b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 1 2 5 e1 3 3

Available online at www.sciencedirect.com

http://www.elsevier.com/locate/biombioe

Experimental methods for laboratory-scale ensilage

of lignocellulosic biomass
Deepti Tanjore, Tom L. Richard*, Megan N. Marshall
Pennsylvania State University, Department of Agricultural and Biological Engineering, 249 Agricultural Engineering Building,
University Park, PA 16802, USA

article info

abstract

Article history:

Anaerobic fermentation is a potential storage method for lignocellulosic biomass in biofuel

Received 8 June 2011

production processes. Since biomass is seasonally harvested, stocks are often dried or

Received in revised form

frozen at laboratory scale prior to fermentation experiments. Such treatments prior to

13 September 2012

fermentation studies cause irreversible changes in the plant cells, influencing the initial

Accepted 17 September 2012

state of biomass and thereby the progression of the fermentation processes itself. This

Available online 23 October 2012

study investigated the effects of drying, refrigeration, and freezing relative to freshly
harvested corn stover in lab-scale ensilage studies. Particle sizes, as well as post-ensilage

Keywords:

drying temperatures for compositional analysis, were tested to identify the appropriate

Lignocellulosic biomass

sample processing methods. After 21 days of ensilage the lowest pH value (3.73
0.03),

Anaerobic fermentation

lowest dry matter loss (4.28
0.26 g. 100 g-1DM), and highest water soluble carbohydrate

Biofuel

(WSC) concentrations (7.73
0.26 g. 100 g-1DM) were observed in control biomass (stover

Ensilage

ensiled within 12 h of harvest without any treatments). WSC concentration was signifi-

Storage

cantly reduced in samples refrigerated for 7 days prior to ensilage (3.86
0.49 g. 100 g1

Pretreatment

DM). However, biomass frozen prior to ensilage produced statistically similar results to the
fresh biomass control, especially in treatments with cell wall degrading enzymes. Grinding
to decrease particle size reduced the variance amongst replicates for pH values of individual reactors to a minor extent. Drying biomass prior to extraction of WSCs resulted in
degradation of the carbohydrates and a reduced estimate of their concentrations. The
methods developed in this study can be used to improve ensilage experiments and thereby
help in developing ensilage as a storage method for biofuel production.
Published by Elsevier Ltd.

1.

Introduction

Biofuels from lignocellulosic biomass are fast becoming

a commercially viable alternative to gasoline as a transportation fuel. Several biorefinery unit operations and pathways have been developed to pretreat and convert cellulose
and hemicellulose to biofuels including ethanol, jet fuel, etc.
Although much less attention has been given to harvest,
storage, and transport, these upstream supply-chain unit
operations have the potential for very large impacts on the

carbohydrate content of biomass, and thereby final product

yields and costs [1e3]. Appropriate biomass storage can be
a key upstream process to preserve polysaccharides and
minimize the costs involved in ethanol production [3].
Traditionally, corn stover is field dried [4] and then stored
in bales such that the water activity and thereby the microbial
activity is minimal. However, bales stored outdoors or in
humid environments can get moist, allowing microbial
activity and aerobic degradation to increase [5,6]. In addition
to dry matter loss, catastrophic fires have been observed in

* Corresponding author. Tel.: 1 814 865 3722; fax: 1 814 863 1031.
E-mail addresses: deeptitanjore@gmail.com (D. Tanjore), trichard@psu.edu (T.L. Richard).
0961-9534/$ e see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.biombioe.2012.09.050

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traditional dry storage [4,5,7]. These problems can be considerably reduced through anaerobic wet storage strategies, such
as ensilage. Ensilage has been attracting interest as a low-cost,
low-risk, and efficient storage method for lignocellulosic
biomass [5e9]. The anaerobic microbial activity in ensiled
stover produces organic acids that reduce pH of the biomass,
and may reduce the acid requirement during pretreatment
process. When wet plants are baled for storage, it has been
observed that anaerobic conditions lead to lower dry matter
loss (0.2e0.9%) compared to aerobic conditions (7.4e22.0%) [5].
Drying leads to cell wall pore collapse, which can possibly lead
to recalcitrance during enzymatic hydrolysis for sugar release
[10]. Also, partial hydrolysis of cellulose and hemicellulose
observed during ensilage may decrease the recalcitrance of
lignocellulosic biomass during downstream processing
[3,6,11]. These potential advantages are attracting increasing
amounts of biomass ensilage research, both to better manage
the storage process, and to explore interactions with other
unit operations. In this context it is important to develop
standard methods that will be relevant to biomass, which will
be ensiled immediately after harvest for industrial scale
production, and to assure that conclusions can be generalized.
The biomass used for laboratory silage studies is usually
harvested in advance, sometimes up to several months prior
to the ensilage experiment. Many factors including preensilage biomass conditions, particle size, moisture, and
temperature influence ensilage process and could potentially
interfere with inferences about treatments. In previous laboratory studies plant material has often been frozen to minimize any microbial activity [6,9,12,13]. Alternatively, plant
material has been dried before ensilage [7]. To extrapolate the
results from lab-scale ensilage experiments to real world
scenarios, researchers must assume that these stored plant
materials behave similarly to freshly harvested materials.
However, cell wall rupture or pore collapse due to freezing or
drying can significantly affect the physical properties of
biomass and thereby further treatments on biomass [14e18].
Particle size reduction can also alter physical properties of
biomass and thereby downstream processes [19]. A medium
(<10 mm) sized biomass particle has been shown to be the
largest size of corn stover to ensile without any detrimental
effect on subsequent silage quality [9]. However, smaller
particle sizes can potentially reduce variability between
replicates in small reactors. Thus understanding the influence
of particle size on ensilage is vital, analogous to particle size
studies in other biofuel unit operations [20]. Influence of
sample preparation on key process parameters, such as pH,
fiber content (cellulose, hemicellulose, and lignin), and water
soluble carbohydrates (WSC) content, are also important to
establish, as the analysis methods of some of these parameters require drying and milling of the plant material [21,22].
Sample drying temperatures or extraction methods can
influence the assessment of these parameters [23,24].
With the advent of ensilage as a unit operation in the
biofuel production chain, there are thus several reasons to
establish standard laboratory-scale methods to ensure
repeatability and reliability of the data and to quantify treatment effects. Appropriate laboratory ensilage methods should
improve our understanding of test variables and reduce
interference from unintended factors. In this study, we tested

pre-ensilage biomass storage conditions, particle size reduction, and post-ensilage solubles extraction to understand their
influence on the ensilage process and to contribute to the
development of standard laboratory ensilage protocols. We
tested these methods by ensiling biomass with and without
supplemental enzymes, to investigate enzyme addition as
a possible method to improving biomass quality. Since
enzymes are expensive biocatalysts, microbial silage inoculants such as those currently used in animal feed silage
operations can be considered to improve the quality of
biomass during ensilage [25,26]. The results of this evaluation
can provide a basis for the development of standard methods
in this important and emerging field.

2.

Materials and methods

2.1.

Biomass collection

Stover (stalks, leaves, and husks) from field grown maize (Zea
mays) was harvested on September 29 and November 23, 2005
from a plot in Rock Springs, Pennsylvania. To determine
moisture content, a representative sample of the biomass was
weighed and dried in an oven at 105  C until a constant weight
was achieved. The moisture contents of the stover from
September and November harvests were observed to be
42.13
0.46 and 17.33
0.23 (g. 100 g1 wet biomass),
respectively.

2.2.

Experimental design

The three factors explained in the introduction, stover treatment prior to ensilage, particle size, and sample drying
temperature, were tested in three different experiments. A
split plot design was used in all the experiments with noenzyme and enzyme treatment forming the split. The three
experiments are explained below:
(i) The stover treatment experiment was conducted in two
parts as two separate studies, where biomass was frozen,
dried, refrigerated at 20  C, 105  C, and 4  C, respectively
prior to ensilage. The first study was conducted on
biomass harvested in September 2005 for a treatment
period of 7 days. This study did not include a control,
biomass ensiled immediately after harvest without any
treatments. Refrigeration for 7 days led to fungal growth
and high pH values, as elucidated in Section 3.1.1. To
avoid fungal growth, the second study was performed for
2 days on biomass harvested in November 2005, with
a control of stover ensiled within 12 h of harvest. All
samples had water added after these initial storage
treatments to adjust moisture to 60% (wet basis) for
ensilage. This water provided a vehicle for enzyme addition and also facilitated grinding to reduce particle size
required for the following study.
(ii) Two particle sizes, around <5 mm and <2 mm, were
tested in the ensilage systems as treatments. The
biomass was initially chopped to <5 mm particle size
during harvest. A food processor, (Buffalo Chopper model
81481D, Hobart Mfg. Co., Troy, OH) was used to reduce

b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 1 2 5 e1 3 3

biomass size to around <2 mm after addition of water and

15 min chopping with knives in a rotating bowl. One batch
of ground biomass was measured to characterize the
particle size reduction and the noted time was used to
chop the other samples. Particle size was controlled by
selecting biomass passing through VWR testing sieves
(Radnor, PA) for 5 and 2 inch sized screens on a shaker for
15 min.
(iii) Sample analysis protocols typically require drying of
post-ensilage samples to determine fiber and water
soluble carbohydrate (WSC) composition. Two drying
temperatures, 60  C and 105  C, were tested along with
a no-drying control treatment. Also, two different WSC
analytical methods, the original [22] and modified phenolsulfuric acid methods [27], were tested for the two drying
temperatures.
Experiments (ii) and (iii) were conducted on stover obtained from the November 2005 harvest, frozen in 20  C
freezer until May 2006, and thawed for 24 h prior to chopping
and ensilage.

2.3.

Silage preparation

Deionized water was added to the stover prior to ensilage to

achieve 60% moisture content [8]. Multifect A40 (Genencor,
Palo Alto, CA) was the enzyme used in enzyme treatments [8].
Enzyme activity measurements for carboxymethyl cellulase
(CMCase) and xylanase were conducted with carboxymethyl
cellulose and 1.0% birchwood 4-O-methyl glucuronoxylan
(Roth 7500) as substrates, respectively [29,30]. The ratio of
CMCase to xylanase activities was observed to be 5.29. The
enzyme loading rate was 5.0 IU/g dry matter (DM), calculated
as the sum of CMCase and xylanase activities, which has been
observed to be the minimum effective rate [9]. The enzyme
was mixed with water prior to adjusting moisture contents to
60%.
The silage reactors were plastic bags, vacuum sealed using
a Foodsaver (bags and Foodsaver produced by Jarden Corporation, Rye, NY). Wet stover samples of approximately 500 g
were double-bagged, first vacuumed and sealed in an inner
plastic bag, which was in turn vacuumed and sealed in an
outer plastic bag. The double-bagging system was used to
avoid the possibility of aerobic conditions due to leakage from
a bad seal in a single bag. Each of these bagged biomass
samples can be considered as an independent ensilage
reactor. Each treatment preparation was ensiled in triplicate
in separate reactor bags for 0, 1, 7, and 21 days at an incubation
temperature of 37  C [8]. Biomass before and after ensilage
was weighed to calculate loss of dry matter, after adjusting for
moisture content (see Section 2.4). All variables mentioned
below in Section 2.4 were measured on both initial (0 days of
ensilage) and final (21 days of ensilage) samples.

2.4.

Sample analysis

To measure the pH of biomass, 10 g of wet biomass was

soaked in 100 ml deionized water for 30 min. The pH value of
the solution was measured and noted for each biomass
sample. This solution was filtered with a 0.2 mm syringe filter

127

and tested for monosaccharide concentrations (glucose,

xylose, arabinose, mannose, and galactose) using anion
exchange chromatography (ICS 3000, Dionex, Sunnyvale, CA).
Separation was performed using CarboPac PA20 guard
(3  30 mm) and analytical (3  150 mm) columns. Monosaccharides were detected by pulsed amperometry with a gold
working electrode. Each analysis consisted of column cleaning for 20 min with 200 mM NaOH, equilibration for 15 min
with 10 mM NaOH, sample injection, and then sample running
for 25 min with 10 mM NaOH. A flowrate of 0.5 mL/min and
temperature of 30  C were held constant throughout the tests.
A representative sample of biomass from each replicate of
each treatment was dried at 60  C until constant weight to
determine moisture content. The dried biomass was ground to
<1 mm in a Wiley Mill (Thomas-Wiley Laboratory Mill, Model
4, Arthur H. Thomas Company, Philadelphia, PA) and tested
for fiber analysis using the ANKOM method [21]. Cellulose and
hemicellulose fractions were calculated as the difference
between Neutral Detergent Fiber (NDF), Acid Detergent Fiber
(ADF), and Acid Detergent Lignin (ADL) values, respectively.
Both the dried ground and the undried wet biomass were
soaked in water (10% w/v) in a conical flask and placed on
a shaker for 30 min at 200 rpm. The solution was filtered with
Whatman filters (grade 40), and water soluble carbohydrates
(WSC) were measured using the phenol-sulfuric acid method
[22]. A modified phenol-sulfuric acid method [27] was also
used with biomass that had been dried and wet samples and
compared with the original method [22].

2.5.

Statistical analysis

The statistical analysis was performed with SAS software (SAS

Institute Inc., Cary, NC) using PROC MIXED and LSMEANS
commands. Difference between treatments was determined
by Tukeys test. Prior to interpreting the results from ANOVA,
three assumptions were confirmed: the normality of the
residual, equal variance of residuals, and independence of
residuals. A logarithm of the responses was used to test
ANOVA when equal variances were not observed. Treatment
effects were considered significant at p-value < 0.05.

3.

Results and discussion

3.1.
Effects of treatments (drying, freezing, and
refrigeration) followed by ensilage on biomass quality
To extrapolate the results from lab-scale experiments to real
world scenarios, researchers assume that frozen and thawed
or dried and remoistened plant material is similar to freshly
harvested material [12,13]. Sometimes, low moisture biomass
is milled and stored in refrigerator (4  C) for prolonged periods
of time prior to re-moistening for experimentation [28].
However, it is known that the assumption of the stored
biomass being similar to freshly harvested material does not
always hold. Freezing, for example, can physically damage
plant cell walls, making them dissimilar from freshly harvested materials. Freezing too quickly can break cell walls due
to expansion of water molecules to ice, while freezing too
slowly can collapse the walls due to osmotic dehydration

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[14e17]. Drying plant material can lead to pore collapse [16,18].

In this study, we attempted to quantify the effects of drying,
freezing, or refrigeration of biomass followed by ensilage on
biomass quality. Biomasses harvested in two different
seasons were used for the study. The first set of experiments
was conducted on stover harvested in September with an
extended treatment (drying, freezing, or refrigeration) time of
7 days prior to ensilage. This study was carried out without
a control, stover ensiled immediately after harvest. The
second study, with corn stover harvested in November, was
performed with treatment (drying, freezing, or refrigeration)
time up to only 2 days prior to the set-up of ensilage experiments. The second study included a control, i.e. biomass
ensiled fresh within 12 h of harvest. Due to the difference in
treatment time periods, crop maturity, and original moisture
content of the biomass, different trends were observed in final
biomass quality from the two studies. This difference was
assessed by measuring several parameters of interest
including pH, dry matter loss, water soluble carbohydrate
content, and cell wall composition.

3.1.1.

Changes of pH values during ensilage

The initial pH levels (prior to ensilage) of refrigerated biomass

from the first study (8.43
0.06) were much higher than dried
and frozen (5.79
0.11, and 7.35
0.11, respectively)
biomasses; see Fig. 1(a). Some fungal growth was observed on
the refrigerated biomass due to high moisture and extended
treatment period of 7 days. After ensilage for 21 days, pH
values of frozen biomass were lower (4.19
0.12) than
refrigerated or dried biomasses (4.58
0.04 and 4.47
0.09),
but not significantly different ( p > 0.05). Initial pH values from
the second study were similar across all treatments and
exhibited no significant differences ( p > 0.05); see Fig. 1 (b).
After ensilage, biomass was much more acidic and similar
across all treatments, including the control, except for
refrigerated biomass, which was significantly higher ( p > 0.05)
at 5.00
0.15. Lower pH reduces unwanted microbial metabolism, thereby enhancing preservation and minimizing dry
matter loss. Because refrigeration prior to ensilage resulted in

a higher initial and pH, this feedstock holding strategy seems

likely to have a negative influence on subsequent experimental results. Unlike drying and freezing where feedstock
could be stored for many months, refrigeration would at best
be a temporary strategy in any case.

3.1.2.

Dry matter loss observed during ensilage

Though no statistically significant effect was observed on dry

matter loss due to any of the treatments or enzyme for both
studies, see Fig. 2(a) and (b), refrigerated and dried biomass
seemed to be dissimilar from the control in the second study,
see Fig. 2(b). The stover harvested in November had already
experienced considerable field drying, leading to a minimized
difference among treatments dry matter losses. The frozen
biomass behaved most similar to the control with low dry
matter loss, possibly due to reduced microbial activity prior to
and during ensilage due to lower temperature and pH,
respectively.
Comparing the two studies, dry matter losses were lower
for all treatments in the first study. Since the first study was
conducted almost 2 months prior to the second, the higher
original moisture content in the biomass may have allowed
higher microbial conversion of readily degradable dry matter
during the holding period, leading to lower possible loss
during ensilage. This could have especially occurred in
refrigerated biomass, which exhibited higher final pH values,
possibly from lower organic acid production.

3.1.3. Enzyme addition during ensilage as a possible solution

to resuscitate biomass
While the refrigerated biomass in first harvest had the lowest
final (after ensilage) Water Soluble Carbohydrate (WSC) levels
(3.29
0.39 g. 100 g1 DM), the lowest change in WSC after
ensilage was observed consistently in dried biomass for both
the harvests. Also, refrigerated and frozen biomasses
produced very little WSC after ensilage; see Fig. 3(a) and (b).
From Fig. 3(b), it is clear that the WSC production for all the
treated biomasses during ensilage is dissimilar to the control.
Among all the biomass holding strategies, the freshly ensiled

Fig. 1 e Effect of drying, refrigeration, and freezing treatments on pH values of subsequently ensiled corn stover biomass.

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129

Fig. 2 e Effect of drying, refrigeration, and freezing treatment on dry matter loss of subsequently ensiled corn stover
biomass.

control samples had the highest final WSC levels (8.03
1.50 g.
100 g-1DM) for both the enzyme and non-enzyme groupings
( p < 0.0005). Though the final WSC levels were much lower for
the treatments, when these biomasses were ensiled with
enzyme, all the treatments exhibited a WSC production
similar to that control. Use of enzyme during ensilage
produced negated the effects of treatments in biomass quality
with respect to WSC production. However, addition in enzyme
did not improve the WSC content of dried biomass in first
study indicating that irreversible pore collapse [10] occurred
during prolonged treatment period. On the other hand, WSC
was much higher in enzyme treated frozen biomass (final

value of 8.03
1.50 g. 100 g1 DM), possibly due enhanced

action of enzyme on burst cell walls with increased surface
area. Unfortunately, without a control for this study, it is not
possible to identify if this treatment is most similar to a real
world scenario. But since frozen biomass ensiled with enzyme
exhibited high WSC release in both harvests, this treatment
should be chosen for further processing of biomass where
pore wall collapse and sugar release from enzymatic treatments play a critical role.
Dried, frozen, or refrigerated biomass were not significantly different with respect to biomass fiber content
(hemicellulose and cellulose) ( p < 0.05) in the second study

Fig. 3 e Effect of drying, refrigeration, and freezing treatment ensiled with and without enzyme (Enzyme Loading Rate [ 5.0
IU/g initial dry matter) during ensilage on Water Soluble Carbohydrate content of biomass.

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Table 1 e Cellulose and hemicellulose content (g. 100 gL1dry matter) in corn stover biomass after a holding treatment
(drying, freezing, or refrigeration) followed by ensilage for 21 days on stover harvested in November (second study) with
moisture content [ 17.33 0.23 g. 100 gL1 wet biomass and particle size < 5 mm.
Treatments

No enzyme
After treatment

Enzyme

After treatment ensilage

After treatment

After treatment ensilage

Cellulose
Control
Drying
Refrigeration
Freezing
LSD ( p < 0.05)

43.45
42.13
45.10
43.07
29.15

3.60b

1.59

0.49

8.52b

49.85
45.20
44.78
47.38
10.17

1.46

1.66

2.21

0.98

45.80
43.04
40.71
43.45
14.91

2.39
1.50a
3.10b
2.36

45.17
43.01
42.51
39.13
10.35

Hemicellulose
Control
Drying
Refrigeration
Freezing
LSD ( p < 0.05)

20.82
22.47
23.33
25.02
17.73

3.95b

1.11

0.54

3.95b

22.63
18.79
19.23
20.14
6.10

1.85

0.55

0.37

0.02

23.79
21.93
22.60
22.99
8.34

1.74
1.37a
1.46
0.48

20.75
1.75
22.83
1.90
19.77
0.76
17.1.
0.36
8.40

0.34

0.42

2.40

2.26

a Data obtained after 24 h of ensilage.

b High standard deviations increased LSD and reduced chances of obtaining good inferences of the treatment effects.

where the treatments were conducted for 2 days, see Table 1.

These fiber content results are consistent with those previously observed, i.e. no significant effect on the fiber
composition of corn stover even after 96 h of drying up to
100  C [31]. But significant differences were observed
between dried and both refrigerated and frozen biomass in
the first study, where the treatment period was extended up
to 1 week. Enzyme treated biomass showed higher final WSC
levels for all treatments ( p < 0.0005) (Fig. 3), and lower
cellulose concentrations (Table 1), indicating that these
enzymes are converting cellulose to sugar as expected.
Noting that this enzyme mixtures xylanase activity was less
than 20% of it CMCase activity, it is also not surprising that
the impact of enzymes on hemicellulose concentration was
mixed (Table 1).
Considering all the above results, the ideal method of
handling biomass for realistic wet storage experiments would
be to ensile it within 12 h of harvest, i.e. the control treatment.
When biomass does need to be stored for longer periods,
freezing appears to offer advantages, especially for the wetter
biomass harvested earlier in the season, while drying may
also be appropriate for later harvests of senesced biomass that
has already partially dried in the field.

3.2.

Effect of particle size

The standard deviations in Table 1 and Fig. 2 provide evidence

that variation among ensilage replicates can make it difficult
to distinguish among treatment affects. Reducing the particle
size of corn stover prior to ensilage results in a more homogenous feedstock, which may lead to decreased variability
between different reactors acting as replicates, both for initial
and post-ensilage sampling.
Stover particle size has previously been shown to impact
silage quality, with particles less than 10 mm considered
small enough to maintain homogeneous silage quality [9].
However, the smallest particle size considered in the test was

< 5 mm [9]. The present study considered two particle sizes,

<5 mm and <2 mm to study the effect of even smaller particles on variance among sample replicates. Reducing stover
particle size from 5 mm to 2 mm did not significantly affect
the mean final pH values of the ensilage process ( p > 0.07)
(Fig. 4a) but it did decrease the variance within replicates for
pH (Fig. 4b). Unfortunately, there was no similar pattern of
reduced variance observed for cellulose, hemicellulose, or
WSC content (see Supplementary data). Since reducing
particle size from 5 mm to 2 mm only significantly reduced the
variability of one of four measured parameters, additional
grinding may not be justified for improving replicability in
laboratory ensilage experiments. As with any heterogeneous
material, appropriate techniques for sample splitting and
subsampling and increased replication of measurements can
help achieve greater statistical power.

3.3.

Post-ensilage extraction of solubles

Post-ensilage drying temperatures can influence the

measurement of several parameters, especially freely available sugars [23]. Oven-drying biomass at 105  C prior to
washing completely deactivates enzymes that would otherwise have the potential to degrade carbohydrates [24].
However, temperatures of 105  C could interfere with
measurements of monosaccharides and carbohydrates due to
the Maillard reaction [23]. If plant material is dried at a lower
temperature, say 60  C, in unsterile conditions at a lower
temperature biological degradation akin to composting may
occur during the early stages of drying while the samples are
still moist. Composting is a natural aerobic microbial process
occurring at around 50e60  C in unsterile biomass, and can
rapidly metabolize sugars and carbohydrates [32]. In the case
of ensiled biomass, the low pH of ensiled biomass might
prevent microbial activity at these lower temperatures,
resulting in similar or lower carbohydrate loss than occurs at
higher temperatures. Based on these considerations drying

b i o m a s s a n d b i o e n e r g y 4 7 ( 2 0 1 2 ) 1 2 5 e1 3 3

131

Fig. 5 e Effect of post-ensilage drying temperatures on

Water Soluble Carbohydrates compared to wet plant
material.

Fig. 4 e Effect of particle size and enzyme treatment on

(a) Means and (b) Standard deviations of the pH values of
biomass harvest on 09/29/2005 with moisture content of
17.33 0.23%.

temperatures of both 60  C and 105  C were applied prior to

extraction of solubles, and the results compared with a direct
method of carbohydrate extraction by washing fresh plant
material.
Biomass dried at 60  C resulted in higher WSC measurements than 105  C (Fig. 5). Drying at the higher temperature
may have led to Maillard reactions where glucose forms
a complex through cross linking with proteins [23]. Consistent
with the observations of Kerepesi et al. [23], wet plant material
(no-drying) produced the most WSC, and this was significantly
higher than either drying temperature for the enzyme treatment (6.27
0.63 g. 100 g1 DM). However, wet extraction on
fresh biomass was not significantly different than the 60  C
dried biomass extraction (2.18
0.12 g. 100 g1 DM) for the
non-enzyme treatment (Fig. 5). In the biomass with enzyme
dried at 60  C, higher availability of sugars in the enzyme
treatment may have increased the chances of the Maillard
reaction in wet biomass leading to decreased sugar levels
(4.36
0.61 g. 100 g1 DM).
These same temperature treatments were used to compare
the original Dubois method to the modified phenol-sulfuric
acid method for measuring WSCs of both post-ensilage

drying treatments and control samples [22,27]. The modified

phenol-sulfuric acid method provides similar trends of results
for all the treatments, but much higher numerical values
(Fig. 6). The modified method uses an ice bath to hold the test
tubes before adding sulfuric acid to avoid heat and the associated destruction or degradation of carbohydrates [27]. The
modified method did achieve significantly higher WSCs for all
temperature/extraction treatments, so appears to be an
effective way to avoid both types of interference.
Based on this set of experiments, the modified phenolsulfuric acid method is recommended for WSC analysis, and
should ideally be applied to wet ensiled material. However, for
samples with lower WSC levels, drying at 60  C may also be
acceptable.

Fig. 6 e Comparison of original phenol-sulfuric acid and

the modified methods for Water Soluble Carbohydrate
values of biomass dried at different post-ensilage drying
temperatures.

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4.

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Conclusions

With increased interest in the effects of feedstock supply chain

logistics and storage on biomass characteristics and downstream performance, it is critical that laboratory experiments
use methods that adequately simulate real process and provide
accurate results. For experimentalists needing to store
seasonally harvested biomass, refrigeration is an ineffective
strategy as results begin to deviate from control material within
the first couple of days. For early season feedstocks that are
moist, fresh biomass can be frozen prior to storage trials and
further testing. For feedstocks harvested later in the season
after they have substantially dried in the field, dry storage of
biomass can also be considered a suitable holding strategy prior
to ensiled storage and other downstream experiments.
Biomass is a heterogeneous material and methods are
needed to reduce variability and improve reproducibility to
discern patterns in analytical results. Unfortunately, reducing
particle size to <2 mm did not consistently improve replicability in ensilage treatments. While wet grinding reduced the
variance in pH values of replicates within same treatment,
a similar effect was not observed for the other three tested
parameters: WSC levels, cellulose, and hemicellulose content.
Based on these results, there is little justification for particle
size reduction to aid in the interpretation of ensilage experiments. However, other strategies, including appropriate
sample splitting and increased numbers of measurements,
should also be tried.
Because of potential chemical interferences from both high
and low temperature drying, different drying temperatures
and extraction methods were evaluated. Drying at 105  C
significantly decreased the WSC levels ( p < 0.05) compared to
drying at 60  C or washing wet biomass. It is advisable to wash
wet plant material and test with the modified phenol-sulfuric
acid method to retrieve WSCs prior to oven-drying. If WSCs
valued in samples are likely to be below 4 g/100 g dry matter, it
appears that drying at 60  C would also be suitable.
The methods developed in this study can act as a guide in
experimental design, and pre-and post- experimental techniques in wet storage ensilage research. More methods need
to be developed to support the research protocols used to
understand and improve ensilage as a biomass storage
method for biofuel production.

Acknowledgements
The authors would like to thank Penn State University and the
USDA-DOE Biomass Research and Development Initiative,
Contract # 68-3A75-4-137 for their funds to support this work.
The authors would also like to thank Drs. Haiyu Ren, Dr. Qin
Chen, Kay Marie Dimarco, and Jessica Schwartz for their
contribution to the project.

Appendix A. Supplementary data

Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.biombioe.2012.09.050.

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