Method for purifying tetracycline and producing oil by microalgae across temperature zones

Abstract

The application relates to a method for purifying tetracycline and producing oil through microalgae across temperature zones, and belongs to the field of microalgae bioengineering. The application aims to solve the technical problem of tetracycline pollution in the current cold region water environment. The method comprises the following steps: preparing seed liquid of microalgae culture, inoculating the seed liquid into BG-11 culture medium containing tetracycline, culturing to the late logarithmic growth phase of the microalgae, centrifuging to collect the algal cells, freeze-drying and weighing, and extracting oil in the dry algal powder by using ultrasonic crushing combined with an organic solvent method. The method is simple in operation, can treat low-temperature conditions to produce biological energy, adsorb and degrade tetracycline, improve the stress resistance of microalgae under low-temperature conditions, regulate the permeability of algal cells, and promote the production of microalgae biological energy. The method can be used for purifying tetracycline and producing oil across temperature zones.

Description

Technical Field

[0001] This invention belongs to the field of microalgae bioengineering, specifically relating to a method for cross-temperature zone microalgae to purify tetracycline and produce lipids. Background Technology

[0002] As a broad-spectrum antibiotic, tetracycline has been widely used in medicine, animal husbandry, and aquaculture since its discovery in the mid-20th century due to its broad antibacterial spectrum, well-defined antibacterial mechanism, and low price, playing an important role in the prevention and treatment of infectious diseases. However, with the large-scale production and widespread use of tetracycline, tetracycline and its metabolites have entered the environment through wastewater discharge and agricultural runoff. Due to the long and harsh winters in cold regions, the purification and treatment of tetracycline is extremely difficult. Summary of the Invention

[0003] In order to solve the technical problem of tetracycline pollution in cold-region aquatic environments, this invention provides a method for purifying tetracycline and producing oil efficiently through microalgae across temperature zones.

[0004] This invention provides a method for efficient tetracycline removal and oil production using microalgae across temperature zones. A single oil-rich microalga, R-3, is cultivated. The growth status and oil accumulation of the microalga vary in different regions, particularly in cold regions. This invention addresses the tetracycline and oil accumulation issues observed by the microalgae in different temperature zones, achieving efficient production of both tetracycline and energy using microalgae in various temperature areas.

[0005] A method for purifying tetracycline and efficiently producing oil using microalgae across temperature zones is specifically carried out according to the following steps:

[0006] I. Preparation of seed culture for microalgae;

[0007] 2. Inoculate the seed culture of microalgae prepared in step 1 into BG-11 medium containing tetracycline, and culture at a temperature of 15~35℃.

[0008] 3. Culture the culture medium described in step 2 to the late logarithmic growth stage, centrifuge to collect algal cells, then freeze-dry to obtain freeze-dried algal powder, weigh it, and extract the oil using ultrasonic disruption combined with organic solvent method to complete the process.

[0009] Furthermore, the microalgae mentioned in step one are... Parachlorellakessleri R-3 (18S OM428206; ITSOM432154) was obtained from and preserved in the laboratory of Harbin Institute of Technology.

[0010] Furthermore, the specific steps for preparing the seed culture medium in step one are as follows: inoculate the microalgae into BG-11 medium and culture them until the logarithmic growth phase of the microalgae, which is the seed culture medium.

[0011] Furthermore, the seed culture solution for the microalgae uses glucose as the sole carbon source, with a glucose concentration of 10 g / L.

[0012] Furthermore, the culture conditions for step two are as follows: the pH of the culture medium is 6.8-7.0, the light intensity is 3500-4000 lux, the light-dark time ratio is 12 h:12 h, and the shaking speed is 140-160 r / min.

[0013] Furthermore, the concentration of tetracycline in step two is 1 mg / L.

[0014] Furthermore, the late logarithmic growth phase described in step three refers to 7-9 days after entering the logarithmic growth phase.

[0015] Furthermore, in step three, the centrifugation process for collecting algal cells involves centrifuging at a speed of 8000-10000 r / min for 3-5 minutes to collect the algal cells.

[0016] Furthermore, in step three, the freeze-drying process involves washing the collected algal cells with distilled water, placing them in a -80°C freezer for 12 hours, and then freeze-drying them under vacuum for 48 hours to obtain freeze-dried algal powder.

[0017] Furthermore, in step three, the oil extraction process using ultrasonic disruption combined with organic solvents involves adding freeze-dried algal powder to a chloroform-methanol solution with a volume ratio of chloroform to methanol of 2:1. The algae are ultrasonically disrupted at a power of 200 W until they turn white, and then the supernatant of the organic phase is collected by centrifugation, dried, and weighed.

[0018] Microalgae, as widely distributed tiny organisms in nature, possess advantages such as being environmentally friendly and renewable, growing rapidly, highly adaptable, and capable of efficiently utilizing light energy and fixing carbon, making them a promising candidate for treating tetracycline wastewater. The mechanisms by which microalgae remove tetracycline mainly include abiotic processes (photolysis and hydrolysis) and biological processes (biosorption, biodegradation, and co-metabolism). Biosorption is a crucial mechanism for tetracycline removal, primarily through the secretion of extracellular polymeric substances (EPS) that adsorb tetracycline molecules using physical adsorption methods such as electrostatic attraction. Biodegradation is another important mechanism, where tetracycline undergoes a series of chemical reactions catalyzed by enzyme systems within algal cells, such as decarboxylation, deamination, bond cleavage, and hydroxylation, ultimately breaking down tetracycline into smaller molecules. These degradation products are typically non-toxic or have low toxicity and are further degraded or removed by the environment. Besides treating tetracycline, microalgae can also efficiently produce oil under tetracycline stress, thus achieving clean energy production.

[0019] Beneficial effects of this invention:

[0020] The method of this invention is simple to operate and can utilize microalgae to produce bioenergy under low-temperature conditions, adsorb and degrade tetracycline, while simultaneously improving the stress resistance of microalgae under low-temperature conditions and regulating the permeability of algal cells, thus promoting microalgal bioenergy production. This provides new insights into the use of microalgae for bioenergy production and the treatment of tetracycline pollutants in cold regions.

[0021] This invention provides a method for purifying tetracycline and efficiently producing oil from microalgae across temperature zones, simultaneously achieving bioenergy production and tetracycline removal. This opens up a new perspective and provides a practical solution for the continuous progress and innovative development of the microalgae industry, thereby powerfully promoting the widespread application and in-depth development of microalgae in several key areas such as biomass energy development and environmental protection.

[0022] This invention utilizes microalgae Parachlorellakessleri R-3 provides data and theoretical support for cross-regional energy production and tetracycline removal. Verification showed that efficient oil accumulation was achieved under low-temperature and TC treatments, with the highest oil content reaching 71.25%. This demonstrates that utilizing microalgae to remove tetracycline pollution and produce clean energy across temperature zones is a feasible, economical, and effective method, meeting the basic needs for bioenergy production and water treatment using microalgae in different temperature regions.

[0023] The method of this invention is used to purify tetracycline across temperature zones and produce oil efficiently. Attached Figure Description

[0024] Figure 1 The graph shows the changes in microalgal biomass and biomass yield in the example and control groups;

[0025] Figure 2 The graph shows the oil content and oil yield of microalgae in the examples and control group;

[0026] Figure 3 The graph shows the efficiency of microalgae in removing tetracycline in the examples and control group. Detailed Implementation

[0027] Specific Implementation Method 1: This implementation method describes a method for purifying tetracycline and efficiently producing oil from microalgae across temperature zones, specifically carried out according to the following steps:

[0028] I. Preparation of seed culture for microalgae;

[0029] 2. Inoculate the seed culture of microalgae prepared in step one into BG-11 medium containing tetracycline, and culture at a temperature of 14~36℃.

[0030] 3. Culture the culture medium described in step 2 to the late logarithmic growth stage, centrifuge to collect algal cells, then freeze-dry to obtain freeze-dried algal powder, weigh it, and extract the oil using ultrasonic disruption combined with organic solvent method to complete the process.

[0031] Specific Implementation Method Two: This implementation method differs from Implementation Method One in that the microalgae mentioned in step one are... Parachlorellakessleri R-3. Everything else is the same as in Specific Implementation Method 1.

[0032] Specific Implementation Method 3: This implementation method differs from Implementation Method 1 or 2 in that: Step 1, preparing the seed culture medium for microalgae cultivation, specifically involves inoculating microalgae into BG-11 medium and culturing them until the logarithmic growth phase of the microalgae, which yields the seed culture medium. Everything else is the same as in Specific Implementation Method 1 or 2.

[0033] Specific Implementation Method Four: This implementation method differs from one of Implementation Methods One to Three in that the carbon source used in the seed culture of the microalgae is glucose, and the concentration of glucose is 10 g / L. Everything else is the same as in one of Specific Implementation Methods One to Three.

[0034] Specific Implementation Method Five: This implementation method differs from one of Implementation Methods One to Four in that the culture conditions in step two are: culture medium pH 6.8-7.0, light intensity 3500-4000 lux, light-dark duration ratio 12 h:12 h, and shaking speed 140-160 r / min. All other conditions are the same as in one of Specific Implementation Methods One to Four.

[0035] Specific Implementation Method Six: This implementation method differs from one of Implementation Methods One to Five in that the concentration of tetracycline in step two is 1 mg / L. Everything else is the same as in one of Specific Implementation Methods One to Five.

[0036] Specific Implementation Method Seven: This implementation method differs from one of Implementation Methods One to Six in that the late logarithmic growth phase described in step three is 7-9 days after entering the logarithmic growth phase. Everything else is the same as in one of Specific Implementation Methods One to Six.

[0037] Specific Implementation Method Eight: This implementation method differs from one of Implementation Methods One to Seven in that: in step three, the centrifugation process for collecting algal cells involves centrifuging at a controlled speed of 8000-10000 r / min for 3-5 minutes to collect the algal cells. Everything else is the same as in one of Specific Implementation Methods One to Seven.

[0038] Specific Implementation Method Nine: This implementation method differs from Implementation Methods One to Eight in that: the freeze-drying in step three involves washing the collected algal cells with distilled water, placing them in a -80°C freezer for 12 hours, and then vacuum freeze-drying for 48 hours to obtain freeze-dried algal powder. Everything else is the same as in Specific Implementation Methods One to Eight.

[0039] Specific Implementation Method Ten: This implementation method differs from Implementation Methods One to Nine in that: Step Three uses ultrasonic disruption combined with organic solvent extraction of oil. The procedure is as follows: freeze-dried algal powder is added to a chloroform-methanol solution with a chloroform to methanol volume ratio of 2:1. The algae are ultrasonically disrupted at 200 W until they turn white, then centrifuged, the organic supernatant is collected, dried, and weighed. The rest is the same as in Specific Implementation Methods One to Nine.

[0040] The scope of this invention is not limited to the above-described embodiments; a combination of one or more specific embodiments can also achieve the purpose of the invention.

[0041] Example 1:

[0042] A method for purifying tetracycline and efficiently producing oil using microalgae across temperature zones is specifically carried out according to the following steps:

[0043] I. Preparation of seed culture for microalgae: Using 10 g / L glucose as the carbon source, inoculate microalgae into BG-11 medium and culture until the logarithmic growth phase of the microalgae; this is the seed culture. Parachlorellakessleri R-3;

[0044] 2. Inoculate the seed culture prepared in step 1 into BG-11 medium containing tetracycline at a volume ratio of 10%. Control the culture temperature at 14-16℃, the pH of the medium at 6.8-7.0, the light intensity at 3500-4000 lux, the light-dark ratio at 12 h:12 h, and the shaking speed at 140-160 r / min for culture.

[0045] 3. Culture the culture medium described in step 2 to the late logarithmic growth stage, centrifuge to collect algal cells, then freeze-dry to obtain freeze-dried algal powder, weigh it, and extract the oil using ultrasonic disruption combined with organic solvent method to complete the process.

[0046] The results showed that in the 15℃-TC group of this example, the microalgal biomass was 0.70 g / L, the obtained oil content was 71.25%, and the oil yield was 55.33 mg / L·d. The TC removal efficiency was 90.79%.

[0047] Example 2:

[0048] The difference between this embodiment and Embodiment 1 is that the culture temperature is controlled at 24~26℃ in step two. Everything else is the same as in Embodiment 1.

[0049] The results showed that in the 25℃-TC group of this example, the biomass of microalgae was 7.52 g / L, the obtained oil content was 51.57%, and the oil yield was 431.03 mg / L·d. The TC removal efficiency was 100%.

[0050] Example 3:

[0051] The difference between this embodiment and Embodiment 1 is that the culture temperature is controlled at 34~36℃ in step two. Everything else is the same as in Embodiment 1.

[0052] The results showed that in the 35℃-TC group of this example, the microalgal biomass was 6.83 g / L, the obtained oil content was 48.20%, and the oil yield was 365.65 mg / L·d. The TC removal efficiency was 100%.

[0053] Control group 1:

[0054] The difference between this control group and Example 1 is that in step two, the seed culture prepared in step one was inoculated into BG-11 medium (which does not contain tetracycline). Everything else is the same as in Example 1.

[0055] The results showed that in the control group (15℃-control group), the biomass of microalgae was 0.73 g / L, the obtained oil content was 51.90%, and the oil yield was 41.97 mg / L·d.

[0056] Control group 2:

[0057] The difference between this control group and control group 1 is that the culture temperature in step two was controlled at 24~26℃. Everything else was the same as control group 1.

[0058] The results showed that in the control group (25℃-control group), the biomass of microalgae was 7.47 g / L, the oil content was 42.37%, and the oil yield was 351.70 mg / L·d.

[0059] Control group 3:

[0060] The difference between this control group and control group 1 is that the culture temperature in step two is controlled at 34~36℃. Everything else is the same as control group 1.

[0061] The results showed that in the control group (35℃-control group), the biomass of microalgae was 6.91 g / L, the oil content was 40.90%, and the oil yield was 314.09 mg / L·d.

[0062] The above results indicate that the biomass at a culture temperature of 15 °C was significantly lower than that at 25 °C and 35 °C, with the highest biomass reaching only 0.73 g / L. The biomass in Example 1 also only reached 0.7 g / L. -1There was no significant difference compared to control group 1. In both the control and TC-treated groups, microalgae growth was relatively slow at 15℃, indicating that this temperature resulted in slow algal cell metabolic activity. Biomass was higher at 25℃ than at 35℃. This may be because high temperatures can affect related enzyme activities, leading to a decrease in growth rate; in comparison, 25℃ is the more suitable temperature for microalgae growth. Regarding lipid content, especially at 15℃, Example 1 showed the highest lipid content (71.25%) compared to control group 1. Furthermore, lipid content was lower at both 25℃ and 35℃ than at 15℃. However, it is noteworthy that the lipid content in the TC-treated group was higher than in the control group at both 25℃ and 35℃, possibly because TC directly or indirectly activated metabolic pathways related to lipid synthesis. At both 25℃ and 35℃, TC was completely removed (below the detection limit) on day 9. Even at the low-temperature environment of 15℃, the TC removal efficiency reached 90.79%, indicating a significant effect of microalgae in TC removal.

[0063] Therefore, through Figures 1 - 3 The results regarding the biomass / biomass yield, lipid content / lipid yield, and TC removal efficiency of microalga R-3 provide data and theoretical support for utilizing microalga R-3 to achieve cross-regional energy production and tetracycline removal. Furthermore, Example 1 shows that high-efficiency lipid accumulation was achieved under low temperature and TC treatment, with the highest lipid content reaching 71.25%. Therefore, utilizing microalgae to remove tetracycline pollution across temperature zones and produce clean energy is a feasible, economical, and effective method, meeting the basic needs for bioenergy production and water treatment using microalgae in different temperature regions.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not impose any limitations on the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications, alterations, substitutions and imitations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for microalgae to purify tetracycline and produce lipids, characterized in that... This method is specifically carried out in the following steps: Step 1: Prepare seed culture solution for microalgae culture; Step 2: Inoculate the seed culture of microalgae prepared in Step 1 into BG-11 medium containing tetracycline, and culture at a temperature of 14~16℃ to remove tetracycline.

3. Culture the culture medium described in step 2 to the late logarithmic growth stage, centrifuge to collect algal cells, then freeze-dry to obtain freeze-dried algal powder, weigh it, and extract the oil using ultrasonic disruption combined with organic solvent method to complete the process. The microalgae mentioned in step one are Parachlorella kessleri R-3.

2. The method for purifying tetracycline and producing oils using microalgae according to claim 1, characterized in that... Step 1, preparing the seed culture medium for microalgae culture, involves inoculating microalgae into BG-11 medium and culturing them until the logarithmic growth phase of the microalgae, which yields the seed culture medium.

3. The method for purifying tetracycline and producing oils using microalgae according to claim 2, characterized in that... The seed culture solution for the microalgae uses glucose as the carbon source, with a glucose concentration of 10 g / L.

4. The method for purifying tetracycline and producing oils using microalgae according to claim 1, characterized in that... The culture conditions for step two are as follows: pH of the culture medium is 6.8-7.0, light intensity is 3500-4000 lux, light-dark time ratio is 12 h:12 h, and shaking speed is 140-160 r / min.

5. The method for purifying tetracycline and generating oils using microalgae according to claim 1, characterized in that... The concentration of tetracycline mentioned in step two is 1 mg / L.

6. The method for purifying tetracycline and producing oils using microalgae according to claim 1, characterized in that... Step 3 describes the late logarithmic growth phase, which lasts 7-9 days after the start of the logarithmic growth phase.

7. The method for purifying tetracycline and producing oils using microalgae according to claim 1, characterized in that... Step 3, the centrifugation process for collecting algal cells, involves centrifuging at a speed of 8000-10000 r / min for 3-5 min to collect the algal cells.

8. The method for purifying tetracycline and producing oils using microalgae according to claim 1, characterized in that... Step 3, freeze-drying, involves washing the collected algal cells with distilled water, placing them in a -80°C freezer for 12 hours, and then freeze-drying them under vacuum for 48 hours to obtain freeze-dried algal powder.

9. The method for purifying tetracycline and producing oils using microalgae according to claim 1, characterized in that... Step 3 involves extracting oil using an ultrasonic disruption combined with an organic solvent method. The freeze-dried algal powder is added to a chloroform-methanol solution with a volume ratio of chloroform to methanol of 2:

1. The algae are ultrasonically disrupted at a power of 200 W until they turn white. The supernatant of the organic phase is then collected by centrifugation, dried, and weighed.

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