A comparative analysis method and system for functional differentiation of denitrification microbial aggregates in aquaculture wastewater

Abstract

This invention discloses a comparative analysis method and system for the functional differentiation of microbial aggregates in aquaculture wastewater denitrification. Two parallel systems, a bioflocculation reactor and an activated sludge reactor, are constructed with equal inoculation volumes. The bioflocculation reactor operates within a first C / N ratio range and in a continuous aeration mode, while the activated sludge reactor operates within a second C / N ratio range and in a sequential batch operation mode. The first C / N ratio range is higher than the second C / N ratio range. Water samples and microbial aggregate samples are collected. Multi-dimensional comparative analysis of denitrification performance, physical properties, metabolic characteristics, and community characteristics is conducted. The analysis results are integrated to establish a correlation model of environmental pressure, community structure, functional characteristics, and denitrification performance, characterizing the mechanism of microbial aggregate functional differentiation. This invention also discloses a comparative analysis system for implementing the above method, providing a theoretical basis for the scientific selection and precise optimization of aquaculture wastewater denitrification treatment technologies.

Description

Technical Field

[0001] This invention relates to the field of environmental protection and biological wastewater treatment technology, specifically to a comparative analysis method and system for functional differentiation of denitrification microbial aggregates in aquaculture tailwater. Background Technology

[0002] Aquaculture is an important source of high-quality protein globally, but nitrogenous pollutants (ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen) generated during intensive aquaculture can easily lead to eutrophication if discharged directly without effective treatment. Biological denitrification technology, due to its high efficiency and environmental friendliness, has become the mainstream technology for treating aquaculture wastewater.

[0003] Microbial aggregates are the core function of biological nitrogen removal technology. Currently, two typical types of microbial aggregates have been developed in research and application: one is bioflocs formed under high C / N ratio (C / N=10-20) and continuous aeration conditions, which have a relatively loose structure and large particle size (usually 200-300 μm), and mainly rely on the ammonia assimilation of heterotrophic microorganisms to achieve nitrogen removal; the other is activated sludge formed under low C / N ratio (C / N=3-8) and sequencing batch reactor (SBR) operation (alternating aeration / stopping), which have a dense structure and small particle size (usually 50-100 μm), and complete nitrogen removal through the synergistic effect of nitrification and denitrification.

[0004] However, existing technologies for studying these two types of aggregates have significant limitations. On the one hand, existing studies mostly focus on isolated optimization of single aggregates. Differences exist between studies in reactor specifications, influent water quality, operating parameters, and detection methods, making cross-sectional comparisons difficult and hindering objective evaluation of the advantages, disadvantages, and applicable scenarios of the two technologies. On the other hand, existing technologies fail to clearly reveal how different environmental pressures (C / N ratio, operating modes) drive the differentiation of microbial community structure and function, and the intrinsic relationship between this differentiation and final nitrogen removal performance. Furthermore, existing analyses largely remain at the level of water quality indicators and macroscopic physical properties, lacking a systematic method for integrating and analyzing multi-dimensional information such as nitrogen removal performance, aggregate properties, extracellular polymer metabolism, microbial community, and functional gene expression.

[0005] For example, Chinese patent application CN119811508A discloses a statistical analysis-based method for microbial community analysis. This method uses high-throughput sequencing combined with diversity analysis, similarity analysis, and difference analysis to analyze the structural composition and characteristics of microbial communities in aquatic environments. However, this method only focuses on the structural characteristics of the microbial community itself, failing to integrate community analysis with denitrification performance, aggregate physicochemical properties, and extracellular polymeric metabolic characteristics in a multi-dimensional manner. Furthermore, it does not establish a correlation model between environmental stress and community functional differentiation. Therefore, it cannot provide direct guidance for the technical selection and process optimization of microbial aggregates under different operating conditions.

[0006] Due to insufficient understanding of the aforementioned mechanisms, in practical engineering, the selection of effluent treatment technologies for specific water qualities mainly relies on experience and lacks scientific theoretical basis, resulting in unclear directions for process optimization.

[0007] Therefore, how to establish a method and system that can systematically compare and analyze the functional differentiation characteristics of bioflocs and activated sludge, understand their underlying mechanisms, and provide scientific guidance for engineering applications is an urgent technical problem that needs to be solved. Summary of the Invention

[0008] To address the technical problems existing in the prior art, the first objective of this invention is to provide a comparative analysis method for the functional differentiation of microbial aggregates used in aquaculture wastewater denitrification. By establishing a standardized parallel comparative research system, this invention systematically reveals the essential differences and correlation mechanisms between bioflocs and activated sludge formed under different environmental pressures in terms of denitrification performance, structural characteristics, EPS metabolism, microbial communities, and functional genes. It also establishes a correlation model of environmental pressure, community structure, functional characteristics, and denitrification performance, thereby providing a theoretical basis for the scientific selection and precise optimization of aquaculture wastewater denitrification treatment technologies.

[0009] The second objective of this invention is to provide a comparative analysis system for the functional differentiation of microbial aggregates used in aquaculture wastewater denitrification, which is used to implement the above-mentioned comparative analysis method. In this system, the parallel reactor unit, control unit, analysis unit, and correlation model establishment unit work together to complete the construction of the parallel reaction system, the control of differentiated operating conditions, the collection and analysis of multi-dimensional data, and the establishment of the correlation model in an automated and modular manner, thereby providing hardware support for the implementation of the method.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A comparative analysis method for the functional differentiation of denitrifying microbial aggregates in aquaculture wastewater includes the following steps: A parallel comparative reaction system was constructed, with two parallel systems: a biofloc reactor and an activated sludge reactor. The two reactors were inoculated with equal amounts of total suspended solids to ensure consistent initial biomass. Differentiated operating conditions were set: the biofloc reactor operated in the first carbon-nitrogen ratio range and continuous aeration mode, while the activated sludge reactor operated in the second carbon-nitrogen ratio range and sequencing batch operation mode, wherein the first carbon-nitrogen ratio range was higher than the second carbon-nitrogen ratio range. Samples were collected from the biofloc reactor and the activated sludge reactor, including water samples and microbial aggregate samples. A multi-dimensional comparative analysis was conducted, including at least the analysis of denitrification performance, physical properties, metabolic characteristics, and community characteristics of the collected samples. A correlation model was established, and the results of multi-dimensional comparative analysis were integrated to obtain a correlation model of environmental pressure, community structure, functional characteristics, and denitrification performance, so as to characterize the mechanism of functional differentiation of microbial aggregates.

[0011] According to one example, the first carbon-nitrogen ratio ranges from 10 to 20, and the second carbon-nitrogen ratio ranges from 3 to 8.

[0012] According to one example, in the constructed parallel comparative reaction system, the biofloc reactor and the activated sludge reactor are of the same size, with an effective volume of 2.0-100.0 L; after inoculation according to the principle of equal total suspended solids, the initial biomass is controlled at 1000-1200 mg / L of total suspended solids.

[0013] According to one example, in the setting of differentiated operating conditions, the dissolved oxygen concentration of the biofloc reactor is 6-8 mg / L; the activated sludge reactor adopts a sequential batch operation mode with alternating aeration and shutdown, and the dissolved oxygen concentration during the aeration stage is 6-8 mg / L.

[0014] According to one example, the comparative analysis of denitrification performance includes determining the concentrations of total nitrogen, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and chemical oxygen demand, and calculating the removal rate, removal speed, and carbon source utilization efficiency for each form of nitrogen.

[0015] According to one example, the comparative analysis of the physical properties includes: determining the particle size distribution using a laser particle size analyzer; Settlement performance was determined through settling experiments; The distribution of live and dead cells in microbial aggregates was analyzed using confocal laser scanning microscopy combined with live / dead cell staining.

[0016] According to one example, the comparative analysis of the metabolic characteristics includes: extracting loosely bound extracellular polymers, tightly bound extracellular polymers, and total extracellular polymers; determining the protein and polysaccharide content and calculating the protein to polysaccharide ratio; and using three-dimensional fluorescence spectroscopy to analyze the compositional characteristics of organic matter in the extracellular polymers.

[0017] According to one example, the comparative analysis of community characteristics includes metagenomic sequencing of collected microbial aggregate samples, analysis of α- and β-diversity of the microbial community, comparison of differences in microbial composition at the phylum and genus levels, analysis of the abundance of key functional genes in nitrogen and carbon metabolism pathways based on the KEGG database, and species-functional contribution analysis.

[0018] According to one example, within the first carbon-to-nitrogen ratio range and under continuous aeration mode, bioflocs are enriched to Nakamurella The dominant functional microbial community achieves nitrogen transformation through the GS-GOGAT ammonia assimilation pathway; Under the second carbon-to-nitrogen ratio range and sequencing batch operation mode, the activated sludge maintains a multi-microbial synergistic metabolic network, achieves ammonia assimilation through the GDH pathway, and utilizes extracellular polymers as an endogenous carbon source to provide energy for the denitrification process.

[0019] A comparative analysis system for functional differentiation of denitrifying microbial aggregates in aquaculture wastewater to achieve the above method includes: Parallel reactor unit, used to set up two parallel systems: a biofloc reactor and an activated sludge reactor; The control unit, connected to the parallel reactor unit, is used to control the first carbon-nitrogen ratio range and continuous aeration mode of the biofloc reactor, and to control the second carbon-nitrogen ratio range and sequential batch operation mode of the activated sludge reactor. An analysis unit, connected to the parallel reactor unit, includes a denitrification performance detection module, a physical property analysis module, a metabolic characteristic analysis module, and a community characteristic analysis module; The correlation model building unit is connected to the analysis unit and is used to integrate multi-dimensional analysis results and establish a correlation model of environmental pressure, community structure, functional characteristics and denitrification performance.

[0020] The present invention has the following advantages: In terms of methodological innovation, this invention proposes for the first time a standardized and systematic comparative research paradigm. By constructing parallel reactors, adhering to the principle of equal inoculation, and designing differentiated operating conditions, it ensures the scientific nature of the comparative analysis and the reliability of the experimental results, providing a replicable and comparable new method for the study of functional differentiation of microbial aggregates.

[0021] In terms of the depth of mechanism elucidation, this invention systematically reveals the essential differences in functional differentiation between bioflocs and activated sludge, two typical microbial aggregates, from five levels: denitrification performance, physicochemical structure, extracellular polymer dynamics, community composition, and metagenomic metabolic network. Driven by a high carbon-to-nitrogen ratio (C / N=15) and continuous aeration, the biofloc system forms... Nakamurella The nitrogen removal mode, dominated by bacteria and centered on the GS-GOGAT ammonia assimilation pathway, has a total nitrogen removal rate of about 70% in 24 hours. Under low carbon-to-nitrogen ratio (C / N=5) and sequencing batch reactor operation conditions, the activated sludge system forms a diversified nitrogen removal mode characterized by multi-bacterial synergy, GDH ammonia assimilation pathway and complete nitrification-denitrification coupling, and its carbon source consumption is about one-third of that of the biofloc system.

[0022] In terms of application guidance, this invention transforms the aforementioned mechanistic understanding into technical standards that can directly guide engineering practice. On the one hand, it clarifies the basis for technology selection based on the carbon-nitrogen ratio of effluent, namely, biofloc technology is selected when the carbon-nitrogen ratio is ≥10, and activated sludge technology is selected when the carbon-nitrogen ratio is ≤6, thus elevating technology selection from experience-based judgment to scientific decision-making. On the other hand, it specifically proposes process optimization directions for two types of technologies, such as enhancing the carbon source supply of bioflocs to maintain their structural stability, and utilizing the endogenous carbon metabolism characteristics of activated sludge to save on external carbon source addition.

[0023] In terms of economic and social benefits, the application of this invention can shorten the treatment time of high carbon-to-nitrogen ratio wastewater by more than 50%, reduce the operating cost of low carbon-to-nitrogen ratio wastewater by about 30%, and effectively improve the scientific and precise level of aquaculture wastewater treatment, thus having significant economic and environmental benefits. Attached Figure Description

[0024] Figure 1 This is a flowchart of the comparative analysis method for the functional differentiation of denitrification microbial aggregates in aquaculture wastewater according to the present invention.

[0025] Figure 2 This is a comparison chart of the nitrogen removal performance of the biofloc reactor and the activated sludge reactor according to the present invention. The biofloc reactor achieved an ammonia nitrogen removal rate of over 99% within 12 hours and a total nitrogen removal rate of 69.92% within 24 hours. The activated sludge reactor achieved an ammonia nitrogen removal rate of 97.82% within 24 hours and a total nitrogen removal rate of 45.22%, with its carbon source consumption being approximately one-third that of the biofloc reactor.

[0026] Figure 3 This is a comparison chart of the physicochemical properties (including particle size distribution and settling performance) of the bioflocs and activated sludge of this invention. The average particle size of the bioflocs is 224 μm, with the particle size concentrated in the range of 80-502 μm, accounting for 86% of the total. The average particle size of the activated sludge is 80 μm, with a particle size distribution range of 32-564 μm, accounting for 85% of the total. Settling performance tests show that the floc volume index of the activated sludge is significantly lower than that of the bioflocs (P<0.001), indicating that it has superior settling performance.

[0027] Figure 4This is a comparison chart of the changes in extracellular polymeric substances (EPS) content and composition between bioflocs and activated sludge in this invention. The total EPS content of the bioflocs significantly increased from an initial 1412.76 mg / L to 1611.57 mg / L (P<0.001), with tightly bound EPS increasing from 608.37 mg / L to 841.91 mg / L, and polysaccharide content showing a significant increase. Conversely, the total EPS content of the activated sludge significantly decreased from an initial 1376.78 mg / L to 1253.25 mg / L (P<0.001), with both loosely bound and tightly bound EPS showing a significant decrease.

[0028] Figure 5 This is a comparative diagram of the microbial community structure of the bioflocs and activated sludge of this invention (e.g., horizontal composition). Specifically, in the biofloc reactor... Nakamurella The genus was the absolute dominant bacteria, with an initial relative abundance of 39.41% and a final abundance of 35.94%. Activated sludge reactors exhibited higher microbial diversity, with dominant genera including... Candidatus Promineifilum, Pseudoxanthomonas , Caldilinea wait.

[0029] Figure 6 This is a comparison chart of the abundance of key functional genes related to nitrogen metabolism in bioflocs and activated sludge according to the present invention. The biofloc reactor shows significant enrichment of ammonia assimilation-related genes. glnA , gltB , gltD (GS-GOGAT pathway); activated sludge reactors significantly enrich low-energy ammonia assimilation genes. gdhA (GDH pathway), nitrification genes amoABC Complete denitrification genes napA , nirKS , norB , nosZ . Detailed Implementation

[0030] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention are all commercially available.

[0031] Example 1

[0032] Reference Figure 1 This embodiment provides a comparative analysis method for the functional differentiation of denitrification microbial aggregates in aquaculture wastewater, specifically including the following steps: (1) Construction of parallel comparative reaction system and domestication and maturation This embodiment sets up two parallel control reaction systems: a bioflocculation reactor (BFR) and an activated sludge reactor (ASR). The reactors are made of cylindrical polyethylene with an inner diameter of 99 mm, a height of 450 mm, and an effective volume of 2.5 L. Three parallel samples are set up for each reactor. The three parallel samples for the bioflocculation reactor are designated BFR-1, BFR-2, and BFR-3, respectively, and the three parallel samples for the activated sludge reactor are designated ASR-1, ASR-2, and ASR-3, respectively, to eliminate random errors.

[0033] The biofloc reactor was inoculated with bioflocs matured under high C / N ratio conditions (C / N = 15) and cultured under continuous aeration. The activated sludge reactor was inoculated with activated sludge matured under low C / N ratio conditions (C / N = 5) and cultured under sequencing batch reactor (SBR) operation, i.e., 3 hours of aeration followed by 1 hour of aeration stoppage.

[0034] Both reactors were inoculated with equal amounts of total suspended solids, and the initial biomass was controlled within the range of 1000–1200 mg / L. Specifically, the initial total suspended solids concentration of the biofloc reactor was 1100 ± 80 mg / L, and the initial total suspended solids concentration of the activated sludge reactor was 1050 ± 50 mg / L.

[0035] The criteria for determining acclimatization maturity are as follows: The inoculated bioflocs and activated sludge are operated separately under the formal experimental conditions of their respective reactors. Specifically, the biofloc reactor uses a C / N ratio of 15 with continuous aeration, while the activated sludge reactor uses a C / N ratio of 5 with sequential batch operation. The ammonia nitrogen concentration and chemical oxygen demand (COD) in the effluent are continuously monitored. Acclimatization maturity is considered achieved when the effluent indicators remain stable for three consecutive days, i.e., the ammonia nitrogen removal rate fluctuates by less than 5% and the COD removal rate fluctuates by less than 5%. The acclimatization period is one week.

[0036] (2) Setting differentiated operating conditions The biofloc reactor operates in a continuous aeration mode, with dissolved oxygen concentration controlled at 6–8 mg / L via an air pump. The activated sludge reactor operates in a sequencing batch reactor mode, with each operating cycle lasting 4 hours, alternating between 3 hours of aeration and 1 hour of aeration stoppage, with dissolved oxygen concentration also controlled at 6–8 mg / L during the aeration phase.

[0037] Both reactors used the same formula for simulated aquaculture wastewater, with the following basic components: NH4Cl 76.43 mg / L, KNO3 216.43 mg / L, NaNO2 4.93 mg / L, and KH2PO4 43.87 mg / L. During influent preparation, glucose was added to the influent of the biofloc reactor to adjust the C / N ratio to 15; glucose was also added to the influent of the activated sludge reactor to adjust the C / N ratio to 5.

[0038] During operation, the reactor temperature was controlled at 20-25℃, the pH value was maintained at 7.0-8.0 through the NaHCO3 buffer system, and the hydraulic retention time (HRT) was set to 24 hours.

[0039] (3) Sample collection and pretreatment After the animals have matured, formal experiments will begin, and this moment will be recorded as 0 h.

[0040] Water sampling: Every 2 hours, 50 mL water samples were independently collected from each parallel sample in each reactor and stored in a 4°C refrigerator for subsequent water quality index determination. After each sampling, an equal amount of simulated aquaculture wastewater was added to the reactor to maintain a constant working volume of the reactor.

[0041] Microbial aggregate sample collection: At the end of the experiment, 100 mL of activated sludge or biofloc sample was independently collected from each parallel sample in each reactor. The collected sample was divided into two parts: one part was the original mixed liquid sample, used for particle size distribution analysis and confocal laser scanning microscopy (CLSM) observation; the other part was the precipitate sample obtained after settling, used for extracellular polymeric substances (EPS) extraction and determination. The collected sample was allowed to settle, and the supernatant was separated to obtain the precipitate sample. Another 50 mL of the original mixed liquid sample was filtered through a 0.22 μm sterile polyethersulfone filter membrane to enrich the bacterial cells. The filter membrane was stored at -80℃ for later use in subsequent metagenomic sequencing.

[0042] Sample preservation and labeling: All samples should be labeled with reactor type, sampling time point and parallel sample number to ensure sample traceability.

[0043] (4) Multi-dimensional comparative analysis Data presentation: All parameters were measured independently for three parallel samples from each reactor. The experimental data are expressed as the mean ± standard deviation (Mean ± SD) of the three parallel samples.

[0044] Comparative analysis of denitrification performance Water samples collected every 2 hours were filtered through a 0.45 μm filter membrane, and ammonia nitrogen (NH3) was determined using the national standard method. 4+ -N), nitrite nitrogen (NO) 2--N), nitrate nitrogen (NO) 3- The concentrations of nitrogen (N), total nitrogen (TN), and chemical oxygen demand (COD) were measured independently for each parallel sample, and the removal rate and removal efficiency of each form of nitrogen in each reactor group were calculated.

[0045] Reference Figure 2 The results showed that the biofloc reactor achieved an ammonia nitrogen removal rate of over 99% within 12 hours and a total nitrogen removal rate of 69.92% within 24 hours; the activated sludge reactor achieved an ammonia nitrogen removal rate of 97.82% and a total nitrogen removal rate of 45.22% within 24 hours, and its carbon source consumption was about one-third that of the biofloc reactor.

[0046] Comparative analysis of the physicochemical properties and structure of aggregates Particle size distribution: The particle size of each sample was measured using a laser particle size analyzer. Each sample was measured three times and the average value was taken.

[0047] Settling performance: Take 1000 mL of the mixture and place it in a 1 L Imhoff settling cone. After standing for 30 min, record the volume of the settled flocs and calculate the floc volume index (FVI) according to the following formula: FVI = floc volume (mL) / total suspended solids concentration (g / L).

[0048] Live / dead cell staining: The distribution of live / dead cells was observed using a confocal laser scanning microscope combined with SYTO 9 / PI double staining. Ten fields of view were randomly selected from each sample for observation.

[0049] Reference Figure 3 The results showed that the average particle size of microbial aggregates in the biofloc reactor was 224 μm, with the majority of particles ranging from 80 to 502 μm, accounting for 86% of the total. In the activated sludge reactor, the average particle size of microbial aggregates was 80 μm, with a range of 32 to 564 μm, also accounting for 85% of the total. Settling performance tests indicated that the floc volume index of activated sludge was significantly lower than that of bioflocs (P<0.001), demonstrating superior settling performance. Confocal laser scanning microscopy images showed that bioflocs were predominantly composed of living cells, mainly distributed on the surface of the aggregates; activated sludge contained a large number of dead cells, exhibiting a core-shell structure.

[0050] Comparative analysis of extracellular polymer metabolic characteristics Microbial aggregate samples collected at 0 h and 24 h were allowed to settle and precipitate. Extracellular polymers were extracted and measured independently for each parallel sample.

[0051] Extracellular polymer extraction: Loosely bound extracellular polymers, tightly bound extracellular polymers, and total extracellular polymers were extracted using thermal extraction.

[0052] Component determination: Protein content was determined using the modified Lowry method, and polysaccharide content was determined using the anthrone-sulfuric acid method. The protein / polysaccharide ratio was also calculated.

[0053] Fluorescence spectroscopy analysis: Three-dimensional fluorescence spectroscopy is used to analyze the compositional characteristics of organic matter in tightly bound extracellular polymers.

[0054] Reference Figure 4 The results showed that the total extracellular polymeric substance (ECP) content of microbial aggregates in the biofloc reactor significantly increased from the initial 1412.76 mg / L to 1611.57 mg / L (P<0.001), with the content of tightly bound ECP increasing from 608.37 mg / L to 841.91 mg / L, and the polysaccharide content also increasing significantly. In the activated sludge reactor, the total ECP content of microbial aggregates significantly decreased from the initial 1376.78 mg / L to 1253.25 mg / L (P<0.001), with both loosely bound and tightly bound ECP contents decreasing significantly.

[0055] Three-dimensional fluorescence spectroscopy analysis showed that the fluorescence intensity of tryptophan-like proteins and soluble microbial products of tightly bound extracellular polymers was significantly reduced in the activated sludge reactor; while the fluorescence intensity of soluble microbial products and fulvic acid-like substances of tightly bound extracellular polymers was significantly enhanced in the biofloc reactor.

[0056] Comparative analysis of microbial communities and metabolic networks Take filter membrane samples collected at 0 h and 24 h and stored at -80℃ (filtered through a 0.22 μm sterile polyethersulfone filter membrane to enrich bacterial cells), and perform the following operations independently for each parallel sample: DNA extraction: Total DNA was extracted using a soil DNA extraction kit, and DNA quality was assessed by 1% agarose gel electrophoresis.

[0057] Metagenomic sequencing: PE150 sequencing was performed using the Illumina NovaSeq platform, producing no less than 10 Gb of clean data per sample.

[0058] Data Analysis: Raw data underwent quality control using Fastp software, assembly using MEGAHIT software, and gene prediction using Prodigal software. Functional annotation was then performed by comparing the data with the KEGG database. Differences in microbial composition were compared at the phylum and genus levels, and the abundance of key functional genes in nitrogen metabolism pathways was analyzed. The abundance of functional genes for each sample is expressed as RPKM (Reads Per Kilobaseper Million mapped reads), and the average of three parallel samples was used.

[0059] The conclusions are as follows: Reference Figure 5 Community structure: In a biofloc reactor, Nakamurella This genus was the absolute dominant bacterial genus, with an initial relative abundance of 39.41% and a final abundance of 35.94%. The activated sludge reactor exhibited higher microbial diversity, with dominant genera including... Candidatus Promineifilum, Pseudoxanthomonas , Caldilinea wait.

[0060] Reference Figure 6 Nitrogen metabolism genes: Biofloc reactors significantly enrich ammonia assimilation-related genes. glnA , gltB , gltD This refers to the GS-GOGAT pathway; activated sludge reactors significantly enrich low-energy ammonia assimilation genes. gdhA (GDH pathway), nitrification genes amoABC and complete denitrification genes napA , nirKS , norB , nosZ .

[0061] (5) Verification of technical effects and mechanism analysis Based on the above analysis results, this embodiment establishes a correlation model of "environmental pressure - community structure - functional characteristics - denitrification performance".

[0062] Under the environmental pressure of a high carbon-to-nitrogen ratio (C / N=15) and continuous aeration, the biofloc system enriched with […]. Nakamurella This is a dominant functional bacterial community. This community achieves efficient ammonia assimilation through the highly abundant GS-GOGAT pathway, while storing carbon sources and maintaining structural stability by secreting large amounts of extracellular polymers (especially tightly bound extracellular polymers). Ultimately, it exhibits a denitrification characteristic with a high total nitrogen removal rate (69.92%) but a large carbon source consumption.

[0063] Under the environmental pressures of a low carbon-to-nitrogen ratio (C / N=5) and sequencing batch reactor (SBR) operation (alternating aerobic / anoxic conditions), the activated sludge system maintained high microbial diversity, forming a synergistic metabolic network of multiple microbial communities: Candidatus Nitrosocosmicus Nitrifying bacteria perform the function of ammonia oxidation. Pseudoxanthomonas When denitrifying bacteria take on the function of nitrate reduction, Candidatus Promineifilum The bacteria achieve low-energy ammonia assimilation via the GDH pathway. In this system, extracellular polymers are degraded and utilized as an endogenous carbon source, providing energy for the denitrification process. Ultimately, the denitrification process exhibits a moderate total nitrogen removal rate (45.22%) but significantly high carbon source utilization efficiency.

[0064] This method establishes a standardized process of "parallel system construction → differentiated system operation → sample collection and pretreatment → multi-dimensional measurement → data integration and analysis → application guide output," and systematically reveals the essential differences in the denitrification performance and intrinsic mechanisms of two typical microbial aggregates (i.e., bioflocs formed under high carbon-nitrogen ratio and continuous aeration conditions, and activated sludge formed under low carbon-nitrogen ratio and sequencing batch operation conditions) in aquaculture effluent.

[0065] Based on the aforementioned differences, this method clearly defines the technology selection criteria based on the carbon-to-nitrogen ratio of the effluent: when the carbon-to-nitrogen ratio is ≥10, biofloc technology is selected; when the carbon-to-nitrogen ratio is ≤6, activated sludge technology is selected. Simultaneously, it provides recommended process optimization parameters for both types of technologies, including carbon source replenishment strategies, aeration modes, and functional microbial enrichment directions. These outputs can provide a scientific basis for technology selection, operational optimization, and cost control in aquaculture effluent treatment projects, and have clear practical value.

[0066] Application Validation Example 1

[0067] This application verification example provides a practical application verification of the method of the present invention in the selection of aquaculture wastewater treatment technology.

[0068] The effluent quality characteristics of a certain aquaculture farm are as follows: carbon-to-nitrogen ratio (C / N) of 12, ammonia nitrogen concentration of 25 mg / L, and total nitrogen concentration of 60 mg / L. Based on the functional differentiation patterns of the two types of microbial aggregates (bioflocs and activated sludge) revealed in Example 1, when the effluent C / N ratio is ≥10, biofloc technology exhibits a significant advantage due to its higher total nitrogen removal efficiency. Therefore, it is recommended that this aquaculture farm adopt the biofloc process for effluent treatment.

[0069] Actual operation results show that, under continuous aeration and a carbon-to-nitrogen ratio maintained at 12, the system achieved a total nitrogen removal rate of 68% within 24 hours, which is highly consistent with the nitrogen removal performance of the biofloc reactor in Example 1 (69.92% total nitrogen removal rate within 24 hours). This result verifies the scientific guiding role of the method of this invention in the selection of aquaculture wastewater treatment technologies.

[0070] Application Validation Example 2

[0071] This application verification example provides a practical application verification of the method of the present invention in the optimization of aquaculture wastewater treatment process.

[0072] A wastewater treatment plant using the activated sludge process to treat aquaculture effluent with a low C / N ratio (C / N=4) experienced nitrate accumulation during operation. Based on the analysis of extracellular polymer metabolism characteristics of the activated sludge system in Example 1, the activated sludge can degrade extracellular polymers under low C / N ratio conditions, providing an endogenous carbon source for the denitrification process. Based on this mechanism, it is recommended to extend the anaerobic phase duration to enhance endogenous denitrification.

[0073] The anaerobic phase of the original sequential batch reactor (SBR) process was extended from 1 hour to 1.5 hours, i.e., the operating mode was adjusted to 2.5 hours of aeration followed by 1.5 hours of shutdown, while other operating parameters remained unchanged. After process optimization, the nitrate concentration in the effluent decreased from 45 mg / L to 18 mg / L, significantly improving the nitrate accumulation problem. This result confirms the guiding value of the method of this invention for the precise optimization of aquaculture wastewater treatment processes.

[0074] This application also provides a comparative analysis system for the functional differentiation of microbial aggregates used in aquaculture wastewater denitrification, which generally includes a parallel reactor unit, a control unit, an analysis unit, and a correlation model establishment unit.

[0075] The parallel reactor unit is used to construct a parallel comparative reaction system, comprising two independent reactors: a bioflocculation reactor and an activated sludge reactor. Multiple parallel samples are set up in each reactor group to eliminate random errors. The bioflocculation reactor is inoculated with matured bioflocculation under high C / N ratio and continuous aeration conditions; the activated sludge reactor is inoculated with matured activated sludge under low C / N ratio and sequencing batch reactor (SBR) operation conditions. Both reactors are inoculated with equal total suspended solids, and the initial biomass is controlled within the range of 1000–1200 mg / L.

[0076] The control unit is connected to the parallel reactor unit and is used to control the differentiated operating conditions of the two reactors respectively. Specifically, it includes a first control module and a second control module. The first control module is used to control the carbon-nitrogen ratio of the biofloc reactor to be high and adopt a continuous aeration operation mode. The second control module is used to control the carbon-nitrogen ratio of the activated sludge reactor to be low and adopt a sequencing batch operation mode.

[0077] Optionally, the high carbon-to-nitrogen ratio is C / N=15, and the low carbon-to-nitrogen ratio is C / N=5; the dissolved oxygen concentration is controlled at 6-8 mg / L in continuous aeration mode; in the sequential batch operation mode, each cycle is 4 hours, including alternating 3 hours of aeration and 1 hour of aeration stop, and the dissolved oxygen concentration is controlled at 6-8 mg / L during the aeration stage.

[0078] The analysis unit is connected to the parallel reactor unit for multi-dimensional comparative analysis of microbial aggregates in the two reactors. The analysis unit includes a denitrification performance detection module, a physical property analysis module, a metabolic characteristic analysis module, and a community characteristic analysis module. Specifically, the denitrification performance detection module measures the concentrations of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, and chemical oxygen demand in the water sample, and calculates the removal rate and extent of each nitrogen form. The physical property analysis module measures the particle size distribution, sedimentation performance, and live / dead cell distribution of the microbial aggregates. The metabolic characteristic analysis module extracts and measures extracellular polymers from the microbial aggregates, including loosely bound extracellular polymers, tightly bound extracellular polymers, and total extracellular polymers, measures protein and polysaccharide content, and performs three-dimensional fluorescence spectroscopy analysis. The community characteristic analysis module performs metagenomic sequencing and data analysis on the microbial aggregates, compares differences in microbial composition at the phylum and genus levels, and analyzes the abundance of key functional genes in nitrogen metabolism pathways.

[0079] The correlation model is established by connecting the unit and the analysis unit to integrate the multi-dimensional analysis results of denitrification performance, physical properties, metabolic characteristics and community characteristics. The correlation model of "environmental pressure - community structure - functional characteristics - denitrification performance" is established to reveal the essential differences between the two types of typical microbial aggregates in the denitrification performance and internal mechanism of aquaculture tailwater.

[0080] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A comparative analysis method for the functional differentiation of microbial aggregates used in aquaculture wastewater denitrification, characterized in that, Includes the following steps: A parallel comparative reaction system was constructed, with two parallel systems: a biofloc reactor and an activated sludge reactor. The two reactors were inoculated with equal amounts of total suspended solids to ensure consistent initial biomass. Differentiated operating conditions were set: the biofloc reactor operated in the first carbon-nitrogen ratio range and continuous aeration mode, while the activated sludge reactor operated in the second carbon-nitrogen ratio range and sequencing batch operation mode, wherein the first carbon-nitrogen ratio range was higher than the second carbon-nitrogen ratio range. Samples were collected from the biofloc reactor and the activated sludge reactor, including water samples and microbial aggregate samples. A multi-dimensional comparative analysis was conducted, including at least the analysis of denitrification performance, physical properties, metabolic characteristics, and community characteristics of the collected samples. A correlation model was established, and the results of multi-dimensional comparative analysis were integrated to obtain a correlation model of environmental pressure, community structure, functional characteristics, and denitrification performance, so as to characterize the mechanism of functional differentiation of microbial aggregates.

2. The method according to claim 1, characterized in that, The first carbon-nitrogen ratio ranges from 10 to 20, and the second carbon-nitrogen ratio ranges from 3 to 8.

3. The method according to claim 1, characterized in that, In the constructed parallel comparative reaction system, the biofloc reactor and the activated sludge reactor are of the same size, with an effective volume of 2.0-100.0 L; After inoculation according to the principle of equal total suspended solids, the initial biomass should be controlled at 1000-1200 mg / L of total suspended solids.

4. The method according to claim 1, characterized in that, In the aforementioned differentiated operating conditions, the dissolved oxygen concentration in the biofloc reactor is 6-8 mg / L; The activated sludge reactor adopts a sequential batch operation mode with alternating aeration and shutdown, and the dissolved oxygen concentration during the aeration stage is 6-8 mg / L.

5. The method according to claim 1, characterized in that, The comparative analysis of denitrification performance includes measuring the concentrations of total nitrogen, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and chemical oxygen demand, and calculating the removal rate, removal speed, and carbon source utilization efficiency of each form of nitrogen.

6. The method according to claim 1, characterized in that, The comparative analysis of the physical properties includes: measuring the particle size distribution using a laser particle size analyzer; Settlement performance was determined through settling experiments; The distribution of live and dead cells in microbial aggregates was analyzed using confocal laser scanning microscopy combined with live / dead cell staining.

7. The method according to claim 1, characterized in that, The comparative analysis of the metabolic characteristics included: extraction of loosely bound extracellular polymers, tightly bound extracellular polymers, and total extracellular polymers; Determine the protein and polysaccharide content, and calculate the protein to polysaccharide ratio; The compositional characteristics of organic matter in extracellular polymers were analyzed using three-dimensional fluorescence spectroscopy.

8. The method according to claim 1, characterized in that, The comparative analysis of community characteristics includes metagenomic sequencing of collected microbial aggregate samples, analysis of α- and β-diversity of microbial communities, comparison of differences in microbial composition at the phylum and genus levels, analysis of the abundance of key functional genes in nitrogen and carbon metabolism pathways based on the KEGG database, and species-functional contribution analysis.

9. The method according to claim 1, characterized in that, Within the first carbon-to-nitrogen ratio range and under continuous aeration mode, biofloc enrichment is achieved. Nakamurella The dominant functional microbial community achieves nitrogen transformation through the GS-GOGAT ammonia assimilation pathway; Under the second carbon-to-nitrogen ratio range and sequencing batch operation mode, the activated sludge maintains a multi-microbial synergistic metabolic network, achieves ammonia assimilation through the GDH pathway, and utilizes extracellular polymers as an endogenous carbon source to provide energy for the denitrification process.

10. A comparative analysis system for functional differentiation of microbial aggregates used in aquaculture wastewater denitrification to implement the method described in any one of claims 1-9, characterized in that, include: Parallel reactor unit, used to set up two parallel systems: a biofloc reactor and an activated sludge reactor; The control unit, connected to the parallel reactor unit, is used to control the first carbon-nitrogen ratio range and continuous aeration mode of the biofloc reactor, and to control the second carbon-nitrogen ratio range and sequential batch operation mode of the activated sludge reactor. An analysis unit, connected to the parallel reactor unit, includes a denitrification performance detection module, a physical property analysis module, a metabolic characteristic analysis module, and a community characteristic analysis module; The correlation model building unit is connected to the analysis unit and is used to integrate multi-dimensional analysis results and establish a correlation model of environmental pressure, community structure, functional characteristics and denitrification performance.

Images