LP

HB

a

ARRAA

KTLBS

1

abomh(aspSleeio

ARf

0d

Industrial Crops and Products 37 (2012) 170– 177

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l h o me pag e: www.elsev ier .com/ locate / indcrop

ignin modification improves the biofuel production potential in transgenicopulus tomentosa

ongzhi Wang1, Yingxi Xue1, Yajuan Chen, Ruifen Li, Jianhua Wei ∗

eijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China

r t i c l e i n f o

rticle history:eceived 30 August 2011eceived in revised form 6 December 2011ccepted 14 December 2011vailable online 11 January 2012

eywords:ransgenic poplarsignin modification

a b s t r a c t

Lignin has been recognized for its negative impact on forage digestibility, tree pulping properties, andcellulosic biofuel production, although it is the major structural component of the secondarily thickenedcell walls of vascular plants. Earlier studies have demonstrated that lignin modification improves foragedigestibility and poplar pulping properties. To determine whether lignin modification has beneficialeffect on saccharification of lignocellulosic biomass, we pretreated and then enzymatically hydrolyzedthe mature wood from transgenic poplar plants that expressed the antisense transgenes of monolignolbiosynthesis genes 4-coumarate: CoA ligase (4CL) or caffeoyl CoA 3-O-methyltransferase (CCoAOMT).Firstly, a long-term field trial was set up for the transgenic plants. Over five years, the reduced trend

iofuelsaccharification

of lignin content remained stable in all transgenic lines. And a total lignin reduction of up to 10% didnot alter the growth rate or biomass yield of the transgenic poplars. In the mature wood, suppressionof CCoAOMT increased saccharification potential, but 4CL down-regulation had no significantly positiveeffect on saccharification. Sugar yield were negatively correlated with soluble lignin content of dried,extractive-free stem biomass. These results imply that lignin modification can facilitate the process of

el pro

saccharification for biofu

. Introduction

Renewable and sustainable liquid biofuels must be explored aslternatives to petroleum-based fuels if the world energy crisis is toe solved (Kerr and Service, 2005; Schubert, 2006). Bioconversionf cellulosic biomass to fuel is considered a particularly attractiveeans of generating low-cost, sustainable biofuels, with enzymatic

ydrolysis of cellulose to glucose and then fermentation to ethanolWyman, 1999). Lignocellulosic biomass from non-food crops, suchs trees and grasses, among a variety of other biofuel feedstockources, has been proposed as a potential raw material source forroducing cellulosic ethanol (Demirbas, 2005; Lynd et al., 1991;omerville, 2006, 2007). Currently, producing biofuels derived fromignocellulosic biomass is expensive and difficult due to the inher-nt complexity of plant cell walls (Himmel et al., 2007; Somerville

t al., 2004). Lignin is one major component of the plant cell wall ands known to be especially problematic with regard to bioconversionf cellulosic biomass to fuels because it interferes with the ability

∗ Corresponding author at: Beijing Agro-Biotechnology Research Center, Beijingcademy of Agriculture and Forestry Sciences, No. 9, Shuguang Huayuan Middleoad, Haidian District, Beijing 100097, China. Tel.: +86 10 51503830;

ax: +86 10 51503980.E-mail addresses: weijianhua@baafs.net.cn, cauwhz@yahoo.com (J. Wei).

1 These two authors contributed equally to this paper.

926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2011.12.014

duction in tree crops.© 2011 Elsevier B.V. All rights reserved.

of hydrolytic enzymes to access the cellulose polymer and adsorbshydrolytic enzymes (Chen and Dixon, 2007; Simmons et al., 2010;Keating et al., 2006). Loosening lignin’s grip on biofuel productionhas become the new aim of lignin engineering of plants (Chappleet al., 2007).

During the last two decades, most reports on lignin engineer-ing have involved in improving forage digestibility and pulpingperformance. Recently, there have been a few reports in whichthe aim of lignin modification was to improve biofuel production.Chen and Dixon (2007) analyzed the biomass digestibility of sixlignin-modified alfalfa lines by cellulase/cellobiase with and with-out dilute acid pretreatment. There were significant differences inenzymatic hydrolysis efficiency in biomass of the transgenic alfalfalines. Some lines showed enzymatic efficiencies double those of thewild-type control plant. These results indicate that lignin modifi-cation can facilitate the bioconversion of lignocellulosic biomass tobiofuel in alfalfa. Davison et al. (2006) analyzed the saccharificationefficiency of poplar biomass in five genetic variants from a singlesegregating F2 hybrid family via acid hydrolysis. Results indicatedthat both lignin content and S/G ratio significantly affected the yieldof 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 saccharificationpotential of transgenic poplars with 4CL down-regulation.

To investigate the agronomic and enzymatic saccharifica-tion performance of lignin-modified poplars and to evaluate the

ps and

cdfipCp

2

2

tdB2erFtw1

ofdaZcdt

2

mSeftattsfiCt

2

iPpb

2

aR1hl1u

H. Wang et al. / Industrial Cro

ommercial value of the mature wood of 4CL- or CCoAOMT-eficient transgenic poplars for biofuel production, a long-termeld trial was set up. The resulting biomass was subjected toretreatment and enzymatic hydrolysis. Our results showed thatCoAOMT deficiency significantly improved the saccharificationotential of poplar biomass.

. Material and methods

.1. Plant materials and sample collection

The study was performed on transgenic poplar (Populus tomen-osa) lines B4CL28, B4CL86, BCOA264, and BCOA133, all previouslyescribed and known to be deficient in either 4CL (B4CL28 and4CL86) or CCoAOMT (BCOA264 and BCOA133) activity (Jia et al.,004; Zhao et al., 2004). Shoots were multiplied from each line byxcising nodal segments and allowing axillary buds to elongate andoots to regenerate. They were then acclimatized in a greenhouse inebruary 2004. In April 2004, these lines, along with wild-type con-rol plants, were planted in the field. Agronomic traits of all treesere investigated, and stem height (main stem) and diameter (at

.3 m high) were measured annually.Five-year-old plants were harvested prior to dormancy in the fall

f 2009. Stems of two different plants from each line at 1.0–1.4 mrom the ground were collected, debarked, chipped, mixed, andried overnight in an oven at 60 ◦C. They were then ground in

mill with a sieve diameter of 1.00 mm (DFT-2000, Wenling,hejiang, China). Soluble extractives were removed by three suc-essive extractions with methanol at room temperature. Theseried, extractive-free biomass samples were used for the composi-ional analyses and saccharification experiments.

.2. Determination of lignin and cellulose content

Acid-insoluble lignin (i.e. Klason Lignin) content was deter-ined using a modified method derived from the China National

tandard Method GB/T 2677.8-94. Briefly, 1.0 g of methanol-xtracted ground stem sample was treated with 4 mL of 72% H2SO4or 1 h at 30 ◦C with mixing taking place every 10 min. This mix-ure was diluted with 142 mL of deionized water to achieve a finalcid concentration of 3% H2SO4 and transferred to a Schott bot-le. The solution was then autoclaved at 100 ◦C for 1 h and filteredhrough a crucible for determination of acid-insoluble lignin. Acid-oluble lignin was quantified by spectrophotometric analysis of theltrate at 205 nm (National Standard Method GB/T 10337-2008).ellulose content was determined using nitric acid–ethanol mix-ure methodology (Liu, 2003).

.3. Chemical pretreatment

Dried, milled, and extractive-free stem material at a solid load-ng of 10% (w/v) was mixed with 1% or 3% sodium hydroxide.retreatment was run in an autoclave at 120◦C for 30 min. Afterretreatment, residual biomass was separated from supernatanty filtration and washed with water.

.4. Enzymatic hydrolysis

Enzymatic hydrolysis of pretreated residues was conductedccording to the laboratory analytical procedures of the Nationalenewable Energy Laboratory (LAP-009) (Brown and Torget,996). After filtering and washing, 0.5-g biomass samples were

ydrolyzed with mixtures of cellulase (Celluclast 1.5 L) and cel-

obiase (Novozyme 188) in a total volume of 30 mL at 50 ◦C and50 rpm for 72 h. The activity of enzyme was 94.9 filter papernits (FPU) mL−1 for cellulase and 101 CBU mL−1 for cellobiase.

Products 37 (2012) 170– 177 171

Equal volumes of the two enzymes were mixed, and the loadingsof enzyme mixture were in excess (about 90 FPU per g cellulose).Sodium citrate buffer (0.05 M, pH 4.8) was used to maintain thepH at 4.8. Tetracycline and cycloheximide were added to protectthe reaction mixture from microbes. Enzyme blanks were set upalongside the sample.

2.5. Sugar content

The enzymatic hydrolysate was centrifuged for 2 min. The sus-pernatant was then filtrated with 0.45 �m filter. The filtrate wassubjected to glucose and xylose analysis by HPLC (Agelent 1100Series). An Aminex colum (Model HPX-87P, Bio-Rad, Sunnyvale,CA, U.S.) was used to separate the sugars.

2.6. Statistical analyses

Significance (ANOVA) was tested at p < 0.05 using SPSS (SPSS Inc,1998), to evaluate the statistical differences in lignin and cellulosecontent and the amounts of sugar released in different lines. Twoplants for each independent line were analyzed, with the exceptionof transgenic line B4CL28, only one plant of which was analyzed.All compositional and saccharification analyses were performed intriplicate for each individual plant biomass. Regression analysis wasperformed using the statistics program in Microsoft Office Excel2003.

3. Results

3.1. Growth, lignin content, and cellulose content of transgenicpoplars

A long-term field trial of transgenic poplar lines was set up toevaluate the effects of lignin modification on plant growth andwood properties. Five different lines, including the untransformedwild-type line (Populus tomentosa), two antisense lines deficient in4CL, B4CL28 and B4CL86 (Jia et al., 2004), and two antisense linesdeficient in CCoAOMT, BCOA264 and BCOA133 (Zhao et al., 2004),were evaluated. The field trial began in 2004 in Beijing, China. Treegrowth parameters were investigated annually. None of transgeniclines showed any significant difference from the untransformedwild-type line in height or trunk diameter (Fig. 1A and B).

Lignin and cellulose content were analyzed in stem biomassfrom 5-year-old trees. Wild-type plants contained approximately21% insoluble lignin, while transgenic lines contained approxi-mately 19%. All transgenic plants showed significant reductionsin insoluble lignin content, about 7–10% relative to wild-type(Table 1). Transgenic lines B4CL28 and B4CL86 had levels of solublelignin similar to those of control plants, while lines with CCoAOMTdown-regulation showed significant reductions in levels of solublelignin (Table 1), about 14–16% below that of controls. This suggeststhat CCoAOMT might be involved in the formation of soluble lignin.Together, total lignin content of wild type tree was found to be24.12%, whereas that of the transgenic lines ranged from 21.59 to22.68% (Table 1), with a decrease of 6–10%. All the tested transgeniclines showed a significant increase in cellulose content, up to about3–8%. These results indicate that increased cellulose compensatedthe reduction of lignin, which is consistent with the observationreported by Hu et al. (1999) and Li et al. (2003).

3.2. Effects of lignin modification on fermentable sugar yields for

biofuel production

Dried, milled, extractive-free stem biomass samples were pre-treated 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) 170– 177

Table 1Soluble lignin, insoluble lignin, and cellulose content in five-year-old poplar wood.

Soluble lignin (%) Insoluble lignin (%) Total lignin (%) Cellulose (%)

Mean SD Reductiona Mean SD Reductiona Mean SD Reductiona Mean SD Increase b

Control 3.19a 0.12 – 20.92a 1.00 – 24.12a 1.09 – 47.94a 0.76 –B4CL28 3.19a 0.10 0.00 18.93b 0.01 9.51 22.16b 0.11 8.13 51.53d 0.85 7.49B4CL86 3.25a 0.09 −1.88 19.38b 0.52 7.36 22.68b 0.50 5.97 49.58b 0.40 3.42BCOA264 2.73b 0.09 14.42 18.91b 0.53 9.61 21.59b 0.46 10.49 51.75d 0.62 7.95BCOA133 2.69b 0.15 15.67 19.21b 0.43 8.17 21.80b 0.42 9.62 50.41c 0.53 5.15

Means with the same letter are not significantly different from each other at the 0.95 confidence level.aReduction 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 wildbIncrease refers to percent increase of cellulose in transgenic lines comparin(cellulose content of transgenic line − cellulose content of wild type plant)/cellulose conte

Fig. 1. Height and diameter of lignin-modified poplars grown in field. (A) Aver-aA

hm7aNiTyot

ehfnts

ear correlations between the two pretreatments were significant

ge diameter of transgenic lines from 2005 to 2008, measured at 1.3 m height. (B)verage height. Error bars represent the SD from the mean. n = 5 per line.

ydrolysate were separated. Solid residue remaining after pretreat-ent decreased with higher concentrations of chemical, averaging

5–81% of stem biomass residue for tested lines with 1% NaOHnd 63–69% stem biomass residue after pretreatment with 3%aOH. This indicates that the removal of lignin and hemicellulose

ncreased with higher concentrations of pretreatment chemical.he residue after pretreatment was subjected to enzymatic hydrol-sis with cellulase and cellobiase alongside the untreated biomassf tested lines. Data on glucose and xylose yield were normalizedo dried, extractive-free biomasses.

The yields of glucose and xylose and enzymatic hydrolysisfficiency of cellulose after alkali pretreatment and enzymaticydrolysis are shown in Table 2. Little glucose was released

rom untreated biomasses subjected to enzymatic digestion, and

o xylose was detectable in enzymatic hydrolysates, suggestinghat lignocellulosic biomass must be pretreated to open up thetructure of the plant cell wall and improve the accessibility of

type plant × 100.g to that of wild type plant, which was calculated as: Increase (%) =

nt of wild type plant × 100.

polysaccharides to enzymatic digestion. Alkali pretreatmentincreased the glucose yield as well as enzymatic hydrolysis effi-ciency of cellulose at accumulative chemical amount of 1% NaOHand 3% NaOH (Table 2), and the increase of the enzymatic hydrolysisefficiency (i.e. pretreatment efficiency) reached to 43–50% with 1%NaOH and 50–64% with 3% NaOH pretreatment, respectively, com-pared to that of untreated biomass (Table 3). Pretreatment with3% NaOH resulted in significantly higher (p < 0.05) glucose yieldsand cellulose digestion efficiency than did pretreatment with 1%NaOH in all the tested lines except transgenic line BCOA264 (Table 2and Fig. 2B). However, the more severe pretreatment resulted insignificantly lower (p < 0.05) xylose yields from enzymatic sac-charification for all lines tested except transgenic line B4CL86(Table 2 and Fig. 2C), due to the improved xylan solubilization dur-ing pretreatment with higher concentrations of chemicals (datanot shown). The much greater increase in the digestion efficiency(i.e. pretreatment efficiency) with 3% NaOH, compared to 1% NaOHpretreatment, took place in wild-type plants and transgenic linesB4CL28 and B4CL86 (reaching to 12.98–15.41%), which showed thelowest level of release of glucose in the enzymatic hydrolysates ofuntreated biomass (Table 3). This suggests that harsher pretreat-ment is more effective for the biomasses with strong resistance tosaccharification.

To evaluate the impact of lignin modification on biomass saccha-rification, the glucose and xylose yields were compared betweentransgenic lines and wild-type plants (Fig. 2). The yield of glu-cose and total glucose and xylose showed significant increasesin transgenic lines BCOA264 and BCOA133 under 1% NaOH pre-treatment. With 3% NaOH pretreatment, significant alterations inthe yield of glucose and total glucose and xylose were observedonly in transgenic line BCOA133 (Fig. 2A and B), suggesting thatpretreatment with harsher chemicals renders the positive effectsof lignin modification on saccharification efficiency less obvious.Unlike the alternations in glucose yields among the tested lines,enzymatic hydrolysis efficiency of cellulose with 1% NaOH pre-treatment increased significantly only in transgenic line BCOA133,excluding line BCOA264, in which the hydrolysis efficiency wascorrected and relatively decreased to its highest cellulose content(Tables 3 and 1). In contrast, 4CL down-regulated transgenic treesdid not show significant alterations in sugar yield relative to wildtype (Fig. 2A and B).

3.3. Correlations between composition and saccharificationunder pretreatment conditions

Differences in glucose and xylose yield associated with the twotypes of pretreatment are shown in Fig. 2B and C and Table 2. Lin-

for glucose released (r = 0.7826, p < 0.05) (Fig. 3A). Xylose releaseshowed no correlation with NaOH concentration during pretreat-ment (Fig. 3B).

H. Wang et al. / Industrial Crops and Products 37 (2012) 170– 177 173

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

Pretreatment Sugar released by enzymatic hydrolysis [mg/g DW] Enzymatic hydrolysis efficiency of cellulose %a

Xylose Glucose Total sugars

Control Untreated 0a 21a 21a 4.27a1% NaOH 77c 255b 331b 53.09b3% NaOH 59b 328c 387c 68.50c

B4CL28 Untreated 0a 27a 27a 5.32a1% NaOH 78c 279b 356b 53.85b3% NaOH 58b 354c 412b 69.23c

B4CL86 Untreated 0a 18a 18a 3.57a1% NaOH 72b 255b 326b 51.33b3% NaOH 66b 319c 385c 64.31c

BCOA264 Untreated 0a 87a 87a 16.79a1% NaOH 85c 310b 395b 59.91b3% NaOH 49b 341b 391b 67.11b

BCOA133 Untreated 0a 86a 86a 17.15a1% NaOH 92c 339b 430b 67.17b3% NaOH 83b 388c 471c 77.01c

Means not sharing a common letter, within individual tested lines, differ in response to pretreatment.a Enzymatic hydrolysis efficiency (%) means total cellulose digested as a percentage of total cellulose in the dried, extractive-free biomass subjected to pretreatment

a as recm , and i0

ndaeacxct3ott

4

ttapsDc

TE

M

am0

ld

nd enzymatic hydrolysis with cellulose and cellobiase. Total cellulose digested wolecule added upon hydrolysis of the cellulose polymer (Brown and Torget, 1996)

.9/gram cellulose added × 100.

Under both pretreatments, the amount of glucose released wasegatively correlated with the soluble lignin concentration of thery, extractive-free biomass (r = 0.8097 for 1% NaOH pretreatmentnd r = 0.5801 for 3% NaOH pretreatment, p < 0.05) (Fig. 4A). How-ver, different correlation patterns were observed between themount of xylose released and soluble lignin with response to theoncentration of NaOH used during pretreatment. The amount ofylose released from the enzymatic hydrolysis step was negativelyorrelated with soluble lignin concentration under 1% NaOH pre-reatment (r = 0.933, p < 0.05), but no correlation was found under% NaOH pretreatment (Fig. 4B). Moreover, no correlation wasbserved between the amounts of glucose or xylose released andhe concentration of insoluble lignin or cellulose with either pre-reatment (Fig. 4C–F).

. Discussion

Generally speaking, the reduced trend of insoluble lignin con-ent in transgenic trees remained stable over time, althoughhe content of lignin increased throughout development, havingpproximate 19% of insoluble lignin in the five-year-old transgenic

lants (Table 1) as opposed to 10–15% in their one-year-old clonaliblings reported previously (Jia et al., 2004; Zhao et al., 2004).uring wood formation, the relative reduction in insoluble ligninontent became less, with a reduction of 7–10% in five-year-old

able 3fficiency of pretreatment with different concentrations of NaOH.

Line Enzymatic digestion efficiency of cellulose [%]a Pretreat

Untreated 1% NaOH 3% NaOH 1% NaO

Control 4.27a 53.09a 68.50a 48.82

B4CL28 5.32a 53.85a 69.23a 48.53

B4CL86 3.57a 51.33a 64.31a 47.76

BCOA264 16.79b 59.91ab 67.11a 43.12

BCOA133 17.15b 67.17b 77.01b 50.02

eans with the same letter are not significantly different from each other at the 0.95 cona Enzymatic hydrolysis efficiency (%) means total cellulose digested as a percentage

nd enzymatic hydrolysis with cellulose and cellobiase. Total cellulose digested was recolecule added upon hydrolysis of the cellulose polymer (Brown and Torget, 1996), and i

.9/gram cellulose added × 100.b Pretreatment efficiency (%) means the increased enzymatic hydrolysis efficiency (%) by

ower concentration. For example: Pretreatment efficiency % of 3% NaOH vs. 1% NaOH = enzigestion efficiency of cellulose (%) with 1% NaOH pretreatment.

overed by multiplying the total glucose released with 0.9 to correct for the watert was calculated as: Enzymatic hydrolysis efficiency (%) = grams glucose released ×

plants (Table 1) compared to 16–40% in their one-year-old clonalsiblings (Jia et al., 2004; Zhao et al., 2004). These changes weresupported by the well known fact that trees naturally regulatethe synthesis of cell wall during wood formation (Scurfield, 1973;Wardrop and Davies, 1964). Thus, the increase in lignin could sus-tain mechanical strength in highly lignified and developed trees.The augmentation of cellulose as compensation for the loss of lignin(Table 1) imply another specific adaptation to strengthen mechan-ical support in lignin-deficient xylem cells, and suggest that therewould be intrinsic crosstalk between the synthesis pathways ofthese two major cell wall components. Reflecting the real statesin the mature wood intended for biofuel production, our resultssuggest a need for long-term field trials of transgenic lines toevaluate the effects of genetic modification and industrial appli-cation of tree crops.

Pretreatment is necessary for lignocellulosic biomass to be effec-tively hydrolyzed to fermentable sugar (Mosier et al., 2005). Sodiumhydroxide pretreatment used here promoted delignification aswell as xylan solubilization, although xylan solubilization due tosodium hydroxide pretreatment was lower than that by dilute sul-furic acid pretreatment (data not shown), which was consistentwith the observation reported by Silverstein et al. (2007). And the

delignification and xylan solubilization were associated with pre-treatment severity, with insoluble lignin content of 12–16% in thebiomass residue and 60–90 mg/g xylose dissolved in the filtrate

ment efficiency [%]b

H vs. untreated 3% NaOH vs. untreated 3% NaOH vs. 1% NaOH

64.23 15.4163.91 15.3860.74 12.9850.32 7.2059.86 9.84

fidence level.of total cellulose in the dried, extractive-free biomass subjected to pretreatmentovered by multiplying the total glucose released with 0.9 to correct for the watert was calculated as: Enzymatic hydrolysis efficiency (%) = grams glucose released ×

indicated pretreatment, compared to the untreated or chemical pretreatment withymatic digestion efficiency of cellulose (%) with 3% NaOH pretreatment − enzymatic

174 H. Wang et al. / Industrial Crops and Products 37 (2012) 170– 177

Fig. 2. Sugar released from poplars via enzymatic digestion of pre-treated anduntreated biomass by a mixture of cellulase and cellobiase. (A) Total glucose andxylose released during saccharification. (B) Glucose released during saccharifica-tion. (C) Xylose released during saccharification. Means not sharing common lettersor numbers, within each pretreatment, differ in response to the individual testedlines. Lignified stems (from at 1.0 to 1.4 m off the ground) were harvested fromfive-year field-grown poplars. Dried, milled, extractive-free stems were untreatedor pretreated with alkaline substances and then subjected to enzymatic hydroly-sis with cellulase and cellobiase. Control, untransformed control Populus tomentosa;B4CL28 and B4CL86: two transgenic lines down-regulated for 4CL activity; BCOA264am

aar2h

A

y = 0.5854x + 179.58R2 = 0.7826

300

320

340

360

380

400

400350300250200Glucose yield under pretreatment with 1% NaOH (mg/g)

Glu

cose

yie

ld u

nder

pre

treat

men

t w

ith 3

% N

aOH

(mg/

g)

B

40

50

60

70

80

90

10090807060

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

Xyl

ose

yiel

d un

der p

retre

atm

ent

with

3%

NaO

H (m

g/g)

Fig. 3. Sugar yield after pretreatment with different concentrations of alkaline

nd BCOA133: two lines down-regulated for CCoAOMT activity. Sugar levels wereeasured by HPLC.

fter 3% NaOH pretreatment as opposed to left lignin of 14–18%

nd 48–68% mg/g dissolved xylose for 1% NaOH pretreatment. Aseported (Jeffries et al., 2007; Várnaia et al., 2010; Watanabe et al.,007), here it was found that the release of glucose in the enzymaticydrolysate increased in proportion to the increase of xylose with

chemical. Each point represents an individual wild-type or transgenic plant. (A)Glucose. (B) Xylose.

1% NaOH pretreatment (Fig. 4G), which was negatively correlatedwith soluble lignin content (Fig. 4B). This could be explained by thefact that removal of xylan improves the accessibility of biomass tothe enzyme due to changes in the structure of plant cell walls, suchas increases in pore volume and specific surface area (Grohmannet al., 1986).

In this study, we found that the characteristic of wood biomassthat most dramatically influenced the glucose and xylose yieldsin enzymatic hydrolysis was soluble lignin content, and it nega-tively correlated with the amount of glucose and xylose releaseddespite the insoluble lignin and cellulose content (Fig. 4A–F). How-ever, these results were against from our initial expectation. Asreported, lignin solubility is positively correlated with syringyl(S) monomer content, and higher S content were associated withhigher pulp yield (Huntley et al., 2003; Magaton et al., 2009).With the less S units here, more guaiacyl (G) units involved inlignin chain would give more potential for covalent crosslinkingand would cause harder and less hydrolysis. But we found thatthe less the soluble lignin (less S units) in poplar wood, the moreefficient saccharification occurred (Fig. 4A and B). And in 4CL down-regulated poplar with non-changed soluble lignin content, glucoseyield did not increase significantly (Table 1 and Fig. 2B). Simi-lar result has been observed in dilute acid hydrolysis of populusbiomass, and lower S/G ratio significantly increased the release ofxylose (Davison et al., 2006). Likewise, Corredor et al. (2009) havereported that forage sorghums with a low S/G ratio were easy tohydrolyze after pretreatment despite the initial lignin content. Inanimal digestibility studies, it also has been observed that lowerS 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 withreduced lignin content, a largely varied S/G ratio and rich phenolicextractives, and found that there were no substantially increase of

H. Wang et al. / Industrial Crops and Products 37 (2012) 170– 177 175

A

y = -135.28x + 686.82R2 = 0.8097

y = -75.781x + 571.46R2 = 0.5801

200

250

300

350

400

2.5 2.7 2.9 3.1 3.3 3.5Soluble lignin (%)

Glu

cose

(mg/

g)

Pretreated with 1% NaOH Pretreated with 3% NaOH

By = -29.695x + 170.06

R2 = 0.933

40

50

60

70

80

90

100

2.5 2.7 2.9 3.1 3.3 3.5Soluble Lignin (%)

Xyl

ose

(mg/

g)

C

200

250

300

350

400

18.5 19 .0 19 .5 20.0 20.5 21.0 21 .5

Insoluble lignin (%)

Glu

cose

(mg/

g)

G

y = 4.3822x - 73.812R2 = 0.8029

200

250

300

350

400

40 50 60 70 80 90 100Xylose (mg/g)

Glu

cose

(mg/

g)

D

40

50

60

70

80

90

100

18.5 19.0 19.5 20 .0 20.5 21.0 21.5Insoluble lignin (%)

Xyl

ose

(mg/

g)

E

200

250

300

350

400

47 48 49 50 51 52 53Cellulose percent dry weight (%)

Glu

cose

(mg/

g)

F

40

50

60

70

80

90

100

47 48 49 50 51 52 53

Cellulose percent dry weight (%)

Xyl

ose

(mg/

g)

Fig. 4. Relationships between composition of biomass and saccharification. Each point represents an individual wild type or transgenic plant. (A) Amount of glucose releasedi tmenc t of gle t of xy

sctc

s shown as a function of the soluble lignin content of the biomass with both pretreaontent with the 1% NaOH pretreatment. (C–F) No correlation between the amounither pretreatment. (G) The amount of glucose released was parallel to the amoun

accharification potential. On the contrary, statistically signifi-ant decrease in total glucose and xylose release was shown inwo lines with significant lower S/G ratio and higher extractiveontent. These data recommended that phenolic fragment from

ts. (B) Linear correlation between the amount of xylose released with soluble ligninucose or xylose released and insoluble lignin or cellulose content was found withlose in the hydrolysate with the 1% NaOH pretreatment.

soluble and/or insoluble lignin and other cell wall extractivescould acted as inhibitory components for cellulase in enzymatichydrolysate, as reported by Berlin et al. (2006) and Chundawat et al.(2007).

1 ps and

gifisfiiiwfietalfpaeciba

5

biepepwspt

1

2

3

mto

A

(t

R

B

B

76 H. Wang et al. / Industrial Cro

Here we demonstrate the feasibility of generating, throughenetic engineering with CCoAOMT down-regulation, a wood eas-ly susceptible to saccharification in biofuel production. Analysis ofve-year-old trees does mean applicable to commercial projectsince the Chinese white poplars could be harvested by the end ofve growing seasons. Lignin modification presents a substantial

mprovement for biofuel production as the result of the increasen digestion efficiency of cellulose (Tables 2 and 3) combined

ith non-reduction in biomass yield (Fig. 1). And it will bene-t bioconversion by using less severe chemical pretreatment. Forxample, about 330 mg/g glucose was recovered from a wild-typeree pretreated with 3% NaOH, while transgenic lines BCOA264nd BCOA133 achieved comparable or greater glucose yield at aower NaOH concentration of 1% (Table 2). However, the release ofermentable sugar from lignocellulosic biomass was affected com-licatedly by a number of factors, such as cellulose crystallinity,ccessible surface area, biomass composition and structure (Mosiert al., 2005). A report recently showed diverse influences of ligninontent and S/G ration on sugar release from Populus wood, imply-ng that a deeper understanding of cell wall structure is neededefore plants can be rationally engineered for reduced recalcitrancend efficient biofuels production (Studer et al., 2011).

. Conclusion

Although many studies have investigated the relationshipetween cell wall composition or structure and cellulose digestibil-

ty in poplar wood (Grethlein, 1985; Grohmann et al., 1989; Kongt al., 1992; Studer et al., 2011) and two-year-old lignin-engineeredoplar wood (Voelker et al., 2010), very little is known about theffects of lignin engineering on cellulose hydrolysis from matureoplar wood. We set up a long-term field trial of transgenic poplarsith either 4CL or CCoAOMT down-regulation, which was then

ubject to pretreatment and enzymatic hydrolysis to evaluate theotential application of lignin-modified poplars in biofuel produc-ion. The results are as following:

. 4CL and CCoAOMT down-regulation of poplars resulted in a6–10% reduction in total lignin but did not cause altered growthrates or biomass yields.

. Soluble-lignin-reduced transgenic lines with CCoAOMT down-regulation showed significant increases in glucose yieldbeneficial to biofuel production. There was a strong negative cor-relation between soluble lignin content and amount of glucosereleased by enzymatic hydrolysis.

. Harsher pretreatment rendered the positive effects of ligninmodification on saccharification efficiency and sugar yield lessobvious.

In conclusion, data presented here demonstrate that ligninodification does indeed improve saccharification of biomass in

ransgenic Populus tomentosa, implying the potential applicationf lignin-modified poplar in biofuel production.

cknowledgements

This work was supported by the State 863 High-Tech ProgramGrant Nos. 2009AA10Z101 and 2011AA100201) and 948 Project ofhe State Forestry Administration of China (Grant No. 2008-4-26).

eferences

erlin, A., Balakshin, M., Gilkes, N., Kadla, J., Maximenko, V., Kubo, S., Saddler, J., 2006.Inhibition of cellulase, xylanase and beta-glucosidase activities by softwoodlignin preparations. J. Biotechnol. 125, 198–209.

rown, L., Torget, R., 1996. Enzymatic saccharification of lignocellulosic biomass, no.LAP 009. National Renewable Energy Laboratory, Golden, CO.

Products 37 (2012) 170– 177

Chapple, C., Ladisch, M., Meilan, R., 2007. Loosening lignin’s grip on biofuel Produc-tion. Nat. Biotechnol. 25, 746–748.

Chen, F., Dixon, R.A., 2007. Lignin modification improves fermentable sugar yieldsfor biofuel production. Nat. Biotechnol. 25, 759–761.

Chundawat, S.P., Venkatesh, B., Dale, B.E., 2007. Effect of particle size based sepa-ration of milled corn stover on AFEX pretreatment and enzymatic digestibility.Biotechnol. Bioeng. 96, 219–231.

Corredor, D.Y., Salazar, J.M., Hohn, K.L., Bean, S., Bean, B., Wang, D., 2009. Evalua-tion and characterization of forage sorghum as feedstock for fermentable sugarproduction. Appl. Biochem. Biotechnol. 158 (1), 164–179.

Davison, B.H., Drescher, S.R., Tuskan, G.A., Davis, M.F., Nghiem, N.P., 2006. Variationof S/G ratio and lignin content in a Populus family influences the release of xyloseby dilute acid hydrolysis. Appl. Biochem. Biotechnol. 129–132, 427–435.

Demirbas, A., 2005. Bioethanol from cellulosic materials: a renewable motor fuelfrom biomass. Energy Sources 27, 327–337.

Fontaine, A.S., Bout, S., Barriere, Y., Vermerris, W., 2003. Variation in cell wallcomposition among forage maize (Zea mays L.) inbred lines and its impacton digestibility: analysis of neutral detergent fiber composition by pyrolysis-gas chromatography–mass spectrometry. J. Agric. Food Chem. 51, 8080–8087.

Grethlein, H.E., 1985. The effect of pore-size distribution on the rate of enzymatic-hydrolysis of cellulosic substrates. Nat. Biotechnol. 3, 155–160.

Grohmann, K., Mitchell, D.J., Himmel, M.E., 1989. The role of ester groups in resis-tance of plant-cell wall polysaccharides to enzymatic-hydrolysis. Appl. Biochem.Biotechnol. 20/21, 45–61.

Grohmann, K., Torget, R., Himmel, M.E., 1986. Optimization of dilute acid pretreat-ment of biomass. Biotechnol. Bioeng. Symp. 15, 59–80.

Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W., Foust,T.D., 2007. Biomass recalcitrance: engineering plants and enzymes for biofuelsproduction. Science 315 (5813), 804–807.

Hu, W.J., Harding, S.A., Lung, T., 1999. Repression of lignin biosynthesis promotescellulose accumulation and growth in transgenic trees. Nat. Biotechnol. 17,808–812.

Huntley, S.K., Ellis, D., Gilbert, M., Chapple, C., Mansfield, S.D., 2003. Significantincreases in pulping efficiency in C4H-F5H-transformed poplars: improvedchemical savings and reduced environmental toxins. J. Agric. Food Chem. 51,6178–6183.

Jeffries, T.W., Grigoriev, I.V., Grimwood, J., Laplaza, J.M., Aerts, A., Salamov, A.,Schmutz, J., Lindquist, E., Dehal, P., Shapiro, H., Jin, Y.S., Passoth, V., Richardson,P.M., 2007. Genome sequence of the lignocelluloses-bioconverting and xylose-fermenting yeast Pichia stipitis. Nat. Biotechnol. 25, 319–326.

Jia, C.H., Zhao, H.Y., Wang, H.Z., Xing, Z.F., Du, K.J., Song, Y.R., Wei, J.H., 2004. Obtainingthe transgenic poplars with low lignin content through down-regulation of 4CL.Chin. Sci. Bull. 49, 905–909.

Keating, J.D., Panganiban, C., Mansfield, S.D., 2006. Tolerance and adaptation ofethanologenic yeasts to lignocellulosic inhibitory compounds. Biotechnol. Bio-eng. 93, 1196–1206.

Kerr, R.A., Service, R.F., 2005. What can replace cheap oil- and when? Science 309,101–101.

Kong, F.R., Engler, C.R., Soltes, E.J., 1992. Effects of cell-wall acetate, xylan backbone,and lignin on enzymatic-hydrolysis of aspen wood. Appl. Biochem. Biotechnol.34, 23–35.

Li, L.G., Zhou, Y.H., Cheng, X.F., Sun, J.Y., Marita, J.M., Ralph, J., Chiang, V.L., 2003.Combinatorial modification of multiple lignin traits in trees through multigenecotransformation. Proc. Natl. Acad. Sci. U. S. A. 100, 4939–4944.

Liu, S.C., 2003. Assay and Examination of Pulp Manufacture and Papermaking, thirded. China Chemistry Industry Press, Beijing.

Lynd, L.R., Cushman, J.H., Nichols, R.J., Wyman, C.E., 1991. Fuel ethanol from cellulosicbiomass. Science 251, 1318–1323.

Magaton, A.D., Colodette, J.L., Gouvea, A.D.G., Gomide, J.L., Muguet, M.C.D., Pedrazzi,C., 2009. Eucalyptus wood quality and its impact on kraft pulp production anduse. TAPPI J. 8 (8), 32–39.

Mosier, N., Wyman, C.E., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., Ladisch, M.,2005. Features of promising technologies for pretreatment of lignocellulosicbiomass. Bioresour. Technol. 96, 673–686.

Schubert, C., 2006. Can biofuels finally take center stage. Nat. Biotechnol. 24,777–784.

Scurfield, G., 1973. Reaction wood—its structure and function. Science 179 (4074),647–655.

Silverstein, R.A., Chen, Y., Sharma-Shivappa, R.R., Boyette, M.D., Osborne, J., 2007. Acomparison of chemical pretreatment methods for improving saccharificationof cotton stalks. Bioresour. Technol. 98 (16), 3000–3011.

Simmons, B.A., Loque, D., Ralph, J., 2010. Advances in modifying lignin for enhancedbiofuel production. Curr. Opin. Plant Biol. 13, 313–320.

Somerville, C., Bauer, S., Brininstool, G., Facette, M., Hamann, T., Milne, J., Osborne, E.,Paredez, A., Persson, S., Raab, T., Vorwerk, S., Youngs, H., 2004. Toward a systemsapproach to understanding plant cell walls. Science 306, 2206–2211.

Somerville, C., 2006. The billion-ton biofuels vision. Science 312, 1277–1277.Somerville, C., 2007. Biofuels. Curr. Biol. 17, R115–R119.SPSS Inc., 1998. SPSS Base 8.0 for Windows User’s Guide. SPSS Inc., Chicago.Studer, M.H., DeMartini, J.D., Davis, M.F., Sykes, R.W., Davison, B., Keller, M., Tuskan,

G.A., Wyman, C.E., 2011. Lignin content in natural Populus variants affects sugarrelease. Proc. Natl. Acad. Sci. U. S. A. 108, 6300–6305.

Várnaia, A., Siika-ahob, M., Viikari, L., 2010. Restriction of the enzymatic hydrolysis ofsteam-pretreated spruce by lignin and hemicellulose. Enzyme Microb. Technol.46, 185–193.

ps and

V

W

H. Wang et al. / Industrial Cro

oelker, S.L., Lachenbruch, B., Meinzer, F.C., Jourdes, M., Ki, C., Patten, A.M.,Davin, L.B., Lewis, N.G., Tuskan, G.A., Gunter, L., Strauss, S.H., 2010. Anti-sense down-regulation of 4CL expression alters lignification, tree growth,

and saccharification potential of field-grown poplar. Plant Physiol. 154, 874–886.

ardrop, A.B., Davies, G.W., 1964. The nature of reaction wood. VIII. Thestructure and differentiation of compression wood. Aust. J. Bot. 12 (1),24–38.

Products 37 (2012) 170– 177 177

Watanabe, S., Saleh, A.A., Pack, S.P., Annaluru, N., Kodaki, T., Makino, K., 2007. Ethanolproduction from xylose by recombinant Saccharomyces cerevisiae express-ing protein-engineered NADH-preferring xylose reductase from Pichia stipitis.

Microbiology 153, 3044–3054.

Wyman, C.E., 1999. Biomass ethanol: technical progress, opportunities, and com-mercial challenges. Annu. Rev. Energy Environ. 24, 189–226.

Zhao, H.Y., Wei, J.H., Lu, J., Shi, C., Jia, C.H., Song, Y.R., 2004. The study of transgenicpoplar expressing antisense CCoAOMT. Progr. Nat. Sci. 14, 49–53.