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.
2.
3.
4.
Introduction ....................................................................................................... 3
Wood as natural resource ................................................................................... 3
Wood chemistry ................................................................................................ 5
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
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 biochemical 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.
3
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)
4
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.
5
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 carbohydratebased polymers, which are heteropolysaccharides and are clearly less well-defined
than cellulose. The building units of hemicelluloses are hexoses, pentoses or
6
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.
7
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
8
solvents including dioxane, acetone, methyl cellosolve, tetrahydrofuran, dimethylformamide 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)
9
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.
4.1
Wood extractives
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.
10
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
Gums (polysaccharides)
Terpenes and terpenoids
(including resin acids and steroids)
Esters of fatty acids
(fats and waxes)
Fatty acids and alcohols
Alkanes
Phenolic compounds
Linear structure
Branched structure
Branch-on-branch structure
Simple phenols
Stilbenes
Lignans
Isoflavones
Condensed tannins
Flavonoids
Hydrolyzable tannins
Sugars
Cyclitols
Tropolones
Amino acids
Alkaloids
Coumarins
Quinones
Other compounds
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 carboncarbon 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.
11
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
1
C10H16
Sesquiterpenes
1.5
C15H24
Diterpenes
2
C20H32
Triterpenes
3
C30H48
Polyterpenes
4
C40H64
12
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.
13
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 woodbased 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
14
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
15
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
16
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.
17
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
Subclasses
Monoterpenoids
Resin acids
Other terpenoids
Triglyceride
Fatty acids
steryl esters
sterols
Function in
the tree
Protection
Physiological
Protection
Occurrence
Oleoresin
canals
Heartwood
Knots
Bark
Softwoods
Parenchyma
Cell
Heartwood
Knots
Bark
Foliage
All wood
species,
Especially
softwoods
Tree
species
All wood
species
Phenolic
substances
Lignans
Flavonoid
Stibenes
Tannins
Carbohydrates
Glycosides
Sugars
Starch
Proteins
Gum
Pectins
Biosynthesis
Nutrient reserve
Protection
Inorganic
Various
salts
Sapwood
Cambium
Heartwood
Photosynthesis
Biosynthesis
Sapwood
Sap in
inner bark
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,
18
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.
19
Table 4 Solubility of wood extractives (Sjöström, 1998)
Extractives
Terpenoids
Fats
Carbohydrate
+++
Phenolic
substances
0
Alkanes
+++
Diethyl ether
+++
+++
++
0
Dichloro
+++
+++
++
0
Acetone
+++
+++
+++
++
Ethanol
++
++
+++
+
Water
0
0
+
+++
Solubility
Non-polar or
Non-polar or
Polar (water)
Water
Polar solvent
Polar solvent
solvent
0
-methane
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
20
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 samplecontaining 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
21
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
22
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.
23
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
24
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 nonrenewable raw materials, in combination with public concern over global
environmental issues, it is time not only to develop new ways of producing bioenergy 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 valueadded chemicals, as shown in Appendix 16 - 17.
25
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.
26
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 greybrown 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
27
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
28
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
29
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
30
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)
(D)
(C)
(E)
(F)
31
Appendix 3
The structure of wood stem (a) and wood bark (b) (Sjöström, 1993)
(a)
(b) young stem (A) and mature bark (B)
32
Appendix 4
The chemical composition of various wood species (Sjöström, 1993)
33
Appendix 5
The sugar moieties of wood hemicelluloses (Stenius, 2000)
34
Appendix 6
Major structures and frequencies of the linkages in softwood and hardwood lignins
(Stenius, 2000)
35
Appendix 7
Examples of the common terpenes and terpenoids in wood (Stenius, 2000)
36
Appendix 8
Examples of the aromatic extractives in wood (Stenius, 2000)
37
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
38
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-CH2CH2-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-CH2O-\
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-CH 2-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
39
Appendix 11
Extraction equipments (a) Soxhlet extractor (b) Soxtec extractor (Dean, 2009)
(a)
(b)
40
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)
41
Appendix 12
GC column selection (Document from Agilent Company)
42
Appendix 13
Guide to Derivatization Reagents for GC (Bulletin 909A, 1997)
43
Appendix 14
GC analysis step for extractives (Sjöström, 1998)
44
Appendix 15
Principal conversion routes for cellulosic biomass to produce various energy sources
(Alén, 2011)
45
Appendix 16
Examples of fermentation products from glucose (Alén, 2011)
46
Appendix 17
Examples of chemical products from glucose by common chemical treatments (Alén,
2011)
47