CN105132404B - Method for rapidly accumulating grease by utilizing ultrasonic stimulation microalgae - Google Patents
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Abstract
The invention belongs to the research field of microalgae bioenergy, and relates to a method for rapidly accumulating grease by utilizing ultrasonic stimulation of microalgae. Culturing microalgae in a reactor until the growth of the microalgae enters the late logarithmic phase, treating the microalgae in an ultrasonic environment for 20-30min, culturing in a culture medium for 2-3 days, and centrifuging to obtain microalgae powder. The method has the advantages of simple operation, short time consumption, high efficiency and easy realization; the concentration of Scenedesmus quadricauda biomass is not influenced, and the yield of grease is improved; the Scenedesmus tetracaudatus can promote the Scenedesmus tetracaudatus to quickly accumulate grease, and the high-grease microalgae produced by the method can be used for refining gasoline, diesel oil and aviation fuel oil and can be used as raw materials of plastic products and medicines.
Description
Technical Field
The invention belongs to the field of development of biomass energy, and relates to a method for quickly accumulating grease by utilizing ultrasonic stimulation microalgae.
Background
As the amount of carbon dioxide in the atmosphere is continuously increased, the greenhouse effect is also intensified, and the combustion of fossil energy accelerates the process, a new sustainable renewable energy source is searched, and the emission of carbon dioxide to the atmosphere can be effectively reduced. The renewable biomass raw materials which can be used for producing the biodiesel comprise biomass raw materials such as jatropha curcas, rapeseeds, palms, kitchen waste and microalgae, compared with other raw materials, the microalgae has the advantages of high growth speed, carbon neutrality, extremely strong environmental adaptability and the like, and the nutritional value, the pharmaceutical value and the biological value of the microalgae are higher, so that the microalgae can be applied to certain biological and pharmaceutical industries, and can also be used for producing high value-added nutrients, fish bait and the biodiesel.
The method for producing the biodiesel by using the microalgae as the raw material can reduce the floor area to a great extent, even can utilize land which is not suitable for cultivation, and can effectively improve the production efficiency and save time due to the advantages of short reproduction period, high oil content and the like, so the microalgae is a renewable biomass resource with development prospect, and the method can be used for the development and research of microalgae accumulation biomass and oil. Scenedesmus tetracaudatus belongs to the phylum Chlorophyta, the order Chlorococcales, the family Scenedesmaceae, the genus Scenedesmus, generally exists in various freshwater environments, and a great deal of research shows that the Scenedesmus tetracaudatus grows rapidly and is easy to culture, has excellent performance of producing biomass and grease, and can be used as an ideal raw material for producing biodiesel. In the growth process of microalgae, biomass and grease cannot be synchronously and efficiently accumulated, so that two-stage culture is regarded as a reasonable solution, the microalgae is cultured to the end of a logarithmic phase under the optimal condition in the first stage, the microalgae cells reach the maximum concentration at a higher growth rate, and the microalgae is cultured under the stimulation and stress of an external adverse environment in the second stage to promote the microalgae to synthesize and accumulate the grease. Research shows that the ultrasonic action can affect intracellular components in a short time, the content of intracellular chlorophyll is changed by carrying out ultrasonic stimulation on microalgae, and the ultrasonic treatment can affect the content of cell membrane lipid. Research shows that the ultrasonic wave can generate cavitation in water, the formed micro bubbles can generate instantaneous high-temperature and high-pressure environment when being exploded inwards, and can generate stimulation to microalgae cells, so that the synthesis and conversion of chemical components in cells are influenced, particularly, the high temperature can form a stress environment, and the improvement of the oil content is facilitated.
Disclosure of Invention
The invention aims to overcome the contradiction between microalgae biomass growth and grease accumulation, and provides a method for promoting rapid accumulation of microalgae grease, which is simple to operate, short in time consumption, high in efficiency and easy to realize, by utilizing a step-by-step culture scheme. The ultrasonic wave is utilized to realize a rapid and convenient environmental stress scheme, the high-efficiency accumulation of grease in the microalgae at the late logarithmic phase is promoted in a short time, the biomass concentration of the microalgae is not influenced, the purpose of improving the yield of the grease is achieved, and the method has very important practical significance and application prospect.
In order to achieve the purpose, the invention adopts the following specific technical scheme
A method for stimulating microalgae to quickly accumulate oil by using ultrasonic waves comprises the step of treating and culturing microalgae entering the late logarithmic phase in an ultrasonic environment to obtain the microalgae with high oil content.
Further, the culture conditions of the microalgae before entering the late logarithmic growth phase are as follows: BG11 is used as a culture medium, and microalgae is cultured for 10-15 days under the conditions of 20-25 ℃, aeration rate of 500-. Provides proper culture conditions to ensure the rapid growth of the microalgae.
Further, the BG11 medium components (g/L) are: NaNO3，1.5；K2HPO4，0.04；MgSO4·7H2O，0.075；CaCl2·2H2O, 0.036; citric acid, 0.006; ammonium ferric citrate, 0.006; EDTA-2 Na, 0.001; na (Na)2CO30.02; and 1mL/L of A5.
Further, the A5 component (g/L) is: h3BO3，2.86；MnCl2·4H2O，1.86；ZnSO4·7H2O，0.22；Na2MoO4·2H2O，0.39；CuSO4·5H2O，0.08；Co(NO3)2·6H2O，0.05。
Further, the microalgae is Scenedesmus quadricauda, purchased from research center of environmental science engineering and technology in Shandong province, the algal species are preserved in freshwater algal species bank of Chinese academy of sciences with the preservation number of FACHB-1768, and the preservation date is as follows: 12 months in 2013. The preferable microalgae species is Scenedesmus quadricaudatus, but the species is not particularly limited.
Further, after the growth of the microalgae enters the end period of logarithmic growth period, the biomass concentration of the microalgae is 0.99-1.11 g/L, and the oil content is 22.3% -25.3%. Judging the growth condition and the oil content of the microalgae, and providing a basis for selecting subsequent treatment conditions. Further, the ultrasonic environmental condition is 150-. The ultrasonic treatment used in the invention is only carried out once at the end of logarithmic phase of microalgae growth, namely single operation, and compared with intermittent continuous ultrasonic, the ultrasonic treatment method has the advantages of short operation time, simplicity and easiness in implementation. Meanwhile, in order to realize the stimulation effect on the oil production of the microalgae, higher ultrasonic power is selected.
When the ultrasonic power is less than 150W and the frequency is less than 30kHz, the stress reaction of the microalgae cannot be effectively stimulated, and the oil accumulation effect is poor; when the ultrasonic power is more than 200W and the frequency is more than 40kHz, the activity of the enzyme related to the synthesis of the grease is reduced, even the enzyme is partially inactivated, and the biomass and the grease accumulation efficiency are in a descending trend along with the continuous increase of the ultrasonic power and the frequency.
Further, the microalgae are subjected to ultrasonic treatment and culture, and then are centrifuged for 5-10min at the temperature of 4-6 ℃ and the speed of 3500-. The low-temperature centrifugation ensures that the biomass components of the microalgae are not damaged, and the microalgae rich in grease is rapidly deposited by controlling the centrifugal rotating speed.
Due to the adoption of ultrasonic treatment, the high oil content of the microalgae is remarkably improved to 37.1-39.6% on the premise of not influencing the biomass concentration of the microalgae, and the requirement of the microalgae as a biodiesel fuel can be met.
In some embodiments of the present invention, the microalgae can be rapidly grown and propagated under a certain culture device and suitable external environmental control. The term "certain culture apparatus" may be a closed photobioreactor (e.g., glass bubble column reactor), an open raceway culture pond, or a biofilm culture apparatus (e.g., semi-dry solid adherent culture apparatus (CN201210051158.2)), which may be used in series in this order or individually. The term "suitable external environment" refers to a combination of temperature, light, nutrient salts and carbon dioxide, which are suitable for the growth of microalgae.
The microalgae with high oil content can be used for refining gasoline, diesel oil and aviation fuel oil, and can be used as raw materials of plastic products and medicines.
Compared with the prior art, the invention has the advantages that:
1. the method has the advantages of simple operation, short time consumption, high efficiency and easy realization.
2. The invention can enable the biomass concentration of Scenedesmus quadricaudatus to be not influenced and realize the rapid accumulation of grease within 2 days.
3. The stimulation scheme used by the invention is convenient for expanding implementation and has better applicability.
4. The invention can promote Scenedesmus quadricauda to quickly accumulate grease at the late logarithmic phase, and can provide an effective scheme for producing biodiesel by utilizing microalgae or developing other biomass energy sources.
Drawings
FIG. 1 shows the changes of biomass concentration and oil content with the time of ultrasonic stimulation after further culturing for 2 days for Scenedesmus quadricauda at the end of log phase by ultrasonic stimulation at different times. The ultrasonic environment is 200W and 40 kHz.
FIG. 2 shows the variation of Scenedesmus quadricauda biomass concentration with the time of continuous culture after 20 minutes of ultrasonic stimulation of Scenedesmus quadricauda at the end of log phase. The ultrasonic environment is 200W and 40 kHz.
The specific embodiment is as follows:
example 1
The invention relates to a microalgae culture method for rapidly accumulating grease and improving the grease yield by stimulating microalgae with ultrasound.
Culturing microalgae by using BG11 culture medium under continuous illumination conditions of 25 ℃ temperature, 600mL/min aeration rate and 6000lux illumination intensity. The cultured microalgae is Scenedesmus quadricauda, is from research center of environmental science engineering technology in Shandong province, and has been deposited in freshwater algae seed bank of Chinese academy of sciences with accession number of FACHB-1768. After 15 days of culture, the biomass concentration of the microalgae reaches the highest 0.99-1.11 g/L and the oil content is 22.3% -25.3% at the end of logarithmic growth phase.
Optimizing ultrasonic stimulation conditions, namely placing Scenedesmus quadricauda at the end of log phase in an ultrasonic environment (200W, 40kHz), performing ultrasonic treatment for different times (0, 5, 10, 15, 20 and 40 minutes), then placing the microalgae under the culture conditions of the same step 1 again for continuous culture for 2 days, and detecting the biomass concentration and the oil content of the microalgae after harvesting, wherein the result is shown in figure 1, so that the optimal ultrasonic stimulation time is determined. From fig. 1, it can be seen that after the microalgae is cultured for 2 days by ultrasonic stimulation, the oil content increases with the increase of the ultrasonic time, the maximum value is reached under the condition of ultrasonic for 20 minutes, and the oil content is reduced by continuing to prolong the ultrasonic time, probably because the cells are damaged to influence the oil synthesis. The different ultrasonic time does not cause obvious reduction of microalgae biomass concentration, and the microalgae biomass concentration is the lowest under the condition of the longest ultrasonic time of 40 minutes. Therefore, the oil yield of the microalgae is the maximum under the condition of 20 minutes, and the optimal ultrasonic stimulation time is obtained.
After placing Scenedesmus quadricauda at the end of log phase in an ultrasonic environment (200W, 40kHz) for processing for 20 minutes (optimal time ultrasonic time), then placing the microalgae under the condition of the same step 1 again for continuous culture for 6 days, carrying out centrifugal harvesting every day, and detecting the biomass concentration and the oil content of the microalgae, wherein the result is shown in figure 2, thereby determining the optimal continuous culture time. As can be seen from fig. 2, the biomass concentration of microalgae increases slowly with the extension of the continuous culture time, which indicates that the ultrasound does not adversely affect the microalgae, the oil content of the microalgae reaches the maximum value (37.1% -39.6%) at day 2, the continuous culture causes the reduction of the oil content, the oil content obtained after 6 days is 21.8% -23.5%, and the oil yield is also reduced, thereby determining that the optimal continuous culture time is 2 days.
According to the experimental result, the highest oil content (37.1-39.6%) is finally obtained by the scheme of ultrasonic stimulation for 20 minutes and continuous culture for 2 days, and is improved by about 67.8% compared with the late logarithmic phase; the oil yield is 25.3-25.9 mg/L at most, and is improved by about 48.7% relative to the logarithmic phase end. In the embodiment, a blank control group is set, namely the oil content of microalgae which is not subjected to ultrasonic stimulation is 26.2-27.5% after being cultured for 6 days, which indicates that the efficiency of oil accumulation is low when the microalgae grows in a stationary phase.
The method for determining the oil content of the microalgae in the embodiment comprises the following steps:
centrifuging the algae liquid at 4000r/min at 4 ℃ by using a centrifugal machine, freezing the obtained algae mud into blocks in a refrigerator, freeze-drying the blocks by using a freeze dryer, grinding the dried microalgae into powder in a mortar, and drying the powder in a drying oven for later use.
1. Weighing about 0.1g of dried algae powder (m)1) Adding 10ml chloroform/methanol (2:1) solution into a 50ml centrifuge tube, and crushing (treating) for 10min (frequency is 20%) by using an ultrasonic cell crusher;
2. centrifuging the mixture at 4000r/min for 10min, separating the mixture into two phases after centrifugation is finished, transferring the supernatant into a 60mL separating funnel, adding 10mL chloroform/methanol (2:1) solution into the centrifuge tube, and repeating the whole extraction process according to steps 1 and 2 by using ultrasound and centrifugation;
3. adding 0.9% sodium chloride solution (added with 1/5 of the volume of the sodium chloride solution which is about 3-4 mL of the sodium chloride solution of the volume of the grease extracting solution), fully shaking for 1min, and standing for 15min until the liquid is layered;
4. the volume V of the low-phase solution was measured, and 5mL of the low-phase solution was placed in a 10mL glass test tube (m)0) Drying with nitrogen, placing the glass tube in an oven at 60 deg.C, and drying to constant weight (m)2About 30 minutes).
Oil content: LW (%) ═ (m)2-m1)×V×100/(5×m1)；
In the formula: LW-oil content% on a dry weight basis, g/g;
m1-dry weight of algae meal, g;
m2-10 mL glass tube dry weight with grease, g;
m010mL glass tube dry weight, g;
v-volume of low phase oil, mL.
The final experimental data show that the method for promoting scenedesmus tetracauda to accumulate grease at the late logarithmic phase by utilizing ultrasonic stimulation can realize quick accumulation of grease within 2 days, obviously improve the grease content, ensure that the biomass concentration of microalgae is not influenced, achieve the maximum grease yield and be used as a raw material for producing biodiesel or other biological energy sources. The microalgae cultured by the method can greatly improve the oil content, and the produced biodiesel does not contain sulfur, does not discharge toxic gas during combustion, can be degraded by microorganisms when discharged into the environment, and does not pollute the environment.
Example 2
The invention relates to a microalgae culture method for rapidly accumulating grease and improving the grease yield by stimulating microalgae with ultrasound.
Culturing microalgae by using BG11 culture medium under continuous illumination conditions of 20 ℃ of temperature, 500mL/min of aeration amount and 5000lux of illumination intensity. The cultured microalgae is Scenedesmus quadricauda, is from research center of environmental science engineering technology in Shandong province, and has been deposited in freshwater algae seed bank of Chinese academy of sciences with accession number of FACHB-1768. After 10 days of culture, the biomass concentration of the microalgae reaches the highest 0.99-1.11 g/L and the oil content is 22.3% -25.3% at the end of logarithmic growth phase.
Optimizing ultrasonic stimulation conditions, placing Scenedesmus quadricauda at the end of logarithmic phase in an ultrasonic environment (150W, 30kHz), performing ultrasonic treatment for different time (0, 5, 10, 15, 20 and 40 minutes), then placing the microalgae again in the same culture condition of the step 1 to continue to culture for 2 days, detecting the biomass concentration and the oil content of the microalgae after harvesting, after treating for 30 minutes, placing the microalgae again in the same condition of the step 1 to continue to culture for 2 days, wherein the oil content reaches the maximum value.
The method for determining the oil content of the microalgae in the embodiment comprises the following steps:
centrifuging the algae liquid at 3500r/min and 6 deg.C by using centrifuge, freezing the obtained algae mud into blocks in refrigerator, freeze-drying with freeze dryer, grinding the dried microalgae into powder in mortar, oven drying, and storing for use.
Example 3
The invention relates to a microalgae culture method for rapidly accumulating grease and improving the grease yield by stimulating microalgae with ultrasound.
Culturing microalgae by using BG11 culture medium under the continuous illumination conditions of 25 +/-1 ℃, aeration rate of 600mL/min and illumination intensity of 5000 lux. The cultured microalgae are anabaena, and are purchased from Wuhan freshwater algae seed bank of Chinese academy of sciences. After 11 days of culture, the biomass concentration of the microalgae reaches the highest 1-1.2 g/L at the end of logarithmic growth phase, and the oil content is 28% -31%.
Optimizing ultrasonic stimulation conditions, placing anabaena at the end of log phase in ultrasonic environments (200, 350 and 500W, 40kHz) with different powers, processing for 10min, placing the microalgae in the environment again to continue culturing for 3 days, and detecting the biomass concentration and the grease content of the microalgae after harvesting. Then ultrasonic treatment is selected for different time (5, 10, 20 and 40 minutes), the ultrasonic power is fixed at 200W and 40kHz, the treated microalgae are placed under the culture condition of the embodiment again to be continuously cultured for 3 days, and the biomass concentration and the oil content of the microalgae are detected after harvesting. Finally, the oil content reaches the maximum value of 45-47 percent under the conditions of 200W of ultrasonic power, 5 minutes of ultrasonic time and 1 day of continuous culture time. And the biomass concentration is not obviously influenced to be 1.3-1.45 g/L.
The method for determining the oil content of the microalgae in the embodiment comprises the following steps:
centrifuging the algae liquid at 5000r/min at 4 deg.C with a centrifuge, freezing the obtained algae mud into blocks in a refrigerator, freeze-drying with a freeze dryer, grinding the dried microalgae into powder in a mortar, oven-drying in an oven, and storing in a dryer for use.
The above embodiments describe specific embodiments of the present invention, but do not limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications or variations can be made without inventive effort by those skilled in the art based on the technical solutions of the present invention, and still fall within the scope of the present invention.
Claims (1)
1. A method for stimulating microalgae to quickly accumulate oil by using ultrasound is characterized in that BG11 culture medium is used at the temperature of 25 ℃,culturing the microalgae under the continuous illumination condition with the aeration rate of 600mL/min and the illumination intensity of 6000lux, and after 15 days of culture, when the microalgae reaches the end of logarithmic phase, measuring that the biomass concentration of the microalgae reaches the maximum of 0.99-1.11 g/L and the oil content is 22.3% -25.3%; placing the microalgae entering the late logarithmic phase in an ultrasonic environment for treatment, then placing the microalgae in the culture medium again for continuous culture for 2 days, and centrifuging and harvesting the microalgae liquid at 4000r/min and 4 ℃ by using a centrifugal machine to obtain the microalgae with high oil content; the microalgae is Scenedesmus tetracaudatus (Scenedesmus quadricaudatus)Scenedesmus quadricauda) The algae seeds are from the research center of environmental science engineering technology in Shandong province and are preserved in the fresh water algae seed bank of Chinese academy of sciences with the accession number of FACHB-1768; the ultrasonic condition is 200W and 40kHz, and the ultrasonic stimulation is carried out for 20 min; the BG11 culture medium comprises the following components: NaNO3，1.5g/L；K2HPO4，0.04g/L；MgSO4·7H2O，0.075g/L；CaCl2·2H2O, 0.036 g/L; citric acid, 0.006 g/L; ammonium ferric citrate, 0.006 g/L; EDTA-2 Na, 0.001 g/L; na (Na)2CO30.02 g/L; and 1mL/L of A5; the A5 comprises the following components: h3BO3，2.86g/L；MnCl2·4H2O，1.86g/L；ZnSO4·7H2O，0.22g/L；Na2MoO4·2H2O，0.39g/L；CuSO4·5H2O，0.08g/L；Co(NO3)2·6H2O, 0.05 g/L; the oil content of the microalgae with high oil content is 37.1-39.6%.
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