isolation, selection and identification bacillus subtilis from muddreg of beer

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


produced less bubbling (6.25%) and 4 strains without bubbling,
that’s mean no catalase activity, accounting for 25%. Reaction
2H2O2

catalase

2H2O + O2

Bacillus subtilis are capable of producing enzyme catalase.
When exposed to H2O2 is generated O2 bubbling in previous
studies on the possibility of creating catalase by Nguyen Duc
Luong and Nguyen Thi Thuy Duong (2003), Slepecky and
Hemphill (2006), and Sharmin Rahman (2007). Therefore,
selecting 12 strains to do the next reaction.
3.2.2.Amylase:
In total 16 strains, 6 strains discoloration of starch and iodine
immediately accounted for 37.5%, these strains are strong
amylase activity, 7 strains of discoloration starch and iodine after

a period accounted for 18.75%. All bacteria strain ha capable of
synthesizing amylase consistent with the study by Nguyen Duc
Luong and Nguyen Thi Thuy Duong (2003), Slepecky and
Hemphill (2006).
3.2.3.Protease:
After incubate at 37°C for 24h. 8 strains had halo round, can
create protease enzyme, accounted for 66.7%. Besides, BN5 get
highest halo diameter (1.25cm) and BN9 (1.2cm) took the second
range.

16


Figure 10: diameter of Halo on skim milk agar
In the same column mean values followed by the same letter are
not significantly different at= 5%
3.2.4.Methyl Red
When driped MR into 1ml of bacteria, making color changing
3 strains made medium turn into orange accounted for 25%, 8
strains made medium turm in to pale orange (50%). Another
could not make change medium color. Therefore, 8 strains are
chosen. Similar studies of Bacillus subtilis of Sharmin Rahman
(2007) and Tran Linh Thuoc (2008).
3.2.5.Stained bacterial spore:
When observed under the microscope at 400 times
magnification, 8 strains bacterial spores form oval, accounting for
50%, they stared green of malachite green solution. This result
the same with studies of Stainer et al.,(1999), Slepecky and
17

Hemphill (2006) and Luong Bich Dung (2008). The remaining 8
strains had red color from safranin (50%)
3.3. PCR product:
After done bacterial chemical reaction. There are 8 strains
suitable to do electrophoresis:
No

strain

Catalase

MR

Amylase

Protease

spore

1

Bs1

+++

+

+++

+

+

2

Bs2

+++

+

+++

+

+

3

Bs4

+++

+

++

+

+

4

BN1

+++

++

+++

+

+

5

BN2

+++

+

+++

+

+

6

BN5

++

+

++

+

+

7

BN9

++

+

+++

+

+

8

BN12

+++

++

+++

+

+

Table 4: Selecting 8 strains to do electrophoresis

18


1500bp

Figure 13: DNA profiles of 9 well, others lanes are follows
Bs1; Bs2; Bs4; BN1; BN2; BN5; BN9; BN12; control (+);
control(-)
3.4.Sequencing:
After

sequencing

by

NUCLEOTIDE

BLAST

( . Two strains have been identified
Bacillus subtilis (denoted as Bs4 and BN5) with 99% and 87%.
The nucleotide sequence result can be seen in appensix.
-Three Bacillus subtilis strains identical with Bs4 and BN5:
+Bacillus

subtilis

strain

S20

was

identified

by

Mohammadou, B.A., Mbofung, C.M. and Barbier, G. in a
research “Genotypic and phenotypic diversity among Bacillus
species isolated”. It was published on Food engineering and
19


Quality journal in 2012, at University Institute of Technology,
Cameroon, Africa.
+Bacillus subtilis strain OSS 42 was sequenced in

“Comparison of microbial communities native to three differently
polluted elogical niches in the industrial site of Bagnoil” research,
author: Sprocati, A.R., Alisi, C. And Tasso, F. and published on
Modern multidisciplinary applied microbiology journal, in Italia
(2006).
+Bacillus subtilis strain E9-1was identified in “Isolation,
identification, and characterrization of bacterial isolates degrading
chlorophenols

and

absorbable

organic

halides”,

author

:Dhakephalkar, P.K., Lapsiya, K.L., Savant, D.V. and Ranade,
D.R.. It was published on Microbial science Division in 2008, at
India.
-Bs2 and BN9 strains were consistent with Bacillus
megaterium (99%). Bs2 sample is isolated from “mud-dreg” and
BN9 from waste water. Bacillus megaterium is the one of bacteria
that is commonly used in the field of environment treatment.
4.Conclusion and Suggestion:
From “Mud-dreg” and waste water, there had 21 bacteria
strains were isolated. Through out gram staning, spore staining,
biochemistry test and biomolecular method, 2 strains are

identified as Bacillus subtilis (99%, 87% identical).

20