Structural Modifications of Lignosulphonates
Dimitri Areskogh Doctoral Thesis Royal Institute of Technology School of Chemical Science and Engineering Department of Fibre & Polymer Technology Division of Wood Chemistry and Pulp Technology Stockholm 2011
AKADEMISK AVHANDLING Som med tillstånd av Kungliga Tekniska Högskolan framläggs till offentlig granskning för avläggande av teknologie doktorsexamen, fredagen den 13 maj 2011 kl. 10.00 i sal D3, Lindstedsvägen 3, KTH, Stockholm. Avhandlingen försvaras på engelska. Fakultetsopponent: Prof. Arthur J Ragauskas Georgia Institute of Technology, Atlanta, Georgia, USA © Dimitri Areskogh Stockholm 2011 TRITA‐CHE Report 2011:26 ISSN 1654‐1081 ISBN 978‐91‐7415‐923‐3
“Reading is the basics for all learning.” —Presidential candidate George W. Bush, Reston, Virginia, March 28, 2000
Abstract Lignosulphonates are by‐products from the sulphite pulping process for the manufacture of specialty dissolving pulps and paper. During the liberation of the cellulose, the lignin is fractionated and solubilised through covalent addition of sulphonic acid groups at various positions in the structure. The formed sulphonated lignin, lignosulphonate is then further isolated and refined. The amphiphilic nature of lignosulphonates has enabled them to be used as additives to various suspensions to improve their dispersion and stability. The by far largest utilisation of lignosulphonates is as dispersants in concrete. Here, lignosulphonates act by dispersing cement particles to prevent flocculation, un‐even particle distribution and reduced strength development. The dispersion is achieved through steric and electrostatic repulsion of the cement particles by the lignosulphonate polymer. This behaviour is intimately linked with the overall size and amount of charged groups in the dispersing polymer. Traditional modifications of lignosulphonates have been limited to removal of sugars, filtration and fractionation. These modifications are not sufficient for utilisation of lignosulphonates in high‐strength concrete. Here synthetic dispersants and superplasticisers are used which are considerably more efficient even at low dosages. To compete with these, additional modifications of lignosulphonates are likely to be necessary. The molecular weight and functional group composition have been identified and described as the most interesting parameters that can be modified. Currently, no suitable method exists to increase the molecular weight of lignosulphonates. Oxidation by the natural radical initiating enzyme laccase is an interesting tool to achieve such modifications. In this thesis several aspects of the mechanism through which this enzyme reacts with lignin and lignosulphonate structures have been elucidated through model compound studies. Further studies showed that laccase alone was a highly efficient tool for increasing the molecular weight of commercial lignosulphonates at low dosages and in short incubation times. Immobilisation of the laccase to a solid support to enable re‐utilisation was also investigated. Modification of functional group composition of lignosulphonates was achieved through ozonolysis and the Fenton’s reagent, a mixture of hydrogen peroxide and iron(II)acetate. Introduction of charged carboxylic groups was achieved through opening of the benzyl rings of lignosulphonates. It was found that a two‐stage process consisting of laccase oxidation followed by ozonolysis was an efficient technique to create a polymer enriched with carboxylic acid groups with a sufficient molecular size. Oxidation by the Fenton’s reagent was shown to yield similar modifications as the combined laccase/ozonolysis treatment albeit with less pronounced results but with a large level of control through variation of a number of reaction parameters. The Fenton’s reagent can therefore be an interesting alternative to the aforementioned two‐stage treatment. These modifications are interesting for large‐scale applications not only because of their simplicity in terms of reaction parameters but also because of the ubiquity of the used enzyme and the chemicals in the pulp and paper industry.
Sammanfattning Lignosulfonater är bi‐produkter från sulfitprocessen som används för framställning av ren dissolvingmassa för vidare produktion av regenererad cellulosa. Under denna process löses ligninet upp i kokvätskan genom introduktion av sulfonsyragrupper, vilket medför att ligninet blir vattenlösligt och kan därför separeras från massan. Den amfifatiska strukturen hos lignosulfonaterna som innehåller både hydrofila och hydrofoba grupper har gett lignosulfonaterna unika egenskaper. Lignosulfonater används idag som dispergeringsmedel för olika typer av suspensioner för att förbättra deras dispersion och stabilitet. Det överlägset största användningsområdet av lignosulfonater är som dispergeringsmedel för betongtillverkning. Här används lignosulfonaterna för att dispergera cementpartiklarna för att ge betongen bra flyt och undvika partikelaggregation. Denna dispersion sker främst genom sterisk och elektrostatisk repulsion av de laddade cementpartiklarna. Dessa två fenomen är intimt förknippade med storleken och laddningen hos den dispergerande polymeren. De traditionella modifieringarna av lignosulfonater har varit begränsade till eliminering av socker, filtrering och fraktionering. I högstyrke‐betong ställs andra krav på dispersion vilket medför att man använder syntetiska dispergeringsmedel med väldigt specifika egenskaper vilka är avsevärt effektivare. För att konkurrera med dessa, är ytterligare modifieringar av lignosulfonater nödvändiga. Molekylvikten och andelen laddade grupper har därför identifierats som de två mest lämpliga egenskaperna för modifiering av lignosulfonater. För tillfället finns det inga metoder för att öka molekylvikten hos lignosulfonater. En intressant metod är polymerisering genom enzymatisk oxidering med hjälp av det radikal‐initierande enzymet lackas. I denna avhandling har flera aspekter av reaktionsmekanismen hos detta enzym undersökts och kartlagts via studier med modellkomponenter. Oxidation av tekniska lignosulfonater visade att detta enzym är mycket kapabelt till att öka molekylvikten markant även vid låga doseringar. Tekniker för att tillåta återanvändning av enzymet genom immobilisering till en support har undersökts. Modifiering av mängden laddade grupper hos lignosulfonater skedde genom ozonering och reaktion med Fentons reagens, en blandning av väteperoxid och järn(II)acetat. Laddade karboxylgrupper visade sig bildas genom reaktioner där bensylgrupper i lignosulfonaterna bröts upp. En kombination av oxidation med lackas följt av ozonering visade sig vara en mycket intressant tvåstegsmodifiering vilket gav upphov till en högmolekylär polymer berikad med laddade karboxylgrupper. Oxidering med Fentons reagens visade sig ge liknande resultat som en kombinerad lackas och ozonbehandling med märkbart lägre effektivitet men dock med en större grad av kontroll. Detta reagens skulle kunna vara ett intressant alternativ till den ovan nämnda tvåstegsmodifieringen. Dessa modifieringar är intressanta för storskalig användning då dessa är bör vara lätta att implementera men också eftersom både enzymet och kemikalierna är väl bekanta för pappers‐ och massaindustrin.
List of Publications This thesis is based on the following papers:
I.
Oxidative polymerisation of models for phenolic lignin end‐groups by laccase Areskogh, D.; Li, J.; Nousiainen, P.; Gellerstedt, G;, Sipilä, J. and Henriksson, G. Holzforschung, 2009, 64, 21–34.
II.
Sulfonation of phenolic end groups in lignin directs laccase‐initiated reactions towards cross‐linking. Areskogh, D,; Li, J.; Nousiainen, P.; Gellerstedt, G.; Sipilä, J. and Henriksson, G. Industrial Biotechnology 2010, 6, 50‐59.
III.
Investigation of the Molecular Weight Increase of Commercial Lignosulfonates by Laccase Catalysis. Areskogh, D.; Li, J.; Gellerstedt, G. and Henriksson, G.
Biomacromolecules, 2010, 11, 904–910.
IV.
Structural modification of commercial lignosulphonates through laccase catalysis and ozonolysis. Areskogh, D.; Li, J.; Gellerstedt, G. and Henriksson, G.
Industrial Crops and Products 2010, 32, 458‐466.
V.
Immobilisation of laccase for polymerisation of commercial lignosulphonates. Areskogh, D. and Henriksson, G.
Process Biochemistry 2011, 46, 1071‐1075
VI.
Fenton’s reaction: a simple and versatile method to structurally modify commercial lignosulphonates. Areskogh, D. and Henriksson, G.
Nordic Pulp & Paper Research Journal, 2011, 26, 90‐98
Author’s Contribution Paper I‐VI: Principal author. Formulated research strategies with Prof. Gunnar Henriksson.
Abbreviations In alphabetical order: C3A
Calcium aluminate, (CaO)3Al2O3.
C2S
Dicalcium silicate, (CaO)2SiO2
C3S
Tricalcium silicate, (CaO)3SiO2
C‐S‐H
Calcium silicate tetrahydrate (CaO)3(SiO2)2∙4∙H2O
ECF
Elemental Free Chlorine
FT‐IR
Fourier Transform Infrared Spectroscopy
GC/MS
Gas Chromatography/Mass Spectrometry
Gypsum
Calcium sulphate dihydrate, CaSO2⋅2∙(H2O)
HSQC‐NMR
Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance
LCC
Lignin‐carbohydrate complex
LMS
Laccase‐mediatory system
MALDI‐TOF MS
Matrix‐Assisted Laser Desorption/Ionisation Time‐of‐Flight Mass Spectrometry
Me‐
Methyl group, CH3‐
MeO‐
Methoxy group, CH3O‐
MtL
Myceliophthora thermophila laccase
SEC
Size Exclusion Chromatography
TCF
Totally Chlorine Free
TvL
Trametes villosa laccase
Table of Contents I
Introduction .......................................................................................................................................... 1 I.1
II
Aim ................................................................................................................................................ 2
Background ........................................................................................................................................... 3 II.1
Structure and Chemistry of Lignin ........................................................................................... 3
II.2
Lignosulphonates ........................................................................................................................ 4
II.2.1
Production and Structural Properties ............................................................................... 4
II.2.2
Utilisation ............................................................................................................................. 6
II.2.3
Concrete Admixtures .......................................................................................................... 6
II.2.4
Specialty Markets .............................................................................................................. 10
II.2.5
Modified lignosulphonates .............................................................................................. 10
II.3
Laccase ........................................................................................................................................ 11
II.3.1
A Blue Multi‐Copper Oxidase ......................................................................................... 11
II.4
Structure and Catalytic Properties .......................................................................................... 11
II.5
Occurrence and Role in Nature ............................................................................................... 13
II.6
The laccase‐mediator system ................................................................................................... 14
II.7
Applications ............................................................................................................................... 15
II.7.1
Laccase in the pulp and paper industry ......................................................................... 16
II.7.2
Laccase in the textile industry ......................................................................................... 16
II.7.3
Laccase in alternative applications ................................................................................. 17
III Experimental ....................................................................................................................................... 18 III.1
Materials ..................................................................................................................................... 18
III.1.1
Enzymes .............................................................................................................................. 18
III.1.2
Lignosulphonates .............................................................................................................. 18
IV Results and Discussion ...................................................................................................................... 19 IV.1
Model compound studies of the laccase reaction mechanism (Paper I & II) .................... 19
IV.1.1
The 1‐position ..................................................................................................................... 20
IV.1.2
The Cα position .................................................................................................................. 23
IV.1.3
Conclusions from the model compound studies .......................................................... 25
Laccase oxidation of lignosulphonates (Paper III) ............................................................................. 26 IV.1.4
The influence of lignosulphonate concentration and structure .................................. 27
IV.1.5 IV.2
The influence of the enzyme ............................................................................................ 29
Structural modifications of lignosulphonates through laccase oxidation and ozonolysis
(Paper IV) ................................................................................................................................................ 31 IV.2.1
Laccase oxidation of DP401 and DP795 ......................................................................... 31
IV.2.2
Ozonolysis of DP401 and DP795 after laccase oxidation ............................................. 32
IV.3
IV.3.1
Oxidation of DP851 by immobilised TvL ....................................................................... 36
IV.3.2
Lignosulphonate adsorption ............................................................................................ 38
IV.3.3
Deactivation of the immobilised TvL ............................................................................. 39
IV.4
V
Immobilisation of TvL (Paper V) ............................................................................................. 35
Lignosulphonate structural modification by the Fenton’s reagent (Paper VI) ................. 40
IV.4.1
Fenton’s reagent at acidic pH .......................................................................................... 41
IV.4.2
Which cation does what? .................................................................................................. 43
IV.4.3
Fenton’s reagent at alkaline pH ....................................................................................... 43
IV.4.4
The effect of lignosulphonate concentration ................................................................. 44
IV.4.5
The effect of hydrogen peroxide concentration ............................................................ 45
Conclusions and Further Perspectives ............................................................................................ 47
VI Acknowledgements ........................................................................................................................... 49 VII References ........................................................................................................................................... 50 VIII Errata‐list ............................................................................................................................................. 55
I
Introduction
The industrial utilisation of cellulose for papermaking from lignocellulosic plants has in many ways transformed the role of lignin from a necessity in nature to a reject in the industry. Not only is lignin one of the most abundant biopolymers on Earth, second only to cellulose and possibly chitin 1, it is also a bafflingly complex plant constituent of wood. While the other major wood constituent, cellulose occupies a prominent place as a valued industrial product, lignin has been reduced to an obscure role as a low‐quality fuel or chemical. The reborn realisation in the pulp and paper industry that wood can be used for a variety of products besides paper and fuel has led to the adoption of the biorefinery concept, where the biomass feedstock is processed into a spectrum of products such as fuel, chemicals, materials and energy through sustainable processes. This concept is nothing new to the petroleum industry which has been producing fuels, power and chemical products from petroleum for the last two centuries 2. Ironically, the idea of maximising the utilisation of the wood feedstock was exploited already in 1898 by Simonsen in his efforts to convert sawdust to ethanol 3. Over the decades, this knowledge was however forgotten, but the concept of a biorefinery experienced a rebirth some 30 years ago and it is emerging today as a future model of operation for the pulp and paper industry to expand its business into new fields. The National Renewable Energy Laboratory (NREL), a facility of the U.S. Department of Energy for research and development in renewable energy and energy efficiency, states that “to maximize the value generated from a heterogeneous feedstock, refineries make use of all component fractions, producing a variety of co‐products in the process … and also has tremendous potential to benefit society” 4. The role of lignin in a biorefinery is yet to be defined, but it is clear that is essential to exploit its value. Although the use of lignin as a replacement or complement to fossil fuels makes sense in a traditional paper mill, the concept of a biorefinery is likely to change this. An excellent example of this is the water‐soluble lignosulphonates, a by‐product from the sulphite pulping process. Their unique properties have led to utilisation of lignosulphonates in a variety of fields as additives or binders and as a precursor for further chemical processing. Due to the limited quality and performance, lignosulphonates have faced significant competition from synthetic plasticisers as concrete additives. Traditional modifications of lignosulphonates to improve their performance have been limited to filtration and fractionation to obtain lignosulphonates in more specific molecular weight ranges. Although sufficient to give moderate improvements in performance, the improvements are not enough to compete with their synthetic equivalents as concrete dispersing additives. Other modifications are likely to be necessary. No methods suitable for industrial integration to increase the average molecular weight of lignosulphonates exist to date. To achieve such an increase, it might be interesting to utilise biotechnical tools such as enzymes. One particular group appear promising, the natural radical‐
1
initiating laccases. The recent rapid advancements in heterologous expression and production have solved the problem of the low availability of the enzyme for industrial applications. What remains to be solved is how laccases can be applied on lignosulphonates and what benefits are to be gained.
I.1
Aim
The goal of this work described in this thesis is to investigate the various modifications of lignosulphonates achieved by laccase and conventional bleaching chemicals. Several aspects of laccase oxidation of lignosulphonates are explored through studies with model compounds and experiments with lignosulphonates. The main targets for modification are the average molecular weight and functional group composition. The proposed modifications of technical lignosulphonates are potentially important for improving their performance as additives to concrete and also for expanding their industrial utilisation.
2
II
Background
II.1 Structure and Chemistry of Lignin Lignin is next to cellulose the most abundant biopolymer on Earth, manifesting its presence on all corners of the planet Earth, on land as well as sea 5. Fortification of the cell wall by lignin is considered to be the key innovation in the evolution of terrestrial plants from their aquatic ancestors nearly half a billion years ago. The heterogeneous and highly complex aromatic polymer loosely termed “lignin” encrusts cellulose microfibrils and other components in the cell wall preventing collapse and lending biomechanical support to the cell wall and allowing plants to overcome gravity and rise above the ground. The widespread colonisation of terrestrial eco‐ regions by plants with lignified cell walls is excellent evidence of the success of such a design. No exact primary chemical and structure of lignin currently exists. In a broader sense, lignin can be described as an aromatic polymer of methoxylated phenylpropanoid units linked to each other through carbon‐carbon and and ether bonds. Several models of lignins have been proposed over the years 6‐9. The fact that only models and no actual structural determination of lignin in situ are available indicates not only the complexity of lignin but also the lack of methods to extract lignin in its’ native form. With current techniques, only fragments can be extracted and this means that the current knowledge of lignin can be considered at best fragmental. Attempts at obtaining the full picture of the structure of lignin have so far yielded only a determination of spruce secondary wall with Raman spectroscopy 8. The lignin polymer is optically inactive 10 although it contains several chiral centres and is generally considered to be branched to facilitate cross‐linking to other cell wall components to form lignin‐carbohydrate complexes (LCCs)11,12. The LCCs serve as anchoring points where the carbohydrates and lignin are covalently bonded to each other and are important for the unique physical properties that wood exhibits as a natural composite material. The lignin polymer is synthesized by the polymerisation mainly of three different cinnamyl alcohols; p‐coumaryl, coniferyl and sinapyl alcohol. These lignin monomers, monolignols, are produced within the wood cell and exported to the cell wall where they are polymerised. The monolignols are the product of the phenylpropanoid pathway starting from the amino acid phenylalanine. The end product of the monolignol polymerisation is the lignin polymer (Scheme 1). The hydroxylation and methylation reactions in the phenylpropanoid pathway ultimately determine which monolignols is formed because the three lignols differ only in the number of methoxyl groups.
3
Lignin O
HO HO
OH OH
Lignin
OH
O HO
CH3
HO
OH
O
O
OH
p-coumaryl alcohol O
OMe
MeO
HO
O
O OH O
H2N OH
MeO
OMe
phenylalanine
OMe O
OH
O
coniferyl alcohol
OH OH
MeO
HO
MeO O
OH
O
OMe
HO HO
OH HO
MeO
OMe
OH MeO
OH
OH
O OMe
sinapyl alcohol
HO
Scheme 1: Phenylpropanoid pathway for lignin biosynthesis in vascular plants.
II.2 Lignosulphonates II.2.1 Production and Structural Properties Lignosulphonates are by‐products of the sulphite pulping process for the manufacture of specialty dissolving pulps and paper. The wood chips are digested with acidic calcium bisulphite for 6‐10 hours at 100‐130 °C in a batch‐wise cooking process. During this process, the native lignin is broken down through the degradation of the randomly distributed ether bonds throughout the structure 13. The fragments are solubilised in the cooking liquor through covalent addition of sulphonic acid groups at various positions in the lignin structure (Figure 1). The sulphonic acid groups are stabilised by the presence of calcium ions. After the competition of the cooking stage, the insoluble cellulose is separated from the solubilised lignin by filtration. Further processing of the cooking liquor involves precipitation of the sulphonated lignin through the addition of excess calcium hydroxide (Howard process), the evaporation of water and residual sulphite (in the form of sulphur dioxide) and dilution with fresh water followed by ultrafiltration to remove low‐molecular‐weight fractions and sugar monomers. The purified calcium lignosulphonate is pH‐adjusted to a specific pH and evaporated to a dry matter content suitable for spray drying and packaging (Scheme 2). Ca(OH)2
Spent cooking liquor
pH adjustment
Spray drying
Evaporation
Evaporation
Precipitation (Howard process)
Dilution
pH adjustment
Ultrafiltration
Scheme 2: Simplified flow chart of lignosulphonate production from spent cooking liquor from the sulphite process.
4
The co‐existence of the hydrophilic sulphite groups and hydrophobic aromatic structures provide lignosulphonates with unique amphiphilic properties. The overall structure of a lignosulphonate is not known. Several models have been proposed over the years suggesting that lignosulphonate behaves as a coiled or expanded polyelectrolyte at either high or low concentration 14. These polyelectrolytes are likely to associate in solution and the high‐molecular weight fractions are more branched than the low‐molecular weight ones. LS
LS
OMe
2+
O
-
OH O S
Ca
O
O
2+
Ca O
-
S
OH
HO
LS
+
Ca
O O
O
HO
MeO
O
OMe
OH
S O
O OH
S
OMe
O
O
O
2+
MeO
Ca
OMe
O O LS
O
O
-
S
2+
HO
O
Ca O
OH
OMe
Ca
O
HO HO O
S
MeO
2+
O
-
HO
-
LS
S
HO
O OH
O
O
O
OH O OMe
O
OH
S
OH
OH
O MeO
MeO
O
OMe O
S
O
2+
-
O
2+
Ca
LS
Ca
Figure 1: Tentative calcium lignosulphonate structure. The sulphonic acid groups are stabilised by calcium ions. Residual lignosulphonate chains are denoted as LS.
This model has been expanded to describe lignosulphonates as micelle‐type microgels 15 with a non‐charged core consisting of cross‐linked aromatic chains with all the charged groups relocated to or near the surface to facilitate interactions with the aqueous surroundings (Figure 2A) . These particles are likely to exist in solution as irregularly shaped in a wide range of sizes 15. This model has been further elaborated and supported with high‐resolution microscopic imagery 16‐19. An expansion of this model was proposed following the discovery of a monolayer formation of various lignins when spread on a liquid surface 20 and the sandwich‐like arrangement of lignins in the secondary cell wall of wood cells 21. The earlier model of a spherical micelle was revised to a flat disc‐like molecule to better conform to these findings 22. No efforts to relate this new structure to the well‐known behaviour of lignosulphonates in solution have been made to date. Additional studies of lignosulphonates under electric fields displayed a conformational change from a compact sphere to a non‐free unwinding coil 23. A recent investigation suggests that lignosulphonates are randomly branched cross‐linked polyelectrolytes 24 (Figure 2B). According to this suggestion, lignosulphonates consist of a long continuous chain acting as a backbone with short side chains. The side chains are possibly further branched and may be reconnected to the backbone forming closed loops 24. The breaking of ether bonds that occurs during the cooking stage due to acidic hydrolysis and the subsequent
5
introduction of sulphonate groups to the structure is assumed to occur at positions that form the short side chains. A)
B)
C)
Figure 2: Proposed lignosulphonate structural models as globular micellar-type microgels 15 (A), randomly branched polyelectrolytes 24 (B) and ellipsoidal flat particles 25 (C).
It is therefore assumed that the longer backbone is more hydrophobic while side-chains are hydrophilic due to the presence of covalently linked sulphonate groups. This model was based on scaling analysis that showed how the intrinsic viscosity changed with molecular weight, something which is not valid for the microgel model where the gel increases in size but more or less keeps its overall shape with no change in intrinsic viscosity. A randomly branched polyelectrolyte will change from a spherical to an elongated shape as the molecular weight is increases with subsequent changes in intrinsic viscosity. Recent small angle X-ray scattering and rheological studies of lignosulphonates in aqueous solutions have shown that low-molecularweight lignosulphonates form ellipsoidal compact and flat particles 25 (Figure 2C).
II.2.2 Utilisation The annual global production of lignosulphonates amounted to 1.8 million metric tonnes in 2005 , but this accounts for less than 2% of all the lignin produced in the pulp and paper industry.
26
The by far largest utilisation of lignosulphonates worldwide which accounts for as much as 90% of the worldwide production is as concrete admixtures 27 and for energy production during the pulping process 28.
II.2.3 Concrete Admixtures Chemical admixtures have been used in concrete formation throughout the history of construction. As early as several hundred years B.C., Roman masons added blood and eggs to cement and water pastes to improve their mixing 29. The modern cement formula, Portland cement, was formulated during the mid-1800’s.
6
Despite the use of cement since the industrial revolution in the early 1800’s, the mechanism of the cement setting when mixed with water is still only partially understood. This process involves a series of complicated hydration and crystallisation reactions where the cement particles are interlocked and form a strong water‐insoluble binder. The actual benefits of admixtures in concrete in modern times were unintentionally discovered in the United States after the Second World War in two separate events. One event tells of a leaking bearing that released heavy oil into a grinding mill where concrete was produced. This resulted in the discovery of air‐entraining compounds 29. The second event tells of an employee at the Department of Transportation who had the idea of colouring the concrete in the black central lane of a three‐lane‐highway so that the drivers would notice when they were switching lanes. The Department of Transportation contractor chose, after a recommendation by a sub‐contractor, to use a lignosulphonate‐based dispersant to improve dispersion of the Carbon Black, the colouring agent used to obtain the distinct black colour. After several years it was observed that the state of the central lane on this particular three‐way highway was significantly better than that of the two outer lanes 30. Microscopic studies of the concrete revealed evenly distributed air‐ bubbles throughout the concrete which provided the concrete with greatly improved durability against freezing and thawing. Further studies revealed that lignosulphonates also reduced the tendency of the cement particles to flocculate without any further addition of water. The most important components of Portland cement are calcium silicates C2S and C3S and calcium aluminates C3A. They comprise over 80% of the total content of Portland cement and are essential for the strength development in the formed concrete. Both silicates react with water during the hydration process to form calcium hydroxide and calcium silicate hydrate gel, C‐S‐H. These three components play an instrumental role during the initial steps of Portland cement hydration further setting and strength development. This process can be broken down to five steps 31: 1. Initial mixing (0‐10 min). The cement particles enter into solution and hydration reactions with C2S, C3S, C3A and water start, as a result of which C‐S‐H is formed: C2S: 2 (CaO)2(SiO2) + 5 H2O → (CaO)3(SiO2)2 · 4 H2O + Ca(OH)2 C3S: 2 (CaO)3(SiO2) + 7 H2O → (CaO)3(SiO2)2 · 4 H2O + 3 Ca(OH)2 C3A: 2 (CaO)3(Al2O3) + 21 H2O → (CaO)4(Al2O3) · 13 H2O + Ca(O)2(Al2O3) · 8 H2O
If the rapid C3A hydration reactions are allowed to proceed unhindered, setting occurs too quickly and the formed concrete is not able to develop strength. Therefore, gypsum is added to the Portland cement composition to slow down the C3A hydration. 2. Dormant period (10 min – 3 h). The hydration reactions are significantly slowed down. Flocculation of unhydrated silicates occurs at this stage. 3. Initial setting (3h – 9h). The hydration reactions are rapidly started again as silicates start to precipitate.
7
4. Hardening. (9h – 15h) Most of the hydrate gel formation occurs in this highly exothermic stage which in turn accelerates further hydration reactions. 5. Slowdown (15h – 2 days). The hydration proceeds more slowly due to the extensive covering of the cement particles by the hydrate gel which hinders water from penetratating the calcium hydrate silica gel to reach unhydrated parts of the particles. When the water can no longer reach unhydrated areas or when it is consumed, the hydration stops. Water plays a crucial role in concrete preparation; it provides the cement mixture with certain rheological properties and takes part in hydration. Cement particles with their surface charges are highly prone to flocculation when in contact with a polar solvent such as water. Flocculation is detrimental not only for the uniformity of the cement mixture but also for the hydration process which begins as soon as the mixture comes into contact with water. The formation of flocculation aggregates entraps certain amounts of water which become unavailable to lubricate and ensure a flowability of the mixture. To achieve a certain workability of the concrete mixture, more water must be added than is necessary to achieve full hydration of the cement particles 31. When the excess water that does not participate in the hydration evaporates, porous cavities are formed within the paste and these lead to weakening of the mechanical properties and durability of the concrete. The addition of plasticisers and water‐reducing agents is therefore crucial. Approximately 50% of the lignosulphonates produced worldwide are used as concrete admixtures. Here lignosulphonates serve several purposes; they achieve workability of the concrete mixture through dispersion of concrete particles, they reduce amount of water necessary to achieve a certain workability of the mixture to improve the strength of the set concrete and they accelerate or retard the setting and they entrain air in the concrete. Lignosulphonates actively participate in the hydration of the cement minerals in Portland cement by irreversible adsorption to and incorporation into the calcium silicate hydrate gels 32,33. The adsorption retards the C2S, C3S and C3A hydration 34. The initial setting phase can thus be significantly prolonged. Retardation of the C3S and C3S hydration is very pronounced even at low lignosulphonate concentrations. C3A hydration is highly important as this process is rapid and it significantly affects the early hydration and setting of the cement. In the final stage of hydration, lignosulphonates are incorporated into the structure of the calcium silicate hydrate gel and are thus removed from the solution 34. As a consequence of the lignosulphonate adsorption to the cement particle surfaces, steric and electrostatic repulsion between the individual particles occurs. Steric repulsion prevents particle flocculation by forcing the particles apart. The electrostatic repulsion is being achieved through presence of charged groups in the lignosulphonate structure (Figure 3). This mode of action of dispersants was first described by Uchikawa et al. 35 and has been generally accepted. Using lignosulphonates, concrete with a compressive strength of 40‐50 MPa can be produced 36. With the rapidly increasing demands of high‐strength concrete with a compressive strength of 100 MPa and above (during the construction of The Petronas Towers in Kuala Lumpur 120 MPa
8
silica fume concrete was used 37), lignosulphonates are being displaced by expensive synthetic dispersants and water-reducing agents which achieve significantly greater water reduction and compression strength while maintaining low levels of air entrainment and high workability of the concrete. The entrainment of air and excessive retardation are two problems limiting the increase of dosage lignosulphonate in a cement mixture. Current-generation lignosulphonates differ significantly in their purity and sugar content to minimise the unwanted reactions in concrete and to allow for further water reduction 38.
Entrapped water
Figure 3: Cement particle flocculation and entrapment of water within the floc. Steric and electrostatic repulsion of the particles is achieved through rearrangement of the charged additive in relation to the cement particle surface charges. The individual cement particles are pushed apart and the integrity of the floc is compromised resulting in liberation of the entrapped water.
The synthetic equivalents, the so-called superplasticisers, are based mainly on two groups of nonrenewable petrochemicals. Some examples of the most utilised superplasticisers are polymelamine formaldehyde sulphite (PMS), β-naphthalene sulphonic acid formaldehyde (BNS) and methacrylic acid–methacrylate ω-methoxypolyethylenglycol 39 (Figure 4). SO3H H 3C
NH
N N
NH N
O
CH3 CH3
H3C
H n
n
H3C
CH3 a
COOH O
HN SO3H
CH3 b
CH3
c
O
n
HO 3S O
1
2
H 3C
3
Figure formaldehyde and (3) methacrylic acid–methacrylate ω-methoxypolyethylenglycol superplasticisers. The ratio of monomeric composition of (3) is indicated by a, b and c.
The high efficiency of superplasticisers is attributed to the large amount of charged groups, up to 0.4 per available position, 31 and their tailored molecular weight, which increases the repulsive forces by ensuring that there is a constant surplus of charged groups present on the superplasticiser molecule, despite the number of active sites on the cement particle 40-42. Synthetic superplasticisers allow a water reduction of up to 40% and are far superior to lignosulphonates, which allow only a 15% reduction 43. The difference in efficacy between superplasticisers and
9
lignosulphonates, new standards and a constant production of new‐generation superplasticisers has resulted in a decrease in the use of lignosulphonates 44. The ability of lignosulphonates to function as plasticisers, dispersing agents or surfactants is intimately linked with their molecular weight of the lignosulphonate and the presence of charged groups within the macromolecule. An increase in molecular weight and purity of the lignosulphonate enhances the plasticising effect as well as reducing the viscosity of the concrete 45
. High molecular weight lignosulphonates are highly efficient in this sense, even at very low
dosages. These lignosulphonates could be a potential plasticising admixture for self‐compacting concrete 45, a form of concrete that does not require vibration for placing and compaction, but is able to flow under its own weight, completely filling formworks and achieving full compaction 46.
II.2.4 Specialty Markets The use of lignosulphonates as a precursor for the production of chemicals has had only limited success. The conversion of lignosulphonates to vanillin is presently still the most successful process of this kind, despite the fact that it was discovered as early as 1874 47. This process was a prerequisite for introducing vanillin as a bulk ingredient in the food industry but, in comparison with the amount of lignosulphonates produced by the pulping industry and considering the global demand for vanilla, the production of vanillin from lignosulphonates accounts for a far too small market to be able to absorb the vast amount of produced lignosulphonates 48. The current synthetic production of vanillin from guaiacol is more than sufficient to saturate the existing markets. The dispersing and binding properties of lignosulphonates have been explored in various fields such as animal food pellet formation, gypsum board production, pigment dispersion, complexing agents, sludge containers, scale‐inhibitors in wastewater treatment, emulsion stabilisers in oil drilling muds and expanders in lead acid batteries 27.
II.2.5 Modified lignosulphonates The modification of lignosulphonates has traditionally been limited to the purification, ultrafiltration and sugar removal performed during the production. The structural similarities to conventional lignins extracted during Kraft pulping clearly suggest that the traditional bleaching chemicals used in a modern pulp mill can be employed to achieve various modifications in terms of molecular size and functional group composition. The toolbox of bleaching chemicals and the understanding of their mechanisms on lignin structures is extensive and relatively well understood, and this permits specific modifications should they be employed on lignosulphonates. Significantly fewer enzymatic tools are available for lignin modification due to the nature of the lignin itself. The most promising is the oxidoreductive radical‐initiating enzyme laccase.
10
II.3 Laccase II.3.1 A Blue Multi‐Copper Oxidase The laccase belongs to the group of oxidoreductases, the first class of enzymes in the enzyme classification system (E.C. 1). Strictly defined, oxidoreductases are capable of performing electron transfer from a donor to an acceptor. Further classification of oxidoreductases divides this class into sub‐groups based on the donor and the acceptor. Laccase is found in the group of oxidoreductases active on diphenols and similar substrates (E.C. 1.10), utilising oxygen as electron acceptor (E.C. 1.10.3). The members of the oxidoreductases share one unique feature; they all belong to the group of multi‐copper oxidases with several centres where copper atoms are stabilised 49. Copper is one of the most widespread transition metals in nature. The rich abundance in nature is intimately linked to its oxidation/reduction potential and the key role which oxidation/reduction reactions play in nature. Due to the high toxicity, even at a very low concentration, elaborate stabilisation of copper is required for it to reside in living organisms. It is the presence of copper and the interplay between the Cu(I) and Cu(II) oxidation states that allows the organisms in which it resides to perform complicated oxidation and reduction reactions. Multi‐copper oxidases contain two mono‐nuclear centres (type‐1 and ‐2, containing one copper ion) and one di‐nuclear centre (type‐3, containing two copper ions), each having unique spectroscopic features. The type‐1 centre shows high absorption in the visible region (giving the enzyme a distinct blue colour when in solution), type‐2 centre has undetectable absorption and the type‐3 centre with its pair of copper ions coordinated anti‐ferromagnetically shows strong absorption in the near‐ultraviolet region 50. Multi‐copper oxidases are found in all three life domains, prokaryotes, eukaryotes and archae. In plants and fungi, they are involved in lignin formation and degradation and in yeast and mammals with iron metabolism 51.
II.4 Structure and Catalytic Properties The majority of fungal laccases are monomeric, dimeric or tetrameric glycosylated protein complexes. Glycosylation is believed to play an important role during its secretion, susceptibility towards degradation and thermal stability 52. Besides glycosylation, laccases contain covalently linked carbohydrate units (ranging from 10‐45% of the total molecular mass) which are assumed to contribute to the conformational stability by protecting them from proteolysis and deactivation by radicals 53,54. The overall molecular weight of laccases varies from 50 to 100 kDa. Several three‐dimensional crystal structures of different fungal laccases have been determined 55‐ 60
. They all show a striking structural homology but with some minor differences in loop
organisation and in the appearance of the substrate‐binding pocket. The overall three‐
11
dimensional structure of a laccase consists of three consecutively connected cupredoxin-like domains twisted in a tight globule (Figure 5A). The copper ion binding site T1 is located in the third domain and the T2/T3 site is located between the first and third domains (Figure 5B). This site contains amino acid ligands from both domains 61. Structural sequence comparison of more than 100 laccases identified four conservative regions which are specific to all laccases containing a cysteine and ten histidine residues 62 (Figure 5C). Together they form a compartment in which copper ions are located. Multiple isoforms of the same laccase are known to be secreted by several white-rot fungi
63,64
.
These can differ significantly in stability, optimal pH and temperature and substrate affinity. They are encoded by gene families and can be either constitutively expressed or induced by external factors 65,66. Laccases have been shown to have remarkably low substrate specificity and large differences in range of oxidised substrates can be found in different laccases. These enzymes are all capable of oxidising of wide array of organic and inorganic substrates, including polyphenols, various substituted phenols and aromatic amines. H N
His 395
A)
B)
T1 Cu
C)
N
H N
N
T1 Cu Cys 453
His 458
SH
His 454 NH
His 452 N
N
H N
His 400
T2 Cu
N
T2 Cu
T3 Cu
N H
T4 Cu
O
T3 Cu
His 398
N
H
NH
N
His 111
His 109
H
N
His 66
T4 Cu NH
N H N
N
N H
His 64
NH
Figure 5: (A) Ribbon diagram of Trametes hirsuta laccase determined by X-ray chrystallography at 1.8 Å resolution 61 with the three-domain organisation and copper ions clearly distinguishable. (B) The active site consisting of four copper ion sites (T1 through T4) is located between the domains. (C) The four copper ions are stabilised almost excusively by adjacent histidine residues.
The active site of laccases consists of three copper ion sites designated type-1, -2 and 3 (T1 through T3). The type 2 and 3 sites forms a type 2/3 (T2/T3) cluster. The four copper ions located in these sites are consequently designated Cu T1 through Cu T4. A two-site ping-pong bi-bi reaction mechanism for laccase has been proposed 67. The reaction begins with the oxidation of substrate by the Cu T1 which accepts one electron from the substrate and transfers it to the T2/T3 cluster where dioxygen binds. Upon binding to the T2/T3 cluster, dioxygen accepts the electrons from T1 and is reduced to two molecules of water. This reduction requires four electrons which mean that four one-electron transfers have to be made from the T1 site (Figure 6). The extraordinary ability of laccase to reduce oxygen to water without the formation of radicals or anions is unusual in biological systems.
12
T1 Cu(II)
OH
Lignin
4x
T3 Cu(II)
T2 Cu(II)
H2O
OMe OH
T4 Cu (II)
T1 Cu(I)
OH
Lignin
T2 Cu(I)
T3 Cu(I)
O2
4x OMe
T4 Cu (I)
O
Figure 6: Schematic representation of the laccase reaction mechanism. In order to complete the full reduction of one molecule of dioxygen to water, four substrate molecules are oxidised.
The key to laccase activity is the redox potential of the T1, T2/T3 copper sites. The potential of T1 site has been determined for a large number of laccases to be within 400‐800 mV 68. The potentials for T2 and T3 sites are less investigated and are so far known only for plant laccase from Rhus vernicifera (390 mV and 460 mV for T2 and T3 respectively) and fungal laccase from Trametes hirsuta (400 mV for T2) 69,70. Based on the potential of the T1 site, laccases are divided into low‐, medium‐ and high‐redox potential enzymes.
II.5 Occurrence and Role in Nature The first known isolation of laccase was performed by Yoshida in 1883 from the sap of the lacquer tree Rhus vernicifera 71. He was able to isolate a thermolabile compound with what he referred to as diastatic properties which consumed oxygen when it was mixed with urushiol. He drew the conclusion that this diastase‐like compound was responsible for the lacquer drying process that occurs naturally. More than a decade later, the name “laccase” was coined by Bertrand who isolated and purified this compound from the sap of the lacquer tree 72 and also for the first time demonstrated their presence in fungi 73 . He also introduced the concept of metalloenzymes by erroneously claiming that this newly discovered laccase contained manganese, a conclusion he drew from the large abundance of manganese in the sap itself. It was however later shown that laccase in fact contained copper 74,75. Laccases are widely distributed among terrestrial life forms such as plants and fungi. While their role in fungi has been extensively studied and is still a topic under discussion, laccases in plants have been less studied. It is well known that in lacquer trees grown in Eastern Asia laccase is involved in various defence mechanisms as a response to physical injury to the bark of the tree. A white sap (lacquer) is excreted which is oxidised when in contact with oxygen with the subsequent polymerisation of the phenols in the sap so that a highly resistant protective structure is formed. Laccase has been isolated not only from trees such as sycamore and loblolly pine 76 but also from a variety of vegetables and fruits 77 and green shoots of tea 78.
13
In the fungi kingdom, laccases are present in all seven phylums. The enzyme has been attributed a role in a variety of cellular processes including delignification, sporulation, plant pathogenesis, pigment production and fruit‐body ripening 79. The most widely studied laccases originate from the white‐rot basidiomycetes which are highly efficient lignin degraders. It is due to their abundant presence in the white‐rot fungi that laccases traditionally have been regarded as an important participant in the lignin‐degradation process. Numerous studies of wood decay have identified other enzymes that participate in the delignification process. Among these are lignin peroxidases and manganese peroxidases, responsible for oxidation of both phenolic and non‐ penolic units, glucose oxidase and glyoxal oxidases for H2O2 production and cellobiose‐quinone oxidoreductase for quinine reduction and hydroxyl radical generation 80. It has been demonstrated that there is no unique mechanism for lignin degradation and that the setup of the required enzymatic tools differs greatly between various microorganisms. Laccases have also been associated with lignin biosynthesis participating as an oxidant of monolignols, a role traditionally reserved solely for peroxidases81‐83. This theory was first proposed by Freudenberg et al. 83 who saw that laccase was able to oxidise lignin monomers to form dimers of similar structure to those produced through the chemical degradation of lignins. Their theory was abandoned when no successful detection of laccases in plant tissues was achieved. Later investigations were however able to reignite this discussion when it was demonstrated that laccase alone could polymerise monolignins in the complete absence of peroxidases 84. It was also suggested that laccases were involved in the very early stage of lignin biosynthesis and that peroxidases were involved at later stages 84. The investigation of lignin biosynthesis (extensively reviewed by Lewis et al. 85) has provided indirect evidence of the involvement of a variety of peroxidases, laccases and other oxidases, suggesting that such a complex reaction as lignin biosynthesis cannot simply involve one specific group of oxidases. This question remains open and is still being widely discussed. Although many enzymes are able to oxidise monolignols in vitro, no unequivocal proof of the involvement of any specific group of oxidases in lignin biosynthesis in vivo has been presented through loss‐of‐fuction experiments in transgenic trees 86. Laccases active on lignin have been reported to be mostly extracellular, although the occurrence of intracellular laccases in white‐rot fungi has been demonstrated 87,88. Froehler and Ericsson also proposed a role for the intracellular as a precursor to the extracellular laccase with little or no difference between them other than their location in the cell 87.
II.6 The laccase‐mediator system The heterogeneity and complexity of lignin together with the low redox potential of laccases allow them, in contrast to other lignolytic enzymes, to oxidise only those phenolic units in lignin which have an ionisation potential within the range of the T1 site 89,90. To explain how laccase is still able to be active on such a complex polymer, the laccase mediator theory was developed. Earlier it was believed that laccase was involved only in lignin degradation by oxidising phenolic end groups in lignin to phenoxy radicals with subsequent linkage breakage. In later experiments,
14
the presence of low‐molecular‐weight co‐substrates, the degradation of non‐phenolic structures was observed 63. The term mediator was soon coined to describe the role of these co‐substrates. Their significantly higher oxidation potential (>900 mV) was shown to be essential for expanding the activity of laccase on non‐phenolic substrates. Since the first experiment where ABTS, diammonium salt of 2,2ʹ‐azinebis(3‐ethylbenzothiazoline‐6‐sulfonic acid) was demonstrated to enhance the enzymatic ability of laccase 63, the number of compounds that can be converted by laccase has increased significantly. Over the years, a large number of synthetic and naturally occurring mediators have been proposed and successfully tried with laccase. Consequently, the role of laccase in nature has also been expanded or revised 63,91. A laccase mediator participates in a cyclic reaction with laccase (Figure 7) where a high‐potential mediator intermediate is formed through laccase oxidation. This intermediate participates in non‐ enzymatic reactions with other substrates not accessible to or oxidisable by laccase alone. Upon oxidising the substrate, the mediator returns to its reduced state, thus closing the cycle 92. This cycle should, ideally, be repeated a number of times before the mediator is degraded. It should be noted that the chemistry by which laccase reacts with the mediator is significantly different to the mediator‐substrate chemistry. The misconception that the redox potential of the laccase increases when a mediator is used is thus not valid 93. O2
Laccase
H2O
Laccaseox
Mediatorox
Mediator
Substrate
Substrateox
Figure 7: The laccase mediator cycle as first proposed by Bourbonnais 63.
In‐depth studies of a number of mediators such as ABTS 94,95 various N‐O‐ and N‐OH‐containing mediators such as HBT (1‐hydroxybenzotriazole), violuric acid, N‐hydroxyacetanilide and various polyoxymetals (such as [SiW11V1O40]5–)92,96‐98 show that no compounds currently fit the criteria of an ideal mediator. All the proposed compounds either exhibit low stability during the enzymatic activation or show no or very low ability to regenerate and thus participate in a series of cycles. Despite the recent findings that a few compounds are actually able to participate in a substantial amount of cycles 99,100 it appears that a true laccase redox mediator is yet to be discovered.
II.7 Applications The potential of laccase as an industrial biocatalyst appears to be significant. Laccase is one of the few oxidoreductive enzymes that do not require expensive co‐factors other than dioxygen and recent developments in heterologous expression have enabled the large‐scale production of the enzyme. Consequently, the enzyme has found its way into a number of industrial processes including paper processing, the prevention of wine decolouration, bioremediation and textile dye oxidation. Extensive research into the laccase‐mediator system has resulted in a massive effort to find technical and industrial applications for such systems.
15
II.7.1 Laccase in the pulp and paper industry Lignocellulose is a natural substrate for laccase and, provided that mediators are used, laccases have the potential for breaking non‐phenolic units in lignin without disrupting the integrity of the cellulose which is closely linked to lignin in native wood 11. This is particularly interesting in the pulp and paper industry. The enormous global production volumes of this industry means that even a minor improvement achieved by a LMS process step would have huge implications. The current industrial preparation of pulp and paper, the Kraft and sulphite processes relies on the separation and degradation of lignin from cellulose through cooking and bleaching with conventional chemicals. While the recovery and reuse of the chemicals in the cooking stage is practised, the negative environmental impact of the bleaching stage is well documented. Although recent developments have yielded significantly less detrimental processes with the abolition of elemental chlorine in the early 1990’s and the development of the ECF and TCF bleaching processes there appears still to be a need to develop new methods based on laccase. Extensive studies have been performed to develop and evaluate LMS for Kraft pulp bleaching 101‐ 103
. The results appear promising and they have already found practical application in the
Lignozym© process 104. The use of LMS with flax pulps has also been explored with successful results 105, displaying the versatility of these systems. There is however one major obstacle for the successful implementation of LMS in a modern paper mill; the mediator. There is, to date, no readily available and cheap mediator that performs several oxidation cycles. In addition, large quantities of mediator are required to achieve a bleaching performance comparable to that of conventional chemical processes. For instance, the Lignozym© process 104 requires a mediator amount of up to 2% of the dry weight of the pulp. In a normal Kraft mill producing 1 000 ‐ 3 000 tonnes of pulp per day, a daily mediator consumption of 60 tonnes is required. Considering the significant cost of the enzyme and the mediator as well as the minimal environmental footprint of a modern pulp mill, the incentive to replace conventional bleaching chemicals with a LMS process step is low. Despite more than two decades of research and development of a LMS bleaching stage there has as yet been no commercial adaptation of this process on an industrial scale. The ability of laccase to form reactive radical species on lignin end groups can also be utilised for fibre modification. It has been successfully demonstrated that laccase facilitates adhesion of fibres during the manufacturing of wood composite materials such as fibreboards 106,107. Functionalisation of lignocellulotic fibres by grafting various phenolic acid derivatives onto Kraft pulp fibres is an another possibility use for laccase 108,109. Although promising, these are areas of laccase utilisation that have not yet reached industrial levels.
II.7.2 Laccase in the textile industry The textile industry consumes large volumes of water and chemicals for textile processing. Each year, more than 700 000 tonnes of dyestuff are produced 110 which are resistant to a variety of chemicals making them difficult to decolourise and detoxify. Current ways of treating dye waste 16
water are ineffective and expensive 111 making them an ideal target for processes based on laccase oxidation which have been demonstrated to be very capable of degrading dyes of various structures 112,113. Utilisation of laccase in the textile industry is an expanding field and covers not only effluent treatment but also textile bleaching and dye synthesis 114. In 1996, Novozymes (Bægsverd, Denmark) launched the first laccase preparation to be used in the fabric industry under the name DeniLite®. This preparation utilises a redox mediator (phenothiazine‐ propionate). The product was followed by the laccase‐mediator formulation under the trade name Zylite® (Zytex Pvt. Ltd., Mumbai, India). Both formulations are used for the removal of indigo from denim jeans clothing.
II.7.3 Laccase in alternative applications In addition to the previously discussed industrial applications, laccase is of interest in a variety of fields and applications. In the food industry, laccase can be used for drink clarification and as biosensor for the monitoring of phenol formation 115. In the baking industry, laccase is interesting for its ability to cross‐link biopolymers in various doughs 116. Other highly interesting applications of laccases are in nanobiotechnology for electroimmunoassay sensors 117, organic synthesis 118 , biofuel production 117 and keratinous fibre dying 119.
17
III Experimental Detailed description of the materials, methods and analytical apparatus is given in the related papers.
III.1 Materials III.1.1 Enzymes Two laccases provided by Novozymes (Bagsværd, Denmark) were used in all the experiments. The laccases, denoted as NS51002 and NS51003, originated from Trametes villosa (TvL) and Myceliophthora thermophila (MtL) respectively. The two enzymes had different temperature and pH optima as well as redox potentials E0 (50°C, pH 5, E0 780 mV for TvL, 40°C, pH 7.5, E0 480 mV for MtL.
III.1.2 Lignosulphonates Four lignosulphonate salts supplied by Borregaard LignoTech (Sarpsborg, Norway) were used in all the experiments. The salts were characterised by the supplier by standard methods (Table 1). The lignosulphonates were used without further purification. Table 1: Characteristics of the lignosulfonate salts used in laccase oxidation experiments described in Papers III-VI.
Mw(Da)
Phenolic content (%)
Organic sulphur (%)
2+
28 400
1.9
5.7
Na
+
46 500
2.1
6.2
Ultrafiltered Desulphonated Oxidised
Na
+
9 000
1.9
3
Filtered Heat treated
Ca
2+
5 900
1.4
4.7
Salt
Raw Material
Process
Counter-ion
DP398
Softwood
Filtered
Ca
DP399
Softwood
Ultrafiltered Ion exchanged
DP400
Softwood
DP401
Hardwood
The lignosulphonate DP398 was provided from different batches and was thus also denoted DP795 and DP851.
18
IV Results and Discussion IV.1 Model compound studies of the laccase reaction mechanism (Paper I & II) The reaction mechanism of laccase has been extensively studied over the years both in lignin degradation 104,120‐124 and in biosynthesis 83‐85. Model compound studies of laccases have demonstrated the efficacy of the laccase‐mediator systems with an array of mediators, both natural and synthetic. These experiments were however aimed to understand and expand the degradative aspects of laccase activity, and the potential of laccase for polymerisation has attracted significantly less attention. Experiments with mediators such as ABTS 125, polyoxalates 126
, ferulic acid 127, HBT 128 and also in the absence of mediators 129‐132 have clearly demonstrated
that laccase is a highly potent tool for the polymerisation of phenol containing compounds. To elucidate the polymerisation mechanisms of laccase in the present work, a number of lignin end‐group compounds were subjected to oxidation by two laccases, TvL and MtL (Figure 8). The oxidation experiments were conducted under the optimal conditions for the respective enzymes in Tris‐buffer under constant oxygen saturation. The products were analysed with a variety of mass spectrometric and spectroscopic tools. OH
1
OH
Me
1
6
2
OH
Me 1
1
6
2
6
6
2
2
5 3 OMe
4
5 4
MeO
3 OMe
MeO
5
3 OMe
4
5 4
MeO
3 OMe
OH
OH
OH
OH
1
2
3
4
Vanillyl alcohol
Syringyl alcohol
-methylsyringyl alcohol
4-methyl syringol
(4-hydroxy-3-methoxybenzyl alcohol)
(4-hydroxy-3,5-dimethoxybenzyl alcohol)
(4-(1-hydroxyethyl)-2,6-dimethoxy phenol)
(4-methyl-2,6-dimethoxy phenol)
OH
O
1 6 5
6
2 3
4
OMe
2
MeO 5 4
OH
Me 1
1
3
6 OMe
MeO
6
2
5 4
OMe
SO 3H
1
3 OMe
OMe
2
5 4
3
OMe
OH
5
6
7
8
Vanillin
3,4,5-trimethoxybenzyl alcohol
1,2,3-trimethoxy-5-methyl benzene
Vanillyl sulfonic acid
(4-hydroxy-3-methoxy benzaldehyde) SO3H
Me
Me
6
6
2 3
OMe
OH
5
6
2
4
SO 3H 1
1
1
MeO 5 4
SO3H
(4-hydroxy-3-methoxyphenylmethanesulfonic acid)
3
2
MeO 5 4
OMe
3
OMe
OH
OH
9
10
11
Syringyl sulfonic acid
-Methylvanillyl sulfonic acid
-Methylsyringyl sulfonic acid
(4-hydroxy-3,5-dimethoxyphenyl - methanesulfonic acid)
(1-(4-hydroxy-3-methoxyphenyl) ethanesulfonic acid)
(1-(4-hydroxy-3,5-dimethoxyphenyl) ethanesulfonic acid)
Figure 8: Model compounds oxidised by MtL and TvL in Papers I and II. The nomenclature is as follows; the aromatic carbons in the benzyl ring are numbered clockwise from 1 to 6 starting with the carbon to which the side‐chain is attached. The side‐chain carbons are denoted with greek letters α, β and γ. To distinguish atoms on a secondary monolignol, an index ‘ (prime) is added.
19
The end groups in lignin occupy several interesting positions which to varying degrees affect the coupling pattern when they are subjected to oxidation reactions by laccases. The traditional understanding of the polymerisation mechanism of laccase can be summarised in three steps: oxidation of a phenolic substrate through one‐electron abstraction, generation of a resonance‐ stabilised phenoxy radical, and thereafter a spontaneous, non‐enzyme catalysed coupling and rearrangement. The phenoxy radical formed in the first step is characterised by a relatively long life‐time and stability due to the delocalisation of the unpaired electron to various positions along the benzylic ring and side‐groups. As a consequence, the phenoxy radical can participate in a variety of reactions depending on the position of the unpaired electron. The generally low redox potential of laccases limits their action to phenolic substrates. This was evident as neither of the two laccases TvL and MtL was able to alone oxidise the non‐phenolic trimethoxy benzyl alcohol 6. Although the laccase‐mediator system (LMS) has been demonstrated to be a highly efficient tool for oxidation of non‐phenolic lignin units 63,91, the chemistry of those reactions is not within the scope of this thesis and has not been investigated. It should however be noted that the mechanisms of the LMS are significantly different from those of laccase alone. Whereas laccase oxidation relies on the abstraction of phenolic hydrogen, the LMS is able to react with more inert hydrogens such as Hα and Hβ.
IV.1.1 The 1‐position Laccase oxidation of vanillyl alcohol 1 enables the unpaired electron to delocalise to 4‐OH, 5, 1 and 3‐positions in the benzyl ring (Scheme 3). Subsequent radical‐radical coupling is thus expected to occur between these positions. GC/MS analysis revealed the formation of 4‐O‐5’ and 5‐5’ coupling products. OH
1 6
2
Laccase
5 4
3 OMe
O2
1
1 6
6
2
5 3
4
OH
OMe
3 OMe
O
2
2
6
3 OMe
4
C 3 OMe
O
2+2
OH
HO
OMe
OH
1 5
4
O
OH
OH
1 C
OH
5
1+2
O
6
2
HC 5 4
O
OMe
OH
OH
4
3
2
1
OH
MeO
OMe OH
OH
5-5 dimer
4-O-5' dimer
Scheme 3: Delocalisation of the unpaired electron and proposed reaction mechanism for 4‐O‐5’ and 5‐5’ dimerisation of vanillyl alcohol when oxidised by MtL and TvL. The various resonance forms of the oxidised vanillyl alcohol are labelled 1 through 4.
20
Interestingly, the amounts of 4‐O‐5’ and 5‐5’ coupling products obtained were shown to be related to the pH at which the oxidation was performed. At pH 5, the optimal pH for TvL, the formation of 4‐O‐5’ coupling was promoted, whereas formation of 5‐5’ coupling products dominated at pH 7 (the optimal pH for MtL) (Figure 9A, B). This phenomenon was attributed exclusively to pH when the same outcome was observed when TvL was run at the optimal pH for MtL and vice versa. No explanation of the pH‐dependence of the reaction has yet been proposed but the deprotonation of the phenol group of the substrate prior to electron abstraction by laccase can provide some explanation. HO
A)
HO
OH
B)
OH
MeO
OMe OH
MeO
OMe OH
42.77
100
5-5’ dimer
2.50
OH
OH
5-5’ dimer OH
OH
90
OH
OH
2.00 O
O
70 OMe
60
OMe
OMe OH
AU
Relative Abundance
80
1.50
OMe OH
4-O-5’ dimer
4-O-5’ dimer
50 1.00
40
43.10
30 0.50
20 10 0.00
0 42.6
42.8
43.0
43.2
10.00
43.4
15.00
152
122
152
122
152
122
152
122
122
152
122
152
x 104
603.183
3.0
893.373
877.357
755.282
771.294
619.174
741.194
1.0
497.078
467.089
344.949
2.0
481.090
328.964
intens. [a.u.]
C)
20.00
Minutes
Time (min)
0.0 300
400
500
600
700
800
900 m/z
Figure 9: (A) GC, (B) HPLC and (C) MALDI‐TOF MS spectrograms of vanillyl alcohol 1 oxidation by MtL, (dashed) and TvL (solid).
Further probing of the oxidation results with MALDI‐TOF MS analysis revealed formation of oligomeric coupling products consisting of up to seven units separated by masses of 152 and 122 Da (Figure 9C). The mass difference of every second monomer unit suggests a loss of 30 Da from every second monomeric unit during polymerisation. HPLC analysis showed that this loss was due to a rearrangement and release of the side‐chain at the 1‐position of vanillyl alcohol as formaldehyde (Scheme 4). These findings are in line with previous studies on lignin model compounds where side‐chain elimination reactions were observed during oxidation 133‐138. It is likely that the 4‐O‐5’ and 5‐5’ dimers serve as initiation points for the oligomer which growths through the addition of single monomers or dimers. 21
2
6 R
3
4
6
O2
OMe
6
2
5
5 R
OH
OH
3 OMe
4
2
1 6
2
C
3
4
R
OMe
4
R
3 OMe
2+3
C 3 OMe
4 O
O
O
O
2
5
5
R
OH
OH
1 6 C
1
1
Laccase
5
OH
1
4
3
2
1 OH
1+3
OH
OH
OMe
OMe
HO
HO
MeO OMe
OH
OH
- CH2O
O R
- CH2O
O
OH R
MeO
R
OH
O
MeO
OH
OMe
R
O
OH OMe
1-5' bond
1-O-4' bond
Scheme 4: Proposed mechanism for formation of 1‐5’ and 1‐O‐4’ bonds when vanillyl alcohol is oxidised by MtL and TvL. Formation of these couplings predominates when the 5‐position of the substrate is occupied (denoted R). The various resonance forms of the oxidised vanillyl alcohol are labelled 1 through 4.
The repetitive pattern of alternating masses observed in the MALDI‐TOF analysis suggests that every second unit is incorporated through the elimination of the side‐group at the 1‐position. Since no 1‐O‐5’ or 1‐5’ dimers were identified, it is assumed that coupling to the 1‐position occurs only when either the 4‐OH or 5‐position is occupied. It has been postulated that coupling to 1‐ position occurs in natural lignin, albeit with a low occurence 139,140. Nonetheless, these findings are relevant to establish the reactivity of the 1‐position. Replacing the hydroxyl group with a sulphonic acid group (SO3H) at the Cα position of vanillyl alcohol (vanillyl sulphonic acid 8), did not inhibit formation of oligomeric material. It did however effectively prevent side‐chain elimination as no traces of elimination products were detected (Scheme 5). The introduction of C (α‐methylvanillyl sulphonic acid 10) yielded similar results. Me
Me
1 6
2
5 4 OH
3
Laccase OMe
O2
SO 3H 2
5 4
Me
3 OMe
SO 3H
6
1+2
Me 6
2
HC 5 4
3
2+2
4-O-5' and 5-5' bonds
OMe
SO 3H
Me
4 SO 3H 1
1C
6
2
2
5
5
O
O
1
1 6
3
2
1
SO 3H
4
3
OMe
4
C 3 OMe
O
O
1+3 2+3
1-5' and 1-O-4' bonds
Scheme 5: Sulphonation of Cα effectively prevents couplings to the 1‐position when the substrate is oxidised by laccase. The various resonance forms of the oxidised sulphonated end group model are labelled 1 through 4.
22
IV.1.2 The Cα position Model compound experiments with laccase revealed the importance of the Cα position in both lignin and lignosulphonates. Based on previous experience of laccase oxidation of syringyl alcohol 2 and α‐methyl syringyl alcohol 3 with methoxy groups at the 3‐ and 5‐positions, only the 4‐OH and the 1‐position are expected to participate in any eventual coupling reactions. Interestingly, only dimeric products in small amounts were detected. The main oxidation products detected were Cα oxidation products (Figure 10). This oxidation is assumed to occur in two steps; disproportionation and tautamerisation. While the disproportionation involves the reaction of two radicals, one being reduced back to a phenolate and the other being oxidised to a quinone methide, the subsequent tautamerisation is a rearrangement of the oxidised quinone methide where the Cα is rearranged to a carbonyl (Scheme 6). A)
B)
Me
O
O
OH MeO
3.00
3.00
MeO
OH
OH
OMe
MeO
Me
HO
OMe
OMe
OH
syringyl alcohol 2
1.00
0.50
0.50
15.00
OMe
4-O-1’ dimer
OH
1.50
4-O-1’ dimer
OMe
HO MeO
1.00
10.00
MeO
2.00
AU
AU
MeO
OH
O
MeO
O
OMe
OH
2.00
0.00
MeO
2.50
Syringyl aldehyde
1.50
OMe
Acetylsyringone
OH
2.50
Me
OMe
α-methylsyringyl alcohol 3
0.00
20.00
10.00
Minutes
15.00
20.00
Minutes
Figure 10: HPLC spectrograms of oxidation product formed from oxidation of (A) syringyl alcohol 2 and (B) α‐ methyl syringyl alcohol 3 by TvL (dashed) and MtL (solid).
2
1
OH
1 6
2
5 MeO
4
3
OMe
OH
1
1
Laccase
6
O2
5 MeO
OH
6
2
C
OH
OH
OH
Disproportionation
2
+
5 4
3 OMe
MeO
4
3
OMe
O
O
MeO
OMe
MeO
OH
OMe O
1+2
Tautamerisation
OH O
MeO
O OMe MeO
OMe OH
MeO
OMe
Syringaldehyde
OH
1-O-4' bond
Scheme 6: Possible reaction routes of syringyl alcohol after oxidation by laccase. The various resonance forms of the oxidised vanillyl alcohol are labelled 1 through 4.
23
The pH was again shown to influence the oxidation reaction as larger amounts of syringaldehyde and acetylsyringone were formed at the higher pH (Figure 10). Oxidation of Cα is a potential terminal reaction where the oxidation potential of the oxidised syringaldehyde is raised to a level close to or above the E0 of MtL 141. This was evident when vanillin 5 with a Cα carbonyl was oxidised by TvL and MtL. While the high‐E0 TvL generated oligomeric materials, the low‐E0 MtL left the vanillin untouched. Laccase oxidation of syringyl sulphonic acid 9 and of α‐methylsyringyl sulphonic acid 11 in which the Cα hydroxyl was replaced with a sulphonic acid group left the model compounds untouched, suggesting that Cα oxidation is inhibited by the presence of a sulphonic acid group. On the other hand, the lack of a non‐occupied 5‐position effectively prevents any coupling reactions between these model compounds. These findings are important as they illustrate the detrimental outcome of laccase oxidation of lignin end groups containing Cα hydroxyls. The presence of end groups containing reduced Cα has been postulated to exist in lignin 142,143. To investigate their influence, the model compound 4‐methyl syringol, 4 was oxidised by laccase. Lacking a non‐occupied 5‐position and a suitable leaving group at the 1‐position, the possibilities for polymerisation appeared less obvious. Nonetheless, oligomeric compounds consisting of up to three monomeric units were discovered with MALDI‐TOF MS analysis (Figure 11).
Intens. [a.u.]
A) 166
x 104
166
166
166
166
166
166
166
871.176
705.045
855.189
2
721.049
554.907
689.057
522.911
538.899
372.748
388.735
4
356.755
6
0 400
500
600
700
800
900 m/z
x 104
166
871.161
689.048
522.916
2
166
705.039
166
538.904
166 356.842
4
166
855.173
166 6
372.852
Intens. [a.u.]
B)
0 400
500
600
700
800
900 m/z
Figure 11: MALDI‐TOF MS spectrograms of 4‐methyl syringol 4 oxidation by (A) MtL and (B) TvL.
The high degree of substitution of the model compound limits the theoretically possible number of linkages to two; α‐α’ and 4‐O‐α’. This implies that delocalisation of the unpaired electron to the Cα position occurs. Two probable explanations may be considered; direct electron abstraction from the Cα position by laccase or an indirect electron shuttle effect. The inability of laccase to
24
directly oxidise the Cα was clearly demonstrated when the non‐phenolic model compound 3,4,5‐ trimethoxybenzyl alcohol 6 (Figure 8) remained unchanged after treatment with TvL and MtL. The shuttle effect was investigated by oxidising a 1:1 (mol:mol) mixture of 4‐methyl syringol 4 and its non‐phenolic equivalent trimethoxy benzene 7. Dimers of the model compounds 4 and 7 were indeed discovered, strengthening the electron shuttle theory (Scheme 7). Me
Me
Me Laccase
MeO
OMe OH
O2
CH2
Me
+
+ MeO
OMe O
4-methylsyringol 4
MeO
OMe OMe
MeO
OMe OH
MeO
OMe OMe
trimethoxy benzene 6
Scheme 7: Localisation of the unpaired electron to the Cα position of trimethoxy benzene through electron abstraction by the 4‐methylsyringol radical formed by laccase oxidation.
It appears that a level of self‐mediation occurs in this reaction. Whether an end group in a polymer chain is able to perform similar reactions remains to be answered. Although unusual, the existence of 4‐O‐α’ linkages in native lignin 144 and oligomeric oxidation products of highly substituted phenols has been previously been reported 145‐147.
IV.1.3 Conclusions from the model compound studies Laccase has clearly been demonstrated as a very potent tool for the polymerisation of various hardwood and softwood lignin end‐group models. The outcome of the reactions is largely governed by the structure of the substrate and the pH during the reactions rather than by the structure of the laccase. This is expected since radical coupling is not an enzyme‐catalysed reaction. The availability of free 5‐positions on the benzyl ring clearly favours the more abundant 5‐5’ and 4‐O‐5’ couplings. These bond forms new and not previously existing linkages between different lignin end groups and are thus termed “productive”. When the 5‐position is occupied, as in hardwood lignin or due to inter‐monolignol bonds, additional side‐chain oxidation reactions and/or coupling to the 1‐ or α‐position of the substrate occur. These reactions are termed “unproductive” since they either raise the redox potential of the end group or are formed through the breakage of existing bonds. The aryl‐ether and aryl‐aryl couplings are the most abundant in lignin and as such are easily formed as a consequence of laccase oxidation but the occurrence of couplings to 1‐ and Cα positions, albeit relatively scarce in lignin, has nonetheless been demonstrated. Sulphonation of the Cα‐ lignin is a relatively easily achieved modification of lignin that occurs in sulphite pulping. It does not only increase the solubility of lignin, but it also directs the laccase‐ initiated end‐group reactions towards specific types of couplings (Scheme 8).
25
If laccase is to be used for polymerisation of lignins, it is imperative that the reactions are directed towards the formation of new inter‐linkages and not breakage of existing ones. Sulfonated lignin end group
Native lignin end group L
L
OH
SO3
-
OMe
OMe OH
OH
OH
L
OH L OH OMe
OMe
O O
L
O
OH
OH
L H
Cα -oxidation
MeO
OH
5-5'-couplings
OMe
1-couplings
OH
OMe
O
OMe
OH
L HO
MeO
OH
L
MeO HO
SO3
L
SO3
L
MeO
OH O
MeO
OMe OH
MeO
L
SO3
5-5'-couplings
L
SO3
4-O-5'-couplings HO
Unproductive couplings
L
HO
OH
L L
MeO
4-O-5'-couplings
OH
α-couplings
Productive couplings
Scheme 8: Possible reaction pathways of laccase oxidation of phenolic end groups in native lignin and lignosulphonates.
Laccase oxidation of lignosulphonates (Paper III) Four commercial lignosulphonate salts DP398‐DP401 (Table 1) at various concentrations were subjected to oxidation by different activity units of TvL and MtL under constant oxygen saturation for 24h (Figure 12). In all cases, an increase in the average molecular weight as well as a decrease in the phenolic content was observed. Based on previous model compound studies, it is concluded that the polymerisation reactions are a consequence of coupling reactions by phenoxy radicals generated by laccase oxidation of phenolic end groups.
26
DP398
A)
B)
DP398 1.40
Ph-OH (mmol/g)
400 000
_ M
w
300 000 200 000 100 000 0 0
4
8
12 Time (h)
16
20
1.20 1.00 0.80 0.60 0.40 0.20 0.00
24
0
4
8
DP401
20
24
16
20
24
DP401 Ph-OH (mmol/g)
125 000 w
100 000 _ M
16
1.60
150 000
75 000 50 000 25 000 0
12 Time (h)
1.20 0.80 0.40 0.00
0
4
8
12 16 Time (h)
20
0
24
4
8
12 Time (h)
Figure 12: (A) Molecular weight averages and (B) phenolic content of lignosulphonates DP398 andDP401 after oxidation by TvL at various lignosulphonate concentrations and laccase activity units. The following denotions are used; (1 g/l and 50U), (10 g/l and 50U), (100 g/l and 50U), (1 g/l and 500U), (10 g/l and 500U) and (100 g/l and 500U). Similar reaction profiles were obtained with MtL as well as with the other lignosulphonate salts DP399 and DP400.
IV.1.4 The influence of lignosulphonate concentration and structure The laccase oxidation experiments were performed at three different lignosulphonate concentrations; 1, 10 and 100 g/l. Two striking observations were made; the average molecular weight increased significantly at high lignosulphonate concentrations and the reaction was more or less completed during the initial 4 hours. At lignosulphonate concentrations below 100 g/l, only minor changes were observed. The greatest increase in the average molecular weight was observed in the oxidation of DP401 by TvL where a roughly 20‐fold increase was observed. Interestingly, the lowest increase was seen in DP400 with only a 2‐fold increase after 24 hours. The decrease in the phenolic content was essentially unaffected by the lignosulphonate concentration suggesting that there was no direct relationship between these two. The consumed phenols probably either form quinone structures or participate in intramolecular coupling reactions which are not detected as an overall increase in the molecular weight. Only at higher lignosulphonate concentrations does there appear to be a near‐inverse linear relationship between the consumption of phenols and the average molecular weight increase. This relationship is most likely related to the increased probability at higher concentrations of intermolecular reactions where end groups from two individual lignosulphonate chains are coupled together (Figure 13).
27
OMe HO 3S
OMe
O
OH
HO
SO 3H
SO 3H
O M eO
M eO
O
SO 3H
M eO M eO
SO3H
OM e
OH
HO 3S
OM e
O
HO
HO
O
O HO
OH
O
OM e
O
HO 3S OH
OH
O
OH
HO
HO 3S
HO
OMe
O
OM e OH
HO
SO 3H O
M eO
O
M eO
HO 3S
OH
HO
OH OM e
O
SO 3H
M eO
O
OH
OMe
OH
O
O
HO
HO 3S
M eO
HO 3S
HO 3S
O
OMe
OMe
OMe
OM e
O OH
OH HO
HO 3S
O
OH
O
HO OMe OH
M eO O
SO 3H
O
HO
M eO
O HO 3S
OH
HO 3S
OMe
OH
SO 3H
OMe
OM e OH
SO 3H
OMe
SO 3H
O
OMe
SO3 H
O SO 3H
OH
HO
O
OH
HO
HO M eO
OH
HO HO
M eO
OMe
O SOH3H
M eO OM e
SO3H
M eO
O
HO 3 S
OH
M eO
O
M eO
HO HO SO 3H
OM e
HO 3S
O
OH
SO3H
SO 3H OH
SO 3H
O O
O O
OM e
SO 3H M eO
OH
HO 3S M eO HO 3S
OH SO3H M eO
OMe
M eO
O
O SO 3H
O
M eO
OH
M eO O
OH HO
OMe
M eO OH
O
OH
M eO
HO
HO
HO 3S
HO
O
HO3S
SO 3H
OM e
M eO
HO
HO OM e
OMe
SO 3H
HO
HO
HO 3S O
HO3S
M eO
HO
HO 3S
O
OH
OM e
OM e M eO
SO3 H
O
O
O
SO 3H
OH O
HO 3S
SO 3H
HO
O
M eO
O
M eO
OM e
O
M eO
OH
HO 3S HO
O
O
OH
OMe
OH
OH
M eO
OH
OH
M eO OMe
OH M eO
OMe M eO
M eO O
O
HO 3S
O
HO 3S HO
SO3H
HO OM e
HO 3S OH
O
HO
O
OM e O OH
O
M eO
SO3H
HO 3S
HO M eO HO
OH
HO3S
OH
O
M eO
OH
SO 3H OH OM e
SO 3H
SO3H
OH OMe OMe
HO
HO
O
O
OH
HO
OM e
O
O O
HO3S
HO 3S
OMe
HO3S
HO 3S
HO
M eO
OH
OH
M eO HO HO3S
O
M eO
SO 3H
HO SO3 H
OM e
OH
O
O
O
OM e SO 3H
High lignosulphonate concentration
Low lignosulphonate concentration
Figure 13: The importance of concentration to avoid intramolecular cross‐coupling of individual chains during laccase oxidation of lignosulphonates.
Of the four lignosulphonate salts, only DP400 exhibited low or no susceptibility to laccase oxidation. This lignosulphonate, although containing significant amounts of phenols contained only low amounts of organic sulphur. The low sulphur levels can be related to the previous model compound experiments which demonstrated the importance of the sulphonic acid group on the α‐position of a lignosulphonate end group. While the phenols are consumed at a rate similar to that of the other salts, no significant increase in average molecular weight of DP400 was observed at any concentration. This can be related to the results with model compound where it was found that the laccase oxidation of phenolic end groups yielded different results depending on the nature of the Cα. A lack of a sulphonic acid group at this position promotes non‐ productive reactions such as a coupling to the 1‐position with the subsequent breakage of the Cα side‐chain and oxidation of the Cα hydroxyl to a carbonyl group. These reactions are expected to occur on non‐sulphonated end groups in lignosulphonates and, as consequence of a low degree of sulphonation, they are dominant over the productive aryl‐aryl and ethyl‐ether couplings. The polydispersity index was shown to increase significantly with high laccase amounts (Figure 14). This is to be expected despite the the nature of the non‐selective radical‐radical couplings of the phenoxy radicals. These reactions, although not enzyme‐catalysed, are expected to correlate with enzyme amount since the radicals themselves are formed by the enzyme. The following coupling reactions occur spontaneously with little or no control, resulting in an increase in the polydispersity index. FT‐IR‐analysis of the lignosulfonates DP398‐DP401 prior to and after oxidation by TvL (Figure 14) revealed only minor differences, suggesting that no structural changes occurred in the lignosulphonates other than the increase in molecular weight and reduction of phenols. The significant drop in the reaction rate after 4 hours of reaction time can be partly explained by the lignosulphonate macromolecular structure. Several such structures have been proposed (see Introduction) and these experiments validate some of these, most notably the suggested micelle‐ like structures that lignosulphonates form in solutions with a hydrophilic surface and a hydrophilic core 15.
28
DP398 6
_ _ Mw / Mn
5
A
4 3 2 1 0 0
4
8
12
16
20
4000
24
3000
2000
Time (h)
cm
1500
1000
600
1500
1000
600
-1
DP401 3.5 3.0
_ _ Mw / Mn
2.5
A
2.0 1.5 1.0 0.5 0.0 0
4
8
12
16
20
24
Time (h)
4000
3000
2000
cm
-1
Figure 14: Polydispersity indeces and FT‐IR spectrograms of DP398 and DP401 at 100 g/l during oxidation by MtL (solid) and TvL (dotted) at 50 ( ) or 500 ( ) activity unit. The black dashed lines in the FT‐IR spectrograms represent untreated lignosulphonate salts.
Due to steric constraints, the enzyme can only access the phenolic end groups located on the surface of the lignosulphonate micelle. It is therefore probable that the molecular weight increase levels out when the phenolic end groups available to the enzyme for oxidation are consumed while the remaining groups buried within the micelle remain untouched. At that point, the polymerisation reactions are slowed down and the molecular weight increase and the concurrent consumption of phenols levels out.
IV.1.5 The influence of the enzyme The difference in redox potential between TvL and MtL was manifested during the oxidation of the lignosulphonates DP398‐DP401. A tenfold increase in activity units (U) of MtL was required to match the average molecular weight increase achieved by TvL under identical conditions. At low MtL activity units (50U), no change in the average molecular weight was observed at any of the investigated lignosulphonate concentrations. The most probable explanation is the ability of TvL to oxidise a broader range of phenolic end groups due to its significantly higher redox potential. To investigate the influence of enzyme addition to lignosulphonate, two separate experiments were conducted where equal amounts of activity units of TvL was added batch‐wise (at time = 0h) and continuously (each hour) to a 100 g/l lignosulphonate solution during 4 hours of reaction time (Figure 15).
29
3.0
200 000
2.5
Ph-OH (mmol/g)
250 000
150 000
_ M
w
Batch-wise addition
100 000 50 000 0
2.0 1.5 1.0 0.5 0.0
0
1
2 Time (h)
3
4
0
1
2 Time (h)
3
4
1
2 Time (h)
3
4
Continuous addition 3.0
Ph-OH (mmol/g)
250 000
150 000
_ M
w
200 000
100 000 50 000
2.5 2.0 1.5 1.0 0.5 0.0
0
0
1
2 Time (h)
3
0
4
Figure 15: Average molecular weight increase (left) and phenolic content decrease (right) of batchwise and continuous addition of 50U TvL to 100 g/l of DP398 ( ), DP399 ( ), DP400 ( ) and DP401 ( ).
While the batch‐wise experiment reached a plateau after the first hour with apparently no further reactions, the addition of Tv each hour resulted in continuing polymerisation reactions under the 4 hour duration time. This experiment demonstrates an interesting observation; the ability to control the reaction rate through the continuous addition of the catalyst. It could also suggest that the life span of laccase is reduced by the presence of lignosulphonates, possibly through enzyme deactivation. In that sense, the unique feature of enzymes, their reusability, is lost after only one hour of reaction but the reaction can be restarted if fresh laccase is added. This is however only possible as long as there is phenols available for oxidation.
30
IV.2 Structural modifications of lignosulphonates through laccase oxidation and ozonolysis (Paper IV) Earlier efforts with technical lignosulphonates and laccases clearly confirmed that a significant increase in the average molecular weight can be obtained already after 4 hours of reaction time. Laccase oxidation of lignosulphonates serves however only to increase the average molecular weight. It does not solve the question of how to change the functional group composition, other than the phenol content. To introduce additional charged groups and potentially increase the performance of lignosulphonates as dispersants, significant modifications of the lignin backbone have to be achieved. Traditional ozone bleaching has the potential to introduce charged muconic‐ acid‐like structures through ring‐opening of the lignin benzyl ring (Figure 16). While the purpose of bleaching is lignin degradation to liberate the cellulose, applying bleaching techniques onto lignosulphonates serves a different purpose, viz.: to introduce of charged groups into the polymer. L
-
SO 3
L R
O3
1 6
2
HO
-
SO3 1
4 5
2 6
5
R
4
3
3
O
OMe
O
OMe
OH
Figure 16: Ring‐opening reactions performed by ozone on a lignosulphonate end group. The R‐ group designates H, OMe or cross‐coupling in DP795, DP401 and laccase oxidised DP795 respectively.
Studies in which ozonolysis was employed to solubilise technical lignins 148,149, a significant increase in the amount of carboxylic groups was observed. This occurred however at the expense of a reduction in molecular weight, which is potentially detrimental to the performance of lignosulphonates as dispersants. By combining laccase catalysis and ozonolysis, a high‐molecular weight lignosulphonate polymer enriched with carboxyl groups can be produced. For these investigations, two lignosulphonates DP795 (different batch of DP398), DP401 and the MtL were chosen.
IV.2.1 Laccase oxidation of DP401 and DP795 DP401 and DP795 at 100 g/l were oxidised by 500AU of MtL over a period of 4 hours under constant oxygen saturation. As a consequence of the previously reported radical‐radical coupling reactions, both the average molecular weight and the polydispersity of the lignosulphonates showed an almost 3‐fold increase (Table 2).
31
Table 2: Molecular mass distributions of lignosulphonates DP401 and DP795 after oxidation by 500U MtL. DP401
DP795
Time (min)
Mw (Da)
Mw / Mn
Mw (Da)
Mw / Mn
0
12 700
2.4
41 700
2.8
60
16 500
3.2
52 200
3.5
120
26 900
3.4
78 900
4.6
180
32 200
3.8
92 500
5.7
240
37 800
4.1
122 300
6.5
FT-IR spectroscopy analysis of the oxidised lignosulphonates revealed only minor changes after laccase oxidation (Figure 17). The distinct peak at 647 cm-1 (associated with the stretching of S-O bond) n DP401 and DP795 remained after oxidation (Figure 17). DP401
DP795 1500 - 1300
1500 - 1300 1200 - 1000
1200 - 1000 1592 647
1589
647
A
1717
A
4000
3200
2400
1800
1400
1000
4000
600
3200
2400
1800
1400
1000
600
Wavenumber (cm-1)
Wavenumber (cm-1)
Figure 17: FT-IR spectra of lignosulphonates DP401 and DP795 prior to (solid) and after (dashed) 4 hours of oxidation by MtL.
IV.2.2 Ozonolysis of DP401 and DP795 after laccase oxidation The ozonolysis reaction was followed by SEC, FT-IR and 13P NMR analysis. As expected, a significant reduction in the average molecular weight continued throughout the reaction (Table 3). Table 3: Molecular mass distributions of lignosulphonates DP401 and DP795 after ozonolysis. DP401
DP795
Time (min)
Mw (Da)
Mw / Mn
Mw (Da)
Mw / Mn
0
37 800
4.1
122 300
6.5
20
30 300
2,7
66 900
3.8
40
27 700
2.4
51 400
3.2
60
24 100
2
-
-
80
21 700
1.8
-
-
32
FT‐IR analysis revealed emerging carbonyl stretching peaks (1717 cm‐1). The skeletal C‐C, aromatic C‐H and C=C vibrational peaks (1510‐1420 and 1590 cm‐1 respectively) were clearly distorted and reduced after the completion of the ozonolysis (Figure 18). This distortion and reduction of the aromatic vibrational peaks was clearly related to a simultaneous increase in the carbonyl stretching peaks as the mechanisms through benzyl rings are degraded generate carbonyls such as carboxylic‐ and muconic‐acid‐like structures. Consequently, an increase in the C=O stretching signal occurs at the expense of a reduction in the C=C and C‐C ring skeletal vibration signals. DP795
DP401
1717
1589
1419
1592 1510
1453
1420 1451
1717 1502
40 min 80 min 30 min
A
A
60 min 40 min
20 min
20 min
10 min
0 min
0 min
1800
1700
1600
1500
1400
1800
Wavenumber (cm-1)
1700
1600
Wavenumber (cm-1)
1500
1400
Figure 18: FT‐IR spectra of DP401 and DP795 during ozonolysis. The increase in the carbonyl stretching peaks at 1717 cm‐1 is clearly visible in both lignosulphonates.
Two‐dimensional HSQC further confirmed the existence of muconic‐ and carboxylic‐acid‐type structures. The cross peak of the methoxy C/H (Figure 19A) of the partially methylated muconic acids as well as the α‐ and β‐vinyl C/H (Figure 19B) were found in the HSQC spectra of DP401 and DP795 after laccase oxidation followed by ozonolysis. The shifts were in good agreement with previous data from the 2D‐NMR analysis of muconic‐ acid‐type structures in lignin 150. The absence of both the α‐ and β‐cross peaks after MtL oxidation and prior to ozonolysis suggests that their formation is a direct consequence of the ring‐opening reactions by ozonolysis. Quantification of the functional groups through 31P‐NMR showed a significant increase in carboxyls (Figure 6), as well as a strong decrease in phenols in both DP401 and DP795 during ozonolysis (Figure 20).
33
DP401 (A)
35.0
Me O L
40.0
O
L
DP795 (A)
SO3-
OH OH
O
Me
O
40.0
SO3-
O
45.0
Me O
O
45.0 50.0 50.0 55.0
55.0
L
S O3-
L
SO3-
60.0 Me
Me
O
O
60.0 Me
65.0
O
OH
4.50 ppm (f2)
4.00
3.50
65.0
OH
3.00
70.0 ppm (f1)
ppm (t1) ppm (t2)
4.00
3.00
3.50
100 Me
DP401 (B)
O L
α
O
O OH
O
SO3-
α
100.0
β
OH
L
DP795 (B)
95.0
S O3-
O
α
β
110
Me O
O
105.0
α 120
110.0 β
115.0
β
130
120.0
α 140
125.0 ppm (f1) ppm (f2)
7.00
6.50
ppm (t1)
6.00
7.00
ppm (t2)
6.50
6.00
Figure 19: 2D‐HSQC NMR spectra of (A) aromatic and (B) aliphatic regions of lignosulphonates DP401 and DP795.
The largest increase in carboxyls was observed in DP401, in which an increase from 0.13 to 0.46 carbonyl groups per aromatic ring was recorded. The formation of carbonyls is desirable, but the degradation of phenols has several implications, the most important being the possibility of polymerising the ozonated lignosulphonates with laccase. As previously investigated, laccase treatment of these lignosulphonates consumes up to two thirds of the available phenols to enable polymerisation reactions. Carboxylic groups
Phenolic groups 1.20
1.80
Amount (mmol/g)
Amount (mmol/g)
1.60 1.40 1.20 1.00 0.80 0.60 0.40
1.00 0.80 0.60 0.40 0.20
0.20 0.00
0.00 0
10
20
30
40
50
60
70
0
80
10
20
30
40
50
60
70
80
Ozonolysis time (min)
Ozonolysis time (min)
Figure 20: 31P‐NMR quantification of carboxylic and phenolic groups during ozonolysis of lignosulphonates DP401 ( ) and DP795 ( ).
During these experiments, approximately one‐third of the phenols were consumed during the laccase oxidation. Further reduction of phenols by ozonolysis effectively prevents oxidation and polymerisation of the lignosulphonates once they have been ozonated. To take full advantage of the enzymatic treatment, laccase oxidation should therefore preferably be performed as a first stage in a two‐stage treatment of lignosulphonates.
34
The purpose of a two‐stage treatment to modify lignosulphonates is to generate plasticisers structurally similar to those currently available. Among the more interesting are the polynapthalene sulphonate and polycarboxylate‐polysulphonate superplasticisers (Figure 21). OH OH
O
H
H3C n
S O
HO
O H3C
S
O CH3
n
m
OH n
MeO
1
OH
HO 3S
O
2
OH
3
Figure 21: Structures of (1) lignosulphonate plasticiser, (2) polynapthalene sulphonate superplasticiser and (3) polycarboxylate‐polysulphonate superplasticiser.
The structural lignosulphonate modifications demonstrated will probably yield lignosulphonate structures similar to the aforementioned superplasticisers. It is however uncertain how these modified lignosulphonates would behave in practical applications. To evaluate this question, thorough dispersing and plasticising studies need to be performed.
IV.3 Immobilisation of TvL (Paper V) To investigate the possibility of reusing laccase for the polymerisation of lignosulphonates, TvL was chosen for immobilisation. Reusing of the catalyst is undeniably an efficient method not only of reducing the cost of the catalyst but also of fully harnessing the inherent properties of a true catalyst i.e. the ability to perform multiple reaction cycles without being consumed. The cost of the catalyst is likely to represent a significant portion of the total cost and methods of reducing the cost are of great interest. The question of whether an immobilised laccase is able to polymerise a high‐Mw polymer as well as the non‐immobilised equivalent remains to be answered. Previous experiments with immobilised laccase have all used low‐Mw substrates in dilute solution which enables them to easily diffuse to and from the immobilised enzyme 151,152. A high‐Mw polymer such as lignosulphonate with its unique amphiphilic properties at high concentration is a different matter, and this has not previously been investigated. In the literature, the efficacy of immobilised laccases on a variety of supports and through a number of immobilisation techniques has been thoroughly demonstrated and evaluated, both in terms of immobilisation yield and catalytic properties 153. For the purpose of the present investigations, the covalent immobilisation technique based on the reaction between 3‐aminopropyltriethoxysilane (APTES) and glutaraldehyde (GLUTAL) was chosen. This reaction involves a surface derivatised with 3‐aminopropyltriethoxysilane and the N‐terminus of a protein residue in the presence of glutaraldehyde (Figure 22). The carbonyl group of glutaraldehyde activates the amine group of the N‐terminus of a protein residue. In the subsequent reaction, the amine groups on the APTES‐surface generate a Schiff base 154 and most notably a covalent linkage between the surface and the protein.
35
The APTES/GLUTAL immobilisation technique has proven to be a versatile immobilisation technique retaining most of the activity while significantly improving the thermal stability of the bound catalyst 153. O
HO
Protein residue
NH2
N
O NH2 O
Si
OH
O
N-terminus
O
Si
O
O
O
O
O
APTES-activated surface
Figure 22: Protein immobilisation on a 3‐aminopropyltriethoxysilane‐activated surface.
TvL was immobilised on two different supports, APTES‐activated controlled‐porocity carriers (CPC) and aluminium oxide (Al2O3) pellets. Different immobilisation yields were obtained depending on the porosity of the carriers. The high‐porosity CPC could retain significantly higher amounts of immobilised TvL (Table 4). Table 4: Yields of protein amount and activity of TvL immobilised onto controlled porosity carrier beads (CPC and CPC‐LS) and aluminium oxide pellets (Al2O3) relative the amounts available in the stock solution. CPC
CLC-LS
Al2O3
Activity (%)
86.2
43.8
18.7
Protein amount (%)
88.6
68.2
20.2
The activity of the immobilised TvL was determined in up to five consecutive cycles using the ABTS assay. The green ABTS solution was adsorbed onto the surface of the immobilised carriers, but could be completely removed after washing with buffer solution after each oxidation cycle.
IV.3.1 Oxidation of DP851 by immobilised TvL The TvL immobilised on CPC was loaded onto a tubular column reactor. Lignosulphonate DP851 dissolved in acetate buffer (pH 5) to a concentration of 100 g/l was oxidised in five consecutive cycles where 50‐ml batches were recirculated through the column under constant oxygen saturation.
36
O2
DP851
Figure 23: Immobilised TvL loaded on a column through which a 100 g/l solution of DP851 was recirculated under constant oxygen saturation.
After 4h of reaction time, the lignosulphonate solution was removed and the column was washed with buffer solution until no discolouration of the residual solution was visible and the oxidation was repeated. While the adsorption of the ABTS assay was shown to be reversible, this was not the case when DP851 was oxidised. Already after the first cycle, extensive discolouration was observed as a consequence of lignosulphonate adsorption onto the CPC. The adsorption appeared to be irreversible, as it remained after the following washing step and no leaching of the lignosulphonates into the residual water occurred. The discolouration remained throughout the next two cycles until the beads were completely saturated with lignosulphonates. SEC analysis of the oxidised lignosulphonate DP851 after each cycle showed a reduction in the efficacy of the immobilised TvL to polymerise. After the first two cycles, the average molecular weight of the oxidised DP851 significantly dropped and became stable during the remainder of cycles at a level close to the initial average molecular weight of the DP851 (Figure 24, legend ). 350 000 1.20
300 000
1.00 AU
_ MW
250 000 200 000
0.80 0.60
2 th cycle
150 000 0.40 100 000
1th cycle
0.20 50 000
3nd, 4rd , 5th cycle
0.00 0
_ Initial M W
14.00 1
2
3
4
5
16.00
18.00
20.00
22.00
24.00
26.00
Minutes
O x id a tio n c y c le
Figure 24: Average molecular weight (left) and SEC eluograms (right) of DP851 oxidation by TvL immobilised on CPC ( ).
There are a number of possible explanations of this behaviour such as lignosulphonate adsorption, leaching of the immobilised TvL into lignosulphonate solution, and TvL deactivation.
37
IV.3.2 Lignosulphonate adsorption Irreversible adsorption of lignosulphonates onto the highly porous CPC surface may be providing an obstacle for the lignosulphonate substrate to diffuse to the immobilised TvL. To evaluate the impact of this adsorption, two different strategies were employed; immobilisation onto a less porous carrier to facilitate easier removal of adsorbed lignosulphonates and immobilisation onto pre‐saturated CPC surface. Aluminium oxide (Al2O3) pellets and CPC‐LS (pre‐saturated with DP851 for 24 hours at room temperature followed by extensive washing) were chosen for the first and second approach respectively. As expected, the amount of immobilised TvL was considerably reduced (Table 4). The adsorption was investigated by immersing the immobilised Al2O3, CPC and CPC‐LS in a lignosulphonate solution at room temperature for 24 hours. Nearly all the discolouration could be removed from the Al2O3 carriers after buffer washing, whereas almost no adsorption occurred on the pre‐saturated CPC‐LS. Activity was investigated with the ABTS assay for five consecutive
35
350 000
30
300 000
25
250 000
20
200 000
_ MW
A c tiv ity (U )
cycles (Figure 25, left insert).
15
150 000
10
100 000
5
50 000
0
0 1
2
3
4
5
A B T S o x id a tio n c y c le
_ Initial M W
1
2
3
4
5
O x id a tio n c y c le
Figure 25: ABTS activity (left) and average molecular weight of DP851 (right) after five oxidation cycles by TvL immobilised onto CPC ( ), CPC‐LS ( ) and Al2O3 ( ) carriers. Error bars represent range of deviation of three individual measurements. The ABTS activity measurements were repeated for CPC and Al2O3 pellets ( and respectively) after immersion in lignosulphonates for 24 at room temperature. Lignosulphonate oxidation was also performed with CPC at 30° ( ).
Much lower activities were recorded with CPC before and after immersion in lignosulphonates (symbols and , Figure 25). Contrary, to CPC, the activity of Al2O3 carriers was essentially equal before and after lignosulphonate saturation (symbols and , Figure 25). These two experiments demonstrate that the adsorption of lignosulphonates does indeed present an obstacle for the immobilised laccase to access its substrate. By choosing an immobilisation surface with low lignosulphonate‐adsorption properties, this problem can be addressed. This occurs however at the expense of lower enzyme immobilisation yield due to the significantly reduced capacity of a low‐adsorption surface such as Al2O3 pellets used here. The low TvL immobilisation yield was also evident when the carriers were employed to oxidise DP851 (Figure 25, left, symbols , and for CPC, CPC‐LS and Al2O3 respectively). While the Al2O3 pellets showed little or no ability to increase the average molecular weight of DP851, the CPC‐LS was able to polymerise DP851 in 5 consecutive cycles, albeit at a lower rate in each successive cycle. Other mechanisms than lignosulphonate adsorption are evidently in play.
38
Leaching of the immobilised TvL into the lignosulphonate solution, although possible, can be ruled out as it was demonstrated by the ABTS assay experiments that the enzyme remained bound to the immobilisation surface after a several oxidation cycles despite extensive buffer washing between the cycles (Figure 25).
IV.3.3 Deactivation of the immobilised TvL The rapid reduction in enzyme activity on DP851 after the first two cycles cannot be explained solely by adsorption onto the carrier surface. It is hypothesised that lignosulphonates at high reaction temperatures deactivate the immobilised laccase by irreversible adsorption or denaturation. To investigate this, the oxidation of DP851 by CPC was carried out at 30°C instead of 50°C (Figure 25, left, symbol ). There was a similar tendency for the oxidation reaction to slow down after the first two cycles, but the rate at which this occurred was significantly slower, suggesting that the enzymatic activity is greater at reduced oxidation temperatures. The hypothesis that lignosulphonates deactivate the immobilised TvL at high temperatures appears therefore to be supported. The deactivation can be retarded somewhat by lowering the temperature. A number of articles in the scientific literature report no changes or in some cases greater catalytic properties and thermal stability of the immobilised laccase 155,156. To investigate the catalytic properties of the immobilised TvL, DP851 was oxidised by equal amounts of immobilised and free laccase. The SEC eluogram obtained clearly showed that immobilised TvL did indeed retain the catalytic activity of the non‐immobilised TvL to polymerise DP851 (Figure 26). 2.80 2.60 2.40 2.20
Starting material
600U TvL
2.00
AU
1.80 1.60 1.40
600U TvL on CPC
1.20 1.00 0.80 0.60 0.40 0.20 0.00 13.00
14.00
15.00
16.00
17.00
18.00
19.00
20.00
21.00
22.00
23.00
24.00
25.00
26.00
Figure 26: SEC Eluogram of DP851 oxidation by free (600 U TvL) and immobilised TvL (600U TvL on CPC). Minutes
While the lignosulphonate adsorption can be addressed by choosing a different immobilisation support, the problem of the deactivation of the laccase appears to be more difficult to solve since the deactivation mechanisms are still not understood. A more structurally stable laccase could possibly withstand this deactivation. Before implementing this technique on any large scale, attention should be directed to academia and the on‐going pursuit of increased laccase stability through e.g. directed evolution and high‐throughput screening 157.
39
The observed deactivation of the immobilised TvL is an interesting observation since it confirms the observations in earlier experiments. When the lignosulphonates DP398‐DP401 were oxidised by free TvL (described in Paper III), the reaction was more or less halted after the initial 4 hours (Figure 12). In the light of the later findings, it is clear that deactivation of the TvL occurred in the earlier experiments and this was the probable explanation of why only a minor increase in the average molecular weight of lignosulphonates is observed after the first initial 4 hours of oxidation with laccase.
IV.4 Lignosulphonate structural modification by the Fenton’s reagent (Paper VI) Although laccase has been demonstrated to be a highly efficient tool to most notably increase the molecular weight of lignosulphonates, the cost of the enzyme itself cannot be ignored. The successful effects of a conventional ozone treatment on lignosulphonates have been reported in Paper IV. However, a disadvantage of employing ozone is the degradation of the lignosulphonates. To achieve polymerisation, laccase oxidation must be applied, a treatment that require mild and controlled conditions to fully take advantage of the enzyme. Although this is generally preferred, the use of traditional bleaching chemicals might be advantageous in certain situations. Accordingly, alternative methods of increasing the molecular weight of lignosulphonates were investigated using the Fenton’s reagent, a mixture of hydrogen peroxide and a ferrous salt capable of generating highly potent radical species (Scheme 9) 158.
H2O2 + Fe(II)
OH• + OH- + Fe(III)
(1)
H2O2 + Fe(III)
O2•- + 2H+ + Fe(II)
(2)
O2•- + Fe(III)
O2 + Fe(II)
(3)
H 2O 2
OOH- + H+ (pKa 11.6)
(4)
OH•
O•- + H+ (pKa 11.9)
(5)
H 2O 2 + H +
H3O 2+
H2O + OH+
(6*)
Scheme 9: Different pathways leading to the formation of reactive species (bold) in Fenton’s reaction. * The existence of the hydroxyl cation (OH+, eq. 6) is not uncontroversial and the question of its existence remains open.
( )
These species react with a variety of organic compounds including several wood components during lignin biodegradation by wood‐degrading fungi 159‐161. In the pulp and paper industry, Fenton’s reagent is closely associated with conventional hydrogen peroxide bleaching 162. This section describes the action of Fenton’s reagent on lignosulphonates at acidic and alkaline pH and how this powerful radical‐generating system can be used for structural modifications.
40
IV.4.1 Fenton’s reagent at acidic pH Lignosulphonates were oxidised by Fenton’s reagent (a mixture of hydrogen peroxide and iron(II) acetate) at pH 5 for 1h. It has previously been reported that a low pH is optimal for Fenton’s reaction to reach completion 158,163. During the oxidations of lignosulphonates, the pH decreased somewhat, probably through the formation of acidic groups. SEC analysis of the oxidised lignosulphonates revealed a drastic increase in the average molecular weight, with the exception of DP400 where degradation was observed (Table 5). Table 5: Average molecular weight, polydispersity and functional group composition of lignosulphonate DP398-DP401 after 1h of oxidation by the Fenton’s reagent at acidic conditions. Mw (Da)
Mw / Mn
Carboxyl -OH (mmol/g)
Phenol -OH (mmol/g)
DP398 Reference
38 900
2.0
0.11
1.08
Fenton’s Reaction 1h
72 300
2.3
0.29
0.93
without Fe(II)
32 900
1.9
0.18
1.18
DP399 Reference
46 800
1.8
0.09
1.01
Fenton’s Reaction 1h
90 000
2.4
0.20
0.96
without Fe(II)
40 900
1.7
0.21
1.40
DP400 Reference
9 700
1.8
0.18
2.49
Fenton’s Reaction 1h
8 800
1.8
0.33
2.06
without Fe(II)
7 400
1.3
0.30
2.90
0.26
1.50
DP401 Reference
12 300
1.3
Fenton’s Reaction 1h
21 400
1.7
0.34
0.74
without Fe(II)
9 100
1.2
0.31
1.72
These findings are unexpected in the light of the traditional understanding of Fenton’s reagent and the degradative lignin reactions due to the aggressive hydroxyl radicals (OH•), superoxide anion radicals (O2•-) and secondary radical and ionic species such as hydroperoxide anions (HOO-) and oxyl anion radicals (O•-) formed by the reagent (Scheme 9, equations 1 and 5). It is proposed that the hydroxyl (OH•) and the superoxide anion radical (O2•-) act through different pathways when in contact with lignosulphonate structures, that the hydroxyl radical initiates radicalisation of a phenolic lignosulphonate end-group with subsequent coupling reactions, and that the superoxide anion radical, through addition to the benzyl ring, achieves ring-opening reactions transforming phenolic hydroxyls to their carbonylic counterparts (Scheme 10, right pathway).
41
If the reaction occurs on a cross‐linked lignosulphonate unit, it is suggested that depolymerisation and ring‐opening reactions (Scheme 10, left pathway) occur simultaneously.
SO3
L
-
SO3
L
OH
OH
-
L
HO
OH
SO 3
-
L
1
OMe
L
OH
2
O
Depolymerization
-
HC
OMe 5
L
3
4
SO3
L
R=H
OMe HO
OH
OH
SO3
-
SO3
L
-
H2O
HO
6
R=L OH
3
2
1 OH
O
OMe
C
OMe
OMe
O
O
O2
OR O2
2+2
1+2 OH OH
SO3
L
-
L
OH
OH SO3
-
SO3 CH3
-
-
HO L
L
MeO
O
O
OH L
O
O
SO3
-
MeO
O
OH -
OH
OH
O3 S
HO
HO
Ring-opening
OMe
Ring-opening
SO3 OMe
L
OMe
Cross-linking through 5-5' and 4-O-5' bonds
•‐
Scheme 10: Proposed reaction of the hydroxyl (OH•) and the superoxide anion radical (O2 ) with a lignosulphonate structure. Two pathways are proposed; reactions with a phenolic end‐group (R = H, right reaction arrow) and a cross‐linked lignosulphonate unit (R = L, left reaction arrow). The various resonance forms of the oxidised lignosulphonate structures are labelled 1 through 3.
If iron(II) acetate is omitted, radical formation is reduced and it is expected that no structural changes occur. A significant increase in phenolic hydroxyls was nevertheless recorded as well as a slight decrease in the average molecular weight. These reactions are probably a result of the activity of cationic oxidation species (H2O3+) on lignin structures (Scheme 11). They are likely to be formed by the protonation of hydrogen peroxide under acidic conditions (Scheme 9, equation 6) 164.
O
L
OH
OH
H2O OMe
H 3O 2
6
H3 O 2
2
R=L
Depolymerization
+
L
OH
L
OMe O
H 2O O
OMe
OMe
R=H 3
4
OH OH H2O
5
L
OH
L
1
+
OH
OH
L H
O
OH
OH
O
OH
OH
O
OMe
OH
Ring-opening
O
L +
Scheme 11: Proposed reaction of the cationic oxidation species (H2O3 ) and lignin end group at acidic conditions in absence of iron(II) acetate. Two pathways are proposed; reactions with a phenolic end‐group (R = H, right reaction arrow) and a cross‐linked lignosulphonate unit (R = L, left reaction arrow).
Despite the absence of iron(II) acetate, radical species are probably still formed due to trace amounts of transitions metal ions in amounts sufficient to catalyse Fenton‐type chemistry and the subsequent reactions by those radicals. Omission of hydrogen peroxide did not yield any notable differences (data not shown), emphasising the importance of this species for Fenton’s reagent.
42
IV.4.2 Which cation does what? The chemistry through which the cationic species H2O3+ reacts with lignosulphonate structures can be related to the chemistry involved in peracetic acid bleaching. Here, ring-opening products as well as quinone structures are generated through the action of the hydroxyl cation OH+ which is generated by the acid-catalysed decomposition of peracetic acid (Scheme 12) 165. O 3C
HO
OH
+
H
O
+ 3C
HOH
+
OH
+
Scheme 12: Decomposition of peracetic acid to acetic acid and hydroxyl cation.
It has been suggested that OH+ is the active species during peracetic acid bleaching 165. This species is formed during protonation of hydrogen peroxide (Scheme 9, equation 6), but it is assumed for the sake of simplicity that the intermediate protonated hydrogen peroxide H2O3+ is the reactive species during these reactions. This species is expected to react with lignosulphonate structures before it decomposes to OH+ and H2O. It should also be noted that the reactions of both the OH+ and H2O3+ cations are expected to lead so the same end products, so the involvement of the hydroxyl cation cannot be ruled out. This is however still an open question as the existence of both the H2O3+and OH+ is yet to be established.
IV.4.3 Fenton’s reagent at alkaline pH For comparison with reactions under acidic conditions, the oxidation of DP398 by Fenton’s reagent was repeated at an alkaline pH. Table 6: Average molecular weight, polydispersity and functional group composition of lignosulphonate DP398 after 1h oxidation by Fenton’s reagent under alkaline conditions. Mw (Da)
Mw / Mn
Carboxyl -OH (mmol/g)
Phenol -OH (mmol/g)
Reference
38 900
2.0
0.11
1.08
Fenton’s Reaction 1h
43 000
1.9
0.21
0.78
without Fe(II)
41 800
2.0
0.26
2.39
The pH of the reaction was set close to the pKa of hydrogen peroxide to promote the formation of perhydroxyl anion species HOO- (Scheme 9, equation 4). This highly reactive nucleophile is traditionally considered to be the main agent during the alkaline hydrogen peroxide bleaching of residual lignin. The mechanisms of these reactions have been thoroughly investigated and reviewed over the years 162,166-168. As under acidic conditions, polymerisation was observed, albeit at a much slower rates. The moderate increase in the average molecular weight at alkaline conditions is probably a consequence of competing degradation reactions by the perhydroxyl anions and polymerisation
43
reactions by the previously discussed radicals (OH• and O2•‐). It is also probable that the lignosulphonate degradation achieved by the perhydroxyl anions would be greater at higher reaction temperatures, as during alkaline hydrogen peroxide pulp bleaching.
IV.4.4 The effect of lignosulphonate concentration Oxidation of DP398 by Fenton’s reagent was conducted at both acidic and neutral pH. The concentration of DP398 was varied from 6.25 to 100 g/l (Figure 27). Interestingly, at low lignosulphonate concentrations (below 25 g/l) extensive depolymerisation occured. This depolymerisation is likely due to ring‐opening reactions where phenolic hydroxyls are transformed to carboxylic ones. An increase in the lignosulphonate concentration promotes polymerisation reactions through radical‐radical coupling. Radicalisation of phenolic end groups probably occurs at both low and high lignosulphonate concentrations, but since coupling requires at least two radicals in close proximity, dilution of the lignosulphonates significantly reduces the probability that this will occur. The observation of the effect of lignosulphonate concentration is interesting in the light of the earlier laccase oxidation experiments. Both sets of experiments showed the importance of the lignosulphonate concentration, which is an integral factor during radical‐radical polymerisation. At low concentrations, decomposition of the radicals dominates with few radical‐radical reactions due to the infrequent of interactions between two individual species. The opposite is valid at high concentrations where decomposition of radicals is probably suppressed by the frequently occurring coupling reactions. pH 5
1 80 000
2.4
80 000
2.4
70 000
2.2
70 000
2.2
60 000
60 000
1.8
40 000 1.6
30 000
1.4
20 000
0
1.0
0
25
50
1.4
20 000 10 000
12.5
1.6
30 000
1.2 6.25
1.8
40 000
10 000 Reference
2.0
50 000
100
1.2 1.6 Reference
6.25
[DP398] (g/l)
0.3
0.4
0.2 0.1 0.0
Carboxyl -OH (mmol/g)
Carboxyl -OH (mmol/g)
0.4
50
1.2
0.7 0.6 0.8
0.5 0.4 0.3
0.4
0.2 0.1 0.0
0.0 25
100
100
0.0 Reference
[DP398] (g/l)
Phenol -OH (mmol/g)
0.8
0.5
Phenol -OH (mmol/g)
0.6
12.5
50
0.8
1.2
0.7
6.25
25
pH 11
2a
0.8
Reference
12.5
[DP398] (g/l)
pH 5
1a
_ _ Mw / Mn
50 000
_ Mw
2.0
_ _ Mw / Mn
_ Mw
pH 11
2
6.25
12.5
25
50
100
[DP398] (g/l)
Figure 27: Lignosulphonate concentration versus molecular mass distribution (insert 1, , left y‐axis), polydisperisy (insert 2, , right y‐axis), carboxyl ‐OH (insert 1a, , left y‐axis) and phenol ‐OH (insert 2a, , right y‐axis) of DP398 after Fenton’s reaction of DP398. The refence values are displayed as dashed horizontal lines.
44
The pH during the oxidation reaction was shown to play an important role. Both polymerisation and ring‐opening reactions were promoted under acidic pH conditions.
IV.4.5 The effect of hydrogen peroxide concentration Fenton’s reaction on DP398 was conducted under both acidic and alkaline pH using different amounts of hydrogen peroxide. The lignosulphonate concentration was set to 100 g/l and the hydrogen peroxide concentration was varied from 2 to 32 mM. Under both acidic and alkaline conditions, polymerisation reactions with the concurrent consumption of phenolic hydroxyls predominated up to 8 mM of added H2O2. Above 8 mM H2O2, extensive depolymerisation occurred, probably through ring‐opening reactions as the carboxylic group levels increased. Reactions at alkaline pH were as before much less pronounced (Figure 28). One explanation of these observations is that hydrogen peroxide dissociates to H3O2+ and HOO‐ at acidic and alkaline pH respectively. These ionic species become the predominant reactant with increasing addition of hydrogen peroxide. The formation of radicals proceeds through the oxidation of Fe(II) to Fe(III) and to sustain this reaction, the Fe(III) must be reduced back to Fe(II) (Scheme 9, equation 3). The dissociation of H2O2 to H3O2+ and HOO‐ does however proceed faster than these Fe(II) oxidation/reduction reactions. Consequently, products from the reactions with these ionic species should dominate. pH 5
1 120 000
120 000
3.0
100 000
3.0
100 000
2.6
2.6
80 000
20 000 0 0
2
4
8
16
2.2 60 000 1.8 40 000
1 .4
20 000
1 .0
0
1.4 1.0
32
0
2
4
[H2O2] (mM)
0.8
0.2 0.4
0.1 0.0
Carboxyl -OH (mmol/g)
Carboxyl -OH (mmol/g)
1.2
0.3
16
1.6
0.5 0.4
1.2
0.3
0.8
0.2 0.4
0.1 0.0
0.0 8
2.0
0.6
32
0.0 0
[H2O2] (mM)
Phenol -OH (mmol/g)
0.4
Phenol -OH (mmol/g)
1.6
0.5
4
32
0.7
2.0
0.6
2
16
pH 11
2a
0.7
0
8
[H2O2] (mM)
pH 5
1a
_ _ Mw / Mn
1.8 40 000
_ _ Mw / Mn
2.2 60 000
_ Mw
80 000
_ Mw
pH 11
2
2
4
8
16
32
[H2O2] (mM)
Figure 28: Hydrogen peroxide concentration versus molecular mass distribution (1, , left y‐axis), polydispersity (2, , right y‐axis), carboxyl ‐OH (1a, , left y‐axis) and phenol –OH (2a, , right y‐axis) of DP398 after Fenton’s reaction on DP398. The refence values are displayed as dashed horizontal lines.
Fenton’s reagent has proven to be a versatile tool for the modification of lignosulphonates under acidic and alkaline conditions. Lignosulphonate concentration, reaction pH and, hydrogen peroxide dosage can be used to control the reaction towards a desired results. While some of the
45
reactions performed by the reagent were previously known, there are aspects of Fenton’s reagent and its reactions with lignosulphonates discovered here have not previously been investigated. The chemicals required for Fenton’s reagent, mainly hydrogen peroxide, are ubiquitous in the pulp and paper industry and there no significant environmental issues are expected, either in processing or in effluent treatment. The versatility of this system for modification of lignosulphonates is extraordinary. By adjusting a series of parameters (lignosulphonate concentration, pH, peroxide dosage etc.) different results can be obtained. The studies described here are far from complete suggesting that there is plenty of room for optimisation. Therefore, the possibilities of scaling up this method appear promising.
46
V
Conclusions and Further Perspectives
This thesis describes various methods of modifying commercial lignosulphonates in order to enhance their performance as technical dispersants or to expand their utilisation to new areas. The challenge of increasing the molecular weight of lignosulphonates as well as introducing charged groups to the polymer has been largely solved using both enzymatic and chemical tools. Much effort has been put into achieving the mechanistic understanding of the oxidoreductive enzyme laccase reactions with both lignin and lignosulphonates. Several key features were identified as essential to achieve polymerisation of lignosulphonates under reaction conditions suitable for large‐scale applications. Successful attempts have also been made to achieve multiple usage of the laccase through immobilisation. In order to fully investigate the impacts of these modifications on the performance of lignosulphonates, further studies of the dispersing properties are critical. There is support for the improved dispersing power of lignosulphonates oxidised by laccase 169 as well as the improved dispersion of ozonated Kraft lignins 170 in the scientific literature showing that modification of lignosulphonates has a significant impact on their final application. This needs however to be fully investigated and thoroughly evaluated Should the modifications described here be successful, it is not difficult to envision an implementation of these methods in an existing sulphite plant. The installation of a reactor with immobilised laccase and an ozone generator prior to a pH adjustment (to optimal pH for the laccase) as well as an additional ultrafiltration stage (to fractionate the oxidised polymers) should suffice (Scheme 13). Ca(OH)2
Spent cooking liquor
pH adjustment
Evaporation
Precipitation (Howard process)
Dilution
pH adjustment
Ultrafiltration
Ozonolysis
Spray drying
Evaporation
Ultrafiltration
Immobilised laccase reactor
Scheme 13: A suggested production flow chart of a new generation of lignosulphonates where modification through laccase oxidation and ozonolysis are implemented.
The global concrete production is expected to approach volumes of 4 billion tonnes annually, largely driven by the infrastructure investments in the developing world171. The need for high‐ performance concrete is here expected to be lower than in the advanced economies, and the need for lignosulphonates as additives to medium‐to‐low strength concrete should be ample. Therefore, any modifications that would improve the performance even marginally are potentially interesting. The position that lignosulphonates holds should not however be taken for
47
granted. This is clearly illustrated in the on‐going displacement of lignosulphonates from the high‐performance concrete additive market in favour of a new generation of synthetic equivalents. This speed of such development is troublesome, and it indicates the importance of the ability to tailor an additive to a specific need. So far, tailoring of lignosulphonates has been limited while the synthetic equivalents can be tailored almost without limit. Most lignosulphonate utilisation is based on the concrete additive market and a complete elimination of lignosulphonates from this market would be a major setback and would leave the producers with enormous quantities of lignosulphonates with no apparent usage. In the current concept of a biorefinery, the role that lignin plays is far from being defined. Two main alternatives have however been identified; fuel or chemicals. To use lignin as fuel in a paper mill is tempting, but as long as it is economically viable to upgrade lignin, it makes little sense. With constantly rising crude oil prices, increasing energy demands and the lack of a stable and reliable means to meet this demand, coupled with a reduction in the utilisation of lignin for chemicals, the fate of lignin lies undoubtedly in the fuel market. The fact that this source of fuel is abundant, essentially renewable and is more or less carbon dioxide neutral makes utilisation of this fuel even more tempting. To oppose this trend, it is therefore imperative that research on lignosulphonates continues, not only to enhance its performance but also to expand its utilisation to new areas. Although, the challenge is daunting, given the inherent complexity of such a biopolymer, lignosulphonates as well as lignins in general are far too interesting biopolymer to be doomed to a fate as a low quality fuel. With regard to the specific use of lignosulphonates as additives to cement, this thesis has clearly demonstrated that significant modifications can be achieved using both enzymes and conventional bleaching chemicals. These tools have proven to be versatile enough to obtain lignosulphonates with specific properties and this thesis should provide a good foundation for further investigations into the specific mechanisms of lignosulphonate/cement interactions. It is the hope of the author that this thesis will serve as a contribution to the current knowledge of lignosulphonates as well as raise the awareness and understanding of the possibilities to modify lignosulphonates through relative simple methods.
48
VI Acknowledgements First and foremost I would like to extend my gratitude to Gunnar Henriksson for supervising me over the years and for being an unlimited resource of new ideas and suggestions. Göran Gellersted and Jiebing Li are also thanked for good co‐operation within the Biorenew research project. Partners within the Biorenew project, most notably the LignoTech division of Borregaard, are acknowledged for their contribution to this work. Financial support from the Biorenew program (EU grant FP6‐NMP2‐CT 2006‐26456) which made this work possible is acknowledged. All co‐authors are thanked for their efforts. The administrative personnel at the Fibre & Polymer department, most notably Inga Persson and Mona Johansson are thanked. I truly believe that without you there would be no department to speak of. Former and present colleagues at the department are thanked for their contribution to the general atmosphere and for interesting and fruitful discussions about everything between heaven and earth. And finally, family and past and present friends are thanked for providing the necessary distraction from the scientific work. You are too numerous to be mentioned here by name but your contribution to my efforts to remain somewhat sane cannot be overstated.
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VIII
Errata‐list
Paper I: Page 22, Section ”Enzymes”, lines 5 and 6: “…0.4 for NS51002 and 0.7 for NS51003…” should read “…0.7 for NS51002 and 0.5 for NS51003…”. Page 25, Table 1: Compound III should not contain a methyl group at the Cα carbon. Page 25, left column, line 1: …low pH will lead to…” should read “…high pH will lead to…”. Page 28, left column, lines 16‐17: “…high‐oxidation potential NS51003...” should read “…low‐redox potential NS51003...”. Page 28, left column, lines 18‐19: “…low‐oxidation potential NS51002...” should read “…high‐redox potential NS51002...”.
Paper III: Page 907, right column, line 2: “… oxidize non‐phenolic end groups…” should read “…oxidize phenolic end groups with high oxidation potential…” Page 907, right column, line 4: “…only phenolic end groups…” should read “…only low‐oxidation potential phenolic end groups…” Page 908, right column, line 1: “…phenolic and nonphenolic end groups…” should read “…high‐ and low‐oxidation potential phenolic end groups…”
Paper IV: Figure 10, compound 3: H
H
H
H
H CH3
H3C H m H n COOH COO-SO 3H
3
H
H
CH3 H m H n COOH SO 3H
should be
3
55
H
H3C
