CN105132404A - Method for fast accumulating grease through ultrasonic stimulation micro algae - Google Patents
Method for fast accumulating grease through ultrasonic stimulation micro algae Download PDFInfo
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Abstract
The invention belongs to the field of study of micro algae biological energy sources, and relates to a method for fast accumulating grease through ultrasonic stimulation micro algae. Micro algae are cultured in a reactor till the micro algae grow to enter the last phase of the logarithmic phase, the micro algae are treated for 20-30 min in an ultrasonic environment and cultured for 2-3 days in a culture medium, centrifugation is carried out, and micro algae powder is obtained. The method is easy to operate, short in consumed time, high in efficiency and easy to achieve. The concentration of scendesmus quadricauda biomass is not influenced, and the productivity of the grease can be improved. Sendesmus quadricauda is promoted to accumulate the grease fast, and the high-grease micro algae produced through the method can be used for refining gasoline, diesel and aviation fuel, and can be used as raw materials of plastic products and medicine.
Description
Technical field
The invention belongs to the development field of biomass energy, relate to and utilize ultrasound stimulation micro-algae Rapid Accumulation oil and fat method.
Background technology
Because in air, the amount of carbonic acid gas constantly increases, Greenhouse effect are also in aggravation, and the burning of fossil energy accelerates this process, therefore finds the new continuable renewable energy resources, effectively can reduce and discharge carbonic acid gas in air.The recyclable organism raw material that can be used to production biofuel has multiple, comprising biomass materials such as manioca, Semen Brassicae campestris, palm tree, changing food waste and micro-algaes, compare other raw materials, micro-algae has the advantages such as Seedling height speed, neutral carbon and extremely strong environmental compatibility, and the trophology of micro-algae, pharmacy and biological value are higher, some biological and pharmaceutical industries can be applied to, also can be used to production high added value nutritious prod, fish food and biofuel.
Utilize micro-algae as raw material production biofuel, floor space can be reduced to a great extent, even can utilize the soil being not suitable for cultivating, because its breeding cycle is short, fat content advantages of higher, effectively can enhance productivity, save time, therefore micro-algae is a kind of renewable biomass resource having DEVELOPMENT PROSPECT, and the present invention can be used for the development research of micro-algae accumulates biomass and grease.Scenedesmus quadricauda belongs to Chlorophyta, Chlorococcale, Shan Zao section, Scenedesmus, is prevalent in various fresh water environment, and existing large quantity research shows that its growth is rapid, is easy to cultivate, there are excellent production biomass and the performance of grease, can be used as the desirable feedstock of production biofuel.Because micro-algae is in process of growth, biomass and grease can not realize synchronous high-efficiency accumulation, therefore two benches is cultivated and is considered to the more rational solution of one, micro-algae is cultured to logarithmic phase latter stage by the first stage under optimum condition, microalgae cell is made to reach peak concentration with higher growth velocity, subordinate phase is cultivated under the stimulation of extraneous adverse environment is coerced, and impels micro-algae dynamic accumulation grease.Have research display, ultrasonication can have an impact to intracellular members at short notice, and by carrying out ultrasound stimulation to micro-algae, its born of the same parents' inner chlorophyll content creates change, and also have research display, supersound process can affect the content of cell membrane lipid.Research shows, ultrasonic wave can produce cavatition in water, instantaneous high temperature and high pressure environment can be produced during the inside explosion of micro-bubble formed, hormesis can be produced to microalgae cell, and then affect synthesis and the conversion of chemical composition in born of the same parents, especially high temperature can form stressful environmental, is beneficial to the raising of fat content.
Summary of the invention
The object of the invention is to overcome the contradiction between microalgae biomass growth with oil and fat accumulation, utilize substep culture scheme, provide a kind of simple to operate, consuming time short, efficiency is high, be easy to the method for the promotion microalgae grease Rapid Accumulation realized.Ultrasonic wave is utilized to realize one fast and easily environment-stress scheme, micro-algae in logarithmic phase latter stage is impelled to realize the efficient accumulation of grease at short notice, do not affect microalgae biomass concentration simultaneously, reach the object improving grease productive rate, there is very important realistic meaning and application prospect.
For achieving the above object, the present invention adopts following concrete technical scheme
Utilize a method for ultrasound stimulation micro-algae Rapid Accumulation grease, the micro-algae entering logarithmic phase latter stage is processed, cultivates in ultrasound environments, obtains the micro-algae of high fat content.
Further, described micro-algae entering the culture condition before logarithmic phase latter stage is: be substratum with BG11, at temperature 20-25 DEG C, cultivates micro-algae 10-15 days under aeration rate 500-600mL/min, intensity of illumination 5000-6000lux condition.Suitable culture condition is provided, ensures that micro-algae grows fast.
Further, described BG11 medium component (g/L) is: NaNO
3, 1.5; K
2hPO
4, 0.04; MgSO
47H
2o, 0.075; CaCl
22H
2o, 0.036; Citric acid, 0.006; Ferric ammonium citrate, 0.006; EDTA2Na, 0.001; Na
2cO
3, 0.02; And the A5 of 1mL/L.
Further, described A5 composition (g/L) is: H
3bO
3, 2.86; MnCl
24H
2o, 1.86; ZnSO
47H
2o, 0.22; Na
2moO
42H
2o, 0.39; CuSO
45H
2o, 0.08; Co (NO
3)
26H
2o, 0.05.
Further, described micro-algae is Scenedesmus quadricauda, and purchased from Shandong Province's environmental science Engineering Technical Research Centre, algae kind has been preserved in Chinese Academy of Sciences's algae kind storehouse, and preserving number is FACHB-1768, preservation date: in December, 2013.Preferred micro-algae kind is Scenedesmus quadricauda, but there is no particular determination to its kind.
Further, described micro-algae grow into logarithmic phase latter stage after, the biomass concentration of micro-algae is 0.99 ~ 1.11g/L, and fat content is 22.3% ~ 25.3%.Judge upgrowth situation and the fat content of micro-algae, for the selection of subsequent disposal condition provides foundation.Further, described ultrasound environments condition is 150-200W, 30-40kHz.The supersound process used in the present invention is only carried out once in the logarithm end of term of micro algae growth, and be single operation, compared with interval type continuous ultrasound, the operating time of the present invention is short, more simply, more easily realizes.Meanwhile, in order to realize the hormesis to micro-algae produce oil, higher ultrasonic power has been selected.
Ultrasonic power is less than 150W, when frequency is less than 30kHz, cannot produce stress reaction, oil and fat accumulation weak effect by the micro-algae of effective stimulus; Ultrasonic power is greater than 200W, when frequency is greater than 40kHz, declines, even part inactivation with oil synthesis involved enzyme vigor, and biomass and oil and fat accumulation efficiency raise on a declining curve along with the continuation of ultrasonic power and frequency.
Further, described micro-algae through supersound process, cultivate after, in 4-6 DEG C, centrifugal 5-10min under 3500-4000r/min.Low-temperature centrifugation ensures that the components of biomass of micro-algae is not destroyed, and by the control of centrifugal rotational speed, makes rich grease-contained micro-algae fast deposition.
Owing to have employed supersound process, under the prerequisite of biomass concentration not affecting micro-algae, micro-algae height fat content significantly improves, and can reach 37.1% ~ 39.6%, can meet the requirement as biodiesel fuel.
In certain embodiments of the present invention, described micro-algae under certain culture apparatus and suitable outside atmosphere control, can carry out growth and breeding fast.Term used " certain culture apparatus " can be Closed photobioreactor (such as glass blister column type reactor), open runway cultivation pool or biological film culture device (as half-dry solid-state adherent culture device (CN201210051158.2)), and wherein said apparatus can use according to order series connection or be used alone.Term used " suitable external environment " refer to the temperature of suitable micro algae growth, illumination, the factor such as nutritive salt and carbonic acid gas comprehensive.
The micro-algae of high fat content of the present invention can be used for extracting gasoline, diesel oil, Aviation Fuel, and as the raw material etc. of plastics and medicine.
Compared with prior art, advantage of the present invention is:
1. the present invention simple to operate, consuming time short, efficiency is high, be easy to realize.
2. the present invention can make the biomass concentration of Scenedesmus quadricauda unaffected, realizes the Rapid Accumulation of grease within 2 day time.
3. the present invention's stimulation protocol used is convenient to expand and is implemented, and has good applicability.
4. the present invention can impel the Scenedesmus quadricauda Rapid Accumulation grease in logarithmic phase latter stage, can be and utilizes micro-algae production biofuel or exploitation other biological mass-energy source to provide effective scheme.
Accompanying drawing explanation
Fig. 1 is that the Scenedesmus quadricauda in logarithmic phase latter stage continues cultivation after 2 days again through the ultrasound stimulation of different time, and biomass concentration and fat content are with the change of ultrasound stimulation time.Ultrasound environments is 200W, 40kHz.
Fig. 2 be the Scenedesmus quadricauda in logarithmic phase latter stage after ultrasound stimulation in 20 minutes, Scenedesmus quadricauda biomass concentration is with the change continuing incubation time.Ultrasound environments is 200W, 40kHz.
Specific embodiment is as follows:
Embodiment 1
The present invention relates to a kind of microalgae culture method utilizing ultrasound stimulation micro-algae Rapid Accumulation grease, improve grease productive rate.
Utilize BG11 substratum temperature 25 DEG C, under the continuous illumination condition of aeration rate 600mL/min, intensity of illumination 6000lux, cultivate micro-algae.Micro-algae of cultivating is Scenedesmus quadricauda, and from Shandong Province's environmental science Engineering Technical Research Centre, be preserved in Chinese Academy of Sciences's algae kind storehouse, preserving number is FACHB-1768.After 15 days cultivate, micro-algae reaches logarithmic phase latter stage, and now the biomass concentration of micro-algae reaches and is up to 0.99 ~ 1.11g/L, and fat content is 22.3% ~ 25.3%.
Carry out the optimization of ultrasound stimulation condition, the Scenedesmus quadricauda in logarithmic phase latter stage is positioned over (200W in ultrasound environments, 40kHz), supersound process different time (0,5,10,15,20 and 40 minute), then micro-algae is reapposed and continue cultivation 2 days under the culture condition of same step 1, and after results, detecting biomass concentration and the fat content of micro-algae, result as shown in Figure 1, determines the best ultrasound stimulation time thus.As can be seen from Figure 1 micro-algae is continuing cultivation after 2 days through ultrasound stimulation, fat content improves along with the increase of ultrasonic time, condition at ultrasonic 20 minutes is issued to maximum value, continuing the decline that prolongation ultrasonic time then causes fat content, may be because cell is damaged and have impact on oil synthesis.Different ultrasonic times does not cause the obvious decline of microalgae biomass concentration, and under the longest ultrasonic time condition of 40 minutes, microalgae biomass concentration is minimum.Therefore maximum at the grease productive rate of 20 minutes micro-algaes of condition, be the best ultrasound stimulation time.
The Scenedesmus quadricauda in logarithmic phase latter stage is positioned over (200W in ultrasound environments, 40kHz) process 20 minutes after (Best Times ultrasonic time), then micro-algae is reapposed and continue cultivation 6 days under the condition of same step 1, and carry out harvested by centrifugation every day, detect biomass concentration and the fat content of micro-algae, result as shown in Figure 2, determines best continuation incubation time thus.As can be seen from Figure 2, microalgae biomass concentration slowly raises along with the prolongation continuing incubation time, illustrate and ultrasonicly micro-algae not to be had a negative impact, its fat content reached maximum value (37.1% ~ 39.6%) the 2nd day time, continue to cultivate the reduction that then result in fat content, the fat content obtained after 6 days is 21.8% ~ 23.5%, and grease productive rate also can reduce, and determines that best continuation incubation time is 2 days thus.
Experimentally result determines the ultrasound stimulation through 20 minutes, then the fat content the highest (37.1% ~ 39.6%) that the scheme continuing the 2 day time of cultivation finally obtains, and compares late log phase and improves about 67.8%; Grease productive rate is 25.3 ~ 25.9mg/L to the maximum, improves about 48.7% latter stage relative to logarithmic phase.In an embodiment, set blank group, namely not through the microdisk electrode of ultrasound stimulation after 6 days fat content be 26.2 ~ 27.5%, illustrate by micro-algae lower from carrying out oil and fat accumulation efficiency in growth stationary phase.
The method measuring microalgae grease content in above-described embodiment is as follows:
Utilize whizzer by algae liquid in 4000r/min, harvested by centrifugation at 4 DEG C, the algae mud of acquisition is frozen into block in refrigerator, then carries out lyophilize with freeze drier, is clayed into power by dried micro-algae in mortar, and preserves stand-by after putting into baking oven oven dry.
1. take about 0.1g dry algae powder (m
1) in 50ml centrifuge tube, add 10ml chloroform/methanol (2:1) solution, with ultrasonic cell disruption instrument fragmentation (process) 10min (frequency is 20%);
2. by mixture centrifugal 10min under 4000r/min, after centrifugal end, mixture divides two-phase, supernatant liquor is transferred in the separating funnel of 60mL, then in centrifuge tube, adds 10ml chloroform/methanol (2:1) solution, and utilize and ultrasonic and centrifugally repeat whole leaching process by step 1,2;
3. according to grease extracting liquid volume, add the sodium chloride solution (adding 1/5, the about 3 ~ 4mL sodium chloride solution that sodium chloride solution volume is grease extracting liquid volume) of 0.9%, fully shake up 1min, and standing 15min treats liquid layered;
4. measure low phase liquor capacity V, and get the low phase solution of 5mL in 10mL glass test tube (m
0) in, dry up with nitrogen, Glass tubing is positioned in 60 DEG C of baking ovens the (m that dries to constant weight
2, about 30 minutes).
Fat content: LW (%)=(m
2-m
1) × V × 100/ (5 × m
1);
In formula: LW-based on the fat content % under dry weight, g/g;
M
1-algae dried bean noodles weight, g;
M
2-with the 10mL Glass tubing dry weight of grease, g;
M
0-10mL Glass tubing dry weight, g;
The volume of V-low phase grease, mL.
Final experimental data shows, the method of the Scenedesmus quadricauda accumulation grease utilizing ultrasound stimulation to promote logarithmic phase latter stage provided by the present invention, grease Rapid Accumulation in 2 days can be realized, fat content is significantly improved, the biomass concentration not affecting micro-algae can be ensured simultaneously, reach maximum grease productive rate, can as the raw material of production biofuel or other biological Energy production.Micro-algae that the inventive method is cultivated is owing to can increase substantially its fat content, and the biofuel not sulfur-bearing of production, does not discharge toxic gas during burning, enter in environment and can be degraded by microorganisms yet, free from environmental pollution.
Embodiment 2
The present invention relates to a kind of microalgae culture method utilizing ultrasound stimulation micro-algae Rapid Accumulation grease, improve grease productive rate.
Utilize BG11 substratum temperature 20 DEG C, under the continuous illumination condition of aeration rate 500mL/min, intensity of illumination 5000lux, cultivate micro-algae.Micro-algae of cultivating is Scenedesmus quadricauda, and from Shandong Province's environmental science Engineering Technical Research Centre, be preserved in Chinese Academy of Sciences's algae kind storehouse, preserving number is FACHB-1768.After 10 days cultivate, micro-algae reaches logarithmic phase latter stage, and now the biomass concentration of micro-algae reaches and is up to 0.99 ~ 1.11g/L, and fat content is 22.3% ~ 25.3%.
Carry out the optimization of ultrasound stimulation condition, the Scenedesmus quadricauda in logarithmic phase latter stage is positioned over (150W in ultrasound environments, 30kHz), supersound process different time (0,5,10,15,20 and 40 minute), then micro-algae is reapposed and continue cultivation 2 days under the culture condition of same step 1, and after results, detect biomass concentration and the fat content of micro-algae, process after 30 minutes, then reapposed by micro-algae and continue cultivation 2 days under the condition of same step 1, its fat content reaches maximum value.
The method measuring microalgae grease content in above-described embodiment is as follows:
Utilize whizzer by algae liquid in 3500r/min, harvested by centrifugation at 6 DEG C, the algae mud of acquisition is frozen into block in refrigerator, then carries out lyophilize with freeze drier, is clayed into power by dried micro-algae in mortar, and preserves stand-by after putting into baking oven oven dry.
Embodiment 3
The present invention relates to a kind of microalgae culture method utilizing ultrasound stimulation micro-algae Rapid Accumulation grease, improve grease productive rate.
Utilize BG11 substratum temperature 25 ± 1 DEG C, under aeration rate 600mL/min, intensity of illumination 5000lux continuous illumination condition, cultivate micro-algae.Micro-algae of cultivating is anabena, buys in algae kind storehouse, Chinese Academy of Sciences Wuhan.After 11 days cultivate, micro-algae reaches logarithmic phase latter stage, and now the biomass concentration of micro-algae reaches and is up to 1 ~ 1.2g/L, and fat content is 28% ~ 31%.
Carry out the optimization of ultrasound stimulation condition, the anabena in logarithmic phase latter stage to be positioned in the ultrasound environments of different capacity (200,350 and 500W, 40kHz), after process 10min, micro-algae is reapposed and continue cultivation 3 days in environment described in the present embodiment, and after results, detect biomass concentration and the fat content of micro-algae.Select supersound process different time (5,10,20 and 40 minutes) again, fixing ultrasonic power is 200W, 40kHz, then the micro-algae after process is reapposed and continue cultivation 3 days under culture condition described in the present embodiment, and after results, detect biomass concentration and the fat content of micro-algae.Finally under ultrasonic power 200W, ultrasonic time 5 minutes, the continuation incubation time condition of 1 day, its fat content reaches maximum value 45% ~ 47%.And biomass concentration is not subject to obviously impact is 1.3 ~ 1.45g/L.
The method measuring microalgae grease content in above-described embodiment is as follows:
Utilize whizzer by algae liquid in 5000r/min, harvested by centrifugation at 4 DEG C, the algae mud of acquisition is frozen into block in refrigerator, then carries out lyophilize with freeze drier, dried micro-algae is clayed into power in mortar, and it is stand-by to be stored in moisture eliminator after putting into baking oven oven dry.
Above-described embodiment is described the specific embodiment of the present invention; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention; those skilled in the art do not need to pay creative work; the various amendment that can make or distortion, still within protection scope of the present invention.
Claims (7)
1. utilize a method for ultrasound stimulation micro-algae Rapid Accumulation grease, it is characterized in that, the micro-algae entering logarithmic phase latter stage is processed, cultivates in ultrasound environments, obtains the micro-algae of high fat content.
2. the method for claim 1, it is characterized in that, described micro-algae entering the culture condition before logarithmic phase latter stage is: be substratum with BG11, at temperature 20-25 DEG C, micro-algae 10-15 days is cultivated under aeration rate 500-600mL/min, intensity of illumination 5000-6000lux condition.
3. the method for claim 1, is characterized in that, described micro-algae is Scenedesmus quadricauda, and from Shandong Province's environmental science Engineering Technical Research Centre, algae kind has been preserved in Chinese Academy of Sciences's algae kind storehouse, and preserving number is FACHB-1768.
4. the method for claim 1, is characterized in that, described micro-algae grow into logarithmic phase latter stage after, the biomass concentration of micro-algae is 0.99 ~ 1.11g/L, and fat content is 22.3% ~ 25.3%.
5. the method for claim 1, is characterized in that, described ultrasound environments condition is 150-200W, 30-40kHz.
6. the method for claim 1, is characterized in that, described micro-algae through supersound process, cultivate after, in 4-6 DEG C, centrifugal 5-10min under 3500-4000r/min.
7. the method for claim 1, is characterized in that, the fat content of the micro-algae of described high fat content is 37.1% ~ 39.6%.
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