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Industrial Crops and Products 37 (2012) 170177

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Industrial Crops and Products


journal homepage: www.elsevier.com/locate/indcrop

Lignin modication improves the biofuel production potential in transgenic


Populus tomentosa
Hongzhi Wang 1 , Yingxi Xue 1 , Yajuan Chen, Ruifen Li, Jianhua Wei
Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China

a r t i c l e

i n f o

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

Corresponding author at: Beijing Agro-Biotechnology Research Center, Beijing


Academy of Agriculture and Forestry Sciences, No. 9, Shuguang Huayuan Middle
Road, Haidian District, Beijing 100097, China. Tel.: +86 10 51503830;
fax: +86 10 51503980.
E-mail addresses: weijianhua@baafs.net.cn, cauwhz@yahoo.com (J. Wei).
1
These two authors contributed equally to this paper.
0926-6690/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2011.12.014

of hydrolytic enzymes to access the cellulose polymer and adsorbs


hydrolytic enzymes (Chen and Dixon, 2007; Simmons et al., 2010;
Keating et al., 2006). Loosening lignins grip on biofuel production
has become the new aim of lignin engineering of plants (Chapple
et al., 2007).
During the last two decades, most reports on lignin engineering have involved in improving forage digestibility and pulping
performance. Recently, there have been a few reports in which
the aim of lignin modication was to improve biofuel production.
Chen and Dixon (2007) analyzed the biomass digestibility of six
lignin-modied alfalfa lines by cellulase/cellobiase with and without dilute acid pretreatment. There were signicant differences in
enzymatic hydrolysis efciency in biomass of the transgenic alfalfa
lines. Some lines showed enzymatic efciencies double those of the
wild-type control plant. These results indicate that lignin modication can facilitate the bioconversion of lignocellulosic biomass to
biofuel in alfalfa. Davison et al. (2006) analyzed the saccharication
efciency of poplar biomass in ve genetic variants from a single
segregating F2 hybrid family via acid hydrolysis. Results indicated
that both lignin content and S/G ratio signicantly affected the yield
of xylose when biomass was hydrolyzed with dilute sulfuric acid.
In contrast, Voelker et al. (2010) reported that reduced AcBr lignin
and MBMS lignin did not substantially improve the saccharication
potential of transgenic poplars with 4CL down-regulation.
To investigate the agronomic and enzymatic saccharication performance of lignin-modied poplars and to evaluate the

H. Wang et al. / Industrial Crops and Products 37 (2012) 170177

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.

2. Material and methods

2.5. Sugar content

2.1. Plant materials and sample collection

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.

2.6. Statistical analyses


Signicance (ANOVA) was tested at p < 0.05 using SPSS (SPSS Inc,
1998), to evaluate the statistical differences in lignin and cellulose
content and the amounts of sugar released in different lines. Two
plants for each independent line were analyzed, with the exception
of transgenic line B4CL28, only one plant of which was analyzed.
All compositional and saccharication analyses were performed in
triplicate for each individual plant biomass. Regression analysis was
performed using the statistics program in Microsoft Ofce Excel
2003.
3. Results
3.1. Growth, lignin content, and cellulose content of transgenic
poplars
A long-term eld trial of transgenic poplar lines was set up to
evaluate the effects of lignin modication on plant growth and
wood properties. Five different lines, including the untransformed
wild-type line (Populus tomentosa), two antisense lines decient in
4CL, B4CL28 and B4CL86 (Jia et al., 2004), and two antisense lines
decient in CCoAOMT, BCOA264 and BCOA133 (Zhao et al., 2004),
were evaluated. The eld trial began in 2004 in Beijing, China. Tree
growth parameters were investigated annually. None of transgenic
lines showed any signicant difference from the untransformed
wild-type line in height or trunk diameter (Fig. 1A and B).
Lignin and cellulose content were analyzed in stem biomass
from 5-year-old trees. Wild-type plants contained approximately
21% insoluble lignin, while transgenic lines contained approximately 19%. All transgenic plants showed signicant reductions
in insoluble lignin content, about 710% relative to wild-type
(Table 1). Transgenic lines B4CL28 and B4CL86 had levels of soluble
lignin similar to those of control plants, while lines with CCoAOMT
down-regulation showed signicant reductions in levels of soluble
lignin (Table 1), about 1416% below that of controls. This suggests
that CCoAOMT might be involved in the formation of soluble lignin.
Together, total lignin content of wild type tree was found to be
24.12%, whereas that of the transgenic lines ranged from 21.59 to
22.68% (Table 1), with a decrease of 610%. All the tested transgenic
lines showed a signicant increase in cellulose content, up to about
38%. These results indicate that increased cellulose compensated
the reduction of lignin, which is consistent with the observation
reported by Hu et al. (1999) and Li et al. (2003).
3.2. Effects of lignin modication on fermentable sugar yields for
biofuel production
Dried, milled, extractive-free stem biomass samples were pretreated with 1% or 3% NaOH at 120 C for 30 min, and the residue and

172

H. Wang et al. / Industrial Crops and Products 37 (2012) 170177

Table 1
Soluble lignin, insoluble lignin, and cellulose content in ve-year-old poplar wood.
Soluble lignin (%)

Control
B4CL28
B4CL86
BCOA264
BCOA133

Insoluble lignin (%)

Total lignin (%)

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

polysaccharides to enzymatic digestion. Alkali pretreatment


increased the glucose yield as well as enzymatic hydrolysis efciency of cellulose at accumulative chemical amount of 1% NaOH
and 3% NaOH (Table 2), and the increase of the enzymatic hydrolysis
efciency (i.e. pretreatment efciency) reached to 4350% with 1%
NaOH and 5064% with 3% NaOH pretreatment, respectively, compared to that of untreated biomass (Table 3). Pretreatment with
3% NaOH resulted in signicantly higher (p < 0.05) glucose yields
and cellulose digestion efciency than did pretreatment with 1%
NaOH in all the tested lines except transgenic line BCOA264 (Table 2
and Fig. 2B). However, the more severe pretreatment resulted in
signicantly lower (p < 0.05) xylose yields from enzymatic saccharication for all lines tested except transgenic line B4CL86
(Table 2 and Fig. 2C), due to the improved xylan solubilization during pretreatment with higher concentrations of chemicals (data
not shown). The much greater increase in the digestion efciency
(i.e. pretreatment efciency) with 3% NaOH, compared to 1% NaOH
pretreatment, took place in wild-type plants and transgenic lines
B4CL28 and B4CL86 (reaching to 12.9815.41%), which showed the
lowest level of release of glucose in the enzymatic hydrolysates of
untreated biomass (Table 3). This suggests that harsher pretreatment is more effective for the biomasses with strong resistance to
saccharication.
To evaluate the impact of lignin modication on biomass saccharication, the glucose and xylose yields were compared between
transgenic lines and wild-type plants (Fig. 2). The yield of glucose and total glucose and xylose showed signicant increases
in transgenic lines BCOA264 and BCOA133 under 1% NaOH pretreatment. With 3% NaOH pretreatment, signicant alterations in
the yield of glucose and total glucose and xylose were observed
only in transgenic line BCOA133 (Fig. 2A and B), suggesting that
pretreatment with harsher chemicals renders the positive effects
of lignin modication on saccharication efciency less obvious.
Unlike the alternations in glucose yields among the tested lines,
enzymatic hydrolysis efciency of cellulose with 1% NaOH pretreatment increased signicantly only in transgenic line BCOA133,
excluding line BCOA264, in which the hydrolysis efciency was
corrected and relatively decreased to its highest cellulose content
(Tables 3 and 1). In contrast, 4CL down-regulated transgenic trees
did not show signicant alterations in sugar yield relative to wild
type (Fig. 2A and B).
3.3. Correlations between composition and saccharication
under pretreatment conditions
Differences in glucose and xylose yield associated with the two
types of pretreatment are shown in Fig. 2B and C and Table 2. Linear correlations between the two pretreatments were signicant
for glucose released (r = 0.7826, p < 0.05) (Fig. 3A). Xylose release
showed no correlation with NaOH concentration during pretreatment (Fig. 3B).

H. Wang et al. / Industrial Crops and Products 37 (2012) 170177

173

Table 2
Sugar released by enzymatic hydrolysis of biomass samples pretreated with 1% and 3% NaOH.
Pretreatment

Control

B4CL28

B4CL86

BCOA264

BCOA133

Enzymatic hydrolysis efciency of cellulose %a

Sugar released by enzymatic hydrolysis [mg/g DW]

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.

Under both pretreatments, the amount of glucose released was


negatively correlated with the soluble lignin concentration of the
dry, extractive-free biomass (r = 0.8097 for 1% NaOH pretreatment
and r = 0.5801 for 3% NaOH pretreatment, p < 0.05) (Fig. 4A). However, different correlation patterns were observed between the
amount of xylose released and soluble lignin with response to the
concentration of NaOH used during pretreatment. The amount of
xylose released from the enzymatic hydrolysis step was negatively
correlated with soluble lignin concentration under 1% NaOH pretreatment (r = 0.933, p < 0.05), but no correlation was found under
3% NaOH pretreatment (Fig. 4B). Moreover, no correlation was
observed between the amounts of glucose or xylose released and
the concentration of insoluble lignin or cellulose with either pretreatment (Fig. 4CF).
4. Discussion
Generally speaking, the reduced trend of insoluble lignin content in transgenic trees remained stable over time, although
the content of lignin increased throughout development, having
approximate 19% of insoluble lignin in the ve-year-old transgenic
plants (Table 1) as opposed to 1015% in their one-year-old clonal
siblings reported previously (Jia et al., 2004; Zhao et al., 2004).
During wood formation, the relative reduction in insoluble lignin
content became less, with a reduction of 710% in ve-year-old

plants (Table 1) compared to 1640% in their one-year-old clonal


siblings (Jia et al., 2004; Zhao et al., 2004). These changes were
supported by the well known fact that trees naturally regulate
the synthesis of cell wall during wood formation (Scureld, 1973;
Wardrop and Davies, 1964). Thus, the increase in lignin could sustain mechanical strength in highly lignied and developed trees.
The augmentation of cellulose as compensation for the loss of lignin
(Table 1) imply another specic adaptation to strengthen mechanical support in lignin-decient xylem cells, and suggest that there
would be intrinsic crosstalk between the synthesis pathways of
these two major cell wall components. Reecting the real states
in the mature wood intended for biofuel production, our results
suggest a need for long-term eld trials of transgenic lines to
evaluate the effects of genetic modication and industrial application of tree crops.
Pretreatment is necessary for lignocellulosic biomass to be effectively hydrolyzed to fermentable sugar (Mosier et al., 2005). Sodium
hydroxide pretreatment used here promoted delignication as
well as xylan solubilization, although xylan solubilization due to
sodium hydroxide pretreatment was lower than that by dilute sulfuric acid pretreatment (data not shown), which was consistent
with the observation reported by Silverstein et al. (2007). And the
delignication and xylan solubilization were associated with pretreatment severity, with insoluble lignin content of 1216% in the
biomass residue and 6090 mg/g xylose dissolved in the ltrate

Table 3
Efciency of pretreatment with different concentrations of NaOH.
Line

Control
B4CL28
B4CL86
BCOA264
BCOA133

Enzymatic digestion efciency of cellulose [%]a

Pretreatment efciency [%]b

Untreated

1% NaOH

3% NaOH

1% NaOH vs. untreated

3% NaOH vs. untreated

3% NaOH vs. 1% 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.

H. Wang et al. / Industrial Crops and Products 37 (2012) 170177

Glucose yield under pretreatment


with 3% NaOH (mg/g)

174

400

380

y = 0.5854x + 179.58
R2 = 0.7826

360
340
320
300
200

250

300

350

400

Xylose yield under pretreatment


with 3% NaOH (mg/g)

Glucose yield under pretreatment with 1% NaOH (mg/g)

90
80
70
60
50
40
60

70

80

90

100

Xylose yield under pretreatment with 1% NaOH (mg/g)


Fig. 3. Sugar yield after pretreatment with different concentrations of alkaline
chemical. Each point represents an individual wild-type or transgenic plant. (A)
Glucose. (B) Xylose.

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.

after 3% NaOH pretreatment as opposed to left lignin of 1418%


and 4868% mg/g dissolved xylose for 1% NaOH pretreatment. As
reported (Jeffries et al., 2007; Vrnaia et al., 2010; Watanabe et al.,
2007), here it was found that the release of glucose in the enzymatic
hydrolysate increased in proportion to the increase of xylose with

1% NaOH pretreatment (Fig. 4G), which was negatively correlated


with soluble lignin content (Fig. 4B). This could be explained by the
fact that removal of xylan improves the accessibility of biomass to
the enzyme due to changes in the structure of plant cell walls, such
as increases in pore volume and specic surface area (Grohmann
et al., 1986).
In this study, we found that the characteristic of wood biomass
that most dramatically inuenced the glucose and xylose yields
in enzymatic hydrolysis was soluble lignin content, and it negatively correlated with the amount of glucose and xylose released
despite the insoluble lignin and cellulose content (Fig. 4AF). However, these results were against from our initial expectation. As
reported, lignin solubility is positively correlated with syringyl
(S) monomer content, and higher S content were associated with
higher pulp yield (Huntley et al., 2003; Magaton et al., 2009).
With the less S units here, more guaiacyl (G) units involved in
lignin chain would give more potential for covalent crosslinking
and would cause harder and less hydrolysis. But we found that
the less the soluble lignin (less S units) in poplar wood, the more
efcient saccharication occurred (Fig. 4A and B). And in 4CL downregulated poplar with non-changed soluble lignin content, glucose
yield did not increase signicantly (Table 1 and Fig. 2B). Similar result has been observed in dilute acid hydrolysis of populus
biomass, and lower S/G ratio signicantly increased the release of
xylose (Davison et al., 2006). Likewise, Corredor et al. (2009) have
reported that forage sorghums with a low S/G ratio were easy to
hydrolyze after pretreatment despite the initial lignin content. In
animal digestibility studies, it also has been observed that lower
S content correlated with improved degradability and more milk
and meat production (Fontaine et al., 2003). However, Voelker et al.
(2010) checked a number of 4CL down-regulated poplar lines with
reduced lignin content, a largely varied S/G ratio and rich phenolic
extractives, and found that there were no substantially increase of

H. Wang et al. / Industrial Crops and Products 37 (2012) 170177

Pretreated with 1% NaOH

Pretreated with 3% NaOH

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

Soluble lignin (%)


400

175

2.9

3.1

3.3

3.5

Soluble Lignin (%)

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

Insoluble lignin (%)

20 .0

20.5

21.0

21.5

52

53

Insoluble lignin (%)

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

Cellulose percent dry weight (%)

Cellulose percent dry weight (%)

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

soluble and/or insoluble lignin and other cell wall extractives


could acted as inhibitory components for cellulase in enzymatic
hydrolysate, as reported by Berlin et al. (2006) and Chundawat et al.
(2007).

176

H. Wang et al. / Industrial Crops and Products 37 (2012) 170177

Here we demonstrate the feasibility of generating, through


genetic engineering with CCoAOMT down-regulation, a wood easily susceptible to saccharication in biofuel production. Analysis of
ve-year-old trees does mean applicable to commercial projects
since the Chinese white poplars could be harvested by the end of
ve growing seasons. Lignin modication presents a substantial
improvement for biofuel production as the result of the increase
in digestion efciency of cellulose (Tables 2 and 3) combined
with non-reduction in biomass yield (Fig. 1). And it will benet bioconversion by using less severe chemical pretreatment. For
example, about 330 mg/g glucose was recovered from a wild-type
tree pretreated with 3% NaOH, while transgenic lines BCOA264
and BCOA133 achieved comparable or greater glucose yield at a
lower NaOH concentration of 1% (Table 2). However, the release of
fermentable sugar from lignocellulosic biomass was affected complicatedly by a number of factors, such as cellulose crystallinity,
accessible surface area, biomass composition and structure (Mosier
et al., 2005). A report recently showed diverse inuences of lignin
content and S/G ration on sugar release from Populus wood, implying that a deeper understanding of cell wall structure is needed
before plants can be rationally engineered for reduced recalcitrance
and efcient biofuels production (Studer et al., 2011).
5. Conclusion
Although many studies have investigated the relationship
between cell wall composition or structure and cellulose digestibility in poplar wood (Grethlein, 1985; Grohmann et al., 1989; Kong
et al., 1992; Studer et al., 2011) and two-year-old lignin-engineered
poplar wood (Voelker et al., 2010), very little is known about the
effects of lignin engineering on cellulose hydrolysis from mature
poplar wood. We set up a long-term eld trial of transgenic poplars
with either 4CL or CCoAOMT down-regulation, which was then
subject to pretreatment and enzymatic hydrolysis to evaluate the
potential application of lignin-modied poplars in biofuel production. The results are as following:
1. 4CL and CCoAOMT down-regulation of poplars resulted in a
610% reduction in total lignin but did not cause altered growth
rates or biomass yields.
2. Soluble-lignin-reduced transgenic lines with CCoAOMT downregulation showed signicant increases in glucose yield
benecial to biofuel production. There was a strong negative correlation between soluble lignin content and amount of glucose
released by enzymatic hydrolysis.
3. Harsher pretreatment rendered the positive effects of lignin
modication on saccharication efciency and sugar yield less
obvious.
In conclusion, data presented here demonstrate that lignin
modication does indeed improve saccharication of biomass in
transgenic Populus tomentosa, implying the potential application
of lignin-modied poplar in biofuel production.
Acknowledgements
This work was supported by the State 863 High-Tech Program
(Grant Nos. 2009AA10Z101 and 2011AA100201) and 948 Project of
the State Forestry Administration of China (Grant No. 2008-4-26).
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