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

Figure 3.46 shows the experimental results showing that the density of nitrite-metabolizing bacteria (NOB) fluctuated from 10 2 CFU/mL – 10 5 CFU/mL after 30 days of shrimp farming. In the control treatment, NOB bacteria appeared at the 4th sampling time (13 days after farming), with a density of 3.1 x 10 2 CFU/mL and gradually increased to 6 x 10 4 CFU/mL at the end of the experiment . In treatment 1, the density of NOB bacteria was 10 2 – 10 5 CFU/mL and nitrite-metabolizing bacteria appeared on the 10th day. In the NT2 and NT3 experiments, the NOB bacteria density ranged from 10 3 to 10 6 CFU/mL, and the nitrite-metabolizing bacteria density was 10 6 CFU/mL at the 10th, 11th, and 12th sampling times. It is possible that near the end of the experiment, organic matter from excess food accumulated at the bottom of the tank, bacteria were added continuously every 6 days, and the nitrite bacteria group became accustomed to the operating conditions of the new environment. They converted NH 4 + to NO 2 - more strongly, so the bacteria density increased.

Evaluation of shrimp growth and survival rate

The results of growth rate and survival rate of shrimp in the treatments supplemented with probiotics were higher than the control treatment (Table 3.14 - Appendix 3.2). Factors such as shrimp survival rate, average shrimp weight gain, shrimp growth rate were proportional to the rate of probiotics added to the tank, proving that the probiotics added in large quantities helped the system function better, reduce harmful bacteria, and help shrimp grow.

Author Far et al. (2009) summarized a number of authors who have demonstrated the benefits of applying beneficial bacteria in shrimp farming. The role of these bacteria is to help balance the intestinal microflora, leading to increased food absorption and increased weight gain of livestock (Fuller, 1998; Rengpipat et al., 1998). Many studies have shown that the number of beneficial bacteria is the only factor promoting increased weight gain and survival rate of farmed shrimp (Zhou et al., 2009). The researched nitrifying biological product has achieved an ammonia conversion efficiency of over 95% in the filtration system under laboratory conditions. This product has also been successfully applied in aquaculture ponds and lagoons in Thanh Hoa and Soc Trang provinces, the total ammonia (TAN) content is always lower than 0.1 mg/L when using the researched nitrifying product (Hoang Phuong Ha et al., 2017). Author Xue Li et al. (2021) examined a mixture of bacteria Bacillus megaterium and Bacillus subtilis with a density of 6 x 10 5 CFU/g, treated

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ammonia, nitrite, nitrate in a carp farming system for 15 days, showing that the conversion efficiency of ammonia was 46.3%, nitrite was 76.3% and nitrate was 35.5%. According to author Vo Van Nha et al. (2021), it shows that using a dose of 5ppm of microbial products, frequency 7 days/time, for 3 times, can treat wastewater from intensive whiteleg shrimp farming ponds in sand ponds.

The above results show that when adding probiotics to whiteleg shrimp tanks, the content of NH 4 + , NO 2 - , NO 3 - is well controlled, the density of Vibrio bacteria is also limited, thereby helping shrimp grow and develop better, helping to reduce the mortality rate. In this study, it is proposed that the ratio of supplemented bacterial products is 0.4% or 0.5% with a density of 10 8 CFU/g in whiteleg shrimp tanks periodically every 6 days/1 time can limit and reduce the content of NH 4 + , NO 2 - , NO 3 - in the tank.

The product achieved in the thesis is the isolation and selection of three bacterial strains, Bacillus licheniformis B85, Rhodococcus rhodochrous T 9, and Pseudomonas stutzeri KL15, from the bottom of the lobster farming area with the best ability to metabolize ammonia and nitrite. The above bacterial strains are heterotrophic nitrifying bacteria, naturally occurring in the seawater environment, participating in the cycle of converting N compounds to treat excess waste in the bottom of the lobster farming area. Domestic studies have always been interested in and selected the group of autotrophic nitrifying bacteria because they metabolize ammonia and nitrite quite well in the N cycle (Rajta, 2019 ), but in this study, heterotrophic bacterial strains living in the bottom of Xuan Dai Bay, tolerant to high salinity, were selected. Author Rajta, (2019) has shown the role of heterotrophic bacteria in the metabolism of ammonia, nitrite, nitrate, especially the Bacillus and Pseudomonas bacteria . In addition, the author also believes that heterotrophic bacteria can metabolize ammonia, nitrite and nitrate in wastewater containing heavy metals and water with high NaCl salt concentrations. This proves that in the nitrogen metabolism cycle, in addition to autotrophic nitrifying bacteria, there is still the parallel existence of heterotrophic nitrate bacteria. According to author Nguyen Lan Dung et al. (2002), heterotrophic microorganisms use carbon sources as a source of nutrition and energy. In addition, for heterotrophic bacteria to grow quickly and strongly, they must simultaneously use carbon, nitrogen and

Mineral elements for food, however, the food requirements for growth of each group of bacteria are not the same. Therefore, in this study, the above characteristics of heterotrophic bacteria were applied to initially create liquid and powdered ammonia and nitrite-converting microbial preparations. The thesis investigated the conditions for microbial culture, optimized the components of the liquid medium to create liquid microbial preparations and investigated the factors for creating powdered microbial preparations.

Author Nguyen Lan Dung and colleagues (2002) showed that the nitrogen source that microorganisms can most easily absorb is NH 3 and NH 4 + and convert it into nitrite and nitrate in the N cycle. Because of that characteristic, the study applied microbial preparations to the conversion of N compounds for application in aquaculture water. The thesis evaluated the effectiveness of microbial preparations on the model of white-leg shrimp nursery tanks at the post-5 stage, showing that when the preparation was added periodically every 6 days, the ammonia, nitrite, and nitrate levels were all well controlled. Currently, lobsters are a type of aquatic product with high economic value, raised in cages in the bay waters with high salinity, natural seeds and using fresh food, so the treatment of the water environment is very difficult and completely depends on the carrying capacity of the water body (Directorate of Fisheries, 2015). In addition, according to the lobster farming planning report of the Directorate of Fisheries (2015), oriented for development from 2020 - 2030, lobster farming is being studied in tanks or ponds on land, converting fresh food into industrial food and applying microbial products in the lobster farming process to treat the water environment. Therefore, the study of supplementing microbial products for the whiteleg shrimp farming environment in 1m3 tanks at the post-5 rearing stage aims to evaluate the effectiveness of microbial products in treating the brackish water farming environment and also lays the foundation for the study of applying microbial products to treat N for the lobster farming water environment in tanks in the future.

Thus, the thesis has contributed to the selection and manufacture of microbial preparations from heterotrophic bacteria originating from the lobster farming environment in the sea, salinity 30-35 ppt, capable of metabolizing N compounds well in the whiteleg shrimp farming environment in tanks, orienting the application of microbial preparations to treat ammonia, nitrite, nitrate compounds for lobster farming tank environment in the future.

CONCLUSION AND RECOMMENDATIONS

1. Conclusion

- Isolation and selection of 3 bacterial strains Bacillus licheniformis B85, Pseudomonas stutzeri KL15, Rhodococcus rhodochrous T 9 with the ability to metabolize ammonia and nitrite as follows: (1) Bacillus licheniformis B85 has an ammonia conversion efficiency of 98.8% in 24 hours, nitrite conversion of 96.97% and nitrate conversion of 89.63% in 4 days; (2) Pseudomonas stutzeri KL15 has an ammonia conversion efficiency of 83.87% in 7 days, nitrite conversion of 99.06% and nitrate conversion of 98.02% in 4 days; (3) Rhodococcus rhodochrous T 9 has an ammonia conversion efficiency of 86.21

% in 5 days, nitrite conversion 95.01% in 3 days, nitate conversion 81.24% in 4 days.

- Determine the isolation process for each bacterial strain in the lobster farming bottom. Each process has differences in environment, isolation conditions and growth environment.

- Optimized the composition of liquid biomass medium for 3 bacterial strains to produce liquid microbial products: (1) The density of B.licheniformis B85 bacteria is 3.14 x 10 11 CFU/mL with the medium composition including 3.94 g/L molasses, 15.56 g/L yeast extract and 1.13 g/L NaCl; (2) 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; (3) The bacterial density of R.rhodochrous T 9 was 2.52 x 10 10 CFU/mL with the medium composition of 7.93 g/L glucose, 6.1 g/L peptone and 2.95 g/L NaCl.

- Determine factors such as carrier, seed ratio, humidity and time affecting the semi-solid biomass production process of 3 bacterial strains to produce powdered microbial products: (1) Bacillus licheniformis B85: carrier is soybean residue, seed ratio 5%, humidity 55% and fermentation time is 60 hours; (2) Pseudomonas stutzeri KL15: corn bran, seed ratio 7.5%, humidity 55% and fermentation time is 72 hours.

hours; (3) Rhodococcus rhodochrous T 9 rice bran, seed ratio 5%, humidity 55% and fermentation time 72 hours.

- The probiotic product contains 3 strains of bacteria Bacillus licheniformis B85, Pseudomonas stutzeri KL15, Rhodococcus rhodochrous T 9 that are capable of metabolizing ammonia and nitrite in the water environment for rearing whiteleg shrimp larvae (post 5) at a density of 10 8 CFU/gram, periodically every 6 days.

2. Suggestion

Study on genetic diversity of ammonia and nitrite metabolizing bacteria groups in lobster farming bottom.

Further research on conditions and optimization of semi-solid media for large-scale production of microbial products.

Evaluation of the antagonistic ability of three bacterial strains Bacillus licheniformis B85, Pseudomonas stutzeri KL15, Rhodococcus rhodochrous T 9 mixed to produce large-scale microbial products.

Evaluation of the effectiveness of water environment treatment of microbial products from 3 bacterial strains Bacillus licheniformis B85, Pseudomonas stutzeri KL15, Rhodococcus rhodochrous T 9 in actual ponds for raising whiteleg shrimp, tanks or ponds for raising lobsters or other aquatic species raised in saltwater and brackish water environments.

Comparative study on the effectiveness of microbial products from 3 bacterial strains Bacillus licheniformis B85, Pseudomonas stutzeri KL15, Rhodococcus rhodochrous T 9 with products for treating brackish water shrimp farming environment on the market.

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