production of bioethanol from fermented sugars

8/17/2019 Production of Bioethanol From Fermented Sugars

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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 sugar-

cane, miscanthus) [8]. The major components of lignocellulosics are cellulose (polymers of hexose sugars, 35 – 50 %), hemicelluloses (polymers of pentose sugars, 20 – 35 %), and lignin

(polyphenols, 10 – 25 %) [8, 9]. The large-scale use of lignocelluloses for the production of

biofuels or other value-added products depends on the breakdown of cellulose, hemicellu-

loses, 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 lignocel-

lulosics. 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 cost-

effective 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 lignocellulosicmaterials. 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 enzymestiter 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 cellulase-

producing microorganisms [20]. Nonetheless, none of these microorganisms are efficient at cellulolytic, hemicellulolytic, and ligninolytic activities simultaneously, rendering the opportu-

nity 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


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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 40 – 60 % 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 40 – 42 % cellulose, 24 – 28 %

hemicelluloses, and 10 – 12 % 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 Saccharo-

myces cerevisiae.

Materials and Methods

Chemicals

Carboxymethylcellulose (medium viscosity, 400 – 800 cP), cellulose powder (Sigma cell

Cellulose, Type 20; particle size 20 μ 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,



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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 5.0 – 10.0, temperature ambient from 30 to 50 °C, and salinity

tolerance from 1 to 16 % sodium chloride, were carried out. Results were analyzed as per

Bergey’s 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 50±5 °C to attain 10 % moisture content (untreated material) and steam-

exploded 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; K 2HPO4, 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 μ l of enzyme

solution, 400 μ l of 1 % (w/ v ) corresponding substrate, and 500 μ 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 μ mol/ml/min of reducing sugar under the standard assay

conditions. The protein concentration was measured according Lowry’s 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 (20 – 60 °C), different

initial pH values using 50 mM adequate buffers (4 – 6, acetate buffer; 7 – 8, phosphate buffer;


http://www.ncbi.nlm.nih.gov/nuccore/JQ312121http://www.ncbi.nlm.nih.gov/nuccore/JQ312121


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and 9 – 12, glycine – NaOH buffer), and NaCl concentrations, 0 – 16 % (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. Hydro-

lysis 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 (4.1×300 mm). Samples (20 μ l)

were injected and eluted with acetonitrile – water (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 supplemen-

tation 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 andincubated 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



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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

50 – 60 °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 μ 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 statisticallyevaluated 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, 0.5 –

1.06×2 –

10 mm, occur-ring 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 5.0 – 10.0 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



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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 tothe 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 (7 – 10) with an optimum at 9.0. There was

low growth at pH 7, whereas high growth was noticed at pH 8 – 10. Maximum growth was

observed in the temperature range of 30 – 50 °C with optimum at 37 °C. The Exiguobacterium

sp. strain VSG-1 was able to grow in a broad-range NaCl concentration (1 – 16 %) 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



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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 exper-

imental 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 42.6±0.1* 57.3±0.2* 58.8±0.1*

Hemicelluloses 24.7±0.2* 14.8±0.1* 13.5±0.2*

Lignin 22.3±0.1* 23.8±0.2* 23.4±0.1*Ash 1.5±0.3* 2.2±0.1* 2.8±0.1*

Extractives 5.6±0.1* 1.2±0.2* 1.1±0.2*

Total 96.7±0.1* 98.1±0.1* 98.5±0.1*

± standard deviation

* P


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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

(5.0 – 6.0) with an optimum at 5.5 and the temperature range of 25 – 35 °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

0

20

40

60

80

100

0 2 4 6 8 10 12

G l u c o s e c o n c e n t r a t i o n ( g / l )

Reaction time (h)

2 mg/g 5 mg/g 10 mg/g

20 mg/g 30 mg/g 40 mg/g

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

Table 2 Estimation of total sugars, reducing sugars, and polyphenols after pretreatment of steam explosion

(hydrothermal) and enzymatic hydrolysis of SCB

Untreated SCB Steam-exploded SCB Enzymatic hydrolyzed SCB

Total sugars (mg/ml) 17.5±0.1* 67±0.2* 640±0.1*

Reducing sugars (mg/ml) 1.5±0.2* 8.2±0.1* 45±0.2*

Polyphenols (mg/ml) 7.4±0.1* 6.2±0.2* 4.6±0.1*


* P


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of total sugars, reducing sugars, and polyphenols was calculated from the hydrolyzate of steam-

exploded 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 chromatogra-

phy and identified as ethyl alcohol (Fig. 6).

Discussion

Temperature is a major environmental condition that affects microbial physiology andgrowth. Every bacterial sp. has its own optimum temperature and cultural conditions for

Minutes

3 4 5 6 7 8 9 10

0.000

0.002

0.004

4 .

3 6 7

4 .

7 2 5

5 .

4 0 0

4.3-glucose

4.7-xylose

5.4-galactose

Fig. 4 HPLC profile of sugars present in hydrolysed SCB before fermentation

0

2

4

6

8

0

20

40

60

80

100

0 24 48 72 96

A l c o h o l c o n t e n t ( % v / v )

G l u c o s e c o n c e n t r a t i o n ( % )

Fermentation days (h)

Glucose concentration (%)

Alcohol content (%v/v)

Fig. 5 Changes in sugar concen-

tration and alcohol contents of

SCB sugar syrup over the

fermentation period



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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, espe-

cially 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. Asa 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 53 – 79 % delignification with75 – 97 % removal of hemicelluloses and 4 – 11 % removal of cellulose from corn stover by a two

stage hot water and ammonia recycle percolation process at high temperature (170 – 210 °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 3 Calculation of total sugars, reducing sugars and polyphenols before and after fermentation of alcohol

Before fermentation After fermentation

Total sugars(mg/ml) 640.6±0.1* 58.8±0.1*

Reducing sugars (mg/ml) 45.2±0.1* 8.5±0.1*Polyphenols (mg/ml) 3.8±0.1* 0.5±0.1*


* P


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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/complex’s ability to bind andthus 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 [42 – 44]. 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 sacchar-

ification of steam explosion pretreated Eucalyptus viminalis chips was obtained using a cellulose mixture of commercial celluclast and novozyme preparations [46]. Use of com-

mercial 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

Fig. 6 Gas chromatogram of alcohol from fermented SCB. Oven temperature, 180 °C; injector and detector

temperatures, 220 and 230 °C, respectively



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of sugarcane bagasse (225 g glucose/kg dry and 62 g xylose/kg biomass) [47] and sorghum

(glucose, 400 – 470 g/kg; xylose, 130 – 170 g/kg dry sorghum) [48] pretreated with ammonia – water.

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 theethanol 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. Sugar-

cane bagasse and sorghum are grass plants and have the similar composition of lignocellu-

losic 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 pretreatedSCB 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, theGovernment 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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