Immobilized adsorption microspheres and their application in treatment of glutamic acid high-ammonia fermentation wastewater

Abstract

The application belongs to the field of environmental protection technology of biological repair of wastewater, and discloses a kind of immobilized adsorption microspheres, which comprise sea kobe bacteria, Candida guilliermondii and European nitrosomonas bacteria, and are prepared by immobilized embedding technology. The microspheres of the application can effectively repair glutamic acid high ammonia nitrogen fermentation wastewater.

Description

Immobilized adsorption microspheres and their application in the treatment of high ammonia nitrogen fermentation wastewater of glutamic acid Technical Field

[0001] This invention belongs to the field of environmental protection technology of bioremediation of wastewater, and relates to an immobilized adsorption microsphere and its application in the treatment of glutamic acid high ammonia nitrogen fermentation wastewater. Background Technology

[0002] Industrial production, especially in glutamic acid production, generates large amounts of wastewater with high ammonia nitrogen levels. Natural water bodies are suffering from severe nitrogen pollution from this wastewater, and eutrophication is becoming increasingly prominent. Ammonia nitrogen has become a key limiting indicator for total pollution control in my country. Glutamic acid wastewater with high ammonia nitrogen levels has a complex composition. If the ammonia nitrogen in it is discharged directly into the environment without effective treatment, it will lead to the proliferation of algae and other plankton in the water, consuming dissolved oxygen, causing hypoxia, and consequently affecting the survival of aquatic organisms and disrupting the aquatic ecological balance. At the same time, high ammonia nitrogen wastewater can also cause problems such as unpleasant odors and discoloration in water bodies, seriously affecting the utilization value of water resources and the quality of the surrounding environment.

[0003] Wastewater denitrification methods mainly include physicochemical methods, advanced oxidation methods, and biological methods.

[0004] Biological denitrification is an effective technology for removing ammonia nitrogen from wastewater through microbial metabolic processes. Its main advantages include less secondary pollution, high treatment efficiency, simple operation, and relatively low cost. Biological denitrification can effectively remove ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen, reducing the chemical reagents and energy consumption required by traditional chemical treatment methods. This technology also features low sludge production and good environmental adaptability, allowing for stable operation under varying wastewater concentrations and flow rates. Furthermore, biological denitrification can achieve high nitrogen removal efficiency by controlling and optimizing the growth and metabolic processes of microorganisms, demonstrating good economic viability and sustainability.

[0005] Traditional biological denitrification of wastewater, based on nitrification and denitrification processes, is widely used in wastewater treatment and has achieved good denitrification results. However, with the increasing stringency of wastewater discharge standards, these processes have also revealed their shortcomings. For example, when treating wastewater with high ammonia nitrogen and high organic matter content, they suffer from drawbacks such as long process flow, high oxygen consumption, insufficient carbon source for denitrification, and low denitrification efficiency.

[0006] Chinese patent CN118026423A discloses a method for treating glutamic acid fermentation wastewater, which includes the following steps: mixing Bacillus subtilis and Bacillus flavus to obtain a compound bacterial agent, adding it to the glutamic acid fermentation wastewater, and treating for more than 24 hours. This compound bacterial agent has a reasonable formulation and good water purification effect, but it has poor tolerance to high-concentration ammonia nitrogen wastewater (above 1000 mg / L), and its activity is somewhat reduced.

[0007] Chinese patent CN105833832A discloses a biochemical agent for treating glutamic acid fermentation wastewater, comprising a physical adsorbent and a composite microbial agent. The composite microbial agent comprises the following raw materials in parts by weight: 6-8 parts of *Azotobacter chroococcus*, 4-6 parts of *Rhodococcus*, 4-6 parts of *Bacillus amyloliquefaciens*, 3-5 parts of *Clostridium papillosum*, 3-5 parts of *Penicillium ochraceus*, 3-4 parts of *Pseudomonas*, and 2-3 parts of *Bacillus cereus*. It contains multiple microorganisms with excellent degradation capabilities for recalcitrant pollutants. However, the above biochemical agent contains too many strains, making compatibility difficult, increasing the possibility of strain contamination, and also increasing the cost of cultivating the bacteria.

[0008] In 2024, Ding Jiale et al. published "A Study on the Effect of Yeast and its Combination with Bacillus on Removing Ammonia Nitrogen from Foaming Manure Sewage". They used a combination of yeast and Bacillus, which can be used for the treatment of ammonia nitrogen in foaming manure sewage. The combination is more efficient in removing ammonia nitrogen than yeast alone. However, the ammonia nitrogen removal rate is reduced to less than 50%, which cannot meet the needs of practical applications.

[0009] Traditional biological technologies for treating ammonia nitrogen have many drawbacks. To address these, researchers have developed several novel biological nitrogen removal processes with low energy consumption and high efficiency, the most typical being anaerobic ammonia oxidation (ANAO). However, ANAO bacteria have low growth rates, require demanding cultivation conditions, and are sensitive to environmental conditions. High organic matter content or the presence of toxic or harmful substances in wastewater can interfere with their metabolic processes, reducing nitrogen removal efficiency. Furthermore, they are difficult to isolate and purify, significantly limiting their applicability. Compared to the bacteria involved in traditional nitrification-denitrification processes, ANAO bacteria require a much longer time to reach sufficient numbers and activity to achieve efficient nitrogen removal, greatly restricting the rapid application and promotion of this technology in practical engineering. Coupled ammonification and nitrification-denitrification processes can leverage their respective advantages to improve nitrogen removal efficiency. However, this coupled process also faces challenges. First, competition between strains can easily occur, leading to system instability or reduced efficiency. Second, when the nitrate nitrogen concentration in the system is too high, it is difficult to maintain a balance between different strains, affecting the overall treatment effect.

[0010] Solving the above problems requires selecting and optimizing suitable strain combinations, optimizing the synergistic effect of the bacterial community, and ensuring that different bacterial communities can coexist well in the system, thereby achieving efficient ammonia nitrogen removal. Summary of the Invention

[0011] The purpose of this invention is to address the shortcomings of traditional processes by providing an immobilized adsorption microsphere and its application in the treatment of high ammonia nitrogen fermentation wastewater from glutamic acid.

[0012] The present invention is achieved through the following technical solution.

[0013] An immobilized adsorbent microsphere comprising Betella hygrophytes, Candida pruriens, and Nitrosomonas cerevisiae.

[0014] Furthermore, the immobilized adsorption microspheres are prepared according to the following steps:

[0015] Step 1) Take polyvinyl alcohol, dissolve it in hot water, then add sodium alginate and stir to obtain a uniform and transparent mixed solution. Cool it down to 30°C, then add modified diatomaceous earth and Helicobacter erythrorhizon bacterial solution, stir evenly, and then add saturated boric acid solution containing calcium chloride dropwise to crosslink and obtain immobilized microspheres.

[0016] Step 2) Mix the Candida albicans bacterial solution and Nitrosomonas molluscioides bacterial solution, then mix with zeolite powder, add to the immobilized microspheres from Step 1), stir, and finally dry and granulate at low temperature to obtain the final product.

[0017] Furthermore, the immobilized adsorption microspheres are prepared according to the following steps:

[0018] Step 1) Take 50g of polyvinyl alcohol and dissolve it in 1L of hot water at 90℃. Then add 10g of sodium alginate and stir at 300rpm for 5min to obtain a homogeneous and transparent mixed solution. Cool down to 30℃, then add 4g of modified diatomaceous earth and 30ml of Hericium erinaceus bacterial solution and stir evenly. Then add a saturated boric acid solution containing 50g / L calcium chloride and crosslink at 4℃ for 6 hours to obtain immobilized microspheres with a particle size of 3-4mm.

[0019] Step 2) Mix the Candida albicans bacterial solution and Nitrosomonas nitrosamine bacterial solution in equal volume ratio, then mix them with zeolite powder in a 1:1 volume-to-mass ratio, let stand for 30 minutes, then add them to an equal weight of immobilized microspheres, stir at 200 rpm for 3 minutes, and finally dry and granulate at low temperature, controlling the particle size to 10-20 mm and the water content to 6%, thus obtaining immobilized adsorption microspheres.

[0020] Preferably, the modified diatomaceous earth is obtained according to the following process:

[0021] Diatomaceous earth was soaked in a 5% hydrochloric acid solution and stirred for 30 minutes. The diatomaceous earth was then collected by centrifugation and calcined at 300°C for 2 hours under a nitrogen atmosphere. After removal and natural cooling, modified diatomaceous earth was obtained.

[0022] More preferably, the method for preparing the *Hydrocotyle hygrophytes* bacterial culture includes: picking colonies from a solid agar medium and inoculating them into a seed culture medium, culturing at 28°C for 48 hours to obtain a seed culture; transferring the seed culture at 10% of the inoculation amount to a fermentation medium, and culturing at 30°C until the bacterial culture has an OD600 of 3.0.

[0023] More preferably, the method for preparing the Candida pruriens bacterial culture includes: activating Candida pruriens, then inoculating it into a seed culture medium and culturing it at 30°C and 200 rpm for 24 h to obtain a seed culture; transferring the seed culture to a fermentation medium at an inoculation rate of 15% and culturing it at 30°C until the bacterial concentration OD600 is 4.5.

[0024] More preferably, the method for preparing the European Nitrosomonas bacterial solution includes: picking European Nitrosomonas colonies and inoculating them into a seed culture medium, culturing them in a shaker at 30°C and 100 rpm in the dark for 48 hours, and then inoculating them into a fermentation culture medium at an inoculation rate of 15% and culturing them until the OD600 is 2.0.

[0025] As another aspect of the present invention, the application of immobilized adsorption microspheres in the remediation of glutamic acid high ammonia nitrogen fermentation wastewater is also involved.

[0026] The beneficial effects achieved by this invention mainly include, but are not limited to, the following points.

[0027] Glutamic acid fermentation wastewater is characterized by high ammonia nitrogen, high salinity, and high organic matter content, making it difficult to treat with a single microorganism. Excessive organic matter content in the wastewater can interfere with the nitrification-denitrification metabolic process, reducing nitrogen removal efficiency. This invention utilizes a double-layer encapsulation technology, encapsulating *Betterella hygroscopica*, a bacterium capable of denitrification under anaerobic or anoxic conditions, inside microspheres. *Candida przewalskii* and *Nitrosomonas cerevisiae* are then encapsulated secondary over a secondary layer, primarily attached to the outside of the microspheres. *Candida przewalskii* can ferment and utilize organic matter as an energy source under aerobic conditions, rapidly reducing the organic matter content in the wastewater. *Nitrosomonas cerevisiae*, as an aerobic nitrifying bacterium, exhibits strong nitrification capabilities under low pH and high salinity conditions, rapidly degrading ammonia nitrogen in the wastewater.

[0028] The three strains of this invention exhibit good symbiotic synergistic effects, and the prepared immobilized microspheres have stable performance, realizing the wastewater treatment process of organic matter-nitrification-denitrification. It has broad application prospects in the treatment of wastewater containing complex organic matter.

[0029] This invention utilizes diatomaceous earth that has undergone acid treatment and calcination to increase its specific surface area, pore volume, and pore size, thereby enhancing the adhesion ability of microorganisms. The secondary encapsulation uses zeolite powder, whose pore structure consists of interconnected tetrahedrons and hexahedrons with uniform and moderate pore size. This structure allows the zeolite to rapidly adsorb substances such as ammonia nitrogen from wastewater, thus improving the degradation efficiency of microorganisms. Attached Figure Description

[0030] Figure 1: Degradation rate of various pollutants by the microspheres of Example 1 in 48-96 hours.

[0031] Figure 2: Effect of different amounts of microspheres on pollutant degradation rate.

[0032] Figure 3: Degradation rate of each pollutant by the microspheres of Example 1 and Comparative Examples 1-4 after 96 hours. Detailed Implementation

[0033] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be described more clearly and completely below in conjunction with specific embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0034] The bacterial strains used in this invention are all conventional strains in the art and are commercially available. The bacterial culture is not limited to the cultivation method described in the specific embodiments; it can also be obtained through other conventional cultivation methods. Specifically, the strains used in the embodiments of this invention are *Candida pruriens* ATCC42402, *Nitrosomonas cerevisiae* ATCC25978, and *Betterlia hygroscopica* ATCC25374. It should be noted that the invention is not limited to the specific strains mentioned above; other strains of the same genus can also be used to implement the technical solution of this invention.

[0035] Example 1

[0036] An immobilized adsorbent microsphere comprising Betella hyacinthii ATCC25374, Candida pruriens ATCC42402, and Nitrosomonas cerevisiae ATCC25978.

[0037] Specifically, it is prepared according to the following method:

[0038] Diatomaceous earth was soaked in a 5% hydrochloric acid solution and stirred for 30 minutes. The diatomaceous earth was then collected by centrifugation and calcined at 300°C for 2 hours under a nitrogen atmosphere. After removal and natural cooling, modified diatomaceous earth was obtained.

[0039] Take 50g of polyvinyl alcohol and dissolve it in 1L of hot water at 90℃. Then add 10g of sodium alginate and stir at 300rpm for 5min to obtain a homogeneous and transparent mixed solution. Cool down to 30℃, then add 4g of modified diatomaceous earth and 30ml of Hericium erinaceus bacterial solution and stir evenly. Then add dropwise a saturated boric acid solution containing 50g / L calcium chloride and crosslink at 4℃ for 6 hours to obtain immobilized microspheres with a particle size of 3-4mm.

[0040] The *Candida przewalskii* and *Nitrosomonas flavonoids* cultures were mixed in equal volume ratios, then mixed with zeolite powder at a 1:1 volume-to-mass ratio (ml:g). The mixture was allowed to stand for 30 minutes, then added to an equal weight of immobilized microspheres. The mixture was stirred at 200 rpm for 3 minutes, and finally dried and granulated at low temperature to control the particle size to 10-20 mm and the moisture content to 6% (mass ratio), thus obtaining the immobilized adsorption microspheres.

[0041] The above bacterial solution was prepared by the following method:

[0042] *Hydrocarpus hainanensis* bacterial culture: Colonies were picked from solid agar medium and inoculated into seed culture medium, and cultured at 28°C for 48 h to obtain seed culture. The seed culture was then transferred to fermentation medium at 10% (v / v) and cultured at 30°C until an OD600 of 3.0 was achieved. Seed culture medium (g / L): tryptone 15, yeast extract 3, glucose 2, sodium chloride 10, sodium nitrate 10. Fermentation medium (g / L): glucose 5, tryptone 10, sodium chloride 10, sodium nitrate 10, sodium sulfate 1, magnesium sulfate heptahydrate 0.5.

[0043] Candida pruriens culture: Activated Candida pruriens was inoculated into a seed culture medium and cultured at 30℃ and 200 rpm for 24 h to obtain the yeast seed culture. The seed culture was then transferred to the fermentation medium at 15% (v / v) and cultured at 30℃ until the OD600 concentration reached 4.5. Seed culture medium (g / L): yeast extract 10, peptone 10, glucose 15. Fermentation medium (g / L): yeast extract 15, peptone 20, glucose 50, potassium dihydrogen phosphate 2, dipotassium hydrogen phosphate 2, magnesium sulfate heptahydrate 1, sodium chloride 1.

[0044] *Nitrosomonas cerevisiae* inoculum: Colonies of *Nitrosomonas cerevisiae* were picked and inoculated into seed culture medium. The culture was incubated at 30°C and 100 rpm in the dark for 48 hours. Then, 15% of the inoculum was added to the fermentation medium and cultured until the OD600 reached 2.0. Seed culture medium (g / L): Ammonium sulfate 5 g, potassium dihydrogen phosphate 2 g, sodium carbonate 0.5 g, magnesium sulfate heptahydrate 0.1 g, manganese sulfate monohydrate 0.03 g. Fermentation medium (g / L): Ammonium sulfate 10 g, potassium nitrate 5 g, sodium chloride 3 g, potassium dihydrogen phosphate 2 g, magnesium sulfate heptahydrate 0.2 g.

[0045] Example 2

[0046] The glutamic acid fermentation wastewater from the production workshop was analyzed. The main pollutants were: organic matter (COD) 4890 mg / L, ammonia nitrogen 1826 mg / L, and sulfate 751 mg / L. 20 g of the immobilized adsorption microspheres prepared in Example 1 were added per m³ of wastewater, and the treatment time ranged from 48 to 96 hours. As shown in Figure 1, with increasing treatment time, COD, ammonia nitrogen, and sulfate rapidly degraded. After 96 hours, over 90% of each pollutant was degraded, with ammonia nitrogen reaching a degradation rate of 98%.

[0047] Example 3

[0048] The experimental sample used was the same batch of wastewater as in Example 2. Immobilized adsorption microspheres prepared in Example 1 were added at concentrations ranging from 0 to 40 g per m³ of wastewater, with five addition concentrations: 0, 5, 10, 20, and 40 g. The treatment time was 96 hours. As shown in Figure 2, with increasing addition of immobilized adsorption microspheres, the degradation rates of COD, ammonia nitrogen, and sulfate significantly increased, reaching a peak at an addition of 20 g / m³. Further increasing the addition to 40 g / m³ did not result in significant changes to any of the indicators.

[0049] Comparative Example 1

[0050] Same as Example 1, except that no Candida albicans culture was added.

[0051] Comparative Example 2:

[0052] Same as Example 1, except that no European Nitromonas bacterial solution was added.

[0053] Comparative Example 3

[0054] The microspheres are prepared using different processes, as follows:

[0055] The bacterial cultures of Betella hygrophytes, Candida przewalskii, and Nitrosomonas molluscioides were mixed in equal volume ratios, and then mixed with zeolite powder in a 1:1 volume-to-mass ratio (ml:g). The mixture was then dried at low temperature.

[0056] Comparative Example 4

[0057] The microspheres are prepared using different processes, as follows:

[0058] Take 50g of polyvinyl alcohol and dissolve it in 1L of hot water at 90℃. Then add 10g of sodium alginate and stir at 300rpm for 5min to obtain a homogeneous and transparent mixed solution. Then cool it down to 30℃ and add 4g of modified diatomaceous earth and 30ml of mixed bacterial solution (Hydrocarpus natans, Candida albicans, and Nitrosomonas cerevisiae are mixed in equal volume ratio). Stir evenly and then add a saturated boric acid solution containing 50g / L calcium chloride. Crosslink at 4℃ for 6 hours to form immobilized microspheres with a particle size of 3-4mm. Dry at low temperature.

[0059] Example 4

[0060] Using the same batch of wastewater and the same treatment method as in Example 2, the pollutant degradation rate of Example 1 and Comparative Examples 1-4 was verified after 96 hours of treatment.

[0061] As shown in Figure 3, the method of bacterial encapsulation and the combination of bacterial strains have a significant impact on wastewater treatment. Comparative Examples 1 and 2, which used two different bacterial strains, only achieved ammonia nitrogen degradation rates of approximately 60-70%, failing to meet wastewater treatment standards. This demonstrates the crucial importance of the combination of *Candida przewalskii* and *Nitrosomonas cerevisiae*. Both strains primarily attach to the exterior of the microspheres. *Candida przewalskii* can ferment and utilize organic matter as an energy source under aerobic conditions, rapidly reducing the organic matter content in wastewater and avoiding damage to nitrifying bacteria from high concentrations of organic matter. *Nitrosomonas cerevisiae*, as an aerobic nitrifying bacterium, still exhibits strong nitrification capabilities under low pH and high salinity conditions, rapidly degrading ammonia nitrogen in wastewater. Comparative Examples 3-4 used a combination of three strains, but the pollutant degradation effect was still not good. The main reason may be that Candida pruriens and Nitromonas cerevisiae have reduced organic matter degradation and nitrification activity under anaerobic or hypoxic conditions, while Betella hygroscopica cannot exert its denitrification performance under aerobic conditions, thus reducing the ammonia nitrogen degradation efficiency.

[0062] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. An application of immobilized adsorption microspheres in the remediation of high ammonia nitrogen fermentation wastewater containing glutamic acid, characterized in that, The application includes: treating high-ammonia nitrogen fermentation wastewater containing glutamic acid with immobilized adsorption microspheres for more than 48 hours, wherein the amount of immobilized adsorption microspheres added is 5-40 g / m³, and the immobilized adsorption microspheres include *Bebrillariae hygroscopica*, *Candida pruriens*, and *Nitrosomonas cerevisiae*; the immobilized adsorption microspheres are prepared according to the following steps: Step 1) Take 50 g of polyvinyl alcohol, dissolve it in 1 L of hot water at 90 °C, then add 10 g of sodium alginate, stir at 300 rpm for 5 min to obtain a homogeneous and transparent mixed solution, cool to 30 °C, and then add 4 g of modified... Diatomaceous earth and 30 ml of *Hydrocotyle spp.* bacterial solution were stirred evenly, and then a saturated boric acid solution containing 50 g / L calcium chloride was added dropwise. Crosslinking was carried out at 4°C for 6 hours to obtain immobilized microspheres with a particle size of 3-4 mm. Step 2) *Candida spp.* bacterial solution and *Nitrosomonas spp.* bacterial solution were mixed in equal volume ratio, and then mixed with zeolite powder in a volume-to-mass ratio of 1:

1. The mixture was allowed to stand for 30 min, and then added to an equal weight of immobilized microspheres. The mixture was stirred at 200 rpm for 3 min, and finally dried and granulated at low temperature to control the particle size to 10-20 mm and the water content to 6%, thus obtaining immobilized adsorption microspheres.

2. The application according to claim 1, characterized in that, The modified diatomaceous earth is obtained by the following process: diatomaceous earth is soaked in a 5% hydrochloric acid solution and stirred for 30 minutes. Then, the diatomaceous earth is collected by centrifugation and calcined at 300°C for 2 hours under a nitrogen atmosphere. After removal and natural cooling, the modified diatomaceous earth is obtained.

3. The application according to claim 1, characterized in that, The method for preparing the *Hydrocotyle hygrophytes* bacterial suspension includes: picking colonies from a solid agar medium and inoculating them into a seed culture medium, culturing at 28°C for 48 hours to obtain a seed culture; transferring the seed culture at 10% of the inoculation amount to a fermentation medium, and culturing at 30°C until the OD600 is 3.

0.

4. The application according to claim 1, characterized in that, The method for preparing the Candida pruriens bacterial culture includes: activating Candida pruriens, then inoculating it into a seed culture medium and culturing it at 30°C and 200 rpm for 24 hours to obtain the seed culture; transferring the seed culture to the fermentation medium at an inoculation rate of 15% and culturing it at 30°C until the bacterial concentration OD600 is 4.

5.

5. The application according to claim 1, characterized in that, The preparation method of the European Nitrosomonas bacterial culture includes: picking European Nitrosomonas colonies and inoculating them into a seed culture medium, culturing them in a shaker at 30°C and 100 rpm in the dark for 48 hours, and then inoculating them into a fermentation culture medium at an inoculation rate of 15% and culturing them until the OD600 is 2.0.

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