Method for in-situ enrichment of anaerobic ammonium oxidation bacteria by using bacterial strain with quorum sensing behavior

Abstract

The invention discloses a method for in-situ enrichment of anaerobic ammonium oxidation bacteria by using strains with quorum sensing behaviors, and belongs to the technical field of biological sewage treatment. The method for in-situ enrichment of anaerobic ammonium oxidation bacteria comprises the following step: co-culturing anaerobic ammonium oxidation bacteria and pseudomonas aeruginosa. According to the method disclosed by the invention, a small amount of Pseudomonas aeruginosa is added, so that the abundance of the anaerobic ammonium oxidation bacteria (Candidate Brocadium) can be improved, and the starting time of an anaerobic ammonium oxidation process is shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of sewage biological treatment, and in particular to a method for in-situ enrichment of anaerobic ammonia-oxidizing bacteria using strains with quorum sensing behavior. Background Art

[0002] Anaerobic ammonium oxidation refers to a biological process in which anaerobic ammonium oxidizing bacteria use nitrite nitrogen as an electron acceptor and ammonia as an electron donor to produce nitrogen gas and partially generate nitrate nitrogen under anaerobic or anoxic conditions. Anaerobic ammonium oxidizing bacteria have very broad application prospects and potential. Compared with traditional denitrification processes, their unique denitrification pathways have significant advantages. In fact, the demand for organic carbon in the anaerobic ammonium oxidation process is reduced by 100%, the aeration demand is reduced by about 60%, and the sludge production is reduced by about 90%. However, due to the slow growth of anaerobic ammonium oxidizing bacteria (doubling time is 10-14 days), it is difficult to enrich them, and anaerobic ammonium oxidizing bacteria are susceptible to many environmental conditions, such as pH, substrate concentration, temperature and the presence of heavy metals, which makes the stable operation of the anaerobic ammonium oxidation process difficult. The ability of microorganisms to resist these inhibitory factors is mainly achieved after forming a tight biofilm. Some typical pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, etc., can form a strong biofilm and acquire strong drug resistance. The study found that the efficient biofilm formation of these bacteria is related to a bacterial regulatory activity called quorum sensing. When the concentration of signal molecules in the environment reaches a threshold, bacteria will regulate the expression of related genes and trigger corresponding behaviors, including biofilm formation, protease synthesis, and bioluminescence.

[0003] Acyl-homoserine lactones (AHLs) can affect the growth of anaerobic ammonium oxidizing bacteria by regulating metabolism, and quorum sensing regulation also shows great potential in improving the activity of anaerobic ammonium oxidizing bacteria. In addition to improving the activity and growth rate of anaerobic ammonium oxidizing bacteria, quorum sensing technology also increases the secretion of EPS, thereby accelerating the aggregation of anaerobic ammonium oxidizing bacteria in the initial startup stage. Existing technologies usually add exogenous signal molecules, and the price of commercial homoserine lactones is very expensive (>100,000 yuan / g). Therefore, the best way to introduce quorum sensing enhancement methods for treating actual wastewater is to co-culture anaerobic ammonium oxidizing bacteria with bacteria with quorum sensing functions. Summary of the invention

[0004] In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a method for in situ enrichment of anaerobic ammonia-oxidizing bacteria using strains having quorum sensing behavior; the method of the present invention adds a small amount of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) can make anaerobic ammonium oxidizing bacteria ( Candidate Brocadia) abundance is increased; the present invention can effectively enrich anaerobic ammonium-oxidizing bacteria and shorten the start-up time of the anaerobic ammonium-oxidizing process.

[0005] The present invention firstly provides a method for in-situ enrichment of anaerobic ammonia oxidizing bacteria, comprising the following steps: co-culturing the anaerobic ammonia oxidizing bacteria and Pseudomonas aeruginosa.

[0006] Specifically, the method comprises the following steps: mixing anoxic sludge from a pig farm, Pseudomonas aeruginosa and water containing ammonia nitrogen and nitrite nitrogen for culturing.

[0007] In the above method, the Pseudomonas aeruginosa is added in the form of bacterial cells; the amount of the bacterial cells added is 2.5-3.5 mg of bacterial cells per 10L culture system; specifically, the Pseudomonas aeruginosa bacterial cells are taken from the logarithmic growth phase, OD 600 In the bacterial solution of 0.6~0.8; The preparation method of the bacterial cell comprises the following steps: culturing Pseudomonas aeruginosa, taking the logarithmic growth phase, OD 600 The bacterial solution was centrifuged at 0.6-0.8 to obtain the bacterial cells.

[0008] In the culture system, the concentration of the anoxic sludge from the pig farm was 114-117 mg / L.

[0009] The culture system refers to a mixed system of anoxic sludge from a pig farm, Pseudomonas aeruginosa and water containing ammonia nitrogen and nitrite nitrogen.

[0010] In the above method, the Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) is recorded in the literature as Significant enhancement of nitrous oxide energy yields from wastewater achieved by bioaugmentation with a recombinants train of Pseudomonas aeruginosa [J]. Scientific reports,2018,8 . aeruginosa in.

[0011] In the above method, in the water containing ammonia nitrogen and nitrite nitrogen, the concentration of ammonia nitrogen is 100±10 mg / L; the concentration of nitrite nitrogen is 132±13 mg / L; Inorganic salts and trace elements required by anaerobic ammonia-oxidizing bacteria are also added to the water containing ammonia nitrogen and nitrite nitrogen.

[0012] In the above method, the inorganic salt composition and concentration in the water containing ammonia nitrogen and nitrite nitrogen are as follows: 0.25-0.35 g / L CaCl2, 0.16-0.17 g / L MgCl2, 1-1.5 g / L NaHCO3, 0.05-0.1 g / L KH2PO4; The trace elements include trace element I solution and trace element II solution; wherein the dosage of the trace element I solution and the trace element II solution in the water containing ammonia nitrogen and nitrite nitrogen is 1 mL / L; The composition and concentration of the trace element I solution are as follows: 6-6.5 g / L EDTA·2Na, 4.5-5.5 g / L FeSO4·7H2O; The composition and concentration of the trace element II solution are as follows: 19~19.5 g / L EDTA·2Na, 0.01~0.05 g / L H3BO3, 0.4~0.6 g / L ZnSO4·7H2O, 0.2~0.3 g / L CoCl2·6H2O, 0.5~1.5 g / L MnCl2·4H2O, 0.2~0.3 g / L CuSO4·5H2O, 0.1~0.3 g / L NiCl2·6H2O, and 0.15~0.25 g / L NaMoO4·2H2O.

[0013] In the above method, the culturing is carried out under hypoxic conditions; The culture temperature is 30±1°C.

[0014] Furthermore, the present invention also provides a method for shortening the start-up time of an anaerobic ammonium oxidation process, comprising the following steps: adding Pseudomonas aeruginosa during the start-up phase of the anaerobic ammonium oxidation system and culturing the bacteria.

[0015] In the above method, the Pseudomonas aeruginosa is added in the form of bacteria; the amount of bacteria added is 2.5-3.5 mg of bacteria per 10L system; Specifically, the Pseudomonas aeruginosa cells were taken from the logarithmic growth phase, OD 600 At 0.6~0.8 of bacterial solution.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The Pseudomonas aeruginosa used in the present invention is a natural quorum sensing functional strain and is easy to obtain; (2) In the present invention, Pseudomonas aeruginosa is co-cultured with anaerobic ammonium oxidizing bacteria, which promotes the secretion of signal molecules in the system, thereby enriching anaerobic ammonium oxidizing bacteria in situ and increasing their abundance; (3) The method of the present invention can effectively enrich anaerobic ammonium-oxidizing bacteria in situ, thereby shortening the start-up time of the anaerobic ammonium-oxidizing process. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the anaerobic ammonium oxidation process device of the present invention; Figure 2 The concentrations of signal molecules in the two reactors of AS and AS+P at the 20th and 64th reaction cycles; Figure 3 The abundance of anaerobic ammonium oxidizing bacteria in the AS and AS+P reactors during the 20th reaction cycle. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0019] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0020] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0021] Pseudomonas aeruginosa in the following examples ( Pseudomonas aeruginosa ) recorded in the literatureSignificant enhancement of nitrous oxide energy yields from wastewaterachieved by bioaugmentation with a recombinantstrain of Pseudomonas aeruginosa [J]. Scientific reports,2018,8 . The public can obtain it from the Energy Research Institute of Jiangxi Academy of Sciences (i.e. the applicant). The above-mentioned biological materials are only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0022] Example 1 The present invention sets two groups of 10 L SBR reactors, and the reactor wall is provided with 5 equally spaced water outlets for sampling, drainage, water inlet, etc. The water inlet and drainage are connected to the water outlet of the SBR reactor through a peristaltic pump, and an agitator is provided. The temperature of the SBR reactor is controlled at 30°C through a circulating pump connected to a water bath (see the schematic diagram of the device). Figure 1 ).

[0023] (1) Pour 300 mL of anoxic sludge (sludge concentration of 3850 mg / L) from Muyuan Farm No. 2 in Guangfu Town, Nanchang County, Nanchang City, Jiangxi Province into each of the two reactors, and add water to make the effective volume of the reactor 10 L.

[0024] (2) Pseudomonas aeruginosa The bacterial suspension was transferred to Luria-Bertani (LB) culture medium and cultured in a 37°C constant temperature incubator. Cultured until OD 600 When the value is about 0.7, 100 mL of bacterial solution is centrifuged at 5000 r / min for 10 min to obtain 3 mg of bacterial cells; the bacterial cells are added to one of the reactors in step (1) and counted as the AS+P group; no Pseudomonas aeruginosa The reactor with bacterial liquid was counted as AS group.

[0025] (3) Both groups of reactors are equipped with three reaction stages: Stage I (1-5 cycles): the reactor is not equipped with water inlet and outlet, and only 0.132 g NaNO2 and 0.768 g NH4Cl powder are added in each cycle to supplement the nitrogen source; each reaction cycle is 24 hours; Stage II (6-14 cycles): 2 L of water is inlet and outlet per cycle, the water discharge ratio is 20% (volume percentage), and each reaction cycle is 24 hours; Stage III (15-65 cycles): 2 L of water is inlet and outlet per cycle, the water discharge ratio is 20% (volume percentage), but each reaction cycle is shortened to 12 hours.

[0026] The water for the two groups of reactors is prepared with ammonia nitrogen and nitrite nitrogen as the matrix, and appropriate amounts of inorganic salts and trace elements required by anaerobic ammonium oxidizing bacteria are added at the same time. In Table 1, the concentrations of matrix and inorganic salts refer to the concentrations of each component in the prepared water; the concentrations of trace elements I and trace elements II refer to the concentrations of each component in trace element I solution and trace element II solution; the amount of trace element I solution and trace element II solution added to the prepared water is 1 mL / L. The prepared water is prepared with this concentration ratio, and the concentration of ammonia nitrogen in the prepared water is 100 mg / L; the concentration of nitrite nitrogen is 132 mg / L.

[0027] Table 1 Water composition and concentration

[0028] At the 20th and 64th reaction cycles, water was taken from the two reactors and 50 mL of water sample was filtered with a 0.22 mm filter head, and then completely extracted twice with 50 mL of 0.1% (volume ratio) formic acid acidified ethyl acetate solution. The extracted ethyl acetate solution was evaporated to 2 mL at 40°C using a rotary evaporator, and then evaporated to 400 µL with nitrogen at 40°C and stored in a -20°C refrigerator for subsequent analysis. C6-HSL and C8-HSL in the aqueous phase were determined by liquid chromatography-mass spectrometry (Thermo ULTIMATE 3000 UHPLC-QExactive). The LC parameters were set as follows: mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, and the injection volume was 50 µL. The MS parameters were as follows: ion source was HESI, the flow rate was 40 mL / min, the capillary temperature was 300°C, and the spray voltage was 3800 V. The results are shown in Tables 2 and Figure 2 .

[0029] Table 2 Composition and concentration of signal molecules in sampled water

[0030] From Table 2 and Figure 2 It can be seen that in the 20th reaction cycle, the C6-HSL concentrations in the AS group and the AS+P group were 5.84 μg / L and 9.68 μg / L, respectively; in the 64th reaction cycle, the C6-HSL concentrations in the AS group and the AS+P group were 0.64 μg / L and 0.96 μg / L, respectively. Pseudomonas aeruginosa ) in the AS+P group increased by 65.75% and 50% respectively. C6-HSL enhances the metabolic activity of anaerobic ammonium oxidizing bacteria and improves the activity of anaerobic ammonium oxidizing bacteria in the reactor. Candidate Brocadia ) abundance.

[0031] At the 20th and 64th reaction cycles, part of the sludge was taken from the two groups of reactors and centrifuged at 5000 rpm for 10 min before high-throughput sequencing and microbial community structure analysis.

[0032] According to the high-throughput sequencing results, the microbial community structure was analyzed. In the 20th reaction cycle, the anaerobic ammonium oxidizing bacteria ( Candidate Brocadia ) abundances were 0.45% and 1.63%, respectively, and the absolute abundance increased by 1.18%; in the 64th reaction cycle, the anaerobic ammonia oxidizing bacteria in the AS group and the AS+P group ( Candidate Brocadia ) were 1.89% and 2.01% respectively, and the absolute value of abundance increased by 0.12%. Pseudomonas aeruginosa It is rich in anaerobic ammonium oxidizing bacteria ( Candidate Brocadia ) function.

Claims

1. A method for in situ enrichment of anaerobic ammonia oxidizing bacteria, comprising the following steps: co-culturing anaerobic ammonia oxidizing bacteria and Pseudomonas aeruginosa.

2. The method according to claim 1, characterized in that: The method comprises the following steps: mixing anoxic sludge from a pig farm, Pseudomonas aeruginosa and water containing ammonia nitrogen and nitrite nitrogen for culturing.

3. The method according to claim 2, characterized in that: The Pseudomonas aeruginosa is added in the form of bacterial cells; the amount of the bacterial cells added is 2.5-3.5 mg of bacterial cells per 10 L of culture system; In the culture system, the concentration of the anoxic sludge from the pig farm was 114-117 mg / L.

4. The method according to claim 3, characterized in that: The cells of Pseudomonas aeruginosa were taken from the logarithmic growth phase, OD 600 At 0.6~0.8 of bacterial solution.

5. The method according to any one of claims 2 to 4, characterized in that: In the water containing ammonia nitrogen and nitrite nitrogen, the concentration of ammonia nitrogen is 100±10 mg / L; the concentration of nitrite nitrogen is 132±13 mg / L; Inorganic salts and trace elements required by anaerobic ammonia-oxidizing bacteria are also added to the water containing ammonia nitrogen and nitrite nitrogen.

6. The method according to claim 5, characterized in that: In the water containing ammonia nitrogen and nitrite nitrogen, the composition and concentration of the inorganic salts are as follows: 0.25-0.35 g / L CaCl2, 0.16-0.17 g / L MgCl2, 1-1.5 g / L NaHCO3, 0.05-0.1 g / L KH2PO4; The trace elements include trace element I solution and trace element II solution; wherein the dosage of trace element I solution and trace element II solution in water containing ammonia nitrogen and nitrite nitrogen is 1 mL / L; The composition and concentration of the trace element I solution are as follows: 6-6.5 g / L EDTA·2Na, 4.5-5.5 g / L FeSO4·7H2O; The composition and concentration of the trace element II solution are as follows: 19~19.5 g / L EDTA·2Na, 0.01~0.05 g / LH3BO3, 0.4~0.6 g / L ZnSO4·7H2O, 0.2~0.3 g / L CoCl2·6H2O, 0.5~1.5 g / L MnCl2·4H2O, 0.2~0.3 g / L CuSO4·5H2O, 0.1~0.3 g / L NiCl2·6H2O, and 0.15~0.25 g / L NaMoO4·2H2O.

7. The method according to any one of claims 2 to 6, characterized in that: The culturing is carried out under hypoxic conditions; The culture temperature is 30±1°C.

8. A method for shortening the start-up time of an anaerobic ammonium oxidation process, comprising the following steps: adding Pseudomonas aeruginosa during the start-up phase of the anaerobic ammonium oxidation system and culturing it.

9. The method according to claim 8, characterized in that: The Pseudomonas aeruginosa is added in the form of bacterial cells; the amount of the bacterial cells added is 2.5-3.5 mg of bacterial cells per 10 L of the system.

10. The method according to claim 9, characterized in that: The cells of Pseudomonas aeruginosa were taken from the logarithmic growth phase, OD 600 At 0.6~0.8 of bacterial solution.

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