MINISTRY OF EDUCATION AND TRAINING
HO CHI MINH CITY UNIVERSITY OF AGRICULTURE AND FORESTRY
TRUONG PHUOC THIEN HOANG
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RESEARCH ON NITROGEN-METABOLIC BACTERIA IN THE BOTTOM OF LOBSTER CULTIVATION AREA ( Panulirus sp.)
AQUACULTURE SERVICES
Major: Biotechnology Code: 9.42.02.01
DOCTORAL THESIS IN BIOTECHNOLOGY
Ho Chi Minh City - 2022
MINISTRY OF EDUCATION AND TRAINING
HO CHI MINH CITY UNIVERSITY OF AGRICULTURE AND FORESTRY
TRUONG PHUOC THIEN HOANG
RESEARCH ON NITROGEN-METABOLIC BACTERIA IN THE BOTTOM OF LOBSTER CULTIVATION AREA ( Panulirus sp.)
AQUACULTURE SERVICES
Major: Biotechnology Code: 9.42.02.01
DOCTORAL THESIS IN BIOTECHNOLOGY
Scientific supervisor: Associate Professor, Dr. Nguyen Phu Hoa
Assoc.Prof.Dr. Pham Cong Hoat
Ho Chi Minh City - 2022
ACKNOWLEDGEMENTS
During the time of studying, implementing and completing this thesis, I have received enthusiastic guidance and support from the collective of teachers, agencies, brothers and sisters and friends. With deep respect and gratitude, I would like to express my sincere thanks to:
Associate Professor, Dr. Nguyen Phu Hoa and Associate Professor, Dr. Pham Cong Hoat have enthusiastically guided, helped, and imparted valuable experiences and knowledge to help me complete my thesis successfully.
Dr. Hoang Quoc Khanh and Associate Professor Dr. Nguyen Bao Quoc encouraged and enthusiastically supported us professionally.
The Board of Directors of Ho Chi Minh City University of Agriculture and Forestry, Institute of Biotechnology and Environment, Faculty of Biological Sciences, Faculty of Aquaculture, and Department of Postgraduate Training have helped and created the best conditions to help me during the process of doing my thesis.
The group of researchers, graduate students, and students from the Microbiological Technology Laboratory, Institute of Biotechnology and Environment, Ho Chi Minh City University of Agriculture and Forestry have supported and helped me to study, carry out, and complete my thesis well.
All friends and colleagues who always encourage and sincerely help me during the thesis process.
My parents and relatives, husband and children have always supported, encouraged and been my spiritual support throughout the process of doing my thesis.
PhD student
Truong Phuoc Thien Hoang
COMMITMENT
I certify that the publications in this thesis are truthful and a part of the research results of the state-level project code DTĐL.CN-60/15 chaired by Associate Professor, Dr. Nguyen Phu Hoa. The data in the thesis is allowed to be published with the consent of the project manager. All data and results presented in the thesis have not been previously published by other authors.
TRUONG PHUOC THIEN HOANG
SUMMARY
Research on nitrogen-metabolizing bacteria in the bottom of lobster farming areas ( Panulirus sp.) for aquaculture was conducted with the following contents:
(1) The study isolated and identified bacteria from mud samples taken from the bottom of lobster cages in Xuan Dai Bay, Phu Yen province over a period of 12 months; (2) The study created a suitable liquid and semi-solid fermentation environment for bacterial strains capable of creating biological products for environmental treatment; (3) Evaluation of biological products for environmental treatment in the post-5 white-leg shrimp rearing model at a scale of 1m 3 .
The research results isolated bacterial strains capable of metabolizing ammonia and nitrite. The bacterial strains were identified by morphological and biochemical tests using API 20E, 20NE kits, 16S-rRNA region sequencing method and determined the ability to metabolize ammonia and nitrite; in which 3 bacterial strains Bacillus licheniformis B85, Pseudomonas stutzeri KL15, Rhodococcus rhodochrous T 9 had the best ability to metabolize ammonia and nitrite.
The thesis has studied the liquid medium composition suitable for the growth of 3 bacterial strains on the Box - Behnken model as follows: the medium composition for B.licheniformis B85 bacteria at a density of 3.14 x 10 11 CFU/mL includes 3.94 g/L molasses, 15.56 g/L yeast extract and 1.13 g/L NaCl; The density of P.stutzeri KL15 bacteria is 2.37 x 10 11 CFU/mL with the medium composition including 4.95 g/L molasses, 19.08 g/L yeast extract and 1.13 g/L MgSO 4; For R.rhodochrous strain T 9, the medium composition was 7.93 g/L glucose, 6.1 g/L peptone and 2.95 g/L NaCl with a bacterial density of 2.52 x 10 10 CFU/mL.
The three bacterial strains above were cultured on semi-solid medium with appropriate seed ratio, time and humidity, then dried and finely ground to create powdered microbial preparations with bacterial density of 109 CFU /g. Powdered microbial preparations were stored at two temperature ranges: cold temperature 4 - 8 o C and room temperature 28-32 o C. At
At 4 - 8 o C, the bacterial density was better preserved. After 360 days, the density of P. stutzeri KL15 and R. rhodochrous T 9 bacteria decreased from 10 9 CFU/g to 10 6 CFU/g, and B. licheniformis B85 bacteria decreased from 10 9 CFU/g to 10 7 CFU/g. For storage at room temperature of 28-32 o C, after 360 days, the density of P. stutzeri KL15 and R. rhodochrous T 9 bacteria decreased from 10 9 CFU/g to 10 5 CFU/g, and the density of B. licheniformics B85 bacteria decreased from 10 9 CFU/g to 10 6 CFU/g. B. licheniformics B85 is a spore-forming bacteria group, so it has a higher bacterial density than the other two bacterial strains.
The results of evaluating the environmental treatment efficiency of microbial preparations on the white-leg shrimp nursery model at postlarvae stage 5 in a 1m 3 cement tank showed the ability to control well the content of TAN , NO 2 and NO 3 with the preparation ratio of 0.5% with a density of 10 8 CFU/g, used periodically every 6 days/1 time.
SUMMARY
The study on nitrogen-metabolizing bacteria in the bottom of lobster ( Panulirus sp.) culture area for aquaculture was carried out with the following contents: (1) The study was conducted to isolate and identify bacteria from sludge samples which were taken from the bottom of lobster cages in Xuan Dai Bay, Phu Yen province during 12 months (2) Research on creating suitable liquid and semi-solid media for bacterial strains to make biological products to treat the environment; (3) Experimenting with the use of microbiological products for environmental treatment in the 5-day old postlarvae of white leg shrimp rearing in 1m 3 - cement tanks.
The results shown that bacterial strains capable of metabolizing ammonia and nitrite in the bottom sludge, strains were identified by morphology, chemistry and DNA marker characterization by API 20E, 20NE kit and 16S - rRNA region sequencing method and determined the ability to metabolize ammonia, nitrite . Three strains of Bacillus licheniformis B85, Pseudomonas stutzeri KL15, and Rhodococcus rhodochrous T9 were able to metabolize ammonia and nitrite in highest efficiency. The thesis has optimized the composition of liquid medium of 3 bacterial strains on the Box - Behnken model as follows: the composition of the medium of Bacillus licheniformis B85 at the density of 3.14 x 10 11 CFU/mL includes molasses, yeast extract and NaCl. The density of Pseudomonas stutzeri KL15 was 2.37 x 10 11 CFU/mL with the media composition including molasses, yeast extract and MgSO4. For the strain Rhodococcus rhodochrous T9, the media composition was glucose, peptone and NaCl with a bacterial density of 2.52 x 10 10 CFU/mL.
The above three bacterial strains were cultured on semi-solid media with suitable time and humidity, then dried and ground to produce a powdered probiotic product with a bacterial density of 10 9 CFU/gram. Powder microbiology mode is stored at two temperature ranges: cold temperature 4-8 o C and room temperature 28- 32 o C. At a temperature of 4 - 8 o C, the bacterial density was better preserved, after
360 days, the density of P.stutzeri KL15, R.rhodochrous T9 decreased from 10 9 CFU/g to 10 6 CFU/g, B.licheniformis B85 reduced from 10 9 CFU/g to 10 7 CFU/g. For storage at temperature 28-32 o C, after 360 days, the density of P.stutzeri KL15 and R.rhodochrous T9 decreased from 10 9 CFU/g to 10 5 CFU/g, the density of B.licheniformics B85 reduced from 10 9 CFU/g to 10 6 CFU/g. B. licheniformics B85 is a group of spore-forming bacteria, so it has a higher concentration of bacteria than P.stutzeri KL15 and R.rhodochrous T9.
The results of evaluation of the environmental treatment efficiency of probiotic products in water of the white leg shrimp nursing cement tank showed the ability to control the content of TAN, NO2 and NO3 well with a density of 10 8 CFU/g, used periodically every 6 days. days.