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UPTEC X 08 047 Examensarbete 30 hp November 2008 Thermochemical pretreatment and enzymatic saccharification of lignocellulose for biofuel production Jesper Svedberg

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Page 1: Thermochemical pretreatment and enzymatic saccharification ...files.webb.uu.se/uploader/ibg.uu.se/examensarbete-js.pdf · Syftet med detta projekt var att undersöka hur effektiva

UPTEC X 08 047

Examensarbete 30 hpNovember 2008

Thermochemical pretreatment and enzymatic saccharification of lignocellulose for biofuel production

Jesper Svedberg

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Molecular Biotechnology Programme

Uppsala University School of Engineering

UPTEC X 08 047 Date of issue 2008-11

Author

Jesper Svedberg

Title (English)

Thermochemical pretreatment and enzymatic saccharification of lignocellulose for biofuel production

Title (Swedish)

Abstract The efficiency of five different thermochemical pretreatment methods has been evaluated for efficiency and suitability for laboratory scale use with the purpose of facilitating enzymatic saccharification of aspen sawdust and oat straw. The ground substrates were first pretreated, while process conditions such as temperature, time, catalyst concentration and substrate loading were varied. The pretreated substrates were then saccharified using a commercially available enzyme mixture and finally the composition and concentration of solubilized sugars were determined using a high performance anion exchange chromatography system with pulsed amperometric detection (HPAE-PAD).

The most efficient pretreatment method appears to be dilute acid and least efficient hot water, but problems achieving stable measurements in the analysis step limits the quality of the results and further studies are needed.

Keywords Lignocellulose, ethanol, cellulases, thermochemical pretreatment

Supervisors

Jerry Ståhlberg and Mats Sandgren Dept. of Molecular Biology, Swedish University of Agricultural Sciences

Scientific reviewer

Sherry Mowbray Swedish University of Agricultural Sciences

Project name Sponsors Language

English

Security

ISSN 1401-2138

Classification

Supplementary bibliographical information Pages

49

Biology Education Centre Biomedical Center Husargatan 3 Uppsala

Box 592 S-75124 Uppsala Tel +46 (0)18 4710000 Fax +46 (0)18 555217

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Thermochemical pretreatment and enzymatic saccharification of lignocellulose for biofuel production

Jesper Svedberg

Populärvetenskaplig sammanfattning

Den etanol som används som biobränsle idag tillverkas till stor del av jordbruksgrödor som innehåller stora mängder socker eller stärkelse. Ett råmaterial som dock finns tillgängligt i betydligt större mängder är den cellulosa som är en del av växters cellväggar och som kan brytas ner till jäsbar glukos. Detta kan göras med hjälp av särskilda enzymer som finns naturligt i vissa bakterier och svampar, men för att då uppnå en effektiv nedbrytning krävs först att man luckrar upp strukturen på cellväggarna med hjälp av höga temperaturer och korrosiva kemikalier. Denna process kallas för termokemisk förbehandling.

Syftet med detta projekt var att undersöka hur effektiva fem olika förbehandlingsmetoder (förbehandling med utspädd syra, kalk, alkalisk väteperoxid, hett vatten samt ångexplosion) var på att underlätta enzymatisk nedbrytning av havrehalm och aspspån. Halmen och spånen maldes och förbehandlades med de olika metoderna, varefter de behandlades med enzym i upp till 72 timmar. Slutligen bestämdes mängden och sammansättningen av det socker som lösts ut med hjälp av ett HPLC-system.

Förbehandling med utspädd syra verkar ge de bästa sockerutbytena och hett vatten de sämsta, men problem med att få stabila mätvärden vid analysen av sockermängderna innebär tyvärr att ytterligare studier för att bestämma mer tillförlitliga värden krävs.

Examensarbete 30hp

Civilingejörsprogrammet Molekylär bioteknik

Uppsala universitet November 2008

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Contents

CONTENTS ........................................................................................................................................................... 1

INTRODUCTION ................................................................................................................................................ 4 LIGNOCELLULOSE ............................................................................................................................................................. 4 THE ETHANOL-FROM-LIGNOCELLULOSE PROCESS ...................................................................................................... 5 RESIDUAL MATERIAL ....................................................................................................................................................... 7 THERMOCHEMICAL PRETREATMENT ............................................................................................................................ 7

Dilute acid ............................................................................................................................................................................ 8 Lime ........................................................................................................................................................................................ 8 Alkaline Peroxide .............................................................................................................................................................. 9 Hot water ............................................................................................................................................................................. 9 Steam explosion ..............................................................................................................................................................10 Ammonia Fiber Explosion (AFEX) ..........................................................................................................................10 Organosolv.........................................................................................................................................................................10 Biological pretreatment ..............................................................................................................................................11

CARBOHYDRATE ANALYSIS ........................................................................................................................................... 11 Colorimetry .......................................................................................................................................................................11 HPLC/HPAE-PAD ............................................................................................................................................................11

OBJECTIVES ...................................................................................................................................................................... 12

MATERIALS AND METHODS ...................................................................................................................... 12 LIGNOCELLULOSE MATERIALS ...................................................................................................................................... 12 PRETREATMENT ............................................................................................................................................................. 12

Dilute acid ..........................................................................................................................................................................12 Lime ......................................................................................................................................................................................13 Hot water ...........................................................................................................................................................................13 Alkaline peroxide ............................................................................................................................................................13 Steam explosion ..............................................................................................................................................................14

ENZYMATIC SACCHARIFICATION .................................................................................................................................. 14 FILTRATION ..................................................................................................................................................................... 15 ANALYSIS ......................................................................................................................................................................... 15

DNS .......................................................................................................................................................................................15 PAHBAH ..............................................................................................................................................................................16 GOD/POD............................................................................................................................................................................16 HPLC/HPAE-PAD ............................................................................................................................................................16

RESULTS AND DISCUSSION ........................................................................................................................ 17 THERMOCHEMICAL PRETREATMENT .......................................................................................................................... 17

Alkaline hydrogen peroxide pretreatment .........................................................................................................17 Lime pretreatment.........................................................................................................................................................20 Hot water pretreatment..............................................................................................................................................21

CRITICAL ANALYSIS OF THE RESULTS .......................................................................................................................... 23 KINETICS OF THE ENZYMATIC SACCHARIFICATION ................................................................................................... 24 SUGAR CONTENT OF THE ACCELERASE ENZYME SOLUTION .................................................................................... 25 TABULATED SACCHARIFICATION RATES AND ETHANOL POTENTIALS ................................................................... 25

Colorimetric sugar measurements .........................................................................................................................26 LESSONS LEARNED, IMPROVEMENTS AND REMAINING PROBLEMS ........................................................................ 27 FUTURE PERSPECTIVES .................................................................................................................................................. 27

REFERENCES ................................................................................................................................................... 29

ACKNOWLEDGEMENTS ............................................................................................................................... 31

APPENDIX A ..................................................................................................................................................... 32

APPENDIX B ..................................................................................................................................................... 39

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APPENDIX C ..................................................................................................................................................... 41

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Introduction

Over the last decade ethanol has increased dramatically in importance as a fuel for motorized vehicles. Today it is one of the most widely used biofuels and because of present concerns over climate change and the diminishing reserves of fossil fuel, there are widespread efforts to increase production even further.

The ethanol produced today is largely made from crops containing high levels of sugar and starch, such as sugarcane, maize and wheat. Production of ethanol from such crops is a mature process, which can be established relatively easily on an industrial scale using commercially available technology. Despite this, there are reasons to look for alternative raw materials. Concerns have, for instance, been raised over the fact that this type of ethanol production competes with the food and feed industries for the same raw materials, and so an increased ethanol production may in turn lead to an increase in food prices and potentially starvation. Also, when producing ethanol from sugar and starch, only a part of the energy stored in the plant through photosynthesis is transferred into the fuel, making the process inefficient. However, if the parts of the plants that consist of lignocellulose are used as well, it is actually possible to take advantage of a much larger part of that stored energy.

Lignocellulose

Plant cell walls are the most common organic material in the biosphere. At an estimated annual production of 5-25 billion tons, it accounts for approximately 50% of the total biomass production in the world [Claassen et. al., 1999]. These cell walls consist primarily of three types of polymers, cellulose, hemicellulose and lignin, which form a tight and resilient structure. This complex is often referred to as lignocellulose [Claassen et. al., 1999].

Cellulose is a polysaccharide that consists of D-glucose bound in chains by β-(1,4)-glycosidic bonds. These chains bind to each other with hydrogen bonds to form strong, crystalline microfibrils. Hemicellulose is collective name for other polysaccharide components present in plant cell walls; it is a heterogeneous and complex mixture that contains many different types of monomeric sugars, such as glucose, xylose, mannose, galactose and arabinose. Lignin is a hydrophobic polymer consisting of various organic moieties - such as aromatic rings - which are polymerized in a complex and unpredictable manner. Some plants also contain a significant amount of pectin, which is another kind of heterogeneous carbohydrate polymer [Taherzadeh and Karimi, 2007]. The composition of the lignocellulose varies between plant species, but a general ratio is 30-50% cellulose, 10-30% hemicellulose and 10-30% lignin. A more detailed breakdown of the composition for a few important plant species can be seen in Table 1.

In order to use lignocellulose as a raw material for ethanol production the different sugars bound in the cellulose and hemicellulose must first be released as monomers, which makes them accessible to the microorganisms that ferment them into ethanol. Hexoses - or six carbon sugars - such as glucose and mannose can be fermented using normal baker’s yeast (Saccharomyces cerevisiae), but in order to utilize the different pentose sugars found in the hemicellulose, other forms of microorganisms, which carry

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the necessary metabolic enzymes, will be needed. Such organisms exist, but have yet to be used on a scale comparable to that of baker’s yeast [Taherzadeh and Karimi, 2007].

Table 1 Dry weight percentage of the main components of some model lignocellulosic materials.

Material Cellulose Hemicellulose Lignin Corn stover 37.5 22.4 17.6 Pine wood 46.4 8.8 29.4 Poplar 49.9 17.4 18.1 Wheat straw 38.2 21.2 23.4 Switch grass 31.0 12.4 17.6

Percentages do not add up to 100%, since minor components have not been listed. All data taken from Mosier et. al., 2005.

The process in which monosaccharides bound in the cellulose and hemicellulose are released is referred to as saccharification. It can be performed using concentrated or dilute acids at high temperatures, but a method that has received a greater amount of attention in recent years is enzymatic saccharification. Here enzymes found in some bacteria and fungi are used to break down the cellulose and hemicellulose polymers. [Taherzadeh and Karimi, 2007].

In order to achieve an efficient hydrolysis of a lignocellulosic material it is necessary to use a mixture of several different types of enzymes that work synergistically on the cellulose and hemicellulose. These include cellulases, hemicellulases and -glucosidases. There are two main types of cellulases which degrade cellulose in different ways: endoglucanases, which can make a cut in the middle of a cellulose chain and exoglucanases, which cut off cellobiose units (a disaccharide consisting of two glucose units) at the end of the cellulose chain, or at the points where the endoglucanase has cut the chain. The presence of both types of enzyme will speed up the hydrolysis to a degree that further addition of just one of the types alone would not. The hemicellulases are a large group of different enzymes (among others, endo-1,4- -D-xylanases, exo-1,4- -D-xylanases, -glucuronidases and acetyl xylan esterases) that are needed to degrade the very complex hemicellulose polymers. Finally, -glucosidases are necessary to break the glucoside bond in cellobiose and release the two glucose molecules [Taherzadeh and Karimi, 2007].

There are many different species of bacteria and fungi that produce cellulases and hemicellulases, but the one that has been the subject of most research is the fungus originally designated Trichoderma reesei (or Hypocrea jecorina as it should be called now). This is also the microorganism that is primarily used to produce cellulases on an industrial scale [Taherzadeh and Karimi, 2007].

The ethanol-from-lignocellulose process

When going from a lignocellulosic raw material to ethanol, a process consisting of several steps is necessary for an efficient degradation and conversion into ethanol. At the most basic level the substrate is treated with degrading enzymes, the monomeric sugars then released are put it into a fermentation vessel where microorganisms use it to produce ethanol, which is finally separated from the culture solution by for instance distillation. In reality a more complex process is required (Figure 1).

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Since the lignocellulose in the plant cell walls has a compact structure, which is highly resistant to degradation, it is necessary to make the cellulose and hemicellulose more accessible to the enzymes used in the saccharification. This is done first through size reduction, where the size of the substrate particles is reduced by cutting, chipping or milling the material (this also makes the material easier to handle in an industrial process) and then through thermochemical pretreatment, where high temperatures and corrosive chemicals are used to open up the structure of the lignocellulose.

The particle size can vary from a sub-millimeter size up to a few centimeters. Generally smaller particles mean larger surface area and a more easily degradable substrate, but even though a smaller particle size may lead to a higher degree of hydrolysis, it might still be economically sound to use larger particles, since milling often is a highly energy intensive process.

The part of the process with the greatest potential impact on maximizing the final sugar yield is probably the thermochemical pretreatment. Where an untreated substrate may produce a sugar yield of less than 20% of the theoretical maximum after enzymatic saccharification, the leading pretreatment methods regularly give rise to yields of 70-90% [Mosier et. al., 2005]. Many different pretreatment methods, where reaction time, temperature and chemical mechanisms differ, have been tested over the last thirty years; some of the more important alternatives are discussed here.

From a process perspective, the enzymatic saccharification is a relatively straightforward procedure. Substrate, enzymes and water are mixed in a stirred vessel where temperature and pH are kept on a controlled level (30-60 C, pH 4-5). The enzymes are allowed to act upon the substrate for a period of 1-3 days, after which almost all sugars that can be recovered from a particular substrate will be solubilized.

When fermenting hexose sugars (such as glucose and mannose) into ethanol, baker’s yeast can be used. This is done when ethanol is produced from sugar or starch containing crops and this method can also be used to ferment the hexose sugars found in lignocellulosic crops. However, since such crops also will contain pentose sugars found in the hemicellulose, baker’s yeast will not be able to fully realize the ethanol potential of the crop. Researchers have therefore looked for, or tried to create, microorganisms that can ferment pentose sugars to ethanol and today there are several alternatives where pentose hydrolyzing metabolic pathways have been introduced in well known microorganisms, such as Escherichia coli and Zymomonas mobilis. While these designed

Figure 1: Schematic representation of the lignocellulose-to-ethanol process.

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organisms show promising results, they have yet to be tried and proven useful in large-scale ethanol production projects [Galbe and Zacchi, 2002].

A somewhat different procedure compared to the one outlined above is simultaneous saccharification and fermentation (SSF), where the enzymatic saccharification and the fermentation takes place together in the same vessel. The main advantage of this procedure is that the continuous metabolization of the enzymatically released sugars by the fermenting microorganisms reduces product inhibition in the enzymatic process (it has been reported that –glucosidase loses 75% of its activity at glucose levels of 3 g/l [Philippidis and Smith, 1995; Philippidis et. al., 1993]). The main disadvantage is the necessity to keep the temperature and pH at levels that are acceptable to the fermenting microorganisms, which are not necessarily ones that are optimal for the enzymes. The enzymatic saccharification should optimally be performed at 50-60 C, whereas an organism like S. cerevisiae prefers a working temperature of 30-35 C. Experiments have shown that 38 C is the optimal compromise and since product inhibition may be severe the catalytic efficiency of the enzymes in SSF still shows a superior efficiency, compared to a separated saccharification and fermentation, even at such a low temperature. Usage of thermotolerant bacteria and yeast, such as Candida acidothermophilum and Kluyveromyces marxianus as fermenting microorganisms may in the future allow for higher working temperatures in an SSF process, but the ongoing work aimed at minimizing product inhibition of cellulases by directed mutagenesis and protein engineering might instead lessen the need for an SSF process [Taherzadeh and Karimi, 2007].

Residual material

After the saccharification and fermentation there will still be some material left that has not been metabolized into ethanol or CO2 by the fermenting microorganisms. The lignin in the lignocellulosic raw material cannot be utilized in the process and there will also be undigested cellulose and hemicellulose polymers left since it is generally very difficult to achieve a complete saccharification of these. In order to utilize the raw material to its full capacity, this residual material can be treated in several different ways. Incineration is an attractive alternative, since it is a simple process that generates products (heat and electricity) for which there is a constant demand. Another alternative is to utilize the residual material in an anaerobic digestion process where biogas is produced. A third is to use the material to produce some other product of higher value, such as plastics that can be derived from lignin [Galbe and Zacchi, 2002].

The most efficient way to utilize the raw material will depend on what type of material it is, the location of the processing plant and other variations in economical conditions. It is also necessary to choose pretreatment methods appropriate to the usage the leftover products are intended for. If the lignin is intended to be refined for production of other high value chemicals, it is unsuitable to use a pretreatment method where the lignin is oxidized [Taherzadeh and Karimi, 2007].

Thermochemical pretreatment

Due to the recalcitrant nature of lignocellulose a pretreatment step is necessary before any lignocellulosic material can be enzymatically hydrolyzed with an adequate level of efficiency. The pretreatment should break up the tight structure of the plant cell walls and expose the cellulose and hemicellulose to the enzymes, either by dissolving a large

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part of the material or by “loosening up” the lignin-cellulose-hemicellulose matrix and thereby creating space for the enzymes to access carbohydrate polymers [Galbe and Zacchi, 2002].

During the last twenty to thirty years, large efforts have been put into developing efficient pretreatment methods, and today there exists a plethora of different alternatives. These range in mechanism from simple grinding of the lignocellulosic biomass to the usage of specialized microorganisms for delignification of the feedstock, but the majority of the methods that appear to produce the best results either use high temperatures, corrosive chemicals, or more often a combination of the two, when preparing the substrates for enzymatic saccharification. This is why one often speaks of “thermochemical” pretreatment [Taherzadeh and Karimi, 2007].

Dilute acid

Dilute acid is, together with steam explosion, the pretreatment method most popular among the many commercial lignocellulosic ethanol projects that have recently started or reached the planning stages [Taherzadeh and Karimi, 2007]. The lignocellulosic biomass is mixed with water and acid is added to a low concentration (0.5-2% v/v) in a pressurized reactor. The reaction mixture is heated to 140-240 C and is kept there for a time period of a few minutes up to an hour. Sulphuric acid (H2SO4) is the most commonly used catalyst, but others such as hydrochloric acid and phosphoric acid have also been investigated [Mosier et. al., 2005].

The basic catalytic function of dilute acid pretreatment is hydrolysis of hemicellulose. This not only releases sugars that can be fermented to ethanol, but also breaks up the structure of the lignocellulose and makes the cellulose fibers more accessible to the cellulases. It is a fairly efficient and cheap method, but there are a few problems. Most significantly, the degradation of the hemicellulose does not limit itself to the release of the monomeric sugars, but these are in turn further degraded into HMFs (hydroxymethylfurfurals) and furfurals, which both act as inhibitors in the fermentation step [Mosier et. al., 2005]. It is therefore often common to wash away the liquid fraction from the solid biomass and only use the solids in the enzymatic step [Taherzadeh and Karimi, 2007]. Unfortunately this not only removes the inhibitory compounds but also the sugars that have been released from the hemicellulose, thus limiting the final amount of ethanol produced. Beyond this, the acid is also a corrosive agent, which leads to higher equipment costs; it is also necessary to neutralize it before the saccharification, which is an extra cost as well [Mosier et. al., 2005].

Lime

Lime (calcium hydroxide, “slaked lime” or Ca(OH)2) is also a popular pretreatment catalyst. It is added to the reaction mixture to about 10% of the weight of the biomass (w/w) in water; further addition of lime does not seem to significantly increase its catalytic efficiency. The lime pretreatment is generally performed at a lower temperature than when acid is used, and a range between 80 C and 120 C can be found in the literature; accordingly the pretreatment time is also significantly longer at 1-24 hours [Chang et. al, 1997].

Lime pretreatment works primarily by two mechanisms: firstly it solubilizes lignin and breaks up the structure of the lignocellulose; secondly it removes acetyl and uronic acid

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substitutions from the hemicellulose, which facilitates the access of the cellulases to the hemicellulose and cellulose. Lime pretreatment works better on substrates with a lower lignin content (such as straw materials and corn stover), but if oxygen is added during the process, lignin is more easily depolymerized and more lignin-rich substrates, such as wood, can also be efficiently pretreated [Mosier et. al., 2005; Chang et. al., 1997].

Other alkaline catalysts, such as sodium hydroxide, ammonia and urea will also affect lignocellulose in a similar manner, but lime has a few advantages that give it a comparative edge. The biggest one is the price; lime is significantly cheaper than other alkalis. The other main advantage is the possibility of recovering the lime by bubbling CO2 through the substrate mixture; this will cause the lime to react with the CO2 and form calcium carbonate, which can then be turned back into calcium hydroxide using classical limekiln technology [Chang et. al, 1997]. Compared to other leading pretreatment methods, lime shows good efficiency and a low price, but it has yet to be tried on a large scale.

Alkaline Peroxide

Using hydrogen peroxide (H2O2) in an alkaline environment (pH 11-13) is another efficient way of pretreating lignocellulose. Using a H2O2 concentration of about 2% (v/v) and pretreatment temperatures as low as 35-50 C, after incubation times of 12-24 hours, this method can contribute to a production of monomeric sugars after enzymatic saccharification at a level very close to the theoretical limit.

The primary mechanism of H2O2 action derives from its ability to oxidize lignin, but it also solubilizes hemicellulose to some extent. The oxidation of lignin is a highly efficient way of loosening up the structure of the lignocellulose, but unfortunately it also means that the lignin can no longer be used for further conversion into high value end products. This, together with the fact that hydrogen peroxide is a comparatively expensive chemical makes this method better suited for laboratory work than for industrial scale usage [Saha and Cotta, 2006; Fang et. al., 1999].

Hot water

It is possible to use plain water, without any corrosive chemicals added, when pretreating lignocellulose. When heating water to very high temperatures (200-230 C), the pH of the water will decrease and it will, together with compounds such as acetyl groups released from the hemicellulose, act upon the lignocellulose in a similar manner to dilute acid. Both the catalytic mechanism (primarily depolymerization of hemicellulose) and the unwanted formation of inhibitory compounds (HMFs and furfurals) are the same in hot water pretreatment as in dilute acid pretreatment, even if it is noticeable to a lesser extent in both cases. Hot water pretreatment has the added advantages of being cheaper and more environmentally friendly compared to dilute acid pretreatment and the products do not need to be neutralized before the saccharification step [Mosier et. al., 2005; Taherzadeh and Karimi, 2007].

If oxygen is continuously added during the hot water pretreatment, a process known as wet oxidation takes place. In this process the pretreatment works by oxidizing the lignin instead of acting on the hemicellulose, and it is therefore better suited for more lignous substrates [Taherzadeh and Karimi, 2007].

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Steam explosion

In the previously described pretreatment methods the lignocellulosic raw material is mixed with a significantly larger amount of water (a substrate load of 5-10% of the water content is common) and the mixture is then heated to the final temperature. An alternative procedure is to limit the amount of water (to about 50% of the total substance amount), and then heat the water and substrate by adding steam, which also raises the pressure. The temperature and pressure are kept at a high level for a short period of time (seconds to minutes), during which the water, or added catalysts such as sulphuric acid or sulphur dioxide acts upon the lignocellulose. At this point the pressurized vessel is opened and an explosive depressurization occurs, which in turn leads to the expulsion of the material into another vessel.

This procedure is known as steam explosion and it is perhaps the pretreatment method that is most popular today. As with dilute acid and water pretreatment, the high temperature (160-260 C) and a high pressure, acting together with a catalysts or without it, hydrolyzes the hemicellullose and thus breaks up the structure. The main difference is the more limited use of water, which also makes it possible to limit the use of catalysts and lower the total price and energy usage. The explosive decompression is often said to contribute to a further loosening of the structure, but it appears to be of a more limited importance compared to the removal of the hemicellulose [Mosier et. al., 2005; Galbe and Zacchi, 2002].

Both H2SO4 and SO2 are efficient catalysts and the optimal choice may differ between different substrates. H2SO4 is more corrosive and more difficult to apply to the substrate, but SO2 is a highly toxic gas, which might be difficult to handle. However, there are already industrial processes where SO2 is used, so this may not be a major problem [Galbe and Zacchi, 2002].

Steam explosion is a method that is commonly used in the production of fiber boards (the Masonite method) and it has been the subject of very intensive research regarding its use as a pretreatment method. All of this, together with a reasonably high efficiency and a relatively low cost, contributes to its considerable popularity [Mosier et. al., 2005].

Ammonia Fiber Explosion (AFEX)

Ammonia fiber explosion is basically the same method as steam explosion, but with ammonia is as the catalytic agent. It works at lower temperatures compared to steam explosion and the pretreatment mechanism is primarily delignification and the removal of lignin-hemicellulose bonds. This is a very efficient pretreatment method for agricultural byproducts and other materials with a lower lignin content, which makes it an attractive option, despite the relatively high process costs associated with the price, and necessary recycling, of ammonia [Taherzadeh and Karimi, 2007].

Organosolv

In the organosolv process, the various components in the lignocellulosic material are separated into different fractions using organic solvents and catalysts that solubilize the cellulose and hemicellulose. The lignin will be collected in the organic fraction and the cellulose and hemicellulose in the water fraction, which may then be separated further.

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The process can be performed from ambient temperature up to over 200 C, depending on which organic solvents and catalysts are used.

Organosolv is a potentially very attractive pretreatment alternative. Some versions show a high level of efficiency, and the fractionation of the different lignocellulosic parts makes the process easier to manage downstream. For instance, the relatively pure lignin in the organic fraction will be much easier to refine for further use. Industrial scale trials have yet to be performed though, which makes it difficult to assess its economic feasibility [Taherzadeh and Karimi, 2007].

Biological pretreatment

Biological pretreatment methods have also been considered and tried. Here, the idea is to utilize microorganisms such as white rot fungi, which have the natural ability to degrade lignin, in order to make the cellulose and hemicellulose more accessible. The procedure is very simple: the lignocellulosic material is mixed with spores from the fungus and is kept for a few weeks or months until a sufficient amount of lignin has been removed [Taherzadeh and Karimi, 2007].

Carbohydrate analysis

Many different methods for carbohydrate analysis exist. Here the two main types of analytic methods that were used for measuring sugar content during this project will be described.

Colorimetry

There are several different types of colorimetric methods, but they all share the same basic principle: the sample is mixed with a chemical substance that changes color in the presence of sugar, and the intensity of the color, which should be proportional to the sugar content, can then be measured using a spectrophotometer. Three different colorimetric analysis methods were evaluated: DNS (where the active chemical is dinitrosalicylic acid) [Miller, 1959], PAHBAH (p-hydroxy benzoic acid hydrazide) [Lever, 1972] and GOD/POD (glucose oxidase/peroxidase) [McCleary and Codd, 1991]. DNS and PAHBAH methods measure the total amount of reducing sugar, whereas GOD/POD is able to measure only the glucose using the glucose specific enzyme glucose oxidase in combination with peroxidase in order to cause a change in color of another substance.

Colorimetric methods have the advantage that they are simple, fast and easy to use on large sample sets, but they are limited by their ability to measure only total reducing sugar or, in the case of GOD/POD glucose, and the presence of other redox-active compounds may influence the response. If one has a complex carbohydrate mixture and wants to quantify the components individually, other methods are needed.

HPLC/HPAE-PAD

HPLC (High Performance Liquid Chromatography) is a form a column chromatography performed at higher flow rates and pressures than with standard liquid chromatography. Dionex (Sunnyvale, CA, USA) has developed a type of high performance anion exchange chromatography (HPAE) that together with a pulsed amperometric detector (PAD) can separate mono- and oligosaccharides with high resolution and detect and quantify them down to a nanomolar scale [Dionex, 2006].

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Neutral sugars can be separated on an anion exchange column when a strongly alkaline mobile phase is used, due to the fact that they are weak acids and will become partially or completely ionized at a high pH. Detection is performed by measuring the electrical currents generated by the oxidation of carbohydrates on the surface of a gold electrode, a process that is also facilitated in an alkaline environment.

Objectives

One long-term goal for the project described in this master’s thesis is to make biofuel production from lignocellulose more efficient. In order to achieve this goal it is necessary to develop methods for optimization of pretreatment and saccharification conditions for different lignocellulose substrates. Specific goals for this study were to:

Establish efficient laboratory-scale routines for thermochemical pretreatment and enzymatic saccharification of lignocellulose substrates, as well as measurement of fermentable sugar yields.

Make a literature survey of pretreatment methods that has been tried and suggest which methods that may be best suited for laboratory-scale pretreatments of lignocellulosic plant material.

Perform selected pretreatments of cellulose model substrates (aspen sawdust and oat straw) followed by enzymatic saccharification and sugar yield measurements.

Evaluate the five pretreatment methods for efficiency and suitability for laboratory scale use, and compare the yields with methods developed for industrial scale usage.

Materials and Methods

Lignocellulose materials

Oat straw from the region around Sala was provided by Sala-Heby Energi AB (Sala, Sweden). Aspen sawdust, with a particle size of up to a few millimeters, was received from a small sawmill outside of Uppsala. The sawdust came from planks consisting mainly of highly lignified aspen heartwood, which had been sawed with the specific intention of creating sawdust. The oat straw was cut up into 5-15 cm pieces. Both materials were dried at 60 C for 24 hour in preparation for milling. The milling was performed in a hammer mill (provided by The Department of Animal Nutrition and Management at the Swedish University of Agricultural Sciences in Uppsala) until all material could pass through a 1 mm sieve (Figure 2). Most of the particles of both oat straw and aspen were significantly smaller than 1 mm after the milling.

The dry weight was measured by weighing the samples before and after placing them samples in an oven at 105 C for 24 hours.

Pretreatment

Oat straw and aspen sawdust was pretreated with the following pretreatment procedures: dilute acid, lime, alkaline peroxide, hot water and steam explosion.

Dilute acid

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2.5 g or 5.0 g of lignocellulosic substrate was put into 100 ml Pyrex bottles. Water and concentrated sulphuric acid was added to a final volume of 50 ml and a final acid

Figure 2: The hammer mill used for sample preparation.

concentration of 0.5 or 1.0 %. The bottles were put in a Certoclave tabletop autoclave and heated to 140 C for four different time periods ranging from 15 to 90 minutes.

These times refer to the time spent at 140 C; it generally took about 20 minutes to reach this temperature and about 40 minutes to cool down enough to open the autoclave. The bottles were then cooled down to room temperature in a water bath and the pH was set to 5 using 10 M NaOH before they were frozen at -20 C, awaiting enzymatic saccharification.

Lime

5 g of substrate was put into 250 ml Pyrex bottles together with 0.5 or 0.75 g of lime (calcium hydroxide, Ca(OH)2). 50 ml of 90-100 C water was added and the bottles were then put in a shaking incubator at 80 or 95 C. Bottles were then removed after 1, 3, 6 or 24 hours and cooled down to room temperature in a water bath. The pH was set to 5 using 25 % hydrochloric acid after which the bottles were finally frozen at -20 C.

Hot water

Hot water pretreatment was performed in the same manner as lime pretreatment, with the exception that no lime was added and that the pH was not changed before freezing.

Alkaline peroxide

5 g or 10 g of substrate was added to 250 ml bottles, together with water and 7.5 ml 30 % hydrogen peroxide. The pH was set to 11.5 using 10 M NaOH and the volume was adjusted to 100 ml. The samples were kept in a shaking incubator at 35 C for 24 hours,

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after which the pH was set to 5 using 25 % hydrochloric acid and the bottles were frozen.

Figure 3: Oat straw and aspen, after pretreatment using sulphuric acid.

Steam explosion

Steam explosion was performed at the Department of Chemical Engineering at Lund University, Sweden, where they have the necessary equipment for the procedure. The “steam gun” used in the pretreatment may clog if the substrate particles are too small and the hammer-milled material could therefore not be used. Instead the oat straw was cut into 3-5 cm pieces using a household mixer and the aspen sawdust was used directly.

The dry weight of the substrates was measured. The oat straw was sprayed with diluted sulphuric acid to a final dry weight of 50 % and an acid concentration of 0.2 % and was left to soak overnight at room temperature. The aspen sawdust was then put in a plastic bag and infused with 2.5 % of sulphur dioxide (SO2) for 30 minutes.

Approximately 1 kg of material was put into the reactor of the steam gun and the temperature was raised to 200 C for the aspen sawdust and 190 C for the oat straw. After 5 minutes for the aspen and 10 minutes for the oat straw, a valve was opened into a collecting vessel, which caused the explosive expulsion of the substrate from the reactor. The substrate was then collected and the dry weight measured again.

Enzymatic Saccharification

For the enzymatic saccharification a commercially available enzyme preparation called Accelerase 1000 was provided as a kind gift by Genencor - A Danisco Division (Palo Alto, USA), consisting of biomass degrading enzymes from Hypocrea jecorina, supplemented with additional -glucosidases. The accompanying product statement declared that it had a measured cellulase activity of 2707 CMCU/g (1 CMCU defined as 1 µmol of reducing sugar released per minute when hydrolyzing carboxymethylcellulose

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at 55 C and pH 4.8) and a -glucosidase activity of 403 U/g (U defined as 1 µmol of nitrophenol liberated from para-nitrophenyl-B-D-glucopyranoside in 10 minutes at 50 C and pH 4.8). Saccharification of the pretreated lignocellulosic samples was performed according to the recommendations of the manufacturer, at 55 C, pH 4.5 and with an enzyme loading of 0.25 g of Accelerase solution per g of substrate (dry weight). This is an enzyme loading at the upper limit of the recommended loading range.

Bottles with pretreated lignocellulosic substrate were taken from the freezer and defrosted. New bottles were weighed empty and the contents of each bottle with pretreated material were transferred into the new bottle. The old bottles were rinsed with a small amount (~30 ml) of water, which was also transferred into the new bottles in order to recover as much as possible of the pretreated material. Citrate buffer was added to a final concentration of 50 mM, the pH was set to 4.5 using NaOH or hydrochloric acid and water was added to a total weight of 100 g (excluding the bottle). 0.5 ml samples were taken in triplicate from each bottle and saved. These are the zero-hour saccharification samples. The bottles were put in a shaking incubator at 55 C for at least one hour in order to reach the process temperature. Enzymes were then added in varying amount, depending on dry weight and initial amount of substance added, and the bottles were put back in the incubator.

0.5 ml samples were taken in triplicate at 30 minutes, 1, 3, 6, 24, 48 and 72 hours for carbohydrate analysis. The samples taken were loaded on 1.5 ml microtiter plates, which were sealed using silicone lids or adhesive film and put into a water bath set at 95-100 C. This was done in order to inactivate the cellulases and stop the hydrolysis of the cellulose and hemicellulose. The microtiter plates containing the samples were then frozen at -20 C, awaiting filtration.

The zero-hour samples were also put in a 95-100 C water bath and a small amount of enzymes was added, corresponding to the enzyme concentration in the saccharification bottles. This control was done because the enzyme solution contains a small, but potentially significant, amount of sugars, which may need to be subtracted from all samples in order to accurately estimate the increase caused by the hydrolysis of the substrate. The enzymes were added to the hot samples in order to destroy them immediately, before they could act upon the substrate.

After 72 hours the bottles were removed from the incubator, boiled for twenty minutes and frozen.

Filtration

Before any type of analysis could be performed on the saccharification samples, the remaining substrate particles were removed using a vacuum manifold filtration unit for microtiter plates, with 1 µm glass fiber filters. After filtration, the samples were either frozen again or immediately prepared for further analysis.

Analysis

DNS

DNS (dinitrosalicylic acid) reagent was prepared according to three slightly differing protocols from Methods in Enzymology 160, Wang [Wang, 2008], and the Accelerase manual. Equal amounts of DNS reagent and sample were added to a 10 ml Falcon tube,

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which was then boiled for 10 minutes. After cooling, the boiled sample was measured in a spectrophotometer at 575 nm and the reducing sugar concentrations quantified using a calibration curve made with glucose standards.

PAHBAH

PAHBAH (p-hydroxybenzoic acid hydrazide) reagent was prepared according to instructions from Megazyme [Megazyme, 2008]. Reagent and sample were mixed in a ratio of 5 to 1 and then boiled for 10 minutes. The absorbance was measured at 410nm using a spectrophotometer; reducing sugar concentrations were quantified by comparison to glucose standards of known concentrations.

GOD/POD

A GOD/POD assay kit was bought from Megazyme (K-GLUC) and reagents were prepared according to the manual. The reagent was mixed with the sample and incubated at 50 C for 20 minutes and absorption was measured at 510 nm.

Figure 4: The Dionex ICS 3000 HPAE-PAD carbohydrate analysis system. From left to right: autosampler, column oven and detector compartment, and pumps. On top: a fraction collector and eluent bottles.

HPLC/HPAE-PAD

A Dionex (Sunnyvale, CA, USA) ICS-3000 HPAE-PAD system (Figure 4) was used to separate arabinose, galactose, glucose, xylose, mannose and cellobiose and measure their concentrations in the samples taken from the enzymatic saccharification. A CarboPac PA10 (4x250mm) (Dionex) anion exchange column was used at 30 C and detection of the separated sugars was performed on a pulsed amperometric detector (PAD), using the “Standard Quad” waveform.

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The flow rate through the column was 1.0 ml/min. Arabinose, galactose, glucose, xylose and mannose were eluted isocratically using 100% water, and a gradient up to 200 mM NaOH and 70 mM sodium acetate was then initiated in order to elute the cellobiose and other oligosaccharides that might otherwise remain on the column. Table 2 shows a detailed description of all gradient steps. 300 mM NaOH at 0.5 ml/min was added after the column, in order to keep the pH sufficiently high for an efficient detection by the PAD detector.

Table 2 Analytical program for the Dionex HPEA-PAD system.

Time (minutes) Eluent composition -12 to -7 Regeneration: 200 mM NaOH and 70 mM sodium acetate

isocratically. -7 to -6 Gradient down to 100% water. -6 to 17 100% water isocratically. 0 Injection of samples. 17 to 35 Gradient up to 200 mM NaOH and 70 mM sodium acetate.

Enzymatically treated samples were diluted 200 or 400 times in water before analysis. Quantification was performed by comparing peak areas of sugars in the samples to those of five different standard solutions containing all six sugar types, with known concentrations ranging from 2 to 80 mg/l. The injection volume was at all times 25 µl.

Calculations of the sugar concentrations of the samples were done automatically using the Chromeleon software controlling the HPLC machine and data were later compiled and processed further using Microsoft Excel.

Results and discussion

The dry weight of the aspen and oat straw substrates was determined to be 96.4 % and 94.5 % of the initial weight, respectively.

Thermochemical pretreatment

The comparative efficiency of different pretreatment methods and different conditions of a particular pretreatment method was determined by measuring the sugar content and sugar composition of samples taken after saccharification of the pretreated material. In order to compare the full potential of the different pretreatment methods, sugar composition of the 72-hour samples was analyzed using the Dionex HPAE-PAD equipment for all saccharifications. The impact of the pretreatment on the kinetics of the enzymatic saccharification was also investigated on a smaller number of samples, by analyzing sugar composition of samples taken at all time points (0-72 hours).

Alkaline hydrogen peroxide pretreatment

Alkaline hydrogen peroxide pretreated samples showed saccharification yields of 0.25-0.50 g total sugar per gram of pretreated substrate (wet weight) for aspen (Figure 5) and 0.37-0.40 g sugar per g substrate for oat straw (Figure 6) with substrate load as the variable pretreatment condition. Aspen gave a significantly lower saccharification yield

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for a 10 % (weight of substrate/volume of liquid) substrate load compared to a 5 % substrate load. This might be due to the fact that aspen has a high lignin content and there might not be enough H2O2 available in the pretreatment mixture for a sufficient oxidation of the lignin to effectively break up the lignocellulose matrix at a substrate load of 10 %. In contrast, oat straw shows a much smaller variation in saccharification rate between 5 and 10 % substrate load. This is unsurprising if the ratio of H2O2 to lignin is the determining factor of catalytic efficiency, since straw materials have a lower lignin content than hard woods.

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Figure 5: Alkaline hydrogen peroxide pretreatment of aspen, where substrate loading has been varied between 5 and 10 %. The bars show the average sugar amounts based on triplicate samples; the error bars show the highest and lowest total amount of sugars of the triplicate samples.

Oat straw, hydrogen peroxide pretreatment

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Figure 6: Alkaline hydrogen peroxide pretreatment of oat straw, where substrate loading has been varied between 5 and 10 %.

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Lime pretreatment

The impact of varying incubation time, temperature and amount of lime was investigated for lime pretreatment. Lime pretreated aspen shows saccharification yields of 0.075-0.18 g sugar / g substrate (Figure 7). It is difficult to find a pattern in the saccharification rates, when varying the above-mentioned factors. When time was

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Figure 7: Lime pretreatment of aspen. Substrate loading, lime loading, time and temperature have been varied.

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Figure 8: Lime pretreatment of oat straw. The same parameters have been varied as with aspen.

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varied from 1 to 24 hours, no major differences can be seen and in fact both the lowest and highest saccharification yields were obtained in samples pretreated for 24 hours. The variation of lime from 1 % to 1.5 % and of temperature from 80 to 95 C shows no clearer patterns either. The variations in the sugar analysis seem to be larger than any variations caused by different pretreatment conditions.

Oat straw gave saccharification yields of 0.15-0.35 g sugar / g substrate (Figure 8), though apart from one sample (1 % lime, 10% substrate, 3 hours at 80 C) no samples gave yields below 0.25 g/g. The same issues with interpreting the results as with aspen apply here and it is difficult to draw any conclusions regarding optimal conditions. The general levels of saccharification are higher for oat straw compared to aspen. This is in line with results found in the literature, where a generally higher catalytic efficiency of lime pretreatment on substrates with a lower lignin content has been reported [Mosier et. al., 2005], but the total rates are still comparatively low.

Hot water pretreatment

Pretreatment time and temperature were varied when saccharification rates were measured for hot water pretreatment. Pretreated aspen samples gave yields of 0.040-0.045 g sugar / g substrate and oat straw 0.10-0.12 g sugar / g substrate (Figures 9 and 10). The highest conversion rates were found after 24 hours pretreatment at 95 C for both aspen and oat straw, but these were not significantly higher than pretreatment carried out at 80 C or for 3 hours. The total conversion levels are noticeably lower than for the other methods investigated in this study and it is likely that much higher temperatures (from 150 C and upwards) are necessary in order to reach competitive levels of efficiency using hot water pretreatment.

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Figure 9: Hot water pretreatment of aspen sawdust.

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Oat straw, hot water pretreatment

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Figure 10: Hot water pretreatment of oatstraw shows somewhat higher saccharification rates compared to aspen sawdust, but both substrates show significanly lower rates when treated with water compared to other pretreatment methods.

Diluted sulphuric acid pretreatment

Pretreatment time, substrate loading and acid loading were investigated. Acid pretreatment produced saccharification yields of 0.35-0.55 g sugar / g substrate of aspen and 0.25-0.90 g/g of oat straw (Figures 11 and 12). In this case it is also difficult to draw any conclusions regarding how the variable factors influence the saccharification rates. 1 % acid seems to lead to higher rates than 0.5 %, but the differences are not significant. The oat straw sample producing a saccharification rate of 0.9 g/g (0.5 % acid, 5 % substrate, 30 minutes) is unreasonably high, since it is probably impossible the reach such high levels due to lignin content etc. It is probably the result of a faulty measurement, but even so it seems clear that acid pretreatment and particularly acid pretreatment of oat straw produces the highest final sugar concentrations.

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Aspen, sulphuric acid pretreatment

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Figure 11: Dilute sulphuric acid pretreatment at 140 C of aspen sawdust. There are only small differences between the varying conditions, but the general level is higher than for other pretreatments of aspen.

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Figure 12: Dilute sulphuric acid pretreatment at 140 C of oat straw appears to be very efficient. However saccharification rates above 0.6-0.7 are generally not possible, since the total amount of cellulose and hemicellulose does not exceed 60-70 % of the dry weight of most lignocellulosic materials.

Critical analysis of the results

Figure 13 shows a compilation of all pretreatment methods investigated, including steam explosion, for both aspen sawdust and oat straw. The conditions producing the highest saccharification rates were chosen in order to visualize the comparative catalytic potential. For both oat straw and aspen sawdust, sulphuric acid pretreatment

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produced the highest conversion rates, but with the inhibitory compounds that may be formed during acid pretreatment this method might still prove to be an inferior option. For aspen sawdust, both alkaline hydrogen peroxide and steam explosion pretreatment show comparable results, and they may prove to be better options. For oat straw, alkaline peroxide and steam explosion pretreatment show somewhat lower final sugar levels compared to aspen, but lime shows higher conversion rates. Saccharification yields above 0.60-0.75 g sugar/ g substrate are unreasonable, since there is not that much cellulose and hemicellulose available in a substrate to reach such conversion levels (see Table 1) and some of the values reported for acid pretreatment of oat straw therefore appear to be inherently untrustworthy. Hot water pretreatment gives conversion levels that are significantly lower than other methods and they do not appear to be much higher than reported values for untreated materials.

Most of the pretreatment methods tested in this study show lower rates of saccharification compared to values reported in the literature for similar substrates [Saha et. al., 2005; Saha and Cotta, 2006; Chang et. al., 1997; Galbe and Zacchi, 2002; Taherzadeh and Karimi, 2007; Mosier et. al, 2005]. There are many possible explanations for this; equipment used for pretreatment and saccharification could differ, with for instance better mixing during the different incubation steps, the enzymes could be more active or the analytical methods could differ.

Comparision of pretreatment methods for

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Figure 13: Summary of the most efficient example of each pretreatment method for oat straw and aspen. Dilute sulphuric acid appears to be the most efficient method for oat straw, but for aspen steam explosion and hydrogen peroxide are comparable.

Kinetics of the enzymatic saccharification

Figures A1 to A19 in Appendix A show complete curves of the saccharification, with samples taken at 0, 0.5, 1, 3, 6, 24, 48 and 72 hours. A majority of the curves show that the final sugar concentrations are reached after 6-24 hours and that sugar levels remain

Oat Straw Aspen

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basically constant after that. The exception is oat straw, pretreated with 1.5% lime for 24 hours at 95 C (Figure A7), which appears to show a constantly increasing sugar concentration up to 72 hours. It is difficult to say whether this is caused by variations in the analysis or by an actual increase in sugar levels, but a repeated saccharification of this pretreated substrate with samples taken up to five days (120 hours) would hopefully clarify the situation.

Certain saccharification curves appear to reach a clear maximum after 3 to 6 hours of incubation and then decrease (examples can be seen in Figures A4 and A14). This is an unreasonable result, since there is no obvious reason why sugar should disappear from the solution, but there are a few possible explanations. The first is a bacterial infection, which would cause sugars to be consumed during the saccharification. This explanation can be probably disregarded though, since the bacteria in all likelihood would consume the glucose first and then go over to other sugars, and there is no indication of this. Instead one can generally see a proportional variation of all sugars between time points. Therefore a likelier explanation is variations in sensitivity during the HPLC measurements or errors in the dilution during the preparation of the samples. Since all of the samples that show this pattern were saccharified and analyzed together and each time point for all of these samples was analyzed in a group, it is difficult to decide which explanation that is correct, but the fact that a pipette was found to be broken and was replaced somewhere around the time when these samples were analyzed, in combination with the fact that the 3 and 6 hour samples seem to be unreasonably high, may indicate that dilution errors might have caused the problem. A repeated analysis of these samples was performed, but unfortunately this coincided with further problems with the sensitivity of the PAD detector on the Dionex system and the results had to be discarded. At this point it was not possible to perform any further carbohydrate measurements due to lack of time within this thesis project and the solution of this problem had to be postponed until a later stage.

Sugar content of the Accelerase enzyme solution

The Accelerase enzyme solution is known to contain sugar, which comes from the fungal culture solution. In order to determine how much sugar that was added to the saccharification process together with the enzymes the sugar content of the Accelerase enzyme mixture was measured using the same method as for the saccharification samples. These measurements showed that there were detectable levels of galactose, glucose and mannose present (Figure 14), but they were not high enough to significantly influence final yields, since the sugar added with Accelerase would correspond to about 0.1 % of the total sugar content of most samples.

Tabulated saccharification rates and ethanol potentials

Calculated saccharification rates are tabulated in Appendix B. The rates are reported in g sugar / g substrate for glucose, xylose and the combined values for the other sugars (arabinose, galactose, mannose and cellobiose). Theoretical estimations of ethanol production from the released sugars in each sample are also listed. These values are

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0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0

-10

13

25

38

50

63

75

88

100080630 #416 accelerase-2 ED_1nC

min

1 - A

rabino

s - 9,

075 2 -

Gala

ktos -

10,32

5

3 - G

lukos

- 12,3

50

4 - M

anno

s - 16

,600

6 - C

ellob

ios - 2

8,500

Figure 14: An example of the Accelerase cellulose solution measured on the Dionex HPAE-PAD system. The levels measured correspond to a total sugar amount of 10-25 mg sugar / l the samples measured.

calculated using reported conversion rates of 0.375 g ethanol / g glucose and 0.25 g ethanol / g xylose, in addition to an estimation of 0.32 g / g for “other sugars” [Badger, 2002].

Appendix C contains all samples analyzed for soluble sugar content.

Colorimetric sugar measurements

It was initially planned to do large scale colorimetric measurements of the total reducing sugar contents of the samples taken from the saccharification, in order to identify the most interesting pretreatment methods and conditions which could then be analyzed using the HPAE-PAD system. The DNS and PAHBAH colorimetric methods for measuring total reducing sugar were evaluated together with the GOD/POD glucose measurement procedure, but when the HPAE-PAD system appeared to be easier to use than initially thought it was decided to use it for all sugar measurements.

The DNS method is widely used for measuring total reducing sugar and it was the first method to be evaluated. Unfortunately, it was found to be very difficult to get stable measurements, and the absorbance was found to increase significantly within minutes when a sample was put into a spectrophotometer. This phenomenon does not appear to be mentioned anywhere in the literature, and despite varying absorbance wavelength and trying several different analytical protocols the problem remained.

The PAHBAH procedure was tested and found to be a superior option. Even though it is slightly more complicated to perform, the measurements were much more stable and reproducible, which is clearly a great advantage.

GOD/POD was also found to be a stable and easily performed method for measuring glucose specifically. If the glucose potential of a substrate or a pretreatment method were the only factor of interest, this method would be the recommended analytical option.

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Lessons learned, improvements and remaining problems

Since one of the objectives of this project was to develop methods and procedures for thermochemical pretreatment, saccharification and sugar analysis, some words regarding the actual methods are relevant.

The difficulty of interpreting how varying conditions affect saccharification rates makes it hard to evaluate the actual procedures, but they are in general very simple and possible improvements would in all likelihood require the purchase of expensive equipment, such as stirred, pressurized vessels. The saccharification process seems also sufficiently mature, but it would be of interest to investigate the possibility of bacterial or fungal infections by adding an antibiotic agent, such as sodium azide, to the substrate solution. The preparation and analysis of the saccharification samples on the HPAE-PAD system should also be transferred entirely to a microtiter plate system, since this would both decrease analysis costs and, by always using multichannel pipettes, speeding up times for sample preparation.

The main issue that has to be dealt with is the large variations in sugar measurements. The Dionex HPAE-PAD system used during this project is completely new and stable running protocols for sugar analysis and quantification have not yet been established in the lab. Steps must be taken to clarify what parameters might cause drift in signal strength of the PAD detector and to investigate whether the large variations seen between triplicates of the same sample and between different analysis rounds are caused by inaccuracy when diluting or by variations in the analytical method. Repeated measurements of sugar standards do show stability, but systematical repetitions of the same sample, using the same or new dilutions, have not been performed and doing so would improve understanding the HPAE-PAD system and hopefully lead to measurements of higher quality. If it turns out that the problem has been caused by the dilution of the samples, stricter protocols must be established to make sure that the highest possible precision is achieved.

Future perspectives

Beyond the need to stabilize the analytical procedures, the research on pretreatment methods and saccharification could be taken in many different directions. Theoretical estimations of the ethanol potential of the saccharified material have here been calculated, but it is necessary to perform actual fermentations of the material for trustworthy ethanol potential values to be acquired, since the presence of inhibitors and other chemical substances in the saccharified solutions might influence the efficiency of the fermentation process greatly. It would also be of interest to set up an SSF (Simultaneous Saccharification and Fermentation) process, since the reported efficiency is greater than that of separate saccharification and fermentation.

During the course of this project oat straw and aspen sawdust have acted as model substrates, representing the categories of straw and hardwood. In reality, there will be variations between different types of substrates, even within theses categories and there is an endless variety of different types of lignocellulosic materials whose potential as a feedstock for ethanol production can be investigated.

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A different direction, where another master’s student has already started investigations, is to look into how the pretreatment methods here evaluated affect the biogas production rates in an anaerobic digestion process. Since the bacteria present in this process will also use extracellular enzymes to break down the cellulose, a loosening of the lignocellulosic structure may also lead to an increased rate of biogas production.

The original plan for this master’s project was to focus on the optimization of the enzymatic saccharification and the pretreatment was just a minor problem that had to be solved before this could be done. Soon it became clear that the pretreatment methods represent a much wider and more important area than initially thought and focus shifted towards this part. However, now that the groundwork is laid the focus should again shift to looking primarily at the enzymatic process since it may be important to optimize this step in the lignocellulose-to-ethanol process, by varying enzyme composition or catalytic conditions, for all different substrates.

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References

P. C. Badger. Ethanol from cellulose: A general review. Trends in new crops and new uses. J. Janick and A. Whipkey (eds.), 2002, ASHS Press, Alexandria, VA.

V. Chang, B. Burr, and M. Holtzapple. Lime pretreatment of switchgrass. Applied Biochemistry and Biotechnology, 63-65(18):3–19, 1997.

P. A. M. Claassen, J. B. van Lier, A. M. Lopez Contreras, E. W. J. van Niel, L. Sijtsma, A. J. M. Stams, S. S. de Vries, and R. A. Weusthuis. Utilisation of biomass for the supply of energy carriers. Applied Microbiology and Biotechnology, 52(6):741–755, 1999.

Dionex. Dionex Reference Library, 2006. http://www.dionex.com/ (2008-08-08).

J. Fang, R. Sun, D. Salisbury, P. Fowler, and J. Tomkinson. Comparative study of hemicelluloses from wheat straw by alkali and hydrogen peroxide extractions. Polymer Degradation and Stability, 66:423–432, 1999.

M. Galbe and G. Zacchi. A review of the production of ethanol from softwood. Applied Microbiology and Biotechnology, 59(6):618–628, 2002.

M. Lever. A new reaction for colorimetric determination of carbohydrates. Analytical Biochemistry, 47(1):273–279, 1972.

B. V. McCleary and R. Codd. Measurement of (1,3),(1,4)-beta-d-glucan in barley and oats: A streamlined enzymic procedure. Journal of the Science of Food and Agriculture, 55(2):303–312, 1991.

Megazyme. PAHBAH Reducing Sugar Procedure. http://secure.megazyme.com/GetAttachment.aspx?id=6402a20b-971f-48ca-8ebe-c9d10db00b4f (2008-04-20).

G. L. Miller. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31:426–428, 1959.

N. Mosier, C. Wyman, B. Dale, R. Elander, Y. Y. Lee, M. Holtzapple, and M. Ladisch. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology, 96(6):673–686, 2005.

G. Philippidis and T. Smith. Limiting factors in the simultaneous saccharification and fermentation process for conversion of cellulosic biomass to fuel ethanol. Applied biochemistry and biotechnology, 51/52:117–124, 1995.

G. P. Philippidis, T. K. Smith, and C. E. Wyman. Study of the enzymatic hydrolysis of cellulose for production of fuel ethanol by the simultaneous saccharification and fermentation process. Biotechnology and Bioengineering, 41(9):846–853, 1993.

B. C. Saha and M. A. Cotta. Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw. Biotechnology Progress, 22(2):449–453, 2006.

B. C. Saha, L. B. Iten, M. A. Cotta, and Y. V. Wu. Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol. Process Biochemistry, 40(12):3693–3700, 2005.

M. J. Taherzadeh and K. Karimi. Acid-based hydrolysis processes for ethanol from

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lignocellulosic materials: A review. BioResources, 2(3):472–499, 2007.

N. S. Wang. Glucose Assay by Dinitrosalicylic Colorimetric Method. http://ww.glue.umd.edu/~NSW/lab4a.htm (2008-04-02).

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Acknowledgements

I would like to thank my supervisors Jerry Ståhlberg and Mats Sandgren for all their advice and help, Majid Haddad for an extra pair of hands during the end of my project, Sherry Mowbray for her input on this report, The Department of Animal Nutrition and Management at the Swedish University of Agricultural Sciences in Uppsala for letting me use their milling equipment and Christian Roslander at The Department of Chemical Engineering at Lund University for showing me their steam explosion equipment.

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Appendix A

These curves show the progress of the saccharification process by visualizing the amount of released sugars over time. Total sugar, glucose, xylose and other sugars are presented in g of measured sugar in the solution per g of substrate added to the saccharification.

Oat straw, water, 10% substrate , 24h, 80°C

0,0000

0,0200

0,0400

0,0600

0,0800

0,1000

0,1200

0,1400

0,1600

0,1800

0,2000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A1

Oat straw, 1% acid, 10% substrate, 90 min

0,0000

0,1000

0,2000

0,3000

0,4000

0,5000

0,6000

0,7000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A2

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Oat straw, 0.5% acid, 5% substrate, 90 min

0,0000

0,2000

0,4000

0,6000

0,8000

1,0000

1,2000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A3

Oat straw, 0.5% acid, 5% substrate,15 min

0,0000

0,2000

0,4000

0,6000

0,8000

1,0000

1,2000

1,4000

1,6000

1,8000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A4

Oat straw, 0.5% acid, 5% substrate, 60 min

0,0000

0,1000

0,2000

0,3000

0,4000

0,5000

0,6000

0,7000

0,8000

0,9000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A5

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Oat straw, lime 1.5%, 10% substrate, 3h, 80°C

0,0000

0,0500

0,1000

0,1500

0,2000

0,2500

0,3000

0,3500

0,4000

0,4500

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A6

Oat straw, lime 1.5%, 10% substrate, 6h, 80°C

0,0000

0,0500

0,1000

0,1500

0,2000

0,2500

0,3000

0,3500

0,4000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A7

Oat straw, lime 1.5%, 10% substrate, 24h, 95°C

0,0000

0,0500

0,1000

0,1500

0,2000

0,2500

0,3000

0,3500

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A8

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Oat straw, hydrogen peroxide, 10% substrate

0,0000

0,0500

0,1000

0,1500

0,2000

0,2500

0,3000

0,3500

0,4000

0,4500

0,5000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A9

Oat straw, hydrogen peroxide, 5% substrate

0,0000

0,2000

0,4000

0,6000

0,8000

1,0000

1,2000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A10

Oat straw, steam explosion

0,0000

0,0500

0,1000

0,1500

0,2000

0,2500

0,3000

0,3500

0,4000

0,4500

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A11

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Aspen, water, 10% substrate, 24h, 80°C

0,0000

0,0200

0,0400

0,0600

0,0800

0,1000

0,1200

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A12

Aspen, 1% acid, 10% substrate, 90 min

0,0000

0,1000

0,2000

0,3000

0,4000

0,5000

0,6000

0,7000

0,8000

0,9000

1,0000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A13

Aspen, lime 1%, 10% substrate, 24h, 80°C

0,0000

0,0500

0,1000

0,1500

0,2000

0,2500

0,3000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A14

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Aspen, lime 1%, 10% substrate, 6h, 80°C

0,0000

0,0500

0,1000

0,1500

0,2000

0,2500

0,3000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A15

Aspen, lime 1%, 10% substrate, 1h, 80°C

0,0000

0,0500

0,1000

0,1500

0,2000

0,2500

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A16

Aspen, lime 1.5%, 10% substrate, 24h, 95°C

0,0000

0,0200

0,0400

0,0600

0,0800

0,1000

0,1200

0,1400

0,1600

0,1800

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A17

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Aspen, hydrogen peroxide, 10% substrate

0,0000

0,0500

0,1000

0,1500

0,2000

0,2500

0,3000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A18

Aspen, steam explosion

0,0000

0,1000

0,2000

0,3000

0,4000

0,5000

0,6000

0,7000

0,8000

0 10 20 30 40 50 60 70 80

Time (h)

g s

ug

ar

/ g

su

bstr

ate

Total Sugar

Glucose

Xylose

Other sugars

Figure A19

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Appendix B

A table of all 72 hour samples with results from HPAE-PAD analysis. “Volume” refers to the volume in l of the saccharification vessel, “Substrate” to amount of substrate (in g) added to the saccharification and “Dry weight” to the fraction of dry weight substance in the added substrate. “Glucose”, “Xylose” and “Other sugars” lists the amount of sugar released during the saccharification in g sugar / g substrate; “Ethanol” lists an theoretical estimation of the amount of ethanol in l / g substrate that one can expect after fermentation of the released sugar.

Substrate and pretreatment Sample ID Volume Substrate Dry weight Glucose Xylose Other sugars Ethanol

Aspen, h2o2, 10% s3-02-72h 0.15 10 0.964 0.148 0.094 0.007 0.103

Aspen, h2o2, 5% s1-01-72h 0.15 5 0.964 0.309 0.166 0.006 0.202

Aspen, h2so4 0.5%, 10%, 30m s5-05-72h 0.1 5 0.964 0.207 0.203 0.031 0.175

Aspen, h2so4 0.5%, 10%, 90m s4-06-72h 0.1 5 0.964 0.182 0.151 0.029 0.146

Aspen, h2so4 0.5%, 5%, 15m s4-09-72h 0.1 2.5 0.964 0.155 0.165 0.025 0.136

Aspen, h2so4 0.5%, 5%, 30m s5-01-72h 0.1 2.5 0.964 0.174 0.187 0.029 0.154

Aspen, h2so4 0.5%, 5%, 60m s5-02-72h 0.1 2.5 0.964 0.142 0.179 0.029 0.136

Aspen, h2so4 0.5%, 5%, 90m s5-03-72h 0.1 2.5 0.964 0.198 0.177 0.030 0.162

Aspen, h2so4 1%, 10%, 30m s5-06-72h 0.1 5 0.964 0.206 0.194 0.032 0.172

Aspen, h2so4 1%, 10%, 60m s1-02-72h 0.1 5 0.964 0.254 0.176 0.030 0.189

Aspen, h2so4 1%, 5%, 15m s4-01-72h 0.1 2.5 0.964 0.211 0.187 0.029 0.171

Aspen, h2so4 1%, 5%, 30m s5-04-72h 0.1 2.5 0.964 0.153 0.180 0.025 0.140

Aspen, h2so4 1%, 5%, 60m s4-08-72h 0.1 2.5 0.964 0.336 0.180 0.033 0.230

Aspen, h2so4 1%, 5%, 90m s4-10-72h 0.1 2.5 0.964 0.345 0.150 0.030 0.224

Aspen, lime 1%, 10% 1h, 80c s3-03-72h 0.1 5 0.964 0.077 0.054 0.001 0.054

Aspen, lime 1%, 10% 24h, 80c s3-05-72h 0.1 5 0.964 0.105 0.073 0.003 0.074

Aspen, lime 1%, 10% 6h, 80c s3-04-72h 0.1 5 0.964 0.080 0.056 0.001 0.056

Aspen, lime 1.5%, 10%, 24h, 80c s1-04-72h 0.1 5 0.964 0.048 0.033 0.001 0.034

Aspen, lime 1.5%, 10%, 24h, 80c s7-09-72h 0.1 5 0.964 0.091 0.062 0.001 0.063

Aspen, lime 1.5%, 10%, 24h, 95c s6-06-72h 0.1 5 0.964 0.085 0.056 0.002 0.059

Aspen, lime 1.5%, 10%, 3h, 80c s7-10-72h 0.1 5 0.964 0.066 0.046 0.001 0.047

Aspen, lime 1.5%, 10%, 3h, 95c s6-05-72h 0.1 5 0.964 0.096 0.067 0.002 0.068

Aspen, lime 1.5%, 10%, 6h, 80c s7-04-72h 0.1 5 0.964 0.108 0.074 0.003 0.076

Aspen, steam exp s2-01-72h 0.1 5 0.964 0.248 0.091 0.006 0.149

Aspen, water, 10%, 24h, 95c s6-08-72h 0.1 5 0.964 0.037 0.009 0.001 0.021

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Aspen, water, 10%, 3h, 80c s7-08-72h 0.1 5 0.964 0.037 0.006 0.001 0.020

Aspen, water, 10%, 3h, 95c s6-07-72h 0.1 5 0.964 0.034 0.006 0.001 0.018

Oat straw, h2o2, 10% s3-01-72h 0.15 10 0.945 0.258 0.118 0.021 0.169

Oat straw, h2o2, 5% s2-03-72h 0.15 5 0.945 0.261 0.105 0.014 0.163

Oat straw, h2so4 0.5%, 10%, 30m s5-07-72h 0.1 5 0.945 0.187 0.186 0.043 0.165

Oat straw, h2so4 0.5%, 10%, 60m s5-10-72h 0.1 5 0.945 0.235 0.205 0.048 0.196

Oat straw, h2so4 0.5%, 10%, 90m s5-09-72h 0.1 5 0.945 0.196 0.175 0.038 0.164

Oat straw, h2so4 0.5%, 5%,15m s3-08-72h 0.1 2.5 0.945 0.312 0.289 0.061 0.265

Oat straw, h2so4 0.5%, 5%,30m s4-03-72h 0.1 2.5 0.945 0.421 0.406 0.089 0.365

Oat straw, h2so4 0.5%, 5%,60m s3-09-72h 0.1 2.5 0.945 0.358 0.268 0.060 0.279

Oat straw, h2so4 0.5%, 5%,90m s3-10-72h 0.1 2.5 0.945 0.295 0.202 0.046 0.223

Oat straw, h2so4 1%, 10%, 30m s4-04-72h 0.1 5 0.945 0.120 0.099 0.022 0.097

Oat straw, h2so4 1%, 5%, 15m s7-07-72h 0.1 2.5 0.945 0.247 0.240 0.053 0.215

Oat straw, h2so4 1%, 5%, 30m s5-08-72h 0.1 2.5 0.945 0.223 0.195 0.044 0.186

Oat straw, h2so4 1%, 5%, 60m s4-05-72h 0.1 2.5 0.945 0.285 0.182 0.044 0.211

Oat straw, lime 1%, 10%, 1h, 80c s7-05-72h 0.1 5 0.945 0.159 0.089 0.014 0.110

Oat straw, lime 1%, 10%, 24h, 80c s7-02-72h 0.1 5 0.945 0.191 0.117 0.022 0.137

Oat straw, lime 1%, 10%, 6h, 80c s7-01-72h 0.1 5 0.945 0.186 0.114 0.021 0.133

Oat straw, lime 1.5%, 10%, 24h, 80c s7-03-72h 0.1 5 0.945 0.195 0.125 0.023 0.142

Oat straw, lime 1.5%, 10%, 24h, 95c s6-02-72h 0.1 5 0.945 0.151 0.096 0.025 0.112

Oat straw, lime 1.5%, 10%, 3h, 80c s3-06-72h 0.1 5 0.945 0.152 0.092 0.019 0.109

Oat straw, lime 1.5%, 10%, 3h, 95c s6-01-72h 0.1 5 0.945 0.140 0.088 0.020 0.103

Oat straw, lime 1.5%, 10%, 6h, 80c s3-07-72h 0.1 5 0.945 0.189 0.116 0.026 0.137

Oat straw, steam exp s2-02-72h 0.1 5 0.945 0.099 0.050 0.003 0.064

Oat straw, water, 10%, 24h, 95c s6-04-72h 0.1 5 0.945 0.097 0.018 0.004 0.053

Oat straw, water, 10%, 3h, 80c s7-06-72h 0.1 5 0.945 0.093 0.015 0.006 0.051

Oat straw, water, 10%, 3h, 95c s6-03-72h 0.1 5 0.945 0.084 0.014 0.003 0.046

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Appendix C

A table listing all samples measured on the Dionex HPAE-PAD system. All sugar values are listed in mg/l.

Substrate and pretreatment Sample ID Arabinose Galactose Glucose Xylose Mannose Cellobiose Total Time (h)

Aspen, h2o2, 5% s1-01-000h 0.0 0

s1-01-000h 85.6 19.3 141.9 246.8 0

s1-01-000h 76.5 33.2 109.7 0

s1-01-000h 126.7 55.6 182.3 0

s1-01-005h 964.3 361.9 41.5 1367.7 0.5

s1-01-005h 16.9 1126.2 392.3 155.0 58.6 1749.0 0.5

s1-01-005h 673.4 252.0 925.4 0.5

s1-01-005h 1324.4 487.1 56.2 1867.7 0.5

s1-01-010h 3694.6 1171.9 69.8 232.2 5168.5 1

s1-01-010h 17.9 2496.1 723.3 153.7 164.3 3555.3 1

s1-01-010h 2932.8 940.6 56.0 176.7 4106.1 1

s1-01-010h 2315.1 747.7 43.2 139.4 3245.4 1

s1-01-030h 4824.8 2321.0 58.6 292.4 7496.8 3

s1-01-030h 22.3 4963.2 2155.8 200.7 408.1 7750.1 3

s1-01-030h 5656.0 2685.0 69.0 353.7 8763.7 3

s1-01-030h 32.3 6536.9 3078.6 78.4 490.4 10216.7 3

s1-01-060h 6267.2 3426.0 63.7 403.1 10160.0 6

s1-01-060h 22.7 6424.5 3218.7 211.5 519.0 10396.3 6

s1-01-060h 3494.7 1966.4 36.5 213.3 5710.9 6

s1-01-060h 45.8 7465.2 4007.1 77.4 496.6 12092.1 6

s1-01-24h 33.6 11152.6 6052.5 118.5 572.7 17930.0 24

s1-01-24h 33.4 11769.0 6130.7 323.9 350.9 18607.8 24

s1-01-24h 10101.4 5520.4 104.5 511.2 16237.5 24

s1-01-24h 34.7 11322.6 6165.3 123.2 578.0 18223.8 24

s1-01-48h 39.4 13655.5 7411.5 157.3 275.0 21538.7 48

s1-01-48h 27.2 10012.1 5174.5 301.3 212.9 15727.9 48

s1-01-48h 35.1 12015.7 6555.7 140.4 238.1 18985.1 48

s1-01-48h 319.7 319.7 48

s1-01-48h 8572.7 4715.7 98.8 301.1 13688.3 48

s1-01-72h 4014.1 2147.5 55.6 80.1 6297.2 72

s1-01-72h 0.0 72

s1-01-72h 35.6 12737.8 6968.4 155.6 19897.5 72

s1-01-72h 23.4 8918.3 4734.9 213.4 108.0 13998.0 72

s1-01-72h 30.9 11314.2 6238.5 138.3 17722.0 72

s1-01-72h 36.4 12608.6 6635.8 151.6 148.7 19581.1 72

Aspen, h2so4 1%, 10%, 60m s1-02-000h 310.5 446.9 2435.4 10239.9 975.9 14408.7 0

s1-02-000h 267.0 360.3 2022.0 8628.0 900.5 12177.8 0

s1-02-000h 242.6 342.2 1798.9 7828.2 753.1 10965.0 0

s1-02-000h 420.4 618.3 3237.0 13801.3 1348.5 19425.6 0

s1-02-005h 232.8 338.7 2597.5 7629.8 745.0 656.6 12200.4 0.5

s1-02-005h 316.4 414.7 3239.0 9684.8 913.8 896.2 15464.7 0.5

s1-02-005h 220.6 309.2 2339.0 7049.4 678.5 547.7 11144.3 0.5

s1-02-005h 302.2 425.9 3241.3 9706.7 937.6 773.5 15387.2 0.5

s1-02-010h 266.0 381.0 4183.3 8686.5 842.1 1102.7 15461.5 1

s1-02-010h 338.6 443.0 4917.1 10365.4 1060.0 1322.3 18446.4 1

s1-02-010h 259.1 365.3 4024.8 8319.0 804.8 1044.3 14817.3 1

s1-02-010h 244.4 337.7 3698.0 7777.8 748.1 934.1 13739.9 1

s1-02-030h 165.0 233.5 4797.9 5471.1 515.7 245.5 11428.7 3

s1-02-030h 333.7 433.1 8842.6 10243.4 421.5 502.3 20776.5 3

s1-02-030h 238.7 330.6 6790.6 7686.0 731.9 358.2 16136.0 3

s1-02-030h 313.6 444.4 9111.6 10144.5 979.4 498.3 21491.8 3

s1-02-060h 324.7 456.8 11744.0 10476.0 1005.6 238.0 24245.2 6

s1-02-060h 294.3 401.9 10250.6 9581.0 378.7 230.2 21136.6 6

s1-02-060h 272.5 384.4 10062.4 8793.4 845.6 185.6 20543.8 6

s1-02-060h 277.9 388.7 10197.1 9009.8 857.1 182.4 20913.1 6

s1-02-24h 335.2 449.5 14435.0 10759.6 371.4 26350.6 24

s1-02-24h 157.5 219.6 7207.0 5120.8 550.2 13255.1 24

s1-02-24h 321.0 464.9 15304.9 10730.0 1033.3 27854.0 24

s1-02-24h 0.9 1.1 37.8 27.5 2.7 70.1 24

s1-02-24h 266.3 382.4 12565.0 8894.0 852.5 22960.2 24

s1-02-48h 152.9 216.1 7264.8 4965.0 545.1 13143.9 48

s1-02-48h 297.9 396.0 13054.7 9519.8 352.3 23620.8 48

s1-02-48h 271.3 391.0 13205.8 9157.1 876.8 23902.1 48

s1-02-48h 276.9 397.9 13386.0 9242.9 888.4 24192.1 48

s1-02-48h 200.2 287.8 9670.7 6588.8 709.7 17457.1 48

s1-02-48h 263.0 380.0 12818.0 8890.2 851.4 23202.7 48

s1-02-48h 175.5 245.6 8220.6 5647.6 609.8 14899.1 48

s1-02-72h 130.8 260.6 6335.9 4495.9 435.3 11658.6 72

s1-02-72h 255.8 361.0 12098.9 8261.4 877.6 21854.7 72

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s1-02-72h 327.2 434.9 14411.0 10479.3 849.9 26502.4 72

s1-02-72h 323.3 467.5 15865.7 10828.1 1043.6 28528.2 72

s1-02-72h 256.8 365.8 12436.0 8647.8 824.9 22531.3 72

s1-02-72h 154.8 216.0 7299.9 5016.4 558.5 13245.7 72

s1-02-72h 347.7 506.0 17220.6 11769.6 1134.8 30978.8 72

Aspen, lime 1.5%, 10%, 24h, 80c s1-04-000h 56.1 56.1 0

s1-04-005h 986.6 229.4 87.4 1303.5 0.5

s1-04-010h 18.6 1799.2 544.8 68.6 2431.2 1

s1-04-030h 18.3 2360.7 1203.6 82.3 3664.8 3

s1-04-060h 20.5 2851.0 1747.1 86.5 4705.2 6

s1-04-24h 19.7 2772.1 1700.6 83.7 4576.0 24

s1-04-48h 20.7 3132.5 2142.7 50.1 5346.0 48

s1-04-72h 1131.0 800.7 1931.7 72

s1-04-72h 23.4 3512.2 2411.6 56.5 6003.6 72

Aspen, steam exp s2-01-010h 86.8 1904.0 869.5 82.8 2943.1 1

s2-01-010h 95.7 2086.0 959.4 94.3 3235.4 1

s2-01-010h 69.2 106.6 2158.8 1015.4 97.9 3447.9 1

s2-01-030h 62.0 103.5 4048.5 1449.4 81.4 317.7 6062.5 3

s2-01-030h 79.4 138.5 5326.7 1894.0 108.0 307.8 7854.4 3

s2-01-030h 83.2 142.2 5546.5 1966.3 112.9 436.3 8287.6 3

s2-01-060h 58.2 105.6 6029.3 2093.0 80.1 207.6 8573.7 6

s2-01-060h 43.2 78.1 4500.8 1584.1 58.0 146.0 6410.1 6

s2-01-060h 66.7 121.7 6900.4 2412.9 91.7 234.6 9827.9 6

s2-01-24h 76.8 126.5 13880.5 4941.1 126.3 19151.3 24

s2-01-24h 68.5 99.3 12305.5 4422.1 112.8 17008.2 24

s2-01-24h 80.9 133.0 14746.3 5242.4 135.2 20337.7 24

s2-01-48h 66.2 99.8 12799.2 4645.6 119.7 17730.6 48

s2-01-48h 80.7 135.1 15396.4 5582.6 145.5 21340.2 48

s2-01-48h 78.3 131.9 15189.3 5483.3 140.7 21023.5 48

s2-01-72h 46.9 90.1 8595.4 3179.1 93.7 68.1 12073.3 72

s2-01-72h 71.0 108.7 13893.9 5109.2 133.3 19316.2 72

s2-01-72h 66.5 99.7 12803.3 4725.5 121.7 17816.7 72

s2-01-72h 65.1 97.2 12458.4 4598.5 115.9 17335.2 72

Oat straw, steam exp s2-02-010h 92.2 2154.6 309.6 2556.4 1

s2-02-010h 90.3 2035.8 284.1 2410.2 1

s2-02-010h 90.7 2075.3 283.9 2450.0 1

s2-02-030h 139.9 5101.3 1949.0 7190.2 3

s2-02-030h 94.3 2795.7 761.1 3651.0 3

s2-02-030h 106.8 3162.6 855.3 4124.7 3

s2-02-060h 96.0 2838.7 1268.2 83.3 4286.2 6

s2-02-060h 98.5 3434.6 1415.3 109.3 5057.7 6

s2-02-060h 92.9 3230.5 1343.3 103.5 4770.2 6

s2-02-24h 85.1 3764.0 1870.3 100.8 5820.3 24

s2-02-24h 89.0 3808.2 1897.5 101.8 5896.5 24

s2-02-24h 91.9 3998.5 1985.1 107.2 6182.7 24

s2-02-48h 137.3 32.8 6424.7 3148.4 135.9 9879.1 48

s2-02-48h 101.3 4646.5 2312.5 95.2 7155.5 48

s2-02-48h 111.5 5116.4 2544.2 106.2 7878.3 48

s2-02-72h 115.8 5489.8 2713.7 8319.3 72

s2-02-72h 90.7 4316.2 2148.6 6555.5 72

s2-02-72h 91.7 4286.0 2152.9 70.4 6601.0 72

Oat straw, h2o2, 5% s2-03-010h 92.0 2098.0 290.1 2480.0 1

s2-03-010h 1281.3 438.6 8474.7 8625.4 548.1 19368.1 1

s2-03-010h 334.1 6557.2 1012.9 527.2 8431.4 1

s2-03-030h 407.7 11100.4 2440.7 54.2 480.2 14483.2 3

s2-03-030h 328.9 8946.6 1973.4 387.9 11636.8 3

s2-03-030h 295.3 8043.0 1766.5 351.1 10455.8 3

s2-03-060h 353.0 11410.5 3547.4 44.5 65.2 15420.6 6

s2-03-060h 400.1 36.8 12885.6 4012.1 50.3 73.6 17458.4 6

s2-03-060h 394.5 36.9 12806.2 3968.8 50.4 75.5 17332.4 6

s2-03-72h 225.3 19.5 8219.8 3310.2 31.4 177.8 11984.0 72

Oat straw, 1% h2so4, 10%, 60m s2-04-010h 1215.1 423.1 6824.6 8463.0 539.1 17464.9 1

s2-04-010h 0.0 1

s2-04-010h 1144.9 395.3 6511.3 7999.1 498.9 16549.5 1

s2-04-030h 1231.3 412.5 8450.0 8572.4 523.3 612.9 19802.5 3

s2-04-030h 1259.9 422.9 8700.4 8791.3 541.0 646.0 20361.5 3

s2-04-030h 1359.8 459.8 9379.1 9482.8 582.3 688.7 21952.5 3

s2-04-060h 1342.9 442.8 10633.1 9627.3 564.2 226.3 22836.6 6

s2-04-060h 1430.9 475.9 11586.2 10274.4 603.8 243.8 24615.0 6

s2-04-060h 1345.3 444.8 10683.1 9630.9 565.5 226.3 22895.8 6

Aspen, 1% h2so4, 10%, 24h s2-05-010h 244.0 538.8 6143.7 7975.0 875.4 15777.0 1

s2-05-010h 283.6 626.2 6928.7 9145.9 1008.6 17992.8 1

s2-05-010h 293.7 651.6 7043.3 9456.8 1056.6 18502.1 1

s2-05-030h 240.6 516.5 7854.8 7845.4 832.3 875.9 18165.6 3

s2-05-030h 219.7 476.1 7221.0 7241.3 763.9 805.1 16727.3 3

s2-05-030h 262.1 567.5 9000.3 8852.2 995.8 1014.4 20692.5 3

s2-05-060h 261.1 559.1 10627.2 8683.3 892.4 375.2 21398.2 6

s2-05-060h 241.8 512.1 9752.9 8077.2 814.5 328.4 19726.9 6

s2-05-060h 267.6 571.6 10858.3 8916.3 906.6 503.5 22023.9 6

Oat straw, 1% h2so4, 10%, 90m s2-06-010h 1493.8 543.3 9429.7 10109.7 687.0 22263.5 1

s2-06-010h 1485.3 533.6 9117.2 10044.6 674.8 21855.5 1

s2-06-010h 1224.7 435.0 7466.7 8351.3 547.5 18025.2 1

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s2-06-030h 1203.2 410.9 9071.1 8186.7 521.1 649.0 20042.1 3

s2-06-030h 1036.5 351.4 7783.9 7058.8 445.7 557.7 17234.0 3

s2-06-030h 1093.6 370.5 8226.2 7446.0 469.4 596.4 18202.0 3

s2-06-060h 1382.3 467.8 11873.9 9644.0 596.6 247.3 24211.8 6

s2-06-060h 1337.9 446.8 11314.0 9302.5 566.1 224.8 23192.1 6

s2-06-060h 1398.7 466.6 11823.3 9727.7 599.0 233.4 24248.6 6

Aspen, water, 10%, 24h, 80c s2-07-010h 1132.4 180.2 1312.5 1

s2-07-010h 1251.4 191.6 1443.1 1

s2-07-010h 1451.0 222.7 76.7 1750.4 1

s2-07-030h 1734.9 273.6 2008.5 3

s2-07-030h 2324.8 388.8 88.9 2802.5 3

s2-07-030h 2124.1 335.0 81.3 2540.5 3

s2-07-060h 1791.8 279.4 58.7 2129.9 6

s2-07-060h 2029.8 294.7 65.7 2390.2 6

s2-07-060h 1648.5 243.4 54.7 1946.6 6

Oat straw, lime 1.5%, 10%, 24h, 80c s2-08-010h 388.3 5544.4 817.0 479.1 7228.8 1

s2-08-010h 311.8 4865.2 694.6 437.3 6308.9 1

s2-08-010h 358.6 5301.6 783.2 465.7 6909.2 1

s2-08-030h 660.1 8300.3 2046.0 586.7 11593.1 3

s2-08-030h 608.6 7609.2 1879.0 516.3 10613.1 3

s2-08-030h 637.5 7999.2 1976.5 548.4 11161.5 3

s2-08-060h 582.0 8428.4 2881.5 44.4 11936.2 6

s2-08-060h 529.4 7591.7 2615.4 10736.4 6

s2-08-060h 581.7 39.0 8406.4 2876.1 11903.2 6

Oat straw, water, 10%, 24h, 80c s2-09-010h 75.3 3166.5 321.9 3563.7 1

s2-09-010h 84.1 3251.0 338.6 3673.7 1

s2-09-010h 81.2 3382.1 342.5 3805.8 1

s2-09-030h 123.5 4899.4 591.3 5614.2 3

s2-09-030h 129.7 5130.0 618.7 5878.4 3

s2-09-030h 111.6 4367.8 536.4 5015.8 3

s2-09-060h 94.6 4105.6 509.4 49.3 4758.9 6

s2-09-060h 93.9 4052.9 494.2 46.0 4687.0 6

s2-09-060h 101.2 40.9 4398.2 535.1 51.4 5126.9 6

Oat straw, lime 1%, 10%, 3h, 80c s2-10-010h 296.6 4951.4 766.8 365.6 6380.5 1

s2-10-010h 325.3 5550.0 914.7 418.0 7208.0 1

s2-10-010h 291.5 4926.2 812.3 372.3 6402.3 1

s2-10-030h 652.6 8906.4 2446.4 567.5 12572.7 3

s2-10-030h 583.1 7890.3 2159.8 492.4 11125.6 3

s2-10-030h 595.4 8041.5 2203.8 503.5 11344.3 3

s2-10-060h 413.3 6369.2 2424.2 9206.7 6

s2-10-060h 377.0 5755.7 2213.8 8346.5 6

s2-10-060h 394.4 6053.3 2314.2 8761.9 6

s2-10-72h 250.5 19.6 3850.5 2363.2 22.7 158.1 6664.6 72

Oat straw, h2o2, 10% s3-01-000h 253.2 253.2 0

s3-01-000h 367.2 367.2 0

s3-01-000h 288.9 288.9 0

s3-01-005h 172.2 34.0 3660.8 548.2 50.8 296.6 4762.7 0.5

s3-01-005h 147.4 3085.2 466.8 43.3 247.4 3990.1 0.5

s3-01-005h 151.6 3170.5 494.4 42.6 261.6 4120.7 0.5

s3-01-01h 212.2 4285.0 870.1 36.3 282.9 5686.4 1

s3-01-01h 228.1 31.9 4765.6 942.1 38.6 325.5 6331.9 1

s3-01-01h 212.2 4420.5 918.7 36.9 306.3 5894.6 1

s3-01-03h 341.4 38.3 9153.3 2233.2 46.1 78.9 11891.3 3

s3-01-03h 379.7 43.4 10312.7 2561.3 51.3 1339.6 14688.0 3

s3-01-03h 334.7 37.4 8905.1 2252.3 43.5 1274.3 12847.3 3

s3-01-06h 545.2 62.7 18246.9 5851.4 206.8 2238.0 27151.0 6

s3-01-06h 504.9 59.0 16830.6 5429.8 312.6 2019.2 25156.1 6

s3-01-06h 377.8 43.8 12556.8 3885.5 48.1 1486.4 18398.3 6

s3-01-24h 292.4 38.2 13396.8 5728.3 37.1 1012.9 20505.6 24

s3-01-24h 266.9 45.2 16043.9 6829.7 58.8 1247.1 24491.4 24

s3-01-24h 4964.3 3320.3 62.8 138.9 8486.3 24

s3-01-48h 307.3 40.2 14767.4 6661.7 54.5 896.8 22727.8 48

s3-01-48h 301.9 38.9 14376.5 6454.2 42.5 863.2 22077.1 48

s3-01-48h 306.1 39.3 14636.4 6624.4 53.1 880.8 22540.1 48

s3-01-72h 251.1 42.3 15362.7 7033.8 55.0 946.3 23691.2 72

s3-01-72h 266.5 44.4 16233.8 7449.9 55.4 986.6 25036.6 72

s3-01-72h 279.9 46.6 17106.8 7835.8 59.7 1027.6 26356.2 72

Aspen, h2o2, 10% s3-02-000h 203.6 203.6 0

s3-02-000h 196.3 196.3 0

s3-02-000h 280.0 280.0 0

s3-02-005h 2044.5 499.9 45.1 172.3 2761.8 0.5

s3-02-005h 1933.4 495.1 42.5 174.1 2645.1 0.5

s3-02-005h 1925.5 500.1 43.5 168.0 2637.1 0.5

s3-02-01h 3243.7 1075.8 52.2 251.4 4623.1 1

s3-02-01h 3064.5 989.6 47.3 236.0 4337.4 1

s3-02-01h 2984.2 919.7 46.8 228.4 4179.1 1

s3-02-03h 6241.0 2726.2 69.2 550.8 9587.2 3

s3-02-03h 32.5 6476.4 2869.5 72.5 568.0 10018.9 3

s3-02-03h 5659.9 2547.7 62.7 486.3 8756.6 3

s3-02-06h 38.0 8861.0 4613.4 86.3 681.0 14279.6 6

s3-02-06h 39.0 9616.1 5083.5 232.8 733.6 15704.9 6

s3-02-06h 39.9 9329.8 4795.9 90.3 728.1 14984.0 6

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s3-02-24h 35.1 10115.8 6219.4 112.0 630.9 17113.3 24

s3-02-24h 36.0 10503.5 6429.2 116.1 660.5 17745.3 24

s3-02-24h 34.3 10425.5 6400.9 114.7 652.1 17627.6 24

s3-02-48h 8708.1 5584.8 108.2 371.8 14772.9 48

s3-02-48h 8644.8 5534.6 100.7 370.8 14650.9 48

s3-02-48h 8630.9 5536.7 108.2 370.8 14646.6 48

s3-02-72h 34.8 10613.5 6631.7 126.1 336.4 17742.4 72

s3-02-72h 8915.8 5708.2 112.5 272.2 15008.7 72

s3-02-72h 8997.7 5756.8 112.4 275.2 15142.1 72

Aspen, lime 1%, 10% 1h, 80c s3-03-000h 144.5 144.5 0

s3-03-000h 212.6 212.6 0

s3-03-000h 149.1 149.1 0

s3-03-005h 1461.2 428.2 40.1 1929.4 0.5

s3-03-005h 1573.3 456.5 44.1 89.1 2163.0 0.5

s3-03-005h 1720.6 505.4 48.9 84.3 2359.1 0.5

s3-03-01h 1978.8 809.8 43.5 76.0 2908.1 1

s3-03-01h 2039.6 822.7 45.3 77.2 2984.7 1

s3-03-01h 1991.2 800.5 43.4 75.5 2910.6 1

s3-03-03h 3863.3 2290.0 62.7 178.2 6394.2 3

s3-03-03h 4435.2 2668.0 74.9 206.7 7384.7 3

s3-03-03h 2323.1 1457.1 38.8 97.2 3916.2 3

s3-03-06h 3975.8 2638.4 63.8 146.8 6824.8 6

s3-03-06h 40.1 6081.9 3951.5 97.2 236.4 10407.2 6

s3-03-06h 36.0 5308.4 3412.1 84.5 209.2 9050.2 6

s3-03-24h 3506.5 2413.0 59.5 5979.0 24

s3-03-24h 3738.6 2532.8 63.6 65.6 6400.6 24

s3-03-24h 4283.3 2906.1 72.0 74.0 7335.4 24

s3-03-48h 3494.9 2449.6 62.3 6006.8 48

s3-03-48h 3442.6 2415.6 61.6 5919.9 48

s3-03-48h 3827.0 2672.6 66.5 6566.1 48

s3-03-72h 3398.9 2392.5 62.8 5854.2 72

s3-03-72h 4313.7 3012.6 77.9 7404.2 72

s3-03-72h 3395.8 2395.2 62.3 5853.3 72

Aspen, lime 1%, 10% 6h, 80c s3-04-000h 178.1 178.1 0

s3-04-000h 130.4 130.4 0

s3-04-000h 195.6 195.6 0

s3-04-005h 1804.5 510.2 43.9 61.5 2420.1 0.5

s3-04-005h 2062.7 575.7 51.4 69.3 2759.0 0.5

s3-04-005h 2057.9 582.3 51.5 71.9 2763.6 0.5

s3-04-01h 2265.2 885.3 42.6 93.4 3286.5 1

s3-04-01h 2265.8 852.5 45.5 94.6 3258.3 1

s3-04-01h 2263.2 844.4 45.3 91.1 3244.1 1

s3-04-03h 32.1 4578.6 2567.5 68.3 226.3 7472.8 3

s3-04-03h 47.0 6655.6 3754.5 147.1 356.3 10960.5 3

s3-04-03h 36.1 5055.4 2887.9 75.7 254.5 8309.6 3

s3-04-06h 47.5 7339.9 4652.3 175.1 324.8 12539.7 6

s3-04-06h 40.1 6092.3 3866.0 84.5 260.1 10343.1 6

s3-04-06h 37.2 5883.2 3674.5 82.1 258.7 9935.7 6

s3-04-24h 4209.1 2808.8 61.7 91.8 7171.4 24

s3-04-24h 4706.6 3139.8 68.9 103.5 8018.9 24

s3-04-24h 5105.1 3381.8 74.9 114.4 8676.3 24

s3-04-48h 4075.7 2819.0 62.7 6957.3 48

s3-04-48h 4032.5 2785.7 62.1 6880.3 48

s3-04-48h 3926.3 2719.6 60.7 6706.6 48

s3-04-72h 3524.4 2468.9 56.0 6049.2 72

s3-04-72h 3682.6 2562.8 57.7 6303.1 72

s3-04-72h 4330.0 3014.2 69.1 7413.3 72

Aspen, lime 1%, 10% 24h, 80c s3-05-000h 165.1 165.1 0

s3-05-000h 127.2 127.2 0

s3-05-000h 159.4 159.4 0

s3-05-005h 2075.3 490.8 41.9 84.7 2692.7 0.5

s3-05-005h 1965.7 493.3 42.2 77.7 2579.0 0.5

s3-05-005h 2440.6 594.7 52.2 99.1 3186.5 0.5

s3-05-01h 2408.7 829.6 40.8 105.8 3384.8 1

s3-05-01h 1075.9 361.8 8165.5 7375.8 460.5 77.1 17516.7 1

s3-05-01h 2388.4 784.1 40.9 104.7 3318.2 1

s3-05-01h 37.0 4055.0 1307.5 68.1 177.1 5644.8 1

s3-05-03h 4643.7 2375.7 58.8 236.2 7314.4 3

s3-05-03h 35.3 5283.0 2747.0 65.9 272.4 8403.6 3

s3-05-03h 4787.1 2496.3 59.1 247.9 7590.5 3

s3-05-06h 40.4 6454.4 3922.0 135.2 310.7 10862.7 6

s3-05-06h 45.1 7136.2 4260.9 82.6 346.2 11870.9 6

s3-05-06h 40.1 6283.8 3768.3 132.1 306.9 10531.2 6

s3-05-24h 35.9 5880.7 3795.8 71.4 178.5 9962.4 24

s3-05-24h 4862.4 3181.4 59.8 141.3 8245.0 24

s3-05-24h 41.9 6774.2 4373.4 83.7 200.7 11473.9 24

s3-05-48h 4034.3 2757.6 52.8 6844.7 48

s3-05-48h 4782.8 3268.1 61.7 64.2 8176.7 48

s3-05-48h 3717.5 2541.1 46.5 6305.1 48

s3-05-72h 47.3 7663.5 5245.7 153.2 67.0 13176.7 72

s3-05-72h 3439.8 2401.4 45.5 5886.7 72

s3-05-72h 4139.7 2879.7 55.8 7075.3 72

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Oat straw, lime 1.5%, 10%, 3h, 80c s3-06-000h 314.1 126.6 440.8 0

s3-06-000h 329.3 111.5 440.8 0

s3-06-000h 204.5 204.5 0

s3-06-005h 217.9 3765.4 447.4 39.5 343.7 4813.9 0.5

s3-06-005h 245.4 4128.5 501.5 45.4 410.9 5331.6 0.5

s3-06-005h 275.5 4738.1 562.3 49.4 276.6 5901.8 0.5

s3-06-01h 405.2 4399.8 737.4 34.9 236.3 5813.6 1

s3-06-01h 347.5 4760.4 768.2 39.6 254.0 6169.7 1

s3-06-01h 597.3 50.2 8497.2 1376.8 121.3 467.0 11109.9 1

s3-06-03h 672.1 41.9 8217.5 2301.5 58.3 631.0 11922.4 3

s3-06-03h 765.2 46.9 9294.5 2621.1 65.6 703.7 13497.0 3

s3-06-03h 765.3 47.8 9324.0 2617.9 65.1 709.6 13529.7 3

s3-06-06h 898.9 57.4 11890.8 4563.0 180.5 981.2 18571.9 6

s3-06-06h 888.2 58.6 11708.1 4509.2 180.5 954.4 18299.1 6

s3-06-06h 707.7 46.3 9316.7 3450.1 60.6 779.6 14360.9 6

s3-06-24h 538.5 36.4 7406.9 4175.6 50.9 548.1 12756.3 24

s3-06-24h 577.7 36.6 8032.9 4462.8 51.1 559.1 13720.2 24

s3-06-24h 545.1 35.1 7611.6 4238.4 49.9 577.7 13057.8 24

s3-06-48h 437.1 5971.0 3541.8 36.9 307.2 10293.9 48

s3-06-48h 376.1 6105.5 3614.1 38.5 317.2 10451.3 48

s3-06-48h 379.2 6154.7 3646.7 37.7 320.1 10538.4 48

s3-06-72h 447.0 6108.2 3678.0 40.5 268.4 10542.1 72

s3-06-72h 505.9 32.8 6936.4 4173.6 44.3 306.7 11999.6 72

s3-06-72h 618.1 39.6 8493.8 5116.9 56.5 374.2 14699.1 72

Oat straw, lime 1.5%, 10%, 6h, 80c s3-07-000h 244.0 244.0 0

s3-07-000h 0.0 0

s3-07-000h 191.6 191.6 0

s3-07-005h 210.2 3757.1 425.6 35.0 201.2 4629.2 0.5

s3-07-005h 227.0 3984.8 456.3 38.9 214.7 4921.8 0.5

s3-07-005h 287.6 5164.3 578.8 49.5 281.3 6361.6 0.5

s3-07-01h 405.0 4471.9 690.6 33.2 247.2 5847.8 1

s3-07-01h 342.6 4673.3 701.4 36.1 248.3 6001.8 1

s3-07-01h 498.4 39.2 6900.1 1029.2 53.4 363.1 8883.5 1

s3-07-03h 617.6 36.1 7592.0 1871.4 48.9 520.7 10686.8 3

s3-07-03h 701.1 42.9 8597.8 2116.9 56.0 632.8 12147.6 3

s3-07-03h 798.9 48.7 9835.5 2413.0 64.6 729.8 13890.4 3

s3-07-06h 608.3 36.2 7884.2 2668.9 47.0 584.7 11829.4 6

s3-07-06h 589.0 36.6 7755.2 2588.7 48.2 598.1 11615.7 6

s3-07-06h 722.1 44.0 9735.1 3177.6 58.9 784.1 14521.9 6

s3-07-24h 433.8 5984.5 3288.7 462.5 10169.4 24

s3-07-24h 528.3 34.9 7374.2 3985.3 43.4 599.6 12565.7 24

s3-07-24h 558.1 36.8 7861.6 4237.0 44.7 641.1 13379.4 24

s3-07-48h 390.9 6454.4 3846.6 36.7 393.6 11122.2 48

s3-07-48h 406.0 32.0 6719.9 4010.0 39.5 398.6 11606.0 48

s3-07-48h 385.8 6364.1 3797.3 35.8 385.7 10968.7 48

s3-07-72h 688.3 44.4 9648.1 5979.9 135.3 521.5 17017.5 72

s3-07-72h 664.1 42.7 9209.0 5655.2 56.9 496.4 16124.3 72

s3-07-72h 568.4 36.8 7874.7 4831.8 46.3 421.3 13779.3 72

Oat straw, h2so4 0.5%, 5%,15m s3-08-000h 461.5 175.1 433.1 3597.9 232.1 4899.7 0

s3-08-000h 538.5 188.6 403.5 4190.5 230.4 5551.4 0

s3-08-000h 576.5 203.4 427.8 4521.5 257.5 5986.7 0

s3-08-005h 702.4 279.2 3608.9 5768.4 338.8 10697.8 0.5

s3-08-005h 711.9 277.4 3593.7 5852.9 340.7 10776.6 0.5

s3-08-005h 612.9 236.9 3072.2 5045.2 289.9 9257.2 0.5

s3-08-01h 525.5 206.6 3246.3 4398.1 250.5 8627.0 1

s3-08-01h 780.1 308.8 4801.4 6451.2 374.4 12715.8 1

s3-08-01h 898.2 359.9 5585.7 7395.4 435.5 47.3 14722.0 1

s3-08-03h 765.3 275.0 5997.1 6310.7 373.1 59.8 13781.0 3

s3-08-03h 879.5 318.1 6909.1 7256.8 428.7 80.7 15872.8 3

s3-08-03h 739.2 265.2 5765.7 6091.4 359.0 13220.5 3

s3-08-06h 2042.7 733.3 18448.7 16880.8 1410.2 137.1 39653.0 6

s3-08-06h 1899.8 707.6 16176.5 16005.5 924.3 166.2 35879.9 6

s3-08-06h 1177.5 442.4 10195.4 10089.1 583.3 97.6 22585.3 6

s3-08-24h 639.8 245.2 6048.2 5635.4 323.4 12892.0 24

s3-08-24h 738.2 283.2 7055.1 6479.2 373.4 14929.1 24

s3-08-24h 671.9 258.0 6438.4 5898.5 339.7 13606.4 24

s3-08-48h 508.6 189.7 4813.7 4448.0 251.1 10211.0 48

s3-08-48h 541.2 203.6 5133.9 4751.4 268.1 10898.2 48

s3-08-48h 512.1 193.1 4837.6 4494.0 254.0 10290.7 48

s3-08-72h 681.1 255.6 6468.8 5996.4 339.4 13741.3 72

s3-08-72h 774.2 293.5 7449.5 6862.5 390.3 15770.1 72

s3-08-72h 862.0 324.2 8215.6 7617.7 430.4 17449.9 72

Oat straw, h2so4 0.5%, 5%,60m s3-09-000h 612.0 230.5 760.6 4653.0 308.3 6564.4 0

s3-09-000h 603.0 211.9 695.6 4595.4 271.2 6377.1 0

s3-09-000h 562.4 186.7 601.8 4316.1 233.8 5900.8 0

s3-09-005h 745.4 270.7 3885.0 5858.0 322.9 53.5 11135.4 0.5

s3-09-005h 747.2 274.4 3903.3 5966.6 326.2 86.3 11304.0 0.5

s3-09-005h 745.8 274.5 3872.1 5945.2 325.7 134.4 11297.7 0.5

s3-09-01h 560.2 210.1 3702.0 4504.6 246.8 9223.6 1

s3-09-01h 765.5 288.4 5066.4 6101.3 339.7 12561.3 1

s3-09-01h 1073.9 398.9 6984.7 8253.7 467.5 17178.8 1

s3-09-03h 998.7 334.4 8315.9 7597.3 444.6 17690.9 3

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s3-09-03h 1031.1 350.5 8766.7 7971.8 463.6 18583.7 3

s3-09-03h 989.7 326.4 8186.7 7473.9 432.2 17409.0 3

s3-09-06h 933.3 317.2 8652.3 7152.3 410.7 17465.8 6

s3-09-06h 739.0 259.1 7082.0 5813.2 335.8 14229.2 6

s3-09-06h 742.7 260.7 7130.3 5845.0 336.5 14315.1 6

s3-09-24h 913.7 331.6 9787.8 7379.6 428.5 18841.2 24

s3-09-24h 726.8 258.0 7760.2 5791.0 333.3 14869.3 24

s3-09-24h 749.1 266.3 7999.2 5988.3 343.4 15346.2 24

s3-09-48h 563.9 198.6 6008.9 4505.7 255.7 11532.7 48

s3-09-48h 577.8 202.6 6153.2 4611.7 262.7 11808.0 48

s3-09-48h 582.9 203.4 6218.7 4658.6 265.3 11928.8 48

s3-09-72h 709.0 250.6 7599.4 5696.2 324.6 14579.7 72

s3-09-72h 707.4 249.6 7573.5 5676.7 321.4 14528.6 72

s3-09-72h 942.0 335.6 10167.7 7587.2 434.4 19466.8 72

Oat straw, h2so4 0.5%, 5%,90m s3-10-000h 604.2 221.9 776.6 4534.4 293.4 6430.4 0

s3-10-000h 920.5 349.5 1217.3 6891.1 463.2 9841.6 0

s3-10-000h 929.8 315.6 1093.1 7019.5 390.4 9748.3 0

s3-10-005h 666.5 241.2 3711.4 5246.2 282.2 77.5 10224.9 0.5

s3-10-005h 737.4 266.4 4010.7 5779.7 313.0 173.1 11280.3 0.5

s3-10-005h 763.9 277.8 4243.4 5989.1 323.6 111.1 11708.9 0.5

s3-10-01h 564.0 209.9 3954.6 4442.2 241.6 9412.3 1

s3-10-01h 550.4 205.1 3817.0 4331.4 236.1 9140.0 1

s3-10-01h 601.3 224.9 4191.9 4729.8 259.1 10006.9 1

s3-10-03h 871.0 292.6 7877.9 6566.9 379.5 15987.9 3

s3-10-03h 1264.1 417.4 11211.3 9307.8 539.7 22740.4 3

s3-10-03h 1303.6 436.9 11759.8 9680.2 564.6 23745.0 3

s3-10-06h 1162.6 399.5 11781.9 8759.7 506.3 22610.0 6

s3-10-06h 887.8 304.9 8987.9 6689.6 386.3 17256.4 6

s3-10-06h 967.6 332.2 9806.2 7285.3 421.1 18812.4 6

s3-10-24h 943.7 329.5 10804.1 7197.6 418.0 19692.9 24

s3-10-24h 767.1 265.0 8655.3 5849.3 336.6 15873.2 24

s3-10-24h 763.6 265.1 8642.3 5814.0 334.5 15819.6 24

s3-10-48h 576.7 201.0 6628.5 4483.1 253.7 12142.9 48

s3-10-48h 541.9 186.5 6218.7 4210.4 240.2 11397.8 48

s3-10-48h 581.4 203.0 6687.4 4524.0 257.2 12253.0 48

s3-10-72h 688.7 240.0 7905.2 5386.6 305.4 14525.8 72

s3-10-72h 518.3 176.8 5832.9 4033.0 226.0 10787.1 72

s3-10-72h 625.4 216.3 7194.6 4897.4 278.2 13212.0 72

Aspen, h2so4 1%, 5%, 15m s4-01-000h 306.7 418.0 1072.8 7003.2 554.0 9354.6 0

s4-01-000h 232.5 328.0 766.1 5456.1 438.7 7221.4 0

s4-01-000h 152.0 297.2 723.0 4853.2 403.0 6428.4 0

s4-01-005h 225.0 298.2 2432.1 5034.8 405.2 8395.4 0.5

s4-01-005h 232.4 313.3 2908.7 5288.3 422.7 9165.4 0.5

s4-01-005h 221.4 296.5 2522.5 5009.5 399.2 8449.1 0.5

s4-01-72h 121.9 228.9 4819.8 4285.5 321.9 9778.0 72

s4-01-72h 127.7 240.8 5055.4 4485.2 337.3 10246.3 72

s4-01-72h 135.1 253.1 5343.0 4744.5 357.8 10833.5 72

Oat straw, 1% h2so4, 5%, 90m s4-02-000h 823.0 288.6 1424.3 5670.6 360.3 8566.8 0

s4-02-000h 666.6 231.0 1206.0 4422.1 294.1 6819.7 0

s4-02-000h 595.9 203.3 1056.6 3959.5 258.7 6074.0 0

s4-02-005h 772.5 265.1 6025.1 5177.9 334.9 118.0 12693.4 0.5

s4-02-005h 773.9 266.5 5904.3 5180.7 337.4 94.1 12557.0 0.5

s4-02-005h 826.1 284.4 6324.9 5525.1 360.5 93.5 13414.4 0.5

Oat straw, h2so4 0.5%, 5%,30m s4-03-000h 1185.0 396.7 1354.4 9154.1 509.7 12599.8 0

s4-03-000h 1149.9 375.5 1235.3 8649.4 481.9 11891.8 0

s4-03-000h 1416.9 456.1 1462.0 10764.4 591.6 14691.0 0

s4-03-005h 1319.9 436.0 6862.0 10012.2 558.8 241.9 19430.7 0.5

s4-03-005h 1609.0 532.1 8245.7 12271.8 681.8 249.3 23589.8 0.5

s4-03-005h 1591.3 526.1 8178.2 12121.4 672.7 231.4 23321.1 0.5

s4-03-72h 1230.1 408.1 10237.9 9911.9 530.0 22318.0 72

s4-03-72h 1267.5 416.9 10470.8 10123.9 540.7 22819.8 72

s4-03-72h 1087.7 362.0 9089.9 8767.2 469.4 19776.2 72

Oat straw, h2so4 1%, 10%, 30m s4-04-000h 645.0 236.4 736.8 5060.1 315.2 6993.6 0

s4-04-000h 604.9 210.8 636.3 4747.9 277.2 87.8 6564.9 0

s4-04-000h 658.3 238.2 734.5 5155.5 313.8 7100.4 0

s4-04-005h 726.7 255.5 4831.1 5622.9 334.3 11770.4 0.5

s4-04-005h 731.7 267.4 4896.8 5887.9 350.5 12134.2 0.5

s4-04-005h 1081.9 383.8 7160.0 8425.6 500.8 17552.2 0.5

s4-04-72h 577.6 198.5 6036.9 4608.3 263.9 11685.2 72

s4-04-72h 519.9 183.3 5079.2 4428.9 243.8 10455.1 72

s4-04-72h 592.1 209.6 5829.2 5048.4 278.7 11957.9 72

Oat straw, h2so4 1%, 5%, 60m s4-05-000h 582.9 194.3 882.8 4046.1 246.1 5952.3 0

s4-05-000h 790.2 267.9 1248.4 5484.8 341.6 8133.0 0

s4-05-000h 857.4 292.3 1353.7 5982.6 371.3 8857.2 0

s4-05-005h 1054.2 356.3 8010.8 7238.6 452.9 67.9 17180.7 0.5

s4-05-005h 925.9 308.3 6968.3 6326.8 390.9 60.4 14980.7 0.5

s4-05-72h 619.8 212.7 6858.9 4700.3 276.2 12668.0 72

s4-05-72h 563.0 186.4 6453.2 3977.8 241.2 11421.6 72

s4-05-72h 596.5 198.4 6878.4 4230.4 256.7 12160.4 72

Aspen, h2so4 0.5%, 10%, 90m s4-06-000h 326.4 505.9 1522.7 8587.9 737.3 11680.2 0

s4-06-000h 421.5 672.5 1995.1 11373.9 973.9 15436.9 0

s4-06-000h 358.5 551.7 1617.5 9555.2 898.2 12981.0 0

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47

s4-06-005h 419.9 652.9 6583.7 11109.1 949.6 197.8 19912.9 0.5

s4-06-005h 475.8 736.4 7125.0 12569.3 1064.5 154.0 22124.9 0.5

s4-06-005h 473.7 738.4 7887.5 12776.5 1274.5 204.2 23354.9 0.5

s4-06-72h 235.6 460.4 9190.6 8447.5 854.6 19188.7 72

s4-06-72h 649.1 216.1 7436.0 4599.7 278.7 13179.8 72

s4-06-72h 249.1 487.1 9688.8 8834.0 730.4 19989.4 72

Aspen, h2so4 0.5%, 10%, 60m s4-07-000h 331.8 488.8 1349.7 8480.1 695.7 11346.0 0

s4-07-000h 391.9 587.7 1590.2 10180.5 838.5 13588.9 0

s4-07-000h 448.9 688.8 1873.6 11680.9 979.0 15671.3 0

s4-07-005h 473.7 702.7 5952.0 12157.1 988.5 20273.9 0.5

s4-07-005h 425.9 621.4 5656.8 10818.7 884.7 119.2 18526.7 0.5

s4-07-005h 468.4 694.3 5696.5 12066.3 988.0 19913.6 0.5

s4-07-72h 268.0 513.6 9174.6 9548.4 754.3 20258.9 72

s4-07-72h 296.9 571.7 10184.2 10607.6 838.6 22498.9 72

s4-07-72h 251.8 482.6 8635.8 9099.8 874.1 19344.2 72

Aspen, h2so4 1%, 5%, 60m s4-08-000h 120.3 267.7 1045.2 4257.5 414.6 6105.3 0

s4-08-000h 128.2 287.1 1125.4 4460.5 445.6 6447.0 0

s4-08-000h 143.6 319.8 1250.6 4982.5 495.7 7192.2 0

s4-08-005h 132.2 296.3 3900.1 4691.6 457.3 80.0 9557.5 0.5

s4-08-005h 141.4 315.5 4104.9 5012.7 486.7 70.2 10131.4 0.5

s4-08-005h 129.1 286.2 3950.1 4575.1 443.9 87.7 9472.1 0.5

s4-08-72h 118.5 253.4 7835.4 4176.3 398.9 12782.5 72

s4-08-72h 128.3 274.5 8507.1 4535.4 432.5 13877.8 72

s4-08-72h 120.6 257.1 7961.4 4268.6 410.1 13017.7 72

Aspen, h2so4 0.5%, 5%, 15m s4-09-000h 201.7 363.2 749.7 5225.0 431.0 6970.5 0

s4-09-000h 217.9 375.3 665.2 5573.3 413.6 7245.4 0

s4-09-000h 200.0 345.8 630.5 5091.5 385.8 6653.6 0

s4-09-005h 170.3 297.5 1903.5 4593.1 338.6 7303.0 0.5

s4-09-005h 164.3 285.8 1710.4 4426.3 326.3 6913.1 0.5

s4-09-005h 156.3 272.6 1500.5 4207.8 310.2 6447.4 0.5

s4-09-72h 136.9 233.4 3955.3 4211.0 281.6 8818.1 72

s4-09-72h 122.9 208.9 3540.5 3778.1 251.0 7901.5 72

s4-09-72h 128.8 217.9 3699.4 3924.2 260.2 8230.5 72

Aspen, h2so4 1%, 5%, 90m s4-10-000h 131.7 304.1 1428.0 4487.3 479.6 6830.7 0

s4-10-000h 130.7 302.4 1405.7 4449.5 478.0 6766.3 0

s4-10-000h 140.6 327.1 1514.9 4774.4 517.3 7274.3 0

s4-10-005h 164.7 384.6 5260.6 5637.7 605.2 116.9 12169.6 0.5

s4-10-005h 142.1 324.7 4094.3 4796.8 512.7 9870.6 0.5

s4-10-005h 141.6 329.7 4131.5 4860.0 520.2 9983.0 0.5

s4-10-72h 111.7 252.1 8825.7 3804.3 405.4 13399.2 72

s4-10-72h 97.9 217.7 7503.0 3349.8 350.5 11518.9 72

s4-10-72h 109.7 244.6 8589.4 3712.0 394.5 13050.2 72

Aspen, h2so4 0.5%, 5%, 30m s5-01-72h 134.6 249.6 4388.9 4727.4 342.6 9843.1 72

s5-01-72h 130.9 240.7 4267.4 4599.2 334.8 9573.0 72

s5-01-72h 119.5 221.6 3905.7 4205.3 301.2 8753.2 72

Aspen, h2so4 0.5%, 5%, 60m s5-02-72h 114.0 226.3 3286.3 4141.8 337.1 8105.6 72

s5-02-72h 114.8 228.3 3313.5 4170.6 338.8 8165.9 72

s5-02-72h 127.9 255.5 3684.2 4631.3 378.3 9077.3 72

Aspen, h2so4 0.5%, 5%, 90m s5-03-72h 118.1 243.2 4791.9 4285.6 366.1 9804.9 72

s5-03-72h 119.5 245.6 4849.5 4348.8 372.2 9935.5 72

s5-03-72h 114.3 235.1 4647.3 4168.2 358.0 9522.9 72

Aspen, h2so4 1%, 5%, 30m s5-04-72h 119.1 238.6 3756.9 4335.0 351.1 8800.7 72

s5-04-72h 108.4 165.0 273.4 72

s5-04-72h 114.5 223.1 3492.2 4148.4 332.8 8311.1 72

s5-04-72h 123.3 242.1 3782.8 4492.2 412.8 9053.3 72

Aspen, h2so4 0.5%, 10%, 30m s5-05-72h 302.6 528.7 10251.1 715.5 11797.9 72

s5-05-72h 292.4 512.4 9938.3 9853.8 689.9 21286.9 72

s5-05-72h 251.1 440.6 8544.8 8480.3 594.8 18311.5 72

s5-05-72h 324.3 573.2 11124.6 10974.4 772.2 23768.6 72

Aspen, h2so4 1%, 10%, 30m s5-06-72h 247.5 473.6 9400.3 8872.5 701.0 19695.0 72

s5-06-72h 246.0 468.0 9301.6 8788.1 688.3 19492.0 72

s5-06-72h 291.5 558.7 11089.7 10406.5 882.0 23228.4 72

Oat straw, h2so4 0.5%, 10%, 30m s5-07-72h 957.0 354.3 8544.6 8548.1 611.8 19015.9 72

s5-07-72h 961.2 343.9 8338.2 8329.6 595.2 18568.1 72

s5-07-72h 1076.5 395.5 9575.8 9524.1 684.2 68.2 21324.3 72

Oat straw, h2so4 1%, 5%, 30m s5-08-72h 616.9 194.1 5271.1 4613.8 249.6 10945.5 72

s5-08-72h 597.4 192.6 5225.0 4580.5 249.7 10845.2 72

s5-08-72h 606.0 195.7 5296.1 4639.1 251.0 10987.9 72

Oat straw, h2so4 0.5%, 10%, 90m s5-09-72h 952.2 339.2 8928.9 8000.8 432.5 18653.6 72

s5-09-72h 978.0 346.6 9149.8 8181.7 435.8 19091.9 72

s5-09-72h 1034.9 367.8 9691.0 8681.1 471.2 20246.0 72

Oat straw, h2so4 0.5%, 10%, 60m s5-10-72h 1117.0 399.3 10205.3 9512.8 512.3 21746.7 72

s5-10-72h 1232.3 408.9 11380.6 9771.7 526.1 166.5 23486.1 72

s5-10-72h 1267.3 407.6 11729.1 9816.2 523.4 268.6 24012.1 72

Oat straw, lime 1.5%, 10%, 3h, 95c s6-01-72h 351.2 5403.4 3388.2 345.6 9488.5 72

s6-01-72h 511.2 6735.6 4215.3 45.1 433.3 11940.6 72

s6-01-72h 585.6 35.3 7768.2 4840.2 54.6 506.5 13790.5 72

Oat straw, lime 1.5%, 10%, 24h, 95c s6-02-000h 36.4 36.4 0

s6-02-000h 0.0 0

s6-02-000h 0.0 0

s6-02-005h 192.2 2611.7 253.8 518.4 3576.1 0.5

s6-02-005h 207.4 2804.0 268.4 37.5 551.3 3868.7 0.5

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s6-02-005h 223.6 3037.8 292.1 39.8 595.2 4188.5 0.5

s6-02-01h 361.2 3832.6 405.8 39.0 406.8 5045.4 1

s6-02-01h 287.3 3823.6 404.1 40.6 396.5 4952.1 1

s6-02-01h 397.5 3829.2 613.7 39.7 461.4 5341.4 1

s6-02-03h 404.1 5013.2 822.4 37.9 346.2 6623.7 3

s6-02-03h 371.1 4632.5 761.0 36.8 319.2 6120.5 3

s6-02-03h 436.4 5354.6 882.3 41.6 371.5 7086.4 3

s6-02-06h 390.9 5169.5 1216.6 36.6 419.9 7233.5 6

s6-02-06h 385.2 5052.4 1194.4 37.5 411.5 7080.9 6

s6-02-06h 434.9 5663.5 1340.3 40.6 459.0 7938.3 6

s6-02-24h 351.2 4645.2 2139.9 441.9 7578.2 24

s6-02-24h 509.1 36.0 6752.1 3107.9 47.9 639.2 11092.1 24

s6-02-24h 361.7 4742.4 2213.5 35.9 433.9 7787.4 24

s6-02-48h 494.5 33.9 6564.9 3810.1 46.7 598.6 11548.7 48

s6-02-48h 391.9 5230.0 3055.4 40.9 469.5 9187.6 48

s6-02-48h 494.9 34.7 6625.4 3849.2 51.4 602.2 11657.8 48

s6-02-72h 507.3 34.9 6791.2 4291.6 45.9 583.4 12254.2 72

s6-02-72h 488.2 40.6 7796.6 4910.6 53.4 674.2 13963.7 72

s6-02-72h 429.8 34.5 6856.4 4337.4 49.8 580.2 12288.0 72

Oat straw, water, 10%, 3h, 95c s6-03-72h 61.7 3618.8 605.3 43.9 4329.6 72

s6-03-72h 76.2 39.3 4574.8 754.5 55.6 62.0 5562.3 72

s6-03-72h 62.3 3706.6 616.9 44.1 4429.9 72

Oat straw, water, 10%, 24h, 95c s6-04-72h 72.8 40.9 4508.8 841.9 51.1 62.9 5578.4 72

s6-04-72h 62.6 36.1 3912.1 734.9 42.7 59.8 4848.1 72

s6-04-72h 83.2 47.9 5281.7 978.7 60.2 6451.7 72

Aspen, lime 1.5%, 10%, 3h, 95c s6-05-72h 3844.5 2684.7 61.0 6590.2 72

s6-05-72h 38.0 5272.6 3665.1 82.1 9057.8 72

s6-05-72h 35.8 4778.3 3305.4 74.7 8194.2 72

Aspen, lime 1.5%, 10%, 24h, 95c s6-06-000h 0.0 0

s6-06-000h 0.0 0

s6-06-000h 0.0 0

s6-06-005h 1551.9 390.4 36.4 60.6 2039.3 0.5

s6-06-005h 1539.0 387.7 37.3 59.4 2023.3 0.5

s6-06-005h 1611.2 403.2 39.3 59.7 2113.5 0.5

s6-06-01h 2033.6 641.0 38.2 66.4 2779.1 1

s6-06-01h 1984.3 626.3 37.3 65.2 2713.1 1

s6-06-01h 1985.3 621.2 36.3 64.9 2707.7 1

s6-06-03h 2637.1 1263.7 37.2 115.6 4053.7 3

s6-06-03h 2634.0 1266.4 36.2 115.4 4051.9 3

s6-06-03h 2762.8 1323.7 36.0 122.2 4244.6 3

s6-06-06h 3098.1 1835.6 41.0 137.0 5111.8 6

s6-06-06h 3116.9 1866.3 38.8 134.2 5156.1 6

s6-06-06h 3275.7 1940.2 42.4 148.8 5407.1 6

s6-06-24h 3495.0 2302.3 46.4 85.8 5929.5 24

s6-06-24h 3866.9 2534.1 52.0 96.5 6549.6 24

s6-06-24h 34.1 3906.2 2561.0 54.2 97.2 6652.8 24

s6-06-48h 2859.9 1913.7 39.5 4813.1 48

s6-06-48h 34.5 4061.5 2701.2 56.7 60.3 6914.1 48

s6-06-48h 32.2 3797.5 2521.7 51.7 6403.1 48

s6-06-72h 3784.7 2514.4 53.3 6352.4 72

s6-06-72h 33.4 3934.7 2626.0 54.7 6648.8 72

s6-06-72h 38.5 4501.5 2979.5 62.6 7582.2 72

Aspen, water, 10%, 24h, 95c s6-07-72h 1514.4 247.5 61.5 1823.4 72

s6-07-72h 1611.1 263.6 66.0 1940.8 72

s6-07-72h 1748.1 286.6 69.7 2104.4 72

Aspen, water, 10%, 24h, 95c s6-08-72h 1728.7 396.8 62.6 2188.2 72

s6-08-72h 1733.3 399.3 63.2 2195.8 72

s6-08-72h 1937.5 442.4 70.2 2450.0 72

Oat straw, lime 1%, 10%, 6h, 80c s7-01-72h 647.0 43.3 9599.7 5884.2 56.2 397.3 16627.8 72

s7-01-72h 446.5 35.8 7792.8 4784.3 41.4 320.3 13421.1 72

s7-01-72h 508.7 41.5 9017.1 5525.6 53.1 373.5 15519.4 72

Oat straw, lime 1%, 10%, 24h, 80c s7-02-72h 498.9 42.2 9154.7 5610.9 52.9 433.4 15793.0 72

s7-02-72h 610.3 43.8 9435.4 5785.1 54.0 415.0 16343.6 72

s7-02-72h 549.1 39.2 8476.2 5206.1 47.1 373.2 14691.0 72

Oat straw, lime 1.5%, 10%, 24h, 80c s7-03-72h 697.4 44.9 9922.5 6367.9 51.1 387.5 17471.4 72

s7-03-72h 593.3 38.2 8468.0 5452.1 41.6 322.9 14916.0 72

s7-03-72h 643.3 40.9 9228.6 5935.8 51.6 355.8 16256.0 72

Aspen, lime 1.5%, 10%, 6h, 80c s7-04-72h 80.8 39.2 6053.4 4104.5 87.2 62.9 10427.9 72

s7-04-72h 33.1 4834.6 3334.0 79.6 8281.4 72

s7-04-72h 33.1 4726.5 3261.6 79.9 8101.1 72

Oat straw, lime 1%, 10%, 1h, 80c s7-05-72h 383.7 5773.3 3253.3 139.4 9549.7 72

s7-05-72h 469.6 37.8 8537.9 4772.3 47.1 214.8 14079.4 72

s7-05-72h 452.7 36.6 8251.3 4605.4 49.7 190.1 13585.7 72

Oat straw, water, 10%, 3h, 80c s7-06-72h 90.9 35.4 4399.2 710.8 46.6 104.1 5387.1 72

s7-06-72h 78.1 35.7 4224.7 658.9 56.6 71.6 5125.6 72

s7-06-72h 117.8 35.4 4490.4 785.7 45.8 103.0 5578.1 72

Oat straw, h2so4 1%, 5%, 15m s7-07-72h 735.2 241.1 6143.1 5880.1 314.7 13314.2 72

s7-07-72h 759.2 246.3 6125.1 5981.0 323.8 13435.5 72

s7-07-72h 637.0 211.2 5241.5 5155.6 277.4 11522.7 72

Aspen, water, 10%, 3h, 80c s7-08-72h 1859.9 304.9 63.1 2227.9 72

s7-08-72h 1761.6 290.3 60.4 2112.2 72

s7-08-72h 1784.4 301.0 59.1 2144.5 72

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Aspen, lime 1.5%, 10%, 24h, 80c s7-09-72h 4498.0 3051.4 66.5 7615.9 72

s7-09-72h 4481.6 3039.1 64.2 7584.9 72

s7-09-72h 4201.8 2856.4 61.5 7119.8 72

Aspen, lime 1.5%, 10%, 3h, 80c s7-10-72h 3228.3 2273.8 57.9 5559.9 72

s7-10-72h 3093.1 2140.2 57.0 5290.4 72

s7-10-72h 3231.4 2264.1 58.4 5553.8 72