A method for treating antibiotic wastewater using heterotrophic Zofia green algae

Abstract

The invention discloses the application of Zofia chlorophylla in antibiotic degradation and a method for treating antibiotic wastewater using heterotrophic Zofia chlorophylla. The Zofia chlorophylla can grow heterotrophically using organic carbon sources, and its biomass under heterotrophic conditions is dozens or even hundreds of times that of autotrophic microalgae, and has an excellent degradation rate for single antibiotics or mixed antibiotics.

Description

Technical Field

[0001] The invention belongs to the technical field of antibiotic wastewater treatment, and more specifically, relates to a method for treating antibiotic wastewater by utilizing heterotrophic Zofia chlororaphis. Background Art

[0002] Antibiotics are natural or synthetic compounds with antibacterial or bactericidal activity. They can be used to treat and prevent various diseases caused by bacterial infections and are widely used in medicine and animal husbandry. However, neither humans nor animals can fully absorb or metabolize antibiotics. Antibiotics are excreted in urine and feces in the form of parent substances or active metabolites. Currently, most sewage treatment processes have limited ability to degrade antibiotics, and a large amount of undegraded antibiotics enter the water environment with the effluent from sewage treatment plants. The resulting evolution and spread of antibiotic-resistant genes and antibiotic-resistant bacteria will lead to serious antimicrobial resistance, increase the difficulty of drug development, affect food production, and directly endanger the health of humans and other animals. This is the biggest challenge we currently face in the use of antibiotics.

[0003] Currently, physical adsorption, advanced oxidation and photocatalysis are used to treat antibiotics. However, none of the above methods can effectively degrade antibiotics in water bodies, and there are problems such as high treatment costs and easy secondary pollution. The activated sludge method with bacteria as the main body is a typical biological treatment method and is the core process of urban sewage treatment. However, when facing pollutants such as antibiotics with antibacterial effects, in addition to destroying its own stability, it is also easy to cause the growth and spread of resistance genes and resistant bacteria.

[0004] Microalgae refer to a class of organisms that are tiny and between terrestrial microorganisms and plant cells. They grow and reproduce quickly, have strong adaptability, can use organic matter in water as nutrients to grow, and accumulate large amounts of bioactive substances such as proteins, oils, and carotenoids in their cells. The use of microalgae to treat wastewater containing antibiotics will not produce or release toxic substances during the treatment process, will not cause the spread of drug-resistant genes, but can simultaneously obtain a large amount of microalgae biomass. These biomasses can be further processed into high-value-added products such as fertilizers, biofuels, and animal feed, indirectly reducing treatment costs. Therefore, microalgae-based antibiotic remediation technology is considered to be an alternative method that has both ecological and economic benefits.

[0005] All along, algae is considered to be like higher plants and must be photosynthesized to achieve cell biomass synthesis, i.e., photoautotrophic culture mode. At present, research on microalgae treatment of antibiotic-containing wastewater is also mainly concentrated in the autotrophic mode. For example, patent publication number CN114249500A discloses a method for purifying high-concentration antibiotic wastewater by ultraviolet pretreatment and then cultivating microalgae, and patent publication number CN114561293A discloses a method for purifying cefradine wastewater based on microalgae cultivation. However, this autotrophic mode can only "rely on the sky for food", which is subject to external environmental conditions such as temperature, light intensity, sunshine time and other conditions. It is difficult to avoid problems such as light shielding between algae cells, slow growth, low cell concentration, and its biomass is mostly less than 1g / L, which is easily contaminated by other microorganisms, thereby further limiting the degradation efficiency of its pollutants (especially new pollutants such as antibiotics), and large-scale processing is limited.

[0006] In addition, it is not clear whether these algae strains used for antibiotic treatment contain high-value active ingredients in their cells. Patent Publication No. CN117384763A discloses a method for strengthening the coupling of microalgae purification of highly toxic antibiotic wastewater treatment and bioenergy production by a co-matrix strategy. The biomass of microalgae was increased by 1.36 times by adding 0.6mM sodium acetate to the microalgae, but the overall biomass was still at a low level due to light shielding, and real wastewater, due to its high turbidity, often affects light penetration. In addition, current studies have basically only dealt with a single antibiotic, which does not conform to the current situation of the coexistence of multiple antibiotics in actual wastewater. Summary of the invention

[0007] In view of the above existing technical problems, the primary purpose of the present invention is to provide an application of Chromochloris zofingiensis in the degradation of antibiotics. Chromochloris zofingiensis grows heterotrophically in an environment containing an organic carbon source and has an excellent degradation rate for antibiotics.

[0008] The second object of the present invention is to provide a method for treating antibiotic wastewater using heterotrophic Zofia chlororaphis.

[0009] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0010] The present invention claims the use of Zofia chlorophylla in antibiotic degradation, wherein the Zofia chlorophylla is cultured under heterotrophic conditions.

[0011] The inventors have discovered a single-cell microalgae, Zofia, through research, which can use organic carbon sources (such as glucose, sugarcane molasses or mannose, etc.) as energy and carbon sources for heterotrophic growth. Under heterotrophic conditions, the biomass produced by Zofia is dozens or even hundreds of times that of autotrophic microalgae, and its cells can accumulate a large amount of high-value carotenoids, astaxanthin and oil (the oil content reaches 52% of the cell dry weight). Heterotrophic Zofia has an excellent degradation rate not only for a single antibiotic, but also for mixed antibiotics.

[0012] Preferably, culturing under heterotrophic conditions means that the Zofia chlororaphis is cultured in a culture medium containing an organic carbon source.

[0013] Preferably, the concentration of the organic carbon source in the culture medium is 5 to 100 g / L. Further preferably, the concentration of the organic carbon source in the culture medium is 10 to 50 g / L; more preferably, the concentration of the organic carbon source in the culture medium is 20 to 30 g / L.

[0014] Preferably, the organic carbon source is selected from one or more of glucose, sugarcane molasses, mannose, fructose, sodium acetate, and glycerol. Further preferably, the organic carbon source is glucose.

[0015] Preferably, the culture medium can adopt the culture medium conventionally used for green algae cultivation in the art. More specifically, the culture medium is CZ-M1 culture medium. More specifically, CZ-M1 culture medium includes: 500-3000 mg / L NaNO3, 170-175 mg / L KH2PO4, 145-155 mg / L MgSO4·7H2O, 70-80 mg / L K2HPO4, 20-30 mg / L CaCl2·2H2O, 20-30 mg / L NaCl, 3-8 mg / L FeCl3·6H2O, 0.12-0.20 mg / L MnSO4·H2O, 0.1-0.5 mg / L ZnSO4·7H2O, 0.001-0.002 mg / L (NH4)6Mo7O 24 ·7H2O, 0.04~0.08mg / L H3BO3, 0.002~0.005mg / L CuSO4·5H2O, pH adjusted to 6.5.

[0016] Preferably, the culture temperature is 20-40°C. More preferably, the culture temperature is 22-28°C.

[0017] Preferably, the antibiotic is selected from one or more of penicillin antibiotics, cephalosporin antibiotics, macrolide antibiotics, quinolone antibiotics, β-lactam antibiotics, aminoglycoside antibiotics, sulfonamide antibiotics, and tetracycline antibiotics.

[0018] More specifically, the cephalosporin antibiotics can be cephalosporin antibiotics conventionally contained in antibiotic wastewater, such as cephradine, etc.; the quinolone antibiotics can be conventional quinolone antibiotics conventionally contained in antibiotic wastewater, such as norfloxacin, levofloxacin, ciprofloxacin, etc.; the quinolone antibiotics can be conventional quinolone antibiotics conventionally contained in antibiotic wastewater, such as norfloxacin, levofloxacin, ciprofloxacin, etc.; the sulfonamide antibiotics can be conventional sulfonamide antibiotics conventionally contained in antibiotic wastewater, such as trimethoprim, sulfamethoxazole, sulfamethoxazole, etc.; the tetracycline antibiotics can be conventional tetracycline antibiotics conventionally contained in antibiotic wastewater, such as tetracycline, etc.

[0019] Furthermore, the present invention claims the use of Zofia sphaerocephala to improve the degradation rate of sulfamethoxazole, sulfamethoxazole and tetracycline in the degradation of mixed antibiotics. Preferably, the mixed antibiotics include sulfamethoxazole, sulfamethoxazole, norfloxacin, trimethoprim, ciprofloxacin, levofloxacin, cephradine and tetracycline.

[0020] Furthermore, the present invention claims a method for treating antibiotic wastewater using heterotrophic Zofia chlororaphis, comprising the following steps:

[0021] S1. Cultivation under heterotrophic conditions: activating and expanding the Zofia chlororaphis in a culture medium containing an organic carbon source to obtain Zofia chlororaphis seed solution;

[0022] S2. The seed liquid of Zofia green algae is mixed with antibiotic wastewater to treat antibiotics.

[0023] Preferably, the volume mass ratio of the Zofia green algae seed liquid to the antibiotic is 10: 0.01-100 mL / mg. Further preferably, the volume mass ratio of the Zofia green algae seed liquid to the antibiotic is 10: 0.05-1 mL / mg.

[0024] Preferably, the activation conditions are: 50-250 rpm / min, 20-40°C.

[0025] Preferably, the activated algae liquid is inoculated into the culture medium at a ratio of 10% and cultured for 2 to 30 days.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention found that Zofia has the characteristics of heterotrophic growth using organic carbon sources in a dark environment without light, and its biomass is dozens or even hundreds of times that under autotrophic conditions, and its cells accumulate a large amount of high-value carotenoids astaxanthin and oil. The heterotrophic microalgae has an excellent degradation rate for single antibiotics or mixed antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The growth of Zofia sphaeroides when 1 mg / L NOR was added; Figure 1 (a) shows the growth of Zofia chlororaphis under autotrophic conditions; Figure 1 (b) shows the growth of Zofia chlororaphis under heterotrophic conditions.

[0029] Figure 2 The growth of Zofia chlororaphis when 1 mg / LTMP was added; Figure 2 (a) shows the growth of Zofia chlororaphis under autotrophic conditions; Figure 1 (b) shows the growth of Zofia chlororaphis under heterotrophic conditions.

[0030] Figure 3 The growth of Zofia schrenkiana under mixed antibiotic treatment conditions; Figure 3 (a) shows the growth of Zofia chlororaphis under autotrophic conditions; Figure 3 (b) shows the growth of Zofia chlororaphis under heterotrophic conditions. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] Example 1 Activation and expansion of Zofia chlororaphis

[0033] (1) The plate-preserved Chromochloris zofingiensis (ATCC 30412, from the American Type Culture Collection, Rockville, USA) was inoculated into a conical flask containing 20 mL CZ-M1 medium (with 5 g / L glucose added) for activation, and cultured at 150 rpm / min, 2500-2700 lux, 25°C for one week (the color of the algae solution changed significantly). The algae solution cultured for one week was inoculated into a conical flask containing 90 mL CZ-M1 medium at a ratio of 10%, and cultured under the same conditions for about 14 days. The algae solution obtained was the seed solution (cell dry weight greater than 1 g / L).

[0034] The components of the CZ-M1 medium used were: 1500 mg / L NaNO3, 175 mg / L KH2PO4, 150 mg / L MgSO4·7H2O, 75 mg / L K2HPO4, 25 mg / L CaCl2·2H2O, 25 mg / L NaCl, 5 mg / L FeCl3·6H2O, 0.169 mg / L mnSO4·H2O, 0.287 mg / L ZnSO4·7H2O, 0.00124 mg / L (NH4)6Mo7O 24 ·7H2O, 0.061mg / L H3BO3, 0.0025mg / L CuSO4·5H2O, pH adjusted to 6.5.

[0035] Example 2 Degradation of Norfloxacin by Zofia

[0036] 1. Experimental operation

[0037] (1) Weigh 10 mg of norfloxacin (NOR) and dissolve it in 10 mL of solvent (1 mM hydrochloric acid: methanol = 1:1) to prepare an antibiotic stock solution with a concentration of 1 mg / mL, and filter it through a 0.2 μm filter membrane for later use.

[0038] (2) 10 mL of the Zofia seed solution in Example 1 was inoculated into a conical flask containing 90 mL of CZ-M1 medium (without glucose), and 100 μL of the antibiotic stock solution in step (1) was added to obtain an autotrophic experimental group with a final antibiotic concentration of 1 mg / L.

[0039] (3) 10 mL of the Zofia seed solution in Example 1 was inoculated into a conical flask containing 90 mL of CZ-M1 culture medium (with additional 30 g / L glucose), and 100 μL of antibiotic stock solution was added to obtain a heterotrophic experimental group with a final antibiotic concentration of 1 mg / L. The culture group without antibiotics was used as the biological control, and the culture group with only antibiotics but no algae solution was used as the non-biological control group.

[0040] The experimental group was cultured for 14 days at 2500-2700 lux (autotrophic) / darkness (heterotrophic), 150 rpm / min, and 25°C. Every other day, the algae solution of Zofia chlororaphis was collected for monitoring of biomass and antibiotic concentration.

[0041] 2. Experimental results

[0042] The biomass of the autotrophic Zofia green algae was determined by spectrophotometry, specifically: 3 mL of algae solution was taken into a centrifuge tube, and its absorbance at 680 nm was measured by a spectrophotometer. The obtained OD680 was brought into the standard curve to calculate its algae cell dry weight (DCW). The equation of the autotrophic Zofia green algae dry weight-OD680 standard curve is as follows:

[0043] DCW(g / L)=1.0054x-0.025(R 2 =0.9991).

[0044] The biomass of heterotrophic Zofia chlororaphis was determined by dry weight method, specifically: 3 ml of algae solution was placed in a centrifuge tube, centrifuged at 4°C, 6000 rpm / min for 10 min, the supernatant was removed, the precipitate was washed with pure water 3 times, and then filtered with a filter membrane that was previously dried in an oven to constant weight and weighed (weight is m1), and the filter membrane with algae precipitate was obtained, dried at 105°C to constant weight, and finally placed in a drying oven to cool to room temperature and weighed again, and the weight was m2. The cell dry weight was calculated by the following formula:

[0045] DCW (g / L) = (m2-m1) × 1000 / 3.

[0046] The residual NOR concentration in the culture medium was determined by high performance liquid chromatography, with the mobile phase being acetonitrile: 0.025 mol / L phosphoric acid solution (adjusted to pH 3.0 with triethylamine) (13:87, v / v), and the detection wavelength being 278 nm. The peak area of ​​the response of the sample with unknown concentration was detected by high performance liquid chromatography, and the concentration of NOR in the sample could be calculated according to the standard curve equation. The antibiotic removal rate E (remove rate, %) and the degradation rate constant k (d -1 ) is calculated as follows:

[0047] E=[(C0-C t ) / C0]×100%; ln C t =-k t +ln C0

[0048] Where t represents the processing time (d), C0 and C t represent the initial concentration of antibiotic and the concentration on day t, respectively.

[0049] The growth of Zofia Figure 1 As shown in the figure, it can be seen that after adding 1 mg / L of NOR, the growth of autotrophic Zofia was slightly promoted, and the biomass was 1.1 g / L. The growth of heterotrophic Zofia was not significantly affected, and the biomass was as high as 10.3 g / L. The removal of 1 mg / L NOR by Zofia is shown in Table 1. The removal rate of NOR by heterotrophic Zofia is 59.9%, which is significantly better than that of autotrophic Zofia.

[0050] Table 1

[0051] Removal rate E(%) <![CDATA[Degradation reaction rate constant K(d -1 )]]> Non-biological control group 0 0 Autotrophic experimental group 0 0 Heterotrophic experimental group 59.9±0.6 0.07

[0052] Example 3 Degradation of trimethoprim by Zofia chlororaphis

[0053] According to the method in Example 2, a 1 mg / L trimethoprim (TMP) treatment experiment was carried out. The biomass monitoring method of Zofia chlororaphis was the same as in Example 2. The residual TMP concentration in the culture medium was determined by liquid chromatography, the mobile phase was acetonitrile: water (20:80, v / v), and the detection wavelength was 271 nm.

[0054] The growth of Zofia Figure 2 As shown, from Figure 2 It can be seen that the growth of autotrophic Zofia was not significantly affected after adding 1 mg / L TMP, and the biomass was 1.0 g / L. The addition of TMP promoted the growth of heterotrophic Zofia, and the biomass was as high as 11.1 g / L. The removal of 1 mg / L TMP by Zofia is shown in Table 2. The removal rate of TMP by heterotrophic Zofia was 71.1%, which was significantly better than that of autotrophic Zofia.

[0055] Table 2

[0056] Removal rate E(%) <![CDATA[Degradation reaction rate constant K(d -1 )]]> Non-biological control group 0 0 Autotrophic experimental group 0 0 Heterotrophic experimental group 71.1±1.2 0.09

[0057] Example 4: Degradation of antibiotics by Zofia green algae

[0058] According to the method in Example 2, the mother solutions of eight antibiotics including sulfamethoxazole (SMX), sulfamethazine (SMZ), norfloxacin (NOR), trimethoprim (TMP), ciprofloxacin (CIP), levofloxacin (LEV), cephradine (CE) and tetracycline (TC) were prepared, and autotrophic and heterotrophic experimental groups were set up. In each experimental group, 100 μL of each mother solution was added to make the final concentration of each antibiotic in the culture medium 1 mg / L, and the removal of the two sulfonamide antibiotics SMX and SMZ was monitored in particular.

[0059] The biomass monitoring method of Zofia sphaeroides was the same as that in Example 2. The concentrations of the remaining SMX and SMZ in the culture medium were determined by liquid chromatography, the mobile phase was acetonitrile: water (30:70, v / v), and the detection wavelength was 271 nm.

[0060] The growth of Zofia Figure 3 As shown, from Figure 3It can be seen that after adding 1 mg / L of mixed antibiotics, the growth of autotrophic Zofia almost stagnated, and the growth of heterotrophic Zofia was also significantly inhibited, with a biomass of 0.8 g / L. The removal of mixed antibiotics by heterotrophic Zofia is shown in Table 3. It can be seen from Table 3 that heterotrophic Zofia has excellent removal rates for SMX, SMZ and TC in mixed antibiotics.

[0061] Table 3

[0062] SMZ SMX TC Single antibiotic removal rate-autotrophic (%) 0 0 70.9±2.2 Single antibiotic removal rate-heterotrophic (%) 28.4±2.2 38.7±2.9 97.5±0.6 Mixed antibiotic removal rate-autotrophic (%) 27.7±1.5 23.5±1.1 66.6±0.8 Mixed antibiotic removal rate - heterotrophic (%) 97.3±0.8 99.0±1.4 97.2±1.3

[0063] The foregoing examples are merely illustrative and are used to explain some features of the method of the present invention. The appended claims are intended to require the widest possible range that can be imagined, and the embodiments presented herein are demonstrated by the applicant's actual test results. Therefore, the applicant's intention is that the appended claims are not limited by the selection of examples that illustrate the features of the present invention. Some numerical ranges used in the claims also include sub-ranges therein, and changes in these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. The application of Zofia chlororaphis to improve the degradation rate of sulfamethoxazole and sulfadimethoxine in the degradation of mixed antibiotics, characterized in that: The mixed antibiotics include sulfamethoxazole, sulfamethoxazole, norfloxacin, trimethoprim, ciprofloxacin, levofloxacin, cephradine and tetracycline; and the Zofia green algae is cultured under heterotrophic conditions.

2. A method for treating antibiotic wastewater using heterotrophic Zofia chlororaphis, characterized in that: The following steps are involved: S1. Cultivation under heterotrophic conditions: Activating and expanding the Zofia chlororaphis in a culture medium containing an organic carbon source to obtain Zofia chlororaphis seed solution; S2. Zofia green algae seed liquid is mixed with antibiotic wastewater to treat antibiotics; Antibiotic wastewater contains sulfamethoxazole, sulfamethoxazole, norfloxacin, trimethoprim, ciprofloxacin, levofloxacin, cephaladine and tetracycline.

3. The method according to claim 2, characterized in that: The volume-to-mass ratio of the Zofia green algae seed liquid to the antibiotic is 10: 0.01-100 mL / mg.

4. The method according to claim 2, characterized in that: The activation conditions are: 50-250 rpm / min, 20-40°C.

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