b i o m a s s a n d b i o e n e r g y 5 8 ( 2 0 1 3 ) 1 8 0 e1 8 7

Available online at www.sciencedirect.com

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

Relationship to reducing sugar production and

scanning electron microscope structure to
pretreated hemp hurd biomass (Cannabis sativa)

Reinu E. Abraham, Colin J. Barrow, Munish Puri*

Centre for Chemistry and Biotechnology, Geelong Technology Precinct, Deakin University, Geelong, Victoria 3217,
Australia

article info abstract

Article history: Lignocellulosic biomass is a highly rigid and recalcitrant structure which requires pre-
Received 15 August 2012 treatment to loosen chemical bonds to make accessible monomeric sugars for biofuel
Received in revised form production. In this study, locally available biomass, that is hemp (Cannabis sativa), a low
10 June 2013 cost feedstock for ethanol production, has been used for the production of fermentable
Accepted 13 June 2013 sugars. Hemp hurd biomass (HHB) was exposed to five different pretreatments which
Available online 19 July 2013 included dilute acid (H2SO4), alkaline (NaOH), alkaline peroxide, hot water and one stage
dilute acid (H2SO4). Different pretreatments resulted in loosening and degradation of HHB
Keywords: structure thus facilitating enzymatic saccharification at optimized parameters (pHe4.8 and
Biomass 50
C). The changes in the reactive groups (hydroxyl or acetyl) of the HHB were confirmed
Saccharification by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Scan-
Cellulase ning electron microscopy (SEM) was employed to characterize the surface morphology of
Biofuel untreated and treated HHB. Finally, enzymatic saccharification demonstrated maximum
Lignocellulose yield of total sugars (743 mg g1) that are suitable for biofuel production.
Pretreatment 2013 Elsevier Ltd. All rights reserved.

1. Introduction
Biofuels from biomass are a potentially important way of
developing sustainable energy for the future. The demand for
biofuel has increased considerably over the past few years
because of increasing energy consumption globally and
adverse climatic changes due to the pollutants from fossil
fuel. Many countries across the world are replacing some
percentage of their fossil fuel consumption with biofuel,
depending upon their capacity to produce biofuel. The EU is
producing around 70% of its biofuel from wheat and the
remainder from other cereal crops, whereas more than 90% of
biofuel production in the USA takes place from corn [1]. The
Australian biofuel market is in the early development stage
but is set to become more important with the advent of carbon
trading and carbon taxes within Australia. India is replacing
20% of fossil fuel consumption with ethanol and biodiesel
whereas other Southeast Asian countries like Indonesia and
Malaysia are trailing big nations in the production of biofuel
[2,3].
Lignocellulosic biomass is available in abundance. How-
ever, an efficient bioprocessing technology is required for its
conversion to biofuel. The conversion of biomass to biofuel
includes a series of technological hurdles due to the complex

* Corresponding author. Bioprocessing Laboratory, Centre for Chemistry and Biotechnology, Deakin University, Australia. Tel.: 61 3
52272325.
E-mail address: munish.puri@deakin.edu.au (M. Puri).
0961-9534/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biombioe.2013.06.006
 b i o m a s s a n d b i o e n e r g y 5 8 ( 2 0 1 3 ) 1 8 0 e1 8 7
structure of lignocellulose, cellulose, and hemicellulose [4].
The interaction of the biomass molecules makes many
biomass materials tough, rigid and difficult substances to
hydrolyze. The key steps involved in the conversion technol-
ogy are feedstock selection, pretreatment, enzyme sacchari-
fication and fermentation [5]. Among these steps,
pretreatment is a key step since enzymatic hydrolysis of
biomass to fermentable material only occurs with effective
pretreatment phases. The final concentration of ethanol pro-
duced is dependent on the amount of fermentable sugars
obtained after enzymatic hydrolysis [6,7]. Excessive presence
of lignin, wax and volatile substance requires severe condi-
tions to hydrolyze the lignocelluloses, because it forms strong
binding and coiling among the biomass components making it
difficult to access for hydrolysis.
A wide variety of biomass is used worldwide for the pro-
duction of biofuel, including food and non-food crops. Corn,
wheat straw, switchgrass and, miscanthus are largely
explored for ethanol production. However, hemp (Cannabis
sativa) for biofuel production has not been extensively
explored [8]. Hemp is an annual plant mostly cultivated for
strong fibers [9]. Currently hemp has various industrial uses
such as paper and pulp production fiber and textile, and is
now finding application in ethanol production [10]. Further-
more, research has resulted in non-textile applications of
hemp like essential oils, thermal insulation and the produc-
tion of composite materials to be used in the automotive in-
dustry [11,12]. The yield and quality of hemp differs with
growing conditions such as soil quality, nutrition and climatic
changes [13].
Various combinations of pretreatments are used world-
wide for the hydrolysis of biomass and depending upon
treatment conditions the hydrolysis of lignocellulose occurs.
Amongst them are the uses of chemicals like acid, alkaline,
ionic liquids or thermal treatment involving steam explosion,
pyrolysis or combinations of these methods [14]. Dry hemp
has recently been studied for the production of methane [15].
In this study, five different pretreatments ranging from
acidic to alkaline conditions and their comparison were car-
ried out on hemp hurds. The morphological changes occurred
in the structure of hemp are due to pretreatment which
facilitated enzyme saccharification. Structural changes in the
raw material as a result of pretreatment were determined by
attenuated total reflection Fourier transform infrared (ATR-
FTIR) and scanning electron microscopy (SEM) techniques.
Further, enzyme saccharification of pretreated biomass was
optimized to release reducing sugars for biofuel production.
2. Materials and methods
2.1. Materials
The biomass sample examined in this study was a Ukrainian
variety of Hemp (C. sativa) which was grown in Griffith, New
South Wales (NSW), Australia (S34
340 , E146
120 ) and was
harvested in March 2009. This study included only the hemp
hurd biomass (HHB) which was the woody core of industrial
hemp (pith and xylem) obtained as an industrial residue from
Commins stainless manufacturing, Whitton, NSW. The HHB
181
powder was obtained by mechanical decortication by passing
once through a Fritsch Pulverisette 19 Universal Cutting Mill
which was equipped with a 1.0 mm diameter sieve. The
powder was sieved through three mesh sizes 1 mm, 600 mm
and 300 mm. All the experiments were performed using 300 mm
size HHB powder dried in an oven at 70
C until the weight was
constant. Then the HHB was stored in a plastic bag at room
temperature under dry conditions until further use. Sulfuric
acid (98%, AR grade) was obtained from Merck (Australia),
hydrogen peroxide (3%) was purchased from Chem-Supply
(Australia) and rest all the chemicals were procured from
SigmaeAldrich (Australia).
2.2. Pretreatment
Five pretreatments (described below) were performed on
hemp hurd biomass (HHB) in which two pretreatments were
done using sodium hydroxide (NaOH), another two with dilute
sulfuric acid (H2SO4) and the last was performed with hot
water treatment. All experiments were carried out in tripli-
cates and the mean values with standard deviation are
reported.
2.2.1. Alkaline pretreatment
Dried raw material (HHB) weighing 10 g was slurried in NaOH at
a concentration of 0.5 g g1 of dry sample and water loading of
100 g g1 of dry sample. The slurry was autoclaved at 121
C for
60 min and then cooled to room temperature. Second alkaline
pretreatment was performed using 10 g of dried biomass in
NaOH at a loading concentration of 1 g g1 of dry sample and
hydrogen peroxide at 3.3 g g1 of dry sample with a water
loading of 100 g g1 of dry sample. Pretreatment was performed
by incubating the slurry at 90
C for 60 min in a water bath. The
liquor was removed and respective slurries were washed
several times to remove the alkaline traces. The alkaline treated
samples were stored at 4
C for enzymatic hydrolysis [16].
2.2.2. Dilute acid pretreatment
Dilute acid pretreatment on HHB was achieved by modifying
acid pretreatment procedure [17]. Dry HHB weighing 10 g was
added to water with a loading of 100 g g1 of dry sample
containing H2SO4 at a loading concentration of 0.92 g g1 dry
sample and autoclaved at 121
C for 60 min. Another pre-
treatment was achieved by using 10 g of dry sample in H2SO4
at 0.92 g g1 of dry sample concentration and water loading at
100 g g1 of dry sample. This slurry was placed in an oil bath at
140
C for 15 min and then it was cooled and washed once with
water and again placed at 140
C for 10 min with H2SO4 at
concentration of 0.92 g g1 of dry sample with a water loading
of 100 g g1 of dry sample. Both the slurries were allowed to
cool at room temperature and thereafter it was washed
several times to remove residues and traces of acid. Later it
was stored at 4
C for enzymatic hydrolysis.
2.2.3. Hot water treatment
Hot water treatment was performed using 10 g of HHB in
distilled water loading of 100 g g1 of dry sample. HHB was
treated at 121
C for 60 min. Insoluble fraction was recovered
after pretreatment and washed several times with distilled
water before stored at 4
C for enzymatic hydrolysis.
182 b i o m a s s a n d b i o e n e r g y 5 8 ( 2 0 1 3 ) 1 8 0 e1 8 7
2.3. Biomass saccharification
The enzymatic saccharification of biomass was performed by
incubating 5 g of pretreated HHB in 250 ml citrate buffer, pH
4.8 (0.05 mol L1) with cellulase from Trichoderma reesei (Cat
C2730; 700 units g1). The enzyme saccharification was
achieved using optimised enzyme concentration (18 FPU g1
biomass at 50
C), 1.67 Hz for 72 h in an orbital shaker. The
supernatant was removed at 12 h time intervals and imme-
diately heated at 75
C for 10 min in a water bath and then
cooled for 10 min in an ice bath. The collected supernatant
was analyzed for the amount of reducing sugar. Optimised
amount of enzyme loading (data not shown) was used to
better understand the efficiency level of different pre-
treatments. All experiments including pretreatment and
enzyme saccharification were run in triplicate.
2.4. Analytical method
The concentration of reducing sugars produced from enzyme
saccharification was determined using 3, 5-dinitrosalicylic
acid (DNS) method [18] .The activity of cellulase was
measured using FPU method [19]. The yield of enzymatic
saccharification was calculated as:
Hydrolysis yield (%) reducing sugar (g)/polysaccharide in
substrate (g)  100 [20].
The composition of biomass was determined using NREL-
LAP protocols [21e23]. ATR-FTIR measurement of pretreated
HHB was performed using an Alpha FTIR spectrometer (Bruker
Optik GmbH, Germany) equipped with a deuterated triglycine
sulfate (DTGS) detector and a single-reflection diamond ATR
sampling module (Platinum ATR QuickSnap). The results
were analyzed using OPUS 6.0 software suite (Bruker). The
analysis was performed between the wavelength ranges of
375e4000 cm1. Pretreated and untreated biomass samples
were imaged using scanning electron microscope (Zeiss Supra
55 VP-Germany). SEM imaging was done with an accelerating
voltage of 7 kV and 5 kV using secondary electron (SE2)
detector.
3. Result and discussion
3.1. Pretreatment and recovery
The HHB contains 85% mass fraction of total solids and
14.81% of water content. Approximately 8% of HHB (based on
dry mass) constitutes lignin, residues, ash and other extrac-
tive and remainder 77% biomass is composed of hol-
ocellulose (Table .1). The presence of high content of
holocellulose can be a potential source for the production of
biofuel. The weight of dry solids obtained after different
pretreatments varied for all samples. An average of 73%
biomass was obtained after all pretreatments which were
further utilized for enzyme saccharification (Table 2). The
remaining 27% was soluble material which got dissolved as a
result of pretreatment and biomass loss due to washing. Loss
Table 1 e Composition of hemp hurd biomass.
Components Mass fraction (%)
Total solids 85.18
Acid insoluble residue 4.04
Acid soluble lignin 0.50
Ash content 3.10
Moisture content 14.81
Oven dry weight (ODW) 80.15
Holocellulose w77.00
of a significant amount of biomass (20e25%, dry wt. of
biomass) in different pretreatment conditions for wheat
straw was observed using a combination of steam explosion
and various solvents [24]. Earlier studies investigated
different concentration of sodium hydroxide ranging from
7.5 g L1e20 g L1 to determine the efficiency of pretreatment
[25,26], however, this study contained NaOH and H2SO4 to
5 g L1 thus making process economical.
3.2. Enzymatic saccharification
Enzymatic hydrolysis of the pretreated biomass was per-
formed to evaluate the accessibility of the cellulose and thus
efficiency of pretreatment. The use of cellulase from Tricho-
derma reesei for enzymatic saccharification of HHB has not
been reported; hence our studies demonstrated the amount of
reducing sugars produced using various concentration of
enzyme. Further, our interest was to develop a comparative
study of different pretreatment with the economical usage of
single enzyme loading to better understand the efficiency of
each pretreatment for delignification. The effectiveness of all
pretreatments was determined by enzyme saccharification
based on total reducing sugar yield, as shown in Fig. 1. During
72 h of incubation of untreated HHB it released a maximum of
126  0.01 mg g1 of reducing sugar in 24 h of enzymatic hy-
drolysis. The concentration of reducing sugars in all pre-
treated samples gradually increased after 6 h of hydrolysis.
The alkaline treatments (NaOH/alkaline peroxide) enhanced
the digestibility of raw HHB to a greater extent when
compared with acid treatments (one stage and dilute acid).
Our results were in agreement with previous reports which
suggests that pretreatment with sodium hydroxide degrades
lignin and acetyl group from the biomass (switchgrass) and
improves enzyme accessibility [27,28]. Thus alkaline treat-
ment was successful in exposing biomass surface for an
enhanced enzyme digestibility of holocellulose. In particular,
alkaline peroxide treated samples provided maximum
amount of reducing sugar that is 743  3.1 mg g1, among all
the pretreatments. However, both alkaline treated samples
released almost the same amount of reducing sugars with
minimal loading of enzyme. The concentration of enzyme
was kept low to aid in determining the efficiency level of
different pretreatments. Similarly, low concentration of
enzyme was used for the hydrolysis of bagasses pretreated
with lime and alkaline hydrogen peroxide [29]. The concen-
tration of reducing sugars obtained after alkaline treatments
in this study was greater than those on similar biomass where
high enzyme loading but limited pretreatment was used [30].
 b i o m a s s a n d b i o e n e r g y 5 8 ( 2 0 1 3 ) 1 8 0 e1 8 7 183

Table 2 e Yield of reducing sugars with different pretreatments in combination with enzymatic saccharification.
Pretreatment methodsa Recovered biomass (g) Recovery (%) Reducing sugars (mg g1) Hydrolysis yieldb (%)

Alkaline peroxide 3.20  0.70 64.0 743  3 74

Dilute acid 3.86  0.13 77.2 349  4 35
Hot water treatment 4.00  0.57 80.0 299  3 30
Sodium hydroxide 3.36  0.34 67.2 719  3 72
One stage e temperature (140
C) 3.75  0.26 75.0 293  4 29
Control 5 100 126  0.01 13

a Hemp hurd biomass (dry matter, 5 g) used for carrying pretreatment, whereas 5 g was used for enzymatic saccharification experiments.
b Based on the mass fraction of raw material.

In acid treatments, dilute acid pretreated HHB provided a
better yield of reducing sugars when compared to one stage
and hot water pretreatments. The rate of hydrolysis increased
after 36 h of incubation and released a maximum of
349  4 mg g1 reducing sugar in 72 h of incubation. One stage
pretreated samples could produce a maximum of
293  4 mg g1 and hot water treated sample reached up to
299  3 mg g1 in 72 h of saccharification. The amount of
reducing sugars doesnt significantly change after 72 h when
conducting the study on HHB. Therefore, 72 h was used for all
the samples in enzymatic hydrolysis [24]. In general, the
amount of reducing sugars released from enzyme saccharifi-
cation of hot water treated, dilute acid and one stage samples
were considerably low in comparison to alkaline treatments.
Nonetheless, on comparing these observations the perfor-
mance level of alkaline pretreated samples predominates in
terms of sugar recovery demonstrating that loosening of bond
occurred best with alkaline pretreated biomass.
Material balance: The conversion of biomass to reducing
sugar was summarized in Fig. 2. On the basis of 1 g HHB, an
average of 73% of pretreated solids was recovered after pre-
treatment. Furthermore, about 743  3.1 mg g1 of glucose (as
reducing sugar) was recovered from enzymatic hydrolysis of
sodium hydroxide and alkaline peroxide pretreated HHB for
the selected conditions. These pretreatments followed by
enzymatic saccharification resulted in maximum hydrolysis
yield (74%) on comparing with all pretreatments (Table 2).
Although, the material balance indicated loss of some
biomass during pretreatment and enzymatic hydrolysis, the
overall reducing sugar yield was over 74%, confirming that the
selected pretreatment conditions (alkaline) substantially
enhanced the effectiveness of the enzymatic hydrolysis.
3.3. ATR-FTIR
FTIR spectra of HHB showing the difference in peak intensity
at approximately 1760e1030 cm1 after pretreatment are
shown in Fig. 3. A peak at around 1734 cm1 indicates the
presence of carboxylic esters from pectin and wax in un-
treated, hot water treated, dilute acid and one stage pretreated
biomass sample, while reduction of this peak in NaOH and
alkaline peroxide pretreated samples suggests that a consid-
erable degradation of pectin and wax has occurred. In previ-
ous reports, similar observation of pectin and wax reduction
was observed at 1736 cm1 [31]. The occurrence of a strong
peak at 1030 cm1 represents the CeC and CeO stretching at
C-6. The region around 1638 cm1 was stretched in hot water
treated samples in comparison to other samples revealing
absorbance of water after pretreatment [30] The peak
w1230 cm1 in untreated, hot water treated, dilute acid, one
stage pretreated biomass illustrates the presence of guaiacyl
ring & CeO stretch in lignin & xylan but this stretch flattens
for NaOH and alkaline peroxide samples indicating a partial
removal of lignin from the HHB structure. The flattening of
this peak indicates that the delignification by alkaline treat-
ment was remarkable in comparison to other treatments [32].

Fig. 1 e Enzymatic saccharification of pretreated hemp hurd biomass for releasing reducing sugars.
184 b i o m a s s a n d b i o e n e r g y 5 8 ( 2 0 1 3 ) 1 8 0 e1 8 7

(Cellulase-Trichoderma
Pretreatments ressei)
Hemp hurd Alkaline
biomass Mechanical peroxide
Total reducing
Pretreatment Sodium Enzyme hydrolysis
sugars
(Milling & (15% moisture & hydroxide (50C, 1.67
1g HHB (dry Dilute acid
Grinding) 85% total solids) Hz, 72h)
mass) hot water
(Autoclaving)
One stage-
Glucose yield 74 mg g
Temperature
(140 C)

(~ 300 m size)

73 % (Average pretreated 27% (soluble material removed

biomass was obtained after with washing)
washing)

Fig. 2 e Mass balance for pretreated hemp hurd biomass based on enzymatic hydrolysis.

Fig. 3 e ATR-FTIR spectrum of untreated and pretreated hemp hurd biomass.

The untreated sample lacks strong peaks around 1158 and
1104 cm1 which indicates the absence of CeO stretching at C-
3 and CeOeC stretching at the b-(1-4)-glycosidic linkages. This
indicates that with different pretreatments the structure of
biomass was altered exposing the CeO and CeOeC bonds for
further degradation by enzymatic hydrolysis [33]. But while
comparing FTIR of untreated with respect to alkaline it can be
observed that many peaks were either flattened or reduced
which determines that delignification in alkaline pretreated
HHB was more evident and the effect of structural modifica-
tion was seen during enzymatic saccharification. The alkaline
pretreated HHB produced maximum amount of reducing
sugar in comparison to other pretreated HHB. Delignification
in sugarcane bagasse using alkaline pretreatment and hemi-
cellulose removal by 90% was observed previously [26].
3.4. Scanning electron microscopy (SEM)
SEM images of untreated and pretreated HHB with different
magnification and accelerating voltage are shown in
Fig. 4(aef). After pretreatment, the HHB was swollen and the
stacked bundles opened (Fig. 4bef). The SEM of untreated HHB
shows a rough surface with sharp edges and grooves but after
various pretreatments significant and multiple morphological
changes were observed indicating considerable damage after
pretreatment. Ridges and grooves became more prominent
and the roughness of the wood surface increased greatly [34].
In all the pretreatments, major changes such as cracked and
flaked off surface were observed, demonstrating the separa-
tion of compact structure which further enabled enzymatic
degradation. The surface was significantly flaking off in the
 b i o m a s s a n d b i o e n e r g y 5 8 ( 2 0 1 3 ) 1 8 0 e1 8 7 185

Fig. 4 e SEM images of hemp hurd biomass before and after pretreatment at accelerating voltage of 7 and 5 kV and
magnification ranging from 10 to 20 mm. Five different pretreated HHB with untreated are represented from (aef):
(a) untreated (b) dilute acid (c) hot water treatment (d) NaOH (e) alkaline peroxide and (f) one stage.

cases of dilute acid, NaOH, or one stage pretreatments, but in
case of hot water treated or alkaline peroxide pretreatments,
the appearance of holes at regular intervals were very prom-
inent [35]. In alkaline peroxide pretreated HHB, a majority of
the surface has voids and holes at regular intervals and the
size of biomass also is reduced. Similar type of holes on the
structure was observed when corn stover was given lime
pretreatment, indicating a major degradation of lignin and
hemicellulose [36]. The surface of hot water treated HHB was
observed to be uneven and eroded, whereas, after NaOH pre-
treatment, tracheids were clearly observed, indicating the
degradation of lignified cell wall [37]. The tracheids bundles
opened significantly in NaOH pretreatment, thereby
increasing cellulose accessibility for enzyme saccharification.
While comparing three pretreatments alkaline, dilute acid
and hot water treated which took place at same temperature
conditions (121
C, 60 min); it can be seen that the alkaline
treatment made a prominent effect on the structure of
biomass in comparison to other two treatments. It is quite
evident from the SEM images that the loosening and opening
of these bundles are more evident in alkaline treatment with
respect to untreated and other treated biomass. This indicates
that the structure of HHB is modified with the presence of
NaOH and made the structure more accessible to the enzyme
186 b i o m a s s a n d b i o e n e r g y 5 8 ( 2 0 1 3 ) 1 8 0 e1 8 7
to produce reducing sugars by completely opening the
bundles.
4. Conclusion
The digestibility of HHB improved with all pretreatments.
However, a remarkable difference was observed with alkaline
pretreatment, which disrupted the biomass microstructure by
creating distinct holes and opening the stacked holocellulose
bundles and providing very high sugar recovery. Pretreatment
improved the exposure of cellulose, which enhanced enzy-
matic hydrolysis of HHB. Although other treatments like hot
water and acid treatment also improved the solubility of HHB,
these were not as effective in comparison to NaOH/alkaline
peroxide treatment. Among all the pretreatments alkaline
peroxide pretreatment resulted in maximum recovery of
reducing sugar at 743  3 mg on per g of pretreated material.
The FTIR spectrum and SEM images showed a significant
change in structure after various pretreatments. The removal
of lignin, pectin and wax was shown by the absence of their
respective signal in FTIR spectra and the opening of structure
was seen in SEM images. Voids and cracks in the structure of
HHB after NaOH/alkaline pretreatment supported the enzyme
saccharification. This study indicates that NaOH/alkaline
treatment is the most effective pretreatment, when compared
with acid and hot water pretreatments, for the efficient
saccharification of HHB.
Acknowledgments
Authors acknowledge Deakin University, Australia for
providing funding to pursue research on biofuels under ITRI
think tank initiative. Authors are thankful to Dr A Sullivan,
Electron Microscopy facility at Institute for Frontier Materials
(IFM), Deakin University, Australia for SEM work.
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