A microbial composition for enhancing wastewater denitrification based on quorum sensing and its application

Abstract

The present invention proposes a microbial composition for enhancing sewage denitrification based on quorum sensing and its application, which belongs to the technical field of sewage biological treatment, and is composed of a quorum sensing bacterial composition that coexists with anaerobic ammonia oxidation, an adhesive and a biological carrier. Among them, the quorum sensing bacterial composition is composed of three quorum sensing bacteria, namely, Sinorhizobium CH9, Brucella CH10 and Burkholderia CH12. The microbial composition prepared by the present invention can continuously stabilize signal molecules, improve the denitrification efficiency of sewage treatment reactors, shorten the startup time of anaerobic ammonia oxidation reactors, and has broad application prospects in the field of sewage denitrification.

Description

Technical Field

[0001] The invention relates to the technical field of sewage biological treatment, and in particular to a microbial composition for enhancing sewage denitrification based on quorum sensing and an application thereof. Background Art

[0002] The oxygen-ammonia oxidation process is a new type of autotrophic biological denitrification process. This process breaks through the basic theoretical concepts of traditional biological denitrification processes and has the advantages of no need for aeration, low sludge production, and no need for external carbon sources. However, anaerobic ammonium-oxidizing bacteria (AnAOB) are sensitive to the environment, grow slowly, are small in size, and are easily lost, resulting in a long reactor startup time, which affects the promotion and application of the anaerobic ammonium oxidation process. Therefore, how to quickly enrich anaerobic ammonium-oxidizing bacteria in practical applications is an important issue that needs to be solved urgently.

[0003] Quorum sensing (QS) can regulate gene expression according to bacterial population density to improve bacterial growth rate, activity, and denitrification efficiency. Acylhomoserine lactone (AHL) is a common signal molecule that is mainly involved in the regulation of Gram-negative bacteria. It is reported that the introduction of exogenous caproyl-L-homoserine lactone (C6-HSL) and octanoyl-L-homoserine lactone (C8-HSL) can significantly increase the activity and growth rate of anaerobic ammonium-oxidizing bacteria, thereby improving the denitrification performance of the entire anaerobic ammonium-oxidizing system. However, due to the high cost of exogenously added signal molecules, it is difficult to apply. Summary of the invention

[0004] The purpose of the present invention is to provide a microbial composition for enhancing sewage denitrification based on quorum sensing and its application, which has a good sewage denitrification effect.

[0005] The technical solution of the present invention is achieved in this way:

[0006] 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.

[0007] As a further improvement of the present invention, the quorum sensing bacterial composition consists of three strains of quorum sensing bacteria, namely, Sinorhizobium CH9, Brucella CH10 and Burkholderia CH12.

[0008] As a further improvement of the present invention, the quorum sensing bacteria can produce N-acyl homoserine lactone signal molecules.

[0009] As a further improvement of the present invention, 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.

[0010] As a further improvement of the present invention, the quorum sensing bacteria are deposited in the China Center for Type Culture Collection, where CH9 is classified and named Sinorhizobium Ensifer sp. , the deposit number is CCTCC M 20232628, the deposit date is December 22, 2023, the depository is China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072; CH10 classification name is Brucella Brucella sp. , the deposit number is CCTCC M20232629, the deposit date is December 22, 2023, the depository is China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072; CH12 is classified as Burkholderia Burkholderia sp. , the deposit number is CCTCC M 20232630, the deposit date is December 22, 2023, the depositor is China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0011] As a further improvement of the present invention, the concentration of the quorum sensing bacterial composition is OD 600 =0.8-1.2, composed of the following parts by weight of bacterial liquid: 20-60 parts of Sinorhizobium CH9, 20-80 parts of Brucella CH10, and 20-60 parts of Burkholderia CH12.

[0012] As a further improvement of the present invention, the adhesive is a glue solution formed by mixing polyvinyl alcohol and sodium alginate, and the biological carrier is a polyurethane sponge biological carrier; the mass ratio of the polyvinyl alcohol to the sodium alginate is 9-12:1, the pore size of the polyurethane sponge is 4-6 ppi, and the density is 0.4-0.6 g / cm 3 .

[0013] As a further improvement of the present invention, the mixing ratio of the glue solution and the quorum sensing bacteria composition is 3-5:1, which is 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 .

[0014] As a further improvement of the present invention, the bio-carrier adhered with the microbial composition is immersed in a saturated boric acid solution containing 4-5 wt % calcium chloride for curing, and the curing time is 6-8 hours.

[0015] The present invention further protects the use of the above-mentioned microbial composition for enhancing wastewater denitrification in wastewater treatment.

[0016] The present invention has the following beneficial effects:

[0017] The present invention uses polyurethane bio-carriers to adsorb and self-fix the microbial composition, and then puts it into the sewage treatment reactor. This process enhances the anaerobic ammonium oxidation activity by in-situ production of a class of signal molecules called AHL by the microbial composition in the reactor. At the same time, the process also involves partial denitrification, that is, nitrate nitrogen (NO3 - -N) is reduced to nitrite nitrogen (NO2 - -N), thereby improving the total nitrogen removal performance.

[0018] In this process, the main purpose of using polyurethane bio-carriers is to adsorb and fix the microbial composition to ensure that they maintain a stable presence in the reactor. The fixation of the microbial composition on this carrier helps to evenly distribute it throughout the reactor and provides an environment conducive to microbial activity.

[0019] The production of AHL signal molecules is one of the key steps in this experiment. These signal molecules are essential for the enhancement of anaerobic ammonium oxidation activity. Compared with exogenous addition, the in situ production of these signal molecules by microbial compositions can reduce costs, reduce dependence on expensive signal molecules, and is more economical and feasible in practical applications.

[0020] In addition to enhancing the anaerobic ammonium oxidation activity, the present invention also introduces the concept of partial denitrification, which helps to improve the performance of total nitrogen removal by reducing nitrate nitrogen to nitrite nitrogen, and further optimizes the effect of wastewater treatment.

[0021] In general, the present invention designs an integrated wastewater treatment strategy that combines microbiology, bio-carrier technology, and signal molecule regulation to improve the performance of anaerobic ammonium oxidation reactors while reducing operating costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 The results of the quorum sensing activity verification of the present invention (the lower left corner is CV026, the lower right corner is VIR07, and the upper part is the test bacteria). Among them, A is CH9 (Ensifer sp.), B is CH10 (Brucella sp.), C is CH12 (Burkholderia sp.), and D is the positive control Pantoea ananatis SK-1.

[0024] Figure 2 These are photos of colonies of three quorum sensing bacteria of the present invention.

[0025] Figure 3 These are SEM images of three strains of quorum sensing bacteria of the present invention, wherein Figure A is CH9, Figure B is CH10, and Figure C is CH12.

[0026] Figure 4 This is a phylogenetic tree of three quorum sensing bacteria of the present invention.

[0027] Figure 5 This is a TLC identification plate for the signal molecule type of the present invention (H2O is a blank control, C6, C8, C10, C12 are signal molecule standard samples C6-HSL, C8-HSL, C10-HSL, C12-oxo-HSL).

[0028] Figure 6 These are photos of the polyurethane biological carrier of the present invention (A is a blank polyurethane carrier, and B is a polyurethane carrier of a solidified microbial composition).

[0029] Figure 7 This is the denitrification performance of Example 6 of the present invention.

[0030] Figure 8 The effluent water quality index changes during the startup of the anaerobic ammonium oxidation reactor in Example 10 of the present invention (A is the reactor R1 of Comparative Example 1 without adding the microbial composition; B is the reactor R2 with adding the microbial composition of Example 7; C is the reactor R3 with adding the microbial composition of Example 8).

[0031] Fig. 9 is the relative abundance (%) of the 16S V3-V4 rRNA gene sequence of the genus level sample of the Anammox reactor in Example 11 of the present invention (seed sluge is the seed sludge for starting the reactor; R1 is the activated sludge when the reactor of Comparative Example 1 without adding the microbial composition is successfully started; R2 is the activated sludge when the reactor with the addition of the microbial composition of Example 7 is successfully started; R3 is the activated sludge when the reactor with the addition of the microbial composition of Example 8 is successfully started). DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1 Screening and activity verification of quorum sensing bacteria

[0034] In the existing anaerobic ammonium oxidation reactor in the laboratory, red flocculent anaerobic ammonium oxidation activated sludge granules were taken as samples for subsequent separation and purification. 1g of sludge sample was taken, 9mL of sterile deionized water was added, and the mixture was resuspended by shaking for 10min. The concentration was diluted to 10 with sterile water. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Gradient, take 200 μL of sample suspension on the original water culture medium plate, and perform separation and purification in an anaerobic glove box, and set the oxygen concentration to 5% and the culture temperature to 33°C. Pick a single colony, streak it on the same culture medium for purification, and store the pure bacteria in a -80°C refrigerator with 50% glycerol.

[0035] Purple Bacillus ( Chromobacterium violaceum )CV026 and VIR07 can bind to AHLs and express their specific genes, resulting in a color reaction. LB solid culture medium was poured into a 12-well plate, and samples were added in an equilateral triangle, with 1μL of QS bacterial solution to be tested, 1μL of CV026 bacterial solution and 1μl of VIR07 bacterial solution at the three corners. Pantoea ananas ( Pantoea ananatis ) SK-1 was used as a positive control, the plates were sealed, and cultured in an incubator for 1-3 days. Every other day, CV026 and VIR24 were observed and photographed to record whether they were purple. Figure 1 As shown, the positive control SK-1 showed color, indicating that the indicator bacteria had good activity, and CH9, CH10 and CH12 could make VIR07 show color, indicating that the three strains had quorum sensing activity.

[0036] Example 2 Morphological characteristics of three quorum sensing bacteria

[0037] Colony characteristics such as Figure 2 As shown, CH9 is round, white, with a large diameter, smooth surface, convex lens-shaped, transparent, with complete edges, and a white circle in the middle of the colony. CH10 is a translucent, round, milky white, raised, scleroderma-like colony. CH12 is a yellow round colony with neat edges, raised edges, and a small diameter.

[0038] Scanning electron microscope photos Figure 3As shown, the CH9, CH10 and CH12 bacteria are all rod-shaped, with a length of about 1.0-1.6 μm, and the width of all bacteria is relatively consistent, about 0.4-0.6 μm.

[0039] Example 3 Physiological and biochemical characteristics of three quorum sensing bacteria

[0040] The strain was inoculated on LB medium and various physiological and biochemical characteristics were tested. Gelatin liquefaction, nitrate reduction, hydrogen peroxide reduction, starch hydrolysis, cellulose hydrolysis, IMViC test (indole test, methyl red test, volt-Pu test, citrate utilization), ammonia production test, H2S production test, oxidase test and other characteristic analyses were mainly referred to the "Common Bacteria System Identification Manual". The results of physiological and biochemical tests are shown in Table 1.

[0041] Table 1 Physiological and biochemical test results of three quorum sensing bacteria

[0042] Testing items and methods CH9 CH10 CH12 Starch hydrolysis test - - - Gelatin hydrolysis test + + + Cellulose hydrolysis test - + - Glucose fermentation test No acid or gas generation Produces acid but no gas No acid or gas generation Fructose fermentation test No acid or gas generation No acid or gas generation No acid or gas generation Indole test - - - Methyl red test - - - Volt-Pull (VP) test + + + Citrate test - + + Nitrate reduction test (nitrite detection method) + - - Hydrogen peroxide reduction test - - - Oxidase test - - - Ammonia production test - - - <![CDATA[H2S production test]]> - - -

[0043] Note: “-” indicates negative result, “+” indicates positive result. CH9 (Ensifer sp.), CH10 (Brucellas p.), CH12 (Burkholderia sp.).

[0044] Example 4 Determination of 16S rDNA Genes of Three Quorum Sensing Bacteria

[0045] Phylogenetic tree of three quorum sensing bacteria Figure 4 The sequencing results are shown below:

[0046] CH9:

[0047]

[0048] CH10:

[0049]

[0050] CH12:

[0051]

[0052] Example 5 Identification of signal molecule types

[0053] Three strains of quorum sensing bacteria were cultured in LB medium for 5 days, centrifuged, and the supernatant was taken. The signal molecule AHLs in the supernatant was extracted with an equal volume of ethyl acetate. The extract was concentrated by rotary evaporation and dissolved in methanol. Use a pencil to draw a line 1 cm from the bottom of the TLC thin layer plate, and mark the spot every 1 cm. Deionized water was used as a negative control. The concentrations of signal molecules were C6-HSL (100μM), C8-HSL (1μM), C10-HSL (10μM) and 3OC12-HSL (10μM), respectively. 2 μL was spotted on the TLC thin layer plate with a spotting capillary. Place the spotted TLC thin layer plate in a beaker with a developing solution. After fully developing (leaving a little space), evaporate the solvent and place the TLC thin layer plate in a 9*9 square culture dish. Prepare the color development culture medium and pour it into the culture dish with the TLC thin layer plate. Place it in an incubator and culture it for 2 days (30°C). The results are as follows. Figure 5 As shown, the signal molecules produced by CH9 are C6-HSL, C8-HSL and C12-HSL, the signal molecules produced by CH10 are C6-HSL, and the signal molecules produced by CH12 are C6-HSL and C8-HSL.

[0054] Example 6: Verification of CH10 denitrification performance

[0055] like Figure 7 As shown, when NO3 - When CH10 was cultured under the condition of 5% oxygen concentration with -N as the only nitrogen source, NO3 - -N concentration decreased rapidly, and the removal rate reached 76.91%. At 42h, NO3 - -N removal rate reaches 100%. A small amount of NO2 - -N accumulation, its concentration first increased and then decreased, at 48h, NO2 - -N accumulation does not exceed 2 mg / L.

[0056] Example 7 Preparation of microbial composition

[0057] Three strains of quorum sensing bacteria were inoculated into 300 mL LB liquid medium and cultured at 30 °C and 200 rpm / min for 24 h. 600=1.0 bacterial solution in proportion, including 30 parts of CH9; 30 parts of CH10; 40 parts of CH12. Mix and shake the polyvinyl alcohol-sodium alginate (PVA-SA) gel: bacterial solution at a ratio of 4:1, and remove bubbles by low-power ultrasound for 10 minutes. Inject the gel containing the microbial composition into the polyurethane bio-carrier and cross-link it in a 7% H3BO3-4% CaCl2 solution for 7 hours. After the cross-linking is completed, wash the residual primary cross-linking agent with clean water and then use 7.1% Na2SO4 for the second cross-linking. After the two cross-linkings are completed, wash the residual cross-linking agent with clean water, soak it in deionized water and store it in a refrigerator at 4°C. The bio-carrier after adsorption and fixation is as follows Figure 6 shown.

[0058] Embodiment 8:

[0059] The mixing ratio of the bacterial solution is 50 parts of CH9, 30 parts of CH10 and 20 parts of CH12. The other preparation methods are the same as those in Example 7.

[0060] Embodiment 9:

[0061] The mixing ratio of the bacterial solution is 20 parts of CH9, 40 parts of CH10 and 40 parts of CH12. The other preparation methods are the same as those in Example 7.

[0062] Comparative Example 1

[0063] According to the preparation method in Example 7, only polyvinyl alcohol-sodium alginate (PVA-SA) gel was injected into the polyurethane sponge biological carrier, and no bacterial solution was injected.

[0064] Example 10 Application of quorum sensing mixed immobilized bacterial agent during the start-up of anaerobic ammonium oxidation reactor

[0065] The microbial compositions in Examples 7 and 8 were adsorbed and fixed by biological carriers and then added to the laboratory anaerobic ammonium oxidation reactor. The reactor system was 2.2L, and 1.2L of biological filler was added. A total of 3 groups of reactors were set up, R1 added the blank gel carrier of Comparative Example 1, R2 added the microbial composition solidified carrier of Comparative Example 7, and R3 added the microbial composition solidified carrier of Comparative Example 8. The seed sludge for starting the carrier reactor was the denitrification sludge of the anoxic tank of the sewage treatment plant. The hydraulic retention time during the startup period was 24h, and the influent was artificial synthetic wastewater (NO2 - -N 66mg / L,NH4 + -N50mg / L).

[0066] The water quality of the reactor outlet water changes as follows Figure 8As shown in the figure (A is the reactor R1 of comparative example 1 without adding the microbial composition; B is the reactor R2 with adding the microbial composition of example 7; C is the reactor R3 with adding the microbial composition of example 8), R1 was successfully started at the 104th day, R2 was successfully started after running for 54 days, and R3 was successfully started after running for 60 days. The addition of the microbial composition greatly shortened the start-up time of the anaerobic ammonium oxidation reactor.

[0067] Example 11 Analysis of differences in microbial community structure in activated sludge before and after reactor startup

[0068] like Fig. 9 As shown in the figure (seed sluge is the seed sludge for reactor startup; R1 is the activated sludge when the reactor of comparative example 1 without adding microbial composition is successfully started; R2 is the activated sludge when the reactor of example 7 is successfully started; R3 is the activated sludge when the reactor of example 8 is successfully started), at the genus level, the bacterial diversity and abundance are quite different, and the relative abundance of anaerobic ammonia oxidizing bacteria increases significantly with the operation of the reactor. The anaerobic ammonia oxidizing bacteria in the activated sludge mainly come from four genera, Candidatus Brocadia , Candidatus Jorgensenbacteria , Candidatus Kuenenia , Candidatus Saccharimonas ,in Candidatus Brocadia The relative abundance of anaerobic ammonium-oxidizing bacteria in the seed sludge at the start-up of the reactor was 4.62%, of which Candidatus Brocadia and Candidatus Kuenenia After the reactor was successfully started, the relative abundance of anaerobic ammonia-oxidizing bacteria in R1 increased to 11.22%, of which Candidatus Brocadia and Candidatus Kuenenia The relative abundance of anaerobic ammonia-oxidizing bacteria in R2 increased to 21.62%, including Candidatus Brocadia rises to 17.61%, Candidatus Jorgensenbacteria rose to 3.23%, while Candidatus Kuenenia The relative abundance of anaerobic ammonium oxidizing bacteria in R3 increased to 19.75%, among which Candidatus Brocadia rises to 15.61%, Candidatus Jorgensenbacteria Rised to 2.23%.

[0069] Compared with the control group R1, the relative abundance of anaerobic ammonium oxidizing bacteria in R2 and R3 increased significantly, indicating that the addition of the microbial composition promoted the enrichment of anaerobic ammonium oxidizing bacteria and accelerated the start-up of the anaerobic ammonium oxidizing reactor.

[0070] 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, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A microbial composition for enhancing wastewater denitrification based on quorum sensing, characterized in that: The invention is composed of a group sensing bacterial composition that coexists with anaerobic ammonium oxidizer, an adhesive and a biological carrier; the group sensing bacteria are preserved in the China Center for Type Culture Collection, among which CH9 is classified and named as Sinorhizobium Ensifer sp. , the deposit number is CCTCC M20232628, the deposit date is December 22, 2023, the depository is China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072; CH10 classification name is Brucella Brucella sp. , the deposit number is CCTCC M 20232629, the deposit date is December 22, 2023, the depository is China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072; CH12 is classified as Burkholderia Burkholderia sp. , the deposit number is CCTCC M 20232630, the deposit date is December 22, 2023, the depositor is China Center for Type Culture Collection, the address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072; the quorum sensing bacterial composition is composed of three strains of quorum sensing bacteria, namely, Sinorhizobium CH9, Brucella CH10 and Burkholderia CH12, which are mixed according to the following weight parts: 30-50 parts of CH9; 30 parts of CH10; 20-40 parts of CH12.

2. The microbial composition for enhancing wastewater denitrification according to claim 1, characterized in that: The quorum sensing bacteria can produce N-acyl homoserine lactone signal molecules.

3. The microbial composition for enhancing wastewater denitrification according to claim 2, characterized in that: 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.

4. The microbial composition for enhancing wastewater denitrification according to claim 1, characterized in that: The concentration of the quorum sensing bacterial composition is OD 600 =0.8-1.

2.

5. The microbial composition for enhancing wastewater denitrification according to claim 1, characterized in that: The adhesive is a glue 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 size of the polyurethane sponge is 4-6 ppi, and the density is 0.4-0.6 g / cm 3 .

6. The microbial composition for enhancing wastewater denitrification according to claim 5, characterized in that: The mixing ratio of the glue solution and the quorum sensing bacteria composition is 3-5:1, and the mixture is injected into the polyurethane sponge biological carrier. The adsorption density of the quorum sensing bacteria on the polyurethane sponge biological carrier is 0.05-0.45 ml / cm 3 .

7. The microbial composition for enhancing wastewater denitrification according to claim 1, characterized in that: The adhesive is used to solidify the biological carrier with the microbial composition adhered thereto by immersing it in a saturated boric acid solution containing 4-5 wt % calcium chloride for 6-8 hours.

8. Use of the microbial composition for enhancing denitrification of sewage as claimed in any one of claims 1 to 7 in sewage treatment.

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