Ammonia nitrogen degrading bacteria and its application in leather wastewater treatment

Abstract

The invention relates to the technical field of microbial wastewater treatment, and specifically to an ammonia nitrogen degrading bacterium and an application thereof in leather wastewater treatment. The invention screens and cultivates bacterial species, and finally cultivates a strain HZ-N-001 with good ammonia nitrogen degradation performance, which can realize denitrification treatment of a water body in a heterotrophic aerobic environment, and simultaneously remove organic matter in the water body, thereby greatly simplifying the existing water body biological denitrification process steps and reducing costs. In addition, the strain HZ-N-001 prepared by the invention can also effectively reduce sludge production, degrade difficult-to-biodegrade macromolecular substances into small molecular products, thereby improving the physical and chemical properties of wastewater and enhancing the biodegradability of sewage.

Description

Technical Field

[0001] The invention relates to the technical field of microbial wastewater treatment, in particular to an ammonia nitrogen degrading bacterium and application thereof in leather wastewater treatment. Background Art

[0002] With the development and expansion of the leather industry, the harm caused by industrial wastewater from the leather industry to the environment has become increasingly obvious. In particular, the leather industry wastewater has complex components, heavy pollution burden, large sludge output, and is not easy to be decomposed by existing microbial degradation technology. In addition, the ammonia nitrogen content of leather industry wastewater is high, which is easy to hinder the growth of microorganisms and seriously affects the efficiency of biodegradation treatment. Therefore, it is necessary to solve the problems that arise in the biodegradation of leather industry wastewater. Summary of the invention

[0003] The purpose of the present invention is to provide an ammonia nitrogen degrading bacteria and its application in leather wastewater treatment to solve the problems raised in the above background technology.

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions: an ammonia nitrogen degrading bacterium having the following characteristics: the ammonia nitrogen degrading bacterium is the HZ-N-001 strain, and the DNA sequence of the HZ-N-001 strain is shown in SEQ ID NO.1.

[0005] Further, the HZ-N-001 strain is Pseudomonas;

[0006] The preservation number of the HZ-N-001 strain is CGMCC NO.23461, the preservation date is September 22, 2021, and the preservation unit is the General Microbiology Center of China Culture Collection Administration, the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the classification name is Pseudomonas sp.

[0007] Furthermore, the screening method for ammonia nitrogen degrading bacteria comprises the following steps:

[0008] a. Collect the sludge, mix it evenly, separate the sludge by centrifugation, and inoculate the sludge into liquid heterotrophic nitrification medium, culture it in a shake flask at 25-30℃, and test the ammonia nitrogen content in the shake flask every 48h;

[0009] b. When the ammonia nitrogen content in the shake flask drops to 0-0.5 mg / L, add new liquid heterotrophic nitrification medium, continue to cultivate and enrich for 1-2 weeks, and obtain enriched bacterial liquid;

[0010] c. Dilute the enriched bacterial solution to 10 with liquid heterotrophic nitrification medium. -1 -10 -7There are 7 gradients in total. Take 100ul of each gradient and spread it on solid heterotrophic nitrification medium. Make 3-5 parallel groups for each gradient. After culturing for 48 hours at 25-30℃, pick different colonies grown on the plate for streak purification.

[0011] d. After streaking purification, the solid heterotrophic nitrification medium was tested. When the colony growth in the solid heterotrophic nitrification medium was uniform and free of foreign bacteria, the purified strain HZ-N-001 was obtained.

[0012] Furthermore, the liquid heterotrophic nitrification culture medium includes the following components: 0.45-0.5 g / L (NH4)2SO4, 5.6-5.65 g / L sodium acetate, 4.75-4.8 g / L K2HPO4·3H2O, 1.2-1.25 g / L KH2PO4, 8-10 mg / L MgSO4·7H2O, 3.2-3.5 mg / L MnSO4·H2O, 2.5-3.5 mg / L ZnSO4·7H2O, 0.02-0.04 mg / L FeSO4·7H2O, 0.05-0.08 mg / L CaCl2, and the balance is water.

[0013] Furthermore, the solid heterotrophic nitrification culture medium includes the following components: 0.45-0.5 g / L of (NH4)2SO4, 5.6-5.65 g / L of sodium acetate, 4.75-4.8 g / L of K2HPO4·3H2O, 1.2-1.25 g / L of KH2PO4, 8-10 mg / L of MgSO4·7H2O, 3.2-3.5 mg / L of MnSO4·H2O, 2.5-3.5 mg / L of ZnSO4·7H2O, 0.02-0.04 mg / L of FeSO4·7H2O, 0.05-0.08 mg / L of CaCl2, 15-20 g / L of agar powder, and the balance is water.

[0014] Furthermore, when the ammonia nitrogen-degrading bacteria are used in wastewater purification, the ammonia nitrogen-degrading bacteria are prepared as a microbial agent and added to the wastewater for purification;

[0015] In the microbial agent, the live bacteria content of ammonia nitrogen degrading bacteria HZ-N-001 is 1×10 9 -1.5×10 9 CFU / mL.

[0016] The preparation method of the microbial agent comprises: adding 0.3-0.5 g / L NH4Cl and 1.8-2.2 g / L sodium acetate to a basic culture medium, inoculating ammonia nitrogen degrading bacteria HZ-N-001 strain, culturing to a logarithmic phase, centrifuging and separating the bacterial suspension, collecting the bacterial cells, resuspending the bacterial cells in the basic culture medium again, and starving and culturing for 18-24 hours to obtain the microbial agent;

[0017] The basic culture medium includes the following components: 4.75-4.8 g / L K2HPO4·3H2O, 1.2-1.25 g / L KH2PO4, 8-10 mg / L MgSO4·7H2O, 3.2-3.5 mg / L MnSO4·H2O, 2.5-3.5 mg / L ZnSO4·7H2O, 0.02-0.04 mg / L FeSO4·7H2O, 0.05-0.08 mg / L CaCl2, and the rest is water.

[0018] Furthermore, in wastewater with an ammonia nitrogen content of 300-500 mg / L, the volume ratio of the microbial agent added is 0.5-1.5%; in wastewater with a COD content of 2000-3000 mg / L, the volume ratio of the microbial agent added is 0.1-1%; in wastewater with a total nitrogen content of 500-600 mg / L, the volume ratio of the microbial agent added is 0.5-1%.

[0019] Furthermore, when the ammonia nitrogen-degrading bacteria are used in reducing leather wastewater sludge, the volume ratio of the ammonia nitrogen-degrading bacteria added to the leather wastewater is 0.5-1%.

[0020] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: the present invention screens and cultivates bacterial strains, and finally cultivates the strain HZ-N-001 with good ammonia nitrogen degradation performance, which can realize denitrification treatment of water bodies in a heterotrophic aerobic environment, and simultaneously remove organic matter in the water bodies, thereby greatly simplifying the existing water body biological denitrification process steps and reducing costs, and the strain HZ-N-001 prepared by the present invention can also effectively reduce sludge production, degrade difficult-to-biodegrade macromolecular substances into small molecular products, thereby improving the physical and chemical properties of wastewater and enhancing the biodegradability of sewage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0022] Figure 1 It is a scanning electron microscope image of HZ-N-001 prepared by the present invention;

[0023] Figure 2 It is a phylogenetic tree analysis diagram of the strain HZ-N-001 prepared by the present invention;

[0024] Figure 3 This is the process flow chart of a leather wastewater plant in Xinji in Example 5 of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0026] Example 1.

[0027] A method for preparing ammonia nitrogen degrading bacteria comprises the following steps:

[0028] a. Collect activated sludge from the biochemical pool of the municipal sewage treatment system, river sludge, and aerobic pool sludge from the garbage treatment plant, mix them evenly, centrifuge and separate the activated sludge, and inoculate the activated sludge into the liquid heterotrophic nitrification medium at 10% (w / v), culture in shake flasks at 30°C, and detect the ammonia nitrogen content in the shake flasks every 48 hours;

[0029] b. When the ammonia nitrogen content in the shake flask drops to 0 mg / L, add new liquid heterotrophic nitrification medium, continue to cultivate and enrich for 1-2 weeks, and obtain enriched bacterial liquid;

[0030] c. Dilute the enriched bacterial solution to 10 with liquid heterotrophic nitrification medium. -1 -10 -7 There were 7 gradients in total. 100ul of each gradient was spread on solid heterotrophic nitrification medium. Five parallel groups were made for each gradient. After culturing at 30°C for 48h, different colonies grown on the plate were picked for streaking purification.

[0031] d. After 8 generations of streaking purification, the solid heterotrophic nitrification medium was tested. When the colony growth in the solid heterotrophic nitrification medium was uniform and free of foreign bacteria, the purified strain HZ-N-001 was obtained.

[0032] The liquid heterotrophic nitrification culture medium comprises the following components: 0.47 g / L (NH4)2SO4, 5.62 g / L sodium acetate, 7.78 g / L K2HPO4·3H2O, 1.24 g / L KH2PO4, 9 mg / L MgSO4·7H2O, 3.36 mg / L MnSO4·H2O, 3 mg / L ZnSO4·7H2O, 0.03 mg / L FeSO4·7H2O, 0.06 mg / L CaCl2, and the balance is water.

[0033] The solid heterotrophic nitrification medium comprises the following components: 0.47 g / L (NH4)2SO4, 5.62 g / L sodium acetate, 7.78 g / L K2HPO4·3H2O, 1.24 g / L KH2PO4, 9 mg / L MgSO4·7H2O, 3.36 mg / L MnSO4·H2O, 3 mg / L ZnSO4·7H2O, 0.03 mg / L FeSO4·7H2O, 0.06 mg / L CaCl2, 20 g / L agar powder, and the balance is water;

[0034] The strain HZ-N-001 was streaked on beef extract peptone solid medium and cultured at 30°C for 24-48 hours. The colony diameter of HZ-N-001 was 0.5-1.2 mm, and the colony was milky white, raised, smooth and matte, round, with smooth edges, opaque, odorless and tasteless. The strain morphology was observed by electron microscopy. Figure 1 ., It can be seen from the figure that the strain HZ-N-001 obtained by the present invention is a bacillus, 1.5-3μm in length and 0.5-0.8μm in width.

[0035] The DNA sequencing results are as follows:

[0036]

[0037]

[0038] The sequencing results were compared with the known sequences in the Genbank database for similarity. Figure 2 It can be seen that the HZ-N-001 prepared by the present invention has the highest sequence homology with Pseudomonas sp. and is identified as Pseudomonas sp.

[0039] Example 2.

[0040] Physiological and biochemical functions of strain HZ-N-001:

[0041] The strain HZ-N-001 was inoculated into a liquid heterotrophic nitrification medium, and cultured at 30°C with a stirring and shaking rate of 160 r / min for 48 hours to activate the strain. The activated strain liquid was inoculated into 100 ml of liquid heterotrophic nitrification medium at a volume ratio of 1%, and cultured at 30°C with a stirring and shaking rate of 160 r / min for 48 hours. During the culture period, samples were taken every 8 hours to detect the OD value of the culture liquid. 600 (bacteria concentration), NH4 + -N, NO2 - -N、NO3 - -N, COD, TN were tested; the test results are shown in Table 1;

[0042] Table 1. Physiological and biochemical data of HZ-N-001

[0043]

[0044] As can be seen from the above table, HZ-N-001 prepared by the present invention only needs an 8-h lag period to enter the exponential period. The OD value reaches a maximum value of 1.144 at 32 h, but the ammonia nitrogen and total nitrogen degradation rates do not reach the maximum values. The ammonia nitrogen content is the lowest at 40 h, the ammonia nitrogen degradation rate is 92.52%, the total nitrogen degradation rate is 84.19%, and the COD degradation rate is 96.96%.

[0045] Example 3.

[0046] The HZ-N-001 strain is prepared as a microbial agent, specifically comprising the following steps:

[0047] 0.4 g / L NH4Cl and 2 g / L sodium acetate were added to the basic culture medium, and the ammonia nitrogen-degrading bacteria HZ-N-001 strain was inoculated. After culturing to the logarithmic phase, the bacterial suspension was separated by centrifugation, the bacterial cells were collected, and the bacterial cells were resuspended in the basic culture medium again. After starvation culture for 18 hours, a microbial agent was obtained;

[0048] The basic culture medium includes the following components: 4.8 g / L K2HPO4·3H2O, 1.2 g / L KH2PO4, 9 mg / L MgSO4·7H2O, 3.4 mg / L MnSO4·H2O, 3 mg / L ZnSO4·7H2O, 0.03 mg / L FeSO4·7H2O, 0.07 mg / L CaCl2, and the rest is water.

[0049] Add the microbial agent to the artificially prepared wastewater according to the volume ratio in Table 2-4. The COD in the wastewater is 2000-3000 mg / L, the ammonia nitrogen is 300-500 mg / L, and the total nitrogen (TN) is 500-600 mg / L. Treat at 30°C and 160 r / min, and regularly test the COD, ammonia nitrogen and total nitrogen content in the wastewater. The test results are shown in Table 2-4.

[0050] Table 2. Effect of strains on treating ammonia nitrogen in wastewater

[0051]

[0052] As can be seen from the table above, within 48 hours, No. 3, No. 4, No. 5 and No. 6 have the highest degradation efficiency for ammonia nitrogen, all reaching more than 99.4%. Within 24 hours, No. 5 and No. 6 in the experimental group have the highest degradation efficiency. Considering the economy and efficiency of future product applications, the amount of bacterial agent added when treating ammonia nitrogen wastewater is 0.5%-1.5%;

[0053] Table 3. Effect of strains on COD in wastewater

[0054]

[0055] From the above table, we can see that: at 48h, No. 2, No. 3, No. 4, No. 5 and No. 6 have the best COD degradation effect, reaching 96.2%, 96.1%, 96.4%, 96.4% and 96.7% respectively. Compared with 24h, No. 4, No. 5 and No. 6 have the highest degradation efficiency. Considering the economic efficiency of future product applications, when treating high COD sewage, the amount of bacterial agent added to the sewage is 0.1%-1%;

[0056] Table 4. Effect of strains on TN in wastewater

[0057]

[0058]

[0059] From the table above, we can see that at 48h, No. 3, No. 4, No. 5 and No. 6 have the best degradation effect on TN, reaching 93.4%, 93.6%, 93.1% and 94.3% respectively. Compared with 24h, the degradation rates of No. 4, No. 5 and No. 6 are higher than No. 3. Considering the economy and efficiency of the product, the addition ratio of 0.5%-1% is selected to treat total nitrogen wastewater;

[0060] Example 4: Application of strains in sludge reduction;

[0061] The microbial agent prepared in Example 3 was added to leather wastewater at a ratio of 0%, 0.5%, 1% and 1.5% (v / v), the MLSS in the leather wastewater was 8000-12000 mg / L, and aeration treatment was performed at room temperature for 7 days. The change of MLSS in the leather wastewater was detected, and the detection results are shown in Table 5;

[0062] Table 5. Strain sludge reduction results

[0063]

[0064] From the table above, we can see that: MLSS in the table represents the content of suspended solids in wastewater, and is also an indicator used to measure the amount of activated sludge in the aeration tank. It can be seen that after inoculation with different proportions of bacterial liquid, the MLSS in the water samples has decreased, among which 0.5% and 1% inoculation has the best effect on MLSS reduction, and the sludge volume has been reduced by 55.5% and 55.9% respectively, and the sludge reduction has exceeded 55%. However, the MLSS of the wastewater after 1.5% inoculation did not decrease, mainly because the solid matter in MLSS contains the amount of microorganisms, the inoculation amount is large, the strain grows more, and the MLSS is higher than that of 0.5% and 1% inoculation.

[0065] Embodiment 5:

[0066] The leather wastewater inflow of a leather industry company in Xinji City is mainly sheepskin wastewater, cowhide wastewater, sulfur-containing water and other wastewater. The daily water treatment capacity is 2000~3500m 3 , the inlet water quality is as follows:

[0067] Table 6. Water quality parameters of leather wastewater from a leather company in Xinji City

[0068]

[0069] The total designed treatment capacity of a leather wastewater treatment plant in Xinji is 4500m 3 / d, actual operation time 2000~3500m 3 / d, and the amount of water treated fluctuates with the seasonality of production. Among the main structures of the process flow, the effective volume of the aeration regulating tank is 4800m 3 ; The effective volume of the anoxic pool is 2500m 3 ; The effective volume of the aerobic pool is 12500m 3 ; The effective volume of the sedimentation tank is 1600m 3 , set up secondary sedimentation; the effective volume of the sludge digestion tank is 2500m 3 ; Effective volume of sludge thickening tank 300m 3 All tanks are reinforced concrete structures. The aeration regulating tank and aerobic tank use jet aeration as the oxygen supply method. The bottom of the sludge digestion tank and anoxic tank is equipped with plug flow stirring.

[0070] Before the use of bacterial agents, the pollutant concentration of the mixed sewage of the plant was high and fluctuated greatly. The effluent ammonia nitrogen and COD often failed to meet the standards. It was unable to meet the ammonia nitrogen discharge standard of less than 70 mg / L and the COD discharge standard of less than 300 mg / L stipulated in the "Comprehensive Sewage Discharge Standard" (GB 8978-1996), and the daily sludge discharge was 60t (dry sludge) / d;

[0071] The microbial agent prepared in Example 3 was added to the sewage treatment system at 1% of the total water volume, and was added to the aeration regulating tank, sludge digestion tank and aerobic tank respectively. The addition ratio of each tank was 50%, 35% and 15% of the total amount of the microbial agent. After using the agent, the effluent in the leather sewage treatment system was stable for one month, and the effluent data were as follows:

[0072] Table 7. Effluent data of leather wastewater from a leather company in Xinji City after using bacterial agents

[0073] project <![CDATA[COD(mg·L -1 )]]> <![CDATA[NH4 + -N(mg·L -1 )]]> <![CDATA[Sludge production (t·d -1 )]]> No bacteria added to the water 200-800 60-100 60 Water after adding bacteria 100-200 0-30 40

[0074] From the table above, we can see that after using HZ-N-001 bacteria agent, COD degradation rate increased by 3%-14% compared with the time without bacteria, ammonia nitrogen degradation rate increased by 15%-64% compared with the time without bacteria, and ammonia nitrogen and COD both reached the emission standards. The sludge production was reduced by 33%, which greatly saved the company

[0075] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device 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 device.

[0076] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0077]

[0078] .

Claims

1. An ammonia nitrogen degrading bacterium, characterized in that: The ammonia nitrogen degrading bacteria are Pseudomonas ( Pseudomonas sp. )HZ-N-001 strain, the preservation number of the HZ-N-001 strain is CGMCC NO.23461.

2. Use of the ammonia nitrogen degrading bacteria as claimed in claim 1 in the purification of leather wastewater.

3. The use according to claim 2, characterized in that: The ammonia nitrogen-degrading bacteria are prepared as microbial agents and added to leather wastewater for purification; In the microbial agent, the live bacteria content of ammonia nitrogen degrading bacteria HZ-N-001 is 1×10 9 -1.5× 10 9 CFU / mL。 4. The use according to claim 3, characterized in that: The preparation method of the microbial agent comprises: adding 0.3-0.5 g / L NH4Cl and 1.8-2.2 g / L sodium acetate to a basic culture medium, inoculating ammonia nitrogen degrading bacteria HZ-N-001 strain, culturing to a logarithmic phase, centrifuging and separating the bacterial suspension, collecting the bacterial cells, resuspending the bacterial cells in the basic culture medium again, and starving and culturing for 18-24 hours to obtain the microbial agent; The basic culture medium includes the following components: 4.75-4.8 g / L K2HPO4·3H2O, 1.2-1.25 g / L KH2PO4, 8-10 mg / L MgSO4·7H2O, 3.2-3.5 mg / L MnSO4·H2O, 2.5-3.5 mg / L ZnSO4·7H2O, 0.02-0.04 mg / L FeSO4·7H2O, 0.05-0.08 mg / L CaCl2, and the rest is water.

5. The use according to claim 3, characterized in that: In wastewater with an ammonia nitrogen content of 300-500 mg / L, the volume ratio of the microbial agent added is 0.5-1.5%; in wastewater with a COD content of 2000-3000 mg / L, the volume ratio of the microbial agent added is 0.1-1%; in wastewater with a total nitrogen content of 500-600 mg / L, the volume ratio of the microbial agent added is 0.5-1%.

6. Use of the ammonia nitrogen degrading bacteria as claimed in claim 1 in reducing leather wastewater sludge.

Images