Physicochemical Characterization, Modelling and Optimization of

Journal of Environmental Management xxx (2016) 1e13
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Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman
Research article
Physicochemical characterization, modelling and optimization of

ultrasono-assisted acid pretreatment of two Pennisetum sp. using
Taguchi and artificial neural networking for enhanced delignification
Sonali Mohapatra a, Snigdha Jyotsna Dandapat a, Hrudayanath Thatoi b, *
a
Department of Biotechnology, College of Engineering and Technology, Biju Pattnaik University of Technology, Bhubaneswar, 751003, India
b
Department of Biotechnology, North Orissa University, Sriramchandra Vihar, Takatpur, Baripada, 757003, Odisha, India
a r t i c l e i n f o a b s t r a c t
Article history: Acid as well as ultrasono-assisted acid pretreatment of lignocellulosic biomass of two Pennisetum sp.;
Received 27 April 2016 Denanath grass (DG) and Hybrid Napier grass (HNG) have been investigated for enhanced delignification
Received in revised form and maximum exposure of cellulose for production of bioethanol. Screening of pretreatment with
16 August 2016
different acids such as H2SO4, HCl, H3PO4 and H2NO3 were optimized for different temperature, soaking
Accepted 18 September 2016
Available online xxx
time and acid concentrations using Taguchi orthogonal array and the data obtained were statistically
validated using artificial neural networking. HCl was found to be the most effective acid for pretreatment
of both the Pennisetum sp. The optimized conditions of HCl pretreatment were acid concentration of 1%
Keywords:
Lignocellulose
and 1.5%, soaking time 130 and 50 min and temperature 121 C and 110 C which yielded maximum
Delignification delignification of 33.0% and 33.8% for DG and HNG respectively. Further ultrosono-assisted HCl pre-
Ultrasonication treatment with a power supply of 100 W, temperature of 353 K, and duty cycle of 70% has resulted in
Pennisetum sp significantly higher delignification of 80.4% and 82.1% for both DG and HNG respectively than that of acid
Taguchi orthogonal array pretreatment. Investigation using SEM, FTIR and autofloresence microscopy for both acid and ultrasono-
Artificial neural network assisted acid pretreatment lignocellulosic biomass revealed conformational changes of pretreated
lignocellulosic biomass with decreased lignin content and increased exposure of cellulose, with greater
effectiveness in case of ultrasono assisted acid pretreatment condition.
© 2016 Published by Elsevier Ltd.
1. Introduction biomasses holds great promise to be used as biofuels. Among these

plant based sources, the sugary and starchy substrates can be easily
The fast diminution of global fossil fuel reserves and increasing processed for bioethanol production. However, use of food crops is
price of crude oil has become one of the most daunting challenges discouraged as this will lead to food crisis. On the other hand,
that are encountered by the modern world. Adding to it, are the lignocellulosic biomass such as agricultural and forest residues,
concerns of global warming due to increase in consumption of fossil woody and herbaceous energy crops is expected to find its appli-
fuel, which has made it essential for search of renewable alterna- cation as a promising source of biofuel as these do not interfere
tives that can bring the promise of energy security and environ- with the food source of humans (Pan et al., 2014). Recent researches
mental protection (Charles et al., 2007). Biofuels from plant have indicated that, global bioethanol production will be headed by
biomass has become one of the important alternative energy lignocellulosic biomass which produces 1.5 1010 tons/year of
sources which is not only renewable but is also carbon neutral. It biomass with a conversion efficiency of 60% (Chen and Peng, 2013).
has been estimated that by 2050, the use of biofuels must increase Among the lignocellulosic biomass, substrates from grasses could
upto four folds which would lead to a reduction of worldwide CO2 be a promising source of biofuel. The polysaccharides present in
emissions by 50% (Wang et al., 2016). Bioethanol production from grass biomass can be converted to glucose which is subsequently
different plant sources such as sugary, starchy and lignocellulosic fermented to alcohol.
Grass as lignocellulosic biomass have several advantages like
high production, have good suitability for low quality land, cause
lower environmental impacts and have low lignin content (Cha
* Corresponding author.
E-mail address: hn_thatoi@rediffmail.com (H. Thatoi). et al., 2014). However, there are certain constraints for industrial
http://dx.doi.org/10.1016/j.jenvman.2016.09.060
0301-4797/© 2016 Published by Elsevier Ltd.
Please cite this article in press as: Mohapatra, S., et al., Physicochemical characterization, modelling and optimization of ultrasono-assisted acid
pretreatment of two Pennisetum sp. using Taguchi and artificial neural networking for enhanced delignification, Journal of Environmental
Management (2016), http://dx.doi.org/10.1016/j.jenvman.2016.09.060
2 S. Mohapatra et al. / Journal of Environmental Management xxx (2016) 1e13
bioethanol production from grass biomass which include collection of Research for Women in Agriculture (DRWA), Bhubaneswar,
of the raw material and its complicated method of conversion to Odisha during the month of March 2015. The collected substrates
ethanol (Vucurovic et al., 2009). Bioethanol production from i.e the plant biomass except the roots were brought to the micro-
biomass requires enzymatic hydrolysis and fermentation. Never- biology laboratory of Department of Biotechnology, College of En-
theless, lignocellulose biomass is usually complex and highly gineering and Technology, Bhubaneswar and were processed for
resistant to enzymatic hydrolysis because of its recalcitrance. further experiments.
Therefore pretreatment is needed to increase the hydrolysis rates.
Various pretreatment methods have been reported for delignifi-
2.2. Pretreatment methods
cation of biomass using acids, alkalis, ionic solvents, flux treat-
ments, high pressure steam explosion and ultrasonication
2.2.1. Sample preparation and pretreatment
pretreatment, etc which are applied either in single or in combi-
The sample preparation involved the shade drying followed by
nation. Among these processes acid pretreatment has the advan-
grounding of the two Pennisetum sp using a ball mill at the Her-
tage such as disruptions of covalent, hydrogen and van der Waals
barium section of the Institute of Minerals and Material Technol-
forces which leads to low requirement of enzymes in the hydrolysis
ogy, Bhubaneswar, Odisha which were then passed through a sieve
step (Maurya et al., 2015). The intention of pretreatment is to open
of mesh size of 2 mm. The powdered biomass was stored in air tight
or moderately break up its highly resistant lignin-carbohydrate
containers at room temperature. The required amount of biomass
complex and make the cellulose more accessible for further hy-
was taken and treated with different concentrations of acids like
drolysis or saccharification. Subsequently, during saccharification
(dilute) sulphuric acid, phosphoric acid, nitric acid and hydro-
process cellulose degrading enzyme convert cellulose into simpler
chloric acid and autoclaved at different temperature and incubation
sugars, primarily glucose, which can then be utilised by the mi-
time. The screening of acid pretreatment was optimized using
croorganisms for fermentation into bioethanol (Dias et al., 2013;
Taguchi L16 orthogonal array method. Further, the acid pretreated
Healey et al., 2015). Apart from acid, ultrasonication pretreatment
biomass showing greater delignification and higher cellulose
had also been reported to play a positive impact on the pretreat-
exposure was subjected to ultrasonication pretreatment under
ment and subsequent conversion of biomass. The amalgamation of
different parametric conditions like temperatures, duty cycles and
ultrasonic energy and acid pretreatment permits the destruction of
power. After the treatment, the biomass was filtered using double-
the recalcitrant lignocellulosic structure, promotes the solvation
layered muslin cloth and the filtrate and the biomass were kept
and fractionation of biomass components and finally helps in in-
separately. The biomass was repeatedly washed with tap water till
crease of equilibrium yields of sugars (Ravindran and Jaiswal, 2015).
neutral pH of wash water was obtained affirming that no persistent
Besides the ultrasonication pretreatment on the lignocellulosic
chemicals were left on the biomass surface. After the washing the
biomass in an aqueous media produces cavitation. The cavitation
biomass was dried at 105 C in hot air oven for 24 h, and then
leads to decomposition of water molecule into free radical that
analysed for determination of cellulose, hemicellulose and lignin
leads to cleavage in lignin and xylan networks (Subhedar and
content. All the experiments were repeated three times to check
Gogate, 2014). Simultaneously, the collapse of cavities with the
the reproducibility and the average value have been reported in
neighbouring cavities also causes pyrolysis of the molecules trap-
table and figures. Error bars have also been shown to illustrate the
ped inside these cavities (Badve et al., 2014).
variations.
Modelling and optimization are the most important tools in a
biological process to increase the efficiency of the process (Betiku
and Taiwo, 2015). Taguchi orthogonal array (OA) statistical design 2.2.2. Experimental design for optimization of acid pretreatment
is quick and effective in the simultaneous study of many factors, Taguchi orthogonal array (OA) was employed to obtain optimal
with enhanced process performance, giving high yield and pretreatment condition for enhanced delignification and cellulose
improved stability (Taguchi, 1986). On the other hand data analysis production. Acid concentration, temperature and soaking time
tools based on ANNs are now used successfully for solving represented by X1, X2 and X3 respectively were chosen as the
biotechnological complex problems for modelling and optimization factors that affect exposure of cellulose and lignin removal of DG
(Balaraman and Soundar, 2005). Thus, the present investigation and HNG. For each individual factor four levels were considered.
was aimed at screening and optimization of pretreatment with The performance parameters measured in this were: (i) mg cellu-
different acids and different pretreatment parameters using Tagu- lose/gm of biomass (Y1) and (ii) % lignin/gm of biomass (Y2).
chi L16 design for efficient delignification and enhanced cellulose Sixteen combinations were used to create an L16 OA using the first
exposure of two Pennisetum sp. Further optimized results obtained three factors (X1, X2 and X3) into consideration. The sixteen
in Taguchi experiments were validated using ANN model by using combinations were repeated for each sample to create the design
genetic algorithm as the global optimization procedure. The dilute database for the analysis. The influence of the selected parameters
acid showing the best response was subjected to ultrasound on the cellulose, hemicelluloses, and lignin yields were studied to
assisted acid pretreatment for enhanced delignification which was find the optimal condition using the S/N ratio. The desired value for
also optimized for different parameters. Further, untreated (native), the output was termed as ‘signal’ (S) while ‘noise’ (N) represented
acid pretreated and the combined pretreated biomasses (acid and the value that was not needed for the output characteristic. The S/N
ultrasonicated) were analysed by FTIR (Fourier transform infrared ratio i.e the ratio of the signal to the noise was used to quantitate
spectroscopy), SEM (Scanning electron microscopy) and auto- how a specific quality characteristic deviated from the desired
flurosence analyses for assessment of structural changes. value. The mean square deviation (MSD) was calculated from the
log equation of signal to noise ratio. For optimization of Taguchi
2. Materials and methods method any one of the three conditions (Condition 1: For maxi-
mization of the performance, the value of S’/N should be high,
2.1. Materials Condition 2: For minimization of the performance, the value of S”/N
should be low. Condition 3: if a predetermined S/N has to be tar-
Two grass verities of Pennisetum sp (Hybrid Napier grass (variety geted then the value of S000 /N being nominal is better) can be
CO-3) [HNG] and Denanath grass [DG] used as lignocellulosic considered. In the present study Condition 1 was considered. The
biomass for acid pretreatment were collected from the Directorate experimental data from the Taguchi arrays were analysed by
S. Mohapatra et al. / Journal of Environmental Management xxx (2016) 1e13 3
plotting the data and performing a one-way analysis of variance acid (10 ml) was added to the washed residue which was incubated
(ANOVA). The factors in the experimental design that were for 1 h. From this solution, 1 ml of the sample was taken and the
considered to be statistically significant at 95% confidence limit volume was made 100 ml using distilled water. Then from this
were used to determine the ratio (F) and the p-value (p < 0.05) 100 ml solution, 1 ml was taken to which 10 ml of anthrone reagent
(Mohapatra et al., 2015). was added. The resulting solution was incubated in a water bath at
100 C for 10 min and then cooled to room temperature. The
2.2.3. Acid þ Autoclaving (AA) pretreatment absorbance was measured at 630 nm (Updegraff, 1969). Hemicel-
Acid pretreatment was carried out following the analysis by lulose content in the biomass was estimated, by mixing 1 g of the
Taguchi Orthogonal method as described above. The biomass (10 g) dried biomass samples to 10 ml of 3% w/v H2SO4. The mixture was
was taken in 250 mL Erlenmeyer flask and subjected to treatment autoclaved at 121 C with 6.803 kg pressure. After autoclaving the
with different acids (100 ml) such as sulphuric acid, phosphoric samples the pH was adjusted to 7.0 with addition of distilled water
acid, nitric acid and hydrochloric acid with concentrations 0.5%, 1%, making the total volume to 100 ml. 5 ml of p-bromoaniline was
1.5%, 2% (w/v) at temperatures of 100, 110, 115, 121 (0C) for 10, 50, 90 added to the above mixture and the sample was incubated at 70 C
and 130 (minutes) in autoclave condition. The cellulose, hemicel- for 10 min. It was then incubated in the dark for 70 min and the
lulose and lignin from the solid fraction of the AA treated biomass absorbance was taken at 540 nm (Dubois et al., 1956). The ASL and
were analysed following standard methods (Naseeruddin et al., AIL content of the biomass was analysed by using NREL protocol
2013). (Sluiter et al., 2008). Dried biomass (300 mg) was taken in glass
pressure tubes and 3 ml of 72% H2SO4 (w/w) was added and kept at
2.2.4. Artificial neural networking 30 C for 2 h. The samples were mixed properly at 30 min time
A feed-forward back-propagation neural network was gener- intervals for proper acid hydrolysis. Followed by the hydrolysis
ated using SPSS modeller version 15.0 and minitab version 14. The step, the total volume was brought to 87 ml (with double distilled
data were selected using the OA table in the statistical design of the water) and the samples were autoclaved at 121 C for 1 h. Further,
experiments. Back-propagation training method was applied to these samples (after cooling at room temperature) were filtered
train the neural networks in MATLAB. The neural networking was through a vacuum pump using a filtering crucible. The ASL fraction
fed with three inputs (pretreatment conditions) temperature ( C), was determined by measuring the absorbance at 240 nm. The AIL
acid concentration (% w/v), and soaking time (minutes). Three was determined using deionized water to transfer all the residual
outputs were mapped i.e cellulose, hemicelluloses, and ASL þ AIL, solids out of the pressure tube into the filtering crucible. The solids
from the pretreatment. Out of the data obtained from the experi- were rinsed thoroughly with a minimum of 50 mL hot deionized
ment 60% of the data were used for training the network. From the water. The crucible and acid insoluble residue were dried at 105 C
rest 40%, 20% of the data were used for testing the network while until a constant weight was achieved. The samples were kept in the
the other 20% of the data were used for validation. The data that oven and cooled in a desiccator. The weight of the crucible and dry
were fed into the neural network were commanded to normali- residue was measured. The crucibles and residue were kept in the
zation of means and standard deviations. The neural network muffle furnace at 575 ± 25 C for 24 ± 6 h.
consisted of a single hidden layer with six neurons along with a FTIR spectra, SEM and autofloresence analysis were undertaken
sigmoid transfer function. It also consisted of a single output layer in order to inspect the structural variations in the physical and
with one neuron and a linear transfer function. To make sure that chemical features of the untreated and pretreated lignocellulosic
the six hidden layer neurons that were given to the neural network biomasses (HNG and DG). For FTIR analyses, the spectra of the un-
were the best choice, a range of 4e10 neurons were taken over treated and pretreated lignocellulosic biomasses in the KBr phase
which the network was run 100 times to see the effect on the R2 were recorded in a Perklin FTIR spectrophotometer, with a nominal
and Root mean square error (RMSE). In the current investigation resolution of 2 cmone averaging 44 scans. SEM analysis was used
(data not given), 6 neurons reduced the R2 and RMSE values, sup- to investigate the structural characterization of native, acid pre-
porting this choice (Mohapatra et al., 2015). treated and ultrasonication assisted acid pretreated grass varieties.
A SEM (Hitachi, Model: S-3400, USA) was used for the analyses. The
2.2.5. Ultra-sonication assisted acid (UAA) pre-treatment detector used for SEM was Secondary Electron; Semiconductor
The best acid condition responsible for higher release of cellu- Back-scattered Electrons (Quad type)* and Cathodoluminescence
lose with maximum delignification from acid treatment was detector, respectively. The samples were mounted in very thin
further subjected to treatment with ultra-sonication at tempera- layers over the copper sample holder which was gold coated and
tures of 30, 40.50, 60, 70, 80 C [¼293, 313, 333, 353, 373 (K)] for then analysed for the morphological changes. Autofloresence was
varying duty cycles of 30, 40, 50, 60, 70 (%) and power of 50, 60, 70, carried out using Leica DMIL microscope at Institute of Life sciences
80, 90 and 100 (W). These variations were studied with respect to (ILS), Bhubaneswar using LAS V4.2 software.
time variations of 10, 20, 30, 40 and 50 (minutes). The kinetic
parametres were determined using Arrhenius plot which is based 3. Results and discussion
on rate constants obtained at various temperatures. The biomass
treated above was analysed for maximum delignification by Experiment on screening of pretreatment with different acids
following the standard method of Subhedar and Gogate (2014). (H2SO4, HCl, H3PO4 and H2NO3) on both the grass (DG and HNG)
biomass was optimized employing Taguchi orthogonal array for
2.2.6. Analysis soaking time, concentration of acid and temperature. The pre-
The solid fractions of pretreated samples were analysed for treatment with the acid under autoclaving condition has been
cellulose, hemicellulose and acid insoluble lignin (AIL), whereas the denoted as AA (acid þ autoclaving) in rest of the sections. The acid
liquid part obtained during processing of AIL was used for deter- showing the highest cellulose exposure with the maximum
mination of acid soluble lignin (ASL). For cellulose estimation, 1 g of removal of hemicellulose and lignin was further used for a com-
sample was mixed with three millilitres nitric reagent and was bination pretreatment with acid þ autoclave and ultrasonication
placed in a water bath at 100 C for 30 min. The samples in tubes denoted as AAU (acid þ autoclaving þ ultrasonication) in the
were then cooled and centrifuged at 5000 rpm for 15e20 min. The impending sections. The effect of pretreatment was also investi-
residue content was washed with distilled water and 67% sulphuric gated through determination of cellulose, hemicellulose and lignin
content along with structural changes in untreated and pretreated
0.61
0.98
0.40
0.75
0.60
0.92
0.89
0.51
0.75
0.76
0.37
0.85
0.91
0.76
0.75
0.45
biomass.
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
17.2
16.2
16.2
15.8
15.7
13.5
13.4
12.7
14.7
14.0
13.1
13.8
13.6
13.0
12.9
12.1
HCl
3.1. Screening of AA pretreatment using Taguchi analysis

0.65
0.63
0.77
0.64
0.75
0.75
0.90
0.50
0.75
0.76
0.45
0.52
0.80
0.90
0.75
0.86
Screening of acid pretreatments of two grass varities (DG and

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
H2SO4
HNG) were investigated for each of the acids (HNO3, H2SO4, HCl,
17.8
17.8
17.4
15.2
16.6
16.8
14.9
13.5
13.6
12.8
12.3
12.0
14.8
12.3
12.8
12.6
H2PO4) according to the L16 OA of the Taguchi design (Table 1(a and
0.83
0.42
0.66
0.65
0.96
0.68
0.69
0.66
0.81
0.65
0.66
0.75
0.50
0.65
0.60
0.73
b)). Pretreatment for selection of the efficient acid for delignifica-

tion and cellulose exposure from the lignocellulosic biomass has
H2NO3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
20.4
19.1
17.9
16.8
16.7
16.0
15.3
14.9
16.5
15.7
17.2
15.0
17.4
16.7
14.7
14.1
been varied upon the type of acid used. This necessitates the se-
ASL þ AIL (%)
lection of acid for proper hydrolysis of lignocellulosic biomass.

0.45
0.49
0.60
0.95
0.66
0.56
0.55
0.75
0.58
0.55
0.66
0.65
0.98
0.80
0.65
0.95
Pretreatment of lignocellulosic biomass with sulphuric, nitric, or

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
H2PO4
hydrochloric acids have been reported to efficiently remove

19.0
18.2
16.1
15.0
15.8
14.2
14.1
13.9
18.1
17.1
15.9
14.7
14.6
14.0
13.6
12.8
hemicellulose components and expose cellulose for further enzy-

112.1 ± 0.60
176.1 ± 0.56
189.7 ± 0.85
128.0 ± 0.58
156.9 ± 0.62
129.7 ± 0.36
172.6 ± 0.60
131.1 ± 0.55
198.5 ± 0.50
147.6 ± 0.51
141.5 ± 0.76
121.3 ± 0.79
101.1 ± 0.66
105.0 ± 0.67
matic hydrolysis (Schell et al., 2003; Silverstein et al., 2007).

97.2 ± 0.80
94.9 ± 0.95
However effectiveness of pretreatment, also depends on factors

such as temperature, acid concentration and soaking time which
HCl
greatly influences hydrolysis of various components of grass

0.61
0.77
0.50
0.60
0.75
0.57
0.90
0.60
0.51
0.34
0.51
0.34
0.51
0.46
0.50
0.86
biomass during pretreatment (Das et al., 2014). In the present
investigation acid pretreatment was studied considering three
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
H2SO4
289.8
271.6
312.5
372.6
317.6
302.0
206.9
175.0
227.6
190.6
195.9
145.6
153.9
113.8
108.5
101.5
factors i.e. temperature (100 C, 110 C, 115 C, and 120 C), acid
concentration (0.5%, 1%, 1.5%, and 2%), and soaking time (10, 50, 90
0.80
0.70
0.87
0.48
0.77
0.66
0.80
0.58
0.55
0.65
0.80
0.70
0.85
0.57
0.60
0.55
and 130 min) in four levels (level 1 to level 4) in shake flask con-
Hemicellulose (mg/g)
dition. In the experiments, acid pretreatment processes were car-

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
H2NO3
317.9
296.1
261.0
297.3
276.5
312.7
272.6
211.0
198.1
197.6
191.6
138.2
171.1
119.0
177.1
174.9
L16 Orthogonal design for screening of different acids for maximum exposure of cellulose and removal of hemicellulose and lignin of DG.
ried out under low concentration of acids with lower incubation

periods and temperature, because under these conditions by-
0.75
0.91
0.75
0.58
0.50
0.55
0.49
0.57
0.45
0.80
0.50
0.65
0.70
0.51
0.70
0.32
products such as furfural and hydroxyl methyl furfural are formed
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
in very nominal quantities (Chandel et al., 2007). The results from
H2PO4
212.3
236.1
199.7
168.0
246.4
212.6
167.8
115.0
231.5
327.6
231.5
101.3
221.1
225.0
120.2
107.1
the present study showed that out of the four acids used with
different combinations of variables the highest delignification of
0.75
0.77
0.65
0.58
0.80
0.61
0.45
0.75
0.78
0.50
0.81
0.41
0.52
0.66
0.55
0.86
33.0% was achieved using hydrochloric acid (HCl) at a concentra-
tion of 1% at 121 C for 130 min for DG. The pretreatment under ±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
235.7
282.9
273.4
361.0
243.5
314.4
348.5
370.4
361.3
354.5
329.1
317.4
210.2
202.9
215.1
200.0
these conditions enhanced the delignification, by decreasing the HCl
lignin content from 19.0% in untreated DG to 12.7% in pretreated DG
196.7 ± 0.47
191.8 ± 0.84
210.5 ± 0.55
221.6 ± 0.94
116.7 ± 0.80
231.8 ± 0.46
246.3 ± 0.61
289.5 ± 0.45
236.5 ± 0.95
297.8 ± 0.65
241.2 ± 0.41
124.8 ± 0.91
127.5 ± 0.58
127.8 ± 0.83
121.2 ± 0.36
123.6.±0.49
with increased cellulose yield from 161 mg/g in untreated biomass
to 370.4 mg/g in HCl treated DG which was an increase of 56.5%. In
H2SO4
case of HNG maximum delignification of 31.8% delignification was
obtained i.e a reduction from 19.8% in untreated biomass to 13.5%
0.85
0.56
0.75
0.85
0.65
0.50
0.60
0.55
0.58
0.87
0.44
0.90
0.52
0.33
0.85
0.88
using 1.5% HCl at 110 C for 50 min. An increase of 252.7 mg/g of
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
H2NO3
cellulose yield for pretreated biomass as compared to the untreated
126.2
142.1
156.8
221.2
233.2
216.3
248.7
327.5
231.1
270.5
234.6
229.0
210.2
187.5
102.1
101.0
HNG (172.4 mg/g) was also observed in the same condition. The
Cellulose (mg/g)
increase in cellulose yield of 46.6% was observed in HNG which was
0.56
0.14
0.35
0.55
0.35
0.55
0.50
0.75
0.51
0.55
0.85
0.65
1.07
0.95
0.65
0.5
comparatively less than that of DG. Use of low concentration acid
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
leads to an effective pretreatment process with reduced load on
H2PO4
202.1
212.4
287.9
131.0
163.2
274.7
248.4
277.9
165.4
140.4
154.5
205.1
110.2
102.9
100.2
101.2
waste treatment and decreased investment costs. Similarly hemi-
cellulose yield of untreated and pretreated biomass was 298.6 and
131.1 mg/g for DG (56.09% reduction) and 322.3 mg/g and 218.6 mg/
g (32.1% reduction) for HNG respectively. Although other combi-
Soaking time
nations exhibited higher reductions in hemicellulose for grass
(mins)
biomass but those conditions were unsuitable as higher cellulose
130
130
130
130
degradation was also observed under such conditions.
10
50
90
10
50
90
10
50
90
10
50
90
From the literature it is found that hydrochloric (HCl) acid is
extra volatile, easier to recuperate and breaks biomass better than
Temperature
H2SO4 (Demirbas, 2008). Though, dilute HNO3 pretreatment has
been found to give the highest glucose concentration (compared to
( C)
100
110
115
121
100
110
115
121
100
110
115
121
100
110
115
121
dilute H2SO4) in the pretreatment of rye straw (Tutt et al., 2012), yet
the byproducts from nitric acid pretreatment are difficult to remove
by washing from the pretreated biomass (Tutt et al., 2012). Simi-
Sl.No Concentration
larly, H3PO4 pretreatment has been reported for yielding high sugar
levels but it is mostly found beneficial for conversion of xylose
rather than cellulose (Zhang et al., 2012). Considering the better
0.5
0.5
0.5
0.5
1.5
1.5
1.5
1.5
(%)
Table 1(A)
1
1
1
1
2
2
2
2
cellulose yield and greater delignification by HCl as compared to
other acids further studies were confined to the use of HCl for the
10
11
12
13
14
15
16
1
2
3
4
5
6
7
8
9
pretreatment of the Pennisetum sp.
The factors considered previously during the design of taguchi
18.42 ± 0.56
19.0 ± 0.52
18.3 ± 0.71
17.6 ± 0.65
18.4 ± 0.83
17.5 ± 0.60
16.1 ± 0.91
14.2 ± 0.65
13.5 ± 0.76
13.8 ± 0.80
12.9 ± 0.81
14.9 ± 0.95
14.0 ± 0.81
12.9 ± 0.80
12.0 ± 0.81
experiment for screening i.e acid concentration, temperature and

17.±0.89
soaking time taken as X1, X2 and X3 respectively were analysed to

determine the factor responsible for maximum effect on cellulose
HCl
yield and delignification. The factors responsible for reduction in

0.50
0.99
0.95
0.80
0.68
0.50
0.51
0.65
0.66
0.90
0.65
0.91
0.80
0.55
0.73
0.81
hemicellulose yield were also studied (data not shown), for which
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
only the taguchi graphs have been presented. The response values
H2SO4
19.5
19.1
18.0
16.2
17.0
17.1
16.5
15.2
17.4
16.1
15.7
13.1
16.2
16.0
14.6
14.0
in terms of the cellulose and lignin yields during acid pretreatment

under the three factors (X1, X2 and X3) were studied to attain the
0.70
0.91
0.80
0.70
0.50
0.65
0.80
0.81
0.91
0.80
0.95
0.91
0.70
0.85
0.81
0.55
optimum condition. The S/N ratio condition, of larger is better for

H2NO3
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
both the performance parameters i.e cellulose yield (Y1) and lignin
20.3
20.1
19.8
18.1
19.1
18.2
17.7
17.0
18.6
17.0
16.1
15.2
16.2
15.1
14.0
13.4
yield (Y2) were considered. From the experimental results it is

ASL þ AIL (%)
0.95
0.98
0.85
0.80
0.61
0.90
0.90
0.90
0.65
0.90
0.89
0.85
0.75
0.66
0.96
0.85
evident that the X1 was found to be the most important factor with
its maximum at 1.5% and 1% for DG and HNG respectively, beyond
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
H2PO4
21.0
20.2
18.1
16.0
19.0
17.2
17.1
16.9
16.1
15.7
14.9
14.0
15.6
15.8
13.8
13.0
which a sharp decrease in the cellulose concentration was observed

(Table 2(a) and Fig. 1(a)) and (Table 2(b) and Fig. 1(d)). Pretreatment
0.60
0.71
0.72
0.90
0.77
0.56
0.61
0.90
0.86
0.90
0.86
0.80
0.58
0.53
0.75
0.58
of wild grass with dilute acid HCl was undertaken by Li et al. (2007)
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
who found the glucose yield of pretreated substrates to be gov-

276.5
291.9
309.0
312.6
341.4
336.6
293.1
302.1
261.9
218.6
231.8
246.2
207.0
165.8
113.8
100.0
HCl
erned more on acid concentration than on reaction temperature.

The factor that showed the least impact on Y1 was X3 for both DG
0.65
0.78
0.91
0.61
0.80
0.64
0.64
0.60
0.75
0.92
0.55
0.81
0.90
0.81
0.80
0.65
and HNG. Similarly for Y2 i.e maximum delignification, X1 was the

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
H2SO4
main criterion for both DG and HNG (Table 2(a) and Fig. 1(c)) and
241.8
248.8
301.8
307.4
326.6
308.1
321.8
327.7
339.6
341.1
285.0
196.5
191.2
172.1
170.6
141.8
HNG (Table 2(b) and Fig. 1(f)). But interestingly, temperature and
0.70
0.71
0.65
0.60
0.95
0.81
0.55
0.92
0.50
0.51
0.52
0.80
0.61
0.52
0.95
0.57
soaking time for both the Pennisetum sp though not significant, but
had equal effects on Y1 and Y2 for both DG and HNG. The reduction
L16 Orthogonal design for screening of different acids for maximum exposure of cellulose and removal of hemicellulose and lignin of HNG.
Hemicellulose (mg/g)
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
H2NO3
of hemicellulose in pretreatment is also a beneficial factor for

209.5
221.7
258.8
298.7
212.7
284.8
296.5
287.1
254.3
198.7
187.8
210.7
295.4
231.8
279.9
264.0
ethanol production. But, in the present study no particular factor

0.60
0.70
0.70
0.90
0.77
0.90
0.89
0.56
0.92
0.65
0.55
0.92
0.81
0.60
0.90
0.91
was seen to effect the reduction of hemicellulose in both DG and

HNG. Instead all the factors seemed to have equal contribution
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
H2PO4
212.3
236.1
199.7
168.0
246.4
212.6
167.8
115.1
231.5
227.6
231.5
201.3
221.1
225.0
120.2
107.2
(Fig. 1(b) and (e)).

The analysis of variance (ANOVA) for the responses of the fac-
179.6 ± 0.86
195.9 ± 0.78
220.5 ± 0.50
118.6 ± 0.86
101.7 ± 0.90
175.9 ± 0.85
221.8 ± 0.58
210.9 ± 0.70
245.8 ± 0.90
252.7 ± 0.90
129.9 ± 0.85
100.8 ± 0.70
119.7 ± 0.75
104.7 ± 0.85
104.5 ± 0.90
tors X1, X2 and X3 of HCl treated DG and HNG on maximum cel-

92.6 ± 0.60
lulose release was carried out according to the factors contribution

by the Taguchi method (Table 3). From the calculated ratios (F), it
HCl
can be concluded that the factors taken into account are statisti-
214.7 ± 0.65
216.4 ± 0.85
154.3 ± 0.80
128.9 ± 0.70
166.8 ± 0.61
161.1 ± 0.90
172.4 ± 0.56
151.3 ± 0.55
111.9 ± 0.75
132.8 ± 0.85
196.9 ± 0.75
162.8 ± 0.63
138.9 ± 0.86
106.5 ± 0.51
cally significant at 95% confidence limit in the experimental design.

74.8 ± 0.85
49.4 ± 0.55
A model is considered significant if the p-value is lower than 0.05,

H2SO4
indicating only a 5% chance that their respective model could occur

due to noise. In the present study the p-values of the model terms
184.7 ± 0.75
191.4 ± 0.98
148.3 ± 0.61
136.0 ± 0.76
179.4 ± 0.77
182.1 ± 0.61
148.4 ± 0.40
167.9 ± 0.85
185.4 ± 0.89
112.9 ± 0.85
120.2 ± 0.71
117.0 ± 0.85
104.4 ± 0.91
(factor X1 for DG and factor X1 and X2 for HNG) were significant,

94.5 ± 0.87
95.1 ± 0.66
89.2 ± 0.65
i.e. p < 0.05. The statistical outcomes in our research also confirmed
H2NO3
acid concentration as one of the important factor affecting the acid

Cellulose (mg/g)
pretreatment of the Pennisetum sp. Lara et al. (2015) found similar

0.56
0.91
0.79
0.89
0.62
0.75
0.61
0.85
0.61
0.94
0.60
0.55
0.40
0.75
0.76
0.81
results in their study using dilute acid pretreatment for bioethanol

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
production from agave bagasse.
H2PO4
202.1
212.4
287.9
131.0
163.3
274.7
248.4
277.9
165.4
140.4
154.5
305.1
110.2
102.9
100.2
101.2
Table 2
(a) S/N ratio at each level of factors and rank of factors (DG); (b) S/N ratio at each
Soaking time
level of factors and rank of factors (HNG).

S/N ratio corresponding to Y1 S/N ratio corresponding to
(mins)
Y2
130
130
130
130
10
50
90
10
50
90
10
50
90
10
50
90
X1 X2 X3 X1 X2 X3
(a) Level
Temperature
1 152.9 134.4 140.0 24.28 23.66 23.66

2 160.2 139.1 147.5 22.79 23.01 23.01
3 168.9 157.4 151.9 22.86 22.82 22.82
( C)
100
110
115
121
100
110
115
121
100
110
115
121
100
110
115
121
4 106.5 156.7 148.2 22.20 22.64 22.64
Delta 62.4 23.0 11.8 2.08 1.02 1.02
Rank 1 2 3 1 2.5 2.5
Sl.No Concentration
(b) Level
1 164.83 132.10 150.01 25.26 24.35 24.35
2 171.04 129.89 150.45 24.73 23.91 23.91
3 145.18 156.95 140.55 22.67 23.73 23.73
0.5
0.5
0.5
0.5
1.5
1.5
1.5
1.5
(%)
Table 1(B)
1
1
1
1
2
2
2
2
4 88.49 150.59 128.53 22.55 23.21 23.21
Delta 82.55 27.06 21.92 2.71 1.13 1.13
10
11
12
13
14
15
16
1
2
3
4
5
6
7
8
9
Rank 1 2 3 1 2.5 2.5
Fig. 1. Taguchi graphs for DG (a, b, and c) and HNG (d, e, and f).
Table 3
(a) ANOVA for DG; (b) ANOVA for HNG.
Simultaneously, the data was carefully selected from the L16
orthogonal array for the HCl pretreated DG and NG for training the
Factor for Y1 DF Seq SS Adj SS Adj MS F P model. The most important part for selecting an ANN model is the
(a) topology and the architecture of the neural-network. A feed-
X1 3 9278.3 9278.3 3092.78 15.47 0.003 forward back-propagation neural network consisted of a topology
X2 3 1691.3 1691.3 563.77 2.82 0.129
5-4-3 (Fig. 2(a)) for DG and 4-4-3 for HNG (Fig. 2(b)). Equally
X3 3 296.1 296.1 98.69 0.49 0.700
Residual error 6 1199.3 1199.3 199.89 important is, the training of the neural network model which al-
lows the selection of one model from the given set of models.
Total 15 12465.0
Depending on the type and complexity of the task, the selection of
(b) the optimal number of hidden neurons is usually done by trial and
X1 3 16948.8 16948.8 5649.6 36.73 0.000
error. Too many neurons may lead to much freedom for adjustment
X2 3 2165.7 2165.7 721.9 4.69 0.051
X3 3 1273.7 1273.7 424.6 2.76 0.134 of weights, resulting in learning only the noise present in the
Residual error 6 922.8 922.8 153.8 database that is used during training the ANN (Linko et al., 1999).
Total 15 21311.0 Therefore the neural network in this study was composed of one
hidden layer with six neurons and a sigmoid transfer function. It is
also composed of one output layer with one neuron and a linear
3.2. ANN modelling of AA pretreatment transfer function. The transfer function applied, affects the rate of
learning of the neural network (Ebrahimpour et al., 2008). It was
In the present work, network like multiple layer feed-forward apparent from the current study that, ANN was consistently
propagation (MLPs) was used for modelling the data obtained learning the relationship between inputs and outputs, as the R2
from Taguchi orthogonal array for HCl treated DG and HNG. statistic were reasonable compared to the average values.
Fig. 2. Topology of neural network for the estimation of optimum delignification (a) DG and (b) HNG.
The test of significance showed that the p-values of the model 3.3. Optimization of ultrasonicated acid pretreatment
terms were significant, i.e. p < 0.05. Though p-values are
important for determination of significance, but they do not In order to enhance the delignification process further experi-
distinguish between a positive or a negative significance effect on ments were conducted using AAU pretreatment for DG and HNG.
the model. Therefore, the interactions of the variables viz. soaking Optimization of AAU pretreatment of both the grass samples were
time, pH and temperature were inspected using pareto chart carried out under parametres i.e effect of ultrasonication duty cycle,
(Fig. 3). All the positive variables showed a favourable or syner- power and temperature. The % delignification under these param-
gistic effect on delignification. The linear term of concentration eters were determined against different incubation time, rate
had the most significant effect followed by temperature and constants (k/min) and R2 values.
soaking time.
The parity plots for validation of data are depicted in Fig. 4. 3.3.1. Effect of ultrasonication duty cycle on AA samples
Comparison of experimental and predicted values in validating The increase of ultrasonic power, as well as the increase of the
data sets, not only showed effectiveness of ANN in prediction of duration (duty cycle) during ultrasonic treatment, prominently
known data responses but also exhibited the ability of generaliza- influences hydrolysis efficiency. Ultrasound duty cycle is an
tion for unknown data (Ebrahimpour et al., 2008). This implies that important parameter in delignification and is defined as the frac-
empirical models derived from ANN can be used to effectively tion of time for which the ultrasonic system is operated. The
describe the association between the input variables to the cellu- employment of non-stop mode of ultrasound leads to deterioration
lose yield and hemicellulose and lignin delignification. of cellulose and excessive increase in electrical energy consumption
Fig. 3. Pareto chart of standardized effects of factors X1, X2 and X3 on delignification of both the Pennisetum sp.
Fig. 4. ANN graphs showing the actual and predicted values for DG and HNG.
compared to pulsed mode of operation. Hence, intervals between increase in the cavitational bubbles and the implosion formed due
the ultrasonication duty cycles are important for achieving good to the higher exposure time of biomass to ultrasonication (Saini
delignification and duty cycle is expressed in terms of %. For et al., 2015). The formation of cavities in the bulk liquid phase
example a duty cycle of 30% designates to a sonication period 3 s under ultrasonic conditions is an indication of the unavoidable
which is followed with a break (i.e the time where no sonication weakening and collapse of intermolecular interactions between the
occurs) of 7 s. In the present study effect of different ultrasound cell wall components (Ramadoss and Muthukumar, 2016). The re-
duty cycle (30%, 40%, 50%, 60%, and 70%) on AA treated DG and HNG sults obtained in the present investigation are in agreement with
was investigated for maximum delignification (Figs. 5(A) and 6(A)). the results reported for the pretreatment of lignocellulosic raw
The delignification increased from 45.1 to 80.2% and 48.3e71.3% for material using ultrasound aided alkaline pretreatment (Subhedar
DG and HNG respectively, with an increase in duty cycle from 30% and Gogate, 2014). The combination of acid pretreatment with
to 70%. For AAU pretreated DG the optimum duty cycle was taken to sonication has ensued greater delignification in both rice straw
be 70% as significant increase in rate constant of 0.401 K/min was (Belal, 2013) and sugarcane baggase (Esfahani and Azin, 2012) with
observed at the same duty cycle. But in case of AAU pretreated HNG alterations in the duty cycle. Ramadoss and Muthukumar (2016)
the rate constant did not show any significant rise from 60%. Hence, investigated on ultrasound assisted acid pretreatment of sugar-
the optimum duty cycle for HNG was taken as 60%. The rate con- cane bagasse and obtained 78.7% delignification. There has been
stants for the delignification observed in all the duty cycles and reports that the duty cycle ultrasonication is a helpful procedure for
their respective R2 values are given in Table 4. The increased removal of selective hemicellulose and lignin components from the
delignification due to ultrasonication might have resulted from the lignocellulosic biomass (Li et al., 2012; Garcıa et al., 2011).
30 40 50 60 70 A B
100 50 60 70 80 90 100
100
DelignificaƟon (%)
DelignificaƟon (%)
80
80
60
60
40
40
20 20
0 0
10 20 30 40 50 10 20 30 40 50
Time (mins) Time (in mins)
293 313 333 353 373 C

100
DelignificaƟon (%)
80
60
40
20
0
10 20 30 40 50
Time(in mins)
Fig. 5. (A) Effect of Duty cycle, (B) Effect of power and (C) Effect of Temperature on delignification of DG.
30 40 50 60 70 A 100 B
80 50 60 70 80 90 100
DelignificaƟon (%)
80
DelignificaƟon (%)
60
60
40
40
20 20
0 0
10 20 30 40 50 10 20 30 40 50
Time(in mins) Time (in mins)
293 313 333 353 373 C

100
DelignificaƟon (%)
80
60
40
20
0
10 20 30 40 50
Time(in mins)
Fig. 6. (A) Effect of Duty cycle, (B) Effect of power and (C) Effect of Temperature on delignification of HNG.
Table 4 respectively which are significantly higher than delignification

Effect of duty cycle on rate of delignification in ultrasound-assisted process. achieved in AA pretreatment only. Remarkably, ultrasonication
Duty cycle Delignification Rate constant k R2 assisted acid pretreatment has been more effective than AA
(%) (min-1) treated samples, which might be attributed to the high ultra-
DG HNG DG HNG DG HNG sonication power used. In a study conducted on wheat straw
similar results of 78.5% delignification were achieved by using
30 45.1 48.3 0.236 0.29 0.97 0.98
40 51.5 52.1 0.281 0.219 0.99 0.96 ultrasound at 100 W power (Sun et al., 2002), which is in agree-
50 59.4 58.2 0.39 0.258 0.98 0.96 ment with the outcomes of the present study. Subhedar and
60 66.4 62.9 0.398 0.324 0.98 0.96 Gogate (2014) obtained 80.16% delignification at 100 W in
70 80.2 71.3 0.401 0.359 0.98 0.98
70 min from newspaper using ultrasonication assisted alkali pre-
treatment. Previous reports suggest that high power ultrasound
leads to reduced particle size as it destroys the wax layers, silica
3.3.2. Effect of ultrasonication power on AA samples bodies along with the lignins from the surface of lignocellulosic
Optimization of power of ultrasonication plays a crucial role in structures (Zhang et al., 2013). However, unnecessary ultrasonic
delignification of lignocellulosic biomass. Demolition of the power should be avoided as this may lead to other side reactions
microfibril structure of microcrystalline cellulose requires very such as the corrosion of valuable chemicals by active radicals (Luo
high energy and ultrasonication power in the range of 10e100 W et al., 2013). In the present study the rate constant for delgnifi-
provides the appropriate energy which is sufficient for breaking cation of DG increased from 0.28 to 0.42 with the increase in
the cellulose in plants (Lu et al., 2013).The increase in power of sonication power, but similar trend was not observed for HNG
sonication from 50 to 100 W was seen to be directly proportional samples as shown in Table 5.
on the rate of delignification of DG and HNG as evident from
present study (Figs. 5(B) and 6(B)). The highest delignification of 3.3.3. Effect of ultrasonication temperature on AA samples
88.3 and 87.2% was obtained in AAU pretreated DG and HNG Temperature of ulrasonication contributes for efficient
Table 5 3.4. Assessment of pretreatment using FTIR

Effect of power on rate of delignification in ultrasound-assisted process.
Power Delignification Rate constant k R2 The variations in the configuration of lignocellulosic biomass
(%) (min-1) before and after pretreatments can be monitored using FTIR spec-
DG HNG DG HNG DG HNG troscopy. The FTIR spectra of untreated, acid treated and acid along
with ultrasonication pretreatment DG and HNG were assessed. The
50 58.1 46.3 0.28 0.279 0.98 0.95
60 66.9 55.2 0.322 0.417 0.99 0.98 assigned vibrational changes and observed changes of different
70 78.4 60.8 0.341 0.20 0.98 0.98 functional groups of untreated,1%, AA DG, 1% AAU DG, 1.5% AA HNG
80 80.1 73.1 0.374 0.429 0.98 0.97 and 1.5%AAU HNG pretreated Pennisetum sp. in the IR spectrum are
90 85.3 81.7 0.401 0.343 0.99 0.95
summarized in Table 7 and Fig. 8(A and B). The prominent bands
100 88.3 87.2 0.426 0.446 0.99 0.95
around 3346 and 3352 cm1 showed the rupturing of the cellulosic
hydrogen bonds for AAU DG and NG respectively as compared to
the untreated and AA treated biomass. This was in good agreement
delignification of lignocellulosic biomass as it disrupts the cellular with the observed results in Parthenium hysterophorous by Singh
walls making the availability of the intracellular material for sub- et al. (2014). The aromatic ring stretch at 1587, 1517 and 1582 and
sequent enzymatic hydrolysis (Rinco n et al., 2014). It was observed
1509 cm-1 in DG and NG respectively that is related to lignin
in the present study that there was an increase of 58.8% delignifi- removal was absent in untreated grasses, whereas the bands were
cation using AAU pretreatment as compared to AA pretreated DG. clearly visible in AA and AAU grasses. The shift of 1264 cm1 peak of
In case of AAU pretreated HNG 61.2% increase in delignification was AA pretreated DG to 1250 cm1 of AAU pretreated DG could be
observed as compared to AA pretreated HNG. In the present study attributed to C]O absorption that results from cleavage of acetyl
an increase in temperature from 293 to 373 K increased the groups. Similar results were observed in FTIR study of newspaper
delignification from 43.6 to 88.2% in case of DG (Fig. 5(C)), but 353 K by Subhedar and Gogate (2014). But, in case of HNG no such bands
was considered as the optimal temperature where maximum were observed. Many research groups suggested different peak
delignification of 80% was observed. Temperature beyond this pairs for crystalline and amorphous cellulose viz., 1098 cm1 for
resulted in greater loss of biomass. Similar trend was observed for amorphous to crystalline cellulose ratio and 900 cm1 for amor-
HNG where delignificaton increased from 40.8 to 87.5% (Fig. 6(C)). phous cellulose (Shi et al., 2014). In the present study peaks at 1091
Further the rate constant and R2 were calculated, which showed and 900 cm1 were observed for AAU DG whereas peaks at 1086
increase in rate constant from 0.127 to 0.343 and 0.144 to 0.0.324 K/ and 905 cm1 were obtained for AAU HNG. Analogous results were
min for DG and HNG respectively with R2 values in the range of obtained by Das et al. (2014) in steam exploded thatch grass.
0.95e0.99 for DG and HNG respectively as shown in Table 6. Li et al.
(2012) conducted ultrasound-assisted NMMO (N-methylmorpho-
line-N-oxide) treatment on sugarcane bagasse and showed that rise 3.5. Effect of pretreatment on structural morphology
in temperature has a positive impact not only on delignification but
on glucan or cellulose recovery as well. The kinetic parametres for Examination of the structural changes of the untreated, 1% AA
both DG and HNG were determined by plotting an Arrhenius plot DG, 1% AAU DG, 1.5% AA HNG and 1.5% AAU HNG were obtained
which was prepared using the rate constants obtained at the using SEM micrographs. A comparison of the structures of un-
highest delignification temperatures (Fig. 7(a and b)). R2 values of treated and pretreated biomass showed a strong, well-arranged,
0.97 and 0.94 were obtained for the plots of DG and HNG and even surface for untreated biomass (Fig. 9(A) and (D))
respectively. whereas the 1% AA DG, 1% AAU DG, 1.5% AA HNG and 1.5% AAU HNG
appeared to be disrupted and rough in structure (Fig. 9(B and C) and
(E and F)). Both the grasses showed changes in the surface layer
Table 6 with 1% AA DG and 1.5% AA HNG. This indicates removal of lignin
Effect of temperature on rate of delignification in ultrasound-assisted process.
from the surface of biomass. Increase in the external surface area
Temperature (k) Delignification Rate constant k R2 and porosity of the pretreated grass biomass are attributed to the
(%) (min1) removal of external fibres which ultimately leads to high efficiency
DG HNG DG HNG DG HNG of enzymatic hydrolysis of the biomass (Remli et al., 2014). But,
293 43.6 40.8 0.127 0.144 0.98 0.99 significant morphological changes were observed in sono-assisted
313 55.2 52.6 0.195 0.181 0.97 0.97 acid pretreated samples where the samples showed a tendency of
333 66.3 71.8 0.238 0.271 0.95 0.99 crack formation on the outer layer which could be attributed to the
353 80.4 82.1 0.303 0.312 0.97 0.98 mechanical disruption through the force exerted on the particles.
373 88.2 87.5 0.343 0.324 0.98 0.97
This has resulted in reduction in lignin-carbohydrate complexes on
a b
0 0
y = -1341.7x + 2.5792
y = -1341.7x + 2.5792 -0.5 R² = 0.9789
-0.5
R² = 0.9789
-1
-1
-1.5
ln K
-1.5
ln K
-2
-2
-2.5
-2.5
-3
-3
-3.5
-3.5 0.00220.00240.00260.00280.0030.00320.00340.00360.0038
0.00220.00240.00260.00280.0030.00320.00340.00360.0038 1/T(K-1)
1/T(K-1)
Fig. 7. .Relationship between 1/T and ln (K) in a) DG and b) HNG.
Table 7
Assignment of the band positions in IR spectra (Adopted from Kumar et al., 2014) and characterization of untreated and pretreated biomass by FTIR spectroscopy in terms of
change in band position.
Band position (cm- Assignment DG HNG

1)
UDG 1% AA DG 1% AAU UHNG 1.5% AA 1.5%AAU
DG HNG HNG
3348/3414 OeH stretching (related to rupture of cellulose hydrogen bonds) 3510 3455 3412 3480 3453 3442
2900 CeH stretching (related to rupture of methyl/methylene group of 2948 2935 2911 2959 2934 2905
cellulose)
1745 Carbonyl bonds (related to lignin side chain removal) 1701 1712 1751 1749 1751 1777
1738 C¼O stretching due to carbohydrate linked with lignin 1756 1747 1736 1766 1751 1748
1720 Carboxylic acids/ester groups NBO NBO 1722 NBO NBO NBO
1595 Aromatic ring stretch (related to lignin removal) 1560 1581 1587 1578 1579 1582
1508 Aromatic ring vibration (related to lignin removal) NBO 1529 1517 NBO 1525 1509
1428 Band of cellulose NBO 1430 1428 NBO 1441 1418
1378/1071/805 Unconjugated C]O stretching in acetyl group of hemicellulose 1367/1126/ 1375/1076/ 1398/805 1342/1177/ 1387/1093 1383
933 893 956
1260 Ester absorbance (related to removal of uronic acid) 1281 1274 1269 1271 1272 1279
1245 C]O absorption (resulting from acetyl groups cleavage) NBO 1264 1250 NBO NBO NBO
1238 Hemicelluloseelignin linkage NBO NBO NBO NBO NBO NBO
1059 C]O stretching due to carbohydrateelignin linkage 1038 1058 1063 1042 1053 1069
1098 Amorphous to crystalline cellulose ratio 1068 1071 1096 NBO 1076 1086
900 Band of cellulose NBO 871 900 NBO 899 905
96
A B
90
2 2
85
80
3 3
75
%T
70 1
1
65
60
55
52
4000 3500 3000 2500 2000 1500 1000 500 400
cm-1
Fig. 8. FTIR analysis of (A) [1] Untreated DG, [2] 1% AA DG, [3] 1% AAU DG (B) [1] Untreated HNG, [2] 1.5% AA HNG and [3] 1.5% AAU HNG.
the surface of the grass biomass. Sharma et al. (2013), obtained during lambda scanning. During this analysis, the green fluores-
similar results using switchgrass as a substrate for ultrasonication cence is mainly derived from bound ferulic acid in cell walls and
pretreatment. Zhang et al. (2013) observed while working on carbohydrates like cellulose (Meineke et al., 2014). Cellulose is a
developing cellulose fibres that, cellulose nanofibres could be polymer in the plant cell wall and is induced with the 405 nm diode
removed from lignocellulose by the application of high intensity laser (Harris and Hartley, 1976). In this context, a decrease in green
ultrasonication. Furthermore, a substantial opening up of the fluorescence during pretreatment procedures would not only
biomass was observed which confirms the availability of more indicate the hydrolysis of cellulose but also a reduction of cross-
surface area that would allow effective enzymatic hydrolysis during linked polysaccharides (Rancour et al., 2012). In the present study
saccharification. The structural transformations of the biomass due two main factors i.e delignification of plant biomass and maximum
to the combination pretreatment with acid and ultrasonication can exposure of the cellulosic fraction of the plant cell wall were the
be attributed to the liquid solid interface that occurs internally primary objectives. As estimated, the untreated biomass of Penni-
through free radicals. It so happens that as the bonds in the aro- setum species revealed a strong red auto-fluorescence, which
matic rings and side groups are broken leading to formation of indicated a highly lignified biomass (Fig. 10(A and D)). Mostly the
more macro radicals (Rehman et al., 2012). This results in selective autoflurosence of lignin occurs at lexc ¼ 510e560 nm (Pauly and
effects on interactions between lignin and hemicellulosic compo- Keegstra, 2008). Subsequently after the AA pretreatment the pre-
nents thereby leading to weakened cell wall complex resulting in dominant red fluorescence was partially lower but was not
more surface area in biomass for efficient enzymatic hydrolysis completely eliminated as seen in Fig. 10(B and E). This can be
(Izadifar, 2013). related, with the high recalcitrance that the plant cell wall offers
(Pu et al., 2013). Compared to the AA NG, AA DG showed more
3.6. Qualitative analysis of biomass composition by autoflorecence delignfication. Furthermore, the complete dominance of the green
microscopy fluorescence could be easily identified in the AAU DG and AAU HNG
samples as seen in Fig. 10(C and F). This could be attributed to the
Auto-fluorescence is used for the qualitative analysis of the cleavage of linkages between lignin and xylan networks caused by
plant cell walls which is based on its different colour emissions the combination of AAU pretreatment which could lead to greater
Fig. 9. SEM analysis of (A) Untreated DG, (B) 1% AA DG, (C) 1% AAU DG, (D) Untreated HNG, (E) 1.5% AA HNG and (F) 1.5% AAU HNG.
Fig. 10. Autoflorecence images (A) Untreated DG, (B) 1% AA DG, (C) 1% AAU DG, (D) Untreated HNG, (E) 1.5% AA HNG and (F) 1.5% AAU HNG.
exposure of cellulose. The acquired results have much resemblance of pretreatment yields of DG and HNG. Further ultrasonication
with the autoflurosence studies conducted on grass species like pretreatment combined with dilute acid (HCl) showed enhanced
Brachypodium distachyon and Miscanthus sp.by Meineke et al. delignification of 80.4% and 82.1% for DG and HNG respectively
(2014). indicating combination pretreatment could be more effective than
acid pretreatment only. Further studies by SEM, FTIR and auto-
floresence analyses confirmed greater delignification and enhanced
4. Conclusion
cellulose exposure in ultrasonication assisted HCl pretreatment of
biomass as compared to untreated and only HCl treated biomass.
The present study investigated the screening of different acids in
The present study suggests that combination of dilute HCl and
low concentrations for pretreatment of two Pennisetum sp. (DG and
ultrasonication could be an attractive mode of pretreatment for
HNG) for enhanced delignification and maximum cellulose expo-
efficient delignification and cellulose exposure in grasses like DG
sure. Dilute HCl was found to be the best acid for pretreatment of
and HNG which could be exploited for bioethanol production in the
the two grass varities showing 33.0% and 31.8% delignification
future with suitable saccharification and fermentation processes.
along with simultaneous cellulose yield of 56.5% and 46.6% for DG
and HNG respectively. Statistical optimization of acid pretreatment
of DG and HNG by Taguchi OA confirmed that the acid concentra- Acknowledgement
tion was the significant factor which contributed for higher
delignification in the two grass varities. Employment of ANN Authors are grateful to the authorities of college of engineering
exhibited good correlation between the predicted and actual values and technology, BPUT, Bhubaneswar for providing necessary
laboratory facilities to carry out this experiment and to Dr. Anil lignocellulosic ethanol production:a comparative study of Brachypodium dis-
tachyon, wheat,maize,and Miscanthusx giganteus leaf and stem biomass. PLoS
Kumar, Senior scientist-DRWA, Bhubaneswar for kindly providing
One 9.
the two grass varieties. Mohapatra, S., Dash, P.K., Behera, S.S., Thatoi, H., 2015. Optimization of delignifi-
cation of two Pennisetum grass species by NaOH pretreatment using Taguchi
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Journal of Environmental Management xxx (2016) 1e13

Contents lists available at ScienceDirect

Journal of Environmental Management

Physicochemical characterization, modelling and optimization of

1. Introduction biomasses holds great promise to be used as biofuels. Among these

3.1. Screening of AA pretreatment using Taguchi analysis

Screening of acid pretreatments of two grass varities (DG and

b)). Pretreatment for selection of the efﬁcient acid for deligniﬁca-

lection of acid for proper hydrolysis of lignocellulosic biomass.

Pretreatment of lignocellulosic biomass with sulphuric, nitric, or

hydrochloric acids have been reported to efﬁciently remove

hemicellulose components and expose cellulose for further enzy-

matic hydrolysis (Schell et al., 2003; Silverstein et al., 2007).

However effectiveness of pretreatment, also depends on factors

greatly inﬂuences hydrolysis of various components of grass

dition. In the experiments, acid pretreatment processes were car-

ried out under low concentration of acids with lower incubation

experiment for screening i.e acid concentration, temperature and

soaking time taken as X1, X2 and X3 respectively were analysed to

yield and deligniﬁcation. The factors responsible for reduction in

in terms of the cellulose and lignin yields during acid pretreatment

optimum condition. The S/N ratio condition, of larger is better for

yield (Y2) were considered. From the experimental results it is

which a sharp decrease in the cellulose concentration was observed

who found the glucose yield of pretreated substrates to be gov-

erned more on acid concentration than on reaction temperature.

and HNG. Similarly for Y2 i.e maximum deligniﬁcation, X1 was the

of hemicellulose in pretreatment is also a beneﬁcial factor for

ethanol production. But, in the present study no particular factor

was seen to effect the reduction of hemicellulose in both DG and

(Fig. 1(b) and (e)).

tors X1, X2 and X3 of HCl treated DG and HNG on maximum cel-

lulose release was carried out according to the factors contribution

cally signiﬁcant at 95% conﬁdence limit in the experimental design.

A model is considered signiﬁcant if the p-value is lower than 0.05,

indicating only a 5% chance that their respective model could occur

(factor X1 for DG and factor X1 and X2 for HNG) were signiﬁcant,

acid concentration as one of the important factor affecting the acid

pretreatment of the Pennisetum sp. Lara et al. (2015) found similar

results in their study using dilute acid pretreatment for bioethanol

level of factors and rank of factors (HNG).

1 152.9 134.4 140.0 24.28 23.66 23.66

293 313 333 353 373 C

293 313 333 353 373 C

Table 4 respectively which are signiﬁcantly higher than deligniﬁcation

Table 5 3.4. Assessment of pretreatment using FTIR

Fig. 7. .Relationship between 1/T and ln (K) in a) DG and b) HNG.

Band position (cm- Assignment DG HNG

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