Role of laccase as an enzymatic pretreatment method to improve lignocellulosic saccharification

Article abstract of DOI:10.1039/c4cy00046c

The recalcitrant nature of lignocellulose, in particular due to the presence of lignin, is found to decrease the efficiency of cellulases during the saccharification of biomass. The efficient and cost effective removal of lignin is currently a critical bi

Full text of DOI:10.1039/c4cy00046c

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Role of laccase as an enzymatic pretreatment
method to improve lignocellulosic
Cite this: DOI: 10.1039/c4cy00046c
saccharification†
Lucy Heap,a Anthony Green,a David Brown,b Bart van Dongenc
a
*
and Nicholas Turner
The recalcitrant nature of lignocellulose, in particular due to the presence of lignin, is found to decrease
the efficiency of cellulases during the saccharification of biomass. The efficient and cost effective
removal of lignin is currently a critical biotechnological challenge in order to improve the enzymatic
digestibility of cellulose for bioethanol production. In this study the role and reactivity of laccase from
Trametes versicolor (TvL) was assessed with and without mediators for the improved saccharification of
acid-pretreated wheat straw. Lignin model compound studies using veratryl alcohol and β–O–4 dimers
revealed that 1-hydroxybenzotriazole (1-HBT) was the most effective mediator. Combination of TvL and
TvL + 1-HBT treatments with an alkaline-peroxide extraction step increased the released glucose
concentration following hydrolysis by up to 2.3 g L−1 compared to an untreated control. Pyrolysis-gas
chromatography-mass spectrometry (py-GC-MS) with tetramethylammonium hydroxide (TMAH)
thermochemolysis analysis of the extracted lignin revealed structural changes that are consistent with
lignin degradation mechanisms typical of fungi.
Received 13th January 2014,
Accepted 25th February 2014
DOI: 10.1039/c4cy00046c
www.rsc.org/catalysis
activity of these enzymes due to the potential blocking of active
sites and the prevention of cellulose binding.2–4
Introduction
Lignocellulosic substrates are attractive feedstocks for second
generation biofuel production due to their high abundance
and low cost. They are widely considered as waste or
by-products from forestry and agricultural industries and
unlike first generation biofuel feedstocks such as corn, they
do not compete with food.1 The removal of lignin from
lignocellulosic substrates remains a critical issue in biomass
processing for the production of bioethanol. The presence
of residual lignin following conventional pretreatment
methodologies is reported to negatively affect ethanol produc-
tion via several mechanisms:
2) Lignin that remains bound to cellulose following
pretreatment causes a reduction in the surface area of the
cellulose that is available for enzymatic hydrolysis.4
3) Lignin derived products such as aromatic phenols,
acids and aldehydes are often toxic to the yeasts that perform
the ethanol fermentation. Small concentrations of these
inhibitors have been found to destroy the integrity of the
yeast membrane systems preventing growth and sugar
assimilation.5–8
The first two issues directly affect the efficiency of cellu-
lose hydrolysis during biomass processing and contribute to
the recalcitrance-related obstacles that currently make second
1) Lignin can bind non-specifically to the cellulases
employed for saccharification, resulting in the reduced catalytic
generation bioethanol production
a costly and energy
intensive process. There is a need for effective pretreatment
technologies that can remove or reduce this recalcitrance
issue whilst still maintaining cost competitive fuel production.
Biocatalytic approaches to lignin removal are currently
being explored as greener alternatives to pre-existing chemical,
thermal and physical pretreatment strategies. White-rot fungi
from the basidiomycete phyla degrade all components in wood
including lignin. Lignin degradation is carried out via the
oxidative activities of a panel of ligninolytic enzymes from
the fungal secretome. The majority of these enzymes are
peroxidases such as lignin peroxidase (LiP EC 1.11.1.14) and
manganese peroxidase (MnP EC 1.11.1.13), however laccases
a School of Chemistry, Manchester Institute of Biotechnology (MIB), University of
Manchester, 131 Princess street, M17DN, UK. E-mail: Nicholas.Turner@manchester.ac.uk;
Fax: +44 (0)161 275 1311; Tel: +44 (0)161 306 5173
b Shell International Exploration and Production, Westhollow Technology Centre,
333 Highway 6 South, Houston, TX, USA. Tel: +128 1928 3254
c School of Earth, Atmospheric & Environmental Sciences and Williamson Research
Centre for Molecular Environmental Science, The University of Manchester,
Williamson Building, Oxford Rd, Manchester, M139PL, UK
† Electronic supplementary information (ESI) available: See ESI for supporting
experimental results and all full experimental procedures. See DOI: 10.1039/
c4cy00046c
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(EC 1.10.3.2; benzenediol: oxygen oxidoreductase) are also
suggested to play a role in lignin degradation and are attractive
enzymes industrially due to their catalytic dependence on
molecular oxygen as opposed to hydrogen peroxide. This
dependence results in the higher stability of most laccases
compared to peroxidases which suffer from deactivation by
hydrogen peroxide.9 Laccases are phenol-oxidases that catalyse
the one electron oxidation of phenolics, aromatic amines,
di-amines and other electron rich substrates via the four-
electron reduction of oxygen to water.9–11 Interestingly the sub-
strate range of laccase can be expanded through the oxidation
of lower molecular weight molecules known as mediators or
enhancers. Oxidation of these mediators by laccase generates
charged intermediates which are able to act as chemical
oxidants, overcoming the steric and redox limitations that
prohibit laccases from oxidising bulky and/or nonphenolic
substrates.12–14
Interest into the role of laccases on lignin degradation has
developed due to reports of some white rot basidiomycetes
that are found to degrade lignin or lignin model compounds
in the absence or deficiency of LiP and/or MnP.15–18 This
observation suggests that degradation mechanisms by other
enzymes such as laccase or aryl-alcohol oxidase (AAO) can
predominate in lignin depolymerisation. However, a typical
lignin polymer is 10–15% phenolic in composition, rendering
up to 90% of the polymer unreactive to laccase. Fungi are
assumed to use laccase for non-phenolic lignin degradation
through self-generation of reactive phenoxy radicals which
act as natural mediators. This is said to occur from the
laccase catalysed oxidation of phenolic lignin components
and or/existing lignin degradation products18–20 or through
the oxidation of their secreted phenolic metabolites.16
were found to be the best mediators for decolourising RB-5
(see ESI† Table S1). No decolourisation was observed in the
absence of mediators.
The best 13 phenolic compounds from the decolourisation
of RB-5 were further screened against the lignin model
compound veratryl alcohol 1 along with synthetic compounds
1-hydroxybenzotriazole (1-HBT), 2,2′-azinobis(3-ethylbenzthiazoline-
6-sulphonic acid) ABTS, and violuric acid which are reported to
be effective mediators for laccase catalysed oxidations.14,23,24
In addition, several dyes/indicators were screened following
reports of these compounds acting as laccase substrates and
mediators.20,22,25 1 is a widely utilised monomeric lignin model
compound for laccase mediator studies.12,13,24,26–28 Oxidation
leads to the formation of a single product veratryl aldehyde 2,
which is simple to detect by HPLC. Laccase is unable to oxidise
1 in the absence of a mediator making this a suitable candi-
date for assessing the non-phenolic oxidation ability of laccase
mediators. Analysis of the oxidation of 1 over time showed that
1-HBT, violuric acid and ABTS were efficient redox mediators
for this reaction, with conversions to 2 reaching up to 99%
after 24 h under optimal conditions (Table 1). Of the dyes
investigated, only phenol red (PR) and remazol brilliant blue
(RBB) acted as potential mediator substrates for TvL, resulting
in 50 & 52% conversion respectively. No oxidation of 1 was
observed in the absence of mediators.
In agreement with previous studies,19,29 the natural
phenolic mediators were not found to oxidise 1 to 2 in
significant quantities which is in contrast to the results
observed with RB-5. Presumably substrate 1 is less effective
at reducing the intermediate phenoxy radicals produced by
laccase compared with RB-5. This will explain the slower
reaction rate observed with
1 which would promote
In this study we initially investigated the development of
an effective laccase-mediator system (LMS) using laccase
from Trametes versicolor (TvL) in the presence of both
synthetic and natural mediators. Selected systems were there-
after evaluated for their ability to improve the saccharifica-
tion reaction of acid-pretreated wheat straw by analysing the
concentration of glucose released. The mechanism of laccase
and laccase-mediated lignin modification and degradation
was explored using a series of model lignin β–O–4 linked
dimers and py-GC-MS with TMAH.
undesired non-catalytic mechanisms leading to radical
coupling and polymerisation, thus preventing the conversion
Table 1 Oxidation of veratryl alcohol (1) by TvL in the presence of a
redox mediator
Results and discussion
Redox mediator screening for lignin studies
Mediator
% conversion to 2a
A panel of 30 phenolic compounds were selected for assessment
of their suitability as natural redox mediators with Trametes
versicolor laccase (TvL redox potential ~800 mV (ref. 21)). A
preliminary high throughput screening assay was developed
based on the previous work of Camarero et al.,22 using the
recalcitrant dye Reactive Black-5 (RB-5). The data revealed
that under optimised mediator and dye concentrations
most of the 30 phenolic compounds screened were successful
A
B
C
D
1-HBT
ABTS
Violuric acid
Phenol red
Remazol brilliant blue
99
74
31
50
21
94
61
71
19
9
98
87
24
17
22
92
88
74
33
52
Oxidation of 3 mM 1 under four different enzyme-mediator condi-
tions. A: 1.6 U ml−1 TvL, 3 : 1 lac-med ratio, B: 1.6 U ml−1 TvL, 1 : 1,
C: 0.4 U ml−1 TvL, 3 : 1 D: 0.4 U ml−1 1 : 1.a % conversion was deter-
mined by LC-MS after 24 h, optimal conversions are shown in bold.
at decolourising RB-5 to varying degrees within
3 h.
Syringaldehyde, acetosyringone and 2,4,6-trimethylphenol
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of 1 to 2.20,30 Under the assumption that lignin model
compounds such as 1 can provide a reasonable indication of
laccase reactivity towards natural lignin, then naturally
occurring phenolic structures such as those investigated
here would be expected to act poorly as mediators due to
their instability in the catalytic cycle. The rapid decolourisation
of RB-5 suggests a role for these compounds in technologies
linked with dye detoxification/decolourisation in the textile
industry.
The effect of laccase and LMS on lignocellulosic
saccharification
In order to determine the effectiveness of the successful
laccase mediator systems (LMS) investigated with 1 towards a
lignocellulosic bioprocess, laccase and LMS treatments were
carried out on wheat straw that had previously been treated
with dilute sulphuric acid. Dilute acid hydrolysis is
a
commonly employed pretreatment method that effectively
solubilises the hemicellulose component within lignocellu-
lose. Removal of hemicellulose improves the digestibility of
cellulose by increasing the porosity of the substrate.31 Wheat
straw slurries treated with different concentrations of TvL
were initially analysed for free phenol content using the
Folin-ciocalteu total phenol assay.32 In accordance with a previous
study,33 a decrease in soluble phenol content was observed in
wheat straw slurries treated with TvL (see ESI,† Fig. S2)
supporting the role of laccase in the oxidation of phenolic
structures within biomass and the removal of the reactive inter-
mediate phenoxy radicals by polymerisation. A cellulase prepa-
ration (Genencor GC-220) was added to the slurries following
treatment with the different TvL concentrations to allow
hydrolysis of the cellulose fraction. Following quantification
of the released glucose it was revealed that no increase in
glucose concentration was observed in the TvL treated slurries
compared to the laccase free and denatured laccase negative
controls. The reaction was further investigated with TvL and
the addition of synthetic mediator 1-HBT. An increase in
glucose release was not observed following hydrolysis despite
the incorporation of wash steps to remove potential inhibitory
compounds and both the enzyme and mediator. In fact, it
appeared that laccase treated slurries were inhibiting the
saccharification process (Fig. 1 top graph).
Fig. 1 Concentration of glucose released from 0.6 g (d.w) wheat straw
hydrolysed by 2.9FPU GC220 for 48 h following treatment with/without
TvL and TvL + 1-HBT. Top: No APE step after laccase treatment. Bottom:
APE treatment performed after laccase treatment. Error bars represent
standard error of three biological replicates.
in released glucose concentration both in the presence and
absence of laccase (Fig. 1). This can be explained by the addi-
tional biomass handling steps during three consecutive
extractions and subsequent wash steps which results in
biomass loss. In addition, the alkali oxidative environment
can induce undesired cellulose cleavage reactions. The addi-
tion of magnesium sulphate has been reported to minimise
peroxide induced cellulose degradation suggesting the oppor-
tunity to minimise these undesired reactions during process
optimisation and development.35
Optimisation of laccase loading and mediator concentration
revealed that 500 U g−1 d.w WS (units TvL per gram dry weight
of wheat straw) was optimal for increased saccharification
(see ESI,† Fig. S4) however due to commercial enzyme costs,
subsequent experiments were carried out using 150 U g−1
(d.w WS). Use of the redox mediator 1-HBT at 5% (w/w)
with 150 U g−1 TvL proved optimal and lead to an increase
of 1.4 g L−1 (13%) glucose after 64 h hydrolysis compared to
the mediator free control (Table 2).
Based on the mediator screening data, some synthetic and
natural mediators were applied to the optimised bioprocess
to assess their suitability and reactivity on wheat straw lignin.
1-HBT was found to be the most effective mediator, increasing
the concentration of released glucose following hydrolysis
by 2.3 g L−1 (35%) compared to the NLNM negative control
(Table 3). This is in support of previous studies whereby 1-HBT
was reported to be the most efficient mediator in the
A recent study by Gutierrez et al.,34 reported an increase
in polysaccharide hydrolysis for eucalypt wood and elephant
grass treated with T. villosa laccase and 1-HBT after the incor-
poration of an alkaline-peroxide extraction (APE) step. This
step was therefore applied to all wheat straw slurries following
laccase treatments. Quantification of released glucose during
hydrolysis following this additional step revealed that slurries
pretreated with TvL and TvL + 1-HBT now released higher
concentrations of glucose following hydrolysis compared to
the no laccase no mediator negative control (NLNM) (Fig. 1
bottom graph). This LMS can induce a positive effect on the
saccharification process of wheat straw. The results in Fig. 1
also demonstrate that use of an APE step leads to a decrease
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Table
2
Investigating
the
optimum
concentration
of
model compound 1 by both LMS. Surprisingly, the reaction
of TvL with the synthetic mediator ABTS did not improve
saccharification despite the excellent conversion of 1 to 2
using this LMS (Table 1). A similar result was also observed
by Chen,37 who reported little differences between the
measured water soluble carbohydrate concentration released
from hydrolysed ensiled corn stover after TvL and ABTS
treatment. Similarly, the application of PR and RBB as media-
tors failed to result in an increased glucose concentration
compared with the mediator free control (see ESI,† Fig. S6).
These observations suggest that the ability of a LMS to
successfully oxidise the monomeric guaiacyl (G-type) lignin
model compound 1, does not necessarily correlate with an
ability of the LMS to react with lignin to improve cellulose
hydrolysis. Monomeric substrates such as 1 are likely to
represent poor lignin model compounds due to the absence
of β-linked structures that are predominant in natural lignin.
Furthermore, the structural complexity of lignin regarding
the heterogeneously linked G-type, S-type (syringyl) and H-type
(p-hydroxyphenyl) sub-structures highlights the challenges
associated with drawing meaningful conclusions from model
studies.
Interestingly, the incubation of wheat straw with TvL in
the absence of mediator consistently improved saccharification.
This increase in glucose release following laccase treatment
alone has been previously reported.4,34,36 It has been speculated
that the binding of laccase to lignin sites within biomass
competes with, and reduces the non-specific binding of
cellulases therefore improving saccharification. Furthermore,
electron spectroscopy for chemical analysis (ECSA) has been
used to probe surface modifications of spruce lignin following
treatment with Trametes hirsuta laccase.36 The study revealed
an increase in carboxylic acid residues following laccase
treatment and suggests that this modification to lignin may
decrease the non-specific adsorption of negatively charged
cellulases due to electrostatic repulsion. Conversely, the treat-
ment of lignin with laccase has been reported to increase
undesired cellulase binding thus inhibiting saccharification.4
Although the mechanism of this has not been described, this
effect may explain the decrease in saccharification following
TvL and TvL + 1-HBT treatment without APE reported in this
study. Jurado et al., and Tabka et al.,33,41 reported the same
inhibitory effect with steam exploded wheat straw and two
different fungal laccases, however they did not investigate the
use of APE.
1-hydroxybenzotriazole (1-HBT) with TvL to improve wheat straw
saccharification
Biomass treatmenta
Glucose concentrationd/g L−1
150 U g−1 TvLb
10.8 (0.13)
11.0 (0.23)
12.2 (0.37)
11.2 (0.80)
150 U g−1 TvLb + 2.5%b 1-HBTc
150 U g−1 TvLb + 5%b 1-HBTc
150 U g−1 TvLb + 7.5%b 1-HBTc
a
0.5 g d.w wheat straw incubation for 40 h, 28 °C, 200 rpm.
Enzyme units per g dry weight wheat straw biomass. % (g/g)
b
c
d
mediator per dry weight wheat straw biomass.
Glucose
concentration determined by HPLC-RID following 64 h hydrolysis
with 2.9 FPU GC220. Parentheses represent standard error of three
biological replicates. Bold indicates most successful treatment.
Table 3 Screening of synthetic and natural mediators with TvL for the
improvement of wheat straw lignocellulosic saccharification
Biomass treatmenta
Glucose concentrationd/g L−1
NLNM
6.6 (0.03)
7.8 (0.19)
8.9 (0.20)
7.7 (0.22)
8.3 (0.10)
7.7 (0.08)
7.5 (0.25)
150 U g−1 TvL
150 U g−1 TvL 1-HBTb
150 U g−1 TvL ABTSb
150 U g−1 TvL violuric acidb
150 U g−1 TvL syringaldehydec
150 U g−1 TvL acetosyringonec
a
0.5 g d.w wheat straw incubation for 40 h, 28 °C, 200 rpm.
Mediator concentration of 1-HBT was 5% (g/g) d.w biomass, molar
b
concentration of ABTS and violuric matched to molar concentration
c
d
of 1-HBT. Mediator concentration 7 mM. Glucose concentration
determined by HPLC-RID following 42 h hydrolysis with 2.9 FPU
GC220. Parentheses represent standard error of three replicates. Bold
indicates most successful treatment.
delignification and bioleaching of lignocellulose.14,36–38
Violuric acid also proved to be an effective mediator, increasing
glucose concentration by 1.7 g L−1 (26%). 1-HBT and violuric
acid belong the hydroxylamine laccase mediator class and
share the N–OH structural feature. Previous studies have
revealed that these mediators undergo laccase catalysed
oxidation to produce aminoxyl radicals following deproton-
ation as outlined with 1-HBT in Scheme 1. Oxidation reactions
promoted by these aminoxyl radicals are reported to follow a
hydrogen-atom transfer (HAT) mechanism by abstraction of
the benzylic hydrogen of the reduced substrate.28,39
The use of potential TvL mediators acetosyringone and
syringaldehyde did not improve saccharification when
compared to the mediator free control. This result was
expected following the previous unsuccessful oxidation of
The results obtained in this study consistently revealed an
improvement of saccharification following the incubation of
wheat straw with TvL and a successful LMS followed by APE,
demonstrating the reproducibility of this effect even when
different batches of washed substrate were used. To further
investigate reproducibility, the optimised bioprocess with
TvL, 1-HBT and APE was repeated with two other agricultural
residues, corn and sorghum stover. The same trends regarding
the increase in glucose release was observed with both
substrates (see ESI† Fig. S7) demonstrating the success of the
laccase and 1-HBT treatments with alternative substrates.
Scheme
1 The oxidation of 1-hydroxybenzotriazole (1-HBT) by
laccase to produce the corresponding nitroxy radical.
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Investigating the reaction of TvL and TvL + 1HBT with lignin
model β–O–4 linked dimers
identification of degradation products 8–12 (m/z 265, 293, 263,
247 and 291) (Scheme 2, refer to ESI† Fig. S8–S11 for all LC-MS
traces and product characterisation). The production of Cα
oxidation products 6 and 7 from the non-phenolic dimers
4 and 5 may provide a plausible explanation for the role of
APE in the improvement of saccharification following LMS
treatment. During APE, the hydroperoxide anions produced are
reported to react with the carbonyl structures within lignin
resulting in C–C bond cleavage.48,49 The mechanism of
degradation of phenyacylaryl ethers (e.g. structure 6) by hydrogen
peroxide in alkaline media has been studied previously and is
outlined in Scheme 3.50 An increased presence of carbonyl
structures in lignin following treatment with TvL + 1-HBT may
correlate with increased lignin removal by C–C bond cleavage
following APE. These studies conducted with non-phenolic
dimers 4 and 5 may provide important mechanistic insights
into the role of LMS towards lignin degradation.
β–O–4 linked dimers (3–5) were used in an attempt to gain
an understanding of potential structural changes occurring
within lignin as a result of laccase/LMS treatment. Dimers
3 and 4 were commercially available whilst dimer 5 was
synthesised by a modified method of Kawai et al.,40,42
(see ESI† section 1.9). Such structures have been used exten-
sively to study lignin degradation mechanisms due to the
predominance of this β–O–4 linkage within lignin.42–46
All 3 dimers were incubated with TvL both with and without
1-HBT. Immediate sampling of the reaction of the phenolic
dimer 3 with TvL in the absence of 1-HBT and subsequent
analysis by LC-MS revealed a product peak m/z 661, consistent
with oxidative dimerisation of 3. After 24 h, complete consump-
tion of starting material 3 and initially formed dimerisation
product was observed (see ESI,† Fig. S8), presumably as a result
of further polymerisation as anticipated with oxidised phenolic
substrates. As expected, the reactions of non-phenolic dimers
4 and 5 with TvL in the absence of 1-HBT failed to result in the
formation of oxidation products, with only starting material
observed by LC-MS. Consistent with previous studies,13,47
oxidation of dimer 4 with TvL in the presence of 1-HBT led to
the formation of the uncleaved ketone product 6 as the sole
oxidation product, providing evidence for a Cα oxidation
mechanism (Scheme 1). The analogous Cα oxidation product
was also observed when dimer 5 (threo : erythro 5 : 2) was
reacted with TvL + 1-HBT, however, in this instance a complex
mixture of degradation products were also observed by LC-MS
that were not present in the TvL and substrate only negative
controls. Presumably these degradation products are derived
from oxidation of the more electron rich aromatic ring in
dimer 5 which bears an additional methoxy substituent.
Comparison of the m/z values with the suite of oxidation products
characterised by Kawai et al.,44 from the same dimer allowed
Pyrolysis GC-MS with TMAH
Thermally-assisted hydrolysis and methylation using
tetramethylammonium hydroxide (TMAH) thermochemolysis
followed by gas chromatography-mass spectrometry (GC-MS)
has rapidly developed as a tool for characterising the relative
proportions of lignin monomers in plant material.51 Pyrolysis
of lignin with TMAH is reported to induce cleavage of
propyl–aryl–ether bonds (β–O–4) and the methylation of
both aromatic and alkyl side chain hydroxyl groups.52 This
technique has been used by researchers studying the degra-
dation of lignin in the natural environment or by pure
cultures of fungi53–55 however in this study it is explored for
the determination of the action of laccase and LMS on the
lignin structure. The technique was used to investigate and
compare the composition of organosolv extracted wheat straw
lignin following pretreatment with TvL, TvL + 1-HBT and no
enzyme and no mediator (NLNM).
Scheme 2 Reactions of lignin model β–O–4 dimers 3, 4, and 5. (a) Product 6 characterised by MS and NMR following purification. (b) Product 7
characterised by MS and by comparison with an authentic standard. (c) Degradation products were identified by LC-MS and comparison with
degradation products reported in the literature.40
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Scheme 3 The proposed pathway for the degradation of a phenacyl
aryl ether (involving cleavage of the Cα–Cβ) by alkaline–peroxide
treatment as investigated by Gierer et al.50
A
collection of guaiacyl (G) and syringyl (S)
thermochemolysis products were liberated from the lignin
samples. Peaks were identified by library searches and by the
comparison of peak positions and characteristic mass ions
from published data53 (see ESI† Table 2). A peak of high
abundance derived from the p-hydroxylphenyl (H) monolignol
was also observed. Wheat straw lignin is reported to contain
all 3 mono-lignols (G, S and H) with a predominance of G and
S-type lignins (H-type < 10%)56,57 and S/G ratios around
1.2–1.4.53,58 A partial chromatogram displaying the identified
TMAH thermochemolysis products of organosolv extracted
wheat straw lignin without enzymatic pretreatment is pro-
vided in the ESI† (Fig. S12).
The most significant observation from the total ion
currents (TIC) was the differences observed in the peak inten-
sities of methyl 3,4,5-trimethoxybenzoate (S6) and methyl
3,4-dimethoxybenzoate (G6) between the laccase treated
samples and the untreated control (Fig. 2). Oxidative lignin
degradation mediated by white rot fungi such as T. versicolor
is reported to occur via Cα–Cβ side chain cleavage at the Cα
position. This oxidative cleavage leads to the production of
aromatic aldehydes such as 3,4,-dimethoxybenzaldehyde (G4)
and 3,4,5-trimethoxybenzaldehyde (S4) from alcohol groups
Fig. 2 Partial chromatograms of the total ion current (TIC) for the
TMAH thermochemolysis products methyl, 3, 4, 5-trimethoxybenzoate
(S6) (top), and methyl, 3,4-dimethoxybenzoate (G6) and 3,4,5-
trimethoxybenzaldehyde (S4) (bottom) for both laccase and laccase
mediator treated wheat straw lignins and the untreated control.
which are reported to undergo further oxidation to their
carboxylic acids (such as G6 and S6).53,59,60 Researchers
examining the ratio between the G and S unit acids (G6 and S6)
and the aldehydes (G4 and S4) by calculating [Ac/Ald]G
and [Ac/Ald]S have found increased ratios in fungal-degraded
lignin compared to native lignin.53,55 The same ratio increase
Table 4 [Ac/Al] ratios of both G and S units from organosolv extracted wheat straw treated with/without TvL/TvL + 1-HBT
Treatmenta
[Ac/Al]G
[Ac/Al]S
Exp 1
Exp 2
Exp 3
NLNM
0.79
1.22
1.55
0.76
1.67
1.82
1.13
1.67
3.44
4.91
5.69
3.96
5.21
6.97
4.80
5.36
150 U g−1 TvL
150 U g−1 TvL + 1-HBTb
NLNM
150 U g−1 TvL
150 U g−1 TvL + 1-HBT
NLNM
150 U g−1 TvL
150 U g−1 TvL + 1-HBT
1.82
7.62
Average increase in [Ac/Al]G
0.63 (0.15)
Average increase in [Ac/Al]S
1.09 (0.27)
NLNM to TvL
NLNM to TvL + 1-HBT
TvL to TvL + 1-HBT
0.84 (0.11)
0.21 (0.06)
2.69 (0.23)
1.6 (0.43)
a
b
Incubation of wheat straw for 40 h, at 28 °C and 200 rpm. 5% (g/g) d.w biomass. Parenthesis represent standard error of three biological
replicates. Bold represents highest ratio.
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was found when fungal-degraded lignin was analysed via
alternative methods such as solid state 13C-NMR, alkaline
CuO and nitrobenzene oxidation.60–62 Calculation of these
ratios revealed an increase in the [Ac/Ald]G ratio from
0.79 (NLNM) to 1.22 (TvL) and a further increase to 1.55
(TvL + 1-HBT). A similar trend was observed for [Ac/Ald]S
whereby the ratio increased from 3.44 (NLNM) to 4.91 (TvL)
and 5.69 (TvL + 1-HBT) (Table 4). The trend was reproducible
across three separate experiments set up using wheat straw
from different washed batches. The increase in both ratios
following preincubation with TvL and TvL + 1-HBT is consis-
tent with the reported Cα–Cβ oxidative cleavage mechanism
of lignin by fungal enzymes.53,59
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Conclusions
The saccharification of acid-pretreated wheat straw was
successfully improved by the development of a bioprocess
incorporating an alkaline-peroxide extraction step following
the incubation with laccase and a laccase-mediator system.
Mediator screening studies using the lignin model compound
veratryl alcohol revealed that a correlation between the activity
towards the substrate and real lignin could not be established
for all mediators. 1-HBT was proven to be the most successful
mediator for the bioprocess. Studies using non-phenolic β–O–4
dimers with the LMS provided evidence for a Cα oxidation
mechanism and possible β–O–4 cleavage. We suggest that the
increased formation of the Cα oxidised groups within lignin
following LMS treatment positively assists with lignin removal
by alkaline peroxide extractions. Extracted lignin from laccase
and LMS treated wheat straw was found to contain a higher
proportional of syringyl and guaiacyl acid monomers by
py-GC-MS with TMAH thermochemolysis when compared to
the aldehyde counterpart. This observation is consistent with
reported fungal-mediated lignin degradation mechanisms
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