CN112501066B

CN112501066B - Compound strain, microbial inoculum and application thereof

Info

Publication number: CN112501066B
Application number: CN202011424501.4A
Authority: CN
Inventors: 庄绪亮; 徐圣君; 徐立娜; 左佳靓; 张小寒; 姜参参; 杨东敏; 白志辉; 李�瑞; 杨凡
Original assignee: Shenzhen Shenshui Water Resources Consulting Co ltd; Yangtze River Delta Yiwu Ecological Environment Research Center; Research Center for Eco Environmental Sciences of CAS
Current assignee: Shenzhen Shenshui Water Resources Consulting Co ltd; Yangtze River Delta Yiwu Ecological Environment Research Center
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2022-07-08
Anticipated expiration: 2040-12-08
Also published as: CN112501066A

Abstract

A composite bacterial strain, a microbial inoculum and application thereof are provided, the composite bacterial strain is composed of enterobacter C1-3 with the preservation number of CGMCC No.20581 and pantoea A1 with the preservation number of CGMCC No.20582 which are obtained by separating and screening activated sludge, and the composite bacterial strain or the microbial inoculum containing the composite bacterial strain is applied to denitrification treatment, so that the high-efficiency denitrification treatment effect is obtained. The composite strain denitrification system constructed by the invention has good application value in the aspect of denitrification of nitrate (biogas slurry).

Description

Compound strain, microbial inoculum and application thereof

Technical Field

The invention relates to the technical field of denitrification, and particularly relates to a composite strain, a microbial inoculum and application thereof.

Background

Along with the comprehensive control of point source pollution in urban areas, the intensive livestock and poultry breeding in China is gradually highlighted, and the livestock and poultry breeding wastewater contains a large amount of Chemical Oxygen Demand (COD) and ammonia nitrogen; aiming at the characteristic of high organic matter of livestock wastewater, an anaerobic fermentation mode is generally adopted at present to obtain methane with higher value, thereby realizing energy regeneration. The anaerobic fermentation stage realizes the conversion of organic matters to CH₄The biological conversion is carried out, but the removal of ammonia nitrogen (ammonia nitrogen is approximately equal to 500mg/L) is not obvious, so the COD/TN (total nitrogen) ratio of the biogas slurry is obviously reduced, the COD/TN ratio of the current livestock and poultry biogas slurry is about 1.8(COD is approximately equal to 1000 mg/L; TN is approximately equal to 600mg/L) and is obviously lower than 4.0(COD is approximately equal to 350 mg/L; TN is approximately equal to 85mg/L) of the municipal sewage; in order to realize biological denitrification of the livestock and poultry biogas slurry, a full-process nitrification and denitrification process of an urban sewage system is mainly adopted, low C/N of the livestock and poultry biogas slurry influences biological removal of nitrate in the denitrification process, and the effluent total nitrogen can reach the standard generally by means of adding a carbon source and prolonging hydraulic retention time at present, but the operation cost and the residual sludge amount are increased by the method.

Disclosure of Invention

In view of the above, the invention mainly aims to provide a composite strain, a microbial inoculum and application thereof, wherein the composite strain can directionally convert nitrate and nitrite, realize efficient denitrification treatment, and solve the problems of high cost and large sludge amount in the denitrification treatment process of low-C/N livestock biogas slurry.

In order to achieve the purpose, the technical scheme of the invention is as follows:

as a first aspect of the present invention, there is provided a composite strain consisting of Enterobacter (Enterobacter sp.) C1-3 having a accession number of CGMCC No.20581 and Pantoea (Pantoea sp.) A1 having a accession number of CGMCC No. 20582.

As a second aspect of the present invention, there is provided a use of the complex strain as described above in denitrification treatment.

As a third aspect of the present invention, there is provided a microbial agent containing the complex strain as described above.

In some embodiments of the invention, the ratio of the number of the enterobacter C1-3 to the number of the pantoea A1 in the composite strain is 5: 4-6, so that a good denitrification effect can be achieved.

In some embodiments of the invention, the microbial inoculum is a seed solution obtained by inoculating and culturing the compound strain in an anaerobic environment to logarithmic growth phase, and then cleaning and resuspending the seed solution, wherein the OD of the seed solution₆₀₀Preferably 0.6 to 1.

As a fourth aspect of the invention, there is provided a use of the microbial inoculum as described above in denitrification treatment.

As a fifth aspect of the present invention, there is provided a method for denitrification treatment of nitrate wastewater using the composite strain as described above or the microbial inoculum as described above, comprising the steps of:

and (3) putting the composite strain or the microbial inoculum into nitrate wastewater to carry out denitrification reaction.

In some embodiments of the invention, the ratio of Chemical Oxygen Demand (COD) to Total Nitrogen (TN) in the nitrate containing wastewater is not greater than 2.

In some embodiments of the invention, the denitrification reaction is: reacting for 72-80 h at the temperature of 28-32 ℃ and the rotating speed of 120-180 rpm. By limiting the reaction conditions, the denitrification efficiency of the composite bacterial strain can be improved, and the removal rate of total nitrogen in the nitrate wastewater can be improved.

In some embodiments of the invention, the inoculum size of the inoculum is 8-12%; optical density value OD of the microbial inoculum₆₀₀0.6 to 1. Tong (Chinese character of 'tong')OD of over-limited microbial inoculum₆₀₀The inoculation amount and the strain ratio can ensure the strain concentration in the nitrate wastewater and better promote the denitrification reaction of the strains.

Denitrification generally mainly comprises the steps of oxidizing ammonia nitrogen into nitrous acid by nitrification, further oxidizing the nitrous acid into nitric acid, and then reducing the nitrous acid into nitrogen by coupling nitric acid serving as a substrate with an effective carbon source through denitrification so that nitrogenous substances in water finally enter an atmospheric environment; the functional microorganisms play an important role in the biogas slurry denitrification process, the key microorganisms have diversity in nitrogen conversion, and an effective nitrogen conversion network path can be established in a system through the action among microbial communities; in the process of realizing the invention, the inventor finds that the nitrate is converted into the nitrite by the enterobacter C1-3, and the nitrite is reduced into the nitrogen by the Pantoea A1, so that the denitrification treatment can be realized, and the high-efficiency denitrification treatment can be carried out on the livestock biogas slurry with the low COD/TN ratio.

From the above, the composite strain, the microbial inoculum and the application thereof provided by the invention at least comprise the following beneficial effects:

according to the invention, based on the core of two key steps of reducing nitrite and nitrite into nitrogen in biological denitrification, nitrate reducing bacteria and nitrite reducing bacteria (namely enterobacter C1-3 strain and pantoea A1 strain) are obtained by respectively and directionally screening, nitrate is converted into nitrite by enterobacter C1-3 strain, and nitrite is reduced into nitrogen by pantoea A1 strain, so that the high-efficiency denitrification treatment of the livestock and poultry biogas slurry is realized.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a colony morphology of Enterobacter C1-3 provided in an embodiment of the present invention;

FIG. 2 is a colony morphology of Pantoea 1 provided in an example of the present invention;

FIG. 3 shows the nitrate nitrogen removal results of Enterobacter C1-3 according to the experimental examples of the present invention;

FIG. 4 shows the nitrite nitrogen removal results of Pantoea A1 according to the experimental examples of the present invention;

FIG. 5 shows the results of nitrate nitrogen removal by the composite strains of Enterobacter C1-3 and Pantoea A1 provided in the experimental examples of the present invention.

Detailed Description

In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.

The raw materials adopted by the embodiments of the invention are as follows:

LB medium (per liter): consists of 10.0g of trypsin, 5.0g of yeast extract, 10.0g of NaCl, 18.0g of agar and deionized water.

Enrichment medium (EM1) (pH 7.0) contained the following reagents per liter: 0.2g KNO₃0.566g of sodium citrate, 5 percent (mass to volume ratio, g/mL) of trace elements and distilled water, wherein the trace elements consist of 2.5g of MgSO₄·7H₂O、6.5g K₂HPO₄·3H₂O、0.04g MnSO₄·H₂O、2.5g NaCl、0.05g FeSO₄·7H₂And (C) O.

Enrichment medium (EM2) (pH 7.2): each liter contains 5g of glucose, 1g of NaCl, 0.5g of NaNO₂，0.02g MgSO₄、4g KH₂PO₄、6g K₂HPO₄And the volume of the distilled water is up to 1L.

Denitrifying medium (DM1) (pH 7.0-7.3): comprising 0.2g KNO per liter₃、10.55g Na₂HPO₄·12H₂O、1.5g KH₂PO₄、0.1g MgSO₄·7H₂O, 0.566g of sodium citrate, 0.2 percent (mass to volume ratio, g/mL) of trace elements (comprising 50.0g of EDTA-Na2, 2.2g of ZnSO₄、5.5g CaCl₂、5.06g MnCl₂·4H₂O，5.0g FeSO₄·7H₂O，1.57g CuSO₄·5H₂O、1.61g CoCl₂·6H₂O) and distilled water.

Denitrifying medium (DM2) (pH 7.2-7.5): each liter comprises 1g of glucose and 0.02g of NaNO₂、0.02g MgSO₄0.1g of sodium citrate, 0.1g K₂HPO₄And the volume of the distilled water is up to 1L.

The following equipment was used:

example 1

This example is the screening and purification of Enterobacter C1-3 strain and Pantoea A1 strain:

1. constructing a microbial strain library:

1.1, use

The method comprises the following steps that DNA extraction is carried out on an activated sludge sample which is fully mixed and freeze-dried by a SPIN Kit for Soil DNA extraction Kit of Soil DNA (MP Biomedicals, CA, USA), wherein the activated sludge sample is derived from activated sludge sediment in a livestock and poultry biogas slurry treatment device of a livestock and poultry breeding wastewater treatment system of a Jinjing laboratory of the subtropical ecological research institute of the Chinese academy of sciences; and F27 and 1492R are used as primers, the extracted DNA is used as a template for PCR amplification, and a 50ul amplification system is 2 XTaq PCR Master Mix 25ul, F271 ul, 1492R 1ul, DNA template 2ul and ddH₂O21 ul. The amplification procedure comprises pre-denaturation at 94 deg.C for 1min, denaturation at 94 deg.C for 30s, annealing at 57 deg.C for 20s, extension at 72 deg.C for 30s, and final extension at 72 deg.C for 10min

ND-1000spectrophotometer(

Technologies, Wilmington, DE, USA) to determine the total DNA concentration extracted; finally, storing the DNA sample in a refrigerator at the temperature of-20 ℃;

1.2, performing high-throughput sequencing on a 16S rDNA PCR product based on a second-generation gene sequencing platform, and exploring the microbial diversity in an environmental sample; V3-V4 region of the bacterial 16S rDNA gene using primer 338F: 5'-ACTCCTACGGGAGGCAGCAG-3' (SEQ ID NO.1), 806R: 5 '-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO.2) was amplified on a PCR (Eastwin, China) instrument; PCR products were first recovered using 2% agarose Gel, then purified using AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, Calif., USA), and then checked for band unity by agarose Gel electrophoresis and high throughput;

1.3, 16S rRNA gene sequencing data is analyzed by a Meiji biological analysis platform (website address is https:// closed. majorbio. com /), different samples in original data are distinguished according to a barcode sequence, species with the 16S rRNA sequence similarity of more than 97% are classified as an OTU, a RDP classification database is used for classification and annotation, in order to eliminate deviation caused by different sequencing quantities, minimum sequence number flattening is carried out on the data, and the flattened OTU table is adopted for subsequent analysis.

2. Screening and purification of Enterobacter C1-3 Strain

2.1, enrichment culture

Adding 10g of activated sludge sediment (the activated sludge sediment is from a livestock and poultry breeding wastewater treatment system of a Jinjing laboratory station of the subtropical ecological research institute of Chinese academy) in a 250ml anaerobic serum bottle filled with 100ml of sterilized Phosphate Buffer Solution (PBS) and 5-10 glass beads according to the weight-volume ratio of 10% on an ultra-clean workbench, and oscillating for 30min at the speed of 150rpm in a constant temperature oscillator at the temperature of 30 ℃ so as to fully release microorganisms in the substrate sludge; then 10ml of the sediment suspension was transferred to fresh EM1 medium, 10ml of the mixed solution of EM1 and microorganisms was aspirated every 2 days during the enrichment phase and transferred to fresh EM1, which was shake-cultured on a constant temperature shaker (150rpm) at 30 ℃ for 30 days;

2.2, separation and purification

After 30 days of shake culture in EM1, 0.1mL of 10-fold diluted bacterial suspension is inoculated on a DM1 agar plate, coated by a sterile coating rod and cultured in a 30 ℃ incubator, cultured colonies with different appearances and shapes are separated on a fresh DM1 solid plate by using an inoculating loop streak, so that a single colony can be obtained, and the single colony is purified on a DM1 solid culture medium for three generations;

2.3 verification of transformation Capacity

Taking 40mL of bacterial liquid in the logarithmic phase, centrifuging at 5000rpm for 5min, collecting thalli, and rinsing each thalli for 3 times by using sterilized 1 XPBS (phosphate buffered saline) to remove residual culture medium; after rinsing, the thalli are resuspended by 1 XPBS, the mixture is evenly mixed by blowing and sucking, 3mL of the resuspended bacterial liquid is inoculated into 30mL of sterilized DM1, and the degradation condition of the resuspended bacterial liquid to nitrate is measured under the conditions of 150rpm and 30 ℃; 3 parallel groups are respectively arranged on each representative strain, and the strain with the best nitrate degradation is selected as the strain with the serial number of C1-3.

2.4 molecular biology characterization of the strains

The C1-3 strain was streaked on beef extract peptone agar medium for 24h, and the culture results are shown in FIG. 1: the large bacterial colony of the C1-3 bacterial strain is round, neat and moist in edge, smooth in surface, milky, translucent and small in bulge;

gram staining the C1-3 strain to identify the bacterium as gram negative;

extracting the DNA of the C1-3 strain by using a special Kit for extracting bacterial DNA (Fast DNA Spin Kit for Soil, MP bio, USA), and selecting a universal primer F27 of PCR: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.3) and 1492R: 5 '-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO.4) as an amplification primer, a 50. mu.L PCR reaction system (2 XTAQA PCR Master Mix 25. mu.L, F27 (10. mu.M) 1. mu.L, 1492R (10. mu.M) 1. mu.L) was prepared, and the following PCR amplification procedure was initiated later: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and amplification for 30 cycles; final extension at 72 deg.C for 10 min; after PCR amplification is finished, injecting a PCR product into a 1% agarose gel hole, carrying out electrophoresis for 30min under the condition of 120V voltage, and then analyzing the result by using a gel imager; the successfully amplified gene sequence is sent to Beijing Rui Boxing science biotechnology limited company for Sanger sequencing, and is compared with the constructed microbial strain library to determine that the strain belongs to one of the microbial strain libraries, and then the strain belongs to enterobacter by sequence comparison of Blast, wherein the name of the strain is enterobacter C1-3;

2.5 preservation of the Strain

The strains are preserved by adopting a method of slant preservation of beef extract peptone at 4 ℃ and low-temperature freezing preservation of glycerol suspension; the method for low-temperature freezing preservation of the glycerol suspension comprises the following steps: mixing 50% glycerol with strain suspension cultured by LB at a ratio of 1:1, and storing at-80 deg.C with final concentration of glycerol of about 25%. The enterobacter C1-3 strain is delivered to China general microbiological culture collection center for collection in 2020, 9 and 1, with the collection number of CGMCC No. 20581.

3. Screening and purification of Pantoea A1 Strain

3.1, enrichment culture

Adding 10g of activated sludge sediment in a livestock and poultry biogas slurry treatment device into a 250ml anaerobic serum bottle filled with 100ml of sterilized Phosphate Buffer Solution (PBS) and 5-10 glass beads according to the proportion of 10 percent by weight and volume on an ultra-clean workbench, and oscillating for 30min at the speed of 150rpm in a constant temperature oscillator at the temperature of 30 ℃ so as to fully release microorganisms in the substrate sludge; then 10ml of the sediment suspension was transferred to fresh EM2 medium, 10ml of the mixed solution of EM2 and microorganisms was aspirated every 2 days in the enrichment phase and transferred to fresh EM2 and shake-cultured on a 30 ℃ constant temperature shaker (150rpm) for 30 days;

3.2, separation and purification

After 30 days of shake cultivation in EM2, 0.1mL of 10-fold diluted bacterial suspension is inoculated on a DM2 agar plate, a sterile coating rod is used for coating, the bacterial suspension is cultivated in a 30 ℃ incubator, cultivated colonies with different appearances and shapes are separated on a fresh DM2 solid plate by using an inoculating loop streak to obtain a single colony, and the single colony is purified on a DM2 solid medium for three generations;

3.3 verification of transformation Capacity

Taking 40mL of bacterial liquid in a logarithmic phase, centrifuging at 5000rpm for 5min, collecting thalli, rinsing each thalli for 3 times by using sterilized 1 XPBS to remove residual culture medium, after rinsing, re-suspending the thalli by using 1 XPBS, blowing, sucking and mixing uniformly, inoculating the re-suspended bacterial liquid into sterilized 30mL of DM2, culturing at 150rpm and 30 ℃ for 72h, determining the degradation condition of the bacterial liquid on nitrite nitrogen, setting 3 parallel groups for each representative strain respectively, and selecting the strain with the best nitrate degradation as the A1 strain.

3.4 molecular biological identification of the strains

The A1 strain was streaked on beef extract peptone agar medium for 24h, and the culture results are shown in FIG. 2: a1 large bacterial colony of the strain is round, neat and moist, and has a smooth surface, a light yellow color and small bulges;

gram-negative was identified by gram-staining the a1 strain;

extracting DNA of A1 by using a special Kit (Fast DNA Spin Kit for Soil, MP bio, USA) for extracting bacterial DNA, selecting a general primer F27 of PCR: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.3) and 1492R: 5 '-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO.4) as an amplification primer, a 50. mu.L PCR reaction system (2 XTAQQ PCR Master Mix 25. mu.L, F27 (10. mu.M) 1. mu.L, 1492R (10. mu.M) 1. mu.L) was prepared, and the following PCR amplification procedure was initiated later: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and amplification for 30 cycles; final extension at 72 deg.C for 10 min; after the PCR amplification is finished, injecting the PCR product into a 1% agarose gel hole, performing electrophoresis for 30min under the condition of 120V voltage, and analyzing the result by using a gel imager; the successfully amplified gene sequence is sent to Beijing Rui Boxing science biotechnology limited company for Sanger sequencing, and is compared with the constructed microbial strain library to determine that the strain belongs to one of the microbial strain libraries, and then the strain belongs to enterobacter by Blast sequence comparison, and the name is defined as Pantoea A1 strain.

3.5, preservation of the Strain

The strains are preserved by adopting a method of slant preservation of beef extract peptone at 4 ℃ and low-temperature freezing preservation of glycerol suspension; specifically, the low-temperature freezing preservation method of the glycerol suspension comprises the following steps: mixing 50% glycerol with strain suspension cultured by LB at a ratio of 1:1, and storing at-80 deg.C with final concentration of glycerol of about 25%. The Pantoea A1 strain is delivered to China general microbiological culture collection center for collection in 2020, 9.1.with the collection number of CGMCC No. 20582.

Example 2

The embodiment is a microbial inoculum, which comprises a Pantoea 1 strain and an enterobacter C1-3 strain, wherein the strain number ratio of the Pantoea 1 strain to the enterobacter C1-3 strain is 1: 1.

The microbial inoculum is prepared by the following method:

flushing argon into a serum bottle to provide anaerobic conditions for anaerobic denitrifying microorganisms, injecting an enterobacter C1-3 strain and a pantoea A1 strain into 4ml of sterile distilled water according to the proportion of 1:1, injecting the sterile distilled water into the serum bottle filled with 40ml of LB liquid culture medium by using a sterile injector, culturing for 12h, collecting a culture solution in a logarithmic phase, centrifuging the culture solution in a centrifuge at 5000rpm for 5 minutes, flushing the culture solution for 3 times by using sterilized 1 XPBS (phosphate buffered saline), removing residual LB culture medium, and then re-suspending the bacteria by using the 1 XPBS to obtain the microbial inoculum.

Experimental example 1

This example is a study of the biotransformation of nitrate by Enterobacter strain C1-3

Flushing argon gas into a serum bottle to provide anaerobic conditions for anaerobic denitrifying microorganisms, injecting the Enterobacter C1-3 strain into 4ml of sterile distilled water and into a serum bottle containing 40ml of LB liquid medium using a sterile syringe, collecting the culture solution in the logarithmic growth phase after culturing for 12 hours, centrifuging at 5000rpm for 5 minutes in a centrifuge, then flushing 3 times with sterilized 1 XPBS to remove the residual LB medium, then resuspending the bacteria with 1 XPBS, and OD of the resuspended bacterial solution₆₀₀Was 1.42 and inoculated into sterilized DM1 (KNO) at a dose of 10%₃30mg/L as the sole nitrogen source) and cultured at 150rpm at 30 ℃ for 72h, three for each strain being set in parallel;

the nitrate conversion results are shown in FIG. 3, and it can be seen from FIG. 3 that: 0-4h is the lag phase of the strain, 4-24h is the log phase of the strain, and 24-72h is the stationary phase of the strain; nitric acid Nitrogen (NO) in the culture broth in the first 12h₃ ^-N), the degradation rate of the enterobacter C1-3 strain to nitric acid nitrogen reaches 82 percent; after 24h, the conversion rate of the enterobacter C1-3 strain to nitric acid nitrogen is slowed down after a stabilization period;in general, the enterobacter C1-3 strain can completely convert nitric acid nitrogen with the concentration of 28mg/L after 72 hours, and the conversion rate reaches 100%; in addition, during the first 12h, with the rapid conversion of nitric acid nitrogen, nitrous acid Nitrogen (NO)₂ ^-The content of-N) rises rapidly, and the content of the nitrogen nitrite in the culture solution reaches 24.150mg/L by 72 hours.

Experimental example 2

This example is a study of the biotransformation of nitrite by Pantoea A1

Argon gas was flushed into a serum bottle to provide anaerobic conditions for anaerobic denitrifying microorganisms, and Pantoea a1 strain was injected into 4ml of sterile distilled water and into a serum bottle containing 40ml of LB liquid medium using a sterile syringe, and after 12 hours of culture, the culture broth in the logarithmic phase was collected and centrifuged at 5000rpm for 5 minutes in a centrifuge, and then washed 3 times with sterilized 1 XPBS to remove the residual LB medium. The bacterial suspension was then mixed homogeneously with 1 XPBS and the OD of the resuspended bacterial solution was₆₀₀1.37, and inoculated into sterilized DM2 (NaNO) at an inoculum size of 10%₂30mg/L as the sole nitrogen source) and cultured at 150rpm at 30 ℃ for 72h, three for each strain being set in parallel;

the nitrite conversion results are shown in FIG. 4, and it can be seen from FIG. 4 that: 0-6h is the slow phase of the strain, 6-24h is the log phase of the strain, and 24-72h is the stationary phase of the strain; the bacterial strain can rapidly convert nitrite nitrogen after entering a logarithmic growth phase, the content of nitrite nitrogen and TN in the culture medium is 7.3 and 10.54mg/L when 12 hours later, the degradation rate of the Pantoea 1 bacterial strain to nitrite nitrogen is 75.67 percent, and the degradation rate of the Pantoea 1 bacterial strain to TN is 67 percent; then the degradation rate of the nitrite nitrogen tends to be slow, the concentration of the residual nitrite nitrogen in the culture solution is only 2.2mg/L when 72 hours are reached, the degradation rates of the strain to the nitrite nitrogen and TN are respectively 92.67 percent and 81.48 percent, and the Pantoea 1 strain can efficiently remove the nitrite nitrogen in a short time, thereby realizing the conversion of the nitrite nitrogen to nitrogen.

Experimental example 3

The experimental example is a research on the efficient denitrification of the denitrifying composite strains (Pantoea 1 strain and Enterobacter C1-3 strain) by coupling nitrate and nitrite step by step

Flushing argon gas into a serum bottle to provide anaerobic conditions for anaerobic denitrifying microorganisms, injecting Enterobacter C1-3 strain and Pantoea A1 strain into 4ml of sterile distilled water according to the ratio of 1:1, injecting into a serum bottle filled with 40ml of LB liquid medium by using a sterile injector, culturing for 12h, collecting the culture solution in logarithmic growth phase, centrifuging at 5000rpm for 5min in a centrifuge, flushing 3 times with sterilized 1 XPBS to remove residual LB medium, then resuspending the bacteria by using 1 XPBS, and resuspending the OD of the resuspended bacteria solution₆₀₀Was 1.62 and inoculated into sterilized DM1 (KNO) at a dose of 10%₃30mg/L as the sole nitrogen source) and cultured at 150rpm at 30 ℃ for 72h, three for each strain;

the conversion results of nitrate and nitrite are shown in FIG. 5, and it can be seen from FIG. 5 that: the growth of the compound strain is very rapid, the compound strain enters a logarithmic growth phase almost without a lag phase, 0-12h is the logarithmic growth phase of the strain, and 12-72h is the stationary phase of the strain; the conversion rate of the composite strain to the nitric acid nitrogen reaches 90.68% in 12h, compared with the capability of the single strain enterobacter C1-3 strain to degrade the nitric acid nitrogen, the degradation efficiency of the composite strain to the nitric acid nitrogen is improved by 7.32% compared with the degradation efficiency of the single strain enterobacter C1-3 strain, the removal efficiency of the composite strain to TN reaches 76.50%, the highest accumulation amount of the nitrite nitrogen is 4.81mg/L, and the value is far lower than the accumulated nitrite nitrogen content of the single enterobacter C1-3 strain; the compound strain can completely convert nitrate nitrogen by 72 hours and has a removal rate of TN as high as 87.68%, and the cumulative amount of nitrite nitrogen in the culture solution is only 1.3mg/L, which is 2.2mg/L lower than that of a single Pantoea A1 strain and is far lower than that of a single Enterobacter C1-3 strain (24.150 mg/L); therefore, compared with the denitrification of a single strain, the composite strain can efficiently realize the full-process denitrification of nitrate.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A composite strain is characterized by consisting of Enterobacter (Enterobacter sp.) C1-3 with the preservation number of CGMCC No.20581 and Pantoea (Pantoea sp.) A1 with the preservation number of CGMCC No. 20582.

2. Use of the complex strain of claim 1 in denitrification treatment.

3. A microbial preparation comprising the complex strain according to claim 1.

4. The microbial inoculum according to claim 3, wherein the number ratio of the enterobacter C1-3 to the Pantoea A1 strain of the composite strain is 5: 4-6.

5. The microbial inoculum according to claim 3, which is a seed solution obtained by inoculating and culturing the compound strain in an anaerobic environment to a logarithmic growth phase, and then cleaning and resuspending the compound strain.

6. The microbial inoculum according to claim 5, in which the OD of the seed liquid₆₀₀0.6 to 1.

7. Use of the microbial agent of any one of claims 3 to 6 in denitrification treatment.

8. A method for denitrification treatment of nitrate wastewater using the complex strain of claim 1 or the microbial agent of any one of claims 3 to 6, comprising the steps of:

9. The method according to claim 8, wherein the ratio of the Chemical Oxygen Demand (COD) to the Total Nitrogen (TN) in the nitrate containing wastewater is not more than 2.

10. The method according to claim 8 or 9, wherein the denitrification reaction is: reacting for 72-80 h at 28-32 ℃ and 120-180 rpm.

11. The method according to claim 8 or 9, wherein the inoculum size of the inoculum is 8-12%; optical density value OD of the microbial inoculum₆₀₀0.6 to 1.