Study on nitrogen-metabolizing bacteria in the bottom of the lobster farming area Panulirus sp. for aquaculture - 2

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Summary iv

Summary

Table of Contents viii

List of abbreviations xiv

List of tables xv

List of Figures xvii

INTRODUCTION 1

CHAPTER 1 OVERVIEW OF DOCUMENTS 5

1.1. Overview of lobster cage farming situation 5

1.2. Current status of water pollution in lobster farming area of ​​Xuan Dai Bay 6

1.3. Microorganisms in saltwater environment 7

1.4. Diagram of nitrogen metabolism cycle in marine ecosystem 8

1.5. Nitrogen metabolism processes and the role of bacterial groups involved in metabolism 11

1.6. Characteristics of bacterial groups involved in the nitrogen metabolism process 13

1.6.1. Biological characteristics of Bacillus 13 bacteria

1.6.2. Biological characteristics of Rhodococcus 15 bacteria

1.6.3. Biological characteristics of Pseudomonas bacteria 15

1.6.4. Biological characteristics of Stenotrophomonas 16 bacteria

1.6.5. Biological characteristics of other nitrogen-metabolizing bacteria groups 17

1.7. Factors affecting the process of bacterial biomass multiplication 18

1.7.1. Carbon Source 18

1.7.2. Nitrogen Source 18

1.7.3. Source of minerals and vitamins 18

1.7.4. Seed density 19

1.7.5. Culture time 19

1.7.6. Temperature 19

1.7.7. pH 19

1.7.8. Kinetics of Microbial Processes 19

1.8. Overview of Plackett - Burman and Box - Behnken matrices 20

1.8.1. Introduction to response surface methodology (RSM) 20

1.8.2. Plackett - Burman Matrix 20

1.8.3. Box-Behnken Matrix (BBD) 21

1.9. Factors affecting the water environment for whiteleg shrimp farming 21

1.9.1. Total Ammonia (TAN - Total Ammonia Nitrogen) 21

1.9.2. Nitrite and Nitrate 22

1.10. Research status of application of microorganisms in aquaculture 22

1.10.1. Situation of application of microorganisms in domestic aquaculture 22

1.10.2. Situation of application of microorganisms in aquaculture in the world 24

CHAPTER 2 RESEARCH METHODOLOGY 26

2.1. Research diagram 26

2.2. Time and place of research 26

2.3. Method of collecting bottom sludge samples 27

2.3.1. Sampling location 27

2.3.2. Method of physical and chemical analysis of sludge samples 27

2.4. Content 1: Isolation and identification of nitrogen-metabolizing bacteria from the bottom of lobster farming areas. 28

2.4.1. Isolation and identification of ammonia-metabolizing Bacillus sp. bacteria 28

2.4.1.1. Isolation of Bacillus sp. 28

2.4.1.2. Biochemical identification method of Bacillus sp. 28

2.4.1.3. Molecular biological identification method of Bacillus sp. 28

2.4.2. Isolation and identification of ammonia-metabolizing bacteria (AOB) 29

2.4.2.1. Isolation medium for ammonia-metabolizing bacteria (Appendix 1.3.1) 29

2.4.2.2. Steps to isolate ammonia-metabolizing bacteria 29

2.4.2.3. Biochemical identification method of AOB bacteria 31

2.4.2.4. Molecular biological identification method of AOB bacteria 31

2.4.3. Method for isolating nitrite-metabolizing bacteria (NOB) 32

2.4.3.1. Environment for isolating nitrite-metabolizing bacteria 32

2.4.3.2. Steps for isolating nitrite-metabolizing bacteria (NOB) 32

2.4.3.3. Biochemical identification method of NOB 32 bacteria

2.4.3.4. Molecular biological identification method of NOB 33 bacteria

2.4.4. Survey on the metabolism of ammonium, nitrite, nitrate of selected bacterial strains 33

2.4.5. Survey on salt tolerance of bacterial strains 34

2.5. Content 2: Creating liquid and powdered microbial preparations 34

2.5.1. Survey of factors affecting the culture and optimization of fermentation medium composition to create liquid preparations of bacterial strains 34

2.5.1.1. Survey of factors affecting the biomass growth of three bacterial strains...34

2.5.1.2. Survey of factors affecting the process of creating liquid preparations 35

2.5.1.3. Optimization of media components for biomass multiplication of 3 bacterial strains by response surface methodology (RMS). 36

2.5.2. Manufacturing of powdered microbial preparations 42

2.5.2.1. Survey of factors affecting the production conditions of bacterial strains on semi-solid media 42

2.5.2.2. Preservation of powdered microbial preparations 43

2.6. Content 3: Evaluation of nitrogen metabolism of bacterial strains in aquaculture. 43

2.6.1. Evaluation of N metabolism of bacterial strains in whiteleg shrimp pond water at laboratory scale 43

2.6.2. Evaluation of N metabolism of bacterial strains in whiteleg shrimp farming in the nursery stage in 1m3 cement tank scale 44

2.6.3. Evaluation criteria 46

2.7. Statistical processing 46

CHAPTER 3 RESULTS AND DISCUSSION 47

3.1. Content 1: Isolation and identification of nitrogen-metabolizing bacteria from the bottom of lobster farming areas. 47

3.1.1. Environmental indicators in mud samples collected in Xuan Dai Bay Area 47

3.1.2. Bacillus sp. bacteria group metabolizes ammonia 48

3.1.2.1. Results of isolating ammonia-metabolizing Bacillus bacteria 48

3.1.2.2 Selection of ammonia treatment ability of isolated bacteria. 48

3.1.2.3. Biochemical identification results of Bacillus sp. 49 strains

3.1.2.4. Results of molecular biological identification of Bacillus sp. 49 strains

3.1.2.5. Results of evaluating the ammonia metabolism ability of bacterial strains

Bacillus sp. isolated 51

3.1.3. Results of isolating ammonia-metabolizing bacteria (AOB) from sludge sample 53

3.1.3.1. Results of determining ammonia-metabolizing bacteria in sludge sample 53

3.1.3.2. Results of morphological characteristics of isolated bacterial strains 54

3.1.3.3. Results of determining the NH3 metabolism ability of bacterial strains 54

3.1.3.4. Results of biochemical identification of bacterial strains 55

3.1.3.5. Identification results by sequencing the 16S – rRNA region 57

3.1.3.6. Results of evaluating the ammonia metabolism ability of selected bacterial strains 59

3.1.4. Results of isolating nitrite-metabolizing bacteria from sludge sample 61

3.1.4.1. Results of determining nitrite-metabolizing bacteria in sludge samples. 61

3.1.4.2. Results of morphological characteristics of isolated bacterial strains 62

3.1.4.3. Results of determining the NO2- metabolism ability of bacterial strains 62

3.1.4.4. Results of biochemical identification of bacterial strains 63

3.1.4.5. Bacterial identification by 16S rRNA gene sequence analysis 66

3.1.4.6. Results of evaluating the nitrite metabolism ability of selected bacterial strains 70

3.1.5. Investigation of the ability to metabolize nitrogen-containing compounds and the salt tolerance of bacterial groups belonging to the genera Bacillus, AOB and NOB. 71

3.1.5.1. Survey of NO2-, NO3- metabolism ability and salt tolerance of Bacillus and AOB bacteria strains. 71

3.1.5.2. Investigation of the ability to metabolize NH4- and NO3- and the salt tolerance of the NOB bacteria group. 73

3.2. Content 2: Creating liquid and powdered microbial preparations 76

3.2.1. Survey of factors affecting the culture and optimization of fermentation medium composition to create liquid preparations of bacterial strains 76

3.2.1.1. Survey of factors affecting the biomass growth of three bacterial strains...76

3.2.1.2. Survey of factors affecting the process of creating liquid preparations Survey of the effect of seed loading ratio on the process of bacterial biomass multiplication 79

3.2.1.3. Optimization of media components for biomass multiplication of 3 bacterial strains by response surface methodology (RMS). 85

3.2.2. Creating powdered microbial preparations 101

3.2.2.1. Survey of factors affecting the production conditions of bacterial strains on semi-solid medium 101

3.2.2.2. Making powdered bacterial preparations 105

3.3. Content 3: Evaluation of nitrogen metabolism of bacterial strains in aquaculture. 108

3.3.1. Evaluation of N metabolism of bacterial strains in whiteleg shrimp farming water (without shrimp) at laboratory scale 108

3.3.1.1. Evaluation of N metabolism of bacterial strains 108

3.3.1.2. Evaluation of microbial density when adding microbial products 113

3.3.2. Evaluation of N metabolism of bacterial strains in whiteleg shrimp rearing tanks during the nursery stage at a 1m3 cement tank scale 116

CONCLUSION AND RECOMMENDATIONS 128

REFERENCES 130

LIST OF PUBLISHED RESEARCH WORKS 150

APPENDIX 151

LIST OF ABBREVIATIONS

AOB: Ammonia-Oxidizing Bacteria (ammonia oxidizing bacteria) BC: Sludge collected in submerged cage

BT: Sludge collected at hanging cage

BTNMT: Ministry of Natural Resources and Environment

CPVS: Microbiological products

DX : Design Expert

FCR : Feed conversion ratio

KL: Colony

MPN: Most Probable Number (most probable number method) MT: environment

NOB: Nitrite – Oxidizing Bacteria (nitrite oxidizing bacteria) VKHK: Total aerobic bacteria

NT1: Solution 1

NT2: Formula 2

NT3: Formula 3

NT4: Formula 3

NT5: Formula 3

NTDC: Control formula

NTTS: Aquaculture

PCR: Polymerase Chain Reaction

QCVN: Vietnamese Standards

TAN : Total ammonia nitrogen (total ammonia)

TCVN : Vietnam Standard

LIST OF TABLES

PAGE TABLE

Table 2.1. Variables in the Plackett – Burman matrix of two bacterial strains

B.licheniformis B85 and P.stutzeri KL15 37

Table 2.2. Plackett – Burman design matrix of B.licheniformis B85...37

Table 2.3. Plackett – Burman design matrix of P. stutzeri KL15 38

Table 2.4. Variables in the Plackett - Burman matrix of R.rhodochrous T9 bacteria.

................................................................ ................................................................ .........................38

Table 2.5. Plackett – Burman design matrix of R.rhodochrous T9 39

Table 2.6. Elements used in Box – Behnken of Bacillus licheniformis B85

................................................................ ................................................................ ..........................39

Table 2.8. Factors used in Box –Behnken of Pseudomonas stutzeri KL15

................................................................ ................................................................ ..........................40

Table 2.9 Box – Behnken test matrix of Pseudomonas stutzeri KL15 40

Table 2.10. Factors used in Box – Behnken of R.rhodochrous T9. 41

Table 2.11. Box – Behnken test matrix of Rhodococcus rhodochrous T9 42 Table 2.12. Methods for evaluating water quality and microbial indicators used in the experiment 46

Table 3.1 Environmental indicators, microbial density of sludge samples in 12 months 47

Table 3.2. Results of biochemical reactions of 13 strains of Bacillus sp. 49

Table 3.3 16S-rRNA region identification results of 13 strains of Bacillus sp. 50

Table 3.4. Some biochemical reactions of bacterial strains according to API 20NE kit...55

Table 3.5. Some biochemical reactions of bacterial strains according to API 20E 56 kit

Table 3.6. Table of molecular identification results of AOB 57 bacterial groups

Table 3.7. Biochemical reaction results of bacterial strains according to API 20E kit. 63

Table 3.8. Biochemical reaction results of bacterial strains according to API 20NE kit. 64

Table 3.9. Biochemical reaction results of bacterial strains according to API Coryne kit

................................................................ ................................................................ ..........................65