Bioresource Technology xxx (2015) xxxxxx

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Characterization of natural low transition temperature mixtures

(LTTMs): Green solvents for biomass delignication
Chung Loong Yiin a, Armando T. Quitain b, Suzana Yusup a,, Mitsuru Sasaki b, Yoshimitsu Uemura a,
Tetsuya Kida b
a
Biomass Processing Technology Cluster, Center of Biofuel and Biochemical Research, Mission Oriented Research (Green Technology), Chemical Engineering Department,
Universiti Teknologi PETRONAS, Bandar Seri Iskandar, 32610 Tronoh, Perak, Malaysia
b
Department of Applied Chemistry and Biochemistry, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Cheap and commercially available

starting materials.
 Enhancement of lignin solubility
capacity with the presence of water.
 Pretreatment of real biomass was
consistent with biopolymers
solubility of LTTMs.
 Physicochemical properties of LTTMs
were not affected by the presence of
water.
 Green alternative in utilizing the
microwave hydrothermal extracted
malic acid.

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this work was to characterize the natural low transition temperature mixtures (LTTMs) as
Received 22 June 2015 promising green solvents for biomass pretreatment with the critical characteristics of cheap, biodegrad-
Received in revised form 26 July 2015 able and renewable, which overcome the limitations of ionic liquids (ILs). The LTTMs were derived from
Accepted 27 July 2015
inexpensive commercially available hydrogen bond acceptor (HBA) and L-malic acid as the hydrogen
Available online xxxx
bond donor (HBD) in distinct molar ratios of starting materials and water. The peaks involved in the
H-bonding shifted and became broader for the OH groups. The thermal properties of the LTTMs were
Keywords:
not affected by water while the biopolymers solubility capacity of LTTMs was improved with the
Low transition temperature mixture (LTTM)
Water
increased molar ratio of water and treatment temperature. The pretreatment of oil palm biomass was
Physicochemical property consistence with the screening on solubility of biopolymers. This work provides a cost-effective alterna-
Biopolymer solubility tive to utilize microwave hydrothermal extracted green solvents such as malic acid from natural fruits
Pretreatment and plants.
2015 Elsevier Ltd. All rights reserved.

1. Introduction Nilsen et al. (2007) highlighted that unlike other renewable

The global depletion of fossil fuels such as crude oil and natural
gas will leave us with just renewable energy sources to rely on.
Corresponding author. Tel.: +60 53687642; fax: +60 53688204.
E-mail addresses: ychungloong2009@gmail.com (C.L. Yiin), quitain@
kumamoto-u.ac.jp (A.T. Quitain), drsuzana_yusuf@petronas.com.my (S. Yusup),
msasaki@kumamoto-u.ac.jp (M. Sasaki), yoshimitsu_uemura@petronas.com.my
(Y. Uemura), tetsuya@kumamoto-u.ac.jp (T. Kida).
sources of energy such as solar and wind, which merely generates
heat and power, biomass can be converted into liquid, solid and
gaseous fuels. A lignocellulosic biomass contains 3550% cellulose,
35% hemicelluloses, 530% lignin and a few percentages of extrac-
tives. Cellulose is the most abundant biopolymer on earth, thus it is
a valuable source of raw materials for chemical and fuel industries
(Maki-Arvela et al., 2010). An effective separation of these compo-
nents and their subsequent fractionation are required for the

http://dx.doi.org/10.1016/j.biortech.2015.07.103
0960-8524/ 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Yiin, C.L., et al. Characterization of natural low transition temperature mixtures (LTTMs): Green solvents for biomass
delignication. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.07.103
2 C.L. Yiin et al. / Bioresource Technology xxx (2015) xxxxxx
production of high-value products from lignocellulosic biomass
(Hamzeh et al., 2013).
Various technologies for saccharication of lignocellulosic bio-
mass have evolved over the years. Numerous conventional meth-
ods for dissociating biomass into cellulose, hemicelluloses and
lignin bioproducts have been investigated such as dilute acid
hydrolysis, alkaline (Girio et al., 2010), hydrothermal steam explo-
sion (Ruiz et al., 2008) and autohydrolysis (Carvalheiro et al.,
2009). In addition, Morais et al. (2015) presented the role of super-
critical CO2 as a media for pretreatment to improve the enzymatic
digestibility to convert lignocellulosic or starch biomass into biofu-
els and biochemicals. Current processes to convert lignocellulosic
biomass into useful raw materials are still considered inefcient
and making the process cost competitive remains a big challenge.
Some ionic liquids (ILs) were found to triumph as solvents for
the applications in separation and extraction (Lateef et al., 2008).
ILs initially still show restrictions in terms of recoverability, cost
for large scale applications and toxicity (Ventura et al., 2013;
Egorova and Ananikov, 2014). However, the recoverability and
commercialization potential of ILs had been evaluated and
improved recently (da Costa Lopes et al., 2013; Chen et al., 2014).
On the other hand, new green approaches towards the substitution
of volatile organic solvents were taken into consideration. Deep
eutectic solvents (DESs) were suggested by Abbott et al. as suit-
able alternatives for ILs (Abbott et al., 2004). The DESs are synthe-
sized by mixing two components to form easily biodegradable
products without additional solvent or formation of by-products
(Cvjetko Bubalo et al., 2014). DESs are sometimes referred to ana-
logs of ILs (Lloyd et al., 2013) even though they could not be
dened as real ILs as they are not completely comprised of ionic
species and are connected by a hydrogen bonding network.
Instead of DES, in recent years, Francisco et al. (2012) intro-
duced low transition temperature mixtures (LTTMs) as most of
the DESs show glass transition points instead of eutectic melting
points. LTTMs have advantageous qualities such as wide liquid
range, water compatibility, low vapor pressure, non-ammability,
biocompatibility and biodegradability. LTTMs consist of at least
one hydrogen bond donor (HBD) and one hydrogen bond acceptor
(HBA) counterpart that result in the formation of liquid mixture
showing an unusually low freezing point. Natural products are
the ideal source of DESs due to their biodegradable properties,
enormous chemical diversity and pharmaceutically acceptable tox-
icity prole (Dai et al., 2013). The proton afnity (PA)/pKa equaliza-
tion plays a role in strengthening of the H-bond, so the pKa slide
rule was taken into account in the selection of the H-bonding coun-
terparts (Gilli et al., 2009). The potentials of these LTTMs to
degrade the lignocellulosic biomass structure were evaluated
(Francisco et al., 2012). Fewer biomass particles could be noticed
after the pretreatment with malic acidproline mixtures. The mix-
tures prepared from malic acid as the hydrogen bond donor show
much higher solubility in lignin when comparing with other
hydrogen bond donors (Francisco et al., 2012). Hence, natural
abundant resources such as plants and fruits with high malic acid
content have a high potential to be used for the synthesis of LTTMs.
Hydrothermal extraction is a natural and green method using
water at high temperature and pressure condition as extraction
solvent. It has been used to extract polar organic compounds or
to decompose lignocellulosic materials to obtain precious compo-
nents such as saccharides and aromatic organic acids. Microwave
assisted extraction (MAE) has been proven to be a promising tech-
nique for the extraction of phytochemicals from botanicals (Dhobi
et al., 2009; Mandal et al., 2007). This mode of heating can reduce
residence times, increase reaction rates and provide more accurate
control of reaction conditions (Tsubaki et al., 2012) as it provides a
Please cite this article in press as: Yiin, C.L., et al. Characterization of natural low transition temperature mixtures (LTTMs): Green solvents for biomass
delignication. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.07.103
more uniform method of heating with the rotation of dipolar mole-
cules and vibration of ions in solution in an electromagnetic eld.
Thus, removal of water from the extracted solvents is not neces-
sary if it can be proved that the water will bring no effect to the
properties of LTTMs. This helps to save the energy and time con-
sumption for the synthesis of natural and biorenewable green sol-
vents from microwave hydrothermal extracted malic acid.
The aim of this work was to study the effects of molar ratios of
starting materials and water in the preparation of LTTMs by using
L-malic acid as HBD and cheap commercially available materials
(sucrose, monosodium glutamate) as HBA. The physicochemical
properties of the LTTMs such as hydrogen bonding, thermal stabil-
ity, water content and biopolymers solubility were analyzed and
discussed. Preliminary experiment was performed to examine
the solubility of grinded empty fruit bunches (EFBs) from an oil
palm mill in the wet-synthesized LTTM. Additionally, the recover-
ability of LTTM after the extraction of lignin was also considered.
2. Methods
2.1. Raw materials and chemicals
Starting materials for the preparation of LTTMs such as L-malic
acid (P99 wt%) and sucrose (>97 wt%) were obtained from Wako
Pure Chemical Industries Ltd. while the monosodium glutamate
(MSG) with stated purity (P98 wt%) was commercially available.
Cellulose microcrystalline (90%) and starch (corn) were pur-
chased from Merck, Germany and Wako Pure Chemical Industries
Ltd. respectively for the screening of biopolymers solubility. The
lignin (96%, alkali lignin) was obtained from Tokyo Kasei Kogyo
Co., Ltd.
Empty fruit bunches (EFBs) were supplied by palm tree planta-
tion area Kilang Sawit FELCRA Berhad Nasaruddin, KM 37, Jalan
Tronoh, 32,600 Bota, Perak, Malaysia. These EFBs were washed
and dried in an oven at 100 C for 3 days. The dried EFBs were
chopped and cut into smaller pieces. The dried EFBs were then
grounded with FRITSCH Cutting Mill to particle size below
500 lm. The grinded EFBs were sieved to get the desired particle
size of <500 lm. Ultimate analysis of the EFBs were carried out
using LECO 932 CHNS Analyzer. The carbon, hydrogen, nitrogen,
sulphur and oxygen (by difference) content of EFBs were 46.83,
6.28, 0.66, 0.24 and 45.99 wt%, respectively. Higher Heating
Value (HHV) of 19.64 MJ/kg was obtained for the EFBs using IKA
C5000 Bomb Calorimeter.
2.2. Preparation of LTTMs
Starting materials (HBD and HBA) with 1:1 molar ratio were
mixed thoroughly in a three-neck round bottom ask with mag-
netic stirring to promote contact between the solid crystals. The
ask was heated up slowly in an oil bath. The heating process
was stopped as the materials start to melt. The reaction tempera-
ture and time taken to synthesize the LTTMs were recorded. The
experiment was repeated at different molar ratios of starting mate-
rials. The optimum molar ratio of starting materials was used for
the preparation of LTTMs in various amounts of distilled water. 1,
2 and 10 molar ratios of distilled water to starting materials were
added to the mixtures separately in order to study the effects of
water content in the preparation of LTTMs. The excessive water
content in the LTTMs was removed by freeze-drying (EYELA
FDU-1200). Its auto-mode function started the vacuum pump oper-
ation after the trap temperature was cooled to 45 C. The drying
process was stopped once the samples shown constant weight.
 C.L. Yiin et al. / Bioresource Technology xxx (2015) xxxxxx
2.3. Physicochemical properties tests
Fourier transform infrared spectroscopy (FTIR) spectra of the
starting materials and the LTTMs were analyzed using Frontier
FT-IR Spectrometer (PerkinElmer) equipped with attenuated total
reectance (ATR). The data collected was compared in order to
ensure the existence of hydrogen bond between the starting mate-
rials. Thermogravimetric analysis (TGA) was performed using
EXSTAR 6000 TG/DTA6300 from 35 C to 300 C with heating rate
of 20 C /min. The carrier gas used was nitrogen with ow rate of
250 ml/min. Differential scanning calorimetry (DSC) curve was
recorded using EXSTAR 6000 DSC6100 from 75 C to 10 C by
using an electrical cooling system at a rate of 2 C/min. The heat
adjustment process was conducted in nitrogen with ow rate of
70 ml/min. The water content of the freeze-dried LTTMs was mea-
sured using Methohm 870 KF Titrino Plus.
2.4. Biopolymers solubility tests
The solubility of biopolymers (lignin, starch, cellulose) was
determined by cloud point method, following a similar procedure
previously reported (Francisco et al., 2012). Vials containing 2 g
of LTTMs were placed into an oil bath at a constant temperature
of 60 C. A 0.21 mg of sample was added continuously with vigor-
ous stirring. After complete dissolution of sample in LTTMs was
observed, the sample was kept for 24 h. The solubility (wt%) was
registered if the solutes did not dissolve into the solvents. The
experiment was repeated in an oil bath at a consistent temperature
of 100 C.
2.5. Pretreatment of grinded empty fruit bunches (EFBs) and LTTM
regeneration screening
300 mg of grinded EFBs were added to 3 g of LTTM in a bottle
and heated at 100 C in an oil bath with magnetic stirring for
24 h. After cooling the EFBs-LTTM mixture to room temperature,
the mixture was centrifuged after three washing cycles with etha-
nol (>95%) The supernatant was then separated from the precipi-
tate using lter paper (advantec grade 5C ashless quantitative,
<5 lm). The ethanol was removed from the ltered mixture
through rotary evaporator.
3. Results and discussion
3.1. Effects of starting materials molar ratio in the preparation of
LTTMs
Dai et al. (2013) stated distinct ratios of the components of nat-
ural deep eutectic solvent (NADES) may affect stability of NADES in
terms of the mixture remaining in the liquid phase for a prolonged
period. The physicochemical properties of LTTMs prepared at dif-
ferent molar ratios of starting materials were evaluated.
In this study, the molar ratios of HBD to HBA used were 1:1, 1:2
and 1:3. Nevertheless, the aforementioned ratios were only appli-
cable to the condition of sucrose as HBA. The combination of malic
acid and MSG at these molar ratios produced hard solid mixtures.
Thus, a trial experiment with an increased ratio of HBD instead of
HBA (1:1, 2:1, 3:1) was performed but only the molar ratio of 3:1
formed liquid mixtures. The color of LTTMs turned darker with the
increased ratio of sucrose while light yellowish color LTTMs was
synthesized with the molar ratio of 3:1 of malic acid and MSG.
The temperature and time taken for the preparation of LTTMs
without addition of water: sucrose (90 C, 10 min) and MSG
(110 C, 30 min). The melting point of mixtures was lower com-
pared to the raw materials due to the occurrence of charge
Please cite this article in press as: Yiin, C.L., et al. Characterization of natural low transition temperature mixtures (LTTMs): Green solvents for biomass
delignication. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.07.103
3
delocalization (Carriazo et al., 2012). The dark color of the LTTMs
was presumably due to the caramelization of sucrose at high tem-
perature. Both sucrose and MSG were compatible with malic acid
as the LTTMs formed remained in a liquid state when exposed to
room temperature. The hydrogen bonding and van der Waals inter-
actions prevented crystallization of the starting materials, thus
resulting in the mixtures remaining in the liquid state (Francisco
et al., 2013). FTIR analysis was conducted in order to prove the
existence of hydrogen bond in the starting materials, which pre-
vented the LTTMs from crystallization.
3.1.1. FTIR spectra analysis
FTIR spectrometer simultaneously collects spectral data in a
wide spectral range, which enabled a signicant advantage in mea-
suring intensity over a narrow range of wavelengths at a time.
Francisco et al. (2012) stated that the existence of hydrogen bond-
ing can be veried by the shifts in the representative peaks of the
involved bonds in the FTIR spectra.
Based on the FTIR spectra, the representative peak of carboxylic
acid group of malic acid with wavenumber 1710 cm 1 for the C@O
group broaden in all the LTTMs due to the hydrogen bonding. The
hydroxyl group of malic acid had direct interaction with the car-
bonyl group by the formation of a stable dimetric
hydrogen-bonded structure in liquid phase. In addition, MS 1:1
had the broadest peak followed by MS 1:2 and MS 1:3. This was
probably due to the increased ratio of sucrose, which demanded
more C@O group for the formation of hydrogen bond and reduced
the peak.
Besides, the peaks involved in the H-bonding responsible for the
formation of the LTTM were shifted and broaden within the
wavenumber range of 25003700 cm 1 and 22003700 cm 1 con-
sequently, for the OH groups due to the stretching absorption of
hydrogen bonding. The results were comparable with the works
of Francisco et al. based on the hydrogen bonding existed between
malic acid and proline as observed by FTIR analyses (Francisco
et al., 2012). The LTTMs synthesized in the aforementioned molar
ratios of starting materials revealed the spectra which were in
vicinity to each other. Hence, further study such as thermal stabil-
ity is crucial in studying the effects of increased ratio of HBD and
HBA. This test enables the evaluation of the optimum temperature
of LTTMs in delignifying the lignocellulosic biomass.
3.1.2. TG/DTA analysis
The thermal behavior of LTTMs was studied using thermogravi-
metric analysis (TGA). LTTMs have critical drawbacks related to
their thermal stability. The thermal stability of LTTMs is not always
proportional to the thermal stability of the starting materials due
to the presence of hydrogen bonding and electrostatic interactions
(Francisco et al., 2013).
As it can be seen in Fig. 1a, the LTTMs synthesized from sucrose
and malic acid (MS 1:1, MS 1:2, MS 1:3) started to degrade at 400 K
(127 C), 405 K (132 C) and 420 K (147 C) respectively. The LTTMs
had lower degradation temperature compared to the starting
materials. The results were analogous to the outputs of Francisco
et al. in which not all the LTTMs had higher thermal stability com-
pared to the starting materials (Francisco et al., 2013).
In addition, MS 1:3 had the highest degradation temperature
compared to MS 1:2 and MS 1:1. This was to be presumed that
MS 1:3 had more hydrogen bonds to be formed due to the presence
of extra hydroxyl groups. Similar ndings were reported elsewhere
(Dai et al., 2013). Asymmetrically, Fig. 1b shows the LTTMs (MMSG
3:1) decomposed at 405 K (132 C) which also had a lower degra-
dation temperature than raw materials. Besides, the LTTMs with
MSG as HBA decomposed at the higher temperatures compared
to the LTTM with sucrose as HBA (MS 1:1).
4 C.L. Yiin et al. / Bioresource Technology xxx (2015) xxxxxx

(a) 100
(b) 100

80 80

Weight (%)
Weight (%)

60 60

40 MS 1:1 40
MS 1:2 MMSG 3:1
MS 1:3
20 20 Malic acid
Malic acid
Monosodium Glutamate (MSG)
Sucrose
0 0
350 400 450 500 550 600 350 400 450 500 550 600
Temperature (K) Temperature (K)
(c) 100 (d) 100

80 80

Weight (%)
Weight (%)

60 60

MSW 1:1:1 MMSGW 3:1:1

40 40 MMSGW 3:1:2
MSW 1:1:2
MSW 1:1:10 MMSGW 3:1:10
20 Malic acid 20 Malic acid

Sucrose Monosodium Glutamate (MSG)

0 0
350 400 450 500 550 600 350 400 450 500 550 600
Temperature (K) Temperature (K)

Fig. 1. TGA curves. (a) Malic acid, sucrose and LTTMs (MS 1:1, MS 1:2, MS 1:3); (b) Malic acid, MSG and LTTMs (MMSG 3:1); (c) Malic acid, sucrose and LTTMs (MSW 1:1:1,
MSW 1:1:2, MSW 1:1:10); (d) Malic acid, MSG and LTTMs (MMSGW 3:1:1, MMSGW 3:1:2, MMSGW 3:1:10); the vertical line indicates the transition temperature of the
constituents.

3.2. Effects of water in the preparation of LTTMs
Based on the studies in ILs, addition of a small amount of water
can alter their physical behavior (Koddermann et al., 2006) and
degree of solubility (Zakrzewska et al., 2010). Following the key
discoveries of Francisco et al. (2013) and Dai et al. (2013), water
can form part of the mixtures produced as it impedes with the
coordination sphere of the ions, which assisting the interactions
and entropic state. In this study, different molar ratios of water
were added in the preparation of LTTMs in order to study its effects
on the physicochemical properties of mixtures produced.
Transparent color LTTMs were formed in the molar ratio of 1:1
for malic acid and sucrose while light yellowish color LTTMs were
synthesized with the molar ratio of 3:1 for malic acid and MSG.
Addition of water had reduced the temperature for the preparation
of LTTMs from sucrose and MSG to 60 C and 90 C while the reac-
tion time was shortened to 5 min and 15 min respectively. This
was proven by Dai et al. (2013), adding a small amount of water
to a mixture reduced preparation time, temperature and viscosity.
The heating process was stopped once clear mixtures formed. The
LTTMs with MSG as HBA required higher preparation temperature
was probably due to its higher melting point. The excessive water
content in the LTTMs was removed by using a freeze drier.
On top of that, introduction of water also prevented the
caramelization of sucrose at high temperature and yielded clear
LTTMs. Further analyses such as FTIR, TG/DTA, DSC and biopoly-
mers solubility were performed and discussed to prove the water
which remained in the LTTMs after freeze drying process had no
effect on the physicochemical properties of LTTMs.
3.2.1. FTIR spectra analysis
It can be seen in the FTIR spectra obtained that the broad band
with strong intensity in the wavenumber region of 2500
3700 cm 1 and 22003700 cm 1 respectively were assigned to
the OH bond stretching vibration of the alcohol groups. Besides,
the broaden peak at 1710 cm 1 was attributed to the stretching
vibration of C@O group. In accordance with the literature
(Francisco et al., 2012), these peaks indicated the existence of
hydrogen bond in the mixtures. Both spectra for the LTTMs pre-
pared from sucrose and MSG as HBA appeared very similar with
the increased molar ratio of water in the preparation of LTTMs.
Taking a closer inspection to the FTIR spectra, they revealed slight
differences in terms of band intensities within the wavenumber of
25003700 cm 1 and 22003700 cm 1 correspondingly. This phe-
nomenon might be due to the increased ratio of symmetric and
asymmetric H2O stretching vibrations.
Removal of excessive water content from the LTTMs through
freeze drying was crucial as hydration of compounds has an enor-
mous consequence on the spectrum and generally produces a lot of
complexity in the form of additional absorption bands and struc-
ture to the existing band.
3.2.2. Thermal behavior of LTTMs
3.2.2.1. TG/DTA analysis. The thermal decomposition of LTTMs syn-
thesized in the presence of water was studied to determine if this
combination led to the changing of degradation temperature. The
comparison of the TGA curves of LTTMs with different molar ratios
of water is illustrated in Fig. 1. Based on Fig. 1c and d, the TGA
curves of LTTMs derived from sucrose and MSG are almost identi-
cal to each other with the degradation temperature of 400 K
(127 C) and 405 K (132 C) respectively. There was no reduction
of mass at 373 K (100 C) which indicated the remaining water in
the LTTMs formed part of the solvent itself. In addition, their ther-
mal behaviors were similar to the LTTMs without addition of water
during the preparation where the starting materials had higher
degradation temperature than the LTTMs. This was presumably

Please cite this article in press as: Yiin, C.L., et al. Characterization of natural low transition temperature mixtures (LTTMs): Green solvents for biomass
delignication. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.07.103
 C.L. Yiin et al. / Bioresource Technology xxx (2015) xxxxxx 5

20 On the other hand, no thermal circumstances were noticed for

the LTTMs synthesized from MSG and malic acid except for the
18
MMSGW 3:1:10 alteration in the baseline at 235 K ( 38 C). This phenomenon
16 was similar with a material that undergone glass transition but
Heat Flow (W/100 g)

14 with no crystallization upon heating (Dai et al., 2013; Singh

12
10
8
6 seen in Fig. 2, the LTTMs synthesized at different molar ratios of
4
2 MSW 1:3:10
0 properties. Besides, MSW 1:1:10 had a glass transition tempera-
205 215 225
Temperature (K)
Fig. 2. DSC curves for mixtures of malic acidsucrosewater (MSW 1:1:2, MSW
1:1:10, MSW 1:3:10) and malic acidMSGwater (MMSGW 3:1:10)
due to the possible scattered structure during the preparation of
LTTMs that led to the existence of high amorphous fraction and
further reduced the heat-resistivity of LTTMs to decompose at a
lower temperature (Ramesh et al., 2012).
Water is one of the few abundant natural substances that act
both as donor and acceptor due to its high polarity. Thus, water
was assumed to form part of the LTTMs without affecting their
properties (Francisco et al., 2013). It is critical for LTTMs to have
stable degradation temperature after the addition of water as ther-
mal properties regulate the range of temperature at which the
LTTMs can sustain their liquid state and its range of function. In
this context, the maximum temperature used for the study of
biopolymers solubility was based on the degradation temperature
of LTTMs. It was used as the treatment temperature for the pre-
treatment of oil palm biomass sample using LTTMs.
3.2.2.2. DSC analysis. DSC analysis was conducted in order to deter-
mine the phase transition temperature of the LTTMs. The forma-
tion of supramolecular complexes in the mixtures reoriented the
free energy of the solid phase compared to that of the liquid and
resulted in the depression of freezing point to below 0 C
(Hayyan et al., 2012). It is seen in Fig. 2 that the mixtures derived
from sucrose and MSG with water presented glass transition tem-
perature at 230 K ( 43 C) and 235 K ( 38 C) respectively, which
conrmed that these mixtures were supramolecular complexes
with the liquid state over a wide temperature range (Dai et al.,
2013; Florindo et al., 2014). Nonetheless, the LTTMs derived from
sucrose and malic acid showed glass formation, crystallization
and melting point. This result is in agreement with Choi et al.
(2012) that their DSC analysis of the mixtures MS 2:1 had the same
thermal behavior.
et al., 2012; Siongco et al., 2013). Nucleation and crystal growth
during heating were hindered by the high viscosity of liquid at
room temperature (Francisco et al., 2012). Mixtures with this trend
of thermal behavior are known as low transition temperature mix-
tures (LTTMs) (Francisco et al., 2012, 2013). Additionally, as can be
MSW 1:1:2
MSW 1:1:10 water had similar glass transition temperature at 230 K ( 43 C)
when the mixture was heated from 75 C to 10 C. This indicated
that the addition of water had no consequences on the transition
235 245 ture of 230 K ( 43 C) and as the molar ratio of sucrose increased,
the glass transition temperature decreased to 220 K ( 53 C) for
MSW 1:3:10. Similar behavior was reported for the systems of
other LTTMs (Francisco et al., 2012).
The preparation method is very crucial in the formation of
molecular complexes in the liquid phase. The method reported
by Francisco et al. (2013) involved mixing of both the starting
materials in the solid state at room temperature to promote con-
tact between the solid crystals. The vessel containing the mixtures
was sealed and heated up slowly with continuous stirring until a
clear liquid was formed. An alternative novel preparation method
was proposed in this work where both of the HBD and HBA were
mixed with water. The three neck round ask containing the mix-
tures was heated up and stirred in an oil bath. The heating process
was stopped when all the solutes dissolved in the water. The exces-
sive water content of the mixtures was removed by using a freeze
drier. The LTTMs were produced at lower temperature and reduced
time. The main purpose for preparing the LTTMs in water is to
replace the commercial malic acid with microwave hydrothermal
extracted malic acids from natural fruits and plants such as papaya,
cactus, lophatherum herb and luffa cylindrica which contain a high
percentage of malic acid. It is not necessary to remove all the water
through microwave hydrothermal extracted malic acid before it
can be used as a HBD if it is proven that the addition of water dur-
ing the preparation of LTTMs has no effect on their physicochemi-
cal properties.
3.2.3. Water content analysis and screening of biopolymers solubility
The water content of freeze dried LTTMs were proportional to
the increased molar ratio of water as depicted in Table 1. The water
which remained in the LTTMs was assumed to form part of the sol-
vent itself through stronger intermolecular bonds (Francisco et al.,
2013). Besides, the screening is very vital to estimate the capability
of LTTMs to extract the lignin from lignocellulosic biomass. The
most abundant renewable polymers in lignocellulosic biomass
are cellulose and lignin while starch is used to replace the polysac-
charide in our study (Francisco et al., 2012). An overview of the
results of screening is given in Table 1, showing all mixtures had

Table 1
Water content and biopolymers solubility of the LTTMs at different molar ratio of starting materials and water.

Name Water content (wt%) Ttest (C) Lignin (wt%) Starch (wt%) Cellulose (wt%)
MSW 1:1:1 1.88 60 6.38 0.16 0.00
MSW 1:1:2 3.61 60 7.42 0.25 0.11
MSW 1:1:10 7.36 60 9.16 0.31 0.15
MSW 1:2:10 7.54 60 10.58 0.92 0.45
MSW 1:3:10 7.89 60 12.02 2.25 0.63
MMSGW 3:1:1 1.17 60 5.33 0.95 0.16
MMSGW 3:1:2 2.64 60 6.02 1.03 0.19
MMSGW 3:1:10 6.82 60 7.69 1.11 0.25

Please cite this article in press as: Yiin, C.L., et al. Characterization of natural low transition temperature mixtures (LTTMs): Green solvents for biomass
delignication. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.07.103
6 C.L. Yiin et al. / Bioresource Technology xxx (2015) xxxxxx

(a) (b)
16 10
60 60

Solubility of lignin (wt %)

Solubility of lignin (wt %) 14
100 100
8
12

10
6
8

6 4

4
2
2

0 0

Fig. 3. Solubility of lignin in (a) malic acidsucrosewater (MSW 1:1:1, MSW 1:1:2, MSW 1:1:10, MSW 1:2:10, MSW 1:3:10) and (b) malic acidMSGwater (MMSGW 3:1:1,
MMSGW 3:1:2, MMSG 3:1:10). The data is expressed in standard deviation.

higher solubility for lignin compared to starch and cellulose. The
solubility of cellulose was found to be very poor for most of the sol-
vents, which formed opaque liquid after stirring for 24 h. Solvents
with high selectivity are preferable for separating lignin from cel-
lulose and starch (Francisco et al., 2012). Thus the mixtures had
a great potential to be used for the delignication of lignocellulosic
biomass.
In addition, Table 1 shows the ability of mixtures in dissolving
the biopolymers improved with the increased molar ratio of HBA.
Lignin solubility increased in the order of MSW 1:1:10 < MSW
1:2:10 < MSW 1:3:10, which corresponds to the increasing molar
ratio of sucrose. This was probably due to the alteration of solubil-
ity mechanisms of LTTMs with the increment of HBA ratio.
However, a high-resolution magic angle spinning (HR-MAS) NMR
analysis needs to be performed in order to acquire more fore-
knowledge into the solubilizing mechanism of the mixtures (Dai
et al., 2013). On the contrary, the solubilitys efciency of LTTMs
was enhanced with the increased molar ratio of water which
shows the trend of MSW 1:1:1 < MSW 1:1:2 < MSW 1:1:10.
Moreover, the mixtures synthesized from MSG and malic acid
had an identical solubility behavior with the trend of MMSGW
3:1:1 < MMSG 3:1:2 < MMSG 3:1:10. Therefore, the solubility
capacity of the solvents was tuned accordingly to the amount of
water added during preparation. At the same time, an appropriate
amount of water should be taken into consideration for the prepa-
ration of LTTMs as extended dilution would result in the loss of
existing hydrogen bonds and resulted in the rupture of unique
structures in mixtures (Gutierrez et al., 2009, 2010).
3.2.3.1. Effect of treatment temperature on lignin solubility. The treat-
ment temperature was increased from 60 C to 100 C in order to
determine the optimum temperature for the study of delignica-
tion of lignocellulosic biomass. Fig. 3 illustrates the solubility
capacity of LTTMs increased at higher temperature. This phe-
nomenon was discovered by Dai et al. (2013) in which solubility
of compounds in mixtures intensely affected by temperature.
Nevertheless, no further increase of the treatment temperature at
more than 100 C since degradation of mixtures occurred.
3.2.4. Pretreatment of real biomass
Preliminary experiment was performed to test the solubility of
grinded EFBs from oil palm mill. Grinded EFBs was suspended in
the MSW 1:3:10. The wet-synthesized LTTM turned into dark color
upon stirring the biomass suspension for 24 h at 100 C.
Nonetheless, the biomass particles were still visible in the LTTM
after the pretreatment. Besides, ethanol was proved to act as an
anti-solvent for the recoverability of LTTM. 92 wt% of the
non-dissolved biomass could be recovered after three washing
cycles with ethanol. On the other hand, 6.2 wt% and 1.8 wt% of
the added biomass was recovered from the separated precipitate
and supernatant respectively. In this context, 1.8 wt% of the recov-
ered biomass indicates the removal of 1.8 wt% lignin from the EFBs.
4. Conclusions
Novel LTTMs were prepared from cheap materials such as malic
acid, sucrose and MSG. The lignin solubility capacity and degrada-
tion temperature of LTTMs were improved with the increased
molar ratio of HBA. It has been demonstrated that the addition of
water enhanced the lignin solubility capacity without affecting
the thermal behavior of LTTMs. The LTTM had the ability of dis-
solving grinded EFBs as reected in the change of solvents color.
This work overcomes the restrictions of ILs by utilizing microwave
hydrothermal extracted malic acid from fruits and plants for the
synthesis of natural and biorenewable green solvents, LTTMs.
Acknowledgements
The authors would like to acknowledge Kumamoto University
and Universiti Teknologi PETRONAS for the support given to under-
take the research work. The funding support from Japan Society for
the promotion of Science (JSPS) Bilateral Joint Research Program
and Fundamental Research Grant Scheme (FRGS) are also grate-
fully acknowledged.
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