CN108587917B - Method for preparing biodiesel by using chlorella pyrenoidosa cells as main raw material by using cassava residues - Google Patents
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Abstract
The invention belongs to the technical field of biological energy, and discloses a method for preparing biodiesel by using chlorella pyrenoidosa cells with cassava dregs as a main raw material, which comprises the following steps: step 1) mixed culture of microorganisms, step 2) extraction of grease, and step 3) preparation of biodiesel. The method of the invention reduces the cost and improves the oil yield. The biodiesel prepared by the method has high yield and quality, and can be used as an ideal substance of a diesel substitute.
Description
Technical Field
The invention belongs to the technical field of biological new energy, and particularly relates to a method for preparing biodiesel by using chlorella pyrenoidosa cells and cassava residues as main raw materials.
Background
Biodiesel is a long-chain fatty acid ester substance, and is a product obtained by reacting a short-chain alcohol substance (methanol or ethanol) with certain fatty oil substances. The most common production method of biodiesel is transesterification, i.e. adding a certain amount of methanol to vegetable oil and fat, and heating to a certain temperature. Reacting under the action of catalyst (acid or alkali) to produce fatty acid methyl ester, and separating out by-product glycerin. As a clean biological fuel capable of replacing petroleum diesel, the use performance of the biological diesel is basically equivalent to that of the existing petroleum diesel, and the biological diesel has better performance, and comprises the following components: the composite material has the advantages of high biodegradability, good environmental protection performance, good low-temperature engine starting function and lubricating function, high lightning, good safety performance, renewable performance and wide development prospect.
The current production method of biodiesel mainly comprises a physical method and a chemical method, and the chemical method is most commonly used. The chemical method is to chemically convert animal and vegetable oil and fat to change its molecular structure and radically change its flowability and viscosity, so that the prepared biological diesel oil has dynamic and combustion characteristics similar to those of petroleum diesel oil. The source of grease is the focus of current research, and the main sources include: plant origin, animal origin and algal origin. The plant source is that grease extracted from rapeseeds, soybeans, peanuts and various oil crops is used as a raw material; the animal source is animal fat such as lard, beef tallow, mutton fat and the like or waste catering grease; the microalgae is used as a source of algae, a large amount of grease can be synthesized by the microalgae in the growth process, the microalgae grease belongs to single-cell grease, the main components of the microalgae grease are glycerol and fatty acid, the microalgae is synthesized in an algae body by using carbohydrate, hydrocarbon and common grease as carbon sources under a certain condition and mainly used as components of biomembranes, metabolites and energy sources. The development of the biodiesel is restricted due to the plant source, the animal source, the long life cycle of the raw materials, the insufficient total amount of resources, the low economic benefit and the influence on the price of agricultural products, farmland resources and grain safety. Compared with the traditional biodiesel raw materials from plant sources and animal sources, the microalgae have the great advantages of wide distribution, short growth cycle, large biomass, strong environmental adaptability, high oil content, no land competition with grains, no competition with people and the like, are greatly pursued by researchers, and are considered to be one of important ways for solving the problem of insufficient biodiesel raw materials at present. Currently, grease of algae origin is the focus of research. Microalgae have many advantages as a new generation of biodiesel feedstock. Algae are widely distributed in fresh water and seawater. There are tens of thousands of species of microalgae that have been identified worldwide, and their number is increasing. Compared with traditional oil crops, the microalgae have large biomass and short growth period. The growth rate of the microalgae is far higher than that of terrestrial crops, the biomass of the microalgae can be doubled within 24 hours generally, and the biomass doubling time in the exponential growth phase is 3-5 hours generally. The microalgae oil has similar components to vegetable oil, is a substitute of vegetable oil, and can be directly used for producing biodiesel by the prior art. Under normal culture conditions, the oil content of general microalgae can reach 20-50%, part of microalgae can be cultured by seawater, can tolerate extreme environments such as deserts, arid lands, semi-arid lands and the like, and does not occupy cultivated lands, so that the production of grain crops is not threatened. The microalgae can absorb and utilize a large amount of C02 and nitride discharged in industrial and agricultural production or extract nitrogen, phosphorus and the like from waste water, and is beneficial to improving the environment.
The biochemical composition of microalgae can be adjusted by changing environmental conditions, thereby increasing oil content. The algae species in Zhejiang are abundant, and the method has natural favorable conditions for researching the production of biodiesel by the algae. The biggest problem in the current industrialization of producing diesel oil by utilizing algae is how to reduce the culture cost and improve the oil yield. Because of the differences in oil content and growth capacity of different microalgae species, the microalgae species need to be screened before microalgae culture. The selected microalgae strain must have high productivity and high oil content, have strong anti-fouling capability, and be able to adapt to environmental changes. The most common microalgae include chlorella, dunaliella, nannochloropsis, porphyridium, scenedesmus, etc., and are often used for producing microalgae biodiesel due to their high oil content and growth rate. However, there are many researchers to improve the oil content and growth rate of microalgae by changing the growth environment of microalgae or using genetic engineering.
The chlorella pyrenoidosa serving as a renewable energy biomass can accumulate grease under photoautotrophic and heterotrophic culture conditions, can effectively utilize solar energy and organic energy to rapidly grow and accumulate the grease, and can be used as a renewable energy source to prepare biodiesel. However, the existing large-scale culture of the chlorella pyrenoidosa has the problems of slow growth, low biomass and low oil content, and is not beneficial to large-scale culture. CN106754383A discloses a method for co-culturing chlorella and oleaginous yeast to improve oil yield, which comprises inoculating the chlorella and oleaginous yeast into a culture medium at the same time, wherein the total fat content of the mixed culture microorganism dry powder can reach 40.55%, the total fatty acid yield can reach 175.64mg/l/d, which is obviously larger than that of microalgae cells cultured independently and is more than twice of that of yeast cells cultured independently. But still has the defects of increased fat content, higher fermentation cost and long period. The influence of fermentation conditions on the fat content of chlorella pyrenoidosa, which is reported by the academy of Tianjin agriculture, of Wangzhijiang et al, researches the influence of carbon source, nitrogen source, carbon-nitrogen ratio, pH, temperature and the like in a culture medium on the biomass and the fat content of the chlorella pyrenoidosa, adopts glucose as a main raw material, and ensures that the algae content in a fermentation liquid reaches 5.6g/L and the fat content reaches 1.46 g/L. The cassava dregs are byproducts generated after starch is extracted from cassava, the main indexes comprise crude fiber, crude ash and water, the nutrition cost is low, and the cassava dregs are generally used as feed or waste. In Hangzhou areas, a large number of starch factories exist, a large number of cassava residues are generated when cassava is used for processing and producing starch, and the technical problem to be solved is how to effectively utilize the cassava residues. The previous patent technology of the applicant, namely a method for preparing oil by using the cassava dregs as the main raw material by using the chlorella pyrenoidosa cells, improves the oil yield and reduces the fermentation cost by mixed culture and adopting the cassava dregs as the main raw material; on the basis, the applicant continues to research to prepare the grease into the biodiesel.
Disclosure of Invention
The invention aims to overcome the defects of low efficiency, high culture cost and the like of biodiesel produced by algae in the prior art, and provides a method for preparing biodiesel by using cassava residues as main raw materials by using chlorella pyrenoidosa cells.
The invention is realized by the following technical scheme:
the method for preparing the biodiesel by using the chlorella pyrenoidosa cells as the main raw material comprises the following steps: step 1) mixed culture of microorganisms, step 2) extraction of grease, and step 3) preparation of biodiesel.
Specifically, the step 1) of mixed culture of microorganisms comprises the following steps: inoculating the chlorella pyrenoidosa liquid and the trichoderma reesei seed liquid in the logarithmic growth phase into a culture medium containing cassava dregs, continuously illuminating for 24 hours at 28 ℃, wherein the intensity is 6000 plus 8000Lux, the rotating speed is 100rpm, the fermentation time is 2-3 days, then inoculating the aspergillus niger seed liquid, continuously culturing for 3-4 days, and after the culture is finished, centrifuging, washing, freezing and drying to obtain microbial powder; the composition of the medium is as follows: 50-80g/L of cassava dregs, 1-2g/L of sodium nitrate, 0.5-1g/L of potassium dihydrogen phosphate, 0.1-0.2g/L of sodium chloride, 200mg/L of magnesium sulfate heptahydrate, 50-70mg/L of calcium chloride, 20-30mg/L of ferric ammonium citrate and 10-15mg/L of zinc sulfate heptahydrate.
Specifically, the step 2) of extracting the grease comprises the following steps: treating microbial powder by adopting a pulsed electric field, and then adding the powder into a chloroform-methanol mixed solution, wherein the addition amount is 1g of powder: 2ml of chloroform-methanol mixed solution, ultrasonically extracting, then centrifuging, collecting a chloroform phase, blow-drying in nitrogen, and vacuum-drying to obtain the grease.
Specifically, the step 3) for preparing the biodiesel comprises the following steps:
the oil and fat, lipase and methanol are mixed according to the proportion of 5 ml: 1 g: adding 6ml of methanol into a reactor in a volume ratio, carrying out catalytic reaction for 12 hours, adding methanol which accounts for twice the volume of the grease, continuing the catalytic reaction for 20 hours, and stopping the reaction, wherein the catalytic reaction is carried out in a shaking table with the temperature controlled at 45 ℃, and the rotating speed of the shaking table is 100 rpm; centrifuging the mixture of the reaction system at 12000rpm for 10 minutes, removing the upper liquid phase part, and standing for layering; taking out the lower liquid phase after standing and layering, namely the crude biodiesel, centrifuging at 12000rpm for 10 minutes again, taking out the upper liquid phase, adding ultrapure water with the volume of 2 times and the temperature of 50 ℃, fully and uniformly stirring, centrifuging at 12000rpm for 10 minutes again, standing and layering to obtain an upper organic phase, separating and taking out the upper organic phase to obtain the biodiesel.
Preferably, the trichoderma reesei seed solution is prepared according to the following process: inoculating the Trichoderma reesei on a PDA culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain a trichoderma reesei seed solution; the primary seed culture medium and the secondary seed culture medium are both PDA liquid culture media.
Preferably, the protein chlorella solution is prepared by the following process: selecting Chlorella pyrenoidosa, inoculating into a container containing growth medium, culturing at 28 deg.C under illumination intensity of 6000lux, shaking the container 2-3 times per day, and growing to logarithmic phase to obtain Chlorella pyrenoidosa solution; the components of the growth medium are as follows: 10g/L of glucose, 2g/L of ammonium chloride, 1g/L of sodium nitrate, 0.5g/L of monopotassium phosphate, 0.1g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 30mg/L of calcium chloride, 20mg/L of ferric ammonium citrate, 10mg/L of zinc sulfate heptahydrate and 10mg/L of manganese sulfate.
Preferably, the aspergillus niger seed liquid is prepared according to the following process: inoculating Aspergillus niger on a slant culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain an Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the first-stage seed culture medium and the second-stage seed culture medium comprise the following components: 50g/L of corn flour, 10g/L of cane sugar, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate and 1g/L of dipotassium phosphate.
Preferably, the pulsed electric field treatment is: electric field intensity 20kV/cm, pulse width 4us, and processing time 200 us.
Preferably, the ultrasound extraction is: the extraction temperature is 60-65 ℃, the ultrasonic power is 100-.
Preferably, the chloroform-methanol mixed solution is prepared by mixing chloroform and methanol according to the volume ratio of 2: 1.
Compared with the prior art, the invention has the advantages that the invention mainly comprises but is not limited to the following aspects:
the growth amount of algae and the oil yield are not completely in a positive correlation proportion, the growth amount is in a reasonable range by adjusting external factors, and the oil yield is maximized, which is always a technical difficulty. The trichoderma reesei utilizes fermented cassava residues to generate reducing sugar, the reducing sugar can promote the growth rate of algae and increase biomass, aspergillus niger is inoculated after the algae reaches a certain growth amount, and the aspergillus niger can quickly utilize a nitrogen source and part of the reducing sugar so as to compete with the algae, so that the algae can obtain higher grease content through nitrogen limitation or nutrient limitation under the conditions of nitrogen limitation and nutrient deprivation stress. And Aspergillus niger can also generate a large amount of carbon dioxide inorganic carbon source, so that the grease yield of algae is improved. Trichoderma reesei and Aspergillus niger can also produce grease substances, and cassava residues are used as main raw materials in the culture process, so that the price is low, and the enterprise cost is reduced. Oxygen released by photosynthesis of the microalgae in the mixed culture can be utilized by somatic cells, so that the mixed culture system is in an equilibrium state. The strong electric field with proper treatment time can affect polar molecules in cells, generate oscillation on cell membranes, generate unrecoverable destructive action, enhance the permeability of the cell membranes and finally accelerate the exchange efficiency of intracellular and extracellular grease. When ultrasonic wave acts on a liquid reaction system, a large number of tiny bubbles are formed in the liquid due to the cavitation action of the ultrasonic wave, the bubbles are generated and destroyed very quickly, the reaction system can generate local high temperature and high pressure, the effect of destroying cell walls can be achieved, and the contact chance of a solvent and intracellular substances can be increased. The method for extracting the grease by using the pulsed electric field and the ultrasonic wave is an efficient method, combines the advantages of the pulsed electric field and the ultrasonic wave, shortens the reaction time, and can reduce the reaction energy consumption. The biodiesel prepared by the grease has high yield, and the percentage content of C16 and C18 fatty acid methyl ester in the biodiesel component reaches more than 95 percent, so the biodiesel component can be used as an ideal substance of a diesel substitute.
Drawings
FIG. 1: the influence of different pulse electric field time on the oil yield;
FIG. 2: the influence of the ultrasonic power and the ultrasonic time on the oil yield.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
The method for preparing the grease by using the chlorella pyrenoidosa cells as the main raw material comprises the following steps:
inoculating the trichoderma reesei on a PDA culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain a trichoderma reesei seed solution; the primary seed culture medium and the secondary seed culture medium are both PDA liquid culture media;
selecting Chlorella pyrenoidosa, inoculating into a container containing growth medium, culturing at 28 deg.C under illumination intensity of 6000lux, shaking the container 2-3 times per day, and growing to logarithmic phase to obtain Chlorella pyrenoidosa solution; the components of the growth medium are as follows: 10g/L of glucose, 2g/L of ammonium chloride, 1g/L of sodium nitrate, 0.5g/L of monopotassium phosphate, 0.1g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 30mg/L of calcium chloride, 20mg/L of ferric ammonium citrate, 10mg/L of zinc sulfate heptahydrate and 10mg/L of manganese sulfate;
inoculating Aspergillus niger on a slant culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain an Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the first-stage seed culture medium and the second-stage seed culture medium comprise the following components: 50g/L of corn flour, 10g/L of cane sugar, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate and 1g/L of dipotassium phosphate.
Chlorella pyrenoidosa in logarithmic growth phaseInoculating the solution and Trichoderma reesei seed solution into a reaction tank containing culture medium, wherein the inoculation densities of Chlorella pyrenoidosa and Trichoderma reesei are 1 × 106Each/ml and 1X 107cfu/ml, temperature 28 deg.C, 24 hr continuous illumination, intensity 6000Lux, rotation speed 100rpm, fermentation time 3 days, inoculating Aspergillus niger seed solution with inoculation density of 5 × 107cfu/ml, continuously culturing for 3 days, and after the culture is finished, centrifuging, washing and freeze-drying to obtain powder; the specific composition of the culture medium is as follows: 50g/L of cassava dregs, 1g/L of sodium nitrate, 0.5g/L of monopotassium phosphate, 0.1g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 50mg/L of calcium chloride, 20mg/L of ferric ammonium citrate and 10mg/L of zinc sulfate heptahydrate.
The powder was treated with a pulsed electric field having an electric field strength of 20kV/cm, a pulse width of 4us, and a treatment time of 200us, and then added to a chloroform-methanol mixed solution (the volume ratio of chloroform to methanol was 2: 1) in an amount of 1g of the powder: 2ml of chloroform-methanol mixed solution is subjected to ultrasonic extraction at the extraction temperature of 60 ℃, the ultrasonic power of 200W and the extraction time of 60min, then the mixture is centrifuged, the chloroform phase is collected and dried in nitrogen, and the mixture is dried in vacuum to obtain grease;
fats and oils, lipase (1 ten thousand U/g) and methanol were mixed in a ratio of 5 ml: 1 g: adding 6ml of methanol into a reactor in a volume ratio, carrying out catalytic reaction for 12 hours, adding methanol which accounts for twice the volume of the grease, continuing the catalytic reaction for 20 hours, and stopping the reaction, wherein the catalytic reaction is carried out in a shaking table with the temperature controlled at 45 ℃, and the rotating speed of the shaking table is 100 rpm; centrifuging the mixture of the reaction system for 10 minutes at 12000rpm, removing the upper liquid phase part, and standing for layering; taking out the lower liquid phase after standing and layering, namely the crude biodiesel, centrifuging for 10 minutes again at 12000rpm, taking out the upper liquid phase, adding ultrapure water with the volume of 2 times of 50 ℃, fully and uniformly stirring, centrifuging for 10 minutes again at 12000rpm, standing and layering to obtain an upper organic phase, separating and taking out the upper organic phase, and obtaining the biodiesel.
Example 2
The method for preparing the grease by using the chlorella pyrenoidosa cells as the main raw material comprises the following steps:
inoculating the trichoderma reesei on a PDA culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain a trichoderma reesei seed solution; the primary seed culture medium and the secondary seed culture medium are both PDA liquid culture media;
selecting Chlorella pyrenoidosa, inoculating into a container containing growth medium, culturing at 28 deg.C under illumination intensity of 6000lux, shaking the container 2-3 times per day, and growing to logarithmic phase to obtain Chlorella pyrenoidosa solution; the components of the growth medium are as follows: 10g/L of glucose, 2g/L of ammonium chloride, 1g/L of sodium nitrate, 0.5g/L of monopotassium phosphate, 0.1g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 30mg/L of calcium chloride, 20mg/L of ferric ammonium citrate, 10mg/L of zinc sulfate heptahydrate and 10mg/L of manganese sulfate;
inoculating Aspergillus niger on a slant culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain an Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the first-stage seed culture medium and the second-stage seed culture medium comprise the following components: 50g/L of corn flour, 10g/L of cane sugar, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate and 1g/L of dipotassium phosphate.
Inoculating Chlorella pyrenoidosa solution and Trichoderma reesei seed solution in logarithmic growth phase into a reaction tank containing culture medium, wherein the inoculation density of Chlorella pyrenoidosa and Trichoderma reesei is 1 × 106Each/ml and 1X 107cfu/ml, temperature 28 deg.C, 24 hr continuous illumination, intensity 7000Lux, rotation speed 100rpm, fermentation time 3 days, inoculating Aspergillus niger seed solution with inoculation density of 5 × 107cfu/ml, continuously culturing for 4 days, and after the culture is finished, centrifuging, washing and freeze-drying to obtain powder; the specific composition of the culture medium is as follows: 80g/L of cassava residue, 2g/L of sodium nitrate, 1g/L of monopotassium phosphate, 0.2g/L of sodium chloride, 200mg/L of magnesium sulfate heptahydrate, 70mg/L of calcium chloride, 30mg/L of ferric ammonium citrate and 15mg/L of zinc sulfate heptahydrate.
The powder was treated with a pulsed electric field having an electric field strength of 20kV/cm, a pulse width of 4us, and a treatment time of 200us, and then added to a chloroform-methanol mixed solution (the volume ratio of chloroform to methanol was 2: 1) in an amount of 1g of the powder: 2ml of chloroform-methanol mixed solution is subjected to ultrasonic extraction at 65 ℃, the ultrasonic power is 100W, the extraction time is 90min, then centrifugation is carried out, a chloroform phase is collected, the chloroform phase is dried in nitrogen, and vacuum drying is carried out, so as to obtain grease;
fats and oils, lipase (1 ten thousand U/g) and methanol were mixed in a ratio of 5 ml: 1 g: adding 6ml of methanol into a reactor in a volume ratio, carrying out catalytic reaction for 12 hours, adding methanol which accounts for twice the volume of the grease, continuing the catalytic reaction for 20 hours, and stopping the reaction, wherein the catalytic reaction is carried out in a shaking table with the temperature controlled at 45 ℃, and the rotating speed of the shaking table is 100 rpm; centrifuging the mixture of the reaction system for 10 minutes at 12000rpm, removing the upper liquid phase part, and standing for layering; taking out the lower liquid phase after standing and layering, namely the crude biodiesel, centrifuging for 10 minutes again at 12000rpm, taking out the upper liquid phase, adding ultrapure water with the volume of 2 times of 50 ℃, fully and uniformly stirring, centrifuging for 10 minutes again at 12000rpm, standing and layering to obtain an upper organic phase, separating and taking out the upper organic phase, and obtaining the biodiesel.
Comparative example 1
The procedure of example 1 was repeated except that Trichoderma reesei and Aspergillus niger were not added.
Comparative example 2
The procedure of example 1 was followed without adding Aspergillus niger.
Comparative example 3
Chlorella pyrenoidosa, Trichoderma reesei, and Aspergillus niger were added simultaneously, as in example 1.
Example 3
The dry weight content of biomass, the total fat content (as a percentage of the dry weight of biomass) and the oil yield were measured in the examples and comparative examples of the invention. The specific detection results are shown in table 1;
TABLE 1
| Group of | Incubation time d | Dry weight content g/L of biomass | The total lipid content% | Oil yield g/L |
| Example 1 | 6 | 4.37 | 44.5 | 1.94 |
| Comparative example 1 | 6 | 3.02 | 35.2 | 1.06 |
| Comparative example 2 | 6 | 4.19 | 37.8 | 1.58 |
| Comparative example 3 | 6 | 3.74 | 41.6 | 1.56 |
And (4) conclusion: the influence of the strain type and the addition timing on the dry matter content, the total fat content and the oil and fat yield was verified by table 1, and it was found that each index of the dry matter content, the total fat content and the oil and fat yield of example 1 was higher than those of comparative examples 1 to 3; the comparative example 1 has the advantages that no strain is added, only algae culture is adopted, indexes in all aspects are lowest, and the oil yield is reduced by about 40% compared with that of the example 1; comparative example 2 only using trichoderma reesei, which produced reducing sugars that could promote the growth rate of algae and increase biomass by fermenting cassava residue, but reduced the oil content compared to example 1, while maintaining a higher microbial quality with continued adequate nutrition; comparative example 3 aspergillus niger was inoculated at the same time, resulting in the aspergillus niger competing for the carbon source transitionally, causing insufficient nutrients in the algae, resulting in slow growth of the algae; in example 1, after the algae substantially reaches a high growth amount, aspergillus niger is inoculated, wherein the aspergillus niger can rapidly utilize a nitrogen source and part of reducing sugar, so as to compete with the algae, so that the algae can obtain a higher oil content through nutrient limitation under the stress conditions of nitrogen limitation and nutrient deprivation, and the aspergillus niger can also generate a large amount of carbon dioxide inorganic carbon source, so as to improve the oil yield of the algae.
Example 4
Taking example 1 as an example, the influences of different pulse electric field times on the grease yield are respectively set to 0, 100, 200, 400 and 600us, as shown in fig. 1, the grease yield is obviously increased along with the increase of the processing time, and after 200us, the increase of the processing time does not obviously influence the grease yield, so that 200us is most suitable for selection. The strong electric field with proper treatment time can affect polar molecules in cells, generate oscillation on cell membranes, generate unrecoverable destructive action, enhance the permeability of the cell membranes and finally accelerate the exchange efficiency of intracellular and extracellular grease.
Example 5
Influence of ultrasonic power and ultrasonic time on grease yield:
setting the ultrasonic power to be 50w, 100w, 200w, 400w and 800 w; the ultrasonic extraction time is 15, 30, 60, 90 and 120min, as shown in fig. 2, the oil yield is gradually increased along with the increase of the ultrasonic power and the ultrasonic time, and finally, when the ultrasonic power is selected to be 100 and 200W and the treatment time is 60-90min, the oil extraction effect is optimal. When ultrasonic wave acts on a liquid reaction system, a large number of tiny bubbles are formed in the liquid due to the cavitation action of the ultrasonic wave, the bubbles are generated and destroyed very quickly, the reaction system can generate local high temperature and high pressure, the effect of destroying plant cell walls can be achieved, and the contact opportunity of a solvent and intracellular substances can be increased. The method for extracting the grease by using the pulsed electric field and the ultrasonic wave is an efficient method, combines the advantages of the pulsed electric field and the ultrasonic wave, shortens the reaction time, and can reduce the reaction energy consumption.
Example 6
On the basis of example 4, the present inventors also examined the yield of each group of biodiesel (calculation of the yield of biodiesel, yield (Y) ═ biodiesel weight/microalgal oil weight) × 100%, and analyzed the composition of biodiesel by GC-MS, as shown in table 2.
TABLE 2
| Group of | Yield of biodiesel% | Total content of C16% | Total content of C18% |
| Example 1 | 81.6 | 39.2 | 57.7 |
| Comparative example 1 | 82.1 | 28.5 | 60.8 |
| Comparative example 2 | 81.3 | 35.6 | 56.4 |
| Comparative example 3 | 80.1 | 31.7 | 58.1 |
As shown in Table 2, the yields of biodiesel from each group were similar, at around 80%. 16-18C atoms in the diesel oil are proper, the diesel oil has good liquidity and is easy to oxidize, and full combustion can be ensured, in the embodiment 1, the main components C16 and C18 of the biodiesel oil account for 96.9 percent of the total amount of the grease, and no aromatic hydrocarbon exists, so that the full combustion can be ensured, and no carbon black is generated; the percentage content of C16 and C18 is obviously higher than that of comparative examples 1-3, and the diesel oil is similar to the conventional diesel oil and can be used as a more appropriate substitute of the conventional diesel oil.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (7)
1. The method for preparing the biodiesel by using the chlorella pyrenoidosa cells as the main raw material is characterized by comprising the following steps:
step 1) mixed culture of microorganisms: inoculating the chlorella pyrenoidosa solution and the trichoderma reesei seed solution in the logarithmic growth phase into a reaction tank of a culture medium containing cassava residues, continuously illuminating for 24 hours at 28 ℃, wherein the intensity is 6000-plus 8000Lux, the rotating speed is 100rpm, the fermentation time is 2-3 days, then inoculating the aspergillus niger seed solution, continuously culturing for 3-4 days, and after the culture is finished, centrifuging, washing and freeze-drying to obtain microbial powder; the composition of the medium is as follows: 50-80g/L of cassava dregs, 1-2g/L of sodium nitrate, 0.5-1g/L of potassium dihydrogen phosphate, 0.1-0.2g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 50-70mg/L of calcium chloride, 20-30mg/L of ferric ammonium citrate and 10-15mg/L of zinc sulfate heptahydrate;
extracting grease in the step 2): treating microbial powder by adopting a pulsed electric field, and then adding the powder into a chloroform-methanol mixed solution, wherein the addition amount is 1g of powder: 2ml of chloroform-methanol mixed solution is subjected to ultrasonic extraction and then centrifugation, a chloroform phase is collected, the chloroform phase is dried in nitrogen, and vacuum drying is carried out to obtain grease;
the step 3) is to prepare the biodiesel: the oil and fat, lipase and methanol are mixed according to the proportion of 5 ml: 1 g: adding 6ml of methanol into a reactor in a volume ratio, carrying out catalytic reaction for 12 hours, adding methanol which accounts for twice the volume of the grease, continuing the catalytic reaction for 20 hours, and stopping the reaction, wherein the catalytic reaction is carried out in a shaking table with the temperature controlled at 45 ℃, and the rotating speed of the shaking table is 100 rpm; centrifuging the mixture of the reaction system at 12000rpm for 10 minutes, removing the upper liquid phase part, and standing for layering; taking out the lower liquid phase after standing and layering, namely the crude biodiesel, centrifuging at 12000rpm for 10 minutes again, taking out the upper liquid phase, adding ultrapure water with the volume of 2 times and the temperature of 50 ℃, fully and uniformly stirring, centrifuging at 12000rpm for 10 minutes again, standing and layering to obtain an upper organic phase, separating and taking out the upper organic phase, and obtaining the biodiesel.
2. The method according to claim 1, wherein the trichoderma reesei seed solution is prepared by the following process: inoculating the Trichoderma reesei on a PDA culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain a trichoderma reesei seed solution; the primary seed culture medium and the secondary seed culture medium are both PDA liquid culture media.
3. The method of claim 1, wherein the protein chlorella solution is prepared by the following process: selecting Chlorella pyrenoidosa, inoculating into a container containing growth medium, culturing at 28 deg.C under illumination intensity of 6000lux, shaking the container 2-3 times per day, and growing to logarithmic phase to obtain Chlorella pyrenoidosa solution; the components of the growth medium are as follows: 10g/L of glucose, 2g/L of ammonium chloride, 1g/L of sodium nitrate, 0.5g/L of monopotassium phosphate, 0.1g/L of sodium chloride, 100mg/L of magnesium sulfate heptahydrate, 30mg/L of calcium chloride, 20mg/L of ferric ammonium citrate, 10mg/L of zinc sulfate heptahydrate and 10mg/L of manganese sulfate.
4. The method according to claim 1, wherein the Aspergillus niger seed solution is prepared by the following process: inoculating Aspergillus niger on a slant culture medium for culturing by streaking to obtain a single colony; selecting a single colony, inoculating the single colony to a primary seed culture medium for culture, and then performing secondary seed culture medium culture to obtain an Aspergillus niger seed solution; the slant culture medium comprises the following components: 150g/L of potato, 20g/L of cane sugar and 15g/L of agar; the first-stage seed culture medium and the second-stage seed culture medium comprise the following components: 50g/L of corn flour, 10g/L of cane sugar, 5g/L of ammonium sulfate, 1g/L of monopotassium phosphate and 1g/L of dipotassium phosphate.
5. The method of claim 1, wherein the pulsed electric field treatment is: electric field intensity 20kV/cm, pulse width 4us, and processing time 200 us.
6. The method of claim 1, wherein the ultrasonic extraction is: the extraction temperature is 60-65 ℃, the ultrasonic power is 100-.
7. The method as claimed in claim 1, wherein the chloroform-methanol mixed solution is prepared from chloroform and methanol at a volume ratio of 2: 1.
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