Compound microbial agent and application thereof

Abstract

The invention discloses a compound microbial agent and an application thereof, and the compound microbial agent is characterized in that the compound microbial agent is prepared by compounding Escherichia coli SQ-1A, Acinetobacter radiobacter radioreae SQ-1B and Klebsiella michelianensis WF-2 according to a volume ratio of 1: 1: 1, and the compound microbial agent is prepared by compounding the following raw materials in parts by volume: 1: 1, 1: 1, 1: 1, 1: 1: 1, 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1. The invention aims to prepare the compound microbial agent with high ammonia nitrogen degradation rate.

Description

Technical Field

[0001] The invention relates to the technical field of biological degradation of ammonia nitrogen in wastewater, and in particular to a composite microbial agent and application thereof. Background Art

[0002] At present, the concentration of ammonia nitrogen is a key factor in determining the treatment efficiency and effluent quality during wastewater treatment. Among them, excessive ammonia nitrogen concentration will lead to oxygen depletion and inhibition of nitrification reaction, and produce carcinogenic nitrite; while too low ammonia nitrogen concentration will increase nitrate production, limit denitrification, and affect the denitrification effect. Existing denitrification methods include physical denitrification, chemical denitrification and biological denitrification. Compared with physical denitrification and chemical denitrification, biological denitrification has certain advantages in practical applications, namely, it has lower economic cost, higher treatment efficiency, simple operation process and no secondary pollution to the environment. Therefore, biological denitrification has been widely recognized as the most potential and development prospects of denitrification technology. At the same time, in wastewater treatment, the efficiency of ammonia nitrogen degradation by relying on a single strain is limited by the specific metabolic characteristics of the strain and its sensitivity to environmental conditions. Environmental changes may lead to reduced efficiency, and the single strain system is not stable enough and is easily affected by toxic substances and fails.

[0003] The invention patent with the existing patent publication number CN115287209A discloses a composite microbial agent and its application in treating pig urine wastewater. The composite microbial agent used is composed of Bacillus pumilus LZP02 strain, Bacillus megaterium LZP03 strain, Bacillus subtilis SC strain and Bacillus velezensis WB strain. The composite rhizosphere growth-promoting bacterial community constructed by the four strains LZP03, LZP02, WB and SC is used as the fermentation strain to ferment pig wastewater. Although it can reduce the pollution sources threatening environmental safety in pig wastewater such as ammonia nitrogen, organic matter, phosphorus, etc., and realize the degradation of multiple pollution sources, the removal rate of ammonia nitrogen is 58.17%, which is relatively low, and then continue to obtain a new composite microorganism to improve the removal rate of ammonia nitrogen. Summary of the invention

[0004] The main purpose of the present invention is to provide a composite microbial agent and its application, aiming to solve the technical problem that the existing composite microbial agent has a low degradation rate of ammonia nitrogen.

[0005] To achieve the above object, the present invention provides a composite microbial agent, which is composed of Escherichia coli SQ-1A, Acinetobacter radioresistens SQ-1B and Klebsiella michiganensis WF-2. The Escherichia coli SQ-1A is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC NO.23384, the Acinetobacter radioresistens SQ-1B is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC NO.23385, and the Klebsiella michiganensis WF-2 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC NO.23386.

[0006] Optionally, the composite microbial agent is prepared by compounding activated bacterial liquids of Escherichia coli SQ-1A, Acinetobacter radioresistancetens SQ-1B and Klebsiella michiganensis WF-2 in a volume ratio of 1:1:1.

[0007] In addition, in order to achieve the above-mentioned purpose, the present invention also provides the use of the above-mentioned composite microbial agent in treating livestock and poultry breeding wastewater.

[0008] Optionally, the composite microbial agent is added into an SBR reactor to detect the ammonia nitrogen concentration in livestock and poultry breeding wastewater or ammonia nitrogen polluted wastewater.

[0009] In addition, in order to achieve the above purpose, the composite microbial agent is used in the treatment of simulated wastewater.

[0010] Optionally, treating the simulated wastewater comprises the following steps:

[0011] The composite microbial agent is introduced into simulated wastewater at an inoculation rate of 0.8% to 1.2%, and is shaken and cultured at 35°C and 180 r / min for 24 to 36 hours, and the number of bacteria and the ammonia nitrogen concentration in the simulated wastewater are detected every 12 hours, wherein the simulated wastewater contains sodium acetate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, sodium chloride, manganese sulfate, ferrous sulfate and ammonium chloride.

[0012] Optionally, the initial ammonia nitrogen concentration in the simulated wastewater is 100-300 mg / L.

[0013] Optionally, the carbon-nitrogen ratio in the simulated wastewater is (12-15):1, and the pH is 6.8-7.2.

[0014] Optionally, the inoculation amount of the composite microbial agent is 1.0%.

[0015] Optionally, the carbon-nitrogen ratio in the simulated wastewater is 15 and the pH value is 7.

[0016] Beneficial effects:

[0017] The present invention combines the three existing strains of bacteria in pairs, and the ammonia nitrogen degradation rate is lower than that of a single strain combined with the three strains, and the ability of the composite microbial agent obtained by combining the three strains to degrade ammonia nitrogen is basically higher than that of a single bacteria, and the composite microbial agent is mixed in the optimal ratio to form a composite microbial agent named SSF-1. The removal rate of ammonia nitrogen in simulated wastewater by SSF-1 is as high as 98.41%, reflecting a good degradation effect. In addition, SSF-1 also shows excellent performance in actual pig farm wastewater treatment, with a maximum degradation rate of 83.12%. The principle is that the introduction of SSF-1 increases the proportion of Proteobacteria Proteobacter ia, thereby enhancing the richness and diversity of the bacterial community, thereby improving the biological denitrification capacity of activated sludge. Therefore, the SSF-1 composite bacteria obtained in the present invention can be used to remove or reduce the residual ammonia nitrogen in the environment, providing a safe and environmentally friendly microbial degradation method for the problem of residual ammonia nitrogen. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a growth curve of a composite microbial agent SSF-1 of the present invention;

[0019] Figure 2 for Figure 1 The ammonia nitrogen degradation ability of the composite microbial agent SSF-1 is shown in;

[0020] Figure 3 for Figure 1 The degradation rate of the composite microbial agent SSF-1 at different ammonia nitrogen concentrations shown in;

[0021] Figure 4 for Figure 1 The line graph of the dynamic changes of ammonia nitrogen content in the reactor during the treatment of pig farm wastewater by the composite microbial agent SSF-1 shown in FIG.

[0022] Figure 5 for Figure 1 The line graph shown in the figure shows the dynamic changes in ammonia nitrogen concentration in the reactor during the treatment of pig farm wastewater by the composite microbial agent SSF-1.

[0023] Figure 6 for Figure 1 Bar chart of relative abundance of bacterial community composition at phylum classification level in the reactor during the treatment of swine farm wastewater by the composite microbial agent SSF-1 shown in the figure. DETAILED DESCRIPTION

[0024] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0025] The invention provides a composite microbial agent. The composite microbial agent consists of Escherichia coli SQ-1A, Acinetobacter radioresistens SQ-1B and Klebsiella michiganensis WF-2. The Escherichia coli SQ-1A is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC NO.23384, the Acinetobacter radioresistens SQ-1B is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC NO.23385, and the Klebsiella michiganensis WF-2 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC NO.23386.

[0026] Specifically, three existing strains such as Escherichia coli SQ-1A, Acinetobacter radioresistance SQ-1B and Klebsiella michiganensis WF-2 are compounded in a volume ratio of 1:1:1 to prepare the target composite microbial agent.

[0027] Furthermore, the compounding ratio of the three different strains was determined by mixing them in different ratios to form a composite microbial agent, and then the composite microbial agents of different ratios were inoculated at 1% into simulated wastewater with a carbon-nitrogen ratio of 15, a pH value of 7.0, a carbon source of sodium acetate, and an initial ammonia nitrogen concentration of 100 mg / L. The simulated wastewater without inoculation was used as the experimental control group to compare the ammonia nitrogen degradation rate. The results are shown in Table 1. And the calculation method of ammonia nitrogen degradation rate:

[0028] Remova l efficiency(%)=(C 0 -C t ) / C0 *100%, C 0 and C t (mg / L) are the initial concentration of ammonia nitrogen and the concentration at time t, respectively.

[0029] Table 1 Strain compound ratio

[0030] Strain type SQ-1A SQ-1B WF-2 Ammonia nitrogen degradation rate (%) SSF-1 1 1 1 96.1±0.31a SSF-2 1 2 1 90.4±0.62c SSF-3 2 1 1 92.8±0.3b SSF-4 1 1 2 94.38±2.24b SSF-5 1 1 0 51.47e SSF-6 1 0 1 51.75e SSF-7 0 1 1 51.42±0.29e SQ-1A 1 0 0 87.33±0.48d SQ-1B 0 1 0 89.83±0.41c WF-2 0 0 1 90.47±0.49c

[0031] As shown in Table 1 above, the ammonia nitrogen degradation rate of the three strains combined in pairs is lower than that of the single strain combined with the three strains, and the ability of the composite microbial agent obtained by the combination of the three strains to degrade ammonia nitrogen is basically higher than the ammonia nitrogen degradation ability of a single bacteria. In particular, when the three strains are used to construct a composite microbial agent in a ratio of 1:1:1, the ammonia nitrogen degradation rate is the highest, reaching 96.1%. The composite bacteria constructed by this ratio combination is named SSF-1.

[0032] Furthermore, the growth curve and ammonia nitrogen degradation rate of the composite microbial agent obtained above in simulated wastewater were determined.

[0033] Specifically, the composite microbial agent SSF-1 was inoculated into the ammonia nitrogen simulated wastewater at a 1% inoculation rate, and the uninoculated simulated wastewater was used as the blank control group. The culture was shaken at 35°C and 180r / min. The number of bacteria and the ammonia nitrogen concentration in the simulated wastewater were detected every 12 hours, wherein the simulated wastewater included sodium acetate, magnesium sulfate heptahydrate, potassium hydrogen phosphate, sodium chloride, manganese sulfate, ferrous sulfate and ammonium chloride. Growth and ammonia nitrogen degradation ability Figure 1 and Figure 2 As shown. The results show that the composite microbial agent SSF-1 in the simulated wastewater grows rapidly from 0 to 24 hours and reaches its peak at 24 hours. The biomass in the simulated wastewater tends to be flat from 24 hours to 36 hours, indicating that the composite bacteria grows slowly at this time. After 36 hours, the growth of bacteria in the simulated wastewater gradually decreases, and the activity of the bacteria decreases at this time. The change trend of the ammonia nitrogen degradation ability of the composite microbial agent in the simulated wastewater is the same as the growth trend. At 24-36 hours, the ammonia nitrogen content in the simulated wastewater drops to a minimum of only 3.17 mg / L, indicating that the ammonia nitrogen degradation rate of the composite microbial agent SSF-1 is the highest during this period.

[0034] Furthermore, the effect of the initial ammonia nitrogen concentration on the ability of the composite microbial agent to degrade ammonia nitrogen is described below through specific examples.

[0035] Specifically, the composite microbial agent SSF-1 was inoculated at a 1% inoculum into simulated wastewater with different ammonia nitrogen concentrations (100 mg / L, 300 mg / L, 500 mg / L, 800 mg / L, 1000 mg / L), and the ammonia nitrogen concentration in the simulated wastewater was measured after shaking culture at 35°C and 180 r / min for 36 hours, and the degradation rate was calculated. Figure 3 As shown, each composite microbial agent SSF-1 has the ability to degrade ammonia nitrogen in the simulated wastewater within the range of ammonia nitrogen concentration of 100-1000 mg / L. However, with the increase of ammonia nitrogen concentration, the ability of SSF-1 to degrade ammonia nitrogen gradually weakened, and at 500-800 mg / L, there was no significant change in the degradation rate (p<0.05), and when the ammonia nitrogen concentration was 100 mg / L, SSF-1 had the strongest ability to degrade ammonia nitrogen.

[0036] Furthermore, in order to optimize the conditions for the degradation of ammonia nitrogen by composite microbial agents, a 3-factor 3-level L9 (3 4 ) orthogonal experiment, wherein the experimental table is shown in Table 2. The determination of the experimental table is based on the fact that the three strains have the best ammonia nitrogen degradation ability when inoculated with 1% inoculation amount in the simulated wastewater with a carbon-nitrogen ratio of 15 and a pH value of 7.

[0037] Furthermore, corresponding experiments were performed according to the experimental table in Table 2, and the orthogonal test results obtained are shown in Table 3.

[0038] Table 2 Factor and level settings

[0039]

[0040]

[0041] Table 3 SSF-1 orthogonal test results

[0042] Serial number C / N pH Inoculation amount (%) Ammonia nitrogen degradation rate (%) 1 1 1 1 95.44±0.13c 2 1 2 2 95.21±0.07c 3 1 3 3 94.12±0.13d 4 2 1 2 98.11±0.52a 5 2 2 3 96.91±0.45b 6 2 3 1 98.41±0.16a 7 3 1 3 97.03±0.38b 8 3 2 1 97.97±0.14a 9 3 3 2 98.12±0.03a K1 94.92 96.86 97.27 K2 97.81 96.70 97.15 K3 97.71 96.88 96.02 Delta 2.89 0.19 1.25 Rank 1 3 2

[0043] The results in Table 3 above show that when the composite microbial agent SSF-1 is cultured under different C / N, pH value and inoculum size conditions, its ammonia nitrogen degradation efficiency changes significantly (p<0.05). When the C / N in the simulated wastewater is 15:1, the pH value is 7.2, and the inoculum size is 0.8%, the ammonia nitrogen degradation rate of the composite microbial agent SSF-1 reaches the highest value, at which the ammonia nitrogen degradation rate is 98.41%. Among the three factors of C / N, pH value and inoculum size, C / N has the greatest influence on the ability of composite bacteria SSF-1 to degrade ammonia nitrogen, followed by the inoculum size, and the pH value has the least influence on the ability of composite bacteria SSF-1 to degrade ammonia nitrogen. The reason why pH value has little effect on the ammonia nitrogen degradation ability of composite bacteria SSF-1 may be that the composite microbial agent can adapt well under the condition of pH value of 6.8-7.2.

[0044] Furthermore, the results of variance analysis of orthogonal test data are shown in Table 4, which further confirms that the composite bacteria SSF-1 can adapt well under the condition of pH 6.8-7.2. Among the three factors of C / N, pH value and inoculation size, the effect of pH change on the results was not significantly different (p>0.05), while the changes in C / N and inoculation size had significant effects on the results (p<0.05). The optimization results of the degradation conditions of ammonia nitrogen by composite bacteria SSF-1 showed that the degradation efficiency of ammonia nitrogen in simulated wastewater by composite microbial agents was higher than that of single strains, and the ability of composite microbial agents to adapt to the environment was also enhanced compared with single strains.

[0045] Table 4 Analysis of variance of orthogonal test

[0046] source Degrees of Freedom Sequential sum of squares Adjusted mean square F-number P-value C / N 2 16.09 8.06 470.48 0.002 pH 2 0.06 0.03 1.81 0.355 Inoculation amount (%) 2 2.87 1.43 83.52 0.012 Residual error 2 0.03 0.02 sum 8 19.04

[0047] Furthermore, the ammonia nitrogen degradation characteristics of the composite microbial agent in actual wastewater are explained below through specific experiments.

[0048] The composite microbial agent SSF-1 was added into the SBR reactor to detect the ammonia nitrogen concentration in the wastewater and calculate the ammonia nitrogen degradation rate. ▉ is the uninoculated SBR reactor as the experimental control group, and ▲ is the composite microbial agent SSF-1 experimental group. The results are as follows: Figure 4 and Figure 5 As shown, in the SBR reactor inoculated with activated sludge only, the ammonia nitrogen degradation efficiency of the system is stable at about 25%, while in the SBR reactor inoculated with composite microbial agents and activated sludge, the ammonia nitrogen degradation efficiency of the system can reach up to 83.12%.

[0049] Furthermore, the microbial community of the activated sludge in the reactor was analyzed, and it was found that the coverage of all collected samples was very high (>0.999), indicating that the samples in this study effectively represented the diversity of the microbial community.

[0050] Figure 6 The dominant bacterial phyla at different stages with and without the addition of SSF-1 are shown. In the reactor with the addition of SSF-1, Proteobacter ia was dominant, accounting for 68.852%-88.174%, and the ammonia nitrogen content in the corresponding device continued to decline, reaching the maximum degradation rate at 96h. Based on Proteobacter ia as an important bacterial phylum, it occupies an important position in the microbial community due to its environmental adaptability and growth rate. Studies have shown that some bacteria in the Proteobacteria phylum can effectively remove nitrogen compounds from water, which is essential for maintaining the ecological balance of aquatic systems and preventing eutrophication. Overall, the introduction of SSF-1 increased the richness and diversity of the bacterial community, thereby improving the biological denitrification capacity of activated sludge. It shows that the SSF-1 composite microbial agent has good compatibility with the ecosystem in the SBR and plays an important role in the actual sewage treatment process.

[0051] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or system including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or system. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or system including the element.

[0052] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0053] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A composite microbial agent, characterized in that: The composite microbial agent consists of Escherichia coli SQ-1A, Acinetobacter radioresistens SQ-1B and Klebsiella michiganensis WF-2. The Escherichia coli SQ-1A is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC NO.23384, the Acinetobacter radioresistens SQ-1B is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC NO.23385, and the Klebsiella michiganensis WF-2 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC NO.23386.

2. The composite microbial agent according to claim 1, characterized in that The composite microbial agent is prepared by compounding activated bacterial liquids of Escherichia coli SQ-1A, Acinetobacter radioresistens SQ-1B and Klebsiella michiganensis WF-2 in a volume ratio of 1:1:

1.

3. Use of the composite microbial agent described in claim 1 or 2 in treating livestock and poultry breeding wastewater or ammonia nitrogen polluted wastewater.

4. The use according to claim 3, characterized in that The composite microbial agent is added into the SBR reactor to detect the ammonia nitrogen concentration in the livestock and poultry breeding wastewater.

5. Use of the composite microbial agent according to claim 1 or 2 in treating simulated wastewater.

6. The use according to claim 5, characterized in that Treatment of simulated wastewater includes the following steps: The composite microbial agent is introduced into simulated wastewater at an inoculation rate of 0.8% to 1.2%, and is shaken and cultured at 35°C and 180 r / min for 24 to 36 hours, and the number of bacteria and the ammonia nitrogen concentration in the simulated wastewater are detected every 12 hours, wherein the simulated wastewater contains sodium acetate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, sodium chloride, manganese sulfate, ferrous sulfate and ammonium chloride.

7. The use according to claim 5, characterized in that The initial ammonia nitrogen concentration in the simulated wastewater is 100-300 mg / L.

8. The use according to claim 7, characterized in that The carbon-nitrogen ratio in the simulated wastewater is (12-15):1, and the pH is 6.8-7.

2.

9. The use according to claim 6, characterized in that: The inoculation amount of the composite microbial agent is 1.0%.

10. The use according to claim 8, characterized in that The carbon-nitrogen ratio in the simulated wastewater is 15:1 and the pH value is 7.

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