CN105505555B

CN105505555B - Method for extracting grease from fermentation liquor containing oil-producing microbial cells

Info

Publication number: CN105505555B
Application number: CN201610059892.1A
Authority: CN
Inventors: 姜悦; 陈�峰; 陈璇; 柳泽深
Original assignee: RUNKE BIOENGINEERING (FUJIAN) Co Ltd
Current assignee: RUNKE BIOENGINEERING (FUJIAN) Co Ltd
Priority date: 2016-01-28
Filing date: 2016-01-28
Publication date: 2020-01-03
Anticipated expiration: 2036-01-28
Also published as: CN105505555A

Abstract

The invention belongs to a method for extracting grease, and particularly relates to a method for extracting grease from fermentation liquor containing oil-producing microbial cells. Comprises the process steps of cracking, wall breaking, extraction and refining, recycling of waste acid and the like. The invention solves the problems of high waste acid treatment cost, low acid utilization rate, poor wall breaking effect, low oil extraction rate and the like in the prior art, and has the advantages of high oil extraction rate, low raw material cost and waste acid treatment cost, effective reduction of energy consumption and the like.

Description

Method for extracting grease from fermentation liquor containing oil-producing microbial cells

Technical Field

The invention belongs to a method for extracting grease, and particularly relates to a method for extracting grease from fermentation liquor containing oil-producing microbial cells.

Background

Microbial oil is another new oil resource developed after animal and vegetable oil. In recent years, with the lack of resources for producing functional oils and fats having medical and health care effects and energy oils and fats, cell oils and fats derived from microorganisms have been receiving extensive attention and research. The culture of the microbial cells has the advantages of small occupied area, rapid growth, short period and no influence of climatic conditions, so the method has great development potential. The oil-producing microorganisms are various in types, and mainly comprise microalgae, molds, bacteria, yeasts and the like, common oil-producing microalgae comprise photoautotrophic diatoms (Diatom), Spirulina (Spirulina) and the like, heterotrophic Schizochytrium (Schizochytrium) and Crypthecodinium cohnii and the like; the yeast includes Lipomyces Stachydis (Lipomyces), Rhodotorula glutinis (Rhodotorula glutinis), and Lipomyces oleaginous yeast (Lipomyces adipofer); examples of the mold include Mortierella alpina (Mortierella alpina), Mortierella pusilla (Mortierella isabellina), etc., and these microorganisms can be artificially cultured to obtain a large amount of oil-and-fat-containing biomass.

In the microbial oil production process, oil extraction is an important link, and the general steps are to perform certain treatment on the concentrated and dried fermentation product and then extract the fermentation product by using an organic solvent to obtain the oil. Because microbial cells have cell walls, oil in the cells is difficult to extract through the cell walls only by soaking the microbial cells with an organic solvent, and the oil must be extracted after the cell wall structure is broken. However, the cell wall is a firm structure composed of many high molecular compounds, including peptidoglycan, celloglycan, etc., these glycans are formed by monosaccharide bonded together through glycoside bonds such as β -1,4 or β -1,6, etc., and a fiber network structure is formed on the outer layer of the cell to maintain the cell shape and protect the cell, and the firm cell wall is difficult to be damaged, so the cell wall breaking treatment becomes a difficult point in the microbial oil extraction process.

The currently researched and reported wall breaking methods mainly comprise mechanical grinding, thermal cracking, ultrasonic/microwave assistance, high-pressure homogenization, chemical hydrolysis, biological enzyme hydrolysis and the like. For example, CN102051330B adopts a liquid nitrogen freezing-mechanical grinding method to extract schizochytrium limacinum grease, but in industry, algae liquid contains a large amount of moisture, a large amount of liquid nitrogen is consumed during freezing, and liquid nitrogen is a disposable consumable and cannot be reused, so that the cost is high. CN102051331A and CN102533879B both adopt a biological enzyme method to carry out wall breaking, although the method omits the steps of biomass concentration and drying, the biomass concentration of the fermentation liquor is lower, the volume is huge, a large amount of biological enzyme is required to be added for carrying out cell wall hydrolysis reaction, and a large amount of organic solvent is added for extraction, the operation volume is too large, and the energy consumption is increased, so the fermentation liquor which is not concentrated is directly used as the material in industry, in addition, the price of the biological enzyme preparation is very expensive, the treatment time is longer, and the oil extraction rate is not high. The CN103173273A method is that acid is added into the algae liquid, pH is adjusted to 1.5-3, the algae liquid is processed under the conditions of 100 ℃ and 150 ℃ and pressurization, and because the cell wall can not be completely cracked by single dilute acid and heating treatment, the biological enzyme preparation is added after cooling to further hydrolyze the cell wall. CN101638361A adopts a chemical-physical combined method to extract grease of schizochytrium limacinum, thallus obtained after solid-liquid separation is added with NaOH solution for treatment, then the thallus is sent into a colloid mill to be ground into cell fragments, then the cell fragments are sent into a homogenizing crusher to be crushed under high pressure, the obtained cell slurry is centrifuged to remove water and then extracted by organic solvent. CN102965182B and CN104293473A adjust the pH of the fermentation liquor to acidity, crack the microalgae cell walls under the conditions of dilute acid and heating, and then add an organic solvent to extract oil, but the treatment condition of CN102965182B has low cracking efficiency on the cell walls, and a large amount of oil still exists in the cells and can not be extracted, so that the recovery rate of the total oil is very low; CN104293473A raised the reaction temperature to 260 ℃ in order to improve the cell wall breaking efficiency under dilute acid conditions, but at such high temperature, oxidation of some functional oil is easily caused, and the quality of the extracted oil is damaged. In addition, the waste acid solution after the dilute acid treatment cannot be reused due to low acid concentration, and only can be discharged to cause waste and environmental pollution. In the literature of 'Chinese brewing', 5 th stage 2010, Vanmin and the like, 4mol/L hydrochloric acid is adopted to be treated in a boiling water bath for 8min to extract yeast grease, so that the highest relative yield of grease is obtained, but the cyclic utilization of waste acid liquid is not realized, so that the waste of the waste acid liquid is only caused, and the treatment cost is increased.

In a word, the existing microbial oil extraction method has the problems of poor wall breaking effect, high cost and various steps. Therefore, it is necessary to develop a green oil extraction method with higher extraction efficiency, faster wall breaking speed, simple steps, industrial applicability and no waste discharge.

Disclosure of Invention

The invention aims to provide a method for extracting grease from fermentation liquor containing oil-producing microbial cells, which has high extraction efficiency, high wall breaking speed and less waste discharge.

The overall technical concept of the invention is as follows:

a method for extracting grease from fermentation liquor containing oil-producing microbial cells comprises the following process steps:

A. cracking wall breaking

Carrying out solid-liquid separation on fermentation liquor containing oleaginous microorganisms to obtain wet bacterial sludge with water content of 70-90% by mass, adding a lysate into the wet bacterial sludge, heating to crack and break the wall, and cooling a reactant, wherein the lysate is one of concentrated hydrochloric acid, concentrated sulfuric acid or concentrated phosphoric acid;

B. refining by extraction

Adding an organic solvent into the cooled reactant in the step A to obtain an organic phase and a waste acid water phase, filtering the organic phase, distilling under reduced pressure to obtain crude oil and organic solvent steam, and refining the crude oil to obtain refined oil;

C. cyclic utilization of waste acid

And D, recycling the waste acid liquor generated in the step B as a cracking solution into the step A.

The specific technical concept of the invention is as follows:

the wet bacterial sludge in the step A: the mass ratio of the lysate is 1: 0.6-10.

The temperature raising and cracking conditions in the step A are 40-120 ℃ and 10-100 minutes.

The preferred embodiment comprises the following three technical schemes:

concentrated hydrochloric acid is used as cracking liquid

The process conditions of the process steps are as follows:

A. cracking wall breaking

Carrying out solid-liquid separation on the cultured oleaginous microorganism fermentation liquor to obtain wet bacterial sludge with the water content of 70-90% by mass percent, wherein the weight percentage of the wet bacterial sludge is as follows: adding lysate into wet bacterial sludge according to the mass ratio of the lysate to 1:1-4.5, reacting at 40-80 ℃ for 10-90 minutes, and cooling a reactant, wherein the lysate is concentrated hydrochloric acid with the mass percentage concentration of 37%;

B. refining by extraction

C. cyclic utilization of waste acid

C1, first recycling: according to wet bacterial mud: adding the residual waste acid solution in the step B into the wet bacterial sludge according to the waste acid solution mass ratio of 1:1.3-3.3, carrying out cracking wall breaking reaction for 10-90 minutes at 40-90 ℃, and repeating the step B;

c2, second recycling: according to wet bacterial mud: adding the residual waste acid liquor obtained in the step C1 into the wet bacterial sludge according to the mass ratio of the waste acid liquor being 1:1-2.5, carrying out cracking wall breaking reaction at 50-90 ℃ for 20-90 minutes, and repeating the step B;

c3, third recycling: according to wet bacterial mud: and D, adding the waste acid liquor left in the step C2 into the wet bacterial sludge according to the mass ratio of the waste acid liquor being 1:1.5-2, carrying out a cracking wall breaking reaction at the temperature of 75-90 ℃ for 60-90 minutes, and repeating the step B.

(II) concentrated sulfuric acid is used as cracking liquid

The process conditions of the process steps are as follows:

A. cracking wall breaking

Carrying out solid-liquid separation on the cultured oleaginous microorganism fermentation liquor to obtain wet bacterial sludge with the water content of 70-90% by mass percent, wherein the weight percentage of the wet bacterial sludge is as follows: adding the lysate into the wet bacterial sludge according to the mass ratio of the lysate to 1:0.6-1, reacting at 40-70 ℃ for 10-90 minutes, and cooling the reactant, wherein the lysate is concentrated sulfuric acid with the mass percentage concentration of 98%;

B. refining by extraction

C. cyclic utilization of waste acid

C1, first recycling: according to wet bacterial mud: adding the residual waste acid solution in the step B into the wet bacterial sludge according to the mass ratio of the waste acid solution being 1:2-3.5, carrying out cracking wall breaking reaction for 10-90 minutes at 40-90 ℃, and repeating the step B;

c2, second recycling: according to wet bacterial mud: adding the residual waste acid liquor obtained in the step C1 into the wet bacterial sludge according to the mass ratio of the waste acid liquor being 1:1.5-2.5, carrying out cracking wall breaking reaction at the temperature of 50-90 ℃ for 20-90 minutes, and repeating the step B;

c3, third recycling: according to wet bacterial mud: and D, adding the waste acid liquor left in the step C2 into the wet bacterial sludge according to the mass ratio of the waste acid liquor being 1:0.8-1.5, carrying out cracking wall breaking reaction at the temperature of 75-90 ℃ for 60-90 minutes, and repeating the step B.

(III) concentrated phosphoric acid is used as cracking liquid

The process conditions of the process steps are as follows:

A. cracking wall breaking

Carrying out solid-liquid separation on the cultured oleaginous microorganism fermentation liquor to obtain wet bacterial sludge with the water content of 70-90% by mass percent, wherein the weight percentage of the wet bacterial sludge is as follows: adding the lysis solution into the wet bacterial sludge in a mass ratio of 1:4.5-10, reacting at 70-120 ℃ for 20-100 minutes, performing lysis and wall breaking, and cooling the reactant, wherein the lysis solution is concentrated phosphoric acid with the mass percentage concentration of 85%;

B. refining by extraction

C. cyclic utilization of waste acid

C1, first recycling: according to wet bacterial mud: adding the residual waste acid solution in the step B into the wet bacterial sludge according to the mass ratio of the waste acid solution being 1:4-12, carrying out cracking wall breaking reaction for 50-100 minutes at 90-120 ℃, and then repeating the step B;

c2, second recycling: according to wet bacterial mud: adding the residual waste acid liquor obtained in the step C1 into the wet bacterial sludge according to the mass ratio of the waste acid liquor being 1:2-4, carrying out cracking wall breaking reaction at 90-120 ℃ for 70-100 minutes, and repeating the step B;

c3, third recycling: according to wet bacterial mud: and D, adding the waste acid liquor left in the step C2 into the wet bacterial sludge according to the mass ratio of the waste acid liquor being 1:1-2.5, carrying out the cracking wall breaking reaction at the temperature of 100-120 ℃ for 90-100 minutes, and then repeating the step B.

In the invention, the oil-producing microorganism in the step A is selected from microalgae, saccharomycetes and mould.

The solid-liquid separation method in the step A selects one or a combination of flocculation precipitation, filtration, centrifugation and membrane dehydration.

In order to improve the utilization rate of heat in the present invention, it is preferable that the method further comprises a step a1 after the step a, wherein the heat for raising the temperature in the step a is recycled to the reduced pressure distillation in the step B.

The temperature of reduced pressure distillation in the step B is 35-75 ℃.

And in the step B, the organic solvent is one or a combination of n-hexane, petroleum ether and No. 6 solvent.

And B, using an organic filter membrane, a ceramic filter membrane or a sand plate filter with the pore diameter of 0.5-30 microns when filtering the organic phase in the step B.

In order to improve the utilization rate of the organic solvent in the invention, a preferred technical scheme is that the method further comprises a step B1 after the step B, wherein the step B is to condense and liquefy the organic solvent steam in the step B under pressure to form the organic solvent, and recycle the organic solvent to the extraction in the step B.

In order to realize the effect of the cracking wall-breaking reaction in the invention, the preferable technical scheme is that the mass percentage concentration of the waste acid liquid in the step C is not less than 4%.

In order to improve the utilization effect of the waste acid in the invention and reduce the discharge of waste, the preferable technical scheme is that a step D is further included after the step C, and the step D is to decolor the waste acid liquid left after the recycling in the step C and then use the waste acid liquid for preparing byproducts.

And D, decoloring the waste acid liquor in the step D by adopting an adsorption method, wherein an adsorbent is one or a combination of activated carbon, bentonite, activated clay, diatomite and silica gel.

And the step B of refining the crude oil sequentially comprises decoloring, alkali refining and deodorizing treatment.

And D, preparing a by-product in the step D, namely adding quick lime, limestone or lime milk into the decolored waste acid solution to prepare calcium chloride, calcium sulfate, calcium phosphate and calcium dihydrogen phosphate.

The invention achieves the substantive characteristics and obvious technical progress that:

1. the method adopts a concentrated acid cell wall hydrolysis method to carry out wall breaking treatment, has high wall breaking efficiency, and is proved by comparative experiments carried out by the applicant to effectively improve the extraction rate of the grease.

2. The waste acid liquid after the concentrated acid hydrolysis is recycled, so that the acid waste is avoided, the acid consumption is greatly reduced, and the raw material cost and the waste acid treatment cost are reduced.

3. When the waste acid liquor is used for a plurality of times and the concentration is lower, the waste acid liquor is used for preparing byproducts, thereby realizing waste utilization and avoiding environmental pollution caused by direct discharge.

4. The organic solvent and waste heat in the process are recycled, which is beneficial to reducing energy consumption and production cost.

Detailed Description

The present invention is further described with reference to the following examples, which should not be construed as limiting the scope of the present invention, but the scope of the present invention is defined by the claims, and any equivalent technical means made in the specification may be substituted without departing from the scope of the present invention.

Example 1

Comparison of concentrated acid method with other methods for extracting microbial cell oil

The alga used in the present example is Schizochytrium sp.CGMCC No. 11382.

Seed liquid culture: preparing a seed liquid culture medium by using 20g/L glucose, 1g/L yeast extract, 15g/L sea salt and the balance of sterile water, and culturing in a shaker at 25 ℃ and 200rpm to prepare the seed liquid.

Culturing in a fermentation tank: preparing a fermentation culture medium by using 80g/L glucose, 5g/L corn steep liquor powder, 15g/L sea salt and the balance of sterile water, performing fermentation culture in a 5L fermentation tank at the fermentation temperature of 25 ℃, the stirring speed of 500rpm and the ventilation amount of 2.5L/min, and sampling and collecting fermentation liquor after the fermentation is finished.

Centrifuging the fermentation liquor to obtain bacterial sludge with the water content of 80%, weighing 5g of wet bacterial sludge in a 50ml centrifugal tube, and mixing the wet bacterial sludge: adding concentrated hydrochloric acid with the mass percent concentration of 37% according to the mass ratio of 1:1.5, oscillating and uniformly mixing, and reacting for 10min at 70 ℃; or wet bacterial sludge: adding concentrated sulfuric acid with the mass percent concentration of 98% according to the mass ratio of 1:0.6, oscillating and uniformly mixing, and reacting at 70 ℃ for 10 min; or wet bacterial sludge: adding 85 mass percent concentrated phosphoric acid according to the mass ratio of 1:8, oscillating, uniformly mixing, reacting at 90 ℃ for 20min, and cooling in cold water after the reaction is finished. Adding petroleum ether for extraction for three times, combining organic phases, drying by using nitrogen, and determining the content of cell grease. In this example, the total lipid content was determined by the Bligh-Dyer method, the ultrasound-assisted extraction method, the high pressure homogenization method and the Soxhlet extraction method, which are well known in the art, and the specific method refers to the steps of the method known in the art. The relative yield of the grease is calculated by taking a classical method for measuring the total grease content by Bligh-Dyer as a standard, and the results are as follows:

measurement method	Total lipid content of cells	Relative yield of oil	Average time consumption
				Bligh-Dyer method	58.1±5.1％	100.0％	16h
Ultrasonic assisted extraction process	61.5±4.4％	105.9％	10h
				Soxhlet extraction method	62.7±3.8％	107.9％	12h
Concentrated hydrochloric acid process	66.1±1.9％	113.8％	1h
				Concentrated sulfuric acid process	66.3±2.1％	114.1％	1h
Concentrated phosphoric acid process	65.9±2.3％	113.4％	1h

The results show that the total lipid content of cells obtained by the concentrated acid method is higher than that of the other three methods, compared with the classical Bligh-Dyer method, the relative yield of the lipid is obviously improved, the average time consumption for measuring the total lipid content by the concentrated acid method is far lower than that of the other three methods, and the method has the advantages of high wall breaking speed and higher efficiency.

Example 2

This example uses concentrated sulfuric acid as a lysis solution to extract oil and fat from a fermentation broth containing yeast Cryptococcus fulvus ATCC 20508

Seed liquid culture medium: 20g/L glucose, 3g/L yeast extract, 3g/L malt extract, 5g/L peptone and the balance of sterile water were used to prepare a seed liquid medium, and the pH was adjusted to 5.5.

Fermentation medium: 40g/L glucose, 5g/L yeast extract, 5g/L peptone, 0.5g/L ammonium chloride, 2.7g/L potassium dihydrogen phosphate, 0.95g/L disodium hydrogen phosphate, 1g/L magnesium sulfate heptahydrate, and the balance of sterile water.

Inoculating a tube of preserved yeast Cryptococcus curvatus ATCC 20508 into 50ml of seed culture medium, placing into a 250ml shake flask, culturing at 30 ℃ and 200rpm until the sugar concentration is about 10g/L, transferring into a new seed culture medium according to 5% of the inoculation amount, inoculating into a 100L fermentation tank according to 10% of the inoculation amount after three-stage culture, stirring at 200rpm, controlling the pH to be 5.5-6.5, supplementing glucose when the sugar concentration is reduced to 5g/L, and placing into the tank after 5 days of fermentation.

The method for measuring the total lipid adopts a traditional solvent extraction method, and comprises the steps of weighing a certain amount of freeze-dried cells according to a classical Bligh-Dyer fatty acid extraction and esterification method, stirring the freeze-dried cells and a mixed solution of methanol, chloroform and water in a ratio of 1:2:0.8 for 30 minutes, centrifuging, collecting supernate, repeatedly washing for a plurality of times, combining the supernate, drying the solvent with nitrogen to obtain the lipid of the algae cells, weighing, and measuring the content of the total lipid.

The extraction steps are as follows:

A. cracking wall breaking

Carrying out flocculation sedimentation and membrane filtration on the cultured yeast fermentation broth to obtain wet bacterial sludge, weighing 40kg of wet bacterial sludge with the water content of 90%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated sulfuric acid is 1: slowly adding 24kg of sulfuric acid with the mass concentration of 98% into the reaction kettle according to the proportion of 0.6, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 70 ℃, and carrying out a cracking reaction for 90 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

100L of petroleum ether is added into the reaction kettle and stirred for 5 min. And taking out the organic phase after the two phases are layered, removing solid impurities through a filter, and adding the organic phase into a reduced pressure distillation kettle. Introducing hot water bath released from the reaction kettle into the interlayer of the distillation kettle, controlling the temperature to be 75 ℃, and obtaining crude oil and petroleum ether steam through reduced pressure distillation.

B1 recycling organic solvent steam

The petroleum ether vapor is put into a condensation liquefier and liquefied under the condition of pressurization and condensation. The recovered petroleum ether is returned to the reaction kettle for extraction, and the extraction is repeated for 3 times.

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 30kg of wet bacterial sludge with the water content of 90% according to the mass ratio of the residual waste acid in the step B being 1:2, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing. And (4) heating the hot water subjected to reduced pressure distillation by a water bath heating tank, introducing the heated hot water into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 90min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 58kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step C1 being 1:1.5, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 90min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and D, weighing 174kg of wet bacterial sludge with the water content of 90% according to the mass ratio of the residual waste acid in the step C2 being 1:0.8, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 90min, and repeating the step B.

D. Preparation of by-products

And D, after extraction is finished, taking out the residual waste acid liquor in the step C3, adsorbing and decoloring the residual waste acid liquor by using bentonite, storing the waste acid liquor in a waste acid liquor tank, and adding lime milk in the stirring process to prepare calcium sulfate.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 14.9kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. The total lipid content of the cells measured by the Bligh-Dyer method is 44.7 +/-3.1 percent, and the relative extraction rate of the finally obtained lipid is 110.4 +/-1.2 percent compared with the Bligh-Dyer method.

Example 3

Fermenting and culturing yeast Cryptococcus curvatus ATCC 20508 as strain, and extracting oil from the obtained fermentation liquid. The yeast was cultured in the same manner as in example 2.

The extraction process comprises the following steps:

A. cracking wall breaking

Carrying out flocculation sedimentation and membrane filtration on the cultured yeast fermentation liquor to obtain wet bacteria, weighing 40kg of wet bacteria mud with the water content of 90 percent, adding the wet bacteria mud into a reaction kettle, and mixing the wet bacteria mud: the mass ratio of concentrated sulfuric acid is 1:1, slowly adding 40kg of sulfuric acid with the mass concentration of 98% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 40 ℃, and carrying out a cracking reaction for 10 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. 100L of n-hexane was added to the reaction vessel, and extraction and organic solvent recycling were carried out as described in example 2.

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 31.7kg of wet bacterial sludge with the water content of 90% according to the mass ratio of the residual waste acid in the step B being 1:2.4, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 50 ℃, stirring for 50min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 58.1kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step C1 being 1:1.8, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 75 ℃, stirring for 50min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and D, weighing 156.8kg of wet bacterial sludge with the water content of 90% according to the mass ratio of the residual waste acid in the step C2 being 1:1, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 80 ℃, stirring for 70min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed, resulting in 13.8kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 107.8 +/-2.2%.

Example 4

Fermenting and culturing yeast Cryptococcus curvatus ATCC 20508 as strain, and extracting oil from the obtained fermentation liquid. The yeast fermentation culture was the same as in example 2.

The extraction process comprises the following steps:

A. cracking wall breaking

Carrying out flocculation sedimentation and membrane filtration on the cultured yeast fermentation liquor to obtain wet bacteria, weighing 40kg of wet bacteria mud with the water content of 70 percent, adding the wet bacteria mud into a reaction kettle, and mixing the wet bacteria mud: the mass ratio of concentrated sulfuric acid is 1: 0.7, slowly adding 28kg of sulfuric acid with the mass concentration of 98% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 50 ℃, and carrying out a cracking reaction for 50 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

100L of solvent No. 6 was added to the reaction vessel, and the extraction and organic solvent recycling operations were carried out as described in example 2.

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 20kg of wet bacterial sludge with the water content of 70% according to the mass ratio of the residual waste acid in the step B being 1:2.8, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 40 ℃, stirring for 70min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 35kg of wet bacterial sludge with the water content of 70% according to the mass ratio of the residual waste acid in the step C1 being 1:2, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing the wet bacterial sludge and the wet bacterial sludge. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 60 ℃, stirring for 30min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and C8978.75 kg of wet bacterial sludge with the water content of 70 percent is weighed according to the mass ratio of the residual waste acid in the step C2 being 1:1.2, and is added into the reaction kettle to be stirred and mixed evenly. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 75 ℃, stirring for 60min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 24.8kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 106.3 +/-1.8%.

Example 5

The extraction process comprises the following steps:

A. cracking wall breaking

Carrying out flocculation sedimentation and membrane filtration on the cultured yeast fermentation liquor to obtain wet bacteria, weighing 40kg of wet bacteria mud with the water content of 75 percent, adding the wet bacteria mud into a reaction kettle, and mixing the wet bacteria mud: the mass ratio of concentrated sulfuric acid is 1:0.8, slowly adding 50kg of sulfuric acid with the mass concentration of 98% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 60 ℃, and carrying out a cracking reaction for 30 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

100L of petroleum ether was added to the reaction vessel, and the extractant organic solvent was recycled as described in example 2.

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 26.7kg of wet bacterial sludge with the water content of 75% according to the mass ratio of the residual waste acid in the step B being 1:3, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 60 ℃, stirring for 10min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 43.5kg of wet bacterial sludge with the water content of 75% according to the mass ratio of the residual waste acid in the step C1 being 1:2.3, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 50 ℃, stirring for 20min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and C2, weighing 94.7kg of wet bacterial sludge with the water content of 75% according to the mass ratio of the residual waste acid in the step C2 being 1:1.4, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 77 ℃, stirring for 75min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 25.3kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 110.5 +/-1.5%.

Example 6

The extraction process comprises the following steps:

A. cracking wall breaking

Carrying out flocculation sedimentation and membrane filtration on the cultured yeast fermentation liquor to obtain wet bacteria, weighing 40kg of wet bacterial sludge with the water content of 80 percent, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated sulfuric acid is 1: slowly adding 36kg of sulfuric acid with the mass concentration of 98% into the reaction kettle according to the proportion of 0.9, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to 65 ℃, and carrying out a cracking reaction for 70 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

100L of n-hexane was added to the reaction vessel, and extraction and organic solvent recycling were carried out as described in example 2.

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 19.4kg of wet bacterial sludge with the water content of 80% according to the mass ratio of the residual waste acid in the step B being 1:3.5, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 75 ℃, stirring for 30min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 33.4kg of wet bacterial sludge with the water content of 80 percent according to the mass ratio of the residual waste acid in the step C1 being 1:2.5, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 80 ℃, stirring for 70min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and C2, weighing 73.5kg of wet bacterial sludge with the water content of 80 percent according to the mass ratio of the residual waste acid in the step C2 being 1:1.5, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 85 ℃, stirring for 80min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 15.0kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 100.8 +/-2.2%.

Example 7

Performing fermentation culture by taking Crypthecodinium cohnii ATCC 30556 as strain, and extracting oil from the obtained fermentation liquid.

Fermentation culture of Crypthecodinium cohnii:

inoculating a tube of preserved Crypthecodinium cohnii ATCC 30556 into 50ml seed culture medium, placing into 250ml shake flask, culturing at 25 deg.C and 150rpm for 36h, transferring into new seed culture medium according to 5% inoculum size, inoculating into 100L fermentation tank with 10% inoculum size after three generations of culture, stirring at 200rpm, ventilating at 2.5vvm, controlling pH to 6-7, supplementing glucose when sugar concentration is reduced to 5g/L, fermenting for 4 days, and placing into tank. The culture medium adopted by the culture comprises glucose as a carbon source, yeast extract and corn steep liquor as a nitrogen source, and sodium salt, magnesium salt, phosphate, potassium salt, calcium salt, sulfate, chloride and the like as inorganic salts. The microelements comprise manganese, zinc, cobalt, nickel, iron, etc.

The total lipids were determined as in example 2.

The process for extracting the grease comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured Crypthecodinium cohnii fermentation liquor to obtain wet bacterial sludge, weighing 50kg of wet bacterial sludge with the water content of 90%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated hydrochloric acid is 1:1, adding 50kg of concentrated hydrochloric acid with the mass concentration of 37% into a reaction kettle, slightly stirring to fully and uniformly mix the acid liquid and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 80 ℃, and carrying out a cracking reaction for 10 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 28.8kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step B being 1:3.3, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 30min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 120.9kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step C1 being 1:1, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 90min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and C2, weighing 114.9kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step C2 being 1:2, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 90min, and repeating the step B.

D. Preparation of by-products

And D, after extraction is finished, taking out the residual waste acid liquor in the step C3, adsorbing and decoloring the residual waste acid liquor by using activated carbon, storing the waste acid liquor in a waste acid liquor tank, and adding limestone during stirring to prepare calcium chloride.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 18.0kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. The total lipid content of the cells measured by the Bligh-Dyer method is 48.6 +/-2.9 percent, and the relative extraction rate of the finally obtained lipid is 117.8 +/-1.2 percent compared with the Bligh-Dyer method.

Example 8

Performing fermentation culture by taking Crypthecodinium cohnii ATCC 30556 as strain, and extracting oil from the obtained fermentation liquid. Crypthecodinium cohnii was cultured in the same manner as in example 7.

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured crypthecodinium cohnii fermentation liquor to obtain wet bacterial sludge, weighing 50kg of wet bacterial sludge with the water content of 80%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated hydrochloric acid is 1:4.5 adding 225kg of concentrated hydrochloric acid with the mass concentration of 37% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 40 ℃, and carrying out cracking reaction for 90 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. And (3) extracting, adding 100L of n-hexane into the reaction kettle, and carrying out extraction and organic solvent recycling operation according to the steps described in the embodiment 2.

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 203.9kg of wet bacterial sludge with the water content of 80% according to the mass ratio of the residual waste acid in the step B being 1:1.3, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 75 ℃, stirring for 10min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and D, weighing 171.2kg of wet bacterial sludge with the water content of 80% according to the mass ratio of the residual waste acid in the step C1 being 1:2.5, adding into the reaction kettle, and stirring and uniformly mixing. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 70 ℃, stirring for 30min, and repeating the step B.

C3, third recycling: wet bacterial sludge: weighing 376.7kg of wet bacterial sludge with the water content of 80% according to the mass ratio of the residual waste acid in the step C2 being 1:1.5, adding the wet bacterial sludge into a reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 85 ℃, stirring for 75min, and repeating the step B.

D. Preparation of by-products

And D, after extraction is finished, taking out the residual waste acid liquor in the step C3, adsorbing and decoloring the residual waste acid liquor by using activated clay, storing the waste acid liquor in a waste acid liquor tank, and adding quicklime in the stirring process to prepare calcium chloride.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 83.2kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 106.8 +/-2.5%.

Example 9

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured crypthecodinium cohnii fermentation liquor to obtain wet bacterial sludge, weighing 50kg of wet bacterial sludge with the water content of 70%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated hydrochloric acid is 1:2, adding 100kg of concentrated hydrochloric acid with the mass concentration of 37% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquid and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 70 ℃, and carrying out cracking reaction for 50 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 67.5kg of wet bacterial sludge with the water content of 70% according to the mass ratio of the residual waste acid in the step B being 1:2, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 60 ℃, stirring for 50min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and D, weighing 121.5kg of wet bacterial sludge with the water content of 70% according to the mass ratio of the residual waste acid in the step C1 being 1:1.5, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 80 ℃, stirring for 20min, and repeating the step B.

C3, third recycling: wet bacterial sludge: 167.1kg of wet bacterial sludge with the water content of 70 percent is weighed according to the mass ratio of the residual waste acid in the step C2 being 1:1.6 and added into a reaction kettle, and the mixture is stirred and mixed evenly. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 80 ℃, stirring for 70min, and repeating the step B.

D. Preparation of by-products

And D, after extraction is finished, taking out the residual waste acid liquor in the step C3, adsorbing and decoloring the residual waste acid liquor by using diatomite, storing the waste acid liquor in a waste acid liquor tank, and adding lime milk in the stirring process to prepare calcium chloride.

The extracted four batches of crude oil were collected and weighed to yield 65.1kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 110.1 +/-1.2%.

Example 10

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured crypthecodinium cohnii fermentation liquor to obtain wet bacterial sludge, weighing 50kg of wet bacterial sludge with the water content of 75%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated hydrochloric acid is 1:3, adding 150kg of concentrated hydrochloric acid with the mass concentration of 37% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquid and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 60 ℃, and carrying out cracking reaction for 30 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and D, weighing 78.1kg of wet bacterial sludge with the water content of 75% according to the mass ratio of the residual waste acid in the step B being 1:2.4, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 50 ℃, stirring for 70min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and D, weighing 123.1kg of wet bacterial sludge with the water content of 75% according to the mass ratio of the residual waste acid in the step C1 being 1:2, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 50 ℃, stirring for 50min, and repeating the step B.

C3, third recycling: wet bacterial sludge: 199.1kg of wet bacterial sludge with the water content of 75 percent is weighed according to the mass ratio of the residual waste acid in the step C2 being 1:1.7, added into a reaction kettle, and stirred and mixed evenly. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 75 ℃, stirring for 60min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 61.2kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 111.9 +/-1.9%.

Example 11

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured crypthecodinium cohnii fermentation liquor to obtain wet bacterial sludge, weighing 50kg of wet bacterial sludge with water content of 85%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated hydrochloric acid is 1:3.5 adding 175kg of concentrated hydrochloric acid with the mass concentration of 37% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 50 ℃, and carrying out cracking reaction for 70 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 77.7kg of wet bacterial sludge with the water content of 85% according to the mass ratio of the residual waste acid in the step B to 1:2.8, adding into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 40 ℃, stirring for 90min, and repeating the step B.

C2, second recycling: wet bacterial sludge: 157.5kg of wet bacterial sludge with the water content of 85 percent is weighed according to the mass ratio of the residual waste acid in the step C1 being 1:1.8, added into a reaction kettle and stirred and mixed evenly. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 60 ℃, stirring for 70min, and repeating the step B.

C3, third recycling: wet bacterial sludge: 231.9kg of wet bacterial sludge with the water content of 85 percent is weighed according to the mass ratio of the residual waste acid in the step C2 being 1:1.8, added into a reaction kettle and stirred and mixed evenly. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 77 ℃, stirring for 80min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 43.3kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 114.9 +/-2.6%.

Example 12

In this example, the grease of Schizochytrium sp.CGMCC No.11382 is extracted by concentrated phosphoric acid

Seed liquid culture: the seed liquid was prepared by culturing 20g/L glucose, 1g/L yeast extract, and 15g/L sea salt as seed liquid culture medium in a shaker at 25 deg.C and 200 rpm.

Culturing in a fermentation tank: taking 80g/L glucose, 5g/L corn steep liquor powder and 15g/L sea salt as fermentation culture media, inoculating 10% of inoculum size into a 50L fermentation tank for fermentation culture, controlling the pH to be 5.5-6.5, supplementing glucose when the sugar concentration is reduced to 5g/L, fermenting for 4 days, and then putting into the tank.

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured schizochytrium limacinum fermentation liquor to obtain wet bacterial sludge, weighing 20kg of wet bacterial sludge with the water content of 90%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated phosphoric acid is 1:4.5 adding 90kg of concentrated phosphoric acid with the mass concentration of 85% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 120 ℃, and carrying out the cracking reaction for 20 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 9.0kg of wet bacterial sludge with the water content of 90% according to the mass ratio of the residual waste acid in the step B being 1:12, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 115 ℃, stirring for 60min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 58.1kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step C1 being 1:2, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 120 ℃, stirring for 75min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and C2, weighing 67.3kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step C2 being 1:2.5, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 115 ℃, stirring for 92min, and repeating the step B.

D. Preparation of by-products

And D, after extraction is finished, taking out the residual waste acid liquid in the step C3, adsorbing and decoloring the residual waste acid liquid by using diatomite, storing the waste acid liquid in a waste acid liquid tank, and adding lime milk in the stirring process to prepare the calcium phosphate.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 10.6kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. The total lipid content of the cells measured by the Bligh-Dyer method is 57.9 +/-4.7 percent, and the relative extraction rate of the finally obtained lipid is 118.9 +/-1.7 percent compared with the Bligh-Dyer method.

Example 13

The schizochytrium limacinum of this example was cultured as in example 12.

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured schizochytrium limacinum fermentation liquor to obtain wet bacterial sludge, weighing 20kg of wet bacterial sludge with the water content of 80%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated phosphoric acid is 1:10, adding 200kg of concentrated phosphoric acid with the mass concentration of 85% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 70 ℃, and carrying out the cracking reaction for 100 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 54kg of wet bacterial sludge with the water content of 80% according to the mass ratio of the residual waste acid in the step B being 1:4, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing the wet bacterial sludge and the wet bacterial sludge. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 120 ℃, stirring for 50min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, adding 103.7kg of wet bacterial sludge with the water content of 80% into a reaction kettle in a mass ratio of the residual waste acid in the step C1 being 1:2.5, and stirring and uniformly mixing. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 115 ℃, stirring for 80min, and repeating the step B.

C3, third recycling: wet bacterial sludge: weighing 342.1kg of wet bacterial sludge with the water content of 80% into a reaction kettle according to the mass ratio of the residual waste acid in the step C2 being 1:1, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 120 ℃, stirring for 90min, and repeating the step B.

D. Preparation of by-products

And D, after extraction is finished, taking out the residual waste acid liquor in the step C3, adsorbing and decoloring the residual waste acid liquor by using activated clay, storing the waste acid liquor in a waste acid liquor tank, and adding quicklime in the stirring process to prepare monocalcium phosphate.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 63.6kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 105.6 +/-2.4%.

Example 14

The schizochytrium limacinum of this example was cultured as in example 12.

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured schizochytrium limacinum fermentation liquor to obtain wet bacterial sludge, weighing 20kg of wet bacterial sludge with the water content of 70%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated phosphoric acid is 1:6, adding 120kg of concentrated phosphoric acid with the mass concentration of 85% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 110 ℃, and carrying out cracking reaction for 40 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 13.4kg of wet bacterial sludge with the water content of 70% according to the mass ratio of the residual waste acid in the step B being 1:10, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 100min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 41.0kg of wet bacterial sludge with the water content of 70 percent according to the mass ratio of the residual waste acid in the step C1 being 1:3.5, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 100min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and C2, weighing 114.7kg of wet bacterial sludge with the water content of 70 percent according to the mass ratio of the residual waste acid in the step C2 being 1:1.5, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 105 ℃, stirring for 97min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 35.8kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 109.0 +/-2.2%.

Example 15

The schizochytrium limacinum of this example was cultured as in example 12.

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured schizochytrium limacinum fermentation liquor to obtain wet bacterial sludge, weighing 20kg of wet bacterial sludge with the water content of 75%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated phosphoric acid is 1:8, adding 160kg of concentrated phosphoric acid with the mass concentration of 85% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 100 ℃, and carrying out a cracking reaction for 60 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 29.2kg of wet bacterial sludge with the water content of 75% according to the mass ratio of the residual waste acid in the step B being 1:6, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing the wet bacterial sludge and the wet bacterial sludge. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 110 ℃, stirring for 75min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 65.6kg of wet bacterial sludge with the water content of 75% according to the mass ratio of the residual waste acid in the step C1 being 1:3, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 110 ℃, stirring for 70min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and D, weighing 123.1kg of wet bacterial sludge with the water content of 75% in the mass ratio of the residual waste acid in the step C2 being 1:2, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 110 ℃, stirring for 95min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 36.9kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 107.2 +/-2.8%.

Example 16

The schizochytrium limacinum of this example was cultured as in example 12.

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured schizochytrium limacinum fermentation liquor to obtain wet bacterial sludge, weighing 20kg of wet bacterial sludge with the water content of 85%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated phosphoric acid is 1: 9 adding 180kg of concentrated phosphoric acid with the mass concentration of 85% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 90 ℃, and carrying out a cracking reaction for 80 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 24.6kg of wet bacterial sludge with the water content of 85% according to the mass ratio of the residual waste acid in the step B being 1:8, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 100 ℃, stirring for 90min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 54.5kg of wet bacterial sludge with the water content of 85 percent according to the mass ratio of the residual waste acid in the step C1 being 1:4, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 100 ℃, stirring for 90min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and D, weighing 105.7kg of wet bacterial sludge with the water content of 85% according to the mass ratio of the residual waste acid in the step C2 being 1:2.5, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 100 ℃, stirring for 100min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed, resulting in a total of 20.6kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 115.8 +/-2.1%.

Example 17

The schizochytrium limacinum of this example was cultured as in example 12.

The extraction process comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured schizochytrium limacinum fermentation liquor to obtain wet bacterial sludge, weighing 40kg of wet bacterial sludge with the water content of 90%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated hydrochloric acid is 1:1.5 adding 60kg of concentrated hydrochloric acid with the mass concentration of 37% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 70 ℃, and carrying out cracking reaction for 50 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

100L of petroleum ether was added to the reaction vessel, and the extraction and organic solvent recycling operations were carried out as described in example 2.

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 73.8kg of wet bacterial sludge with the water content of 90% according to the mass ratio of the residual waste acid in the step B to 1:1.3, adding into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 75 ℃, stirring for 60min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and C1, weighing 162.5kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step C1 being 1:1.5, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing the wet bacterial sludge and the wet bacterial sludge. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 90min, and repeating the step B.

C3, third recycling: wet bacterial sludge: and C2, weighing 205.8kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step C2 being 1:2, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 90min, and repeating the step B.

The extracted four batches of crude oil were collected and weighed to give a total of 30.6kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. Compared with the Bligh-Dyer method, the method has the advantage that the relative extraction rate of the finally obtained grease is 109.6 +/-2.3%.

Example 18

The strain used in this example was Mortierella alpina ATCC 16266.

Seed liquid culture: a seed solution was prepared by culturing 30g/L glucose, 10g/L yeast extract, 0.1g/L potassium dihydrogen phosphate and 0.1g/L magnesium sulfate in a seed solution medium at pH 6.5-7.0 in a shaker at 28 ℃ and 120 rpm.

Fermentation culture: 60g/L glucose, 20g/L yeast extract, 2g/L potassium dihydrogen phosphate and 0.5g/L magnesium sulfate are used as fermentation culture medium, and fermentation culture is carried out in a fermentation tank at the fermentation temperature of 28 ℃ and the rotation speed of 150rpm for 3 times, and the culture lasts for 12 days.

The process for extracting the grease comprises the following steps:

A. cracking wall breaking

Centrifuging the cultured fermentation liquor to obtain wet bacterial sludge, weighing 50kg of wet bacterial sludge with the water content of 90%, adding the wet bacterial sludge into a reaction kettle, and mixing the wet bacterial sludge: the mass ratio of concentrated hydrochloric acid is 1:1.5 adding 75kg of concentrated hydrochloric acid with the mass concentration of 37% into the reaction kettle, slightly stirring to fully and uniformly mix the acid liquor and the wet bacterial sludge, introducing hot water for heating, stirring, controlling the temperature to be 70 ℃, and carrying out a cracking reaction for 10 min. And (3) quickly releasing the hot water bath after the reaction is finished, and introducing cooling water to reduce the temperature in the reaction kettle to room temperature.

B. Extraction of

C. Cyclic utilization of waste acid

C1, first recycling: wet bacterial sludge: and B, weighing 36.4kg of wet bacterial sludge with the water content of 90 percent according to the mass ratio of the residual waste acid in the step B being 1:3.3, adding the wet bacterial sludge into the reaction kettle, and stirring and uniformly mixing. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 30min, and repeating the step B.

C2, second recycling: wet bacterial sludge: and D, weighing 152.7kg of wet bacterial sludge with the water content of 90 percent in the mass ratio of the residual waste acid in the step C1 being 1:1, adding the wet bacterial sludge into the reaction kettle, and uniformly stirring. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 90min, and repeating the step B.

C3, third recycling: wet bacterial sludge: 145.1kg of wet bacterial sludge with the water content of 90 percent is weighed according to the mass ratio of the residual waste acid in the step C2 being 1:2, added into a reaction kettle and stirred and mixed evenly. And (4) reheating the hot water after the reduced pressure distillation through a water bath heating tank, introducing into the interlayer of the reaction kettle, controlling the temperature to be 90 ℃, stirring for 90min, and repeating the step B.

D. Preparation of by-products

The extracted four batches of crude oil were collected and weighed, resulting in a total of 15.5kg of crude oil. The crude oil is subjected to deodorization-alkali refining-decoloration treatment by a technology known in the art to obtain refined oil. The total lipid content of the cells measured by the Bligh-Dyer method is 36.3 +/-1.7 percent, and the relative extraction rate of the finally obtained lipid is 111.3 +/-1.8 percent compared with the Bligh-Dyer method.

Claims

1. A method for extracting grease from fermentation liquor containing oil-producing microbial cells is characterized by comprising the following process steps:

A. cracking wall breaking

B. refining by extraction

C. cyclic utilization of waste acid

2. A method for extracting grease from fermentation liquor containing oil-producing microbial cells is characterized by comprising the following process steps:

A. cracking wall breaking

B. refining by extraction

C. cyclic utilization of waste acid

3. A method for extracting grease from fermentation liquor containing oil-producing microbial cells is characterized by comprising the following process steps:

A. cracking wall breaking

B. refining by extraction

C. cyclic utilization of waste acid

4. The method for extracting oil from fermentation broth containing oleaginous microbial cells of any one of claims 1-3, wherein the oleaginous microbe in step A is selected from one of microalgae, yeast and mold.

5. The method for extracting oil from fermentation broth containing oleaginous microbial cells according to any of claims 1-3, wherein the solid-liquid separation method in step A is one or a combination of flocculation precipitation, filtration, centrifugation and membrane dehydration.

6. The method for extracting oil or fat from a fermentation broth containing oleaginous microbial cells according to any of claims 1 to 3, further comprising a step A1 after step A, wherein the heat for raising the temperature in step A is recycled to the reduced pressure distillation in step B.

7. The method for extracting oil from a fermentation broth containing oleaginous microbial cells according to any of claims 1-3, wherein the temperature of the reduced pressure distillation in step B is 35-75 ℃.

8. The method according to any one of claims 1 to 3, wherein the organic solvent in step B is selected from n-hexane, petroleum ether, and solvent No. 6 or a combination thereof.

9. The method for extracting oil from fermentation broth containing oleaginous microbial cells of any of claims 1-3, wherein the organic phase of step B is filtered by using an organic filter membrane, a ceramic filter membrane or a sand filter with a pore size of 0.5-30 μm.

10. The method for extracting oil from the fermentation liquid containing the oleaginous microbial cells of any one of claims 1-3, further comprising a step B1 after the step B, wherein the step B is to condense and liquefy the organic solvent vapor under pressure to form the organic solvent, and recycle the organic solvent vapor to the extraction in the step B.

11. The method for extracting oil from a fermentation broth containing oleaginous microbial cells according to any of claims 1-3, wherein the concentration of the spent acid solution in step C is not less than 4% by mass.

12. The method for extracting oil from fermentation broth containing oleaginous microbial cells according to any of claims 1-3, further comprising a step D after step C, wherein the waste acid liquid left after recycling in step C is decolorized for preparing by-products.

13. The method of claim 12, wherein the step D of decolorizing the spent acid solution is carried out by adsorption using one or more of activated carbon, bentonite, activated clay, diatomaceous earth, and silica gel.

14. The method according to claim 12, wherein the byproduct obtained in step D is calcium oxide, limestone or milk of lime added to the decolorized spent acid solution to prepare calcium chloride, calcium sulfate, calcium phosphate, and calcium dihydrogen phosphate.

15. The method for extracting oil from fermentation broth containing oleaginous microbial cells according to any of claims 1-3, wherein the refining of crude oil in step B comprises decolorization, alkali refining, deodorization treatment in sequence.