Application of a single-cell green algae in removing sulfamethoxazole from water

Abstract

This invention discloses the use of a single-celled green algae for removing sulfamethoxazole from water. This invention is based on the inventors' discovery that Chlamydopodium sp. HS01 can remove sulfamethoxazole. This algae strain can grow in water containing sulfamethoxazole and efficiently remove it, demonstrating its potential for eliminating antibiotics from wastewater.

Description

Technical Field

[0001] The present invention relates to the field of biological treatment of pollutants, and in particular to application of a single-cell green algae in removing sulfamethoxazole from water. Background Art

[0002] Antibiotic contamination is a new environmental issue, posing potential risks to ecosystems and human health. Sulfamethoxazole is a widely used antimicrobial agent, widely used in the prevention and treatment of human and animal diseases. Studies have shown that only a small amount of sulfamethoxazole can be metabolized or absorbed by animals, while approximately 70% is excreted through feces or urine, entering sewage treatment systems or directly into the natural environment, causing pollution. Microalgae-based pollutant removal technology offers advantages such as low carbon emissions, low cost, and eco-friendliness and sustainability, and is considered a promising alternative to traditional wastewater treatment. In recent years, the potential for using microalgae to eliminate antibiotics from wastewater has received widespread attention. Summary of the Invention

[0003] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a single-cell green algae for use in removing sulfamethoxazole from water.

[0004] The purpose of the present invention is achieved through the following technical solution: The use of a single-cell green algae in removing sulfamethoxazole from water is based on the inventor's discovery that the single-cell green algae isolated by themselves has the effect of removing sulfamethoxazole.

[0005] The name of the unicellular green algae is Chlamydopodium sp. HS01, the preservation number is CCTCC NO: M20211104, the preservation date is August 30, 2021, and the preservation unit is the China Type Culture Collection located at Wuhan University, Wuhan, China.

[0006] The use of the unicellular green algae in removing sulfamethoxazole from water comprises the following steps: adding unicellular green algae species or unicellular green algae liquid to the water for culturing to obtain the water from which sulfamethoxazole is removed.

[0007] The unicellular green algae liquid is a unicellular green algae liquid cultured to the logarithmic growth phase or the stable phase; it is preferably obtained by the following steps: inoculating unicellular green algae species into a culture medium and culturing to the logarithmic growth phase or the stable phase.

[0008] The culture is preferably carried out at 25-30°C, 10,000-12,000 lux, and a light-dark cycle of 10-14h:10-14h; more preferably, at 28°C, 10,000±1,000 lux, and a light-dark cycle of 12h:12h.

[0009] The culture medium is preferably BG11 liquid culture medium.

[0010] The composition of BG11 liquid medium is as follows: K2HPO4 . 3H2O 0.04g / L, MgSO4 . 7H2O 0.075g / L, CaCl2 . 2H2O 0.036g / L, citric acid 0.006g / L, ammonium ferric citrate 0.006g / L, EDTA 0.001g / L, Na2CO3 0.02g / L, NaNO3 1.5g / L, trace element A5 1mL.

[0011] The composition of trace elements A5 is as follows: H3BO3 2.860g / L, NaMoO4 . 2H2O 0.021g / L, ZnSO4 . 7H2O0.222g / L, CuSO4 . 5H2O 0.079g / L, MnCl2 . 4H2O 1.810g / L, NiSO4 . 6H2O 0.479g / L.

[0012] The amount of the unicellular green algae species or unicellular green algae liquid added is calculated based on the OD680 of the algae cells in the water body being 0.05 to 0.15; more preferably, it is calculated based on the OD680 of the algae strain in the water body being 0.1.

[0013] The present invention has the following advantages and effects compared to the prior art:

[0014] The present invention discovers that Chlamydopodium sp. HS01 can efficiently remove sulfamethoxazole, so Chlamydopodium sp. HS01 has application potential in eliminating antibiotics in sewage. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a growth curve of three algae species in BG11 medium containing 0.4 mg / L sulfamethoxazole.

[0016] Figure 2 This is the result of the removal of sulfamethoxazole by different algae.

[0017] Figure 3 This is an analytical diagram of the possible degradation pathways of sulfamethoxazole under light control and algae HS treatment. DETAILED DESCRIPTION

[0018] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0019] Example 1: Screening of algae with strong removal ability

[0020] Evaluate the toxicity and removal ability of three strains of algae, Chlamydopodium sp. HS01 (hereinafter referred to as HS), Nostoc sp. J (hereinafter referred to as J, which has been disclosed in the literature "Lu YZ, Zhuo C, Li YJ, Li HS, Yang MY, Xu DN, He HZ (He Hongzhi)*. Evaluation of filamentous heterocystous cyanobacteria for integrated pig-farm biogas slurry treatment and bioenergy production. Bioresource Technology, 2020, 297: 122418, DOI: 10.1016 / j.biortech.2019.122418", and the applicant can provide it), Anabaena variabillis SCAU26 (hereinafter referred to as TS, which has been disclosed in the literature "Zhou YW, Bao JQ, Zhang DH, Li Y, Li HS, He HZ (He Hongzhi)*. Effect of heterocystous nitrogen-fixing cyanobacteria against rice sheath blight and the underlying mechanism. Applied Soil Ecology, 2020, 153: 103580. DOI: 10.1016 / j.apsoil.2020.103580", and the applicant can provide it) in the culture containing sulfamethoxazole; screen out the algal strains with high tolerance and removal ability to sulfamethoxazole. This experiment was to add sulfamethoxazole to the BG11 medium to simulate the sewage containing sulfamethoxazole, and then add the single-celled green algae algal species for cultivation. Among them, HS was isolated by the applicant himself, and the preservation number is CCTCC NO: M20211104, and the preservation unit is the China Center for Type Culture Collection located at Wuhan University, Wuhan, China.

[0021] (1) Inoculate the HS, J, and TS algal species into the sterilized BG11 liquid medium respectively, and culture them in an incubator at a temperature of 28.0 ± 0.5, a light intensity of 10000 ± 1000 lux, and a day-night ratio of 12 h: 12 h. Shake well 4 - 6 times a day to obtain the HS, J, and TS algal solutions in the logarithmic growth phase.

[0022] (2) Place 200 mL of BG11 in a 500 mL glass Erlenmeyer flask, sterilize at 121°C for 16 min, and add sulfamethoxazole after cooling to a final concentration of 0.4 mg / L to prepare the sulfamethoxazole-containing culture medium. Set up five experimental groups, with three replicates in each experimental group: inoculate the algae seed liquid of three algae in the logarithmic growth phase into the sulfamethoxazole-containing culture medium, set the final algae concentration to OD680 = 0.1, and culture at an incubator temperature of 28.0 ± 0.5°C, a light intensity of 10,000 ± 1,000 lux, and a day / night ratio of 12 h:12 h. Shake well 4-6 times a day. At the same time, set up a CK-light group without algae and a CK-dark group without algae and protected from light.

[0023] (3) Samples were taken on the 0th, 4th, and 8th day of the experiment to measure the growth indicators of the three algae and the concentration of sulfamethoxazole in the culture medium. Figure 1 As shown in Table 1, it can be seen that in the medium containing sulfamethoxazole, the growth rate of HS algae species is relatively fast. The concentration of sulfamethoxazole in the culture medium is shown in Table 1 and Figure 2 As shown, it can be seen that the ability of HS algae to remove sulfamethoxazole in the culture medium is much higher than that of algae J and algae TS, reaching 82.2%.

[0024] Table 1 Calculation results of sulfamethoxazole degradation rate after 8-day treatment with different algae

[0025]

[0026] Example 2: Toxicology Experiment

[0027] Evaluate the toxicity of sulfamethoxazole to HS. HS algae culture medium was prepared with sulfamethoxazole concentrations of 0, 4, 8, and 16 mg / L, with three replicates for each treatment. Experimental conditions were the same as in Example 1. Initial and final OD680 values were measured, and EC50 values were estimated based on the inhibitory effect of the antibiotic on algal growth at different concentrations.

[0028] The results are shown in Table 2. The results in Table 2 show that HS algae can tolerate high concentrations of sulfamethoxazole, and the estimated 9h EC 50 The value exceeds 40mg / L.

[0029] Table 2 Changes in algae OD value and inhibition rate calculation after 4 days of treatment with different concentrations of sulfamethoxazole

[0030]

[0031] Example 3: Identifying degradation products and analyzing degradation pathways

[0032] HS algae culture medium was treated with a sulfamethoxazole concentration of 0.4 mg / L, and a sulfamethoxazole-free light control was used. Experimental conditions were the same as in Example 1. On day 6, the culture medium was centrifuged and filtered through a 0.22 μm filter. Half of the filtrate was lyophilized, reconstituted with methanol, and analyzed by UPLC-Q-TOF. The remaining half of the filtrate was extracted with ethyl acetate, the organic phase collected by rotary evaporation, reconstituted with methanol, and analyzed by GC-MS.

[0033] The results are shown in Tables 3, 4, and 5. UPLC-Q-TOF screened eight sulfamethoxazole and its potential degradation products after algae treatment. UPLC-Q-TOF screened three sulfamethoxazole and its potential degradation products after photolysis or hydrolysis. GC-MS screened two potential degradation products, and the results for aniline overlapped with those for TOF.

[0034] Table 3 UPLC-Q-TOF screening results of sulfamethoxazole products degraded by algae

[0035]

[0036] Table 4 UPLC-Q-TOF screening results of sulfamethoxazole photolysis or hydrolysis products

[0037]

[0038] Table 5 GC-MS screening results of sulfamethoxazole photolysis or hydrolysis products

[0039]

[0040] According to the UPLC-Q-TOF and GC-MS results, the possible degradation pathways of sulfamethoxazole under two conditions were analyzed. Figure 3 Four possible degradation products were identified in the light control treatment, primarily through deoxazolylation and sulfonamide bond cleavage. Eight possible degradation products were identified in the algae treatment, with additional degradation pathways, including denitrification, nitrosation, and hydroxylation, on the benzene ring, compared to the light control.

[0041] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. The use of a single-cell green algae in removing sulfamethoxazole from water, characterized by: The name of the unicellular green algae is Chlamydopodium sp. HS01, the preservation number is CCTCC NO: M20211104, the preservation date is August 30, 2021, and the preservation unit is the China Type Culture Collection located at Wuhan University, Wuhan, China.

2. The use of the unicellular green algae in removing sulfamethoxazole from water according to claim 1, characterized in that The method comprises the following steps: adding single-cell green algae species or single-cell green algae liquid into a water body for cultivation to obtain a water body from which sulfamethoxazole has been removed.

3. The use of the unicellular green algae in removing sulfamethoxazole from water according to claim 2, characterized in that: The single-cell green algae liquid is a single-cell green algae liquid cultured to the logarithmic growth phase or the stable phase.

4. The use of the unicellular green algae in removing sulfamethoxazole from water according to claim 3, characterized in that: The unicellular green algae liquid is obtained by the following steps: inoculating unicellular green algae species into a culture medium and culturing them to a logarithmic growth phase or a stationary phase.

5. The use of the unicellular green algae in removing sulfamethoxazole from water according to claim 4, characterized in that: The culture is carried out at 25-30° C., with an illumination of 10,000-12,000 lux and a light-dark time of 10-14 h:10-14 h.

6. The use of the unicellular green algae in removing sulfamethoxazole from water according to claim 4, characterized in that: The culture medium is BG11 liquid culture medium.

7. The use of the unicellular green algae in removing sulfamethoxazole from water according to claim 2, characterized in that: The amount of the unicellular green algae species or unicellular green algae liquid added is calculated based on the OD680 of the algae cells in the water body being 0.05 to 0.15.

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