Preparation method of high-salt-resistant wastewater COD (Chemical Oxygen Demand) degradation complex microbial inoculant

Abstract

The invention discloses a preparation method of a high-salt-resistant wastewater COD (Chemical Oxygen Demand) degradation complex microbial inoculant. According to the invention, a microbial agent is customized according to the characteristics of the wastewater to realize the targeted treatment of target pollutants, and the provided high-salt-resistant wastewater COD (Chemical Oxygen Demand) degradation composite microbial agent is composed of Acinetobacter sp., Comamonas sp. And Bacillus cereus. The research shows that the complex microbial inoculant obtained by mixing the strains Acinetobacter sp., Comamonas sp. And Bacillus cereus with the concentration of 1-10 * 10 < 9 > CFU / mL according to the volume ratio of (0.5-2): (0.5-2): (0.5-2) can achieve the effects that the sewage treatment load is increased by 30% or above, the consumption of an external carbon source is reduced by 20%, the tolerable salinity reaches 5% or above, and the structure does not need to be changed in a biological treatment unit of high-salinity and high-COD wastewater.

Description

Technical Field

[0001] The invention belongs to the technical field of biological treatment of high-salt industrial wastewater, and specifically relates to three strains of high-salt-tolerant and efficient COD-degrading bacteria and a composite bacterial agent thereof, a preparation method and application thereof. Background Art

[0003] High salinity and toxic organic matter significantly inhibit biochemical treatment units. Microorganisms cannot tolerate high salinity, resulting in system failure. The intuitive manifestation of high salinity inhibiting the wastewater biochemical treatment system is that the water treatment process unit shows problems such as a significant decrease in COD removal rate, sludge floating, microbial dehydration and even death. On the one hand, the osmotic pressure imbalance under salt stress leads to microbial community imbalance. Excessive external osmotic pressure leads to cell dehydration, cell wall separation and microbial inactivation. On the other hand, salinity directly affects the diversity and community composition of microbial communities. Under high salinity, the stability of the bacterial network is destroyed and the community interaction is weakened. Salt stress intensifies the competition among microorganisms in the water treatment system.

[0004] The use of biological agents to bio-enhance water treatment units is a low-cost, green and efficient biological treatment method. However, in current applications, the treatment of sewage with biological agents has problems such as long treatment cycle and low treatment efficiency. The main reason is that the degradation efficiency of the selected microbial strains is not long enough or they cannot obtain stable colonization in the natural community after addition. Therefore, the present invention customizes the bacterial agent according to the characteristics of the wastewater to achieve targeted treatment of the target pollutants, which can improve the stability of the microbial community under salt stress and achieve the synergistic transformation of multiple pollutants in high-salt organic wastewater. Summary of the invention

[0005] In view of the shortcomings of the prior art in microbial treatment of high-salinity organic wastewater, the present invention provides three high-salinity tolerant COD degradation bacteria and a composite bacterial agent containing the three bacteria. The strains and composite bacterial agent of the present invention can significantly improve the COD degradation efficiency of high-salinity organic wastewater.

[0006] The first object of the present invention is to provide three high-salt tolerant COD degrading bacteria, which are composed of Acinetobacter sp., Comamonas sp., and Bacillus cereus. Acinetobacter sp. is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 10462; Comamonas sp. is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 20580; and Bacillus cereus is deposited in the China Industrial Microbiological Culture Collection Center with a deposit number of CICC 24945.

[0007] The second object of the present invention is to provide the use of strains Acinetobacter sp., Comamonas sp., and Bacillus cereus in preparing a composite bacterial agent for COD degradation in high-salinity wastewater.

[0008] The third object of the present invention is to provide a composite bacterial agent, which comprises strains of Acinetobacter sp., Comamonas sp., and Bacillus cereus as active ingredients.

[0009] Preferably, the composite bacterial agent is prepared by mixing bacterial solution of Acinetobacter sp., bacterial solution of Comamonas sp. and bacterial solution of Bacillus cereus in a volume ratio of 0.5-2:0.5-2:0.5-2, and the concentration of the bacterial solution is 1-10×10 9 CFU / mL.

[0010] The fourth object of the present invention is to provide a method for preparing a composite bacterial agent, comprising the following steps:

[0011] (1) Primary seed culture: Acinetobacter sp., Comamonas sp., and Bacillus cereus were inoculated into enrichment medium under sterile conditions, and cultured at 30-35° C. and 150-300 rpm for 12-36 h to obtain primary seed solution;

[0012] (2) under sterile conditions, the primary seed culture solution of Acinetobacter sp., Comamonas sp., and Bacillus cereus was inoculated into the enrichment medium at an inoculum amount of 1-5 vol%, and cultured at 30-35° C. and 150-300 rpm for 12-36 h to obtain the secondary seed culture solution;

[0013] (3) Fermentation: After the fermentation medium in the fermentation tank is disinfected, the secondary seed culture solution of Acinetobacter sp., Comamonas sp., and Bacillus cereus obtained in step (2) is inoculated into the fermentation medium at an inoculum amount of 5-10 vol%, respectively, and the fermentation is carried out under the conditions of 0.02-0.03 MPa, the temperature is controlled at 30-35° C., the pH is controlled at 6.8-7.2, the dissolved oxygen is greater than 6 mg / L, and the fermentation is carried out at 150-300 rpm. When the dissolved oxygen begins to rise, the fermentation is stopped to obtain the fermentation solution of each strain;

[0014] (4) Preparation of liquid composite bacterial agent: dilute the fermentation broth of each strain obtained in step (3), mix and bottle according to proportion, and obtain liquid composite bacterial agent.

[0015] Alternatively, a solid composite bacterial agent is prepared: the fermentation broth of each strain obtained in step (3) is centrifuged and mixed with an equal amount of nutrient agent to form bacterial sludge, which is then dried in a vacuum air drying oven at 40 to 50° C., and then crushed to obtain bacterial powder, and the bacterial powders of the three strains are mixed in proportion to obtain a solid composite bacterial agent.

[0016] Preferably, the formula of the fermentation medium is: 10g tryptone, 5g yeast powder, 10g sodium chloride, 1L distilled water, pH 6.5-7.5. The mixed nutrient in the bacterial sludge preparation process includes corn syrup powder, soybean meal powder, broken yeast powder, beef extract, fish meal peptone, glucose, magnesium sulfate, dipotassium hydrogen phosphate, manganese sulfate, sodium chloride, and Tween 80.

[0017] Preferably, the wastewater is high-salinity organic wastewater, including but not limited to biopharmaceutical wastewater.

[0018] The sixth object of the present invention is to provide a method for degrading COD in high-salinity wastewater, comprising the step of mixing a composite bacterial agent with the wastewater.

[0019] Preferably, the method for degrading COD of high-salinity organic wastewater comprises the following specific steps: mixing the composite biological agent with the biopharmaceutical wastewater at a concentration of 500ppm to 5000ppm, and adjusting the mixing multiple as appropriate according to the treatment salinity and treatment effect.

[0020] Specifically, the cells were cultured in a shaker at 35°C with the rotation speed set at 210 rpm for 1 to 7 days;

[0021] When a sequencing batch bioreactor is used, the reactor is set with an aeration time of 6-10 hours, a standing time of 5-9 hours, anoxic stirring of 5-9 hours, and a drainage time of 1-3 hours; the composite bacterial agent prepared by the present invention is added during the stable operation of the reactor, the inoculation ratio of the composite bacterial agent is 0.05-0.5%, and the operation time is more than 7 days. Preferably, 0.05-0.5% of the composite bacterial agent is added every week, and the addition of the bacterial agent is stopped after continuous circulation for 28 days.

[0022] The present invention has the following beneficial effects:

[0023] 1. The microorganisms in the composite bacterial agent provided by the present invention can grow well in high-salt wastewater, occupy a favorable ecological niche, achieve efficient removal of COD, and shorten the high-salt wastewater treatment cycle.

[0024] 2. The biological strain selected in the present invention can stably increase in value and quickly form an efficient and stable degradation community after being put into the water body, thereby realizing the synergistic transformation of multiple pollutants in high-salt organic wastewater.

[0025] 3. The present invention realizes an effective combination of strains. The combined bacterial agent has higher ecological advantages and treatment effects than single bacteria. The bacterial communities coexist in a mutually beneficial manner and grow rapidly to become dominant bacterial communities.

[0026] 4. The composite bacterial agent provided by the present invention has a wide application range and is suitable for salinity exceeding 6%. The selected strains are obtained from the environment and will not cause harm to the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Liquid composite bacterial agent's COD degradation efficiency.

[0028] Figure 2 The COD degradation efficiency of solid composite bacterial agent.

[0029] Figure 3 Solid composite bacterial agent enhances COD degradation performance of SBR reactor.

[0030] Figure 4 COD degradation performance under different solid composite bacterial agent addition methods. Specific implementation methods

[0032] The following examples are provided to further illustrate the present invention, but not to limit the present invention. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0034] 1. The composite bacterial agent provided by the present invention is prepared by mixing bacterial solution of Acinetobacter sp., bacterial solution of Comamonas sp. and bacterial solution of Bacillus cereus in a volume ratio of 0.5-2:0.5-2:0.5-2, preferably 1:1:1, and the concentration of the bacterial solution is 1-10×10 9 CFU / mL.

[0035] 2. Under sterile conditions, Acinetobacter sp., Comamonas sp., and Bacillus cereus are inoculated into enrichment medium respectively, and cultured at 30-35° C. and 150-300 rpm for 12-36 hours to obtain a primary seed solution; then, under sterile conditions, the primary seed culture solutions of Acinetobacter sp., Comamonas sp., and Bacillus cereus are inoculated into enrichment medium at an inoculation rate of 1-5 vol%, and cultured at 30-35° C. and 150-300 rpm for 12-36 hours to obtain a secondary seed culture solution. After the fermentation medium in the fermentation tank is disinfected, the secondary seed culture solution of Acinetobacter sp., Comamonas sp., and Bacillus cereus is inoculated into the fermentation medium at an inoculation rate of 5-10 vol%, and the fermentation medium is fermented under the conditions of 0.02-0.03 MPa, a temperature of 30-35° C., a pH of 6.8-7.2, dissolved oxygen>6 mg / L, and 150-300 rpm. When the dissolved oxygen starts to rise, the fermentation is stopped to obtain the fermentation solution of each strain; 1-10×10 9 CFU / mL bacterial liquid, the three bacterial liquids were mixed in a volume ratio of 1:1:1 to obtain a liquid composite bacterial agent. The formula of the fermentation medium is: 10g of tryptone, 5g of yeast powder, 10g of sodium chloride, 1L of distilled water, pH 6.5-7.5. The components were mixed evenly and sterilized with wet heat at 121°C for 20 to 30 minutes.

[0036] 3. Further, the liquid composite bacterial agent and an equal amount of nutrient agent are mixed to form bacterial mud, which is then placed in a vacuum air drying oven at 40-50°C for drying, and then crushed to obtain bacterial powder, and the bacterial powders of the three strains are mixed in proportion to obtain a solid composite bacterial agent. The mixed nutrient agent in the bacterial mud preparation process includes corn syrup powder, soybean meal powder, broken yeast powder, beef extract, fish meal peptone, glucose, magnesium sulfate, dipotassium hydrogen phosphate, manganese sulfate, sodium chloride, and Tween 80.

[0037] The present invention will be further described below in conjunction with specific embodiments.

[0038] Example 1: Removal of COD from biopharmaceutical high-salinity wastewater by composite bacterial agent

[0039] Add 2.5 mL of liquid composite bacterial agent to a 250 mL triangular flask containing 100 mL of biopharmaceutical wastewater (salinity 3%), seal it with a bottle sealer, and mark it as No. 1; add 2.5 mL of Cinetobacter sp., Comamonas sp., and Bacillus cereus bacterial solution to three 250 mL triangular flasks containing 100 mL of biopharmaceutical wastewater, seal them with a bottle sealer, and mark them as control groups 1, 2, and 3; at the same time, set up a control group No. 0 without bacterial solution. Each group of experiments has three replicates, and is placed in a 35°C shaker for culture, with the speed set to 210 rpm. The COD changes at 0d, 1d, 3d, 5d, and 7d were measured, and the results are as follows: Figure 1 The results show that the removal efficiency of the combined liquid bacterial agent is significantly improved compared with that of single bacteria and sterile agents.

[0040] Add solid composite bacterial agent to a 250mL triangular flask containing 100mL of biopharmaceutical wastewater (salinity of 3%), with a mixing ratio of 0.5g / kg. Seal with bottle sealing film and mark it as No. 1; add Acinetobacter sp., Comamonas sp., Bacillus cereus solid bacterial powder to three 250mL triangular flasks containing 100mL of biopharmaceutical wastewater, seal with bottle sealing film, and mark them as control groups 1, 2, and 3; at the same time, set up a control group No. 0 without bacterial solution. Each group of experiments has three replicates and is cultured in a 35℃ shaker with a rotation speed set to 210rpm. The COD changes at 0d, 1d, 3d, 5d, and 7d are measured, and the results are as follows: Figure 2 The results show that the removal efficiency of the combined solid bacterial agent is significantly improved compared with that of single bacteria and sterile agents.

[0041] Example 2: Detection of COD degradation performance of composite bacterial agent-enhanced biochemical system

[0042] Three groups of sequencing batch bioreactors (SBR) with an effective volume of 2L were used. The reactors were set with an aeration time of 8h, a standing time of 7h, anoxic stirring of 7h, and a drainage time of 2h. At the beginning of the experiment, activated sludge suspension was inoculated into the reactor, and the initial MLSS was 2900mg / L. The reactor influent was leather wastewater with a COD of 5000mg / L and a salinity of 3%. The startup time was 20 days. After the startup period, solid composite bacterial agents were inoculated in reactors No. 2 and No. 3. The ratio of the three bacteria was preferably 1:1:1, and the inoculation ratio was 0.1% (2g). No solid composite bacterial agents were added to reactor No. 1. After 7 days, the removal efficiency of reactors No. 2 and No. 3 began to show a significant increase. Subsequently, 0.1% bacterial agent was added to reactor No. 3 every week, and the addition was stopped after four cycles. The COD removal effect during the full cycle operation is as follows Figure 3 shown.

[0043] After the start-up period, if Figure 4 As shown in the figure, the COD removal efficiency of the three groups of reactors is stable at about 17%. The COD removal efficiency of Reactor No. 2 is about 44% within one month after the one-time addition of solid bacterial agent, and then gradually drops to about 20% after the addition of bacterial agent is stopped; the removal efficiency of Reactor No. 3 gradually increases to 72% within one month after the step-by-step addition of bacterial agent, and after the addition of bacterial agent is stopped, the COD removal efficiency of Reactor No. 3 continues to be stable at about 73%, indicating that under the action of solid bacterial agent, the community structure of the reactor tends to be stable, achieving the purpose of long-term COD removal.

[0044] The above are only preferred implementation strategies of the present invention. It should be noted that the above preferred implementations should not be regarded as limiting the present invention. The scope of protection of the present invention shall be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a high-salinity wastewater COD degradation composite bacterial agent, characterized in that: Each gram or mL of the compound bacterial agent contains Acinetobacter sp. 1~10× 10 9 indivual, Comamonas sp. 1~10× 10 9 indivual, Bacillus cereus 1~10× 10 9 indivual; Among them, the Acinetobacter sp. Deposited in China Industrial Microbiological Culture Collection Center with the deposit number CICC 10462; Comamonas sp. Deposited in China Industrial Microbiological Culture Collection Center, with the deposit number CICC 20580; Bacillus cereus It is deposited in China Industrial Microbiological Culture Collection Center with the deposit number CICC 24945.

2. The method for preparing the composite bacterial agent according to claim 1, characterized in that: The following steps are involved: (1) Primary seed culture: Take the seeds separately under sterile conditions. Acinetobacter sp. , Comamonas sp. , Bacillus cereus Inoculate in enriched medium, culture at 30-35°C and 150-300 rpm for 12-36 hours to obtain primary seed solution; (2) Under sterile conditions, Acinetobacter sp. , Comamonas sp. , Bacillus cereus The first-level seed culture solution is inoculated into the enrichment medium at an inoculation rate of 1-5 vol%, and cultured at 30-35° C. and 150-300 rpm for 12-36 hours to obtain the second-level seed culture solution; (3) Fermentation: After the fermentation medium in the fermentation tank is sterilized, the fermentation medium obtained in step (2) is Acinetobacter sp. , Comamonas sp. , Bacillus cereus The secondary seed culture solution is inoculated into the fermentation medium at an inoculation rate of 5-10 vol%, the pressure is 0.02-0.03 MPa, the temperature is controlled at 30-35°C, the pH is controlled at 6.8-7.2, the dissolved oxygen is greater than 6 mg / L, and the fermentation is carried out at 150-300 rpm. When the dissolved oxygen begins to rise, the fermentation is stopped to obtain the fermentation solution of each strain; (4) preparing a liquid composite bacterial agent: diluting the fermentation broth of each strain obtained in step (3), mixing and filling according to a certain proportion, and obtaining a liquid composite bacterial agent; Alternatively, the fermentation broth of each strain obtained in step (3) is centrifuged and mixed with an equal amount of nutrient agent to form bacterial sludge to prepare a solid composite bacterial agent.

3. The method for preparing the composite bacterial agent according to claim 2, characterized in that: Further comprising the steps of: The solid composite bacterial agent is obtained in the following manner: the fermentation broth of each strain obtained in step (3) is centrifuged and mixed with an equal amount of nutrient agent to form bacterial sludge, the bacterial sludge is then placed in a vacuum air drying oven at 40-50°C for drying, and then crushed to obtain bacterial powder, and the bacterial powders of the three strains are mixed in proportion to obtain a solid composite bacterial agent.

4. The preparation method according to claim 2, characterized in that: The mixed nutrient agent in the bacterial mud preparation process includes corn steep liquor dry powder, soybean meal powder, broken yeast powder, beef extract, fish meal peptone, glucose, magnesium sulfate, dipotassium hydrogen phosphate, manganese sulfate, sodium chloride and Tween 80.

5. The preparation method according to claim 2, characterized in that: The concentration of the fermentation medium is 10g of tryptone, 5g of yeast powder, 10g of sodium chloride, 1L of distilled water, and pH 6.5-7.

5.

6. A high-salinity tolerant wastewater COD degradation composite bacterial agent obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the composite bacterial agent as claimed in claim 6 in degrading COD in wastewater.

8. The use according to claim 7, characterized in that: The wastewater includes biopharmaceutical wastewater.

9. A method for degrading COD in wastewater, characterized in that: The method comprises the step of mixing the composite bacterial agent described in claim 6 with wastewater, specifically biopharmaceutical wastewater.

10. The method according to claim 9, characterized in that The mixing step is to mix the composite biological agent with the wastewater at a concentration of 500ppm to 5000ppm and shake the mixture; Specifically, the cells were cultured in a shaker at 35°C with the rotation speed set at 210 rpm for 1 to 7 days; When a sequencing batch bioreactor is used, the reactor is set with an aeration time of 6-10 hours, a standing time of 5-9 hours, anoxic stirring of 5-9 hours, and a drainage time of 1-3 hours; the composite bacterial agent prepared by the present invention is added during the stable operation of the reactor, the inoculation ratio of the composite bacterial agent is 0.1-5%, and the operation time is more than 7 days. Preferably, 0.1-5% of the composite bacterial agent is added every week, and the addition of the bacterial agent is stopped after continuous circulation for 28 days.

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