Hydrophage strain SNF1, fermentation compound and method for removing sulfamethoxazole in wastewater
By using a photobiological reaction system coupled with *S. nigra* SNF1 and *Neptune cephalopoda*, the problem of efficient degradation of sulfamethoxazole in water was solved, achieving efficient and low-cost water purification.
Patent Information
- Application Number
- CN202511356890.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-22
- Publication Date
- 2026-02-24
AI Technical Summary
Existing technologies are insufficient to efficiently remove sulfamethoxazole from water, and microalgae have low degradation efficiency and long cycles, leading to environmental pollution and increased antibiotic resistance.
The *S. NF1* strain of *Hydrophage* was immobilized and coupled with *Neptunus cephalopoda*, and sulfamethoxazole was degraded through a photobioreactor system. Light, aeration, and algal density conditions were optimized to improve degradation efficiency.
It significantly improved the degradation rate of sulfamethoxazole to over 80%, shortened the degradation cycle, and achieved a degradation rate superior to that of using hydrophage alone, thus realizing efficient and low-cost water purification.
Smart Images
Figure CN121555346A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of water pollution control and relates to a method for using microalgae and bacteria in combination to remove sulfamethoxazole from wastewater. Background Technology
[0002] As a broad-spectrum antibacterial agent, sulfonamide antibiotics exhibit strong antibacterial activity against both Gram-positive and Gram-negative bacteria, with particularly good efficacy against Escherichia coli and Staphylococcus aureus. Sulfamethoxazole, in particular, is widely used due to its cost-effectiveness and stability. However, because biological absorption of sulfamethoxazole is limited, approximately 45-70% of it is excreted into the environment in its original form or as metabolites within 24 hours via urine. Overuse of sulfamethoxazole has led to an increase in the frequency and concentration of its residues detected in soil, wastewater treatment plants, surface water, and groundwater. Reports indicate that the highest concentrations of sulfamethoxazole detected in domestic, municipal, and hospital wastewater have exceeded 100 μg / L, while the highest concentrations detected in livestock wastewater have reached as high as 10 mg / L.
[0003] Sulfonamide antibiotics have a low octanol-water partition coefficient and are highly hydrophilic, making them difficult to remove by adsorption techniques. Their half-life in water is 85 to 100 days or even longer. The accumulation of sulfamethoxazole adversely affects the reproduction and growth of aquatic plants and animals, and influences the biogeochemical cycles of soil and water by inhibiting environmental microorganisms. More importantly, sulfamethoxazole promotes the spread of antibiotic resistance genes and resistant bacteria, increasing the risk of infection and leading to clinical treatment failure. Therefore, we urgently need efficient and safe methods for removing sulfamethoxazole from water bodies.
[0004] Microorganisms play a vital role in almost all biogeochemical cycles on Earth. Compared to physical and chemical methods, biodegradation of sulfamethoxazole is less costly, more environmentally friendly, and more widely applicable. In recent years, the application of microalgae in wastewater treatment has attracted considerable attention. Microalgae can remove organic pollutants, including antibiotics, through enzymatic processes (biodegradation) or physical and energy-driven processes (biosorption and bioaccumulation). In addition to water purification, microalgae-based technologies also offer numerous benefits such as carbon dioxide capture, biofertilizer production, and renewable fuel generation.
[0005] However, microalgae can only tolerate relatively low concentrations of highly hydrophilic sulfonamide antibiotics, and their degradation rates are slow and the degradation cycles are long. The degradation of organic pollutants in the natural environment often requires multiple microorganisms to interact metabolically. Synthetic microbiomes, based on the interactions within microbial communities, can perform more complex tasks with higher efficiency than single microorganisms, and have broad application prospects in industry, health, and the environment. Summary of the Invention
[0006] To overcome the shortcomings of existing technologies, this invention provides a technique for adding degrading bacteria to a microalgae culture system to improve the removal efficiency of sulfamethoxazole in wastewater.
[0007] Firstly, this invention provides a Hydrogenophaga strain SNF1, with accession number CGMCC No. 35006, deposited at the China General Microbiological Culture Collection Center on June 25, 2025, and classified as Hydrogenophaga sp. This strain was isolated from sludge in a wastewater treatment plant and has the ability to degrade sulfamethoxazole.
[0008] The screened strains were amplified by PCR and identified using universal primers: DNA was extracted according to the instructions of the bacterial DNA extraction kit and used as a template. The forward primer was 27f: 5'-AGAGTTTGATCCTGGCTCAG-3' and the reverse primer was 1492r: 5'-TACGGTTACCTTGTTACGACTT-3', respectively. Sequencing showed that the bacterium belonged to Hydrogenophaga sp., and the sequence is shown in SEQ ID No. 1.
[0009] The present invention also provides a preferred immobilization method for the aforementioned SNF1 hydrophage, wherein the hydrophage is cultured in LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) until the OD600 reaches the range of 0.5 to 1.0, the cells are collected by centrifugation, then 2% sodium alginate is added and mixed thoroughly, the mixture is squeezed out into a 2% calcium chloride solution using a syringe, and cross-linked at 4°C for 4 h to prepare immobilized degradative bacterial particles with a particle size of 3-5 mm.
[0010] Preferably, the culture density of the SNF1 hydrophage is between 0.5 and 1.0 at an OD600. Preferably, the immobilization material is 2% sodium alginate.
[0011] This invention incorporates immobilized degrading bacteria into a microalgae photobioreactor system to promote the degradation of sulfamethoxazole.
[0012] Preferably, the microalgae is *Neptune cephalopoda*, purchased from the Wuhan Institute of Hydrobiology, with accession number FACHB-271.
[0013] This invention also provides an optimized scheme for the coupled degradation of sulfamethoxazole by *S. phage* SNF1 and *FACHB-271*: a photobioreactor system is established using *FACHB-271*, with a light intensity of 5000 lux, a light-dark cycle of 12h:12h, an aeration frequency of 10 min / h, and a temperature range of 20-30℃; the microalgae growth density OD680 should be less than or equal to 0.4, equivalent to 4.3 × 10⁶ algal cells / ml, and if the algal density is too high, it should be diluted by drainage and feeding; *S. phage* SNF1 is added to the microalgae photobioreactor system in the form of the above-mentioned immobilized particles, and the dosage is estimated based on the number of viable bacteria, with an OD600 greater than or equal to 0.025, equivalent to 1.0 × 10⁸ cfu / ml.
[0014] The coupled degradation scheme of *S. nigra* SNF1 and *FACHB-271* provided by this invention can degrade 5 mg / L sulfamethoxazole to below 1 mg / L with a degradation rate of over 80%, significantly improving the degradation capacity of microalgal photobioreactor systems for sulfamethoxazole. Simultaneously, microalgae can provide a carbon source for *S. nigra* SNF1, and the degradation effect of the coupled scheme is superior to using *S. nigra* SNF1 alone. Wastewater treatment technology based on microalgae is considered a highly efficient, low-carbon, low-cost, and sustainable green technology. Enhancing the degradation capacity of microalgal systems for new pollutants such as antibiotics makes microalgal technology more comprehensive and integrated in its ability to treat environmental pollution. Attached Figure Description
[0015] Figure 1 The figure shows the effective viable bacteria count when using three different combinations of immobilization materials; Figure 2 The figure shows a comparison of the degradation effects of sulfamethoxazole when three types of microalgae were co-cultured with Hydrophage. Figure 3 The image shows a comparison of the degradation effects of sulfamethoxazole when algae and bacteria are co-cultured and cultured separately. Figure 4 The results show the chromatographic analysis of sulfamethoxazole degradation by algae-bacteria co-culture and by individual bacteria. Detailed Implementation
[0016] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.
[0017] Example 1: Relationship between immobilized materials and the activity of hydrogen phage.
[0018] Preparation of wet cells of SNF1 (2% concentration): Single colonies of SNF1 were picked and placed in 100 ml of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl), and cultured on a shaker until the OD600 reached 0.5-1.0. The cells were collected by centrifugation at 7500 rpm and weighed (in g). Subsequently, the cells were added to the immobilization mixture at a weight-volume ratio of 2%.
[0019] Taking a 30 mL system as an example: Weigh out 0.6 g of sodium alginate, 0.6 g of sodium alginate + 3 g of polyvinyl alcohol, and 0.6 g of sodium alginate + 0.6 g of polyvinyl alcohol respectively, add them to 30 mL of sterile water, add centrifuged wet bacterial cells (2%, 0.6 g), shake well, squeeze out the microspheres through a syringe into a 2% CaCl2 solution, place in a 4°C refrigerator for cross-linking for 4 h, then filter out the CaCl2 solution and wash the microspheres three times with sterile water.
[0020] LB solid medium containing 50 mg / L sulfamethoxazole was prepared. Microspheres prepared from three different material combinations were dissolved in 3% sodium citrate. These were then diluted with sterile physiological saline to form bacterial suspensions with concentration gradients of 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, and 10⁻⁵. The effective viable count of the three immobilization material combinations was calculated using the plate spread method. Data showed that using 2% sodium alginate alone maintained the highest number of viable *Hydrophage* bacteria (see...). Figure 1 ).
[0021] Example 2: The effect of different microalgae and hydrophage in co-degrading sulfamethoxazole.
[0022] Three different microalgae were purchased from the Wuhan Institute of Hydrobiology: *Chlorella sorokinense* (Chinese Academy of Sciences Freshwater Algae Culture Collection, accession number FACHB-24), *Acinetocephala spicata* (Chinese Academy of Sciences Freshwater Algae Culture Collection, accession number FACHB-271), and *Haematococcus pluvialis* (Chinese Academy of Sciences Freshwater Algae Culture Collection, accession number FACHB-874). After expansion culture in BG11 medium, the algal biomass was diluted to OD680 = 0.05. Then, *S. nigra* SNF1, with estimated OD600 values of 0.02 and 0.04 respectively, was added. The concentration of sulfamethoxazole was set at 5 mg / L. The cultures were statically incubated in a light incubator with a 12h:12h light-dark cycle at 25℃. Samples were taken after one day of culture.
[0023] Samples were filtered through a 0.2 μm filter into chromatographic vials and then analyzed using liquid chromatography-mass spectrometry. Data showed that *Plasmodium cephalopoda* and *Hydrophage* exhibited the best co-degradation effect on sulfamethoxazole, and increasing the initial biomass of *Hydrophage* significantly improved degradation efficiency (see [link to data]). Figure 2 ).
[0024] Example 3: Comparison of the degradation effect of sulfamethoxazole in co-culture and separate culture of algae and bacteria.
[0025] Building upon Example 2, to verify that co-culturing algae and bacteria indeed promotes the degradation of sulfamethoxazole, we further compared the degradation effects of co-culturing *Plasmodium simulans* (hereinafter referred to as algae) and *S. nigra* SNF1 (hereinafter referred to as bacteria) with their individual cultures. *Plasmodium simulans* was cultured on BG11 medium and diluted to an algal biomass OD680 = 0.05. Four treatments were established: algae alone (OD680 = 0.05), algae (OD680 = 0.05) + bacteria (OD600 = 0.02), algae (OD680 = 0.05) + bacteria (OD600 = 0.04), and bacteria alone (OD600 = 0.02). The concentration of sulfamethoxazole was set at 5 mg / L. The cultures were statically incubated in a light incubator with a 12h:12h light-dark cycle at 25 ℃. Samples were taken after 1 and 2 days of culture.
[0026] The sample was filtered through a 0.2 μm filter membrane into a chromatographic vial and then analyzed using liquid chromatography-mass spectrometry (LC-MS). Data are shown (see...). Figure 3 The co-culture of *Anomala stolonifera* and *Hydrophage* did indeed promote the degradation of sulfamethoxazole. *Anomala stolonifera* achieved a degradation rate of approximately 20% for sulfamethoxazole, while *Hydrophage* achieved approximately 65%. In contrast, after two days of co-culture, the algae and bacteria had achieved near-complete degradation (over 99%). Furthermore, compared to the single bacteria culture, the co-culture not only increased the degradation rate of sulfamethoxazole (SMX) but also significantly reduced the concentration of intermediate products. Based on this, we hypothesize that *Anomala stolonifera* participates in the co-metabolism of sulfamethoxazole (e.g., ...). Figure 4 (As shown).
[0027] Example 4: Application of immobilized bacteria in algal photobioreactor system.
[0028] First, immobilized particles of SNF1 of Hydrophage were prepared using 2% sodium alginate as described above.
[0029] A photobioreactor system was constructed using the aforementioned *Neptunus cephalopoda*. Each liter of simulated wastewater contained 36 mg ammonium chloride, 20 mg potassium nitrate, 6 mg dipotassium hydrogen phosphate, 1 ml trace element solution, and 5 mg sulfamethoxazole. Each liter of trace element solution contained 1.0 g EDTA, 0.2 g ZnSO4, 0.1 g MnCl2•4H2O, 0.5 g (NH4)6Mo7•4H2O, 0.5 g CuSO4•5H2O, 0.2 g CoCl2•6H2O, with the remainder being water. The light intensity was 5000 lux, the light-dark cycle was 12 h:12 h, the aeration frequency was 10 min / h, and the temperature was 25℃. A control group consisting only of *Nyctaginus spiculatus* and a treatment group consisting of *Nyctaginus spiculatus* plus immobilized bacteria were established. The experiment was conducted for four cycles, each lasting five days. In the first cycle, the OD600 of the added *Hydrophage* was 0.036, and the water was changed once a day, with 1 / 5 of the water volume replaced each time. In the second cycle, the OD600 of the added *Hydrophage* was increased to 0.08. In the third and fourth cycles, the OD600 of the added *Hydrophage* was maintained at 0.08, but the water change frequency was changed to once every two days. Water samples were collected at the end of each cycle.
[0030] The sample was filtered through a 0.2 μm filter membrane into a chromatographic vial, and then detected by liquid chromatography-mass spectrometry. The degradation rate was calculated by comparing the remaining concentration with the added concentration.
[0031] Table 1. Operating conditions and sulfamethoxazole degradation efficiency of the algal photobioreactor Note: Different letters are used to label two data points from the same period to indicate that there is a significant difference between them. As can be seen from the results in Table 1, (1) compared with the control group containing only *Neptunus cephalopoda*, the addition of immobilized bacteria significantly improved the degradation efficiency of sulfamethoxazole. (2) When the optical density OD600 of *Hydrophage* was increased to 0.08, the degradation efficiency of sulfamethoxazole was further increased to over 70%. (3) When the water change frequency was reduced for a period of time, the degradation efficiency of sulfamethoxazole in the control group was also enhanced, while the degradation efficiency in the experimental group was further increased to over 75%.
[0032] The SEQ ID No. 1 serial number is as follows: >Hydrogenophaga sp. SNF1, 16S rRNA gene
[0033] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A *S. genus* SNF1, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35006 and deposit date of June 25, 2025.
2. A complex, characterized in that, It is used to remove sulfamethoxazole from wastewater, and the complex includes the Hydrophage strain SNF1 as described in claim 1 and microalgae.
3. A fermentation complex, characterized in that, The complex comprises sodium alginate and the SNF1 strain of Hydrophage as described in claim 1, wherein the complex is granular.
4. The fermentation complex according to claim 2, characterized in that, The microalgae mentioned are one or a mixture of several of the following: Chlorella sorogenesis, Cyclocarya subcetamol, and Haematococcus pluvialis.
5. A method for removing sulfamethoxazole from wastewater, characterized in that, Using the SNF1 strain of Hydrophage as described in claim 1, a substance containing the SNF1 strain of Hydrophage as described in claim 1 is added to wastewater.
6. A method for removing sulfamethoxazole from wastewater, characterized in that, Use the complex according to any one of claims 2-4.
7. A method for removing sulfamethoxazole from wastewater, characterized in that, The steps are as follows: I. Preparation of the complex according to claim 3; 2. The aforementioned complex is added to wastewater containing sulfamethoxazole, wherein the wastewater contains microalgae and is able to receive light.