Appl Biochem Biotechnol

DOI 10.1007/s12010-013-0366-0

Production of Bioethanol from Fermented Sugars

of Sugarcane Bagasse Produced by Lignocellulolytic
Enzymes of Exiguobacterium sp. VSG-1
S. Vijayalaxmi & K. A. Anu Appaiah &
S. K. Jayalakshmi & V. H. Mulimani & K. Sreeramulu

Received: 3 March 2013 / Accepted: 23 June 2013

# Springer Science+Business Media New York 2013

Abstract Exiguobacterium sp. VSG-1 was isolated from the soil sample and characterized for
the production of lignocellulolytic enzymes. Production of these enzymes by the strain VSG-1
was carried out using steam-exploded sugarcane bagasse (SCB) and found to secrete cellulase,
pectinase, mannanase, xylanase, and tannase. The growth and enzyme production were found
to be optimum at pH 9.0 and 37 C. Upon steam explosion of SCB, the cellulose increased by
42 %, whereas hemicelluloses and lignin decreased by 40 and 62 %, respectively. Enzymatic
hydrolysis of steam-exploded SCB yielded 640 g/l of total sugars. Fermentation of sugars
produced from pretreated SCB was carried out by using Saccharomyces cerevisiae at pH 5.0
and 30 C. The alcohol produced was calculated and found to be 62.24 g/l corresponding to
78 % of the theoretical yield of ethanol. Hence, the strain VSG-1 has an industrial importance
for the production of fermentable sugars for biofuels.
Keywords Exiguobacterium sp. VSG-1 . Lignocellulolytic enzymes . Sugarcane bagasse
(SCB) . Enzymatic hydrolysis . Bioethanol

Introduction
Extensive research has been carried on the conversion of agro waste cellulosic materials to
bioethanol in the last two decades [17]. The demand for ethanol usage as a chemicals
S. Vijayalaxmi : V. H. Mulimani : K. Sreeramulu (*)
Department of Biochemistry, Gulbarga University, Gulbarga 585106, Karnataka, India
e-mail: ksramu@rediffmail.com
K. A. Anu Appaiah
Department of Food Microbiology, Central Food Technological Research Institute,
Mysore 570020, Karnataka, India
S. K. Jayalakshmi
Agricultural Research Station, University of Agricultural Sciences-Raichur,
Gulbarga 585103, Karnataka, India

Appl Biochem Biotechnol

feedstock or as an octane enhancer or petrol additive in the market is increasing day by day.
Lignocellulosics that show potential for ethanol production include agricultural residues
(i.e., corn stover, wheat straw, and rice straw), agricultural by-products (i.e., corn fiber, rice
hull, sugarcane bagasse), energy crops (i.e., switch grass, sweet sorghum, high fiber sugarcane, miscanthus) [8]. The major components of lignocellulosics are cellulose (polymers of
hexose sugars, 3550 %), hemicelluloses (polymers of pentose sugars, 2035 %), and lignin
(polyphenols, 1025 %) [8, 9]. The large-scale use of lignocelluloses for the production of
biofuels or other value-added products depends on the breakdown of cellulose, hemicelluloses, and lignin into their main components. Pretreatment of lignocelluloses is an important
step for an efficient use of biomass for the production of fermentable sugars. Removal of
lignin and hemicelluloses, reduction of cellulose crystallinity, and increase of porosity in
pretreatment process can significantly improve the hydrolysis [10] and avoid the formation
of inhibitors. Several pretreatment methods have been in use for the hydrolysis of lignocellulosics. The advantages of steam explosion pretreatment include lower energy required
compared to mechanical + combination and no recycling of environmental costs. The
conventional methods required 70 % more energy than steam explosion to achieve
the same size reduction [11]. Steam explosion is recognized as one of the most costeffective pretreatment processes for hardwoods and agricultural residues, but it is less
effective for softwoods [12]. Alkaline [13] and acid [14] hydrolysis methods have
been used to degrade lignocelluloses. Weak acids tend to remove lignin but result in poor
hydrolysis of cellulose whereas strong acid treatment occur under relatively extreme
corrosive conditions at high temperature and pH which necessitate the use of expensive
equipment.
Both bacteria and fungi can produce cellulases for the hydrolysis of lignocellulosic
materials. These microorganisms can be aerobic or anaerobc, mesophilic, or thermophilic.
Bacteria belong to Clostridium, Cellulomanas, Bacillus, Thermomonospora, Ruminococcucs,
Bacteroides, Erwinia, Acetovibrio, Microbispora, and Streptomyces can produce cellulases
[15]. Although many cellulolytic bacteria particularly the cellulolytic anaerobes such as
Clostridium thermocellum and Bacteroides cellulosolvens produce cellulase with high specific
activity but they do not produce high enzyme titers [7]. Several fungi have been reported to
produce cellulases [16]. A non-exhaustive list of cellulolytic microorganisms of aerobic and
anaerobic forms isolated from various habitats has been reported [17]. Fungi and yeasts have
frequently been applied in the development of industrial enzymes. However, bacteria have
several advantages over fungi in the production of hydrolytic enzymes, in terms of and enzymes
titer and the time; for example, many strains have short generation times and can be easily
cultured, making the use of bacteria in the biofuel industry more amiable. Additionally, bacteria
also have increased resilience to environmental stresses due to their biochemical versatility (i.e.,
temperature variations, salinity, oxygen limitation, and change in pH) [18]. Researchers have
typically focused on one group of enzymes during isolation, such as cellulases, hemicellulases,
or lignases. For example, white rot fungi are among the greatest microorganisms which can
degrade lignin and the most well studied [19]. However, anaerobic bacterium Clostridium
thermocellum and aerobic fungi Trichoderma reesei are among some of the greatest cellulaseproducing microorganisms [20]. Nonetheless, none of these microorganisms are efficient at
cellulolytic, hemicellulolytic, and ligninolytic activities simultaneously, rendering the opportunity for discovery of better lignocellulase-producing isolates. Thus, greater importance is being
given for the discovery and characterization of new microorganisms that are able to degrade
complex plant biomass more efficiently into fermentable sugars. An organism utilizes for this
purpose would have to express a mixture of several enzymes including cellobiohydrolases,
hemicellulases, and pectinases. Cellulolytic and hemicellulolytic multienzyme complexes have

Appl Biochem Biotechnol

also been reported in Bacillus circulans, Bacillus megaterium [21, 22], and Paenibacillus
curdlanolyticus [23].
Conventionally, the production of enzymes is very expensive and raw material translates
into 4060 % of the production cost [24]. Starch-based substrates such as maize, wheat, oats,
cassava, potato, and rice were the potential sources for the ethanol fermentation by microbial
processes [25]. In USA and Brazil, fuel ethanol is produced by fermentation of corn glucose
and or sucrose [26, 27], but any country with a significant agronomical-based economy can
use current technology for fuel ethanol fermentation. However, diverting food crops and
their produce for ethanol production is a cause of concern for food and nutrition in
developing and under developing countries. Lignocellulose materials which represent the
most abundant alternative and cost-effective source of biomass can be converted into fuel
ethanol.
In this perspective, this study was aimed at the utilization of agro waste, sugarcane
bagasse, as growth substrates for the production of cellulolytic, hemicellulolytic, and
pectinase enzymes. Among the agricultural residues, sugarcane bagasse (SCB) is a substrate
of high potential for biotechnological processes, which comprises 4042 % cellulose, 2428 %
hemicelluloses, and 1012 % lignin.
In this study, we isolated a new Bacillus sp. Exiguobacterium sp. VSG-1 able to hydrolyze
SCB more efficiently to fermentable sugars. There have been no reports on the production of
lignolytic enzymes by Exiguobacterium sp. using sugarcane bagasse as a substrate. Further, the
present study will investigate the saccharification of steam-exposed SCB by the cell-free extract
of Exiguobacterium sp. VSG-1 followed by ethanol fermentation of glucose with Saccharomyces cerevisiae.

Materials and Methods

Chemicals
Carboxymethylcellulose (medium viscosity, 400800 cP), cellulose powder (Sigma cell
Cellulose, Type 20; particle size 20 m), oat spelt xylan, and locust bean gum were purchased
from Sigma Chemical Company (St. Louis, MO, USA). Pectin, tannic acid, starch, and casein
were purchased from Himedia Chemicals, Mumbai, India. All other reagents were of analytical
grade.
Isolation and Screening of Microorganism
Soil samples were collected around grain mills of Gulbarga, India, and suspended in sterile
saline. Aliquots were inoculated on nutrient agar plates of pH 7.0. The plates were incubated
at 37 C for 48 h. Isolated colonies were then purified through a serial streaking method on
nutrient agar. From these plates, isolated colonies were taken and repeatedly streaked on
nutrient agar to obtain pure cultures. This isolated culture was screened for their ability to
produce enzymes like protease, amylase, xylanase, pectinase, cellulase, and tannase. The
pure bacterial cultures were subsequently transferred into nutrient broth.
Microscopic and Biochemical Characterization
Cell morphology, motility, Gram reaction, and the presence of spores and capsule were
studied using standard protocols. Isolated colonies were tested for catalase, oxidase,

Appl Biochem Biotechnol

utilization of sugars, growth at low and high pH, citrate utilization, indole and urease
production, MR, VP, nitrate reduction, starch hydrolysis, and gelatin liquefaction. Further
tests, viz., growth at pH 5.010.0, temperature ambient from 30 to 50 C, and salinity
tolerance from 1 to 16 % sodium chloride, were carried out. Results were analyzed as per
Bergeys Manual of Systematic Bacteriology [28].
16S rRNA-Based Identification
The partial sequence of the amplified DNA was determined by Ocimum Biosolutions,
Hyderabad, India, and deposited in GenBank under the accession number JQ312121
(http://www.ncbi.nlm.nih.gov/nuccore/JQ312121). Related sequences were obtained from
the GenBank database (National Center for Biotechnology Information) using BLAST. A
phylogenetic tree was constructed by the neighbor-joining method using the MEGA 5.0
software.
Sugar Cane Bagasse Pretreatment by the Steam Explosion Process
SCB was collected from local market, India. The SCB was ground and sieved until the SCB
particles were able to pass through a 60 mesh (0.3 mm) sieve and only these particles were
used for the pretreatment experiments. This material was washed with water until to neutral
pH and dried at 505 C to attain 10 % moisture content (untreated material) and steamexploded separately at certain pressures for 10 to 15 min using autoclave under the following
conditions: 150 and 160 C for 10 min at 1:10 w/v solid/liquid ratio with 6 rpm agitation. The
pretreated SCB was washed with water several times for the removal of residual quantities of
hemicellulosic hydrolysate and then the mass yield measured.
Enzyme Production
Bacterium was grown in a medium containing (in gram per liter): raw SCB, 10.0; peptone,
5.0; NaCl, 5.0; K2HPO4, 2.0; MgSO4, 1.0; and yeast extract, 0.5. After incubation at 37 C,
150 rpm for 48 to 72 h, the contents were centrifuged at 8,000 rpm for 10 min and the cell-free
extract was used as an enzyme source.
Enzyme Assay
The cellulase, mannanase, xylanase, and pectinase activities were carried out according to
dinitrosalicylic acid method [29]. The reaction mixture consisted of 100 l of enzyme
solution, 400 l of 1 % (w/v) corresponding substrate, and 500 l of 50 mM phosphate
buffer of pH 9.0. The mixture was incubated at 50 C for 10 min. The enzyme activity was
determined by measuring the release of reducing sugars. One unit (U) of activity was defined
as the amount of enzyme producing 1 mol/ml/min of reducing sugar under the standard assay
conditions. The protein concentration was measured according Lowrys method [30] using
bovine serum albumin as a standard.
Effect of Temperature, pH, and NaCl Concentrations on the Growth and Lignocellulolytic
Enzymes Production by Exiguobacterium sp. VSG-1
This was carried out by growing the organism at different temperatures (2060 C), different
initial pH values using 50 mM adequate buffers (46, acetate buffer; 78, phosphate buffer;

Appl Biochem Biotechnol

and 912, glycineNaOH buffer), and NaCl concentrations, 016 % (w/v). The enzymes
activity and biomass were measured at optimum growth (48 h).
Enzymatic Hydrolysis of Steam-Exploded Materials
Different concentrations (2 to 50 mg) of the cell-free extract of Exiguobacterium sp. VSG-1
grown at 48 h, was added to 500 ml Erlenmeyer flask containing carbonate buffer (50 mM,
pH 9.0) and 10 g of steam-exploded or unexploded SCB so that the slurry concentration
became 10 % w/v. The flasks were gently mixed to make the slurry uniform. Enzymatic
hydrolysis experiments were carried out at 40 C under the static conditions. Liquid loss
from evaporation of the buffer solution was prevented by tightly sealing the flask. Hydrolysis was terminated by boiling at 100 C for 5 min at the end of stipulated time intervals,
filtered, and the filtrate was collected. The enzymatic hydrolyzation was calculated as
percentage of reducing sugars released.
Estimation of Total Sugars, Reducing Sugars, and Polyphenols
The filtrate was assayed for total sugars by sulfuric acid method [31], reducing sugars by
dinitrosalicylic acid method [29], and inhibitory compounds like polyphenols [32].
Estimation of Sugars by HPLC
Sample slurry was centrifuged (8,000 rpm, 4 C, 10 min) and filtered. The glucose, xylose,
and arabinose concentrations were quantified by high-performance liquid chromatography
(HPLC) on a micro Bond pack Amino Carbohydrate column (4.1300 mm). Samples (20 l)
were injected and eluted with acetonitrilewater (70:30 ratio) at a flow rate of 1 ml/min. The
hydrolyzed products were detected using a refractive index detector.
Culture Conditions of S. cerevisiae
S. cerevisiae (MTCC S-170) is a kind gift of Dr. Anu Appaiah, CFTRI, Mysore. The yeast
culture was maintained in YPD agar media containing (in gram per liter): glucose, 20.0;
peptone 20.0; yeast extract 10.0; and agar, 20.0 at pH 5.5, temperature at 30 C.
Inoculum Preparation and Fermentation
Inoculum (25 ml) was prepared in 100 ml Erlenmeyer flask with the following media
composition (in gram per liter): glucose, 20.0; peptone 20.0; and yeast extract 10.0 at pH
5.0 and incubated on a rotary shaker for 24 h (150 rpm) at 30 C. After 24 h, the cells were
recovered by centrifugation. The fermentation medium (100 ml) was prepared in 250 ml
Erlenmeyer flask containing hydrolyzed SCB (about 30 g/l of glucose) with the supplementation of 0.2 % of yeast extract or fish protein as a nitrogen source. The pH of the medium
was adjusted to 5.0 using 1.0 M acetic acid by inoculating with 10 % of 24 h inoculum and
incubated at 37 C in a rotary shaker at 150 rpm for 24 h.
Growth Versus Ethanol Production of S. cerevisiae
Glucose fermentation was carried out to characterize the time-dependent changes of cell
growth, sugar consumption, and ethanol production by the yeast. Thus, the culture was

Appl Biochem Biotechnol

incubated under aerobic conditions at an agitation speed of 150 rpm. Erlenmeyer flasks
(100 ml) plugged with cotton were used for a working volume of 25 ml containing
hydrolyzed SCB (30 g/l), yeast extract (10 g/l), or fish protein (10 g/l) at pH 5.0. The flasks
were incubated at 30 C on a rotary shaker at 150 rpm for 72 h.
Effect of Initial pH on Ethanol Production by S. cerevisiae
The effect of the initial pH of the fermentation medium was studied by using pH values of
4.0, 4.5, 5.0, 5.5, and 6.0. The pH was adjusted with 0.1 M acetic acid. The overnight YPD
culture was inoculated into 25 ml of medium containing 10 g/l yeast extract and 30 g/l of the
desired sugar described above, and incubated at 30 C, 150 rpm for 48 h. Precultured cells
were inoculated as described above.
Estimation of Alcohol
Estimation of alcohol was done according to [33]. In brief, standards and sample (2 ml) were
taken in a distillation flask. Volume was made up to 50 ml with distilled water and distilled at
5060 C. Fifteen milliliters of distillate was collected in a clean conical flask and 25 ml
chromic acid solution was added. Volume was made up to 50 ml with distilled water. Conical
flasks were incubated at 50 C on water bath for 30 min, then the solutions were brought to
room temperature and optical density was read at 600 nm. The ethanol yield was calculated
by modified formula proposed by Gunasekharan and Kamini [34].
Estimation of Alcohol by Gas Chromatography
The alcohols present in the samples were determined using gas chromatography Shimadzu
GC-6A. A Porapak Q column with a temperature of 180 C was used with nitrogen as a
carrier gas along with a flow rate of 40 ml/min. The injection and departure temperature
were 220 and 230 C. Injected into the column were 0.2 l of alcohol standards and sample
distillates. Peaks were identified by comparing the retention time of the standard alcohols.
Statistical Analysis
The results are the means of three independent experiments. The data were statistically
evaluated using parametric statistic program, version 1.01 (Lundon software, Inc., Chagrin
Falls, OH, USA).

Results
Identification of the Bacterial Isolate
Strain VSG-1 is a gram-positive bacteria, non-sporulating rods, 0.51.06210 mm, occurring singly, in pairs, or in short chains, and motile by means of peritrichous flagella. It is
moderately thermophilic (growth between 30 and 50 C, no growth above 50 C, optimum
at 37 C) and halotolerant (growth in the presence of 12 % NaCl, optimum 1 % NaCl) and
pH range for growth is 5.010.0 with optimum at pH 9.0. The isolate produced yellowish
orange, smooth, glossy, and opaque colonies on nutrient agar plate. It was positive for
methyl red, citrate utilization, arginine utilization, catalase and oxidase reactions, but

Appl Biochem Biotechnol

negative for VP reaction, nitrate reduction, indole utilization, and H2S production. Casein,
gelatin, and starch were hydrolyzed. It utilized glucose, fructose, sucrose, maltose, mannitol,
and trehalose as sole carbon sources. Strain VSG-1 was identified by sequence analysis of
the amplified 1,029-bp segment of its 16S rRNA gene. The strains VSG-1 belonged to the
genus Exiguobacterium, order Bacillales, family Bacillaceae and showed 99 % similarity to
the members of Exiguobacterium (Fig. 1).
Enzyme Production and Assay
Lignocellulolytic enzymes production was observed in the fermentation broth as soon as the
bacterium entered the exponential phase (18 h) and reached maximum in the stationary
phase (48 h). The optimum culture conditions for growth and enzymes production were 48
60 h of incubation after which remained more or less stable until 72 h and then decreased with
increase of incubation time (Fig. 2). Cellulase, pectinase, mannanase, and xylanase activities
were recorded as 38.4, 48.2, 26.6, and 22.8 U/ml, respectively, at 48 h of incubation.
Effect of pH, Temperature, and NaCl Concentrations on the Growth of Exiguobacterium
sp. VSG-1
The highest growth was observed in alkaline pH (710) with an optimum at 9.0. There was
low growth at pH 7, whereas high growth was noticed at pH 810. Maximum growth was
observed in the temperature range of 3050 C with optimum at 37 C. The Exiguobacterium
sp. strain VSG-1 was able to grow in a broad-range NaCl concentration (116 %) with
optimum at 1 % NaCl. This clearly indicates the halotolerant nature of the strain VSG-1 (data
not shown).

Fig. 1 Neighbor-joining phylogenetic dendrogram based on 16S rRNA gene sequence data indicating the
position of strain VSG-1 among members of the genus Exiguobacterium. Accession numbers of 16S rRNA
gene sequences of reference organisms are indicated. Bootstrap values from 1,000 replications are shown at
branching points; only values above 60 are shown. Bar, 0.01 substitutions per 100 nt

60

50

2.5

40

30

1.5

20

10

0.5

Biomass at 660nm

Fig. 2 Effect of incubation time on

the growth and lignocellulolytic
enzyme production by
Exiguobacterium sp. VSG-1 at
pH 9.0 and temperature 37 C

Activity (U/ml)

Appl Biochem Biotechnol

0
0

16

24

32

40

48

56

64

Incubation time (h)

Cellulase

Pectinase

Mannanase

Xylanase

Growth

Pretreatment of SCB
The composition of the raw material used in this work is shown in Table 1. The raw SCB
showed a high carbohydrate content (about 42.6 % of cellulose). This table also shows the
proportional increase of the cellulose by 40 %, whereas hemicellulose and lignin content
decreased by 50 and 60 %, respectively, in the pretreated SCB (150 C; 160 C for 10 min)
compared the raw SCB, due to solubilization of the hemicellulosic fractions.
Effect of Enzyme Concentration on the Hydrolysis of Pretreated SCB
Significant increase in the production of reducing sugars was observed as the concentration
of enzyme increases and reached enzyme loading at 40 mg protein/g steam-exploded SCB.
Thus, the enzymes are saturated at 40 mg protein/g steam-exploded SCB under the experimental conditions (Fig. 3).
Estimation of Total Sugars, Reducing Sugars, and Polyphenols
The estimation of total sugars, reducing sugars, and polyphenols were calculated from the
hydrolyzate of steam-exploded SCB and found to be 640, 45, and 4.6 mg/ml, respectively,
Table 1 Chemical composition of SCB before and after pretreatments with steam explosion
Components

Pretreatment conditions of SCB

Raw SCB

150 C/10 min

160 C/10 min

Cellulose

42.60.1*

57.30.2*

58.80.1*

Hemicelluloses
Lignin

24.70.2*
22.30.1*

14.80.1*
23.80.2*

13.50.2*
23.40.1*

Ash

1.50.3*

2.20.1*

2.80.1*

Extractives

5.60.1*

1.20.2*

1.10.2*

96.70.1*

98.10.1*

98.50.1*

Total
standard deviation

*P<0.05 (two paired sample Students t test)

Appl Biochem Biotechnol

Glucose concentration (g/l)

100
80
60
40
20
0

6
8
Reaction time (h)

2 mg/g

5 mg/g

10 mg/g

20 mg/g

30 mg/g

40 mg/g

10

12

Fig. 3 Changes in glucose concentrations during enzymatic hydrolysis of 10 % w/v SCB at different loadings
of extracellular enzymes by Exiguobacterium sp. VSG-1 under static condition. Enzyme loading is in
milligram protein/gram steam-exploded SCB

whereas unexploded SCB hydrolyzate showed low levels of sugars and high levels of
polyphenols (Table 2).
Effect of pH and Temperature on the Growth and Alcohol Fermentation by S. cerevisiae
Maximum growth and alcohol production by S. cerevisiae S-170 were observed in acidic pH
(5.06.0) with an optimum at 5.5 and the temperature range of 2535 C with optimum at
30 C during the alcohol fermentation (data not shown).
HPLC Analysis of Sugars
The products obtained from enzymatic hydrolysis of pretreated SCB were analyzed and
found to be as glucose, xylose, and arabinose (Fig. 4).
Fermentation of Alcohol
Alcohol production was started in the fermentation broth at 48 h and reached maximum at 72 h.
The optimum cultural conditions for the production of alcohol were up to 72 h, which will remain
more or less up to 96 h and then decreased with increase of incubation time (Fig. 5). The estimation
Table 2 Estimation of total sugars, reducing sugars, and polyphenols after pretreatment of steam explosion
(hydrothermal) and enzymatic hydrolysis of SCB
Untreated SCB
Total sugars (mg/ml)

Steam-exploded SCB

Enzymatic hydrolyzed SCB

6400.1*

17.50.1*

670.2*

Reducing sugars (mg/ml)

1.50.2*

8.20.1*

450.2*

Polyphenols (mg/ml)

7.40.1*

6.20.2*

4.60.1*

standard deviation
*P<0.05 (two paired sample Students t test)

Appl Biochem Biotechnol

4.367

0.004

5.400

4.725

0.002

0.000
3

10

Minutes

4.3-glucose
4.7-xylose
5.4-galactose
Fig. 4 HPLC profile of sugars present in hydrolysed SCB before fermentation

of total sugars, reducing sugars, and polyphenols was calculated from the hydrolyzate of steamexploded SCB before and after the fermentation and the results were presented in Table 3.
Estimation and Detection of Alcohol
The alcohol percentage was estimated and found to be 7.8 or 7.2 % when the fermentation
was carried out with the fish protein or yeast extract, respectively, as a nitrogen source
(Table 4). The alcohol obtained from yeast fermentation was analyzed by gas chromatography and identified as ethyl alcohol (Fig. 6).

Discussion

100

80

60
4
40
2

20
0

0
0

24
48
72
Fermentation days (h)
Glucose concentration (%)
Alcohol content (%v/v)

96

Alcohol content ( %v/v)

Fig. 5 Changes in sugar concentration and alcohol contents of

SCB sugar syrup over the
fermentation period

Glucose concentration (%)

Temperature is a major environmental condition that affects microbial physiology and

growth. Every bacterial sp. has its own optimum temperature and cultural conditions for

Appl Biochem Biotechnol

Table 3 Calculation of total sugars, reducing sugars and polyphenols before and after fermentation of alcohol
Before fermentation

After fermentation

Total sugars(mg/ml)

640.60.1*

58.80.1*

Reducing sugars (mg/ml)

45.20.1*

8.50.1*

Polyphenols (mg/ml)

3.80.1*

0.50.1*

standard deviation
*P<0.05 (two paired sample Students t test)

survival. However, Exiguobacterium sp. have been isolated from and molecularly detected
in a wide range of habitats. The unique feature of this bacterial sp. is to grow under extreme
environmental conditions with the temperature ranging from 12 to 55 C with minimum
nutrients. The Exiguobacterium genus comprises psychrotrophic, mesophilic, and moderate
thermophilic species and strain with pronounced morphological diversity (ovoid, rods,
double rods, and chains) depending on the species, strain, and environmental conditions
[35]. Until now, studies of Exiguobacterium sp. mainly focused on characteristics of
resistance to extreme conditions such as high/low temperature, alkaline environment, and
high concentrations of salts [36]. The biotechnological applications of these strains, especially in the biodegradation of lignocellulosic materials have not been explored.
The characterization of cellulolytic and hemicellulolytic bacteria have been given much
attention as readily available abundance of lignocellulosics carbon source, which can be
degraded to fermentable sugars for the production of bioethanol [37]. Therefore, evaluating
these activities in our isolate is pertinent to finding an efficient lignocellulosic bacterium. As
a result, it was important for us to distinguish those strains which can degrade amorphous and
crystalline cellulose by cellulase in addition, to xylanase activity. Hence, we could isolate a
strain producing high titer of lignocellulosic enzymes. Enzymatic activities of lignocellulolytic
suggest their prominent role during the breakdown of SCB. Several bacterial strains have been
isolated and screened for the lignocellulolytic enzymes [38].
Pretreatment is necessary for lignocellulosics to achieve a highly efficient enzymatic
hydrolysis and fermentation. Composition of untreated and steam explosion pretreated SCB
is summarized in Table 1. Considerable amounts of hemicelluloses (50.20 %) and lignin
(65.02 %) were removed during the process. However, the cellulose content is increased by
more than 40 % in the treated SCB. Kim and Lee [39] reported 5379 % delignification with
7597 % removal of hemicelluloses and 411 % removal of cellulose from corn stover by a two
stage hot water and ammonia recycle percolation process at high temperature (170210 C).
The main objective of the steam explosion pretreatment is the delignification (65.02 %) SCB,
which further increases the area and porosity of hemicelluloses and cellulose and its utilization
for production of value-added materials.

Table 4 Ethanol yields for untreated and pretreated SCB

g/l

Theoretical yield (%)

Untreated SCB

12.30.1*

210.1*

Pretreated SCB

62.240.1*

780.1*

standard deviation
*P<0.05 (two paired sample Students t test)

Appl Biochem Biotechnol

Fig. 6 Gas chromatogram of alcohol from fermented SCB. Oven temperature, 180 C; injector and detector
temperatures, 220 and 230 C, respectively

Enzymatic hydrolysis of cellulosic substances was carried out by cellulase enzymes,

which are highly specific [40]. The products of the hydrolysis are usually reducing sugars
including glucose. Utility cost of enzymatic hydrolysis is low compared to acid or alkaline
hydrolysis because enzyme hydrolysis is usually conducted at mild conditions and does not
have any corrosion problem and environmental problems [7]. Untreated and treated SCB
were hydrolyzed for 48 h using cell-free extract of Exiguobacterium sp. VSG-1. The high
percentage of digestibility in the treated material can be attributed to lignin removal. The
crude supernatant preparation had a wide range of activities on insoluble substrates. The
predominant was cellulase, but mannanase and pectinase were also present. Since the
xylanase activity was low, it is advantageous for hydrolysis process as the supernatant
contains low level of pentoses which are not desirable for the fermentation of alcohol. The
structures of multienzyme complexes (MEC) have not been elucidated in detail, but many of
them contain predominantly cellulase activity found in the cellulosomes. In this study, it was
found that the MECs were able to hydrolyze insoluble cellulose. The ability to bind
insoluble substrates has been considered important due to the fact that degradation of
insoluble substrates was inextricably linked to the enzymes/complexs ability to bind and
thus remain in close proximity to the substrate while it is hydrolyzed. Further, binding to
crystalline cellulose has been a feature of the cellulosome as the scaffolding protein of the
cellulosome which contains a CBM3a domain which is able to bind crystalline substrates
[19]. Cellulosomes have been found to bind only very weakly to insoluble xylan [41]. Use of
mixture of cellulases and other enzymes in the hydrolysis of cellulosic materials have been
extensively studied [4244]. A cellulose conversion yield of 90 % was achieved in the
enzymatic saccharification of 8 % alkali-treated sugarcane bagasse when a mixture of
cellulases from Aspergillus ustus and Trichoderma viride [45]. A nearly complete saccharification of steam explosion pretreated Eucalyptus viminalis chips was obtained using a
cellulose mixture of commercial celluclast and novozyme preparations [46]. Use of commercial enzymes for saccharification increases the cost of the process.
The maximum yield of total reducing sugars (640 g/l) was obtained when 10 g of SCB
was mixed with 40 ml of crude enzyme extract. There was not much significant increase of
reducing sugars yield with increasing concentration of crude enzyme extract. The yield of
glucose (395 g/kg) and xylan (135 g/kg) achieved in this study were higher than those sugars

Appl Biochem Biotechnol

of sugarcane bagasse (225 g glucose/kg dry and 62 g xylose/kg biomass) [47] and sorghum
(glucose, 400470 g/kg; xylose, 130170 g/kg dry sorghum) [48] pretreated with ammoniawater.
Several reports have been published on the production of ethanol fermentation by
bacteria, yeast, and fungi [49]. The most commonly used yeast, S. cerevisiae, produced
ethanol as high as 18 % of the fermentation broth and is the preferred one for most of the
ethanol fermentation. This yeast can grow both on simple sugars, such as glucose, and on the
disaccharide, sucrose. Saccharomyces is also generally recognized as safe as a food additive
for human consumption and is therefore ideal for producing alcoholic beverages and for
leavening bread. Ethanol concentration reached its highest peak at 72 h (Figs. 5 and 6) and
no further increase was observed at 96 h (data not shown). At the end of the fermentation
process, ethanol concentrations reached 642 g/kg of pretreated SCB. This yield is higher
than those reported using dilute ammonia-treated sorghum, 250 g/kg [48], and sulfuric acid
pretreated sorghum, 141 g/kg [50]. Mamma [51] reported 115 g/kg dry sorghum ethanol
from sorghum fibers using mixed culture of Fusarium oxysporum and S. cerevisiae. Sugarcane bagasse and sorghum are grass plants and have the similar composition of lignocellulosic materials. Shaibani et al. [52] have reported that the only 50 % ethanol was produced
by simultaneous saccharification and fermentation of sugarcane bagasse with crude enzyme
solutions of Trichoderma longibrachiatum and S. cerevisiae yeast.
Production of fermentable sugars from the cheap agro waste lignocellulosic materials is a
crucial step for the success of alcohol and beverage industries. It is further constrained by costly
inputs, process operation, and time. Overall, on comparison with the other strains of bacteria
and pretreatment methods, the present study demonstrates that the strain of Exiguobacterium sp.
VSG-1 is the most promising candidate, producing high levels of fermentable of sugars from
steam-exploded SCB. The overall time taken to produce 64.2 g/l of ethanol from pretreated
SCB is 5 days.
The newly isolated strain, Exiguobacterium sp. VSG-1, is an excellent source for
lignocellulolytic enzymes for the production of fermentable sugars from SCB. This strain
may have great potential for developing bacterial consortiums in the near future to enhance
the decomposition of lignocellulosic biomass and helps to overcome costly hurdled being
faced in the industrial production of biofuels. Further, we achieved significant removal of
lignin from SCB, which resulted in higher yield of fermentable sugars for the production of
ethanol.
Acknowledgments This research was supported by research grants to KS from DST and UGC-SAP, the
Government of India, New Delhi. VS thank UGC-SAP for providing JRF. We are grateful to Dr. Anu Appaiah
for providing facilities to carry out the experiments in CFTRI.

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