Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin
and Improves Saccharification
Jorge Rencoret
a‡
, Antonio Pereira
a‡
, José C. del Río
a
, Angel T. Martínez
b
, Ana
Gutiérrez
a,
*
a
Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes 10,
E-41012 Seville, Spain
b
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid,
Spain
* Corresponding author: Tel.: +34 954624711, Fax: +34 954624002, E-mail:
anagu@irnase.csic.es (A. Gutiérrez)
‡
These authors contributed equally to this work
Postprint of: BioEnergy Research 9(3) 917-930 (2016)
Abstract Agricultural by-products such as wheat straw are attractive feedstocks for the
production of second generation bioethanol due to their high abundance. However, the
presence of lignin in these lignocellulosic materials hinders the enzymatic hydrolysis of
cellulose. The purpose of this work is to study the ability of a laccase-mediator system
to remove lignin improving saccharification, as a pretreatment of wheat straw, and to
analyze the chemical modifications produced in the remaining lignin moiety. Up to 48%
lignin removal from ground wheat straw was attained by pretreatment with Pycnoporus
cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) as mediator, followed by
alkaline peroxide extraction. The lignin removal directly correlated with increases
(~60%) in glucose yields after enzymatic saccharification. The pretreatment using
laccase alone (without mediator) removed up to 18% of lignin from wheat straw.
Substantial lignin removal (37%) was also produced when the enzyme-mediator
pretreatment was not combined with the alkaline peroxide extraction. Two-dimensional
nuclear magnetic resonance (2D NMR) analysis of the whole pretreated wheat straw
material swollen in dimethylsulfoxide-d
6
revealed modifications of the lignin polymer,
including the lower number of aliphatic side-chains involved in main β-O-4' and β-5'
inter-unit linkages per aromatic lignin unit. Simultaneously, the removal of p-
hydroxyphenyl, guaiacyl and syringyl lignin units and of p-coumaric and ferulic acids,
as well as a moderate decrease of tricin units, was observed without a substantial change
in the wood polysaccharide signals. Especially noteworthy was the formation of Cα-
oxidized lignin units during the enzymatic treatment.
Keywords: Enzymatic delignification, Laccase-mediator, Lignin, wheat straw, 2D
NMR, Bioethanol
Introduction
Agricultural and forestry residues represent an enormous source of readily available
biomass for biofuel production without the need for additional land cultivation. Among
agricultural residues, wheat straw is potentially one of the most favorable feedstocks in
terms of the quantity of biomass available [1]. However, in spite of the availability of
these residues or other lignocellulosic biomass, their variable composition and
recalcitrance represent some technical and economic challenges. Cellulose,
hemicelluloses and lignin are the three main components of lignocellulosic biomass
linked into a complex matrix highly resistant to chemical and biological conversion.
These components are more resistant to being broken down and fermented than starch
and sucrose in the conventional food crops, making the conversion processes more
complicated. Biofuel production from lignocellulosic material requires deconstruction
of the cell-wall into individual polymers, and hydrolysis of the carbohydrates into
monomeric sugars. One of the major factors causing biomass recalcitrance towards
saccharification is correlated with the content and composition of lignin [2-4].
Lignin is a three-dimensional polymer constituted by phenylpropanoid subunits
linked together by different ether and carbon-carbon bonds. Lignin is intimately
interlaced with hemicelluloses in the plant cell wall forming a matrix to cover the
largely crystalline cellulose microfibrils. Its aromatic nature and complex structure
makes lignin degradation very difficult. Lignin has been shown to have a detrimental
effect on the hydrolysis of biomass because it physically hinder the access of cellulases
and also binds them reducing activity [5]. Therefore, biomass pretreatment to remove
lignin is essential for the enzymatic hydrolysis of lignocellulose. Biotechnology can
contribute to plant biomass deconstruction by providing biocatalysts being able to
degrade or modify lignin [6]. Due to the complex structure of lignin, including
heterogeneity of the monomers and linkages, which are not hydrolyzable, these
biocatalysts must be oxidative (i.e., oxidoreductases) and also nonspecific. Among
these, laccases seem to be the most suitable enzymes for industrial application, because
they only require dioxygen as oxidant and also because it can be produced on a large
scale [7].
Laccases (phenoloxidases, EC 1.10.3.2) are multicopper oxidases that oxidize
substituted phenols using molecular oxygen as the final electron acceptor. The direct
action of laccases on lignin is, in principle, restricted to the phenolic units that only
represent a small percentage of the total polymer [8], a fact that limits their
biotechnological application. However, the discovery that some synthetic compounds
can act as electron carriers between the enzyme and the final substrate [9], 1-
hydroxybenzotriazole (HBT) being among the most efficient ones [10], has expanded
the utility of laccases. A number of studies have confirmed the potential of laccase-
mediator systems for paper pulp delignification [11,12], pitch control [13], polymer
modification [14], and other applications in the forest industry [15] and bioethanol
production from pretreated lignocellulose [16-18]. Recently, the ability of fungal
laccases to remove lignin (when applied in combination with redox mediators) from
whole [19,20] and ensiled [21] lignocellulosic biomass, making cellulose accessible to
hydrolysis, was reported. Additionally, the use of laccases in bioethanol production has
also been reported as a biotechnological tool for the removal of phenolic inhibitors
generated during steam explosion of lignocellulosic feedstocks [22], although some
recent work also shows that some laccase-derived compounds can affect negatively the
enzymatic hydrolysis [23].
A previous work on the laccase-mediator treatment of acid-pretreated wheat straw
has been reported [17]. The novelty of the pretreatment described here, based on the use
of a fungal laccase from the basidiomycete Pycnoporus cinnabarinus [24] in
combination with HBT as mediator [25], is that it was applied directly in the ground
wheat straw feedstock (without a previous chemical pretreatment). Additionally, in the
present study, the modification of lignin structure in the pretreated lignocellulosic
material was analyzed in-depth by two-dimensional nuclear magnetic resonance (2D
NMR) spectroscopy of the whole sample at the gel state [26,27]. Besides lignin
modification and removal, the effect of the laccase-mediator on the saccharification
yield from the pretreated wheat straw feedstock was also assessed.
Material and Methods
Lignocellulosic feedstock, enzyme and mediator
Wheat straw (Triticum durum var. Carioca) was harvested from an experimental field in
Seville (Spain). Wheat straw samples were air-dried and grounded in an IKA MF10
cutting mill to pass through a 100-mesh screen and then finely milled using a Retsch
PM100 planetary mill at 400 rev·min
-1
(with 5 min breaks after every 5 min of milling)
using a 500 mL agate jar and agate ball bearings (20×20 mm). The total ball-milling
time for the samples was 5 h. The chemical composition of wheat straw feedstock (as %
dry weight) was: glucose, 39.4 ± 0.7; xylose, 16.0 ± 0.3; arabinose, 3.8 ± 0.2; soluble
acid lignin, 1.5 ± 0.1; Klason lignin, 16.0 ± 0.3).
The laccase was provided by INRA (Marseille, France) and was obtained from a
laccase-hyperproducing strain of the fungus Pycnoporus cinnabarinus (Herpoël et al.
2000). Its activity was measured as initial velocity during oxidation of 5 mM ABTS
from Roche to its cation radical (
436
29300 M
-1
·cm
-1
) in 0.1 M sodium acetate (pH 5) at
24ºC. The laccase activity of the enzyme preparation was 102 U/ml (specific activity
156 U/mg). One activity unit (U) was defined as the amount of enzyme transforming 1
µmol of ABTS per min. HBT (1-hydroxybenzotriazole) from Sigma–Aldrich
(Steinheim, Germany) was used as mediator.
Laccase-mediator treatments
The wheat straw samples were treated with the P. cinnabarinus laccase in the presence
(and absence) of HBT, as mediator (in duplicate experiments). Laccase doses of 13 and
65 U·g
-1
were assayed, together with 5%, 10% and 20% HBT, all referred to straw dry
weight. The treatments were carried out in 200-mL pressurized bioreactors (Labomat,
Mathis) placed in a thermostatic shaker at 170 rev·min
-1
and 50 ºC, using 4 g (dry
weight) samples at 6% (w:w) solids loading in 50 mM sodium tartrate buffer (pH 4)
under O
2
atmosphere (2 bars) for 24 h. Additionally, the treatment with laccase (65 U·g
-
1
) and HBT (20%) was also performed in presence of 0.05% Tween 20, to test the effect
of adding a surfactant in both the enzymatic delignification and enzymatic hydrolysis of
wheat straw [28]. After the treatment, the samples were filtered through a Büchner
funnel and washed with 1 L of water. In some cases, a subsequent alkaline peroxide
extractions step was performed after the enzymatic pretreatment. In this case,
enzymatically treated samples at 6% (w:w) solids loading were submitted to a peroxide-
reinforced alkaline extraction using 1% (w:w) NaOH and 3% (w:w) H
2
O
2
(also with
respect to sample dry weight) at 80 ºC for 90 min, followed by water washing [29]. The
solids loading for the latter step was achieved by determining the moisture content of an
aliquot. Treatments with laccase (65 U·g
-1
) alone (without mediator) and controls
without laccase and mediator, were also performed (followed in both cases by the
corresponding alkaline peroxide extraction). A control with mediator alone was not
included taking into account the results from previous studies. Duplicate experiments of
a representative (65 U·g
-1
laccase and 20% HBT) laccase-mediator treatment (including
control, laccase alone and laccase-HBT) were performed to estimate the variability in
biological replicates (as shown in Table 1 footnote). A one-way analysis of variance
(ANOVA) was conducted to compare the effects of the different enzymatic treatments
on the lignin removal, and on the releases of glucose and xylose. Post hoc pairwise
comparisons, using the Tukey HSD test, were performed in order to determine which
means are significantly different from each other. Klason lignin content was estimated
(in triplicate measurements) according to T222 om-88 [30]. The data from both
biological and technical replicates were averaged. Weight loss (%) were determined for
all the treatments (Table 1) with respect to the control without enzyme-mediator and
alkaline extraction: Laccase (65 U·g
-1
), 2.3%; Laccase (65 U·g
-1
)-HBT (20%), 6.1%;
Control/alkaline peroxide, 0.8%; Laccase (65 U·g
-1
)/alkaline extraction, 4.8%; Laccase
(13 U·g
-1
)-HBT (10%)/alkaline extraction, 7.3%; Laccase (65 U·g
-1
)-HBT (5%)/alkaline
extraction, 7.7%; Laccase (65 U·g
-1
)-HBT (20%)/alkaline extraction,13.4%; Laccase
(65 U·g
-1
)-HBT (20%)/T20/alkaline extraction, 13.5%. The weight loss of control
without enzyme-mediator and alkaline extraction with respect to initial wheat straw was
12.7%.
Saccharification of treated wood
The laccase-pretreated samples were hydrolyzed with a cocktail containing commercial
enzymes (from Novozymes, Bagsvaerd) with cellulase (Celluclast 1.5 L; 2 FPU·g
-1
) and
β-glucosidase (Novozym 188; 6 U·g
-1
) activities, at 1% solids loading in 3 mL of 100
mM sodium citrate (pH 5) for 72 h at 45 ºC, in a thermostatic shaker at 170 rev·min
-1
(in triplicate experiments). The specific activities of Celluclast 1.5L and β-glucosidase
are 700 EGU/g and 250 CBU/g, respectively.
The different monosaccharides released were determined as alditol acetates [31] by
GC. An HP 5890 gas chromatograph (Hewlett-Packard, Hoofddorp, The Netherlands)
equipped with a split−splitless injector and a flame ionization detector was used. The
injector and detector temperatures were set at 225 and 250 °C, respectively. Samples
were injected in the split mode (split ratio 10:1). Helium was used as the carrier gas.
The capillary column used was a DB-225 (30 m × 0.25 mm i.d., 0.15 μm film thickness;
Agilent J&W). The oven was temperature-programmed from 220 °C (held for 5 min) to
230 °C (held for 5 min) at 2 °C min
−1
. Peaks were quantified by area and glucose and
xylose were used as standards to elaborate calibration curves. The data from both
biological and technical replicates were averaged.
2D NMR spectroscopy
For gel-state NMR experiments, ∼70 mg of finely divided (ball-milled) wheat straw
samples were directly transferred into 5-mm NMR tubes, and swelled in 1 mL of
DMSO-d
6
, forming a gel inside the NMR tube [26,27].
HSQC 2D-NMR spectra were acquired at 25ºC on a Bruker AVANCE III 500 MHz
spectrometer fitted with a cryogenically cooled 5 mm TCI gradient probe with inverse
geometry (proton coils closest to the sample). The 2D
13
C-
1
H correlation spectra were
carried out using an adiabatic HSQC pulse program (Bruker standard pulse sequence
‘hsqcetgpsisp2.2’), which enabled a semiquantitative analysis of the different
13
C-
1
H
correlation signals. Spectra were acquired from 10 to 0 ppm (5000 Hz) in F2 (
1
H) using
1000 data points for an acquisition time (AQ) of 100 ms, an interscan delay (D1) of 1 s,
and from 200 to 0 ppm (25,168) in F1 (
13
C) using 256 increments of 32 scan, for a total
acquisition time of 2 h 34 min. The
1
J
CH
used was 145 Hz. Processing used typical
matched Gaussian apodization in
1
H and a squared cosine bell in
13
C. The central
solvent peak was used as an internal reference (δ
C
/δ
H
39.5/2.49). The
13
C-
1
H correlation
signals from the aromatic region of the spectrum were used to estimate the content of
lignin, p-coumaric acid, ferulic acid and tricin (compared with the amorphous
polysaccharide content, estimated from the anomeric xylose and glucose signals), and
the lignin composition in terms of G, S and oxidized S (S') units. Correlations in the
aliphatic-oxygenated region were used to estimate the inter-unit linkage and end-unit
abundances in lignin. The intensity corrections introduced by the adiabatic pulse
program permits to refer the side-chain integrals to the previously obtained number of
lignin units.
Results and discussion
Delignification of wheat straw by laccase-HBT
Wheat straw lignin is a guaiacyl–rich lignin [32] that usually is reported to be more
resistant to degradation than the syringyl type [33]. For this reason, and also based on
