UNIVERSITY OF COPENH AGEN FACULTY OF SCIENCE

Master Thesis Valentin Waschulin

The Haloperoxidase-DABCO System

Exploring a Novel Method of Enzymatic Pulp Bleaching Name of department: Department of Geosciences and Natural Resource Management, Forest, Nature and Biomass

Author: Valentin Waschulin

Title / Subtitle: The Haloperoxidase-DABCO System: Exploring a Novel Method of Enzymatic Pulp Bleaching

Academic advisor: Prof. Claus Felby

Industrial advisors: Pedro Loureiro

Submitted: 18. September 2016

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Acknowledgements

I want to express my thanks to Prof. Claus Felby for the opportunity of performing my Master Thesis at his chair, and Henrik Lund for providing me with the possibility of performing the thesis work at the Technical Industries department at Novozymes A/S, Denmark. I want to thank Pedro Loureiro for providing supervision, detailed expertise and great scientific discussions throughout the time of the thesis. I also want to thank all the scientists and associates at the department for discussion, advice and hands-on help in the laboratory; in particular Owik Herold-Majumdar for guidance and discussion concerning the model compound work. Finally, I want to thank my parents Susanne and Georg and my sister Laura for supporting me throughout my studies in Denmark.

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Abstract A novel approach for cellulosic pulp bleaching and hexenuronic acid (HexA) removal using a haloperoxidase (Hap) and a tertiary amine catalyst (DABCO) was tested on oxygen-delignified eucalypt kraft pulp. In a buffered system (pH 4,5, 60°C), high brightness gain, good response to peroxide-reinforced alkaline extraction and more than 80% HexA removal were achieved, while preserving cellulose integrity. In an optimized, but non-buffered system, Hap/DABCO was compared to the non-enzymatic HOCl treatment and an inferior performance of the former was found. At the same brightness level, HOCl treatment performed superior in terms of HexA and kappa number reduction. Hydrogen peroxide was proposed as most likely playing a role in contributing to the brightening during Hap/DABCO treatment. Organochlorine formation was measured and found to be much higher than reference chlorine dioxide treatments. It was concluded that the problem of organochlorine formation must be solved for this technology to be used in the future.

Danish Abstract I denne afhandling undersøges en ny enzymatisk proces til blegning af cellulosepulp og fjernelse af hexenuronic acid (HexA). I processen indgår en haloperoxidase (Hap) og en tertiær amin (DABCO) der testes på oxygen-delignificeret eukalyptus kraft pulp. Ved at køre processen ved konstant pH (pH 4,5; 60°C) sås en øget lyshed samt en god respons på peroxid-alkalisk ekstraktion hvor mere end 80% HexA fjernelse opnået, uden at cellulose strukturen blev nedbrudt. I et optimeret, men ikke-bufret system, blev Hap/DABCO-systemet sammenlignet med en ikke- enzymatisk HOCl behandling, og en mindre effekt af Hap/DABCO-systemet blev fundet. På samme niveau af lyshed var HOCl behandlingen overlegen i forhold til reduktion af HexA og kappanummer. Hydrogenperoxid og ikke kun den enzymatiske oxidation kan være medvirkende til blegningen under Hap/DABCO behandling. Organochlor-dannelse blev målt og vurderet at være meget højere end reference-behandlinger med chloridoxid. Det blev konkluderet, at problemet med dannelse af organochlor skal løses før en fremtidig anvendelse af Hap/DABCO teknologien.

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List of abbreviations List of abbreviations mM Millimolar mmol Millimole ISO International Organization for Standardization DABCO 1,4-diazabicyclo[2.2.2]octane ECF Elemental chlorine free TCF Totally chlorine free O Oxygen stage D Chlorine dioxide stage E Alkaline extraction

Ep Alkaline extraction reinforced with hydrogen peroxide COD Chemical oxygen demand mg Milligram MQ Milli-Q water dH2O Deionized water LC Liquid chromatography GC Gas chromatography MS Mass spectrometry odp Oven dry pulp % odp Weight percentage of the amount of oven dry pulp odt Oven dry ton (of pulp) OX Organic halogens AOX Adsorbable organic halogens cP Centipoise T Temperature DM Dry matter DM% Percentage of dry matter HexA Hexenuronic acid °C Degree Celsius T Temperature Hap Haloperoxidase PET Polyethylene terephthalate BSTFA N,O-Bis(trimethylsilyl)trifluoroacetamide TMCS Trimethylchlorosilane DHBQ Dihydroxybenzoquinone; IUPAC: 2,5-dihydroxycyclohexa-2,5-diene-1,4-dione SAL Sinapaldehyde IUPAC: 3-(4-Hydroxy-3,5-dimethoxyphenyl)prop-2-enaI

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List of abbreviations

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Table of Contents Table of Contents Abstract ...... 4 Danish Abstract ...... 4 List of abbreviations ...... 5 Table of Contents ...... 7 1 Aim and Scope ...... 9 2 Introduction and background ...... 11 2.1 The pulp and paper industry ...... 11 2.2 The Raw Material: Cellulose and Lignin ...... 11 2.3 Kraft Pulping ...... 13 2.4 Bleaching: Development and current technology ...... 14 2.4.1 D-stage chemistry ...... 14

2.4.2 Ep chemistry ...... 15 2.5 Hexenuronic acids: A side product of kraft pulping...... 16 2.6 Catalytic bleaching ...... 17 2.6.1 Tertiary amine catalysis of HOCl reactions ...... 17 2.6.2 Catalyic bleaching and removal of hexenuronic acid ...... 19 2.7 Pulp mill effluents and the environment ...... 21 2.7.1 Organochlorine compounds ...... 22 2.8 Selected paper and pulp properties ...... 23 2.8.1 Brightness ...... 23 2.8.2 Strength ...... 24 2.8.3 Viscosity ...... 24 2.8.4 Yield ...... 24 2.9 Enzyme applications in pulp and paper ...... 24 2.9.1 Hydrolytic enzymes ...... 25 2.9.2 Laccases and peroxidases ...... 25 2.10 Haloperoxidases ...... 26 2.10.1 Vanadium haloperoxidases ...... 27 2.10.2 Curvularia haloperoxidases ...... 28 2.10.3 Biotechnological applications of VHPOs ...... 28 2.11 The haloperoxidase – DABCO system ...... 29 3 Materials and Methods ...... 30 3.1 Materials ...... 30 3.1.1 Enzyme ...... 30 3.1.2 Equipment ...... 30 3.2 Methods ...... 31 3.2.1 Enzyme treatment medium consistency ...... 31 3.2.2 D-stage in medium consistency ...... 32

3.2.3 Ep stage in medium consistency ...... 33 3.2.4 A-stage in medium consistency ...... 33 3.2.5 Filtering the pulp ...... 33

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Table of Contents

3.2.6 Handsheet making ...... 34 3.2.7 Handsheet brightness measuring ...... 34 3.2.8 Measurement of dry matter content ...... 34 3.2.9 Preparation of chlorine dioxide ...... 34 3.2.10 Titration of chlorine dioxide, sodium hypochlorite and hydrogen peroxide ...... 34 3.2.11 Kappa number ...... 35 3.2.12 Miniscale assay ...... 35 3.2.13 Mini handsheet making ...... 35 3.2.14 Brightness of mini sheets ...... 36 3.2.15 Hexenuronic acid content of pulp ...... 37 3.2.16 AOX ...... 37 3.2.17 OX ...... 38 3.2.18 Viscosity measurement ...... 38 3.2.19 Model compound studies ...... 38 4 Results and Discussion ...... 39 4.1 Model compound experiments ...... 39 4.2 Pulp: Experimental Rationale and Initial Experiments ...... 40 4.3 Miniscale experiments ...... 41 4.3.1 Optimum reaction pH with DABCO ...... 42 4.3.2 NaCl dosage optimum ...... 44 4.3.2.1 Other dosage experiments [confidential] ...... 46 4.3.3 pH shift in non-buffered system ...... 47 4.3.4 Replication and modification of catalytic bleaching experiments ...... 49 4.4 Medium consistency experiments ...... 51 4.4.1 Experiment A: First high dosage trial and kinetics of brightness gain and HexA degradation ...... 51 4.4.2 Experiment B: Incorporation of Hap/DABCO stage into bleaching sequences and comparison with A stages. Assessment of brightness and viscosity ...... 54 4.4.3 Experiment C: Non-buffered reaction and comparison with HOCl addition. Assessment of brightness, HexA, kappa number and chlorination...... 57 5 Conclusions ...... 70 6 Outlook [confidential] ...... 71 7 References ...... 72 8 Figures ...... 74 9 Tables ...... 75 10 Appendix ...... 76

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Aim and Scope 1 Aim and Scope In the course of the centuries, paper has evolved from a luxury product to a cheap, high-quality product available to everyone. This progress has been made possible by industrialization of the papermaking process, leading to what is now known as the pulp and paper industry. Dozens of millions of tons of pulp are produced, of which a large portion is bleached and made into paper each year.

Efforts to reduce the energy consumption, environmental impact as well as chemical usage have led to very efficient processes, in particular the prevalent elemental chlorine free (ECF) bleaching greatly based on the use of chlorine dioxide. While other industries that use biomass as raw material have profited greatly from the introduction of enzymes into the processes, their usage in the pulp and paper industry is still low. This thesis sought to explore a novel enzyme application for pulp bleaching and hexenuronic acid removal, involving a haloperoxidase and a tertiary amine catalyst.

Hexenuronic acid is a xylan-derived compound that is produced during kraft pulping. It causes brightness reversion in paper and consumes big amounts of oxidation power in the bleaching of kraft pulp. Since its discovery in the 1990s, various methods such as hot acid stages have been developed to reduce HexA content, but these methods come at the price of cellulose hydrolysis and therefore yield and/or strength loss. A method to reduce HexA content while preserving cellulose integrity could greatly impact bleaching efficiency.

Recently, the use of hypochlorite combined with DABCO (1,4-diazabicyclo[2.2.2]octane), a tertiary amine catalyst, was reported to lead to quick and efficient bleaching and HexA removal in what is known as catalytic bleaching. Based on this, it was proposed to adapt this system using a haloperoxidase, an enzyme that catalyzes the oxidation of halides to their respective hypohalous acids. Based on the promising results from first experiments carried out at Novozymes and laid out in a patent (Lund et al., 2015), it was decided to further explore the technology in the course of this master’s project.

Depending on the extent of HexA removal and bleaching level attainable, a Hap/DABCO-stage could potentially replace A stages as well as complement or replace chlorine dioxide stages, thereby reducing chemical usage. Key factors would be the preservation of cellulose integrity and thereby preservation of product strength and production yield, as well as parity to or an advantage over the conventional systems concerning environmental effects, besides cost in use.

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Aim and Scope

The initial focus of the thesis was, on one hand, the optimization and a feasibility study of the Hap/DABCO system with regards to bleaching, HexA removal, cellulose integrity and organohalogen production, and, on the other hand, further insights into the mechanism behind the effect. The optimization was planned to be carried out through bleaching experiments with oxygen-delignified eucalypt pulp that would include Hap/DABCO stages as well as chlorine dioxide and alkaline extraction treatments. Parameters like brightness and HexA level, but also kappa number and viscosity would be assessed. A small-scale, low-consistency assay would be used to rapidly test different conditions, while larger-scale, medium consistency assays would provide a more realistic setting. The extent of chlorination would be assessed by the means of OX and AOX measurements. The mechanistic insights would be obtained through data gained from the pulp experiments, but also through lignin and pulp chromophore model compound experiments and the identification and quantification of the reaction products with GC-MS and LC-MS. In addition, the Hap/DABCO system was compared with non-enzymatic systems to fully ascertain its potential use in pulp bleaching versus benchmark technologies.

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Introduction and background 2 Introduction and background Since the work in this thesis was heavily focused on paper bleaching, this introduction aims at giving an overview of pulp and paper technology, especially the bleaching of kraft pulp. Catalytic bleaching as well as haloperoxidases and their applications will be discussed, and the initial work done on pulp bleaching and HexA removal with haloperoxidases will be presented.

2.1 The pulp and paper industry The term “pulp and paper” refers to the industry which uses plant fibers to produce paper and paper-related products such as cardboard. Most of the paper today uses wood as the fiber source, which is known for its recalcitrance, highly organized fiber structure and brown color. To get from this material to paper, which is usually white and mostly consists of cellulose, a series of treatments is necessary.

The first step in this series is the pulping process, which can be a thermal and mechanical, chemical or a combination of both mechanical and chemical disintegration of the raw material into a fibrous mass called pulp. The pulping makes the biomass amenable to further processing and is conducted in so-called pulp mills that process thousands to millions of tons of wood chips per year. The most popular pulping process is the chemical kraft process, which removes most of the lignin in the wood. The finished pulp is either sold, or further processed on site. In the case of paper production, the further processing comprises one or a series of bleaching and washing steps conducted in a bleach plant. Today, bleach plants mostly operate elemental chlorine free (ECF) sequences involving chlorine dioxide as the main oxidant for lignin removal and bleaching (Sixta, 2008, Suess, 2010).

In 2000, 334 million tons of pulp were produced globally, of which 187 million tons were virgin fiber, meaning that the fibers did not come from recycled paper. With 117 million tons, kraft pulping prevailed as the dominant pulping technology. ECF bleaching accounted for more than 64 million tons of bleached pulp in 2002, 70% of the world production of bleached chemical pulp (Sixta, 2008).

2.2 The Raw Material: Cellulose and Lignin Cellulose, the main constituent of paper, is an integral part of plant cell walls and the most abundant renewable polymer source on earth. It consists of β-1,4-linked D-glucose units, making the dimer cellobiose the repeating structural unit of the polymer (see Fig.1). The outermost glucose units with and without a free anomeric carbon are the non-reducing and reducing end, respectively.

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Figure 1: The molecular structure of cellulose. From Sixta, 2008

In the plant cell wall, cellulose chains are assembled into crystalline microfibrills, which derive their stability from intermolecular hydrogen bonding, although unordered amorphous regions are present too. Microfibrils are associated with hemicelluloses, which are amorphous and branched polymers composed of various carbohydrate and carbohydrate-derived moieties. Hemicellulose composition varies heavily between plant families, and also shows variation among species. For woody biomass, there is a clear distinction between softwoods and hardwoods. Softwoods contain a higher proportion of mannose and galactose units, while hardwoods contain more xylose and glucoronic acids and show more acetylation of hydroxyl groups. The microfibrils assemble into macrofibrils, between and around which hemicellulose and lignin are deposited.

Lignin is a heterogenous polymer derived from the three basic units: p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, which are randomly polymerized by radical coupling and thereby becoming p-hydroxyphenylpropane (H), guaiacylpropane (G) and syringylpropane (S) subunits. Consequently, different kinds of bonds are formed explaining its heterogeneous nature. The most prevalent bonds are β-O-4 ether bonds, but β-5 bonds, β-β bonds and other types of C-O and C-C bonds are also formed. The resulting complex, hydrophobic polymer is recalcitrant and confers degradation resistance as well as brown color to the wood. Wood can contain 20-40% of lignin depending on the species. For paper making, lignin is removed by pulping and bleaching to different extents depending on the final product requirements. The proportions of the three subunits, determining the chemical reactivity of the lignin, depend on the species, type and age of wood. While softwood lignin mainly consists of G units, hardwood additionally contains 20-60% of S untis. Both hardwood and softwood contain small amounts of H units, which is the dominant lignin monomer in grasses. An example of the structure of softwood lignin is shown in Fig. 2. An important example of how lignin composition influences papermaking processes is the fact that S subunits are less prone to cross-linking due to their two methoxy groups, leading to a faster delignification in kraft cooking of hardwood and also improved bleachability.

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Figure 2: Example softwood lignin structure and lignol monomers. From (Christopher et al., 2014).

2.3 Kraft Pulping Of all the different technologies to transform wood into pulp, Kraft pulping remains the dominant process. The basic concept is cooking wood chips at 140-175°C with high concentrations of sodium hydroxide and sodium sulfide. Under these conditions, lignin α-O-4 and β-O-4 ether bonds are cleaved and the solubilized lignin is removed as so-called black liquor, but most of the hemicellulose and some cellulose is also degraded. Acetate side groups are also removed. The cooking is stopped before carbohydrate degradation becomes dominant over delignification. Kraft pulping has almost completely replaced sulfite pulping, which is now mainly being used for dissolving pulp (pulp for the production of textile fibers and cellulose derivatives).

The residual lignin from the kraft cooking is removed and bleached by more selective treatments that remove lignin without compromising yield or viscosity to an unreasonable extent. The current standard treatment after kraft cooking is an oxygen delignification stage (O), where the combination of NaOH, oxygen and a temperature of about 90°C yield a removal of about 60% of the residual lignin in hardwood kraft pulps. An important process parameter is pulp consistency, which describes at which % dry matter the process is carried out. The pulp can absorb multiple times its own weight in water and changes its fluid behavior as the dry matter % is increased. Typically, pulp mills run at either low, medium

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Introduction and background or high consistency. Low consistency (3-4% DM) leads to a water-like behavior, medium consistency (6-14% DM) shows non-Newtonian fluid behavior and high consistency (30-40% DM) leads to a firm mat of pulp. Medium consistency is the most popular choice in pulp mills.

2.4 Bleaching: Development and current technology Historically, fibers, often from rags, were bleached by time-intensive processes using sunlight, fermentation processes and alkali. In 1792, the process of bleaching fibers with chlorine or hypochlorite was patented in France. Hypochlorite (H-stage) was used exclusively until the 1920s, when molecular chlorine (C-stage) followed by an alkaline extraction (E-stage) and a final, light hypochlorite stage came into use. This CEH sequence became the standard bleaching sequence and could bleach pulps up to 85% brightness. Chlorine dioxide (D-stages) came into use after the Second World War, leading to >90% brightness in CEHD and CEHDED sequences. In the 60s and 70s, oxygen delignification became more and more widespread due to technological development. At the same time, concerns about the pollution and environmental problems caused by pulp mill effluents in general, and chlorinated organic compounds in particular, were raised. This resulted on one hand in increased closure of the pulp mills and on the other hand in the adoption of elemental chlorine free (ECF) and totally chlorine free (TCF) processes.

ECF sequences employ a combination of ClO2 treatments (D) and alkaline extractions with or without reinforcement with hydrogen peroxide or oxygen (E, Ep Eo, Eop). TCF sequences employ ozone (Z-stage) and alkaline hydrogen peroxide (P-stage). Applications of Z and P stages in ECF sequences are commonplace too. The usage of elemental chlorine and hypochlorite stages has been steadily declining, but is still found in less developed countries. On a worldwide scale, ECF dominates as a bleaching process and is considered the best available technology (Suhr et al., 2015).

2.4.1 D-stage chemistry After kraft cooking and oxygen delignification, 95% of the lignin has been removed. To further oxidize and depolymerize the lignin, chlorine dioxide is applied at acidic pH in a first D stage, the D0-stage. Subsequent D-stages are referred to as D1, D2, etc. Chlorine dioxide is a toxic and highly explosive gas that must be generated on-site at the pulp mill. It is a resonance-stabilized radical and its oxidation state is +4, thereby consuming 5 electrons to become chloride. From studies with model compound, it could be deducted that chlorine dioxide reacts with lignin by electrophilic addition to aromatic nuclei and immediate eliminiation of chlorous acid, leaving an organic radical. This radical will react with chlorine dioxide again, and yield different quinone or muconic acid compounds (Fig. 3 shows the various

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Introduction and background products from the reaction of ClO2 with a lignin-model compound). Hypochlorous acid and chlorous acid are formed as byproducts. Chlorous acid does not react with lignin, but regenerate chlorine dioxide in a reaction with hypochlorous acid or elemental chlorine. The latter are in equilibrium and both oxidize and chlorinate lignin, yielding chloride ions and chlorinated organic compounds. The conversion of hypochlorous acid to elemental chlorine is facilitated by low pH and high chloride concentration.

Hypochlorous acid will also react with chlorine dioxide to chlorate, which is considered a waste product, since it does not bleach nor react any further.

Figure 3: Cleavage reactions of non-phenolic model compound (4-methyl-2.3’.4’-tri-methoxy- diphenylether) with chlorine dioxide. From (Suess, 2010).

2.4.2 Ep chemistry A complete oxidation of lignin to carbon dioxide is neither possible nor economic, therefore the depolymerization and subsequent removal of the fragments is the primary goal. Chlorine dioxide oxidation of lignin leads to depolymerization of the insoluble polymer into smaller, water-soluble compounds. Moreover, carboxylic acid structures such as muconic acids are generated in the remaining lignin. Typically, after chlorine dioxide treatment, an alkaline extraction stage (E) at high pH is used to ionize and dissolve carboxylic acids and phenols, thereby removing these compounds from the pulp more effectively than a neutral washing stage. Moreover, chlorine 15/79

Introduction and background atoms can be removed from organic compounds by nucleophilic substitution, thereby reducing the amount of organochlorine compounds in the pulp.

Hydrogen peroxide is often used to reinforce extraction stages (Ep-stage) and provide additional brightening and, to a small extent, delignification. At high pH, hydrogen peroxide is in equilibrium with the perhydroxyl anion (HOO-), which is the reactive compound in peroxide bleaching. It is a nucleophile and adds to quinone structures and conjugated carbonyl groups, leading to the formation of more hydrophilic carboxylic acids, side chain cleavage and chromophore destruction (Suess, 2010).

In ECF bleaching, the combination of D and E stage is repeated with reduced chemical loadings, since a smaller amount of lignin needs to be oxidized or removed. The finishing stage that brings the brightness to the final level (>90% for printing and writing grades) is traditionally a D stage, leading to the classical sequence DEDED. However, the present state of the art is to introduce peroxide (P) or, to a smaller extent, ozone (Z) stages into an ECF sequence.

2.5 Hexenuronic acids: A side product of kraft pulping To measure the remaining lignin in pulp, a titration of the so-called kappa number is performed. However, in the 1990s it was discovered the kappa number also measures “false lignin”, i.e. compounds also oxidizable by potassium permanganate. This false lignin was later discovered to be mostly composed of hexenuronic acid (HexA). It was also discovered that the remaining HexA in fully bleached pulp will contribute to brightness reversion, e.g. yellowing.

HexA does not occur naturally in wood. It is produced during kraft pulping, where the 4-O- methyl-glucuronic acids in glucuronoxylan side chains undergo demethylation and formation of a conjugated double bond. Since the amount of glucuronoxylans is much higher in hardwoods than in softwoods, hardwood kraft pulps also contain more hexenuronic acids (20-70 mmol/kg) than softwood kraft pulps (<20 mmol/kg) (Sixta, 2008).

It was found that HexA was responsible for e.g. 1/3 of the kappa number of unbleached and 60- 70% of the kappa number of oxygen-delignified birch kraft pulp. This is caused by the fact that hexenuronic acid is left virtually untouched by oxygen delignification, as well as by alkaline peroxide treatment. Alkaline extractions don’t affect HexA content either (Li and Gellerstedt, 1997).

It is in the D-stages where HexA has a high effect: One mole of HexA was estimated to consume 1,5 moles of ClO2 (Törngren and Ragnar, 2002), and an additional consumption of

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Introduction and background about 0,68kg ClO2 per ton odp in a D0E1D1 sequence was stated for an O2-delignified birch pulp with a HexA content of 50 mmol/kg (Vuorinen et al., 1999).

Although chlorine dioxide is able to react directly with HexA, in actual bleaching reactions HexA is degraded via chlorination by hypochlorous acid and subsequent reactions with chlorous acid, hypochlorous acid and elemental chlorine. Chlorine dioxide was claimed to react twice as fast with lignin as with HexA (Brogdon, 2009).

To solve this problem of wasting oxidation power, the susceptibility of HexA towards acid hydrolysis is exploited: A hot acid stage before the D0 stage can be used to significantly reduce

HexA content, as well as a so-called “hot D0”-stage, a combination of hot acid and D0 stage. The drawback, however, is yield and viscosity loss due to cellulose hydrolysis. Combined with the high investment costs and the moderate savings in chemicals, the use of A and hot D0 is not widespread in pulp mills (Suess, 2010).

In addition to chlorine dioxide bleaching, HexA also impacts ozone bleaching. It has for example been discovered that HexA removal with a hot acid stage leads to a lower viscosity loss during ozone treatment.

2.6 Catalytic bleaching Recently, a novel approach to pulp bleaching was proposed by researchers at Aalto University. In their work, they described the application of hypochlorite together with DABCO, a tertiary amine catalyst, leading to good bleaching performance and HexA degradation as well as acceptable levels of chlorination.

2.6.1 Tertiary amine catalysis of HOCl reactions HOCl is a powerful oxidizer and chlorination agent. It is in equilibrium with the chloronium ion (1), which acts as an electrophile.

+ + 퐻푂퐶푙 + 퐻 ↔ 퐻2푂 + 퐶푙 (1)

HOCl can undergo different reactions with organic substrates. HOCl reacts e.g. with  alkenes, forming chlorohydrins.  alkanes, forming chloroalkanes and water  alcohols, forming chloroalkanes and hydrogen peroxide  enol structures of aldehydes, resulting in polychlorinated compounds

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It was shown that catalytic amounts of certain tertiary amines are able to speed up the reactions of HOCl with salicylic acid, alkenes and nucleotides by several orders of magnitude. This was explained by the formation of an instable quaternary chlorammonium cation intermediate (Fig. 4). On the one hand, the reaction was found to be favorable when the amine group was unprotonated and therefore available for the formation of the intermediate. On the other hand, the stability of the formed species needed to be relatively high so that autocatalytical decomposition would occur slower than the reaction with the substrate. Quinuclidine was suggested to produce a stable quaternary ammonium cation, but its high pkA of 12 would not allow for efficient catalysis at a neutral pH. Other tertiary amines efficiently catalyzed the reaction at neutral pH, but decomposed very fast (Prutz, 1998).

Figure 4: Proposed reaction scheme of the DABCO-catalyzed oxidation of substrates with hypochlorous acid. Adapted from Chenna et al., 2013

1,4-Diazabicyclo[2.2.2]octane (DABCO, Fig. 5) is a bicyclic tertiary amine that is used as a catalyst in polyurethane manufacture. It structurally resembles quinuclidine, but contains another nitrogen atom in place of a carbon. Its two amine groups have pKas of 3 and 8.8, respectively, making it available for the formation of a quaternary chlorammonium cation at neutral pH. It was suggested that DABCO as well as quinuclidine form relatively stable quaternary chlorammonium cations because of their ring structure, which does not allow them to take the conformation necessary for rapid decay (Vuorinen et al., 2013). It has, however, been shown that while the quaternary chlorammonium cation of quinuclidine is stable, the one of DABCO decomposes in a matter of seconds in the absence of substrate to react with (Rosenblatt et al., 1972). Figure 5: The structure of 1,4- Diazabicyclo[2.2.2]octane (DABCO)

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2.6.2 Catalyic bleaching and removal of hexenuronic acid Hexenuronic acid is known to be oxidized by hypochlorous acid (Brogdon, 2009). Researchers at Aalto University have designed a novel approach to delignification and hexenuronic acid removal which relies on the catalytic activity of DABCO with HOCl in a so-called Hcat stage. Experiments were carried out with oxygen-delignified eucalyptus kraft pulp at 1% consistency, HOCl dosage of 1% active chlorine odp and a DABCO dosage of 0,1% odp. More than 80% of hexenuronic acid and more than half of the lignin were removed, and remarkably, the reaction was completed in a matter of minutes and at room temperature. The observed optimum initial pH was 5, although a rapid increase to pH 10 was observed and tentatively attributed to the rapid and quantitative formation of the chlorammonium cation, effectively increasing the concentration of OH- by the concentration of DABCO (Chenna et al., 2013).

In the experiments exploring catalytic oxidation with tertiary amines, only the removal of target species, e.g. ascorbic acid, lignin or HexA was measured, not the formation of the reaction products. This means that it has not been shown whether the reactions of the quaternary chlorammonium cation are identical to the ones of hypochlorous acid, nor if the specificity or nature of the reaction is changed. It has been suggested though that the specificity and nature of the reactions should not be different from HOCl since the Cl+ electrophile is the same active species (Chenna et al., 2016).

The formation of chlorinated organic compounds during catalytic bleaching was also investigated. In the tested conditions (10 minutes of treatment, 1% active chlorine and 0,1% DABCO odp, 40C), the catalytic bleaching stages showed AOX formation on par or higher than the reference D stage at pH 3 and pH 10.5. At pH 3.5 to 8.5, the AOX values were lower than the reference value (470g/t). A sample with an increased temperature of 55C showed increased AOX formation (600g/t). OX results showed a similar behavior. A low level of chloroform production was also found and primarily assigned to HexA degradation by the chlorammonium cation. A mechanism of HexA degradation to chloroform was proposed (Fig. 6).

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Introduction and background

Figure 6: Proposed mechanism of chloroform formation from HexA. From Chenna et al., 2013.

No brightness values were disclosed for catalytic bleaching technology, except for 88% as the end ISO brightness of an Hcat-Z-P sequence.

Hypochlorous acid reacts with lignin compounds by addition of the chloronium ion to electron- rich structures such as aromatic rings or alkenes. Depending on the site of the reaction, the product may be stable or react further. If further reactions occur, an elimination of Cl- leads to an oxidized, unchlorinated compound. Other reactions include the elimination of side chains or methoxy groups. HOCl can generate phenolic groups and quinone structures as well as cleave the propyl unit from the phenyl unit (Fig. 7 and 8).

Figure 9: Generation of phenolic groups by hypochlorous acid (chloronium ion). From Sixta, 2008.

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Figure 10: Generation of halogenated (chlorinated) compounds in hypochlorous acid (chloronium ion) reactions and HCl addition to quinone structures. From Sixta, 2008.

The reaction rate of HOCl with HexA model compounds was found to be tenfold higher than the reaction rate of HOCl with lignin-like structures, indicating that HexA is degraded preferentially in the pulp (Brogdon, 2009).

Hypochlorite bleaching is carried out at high pH (10-12), meaning that it is exclusively the hypochlorite ion OCl- that is present. At a lower pH (<9), small amounts of HOCl can be found. This is known to lead to higher brightness gain, but also to the oxidation of cellulose. Alcohol groups are converted to aldehydes, which under the slightly alkaline conditions results in chain cleavage leading to strength loss, and higher brightness reversion due to chromophore formation from the oxidized cellulose.

2.7 Pulp mill effluents and the environment A pulp mill can process several hundreds to thousands of tons of wood each year, generating vast amounts of effluents that are discharged into rivers. Continued closure and recycling efforts as well as the introduction of aerated lagoons where sedimentation and biodegradation takes place have reduced the amount of wastewater and the degree of pollution.

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Introduction and background

Still, pulp mill effluent is an environmental concern due to the amount of organic carbon discharged (chemical oxygen demand, COD) and the nature of pollutants. Pulp mill effluents can cause elevated levels of steroids and developmental effects in fish, such as developmental retardation. Moreover, elevated activity of P450 oxidases can be observed in fish, which is an indicator for aquatic pollution (Solomon, 1996).

2.7.1 Organochlorine compounds Historically, organochlorine compounds, especially chlorinated dioxins and furans, were one of the most prominent issues about environmental pollution by pulp mills. These environmental concerns eventually led to the abandonment of elemental chlorine bleaching in favor of ECF and TCF bleaching.

While about 10% of elemental chlorine used in bleaching ends up in organochlorine, this number is about 5% for hypochlorite and only 2% for ClO2. This can be explained by the fact that the oxidation state of the Cl in ClO2 is +4, while it is 0 in Cl2. Both are fully reduced to -1 in chloride or organochlorine compounds. Therefore, more oxidation per atom of Cl can occur with chlorine dioxide. Additionally, the nature of the chlorinated compounds is different. Cl2 produces dichlorinated products in the reaction with alkenes. ClO2 does not chlorinate lignin, only the reaction byproduct HOCl does. HOCl, however, preferably forms chlorohydrins in reaction with alkenes. These compounds logically contain less chlorine, and they are also more easily removed in subsequent alkaline extraction stages (Solomon, 1996).

Chlorinated compounds in pulp mill effluents originate from natural sources, i.e. the wood, but also bleaching stages based on chlorine compounds. These substances can be problematic in more than one way. The most obvious one is their toxicity and mutagenicity. 2,3,7,8- Tetrachlorodibenzodioxin for example is a well-known toxic, carcinogenic and teratogenic compound that was typically found in pulp mill effluents before the substitution of Cl2 with ClO2. The structurally similar polychlorinated dibenzofurans are also classified as carcinogenic. An array of other toxic, mutagenic or carcinogenic substances has been found in the effluent of C- stages (Murray, 1992).

Most non-chlorinated, but also monochlorinated organic compounds originating from paper mills can be easily degraded by microorganisms present in the lagoons. Increasing substitution of organic compounds with chlorine, however, decreases their reactivity and makes them more hydrophobic. Highly chlorinated, non-polar compounds such as polychlorinated dibenzodioxins and dibenzofurans are therefore difficult to degrade and have an expected half-life of several years in the environment. The excretion mechanisms of higher animals are inefficient for these compounds, making them persistent in body tissue. Moreover, the lipophilicity of these 22/79

Introduction and background compounds causes bioaccumulation in the fatty tissues of fish, which can eventually be transferred to humans by ingestion (Solomon, 1996).

Polychlorinated dibenzodioxins and furans have been virtually eliminated from pulp mill effluents by the adoption of ECF bleaching. ECF bleaching effluent still contains a number of chlorinated compounds and adverse effects of treated ECF pulp mill effluents on aquatic organisms are still observable.

Pulp mills produce a big diversity of chlorinated organic compounds, where each compound is often present in very small amounts. This makes detection, quantification and especially regulation difficult. To account for this problem, chlorinated organic compounds are regulated via the measurements of AOX (Adsorbable Organic Halogen) for liquid samples, meaning the effluent, and OX (organic halogen) for solid samples, usually meaning the bleached pulp or paper product. The values give an indiscriminate measurement of all organically bound halogens and do not account for the different properties of different chlorinated compounds. For this reason, they have been criticized for not being a relevant measurement of environmental hazards (Muller, 2003).

It has been shown that the removal of HexA during ClO2 stages is correlated with an increase in AOX in the effluent, caused by the soluble, chlorinated degradation products of HexA that are easily removed from the pulp. If the same effluent is measured after a week of storage at 37C, this portion of AOX has been completely degraded, indicating easy degradation of this compound also in real pulp mill effluents (Björklund et al., 2004).

2.8 Selected paper and pulp properties There are several pulp and paper properties which relate to processing and the desired application of the end product. High strength is important for e.g. linerboard and shopping bags, while brightness is of high importance for printing and writing paper. Some key parameters will be outlined here in the context of pulp bleaching.

2.8.1 Brightness ISO brightness is important for paper applications that require high brightness, e.g. printing paper. It is measured as the diffuse reflectance of light with a wavelength of 457 nm, and expressed as a percentage of a 100% standard (ISO 2469). Fully bleached kraft pulp should reach a final brightness of >90%. Brightness is influenced by light-absorbing compounds such as residual lignin and lignin- and carbohydrate-derived chromophores, but also by intrinsic structural features of the fiber matrix.

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Introduction and background

Generally, gaining a point in brightness gets more difficult (i.e. more chemically demanding) at the end of the bleaching sequence. A decrease in brightness after the pulp has been fully bleached, e.g. through yellowing by hexenuronic acids, is called brightness reversion.

2.8.2 Strength Paper strength is measured as resistance to different kinds of mechanical stress, e.g. tensile, breaking and strain at rupture. On a fiber level, strength is dependent on the crystallinity of the cellulose, which can be impeded by chain scissions, chemical modifications and amorphous regions. The strength of a paper sheet additionally depends on fiber length, cellulose:hemicellulose ratio, inter-fiber hydrogen and van der Waals bonds as well as intertwining of fibers.

2.8.3 Viscosity Viscosity is measured as the flow of a solubilized pulp sample through a capillary viscometer. A solubilized polymer increases the viscosity of its solvent, and the longer average chain length, the higher the viscosity. Therefore, this parameter is used to describe the average degree of polymerization (DP) of the cellulose in the pulp. The average DP can sometimes correlate with pulp strength, and a loss in viscosity after e.g. a bleaching stage indicates poor selectivity of the stage, leading to cellulose degradation. For fully bleached softwood kraft pulp, the lowest acceptable viscosity value has been proposed to be 850mLg-1, since pulp strength seriously deteriorated below this value (Sixta, 2008). The ratio of brightness gain to the loss of viscosity is referred to as selectivity.

2.8.4 Yield Yield is measured as the percentage of dry pulp remaining after a treatment. It is an important factor for bleaching processes and depends on the specificity of the employed bleaching process.

2.9 Enzyme applications in pulp and paper Enzymes have been used in the pulp and paper industry since the 1980s. An abundance of organisms thriving on lignocellulosic biomass provide a wide range of potentially useful enzymes to choose from. However, the extreme conditions (temperature, pH) often employed in pulp and paper processes are a challenge for enzyme applications that should fit within existing setups (Bajpai, 2015, Torres et al., 2012).

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2.9.1 Hydrolytic enzymes Cellulases and xylanases can be used to increase drainage. Xylanases can be also used in bleach boosting, where they degrade precipitated xylan from the cooking process and recalcitrant lignin-carbohydrate complexes prior to bleaching and thereby enhance pulp bleachability. This reduces the demand for bleaching chemicals by removing xylan-bound lignin and HexA as well as by making the fiber more accessible to bleaching chemicals. A general consideration about hydrolytic enzymes is that they can possibly decrease yield depending on the conditions and type of product.

2.9.2 Laccases and peroxidases These enzymes have the potential to modify or degrade lignin. Laccases, supplied with a suitable mediator and oxygen, perform one-electron oxidations that drive radical depolymerization of the lignin. Peroxidases oxidize lignin structures by introduction of hydroxyl, carbonyl or carboxyl groups with hydrogen peroxide.

Various other enzymes have been investigated for their potential to improve the papermaking process with regards to biofilm control, extractives mitigation, pulp refining, environmental impact and others.

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Introduction and background 2.10 Haloperoxidases Haloperoxidases are enzymes that catalyze the two-electron oxidation of a halide with hydrogen peroxide, resulting in the production of the corresponding hypohalous acid (2).

− − 푋 + 퐻2푂2 → 퐻푂푋 + 푂퐻 (2)

The hypohalous acid will either react with organic substrates as an electrophile (3), or react with hydrogen peroxide to yield a halide, water, a proton and singlet oxygen (4).

퐻푂푋 + 푅퐻 → 푅푋 + 퐻2푂 (3) − + 1 퐻푂푋 + 퐻2푂2 → 퐻2푂 + 푋 + 퐻 + 푂2 (4)

Haloperoxidases can be divided into iodoperoxidases (IPO), bromoperoxidases (BPO) and chloroperoxidases (CPO) according to the most electronegative halide that they are able to oxidize. A chloroperoxidase is able to oxidize chloride, bromide and iodide, while a BPO is able to oxidize bromide and iodide, and a IPO only oxidizes iodide (Messerschmidt, 2001). No fluoroperoxidases have been identified so far (Liang et al., 2013).

Haloperoxidases can furthermore be classified according to their catalytic moiety into heme- dependent haloperoxidases, vanadium-dependent haloperoxidases (VHPO) and non-heme, non-metal haloperoxidases (Daniela Gamenara, 2012).

Heme haloperoxidases have a ferric heme (protoporphyrin IX) as prosthetic group. They are also called heme-thiolate haloperoxidases, because the fifth ligand coordinating the iron is a cysteine and not a histidine, as in other heme peroxidases. The catalytic cycle involves an oxidation of the iron from iron III to iron IV in the formation of compound I (Fe=O) and the formation of a heme-oxygen-halide intermediate (Fe-O-X), which releases the hypohalous acid. Heme haloperoxidases, being a subclass of the wider heme peroxidase family, are capable of performing peroxidase reactions on organic substrates too. While heme-dependent haloperoxidases are relatively sensitive to oxidation due to their prosthetic group and get rapidly inactivated by hypohalous acids, at least two VHPOs have been shown to be much more resistant towards oxidation (Renirie et al., 2003).

Non-heme non-metal haloperoxidases are found in bacteria and seem to be related to the α/β- hydrolase family. Their catalytic mechanisms are unclear. While a conserved catalytic triad has been found, it has also been suggested that organic acids are necessary as a cofactor for haloperoxidase activity (Hofmann et al., 1998, Pelletier et al., 1995, Xu and Wang, 2016).

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Introduction and background

Just as halogenated organic species can be found throughout nature, haloperoxidases are found in a wide variety of organisms, where they perform different functions. For example, haloperoxidases from marine algae are supposed to be involved in the biosynthesis of the halogenated metabolites characteristic for these organisms as well as in defense, haloperoxidases found in fungi are thought to be part of the oxidative breakdown of the plant cell wall, and haloperoxidases found in human thyroid tissue catalyze the iodation of thyroid hormones (Messerschmidt, 2001, Hofrichter and Ullrich, 2006).

2.10.1 Vanadium haloperoxidases Vanadium haloperoxidases (VHPOs; including VIPOs, VBPOs and VCPOs) are characterized −4 by using covalently bound vanadate (VO3 ) as a co-factor involved in the catalysis. The catalysis involves first the binding of hydrogen peroxide and the formation of a peroxo- intermediate. The subsequent nucleophilic attack of the halide ion on a peroxide oxygen leads to the breaking of the peroxide bond and the formation of the O-X bond. After protonation by a water molecule, HOX leaves the catalytic site. The catalytic cycle does not involve a change in oxidation state of the vanadate. Site-directed mutagenesis studies of VHPOs from Curvularia inaequalis and Corallina pilulifera showed that specificity for the accepted halides can be changed by a single amino acid substitution, leading to wild-type CPOs becoming BPOs, and vice versa (Messerschmidt, 2001, Ohshiro et al., 2004).

The vanadium binding sites of VHPOs show sequence and structure similarities with several families of phosphatases. Phosphate and vanadate have a similar structure, and several phosphatases can be inhibited by vanadate. It is also known that vanadate in the active site can be replaced by phosphate, leading to a loss of haloperoxidase function. Furthermore, VHPO apoenzymes have been shown to act as phosphatases, and vice versa. This suggests an evolutionary relationship between these two enzyme classes (Winter and Moore, 2009)

Most of the naturally occurring brominated compounds are of marine origin. The first VHPO, a VBPO, was isolated from the marine brown algae Ascophyllum nodosum in 1984 and since then, VBPOs have been identified in all major classes of marine algae. In vitro studies suggest a role in the biosynthesis of brominated metabolites including stereo- and regioselective bromination. At the same time, a role of hypohalous acid production as an antimicrobial defense mechanism has been suggested, a claim which is supported by the presence of BPOs on the surface of some algae (Messerschmidt, 2001).

VCPOs have been found in a class of fungi known as dematiaceous hyphomycetes as well as in several prokaryotes. Fungal VCPOs have been found in the genera Curvularia, Embellisia, 27/79

Introduction and background

Drechslera and Ulocladium and have been shown to be localized on the surface of the fungal hyphae as well as in the growth medium, leading to the suggestion that they are involved in the oxidative breakdown of the lignocellulosic plant cell wall. A bacterial VCPO from Streptomyces sp. has been shown to be able to perform a stereoselective bromination-cyclisation reaction on a terpene (Winter and Moore, 2009, Fournier et al., 2014).

For a long time, it was believed that VHPOs acted by producing freely diffusable hypohalous acid which then halogenated substrates. However, proven stereoselectivity and preferred utilization of certain substrates over others, a behavior different from that of free hypohalous acids, shows that this is not the case for all VHPOs (Winter and Moore, 2009). This makes sense in the light of the different suggested roles of VHPOs in nature. Selectivity was proposed to be conferred by the enzyme binding an organic substrate near its active site, leading to its regio- or stereospecific halogenation (Butler and Carter-Franklin, 2004).

2.10.2 Curvularia haloperoxidases In the present study, a VCPO from the fungus Curvularia verruculosa was used. Its amino acid sequence is 90.9% identical to that from Curvularia inaequalis, but it exhibited a fourfold higher specific activity in the chlorination of monochlorodimedone (Fuglsang et al., 1997). The better- studied C. inaequalis VCPO was shown to be an extremely stable enzyme: it retained its activity when exposed to 500 mM of hydrogen peroxide, while producing singlet oxygen for one hour from the reaction of HOBr with H2O2 (Renirie et al., 2003). It also retained its activity under HOCl concentrations of up to 2.5mM as well as organic solvent concentrations of up to 40%.

Furthermore, it showed a high thermostability with a Tm of 90°C. For C. verruculosa VCPO, less data is available. At pH 5, an optimum temperature of 60°C was found (Fuglsang et al., 1997). The enzyme has an average monomer mass of 64,5kDa, as determined by mass spectrometry (Fuglsang et al., 1997). No information on its state of association is available, but C. inaequalis VCPO was reported to be a monomer (Winter and Moore, 2009).

2.10.3 Biotechnological applications of VHPOs To this date, no established commercial biotechnological application involving a haloperoxidases is known to the author. Due to their high tolerance concerning temperature, organic solvents and oxidation, VHPOs seem to be good candidates for industrial use (Van Schijndel et al., 1994, Tromp et al., 1990). Due to their stereo- and regioselective halogenation potential, VHPOs have been proposed to be useful in the production of pharmaceuticals and fine chemicals (Winter and Moore, 2009, Wong et al., 1996).

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Introduction and background

Some proposed applications are based on the potential of the produced hypohalous acids as antimicrobial and antiviral agents, shown by patent applications for the use of VHPOs for inhibiting the growth of mycobacteria, killing viruses, replacing formaldehyde in the production of vaccines, reducing microbial growth in animal feed and reducing malodour in hygiene products and animal litter (Danielsen, 2004, Rikke and Gjermansen, 2010, Gjermansen et al., 2010, Kirk et al., 1999, Klausen and Lassen, 2014). A remarkable biomimetic approach is the application of haloperoxidase in the paint of ship hulls, where they would use the naturally occurring hydrogen peroxide and halogenides to generate hypohalous acid, which could prevent fouling. This is based on the idea that algae express haloperoxidases on their surface for protection. This could replace current anti-fouling-paints, which are the object of environmental concerns (Wever et al., 1995). A patent application for using VHPOs in pulp bleaching is its application in chlorine dioxide treatment, where it can generate hypochlorous acid from chloride ions and hydrogen peroxide formed in situ during chlorine dioxide treatment (Xu et al., 2006). Other propositions in industrial processes are based on the high reactivity and oxidizing potential of hypohalous acids, such as the recovery of liquid hydrocarbons from bitumen and tar sand, the bleaching of stains on fabric, and the oxidation of sulfide minerals in mining processes (Jump, 2010, Lassen, 2013, Beggs et al., 1998).

2.11 The haloperoxidase – DABCO system The haloperoxidase – DABCO system was conceived as a fusion of the catalytic bleaching approach and enzymatic treatments of pulp. The haloperoxidase would produce hypochlorous acid, the effect of which could be potentially magnified by the DABCO addition (Hap/DABCO- stage). The results of preliminary experiments were published in patent WO2015018908 (Lund et al., 2015).

The system was tested on OD0Ep-bleached aspen pulp and with NH4Cl and NaCl as chloride sources, and brightness and HexA levels were measured as outcomes. The effect of the haloperoxidase without DABCO (Hap-stage) was also assessed. Both the Hap and Hap/DABCO stage produced a brightness increase of 2-3%, with the DABCO seemingly conferring a slight increase. While the Hap-stage produced a decrease in HexA of about 30% with NaCl, the Hap-DABCO stage showed about 50% decrease for both NH4Cl and NaCl. The

Hap-stage with NH4Cl did not decrease HexA.

The employed conditions were 45C, 60mg of enzyme per kg odp, 6mM H2O2, 6mM NaCl/NH4Cl, 6mM DABCO and acetate buffer at pH 4,5.

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These results showed an obvious effect of DABCO on HexA removal, and a slight effect on brightness gain (Data in appendix). The difference in HexA removal by NaCl and NH4Cl was explained by the formation of chloramines by the latter, which were assumed to have different reactivity towards HexA than HOCl. With DABCO, these effects were not visible, indicating that the reaction with DABCO outcompeted the chloramine formation, which is in line with the reported fast formation and high reactivity of the chlorammonium cation (Chenna et al., 2013).

3 Materials and Methods 3.1 Materials 3.1.1 Enzyme Vanadium haloperoxidase from Curvularia verruculosa (referred as “Hap”), as disclosed in (Lund et al., 2015).

3.1.2 Equipment UV/VIS Spectrophotometer 8453, HP/Agilent, USA Spectroquant Photometer NOVA 60, Merck, USA Ultrapure Water Milli-Q Q-POD, Millipak Express 20 (0512) Filter Balance Mettler Toledo, NewClassic MF, MS6001S/01, Switzerland Mettler Toledo, XPE304, Switzerland Mettler Toledo, MS 204, Switzerland pH Meter pHM220, MeterLab, Denmark Water Bath Grant T100, UK Moisture Analyzer HX204, Metter Toledo, Switzerland Automatic Sheet Press Model 400-1, Labtech Inc, Canada Vacuum Pump Pfeiffer Vakuum Duo 2.5, Germany Centrifuge 5810, Eppendorf, Germany Labomat BFA24 with 16 beakers, Mathis, Switzerland Pulp Disintegrator App 03 Type 8-3, AB Lorentzen & Wettre, Sweden Freeze Dryer CoolSafe 9515 Pro Superior XL, Labogene, Denmark Thermo Reactor TR620, Merck, USA Titrator system Tim900 Titration Manager System with 30/79

Materials and Methods

ABU091 Autoburette and TimTalk titration software, Radiometer, Denmark DL53 Titrator System with LabX light titration software, Mettler Toledo, Switzerland Heating and stirring unit ThermoModul 40ST, VarioMag, USA LC-MS system 1260 Infinity, Agilent, USA 1260 μ-Degaser, G1379B, Agilent, USA 1260 HiP ALS Autosampler, G1367E, Agilent, USA 1290 Thermostated column compartment, G1316C, Agilent, USA 6120 Quadropole LC/MS Detector, Agilent, USA GC-MS system 7890A GC System, Agilent, USA 5975C inert MSD, Agilent, USA Pipettes m3, m100, m200, 1000, 5000, 10 ml, Sartorius, Germany Diffuse reflectance spectrophotometer Colortouch PC, Technidyne, USA

3.2 Methods 3.2.1 Enzyme treatment medium consistency Pulp of known dry matter content was weighed into plastic bags. Buffer or dH2O was added to reach the targeted consistency, minus the volume of the other reactants and H2SO4 (for the non-buffered experiments).

If water was added, the pH was adjusted by adding H2SO4 until the liquid phase had the desired pH. After 15 to 30 minutes of equilibration time, the process was repeated until the pulp stayed at the desired pH. dH2O was added to reach exactly the desired volume minus the volume of the other reactants. NaCl and DABCO were added and the pulp was mixed by hand. The pulp was pre-heated in the water bath to assure the correct temperature of the pulp. The enzyme (for the control: dH2O) was added and the pulp was mixed by hand. Lastly, the hydrogen peroxide was added and the pulp was thoroughly mixed by hand. The bag was thermally sealed or sealed with a plastic clamp if samples were to be taken continuously. The bag was placed in the water bath for the scheduled incubation time. After the incubation time, the pulp was either put in an ice-water bath to cool down and thereafter filtered, or filtered directly.

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Since pulp experiments in medium consistency consume a vast amount of time per sample, no duplicates were done. Since the amounts of pulp are so big, background error due to e.g. pipetting or weighing inaccuracy can be assumed irrelevant. Human error, however, cannot be excluded. Therefore, experiments were performed with diligence and care, leading to only one apparent pipetting mistake in the work that is presented here (Experiment C, HOCl sample, Ep stage: No H2O2 added).

Table 1: Conditions of enzyme treatment at medium consistency. Variable conditions are referred to in the Results and Discussion section.

Treatment pH Buffer T t H2O2 NaCl DABCO Hap (mM) (°C) (min) (mM) (mM) (mM) (mg/kg odp) Hap 4,5 0/100 60 120- 90- 80- 0 60 180 100 100 Control (Hap) 4,5 0/100 60 120- 90- 80- 0 0 180 100 100 Hap/DABCO 4,5 0/100 60 120- 90- 80- 6 60 180 100 100 Control/DABCO 4,5 0/100 60 120- 90- 80- 6 0 180 100 100

3.2.2 D-stage in medium consistency Pulp of known dry matter content was weighed into plastic bags. dH2O that had been brought to the desired pH with H2SO4 was added until the desired volume, minus the volume of the ClO2 and extra H2SO4, was reached. pH was measured and further adjusted by adding H2SO4 until the supernatant had the desired pH. After 15 to 30 minutes of equilibration time, the process was repeated until the pulp stayed at the desired pH. dH2O was added to reach exactly the desired volume minus the volume of ClO2. The plastic bag was sealed thermally and a small hole was cut into it. Through this hole, ClO2 was added, the pulp was mixed, and the bag was sealed thermally again. The pulp was placed in the water bath for the scheduled incubation time. After the incubation time, the pulp was either put in an ice-water bath to cool down and thereafter filtered, or filtered directly. The conditions of the D-stage were:

Table 2: Conditions of D0 stages at 10% consistency Treatment pH T t ClO2 (°C) (min) (% odp)

D0 2,5 85 120 0,6/1,14 0,3/0,55/1,3

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3.2.3 Ep stage in medium consistency Pulp of known dry matter content was weighed into plastic bags. dH2O was added until the desired volume, minus the volume of the NaOH and H2O2 solutions, was reached. The bag was placed into the water bath until the desired temperature was reached. NaOH solution was added and the pulp was mixed by hand. H2O2 solution was added, the pulp was mixed again and the bag was thermally sealed. The Pulp was placed in the water bath for the scheduled incubation time. After the incubation time, the pulp was either put in an ice-water bath to cool down and thereafter filtered, or filtered directly.

Table 3: Conditions of Ep stages at 10% consistency Treatment T t NaOH H2O2 (°C) (min) (% odp (% odp)

Ep 70 80 1,2 0,3

3.2.4 A-stage in medium consistency Pulp of known dry matter content was weighed into plastic bags. dH2O that had been brought to the desired pH with H2SO4 was added until the desired volume, minus the volume of extra

H2SO4, was reached. pH was measured and further adjusted by adding H2SO4 until the supernatant had the desired pH. The bag was thermally sealed and placed in the water bath for the scheduled incubation time. After the incubation time, the pulp was either put in an ice-water bath to cool down and thereafter filtered, or filtered directly. The specific conditions for the A stage were the following:

Table 4: Conditions of A-stages at 10% consistency pH T t Treatment (°C) (min)

A1 3,5 90 120 A2 3 90 120 A3 3,5 95 120

3.2.5 Filtering the pulp After the incubation time, the pulp was either put an ice-water bath to cool down and thereafter filtered, or filtered directly in plastic Buchner funnels with 40µM mesh inside and a plastic glove

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Materials and Methods on top to ensure proper dewatering. The filtrate was collected. The pulp was then washed three times with 1L warm tap water and once with 1L deionized water. In case of large samples, the washing was done with 2L. The pulp was transferred into plastic bags and crumbled by hand to ensure an equal distribution of moisture. After overnight equilibration at 5C, the DM% could be measured.

3.2.6 Handsheet making 4g of odp were weighed, suspended in 2L of dH2O and disintegrated in a Pulp Disintegrator with 10000 revolutions. The fully desintegrated was adjusted to pH 5 with NaOH and/or H2SO4. The suspension was divided into two times 1L, and filtrated Buchner funnel fitted with a filter paper, resulting in two 200g/m2 handsheets. The sheets were placed between four layers of blotter paper and pressed at 300kpA for 2 min 30s, afterwards left to air-dry without the blotter papers in the climate room overnight. The following day, the sheets were pressed again to flatten out any irregularities caused by drying and then left to equilibrate for one hour in the climate room.

3.2.7 Handsheet brightness measuring For the measurement of ISO brightness, the sheets were stacked and the Technidyne Color Touch PC with standard aperture was used to perform three measurements per handsheet. The brightness measurement corresponded to ISO 2470-2:2008.

3.2.8 Measurement of dry matter content For the measurement of dry matter, it was made sure that the pulp was fully crumbled and equilibrated for a long enough time, so that the moisture content was even in the container. Then, about 0,5 to 1,0g odp were placed into the moisture analyzer and the drying program was started.

3.2.9 Preparation of chlorine dioxide Chlorine dioxide was generated by reacting sodium chlorite solution with 1M sulfuric acid in a nitrogen atmosphere. The generated gas was bubbled through ice-cold MQ and thereby solubilized, giving a concentration of 5-8g/L.

3.2.10 Titration of chlorine dioxide, sodium hypochlorite and hydrogen peroxide Before use, concentration of chlorine dioxide, sodium hypochlorite and hydrogen peroxide was determined by iodometric titration with sodium thiosulfate using a Tim900 Titration Manager System.

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3.2.11 Kappa number Determination of Kappa number was carried out according to ISO 302:2004 DL53 Titrator System. Double determinations were done for each sample.

3.2.12 Miniscale assay 60mg of pulp with a known dry matter content was weighed directly into screw-top 15ml glass tubes with an error tolerance of +/- 2,5%. The pulp containing glass tubes were sealed with the screw cap and either used immediately, or stored e.g. overnight. The glass tubes were placed in a combined heating and stirring unit controlled by a Holm &

Halby Thermo Module 40ST. For an enzyme reaction, all reaction compounds except for H2O2 and enzyme were added. For reactions with NaOCl, all reaction compounds, except for NaOCl, were added. Cross-shaped magnets were added to the tubes and they were stirred for 30 minutes at room temperature to ensure good fiber swelling and mixing. After this, the heating was switched on. As soon as the desired temperature was achieved in a separate glass tube filled with water, the remaining compounds were added. In the case of enzymes, this was first the enzyme, then H2O2. Each tube was taken out of the heating block, vortexed for a few seconds and then put back. After the reaction time, tubes were taken out the same order and timeframe to ensure equal treatment durations. The tubes were placed in an ice-water bath to stop the reactions. The magnets were removed and the tubes were centrifuged for 5 minutes at 5000g. The supernatants were removed by decanting and kept on ice for further measurements. MQ was added to the remaining pulp, the tubes were vigorously shaken and centrifuged again. This washing step was repeated 3x for each sample. After the last washing step, the pulp was either transferred to handsheet making immediately, or stored overnight at 4C. Triplicates of each experiment were performed.

Table 5: Miniscale assay general conditions. Variable conditions are referred to in the Results and Discussion section. pH Buffer T t H2O2 NaCl DABCO Hap (mM) (°C) (min) (mM) (mM) (mM) (mg/mL)

4 – 0 - 60 120 100 60 - 6 0,00666 7,5 100 100

3.2.13 Mini handsheet making 200g/m2 handsheets with a diameter of 2cm were made by suspending the pulp in about 50ml dH2O in a special sheetmaking device constructed from a PET bottle and 40µM nylon mesh.

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Vacuum suction was applied and the sheet was formed immediately. It was transferred from the device to a sheet of blotter paper. As soon as there were 12 sheets on the blotter paper, they were pressed for 5m 30s at 350kPa with blotter paper to absorb the moisture. After the pressing, the blotter papers were exchanged for new ones, except for the one that the sheets were placed on. The stacks were transferred to the climate room and they were dried overnight with a fan on to ensure equilibration with the surrounding air. The next day, the sheets were pressed again at 350kPa for 2min 30s to counter any irregularities caused by drying. The blotter papers (except for the one that the sheets were lying on) were removed and the sheets were left to dry in the climate room for one to four hours before measurement. One sheet was made per sample and one measurement performed on it.

3.2.14 Brightness of mini sheets For the mini sheets, the Technidyne ColorTouch PC was equipped with 7,5mm aperture and optics supplied by the manufacturer. The sheets were stacked and the side in contact with the blotter paper was measured. Conversion of brightness values Since a change of aperture and optics requires a re-calibration of the instrument to give an output that fits the ISO brightness scale, it was decided to manually measure a calibration curve for the sake of convenience. Several handsheets and manufacturer-provided calibration samples were measured with the standard aperture that the instrument was calibrated for as well as with the 7,5mm aperture. A linear regression was made and the obtained equation was used to convert the output to the corresponding “ISO” brightness. The quotation marks indicate that the value is not actual ISO brightness, since it is not in compliance with the official ISO document. The obtained equation (R² = 0,99997) was “퐼푆푂” 푏푟𝑖𝑔ℎ푡푛푒푠푠 = 1,13802 ∗ (7,5푚푚 푏푟𝑖𝑔ℎ푡푛푒푠푠 표푢푡푝푢푡) − 14,5117

100

90

80 70 60 50 y = 1,13802x - 14,51179 40 R² = 0,99997 30

20 output (ISO (ISO outputbrightness %)

Standardaperature brightness 10 0 40 50 60 70 80 90 100 7,5mm aperature brightness output

Figure 11: Correlation between brightness measurements made with standard aperture (Y-axis) versus 7,5mm aperture (X-axis) and correspondent optical 36/79 components. The regression was made with Excel. Materials and Methods

3.2.15 Hexenuronic acid content of pulp The HexA content of the pulp was assessed with a modified HUT method originally developed by (Vuorinen et al., 1999). The fundamental difference is the lack of nitrogen atmosphere during hydrolysis. 2 to 2,5g odp were weighed and transferred into 200mL stainless-steel labomat vessels. 150mL of 10mM sodium formate buffer pH 3,5 were added and the vessels were sealed. The vessels were placed in the labomat, heated up and kept at 110°C for 60 minutes. After the hydrolysis time, the vessels were placed in a water-ice bath to cool down and settle. The supernatant was filtered using a 45µm filter and diluted 10x with MQ. Absorbance was measured at 245nm and 480nm. The HexA concentration was calculated as follows: 푛 퐶 푉 퐴 푉 (0,15 + 10−3퐶퐹) 퐴 푯풆풙푨 (풎풎풐풍⁄풌품 풐풅풑) = 10−3 ( 퐻푒푥퐴 = 퐻푒푥퐴 = ) = × 퐷퐹 푤 푤 휀 푙 푤 8,7 푤 −ퟐ (ퟏ, ퟓ + ퟏퟎ 푪푭)(푨ퟐퟒퟓ − 푨ퟒퟖퟎ) = ퟖ, ퟕ 풘 w = weight of oven-dry pulp sample (kg); V = 150 mL + CF; CF = correction factor accounting for the amount of water in the pulp (mL); DF = dilution factor = 10; A = absorbance at 245 nm (2-furoic acid) with background correction at 480 nm; ε = 8,7 mM-1cm-1 – molar absorption coefficient of 2-furoic acid at 245 nm with respect to HexA in hexenuronoxylo-oligosacharides; l = 1 cm – cell path length.

After the first determinations, it became clear that there was a source of bias in the procedure, since there were big differences in the controls. After some investigation, it was found that it was the rotation settings of the labomat that led to unequal heating and therefore unequal and incomplete hydrolysis of the samples. Based on untreated samples run with these rotation settings, correction factors were calculated and applied to the data already recorded (Experiment A). For the new treatment, the rotation settings were adjusted to ensure equal hydrolysis. For more detailed explanation, see appendix.

3.2.16 AOX AOX was measured with the AOX spectroquant cell test by Merck. The samples were diluted 10 or 100 times to fall into the measurement range of 0,05 to 2,5 mg/L. They were than adsorbed to activated carbon (analogous to EN ISO 9562), washed, and digested by a non-disclosed proprietary reaction. The resulting halides were determined photometrically. As a standard, p- chlorophenol was used.

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Materials and Methods

3.2.17 OX OX measurement was performed according to SCAN-CM 52 by MoRe Research Lab, Örnsköldsvik, Sweden.

3.2.18 Viscosity measurement Viscosity measurement was carried out according to TAPPI T230 om-94. The pulp sample was solubilized in 0,5M cupriethylenediamine and viscosity was measured in a capillary viscometer. The measurements were carried out in duplicates.

3.2.19 Model compound studies Reactions were carried out in a volume of 1ml in LC vials at room temperature, using 10mM sodium acetate buffer pH 4,5 and 10% acetonitrile. Model compound, hydrogen peroxide and NaCl concentrations were 1mM, DABCO concentration was 0,1mM. For GC-MS analysis, the reaction products were freeze-dried, re-solubilized in pyridine and derivatized with an excess of BSTFA + TMCS 99:1.

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Results and Discussion 4 Results and Discussion 4.1 Model compound experiments Dihydrodxybenzoquinone (DHBQ) and Sinapaldeyhde (SAL) were treated with Hap and Hap/DABCO and identification and quantification of the compounds was attempted using LC- MS and GC-MS. We hoped that the results would give us an indication of the reactions taking place as well as the role of DABCO. We expected chlorinations, oxidations and possibly cleavage of the model compounds, as reported earlier (Ortiz-Bermúdez et al., 2003).

Figure 12: The structure of Figure 13: The structure of sinapaldehyde (3-(4- dihydroxybenzoquinone (2,5- Hydroxy-3,5-dimethoxyphenyl)prop-2-enal) dihydroxycyclohexa-2,5-diene-1,4- dione)

DHBQ is considered a carbohydrate-derived chromophore found in kraft pulps that gives rise to a multitude of other chromophores and is resistant to degradation because of its resonance- stabilized structure (Rosenau et al., 2007). SAL was chosen on the grounds that it could be produced in hardwood kraft pulping in the way that coniferyl aldehyde is and because there would be several different reactions possible, such as chlorohydrin formation on the double bond, oxidation of the aldehyde to an acid, de-methoxylation, as well as chlorination or hydroxylation of the aromatic ring.

A general observation was that reactions with DABCO were notably faster, noticeable by the faster color change as well as by the depletion of the original compound when analyzed by LC- MS. Another observation was that, at the same amount of depletion of the original compound, product patterns, according to chromatograms and ion spectra did not seem different in the Hap and Hap/DABCO stages. Lastly, gas formation could be observed on the surfaces of the reaction vessels when using higher concentrations of hydrogen peroxide. This can be attributed to the oxygen forming reaction between hydrogen peroxide and hypochlorous acid. 39/79

Results and Discussion

The experiments with SAL led to depletion of the original compound. It was expected to find chlorinated, oxidized and fragmented derivatives of the original compound. Instead, a red precipitate was formed that was insoluble in water and slightly soluble in different organic solvents. The LC-MS results showed a multitude of partly overlapping peaks with high masses. None of the peaks could be identified, and also when using it in GC-MS, no peak could be identified except for benzaldehyde, which possible was an artifact. Sinapic acid, of which a standard was available, could not be found. Treating sinapic acid with Hap/DABCO lead to a similar result in terms of precipitate formation and multitude of peaks. It was hypothesized that a radical polymerization could be taking place, but using the radical scavengers mannitol and ascorbic acid, the former did not influence the reaction and the latter was oxidized instead of the substrate. No further conclusions could be drawn from the results.

DHBQ experiments proved to show better results: Both chloranilic acid and tetrahydroxybenzoquinone, of which there were standards available, were found, meaning that, in accordance with what was expected, both chlorination and oxidation were taking place.

For both compounds, there were significant problems with product solubilization and proper separation on different LC columns, making a reliable quantification difficult. This in conjunction with extended equipment downtime was an incentive for focusing on the application side of the project.

4.2 Pulp: Experimental Rationale and Initial Experiments The initial approach was to replicate the previously documented effects observed in Novozymes with another pulp and carry out an optimization study to understand how far they could be taken in medium consistency, while measuring important outcomes like brightness and HexA content. The very first experiments did not yield any results in brightness or HexA (data not shown), since the pulp used had an abnormally high amount of carryover due to the fact that the mill supplying the pulp was operating with a very closed filtrate recirculation system. After thoroughly washing the pulp with tap water, the results could be replicated (data not shown). It was decided to continue the experiments with NaCl and not NH4Cl, since it was the more realistic choice for actual mill applications. An experiment was conducted to measure hydrogen peroxide consumption along time and check whether another dose of peroxide would be needed in case it was depleted. The experiment conducted at 45°C and 60°C, at dosages 6mM and 25mM of H2O2/NaCl, with and without DABCO addition showed that peroxide was depleted faster at 60°C and with DABCO

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Results and Discussion addition when compared to 45°C and without DABCO. In addition, a higher dosage led to lower HexA and higher brightness (data not shown).

Another observation was the production of gas in the airtight bags in which the treatment was done. This was assumed to be oxygen created by the reaction of HOCl and H2O2, which is an undesired side reaction of the available H2O2:

− − Enzyme reaction: 푋 + 퐻2푂2 → 퐻푂푋 + 푂퐻 (2) − + 1 Spontaneous reaction: 퐻푂푋 + 퐻2푂2 → 퐻2푂 + 푋 + 퐻 + 푂2 (4) 1 Net reaction: 2 퐻2푂2 → 2 퐻2푂+ 푂2 (5)

The following experiment sought to take the hydrogen peroxide and NaCl dosages even higher (90mM, experiment A) and measure the development of brightness and HexA along the way. After establishing that the enzyme was indeed active at very high dosages of peroxide, the experiment B was carried out with 100mM hydrogen peroxide and NaCl. This experiment explored the role of Hap/DABCO stage in combination with D0 stages and Ep stages while comparing its performance to A stages as the benchmark. Pulp viscosity was measured to assess cellulose integrity. All experiments until this point were done using buffer at pH 4,5.

The next step was the optimization of pH and NaCl dosage at low consistency, as well as the observation of pH development during the unbuffered reaction. The catalytic bleaching experiments (as conducted at Aalto University) were replicated and modified in order to gain insights into the underlying bleaching mechanism and understand the pros and cons that both approaches have (enzymatic HOCl generation vs. NaOCl usage).

Lastly, under optimized and non-buffered conditions (starting pH 5, reduced NaCl dosage) a medium consistency assay was carried out, measuring multiple outcomes such as brightness, HexA, kappa number and chlorination. A sample using simple HOCl addition was also generated for comparison.

In this following section, the low consistency assays (pH optimum, NaCl dosage, catalytic bleaching experiments) will be presented and discussed first. After that, the medium consistency experiments will be presented and discussed.

4.3 Miniscale experiments In order to optimize the conditions of the reaction, miniscale experiments were carried out using 60mg of pulp at a consistency of 1,5%. The miniscale assay allows a rapid testing of different

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Results and Discussion conditions that would take a much longer time in larger-scale experiments at medium consistency. A test run was carried out to test for variability and correlation between the both pulp consistencies (low at 1.5% vs. medium at 10%). An acceptable standard deviation and similar results from both types of trials were found. The measurement of the very small handsheets required a change in aperture of the Technidyne ColorTouch PC, leading to a change in brightness values obtained. Instead of calibrating the Technidyne ColorTouch PC every time the aperture was changed, a calibration curve was made using a set of handsheets with different brightness values. The equation of an almost perfect linear regression was used to calculate “ISO” brightness from the obtained values (as described in the Methods section).

4.3.1 Optimum reaction pH with DABCO In order to test for the pH optimum of the reaction with respect to bleaching, buffers with pH from 4,0 to 7,5 were prepared. In the lower range (4,0; 4,5; 5,0; 5,5; 6,0), acetate buffer was used, and in the higher range (6,0; 6,5; 7,0; 7,5), phosphate buffer was used. Since the addition of 6 mM DABCO shifted the pH of the solution in spite of the buffer, and since this shift was different depending on the pH of the buffer, it was chosen to measure the pH at the end of the reaction. This brings the advantage of knowing the exact pH, but makes the data less comparable. As can be seen from the controls, the reaction itself did not shift the pH significantly.

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Results and Discussion

13,9 14,0 Hap/DABCO, 12,0 acetate 10,1 Hap/DABCO, 9,7 10,0 phosphate Hap, acetate

8,0 Hap, phosphate 6,1 5,6 6,0 5,0 4,6 5,1

"ISO" "ISO" brightness(%) 4,9 3,7 4,4 4,0

1,8 3,1 1,6 2,0 1,2 1,0 1,2 0,0 0,7 3,5 4,0 4,5 5,0 5,5 6,0 6,5 7,0 7,5 8,0 pH

Figure 14: Brightness gain (controls subtracted) of Hap/DABCO and Hap treated samples with 100mM acetate and phosphate buffer at 100mM hydrogen peroxide and NaCl,1,5% consistency. Error bars represent the standard deviation of the difference of the means.

As can be seen in Fig. 12, in the Hap/DABCO reaction, there was a steep slope with a peak at pH 5,19. Above that, the brightness decreased. Interestingly, the type of buffer seemed to have an effect: the acetate buffer lead to a higher brightness gain than the phosphate buffer at pH 6 or above. When looking at the brightness gain instead of the absolute brightness, however, this difference between buffers could only be seen at pH 6. A minimum was seen at pH 7 and 7,5. Generally, the brightness in the controls increased as pH increased.

In the Hap reaction (without DABCO), the brightness of the controls also increased with pH, but no buffer effect could be observed. When looking at the brightness gain, the increase was less steep than with DABCO, and a maximum could be observed at pH 5. At pH 5,5 and 6, the brightness gain was almost the same, but It decreased rapidly above that until a minimum was found at pH 7,5.

The most striking difference between Hap and Hap/DABCO is the much higher increase in brightness, showing the impact of the DABCO treatment. In the Hap treatment, there is a 43/79

Results and Discussion difference of 0,6% between pH 5 and pH 6, while in the Hap/DABCO sample the brightness difference between pH 5,19 and 5,94 is almost 4%. This indicates that while the effect of the enzyme alone is somewhat stable in the range of pH 5 to 6, the efficiency of the reaction with DABCO is highly dependent on pH with a peak around 5,0 – 5,5. This is curious, since data from catalytic bleaching experiments shows that at an initial pH of 5 to 6, there was less delignification and HexA removal than at initial pH ≤4 and ≥6 (Chenna et al., 2016). This difference could be due to the pH rapidly changing in the catalytic bleaching setup. A possible explanation could be that the enzyme is inhibited by its product. While the right pH optimizes the enzymatic catalysis and increases the theoretically possible rate of enzymatic reaction, the actual increase in brightness is not that pronounced, since there is an accumulation of HOCl in the liquid phase, leading to inhibition. If DABCO is added (even if the pH is not optimal for the reaction of DABCO+Cl with the pulp), the speed of HOCl removal is sharply increased, leading to less HOCl in the liquid phase and thus, less inhibition. Since there is more pulp and therefore substrate present to react with in medium consistency than in low consistency, the effect could be weaker in the former.

The increase in brightness of the controls could be due to increased presence of the HOO- ion, which reacts with lignin and bleaches it. It could, however, also be a direct effect of pH on the brightness (leaching of low molecular weight chromophore compounds). It is unclear why this effect seems to be more pronounced with acetate buffer than with phosphate buffer. It has been recommended not to use phosphate buffer with VHPOs, since the phosphate can replace the vanadate and thereby inhibit the enzyme (Faber et al., 2015). Except for one data point (Hap/DABCO, phosphate buffer pH 6,14), this alleged effect could not be observed.

4.3.2 NaCl dosage optimum It has been reported that high amounts of chloride can inhibit the enzyme (Van Schijndel et al., 1994), so the reduction of NaCl dosage was tried in an experiment. It was also assumed that the bulk of oxidized chloride would be in turn reduced back to chloride ions again, making this effect translatable to medium consistency.

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Results and Discussion

15,0

14,5

14,5

14,0 13,9 13,7

13,5

13,1

13,0

"ISO" "ISO" brightness(%) gain

12,5

12,0 100 80 60 40 NaCl mM

Figure 15: Brightness gain (controls subtracted) after Hap/DABCO treatment with different concentrations of NaCl, with 100mM acetate buffer pH 5, 100mM H2O2. Error bars represent the standard deviation of the difference of the means.

As is evident from Fig. 13, from 40 to 80mM, the brightness gain increased, while it was below that level at 100mM. This observation lends itself to the conclusion that the high NaCl concentration inhibited the enzyme, as has been observed for the VCPO from C. inaequalis (Van Schijndel et al., 1994).

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Results and Discussion

4.3.2.1 Other dosage experiments [confidential] This part of the thesis has been removed from the publicly available version for confidentiality reasons.

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Results and Discussion

4.3.3 pH shift in non-buffered system (Chenna et al., 2013) noticed an increase in pH during the reaction that they ascribe to the formation of the quaternary chlorammonium cation, since the reaction of Cl+ leaves an OH- in solution (Eq. 5):

퐷퐴퐵퐶푂 + 퐻푂퐶푙 → 퐷퐴퐵퐶푂+퐶푙 + 푂퐻− (5)

In addition to that, Cl+ that adds to lignin/HexA or that is reduced to Cl- is incapable of neutralizing the generated OH-. However, aromatic substitution of H+ and chlorohydrin formation + should be able to lower the pH due to release of H . In ClO2 bleaching, pH drops due to formation of muconic acids and small organic acids. This reaction is not a well-documented reaction of HOCl with lignin though.

A pH shift of the magnitude reported by Chenna et al. (2013), from pH 5 to pH 10, in a matter of seconds, would be detrimental to enzymatic activity. However, also a smaller shift could impair bleaching efficiency considerably, as suggested by the pH optimum experiments. Keeping an acceptable pH in the unbuffered system is of paramount importance, since buffers are not an option in a pulp mill. To assess the impact of this effect, an experiment was conducted to test the pH shift along time. It was an upscaling of the miniscale experiment by a factor of 20 at the same consistency and performed in beakers heated and stirred in a water bath. The pH was adjusted to 4,5 before the start to account for the expected rise in pH.

The unprocessed pH measurement data showed that there were big differences in the behavior of Hap/DABCO vs. Hap. However, since there were big fluctuations in pH that could be observed in the samples as well as in all the controls, it was very difficult to draw any conclusions from this data. It was concluded that these fluctuations were due to equipment problems and could be disregarded in the interpretation of the data. Therefore, to obtain smoother curves (Fig. 14), the data values of the “pulp and MQ” control were subtracted from the rest of the data set, thereby eliminating the fluctuation and centering the data around the X- axis. Then, the respective starting pH of each sample was re-added to the data set. The original curves can be seen in the appendix.

The relative differences to the baseline showed an interesting effect: Over the course of the two hour reaction, the pH decreased by 0,3 and 0,5 pH units in the two controls containing DABCO (black and blue lines). This effect could also be seen in the Hap/DABCO reaction (green line).

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Results and Discussion

The Hap/DABCO sample showed a steep increase from pH 4,5 up to pH >5,5 in the first 15 minutes. Thereafter, it fell again, being at 5,35 at the end of the reaction time. The Hap curve (purple) showed a high starting pH of 5 (due to insufficient pH adjustment) and a slow increase that did not seem to be finished at the end of the reaction, where it reached pH 5,3. The brightness gains versus the control were 11,7% and 6,3% for DABCO/Hap and Hap, respectively.

6

5,5

5 DABCO H2O2 Hap

DABCO H2O2

4,5 DABCO Hap pH H2O2 Hap H2O2 4

3,5

3 0 20 40 60 80 100 120 incubation time (minutes)

Figure 16: Development of the non-buffered pH of different pulp samples and controls at 1,5% consistency over the course of two hours. H2O2 (if applicable) was 100mM. All the samples contained 100mM NaCl.

For both reactions, the pH stayed in a zone where the enzyme was active and showed a bleaching effect according to the previous experiments. The Hap/DABCO reaction, however, did not stay in the optimal region of around pH 5. This fits with the fact that the obtained brightness was significantly lower than in the same experiment carried out using buffer at pH 5. Assuming that the reactions of HOCl and DABCO+Cl are of the same nature, but of a different speed, the approximately linear Hap curve could correspond to the linear phase (first 15 minutes) of the Hap/DABCO curve. This fits well with the difference in brightness gain between Hap/DABCO and Hap. This could imply that the flattening of the Hap/DABCO pH curve corresponds to a flattening of a “bleaching curve”, and that pH increase is directly related to bleaching effect. Further experiments are necessary to confirm this hypothesis.

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Results and Discussion

The decrease in pH in the Hap/DABCO sample seemed to be caused by DABCO, as it could also be observed in the DABCO-containing controls. The reason for this decrease is unclear, but a breakdown of DABCO into less basic compounds is a possibility. This breakdown would, however, not be related to the formation of the quaternary chlorammonium cation, since the pH decreased at the same rate in both Hap/DABCO and controls. Another option would be the reaction of DABCO with compounds in the pulp, which is likely since DABCO is a highly nucleophile catalyst used in a variety of reactions. When taking this possibility into account, the question arises whether the reason behind the increased bleaching effect of Hap/DABCO vs Hap could be the action of DABCO on the pulp enhancing bleaching response to HOCl, and not the quaternary chlorammonium cation itself. While the former cannot be fully excluded, it seems unlikely due to the well-documented effect of DABCO+Cl and the fact that it is formed in a matter of milliseconds, while the potential reaction of DABCO with the pulp seems to proceed slowly over time.

If it truly is the bleaching reactions that cause the rise in pH, then this effect should be more pronounced in medium consistency, since the substrate (pulp) concentration rises from 1,5% to 10%.

4.3.4 Replication and modification of catalytic bleaching experiments It was of interest to compare the catalytic bleaching experiments as done at Aalto university with the Hap/DABCO system. A first experiment was conducted that employed the conditions used in the enzyme trials (60°C, 2h, 1,5% consistency, buffers ranging from pH 4 to 7,5) with an NaOCl dosage of 3,3% odp active chlorine and a DABCO dosage of 0,33% odp – so the three- fold of the original experiments. The achieved brightness with NaOCl alone was up to 70%, but the NaOCl/DABCO samples showed a surprisingly much lower brightness – only around 60% (data not shown)

Since the positive effect of DABCO could not be found, a second experiment was sought to exactly replicate the catalytic bleaching experiments as conducted by Chenna et al. (2013), and to explore the effect of modifying the conditions. The temperature and time were reduced to 30C and 10 minutes, respectively, and the consistency was reduced to 1%. The NaOCl and DABCO dosages were reduced to 1% active chlorine odp and 0,1% odp, respectively. The pulp suspension was adjusted to pH 5 before the addition of NaOCl and no buffer was used. To check for the effect of variables, one sample was incubated for 1h instead of 10 minutes, another sample received 100mM acetate buffer pH 5, and yet another one received three times the dosage of both NaOCl and DABCO. The achieved brightness values can be seen in Figure 12.

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Results and Discussion

Brightness reached with NaOCl/DABCO 65,0 61,8

60,0

55,2 55,0

50,8 50,0 47,8 48,2 46,6 "ISO" "ISO" brightness(%) 45,8 45,2 45,0

41,1 40,6 41,2 40,8

40,0

NaOCl

DABCO

NaOCl1h

buffer

DABCO1h

3x dosage 3x

NaOCl buffer NaOCl

DABCObuffer

NAOClDABCO NAOClDABCO NAOClDABCO

NaOCl3x dosage

NAOClDABCO 1h DABCO3x dosage

Figure 17: “ISO” brightness (%) reached with different variations of the NaOCl/DABCO system. NaOCl and DABCO: 1% active chlorine odp and 0,1% odp, or threefold in the 3x sample. The duration of each treatment was 10 minutes, except for the 1h experiment. Error bars represent the standard deviation.

The exact replica showed a slight effect of DABCO addition. It could be observed with the naked eye that the brightness difference was bigger directly after the end of the incubation than after centrifugation and washing. The reaction most likely continued when it was cooled down and centrifuged. The sample incubated for 1h instead of 10 minutes showed a pronounced negative effect of DABCO addition, leading to 2,5% less brightness. The sample with buffer also showed a slightly negative effect of DABCO, but the effect was minimal. The sample receiving the three-fold dose of NaOCl and DABCO showed a clear 6% advantage of NaOCl/DABCO.

The 1h sample could explain why the first experiment showed a negative effect of DABCO – the incubation time was too long. Chenna et al. (2013) showed that at starting pH 8 and a dose of 1% odp active chlorine, 0,1% odp active chlorine remained after 10 minutes of treatment. Assuming that this was also the case at a starting pH of 5, it could be assumed that these last 0,1% were completely consumed after 1h both in the NaOCl and in the NaOCl/DABCO sample. However, since DABCO can also be oxidized and degraded by HOCl (Rosenblatt et al., 1972), some oxidation power was lost on DABCO. This fits with the assumption that DABCO does not change the substrate of HOCl, but just speeds up the reaction.

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Results and Discussion

The effect of the buffer – it negated the positive effect of the DABCO – could be related to DABCO protonation. In the NaOCl/DABCO samples without buffer, end pH was significantly higher, around 9. This elevated pH was also observed by Chenna et al. (2013) and explained by formation of the quaternary chlorammonium cation, which leaves OH- in solution. This rise in pH would lead to less DABCO protonation and therefore higher availability to form the quaternary chlorammonium cation. This availability was shown to be an important factor for efficiency of catalysis (Prutz, 1998). If the pH is kept low with a buffer, significantly less DABCO is available, and therefore the reaction proceeds slower. The reason why DABCO still showed a big effect at low pH in the Hap/DABCO experiments could be that the dose was much higher, so the fraction available was still sufficient. The brightness achieved in the buffered NaOCl sample was also slightly lower than the one in the non-buffered sample. This difference could be a result of experimental error, but an effect of the buffer on the reaction cannot be excluded.

4.4 Medium consistency experiments 4.4.1 Experiment A: First high dosage trial and kinetics of brightness gain and HexA degradation An experiment was carried out to test how the pulp responded to a very high dosage (90mM) of

H2O2/NaCl, as well as to test whether a stepwise addition of H2O2/NaCl would show an advantage over one-time dosing of the same dose (90mM in 9x equivalent of 10mM). The latter was deemed reasonable, since a lower H2O2 concentration would lead to less consumption of

H2O2 in reaction with HOCl, and therefore less waste of H2O2. The concern that a lower concentration would lead to a much lower reaction speed was considered negligible, since previous experiments showed that the complete consumption of 6mM H2O2 happened within the first 15 minutes of the reaction, indicating a high affinity for H2O2. In the literature, a very small

Km of 1 µM for H2O2 had been found for the VCPO from the close relative species C. inaequalis (Van Schijndel et al., 1994). Time-dependent profiles of brightness evolution (bleaching reactions) (Fig. 17 and 19) and HexA degradation (Fig. 16 and 18) were obtained to get a kinetic picture of these key reactions.

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Results and Discussion

45 42,2 40,3 39,3 40,1 40

35

30

25 22,0

20 15,6 15 11,9 10 5,1 HexA (mmol / kg odp) kg / (mmol HexA 5 3,4

0 30min 60min 120min 180min 30min 45min 60min 120min 180min

Figure 18: HexA content over time after one-time addition of 90mM H2O2 and NaCl. 100mM acetate buffer pH 4,5. Black: Control; Grey: Treated Error bars represent the standard deviation.

60 58 57,3 56,1 56

54 52,2 52 50,9 50 48

ISO brightnes (%) brightnes ISO 46

44 42,8 42,8 41,7 42 40 30 45 60 120 180 Minutes of incubation

Figure 19: ISO brightness (%) over time after one-time addition of 90mM H2O2 and NaCl. 100mM acetate buffer pH 4,5.Black: Control; Grey: Treated. Due to errors during sampling, no sample was taken at 30 minutes, and no control sample was taken at 45 minutes. Error bars represent the standard deviation.

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Results and Discussion

45 40,7 40 38,7 38,9 38,9

35

30

25 22,9

20

15 12,1

10 6,5

HexA (mmol / kg odp) kg / (mmol HexA 4,7 5

0 30min 60min 120min 180min 30min 60min 120min 180min

Figure 20: HexA content over time with stepwise addition of (in total) 90mM H2O2 and NaCl. 100mM acetate buffer pH 4,5. Black: Control; Grey: Treated Error bars represent the standard deviation.

56 54,8 54 52,5

52

50 48,9 48 46,0

46 ISO brightnes (%) brightnes ISO 44 42,3 41,9 42,0 42 41,5

40 30 60 120 180 Minutes of incubation

Figure 21: ISO brightness (%) over time with stepwise addition of (in total) 90mM H2O2 and NaCl. 100mM acetate buffer pH 4,5. Black: Control; Grey: Treated Error bars represent the standard deviation.

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Results and Discussion

It was found that there was virtually no difference in HexA degradation patterns in the 90mM one-time addition vs. continuous addition samples. In both, about half of the HexA was degraded after half an hour (corresponding to a dosage of 30mM in the continuous addition sample). After another 30 minutes, this value was halved again. After 120 minutes, the value was halved again and after 180 minutes, only a minor decrease could be noted. The 90mM one-time addition had the highest brightness gain, with a noticeable advantage seen already at 60 minutes. At the end, the one-time addition sample yielded a 2,5% higher brightness.

Since there was a higher brightness with a one-time addition of H2O2 and no difference in HexA degradation, it was chosen to continue the experiments with one-time additions. This is also the most feasible way to implement such a solution in existing pulp mills. Medium-consistency pulp mills operate by mixing the pulp with the bleaching chemicals, after which the pulp moves in a plug flow, where no further mixing occurs until the next stage. Since there only was a marginal difference in brightness and HexA content when comparing 120 and 180 minutes, it was decided to limit the future trials to 120 minutes.

The reasons for this result can only be speculated about, but could include a direct or indirect effect of high concentrations of hydrogen peroxide on the pulp or the enzyme system.

4.4.2 Experiment B: Incorporation of Hap/DABCO stage into bleaching sequences and comparison with A stages. Assessment of brightness and viscosity In order to compare the Hap/DABCO-stage with the current state of the art, an A-stage followed by a D-stage was conducted alongside the Hap/DABCO stage. The Hap/DABCO stage was evaluated alone, as a pre-bleaching step followed by D0 (0,6% odp) and Ep stages, or as a bleaching stage followed by an Ep stage. A sample of D0-Ep bleached pulp with a higher dosage (1,14% odp) was also included as a reference. Brightness and viscosity were assessed (Fig. 20) and the selectivity parameter was calculated as units of increase in brightness per units of decrease of viscosity (with reference to the untreated pulp; Fig. 21).

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90 82,5 78,9 80 74,7 75,3 74,8 76

69,3 70

57,2 60 54,9

50 44,9 44,6 40,1 41,7 41,9 40 ISO Brightness 36,3 34,9 29,4 28,7 29,8 30 26,0 25,6 26,7 Viscosity

20 ISO ISO Brightness Viscosity or(%) (cP)

10

0

Figure 22: ISO brightness (%; black) and viscosity (cP, grey) after different treatments. Y axis represents both ISO brightness and viscosity. Enzyme treatment: 100mM H2O2 and NaCl, 100mM acetate buffer pH 4,5 120min. Error bars represent the standard deviation.

6,0 5,3

5,0

4,0 3,4

2,7 3,0 2,6 2,2 1,9 2,0 1,8 1,8

1,5 1,5 dViscosity/dBrightness 1,0

0,0

Figure 23: Selectivity of different treatments, calculated as the decrease in viscosity divided by the increase of brightness from the original pulp.

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Results and Discussion

While the three A-D0- Ep samples each reached a brightness of about 75%, comparable to a D0-

Ep sample with 0,76% odp ClO2 (appendix), the Hap/DABCO-D0-Ep sample reached a brightness of 82,5%, clearly showing a superior effect of the Hap/DABCO stage vs. the A stage on bleaching. The Hap/DABCO-D0-Ep sample also showed a 3,5% higher brightness than the reference D0-Ep sample. This is especially interesting considering that the reference sample employed almost twice the amount of ClO2 in the D0 stage. With 69% of brightness, the

Hap/DABCO-Ep sample showed less brightness than the D0 samples, and is comparable with a

D0-Ep sample using 0,38% ClO2 odp (data not shown). The Hap/DABCO sample shows a brightness of 57%.

All the samples showed decrease in viscosity from the untreated pulp, but the viscosity of the A-

D0-Ep samples was the lowest. It was lower than both the reference D0-Ep sample and the

Hap/DABCO-D0-Ep sample. The effect became even clearer when the selectivity was calculated: Per unit of viscosity lost, fewer units of brightness were gained in the A-D0-Ep samples than all the other samples (except for the controls). The Hap/DABCO sample showed the same minor viscosity loss as the control, indicating that no viscosity loss was caused by the enzymatic system. Indeed, its selectivity quotient was by far the highest among all the samples (5,3), followed by the Hap/DABCO-Ep sample (3,4).

The A stage showed the expected effect: Increased bleachability due to HexA removal (HexA was not measured, but the effect is well-documented) shown by the fact that the brightness achieved with A-D0-Ep was on par with that of a D0-Ep reference pulp with a higher chlorine dioxide dosage. At the same time, viscosity was decreased by cellulose hydrolysis.

A concern when using HOCl-based bleaching is the possible cellulose oxidation and subsequent cleavage, leading to viscosity loss. From this experiment, however, it is very clear that under the conditions employed, there was no significant decrease in viscosity in the Hap/DABCO stage compared to the control. As the HOCl-caused cellulose depolymerization in

H stages (Fig. 22) happens under alkaline conditions, one could think that the alkaline Ep stage could potentially act on cellulose modifications inflicted during the Hap/DABCO stage, thereby delaying the cellulose depolymerization to the next stage. The Hap/DABCO-Ep sample, however, did not show a viscosity loss when compared to the Control- Ep sample. Therefore, no cellulose depolymerization was caused by the Hap/DABCO treatment.

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Results and Discussion

Figure 24: Cleavage of the cellulose chain by β-elimination from a carbonyl group (OR = chain parts). This can be caused by HOCl under alkaline conditions. From Sixta, 2008

The results from this experiment show that the Hap/DABCO stage has a clear advantage over the A stage in terms of bleaching and preservation of cellulose integrity.

The selectivity quotients of Hap/DABCO-D0-Ep and of the D0-Ep reference were very close to each other. This, however, must be seen in the light of a 3,5% brightness difference meaning a lot at this high level of brightness. A conventionally bleached pulp would most likely show a lower viscosity at 82,5% brightness.

All in all, the results show that the Hap/DABCO stage leads to very selective delignification and HexA removal.

4.4.3 Experiment C: Non-buffered reaction and comparison with HOCl addition. Assessment of brightness, HexA, kappa number and chlorination. To implement the results obtained from the previous experiments and test the system under realistic conditions, a medium consistency experiment was conducted without buffer.

Hap/DABCO and Hap stages were followed by Ep and D0-Ep and samples were taken after each step. In order to compare the Hap/DABCO system with the simple addition of HOCl, a sample was prepared in which the pH was adjusted to a very low value with H2SO4, so that upon the addition of the alkaline NaOCl solution, the pH would rise to around 5. To accomplish this, a study was done with several different starting pHs and pH 2,5 was found to be appropriate to keep the reaction within the appropriate pH range where HOCl is the dominating species (pH 2 to 7) (Deborde and von Gunten, 2008). A NaOCl/DABCO sample was not included, since DABCO had already been shown to have a negative effect on brightness under these conditions (see Replication and modification of catalytic bleaching experiments). A different, unwashed oxygen-delignified eucalypt kraft pulp (Pulp 2) with a higher starting brightness was tested as well. Three D0-Ep samples were included as a

Table 6: Overview of all samples generated in experiment C First treatment Ep D0 Ep Reference samples samples samples samples

Hap +Ep + D0 – Ep D0 0,3% – Ep

Control (Hap) +Ep + D0 – Ep D0 0,55% – Ep

Hap/DABCO +Ep + D0 – Ep D0 1,3% – Ep

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Results and Discussion

Control/DABCO +Ep + D0 – Ep

Hap/DABCO Pulp 2 +Ep + D0 – Ep Control/DABCO +E + D – E Pulp 2 p 0 p

HOCl +Ep + D0 – Ep

Control (HOCl) +Ep + D0 – Ep

Brightness was measured in all samples; HexA, AOX and OX were assessed in specific samples.

As seen in Fig. 23, the Hap and Hap/DABCO brightness values were about 1% lower than in the low consistency pH experiment under the same conditions. After Ep, the brightness difference compared to the control increased. After D0-Ep, the brightness difference between control and treated pulp decreased. The Hap/DABCO Pulp 2 samples had a higher starting brightness. The brightness gains showed a comparable pattern as with Pulp 1, but the gains were lower. The HOCl sample showed a brightness corresponding to that of the Hap/DABCO sample after the first treatment. The brightness gain as measured compared to the control was higher, since the control was 2,5% darker. The brightness after Ep increased substantially to an 11 point higher level than the Hap/DABCO sample. This advantage could also be seen after D0-Ep, where the brightness gain was twice as big.

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Results and Discussion

90,0 86,2 85,7 84,0 85,0 82,1 80,8 80,0 77,7 76,8 75,6 75,5 74,7 75,3

75,0 73,9

70,0 67,9 67,6 64,9 65,0 63,5 59,8 58,3 60,0 First stage ISO ISO brightness(%) 54,5 54,8 +Ep 55,0 52,2 52,0 49,6 +D0-Ep 50,0 44,4 44,2 44,7 45,0 41,8 40,0

Figure 25: Brightness values of pulps after different first treatments (dark grey), first treatments and Ep (grey), and first treatments followed by D0 and Ep (light grey). Enzyme treatment: non-buffered, 100mM H2O2, 80mM NaCl, 120min. HOCl treatment: initial pH 2,5; NaOCl 2% act. chlorine odp, 60°C, 120min. Error bars represent the standard deviation.

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Results and Discussion

45,0

40,0 38,1 37,5 38,4 34,6 35,1 35,0

30,0 25,6 25,0 22,1 22,4

20,0

HexA HexA (mmol/kg) 15,0 9,9 10,0 6,1 5,0

0,0

Figure 26: HexA content (mmol/kg) of selected samples after different treatments. Error bars represent the standard deviation.

HexA quantification was performed on samples after the first treatment and after Ep as well as of the reference D0-Ep samples (Fig. 24). The Hap and Hap/DABCO samples showed a reduction of about 9% and 41%, respectively, the HOCl sample showed a reduction of 84%.

The D0-Ep reference samples showed a 27%, 36% and 72% reduction when compared to the untreated sample at 0,30%, 0,55% and 1,30% ClO2, respectively. As expected, the samples after Ep did not show a change in HexA levels (not shown).

Kappa number was measured and the contribution of HexA was calculated from the measured amount of HexA, according to the finding that 11,9 mmol/kg of HexA correspond to 1 Kappa unit (Li and Gellerstedt, 1997). The HOCl control sample could not be measured due to an insufficient amount of pulp left.

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Results and Discussion

14,0

Kappa from 10,1 12,0 rest 9,1 9,0

10,0 Kappa from 7,0 7,4 HexA 7,2 6,7 8,0 5,7 5,5 6,0 4,3

4,0 2,2 3,2 2,9 2,9 3,2 3,1 3,2 3,0 2,0 2,1 2,8 1,9 1,7 1,9 0,9 0,0 0,5 0,3

Figure 27: Kappa number composition of pulps after different treatment. Light grey: HexA contribution; Dark grey: Non-HexA contribution (mostly lignin). Error bars represent the standard deviation of the total kappa measurement. Control (HOCl) was not measured due to lack of pulp. For Hap/DABCO, Control/DABCO, HOCl-Ep and D0-Ep (0,55%), only one sample could be measured.

Table 7: Total kappa numbers of pulps after different treatments. untreated 13,1 Hap/DABCO 7,3 Control/DABCO 12,2 HOCl 2,7 Control (HOCl) x

Hap/DABCO-Ep 6,1 Control/DABCO-

Ep 9,8

HOCl-Ep 1,1

Control-Ep 10,4

D0-Ep (0,55%) 4,1

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Results and Discussion

The kappa number measurements (Fig. 25; Table 7) showed that the Hap/DABCO treatment significantly decreased kappa number from about 13 of the untreated sample to 7,3. The HOCl sample showed an even lower kappa with 2,7.

The Ep stage reduced Kappa number by one to three units, but affected almost only the non-

HexA part, which is assumed to be residual lignin for the most part. The D0-Ep reference sample with 0,55% chlorine dioxide charge showed a kappa number of 4.

0,60 0,55

0,50

0,40

0,30 A280 0,21 0,19 0,20 0,13 0,11 0,10 0,10 0,10 0,08

0,00

Figure 28: 280nm absorbance of different filtrates, diluted 1:10 with MQ

After the first treatment, the absorbance at 280 nm (Fig. 26), a common wavelength for aromatic lignin measurement, showed that the Hap and Hap/DABCO treatment resulted in a doubling of the filtrate absorbance when compared to the control. The HOCl treatment, however, resulted in a five-fold increase thereby indicating extensive delignification.

The HOCl stage left the pulp at the same brightness level as the Hap/DABCO stage, but much lower HexA content and lower Kappa, which also fits with the higher A280 of the effluent. The lower Kappa number and HexA content explain why the further bleaching results were so much better for HOCl after Ep and D0-Ep. It is, however, not clear why the brightness level was the same after the first stage. It is evident that in the Hap/DABCO reaction, more bleaching took place with less lignin removal. A reason could be that the chemical reaction with the lignin itself is different in the Hap/DABCO and HOCl reactions. This, however, would contradict the assumption that the

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Results and Discussion reactions of the quaternary chlorammonium cation and free hypochlorous acid are fundamentally the same, unless the active species was different. This could be the case, provided that the pH was sufficiently low to allow for the presence of substantial amounts of molecular chlorine. In the setup used, the pulp was equilibrated to pH 2,5 to counter the alkalinity of the sodium hypochlorite solution, leading to a pH between 3 and 5, changing as the reaction proceeded. The pH was measured in the supernatant by applying pressure to the soaked pulp. It cannot be ruled out that the pH was lower in the water more tightly bound to the fiber, leading to an increased presence of molecular chlorine. This possibility is supported by the common observation that when adjusting pH of pulp by hand, equilibration time is needed. At pH 2,5, however, the equilibrium is still mostly on the side of HOCl (Deborde and von Gunten, 2008).

A more likely explanation is that the amount of hypochlorous acid produced by the Hap did not match the amount of hypochlorous acid added in the HOCl stage, or the hypochlorous acid was neutralized by the hydrogen peroxide before it could react with the pulp. At the same time, the hydrogen peroxide was responsible for bleaching, but not delignification of the pulp.

We could also observe that controls with H2O2 showed a slightly higher brightness (2%) than controls without H2O2 in 1,5% consistency assays (data not shown). This indicates that there was a bleaching effect of the hydrogen peroxide on the untreated pulp. This effect should also be found in the treated samples, but it wouldn’t able to explain the big discrepancy in brightness and lignin removal. It could, however, be possible that the effect was not purely additive, but that there was a synergy: Chromophores were formed by DABCO+Cl in the Hap/DABCO stage, and instantly degraded by hydrogen peroxide. In the HOCl stage, they were also generated, but not degraded. It therefore showed the same brightness while having much lower lignin content.

It is unclear, however, which species of hydrogen peroxide could be responsible for this bleaching effect, since hydrogen peroxide is relatively stable at near-neutral pH. Hydrogen peroxide bleaching reactions take place at high pH (>11), where the highly reactive HOO- species is dominating. At near-neutral conditions, this species is practically non-existent due to its pKa of 11,75 at 25°C.

However, under acidic conditions, non-ionized H2O2 has been shown to be a nucleophile acting on different lignin model compounds with reactivity increasing as pH decreased (Kishimoto et al., 2005).

Another possibility could be the presence of hydroxyl radicals. These can be generated e.g. in a Fenton reaction, a transition metal-catalyzed degradation of hydrogen peroxide. Other possible sources are the temperature-dependent homolytic cleavage of hydrogen peroxide and the reaction of hydrogen peroxide with hypochlorous acid (Castagna et al., 2008). Hydroxyl radicals 63/79

Results and Discussion are known to attack various structures present in pulp, leading to delignification and bleaching, but also to depolymerization of cellulose. Since no drop in viscosity could be noted in the buffer- controlled experiments, it is not assumed that this is the case.

The reaction of hydrogen peroxide and hypochlorous acid produces singlet oxygen, which has been shown to react with lignin model compounds (D’Auria and Ferri, 2003). However, DABCO is known to quench singlet oxygen (Enko et al., 2013).

Other possibilities include other unknown interactions between hypochlorous acid and hydrogen peroxide as well as interactions between DABCO and hydrogen peroxide. Hydrogen peroxide can form a solid complex with DABCO that has been used in organic synthesis, but this complex was merely considered an anhydrous form of hydrogen peroxide by the authors (Cookson et al., 1975). Possible unknown side activities of the enzyme solution cannot be excluded either. The only known side activity is superoxide dismutase.

It was hypothesized that if the increased bleaching effect was based on the removal of chromophores generated by HOCl or DABCO+Cl during the Hap/DABCO stage in a manner independent from HOCl, it should be possible to further increase the brightness of the HOCl- stage pulp by subjecting it to a pseudo Hap/DABCO stage without enzyme. The results (Fig. 27) showed a six percent brightness increase compared to the control that could not be observed in the non-HOCl treated sample. This indicates that, indeed, the brightness and kappa number discrepancy was not due to action of the enzyme or its product, but most likely due to hydrogen peroxide (in some form) or an interaction of hydrogen peroxide and DABCO. Unfortunately, due to time constraints, the cause could not be narrowed down further.

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Results and Discussion

65,0

61,4

60,0

55,7 55,0

50,0 "ISO" "ISO" brightness%

45,0 42,8 42,0

40,0 HOCl - H2O2 HOCl - Control Control - H2O2 Control - Control

Figure 29: “ISO” brightness (%) of pulps after HOCl or Control treatment followed by a pseudo Hap stage without enzymes. The first part of the sample name designates the treatment in the previous experiment (with HOCl or without), the second part designates the treatment in the pseudo Hap stage (with H2O2 or without) Error bars represent the standard deviation.

The big difference in brightness after Ep when comparing Hap/DABCO (+10%) and HOCl (+20%) is also at least partly explained by this hypothesis. In the Hap/DABCO stage, less HOCl created less chromophores, and at the same time, a part of them was degraded. Therefore, the potential for brightness gain in Ep was limited.

There is, however, another factor to consider: The residual hydrogen peroxide in the Ep stage showed no difference in the D0 treated samples. Hap and Hap/DABCO samples showed depletion, except for Pulp 2, which showed a residual both in control and in the treated sample. The HOCl sample showed a residual while the control showed depletion.

While they reached similar brightness values after the first respective stage, the D0 (0,55%),

HOCl and Hap/DABCO samples showed different peroxide consumption: The D0 (0,55%) sample showed a residual of 30mg/L, the HOCl sample 10mg/L, and the Hap/DABCO sample 0mg/L.

After an Ep stage, there should be some hydrogen peroxide left in the effluent to prevent alkaline darkening. A complete degradation is either caused by an insufficient charge, or by the presence of transition metal ions that catalyze hydrogen peroxide decomposition even at low concentrations. Since it seems unlikely that a large amount of structures susceptible to 65/79

Results and Discussion hydrogen peroxide was created in the Hap/DABCO stage, transition metals seem more probable. Therefore, a factor for the difference in brightness between HOCl (+20%) and Hap/DABCO

(+10%) samples after the Ep stage could be hydrogen peroxide decomposition in Ep. Hexenuronic acid is known to complex transition metals such as manganese, iron or copper, and its removal leads to the removal of the complexed transition metals (Vuorinen et al., 1999). Therefore, a pulp that has more HexA removed could be less prone to transition metal catalyzed hydrogen peroxide decomposition in the Ep stage. To assess the extent of its effect, the treatment could be repeated with chelating agents to bind any transition metals present. Due to time constraints, this was not done.

The better delignification in the HOCl stage is also suggested by the much higher absorbance of the HOCl stage filtrate at 280 nm.

2500,0

26,5 2000,0

17,9 1500,0 5,0

1990,3 1000,0 AOX from Ep

AOX AOX odp)(g/ton 1529,4 AOX from first stage 1272,1 500,0 3,9 0 0,0 0,7 0,0 0,0 0 328,8 0,0

Figure 30: AOX composition of selected samples. The contribution from first treatment stage is seen in light grey, the contribution from the Ep stage is shown in dark grey.

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Results and Discussion

450 420

400

350

300

250 250

200 OX OX (g/ton) 150 150 120 120

100 80 80

50 9 9 10 2 8 0

Figure 31: OX results of pulp handsheets after different treatments.

The AOX results (Fig. 28) showed that the bulk of the AOX was produced in the first stage

(Hap/HOCl/D0), while a much smaller contribution was made by the Ep stage.

The total AOX produced by the HOCl treatment was highest, followed by the D0 with 1,3% chlorine dioxide and by Hap/DABCO. The 0,55% D0 treatment showed low AOX, and no AOX could be detected for the 0,3% D0 stage.

The OX results (Fig. 29) also reflect the results obtained from AOX, with the HOCl samples showing the most chlorination. D0-Ep (1,3%) and HOCl samples without Ep were not included in this analysis.

The higher AOX value for the HOCl stage fits with the rest of the observations, since a higher amount of hypochlorous acid would lead to more chlorination. It has been shown that a large amount of the AOX produced in D stages stems from chlorinated hexenuronic acid products. It is assumed that the hexenuronic acid derived AOX corresponds to the “unstable” part of D-stage AOX, which easily degrades by incubating the filtrate at 35°C for 10 days. Lignin-derived stable AOX, which also correlates with OX, is harder to degrade (Björklund et al., 2004).

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Results and Discussion

The OX results mirror the pattern of the AOX results. The high values of Hap/DABCO and HOCl when compared to the D0-Ep samples correspond well to the previous finding that at least 86% of OX in chlorine dioxide bleaching was produced by hypochlorous acid (Ni et al., 1993).

The D0-Ep sample OX and AOX are in the same range as previous results from chlorine dioxide treatment of oxygen-delignified hardwood (Björklund et al., 2004). Pulp bleached with ECF processes can have a residual in the range of 100 g/t to 250 g/t, which fits with these measurements (Suess, 2010).

Adding the AOX results from the HOCl-stage and the following Ep stage and the OX results from

HOCl-Ep pulp, 9,5% of the added HOCl was converted to organically bound chlorine. This number is higher than usual in alkaline hypochlorite bleaching (about 5%) and more like elemental chlorine bleaching (10%) (Suess, 2010). There is, however, no literature data available on HOCl bleaching at acidic pH. Due to the unknown amount of hydrogen peroxide actually reacting with the enzyme, such a percentage cannot be calculated for the Hap/DABCO samples.

As for comparison with the extent of chlorination measured in the catalytic bleaching experiments at Aalto University, 5% to 13% of NaOCl added was converted into AOX or OX. A comparison the sum of OX and AOX divided by the reduction of kappa number for Hap/DABCO, HOCl and the catalytic bleaching stages shows that the numbers are similarly high for Hap/DABCO and HOCl (Table 8). The catalytic bleaching stages, on the other hand, show up to five times less organochlorine formation per decrease in kappa number. The similar numbers for Hap/DABCO and HOCl support the idea that the chemistry of HOCl and DABCO+Cl (or the Hap/DABCO system, for that matter), is the same. Since the absolute Δkappa was different for the samples, the possibility that AOX+OX per Δkappa increased with higher Δkappa was taken into consideration, but no correlation was found.

Table 8: Total organohalogens (AOX+OX) after first stage, divided by the decrease in kappa number (Δkappa). Catalytic bleaching numbers are taken from Chenna et al. (2016) AOX+OX Δkappa

(g/odt) per Starting pH Δkappa 3 81,5 7,4 3,5 48,5 5,2 4 43,8 6,1 4,5 (55°C ) 88,3 7,8

5 (40°C ) 46,4 7,7 Catalytic bleaching Catalytic 6 47,2 6,7 7 88,9 5,5

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Results and Discussion

8,5 97,4 3,4 4,6 10,5 150,7 AOX+OX Δkappa

(g/odt) per Sample Δkappa Hap/DABCO 266,9 5,7

Control/DABCO 10,3 0,9 Hap/DABCO HOCl 258,7* 10,4 Control (HOCl) 1,5 0,5

*OX value of HOCl treated sample was not available and therefore estimated from HOCl-Ep treated sample, based on the reduction in OX seen from Hap/DABCO to Hap/DABCO-Ep.

The reasons for this could lie in the short reaction time (10 minutes) in the catalytic bleaching experiments. As reported before by Chenna et al. (2013), not all hypochlorite is consumed after 10 minutes of reaction. In our reproduction of the experiments, we could also see that the positive effect of DABCO could only be observed after 10 minutes, but not after an hour. It could be the case that this short reaction time is the key to keeping chlorination at bay. It has, however, been shown that 86% of the AOX generated in chlorine dioxide bleaching is produced by hypochlorous acid generated in situ, within the first ten minutes of the reaction (Ni et al., 1993). This leads again to the question whether HOCl and DABCO+Cl truly lead to the same reactions in pulp. More experiments are needed to investigate the relationship between treatment duration and chlorination. In an enzyme-driven reaction, a short reaction time is hard to achieve, since the enzyme needs time to produce adequate amounts of hypochlorous acid and keeps producing it constantly.

The latest best available technique reference document by the European Commission states a yearly AOX average of 0 to 0,2 kg/t air dry pulp as the BAT-associated emission level (BAT- AEL) for kraft pulp mills. Air-dry pulp is defined as 90% dry, which means that for measurements referring to odp, the levels are a bit higher. Even though BAT-AELs function more as guidelines then as strict upper limits, it is important for new permits to stay at or below the BAT-AEL (Suhr et al., 2015).

Kraft pulp mill effluent treatment in aerated lagoons typically reduces AOX content by 20% to 45%, while activated sludge reduces it by 40% to 65% (Suhr et al., 2015).

The sum of AOX for the Hap/DABCO stage is already over the limit (about 1,3kg/odt) and following D-stages would generate even more organochlorine compounds. The use of ozone

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Conclusions instead could keep this at bay and also degrade some of the organochlorine compounds formed (Karim, 2011). However, with an AOX load six times over the BAT limit, the reduction afforded by ozonation and wastewater treatment most likely is not enough to sufficiently lower the effluent load. Therefore, the Hap/DABCO technology, as used in this study, does not fulfill the environmental criteria a new bleaching technology should be able to meet. To be able to be considered an alternative technology for pulp bleaching, this problem needs to be addressed.

5 Conclusions In this study, the application of a haloperoxidase and DABCO for bleaching and HexA degradation was tested on oxygen-delignified eucalypt kraft pulp. An optimum reaction pH of 5 to 5,5 was determined. High-dose experiments using buffer showed a >80% reduction in hexenuronic acid and an impressive bleaching effect (from 40% to 57%), which was magnified by applying a peroxide-reinforced alkaline extraction (69,3%, control 54,9%). When applying a subsequent chlorine dioxide stage and peroxide-reinforced alkaline extraction, brightness was significantly increased (82,5%, control 76%). Viscosity was not decreased by the Hap/DABCO stage.

Using a non-buffered system, the brightness results could be replicated, while the HexA degradation was not as pronounced. A comparison with the simple addition of HOCl in terms of brightness, HexA reduction, kappa number among others showed that, at the same achieved brightness, the Hap/DABCO system did not reduce the kappa number or HexA content as efficiently as HOCl. This was attributed to a brightening effect of the hydrogen peroxide in the Hap/DABCO stage. This hypothesis was subsequently supported by an additional experiment.

Measurements of AOX and OX showed that, both in Hap/DABCO and HOCl stages, the amount of organochlorine formation was a multiple of what was measured with chlorine dioxide, and far beyond the limit set for the best available technique guidelines. This makes the industrial application of this system in its current form unlikely.

The side reaction of hydrogen peroxide and hypochlorous acid, yielding chloride, water, and oxygen, could be observed as gas formation and is likely to waste a big amount of oxidation power.

The catalytic bleaching technology, developed at the university of Aalto, was replicated and compared to the Hap/DABCO system. The short duration of the reaction was identified as central for the positive effect of DABCO and likely also for the little amount of chlorination.

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Outlook [confidential]

The model compound studies were abandoned in favor of the pulp experiments, but results showed oxidation and chlorination of dihydroxybenzoquinone, a pulp chromophore.

6 Outlook [confidential] This part of the thesis has been removed from the publicly available version for confidentiality reasons.

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LI, J. & GELLERSTEDT, G. 1997. The contribution to kappa number from hexeneuronic acid groups in pulp xylan. Carbohydrate Research, 302, 213-218. LIANG, T., NEUMANN, C. N. & RITTER, T. 2013. Introduction of fluorine and fluorine-containing functional groups. Angew Chem Int Ed Engl, 52, 8214-64. LUND, H., LASSEN, K. S., CASSLAND, B. L. P. A. & LOUREIRO, P. E. G. 2015. Reducing Content of Hexenuronic Acids in Cellulosic Pulp. WO2015018908. MESSERSCHMIDT, A. 2001. Handbook of metalloproteins, Chichester, Wiley. MULLER, G. 2003. Sense or no-sense of the sum parameter for water soluble "adsorbable organic halogens" (AOX) and "absorbed organic halogens" (AOX-S18) for the assessment of organohalogens in sludges and sediments. Chemosphere, 52, 371-9. MURRAY, W. 1992. Pulp and Paper: The Reduction of Toxic Effluents. In: BRANCH, L. O. P. R. (ed.). Ottawa, Canada. NI, Y., VAN HEININGEN, A. R. P. & KUBES, G. J. 1993. Mechanism of formation of chloro-organics during chlorine dioxide prebleaching of kraft pulp. Nordic Pulp and Paper Research Journal, 8, 350-351. OHSHIRO, T., LITTLECHILD, J., GARCIA-RODRIGUEZ, E., ISUPOV, M. N., IIDA, Y., KOBAYASHI, T. & IZUMI, Y. 2004. Modification of halogen specificity of a vanadium-dependent bromoperoxidase. Protein Sci, 13, 1566-71. ORTIZ-BERMÚDEZ, P., SREBOTNIK, E. & HAMMEL, K. E. 2003. Chlorination and Cleavage of Lignin Structures by Fungal Chloroperoxidases. Appl Environ Microbiol, 69, 5015-8. PELLETIER, I., ALTENBUCHNER, J. & MATTES, R. 1995. A catalytic triad is required by the non-heme haloperoxidases to perform halogenation. Biochim Biophys Acta, 1250, 149-57. PRUTZ, W. A. 1998. Reactions of hypochlorous acid with biological substrates are activated catalytically by tertiary amines. Arch Biochem Biophys, 357, 265-73. RENIRIE, R., PIERLOT, C., AUBRY, J.-M., HARTOG, A. F., SCHOEMAKER, H. E., ALSTERS, P. L. & WEVER, R. 2003. Vanadium Chloroperoxidase as a Catalyst for Hydrogen Peroxide Disproportionation to Singlet Oxygen in Mildly Acidic Aqueous Environment. Advanced Synthesis & Catalysis, 345, 849--858. RIKKE, F. & GJERMANSEN, M. 2010. Methods for Killing or Inhibiting Growth of Mycobacteria. WO2010122100. ROSENAU, T., POTTHAST, A., KOSMA, P., SUESS, H. U. & NIMMERFROH, N. 2007. Chromophores in Aged Hardwood Pulp - their structure and degradation potential. ISWFPC. Durban, South Africa. ROSENBLATT, D. H., DEMEK, M. M. & DAVIS, G. T. 1972. Oxidations of Amines. XI. Kinetics of Fragmentation of Triethylenediamine Chlorammonium Cation in Aqueous Solution. J. Org. Chem, 37, 4148-4151. SIXTA, H. 2008. Handbook of Pulp, Wiley. SOLOMON, K. R. 1996. Chlorine in the Bleaching of Pulp and Paper. Pure and Applied Chemistry, 68, 1721-1730. SUESS, H. U. 2010. Pulp Bleaching Today, de Gruyter. SUHR, M., KLEIN, G., KOURTI, I., GONZALO, M. R., SNTONJA, G. G., ROUDIER, S. & SANCHO, L. D. 2015. Best Available Techniques (BAT) Reference Document for the Production of Pulp, Paper and Board. Seville, Spain: European Commission. TORRES, C. E., NEGRO, C., FUENTE, E. & BLANCO, A. 2012. Enzymatic approaches in paper industry for pulp refining and biofilm control. Appl Microbiol Biotechnol, 96, 327-44. TROMP, M. G., OLAFSSON, G., KRENN, B. E. & WEVER, R. 1990. Some structural aspects of vanadium bromoperoxidase from Ascophyllum nodosum. Biochim Biophys Acta, 1040, 192-8. TÖRNGREN, A. & RAGNAR, M. 2002. Hexenuronic acid reactions in chlorine dioxide bleaching - aspects on in situ formation of molecular chlorine. Nordic Pulp and Paper Research Journal, 17, 179-182. VAN SCHIJNDEL, J. W., BARNETT, P., ROELSE, J., VOLLENBROEK, E. G. & WEVER, R. 1994. The stability and steady-state kinetics of vanadium chloroperoxidase from the fungus Curvularia inaequalis. Eur J Biochem, 225, 151-7. VUORINEN, T., FAGERSTRÖM, P., BUCHERT, J. & TELEMAN, A. 1999. Selective Hydrolysis of Hexenuronic Acid Groups and its Application in ECF and TCF Bleaching of Kraft pulps. Journal of Pulp and Paper Science, 25, 155-162. VUORINEN, T., JÄÄSKELÄINEN, A.-S. & LINDBERG, A. 2013. A method for bleaching pulp. WO2013150184. WEVER, R., DEKKER, H. L., SCHIJNDEL, J. W. P. M. V. & GEZINA MARIA VOLLENBROEK, E. 1995. Antifouling paint containing haloperoxidases and method to determine halide concentrations. WINTER, J. M. & MOORE, B. S. 2009. Exploring the Chemistry and Biology of Vanadium-dependent Haloperoxidases. The Journal of Biological Chemistry, 284, 18577-18581. WONG, B., L., SHEN, Y.-Q. & CHEN, Y.-P. 1996. Enzymatic Production of Halogenated Cephalosporin. WO1996019569. 73/79

Figures

XU, G. & WANG, B. G. 2016. Independent Evolution of Six Families of Halogenating Enzymes. PLoS One, 11, e0154619. XU, H., BLOOMFIELD, K. & LUND, H. 2006. Chlorine Dioxide Composition and Processes. PCT/US2005/015577.

8 Figures Figure 1: The molecular structure of cellulose. From Sixta, 2008 ...... 12 Figure 2: Example softwood lignin structure and lignol monomers. From (Christopher et al., 2014)...... 13 Figure 3: Cleavage reactions of non-phenolic model compound (4-methyl-2.3’.4’-tri-methoxy- diphenylether) with chlorine dioxide. From (Suess, 2010)...... 15 Figure 4: Proposed reaction scheme of the DABCO-catalyzed oxidation of substrates with hypochlorous acid. Adapted from Chenna et al., 2013 ...... 18 Figure 5: The structure of 1,4-Diazabicyclo[2.2.2]octane (DABCO) ...... 18 Figure 6: Proposed mechanism of chloroform formation from HexA. From Chenna et al., 2013...... 20 HOCl can generate phenolic groups and quinone structures as well as cleave the propyl unit from the phenyl unit (Fig. 7 and 8)...... 20 Figure 7: Generation of phenolic groups by hypochlorous acid (chloronium ion). From Sixta, 2008...... 20 Figure 8: Generation of halogenated (chlorinated) compounds in hypochlorous acid (chloronium ion) reactions and HCl addition to quinone structures. From Sixta, 2008...... 21 Figure 9: Correlation between brightness measurements made with standard aperture (Y-axis) versus 7,5mm aperture (X-axis) and correspondent optical components. The regression was made with Excel...... 36 Figure 10: The structure of dihydroxybenzoquinone (2,5-dihydroxycyclohexa-2,5-diene-1,4- dione) ...... 39 Figure 11: The structure of sinapaldehyde (3-(4-Hydroxy-3,5-dimethoxyphenyl)prop-2-enal) ... 39 Figure 12: Brightness gain (controls subtracted) of Hap/DABCO and Hap treated samples with 100mM acetate and phosphate buffer at 100mM hydrogen peroxide and NaCl,1,5% consistency...... 43 Figure 13: Brightness gain (controls subtracted) after Hap/DABCO treatment with different concentrations of NaCl, with 100mM acetate buffer pH 5, 100mM H2O2...... 45 Figure 14: Development of the non-buffered pH of different pulp samples and controls at 1,5% consistency over the course of two hours. H2O2 (if applicable) was 100mM. All the samples contained 100mM NaCl...... 48 Figure 15: “ISO” brightness (%) reached with different variations of the NaOCl/DABCO system. NaOCl and DABCO: 1% active chlorine odp and 0,1% odp, or threefold in the 3x sample. The duration of each treatment was 10 minutes, except for the 1h experiment...... 50 Figure 16: HexA content over time after one-time addition of 90mM H2O2 and NaCl. 100mM acetate buffer pH 4,5. Black: Control; Grey: Treated ...... 52 Figure 17: ISO brightness (%) over time after one-time addition of 90mM H2O2 and NaCl. 100mM acetate buffer pH 4,5.Black: Control; Grey: Treated. Due to errors during sampling, no sample was taken at 30 minutes, and no control sample was taken at 45 minutes...... 52 Figure 18: HexA content over time with stepwise addition of (in total) 90mM H2O2 and NaCl. 100mM acetate buffer pH 4,5. Black: Control; Grey: Treated...... 53 Figure 19: ISO brightness (%) over time with stepwise addition of (in total) 90mM H2O2 and NaCl. 100mM acetate buffer pH 4,5. Black: Control; Grey: Treated ...... 53 Figure 20: ISO brightness (%; black) and viscosity (cP, grey) after different treatments. Y axis represents both ISO brightness and viscosity. Enzyme treatment: 100mM H2O2 and NaCl, 100mM acetate buffer pH 4,5 120min. Error bars represent the standard deviation...... 55 Figure 21: Selectivity of different treatments, calculated as the decrease in viscosity divided by the increase of brightness from the original pulp...... 55 Figure 22: Cleavage of the cellulose chain by β-elimination from a carbonyl group (OR = chain parts). This can be caused by HOCl under alkaline conditions. From Sixta, 2008 ...... 57 74/79

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Figure 23: Brightness values of pulps after different first treatments (dark grey), first treatments and Ep (grey), and first treatments followed by D0 and Ep (light grey)...... 59 Figure 24: HexA content (mmol/kg) of selected samples after different treatments...... 60 Figure 25: Kappa number composition of pulps after different treatment. Light grey: HexA contribution; Dark grey: Non-HexA contribution (mostly lignin)...... 61 Figure 26: 280nm absorbance of different filtrates, diluted 1:10 with MQ ...... 62 Figure 27: “ISO” brightness (%) of pulps after HOCl or Control treatment followed by a pseudo Hap stage without enzymes. The first part of the sample name designates the treatment in the previous experiment (with HOCl or without), the second part designates the treatment in the pseudo Hap stage (with H2O2 or without)...... 65 Figure 28: AOX composition of selected samples. The contribution from first treatment stage is seen in light grey, the contribution from the Ep stage is shown in dark grey...... 66 Figure 29: OX results of pulp handsheets after different treatments...... 67

9 Tables Table 1: Conditions of enzyme treatment at medium consistency. Variable conditions are referred to in the Results and Discussion section...... 32 Table 2: Conditions of D0 stages at 10% consistency ...... 32 Table 3: Conditions of Ep stages at 10% consistency ...... 33 Table 4: Conditions of A-stages at 10% consistency ...... 33 Table 5: Miniscale assay general conditions. Variable conditions are referred to in the Results and Discussion section...... 35 Table 6: Overview of all samples generated in experiment C ...... 57 Table 7: Total kappa numbers of pulps after different treatments...... 61 Table 8: Total organohalogens (AOX+OX) after first stage, divided by the decrease in kappa number (Δkappa). Catalytic bleaching numbers are taken from Chenna et al., 2016 ...... 68

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Appendix 10 Appendix

Hap/DABCO: Results from previous studies

Table S1: Brightness and HexA results from the first Hap and Hap/DABCO-stage experiments using OD0Ep-birch pulp. From (Lund et al., 2015). HexA content ISO brightness Experiment (mmol/kg odp) (%) Untreated 26,3 76,8 Control NaCl 24,8 76,1 (no enzyme) Hap (NaCl) 18,9 79,5 Control NH Cl 4 26,8 77,7 (no enzyme)

Hap (NH4Cl) 26,1 79,8 Control DABCO, NaCl 27,8 77,3 (no enzyme) Hap/DABCO (NaCl) 15,3 79,8 Control DABCO, NH Cl 4 27,9 77,4 (no enzyme)

Hap/DABCO (NH4Cl) 12,1 80,4

Labomat troubleshooting The labomat heats up the pulp samples, which are arranged in a circle, by the means of two heaters in the upper corners of the instrument. To ensure equal heating, the vessels rotate. In the original program, the labomat was set to alternating between clockwise and counter- clockwise rotation, leading to unequal heating of the samples. A set of samples containing the same, untreated pulp was done. The obtained values were divided by the average, yielding a correction factor. This correction factor was multiplied with the raw data from Experiment A, 90mM to yield corrected values.

Table S2: Results of HexA measurement using identical samples of untreated pulp. The position correction factor was obtained by dividing the result by the mean results obtained from all the samples. Position Labomat HexA correction position (mmol/kg) factor

1 34,683 0,864

2 35,790 0,891

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3 37,813 0,942

4 40,167 1,000

5 42,188 1,051

6 43,739 1,089

7 43,155 1,075

8 42,705 1,064

9 41,899 1,044

10 43,061 1,073

11 42,015 1,046

12 40,466 1,008

13 38,190 0,951

14 39,717 0,989

15 40,093 0,999

16 36,697 0,914

Table S3: The original HexA data from experiment A was divided by the correction factor from Table S2, leading to a corrected HexA estimate, which was used in the figures presented in the Results and Discussion part. HexA Labomat HexA raw Correction Sample corrected position (mmol/kg) factor (mmol/kg) Control 1 30min 37,1 0,864 42,9 2 no sample

3 60min 38,1 0,942 40,5 4 60min 38,3 1,000 38,3 5 120min 42,5 1,051 40,4 6 120min 43,3 1,089 39,7 7 180min 42,6 1,075 39,6 8 180min 43,6 1,064 41,0 Treated 9 30min 23,3 1,044 22,3 10 45min 16,5 1,073 15,4 11 60min 11,3 1,046 10,8 12 60min 13,1 1,008 13,0 13 120min 5,3 0,951 5,6

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14 120min 4,5 0,989 4,6 15 180min 3,7 0,999 3,7 16 180min 2,7 0,914 3,0

Replication and modification of catalytic bleaching experiments Brightness reached with NaOCl/DABCO employing Hap/DABCO-stage conditions 75,0 70,0 70,6 70,9 68,1 68,2 68,7 68,7 70,0 67,6 67,2 63,1 65,0 60,3 61,3 61,1 60,4 60,6 59,8 59,6 59,6 60,0 55,0 50,0 45,0 42,2 41,4 41,8 41,5 40,5 40,9 40,9 40,4 39,4 "ISO" "ISO" brightness(%) 40,0

35,0

NaOClpH 5 NaOClpH 6 NaOClpH 7

DABCOpH4

DABCOpH 5 DABCOpH 6 DABCOpH 7

NaOCl pH NaOCl 7,5 NaOClpH 4,5 NaOClpH 4,5 NaOClpH 5,5 NaOClpH 6,5

DABCOpH 4,5 DABCOpH 5,5 DABCOpH 6,5 DABCOpH 7,5

NAOClDABCO 4 pH NAOClDABCO 5 pH NAOClDABCO 6 pH NAOClDABCO 6… pH NAOClDABCO 7 pH

NAOClDABCO 4,5 pH NAOClDABCO 5,5 pH NAOClDABCO 6,5 pH NAOClDABCO 7,5 pH NaOClpH 6 phosphate DABCOpH 6 phosphate

Figure S1: Brightness reached using NaOCl (light grey), DABCO (dark grey) or NaOCl/DABCO (grey) when employing the conditions of Hap/DABCO stages: 6mM DABCO and 100mM buffer (sodium acetate pH 4,5 to pH 6 and sodium phosphate pH 6 to pH 7,5). The negative effect of DABCO on bleaching is clearly visible.

Unprocessed data of the pH development during incubation.

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6

5,5

5 DABCO H2O2 Hap

DABCO H2O2 4,5 pH DABCO Hap H2O2 Hap 4 H2O2 untreated 3,5

3 0 20 40 60 80 100 120 incubation time (minutes)

Figure S2: Development of the pH of different samples and controls at 1,5% consistency over the course of two hours. Unprocessed data.

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