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production processes. Since biomass is seasonally harvested, stocks are often dried or
13 September 2012
fermentation studies cause irreversible changes in the plant cells, influencing the initial
state of biomass and thereby the progression of the fermentation processes itself. This
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
* 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.
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
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2.3.
Silage preparation
2.4.
Sample analysis
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2.5.
Statistical analysis
3.
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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3.1.1.
3.1.2.
Fig. 1 e Effect of drying, refrigeration, and freezing treatments on pH values of subsequently ensiled corn stover biomass.
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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
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
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
3.2.
3.3.
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4.
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Conclusions
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.
references
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