Methods for Rapid Cultivation of Stable Nitrifying Bacteria in Land-Based Circulating Water Systems

Abstract

This invention relates to the field of water treatment technology for recirculating aquaculture systems (RAS), and discloses a method for rapidly cultivating stable nitrifying bacteria in a land-based recirculating aquaculture system. The method includes the following steps: Step S1, system preparation and enrichment of ammonia-oxidizing bacteria; Step S2, oxygen deprivation and regulation to inhibit ammonia-oxidizing bacteria and create a growth window for nitrite-oxidizing bacteria; Step S3, restarting oxygenation to promote the establishment of nitrite-oxidizing bacteria dominance; Step S4, stable system operation. This method for rapidly cultivating stable nitrifying bacteria in a land-based recirculating aquaculture system achieves rapid startup of the biofilter and synchronization with aquaculture production, greatly improving facility utilization. This invention completely changes the traditional two-stage model of "cultivating bacteria first, then releasing fish." By simultaneously releasing low-density aquaculture organisms after system water injection, the bacteria cultivation process is fully integrated into the early stage of aquaculture. This allows the biofilter to reach a stable operating state within 25-30 days after the start of aquaculture, significantly shortening the system idle period.

Description

Technical Field

[0001] This invention relates to the field of water treatment technology for recirculating aquaculture systems (RAS), specifically a method for rapidly cultivating stable nitrifying bacteria in a land-based recirculating aquaculture system. Background Technology

[0002] In marine recirculating aquaculture systems, maintaining stable water quality hinges on the effective operation of the biofilter. The beneficial microbial community enriched within the biofilter, particularly ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), plays a crucial role in oxidizing the toxic ammonia nitrogen (NH3-N) produced by fish metabolism into nitrite (NO2⁻-N), and further oxidizing it into the less toxic nitrate (NO3⁻-N). Therefore, rapidly and stably establishing a fully functional nitrifying bacterial community is the primary step in ensuring the safe operation of the system and reducing stress and mortality risks in farmed organisms. Currently, the industry mainly relies on the following technical solutions for the construction (i.e., "cultivation") of microbial communities in biofilters: Natural acclimatization method: This method involves running the prepared aquaculture seawater in a recirculating water system for an extended period without load, or by introducing only a very small number of fish fry. The system relies on the slow proliferation and enrichment of a small number of naturally occurring microorganisms to eventually develop nitrification capacity. This method is simple to operate and requires no additional investment. However, its core drawback is that the start-up period is extremely long, usually requiring several weeks or even more than two months to establish an effective nitrification function. During this process, the system cannot support normal stocking densities, and the water quality (ammonia nitrogen and nitrite concentrations) fluctuates drastically, making the effect uncontrollable and seriously affecting the planning and economic efficiency of aquaculture production. Exogenous bacterial agent inoculation method: To shorten the start-up time, a common practice is to directly add commercial nitrifying bacteria agents to the system. Although this method is simple to operate, it has significant problems: First, many commercial agents are freshwater or euryhaline strains, which have poor activity, adaptability and colonization ability in the high salinity environment of seawater. They are easy to be inactivated or cannot effectively attach to biological filter media after addition. Second, the composition and activity of different brands of agents vary greatly, and the effect is unstable, which may lead to the disorder of the system's microbial community structure. Finally, long-term reliance on exogenous bacterial agents also increases the aquaculture cost. Independent bioreactor cultivation method: This scheme attempts to pre-cultivate high concentrations of nitrifying bacteria in an independent bioreactor outside the system. After they mature, they are then introduced into the main circulation system. This method aims to quickly provide a large amount of bacteria source, but its disadvantages are: it requires the addition of independent reactors, temperature control and aeration equipment, etc., resulting in high investment and operating costs. More importantly, the bacteria cultured under optimized conditions often face the problem of "acclimatization" after being transferred to the actual aquaculture system. The bacterial community structure will change drastically, the pre-cultivation effect will be greatly reduced, and the system will still need a long time to re-stabilize. Therefore, there is an urgent need to develop a biofilter cultivation method for recirculating aquaculture systems that is fast to start up, has stable microbial colonization, is easy to operate, and has controllable costs, in order to overcome the shortcomings of existing technologies. Summary of the Invention

[0003] The purpose of this invention is to provide a method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system, in order to solve the problems mentioned in the background art, such as long start-up period, low and unstable colonization efficiency of bacteria, high water quality safety risk, and complex or high cost of operation.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system, comprising the following steps: Step S1, System Preparation and Ammonia Oxidizing Bacteria Enrichment: Inject aquaculture water into the aquaculture system and introduce aquaculture organisms, start the system circulation and continuously oxygenate the biological filter, so that the ammonia oxidizing bacteria inherent in the system can be activated and reproduced using the ammonia nitrogen produced by the metabolism of aquaculture organisms. Step S2, oxygen deprivation regulation to inhibit ammonia-oxidizing bacteria and create a growth window for nitrite-oxidizing bacteria: When the ammonia nitrogen concentration in the system drops to a preset threshold and the nitrite concentration accumulates to its peak, oxygenation of the biofilter is stopped to maintain water circulation and create an anaerobic environment to inhibit the activity of ammonia-oxidizing bacteria. Step S3: Restart oxygenation to promote the establishment of nitrite-oxidizing bacteria dominance: After the preset time of oxygenation shutdown, restart the oxygenation of the biofilter to enable the inherent nitrite-oxidizing bacteria in the system to rapidly proliferate using the accumulated nitrite. Step S4, Stable System Operation: Adjust water quality by changing water and gradually increase the amount of feed for aquaculture until the system's nitrification function is stable, and then switch to regular aquaculture.

[0005] Preferably, in step S1, the initial density of the cultured organisms is 10% to 30% of the planned final culture density.

[0006] By adopting the above technical solution, sufficient ammonia nitrogen substrate can be provided in the early stage of bacterial cultivation to stimulate the reproduction of ammonia-oxidizing bacteria, while controlling the biological load of aquaculture within a safe range, effectively balancing rapid bacterial cultivation with aquaculture risks.

[0007] Preferably, in step S1, when the biofilter is continuously aerated, its dissolved oxygen concentration is controlled to be greater than 5 mg / L.

[0008] The above technical solution creates an optimal growth and metabolic environment for strictly aerobic ammonia-oxidizing bacteria, ensuring that they can quickly and efficiently convert ammonia nitrogen into nitrite, completing the first step of the nitrification process.

[0009] Preferably, in step S2, the preset threshold is that the ammonia nitrogen concentration is consistently below 0.1 mg / L.

[0010] The above technical solution provides a clear and operable trigger point, indicating that the ammonia-oxidizing bacteria have basically completed the initial ammonia nitrogen conversion task. At this time, the accumulated nitrite concentration has also reached a peak suitable for subsequent regulation, ensuring the accuracy of time-series regulation.

[0011] Preferably, in step S2, the preset duration of oxygen cessation is 24 to 72 hours.

[0012] Using the above technical solution, the duration is sufficient to significantly inhibit the activity and competitive advantage of strictly aerobic ammonia-oxidizing bacteria, creating a growth stagnation window for them, while avoiding the complete collapse of biofilm function due to prolonged hypoxia, thus balancing the inhibitory effect and system safety.

[0013] Preferably, in step S4, the concentration of nitrite in the water is controlled to drop below 0.15 mg / L by diluting the water by changing 5% to 10% of the water daily.

[0014] By adopting the above technical solution, the concentration of nitrate produced by nitrite-oxidizing bacteria can be gradually and steadily reduced, preventing its excessive accumulation, while maintaining the ion balance of the water body, thus preparing the water quality for the system to enter a stable aquaculture period.

[0015] Preferably, in step S4, when gradually increasing the amount of feed for aquaculture, the increase is 10% to 15% of the current amount each time, and the system remains stable for 2 to 3 days after each increase.

[0016] By adopting the above technical solution and employing a gradual enhancement strategy, the nitrification capacity of the biofilter can be matched with the gradually increasing pollution load of aquaculture, which is conducive to the consolidation and adaptation of the microbial community, thereby cultivating a robust and stable microbial community that can cope with load fluctuations.

[0017] Preferably, in step S1, after the cultured organisms are introduced, they are initially fed at a rate of 0.5% to 1.0% of their body weight.

[0018] The above technical solution can not only meet the basic nutritional needs of farmed organisms and ensure their health, but also produce stable and appropriate amounts of organic matter and ammonia nitrogen, providing a continuous and controllable "nutrient source" for the enrichment of functional microbial communities.

[0019] Preferably, the aquaculture water is seawater, and the aquaculture organisms are marine fish.

[0020] By employing the above technical solution, it is clear that the method of this invention is particularly suitable for high-salinity marine aquaculture environments. This method activates the inherent, adapted indigenous bacterial communities within the system, overcoming the problems of poor activity and colonization difficulties of exogenous freshwater bacterial agents in marine systems. It is more targeted and has a higher success rate.

[0021] Compared with the prior art, the beneficial effects of the present invention are: a method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system. 1. This invention enables the rapid start-up of the biofilter bed and the synchronization of aquaculture production, greatly improving the utilization rate of the facility. This invention completely changes the traditional two-stage model of "cultivating bacteria first and then releasing fish". By simultaneously releasing low-density aquaculture organisms after the system is filled with water, the bacteria cultivation process is fully integrated into the early stage of aquaculture. This allows the biofilter bed to reach a stable operating state within 25-30 days after the start of aquaculture, significantly shortening the system idle period. 2. Through active temporal regulation, the orderly succession of nitrifying bacteria is precisely and efficiently guided. The core regulation strategy of this invention, "promoting AOB → inhibiting AOB → promoting NOB", uses the deoxygenation operation at key stages to artificially create an ecological window conducive to the growth of nitrite oxidizing bacteria (NOB). This method overcomes the lag or imbalance caused by bacterial competition in natural succession, can quickly and reliably establish complete nitrification function, and shortens and controls the "nitrite peak" period of the system within a certain range, significantly reducing the risk of poisoning of farmed organisms and improving the success rate and predictability of system startup. 3. It relies entirely on and activates the system's native microbial community, making it simple to operate, low in cost, and ecologically safe. The entire startup process does not rely on any exogenous commercial microbial agents. It only activates and guides the inherent microbial community within the system by regulating basic operating parameters such as dissolved oxygen and water flow. This not only saves on microbial agent costs and avoids the problems of poor adaptability and unstable effects of exogenous strains, but also ensures that the cultivated microbial community is highly adapted to local water quality conditions, has more stable colonization, eliminates the safety risks of introducing exogenous organisms, and is more environmentally friendly. 4. Enhanced long-term stability and resistance to shock loads: In the later stages of startup, this invention adopts a "gradually increasing feeding amount" enhancement method to match the nitrification capacity of the biofilter with the aquaculture load and gradually improve it. During this process, the microbial community structure is consolidated and strengthened, forming a more robust microbial community that matches the aquaculture load. This enables the system to better cope with water quality shocks caused by feeding fluctuations and disease treatments during subsequent normal aquaculture, and maintain long-term water quality stability. 5. Energy saving and consumption reduction, easy to integrate and implement in existing systems. The core of the control method of this invention is only the start and stop of aeration in the biological filter and routine water exchange operations, which are the most basic functions of the recirculating aquaculture system. No special or expensive equipment needs to be added. The critical aeration period is short, and the survival needs of fish are guaranteed by maintaining the main circulation of the system and aeration in the aquaculture pond. The overall energy consumption is low. The method is simple, highly operable, and easy to promote and apply in various existing or newly built recirculating aquaculture systems. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the process for organizing the present invention; Figure 2 The composition of the system strains cultured using traditional methods; Figure 3 The composition of the system strains cultured by the method of the present invention. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Please see Figures 1-3 This invention provides a technical solution: a method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system.

[0025] Example: This example is implemented in an actual marine grouper recirculating aquaculture system with a total water volume of 100 cubic meters. The system is equipped with standard units such as physical filtration (roller microfilter), biological filter (moving bed biofilm reactor, MBBR), protein separator, and ultraviolet sterilizer.

[0026] The implementation steps are as follows: Step 1: System preparation and AOB (ammonia oxidizing bacteria) enrichment; After thoroughly cleaning and disinfecting the aquaculture system (including pipes, aquaculture ponds, and biofilters) with potassium permanganate solution, drain it.

[0027] Fresh seawater, filtered and conditioned (to 26°C), with a salinity of 30‰, is injected.

[0028] Simultaneous stocking of fish fry: Healthy pearl grouper fry with an average weight of 50 grams are directly stocked into the breeding pond. The initial stocking density is 20% of the planned final breeding density (25 kg / m³), that is, the total stocking amount is about 500 kg. This step breaks through the traditional model of "cultivating bacteria first and then releasing fish", and uses the fish fry's metabolic products directly as the substrate for cultivating bacteria.

[0029] The entire system is circulated, and the water pump runs 24 hours a day.

[0030] Start-up microbial cultivation: Continuously and strongly aerate the biological filter to maintain the dissolved oxygen concentration at 6.0-7.0 mg / L. Begin feeding the fish daily at 0.8% of their total body weight (high-quality formulated feed). This stage aims to rapidly enrich the ammonia-oxidizing bacteria (AOB) inherent in the system, relying entirely on the native microbial community without adding any exogenous microbial agents.

[0031] Water quality is monitored daily, with a focus on ammonia nitrogen (NH3-N) and nitrite (NO2⁻-N) concentrations. During this stage, ammonia nitrogen is produced by the metabolic activities of fish, and the inherent AOB in the system is activated and multiplies rapidly, oxidizing ammonia nitrogen into nitrite. The goal is to promote a continuous decrease in ammonia nitrogen concentration and stabilize it at a level close to 0 mg / L.

[0032] Step 2: Oxygen deprivation regulation to inhibit AOB and create a growth window for NOB (nitrite oxidizing bacteria). Triggering conditions: When the system ammonia nitrogen concentration is consistently below 0.1 mg / L for three consecutive days, and the nitrite concentration rises and reaches a peak (5.2 mg / L in this example), the critical operation is executed.

[0033] Perform oxygen deprivation: Immediately stop the blowers supplying air to the biofilter, but keep the water circulation pump running continuously to ensure water flow and dissolved oxygen in the aquaculture pond. This operation creates an anoxic / anaerobic microenvironment in the biofilm carrier area within the biofilter. This operation is the core step of the invention and aims to artificially create ecological selection pressure.

[0034] Duration of oxygen deprivation: The oxygen was deprived for 48 hours. During this period, the activity of strictly aerobic AOB was significantly inhibited, and its competitive advantage decreased. Meanwhile, high concentrations of nitrite accumulated in the water, creating a non-competitive and substrate-rich growth window for NOB.

[0035] Step 3: Restart oxygenation to promote NOB dominance. After the 48-hour aeration period ended, the blower of the biological filter was immediately restarted to restore strong aeration, and the dissolved oxygen quickly recovered to above 6.0 mg / L.

[0036] At this point, due to the phased inhibition of AOB, a large amount of nitrite exists, providing sufficient substrate and ecological niche for the inherent nitrite-oxidizing bacteria (NOB) in the system. NOB begins to proliferate rapidly, oxidizing nitrite to nitrate (NO3⁻-N).

[0037] Monitoring showed that nitrite concentrations began to decline significantly on the 5th day after oxygenation was restored.

[0038] Step 4: Fine-tuning of water exchange and system stabilization After the nitrite concentration begins to decline, a daily water exchange operation is initiated, with a daily water exchange volume of 8% of the total water volume in the system, in order to gradually dilute the accumulated nitrite and nitrate in the water.

[0039] Through water exchange and continuous operation of NOB, the nitrite concentration was steadily reduced to a safe level below 0.1 mg / L.

[0040] System enhancement: Once the nitrite concentration is stably below 0.15 mg / L, gradually increase the daily feeding amount, with each increase being approximately 12% of the current feeding amount. After each increase, observe the system for 3 days to ensure that there are no abnormal fluctuations in water quality (ammonia nitrogen, nitrite) before proceeding with the next increase. This gradual enhancement process helps cultivate a robust microbial community that matches the aquaculture load.

[0041] Through a gradual increase in feeding over approximately 20 days, the system's feeding amount was increased to 2.0% of the fish's total body weight, while the system's water quality (ammonia nitrogen <0.05 mg / L, nitrite <0.1 mg / L) remained stable throughout.

[0042] Step 5: Normal breeding operation When the system can maintain the concentrations of ammonia nitrogen and nitrite at extremely low levels (both below 0.1 mg / L) for a long period (more than a week) under the target feeding amount, the biofilter bed is considered to be fully mature and stable, and then it can be transferred to the regular aquaculture management process.

[0043] Implementation effect verification and comparative analysis: Start-up efficiency: From the start of seedling introduction to the system's ability to stably handle the target load, the total time is about 28 days. In contrast, the start-up cycle of the system using the traditional natural acclimatization method usually takes 50-60 days or even longer. The method of this invention shortens the start-up time by more than 50%.

[0044] Water quality safety and aquaculture performance: Throughout the start-up process, through active regulation of "promoting AOB → inhibiting AOB → promoting NOB", the peak periods of ammonia nitrogen and nitrite were effectively controlled and shortened, without causing visible toxicity or stress to the grouper fry, and the survival rate was as high as 98.5%.

[0045] Microbial community structure optimization: Microbiome analysis was performed on stable systems cultured using the method of this invention, and the results were compared with those of systems cultured using conventional methods. The results were significant. Regarding the control of harmful bacteria: In the system cultured by the method of this invention, harmful bacteria genera such as Vibrio and Photobacterium did not become the dominant bacterial group. Figure 2 As shown, in systems cultured using traditional methods, Vibrio and Luteobacterium are the relatively dominant genera, and the enrichment of these opportunistic pathogens increases the risk of diseases in aquaculture.

[0046] Regarding beneficial functional bacteria: the system cultured by the method of this invention, such as... Figure 3As shown, the dominant bacterial genera are Neptunomonas, Rhodovulum, and Marinacella. Among them, Rhodovulum participates in ammonia nitrogen degradation, while Neptunomonas has nitrogen fixation capabilities. The bacterial community structure is significantly biased towards water purification functions.

[0047] Regarding community stability: In the systems cultured using the method of this invention, the "Others" group (low-abundance unclassified microorganisms) accounts for over 80%, indicating extremely high community diversity, a complex yet stable ecosystem, and strong resistance to disturbance. Figure 2 As shown, while traditional schemes also mention that the "Others" group is the basis of diversity, they do not provide specific data, and its overall community is more affected by potential pathogens.

[0048] Nutritional type: Both methods produce systems dominated by heterotrophic microorganisms, but the method of this invention has directionally cultivated a more functional heterotrophic microbial community.

[0049] Cost and operability: This invention does not use any commercial bacterial agents throughout the process. It is achieved only through conventional operations such as controlling the start and stop of aeration and water changes. It is simple to operate, low in cost, and the cultivated bacterial community is a native bacteria that is completely adapted to the water quality of this system, resulting in more stable colonization.

[0050] Conclusion: This embodiment, combined with comparative experimental data, fully demonstrates that the "time-sequence controlled oxygenation-de-oxygenation regulation" method of the present invention can not only significantly shorten the system start-up cycle from the traditional 50-60 days to about 28 days, achieving synchronization of bacterial cultivation and aquaculture, but more importantly, it can directionally create a healthy microbial ecosystem with clearly defined beneficial functional bacteria, high diversity, strong stability, and extremely low risk of potential pathogens. This is in stark contrast to the result of traditional methods that easily lead to harmful bacteria becoming the dominant bacterial population, fundamentally improving the biosafety, operational efficiency, and success rate of recirculating aquaculture systems.

[0051] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system, characterized in that: Includes the following steps: Step S1, System Preparation and Ammonia Oxidizing Bacteria Enrichment: Inject aquaculture water into the aquaculture system and introduce aquaculture organisms, start the system circulation and continuously oxygenate the biological filter, so that the ammonia oxidizing bacteria inherent in the system can be activated and reproduced using the ammonia nitrogen produced by the metabolism of aquaculture organisms. Step S2, oxygen deprivation regulation to inhibit ammonia-oxidizing bacteria and create a growth window for nitrite-oxidizing bacteria: When the ammonia nitrogen concentration in the system drops to a preset threshold and the nitrite concentration accumulates to its peak, oxygenation of the biofilter is stopped to maintain water circulation and create an anaerobic environment to inhibit the activity of ammonia-oxidizing bacteria. Step S3: Restart oxygenation to promote the establishment of nitrite-oxidizing bacteria dominance: After the preset time of oxygenation shutdown, restart the oxygenation of the biofilter to enable the inherent nitrite-oxidizing bacteria in the system to rapidly proliferate using the accumulated nitrite. Step S4, Stable System Operation: Adjust water quality by changing water and gradually increase the amount of feed for aquaculture until the system's nitrification function is stable, and then switch to regular aquaculture.

2. The method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system according to claim 1, characterized in that: In step S1, the initial density of the cultured organisms is 10% to 30% of the planned final culture density.

3. The method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system according to claim 1, characterized in that: In step S1, when the biofilter is continuously oxygenated, its dissolved oxygen concentration is controlled to be greater than 5 mg / L.

4. The method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system according to claim 1, characterized in that: In step S2, the preset threshold is that the ammonia nitrogen concentration is consistently below 0.1 mg / L.

5. The method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system according to claim 1, characterized in that: In step S2, the preset duration of oxygen cessation is 24 to 72 hours.

6. The method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system according to claim 1, characterized in that: In step S4, the concentration of nitrite in the water is controlled to drop below 0.15 mg / L by diluting the water by changing 5% to 10% of the water daily.

7. The method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system according to claim 1, characterized in that: In step S4, when gradually increasing the amount of feed for aquaculture, each increase is 10% to 15% of the current amount of feed, and the operation is stable for 2 to 3 days after each increase.

8. The method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system according to claim 1, characterized in that: In step S1, after the cultured organisms are released, they are initially fed at a rate of 0.5% to 1.0% of their body weight.

9. The method for rapidly cultivating stable nitrifying bacteria in a land-based circulating water system according to claim 1, characterized in that: The aquaculture water is seawater, and the aquaculture organisms are marine fish.

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