Mysore J. Agric. Sci.

, 56 (4) : 244-250 (2022)

Bioethanol Production from Foxtail Straw

K. C. KIRAN AND K. T. PRASANNA

Department of Forestry and Environmental Science, College of Agriculture, UAS, GKVK, Bengaluru - 560 065
e-Mail : chanakya.kirankc@gmail.com

AUTHORS CONTRIBUTION ABSTRACT

K. C. KIRAN :
Biofuel is a good substitute for fossil fuel as it is economical, renewable and environment
Conducting research, draft
preparation and data friendly emitting about 90 per cent fewer greenhouse gases (GHGs) than gasoline. The
analysis; long-term viability of bioethanol produced from first-generation feedstock is in question
K. T. PRASANNA : because it will ultimately lead to food insecurity. Therefore, to produce bioethanol,
Guidance and data curation second-generation processes that include lignocellulosic materials are gaining
importance. The current study aimed to produce the ethanol from lignocellulosic biomass
such as Foxtail straw. The Foxtail straw was pretreated with different combination of
Corresponding Author : acid and alkali to breakdown the cell wall composition of the straw. Then the pretreated
K. C. KIRAN samples were subjected to hydrolysis method such as, Simultaneous Saccharification
Department of Forestry & and fermentation (SSF) and Separate hydrolysis and fermentation (SHF) using the fungi
Environmental Science,
Saccharomyces cerevisiae with commercial enzyme. With different combination of acid
College of Agriculture,
UAS, GKVK, Bengaluru and alkali, the combination of NaOH and H2O2 pretreatment was considered as the
most suitable pretreatment because highest delignification (39%) was observed. NaOH
alone and combination of NaOH and H2O2 with Simultaneous Saccharification and
fermentation (SSF) method of hydrolysis and fermentation of Foxtail straw yields high
ethanol of 11.31 g/L and 11.00 g/L, respectively, which is four times higher than the
Received : February 2022
control. Significant structure and chemical bond changes in the feedstock after
Accepted : November 2022 pretreatment were found.

Keywords : Bioethanol, Foxtail Straw, Simultaneous Saccharification and fermentation, Separate hydrolysis and
fermentation, Saccharomyces cerevisiae

P RODUCTION of biofuels from renewable energy

sources is gaining importance in the light of the
increase in dependency on non renewable resources
consumption is contributed by renewable energy, out
of which bioenergy accounts for about 10 per cent.
The energy content in solid, liquid and gaseous
The Mysore Journal of Agricultural Sciences

due to the advancement of technology, depleting products derived from biomass is referred to as
oil reserve, rising fossil fuel prices and increasing Bioenergy (IEA, 2010).
greenhouse effect associated with the use of fossil
fuels. There are several advantages of biofuels such Bioethanol is a good substitute for fossil fuel as it
as environmental friendliness, biodegradability and is economical, renewable and environment friendly
high potential for local production from various emitting about 90 per cent fewer greenhouse gases
feedstocks. There is a renewed interest in using (GHGs) than gasoline. Bioethanol can be produced
sugar rich agricultural crops as feedstock for the by the process of fermentation using different sugars
biofuel production (Shalini et al., 2019) Ethanol such as starch, glucose, xylose, etc. The feedstock for
accounts for 90 per cent of total biofuels production bioethanol production is mainly divided into three
and is used in different parts of the world. More over, categories, i.e., starch-based, sugar-based (First
global crude oil production is predicted to decline. generation) and lignocellulosic material (Second
Today, about 13 per cent of the total energy generation) (Choudhary et al., 2013).

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Mysore J. Agric. Sci., 56 (4) : 244-250 (2022) K. C. KIRAN AND K. T. PRASANNA

India’s National Policy on Biofuels, 2018 sets MATERIAL AND METHODS

ambitious biofuel blending targets and aims to source
biofuels only from sustainable feedstocks. Feedstocks Collection and Preparation of Raw Material
are primarily defined as non-food feedstocks that do The Foxtail straw (FS) was collected from the
not threaten food security. Specifically, India intends threshing yard of the zonal agriculture research
to build upon its previous ethanol mandate by station (ZARS), UAS, GKVK, Bengaluru. The
expanding ethanol blending to 20 per cent by 2030; collected FS cut into small pieces was shade
the policy also adds a supplemental biodiesel dried and oven-dried (80ºC) for 48 hours. Then
mandate of 5 per cent. they were grounded and sieved using 2 mm sieve.
They are stored at room temperature in air-tight
The long-term viability of bioethanol produced bags for further use.
from first-generation feedstock such as sugarcane
and starch-rich feedstocks such as potato, corn, etc., Characterization of Biomass
is in question because it will ultimately lead
The composition of biomass was analyzed prior
to food insecurity by significantly increased
to the different pretreatment of FS. The FS was
dependence of amounts of cultivatable land and a
characterized for physicochemical properties
significant hike in food prices. This indicated that
such as cellulose, hemicellulose, lignin, ash
first-generation biofuel is not sufficient to meet the
content, carbon, nitrogen. The cellulose and
global energy demand. Therefore, to produce
hemicellulose were estimated by the procedure
bioethanol, second-generation processes that
outlined by Fruedenburg (1955), Lignin was
include lignocellulosic materials are gaining
estimated by the procedure given by Pandey et al.
importance. The production of ethanol from
(2007), the ash content determined by standardized
lignocellulosic biomass such as agriculture
method. The ADL was determined by the formula
residues (straw, cobs, wood chips, hull, sugarcane
given by Raffrenato and Van Amburgh (2011) and
bagasse, etc.) has become one of the best
the total carbon and nitrogen were estimated using
alternatives because of their widespread abundance
the CN analyzer (LECO Truspec, USA 2009).
and the procurement cost is relatively economical
(Joshi et al., 2011). Pretreatments

The FS with solid loading 8 per cent (w/v) was

Even though the plenty of lignocellulosic biomass,
pretreated with the following chemicals (Table 1).
the commercialization of the bioethanol production
Samples were autoclaved for 1 hour at 121°C
process is limited due to insufficient research,

The Mysore Journal of Agricultural Sciences

(at 15 psi pressure) and after the pretreatment,
related to minimization of the production cost.
samples were filtered, solid part was collected,
Bioethanol production from lignocellulosic materials
oven-dried and stored at room temperature in
relies on technologies that will efficiently hydrolyze
air-tight bags.
cellulosic biomass to fermentable sugars (Joshi et al.,
2011). Inoculum Preparation

One such residue is Foxtail straw which is available The Saccharomyces cerevisiae fungal culture
in plenty, unexplored and it is cheaper for large-scale was obtained from the Department of Agricultural
production of bioethanol. The present investigation Microbiology, UAS, GKVK, Bengaluru and
was undertaken to evaluate the potential of Foxtail maintained on MGYP medium (Composition: Malt
straw in the production of bioethanol through extract 3g, Glucose 10g, Yeast extract 3g,
different physicochemical pretreatment and hydrolysis Peptone 5g, Agar 20g, distilled water 1000 mL,
methods. pH to 4.4-4.6).

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 Mysore J. Agric. Sci., 56 (4) : 244-250 (2022) K. C. KIRAN AND K. T. PRASANNA

TABLE 1 Estimation of Ethanol

Pretreatments of feedstock
One ml of SHF and SSF samples drawn from
Pretreatments Treatment Concentration
each bottle and diluted with 35 ml of distilled
Hydrogen peroxide FSO1 5 % H2 O2 water. Each sample was distilled at 70°C and
FSO2 10 % H2 O2 the distillate containing alcohol was collected
Dilute alkali and FSN1O1 2 % NaOH + 5 % H2 O2 in 25 ml of 0.23 N K 2Cr2O7 solution, till a total
hydrogen peroxide FSN1O2 2 % NaOH + 10 % H2 O2
volume of 45 ml was obtained. Similarly, ethanol
standards (10-200 mg ethanol) were prepared
FSN2O1 4 % NaOH + 5 % H2 O2
separately using ethyl alcohol. These samples and
FSN2O2 4 % NaOH + 10 % H2 O2
standards were kept in water bath at 60°C for
Dilute acid and FSH1O1 1 % H2SO4 + 5 % H2 O2
30 min and were cooled, volume was made up to
hydrogen peroxide FSH1O2 1 % H2 SO4 + 10 % H2 O2
50 ml with distilled water and optical density was
FSH2O1 2 % H2 SO4 + 5 % H2 O2
measured at 600 nm using a spectrophotometer
FSH2O2 2 % H2 SO4 + 10 % H2 O2
(Multiskan Sky, Thermoscientific). The standard
Dilute alkaline FSN1 2 % NaOH curve was plotted considering the concentration
FSN2 4 % NaOH against absorbance. From the standard graph, the
Dilute acid FSH1 1 % H2 SO4 amount of ethanol in the sample was calculated
FSH2 2 % H2 SO4 (Caputi et al., 1968).
Control FSC Soaking in water
for 24 hours Statistical Analysis

The data were analyzed statistically for ethanol

Fermentation yield by factorial design using R software (version
100 g of FS sample in 1 L fermenting bottle, 1:10 4.1.0). Means for pretreatment and hydrolysis
ratio of solution containing the media (composition : and fermenting methods were considered to be
3.5 gL-1 K 2HPO4; 7.5 gL-1 (NH4) 2SO4; 0.75 gL -1 statistically significant at (P<0.05) level of
MgSO4.7H2O; 1 gL-1 CaCl2.2H2O; 5 gL-1 yeast extract) significance.
and citrate buffer (0.05M) were added, the pH was RESULTS AND DISCUSSION
adjusted to 4.5 and autoclaved for 20 minutes at
121°C, 15 psi and cooled to room temperature. These Raw Material Characterization
samples were used for fermentation.
The Foxtail straw was characterized to determine
the components of feedstock were presented
The Mysore Journal of Agricultural Sciences

Separate Hydrolysis and Fermentation (SHF) : Before

in Table 2. Cellulose accounts for 35.9 ± 0.06
fermentation, the samples were hydrolyzed using 1
per cent v/v of commercial enzyme and maintained at TABLE 2
30°C. The samples were allowed for enzymatic Feedstock composition of Foxtail straw
hydrolysis for four days. After that, 10 per cent v/v Parameters Composition (%)
inoculum was added and these bottles were incubated
at 30°C for fermentation for seven days. Cellulose 35.9 ± 0.06
Hemicellulose 24.6 ± 0.95
Simultaneous Saccharification and Fermentation Lignin 26.3 ± 0.20
(SSF) : 10 per cent v/v inoculum and 1 per cent v/v of Ash 17.7 ± 0.05
the commercial enzyme, was added at a time and these Total carbon 37.3 ± 0.07
bottles were incubated at 30°C for 12 days for Nitrogen 1.3 ± 0.01
fermentation. C/N ratio 29.9 ± 0.32

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Mysore J. Agric. Sci., 56 (4) : 244-250 (2022) K. C. KIRAN AND K. T. PRASANNA

per cent to the dry weight of raw material and and hemicellulose to reduce the crystallinity
hemicellulose content was found to be 24.6 ± 0.95 of cellulose (Tan et al., 2021). The suitable
per cent of dry biomass. The presence of high pretreatment and condition usually depend on the
holocellulose (Hemicellulose + Cellulose) content type of the lignocellulosic content present in the
(60.5±1.01%) in the cell wall of Foxtail straw, raw material (Taherzadeh and Karimi, 2008).
provides a potential feedstock for bioethanol
production. Foxtail straw contains 26.3±0.20 The composition of Foxtail straw after pretreatment
per cent lignin, 17.7±0.05 per cent ash content, was presented in the Table 3. The combination of
37.3±0.07 per cent of total carbon and 1.3±0.01 pretreatment showed the highest content of cellulose
per cent of nitrogen content. The similar results (65.90%), ADL (24.8%), lignin content (10.80%) and
were reported by Zhang et al. (2019) in millet low content of Hemicellulose were reported in
straw i.e., cellulose 36.68 per cent and lignin 19.38 pretreatment FSN2O1 (4% NaOH and 5% H2O2). The
per cent, hemicellulose content 17.3 per cent and same trend was observed in all the pretreatment
ash content 4.5 per cent. The difference in the cell with NaOH and H 2 O 2 combination. Fig. 1,
wall composition was due to heterogeneity in raw shows the variation in the composition of Foxtail
material, geographical location, season, processing straw after pretreatment, that lower the lignin
methods and analytical methods used for chemical higher the cellulose content in all the pretreatment
composition (Silverstein et al., 2007 and Binod methods. Dilute NaOH pretreatment of the
et al., 2012). lignocellulosic material was found to cause
swelling, leading to an increase in internal surface
Effect of Pretreatments on the Feedstock area of the Foxtail straw and rupture of lignin
Composition of Foxtail Straw structure. Silverstein et al. (2007) reported that
The pretreatments were imposed on the Foxtail 2 per cent of NaOH in 90 min at 121°C was the best
straw for the removal or to breakdown the lignin condition, resulting in 65 per cent of lignin removal.
TABLE 3
Feedstock composition of Foxtail straw after pretreatment
Pretreatments Cellulose (%) Hemicellulose (%) ADL (%) Lignin (%) Ash (%)

FS_CONTROL 34.60 # 20.90 * 43.80 * 27.80 * 16.00 *

FSH1 50.70 4.00 39.40 25.40 14.00

FSH1O1 59.60 7.80 30.80 18.80 12.00

The Mysore Journal of Agricultural Sciences

FSH1O2 46.50 6.40 35.60 23.60 12.00
FSH2 52.50 3.00 # 33.60 21.60 12.00
FSH2O1 48.10 4.90 35.00 25.00 10.00
FSH2O2 51.50 4.40 30.20 20.20 10.00
FSN1 64.80 6.70 28.40 18.40 10.00
FSN1O1 60.60 10.10 29.00 19.00 10.00
FSN1O2 53.10 5.60 30.80 16.80 14.00
FSN2 61.60 4.80 22.80 # 12.80 10.00
FSN2O1 65.90 * 5.30 24.80 10.80 # 14.00
FSN2O2 65.20 6.30 26.40 12.40 14.00
FSO1 52.80 14.20 25.40 23.40 2.00 #

FSO2 51.00 12.60 28.00 22.00 6.00

Note : * – Highest, # – Lowest

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of NaOH and H2O2. But the dilute NaOH pretreatment

was beneficial for the enzymatic hydrolysis of crop
residues, which was supported by the results of
Keshav et al. (2016); Prabu & Murugesan (2011) and
McIntosh & Vancov (2011). Thus, delignification
increases the effectiveness of enzymes access to
cellulose and hemicellulose for further
saccharification process (Bensah and Mensah, 2013).
Ethanol Yield from different Pretreatments and
Fig. 1 : Variation in composition of Foxtail straw Fermentation Methods
after pretreatment
The methods used for hydrolysis and
In this study, all the pretreatments were conducted at fermentation of Foxtail straw after pretreatments
121°C, 15 psi for 60 min, which resulted in the were i) Simultaneous Saccharification and
removal of 52 per cent and 39 per cent, ADL and Fermentation (SSF), ii) Separate Hydrolysis and
lignin, respectively in the pretreatment combination, Fermentation methods (SHF). The ethanol yield
was found to be significant in fermentation methods
TABLE 4
for all the pretreatments, but highest ethanol yield
Average ethanol yield from different pretreatments (11.31 g/L) was recorded in the SSF fermentation
and fermentation methods of Foxtail straw
method of raw material which is pretreated with 2
Average Ethanol Yield (g/L) per cent NaOH (FSN1) (Table 4) and average ethanol
Pre-treatments
SSF SHF Average
yield (9.10%), followed by the combination of NaOH
and the H2O2, lowest ethanol yield was observed in
FSN1 11.31 a
6.89 h
9.10 a
the H2O2 alone, which is lesser than that of control
FSN1O1 11.00 b
5.04 s
8.02 b
(Table 4). Whereas in the SHF fermentation method
FSN2O1 8.46 e
6.64 j
7.55 c
highest ethanol yield (7.80 g/L) was recoded in raw
FSN1O2 9.86 c
4.71 v
7.29 d
material pretreated with 2 per cent H2SO4 and 5 per
FSN2O2 8.60 d
5.53 o
7.06 e
cent H2O2 (FSH2O1) and lowest was observed in the
FSH2O1 6.04 m
7.80 f
6.92 f
H2O2 alone pretreatment (Table 4). Similar results were
FSN2 7.47 g
4.73 u
6.10 g
observed in the sweet sorghum bagasse (6 .12 g/L)
FSH1O1 5.25 q
6.75 i
6.00 h
(Cao et al., 2012) and higher ethanol production
FSH1 5.10 r
6.28 k
5.69 i
observed in the cotton stalk (23.17 g/L) (Keshav
FSH2O2 6.24 4.86 5.55 et al., 2016 and Govumoni et al., 2013). From the
The Mysore Journal of Agricultural Sciences

l t j

FSH2 4.71 v
5.72 n
5.21 k
Table 5, it was clear that SSF method has highest
FSH1O2 5.43 p
2.21 z
3.82 l

FS_CONTROL 3.15 w
2.34 y
2.74 m TABLE 5
FSO2 1.91 A
3.15 w
2.53 n Average ethanol yield from different fermentation
FSO1 1.59 B
2.51 x
2.05 o methods of Foxtail straw
Average 6.41 5.01 5.71 Fermentation methods Ethanol (g/L)
STDEV 3.03 1.78 2.11 SSF 6.41 a
SE ± 0.78 0.46 0.54 SHF 5.01 b
CD @0.05% 0.01 0.01 Average 5.71
Average 5.71 STDEV 0.99
STDEV 2.54 SE ± 0.70
SE ± 0.46 CD @0.05% 0.004

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Mysore J. Agric. Sci., 56 (4) : 244-250 (2022) K. C. KIRAN AND K. T. PRASANNA

ethanol yield than the SHF method. Similar results times higher than the control. Significant structure and
were recorded by Zhu et al., (2012); Alejo et al., chemical bond changes in the feedstock after
(2020). pretreatment were found. Further research is needed
on improving the delignification and ethanol yield,
Correlation of Compositional Parameters of
as this work provides the foundation for future work.
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