Luận án nghiên cứu ứng dụng công nghệ xử lý nitơ vô cơ trong quản lý môi trường nuôi trồng thủy sản ven biển1

MINISTRY OF EDUCAITON AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRANDUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY Le Thanh Huyen RESEARCH AND APPLICATION OF INORGANIC NITROGEN TREATMENT TECHNOLOGY IN ENVIRONMENT MANAGEMENT OF COASTAL AQUACULTURE Specialty: Natural Resources and Environmental Management Code: 9850101 SUMMARY OF DOCTORAL THESIS HA NOI – 2022 This Thesis was completed at: ACADEMY OF SCIENCE AND TECHNOLOGY VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY Science Instructor 1: Dr Do Manh Hao Science instructor 2: Prof Dr Le Mai Huong Reviewer 1: Reviewer 2: Reviewe 3: The thesis will be defended in front of the Academy-level Doctoral Thesis Evaluation Council, meeting at: The Academy of Science and Technology Vietnam Academy of Science and Technology At hour …, date month … in 2022 The thesis can be found at: - Vietnam National Library - Library of Academy of Science and Technology INTRODUCTION The necessity of the thesis With the trend of growing in marine economic development, socio-economic activities in coastal areas are daily discharging a large amount of waste into the environment, causing the risk of environmental pollution In particular, aquaculture is one of the sources of pollution of concern, which should be strictly controlled One of the important solutions to effectively manage the coastal aquaculture environment and overcome environmental pollution in coastal areas is the development and application of advanced aquaculture technologies The technology of recirculating aquaculture system (RAS) is one of the technological solutions that can reduce environmental pollution in the most radical way With the recirculating culture system, all culture water will be treated, and will be re-supplied the farming system without discharging into the environment Applying RAS technology is one of the appropriate technological solutions to help solve the problem of environmental pollution in aquaculture However, ammonia and nitrite treatment requirements are difficult because the nitrification process is limited due to the growth rate of nitrifying bacteria is estimated to be about 5-10 times slower than other groups of bacteria Moreover, the growth and activity of this group of bacteria are easily inhibited under unfavorable environmental conditions such as lack of air and abundant organic matter Because of the above reasons, the PhD student chose the research topic: "Research and application of inorganic nitrogen treatment technology in environmental management of coastal aquaculture" with the desire to research and offer solutions to treat ammonia and nitrite in the circulating water of aquaculture systems, to minimizing negative impacts on the environment, improving productivity and product quality of the aquaculture industry Objectives Research and apply technology to treat inorganic nitrogen by nitrification membrane to treat circulating water in circulating aquaculture system, contributing to the management of coastal aquaculture environment 4.Content Content 1: Research on the enrichment of nitrifying bacterial communities from the coastal environment Content 2: Research on the formation of nitrification film on the substrate to orient the application in brackish water aquaculture Content 3: Evaluation of the efficiency of TAN and nitrite treatment of nitrification membranes in a recirculating aquaculture system with a scale of 1m3 Content 4: Evaluation of TAN and nitrite treatment efficiency of nitrification membranes in a recirculating aquaculture system with a scale of 100m3 CHAPTER OVERVIEW 1.1 Current status of brackish water aquaculture in the world and in Vietnam Aquaculture in general and shrimp farming in particular in the world has been creating effective transformation and bringing high economic value to many countries, bringing many practical benefits to farmers Vietnam's aquaculture industry is creating an effective transformation and bringing high economic value to many countries, bringing many practical benefits to farmers In Vietnam, in the last two decades, we are witnessing the aquaculture industry, especially the brackish water aquaculture industry, which is developing at a very fast rate However, at present, the shrimp farming industry are being severely affected by environmental pollution One of the main causes of shrimp production reduction is disease and environmental pollution To meet practical needs, 85% of shrimp production is intensively cultured, most notably with high density and super load of feed, thus, 40% of ponds will have to change water after a few days days to remove the hazardous substance The water change in coastal aquaculture ponds has caused eutrophication for the aquatic environment Depending on the density of shrimp farming, the total amount of pollutants such as phosphorus, nitrogen and suspended solids is up to 321; 668 and 215,000 kg/ha, respectively, directly affect the culture environment 1.2 Pollution of inorganic nitrogen compounds in brackish water aquaculture Aquaculture in our country is currently mainly on an industrial scale, leading to the abuse of feed to improve productivity, which has made the lagoon environment and shrimp farming areas seriously polluted and diseases developed develop fast Pollution in shrimp ponds is mainly caused by leftover food and shrimp excreta accumulating on the pond bottom causing organic pollution On average, hectare of pond water surface for a crop of months will produce an amount of algae about 18,000 kg of organic matter/ha, hectare of black tiger shrimp farming with an output of 6-8 tons/crop will release about 3.6 - ,8 tons of waste Therefore, hectare of black tiger shrimp farming will release to the environment about 22 tons of waste (including dead algae biomass) These wastes mostly accumulate in the pond bottom causing organic pollution depending on the method of shrimp farming 1.3 The role of microorganisms in the metabolism of inorganic nitrogen pollutants In the living environment of microorganisms, when there is a substrate, microorganisms will adhere to the surface of the growing medium and develop to form a mucous membrane The growth and development of microorganisms will consume the substrate in the wastewater and purify the wastewater Based on the operating principle of microbial media, people often use media in wastewater treatment to increase the efficiency of the biodegradation process and reduce the amount of sludge generated Increase the efficiency and stable operation of the system Minimizes odors caused by biodegradation of organic compounds Currently, the main raw materials used as substrates include coir, zeolite, coral debris, gravel, sand, ceramic, plastic Individual shapes are usually made in the form of hollow spheres, long bars etc Media types are playing a very active role in enhancing the performance of biological systems and are being applied a lot in wastewater treatment systems However, at present, no research has been conducted specifically on the materials available in Hai Phong city to make the substrate to ensure high adhesion, effective filmforming process, and growth , well developed for the group of nitrifying microorganisms 1.4 Solutions to treat inorganic nitrogen pollutants in brackish water aquaculture ponds by microorganisms 1.4.1 Bio-enhanced solution Bio-enhanced solution is the use of highly biologically active strains of nitrifying and denitrifying bacteria that have been isolated and enriched from nature (Bioproducts) to add to the ponds in order to improve the quality of life increase the selfremoval of pollutants TAN, N-NO2 and N-NO3 According to the results of this study, nitrifying bacteria strains can reduce ammonia, nitrite thereby increasing the survival and growth rate of farmed shrimp The study on creating indigenous nitrifying bacterial communities by the improved enrichment method in this thesis is the first research conducted in Vietnam will contribute to overcoming the difficulties in the treatment of inorganic nitrogen compounds by current biological products 1.4.2 Biological Stimulation Solution Biostimulation is the modification of environmental conditions in a way that stimulates the ability of native microorganisms to remove nitrogen pollutants According to studies, substrate concentration is one of the most important factors affecting the growth and removal capacity of inorganic nitrogen pollutants For the ammonia oxidizing bacteria the substrate is TAN and for the nitrite oxidizing bacteria the substrate is nitrite Under the condition of limited substrate concentration, the nitrification rate of pure bacterial strains or the whole bacterial community is often proportionally proportional to the substrate concentration But when the substrate concentration is high, the growth and nitrification rate of bacteria are inhibited 1.4.3 Biological filtration solutions The available studies show that the solution of inorganic nitrogen treatment by filtration technology has been interested However, one of the biggest challenges of treating inorganic nitrogen compounds by filtration technology is the low treatment efficiency and high cost The reason comes from the nature of nitrifying bacteria, which have a very slow growth rate, and the conditions for these microorganisms to grow are quite strict The researchers are only interested in the design of the filtration system, the operating method of the system, and the assessment of the mechanism of influence of environmental factors on the treatment efficiency There has not been any research that is comprehensive, forming an overall technology solution that can be deployed and applied in accordance with reality, with high applicability In order to overcome the above-mentioned disadvantages, the thesis will conduct research in the direction of focusing on the following contents: (1) creating a group of bacteria with high adaptability, good growth rate; (2) Selecting the type of substrate for effective membrane formation, in which, determining the types of media that help nitrifying microorganisms could grow and develop well; (3) Forming a filter layer by nitrate microorganisms like mini treatment plants with stable and efficient operation; (4) Operate systems at laboratory scale and real scale for solutions to effectively apply these membranes in practice The specific research contents will be presented in the next part of the thesis CHAPTER RESEARCH DOCUMENTS AND METHODS Location, time and research object 2.1.1 Research location - Water samples to enrich nitrifying bacteria were collected in mangrove forests of Phu Long commune, Cat Hai district, Hai Phong city - The process of enriching and concentrating the preparation was carried out at Do Son Marine Research Station - Institute of Marine Resources and Environment - Morphological characterization analysis was conducted at the National Institute of Hygiene and Epidemiology, Hanoi - Genetic diversity analysis of enrichment microbial communities at Biodiversity Research Center, Academia Sinica, Taiwan - Testing the treatment efficiency of nitrification preparations at the experimental area, Do Son Marine Research Station - Institute of Marine Resources and Environment - Trial the model of shrimp farming and tilapia farming on a trial scale at the experimental area, Do Son Marine Research Station - Institute of Marine Resources and Environment - Experimenting with a model of recirculating shrimp farming with a scale of 100m in Tam Da commune, Vinh Bao district, Hai Phong city 2.1.2 Research time - The study was carried out for a period of years (2017 - 2022) 2.1.3 Research subjects - Nitrifying bacteria treat ammonia and nitrite pollutants - Aquaculture recirculating filtration system (RAS) 2.2 Research Methods 2.2.1 Research steps First, sediment samples collected in the field will be transferred to the laboratory for multi-step enrichment in a selective medium The nitrifying bacterial biomass was concentrated from the enrichment suspension by centrifugation After that, the concentrated microbial community will be further studied in different directions to evaluate the diversity of the microbial community and its applicability to brackish water aquaculture + Analysis of morphological characteristics of the microbial community Analysis of genetic diversity of microorganisms + Evaluation of ammonia and nitrite treatment efficiency of nitrate bacteria communities in coastal aquaculture 2.2.2 Retrospective method, inheritance of related documents 2.2.3 Enrichment and film formation on the attachment media of nitrifying bacteria, oriented for application in aquaculture - Collecting specimens: To enrich the nitrifying microbial communities for biofilm research, we collected samples from Phu Long mangrove forest area, Cat Hai district, Hai Phong city - Enrichment of nitrifying bacterial communities: Enrichment of the bacterial group is performed under the condition of regularly adding substrates to the experimental flasks containing the group of bacteria that have been collected - Increasing the microbial community biomass in the fermentation system: Increasing the nitrate microbial community biomass was carried out in a liter capacity fermentation system (BIOFLO 2000) The medium used for culture in the fermentation system included: sea water 1000 ml; (NH4)2SO4 4.72 mg or NaNO2 4.93 mg; K2HPO4 0.88 mg; pH - - Analysis of cell morphological characteristics of the enriched bacterial community: analyzed by staining method, cells after fixation on the slide will be observed under phase contrast microscope and microscope scanning electron microscopy (SEM) - Analysis of genetic diversity of enriched nitrifying bacteria communities: Bacterial biomass before being extracted by Cetyltrimethyl ammonium Bromide method was then analyzed for genetic diversity - Analysis of the surface structure of the material as an attachment medium: The surface shape of the filter material was determined by scanning electron microscopy The surface area of the filter media is calculated using specialized software - Forming membranes of bacterial communities on adhesion substrates: Inoculate nitrification preparations into a 500 mL conical flask containing aqueous medium at a ratio of 1:10 (v/v) The initial substrate concentration in the inoculant activation medium was 10 mg N/l Culture flasks were grown in shaker, in the dark, and pH was adjusted - Selection of substrate: To check the adhesion of the bacterial community to the carrier, it will be examined by scanning electron microscope (SEM) - Creating membranes of bacterial communities on attachment media: This filter will be tested for ammonia and nitrite removal efficiency by culturing in the same liquid medium as the enrichment culture medium 2.2.4 Evaluation of ammonia and nitrite removal efficiency of nitrification membranes in a pilot-scale recirculating aquaculture system - Experimental design: Building 06 water circulation systems with a scale of 1m3/system The experiment was designed with 02 treatments, namely control treatment (DC) and experimental treatment (TN), each treatment repeated times'; Experimental subjects: 21-day-old monogastric perch and vannamei post 12 - Activate the biological filter, operate and monitor: After adding the inoculant to the biological filter tank of the TN treatment, the system is circulated without load for 30 days, the substrate is added regularly 2.2.5 Evaluation of ammonia and nitrite removal efficiency of nitrification membranes in a recirculating aquaculture system with a scale of 100m3 - Experimental design: The experiment was designed with 02 treatments: Experiment (TN1) and Experiment (TN2) Each treatment was a circulating filtration system for white-leg shrimp culture 50 m3/system size - Activate the biological filter, operate and monitor: After adding the inoculant to the biological filter tank of the TN treatment, the system is circulated without load for a period of 30 days, the substrate is added regularly - Operation: Experimental period: months (May 2019 - July 2019); Density: 300 fish/m CHAPTER 3: RESULTS AND DISCUSSION 3.1 Research on enrichment and film formation on the attachment media of nitrifying bacteria with orientation for application in aquaculture 3.1.1 Enrich the nitrifying bacteria community First step enrichment process: 30 0.120 25 0.100 20 0.080 15 0.060 10 0.040 0.020 0.000 10 15 20 25 30 35 Figure 3.1 Substrate consumption and substrate removal rate of nitrifying microbial complex during step enrichment Figure 3.2 Substrate consumption and substrate removal rate of nitrifying microbial complex during step enrichment 11 3.1.2 Morphological diversity of nitrifying bacteria communities Table 3.1 Morphological characterization of the enriched nitrifying bacteria complex No Cell shape Size (µm) Cell surface Gram react 0,5 x 1,9 Less rough surface - Long slender curved bar with round tip Straight bar with round end 0,6 x 1,4 Less rough surface + Short rod with pointed end 0,7 x 1,1 Less rough surface + Straight rod with round tip 0,9 x 1,9 Raw calves - Curved stick with round tip 0,5 x 0,9 Raw calves - Curved stick with round tip 0,5 x 1,4 Raw calves - Globular 0,7 Raw calves ND Long slender curved bar with pointed end Slim curved bar with pointed tip 10 Straight bar with round end Less rough surface 0,3 x 1,3 ND 0,2 x (1,2 -1,6) Less rough surface ND 0,2 x 0,7 Less rough surface ND Figure 3.7 Diversity in cell morphology of microbial complexes enriched in suspension 12 Figure 3.8 Diversity in cell shape of the microbial complex enriched in: a-coconut; b&c - coir-mounted umbrella; d - sponge; e&f - sponge-mounted umbrella 3.1.3 Diversity of species Table 3.2 Diversity of species Mẫu Shannon SI Cd Trình tự gen OTUs CU1 3,97 0,49 0,12 21.531 94 CU2 2,67 0,49 0,42 40.818 137 Figure 3.9 Diversity of species arising from nitrifying bacterial communities after concentration (a): Condensation ice deposition method; (b): concentrate by centrifugation 13 3.1.4 Concentrates The original probiotics were stored at 4oC when diluted with 10 times culture medium capable of oxidizing 0.73 mg/L/hr of substrate to nitrate The dry biomass of microorganisms in the preparation reached 620 mg/l The total microbial biomass in the preparation was quantified by analytical balance with accuracy of 0.001g, centrifuged inoculum and culture medium at 7,000 rpm for 30 minutes, decanted the liquid layer in upper and weigh the lower biomass after drying overnight in an oven at 37°C The inoculum biomass is the difference between the precipitate of the inoculum and the precipitate of the culture medium 3.1.5 Research on selection of media In this study, the author selected 05 different materials to evaluate the effectiveness of their adhesion as well as ammonia and nitrite treatment, including dead coral rock, coir, small pebbles, sponge and plastic beads kaldness After adhesion of microorganisms on the substrate, the media (biofilter) was transferred to a triangular flask with available capacity of mineral medium with initial substrate concentration of 10 mg/L The monitoring results of TAN and nitrite indicators showed that after 15 days, the concentrations of ammonia and nitrite in the treatments with different substrates were all consumed Figure 3.10 Evaluation of the treatment efficiency of TAN and nitrite of different biological filters After days, the removal rates of TAN and nitrite of dead coral, coir, pebbles and kaldness resins were no different and were all higher than those of pebbles 14 Table 3.3 Compare and evaluate some important characteristics of biofilter media TT Criteria 10 11 12 Dead Coral Surface cross-section Porosity Drain diameter Anti-clogging trend Inert material structure Mechanical strength Light-weight Sanitize and disinfect Easy maintenance Power consumption Water permeability Absorption capacity total score Coir Kaldnes Sponge Pebbles 3 3 1 2 3 1 2 3 3 3 1 3 1 1 1 1 3 1 1 20 16 32 23 21 3.2 Research and evaluate the efficiency of ammonia and nitrite treatment in a small-scale aquaculture circulating filtration system 3.2.1 Activate the nitrification membrane After 30 days of activating the nitrification membrane in the biofilter, the TAN content decreased from 20 mg/L in the first day to 0.0 mg/L The trend of Variations in TAN content can be divided into periods: the first days, the next 10 days and the last days Stage 1, TAN decreased slowly with a rate of only about 0.41 mg/L/day Stage 2, TAN decreased fastest at the rate of 1.53 mg/L/day Stage 3, TAN decreased at the lowest rate of 0.075 mg/L/day 3.2.2 Research results on vannamei shrimp 3.2.2.1 Total Ammonia (TAN) 8.00 7.00 TAN (mg/L) 6.00 5.00 4.00 3.00 2.00 1.00 00 10 20 30 40 50 60 70 80 Thời gian (ngày) Figure 3.11 Variation of TAN concentration in vannamei ponds 90 15 3.2.2.2 Nitrite (N-NO2) 7.00 N-NO2 (mg/L) 6.00 5.00 4.00 3.00 2.00 1.00 00 10 20 30 40 50 TB ĐC Thời gian (ngày) 60 70 80 90 Figure 3.12 Variation of Nitrite concentration in vannamei ponds 3.2.2.3 Nitrate (N-NO3) 25 NO3 (mg/L) 20 15 10 0 20 40 60 80 100 Thơi gian (ngày) TN ĐC Figure 3.13 Variation of Nitrate concentration in vannamei ponds 3.2.2.4 Shrimp survival rate The test results showed that the survival rate of shrimp in the control tank in the first 10 days was high and reached 95-97.0% After 36 days of culture, the survival rate of shrimp reached 90.0% 16 3.2.3 Research results for tilapia 3.2.3.1 Total Ammonia (TAN) 12 TAN (mg/L) 10 -2 10 20 30 Đối chứng 40 50 Thí nghiệm Ngày Figure 3.14 Variation of TAN concentration (mg/L) during tilapia culture N-NO2 (mg/L) 3.2.3.2 Nitrite (N-NO2-) -1 10 20 30 Đối chứng 40 50 Thí nghiệm Ngày Figure 3.15 Variation of N-NO2 concentration (mg/L) during tilapia culture (mg/L) 3.2.3.3 Tilapia survival rate % sống sót 120.000 100.000 80.000 60.000 40.000 20.000 000 10 Đối chứng 20 30 Thí nghiệm Figure 3.16 Survival rate (%) of tilapia during stocking 40 50 Ngày 17 General comment Survival rate (%) of tilapia in stocking period Experiment results to evaluate the effect of nitrification products through culture of brackish water tilapia (15‰) and vannamei shrimp in a recirculation system The scale of 1m3 showed that the experimental batch was effective in handling TAN, nitrite and the survival rate of both vannamei shrimp and tilapia were significantly higher than that of the control group The content of TAN and N-NO2 in the control group increased after the experiment cycle, TAN and nitrite for whiteleg shrimp were 5.4 mg/L and 5.2 mg/L respectively, and for tilapia were 5.4 mg/L and 5.2 mg/L respectively 10.0 mg/L and 5.2 mg/L respectively 3.3 Evaluation of ammonia and nitrite removal efficiency of nitrification membranes in a recirculating aquaculture system with a scale of 100m3 3.3.1 Activate the nitrifying membrane (break in) After putting the nitrification product into the biological filter module, activate the aeration mode and the circulation pump to supply oxygen and water is circulated inside the biological filter module Add substrate (NH4Cl) to the filter tank to reach a concentration of 20 mg/L and periodically monitor the parameters of TAN, nitrite and nitrate When the concentration of TAN and nitrite is exhausted, the biological filter has been working effectively and can be connected to the culture tank 30 25 mg/L 20 15 10 0 10 TAN 15 N-NO2 20 25 N-NO3 30 Ngày Figure 3.17 Variations of TAN, nitrite and nitrate levels during biofilter activation3.3.2 Chất lượng môi trường 3.3.2.1 Total Ammonia (TAN) TAN concentrations ranged from 0.0 to 0.7 mg/L, with an average of 0.48 mg/L TAN tends to increase rapidly in the first 18 days with an average rate of 28 µg/L/day Meanwhile, in the following days, TAN tends to increase slightly at a rate of about µg/L/day If only the last 22 days, TAN tends not to increase but always remains at a concentration of 0.6 - 0.7 mg/L/day (Figure 3.18) 18 0.8 0.7 TAN (mg/L) 0.6 0.5 0.4 0.3 0.2 0.1 0 10 20 30 40 50 60 70 Figure 3.18 Variation of TAN concentration in vannamei ponds 3.3.2.2 Nitrite (N-NO2-) The nitrite concentration ranged from to 0.45 mg/L, with an average of 0.25 mg/L Nitrite tends to increase rapidly in the first 12 days and from 24 to 36 days Nitrite tends to be stable from 12 to 24 and in the last 20 days (Figure 3.19) 0.5 0.45 N-NO2 (mg/L) 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 10 20 30 40 Thời gian (ngày) NO2-Bể 50 60 70 NO2-Bể Figure 3.19 Variation of nitrite concentration in vannamei ponds 3.3.2.3 Nitrate (N-NO3-) The nitrate concentration ranged from 0.26 mg/L to 7.1 mg/L, with an average of 5.37 mg/L N-NO3- tends to increase rapidly in the first 27 days with an average rate of 0.27 mg/L/day During the next 30 days, the concentration of N- NO3- tended not to increase and always remained at the concentration of 7.17 mg/L/day (Figure 3.20) Although the biological filtration system does not use a denitrification module, the concentration of N- NO3- in the tank is not high and is especially stable during the last 30 days The cause may be due to the photosynthetic activity of algae and photosynthetic bacteria that used N- NO3- as a nitrogen source for photosynthesis 19 N-NO3 (mg/L) 0 10 20 30 40 NO3-Bể 50 60 70 NO3-Bể Figure 3.20 Changes in nitrate concentration in vannamei ponds 3.3.2.4 Chemical oxygen demand (COD) 10 COD (mg/L) 0 10 20 30 40 COD-Bể 50 60 COD-Bể BOD5 (mg/L) Figure 3.21 Variations in COD concentration in vannamei ponds 3.3.2.5 Biological oxygen demand (BOD5) 0 10 20 30 BOD5-Bể 40 50 60 70 BOD5-Bể Figure 3.22 Variation of BOD5 concentration in vannamei ponds 3.3.2 Total bacteria 70 Vi khuẩn tổng số (CFU/ml) 20 1.800E+06 1.600E+06 1.400E+06 1.200E+06 1.000E+06 8.000E+05 6.000E+05 4.000E+05 2.000E+05 0.000E+00 10 20 30 40 50 60 70 Figure 3.23 Variation of total bacterial density in vannamei ponds 3.3.2.7 Vibrio 200 CFU/ml 150 100 50 0 10 20 30 40 50 60 70 Thời Vibrio-Bể gian (ngày) Vibrio-Bể 3.4 Discussion Multistep enrichment may allow us to stimulate the multiplication of desirable microbial populations while inhibiting the growth of undesirable microbial groups Furthermore, through the process of inoculating the enrichment culture into the new medium, many unidentified intermediate compounds produced during the enrichment process that can inhibit the growth of the nitrifying bacteria group can be reduced go, as a result the growth of the desired microorganism is improved In this study, the enrichment process also showed that the amount of substrate consumed increased gradually during the enrichment process If in the first step of enrichment the cumulative substrate consumption is 28 mg/l, but in the fifth enrichment step the cumulative substrate consumption is 495 mg/l Simultaneously, the substrate removal rate also increased from 0.083 mg/l/hr in the first enrichment step to 3.65 mg/l/h in the 5th enrichment step Previous research results also showed that the rate of substrate removal increased oil after each enrichment The batch enrichment results of Shan and Obbard showed that the TAN removal rate increased from mg/l/day in the 1st generation to mg/l/day in the 9th generation Kim et al enriched with ammonia oxidizing bacteria (AOB) from activated sludge samples of wastewater, AOB bacteria 21 group was enriched in batches in selective medium with replicates The results also showed that the oxidation rate increased from 0.017 mg/MLSS/day in the first enrichment to 0.810 mg/MLSS/day in the 5th enrichment Yao et al also enriched the oxidizing bacteria group, ammonia AOB from activated sludge, AOB was grown in an automatic fermentation system (minifors, INFORS Corp., Switzerland) with a useful volume of 3.5 L During the enrichment process, the ammonia substrate and the carbon source in the form of Na2CO3 were continuously added continue to the fermentation system The results showed that the maximum ammonia oxidation rate exceeded 170 mg/l/h Comparison of water environment monitoring indicators in intensive shrimp farming areas in Hai Phong from 2017 (6 months), 2019 (5 months), 2020 (2 months) and monitoring indicators The water environment in the models applying RAS technology using nitrate membranes in the thesis's research shows that all the indicators in the intensive farming areas have much higher values With the models applying RAS technology using nitrification membranes from the research results of the thesis, both the 1m3 scale model and the 100m3 scale model show that the indicators are much lower and always meet the regulations on water quality according to Vietnam Standard TCVN 11041-8:2018 on production in the field of aquaculture for organic shrimp farming In which, the indicators of BOD and COD ranged from 6-8 mg/l; N- NHL3 ranges from 0.15 to 0.25 mg/l; Vibrio concentrations ranged from 150 to 200 CFU/ml The comparison results highlight the advantages of the recirculation farming technology using the nitrification membrane of the thesis, of which the most important is stabilizing the quality of cultured water, ensuring productivity and quality of farming absolutely no waste water and sludge Especially, not discharging wastewater from farming areas also avoids the spread of diseases between farming facilities; The increased application of this technology is one of the optimal solutions to effectively manage the coastal aquaculture environment, contributing to reducing a large source of emissions to the environment from aquaculture activities, preserving protect water quality in coastal waters 22 CONCLUSIONS AND RECOMMENDATIONS Conclusion (1) Regarding the enrichment and film forming process: The thesis has developed a multi-step enrichment process to successfully multiply both ammonia and nitrite oxidizing bacteria from water samples collected in Bang La mangrove forest, district Cat Hai, Hai Phong city After the enrichment process, the substrate removal rate increased from 0.114 mgN/L/h to 3.58 mgN/L/h, the total amount of substrate consumed during the whole enrichment process was 5,665 mg/L The enrichment process studied by this thesis has many new points compared to existing studies These new points are reflected in the following contents: Selection of enriched microorganisms from native natural microorganisms; selection of enrichment process is a 5-step process; selection of simultaneous enrichment of both ammonia and nitrite oxidizing bacteria groups; select medium with low substrate concentration for enrichment The successful enrichment results created a very diverse post-enrichment microbial community, including 17 different phyla: Actinobacteria, Acidobacteria, Proteobacteria, Bacteroidetes, Chlamydiae, Chlorobi, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Hydrogenedentes, Nitrospirae , Parcubacteria, PAUC34f, Planctomycetes, SBR1093 and Verrucomicrobia The genera taxa showed that Nitrospira, Pseudohongiella, Marinobacter, and Denitromonas were dominant genera in the enrichment community (2) Regarding the ammonia and nitrite treatment efficiency of nitrification membranes in the pilot-scale circulating seafood system: After building the enrichment process and selecting an integrated medium, the thesis has applied Experimental application to the recirculation system for tilapia and vannamei shrimp farming with a scale of m3 showed that the efficiency of TAN, nitrite and survival rates of the experimental group were significantly higher than that of the control group (3) On the basis of the results of evaluating the efficiency of ammonia and nitrite treatment of nitrification membranes in a pilot-scale circulating seafood system, the thesis implements and evaluates the efficiency of ammonia and nitrite treatment of membranes Filtering nitrification in the recirculating aquaculture system with a scale of 100m3: The results of the trial of nitrification preparations in the recirculating system of whiteleg shrimp with a scale of 100 m3 showed that the TAN and nitrite 23 parameters were maintained at low concentration, survival rate is 83-86%, yield is 3.73.9 kg/m3 The nitrification rate of the biological filtration system of the model reached 0.184 gTAN/m2/day Thus, the thesis has completed the research and development of the microbial enrichment process, selected the appropriate type of substrate to ensure the best filmforming process of microorganisms, carried out experimental research on this scale filot scale and on a production scale with a capacity of 100m3 Request Currently, the circulating aquaculture systems are still expensive, so they have not been widely applied in aquaculture practice Therefore, research into technological solutions to create effective filtration systems, contributing to the creation of environmentally-friendly and low-cost farming systems is essential for the development of the aquaculture industry of Vietnam, contributing to achieving the dual goals of economic development while protecting the environment and saving water resources On the basis of research results, the topic recommends scientists, managers, organizations and individuals in the field of aquaculture to continue researching and clarifying the following contents to complete the following: technological process of circular aquaculture with low cost and high economic efficiency, contributing to promoting a key economic sector of the country to develop in a sustainable way: - Testing and perfecting the technological process for the production of nitrification preparations for high and stable treatment efficiency - Research and develop intelligent management and control modules, waste sludge treatment modules from filter drums, foam separators and blowdown blowers - Implement pilot production projects to continue to perfect the technology, expand the scale to improve the economic - technical and technological indicators and gradually put the shrimp farming technology by RAS technology into practice manufacturing 24 NEW CONTRIBUTIONS OF THE THESIS This is a relatively systematic and complete research work to develop nitrification membranes applied in coastal aquaculture In which, there are 03 new research points as follows: (1) Developing a multi-step enrichment process to successfully breed nitrifying bacteria from indigenous microflora in Phu Long mangrove forest, Cat Hai district, Hai Phong city, to create inoculant native nitrification (2) Create a nitrification filter from a multi-step process enriched microbial community and available media (3) Evaluation of TAN and nitrite removal efficiency of nitrification membranes in RAS system for tilapia and vannamei shrimp culture at experimental and pilot scale (1 m3, 100 m3) LIST OF RELATED RESEARCH WORKS 1.Le Thanh Huyen, Dao Thi Anh Tuyet, Le Minh Hiep, Nguyen Tien Dat Ha Thi Binh, Do Trung Sy, Do Manh Hao, Density and nitrateation potention of indigenous microoganism system in mangroves ang Seagrass of Northern ofVietNam, Vietnam journal of Marine Science and Technology, 2021, Hà Nội, Vol 20, No 4; 2020:347 – 445 Le Thanh Huyen, Dao Thi Anh Tuyet, Le Minh Hiep, Nguyen Tien Dat, Ha Thi Binh, Do Trung Sy, Do Manh Hao, Density and de-nitrification potention of indigenous microoganism system in mangroves ang Seagrass of Northern of VietNam, Vietnam journal of Marine Science and Technology, 2021, Hà Nội, Vol 21, No 1; 2021:268-272 Do Manh Hao, Dao Thi Anh Tuyet, Le Minh Hiep, Ha Thi Binh, Le Thanh Huyen, Sen-Lin Tang, Pei-Wen Chiang, A multi-step nitrifying microbial enrichment to remove ammonia and nitrite in brackish aquaculture systems, Biodegradation, Springer Nature B.V, May https://doi.org/10.1007/s10532-022-09988-9 2022, BIOD-D-21-00142R1, ... -2 10 20 30 Đối chứng 40 50 Thí nghiệm Ngày Figure 3.14 Variation of TAN concentration (mg/L) during tilapia culture N-NO2 (mg/L) 3.2.3.2 Nitrite (N-NO2-) -1 10 20 30 Đối chứng 40 50 Thí nghiệm... 3.17 Variations of TAN, nitrite and nitrate levels during biofilter activation3.3.2 Chất lượng môi trường 3.3.2.1 Total Ammonia (TAN) TAN concentrations ranged from 0.0 to 0.7 mg/L, with an average... minutes, decanted the liquid layer in upper and weigh the lower biomass after drying overnight in an oven at 37°C The inoculum biomass is the difference between the precipitate of the inoculum and the