Algae species with efficient denitrification and high free ammonia tolerance and application of algae species

Abstract

The invention discloses an algae species with both efficient denitrification and high free ammonia tolerance and application thereof. The invention provides an inflatedfruit senna herb which is preserved in China General Microbiological Culture Collection Center (CGMCC) on December 11, 2024, and the preservation number of the inflatedfruit senna herb is CGMCC No.46280. The inflatedfruit senna herb has the advantages that the inflatedfruit senna herb is preserved in the China General Microbiological Culture Collection Center (CGMCC); the inflatedfruit senna provided by the invention has efficient assimilation removal capability on NO3 <->-N, NO2 <->-N and NH4 < + >-N, and has relatively strong tolerance on high-concentration free ammonia (FA). A bacterial-algae symbiotic system is constructed by the inflator and activated sludge, and is coupled with a dynamic membrane process, so that the denitrification efficiency of the nitrogen-containing wastewater can be remarkably improved, the addition of exogenous organic matters, the aeration energy consumption and the N2O emission are reduced, meanwhile, high-value microalgae biomass is obtained, and efficient, stable and resourceful treatment of the nitrogen-containing wastewater is realized.

Description

Technical Field

[0001] This invention relates to the field of microalgae biotechnology, specifically to an algal species that combines efficient denitrification and high tolerance to free ammonia, and its applications. Background Technology

[0002] Large quantities of wastewater with high ammonia nitrogen levels are discharged from agricultural production, livestock and poultry farming, landfilling, coking, and fertilizer industries. This type of wastewater contains ammonia nitrogen (NH4+). + High ammonia nitrogen (HNO3) concentrations often reach hundreds to thousands of milligrams per liter, and are frequently characterized by high organic matter, high phosphorus, high turbidity, and a low carbon-to-nitrogen ratio (C / N). Direct discharge of such wastewater into the environment can easily lead to eutrophication, disrupt the ecological balance, and threaten human health. Currently, the main treatment methods for high ammonia nitrogen wastewater include physicochemical and biological methods. Physicochemical methods, such as stripping, breakpoint chlorination, ion exchange, and chemical precipitation (e.g., struvite), while effective to some extent, generally suffer from high energy consumption, high reagent costs, and the potential for secondary pollution or chemical sludge. Therefore, biological denitrification technology has become the mainstream due to its thorough treatment and relatively low cost.

[0003] Traditional biological nitrogen removal relies on a nitrification-denitrification process. This process first oxidizes ammonia nitrogen to nitrate under aerobic conditions, and then denitrifies the nitrate back to nitrogen gas under anoxic conditions. However, this technology has significant drawbacks when applied to wastewater with high ammonia nitrogen levels: First, the denitrification process requires a large amount of organic carbon source as an electron donor, and wastewater with a low C / N ratio must have additional carbon source added, significantly increasing costs; second, the nitrification process requires strong aeration, resulting in huge energy consumption; and third, high concentrations of ammonia nitrogen and its conversion product, free ammonia (FA), have a strong inhibitory toxicity on nitrifying bacteria and other microorganisms, affecting system stability.

[0004] Algal-microbe symbiotic systems, as a green and low-carbon technology, have attracted much attention in recent years. Pollution removal is achieved through the synergistic effect of microalgae and bacteria: microalgae produce oxygen through photosynthesis, supplying the metabolic needs of aerobic bacteria (such as nitrifying bacteria) and reducing aeration energy consumption; the carbon dioxide produced by bacterial respiration provides an inorganic carbon source for microalgae, promoting their growth and assimilation of nutrients such as nitrogen and phosphorus. Theoretically, this technology can effectively reduce the energy consumption and carbon source requirements of traditional processes, achieving biomass resource recovery. However, directly applying this technology to the treatment of high ammonia nitrogen wastewater still faces three major bottlenecks: a lack of highly efficient functional algae species, as commonly used algae species (such as Chlorella) have poor tolerance to high concentrations of free ammonia, which can damage their photosynthetic system, inhibit growth, and lead to system failure; limited light mass transfer, as the high turbidity and high color of the wastewater severely hinder light penetration, resulting in insufficient light energy acquisition by microalgae, low photosynthetic efficiency, and disruption of the synergistic balance between bacteria and algae; and poor long-term system stability, as the complexity of actual wastewater, water quality fluctuations, and toxin accumulation can easily lead to microbial community imbalance, a sharp decline in treatment performance, and difficulty in achieving long-term stable operation in an engineered manner.

[0005] Therefore, there is an urgent need in this field for a specialized algal strain that can tolerate high levels of free ammonia and has excellent denitrification performance. Summary of the Invention

[0006] This invention provides an algal species that combines high efficiency in denitrification and high tolerance to free ammonia, and its applications.

[0007] In a first aspect, the present invention provides a microalgae, wherein the microalgae is a microalgae. Micractinium little bit The microalgae Micractinium small It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46280.

[0008] The microalgae described above are active in an environment containing free ammonia at a concentration of 20-100 mg / L.

[0009] In a second aspect, the present invention provides a composition comprising the above-mentioned live microalgae, dormant bodies, or cultures of the microalgae.

[0010] The composition as described above includes activated sludge.

[0011] Thirdly, the present invention provides the application of any of the microalgae or compositions described above, wherein the application is selected from any one of A1)-A4): A1) Application in the treatment of nitrogen-containing wastewater; A2) Application in the preparation of apparatus for treating nitrogen-containing wastewater; A3) Application in the prevention and control of eutrophication in water bodies; A4) Application in microalgae biomass production.

[0012] As described above, the nitrogen in the nitrogen-containing wastewater exists in the form of ammonia nitrogen.

[0013] Fourthly, the present invention provides an apparatus for treating nitrogen-containing wastewater, wherein the apparatus is inoculated with any of the aforementioned microalgae or compositions.

[0014] The device described above is a dynamic membrane bioreactor, wherein the biofilm of the dynamic membrane bioreactor is inoculated with the aforementioned microalgae or composition.

[0015] Fifthly, the present invention provides a method for treating nitrogen-containing wastewater, comprising treating nitrogen-containing wastewater using any of the microalgae or compositions described above.

[0016] As described above, the growth conditions for the *Microcystis aeruginosa* include: a light intensity of 8-12 kLux, a light-dark cycle of 12h:12h to 16h:8h, and a temperature of 25-35℃. Further, the growth conditions for the *Microcystis aeruginosa* include: a light intensity of 10 kLux and a light-dark cycle of 14h:10h.

[0017] The microalgae provided by this invention possess broad-spectrum and highly efficient denitrification capabilities (effective against nitrate, nitrite, and ammonia nitrogen) and tolerance to free ammonia, making it suitable for treating pig farm biogas slurry. It exhibits significant synergistic effects, with the resulting algae-bacterial symbiotic system achieving high denitrification efficiency (total nitrogen removal rate > 73%), a complete denitrification pathway, and effectively reducing the emission of the strong greenhouse gas N2O, making it more environmentally friendly. The system demonstrates strong stability; the dynamic membrane acts as a pre-barrier, effectively ensuring the influent water quality of the algae-bacterial symbiotic system and solving the problem of high turbidity and high color inhibiting microalgae, enabling the system to operate stably for a long period. It also possesses great resource potential, obtaining rapidly growing microalgae biomass (biomass can increase 3.2 times) while treating wastewater, which can be used to produce biodiesel, feed, etc., realizing "treating waste with waste and turning waste into treasure." Attached Figure Description

[0018] Figure 1 An optical microscope image of microalgae provided by the present invention; Figure 2 Microalgae ( M.pusillum Chlorella ( C. vulgaris Chlorella ( C. tenuitheca )and Mychonastes afer ( M.afer The simulated wastewater was treated for 8 days, and NH4... + -N concentration change curve; Figure 3 Microalgae ( M.pusillum Chlorella ( C. vulgaris Chlorella ( C. tenuitheca )and Mychonastes afer ( M.afer The simulated wastewater was treated for 8 days, and NO3... - -N concentration change curve; Figure 4 Microalgae ( M.pusillum Chlorella ( C. vulgaris Chlorella ( C. tenuitheca )and Mychonastes afer ( M.afer The simulated wastewater was treated for 8 days, and NO2... - -N concentration change curve; Figure 5 Microalgae ( M.pusillum Chlorella ( C. vulgaris Chlorella ( C. tenuitheca )and Mychonastes afer ( M.afer The total phosphorus concentration change curves were obtained by treating simulated wastewater for 8 days. Figure 6 Microalgae ( M.pusillum Chlorella ( C. vulgaris Chlorella ( C. tenuitheca )and Mychonastes afer ( M.afer ) respectively for those containing NH4 + The curve showing the change in algal biomass content over 8 days after treating simulated wastewater with -N; Figure 7 Microalgae ( M.pusillum Chlorella ( C. vulgaris Chlorella ( C. tenuitheca )and Mychonastes afer ( M.afer ) respectively for those containing NO3 - The curve showing the change in algal biomass content over 8 days after treating simulated wastewater with -N; Figure 8 Microalgae ( M.pusillum Chlorella ( C. vulgaris Chlorella ( C. tenuitheca )and Mychonastes afer ( M.afer ) respectively for those containing NO2 - The curve showing the change in algal biomass content over 8 days after treating simulated wastewater with -N; Figure 9 Microalgae ( M.pusillum ) and Chlorella ( C. vulgaris Growth curves of *Microcystis aeruginosa* in simulated wastewater containing different concentrations of free ammonia (FA); where (a) represents the growth curve of *Microcystis aeruginosa*. M.pusillum (b) is Chlorella ( C. vulgaris ); Figure 10 Microalgae ( M.pusillum ) and Chlorella ( C. vulgaris Wastewater was treated under different light intensities for 12 days, resulting in NH4 levels. + -N concentration change curves and ammonia nitrogen conversion rate curves; where (a) is NH4+. + (b) is the concentration change curve of -N and the ammonia nitrogen conversion rate curve; Figure 11 Microalgae ( M.pusillum ) and Chlorella ( C. vulgaris (a) The nitrogen concentration change curves of wastewater treated under different light cycles; where (a) is NH4+. + The concentration change curve of -N, (b) is NO3. -The concentration change curve of -N, (c) is NO2. - -N concentration change curve; Figure 12 The results of the analysis of the bacterial communities contained in the sequencing batch reactors of the two wastewater treatment systems provided in Example 6. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0021] Example 1, Micrococcus ( Micractinium small Acquisition and preservation of ) Mixed algae samples were collected from the natural waters of Yuweizhou Wetland Park in Nanchang City, Jiangxi Province, China. A single microalgae strain was isolated using a plate coating method. The isolated microalgae was then observed under an optical microscope. The results are as follows: Figure 1 As shown.

[0022] The 18S rDNA of this algal strain was sequenced, and the sequencing results are shown in SEQ ID NO:1. Combined with optical microscopy observation and sequencing results, the algal strain was identified as *Microcystis aeruginosa*. Micractinium small ).

[0023] SEQ ID NO:1 is shown below: 5'--3'.

[0024] The separated microalgae ( Micractinium small It was deposited on December 11, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 46280, hereinafter referred to as Micrococcus micrantha CGMCC No. 46280.

[0025] Example 2: Verification of the nitrogen and phosphorus removal performance of Micrococcus pluvialis CGMCC No. 46280 Micrococcus granulosus CGMCC No. 46280 was inoculated into simulated wastewater and cultured under conditions of 25°C, 10 kLux light intensity, and 1% CO2-containing air circulation. Simultaneously, Chlorella vulgaris was used... Chlorella vulgaris (Purchased from the National Aquatic Germplasm Bank of the Institute of Hydrobiology, Chinese Academy of Sciences, internal number FACHB-2338), Chlorella Coelastrella thin (Separated from the water body of Aixi Lake in Nanchang City) and Mychonastes afer Three microalgae strains (isolated from Aixi Lake in Nanchang City) were used as controls. On days 2, 4, 6, and 8 of cultivation, the NH4+ in the wastewater was measured using spectrophotometry. + -N, NO3 - -N, NO2 - The content of -N and total phosphorus (TP) was calculated and plotted as follows: Figure 2-Figure 5 The content change curve shown is illustrated. According to... Figure 2-Figure 5 It can be seen that *Microcystis aeruginosa* CGMCC No. 46280 exhibits good denitrification performance for various forms of nitrogen, especially after 8 days, *Microcystis aeruginosa* CGMCC No. 46280 shows good denitrification performance for NH4+. + The removal rate of -N reached 70.03%, significantly higher than that of Chlorella (46.95%) under the same conditions; meanwhile, Micrococcus microcarpa CGMCC No.46280 also exhibited good phosphorus removal performance, with phosphorus removal efficiency second only to Chlorella. Chlorella common The figure reached 68.2%.

[0026] Example 3: Validation of the biomass synthesis performance of *Microcystis aeruginosa* CGMCC No. 46280 Micrococcus granulosus CGMCC No. 46280 was inoculated into simulated wastewater containing different nitrogen sources and cultured under conditions of 25°C, 10 kLux light intensity, and 1% CO2 air circulation. Meanwhile, Chlorella vulgaris was used... Chlorella common (Purchased from the National Aquatic Germplasm Bank of the Institute of Hydrobiology, Chinese Academy of Sciences, internal number FACHB-2338), Chlorella Coelastrella tenuitheca (Separated from the water body of Aixi Lake in Nanchang City) and Mychonastes afer Three microalgae strains (isolated from Aixi Lake in Nanchang City) were used as controls. On days 2, 4, 6, and 8 of cultivation, the biomass content of the algal strains under different nitrogen sources was tested. The results are as follows: Figure 6-Figure 8 As shown.

[0027] according to Figure 6-Figure 8It can be seen that, in terms of biomass synthesis, Micrococcus micrantha CGMCC No.46280 has the greatest growth advantage under the condition of ammonia nitrogen as nitrogen source, with the highest biomass content reaching 1.2 mg / L, while it ranks second in biomass content under the condition of nitrate nitrogen and nitrite nitrogen as nitrogen source.

[0028] Example 4: Tolerance test of free ammonia (FA) to the microalgae CGMCC No. 46280 By adjusting the pH to control the concentration of free ammonia (FA) in the wastewater, simulated wastewater with different free ammonia concentrations (20, 40, 60, 80, 100 mg / L) was obtained. Microalgae CGMCC No. 46280 was inoculated into these simulated wastewaters with different free ammonia concentrations and cultured under conditions of 25℃, 10 kLux light intensity, and 1% CO2 air circulation. Its tolerance was verified by testing its OD750. Simultaneously, Chlorella was inoculated into the simulated wastewater. Chlorella vulgaris As a control, the results are as follows. Figure 9 As shown, it can be seen that the initial biomass of *Microcystis aeruginosa* CGMCC No. 46280 decreased under a 100 mg / L FA shock, but it began to recover and grow again after 4 days, demonstrating strong adaptability; while *Chlorella vulgaris*... Chlorella vulgaris Discomfort may occur when FA is greater than 80 mg / L.

[0029] Example 5: Construction of a bacterial-algae symbiotic system and optimization of light conditions Micrococcus micrantha CGMCC No. 46280 was mixed with acclimatized aerobic activated sludge from a pig farm (from the aerobic unit of Plant 2 of Nanchang Muyuan Agricultural and Animal Husbandry Co., Ltd.) at a ratio of 1:5 (dry weight) and placed in a reactor containing simulated pig farm biogas slurry. The denitrification effect was compared under different light intensities (6, 8, 10 kLux) and light-dark cycles (10:14, 12:12, 14:10); Chlorella vulgaris was also included in the mixture. Chlorella vulgaris As a comparison. According to Figure 10-Figure 11 The results show that under a light intensity of 10 kLux and a light-dark cycle of 14h:10h, the ammonia nitrogen conversion rate of the system is the fastest (978.21 mg / (L·h·g)), and the final product is mainly NO3. - -N, with the lowest cumulative N2O content, is determined to be the optimal operating condition.

[0030] Example 6: Long-term operation of a microbial-algae symbiotic biofilm system for treating biogas slurry from a real pig farm Two wastewater treatment systems were constructed: System A consisted of a Dynamic Membrane Bioreactor (DMBR) and a Sequencing Batch Reactor (SBR), with activated sludge inoculated onto the biofilm of the DMBR; System B used the same equipment, but the DMBR biofilm was inoculated with activated sludge and algae, specifically *Mammillaria* CGMCC No. 46280 and *Chlorella*, respectively. Coelastrella tenuitheca and Mychonastes afer The microorganisms in the SBR are the same as those in System A. The activated sludge inoculated on the DMBR biofilm was taken from the aerobic tank of the Qingshanhu Wastewater Treatment Plant in Nanchang City, with a sludge concentration of 4564±50 mg VSS / L and a total nitrogen removal rate of 123.2±8.2 mg TN / (L·d). The main dominant denitrifying bacteria included... Burkholderia (10%) Comamonas (4%) Pseudomonas (3%) Nitrospira (3%) Before the experiment, the activated sludge was acclimatized with simulated biogas slurry. The simulated biogas slurry composition was NH4Cl 1.146 g / L, CH3COONa 1.282 g / L, NaHCO3 1.5 g / L, CaCl2 0.1 g / L, MgSO4·7H2O 0.2 g / L, FeSO4 0.1 g / L, and 1 mL / L of trace element mother liquor was added. The trace element mother liquor composition is as follows: EDTA·2Na 19.11 g / L, H3BO3 0.014 g / L, ZnSO4·7H2O 0.43 g / L, CoCl2·6H2O 0.24 g / L, MnCl2·4H2O 0.99 g / L, CuSO4·5H2O 0.25 g / L, NiCl2·6H2O 0.19 g / L, and NaMoO4·2H2O 0.22 g / L.

[0031] A 5-fold diluted biogas slurry from a real pig farm (TN≈250 mg / L) was input into the DMBR system and filtered through a biofilm before entering the SBR reactor. After 30 days of system operation, the average total nitrogen removal rate in systems A and B was measured. The results showed that system B, inoculated with microalgae CGMCC No. 46280, maintained a stable average total nitrogen removal rate of 73.6%, higher than system A's 56.4%. The microalgae biomass in the post-SBR of system B increased 3.2 times, and the dominant bacterial community was heterotrophic nitrifying-aerobic denitrifying bacteria. It also specifically enriched functional bacteria such as Novosphingobium (side carrier), Acidovorax (IAA producer), and Reyranella (denitrifier), forming a mutually beneficial micro-ecosystem with a more complete nitrogen removal pathway. Figure 12 ).

[0032] After 31-45 days of system operation, the pre-DMBR was shut down, and the diluted biogas slurry was directly pumped into the post-SBR. The nitrogen removal efficiency of System B dropped sharply to 18.3%, and a large number of microalgae died (mortality rate of 82.1%), proving that the pre-biofilm is crucial for maintaining the stable operation of the microbial-algae symbiotic system. In addition, the biofilm in System B had a turbidity removal rate of more than 95%, and its retention effect on color and macromolecular organic matter was better than that of System A. Although its membrane fouling cycle (18 days) was shorter than that of System A (27 days), the membrane flux could be effectively restored after simple backwashing, and the regeneration performance was good.

[0033] Example 7: Long-term operation and energy consumption analysis of a bacterial-algae symbiotic-biofilm system for treating actual high ammonia nitrogen wastewater. Based on the two wastewater treatment systems constructed in Example 6, the SBR unit after System A adopts high-intensity aeration throughout the process (80 mL / L·min), and the SBR unit after System B adopts an intelligent aeration strategy based on the light cycle: during the 14-hour light period, only low-intensity stirring aeration (25 mL / L·min) is turned on; during the 10-hour dark period, in order to maintain the anoxic environment required for denitrification and prevent the system from becoming completely anaerobic, extremely low-intensity intermittent aeration (10 mL / L·min, 10 min on / 20 min off) is used.

[0034] The diluted high-ammonia nitrogen wastewater (TN≈250 mg / L) was fed into the two wastewater treatment systems mentioned above and operated for 30 days. The results showed that the average total nitrogen removal rate of system B remained stable at 73.6%, which was better than the 56.4% of system A. Through monitoring and calculation of the entire system (including all energy-consuming units such as booster pumps, aeration, and stirring), the comprehensive energy consumption per unit volume of water treated by system B was reduced by 40%-50% compared to system A. The majority of the energy saving contribution came from the significant reduction in aeration energy consumption of the post-SBR unit. This not only verifies the effectiveness of photosynthetic oxygen supply by *Microcystis aeruginosa*, but also proves that the entire process of this invention has significant energy-saving benefits and economic advantages in long-term operation.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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; and these 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 the present invention.

Claims

1. Microcystis, characterized by, The micro-bright algae is micro-bright algae. Micractinium pusillum The microalgae Micractinium pusillum It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 46280.

2. The microalgae according to claim 1, characterized in that, The microalgae are active in an environment containing free ammonia at a concentration of 20-100 mg / L.

3. The composition, characterized in that, This includes the live algae, dormant cells, or cultures of the microalgae described in any one of claims 1-2.

4. The composition according to claim 3, characterized in that, The composition includes activated sludge.

5. The application of the microalgae according to any one of claims 1-2 or the composition according to any one of claims 3-4, characterized in that, The application is selected from any one of A1)-A4): A1) Application in the treatment of nitrogen-containing wastewater; A2) Application in the preparation of apparatus for treating nitrogen-containing wastewater; A3) Application in the prevention and control of eutrophication in water bodies; A4) Application in microalgae biomass production.

6. The application according to claim 5, characterized in that, The nitrogen in the nitrogen-containing wastewater exists in the form of ammonia nitrogen.

7. An apparatus for treating nitrogen-containing wastewater, characterized in that, The device is inoculated with the microalgae according to any one of claims 1-2 or the composition according to any one of claims 3-4.

8. The apparatus according to claim 7, characterized in that, The device is a dynamic membrane bioreactor, and the biofilm of the dynamic membrane bioreactor is inoculated with the microalgae as described in any one of claims 1-2 or the composition as described in any one of claims 3-4.

9. A method for treating nitrogen-containing wastewater, characterized in that, This includes treating nitrogen-containing wastewater using the microalgae described in any one of claims 1-2 or the composition described in any one of claims 3-4.

10. The method according to claim 9, characterized in that, The growth conditions for the microalgae include: light intensity of 8-12 kLux, light-dark cycle of 12h:12h to 16h:8h, and temperature of 25-35℃.

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