Assessment of leaf/stem ratio in wheat straw feedstockand impact on enzymatic conversionHENG ZHANG* , JONATAN U . FANGEL † , W I LL IAM G .T . W ILLATS † , M ICHAEL J . S EL IG * ,

JANE L INDEDAM* , HENN ING JØRGENSEN* and CLAUS FELBY*

*Forest and Landscape, Faculty of Sciences, University of Copenhagen, Rolighedsvej 23, Frederiksberg C, DK-1958, Denmark,

†Department of Plant and Environmental Sciences, Faculty of Sciences, University of Copenhagen, Thorvaldsensvej 40,

Frederiksberg C, DK-1871, Denmark

Abstract

The composition of wheat straw leaf and stem fractions were characterized using traditional strong acid hydro-

lysis, and monoclonal antibodies using comprehensive microarray polymer profiling (CoMPP). These results are

then related to high throughput lignocellulose pretreatment and saccharification screening data. Pure leaf frac-tion of wheat straw was the least recalcitrant compared to pure stem and easily digested by commercial cellulas-

es after moderate hydrothermal pretreatment; 63% and 31% (w/w) of glucan, 88% and 61% of xylan were

released from the leaf and stem fractions, respectively. By preparing samples of various leaf-to-stem (L/S) ratios,

we found shifting conversion behavior as processing parameters were modified. Increasing the enzyme dosage,

pretreatment temperature and pretreatment time all significantly improved conversion rates in samples with

more than 50% leaf content, whereas less impact was observed on samples with less than 50% leaf content.

Enzyme affinity, desorption and readsorption with leaf and stem fractions may affect the sugar yield in wheat

straw saccharification. The data suggest that the L/S ratio is an important parameter when adjusting or optimiz-ing conversion processes and additionally in feedstock breeding. Furthermore, this highlights the need for rapid

techniques for determining L/S ratio in wheat straw harvests. The CoMPP data on specific carbohydrates and

leaf pectin highlight carbohydrate epitopes that may be useful as markers in the development of novel screening

techniques; especially pectin or arabinogalactan proteins related epitopes are promising.

Keywords: composition, enzymatic saccharification, leaf, leaf-to-stem ratio, recalcitrance, stem, wheat straw

Received 13 October 2012 and accepted 3 January 2013

Introduction

Wheat (Triticum aestivum L.) is one of our largest cereal

crops with an annual grain harvest of nearly 700 million

tons. Consequently, the residual straw biomass is a

potential feedstock for large-scale second-generation

biofuel production. To date, there has been significant

research on conversion technologies for extracting fer-

mentable sugars from lignocellulosic materials such as

wheat straw. However, the recalcitrant nature of ligno-

cellulose is still the bottleneck of modern conversion

processes (Himmel et al., 2007). Numerous factors con-

tribute to the recalcitrance of wheat straw during pre-

treatment and enzymatic saccharification. One of these

factors is the histological variation between different

plant tissues and organs. The wheat straw epidermis is

thin, but has dense and thick-walled cells with an outer

wall coated with a waxy film of cutin-cuticle. The vas-

cular system has xylem tissue with dense lignified

structures in the secondary wall, surrounded by a

strong sheath of sclerenchyma cells, which have elon-

gated thick lignified cell walls resistant to microbial

degradation (Hansen et al., 2011).

Wheat straw has two major anatomical fractions:

leaves and stem, with a share of 20–40% and 60–80% of

the straw, respectively (�Aman & Nordkvist, 1983). Some

work has been done on the chemical composition of

wheat straw anatomical fractions (Hess et al., 2003;

Thompson et al., 2003; Duguid et al., 2007). The propor-

tion of major components (glucan, xylan, lignin, ash)

varies between different anatomical fractions. Duguid

et al. (2007) reported the distribution of major cell wall

components of wheat stover in the anatomical fractions.

Internodes had the highest glucan content compared

with chaff, leaves, and nodes. All anatomical fractions

had similar amount of xylan. More lignin was present

in the node and internode fractions. Ash content was

higher in chaff and leaves fractions than for nodes and

internodes. Thus, compositional and structural variation

in wheat straw anatomical fractions may affect the effi-

ciency of pretreatment and saccharification.Correspondence: Heng Zhang, tel. + 45-35331711, fax + 45-35331508,

e-mail: heng@life.ku.dk

© 2013 Blackwell Publishing Ltd 1

GCB Bioenergy (2013), doi: 10.1111/gcbb.12060

Although many studies have investigated enzymatic

saccharification processes on all fractions of raw or pre-

treated wheat straw, only few have been published on

individual wheat straw fractions. Ramanzin et al. (1991)

found wheat leaves significantly more degradable than

stems in cow rumen, but the degradability was also

highly correlated with wheat varieties. Duguid et al.

(2007) addressed the wheat stover anatomical fractions

and discussed the composition variation among differ-

ent fractions and subsequent performance in enzymatic

saccharification after alkaline or acid treatment. A recent

study by Lindedam et al. (2012) found that wheat straw

sugar yields were more influenced by cultivar varieties

than L/S ratio. Thus, the magnitude of L/S ratio on

wheat straw conversion still remains unclear.

To better understand the structural profile at the

polymer level of stem and leaves, we applied an anti-

body-based screening method, comprehensive micro-

array polymer profiling (CoMPP) for systematic

mapping of cell wall carbohydrates in wheat straw leaf

and stem fractions (Moller et al., 2007). Sequential

extraction using CDTA, NaOH, and Cadoxen solubilizes

pectin, noncellulosic polysaccharides, and celluloses

from plant cell walls, respectively (Dumville & Fry,

2000). The extracts of glycans from plant cell walls are

probed with monoclonal antibodies (mAbs) or cellulose

binding module (CBM), hence to provide rapid map-

ping of specific plant cell wall structure (Moller et al.,

2007).

The aim of this study was to investigate the enzy-

matic saccharification performance of the stem and leaf

fractions from wheat straw under conditions mimicking

industrial processing conditions. Furthermore, we want

to better understand the characteristics contributing to

discrepancies that could additionally be utilized to track

feedstock ratios during harvest. For this, we apply an

antibody-based method for detection and quantitation

of cell wall oligomers and polymers. The performance

of leaves and stem from different wheat samples in

hydrothermal pretreatment and enzymatic saccharifica-

tion were identified using a high throughput pretreat-

ment and enzymatic saccharification platform. Data

from these analyses were linked to compositional struc-

tures to better elucidate the structural characteristics

contributing to recalcitrance.

Materials and methods

Wheat harvest, fractionation, and experimental plan

Nine winter wheat straw samples were collected after grain

harvest in 2006 from two Danish test sites Abed (54o49′40.05″

N and 11o19′30.62″ E) and Sejet (55o49′12.43″ N and 9o55′21.82″

E). Whole air-dry plants were sealed in plastic bags and kept in

ambient condition prior to use. Cultivars were Northern Euro-

pean breeds, namely, Samurai (only from Abed), sj05-21, sj05-

22, Tukan, and Asano. Plants were fractionated into a leaf frac-

tion, containing leaf sheath and leaf blade, and a stem fraction,

containing nodes and internodes. Carbohydrates and lignin in

stem and leaf fractions of all samples were analyzed with

strong acid hydrolysis and polymer and oligomer profiles were

measured by CoMPP on the stem and leaf fraction from Abed

Samurai. Sugar conversions were measured on batches of vary-

ing L/S ratios (0%, 20%, 50%, 80%, and 100% leaf) made with

Abed Asano and Sejet Asano. In addition, sugar conversions

were measured on L/S batches from Abed Asano, varying

enzyme load (1, 2, 5, 10, 20, 40, and 60 filter paper unit (FPU)

g dm�1), pretreatment time (5 min or 10 min), and incubation

time (6 h or 24 h).

Strong acid hydrolysis for compositional analysis ofwheat straw leaves and stems

Dry matter content (DM) of air-dried wheat samples was deter-

mined using a Sartorius MA 30 (Sartorius AG, Goettingen, Ger-

many) moisture analyzer at 105 °C. Residual starch content

that may contribute to the saccharification yield was

determined as glucose after treatment with Liquozyme� SC DS

(a-amylase) and Spirizyme� (Novozymes A/S, Bagsvaerd,

Denmark) fuel (amyloglycosidase) enzymes to degrade the

starch (www.novozyme.com). 100 mg of wheat straw sample

was mixed with 10 ml 50 mM sodium citrate buffer at pH 5.0.

First, 0.04% (weight of enzyme to weight of wheat sample) (w/

w) a-amylase was added followed by 2 h incubation at 82 °C.

Then, 0.1% (w/w) amyloglycosidase was added followed by

2 h incubation at 65 °C. Samples were centrifuged and the

supernatant was filtrated through 0.45 lm filter and analyzed

using high performance liquid chromatography (HPLC).

The composition of carbohydrates and lignin was deter-

mined using a modified Klason lignin method derived from

the TAPPI Standard method T222 om-98 (TAPPI 5960). Briefly,

0.3 g of dried sample was incubated with 3 ml of 72% H2SO4

for 1 h at 30 °C with mixing. The sample was then diluted with

deionized water to a final acid concentration of 4%. The solu-

tion was autoclaved for 1 h at 121 °C and filtered through a

medium coarseness sintered-glass filter for gravimetric deter-

mination of acid insoluble lignin. Each sample was analyzed in

triplicate. The concentration of sugars in the filtrate was deter-

mined by HPLC with quantitation with reference to standards,

which was also autoclaved in 4% H2SO4 to compensate for deg-

radation. Composition dataset was analyzed using GLM proce-

dure on the SAS system. (SAS Institute, SAS/STAT User’s

Guide, Release 6.03 Edition, Cary, NC, USA)

Comprehensive microarray polymer profiling

Abed Samurai sample was washed with 70% ethanol (v/v) five

times and once with acetone and air dried to isolate cell wall

material prior to analysis. Cell wall glycans were sequentially

extracted from each sample by three solvents: (i) 50 mM trans-

1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA) pH

7.5; (ii) 4 M NaOH with 0.1% (v/v) NaBH4; and (iii) cadoxen

© 2013 Blackwell Publishing Ltd, GCB Bioenergy, doi: 10.1111/gcbb.12060

2 H. ZHANG et al.

(31%, v/v 1,2-diaminoethane with 0.78 M CdO), which are

known to extracts pectin, hemicellulose, and cellulose (Fry,

2000). 10 mg of each sample was weighted out and 300 ll ofCDTA was added. Samples were shaken for 2 h at room tem-

perature before centrifugation at 2500 g for 10 min. Superna-

tants containing solubilized cell wall polymers were collected

and the procedure was repeated with NaOH and Cadoxen.

The three extracts from each sample were diluted fivefold and

125-fold in Phosphate buffered saline (PBS) buffer (140 mM

NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.5)

and printed with the undiluted extracts in triplets onto nitrocel-

lulose membranes making each sample represented by nine

spots. Printing was performed using four split pins in a micro-

array robot (Microgrid II, Genomic Solutions, Digilab, Inc.,

Marlborough, MA, USA). Pins were washed twice in water

after deposition of each sample. Arrays were probed with

mAbs or CBMs, as described by (Willats et al., 1998; McCartney

et al., 2005). Arrays were blocked with PBS buffer containing

5% fat-free milk powder for 45 min, before incubation with

primary monoclonal antibodies (mAbs) or cellulose-binding

modules (CBM) for 2 h. The mAbs and CBMs used are listed

in Table 1. After washing, arrays were probed with a second-

ary antibody conjugated with alkaline phosphatase for 2 h.

The arrays were washed and developed using a BCIP/NBT

(5-bromo-4-chloro-3′-indolyphosphate/nitro-blue tetrazolium

chloride) substrate. The arrays were scanned using a flatbed

scanner at 1200 dpi, and converted to negative image 16-bit

gray scale TIFFs. TIFF files were uploaded into ImaGene 6.0

microarray analysis software and analyzed data were con-

verted into a heatmap with a cut-off of five (Moller et al., 2007).

Pretreatment and enzymatic saccharification

Pretreatment and enzymatic saccharification experiments were

carried out using a high throughput screening platform mod-

eled after previously published systems (Selig et al., 2010;

Studer et al., 2010). The system consisted of sample grinding

and dispensing using an automated sample preparation robotic

system, microscale pressurized heat treatment mimicking

large-scale pretreatment, enzymatic saccharification using com-

mercial enzyme products, and monomer sugars determination

by HPLC. The entire process was done in a 96-well plate for-

mat. The automated plant material grinding and dispensing

system was manufactured by Labman Automation Ltd. at

Stokesley, North Yorkshire, UK (http://www.labman.co.uk/).

The pressurized heating system was an in-house build system.

Asano samples from Abed and Sejet sites were ground into

fine powder, and 27.7 mg of each sample was dispensed to alu-

minum microtiter plates followed by addition of 50 mM sodium

citrate buffer (pH 4.8). The plate was covered by Teflon tape

with a little hole for each well. The plate was then placed on a

heating block and covered with a thin aluminum plate, teflon

plate, and thick aluminum plate. The plate was heated up to

190 °C at a stable pressure for 10 min for pretreatment. The

heat treatment apparatus was cooled down to room tempera-

ture by a water cooling system. The microtiter plate was cov-

ered with a new teflon tape after addition of enzyme to each

well. Celluclast 1.5 l and Novozyme 188 were used in a weight

ratio of 5 : 1 (Novozymes A/S, Bagsværd, Denmark). Then, the

plate was incubated on a shaking incubator for enzymatic sac-

charifiation at 50 °C, 450–600 rpm for 24 h. Hydrolysis was ter-

minated by heating up to 90 °C for 30 min. Samples were

filtrated through 0.45 lm plate filter and loaded to HPLC for

sugar determination.

Carbohydrate analysis

Monosaccharides (D-glucose, D-xylose, and L-arabinose)

released from enzymatic saccharification were measured on a

Dionex Ultimate HPLC system. The separation was done in a

column at 80 °C with 5 mM H2SO4 as eluent at a flow rate of

0.6 ml min�1 and quantitated by RI-detector. The samples were

diluted in eluent, filtered through a 0.45 lm nylon filter before

injection.

Monosaccharides from compositional analysis (arabinose,

galactose, glucose, mannose, and xylose) were determined

using an ICS 5000 system from Dionex (Sunnyvale, CA, USA).

The separation was performed in a Dionex CarboPac PA1 col-

umn at 30 °C with a flow rate of 1 ml min�1 of MQ-water and

using fucose as internal standard. Detector sensitivity was opti-

mized by postcolumn addition of 0.2 M NaOH at a flow rate of

0.5 ml min�1. The column was cleaned after each sample with

0.25 M NaOH for 5 min and then reconditioned by MQ-water

for 5 min. The samples were diluted in MQ-water, mixed with

internal standard, and filtered through a 0.45 lm nylon filter

before injection.

Results

Characterization of wheat leaves and stems

The average DM content of leaves and stems in all sam-

ples was identical, which is 91.3% and 91.9%, respec-

tively. Treatment of the samples with starch-degrading

enzymes revealed that the residual starch content was

negligible in both leaves and stems.

The composition of wheat leaves and stems from the

nine samples as determined by strong acid hydrolysis is

summarized in Table 2. In general, stem fractions con-

tained more lignin and glucan than leaf fractions,

whereas leaf had a higher content of ash than stem.

Xylan contents in both fractions are around 18%.

Statistical analysis (Table 2 GLM model) showed that

Table 1 mAbs and CBMs applied in this study

Specificity Reference

LM6 a-(1,5)-arabinan Willats et al. (1998)

LM11 b-(1,4)-D-xylan McCartney et al. (2005)

LM15 Nonfucosylated xyloglucan Marcus et al. (2008)

LM18 Homogalacturonan (HG) Verhertbruggen (2009)

LM19 Homogalacturonan (HG) Verhertbruggen (2009)

LM21

JIM13

Mannan

Arabinogalactan proteins

Marcus et al. (2010)

Yates et al. (1996)

CBM3a Crystalline cellulose Tormo et al. (1996)

© 2013 Blackwell Publishing Ltd, GCB Bioenergy, doi: 10.1111/gcbb.12060

HISTOLOGICAL VARIATION IN WHEAT STRAW HYDROLYSIS 3

anatomical fractions had a highly significant effect on

the wheat straw composition, while sites and cultivars

were not correlated with the composition results.

The CoMPP data for Abed Samurai are presented as

a heatmap, in which the color intensity is proportional

to the signal values (Fig. 1). Leaf and stem fractions

were profiled using several mAbs and CBMs (Table 1).

LM18 and LM19, which bind specifically to homogalac-

turonan (HG), showed no interaction with stem, but a

mild signal to leaf fraction. LM11, specific to b-(1, 4)-D-

xylan, showed slightly higher affinity to leaf than stem.

LM6, specific to arabinan; LM15, specific to nonfucosy-

lated xyloglucan; LM21, specific to mannan, all had

higher signal intensities to leaf than stem. Cellulose was

probed by CBM3a, which indicated low amount of

cellulose in both leaf and stem fractions. JIM13, arabino-

galactan proteins (AGPs) specific mAb, showed stronger

binding to leaf than stem indicating higher AGPs

presence in leaf fraction.

Enzymatic saccharification

Wheat straw leaf and stem fractions differed in compo-

sition profiles and were found to behave differently in

enzymatic saccharification. Pure leaf was hydrolyzed

significantly better than pure stem. In mixtures, higher

L/S ratios always gave a better sugar conversion rate

after saccharification (Fig. 2).

More glucose and xylose were generated in samples

with leaf content ranging from 20% to 80% by increas-

ing enzyme loading (Fig. 3). However, higher enzyme

loading did not help releasing more sugar from pure

stem and pure leaf samples (Fig. 3). Longer pretreat-

ment time gave higher xylose yield in all sample prepa-

rations except pure leaves (Fig. 4). An interesting

phenomenon was observed in pure stem and pure leaf

samples: pure stems produced more glucose at shorter

treatment duration; pure leaves generated about the

same level of glucose at both treatment conditions

(Fig. 4). All sample preparations gave higher sugar

yield at longer incubation time with an exception of

xylose yield of pure stems (Fig. 5). Glucose yield

increased dramatically from all samples by prolonging

the incubation time from 6 h to 24 h (Fig. 5). The high-

est sugar yield was obtained by 80% leaf content sam-

ples (63% glucose, 88% xylose) (Figs 3–5). Fig. 6

shows the effect of wide-range enzyme loadings

Table 2 Proportion of tested compounds in wheat straw leaves and stems determined by strong acid hydrolysis method [data pre-

sented are mean values of nine samples. Standard deviations are given in parentheses (n = 9)]

Component (%)

Acid insoluble lignin Arabinan Galactan Glucan Xylan Mannan Ash

Leaf 23.2 (4.3) 3.4 (0.3) 1.3 (0.3) 32.4 (1.8) 18.5 (2.9) 1.1 (0.4) 7.8 (2.8)

Stem 28.1 (5.1) 2.1 (0.1) 0.5 (0.0) 41.0 (1.6) 17.7 (3.8) 0.4 (0.1) 2.5 (1.7)

Fig. 1 Heatmap of CoMPP analysis of Abed Samurai raw

leaves and stem fractions after extraction with CDTA, NaOH,

and Cadoxen. The numbers in the heatmap matrix indicate the

signal intensity (0?100, signal from low to high). The specifici-

ties of mAbs are listed in Table 1.

Fig. 2 Sugar conversion rate for L/S ratio defined Asano sam-

ples from Abed and Sejet sites. Data are shown as released

sugar relative to the theoretical glucan and xylan contents.

10 min pretreatment, 10 filter paper unit g DM�1, and 24 h

hydrolysis were applied to the samples. Error bars are standard

deviation. ( , glucan yield of Abed Asano samples; , glu-

can yield of Sejet Asano samples; , xylan yield of Abed Asa-

no samples; , xylan yield of Sejet Asano samples).

© 2013 Blackwell Publishing Ltd, GCB Bioenergy, doi: 10.1111/gcbb.12060

4 H. ZHANG et al.

(5–60 FPU g DM�1) to leaf and stem fractions in 24 h

saccharification. Unlike the low enzyme loadings

(� 10 FPU g DM�1), 20–60 FPU g DM�1 could generate

significant amount of glucose in both leaf and stem

fractions (Fig. 6). However, glucose and xylose yield

seems to reach a plateau at 20 FPU g DM�1, at which

78% and 41% of glucan; 88% and 77% of xylan were

converted in leaf and stem samples, respectively

Fig. 3 Sugar conversion rate for L/S ratio defined Asano sam-

ples from Abed site for two different enzyme loadings. Data

are shown as the ratio of released sugar and theoretical glucan,

xylan contents, respectively. Two enzyme loadings (5 filter

paper unit (FPU) g DM�1, 10 FPU g DM�1), 10 min pretreat-

ment, and 24 h hydrolysis were applied to the samples. Error

bars are standard deviation. ( , glucose yield at 5 FPU g DM�1

enzyme loading; , glucose yield at 10 FPU g DM�1 enzyme

loading; , xylose yield at 5 FPU g DM�1 enzyme loading; ,

xylose yield at 10 FPU g DM�1 enzyme loading).

Fig. 4 Sugar conversion rate for L/S ratio defined Asano sam-

ples from Abed site for two different pretreatment periods.

Data are shown as the ratio of released sugar and theoretical

glucan, xylan contents, respectively. Two pretreatment periods

(5 min, 10 min), 10 filter paper unit g DM�1 enzyme loading,

and 24 h hydrolysis were applied to the samples. Error bars

are standard deviation. ( , glucose yield at 5 min pretreatment

time; , glucose yield at 10 min pretreatment time; , xylose

yield at 5 min pretreatment time; , xylose yield at 10 min pre-

treatment time).

Fig. 5 Sugar conversion rate for L/S ratio defined Asano sam-

ples from Abed site for two different incubation periods. Data

are shown as the ratio of released sugar and theoretical glucan,

xylan contents, respectively. Two incubation periods (6 h, 24 h),

10 min pretreatment, and 10 filter paper unit g DM�1 enzyme

loading were applied to the samples. Error bars are standard

deviation. ( , glucose yield under 6 h incubation time; , glu-

cose yield under 24 h incubation time; , xylose yield under 6 h

incubation time; , xylose yield under 24 h incubation time).

Fig. 6 Sugar conversion rate for Asano leaf and stem samples

from Abed site for seven different enzyme dosages. Data are

shown as the ratio of released sugar and theoretical glucan,

xylan contents, respectively. Seven enzyme loadings (1, 2, 5, 10,

20, 40, 60 filter paper unit g DM�1) were applied to the sam-

ples. Error bars are standard deviation. ( , glucan yield of

Abed Asano leaf samples; , glucan yield of Abed Asano

stem samples; , xylan yield of Abed Asano leaf samples;

, xylan yield of Abed Asano stem samples).

© 2013 Blackwell Publishing Ltd, GCB Bioenergy, doi: 10.1111/gcbb.12060

HISTOLOGICAL VARIATION IN WHEAT STRAW HYDROLYSIS 5

(Fig. 6). Structural carbohydrates in leaf were com-

pletely hydrolyzed at the highest enzyme load 60 FPU

g DM�1, at which stem had a glucan conversion of 63%

and a xylan conversion of 95% (Fig. 6). A synergetic

effect of leaf and stem mixed samples was observed in

20%, 50%, and 80% leaf content samples that the stem

carbohydrate was boosted to a higher conversion level

compared with pure stem (Fig. 3–5).

Discussion

The three major cell wall components of wheat straw

are cellulose, hemicellulose, and lignin. Cereal grasses,

such as wheat, are rich in arabinoxylan and mixed-

linked glucans (Carpita, 1996). Chemical composition

determined with strong acid hydrolysis provides quan-

titative profiles of wheat straw leaf and stem fractions,

whereas CoMPP characterizes fractions in a qualitative

and high throughput setup, and is thus well suited for

analysis of large populations.

The CoMPP data in this study showed variations in

the relative levels of some cell wall polymers between

wheat straw leaves and stems. Leaf fraction had higher

hemicellulose content, mainly arabinan, xylan, mannan,

and xyloglucan than stem fraction indicated by heatmap

signals (Fig. 1), which was consistent with the chemical

compositional analysis results (Table 2). Hemicellulose

has been marked as one of the major impediments

decreasing the accessibility of the cellulose to the

enzymes during the enzymatic saccharification of corn

stover and wheat straw (Kim & Holtzapple, 2006;€Ohgren et al., 2007). The microscale pressurized heating

treatment applied in this study simulates the large-scale

steam explosion process, which has been demonstrated

as an efficient method for hemicellulose removal (Mo-

sier et al., 2005). Low severity pretreatment conditions

were applied in this study, as we believe that too harsh

conditions may veil the different characterizations of

leaves and stems. The CoMPP data also gave informa-

tion on the relative amount of pectin and AGPs in

leaves and stems. Pectin, mainly HG in wheat straw,

was reported to be more rapid and extensively digest-

ible than hemicellulose and cellulose (Hatfield & Wei-

mer, 1995; Jung & Engels, 2002). There are no reports

indicating the impact of AGPs on cellulase saccharifica-

tion efficiency. However, pectin and AGPs signals given

by LM 18, LM 19, and JIM 13 were significantly stronger

in leaves than stem, which may be applied as indicators

to estimate L/S ratio of a wheat straw feedstock. Thus,

the CoMPP technique may be further developed to

quantitate L/S ratio of wheat straw, to predict feedstock

potential yield.

Glucose and xylose yield from pure leaf samples did

not change much by prolonging treatment duration

from 5 min to 10 min (Fig. 4) or increasing enzyme

loading from 5 FPU g DM�1 to 10 FPU g DM�1

(Fig. 3), which indicates that accessibility of carbohy-

drates for the enzymes is not a limiting factor in conver-

sion of wheat straw leaves. 15% more xylan was

hydrolyzed from 20% leaf samples by elevating pre-

treatment time, but the removal of hemicellulose did

not correspondingly increase the digestibility of cellu-

lose fibers, which leads us to think that the presence of

lignin is a more influential parameter (Fig. 4). A similar

phenomenon was observed at high enzyme loadings

(Fig. 6).

Structural carbohydrates in leaf and stem fractions

are organized differently. The glucan portion hydro-

lyzed at � 20 FPU g DM�1 enzyme loading could be

marked as ‘easy glucan’ in leaf and stem fractions,

which are 71% and 41%, respectively (Fig. 6). The

compact cellulose fibril structure or the limited binding

surface for cellulases might lead to slow hydrolysis pro-

gressing rate. However, leaf glucan was a much easier

substrate for the enzymes to convert than stem glucan

after removal of xylan at high enzyme loadings. The

easy glucan component is not an absolute concept in a

certain wheat straw feedstock, which is affected highly

by pretreatment methods and saccharification condi-

tions. We hypothesized that enzymes initially have a

higher chance to bind to stem fraction at low L/S ratio

samples (� 20% leaf content). Due to the fact that wheat

stem is a more recalcitrant substrate than leaf for

enzymes, the enzymes progressed slowly on stem frac-

tion and did not work much on leaf fraction (Figs 3–5).

Contrarily, in the samples of higher leaf content,

enzymes were initially more associated with leaf fibers,

which they processed rapidly and then moved on to

part of the stem carbohydrates. The transferal or succes-

sion of enzymes interactions with leaf and stem frac-

tions might be one of the more important factors

determining the sugar yield from wheat straw feed-

stocks. Enzyme absorption, desorption, and readsorp-

tion vary with anatomical fractions. Further

investigation is demanded to verify the hypothesis.

Higher lignin content in stem fractions seems to be

another factor that restricts the enzymatic saccharifica-

tion. Hansen et al. (2011) reported that lignin droplets

accumulated on the wheat straw cavity surface during

hydrothermal treatment and enzymatic hydrolysis pro-

cesses. Relocation and accumulation of lignin and

exposed lignin residuals might hinder the accessibility

of enzymes to cellulose fibers and lead to unproductive

binding of enzymes to lignin, hence decrease the hydro-

lysis rate during enzymatic hydrolysis (Mosier et al.,

2005; Hansen et al., 2011; Heiss-Blanquet et al., 2011).

Our results showed that a longer hydrolysis time did

not increase the glucose and xylose yield of pure stem

© 2013 Blackwell Publishing Ltd, GCB Bioenergy, doi: 10.1111/gcbb.12060

6 H. ZHANG et al.

as much as samples with higher leaf content (Fig. 5),

which might be due to a higher level of lignin accumu-

lation in pure stem.

Our study showed that leaves had lower lignin and

celluloses content and higher hemicelluloses and ashes

content compared with stems. Enzymatic saccharifica-

tion data suggested that wheat straw with higher leaf

content could generate more monosaccharides, mainly

glucose and xylose. Polysaccharides in wheat leaves

could be released up to 63% of glucan and 88% of xylan

under moderate conditions (5 or 10 FPU g DM�1, 5 or

10 min, 24 h), which shows low recalcitrance of the leaf

fraction. Under the applied conditions, the best sugar

yields of pure stem samples were only 31% of glucan

and 61% of xylan. Results suggest that increasing L/S

ratio may increase the sugar yield from wheat straw

feedstock synergistically during enzymatic saccharifica-

tion (Fig. 5); however, this will depend on the pretreat-

ment and processing approaches. The enzyme

performed very differently with pure leaf, pure stem,

and mixed samples, which implies that enzyme succes-

sion or different enzyme affinity on leaf and stem frac-

tions might be one of the factors affecting the sugar

yield.

The actual leaf content of the collected wheat residues

ranged from 20% to 40%, which is less than the leaf con-

tent of the best scenario in this study (leaf content 80%).

However, much milder conditions during pretreatment

and enzymatic hydrolysis could be applied while still

obtaining the same or even higher sugar yield when

processing wheat straw feedstock with high L/S ratio

compared with low L/S ratio. Therefore, prior knowl-

edge of L/S ratio by applying CoMPP as a screening

method could be used to adjust the processing condi-

tions to save energy and enzyme. Simple changes to

agricultural machinery or selection of wheat cultivars

with higher L/S ratio could ultimately also help biore-

fineries. The observations on leaf and stem degradabili-

ty may be expanded to other biomass types.

Acknowledgements

This project is supported by Bio4Bio, which is a strategicresearch center funded by the Danish Council for StrategicResearch and the University of Copenhagen strategic researchinitiative Fuel for Life.

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© 2013 Blackwell Publishing Ltd, GCB Bioenergy, doi: 10.1111/gcbb.12060

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