Changes in the supramolecular structures of cellulose after hydrolysis studied by terahertz spectroscopy and other methods

Article abstract of DOI:10.1039/c4ra08314h

Hydrogen bonding is one of dominant forces in crystalline cellulose and lignocellulose, however, it is still a big challenge to evaluate the changes in the hydrogen bonding strength. Here, we reported a new method for measuring the changes in the hydrogen

Full text of DOI:10.1039/c4ra08314h

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Changes in the supramolecular structures of
cellulose after hydrolysis studied by terahertz
Cite this: RSC Adv., 2014, 4, 57945
spectroscopy and other methods†
a
ad
Hong Guo,a Mingxia He,b Renliang Huang,c Wei Qi,ad Weihua Guo, Rongxin Su
*
and Zhimin Hea
Hydrogen bonding is one of dominant forces in crystalline cellulose and lignocellulose, however, it is still a
big challenge to evaluate the changes in the hydrogen bonding strength. Here, we reported a new method
for measuring the changes in the hydrogen bonding strength of cellulose-based materials by terahertz time
domain spectroscopy (THz-TDS). Avicel, corncob and their residual substrates after enzymatic hydrolysis
were chosen as the targeted cellulose to demonstrate this method. THz adsorption in the range 0.5–2.5
THz and refractive index in the range 0.5–1.0 THz provided a direct signal corresponding to an increase
in the hydrogen bonding strength for the residual samples (Avicel and corncob) after enzymatic
hydrolysis. The THz results were further compared with those obtained from X-ray diffraction (XRD) and
Fourier transform infrared spectroscopy (FTIR) analysis. As a quick and non-invasive technique, THz
spectroscopy provides unique information about the changes in the hydrogen bonding strength of
cellulose-based materials. Therefore, it is a promising way to directly evaluate the hydrogen bonding
strength of cellulosic macromolecules.
Received 7th August 2014
Accepted 17th October 2014
DOI: 10.1039/c4ra08314h
www.rsc.org/advances
understand the enzyme–substrate interactions and to improve
the process of enzymatic hydrolysis, it is essential to measure
Introduction
the changes in the hydrogen bond strength, which is the
accumulated strength of hydrogen bond, during the hydrolysis
process.
Fourier transform infrared spectroscopy (FTIR) shows a high
sensitivity to various conformations and microenvironment of
chemical groups such as hydroxyl and glycosidic bonds present
in cellulose chains, Therefore, it has oen been used to evaluate
the hydrogen bonding strength in cellulosic substrates.12 In
previous studies, the ratio of the absorbance at some charac-
teristic peaks were introduced as the empirical hydrogen bonds
Lignocellulose, the most abundant and renewable biomass, has
a great potential to serve as a sustainable supply of fuels and
chemicals. In recent years, a large number of research efforts
have focused on the bioconversion of lignocellulose into bio-
fuels, such as ethanol and butanol.1–3 During this process,
effectively breaking the recalcitrant structure and converting
the polysaccharides to fermentable sugars are the major
barriers in the commercialization of cellulosic biofuels. The
recalcitrance of the crystalline cellulose in biomasses to enzy-
matic hydrolysis is mainly because of the strong intermolecular
interactions, including hydrogen bonding and van der Waals
force. The hydrogen bonds within the cellulose molecule and
the adjacent cellulose chains provide a big barrier for cellulose
deconstruction and enzymatic hydrolysis.4–11 To better
intensity (or the degree of destruction), such as the absorbance
10,13–15
ratios of A4000–2995/A1337
, A1430/A897, and A897/A2900.
However, the other components (e.g., lignin, hemicellulose) in
lignocellulose also have the similar chemical groups (e.g., OH,
C–H) that signicantly inuence the IR adsorption of charac-
teristic groups derived from cellulose and thus renders a chal-
lenge to obtain exact hydrogen bonding strength. Deuterium
exchange coupled with FTIR was used to monitor the structural
change of lignocellulose during various treatments by following
the change in the retention of the OD group at around 2500
cmꢀ1 in the FTIR spectrum, including cellulose accessibility in
aqueous systems and irreversible microbril aggregation.16,17
Moreover, X-ray diffraction (XRD) is another auxiliary technique
to evaluate the change in the hydrogen bonds within cellulose-
based materials by detecting the amount of crystalline cellulose,
which possesses strong hydrogen bonds.7,9,10,18,19 The crystallinity
aState Key Laboratory of Chemical Engineering, Tianjin Key Laboratory of Membrane
Science and Desalination Technology, School of Chemical Engineering and
Technology, Tianjin University, Tianjin 300072, P. R. China. E-mail: surx@tju.edu.
cn; Fax: +(86)022-27407599; Tel: +(86)022-27407599
bCollege of Precision Instrument and Optoelectronics Engineering, Tianjin University,
Tianjin 300072, China
cSchool of Environmental Science and Engineering, Tianjin University, Tianjin 300072,
China
dCollaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Tianjin 300072, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra08314h
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index obtained from XRD represents the relative amount of unit (FPU) mLꢀ1 and 926 cellobiase unit (CBU) mLꢀ1, respec-
crystalline cellulose in the lignocellulosic substrate. Therefore, tively. All other reagents were of analytical grade and were
XRD results cannot reect the actual crystallinity of cellulose in obtained from commercial sources.
the presence of amorphous components (hemicellulose and
lignin) other than cellulose. Previous studies also demonstrated
that the crystalline index from XRD yields contradictory results
Samples preparation
Corncob was soaked in aqueous ammonia (15 wt%) at a solid-
regarding its effect on the enzymatic hydrolysis in different
to-liquid ratio (w/v) of 1 : 6. The solid/liquid slurry was then
substrate systems.1,8 As well known, the crystallinity of cellulose
incubated in a water bath at 60 ꢁC for 6 h without any agitation.
Aer treatment, the corncob was separated by ltering and
is directly related to the intra- and intermolecular hydrogen
bonds. Therefore, it might be more accurate to employ
washed with distilled water to remove the residual ammonia
hydrogen bonding strength to characterize the cellulose
until the pH became neutral. The resulting solid was oven-dried
crystallinity.
at 100 ꢁC for 12 h and collected for subsequent experiments.
Terahertz time domain spectroscopy (THz-TDS) is a powerful
The carbohydrate and lignin contents were determined by
tool for analyzing the structural and constitutional changes in
following the National Renewable Energy Laboratory (NREL)
materials, because of its high signal-to-noise ratio and broad
chemical analysis and standard testing procedure.
To collect the residual cellulosic samples, Avicel and pre-
treated corncob were added into the citrate buffer (50 mM, pH
spectra band constituting individual THz pulse.20 For example,
the THz-TDS technology has been successfully applied in
monitoring the conformation transition of protein,21 in the
4.8) solution at a solid loading of 5% (w/v). The enzyme loadings
detection of PCR-amplied DNA22 and antibiotic residues in
for Avicel were 40 FPU gꢀ1-glucan Spezyme CP, 60 CBU gꢀ1
-
food industry.23 In general, the absorption features in the ter-
ahertz region are dominated by the intermolecular vibrations,
which are related to the movement of atoms and/or molecules.
In particular, the low-frequency vibrations between 0.5 and
3.0 THz are dominated by non-covalent intermolecular inter-
actions such as hydrogen bonds, electrostatic force, and Van der
Waals force. Previous studies have demonstrated that THz-TDS
is a promising technology to determine these weak interactions
in purine, insulin, and inks.24–26 As a quick and non-invasive
technique, THz-TDS provides unique information about the
changes in weak interactions. As mentioned above, crystalline
cellulose contains strong hydrogen bonds. However, to date, no
report has discussed the utilization of THz-TDS in determining
the hydrogen bonding strength in lignocellulosic substrates.
glucan Novozyme 188, whereas 30 FPU gꢀ1-glucan and 30 CBU
gꢀ1-glucan for corncob. The enzymatic hydrolysis was carried
ꢁ
out in an air shaker at 150 rpm and at 50 C. Aer enzymatic
hydrolysis, the soluble sugars in the supernatant were deter-
mined by high-performance liquid chromatography (HPLC)
using an Aminex HPX-87H column (Bio-Rad, Hercules, CA,
USA). All the solid residues were separated and incubated in a
citrate buffer (50 mM, pH 7.0) with a papain–trypsin (2 mg/2
mg) mixture to remove the bound enzymes. These reactions
were carried out at 50 ꢁC with constant shaking for 12 h; and the
solids were ltered out, washed with distilled water three times
and, thereaer, oven-dried for the successive analysis.
In this study, we introduced the application of THz-TDS in Terahertz time domain spectroscopy (THz-TDS)
evaluating the hydrogen bonding strength of cellulose. Avicel
The THz measurements were performed at room temperature
and corncob were chosen as the target substrates, because they
in a photoconductive switch-based terahertz time-domain
are known to contain crystalline cellulose. Moreover, we also
system, which consists of four parabolic mirrors in an 8-F
analyzed the changes in the hydrogen bonding strength of their
confocal geometry, with a useful bandwidth of 0.1–2.5 THz and
residual solids aer enzymatic hydrolysis. To further analyze
an amplitude signal-to-noise ratio greater than 1.5 ꢂ 104 : 1.
the THz results, these cellulosic samples were characterized
The 8-F confocal system compressed the terahertz beam to a
using XRD and FTIR techniques, to obtain the complementary
frequency-independent diameter of 3.5 mm, and the THz
structural information. In addition, we also discuss the results
radiation was excited by optical pulses from a mode-locked Ti-
from size-exclusion chromatography with multi-angle laser
light scattering (SEC-MALLS), as reported in our previous
studies.27,28
sapphire laser (l ¼ 800 nm, TFWHM ¼ 25 fs). The cellulosic
samples were naturally placed, without applying additional
pressure, in a silicon cell composed of two parallel, 0.64 mm
spaced and 0.64 mm thick windows. An identical empty cell was
Materials and methods
Materials
used as a reference during the data acquisition.
X-ray powder diffraction (XRD)
Microcrystalline cellulose (Avicel PH101) was purchased from
Sigma (MO, USA). Air-dried corncob was collected from a local The XRD measurements were performed on an X'Pert Pro X-ray
farm (Tianjin, China), pre-milled and screened to a nominal diffractometer (PANalytical, Holland). The diffracted intensity
˚
size of 20–80 meshes. Cellulase (Spezyme CP) derived from of the Co Ka radiation (l ¼ 1.789 A, 30 kV, 30 mA) was measured
Trichodermareesei was a kind gi from Genencor International in a 2q range between 5ꢁ and 50ꢁ at the scan rate of 12ꢁ minꢀ1
.
(CA, USA) in 2009. b-Glucosidase (Novozyme 188) was The crystallinity index (CrI) was calculated according to the
purchased from Sigma (St. Louis, MO, USA). The average method adopted by Segal et al.29 The formula used for the above
activities of Spezyme CP and Novozyme 188 were 117 lter paper calculation is as follows:
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Table 1 Chemical compositions (based on the initial dry matter) of
corncob and Avicel before and after 48 h enzymatic hydrolysis
I002 ꢀ Iam
CrI ¼
ꢂ 100%
(1)
I002
Materials
Glucan (%)
Xylan (%)
Lignin (%)
where I002 is the peak intensity on the 002 lattice plane, and Iam
is the peak intensity of the amorphous zone diffraction.
Corncob
Residual corncob
Avicel
45.79
20.1
96.2
96.5
37.01
37.5
3.8
11.14
42.4
Fourier transform infra-red (FTIR)
Residual avicel
3.5
The FTIR spectra were recorded on a Nicolet-560E.S.P FTIR
(Thermal Nicolet Co, USA) with a KBr pellet method in the range
of 400–4000 cmꢀ1. A total of 16 scans were accumulated with a
resolution of 4 cmꢀ1 for each spectrum. The relative absorbance
(Arl) for the band l attributed to the hydrogen bonds was
calculated using the C–H stretching band (2900 cmꢀ1 for Avicel,
2920 cmꢀ1 for corncob) as an internal standard, according to
the empirical formula described in a previous study.13 The
formula used for this calculation is as follows:
corncob. In terms of Avicel, no evident change in the glucan and
hemicellulose contents was found aer enzymatic hydrolysis
(Table 1).
XRD analysis
Prior to the evaluation of hydrogen bonds, we employed XRD to
characterize the samples, aiming to investigate the structural
change in the cellulosic substrates due to enzymatic hydrolysis.
As shown in Fig. 2a, the initial and residual Avicel showed
similar XRD spectra with similar crystallinity indices (CrI,
76.5% vs. 75.2%). There was no signicant change in the rela-
tive crystallinity between the two samples. Although it is not
suitable to estimate the amount of crystalline and amorphous
Al
Arl ¼
(2)
ACꢀH
Results and discussion
Changes in the chemical structure upon hydrolysis
The chemical compositions of these test materials, as a key
factor inuencing hydrogen bonds, were determined in this
work. For Avicel, aer 48 h of enzymatic hydrolysis, approxi-
mately 20% of the initial cellulose was hydrolyzed to glucose,
leaving the residual cellulose for subsequent analysis. For
corncob, enzymes catalyzed the hydrolysis of glucan and xylan,
as shown in Fig. 1. Within the rst 12 h, the glucan content in
corncob signicantly decreased from 45.79–18.4%, whereas the
xylan content decreased from 37.01–18.4%. Subsequently, a
slow degradation was observed up to 48 h.
Table 1 summarizes the main components of corncob before
and aer the 48 h enzymatic hydrolysis. The relative content of
the glucan in the residual corncob was just 20.1%, whereas the
xylan and lignin were 37.5% and 42.4%, respectively. In other
words, approximately 10%, 23% and 88% of the initial glucan,
xylan and lignin, respectively, were retained in the residual
Fig. 2 XRD profiles of Avicel (a) corncob (b) before and after the
Fig. 1 The contents of glucan and xylan (based on the initial dry enzymatic hydrolysis. The inset in Fig. 2b shows the change in the
matter) in the residual corncob during the enzymatic hydrolysis.
crystalline index (CrI) of corncob during the enzymatic hydrolysis.
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cellulose in the lignocellulosic samples, the XRD peaks are
useful for comparing the relative differences among the various
cellulosic samples. The CrI values of Avicel in the previously
studied reports were mostly shown in a wide range of 62–87.6%,
using XRD peaks, whereas a recent research report even showed
an extreme high value (higher than 90%).30 As reported in
previously studied reports, no evident structural change in
Avicel was observed under mild conditions.16 Here, we adopted
the average value of three samples as the presenting CrI, and it
showed consistency with the data of the references. In our
previous study,28 using SEC-MALLS, we found that the residual
Avicel had considerably higher average molecular weight than
the original Avicel (Mw, 103 kDa vs. 59 kDa) and radii of gyration
(Rg, 49.5 nm vs. 23.4 nm), indicating the presence of longer
cellulose chains in the residual substrate. We thus speculated
that the residual cellulose should have different hydrogen bond
strengths than that in the initial cellulose. Unfortunately, this
speculation could not be conrmed by XRD analysis.
The XRD patterns of corncob during the enzymatic hydro-
lysis are shown in Fig. 2b. The diffraction patterns of the
samples shared similar shapes within 0–48 h, whereas a
signicant decrease in the intensity of the I002 peak was found at
96 h. The inset gure shows the change in the CrI, as calculated
from the diffraction patterns. At the initial stage (0–0.5 h), the
CrI increased rapidly from 38.7–43.1%, indicating an increased
content of the crystalline components in corncob. Further
Fig. 3 The absorption coefficient (a) and refractive index (b) of Avicel
increase in the CrI up to 45% was found in the following 11.5 h. before (0 h) and after 48 h of enzymatic hydrolysis in the range 0.1–2.5
THz at 293 K.
The increased CrI should be mainly attributed to the hydrolysis
of the disordered cellulose and hemicellulose. As the reaction
proceeded, the CrI decreased to 35.1% at 48 h and to 22% at
intensity in the range of 1.5–2.5 THz. Specically, the residual
cellulose had an increased absorbance (e.g., from 13.5–18.8 at
1.5 THz and from 30.2–55.1 at 2.5 THz) in this spectral range.
Fig. 3b shows the refractive indexes of the initial and residual
Avicel in the range 0.1–2.5 THz. Both of them showed steady
refractive indexes of 1.12 and 1.25, respectively. As shown in
Fig. 3b, the residual cellulose presents a higher refractive index.
Low frequency vibrational modes in THz-TDS were related to
hydrogen bonding and other weak interactions.24–26 Particu-
larly, a higher absorption and refractive index could be
observed in self-assembled insulin nanobers,25 as well as in
purine and adenine at a lower temperature.24 As we know, both
the self-assembly process and the decreased temperature could
enhance the intermolecular interaction, such as p–p stacking
and hydrogen bonding.41–43 Therefore, an increase in the
absorption intensity for the residual cellulose should be
regarded as a signal an enhancement of the intermolecular
interactions (hydrogen bonding strength and van der Waals
forces). In our previous study, the SEC-MALLS results suggested
that the celluloses with a low molecular weight (Mw) were
preferentially hydrolyzed to glucose, leaving behind the
residual celluloses with a higher Mw.28 Based on these ndings,
we expected that the residual cellulose with higher absorption
and refractive index value at low THz frequency should exhibit a
stronger hydrogen bonding strength. This recalcitrant structure
could thus hinder enzyme access to the residual cellulose for
168 h, suggesting that the crystalline cellulose was degraded or
destructed gradually at this stage. The decrease in the glucan
content of the residual corncob from 12–48 h (Fig. 1) was also in
agreement with this deduction.
Previous studies had also demonstrated that the removal of
amorphous components (e.g., hemicellulose and lignin) could
result in an increased CrI of lignocellulosic substrates.31–34 However,
the degradation of the disordered cellulose and hemicellulose has
little effect on the ordered structure of the crystalline cellulose in
the initial reaction.35 On the other hand, the hydrolysis of crystalline
cellulose could lead to a decrease in CrI of the substrate, but it can't
reect the crystalline change in the residual cellulose of the
substrate. Overall, although XRD is oen used to determine the
crystalline structure and crystallinity index of cellulosic substrates,
it is difficult to determine the actual crystallinity and hydrogen
bonding strength in cellulosic substrates, particularly in the pres-
ence of amorphous hemicellulose and lignin.36,37
THz spectroscopy analysis
The absorption coefficients and refractive indices of Avicel
before and 48 h aer the enzymatic hydrolysis in the range of
0.1–2.5 THz are shown in Fig. 3. It can be clearly seen that these
two samples shared the similar spectral characteristics, lacking
a prominent absorption peak, which was also observed in some
other macromolecules, such as BSA and cytochrome c.38–40
However, they presented signicantly different absorption
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further hydrolysis, leading to a low cellulose conversion results, the residual cellulose in corncob aer the enzymatic
hydrolysis should have a higher degree of polymerization and
(ꢃ20%).
To further evaluate the hydrogen bonding strength in stronger hydrogen bonding strength compared to the original
lignocellulose, we characterized the initial and residual cellulose.
corncob aer 48 h of enzymatic hydrolysis by THz-TDS. The
Based on the THz spectra from single cellulose and cellulose
corncob showed a featureless absorption prole with monot- complex, we proposed that the adsorption in the range of 0.5–
onously increasing absorption at high frequencies (Fig. 4a), 2.5 THz and the refractive index in the range of 0.5–1.0 THz
which is similar to that in the case of Avicel. Compared to the could be used for evaluating the change in the hydrogen
initial corncob, the residual corncob showed a higher absor- bonding strength of the cellulose materials during various
bance in the range of 0.5–2.5 THz. Fig. 4b shows the change in processing treatments. The increased adsorption and refractive
the refractive index of corncob upon enzymatic hydrolysis. The index indicated an increase in the hydrogen bonding strength.
refractive indexes of the initial and residual corncob were
stabilized at about 1.24 and 1.20 in the range of 0.5–1.0 THz,
respectively (as shown in the inset of Fig. 4b). A minor increase
in the refractive index of the residual corncob at low frequency
might be attributed to the removal of the low molecular-weight
or disordered glucan with weak hydrogen bonding strength. A
slight decrease in the refractive index of the residual corncob
at a high frequency might be attributed to lignin accumulation
upon enzymatic hydrolysis.44 In our previous study, we found
that the cellulose in the residual corncob possessed a
considerably higher Mw (1130 kDa) as compared to that (680
kDa) in the case of the initial corncob.27 The increased Mw
indicated that some long chain celluloses in the residual
substrate were still not attacked even aer 48 h of hydrolysis,
whereas the short chain cellulose was degraded preferentially
in this process. According to the THz-TDS and SEC-MALLS
FTIR analysis
To further conrm the feasibility of determining the hydrogen
bonding strength using THz-TDS, we employed FTIR to char-
acterize the samples to verify the above deduction.
Fig. 5 shows the FTIR spectra of Avicel samples before and 48
h aer the enzymatic hydrolysis over a wavenumber range of
4000–2800 cmꢀ1 and 1800–600 cmꢀ1. In general, the peaks at
3200–3600 cmꢀ1 were related to the O–H stretching vibration, in
which the adsorption at 3455–3410 cmꢀ1, 3375–3340 cmꢀ1, and
3310–3230 cmꢀ1 was attributed to the intramolecular O2–H/O6,
O3–H/O5, and intermolecular O6–H/O3 hydrogen bonds,
respectively.45 As shown in Fig. 5a and S1,† aer enzymatic
hydrolysis, the peaks at 3455–3410 cmꢀ1 and 3375–3340 cmꢀ1
(derived from the residual Avicel), shied to lower wave-
numbers and showed a stronger adsorption, indicating an
increase in the intramolecular hydrogen bonding strength
within cellulose, whereas the peak related to the intermolecular
Fig. 4 The absorption coefficient (a) and refractive index (b) of
corncob before (0 h) and after 48 h of enzymatic hydrolysis in the Fig. 5 FTIR spectra of Avicel before and after enzymatic hydrolysis in
range 0.1–2.5 THz at 293 K.
the range of (a) 3800–2800 cmꢀ1 and (b) 1800–600 cmꢀ1
.
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hydrogen bonding showed a weak absorption, which might be
resulting from the degradation of Avicel upon hydrolysis. On
the other hand, the peak at 897 cmꢀ1 originated from the
b-glycosidic linkages associated with C1–H deformation and
O–H bending. The bands 2920 cmꢀ1, 1372 cmꢀ1 and 1423 cmꢀ1
were assigned to the C–H stretching in the methylene groups,
aliphatic C–H stretching, and aromatic skeletal vibrations
combined with C–H in plane deformations, respectively.46,47
According to the empirical formula, the absorbance ratio of the
bands at 897 cmꢀ1 (Ar897), 1375 cmꢀ1 (Ar1375), and 1430 cmꢀ1
(Ar1430) to 2915 cmꢀ1 corresponded to the degree of hydrogen
bonds destroyed.13 As shown in Table 2, these parameters for
the residual Avicel decreased as compared to the original
substrate. These results indicated that the hydrogen bonding
strength in cellulose increased aer enzymatic hydrolysis. The
FTIR results were highly consistent with those obtained from
the THz analysis. In addition, the characteristic adsorption
peak of crystalline cellulose at 1430 cmꢀ1 became weaker for
Avicel aer enzymatic hydrolysis, suggesting that the crystalline
cellulose was partially degraded to glucose in this process.
Based on the THz and FTIR results, it was evident that the
enzymes could degrade part of cellulose but lead to an increase
in the accumulated hydrogen bonding strength for the residual
substrate.
Fig. 6 FTIR spectra of corncob before and after enzymatic hydrolysis
in the range of (a) 3800–2800 cmꢀ1 and (b) 1800–600 cmꢀ1
.
Fig. 6 shows the FTIR spectra of the corncob samples before
and 48 h aer the enzymatic hydrolysis. The infrared charac-
teristic peaks and attribution of lignocellulose are shown in
Table 3. The band 3420 cmꢀ1, which represented the strong bonding strength by FTIR technique. In other words, the FTIR
O–H stretching and bending vibrations of the intra- and inter- results provided some useful information in support of the
molecular hydrogen bonds in cellulose, shied to a lower observation that the THz-TDS technique could be an effective
wavelength of 3408 cmꢀ1 and yielded an increase in the inten- and credible method to evaluate the hydrogen bonding strength
sity aer enzymatic hydrolysis. It suggested an increase in the within cellulose.
intra- and intermolecular hydrogen bonding strength for the
The inter- and intramolecular hydrogen bonds in cellulose
residual corncob. The absorbance ratios, Ar897, Ar1375 and were highly related to the crystalline structure. As mentioned
Ar1430, were also used to evaluate the degree of hydrogen bond above, aer enzymatic hydrolysis, the residual cellulosic
destruction in corncob.13 As shown in Table 2, the decreased substrate had an increased hydrogen bonding strength,
values of Ar897 and Ar1375 indicated an increase in the hydrogen possibly due to the preferential degradation of cellulose with
bonding strength derived from the residual corncob, which was lower crystallinity and degrees of polymerization (DP). The
in accordance with the results from THz and SEC-MALLS change in the cellulose crystallinity and DP were interrelated
analysis.27 However, the value of Ar1430 in the corncob with the hydrogen bonding strength, which was an important
increased from 0.981 to 0.988, which may be attributed to the factor for understanding the enzymatic mechanism. The THz-
other functional groups from lignin or from the residual TDS analysis demonstrated in this study provided clear
hemicellulose. It showed the difficulty of measuring hydrogen evidence for describing the change in the hydrogen bonding
strength of cellulose, which was highly consistent with that in
FTIR and SEC-MALLS analysis,27 as well as with the results
reported in previous studies.31,48
Table 2 The value of Ar (the ratio of adsorption at wavenumber and
In this study, THz-TDS, as a quick and non-invasive
2920 cmꢀ1) before and after the enzymatic hydrolysis for Avicel and
measurement technique, provided a new method for detecting
corncob
the change in hydrogen bonding strength within cellulosic
substrates. Till date, the THz-TDS had been employed to char-
acterize the protein and bimolecular structures. Here, we
attempted to use this technique for evaluating hydrogen
bonding strength in cellulosic molecules for the rst time, to
our knowledge. Some key issues, such as the effects of lignin
and hemicellulose on the THz spectra, and the quantitative
Avicel
0 h
Corncob
0 h
48a h
48a h
Ar897
Ar1375
Ar1430
0.941
1.193
1.249
0.366
0.85
0.786
0.69
0.938
0.981
0.356
0.573
0.988
a
The sample aer enzymatic hydrolysis for 48 h.
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Table 3 The infrared characteristic peaks and attribution of lignocellulose46
Wavenumber (cmꢀ1
Pretreated corncob
)
Attribution of characteristic peak
Hydrolyzed-corncob
O–H stretching in hydroxyl groups
C–H stretching in methyl and methylene groups
C–H deformations (asymmetry in methyl groups, –CH3– and –CH2–)
Aromatic skeletal vibrations combined with C–H in plane deformations
Aliphatic C–H stretching in methyl and phenol OH
C–H out-of-plane in positions 2 and 6 (S units)
3420
2920
1462
1425
1373
896
3408
2920
1463
1423
1371
896
´
determination of the hydrogen bonding strength, need to be
examined in future studies.
7 M. Monrroy, I. Ortega, M. Ramırez, J. Baeza and J. Freer,
Enzyme Microb. Technol., 2011, 49, 472–477.
8 C. M. Mooney, S. D. Manseld and J. N. Saddler, Biotechnol.
Prog., 1999, 15, 804–816.
Conclusions
9 S. Park, J. O. Baker, M. E. Himmel, P. A. Parilla and
D. K. Johnson, Biotechnol. Biofuels, 2010, 3, 10.
10 R. Huang, R. Su, W. Qi and Z. He, Biotechnol. Prog., 2010, 26,
384–392.
11 B. Medronho, A. Romano, M. G. Miguel, L. Stigsson and
B. Lindman, Cellulose, 2012, 19, 581–587.
In summary, we have demonstrated the terahertz spectroscopy
as an efficient technique for directly evaluating the hydrogen
bonding strength within cellulose. The increased THz adsorp-
tion and refractive index provided a clear signal of an increase
in the hydrogen bonding strength in the residual Avicel and
corncob aer enzymatic hydrolysis. The THz results were
further compared with the XRD and FTIR analysis, as well as
with the SEC-MALLS results reported in our previous study. The
THz method demonstrated in this study opens a new avenue for
evaluating the change in the hydrogen bonding strength in
cellulose and thus provides some molecular information for
understanding the mechanism of cellulose degradation.
´
12 Y. Marechal and H. Chanzy, J. Mol. Struct., 2000, 523, 183–
196.
13 K. Kamide, K. Okajima and K. Kowsaka, Polym. J., 1992, 24,
71–86.
14 S. Y. Oh, D. I. Yoo, Y. Shin and G. Seo, Carbohydr. Res., 2005,
340, 417–428.
15 X. Colom and F. Carrillo, Eur. Polym. J., 2002, 38, 2225–2230.
16 R. Ponni, E. Kontturi and T. Vuorinen, Carbohydr. Polym.,
¨
2013, 93, 424–429.
17 M. Suchy, E. Kontturi and T. Vuorinen, Biomacromolecules,
2010, 11, 2161–2168.
18 M. Zhang, W. Qi, R. Liu, R. Su, S. Wu and Z. He, Biomass
Bioenergy, 2010, 34, 525–532.
19 M. Wada, L. Heux, Y. Nishiyama and P. Langan, Cellulose,
2009, 16, 943–957.
20 M. Hangyo, M. Tani and T. Nagashima, Int. J. Infrared
Millimeter Waves, 2005, 26, 1661–1690.
21 C. Yan, B. Yang and Z. C. Yu, Analyst, 2014, 139, 1967–1972.
22 A. Arora, T. Q. Luong, M. Kruger, Y. J. Kim, C. H. Nam,
A. Manz and M. Havenith, Analyst, 2012, 137, 575–579.
23 A. Redo-Sanchez, G. Salvatella, R. Galceran, E. Roldos,
J. A. Garcia-Reguero, M. Castellari and J. Tejada, Analyst,
2011, 136, 1733–1738.
24 Y. C. Shen, P. C. Upadhya, E. H. Lineld and A. G. Davies,
Appl. Phys. Lett., 2003, 82, 2350.
25 R. Liu, M. He, R. Su, Y. Yu, W. Qi and Z. He, Biochem. Biophys.
Res. Commun., 2010, 391, 862–867.
Acknowledgements
We acknowledge the nancial supports received from the
National Natural Science Foundation of China (no. 21276192),
the Ministry of Science and Technology of China (no.
2012BAD29B05 and 2013AA102204), Open Funding Project of
the State Key Laboratory of Chemical Engineering (no. SKL-ChE-
11B01), and the Ministry of Education (no. NCET-11-0372,
20110032130004, and B06006).
Notes and references
1 R. Huang, R. Su, W. Qi and Z. He, BioEnergy Res., 2011, 4,
225–245.
2 H. Jørgensen, J. B. Kristensen and C. Felby, Biofuels, Bioprod.
Bioren., 2007, 1, 119–134.
3 H. Chen and W. Qiu, Biotechnol. Adv., 2010, 28, 556–562.
4 S. P. S. Chundawat, G. Bellesia, N. Uppugundla, L. D. Sousa,
D. H. Gao, A. M. Cheh, U. P. Agarwal, C. M. Bianchetti,
G. N. Phillips, P. Langan, V. Balan, S. Gnanakaran and
B. E. Dale, J. Am. Chem. Soc., 2011, 133, 11163–11174.
5 L. T. Fan, Y.-H. Lee and D. H. Beardmore, Biotechnol. Bioeng.,
1980, 22, 177–199.
26 T. Bardon, R. K. May, P. F. Taday and M. Strlic, Analyst, 2013,
138, 4859–4869.
27 R. Y. Du, R. L. Huang, R. X. Su, M. J. Zhang, M. F. Wang,
J. F. Yang, W. Qi and Z. M. He, RSC Adv., 2013, 3, 1871–1877.
28 M. Zhang, R. Su, W. Qi, R. Du and Z. He, Chin. J. Chem. Eng.,
2011, 19, 773–778.
6 B. Yang and C. E. Wyman, Biotechnol. Bioeng., 2004, 86, 88–
98.
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 57945–57952 | 57951

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RSC Advances
Paper
29 L. Segal, J. J. Creely, A. E. Martin and C. M. Conrad, Text. Res. 39 J. Xu, K. W. Plaxco and S. J. Allen, Protein Sci., 2006, 15, 1175–
J., 1959, 29, 786–794. 1181.
30 M. Hall, P. Bansal, J. H. Lee, M. J. Realff and 40 J. Y. Chen, J. R. Knab, J. Cerne and A. G. Markelz, Phys. Rev. E:
A. S. Bommarius, FEBS J., 2010, 277, 1571–1582. Stat., Nonlinear, So Matter Phys., 2005, 72, 040901.
31 L. Wang, Y. Zhang, P. Gao, D. Shi, H. Liu and H. Gao, 41 A. M. Smith, R. J. Williams, C. Tang, P. Coppo, R. F. Collins,
Biotechnol. Bioeng., 2006, 93, 443–456.
32 S. Yang, J. Li, Z. Zheng and Z. Meng, Int. Biodeterior.
Biodegrad., 2009, 63, 569–575.
33 L. Hao, R. Wang, L. Zhang, K. Fang, Y. Men, Z. Qi, P. Jiao,
J. Tian and J. Liu, Cellulose, 2013, 21, 777–789.
34 H. Kargarzadeh, I. Ahmad, I. Abdullah, A. Dufresne,
M. L. Turner, A. Saiani and R. V. Ulijn, Adv. Mater., 2008, 20,
37–41.
42 R. Huang, S. Wu, A. Li and Z. Li, J. Mater. Chem. A, 2014, 2,
1672–1676.
43 Y. Lu, G. Zhang, M. Feng, Y. Zhang, M. Yang and D. Shen, J.
Polym. Sci., Part B: Polym. Phys., 2003, 41, 2313–2321.
S. Y. Zainudin and R. M. Sheltami, Cellulose, 2012, 19, 44 M. Walther, B. M. Fischer and P. Uhd Jepsen, Chem. Phys.,
855–866. 2003, 288, 261–268.
35 H. Zhao, J. Kwak, Z. Conradzhang, H. Brown, B. Arey and 45 S. Y. Oh, D. I. Yoo, Y. Shin, H. C. Kim, H. Y. Kim, Y. S. Chung,
J. Holladay, Carbohydr. Polym., 2007, 68, 235–241.
36 S. Kim and M. T. Holtzapple, Bioresour. Technol., 2006, 97,
583–591.
W. H. Park and J. H. Youk, Carbohydr. Res., 2005, 340, 2376–
2391.
46 T. Kondo and C. Sawatari, Polymer, 1996, 37, 393–399.
37 L. Zhu, J. P. O'Dwyer, V. S. Chang, C. B. Granda and 47 A. Bazargan, J. Tan, C. W. Hui and G. McKay, Cellulose, 2014,
M. T. Holtzapple, Bioresour. Technol., 2008, 99, 3817–3828. 21, 1679–1688.
38 H. Yoneyama, M. Yamashita, S. Kasai, K. Kawase, R. Ueno, 48 Z. Liao, Z. Huang, H. Hu, Y. Zhang and Y. Tan, Bioresour.
H. Ito and T. Ouchi, Phys. Med. Biol., 2008, 53, 3543. Technol., 2011, 102, 7953–7958.
57952 | RSC Adv., 2014, 4, 57945–57952
This journal is © The Royal Society of Chemistry 2014