OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)

OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)OPTIMIZATION OF THE FERMENTATION MEDIUM FOR BACILLUS SUBTILIS STRAIN VTCCB51 TO ACHIEVE HIGH BIOMASS YIELD (LV tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY, HANOI

VNU UNIVERSITY OF SCIENCE

FACULTY OF BIOLOGY

Nguyen Thi Ngoc

OPTIMIZATION OF THE FERMENTATION

MEDIUM FOR BACILLUS SUBTILIS STRAIN

VTCC-B-51 TO ACHIEVE HIGH BIOMASS

YIELD

Submitted in partial fulfillment of the requirements for the degree of

Bachelor of Science in Microbiology(International Standard Program)

Hanoi – 2017

VIETNAM NATIONAL UNIVERSITY, HANOI

VNU UNIVERSITY OF SCIENCE

FACULTY OF BIOLOGY

Nguyen Thi Ngoc

OPTIMIZATION OF THE FERMENTATION

MEDIUM FOR BACILLUS SUBTILIS STRAIN

VTCC-B-51 TO ACHIEVE HIGH BIOMASS

YIELD

Submitted in partial fulfillment of the requirements for the degree of

Bachelor of Science in Microbiology (International Standard Program)

Supervisor : MSc Hoang Van Thai

Dr Pham The Hai

Hanoi - 2017

Graduation thesis Nguyen Thi Ngoc- K58 International Biology

Furthermore, my sincere thanks also go to Dr Pham The Hai who alsogave me orientations, always willing to answer all my questions about theprofessional issues during my good thesis industry

Besides, I am highly thankful to teachers, staff members and friends atDepartment of of Biology, Siblings in the Department of Biochemistry andBiochemistry Development, the teachers in the Institute of Microbiology -Hanoi National University, VNU University of Science for their love andkindness, insightful comments and suggestions

Last but not least, I would like to thank my family for encouraging andsupporting me spiritually throughout my life

StudentNguyen Thi Ngoc

Graduation thesis Nguyen Thi Ngoc- K58 International Biology

PREFACE

1 Reasons for choosing themes

Currently, in Vietnam as well as in the world, most people preferbioproducts Bioproducts known for their use for controlling pathogens are agrowing boom in developing countries, where products are cultivated oradded to foods to enhance digestive balance Of particular interest is thatbiopreparations 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 greatemphasis on livestock and farming In order to increase the productivity andquality 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 (stirringspeed, air, )

Graduation thesis Nguyen Thi Ngoc- K58 International Biology

Graduation thesis Nguyen Thi Ngoc- K58 International Biology

List of tables

Table 1: Biochemical reactions of B subtilis (Holt, 1992) 7

Table 2: Composition of the culture medium (for 1 liter) 15

Table 3: The pH of the culture medium tested for B subtilis 16

Table 4: Test results of B subtilis culture in triangular flasks 20

Table 5: Test results for B subtilis culture at different temperatures levels 22

Table 6: Results of count culturing obtained at OD different levels 23

Table 7: Parameters and results of series (i) fermentation on a 10 liter fermenter 24

Table 8: Parameters and results of batches (ii) fermentation on a 10 liter fermenter 26

Table 9: Parameters and results of batches (iii) fermentation on a 10 liter fermenter 27

Graduation thesis Nguyen Thi Ngoc- K58 International Biology

List of figures

Figure 1: Cells of B subtilis bacteria 2

Figure 2: Colony of B subtilis 3

Figure 3: Colony of B subtilis 3

Figure 4: Diagrams of internal structure bacterial spores (Nguyen Lan Dung)4 Figure 5: B subtilis spores are stained with malachite green 5

Figure 6: The process of spore formation 6

Figure 7: The fermentation system in the laboratory 13

Figure 8: Main form of fermentation batch cooker 14

Figure 9: Filtration system concentrates B subtilis biomass 19

Figure 10: Relationship between pH and growth capacity of 21

Figure 11: Relationship between temperature and growth capacity of 22

Figure 12: Standard line OD and density 23

Graduation thesis Nguyen Thi Ngoc- K58 International Biology

Contents

CHAPTER 1: LITERATURE REVIEWS 1

1.1. Overview for B subtilis 1

1.1.1 History of development 1

1.1.2 Classification 1

1.1.3 Distribution 1

1.1.4 Cell morphology 2

1.1.5 Colony morphology 2

1.1.6 The process of spore formation 3

1.1.7 Spore composition 5

1.1.8 Biochemical characteristics of B subtilis 5

1.2. Research and application of B subtilis bacteria in biological products in the world 7

1.2.1 In medicine 7

1.2.2 In agriculture 7

1.3. Research and application of B subtilis bacteria in biological products in Vietnam 8

1.3.1 In medicine 8

1.3.2 In agriculture 8

1.4 Fermentation process 8

1.4.1 Propagation 8

1.4.2 Fermentation 9

CHAPTER 2: MATERIALS AND METHODS 10

2.1 Materials, chemicals and equipment 10

2.1.1 Material 10

2.1.2 Chemicals 10

2.1.3 Equipment 11

2.1.4 The fermentation system: 11

Graduation thesis Nguyen Thi Ngoc- K58 International Biology

2.2 Methods 14

2.2.1 The physiological properties of B subtilis 14

2.2.2 Determination of the cell density changes 16

2.2.3 Optimization of fermentation conditions on a 10 liter fermenter.16 2.2.4 Culturing steps 16

CHAPTER 3: RESULTS AND DISCUSSION 19

3.1 Results 19

3.1.1 Physiological properties of B subtilis 19

3.1.1 Environmental temperature suitable for the growth of the strain.20 3.2 Standard OD line for cell density measurement 22

3.3 Culture parameters for a 10 liter fermenter 23

CONCLUSIONS AND PROPOSALS 29

1 Conclusions 29

2 Proposals 29

REFERENCES 30

CHAPTER 1: LITERATURE REVIEWS 1.1. Overview for B subtilis

1.1.1 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 illnessfor 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, colitisand diarrhea due to digestive disorders

Today, this bacterium is widely applied, thanks to the development ofbiotechnology( as medicine, food and animal husbandry purposes)

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].

1.1.4 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 alonem x 1.5-3.0 μm x 1.5-3.0 μm; usually standing alonem; 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.

1.1.5 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 oftenreferred to as concentric rings and clinging to the agar surface

Figure 2: Colony of B subtilis.

Figure 3: Colony of B subtilis.

1.1.6 The process of spore formation

Spores are a thickened solid biomass, the outermost of the spore is amembrane Crustaceans have many layers, which inhibit the penetration of

water and water soluble substances Under the shell is the inner membrane ofthe spore and in the same is a homogeneous mass of cytoplasm[1].

The outer membrane (Exosporium)

Sheathed (Tunique externe)

Covered in (Tunique interne)

B subtilis spores are elliptical to spherical, measuring 0.6-0.9 μm x 1.5-3.0 μm; usually standing alonem

x1.0-1.5μm x 1.5-3.0 μm; usually standing alonem, surrounded by multiple layers of lipoprotein, peptidoglycan,

With the ability to create spores, bacteria can survive under adverseconditions (depleted nutrients in the environment, the accumulation ofharmful metabolites and high temperatures, etc )[7]

Each bacterium produces only one spore When conditions arefavorable, 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 newliving 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 dipicolinicacid (this acid accounts for 5-12% of the dry weight of a spore) The role ofthis acid is to make the spores resistant to high temperatures Water content inspores 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

1.1.8 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)

1.2. Research and application of B subtilis bacteria in biological

products in the world

1.2.1 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 inpowder 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 withother lactic acid bacteria that are effective in treating intestinal dysplasia

In 2013, the objective of this study was to evaluate the effect of dietary

supplementation of a probiotic bacterium, Bacillus subtilis, on the growth, immune response and antioxidant activities of shrimp ((Litopenaeus vannamei) by Wen-Ying Shen, Ling- Lin Fu, Wei- Fen Li and Yao- Rong

Zhu[9]

In 1940, Norio Kimura Yokohamo used B subtilis to suppress the growth and toxin production of Asperligus flavus, Aspergilus paraciticus.

Roman and colleagues studied B subtilis (ATCC_6633, ATCC_9372)

which reduced 40-50% of aflatoxin in aflatoxin- fluid for 20 days[8]

1.3 Research and application of B subtilis bacteria in biological

products in Vietnam

1.3.1 In medicine

B subtilis has been studied for its properties, safety and viability and

development in the intestine (Hong et al., 2009)

In 1958-1960, Dr Pham Ngoc Thach produced a series of B subtilis

preparations for intestinal diseases

The Department of Medical Hygiene of Bach Mai Hospital in Hanoi

has researched and produced B subtilis for human diarrhea.

In 1971, Tran Minh Hung, Le Thi Ba, Nguyen Van Hung studied the

production of B subtilis in the culture of soybean, crab steamed with

starch This preparation is for pigs 0.5-1.0 g / kg body weight Results of pigs

after using B subtilis gained fast weight gain.

1.3.2 In agriculture

The Biological Center of the Ministry of Agriculture in Ho Chi Minh

City has studied the production of Bactophyl from B subtilis to control

fungus on vegetables

Ho Thi My Hong, Nguyen Thanh Binh and the Hanoi Center for

Biological Production of B subtilis to prevent disease in corn Ostrinia

furnacalis

In 1982, Vu Van Ngu and his collaborators produced coliforms thatreduced recurrence due to diarrhea in pigs compared to antibiotic treatment,resulting in rapid weight gain[8]

1.4 Fermentation process

1.4.1 Propagation

The purpose of propagation is to produce sufficiently high biomass forinclusion in the production process Propagation stage must be performed insterile cabinet to avoid infection Then transferred to the heat shakingmachine for culture Shaking speed is an important factor affecting the growth

of strains during propagation[1]

When the breeding process is finished, it is necessary to check thestrains to make sure the strains are not contaminated before production

1.4.2 Fermentation

This is the main stage of the fermentation process, so it is necessary toclosely control the conditions (eg: breeding rate, temperature, pH, air flow,dissolved oxygen DO ) the highest fermentation effect

Sterility conditions during fermentation:

-Use the seeds not contaminated

-Disinfect the environment and fermenter before use

-Sterilize ingredients to be added to the fermenting pot duringfermentation

-Maintain sterile conditions during fermentation

During fermentation, cell growth should be monitored to adjust theparameters accordingly to achieve high biomass yield Tracking methodsinclude:

-Take a microscope stain on the microscope to evaluate spore quality

-Measure pH and measure OD to build the calibration curve

-The colony count on the agar plate contains the appropriate medium

-Add the appropriate gas stirring time from time to time

Note: During the fermentation process, the person wearing the mask

(should wear a blue shirt) and always have to sterilize his hands thoroughlywith 70 ° alcohol to avoid infection

CHAPTER 2: MATERIALS AND METHODS 2.1 Materials, chemicals and equipment

2.1.1 Material

Microbial strain: B subtilis strain VTCC-B-51 was provided by the

Vietnam Type Culture Collection of the IMB – Viet Nam NationalUniversity

2.1.2 Chemicals

Shaking Machine Germany

OD gauge

And other common tools in the lab

2.1.4 The fermentation system:

Manufacturer: Marubishi – Japan

Product code: MDL

Features:

+Accuracy, easy handing and flexibility

+Six kinds of different size are avaiable (100, 200, 300, 500, 700 and

1000 ml) as standard

+Accurate control

+Highly effective aeration and stirring

+MDL- 8 C controller realizes your highly sophisticated cultivationwork with max 8 channel.

fermentation system in the laboratory.

Figure 8: Main form of fermentation batch cooker.

The main construction of the fermentation system includes:

- Air injection unit

- Stirrer

- Stirring shaft

- Partition

- PH electrode