Strain for synergistically strengthening removal of cyclamate, bacteria-algae symbiotic system and application

Abstract

The application discloses a strain for synergistically strengthening removal of cyclamate, a bacteria-algae symbiotic system and application, the strain is a nelsonia strain NCU J5, which is classified and named as Niallia nealsonii ​ NCU J5, and the preservation number is CCTCC NO: M 20251657. The nelsonia strain NCU J5 has a growth characteristic of taking cyclamate as the only carbon source; the chlorella algae strain has the ability of assimilative utilization of cyclamate; the strain and the algae strain realize metabolic cooperation through interface interaction, form a synergistic degradation mechanism, the whole system is easy to construct, can also reduce operation cost, avoid secondary pollution to the environment, and is suitable for purification treatment of water bodies such as industrial wastewater, domestic sewage, livestock and poultry breeding wastewater containing cyclamate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a strain, a bacterial-algal symbiotic system, and its application that synergistically enhances the removal of cyclamate. Background Technology

[0002] Artificial sweeteners (ASs), as sugar substitutes, are widely used in the pharmaceutical, food, and livestock industries. However, due to their prevalent detection in rivers, groundwater, livestock wastewater, and sludge, and their potential ecological risks, they are considered emerging pollutants. Wastewater treatment plants and livestock farms struggle to effectively remove ASs, making them a major source of AS emissions. ASs entering the aquatic environment can pose potential ecological risks to aquatic ecosystems. Studies have shown that ASs can promote the cross-species transmission and generation of antibiotic resistance genes (ARGs) in pure cultures of bacteria, mouse gut microbiota, and anaerobic digestion systems. Therefore, developing efficient and cost-effective AS removal technologies is crucial for mitigating their potential ecological risks.

[0003] Currently, the removal of persistent organic pollutants (ASs) mainly relies on physical methods (such as flocculation and adsorption), chemical methods (such as ozone oxidation and chlorination), and biological methods (such as activated sludge). However, studies have found that due to the structural stability and degradation resistance of ASs such as acesulfame potassium, sucralose, sodium saccharin, and sodium cyclohexylsulfamate (CYC), physical and chemical methods have limited effectiveness in removing these pollutants. The persistent detection of ASs in the aquatic environment and their ecological risks highlight the urgent need to upgrade wastewater treatment technologies.

[0004] Existing studies have confirmed that microalgae can remove sulfonamide antibiotics, microplastics, and perfluorooctanoic acid (PFOA) through biosorption, bioaccumulation, and biodegradation. They also possess the ability to capture carbon and produce high-value-added byproducts, making them applicable to pollutant removal and resource utilization. Furthermore, the synergistic effect between microalgae and bacteria helps alleviate the sensitivity of single-strain systems to persistent organic matter and stress conditions, thereby improving the removal efficiency of recalcitrant pollutants and the production capacity of high-value byproducts. By enhancing photosynthetic activity, promoting microalgae growth, accelerating the co-absorption of nutrients and pollutants, and improving the tolerance of microorganisms to pollution stress, algae-bacteria symbiotic systems outperform single-microalgae systems in removing pollutants such as tetracyclines, sulfadiazine, and sulfamethoxazole. Since CYCs (cyclamides) and sulfonamide antibiotics share a similar sulfonyl group structure, algae-bacteria symbiotic systems can also efficiently remove CYCs. Summary of the Invention

[0005] The purpose of this invention is to address the problem of long-term and large-scale residues of artificial sweeteners, especially cyclamate, in industrial wastewater, and the low efficiency and secondary pollution caused by conventional treatment methods. This invention proposes a strain, a bacterial-algae symbiotic system, and its application for synergistically enhanced removal of cyclamate. This bacterial-algae symbiotic system can completely remove 30 mg / L of cyclamate within 12 days. The entire system is easy to construct, reduces operating costs, and avoids secondary pollution to the environment. It is suitable for the purification and treatment of water bodies containing cyclamate, such as industrial wastewater, domestic sewage, and livestock and poultry breeding wastewater.

[0006] The technical solution of the present invention is as follows:

[0007] A strain for synergistically enhancing the removal of cyclamate, namely *Nilsenia* NCU J5, has been deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251657, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on July 21, 2025. The suggested classification name is... Niallia nealsonii NCU J5. This strain was autonomously isolated and screened from a water sample in a pond near the Engineering Research Center for Biomass Conversion of the Ministry of Education at Nanchang University, Nanchang City, Jiangxi Province in March 2024.

[0008] Preferably, the 16S rRNA gene sequence of the Nelsenia bacterium NCU J5 is shown in SEQ ID NO: 1.

[0009] Preferably, single colonies of Nelsenia bacillus NCU J5 are obtained by dilution and plating, and single colonies are picked and placed in 50 mL of LB medium and preserved in a refrigerator at 4°C.

[0010] Preferably, the culture conditions for Nelsenia bacillus NCU J5 are as follows: cultured in LB medium at a temperature of 28°C and shaken at 200 r / min.

[0011] Preferably, the main biological characteristics of the Nelsenia bacterium NCU J5 are: the colonies are grayish-white, round, smooth, and sticky.

[0012] The present invention also provides a microbial preparation comprising the aforementioned Nelsenia bacterium NCU J5.

[0013] The present invention also provides a cyclamate degrading agent, comprising the aforementioned Nilssonia bacteria NCU J5.

[0014] This invention also provides a symbiotic system of bacteria and algae, comprising a *Scenedesmus* species and *Nilsenia* NCU J5, with a mass ratio of (10-20):1. *Nilsenia* NCU J5 exhibits growth characteristics with cyclamate as its sole carbon source; the *Scenedesmus* species possesses the ability to assimilate and utilize cyclamate; the bacterial strain and algae achieve metabolic cooperation through interfacial interaction, forming a synergistic degradation mechanism.

[0015] Preferably, the *Scenedesmus* strain is *Scenedesmus oblique* (…). Oblique scene ) or Scenedesmus tetra-tailed ( Scenedesmus quadricaud More preferably, the *Scenedesmus* strain is *Scenedesmus oblique*. S. oblique .

[0016] This invention also provides a method for constructing a fungal-algae symbiotic system, comprising the following steps:

[0017] (1) Culture the Scenedesmus genus algae into a Scenedesmus genus algae inoculation stock solution;

[0018] (2) Nelsenia bacillus NCU J5 was cultured to prepare Nelsenia bacillus NCU J5 inoculation stock solution;

[0019] (3) Mix the inoculation stock solution of Scenedesmus genus and the inoculation stock solution of Nilsneria NCU J5, and then inoculate it in the target environment.

[0020] Preferably, the concentrations of the inoculation mother liquor of the Scenedesmus genus and the inoculation mother liquor of Nilsneria NCU J5 are both 0.5~5g / mL, and the wet weight ratio of the two is (10~20):1.

[0021] Preferably, the method for constructing the algal-microbe symbiotic system includes the following steps:

[0022] (1) The *Scenedesmus* strain was inoculated into BG11 medium and cultured under light for 24 h, with the culture temperature maintained at 26±1℃ and the light intensity at 2.50±0.1 Klux. The culture was expanded 2-3 times, followed by centrifugation at 5000 rpm for 5 min. The supernatant was discarded, and the mixture was washed 3 times with BG11 medium. The algal sludge was collected. A certain amount of BG11 medium was then added to obtain a 0.5-5 g / mL *Scenedesmus* strain inoculation stock solution.

[0023] (2) Inoculate a single colony of Nelsenia bacillus NCU J5 into LB medium and expand it to the logarithmic growth phase. Culture at 28℃ and 200r / min on a shaker. Centrifuge the bacterial solution under these conditions at 5000rpm for 5min, discard the supernatant, add BG11 medium and wash 3 times, and collect the bacterial sludge. Add a certain amount of BG11 medium to obtain a 0.5~5g / mL Nelsenia bacillus NCU J5 inoculation stock solution;

[0024] (3) Mix the inoculation mother solution of the Scenedesmus strain obtained in step (1) and the inoculation mother solution of Nelsenia bacillus NCU J5 obtained in step (2) at a wet weight ratio of (10~20):1, and then inoculate it in the target environment.

[0025] This invention also provides an application of Nilsonia bacterium NCU J5, Scenedesmus strains, or a bacterium-algae symbiotic system in wastewater treatment.

[0026] Preferably, the wastewater is wastewater containing cyclamate, and the concentration of cyclamate in the wastewater is 1~200 mg / L.

[0027] This invention also provides the application of Nilssonia bacterium NCU J5, Scenedesmus strains, or a bacterial-algal symbiotic system in the degradation of cyclamate.

[0028] Preferably, the inoculum amount of Nilsonia bacterium NCU J5, Scenedesmus strain, or bacterium-algae symbiotic system is 0.9~1.2 g / L.

[0029] Preferred conditions for wastewater treatment or degradation of cyclamate include: light intensity of 20,000 to 28,000 Lux, light cycle of 12 to 24 h / d, temperature of 24 to 28 °C, culture period of 10 to 15 days, and mechanical shaking and mixing every 6 to 12 hours during the treatment.

[0030] Preferably, the process of degradation of cyclamate by the algae-bacteria symbiotic system is as follows:

[0031] 1) The strain (Nilsenia bacillus NCU J5) and the algal strain of Scenedesmus accumulate cyclamate through extracellular adsorption;

[0032] 2) The bacterial strain and algae absorb and transport cyclamate into the cell, and remove cyclamate mainly through intracellular assimilation and degradation. The algae promote the absorption and metabolism of organic sulfur in the bacterial strain, thereby enhancing its assimilation and degradation of cyclamate. The bacterial strain promotes the heterotrophic metabolism of the algae through secretion of indoleacetic acid and other pathways, thereby enhancing its assimilation and degradation of cyclamate.

[0033] 3) Compared to single bacteria and single algae, the bacterial-algae symbiotic system mainly enhances the removal of CYC by strengthening its own biosorption and assimilation degradation of CYC. The biosorption and assimilation degradation pathways are enhanced by 129% and 69%, respectively.

[0034] 4) Ultimately, cyclamate is 100% degraded and removed, effectively preventing the generation and spread of resistance genes that may result from its entry into the ecological environment.

[0035] The beneficial effects of this invention are:

[0036] 1. The Nilssonia bacterium NCU J5 of the present invention has the growth characteristics of using cyclamate as the sole carbon source, and can metabolize the artificial sweetener cyclamate, thereby achieving the purpose of degrading cyclamate.

[0037] 2. The *Scenedesmus* strain of the present invention has the ability to assimilate and utilize cyclamate, and can achieve heterotrophic metabolic assimilation of cyclamate, thereby effectively removing cyclamate.

[0038] 3. The combination of bacterial and algal species in this invention exhibits synergy: The symbiotic combination of Nilsneria nigra NCU J5 and Scenedesmus algae constructs a bacterial-algae symbiotic system. The synergistic degradation mechanism of the bacterial-algae system is as follows: metabolic cooperation is achieved through the bacterial-algae symbiotic interface, which enhances the absorption and assimilation of organic sulfur by bacteria, thereby promoting the utilization and removal of cyclamate, a sulfur-containing organic compound. At the same time, the bacteria enhance the secretion of substances such as indoleacetic acid, which promote the growth and reproduction of Scenedesmus algae, thus promoting the heterotrophic metabolic assimilation of cyclamate by Scenedesmus algae. The combined effect of the two promotes the biosorption and assimilation (biodegradation) of cyclamate by the bacterial-algae symbiotic system, which are enhanced by 129% and 69%, respectively, breaking the limitations of traditional physicochemical methods. Its assimilation and degradation effect is increased by 35.91% and 81.30% compared with single algae and single bacteria, respectively. Its high-efficiency treatment performance is as follows: under the condition of 10:1 algae-bacteria inoculation ratio, the removal efficiency of the bacterial-algae symbiotic system is increased by 81.9% and 25.6% respectively compared with single bacteria and single algae, and 30 mg / L of cyclamate can be completely removed within 12 days.

[0039] 4. The main degradation pathways of CYC treated by the algae-bacterial symbiotic system constructed in this invention are biosorption and assimilation degradation, avoiding the formation of the toxic byproduct cyclohexylamine and reducing the risk of secondary pollution. This algae-bacterial symbiotic system demonstrated excellent CYC treatment performance in actual wastewater such as municipal sewage and anaerobic digestion broth from pig manure. The removal rates of CYC in municipal wastewater and anaerobic fermentation broth from pig manure reached 27% and 21%, respectively, overcoming the limitations of existing technologies in complex water quality. Simultaneously, transcriptome analysis confirmed the synergistic effect of enhanced heterotrophic metabolism in *Scenedesmus obliquus* and the uptake and utilization of organic sulfur by *Nilsneria*, ensuring the stability and repeatability of the removal efficiency. Furthermore, the obtained biomass can be used as a high-value byproduct (such as biofuel, feed, etc.), achieving a dual function of "wastewater treatment - resource recovery". Attached Figure Description

[0040] Figure 1 The colony morphology of N. nielsoniae NCU J5;

[0041] Figure 2 Phylogenetic tree diagram of N. nielsensis NCU J5 (J5);

[0042] Figure 3The content of live and dead cells under different inoculation ratios of Scenedesmus obliquus and Nilsonia;

[0043] Figure 4 Ultra-high performance liquid chromatography-time-of-flight tandem mass spectrometry (UPLC-TOF-MS / MS) spectral analysis of CYC standards (a) and the derivatization mechanism of cyclohexylamine (b).

[0044] Figure 5 for S. oblique Group, NCU J5 group and S. obliquus- The ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS / MS) analysis chromatograms of CYC in the NCU J5 symbiotic system group (a) and the quantitative results of cyclohexylamine in each group (b);

[0045] Figure 6 To reveal different cellular conditions through pathway enrichment analysis (a: S. oblique -NCU J5 symbiotic system and S. oblique Single culture comparison; b~c: S. oblique - Comparison of NCU J5 symbiotic system and NCU J5 single culture). Detailed Implementation

[0046] The technical solutions in 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. 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.

[0047] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0048] The *Scenedesmus obliquus* of this invention was purchased from the Freshwater Algae Gene Bank of the National Aquatic Germplasm Bank, Institute of Hydrobiology, Chinese Academy of Sciences, at No. 7, East Hunan Road, Luojia Mountain, Wuhan, with accession number FACHB-12, and classified as follows: Oblique scene The following examples are abbreviated as follows: S. oblique .

[0049] The *Scenedesmus tetracaudus* of this invention was purchased from the Freshwater Algae Gene Bank of the National Aquatic Germplasm Bank, Institute of Hydrobiology, Chinese Academy of Sciences, at No. 7, East Hunan Road, Luojia Mountain, Wuhan, with accession number FACHB-44, and classified as follows: Scenedesmus quadricaud The following examples are abbreviated as follows: S. quadricauda .

[0050] The BG11 culture medium formula of the present invention is as follows: sodium nitrate (NaNO3) 1500 mg / L, dipotassium hydrogen phosphate (K2HPO4) 40.00 mg / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 75.00 mg / L, calcium chloride dihydrate (CaCl2·2H2O) 36.00 mg / L, citric acid (C6H8O7) 6.00 mg / L, ferric ammonium citrate (C6H8FeNO7) 6.00 mg / L, disodium ethylenediaminetetraacetate (EDTA·2Na) 1.00 mg / L, sodium carbonate (Na2CO3) 0.02 g / L, and trace element A5 1.00 mL / L.

[0051] The A5 micronutrient formula is as follows: boric acid (H3BO3) 2.86 g / L, manganese chloride tetrahydrate (MnCl2·4H2O) 1.81 g / L, zinc sulfate (ZnSO4) 0.222 g / L, sodium molybdate (Na2MoO4) 0.39 g / L, copper sulfate pentahydrate (CuSO4·5H2O) 0.079 g / L, and cobalt nitrate hexahydrate (Co(NO3)2·6H2O) 0.0494 g / L. The BG 11 expansion medium is based on the BG 11 medium with the additional additions of ammonium chloride (NH4Cl) 1.0 g / L, sodium acetate (CH3COONa) 1.0 g / L, dipotassium hydrogen phosphate (K2HPO4) 0.252 g / L, and potassium dihydrogen phosphate (KH2PO4) 0.25 g / L.

[0052] The LB medium formulation of this invention is as follows: 10.0 g / L peptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride, and pH adjusted to 7.0 using 1 mol / L NaOH.

[0053] Example 1: Acquisition and Identification of Nelsenia bacillus NCU J5

[0054] Water samples were collected in March 2024 from the Engineering Research Center for Biomass Conversion of the Ministry of Education at Nanchang University, Nanchang City, Jiangxi Province. 50 mL of water sample was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the bottom solids were added to 50 mL of LB medium for enrichment. The medium was incubated at 28℃ and 200 rpm for 48 h with shaking. After enrichment, the sample was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the sample was washed three times with deionized water. The resulting bacterial cells were inoculated into 500 mL of the screening medium shown in Table 1 (using cyclamate as the sole carbon source) for further culture. Acclimation: During the screening period, the bacterial culture was expanded using the screening medium every 5 days for 30 days. After acclimation, a second screening of the bacterial strains was performed. 20 μL of the bacterial solution was spread and streaked on a screening medium plate to select the desired strains. Figure 1A cyclamate-degrading strain was screened out. Its colonies were grayish-white, round, smooth, and sticky.

[0055] Table 1 Screening Culture Media

[0056]

[0057] The bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (D1600, Solarbio Science & Technology Co., Ltd., Beijing) according to the manufacturer's instructions. This extracted genomic DNA was then used as a template in the remaining components and amplified in a PCR instrument. The PCR reaction system and amplification program are shown in Tables 2 and 3.

[0058] Table 2 PCR reaction system

[0059]

[0060] Table 3 PCR Amplification Procedure

[0061]

[0062] After PCR amplification, bright bands were detected in agarose gel electrophoresis. The amplified products were sequenced using the Applied Biosystems™ 3730XL first-generation sequencing platform. The results were compared with the NCBI database, and sequences with high homology were selected. A phylogenetic tree of the target strain was constructed using MEGAX64 software (Neighbor-Joining method). Figure 2 As shown.

[0063] 16S rRNA sequencing showed that this strain was related to Niallia nealsonii strain DSM 15077 is in the same branch, and the Bootstrap support rate is 99%, indicating that the two are highly similar in phylogenetic relationship (see Figure 2 Based on 16S rRNA gene sequence similarity analysis (>99%), it was identified as *Nilsenia*. Niallia nealsonii ), named Niallia nealsonii NCU J5. Its 16S rRNA gene sequence (sequence listing as SEQ ID NO: 1) has been submitted to the NCBI database, registration number PV422585. This strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251657, deposit date July 21, 2025, address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province.

[0064] Example 2: Comparison of the effects of Nilssonia bacillus NCU J5 treatment with different concentrations of cyclamate (CYC)

[0065] Single colonies of NCU J5 were picked from LB solid medium and inoculated into 50 mL of LB medium. The culture temperature was 28℃ and the culture was shaken at 200 r / min. After three expansion cultures, the colonies were centrifuged at 5000 rpm for 5 min to collect NCU J5 bacteria that had reached the logarithmic growth phase. The collected bacterial sludge was washed three times with sterile BG11 medium. The collected bacterial sludge was then inoculated into 2 L of sterilized BG11 medium (121℃, 20 min) at a wet weight of 1 g / L. After thorough mixing, the culture was dispensed into 12 500 mL Erlenmeyer flasks, with each flask containing 245 mL. To evaluate the treatment effect of NCU J5 on CYC wastewater, different concentrations of CYC (0 mg / L, 10 mg / L, 50 mg / L, 200 mg / L) were added to dispensed conical flasks, forming three experimental groups (CYC10, CYC50, CYC200) and one blank control group, with three replicates per group. The experiment lasted 12 days, with a temperature of 26±1℃ and a light intensity of 2.50±0.10 Klux. The flasks were continuously illuminated for 24 hours, and the water was shaken evenly three times daily during the incubation period. CYC concentration was monitored every 6 days, and the CYC removal rate was calculated. The CYC removal rate (%) was calculated as 100% - 100% × CYC residue rate, where CYC residue rate = C t / C0,C t C0 is the current concentration of CYC, and C0 is the initial concentration of CYC.

[0066] The results are shown in Table 4. Nelsenia bacillus NCU J5 has a certain CYC removal effect in CYC wastewater of different concentrations. Among them, NCU J5 has a significant removal effect in CYC wastewater of 50 mg / L compared with 10 mg / L and 200 mg / L, and the removal rate reaches 21.700% on the 12th day.

[0067] Table 4. Removal rate (%) of Nelsonia bacillus NCU J5 in different concentrations of CYC

[0068]

[0069] Example 3: Comparison of the effects of two types of Scenedesmus on different concentrations of cyclamate.

[0070] Scenedesmus obliqueis was picked from the solid culture medium containing the stored algal strains. S. oblique FACHB-12) and Scenedesmus tetracoccinea ( S. quadricaudaThe FACHB-44 algal strain was first inoculated into 50 mL of BG11 medium and cultured at 26±1℃, 2.50±0.10 Klux light, and 150 r / min shaking for 3 days. After three expansion cultures, the microalgae were centrifuged at 5000 rpm for 5 min to collect the algae that had reached the logarithmic growth phase. The resulting algal sludge was washed three times with sterile BG11 medium to remove residual nutrients. The collected algal sludge was then inoculated into 4 L of sterilized (121℃, 20 min) BG11 medium at a wet inoculation rate of 1.02 g / L. After thorough mixing, the mixture was dispensed into 15 500 mL Erlenmeyer flasks, each containing 245 mL. To evaluate the treatment effects of two *Scenedesmus* species on cyclamate wastewater, different concentrations of cyclamate (0 mg / L, 1 mg / L, 10 mg / L, 50 mg / L, 200 mg / L) were added to dispensed Erlenmeyer flasks, forming four experimental groups (CYC1, CYC10, CYC50, CYC200) and one control group, with three replicates per group. The experiment lasted 12 days, with a temperature of 26±1℃ and a light intensity of 2.50±0.10 Klux. The flasks were continuously illuminated for 24 hours, and the liquid was shaken evenly three times daily during the incubation period. The CYC removal rate was measured after 12 days.

[0071] The results are shown in Table 5. In terms of CYC removal rate, both *Scenedesmus tetracauda* and *Scenedesmus obliquus* showed certain CYC removal effects in wastewater with different concentrations of CYC. Among them, *Scenedesmus tetracauda* had limited removal capacity, while *Scenedesmus obliquus* was significantly better, achieving a removal rate of 90.970% after 12 days at low concentrations. Moreover, the removal efficiency of both *Scenedesmus tetracauda* and *Scenedesmus obliquus* was more stable at low concentrations.

[0072] Table 5. Removal rates (%) of two types of Scenedesmus at different concentrations of CYC

[0073]

[0074] Example 4: Construction of the bacterial-algal symbiosis and screening of algal strains

[0075] The results of Example 3 show that *Scenedesmus obliquus* S. oblique The ability to degrade cyclamate is significantly higher than that of Scenedesmus tetracauda. S. quadricauda Here, we constructed symbiotic relationships between *Scenedesmus obliquus* and NCU J5, and between *Scenedesmus tetracoccoides* and NCU J5, respectively, to determine the optimal algal strain for the symbiotic relationship.

[0076] (1) Scenedesmus obliquely S. oblique Scenedesmus tetra-tailed S. quadricaudaAlgal strains were inoculated into 50 mL of BG11 liquid medium and cultured at 26±1℃, 2.50±0.10 Klux light, and 150 r / min on a shaker for 3 days. The culture was expanded 2-3 times until the biomass reached 0.7-0.8 g / L (wet weight). The algal culture under these conditions was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the algal sludge was washed three times with BG11 medium. A certain amount of BG11 medium was then added to obtain 1 g / mL *Scenedesmus obliquus* inoculation stock solution and 1 g / mL *Scenedesmus tetraculosus* inoculation stock solution, respectively.

[0077] (2) Inoculate a single colony of Nelsenia bacillus NCU J5 into LB medium and culture it to the logarithmic growth phase at 28℃ and 200 r / min on a shaker. After 2-3 expansion cultures, centrifuge the bacterial solution under these conditions at 5000 rpm for 5 min, discard the supernatant, add BG11 medium and wash 3 times, and collect the bacterial sludge. Add a certain amount of BG11 medium to obtain a 1 g / mL NCU J5 inoculation stock solution.

[0078] (3) The *Scenedesmus obliquus* inoculation stock solution and *Scenedesmus tetrapoda* inoculation stock solution obtained in step (1) were respectively inoculated with the *NCU J5* inoculation stock solution obtained in step (2) at a wet weight ratio of 10:1 into sterilized BG11 medium containing 30 mg / L CYC. The total wet weight of inoculation was 1.08 g / L. They were labeled as follows: S. oblique -NCU J5、 S. quadricauda- NCU J5 was used as a control, with 5 mL of sterilized BG11 medium added. Separate cultures of *Scenedesmus obliquus*, *Scenedesmus tetracauda*, and NCU J5 containing only *N. obliquus* were also set up as controls. The experimental period was 12 days, the temperature was 26±1℃, the light intensity was 2.50±0.10 Klux, and the culture was continuously illuminated for 24 h. During the culture, the cells were shaken evenly three times a day at regular intervals. Microalgal growth (biomass), photosynthetic activity (Fv / Fm), cell viability (SOD), and CYC residue (C) were assessed. t / C0,C t Using indicators such as the current concentration of CYC and the initial concentration of CYC (C0 is the initial concentration of CYC), we screened out the superior bacterial and algal combinations that are highly effective in removing CYC.

[0079] The results are shown in Tables 6-9. *Scenedesmus obliquus* S. oblique Symbiosis with NCU J5 showed significant advantages. Specifically, in terms of biomass, the biomass of all bacterial-algal symbiotic groups was higher in the later stage (day 12) than that of the single-algae control group. S. oblique - The NCU J5 group reached 1.417 g / L, which is... S. quadricauda -1.5 times that of NCU J5, Scenedesmus obliqueis S. oblique Synergistically with N. nielsoniae NCU J5, it significantly promotes biomass accumulation; in terms of photosynthetic activity (Fv / Fm), S. oblique -NCU J5 group was consistently lower S. oblique However, maintaining a certain level, algal-bacterial symbiosis can effectively maintain photosynthetic performance; S. quadricauda , S. quadricauda - The Fv / Fm ratio decreased in the later stages of NCU J5 treatment, possibly due to environmental stress. Regarding oxidative stress levels, the NCU J5 treatment group had the highest SOD activity, indicating higher environmental stress, with a slight decrease in oxidative stress levels later. Microalgae-bacteria co-culture enhanced environmental stress recovery; overall, the symbiotic group showed lower oxidative stress levels compared to the single-algae group. S. oblique - NCU J5 group had the lowest SOD accumulation; in terms of CYC removal rate... S. obliquus- The NCU J5 group was significantly better than other groups, with CYC completely eliminated by day 12. S. four-tailed -NCU J5 had a CYC residue rate of 58%, and NCU J5 and Scenedesmus obliquus... S. oblique Co-culturing showed the best removal effect on CYC, therefore it was chosen. S. obliquus- NCU J5 is the dominant bacterial and algal combination.

[0080] Table 6 Biomass (g / L) of each group

[0081]

[0082] Table 7 Fv / Fm for each group

[0083]

[0084] Table 8. SOD (U / g) for each group

[0085]

[0086] Table 9 CYC Residue Rates in Each Group

[0087]

[0088] Example 5: Under different bacterial and algal inoculation ratios S. obliquus- Comparison of the degradation effects of NCU J5 algae-bacterial symbiotic system on cyclamate

[0089] 1 g / mL Scenedesmus obliquus inoculation stock solution and 1 g / mL Nilssonia NCUJ5 inoculation stock solution were prepared according to the method in Example 4.

[0090] Seven experimental groups were set up: a single algae culture group of *Scenedesmus obliquus*, a single bacterium culture group of NCU J5, and five different inoculation ratios. S. obliquus- The NCU J5 algae-bacterial symbiotic system was cultured in groups of 100:1, 10:1, 1:1, 1:10, and 1:100 (algae:bacteria). All groups were inoculated into BG11 medium supplemented with 30 mg / L CYC, with a total inoculum of 1.08 g / L (wet weight), and each group was replicated in triplicate. The experiment was conducted under continuous light at 26±1℃ and 2.50±0.10 Klux for 24 h, with the medium shaken three times daily during the culture period. The experiment lasted 12 days, and microbial growth (biomass), photosynthetic activity (Fv / Fm), cell viability (SOD), and the removal and residue of CYC were monitored to screen for the optimal inoculum ratio.

[0091] As shown in Tables 10-13, the proportion of high algae... S. obliquus- The biomass of the NCU J5 groups (100:1, 10:1, 1:1) on day 12 was 1.528 g / L~1.638 g / L, significantly higher than other groups, indicating that adding suitable bacteria can enhance the algal-bacterial interaction. With the increase of the Nelsenia bacterium NCU J5 ratio, the Fv / Fm ratio showed a decreasing trend. The introduction of bacteria promoted the metabolic transition of microalgae from autotrophic to mixed culture. Only under the 100:1 and 10:1 conditions did the Fv / Fm recover on day 12; excessively low algal ratios can damage the photosynthetic system. Combined with the SOD content data on days 6 and 12 (see Table 12), it can be concluded that the SOD activity was relatively lower in the 1:1 and 10:1 algal-bacterial ratios, indicating less oxidative damage in the symbiotic system. Through further analysis... S. obliquus- NCU J5 symbiotic system culture group and S. oblique Fluorescence staining analysis of live / dead cells in single algal culture groups ( Figure 3 ,in S. obliquus- Two images were extracted from the NCU J5 group at the same time for each type of algae / bacteria inoculation ratio. S. oblique (One image was captured at the same time in the group). Regardless of the inoculation ratio, the addition of N. nigra NCU J5 promoted the formation of bacterial-algal flocs. Among them, the bacterial-algal flocs were tightly aggregated and had the best flocculation effect at an algae-to-bacteria ratio of 10:1.

[0092] Nilsonia nigra (NCU J5) promotes flocculation by secreting EPS and regulating the pH of the co-culture system to release specific metabolites (such as indoleacetic acid). This process enhances the stability of the co-culture system and generates a positive feedback effect on CYC degradation. Comparing the CYC removal efficiency at different inoculation ratios, the 10:1 algae-bacteria ratio showed the best performance, completely removing 30 mg / L of CYC by day 12. Compared with the single-bacteria and single-algae groups, this improved the CYC removal efficiency by 81.9% and 25.6%, respectively. The ability of bacteria or microalgae alone to degrade CYC was limited. Overall, the 10:1 algae-bacteria ratio demonstrated superior performance in biomass accumulation, Fv / Fm, cell activity, algae-bacteria flocculation, and CYC removal rate, balancing efficiency and stability.

[0093] Table 10 Biomass (g / L) of each group under different inoculation ratios

[0094]

[0095] Table 11 Fv / fm of each group under different inoculation ratios

[0096]

[0097] Table 12 SOD content (U / g) in each group under different inoculation ratios

[0098]

[0099] Table 13 CYC residual rate in each group under different inoculation ratios

[0100]

[0101] Example 6 S. obliquus- Mechanism analysis of cyclamate degradation in the NCU J5 algal symbiotic system

[0102] This embodiment evaluates the abiotic removal efficiency of CYC (photolysis and hydrolysis). CYC was inoculated into sterile BG11 medium at a concentration of 30 mg / L, and a photolysis + hydrolysis group and a hydrolysis group were set up. The photolysis + hydrolysis group was continuously cultured for 24 h at a light intensity of 2.5 ± 0.1 Klux and 26 ± 1℃, while the hydrolysis group was wrapped in aluminum foil and cultured in the dark under the same conditions. The experimental period was 12 days, and the CYC content in the water samples was monitored every 2 days. Simultaneously, a single-strain NCU J5 group was also set up. S. oblique Single algae group and S. obliqueThe NCU J5 algae-bacterial symbiotic system (10:1, algae:bacterium) was used to investigate the metabolic pathways of CYC in single-bacterial, single-algae, and algae-bacterial co-culture systems. The CYC inoculum was 30 mg / L. The microbial inoculum, culture conditions, and experimental period were consistent with those in Example 4. Samples were taken on days 4, 8, and 12 to determine the CYC content in the water, extracellular polymeric substances (EPS), and intracellular organic matter (IPS). After the experiment, samples were collected from the single-algae group, single-bacterial group, and... S. oblique The supernatant of the culture medium, IPS, and EPS of the NCU J5 algal symbiotic system were analyzed using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS / MS) to analyze the degradation products of CYC and to infer its degradation pathway.

[0103] The results are shown in Tables 14 and 15. Table 14 shows that the CYC removal rate did not change significantly under both photolysis + hydrolysis and hydrolysis conditions, thus excluding the interference of environmental factors. Table 15 shows that CYC removal in the NCU J5 group mainly relied on biodegradation, with biodegradation accounting for 16.103% on day 12. S. oblique Groups and S. oblique - In the NCU J5 algae-bacterial symbiotic system, CYC removal was mainly achieved through biodegradation and biosorption, with biodegradation being the primary removal pathway. (Day 12) S. oblique Groups and S. oblique The biodegradation rates of -NCU J5 increased to 61.499% and 97.407%, respectively. However, S. oblique The group could not completely remove CYC at 30 mg / L, with a residual rate of 21.8% on day 12. However, when the algae-bacteria inoculation ratio was 10:1, CYC was completely removed within 12 days.

[0104] Through contribution metric analysis, compared S. oblique Cultured in single-cell NCU J5 culture. S. oblique The enhancement rates of CYC removal efficiency under the bioadsorption and biodegradation mechanisms of the -NCU J5 symbiotic system were 129% and 69%, respectively, indicating that... S. obliqueIt can synergistically enhance the removal of CYC with NCU J5. The contribution quantification analysis and the enhancement rate calculation method are described in reference 1 [Yan H, Lu R, Liu Y, et al. Development of microalgae-bacteria symbiosis system for enhanced treatment of biogas slurry[J]. Bioresource Technology,2022, 354: 127187.] and reference 2 [Lu R, Yan H, Liu Y, et al. Enhancement of nutrients recovery and cell metabolism in piggery anaerobic digestate by the co-cultivation of indigenous microalgae and bacteria[J]. Journal of CleanerProduction, 2022, 375: 134193.].

[0105] Further clarify CYC in S. oblique Single culture, NCU J5 single culture and S. oblique Degradation pathways in the NCU J5 co-culture system, results are shown below. Figure 4 , Figure 5 CYC exists in aqueous solution in ionic form, with hydrogen atoms on cyclohexane replaced by aminosulfonic acid groups. UPLC-QTOF-MS / MS was used to analyze the potential transformation products resulting from the degradation of CYC, such as... Figure 4 As shown in a, CYC (m / z 178.0545) breaks down into the characteristic ionic fragment O3S. - (m / z 79.9548), the CYC of each treatment group after O3S degradation - Characteristic ion fragments disappear ( Figure 5 (a) Studies have shown that certain human gut bacteria may metabolize CYC into cyclohexylamine, a more toxic carcinogen. Therefore, dansyl chloride derivatization-UPLC-MS / MS was used for specific detection of cyclohexylamine. Its derivatization mechanism is as follows: Figure 4 As shown in b, however, no cyclohexylamine was generated in any of the treatment groups. Figure 5 b) indicates that *Scenedesmus obliqueus* S. oblique Nilsenia NCU J5 and S. oblique-NCU J5 can directly absorb and utilize CYC, and does not convert it into the toxic intermediate product cyclohexylamine, but directly mineralizes it into bacterial and algal biomass components, thus reducing ecological risks.

[0106] Table 14 CYC Residue Rate of Each Group

[0107]

[0108] Table 15 NCU J5, S. oblique and S. oblique -CYC removal rate of NCU J5 (%)

[0109]

[0110] Example 7 NCU J5, S. oblique and S. oblique Transcriptomic analysis of microorganisms after NCU J5 treatment of CYC-containing wastewater

[0111] To elucidate the synergistic degradation mechanism of CYC driven by microbial communities, Nilsonia bacillus NCU J5 and Scenedesmus obliquus were investigated. S. oblique and Scenedesmus obliqueis-Nilsenia S. oblique Transcriptome sequencing analysis was performed on the NCU J5 symbiotic system, and the specific steps are as follows:

[0112] Scenedesmus oblique S. oblique Nilsenia NCU J5 and S. obliquus- The NCU J5 symbiotic system (10:1 inoculum ratio, algae:bacteria, containing 30 mg / L cyclamate) was cultured for 6 days as described in Example 4. Collected samples were stored at -80°C, and total RNA was subsequently extracted using TRIzol® Reagent (Thermo Fisher Scientific, USA). Transcriptome sequencing, assembly, and analysis were performed by Shanghai Meiji Biotechnology Co., Ltd., including total RNA extraction, rRNA deletion, mRNA fragmentation, reverse transcription, and adapter ligation.

[0113] Comparative transcriptome analysis was performed in the following steps: (1) Based on the reference genome, prokaryotes were analyzed and compared. S. oblique- (2) Analysis and comparison of NCU J5 symbiotic system and NCU J5 monoculture system based on non-reference genomes of eukaryotes. S. obliquus- NCU J5 symbiotic system and S. oblique Single training system.

[0114] The results are as follows Figure 6 As shown, relative to S. oblique NCU J5 single culture, S. obliqueIn the NCU J5 symbiotic system, pathways related to heterotrophic metabolism in microalgae are upregulated. These include pathways that enhance circulating electron flow, regulate energy distribution within the photosynthetic system, and maintain basal ATP synthesis, such as the upregulation of ferrore-reducing protein NADP. + reductase pet Upregulated genes; key genes associated with glycolysis / gluconeogenesis, citric acid cycle, pyruvate metabolism, and oxidative phosphorylation ( pckA、por、ndh Upregulation of these pathways indicates that the enhancement of these pathways suggests S. oblique -NCU J5 symbiotic system S. oblique It maintained sustained carbon metabolism activity. Figure 6 a). In the symbiotic system, the pathways related to the transport of organic sulfur into the cell and the intracellular metabolism of organic sulfur by Nelsenia bacillus NCU J5 are upregulated ( Figure 6 (bc in the text), such as symbiotic system-specific secretion genes ( gspG、gspC、vgrG Upregulation of NCU J5 enhances the assembly of type II secretory system (T2SS) protein complexes, promotes efficient protein efflux and biomembrane formation, and enables NCU J5 to actively attach to... S. oblique Surface, and S.obliquus -The fluorescence microscopy images of the NCU J5 symbiotic system are consistent ( Figure 3 ),and vgrG Gene upregulation can promote the exchange of substances between bacteria and algae, as confirmed by research. S. oblique It can secrete indoleacetic acid to promote microalgal growth, as shown in Table 10. S. oblique The significant biomass accumulation in the -NCU J5 symbiotic system also provides physiological validation. S. oblique Genes encoding the substrate-binding protein of the ABC transport system and the alkyl sulfonate permease subunit in the -NCU J5 symbiotic system ( ssuA, ssuC Upregulation of NCU J5 indicates that NCU J5 enhances the uptake of alkyl sulfonates in the symbiotic system. For the crucial sulfur metabolism transition, the inorganic sulfate assimilation gene (… cysH、cysK Downregulation and organosulfur acquisition / degradation genes ( Yes, cysI, cysJ The simultaneous upregulation confirmed the bioavailability of CYC by NCU J5. Furthermore, the sulfate / thiosulfate transporter (… cypP Gene upregulation, maintenance S. oblique The NCU J5 symbiotic system continuously supplies inorganic sulfur from the environment. The Nelsenia bacterium NCU J5 in the symbiotic system shifts from the energy-intensive sulfate assimilation pathway to utilizing CYC as a carbon source, thus enhancing... S. oblique- The NCU J5 symbiotic system demonstrates the biosorption and biodegradation of CYC. These results further prove that both *N. nielscens* and *Scenedesmus obliquus* directly assimilate and utilize CYC. Combined with the results of Example 6 (the main removal pathway of CYC is biodegradation), the transcriptome results also indicate that the bacterial-algal symbiosis achieves metabolic cooperation through the interface—the organic sulfur absorption and metabolic assimilation pathway of the bacterium *N. nielscens* NCU J5 is enhanced, thereby promoting the utilization and removal of cyclamate, a sulfur-containing organic compound. Simultaneously, the bacterium *N. nielscens* enhances the secretion of substances such as indoleacetic acid, which promote the growth and reproduction of *Scenedesmus obliquus*, thus promoting the heterotrophic metabolic assimilation of cyclamate by the microalgae. The combined effect of these two factors promotes the synergistic and enhanced removal of cyclamate.

[0115] Example 8: NCU J5 in anaerobic fermentation biogas slurry from actual municipal sewage and actual livestock and poultry manure. S. oblique and S. oblique - Comparison of CYC removal effects of NCU J5

[0116] (1) Pretreatment of municipal wastewater (MW) and anaerobic digestion broth (PMADE): The municipal wastewater was collected from a sewage treatment plant in Xiaolan Economic Development Zone, Nanchang County, Nanchang City, Jiangxi Province, and the anaerobic digestion broth was collected from the biogas station of Zhenghe Ecological Environment Agriculture Co., Ltd., Xinyu City, Jiangxi Province. The municipal wastewater was directly used after being filtered through a 0.45µm aqueous polyether membrane. The anaerobic digestion broth was first filtered through 8 layers of 100-mesh gauze to remove most of the suspended solids, and finally clarified by filtration through a 6kDa hollow fiber membrane. The clarified wastewater was stored in a refrigerator at -20℃ for later use. The main physicochemical indicators of the municipal wastewater and anaerobic digestion broth are shown in Table 16 below.

[0117] Table 16 Main Physicochemical Indicators of Municipal Wastewater and Anaerobic Digestion Liquid from Pig Manure

[0118]

[0119] Note: TC represents total carbon; TOC represents total organic carbon; IC represents organic carbon; TN represents total nitrogen; NH4 + -N represents ammonia nitrogen; TP represents total phosphorus.

[0120] (2) The municipal wastewater and pig manure anaerobic digestion liquid obtained in step (1) were filtered through 0.45 µm and 0.22 µm filter membranes for sterilization. In addition, the pig manure anaerobic digestion liquid was diluted 40 times with sterile deionized water.

[0121] (3) Following the method and conditions of Example 4, *Scenedesmus obliquus* and *Nilsonia* were cultured to the logarithmic growth phase. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the samples were washed three times with BG11 medium. Algal sludge and bacterial sludge were collected separately, and a certain amount of BG11 medium was added to prepare 1 g / L *Scenedesmus obliquus* inoculation stock solution and 1 g / L *Nilsonia* inoculation stock solution. These were then inoculated into municipal wastewater and anaerobic fermentation broth diluted 40 times with the optimal inoculation ratio of 10:1 (algae:bacteria), respectively, with a total wet weight of 1 g / L. Simultaneously, cyclamate was added to bring the cyclamate concentration in the wastewater to 30 mg / L.

[0122] Experimental setup: NCU J5 single bacteria group, S. oblique Single algae group and S. oblique The NCU J5 algal symbiotic system was divided into three replicates. It was cultured under continuous light for 24 hours at 26±1℃ and 2.50±0.10 Klux light intensity, with shaking performed three times daily during the culture period. The experiment lasted 12 days, and the biomass yield, photosynthetic activity, and removal of total organic carbon, ammonia nitrogen, total phosphorus, and CYC from the wastewater were monitored.

[0123] The results are shown in Tables 17-22. In municipal wastewater and anaerobic fermentation broth of pig manure... S. oblique The NCU J5 symbiotic system exhibited higher CYC removal rates than the single algae and single bacteria groups, reaching 27% and 21%, respectively. Although this removal rate was lower than the complete removal in BG11 medium in the aforementioned examples, the symbiotic system still maintained a high CYC removal capacity in complex wastewater environments compared to the monoculture system. S. oblique In the NCU J5 symbiotic system, the anaerobic fermentation broth of pig manure initially showed decreased photosynthetic activity and hindered biomass accumulation due to high free ammonia toxicity and organic acids. However, after 8 days, the system showed adaptive recovery, with Fv / Fm returning to 74% of the initial level, indicating that the symbiotic system enhanced its environmental adaptability through metabolic reorganization. Municipal wastewater and anaerobic fermentation broth of pig manure contain abundant nutrients. Tables 20-22 show that the symbiotic system, compared to the single-culture system, exhibited significant removal effects on total carbon (TC), inorganic carbon (IC), total nitrogen (TN), and total phosphorus (TP). However, this also led to substrate competition between these nutrients and CYC, making it difficult for the aminosulfonic acid groups (-NHSO3⁻) in the CYC molecule to serve as the preferred carbon source, resulting in a decrease in removal rate. In practical wastewater applications, efforts should be made to reduce ammonia toxicity and nutrients to enhance the removal effect of CYC.

[0124] Table 17 NCU J5 in different actual wastewater S. oblique and S. oblique -Biomass of NCU J5 (g / L)

[0125]

[0126] Table 18 Different Actual Wastewater S. oblique and S. oblique -NCU J5's Fv / Fm

[0127]

[0128] Table 19 CYC Residue Rates in Different Actual Wastewaters

[0129]

[0130] Table 20. Changes in physicochemical indicators (mg / L) of NCU J5 group in different actual wastewater samples.

[0131]

[0132] Table 21 Different Actual Wastewater S. oblique Changes in physicochemical parameters of the group (mg / L)

[0133]

[0134] Table 22 Different Actual Wastewater S. oblique Changes in physicochemical parameters (mg / L) of NCU J5 group

[0135]

[0136] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Nystatinia ( Niallia nealsonii NCU J5, characterized in that: Its accession number is CCTCCNO:M 20251657.

2. A microbial preparation, characterized in that: Includes N. nielsoniae NCU J5 as described in claim 1.

3. A cyclamate degrading agent, characterized in that: Includes N. nielsoniae NCU J5 as described in claim 1.

4. A fungal-algae symbiotic system, characterized in that: It includes a *Scenedesmus* strain and the *Nilsenia* strain NCU J5 as described in claim 1, with a mass ratio of (10~20):

1.

5. The symbiotic system of bacteria and algae according to claim 4, characterized in that: The *Scenedesmus* species mentioned are either *Scenedesmus oblique* or *Scenedesmus tetracaudus*.

6. A method for constructing a fungal-algae symbiotic system according to claim 4, characterized in that: Includes the following steps: (1) Culture the Scenedesmus genus algae into a Scenedesmus genus algae inoculation stock solution; (2) Nelsenia bacillus NCU J5 was cultured to prepare Nelsenia bacillus NCU J5 inoculation stock solution; (3) Mix the inoculation stock solution of Scenedesmus genus and the inoculation stock solution of Nilsneria NCU J5, and then inoculate it in the target environment.

7. The method for constructing the bacterial-algae symbiotic system according to claim 6, characterized in that: The concentrations of the inoculation mother liquor for the Scenedesmus genus strain and the inoculation mother liquor for Nilsneria NCU J5 were both 0.5~5 g / mL, and the wet weight ratio of the two was (10~20):

1.

8. The application of the *Nilsenia* NCU J5 as described in claim 1, or the bacterium-algae symbiotic system as described in claim 4 or 5, in wastewater treatment, characterized in that: The wastewater is any one or more of the following: wastewater containing cyclamate, municipal sewage, and anaerobic fermentation biogas slurry from livestock and poultry manure.

9. The application of the Nilssonia bacterium NCU J5 as described in claim 1, or the bacterial-algae symbiotic system as described in claim 4 or 5, in the degradation of cyclamate.

10. The application according to claim 8 or 9, characterized in that: The inoculum amount of Nilssonia bacillus NCU J5 or the bacterial-algal symbiotic system is 0.9~1.2 g / L; The conditions for wastewater treatment or degradation of cyclamate include: light intensity of 20,000-28,000 Lux, photoperiod of 12-24 h / d, temperature of 24-28 ℃, and culture period of 10-15 days.

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