Method for identifying functional microorganisms in a system for iron autotrophic and heterotrophic mixotrophic denitrification

Abstract

This invention relates to a method for identifying functional microorganisms in a mixed nutrient denitrification system of iron autotrophic and heterotrophic components, belonging to the field of wastewater biological treatment technology. The invention first constructs a mixed nutrient denitrification system, and then conducts microenvironmental culture by setting up a control group, an autotrophic labeled group, and a heterotrophic labeled group. Separation is then performed using cesium chloride density gradient centrifugation. 13 C-labeled heavy-chain DNA was used to determine the heavy-chain position using real-time quantitative PCR, followed by 16S rRNA gene sequencing to accurately identify iron-autotrophic and heterotrophic functional microorganisms. Based on the identification results, the response patterns and interaction mechanisms of different trophic microorganisms were analyzed by regulating the COD / TN ratio of the system and constructing a molecular ecological network. This invention achieves accurate identification and interaction analysis of functional microorganisms in complex mixed trophic systems, providing a reliable technical means for optimizing wastewater denitrification processes.

Description

Technical Field

[0001] This invention relates to the field of wastewater biological treatment technology, specifically to a method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system. Background Technology

[0002] The removal of excess nitrates from water bodies is a key issue in environmental engineering, and denitrification technology is widely used due to its high efficiency and economy. In the treatment of low C / N ratio wastewater, autotrophic denitrification technology shows significant advantages, with iron-autotrophic denitrification systems using Fe(II) as electron donors attracting considerable attention due to their characteristics of requiring no organic carbon source and producing low sludge production. However, in practical engineering applications, wastewater often contains both organic and inorganic carbon sources, forming complex iron-autotrophic and heterotrophic mixed-nutrient denitrification systems. In such systems, iron-autotrophic microorganisms and heterotrophic microorganisms coexist and interact, jointly affecting the system's denitrification efficiency and stability.

[0003] Currently, research on mixed-nutrient denitrification systems largely focuses on optimizing process parameters and assessing macroscopic performance. However, significant technical bottlenecks remain in accurately identifying functional microorganisms within the system and elucidating their interaction mechanisms. Traditional microbiological research methods, such as pure culture techniques, struggle to reflect the physiological state and function of microorganisms in real-world environments. While 16S rRNA gene sequencing can reveal species composition, it cannot distinguish between microbial groups with the same 16S sequence but vastly different metabolic functions, let alone achieve in-situ differentiation between autotrophic and heterotrophic microorganisms. Although stable isotope probe technologies (such as DNA-SIP) have shown potential in tracking specific metabolic pathways, their application in iron-autotrophic / heterotrophic mixed systems still faces challenges, including designing effective isotope labeling strategies to simultaneously differentiate between two carbon source utilization pathways and how to correlate microbial identification with their ecological functions.

[0004] Therefore, developing a method that can accurately identify autotrophic and heterotrophic functional microorganisms in a mixed nutrient denitrification system and deeply analyze their interaction mechanism is of great theoretical value and engineering guiding significance for revealing the stable operation mechanism of the system and optimizing process control strategies. Summary of the Invention

[0005] To address the aforementioned problems, this invention first constructs a mixed-nutrient denitrification system, and then cultivates it in a microcosm by setting up a control group, an autotrophic labeled group, and a heterotrophic labeled group, and uses centrifugation with a cesium chloride density gradient for separation. 13C-labeled heavy-chain DNA was used to determine the heavy-chain position using real-time quantitative PCR, followed by 16S rRNA amplicon sequencing to accurately identify iron-autotrophic and heterotrophic functional microorganisms. Based on the identification results, the response patterns and interaction mechanisms of different trophic microorganisms were analyzed by regulating the COD / TN ratio of the system and constructing a molecular ecological network. This invention achieves accurate identification and interaction analysis of functional microorganisms in complex mixed trophic systems, providing a reliable technical means for optimizing wastewater denitrification processes.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The purpose of this invention is to provide a method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system, comprising the following steps: (1) Construction and start-up of iron autotrophic and heterotrophic mixed nutrient denitrification system: Iron autotrophic denitrification sludge was inoculated into an anaerobic sequencing batch reactor, artificial water was added, and the iron autotrophic denitrification system was started and operated to obtain an iron autotrophic denitrification system; after the iron autotrophic denitrification system was running stably, an organic carbon source was added to construct an iron autotrophic and heterotrophic mixed nutrient denitrification system; the iron autotrophic and heterotrophic mixed nutrient denitrification system was started and operated stably; the stable operation process of the iron autotrophic and heterotrophic mixed nutrient denitrification system was divided into four stages, namely stage I, stage II, stage III and stage IV; (2) Isotope-labeled microcosm culture: Collect the sludge from the stable operation of the iron autotrophic and heterotrophic mixed nutrient denitrification system during stage IV, and add the corresponding sludge to the microcosm culture. 12 Sodium C-acetate and 12 C-Sodium bicarbonate, 13 Sodium C-acetate and 12 C-Sodium bicarbonate, 13 C-sodium bicarbonate and 12 Sodium C-acetate was used in isotope microcosm experiments, and the samples were cultured separately. (3) Nucleic acid separation and density gradient centrifugation: After the culture is completed, the sludge DNA sample of the isotope microcosm experiment is extracted; the sludge DNA sample is mixed with the cesium chloride solution prepared with GB buffer to obtain a mixture, and ultra-high speed density gradient centrifugation is performed. The centrifuged sample is further purified by layer to obtain DNA fractions of different density levels. (4) Identification and functional differentiation of heavy chain DNA: The DNA fraction was purified, and the narG and nirS genes in the DNA fraction were quantitatively analyzed by qPCR; by comparing the distribution of the narG and nirS genes, the heavy chain DNA was identified. 13 The enrichment location of C-DNA in the microbial community is the heavy chain DNA fraction in the DNA fraction. The microorganisms enriched in the heavy chain DNA fraction are evaluated as iron autotrophic denitrifying microorganisms or heterotrophic denitrifying microorganisms. (5) Functional microbial composition analysis: 16S rRNA gene amplicon sequencing was performed on the heavy chain DNA fraction. The raw data obtained from the sequencing was split, and then subjected to quality control filtering and paired-end sequence splicing. The high-quality spliced ​​sequences were clustered into operational taxonomic units (OTUs) at a similarity level of 97%, and chimeric sequences were removed. The representative OTU sequences were compared with the SILVA 16S rRNA database to complete species annotation. Based on the annotation results, the taxonomic information and relative abundance of the microorganisms that played a role in the three sets of isotope microcosm experiments at the genus level were statistically analyzed, thereby analyzing the genus-level community composition and abundance distribution of iron autotrophic and heterotrophic functional microorganisms.

[0007] The beneficial effects of the present invention are: (1) through design 13 C-Sodium bicarbonate+ 12 Sodium C-acetate and 12 C-Sodium bicarbonate+ 13 The parallel labeling scheme of C-sodium acetate established a specific identification system for iron autotrophic and heterotrophic microorganisms, solving the problem of distinguishing functional microorganisms in mixed nutrient systems.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, the concentration of the iron-autotrophic denitrifying sludge is 4000 mg / L-5000 mg / L.

[0010] Furthermore, the artificial water preparation in step (1) uses Fe(II) as an inorganic electron donor, sodium bicarbonate as an inorganic carbon source, and sodium nitrate as the sole nitrogen source; The ferrous iron and NO3 in the iron autotrophic and heterotrophic mixed nutrient denitrification system mentioned above - The molar ratio of -N is controlled at 4~6:1, the mass concentration ratio of COD to total inorganic nitrogen in the artificially prepared water is controlled in the range of 1.0-3.0:1, and the amount of sodium bicarbonate added is 0.5 g / L-1.0 g / L.

[0011] Among them, NO3 - -N represents the nitrate concentration (mg / L) measured in nitrogen elemental mass, NO3 - -N mass = NaNO3 mass × (N atomic weight / NaNO3 molecular weight); This invention supplements the nitrogen source by adding sodium nitrate, therefore the total inorganic nitrogen mass = sodium nitrate mass × (N atomic weight / NaNO3 molecular weight); In this invention, sodium acetate is used to adjust the carbon-to-nitrogen ratio of the influent. The sodium acetate is anhydrous sodium acetate (CH3COONa, molecular weight 82.03), and its theoretical chemical oxygen demand (COD) is calculated based on the complete oxidation reaction: CH3COONa + 2O2 → 2CO2 + NaOH + H2O. Therefore, it is determined that the complete oxidation of each gram of anhydrous sodium acetate consumes 0.78 grams of oxygen, meaning that 1 g of anhydrous sodium acetate contributes 780 mg of COD.

[0012] Based on the target COD increase, the sodium acetate dosage is calculated using the following formula: m 乙酸钠 =COD 目标 / 0.78, meaning 1g sodium acetate = 0.78g COD.

[0013] The beneficial effects of adopting the above-mentioned further scheme are: through the synergistic regulation of multiple parameters, a suitable ecological niche for the coexistence and complementary functions of iron autotrophic and heterotrophic microorganisms was constructed, providing a stable and controllable experimental platform for subsequent accurate analysis of the community succession patterns and interaction mechanisms of the two types of microorganisms under different COD / TN ratios using DNA-SIP technology.

[0014] Furthermore, the solvent for the GB buffer solution in step (3) is deionized water; the buffer solution is used after being filtered through a 0.2 μm filter membrane and sterilized.

[0015] Furthermore, the parameters of the ultra-high speed centrifugation in step (3) are set as follows: centrifugation at 45,000 rpm for 44 hours at 20°C; the centrifuge is started with maximum acceleration and brakes are not used when it is terminated, so that the rotor stops naturally by its own friction force, so as to minimize the disturbance to the density gradient that has been formed.

[0016] The beneficial effect of adopting the above-mentioned further scheme is that ultra-high speed centrifugation can maximize the stability of the density gradient and the accuracy of stratification.

[0017] Further, the purification in step (4) specifically involves: precipitating DNA with polyethylene glycol 6000 solution, washing the DNA precipitate with 70% ethanol solution (by volume), and finally adding 30 μL of TE buffer to dissolve the purified DNA.

[0018] Furthermore, the organic carbon source in step (1) is sodium acetate; the operating conditions of the anaerobic sequencing batch reactor are: hydraulic retention time 24h-48h, temperature 25℃-35℃, pH 7.5-8.0.

[0019] The beneficial effects of adopting the above-mentioned further approach are: a complete quality control system is constructed from sample pretreatment to bioreaction, providing a suitable synergistic metabolic environment for the two types of functional microorganisms. These approaches collectively support the core objective of this invention: accurately elucidating the composition and interaction mechanisms of ferroautotrophic and heterotrophic microorganisms.

[0020] Further, the GB buffer in step (1) includes Tris-HCl, KCl, and EDTA; the concentration of Tris-HCl in the GB buffer is 0.1 M, the concentration of KCl is 0.1 M, and the concentration of EDTA is 1.0 mM. The pH value of the GB buffer solution is 7.8~8.2.

[0021] The beneficial effects of the above-mentioned further scheme are as follows: the buffer solution is prepared with deionized water, filtered, and sterilized before use, effectively removing impurities and nuclease contamination; 0.1 M Tris-HCl and pH 7.8-8.2 synergistically maintain the weakly alkaline environment of the centrifugation system, preventing acid / base catalytic damage to DNA during prolonged centrifugation; 0.1 M KCl provides stable ionic strength, forming a uniform density gradient with CsCl to ensure effective separation of light and heavy DNA; 1.0 mM EDTA chelates trace metal ions, inhibiting potential metal-dependent nuclease activity and protecting the integrity of DNA molecules. This scheme lays a reliable foundation for subsequent precise stratified sampling and functional microbial identification.

[0022] Furthermore, the number of days of stable operation mentioned in step (1) is 200 to 250 days; the stable operation specifically means that the iron autotrophic denitrification system is stable when the nitrate nitrogen removal rate of the system effluent is maintained above 90% for 15 consecutive days. The stable operation of the iron autotrophic and heterotrophic mixed nutrient denitrification system is specifically defined as follows: when the nitrate nitrogen removal rate of the effluent from the iron autotrophic and heterotrophic mixed nutrient denitrification system is maintained above 90% for 15 consecutive days, it is considered to have reached a stable operating state.

[0023] The beneficial effects of adopting the above-mentioned further scheme are: by ensuring that the iron autotrophic and heterotrophic mixed nutrient denitrification system reaches a state of functional maturity and high stability during long-term operation, it provides a unified and reliable benchmark platform for subsequent experiments on the regulation of different COD / TN ratios and DNA-SIP isotope labeling studies, effectively eliminating the interference of the system's start-up or fluctuation period on the microbial community response, thereby ensuring the representativeness and comparability of the subsequent functional microbial analysis results.

[0024] Furthermore, the criterion for division in step (1) is the influent COD / TN, and the COD / TN values ​​corresponding to stage I, stage II, stage III, and stage IV are 0, 2.5, 1.5, and 2.0, respectively.

[0025] Furthermore, in step (2), add 12 Sodium C-acetate and 12 The treatment with C-sodium bicarbonate served as a control group; [Addition]13 C-sodium bicarbonate and 12 Treatment with C-acetic acid sodium as an autotrophic marker group; addition 12 C-sodium bicarbonate and 13 Treatment with C-acetic acid sodium was used as a heterotrophic marker group; The operating conditions for the control group, the autotrophic labeling group, and the heterotrophic labeling group were: hydraulic retention time of 24-48 hours, temperature of 25-35°C, and pH of 7.5-8.0.

[0026] The beneficial effect of adopting the above-mentioned further solutions is that, through design... 13 C-Sodium bicarbonate+ 12 Sodium C-acetate and 12 C-Sodium bicarbonate+ 13 Parallel labeling scheme for C-acetic acid sodium, combined with the whole 12 The control group (C) constructed a complete and stable isotope tracing system. Utilizing the fundamental differences in carbon source utilization between autotrophic and heterotrophic bacteria, it achieved the specific differentiation and tracking of iron-autotrophic and heterotrophic functional microorganisms. All three treatments were run synchronously under identical operating conditions, ensuring the consistency of the environmental background. This allowed subsequent microbial community analysis results to be fully attributed to differences in carbon source type, providing a reliable experimental basis for accurately revealing the succession patterns and interaction mechanisms of these two types of functional microorganisms under different carbon source conditions.

[0027] Furthermore, the target refractive index of the mixture in step (3) is 1.4027~1.4031, and the buoyancy density is 1.723 g / mL~1.728 g / mL.

[0028] The beneficial effect of adopting the above further scheme is that it ensures that the CsCl density gradient is within the range of separated light chains ( 12 c-DNA) and heavy chain ( 13 The optimal range for c-DNA is determined. This density range allows the minute buoyancy density difference (approximately 0.015 g / mL) generated by isotope labeling to be converted into a resolvable physical displacement, ensuring that labeled and unlabeled DNA form clearly separated bands after ultracentrifugation. This significantly improves the accuracy of subsequent stratified sampling and the recovery efficiency of the target fraction, providing a reliable separation basis for accurately analyzing the community composition of iron-autotrophic and heterotrophic functional microorganisms.

[0029] Furthermore, the purification in step (4) specifically involves: precipitating the DNA fraction with polyethylene glycol 6000 solution and washing the precipitate with 70% ethanol solution by volume.

[0030] The advantages of using the above-mentioned further approach are: DNA purification is achieved by using PEG6000 precipitation combined with 70% ethanol washing, which efficiently removes CsCl and impurities and dissolves them in TE buffer, ensuring downstream sequencing sensitivity.

[0031] This invention also provides a method for elucidating the functional microbial interaction mechanism in a mixed nutrient denitrification system of iron autotrophy and heterotrophy, comprising the following steps: (1) System regulation and sample collection: Adjust the influent COD / TN ratio of the iron autotrophic and heterotrophic mixed nutrient denitrification system. After the autotrophic and heterotrophic mixed nutrient denitrification system is running stably, collect sludge samples from the autotrophic and heterotrophic mixed nutrient denitrification system and extract DNA. Perform high-throughput sequencing on the V3-V4 region of the 16S rRNA gene to obtain microbial community data. (2) Nutritional response analysis and network construction: Based on the identification results of microbial nutritional types by DNA-SIP experiments, namely the genus-level community composition and abundance distribution of iron-autotrophic and heterotrophic functional microorganisms, the response law of the relative abundance of iron-autotrophic and heterotrophic functional microorganisms with the change of COD / TN ratio was analyzed; at the same time, based on the relative abundance of microorganisms at the genus level, a molecular ecological network was constructed based on the random matrix theory. Significant interaction relationships were screened by Spearman correlation matrix and RMT automatic identification threshold (chi-square test, p<0.001), and the network topology parameters were calculated. (3) Quantitative characterization of interaction mechanism: The molecular ecological network was visualized using Cytoscape software. Based on the identification results of microbial nutrition types by DNA-SIP experiment, namely the genus-level community composition and abundance distribution of iron autotrophic and heterotrophic functional microorganisms, the microbial nodes in the molecular ecological network were divided into iron autotrophic microbial modules, heterotrophic microbial modules and mixed trophic microbial modules. By identifying the key nodes connecting the iron autotrophic microbial modules and the heterotrophic microbial modules and the highly connected nodes within the modules, and combined with the network topology parameters, the synergistic and competitive relationship between iron autotrophic and heterotrophic microorganisms was quantitatively analyzed.

[0032] The beneficial effects of adopting the above-mentioned further scheme are: (1) combining the microbial trophic information obtained by DNA-SIP identification with molecular ecological network analysis, and realizing the technical integration from microbial identification to interaction mechanism analysis through module division and topological parameter calculation; (2) Based on the identification of different trophic microbial compositions and their interaction patterns, direct microbiological basis was provided for optimizing key operating parameters of the denitrification system, such as the COD / TN ratio and Fe(II) dosage.

[0033] Furthermore, the influent COD / TN ratios are 0, 1.5, 2.0, and 2.5, respectively; Furthermore, by adjusting the concentration of sodium acetate added to the influent, the COD / TN ratio of the influent can be controlled in stages.

[0034] Furthermore, the stable operation means that the nitrate removal efficiency of the autotrophic and heterotrophic mixed nutrient denitrification system is consistently above 90% for 15 consecutive days.

[0035] Furthermore, the key node is a node that has direct edge connections to both the autotrophic and heterotrophic iron modules, and whose betweenness centrality is greater than 0.1 times the average betweenness of the network. The highly connected nodes within a module are those that rank in the top 10% of node degree within each module. The network topology parameters include at least one of degree centrality, connectivity, betweenness centrality, and degree distribution. Attached Figure Description

[0036] Figure 1 This is a graph showing the change in nitrogen removal performance of the mixed nutrient denitrification system in Example 1 of the present invention; Figure 2 This is a graph showing the change in nitrogen removal efficiency during the isotope-labeled microcosm experiment in Embodiment 1 of the present invention; Figure 3 This is the quantitative analysis result of the denitrification functional genes narG and nirK in nucleic acid fractions of each density zone based on real-time PCR technology in Example 1 of the present invention; Figure 4 The results of high-throughput sequencing analysis of heavy chain DNA in Example 1 of this invention are shown, wherein: (a) the genus-level microbial community structure is shown, and (b) the response law of the relative abundance of functional microorganisms identified based on DNA-SIP as a function of C / N ratio is shown. Figure 5 This is a molecular ecological network diagram constructed based on DNA-SIP identification results in Embodiment 1 of the present invention, wherein: squares represent autotrophic microorganisms, rhombuses represent mixed-trophic microorganisms, circles represent heterotrophic microorganisms, dashed lines represent positively correlated microorganisms, and solid lines represent negatively correlated microorganisms. Detailed Implementation

[0037] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0038] Example 1: (1) Construction and start-up of iron autotrophic and heterotrophic mixed nutrient denitrification system: The biofilm of the iron autotrophic denitrification biological filter was used as inoculum sludge, with an initial inoculum sludge concentration of 4000-5000 mg / L; artificial water was prepared with Fe(II) as inorganic electron donor, sodium bicarbonate as inorganic carbon source, and sodium nitrate as the only nitrogen source. The inorganic electron donors Fe(II) and NO3 participating in the reaction were... - The molar ratio of -N was controlled within the range of (4:1) to (6:1), the mass concentration ratio of COD to total inorganic nitrogen (TIN) in the artificially prepared water was controlled within the range of 1.0-3.0, and the amount of sodium bicarbonate added was 0.5-1.0 g / L. An anaerobic sequencing batch reactor with a working volume of 4 L was started, and the reactor operating conditions were controlled as follows: hydraulic retention time (HRT) 24-48 hours, temperature 25-35℃, pH 7.5-8.0. An iron autotrophic denitrification system was constructed. When the nitrate nitrogen removal rate of the system effluent was maintained above 90% for 15 consecutive days, it was considered to have reached a stable denitrification state. After the system was in stable operation, sodium acetate was added as an organic carbon source to construct an iron autotrophic and heterotrophic mixed nutrient denitrification system. The system was operated for a total of 220 days and was divided into 4 operating stages according to the influent COD / TN ratio. The stages are designated as Stage I, Stage II, Stage III, and Stage IV, with corresponding COD / TN ratios of 0, 2.5, 1.5, and 2.0, respectively. The changes in influent and effluent nitrate nitrogen and nitrite nitrogen concentrations and denitrification efficiency during each stage of the entire operation are shown below. Figure 1 As shown.

[0039] (2) Isotope-labeled microcosm experiment: Sludge samples were collected during the stable operation of the iron autotrophic and heterotrophic mixed nutrient denitrification system in stage IV. After washing with distilled water to remove residual substrates, the samples were evenly divided into three anaerobic microcosm reaction flasks with a working volume of 250 mL. The following three parallel experiments were set up: control group added 12 Sodium C-acetate (based on COD / TN=2.0, TN=80mg / L, added concentration 149.5 mg / L) and 12 C-sodium bicarbonate (0.8 g / L) was used as a carbon source; autotrophic labeled groups were supplemented with 13 C-sodium bicarbonate (0.8 g / L) and 12 Sodium C-acetate (149.5 mg / L) was used as the carbon source; heterotrophic labeled groups were supplemented with 12 C-sodium bicarbonate (0.8 g / L) and 13 Sodium C-acetate (149.5 mg / L) was used as the carbon source. Fe(II) (1500 mg / L) was supplemented as the inorganic electron donor in all three experiments. The culture conditions of each microcosm system were consistent with those of the system constructed in step (1). The experiment was conducted for a total of 8 cycles, and the changes in nitrate nitrogen concentration in the influent and effluent during each cycle are as follows: Figure 2 As shown; (3) Nucleic acid isolation and density gradient centrifugation: After the 8th culture cycle, 50 mL of mud-water mixture samples from each micro-universe were taken, washed, and the supernatant was removed. The sludge samples were then freeze-dried at -50℃ for 72 hours, and DNA was extracted. 5 μg of DNA from each of the control group, autotrophic labeling group, and heterotrophic labeling group was mixed with a cesium chloride solution (1.85 g / mL) prepared with GB buffer (containing 0.1 M Tris-HCl, 0.1 M KCl, and 1.0 mM EDTA, with deionized water as the solvent and a pH of 8.0; the buffer was filtered through a 0.2 μm filter and sterilized before use). The refractive index of the mixture was adjusted to 1.4029 ± 0.0002, corresponding to a buoyancy density of 1.725 g / mL, and transferred to ultracentrifuge tubes. Ultracentrifugation density gradient centrifugation was performed at 45,000 rpm for 44 hours at 20℃. During centrifugation, maximum acceleration is used at startup, and braking is not applied during termination, allowing the rotor to stop naturally due to its own friction, thus minimizing disturbance to the established density gradient. After centrifugation, DNA fractions from 14 different density zones are collected sequentially from bottom to top of the tube. (4) Identification of heavy chain DNA and differentiation of functional microorganisms: DNA fractions were precipitated using polyethylene glycol 6000 solution, and the precipitate was washed with 70% ethanol solution to purify the DNA in each density fraction. Finally, 30 μL of TE buffer was added to dissolve the DNA. Real-time quantitative PCR was used to quantify the denitrification functional genes narG and nirK in DNA of each density zone. By comparing the distribution of functional genes in different density zones of each group, the DNA was identified as a functional gene. 13 Locations of c-DNA enrichment. For example... Figure 3 As shown, 13 c-DNA is mainly enriched in the band with a buoyant density of 1.73 g / mL to 1.75 g / mL. (5) Analysis of functional microbial composition: DNA corresponding to the buoyancy density bands of 1.73 g / mL-1.75 g / mL was merged and 16S rRNA gene amplicon sequencing was performed. The raw data obtained from sequencing was split, filtered for quality control and spliced ​​with paired-end sequences. The high-quality spliced ​​sequences were clustered into operational taxonomic units (OTUs) at a similarity level of 97%, and chimeric sequences were removed. The representative OTU sequences were compared with the SILVA 16S rRNA database to complete species annotation. Based on the annotation results, the taxonomic information and relative abundance of each sample at the genus level were statistically analyzed to analyze the genus-level community composition and abundance distribution of iron autotrophic and heterotrophic functional microorganisms.

[0040] Microorganisms significantly enriched in the autotrophic marker heavy chain DNA were identified as iron-autotrophic denitrifying microorganisms, and microorganisms significantly enriched in the heterotrophic marker heavy chain DNA were identified as heterotrophic denitrifying microorganisms. For example... Figure 4 As shown in a, in the iron-autotrophic and heterotrophic mixed nutrient denitrification system, Thaurea and Ignavibacterium are heterotrophic functional bacteria, while Thiobacillus and Hydrogenophilaceae are autotrophic functional bacteria.

[0041] Example 2: (1) DNA was extracted from the reactor sludge operating under C / N ratios of 0, 1.5, 2.0, and 2.5. High-throughput sequencing was performed on the V3-V4 region of the 16S rRNA gene. Based on the functional microbial nutrient types determined in step (4) of Example 1, the response of the relative abundance of Thaurea, Ignavibacterium, Thiobacillus, and Hydrogenophilaceae to changes in C / N ratio was analyzed. Figure 4 As shown in b, the relative abundance of heterotrophic bacteria Thaurea and Ignavibacterium increases with increasing C / N ratio, while the relative abundance of autotrophic bacteria Thiobacillus and Hydrogenophilaceae decreases.

[0042] (2) Molecular ecological network construction and key species identification: Based on stochastic matrix theory, a molecular ecological network was constructed at the genus level, and network visualization was achieved using Cytoscape software. Iron-autotrophic, heterotrophic, and mixed-trophic microorganisms were divided into three independent functional modules. For example... Figure 5As shown, in the network diagram, square nodes represent autotrophic microorganisms, circular nodes represent heterotrophic microorganisms, and diamond nodes represent mixed-trophic microorganisms. Nodes represent microbial species, and the edges connecting nodes represent inter-species interactions, with dashed lines indicating positive correlations and solid lines indicating negative correlations. Node size is proportional to its degree centrality, reflecting the extent of direct interaction between the species and other microorganisms. Larger nodes indicate higher degree centrality and are considered key species in the network. In this embodiment, the node with the highest degree centrality in the autotrophic module is Aquabacterium, followed by Hydrogenophilaceae. While Thiobacillus, identified by DNA-SIP, is a key autotrophic functional bacterium, its low degree centrality indicates weaker interactions with other microorganisms, which may be a significant reason for its exclusion in the mixed-trophic system. In the heterotrophic module, in addition to Thaurea and Ignavibacterium, quantitative analysis of node topology attributes revealed that Denitratisoma also plays a crucial role. In the mixed nutrient module, Limnobacter, Gemmatimonadaceae, and Sutterellaceae were identified as key species.

[0043] (3) Key Species Interaction Analysis: Taking the identified key species Ignavibacterium as an example, its interactions with other microorganisms were analyzed. The results showed that Ignavibacterium was positively correlated with Hydrogenophilaceae in the autotrophic module, possibly indicating a metabolic synergistic effect; within the heterotrophic module, it was positively correlated with Thaurea but negatively correlated with Denitratisoma, reflecting functional differentiation within the heterotrophic microbial community; furthermore, Ignavibacterium showed negative correlations with Gemmatimonadaceae and Limnobacter in the mixed trophic module, suggesting competition or inhibition among microbial communities of different trophic types. These results further validated the effectiveness of molecular ecological networks in resolving complex microbial interactions.

[0044] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system, characterized in that, Includes the following steps: (1) Construction and start-up of iron autotrophic and heterotrophic mixed nutrient denitrification system: Iron autotrophic denitrification sludge was inoculated into an anaerobic sequencing batch reactor, artificial water was added, and the iron autotrophic denitrification system was started and operated to obtain an iron autotrophic denitrification system; after the iron autotrophic denitrification system was running stably, an organic carbon source was added to construct an iron autotrophic and heterotrophic mixed nutrient denitrification system; the iron autotrophic and heterotrophic mixed nutrient denitrification system was started and operated stably; the stable operation process of the iron autotrophic and heterotrophic mixed nutrient denitrification system was divided into four stages, namely stage I, stage II, stage III and stage IV; (2) Isotope-labeled microcosm culture: Collect the sludge in the stable state of the iron autotrophic and heterotrophic mixed nutrient denitrification system, and add the corresponding ingredients. 12 Sodium C-acetate and 12 C-Sodium bicarbonate, 13 Sodium C-acetate and 12 C-Sodium bicarbonate, 13 C-sodium bicarbonate and 12 Sodium C-acetate was used in isotope microcosm experiments, and the samples were cultured separately. (3) Nucleic acid separation and density gradient centrifugation: After the culture is completed, the sludge DNA sample of the isotope microcosm experiment is extracted; the sludge DNA sample is mixed with the cesium chloride solution prepared with GB buffer to obtain a mixture, and ultra-high speed density gradient centrifugation is performed. The centrifuged sample is further purified by layer to obtain DNA fractions of different density levels. (4) Identification and functional differentiation of heavy chain DNA: The DNA fraction was purified, and the narG and nirS genes in the DNA fraction were quantitatively analyzed by qPCR; by comparing the distribution of the narG and nirS genes, the heavy chain DNA was identified. 13 The enrichment location of C-DNA in the microbial community is the heavy chain DNA fraction in the DNA fraction. The evaluation is to determine whether the microorganisms enriched in the heavy chain DNA fraction are iron-autotrophic denitrifying microorganisms or heterotrophic denitrifying microorganisms. (5) Functional microbial composition analysis: The V3-V4 region of the 16S rRNA gene of the heavy chain DNA fraction was sequenced to complete species annotation; Based on the results of the species annotation, the taxonomic information and relative abundance of the microorganisms that play a role in the three sets of isotope microcosm experiments at the genus level were statistically analyzed, thereby resolving the genus-level community composition and abundance distribution of iron autotrophic and heterotrophic functional microorganisms.

2. The method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system according to claim 1, characterized in that, The concentration of the iron-autotrophic denitrifying sludge is 4000 mg / L-5000 mg / L.

3. The method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system according to claim 1, characterized in that, The artificial water preparation in step (1) uses ferrous iron as an inorganic electron donor, sodium bicarbonate as an inorganic carbon source, and sodium nitrate as the sole nitrogen source. The ferrous iron and NO3 in the iron autotrophic and heterotrophic mixed nutrient denitrification system mentioned above - The molar ratio of -N is controlled at 4~6:1, the mass concentration ratio of COD to total inorganic nitrogen in the artificially prepared water is controlled in the range of 1.0-3.0:1, and the amount of sodium bicarbonate added is 0.5 g / L-1.0 g / L.

4. The method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system according to claim 1, characterized in that, In step (1), the organic carbon source is sodium acetate; the operating conditions of the anaerobic sequencing batch reactor are: hydraulic retention time 24h-48h, temperature 25℃-35℃, pH 7.5-8.

0.

5. The method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system according to claim 1, characterized in that, The GB buffer solution in step (1) includes Tris-HCl, KCl, and EDTA; the concentration of Tris-HCl in the GB buffer solution is 0.1 M, the concentration of KCl is 0.1 M, and the concentration of EDTA is 1.0 mM. The pH value of the GB buffer solution is 7.8~8.

2.

6. The method for identifying functional microorganisms in a mixed nutrient denitrification system of iron autotrophic and heterotrophic as described in claim 1, characterized in that, The iron autotrophic and heterotrophic mixed nutrient denitrification system described in step (1) operates stably for 200 to 250 days; the iron autotrophic denitrification system operates stably specifically when the nitrate nitrogen removal rate of the system effluent is maintained above 90% for 15 consecutive days. The stable operation of the iron autotrophic and heterotrophic mixed nutrient denitrification system is specifically defined as follows: when the nitrate nitrogen removal rate of the effluent from the iron autotrophic and heterotrophic mixed nutrient denitrification system is maintained above 90% for 15 consecutive days, it is considered to have reached a stable operating state.

7. The method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system according to claim 1, characterized in that, The criterion for division in step (1) is the influent COD / TN, and the COD / TN values ​​corresponding to stage I, stage II, stage III and stage IV are 0, 2.5, 1.5 and 2.0 respectively.

8. The method for identifying functional microorganisms in a mixed nutrient denitrification system of iron autotrophic and heterotrophic as described in claim 1, characterized in that, Add in step (2) 12 Sodium C-acetate and 12 The treatment with C-sodium bicarbonate served as a control group; [Addition] 13 C-sodium bicarbonate and 12 Treatment with C-acetic acid sodium as an autotrophic marker group; addition 12 C-sodium bicarbonate and 13 Treatment with C-acetic acid sodium was used as a heterotrophic marker group; The operating conditions for the control group, the autotrophic labeling group, and the heterotrophic labeling group were: hydraulic retention time of 24-48 hours, temperature of 25-35°C, and pH of 7.5-8.

0.

9. The method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system according to claim 1, characterized in that, The target refractive index of the mixture in step (3) is 1.4027~1.4031, and the buoyancy density is 1.723 g / mL~1.728 g / mL.

10. The method for identifying functional microorganisms in a mixed ferroautotrophic and heterotrophic denitrification system according to claim 1, characterized in that, The purification process in step (4) specifically involves precipitating the DNA fraction with polyethylene glycol 6000 solution and washing the precipitate with 70% ethanol solution (by volume).

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