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2006
Chemical composition and response to dilute-acid pretreatment Chemical composition and response to dilute-acid pretreatment
and enzymatic sacchari>cation of alfalfa, reed canarygrass, and and enzymatic sacchari>cation of alfalfa, reed canarygrass, and
switchgrass switchgrass
Bruce S. Dien
USDA-ARS National Center for Agricultural Utilization Research
, Bruce.Dien@ars.usda.gov
Hans-Joachim G. Jung
USDA-ARS
Kenneth P. Vogel
University of Nebraska-Lincoln
, kvogel1@unl.edu
Michael D. Casler
USDA-ARS
, michael.casler@ars.usda.gov
JoAnn F. S. Lamb
Plant Science Research Unit
See next page for additional authors
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Dien, Bruce S.; Jung, Hans-Joachim G.; Vogel, Kenneth P.; Casler, Michael D.; Lamb, JoAnn F. S.; Iten,
Loren; Mitchell, Robert C.; and Sarath, Gautum, "Chemical composition and response to dilute-acid
pretreatment and enzymatic sacchari>cation of alfalfa, reed canarygrass, and switchgrass" (2006).
Agronomy & Horticulture -- Faculty Publications
. 1032.
https://digitalcommons.unl.edu/agronomyfacpub/1032
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Biomass and Bioenergy 30 (2006) 880–891
Chemical composition and response to dilute-acid pretreatment and
enzymatic saccharification of alfalfa, reed canarygrass, and switchgrass
Bruce S. Dien
a,
, Hans-Joachim G. Jung
b
, Kenneth P. Vogel
c
, Michael D. Casler
d
,
JoAnn F.S. Lamb
b
, Loren Iten
a
, Robert B. Mitchell
c
, Gautum Sarath
c
a
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA
1
,
Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA
b
Plant Science Research Unit, 411 Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55108–6026, USA
c
Wheat, Sorghum, and Forage Research Unit, 344 Keim Hall, University of Nebraska, Box 830937, Lincoln, NE 68583-0937, USA
d
U.S. Dairy Forage Research Center, 1925 Linden Dr. West, Madison, WI 53706-1108, USA
Received 24 June 2005; received in revised form 22 February 2006; accepted 23 February 2006
Available online 2 May 2006
Abstract
Alfalfa stems, reed canarygrass, and switchgrass; perennial herbaceous species that have potential as biomass energy crops in
temperate regions; were evaluated for their bioconversion potential as energy crops. Each forage species was harvested at two or three
maturity stages and analyzed for carbohydrates, lignin, protein, lipid, organic acids, and mineral composition. The biomass samples were
also evaluated for sugar yields following pretreatment with dilute sulfuric followed by enzymatic saccharification using a commercial
cellulase preparation. Total carbohydrate content of the plants varied from 518 to 655 g kg
1
dry matter (DM) and cellulose
concentration from 209 to 322 g kg
1
DM. Carbohydrate and lignin contents were lower for samples from early maturity samples
compared to samples from late maturity harvests. Several important trends were observed in regards to the efficiency of sugar recovery
following treatments with dilute acid and cellulase. First, a significant amount of the available carbohydrates were in the form of soluble
sugars and storage carbohydrates (4.3–16.3% wt/wt). Recovery of soluble sugars following dilute acid pretreatment was problematic,
especially that of fructose. Fructose was found to be extremely labile to the dilute acid pretreatments. Second, the efficiency at which
available glucose was recovered was inversely correlated to maturity and lignin content. However, total glucose yields were higher for the
later maturities because of higher cellulose contents compared to the earlier maturity samples. Finally, cell wall polysaccharides, as
determined by the widely applied detergent fiber system were found to be inaccurate. The detergent fiber method consistently over-
estimated cellulose and hemicellulose and underestimated lignin by substantial amounts.
Published by Elsevier Ltd.
Keywords: Medicago sativa L.; Phalaris arundinacea L.; Panicum virgatum L.; Bioethanol; Biomass; Bioenergy
1. Introduction
Biomass can be converted into energy by thermo-
chemical processes, including combustion, pyrolysis, and
gasification [1], or by fermentation of carbohydrates to
produce methane and ethanol [1,2]. Sources of lignocellu-
losic biomass include wood, paper waste, crop residues,
and herbaceous energy crops. Perennial herbaceous energy
crops have much to recommend them as a feedstock
because once established they do not require annual re-
seeding, they require lower energy inputs (i.e., fertilizer and
pesticides) than annual crops, and they can often be grown
on more marginal cropland [3–5]. They also have environ-
mental benefits including reduced soil erosion, enhanced
carbon sequestration, and providing wildlife habitat
[4,6–9]. Both the US and EU have supported research on
herbaceous en ergy crops since the mid-1980s. Thirty-five
herbaceous perennial species were screened by the US
ARTICLE IN PRESS
www.elsevier.com/locate/biombioe
0961-9534/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.biombioe.2006.02.004
Corresponding author. Tel.: +1 309 681 6270; fax: +1 309 681 6427.
E-mail address: dienb@ncaur.usda.gov (B.S. Dien).
1
Mention of trade names or commercial products in this article is solely
for the purpose of providing specific information and does not imply
recommendation or endorsement by the US Department of Agriculture.
This document is a U.S. government work and
is not subject to copyright in the United States.
Department of Energy and switchgrass (Panicum virgatum
L.) was selected for intensive study [10,11]. The EU
investigated 20 perennial grasses and selected 4 as the
most promising: miscanthus (Miscanthus spp. Anderss.),
reed canarygrass (Phalaris arundinacea L.), giant reed
(Arundo donax L.), and switchgrass [12]. Alfalfa (Medicago
sativa L.) has also been considered for use as an energy
crop in the US [13].
Three forage crops were selected for this study: alfalfa
(only stems), reed canarygrass, and switchgrass. Selection
of the three forage crops evaluated in this study was based
upon high yield potential and other agronomic consider a-
tions. All of these species are broadly adapted to a range of
environmental regions, but each species is also uniquely
suited to special situations. For example, reed canarygrass
is a cool-season grass that is very tolerant of flooding and
its productivity is very responsive to high levels of nitrogen
fertilization, making it a useful crop for disposal of manu re
from livestock operations [14]. In contras t, switchgrass is a
warm-season grass that requires higher growth tempera-
tures for maximum productivity, but this species is
extremely drought tolerant and productive with minimal
fertilizer inputs [10]. Alfalfa’s unique traits include the fact
that this legume does not need nitrogen fertilizer and the
leaves are a valuable supplemental protein feed for
livestock, providing another revenue stream from the use
of this species as a biomass crop [15]. Of the three forage
species evaluated in the current study, alfalfa may be best
suited for use on land suitable for row cropping because
alfalfa’s productivity declines after 3–5 years and alfalfa
can provide the majority of the nitrogen fertilizer require-
ments for 2 years of maize (Zea mays L.) production after
the alfalfa stand is plowed down. Switchgrass and reed
canarygrass remain productive for longer periods of time
and are more suited to marginal cropland because these
perennial grasses are more effective at controlling erosion
and nutrient leaching. Clearly, choice of biomass crops
must include their applicability to farming systems and
characteristics of the land base available.
The efficiency of conversion of biomass to ethanol
depends upon feedstock characteristics and composition,
pretreatment processes, and the fermentation technologies
that are utilized [1,2,16]. Feedstock quality for herbaceous
energy crops has been extensively studied for use as
livestock feed but not for ethanol conversion. Legumes,
grasses with the C
3
photosynthesis system, and grasses with
the C
4
photosynthesis system differ in plant anatomical
characteristics which affect their chemical composition and
utilization by ruminant animals [17]. Other important
factors that are known to strongly impact chemical
composition and digestion by ruminant animals include
forage genotype, maturity, and growth environment, as
well as, interaction among these factors [18]. This study
focused on the influence of plant-type and maturity. The
forages selected for this study include a legume (alfalfa), C
3
grass (reed canarygrass), and C
4
grass (switchgrass) each of
which was harvested at two or three maturities. Biomass
samples were characterized for total chemical composition,
including carbohydrates, protein, lipids, Klason lignin, ash,
etc. Next, recoverable sugar yields wer e evaluated by
measuring monosaccharides released from the cell-wall
matrix following treatment with dilute sulfuric-acid (at 121
and 150 1C) and enzymatic saccharification with a com-
mercial cellulase. Finally, the compositional and yield data
were combined to calculate the relative amount of
recoverable sugars for each sample. The results showed
clear distinctions among the samples based upon both
plant-type and harvest maturity.
2. Materials and methods
2.1. Plant material
Herbaceous biomass crop samples were grown and
harvested in 2003. The two alfalfa samples were created
by harvesting and bulking numerous individual plants
from several genetic nurseries at Rosemount and Becker,
MN. These nurseries were established in 2001 and consis-
ted of mature plants derived from intercrossing commercial
alfalfa varieties. The reed canarygrass plant material was
derived from a low-alkaloid population selected for
improved establishment capacity that was planted at
Arlington, WI. Switchgrass samples were collected from
an established stand of the variety Cave-in-Rock located at
Mead, NE. All field plots were fertilized for high pro-
ductivity under local soil conditions. Plan t mate rials were
harvested at a 10 cm stubble height. The specific maturity
stages and morphological description of the samples are
detailed in Table 1. Following harvest, the biomass was air
dried on greenhouse benches (switchgrass) or in forced-air
ovens at 60 1C (alfalfa and reed canarygrass). The dried
ARTICLE IN PRESS
Table 1
Description of biomass samples used for pretreatment experiments
Species Maturity
a
Sample description
Alfalfa (Medicago sativa L.)
Bud (KF3) Stems, flower buds
present, no open flowers
Full flower (KF6) Stems, open flowers on all
stem shoots
Reed canarygrass (Phalaris arundinacea L.)
Vegetative (V3) Leaf blades and sheaths,
no stem elongation
Ripe seed (S5) Whole herbage, ripe seed
Switchgrass (Panicum virgatum L.)
Pre-boot (E3) Leaf blades and sheaths,
elongated stems
Anthesis (R4) Whole herbage, flower
panicle on stems open
Post-frost (S5+) Whole herbage, ripe seed,
senescent, post-frost
a
Alfalfa maturity stage designations follow [19]. Maturity stage system
for grasses is based on [20].
B.S. Dien et al. / Biomass and Bioenergy 30 (2006) 880–891 881
alfalfa was hand separated into leaf and stem components.
Total sample sizes were 12 kg for each of the alfalfa stem
and reed canarygrass herbage harvests and 100 kg for the
switchgrass herbage harvests. The switchgrass herbage and
alfalfa stem samples were ground through a 2-mm screen in
a Wiley mill. The reed canarygrass samples were ground
using a 1-mm screen in a Wiley mill. Biomass samples
were subsequently re-ground in a cyclone-type mill to pa ss
a 1-mm screen for the compo sitional analyses, but not for
the conversion experiments.
2.2. Compositional analysis
A complete compositional analysis was done for the
biomass samples. Nitrogen content was determined by
combustion, and crude protein concentration was esti-
mated as N 6.25 [21]. Lipid content was determined by
exhaustive extraction with diethyl ether [22]. Organic acids
were extracted with water and analyzed by HPLC with a
refractive index detector [23]. Total ash content was
measured as loss of weight after combustion at 450 1C for
16 h in a muffle furnace. Major mineral components in
the biomass samples were determined using procedures
described by Knudsen et al. [24].
Carbohydrates and lignin were determined using a
sequential procedure. Soluble carbohydrates were ex-
tracted with 80% vol/vol ethanol at 60 1C overnight [25].
The supernatant was analyzed by HPLC for monosacchar-
ides (glucose and fructose) and oligosaccharides (sucrose,
stachyose, and raffinose). The alcohol-insoluble residues
were extracted with water at 4 1C overnight to remove
fructans [25]. Fructans in the water-extract supernatan t
were determined using the ketose assay of Boratynski [26].
The water-insoluble residue was treated with heat-stable a-
amylase and amyloglucosidase in 0.1 M acetate buffer, pH
5, to release glucose from starch [27] . Sufficient 95%
vol/vol ethanol was added to reach an alcohol concentra-
tion of 80%, after which the supernatant was removed and
analyzed by HPLC for glucose released from starch. The
remaining crude, alcohol–insoluble cell wall residue was
subjected to a two-stage sulfuric acid hydrolysis using the
Uppsala Total Dietary Fiber Method [27]. An aliquot from
the first stage of the acid hydrolysis was analyzed for
uronic acids [28], using galacturonic acid as the reference
standard for alfalfa and glucuronic acid as the standard for
the two grasse s. Neutral sugars from the two-stage acid
hydrolysis were analyzed as alditol–acetate derivatives by
GC-FID. The acid-insoluble residue provided the Klason
lignin concentration estimate after correction for ash.
The biomass samples were also analyzed for cellulose,
hemicellulose, and lignin using the detergent fiber system
[29]. Neutral detergent fiber (NDF), acid detergent fiber
(ADL), and acid detergent lignin (ADL) were determined
sequentially using the Ankom (Ankom Technology Cor-
poration, Fairport, NY) Filter Bag method [30]. Cellulose
content was calculated as ADF minus ADL and hemi-
cellulose as the difference between NDF and ADF values .
Gross energy content of the biomass samples was
determined by bomb calorimet ry using benzoic acid as
the standard.
2.3. Dilute acid pretreatment
Two dilute-acid pretreatment methods were evaluated;
121 1C in an autoclave and 150 1C in a pipe reactor. Plant
samples (2 g) were mixed with 18 ml dilute sulfuric acid
solution (0–2.5% wt/vol) in a glass vial capped with a screw
cap lid and heated for 1 h in an autoclave set at 121 1C; the
autoclave vented within 10 min following the end of the
cycle. Alternately, plant samples were pretreated using steel
pipe reactors and a fluidized heating sand bath as
previously described [31]. Each plant sample (2 g) was
mixed with 18 ml of a dilute sulfuric acid solution in a pipe
reactor. The samples were heated to 150 1C, incubated for
20 min, and rapidly cooled by plunging the react or in a
cold-water bath; the time required to heat the samples was
approximately 10 min. The syrups resulting from the two
dilute-acid pretreatments were subsampled and analyzed
for monomeric and total soluble carbohydrates. The
remaining syrup and solid pretreatment residues were
enzymatically hydrolyzed.
2.4. Enzymatic hydrolysis
A modified version of the NREL Laboratory analytical
procedure 9 was used to determine cellulose digestibility
[32]. Acid-pretreated samples were diluted with 10 ml
water, neutralized with 4 M KOH to pH 4.5, and buffered
by adding 2.5 ml of 1 M citric acid (pH 4.8). The contents
were transferred to a 125 ml Erlenmeyer flask using two
7.5 ml washes with water to insure complete transfer of
solids. Cellulase (1 ml) and thymol (40 mlofa50gl
1
solution in 70% vol/vol ethanol) were added and the
contents incubated for 72 h in a shaker incubator set at
45 1C and 125 rpm. The cellulase preparation used was an
equal volume mixture of Celluclast 1.5 l and 188 b-gluco-
sidase (Novozyme, Denma rk). The cellulase mixture had
an activity of 50 filter pap er units ml
1
, as measured by the
previously described procedure of Ghose [33]. Incubation
supernatants were analyzed for soluble carbohydrates.
2.5. Measurement of released sugars
Total soluble carbohydrates were analyzed by HPLC,
after being hydrolyzed by treating with 2 M TFA for
60 min at 100 1C [34]. Samples were analyzed for sugars
and acetic acid using a HPLC equipped with an organic
acids column (Bio-Rad Laboratories, CA) and a refractive
index detector, as previously described [31].
2.6. Statistical analysis
All compositional analyses were done in triplicate, and
data were corrected to a 100% dry matter (DM) basis.
ARTICLE IN PRESS
B.S. Dien et al. / Biomass and Bioenergy 30 (2006) 880–891882
