CN118006593A - Microbial composition for enhancing sewage denitrification based on quorum sensing and application thereof - Google Patents
Microbial composition for enhancing sewage denitrification based on quorum sensing and application thereof Download PDFInfo
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- CN118006593A CN118006593A CN202410353164.6A CN202410353164A CN118006593A CN 118006593 A CN118006593 A CN 118006593A CN 202410353164 A CN202410353164 A CN 202410353164A CN 118006593 A CN118006593 A CN 118006593A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/341—Consortia of bacteria
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/093—Polyurethanes
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/163—Nitrates
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
- C02F2101/166—Nitrites
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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Abstract
The invention provides a microbial composition for enhancing sewage denitrification based on quorum sensing and application thereof, belongs to the technical field of sewage biological treatment, and consists of a quorum sensing bacterial composition symbiotic with anaerobic ammonia oxidation, an adhesive and a biological carrier. The quorum sensing bacterial composition consists of three quorum sensing bacteria, namely Sinorhizobium japonicum CH9, brucella melitensis CH10 and Burkholderia berk CH12. The microbial composition prepared by the invention can continuously stabilize signal molecules, improve the denitrification efficiency of the sewage treatment reactor, shorten the starting time of the anaerobic ammonia oxidation reactor, and has wide application prospect in the sewage denitrification field.
Description
Technical Field
The invention relates to the technical field of sewage biological treatment, in particular to a microbial composition for enhancing sewage denitrification based on quorum sensing and application thereof.
Background
The ammonia oxidation process is a novel autotrophic denitrification process. The process breaks through the basic theoretical concept in the traditional biological denitrification process, and has the advantages of no need of aeration, low sludge yield and no need of additional carbon source. However, anaerobic ammonia oxidizing bacteria (AnAOB) are sensitive to the environment, slow in growth, small in size and easy to run off, so that the starting time of the reactor is long, and the popularization and application of the anaerobic ammonia oxidation process are affected. Therefore, how to rapidly enrich anammox bacteria in practical application is an important problem to be solved.
Quorum sensing (quorum sensing, QS) can regulate gene expression according to flora density to improve bacterial growth rate, activity, denitrification efficiency, etc. Acyl Homoserine Lactones (AHLs) are a relatively common signaling molecule that is primarily involved in the regulation of gram-negative bacteria. It has been reported that the introduction of exogenous hexanoyl-L-homoserine lactone (C6-HSL) and octanoyl-L-homoserine lactone (C8-HSL) can significantly increase the activity and growth rate of anammox bacteria, thereby improving the denitrification performance of the whole anammox system. However, the exogenous addition of signal molecules is difficult to apply due to high cost.
Disclosure of Invention
The invention aims to provide a microbial composition for enhancing sewage denitrification based on quorum sensing and application thereof, and the microbial composition has a good sewage denitrification effect.
The technical scheme of the invention is realized as follows:
The invention provides a microbial composition for enhancing sewage denitrification based on quorum sensing, which consists of a quorum sensing bacterial composition symbiotic with anaerobic ammonia oxidation, an adhesive and a biological carrier.
As a further improvement of the present invention, the quorum sensing bacterial composition consists of three strains of quorum sensing bacteria, namely Sinorhizobium sp CH9, brucella sp CH10 and Burkholderia sp CH12.
As a further improvement of the present invention, the quorum sensing bacteria are capable of producing N-acyl homoserine lactone signaling molecules.
As a further improvement of the invention, the signal molecules generated by the sinorhizobium CH9 are C6-HSL and C12-HSL, the signal molecules generated by the Brucella CH10 are C6-HSL, and the signal molecules generated by the Burkholderia CH12 are C6-HSL and C8-HSL.
As a further improvement of the present invention, the quorum sensing bacteria are preserved in the China center for type culture collection, wherein CH9 classification is named as sinorhizobium Ensifer sp, the preservation number is CCTCC M20232628, the preservation date is 2023, 12 months and 22 days, and the preservation unit is China center for type culture collection, address: eight 299 routes of Wuchang district of Wuhan, hubei province, post code: 430072; CH10 classification is named Brucella sp, the preservation number is CCTCC M20232629, the preservation date is 2023, 12 months and 22 days, the preservation unit is China center for type culture Collection, and the address is: eight 299 routes of Wuchang district of Wuhan, hubei province, post code: 430072; CH12 classification is named Burkholderia sp., with the preservation number of CCTCC M20232630, the preservation date of 2023, 12 months and 22 days, and the preservation unit: china center for type culture Collection, address: eight 299 routes of Wuchang district of Wuhan, hubei province, post code: 430072.
As a further improvement of the invention, the concentration of the quorum sensing bacterial composition is OD 600 =0.8-1.2, and the quorum sensing bacterial composition consists of the following bacterial liquids in parts by weight: 20-60 parts of Sinorhizobium, 10-80 parts of Brucella, and 20-60 parts of Burkholderia CH.
As a further improvement of the invention, the adhesive is a peptized solution formed by mixing polyvinyl alcohol and sodium alginate, and the biological carrier is a polyurethane sponge biological carrier; the mass ratio of polyvinyl alcohol to sodium alginate is 9-12:1, the pore diameter of the polyurethane sponge is 4-6 ppi, and the density is 0.4-0.6 g/cm 3.
As a further improvement of the invention, the mixing ratio of the gum solution and the quorum sensing bacterial composition is 3-5:1, the gum solution and the quorum sensing bacterial composition are injected into the polyurethane sponge biological carrier, and the adsorption density of the quorum sensing bacteria on the polyurethane sponge biological carrier is 0.05-0.45mL/cm 3.
As a further improvement of the present invention, the adhesive cures the biological carrier to which the microbial composition is adhered by immersing the biological carrier in a saturated boric acid solution of 4-5wt% calcium chloride for 6-8 hours.
The invention further protects the application of the microorganism composition for enhancing sewage denitrification in sewage treatment.
The invention has the following beneficial effects:
The invention utilizes polyurethane biological carrier to absorb and self-fix the microorganism composition, and then puts the microorganism composition into a sewage treatment reactor. This process enhances anammox activity by the microbial composition generating a class of signaling molecules known as AHLs in situ in the reactor. At the same time, the process involves performing a partial denitrification, i.e., reducing nitrate nitrogen (NO 3 - -N) to nitrite nitrogen (NO 2 - -N), thereby improving total nitrogen removal performance.
In this process, the main purpose of using polyurethane biovectors is to adsorb and immobilize the microbial compositions to ensure their stable presence in the reactor. The immobilization of the microbial composition on this carrier aids in uniform distribution throughout the reactor and provides an environment conducive to microbial activity.
The generation of AHL signaling molecules is one of the key steps in this experiment. These signal molecules are critical for the enhancement of anammox activity. The in situ generation of these signal molecules by the microbial composition can reduce cost, reduce reliance on expensive signal molecules, and be more economically viable in practical applications than exogenous addition.
In addition to enhancing anaerobic ammoxidation activity, the present invention introduces the concept of partial denitrification. The nitrate nitrogen is reduced to nitrite nitrogen, so that the total nitrogen removal performance is improved, and the wastewater treatment effect is further optimized.
In general, the invention designs a comprehensive wastewater treatment strategy, combines microbiology, biological carrier technology and signal molecule regulation and control, improves the performance of the anaerobic ammonia oxidation reactor, and reduces the running cost.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 shows the results of the quorum sensing activity test of the present invention (CV 026 in the lower left corner, VIR07 in the lower right corner, and the test bacteria in the upper side). Wherein A is CH9 (Ensifer sp.), B is CH10 (Brucella sp.), C is CH12 (Burkholderia sp.), and D is Pantoea ananatis SK-1 as a positive control.
FIG. 2 is a photograph of colonies of three quorum sensing bacteria of the present invention.
FIG. 3 is an SEM image of three quorum sensing bacteria of the present invention, wherein, A is CH9, B is CH10, and C is CH12.
FIG. 4 is a phylogenetic tree of three quorum sensing bacteria according to the present invention.
FIG. 5 shows a TLC identification plate of the type of signal molecule of the invention (H 2 O is blank, C6, C8, C10, C12 is standard sample of signal molecule C6-HSL, C8-HSL, C10-HSL, C12-oxo-HSL).
FIG. 6 is a photograph of a polyurethane biovector of the present invention (A is a blank polyurethane carrier and B is a polyurethane carrier for a cured microbial composition).
FIG. 7 shows denitrification performance of example 6 of the present invention.
FIG. 8 shows the change in effluent quality index during the start-up of the anaerobic ammonium oxidation reactor in example 10 of the present invention (A is the reactor R1 of comparative example 1 to which no microbial composition was added; B is the reactor R2 to which the microbial composition of example 7 was added; C is the reactor R3 to which the microbial composition of example 8 was added).
FIG. 9 shows the relative abundance (%) of the 16S V3-V4 rRNA gene sequence of the Anamox reactor horizontal sample in example 11 of the present invention (seed sluge is seed sludge for reactor start-up; R1 is activated sludge for successful reactor start-up in comparative example 1 without microbial composition added; R2 is activated sludge for successful reactor start-up with microbial composition added in example 7; and R3 is activated sludge for successful reactor start-up with microbial composition added in example 8).
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 population-sensing bacterial screening and Activity validation
In the existing anaerobic ammoxidation reactor in the laboratory, red flocculent anaerobic ammoxidation activated sludge granular sludge is taken as a sample for subsequent separation and purification. 1g of sludge sample is taken, 9mL of sterile deionized water is added, shaking and resuspension are carried out for 10min, the concentration is diluted to 10 -1、10-2、10-3、10-4、10-5 gradient by using sterile water, 200 mu L of sample suspension is taken and put on a raw water culture medium flat plate, separation and purification operation is carried out in an anaerobic glove box, the oxygen concentration is set to be 5%, and the culture temperature is set to be 33 ℃. Single colonies were picked, streaked on the same medium and purified, and the obtained pure bacteria were stored in a-80 ℃ refrigerator with 50% glycerol.
Purple bacillus (Chromobacterium violaceum) CV026 and VIR07 can be combined with AHLs and express specific genes thereof, a chromogenic reaction occurs, LB solid culture medium is poured into a 12-well plate, a regular triangle is added, three corners are respectively 1 mu L of QS bacterial liquid to be detected, 1 mu L of CV026 bacterial liquid and 1 mu L of VIR07 bacterial liquid, pantoea ananatis SK-1 is used as positive control, a sealing plate is adopted, an incubator is used for culturing for 1-3d, and whether CV026 and VIR24 appear purple is observed and photographed every other day. As shown in FIG. 1, the positive control SK-1 was developed all the way, indicating that the indicator bacteria were active well, and CH9, CH10 and CH12 were able to develop VIR07, indicating that three strains had quorum sensing activity.
Example 2 morphological characterization of three populations of sensing bacteria
As shown in FIG. 2, the colony features are circular, white, large in diameter, smooth in surface, convex lens-shaped, transparent, complete in edge and provided with a white ring in the middle. CH10 is a translucent, round, milky white, raised, scleroderma-like colony. CH12 is a yellow circular colony, the edges are neat, the ridges are raised, and the diameter is smaller.
As shown in FIG. 3, the CH9, CH10 and CH12 cells are rod-shaped, the length of the cells is about 1.0-1.6 μm, the width of all the cells is uniform, and the width of the cells is about 0.4-0.6 μm.
Example 3 physiological Biochemical characteristics of three populations of sensing bacteria
Strains are inoculated on LB culture medium, and various physiological and biochemical characteristics are detected respectively. The characteristic analysis of gelatin liquefaction, nitrate reduction, hydrogen peroxide reduction, starch hydrolysis, cellulose hydrolysis, IMViC test (indole test, methyl red test, volt-Pr (V-P) test, citrate utilization), ammonia production test, H2S production test, oxidase test and the like is mainly referred to the "common bacteria System identification Manual". The results of the physiological and biochemical tests are shown in Table 1.
TABLE 1 results of physiological and biochemical tests of three quorum sensing bacteria
| Detection item and method | CH9 | CH10 | CH12 |
| Starch hydrolysis test | - | - | - |
| Gelatin hydrolysis test | + | + | + |
| Cellulose hydrolysis test | - | + | - |
| Glucose fermentation test | Does not produce acid nor gas | Acid production without gas production | Does not produce acid nor gas |
| Fructose fermentation test | Does not produce acid nor gas | Does not produce acid nor gas | Does not produce acid nor gas |
| Indole test | - | - | - |
| Methyl Red test | - | - | - |
| Volt-Pr (V-P) test | + | + | + |
| Citrate test | - | + | + |
| Nitrate reduction test (nitrite detection method) | + | - | - |
| Hydrogen peroxide reduction test | - | - | - |
| Oxidase test | - | - | - |
| Ammonia production test | - | - | - |
| H 2 S production test | - | - | - |
Note that: "-" indicates a negative result, and "+" indicates a positive result. CH9 (Ensifer sp.), CH10 (Brucella sp.), CH12 (Burkholderia sp.).
Example 4 16S rDNA Gene determination of three populations of sensing bacteria
The phylogenetic tree of the three quorum sensing bacteria is shown in FIG. 4. The sequencing results are shown below:
CH9：
CGACGCTGCGGCAGGCTTAACACATGCAAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAATCTACCCTTTTCTACGGAATAACGCAGGGAAACTTGTGCTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGGGAAAGGATGAGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGACATTTAAGTCAGGGGTGAAATCCCAGAGCTCAACTCTGGAACTGCCTTTGATACTGGGTGTCTAGAGTATGGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTTTACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATCCCGATCGCGGATTACGGAGACGTTTTCCTTCAGTTCGGCTGGATCGGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCAGATCAGCATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGGAGTTGGTTCTACCCGAAGGTAGTGCGCTAACCGCAAGGAGGCAGCTAACCACGGTAGGGTCAGCGACTGGG
CH10：
CGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAACGTACCTTTTGCTACGGAATAACTCAGGGAAACTTGTGCTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGGCAAAGGATCGGCCCGCGTTGGATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGATTTACTGGGCGTAAAGCGCACGTAGGCGGACTTTTAAGTCAGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTTGATACTGGAAGTCTTGAGTATGGTAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGACCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTTGGGGAGTTTACTCTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATACCGGTCGCGGACACAGAGATGTGTCTTTCAGTTCGGCTGGACCGGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGCACGCGAGTGTGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGGAGTTGGTTTTACCCGAAGGCGCTGTGCTAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGAACAGG
CH12：
GAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCTACGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTTGGCTCTAATACAGTCGGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGCTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGACTGGCAGGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGCCGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAATCCCGCTGAGAGGTGGGAGTGCTCGAAAGAGAACCGGCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTGGCTAGTCTAACCGCAAGGAGGACGGTCACCACGGTAGGATTCATGACTGGGGTGAAGTCGTAACAGG
example 5 identification of Signal molecule types
Culturing 3 strains of quorum sensing bacteria for 5 days by using an LB culture medium, centrifuging, taking supernatant, extracting signal molecules AHLs in the supernatant by using an equal volume of ethyl acetate, and dissolving the extract by using methanol after rotary evaporation concentration. The sample spots were marked every 1: 1 cm by scribing with a pencil at the lower end 1 cm of the TLC thin-layer plate. Deionized water was used as a negative control, and the concentration of signal molecules was C6-HSL (100. Mu.M), C8-HSL (1. Mu.M), C10-HSL (10. Mu.M) and 3OC12-HSL (10. Mu.M), respectively, and 2. Mu.L was spotted on TLC thin-layer plates using spotting capillaries, respectively. The spotted TLC thin layer plate was placed in a beaker with developing solution, after sufficient development (leaving a small space), the solvent was evaporated, and the TLC thin layer plate was placed in a 9*9 square petri dish. The chromogenic medium was prepared and poured into a petri dish with a thin layer plate of TLC. Culturing in incubator for 2 days (30 ℃). As a result, as shown in FIG. 5, the signal molecules produced by CH9 were C6-HSL, C8-HSL and C12-HSL, the signal molecules produced by CH10 were C6-HSL and the signal molecules produced by CH12 were C6-HSL and C8-HSL.
EXAMPLE 6 CH10 denitrification Performance validation
As shown in FIG. 7, when CH10 was cultured under the condition of 5% oxygen concentration using NO 3 - -N as the sole nitrogen source, the concentration of NO 3 - -N was rapidly decreased for the first 24 hours, the removal rate reached 76.91%, and the removal rate of NO 3 - -N reached 100% by 42 hours. During denitrification, a small amount of NO 2 - -N is accumulated, the concentration of NO 2 - -N is reduced after the concentration is increased, and the accumulated amount of NO 2 - -N is not more than 2 mg/L at 48 hours.
EXAMPLE 7 preparation of microbial composition
3 Strains of quorum sensing bacteria are respectively inoculated in 300mL LB liquid culture medium, and shaking culture is carried out at 30 ℃ and 200 rpm/min for 24 h. Mixing bacterial solutions with OD 600 =1.0 in proportion, wherein CH9 is 30 parts; CH10 30 parts; CH12 40 parts. Gel with polyvinyl alcohol-sodium alginate (PVA-SA): bacterial liquid=4:1 is mixed and shaken uniformly, and low-power ultrasonic degassing bubbles 10 min are removed. The gel containing the microbial composition was injected into the polyurethane biovector and crosslinked in 7%H 3BO3 - 4%CaCl2 solution for 7h. After the crosslinking is completed, the residual primary crosslinking agent is washed by clean water, and then the secondary crosslinking is performed by using 7.1% Na 2SO4. After the two times of crosslinking are finished, cleaning the residual crosslinking agent by using clean water, soaking the residual crosslinking agent in deionized water, and preserving the residual crosslinking agent in a refrigerator at the temperature of 4 ℃. The biological carrier after adsorption fixation is shown in FIG. 6.
Example 8:
The mixing proportion of the bacterial liquid is CH9 50 parts; CH10 30 parts; CH12, 20 parts. Other preparation methods were the same as in example 7.
Example 9:
The mixing proportion of the bacterial liquid is CH9 20 parts; CH10 parts; CH12 40 parts. Other preparation methods were the same as in example 7.
Comparative example 1
According to the preparation method in example 7, polyvinyl alcohol-sodium alginate (PVA-SA) gel is injected only into the polyurethane sponge biological carrier, and bacterial liquid is not injected.
Example 10 use of quorum sensing hybrid immobilized microbial agents during start-up of an anaerobic ammonia oxidation reactor
The microbial compositions of example 7 and example 8 were loaded into a laboratory anaerobic ammoxidation reactor after being adsorbed and immobilized by a biological carrier. The reactor system was 2.2L and 1.2L of the biofilm was added. A total of 3 sets of reactors were set up, R1 was charged with the blank gel carrier of comparative example 1, R2 was charged with the microorganism composition solidifying carrier of comparative example 7, and R3 was charged with the microorganism composition of comparative example 8 to solidify. The seed sludge started by the carrier reactor is denitrified sludge in an anoxic tank of a sewage treatment plant, the hydraulic retention time is 24 hours during the starting period, and the influent water is artificially synthesized wastewater (NO 2 --N 66mg/L,NH4 + -N50 mg/L).
The change in reactor effluent quality is shown in FIG. 8 (A is the reactor R1 of comparative example 1 to which no microbial composition was added; B is the reactor R2 to which the microbial composition of example 7 was added; C is the reactor R3 to which the microbial composition of example 8 was added), R1 was successfully started at 104d, R2 was successfully started after 54d of run, and R3 was successfully started after 60d of run. The addition of the microbial composition greatly shortens the start-up time of the anaerobic ammonia oxidation reactor.
Example 11 differential analysis of microbial community structure in activated sludge before and after reactor startup
As shown in FIG. 9 (seed sluge is seed sludge from reactor start-up; R1 is activated sludge from successful reactor start-up of comparative example 1 without added microbial composition; R2 is activated sludge from successful reactor start-up with added microbial composition of example 7; R3 is activated sludge from successful reactor start-up with added microbial composition of example 8), at the generic level, the bacterial diversity and abundance are greatly different, and the relative abundance of anammox bacteria increases significantly with reactor operation. Anaerobic ammonia oxidizing bacteria in activated sludge are mainly from four genera, candidatus Brocadia, candidatus Jorgensenbacteria, candidatus Kuenenia, candidatus Saccharimonas, with Candidatus Brocadia being dominant in the community. The relative abundance of anaerobic ammonia oxidizing bacteria in the seed sludge started by the reactor was 4.62%, with Candidatus Brocadia and Candidatus Kuenenia accounting for 3.61% and 0.78%, respectively. After successful reactor start-up, the relative abundance of anammox bacteria in R1 increased to 11.22%, with Candidatus Brocadia and Candidatus Kuenenia increasing to 8.08% and 2.71%, respectively. The relative abundance of anammox bacteria in R2 increased to 21.62%, with Candidatus Brocadia rising to 17.61%, candidatus Jorgensenbacteria rising to 3.23%, and Candidatus Kuenenia declining slightly. The relative abundance of anammox bacteria in R3 increased to 19.75%, with Candidatus Brocadia up to 15.61% and Candidatus Jorgensenbacteria up to 2.23%.
Compared with the control group R1, the relative abundance of the anaerobic ammonia oxidation bacteria in R2 and R3 is obviously increased, which proves that the addition of the microbial composition promotes the enrichment of the anaerobic ammonia oxidation bacteria and accelerates the starting of the anaerobic ammonia oxidation reactor.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A microbial composition for enhancing sewage denitrification based on quorum sensing, which is characterized by comprising a quorum sensing bacterial composition symbiotic with anaerobic ammonia oxidation, an adhesive and a biological carrier.
2. The microbial composition for enhancing denitrification of wastewater according to claim 1, wherein the quorum sensing bacterial composition is composed of three strains of quorum sensing bacteria, namely sinorhizobium CH9, brucella CH10 and burkholderia CH12.
3. The microbial composition for enhancing denitrification of wastewater according to claim 2, wherein said quorum sensing bacteria are capable of producing N-acyl homoserine lactone signaling molecules.
4. The microbial composition for enhancing denitrification of sewage according to claim 2, wherein the signal molecules produced by sinorhizobium CH9 are C6-HSL and C12-HSL, the signal molecules produced by brucella CH10 are C6-HSL, and the signal molecules produced by burkholderia CH12 are C6-HSL and C8-HSL.
5. The microbial composition for enhancing denitrification of wastewater according to claim 2, wherein the quorum sensing bacteria are deposited in China center for type culture Collection, wherein CH9 class designation is Sinorhizobium Ensifer sp., deposit number is CCTCC M20232628, deposit date is 2023, 12 months and 22 days, deposit unit is China center for type culture Collection, address: eight 299 routes of Wuchang district of Wuhan, hubei province, post code: 430072; CH10 classification is named Brucella sp, the preservation number is CCTCC M20232629, the preservation date is 2023, 12 months and 22 days, the preservation unit is China center for type culture Collection, and the address is: eight 299 routes of Wuchang district of Wuhan, hubei province, post code: 430072; CH12 classification is named Burkholderia sp., with the preservation number of CCTCC M20232630, the preservation date of 2023, 12 months and 22 days, and the preservation unit: china center for type culture Collection, address: eight 299 routes of Wuchang district of Wuhan, hubei province, post code: 430072.
6. The microbial composition for enhancing denitrification of sewage according to claim 1, wherein the concentration of the quorum sensing bacterial composition is OD 600 = 0.8-1.2, and consists of the following bacterial liquids in parts by weight: 20-60 parts of Sinorhizobium, 10-80 parts of Brucella, and 20-60 parts of Burkholderia CH.
7. The microbial composition for enhancing sewage denitrification according to claim 1, wherein the adhesive is a peptized solution formed by mixing polyvinyl alcohol and sodium alginate, and the biological carrier is a polyurethane sponge biological carrier; the mass ratio of polyvinyl alcohol to sodium alginate is 9-12:1, the pore diameter of the polyurethane sponge is 4-6 ppi, and the density is 0.4-0.6 g/cm 3.
8. The microbial composition for enhancing denitrification of wastewater according to claim 7, wherein the mixing ratio of the peptic solution to the quorum sensing bacterial composition is 3-5:1, the peptic solution is injected into the polyurethane sponge biological carrier, and the adsorption density of quorum sensing bacteria on the polyurethane sponge biological carrier is 0.05-0.45mL/cm 3.
9. The microbial composition for enhancing denitrification of sewage according to claim 1, wherein the adhesive cures the biological carrier to which the microbial composition is adhered by immersing in a saturated boric acid solution of 4 to 5wt% calcium chloride for 6 to 8 hours.
10. Use of a microbial composition for enhancing denitrification of sewage according to any one of claims 1 to 9 in sewage treatment.
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