Method for treating high ammonia-nitrogen wastewater by using algal-bacterial mixed system and application thereof

Abstract

This invention relates to the field of resource and environmental technology, specifically to a method and application of an algae-bacteria hybrid system for treating high ammonia nitrogen wastewater. By regulating nutrient and operational parameters, a functional community is constructed and maintained, dominated by microalgae and supplemented by bacteria and cyanobacteria, with their relative abundance within a preset optimized range. A clear interspecific interaction network forms within the system: microalgae such as *Nostoc* and *Chlorella* exhibit mutualistic symbiosis, while bacteria such as *Flavobacterium* show antagonistic relationships with these microalgae. Positive and negative feedback mechanisms maintain community balance and system robustness. The three components complement each other: microalgae primarily drive photocatalytic nitrogen and phosphorus removal, cyanobacteria provide nitrogen fixation and stability, and functional bacteria participate in organic matter degradation and nitrogen cycling, achieving synergistic metabolism through material exchange. Microbial ecological analysis confirms that a stable community structure and internal interaction mechanisms are the core of efficient and stable ammonia nitrogen reduction. This technology offers precise regulation, stable operation, and is environmentally friendly and economical, and can be widely applied to the treatment of various types of ammonia nitrogen-polluted water bodies.

Description

Technical Field

[0001] This invention relates to the field of resources and environmental technology, specifically to a method and application of a mixed algae and bacteria system for treating high ammonia nitrogen wastewater. Background Technology

[0002] High ammonia nitrogen wastewater typically refers to industrial or agricultural wastewater with an ammonia nitrogen concentration higher than 500 mg / L. It is characterized by high discharge concentration, large volume, and complex pollution sources, posing a serious threat to the aquatic ecological environment and is a key and challenging issue in the field of water treatment.

[0003] Microalgae, as a type of photosynthetic microorganisms, can synthesize their own biomass using inorganic nutrients such as carbon, nitrogen, and phosphorus in wastewater. While purifying wastewater, they can also realize the resource utilization of biomass, such as producing biodiesel and feed protein, showing broad application prospects. However, in existing technologies, conventional pure algae systems have limited tolerance to high ammonia nitrogen levels, generally only able to treat ammonia nitrogen wastewater below 300–500 mg / L. They are easily inhibited in high ammonia nitrogen environments, resulting in insufficient system stability. Even if a single algal strain with strong tolerance is obtained through separation and purification, it must be carried out under sterile conditions, making practical engineering applications difficult. Furthermore, the lack of synergistic effects with functional bacteria limits the efficiency of nitrogen and phosphorus removal.

[0004] In algae-bacteria symbiotic systems, some bacteria can form a synergistic relationship with microalgae through metabolic complementarity and material exchange, enhancing the system's resistance to ammonia nitrogen shocks. For example, bacteria can degrade algal secretions or convert ammonia nitrogen to reduce ammonia toxicity, while the oxygen released by microalgae photosynthesis can provide reaction conditions for aerobic bacteria, promoting processes such as nitrification. However, complex interactions exist between different bacteria and microalgae, including synergy, antagonism, and competition. If the species ratio and metabolic balance are out of control, the system is prone to collapse. Currently, there are no reports on technologies that achieve efficient and stable treatment of high-ammonia nitrogen wastewater under non-sterilization conditions by precisely controlling the composition, ratio, and key metabolic indicators of algae-bacteria-cyanobacteria. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for treating high ammonia nitrogen wastewater using an algae-bacteria hybrid system. By regulating the composition of the microbial community, the nutrient ratio, and operating parameters, the method achieves efficient and stable treatment of high ammonia nitrogen wastewater, reduces treatment costs, and provides a new technical path for the recycling of high ammonia nitrogen wastewater resources.

[0006] To achieve the above objectives, the technical solution of this application is: a method for treating high ammonia nitrogen wastewater using an algae-bacteria mixed system, comprising: A functional microbial system was constructed, which included microalgae of the genera *Chlorella*, *Micractinium*, *Auxenochlorella*, and *Coccomyxa*, cyanobacteria of the genus *Nostoc*, and bacteria of the genera *Flavobacterium*, *Reyranella*, and *Hydrogenophaga*. Through real-time feedback regulation, the relative abundance of microalgae, bacteria and cyanobacteria in the system is stabilized within the range of 0.4~0.6:0.3~0.5:0.1; Adjust the light-related parameters to achieve a chlorophyll / MLSS ratio of 0.2–0.6:1, a chlorophyll A / chlorophyll B ratio of 1.1–1.7:1, and an extracellular polymeric protein / polysaccharide ratio of 1.8–2.5:1. Through the synergistic regulation of the above steps, the metabolic complementarity and material exchange among microorganisms are enhanced, so that the TN removal rate of high ammonia nitrogen wastewater is ≥85% and the TP removal rate is ≥60%.

[0007] In another implementation of the present invention, the N / P ratio of the high ammonia nitrogen wastewater to be treated is controlled at 5~8:1, and the alkalinity / total phosphorus ratio is controlled at 5~8:1.

[0008] In another implementation of the present invention, the illumination-related parameters include illumination intensity and light-dark ratio, wherein the illumination intensity is 2500~15000lx and the light-dark ratio is 8~12:12~16h.

[0009] In another implementation of the present invention, the alkalinity of the wastewater is adjusted to a range of 5~8:6~7:1 for ammonia nitrogen to total phosphorus.

[0010] In another implementation of the present invention, the initial concentration of Chlorella microalgae is set at 2500 mg / L, and the concentration is maintained at >4500 mg / L after the reactor stabilizes.

[0011] In another implementation of the present invention, the Nostoc cyanobacteria form a stable interaction relationship with Chlorella and Micractinium microalgae.

[0012] In another implementation of the present invention, the Flavobacterium and Reyranella bacteria form a stable antagonistic relationship with the Chlorella and Micractinium microalgae.

[0013] In another implementation of the present invention, the relative abundance of microalgae, bacteria and cyanobacteria is determined by metagenomic analysis and maintained within a set range by real-time monitoring and feedback control.

[0014] The present invention also provides an application of the above-mentioned algae-bacteria mixed system for treating high ammonia nitrogen wastewater in the treatment of high ammonia nitrogen wastewater in fertilizer production, livestock and poultry breeding, food processing or fermentation industries.

[0015] By adopting the above technical solution, the present invention can achieve the following technical effects: 1. This invention constructs a synergistic algae-bacteria hybrid system by precisely screening functional microalgae such as Chlorella and Micractinium, cyanobacteria such as Nostoc, and bacteria such as Flavobacterium. Combined with the fine-tuning of nutrient ratios, light parameters, and metabolic indicators, it can efficiently treat high ammonia nitrogen wastewater with ammonia nitrogen concentration ≤1000mg / L, ultimately achieving a TN removal rate ≥85% and a TP removal rate ≥60%, which is significantly better than conventional pure algae systems and traditional algae-bacteria symbiotic systems without precise control, thus solving the industry pain point of low treatment efficiency for high ammonia nitrogen wastewater.

[0016] 2. This invention uses metagenomic analysis to monitor and regulate the relative abundance of microalgae, bacteria, and cyanobacteria in real time, stabilizing them within an optimized range of 0.4–0.6:0.3–0.5:0.1. Simultaneously, it utilizes the stable interactions between *Nostoc* cyanobacteria and core microalgae, and the stable antagonistic relationships between *Flavobacterium* and other bacteria, constructing a micro-ecosystem of metabolic complementarity and competitive balance, effectively preventing system collapse caused by the overgrowth of a single bacterial community. Furthermore, it can operate stably without sterile conditions, significantly improving the technology's resilience in practical engineering applications and adapting to complex and varied industrial and agricultural high-ammonia nitrogen wastewater treatment scenarios.

[0017] 3. During the process of degrading nitrogen and phosphorus pollutants in wastewater, microalgae convert inorganic nutrients into their own biomass. The resulting microalgal biomass can be further utilized for resource recovery, such as producing biodiesel and feed protein. This achieves a synergistic effect of "reduction" and "resource recovery" of pollutants, breaking the limitations of traditional high ammonia nitrogen wastewater treatment that only focuses on pollutant removal and ignores resource recovery. It significantly reduces the overall cost of wastewater treatment and improves the economic feasibility of the technology.

[0018] 4. This invention clarifies the core technical path of "construction of functional microbial system - regulation of nutrient ratio - maintenance of community balance - optimization of light and metabolic indicators". Each regulation parameter is specific and easy to control, without the need for complex equipment investment and process operation, which facilitates large-scale engineering application and promotion. It is applicable to high ammonia nitrogen wastewater treatment scenarios in multiple industries such as fertilizer production, livestock and poultry breeding, food processing, and fermentation.

[0019] 5. This invention, by regulating key metabolic indicators such as chlorophyll / MLSS, chlorophyll A / chlorophyll B, and extracellular polymeric proteins / polysaccharides, can create a "metabolic window" with the highest pollutant degradation rate, and enhance the exchange of glutamate and cobaltamide between microalgae and bacteria, revealing the mechanism of synergistic and efficient nitrogen and phosphorus removal by algae and bacteria from a physiological perspective. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 A diagram showing the dynamic changes of key water quality parameters and biomass. Figure 2 This is a dynamic change diagram of key physiological and ecological parameters during system operation; Figure 3 A diagram illustrating the composition and interspecific relationships of the system's microbial community during stable operation. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] Example 1: Wastewater preparation: High ammonia nitrogen wastewater was simulated using NH4Cl, Na2CO3, NaHCO3, and K2HPO4. The ammonia nitrogen concentration, alkalinity, and total phosphorus concentration in the simulated wastewater were controlled to be 1000 mg / L, 1000 mg / L, and 140 mg / L, ensuring that the N / P ratio was 7.14:1 and the alkalinity / total phosphorus ratio was 7.14:1, which met the set ratio requirements.

[0025] System construction: 300ml conical flasks were used, each containing 200ml of the simulated wastewater described above. A mixture of microalgae, bacteria, and cyanobacteria was inoculated, with an initial concentration of microalgae of 2500mg / L and a relative abundance of microalgae, bacteria, and cyanobacteria of 0.5:0.4:0.1.

[0026] Operating parameter control: Place the conical flask in a light-temperature incubator, set the light intensity to 2700 lx, the temperature to 26℃, the light-dark ratio to 12:12h, and the pH to be maintained naturally in the range of 7-8.

[0027] Monitoring and testing: Water samples were collected every 2 days, and indicators such as ammonia nitrogen, total phosphorus, and alkalinity were tested according to standard methods; at the end of the experiment, microbial samples were collected for metagenomic sequencing.

[0028] The results show that: Figure 1 As shown, after 20 days of cultivation, the ammonia nitrogen concentration decreased from 1000 mg / L to 147.88 mg / L, with a removal rate of 85.21%; the total phosphorus concentration decreased from 140 mg / L to 53.72 mg / L, with a removal rate of 61.63%; and the alkalinity decreased significantly from 1000 mg / L to 77.91 mg / L, with a removal rate as high as 92.21%. Throughout the reaction cycle, the microalgal biomass in the system showed a continuous growth trend, which was significantly negatively correlated with the decreasing trend of ammonia nitrogen and total phosphorus concentrations, but clearly positively correlated with the pollutant removal rate. In particular, around day 5 of the reaction, the system entered the logarithmic growth phase of rapid microalgal proliferation, which corresponds precisely to the peak of the ammonia nitrogen and total phosphorus removal rates, indicating a close coupling relationship between the pollutant degradation process and microalgal growth and metabolism. The synchronicity and rate coupling characteristics of the above-mentioned pollutant removal and biomass growth confirm that the removal of pollutants in this system mainly depends on the reproduction and metabolic activities of functional microalgae. Microalgae assimilate ammonia nitrogen and phosphate in wastewater through photosynthesis and convert them into their own biomass, thereby achieving efficient removal and resource conversion of pollutants.

[0029] Example 2 This embodiment focuses on the effects of chlorophyll and extracellular polymeric substances (EPS) on the degradation of high ammonia nitrogen pollutants by microalgae.

[0030] Chlorophyll plays a central driving and regulatory role in the degradation of ammonia nitrogen by microalgae. It not only provides energy for microalgal growth but also indicates the health and activity levels of the community, forming the physiological basis for efficient ammonia nitrogen degradation. Extracellular polymers (EPS) are polymers synthesized and secreted by microorganisms during their growth and metabolism. They protect cells from external toxins and provide energy for microorganisms under adverse conditions. Furthermore, the abundant functional groups in EPS can adsorb various chemical substances. Currently, EPS is generally recognized to consist mainly of LB-EPS (loose extracellular polymers) and TB-EPS (tight extracellular polymers).

[0031] The experimental setup was the same as in Example 1. During system operation, the ratio of total chlorophyll to suspended solids in the mixed liquor (Chl / MLSS), the ratio of protein to MLSS in EPS (EPS protein / MLSS), the ratio of polysaccharides to MLSS in EPS (EPS polysaccharides / MLSS), and the ratio of extracellular polymeric protein to polysaccharides were monitored.

[0032] like Figure 2 As shown, on days 4 and 5, Chl / MLSS, EPS protein / MLSS, and EPS polysaccharide / MLSS simultaneously reached their peak values, with values ​​of 1.82, 0.036, and 0.09, respectively. The extracellular polymeric protein / polysaccharide ratio was 2.1:1, which is within the range of metabolic indicators set in this invention. During this stage, the microalgal community is in a period of rapid growth and strong photosynthesis, providing sufficient energy (ATP) for the assimilation of ammonia nitrogen. The large-scale secretion and accumulation of EPS is direct evidence of microbial aggregation, biofilm formation, and enhanced interspecific physical contact and material exchange. The appearance of the synergistic peak of key parameters is not an isolated phenomenon; it clearly indicates that this stage is not only the peak of individual physiological activities of microalgae, but also the period of the most frequent and close interaction between algae and bacteria communities: highly active microalgae produce oxygen and organic matter through photosynthesis, which can stimulate bacterial metabolism and EPS secretion; while the EPS and its metabolites (such as vitamins and signaling molecules) secreted by bacteria can in turn promote the growth and activity of microalgae. This strong positive interaction jointly drives the overall increase in system metabolic flux. Therefore, the rapid decrease in ammonia nitrogen concentration observed on days 4-5 is the external manifestation and direct result of this series of highly synergistic physiological and ecological processes. This result strongly verifies the core of the technical solution of this invention: by regulating the system operating conditions, guiding and optimizing microalgal activity (indicated by Chl / MLSS) and algal-bacterial interaction intensity (indicated by EPS components and ratios), it is possible to directionally create a "metabolic window" with the highest pollutant degradation rate, thereby achieving efficient and rapid removal of high ammonia nitrogen wastewater.

[0033] Example 3: Microbial community composition and interspecies association analysis were performed on the system during the stable operation period in Example 1.

[0034] like Figure 3 As shown, the microbial community structure within the system exhibits good stability, with the relative abundance ratio of microalgae, bacteria, and cyanobacteria being approximately 6:3:1. This ratio falls within the optimization range set in this invention (microalgae:bacteria:cyanobacteria = 0.4~0.6:0.3~0.5:0.1), confirming that through precise control of nutrient conditions and operating parameters, a functional microbial community dominated by microalgae and with bacteria and cyanobacteria as key synergistic units was successfully constructed and maintained.

[0035] Further interspecific correlation network analysis revealed a significant positive correlation between Nostoc cyanobacteria and microalgae such as Chlorella and Micractinium (solid lines in the figure), forming a stable mutualistic symbiotic module. Meanwhile, bacteria such as Flavobacterium and Reyranella showed a negative correlation with the aforementioned Chlorella and Micractinium microalgae (dashed lines in the figure). This antagonistic effect helps maintain the dynamic balance of the community and enhances the robustness of the system.

[0036] The stable community ratio structure and clear interspecific interaction network mentioned above jointly construct a micro-ecosystem with complementary metabolic functions and self-regulation capabilities: microalgae dominate the light-driven nitrogen and phosphorus removal process, cyanobacteria undertake nitrogen fixation and provide support for system stability, and functional bacteria participate in organic matter degradation and nitrogen cycling. The three achieve efficient functional synergy through material exchange.

[0037] In summary, this embodiment demonstrates from a microbial ecology perspective that the algae-bacteria-cyanobacteria complex system constructed by precise regulation in this invention has a stable community structure and internal positive and negative feedback regulation mechanism, which are the core reasons for the system's efficient and stable degradation of ammonia nitrogen. This further verifies the scientific nature and advancement of the technical solution of this invention.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A method for treating high ammonia nitrogen wastewater using an algae-bacteria hybrid system, characterized in that, include: A functional microbial system was constructed, which included microalgae of the genera *Chlorella*, *Micractinium*, *Auxenochlorella*, and *Coccomyxa*, cyanobacteria of the genus *Nostoc*, and bacteria of the genera *Flavobacterium*, *Reyranella*, and *Hydrogenophaga*. Through real-time feedback regulation, the relative abundance of microalgae, bacteria and cyanobacteria in the system is stabilized within the range of 0.4~0.6:0.3~0.5:0.1; Adjust the light-related parameters to achieve a chlorophyll / MLSS ratio of 0.2–0.6:1, a chlorophyll A / chlorophyll B ratio of 1.1–1.7:1, and an extracellular polymeric protein / polysaccharide ratio of 1.8–2.5:

1. Through the synergistic regulation of the above steps, the metabolic complementarity and material exchange among microorganisms are enhanced, so that the TN removal rate of high ammonia nitrogen wastewater is ≥85% and the TP removal rate is ≥60%.

2. The method for treating high ammonia nitrogen wastewater using an algae-bacteria mixed system according to claim 1, characterized in that, The N / P ratio of the high ammonia nitrogen wastewater to be treated should be controlled at 5~8:1, and the alkalinity / total phosphorus ratio should be controlled at 5~8:

1.

3. The method for treating high ammonia nitrogen wastewater using an algae-bacteria mixed system according to claim 1, characterized in that, The illumination-related parameters include illumination intensity and light-dark ratio, wherein the illumination intensity is 2500~15000lx and the light-dark ratio is 8~12:12~16h.

4. The method for treating high ammonia nitrogen wastewater using an algae-bacteria mixed system according to claim 1, characterized in that, Adjust the alkalinity of wastewater to a range of 5~8:6~7:1 for ammonia nitrogen to total phosphorus.

5. The method for treating high ammonia nitrogen wastewater using an algae-bacteria mixed system according to claim 1, characterized in that, The initial concentration of Chlorella microalgae was set at 2500 mg / L, and the concentration was maintained at >4500 mg / L after the reactor stabilized.

6. The method for treating high ammonia nitrogen wastewater using an algae-bacteria mixed system according to claim 1, characterized in that, The Nostoc cyanobacteria form a stable interaction relationship with the Chlorella and Micractinium microalgae.

7. The method for treating high ammonia nitrogen wastewater using an algae-bacteria mixed system according to claim 1, characterized in that, The bacteria of the genera *Flavobacterium* and *Reyranella* form a stable antagonistic relationship with the microalgae of the genera *Chlorella* and *Micractinium*.

8. The method for treating high ammonia nitrogen wastewater using an algae-bacteria mixed system according to claim 1, characterized in that, The relative abundance of microalgae, bacteria, and cyanobacteria was determined by metagenomic analysis and maintained within a set range through real-time monitoring and feedback control.

9. The method for treating high ammonia nitrogen wastewater using the algae-bacteria mixed system according to any one of claims 1-7, applied in the treatment of high ammonia nitrogen wastewater in fertilizer production, livestock and poultry breeding, food processing, or fermentation industries.

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