Domestication method of half-process denitrifying flora

Abstract

The invention discloses a half-process denitrifying flora domestication method which comprises the following steps: S1, mixing activated sludge and a sulfur-rich nutrient solution, and carrying out enrichment culture and separation to obtain a bacterial colony; s2, inoculating the bacterial colony into an activation culture medium, and carrying out activation culture until the OD600 of the culture medium is greater than or equal to 0.8, so as to obtain an activated mixed bacterial solution; and S3, inoculating the activated mixed bacterial liquid into a reactor, adding a nutrient solution to enable the concentration of nitrate nitrogen in the reactor to be 110-150mg / L and the mass ratio of carbon to nitrogen to be (1.02-2.04): 1, and domesticating under an anoxic condition to obtain a half-process denitrifying flora. The half-process denitrification flora disclosed by the invention not only can adapt to the growth environment of anaerobic ammonia oxidation coupling, but also can stably and efficiently carry out a half-process denitrification reaction; when the catalyst is applied to treatment of high-concentration nitrate nitrogen wastewater, high-efficiency half-process denitrification can be realized without adding an organic carbon source.

Description

Technical Field

[0001] The invention belongs to the technical field of biological treatment of nitrogen-containing sewage, and specifically relates to a method for acclimating a semi-denitrifying bacterial community. Background Art

[0002] Nitrate is the main monitoring item for nitrogen pollution in the water environment. Its enrichment will not only lead to eutrophication of water bodies and cause red tides, but also infiltrate into the soil and seriously affect the growth and development of plants, pollute groundwater, and accumulate in the food chain, causing serious harm to human production and life. my country's drinking water standards (GB5749-85) also stipulate the nitrate content (<20mg / L). Among the current biological denitrification treatment methods, the anaerobic ammonium oxidation process can effectively save aeration and carbon source costs and greatly shorten the intermediate steps for efficient denitrification. However, how to provide stable nitrite for anaerobic ammonium oxidizing bacteria has always been a bottleneck problem.

[0003] In 1990, the Delft University of Technology in the Netherlands proposed to use a partial nitrification process (PN / A process) to solve this problem, that is, the nitrification reaction is only halfway through, and the generated nitrite is supplied to anaerobic ammonium oxidizing bacteria as an electron acceptor when nitrite is generated from nitrate. However, this also has some disadvantages, such as how to control the reaction to only stay at the step of generating nitrite, and to minimize the competition between nitrite oxidizing bacteria (NOB) and anaerobic ammonium oxidizing bacteria (AnAOB) for nitrite electron acceptors, which is difficult; secondly, in the equation, both the anaerobic ammonium oxidation reaction and the nitrification reaction will eventually have residual nitrates that cannot be removed. Studies have shown that in the PN / A system, the final nitrate nitrogen content is as low as 11% of the total nitrogen, that is, the removal rate is as high as 89%. In this case, the proposal of the half-denitrification process (PD / A) provides a better way to solve the problem.

[0004] The half-denitrification process can provide nitrite as an electron acceptor for anaerobic ammonium oxidizing bacteria, and at the same time, it can use the nitrate generated by the anaerobic ammonium oxidation process as a reaction substrate, which can theoretically achieve 100% nitrogen removal without causing pollution, and can also reduce the aeration energy consumption of the nitrification reaction. It can be said to be an environmentally friendly, efficient and economical treatment method. Therefore, how to domesticate a bacterial community that can not only adapt to the growth environment of anaerobic ammonium oxidation coupling, but also stably and efficiently perform half-denitrification is an urgent problem that needs to be solved. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for acclimating a half-denitrifying bacterial community, which can effectively assist the deep denitrification application of half-denitrification-anaerobic ammonia oxidation.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0007] A method for acclimating a semi-denitrifying bacterial community comprises the following steps:

[0008] S1. Mixing activated sludge and sulfur-rich nutrient solution for enrichment culture, and separating by streaking on a plate to obtain bacterial colonies;

[0009] S2, inoculating the bacterial colony obtained in step S1 into an activation culture medium for activation culture until the OD600 of the culture medium is ≥ 0.8, thereby obtaining an activated mixed bacterial solution;

[0010] S3. Inoculate the activated mixed bacterial solution obtained in step S2 into a reactor, add nutrient solution to make the concentration of nitrate nitrogen in the reactor be 110 mg / L-150 mg / L, and the carbon-nitrogen mass ratio be 1.02-2.04:1, and perform acclimatization under anoxic conditions to obtain a semi-denitrifying bacterial community.

[0011] The above acclimation method is further improved, in step S3, the concentration of nitrate nitrogen in the reactor is 115 mg / L to 130 mg / L.

[0012] The above acclimation method is further improved, in step S3, the nutrient solution contains the following ingredients: 0.636 g / L KCl, 0.728 g / L to 0.911 g / L NaNO 3 , 1.329 g / L~10.637 g / L of Na 2 S 2 O 3 ·5H 2 O, 1g / L to 2g / L NaHCO 3 .

[0013] The above-mentioned acclimation method is further improved, in step S3, the nutrient solution also contains one or more of Ca element, P element, Mg element, and Fe element, and the nutrient solution includes one or more of ethylenediaminetetraacetic acid and ammonium chloride.

[0014] The above domestication method is further improved, in step S3, the preparation method of the nutrient solution is specifically: Na 2 S 2 O 3 ·5H 2 O, NaHCO 3 The other components were packed in three different devices, sealed after nitrogen or inert gas was introduced, and sterilized at 121℃ for 20min. 2 S 2 O 3 ·5H 2O, NaHCO 3 The nutrient solution is prepared by mixing with other components.

[0015] The above acclimation method is further improved, in step S1, the sulfur-rich nutrient solution contains the following ingredients: 0.72 g / L KNO 3 , 1000 mg / L NaHCO 3 , 136 mg / L CaCl 2 ·2H 2 O, 5g / L to 10g / L sulfur powder; the sulfur-rich nutrient solution also contains one or more of Fe, P and Mg; the enrichment culture time is 30 to 35 days.

[0016] The above-mentioned acclimation method is further improved, in step S2, the activation culture medium contains the following ingredients: 10.0 g / L peptone, 5.0 g / L yeast powder, 5.0 g / L sodium chloride; the pH value of the activation culture medium is 6.8-7.2.

[0017] The above acclimation method is further improved, in step S2, the OD600 of the activated mixed bacterial solution is 0.8-1.0.

[0018] The above-mentioned acclimation method is further improved. In step S2, the temperature of the activation culture is 25°C to 29°C, the pH value is controlled to be 6.5 to 7.5 during the activation culture process, and the activation culture time is 3 to 5 days.

[0019] The above-mentioned acclimation method is further improved. In step S3, the volume ratio of the activated mixed bacterial liquid to the nutrient solution is 0.1-0.15:1, the anoxic condition is the introduction of nitrogen, the acclimation time is ≤100h, the acclimation temperature is 25°C-29°C, and the pH value is controlled at 6.5-8.5 during the acclimation process.

[0020] The above acclimation method is further improved, in step S3, the acclimation time is 36h to 98h.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] In view of the defects of the existing wastewater denitrification treatment process, such as high residual nitrate nitrogen, complex denitrification process, high cost of external carbon source consumption, insufficient supply of electron acceptors, etc., the present invention creatively proposes a method for taming a half-denitrifying bacterial community, wherein activated sludge and sulfur-rich nutrient solution are mixed for enrichment culture, separated by streaking on a plate to obtain colonies; the obtained colonies are then activated and cultured to obtain an activated mixed bacterial solution; then, the activated mixed bacterial solution is tamed in a nutrient solution with high concentration of nitrate nitrogen and low carbon-nitrogen ratio, so that the concentration of nitrate nitrogen in the reactor is 110 mg / L-150 mg / L and the carbon-nitrogen mass ratio is 1.02-2.04:1, thereby obtaining a half-denitrifying bacterial community. The existing denitrifying bacteria are usually adapted to wastewater with a nitrate nitrogen concentration of 40 mg / L but cannot adapt to wastewater with a higher concentration of nitrate nitrogen. The half-denitrifying bacteria of the present invention can not only adapt to wastewater with a nitrate nitrogen concentration of up to 110 mg to 150 mg / L, but also adapt to the growth environment of anaerobic ammonia oxidation coupling, so that the half-denitrification reaction can be carried out stably and efficiently. When it is applied to the treatment of high-concentration nitrate nitrogen wastewater, no external organic carbon source is required, that is, efficient half-denitrification can be achieved (the half-denitrification conversion rate is more than 80%, and the average conversion rate is higher than 10 mg·L -1 ·h -1 ), thereby providing a stable and efficient electron donor for the half-denitrification-anaerobic ammonium oxidation (PD / A) process, which has broad application potential and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing changes in nitrate nitrogen degradation and nitrite nitrogen generation during the acclimation process of the semi-denitrifying bacteria community in Example 1 of the present invention.

[0024] Figure 2 This is a graph showing changes in nitrate nitrogen degradation and nitrite nitrogen generation during the acclimation process of the semi-denitrifying bacteria community in Example 2 of the present invention.

[0025] Figure 3 This is a graph showing changes in nitrate nitrogen degradation and nitrite nitrogen generation during the acclimation process of the semi-denitrifying bacteria community in Example 3 of the present invention.

[0026] Figure 4 This is a graph showing changes in nitrate nitrogen degradation and nitrite nitrogen generation during the acclimation process of the semi-denitrifying bacteria community in Example 4 of the present invention.

[0027] Figure 5 This is a graph showing changes in nitrate nitrogen degradation and nitrite nitrogen generation during the acclimation process of the semi-denitrifying bacteria community in Example 5 of the present invention.

[0028] Figure 6 This is a diagram showing the changes in community composition of the half-way denitrifying bacteria community in Example 5 of the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby. The materials and instruments used in the following embodiments are all commercially available.

[0030] Embodiment 1:

[0031] A method for acclimating a semi-denitrifying bacterial community of the present invention comprises the following steps:

[0032] (1) Take activated sludge from the biochemical pool of the leather wastewater treatment plant, add sulfur-rich culture medium, and carry out enrichment culture for 35 days. Take 1 mL of the mixed enrichment and perform gradient dilution. Separate by streaking to obtain colonies. The sulfur-rich culture medium contains the following components: KNO 3 0.72mg / L, NaHCO 3 1000mg / L, CaCl 2 ·2H 2 O 136mg / L, elemental sulfur powder: 10g / L, Trace elementⅠ1mL / L, Trace elementⅡ1mL / L; Trace elementⅠ: ethylenediaminetetraacetic acid (EDTA) 5000mg / L, FeSO 4 5000mg / L, Trace elementⅡ: KH 2 PO 4 27mg / L, MgSO 4 7H 2 O 20mg / L.

[0033] (2) Inoculate the above-mentioned colony into the activation medium, and culture it independently at 27°C for 3 to 5 days. During the culture process, the pH value is controlled to be 6.5 to 7.5, that is, the OD600 of the culture medium is ≥ 0.8, to obtain an activated mixed bacterial solution. The activation medium contains the following components: peptone 10.0 g / L, yeast powder 5.0 g / L, sodium chloride 5.0 g / L; the pH of the activation medium is 7.0 ± 0.2.

[0034] (3) Take Na 2 S 2 O 3 ·5H 2 O and NaHCO 3 Dispense in different crimp bottles, then take KCl, NaNO 3 , CaCl 2 ·2H 2 O.KH 2 PO 4 MgSO 4 7H 2 O, EDTA, FeSO 4 7H2 O were placed in the same crimp bottle; then N 2 After 20 minutes, seal the bottle and sterilize it at 121℃ for 20 minutes. Then, use quantitative injection to mix the components according to the preset concentration to obtain nutrient solution. The nutrient solution contains the following ingredients: KCl 0.636g / L, NaNO 3 0.728g / L, CaCl 2 ·2H 2 O0.136g / L, KH 2 PO 4 0.027g / L, MgSO 4 7H 2 O 0.020g / L, Na 2 S 2 O 3 ·5H 2 O 9.675 g / L, NaHCO 3 1g / L, EDTA 5mg / L, FeSO 4 7H 2 O 9.2mg / L.

[0035] (4) The volume ratio of the activated mixed bacterial solution to the nutrient solution was 0.1:1. The activated mixed bacterial solution with an OD600 value of 0.8 to 1.0 was inoculated into the reactor, and the above nutrient solution was added to make the nitrate nitrogen concentration in the reactor 117.017 mg / L and the carbon-nitrogen mass ratio (i.e., NaHCO 3 -C and NaNO 3 The mass ratio of N to N was 1.22:1; 2 , and acclimatized at 27℃ for 98h. During the acclimatization process, the pH value of the system was controlled at 7.0-8.0 to obtain a semi-denitrifying bacterial community.

[0036] During the acclimation process, the concentrations of nitrate nitrogen and nitrite nitrogen in the reactor were measured.

[0037] Figure 1 This is a graph showing the changes in nitrate nitrogen degradation and nitrite nitrogen generation during the acclimation of the semi-denitrifying bacteria in Example 1 of the present invention. Figure 1 It can be seen that the concentration of nitrate nitrogen decreased from 117.017 mg / L to 5.926 mg / L, while the concentration of nitrite nitrogen was 106.50 mg / L, and the half-way denitrification conversion rate was 95.87%. The calculation formula for the half-way denitrification conversion rate is:

[0038] Half-way denitrification conversion rate = (C1-C0) / (c 0 -c 1 )(1)

[0039] In formula (1), C0 is the initial concentration of nitrite nitrogen, C1 is the concentration of nitrite nitrogen, and c 0 is the initial concentration of nitrate nitrogen, c 1 is the nitrate nitrogen concentration.

[0040] Embodiment 2:

[0041] A method for acclimating a half-way denitrifying bacterial community is basically the same as the method for acclimating a half-way denitrifying bacterial community in Example 1, except that: in step (3), NaNO 3 The nitrate nitrogen concentration in the reactor in step (4) is 139.300 mg / L, and the carbon-nitrogen mass ratio is 1.03:1.

[0042] Figure 2 This is a graph showing the changes in nitrate nitrogen degradation and nitrite nitrogen generation during the acclimation of the semi-denitrifying bacteria in Example 2 of the present invention. Figure 2 It can be seen that the nitrate nitrogen concentration decreased from 139.300 mg / L to 7.075 mg / L, while the nitrite nitrogen concentration was 108.75 mg / L, and its half-way denitrification conversion rate was 82.25%.

[0043] Embodiment 3:

[0044] A method for acclimating a half-way denitrifying bacterial community is basically the same as the method for acclimating a half-way denitrifying bacterial community in Example 2, except that: in step (3), NaHCO 3 The nitrogen content of nitrate in the reactor in step (4) is 140.275 mg / L, and the carbon-nitrogen mass ratio is 2.04:1.

[0045] Figure 3 This is a graph showing the changes in nitrate nitrogen degradation and nitrite nitrogen generation during the acclimation of the semi-denitrifying bacteria in Example 3 of the present invention. Figure 3 It can be seen that the nitrate nitrogen concentration decreased from 140.275 mg / L to 7.900 mg / L, while the nitrite nitrogen concentration was 108.75 mg / L, and its half-way denitrification conversion rate was 82.15%.

[0046] Embodiment 4:

[0047] A method for acclimating a half-denitrifying bacterial group is basically the same as the method for acclimating a half-denitrifying bacterial group in Example 2, except that: in step (3), a group of components, namely, NH 4 Cl 0.2g / L, so that the nitrate nitrogen concentration in the reactor in step (4) is 136.950mg / L, and the carbon-nitrogen mass ratio is 1.04:1.

[0048] Figure 4This is a graph showing the changes in nitrate nitrogen degradation and nitrite nitrogen production during the domestication process of the semi-denitrifying bacterial community in Example 4 of the present invention. From Figure 4 It can be seen that the nitrate nitrogen concentration decreased from 136.950 mg / L to 9.575 mg / L, while the nitrite nitrogen production concentration was 112.50 mg / L, and the semi-denitrification conversion rate was 88.32%.

[0049] Example 5:

[0050] A method for domesticating a semi-denitrifying bacterial community is basically the same as the method for domesticating the semi-denitrifying bacterial community in Example 2, except that: in step (3), the amount of Na 2 S 2 O 3 ·5H 2 O is 2.658 g / L, so that the nitrate nitrogen concentration in the reactor in step (4) is 128.325 mg / L, and the carbon-nitrogen mass ratio is 1.11:1.

[0051] Figure 5 This is a graph showing the changes in nitrate nitrogen degradation and nitrite nitrogen production during the domestication process of the semi-denitrifying bacterial community in Example 5 of the present invention. From Figure 5 It can be seen that the nitrate nitrogen concentration decreased from 128.325 mg / L to 3.750 mg / L, while the nitrite nitrogen production concentration was 114.50 mg / L, and the semi-denitrification conversion rate was 91.91%.

[0052] The activated mixed bacterial liquid, the mixed bacterial liquid in the early stage of domestication (i.e., domesticated for 48 h), and the mixed bacterial liquid in the later stage of domestication (i.e., domesticated for 96 h) were filtered by suction, frozen at -80 °C for 24 h, and then subjected to 16S rDNA sequencing. The results are as Figure 6 shown. From Figure 6 It can be seen that in the mixed bacterial liquid in the later stage of domestication, the main genera of bacteria are Acinetobacter, Citrobacter, and Ciceribacter. Through analysis, it is obtained that Citrobacter is a facultative semi-denitrifying bacterium, and together with Acinetobacter, they belong to the heterotrophic nitrification-aerobic denitrification bacterial community. Ciceribacter is an autotrophic genus of bacteria, and all three genera of bacteria are facultative anaerobes.

[0053] In this example, the semi-denitrification conversion of the semi-denitrifying bacterial community in high-sulfur and high-nitrogen wastewater was also tested, that is, the semi-denitrifying bacterial community obtained in Examples 1 to 4 was used to treat high-sulfur and high-nitrogen wastewater, including the following steps:

[0054] The semi-denitrifying bacterial community obtained in Examples 1 to 4 was respectively inoculated into a reactor, and high-sulfur and high-nitrogen wastewater was added. The sulfur-nitrogen molar ratio M(S 2 O 32- ) / M(NO 3 - ) are 0.675, 1.25, 2.5, and 5 respectively, and N 2 , carry out half-denitrification reaction for 24 hours to achieve the treatment of high-sulfur and high-nitrogen wastewater.

[0055] As shown in Table 1, the half-denitrification reaction lasts for 24 hours. The half-denitrifying bacteria of the present invention can convert nitrate nitrogen in high-sulfur and high-nitrogen wastewater with different sulfur-nitrogen molar ratios into nitrite nitrogen, which shows that the half-denitrifying bacteria of the present invention has good half-denitrification ability. In particular, after being treated with the half-denitrifying bacteria of Examples 1 to 4 of the present invention for 24 hours, the average half-denitrification conversion rate of different high-sulfur and high-nitrogen wastewaters is 92.07%.

[0056] Table 1 Nitrate nitrogen removal and nitrite nitrogen accumulation when the half-way denitrifying bacteria treated high-sulfur and high-nitrogen wastewater

[0057]

[0058] In summary, the domestication method of the half-denitrifying bacterial community of the present invention is to domesticate the activated mixed bacterial solution in a nutrient solution with high concentration of nitrate nitrogen and low carbon-nitrogen ratio (the concentration of nitrate nitrogen in the reactor is 110mg / L-150mg / L, and the carbon-nitrogen mass ratio is 1.02-2.04:1) to obtain a half-denitrifying bacterial community. The half-denitrifying bacterial community of the present invention can not only adapt to the growth environment of anaerobic ammonia oxidation coupling, but also stably and efficiently carry out half-denitrification reaction, and the half-denitrifying bacterial community still has good half-denitrification conversion performance after passage; when it is applied to the treatment of high-concentration nitrate nitrogen wastewater, no external organic carbon source is required, and efficient half-denitrification can be achieved, which has wide application potential and value.

[0059] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for acclimating a semi-denitrifying bacterial community. It is characterized in that The following steps are involved: S1. Mixing activated sludge and sulfur-rich nutrient solution for enrichment culture, and separating by streaking on a plate to obtain bacterial colonies; S2, inoculating the bacterial colony obtained in step S1 into an activation culture medium for activation culture until the OD600 of the culture medium is ≥ 0.8, thereby obtaining an activated mixed bacterial solution; S3. Inoculate the activated mixed bacterial solution obtained in step S2 into a reactor, add nutrient solution to make the concentration of nitrate nitrogen in the reactor be 110 mg / L-150 mg / L, and the carbon-nitrogen mass ratio be 1.02-2.04:1, and perform acclimatization under anoxic conditions to obtain a semi-denitrifying bacterial community.

2. The method for acclimating a half-way denitrifying bacteria group according to claim 1, It is characterized in that In step S3, the concentration of nitrate nitrogen in the reactor is 115 mg / L to 130 mg / L.

3. The method for acclimating a half-way denitrifying bacteria group according to claim 2, It is characterized in that In step S3, the nutrient solution contains the following ingredients: 0.636 g / L KCl, 0.728 g / L to 0.911 g / L NaNO 3 , 1.329 g / L~10.637 g / L of Na 2 S 2 O 3 ·5H 2 O, 1g / L to 2g / L NaHCO 3 .

4. The method for acclimating a half-way denitrifying bacteria group according to claim 3, It is characterized in that In step S3, the nutrient solution further contains one or more of the elements Ca, P, Mg, and Fe, and the nutrient solution includes one or more of ethylenediaminetetraacetic acid and ammonium chloride.

5. The method for acclimating a half-way denitrifying bacteria group according to claim 4, It is characterized in that In step S3, the preparation method of the nutrient solution is specifically as follows: 2 S 2 O 3 ·5H 2 O, NaHCO 3 The other components were packed in three different devices, sealed after nitrogen or inert gas was introduced, and sterilized at 121℃ for 20min. 2 S 2 O 3 ·5H 2 O, NaHCO 3 The nutrient solution is prepared by mixing with other components.

6. The method for acclimating a semi-denitrifying bacteria group according to any one of claims 1 to 5, It is characterized in that In step S1, the sulfur-rich nutrient solution contains the following ingredients: 0.72 g / L KNO 3 , 1000 mg / L NaHCO 3 , 136 mg / L CaCl 2 ·2H 2 O, 5g / L to 10g / L sulfur powder; the sulfur-rich nutrient solution also contains one or more of Fe, P and Mg; the enrichment culture time is 30 to 35 days.

7. The method for acclimating a semi-denitrifying bacteria group according to any one of claims 1 to 5, It is characterized in that In step S2, the activation medium comprises the following components: 10.0 g / L of peptone, 5.0 g / L of yeast powder, and 5.0 g / L of sodium chloride; the pH value of the activated culture medium is 6.8-7.

2.

8. The method for acclimating a semi-denitrifying bacteria group according to any one of claims 1 to 5, It is characterized in that In step S2, the OD600 of the activated mixed bacterial solution is 0.8-1.

0.

9. The method for acclimating a half-way denitrifying bacteria group according to claim 8, It is characterized in that In step S2, the temperature of the activation culture is 25°C to 29°C, the pH value is controlled to be 6.5 to 7.5 during the activation culture process, and the activation culture time is 3 to 5 days.

10. The method for acclimating a semi-denitrifying bacteria group according to any one of claims 1 to 5, It is characterized in that In step S3, the volume ratio of the activated mixed bacterial liquid to the nutrient solution is 0.1-0.15:1, the anoxic condition is the introduction of nitrogen, the acclimation time is ≤100h, the acclimation temperature is 25°C-29°C, and the pH value is controlled at 6.5-8.5 during the acclimation process.

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