Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin and Improves Saccharification
Summary (2 min read)
Introduction
- Agricultural and forestry residues represent an enormous source of readily available biomass for biofuel production without the need for additional land cultivation.
- In spite of the availability of these residues or other lignocellulosic biomass, their variable composition and recalcitrance represent some technical and economic challenges.
- 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].
- 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].
- 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
- 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 laccase was provided by INRA (Marseille, France) and was obtained from a laccase-hyperproducing strain of the fungus Pycnoporus cinnabarinus (Herpoël et al. 2000).
- One activity unit (U) was defined as the amount of enzyme transforming 1 µmol of ABTS per min.
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).
- 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 one-way analysis of variance was conducted to compare the effects of the different enzymatic treatments on the lignin removal, and on the releases of glucose and xylose.
- 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-d6, 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 13 C1 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.
- 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
- Wheat straw lignin is a guaiacyl–rich lignin [32] that usually is reported to be more resistant to degradation than the syringyl type [33].
- Additionally, the effect of the combination of the enzymatic treatment with a subsequent alkaline peroxide extraction was studied.
- Moreover, a similar improvement in saccharification yield was produced after the laccase-mediator treatment alone (without a subsequent alkaline extraction) unlike in a recent study of a laccase-HBT treatment of acid-pretreated wheat straw [17].
- The lignin modification observed in the pretreatments with laccase alone could be due to the action of laccase catalyzing the oxidation of the phenolic moiety (less than 20%) of wheat straw lignin since laccase alone is not able to catalyze the oxidation of non-phenolic lignin units.
Conclusions
- The delignification can be improved up to 48% if a subsequent alkaline peroxide extraction step is combined with the enzymatic treatment.
- Noteworthy was the lignin reduction reached (up to 38%) in lower doses treatments, which would be more industrially attractive.
- In all cases assayed, the pretreated wheat straw was hydrolyzed with higher efficiency than the untreated material releasing higher yields of glucose and xylose by using relatively low doses of cellulases.
- The 2D NMR spectra of whole straw samples (at the gel stage) showed the selective action of laccase-mediator on the lignin moiety, while the polysaccharide signals remained unchanged with respect to the controls.
- This included the breakdown of inter-unit linkages and removal of lignin S and G units, although tricin remained in the residual lignin.
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Citations
192 citations
Cites methods from "Laccase-Mediator Pretreatment of Wh..."
..., 2015) and non-woody (Rencoret et al., 2016) lignocellulosic feedstocks....
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...Mimicking those changes caused by lignin-degrading fungi (van Kuijk et al., 2016), isolated oxidoreductases have been applied as milder and more environmentally-friendly pretreatments of woody (Rico et al., 2015) and non-woody (Rencoret et al., 2016) lignocellulosic feedstocks....
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132 citations
90 citations
Cites background or methods from "Laccase-Mediator Pretreatment of Wh..."
...cinnabarinus laccase followed by alkaline peroxide extraction 18% decrease in lignin after sequential treatment 24–25% increase in glucose and xylose production [110]...
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...[110] also reported lignin structural variations in milled wheat straw treated with the same laccase-mediator system....
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...cinnabarinus laccase followed by an alkaline peroxide extraction [110]....
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82 citations
65 citations
References
2,556 citations
"Laccase-Mediator Pretreatment of Wh..." refers background in this paper
...Generation of oxidized lignin structures is congruent with the nature of the lignin biodegradation process, which has been described as an “enzymatic combustion” where fungal oxidoreductases play a central role [43]....
[...]
1,811 citations
"Laccase-Mediator Pretreatment of Wh..." refers background in this paper
...Among agricultural residues, wheat straw is potentially one of the most favorable feedstocks in terms of the quantity of biomass available [1]....
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1,408 citations
"Laccase-Mediator Pretreatment of Wh..." refers background in this paper
...first, oxidizing Cα and by splitting the aryl-alkyl bond [48]....
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1,296 citations
"Laccase-Mediator Pretreatment of Wh..." refers background in this paper
...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....
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651 citations
"Laccase-Mediator Pretreatment of Wh..." refers background or result in this paper
...4 and a large predominance of β-O-4′ ether linkages, together with some phenylcoumarans and resinols, were estimated for lignin in wheat straw feedstock, in agreement with previous studies [32]....
[...]
...related with the predominance of syringyl lignin units in eucalypt wood with respect to elephant grass and wheat straw [32, 34, 39, 40]....
[...]
...Additionally, several signals corresponding to the flavone tricin (T) recently reported in wheat-straw lignin [32] were observed....
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...Wheat straw lignin is a guaiacyl-rich lignin [32] that is usually reported to be more resistant to degradation than the syringyl type [33]....
[...]
...The selection of these doses was based on previous studies on the pretreatment of another nonwoody feedstock (elephant grass) [19] with a lignin having also a high proportion of guaiacyl units although not as high as that of wheat straw [32, 34]....
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Frequently Asked Questions (13)
Q2. What are the main components of lignocellulosic biomass linked into a?
hemicelluloses and lignin are the three main components of lignocellulosic biomass linked into a complex matrix highly resistant to chemical and biological conversion.
Q3. What is the mechanism of lignin modification observed in wheat straw?
The lignin modification observed in the pretreatments with laccase alone could be due to the action of laccase catalyzing the oxidation of the phenolic moiety (less than 20%) of wheat straw lignin since laccase alone is not able to catalyze the oxidation of non-phenolic lignin units.
Q4. What is the main reason why lignin is harmful to the hydrolysis of biomass?
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].
Q5. What is the role of laccases in biotechnological applications?
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.
Q6. How can a basidiomycete laccase delignify wheat straw?
Wheat straw can be delignified by a basidiomycete laccase in the presence of HBT, directly on the ground lignocellulosic material (i.e. without a previous chemical pretreatment) attaining a lignin removal up to 37%.
Q7. What is the effect of the steam explosion on the saccharification yields of wheat straw?
The sugar degradation and generation of inhibitory compounds during steam explosion that affect the hydrolysis can explain the lower saccharification yields attained in steam-exploded wheat straw.
Q8. What is the novelty of the pretreatment described here?
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).
Q9. What was the lignin content in the aromatic region of the spectrum?
The 13 C1 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.
Q10. What is the effect of laccase on the delignification of woody feedstocks?
when the latter pretreatment was applied to eucalypt wood, higher delignification degrees (up to 48%) were attained [19,37,38], showing that some woody feedstocks can be competitive for bioethanol production.
Q11. What is the S/G ratio of the lignin in wheat straw?
From the integrals of the above signals an S/G ratio around 0.4, and a large predominance of β-O4' ether linkages, together with some phenylcoumarans and resinols, were estimated for lignin in wheat straw feedstock, in agreement with previous studies [32].
Q12. What are the prominent signals in the HSQC spectrum?
Other less prominent signals corresponding to phenylcoumaran (B) and resinol (C) substructures were also observed in the HSQC spectrum.
Q13. What are the main signals in the aromatic/unsaturated region of the HSQC?
The main signals in the aromatic/unsaturated region of the HSQC spectrum (Fig. 2d) correspond to the benzenic rings and unsaturated side chains of H, G and S lignin units, p-coumaric acid (PCA) and ferulic acid (FA).