Tinonematode algal strain and application thereof in degradation of tetracycline in water body

Abstract

The invention relates to the technical field of environmental microbial remediation, and in particular discloses a Leuconoidea algal strain and application thereof in degradation of tetracycline in water, the Leuconoidea algal strain is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20252177. The invention discloses a new application of the algal strain and the preparation thereof in removing or degrading tetracycline in a water body. The strain has remarkable tolerance to high-concentration tetracycline, the removal rate of 100 mg / L of tetracycline can reach up to 96.60% within 48 hours under the optimal condition, and the removal mechanism of the strain is mainly biodegradation. The invention provides an efficient and environment-friendly novel bioremediation resource and technology for treating tetracycline polluted water.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of environmental microbial remediation, in particular to a strain of Oedogonium and its application in degrading tetracycline in water. BACKGROUND

[0002] Tetracycline antibiotics are a kind of broad-spectrum antibiotics with a basic skeleton of naphthacene. Due to their high efficiency and low cost, they are widely used in human medical treatment, veterinary clinical treatment, and livestock and aquaculture industries, and even as feed additives to promote animal growth. However, this kind of antibiotic is not completely absorbed and metabolized in the body, about 30% ~ 90% of which will be excreted out of the body in the form of original shape or active metabolites with feces and urine, resulting in a large amount of residues in the environment. Tetracycline antibiotics are frequently detected in hospital wastewater, pharmaceutical wastewater, livestock and poultry breeding wastewater, and surface water and groundwater contaminated by feces.

[0003] Tetracycline residues in the environment can induce the production of antibiotic resistance genes in microorganisms, accelerate the emergence and spread of “super bacteria”, and pose a serious threat to the ecosystem and human health. Therefore, developing efficient, economical and environmentally friendly remediation technologies for tetracycline-contaminated water bodies has become an important issue to be solved in the field of environmental science and engineering.

[0004] At present, the methods for treating tetracycline in water mainly include physical method, chemical method and biological method.

[0005] Physical method: such as adsorption method (using activated carbon, biochar, montmorillonite, etc.) and membrane separation method. This kind of method usually only transfers or enriches the pollutants from the water body, and does not really degrade the tetracycline molecules, and there are problems such as difficulty in regenerating adsorbents, membrane pollution, high operation cost and generation of concentrated waste liquid which needs secondary treatment.

[0006] Chemical method: such as advanced oxidation method (photocatalytic oxidation, Fenton reaction, ozone oxidation, etc.). This kind of method can effectively degrade tetracycline, but usually needs to add chemical reagents, has high energy consumption, harsh reaction conditions, and may produce more toxic or unknown intermediate products, which has the risk of secondary pollution.

[0007] Biological method: mainly using microorganisms (such as bacteria, fungi) or microalgae to biodegrade or biosorb tetracycline. Compared with traditional methods, biological method has potential advantages such as low cost, environmental friendliness, and no secondary pollution, and thus is concerned. Among them, microalgae remediation technology shows unique potential, because microalgae can not only remove pollutants through biosorption and bioaccumulation, but also provide energy and reducing power for the degradation process by using its photosynthetic system, realizing the synergistic process of "purification-growth". However, most ordinary microalgae have poor tolerance to high concentration of tetracycline, and their growth will be severely inhibited, resulting in limited degradation efficiency. Therefore, screening and obtaining specific microalgae strains with high tolerance to tetracycline and high degradation efficiency is the key to the application of this technology. SUMMARY

[0008] The purpose of the present application is to provide a strain of Stigonema and its application in degrading tetracycline in water, to solve the following problems in the treatment of tetracycline wastewater by microalgae at present: 1. Limited degradation efficiency and long cycle: The removal rate of higher concentration of tetracycline is not ideal, and a long treatment time is needed to achieve good results, which is difficult to meet the demand of efficient wastewater treatment.

[0009] 2. Poor tolerance of algae strains: Common ordinary microalgae have low tolerance to tetracycline. Under high concentration of tetracycline stress, the growth of algal body will be significantly inhibited, which in turn limits the full play of its degradation capacity, forming a vicious cycle.

[0010] 3. Unknown mechanism and weak targeting: The used algae strains are not specifically selected for degrading tetracycline, and their removal of tetracycline may be mainly by adsorption, the path and efficiency of biological degradation are not optimized and deeply analyzed, and there is a lack of efficient specific strains.

[0011] The above purpose of the present application is achieved as follows: The present application provides a strain of Stigonema, which is named Nodosilinea sp. ZL-SQ, the preservation number of which is CCTCC M 20252177, and the preservation unit is China Center for Type Culture Collection, the preservation address is Wuhan University Preservation Center, and the preservation time is September 29, 2025.

[0012] The present application also provides a microbial preparation comprising the Stigonema strain and / or its culture according to claim 1.

[0013] The present application also provides the use of the Stigonema strain in degrading tetracycline antibiotics in water.

[0014] Further, the initial concentration of tetracycline in the water body is 10 mg / L to 200 mg / L.

[0015] Further, the use is carried out under the conditions of illumination intensity of 10000 lux to 15000 lux, light-dark cycle of 10 to 14 hours of light: 10 to 14 hours of darkness.

[0016] Further, the use is carried out at a temperature of 25 to 30 DEG C.

[0017] Further, the use amount of the algal strain is such that the initial OD value of the algal cells in the water body is 0.1 to 0.3. 680 Further, the use amount of the algal strain is such that the initial OD value of the algal cells in the water body is 0.1 to 0.3.

[0018] The scheme of the present application also provides the use of the above-mentioned microbial preparation in removing or degrading tetracycline in a water body.

[0019] Further, the initial concentration of tetracycline in the water body is 10 mg / L to 200 mg / L.

[0020] The scheme of the present application also provides the use of the above-mentioned microbial preparation in removing or degrading tetracycline in a water body.

[0021] In the above-mentioned scheme of the present application, the use of the above-mentioned Zygnema in removing tetracycline in a water body comprises the following steps: adding Zygnema spores or Zygnema liquid culture into a tetracycline-polluted water body for culture to remove tetracycline. The Zygnema liquid culture is a Zygnema liquid culture cultured to the logarithmic growth phase or stationary phase; preferably obtained by the following steps: inoculating Zygnema spores into a culture medium and culturing to the logarithmic growth phase or stationary phase.

[0022] The culture is preferably carried out at 25 to 30 DEG C, illumination of 10000 to 15000 lux, and light-dark time of 10 to 14h: 10 to 14h; more preferably at 28 ± 0.5 DEG C, illumination of 10000 ± 1000 lux, and light-dark time of 14h: 10h.

[0023] The culture medium is preferably BG11 liquid medium. The composition of the BG11 liquid medium is as follows: K2HPO4·3H2O 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, EDTA 0.001 g / L, Na2CO3 0.02 g / L, trace element A5 51 mL. The composition of trace element A5 is as follows: H3BO3 2.860 g / L, NaMoO4·2H2O 0.021 g / L, ZnSO4·7H2O 0.222 g / L, CuSO4·5H2O 0.079 g / L, MnCl2·4H2O 1.810 g / L, NiSO4·6H2O 0.479 g / L.

[0024] The addition amount of the microalgae strain or microalgae liquid is calculated according to the OD 680 of the algal cells in the water body, preferably 0.1-0.2; more preferably calculated according to the OD 680 of the algal strain in the water body, preferably 0.2.

[0025] Compared with the prior art, the present application has the following beneficial effects: 1. The algal strain of the present application significantly improves the tetracycline degradation efficiency and speed. The removal rate of ordinary microalgae in the prior art to tetracycline is limited (60-80%) and the removal period is long (4-7 days), while the algal strain of the present application (ZL-SQ) has a removal rate of up to 96.60% to high-concentration tetracycline (100 mg / L) within 48 hours; this proves that the algal strain of the present application has high-efficiency and rapid tetracycline degradation capacity, can greatly shorten the wastewater treatment period, improve the treatment efficiency, and provides a more optimal solution to the problem of tetracycline residue. Nodosilinea sp . ZL-SQ) has excellent tetracycline tolerance, which ensures the stability of the treatment system. The microalgae in the prior art will be severely inhibited in growth under high-concentration tetracycline stress, resulting in that the degradation capacity cannot be sustained; although the initial inhibition rate of the algal strain of the present application is higher (40.77%), the algal strain of the present application can still maintain a high tetracycline degradation rate after 48 hours of treatment, which proves that the algal strain of the present application has excellent tetracycline tolerance and can ensure the stability of the treatment system.

[0026] 2. The algal strain (ZL-SQ) of the present application has excellent tetracycline tolerance, which ensures the stability of the treatment system. The microalgae in the prior art will be severely inhibited in growth under high-concentration tetracycline stress, resulting in that the degradation capacity cannot be sustained; although the initial inhibition rate of the algal strain of the present application is higher (40.77%), the algal strain of the present application can still maintain a high tetracycline degradation rate after 48 hours of treatment, which proves that the algal strain of the present application has excellent tetracycline tolerance and can ensure the stability of the treatment system. Nodosilinea sp. ZL-SQ) has excellent tetracycline tolerance, which ensures the stability of the treatment system. The microalgae in the prior art will be severely inhibited in growth under high-concentration tetracycline stress, resulting in that the degradation capacity cannot be sustained; although the initial inhibition rate of the algal strain of the present application is higher (40.77%), the algal strain of the present application can still maintain a high tetracycline degradation rate after 48 hours of treatment, which proves that the algal strain of the present application has excellent tetracycline tolerance and can ensure the stability of the treatment system. Nodosilinea sp .ZL-SQ can still grow in the environment of 100 mg / L tetracycline, and the inhibition rate gradually decreases to a stable level of 28%~32% with the treatment time; this shows that the strain has strong tolerance and adaptability; its growth is not completely inhibited by high concentration of antibiotics, but creates a more favorable growth environment for itself by degrading toxins, and this stability is the fundamental guarantee for its efficient and continuous degradation, overcoming the defects of sudden decrease in efficiency of the existing technology due to inhibition of the strain.

[0027] 3, The present application constructs a dynamic balance system, in which the efficient degradation of antibiotics creates a favorable environment for the growth of microalgae, and the continuous and healthy growth of microalgae in turn guarantees the long-term and stability of the degradation efficiency. The removal mechanism of the prior art may be mainly adsorption, but through the analysis of metabolic products in the present application, it is confirmed that Nodosilinea sp. ZL-SQ can completely destroy the molecular structure of tetracycline through biodegradation (such as oxidation, ring opening, etc.), which is not only simple adsorption enrichment, but also complete decomposition and conversion; the strain utilizes and decomposes toxic tetracycline as "nutrient", maintains its biomass growth (see Figure 3 and Table 1 of the examples) while degrading pollutants, forming a self-sustaining and environment-friendly benign treatment system. Compared with the traditional degradation method which depends on physical adsorption or inhibits biological growth, the present application realizes a self-sustaining and continuous efficient biodegradation process, which has more technical advantages in degradation efficiency and system self-sustaining compared with the treatment method which only depends on surface adsorption or inhibits microbial activity. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is the cell morphology of the algae Nodosilinea sp . ZL-SQ in the embodiment of the present application; Figure 2 is the phylogenetic tree of the algae Nodosilinea sp . ZL-SQ in the embodiment of the present application; Figure 3 is the growth curve of the algae Nodosilinea sp . ZL-SQ in the BG11 medium containing 100 mg / L tetracycline in the embodiment of the present application; Figure 4 is the removal effect result graph of tetracycline by the algae Nodosilinea sp . ZL-SQ in the embodiment of the present application; Figure 5 is the possible degradation path analysis graph of tetracycline under the treatment of the algae Nodosilinea sp . ZL-SQ in the embodiment of the present application. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the following description is provided in conjunction with the appendix. Figures 1-5 The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0030] The implementation of the present invention will be described in detail below with reference to specific embodiments.

[0031] Example 1: Activation and propagation of Nematocystis jirovecii The *Nematocystis* isolated, purified, and identified in this laboratory Nodosilinea sp. ZL-SQ was preserved on BG11 solid agar plates. The *Zygium* species preserved on the plates (… Nodosilinea sp. ZL-SQ was inoculated into 100 mL of BG11 medium (the composition of BG11 liquid medium is as follows: K2HPO4·3H2O 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, EDTA 0.001 g / L, Na2CO3 0.02 g / L, and trace element A5 1 mL. The composition of trace element A5 is as follows: H3BO3 2.860 g / L, NaMoO4·2H2O 0.021 g / L, ZnSO4·7H2O 0.222 g / L, CuSO4·5H2O 0.079 g / L, MnCl2·4H2O 1.810 g / L). Activation was performed in Erlenmeyer flasks containing 0.479 g / L NiSO4·6H2O and 0.479 g / L NiSO4·6H2O. The flasks were incubated at 28.0 ± 0.5 ℃ under incubator conditions of 10000 ± 1000 lux light and a day-night ratio of 12h:12h, with the mixture shaken three times daily. At a ratio of 10%, the algal culture from one week was inoculated into Erlenmeyer flasks containing 90 mL of BG11 medium. The flasks were then incubated under the same conditions for approximately 14 days to obtain algae in the logarithmic growth phase. Nodosilinea sp. ZL-SQ algal solution.

[0032] Example 2: Evaluation of algal growth in tetracycline Weigh 0.1 g of tetracycline hydrochloride and dissolve it in 100 ml of pure water to prepare a 1 g / L tetracycline hydrochloride stock solution. Filter the solution through a 0.22 μm filter membrane for later use.

[0033] Inoculate 10 ml of sterile BG11 liquid culture medium. Nodosilinea sp. ZL-SQ algal strain, with a final algal concentration set at OD.680 =0.10 ~ 0.20, add 10 ml of tetracycline hydrochloride stock solution to obtain the experimental group with a final tetracycline concentration of 100 mg / L. Incubate under the conditions of 28.0 ± 0.5℃, 10000 ± 1000 lux light, and a day-night ratio of 12h:12h. Samples were taken at 0, 1, 2, 3, 4, 5, and 6 days. 10 ml of each sample was centrifuged at 8000 rpm at 20℃ for 10 min, and the supernatant was discarded. The sample was resuspended in 10 ml of BG11 medium, shaken, and mixed thoroughly before measuring the OD at each time point. 680 The inhibition rate of tetracycline on algal growth was calculated. The results are shown in Table 1 below. Nodosilinea sp. ZL-SQ algae is less affected by tetracycline stress. Over time, it removes some of the tetracycline and reduces its toxicity, providing a favorable environment for its own growth.

[0034] Table 1. Tetracycline stress Nodosilinea sp. OD of ZL-SQ 680 and growth inhibition rate CK OD 680 ]]> Tetracycline stress OD 680 ]]> Inhibition rate 0d 0.166±0.005 0.098±0.003 40.77±0.17 1d 0.184±0.006 0.118±0.004 36.17±0.88 2d 0.222±0.008 0.146±0.003 34.45±1.00 3d 0.235±0.011 0.165±0.004 29.51±3.27 4d 0.248±0.006 0.177±0.002 28.50±1.48 5d 0.293±0.005 0.200±0.004 31.93±0.52 6d 0.316±0.003 0.216±0.003 31.82±0.74 Example 3: Evaluation of the tetracycline removal capacity of algae Inoculate 10 ml of sterile BG11 liquid culture medium. Nodosilinea sp. ZL-SQ algal strain, with a final algal concentration set at OD. 680 =0.20 ~ 0.30, and at the same time, a light group without algae and a dark treatment group were set up. 10 ml of tetracycline hydrochloride stock solution was added to obtain an experimental group with a final tetracycline concentration of 100 mg / L. The group was cultured under the conditions of incubator temperature of 28.0 ± 0.5℃, light intensity of 10000 ± 1000 lux, and day-night ratio of 12h : 12h. Samples were taken at 0, 6, 12, 24, 36, and 48 h.

[0035] Nodosilinea The tetracycline removal rate of sp. ZL-SQ algae is as follows: Figure 4 As shown, its removal rate of tetracycline reached 96.60% after 48 hours, while photolysis and hydrolysis accounted for a small proportion of tetracycline removal, indicating that... Nodosilinea sp. ZL-SQ algae has a strong ability to biodegrade tetracycline.

[0036] Example 4: Degradation products and their pathways In Example 3 NodosilineaThe sample for removal of tetracycline by sp. ZL-SQ was collected at 2d, and tetracycline and its degradation products were qualitatively analyzed by an ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-TOF-MS, Waters Synapt XS) system. The liquid chromatography conditions are as follows: a C18 reversed-phase chromatographic column (Waters Acquity UPLC BEH, 3.0 × 50 mm, 1.7 μm) is used, the column temperature is 40℃, the sample amount is 5 μL, and the mobile phase is 0.1% formic acid aqueous solution (A phase) and acetonitrile (B phase), and the flow rate is 0.3 mL / min. The gradient elution program is as follows: 0-3 min 95% A + 5% B, 3-4 min 100% B, and 4-6 min 95% A + 5% B. Mass spectrometry detection uses a heated electrospray ionization source (HESI), a positive ion mode ([M+H] + ), a capillary voltage of 2000 V, a cone hole gas flow rate of 50 L / h, an ion source temperature of 100℃, and a mass scan range of 50-800 m / z.

[0037] Possible degradation products of tetracycline and its pathways are shown in Figure 5 Under the biodegradation of sp. ZL-SQ, tetracycline mainly has three degradation pathways, and oxidation, ring opening and other reactions occur. Nodosilinea

[0038] Through the above embodiments of the present application, the strain of Phormidium provided by the present application has the following advantages: 1. Improve the removal efficiency of tetracycline: the high-efficiency biodegradation ability of the microalgae to tetracycline is used, and under experimental conditions (such as the removal rate of 100 mg / L tetracycline within 48 hours can reach 96.60%), the removal effect of tetracycline in water body is significantly improved, and the problem of low efficiency of traditional methods is overcome.

[0039] 2. Avoid secondary pollution: the microalgae remediation technology is based on the biodegradation process, is environmentally friendly, and does not introduce harmful chemicals, thereby eliminating the risk of secondary pollution that may be caused by traditional physical and chemical methods.

[0040] In summary, the strain of Phormidium ZL-SQ and the microbial preparation thereof provided by the present application can be used for treating various water bodies polluted by tetracycline, such as livestock breeding wastewater, aquaculture wastewater and pharmaceutical industry wastewater. By applying the use disclosed in the present application, tetracycline residues in water bodies can be efficiently, economically and greenly removed, and the present application has a broad industrial application prospect.

[0041] The above merely describes preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. A strain of the genus Anabaena, characterized in that, The algal strain is named Nodosilinea ZL-SQ, with the preservation number CCTCC M 20252177, and the preservation unit is the China Center for Type Culture Collection, the preservation address is the Wuhan University Preservation Center, and the preservation time is September 29, 2025.

2. A microbial preparation, characterized in that, The Nodosilinea strain of claim 1 and / or culture thereof.

3. Use of the Nodosilinea strain of claim 1 in degrading tetracycline antibiotics in a water body.

4. Use according to claim 3, characterized in that, The initial concentration of tetracycline in the water body is 10 mg / L to 200 mg / L.

5. Use according to claim 3, characterized in that, The use is carried out under the conditions of illumination intensity of 10000 lux to 15000 lux, light-dark cycle of 10 to 14 hours light: 10 to 14 hours dark.

6. Use according to claim 3, characterized in that, The use is carried out at a temperature of 25℃ to 30℃.

7. Use according to claim 3, characterized in that, The amount of algae used is to achieve the initial OD of algal cells in the water. 680 The value is 0.1 to 0.

3.

8. Use of the microbial preparation of claim 2 in removing or degrading tetracycline in a water body.

9. Use according to claim 8, characterized in that, The initial concentration of tetracycline in the water body is 10 mg / L to 200 mg / L.

10. Use of the microbial preparation of claim 2 in preparing a bioremediation agent for treating a tetracycline-contaminated water body.

Images