Progressive Report

Production cellulases by isolated

indigenous bacteria

Name

Hikmal Fizar bin Jamaludin

Matric Number

SEQ 110003

Supervisor

Dr. Khanom Simarani

Dateline

29th December 2014

Contents
1.

Introduction............................................................................................................................3

2.

Aim...........................................................................................................................................4

3.

Review of relevant literature.................................................................................................4

4.

Materials and methods...........................................................................................................7

5.

Results and Discussion.........................................................................................................11

6.

Appendices............................................................................................................................14

7.

References.............................................................................................................................17

1. Introduction
Sugarcane bagasse
Sugarcane bagasse is a plentiful lignocellulosic waste typically found in tropical countries that
involved in sugarcane-processing industry such as Brazil, India, and Cuba (Samantha &
Khakifrooz, 2011). The sugarcane stalk consists of two parts, an inner pith containing most of the
sucrose and an outer rind with lignocellulosic fibers. During sugar processing, the sugarcane
stalk is crushed to extract sucrose (Boophthy, 2004). This procedure yields a large volume of
residue, the bagasse which contains both crushed rind and pith fibers.
Bagasse, the sugarcane residue after the sugar extraction is one of the most available agricultural
by product that have various kind of applications. They are used as sources of animal feed, pulp,
paper and boards (Banerjee & Pandey, 2002). In addition, the sugarcane bagasse, SCB is also
have a great potential in production of bioethanol (Sanchez & Cardona, 2008). Generally, SCB
consists of cellulose, hemicellulose and lignin. The table below shows the percentage
composition of SCB.
Percentage composition of sugarcane bagasse 9%)
Cellulose
Hemicellulose
Lignin
43.6
33.5
18.1
50
25
25
40-45
30-35
20-30

Reference
Sun et. al. , 2004
Pandey et. al., 2000
Peng et. al. , 2009

Other than that, the SCB also can be also be applied to produce soluble sugars. The formation of
simple sugars from cellulose and hemicellulose of SCB require a series of combination action of
cellulolysis such as exoglucanase, endoglucanase and -glucosidase that derived from the
3

cellulolytic microorganisms. In this study, the known indigenous bacteria of sugarcane

environment was tested on the synthetic and carboxymethylcellulose (CMC) medium in order to
analyze the production of simple sugar that produced. In addition, the ATCC strain is also used
and tested in order to analyze the cellulase activity between the known and ATCC strain in same
media.

2. Aim
The purpose of this study is to determine the production of cellulase from the isolated bacteria
and ATCC strain by using the synthetic and CMC medium. In addition, we are also determine the
potential use of bagasse as a carbon source for bacterial growth and cellulase production.

3. Review of relevant literature

3.1 Lignocellulosic material
Lignocellulose is the major structural component of woody plants such as grass and represents a
major source of renewable organic matter (Howard et. al, 2004). Lignocellulose consists of
4

lignin, hemicellulose and cellulose. Large amounts of lignocellulosic waste are generated
through forestry and agricultural practices, paper-pulp industries, timber industries and many
other agro-industries (Malherbe & Cloete, 2002). The table below summarized the source and the
potential uses of lignocellulosic material.
Lignocellulosic material
Grain harvesting
-Wheat, rice, oats, barley and
corn
Processed grains
-corn, wheat, rice and
soybean
Fruit and vegetable
harvesting
Fruit and vegetable
processing
Sugar cane other sugar
products
Oils and oilseed plants
-Nuts, cotton seeds, olives
and soybean
Animal waste
Forestry paper and pulp
-harvesting of logs
Saw and plywood waste
Pulp and paper mills

Residues
Straw, husks, stalks, cobs

Competing use
Animal feed, burnt as fuel,
compost and soil conditioner

Wastewater, bran

Animal feed

Seeds, peels, husks, stones,

rejected whole fruit and juice
Seeds, peels, wastewater,
husks, shells, stones and
rejected whole fruit and juice
bagasse
Shells, husks, lint, fiber,
sludge, press cake and
wastewater
Manure, other waste
Wood residuals, bark, leaves,
etc
Woodchips, wood shavings,
saw dust
Fiber waste and sulfite liquor

Lignocellulose waste from

communities

Animal and fish feed, some

seeds for oil extraction
Burnt as fuel
Animal feed, fertilizer, burnt
fuel
Soil conditioners
Soil conditioner, burnt
Pulp and paper industries and
fiber board
Reused in pulp and board
industry as fuel
Small percentage recycle,
others burnt

Old newspaper, paper,

cardboard, old boards,
disused furniture
grass
Unutilized grass
burnt
Table 3.1.1: Types of lignocellulosic materials and their current uses (Malherbe & Cloete, 2003)
3.2 Cellulose
Cellulose is the substance that makes up most of a plants cell wall and account for the most
abundant organic compound on Earth. Chemically, cellulose is a linear natural polymer of
anhydroglucose units that linked at the one and four carbon atoms by -glycosidic bonds.
5

The fact of the chemical structure of the cellulose is prove by the presence of three hydroxyl
groups with difference in aspect of acidity or reactivity, secondary OH at carbon number 2
and 3 and primary OH at the C-6 position respectively by the formation of strong various
inter and intramolecular hydrogen bonds (Kadla & Gilbert, 2000). In plant, the cellulose is
organized into fibrils which are surrounded by a matrix of lignin and hemicelluloses.
3.3 Hemicellulose
Hemicellulose,

biological

compound

that

contributes

second

most

common

polysaccharides in nature. It represents about 20-35% of lignocellulosic biomass (Saha,

2003). Hemicelluloses are heterogeneous polymers of pentoses, hexoses and sugar acids.
The composition of hemicellulose can be categorized into two species, hardwood and
softwood hemicellulose. The difference between this two species can be observed by the
abundance of the composition within it. The hardwood hemicellulose are rich of xylans
while softwood hemicellulose are rich in glucomannans (McMillan, 1994). Hemicelluloses
have lower degree of polymerization (DP), that is only 50-300 with side groups on the chain
molecule and are essentially amorphous (Perez et. al., 2002).

3.4 Lignin
Lignin is the most abundant aromatic polymer in nature. It present in the cellular cell wall,
conferring structural support, impermeability and resistance against microbial attack and
oxidative stress. Lignin is an amorphous heteropolymer, hydrophobic and optically inactive.
It consists of phenylpropane units joined together by different types of linkage. The polymer

is synthesized by the generation of free radical, which are released in the peroxidasemedated dehydration of three phenyl propionic alcohols (Sjostrom, 1993).
3.5 Pre-treatment of bagasse
The main purposes of having the pre-treatment of bagasse are to improve its digestibility
and easy access for microbial attack by removing the core and noncore lignin fractions
(Alan & Smith, 1988 and Doran et. al.,1994). They are several physical and chemical
methods that applied for the pre-treatment of SCB which include steam explosion,
gamma radiation, treatment with alkali, hydrogen peroxide and solvents. The pretreatment with alkali such as sodium hydroxide is the most effective and economical way
to treat the SCB. A study by Rodriguez-Vazguez et. al. in 1992 that treated bagasse with
NaOH solution in a dry pre-treatment ( dry pretreatment refers to low or no free liquid
was present) shows that maximum digestibility of 75% from the biomass production.
Bravo et. al. (1994) that also used alkali pre-treatment at 3 liquid:1 solid ratio revealed
that the treatment significantly enhanced fungal growth compared to nontreated bagasse.
Du-Toit et. al. (1984) compared the pre-treatments of bagasse with dilute alkali and acid
for the determination of the monosaccharides present In bagasse hemicellulose. In their
study, the pentosan fraction of the bagasse was successfully hydrolyzed and extracted
with 5% (m/v)HCL. The treatment with alkali resulted in 39.8% solubilization of bagasse
and about 72% of the available hemicellulose be extracted.

4. Materials and methods

4.1 Sampling
The sugarcane bagasse are collected from the sugarcane juice stall at the area of Pantai
Dalam, Kuala Lumpur. The sugarcane bagasse, SCB next are washed with tap water and
7

dried under sunlight for a day. Later, the bagasse were cut into smaller pieces (about 1-2
cm in size) and oven dried in the incubator at 60C temperature overnight.
4.2 Pre-treatment of bagasse
The dried bagasse is grinded by using the mechanical grinder in order to obtain the
powder form of bagasse. Later, the grinded bagasse was sieved using siever (1mm of
pore size) to obtain fine powder of bagasse. Next, the bagasse is soaked in 1% (w/v) ash
solution by adding 5g of bagasse sample to 250 ml of 1% (w/v) ash solution in a beaker
for an hour. After that, the bagasse sample were washed with distilled water until the pH
of the filtrate reach pH 7 in order to remove the alkalinity in the sample. Next, the sample
was then dried at 50C overnight and kept in plastic bag prior to use.
4.3 The production of cellulolytic enzyme
4.3.1 Preparation of inoculum
Firstly, two loopful of pure colony from Bacillus aerius strain 5.2 were inoculated into
50 ml nutrient broth in 250 ml of conical flask. Next, the flask is incubated overnight
(25C, 100 rpm). Later, 50 ml of grown cell were transferred into sterile Falcon tube and
centrifuged (10000 rpm, 4C) for 10 minutes. The cell pellet was resuspend with sterile
0.08% (v/v) phosphate saline buffer until the optical density of the cell suspension
4.3.2

reached at 0.8A at 600nm.

Batch fermentation
10% (v/v) of inoculum was transferred into two different of 500 ml of conical flasks that
contain 250 ml of synthetic medium and CMC medium respectively. Then, the flasks are

4.3.3

incubated (32C, 100rpm) for 48 hours.

Collection of samples
The samples were collected for both CMC and synthetic medium at different intervals (4,
8, 12, 24, 36,48h). Next, the samples were centrifuged (10000rpm) for 15 minutes. The
supernatant was collected and stored in 4C room for further assay.

4.4 Enzyme assay

4.4.1 Filter paper Enzyme Assay (FPase)
The FPase activity was assayed by adding 0.2mL of enzyme sample into test tube that
contained 1.8mL of 0.05M sodium citrate buffer (pH 4.8) and Whatman filter paper
(1.0x 3.0 cm). Next, the mixture was homogenized using vortex mixer and incubated
in water bath 40C) for 60 minutes. Later, 3ml of DNS reagent was added into the
tubes and incubated at boiling water bath (100C) for 3 minutes to stop the enzymatic
and DNS reaction. The reducing sugars liberated were determined by using DNS
reagent (Wood & Bhat, 1988). After that, 1 ml of Rochelle salt was added and the
absorbance was read using spectrophotometer at 575nm. The glucose standard curve
was used to determine the amount of reducing sugar that being released.
One unit of FPase activity was defined as 1mole of glucose liberated per mL
enzyme per minute calculated as shown in Equation 1.0
Calculation:
OD x 1000
Reducing sugar (mole) =
m x GMR
Where:
OD= Absorbance of the sample at 575nm
M= Slope of glucose standard curve
GMR= Glucose molecular weight
GMR of glucose= 180
FPase activity ( U/mL) =

Reducing sugar ( mole)

Volume of sample ( mL ) x incubation time(min)

1.0)

...(Eq

FPase activity is defined as 1 mol reducing sugar released per mL of enzyme per
min.
4.4.2

Carboxymethylcellulose Assay (CMCase)

The CMCase activity was determined by measuring the reducing sugar produced by
using 1.0 % (v/v) CMC as a substrate. 1.8 ml of 1.0 % (v/v) CMC solution was added
in the test tube that contained 0.2 ml of sample. The tube then incubated at 40C
water bath for 60 minutes. Next, 3.0 ml of DNS reagent was added and the
suspension was mixed using vortex mixer. Later, the tubes was incubated in boiling
water bath (100C) for 15 minutes in order to stop the enzymatic and DNS reaction.
Next, 1ml of Rochelle salt was added and the absorbance was read using
spectrophotometer at 575nm (Wood & Bhat, 1988).
The reducing sugar liberated from enzymatic reaction were determined using DNS
reagent (Wood and Bhat, 1988) and calculated using Equation 2
Calculation:
Reducing sugar (mole) =

OD x 1000
m x GMR

Where:
OD= Absorbance of the sample at 575 nm
M= Slope of glucose standard curve
GMR= Glucose molecular weight
GMR of glucose= 180
CMCase activity ( U/mL) =

Reducing sugar ( mole)

Volume of sample ( mL ) x incubation time (min) ...(Eq

2.0)

10

CMCase activity is defined as 1 mol reducing sugar released per mL of enzyme per
min.

5. Results and Discussion

5.1 Pre-treatment of sugarcane bagasse
In this process, the weight of bagasse before and after the pre-treatment were measured to
determine the amount of weight loss during the treatment. The result are as follow.
Table 5.1
The weight of pre-treatment bagasse (before and after the pre-treatment)
Sam
Weight of bagasse (g)
Mean standard
ple
deviation
Before preAfter
Weight loss,
treatment
pretreatment
W3=(W1(W1)
(W2)
W2)
1 5
2.5
2.5
2.410.27
2 5
2.45
2.55
3 5
2.42
2.58
4 5
2.99
2.01
Total 20
10.36
9.64
weig
ht
Therefore, 48.2% of initial weight of bagasse was lost during the pre-treatment of the bagasse
5.2 Glucose concentration in the different medium
The amount of the glucose before addition of the inoculum was analyzed using DNS
assay. The DNS assay was carried out by adding 1ml of medium and 9ml of 0.05M
sodium citrate buffer in the test tubes. Next, the tube was incubated at 40C water bath
for 60 minutes. Later, the DNS reagent was added and the tube was incubated in boiling
water bath (100C) for 3 minutes. 3 ml of Rochelle salt was then added and the
absorbance was read using spectrophotometer at 575nm. The glucose standard curve was
used as standard comparison. The results are as follow:

11

Table 5.2

Glucose concentration in three different media and control

Medium

CMC
Pre-treated
bagasse +
autoclaved
Untreated
bagasse +
autoclaved
Control

Absorbance
(575nm)

Concentration of
glucose,x={y/0.5272}
(mg/ml)

Mea
n

Standa
rd
deviati
on

0.19
3
0.19
8

0.0443

1
0.085

2
0.118

1
0.161

2
0.224

0.117

0.092

0.222

0.175

0.405

0.386

0.768

0.732

0.75
0

0.0255

0.191

0.239

0.362

0.453

0.40
8

0.0644

0.0335

From the result above, the untreated bagasse shows the highest concentration of glucose that it
0.750 mg/ml.
5.3 FPase titer produced by Bacillus aerius strain 5.2 in different culture media

Fpase activity
0.250
0.200
0.150

Fpase activity (U/mL)

0.100
0.050
0.000

12

16

20

24

28

32

36

40

44

Incubation period (h)

CMC

Pretreated bagasse

Untreated bagasse

Figure 5.1
: Fpase activity of Bacillus aerius strain 5.2
The purpose of having the FPase assay is to determine the total cellulase activity and
glucose obtained from the cellulolysis process. In diagram above, the highest cellulase
activity recorded by Bacillus aerius strain 5.2 in pre-treated bagasse is 0.187 U/ml after 8
hours of incubation. This is due to the highest enzyme-substrate reaction that occurred at

12

48

that particular time. For the Fpase activity using untreated bagasse and CMC, the highest
cellulase activity recorded are 0.132 U/ml and 0.132 U/ml respectively. However, the
Fpase activity recorded for these three synthetic media show no significance difference
after 24 hours of incubation.
5.4 CMCase titer produced by Bacillus aerius strain 5.2 in different culture media

CMCase activity
0.400
0.300
0.200
0.100
0.000

12

16

20

24

28

32

36

40

44

48

Incubation period (h)

CMC

Pretreated bagasse

Untreated bagasse

Figure 5.3.2: Fpase activity of Bacillus aerius strain 5.2

The purpose of CMCase test is to measure and determine the endoglucanase activity by using
carboxymethylcellulose solution as a substrate. In the diagram above, the highest endoglucanase
activity recorded for Bacillus aerius strain 5.2 is in untreated bagasse that is 0.287 U/ml after 4
hours of incubation. However, the highest cellulase activity recorded for both CMC and pretreated bagasse are 0.147 U/ml (after 36 hours) and 0.111 U/ml (after 12 hours of incubation).

13

6. Appendices
6.1 Glucose standard curve
The determination of FPase and CMCase activity in the culture medium are obtained by
measuring the amount of reducing sugar liberated in a given period of time. The
determination of reducing sugar is using thus using the gradient of the glucose standard
curve. The procedure of glucose standard curve eventuated by preparation of glucose
stock concentration. 0.25g of anhydrous glucose was diluted with 250ml of distilled
water to make 0.1mg/ml glucose stock concentration. Next, the glucose serial dilution is
prepared as table below. All tubes were incubated at 40C water bath for 60 minutes.
Later, 3.0 ml of DNS reagent was added and further incubation in 100C boiling water
bath for 5 minutes to stop the reaction. After that, let all the tubes to cool down under
room temperature before vortex it using vortex mixer. Lastly, read the absorbance of the
solution by using spectrophotometer under 575nm wavelength.

Test
tube
A
B
C
D
E
F
G
H
I
J

Volume
of
glucose
stock
(ml)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50

Volume of
distilled
water (ml)
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50

Glucose
concentration
(mg/ml)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50

14

5.00

0.00

5.00

Data for the glucose standard curve

Table 6.1.1: Data of Glucose standard curve
Tes
t
tub
e

Volume
of
glucose
stock
(ml)

Volume of
distilled
water (ml)

Glucose
concentrati
on (mg/ml)

0.00

5.00

0.00

0.50

4.50

0.50

1.00

4.00

1.00

1.50

3.50

1.50

2.00

3.00

2.00

2.50

2.50

2.50

3.00

2.00

3.00

3.50

1.50

3.50

4.00

1.00

4.00

4.50

0.50

4.50

5.00

0.00

5.00

15

Absorbance
(575nm)
1
2
0.0
0.00
0
0.1
0.11
5
0.4
0.45
9
0.9
1.08
6
1.1
1.24
2
1.3
1.60
2
1.8
1.80
0
1.9
1.93
2
2.1
2.18
1
2.1
2.35
4
2.1
2.56
8

Total
absorban
ce
(575nm)
0.00
0.26
0.94
2.04
2.36
2.92
3.60
3.85
4.29
4.49
4.74

Average
absorban
ce
(575nm)
0.00
0.13
0.47
1.02
1.18
1.46
1.80
1.93
2.14
2.25
2.37

2.50

f(x) = 0.53x
R = 0.99

2.00

1.50

Absorbance (575nm)
1.00

0.50

0.00
0.00

1.00

2.00

3.00

4.00

5.00

6.00

Glucose concentration (mg/ml)

Graph 6.1.1: The straight line of glucose standard curve

6.2 Preparation of synthetic and CMC media
Chemical

% composition

Preparation in 1000

composition
K2HPO4

0.08

ml (g)
0.80

KH2PO4

0.02

0.20

MgSO4.7H2O

0.02

0.20

NaCl

0.02

0.20

NaNO3

0.1

1.0

CaCO3

0.001

0.01

Yeast extract

0.05

0.5

CMC/Bagasse

0.5

5.0/12.5

16

The composition above is then mixed with 1000ml distilled water. The pH was adjusted
to pH 7 and sterilized at 121C for 15 minutes.

7. References
Samariha A, & A, K. (2011). NSSC for bagasse. BioResources, 6(3), 3313-3323.
Boophthy, R. (2004). Use of post-harvest sugarcane residue in coastal reclamation: A feasibility
study. SugarCane International, Jun/Feb, 9-13.
Banerjee, R., & Pandey, A. (2002). Bio-industrial applications of sugarcane bagasse: a
technological perspective. International sugar journal, 104(1238), 64, 66-67.
Sanchez, O. J., & Cardona, C. A. (2008). Trends in biotechnological production of fuel ethanol
from different feedstocks. Bioresource technology, 99(13), 5270-5295.
Sun, J., Sun, X., Zhao, H., & Sun, R. (2004). Isolation and characterization of cellulose from
sugarcane bagasse. Polymer Degradation and Stability, 84(2), 331-339.
Pandey, A., Soccol, C. R., Nigam, P., & Soccol, V. T. (2000). Biotechnological potential of agroindustrial residues. I: sugarcane bagasse. Bioresource technology, 74(1), 69-80.
Peng, F., Ren, J.-L., Xu, F., Bian, J., Peng, P., & Sun, R.-C. (2009). Comparative study of
hemicelluloses obtained by graded ethanol precipitation from sugarcane bagasse. Journal
of agricultural and food chemistry, 57(14), 6305-6317.

17

Howard, R., Abotsi, E., Van Rensburg, E. J., & Howard, S. (2004). Lignocellulose
biotechnology: issues of bioconversion and enzyme production. African Journal of
Biotechnology, 2(12), 602-619.
Malherbe, S., & Cloete, T. E. (2002). Lignocellulose biodegradation: Fundamentals and
applications. Reviews in Environmental Science and Biotechnology, 1(2), 105-114.
Kadla, J. F., & GILBERT, R. D. (2000). Cellulose structure: A review. Cellulose chemistry and
technology, 34(3-4), 197-216.
Saha, B. C. (2003). Hemicellulose bioconversion. Journal of Industrial Microbiology and
Biotechnology, 30(5), 279-291.
McMillan, J. D. (1994). Pretreatment of lignocellulosic biomass. Paper presented at the ACS
symposium series (USA).
Prez, J., Munoz-Dorado, J., de la Rubia, T., & Martinez, J. (2002). Biodegradation and
biological treatments of cellulose, hemicellulose and lignin: an overview. International
Microbiology, 5(2), 53-63.
Sjstrm, E. (1993). Wood chemistry: fundamentals and applications: Gulf Professional
Publishing.
AlAni, F., & Smith, J. (1988). Effect of chemical pretreatments on the fermentation and ultimate
digestibility of bagasse by Phanerochaete chrysosporium. Journal of the Science of Food
and Agriculture, 42(1), 19-28.
Doran, J. B., Aldrich, H., & Ingram, L. (1994). Saccharification and fermentation of sugar cane
bagasse by Klebsiella oxytoca P2 containing chromosomally integrated genes encoding
the Zymomonas mobilis ethanol pathway. Biotechnology and bioengineering, 44(2), 240247.

18

Rodriguez-Vazquez, R., Villanueva-Ventura, G., & Rios-Leal, E. (1992). Sugarcane bagasse pith
dry pretreatment for single cell protein production. Bioresource technology, 39(1), 17-22.
Bravo, O., Ferrer, A., Aiello, C., Ledesma, A., & Dvila, M. (1994). Growth of Chaetomium
cellulolyticum in solid-state fermentation of sugar cane bagasse treated with water and
alkali at several liquid/solid ratios. Biotechnology letters, 16(8), 865-870.
Du Toit, P., Olivier, S., & Van Biljon, P. (1984). Sugar cane bagasse as a possible source of
fermentable carbohydrates. I. Characterization of bagasse with regard to monosaccharide,
hemicellulose, and amino acid composition. Biotechnology and bioengineering, 26(9),
1071-1078.
Wood, T. M., & Bhat, K. M. (1988). Methods for measuring cellulase activities. Methods in
enzymology, 160, 87-112.

19