A mangrove sediment mediated method for treating effluent from seawater culture

Abstract

A mangrove sediment-mediated seawater aquaculture tail water treatment method, comprising the following steps: pretreating seawater aquaculture tail water to remove large-particle impurities in the seawater aquaculture tail water; then introducing into an SBR tank for heterotrophic nitrification treatment; before the heterotrophic nitrification treatment, introducing mangrove sediment into the SBR tank and domesticating to enrich AOB bacteria; then introducing into an upflow denitrification filter for aerobic denitrification treatment, before the aerobic denitrification treatment, introducing mangrove sediment into the upflow denitrification filter and domesticating to enrich nirS type denitrifying bacteria; finally, disinfecting to remove pathogens in the seawater aquaculture tail water. The present application is based on a sequencing batch reactor and an upflow denitrification filter with domesticated mangrove sediment as activated sludge, and constructs a set of seawater aquaculture tail water treatment process, which can realize efficient removal of NH4 + -N, NO3 ‑ -N, COD and the like, and can maintain long-time operation.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, and in particular to a mangrove sediment-mediated SBR method for treating marine aquaculture tailwater. Background Technology

[0002] The booming development of mariculture has been accompanied by increasingly prominent environmental problems, one of which is the significant eutrophication of aquaculture waters. Surveys show that excessive levels of inorganic nitrogen and reactive phosphate are common in many important natural fishery waters, including oceans and rivers. The widespread use of antibiotics in aquaculture also poses potential threats to aquatic organisms and humans. In pursuit of superior water quality, large amounts of freshwater are extracted to maintain pond levels, while aquaculture wastewater is discharged into surrounding waters, leading to water quality deterioration, damage to sensitive aquatic environments such as wetlands and mangroves, and threats to the health of other aquatic animals. Mariculture wastewater refers to water generated by mariculture activities and discharged into receiving sea areas. This water contains a variety of substances, with key water quality indicators including pH, suspended solids (TSP), chemical oxygen demand (COD), reactive phosphate, inorganic nitrogen (nitrite nitrogen, ammonia nitrogen, nitrate nitrogen), and heavy metals. Due to its high salinity and poor biodegradability, mariculture wastewater is difficult to treat, and a mature mariculture wastewater treatment model is currently lacking. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a method for treating marine aquaculture tailwater mediated by mangrove sediments.

[0004] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0005] A method for treating marine aquaculture wastewater mediated by mangrove sediments includes the following steps:

[0006] S1: Pre-treat the wastewater from marine aquaculture to remove large particulate impurities.

[0007] S2: The pretreated marine aquaculture tailwater is introduced into the SBR tank for heterotrophic nitrification treatment, with an aeration rate of 1.0-1.5 L / min; before heterotrophic nitrification treatment, mangrove sediment is introduced into the SBR tank and the mangrove sediment is acclimated to enrich AOB bacteria.

[0008] S3: The seawater aquaculture tailwater treated by heterotrophic nitrification is introduced into an upflow denitrification filter for aerobic denitrification treatment. Before aerobic denitrification treatment, mangrove sediment is introduced into the upflow denitrification filter and the mangrove sediment is acclimated to enrich nirS type denitrifying bacteria.

[0009] S4: Disinfect the effluent from aquaculture that has undergone aerobic denitrification treatment to remove pathogens from the effluent.

[0010] Preferably, the process of acclimatizing mangrove sediments in the SBR tank is as follows:

[0011] First, a phased increase in simulated seawater NH4 was adopted. + The -N concentration method was used to acclimatize mangrove sediments in SBR tanks to ammonia nitrogen tolerance. The initial NH4 content in the SBR tanks simulated seawater. + The -N concentration is 15-25 mg / L, and the NH4+ in the SBR tank is used to simulate the NH4+ in seawater. + -N concentration was 100-120 mg / L; then, the mangrove sediments in the SBR tank were subjected to the first salt tolerance acclimatization by gradually increasing the salinity of the simulated seawater. The initial salinity of the simulated seawater in the SBR tank was 0.8%-1.2%, and the salinity of the simulated seawater in the SBR tank was 3%-3.5%.

[0012] Preferably, the method of using staged increases in simulated seawater NH4 + The method of -N concentration for acclimation of mangrove sediments in SBR tanks to ammonia nitrogen tolerance specifically includes:

[0013] Simulated seawater with an ammonia nitrogen concentration of 15-25 mg / L was used as the culture medium for the first stage of ammonia nitrogen tolerance acclimatization. In subsequent stages, the ammonia nitrogen concentration of the simulated seawater increased by 8-12 mg / L. The simulated seawater from each stage of ammonia nitrogen tolerance acclimatization was sequentially introduced into an SBR tank, with a hydraulic retention time of 38-48 h. The NH4+ content of the simulated seawater after each stage of ammonia nitrogen tolerance acclimatization was measured. + -N concentration, up to NH4 + When the -N removal rate stabilizes at 70-80%, the next stage of ammonia nitrogen tolerance acclimatization begins; during the ammonia nitrogen tolerance acclimatization process, continuous aeration is used, with an aeration rate of 0.5-1.5 L / min.

[0014] Preferably, the method of gradually increasing the salinity of simulated seawater to perform the first salt tolerance acclimatization on mangrove sediments in the SBR tank specifically includes:

[0015] With a salinity of 0.8%-1.2% and NH4+ + Simulated seawater with a nitrogen concentration of 60-90 mg / L was used as the culture medium for the first stage of the first salt tolerance acclimatization. In subsequent stages of the first salt tolerance acclimatization, the salinity of the simulated seawater increased sequentially by 0.4%-0.6%, while the ammonia nitrogen concentration remained constant. The simulated seawater from each stage of the first salt tolerance acclimatization was introduced into the SBR tank reaction vessel in sequence, with the hydraulic retention time controlled at 20-24 hours until NH4+ concentration reached a certain level. +When the -N removal rate is stable at over 80%, the next stage of cultivation begins; during the first salt tolerance acclimatization process, continuous aeration is used, with an aeration rate of 0.2-0.5 L / min and a stirring intensity of 30-60 r / min.

[0016] Preferably, the process of acclimating mangrove sediments in the upflow denitrification filter is as follows: firstly, the simulated seawater NO3 is increased in stages. - The method of NO3- concentration was used to acclimate mangrove sediments to nitrate in an upflow denitrification filter, simulating the NO3 concentration in seawater in the upflow denitrification filter. - -N concentration was 220-280 mg / L; then, the mangrove sediments in the upflow denitrification filter were subjected to a second salt tolerance acclimatization by gradually increasing the salinity of the simulated seawater until the salinity of the simulated seawater in the upflow denitrification filter was 3%-3.5%.

[0017] Preferably, the method of using staged increases in simulated seawater NO3 - The method of N concentration for nitrate tolerance acclimation of mangrove sediments in upflow denitrification filters specifically includes:

[0018] With NO3 - Simulated seawater with an NO3- concentration of 50-80 mg / L was used as the culture medium for the first stage of nitrate tolerance acclimatization. Subsequent stages of nitrate tolerance acclimatization involved using simulated seawater with NO3- concentrations... - The NO3- concentration was increased sequentially by 45-55 mg / L. Simulated seawater at each stage of nitrate tolerance acclimation was introduced into an upflow denitrification filter, with the hydraulic retention time controlled at 48-56 hours, until NO3- concentration reached the target level. - When the -N removal rate is stable at over 80%, the next stage of nitrate tolerance acclimatization begins; the nitrate tolerance acclimatization process uses a suffocating cultivation method.

[0019] Preferably, the method of gradually increasing the simulated seawater salinity to conduct a second salt tolerance acclimatization of mangrove sediments in an upflow denitrification filter specifically includes:

[0020] With a salinity of 0.8%-1.2% and NO3- - Simulated seawater with a NO3- concentration of 220-280 mg / L was used as the culture medium for the first stage of the second salt tolerance acclimatization. In subsequent stages of the second salt tolerance acclimatization, the salinity of the simulated seawater increased sequentially by 0.4%-0.6%. - With NO3- concentration kept constant, simulated seawater from each stage of the second salt tolerance acclimatization process is sequentially introduced into an upflow denitrification filter, controlling the hydraulic retention time to be 12–20 hours, until NO3- concentration is maintained. - When the -N removal rate is stable at over 80%, the next stage of cultivation begins; the second salt tolerance acclimatization process uses a suffocating cultivation method.

[0021] Preferably, in the SBR tank, the amount of mangrove sediment added is 70%-80% of the SBR tank volume; in the upflow denitrification filter, the amount of mangrove sediment added is 70%-80% of the upflow denitrification filter volume.

[0022] Preferably, the disinfection process specifically includes: introducing the aerobic denitrification treated seawater aquaculture wastewater into a disinfection tank, turning on the chlorine dioxide generator in the disinfection tank, and the retention time of the seawater aquaculture wastewater in the disinfection tank is 5-10 hours.

[0023] Preferably, the pretreatment specifically includes: subjecting the marine aquaculture wastewater to coarse filtration, homogenization, boosting, and fine filtration in sequence.

[0024] Compared with the prior art, the advantages of the present invention are as follows:

[0025] This invention is based on the Sequencing Batch Reactor Activated Sludge Process (SBR) and designs an acclimation tank reactor using mangrove sediment as activated sludge. Through a specially designed acclimation process and optimized process parameters, ammonia-oxidizing bacteria (AOB) in the mangrove sediment are enriched to treat excess nitrogen in pretreated marine aquaculture effluent, converting it into nitrates and nitrites. An upflow denitrification filter is the main nitrate nitrogen treatment process. Mangrove sediment is added to the upflow denitrification filter and undergoes a special gradient acclimation process to enrich the nirS-type denitrifying bacteria in the mangrove sediment. This bacteria can efficiently convert harmful environmental substances in aquaculture effluent—nitrates and nitrites—into harmless nitrogen gas, thus significantly reducing the adverse impact on the surrounding marine ecosystem and protecting the aquatic environment. Practice shows that this invention can achieve NH4+ treatment. + -N, NO3 - It effectively removes nitrogen, COD, and other pollutants, and can maintain long-term operation. Attached Figure Description

[0026] Figure 1 This is a process flow diagram of a mangrove sediment-mediated mariculture wastewater treatment method according to an embodiment of the present invention.

[0027] Figure 2 This is a phylogenetic tree of AOB microbiota based on major OTUs.

[0028] Figure 3 Phylogenetic tree of the nirS gene of denitrifying bacteria from the bottom sediments of a domesticated upflow denitrifying filter. Detailed Implementation

[0029] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0030] Example 1

[0031] The initial simulated seawater composition of this embodiment is shown in Table 1:

[0032] Table 1 Initial simulated seawater ratio

[0033] <![CDATA[NaHCO3]]> 1.25 g <![CDATA[MgSO4·7H2O]]> 0.3 g <![CDATA[Anhydrous CaCl2·H2O]]> 180 mg <![CDATA[KH2PO4]]> 27 mg <![CDATA[(NH4)2SO4]]> 20 mg (initial dose, with subsequent doses increased in gradients) <![CDATA[NaNO2]]> 9.1 mg <![CDATA[KNO3]]> 50 mg / L (initial dose, with subsequent doses increased in a gradient) Fe-EDTA solution <![CDATA[1 ml (solution composition mg·L -1 : EDTA: 5000; FeSO4•7H2O 5000)]]> Trace element solution <![CDATA[1 ml (solution composition mg·L -1 : ZnSO4•7H2O: 430; CoCl2•6H2O: 240; MnCl2•4H2O: 990; HBO3: 314; CuSO4•5H2O: 250; NiCl2•6H2O: 190)]]>

[0034] like Figure 1 As shown, this embodiment provides a method for treating marine aquaculture wastewater mediated by mangrove sediments, including the following steps:

[0035] 1) Pre-treat the effluent from marine aquaculture to remove large particulate impurities.

[0036] In this embodiment, the pretreatment specifically includes: passing the marine aquaculture wastewater through coarse filtration, homogenization, boosting, and fine filtration in sequence.

[0037] 1.1) Coarse filtration

[0038] Coarse screens are selected for the coarse filtration stage. A screen is a pretreatment device that protects subsequent wastewater treatment equipment from clogging. Its main function is to intercept and remove particles and substances in the wastewater that may clog pumps, pipes, valves, and other subsequent treatment equipment, reducing treatment efficiency. For wastewater treatment plants employing treatment processes with specific requirements for influent water quality, such as AAO, oxidation ditch, SBR, and their improved processes, the screen system plays a crucial role and is one of the key steps ensuring the normal operation of the entire wastewater treatment system. Screens are mainly classified according to their filtration precision, i.e., the spacing between the screen bars, and can be divided into three categories: coarse screens, fine screens, and micro screens. This design selects coarse and fine screens.

[0039] Coarse screens effectively remove larger solids from water, such as tree branches and leaves, plastic products, and paper, ensuring the proper functioning of subsequent wastewater treatment equipment. Coarse screens require cleaning and maintenance to ensure proper operation and filtration efficiency. Typically, when mechanical cleaning is used, the filtration precision is selected as 16-25 mm; while when manual cleaning is used, the filtration precision is usually 25-40 mm.

[0040] 1.2) Homogeneous water quality

[0041] To balance water quality and quantity, a buffer zone—an equalization tank—is incorporated into the wastewater treatment system for pretreatment. This homogenizes the incoming wastewater, improving the system's resilience to shock loads, reducing design size, and lowering costs. Furthermore, if other equipment in the process suspends wastewater discharge, the equalization tank ensures a continuous supply, guaranteeing the normal operation of the entire system.

[0042] 1.3) Improvement

[0043] The lifting is primarily achieved by a lift pump station. As is known in conventional wastewater treatment processes, the lift pump station is a crucial power source for wastewater treatment. The main function of the lift pump is to stably and efficiently lift wastewater from lower structures to higher or more distant structures, while also promoting wastewater mixing for subsequent treatment and discharge. In the SBR process, due to the fixed sequence of treatment processes, the operation of the lift pump must be coordinated with these stages. Introducing a PLC automatic control system ensures that the lift pump operates under the correct and appropriate time and conditions, maximizing the process's effectiveness. In this design, the lift pump station is constructed together with the equalization tank.

[0044] 1.4) Fine filtration

[0045] Fine filtration is achieved by fine screens. The function of fine screens is to remove smaller floating debris and small particulate matter missed by the coarse screen, building upon the coarse screen's filtering of larger particles. This reduces the operating load on subsequent wastewater treatment equipment and improves the overall efficiency of the wastewater treatment process. Their filtration precision is typically 2-15 mm. Although there are similarities in control methods between fine and coarse screens, fine screens require higher operational precision. Operators need to make judgments and adjustments based on real-time conditions. Manual cleaning ensures that the fine screen achieves optimal results in filtering small particulate matter and avoids potential misjudgments by the automatic control system. Therefore, manual operation and control are required.

[0046] 2) The pretreated seawater aquaculture wastewater is introduced into the SBR tank for heterotrophic nitrification treatment, with a hydraulic retention time of 30 hours.

[0047] In this embodiment, the acclimatization tank device includes an acclimatization tank, an aeration mechanism, a stirring mechanism, a temperature detection instrument, and a pH detection instrument located inside the acclimatization tank. It uses a PLC controller as the core and control unit, connecting monitoring equipment such as the temperature detection instrument, pH detection instrument, and dissolved oxygen meter, as well as execution equipment such as the aeration mechanism and stirring mechanism.

[0048] Specifically, this embodiment adopts a continuous aeration culture method with an aeration rate of 1.0-1.5 L / min, using aeration sand heads as aerators, and using an aeration pump connected to a rotor flow meter to regulate oxygen supply.

[0049] This embodiment uses mechanical stirring, with the mechanical stirrer rotating at 100 r / min.

[0050] This embodiment uses a double-layer structure of a 100 W electric heating tube and an acclimatization tank, and is equipped with a temperature sensor to maintain a relatively stable temperature inside the tank.

[0051] In this embodiment, an alkaline solution tank is connected to a constant flow pump and equipped with a pH meter to maintain the relative stability of the acidity and alkalinity of the solution in the tank.

[0052] In this embodiment, a sludge activation tank is connected to an SBR acclimatization tank. The sludge activation tank is used to recover part of the sludge from the acclimatization tank, with a sludge return ratio of 20% to 50%, and aeration activation is performed, thereby shortening the sludge acclimatization cycle and improving the acclimatization and effluent treatment efficiency.

[0053] Specifically, before heterotrophic nitrification, mangrove sediments are added to the SBR tank and acclimated to enrich AOB bacteria. The amount of mangrove sediments added is 80% of the volume of the SBR tank.

[0054] In this embodiment, the process of collecting mangrove sediments is as follows:

[0055] The study area was selected as the mangrove ecological reserve in Yangjiang City, Guangdong Province (21°45'18"-21°46'1"N, 111°44'26"-111°45'3"E). Sediment samples were collected on-site and packaged in sterile sealed bags, then placed in a 4°C refrigerator.

[0056] Specifically, the mangrove sediments are placed in an SBR acclimatization tank, and the process of acclimatizing the mangrove sediments in the SBR tank is as follows:

[0057] I. Gradually increase ammonia nitrogen levels to acclimate AOB bacteria

[0058] (1) Initial stage: This design is based on the SBR process and the acclimatization tank is designed. The parameters (pH, DO, temperature, etc.) are controlled by a programmable logic controller (PLC). It is divided into five stages: water inlet, aeration, sedimentation, drainage, and standby. Mangrove sediments are filled into the SBR reactor and NH4 is introduced into the water inlet. + Simulated seawater aquaculture wastewater with a -N concentration of 20 mg / L was treated with a hydraulic retention time of 42 h and an initial aeration rate of 0.5 mg / L. The NH4 content of the treated water was then measured. + -N, NO2 - -N, COD, and pH, when NH4 in the effluent + -N removal rate stabilizes at around 80%, running for about 35-35 days, completing the initial stage of SBR acclimatization.

[0059] (2) Ammonia nitrogen loading increase stage: The simulated effluent with the same composition and ratio as the initial stage of seawater aquaculture effluent is the first stage, and the NH4 load is gradually increased. + The NH4+ content was determined by passing simulated seawater aquaculture wastewater into an SBR reactor, controlling the hydraulic retention time at 38–48 h and the aeration rate at 1.0 mg / L. The NH4+ content of the treated water was measured at each stage. + With the -N removal rate stabilizing at around 70%-80%, the next stage of cultivation begins. In this next stage, the NH4+ in the wastewater... + The concentration of -N was 8–12 mg / L higher than in the previous stage; each stage lasted 7–10 days, until the NH4+ concentration in the simulated aquaculture wastewater was reduced. + -N and NO2 - The concentration of -N is 100–120 mg / L, NH4 + -N and NO2 - -N removal rate stabilized at around 70-80%, completing the domestication process of AOB bacteria in mangrove sediments.

[0060] II. Gradually increase salinity to acclimate AOB bacteria

[0061] (1) Initial stage of salinity acclimatization: The salinity is 1%, NH4+ + The initial salinity of the simulated seawater in the inlet tank, with a -N concentration of 60-90 mg / L, is close to the original salinity of the mangrove sediments (around 1%). The SBR tank is started for continuous aeration, with an initial aeration rate of 0.3 L / min and a stirring intensity of 50 r / min, to ensure sufficient contact between the mangrove sediments and the wastewater. The changes in the mangrove sediment sludge are observed to ensure that AOBs can grow and reproduce normally in a low-salinity environment.

[0062] (2) Gradually increase salinity: After the microorganisms adapt to the low salinity environment, gradually increase the salinity of the wastewater in the SBR reactor. Control the salinity at 1% in the first stage, and then increase it by 0.5% each time. The salinity content of each stage increases sequentially. Detect the NH4 content of the water after each stage of treatment. + -N, NO2 - The degradation efficiency of ammonia nitrogen (AOB) in mangrove sediments is monitored, with each stage lasting 10–15 days. Once the data stabilizes, the next stage of cultivation begins. The hydraulic retention time for each stage is 20–24 hours. Ambient oxygen (AOB) degradation efficiency in mangrove sediments is closely monitored to ensure that AOBs can adapt to the new salinity level. The next stage of cultivation begins when the degradation efficiency is consistently maintained above 80%. If the AOB degradation efficiency in mangrove sediments decreases, the rate of salinity increase should be appropriately reduced, and more time should be given for the AOBs to adapt to the salinity change.

[0063] (3) Stabilization phase: After the mangrove sediments AOB have adapted to the current salinity level, the salinity should be maintained for 10-12 days to consolidate the acclimatization results. During this period, the ammonia nitrogen concentration (60-90 mg / L) in the SBR reactor should remain stable to stabilize the ammonia nitrogen treatment capacity of the SBR reactor.

[0064] Genomic DNA extraction from sediments in SBR tanks after domestication

[0065] Following the steps described in the MagicPure® Soil Genomic DNA Kit, take 0.25 g of the domesticated sediment from the domestication tank and extract microbial genomic DNA from the mangrove sediment. The specific operating steps are as follows:

[0066] (1) Place 0.25 g of sediment sample in a Lysis Tube, add 550 μL LB 35S and 25 μL Proteinase K, and vortex on a vortex oscillator for 15 min.

[0067] (2) Add 200 μL Lysis Enhancer, mix well, heat in a 70℃ water bath for 10 min, and centrifuge at 12000×g for 2 min.

[0068] (3) Take 500 μL of the supernatant after centrifugation, transfer it to a brand new 1.5 mL centrifuge tube, add 200 μL of PB 35, incubate on ice for 5 min, and then centrifuge at 12000×g for 3 min.

[0069] (4) Transfer 500 μL of supernatant to a 1.5 mL centrifuge tube, add 500 μL of BB31 and mix well; mix MagneticSoil Beads, add 15 μL of magnetic beads, vortex for 5 min, and place the centrifuge tube on a magnetic rack until the solution is clear.

[0070] (5) Discard the supernatant, add 800 μL CB 31, vortex for 5 min, and place the centrifuge tube on a magnetic rack until the solution is clear.

[0071] (6) Discard the supernatant, add 700 μL of WB 31, vortex for 3 min, and place the centrifuge tube on a magnetic rack until the solution is clear. Repeat this step once.

[0072] (7) While avoiding the magnetic beads, remove the supernatant as much as possible and dry it at room temperature for 8 min to allow the ethanol in the reagents in the previous steps to fully evaporate.

[0073] (8) Add 75 μL of ultrapure water (ddH2O), vortex for 30 s, and incubate in a 65℃ water bath for 5 min. During this period, remove the vortex once every 1.5 min, and vortex a total of 3 times.

[0074] (9) Let the magnetic rack stand to clarify, and dispense the supernatant into two centrifuge tubes. Store 40 μL of one tube in a -80℃ refrigerator, and store the remaining extract in a -20℃ refrigerator for the next experiment.

[0075] (10) Take 1 μL of the supernatant stored at -20℃ and determine the concentration and mass of DNA in the supernatant.

[0076] AOB functional gene PCR amplification

[0077] Using the DNA extracted in 3.2.3 as a template, the functional gene of AOB was amplified by PCR using specific primers amoA-2R (CCCCTCKGSAAAGCCTTCTTC) and amoA-1F (GGGGTTTCTACTGGTGGT).

[0078] The PCR amplification system is shown in Table 2:

[0079] Table 2 PCR reaction system

[0080] 2×Go-Taq PCR Mix 12.5 Positive primer amoA-1F 1 Anti-primer amoA-2R 1 template 1 Ultrapure water 9.5 Total 25

[0081] The optimal PCR amplification conditions for AOB in the mangrove sediment sample were determined as follows: 95℃, 3 min; 95℃, 5 s; 55℃, 30 s; 72℃, 1 min; run for 35 cycles, followed by a 72℃ extension for 10 min, and finally storage at 4℃.

[0082] After PCR amplification of the functional gene, the product was mixed with GV dye and subjected to gel electrophoresis. After running the gel for about 30 minutes, the electrophoretic bands were observed, and then the target gene was recovered and purified.

[0083] Recovery, purification, and high-throughput sequencing of the target gene

[0084] The PCR products from multiple samples were mixed and subjected to gel electrophoresis. Using a THBC-470 blue light gel cutter, the target band was carefully excised while minimizing the removal of DNA-free portions. The gel was then immediately recovered and purified. The procedure can be summarized as follows:

[0085] Transfer the cut gel strip into a centrifuge tube — add Buffer MB to the gel block — heat in a 37°C water bath to dissolve the gel block — let stand at room temperature — centrifuge and discard the filtrate — add Buffer WB and centrifuge, discard the filtrate — repeat the previous step once — transpose and centrifuge — transpose and add 50 μL of Elution Buffer — let stand at room temperature and centrifuge — obtain the eluted DNA.

[0086] The DNA sample is mixed with a loading buffer, and the target gene is detected. The electrophoretic bands are then observed. If the purified product meets the requirements and clearly separated DNA bands are visible, it is sent to Genewiz Biotechnology Co., Ltd. for high-throughput sequencing.

[0087] High-throughput sequencing data analysis

[0088] Using Mothur v.1.40.5 software, the sequences obtained from the raw data returned from the test were processed. The cutoff threshold was set to 0.09, and Alpha diversity and Beta diversity analyses were performed on the samples. Then, the samples were clustered, representative sequences were selected, a phylogenetic tree was constructed, and the classic tree diagram was beautified into a circle tree on the iTOL website.

[0089] Validation Analysis

[0090] AOB phylogenetic tree and community structure characteristics

[0091] A phylogenetic tree of AOB community in mangrove sediments was drawn based on the main OTUs, as shown below. Figure 2As shown: Based on the phylogenetic tree classification, all OTUs can be divided into three clusters. Cluster 1 has the highest sequence count, with 113,386 sequences, accounting for 41.94%. Fourteen OTUs—OTU1, OTU4, OTU36, OTU98, OTU29, OTU67, OTU57, OTU5, OTU17, OTU3, OTU26, OTU2, OTU14, and OTU28—belong to cluster 1. These are related to AOB (MK487377.1) found in the Gulf of Mexico salt marsh sediments and AOB (EU6) found in the San Francisco Bay Estuary. Similar to 51323.1); cluster 2 contained 13788 sequences, accounting for 5.10%, with OTU24, OTU69, OTU19, OTU13, and OTU16 belonging to cluster 2, whose species are yet to be determined; while cluster 3 had the lowest number of sequences, with 11497, accounting for 4.25%, with OTU8, OTU11, and OTU15 belonging to cluster 3, similar to AOB (MW940588.1) found in mangrove sediments in China. Overall, most OTUs in mangrove sediment AOBs clustered with sequences from uncultured strains in the environment, mainly showing close phylogenetic relationships with sequences from the Gulf of Mexico, San Francisco Bay Estuary, and mangrove sediments in China. These clustering data indicate that the sampled mangrove sediments exhibit a relatively rich diversity of AOB types.

[0092] The analysis ultimately determined that the microbial components and their percentage content in the SBR acclimation tank after gradient acclimation were as follows: AOB belonged to the genus *Nitrosomonas* (…). Nitrosomonas ) and Nitrosporioides ( Nitrosospira ): Nitrosomonas genus related ( Nitrosomonas- Related species accounted for 28.7%, and were further divided into three subclades: oligotrophic nitrosomonas / urea nitrosomonas (…). Nitrosomonas oligotropha / ureae) European nitrosomonas ( Nitrosomonas europaea ) and similar to nitrosomonas ( Nitrosomonas-like ). Nitrosomonas- like Branch B contained the majority of AOB in the sample, accounting for 69.3%, and Nitrosporioides ( ). Nitrosospira () accounted for 2.0%.

[0093] In this step, the SBR acclimation tank is the core of the process. Under intermittent aeration, mangrove sediments are fed into the acclimation tank, which integrates functions such as equalization and biodegradation. This enriches AOB with denitrification capabilities, treating excess nitrogen in the effluent from marine aquaculture and converting it into nitrates and nitrites.

[0094] Practice has shown that the SBR process based on mangrove sediment AOB mediated in this step can effectively treat high ammonia nitrogen and high salinity wastewater generated from marine aquaculture, and can provide important technical support for the sustainable development of aquaculture.

[0095] 3) The seawater aquaculture tailwater that has undergone heterotrophic nitrification treatment is introduced into an upflow denitrification filter for aerobic denitrification treatment.

[0096] Denitrification filters are wastewater treatment processes that integrate biological nitrogen removal and filtration. They combine biofilm treatment and physical treatment technologies. The water flow direction in denitrification filters is either upflow or downflow. Upflow denitrification filters have influent from the bottom and effluent from the top, while downflow denitrification filters have the opposite influent and effluent directions. Upflow biological filters can achieve higher filtration rates, require less space, are more resistant to hydraulic load shocks, and allow for higher filter media packing heights.

[0097] The main function of the filter media in a biological filter is as a biological carrier and to intercept suspended solids. The choice of filter media is a crucial factor affecting the treatment efficiency of a denitrification filter. Currently, commonly used filter media include ceramsite and quartz sand. In terms of nitrogen removal efficiency, ceramsite is superior to quartz sand. Ceramsite has a larger specific surface area and higher adsorption capacity than quartz sand, thus allowing for a larger biofilm to adhere and resulting in better nitrogen removal. However, in terms of filtration efficiency, quartz sand is superior to ceramsite. Considering nitrogen removal efficiency, this design selects ceramsite as the filter media for the denitrification filter.

[0098] Specifically, before aerobic denitrification, mangrove sediments are introduced into the upflow denitrification filter and the mangrove sediments are acclimatized to enrich nirS-type denitrifying bacteria.

[0099] Specifically, the process of acclimating mangrove sediments in an upflow denitrification filter is as follows:

[0100] I. The denitrification filter adopts inoculation and biofilm formation to acclimate Nirs type denitrifying bacteria to nitrate tolerance. The acclimatization is divided into two stages:

[0101] (1) The first stage is biological inoculation and initial cultivation. Mangrove soil sediment is introduced into the denitrification filter. The mangrove soil sediment contains denitrifying bacteria. In order to promote the growth and reproduction of denitrifying bacteria, simulated marine aquaculture tailwater is introduced into the filter. - The -N concentration is 60 mg / L, the hydraulic retention time is 36-48 h, the reactor is kept in a low oxygen state and no agitation is performed to allow the denitrifying bacteria to multiply in large quantities. When the denitrifying bacteria multiply to a certain amount, they are acclimatized to make them more adaptable to the high salinity and high ammonia nitrogen wastewater treatment environment.

[0102] (2) The second stage is to increase the nitrate treatment load of the denitrification filter. The community structure of Nirs type denitrifying bacteria is understood by high-throughput sequencing. Then, the nutrient dosage is adjusted, and the potassium nitrate concentration is increased in stages. The concentration of potassium nitrate in the synthetic seawater aquaculture effluent is gradually increased and introduced into the denitrification filter reaction tank. The hydraulic retention time is controlled at 48-56 hours. The NO3 in the water after each stage of treatment is detected. - -N, COD, and pH, to NO3 in marine aquaculture tailwater + The concentration of -N was 220-280 mg / L, followed by NO3 in the effluent from marine aquaculture. + The -N concentration increased by 50 mg / L compared to the previous stage; each stage lasted 15–20 days. Based on the adaptation of Nirs-type denitrifying bacteria in mangrove sediments and the nitrate degradation efficiency (maintained above 80%), it was ensured that Nirs-type denitrifying bacteria in mangrove sediments could efficiently degrade the gradually increasing nitrate concentration, thereby improving the nitrate nitrogen removal efficiency of the denitrification filter.

[0103] II. The denitrification filter employs inoculation with biofilm formation to acclimate Nirs-type denitrifying bacteria to high salinity.

[0104] The main purpose of salinity acclimatization is to improve the adaptability of denitrifying bacteria in high-salt environments and their ability to remove nitrate nitrogen, so as to ensure that the upflow denitrification filter can maintain stable and efficient operation when treating high-salt wastewater.

[0105] (1) Set the initial salt concentration to 1% and NO3- to 1%. - Simulated seawater with a nitrogen concentration of 220-280 mg / L was introduced into an upflow denitrification filter. That is, the filter was operated under the condition of an initial salt concentration of 1%, with low oxygen conditions, no aeration, and no stirring. The growth of denitrifying bacteria and the effect of nitrate removal in the sediment were observed. If the denitrifying bacteria could adapt and stably remove 90% of the nitrate nitrogen, the next stage was carried out.

[0106] (2) Second stage: Gradually increase the salt concentration in the simulated seawater aquaculture tailwater, increasing by 0.5% each time, NO3 - With the -N concentration kept constant, observe for 5-10 days, controlling the hydraulic retention time to 12-20 hours to ensure that denitrifying bacteria can adapt to the new salinity environment. Measure the NO3 content in the water after each stage of treatment. - -N, COD and pH, up to NO3 - When the -N removal rate is stable above 80%, the next stage of cultivation begins; if the removal effect decreases or the growth of denitrifying bacteria is inhibited, the salt concentration is appropriately reduced and observation continues.

[0107] (3) Third stage: When the denitrifying bacteria can adapt to the high salt concentration of 3-4% and stably remove nitrate nitrogen, the NO3 in the effluent is continuously monitored. - The acclimatization process is considered complete when the denitrifying bacteria adapt to the final salt concentration and stably remove nitrate nitrogen, based on the levels of nitrogen (N), COD, pH, and the growth of denitrifying bacteria. At this point, the upflow denitrifying filter can operate stably at higher salt concentrations.

[0108] The system was constructed based on 50 core OTUs selected from the bottom sediments of the aforementioned acclimatized upflow denitrification filter at the OUT level. nirS Gene phylogenetic tree such as Figure 3 As shown, the tree structure is subdivided into 10 different clusters based on sequence clustering. Cluster 1 has the largest number of sequences, totaling 29,303, accounting for 19.88% of the total, while cluster 8 contains the fewest sequences, with only 3,810, accounting for 2.58%. The remaining clusters cover sequences ranging from 3.49% to 16.78%.

[0109] Cluster 1 contains OTU1, OTU7, OTU13, OTU16, OTU27, OTU30, OTU39, OTU48, and OTU49, indicating that they originate from sediments in the Pearl River Estuary. nirS The nirS-type denitrifying bacteria sequences (HQ882414 and HQ882417.1) are closely related. On the other hand, OTU5 and OTU33 are grouped into cluster 3, similar to the aforementioned Pearl River Estuary sequences; cluster 2 includes OTU10, OTU33, and OTU40, which show high similarity to the denitrifying bacteria sequence (KU995374.1) found in native coastal wetlands. Furthermore, cluster 4 comprises OTU2, OTU3, OTU15, and OTU20, matching the known nirS-type denitrifying bacteria sequence (JX002733) in mangrove ecosystems. Cluster 5 includes OTUs 19 through 47, which are highly similar to denitrifying bacteria sequences in various environments, such as potato fields (FJ853966.1), the South China Sea (HQ666561), New England salt marshes (KF896049), and Bahia del Tobal, Mexico (KC614388.1). Cluster 6 includes OTUs 8, 21, 29, 32, and 44, which are similar to those found in oil-bearing salt marsh environments along the Chesapeake Bay and Gulf Coast. nirSThe sequences of denitrifying bacteria (such as KC2934453, DQ676190, and KX388846.1) show a phylogenetic relationship. Members of cluster 7, OTU6, OTU9, OTU22, OTU41, and OTU46, are related to those found in intertidal marshes and river sediments in Fujian. nirS The strains (e.g., KT444053, JX941775) showed high sequence identity. In cluster 8, OTU11 and OTU45 were associated with denitrifying bacteria sequences (KX510628) derived from beach sediments; while cluster 9, including OTU4 and OTU23, was associated with oil-bearing salt marshes along the Gulf Coast. nirS The strain sequence is similar to that of type KX389087. This reveals the presence of similar strains in the mangrove ecosystem. nirS Type denitrifying bacteria exhibit high diversity and wide distribution.

[0110] The final determination of the microbial composition and percentage content of mangrove sediments after acclimation treatment in an upflow denitrification filter was as follows: Proteobacteria 85.22%, Chlorophyta 10.22%, Aquatic Bacteria 3.45%, and other phyla, including Actinobacteria, Planctomycetes, and Bacteroidetes, accounting for 1.11% of the total.

[0111] After the upflow denitrification filter has been acclimated with mangrove sediments, the seawater effluent that has undergone heterotrophic nitrification can be introduced into the upflow denitrification filter acclimated with mangrove sediments for aerobic denitrification. The hydraulic retention time for aerobic denitrification is 25-30 hours, and the aerobic denitrification process adopts a simmering culture method.

[0112] 4) Disinfect the effluent from aquaculture that has undergone aerobic denitrification treatment to remove pathogens from the effluent.

[0113] In this embodiment, the disinfection process specifically includes: introducing the seawater aquaculture tailwater treated by the SBR acclimation process into the disinfection tank, turning on the chlorine dioxide generator in the disinfection tank, and the seawater aquaculture tailwater staying in the disinfection tank for 5-10 hours.

[0114] Chlorine dioxide disinfection technology, after years of development, has proven to be a stable and effective treatment method, widely used in various fields, and favored for its simple operation and management. This technology excels in eliminating bacteria such as E. coli, typhoid bacilli, and tubercle bacilli, as well as viruses, including hepatitis A and B viruses. Furthermore, the economic efficiency of chlorine dioxide disinfection is a major advantage, as it is not only affordable as a disinfectant but also consumes little electricity during operation, thus reducing overall operating costs. In terms of equipment, it also boasts advantages such as simple structure, reliability, durability, and low cost, and has achieved complete domestic production, making maintenance and repair more convenient. Finally, the disinfection characteristics of chlorine dioxide also include a special residual chlorine effect, ensuring that treated water retains its disinfection effect after discharge, effectively preventing secondary pollution.

[0115] The treatment effect of the seawater aquaculture wastewater treated in this embodiment is shown in Table 3. It can be seen that the treatment process in this embodiment can achieve NH4+ treatment. + -N, NO3 - It effectively removes nitrogen, COD, and other pollutants, and can stably and continuously treat marine aquaculture wastewater for about 120-150 days.

[0116] Seawater aquaculture tailwater treatment effect

[0117] <![CDATA[NH4 + -N]]> 20.8~32.2 3.5~8.5 73.6~83.2 Good removal effect <![CDATA[NO2 - -N]]> 9.5~14.5 0.88~1.52 89.5~90.7 Good removal effect <![CDATA[NO3 - -N]]> 105.5~139.8 10.5~13.8 90.1~95.0 Good removal effect COD 91.2~110.5 12.5~13.9 86.3~87.4 Good removal effect pH 7.7~8.2 7.3~7.8 - Stable water quality

[0118] The above description is merely a preferred embodiment of this application and does not constitute any limitation on this application. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any modifications or alterations made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for treating marine aquaculture wastewater mediated by mangrove sediments, characterized in that, Includes the following steps: S1: Pre-treat the wastewater from marine aquaculture to remove large particulate impurities. S2: The pretreated marine aquaculture tailwater is introduced into the SBR tank for heterotrophic nitrification treatment, with an aeration rate of 1.0-1.5 L / min; before heterotrophic nitrification treatment, mangrove sediment is introduced into the SBR tank and the mangrove sediment is acclimated to enrich AOB bacteria. S3: The seawater aquaculture tailwater treated by heterotrophic nitrification is introduced into an upflow denitrification filter for aerobic denitrification treatment. Before aerobic denitrification treatment, mangrove sediment is introduced into the upflow denitrification filter and the mangrove sediment is acclimated to enrich nirS type denitrifying bacteria. S4: Disinfect the effluent from aquaculture that has undergone aerobic denitrification treatment to remove pathogens from the effluent. The process of acclimatizing mangrove sediments in the SBR tank is as follows: First, a phased increase in simulated seawater NH4 was adopted. + The -N concentration method was used to acclimatize mangrove sediments in SBR tanks to ammonia nitrogen tolerance. The initial NH4 content in the SBR tanks simulated seawater. + The -N concentration is 15-25 mg / L, and the NH4+ in the SBR tank is used to simulate the NH4+ in seawater. + -N concentration was 100-120 mg / L; then, the mangrove sediments in the SBR tank were subjected to the first salt tolerance acclimatization by gradually increasing the salinity of the simulated seawater. The initial salinity of the simulated seawater in the SBR tank was 0.8%-1.2%, and it was increased to 3%-3.5%. The process of acclimating mangrove sediments in the upflow denitrification filter is as follows: First, simulated seawater NO3 is increased in stages. - The method of NO3- concentration was used to acclimate mangrove sediments to nitrate in an upflow denitrification filter, simulating the NO3 concentration in seawater in the upflow denitrification filter. - -N concentration was 220-280 mg / L; then, the mangrove sediments in the upflow denitrification filter were subjected to a second salt tolerance acclimatization by gradually increasing the salinity of the simulated seawater until the salinity of the simulated seawater in the upflow denitrification filter was 3%-3.5%.

2. The method for treating marine aquaculture wastewater mediated by mangrove sediments according to claim 1, characterized in that, The stage of improving simulated seawater NH4 + The method of -N concentration for acclimation of mangrove sediments in SBR tanks to ammonia nitrogen tolerance specifically includes: Simulated seawater with an ammonia nitrogen concentration of 15-25 mg / L was used as the culture medium for the first stage of ammonia nitrogen tolerance acclimatization. In subsequent stages, the ammonia nitrogen concentration of the simulated seawater increased by 8-12 mg / L. The simulated seawater from each stage of ammonia nitrogen tolerance acclimatization was sequentially introduced into an SBR tank, with a hydraulic retention time of 38-48 h. The NH4+ content of the simulated seawater after each stage of ammonia nitrogen tolerance acclimatization was measured. + -N concentration, up to NH4 + When the -N removal rate stabilizes at 70-80%, the next stage of ammonia nitrogen tolerance acclimatization begins; during the ammonia nitrogen tolerance acclimatization process, continuous aeration is used, with an aeration rate of 0.5-1.5 L / min.

3. The method for treating marine aquaculture wastewater mediated by mangrove sediments according to claim 1, characterized in that, The method of gradually increasing the salinity of simulated seawater to perform the first salt tolerance acclimatization of mangrove sediments in the SBR tank specifically includes: With a salinity of 0.8%-1.2% and NH4+ + Simulated seawater with a nitrogen concentration of 60-90 mg / L was used as the culture medium for the first stage of the first salt tolerance acclimatization. In subsequent stages of the first salt tolerance acclimatization, the salinity of the simulated seawater increased sequentially by 0.4%-0.6%, while the ammonia nitrogen concentration remained constant. The simulated seawater from each stage of the first salt tolerance acclimatization was introduced into the SBR tank reaction vessel in sequence, with the hydraulic retention time controlled at 20-24 hours until NH4+ concentration reached a certain level. + When the -N removal rate is stable at over 80%, the next stage of cultivation begins; during the first salt tolerance acclimatization process, continuous aeration is used, with an aeration rate of 0.2-0.5 L / min and a stirring intensity of 30-60 r / min.

4. The method for treating marine aquaculture wastewater mediated by mangrove sediments according to claim 1, characterized in that, The stage of increasing simulated seawater NO3 - The method of N concentration for nitrate tolerance acclimation of mangrove sediments in upflow denitrification filters specifically includes: With NO3 - Simulated seawater with an NO3- concentration of 50-80 mg / L was used as the culture medium for the first stage of nitrate tolerance acclimatization. Subsequent stages of nitrate tolerance acclimatization involved using simulated seawater with NO3- concentrations... - The NO3- concentration was increased sequentially by 45-55 mg / L. Simulated seawater at each stage of nitrate tolerance acclimation was introduced into an upflow denitrification filter, with the hydraulic retention time controlled at 48-56 hours, until NO3- concentration reached the target level. - When the -N removal rate is stable at over 80%, the next stage of nitrate tolerance acclimatization begins; the nitrate tolerance acclimatization process uses a suffocating cultivation method.

5. The method for treating marine aquaculture wastewater mediated by mangrove sediments according to claim 1, characterized in that, The method of gradually increasing simulated seawater salinity to conduct a second salt tolerance acclimatization of mangrove sediments in an upflow denitrification filter specifically includes: With a salinity of 0.8%-1.2% and NO3- - Simulated seawater with a NO3- concentration of 220-280 mg / L was used as the culture medium for the first stage of the second salt tolerance acclimatization. In subsequent stages of the second salt tolerance acclimatization, the salinity of the simulated seawater increased sequentially by 0.4%-0.6%. - With NO3- concentration kept constant, simulated seawater from each stage of the second salt tolerance acclimatization process is sequentially introduced into an upflow denitrification filter, controlling the hydraulic retention time to be 12–20 hours, until NO3- concentration is maintained. - When the -N removal rate is stable at over 80%, the next stage of cultivation begins; the second salt tolerance acclimatization process uses a suffocating cultivation method.

6. The method for treating marine aquaculture wastewater mediated by mangrove sediments according to any one of claims 1-5, characterized in that, In SBR tanks, the amount of mangrove sediment added is 70%-80% of the SBR tank volume; in upflow denitrification filters, the amount of mangrove sediment added is 70%-80% of the upflow denitrification filter volume.

7. The method for treating marine aquaculture wastewater mediated by mangrove sediments according to any one of claims 1-5, characterized in that, The disinfection process specifically includes: introducing the aerobic denitrification treated seawater aquaculture wastewater into a disinfection tank, turning on the chlorine dioxide generator in the disinfection tank, and the retention time of the seawater aquaculture wastewater in the disinfection tank is 5-10 hours.

8. The method for treating marine aquaculture wastewater mediated by mangrove sediments according to any one of claims 1-5, characterized in that, The pretreatment specifically includes: passing the marine aquaculture wastewater through coarse filtration, homogenization, boosting, and fine filtration in sequence.

Images