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Materials Today: Proceedings 5 (2018) 23128–23135 www.materialstoday.com/proceedings

ICAER-2015

Subcritical water hydrolysis of spent Java Citronella biomass for

production of reducing sugar
Robinson Timunga, Vaibhav V. Gouda, * F0F

a
Department of Chemical Engineering, IIT Guwahati, Guwahati-781039, India

Abstract

The present study investigate the production of reducing sugars from spent Java citronella (Cymbopogon winterianus Jowitt)
biomass using subcritical water (SCW) treatment in a batch reactor. The effect of process parameters such as temperature (140-
220 °C) and reaction time (5-40 min) on total reducing sugar (TRS) yield was studied. The production of TRS increased with an
increase in the temperature from 140-160 °C and decreased thereafter. The maximum amount of TRS 132.09 mg.g-1 biomass
was obtained at 160°C and 30 min reaction time. The formation of sugar inhibitors (5-HMF or Furfural) was almost insignificant
upto 160°C, but increased subsequently with an increase in the reaction temperature and time. The maximum inhibitors
concentration of 16.70 mg.g-1 was obtained at 220°C and 30 min reaction time, which includes 5.77 mg.g-1 of 5-HMF and 10.93
mg.g-1 of furfural. The crystallinity index (CI) value of biomass sample increased from 35.30% to 52.68% with an increase in
the temperature from 140-180°C, further increased in temperature to 220°C decreases the CI abruptly to 20.15% thereby altering
the biomass to relatively amorphous. The porous external surface as observed in scanning electron microscopic analysis of the
sample after SCW treatment showed significant removal of sugars and lignin.
© 2018 Elsevier Ltd. All rights reserved.
Selection and Peer-review under responsibility of the Conference Committee Members of International Conference on Advances in Energy
Research 2015 (ICAER-2015).

Keywords: Subcritical water; Java citronella; Reducing sugars; Inhibitors; X-ray diffraction.

1. Introduction

Biofuels derived from lignocellulosic biomass being sustainable fuels are potential feedstock for bioenergy
production to meet the increasing energy demand. There has been worldwide extensive research on the utilization of

* Corresponding author.
E-mail address: vvgoud@iitg.ernet.in

2214-7853 © 2018 Elsevier Ltd. All rights reserved.

Selection and Peer-review under responsibility of the Conference Committee Members of International Conference on Advances in Energy
Research 2015 (ICAER-2015).
 Timung and Goud / Materials Today: Proceedings 5 (2018) 23128–23135 23129

lignocellulosic biomass for the production of biofuels such as bioethanol, biodiesel and biogas, alongside reducing
greenhouse-gas emissions [1]. Additionally aromatic spent biomasses such as citronella, mentha etc. are the most
abundant and underutilized biological resources due to its cooked condition during essential oil extraction (mostly
distillation), and also a non-cattle feed. The spent biomass of aromatic plants are generally burnt haphazardly or
disposed as waste, leading to environmental problems. India alone produces more than 6.0 million tons per annum
of aromatic spent biomass [2]. Lignocellulosic biomass are composed of structural polysaccharides; cellulose and
hemicellulose wrapped inside lignin. The production of fuels from biomass requires pretreatment for conversion of
holocellulose to their constituent sugars and removal of lignin prior to hydrolysis and fermentation. Pre-treatment is
necessary before hydrolysis in order to provide easy access for enzymes to convert cellulose to chains of glucose
and its isomers, and hemicellulose to xylose and arabinose [3]. The efficiency of conversion of biomass to reducing
sugars depends on the choice of biomass and pretreatment method employed. An ideal biomass for the production of
bioenergy should possess high holocellulose content and calorific value [4]. Different pretreatment methods such as
chemical (acid, alkali, organosolv), physical (ultrasound, milling), physico-chemical (steam explosion, AFEX, liquid
hot water) and biological (using microorganisms such as fungi and bacteria) techniques have been practiced since
decades [5], and in this work subcritical water (SCW) extraction technique was investigated. SCW is defined as hot
water in a liquid state at temperature between 100°C to 374°C maintained under high pressure. SCW hydrolysis
technique is an efficient and one step process for converting biomass to reducing sugars. SCW technology is
considered as a green technique and provides extensive advantages over conventional methods. It is a one-step auto
hydrolysis process with less reaction time, non-toxic, non-corrosive etc. and provides wide range of dielectric
constant, density and viscosity to extract desired polar and non-polar compounds by tuning the operational
temperature and pressure. The water at subcritical condition targets the cellulose and hemicellulose fractions of
biomass to solubilize and liberate hexose and pentose sugars, while disrupting lignin and crystalline structure of
cellulose [6]. However the hydrolysis rate and sugar yield depends on the biomass cell structure and composition.
SCW extraction has gained significant interest towards extraction of saccharides from different feedstocks such as
cellulose, oil palm fronds, coconut meal, bamboo, sugarcane bagasse, cotton stalk, tobacco stalk, wheat stalk and
sunflower stalk etc. [7-11]. In the SCW treatment of biomass, polysaccharides degrades to oligosaccharides (Degree
of polymerization (DP) <10) or monosaccharides, and monosaccharides decompose to degradation products
depending on the operational temperature and reaction time [12]. This work is an endeavour to evaluate the
importance of spent biomass of aromatic plants. Spent citronella biomass has been chosen due to its abundance as
raw material in the North Eastern India. The present work aims at optimizing the process parameters of SCW
hydrolysis of spent citronella biomass to produce maximum reducing sugars. The physical and chemical changes in
the biomass sample after SCW treatment were evaluated. The results obtained in this study will also helps in
understanding the potential usage of spent aromatic biomass as a feedstock for TRS production.

2. Materials and Methods

2.1. Materials

Spent Java citronella biomass (after citronella oil extraction) obtained from Karbi Anglong, Assam (India) were
initially dried, chopped into small pieces, grinded, sieved to 1 mm sizes using 16 BSS mesh screen and then stored
in zipped plastic bags at ambient temperature until used. High pressure bath reactor (Model: 1734, M/s Amar
equipment Pvt. Ltd., India) equipped with stirrer and reactor vessel (500 ml) made of stainless steel with 160 mm
height and 75 mm internal diameter was used. The reactor was equipped with pressure gauge and thermocouple. The
standard sugars such as Maltohexaose, Maltopentaose, Raffinose, Cellobiose, Glucose, Xylose, Arabinose and
Erythrose, as well as HMF and furfural (all ≥ 95% purity) were obtained from M/s Himedia and M/s Sigma Aldrich.
Nitrogen gas with 98% purity was purchased from Guwahati, Assam.
2.2. Methodology

Hydrolysis of biomass was carried out in a 500 ml capacity high pressure batch reactor (maximum working
temperature and pressure of 500°C and 350 kg.cm-1respectively) using the procedure as adapted by Mohan et al.,
23130 Timung and Goud/ Materials Today: Proceedings 5 (2018) 23128–23135

(2015) [7], at various temperature ranging from 140-220°C, biomass loading (3 wt%) and reaction time 5-40 min.
The nitrogen gas was used to maintain the required pressure such as 52, 89, 145, 225, 335 psi or slightly more for
140, 160, 180, 200 and 220 °C respectively, to keep water at liquid state and also to prevent unnecessary reaction in
the process. Before achieving a set temperature, stirring was performed to disperse the biomass uniformly in the
water. The extraction time was considered from the moment desired reaction temperature was achieved. The
hydrolysate samples were collected through sample outlet at subsequent 5 min interval. At the completion of
reaction, the whole system was cooled to room temperature; residual biomass was collected, dried at 65°C for 48 hr
and then characterized using X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR) and
Scanning electron microscope (SEM). The collected liquid samples were filtered using 0.2 µm nylon filter paper and
analysed for sugar content in High Performance Liquid Chromatography (HPLC). The experiments at specified
conditions were repeated twice and the average values were reported. At the end of every experiment, the whole
system was flashed using millipore water at approximately 220 psi to prevent choking of sample port and gas inlet
port by residual biomass.

2.2.1. HPLC analysis of hydrolysate

The hydrolysate were analysed for the estimation of sugars such as maltohexaose, maltopentaose, raffinose,
cellobiose, glucose, xylose, arabinose etc. and inhibitors such as 5-HMF and furfural using HPLC (Perkin Elmer
Series 200) equipped with RI detector, vacuum degasser and network chromatography interface (NCI) 900.
SUPECOGEL Ca column (dimensions, 300 mm length and 7.8 mm inner diameter) connected to Ca* guard column
purchased from Sigma-Aldrich was used. The system was operated at 80°C using a column oven. Milli-Q water was
used as mobile phase at flow rate of 0.8 ml.min-1 and retention time of 30 min. The peaks obtained were identified
by comparing the retention time of standard sugars and quantified using the calibration plots standardized with at
least 5 points of different concentrations.

2.2.2. X-ray diffraction (XRD) analysis

The biomass crystallinity was estimated using wide angle X-ray diffractometer (Bruker D8 Advanced X-ray
diffraction measurement systems) at the radiation supplied by the accelerated current of 40 mA and voltage of 40
kV. The diffraction angle (2θ) scan range was 10-28° at a scan speed 1°.min-1 and step size of 0.05° [7].
Crystallinity Index (CI) was calculated based on the intensity of amorphous region at ~18.5±0.05° of 2θ (Iamp) and
the maximum intensity obtained from crystalline fraction at ~ 22.2±0.1°of 2θ (I002) according to Eq. (1).
I -I (1)
C I (% ) = 0 0 2 a m p × 1 0 0
I 002

2.2.3. Fourier Transform Infrared (FTIR) analysis

FTIR spectra of biomass samples were obtained from FTIR spectrometer “IR Affinity1” (Shimadzu
Corporation, Japan), measured at the range between 4000-400 cm-1 with nominal resolution of 2 cm-1 and 30 scans
per spectrum. IR grade potassium bromide (KBr) was used as reference. 20 mg of biomass sample was mixed with
30 mg of spectroscopic grade dried KBr, grinded well for proper mixing and directly taken for the FTIR analysis.
The baseline corrections and variations of frequency was applied using Shimadzu IR solution 1.5 software supplied
with the equipment [7, 13]

2.2.4. SEM analysis

The dry biomass samples were fixed with a conducting adhesive carbon tape on an aluminum sample holder.
Sputter gold coating was performed 2–3 times to prevent charging. All the specimens were qualitatively examined
in scanning electron microscope (LEO 1430vp, Germany) [7], under vacuum condition at accelerating voltage of 8
kV and 10 kV, and the working distance of 15 mm or 13 mm. The image was taken at the magnification of 1.78 KX.
 Timung and Goud / Materials Today: Proceedings 5 (2018) 23128–23135 23131

3. Results and Discussion

The spent aromatic biomass with cellulose and hemicelluloses as major components in its compositions was
hydrolysed using SCW treatment at varying conditions to produce TRS. Different oligo and mono saccharides such
as maltohexaose, maltopentaose, raffinose, cellobiose, glucose, xylose, arabinose and erythrose were released at
different reaction conditions. The release of total sugars initially increased with an increase in the hydrolysis time
and temperature up to 30 min and 160°C, but decreased further. This may be attributed to the fact that
oligosaccharides and monosaccharides degrades much faster than the cellulose or hemicellulose or lignin [14].
Hence before the complete conversion of total sugars from holocellulose, the oligosaccharides or monosaccharides
which were released disintegrate to other degradation products [15]. The overall degradation profile can be
explained as – polysaccharides (cellulose and hemicellulose) converts into oligosaccharides (maltohexaose,
maltopentaose, raffinose, cellobiose etc.) and monosaccharides (glucose, xylose, arabinose etc.), afterwards as
temperature increases the oligosaccharides degrades to di, tri or monosaccharides and later monosaccharides
degrades to sugar inhibitors (5-HMF, Furfural etc.) with further increase in the reaction temperature and time
(Fig.1).

Fig. 1. HPLC chromatogram of hydrolysate samples for different temperatures at 30 min reaction time; A: Maltohexaose, B: Maltopentaose, C:
Raffinose, D: Cellobiose, E: Glucose, F: Xylose, G: Arabinose, H: Erythrose, I: 5-HMF, J: Furfural.
23132 Timung and Goud/ Materials Today: Proceedings 5 (2018) 23128–23135

Temperature remains the most influencing factor in the SCW hydrolysis process. The increase in reaction
temperature provides higher mass transfer and reduces surface tension of water thereby increases the solubility of
the targeted compounds and provides high diffusivity through the sample matrix [8, 16]. The increase in TRS
production with an increase in the temperature from 140-160 °C revealed that hydrolysis rate increases with an
increase in the temperature. However, with further increase (180°C up to 220°C) in the temperature TRS production
decreased, which suggests that the degradation rate of monosaccharides was higher than the conversion rate of
saccharides from the lignocellulosic material at higher temperature. Reaction time is another important factor which
affects the TRS formation and is dependent on the temperature and matrix of targeted compounds [17]. The effect of
reaction time (i.e. 5, 10, 15, 20, 25, 30, 35 and 40 min) on the release of reducing sugars was evaluated. The
degradation of polysaccharide to oligosaccharide and monosaccharide at lower temperature increases the production
of TRS, but further increase in the reaction time and temperature resulted to decrease in TRS production which
might be due to the degradation of saccharides to inhibitors (i.e. 5-HMF and furfural) as can be seen from Table 1.

Table 1. . Effect of time and temperature on release of TRS and total inhibitors (TI)

Time 140°C 160°C 180°C 200°C 220°C

(min)
TRS TI TRS TI TRS TI TRS TI TRS TI
5 68.38 0 70.82 0 80.83 1.47 40.22 4.30 34.43 10.23
10 72.96 0 81.60 0 97.15 2.73 52.76 9.47 38.15 13.20
15 81.84 0 92.93 0 99.34 5.07 71.58 10.97 45.55 13.67
20 89.52 0 105.89 0 108.49 6.07 86.60 11.73 59.87 14.17
25 95.57 0 119.16 0 119.83 7.00 90.68 12.80 82.83 15.43
30 97.99 0 132.09 0 124.97 7.97 89.53 13.10 81.18 16.70
35 95.73 0 108.83 0 123.51 6.83 88.55 11.07 73.59 14.93
40 72.25 0 85.60 0 102.45 6.13 85.75 10.17 66.84 11.47

The maximum TRS (132.09 mg.g-1) was obtained at 160°C and 30 min, which includes 52.24 mg.g-1 of
maltohexaose, 0.70 mg.g-1 of maltopentaose, 1.13 mg.g-1 of raffinose, 10.37 mg.g-1 of cellobiose, 26.01 mg.g-1 of
glucose, 26.33 mg.g-1 of xylose (maximum release amount of xylose) and 15.33 mg.g-1 of arabinose. The release of
glucose increases with temperature producing maximum (27.01 mg.g-1) at 200°C and 30 min. Maximum
maltohexaose, maltopentaose, raffinose, cellobiose and arabinose obtained were 54.10 mg.g-1 (at 160°C, 25 min),
5.86 mg.g-1 (at 180°C, 15 min), 7.85 mg.g-1 (at 160°C, 5 min), 25.73 mg.g-1 (at 160°C, 30 min) and 20.67 mg.g-1
(at 160°C, 30 min) respectively. On the other hand, maximum inhibitors (16.70 mg.g-1) were produced at 220°C
and 30 min reaction time, which includes 5.77 mg.g-1 of 5-HMF and 10.93 mg.g-1 of furfural. Decreased in the
concentration of 5-HMF and furfural after 30 min reaction time suggested that these compounds are not stable up to
longer time in the reaction mixture and degrades to levulinic acid and 1-2-4 benzenenitrol [18].

3.1. XRD Analysis

The XRD analysis was performed to estimate the crystallinity index of hydrolysed biomass. The degradation of
hemicellulose and amorphous cellulose occurs at lower temperature followed by higher degradation of crystalline
cellulose at higher temperature [19]. The XRD pattern and CI (%) of SCW treated sample at different temperature
can be observed from Fig. 2. The CI (%) of spent citronella biomass was 34.91%. The CI of SCW treated biomass
sample increased from 35.30% to 52.68% with an increase in the temperature from 140-180°C. This may be
attributed to the effective removal of certain amount of lignin and hemicellulose from biomass and not necessarily
due to changes in the crystalline structure of the spent biomass. Further increase in the temperature decreases the CI,
which indicates the disruption of crystalline structure of cellulose. The CI at 200°C and 220°C were 29.57% and
20.15% respectively.
 Timung and Goud / Materials Today: Proceedings 5 (2018) 23128–23135 23133

Fig. 2. XRD pattern of SCW treated sample at different temperature.

3.2. FTIR Analysis

Fig. 3. FTIR analysis of Citronella biomass (Untreated biomass, spent biomass and SCW treated biomass at 140°C, 180°C and 220°C).

FTIR of untreated, spent and SCW treated citronella biomass (at 140°C, 180°C and 220°C) was performed to
compare the chemical changes in distribution of different functional groups which might have altered due to
degradation of the biomass (Fig. 3). There was an intense strong and sharp peak in the range of 3800-3200 cm-1
particularly at 3764.42 in all samples, which mostly represents the polymeric free hydroxyl (O-H) stretch. Another
intense and diverged peak observed in the range of 2935-2915 cm-1 corresponds to the aliphatic alkyl group C-H
methyl and methylene asymmetric stretch. Terminal aldehydic C-H stretch was found at around 2830 cm-1. A
characteristic medium peak was noticed at around 2372.28 cm-1 which may corresponds to certain cyano carbons.
Another distinct and sharp peak at 1750-1705 cm-1 in all the samples signifies un-conjugated xylan as well as aldo,
keto, estero and or acido (C=O) stretch. A medium peak at 1529.47 cm-1 corresponding to aromatic C=C ring and
lignin ring stretch was also observed. On the other hand, methylene C-H stretch (1485-1445 cm-1) and methyl C-H
symmetric band in cellulose and hemicellulose (1380-1371 cm-1) is present in all the samples. Moreover CH=CH
trans-unsaturated (910-860 cm-1) functional group with sharp peaks are also observed. Boeriu et al., (2004) reported
most of the above stretches for lignin, which indicates that lignin is dispersed in the residue [20]. The peak at ~1450
23134 Timung and Goud/ Materials Today: Proceedings 5 (2018) 23128–23135

and ~894 cm-1 represents the crystalline and amorphous fractions of cellulose respectively [13, 21]. Miniscule
changes was observed in all the samples except the disappearance of the peaks at 899 cm-1 at 200°C and 220°C
revealed the maximum degradation of cellulose fractions to sugars.

3.3. SEM Analysis

Fig. 4. SEM images of Citronella biomass; (a) Untreated (b) Spent biomass and (c) after SCW treatment.

SEM analysis of the samples showed significant morphological changes after subsequent treatment. SEM
images of untreated plant material showed smooth surface, and the cells of untreated plant material are intact (Fig. 4
(a)). After the extraction of oil it was observed that the cellular content of plant tissues distorted due to extensive
thermal stress on the oil glands causing rapid expansion extending to disruption and releasing the oil as represented
in Fig. 4 (b). Comparing the structural changes of untreated sample image with the insoluble fraction of treated
sample after SCW treatment, exposed pores can be seen on the surface of hydrolysed biomass as depicted in Fig. 4
(c), revealed de-polymerization of holocellulose and removal of significant amount of lignin [22].

4. Conclusions

In the SCW hydrolysis of spent citronella biomass, the released of oligosaccharides such as maltohexaose,
maltopentaose, cellobiose etc. increased with reaction time at lower temperatures (140-160°C), but decreased at
higher temperature, because of its disintegration to monosaccharides (glucose, xylose and arabinose). Further
increased in the reaction temperature and time degraded the monosaccharides to sugar inhibitors such as 5-HMF,
furfural etc. SCW technology which is relatively simple, efficient and environment friendly remains a promising
process for the production of TRS from lignocellulosic biomass.
 Timung and Goud / Materials Today: Proceedings 5 (2018) 23128–23135 23135

Acknowledgement

The work reported in this article was financially supported by a research grant received under Fast Track Scheme
from Department of Science and Technology (DST), Government of India (No. SR/FTP/ETA-0018/2010). Authors
would like to thank Mr Mood Mohan and Mr Narendra Naik for their help during this work. The authors also wish
to thank the Central Instrument Facilities of Indian Institute of Technology Guwahati for providing analytical
facilities.

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