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Article history:
Received 30 August 2011
Received in revised form 6 December 2011
Accepted 14 December 2011
Available online 11 January 2012
Keywords:
Transgenic poplars
Lignin modication
Biofuels
Saccharication
a b s t r a c t
Lignin has been recognized for its negative impact on forage digestibility, tree pulping properties, and
cellulosic biofuel production, although it is the major structural component of the secondarily thickened
cell walls of vascular plants. Earlier studies have demonstrated that lignin modication improves forage
digestibility and poplar pulping properties. To determine whether lignin modication has benecial
effect on saccharication of lignocellulosic biomass, we pretreated and then enzymatically hydrolyzed
the mature wood from transgenic poplar plants that expressed the antisense transgenes of monolignol
biosynthesis genes 4-coumarate: CoA ligase (4CL) or caffeoyl CoA 3-O-methyltransferase (CCoAOMT).
Firstly, a long-term eld trial was set up for the transgenic plants. Over ve years, the reduced trend
of lignin content remained stable in all transgenic lines. And a total lignin reduction of up to 10% did
not alter the growth rate or biomass yield of the transgenic poplars. In the mature wood, suppression
of CCoAOMT increased saccharication potential, but 4CL down-regulation had no signicantly positive
effect on saccharication. Sugar yield were negatively correlated with soluble lignin content of dried,
extractive-free stem biomass. These results imply that lignin modication can facilitate the process of
saccharication for biofuel production in tree crops.
2011 Elsevier B.V. All rights reserved.
1. Introduction
Renewable and sustainable liquid biofuels must be explored as
alternatives to petroleum-based fuels if the world energy crisis is to
be solved (Kerr and Service, 2005; Schubert, 2006). Bioconversion
of cellulosic biomass to fuel is considered a particularly attractive
means of generating low-cost, sustainable biofuels, with enzymatic
hydrolysis of cellulose to glucose and then fermentation to ethanol
(Wyman, 1999). Lignocellulosic biomass from non-food crops, such
as trees and grasses, among a variety of other biofuel feedstock
sources, has been proposed as a potential raw material source for
producing cellulosic ethanol (Demirbas, 2005; Lynd et al., 1991;
Somerville, 2006, 2007). Currently, producing biofuels derived from
lignocellulosic biomass is expensive and difcult due to the inherent complexity of plant cell walls (Himmel et al., 2007; Somerville
et al., 2004). Lignin is one major component of the plant cell wall and
is known to be especially problematic with regard to bioconversion
of cellulosic biomass to fuels because it interferes with the ability
171
commercial value of the mature wood of 4CL- or CCoAOMTdecient transgenic poplars for biofuel production, a long-term
eld trial was set up. The resulting biomass was subjected to
pretreatment and enzymatic hydrolysis. Our results showed that
CCoAOMT deciency signicantly improved the saccharication
potential of poplar biomass.
Equal volumes of the two enzymes were mixed, and the loadings
of enzyme mixture were in excess (about 90 FPU per g cellulose).
Sodium citrate buffer (0.05 M, pH 4.8) was used to maintain the
pH at 4.8. Tetracycline and cycloheximide were added to protect
the reaction mixture from microbes. Enzyme blanks were set up
alongside the sample.
The enzymatic hydrolysate was centrifuged for 2 min. The suspernatant was then ltrated with 0.45 m lter. The ltrate was
subjected to glucose and xylose analysis by HPLC (Agelent 1100
Series). An Aminex colum (Model HPX-87P, Bio-Rad, Sunnyvale,
CA, U.S.) was used to separate the sugars.
The study was performed on transgenic poplar (Populus tomentosa) lines B4CL28, B4CL86, BCOA264, and BCOA133, all previously
described and known to be decient in either 4CL (B4CL28 and
B4CL86) or CCoAOMT (BCOA264 and BCOA133) activity (Jia et al.,
2004; Zhao et al., 2004). Shoots were multiplied from each line by
excising nodal segments and allowing axillary buds to elongate and
roots to regenerate. They were then acclimatized in a greenhouse in
February 2004. In April 2004, these lines, along with wild-type control plants, were planted in the eld. Agronomic traits of all trees
were investigated, and stem height (main stem) and diameter (at
1.3 m high) were measured annually.
Five-year-old plants were harvested prior to dormancy in the fall
of 2009. Stems of two different plants from each line at 1.01.4 m
from the ground were collected, debarked, chipped, mixed, and
dried overnight in an oven at 60 C. They were then ground in
a mill with a sieve diameter of 1.00 mm (DFT-2000, Wenling,
Zhejiang, China). Soluble extractives were removed by three successive extractions with methanol at room temperature. These
dried, extractive-free biomass samples were used for the compositional analyses and saccharication experiments.
2.2. Determination of lignin and cellulose content
Acid-insoluble lignin (i.e. Klason Lignin) content was determined using a modied method derived from the China National
Standard Method GB/T 2677.8-94. Briey, 1.0 g of methanolextracted ground stem sample was treated with 4 mL of 72% H2 SO4
for 1 h at 30 C with mixing taking place every 10 min. This mixture was diluted with 142 mL of deionized water to achieve a nal
acid concentration of 3% H2 SO4 and transferred to a Schott bottle. The solution was then autoclaved at 100 C for 1 h and ltered
through a crucible for determination of acid-insoluble lignin. Acidsoluble lignin was quantied by spectrophotometric analysis of the
ltrate at 205 nm (National Standard Method GB/T 10337-2008).
Cellulose content was determined using nitric acidethanol mixture methodology (Liu, 2003).
2.3. Chemical pretreatment
Dried, milled, and extractive-free stem material at a solid loading of 10% (w/v) was mixed with 1% or 3% sodium hydroxide.
Pretreatment was run in an autoclave at 120 C for 30 min. After
pretreatment, residual biomass was separated from supernatant
by ltration and washed with water.
2.4. Enzymatic hydrolysis
Enzymatic hydrolysis of pretreated residues was conducted
according to the laboratory analytical procedures of the National
Renewable Energy Laboratory (LAP-009) (Brown and Torget,
1996). After ltering and washing, 0.5-g biomass samples were
hydrolyzed with mixtures of cellulase (Celluclast 1.5 L) and cellobiase (Novozyme 188) in a total volume of 30 mL at 50 C and
150 rpm for 72 h. The activity of enzyme was 94.9 lter paper
units (FPU) mL1 for cellulase and 101 CBU mL1 for cellobiase.
172
Table 1
Soluble lignin, insoluble lignin, and cellulose content in ve-year-old poplar wood.
Soluble lignin (%)
Control
B4CL28
B4CL86
BCOA264
BCOA133
Cellulose (%)
Mean
SD
Reductiona
Mean
SD
Reductiona
Mean
SD
Reductiona
Mean
SD
Increase b
3.19a
3.19a
3.25a
2.73b
2.69b
0.12
0.10
0.09
0.09
0.15
0.00
1.88
14.42
15.67
20.92a
18.93b
19.38b
18.91b
19.21b
1.00
0.01
0.52
0.53
0.43
9.51
7.36
9.61
8.17
24.12a
22.16b
22.68b
21.59b
21.80b
1.09
0.11
0.50
0.46
0.42
8.13
5.97
10.49
9.62
47.94a
51.53d
49.58b
51.75d
50.41c
0.76
0.85
0.40
0.62
0.53
7.49
3.42
7.95
5.15
Means with the same letter are not signicantly different from each other at the 0.95 condence level.
a
Reduction refers to percent reduction of lignin in transgenic lines comparing to that of wild type plant, which was calculated as: Reduction (%) =
(lignin content of wild type plant lignin content of transgenic line)/lignin content of wild type plant 100.
b
Increase refers to percent increase of cellulose in transgenic lines comparing to that of wild type plant, which was calculated as: Increase (%) =
(cellulose content of transgenic line cellulose content of wild type plant)/cellulose content of wild type plant 100.
Fig. 1. Height and diameter of lignin-modied poplars grown in eld. (A) Average diameter of transgenic lines from 2005 to 2008, measured at 1.3 m height. (B)
Average height. Error bars represent the SD from the mean. n = 5 per line.
hydrolysate were separated. Solid residue remaining after pretreatment decreased with higher concentrations of chemical, averaging
7581% of stem biomass residue for tested lines with 1% NaOH
and 6369% stem biomass residue after pretreatment with 3%
NaOH. This indicates that the removal of lignin and hemicellulose
increased with higher concentrations of pretreatment chemical.
The residue after pretreatment was subjected to enzymatic hydrolysis with cellulase and cellobiase alongside the untreated biomass
of tested lines. Data on glucose and xylose yield were normalized
to dried, extractive-free biomasses.
The yields of glucose and xylose and enzymatic hydrolysis
efciency of cellulose after alkali pretreatment and enzymatic
hydrolysis are shown in Table 2. Little glucose was released
from untreated biomasses subjected to enzymatic digestion, and
no xylose was detectable in enzymatic hydrolysates, suggesting
that lignocellulosic biomass must be pretreated to open up the
structure of the plant cell wall and improve the accessibility of
173
Table 2
Sugar released by enzymatic hydrolysis of biomass samples pretreated with 1% and 3% NaOH.
Pretreatment
Control
B4CL28
B4CL86
BCOA264
BCOA133
Untreated
1% NaOH
3% NaOH
Untreated
1% NaOH
3% NaOH
Untreated
1% NaOH
3% NaOH
Untreated
1% NaOH
3% NaOH
Untreated
1% NaOH
3% NaOH
Xylose
Glucose
Total sugars
0a
77c
59b
0a
78c
58b
0a
72b
66b
0a
85c
49b
0a
92c
83b
21a
255b
328c
27a
279b
354c
18a
255b
319c
87a
310b
341b
86a
339b
388c
21a
331b
387c
27a
356b
412b
18a
326b
385c
87a
395b
391b
86a
430b
471c
4.27a
53.09b
68.50c
5.32a
53.85b
69.23c
3.57a
51.33b
64.31c
16.79a
59.91b
67.11b
17.15a
67.17b
77.01c
Means not sharing a common letter, within individual tested lines, differ in response to pretreatment.
a
Enzymatic hydrolysis efciency (%) means total cellulose digested as a percentage of total cellulose in the dried, extractive-free biomass subjected to pretreatment
and enzymatic hydrolysis with cellulose and cellobiase. Total cellulose digested was recovered by multiplying the total glucose released with 0.9 to correct for the water
molecule added upon hydrolysis of the cellulose polymer (Brown and Torget, 1996), and it was calculated as: Enzymatic hydrolysis efciency (%) = grams glucose released
0.9/gram cellulose added 100.
Table 3
Efciency of pretreatment with different concentrations of NaOH.
Line
Control
B4CL28
B4CL86
BCOA264
BCOA133
Untreated
1% NaOH
3% NaOH
4.27a
5.32a
3.57a
16.79b
17.15b
53.09a
53.85a
51.33a
59.91ab
67.17b
68.50a
69.23a
64.31a
67.11a
77.01b
48.82
48.53
47.76
43.12
50.02
64.23
63.91
60.74
50.32
59.86
15.41
15.38
12.98
7.20
9.84
Means with the same letter are not signicantly different from each other at the 0.95 condence level.
a
Enzymatic hydrolysis efciency (%) means total cellulose digested as a percentage of total cellulose in the dried, extractive-free biomass subjected to pretreatment
and enzymatic hydrolysis with cellulose and cellobiase. Total cellulose digested was recovered by multiplying the total glucose released with 0.9 to correct for the water
molecule added upon hydrolysis of the cellulose polymer (Brown and Torget, 1996), and it was calculated as: Enzymatic hydrolysis efciency (%) = grams glucose released
0.9/gram cellulose added 100.
b
Pretreatment efciency (%) means the increased enzymatic hydrolysis efciency (%) by indicated pretreatment, compared to the untreated or chemical pretreatment with
lower concentration. For example: Pretreatment efciency % of 3% NaOH vs. 1% NaOH = enzymatic digestion efciency of cellulose (%) with 3% NaOH pretreatment enzymatic
digestion efciency of cellulose (%) with 1% NaOH pretreatment.
174
400
380
y = 0.5854x + 179.58
R2 = 0.7826
360
340
320
300
200
250
300
350
400
90
80
70
60
50
40
60
70
80
90
100
Fig. 2. Sugar released from poplars via enzymatic digestion of pre-treated and
untreated biomass by a mixture of cellulase and cellobiase. (A) Total glucose and
xylose released during saccharication. (B) Glucose released during saccharication. (C) Xylose released during saccharication. Means not sharing common letters
or numbers, within each pretreatment, differ in response to the individual tested
lines. Lignied stems (from at 1.0 to 1.4 m off the ground) were harvested from
ve-year eld-grown poplars. Dried, milled, extractive-free stems were untreated
or pretreated with alkaline substances and then subjected to enzymatic hydrolysis with cellulase and cellobiase. Control, untransformed control Populus tomentosa;
B4CL28 and B4CL86: two transgenic lines down-regulated for 4CL activity; BCOA264
and BCOA133: two lines down-regulated for CCoAOMT activity. Sugar levels were
measured by HPLC.
300
y = -135.28x + 686.82
R2 = 0.8097
200
2.5
2.7
2.9
y = -29.695x + 170.06
R2 = 0.933
90
350
250
100
y = -75.781x + 571.46
R2 = 0.5801
Xylose (mg/g)
Glucose (mg/g)
400
80
70
60
50
3.1
3.3
40
2.5
3.5
2.7
175
2.9
3.1
3.3
3.5
100
Xylose (mg/g)
Glucose (mg/g)
90
350
300
250
80
70
60
50
200
18.5
19 .0
19 .5
20.0
20.5
21.0
40
18.5
21 .5
19.0
20 .0
20.5
21.0
21.5
52
53
100
90
350
Xylose (mg/g)
Glucose (mg/g)
400
19.5
300
250
80
70
60
50
40
47
200
47
48
49
50
51
52
53
400
48
49
50
51
Glucose (mg/g)
350
300
250
200
40
y = 4.3822x - 73.812
R2 = 0.8029
50
60
70
80
90
100
Xylose (mg/g)
Fig. 4. Relationships between composition of biomass and saccharication. Each point represents an individual wild type or transgenic plant. (A) Amount of glucose released
is shown as a function of the soluble lignin content of the biomass with both pretreatments. (B) Linear correlation between the amount of xylose released with soluble lignin
content with the 1% NaOH pretreatment. (CF) No correlation between the amount of glucose or xylose released and insoluble lignin or cellulose content was found with
either pretreatment. (G) The amount of glucose released was parallel to the amount of xylose in the hydrolysate with the 1% NaOH pretreatment.
saccharication potential. On the contrary, statistically signicant decrease in total glucose and xylose release was shown in
two lines with signicant lower S/G ratio and higher extractive
content. These data recommended that phenolic fragment from
176
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