CN102505026A - Method for extracting microalgae oil membrane substrate in situ - Google Patents

Abstract

The invention discloses a method for in-situ extraction of microalgae oil film base. Oil-producing algae are cultivated in a suitable medium to a stable growth period, and the algae liquid after accumulating oil is input into the extraction layer, and the in-situ extraction method is used to establish extraction The two-phase system of solvent and algae liquid blending makes the algae body oil continuously extracted to the solvent phase, the extraction solvent and algae liquid are gradually layered and clarified, the organic solvent in the upper layer is recycled, and the algae liquid in the extraction layer is recycled to the continuous cultivation step. The present invention combines microalgae continuous culture technology, membrane technology and microalgae oil extraction technology, and proposes an integrated method for in-situ extraction of microalgae oil, which avoids the processes of collecting, drying and breaking microalgae cells, and is different from conventional methods. Compared with the existing microalgae oil extraction technology, the process of the present invention has the characteristics of simple process, high oil extraction rate, low process cost, and environmental friendliness.

Description

A method for in-situ extraction of microalgae oil film base

technical field

The invention relates to the fields of bioengineering and bioenergy, in particular to a microalgae oil extraction method which integrates microalgae cultivation, membrane technology and microalgae oil extraction technology.

Background technique

Biodiesel is an important product of bioenergy, but the development of the industry is severely restricted due to the shortage of raw materials. Among the raw materials for preparing biodiesel (oil crops, forest trees and oil-producing microalgae), microalgae grow fast and have high oil content, which is a promising bulk raw material for biodiesel. At present, the bottleneck problem of microalgae production of biodiesel is that the cost is too high, which leads to the unguaranteed economic efficiency, especially the oil extraction from microalgae accounts for about 50% of the cost of biodiesel. Conventional microalgae oil extraction techniques include organic solvent extraction, supercritical fluid extraction, thermal cracking, etc. These methods require the algae to be dry powder, and the dry matter content in the algae liquid is usually less than 1%. The pretreatment of concentrated drying not only prolongs the The biodiesel production cycle also affects the oil extraction efficiency.

The in-situ extraction method refers to the establishment of a two-phase system in which a biocompatible organic solvent and algae liquid are blended, so that the oil of the algae is continuously extracted to the solvent phase, avoiding the dehydration and drying steps of the algae, and realizing the double extraction of oil on-line and increasing the yield. Target.

At present, the extraction methods of microalgae oil mainly include organic solvent extraction, supercritical and subcritical fluid extraction and other methods. For example: Mixing co-solvent extraction refers to a single-phase system composed of a polar solvent and a non-polar solvent to extract algae oil. Bligh and Dyer in 1959 (Bligh EG, Dyer WJ.A rapid method of total lipid extraction and purification "a rapid extraction and purification method of total lipids". Canadian Journal of Biochemistry and Physiology "Canadian Journal of Biochemistry and Physiology", 1959, 37(8):911-917), the methanol/chloroform system proposed is still the most commonly used microalgae oil extraction method. Based on the principle of "like dissolves like", the algal body is in full contact with the mixed solvent of methanol/chloroform, and the polar solvent methanol combines with the polar lipid of the cell membrane, thereby destroying the hydrogen bond and electrostatic interaction between the lipid and protein molecules, making the non-polar The solvent chloroform enters the cells and dissolves the hydrophobic neutral lipid components in the cells. After full extraction, water is added to the system. Methanol dissolves in the water phase and separates with the oil-containing chloroform phase. After the chloroform volatilizes, a crude lipid extract is obtained. However, due to the neurotoxicity of chloroform, some low-toxic dual-solvent systems are also tried for oil extraction, such as n-hexane/isopropanol, DMSO/petroleum ether, n-hexane/ethanol, etc.

In 1996, Richter et al (Richter BE, Jones BA, Ezzell JL, Porter NL. Accelerated solvent extraction: a technique for sample preparation "accelerated solvent extraction: a sample preparation technique". Analytical Chemistry "Analytical Chemistry", 1996, 68( 6): 1033-1039) proposed Accelerated solvent extraction (ASE), which is a method of extracting solids with solvents at higher temperatures (50-200°C) and higher pressures (10.3-20.6MPa). Or the processing method of semi-solid samples. The principle of this method for extracting oil from microalgae is that high temperature and high pressure help to increase the mass transfer rate so that the solvent can quickly penetrate into the algal cells, and at the same time reduce the dielectric constant of the solvent so that its polarity is close to that of oil, thereby improving the extraction efficiency of the solvent. Commonly used solvents Systems include methanol/chloroform, isopropanol/n-hexane, etc. Compared with the double-solvent extraction method, the ASE method has the advantages of short action time (5-10 min), less solvent consumption and high oil extraction rate. For example, traditional Folch method (chloroform/methanol, 2: 1v/v)) to the oil extraction rate of green alga Rhizoclonium hieroglyphicum 44～55%, and with equal amount of solvent at pressure 10.3MPa, 120 ℃, only extract 5min The oil extraction rate can reach 85-95%.

Supercritical fluid extraction (Supercritical fluid extraction) is a new extraction technology, which means that when the critical temperature and pressure are exceeded, the fluid is between the gaseous state and the liquid state, and it has the diffusion and mass transfer properties of gas and the solubility of liquid. The extraction efficiency of algae oil is much higher than that of ordinary organic solvents. Due to the advantages of mild critical conditions (pressure 7.4MPa, temperature 31.1°C), non-toxicity, and chemical inertness, _CO2 is the most widely used. In addition, the use of methanol, ethanol, water, nitrogen dioxide, and sulfur hexafluoride has also been reported. Supercritical CO ₂ extraction (Supercritical carbon dioxide extraction, SCCO ₂ ) microalgae oil action conditions are 40-50°C, 24.1-37.9MPa, after extraction, the oil is dissolved in supercritical liquid CO ₂ , only need to control the temperature and pressure during recovery Grease can be separated by returning _CO2 to a gaseous state. Supercritical extraction is an environmentally friendly and green extraction technology, but because it involves temperature and pressure control, the treatment process has high energy consumption and poor economy, and is not suitable for the extraction of bulk chemicals.

Subcritical water extraction (Subcritical water extraction, SWE) means that the polarity of water decreases when it is slightly lower than the critical temperature, so it has properties similar to organic solvents, and the solubility of oil is also greatly improved. At the same time, high pressure is used to maintain water at Liquid state, high temperature promotes water to quickly enter the cells, and the intracellular lipids are extracted to the water phase. When the system is cooled to room temperature, the polarity of the water increases, and the oil and water dissolved in the water phase are quickly separated for easy collection. In addition, subcritical ethanol can also be used to extract pigments such as carotene from Haematococcus pluvialis and Dunaliella salina. The advantage of this method is that it can directly treat the algae liquid without adding organic solvents to the system. However, due to the existence of high energy consumption steps such as temperature and pressure, the industrial application of this method is also limited.

The above-mentioned different extraction methods of microalgae oil include high-energy processing steps such as algae body drying, high temperature and high pressure control, and the like. At present, the main methods of drying algae include sun drying, spray drying and freeze drying. When dealing with a large number of algae, the drying method is the most economical, but it has certain requirements for the drying area and time. Therefore, in drying, temperature and pressure control The technical improvement of other links can effectively reduce the energy consumption of oil extraction.

Since the beginning of the 1980s, the research on membrane technology has become an important part of the research field of new separation technology. The dispersion extraction process based on microporous membrane has the advantages of both membrane technology and extraction process. The membrane medium itself has no separation effect on the mixture to be treated. It mainly uses the porosity, hydrophilicity or hydrophobicity of the membrane to provide a larger two-phase transfer. The stable phase contact surface can overcome the effects of liquid flooding and back mixing in conventional separation, and has attracted much attention in the past ten years. Using the hollow fiber membrane to promote the dispersion effect of the organic solvent in the algae liquid, and at the same time to couple the microalgae culture and oil extraction process is a new method to realize the continuous synthesis and online extraction of microalgae oil.

Contents of the invention

The invention combines the microporous membrane technology to uniformly disperse the biocompatible extraction solvent into the algae liquid, promote the extraction efficiency of the microalgae oil by the solvent, and propose a brand-new integrated technology for the continuous culture of the microalgae and the in-situ extraction of the oil.

A method for in-situ extraction of microalgae oil film base, comprising the following steps:

(1) Continuous culture of microalgae: culture the oleaginous algae species in a suitable medium until the growth is stable, and the cells of the oleaginous algae species gradually accumulate oil;

(2) In-situ extraction of oil: input the algae liquid after oil accumulation into the extraction system, and at the same time input the extraction solvent, and use the in-situ extraction method to establish a two-phase system in which the extraction solvent and algae liquid are blended, and the algal oil is continuously extracted to the solvent phase The extraction solvent and the algae liquid are gradually layered, the upper layer of the extraction solvent is recycled, and the lower layer of the algae liquid is recycled to the continuous cultivation step; by adjusting the flow rate of the extraction solvent and the algae liquid pumped in and out of the extraction module, the volume ratio of the extraction solvent in the extraction system is maintained Not more than 10% of the total volume.

(3) Real-time detection of the oil content in the extraction solvent. If the oil concentration does not increase significantly, the oil extraction tends to be saturated. Remove the oil-saturated extraction solvent and add a new extraction solvent.

As a preferred solution, a hollow fiber membrane module is set in the extraction system, the algae liquid flows along the shell of the hollow fiber membrane module, the extraction solvent enters the hollow fiber membrane tube with the end sealed, and then passes through the hollow fiber membrane tube The micropores of the wall are dispersed into tiny droplets into the algae liquid in the shell side, and fully mixed with the algae liquid, so that the oil of the algae body is continuously extracted to the solvent phase.

The extraction solvent is a biocompatible organic solvent, generally a hydrophobic organic solvent with log P > 5.5, especially alkane solvents with a carbon chain length of C ₁₂ -C ₁₆ have good biocompatibility.

The method for extracting oil from microalgae in situ with membrane dispersion in the present invention combines the advantages of membrane technology and extraction process, is a new high-efficiency mass transfer process, and has the following main features and advantages:

(1) The extraction solvent is dispersed into the algae liquid in the form of tiny droplets through the micropores of the membrane, and the extraction solvent and the algae liquid are fully mixed to significantly improve the extraction efficiency.

(2) The method does not need to break the microalgae cells, and the microalgae cultivation can be carried out continuously. When the oleaginous algae species are selected to be resistant to organic solvents, since the microalgae cells and organic solvents have a good affinity, the organic solvents are hardly affected by the activity of the microalgae during the in-situ extraction of oil by membrane dispersion. Influence.

(3) When the microalgae culture is controlled to be in a stable period, the oil synthesis of the microalgae is vigorous at this time, and the growth of the microalgae and the oil extraction can be carried out simultaneously, and the oil can be extracted in situ online. This method can break through the lengthy process of harvesting, drying, crushing and extracting microalgae, thereby significantly reducing production costs.

Description of drawings

Fig. 1 is the schematic diagram of the membrane dispersion organic solvent extraction device that is used in the embodiment 1 that is used to realize the method of the present invention, illustration: ① extraction solvent storage tank ② extraction layer ③ dead-end hollow fiber membrane module ④ algae liquid storage bottle ⑤ Air-lift photobioreactor⑥peristaltic pump⑦air compressor pump.

Fig. 2 is the result of the oil content in the algae liquid and the extractant changing with time in Example 1 of the present invention.

Fig. 3 is the change trend of cell activity of Botryococcus braunii oil extracted by membrane dispersion organic solvent in Example 1 of the present invention.

Detailed ways

Example 1:

(1) Cultivate oleaginous algae Botryococcus braunii, BG11 medium (common medium for freshwater algae) in an airlift photobioreactor ⑤ with a volume of 7L, at 25±1°C, under continuous light (light intensity 8k lux) cultured for 3 weeks to the stationary phase.

(2) The algae liquid in the air-lift photobioreactor ⑤ passes through the peristaltic pump into the shell layer of the dead-end PES hollow fiber membrane module ③, and the volume of the algae liquid is controlled to 400ml. Tetradecane, a biocompatible solvent of Botryococcus braunii, is passed into the tube layer of the dead end (a module with one end of the membrane fiber sealed) at a flow rate of 10ml ^min The droplets enter the algae liquid, and the dense solvent droplets gradually float up due to their low density. During the ascent process, they fully contact with the algae cells and extract the oil from the algae.

The tetradecane solvent in the extraction layer is layered with the algae liquid, and the upper solvent phase is pumped back into the organic solvent storage tank ① by a peristaltic pump at a flow rate of 10ml min ^-1 for recycling. In order to reduce the cytotoxicity of the solvent, keep the extraction The volume ratio of the solvent in the layer does not exceed 10% of the total volume.

(3) Detect the oil content of the solvent phase during the extraction process. If the oil concentration does not increase significantly, the oil extraction tends to be saturated. Remove the oil-saturated extraction solvent and add new extraction solvent to maintain a certain oil extraction rate.

Comparative example:

Except that the extraction agent tetradecane in the step (2) is directly pumped into the algae liquid without passing through the membrane module, the rest is the same as that of Example 1.

The specific operation mode of the oil content determination part is:

Determination of intracellular lipid content:

Dilute the algae liquid in proportion and measure its absorbance at 680nm with a UV spectrophotometer (GS-54, Shanghai Prism Light), and keep the OD680 at about 0.5. Take 3ml of the diluted algae liquid and add 3μl of Nile Red dye solution (concentration: 1mg ml ^{- 1} , solvent acetone), dyed in a water bath at 37°C for 10 minutes in the dark, and measured the fluorescence intensity with a fluorescence spectrophotometer (F96-PRO, Shanghai Prismlight), with an excitation wavelength of 480nm, a divergence wavelength of 580nm, and a gain of 10. Fluorescence intensity/OD680 was substituted into the following formula to calculate the oil content of algae.

Y=0.16X+12.49 (X: fluorescence intensity, Y: oil content%)

Determination of oil content in organic solvent (tetradecane):

Use tetradecane (Aladdin, USA) as the solvent and triolein (Aladdin, USA) as the standard to prepare solutions with different concentrations, take 3ml of each and add 5μl of Nile Red dyeing solution (1mgml-1, dissolved in acetone) , dyed in a water bath at 37°C in the dark for 10 minutes, measured the fluorescence intensity with a fluorescence spectrophotometer, the excitation wavelength was 480nm, the divergence wavelength was 570nm, and the gain was 1 file, and the corresponding equation of the standard curve was obtained as follows:

Y＝0.06X+30.07 (Y fluorescence intensity, X oil concentration mg L ^-1 )

When measuring the oil content in tetradecane after extraction, take 3ml of tetradecane and add 5μl of Nile red dye solution (1mg ml ^-1 , dissolved in acetone), stain in a water bath at 37°C in the dark for 10min, and use fluorescence spectrophotometry Measure the fluorescence intensity with a meter, the excitation wavelength is 480nm, the divergence wavelength is 570nm, and the gain is 1 file. Substituting the measured data into the standard curve equation, the oil concentration can be obtained.

Determination of cell viability by flow cytometry

Fluorescein diacetate (FDA) was used as a fluorescent dye for living cells to characterize cell viability during oil extraction. When active cells have intact cell membranes, FDA-hydrolyzed fluorescein will continue to accumulate in the cells, making the cells emit green fluorescence under blue excitation light. When the cell membrane is damaged and the cell loses its activity, the FDA-hydrolyzed fluorescein cannot accumulate in the cell, so that the cell cannot fluoresce. Cells with high activity emit strong green fluorescence, and cells with low activity emit weaker light. When the fluorescent substance fills the entire cell, it makes the cell glow bright green.

The Botryococcus braunii algae fluid was passed through a 300-mesh cell sieve to remove impurities and cell agglomerates, 1ml of the algae fluid was centrifuged and the supernatant was discarded, the algae were washed three times with PBS buffer (phosphate buffered saline), and an appropriate amount of PBS buffer was used to suspend the algae to increase the cell density of the algae. Not less than 10 ⁶ ml ^-1 , add FDA (Sigma, USA) to a final concentration of 100 μg ml ^-1 , stain in the dark at room temperature for 7 minutes, analyze by flow cytometry (Beckman FC 500MCL, USA), flow rate 100-400 cells sec ^{- 1.} The total number of cells is greater than 1000 during data collection, or the time is greater than 2 minutes. After the test is completed, save the analysis results and draw a graph.

As shown in Figure 2, the changes of the organic solvent phase and the oil content in Botryococcus braunii cells during the extraction process (400ml of algae liquid, 40ml of tetradecane, and 10ml min ^-1 circulation speed of both algae liquid and organic solvent), the experimental results show that, In Example 1, after 2 days of extraction, the oil concentration in the tetradecane extractant had reached the maximum value of 520 mg L ^-1 , and the oil extraction rate reached 50.15%, while the extraction rate of the control group was 38.05%. After 2 days, the oil content in tetradecane did not change much, but the oil content in algae liquid recovered gradually. From the analysis of the experimental results, it can be concluded that the dispersion of membrane pores can effectively promote the mixing of solvent and algae liquid, increase the contact area between algae and solvent, and improve the oil extraction rate.

As shown in Figure 3, it is the detection of viability of botrytis oil cells extracted by organic solvent of membrane bubbling (100ugml ^-1 FDA staining for 10min, and the C2 quadrant is the living cell area of FDA staining. Flow cytometry in Example 1 detects 96h in situ extraction process , the cell survival rate remained above 90%, the results showed that the introduction of the PES organic membrane in the system had no effect on cell activity, and it was suitable for the continuous extraction process of oil.

Claims (6)

1. microalgae grease film base original position method of extraction may further comprise the steps:

(1) little algae cultured continuously: produce oil algae kind is cultured to the stationary phase of growing in substratum, accumulates grease gradually in the cell of produce oil algae kind;

(2) original position oil and grease extracting: the algae liquid after cultured continuously accumulates grease is imported extraction system, import extraction solvent and the blend of algae liquid simultaneously and form two-phase system, the frond grease constantly is extracted to solvent phase; Extraction solvent and the layering gradually of algae liquid, the upper strata extraction solvent recycles, and lower floor's algae liquid is circulated to the cultured continuously step;

(3) detect fat content in the extraction solvent in real time, if grease concentration does not have remarkable increase, then oil extraction is tending towards saturated, removes the saturated extraction solvent of grease, adds new extraction solvent.

2. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 1 position method of extraction is characterized in that: described produce oil algae kind is the algae kind of organic solvent-resistant.

3. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 1 position method of extraction, it is characterized in that: described solvent is the biocompatibility organic solvent.

4. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 3 position method of extraction, it is characterized in that: described biocompatibility organic solvent is the alkanes of hydrophobic value logP＞5.5.

5. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 4 position method of extraction is characterized in that: its described biocompatibility organic solvent is C ₁₂～C ₁₆Alkane solvents.

6. microalgae grease pleurodiaphragmatic in terspace as claimed in claim 1 position method of extraction, it is characterized in that: the solvent volume that described extraction system contains is no more than 10% of TV.

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