Comparison between using stem cross-sections and powder for determining the chemical composition of Salix wood samples
Most commonly, entire plant stems and wood samples are dried and milled during processing steps for chemical compositional analysis. The approach is well standardized (e.g. Sluiter et al. [34]) and provides a mean value of the sugars/lignin present in a fixed volume / weight of plant material. With this approach large quantities of plant material are frequently used during the initial phase of processing (i.e. milling) in order to give a homogeneous plant material that can be later sampled to provide statistical valid results that can be related to the original whole plant material. In addition, with Salix, the bark is also frequently included during the milling of stems [35]. The milling approach is however, more difficult to apply where interests are related to specific and smaller morphological plant tissue regions. Since in the present study, we wanted to compare the chemical composition of selected stem regions (debarked and depithed) in different Salix clones, we applied a stem cross-sectional approach for compositional analysis. Using stem cross-sections has an advantage in that analyses can be made on defined material originating from specific regions along a stem, allowing direct comparisons between stem regions and clones.
In order to test the viability of using stem sections for chemical analysis of Salix samples, we conducted preliminary studies on both homogenized stems regions (i.e. powder) and semi-thin sections from one clone “Loden” with comparative samples taken along the stem (Fig. 1, Table 1). To aid comparisons, the Salix sections and homogenized powder came from adjacent 5 cm stem regions taken at four points along the stem (Fig. 1). The percentage total glucose (T-glucose; i.e. total cell wall and tension wood glucose) determined using HPLC ranged from 49.1–49.4% and 44.0–46.6% from the analysis of the Salix sections and powder, respectively (Fig. 1, Table 1). The percentage total xylose ranged between 23.8–28.4% for sections and 20.2–23.9% for the powder. This represents a variation in total glucose and xylose between the sections and the powder of ca. 1.7–5.7% and 2.8–5.2%, respectively, with slightly higher glucose levels found in sections than in powder form at the different stem heights (Fig. 1, Table 1). The results are similar and well within that published for Salix sugar compositions [30, 36–39]. No significant differences were observed for mannose, galactose, and arabinose (p = 0.05, 0.9 and 0.2, respectively, according to paired t-test). The lignin content in sections and powder varied from ca. 20–25% and 22–24%, respectively (Table 1; Fig. 3). However, the mean value for the whole Loden stem (i.e. mean of the 4 samples points) was 22.5% for sections and 23.6% for powder (Table 1). Comparing results between sections and powder (p = 0.5, by one-way ANOVA) and within groups at different stem heights (according to paired t-test) gave no significant differences in the lignin values.
Table 1
Sugar analysis of Salix clone Loden using powder and serial sections from adjacent stem regions and different stem heights. Composition of Salix expressed as % of dry raw material.
Salix
|
Stem height
cm
|
T-Glucose
%
|
Xylose
%
|
Galactose
%
|
Arabinose
%
|
Mannose
%
|
ASL
%
|
AIL
%
|
Total lignin
%
|
Loden-sections
|
280
|
49.1
|
23.8
|
1.5
|
0.3
|
2.4
|
3.5
|
16.1
|
19.6
|
210
|
49.1
|
26.9
|
2.1
|
0.4
|
2.0
|
3.2
|
19.5
|
22.6
|
130
|
49.4
|
28.4
|
2.9
|
0.4
|
2.2
|
2.8
|
20.0
|
22.8
|
50
|
49.2
|
25.6
|
1.8
|
0.2
|
1.7
|
3.1
|
21.6
|
24.8
|
|
|
|
|
|
|
|
|
|
Mean: 22.5
|
Loden-powder
|
280
|
44.7
|
20.2
|
2.5
|
0.1
|
2.1
|
2.9
|
21.1
|
23.9
|
210
|
45.2
|
23.3
|
2.3
|
0.4
|
1.7
|
1.7
|
22.7
|
24.4
|
130
|
46.6
|
22.6
|
2.0
|
0.3
|
1.8
|
2.9
|
21.1
|
24.0
|
50
|
44.0
|
23.9
|
1.6
|
0.2
|
1.7
|
3.5
|
18.6
|
22.1
|
|
|
|
|
|
|
|
|
|
Mean: 23.6
|
The small variations in total glucose and xylose between the section vs powder approaches may reflect slight differences in manner of processing, sample size and native origin of sample. Possible explanations include: i) Slight differences in the amount of TW along the 10 cm stem cylinder used and thus differences in original tissue composition (i.e. fibres vs vessels) and thereby in native chemical composition; ii) Effects on G-layer cellulose during milling to produce the homogenized powder. During milling of dry Salix wood the abrupt change in cellulose microfibril angle between the G-layer and outer S2 layer would cause the latter to be easily detach from the S2 layer which together with the known high crystallinity of G-layers [18] could cause it to break up into very small particles not retained on the sieves during the initial stages of sample preparation. The milled powder for HPLC analysis was fractionated according to the standard particle size using Retsch GmbH sieves. According to Sluiter et al. [34], only fractions passing through a 40-mesh (ca. 425 µm) and retained on a 60-mesh (ca. 250 µm) should be used for standard HPLC analysis of plant (wood) materials and other fractions excluded. Thus, the percentage of the chemical components using HPLC may be slightly over or under estimated compared to the true native wood material. In contrast, the stem cross-sections were cut using a sliding microtome where section thickness is controlled within the range ca. 30-45 µm and used directly for chemical analysis. Our present results and previous light microscopy studies have further shown the G-layer is retained within the G-fibre structure during microtome sectioning [30, 33] .
The complete acid hydrolysis and sugar quantification process for sections was adapted according to Sluiter et al. [34] by replacing the stem powder with exactly the same amount of completely dried (103±2 ℃) sections from debarked and depithed stems. Thus, by using sections instead of powder for chemical compositional analyses, the number of steps and variations during processing can be reduced and a direct morphological relationship maintained. The precision of the section method was further verified by determining the variation in both the retention times of sample peaks with standards and concentration values of each sugar component during analysis with reported values.
In summary, despite some slight variations between sections and powder along stems, the percentage of different sugars from the sections was similar to values reported in the literature [39], indicating that our modified method using sections was precise and consistent for the sugars/lignin, and thus may be considered as having potential for use as a standard method for chemical analysis of small targeted plant biomass regions.
Estimation of tension wood in Salix stem cross-sections using image analysis
Representative sections used for chemical analysis were double stained by chlorazol E black and safranin (See Materials and Methods and Fig. 2) to visualize and quantify the tension wood present in cross-sections from the four Salix clone stems at different heights (Fig. 2). The percentage TW in stem cross-sections varied between both the four clones and with stem height (Fig. 2). The % tension area in Tora (Fig. 2a) ranged from ca. 10% at 50 cm to ca. 39% at 350 cm with corresponding values in Björn (Fig. 2b) of ca. 22% (50 cm) and ca. 34% (330 cm) for similar heights (Fig. 2). Stems of Jorr (Fig. 2c) and Loden (Fig. 2d) had calculated % tension areas varying from ca. 11–22% and ca. 6–35% respectively. These results confirm our previous results of the general trend of increasing TW with increasing stem height and also variations along the stem of the two-year-old fast growing Salix stems [33].
Compositional structure of Salix stems and sections
1. Chemical composition of Salix clones at different stem heights
The chemical composition of different Salix spp. as biomass for biofuel production has been studied for many years, although no specific studies appear to have been conducted on the spatial chemical distribution of components along the stem. In the present work, the distribution of sugars (i.e. glucose, xylose, mannose, galactose and arabinose) and lignin was determined for the four two-year-old Salix (Tora, Björn, Jorr and Loden) stems from bottom to the top (Table 2). All four Salix clones examined showed predominantly glucose (41–56%) and xylose (22–28%) as major sugars. The other sugars were relatively minor components, ranging from 0.2–3.9% for galactose, 0.7–2.4% for mannose and 0-0.5% for arabinose. By considering the mean value of each sugar (i.e. as % dry wt.) along the stem; the most abundant fraction was glucose with 47.7, 52.8, 49.2, and 47.1% for Tora, Björn, Loden, and Jorr, followed by xylose with 24.9, 24.1, 26.2, and 23.0%, respectively (Table 2). These values concur with most published reportes on the sugar composition of Salix species [26, 35, 36, 39]. From a statistical aspect, the result showed some variability in glucose, both between clones (paired t-test p < 0.05) and at different heights (significantly different at p < 0.01 level, one-way ANOVA) with Tora and Björn showing increased % of glucose with stem height (results not shown). At the top of stems, Tora and Björn had 53.1 and 56.4% glucose at 350 and 330 cm stem height, respectively, which is ca. 12 and 6% higher than at 50 cm height (41.3 and 50.7%, respectively, Table 2).
Table 2
Sugar (HPLC) analysis of sections from four Salix clones taken at four stem heights made before enzyme treatment.
Salix
|
Stem height
cm
|
Glucose
%
|
Xylose
%
|
Galactose
%
|
Arabinose
%
|
Mannose
%
|
Tora
|
350
|
53.1
|
24.2
|
3.7
|
0.5
|
2.2
|
|
250
|
49.9
|
25.3
|
2.9
|
0.4
|
2.1
|
|
150
|
46.6
|
24.7
|
2.4
|
0.4
|
1.8
|
|
50
|
41.3
|
25.5
|
2.2
|
0.3
|
1.4
|
|
Mean
|
47.7
|
24.9
|
2.8
|
0.4
|
1.9
|
Björn
|
330
|
56.4
|
26.1
|
3.9
|
0.4
|
2.2
|
|
250
|
52.5
|
23.7
|
3.3
|
0.4
|
2.1
|
|
150
|
51.7
|
24.2
|
3.0
|
0.3
|
1.7
|
|
50
|
50.7
|
24.0
|
2.7
|
0.3
|
1.6
|
|
Mean
|
52.8
|
24.1
|
3.2
|
0.4
|
1.9
|
Jorr
|
200
|
47.1
|
22.0
|
1.7
|
0.3
|
2.3
|
|
150
|
45.6
|
23.0
|
0.8
|
0.0
|
2.1
|
|
100
|
46.5
|
23.4
|
0.2
|
0.0
|
1.5
|
|
50
|
49.1
|
23.6
|
1.4
|
0.0
|
2.2
|
|
Mean
|
47.1
|
23.0
|
1.0
|
0.1
|
2.0
|
Loden
|
280
|
49.1
|
23.8
|
1.5
|
0.3
|
2.4
|
|
210
|
49.1
|
26.9
|
2.1
|
0.4
|
2.0
|
|
130
|
49.4
|
28.4
|
2.9
|
0.4
|
2.2
|
|
50
|
49.2
|
25.6
|
1.8
|
0.2
|
1.7
|
|
Mean
|
49.2
|
26.2
|
2.1
|
0.3
|
2.1
|
Field Salix
|
|
48.0
|
26.6
|
1.6
|
0.0
|
0.7
|
2) Enzymatic saccharification of clone stem cross-sections from different heights
To estimate G-layer glucose (G-glucose) in clones at different stem heights, sections were subjected to 3-days enzymatic (cellulase) saccharification using Cellic CTec2 (See Material and methods). After saccharification, sections were randomly selected, double stained with chlorazol black and safranin and examined using light microscopy. As shown in Figure 2a-d, all the darkly coloured tension wood bands were removed during saccharification verifying the complete removal of the G-layer from the tension wood fibres. G-glucose released due to enzyme saccharification varied along the stems. Results showed an increase in D-glucose release with stem height, with highest and lowest G-glucose release from the cross-sections for all clones recorded at the top (i.e. 14.7% for Tora at 350 cm; 13.4% for Björn at 330 cm; 9.4%, Jorr for 200 cm; and 11.8% for Loden at 280 cm) and bottom (3.7% for Tora at 50 cm; 9.5% for Björn at 50 cm; 7.6% for Jorr at 100 cm; and 4.0% for Loden at 50 cm) of the stems respectively, apart from clone Jorr where the lowest D-glucose level was recorded at 100 cm stem height (Table 4). Additional analysis of D-glucose release from an 8-year-old field Salix sample with mostly tension wood was 11.9% (Table 4).
Table 3. Sugar (HPLC) analysis of sections from four Salix clones taken at four stem heights made after enzyme treatment.
Salix
|
Stem height
cm
|
Glucose
%
|
Xylose
%
|
Galactose
%
|
Arabinose
%
|
Mannose
%
|
Tora-E
|
350
|
45.3
|
31.7
|
2.4
|
0.1
|
1.6
|
|
250
|
44.9
|
29.8
|
2.5
|
0.2
|
1.6
|
|
150
|
40.6
|
25.0
|
2.8
|
0.4
|
1.5
|
|
50
|
36.9
|
23.7
|
2.2
|
0.4
|
1.2
|
|
Mean
|
41.9
|
27.5
|
2.5
|
0.3
|
1.5
|
Björn-E
|
330
|
49.8
|
32.5
|
3.2
|
0.3
|
1.4
|
|
250
|
51.2
|
31.0
|
3.4
|
0.3
|
1.5
|
|
150
|
47.8
|
29.8
|
3.7
|
0.4
|
1.5
|
|
50
|
47.5
|
31.3
|
3.5
|
0.4
|
1.7
|
|
Mean
|
49.1
|
31.1
|
3.5
|
0.4
|
1.5
|
Jorr-E
|
200
|
41.1
|
24.1
|
1.1
|
0.0
|
2.0
|
|
150
|
42.8
|
24.3
|
1.1
|
0.0
|
1.9
|
|
100
|
42.8
|
24.9
|
0.7
|
0.0
|
1.7
|
|
50
|
41.7
|
25.4
|
0.0
|
0.0
|
1.5
|
|
Mean
|
42.1
|
24.7
|
0.7
|
0
|
1.8
|
Loden-E
|
280
|
44.7
|
29.2
|
2.7
|
0.2
|
2.1
|
|
210
|
49.1
|
26.9
|
2.1
|
0.4
|
2.0
|
|
130
|
46.1
|
27.6
|
2.1
|
0.2
|
2.1
|
|
50
|
45.9
|
27.1
|
1.8
|
0.2
|
2.1
|
|
Mean
|
46.5
|
27.7
|
2.2
|
0.3
|
2.1
|
Field Salix-E
|
|
41.6
|
28.3
|
1.6
|
0.0
|
0.4
|
Table 4
Comparison of total glucose (T-glucose) using HPLC with G-glucose from enzymatic hydrolysis (as % of starting raw material) for four Salix clones at different stem heights together with % tension wood at different stem heights
Salix
|
Stem
height
|
T-glucose
|
G-glucose
|
Theoretical
CW-glucose
|
|
Theoretical
CW-glucose
|
HPLC measured
CW-glucose
|
Tension
area
|
Stem
diameter
|
Tension volume
|
|
cm
|
% raw material
|
|
% enzymatic hydrolyzed material
|
%
|
cm
|
%
|
Tora
|
350
|
53.1
|
14.7
|
38.4
|
|
45.1
|
45.3
|
38.9
|
1.4
|
24.2
|
|
250
|
49.9
|
9.6
|
40.3
|
|
44.5
|
44.9
|
36.2
|
1.8
|
|
150
|
46.6
|
7.5
|
39.1
|
|
42.3
|
40.6
|
23.6
|
2.2
|
|
50
|
41.3
|
3.7
|
37.6
|
|
39.0
|
36.9
|
9.7
|
2.6
|
|
Mean
|
47.7
|
8.9
|
38.9
|
|
42.7
|
41.9
|
|
|
|
Björn
|
330
|
56.4
|
13.4
|
43.0
|
|
49.6
|
49.8
|
33.9
|
1.6
|
28.2
|
|
250
|
52.5
|
12.4
|
40.1
|
|
45.7
|
51.2
|
38.7
|
1.8
|
|
150
|
51.7
|
10.8
|
40.9
|
|
45.8
|
47.8
|
25.3
|
2.2
|
|
50
|
50.7
|
9.5
|
41.2
|
|
45.5
|
47.5
|
21.7
|
2.7
|
|
Mean
|
52.8
|
11.5
|
41.3
|
|
46.7
|
49.1
|
|
|
|
Jorr
|
200
|
47.1
|
9.4
|
37.7
|
|
41.7
|
41.1
|
20.8
|
1.4
|
15.5
|
|
150
|
45.6
|
8.0
|
37.6
|
|
40.9
|
42.8
|
14.1
|
1.5
|
|
100
|
46.5
|
7.6
|
38.9
|
|
42.1
|
42.8
|
11.0
|
1.7
|
|
50
|
49.1
|
9.2
|
39.9
|
|
43.9
|
41.7
|
21.8
|
1.8
|
|
Mean
|
47.1
|
8.6
|
38.5
|
|
42.2
|
42.1
|
|
|
|
Loden
|
280
|
49.1
|
11.8
|
37.3
|
|
42.3
|
44.7
|
34.9
|
1.1
|
14.0
|
|
210
|
49.1
|
5.3
|
43.8
|
|
46.2
|
49.1
|
16.6
|
1.2
|
|
130
|
49.4
|
5.1
|
44.3
|
|
46.7
|
46.1
|
13.8
|
1.5
|
|
50
|
49.2
|
4.0
|
45.2
|
|
47.1
|
45.9
|
5.8
|
1.9
|
|
Mean
|
49.2
|
6.6
|
42.7
|
|
45.6
|
46.5
|
|
|
|
Field Salix
|
48.0
|
11.9
|
36.1
|
|
41.0
|
41.6
|
|
|
|
T-glucose: total glucose; G-glucose: tension wood glucose; CW-glucose: cell wall structural glucose. |
In agreement with our previous studies [30, 33], after enzymatic hydrolysis of stem sections, we obtained values for G-glucose ranging from 3.7–14.7% of the original raw material weight (as dry wt.), with the top of the two year old Salix stems generally showing higher yields of G-glucose than bottom regions. The more TW present, the more G-glucose, further proving that G-glucose is significantly and positively related to the percentage of TW (Fig. 4, Table 5).
Table 5
Pearson’s correlation analysis for correlation between T-glucose, G-glucose, CW-glucose, % Tension wood, Stem diameter and Stem height.
|
|
T-glucose
|
G-glucose
|
CW-glucose
|
% Tension wood
|
Stem diameter
|
Stem height
|
T-glucose
|
Pearson Corr.
|
1
|
0.71
|
0.57
|
0.66
|
-0.24
|
0.60
|
|
p-value
|
-
|
0.0019
|
0.020
|
0.0050
|
0.37
|
0.01
|
G-glucose
|
Pearson Corr.
|
|
1
|
-0.024
|
0.89
|
-0.25
|
0.74
|
|
p-value
|
|
-
|
0.93
|
0.0000041
|
0.35
|
0.001
|
CW-glucose
|
Pearson Corr.
|
|
|
1
|
0.040
|
-0.15
|
0.12
|
|
p-value
|
|
|
-
|
0.88
|
0.57
|
0.65
|
% Tension wood
|
Pearson Corr.
|
|
|
|
1
|
-0.24
|
0.81
|
|
p-value
|
|
|
|
-
|
0.37
|
0.00015
|
Stem diameter
|
Pearson Corr.
|
|
|
|
|
1
|
-0.61
|
|
p-value
|
|
|
|
|
-
|
0.01
|
Stem height
|
Pearson Corr.
|
|
|
|
|
|
1
|
|
p-value
|
|
|
|
|
|
--
|
Two-tailed test of significance used. |
The total volume of TW was also determined for each clone (Table 4). Tora and Björn clones showed greater amounts of TW (24.2 and 28.2%, respectively) in the test material (debarked and depithed stem volume: ca. 993 and 968 cm3 for Tora at 50–350 cm and Björn at 50–330 cm, respectively), which was almost two times greater than Jorr (14.9%; stem volume at 50–200 cm is ca. 296 cm3) and Loden (15.5%; stem volume at 50–280 cm is ca. 364 cm3) (Table 4). If we consider larger Salix wood volumes, for example 1 m3 of Tora (i.e. ca. 1007 stems at a height 50–350 cm) and Björn (i.e. ca. 1033 stems at a height of 50–330 cm), the stems would theoretically contain ca. 0.24 and 0. 28 m3 of TW. For the corresponding volume of 1 m3, 3383 stems of Jorr (50–200 cm) and 2750 stems from Loden (50–80 cm) would be required, providing 0.16 and 0.14 m3 of TW respectively. This emphasizes the large differences that can be shown in TW development by the four clones studied when the biomass quantity is scaled up. It is also emphasizes the significant role likely played by the presence of TW to the glucose content.
3) Chemical composition (HPLC) of enzymatic hydrolyzed stem sections from clones at four stem heights
In order to verify the differences and methodology, the same stem cross-sections after enzymatic hydrolysis were re-examined using HPLC for chemical composition. Following enzymatic hydrolysis, the glucose (i.e. cell wall structural glucose = CW-glucose) content was reduced (i.e. 36.9–51.2%) as expected while the xylose content increased from 22.0–28.4% to 23.7–32.5% (Tables 2, 4). Assuming the enzyme saccharification process only removed G-glucose from the G-fibres, the calculation for xylose would be: % xylose (before enzyme saccharification) = xylose content / dry wt. of raw material × 100%, and % xylose (theoretical, after enzyme saccharification) = xylose content / (dry wt. of raw material – G-glucose) × 100%. Therefore, the increase in xylose content after enzyme saccharification is likely due to the loss of G-glucose material during the treatment (i.e. as a % of dry material; Table 4). The percentage glucose and xylose showed a similar trend in the 8-year-old field Salix sample as the two-year-old tested Salix sample (Table 4). The glucose reduced from 48 to 41.6%, while the xylose content slightly increased by 1.7% (Tables 2, 3).
To estimate the cell wall structural glucose (CW-glucose), the theoretical value of CW-glucose of enzymatic hydrolyzed samples was calculated by the following equation:
Theoretical value of CW-glucose (i.e. % enzymatic hydrolyzed material) = (T-glucose – G-glucose) / (100 – G-glucose) × 100%
T-glucose: percentage of T-glucose from HPLC analysis (as % of raw material)
G-glucose: percentage of G-glucose released during the enzymatic hydrolysis process (% of raw material)
The calculated theoretical value of CW structural glucose and the measured result from HPLC analysis of the different clones at different stem heights were very similar (Tora, Björn, Jorr and Loden show p = 0.3, 0.1, 0.5 and 1.0, respectively, according to a paired t-test) emphasizing the validity of the method (Table 4). Thus, the correlation between T-glucose, G-glucose, and CW-glucose conforms well to the expected: Total glucose = G-layer glucose + Cell wall glucose.
4) Correlation before and after enzymatic hydrolysis
In order to determine the likely contribution played by TW glucose to total Salix chemical composition and its potential for biofuels, Pearson’s correlation analysis was used to analyze the significant correlation levels among all related factors (Figs. 4, 5; Table 5). A strong and positive association was observed between T-glucose and G-glucose, % tension wood and stem height (Pearson’s correlation coefficient value (Pearson Corr.) r = 0.71, 0.66 and 0.60, respectively, p < 0.01) (Fig. 4, Table 5). As expected: G-glucose also showed a strong and positive correlation with % tension wood and stem height (Pearson Corr. r = 0.89 and 0.74, respectively, p < 0.01) indicating that the amount of freely available glucose from the G-layer is significantly related to % tension wood. The CW-glucose along the stem (from top to bottom) only varied slightly, indicating that structural CW-glucose has no large variations (Fig. 4, Table 5). This confirms that as the TW increases, more G-glucose is available and more T-glucose detected. In this way, it should be possible to give an average value for CW glucose that may be used as a clone related value (i.e. reference) for HPLC determined glucose that could be used as a standard to compare not only different clones but also the same and different clones from different locations. Higher values will indicate not only greater levels of TW and glucose but also more readily available glucose for hydrolysis.
Principal component analysis (PCA) was also used to understand the scale of impact of chemical component parameters and physical measurement on individual Salix stems. For analysis, CW-glucose, T-glucose, G-glucose, Tension %, Stem height and Stem diameter were used as variables. Figure 5a shows the graphical representation of the PCA results where 6 properties are used per observation. The first component PCA1 explains 57.6% of the variation, and the second component PCA2 20.5%. The influence of variables on the observation is indicative by their length, direction and angle with the longer length of the variable indicative of higher influence on conservation and shorter length less influence. Figure 5b displays the relationship between all samples from different stems and different heights. The two principal components’ relation between different stems showed that Tora and Björn are more similar as are Jorr and Loden with respect to physical and chemical analysis used (Fig. 5b).
5) Comparison of Total glucose and G-glucose in different clones
Comparison of total glucose (T-glucose) as a % of starting raw material gave values along the clone stems of ca. 41.3–53.1% (mean 47.7%) for Tora, ca. 50.7–56.4% (mean 52.8%) for Björn, ca. 45.6–49.1% (mean 47.1%) for Jorr and ca. 49.1–49.4% (mean 49.2%) for Loden (Table 4). Comparative values for G-glucose released by enzyme saccharification were ca. 3.7–14.7% (mean 8.9%) for Tora, ca. 9.5–13.4% (mean 11.5%) for Björn, ca. 7.6–9.4% for Jorr (mean 8.6%) and ca. 4.0–11.8% (mean 6.6%) for Loden (Table 4). As a comparison, this indicates a contribution of between ca. 6.6–11.5% G-glucose to the total glucose with Björn > Tora > Jorr > Loden for the clone test material. These values for G-glucose are slightly lower than that determined previously [33], but nonetheless indicates a significant contribution of G-glucose from the presence of TW fibres with gelatinous layers. Previously we calculated the possible effect of G-glucose based on a literature mean value for stem glucose (i.e. cellulose) of 45% [33] without any prior knowledge of the chemical composition of stems. Here however, we make comparisons based on analyses of the same Salix stem materials. It is highly probable that the wide variations reported in the literature of total glucose (cellulose) actually also include a percentage TW and that the variation in actual cell wall structural glucose (cellulose not in the G-layer) is much less. However, by using wet chemical analysis approaches and milling of the entire wood material, it is not possible to distinguish between them.
6) Determination of lignin in Salix clones at different stem heights both before and after enzymatic saccharification.
Stems of the four Salix clones showed a total lignin content [i.e. acid soluble lignin (ASL) + acid insoluble lignin (AIL)] ranging from ca. 18–25% at different stem heights (Table 6) which is consistent with published results. Serapiglia et al. [36] for example reported a 19–22% lignin content for 25 two-year-old debarked willow shrub clones and Dou et al., 22.4% lignin in a debarked four-year-old willow hybrid ‘Karin’ [14].
Table 6
Lignin analysis of sections from four Salix clones taken at four stem heights made before and after enzyme saccharification.
Salix
|
Stem height
cm
|
Before enzyme saccharification
|
|
After enzyme saccharification
|
ASL
%
|
AIL
%
|
Total
%
|
Mean lignin of
the entire stem
%
|
|
ASL
%
|
AIL
%
|
Total
%
|
Mean lignin of
the entire stem
%
|
Tora
|
350
|
2.4
|
22.2
|
24.6
|
22.8
|
|
4.2
|
23.0
|
27.2
|
24.3
|
250
|
3.2
|
20.6
|
23.8
|
|
3.9
|
21.3
|
25.2
|
150
|
3.6
|
17.2
|
20.8
|
|
3.7
|
19.8
|
23.4
|
50
|
4.0
|
18.0
|
22.1
|
|
4.3
|
17.0
|
21.3
|
Björn
|
330
|
2.8
|
20.4
|
23.2
|
22.4
|
|
4.1
|
19.7
|
23.8
|
22.8
|
250
|
3.2
|
18.5
|
21.7
|
|
3.7
|
20.5
|
24.2
|
150
|
3.6
|
18.5
|
22.1
|
|
3.6
|
18.8
|
22.4
|
50
|
3.8
|
18.9
|
22.7
|
|
3.5
|
17.5
|
21.0
|
Jorr
|
200
|
3.9
|
20.1
|
23.9
|
22.5
|
|
3.7
|
20.4
|
24.2
|
22.4
|
150
|
3.9
|
20.3
|
24.2
|
|
4.3
|
18.2
|
22.6
|
100
|
4.3
|
19.5
|
23.8
|
|
4.8
|
16.2
|
21.0
|
50
|
4.4
|
13.6
|
18.0
|
|
4.5
|
17.2
|
21.7
|
Loden
|
280
|
3.5
|
16.1
|
19.6
|
22.4
|
|
3.7
|
20.9
|
24.6
|
23.8
|
210
|
3.2
|
19.5
|
22.6
|
|
3.6
|
19.8
|
23.4
|
130
|
2.8
|
20.0
|
22.8
|
|
3.6
|
18.4
|
22.0
|
50
|
3.1
|
21.6
|
24.8
|
|
3.5
|
21.9
|
25.4
|
Field Salix
|
1.9
|
17.6
|
19.5
|
|
|
2.2
|
21.2
|
23.4
|
|
Before enzyme saccharification, in 3 (i.e. Tora, Björn, Jorr) of the 4 clones, the highest AIL were recorded in the upper parts of the stem in contrast with Loden that showed the opposite result (Table 6). Using Pearson’s correlation analysis, the ASL showed moderate and negative correlation with stem height, T-glucose, and % tension wood (i.e. -0.65, -0.64, and -0.44, 0.01 < p < 0.1) (data not shown in Table 6). Since the percentage of total ASL contributed less than ca. 5% of the total lignin content, the ASL should not significantly affect the compositional results for lignin. By using the mean lignin content of the whole stem (i.e. mean of the four analysis points along the clone stems), we can compare the total mean lignin content of the whole stem which was very similar with a difference less than 0.4% for all clones (Table 6). This is similar to the results of Cui et al. [39] who studied three-year-old Salix integra and found no significant differences in lignin distribution along the stem. At 10 cm above ground they found 21.3% AIL and 4.9% ASL, at 120 cm 20.0% AIL and 4.3% ASL and at 210 cm 19.3% AIL and 4.3% ASL respectively. Results suggest therefore that in Salix biomass, the percentage of TW will not be a significant factor affecting total lignin.
Some slight variation in lignin for the clones were noted for sections before, and after enzyme saccharification with mean stem values ranging from 22.4–22.8% before and 22.4–24.3% after, the main difference noted for clones Tora (1.5%) and Loden (1.4%). Currently, the reason for the slight increase is unknown as all studies were conducted on debarked and depithed stems. Both the ASL and AIL contributed to the slight increase.