Study on nitrifying bacteria in the sediment ò intensive shrimp pond

1

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY
♣♣♣


PHAM THI TUYET NGAN



STUDY ON NITRIFYING BACTERIA
IN THE SEDIMENT OF INTENSIVE
SHRIMP POND (
Penaeus monodon
)



Specialization: Marine/Brackishwater Aquaculture
Field of Study Code: 62 62 70 05

Dissertation of Doctor Degree in Aquaculture

Cần Thơ, 2012

2

- The study was carried-out at: Department of Applied
Hydrobiology, College of Aquaculture and Fisheries,

Can Tho University.

Supervisors:
Ass/Prof. Dr. Nguyen Huu Hiep
Ass/Prof. Dr. Trương Quoc Phu


Examiner 1: Dr. Luu Hong Man
Examiner 2: Dr. Le Hong Phuoc

The dissertation will be defended at the university committee
in meeting room of College of Aquaculture and Fisheries, Can
Tho University at: ….… hr ….… date ……. month ……. year
2012

The disseratation is available in Libraries:
• Learning Resource Center of Can Tho University.
• College of Aquaculture, Can Tho University





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GENERAL DESCRIPTION OF THE THESIS
1. Background and problem statement
Black tiger shrimp (Penaeus monodon) is the fastest
growing reared in aquaculture operations around the world.
The rapid development of shrimp farming has brought jobs to

people and generate income of many countries. However, the
implications of the growth of shrimp farming industry has led
to environmental pollution and disease, so the shrimp farming
industry has encountered major obstacles. Shrimp yields
decline in many countries, greatly affect the economic life of
many people. To solve this problem chemicals and antibiotics
were used in shrimp farming activities (Gomez-Gil et al.,
2000; Graslund and Bengtsson, 2001). The use of antibiotics
has led to improper drug resistance (Weston, 1996). On the
other hand exports of fishery products do not meet standards
due to antibiotic residues, pesticides and pathogenic
microorganisms (Dang Dinh Kim et al., 2006).
Therefore solutions in disease prevention and
treatments have been raised including disease management,
integrated pest management (Li, 2008), especially the use of
beneficial bacteria (probiotics) to improve rearing
environment and increase production. This positive solution
has great potential for micro-management in intensive ponds
to minimize antibiotic for food safety, significantly limits the
amount of organic waste environment and contribute to
aquaculture development in a sustainable way. The study of
selected beneficial bacterial strains from local area as a basis
for the mass production of probiotic is very necessary and
practical significance in the current period to improve
indigenous of aquaculture, limit the environmental pollution

4

and enhance the sustainability of farming. From these reasons
the subject "Study on nitrifying bacteria in the sediment of

intensive shrimp pond (
Penaeus monodon
) has been carried
out.
2. The objective of the study
To establish the beneficial bacteria strains, which isolated
from intensive shrimp ponds to supply the collection of
bacteria for selecting bacterial strains to produce probiotic
products used in aquacultue.
3. The contents of the study
- Isolation and identification of decomposing organic
matter bacteria, nitrifying bacteria from intensive shrimp
ponds through a cycle.
- Determine the variation of water quality and bacterial
through a cycle.
- Assessment the improving water quality of the isolated
bacteria and their role in the laboratory scale.
4. The useful outcomes
The thesis has added a collection of benificial bacteria,
which has better efficiency on water treatment in shrimp
farming and the scientific conclusions about the impact of
these strains to the water quality parameters and growth of
shrimp in the source database of general scientific application
of beneficial bacteria, to serve as the basis for shrimp farming
sustainable development.
5. New findings of the thesis
Several beneficial bacteria in shrimp ponds were isolated
and selected for the improvement of water quality in shrimp

5

ponds which could improve water quality. Bacillus cereus
was the dominant species in intensive shrimp ponds. This
dominant species was not presence in probiotics products (B.
subtilis and B. licheniformis). This finding proved that this
bacteria species originated naturally in ponds, rather than from
probiotics products.
Finally, four species of bacteria B. subtilis (B41), B.
cereus (B8, B9, B37, B38,) and B. amyloliquefaciens (B67)
could be used to produce probiotics.
6. Layout of the thesis
Chapter 1: Introduction 6 pages
Chapter II: Literature review 40 pages
Chapter III: Methodology 30 pages
Chapter IV: Results and discussion 79 pages
Chapter V: Conclusion and recommendation 2 pages
List of author's publication 1 page
References 21 pages
(includes 298 documents, of which 34 Vietnamese documents
and 264 documents in foreign languages)
Appendix 33 pages

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Chapter 1: Literature review
This chapter focused on the understanding and analysis of
important issues such as:
1. Overview of shrimp, environmental status and disease
in black tiger shrimp.
2. Factors affecting the survival rate of shrimp.
3. Current state of environmental pollution and disease in
intensive shrimp farming.
4. The toxins generated from decomposition of waste in
intensive shrimp ponds (NH
3
, H
2
S and NO
2
)
5. Environmental pollution treatment by biological
methods
6. The role of biodegradation organic matter bacteria
7. Biological characteristics of Bacillus.
8. The metabolism of inorganic nitrogen and the role of
the nitrifying bacteria (Nitrosomonas and
Nitrobacter).
9. PCR technique and its application in aquaculture
10. Techniques in DNA nucleotide sequencing
11. Application of PCR techniques to identify bacteria in

aquaculture
In the results of the previous studies, the population of
bacterial which decomposing organic matter and nitrifying
bacteria in intensive shrimp ponds are not studied completely,
particularly the identification of beneficial bacteria
predominate in intensive shrimp ponds. All the research as the
basis to work towards managing water quality by biological
methods, to serve as the basis for shrimp farming sustainable
development.




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Chapter 2:
METHODOLOGY
2.1 Location and time of the study
Sampling period from 3/2008 to 8/2008 at Vinh Chau
District, Soc Trang province. Sample analysis from 3/2008 to
10/2008 at the Department of Applied Hydrobiology, College
of Fisheries, Can Tho University. Time of identification of
bacteria from the 8/2009 - 8/2011 at the Biotechnology
Research and Development Institute, Can Tho University
and Nam Khoa company, Ho Chi Minh City.
2.2 Study subjects:
Black tiger shrimp (Penaeus monodon) larvae and
commercial shrimp, Bacillus sp., Nitrosomonas sp. and
Nitrobacter sp.
2.3 Sampling cycle

Sampling was made before and after the seed stocking and
then every 2 weeks post stocking until the end of the crop (5.5
months).
2.4 Characteristics of shrimp pond, care regime and
probiotic supplementation
2.5 Preparation prior to sampling
2.6 Equipment, tools and chemicals
2.6.1 Equipment and apparatus
2.6.2 Medium for analyse bacteria and chemical
2. Method of isolation and identification of bacteria in the
pond

8

2.7.1 Sediment sampling method (Somsiri et al., 2006)
Sediment samples were collected by strilized PVC pipe
system (with 70% alcohol solution). After sampling, samples
were kept cold in ice and transported to the laboratory within
3-5 hours, then stored at 4°C and processed within 2 hours.
2.7.2 Method of isolation and identification of bacteria
2.7.2.1 Methodology to isolate and identify Bacillus sp.
a) Methodology to isolates of Bacillus:
Selective media for Bacillus isolates was based on
Harwood and Archibald (1990). Methodology to isolated was
based on Nguyen Lan Dung (1983). Subculture method and
store bacteria were done by the method of Dang Thi Hoang
Oanh et al. (2004).
b) The method identified Bacillus sp.
Identification of bacteria by physiological and biochemical
test

Physiological and biochemical tests to identify isolated
bacterial strains were based on Andretta et al , (2004).
Identification of bacteria by molecular methods
(1) The bacterial strains B8, B37, B41 and B67 developed
on agar were sent to Nam Khoa Company - to perform PCR
and identified with primers: 16F (5'-
TCCAGAGTTTCATCCTGGCTGAC-3 ') and 16R (5'-
TACCGCGCCTGCTCGCTG-3').
(2) DNA extracted and PCR reaction of strains B9 and B38
were carried out the by using 16S rRNA primers 16F8 (5'-
AGAGTTTGATCCTGGCTCAG-3 ') and 16R1391
(GACGGGCGGTGWGTRCA-5'-3 ') (Eden et al. in 1991,
Lane, 1991, cited by Kaspari, 2010). The analysis were done
in Biotechnology Research and Development Institute,
University of Can Tho as following steps:
- ADN extraction (based on Boon et al., (2002))

9

- DNA quality control after extraction (Tran Nhan Dung,
2011)
- Amplification of the target gene by PCR
- Checked the PCR product
- Sequencing the amplified genome
- Data Analysis
2.7.2.2 Method of isolation and identification of bacteria
Nitrosomonas sp.
a) Method to isolate Nitrosomonas sp
Selective ammonium-calcium-carbonate media was used to
isolate Nitrosomonas sp. based on Ehrlich, 1975. Mass culture

Nitrosomonas sp. was done based on Lewis and Pramer (1958)
and MacDonad and Spokes (1980).
b) Method of identification of bacteria Nitrosomonas sp.
Methods identified of bacteria by biochemical tests
including 2 tests:
- Test of the ability to transform from NH
3
to NO
2
by bacteria
group AOB: Griess - Ilosvay reagent was used to test the
conversion of NH
3
into NO
2
by Ammonia-Oxidizing-Bacteria
(AOB).
- Check the shape of bacterial cells by Gram staining method.
Identification of baterial cells by molecular biological
technique
- The quality of DNA extracted was test by using the same
steps for Bacillus sp. (Section 2.7.2.1).
- Amplification of the target gene by PCR: PCR was
performed to amplify the target genes of the 16S rRNA amoA
gene with primers AmoA 1F: 5'-GGG GTT TCT ACT GGT
GGT-3' and 2R: 5-CCC CTC KGS AAA GCC TTC-3'
(Rotthauwe et al., 1997).
- DNA sequencing species identification: PCR products of
the bacterial strains were sent to Nam Khoa for identification.

10

2.7.2.3 Method of isolation and identification of bacteria
Nitrobacter sp.
a) Method to isolate Nitrobacter sp.
Selective nitrite-calcium-carbonate media used for isolating
bacteria Nitrobacter sp. based on the method of Ehrlich
(1975). Selective media were used based on Aleem and
Alexander (1960) to grow bacteria Nitrobacter.
b) Method of identification of bacteria Nitrobacter sp.
Identified by biochemical tests including 2 tests
- Test of the ability to transform from NO
2
to NO
3
by
bacteria group NOB: Griess-Ilosvay reagent was used to test
the conversion of NO
2
to NO
3
by Nitrite-oxidizing-bacteria
(NOB).
- Check the shape of bacterial cells by Gram staining
method.
Identified bacteria by molecular technique
- The quality of DNA extracted was test by using the same
steps for Bacillus sp. (Section 2.7.2.1).
- Amplification of the target gene by PCR: perform PCR
with the primer PNGT 1F: 5’- TTT TTT GAG ATT TGC

TAG - 3’. PNTG 2R: 5’- CTA AAA CTC AAA GGA ATT
TGA - 3’ as primer to identify specific groups of Nitrobacter
(Degrange and Bardin, 1995) to amplify the target genes of
16S rRNA genes of bacteria Nitrobacter sp.
- DNA sequencing species identification: The analysis were
done in Biotechnology Research and Development Institute,
University of Can Tho.
2.8 Method of determining of variation of water quality
and bacteria in shrimp ponds
2.8.1 Sampling methods and determination of the water
quality and pond sediment
2.8.1.1 Sampling methods

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Temperature, pH, salinity were measured in the field.
Parameters TAN, TSS, NO
2
, NO
3
-,
TN, H
2
S was collected and
refrigerated storage (4 ºC). DO was be fixed by chemical.
2.8.1.2 Methods of analysis of water quality
All environmental parameters were analyzed by standard
methods (APHA, et al., 1995). The chemicals used in the
analysis originated from Germany (Merck).
2.8.1.3 Methods of determination of bacteria

a) Determination of total bacteria and Vibrio by colony
count method (Baumann et al., 1980).
Selective media Tripticase Soya Agar (TSA) adding 1,5%
NaCl and Thiosulphate Citrate Bile Sucrose Agar (TCBS)
were used to determine total bacteria and Vibrio.
Bacterial density was calculated using the formula:
Colony forming units (CFU/g) = average number of colonies
× dilution × 10.
b) Determination of Bacillus sp. based on Nguyen Lan
Dung, 1983; Harwood and Archibald, 1990. Using the
selective agar media to determine the density of Bacillus.
Bacterial density was calculated as mentioned above.
c) Determination of Nitrosomonas and Nitrobacter
(Ehrlich, 1975).
MPN method based on the principle of statistical
probability distribution of bacteria in the different dilution of
the sample. This is a quantitative method based on quantitative
results of a series of experiments were repeated in a number of
different dilution. Then based on Mac Crady statistics to infer
the value estimated number of microorganisms in the sample.
Density of two groups Nitrosomonas and Nitrobacter were
calculated (MPN/g).
2.9 Experiments evaluating the effectiveness of water
treatment of selected bacteria

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2.9.1 Selected bacteria
Three strains of Bacillus sp., Nitrosomonas sp., Nitrobacter sp.
had potential to decomposing organic matter and converse

TAN, NO
2
-
were selected.
2.9.2 Experimental conditions were arranged the same in all
treatments
2.9.3 Effect of bacterial B37, S8 and N10 on shrimp larvae
in the recirculated system
2.9.3.1 Experimental materials
Selected bacterial species added to the experimental system
including B37 (B. cereus), S8 (N. nitrosa) and N10 (N.
winogradskyi).
2.9.3.2 Experimental set up
The experiment included three treatments with Bacillus
density respectively 10
6
(NT1), 10
5
(NT2), 10
4
CFU/mL
(NT3) and control treatment without added bacteria (DC). In
the recirculated system bacteria Nitrosomonas and Nitrobacter
(density 10
4
MPN/mL for each species) were added. The tank
was conected to the biofilter tanks at Mysis stage 1, the filter
system was run until the end of the experiment. Each treatment
was repeated 3 times.
Nauplius Larvae stage with density of 150 larvae/L was

applied. Samples were collected 3 days/times with the criteria
include: pH, temperature, TAN, NO
3
-, NO
2
-, COD, DO, TSS,
TN, H
2
S and bacterial density. The experiment ended when
the larva has reached to the PL7.
2.9.3.3 Method of mass bacterial culture
a) Bacillus
Luria Bertani culture media was used (LB, Figure 3.7).
bacterial density was determined by measurement of OD at
600 nm wavelength (Leonel et al., 2006).

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b) Nitrosomonas và Nitrobacter
Two strains S8 and N10 were cultured in the same liquid
medium for Nitrosomonas (Lewis and Pramer, 1958) and
MacDonad and Spokes (1980) and for the same Nitrobacter by
Aleem and Alexander (1960).
2.9.2.4 Water quality parameters
The environmental parameters such as pH, DO, COD, TSS,
TAN, NO
2
-
, NO
3

-
and TN, and bacterial criteria such as density
of total bacteria, Vibrio, Bacillus, Nitrosomonas, Nitrobacter
were monitored (3 days/times) until the end of the experiment.
Survival rates were determined at the stage Zoae3, Mysic2,
Poslarvae 1 and 7.
2.9.3.5 Sampling methods and determination of water
quality (APHA et al. (1995)
2.9.3.6 Sampling methods and determine bacterial density
(refered to 2.8.1.3)
2.9.3.7 Shrimp analysis methodology
Survival rate (%) = Poslarvae * 100/ Nauplius
2.9.4 Effect of Bacillus B9, B41, B67 on water quality in
shrimp tank.






Fig 3.7 Mass bacteria culture and experimental system

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2.9.4.1 Experimental materials
Three species of bacteria B9 (B. cereus), B41 (B.
amyloliquefaciens) and B67 (B. subtilis) were introduced into
shrimp tanks. Juveniles original from experimental hatchery in
Can Tho. Sediment was taken from shrimp ponds in Vinh
Chau District, Soc Trang province.

2.9.4.2 Experimental set up
The experiment was conducted with four treatments:
Treatments 1 (DC): No bacterial cultures. Treatments 2 (B41:
B. amyloliquefaciens). Treatments 3 (B67: B. subtilis.
Treatments 4 (B9: B. cereus). Density of adding bacteria was
10
5
-10
6
CFU/mL/treatments. Each tank was provided a layer
of sediment from 4-5 cm at the bottom of the tank. Density of
shrimp were 30 ind./m
2
. Each treatment was repeated 3 times.
During the experiment, samples of bacteria were collected
before adding bacteria and subsequent periodic sampling once
every 2 weeks until the end of the experiment.
2.9.4.3 Method of mass bacterial culture (refered to 2.9.3.3)
2.9.4.4 Methods of determination bacteria density (refered
to 2.9.3.6)
2.9.4.5 Sampling methods and determine water quality
(APHA et al. (1995)).
2.9.4.6 Feeding and management of shrimp culture
experiments
Shrimps were fed with commercial feeds Feed Grow 5
times/day (06, 10, 14, 18, 22 hrs). The weight of shrimp was
determined at the start and the end of the experiment.
2.9.4.7 Methods of analysis of shrimp samples
Method of determining the growth and survival rates of
shrimp was based on the author Robertson et al. (2000), Felix

and Sudharsan (2004); Venkat et al. (2004).

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2.9.5 Effect of bacterial B8, B37 and B38 on water quality
in shrimp tank.
2.9.5.1 Experimental materials
The experiment was arranged in 12 composite tanks 500
L. Three bacterial species B. cereus (B37), B. cereus. (B38),
and B. cereus (B8) were selected. Mean weight of shrimp
about 1 g. The seawater originating from Vinh Chau (Soc
Trang), was mixed with freshwater to achieve 16 ‰ of
salinity.
2.9.5.2 Experimental design
The experiment included four treatments: (1) additional B.
cereus (B37), (2) additional B. cereus (B38) and (3) additional
B. cereus (B8) and (4) control (DC) without additional
bacteria. Bacterial density was added from 10
5
-10
6
CFU/mL
Stocking density of shrimp was 30-35/m
2
. Bacteria added 5
days/times. Shrimp were fed 5 times/day. Samples were
collected before adding bacteria and once every 5 days until
the end of the experiment.
2.9.5.3 Method of mass bacterial culture (2.9.3.3)
2.9.5.4. Parameter following (2.9.4.4 and 2.9.4.5)

2.9.6 Data processing method
Data were processed with Excel and Statistica 6.0 software.
All data was checked for homogeneity and normal distribution
before being put into treatment One-way ANOVA.
Differences between treatments were tested by Duncan test at
significance level 0.05.

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Chapter 3:
RESULTS AND DISCUSSION
3.1 Isolation and identification of bacteria converting
nitrogen from pond sediments
3.1.1 Isolation and identification of Bacillus
3.1.1.1 Isolation of Bacillus
During the investigation, 67 strains of bacteria (B1-B67)
were collected. All these strains were stored in agar at 4°C and
in glycerol at a temperature of -80°C at the College of
Aquaculture and Fisheries.
3.1.1.2 Identification of Bacillus
a) Identified by physiological and biochemical test
- Colony shape: round-shaped, slightly edge, colonies flat,
smooth or slightly wrinkled surface, opaque white to
yellowish. Cells were Gram-positive, short rod-shaped, or long
rod depending on the species.
- spores staining results: cells of Bacillus sporulation after
24 h growth on agar surface. Observed patterns of Bacillus
spores were stained under microscopic, spores had the green
color of Malachite Green dye and the bacterial cell had the red
color of Safranin.

After checking and re-evaluate 67 collected strains based
on the observation form, characteristics, color, size and timing
of colony growth on agar as well as shape and size bacterial
cells on Gram-stained specimens spores and removed the
duplicate strains in the pond and the sampling period. At the
same time the ability to form internal spores and catalase-
positive reaction characteristic of the species of the genus

17

Bacillus were also recorded. Results identified nine different
strains (B2, B6, B7, B8, B9, B37, B38, B41, B67). Nine
bacterial strains were purified with the aim of selecting a strain
capable to decompose organic mater, but there were only 6/9
selected bacterial strains (B8, B9, B37, B38, B41 and B67)
met the requirements. These strains were stored on agar at 4ºC
and in glycerol at -80°C to use for further research.

Fig 4.1: The shape of Bacillus
a. Bacillus colonies
b. Bacillus cells (100 ×
objective)
c. Bacillus spores (100
× objective lens)
b) Identified bacteria by molecular techniques
M 1 2 1 2 M 3 4

(a) (b)
Fig 4.3 Results of PCR product electrophoresis
a) B9 (well 2), negative control (well 1), M: 200 bp DNA standard ladder

(Fermentas), (b) B37, B67, B41, B8 (wells 1-4), M: DNA ladder standard 100bp
(Fermentas))
- The identification of Bacillus by PCR technique
200bp
100bp
500bp
1000 bp

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Two strains B9 and B38 had the PCR product at position
1500 bp based on the standard scale (Figure 4.3a). For the
remaining four strains B8, B37, B41 and B67, PCR product
sizes in the range of 500-600 bp based on the standard scale
(Figure 4.3b)
- Sequenced results of 6 Bacillus strains
Table 4.3: Comparing the sequence of the isolated Bacillus strains
Code

Description of species
Max
score

Total

score

Max
ident
Accession

number
B9 B. cereus BRL02-43 881

904

97%

DQ339674
B38 B. cereus CMST-AP-MSU 894

1024

99%

JN038553
B37 B. cereus F837/76 996

998

100%

CP003187.1
B67 B. subtilis BL10 887

887

99%

GU826165
B41 B.amyloliquefaciens SB 3297 990

990

100%

GU191913.1
B8 B. cereus G9842 894

894

99%

NC_011772.1
3.1.2 Isolation and identification of Nitrosomonas
3.1.2.1 Isolation of Nitrosomonas
Eight isolated strains of Nitrosomonas were listed from S5
- S12. The strains were stored in liquid culture (Lewis and
Pramer, 1958) and shake on shaker. Besides, samples were
stored at deep freezer -80°C for further research.
3.1.2.2 Identification of Nitrosomonas
Identified by biochemical tests
The results from Gram staining showed that these strains
were Gram-negative and short rods. On the other hand the
results showed that ammonium removal capacity of all these
strains were capable of strong reducing ammonium in 10-day
breeding period. As described in the shape of some bacterial
several authors (Meicklejohn, 1950; Engel and Alexander,

19

1958; Watson and Mandel, 1997) combined with the findings
of Lewis and Pramer (1958), Heselsoe et al. (2001) and Hoang
Phuong Ha et al (2008) confirmed that the isolated strains
belonged to groups of Nitrosomonas sp.

Fig 4.5: Biochemical test of Nitrosomonas sp.
a. Isolated Nitrosomonas were
positive for Griess reagent-Ilosvay
b. Nitrosomonas cells after Gram
staining (objective lens 100 X)
Identified by molecular techniques
Three strains S6, S8 and S12 had the strongest ability to
remove ammonium were used for identification by molecular
technique.
- PCR reaction results
PCR products of all three strains S6, S8 and S12 were
located about 400 bp based on the standard scale (Figure 4.6).
This proved that the three strains were Nitrosomonas sp.
1 2 3 4 5 M

Fig 4.6: PCR results application process PCR
identification of bacteria Nitrosomonas
400 bp

200 bp


20

Wells M: 200 bp DNA ladder (Fermentas); Wells 1: For negative control;

Well 2: S6; Wells 3: S8; Wells 4, 5: S12
- Sequenced results of Nitrosomonas
Table 4.9: Comparing the sequence of isolated Nitrosomonas strains
Code

Description of species
Max

score

Total

score

Max

ident

Accession
number
S6 Nitrosomonadaceae 3CC11 432 814 99%

FJ561357
S8 Nitrosomonas nitrosa 409 409 99%

AF272404.1

S12 Nitrosomonas nitrosa 499 499 99%

AF272404.1

3.1.3 2 Isolation and identification of Nitrobacter
3.1.3.1 Isolation of Nitrobacter
Eight strains of Nitrobacter capable of oxidizing nitrite
were isolated in nitrite-calcium-carbonate media. Code of
samples was listed from N5 - N12. The strains were stored in
the selective media on shaker (Aleem and Alexander, 1960).
Besides samples were stored at deep freezer -80°C for further
research.
3.1.3.2 Identification of Nitrobacter
Identified by biochemical tests
This Gram-negative group, short rod-shaped stick on the
substrate particles media. On the other hand, most bacterial
species isolated was completely reduced nitrite in 10-day
period. Based on research findings about the shape of bacteria
NOB group of Stanley and Mandel (1971) combined with
studies of Aleem and Alexander (1960), Bock and Koops
(1992), Grundman and Gappe (2000), Proser and Embley
(2002) Nitrobacter isolated from soil, suggesting that the
isolated bacteria belonged to the genus Nitrobacter sp.

21

Fig. 4.7: Biochemical test of Nitrobacter sp
a. Nitrobacter G-cells, short rods sticking

on the substrate particle environment
b. Isolated Nitrobacter negative for Griess
reagent-Ilosvay
Identified by molecular techniques

- The results of PCR reaction
1 2 3 M

Fig 4.8: Results of PCR Nitrobacter
Wells M: 200 bp DNA ladder (Fermentas); Wells 1: negative control; Well 2: strains
N10 and Wells 3: strains N12
PCR products of 2 strains N10 and N12 were at about 400
bp size based on the standard scale (Figure 4.8). This proved
that 2 isolated strains were Nitrobacter.
- The sequencing results of two strains of Nitrobacter sp.
Table 4.13: Comparing the sequence of isolated Nitrobacter N10 và
N12 on the gene bank using specific primers PNTG 2R and 1F
PNGT
Code

Description of species
Max
score
Total

score
Max
ident
Accession
number
N10 N. winogradskyi ATCC 25381

432 814 99% L35506.1
N12 N.winogradskyi ATCC 25381 409 409 99% L35506.1
200 bp

400 bp

22

3.2 Fluctuations of environmental factors and microbial
factors in the pond
3.2.1 Fluctuations of environmental factors in the pond
a) Total suspended solid (TSS)
TSS fluctuations tended to increase over time culture
(Figure 4.12). TSS concentrations in the pond began to
increase when the shrimp was 60 days. According to Lawson
(1995) TSS concentrations suitable for aquaculture was less
than 80 mg/L. However, in the shrimp ponds studied over 60
days, TSS in most ponds were at relatively high levels (>100
mg/L).
0
50
100
150
200
250
0 1 15 30 45 60 75 90 105 120 135
Ngày
TSS (mg/L)
A1 A2 A3 A4

Fig 4.12: Fluctuations of total suspended solid (TSS)
b) Total ammonium nitrogen TAN-NH
3
/NH

4
+

TAN tended to increase continuously in all ponds (0.01 to
5.89 mg/L), a slight decrease at the end of crop (Figure 4.14).
On the other hand the use of probiotic to create a stable
Bacillus population (Figure 4.21), contributing to the
decomposition of organic matter containing nitrogen to form
nitrogen into the TAN increase.

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0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 1 15 30 45 60 75 90 105 120 135
Ngày
NO
2
-
(mg/L)
Ao 1 Ao 2 Ao 3 Ao 4
2
2.5
3
3.5

4
4.5
5
5.5
6
6.5
7
0 1 15 30 45 60 75 90 105 120 135
Ngày
TN (mg/L)
Ao 1 Ao 2 Ao 3 Ao 4

Fig 4.14: Fluctuations of TAN during sampling period
c) Nitrite (N-NO
2
-
)
N-NO
2
-
increased towards the end of crop and ranged from
a low of 0.08 to 2.4 mg/L (Figure 4.15). At the beginning of
75 days for raising levels of N-NO
2
-
always at a low level <0.2
mg/L. After 90 days of culture, NO
2
-
gradually increased and

reached a peak at the end of crop culture (0.366 mg/L) in pond
3.








Fig 4.15: Fluctuations of NO
2
-

during sampling period
d) Nitrate N-NO
3
-

N-NO
3
-
in ponds were at low levels over 2.5 mg/L (Figure
4.16) and stable during the culture, only increased after 195
days culture. The variation of NO
3
-
among ponds was slightly
from 0.12 to 1.2 mg/L.

24

0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 1 15 30 45 60 75 90 105 120 135
Ngày
N O
3
-
(m g/L )
Ao 1 Ao 2 Ao 3 Ao 4

Fig 4.16: Fluctuations of NO
3
-
during sampling period
3.2.2 Fluctuations of microorganisms in intensived shrimp
ponds
a) Density of total bacteria and Vibrio in the sediment
Density of total bacteria in the sediment was less
fuctuation, the lowest density in the first sampling (5.3 × 10
4

CFU/g). After preparing ponds and shrimp stocking, the total
density of bacteria in the sludge increased rapidly and reached

>10
6
CFU/g, then stabilized until the end of cycle (Figure
4.20). Meanwhile, the results also showed that the density of
Vibrio in mud tends to increase gradually, reaching a density
of greater than 10
4
CFU/g.
1
2
3
4
5
6
7
0 1 15 30 45 60 75 90 105 120 135
Ngày
Log CFU/g
Vibrio
Tổng VK

Fig 4.20: Fluctuations of total bacteria and Vibrio
b) Density of total Bacillus in sediment

25

Bacillus spore density in sludge ranged from 4,3 × 10
4
to
7,9 × 10

5
CFU/g. In the sampling 1, 2 and 3 of the sampling
schedule Bacillus spores ranged from 4,3 × 10
4
to 6,1 × 10
4
CFU/g, but then gradually increase the number of spores and
stability at the highest in the last sampling of 7,9 × 10
5
CFU/g
(Figure 4.21).
c) Density of Nitrosomonas and Nitrobacter
Nitrosomonas density ranged from 7 to 2.6 × 10
3
MPN/g
over the sampling period. Meanwhile Nitrobacter was much
lower, only from 5.5 to 1.9 × 10
3
MPN/g sludge and without
the presence of them in the first, second and third sampled
(Figure 4.21). From the above results showed that the density
of Nitrosomonas and Nitrobacter in pond was very low
compared to the Bacillus group.
1
2
3
4
5
6
0 1 15 30 45 60 75 90 105 120 135

Ngày
Log CFU/g
Nitrosomonas Nitrobacter Bacillus


Fig 4.21: Density of Bacillus, Nitrosomonas and Nitrobacter
3.3 Assessment of the potential of converting protein
potential of isolated bacteria
3.3.1 Effect of bacterial B37, S8 and N10 in shrimp larvae
in the recirculated system
3.3.1.1 Fluctuations of environmental factors
a) COD