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Wood Science and Technology Vol. 3 (1969) p. 117--
The Distribution of Lignin in Sprueewood as
Determined by Ultraviolet ~Iieroseopy
By B. J. FE]~Gus, A. R. PROCTER, J. A. N. SCOTT a n d D. A. I. GORING, Montreal, Quebec
Abstract The distribution of lignin in black spruce has been determined quantitatively by the study of 0.5 ~m transverse sections in a UV microscope. The average lignin concentration in the compound middle lamclla was about twice that in the secondary wM1. The lignin concentra- tion of the middle lamella at the cell corners of adjacent traeheids was nearly four times that in the secondary wM1 but the volume of the secondary wall was much greater than the volume of the middle lamella. Thus, for earlywood, 72% of the total lignin was in the secondary wall leaving only 28% in the compound middle lamella and cell corner middle lamella regions. The corresponding values for latewood were 82~o and 18% respectively. Use of oblique longi- tudinM sections of 0.1 ~m thick permitted the resolution of the compound middle lamella. The lignin concentration in the true middle lamella was found to be equal to that in the cell corner middle lamella and the primary wall lignin content to be about twice that in the secondary wall.
Zusammenfassung
Die Verteilung des Lignins in Fichtenholz wurde quantitativ dureh Untersuchung yon 0,5 ~m dicken Querschnitten unter d e n UV-Mikroskop bestimmt. Die mittlere Ligninkonzen- tration war in der Mittelschicht etwa doppelt so hoeh wie in der Sekund~rwand. Die Lignin- konzentration der Mittelschicht war in den an die Tracheiden anstoBenden Zellecken anna- hernd viermal hbher als in der Sekundi~rwand, wogegen das Volumen der Sekund~rwand wesentlieh gr5ger war als dasder Mittelschicht. Dagegen befand sich beim Friihholz 72% des gesamten Lignins in der Sekundiirwand und nut 28% fanden sich in der Mittelschich$ selbst und in ihren Zelleekbereiehen. Die entspreehenden Werte fiir Sp~tholz betragen 82% bzw. 18%. Die Anwendung yon sehr~gen Langsschnitten yon 0,1 ~m Dieke erlaub~e die Aufl6sung der Mittelschieht. Die Ligninkonzentration in der Mittellamelle war glcich groB wie in der in den Zellzwiekeln befindliehen Mittellamelle und der LigningehMt der Prim~rwand war etwa doppelt so groB wie derjenige in der Sekund~rwand.
Introduction
A l t h o u g h the a n a t o m y a n d s t r u c t u r a l organization of wood have been well d o c u m e n t e d [JANE 1956; WA~D~OP 1962, 1963, 1964], there r e m a i n s some u n - c e r t a i n t y on the q u a n t i t a t i v e d i s t r i b u t i o n of l i g n i n in the cell wall. One of the first to s t u d y this d i s t r i b u t i o n was RITTEI~ [1925], who concluded t h a t 75% of the l i g n i n in wood was located in the middle lamella, the secondary wall contain- ing the residual 25 %. B y use of a m i e r o m a n i p u l a t o r , BAILEY [1936] isolated the middle lamella of Douglas fir, a n d b y chemical analysis f o u n d a lignin c o n t e n t of a p p r o x i m a t e l y 71%. A n essentially similar result was o b t a i n e d b y LANGE [1954, 1958], who used UV-mieroscopy a n d calculated the weight c o n c e n t r a t i o n of ligrtin in the secondary wall a n d c o m p o u n d middle lamella to be 16% a n d 73% re- spectively. LANGE proposed a continuous decrease in lignin c o n c e n t r a t i o n from the middle lamella to the lumen, b u t FREY [1959] also using UV-microseopy, claimed t h a t the i i g n i n d i s t r i b u t i o n across the secondary wall was fairly uniform. 2 Wood Scienceand Technology,Vol. 3
118 B.J. FnRGUS, A. R. PRoc~R, J. A. N. ScoT~ and D. A.I. GORING
Similar uniformity of distribution ill the secondary wall was reported by l%uoI~ and I-IENG)~RTNEtr [1960] who had studied jute fibres. They questioned Lange's techniques on the basis that conical light had been used and that no correction was applied for t h e diffraction at the primary wall/secondary wall interface. However, it now seems likely that the main cause for the poor definition in LA?~GE'Sphotographs was the use of a section thickness of several microns [SCOTT et al. 1968] which was the minimum generally available twenty years ago. The question of lignin distribution has been critically appraised by BERLYN and MARI; [1965], who pointed out that the Bailey-Lange result was diametrically opposed to that of I%ITTEIr Noting t h a t the volume fraction of the compound middle lamella in coniferous wood is only 10 ... 12% of the wood tissue volume, BElCLY~ and MAlCK showed that even if the middle lamella region was composed of 100% Iignin, it could not contain more than 40% of the total lignin in wood. The present investigation deals with the measurement of the distribution of lignin in the black spruce cell wall. The techniques used were a combination of ultraviolet microscopy a n d densitometrie analysis of UV photomicrograph negatives. A recent appraisal of these techniques [ScoTT et al. 1968] has in- dicated that, provided certain experimental conditions are met, the lignin con- centration in various parts of the wood tissue can be determined with reasonable accuracy.
Experimental
Small chips of dry black spruce (Picea mariana Mill.) wood containing the
whole of the 56th or 64th annual ring of a 68-year old log were embedded and sectioned b y the method described earlier [ScoTT et al. 1968]. Either metha- crylate or Epon was used as the embedding medium. Transverse sections were usually 0.5 ~m thick, but a cut of 0.1 ~m was used for oblique longitudinal sections. The ultrathin sections were examined under a Leitz UV-microscope. The con- denser had a numerical aperture of 0.60, and the objective had a magnification of 300 : 1 and an aperture of 0.85. Photographs were taken on K o d a k Spectrum Analysis No. 1-35 m m film. The wavelengths used were 280 nm or 240 nm. The former wavelenggh was used for quantitative measurements, the latter mainly to obtain photographs for display purposes. The photomicrograph negatives were analysed densitometrieally by means of a Joyce, Loebl Mark I I I CS recording microdensitometer to give ghe relative amounts of lignin in the various morphological regions of the wood [ScoTT et al. 1968].
Results and Discussion
An example of a UV photomicrograph of a black spruce tracheid wall, with the corresponding densitometer trace, is shown in Fig. 1. The compound middle lamella stands out as a narrow highly lignified layer with a considerably smaller but uniform lignin concentration across the secondary wall. The compound middle lamella observed here probably consists of the two primary walls and the true middle lamella. Quantitative analysis of the negative revealed differences in the UV absorbance of the various morphological regions. As found by WAI~DROP [1963], the ab-
120 B.J. FEnGUS, A. 1~. PgOCTEg, J. A. N. SCO~Tand D. A. I. GORING
The results are shown in Fig. 2. The annual ring contained 55 cells. Each point on Fig. 2, and subsequent Figs. 3 and 4, represents the average of determinations on at least three adjacent cell walls at the same eel1 number position in the annual ring. No absorption measurements were made on the radial wails as these walls were wrinkled and heavily pitted. I n the case of the tangential walls, the cell dimensions WTt and MTt were obtained directly from the densitometer traces. The radial and tangential tracheid diameters and WT~ were measured on the photomicrograph negatives.
o o o o 0.3-~-~<_ ~ %
;ooo' o
N 0.
=; o
, o O 9 o
OoO
9 Middle[0mella 9 9 o ,,&-t.,-a..ip~ 0. oo __ o
Secondary wa[t
Enrlywood i 0 10 20 30 40 50 Ceil number
Lo~ewood
F i g. 2. V a r i a t i o n of t h e U V - a b s o r b a n c e of t h e t a n g e n t i a l c o m p o u n d m i d d l e l a m e l l a a n d s e c o n d a r y w a l l a c r o s s t h e a n n u a l i n c r e m e n t. C e l l no. 1 is t h e f i r s t e a r l y w o o d cell, cell no. 55 t h e l a s t l a t e w o o d cell. T h e f i l l e d a n d e m p L y circles r e f e r t o t w o d i f f e r e n t s a m p l e s of t h e s a m e a n n u a l r i n g.
c~ c~
o
F i g. 3.
16
k ~
~O 8
8 o o^ o
4o Eorlywocd Latewood 10 20 30 40 50 Cell number V a r i a t i o n of t h e t a n g e n t i a l d o u b l e c e l l w a l l t h i c k n e s s a c r o s s t h e a n n u a l r i n g. T h e f i l l e d a n d e m p t y circles r e f e r t o t w o d i f f e r e n t s a m p l e s of t h e s a m e a m l u a l r i n g.
From Fig. 2 it can be seen that there was a steady decrease in the UV-absorb- anne of the tangential secondary wall in the transition earlywood to latewood. The UV-absorbanee of the secondary wall of the 45th cell was only 78 % of that of the 10th cell. Apart from some irregularity in the first 10 earlywood cells the UV-absorbance of the tangential compound middle lamella remained fairly con- stant over the remainder of the annual ring.
Distribution of Lignin in Wood as Determined by UV-Microscopy 121
The variations of the various cell wall dimensions across the annual ring are shown in Figs. 3 ... 5. I n Fig. 3, WTt increased steadily from a value of about 5 barn for the first earlywood cells, reached a m a x i m u m value of about 14 ~m in the cell interval 45 ... 50 and then decreased rapidly over the last five latewood cells. The variation of W T r (Fig. 4) was similar to t h a t of W T t b u t the size of the change in dimensions, 4.5 ~tm ... 17 ~zm, was slightly greater. Furthermore, there was no marked decrease in WTr over the last five latewood cells. Interestingly, the ratio W T t / W T r was fairly constant at an average value of 1.16, with arithmetic
~m^16
_v
o )$ o ir oEor[yw00d I 10 20 30 40 Cell number
o ~
o~ ~
o./:
Fig. 4. Voziatioa of the radial double cell wall thickness across the annttal ring. The filled and empfiy circles refer to two different s~,mples of the same ~nnu~l ring.
4C !,./Q,,, - ~
o ~ o ##* - 35 ~ o^ Tr0chmd^9 dl0meler^9 o^ o oo c^ o o^ ~^ / ' /^ ** ~'
~o ~ o~OO ~:o - M 2 " / ;
~ o oyoo o~176 oo /,,'VQ ~,\ -,0o *30 / f /,'o oo" \ *
~. I IT0tcil ce[I wcl I / --'// .,4 ~ o N oo'q, o \I Xl =~
~- 25 Itissue ureo/iJ/"I ..,"" X o \ I -
/ _ j.... o\o ,i ,00=
i I" o.d'~ISecondlJry WO[[ h 0 Ill ~" 20 /. , i ' " tissue cire0 " X < %1 ~>
15 /.,.." O ~ o ~-
10 Eorlywood lotewood~_ 10 20 30 40 50 Cell number
Fig. 5. V~riation of radial traeheid diameter and the tissue area of the cell wall across the a n n u a l ring,
deviation of :~ 0.05, for the first 40 cells but then decreased rapidly to 0.38 for the last latewood cell. The radial and tangential tracheid diameters were measured for one sample across the annual ring. The tangential tracheid diameters of adjacent cells across
F i g. 6. C r o s s s e c t i o n of b l a c k s p r u c e l a t e w o o d , e m b e d d e d i n m e t h a e r y l a t c ~ n d p h o t o g r a p h e d a,t 280 ~ m.
F i g. 7. C r o s s s e c t i o n of u n E p o n e m b e d d e d r ~ y P a r e n c h y m a cell. N o t e t h e h i g h U V - a b s o r p t i o n of t h e r ~ y c e l l w a l l w h e n c o m p a r e d ito t h e t r a c h e i d s e c o n d a r y w a l l. ~ = 240 n m.
Z=
9
OO
b~
124 B.J. F~RGvs, A. R. PROCT~R, J. A. N. SCOTTand D. A. 1. GORING
A UV photomicrograph of spruce latewood is shown in Fig. 6. Comparison
with Fig. 1 shows the poorer quality of the latewood print. Considerable effort
towards improving the photomicrographs of latewood met little success. For this
reason the latewood cells were not studied as extensively as the earlywood cells
and hence the latewood data in Table 1 are not as numerous as the earlywood data.
However, the measurements show that CMs/Cs increases for latewood both in the
tangential middle lamella and the cell corner region. This result was expected in
view of the trends in absorbanee shown in Fig. 2.
The appropriate cell wall dimensions arc also listed in Table 1. The standard
deviations in the values of MTt and MTr for earlywood of the methacrylate em-
bedded 64th annum ring were • 0.03 ~m and • 0.04 ~m respectively (23 samples
each). For the same samples the standard deviations in WTt and WTr were
-- 0.6 ~m and ~: 0.7 ~m respectively.
The tangential double cell wall in earlywood was thicker than the radial wall.
This trend may have been due to increased swelling in the tangential direction
[BoUTELJE 1962] which could also produce the lower volume concentration of
lignin in the tangential wall [ScoTT et al. 1968]. In addition, significant differ-
ences were found in the dimensions (Table 1) and UV absorbance [ScoTT et al.
1968, Fig. 4] of the tangential and radial compound middle lamellac. Such morph-
ological detail is of considerable interest and may well prove to be a general
characteristic of conifer woods. However, the present work is more concerned
with establishing quantitatively the broad patterns of lignin distribution. There-
fore the dimensions and lignin concentration ratios of the radial and tangential
walls will be averaged in the subsequent analysis.
From the values of C M~/Cs shown in Table 1, it was possible to calculate the
quantitative distribution of lignin in the various morphological regions of the cell
wall. If uniform density and swelling are assumed throughout the tracheid cell
wall, we may write
(Volume fraction of
a secondary)
CMT, (Volume fraction of the
a ~ - s (r) (t) compound middle lamella)
CML (CC) (Volume fraction of cell
~- a ~ corner middle lamella) = W (1)
in which a and W are the weight concentrations of lignin in g/g of dry secondary
wall and whole wood respectively.
In order to obtain a from Eq. (1) we must first know the relative tissue volumes
of the morphological subdivisions. By assuming a rectangular tracheid and using
the appropriate cell wall dimensions it was possible to calculate geometrically the
tissue area of the secondary wall and compound middle lamella for an average
spruce tracheid. For earlywood cells at approximately the cell no. 10 position the
tissue areas for the secondary wall and compound middle lamella were 312 ~m 2
and 31 ~m ~ respectively. The tissue area of the cell corner middle lamella was
obtained by measuring the corresponding areas off 8 " • 10" prints (final magnifi-
cation 1995 • ) of the photomicrograph negatives. The boundary of the cell corner
was taken at the point at which the thin compound middle lamella expanded
rapidly. For obvious reasons, this measurement was rather approximate. For
96 cell corner middle lamellae the average tissue area per tracheid in earlywood
126 B.J. FERGUS, A. R. PROCTER, J. A. N. SCOTTand D. A. I. GORING
estimate by BERLYN and MARK [1965] that less than 40% of the lignin in wood is located in the compound middle lamclla region. For earlywood, the weight concentration of lignin in the secondary wall is 22.5 % which agrees rather welI with a recent estimate by S T A ~ and SANDERS [1966] based on the density of the wood substance of the loblolly pine. These authors concluded that between 22 and 24% of the earlywood secondary wall substance was lignin. A somewhat unexpected result was that the lignin concentration was virtually the same for the earlywood and latewood secondary walls. This is in apparent contradiction with the trend shown in Fig. 2 for the decrease in UV-absorbance of the tangential secondary wall across the annual ring. As the UV-absorbance is volume dependent, the trend in Fig. 2 could be a reflection of the increased swelling of the cell wall from earlywood to latewood. In fact, this has been observed recently by BOUTELJE [1962], who showed that latewood cells swell some 11% more than carlywood cells. I t is not known whether this increased swelling arises solely from the secondary wall or extends to the compound middle lamclla as well. I t is important to note that the validity of Eq. (1) is not affected provided the swelling is the same in all regions of the tracheid. Failure of this condition could account for the apparent increased weight concentration of ]ignin in the latewood middle lamella, whereas the results in Fig. 2 indicate that the volume concentration of lignin in this region remains approximately constant. The compound middle lamella is composed of two primary walls and the true middle lamella. Hence, the lignin content of 50% in Table 2 is probably an average value for the three layers. The lignin concentration in the cell corner middle lamella was high. Evidently this region was almost pure lignin. As the cell corner middle lamella is an extension of the true middle lamella it might be expected that the latter would have the same high lignin content. The resolution of this question is discussed in a later section of the paper. Table 2. The Distribution o/Lignin in the Spruce Tracheid
W o o d
M o r p h o l o g i c a l d i f f e r e n t i a - t i o n
R e l a t i v e a b s o r b a n c e
Tissue volume Lignin f r a c t i o n (% of t o t a l ) %
L i g n i n c o n c e n t r a t i o n g/g
Earlywood
La$ewood
S Secondar
S 1 87.4 72.
ML(r), (~) 2.21 8.7 15. ML (ce) 3.77 3.9 12. S 1 93.7 81. ML(r), (~) 2.7 4.1 9. NL (cc) 4.5 2.2 8. 7wall, ML Middle lammella, r radial, t tangential, cc cell corner
The Calculation of Lignin Concentrations from the Extinction Coefficient I t was of considerable interest to check the lignin content of the earlywood secondary wall, as calculated b y the tissue volume method, with a direct calcula- tion from the UV-absorbanee data. The lignin concentration in any particular morphological region can be calcu- lated directly from the Beer-Lambert law UV-absorbance = e • C • d (4)
Distribution of Lignin in Wood as Determined by UV-Microscopy 127
where e is the extinction coefficient, C the volume concentration of ligain and d the thickness of the section. A serious limitation on the application of the Beer-Lambert law to wood sections is the lack of any reliable value for the extinction coefficient of lignin in wood. There is a large scatter in the value of s2s0 for various lignins isolated from sprucewood and it is difficult to know which, if any, preparation corresponds to the protolignin. Figures quoted for S2s0 range from 12.8 em -1 1 g 1 for spruce lignin sulphonate [YEAN, GORING 19641 to 18.7 cm -~ 1 g-1 for spruce kraft lignin [MCNAVGHTON et. al. 1967]. Ball milled lignin, prepared by the method of Bj6rkman, is generally regarded as having a chemical structure near to that of the lignin in the wood. However, SAaKANEN and coworkers [19671 found that when milled wood lignins from the sapwood of three softwood species were reduced with sodium borohydride the extinction coefficient at 280 m~ was perceptibly lower. From their data the average value of S2s0 for the three softwoods was decreased from 18.9 ... 15.6 cm - 1 g-1 by this treatment (Table 3). The lowering of e~s0 was attributed to the Table 3. Ultraviolet Absorption Data o/Milled Wood Lignins ~ ]3orohydride Species U n t r e a t e d lignins reduced lignins e2s0(em- t 1 g-~) e~s0(cm-~ 1 g - l ) Douglas-fir Western red cedar Himalayan pine 1 Sarkanen et al. 1967
borohydride reduction of carbonyl groups conjugated with the phenyl nucleus. Conjugated carbonyl absorption contributes heavily to the lignin spectrum above 300~m. This is illustrated by the fact that the average value of S~so/S3os, for the softwoods, is 1.94 for untreated lignin and 3.39 for sodium borohydride reduced lignins. As described in more detail in a subsequent report [FERGVS, GORING in print] lignin in the spruce secondary wall yields a value of e~s0/e305 = 3.8. Thus it seems reasonable to assume that spruce proto-lignin has a UV spectrum similar to that of the reduced milled wood lignins. As softwood lignins are comprised almost entirely of the guaiacylpropane unit and hence will have similar values of e2so, the value of e~so ~ 15.6 cm -1 1 g-1 was used for the calculation of absolute lignin concentrations from Eq. (4). Interestingly, an S2s0 of 15.3 cm -1 1 g-1 can be computed from the UV spectrum published by PEW [1957] for ball milled spruce wood dissolved in aqueous lithium bromide. The UV-absorbanee of the secondary wall (average of the radiM and tangential walls) for earlywood tracheids at about the cell no. 10 position, was 0.154 for a section 0.5 ~m thick [ScoTT et M. 1968]. This UV-absorbance must be reduced by 3 % to correct for the effect of nonparallel illumination. The corrected UV- absorbanee is then 0.149. By substitution of this value and s = 15.6 cm -1 1 g-l, and d = 0.5 ~tm in Eq. (4), the volume concentration of lignin in the spruce early- wood secondary wall is 0,191 g/era 2. To convert to the weight concentration of lignin it is necessary to multiply this volume concentration by the specific volume of the water swollen cell wall. STONE and SCALLAN [1967] have found the specific volume of the water swollen cell wall of black spruce to be 1.07 cm2/g.
F i g. 8. L o n g i t u d i n u l t a n g e n t i a l s e c t i o n (0.1 p+m) c u t u t ~ n u n g l e of 7 ~ f r o m t h e m ~ i n t r a e h e i d u x i s. N o t e t h e h i g h l y U V - a b s o r b i n g m i d d l e l ~ y e r a n d t h e t w o lesser ~ b s o r b i n g l ~ y e r s o n e i t h e r side of i t (see a r r o w ) ~ = 240 n m.
F i g. 11. C r o s s s e c t i o n of e ~ r l y w o o d t r ~ e h e i d s e m b e d d e d i n E p o n. N o t e t h e m e d i u m i n t e n s i t y l ~ y e r b e t w e e n t h e c e l l c o r n e r m i d d l e l ~ m e l l a a n d secondo,ry w ~ l l (see a r r o w s ). ,% ~ 240 n m.
9
~Q
(^9) 9
b~
130 B.J. I~ERGUS,A. ~. PROCTEr, J. A. N. 8COT~ and D. A. I. GORING
traces. The ratio 5.5 : 0.41 is almost identical to the ratio WTt : MTt = 5.5 : 0. (Table I) in transverse cross sections. Hence, the tricomposite layer does appear to correspond to a resolved compound middle lamella. From a comparison of the double cell wall dimensions in transverse and oblique sections it is possible to show that the sections were cut at an angle of sin -1 (5.5)/(46.3), i.e. 7 ~ I t seems reasonable to assume that the highly absorbing middle layer in Fig. 8 is, in fact, the true middle lamella flanked by the less absorbing primary walls. The average of several densitometer traces gave an apparent width of 0.73 ~m for the true middle lamella which corresponded to about 0.1 ~m in transverse sections. Densitometric analysis of 10 oblique tangential walls showed that the true middle Iamella had an absorption 3.64 times that of the secondary wall whereas the ratio of primary wall to secondary wall absorption was 1.97. This oblique sectioning experiment can yield unambiguous results only if the section thickness is less than the width of the true middle lamella. If the middle lamella width is smaller than the section thickness it is not possible to obtain the dimensions and absorbance of the true middle lamella. These two conditions are shown in Fig. 9 which depicts diagrammatically oblique sections, 0.1 ~m in width, cut at 7 ~ to the main tracheid axis. I n Fig. 9a, the true middle lamella is 0.2 ~m thick, while in Fig. 9b it is 0.05 ~m. For the geometries represented in Figs. 9a and 9b, the theoretical traces yield a plateau for the middle lamella. I n Fig. 9a, the absorbance of the middle lamella is given by the height of the plateau and the middle lamella thickness corresponds to the width of the plateau at half-height multiplied by sin 7 ~ However, in Fig. 9b, the width at half-height multiplied by tan 7 ~ gives the section thickness and the height of the plateau corresponds to the average absorbanee arising from equal layers of middle lamella and primary wall. Interestingly, it can be showrt that in the ease of Fig. 9 b, a middle lamella thinner than 0.07 jim would require a lignin concentration greater than 1 g/g in order to satisfy the requirements of the absorbanee data. Fig. 9c represents an oblique section cut at 7 ~ to the tracheid axis and with the true middle lamella width equal to the section width of 0.1 ~m. In the theoret- ical trace the plateau has now been replaced by a peak, the height of which corresponds to the absorbance of the true middle lamella. As with Fig. 9a, the width of the true middle lamella is given b y the width at half-height multiplied by sin 7 ~ An average experimental densitometer trace is given in smoothed form as a dotted line. Apart from the slight depressions between the primary wall and the middle lamella absorption peaks, the experimental trace is very similar to that proposed by consideration of the geometry of the system. However, it must be remembered that no account has been taken of the limitation of optical resolut- ion and non-parMld illumination [SCOTT et al. 1968] in drawing Figs. 9a, b and e. If the geometry in Fig. 9 e is taken to be approximately correct then the middle peak in the densitometer trace corresponds to the true middle lamella absorbance
and (CML/Cs) ( t ) : 3.64. Although only 10 samples were considered the results
indicate that (CML/Cs) (t) is tending to the value of CML/Cs (ca) = 3.77. On the
basis of these measurements it is not unreasonable to conclude that the lignin content of the true middle lamella does not differ from that of the cell corner middle lamella. This experiment also showed that the primary wall lignin content is about one-hali that of the true middle lamella.
132 B.J. FERGUS, A. ~. PROC~R, J. A.N. SCOTTand D. A.I. GORING
revealed by the oblique sections, is about 16% greater than the lignin in the compound middle lamella measured in transverse sections. This is a compara- tively small error. A calculation based on the higher value of lignin content obtained from the oblique sections gives the proportion of lignJn in the secondary wall, compound middle lamella and middle lamella cell corner region to be 70 %, 18 % and 12 % respectively. There is little change from the corresponding figures in Table 2. I t can therefore be claimed that although the compound middle lamel]a is not resolved by UV-microseopy of transverse sections, its dimensions and average lignin content are derived with reasonable accuracy by this method.
w
~
-- 3.
- - 2. 0 8
il Middte lcimello
2~m I l Fig. 10. I d e a l i z e d distribution of lig~nin across the t a n g e n t i a l double cell w a i l of e a r l y w o o d as o b t a i n e d for oblique sections (shaded area) c o m p a r e d w i t h a t y p i c M e x p e r i m e n t a l d e n s i t o m e t e r t r a c e on t r a n s - verse cross sections (dashed llne).
I n some densitometer traces of the oblique walls there was found to be an absorbing layer adjacent to the primary wall. This layer was of varying width and its absorption was about 89more than that of the secondary wall. This was probably the S 1 layer but on account of the uncertainty in its measurement and the lack of a sufficient number of walls no quantitative study was made. Although an S1 layer adjacent to the compound middle Iamella was usually not evident in UV-photomierographs of transverse walls it could often be recognized as an absorbing layer slightly darker than the rest of the secondary wall around a cell corner middle lamclla (Fig. 11).
Other Morphological Features
The heavy lignification in the ray parenehyma cell is apparent in Fig. 7 and a distinct lamellar structure is evident in the cell wall. A lamellar structure in the ray cell wall has also been detected by electron mieroseopy [HAI~ADA 1965].
Distribution of Lignin in Wood as Determined by UV-Microseopy 133
Lamellation of the cell wall was not restricted to ray cells. UV-photomiero-
graphs at 240 ~m of spruce traeheid secondary walls often revealed two or three
broad lamellations parallel to the middle lamella. Lamellation of the cell wall of
various hardwoods and softwoods has been reported by TRAY?CARD et al. [1954]
for tissue delignified by chlorine or nitric acid. PA~E and DE G]~CE [1967] found
that concentric layers were present in commercially pulped softwood fibres that
had been subjected to the mechanical stresses of beating and refining. JAY3IE
and TORGEt~SE?q [1966], using UV-microseopy, observed occasional lamellation in
some spruce latewood tracheids. The lamellations found in the presertt work were
not very marked and could not be detected in micrographs taken at wavelengths
higher than 240 nm. These lamellations were apparent in the original print of
Fig. 1 and can atso be seen as small peaks in the secondary wall plateau in the
corresponding densitometer trace.
A layer of high UV-absorption surrounds the pits (Fig. 12, single arrow). This
layer most probably corresponds to the initial pit border [WA~])I~oP 1964]. There
appears to be a progressive decrease in the UV-absorption in the initial pit border
as the pit aperture is approached (Fig. 12, double arrow). The pit membrane has
been shown to be unlignified [BA3~E~ 1961]. However, the torus, which straddles
the membrane near its centre, shows a very strong UV-absorption (Fig. 13).
If Epon is used as an embedding medium, the high UV-absorption of the torus
disappears and a fine structure consisting of a thin absorbing layer is apparent
within the torus (Fig. 14). The width of this "toms middle lamella" is about
0.10 ~m. Interestingly, this is considerably less than the lower resolution limit of
the microscope, which at ~ = 0.24 ~m is 0.14 ... 0.17 ~xn [ScoT~r et al. 1969].
The extraction of the highly absorbing material from the toms when Epon is
used for embedding can most probably be attributed to the use of propylene oxide
in the solvent exchange procedure. This absorbing material is also rapidly removed
by kraft and sulphite cooking liquors [PRocTEtt et al. 1967].
The Occurrence of Splits and Fissures
An interesting phenomenon is illustrated in Fig. 15 where splits have ocemTed
in the region of the S 1 layer. Such splits were observed in several sections.
WAlCDaOP [1963] has pointed out that mechanical splits usually occur between
81 and 82 whereas chemical splits normally take place within the middle lamella.
From the electron microscopy of black spruce fibre surfaces, separated by tensile
failure, KOraN [1967, 1969] found that the site of failure occurred consistently in
the primary wall and 81 wall layers. Furthermore, the type of splitting was
temperature dependent. The splits observed in the present work are therefore
attributed to mechanical damage of the section during its preparation, and
illustrate the potentially weak area about the 81 layer.
Concluding Remarks
The most important conclusion that emerged from this investigation was that
although the compound middle lamella was highly lignified, with respect to the
secondary wall, it only contained a small percentage of the total lignin in the
3 W o o d Science a n d Technology. Vol. 3
F i g. 14. B o r d e r e d p i t i n E p o n e m b e d d e d b l a c k s p r u c e e a r l y w o o d. T h e t o r u s h a s l o s t a l l i t s h i g h U V - a b s o r p t i o n ( c o m p a r e F i g. 13) mad a f i n e s t r u c t u r e is a p p a r e n t w i t h i n t h e t o r u s (see a r r o w ). ~ = 240 ran.
F i g. 15. C r o s s s e c t i o n of b l a c k s p r u c e s h o w i n g t h e l o c a t i o n o f m e c h a n i c a l s p l i t s. R u p t u r e of t h e m e t h a c r y l a t e e m b e d d i n g m a t e r i a l f r o m t h e c e l l w a l l c a n b e s e e n i n t h e l u m e n of t h e c e l l o n t h e u p p e r l e f t (see a r r o w ).
9
UQ
(^9) 9
2
136 B. J, FERGUS, A. ~. PROCTER, J. A. N. SCOTTand D. A. I. GORING
wood. This was predicted by B]~RLYN and MARK [1965] but until now experi- mental evidence to support this prediction has been scarce. I t is interesting to note that BAILEY [1936] obtained a lignin concentration in the middle lamella not very different from the values found in the present work. I t is in the estimat- ion of the proportion of total lignin in the middle lamella that most errors have crept into the literature. The reliability of the method is best shown by the good agreement between the two calculations of the lignin concentration in the secondary wall. The method involving relative absorbances and tissue volumes is quite distinct from the direct calculation from the UV-absorbance data utilising the Beer-Lambert relationship. A useful extension of the present work would be to check the technique by similar measurements on a wood with a lignin distribution different from that of spruce. Although the study of ray cells and bordered pits was not the primary purpose of the present work, the mierographs revealed certain of their interesting features. The fine structure and chemical composition of the various pit types is not yet fully understood. Possibly, a detailed investigation with the UV-microscope m a y elucidate more fully the distribution of ]ignin around and within these important morphological entities. Finally, it m a y be profitable to speculate on the significance of these findings to the technology of softwood pulping. The purpose of chemical pulping is either to remove as much of the lignin as possible from the wood without excessive degradation of the carbohydrate component or to weaken the middle lamella bond so that reasonably intact fibres can be obtained. The oblique sectioning experi- ment has shown us that the quantity of lignin in the true middle lamella is only about 5 % of the weight of the wood. Thus, the logical way to pulp softwoods is to use a delignifying agent which is highly selective for the almost pure lignin in the middle lamella. The wood is then reduced to separate fibres, highly lignified, and containing over 95 % of the original wood substance. This primary pulp is then used as such, or further delignified to yield the type of fibre required. How- ever, it now appears that most of the commercially-used pulping processes work in the opposite fashion [PROCTER et al. 1967; FERGUS, GORISIG in print], where the lignin is preferentially removed from the secondary wall. By the time the middle lamella is attacked valuable carbohydrates have been degraded in the fibre wall. This not only decreases the fibre strength but obviates the attainment of m a x i m u m yield at a particular degree of lignin removal. The discovery of a middle lamella selective delignifying agent m a y not be possible but it is certainly worth a try.
References BACK, E. : Tracheidal and parcnchymatous cells in picca abies (Karst.) pulpwood and their behaviour in su]pite pulping. Svcnsk Papperstidn. 68 (1960) 695. BAILEY, A. J. : Lignin in Doug]as-fir, composition of the middle lamella. Ind. a. Engin. Chem., Anal. Ed. 8 (1836) 52. BAMBER, 1%.K. : Staining reaction of the pit membrane of wood cells. Nature 191 (1961) 409. BERLYN, G. P., and R. E. MAZCK:Lignin distribution in wood cell walls. ~orest Prod. J. 15 (1965) 140. BLOUT, E. R., G. R. BIRD and D. S. GREY: Infra-red microspec~roscopy. J. Opt. Soc. Amer. 40 (1950) 304.
