Wood chemistry and isolation of extractives from wood Literature study for BIOTULI project Guangyu Yang Pirjo Jaakkola Saimaa University of Applied Sciences Dec. 2011

Contents

1. Introduction ....................................................................................................... 3 2. Wood as natural resource ................................................................................... 3 3. Wood chemistry ................................................................................................ 5 4. Wood extractives ............................................................................................. 10

4.1 Chemistry of wood extractives ................................................................. 10 4.2 Morphological site and function of extractives ......................................... 18 4.3 Isolation of extractives from wood ........................................................... 19 4.4 Identification of extractives ...................................................................... 22

5. Bacterial and antibacterial ................................................................................ 23 6. Extractives from wood with antibacterial function ........................................... 24 7. Bio-refining based on wood materials .............................................................. 25 8. Extractives from Scots pine ............................................................................. 27 9. Summary ......................................................................................................... 28 10. Reference ........................................................................................................ 28

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1. Introduction Wood, as the traditional resources, is used in many ways to support our life, e.g.

energy, building, paper and chemicals etc. With the hot topics in our society

considering energy crisis, environment issue and sustainable development, the bio-

chemical refined from wood become especially important field for the research and

industry development all over the world. Project BIOTULI are carried out with the

aim to find out the compounds from wood material that have antimicrobial

characteristics.

This report focuses on the literature study about wood, wood chemistry and wood

extractives. The knowledge on the wood extractives is specially highlighted, e.g. the

chemistry of extractives, extraction of extractives and identification of extractives.

This report was mainly figured out on the basis of several books considering the

wood industry and also published standard research results.

2. Wood as natural resource

Tree, as perennial and seed-bearing plants, are classified into two broad categories

known commercially as softwoods and hardwoods. Softwoods are also referred to as

coniferous wood (conifers). However, these general names can not be used

exclusively as a measure of hardness because considerable overlap occurs in the

range of average specific gravities of softwood and hardwood. Another classification

is based on the retention of leaves on the tree. Major commercial softwoods are

generally called evergreen trees, i.e. they retain new leaves for several years.

Hardwood are also called deciduous trees, i.e. they commonly shed their broad or

blade-like leaves each fall at the end of the tree’s growing season. There are

differences between wood species in terms of fibre length properties, annual growth,

wood cells, wood pores and also chemistry of woods.

Both softwoods and hardwoods are widely distributed on the earth. The number of

known softwood species is about 1000 and hardwood species is about 30000- 35000.

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In North America, about 1200 species exist naturally and about 100 species are of

commercial importance. In Europe, the corresponding approximate numbers are 100

and 20. Finland is the most extensively forested country in Europe. Forests cover 86

percent of its land area. In all, Finland has about thirty indigenous tree species, as

shown in Appendix 1. Among these trees, almost half of the volume of the timber

stock consists of pine (Pinus sylvestris). The other most common species are spruce

(Picea abies,) downy birch (Betula pubescens) and silver birch (Betula pendula). The

Finnish forest industry is based on the use of these principal tree species.

The main structural parts of a tree include stem, branch, root, bark and foliage and all

of them are suitable as the renewable natural resources. In general, wood is an

anisotropic material, with respect to its anatomical, physical, and chemical properties.

Wood is made up of different kinds of cells, performing the necessary functions of

mechanical support, water transport, and metabolism. Wood is degradable by fungi,

microorganisms and heating. The structure of a tree is shown in Fig. 1 and Appendix

2. The structure of wood stem and wood bark is shown in Appendix 3.

Fig. 1 Structure of a tree (http://www.infovisual.info)

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3. Wood chemistry The moisture content of a living tree varies seasonally and the average values are in

the range of 40%-50% of the total wood mass. The chemical constituents of dry wood

species are so-called structural substances and non-structural substances. Structural

substances are cellulose, hemicelluloses and lignin. Non-structural substances are

mostly low-molecular-mass compounds, e.g. extractives, some water-soluble organics,

and inorganics, as shown in Fig. 2.

Fig. 2 General classification and content of the chemical wood components

(Alén, 2011)

The gross chemical composition of softwood and hardwood is presented in Appendix

4. Softwood differs typically from hardwood with regard to their chemical

composition, for example, in pine and birch. The cellulose content in pine and birch is

more or less the same, but pine usually contains less hemicelluloses and more lignin.

Furthermore, the chemistry of hemicelluloses and lignin also differ for pine and birch.

On the other hand, the cellulose, hemicelluloses and lignin are not uniformly

distributed in wood cells, and their relative mass proportions can vary widely,

depending on the morphological region and age of the wood. In the following text,

the chemistry of cellulose, hemicelluloses and lignin are introduced.

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Cellulose

Cellulose is the world’s most abundant and important biopolymer. Cellulose is a

polydispersed linear homopolysaccharide consisting of -D-glucopyranose moieties

linked together by (1 4)-glycosidic bonds. The degree of polymerization (DP) of

native wood cellulose is of the order of 10000. Because of the strong tendency for

intra- and intermolecular hydrogen bonding, bundles of cellulose molecules aggregate

to microfibrils, which form either highly ordered (crystalline) or less ordered

(amorphous) regions. Microfibrils are further aggregated to fibrils and finally to

cellulose fibers. The structure of cellulose molecule is shown in the Fig. 3.

Fig. 3 A segment of cellulose chain (Sjöström, 1998)

The tight fiber structure created by hydrogen bonds results in the typical material

properties of cellulose, such as high tensile strength and insolubility in most solvents.

X-ray and other diffraction methods have played a decisive role in the analysis of the

crystalline structure of cellulose. It is commonly accepted that native cellulose has

parallel structure. For isolation of cellulose from wood, a direct nitration of wood

yields undegraded cellulose trinitrate, which is soluble in organic solvents. On the

other hand, the glycosidic linkages are easily cleaved by strong mineral acids and

therefore cellulose can be hydrolyzed to simple sugars. However, for a complete

hydrolysis of cellulose, concentrated acid solutions must be used in order to bring

about the necessary swelling and at least a partial destroying of the ordered regions.

Hemicelluloses

Besides cellulose, hemicelluloses are other major naturally occurring carbohydrate-

based polymers, which are heteropolysaccharides and are clearly less well-defined

than cellulose. The building units of hemicelluloses are hexoses, pentoses or

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deoxyhexoses, as shown in Appendix 5. These units exist mainly as six-membered

(pyranose) structures either in the - or -forms.

Hemicelluloses vary among hardwoods and softwoods according to the type and

content. Softwood hemicelluloses mainly include galactoglucomannans and

arabinoglucuronoxylan. Galactoglucomannans are built up of a mainly linear

backbone chain of (1 4)-linked and partially acetylated -D-glucopyranose and -D-

mannopyranose units, which are substituted at C-6 with a variable number of a single

-D-galactopyranose unit. Arabinoglucuronoxylan consists of a linear frame-work of

(1-4)-linked -D-xylopyranose units with branches of both 4-O-methyl- -D-

glucuronic acid and -L-arabinofuranose. Unlike the hardwood xylan, no acetyl

groups are present. Hardwood hemicelluloses mainly include glucuronoxylan and

glucomannan. Glucuronoxylan is composed of the same framework as the

arabinoglucuronoxylan, but it contains much fewer uronic acid substituents. No

arabinose units are present and the xylose residues are partially acetylated.

Glucomannan has the same linear framework as galactoglucomannans, except that it

is unsubstituted and has a higher glucose to mannose ratio. For a special type of wood,

e.g. larches, arabinogalactan content could reach 10-20% by mass. The structures of

the different hemicelluloses are shown in Fig. 4.

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Fig. 4 Partial chemical structure of hemicelluloses from wood (Stenius, 2000)

Hemicelluloses are usually isolated by successive extractions with dimethyl sulfoxide

and aqueous alkali. However, degradation caused by the alkali cannot be completely

avoided. Galactoglucomannans are easily depolymerised by acids and especially the

bond between galactose and the main chain. The acetyl groups are much more easily

cleaved by alkali than by acid. For arabinoglucuronoxylan, the arabinose side chains

can be easily hydrolyzed by acids due to their furanosidic structure which is less

resistant to hydrolysis. Unlike all other wood hemicelluloses, larch arabinogalactan is

extracellular and can be extracted quantitatively from the heartwood with water. The

reliable analysis of hemicelluloses is based on the separate determination of the

polysaccharide constituents using chromatographic method. The extractive-free wood

sample is subjected to an acid hydrolysis after which the liberated monosaccharides

are separated and quantified.

Lignin

Lignin is an amorphous polymer and the chemical structure of lignin is irregular in

the sense that different structural elements are not linked to each other in any

systematical order. In general, lignins are roughly classified into softwood lignin,

hardwood lignin and grass lignin. Besides these native lignin, there are several

methods can be used to separate the lignin, and therefore, various forms of lignin are

available, e.g. milled wood lignin, dioxane lignin or enzymically liberated lignin,

Kraft lignin, alkali lignin etc. Although native lignins behave as an insoluble and

three-dimensional network, the isolated lignins exhibit maximum solubility in

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solvents including dioxane, acetone, methyl cellosolve, tetrahydrofuran, dimethyl-

formamide and dimethyl sulfoxide.

Lignin can be defined as a polyphenolic material arising primarily from enzymic

dehydrogenative polymerization of three phenylpropanoid units (p-hydroxycinnamyl

alcohols), as shown in Fig. 5. The proportions of the precursors in lignins vary with

their botanical origin. Normal structural elements of softwood lignins are derived

principally from trans-coniferyl alcohol (90%) with the remainder consisting mainly

of trans-p-coumaryl alcohol. In contrast, hardwood lignins are mainly composed of

trans-coniferyl alcohol and trans-sinapyl alcohol in varying ratios (about 50% for

each alcohol). The simplified representation of a segment of softwood lignin is shown

in Fig. 6.

Fig. 5 The building units of lignin (Stenius, 2000)

Fig. 6 Simplified representation of a segment of softwood lignin (Sjöström, 1998)

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The structural building blocks of lignin are joined together by ether linkages (C-O-C)

and carbon-carbon bonds (C-C) and the former ones dominate both in softwood and

hardwood. The dominating bond types and frequencies are shown in Appendix 6, and

the frequency of these groups can vary according to the morphological location of

lignin. As its precursors, the lignin polymer contains several function groups in the

side chain, e.g. methoxyl groups, phenolic hydroxyl groups and aldehyde groups.

Only relatively few of the phenolic hydroxyl groups are free because most of them

form linkages to the neighboring phenylpropane units.

Extractives

Extractives can be regards as non-structural wood constituents and usually represent a

minor fraction in wood. Although there are similarities in the occurrence of wood

extractives within families, there are distinct differences in the composition even

between closely related wood species. Furthermore, various parts of the same tree, e.g.

stem, branches, roots, bark and needles, differ markedly with respect to both their

amount and composition of extractives.

The extractives comprise both inorganic and organic components. Generally, content

of extractives is higher in bark, leaves and roots, than that in stem wood. The

inorganic components measured as ash seldom exceeding 1% of the dry wood weight.

However, the ash content of needles, leaves and bark can be much higher. Organic

components are an extraordinarily large number of individual compounds of both

lipophilic and hydrophilic type, and their contents are usually less than 10%, but it

can vary from traces up to 40% of the dry wood weight.

4. Wood extractives

4.1 Chemistry of wood extractives

Organic extractives of wood can be classified into the different groups as shown in

Table 1, i.e. aliphatic and alicyclic compounds, phenolic compounds and other

compounds. The composition of extractives varies widely from species to species,

and the total amount of extractives in a given species depends on growth conditions.

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For example, the typical content of extractives in Scots pine (Pinus sylvestris),

Norway spruce (Picea abies) and silver birch (Betula pendula) is, respectively, in the

range of 2.5 to 4.5, 1.0 to 2.0, and 1.0 to 3.5%, of the wood dry solids.

Table 1 Classification of organic extractives in woods (Stenius, 2000) Aliphatic and alicyclic compounds Terpenes and terpenoids (including resin acids and steroids) Esters of fatty acids (fats and waxes) Fatty acids and alcohols Alkanes

Gums (polysaccharides) Linear structure Branched structure Branch-on-branch structure

Phenolic compounds Simple phenols Stilbenes Lignans Isoflavones Condensed tannins Flavonoids Hydrolyzable tannins

Other compounds Sugars Cyclitols Tropolones Amino acids Alkaloids Coumarins Quinones

After felling of the tree, the content of extractives starts immediately to decrease and

the chemical composition of the fraction changes. Exposure to air affects the carbon-

carbon double bonds in extractives and initiates a chain reaction that generates free

radicals which, in turn, are particularly strong oxidants. Transition metal ions and

light generally accelerate this kind of auto-oxidation. Furthermore, extractives are

oxidized by certain enzymes, and some enzymes also act as catalysts in the hydrolysis

of the esterified components. All these chemical and biochemical reactions are largely

influenced by the conditions prevailing during wood storage and are markedly faster

when the wood is stored in the form of chips instead of logs. It is also known that the

hydrolysis of glycerides leading to free fatty acids and glycerol proceeds faster when

the conditions for wood storage are wet instead of dry. The chemistry and property of

the different types of extractives are introduced as follows.

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Terpenes and terpenoids Terpenes and their derivatives comprise a broad class of compounds (more than 4000

have been isolated and identified), which appearance in the plant. The term

“terpenes” refers generally to pure hydrocarbons, whereas the compounds collectively

called as “terpenoids” bear one or more oxygen-containing functional groups, such as

hydroxyl, carbonyl, and carboxylic acid groups. The basic structural unit of terpenes

is isoprene and they can be divided into subgroups according to the number of

isoprene units linked in a terpene, as shown in Table 2. Mono-, sesqui-, di-, tri- and

polyterpenoids are the most abundant terpenes in wood. In addition to this

classification, terpenes can be generally classified according to the number of rings

within a structure, for example, into acyclic, monocyclic, bicyclic, tricyclic, and

tetracyclic terpenes. Fig. 7 shows typical examples of some common terpenes and

terpenoids. Appendix 7 collects some common terpenes and terpenoids in wood.

Considering the polyterpenoids, acyclic primary alcohols consisting of 6-9 isoprene

units, e.g. betulaprenols, and esterified with various saturated fatty acids are present

in silver birch (Betula pendula) Various polyprenes are also present in certain wood

species in the form of rubber and gutta percha. In these macromolecules, the number

of isoprene units is high.

Table 2 Classification of the main terpene structural types in woody tissues(Stenius,

2000)

Name Number of (C10H16) units Molecular formula

Monoterpenes

Sesquiterpenes

Diterpenes

Triterpenes

Polyterpenes

1

1.5

2

3

4

C10H16

C15H24

C20H32

C30H48

C40H64

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Fig. 7 Chemical structure of some common terpenes and terpenoids (Stenius, 2000)

Monoterpenes and monoterpenoids are volatile compounds and contribute

substantially to the odor of wood. Monoterpenoids occur mainly in softwood

oleoresin, either as hydrocarbons or their derivatives. Certain monoterpenoids, such

as bornyl acetate, are typical compounds of needles, and are more seldom present in

wood. Monoterpenes represent one of the most important constituents of the resin

canal extractives and exudates of softwoods. Although monoterpenes and

monoterpenoids are rare in common hardwood species, some of these compounds are

minor constituents of the oleoresins of tropical hardwoods.

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Sesquiterpenes and Sesquiterpenoids represent a wide variety of compounds, and they

are found as components of canal resin and of the heartwood deposits of softwoods.

They usually represent a minor portion of the volatile substances of certain pines.

Sesquiterpenes and –terpenoids are found in many tropical hardwoods but are rare

components of hardwoods from temperate zones. Since these compounds occur

usually only in small amounts and are therefore commercially less important.

Diterpenes and Diterpenoids constitute a major part of the canal extractives and are of

great industrial importance. Diterpenoids can be divided into acyclic, bicyclic,

tricyclic, tetracyclic and macrocyclic structure types. Diterpenoids are present either

as hydrocarbons or as derivatives with hydroxyl, carbonyl or carboxyl groups. They

seem to be restricted to softwood species mainly in the form of resin acids and only

some diterpenoids have been found in tropical hardwoods. The most common resin

acids are bicyclic, tricyclic, and tetracyclic diterpenoids and they can be classified

into abietane, pimarane, labdane, and phyllocladene type derivatives.

Triterpenes and triterpenoids comprise mainly oxygenated derivatives and are

traditionally treated as two classes of compounds, i.e. triterpenoids and steroids.

Triterpenoids can be roughly divided into three subgroups: tetracyclic lanostane,

pentacyclic lupine, and pentacyclic oleanane derivatives. Steroids are similar

compound groups comparing with triterpenoids, but they differ from some of

tetracyclic terpenoids only by the postcyclization loss of methyl groups in

biosynthesis process from the acyclic squalene precursor. Triterpenoids and steroids

occur mainly as fatty acid esters and as glycosides, but also in the free form.

Triterpenoids and steroids are common in softwoods and generally in relatively small

amounts. A great variety of triterpenes and steroids are also present in many

hardwoods of tropical and temperate zones. In both softwood and hardwood, the most

prominent compound is sitosterol, which is potential raw material for making wood-

based chemicals.

Phenolic extractives

Wood contains a large variety of aromatic extractives reaching from simple phenols

to complex polyphenols and their related compounds. Fig. 8 shows selected examples

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of the chemical structures of some phenolic extractives and more detail information

can be found in Appendix 8. It is often happen that polyphenols are colored

compounds which are accumulated abundantly in the heartwood of many species.

Some of them are probably degradation products of compounds that can be

hydrolyzed during extraction of steam distillation, e.g. glycosides. This kind of

extractive also has fungicidal properties and thus protects the tree against

microbiological attack.

Fig. 8 Chemical structures of some phenolic extractives (Stenius, 2000)

Stilbenes are derivatives of 1, 2 – diphenylethene. These compounds are mainly

located in the heartwood of the Pinus species. In contrast, lignans are distributed

widely in the stem wood of both softwoods and hardwoods. They are basically

formed by oxidative coupling of two phenylpropane units and can be classified

according to their chemical structures into several groups. Hydrolyzable tannins are

esters of a sugar residue (usually D-glucose) with one or more polyphenol carboxylic

acids, e.g. gallic, digallic and ellagic acids. The ester linkages in these structures are

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readily hydrolyzed by acids, alkalis and enzymes. Flavonoids have a typical

diphenylpropane skeletal structure. These compounds are widely distributed in the

stem wood of both softwoods and hardwoods. Isoflavones or isoflavonoids have a

slightly different carbon skeleton from that of flavonoids. Condensed tannins are

polymers of flavonoids consisting mainly of 3-8 flavonoid units. They are distributed

widely in the stem wood of many species.

Aliphatic extractives

Aliphatic extractives contain alkanes, fatty alcohols, fatty acids, fats and waxes. Only

small amounts of alkanes, free alcohols, and free fatty acids occur in woods. The

most common fatty acid constituents belong both to the saturated and unsaturated

compounds. More than 30 fatty acids (or fatty acid moieties) have been identified in

softwoods and hardwoods, as shown in Appendix 9. The major parts of the fatty acids

in wood are esterified with glycerol (i.e. fats) or with higher fatty alcohols and

terpenoids (i.e. waxes). In softwoods, the parenchyma resin is mainly composed of

fats. In hardwoods, the parenchyma resin is virtually the only resin type and contains

a significant proportion of waxes as well as fats. Fats and waxes are hydrolyzed

during wood storage.

Inorganic components

In woods from temperate zones, elements other than carbon, hydrogen, oxygen, and

nitrogen make up between 0.1% and 0.5% of the dry solids in wood, whereas those

from tropical and subtropical regions make up even to 5%. In practice, the total

amount of wood inorganics is measured as ash, which is the residue obtained after the

proper combustion of the organic matter of a wood sample. The ash contains mainly

different metal oxides and average values for the ash content of commercial

softwoods and hardwoods are generally in the range of 0.3% to 1.5% of the wood dry

solids. There is also a significant dependence of the ash content and composition on

the environmental conditions under which the tree has grown and, on the other hand,

on the location within the tree. Some of the inorganic elements present in wood are

essential for wood growth. In many cases, alkali and alkali earth elements such as

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potassium, calcium, and magnesium make up about 80% of the total inorganic

elemental constituents of softwoods and hardwoods.

Chemistry of bark

Bark is the external layer which surrounds the stem, branches, and roots, amounting

to about 10-15% of the total weight of the tree. Bark can roughly be divided into

living inner bark or phloem and dead outer bark or rhytidome. The main components

of inner bark are sieve elements, parenchyma cells, and sclerenchymatous cells. Sieve

elements perform the function for transportation of liquids and nutrients. Parenchyma

cells have the function of storing nutrients. Sclerenchymatous cells have the function

as the supporting tissue. The outer bark, which consists mainly of periderm or cork

layers, protects the wood tissues against mechanical damage and preserves it from

temperature and humidity variations.

The chemical composition of bark varies among the different tree species and also

depends on the morphological elements involved. Many of the constituents present in

wood also occur in bark, but their proportions are different. Bark can roughly be

divided into the following fractions: fibers, cork cells and fine substance including the

parenchyma cells. The fiber fraction consists of cellulose, hemicelluloses, and lignin.

The walls of the cork cells are impregnated with suberin, whereas the polyphenols are

concentrated in the fine fraction.

Bark contains the high content of certain soluble constituents, e.g. extractives, and

mineral content. The total content of both lipophilic and hydrophilic extractives

usually corresponds to 20 – 40% of the dry weight of bark. The lipophilic fraction

consists mainly of fats, waxes, terpenoids, and higher aliphatic alcohols. Terpenoids,

resin acids, and sterols are located in the resin canals present in the bark and also

occur in the cork cells and in the pathological exudate of wounded bark. Triterpenoids

are abundant in bark. The bark contains large amount of phenolic constituents, such

as condensed tannins, monomeric flavonoids, lignans and stilbenes.

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4.2 Morphological site and function of extractives

Wood extractives have their morphological site and function in the tree, as shown in

Table 3. It can be seen that extractives have the wide distribution in the tree. Fats

mainly exist in parenchyma cell. Terpenoids oleoresin canals mainly contain the

terpenoids. Phenolic substances can be found from foliage also. Some carbohydrates,

e.g. glycosides, sugars, starch and proteins can be found in the cambium and growing

zone of tree. In the sapwood or sap in inner bark, inorganics are transported with the

ascending water.

Table 3 Classification of non-structural components in trees (Alén, 2011)

Extractives Terpenoids

Fats

Phenolic substances

Carbo- hydrates

Inorganic

Subclasses Mono-terpenoids Resin acids Other terpe-noids

Triglyceride Fatty acids steryl esters sterols

Lignans Flavonoid Stibenes Tannins

Glycosides Sugars Starch Proteins Gum Pectins

Various salts

Function in the tree

Protection Physi-ological

Protection Biosynthesis Nutrient reserve Protection

Photo-synthesis Bio-synthesis

Occurrence

Oleoresin canals Heartwood Knots Bark

Parenchyma Cell

Heart-wood Knots Bark Foliage

Sapwood Cambium Heartwood

Sapwood Sap in inner bark

Tree species

Softwoods All wood species

All wood species, Especially softwoods

All wood species

All wood species

The wood extractives contribute to wood properties such as color, odor and taste.

Different types of extractives are necessary to maintain the diversified biological

functions of the tree. For example, traces of certain metal ions are present usually as

functional parts of the enzymes which are needed as catalysts for biosynthesis, fats

constitute the energy source of the wood cells, whereas lower terpenoids, resin acids,

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and phenolic substances protect the wood against microbiological damage or insect

attacks.

The extractives in softwood are located both in the ray parenchyma cells and in the

resin canals. In hardwoods, extractives in parenchyma cells dominate because of the

absence of resin canals. The parenchyma resin of both softwoods and hardwoods is

composed of fats and waxes as well as triterpenoids and steroids. Oleoresin from

resin canals is mainly composed of monoterpenoids and diterpenoids (including resin

acids). Softwoods and hardwoods also differ with respect to the composition of the

phenolic extractives. It is noteworthy that even each species within various families

and genera tends to produce its own, specific phenolic substances.

4.3 Isolation of extractives from wood

For isolation of the extractives from wood, the different methods can be used.

Volatile extractives are represented by high-volatile compounds which can be

separated by water distillation. They are mainly composed of monoterpenes and other

volatile terpenes, terpenoids as well as of many different low molecular compounds.

Resin is the name as a collective name for the lipophilic extractives (with the

exception of phenolic substances). Resin extractives can be extracted with organic

solvents. Water-soluble compounds consist of various phenol compounds,

carbohydrates, glycosides, and soluble salts, which can be extracted by cold or hot

water.

Solvents for extraction of wood

Resins are divided into free acids, e.g. resin acid and fatty acid, and neutral

compounds, e.g. fats and waxes. Resin is soluble in organic solvents but insoluble in

water, and therefore it can be extracted with organic solvents, such as hexane,

dichloromethane, diethyl ether, acetone or ethanol. Table 4 shows the solubility of

extractives in the different solvents. It can be seen that the different non-polar and

polar solvents can be selected for isolation of the certain type of extractives from

woods. The common solvents and the related properties are listed in Appendix 10.

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Table 4 Solubility of wood extractives (Sjöström, 1998)

Extractives

Terpenoids Fats

Phenolic substances

Carbohydrate

Alkanes +++ +++ 0 0

Diethyl ether +++ +++ ++ 0

Dichloro

-methane

+++ +++ ++ 0

Acetone +++ +++ +++ ++

Ethanol ++ ++ +++ +

Water 0 0 + +++

Solubility Non-polar or

Polar solvent

Non-polar or

Polar solvent

Polar (water)

solvent

Water

The table shows that the different solvents can be used for isolation of extractives

according to the various purposes. For the lipophilic compounds, e.g. terpenoids and

fats, the non-polar solvents are good choice for the selectively isolation. For the

hydrophilic compounds, e.g. phenolic substances and some carbohydrate, polar

solvents and water could be used in the extraction. It can be seen that the certain type

of solvents, e.g. acetone, have the ability to dissolve all kind of extractives and

therefore they could be used when the total amount of extractives is determined.

Extraction equipments

The different types of extractor can be used for extraction of wood, e.g. Soxhlet

extractor, Soxtec extractor and Accelerated solvent extraction (ASE). Soxhlet or

Soxtec extractor are traditional way for the extraction of wood, and ASE as developed

equipment play the key role with the aim to the efficient extraction, as shown in

Appendix 11.

When Soxhlet extractor is used, the solvent is boiled and its vapor travels upward

through the extraction tube into the condenser tube. The cool water flowing around

the outside of the condenser tube condenses the vapour of solvent and then solvent

drips into the thimble, containing the sample. When the extractives are soluble in

solvents, they move into the condensed solvent as it accumulated in the thimble. The

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solution containing the extractives builds up in the thimble. Once the liquid reaches

the level of the bypass arm, it is siphoned back into the flask. The advantage of the

Soxhlet extractor is that once the extractives are brought into solution, and siphoned

back into the flask, they stay in the flask so that the sample in the extraction thimble

is continuously re-exposed to fresh, heated solvent and thus greatly increasing the

extraction rate.

For Soxtec extractor, a three-stage process is used. In the first stage, a sample-

containing thimble is immersed in boiling solvent for approximately 60 min. In the

second stage, the sample-containing thimble is removed from the solvent and the

process continued as in the Soxhlet extraction approach. This second stage is repeated

for up to 60 min. In the final stage, solvent evaporation takes place within the Soxtec

apparatus, reducing the final extract volume to 1-2 ml in approximately 10 – 15 min.

The advantages of Soxtec over Soxhlet extraction are rapid extraction, smaller solvent

usage and sample concentrated directly within the apparatus. An alternative to Soxtec

is simple to reflux the sample in a flask equipped with a condenser.

Another extraction system called accelerated solvent extraction (ASE) has been

developed. ASE combines elevated temperatures and pressures with the standard

solvents used for Soxhlet extraction. ASE is much faster and requires considerably

less solvent than traditional techniques. ASE system is expensive, but it is the

promising efficient extraction method. ASE technology is a flow-through solvent

extraction system that helps increase productivity and sample throughput while

reducing preparation cost and providing a platform for automation.

Extraction methods

The extraction of wood can be carried out with various solvents and equipments. The

experimental procedure for extraction of wood includes preparation of sample,

extraction process and drying of the residue. The summary of SCAN-CM 49:03 for

content of acetone-soluble matter can be described as follows.

In the sampling procedure, it is important that fresh chips are selected in order to

completely detect the extractives in wood, and also the test portions taken are

22

representative. It is recommended to keep the wood samples in a refrigerator in

polyethylene bags or in packages of aluminium foil. For long-term storage, samples

should be placed in a freezer. In order to carry out efficient extraction work, the wood

chips is disintegrate approximately length of 2 mm and sample temperature should

not exceed 40 C during sample treatment. Meanwhile, the dry matter content in the

wood can be determined. If the dry matter content is below 90%, the whole sample is

allowed to dry overnight in the air at room temperature or in a drying oven at a

temperature not exceeding 40 C.

After the wood sample is put into the extractor, bring the solvent to the boil and

continue the extraction for several hours. The test portion shall be covered with

solvent. After extraction, the solvent can be partially evaporated directly in the

apparatus, e.g. Soxhlet or Soxtec. Then, the residual solvent can be transferred to a

weighed aluminium dish. The sample is evaporated in a fume-cupboard and finally

dried about 30 minutes in a drying oven at 105 C to constant mass. When the

extraction residue is to be further analysed, the drying process might be different

comparing with the above method. In this case, glass flask should be used instead of

aluminium dish and also drying should be carried out at a lower temperature, e.g. at

40 C for 2 hours, to prevent oxidation.

4.4 Identification of extractives

Analysis of extractives can be made at different levels, i.e. gravimetric or

determination of total extractives, determination of different component groups, and

analysis of individual components. Component groups in extracts can be determined

by several chromatographic techniques: gas chromatography (GC), high performance

liquid chromatography (HPLC), size-exclusion chromatography (SEC), supercritical

fluid chromatography (SFC), and thin-layer chromatography (TLC).

For identification of individual components of extractives, gas chromatographic

methods in combination with mass spectrometry play a key role. When GC was used

for analysis, the sample vaporizes in the injector and flows with carrier gas to the

column and then to the detector. The column is in an oven and the temperature of the

column can be controlled. The carrier gas can be helium, hydrogen or nitrogen. The

23

most typical injection techniques are split injection, direct injection and on column

injection. The temperature of the injector is near the highest boiling point of

compounds. In gas chromatography typically capillary columns are used. The column

has an internal diameter of 0.2-0.7 mm and a length of 20-50 m. The stationary phase

is a thin layer in the inner surface of the column and the thickness of the layer is 0.1-1

m. Liquid stationary phases are polymers, typically polysiloxanes. There are long

silicon – oxygen chains in polysiloxane but the properties of the column material

depend on substitution groups in polysiloxane. The substitution groups in the

polysiloxane structure are typically methyl group, phenyl group, cyanopropyl group

or trifluoropropyl group. Using GC for analysis of chemicals, the suitable GC column

should be selected according to the properties of the detected chemicals, as shown in

Appendix 12. A number of detectors are used in gas chromatography. They are flame

ionization detector (FID), electron capture detector (ECD), nitrogen phosphorus

detector (NPD), flame photometric detector (FPD), photo ionization detector (PID),

thermal energy detector (TCD), and mass spectrometer (MS).

When GC is used, sometimes, the derivatization of sample has been considered. A

large number of reagents can be used to prepare derivatives for GC, and normally the

reagents fit into one of three categories: acylation, alkylation or silylation, as shown

in Appendix 13. The GC analysis results depend on equipment parameters and

operating parameters. Equipment selection includes carrier gas, GC column and

detector. Operating parameters include inlet temperature, detector temperature,

column temperature, carrier gas flow rate, injection technique and inlet flow rate etc.

The general procedure for the GC analysis for extractives of wood is shown in

Appendix 14.

5. Bacterial and antibacterial

Bacteria are present in most habitats on Earth, growing in soil, acidic hot springs,

radioactive waste, water, as well as in organic matter and the live bodies of plants and

animals. In all, there are approximately five nonillion bacteria on Earth forming a

biomass that exceeds that of all plants and animals. The vast majority of the bacteria

in the body are rendered harmless by the protective effects of the immune system, and

a few are beneficial. However, a few species of bacteria are pathogenic and cause

infectious diseases, including cholera, syphilis, anthrax, leprosy, and bubonic plague.

24

Bacteria display a wide diversity of shapes and sizes. Bacteria often attach to surfaces

and form dense aggregations called biofilms or bacterial mats. The International

Committee on Systematic Bacteriology (ICSB) maintains international rules for the

naming of bacteria and taxonomic categories and for the ranking of them in the

International Code of Nomenclature of Bacteria.

Antibiotics are chemical compounds used to kill or inhibit the growth of bacteria. The

term antibiotic is now used loosely to include anti-infectives produced from synthetic

and semisynthetic compounds. The term antibiotic may be used interchangeably with

the term antibacterial. However, it is incorrect to use the term antibiotic when

referring to antiviral, antiprotozoal and antifungal agents. Antibiotics can be classified

in several ways. The most common method classifies them according to their

chemical structure as antibiotics sharing the same or similar chemical structure will

generally show similar patterns of antibacterial activity, effectiveness, toxicity and

allergic potential.

Using the tree as the raw materials to obtain the antibacterial chemical compounds,

the study could focus on the chemicals from wood which originally have the

antibacterial function, for example, terpenoids and phenolic substances of wood has

the function to protect tree for sickness, therefore, it might be good way that this type

of extractive could be isolation directly from wood and further purified by the

separation process. On the other hand, it is also possible to use biochemical

conversion method to treat the wood in order to obtain the compounds which have

antibacterial function.

6. Extractives from wood with antibacterial function

With the knowledge of early Section 2, Section 4 and Section 5 as the background,

the certain amount of research work has been done considering extractives from wood

with antibacterial function. The published papers included the research work and

review papers. The research work was carried out with the different plant material

resource, e.g. hardwood, softwood and medicine plant etc. Also, the raw material

from the various locations of wood was detected. For the isolation of extractives, the

different methods are applied, such as, water distillation or water extraction and the

25

different organic solvents extraction etc. For the identification of extractives, the

various chromatographic techniques, e.g. GC and HPLC, are tested. In addition, the

ability of extractives against the different types of bacterial was studied. On the other

hand, the certain amounts of review papers were published considering the group of

study on the certain type of extractives or the certain type of wood etc. This part of

review study will be summarized in the next work report.

7. Bio-refining based on wood materials

We human being has long history to use the renewable resources, but this practice

gradually disappeared with coal-based thermo-chemistry and petroleum-based

industry chemistry. However, increasing competition and rising prices of non-

renewable raw materials, in combination with public concern over global

environmental issues, it is time not only to develop new ways of producing bio-

energy but also to find alternative ways of manufacturing important bio-chemicals.

Using bio-energy as the example, Appendix 15 shows the principal conversion routes

for cellulosic biomass to bio-product. In the following text, the chemical conversion

and thermal conversion of biomass are shortly introduced.

Chemical and biochemical conversion

In conventional conversion of biomass to useful products the most common method is

the pre-treatment of biomass through hydrolysis to glucose and other

monosaccharides, which can be accomplished by chemical (acids) or biochemical

(enzymes) treatments, as shown in Fig. 9. The monosaccharides liberated are suitable

for further fermentation or chemical modification into a wide range of useful value-

added chemicals, as shown in Appendix 16 - 17.

26

Fig. 9 Process scheme for the conversion of biomass-derived carbohydrates (Alén,

2011)

Thermal conversion

Pyrolysis and gasification are two main thermal conversion methods. Pyrolysis refers

to thermal degradation in the complete or near complete absence of an oxidising

agent to provide complex fractions of gases, condensable liquids and char. Whereas,

in gasification, cellulosic materials are converted by heating in the presence of

controlled amount of oxidising agents to primarily provide a simple gaseous phase

(syngas). When molecular oxygen is the oxidising agent, the amount used for

gasification is substantially below that required for stoichiometric combustion. In

additional, the pyrolysis processes are generally carried out at low temperatures than

those of gasification. The thermal conversion of cellulosic biomass can be seen in Fig.

10.

27

Fig. 10 Product groups from thermal conversion of cellulosic biomass (Alén, 2011)

8. Extractives from Scots pine

Pinus sylvestris, commonly known as the Scots Pine, is a species of pine native to

Europe and Asia, ranging from Scotland, Ireland and Portugal in the west, east to

eastern Siberia, south to the Caucasus Mountains, and as far north as well inside the

Arctic Circle in Scandinavia (including Lapland). Scots Pine is the only pine native to

northern Europe, forming either pure forests or alongside Norway Spruce, Common

Juniper, Silver Birch, European Rowan, Eurasian Aspen and other hardwood species.

Scots pine has a dry density around 470 kg/m3 and the bark is thick, scaly dark grey-

brown on the lower trunk, and thin, flaky and orange on the upper trunk and branches.

Scots Pine is an important tree in forestry. The wood is pale brown to red-brown, and

used for general construction work. The wood of pine is used for pulp and sawn

timber products. In Finland and the Scandinavian countries, Scots Pine was used for

making tar in the pre-industrial age. The pine has also been used as a source of rosin

and turpentine.

BIOTULI project was carried out in Saimaa University of Applied Sciences since

2011. The literature and experimental study have been done focusing on the

extraction of extractives from wood with non-polar and polar solvents. The various

species of wood, e.g. pine, birch, spruce and aspen, and also different parts of wood

were test experimentally for the extraction work. The extracted samples were

28

analyzed in Lappeenranta University of Technology in order to identify the interested

chemical compounds. On the other hand, it is also important to make clear the

influence of operating condition, e.g. extraction time, extraction equipment and

extraction temperature, on the extraction results. Therefore, two students (Li yuman

and Zhou yuanlin) in SUAS join in this project for their B.Sc. thesis with the aim to

investigate the operating conditions on the extraction process and the raw material

they used was Scots pine. Furthermore, GC analysis was carried out in their work in

order to preliminary test the extraction solution. Meanwhile, they did the certain

literature study focus on the certain type of extractive from wood.

9. Summary

In this report, the literature study for wood chemistry and bio-refining was introduced.

Considering the BIOTULI project as the background, the report especially focuses on

the extractives of wood, and extraction and identification of extractives. Due to the

extractives of the wood are wide lipophilic and hydrophilic organic compounds, it is

important to focus on the certain types of chemicals which have the antibacterial

function. However, the general wide knowledge about the wood chemistry gives the

good basis for summary of previous research work and also guides the experimental

work. In case the interested chemical compounds were identified, the continuous

work might focus on the scale-up of the process and improvement of productivity of

the process.

10. Reference Alén Raimo, Papermaking science and technology, Book 20, Bio-refining of forest resources, Paperi ja puu, Helsinki, 2011 Dean J. R., Extraction Techniques in Analytical Sciences, John Wiley & Sons, Ltd, UK, 2009 Jennings, W., Mittlefehldt, E., Stremple, P., Analytical Gas Chromatography, ACADEMIC PRESS, USA, 1997 Sjöström, E., Wood Chemistry: Fundamentals and Application. Academic Press, UK, 1993

29

Sjöström, E. and Alén R., Analytical Methods in Wood Chemistry, Pulping, and Papermaking, Springer-Verlag Berlin, 1998 Stenius, P., Papermaking science and technology, Book 3, Forest products chemistry, Fapet, Helsinki, 2000 Scandinavian Pulp, Paper and Board Testing Committee, Content of acetone-soluble matter, SCAN-CM 49:03, 2003 Scandinavian Pulp, Paper and Board Testing Committee, Content of extractable lipophilic matter, SCAN-CM 67:03, 2003

30

Appendix 1 Indigenous tree species in Finland (http://www.forest.fi/) Softwoods Pine (Pinus sylvestris) Spruce (Picea abies) Common juniper (Juniperus communis) Common or european or english yew (Taxus baccata) Hardwoods Silver birch (Betula pendula) Downy birch (Betula pubescens) Common or european alder (Alnus glutinosa) Grey alder (Alnus incana) Aspen (Populus tremula) Rowan, european mountain ash (Sorbus aucuparia) Oaklef mountain ash (Sorbus hybrida) Swedish whitebeam (Sorbus intermedia ) Teodori rowan (Sorbys teodori) European bird cherry (Prunus padus) Littleleaf linden (Tilia cordata) Norway maple(Acer platanoides) Common or english or pedunculate oak (Quercus robur) Common or european ash (Fraxinus excelsior) Saarni Wych elm (Ulmus glabra) European white elm (Ulmus laevis) Crab apple (Malus sylvestris) Common buckthorn (Rhamnus catharticus) Alder or glossy buckthorn (Rhamnus frangula) Hawnthorn (Crataegus monogyna) Goat willow (Salix caprea) Bay or laurel willow (Salix pentandra) Black maul. almond or almond-leaved willow (Salix triandra) Dark-leaved willow (Salix myrsinifolia) No known English name: Crataegus rhipidophylla Salix borealis

31

Appendix 2 Pinus sylvestris (http://www.google.fi/image) (A) tree (B) stem (C) bark (D) stump (E) knots (F) branch and leaves

(A) (B)

(C)

(D)

(E) (F)

32

Appendix 3 The structure of wood stem (a) and wood bark (b) (Sjöström, 1993)

(a)

(b) young stem (A) and mature bark (B)

33

Appendix 4 The chemical composition of various wood species (Sjöström, 1993)

34

Appendix 5 The sugar moieties of wood hemicelluloses (Stenius, 2000)

35

Appendix 6 Major structures and frequencies of the linkages in softwood and hardwood lignins (Stenius, 2000)

36

Appendix 7 Examples of the common terpenes and terpenoids in wood (Stenius, 2000)

37

Appendix 8

Examples of the aromatic extractives in wood (Stenius, 2000)

38

Appendix 9

Examples of fatty alcohols and fatty acids in wood (Stenius, 2000)

FATTY ALCOHOLS

Arachinol or eicosanol (C20), behenol or docosanol (C22)

Lignocerol or tetracosanol (C24)

FATTY ACIDS

Saturated acids:

Lauric or dodecanoic acid, myristic or tetradecanoic acid

palmitic or hexadecanoic acid, stearic or octadecanoic acid

arachidic or eicosanic acid, behenic or docosanoic acid

lignoceric or tetracosanoic acid

Unsaturated acids:

Oleic or cis-9-octadecenoic acid, linoleic or cis,cis-9, 12-octadecadienoic acid

linolenic or cis,cis,cis-9, 12, 15-octadecatrienoic acid

pinolenic or cis,cis,cis-5,9,12-octadecatrienoic acid

eicosatrienoic or cis,cis,cis-5, 11,14-eicosatrienoic acid

39

Appendix 10 Non-polar and polar solvents (http://en.wikipedia.org/wiki/Solvent)

Solvent Chemical formula Boiling point[7]

(°C)

Dielectric constant[8]

Density (g/ml)

Dipole moment

(D)

Non-polar solvents

Pentane CH3-CH2-CH2-CH2-CH3 36 1.84 0.626 0.00

Cyclopentane C5H10 40 1.97 0.751 0.00

Hexane CH3-CH2-CH2-CH2-CH2-CH3

69 1.88 0.655 0.00

Cyclohexane C6H12 81 2.02 0.779 0.00

Benzene C6H6 80 2.3 0.879 0.00

Toluene C6H5-CH3 111 2.38 0.867 0.36

1,4-Dioxane /-CH2-CH2-O-CH2-CH2-O-\ 101 2.3 1.033 0.45

Chloroform CHCl3 61 4.81 1.498 1.04

Diethyl ether CH3CH2-O-CH2-CH3 35 4.3 0.713 1.15

Polar aprotic solvents

Dichloromethane (DCM) CH2Cl2 40 9.1 1.3266 1.60

Tetrahydrofuran (THF) /-CH2-CH2-O-CH2-CH2-\ 66 7.5 0.886 1.75

Ethyl acetate CH3-C(=O)-O-CH2-CH3 77 6.02 0.894 1.78

Acetone CH3-C(=O)-CH3 56 21 0.786 2.88

Dimethylformamide (DMF) H-C(=O)N(CH3)2 153 38 0.944 3.82

Acetonitrile (MeCN) CH3-C N 82 37.5 0.786 3.92

Dimethyl sulfoxide (DMSO) CH3-S(=O)-CH3 189 46.7 1.092 3.96

Polar protic solvents

Formic acid H-C(=O)OH 101 58 1.21 1.41

n-Butanol CH3-CH2-CH2-CH2-OH 118 18 0.810 1.63

Isopropanol (IPA) CH3-CH(-OH)-CH3 82 18 0.785 1.66

n-Propanol CH3-CH2-CH2-OH 97 20 0.803 1.68

Ethanol CH3-CH2-OH 79 24.55 0.789 1.69

Methanol CH3-OH 65 33 0.791 1.70

Acetic acid CH3-C(=O)OH 118 6.2 1.049 1.74

Water H-O-H 100 80 1.000 1.85

40

Appendix 11

Extraction equipments (a) Soxhlet extractor (b) Soxtec extractor (Dean, 2009)

(b)

(a)

41

Schematic of extraction using ASE: 1) load cell into ASE, 2) fill cell with solvent (0.5–1.0 min), 3) heat and pressurize cell (5 min), 4) static extraction (5 min) (repeat if necessary), 5) flush cell with fresh solvent (0.5 min), 6) purge cell with nitrogen (1–2 min), and 7) extract ready. Total extraction time: 12–18 min depending on number of static cycles. (Dorich, B., et al., Accelerated Solvent Extraction with Acid Pretreatment for Improved Laboratory Productivity, the New American Laboratory Website, USA, 2008)

42

Appendix 12

GC column selection (Document from Agilent Company)

43

Appendix 13

Guide to Derivatization Reagents for GC (Bulletin 909A, 1997)

44

Appendix 14

GC analysis step for extractives (Sjöström, 1998)

45

Appendix 15

Principal conversion routes for cellulosic biomass to produce various energy sources

(Alén, 2011)

46

Appendix 16

Examples of fermentation products from glucose (Alén, 2011)

47

Appendix 17

Examples of chemical products from glucose by common chemical treatments (Alén,

2011)