Mixed bacterial agent for denitrification of cyanogen-containing wastewater

Abstract

The invention relates to the technical field of environmental microorganisms, in particular to a mixed bacterial agent for denitrification of cyanogen-containing wastewater. The microbial inoculum is prepared from five strains, namely alcaligenes alcaligenes YH-2C1, sphingosynium CY-10, calcium acetate-acinetobacter TNJ-1, acinetobacter and rhodococcus ruber C1 according to a proportion, the tolerance concentration of the microbial inoculum to CN <-> concentration reaches 40mg / L, and the tolerance concentration to ammonia nitrogen reaches 2000mg / L. The microbial agent for denitrification of the cyanide-containing wastewater, provided by the invention, is mainly applied to the fields of denitrification of high-ammonia-nitrogen wastewater, denitrification of the cyanide-containing wastewater and the like, has the advantages of high removal efficiency, strong endurance capacity, convenience in use and culture and the like, and can quickly remove ammonia nitrogen and total nitrogen in water under the condition of containing high-concentration cyanide.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental microorganism technology, and particularly relates to a mixed bacterial agent for denitrification of cyanide-containing wastewater. BACKGROUND

[0002] Industry is the main body of the national economy, however, a large amount of high ammonia-nitrogen wastewater is generated in the industrial production process, and the high ammonia-nitrogen wastewater generated in the industrial production process is different from domestic sewage and aquaculture wastewater, that is, the high ammonia-nitrogen wastewater generated in the industrial production process usually has high biological inhibition, such as the high ammonia-nitrogen wastewater generated in the production process of coking, electrolyte production, pharmaceutical industry and the like, which often contains highly toxic substances such as trichloroethane and cyanide, and has a high treatment difficulty.

[0003] The treatment of high ammonia-nitrogen wastewater is generally divided into physical and chemical methods and biological methods, compared with the physical and chemical methods, the biological methods have the advantages of low treatment cost and no secondary pollution, and are widely used in the treatment of municipal, aquaculture, chemical and other fields. The traditional biological method mainly relies on autotrophic nitrifying bacteria and heterotrophic denitrifying bacteria, and the nutritional metabolism type of autotrophic nitrifying bacteria and heterotrophic denitrifying bacteria determines that the generation cycle is long, the tolerance is poor, the pollutant removal efficiency is low, and most importantly, the autotrophic nitrifying bacteria and heterotrophic denitrifying bacteria lack tolerance to the above-mentioned cyanide, trichloride and other toxic substances, and the biological treatment technology for high ammonia-nitrogen wastewater containing toxic substances is in urgent need of development. In the actual treatment process, the physical and chemical technology is used for detoxification treatment at the front end, and then biochemical treatment is carried out, however, the physical and chemical treatment technology cannot achieve precise detoxification for complex water quality, and an excessive amount of chemical agents need to be added, which leads to the introduction of new chemical substances while the toxic substances are removed, and the growth of microorganisms and the denitrification effect are inhibited. Existing researches have shown that by compounding different strains, the tolerance of the composite bacterial agent to toxicity can be improved. Under the condition of toxicity inhibition, different strains may exchange tolerance genes through plasmid transfer, transduction or transformation, or produce a group cooperation relationship, so as to improve the tolerance of the whole group to toxic substances. In addition, certain strains may also secrete specific enzymes or metabolic products to enhance the activity of other strains, thereby improving the tolerance to toxic substances. Therefore, by compounding strains with functions of denitrification, cyanide tolerance and organic matter removal into a composite denitrification bacterial agent, and applying the composite denitrification bacterial agent to the denitrification of high ammonia-nitrogen wastewater containing cyanide toxic substances, the biological denitrification effect of wastewater containing cyanide generated in the production of coking, electrolyte production, pharmaceutical industry and the like can be improved.

[0004] Therefore, in order to improve the biological treatment effect of denitrification of cyanide-containing wastewater, the present application provides a mixed bacterial agent for denitrification of cyanide-containing wastewater. SUMMARY

[0005] The present application aims to provide a mixed bacterial agent for denitrification of cyanide-containing wastewater, and to provide a bacterial agent for denitrification of cyanide-containing wastewater and its application, which can significantly improve the biological treatment effect of denitrification of cyanide-containing wastewater through the interaction of multiple microorganisms.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a mixed microbial agent for wastewater treatment, wherein the mixed microbial agent is composed of the following microbial strains:

[0008] Alcaligenes aquatilis YH-2C1, Sphingopyxis sp. CY-10, Acinetobacter calcoaceticus TNJ-1, Acinetobacter sp. TNJ-1, and Rhodococcus ruber C1.

[0009] Furthermore, the Alcaligenes aquatilis YH-2C1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 2022972;

[0010] The sphingopyxis sp. CY-10 strain is deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 2021628.

[0011] The calcium acetate-Acinetobacter calcoaceticus TNJ-1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 2019726;

[0012] The Acinetobacter sp. TNJ-1 is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC 10695;

[0013] The Rhodococcus ruber C1 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2019725.

[0014] Furthermore, the volume ratio of the aquatic alkaloid-producing bacteria (Alcaligenes aquatilis YH-2C1), sphingopyxis sp. CY-10, calcium acetate-acinthiazide-acinthiazide (Acinetobacter calcoaceticus TNJ-1), Acinetobacter sp. TNJ-1, and Rhodococcus ruber C1 is as follows:

[0015] 30%–50%: 30%–50%: 5%–20%: 5%–20%: 5%–20%.

[0016] Furthermore, the volume ratio of the aquatic alkaloid-producing bacteria (Alcaligenes aquatilis YH-2C1), sphingopyxis sp. CY-10, calcium acetate-acinthiazide-acinthiazide (Acinetobacter calcoaceticus TNJ-1), Acinetobacter sp. TNJ-1, and Rhodococcus ruber C1 is as follows:

[0017] 30%:30%:10%:10%:20%.

[0018] This invention provides a method for preparing a mixed microbial agent, comprising the following steps:

[0019] Alcaligenes aquatilis YH-2C1, Sphingopyxis sp. CY-10, Acinetobacter calcoaceticus TNJ-1, Acinetobacter sp. TNJ-1, and Rhodococcus ruber C1 were inoculated onto solid culture medium for activation.

[0020] Use an inoculation loop to take the activated colonies and inoculate them into liquid culture medium.

[0021] Incubate for 3–5 days at 25–30°C and 170 rpm in a constant temperature shaker. OD 600 When the value reaches 1.0 or higher, five types of microbial inoculum solutions are obtained;

[0022] The above bacterial solutions were mixed in a volume ratio of 30%–50%: 30%–50%: 5%–20%: 5%–20% to obtain this mixed bacterial agent.

[0023] Furthermore, the solid culture medium comprises: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 15 g / L agar powder, and 1000 mL purified water; pH is 7.0, and it is autoclaved at 121°C for 30 min.

[0024] Furthermore, the liquid culture medium comprises: 1.89 g / L (NH4) 2 SO4, 11.7 g / L 4H4Na2O4·6H2O, 50 ml / L trace element solution; pH 7.0–7.5, autoclaved at 121℃ for 30 min;

[0025] The trace elements include: K2HPO4 5.0 g / L, NaCl 2.5 g / L, FeSO4·7H2O 0.05 g / L, MnSO4·4H2O 0.05 g / L, MgSO4·7H2O 2.5 g / L, and 1000 mL of purified water; pH is 7.0–7.2.

[0026] This invention provides the mixed microbial agent for any of the following applications:

[0027] (1) Used in the preparation of ammonia nitrogen removal agents, and / or total nitrogen removal agents;

[0028] (2) Used for denitrification in cyanide-containing wastewater.

[0029] Furthermore, the cyanide concentration in the cyanide-containing wastewater is 0–40 mg / L.

[0030] The present invention has at least the following beneficial effects:

[0031] This invention discloses a mixed microbial agent that can still grow rapidly and maintain high activity for a long time in highly toxic wastewater containing cyanide, achieving efficient denitrification under conditions containing cyanide and high ammonia nitrogen, and realizing the simultaneous removal of ammonia nitrogen and total nitrogen. The microbial agent for denitrification of cyanide-containing wastewater provided by this invention is easy to use and requires a small dosage. It can be directly added to existing activated sludge or biofilm processes. The microorganisms in the agent will establish a dominant bacterial community in the activated sludge, enhancing the treatment effect. It has the advantages of high treatment efficiency, low application cost, and convenient application. At the same time, it can solve the problems of poor effect, low treatment efficiency, and substandard effluent in traditional microbial treatment of cyanide-containing wastewater. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 Verification diagram of the cyanide-resistant denitrification effect of the bacterial agent;

[0034] Figure 2 Diagram showing the tolerance of the microbial agent to cyanide concentration;

[0035] Figure 3 Diagram showing the denitrification effect of microbial agents on coking wastewater;

[0036] Figure 4 A diagram showing the denitrification effect of microbial agents on shale gas extraction wastewater. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] The culture medium used in this invention is as follows:

[0039] Solid culture medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 15 g / L agar powder, 1000 mL purified water, pH 7.0, autoclaved at 121℃ for 30 min.

[0040] Liquid culture medium composition: 1.89 g / L (NH4) 2 SO4, 11.7 g / L C4H4Na2O4·6H2O, 50 ml / L trace element solution, pH 7.0–7.5, autoclaved at 121°C for 30 min.

[0041] Trace element composition: K2HPO4 5.0 g / L, NaCl 2.5 g / L, FeSO4·7H2O 0.05 g / L, MnSO4·4H2O 0.05 g / L, MgSO4·7H2O 2.5 g / L, 1000 mL of purified water, pH 7.0~7.2.

[0042] Composition of cyanide-containing culture medium: KCN solution is added to liquid culture medium.

[0043] The remaining reagents were commercially available analytical grade products.

[0044] The detection method used in this embodiment of the invention is as follows: OD of bacterial culture 600 The values ​​were detected using a UV2000 spectrophotometer at a wavelength of 600 nm. The monitoring and analysis methods for each pollutant were based on "Methods for Monitoring and Analysis of Water and Wastewater" (4th Edition, China Environmental Science Press, 2002).

[0045] The Alcaligenes aquatilis YH-2C1, Sphingopyxis sp. CY-10, Acinetobacter calcoaceticus TNJ-1, Acinetobacter sp. TNJ-1, and Rhodococcus ruber C1 mentioned in the following examples are all existing technologies, meaning that these five bacterial species have been disclosed in other literature. Specifically:

[0046] Alcaligenes aquatilis YH-2C1 is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M 2022972; (for prior art, see patent number: CN202211582688.X):

[0047] Sphingopyxis sp. CY-10 is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M 2021628; (for prior art, see patent number: CN202110622139.X)

[0048] Acinetobacter calcoaceticus TNJ-1 is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M 2019726; (Prior art, see patent number: CN201911106185.3)

[0049] Acinetobacter sp. TNJ-1 was purchased from the China Industrial Microbial Culture Collection Center, accession number CICC 10695. (Prior technology, see patent number: CN201510392432.6)

[0050] Rhodococcus ruber C1 is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: M 2019725; (for prior art, see patent number: CN202010965501.9)

[0051] Example 1: Preparation of bacterial agent for denitrification of cyanide-containing wastewater

[0052] Alcaligenes aquatilis YH-2C1, Sphingopyxis sp. CY-10, Acinetobacter calcoaceticus TNJ-1, Acinetobacter sp., and Rhodococcus ruber C1 were taken from the bacterial strain bank and inoculated into solid culture media, then placed in a constant temperature incubator and cultured at 30-32℃ for 3-5 days to obtain colonies of different bacterial species.

[0053] Then, using an inoculation loop, pick the colonies into liquid culture medium and incubate them for 3-5 days at 25-30°C and 170 rpm in a constant temperature shaker. OD 600 When the value reaches 1.0 or higher, five types of microbial inoculum solutions are obtained;

[0054] A bacterial agent for denitrification of cyanide-containing wastewater was prepared by mixing the bacterial solution in the following proportions: Aquatic alkalobacterium YH-2C1: Sphingosine Synthromyces CY-10: Calcium acetate-Acinetobacter TNJ-1: Acinetobacter: Rhodococcus deepis C1 = 30%: 30%: 10%: 10%: 20%.

[0055] Using a micropipette, inoculate 5 ml of the bacterial agent described in this invention into an Erlenmeyer flask containing 100 ml of cyanide-containing culture medium (set to CN). - (Concentration of 10 mg / L) The conical flask was placed in a constant temperature shaker at 30℃ and 170 rpm, and samples were taken every 24 hours to continuously monitor the removal efficiency of ammonia nitrogen and total nitrogen in the culture medium. Figure 1 As shown, in CN - Under the conditions of a nitrogen concentration of 10 mg / L, an initial ammonia nitrogen concentration of 393.0 mg / L, and an initial total nitrogen concentration of 402.0 mg / L, the ammonia nitrogen removal rate reached 97.8%, and the total nitrogen removal rate reached 88.2%, indicating a good denitrification effect.

[0056] Example 2: Experiment on the tolerance of bacterial agent to cyanide

[0057] Prepare 9 portions of cyanide-containing culture medium, each with CN... - The concentrations were 0 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, and 40 mg / L; the initial ammonia nitrogen concentration was 401.3 mg / L, and the initial total nitrogen concentration was 411.1 mg / L. 100 ml of cyanide-containing culture medium was added to each 250 ml Erlenmeyer flask. 5 mL of the bacterial agent from this invention was added to the flask using a micropipette. The Erlenmeyer flask was then placed on a shaker set to 30°C and 170 rpm. -1Under conditioned conditions, the removal of ammonia nitrogen and total nitrogen was measured every 24 hours, and data was collected at 120 hours. Figure 2 As shown, CN - As the concentration gradually increased from 0 mg / L to 40 mg / L, the ammonia nitrogen removal rate was 49.8%–99.3%, and the total nitrogen removal rate was 18.6%–91.6%. Under conditions of high concentrations of ammonia nitrogen, total nitrogen, and cyanide, the compound bacteria could still grow and play a denitrification role.

[0058] Example 3: Comparative experiment on denitrification of bacterial agents and single strains

[0059] Alkali-producing bacilli YH-2C1, sphingosine synergists CY-10, calcium acetate-Acinetobacter TNJ-1, Acinetobacter, and Rhodococcus rubrum C1 were cultured in liquid medium to produce OD 600 Achieve a pH of 1.0–1.5; prepare 6 portions of cyanide-containing culture medium, CN - The concentration was 10 mg / L for all strains. Six conical flasks were filled with 100 ml of cyanide-containing medium, and 5 ml of the above five bacterial strains and the bacterial agent of this invention were inoculated into each flask. The removal of ammonia nitrogen and total nitrogen was measured every 24 hours. Table 1 compares the denitrification effects of single strains and the bacterial agent of this invention under conditions of high CN- concentration, ammonia nitrogen concentration of 402.3 mg / L, and total nitrogen concentration of 411.3 mg / L. After 144 hours, the single strain had the highest ammonia nitrogen removal rate of 65.3% and the highest total nitrogen removal rate of 52.6%. The bacterial agent of this invention had the highest ammonia nitrogen removal rate of 98.6% and the highest total nitrogen removal rate of 89.9%, which is significantly better than that of the single strain.

[0060] Table 1

[0061]

[0062] Example 4: Verification Experiment of the Denitrification Capacity of the Bacterial Agent

[0063] Prepare 10 portions of cyanide-containing culture medium, CN - The concentration was set to 10 mg / L, and the initial ammonia nitrogen concentrations were set to 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L, 1000 mg / L, 1200 mg / L, 1400 mg / L, 1600 mg / L, 1800 mg / L, and 2000 mg / L, respectively. 100 ml of cyanide-containing culture medium was added to each 250 ml Erlenmeyer flask. 5 mL of the bacterial agent from this invention was added to the flask using a micropipette. The Erlenmeyer flask was then placed on a shaker set to 30°C and 170 rpm. -1The bacteria were cultured under specific conditions, and the removal of ammonia nitrogen and total nitrogen by the bacterial culture was measured every 24 hours, with data collected at 120 hours. Table 2 shows that the bacterial agent of this invention was effective in removing high concentrations of nitrogen, finding that when the ammonia nitrogen concentration was below 1000 mg / L, the OD... 600 The value can reach above 1.77, the ammonia nitrogen removal rate is above 93.3%, and the total nitrogen removal rate is above 81.3%; it was found that at an ammonia nitrogen concentration of 2000 mg / L, the OD... 600 The value can still reach 1.53, the ammonia nitrogen removal rate is 49.2%, and the total nitrogen removal rate is 33.3%. This proves that the bacterial agent of this invention can achieve high concentrations of CN... - It can still achieve rapid growth and exert a good denitrification effect even under high concentrations of ammonia nitrogen.

[0064] Table 2

[0065]

[0066] Example 5: Experiment on the treatment of cyanide-containing wastewater with bacterial agents

[0067] A sample of coking wastewater from a chemical plant in Chongqing was taken. The ammonia nitrogen concentration in the wastewater was 710 mg / L, the total nitrogen concentration was 770 mg / L, and the CN concentration was... - The concentration was 20 mg / L, and the COD concentration was 12200 mg / L. 200 ml of coking wastewater was placed in a 500 ml Erlenmeyer flask, and 10 ml of the bacterial agent from this invention was added. The Erlenmeyer flask was then placed on a shaker set to 30°C and 170 rpm. -1 The bacteria were cultured under specific conditions, and then the removal of ammonia nitrogen and total nitrogen by the bacterial culture was measured every 24 hours. Figure 3 As shown in CN - In real wastewater containing high levels of ammonia nitrogen and total nitrogen, the ammonia nitrogen removal rate reached 97.2%, and the total nitrogen removal rate reached 87.7%, which is significantly better than the removal effect of laboratory-prepared culture media.

[0068] Example 6: Experiment on the treatment of shale gas extraction wastewater with microbial agents

[0069] Wastewater from a shale gas extraction site in Chongqing was collected. The wastewater had an ammonia nitrogen concentration of 66.6 mg / L, a COD concentration of 3660 mg / L, and a chloride ion concentration of 16400 mg / L. The wastewater also contained cyanide, mercury compounds, and total metals, all of which are biotoxic. 200 ml of coking wastewater was placed in a 500 ml Erlenmeyer flask, and 10 ml of the bacterial agent described in this invention was added. The Erlenmeyer flask was then placed on a shaker set at 30°C and 170 rpm. -1 The bacteria were cultured under specific conditions, and then the removal of ammonia nitrogen and COD by the bacterial culture was measured every 24 hours. Figure 4The bacterial agent shown achieved an ammonia nitrogen removal rate of 96.2% and a COD removal rate of 49.5% in the treatment of shale gas extraction wastewater, indicating that the bacterial agent still has significant application value in the treatment of wastewater containing high salt and complex toxic substances.

[0070] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A mixed microbial agent, characterized in that, The mixed microbial agent is composed of the following microbial strains: Alcaligenes aquatilis YH-2C1, Sphingopyxis sp. CY-10, Acinetobacter calcoaceticus TNJ-1, Acinetobacter sp. TNJ-1, and Rhodococcus ruber C1.

2. The mixed microbial agent according to claim 1, characterized in that, The Alcaligenes saquatilis YH-2C1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2022972. The sphingopyxis sp. CY-10 strain is deposited at the China Center for Type Culture Collection, accession number CCTCC NO:M 2021628. The calcium acetate-Acinetobacter calcoaceticus TNJ-1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 2019726; The Acinetobacter sp. TNJ-1 is deposited at the China Industrial Microbial Culture Collection Center, with accession number CICC 10695; The Rhodococcus ruber C1 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2019725.

3. The mixed microbial agent according to claim 1, characterized in that, The volume ratio of the following bacteria was: *Alcaligenes saquatilis* YH-2C1, *Sphingopyxis* sp. CY-10, *Acinetobacter calcoaceticus* TNJ-1, *Acinetobacter sp. TNJ-1*, and *Rhodococcus ruber* C1. 30％～50％:30％～50％:5％～20％:5％～20％:5％～20％。 4. The mixed microbial agent according to claim 3, characterized in that, The volume ratio of the following bacteria was: *Alcaligenes saquatilis* YH-2C1, *Sphingopyxis* sp. CY-10, *Acinetobacter calcoaceticus* TNJ-1, *Acinetobacter sp. TNJ-1*, and *Rhodococcus ruber* C1. 30％:30％:10％:10％:20％。 5. A method for preparing a mixed microbial agent, characterized in that, Includes the following steps: Alcaligenes aquatilis YH-2C1, Sphingopyxis sp. CY-10, Acinetobacter calcoaceticus TNJ-1, Acinetobacter sp. TNJ-1, and Rhodococcus ruber C1 were inoculated onto solid culture medium for activation. Use an inoculation loop to take the activated colonies and inoculate them into liquid culture medium. Incubate for 3–5 days at 25–30°C and 170 rpm in a constant temperature shaker. OD 600 When the value reaches 1.0 or higher, five types of microbial inoculum solutions are obtained; The above bacterial solutions were mixed in a volume ratio of 30%–50%: 30%–50%: 5%–20%: 5%–20% to obtain this mixed bacterial agent.

6. The preparation method according to claim 5, characterized in that, The solid culture medium comprises: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 15 g / L agar powder, and 1000 mL purified water; pH is 7.0, and it is autoclaved at 121°C for 30 min.

7. The mixed bacterial agent for denitrification of cyanide-containing wastewater according to claim 5, characterized in that, The liquid culture medium contains: 1.89 g / L (NH4). 2 SO4, 11.7 g / L 4H4Na2O4·6H2O, 50 ml / L trace element solution; pH 7.0–7.5, autoclaved at 121℃ for 30 min; The trace elements include: K2HPO4 5.0 g / L, NaCl 2.5 g / L, FeSO4·7H2O 0.05 g / L, MnSO4·4H2O 0.05 g / L, MgSO4·7H2O 2.5 g / L, and 1000 mL of purified water; pH is 7.0–7.

2.

8. The mixed microbial agent according to any one of claims 1-4, in any of the following applications: (1) Used in the preparation of ammonia nitrogen removal agents, and / or total nitrogen removal agents; (2) Used for denitrification in cyanide-containing wastewater.

9. The application according to claim 8, characterized in that, The cyanide concentration in the cyanide-containing wastewater is 0–40 mg / L.

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