MINISTRY OF EDUCATION & TRAINING
CAN THO UNIVERSITY
BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE
SUMMARY
BACHELOR OF SCIENCE THESIS
THE ADVANCED PROGRAM IN BIOTECHNOLOGY
ISOLATION, SELECTION AND
IDENTIFICATION BACILLUS SUBTILIS FROM
MUD-DREG OF BEER
SUPERVISOR
STUDENT
Assoc. Prof. Dr. TRAN NHAN DUNG
NGUYEN MINH THUY
Student’s code: 3084035
Session: 34
Can Tho, 05/2013
APPROVAL
SUPPERVISOR
STUDENT
Assoc. Prof. Dr. TRAN NHAN DUNG NGUYEN MINH THUY
Can Tho, May, 2013
PRESIDENT OF EXAMINATION COMMITTEE
Abstract
Bacillus subtilis had widely used in many fields such as
medicine, food processing, veterinary, aquaculture and especially
in environmental treatment. It helps disintegrate dreg, mud ...In
this study”Isolation selection and identification of
Bacillus
subtilis from “mud-dreg of beer” 21 strains of bacteria isolated
from ” mud-dreg” and (Bs1, Bs2, Bs3, Bs4, Bs5, Bs6, Bs7, Bs8,
Bs9) waste water of beer (BN1, BN2, BN3, BN4, BN5, BN6,
BN7,BN8, BN9, BN10, BN11, BN12).
There were sixteen strains selected because of Gram
(Gram positive). After testing biochemical activities such as
enzyme Catalase, Amylase, Protease and reaction with Methyl
Red, all results were compared with Bacillus subtilis control from
Biochemical Food laboratory. Eight strains (Bs1, Bs2, Bs4, BN1,
BN2, BN5, BN9, BN12) are selected to do PCR (Polymerase
chain reaction) with primer 16S-9F and 16S-1525R. The result of
PCR perform size band of 8 strains and Bacillus subtilis control
had 1500bp. Sequencing perform that 2 strains (Bs4 and BN5)
were Bacillus subtilis (99% and 98% identical), they present both
of sample “mud-dreg” and waste wate. Besides, Bs2 and BN9
also so found in “mud-dreg” and waste water, were sequenced as
Bacillus megaterium.
Key words: Bacillus subtilis, beer, isolation, mud-dreg, waste
water
ii
CONTENTS
Abstract ..................................................................................i
Content ...................................................................................ii
1.Introduction .........................................................................1
2.Material and Method ...........................................................3
2.1.Material ......................................................................3
2.2.Method .......................................................................4
2.2.1.Isolation Method..................................................4
2.2.2.Observation shape and movement of bacteria ....5
2.2.3.Measured bacteria size ........................................5
2.2.4.Gram stained........................................................6
2.2.5.Spore stained Bacillus subtilis .............................7
2.2.6.Chemical reaction ................................................7
2.2.8.Polymerase chainreaction ....................................8
3.Result and Discussion .........................................................10
3.1.Bacteria Isolation and characteristic ..........................10
3.1.1.Bacteria Isolation .................................................10
3.1.2.Characteristic (size of cell, Gram, movement) ....12
3.1.3.Chemical reaction to test bacterial capacity create
synthesize exoenzyme ............................................................14
iii
3.1.4.Catalase ...............................................................15
3.1.5.Amylase ...............................................................16
3.1.6.Protease ...............................................................16
3.1.7.Reaction with Methyl Red ...................................17
3.1.8.Spore stained .......................................................17
3.3.PCR product...............................................................18
3.4.Sequencing.................................................................19
4.Result and Sugestion ...........................................................20
Reference................................................................................21
iv
1. INTRODUCTION
The genus Bacillus have a great potential for extracellular
enzymes. Many of these enzymes are extracellular enzymes that
hydrolyze large organic molecules. Thus, genus Bacillus has
many application in different fields (R.Gupta et al., 2002), such as
industrial
manufacturing
detergents,
food
industry,
pharmaceuticals and leather processing, especially in minimize
waste pollution (Outtrup H. Et al., 2002). Priest in 1977 had study
demonstrated a Gram positive bacteria, spore forming. Bacillus
subtilis is able to synthesize and produce protease, amylase, and a
number of axoenzyme (extracellular enzyme)... The products
nature of biotechnology, along with the ability to create and spore
germination by Bacillus became hereditary system typical of
Gram positive bacteria. (Le Thi Lan, 1997; Priest, 1993)
Beer is a beverage products are increasingly popular and
common in the holidays in Vietnam. In the process of brewing
beer sludge is discharged into a nutritious environment for
microorganisms to grow. Each day, about 2 tons of sludge
discharged ”mud-dreg” from a beer brewing plant in Bac Lieu
province that does not have the resolution, causing environmental
pollution.
Because of the biological properties of extracellular enzyme
aforementioned Bacillus subtilis of the hydrolysis of large organic
molecules. Beer dregs is a nutrient-rich environment and organic
matter should be decided on the subject of Bacillus subtilis
1
isolated from waste water of beer and beer wallows in the
brewing process.
Objective:
Isolation, selection and identification of Bacillus
subtilis from beer dregs, that is done with the aim of isolating,
identifying characteristic biochemical and morphological methods
in molecular biology of Bacillus subtilis, which have the ability to
stream selected synthetic protease and amylase enzyme highly
active. For the purpose of beer dregs utilize as fertilizer for plants
and micro-organisms to reduce environmental pollution.
2
2. MATERIALS AND METHODS
2.1. Materials
- “Mud-dreg” 2kg; waste water 1 littre
- Bacillus subtilis positive control from Food chemical
labotary
- Forward and reverse primer 16S-9F and 16S-1525r (JangSeu ki et al., 2009)
16S-9F
5’ GAG TTT GAT CCT GGC TAC G 3’
16S-1525r 5’ AGA AAG GAG GTG ATC CAG CC 3’
-Medium: TSA agar, Luribernate liquid
TSA (Tryptic Soy Agar)
Chemical
Concentration (g/l)
Tryptone
15
Soytone
5
NaCl
5
Agarose
15
Starch 0.1%; NaCl 0.1%; Iod 0.02N; Machite Green 5%,
safranin;
H2O2 3%; Methyl Red; Iod; Fushin; Crystal violet;
Ethanol 70%; BiH2O
. Chemicals and equipments in Plant molecular laboratory
3
Medium SMA (Skim milk Agar)
Chemical
Concentrate (g/l)
Pepton
5
Yeast extract
3
Skim milk
100 ml/l
Agarose
20
2.2. Methods
2.2.1. Isolation method
Identical samples: Add about 10g of “mud-dreg” to 90ml
distilled water, these samples were shaked at 150round/per
minute after that incubated in 80°C for 20 minutes.
“Mud-dreg” suspension was dilluted into 5 concentrations
such as 10-1, 10-2, 10-3, 10-4, 10-5. Waste water was diluted
directly. After that spreaded samples on petri dish, incubated at
37°C for one or two days and observed the appearance of
colonies. Cultured seperated colonies on medium until having
isolate strains, cultured isolate strain to LB tube and storaged in
refrigerator.
4
2.2.2. Observation shape and movement of bacteria
After isolation and separation bacteria, observation the
movement and shape in sterile distilled water. Preparation of
baterial samples by pressure drop method.
+Drip 5µl sterile distilled water to lam glass.
+Sterilized wire loop on alcohol lamp.
+Used wire loop to take a few colony and stretch it on the
drop.
+Took a lame cover on the drop.
+Observed the specimen under optical microscope zoom 400.
2.2.3. Measure bacteria size
Measured the diameter of the microscope's field of view.
Using the low power objective, look through the microscope, and
place the ruler under the field of view. Measure the diameter in
millimeters. For example, you may find that the diameter of the
field of view is five millimeters.
Observed the bacteria under the microscope with low power.
Place the bacteria slide on the stage of the microscope, and then
bring it into focus using the fine course adjustment knobs.
Locate a single bacterium. The bacteria slide will typically have
more than one bacterium. Find one bacteria, and then estimate
how many times it will fit across the field of view. For example,
you may find that a single rod-shaped bacteria will fit across the
field of view about three times.
5
Divide the diameter of the field of view by the number of
times that the bacterium fits across the field of view.
2.2.4. Gram-stained
Place a sample of a bacterial culture on a microscope slide.
An inoculation loop can be used to transfer the bacteria to the
slide.
Dry
the
slide
containing
the
bacterial
culture.
Pour crystal violet stain over the bacterial specimen on the slide.
Let the slide stand for approximately 10 to 60 seconds depending
on the thickness of the smear on the slide. Rinse the crystal violet
stain off of the slide with water.
Place Gram's iodine solution on the bacterial smear. Let the
smear stand for another 10 to 60 seconds depending on smear
thickness. Rinse off the extra Gram's iodine solution with more
water.
Add several drops of a decolorizer to the bacterial smear on
the slide. Rinse the decolorizer off of the slide when the
decolorizer is no longer colored by the previous stains as the
decolorizer runs off of the slide. A typical time for this is
approximately 5 seconds.
Put a counterstain, such as basic fuchsin solution, on the slide
over the bacterial smear. Allow the counterstain to remain on the
smear for approximately 40 to 60 seconds and then rinse off the
counterstain with water. Gram-positive bacteria will be colored
purple, and Gram-negative bacteria will have a red or pink color.
6
2.2.5. spore-stain Bacillus subtilis
Staining the spore of bacteria, the firt step like Gram staining,
after that take machite green 5% on specimens. Clean up lam
under water then keep it in safranin liquid about 1minute and
clean up under water. Observing with lam in the oil.
2.2.6. Chemical reaction to identify Bacillus subtilis
a. Catalase reaction:
Drip one drop of H2O2 30% on the lam, take a amount of
colony put into H2O2 30% drop.
+Positive: bubble up
+Negative: Not bubble up
b. Methyl Red Test:
Transfered 1ml of bacteria in LB medium into a tube. Drip 5
drop of Methyl Red into the tube.
+ Positive: turn into light red color
+ Negative: turn into yellow color
c. A capacity to synthesize protease enzyme:
Use micropipett to drop 5µl bacteria from LB liquid medium.
Drip 5µl bacteria on SMA medium repeat 3 times and incubate in
40°C on 24 hours. Bacteria strains have protease enzyme will
create halo round, measure thi round follow formula:
Hydrolyze diameter = halo diameter – drop of bacteria diameter
d. A capacity to synthesize amylase enzyme:
7
Take 1ml bacteria from LB medium in to the tube in which
has 1ml NaCl 0.1%. After that, take 1ml starch and shake the
tube, leave it at 30°C 30 minutes. Taking 1 drop of iod into the
tube and observes the result for:
+ Bacteria have amylase enzyme: loose the color of starch
+ Bacteria do not have amylase enzyme: dark blue color
2.2.7. Electrophoresis PCR products
PCR procedure:
Primers 16S rRNA (16s-9F và 16S-1525r) (Jang-Seu ki et
al.,2009)
Forward primer:
16S-9F
5’GAG TTT GAT CCT GGC TAC G 3’
Reverse primer:
16S-1525r AGA AAG GAG GTG ATC CAG CC 3’
After DNA extracted, PCR reaction with primers above.
BiH2O
9,5µl
Buffer 10X
2µl
MgCl2 25mM
1,6µl
dNTPs
3,2µl
Forward primer (diluted 10 times) 0,6µl
Reverse primer (diluted 10 times)
0,6µl
Taq polymerase
0,5µl
8
DNA
2µl
Total
20µl
*PCR cycle (repeat 35 cycles)
Component of Gel:
Agarose 1%
0,4g
TE1X
45ml
EtBr
0,8µl
9
3. RESULTS AND DISCUSSION
3.1. Bacteria Isolation and characteristic:
3.1.1.Bacteria Isolation:
21 isolates bacteria strains. 9 isolates from “mud-dreg” took
42,85% and 12 from waste water took 57,14%.
No. isolate Sp
Form
Margin
Elevation color
Size
cm
1
Bs1
Mud circular entire
raised
milky 3
2
Bs2
Mud circular entire
umbonate
white
3
3
Bs3
Mud circular curled
umbonate
white
2
4
Bs4
Mud circular entire
raised
milky 3
5
Bs5
Mud circular entire
raised
milky 1
6
Bs6
Mud circular curled
umbonate
milky 1.5
7
Bs7
Mud circular entire
raised
milky 2
8
Bs8
Bùn
circular entire
raised
milky 4
9
Bs9
Bùn
circular entire
raised
milky 1
10
Table 1a: 9 isolates bacteria strains from waste water
Sp Form
Margin
Elevation
N
o
Strain
Color
Size
1
BN1
W
circular
smooth
raised
milky
1
2
BN2
W
circular
smooth
raised
white
1
3
BN3
W
circular
curled
raised
white
1
4
BN4
W
circular
smooth
raised
milky
0.5
5
BN5
W
circular
curled
umbonate
milky
5
6
BN6
W
circular
smooth
raised
milky
0.5
7
BN7
W
circular
smooth
raised
milky
2
8
BN8
W
circular
curcled
umbonate
milky
2
9
BN9
W
circular
smooth
raised
milky
5.5
10
BN10
W
circular
smooth
raised
milky
5
11
BN11
W
circular
smooth
umbonate
milky
2
mm
11
12
BN12
W
circular
smooth
umbonate
milky
5
Table 1b: 12 isolates bacteria strains from waste water
3.1.2.Characteristic (size of cell, Gram, movement)
No
strain
Shape
mobility
(rod)
Gram
length
width
test
(µm)
(µm)
1
Bs1
long
+
+
1.9
0.74
2
Bs2
short
+
+
1.0
0.68
3
Bs3
long
+
+
2.83
0.71
4
Bs4
short
+
+
1.65
0.74
5
Bs5
long
+
+
2.87
0.7
6
Bs6
long
+
-
1.86
0.65
7
Bs7
short
+
+
1.41
0.71
8
Bs8
long
+
+
2.86
0.74
9
Bs9
long
+
-
1.8
0.68
10
BN1
long
+
+
2.86
0.71
12
11
BN2
long
+
+
2.83
0.74
12
BN3
long
+
+
1.83
0.68
13
BN4
short
+
-
1.43
0.71
14
BN5
short
+
+
1.43
0.72
15
BN6
short
+
-
1.6
0.68
16
BN7
long
+
+
2.7
0.74
17
BN8
short
+
-
1.43
0.68
18
BN9
long
+
+
2.83
0.71
19
BN10
short
+
+
1.68
0.7
20
BN11
short
+
+
1.43
0.72
21
BN12
long
+
+
2.9
0.74
Table 2: Characteristic of baterial cell
Notice:Gram(+): gram positive; move + : can move
13
The size of bacterial cell varies between 1.0 to 2.86µm and
width in the range of 0.65 to 0.74µm consistent with the
description of Nguyen Lan Dung (1997).
After Gram stain 21 strains, obtained 16 strains had purple
blue color from crystal violet, positive gram, accounting for
76,19%. Therefore, they suitable description of Bacillus subtilis
are positive gram of Rahnman Sharmin (2007), excluding 5
strains had pink color from Fuchsin, gram negative bacteria.
3.2.Chemical reaction to test bacterial capacity create
synthesize exoenzyme:
No
strain
Catalase
Amylase
Protease
Methyl
Red
spore
1
Bs1
+++
+++
+
+
+
2
Bs2
+++
+++
+
+
+
3
Bs3
-
++
None
None
None
4
Bs4
+++
++
+
+
+
5
Bs5
+
++
-
None
-
6
Bs7
++
+
-
None
-
7
Bs8
-
++
-
None
-
14
8
BN1
+++
+++
+
++
+
9
BN2
+++
+++
+
+
+
10
BN3
-
++
None
None
None
11
BN5
++
++
+
+
+
12
BN7
-
++
None
None
None
13
BN9
++
+++
+
+
+
14
BN10
+++
+
-
None
-
15
BN11
-
+
None
None
None
16
BN12
+++
+++
+
++
+
17
control
+
+++
+
+
+
Table 3: Bacterial chemical characteristic
3.2.1.Catalase:
Seven strains had bubbling stream faster took 43.75%, 3
strains had bubbling at everage range at 18.75%, 1 strain
15
- Xem thêm -