A carrier for culturing anaerobic ammonia-oxidizing bacteria, and its preparation method and application

Abstract

The present invention relates to a carrier for culturing anaerobic ammonia-oxidizing bacteria, as well as a preparation method and application thereof. The method comprises culturing heterotrophic bacteria that utilize an organic carbon source to the late logarithmic growth phase, harvesting bacterial cells, mixing the bacterial cells with chitosan, and then cross-linking with calcium carbonate to prepare a cross-linked chitosan carrier; and modifying the cross-linked chitosan carrier with polyethyleneimine to obtain a carrier for culturing anaerobic ammonia-oxidizing bacteria. The carrier provided by the present invention can provide a suitable growth environment for anaerobic ammonia-oxidizing bacteria, facilitate the adsorption and reproduction of anaerobic ammonia-oxidizing bacteria, prevent bacterial cell loss, and achieve a long-lasting and stable denitrification effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental protection water pollution control, and specifically relates to a carrier for culturing anaerobic ammonia oxidizing bacteria, and a preparation method and application thereof. Background Art

[0002] Anaerobic ammonium oxidizing bacteria (ANAMMOX) are chemoautotrophic microorganisms that do not require oxygen or organic carbon sources for growth, reproduction, or denitrification. Their application in sewage treatment can save electricity and energy, reduce greenhouse gas emissions, and produce no excess sludge. Anaerobic ammonium oxidizing technology can also generate electricity when coupled with other anaerobic technologies. Therefore, ANAMMOX technology has become a research hotspot. However, this technology also has certain limitations, such as the long generation cycle of ANAMMOX bacteria (11 days), low cell yield (0.066 molC / molNH4 + ), are sensitive to and demanding of environmental conditions such as pH, dissolved oxygen, and temperature. Anaerobic ammonium oxidizing bacteria grow slowly, leading to significant process sludge loss and difficulty collecting it, making it difficult to effectively retain the bacteria within the reactor. Therefore, obtaining sufficient anaerobic ammonium oxidizing bacteria and preventing bacterial loss is a major technical bottleneck in achieving and maintaining a stable anaerobic ammonium oxidation process.

[0003] Many researchers have carried out extensive research work on the enrichment and cultivation of anaerobic ammonium oxidizing bacteria, sludge granulation, bacterial immobilization and the form of reactors, among which the enrichment and cultivation of anaerobic ammonium oxidizing bacteria is the basis of all work.

[0004] CN201810441790.5 discloses a method for enriching high-density anaerobic ammonium oxidizing bacteria in a sewage denitrification system, comprising: 1) collecting nitrified sludge containing anaerobic ammonium oxidizing bacteria and preparing an activated sludge solution; inoculating it into an enrichment medium for nitrification acclimation until the conversion rate of ammonia nitrogen is not less than 80%; 2) inoculating the acclimated sludge into a reactor, and adding a large-pore polyurethane filler and a substrate ammonia nitrogen and nitrite nitrogen to start the anaerobic ammonium oxidizing reactor at a low substrate concentration; 3) increasing the amount of substrate added to start the anaerobic ammonium oxidizing reactor at a medium-to-high substrate concentration; 4) continuing to increase the amount of substrate added to enrich high-density anaerobic ammonium oxidizing bacteria. The invention uses a macroporous polyurethane filler to provide a micro-aerobic environment for anaerobic ammonium oxidizing bacteria and can adapt to the agglomerative growth characteristics of anaerobic ammonium oxidizing bacteria, which helps to eliminate bacteria such as AOB and NOB, and can obtain anaerobic ammonium oxidizing bacteria with higher purity and higher abundance. Although the macroporous polyurethane filler used in this invention can provide a micro-oxygen environment for anaerobic ammonia-oxidizing bacteria, which helps to eliminate bacteria such as AOB (ammonia-oxidizing bacteria) and NOB (nitrite-oxidizing bacteria), when the bacteria fill the pores, there will be a risk of blockage, which will affect the long-term performance of the bacteria.

[0005] CN201910961126.8 discloses a method for enriching anaerobic ammonia-oxidizing bacteria, comprising the following steps: S1. Wastewater pretreatment: adding an anaerobic enhancing bacterial agent and / or a composite denitrifying bacterial agent activated at a predetermined volume concentration into the wastewater for anaerobic / anoxic reaction treatment for a predetermined time; S2. Aeration treatment: after the anaerobic effluent, the wastewater is passed into an aeration device, and an aerobic enhancing bacterial agent and an autotrophic nitrifying bacterial agent activated at a predetermined volume concentration are added to remove COD and degrade ammonia nitrogen in the wastewater, and the aeration treatment reaction time is controlled to regulate the ratio of COD, ammonia nitrogen and nitrite; S3. Enrichment treatment: the aerated effluent enters the enrichment device for static reaction until red bacterial flocs appear, and the total nitrogen removal rate is stabilized to complete the enrichment of anaerobic ammonia-oxidizing bacteria. This invention uses wastewater as an enrichment substrate and nutrient source, and uses the addition of microbial agents to treat wastewater at different stages. On the one hand, it can provide the required enrichment and culture conditions for anaerobic ammonium oxidizing bacteria, so that the effluent COD, ammonia nitrogen and nitrite meet the requirements for enriching anaerobic ammonium oxidizing strains; on the other hand, the target strain is synergistically domesticated during the enrichment process, which can improve the growth rate and tolerance of anaerobic ammonium oxidizing bacteria, improve the enrichment effect and total nitrogen removal rate of anaerobic ammonium oxidizing bacteria, and ensure that the strain and device can be quickly started during subsequent applications, thereby accelerating the process of biological denitrification in the anaerobic ammonium oxidizing process. The final abundance of the domesticated target anaerobic ammonium oxidizing bacteria accounts for 6-9% of the total bacterial abundance, which greatly improves the enrichment effect of anaerobic ammonium oxidizing bacteria. However, this enrichment process requires the use of multiple enhancing agents such as anaerobic enhancing agents, aerobic enhancing agents, composite denitrifying agents, autotrophic nitrifying agents, etc., and also requires the use of functional activators, etc., which makes the process more complicated. In addition, the anaerobic ammonium oxidizing bacteria enriched by this method also have the problem of easy loss. Summary of the Invention

[0006] To address the shortcomings of the prior art, the present invention provides a carrier for culturing anaerobic ammonium oxidizing bacteria, as well as its preparation method and application. The carrier provided by the present invention can provide a suitable growth environment for anaerobic ammonium oxidizing bacteria, facilitate the adsorption and reproduction of anaerobic ammonium oxidizing bacteria, avoid bacterial loss, and achieve a long-lasting and stable denitrification effect.

[0007] The present invention provides a method for preparing a carrier for culturing anaerobic ammonia-oxidizing bacteria, comprising the following steps:

[0008] (1) Cultivating heterotrophic bacteria that utilize organic carbon sources to the late logarithmic growth phase and harvesting bacterial cells;

[0009] (2) Mixing bacterial cells with chitosan, and then cross-linking with calcium carbonate to prepare a cross-linked chitosan carrier;

[0010] (3) The cross-linked chitosan carrier was modified with polyethyleneimine to obtain a carrier for cultivating anaerobic ammonia-oxidizing bacteria.

[0011] In the method of the present invention, the heterotrophic bacteria utilizing an organic carbon source in step (1) are microorganisms that reproduce and grow using an organic carbon source under anaerobic or anoxic conditions, and may be at least one of yeast, lactic acid bacteria, and sulfate-reducing bacteria. The yeast may be selected from at least one of Candida, Cryptococcus, Hansenula, Pichia pastoris, Rhodotorula, Torulopsis, or Trichosporon, preferably Candida tropicalis. The lactic acid bacteria may be selected from at least one of Lactobacillus, Bifidobacterium, and Lactococcus. The sulfate-reducing bacteria may be selected from at least one of Desulfomonas and Desulfuromyces.

[0012] In the method of the present invention, the organic carbon source in step (1) is determined according to the selected heterotrophic bacteria, and is generally at least one of the carbon-containing organic substances such as sugars, proteins, organic acids, etc. used in the conventional cultivation of the selected heterotrophic bacteria, and specifically can be at least one of glucose, hexose, xylose, sucrose, starch, etc.

[0013] In the method of the present invention, the culture medium commonly used in the art for culturing the heterotrophic bacteria utilizing an organic carbon source in step (1) is selected based on the bacterial cells. The culture conditions are: a temperature of 20-38°C, preferably 20-30°C, a pH of 6.0-8.5, preferably 6.0-7.0; static culture or shaking culture, with stirring every 30-60 minutes for static culture and a rotation speed of 200-600 r / min for shaking culture. After culturing to the late logarithmic growth phase, generally for 24-80 hours, the bacterial cells can be harvested by filtration, centrifugation, or the like. For example, the supernatant can be discarded and the bacterial cells retained by centrifugation at 10,000-15,000 r / min.

[0014] In the method of the present invention, step (2) involves mixing bacterial cells with chitosan in a mass ratio of 1:1-1:3, and then cross-linking with calcium carbonate to prepare a cross-linked chitosan carrier. The cross-linked chitosan carrier is prepared by conventional preparation methods in the art. The preparation method can adopt a direct cross-linking method, in which the cross-linking agent used is at least one of epichlorohydrin, glutaraldehyde, formaldehyde, crown ethers, and genipin, preferably genipin. Cross-linking is a cross-linking reaction between chitosan and cross-linking agent molecules, which converts the chitosan molecules from straight chains to a network structure. Cross-linking can improve the physical properties of chitosan, such as the specific surface area and pore structure, and effectively improve the stability of chitosan. The specific preparation process is as follows: chitosan is added at a mass volume ratio of 2% and acetic acid is added at a volume ratio of 1% to prepare a 500 mL mixed solution, the bacterial cells prepared in step (1) are added at a mass ratio of bacterial cells to chitosan of 1:1-1:3, and then 10 g of CaCO3 nanoparticles are added, 5 times the volume of edible oil is added, and 10 mL of Span-80 is added and stirred vigorously; then genipin is added to a final concentration of 20 mM in the aqueous phase, stirring is continued for 24 hours, and the precipitate is separated by centrifugation and washed several times with acetone, hot water, and cold water to remove the oil phase and impurities remaining on the surface. Finally, the mixture is dehydrated with acetone and the resulting product is dried at room temperature to obtain a cross-linked chitosan carrier with calcium carbonate as the core and bacterial cells and chitosan uniformly wrapped around it.

[0015] In the method of the present invention, a cross-linked chitosan carrier is modified with polyethyleneimine. Specifically, the cross-linked chitosan carrier is immersed in a polyethyleneimine aqueous solution having a mass fraction of 1% to 10%. The carrier is immersed in the solution for 30 to 90 minutes. After the immersion is completed, the carrier is removed and dried at a temperature of 25 to 40°C for 1 to 5 hours.

[0016] The carrier for culturing anaerobic ammonia-oxidizing bacteria of the present invention is prepared by the above-mentioned method of the present invention. In the prepared carrier, heterotrophic bacteria account for 1%-5% of the carrier mass, and polyethyleneimine accounts for 1%-10% of the carrier mass.

[0017] The present invention also provides an application of the carrier prepared by the present invention, that is, using the carrier to enrich and culture anaerobic ammonia-oxidizing bacteria.

[0018] In the present invention, the carrier is filled to 20%-40% of the reactor's effective volume, and anaerobic ammonium oxidizing bacteria are inoculated for enrichment culture. When the abundance reaches 10% or more, red spherical particles are formed. These particles have a high biomass concentration and good denitrification efficiency, completing the enrichment culture of anaerobic ammonium oxidizing bacteria. The resulting spherical particles can be used directly or stored for later use.

[0019] In the application of the present invention, the enrichment culture of anaerobic ammonia oxidizing bacteria can be carried out by using an anaerobic ammonia oxidizing agent known in the prior art, and the enrichment culture can be carried out after inoculation at an inoculum size of 0.1%-0.5% by volume, or activated sludge rich in anaerobic ammonia oxidizing bacteria can be used, and the enrichment culture can be carried out after inoculation at a sludge concentration of 2-4 g / L. After inoculation in the above manner, the abundance value of the anaerobic ammonia oxidizing bacteria contained in the system at the initial stage of enrichment culture is less than 1%.

[0020] In the application of the present invention, the culture solution used in the enrichment culture has an ammonia nitrogen concentration of 500-2000 mg / L, a mass ratio of ammonia nitrogen to nitrite nitrogen of 1:1-1:1.5, and a COD concentration of 50-200 mg / L.

[0021] In the application of the present invention, the enrichment culture conditions are: temperature 25-38° C., pH 7.5-8.5, and dissolved oxygen concentration lower than 0.2 mg / L.

[0022] In the application of the present invention, the enrichment culture of the anaerobic ammonium oxidizing bacteria can be carried out by batch drainage or batch feeding. The reactor used can be a reactor with a stirring function, such as an IC reactor or a UASB reactor.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) A cross-linked chitosan carrier was prepared by mixing chitosan with heterotrophic bacteria, and then the carrier was modified with polyethyleneimine. The synergistic combination of the three substances provided excellent conditions for the growth and reproduction of anaerobic ammonia-oxidizing bacteria, which was beneficial to the adsorption and reproduction of anaerobic ammonia-oxidizing bacteria, avoided the loss of bacteria, and achieved a long-lasting and stable denitrification effect.

[0025] (2) Using heterotrophic bacteria and chitosan for cross-linking, the use of positively charged chitosan can eliminate the adverse effects of charge on anaerobic ammonium oxidizing bacteria. Heterotrophic bacteria can improve the specific surface area and pore structure of chitosan, and the two play a synergistic role with each other. Moreover, during the growth of anaerobic ammonium oxidizing bacteria, heterotrophic bacteria can degrade dead bacteria, leaving pores that provide a place for anaerobic ammonium oxidizing bacteria. While degrading dead bacteria, carbon dioxide gas is released, which can avoid clogging of the carrier pores and improve mass transfer efficiency.

[0026] (3) The polyethyleneimine used can prevent the influence of oxygen on anaerobic ammonia-oxidizing bacteria, which is beneficial to increasing the reproduction and growth rate of anaerobic ammonia-oxidizing bacteria. DETAILED DESCRIPTION

[0027] The following examples further illustrate the method and effects of the present invention. The examples are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating processes, but the scope of protection of the present invention is not limited to the following examples.

[0028] The experimental methods in the following examples, unless otherwise specified, are all conventional methods in the art. The experimental materials used in the following examples, unless otherwise specified, can all be purchased from biochemical reagent stores.

[0029] In the embodiments of the present invention, the ammonia nitrogen concentration is determined by GB7478-87 "Water quality-Determination of ammonium-Distillation and titration method"; the nitrite nitrogen concentration is determined by GB7493-87 "Water quality-Determination of nitrite nitrogen-Spectrophotometry"; the COD concentration is determined by GB11914-1989 "Water quality-Determination of chemical oxygen demand-Potassium dichromate method"; the abundance value of microorganisms is determined by GB / T40226-2021 "High-throughput sequencing method for metagenomic detection of environmental microorganisms".

[0030] Example 1

[0031] (1) Cultivate xylose-utilizing Candida tropicalis. The xylose concentration was 2 g / L. The culture conditions were: temperature 25°C, pH 6.0-7.0, incubation on a shaker at 200 rpm. After 48 h of culture, the cells were centrifuged at 15,000 rpm, and the supernatant was discarded to retain the bacterial cells.

[0032] (2) Chitosan was added at a mass volume ratio of 2% and acetic acid was added at a volume ratio of 1% to prepare 500 mL of a mixed solution. The bacterial cells prepared in step (1) were added at a mass ratio of bacterial cells to chitosan of 1:2. Then 10 g of CaCO3 nanoparticles, 5 times the volume of edible oil, and 10 mL of Span-80 were added and stirred vigorously. Genipin was added to a final concentration of 20 mM in the aqueous phase, and the mixture was stirred for 24 hours. The precipitate was separated by centrifugation and washed several times with acetone, hot water, and cold water to remove the oil phase and impurities remaining on the surface of the microspheres. The microspheres were dehydrated with acetone twice and the obtained product was dried at room temperature to obtain a cross-linked chitosan carrier with calcium carbonate as the core and bacterial cells and chitosan uniformly wrapped around it.

[0033] (3) The cross-linked chitosan carrier was immersed in a 5% (mass fraction) polyethyleneimine aqueous solution for 60 min, then removed and dried at 35°C for 3 h to obtain a carrier for culturing anaerobic ammonia-oxidizing bacteria. Testing showed that heterotrophic bacteria accounted for 2.5% of the carrier mass, and polyethyleneimine accounted for 5.5% of the carrier mass.

[0034] The laboratory configured 2L of anaerobic ammonia-oxidizing bacteria enrichment culture solution with an ammonia nitrogen concentration of 1000mg / L, a mass ratio of ammonia nitrogen to nitrite nitrogen of 1:1, and a COD concentration of 50mg / , which was divided into two IC reactors with an effective volume of 1L, one of which was filled with the above-mentioned carrier at a ratio of 20% of the effective volume, and the other reactor was a control group without a carrier; the activated sludge rich in anaerobic ammonia-oxidizing bacteria was inoculated into the two reactors according to the sludge concentration after inoculation of 2g / L. The abundance value of anaerobic ammonia-oxidizing bacteria in the activated sludge was tested to be 0.9%. The enrichment culture of anaerobic ammonia-oxidizing bacteria was carried out by batch drainage replacement. The enrichment culture conditions are: temperature 30°C, pH 7.5-8.5, and dissolved oxygen concentration less than 0.2mg / L. After 30 days of culture, the abundance value of microorganisms in the activated sludge was analyzed. The abundance value of anaerobic ammonia-oxidizing bacteria in the reactor inoculated with the carrier reached 12%, which is 5 times that of the uninoculated carrier. Example 2

[0035] (1) Cultivate xylose-utilizing Candida tropicalis. The xylose concentration was 3 g / L. The culture conditions were: temperature 25°C, pH 6.0-7.0, incubation on a shaker at 200 rpm. After 48 h of culture, the cells were centrifuged at 15,000 rpm, and the supernatant was discarded, retaining the bacterial cells.

[0036] (2) Chitosan was added at a mass volume ratio of 2% and acetic acid was added at a volume ratio of 1% to prepare 500 mL of a mixed solution. The bacterial cells prepared in step (1) were added at a mass ratio of bacterial cells to chitosan of 1:1. Then 10 g of CaCO3 nanoparticles, 5 times the volume of edible oil, and 10 mL of Span-80 were added and stirred vigorously. Genipin was added to a final concentration of 20 mM in the aqueous phase, and the mixture was stirred for 24 hours. The mixture was centrifuged and precipitated, and washed several times with acetone, hot water, and cold water to remove the oil phase and impurities remaining on the surface. Finally, the mixture was dehydrated with acetone twice, and the obtained product was dried at room temperature to obtain a cross-linked chitosan carrier with calcium carbonate as the core and bacterial cells and chitosan uniformly wrapped around it.

[0037] (3) The cross-linked chitosan carrier was immersed in a 2% (mass fraction) polyethyleneimine aqueous solution for 30 minutes, then removed and dried at 38°C for 2 hours to obtain a carrier for culturing anaerobic ammonia-oxidizing bacteria. Testing showed that heterotrophic bacteria accounted for 4.5% of the carrier mass, and polyethyleneimine accounted for 3.1% of the carrier mass.

[0038] In the laboratory, 2 L of anaerobic ammonium oxidizing bacteria enrichment culture solution with an ammonia nitrogen concentration of 1000 mg / L, a 1:1 ammonia nitrogen to nitrite nitrogen mass ratio, and a COD concentration of 50 mg / L was prepared and divided into two 1-L IC reactors. One reactor was filled with the aforementioned carrier at a ratio of 20% of the effective volume, and the other served as a control without carrier. Activated sludge enriched with anaerobic ammonium oxidizing bacteria was inoculated into both reactors at a post-inoculation sludge concentration of 2 g / L. The abundance of anaerobic ammonium oxidizing bacteria in the activated sludge was determined to be 0.9%. Anaerobic ammonium oxidizing bacteria enrichment culture was performed using a batch-wise wastewater exchange method. Enrichment culture conditions were: temperature 25°C, pH 7.5-8.5, and dissolved oxygen concentration below 0.2 mg / L. After 30 days of incubation, the abundance of microorganisms in the activated sludge was analyzed. The abundance of anaerobic ammonium oxidizing bacteria in the reactor inoculated with carrier reached 10.9%, 4.7 times that of the reactor without carrier.

[0039] Example 3

[0040] (1) Cultivate xylose-utilizing Candida tropicalis. The xylose concentration was 2 g / L. The culture conditions were: temperature 25°C, pH 6.0-7.0, incubation on a shaker at 200 rpm. After 48 h of culture, the cells were centrifuged at 15,000 rpm, and the supernatant was discarded to retain the bacterial cells.

[0041] (2) Chitosan was added at a mass volume ratio of 2% and acetic acid was added at a volume ratio of 1% to prepare 500 mL of a mixed solution. The bacterial cells prepared in step (1) were added at a mass ratio of bacterial cells to chitosan of 1:3. Then 10 g of CaCO3 nanoparticles, 5 times the volume of edible oil, and 10 mL of Span-80 were added and stirred vigorously. Genipin was added to a final concentration of 20 mM in the aqueous phase, and the mixture was stirred for 24 hours. The precipitate was separated by centrifugation and washed several times with acetone, hot water, and cold water to remove the oil phase and impurities remaining on the surface of the microspheres. Finally, the mixture was dehydrated with acetone twice, and the obtained product was dried at room temperature to obtain a cross-linked chitosan carrier with calcium carbonate as the core and bacterial cells and chitosan uniformly wrapped around it.

[0042] (3) The cross-linked chitosan carrier was immersed in a 10% aqueous solution of polyethyleneimine for 90 minutes, then removed and dried at 25°C for 5 hours to obtain a carrier for culturing anaerobic ammonia-oxidizing bacteria. Testing showed that heterotrophic bacteria accounted for 1.3% of the carrier's mass, and polyethyleneimine accounted for 8.2% of the carrier's mass.

[0043] In the laboratory, 2 L of anaerobic ammonium oxidizing bacteria enrichment culture solution with an ammonia nitrogen concentration of 1000 mg / L, a 1:1 ammonia nitrogen to nitrite nitrogen mass ratio, and a COD concentration of 50 mg / L was prepared and divided into two 1-L IC reactors. One reactor was filled with the aforementioned carrier at a ratio of 20% of the effective volume, and the other served as a control without carrier. Activated sludge enriched with anaerobic ammonium oxidizing bacteria was inoculated into both reactors at a post-inoculation sludge concentration of 2 g / L. The abundance of anaerobic ammonium oxidizing bacteria in the activated sludge was determined to be 0.9%. Anaerobic ammonium oxidizing bacteria enrichment culture was performed using a batch-wise wastewater exchange method. Enrichment culture conditions were: temperature 35°C, pH 7.5-8.5, and dissolved oxygen concentration below 0.2 mg / L. After 30 days of incubation, the abundance of microorganisms in the activated sludge was analyzed. The abundance of anaerobic ammonium oxidizing bacteria in the reactor inoculated with carrier reached 11.1%, 4.5 times that of the reactor without carrier.

[0044] Example 4

[0045] Same as Example 1, except that glucose was used as the organic carbon source and Lactobacillus was used as the heterotrophic bacteria. Testing revealed that the heterotrophic bacteria accounted for 2.3% of the carrier mass, while the polyethyleneimine accounted for 5.2% of the carrier mass. The carrier was used to culture anaerobic ammonium oxidizing bacteria adjacent to the activated sludge. Analysis of the abundance of microorganisms after 30 days of culture revealed that the anaerobic ammonium oxidizing bacteria abundance in the reactor inoculated with the carrier reached 10.7%, 4.4 times that of the uninoculated reactor.

[0046] Example 5

[0047] Same as Example 1, except that sucrose was used as the organic carbon source and Desulfuromonas was used as the heterotrophic bacteria. Testing showed that the heterotrophic bacteria accounted for 2.1% of the carrier mass, and the polyethyleneimine accounted for 5.0% of the carrier mass. The carrier was used to culture anaerobic ammonium-oxidizing bacteria. Analysis of the abundance of microorganisms in the activated sludge after 30 days of culture revealed that the anaerobic ammonium-oxidizing bacteria abundance in the reactor inoculated with the carrier reached 10.4%, 4.2 times that of the uninoculated reactor.

[0048] Example 6

[0049] Same as Example 1, except that the culture conditions were static, with stirring every 60 minutes. Testing revealed that heterotrophic bacteria accounted for 2.4% of the carrier mass, while polyethyleneimine accounted for 5.3% of the carrier mass. The carrier was used to culture anaerobic ammonia-oxidizing bacteria adjacent to the activated sludge. Analysis of microbial abundance in the activated sludge after 30 days of culture revealed that the carrier-inoculated reactor had an anaerobic ammonia-oxidizing bacteria abundance of 10.5%, 4.3 times that of the uninoculated reactor.

[0050] Comparative Example 1

[0051] The same method as Example 1 was used, except that only chitosan was used in the carrier preparation, without bacterial cells. The carrier was used to culture anaerobic ammonium oxidizing bacteria. After 30 days of culture, the abundance of microorganisms in the activated sludge was analyzed. The abundance of anaerobic ammonium oxidizing bacteria in the reactor inoculated with the carrier was 1.5 times that of the reactor without carrier inoculation.

[0052] Comparative Example 2

[0053] The same method as Example 1, except that the cross-linked chitosan carrier was not modified with polyethyleneimine. The carrier was used to culture anaerobic ammonium oxidizing bacteria. After 30 days of culture, the abundance of microorganisms in the activated sludge was analyzed. The abundance of anaerobic ammonium oxidizing bacteria in the reactor inoculated with the carrier was 1.7 times that of the reactor uninoculated with the carrier.

Claims

1. A method for preparing a carrier for culturing anaerobic ammonia-oxidizing bacteria, characterized in that The method comprises the following steps: (1) culturing heterotrophic bacteria that utilize an organic carbon source to the late logarithmic growth phase, and harvesting bacterial cells; the heterotrophic bacteria that utilize an organic carbon source are at least one of yeast, lactic acid bacteria, and sulfate-reducing bacteria that utilize an organic carbon source to reproduce and grow under anaerobic or anoxic conditions; (2) mixing the bacterial cells with chitosan, and then cross-linking with calcium carbonate to prepare a cross-linked chitosan carrier; (3) modifying the cross-linked chitosan carrier with polyethyleneimine, specifically, immersing the cross-linked chitosan carrier in a polyethyleneimine aqueous solution with a mass fraction of 1%-10%, and immersing the carrier in the solution for 30-90 minutes; thereby obtaining a carrier for culturing anaerobic ammonia-oxidizing bacteria.

2. The method according to claim 1, wherein: The yeast is selected from at least one of Candida, Cryptococcus, Hansenula, Pichia, Rhodotorula, Torulopsis or Trichosporon; the lactic acid bacteria is selected from at least one of Lactobacillus, Bifidobacterium and Lactococcus; the sulfate-reducing bacteria is selected from at least one of Desulfomonas and Desulfuromyces.

3. The method according to claim 2, wherein: The yeast is selected from Candida tropicalis.

4. The method according to claim 1, wherein: The organic carbon source in step (1) is determined based on the selected heterotrophic bacteria, and the selected heterotrophic bacteria are conventionally cultured using at least one of carbohydrates, proteins, and organic acids.

5. The method according to claim 4, characterized in that: The organic carbon source in step (1) is at least one of glucose, hexose, xylose, sucrose and starch.

6. The method according to claim 1, wherein: The culture conditions of the heterotrophic bacteria in step (1) are: temperature 20-38°C, pH 6.0-8.5; static culture or shaking culture, stirring every 30-60 minutes for static culture, and a rotation speed of 200-600 r / min for shaking culture.

7. The method according to claim 6, characterized in that: The culture conditions of the heterotrophic bacteria in step (1) are: temperature 20-30°C, pH 6.0-7.

0.

8. The method according to claim 1, wherein: Step (2) The bacterial cells and chitosan are mixed in a mass ratio of 1:1-1:3, and then cross-linked with calcium carbonate to prepare a cross-linked chitosan carrier.

9. The method according to claim 8, characterized in that: The cross-linked chitosan carrier is prepared by a direct cross-linking method, wherein the cross-linking agent used in the direct cross-linking method is at least one of epichlorohydrin, glutaraldehyde, formaldehyde, crown ethers and genipin.

10. The method according to claim 9, characterized in that: The crosslinking agent used in the direct crosslinking method is genipin.

11. The method according to claim 1, 8 or 9, wherein: The preparation of the cross-linked chitosan carrier is specifically as follows: chitosan is added at a mass volume ratio of 2% and acetic acid is added at a volume ratio of 1% to prepare 500 mL of a mixed solution, the bacterial cells prepared in step (1) are added, and then 10 g of CaCO3 nanoparticles are added, 5 times the volume of edible oil is added, and 10 mL of Span-80 is added and stirred vigorously; then genipin is added to a final concentration of 20 mM in the aqueous phase, stirring is continued for 24 hours, the precipitate is separated by centrifugation, and washed several times with acetone, hot water, and cold water to remove the oil phase and impurities remaining on the surface; finally, it is dehydrated with acetone, and the resulting product is dried at room temperature to obtain a cross-linked chitosan carrier with calcium carbonate as the core and bacterial cells and chitosan uniformly wrapped around it.

12. The method according to claim 1, wherein: After the dipping is completed, take out and dry. The drying temperature is 25-40℃ and the drying time is 1-5h.

13. A carrier for culturing anaerobic ammonia-oxidizing bacteria, characterized in that The invention is prepared by the method according to any one of claims 1 to 12.

14. The carrier according to claim 13, characterized in that: In the prepared carrier, heterotrophic bacteria account for 1%-5% of the carrier mass, and polyethyleneimine accounts for 1%-10% of the carrier mass.

15. Use of the carrier prepared according to claim 13, characterized in that The carrier is used to enrich and culture anaerobic ammonia-oxidizing bacteria.

16. The use according to claim 15, characterized in that: The carrier is filled with 20%-40% of the effective volume of the reactor, and anaerobic ammonia oxidizing bacteria are inoculated for enrichment culture. When the abundance value reaches more than 10%, the enrichment culture of anaerobic ammonia oxidizing bacteria is completed.

17. The use according to claim 15 or 16, characterized in that: Anaerobic ammonia oxidizing bacteria are used for enrichment culture, and the inoculation volume ratio is 0.1%-0.5%. Alternatively, activated sludge rich in anaerobic ammonia oxidizing bacteria is used, and the enrichment culture is carried out after inoculation at a sludge concentration of 2-4g / L. After inoculation in the above manner, the abundance value of anaerobic ammonia oxidizing bacteria in the system at the initial stage of enrichment culture is less than 1%.

18. The use according to claim 15 or 16, characterized in that: In the culture solution used in the enrichment culture, the ammonia nitrogen concentration is 500-2000 mg / L, the mass ratio of ammonia nitrogen concentration to nitrite nitrogen is 1:1-1:1.5, and the COD concentration is 50-200 mg / L.

19. The use according to claim 15 or 16, characterized in that: The enrichment culture conditions are: temperature 25-38° C., pH 7.5-8.5, and dissolved oxygen concentration lower than 0.2 mg / L.

20. The use according to claim 15 or 16, characterized in that: The enrichment culture of the anaerobic ammonia oxidizing bacteria adopts a batch drainage replacement or batch feeding method.