Tải bản đầy đủ
Supervisor : MSc. Hoang Van Thai

Supervisor : MSc. Hoang Van Thai

Tải bản đầy đủ

I would like to express my deepest appreciation to all those who have
provided me the possibility to complete this research.
First and foremost, I would like to express my sincere gratitude to my
advisors MSc. Hoang Van Thai who directed me, guided me to take the first
step in scientific research and he always made every effort in terms of
equipment and chemicals in time.
Furthermore, my sincere thanks also go to Dr. Pham The Hai who also
gave me orientations, always willing to answer all my questions about the
professional issues during my good thesis industry.
Besides, I am highly thankful to teachers, staff members and friends at
Department of of Biology, Siblings in the Department of Biochemistry and
Biochemistry Development, the teachers in the Institute of Microbiology Hanoi National University, VNU University of Science for their love and
kindness, insightful comments and suggestions.
Last but not least, I would like to thank my family for encouraging and
supporting me spiritually throughout my life.

Nguyen Thi Ngoc

1. Reasons for choosing themes
Currently, in Vietnam as well as in the world, most people prefer
bioproducts. Bioproducts known for their use for controlling pathogens are a
growing boom in developing countries, where products are cultivated or
added to foods to enhance digestive balance. Of particular interest is that
biopreparations can be commercialized (Fuller, 1987). Therefore, among the
microorganisms used for biological preparation, Bacillus subtilis is a popular
example on the market today.
B. subtilis has many good qualities. They are food safety, have high
economic benefits (in Japan and in European countries). B. subtilis bacteria
stimulate immunity, prevent bacterial pathogens from entering the body, ...
Also they are functional foods to supplement beneficial bacteria. Thus, it can
be said that "B. subtilis is a strain of probiotics that is safe for users".
In addition, Vietnam is a very agricultural country with a great
emphasis on livestock and farming. In order to increase the productivity and
quality of products based on practical needs and promote their strengths, I
conduct research "Optimization of the fermentation medium for B. subtilis
strain VTCC-B-51 to achieve high biomass yield".
2. Objectives of the study
Study on the fermentation parameters of B. subtilis on a 10 liter
fermenter based on environmental factors and culture parameters (stirring
speed, air,...).

B. subtilis


Bacillus subtilis
Luria- Bertani
Strain volume
OD strain
Optical density

List of tables

List of figures



Overview for B. subtilis


History of development

B. subtilis was first discovered in horse dung (1941) by the Nazi
German medical organization. At first, it was mainly used to prevent illness
for German soldiers fighting in North Africa[7].
Treatment had to wait until 1949-1957, when Henrry et al., obtaine the
pure culture of B. subtilis[16]. Since then, the term "subtilistherapie" has been
used to indicate the subtilis treatment of inflammatory bowel disease, colitis
and diarrhea due to digestive disorders.
Today, this bacterium is widely applied, thanks to the development of
biotechnology( as medicine, food and animal husbandry purposes).

According to the classification of Bergey (1974), B. subtilis belongs to:


- Kingdom: Bacteria
- Division: Firmicutes
- Class: Bacilli
- Order: Bacillales
- Family: Bacillaceae
- Genus: Bacillus
-Species: Bacillus subtils

B. subtilis belongs to the intestinal microflora of the intestinal tract,
which is distributed almost exclusively in the wild such as hay, dust, soil....
Most of them exist in the soil, typically cropland soil containing about
10-100 million CFU / g. In poor nutient soil in deserts, wastelands, B. subtilis
is very rare. Water and mud at the estuaries as well as in seawater have the
existence of B. subtilis spores( Vu Thi Thu, 1996 )[7].

Cell morphology

B. subtilis is bacterium with small size and short rod-shaped cells each
having two rounded tips of 0.5-1.0 μm x 1.5-3.0 μm; usually standing alone

or making short strings. The bacterium is a Gram-positive and has a capacity.
B. subtilis is bacterium of producing spores that can be and between the cell.
The bacterium is developed by the germination.

Figure 1: Cells of B. subtilis bacteria.

Colony morphology

After 24 hours implanted on agar medium, B. subtilis usually produces
clear colonies that are: dry, milky white, crease on the surface of the agar,
creased and creamy lobes, in the middle with a knob small convex and often
referred to as concentric rings and clinging to the agar surface.


Figure 2: Colony of B. subtilis.

Figure 3: Colony of B. subtilis.

The process of spore formation

Spores are a thickened solid biomass, the outermost of the spore is a
membrane. Crustaceans have many layers, which inhibit the penetration of
water and water soluble substances. Under the shell is the inner membrane of
the spore and in the same is a homogeneous mass of cytoplasm[1].

The outer membrane (Exosporium)
Sheathed (Tunique externe)
Covered in (Tunique interne)
Shell (Cortex)
Membrane biofilter spore center
Spore wall

Figure 4: Diagrams of internal structure bacterial spores (Nguyen Lan


B. subtilis spores are elliptical to spherical, measuring 0.6-0.9 μm x1.01.5μm, surrounded by multiple layers of lipoprotein, peptidoglycan,....
With the ability to create spores, bacteria can survive under adverse
conditions (depleted nutrients in the environment, the accumulation of
harmful metabolites and high temperatures, etc...)[7].
Each bacterium produces only one spore. When conditions are
favorable, spores will return to the vegetative cell.

Figure 5: B. subtilis spores are stained with malachite green.
The process of spore formation includes:
- Septum forming.
-Creating spore.
- Creating spore shell
- Synthetic spore shell.
- Releasing spores.
- When conditions are favorable, spores will germinate and develop into new
living cells (Nguyen Lan Dung et al., 2003).


Figure 6: The process of spore formation[1].
1.1.7. Spore composition
Spores contain large amounts of calcium, magnesium and dipicolinic
acid (this acid accounts for 5-12% of the dry weight of a spore). The role of
this acid is to make the spores resistant to high temperatures. Water content in
spores very low and exist in the form of link[2].
A spore of B. subtilis is oval-shaped when examined under an opaque
microscope, with its two ends are darker and can not be Gram-stained.

Biochemical characteristics of B. subtilis

B. subtilis fermentation does not produce sugars: glucose, maltose,
saccharose, xylose, mannitol, etc...
Other biochemical characteristics of B. subtilis include: indol (-), nitrate
(-), VP (+), NH3 (+), H2s (-), aerobic (+), catalase (+), amylase (+), casein
(+), citrate (+).
Blood clots: Some strains cause haemorrhage in horses and rabbits due
to the action of hemolysin [8].
Table 1: Biochemical reactions of B. subtilis (Holt, 1992)
Biochemical reaction



Catalase activity
MR (Methyl Red)
VP (Voges- Proskauer
Use citrate
Deodorizing Nitrate
Melt Gelatin
Starch grading



Research and application of B. subtilis bacteria in biological
products in the world


In medicine

In the resistance war against France, B. subtilis preparations was
researched to fight the battlefield to resolve diarrhea.
In 1949, the oral medicinal product containing B. subtilis in the form of
IB 5832 was introduced in France. In 1955, oral medications were given in
powder form and capsules.
In 1962, Guy Albot discovered that B. subtilis is effective in treating
diarrhea due to antibiotic abuse and maxvital colitis, in combination with
other lactic acid bacteria that are effective in treating intestinal dysplasia.

In agriculture

B. subtilis preparations are used to control pathogenic microorganisms
such as fungus Rhizoctonia solani, Fusarium sp, Pyriculariaoryaze and
postharvest products protection.