABTS) was purchased from Sigma–Aldrich Co. (MO,
bottles with airtight screw caps at 50 °C for 72 h in an orbital shaker
USA). Total protein content was determined by Bicinchoninic acid
incubator according to Alcantara et al. [15]. The total solids content
(BCA) protein assay kit from Applichem (Darmstadt, Germany). All
was adjusted to 20% with water (TS; based on the substrate). Before
other reagents were analytical or HPLC grade.
enzyme addition, the pH value of the reaction mixture was adjusted to
5.5 with 25% NH4OH. Samples aliquots were withdrawn at 0 and 72 h
2.2. Biomass of hydrolysis course and processed for HPLC analysis according to
Kristensen et al. [20]. The analytes were quantified in weight/weight
Pretreated sugar cane straw and corn stover (from now on, PSCS (g/Kg). All hydrolysis experiments were performed in triplicate, and the
and PCS) were obtained from Abengoa Bioenergy Biomass Pilot Plant in results were expressed as the mean values.
York, Nebraska, USA. A 1-inch hammer mill screen was used to mill the
pretreated biomass. Pretreatment of the milled or not milled material 2.5.1. Strategy 1: simultaneous laccase treatment and hydrolysis of
was performed according to Alcántara et al. [15]. The final dry matter pretreated biomass
content of PSCS and PCS was 39.9 and 43.3%. Standard analytical Pretreated and milled biomass were used to perform these experi-
procedures (LAP-002, LAP-003, and LAP-019) from the National Re- ments. TS was adjusted to 20% with water. The enzyme loading was
newable Energy Laboratory (NREL) were used to determine the com- 10–11 mg protein/g glucan of WC for PCS hydrolysis and 15–16 mg
position of the lignocellulosic materials of this study (https://www. protein/g glucan of WC cocktail for PSCS hydrolysis. When indicated,
nrel.gov/bioenergy/biomass-compositional-analysis.html). The com- 0.1–1 mg protein/g glucan of FLc (specific activity of 65,000 U/mg) or
positions of PSCS and PCS are given in Table 1. In experiments where BLc (specific activity of 5000 U/mg) laccases were added to the reaction
milled biomass was used, the obtained biomass samples after pre- mixtures. Control assays were performed under the same conditions,
treatment were freeze-dried. In these cases, dry biomass was ground by without the addition of laccase.
mechanical fractionation in a Polymix PX-MFC 90D system (Kinemática
GMBH, Germany). The grinding conditions were 6,000 rpm, room 2.5.2. Strategy 2: prior laccase treatment and subsequent hydrolysis of
temperature and screen size of 0.2 mm [16]. The water content of the pretreated biomass
biomass samples was determined using a heating balance (Shimadzu, Prior to enzymatic hydrolysis, the diluted acid/steam-explosion
Kyoto, Japan). biomass was incubated with and without 1 mg protein/g glucan of FLc

Table 1
Chemical composition of lignocellulosic substrates. All values are in percent of total content on a dry matter basis. Samples were pre-treated using steam explosion method as described in
experimental procedures.

Insoluble fraction composition (% DM) Soluble fraction composition (% DM)

Component PCS PSCS Component PCS PSCS

Cellulose Glucose 1.1 ± 0.1 2.96 ± 0.05

Glucan 35.41 ± 0.25 36.34 ± 0.21 Glucose-oligomer 1.97 ± 0.09 0.15 ± 0.01
Hemicellulose Xylose 10.12 ± 0.16 8.06 ± 0.1
Xylan 6.5 ± 0.45 4.37 ± 0.34 Xylose-oligomer 6.3 ± 0.22 0.27 ± 0.09
Arabinan 0.79 ± 0.06 n.d Acetic Acid 1.19 ± 0.01 2.08 ± 0.07
Mannan 0.54 ± 0.04 n.d Furfural 0.31 ± 0.05 1.1 ± 0.08
Klason lignin 21.52 ± 0.07 18.51 ± 0.09 HMF 0.23 ± 0.07 0.28 ± 0.06
Ash 3.7 ± 0.1 2.41 ± 0.07
Total 68.46 ± 0.89 60.59 ± 0.95

HMF, 5-hydroxymethylfurfural; n.d., not determined.

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J. Rocha-Martín et al. Process Biochemistry 67 (2018) 88–91

(65,000 U/mg) for 24 h at 50 °C in an orbital shaker (150 rpm). When

indicated, 1% (w/w) SGA was added to the mixture. Then, the mixture
was incubated at 70 °C for 2 h to inactivate the enzyme. After laccase
treatment, the hydrolysis of PCS or PSCS started after the addition of
the enzymatic cocktail to the whole slurry. The enzyme loading was
8–9 mg WC/g glucan for PCS hydrolysis and 12–13 mg WC/g glucan for
PSCS hydrolysis. Control assays were performed under the same con-
ditions, without the addition of laccase.

2.6. Analytical methods

Soluble sugars and compounds resulted from their degradation were

analysed by high performance liquid chromatography (HPLC) in an
Agilent 1260 chromatograph (Agilent Technologies, Germany) using an
Aminex HPX-87H 300 mm × 7.8 mm column with 9 μm particle size Fig. 1. Glucose released after 72 h of enzymatic hydrolysis of PCS and PSCS in the pre-
sence/absence of laccase. Enzymatic hydrolysis of PCS (filled bars): Control, 10 mg WC/g
(Bio-Rad, California, USA) according to Rocha-Martín et al. [16].
glucan; FLc, 10 mg WC/g glucan supplemented with 1 mg FLc/g glucan; BLc, 10 mg WC/g
glucan supplemented with 1 mg BLc/g glucan; and WC, 11 mg WC/g glucan. Enzymatic
3. Results and discussion hydrolysis of PSCS (unfilled bars): Control, 15 mg WC/g glucan; FLc, 15 mg WC/g glucan
supplemented with 1 mg FLc/g glucan; BLc, 15 mg WC/g glucan supplemented with 1 mg
3.1. Pretreated biomass composition BLc/g glucan; and WC, 16 mg WC/g glucan. 100% corresponds to the glucose con-
centration released after the enzymatic hydrolysis of standard PCS (68 g/kg) and standard
PSCS (68.9 g/kg).
Table 1 shows the carbohydrate and lignin composition of PCS and
PSCS materials. The pretreatment increased the cellulose proportion of
the WIS fraction (68.5% and 65.94%, respectively) due to the solubi- efficiency in phenol removal and can act on a wider range of substrates.
lization of much of the hemicellulose and degradation. Accordingly, a Therefore, the lower negative effect of BLc enzyme in the enzymatic
lower proportion of the hemicellulose (7.8% and 4.37%) remained in hydrolysis could be due to the lower laccase activity of this preparation.
the fraction of WIS and a high xylose content (16.42% and 8.34% w/w) On the other hand, supplementation with FLc or BLc in the hydrolysis of
was determined in the liquid fraction. Different degradation products both pretreated substrates did not significantly alter the xylose con-
from the biomass due to the pretreatment process were determined in centration (data not shown). This fact contrasts with the result recently
the liquid fractions. Among them, acetic acid was the most pre- obtained by Oliva-Taravilla et al. [23] in which a reduction of xylose
dominant. In lower concentrations, furfural (FF) and 5-HMF (5-(hy- released in the presence of laccase was observed after 72 h. Xylanases
droxymethyl) furfural) were detected (Table 1). FF and HMF are formed mainly act on soluble xylooligosaccharides, and so, these enzymes
from the degradation of pentoses and hexoses, respectively [21]. Acetic could be less susceptible than cellulases to the inhibition by oligomeric
acid is formed by sugar degradation and as a result of the hydrolysis of phenols.
the acetyl groups contained in the hemicellulose structure [21]. It is known that laccases perform an oxidative polymerization pro-
cess producing unstable radicals. These phenolic polymers could have a
3.2. Effect of laccase supplementation on the enzymatic hydrolysis of significant negative effect on the hydrolytic activities. Martin-Sampedro
pretreated lignocellulosic biomass et al. [24] observed that laccase treatment of steam-exploded Eucalyptus
wood chips produced some lignin fractions of low molecular weight and
The effect of laccase addition on the sugar release by enzymatic small lignin fragments linked to carbohydrates which could be solubi-
hydrolysis of milled PCS and PSCS was evaluated (Strategy 1). External lized. Thus, oligomeric phenols formed after the pretreatment of lig-
addition of laccases could remove the phenolic compounds from pre- nocellulosic materials could have a stronger negative effect than soluble
treated lignocellulosic materials boosting the enzymatic hydrolysis. phenols during the enzymatic hydrolysis. Moreover, cellobiose con-
Thus, in the first instance, 1 mg BLc or FLc/g glucan was added to the centration after 72 h of hydrolysis of both materials was analysed. For
enzymatic hydrolysis mixture. In the absence of laccase, glucose con- all laccase-treated assays, cellobiose accumulation was not detected at
centration after 72 h of hydrolysis of PCS was 67.7 g/Kg as shown in 72 h of hydrolysis (lower than 1 g/Kg). Oliva-Taravilla et al. [25] de-
Fig. 1. In contrast, when the hydrolysis was performed with 1 mg FLc/g scribed an adverse effect of laccase on enzymatic hydrolysis of steam-
glucan, glucose concentration decreased approximately to 39.2 g/Kg exploded wheat straw and Sigmacell (crystalline cellulose). This result
regarding the control in the absence of laccase (reduction in glucose was explained by the inhibition of β-glucosidase activity and the
conversion of 42%). When the enzymatic hydrolysis was supplemented competition for cellulose binding sites between cellulase and laccase
with 1 mg BLc/g glucan, glucose concentration after 72 h of hydrolysis molecules. As it is well known, endo- and exoglucanase enzymes are
of PCS was only reduced by 5 g/Kg (Fig. 1). Similar results were ob- acting on cellulosic polymer at the first stage of the hydrolysis, and β-
tained in the PSCS hydrolysis in the presence of 1 mg FLc/g glucan. As glucosidase is sequentially acting afterward [26]. Thus, this result
shown in Fig. 1, enzymatic hydrolysis of PSCS in the presence of FLc led suggests that derived products from the laccase action affected more
to a reduction of glucose concentration from 69 to 53.5 g/Kg compared significantly the cellobiohydrolases and endoglucanases than β-gluco-
to the control in the absence of FLc (reduction in glucose conversion of sidase during the hydrolysis process [9,27].
22%). The addition of only 0.1 mg laccase/g glucan was sufficient to
obtain the same negative effect for both substrates (data not shown). In 3.3. Effect of the separate laccase treatment and hydrolysis of pretreated
contrast, when BLc was added to the hydrolysis reaction, there was not lignocellulosic biomass
any significant change in glucose concentration.
In the light of these results, the laccase activity of the two com- A second strategy (Strategy 2) was tested for detoxification of PCS
mercial preparations and WC was investigated using ABTS as substrate. and PSCS. For this purpose, prior to saccharification step, PCS and PSCS
Although laccase activity was not detected in WC, BLc had 13-fold less were treated with FLc in the presence and absence of SGA (mediator),
activity than FLc. Some researchers have found that bacterial laccases as described in Methods section. After 24 h of incubation, FLc was in-
as BLc have a lower redox potential than fungal laccases (+730 mV activated by heating at 70 °C for 2 h (laccase activity was reduced to less
versus 450 mV) [22]. As a result, fungal laccases display higher than 5% of its initial activity). When PCS was treated with FLc before

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