Method for degrading chloramphenicol by using chlamydomonas reinhardtii-isoleucine metabolism system

Abstract

The invention relates to the technical field of environmental treatment, in particular to a method for degrading chloramphenicol by using a co-metabolism system of chlamydomonas reinhardtii and isoleucine. Isoleucine and chlamydomonas reinhardtii form a co-metabolism system, the degradation efficiency and degradation rate of chlamydomonas reinhardtii on chloramphenicol are greatly improved, high-efficiency degradation of chloramphenicol can be achieved, meanwhile, proliferation and propagation of chloramphenicol drug-resistant genes can be blocked, and the method is a chloramphenicol pollution efficient remediation strategy which is low in price and environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of biodegradation technology, specifically to a method for degrading chloramphenicol using the Chlamydomonas reinhardtii-isoleucine metabolic system. Background Technology

[0002] Chloramphenicol, a highly effective and broad-spectrum chloronitroaromatic antibiotic, has been widely used in human medicine, livestock and poultry farming, and aquaculture. However, related studies have confirmed that chloramphenicol can impair human hematopoietic function, induce aplastic anemia, and has potential carcinogenicity and genotoxicity in humans. In addition, the abuse of chloramphenicol can accelerate the emergence of drug-resistant bacteria and the spread of resistance genes, causing serious negative impacts on the ecological environment.

[0003] Chlamydomonas reinhardtii, a classic model organism among single-celled green algae, possesses significant advantages such as rapid growth rate, simple culture conditions, well-defined genetic background, and the ability to achieve dual transformation between nuclear and chloroplast genomes. It is widely used in basic research fields such as photosynthetic mechanisms, flagellar development regulation, stress physiological responses, and synthetic biology. Meanwhile, this algae is rich in proteins, lutein, polysaccharides, and various bioactive substances, demonstrating broad application potential in industrialization fields such as functional food development, aquatic feed preparation, bioenergy production, environmental remediation, and recombinant medicinal protein expression. It is a high-quality microalgae resource with both significant scientific research value and promising industrialization prospects.

[0004] Traditional methods are ineffective at removing new pollutants such as antibiotics, and the removal of antibiotics using conventional processes is highly complex and uncertain. More seriously, high background levels of antibiotics continuously drive microorganisms in the environment to develop resistance genes, posing a potential threat to ecosystems. Furthermore, the induced multidrug-resistant bacteria can even threaten human health through the food chain. The lag in new drug development and the increasing complexity of drug resistance mechanisms are forcing the academic community to explore remediation pathways for novel antibiotic pollution. Summary of the Invention

[0005] Cometabolism refers to the process by which microorganisms obtain most or all of the carbon and energy sources from other substrates and then degrade organic compounds in the same medium. Based on this, we developed a Chlamydomonas reinhardtii combination formulation, which significantly improves the biodegradation efficiency of chloramphenicol by adding isoleucine to establish a cometabolism system.

[0006] Existing research indicates that traditional processes are not highly effective at removing new pollutants such as antibiotics, and microbial systems that use antibiotics as their sole carbon source also have limited effectiveness in antibiotic removal. Based on this, we developed a Chlamydomonas reinhardtii combination formulation with added isoleucine. Through the co-metabolism system formed by Chlamydomonas reinhardtii and isoleucine, rapid and efficient biodegradation of chloramphenicol is achieved, thereby blocking the generation and spread of antibiotic resistance genes.

[0007] This method can achieve highly efficient degradation of chloramphenicol, and at the same time block the proliferation and spread of antibiotic resistance genes such as chloramphenicol.

[0008] Specifically, the technical solution of the present invention is implemented as follows:

[0009] This invention provides a method for degrading chloramphenicol, wherein the method utilizes a co-metabolism system composed of Chlamydomonas reinhardtii and isoleucine to degrade chloramphenicol.

[0010] Furthermore, the concentration of isoleucine is 0.5 g / L-2 g / L; and the concentration of chloramphenicol is 0.1 mg / L-1 mg / L.

[0011] Furthermore, the application of the aforementioned co-metabolism system in the degradation of chloramphenicol.

[0012] Furthermore, the method for culturing *Chlamydomonas reinhardtii* is as follows: *Chlamydomonas reinhardtii* is cultured in a shaker culture medium under the following conditions: rotation speed 150 rpm, temperature 27 ℃, light-dark ratio 16 h:8 h, and light intensity 10000 lux.

[0013] Furthermore, the culture medium is TAP medium, specifically: 2.420 g of tris(hydroxymethyl)aminomethane, 0.400 g of ammonium chloride, 0.100 g of magnesium sulfate heptahydrate, 0.050 g of calcium chloride dihydrate, 0.108 g of dipotassium hydrogen phosphate, 0.056 g of potassium dihydrogen phosphate, 1.0 mL of trace element stock solution (1000×), and 1.0 mL of glacial acetic acid are added per liter of medium. After sterilization, 1.0 mL of vitamin stock solution (1000×) is added, and the pH value should be 7.0. The vitamin stock solution is prepared in the laboratory. Its ingredients are: Vitamin B12 (0.1 g / L), Inositol (0.1 g / L), Biotin (0.1 g / L), Folic Acid (0.1 g / L), Para-aminobenzoic Acid (1 g / L), Niacin (10 g / L), D-Pantothenic Acid (10 g / L), and Thiamine (20 g / L).

[0014] Furthermore, the cell count of *Chlamydomonas reinhardtii* reached 10. 7 When the cell count is in mL, collect by centrifugation and wash with deionized water before centrifugation.

[0015] Furthermore, the application conditions are as follows: Prepare wastewater containing chloramphenicol at a concentration of 0.1 mg / L-1 mg / L and isoleucine at a concentration of 0.5 g / L-2 g / L, and then wash and centrifuge the *Chlamydomonas reinhardtii* at 10... 5 The cells / mL concentration was inoculated into the co-metabolism system, which, in addition to the usual TAP medium components, carried out the degradation of chloramphenicol at a pH of 6-8 and a temperature of 20-40℃.

[0016] Furthermore, the application conditions are as follows: Prepare wastewater containing chloramphenicol at a concentration of 0.1 mg / L and isoleucine at a concentration of 1 g / L, and then wash and centrifuge the *Chlamydomonas reinhardtii* at a temperature of 10... 5 The cells / mL concentration was inoculated into the co-metabolism system, which, in addition to the usual TAP medium components, carried out the degradation of chloramphenicol at pH 7 and temperature 30°C.

[0017] Furthermore, the cometabolism described in this invention is manifested as follows:

[0018] First, a co-metabolism system was established. *Chlamydomonas reinhardtii* was cultured using TAP medium, prepared as follows: 2.420 g of tris(hydroxymethyl)aminomethane, 0.400 g of ammonium chloride, 0.100 g of magnesium sulfate heptahydrate, 0.050 g of calcium chloride dihydrate, 0.108 g of dipotassium hydrogen phosphate, 0.056 g of potassium dihydrogen phosphate, 1.0 mL of trace element stock solution (1000×), and 1.0 mL of glacial acetic acid were added per liter of medium. After sterilization, 1.0 mL of vitamin stock solution (1000×) was added. The pH of the TAP medium should be 7.0.

[0019] During the cultivation of *Chlamydomonas reinhardtii*, the algal strain was inoculated into 250 mL Erlenmeyer flasks containing 150 mL of TAP medium and placed in a precision combined full-temperature shaking incubator. Specific cultivation conditions were: rotation speed: 150 rpm, temperature: 27℃, light-dark ratio: 16 h:8 h, light intensity: 10000 lux. When the cell count reached 10⁷ cells / mL, the algae were collected by centrifugation and washed with deionized water before centrifugation again.

[0020] To further verify the efficiency of Chlamydomonas reinhardtii in degrading chloramphenicol, this invention selected a total of 22 substrates, including sodium oxalate, sodium bicarbonate, sodium formate, sodium carbonate, glucose, sodium acetate, sucrose, sodium succinate, glutamic acid, glutamine, sodium pyruvate, leucine, arabinose, isoleucine, lysine, histidine, aspartic acid, fructose, asparagine, proline, sodium molybdate, and sodium ascorbate.

[0021] After washing and centrifuging, Chlamydomonas reinhardtii was... 5To establish a cometabolite system for chloramphenicol removal, 22 cometabolite substrates (e.g., isoleucine at 1 g / L) were inoculated at a concentration of cells / mL in different cometabolite systems. In addition to standard TAP medium components, each system contained 22 cometabolite substrates at 1 g / L. Each cometabolite system was exposed to 0.1 mg / L chloramphenicol to verify whether it could enhance the degradation of chloramphenicol by *Chlamydomonas reinhardtii* over a 10-day culture period.

[0022] The results of chloramphenicol degradation efficiency showed that the antibiotic degradation efficiency of the cometabolite system with added isoleucine was significantly improved, increasing the degradation efficiency of chloramphenicol by Chlamydomonas reinhardtii from 62.2% to 99.4%, while other substrates were significantly lower than this percentage.

[0023] Furthermore, the researchers investigated the effect of isoleucine concentration on the degradation of chloramphenicol. They found that by changing the isoleucine concentration in the cometabolite system (0.03 g / L, 0.1 g / L, 0.5 g / L, 1 g / L, 2 g / L), the removal efficiencies of *Chlamydomonas reinhardtii* for 0.1 mg / L chloramphenicol were 69.7%, 74.1%, 85.8%, 99.4%, and 91.6%, respectively. Among these, the highest removal efficiency (99.4%) was observed at an isoleucine concentration of 1 g / L, representing a significant 159% increase compared to the control group.

[0024] Furthermore, to investigate the removal efficiency of chloramphenicol at different concentrations under the optimal isoleucine concentration, this invention screened the removal efficiencies of chloramphenicol concentrations of 0.1 mg / L, 1 mg / L, 10 mg / L, 50 mg / L and 100 mg / L.

[0025] The results of chloramphenicol removal showed that after 6 days of culture with added isoleucine, the removal efficiencies of Chlamydomonas reinhardtii for 0.1 mg / L, 1 mg / L, 10 mg / L, 50 mg / L, and 100 mg / L chloramphenicol were 99.4%, 69.96%, 35.38%, 21.65%, and 19.62%, respectively, which were significantly higher than those in the group without added isoleucine (62.2%, 37.33%, 16.09%, 0.54%, and 4.24%, respectively).

[0026] The algal cell density and chlorophyll content were measured before and after the addition of isoleucine (see attached). Figure 3 Without the addition of isoleucine, the cell densities of *Chlamydomonas reinhardtii* after exposure to different concentrations of chloramphenicol (0.1, 1, 10, 50, 100 mg / L) were: 928.5 × 10⁻⁶. 4 cell / mL, 922.5×10 4 cell / mL, 773.5×10 4cell / mL, 190×10 4 cell / mL and 105×10 4 The cell / mL count and chlorophyll content in the different treatment groups were 24.405 mg / L, 21.949 mg / L, 16.37 mg / L, 3.34 mg / L, and 1.88 mg / L, respectively. However, the addition of isoleucine as a co-metabolite substrate did not increase either the number of algal cells or the chlorophyll content. Therefore, the number of microalgae is not the key driving mechanism affecting the degradation of chloramphenicol by Chlamydomonas reinhardtii promoted by isoleucine; isoleucine may simply enhance the ability of a single alga to remove chloramphenicol.

[0027] The ability of algal cells to remove chloramphenicol was further characterized, and the results showed that the addition of isoleucine significantly promoted the degradation of chloramphenicol at all concentrations. During the six-day culture period, in the experimental group without added co-metabolite substrates, the removal efficiencies of algal cells for chloramphenicol at concentrations of 0.1 mg / L, 1 mg / L, 10 mg / L, 50 mg / L, and 100 mg / L were 0.067% / 102. 5 cells, 0.041% / 10 5 cells, 0.0193% / 10 5 cells, 0.003% / 10 5 cells and 0.041% / 10 5 The removal efficiency of chloramphenicol by a unit algal cell was significantly increased to 0.11% / 10 after the addition of isoleucine. 5 cells, 0.091% / 10 5 cells, 0.048% / 10 5 cells, 0.125% / 10 5 cells, 0.2% / 10 5 Therefore, we believe that the addition of isoleucine improves the biodegradation of chloramphenicol by Chlamydomonas reinhardtii by promoting the removal efficiency per unit algal cell.

[0028] 1) Compared with traditional wastewater treatment processes, the co-metabolism system composed of isoleucine and Chlamydomonas reinhardtii can achieve highly efficient degradation of chloramphenicol, with a degradation efficiency of over 95%.

[0029] 2) The chloramphenicol degradation strategy utilizing a cometabolism system is less prone to secondary pollution and is characterized by low cost and environmental friendliness. It has high potential for environmental remediation.

[0030] 3) Using isoleucine to form a co-metabolism system with Chlamydomonas reinhardtii significantly improves the degradation efficiency and rate of chloramphenicol by Chlamydomonas reinhardtii, enabling high-efficiency degradation of chloramphenicol. At the same time, it can also block the proliferation and spread of chloramphenicol resistance genes, which is a low-cost, environmentally friendly and efficient remediation strategy for chloramphenicol pollution. Attached Figure Description

[0031] Figure 1 Removal kinetics of chloramphenicol (CAP) from Chlamydomonas reinhardtii after addition of 1 g / L co-metabolite substrate.

[0032] Figure 2 Figure A shows the effect of different concentrations (0.03 g / L, 0.1 g / L, 0.5 g / L, 1 g / L, and 2 g / L) of isoleucine on the removal of 0.1 mg / L chloramphenicol by *Chlamydomonas reinhardtii* during a 6-day culture period. It can be seen that isoleucine concentrations between 0.5 g / L and 2 g / L have a more significant effect on the removal of chloramphenicol by *Chlamydomonas reinhardtii*. Figures B, C, D, E, and F show the removal kinetics of different concentrations (0.1 mg / L, 1 mg / L, 10 mg / L, 50 mg / L, and 100 mg / L) of chloramphenicol under conditions with and without 1 g / L isoleucine. It can be seen that chloramphenicol concentrations between 0.1 and 1 mg / L are more effective in removing chloramphenicol from *Chlamydomonas reinhardtii*.

[0033] Figure 3 The effects of different concentrations of chloramphenicol on microalgal cell density (A) and chlorophyll content (B) with and without the addition of isoleucine during a 6-day culture period.

[0034] Figure 4 The removal of different concentrations of chloramphenicol per unit algal cell (10⁵ cells) with and without the addition of 1 g / L isoleucine.

[0035] Figure 5 Targeted metabolomics quantification of chloramphenicol metabolites. Control group consisted of Chlamydomonas reinhardtii alone, 0.1 mg / LCAP group consisted of chloramphenicol plus Chlamydomonas reinhardtii, and 0.1 mg / LCAP plus AA group consisted of chloramphenicol plus co-metabolism system.

[0036] Figure 6 Electrochemical detection: DPV scans of Chlamydomonas reinhardtii exposed to different concentrations of chloramphenicol after the addition of isoleucine. Detailed Implementation

[0037] To make the objectives and technical solutions of this invention clearer, the following embodiments are provided for further explanation. However, the scope of protection of this invention is not limited to these embodiments; the embodiments are merely for illustrative purposes. Those skilled in the art should understand that any changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention.

[0038] Example 1: Cultivation of Chlamydomonas reinhardtii

[0039] Chlamydomonas reinhardtii was cultured using TAP medium. The medium consisted of 2.420 g of tris(hydroxymethyl)aminomethane, 0.400 g of ammonium chloride, 0.100 g of magnesium sulfate heptahydrate, 0.050 g of calcium chloride dihydrate, 0.108 g of dipotassium hydrogen phosphate, 0.056 g of potassium dihydrogen phosphate, 1.0 mL of trace element stock solution (1000×), and 1.0 mL of glacial acetic acid per liter of medium. After sterilization, 1.0 mL of vitamin stock solution (1000×) was added. The pH of the TAP medium should be 7.0. Chlamydomonas reinhardtii was purchased from the Freshwater Algae Culture Collection of the Chinese Academy of Sciences.

[0040] During the cultivation of *Chlamydomonas reinhardtii*, the algal strain was inoculated into 250 mL Erlenmeyer flasks containing 150 mL of TAP medium and placed in a precision combined full-temperature shaking incubator. Specific cultivation conditions were: rotation speed: 150 rpm, temperature: 27℃, light-dark ratio: 16 h:8 h, light intensity: 10000 lux. When the cell count reached 10⁷ cells / mL, the algae were collected by centrifugation and washed with deionized water before centrifugation again.

[0041] Example 2: Investigating the degradation of chloramphenicol by different substrates of Chlamydomonas reinhardtii

[0042] After washing and centrifuging, Chlamydomonas reinhardtii was... 5 The cells / mL were inoculated into different cometabolism systems to establish cometabolism systems for the removal of chloramphenicol. In addition to the standard TAP medium components, each system contained 22 cometabolism substrates at a concentration of 1 g / L.

[0043] The metabolic substrates were sodium oxalate, sodium bicarbonate, sodium formate, sodium carbonate, glucose, sodium acetate, sucrose, sodium succinate, glutamate, glutamine, sodium pyruvate, leucine, arabinose, isoleucine, lysine, histidine, aspartic acid, fructose, asparagine, proline, sodium molybdate, and sodium ascorbate. Each co-metabolite system was exposed to 0.1 mg / L chloramphenicol to verify whether it could improve the degradation of chloramphenicol by Chlamydomonas reinhardtii over a 10-day culture period.

[0044] The results showed that the co-metabolism system with added isoleucine significantly improved the degradation efficiency of chloramphenicol, increasing the degradation efficiency of chloramphenicol by *Chlamydomonas reinhardtii* from 62.2% to 99.4% (see appendix). Figure 1 ).

[0045] Example 3: Investigating the degradation of chloramphenicol by different concentrations of isoleucine in Chlamydomonas reinhardtii

[0046] By varying the isoleucine concentration in the co-metabolism system (0.03 g / L, 0.1 g / L, 0.5 g / L, 1 g / L, 2 g / L), the removal efficiencies of *Chlamydomonas reinhardtii* for 0.1 mg / L chloramphenicol were 69.7%, 74.1%, 85.8%, 99.4%, and 91.6%, respectively. Among these, the highest removal efficiency (99.4%) was observed at an isoleucine concentration of 1 g / L, representing a significant 159% increase compared to the control group (see Appendix). Figure 2 A).

[0047] Example 4: Investigating the degradation of chloramphenicol by different concentrations of the substrate isoleucine-containing Chlamydomonas reinhardtii.

[0048] The algal cell density and chlorophyll content were measured before and after the addition of isoleucine (see attached). Figure 3 Without the addition of isoleucine, the cell densities of *Chlamydomonas reinhardtii* after exposure to different concentrations of chloramphenicol (0.1 mg / L, 1 mg / L, 10 mg / L, 50 mg / L, and 100 mg / L) were 928.5 × 10⁻⁶. 4 cell / mL, 922.5×10 4 cell / mL, 773.5×10 4 cell / mL, 190×10 4 cell / mL and 105×10 4 The cell / mL count and chlorophyll content in the different treatment groups were 24.405 mg / L, 21.949 mg / L, 16.37 mg / L, 3.34 mg / L, and 1.88 mg / L, respectively. However, the addition of isoleucine as a co-metabolite substrate did not increase either the number of algal cells or the chlorophyll content. Therefore, the number of microalgae is not the key driving mechanism affecting the degradation of chloramphenicol by Chlamydomonas reinhardtii promoted by isoleucine; isoleucine may simply enhance the ability of a single alga to remove chloramphenicol.

[0049] Example 5: Investigating the degradation of chloramphenicol by different concentrations of the substrate isoleucine-containing Chlamydomonas reinhardtii.

[0050] The ability of a single algal cell to remove chloramphenicol was further characterized (see appendix). Figure 4The results showed that the addition of isoleucine significantly promoted the degradation of chloramphenicol at all concentrations. During the six-day culture period, in the experimental group without added co-metabolite substrates, the removal efficiencies per algal cell for 0.1, 1, 10, 50, and 100 mg / L chloramphenicol were 0.067, 0.041, 0.0193, 0.003, and 0.041% / 10⁵ cells, respectively. However, after the addition of isoleucine, the removal efficiencies per algal cell for different concentrations of chloramphenicol significantly increased to 0.11, 0.091, 0.048, 0.125, and 0.2% / 10⁵ cells. Therefore, we conclude that the addition of isoleucine improved the biodegradation of chloramphenicol by *Chlamydomonas reinhardtii* by promoting the removal efficiency per algal cell.

[0051] Example 6: Targeted Metabolomics Analysis of the Co-metabolite System

[0052] A total of 182 metabolites were identified in each group (the control group with only *Chlamydomonas reinhardtii*, the experimental group with *Chlamydomonas reinhardtii* + 0.1 mg / L chloramphenicol, and the experimental group with the co-metabolism system + 0.1 mg / L chloramphenicol). Compared with the control group (the control group with only *Chlamydomonas reinhardtii*), 79 metabolites were significantly upregulated after the addition of isoleucine as a co-metabolism substrate. Among them, 4-citrullinated butyrate, glycine, and folic acid were significantly upregulated by 43.83, 8.52, and 8.46 times, respectively.

[0053] The results also showed that these pathways can significantly promote metabolic degradation into the TCA cycle, thereby achieving complete removal of chloramphenicol (see appendix). Figure 5 ).

[0054] Example 7: Electrochemical detection to verify extracellular current in algal cells

[0055] Chlamydomonas reinhardtii was cultured for 6 days in different concentrations of chloramphenicol (0.1, 1, 10, 50, and 100 mg L⁻¹), followed by the addition of isoleucine. The current values ​​after isoleucine addition were 30.93, 18.49, 39.48, 18.09, and 19.21 μA, respectively, significantly higher than the control group (14.18 μA). The addition of isoleucine improved extracellular electron transport in microalgae (see appendix). Figure 6 ).

Claims

1. A method for degrading chloramphenicol, characterized in that: The method utilizes a co-metabolism system composed of Chlamydomonas reinhardtii and isoleucine to degrade chloramphenicol.

2. The method as described in claim 1, characterized in that: The concentration of isoleucine is 0.5 g / L-2 g / L; the concentration of chloramphenicol is 0.1 mg / L-1 mg / L.

3. The method as described in claim 1, characterized in that: The application of the aforementioned cometabolite system in the degradation of chloramphenicol.

4. The method as described in claim 1, characterized in that: The method for culturing *Chlamydomonas reinhardtii* is as follows: *Chlamydomonas reinhardtii* is cultured in a shaker culture medium under the following conditions: rotation speed 150 rpm, temperature 27 ℃, light-dark ratio 16 h:8 h, and light intensity 10000 lux.

5. The method as described in claim 1, characterized in that: The culture medium is TAP medium, specifically: 2.420 g of tris(hydroxymethyl)aminomethane, 0.400 g of ammonium chloride, 0.100 g of magnesium sulfate heptahydrate, 0.050 g of calcium chloride dihydrate, 0.108 g of dipotassium hydrogen phosphate, 0.056 g of potassium dihydrogen phosphate, 1.0 mL of trace element stock solution (1000×), and 1.0 mL of glacial acetic acid are added per liter of medium. After sterilization, 1.0 mL of vitamin stock solution (1000×) is added. The pH value should be 7.

0.

6. The method as described in claim 5, characterized in that: The number of cells in *Chlamydomonas reinhardtii* reached 10. 7 When the cell count is in mL, collect by centrifugation and wash with deionized water before centrifugation.

7. The method as described in claim 1, characterized in that: The application conditions are as follows: Prepare wastewater with chloramphenicol concentrations of 0.1 mg / L-1 mg / L and isoleucine concentrations of 0.5 g / L-2 g / L, and then wash and centrifuge the *Chlamydomonas reinhardtii* at 10... 5 The cells / mL concentration was inoculated into the co-metabolism system, which, in addition to the usual TAP medium components, carried out the degradation of chloramphenicol at a pH of 6-8 and a temperature of 20-40℃.

8. The method as described in claim 1, characterized in that: The application conditions are as follows: Prepare wastewater containing chloramphenicol at a concentration of 0.1 mg / L and isoleucine at a concentration of 1 g / L, and then wash and centrifuge the *Chlamydomonas reinhardtii* at a temperature of 10... 5 The cells / mL concentration was inoculated into the co-metabolism system, which, in addition to the usual TAP medium components, carried out the degradation of chloramphenicol at pH 7 and temperature 30°C.

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