A commercial laccase-mediator system to delignify and improve saccharification of the fast-growing Paulownia fortunei (Seem.) Hemsl.

TL;DR: The presence of oxidized lignin compounds in the filtrates of the enzymatic treatments – such as vanillin, vanillic acid, syringaldehyde and syringic acid – conclusively demonstrates the ability of L/MeS treatment to oxidize and depolymerize the lign in paulownia wood.

Abstract: 10 paginas.-- 5 figuras.-- 3 tablas.-- 41 referencias.-- El archivo adjunto no es la ultima version postprint

Summary

Introduction

• Its properties such a lightweight, strength, insulation, fast drying, high ignition point and rot resistance makes it popular for house construction and furniture making.
• Paulownia also has a potential use as energy crop for the production of bioethanol (Ye et al. 2016; Domínguez et al. 2017) also by enzymatic hydrolysis (Chandra et al. 2007) and subsequent fermentation by yeasts (Park et al. 2013) or bacteria (Ng et al. 1981) to ethanol.
• Different physical and chemical pretreatments have been proposed for a better saccharification of paulownia wood, such as dilute acid, alkali, ultrasonic-assisted alkali treatments (Ye et al. 2015, 2016), autohydrolysis (Domínguez et al. 2017), and steam explosion (Radeva et al. 2012).
• Methyl syringate (MeS) will serve as mediator, which is obtained from syringic acid present in pulp and paper side-streams (Rosado et al. 2012).

Material and methods

• Paulownia wood, enzymes and mediator: 3-year-old P. fortunei trees were provided by the University of Huelva .
• At the 95% confidence level, the enzymatic experiments are smaller than the differences found between the control, laccase alone, and L/MeS treatments.
• The 2D HSQC NMR experiments were performed at the gel state, which is an in situ analysis of the whole cell-wall (Kim et al. 2008, Rencoret et al. 2009b).
• The 1H-13C correlation signals from the aromatic region of the spectrum were used to estimate the content of lignin (relative to the content of amorphous carbohydrates, estimated from the anomeric xylose and glucose signals), and the lignin composition in terms of G, S and oxidized S (S′) and G (G′) units.
• The extraction process was repeated three times, using fresh chloroform each time and the supernatants were combined.

Result and discussion

• Delignification of paulownia wood by the enzymatic pretreatment The Klason lignin content of the paulownia control, processed as the full enzymatic treatment but without laccase and mediator, was only slightly reduced from 23.8 to 22.0% with respect to the initial paulownia lignin content (Table 1).
• This is the effect of alkaline peroxide extractions.
• The treatment with laccase alone only resulted in a 5% lignin reduction compared to the control sample.
• This low efficiency of laccase alone is well known (Lai, 1992).
• The pretreatment with L/MeS, however, resulted in a lignin decrement up to 24%, with respect to the control, but this delignification is degree is lower than reported about eucalypt wood Page 7 of 23 https://mc.manuscriptcentral.com/holz.

Conclusions

• The lignin in paulownia wood was modified and partially removed by pretreatment with recombinant laccase of M. thermophila in the presence of MeS as phenolic mediator, in spite of its high G-unit content.
• The L/MeS system acts selectively on the lignin polymer, leaving the carbohydrate signals in the HSQC spectra practically unaffected.
• The utilization of The commercially available L/MeS system has a high application potential as pretreatment for the saccharification of paulownia wood.
• The authors thank Dr. Manuel Angulo (CITIUS, University of Seville) for technical assistance with the NMR experiments.

Figures

Table 1. Laccase-mediator treatment of paulownia wood (material recovery and final lignin content) and subsequent Celluclast hydrolysis (amount of glucose and xylose per 100 g of sample)* Samples 1) Laccase-mediator treatment 2) Celluclast hydrolysis (2 FPU) Recovery (%) Lignin (%) Glucose (%) Xylose (%) Initial paulownia 100 (4 g) 23.8 ± 0.2 31.5 ± 0.3 6.7 ± 0.1 Control * 83 (3.32 g) 22.0 ± 0.1 a 37.3 ± 0.2 a 7.3 ± 0.1 a Laccase (50 U g -1 ) † 80 (3.21 g) 20.9 ± 0.3 a 39.4 ± 0.4 b 7.8 ± 0.1 a Laccase (50 U g -1 )-MeS (3%) † 76 (3.03 g) 16.7 ± 0.2 b 51.3 ± 0.4 c 9.8 ± 0.3 b * Means ± StD presented were obtained from triplicate determination. Letters next to the StD, from the Tukey test, show: no significant differences to the control (a), significant differences (b) and significantly different from both the control and the Lalone results (c), at the 0.05 level. * followed by alkaline-peroxide extraction (4 cycles).

Figure 1. 2D-HSQC NMR spectra of paulownia after laccase-mediator treatments followed by alkaline peroxide extractions (4 cycles): Control without enzyme (a), treated with 50 U g-1 laccase (b) and treated with 50 U g-1 laccase and 3% MeS (c). Correlation signals in reddishbrown color largely correspond to lignin, whereas signals in cyan color belong to carbohydrates.

Figure 2. Expanded aliphatic oxygenated (δC/δH 50-110/2.5-6.0, top) and aromatic (δC/δH 100-125/6.0-8.0, bottom) regions of the 2D-HSQC NMR spectra of paulownia samples; Control without enzyme (a, d), treated with 50 U g-1 enzyme (b, e) and treated with 50 U g-1 enzyme and 3% MeS. The lignin and carbohydrate signal assignments are listed in Table 2 and the lignin structures identified are depicted in Figure 3. Correlation signals of MeS in the HSQC spectrum from laccase-mediator treated paulownia are observed at δC/δH 52.1/3.82 (C/H in MeS) and δC/δH 106.4/7.20 (C2/H2 and C6/H6 in the aromatic ring).

Table 2. Assignments of the 1H/13C correlation signals in the HSQC spectra of the whole cellwalls from treated paulownia.

Full text

For Review Only
1
1
Short title: Enzymatic pretreatment of P. fortunei
A commercial laccase-mediator system to delignify and improve
saccharification of the fast-growing Paulownia fortunei
Jorge Rencoret
1,*
, Antonio Pereira
1
, Gisela Marques
1
, José Carlos del Río
1
, Ángel T.
Martínez
2
and Ana Gutiérrez
1
1
Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, E-41012-Seville, Spain
2
Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040-Madrid, Spain
* Corresponding author: (Tel: +34 954624711; E-mail: jrencoret@irnase.csic.es)
Abstract: It was demonstrated for the first time that a laccase-based enzymatic pretreatment
is able to delignify fast-growing paulownia species. The treatment was performed with a
commercial low-redox potential laccase isolated from Myceliophthora thermophila and
methyl syringate (MeS) as a natural phenolic mediator. Up to 24% lignin removal was
attained by the laccase-mediator treatment (L/MeS), followed by alkaline peroxide extraction
in a multistage sequence. The reduction in lignin content was accompanied by a significant
improvement in the subsequent enzymatic saccharification ending up in 38% glucose and
34% xylose yields. The structural modifications of the lignin were analyzed in situ by 2D-
NMR spectroscopy. A considerable removal of guaiacyl and syringyl lignin units with respect
to the carbohydrate signals was visible as well as the cleavage of β-O-4
ʹ, β
-5
ʹ and β
-β
ʹ
linkages leading to elevated amounts of Cα-oxidized guaiacyl and syringyl units. The
presence of oxidized lignin compounds in the filtrates of the enzymatic treatments ─ such as
vanillin, vanillic acid, syringaldehyde and syringic acid ─ conclusively demonstrates the
ability of L/MeS treatment to depolymerize the lignin in paulownia wood.
Keywords: pretreatment; paulownia; lignin; 2D-NMR; laccase; saccharification
Introduction
Paulownia genus comprises of nine species and a few hybrids native to China and East Asian
(Zhu et al. 1986), which are grown commercially for the production of hardwood timber
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(Bergmann 1998), while Paulownia is a fast-growing tree with a high biomass production
(Jiménez et al. 2005). Its properties such a lightweight, strength, insulation, fast drying, high
ignition point and rot resistance makes it popular for house construction and furniture making.
It was also investigated as a raw material for the production of chemical pulp (Jiménez et al.
2005; Caparrós et al. 2007, 2008). Paulownia fortunei is the most suitable species to this
purpose (Rai et al. 2000). P. fortunei shows extraordinary high growth rates under suitable
conditions (Ede et al. 1997), reaching up to 15-20 m high in only 5-7 years, and annual
productions as high as 150 t ha
-1
y
-1
(Jiménez et al. 2005).
Paulownia also has a potential use as energy crop for the production of bioethanol (Ye
et al. 2016; Domínguez et al. 2017) also by enzymatic hydrolysis (Chandra et al. 2007) and
subsequent fermentation by yeasts (Park et al. 2013) or bacteria (Ng et al. 1981) to ethanol.
To this purpose, the cross-linked a macro-molecular assembly of the cell wall must be
submitted to a pretreatment (Chen 2014). Especially lignins limits the enzymatic hydrolysis
by steric hindrance of the enzymes access to the polysaccharides and their inactivation
(Kumar and Wyman 2009; Rahikainen et al. 2013).
Paulownia wood contains ~24% lignin, which is composed of guaiacyl (G) and
syringyl (S) units with an S/G ratio of 0.66 (Rencoret et al. 2009a). Different physical and
chemical pretreatments have been proposed for a better saccharification of paulownia wood,
such as dilute acid, alkali, ultrasonic-assisted alkali treatments (Ye et al. 2015, 2016),
autohydrolysis (Domínguez et al. 2017), and steam explosion (Radeva et al. 2012). Biological
pretreatments are also possible via ligninolytic fungi or their enzymes, but these methods have
not yet been investigated on paulownia.
The aim of the present study is to evaluate the pretreatment of paulownia wood by
commercially available laccase-mediator (L/M) system to improve the subsequent
saccharification. The thermostable laccase from the fungus M. thermophila will be applied
from Novozymes (Bagsvaerd, Denmark) that has been cloned and expressed in Aspergillus
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oryzae (Xu et al. 1996; Berka et al. 1997). Methyl syringate (MeS) will serve as mediator,
which is obtained from syringic acid present in pulp and paper side-streams (Rosado et al.
2012). MeS is cheaper and less toxic than synthetic mediators such as HBT, violuric acid or
ABTS. The L/MeS treatment was proved to be highly effective on eucalypt wood (
Rico et al.
2014), which contains a syringyl-rich lignin (Rencoret et al. 2008) that is easier to degrade
under alkaline conditions (González-Vila et al. 1999; Shimizu et al. 2017). The modification
of the structure of the lignin polymer in the pretreated paulownia will be monitored by 2D-
NMR spectroscopy of the whole sample at the gel state (Kim et al. 2008; Rencoret et al.
2009b), and the effect of the L/MeS treatment on the saccharification yield will be reported.
Material and methods
Paulownia wood, enzymes and mediator: 3-year-old P. fortunei trees were provided by the
University of Huelva (Spain). The wood was manually debarked, chipped, air-dried and
ground in a knife mill IKA MF1O to pass 1 mm screen, and then finely milled in a planetary
mill Retsch PM100 at 400 rpm for 2 h (including pause times to prevent sample heating),
using a 500 mL agate jar and agate balls (20 × 20 mm). The commercial (recombinant) fungal
laccase from M. thermophila (Novozym 51003) was supplied by Novozymes. The enzymatic
activity was measured as initial velocity during oxidation of 5 mM ABTS from Roche to its
cation radical (ε
436
29300 M
-1
cm
-1
) in 0.1 M sodium acetate (pH 5) at 24°C. The laccase
activity was 1000 U mL
-1
. One activity unit (U) is defined as the amount of enzyme
transforming 1 µmol of ABTS per min. MeS (methyl 4-hydroxy-3,5-dimethoxybenzoate from
Alfa Aesar (Karlsruhe, Germany) served as the redox mediator.
L/MeS treatments: A sequence of four L/MeS treatments was applied, each one followed by
an alkaline peroxide extraction step. Laccase doses of 50 U g
-1
were assayed, together with
3% MeS (% is b.o. dry wood). Paulownia samples (4 g) at 6% consistency (w:w) in 50 mM
NaH
2
PO
4
(pH 6.5) were placed into 200 mL pressurized bioreactors (Labomat, Mathis,
Formatted: Font color: Auto
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Switzerland) and treated under O
2
atmosphere (2 bar), in a thermostatic shaker (adjusted at
50°C and 190 rpm), for 24 h. Then, samples were filtered through a Büchner funnel and
washed with 1 L of water. Subsequently, samples at 6% consistency (w:w) were submitted to
an alkaline peroxide extraction wit 1% (w:w) NaOH and 3% (w:w) H
2
O
2
at 80°C for 90 min
in a thermostatic shaker at 140 rpm, followed by water washing. Treatments with laccase
alone (50 U g
-1
), without mediator, and controls without laccase and mediator were also
performed. A control treatment with mediator alone was not included in view of the negative
results of previous studies. The Klason lignin contents (TAPPI Method T222 om-88, 2004)
were corrected for ash.
Saccharification of pretreated samples: After L/MeS treatment, the samples were
hydrolyzed in an enzyme cocktail of commercial enzymes (Novozymes) with cellulase
(Celluclast 1.5 L; 2 FPU g
-1
) and β-glucosidase (Novozym 188; 6 U g
-1
) at 1% solid loading
in 3 mL of 100 mM sodium citrate (pH 5) for 72 h at 45°C, in a shaker bath at 140 rpm. The
released monosaccharides were determined as alditol acetate derivatives by GC (Selvendran
et al. 1979) on an HP 5890 instrument (Hewlett-Packard, Hoofddorp, The Netherlands), as
previously described (Rencoret et al. 2017). Chromatographic peaks were quantified by area,
and different standards (including glucose and xylose, among others) were used to elaborate
calibration curves. Duplicate experiments were performed in terms of of L/MeS treatment
(including control, laccase and laccase-MeS) and glucose and xylose release, and Klason
lignin contents were analysed in triplicate measurements. At the 95% confidence level, the
enzymatic experiments are smaller than the differences found between the control, laccase
alone, and L/MeS treatments. Moreover, ANOVA experiments between subjects were
performed. Post hoc pairwise comparisons, using the Tukey
HSD test, were also performed.
The data from both enzymatic and technical replicates were averaged.
2D-NMR analyses: The 2D HSQC NMR experiments were performed at the gel state, which
is an in situ analysis of the whole cell-wall (Kim et al. 2008, Rencoret et al. 2009b). This
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approach does not require a previous lignin isolation and avoids possible alterations and
material losses during the isolation process. Theseis experiments provided also structural
information on the hemicelluloses in the cell wall. 70 mg of ball-milled samples (and filtrate
samples) were transferred into 5-mm NMR tubes, and swelled in 1 mL of DMSO-d
6
, forming
a gel inside the tube (Kim et al. 2008; Rencoret et al. 2009b). Heteronuclear single quantum
coherence (HSQC) 2D-NMR spectra were recorded at 300 K on a Bruker AVANCE III 500
MHz spectrometer equipped with a 5 mm TCI gradient cryoprobe with inverse geometry. An
adiabatic HSQC pulse sequence (Bruker standard ‘hsqcetgpsisp.2’), which enabled a
semiquantitative analysis of the different
1
H-
13
C correlation signals (Kupče and Freeman,
2007), was utilized. HSQC 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). Lignin and carbohydrate correlation signals in
the HSQC spectra were assigned by comparison with the literature (Rencoret et al. 2009a;
2009b; Kim et al. 2014). The
1
H-
13
C correlation signals from the aromatic region of the
spectrum were used to estimate the content of lignin (relative to the content of amorphous
carbohydrates, estimated from the anomeric xylose and glucose signals), and the lignin
composition in terms of G, S and oxidized S (S′) and G (G′) units. The C
α
-H
α
correlation
signals in the aliphatic-oxygenated region were used to estimate the abundance of the various
lignin inter-unit linkages whereas the C
γ
-H
γ
correlation signals were used to estimate the
abundance of the cinnamyl alcohol end-units. Likewise, S
2,6
(and S′
2,6
) and G
2
(and G′
2
)
signals were used to estimate the relative abundances of the aromatic units – as signals S
2,6
and S′
2,6
involve two proton-carbon pairs, their volume integrals were halved. The content of
Cα-oxidized S-lignin units in the HSQC spectrum of paulownia treated with laccase and
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Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Short title: enzymatic pretreatment of p. fortunei a commercial laccase-mediator system to delignify and improve saccharification of the fast-growing paulownia fortunei" ?

Up to 24 % lignin removal was attained by the laccase-mediator treatment ( L/MeS ), followed by alkaline peroxide extraction in a multistage sequence.