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

Abstract

The invention discloses a method for purifying tetracycline and efficiently producing oil by using microalgae in a cross-temperature zone, and belongs to the field of microalgae bioengineering. The invention aims to solve the technical problem of tetracycline pollution in the current cold region water environment. The method comprises the following steps: preparing a seed solution for microalgae culture, inoculating the seed solution into a BG-11 culture medium containing tetracycline, culturing until the logarithmic growth later stage of microalgae, centrifugally collecting algae cells, freeze-drying and weighing, and extracting grease in dry algae powder by utilizing an ultrasonication combined organic solvent method. The method disclosed by the invention is simple to operate, can be used for producing biological energy and adsorbing and degrading tetracycline under the low-temperature treatment condition by utilizing the microalgae, meanwhile, the stress resistance of the microalgae under the low-temperature condition is improved, the permeability of algae cells is regulated and controlled, and the production of the microalgae biological energy is promoted. The method disclosed by the invention is used for purifying tetracycline in cross-temperature zones and efficiently producing oil.

Description

Technical Field

[0001] The invention belongs to the field of microalgae bioengineering, and in particular relates to a method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae. Background Art

[0002] As a broad-spectrum antibiotic, tetracycline has been widely used in medicine, animal husbandry and aquaculture since it was discovered in the mid-20th century due to its broad antibacterial spectrum, clear antibacterial mechanism and low price. It has played 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, agricultural runoff and other pathways, becoming emerging pollutants. In addition, with the rapid development of the economy, the problem of energy shortage has become increasingly prominent, which not only threatens energy security, but also aggravates greenhouse gas emissions and poses a great threat to the earth's ecological environment. Microalgae biodiesel, as a clean, environmentally friendly and renewable alternative energy source, is gradually becoming a research hotspot in the field of biodiesel production.

[0003] With regard to the tetracycline pollution and resource shortage problems faced by different regions (cold regions, medium-temperature regions, and high-temperature regions), special attention is paid to the cold regions, whose unique geographical structure and climatic conditions make the problem of tetracycline pollution in the water environment more prominent. Due to the long and severe winters in the cold regions, this low temperature environment aggravates the accumulation effect of pollutants and significantly increases the complexity and difficulty of purification treatment. What is more serious is that the water ecosystems in the cold regions are inherently more sensitive and fragile, and the biodiversity is relatively low. Therefore, antibiotic pollutants such as tetracycline are more destructive to such ecosystems and difficult to reverse. In addition, the problem of energy shortage in the cold regions is also one of the main threats facing the cold regions. Summary of the invention

[0004] In order to solve the current technical problem of tetracycline pollution in cold zone water environment, the present invention provides a method for purifying tetracycline and efficiently producing oil by using cross-temperature zone microalgae.

[0005] The present invention provides a method for purifying tetracycline and efficiently producing oil by using microalgae across temperature zones. An oil-rich microalgae R-3 is used for cultivation. The growth state and oil accumulation of microalgae in different regions are different, especially in cold regions. The present invention processes tetracycline and oil accumulation of microalgae in different temperature zones to achieve efficient production of tetracycline removal and energy by using microalgae in different temperature zones.

[0006] A method for purifying tetracycline and efficiently producing oil by cross-temperature microalgae, which is specifically carried out in the following steps:

[0007] 1. preparing seed solution for microalgae culture;

[0008] 2. Inoculate the seed solution of microalgae culture prepared in step 1 into BG-11 medium containing tetracycline, control the temperature to 15-35° C., and culture;

[0009] 3. Cultivate the culture medium in step 2 to the late logarithmic growth stage, collect algal cells by centrifugation, and then freeze-dry to obtain freeze-dried algal powder, weigh it, and extract oil by ultrasonic crushing combined with organic solvent method to complete.

[0010] Furthermore, the microalgae in step 1 is Parachlorella kessleri R-3 (18S OM428206; ITSOM432154), which is obtained from and preserved in the laboratory of Harbin Institute of Technology.

[0011] Furthermore, step 1 of preparing the seed solution for culturing microalgae is specifically as follows: inoculating the microalgae into the BG-11 culture medium and culturing the microalgae until the logarithmic growth phase, which is the seed solution.

[0012] Furthermore, the only carbon source used in the seed solution for culturing the microalgae is glucose, and the concentration of glucose is 10 g / L.

[0013] Furthermore, the culture conditions in step 2 are: the pH of the culture medium is 6.8-7.0, the light intensity is 3500-4000 lux, the light-dark time ratio is 12h:12h, and the shaking speed is 140-160r / min.

[0014] Furthermore, the concentration of tetracycline in step 2 is 1 mg / L.

[0015] Furthermore, the late logarithmic growth period in step 3 is 7d-9d into the logarithmic growth period.

[0016] Furthermore, in step 3, the algae cells are collected by centrifugation for 3-5 minutes at a controlled speed of 8000-10000 r / min to collect the algae cells.

[0017] Furthermore, the freeze-drying in step three is to wash the collected algae cells with distilled water, place them in a -80°C refrigerator for 12 hours, and then vacuum freeze-dry them for 48 hours to collect freeze-dried algae powder.

[0018] Furthermore, step three uses ultrasonic crushing combined with organic solvent method to extract oil. The operation is as follows: freeze-dried algae powder is added to chloroform-methanol solution, the volume ratio of chloroform to methanol in the chloroform-methanol solution is 2:1, ultrasonic crushing is performed at a power of 200 W until the algae turns white, and then the organic phase supernatant is collected by centrifugation, dried and weighed.

[0019] Microalgae, as a microorganism widely found in nature, has the advantages of environmental protection, renewability, fast growth rate, strong adaptability, efficient use of light energy and carbon fixation, and has great potential in treating tetracycline wastewater. The mechanisms of microalgae to remove tetracycline mainly include abiotic processes (photolysis and hydrolysis) and biological processes (biosorption, biodegradation and co-metabolism). Among them, biosorption is an important way to remove tetracycline, mainly through the secretion of extracellular polymers (EPS) to adsorb tetracycline molecules by physical adsorption such as electrostatic attraction; biodegradation is another important mechanism for microalgae to remove tetracycline. Tetracycline is catalyzed by the enzyme system in algae cells to undergo a series of chemical reactions, such as decarboxylation, deamination, bond cleavage and hydroxylation, and finally decomposes tetracycline into small molecules. The degradation products of these small molecules are usually non-toxic or less toxic, and are further degraded or removed by the environment. In addition to treating tetracycline, microalgae can also efficiently produce oil under tetracycline stress and achieve the production of clean energy at the same time.

[0020] Beneficial effects of the present invention:

[0021] The method of the present invention is simple to operate, and can utilize microalgae to produce bioenergy under low temperature conditions, adsorb and degrade tetracycline, and at the same time improve the stress resistance of microalgae under low temperature conditions and regulate the permeability of algae cells, thereby promoting microalgae bioenergy production. This provides new insights into the use of microalgae to produce bioenergy and treat tetracycline pollutants in cold regions.

[0022] The present invention provides a method for purifying tetracycline and efficiently producing oil by cross-temperature microalgae, while achieving the production of bioenergy and the removal of tetracycline, opening up a new perspective and providing a practical solution for the continuous progress and innovative development of the microalgae industry, thereby effectively promoting the extensive application and in-depth development of microalgae in multiple key fields such as biomass energy development and environmental protection.

[0023] The present invention provides data support and theoretical support for realizing cross-region energy production and tetracycline removal by using microalgae Parachlorella kessleri R-3. It has been verified that under low temperature and TC treatment, efficient accumulation of oil is achieved, and the highest oil content can reach 71.25%. It can be seen that the removal of tetracycline pollution and the production of clean energy by using microalgae across temperature zones is a feasible, economical and effective method, which meets the basic needs of using microalgae to produce bioenergy and water treatment in different temperature zones.

[0024] The method of the invention is used for purifying tetracycline across temperature zones and producing oil with high efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a graph showing the changes in microalgae biomass and biomass yield in the embodiment and the control group;

[0026] Figure 2 Graph showing the microalgae oil content and oil yield in the example and control groups;

[0027] Figure 3 Graph showing the efficiency of tetracycline removal using microalgae in the example and control group. DETAILED DESCRIPTION

[0028] Specific implementation method 1: This implementation method is a method for purifying tetracycline and efficiently producing oil by microalgae across temperature zones, which is specifically carried out in the following steps:

[0029] 1. preparing seed solution for microalgae culture;

[0030] 2. Inoculate the seed solution of microalgae culture prepared in step 1 into BG-11 medium containing tetracycline, control the temperature to 14-36° C., and culture;

[0031] 3. Cultivate the culture medium in step 2 to the late logarithmic growth stage, collect algal cells by centrifugation, and then freeze-dry to obtain freeze-dried algal powder, weigh it, and extract oil by ultrasonic crushing combined with organic solvent method to complete.

[0032] Specific embodiment 2: This embodiment is different from the first embodiment in that the microalgae in step 1 is Parachlorella kessleri R-3. The rest is the same as the first embodiment.

[0033] Specific implementation method 3: This implementation method is different from implementation method 1 or 2 in that: Step 1 of preparing the seed solution for microalgae culture is as follows: inoculating the microalgae into BG-11 culture medium and culturing the microalgae to the logarithmic growth phase, which is the seed solution. The rest is the same as specific implementation method 1 or 2.

[0034] Specific embodiment 4: This embodiment differs from Embodiments 1 to 3 in that the carbon source used in the seed solution for culturing microalgae is glucose, and the concentration of glucose is 10 g / L. Other aspects are the same as those of Specific Embodiments 1 to 3.

[0035] Specific embodiment 5: This embodiment differs from Embodiments 1 to 4 in that: the culture conditions in step 2 are: the pH of the culture medium is 6.8-7.0, the light intensity is 3500-4000 lux, the light-dark time ratio is 12h:12h, and the shaking speed is 140-160r / min. The rest is the same as Specific Embodiments 1 to 4.

[0036] Specific implementation method 6: This implementation method is different from implementation methods 1 to 5 in that the concentration of tetracycline in step 2 is 1 mg / L. The rest is the same as implementation methods 1 to 5.

[0037] Specific implementation method 7: This implementation method is different from one of the first to sixth implementation methods in that the late logarithmic growth period in step 3 is 7d-9d into the logarithmic growth period. The rest is the same as one of the first to sixth implementation methods.

[0038] Specific embodiment 8: This embodiment differs from Embodiments 1 to 7 in that: the centrifugal treatment to collect algae cells in step 3 is to collect algae cells by centrifugation at a speed of 8000-10000 r / min for 3-5 minutes. The rest is the same as Specific embodiments 1 to 7.

[0039] Specific embodiment 9: This embodiment is different from the first to eighth embodiments in that the freeze drying in step 3 is to wash the collected algae cells with distilled water, keep them in a -80°C refrigerator for 12 hours, and then vacuum freeze-dry them for 48 hours to collect freeze-dried algae powder. The rest is the same as the first to eighth embodiments.

[0040] Specific embodiment 10: This embodiment differs from Embodiments 1 to 9 in that: Step 3 uses ultrasonic crushing combined with organic solvent method to extract oil and fat as follows: freeze-dried algae powder is added to a chloroform-methanol solution, wherein the volume ratio of chloroform to methanol in the chloroform-methanol solution is 2:1, ultrasonic crushing is performed at a power of 200W until the algae turns white, and then the organic phase supernatant is collected by centrifugation, dried and weighed. The rest is the same as Specific Embodiments 1 to 9.

[0041] The content of the present invention is not limited to the content of the above-mentioned embodiments. The combination of one or several specific embodiments can also achieve the purpose of the invention.

[0042] Embodiment 1:

[0043] A method for purifying tetracycline and efficiently producing oil by cross-temperature microalgae, which is specifically carried out in the following steps:

[0044] 1. Prepare seed solution for microalgae culture; use 10 g / L glucose as carbon source, inoculate microalgae into BG-11 medium, and culture until the microalgae is in logarithmic growth phase, which is the seed solution; the microalgae is Parachlorella kessleri R-3;

[0045] 2. Inoculate the seed solution prepared in step 1 into the BG-11 medium containing tetracycline at 10% of the volume, control the culture temperature to 14-16°C, the medium pH to 6.8-7.0, the light intensity to 3500-4000 lux, the light-dark time ratio to 12h:12h, and the shaking speed to 140-160r / min for cultivation;

[0046] 3. Cultivate the culture medium in step 2 to the late logarithmic growth stage, collect algal cells by centrifugation, and then freeze-dry to obtain freeze-dried algal powder, weigh it, and extract oil by ultrasonic crushing combined with organic solvent method to complete.

[0047] The results showed that in the 15℃-TC group of this example, the biomass of microalgae was 0.70 g / L, the obtained oil content was 71.25%, the oil yield was 55.33 mg / L·d, and the TC removal efficiency was 90.79%.

[0048] Embodiment 2:

[0049] The difference between this embodiment and embodiment 1 is that the culture temperature in step 2 is controlled to be 24-26° C. The rest is the same as embodiment 1.

[0050] 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%, the oil yield was 431.03 mg / L·d, and the TC removal efficiency was 100%.

[0051] Embodiment 3:

[0052] The difference between this embodiment and embodiment 1 is that the culture temperature in step 2 is controlled to be 34-36° C. The rest is the same as embodiment 1.

[0053] The results showed that in the 35℃-TC group of this example, the biomass of microalgae was 6.83 g / L, the obtained oil content was 48.20%, the oil yield was 365.65 mg / L·d, and the TC removal efficiency was 100%.

[0054] Control group 1:

[0055] The difference between this control group and Example 1 is that in step 2, the seed solution prepared in step 1 is inoculated into BG-11 medium (not containing tetracycline). The rest is the same as Example 1.

[0056] 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.

[0057] Control group 2:

[0058] The difference between this control group and control group 1 is that the culture temperature in step 2 is controlled at 24-26° C. The rest is the same as control group 1.

[0059] The results showed that in the control group (25℃-control group), the biomass of microalgae was 7.47 g / L, the oil content obtained was 42.37%, and the oil yield was 351.70 mg / L·d.

[0060] Control group 3:

[0061] The difference between this control group and control group 1 is that the culture temperature in step 2 is controlled at 34-36° C. The rest is the same as control group 1.

[0062] The results showed that in the control group 35℃-control group, the biomass of microalgae was 6.91g / L, the obtained oil content was 40.90%, and the oil yield was 314.09mg / L·d.

[0063] The above results show that when the culture temperature is 15°C, the biomass is significantly lower than that at the culture temperatures of 25°C and 35°C, and the highest biomass is only 0.73 g / L. The biomass of Example 1 is only 0.7 g / L. -1 , and there was no significant difference with the control group 1. Whether it was the control group or the TC treatment group, the growth of microalgae under 15℃ culture was relatively slow, which indicated that under this temperature condition, the metabolic activity of algae cells was slow. When the culture temperature was 25℃, the biomass was higher than that at 35℃. This may be because high temperature may affect the activity of related enzymes, resulting in a decrease in growth rate. In contrast, 25℃ is a more suitable temperature for the growth of microalgae. As for the oil content, especially at 15℃, the oil content of Example 1 can reach the highest, which is 71.25%, compared with the control group 1. In addition, the oil content under the culture conditions of 25℃ and 35℃ was lower than that under the culture condition of 15℃, but it is worth noting that the oil content of the TC treatment group was higher than that of the control group, whether it was 25℃ or 35℃, which may be because TC directly or indirectly activated the metabolic pathways related to oil synthesis. Under the conditions of 25℃ and 35℃, TC was completely removed (below the detection limit) on the 9th day of culture. Even under the low temperature environment of 15℃ culture conditions, the removal efficiency of TC can reach 90.79%, which indicates that microalgae has a significant effect in TC removal.

[0064] Therefore, through Figure 1-3 In the results of the biomass / biomass yield, oil content / oil yield and TC removal efficiency of microalgae R-3, these results provide data support and theoretical support for the use of microalgae R-3 to achieve cross-regional energy production and tetracycline removal. In addition, it can be seen from Example 1 that under low temperature and TC treatment, efficient accumulation of oil is achieved, and the highest oil content can reach 71.25%. It can be seen that the use of microalgae to achieve the removal of tetracycline pollution across temperature zones and the production of clean energy is a feasible, economical and effective method that meets the basic needs of using microalgae to produce bioenergy and water treatment in different temperature zones.

[0065] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications, changes, replacements 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 purifying tetracycline and efficiently producing oil by cross-temperature microalgae, characterized in that The method is specifically carried out in the following steps:

1. preparing seed solution for microalgae culture; 2. Inoculate the seed solution of microalgae culture prepared in step 1 into BG-11 medium containing tetracycline, control the temperature to 14-36° C., and culture; 3. Cultivate the culture medium in step 2 to the late logarithmic growth stage, collect algal cells by centrifugation, and then freeze-dry to obtain freeze-dried algal powder, weigh it, and extract oil by ultrasonic crushing combined with organic solvent method to complete.

2. The method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae according to claim 1, characterized in that The microalgae described in step 1 is Parachlorella kessleri R-3.

3. The method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae according to claim 1, characterized in that Step 1: preparing the seed solution for culturing microalgae. Specifically, the microalgae are inoculated into BG-11 culture medium and cultured until the microalgae are in the logarithmic growth phase, which is the seed solution.

4. The method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae according to claim 3, characterized in that The carbon source used in the seed solution for culturing the microalgae is glucose, and the concentration of glucose is 10 g / L.

5. The method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae according to claim 1, characterized in that The culture conditions of step 2 are: the pH of the culture medium is 6.8-7.0, the light intensity is 3500-4000 lux, the light-dark time ratio is 12h:12h, and the shaking speed is 140-160r / min.

6. The method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae according to claim 1, characterized in that The concentration of tetracycline in step 2 is 1 mg / L.

7. The method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae according to claim 1, characterized in that The late logarithmic growth phase in step 3 is 7d-9d into the logarithmic growth phase.

8. The method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae according to claim 1, characterized in that In step 3, the algae cells are collected by centrifugation for 3-5 minutes at a controlled speed of 8000-10000 r / min to collect the algae cells.

9. The method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae according to claim 1, characterized in that The freeze-drying in step three is to wash the collected algae cells with distilled water, place them in a -80°C refrigerator for 12 hours, and then vacuum freeze-dry them for 48 hours to collect freeze-dried algae powder.

10. The method for purifying tetracycline and efficiently producing oil by using cross-temperature microalgae according to claim 1, characterized in that Step three uses ultrasonic crushing combined with organic solvent method to extract oil. The operation is as follows: add freeze-dried algae powder into chloroform-methanol solution, the volume ratio of chloroform to methanol in the chloroform-methanol solution is 2:1, and ultrasonic crush at a power of 200W until the algae turns white, then centrifuge to collect the organic phase supernatant, dry and weigh.

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