System and method for improving oil yield and cadmium removal rate of microalgae under cadmium stress

Abstract

The invention discloses a system and a method for improving oil yield and cadmium removal rate of microalgae under cadmium stress, and belongs to the technical field of biological engineering. According to the method, a mixotrophic culture mode is adopted, and algae species mixotrophic culture is carried out, so that algae cells rapidly grow in a short time and are cultured to a logarithmic growth expectation for later use; and applying cadmium stress by using the same mixotrophic mode, and simultaneously adding exogenous fulvic acid to induce microalgae to grow and accumulate grease. According to the method, a mixotrophic culture mode is adopted, under the stress of 0.2 mu M Cd, when the concentration of fulvic acid is 40mg / L, the biomass, biomass yield and grease yield of the microalgae are respectively improved by 20.22%, 20.32% and 26.55% compared with those of single cadmium treatment, and the cadmium removal rate is 100% and is improved by 16.6% compared with that of single separation treatment. According to the method, the biomass and grease yield of the microalgae can be improved, meanwhile, removal of cadmium is increased, and a new theoretical basis is provided for co-production of grease by treating cadmium-containing wastewater through the microalgae.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a system and method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress. Background Art

[0002] Biodiesel is currently the most promising new energy source to replace fossil fuels. Microalgae can efficiently use light energy to convert CO2 in the atmosphere into 2 Efficient fixation and conversion into biomass is a highly advantageous biofuel. Monospinella can adapt to a variety of cultivation modes, such as autotrophic, mixotrophic and heterotrophic, and can also accumulate energy storage substances such as oils and carbon water.

[0003] The traditional methods of inducing microalgae to synthesize oils today mainly include abiotic stress and chemical induction. However, abiotic stress usually inhibits the photosynthetic efficiency and growth of algae cells in the process of improving oil synthesis, resulting in a decrease in the overall biomass and oil yield of the microalgae. Cultivating microalgae with heavy metal-containing wastewater can absorb heavy metals and organic matter such as nitrogen and phosphorus in the wastewater, and stimulate microalgae to synthesize oils. However, directly culturing microalgae with culture media containing heavy metals will also inhibit the activity of algae cells and reduce the overall yield. Therefore, it is urgent to find methods to improve the oil yield and stress resistance of microalgae. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the present invention provides a system and method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress comprises the following steps:

[0007] (1) adding Monopterus sphaeroides to BG-11 medium with glucose as carbon base to obtain seed solution;

[0008] (2) adding cadmium and fulvic acid to a BG-11 medium with glucose as the carbon base, and then adding seed solution to obtain a culture solution;

[0009] (3) extracting oil from the algae of the monophyll sp. in the culture solution using an organic solvent to obtain the oil in the algae cells in the culture solution;

[0010] (4) Detect the cadmium concentration in the culture medium and obtain the cadmium removal rate.

[0011] As a preferred embodiment of the present invention, in step (1) and step (2), the culture temperature is 25±1°C, the culture time is 9 days, and the light intensity is 30 μmol m -2 s -1 .

[0012] As a preferred embodiment of the present invention, in step (2), the inoculation concentration of Mononema is 0.1 g / L; the concentration of cadmium is 0.2 μM; and the concentration of fulvic acid is 10-40 mg / L.

[0013] As a preferred embodiment of the present invention, in step (3), the oil extraction specifically includes: centrifuging the culture solution to separate the monotrichia algae, then freeze-drying the monotrichia algae to obtain dry algae powder, and adding a mixed organic solvent of chloroform and methanol to the dry algae powder to extract the oil.

[0014] As a preferred embodiment of the present invention, the volume ratio of chloroform to methanol in the mixed organic solvent of chloroform and methanol is 2:1.

[0015] A system for improving the oil yield and cadmium removal rate of microalgae under cadmium stress, comprising: a microalgae cultivation module, a cadmium and fulvic acid addition module, an oil extraction module, a cadmium removal analysis module and a data analysis module;

[0016] The microalgae cultivation module cultivates microalgae and monitors the growth of microalgae in real time by controlling temperature, lighting, and ventilation;

[0017] The cadmium and fulvic acid adding module adds cadmium and fulvic acid during the process of cultivating microalgae in the microalgae cultivation module;

[0018] The oil extraction module is a microalgae cultivation module that cultivates microalgae for separation, collection, crushing and oil extraction;

[0019] The cadmium removal analysis module detects and analyzes the cadmium in the culture medium after cultivation and calculates the cadmium removal rate;

[0020] The data analysis module collects the growth rate and biomass of microalgae in the microalgae cultivation module, the concentration of cadmium and fulvic acid in the cadmium and fulvic acid addition module, the cadmium removal rate in the cadmium removal analysis module, and the content and yield of oil in the oil extraction module for data analysis and processing.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention adopts a mixotrophic culture mode to make algae cells grow rapidly in a short period of time, and culture them to logarithmic growth before use; then, using the same mixotrophic mode, cadmium stress is applied, and exogenous fulvic acid is added to induce microalgae growth and oil accumulation. The present invention adopts a mixotrophic culture mode. Under 0.2μM Cd stress, when the fulvic acid concentration is 40mg / L, the biomass, biomass yield and oil yield of the microalgae are increased by 20.22%, 20.32% and 26.55% respectively compared with the single cadmium treatment, and the cadmium removal rate is 100%. The present invention can not only increase the biomass and oil yield of microalgae, but also increase the removal of cadmium, providing a new theoretical basis for using microalgae to treat cadmium-containing wastewater and produce oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention is a flow chart of the method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress.

[0023] Figure 2 1-2 and Comparative Examples 1-3 provide a graph showing the effects of different concentrations of fulvic acid coupled with cadmium stress on microalgae biomass and biomass yield.

[0024] Figure 3 1-2 and Comparative Examples 1-3 provide a graph showing the effects of different concentrations of fulvic acid coupled with cadmium stress on the lipid content and lipid yield of microalgae.

[0025] Figure 4 Graph showing the efficiency of microalgae in removing cadmium in Examples 1-2 of the present invention and Comparative Examples 2-3.

[0026] In the figure: the control is comparative example 1, 0.2 μM cadmium is comparative example 3, 0.2 μM cadmium and 20 mg L -1 Fulvic acid is Example 1, 0.2 μM cadmium and 40 mg L -1 Fulvic acid is Example 2, 0.2 μM cadmium and 80 mg L -1 Fulvic acid is comparative example 2. DETAILED DESCRIPTION

[0027] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0028] A system for improving the oil yield and cadmium removal rate of microalgae under cadmium stress, comprising: a microalgae cultivation module, a cadmium and fulvic acid addition module, an oil extraction module, a cadmium removal analysis module and a data analysis module;

[0029] The microalgae cultivation module is used to cultivate microalgae and monitor the growth of microalgae in real time by controlling temperature, lighting, and ventilation;

[0030] The cadmium and fulvic acid adding module is used to add cadmium and fulvic acid during the process of cultivating microalgae, and then cultivate the microalgae in the microalgae cultivation module;

[0031] The oil extraction module is used to separate, collect, crush and extract oil from the microalgae cultivated in the microalgae cultivation module;

[0032] The cadmium removal analysis module is used to detect and analyze the cadmium in the culture medium after cultivation and calculate the cadmium removal rate;

[0033] The data analysis module collects the growth rate and biomass of microalgae in the microalgae cultivation module, the concentration of cadmium and fulvic acid in the cadmium and fulvic acid addition module, the cadmium removal rate in the cadmium removal analysis module, and the content and yield of oil in the oil extraction module for data analysis and processing.

[0034] Example 1

[0035] A method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress comprises the following steps:

[0036] (1) Add BG-11 medium containing 10 g / L glucose as the carbon base into a 500 mL Erlenmeyer flask, and then add Monoraphidium sp. QLZ-3 at 25 ± 1 °C and 30 μmol m -2 s -1 The seed solution was obtained after 9 days of cultivation under light intensity.

[0037] (2) Add 0.2 μM cadmium and 20 mg / L fulvic acid to BG-11 medium with 10 g / L glucose as the carbon base, and then add seed solution at 25 ± 1 °C and 30 μmol m -2 s -1 The culture solution was obtained by culturing for 9 days under the light intensity, and the inoculation amount of Monoraphidium sp. QLZ-3 was 0.1 g / L.

[0038] (3) The culture medium was centrifuged at 7000 r / min for 5 minutes, and the algae were collected in a 50 mL centrifuge tube, frozen at -80 °C overnight, and then freeze-dried in a freeze dryer for 24 hours before weighing; the dry weight of the algae cells was weighed as w (g); the microalgae biomass (dry cell weight) was the dry weight of the algae cells minus the volume of the algae solution. Weigh 0.16g of dry algae powder, then weigh twice the weight of quartz sand and grind it in a mortar; after grinding completely, transfer it to a 10 ml EP tube; add an appropriate volume of chloroform: methanol (v:v, 2:1) mixture to extract oil, shake and extract for 30min, centrifuge at 3500rpm for 5min, collect the supernatant into a weighed (w1) 50 ml centrifuge tube, and extract repeatedly until the algae turns white; then, place the centrifuge tube containing the supernatant at 40℃ to dry to constant weight (w2), and use the weighing method to calculate the oil content of microalgae (% dry weight), which is the oil weight (w2-w1) minus the dry weight of algae powder (0.16g). Collect 5 ml of the cultured medium by centrifugation, and determine the cadmium concentration by graphite furnace atomic absorption according to the national standard GB / T17141-1997, and then subtract the cadmium concentration after culture from the original cadmium concentration and divide it by the original cadmium concentration to obtain the cadmium removal rate (%). Its biomass was 3.07 g / L and its biomass yield was 341.33 mg / L / d, which was significantly higher than that of cadmium stress alone, and increased by 12.87% and 12.91% respectively (see Figure 2 ); In Example 1, the oil content was 48.43%, and the oil yield was 165.04 mg / L / d, which was 19.78% and 8.07% higher than that of the control group and cadmium stress alone (see Figure 3 ); the cadmium removal rate was 95.46%, which was 11.31% higher than that under cadmium stress alone.

[0039] Example 2

[0040] The only difference between the method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress in this embodiment and that in embodiment 1 is that the concentration of fulvic acid in step (2) is 40 mg / L.

[0041] The biomass obtained in Example 2 was 3.27 g / L, and the biomass yield was 363.74 mg / L / d, which were 20.22% and 20.32% higher than those under cadmium stress alone (see Figure 2 ); the oil content in Example 2 was 52.73%, an increase of 23.99% over the control group; the oil yield was 203.85 mg / L / d, an increase of 40.27% and 26.55% over the control group and cadmium stress alone, respectively; the cadmium removal rate was 100%, an increase of 16.6% over cadmium stress alone ( Figure 4 ).

[0042] Comparative Example 1

[0043] A method for increasing the yield of microalgae oil, comprising the following steps:

[0044] (1) Add BG-11 medium containing 10 g / L glucose as the carbon base into a 500 mL Erlenmeyer flask, and then add Monoraphidium sp. QLZ-3 at 25 ± 1 °C and 30 μmol m -2 s -1 The seed solution was obtained after culturing for 9 days under light intensity.

[0045] (2) Add the seed solution to BG-11 medium containing 10 g / L glucose as the carbon base at 25 ± 1 °C and 30 μmol m -2 s -1 The culture solution was obtained by culturing for 9 days under the light intensity, and the inoculation amount of Monoraphidium sp.QLZ-3 was 0.1 g / L.

[0046] (3) The culture medium was centrifuged at 7000 r / min for 5 minutes, and the algae were collected in a 50 mL centrifuge tube, frozen at -80 °C overnight, and then freeze-dried in a freeze dryer for 24 hours before weighing; the dry weight of the algae cells was weighed as w (g); the microalgae biomass (dry cell weight) was the dry weight of the algae cells minus the volume of the algae solution. Weigh 0.16g of dry algae powder, then weigh twice the weight of quartz sand and grind it in a mortar; after complete grinding, transfer it to a 10 ml EP tube; add an appropriate volume of chloroform: methanol (v:v, 2:1) mixture to extract oil, shake and extract for 30 minutes, centrifuge at 3500rpm for 5 minutes, collect the supernatant into a weighed (w1) 50 ml centrifuge tube, and extract repeatedly until the algae turns white; then, place the centrifuge tube containing the supernatant at 40℃ to dry to constant weight (w2), and use the weighing method to calculate the oil content of microalgae (% dry weight), which is the oil weight (w2-w1) minus the dry weight of algae powder (0.16g). Its biomass is 2.73g / L, the biomass yield is 302.85mg / L / d, the oil content is 45.53%, and the oil yield is 137.78mg / L / d.

[0047] Comparative Example 2

[0048] The only difference between the method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress in this embodiment and that in embodiment 1 is that the concentration of fulvic acid in step (2) is 80 mg / L.

[0049] The biomass obtained in Comparative Example 2 was 2.15 g / L, and the biomass yield was 239.15 mg / L / d, which were significantly lower than those under cadmium stress alone (see Figure 2); the oil content in comparative example 2 was 50.3%, which was not significantly different from that under cadmium stress alone; the oil yield was 120.24 mg / L / d, which was 27% lower than that under cadmium stress alone; the cadmium removal rate was 51.32%.

[0050] Comparative Example 3

[0051] The only difference between the method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress in this embodiment and that in embodiment 1 is that the concentration of fulvic acid in step (2) is 0 mg / L.

[0052] The biomass obtained in Comparative Example 3 was 2.72 g / L, the biomass yield was 302.30 mg / L / d, the oil content was 50.5%, the oil yield was 152.72 mg / L / d; and the cadmium removal rate was 85.76%.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress, characterized in that: The steps include: (1) adding Monopterus sphaeroides to BG-11 medium with glucose as carbon base to obtain seed solution; (2) adding cadmium and fulvic acid to a BG-11 medium with glucose as the carbon base, and then adding seed solution to obtain a culture solution; (3) Extracting oil from the algae of Monopteris spp. in the culture medium using an organic solvent to obtain the oil in the algae cells in the culture medium.

2. The method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress as claimed in claim 1, characterized in that: In the steps (1) and (2), the culture temperature is 25±1°C, the culture time is 9 days, and the light intensity is 30 μmol m -2 s -1 .

3. The method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress as claimed in claim 1, characterized in that: In the step (2), the inoculation amount of Monochamus thunbergii is 0.1 g / L, the concentration of cadmium is 0.2 μM, and the concentration of fulvic acid is 10-40 mg / L.

4. The method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress as claimed in claim 3, characterized in that: In the step (3), the oil extraction specifically includes: centrifuging the culture solution to separate the monophyllous algae bodies, then freeze-drying the monophyllous algae bodies to obtain dry algae powder, and adding a mixed organic solvent of chloroform and methanol to the dry algae powder to extract the oil.

5. The method for improving the oil yield and cadmium removal rate of microalgae under cadmium stress as claimed in claim 4, characterized in that: The volume ratio of chloroform to methanol in the mixed organic solvent of chloroform and methanol is 2:

1.

6. A system for improving the oil yield and cadmium removal rate of microalgae under cadmium stress, characterized in that: include: microalgae cultivation module, cadmium and fulvic acid addition module, oil extraction module, cadmium removal analysis module and data analysis module; The microalgae cultivation module cultivates microalgae and monitors the growth of microalgae in real time by controlling temperature, lighting, and ventilation; The cadmium and fulvic acid adding module adds cadmium and fulvic acid during the process of cultivating microalgae in the microalgae cultivation module; The oil extraction module separates, collects, crushes and extracts oil from the microalgae cultivated in the microalgae cultivation module; The cadmium removal analysis module detects and analyzes the cadmium in the culture medium after the microalgae cultivation module is cultivated, and calculates the cadmium removal rate; The data analysis module collects the growth rate and biomass of microalgae in the microalgae cultivation module, the concentration of cadmium and fulvic acid in the cadmium and fulvic acid addition module, the cadmium removal rate in the cadmium removal analysis module, and the content and yield of oil in the oil extraction module for data analysis and processing.

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