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 3 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 6 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. 7 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 [...]... 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 × 104 CFU/g) After preparing ponds and shrimp stocking, the total density of bacteria in the sludge increased rapidly and reached >106 CFU/g, then stabilized... suspended solid (TSS) b) Total ammonium nitrogen TAN-NH3/NH4+ 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 22 Ao 1 Ao 2 Ao 3 Ao... Fluctuations of the density of bacteria on shelter In general, the density of Nitrosomonas and Nitrobacter maintained relatively stable In control treatment, bacterial density on the shelter increased gradually In other treatments bacterial density maintained stable from 3.0 - 5.6 × 103 MPN/cm2 and was significant difference (p 100 mg/L) A1 250 A2 A3 A4 TSS (m g/L) 200 150 100 50 0 0 1 15 30 45 60 75 90 105 120 135 Ngày Fig 4.12: Fluctuations of total suspended... treatment One-way ANOVA Differences between treatments were tested by Duncan test at significance level 0.05 15 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... 4.46: Fluctuations of NO2- d) Nitrate (NO3-) - N 3m O g/L Concentration of NO3- in treatment of B37 increased from day 12 to the end of the experiment (Fig 4.47) Variation of NO3- in B38, B8 treatment was similar to the variation of NO 3in B37 treatment but the development of Nitrobacter in B38, B8 treatment were slower (5 days) In control treatment, the concentration of NO3- increased in day 22th (later . 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. 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 ) . 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