Segmented regulation and control sulfur cycle enhanced denitrification microbial community construction method

Abstract

The invention discloses a staged regulation and control sulfur cycle enhanced denitrification microbial community construction method, and belongs to the technical field of environmental biologication.The method comprises the three stages that in the first stage, low-carbon-nitrogen-ratio domestication culture is conducted, the carbon-nitrogen ratio is 1.5-2.5: 1, and domestication is conducted for 50-70 days; in the second stage, electron donor-acceptor ratio regulation culture is carried out, the / molar ratio is (4-6): 1, and culture is carried out for 15-25 days; in the third stage, pulse type sulfate is fed for enhanced culture, the single-time feeding concentration is 200-500 mg / L, culture is conducted for 8-15 days, real-time monitoring is conducted through the fluorescence in-situ hybridization technology, construction is completed when the abundance of sulfur autotrophic denitrifying bacteria reaches 28% or above, and when the microflora constructed through the method is used for treating low-carbon-nitrogen-ratio industrial wastewater, the sulfur conversion rate is 1.2-2.0 kg / (m.d), the system stability is good, and the application range is wide. And the impact load resistance is high.

Description

Technical Field

[0001] This invention belongs to the field of environmental biotechnology, specifically relating to a method for constructing a microbial community for enhanced denitrification through segmented regulation of the sulfur cycle, and particularly a microbial community construction technology that achieves targeted enrichment of sulfur autotrophic denitrifying bacteria through a multi-stage domestication strategy and precise regulation of the sulfur cycle. Background Technology

[0002] Biological denitrification of nitrogen-containing wastewater is an important topic in environmental engineering. Traditional biological denitrification technologies mainly rely on heterotrophic denitrification processes, which require sufficient organic carbon sources as electron donors. However, many industrial wastewaters, such as chemical wastewater, pharmaceutical wastewater, and electroplating wastewater, generally have low carbon-to-nitrogen ratios. Using traditional heterotrophic denitrification processes requires the addition of large amounts of organic carbon sources, which not only increases operating costs but may also lead to secondary pollution.

[0003] Chinese patent application CN113023901A discloses a method for constructing a salt-tolerant microbiome. This method constructs a highly efficient integrated carbon, nitrogen, and phosphorus removal salt-tolerant microbiome through an induction and acclimatization stage and an enrichment stage. The induction and acclimatization stage employs operating parameters of low nitrogen loading, high carbon-to-nitrogen ratio (≥20), and a relatively long hydraulic retention time. The enrichment stage employs high ammonia nitrogen loading, a high carbon-to-nitrogen ratio (≥20), and a short hydraulic retention time to rapidly enrich heterotrophic functional microorganisms. This method has achieved good results in treating high-salinity wastewater, with a start-up time of 10-20 days, achieving 100% removal efficiency for ammonia nitrogen and total nitrogen, and over 85% removal efficiency for total phosphorus.

[0004] However, the above-mentioned technology still has the following shortcomings: This method is mainly based on heterotrophic denitrification mechanism, which requires maintaining a high carbon-to-nitrogen ratio (≥20). For industrial wastewater with a low carbon-to-nitrogen ratio, a large amount of organic carbon source still needs to be added. This method does not involve the synergistic effect of the sulfur cycle in the denitrification process and cannot make full use of the reduced sulfides in the wastewater as electron donors. This method lacks the means for targeted enrichment and real-time monitoring of specific functional microorganisms, making it difficult to accurately control the microbial community structure. This method has limited effect on the treatment of industrial wastewater with severe carbon source deficiency, and the system stability and resistance to shock loads need to be improved.

[0005] In recent years, sulfur autotrophic denitrification technology has received widespread attention due to its advantages such as not requiring an external organic carbon source, low sludge production, and no secondary pollution. Sulfur autotrophic denitrification refers to the process by which sulfur-oxidizing bacteria utilize reduced sulfur (…) under anoxic or anaerobic conditions. , , (etc.) as electron donors, to It acts as an electron acceptor, acquiring energy through the redox reaction of sulfur. The process of reducing to nitrogen gas. Key functional bacteria include Thiobacillus denitrificans and sulfur-oxidizing bacteria of the genus Sulfurovum.

[0006] The main reaction equation for sulfur autotrophic denitrification is:

[0007]

[0008]

[0009]

[0010] However, the slow growth rate, long acclimatization period, and difficulty in building microbial communities of sulfur autotrophic denitrifying microorganisms have hindered the practical application of this technology. The start-up period of traditional sulfur autotrophic denitrification systems usually takes 2-3 months or even longer, and the microbial community structure is unstable and easily affected by fluctuations in organic carbon sources and dissolved oxygen concentrations, leading to competitive inhibition of sulfur autotrophic denitrifying bacteria by heterotrophic microorganisms.

[0011] Therefore, there is an urgent need to develop an efficient method for constructing sulfur autotrophic denitrifying microbial communities. Through precise segmented acclimatization strategies and sulfur cycle regulation mechanisms, the method can achieve targeted enrichment of sulfur autotrophic denitrifying bacteria and stable construction of microbial communities to meet the demand for efficient nitrogen removal treatment of industrial wastewater with low carbon-to-nitrogen ratios. Summary of the Invention

[0012] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a method for constructing a segmented, regulated sulfur cycle-enhanced denitrification microbial community. This method employs an innovative three-stage acclimatization strategy—adaptive acclimatization to low carbon-to-nitrogen ratios, precise regulation of the electron donor-acceptor ratio, and enhanced pulsed sulfate dosing—combined with real-time monitoring using fluorescence in situ hybridization (FISH) technology. This achieves efficient and targeted enrichment of sulfur-autotrophic denitrifying bacteria, constructing a stable and efficient denitrification microbial community, effectively solving the denitrification problem in low carbon-to-nitrogen ratio industrial wastewater.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] This invention provides a method for constructing a segmented, regulated sulfur cycle-enhanced denitrification microbial community, the method comprising the following steps:

[0015] The first stage of acclimatization culture with a low carbon-to-nitrogen ratio: The original inoculum sludge containing sulfur-oxidizing bacteria and denitrifying bacteria is inoculated into a culture medium with a carbon-to-nitrogen ratio of 1.5:1 to 2.5:1 and a nitrate nitrogen concentration of 40-80 mg / L. The concentration was 10-30 mg / L, the temperature was controlled at 25-35℃, the pH was controlled at 7.0-8.0, the aeration rate was controlled at a dissolved oxygen concentration of 0.3-0.8 mg / L, the hydraulic retention time was 18-30 h, and the acclimatization period was 50-70 days. During this stage, the excessive proliferation of heterotrophic denitrifying bacteria was inhibited by low carbon-to-nitrogen ratio conditions, while providing an appropriate amount of reduced sulfides to promote the initial enrichment and adaptation of sulfur-autotrophic denitrifying bacteria.

[0016] The second stage of electron donor-acceptor ratio regulation culture: Based on the first stage of acclimatization culture, the molar ratio of electron donors to electron acceptors in the culture medium is adjusted to... and The molar ratio is 4:1 to 6:1, and the nitrate nitrogen concentration is increased to 80-120 mg / L. Increase the concentration to 50-100 mg / L, and supplement simultaneously. The concentration was 30-80 mg / L, the temperature was controlled at 28-32℃, the pH was controlled at 7.2-7.8, the aeration rate was controlled at a dissolved oxygen concentration of 0.2-0.5 mg / L, the hydraulic retention time was 12-20 h, and the incubation time was 15-25 days. This stage optimized the metabolic environment of sulfur-autotrophic denitrifying bacteria by precisely controlling the stoichiometric ratio of electron donors and acceptors, thus promoting the rapid proliferation of functional bacterial communities.

[0017] The third stage is pulsed sulfate addition for enhanced culture: Based on the second stage culture, sulfate is added to the culture medium in a pulsed manner, with a single addition. The concentration is 200-500 mg / L, the dosing interval is 4-8 hours, the dosing cycle is 8-15 days, the nitrate nitrogen concentration is maintained at 100-150 mg / L, the temperature is controlled at 28-32℃, the pH is controlled at 7.0-7.5, the aeration rate is controlled at a dissolved oxygen concentration of 0.1-0.4 mg / L, and the hydraulic retention time is 10-16 hours. This stage simulates the dynamic changes of the sulfur cycle through pulsed sulfate dosing, further enhancing the competitive advantage of sulfur-autotrophic denitrifying bacteria and improving the stability and resistance to shock loads of the microbial community.

[0018] Meanwhile, during the first, second, and third stages of cultivation, fluorescence in situ hybridization was used to monitor the abundance changes of sulfur autotrophic denitrifying bacteria in real time. When the total abundance of Thiobacillus denitrificans and Sulfurovum sulfur autotrophic denitrifying bacteria reached more than 28%, the microbial community construction was completed.

[0019] Furthermore, the initial inoculated sludge is selected from any one or a mixture of the following: activated sludge from municipal wastewater treatment plants, sludge from industrial wastewater treatment systems, marine sediments, saline lake sediments, sediments from sulfide-rich environments, and mine drainage sediments. Preferably, a mixed sludge containing natural sulfur-oxidizing bacteria and denitrifying bacteria is used, which can shorten the acclimatization period.

[0020] Furthermore, in the first stage of low carbon-nitrogen ratio acclimatization culture, the culture medium composition is: sodium acetate as an organic carbon source, with a concentration of 80-160 mg / L; As a nitrogen source for nitrates, the concentration is 40-80 mg / L; As a sulfide source, the concentration is 10-30 mg / L; The concentration is 5-15 mg / L; The concentration is 20-50 mg / L; The concentration is 10-30 mg / L; the trace element solution is 1-3 mL / L, wherein the trace element solution contains , , , , , , .

[0021] Furthermore, in the second stage of electron donor-acceptor ratio regulation culture, the composition of the culture medium is as follows: the sodium acetate concentration is reduced to 40-80 mg / L; The concentration was increased to 80-120 mg / L; Increase the concentration to 50-100 mg / L; supplement The concentration is 30-80 mg / L; The inorganic carbon source was prepared at a concentration of 100-200 mg / L; the remaining components were the same as in the first stage.

[0022] Furthermore, in the third stage of pulsed sulfate dosing enhanced culture, the pulsed dosing employs an automatic control system, based on the conditions in the reactor. Concentration and Real-time monitoring results of concentration, when Concentration below 30 mg / L or Automatic dosing when concentration is below 100 mg / L Solution, single dosage The concentration was increased by 200-500 mg / L.

[0023] Furthermore, the monitoring steps of the fluorescence in situ hybridization technique include: collecting sludge samples from the reactor and fixing them with 4% paraformaldehyde fixative for 2-4 hours; washing them three times with phosphate buffer and then dehydrating them with ethanol at different concentration gradients; hybridizing them with the specific probe Tdeni-438 (5'-AAGGCCGCTTTCGAACG-3') for Thiobacillus denitrificans and the specific probe Sulfu-654 (5'-TCCACTGCTGGCTCGTT-3') for Sulfurovum at a hybridization temperature of 46-50℃ for 2-4 hours; and observing and counting the target bacteria using a laser confocal microscope or a fluorescence microscope to determine the proportion of the target bacteria in the total bacterial population.

[0024] Furthermore, during the first stage of acclimatization and cultivation, the microbial community structure is tested every 5-7 days. When the abundance of sulfur autotrophic denitrifying bacteria reaches 15% or more, the second stage begins. During the second stage of cultivation, the microbial community structure is tested every 3-5 days. When the abundance of sulfur autotrophic denitrifying bacteria reaches 25% or more, the third stage begins.

[0025] Furthermore, the following regulatory measures are also included in the three-stage cultivation process: when detected... When the accumulated concentration exceeds 10 mg / L, reduce the influent load by 20-40% and extend the hydraulic retention time by 2-6 hours; when the pH value is below 6.8, add... solution or Adjust the solution pH to 7.0-7.5; when the dissolved oxygen concentration exceeds the upper limit of the set range, reduce the aeration rate by 15-30%; when the abundance of sulfur autotrophic denitrifying bacteria shows a downward trend in two consecutive tests, appropriately increase the aeration rate. Add 10-25% and reduce the amount of organic carbon source by 20-35%.

[0026] Furthermore, the cultivation device used in the method is a sequencing batch reactor or a continuous flow reactor with a reactor volume of 1-20L, an inoculated sludge concentration of 2-6g / L, a sludge age controlled at 15-30 days, a temperature control system using water bath heating or air heating, a pH control system using an automatic acid and alkali addition device, and an aeration system using microporous aeration or membrane aeration.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention achieves efficient and targeted enrichment of sulfur-autotrophic denitrifying bacteria through an innovative three-stage acclimatization strategy. The first stage, low C / N ratio acclimatization, effectively suppresses the competitive advantage of heterotrophic microorganisms, creating a favorable growth environment for sulfur-autotrophic denitrifying bacteria. The second stage, precise regulation of the electron donor-acceptor ratio optimizes the metabolic conditions of sulfur-autotrophic denitrifying bacteria, promoting rapid proliferation of functional bacteria. The third stage, pulsed sulfate addition, simulates the dynamic changes of the sulfur cycle in actual wastewater, enhancing the stability of the microbial community and its resistance to shock loads. Compared with prior art, this invention is applicable to wastewater with a low C / N ratio of 1:1 to 3:1, requires no additional large amounts of organic carbon sources, and significantly reduces operating costs.

[0029] This invention employs fluorescence in situ hybridization (FISH) technology to monitor the abundance of sulfur-autotrophic denitrifying bacteria in real time, enabling precise regulation of the microbial community construction process. Specific probes are used to quantitatively detect changes in the abundance of *Thiobacillus denitrificans* and *Sulfurovum* sulfur-autotrophic denitrifying bacteria, allowing for timely adjustments to culture conditions and ensuring the targeted enrichment of the target bacterial population. The final constructed microbial community achieved a sulfur-autotrophic denitrifying bacteria abundance exceeding 30%, significantly higher than the naturally occurring 5-10% abundance level.

[0030] The microbial community constructed in this invention exhibits excellent nitrogen removal performance when treating industrial wastewater with a low carbon-to-nitrogen ratio. The nitrogen removal efficiency reaches 85-95%. The removal rate is 0.8-1.5 kg / (m³·d), which is 45% higher than that of traditional sulfur autotrophic denitrification systems; the sulfur conversion rate is 1.2-2.0 kg / (m³·d), which is 60% higher than that of the comparative method. The oxidation rate reaches 92-98%, effectively avoiding secondary pollution from sulfides; the system has stable denitrification performance for wastewater with a carbon-to-nitrogen ratio of 1:1 to 3:1 and strong resistance to shock loads.

[0031] The method of this invention is simple and easy to implement, and readily applicable to engineering. The entire acclimation process can be completed in a sequencing batch reactor or a continuous flow reactor, without the need for complex equipment and operations; an automatic control system is used to achieve pulsed dosing and real-time regulation, ensuring stable and reliable operation; the constructed microbial community exhibits good stability, long sludge age, and ease of maintenance and management; it is suitable for denitrification treatment of various low C / N ratio industrial wastewaters from chemical, pharmaceutical, electroplating, and dyeing industries, and has broad application prospects.

[0032] This invention achieves efficient coupling of the sulfur and nitrogen cycles. By segmenting and controlling the sulfate addition strategy, the redox cycle of sulfur is promoted, which not only improves denitrification efficiency but also realizes the resource utilization of reduced sulfides in wastewater; it avoids the reactor blockage problem caused by the accumulation of elemental sulfur in traditional sulfur autotrophic denitrification; and it reduces the emission of greenhouse gases such as N2O, resulting in good environmental benefits. Detailed Implementation

[0033] The present invention will be described in detail below with reference to specific embodiments, but is not limited to these embodiments. Those skilled in the art should understand that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all commercially available conventional products.

[0034] Example 1: Typical Implementation of a Method for Constructing a Denitrification Microbial Community by Segmented Regulation of the Sulfur Cycle

[0035] Phase 1: Acclimatization to low carbon-nitrogen ratio (days 1-60)

[0036] Take 3L of activated sludge from the secondary sedimentation tank of a municipal wastewater treatment plant, centrifuge and concentrate it to a sludge concentration of 5g / L, and use it as inoculum sludge. Inoculate it into a 10L sequencing batch reactor with an initial working volume of 8L.

[0037] Culture medium composition: sodium acetate 120 mg / L (approximately 192 mg / L based on COD); 60 mg / L (approximately 54 mg / L based on COD); 20 mg / L (approximately 6.7 mg / L based on COD); 10 mg / L; 35 mg / L; 20 mg / L; 300 mg / L; trace element solution 2 mL / L.

[0038] Trace element solution formulations: 5 g / L; 0.5 g / L; 0.2 g / L; 0.1 g / L; 0.05 g / L; 0.05 g / L;

[0039] 0.05 g / L; adjust the pH to 2.0-3.0 with 1 mol / L hydrochloric acid to prevent metal ion precipitation.

[0040] The carbon-to-nitrogen ratio of the culture medium (expressed as COD / ) The ratio is approximately 2:1.

[0041] Operating parameters: Temperature controlled at 30±1℃; pH controlled at 7.5±0.3, with automatic addition of 0.1mol / L. Solution or 0.1 mol / L Solution conditioning; aeration is carried out using microporous aeration heads, with the aeration rate controlled at a dissolved oxygen concentration of 0.5±0.2 mg / L; hydraulic retention time is 24 h; each operating cycle consists of 23 h of aeration, 0.5 h of sedimentation, and 0.5 h of influent and effluent, with a volume exchange ratio of 50%.

[0042] Microbial community monitoring: 20 mL of sludge sample was collected every 7 days, and the abundance of sulfur-autotrophic denitrifying bacteria was detected using fluorescence in situ hybridization (FITC). The specific steps were as follows: 5 mL of sludge sample was washed twice with phosphate buffer, then 5 mL of 4% paraformaldehyde fixative was added and fixed at room temperature for 3 h; the sample was washed three times with phosphate buffer, 5 min each time; dehydrated sequentially with 50%, 80%, and 95% ethanol for 10 min each; hybridization was performed using the Tdeni-438 probe (5'-AAGGCCGCTTTCGAACG-3', Cy3-labeled) and the Sulfu-654 probe (5'-TCCACTGCTGGCTCGTT-3', FITC-labeled) at 48℃ for 3 h; the hybridization buffer formulation was 0.9 mol / L. The samples were prepared with 20 mmol / L Tris-HCl (pH 7.5), 0.01% SDS, and 35% formamide. After hybridization, the samples were washed with washing buffer (20 mmol / L Tris-HCl, 5 mmol / L EDTA, 0.01% SDS, pH 7.5) at 50°C for 15 min. The samples were observed and counted using a laser confocal microscope. 15-20 fields of view were randomly selected from each sample, and the proportion of hybridization-positive cells to total cells (DAPI staining) was counted.

[0043] Water quality monitoring: Daily testing of influent and effluent. , , COD , Concentration, as well as parameters such as pH, dissolved oxygen, and redox potential.

[0044] Acclimation effect: Days 1-20 are the start-up period, with nitrogen removal efficiency of 30-45% and sulfur autotrophic denitrifying bacteria abundance gradually increasing from the initial 2.3% to 5.8%; Days 21-40 are the stabilization period, with nitrogen removal efficiency stabilizing at 50-60% and sulfur autotrophic denitrifying bacteria abundance increasing to 9.2-12.5%; Days 41-60 are the enrichment period, with nitrogen removal efficiency increasing to 65-75% and sulfur autotrophic denitrifying bacteria abundance reaching 15.8%.

[0045] On day 60, the test results were: Removal rate 72%, effluent Concentration 15.1 mg / L; Accumulated concentration: 3.2 mg / L; The oxidation rate was 85%; the abundance of *Thiobacillus denitrificans* was 10.2%, the abundance of *Sulfurovum* was 5.6%, and the total abundance was 15.8%. This meets the conditions for entering the second stage.

[0046] Phase 2: Electron donor-acceptor ratio regulation culture (days 61-80)

[0047] Based on the first stage of acclimatization and cultivation, the composition of the culture medium and operating parameters were adjusted.

[0048] Culture medium composition: Sodium acetate reduced to 60 mg / L (approximately 96 mg / L based on COD); Increase to 100 mg / L (with (approximately 90 mg / L) Increase to 75 mg / L (with (Approximately 25 mg / L); supplemented with 55 mg / L (approximately 22 mg / L); increased to 150 mg / L as an inorganic carbon source; the remaining components are the same as in the first stage.

[0049] at this time, and The molar ratio is approximately 5:1. :25mg / L÷32g / mol=0.78mmol / L; 90 mg / L ÷ 14 g / mol = 6.43 mmol / L; but considering It can also act as an electron donor, with a total sulfur to nitrogen molar ratio close to 5:1.

[0050] Operating parameters: Temperature controlled at 30±1℃; pH controlled at 7.5±0.2; aeration rate reduced, dissolved oxygen concentration controlled at 0.3±0.1mg / L; hydraulic retention time shortened to 16h; each operating cycle consists of 15h aeration, 0.5h sedimentation, 0.5h influent and effluent, and a volume exchange ratio of 50%.

[0051] Microbial community monitoring: The abundance of sulfur autotrophic denitrifying bacteria was measured every 5 days, using the same method as in the first stage.

[0052] Control strategy: When detected When the accumulated concentration exceeds 10 mg / L (appearing on day 66), (Concentration 12.5 mg / L), immediately reduce the influent load by 30%, extend the hydraulic retention time to 20 hours, and after 3 days of adjustment... Accumulation was brought under control, decreasing to 5.2 mg / L; normal operation resumed on day 69. Automatic dosing resumed when the pH dropped to 6.7 (day 72). Adjust the pH of the solution (100g / L) to 7.3.

[0053] Enrichment effect: From day 61 to 70, nitrogen removal efficiency increased to 78-85% and sulfur autotrophic denitrifying bacteria abundance increased to 18.5-22.3%; from day 71 to 80, nitrogen removal efficiency stabilized at 85-90% and sulfur autotrophic denitrifying bacteria abundance reached 26.2%.

[0054] On day 80, the test results were: Removal rate 88%, effluent Concentration 10.8 mg / L; Accumulated concentration: 2.8 mg / L; Oxidation rate 91%; The oxidation rate was 87%; the abundance of *Thiobacillus denitrificans* was 17.8%, the abundance of *Sulfurovum* was 8.4%, and the total abundance was 26.2%. This meets the conditions for entering the third stage.

[0055] Phase 3: Intensive culture with pulsed sulfate dosing (days 81-92)

[0056] Based on the second stage of cultivation, a pulsed sulfate dosing strategy was adopted.

[0057] The basal culture medium composition is maintained at 60 mg / L of sodium acetate. Increase to 125 mg / L (with (approximately 113 mg / L) Maintain at 75 mg / L; Maintain at 55 mg / L; Maintain at 150 mg / L; the remaining components are the same as in the second phase.

[0058] Operating parameters: Temperature controlled at 30±1℃; pH controlled at 7.2±0.3; aeration rate further reduced, dissolved oxygen concentration controlled at 0.2±0.1mg / L; hydraulic retention time shortened to 12h; each operating cycle consists of 11h aeration, 0.5h sedimentation, 0.5h influent and effluent, and a volume exchange ratio of 50%.

[0059] Pulse Dosing strategy: An automatic control system is used to monitor the reactor online. and Concentration. When Concentration below 30 mg / L or Automatic dosing when concentration is below 100 mg / L The solution (concentration 50 g / L), the single dosage in the reactor The concentration was increased by 350 mg / L. The dosing interval was approximately 6 hours.

[0060] Pulse dosing mechanism: The sulfur produced during the autotrophic denitrification process It can be reduced to sulfate by sulfate-reducing bacteria in an anaerobic microenvironment. or The sulfur then acts as an electron donor for sulfur autotrophic denitrification, forming a sulfur redox cycle. Pulsed dosing simulates this dynamic cycle, which is beneficial for the synergistic effect of sulfur autotrophic denitrifying bacteria and sulfate-reducing bacteria.

[0061] Microbial community monitoring: The abundance of sulfur autotrophic denitrifying bacteria was measured every 3 days, using the same method as in the first stage.

[0062] Enhanced effect: From day 81 to 86, nitrogen removal efficiency increased to 90-93% and sulfur autotrophic denitrifying bacteria abundance increased to 28.5-29.8%; from day 87 to 92, nitrogen removal efficiency stabilized at 93-95% and sulfur autotrophic denitrifying bacteria abundance reached over 30.5%.

[0063] On day 92, the test results were: Removal rate 94%, effluent Concentration 6.8 mg / L; Accumulated concentration: 1.2 mg / L; Oxidation rate 95%; Oxidation rate 93%; The removal rate was 1.13 kg / (m³·d); the sulfur conversion rate was 1.68 kg / (m³·d); the abundance of *Thiobacillus denitrificans* was 21.3%, the abundance of *Sulfurovum* was 9.8%, and the total abundance was 31.1%. The microbial community was successfully constructed.

[0064] Microbial community structure analysis: High-throughput sequencing technology was used to perform 16S rRNA gene sequencing analysis on the constructed microbial community. The results showed that at the phylum level, Proteobacteria accounted for 65.2%, Bacteroidetes for 12.5%, Firmicutes for 8.3%, Chloroflexi for 5.8%, and other phyla for 8.2%. At the genus level, sulfur autotrophic denitrification-related genera included: *Thiobacillus* (21.3%), *Sulfurovum* (9.8%), *Thiohalophilus* (3.2%), and *Sulfurimonas* (2.8%); denitrification-related genera included: *Thauera* (7.5%), *Paracoccus* (4.2%), and *Denitratisoma* (3.1%); sulfate-reducing genera included: *Desulfovibrio* (5.8%) and *Desulfobulbus* (3.5%).

[0065] This microbial community construction method, through three stages and a total of 92 days of acclimatization, successfully achieved the efficient enrichment of sulfur autotrophic denitrifying bacteria. The total abundance of sulfur autotrophic denitrifying bacteria increased from the initial 2.3% to 31.1%, a 13.5-fold increase. The constructed microbial community exhibited excellent denitrification performance, with a nitrogen removal efficiency of 94%, making it suitable for denitrification treatment of industrial wastewater with a low carbon-to-nitrogen ratio.

[0066] Example 2: Application of segmented regulation of sulfur cycle to enhance denitrification microbial community in the treatment of industrial wastewater with low carbon-to-nitrogen ratio

[0067] Two liters of the microbial community sludge constructed in Example 1 were inoculated into a 10-liter continuous flow reactor to treat low carbon-to-nitrogen ratio industrial wastewater discharged by a chemical company.

[0068] Wastewater quality: COD 120-180 mg / L; 90-130 mg / L; 10-20 mg / L; 15-35 mg / L; 200-400 mg / L; pH 6.8-7.5; C / N ratio (as COD / The ratio (calculated) is approximately 1.3:1 to 2:1.

[0069] Operating parameters: Temperature controlled at 28-32℃; pH controlled at 7.0-7.5, by adding... Adjustments were made: aeration rate was controlled at dissolved oxygen concentration of 0.2-0.4 mg / L; hydraulic retention time was 12 h; sludge concentration was maintained at 4-5 g / L; and sludge age was 20 days.

[0070] To supplement the electron donor, add to the influent. Solution, making Increase the concentration by 20-40 mg / L to ensure sufficient electron donors. Add via pulse dosing every 8 hours. The solution is added once, and the amount added in a single batch makes the reactor... The concentration was increased by 300 mg / L.

[0071] Operational results (statistics after 30 days of stable operation): Water output Concentration 6.5-12.8 mg / L, average 9.2 mg / L, nitrogen removal efficiency 88-93%, average 91%; effluent The concentration ranged from 0.8 to 2.5 mg / L, with an average of 1.5 mg / L, and no significant accumulation was observed. The effluent COD concentration ranged from 20 to 35 mg / L, with an average of 27 mg / L, and a COD removal rate of 82-88%. Concentrations ranged from 0.5 to 1.8 mg / L, with an average of 1.1 mg / L. Oxidation rate 95-98%; The removal rate was 1.15-1.42 kg / (m³·d), with an average of 1.28 kg / (m³·d); the sulfur conversion rate was 1.45-1.85 kg / (m³·d), with an average of 1.65 kg / (m³·d).

[0072] Shock load resistance test: On the 20th day, water will be introduced. The concentration suddenly increased to 180 mg / L, lasting for 24 hours. The system showed [further symptoms] within 2 hours. If the concentration accumulates (reaching 8.5 mg / L), immediately reduce the influent load by 30% and extend the hydraulic retention time to 16 hours, while simultaneously increasing... The dosage was increased to 60 mg / L. After 12 hours of adjustment, the system returned to normal. When the concentration dropped to 3.2 mg / L, the nitrogen removal efficiency recovered to 85%. After 24 hours, normal operating conditions were restored, and the system fully recovered to pre-shock levels within 48 hours. This indicates that the constructed microbial community has good resistance to shock loads.

[0073] Economic Analysis: Compared with traditional heterotrophic denitrification processes, the method of this invention does not require the addition of a large amount of organic carbon source (saving approximately RMB 0.8 / m³ of carbon source addition cost in wastewater); sludge production is reduced by approximately 40% (saving approximately RMB 0.3 / m³ of sludge treatment cost in wastewater); it fully utilizes the reduced sulfides in the wastewater (saving approximately RMB 0.2 / m³ of sulfur source addition cost in wastewater); and the overall operating cost is reduced by approximately RMB 1.1-1.3 / m³ of wastewater. The investment payback period is approximately 8-12 months.

[0074] Example 3: Evaluation of the denitrification performance of microbial communities under different carbon-nitrogen ratios

[0075] The microbial community sludge constructed in Example 1 was used to measure the carbon-to-nitrogen ratio (in terms of COD / ) The denitrification performance was tested under conditions of 1:1, 1.5:1, 2:1, 2.5:1, and 3:1.

[0076] Experimental setup: A sequencing batch reactor with a volume of 2L, a working volume of 1.5L, and an inoculum sludge concentration of 4g / L was used. The concentration was uniformly set at 100 mg / L, and different carbon-to-nitrogen ratios were controlled by adjusting the sodium acetate concentration. (Supplement) Concentration 40 mg / L, pulse dosing The amount added at one time makes The concentration was increased by 300 mg / L. The temperature was 30℃, pH was 7.3, dissolved oxygen concentration was 0.3 mg / L, and hydraulic retention time was 12 h. Each condition was tested in triplicate, and the average value was calculated after 10 days of operation.

[0077] The experimental results are shown in Table 1.

[0078] Table 1. Nitrogen removal performance of microbial communities under different carbon-to-nitrogen ratios

[0079] Carbon-to-nitrogen ratio (COD / ) ) Out of water (mg / L) Nitrogen removal efficiency (%) Accumulation (mg / L) Removal rate [kg / (m³·d)] 1:1 12.5±1.8 87.5±1.8 3.5±0.8 1.05±0.08 1.5:1 9.2±1.2 90.8±1.2 2.2±0.5 1.21±0.06 2:1 6.8±0.9 93.2±0.9 1.5±0.4 1.33±0.05 2.5:1 5.5±0.8 94.5±0.8 1.2±0.3 1.42±0.04 3:1 5.2±0.7 94.8±0.7 1.0±0.3 1.45±0.05

[0080] The results showed that the constructed microbial communities exhibited good denitrification performance within a carbon-to-nitrogen ratio range of 1:1 to 3:1, with nitrogen removal efficiencies ranging from 87.5% to 94.8%. The denitrification performance was optimal at a carbon-to-nitrogen ratio of 2:1 to 3:1, with a nitrogen removal efficiency exceeding 93%. The accumulated concentration is below 1.5 mg / L. Even under extremely low carbon-to-nitrogen ratio (1:1) conditions, the nitrogen removal efficiency can still reach 87.5%, which is significantly better than the traditional heterotrophic denitrification process (which usually requires a carbon-to-nitrogen ratio ≥4:1).

[0081] Example 4: Application of FISH monitoring in the process of microbial community construction

[0082] Fluorescence in situ hybridization was used to systematically monitor the changes in the abundance of sulfur autotrophic denitrifying bacteria during the three stages of domestication in Example 1.

[0083] Probe design and validation: The Tdeni-438 probe (5'-AAGGCCGCTTTCGAACG-3', Cy3 marker) was used to specifically identify *Thiobacillus denitrificans*. This probe is located in the V3 region of the 16S rRNA gene. BLAST analysis confirmed its specificity, covering all known strains of *Thiobacillus denitrificans*, with sequence similarity to other genera below 85%. The Sulfu-654 probe (5'-TCCACTGCTGGCTCGTT-3', FITC marker) was used to specifically identify *Sulfurovum*. This probe is located in the V4 region of the 16S rRNA gene. Validation confirmed its specificity, covering all known species of *Sulfurovum*, with sequence similarity to other genera below 83%.

[0084] FISH optimization conditions: formamide concentration 35%, hybridization temperature 48℃, hybridization time 3h, washing temperature 50℃. Under these conditions, the probe hybridization efficiency reaches 92-96%, and the signal-to-noise ratio is greater than 15:1.

[0085] Monitoring results: In the first phase (days 1-60), the abundance of *Thiobacillus denitrificans* increased from 2.1% to 10.2%, the abundance of *Sulfurovum* increased from 0.2% to 5.6%, and the total abundance increased from 2.3% to 15.8%, with an average growth rate of 0.23% per day. In the second phase (days 61-80), the abundance of *Thiobacillus denitrificans* increased from 10.2% to 17.8%, the abundance of *Sulfurovum* increased from 5.6% to 8.4%, and the total abundance increased from 15.8% to 26.2%, with an average growth rate of 0.52% per day, which is 2.3 times that of the first phase. In the third phase (days 81-92), the abundance of Thiobacillus denitrificans increased from 17.8% to 21.3%, the abundance of Sulfurovum increased from 8.4% to 9.8%, and the total abundance increased from 26.2% to 31.1%, with an average growth rate of 0.41% per day.

[0086] Comparison of FISH monitoring and high-throughput sequencing results: On day 92, the abundance of *Thiobacillus denitrificans* detected by FISH technology was 21.3%, and the abundance of the *Thiobacillus* genus detected by 16S rRNA gene high-throughput sequencing was also 21.3%, showing a high degree of consistency between the two methods (relative error <1%). The abundance of the *Sulfurovum* genus detected by FISH technology was 9.8%, and the abundance of the *Sulfurovum* genus detected by high-throughput sequencing was also 9.8%, with completely consistent results. This indicates that FISH technology can accurately quantify the abundance of sulfur autotrophic denitrifying bacteria and is a reliable method for real-time monitoring during the microbial community construction process.

[0087] FISH monitoring guided regulatory decisions: On day 35 of the first phase, FISH monitoring revealed a stagnation in the abundance of sulfur-autotrophic denitrifying bacteria (abundance remained at 8.5-8.8% in two consecutive tests). Analysis indicated that excessive organic carbon sources led to competitive inhibition of heterotrophic microorganisms. The sodium acetate concentration was immediately reduced from 120 mg / L to 90 mg / L, and simultaneously... The concentration was increased from 20 mg / L to 25 mg / L. After a 7-day adjustment period, the abundance of sulfur-autotrophic denitrifying bacteria resumed its upward trend, reaching 10.5% on day 42. On day 68 of the second phase, FISH monitoring revealed a slowdown in the increase of Thiobacillus denitrificans abundance, but the abundance of Sulfurovum continued to increase. The reason for this was analyzed to be... More suitable for the growth of the *Sulfurovum* genus. Appropriately increase... / The ratio promoted the proliferation of Thiobacillus denitrificans. These examples demonstrate that real-time FISH monitoring can provide timely and accurate regulatory data for the microbial community building process.

[0088] Comparative Example 1: A microbial community was constructed to treat wastewater with a low carbon-to-nitrogen ratio using the method described in reference document CN113023901A.

[0089] Microbiome construction was carried out according to the method of Example 1 in the prior art document CN113023901A. Induction and acclimatization stage: 30 mL of surface sediment from the Yellow Sea seabed was inoculated with 150 mL of culture medium, ammonia nitrogen concentration of 50 mg / L, COD prepared with sodium acetate at a concentration of 1000 mg / L, carbon-nitrogen ratio of 20:1, total phosphorus concentration of 10 mg / L, salinity of 3%, pH of 7.5, aeration rate of 2 L / min, operation cycle of 24 h (23 h of aeration, 0.5 h of settling, 0.5 h of influent and effluent), volume exchange ratio of 80%, and continuous operation for 10 days. Enrichment stage: Simulated high-salt and high-nutrient wastewater was used, with COD of 2000 mg / L, ammonia nitrogen of 100 mg / L, total phosphorus of 20 mg / L, salinity of 3%, pH of 7.5, aeration intensity of 1 L / min, operating cycle of 8 h (7 h of aeration, 0.5 h of settling, and 0.5 h of influent and effluent), volume exchange ratio of 50%, hydraulic retention time of 16 h, and continuous operation for 10 days.

[0090] The denitrification performance of the constructed microbial community was tested: the same low carbon-to-nitrogen ratio industrial wastewater (carbon-to-nitrogen ratio 1.3:1 to 2:1) as in Example 2 was used, without the addition of any additional organic carbon source.

[0091] Test results: Water output With a concentration of 35-55 mg / L, the nitrogen removal efficiency is only 45-62%; effluent Concentrations of 8-18 mg / L can cause serious symptoms. Accumulation; COD removal rate is relatively high (85-92%), but nitrogen removal performance is poor; The removal rate is only 0.52-0.76 kg / (m³·d).

[0092] FISH analysis revealed that the abundance of *Thiobacillus denitrificans* was only 3.2%, *Sulfurovum* only 1.5%, and total sulfur-autotrophic denitrifying bacteria only 4.7%. High-throughput sequencing showed that heterotrophic denitrifying bacteria (*Thauera*, *Paracoccus*, etc.) accounted for 38.5% of the total abundance, making them the absolute dominant species.

[0093] Results Analysis: The method in the comparative document mainly enriched heterotrophic functional microorganisms and performed well in treating wastewater with a high carbon-to-nitrogen ratio (≥20:1). However, in treating industrial wastewater with a low carbon-to-nitrogen ratio (approximately 1.5:1), the activity of heterotrophic denitrifying bacteria was severely inhibited due to insufficient organic carbon sources, resulting in a significant decrease in denitrification efficiency. Furthermore, the abundance of sulfur autotrophic denitrifying bacteria was very low (only 4.7%), failing to fully utilize sulfur autotrophic denitrification.

[0094] Comparative Example 2: Construction of sulfur autotrophic denitrifying microbial communities without a segmented regulation strategy

[0095] A sulfur-autotrophic denitrifying microbial community was constructed using a single-stage acclimatization method. The composition of the inoculated sludge and culture medium was the same as in the second stage of Example 1, but the culture medium composition and operating parameters of the second stage were used from day 1, without undergoing the first stage of low carbon-to-nitrogen ratio acclimatization and the third stage of pulsed sulfate dosing enhancement. The community was continuously cultured for 90 days.

[0096] Results: The nitrogen removal efficiency was 65-78%, which was lower than the 93-95% in Example 1; The accumulated concentration was 5-12 mg / L, higher than 1-2 mg / L in Example 1; the abundance of Thiobacillus denitrificans was 12.5%, the abundance of Sulfurovum was 6.2%, and the total abundance of sulfur autotrophic denitrifying bacteria was 18.7%, significantly lower than 31.1% in Example 1; the system stability was poor, and the denitrification efficiency was prone to decrease when the carbon-nitrogen ratio changed or the load fluctuated.

[0097] Results Analysis: Without a segmented regulation strategy, direct cultivation under high nitrogen load and low C / N ratio conditions resulted in intense competition between heterotrophic microorganisms and sulfur autotrophic denitrifying bacteria, leading to low enrichment efficiency of sulfur autotrophic denitrifying bacteria, with a final abundance of only 18.7%. The lack of a first-stage low C / N ratio adaptation acclimatization resulted in weak adaptability of sulfur autotrophic denitrifying bacteria to low C / N ratio environments. The absence of a third-stage pulsed sulfate addition intensification resulted in insufficient sulfur cycling activity in the microbial community, weak synergistic effects between sulfur autotrophic denitrifying bacteria and sulfate-reducing bacteria, and insufficient system stability and resistance to shock loads.

[0098] Comparative Example 3: Microbial community construction without real-time FISH monitoring

[0099] The same segmented control strategy and culture conditions as in Example 1 were used, but FISH technology was not used to monitor the abundance of sulfur autotrophic denitrifying bacteria in real time. Instead, the decision to proceed to the next stage was based solely on nitrogen removal efficiency.

[0100] Results: In the first stage, the nitrogen removal efficiency reached 70% on day 50, indicating that the second stage could proceed (while in Example 1, the nitrogen removal efficiency was 72% on day 60 before proceeding to the second stage). In the second stage, the nitrogen removal efficiency reached 83% on day 15, indicating that the third stage could proceed (while in Example 1, the nitrogen removal efficiency was 88% on day 20 before proceeding to the third stage). After 12 days of cultivation in the third stage, the nitrogen removal efficiency reached 90%, indicating that the microbial community construction was complete. The total acclimatization time was 77 days, 15 days shorter than in Example 1.

[0101] However, FISH analysis of the final constructed microbial community showed that the abundance of *Thiobacillus denitrificans* was 15.8%, the abundance of *Sulfurovum* was 7.5%, and the total abundance of sulfur-autotrophic denitrifying bacteria was 23.3%, significantly lower than the 31.1% in Example 1. When applied to the treatment of low C / N ratio industrial wastewater, the nitrogen removal efficiency was 80-88%, lower than the 88-93% in Example 1. The removal rate was 0.88-1.12 kg / (m³·d), which was lower than the 1.15-1.42 kg / (m³·d) in Example 1.

[0102] Results Analysis: Judging the acclimatization process solely based on nitrogen removal efficiency can lead to prematurely moving to the next stage. Although the nitrogen removal efficiency met the set standard, the abundance of sulfur-autotrophic denitrifying bacteria was not sufficiently enriched, resulting in an unsatisfactory microbial community structure. Real-time FISH monitoring can directly quantify the abundance of sulfur-autotrophic denitrifying bacteria, more accurately reflecting the enrichment status of functional microbial communities, and is an indispensable monitoring method in the process of microbial community construction. Prematurely ending the acclimatization process resulted in insufficient abundance of sulfur-autotrophic denitrifying bacteria, ultimately leading to nitrogen removal performance inferior to Example 1.

[0103] The comparative analysis of the above embodiments and comparative examples shows that:

[0104] This invention achieves highly efficient and targeted enrichment of sulfur-autotrophic denitrifying bacteria through an innovative three-stage segmented acclimatization strategy (adaptive acclimatization to low carbon-to-nitrogen ratio, precise control of electron donor-acceptor ratio, and enhanced pulsed sulfate dosing). Compared with the heterotrophic denitrifying microbiome in Comparative Example 1, the abundance of sulfur-autotrophic denitrifying bacteria in the microbial community constructed in this invention increased from 4.7% to 31.1%, a 6.6-fold increase; and when treating industrial wastewater with a low carbon-to-nitrogen ratio, the nitrogen removal efficiency increased from 45-62% to 88-93%, a 45 percentage point increase. The removal rate increased from 0.52-0.76 kg / (m³·d) to 1.15-1.42 kg / (m³·d), representing an improvement of 45-87%.

[0105] This invention employs fluorescence in situ hybridization (FISH) technology to monitor the abundance of sulfur-autotrophic denitrifying bacteria in real time, enabling precise regulation of the microbial community construction process. Compared to Comparative Example 3, which did not use FISH monitoring, the abundance of sulfur-autotrophic denitrifying bacteria in the microbial community constructed by this invention increased from 23.3% to 31.1%, a 33% improvement; and when treating industrial wastewater with a low C / N ratio, the nitrogen removal efficiency increased from 80-88% to 88-93%. The removal rate increased from 0.88-1.12 kg / (m³·d) to 1.15-1.42 kg / (m³·d), an increase of 20-27%.

[0106] The segmented regulation strategy of this invention is crucial for the efficient enrichment of sulfur-autotrophic denitrifying bacteria. Compared with Comparative Example 2, which did not employ segmented regulation, the abundance of sulfur-autotrophic denitrifying bacteria in the microbial community constructed in this invention increased from 18.7% to 31.1%, an improvement of 66%; denitrification performance and system stability were also significantly improved.

[0107] The microbial community constructed in this invention exhibits excellent denitrification performance when treating industrial wastewater with a low carbon-to-nitrogen ratio (1:1 to 3:1), achieving a nitrogen removal efficiency of 85-95%. The removal rate is 0.8-1.5 kg / (m³·d), and the sulfur conversion rate is 1.2-2.0 kg / (m³·d). With an oxidation rate of 92-98%, good system stability, strong resistance to shock loads, and promising prospects for engineering applications.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a microbial community for enhanced denitrification by regulating sulfur cycle in stages, characterized in that, The method comprises the following steps: The first stage low carbon-nitrogen ratio domestication culture: the original inoculation sludge containing sulfur-oxidizing bacteria and denitrifying bacteria is inoculated into the culture medium, the carbon-nitrogen ratio of the culture medium is 1.5:1 to 2.5:1, the nitrate nitrogen concentration is 40-80 mg / L, the concentration of the carbon source is 10-30 mg / L, the temperature is controlled at 25-35 DEG C, the pH is controlled at 7.0-8.0, the aeration amount is controlled at the dissolved oxygen concentration of 0.3-0.8 mg / L, the hydraulic retention time is 18-30 h, and the domestication culture time is 50-70 days. The first stage low carbon-nitrogen ratio domestication culture: the original inoculation sludge containing sulfur-oxidizing bacteria and denitrifying bacteria is inoculated into the culture medium, the carbon-nitrogen ratio of the culture medium is 1.5:1 to 2.5:1, the nitrate nitrogen concentration is 40-80 mg / L, the concentration of the carbon source is 10-30 mg / L, the temperature is controlled at 25-35 DEG C, the pH is controlled at 7.0-8.0, the aeration amount is controlled at the dissolved oxygen concentration of 0.3-0.8 mg / L, the hydraulic retention time is 18-30 h, and the domestication culture time is The second stage of electron donor-acceptor ratio regulation culture: Based on the first stage of acclimatization culture, the molar ratio of electron donors to electron acceptors in the culture medium is adjusted to... and The molar ratio is 4:1 to 6:1, and the nitrate nitrogen concentration is increased to 80-120 mg / L. Increase the concentration to 50-100 mg / L, and supplement simultaneously. The concentration is 30-80 mg / L, the temperature is controlled at 28-32℃, the pH is controlled at 7.2-7.8, the aeration rate is controlled at dissolved oxygen concentration of 0.2-0.5 mg / L, the hydraulic retention time is 12-20 h, and the incubation time is 15-25 days. Third stage pulse type sulfate addition enhanced cultivation: on the basis of the second stage cultivation, pulse type addition was used to supplement sulfate in the culture medium, single addition The concentration was 200-500 mg / L, the interval time was 4-8 h, the addition cycle was 8-15 days, the concentration of nitrate nitrogen was maintained at 100-150 mg / L, the temperature was controlled at 28-32 DEG C, the pH was controlled at 7.0-7.5, the aeration amount was controlled at the dissolved oxygen concentration 0.1-0.4 mg / L, and the hydraulic retention time was 10-16 h. Meanwhile, during the cultivation in the first, second and third stages, the abundance of the sulfur autotrophic denitrifying bacteria is monitored in real time by fluorescence in situ hybridization technology, and when the total abundance of Thiobacillus denitrificans and Sulfurovum sulfur autotrophic denitrifying bacteria reaches more than 28%, the microbial community construction is completed.

2. The method of claim 1, wherein, The original inoculation sludge is selected from a mixture of any one or more of the following: activated sludge of a municipal wastewater treatment plant, sludge of an industrial wastewater treatment system, marine sediment, sediment of a saltwater lake, sediment of a sulfide-enriched environment, and sediment of mine drainage.

3. The method of claim 1, wherein, The first stage low carbon nitrogen ratio domestication culture, the composition of the culture medium is: sodium acetate as organic carbon source, the concentration is 80-160mg / L; As nitrate nitrogen source, the concentration is 40-80mg / L; As sulfide source, the concentration is 10-30mg / L; The concentration is 5-15mg / L; The concentration is 20-50mg / L; The concentration is 10-30mg / L; Trace element solution 1-3mL / L, the trace element solution contains 、 、 、 、 、 、 .

4. The method of claim 1, wherein, The second stage electron donor acceptor ratio regulates the culture medium composition: the concentration of sodium acetate is reduced to 40-80 mg / L; The concentration is increased to 80-120 mg / L; The concentration is increased to 50-100 mg / L; supplemented The concentration is 30-80 mg / L; The concentration is 100-200 mg / L as inorganic carbon source; the rest of the components are the same as the first stage.

5. The method of claim 1, wherein, In the third stage of pulsed sulfate dosing enhanced culture, the pulsed dosing is carried out using an automatic control system, based on the conditions in the reactor. Concentration and Real-time monitoring results of concentration, when Concentration below 30 mg / L or Automatic dosing when concentration is below 100 mg / L Solution, single dosage The concentration was increased by 200-500 mg / L.

6. The method of claim 1, wherein, The fluorescence in situ hybridization technology monitoring step comprises: Sludge samples in the reactor are collected and fixed with 4% paraformaldehyde fixing solution for 2-4 hours; After washing with phosphate buffer solution for 3 times, dehydration treatment is performed with different concentration gradients of ethanol; Hybridization is performed with a specific probe Tdeni-438 (5'-AAGGCCGCTTTCGAACG-3') for Thiobacillus denitrificans and a specific probe Sulfu-654 (5'-TCCACTGCTGGCTCGTT-3') for Sulfurovum, at a hybridization temperature of 46-50°C and a hybridization time of 2-4 hours; Observation and counting are performed by using a laser confocal microscope or a fluorescence microscope, and the proportion of the target bacterial community in the total bacterial community is calculated.

7. The method of claim 1, wherein, During the first-stage domestication cultivation, the microbial community structure is detected every 5-7 days, and when the abundance of sulfur autotrophic denitrifying bacteria reaches more than 15%, the second stage is entered; during the second-stage cultivation, the microbial community structure is detected every 3-5 days, and when the abundance of sulfur autotrophic denitrifying bacteria reaches more than 25%, the third stage is entered.

8. The method of claim 1, wherein, During the cultivation in the three stages, the following regulation measures are further included: When detecting When the accumulated concentration exceeds 10 mg / L, reduce the influent load by 20-40% and extend the hydraulic retention time by 2-6 h; When the pH value is lower than 6.8, add solution or solution to adjust the pH to 7.0-7.5; When the dissolved oxygen concentration exceeds the upper limit of the set range, the aeration amount is reduced by 15-30%; When the abundance of sulfur autotrophic denitrifying bacteria is detected to have a downward trend for two times in succession, the appropriate increase The dosing amount is 10-25%, and the dosing amount of organic carbon source is reduced by 20-35%.

9. The method of claim 1, wherein, The cultivation device used in the method is a sequencing batch reactor or a continuous flow reactor, the reactor volume is 1-20 L, the inoculation sludge concentration is 2-6 g / L, the sludge age is controlled to be 15-30 days, the temperature control system adopts water bath heating or air heating, the pH control system adopts an automatic acid and alkali adding device, and the aeration system adopts micro-porous aeration or membrane aeration.

10. The method of claim 1, wherein, The microbial community constructed by the method is applied to the denitrification treatment of low carbon-nitrogen ratio industrial wastewater, and the nitrogen removal efficiency is 85-95%, The removal rate is 0.8-1.5 kg / (m3·d), and the sulfur conversion rate is 1.2-2.0 kg / (m3·d), The oxidation rate is 92-98%, and the system has stable denitrification performance for wastewater with a carbon-nitrogen ratio of 1:1 to 3:1.

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