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Nghiên cứu tuyển chọn vi khuẩn bacillus thuringiensis phục vụ tạo chế phẩm diệt côn trùng bộ hai cánh (diptera) tt tiếng anh

MINISTRY OF EDUCATION

MINISTRY OF AGRICULTURE

AND TRAINING

AND RURAL DEVELOPMENT

VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

PHAM THUY DUONG

STUDY ON SELECTION OF BACILLUS THURINGIENSIS
STRAIN FOR DIPTERAN INSECTIDAL PREPARATION
PRODUCTION

Major: Biotechnology
Major code: 9 42 02 01

SUMMARY OF THE AGRICULTURAL DISSERTATION


Ha Noi – 2019


The work is completed at:
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

Science instructors:
Academic advisor 1: Assoc. Prof. Ngo Dinh Binh
Academic advisor 2: Dr. Le Duc Khanh

Reviewer 1
Reviewer 2
Reviewer 3
The dissertation will be defended before the State-level
thesis judge council meets at ….
………………………………………………………………
………………………………………………………………

The thesis can be found at the library:
- Vietnam National Library
- Library of Vietnam Academy of Agricultural Sciences


INTRODUCTION
1. The urgency of the research
Bacillus thuringiensis (B. thuringiensis) is soil, positive-gram
bacteria.The ability of this bacteria is to produce toxic crystal proteins
against insects but not human, ecology and usful organism.Studying on
pesticidal activites against dipteral species of B. thuringinensis isolated in
Vietnam is essential to find the potential isolates with the native, strong
toxic gene to produce the biological product against dipteral species.
The insect species belonged to dipteral order is one of the largest order
in the insect class with the variety of numbers and species. Diptera order is
very diverse in terms of ecology and mainly harmful agents in agriculture
(fruit flies) or healthy like flies, mosquitoes. They are also the vector of
many diseases between people and people or between animals and people.
Mosquito is highly lethal transmission such as Anopheles gambiae is the
vector of malaria. Aedes aegypti transmits dengue fever. Culex
tritaeniorhynchus infects Japanese encephalitis while flies are the
transmitting agents about 100 diseases but mainly dangerous diseases such
as polio, pain trachoma, hepatitis (A, E), Rickettsiale regression fever,
dysentery, cholera, typhoid, Streptococcus and Staphyloccocus.
Today, there are many different methods for control this kind of insects
which were applied. The most commonmethod is the insecticide. However,
these drugs are quite expensive, are produced from chemical agents
causing pollution and bad health effects on human, animals but not
eradicating the pathogen.
Starting from the above practice we conduct research on the topic:
“Study on selection of Bacillus thuringiensis strain for Dipteran insectidal
preparation production”.
2. Objects
The main purpose of this topic is to increase the knowledge of two
species P. heterotremus and P. westermani, provide a scientific basis for
diagnosis and prevention of lung fluke dieasease, contributie to public
health protection
The specific objects:
2.1. General goal


Selection of indigenous Bacillus thuringiensis strains produce Toxic
crystalline proteins against insects belonging Diptera order; Expression,
purificationcrystal proteinin E. coli bacteria for intensive research; And
produce biological production of Bacillus thuringiensis against house flies
(Musca domestica) from brewery wastes
Screening and collection of Bacillus thuringiensis strains which are
capable of producing cry2A coding genesagainst dipteral species soil and
leaf samples in some provinces of Vietnam and identify
Express and purify recombinant protein (rCry2A) in E.coli BL 21 (DE3).
Find out the method of treating beer malt waste to produce culture
environment for B. thuringiensis bacteria and study of factors affecting
grow and Crystalline protein synthesis of B. thuringiensis when using malt
beer as a culture medium at the laboratory scale. At the same time,
preliminarily evaluatethe effect of insecticidal insecticide of BT product
against dipteral.
3. Research content
- Isolating and identification biological characteristics of some strains B.
thuringiensis (Bt) has insecticidal activity.
- Cloning and sequencing the cry2 gene of Bt strains, isolated from soil,
leaves, having the pesticide activity against dipteral insect.
- Expression Cry2 protein from strain of B. thuringiensis selected.
- Studying methods for beer malt waste treatmentto make the culture
medium for B. thuringiensis fermentation. Optimizing the culture medium
for fermenting B . thuringiensis from beer malt. - Study the factors
affecting growth and cry protein biosynthesis of B. thuringiensis when
using beer malt ascultural medium at the laboratory scale.
- BT fermentation to of biological products against dipteral and evaluated
the insecticidal effectiveness against dipteral insect in laboratory and field.
4. New contributions of the topic
- This study is one of the systematic studies about genes cry2A of B.
thuringiensis in Vietnam.
- Cloning and successful expression of cry2A gene in E. coli BL21 (DE3).
- Using beer malt wastewhich is waste of beverage manufacturing industry
as materials to provide nutrients (carbon, nitrogen, minerals...) for B.
thuringiensis fermentation to production BT bio-pesticides. Optimizing the
cultural condition for bacteria B. thuringiensis from the brewer's malt


waste by response surface methodology to produce biological production
against dipteral insects
5. Scientific and practical meaning of the topic
5.1. Scientific meaning:
A systematic study of the cry2 gene is a reliable theoretical basisfor
further studies on B. thuringiensis in common and cry2 gene in particular.
Based on the evaluation of the effect of the gene cry2 protein on
insecticidal ability against diptera like flies, this study was paved the way
to create biological products.
5.2. Practical meaning: The results of the thesis contribute to the
effective exploitation of microbiological gene sources in general and the
cry2A gene of B. thuringiensis in particular contributed produce biological
productionagainst dipteral insects. On the other hand applying Beer malt a waste of industrial manufacturing asthe material for the fermentation
process is needed. This research is to directly improve the value of beer
malt waste and produce the environmentally friendly products for
sustainable agricultural production
6. Thesis structure
Thesis is 149 pages (excluding references and appendices) computer with
A4 size with 23 tables and 43 figures. The thesis consists of 5 parts:
Introduction (04 pages); literatural review (40 pages); Materials and methods
(19 pages); Result and discussion (60 pages); Conclusion and prepective (02
pages). Referenced 146 documents including 20 Vietnamese documents and
126 English documents.
CHAPTER 1. OVERVIEWAND BASIS OF SCIENCE OF THE
THESIS
The thesis has consulted and summarized the English and Vietnamese
documents related to 7 issue: Overview of B. thuringiensis; overview gene
cry2; Overview of E. coli expression system; Overview of test insects
experience; B. thuringiensis bio-pesticide ; Overview of Malt beer; and the
situation of research and application of B. thuringiensis bacteria in
Vietnam.
In it, Bacillus thuringiensis (B. thuringiensis) is soil, positive gram
bacteria. During the development process, B. thuringiensis is to capably
produce 3 main insecticidal toxin including Cry (crystal endotoxin ), Cyt
(cytolysin) and Vip (vegetative insecticidal protein). The cry2 genes


encode crystal proteins of about 65 - 71 kDa, produced by some subspecies
of B. thuringiensis such as : kurstaki (HD-1, HD-263, NRD-12 and 14
other lines), thurigiensis, tolwwarthi, israelensis, kenyae ... (Dwu et al.,
1991; Ohba and Aizawa, 1986; Yamamoto, 1983). So far, more than 80
cry2
genes
have
been
identity
(http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/ B. thuringiensis
/toxins2.html )
Flies and mosquitoes are parasitic arthropods, belonging to the class of
insects (Insecta), order Diptera. They are many intermediaries vector of
many dangerous diseases between people and people or between animals
and people. Further, weather conditions and climate in Vietnam are very
favorable for their development. There are many different methods applied
to control the harmful insects. The most common method is using chemical
toxin against flies and mosquito. However, these drugs are usually too
expensive. It is also easy to pollute the environment and affect to the health
of humans, animals, but not eradicating the pathogen.
These key points are presented in Chapter 1. Thereby, it is easy to found
that the study about pesticidal activities against dipteral larva of B.
thuringiensis strains isolated in Vietnam is necessary. Furthermore, finding
out strains that carry natural genes having strong activity is essential to
produce biologicalproducts against dipteral insects.
CHAPTER 2
MATERIAL, LOCATION AND METHODS
2.1. Material and location
Soil and leaf samples used to isolate Bacillus thuringiensis were taken
from Dien Bien, Hanoi, Nghe An, Nha Trang (Khanh Hoa) and Lam
regions Dong, Ho Chi Minh City, Kien Giang. Beer malt is taken from the
Saigon - Hanoi brewery belonging to the public park Small and medium
enterprises in Bac Tu Liem district. Larvae of house flies age 2; Fruit flies
received from the Insect Department - Plant Protection Institute. Anopheles
minimus mosquito larvae , Aedes aegypti, Culex quinquefasciatus provided
by the Central Institute of Malaria parasites. E. coli strains DH5α for gene
cloning, E.coli BL21 for the expression of cry2A gene from . The strain is
same provided by Seed Breeding Center and Conservation of
Microbiological Genetic Resources, Public Institute Biology. Sequence of
cry2A amplifiers


Location and practice time
The experiments were performed at the Seed Center laboratory and
Preservation of microbial gene sources, Institute of Biotechnology,
Department of Microbiology School - Institute of Environmental
Technology, Department of Insects - Institute of Real Protection Materials,
Laboratory of Department of Biotechnology and Environment-School
Oriental University. Experimental implementation period from June /
2012-December / 2016.
2.2. Methods
2.3.1. Microbiological method The method of isolating Bacillus
thuringiensis. B. thuringiensis bacteria were isolated soil samples and leaf
samples collected according to the method of Thiery and Frachon (1997).
Classification of B. thuringiensis bacteria by free serum method Translate.
2.3.2. Biological activity testing method Quantify spore and crystal density
The isolated Bt strain was planted with carpet on a peptri containing lips
MPA school. The number of spores is calculated according to the formula
(Ohba et al., 1986) Determine LC50 for test insects Testing the ability to
kill the larvae Two wings on the larvae Musca domestica housefly, fruit fly
larvae age of two, mosquito larvae under Thiery and Frachon's methods at
two concentrations are 10 7 and 10 9 spores / ml with house flies, fruit flies
and 10 8 and 10 6 spores / ml with mosquitoes. Each concentration is
tested Experiment with 3 plastic cups (10 cups per each cup)
The rate of dead larvae is calculated according to Abbott formula (Abbott,
1925).
2.3.3. Molecular biological method Method of extracting plasmid DNA
from bacteria Bacterial plasmid DNA is extracted and purified by kit
GenJET TM Plasmid Miniprep (Fermentas). PCR method for amplifying
cry2A gene DNA biosynthesis was conducted by PCR (Polymerase
technique) Chain Reaction) (Sambrook, 2001). Method of isolating the
cry2 gene stream Cut DNA by cutting enzyme restriction. DNA pairing
with T4 DNA ligase. Transform recombinant plasmid into E. coli DH5 α.
Electrophoresis on agarose gel DNA was detected and relatively quantified
by the above electrophoresis techniqueagarose gel according to the method
of Sambrook & Russel, 2001.
Purify DNA from agarose gel The DNA product is purified from agarose
gel with GenJET kit TM Gel Extraction (Fermentas) according to the


manufacturer's instruction manual. Method of determining the nucleotide
sequence of the cloned gene segment The DNA sequence is determined
according to the method of Sanger and his colleagues. DNA samples were
submitted for reading at Microgen, Korea using primers M13. Processing
sequencing results with Bioedit software, comparing gene sequences
separated the lines with the sequences published in the International
GenBank only online: https://blast.ncbi.nlm.nih.gov/Blast.cgi
Expression vector design Use pET22b (+) vector as an expression vector.
The cry2A gene is given Enter pET22b (+) in the position BamHI and Xho
I. The amino acid sequence of the protein recombinant includes the entire
amino acid sequence of the natural cry2A gene by 6amino Histidine acid at
the beginning of C and 36 amino acids at the beginning of N. Genetic
expression method Activating a colony in LB environment with ampicillin
supplemented The final concentration is 50 μ g / ml, shaking at 37 o C
overnight. Then transplant 1% into 20 ml of MPB medium supplemented
with ampicillin antibiotic concentration of 50 μ g / ml and shaking at 37 o
C for OD to 0.5-0.8. IPTG sensor (100 mM) reached 1mM, raised at 37 o
C for 4 hours. Electrophoresis on polyacrylamide gel 12.6% for product
testing Expressive products. 12.6% polyacrylamide gel electrophoresis
method according to the method by Hoefer et al. (1994).
Western blot method Protein samples after testing with SDS-PAGE on
acryamide 12% gel will transferred to PVDF film. Protein samples will be
incubated with specific antibodies and antibody detection. Then detected
by solution containing substrate peroxidase (Sigma). Recombinant protein
purification method Recombinant protein is purified by chromatography
Ni2+ column agarose according to rules Guidelines for Invitrogen
2.3.4. Optimization of fermentation environment according to the surface
response method Prepare to breed Bacteria are raised on sterile MTCS
environment. Shaking at 30 o C, 200 cycles / minute, breeding time 8-10
hours. Cultured culture (contains cells being in the growth stage) is used to
breed for experiments next (Adjiable et al., 2009; Yezza et al., 2006).
Microbial fermentation method
Bacillus thuringiensis in the triangle Ferment in 500 ml triangle flask at a
temperature of 30 ± 0.1, shaking speed 200 round / minute, 48-hour culture
period (Yezza et al., 2006). Fermentation in a 10-liter fermentation device
Put 10 liters of medium into the fermentation tank, sterilize at 121 o C, 1


at, in Within 30 minutes, cool to room temperature, insert the fermentation
vessel into the device yeast. Set up fermentation parameters: temperature
30 ± 0, 1 o C, pH7 ± 0,1, stirring speed 300 rpm, DO> 25 mg / l, air flow
rate of 2-4 liters / minute (Luong Duc Pham, 2008; Avigone et al, 1992)
Hydrolysis of beer residues The beer residue after being taken to the
laboratory is milled and processed Microbial culture materials by
combined pyrolysis method pH (Nguyen Thi Van Trang et al, 2012; Valo
et al, 2004). Experimental layout The hydrolysis of beer residue after
treatment is taken to analyze the chemical composition follow standard
methods. In which TOC is determined by method SMEWW 5310B-2005;
TN, TP is determined by method EPA-352.1 and EPA-365.2, metal
components are determined by mode France SMEWW 3125-2005.
(Adjiable et al, 2009)
Study the effect of solids concentration on the growth process and
crystalline protein biosynthesis of MSS8.4 strain Determine the additional
nutrient source for the hydrolysis of beer residue Evaluate the independent
impact of nutritional factors on growth and the production of crystalline
proteins Optimize the environmental composition by surface response
response method The description is done according to the method of
Rajesh et al. (2012). After determine the impact range of each element on
the growth and total fertility protein combination of MSS8.4, import data
into expert Design software 10.1. Data processing
CHAPTER 3
RESULT AND DISCUSSION
3.1. Isolation and selection of efficacy resistant strains of double-winged
insects Bacillus thuringiensis strains
3.1.1. Isolation of Bacillus strains
For isolation and selection the Bt strain having insecticidal activity against
Diptera, 317 soil and leaf samples were collected in 7 provinces from 7
regions Vietnam. These sites have never applied Bt product before.
In 317 soil samples and leaves, 198 samples carried Bacillus bacteria and
119 samples were not isolated Bt. Thus the frequency of Bacillus appears
62.4%. Location with high rates of Bacillus in the sample included Kien
Giang (75.0%), Dien Bien (72.8%) and Hanoi (72.8%); The lowest bt
isolation rate is Nghe An (33.3%).


Of the 1,020 colonies having characteristics specific for genus Bacillus,
there are 440 Bt group colonies are based on the ability to form crystals
(accounting for 43%). This ratio depends on the number of samples
collected in the provinces, the highest in the collected samples in Hanoi
(54%) and Ho Chi Minh City (53%), the lowest in the sample collect in
Lam Dong (25%), Dien Bien and Kien Giang provinces for similar ratios
each other (42% and 44%). Soil samples were collected in 7 geographic
regions of Vietnam with Bt frequency higher than previous publications
(Martin and Travers, 1989; Infantry and plus The, 2005). The frequency of
Bt in this study was 62.4% in accordance with previous publications by
Martin and Travers (1989), in the soil sample New Zealand (Chilcott and
Wigley, 1993), in the Korean soil (Lee and partner, 1995) and in the
Japanese sample (Ohba and Aizawa, 1986).
The appearance of Bt is almost everywhere in the living environment,
including: soil agriculture, forest land, urban land, mangrove land, even
desert (Dulmage and Aizawa, 1982; Martin and Travers, 1989; Zhang et al,
2000; Uribe and plus The, 2003). In this study, the appearance of Bt was
concentrated mainly in Hanoi urban land and high population density. This
result is much higher than research of Quesada-Moraga (Quesada-Moraga
et al., 2004), appropriate with the results of Ayyasamy (2012). According
to Ayyasamy, the ratio of Bt in urban land is 80%, however, the highest
frequency appears in agricultural land (Ayyasamy and partner, 2012).
Thus, compared to the data published by the above authors, the frequency
appears Bt and the Bt index that the topic isolates are quite high. It means
that the number of Bt species in Vietnam is very rich. This finding
indicated that Bt collected in Vietnam can provide many precious genetic
resources.
3.1.2. Research on crystalline proteins
The strains of B. Thuringiensis isolated in 7 geographical regions of
Vietnam Male (198 strains), was cultured on MPA environment. After 3
days this samples were staining with fushin dye and directly observated on
optical microscopes in oil glass objectsto determine the crystal shape of
micro strains bacteria. Results from 196 isolates have the ability to produce
crystals in all shapes format. There are strains capable of producing 1-3
types of crystals. Specifically, 186 strains pyramid shaped crystals
accounted for 42.2%; 193 spherical crystals accounting for 43.8%; 46 sets


of crystalline seminarians account for 10.5%; 15 strains Unidentified
crystalline births account for 3.5%.
Study on the crystal shape of Bt strains isolated in the province Chengdu China has an extremely spherical and spherical tower shape, occupying
91.8% (Yu et al., 2015). However, the majority of the crystal structure of
these The Bt strain collected in Tamil Nadu state - India has the form of a
cube (26.9%) and pole shape pyramid (21.2%) (Ramalakshmi and
Udayasuriyan, 2010). Results of observing the crystal shape of collected Bt
strains in Saudi Arabia Arabia concluded, spherical crystal structure
accounted for a large proportion (54%), followed by unformed form (27%)
and pole tower (16%) (El-kresh et al., 2014). The diversity and difference
in crystal shape obtained from isolated strains in this study as well as in
other studies maybe due to the differences in biological characteristics of
Bt strains as well as protein characteristics Cry of the collected strains.
According to published documents around the world, strains carring the
cry2A gene were very variable They can be generated from the subspecies
such as. serovar kurstaki, Bt. serovar sotto, Bt. serovar israelensis, Bt.
serovar kenyae has mainly shape bridge and tower crystal protein types.
Therefore, the strains are capable of producing spherical crystals and the
tower shape selected for genetic classification and screening cry2A by
immune serum reaction.
Classification of strains Bacillus thuringiensis with using immune
identification kit
Throughout the serotype identification, 32 Bt strain (accounting for 71%),
including 5 strains belonging to the Bt serovar. kurstaki , 2 strains
belonging to Bt serovar israelensis (Table 3.1) .
Table 3.1. Results of subspecies classification of Bacillus thuringiensis
strains using immune identification method
ST
Subspecies Bacillus thuringiensis
Sero type
No of
Rate
T
strains
(%)
agglutinat
ion
1

Bacillus
israelensis

thuringiensisserovar 14

2

Bacillus thuringiensis serovar kurstaki

3a,3b

2

6,25

5

15,62


3

Bacillus
sumiyoshiensis

thuringiensisserovar 3d

9

28,13

4

Bacillus
coreanensis

thuringiensisserovar 25

4

12,5

5

Bacillus
thuringiensis
neoleonensis

2

6,25

6

Bacillus thuringiensis serovar aizawai

5

15,63

7

Bacillus
thuringiensis
yunnanensis

1

3,13

8

Bacillus thuringiensisserovar pakistani

13

3

9,38

9

Bacillus
thuringiensis
oswaldocruzi

serovar 38

1

1,39

32

100

serovar 24a, 24b
7

serovar 20a, 20b

Total

Research by Martinez et al. (2005) showed that the gene c ry2 appears in
all strains of B. thuringiensis serovar kurstaki . In addition, strain B.
thuringiensis serovar israelensis is also found commonly in environments
life of mosquitoes (Martinez and Caballero, 2002). Therefore, the strains
belong to these groups will be selected by the topic for subsequent
experiments
3.1.3. Evaluate the ability of the two-wing insect to kill insects
In order to examine the killing effects against ( Diptera ) larvae of Bt
subspecies B. Thuringiensis serovar kurstaki and B. Thuringiensis serovar
israelensis spore were diluted to the concentrations of 109 spores / ml and
tests with two day old larvae of mosquito larvae and fruit fly.
Table 3.2. Results of the activity of the two-winged larvae of Bt strains
after 3 and 5 days of testing
Experiment with Experiment with Experiment with
mosquitos’ larvae house flys’ larvae fruit flys’ larvae
Strain

LD 2.21

Death
Death
Death
rate
rate
rate after
after 3 after 5 3
days
days
days(%) (%)
(%)
66,67
90,33
83,33

Death
rate
after 5
days(%)
100

Death
Death
rate
rate
after 3 after 5
days
days(%)
(%)


LNT 7.12

66,67

96,67

83,33

100

16,67

33,33

LNT 16.6

73,33

83,33

86,67

100

13,33

16,67

MSS 1.1

50

100

66,67

83,33

3,33

10

MSS 4.2

46,67

78,67

60

76,67

0

10

MSS 6.3

63,33

98,33

80

100

13,33

20

MSS 8.4

80

100

93,33

100

20

33,33

4D4

6,67

30

16,67

30

0

0

Pesticide activitiesexamination results showed that B. thuringiensis strains
had the high potential of larvae killing effects. The rate of dead larvae
increased duringexperimental time. Atthe concentration of 109 spores / ml
selected strains provide high mortality rate, reaches 76.67 - 100% after 5
days. However, the ability to kill effect against house flies larvae were
quite low, only 0-33.33% after 7 days of testing.
3.2. Confirm the presence of cry2A related to insect killing characteristics
biplane in isolated strains
3.2.1. Detection of cry2A gene from Bacillus thuringiensis strains by PCR
method
The results indicated that Cry2A were observed in 7 strains belonged
to Bt serovar kurstaki and Bt serovar israelensis with the band of
approximately 1,9 kb.
3.2.2. Cloning and sequencing of Cry2A
The Cry2A genes collected from 2 highest pesticidal activities strains
were amplified and cloned into pCR2.1 cloning vector and transformed
into E. coli DH5alpha
After 14 – 16 hours incubating on selection medium LB agar plus
Ampicillin 50 ug/ml in 37oC, growth colonies were preliminarily examined
by colony pcr method using specific primers. The result showed that all off
4 examined colonies had the expected band of 19 kb
Plasmid extraction and digestion from 3 of 4 selected colonies agreed
with the previous results. To be specific, a upper band of 3,9 kb and lower
band of 1,9 kb corresponding to expected band were observed. It means
that these genes were successfully cloned
These plasmids were sequencing by automated sequencing
(Macrogen, Korea) using specific primers. The sequence were pre-process


by BioEdit ver 7.0.9. These sequence were aligned and compared with
reference cry gene available in Genbank
The results indicated the sequences obtained belonged to Cry2A,
subgroup Cry2Aa. Toxics belonged to Cry2Aa and cry4 were
demonstrated that having potential activities against insect species
belonged to dipteral order.
The sequences of the genes were aligned with other sequences of
cry2Aa subgroup. The result indicated the Cry2Aa gene ofMSS8.4 strain
was 1902 bp in length and had 99% of nucleotide identity with previous
public sequences with 6 different sites 305 (G-A), 500 (G-A), 783 (T-G),
1054 (T-G), 1303 (G-A), 1575 (C-T). The Cry2Aa of LNT7.12 had 100%
nucleotide identity with previous strain. The sequence of Cry2Aa obtained
from MSS8.4 were uploaded to Genbank with accession number of
KM588296.
Comparing the redundant amino acid of 2 sequences by BLAST
indicated that amino acid sequence of Cry2Aa protein of MSS8.4 was
similar with others (99% amino acid identity). To be specific, there were 5
different sites (102 (S-N), 167 (R-Q), 261 (F-L), 352 (W-G), 435 (D-N)).
Nucleotide substitution at 1575 (C-T) was not change the corresponding
amino acid. These results indicated that 2 cry proteins were toxic cry
protein group Cry2A, subgroup Cry2Aa.
Comparing the structure of Cry2A protein of MSS8.4 with others
indicated that it was high similarity (99%) with Cry2Aa of B. thuringiensis
serovar kurstaki (Pdb_id 1: 1I5P_A), determined by Morse et al. (2003) via
X-ray crystallization. Cry2Aa structure were divided into 3 domains: Ntermination included 210 aa (Val-53 to Lys-263); central domain (205 aa
from Leu-267 to Asn-472) and 224 a.a C termination (from Phe-494 to
Asn-628).
3.2.3. Protein expression
3.2.3.1. Construct the recombinant structure encoding Cry2A and
expressed in E. coli BL21 DE3
In order to confirmation the pesticide activity is result by Cry2A,
Cry2A sequence from MSS8.4 were further express and examined the
pesticide activity. 5 randomly chosen colonies were examined by directed
PCR. 4 of them appeared the 1,9 kb bands. Enzyme digestion test by


BamHI and XhoI were also indicated that the Cry2A structure were
successfully constructed.
3.2.3.2. Expression cry2A in E. coli
Preliminary expression of cry2A gene
Result of total protein electrophoresis on SDS-PAGE gel showed that the
induced clone E. coli BL21 (DE3) containing expression vector pET22b(+)-cry2A generated a bold protein band with size approximate 70 kDa,
completely coinciding with the expected size of fusion form of Cry2A
protein. Thus, it can be concluded that the expression of cry2A gene was
successful.
Confirm recombinant Cry2A by Western blot analysis
The results of Figure 3.17 indicated that the fusion protein was expressed a
band with the precise size (about 70 kDa). This can be assured that Cry2A
protein was successfully expressed in pET22b (+).

Figure 3.17. Analysis of expression of Cry2A protein by Western blot. M:
Protein marker (Biobasic); 1: Positive control (Sigma); 2-4: Samples after
induction at 1h, 2h, 5h; 5: Empty vector.
In order to be able to produce Cry2A products that have insecticidal
activity against Diptera, protein products need to be further processed and
evaluate insecticidal activity on insects.
Table 3.3. Bioassay of the insecticidal activity of rCry2A and crystal
protein from B. thuringiensis with M. domestica larvae
CI 95%
Larvae
Protein
LC50 (g)
Min
Max
M.
4D4
442,5
11,3
56,5
domestica
MSS8.4
283,5
17,6
100
rCry2A
264,7
28,3
66,1
MSS8.4+rCry2A 268,6
27,8
59,3


Bioassay of insecticidal activity after 72 hours showed that recombinant
protein was highly active with M. domestica larvae (LC 50 = 264.7 µg).
Simultaneously, the activity of the natural Cry2A protein of MSS8.4 (LC 50
= 283.5 µg) was also higher than that of the 4D4 standard strain (LC 50 =
442.5 µg). The difference in insecticidal ability can be found in the
MSS8.4 strain not only form Cry2A toxin but also from Cry1A toxin.
3.2.4. Study onoptimal gene expression conditions
3.2.4.1. Optimizing the temperature in cry2A gene expression
E. coli BL21 cells were inoculated at different temperature conditions: 28,
30, 37, 40oC. After 4 hours of culture, cellswere harvested, run
electrophoresis on 12.6% polyacrylamide gel. The results showed that at
37°C, the cell expressed the darkest protein band. Therefore,the
temperature of 37oC was chosen for rCry2A expression.
3.2.4.2. Optimizing the concentration of inducer in cry2A geneexpression
Study the concentration of inducer IPTG at 0.1; 0.2; 0.5; 1; 1.5 mM. The
results showed that the most optimal level of Cry2A protein was produced
when induced at 1mM.
3.2.4.3. Optimizingthe inducition time in cry2A gene expression
Fixed the induction temperature at 37oC, an IPTG-induced concentration of
1 mM and investigated recombinant protein expression by time (1, 2, 3, 4,
5 hours). Samples were treated and run electrophoresis on 12.6%
polyacrylamide gel. As a result, the amount of recombinant protein
produced was increasedgradually by time, and got the highest levelafter 4
and 5 hours. Therefore, the induction time of 4 hours was chosen to harvest
samples to ensure product quality and fermentation costs.
3.2.5. Purification of recombinant protein by Probond Nikel Resin affinity
chromatography column
Recombinant strain was expressed at 37oC, 1 mM IPTG, and harvesed after
4 hours. The cell biomass was then collected by centrifugation, treated and
purified.
SDS-PAGE electrophoresis result showed that recombinant Cry2A protein
was purified with size of 70 kDa.
3.2.6. The insecticidal activity against Diptera of rCry2A and the strain
MSS8.4
Table 3.4. Insecticidal activity of crystalline protein from MSS8.4 strain
and recombinant rCry2A protein


Larvae
M. domestica

A. minimus

A. aedes

Protein

LC50 (g)* CI 95%
Min

Max

442.5

11.3

56.5

MSS8.4 283.5

17.6

100

rCry2A

264.7

28.3

66.1

4D4

9.42

53.33

70

MSS8.4 7.34

56.67

83.33

rCry2A

7.03

70

93.33

4D4

19.05

6.67

63.33

MSS8.4 14.52

56.67

73.33

rCry2A

13.33

63.33

80

19.05

36.67

46.67

MSS8.4 17.6

70

90

rCry2A

56.67

86.67

4D4

Cx. quinquefasciatus 4D4

14.52

The insecticidal activity of crystalline proteins was determined based on
the ability of killingDiptera larvae: M. domestica, A. minimus, A. aedes,
Cx. quinquefasciatus. Tested at concentrations ranging from 0.5 to 500
µg/g, the results were determined and analyzed by Probit analysis.
Bioassay results were displayed in Table 3.4.
Thus, the recombinant Cry2A protein has been successfully expressed in E.
coli and revealed insecticidal activity against Diptera larvae higher than the
natural protein as well as the standard strain did.This resultswere much
higher than that of Misra et al., purified Cry2Aa4 protein by affinity
chromatography hadLC50 values in the range of 100-500 mg (Misra et al.,
2002); Yilmaz et al (2017) indicated that Cry2Aa18 expressed from cry2A
gene of B. thuringiensis SY49-1 can kill C. pipiens larvae (LC50 = 630
µg/ml). Compared to the study of Reyaz and Arulsel (2016), recombinant
Cry2AI1protein after purification was active for Spodoptera litura and
Helicoverpa armigera (LC50 = 2,448 µg/ml) and rCry2A activity was much
lower for Helicoverpa armigera (LC50 = 3.374μg/ml).
3.3. Environmental selection and optimal fermentation increase the ability
of crystal protein formation of MSS8.4 strains from beer waste
3.3.1. Treatment beer waste as raw material for culture of MSS8.4


3.3.1.1. Influence of pre-treatment methods of beer wasteas raw material
for fermentation of MSS8.4
When used as raw material for fermenting bacteria MSS8.4, beer wastewas
grounded and adjusted to a concentration of 2% solids. The experiment
was carried out in a triangle flask at 30°C, 48 hours and shaking rate of 200
rpm.The analyses were presented in Table 3.5.
The analytical results showed that using acid hydrolyzate generated the
highest density of TCC, SC and crystal protein, the cell density was
reached 4.9x108 CFU/ml; meanwhile, an experiment using sterile beer
waste (TN3), the TCC only reached 107 CFU/ml. Thus, pretreatment of
beer waste by heat acid and alkaline methods has increased the
concentration of nutrients in the medium which helps MSS8.4 grow better.
This results were also suitable to that of Brar and colleagues, when
hydrolyzing organic compounds at high temperatures, it will increase the
nutrition contents in the medium. Therefore, the cell density and spores of
MSS8.4 bacteria on the heat-acid-method-hydrolyzed beer wastemedium
were higher than in other media (Brar et al, 2005).
The ability of producing toxic crystals ofMSS8.4also differed significantly
in different experiments. In the experiment using sterile beer waste, after
48 hours of fermentation of MSS8.4, delta-endotoxin concentrations
reached 319 mg/l. Meanwhile, in the experiment of using pretreatment beer
waste, delta-endotoxin concentration reached 428 mg/l, which 1.3 times
higher than the experiment using sterile beer waste. Therefore, the heat
acid method was chosen as the pre-treatment method of beer waste as the
culture medium of MSS8.4.
3.3.1.2. Analysis of the composition of the beer waste by using heat acid
method
In order to evaluate the content of substances in the hydrolysis of beer
waste, the beer manure hydrolysis by heat acid method was sent to the
Department of Environmental Quality Analysis, Institute of Environmental
Technology for analysis.
The analytical results show that in the hydrolysis of beer waste by heat
acid method, the contents of C and N (the two most important substrates in
the growth of bacteria) were much higher than that of beer waste
hydrolyzed by the heat alkaline or sterile method. This result was
completely consistent with the above observation, given that the acid agent


in high temperature condition facilitated to increase the ability of
decomposing high molecular compounds, increasing the amount of organic
compounds readily absorbed in hydrolyzate.
Mineral elements are indispensable factors during the growth and
development of bacteria in general and Bt strains in particular. The metal
ions such as Mg, Mn, Fe, Zn, Ca, etc. have important regulating effects on
growth, formation of spores as well as biosynthesis of insecticidal crystal
proteins. Therefore, the synthesis medium of bacteria fermentation is often
supplemented some minerals with the concentration as follows: 0.3%
MgSO4.7H2O; 0.02%o MnSO4.7H2O; 0.02%o FeSO4.7H2O; 0.2%o
ZnSO4.7H2O and 1.0%o CaCO3 (Ngo Dinh Binh, 2000). Considering this
mineral needs of bacteria, no element in all three experiments meets the
mineral needs. Therefore, additional minerals must be added to the beer
waste hydrolysis to improve fermentation medium quality for bacteria
MSS8.4.
However, according to the study of Ozkan and colleagues: at a
concentration of 10-6M, Mn is a key factor which affects the biosynthesis
of Bt bacteria without impacting to other cell processes. In the beer manure
hydrolyzed by heat acid method, Mn concentration of 0.136 mg/l (2.7x10 6
M/l) is the concentration in a suitable rangefor fermenting Bt bacteria to
obtain delta endotoxin toxin (Ozkan et al, 2003).
According to a research by Içgen and colleagues, the growth, formation of
spores and biosynthesis of crystal proteins of Bt bacteria depend not only
on carbon and nitrogen nutritional factors but also greatly influenced by
the mineral factors. Specifically, when supplementing Mg with the
concentration from 8x10-5M to 4x10-3M, cell density and crystal protein
concentration increased strongly (Içgen et al., 2002). According to the
analysis results, the concentration of Mg in beer waste hydrolyzed by heat
acid method was 12 mg/l (2.9x10-3M).It is the concentration of Mg in the
range suitable for growth and producing toxins of Bt bacteria follow the
study of Içgen.
3.3.1.3. Determine the concentration of beer waste for fermenting bacteria
MSS8.4
The fermentation experiments of MSS8.4 using beer waste were carried
out at the following concentrations: 1% TS; 1.5% TS; 2% TS; 2.5% TS;
3% TS; 3.5% TS.


The research results show that the percentage of dry matter of beer waste in
the range of 2-3 was suitable for the development of Bt. At low
concentrations (1% TS) the bacterial density in the sample after 48 hours
of fermentation was only 2.9x107 CFU/ml, while at the concentration of
2.0 - 3.0% TS, cell density after 48 hours reached over 108 CFU/ml. In the
experiment 6, when increasing the concentration of beer waste to 3.5%, the
density of cells obtained after 48 hours of fermentation was not high, only
107 CFU/ml.
Crystal protein is produced simultaneously with spore formation and is a
desirable product in the fermentation of MSS8.4 bacteria. Therefore, this is
a critical indicator of product quality. Results in Table 3.6 showed that at
the concentration of 2.5% beer waste, the crystal protein concentration
achieved the highest. Thus, in terms of overall, the concentration of 2.5%
TS was the most suitable for fermenting MSS8.4 to collect toxins
againstneonatal fly larvae. The concentration of 2.5% TS will be used for
all subsequent studies.
3.3.2. Assessment of the independent effects of nutritional factors added to
the hydrolysis of beer waste to the growth and toxic crystal production of
bacteria MSS8.4.
3.3.2.1. Determine the nutrient source added to the beer waste medium
The experiment was carried out in a flask, fermentation time of 48 hours,
shaking speed of 200 rpm. Determination of cell density, spore and crystal
protein concentration after 48 hours of fermentation.
The results of assessing the effect of additional organic compounds on the
hydrolysis of beer waste showed that rice bran and soybean powder were
two factors that have positive effects on growth as well as the formation of
toxic crystals of the strain MSS8.4. According to research of Nguyen Thi
Hoai Tram and colleagues, soybean powderwas the best source of nutrition
for the growth of Bt to produce toxins (Nguyen Hoai Tram, 2004). This
result is also consistent with the results reported by Nguyen Thi Hoa and
colleagues, when adding rice bran and soybean powder to hydrolysis of bio
mud waste as a medium for fermenting Bt bacteria to obtain toxin (Hoa et
al., 2014). In two nutrient sources of rice bran and soybean powder,
soybean powder has better solubility, so soybean powderwas chosen as an
additional nutrient source for hydrolysis of beer waste.


When considering the crystal protein content in fermentation of MSS8.4
after 48 hours of culture, there was a clear difference when adding
MgSO4.7H2O and MnSO4 to the hydrolysis of beer waste. In two
additional experiments with 0.5 g/l MgSO4.7H2O and 0.05 g/l MnSO4, the
amount of crystal protein obtained after 48 hours of fermentation increased
about 10% compared to the control of hydrolysis of beer waste, deta
endotoxin content reached 469 mg/liter and 461 mg/liter, respectively. This
research result is completely consistent with the results of Braun's study in
2000, Icgen and colleagues when studying the role of Mg2+ in the growth
and biosynthesis of crystal proteins. These authors have shown that the
deficiency of Mg2+ in the fermentation medium will reduce the ability of
growth and spore formation in fermenting fluid, but the most significant
effect is reduced ability to synthesize. Crystal toxin. Mg2+ is the core that
controls enzyme activity, the presence of Mg will affect spore formation
and crystal protein biosynthesis (Braun S, 2000; Icgen and colleagues,
2002).
The results displayed on Table 3.9 showed that Mn2+ is one of two factors
that strongly influence to the ability of crystal protein synthesis. This result
is also consistent with the judgment of Ozkan and colleagues when it is
suggested that Mn is a key factor affecting the biosynthesis of crystal
protein in Bt without affecting other cellular processes (Ozkan et al, 2003).
According to research by Subbiah and colleagues, when studying the
production of mosquito-killing products from B. thuringiensis subsp.
Israelensis, the addition of Mn2+ to the feathered nutrient medium
significantly increased the crystalprotein content of fermented Bt broth
(Subbiah et al., 2012).
Based on the above research results, soybean powder, MgSO4.7H2O and
MnSO4 are selected elements as nutritional factors to improve the
fermentation medium of MSS8.4 on the basis of hydrolysis of beer waste.
3.3.2.2. Evaluation of independent effects of selected factors on growth
and biosynthesis of endotoxin delta of MSS8.4
After assessing the independent effect of selected factors on the growth,
spore formation and crystal protein biosynthesis, the manganese
concentration in a range of 0 - 0.1 g/l, magnesium from 0 - 1,0 g/l; soybean
powder from 0-5 g/l has a strong effect on the growth, biosynthesis of
crystal protein of MSS8.4 when added to hydrolysis of beer waste.


3.3.3. Optimizing the fermentation medium for MSS8.4 strains from beer
waste
3.3.3.1. Building the optimal model via response surface methodology.
The composition of supplementary substances is determined by response
surface methodology with the survey areas of the influencing factors as
follows: concentration of Mn (A) 0.0 - 0.1 g / l; concentration of Mg (B) 0
- 0.8 g / l; concentration of soybean meal (C) 0 - 5 g / l. By Design expert
software ver 10.1 obtained a matrix of 20 experiments for 3 influencing
factors. The experiments were repeated 3 times to get the average results of
the repetitions put into statistical processing by expert design software
software 10.1.
The results of regression analysis of the model show that: p value of
<0.0001 model shows that the model is completely statistically significant
with 99.99% reliability. The significance of the regression coefficients
tested by standard F, values P greater than F is less than 0.05, indicating a
meaningful model. In this case, the factors A, B, and C all have p <0.05, so
all of these three factors represent the factors that affect the crystal protein
content in the fermentation. In Table 3.11,the result also shows that the
combination of AB, BC does not show the effect of expression with P>
0.1but these factors are still kept in the model to perform optimization.
The F standard of model incompatibility is 2.38, indicating that the
incompatibility of the model is not significant, only 18.12% of the chance
of error is caused by noise. The insignificance of the model's incompatible
value suggests that the model is fully compatible with experimentation
(Box and Wilson, 1951; Li et al., 2007).
Equation for calculating the expected crystalline protein concentration of
the model:
Crystal protein concentration = 308,51 + 44,25 * A + 1256,53 * B +
173.96 * C + 21,125 * A * B - 9,053 * A * C + 27,857 * B * C - 7,227 *
A2 - 10343.76 * B2 - 146.87 * C2
The study of the effect of each factor (when other factors at the central
level) on crystalline protein biosynthesis of Bt bacteria showed that all
three factors strongly affected delta toxin biosynthesis. edotoxin of bacteria
MSS8.4. In it, the top is soybean meal then to magnesium and manganese.
3.3.3.2.Examine model compatibility and experiment


To evaluate the results of the method, we carried out the verification in any
of the five formulas. The randomly selected formulaswere 2, 16, 38, 47,
90, where options 16 and 47 are the formulas out-of-optimized area,
options 2, 38 and 90 are formulas located in the optimal area.
Experimental results are presented in Table 3.12.
Table 3.12 Cry protein concentration model-based prediction and
experiment examination
Model
Cry
protein
Concentration (g/l)
Density (CFU/ml)
number
concentration(mg/l)
MnSO4 MgSO4

Soy
TCC
powder

SC

Experiment

Model
prediction

2

0,05

0,45

3,00

4,5x108

4,3x108

529,11±11

525

16

0,076

0,221

3,47

2,8x108

1,3x108

509,24±10

513

38

0,051

0,337

2,292

3,3x108

3,1x108

515,2±10

519

47

0,009

0,237

1,487

1,25x107 1,07x107 457±11

471

90

0,056

0,581

3,093

4,15x108 3,8x108

526

Control (beer waste hydrolysis 1,7x108
medium)
MTCS medium

1,5x108

5,10x108 4,5x108

527,93±10
431±10
530,14±12

-

Results of protein content analysis in experiments showed that there was
no notable difference between the predicted results of the model and the
experiment.
Research results in Table 3.12 also show that: After the environment is
optimized by response surface methodology, fermentation efficiency
increases significantly, especially crystalline protein concentration. Total
cell density, spore and crystalline protein concentration in fermented
cultures after optimizing medium were 4.5 × 108CFU / ml, 4.3x108CFU /
ml and 529.11 mg / l respectively compare to the initial condition of
1.7x108 CFU / ml, 1.5x108 CFU / ml and 431 mg / l. Thus, after optimizing
the fermentation condition by the response surface methodology with the
interaction of three additional factors, MnSO4, MgSO4.7H2O and soybean
meal increased 22.7% recovery efficiency. Crystalline protein in
fermentation fluid.


Today, the optimal method of fermentation environment for
microorganisms has been widely applied due to the simplicity and
efficiency of the method. In 2013, Lee and colleagues applied the response
surface methodology to optimize the medium composition of Bacillussp
fermentation bacteria. LX-1 and found an optimal point to increase αgalactosidase 1 recovery performance by 6.3 times compared to
fermentation in baseline conditions (Lee et al., 2013). Gao and colleagues,
using the same method, also increased the recovery efficiency of laccase to
59.68 times when fermenting the Trichoderma harzianum ZF-2 (Gao et al.,
2013). Linna et al. Used that of method to optimize fermented
environmental components for Paecilomyces tenuipes N45 which
increased biomass recovery efficiency, adenosine, polysaccharide and
cordycep acid, respectively 8.20 ; 3.58; 23.17 and 31.51% (Linna et al.,
2012).
Thus, the fermentation medium for Bt bacteria from beer residues has a
specific composition as follows: beer residue hydrolysis (2.5% Total) +
0.45 g / l MgSO4.7H2O + 0.05 g / l MnSO4 + 3 g / l soybean meal.
3.4 Studying to produce Biological products
3.4.1. Effect of fermentation condition on growth, spore formation and cry
protein production of MSS8.4
3.4.1.1. Effect of initial pH on growth, and delta endotoxin production of
MSS8.4
To determine the suitable pH condition for the growth of Bt, the
experiment conditions were conducted as follow, beer waste at pH 2 were
adjusted to pH 5; 6; 6,5; 7; 7,5; 8, 9. Experiments were conducted in 500
ml conical flask with 100 ml medium, fermentation temperature were set at
30oC; shaking 200 rpm and fermentation duration were 48 hours. The
results were showed in table 3.13
The results indicated that MSS8.4 grew well in neutral condition.
Regarding to the ability of cry protein formation: the cry protein were
formed together with spore formation of MSS8.4; therefore, experiments
with high spore density are also having high delta endotoxin concentration.
The initinal pH of 6,5 – 7,5 provided the highest concentration of delta
endotoxin concentration with 520 mg/L after 48 hours fermentation


During the fermentation process, microorganism produced a number
of other products which alter the pH of medium. Determining the final pH
after 48 hours fermentation indicated that the pH slightly increased
Thus, the initial pH of 6.5 - 7.5 is suitable for growth as well as the
biosynthesis of crystalline protein synthesis of MSS8.4.
3.4.1.2. Effect of temperature on growth, and delta endotoxin production of
MSS8.4
The experiment was performed at temperatures: 20oC, 25oC, 30oC,
35oC, 40oC, 45oC.
The results showed that temperature has a strong influence on the
growth of MSS8.4. The strain MSS8.4 grows most strongly in the
temperature range from 25 - 35oC, at this temperature, the density of cells
and spores reaches 4.39x108 CFU / ml, spore density reaches 4.14x108
CFU / ml.
Similar to the growth capacity, the crystalline protein concentration
of MSS8.4 is also strongly influenced by fermentation temperature, the
highest cry protein concentration was achieved at 30oC (541 mg/L). At low
temperatures (20 ° C), after 48 hours of fermentation, endotoxin delta
concentrations were only 445 mg / l equal to 75% compared to
fermentation at 30oC. At temperatures above 40oC MSS8.4 bacteria grow
slowly, the density of spore were low and the cry protein concentration
were 214 mg/L after 48 hours fermentation at 45oC
The result were consistent with the results of Ngo Dinh Binh et al. about
the effect of temperature on the growth of Bt. According to other studies,
the optimized temperature for Bt growth were 28 ± 1 oC (Ngo Dinh Binh,
2011; Tien et al., 2013; Nguyen Thi Van Trang et al, 2012). At
temperatures below 20oC B. thuringiensis grows slowly. On the other hand,
the temperature above 40oC started inhibiting growth and spore formation
of MSS8.4. At 45oC, MSS8.4 still grows but after 48 hours, spore and cell
density is only 107CFU / ml.
3.4.2. Applied Bt production against house flies’ larvae
3.4.2.1. Pesticide activities of Bt production against Musca domestica at
laboratory scale.
In order to evaluate the biological activities of MM8.4 culture on medium
made from malt beer waste, we conducted experiments with house flies
larvae age 2 days


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