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

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

MINISTRY OF EDUCAITON

AND TRAINING

VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY

GRANDUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY

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

At hour …, date month … in 2022

The thesis can be found at:

- Vietnam National Library

- Library of Academy of Science and Technology

INTRODUCTION

1 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

2 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

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 1 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, 1 hectare of pond water surface for a crop of 6 months will produce an amount of algae about 18,000 kg of organic matter/ha, 1 hectare of black tiger shrimp farming with an output

of 6-8 tons/crop will release about 3.6 - 4 ,8 tons of waste

Therefore, 1 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 film-forming 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 (Bio-products) to add to the ponds in order to improve the quality of life increase the self-removal 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 2 RESEARCH DOCUMENTS AND METHODS

2 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 100m3 in Tam Da commune, Vinh Bao district, Hai Phong city

2.1.2 Research time

- The study was carried out for a period of 5 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 3 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 2 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 - 8

- 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 3 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 1 (TN1) and Experiment 2 (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: 2 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:

Figure 3.1.Substrate consumption and substrate removal rate of nitrifying microbial

complex during step 1 enrichment

Figure 3.2 Substrate consumption and substrate removal rate of nitrifying microbial

complex during step 2 enrichment

0.000 0.020 0.040 0.060 0.080 0.100 0.120

Figure 3.3. Substrate consumption and substrate removal rate of nitrifying microbial

complex during step 3 enrichment

Figure 3.4 Substrate consumption and substrate removal rate of nitrifying microbial

complex during step 4 enrichment

Figure 3.5 Substrate consumption and substrate removal rate of nitrifying microbial

complex during step 5 enrichment

Conclusion: Through 5 steps, both ammonia oxidizing bacteria and nitrite oxidizing bacteria were enriched simultaneously Although substrates for nitrite oxidizing bacteria are not added directly to the culture medium, nitrite produced by ammonia oxidizing bacteria is used as a substrate for nitrite oxidizing bacteria The nitrifying flora consumed 1,238 mgN L-1 in 122 days The amount of substrate consumed increases gradually during the enrichment process In the first enrichment step, the total substrate is consumed about 28 mg L-1, but up to 495 mg L-1 in the fifth enrichment The substrate removal rate increased from 0.083 mg L-1 h-1 in the first step enrichment to 3.65 mg L-1 h-1 in the fifth step enrichment (Figure 3.6)

Figure 3.6. Comparison of the substrate consumption and substrate removal rate of the

binding between the enrichment steps

Thời gian (ngày)

3.1.2 Morphological diversity of nitrifying bacteria communities

Table 3.1 Morphological characterization of the enriched nitrifying bacteria complex

1

Long slender curved bar with round tip

Figure 3.7. Diversity in cell morphology of microbial complexes enriched in suspension