KR101769875B1 - Method of preparing triacylglycerol or biodiesel in microalgae - Google Patents

Abstract

The present invention relates to a method for producing biodiesel from microalgae, comprising: a first step of growing and culturing microalgae for a predetermined period; A second step of applying pressure stress to the microalgae for a certain period of time to convert the polar lipid bound to the cell membrane into the form of triglyceride; And a third step of converting biodiesel from triglyceride to biodiesel.
Since the present invention can accumulate large amounts of triglyceride-containing lipid droplets (LDs), it is possible to greatly reduce the oil extraction cost of the previous step for biodiesel production. Thus, it is possible to produce biodiesel from microalgae at a low cost and industrially applicable scale.

Description

Technical Field [0001] The present invention relates to a method for preparing triglyceride (TAG) or biodiesel in microalgae,

The present invention relates to a method for producing triglyceride (TAG) in microalgae. Specifically, the present invention relates to a technique for converting a lipid component of a microalgae fed with CO ₂ into sunlight using solar energy into biodiesel, which is also useful for CO ₂ treatment and production of fuel for environmentally friendly transportation.

As biofuel production from edible crops has increased steadily, competition for farmland has become more intense between food and fuel production. New feedstocks for biofuels that can replace fossil fuels are inevitably needed to sustain the food supply to an ever-growing population.

Microalgae, such as green algae, are photosynthetic microorganisms that require nitrogen to synthesize proteins necessary for cell growth, producing chemical energy in the form of carbohydrates and fats using sunlight and carbon dioxide. Photosynthesis capacity of microalgae is superior to plants. In addition, marine, coastal or wasteland use is possible, so it is not competitive with edible crops in land land use, and various water resources (sewage, seawater, wastewater) can be used and recycled.

In addition, microalgae can be produced in large quantities through large-scale bioprocesses because the rate of capturing carbon dioxide and the rate of growth of cells are faster than those of other photosynthetic organisms and artificial cultivation is easy. Microalgae is 50-100 times more biomass productivity per unit area than first generation biofuel feedstock (soybean, oilseed rape, etc.).

Many microalgae can grow at high rates and produce large quantities of biomass and can accumulate large amounts of oil that can be easily converted to biodiesel under certain culture conditions. Accordingly, microalgae are attracting attention as an alternative source for the production of renewable green energy.

Under stress conditions (especially nitrogen depletion), microalgae accumulate significant amounts of triglycerides (mainly triglycerol) as cellular organelles called lipid droplets (LDs) do.

The fatty acids needed for TAG synthesis are either de novo synthesized or produced from recycled acyl chains recycled from membrane lipids.

Under culturing conditions widely used in laboratories, externally provided acetate promotes TAG production through a new de novo pathway. The precise contribution of each pathway involved in TAG synthesis under specific conditions is not yet known.

Conventional studies have shown that the TAG content of cells can be significantly increased under appropriate culture conditions such as temperature, light intensity, nitrogen depletion, phosphorus limitation, iron concentration and salinity. Amongst various stress conditions, nitrogen starvation is the most effective way to induce TAG accumulation in algae. However, (i) nitrogen depletion reduces the photosynthetic reaction to reduce overall biomass production, and (ii) in industrial terms, the nitrogen removal process consumes a significant amount of energy, time, and cost, Using nitrogen deficiency conditions is not ideal.

The present invention provides a method for inducing intracellular TAG accumulation that can be applied to various microalgae separately from the step of acquiring a large amount of biomass and the growth of microalgae without including the nitrogen deficiency condition in the cell culture.

According to a first aspect of the present invention, there is provided a method for producing triglyceride (TAG) in microalgae, comprising the steps of: subjecting microalgae to pressure stress to remove polar lipids bound to the cell membrane, To form a triglyceride. ≪ Desc / Clms Page number 7 >

In a second aspect of the present invention, there is provided a method for producing triglyceride (TAG) in a microalgae, comprising: a first step of growing and culturing microalgae for a predetermined period; And a second step of converting the polar lipid bound to the cell membrane into the form of triglyceride by applying pressure stress to the microalgae for a certain period of time, distinguishing the first step from the first step.

In a third aspect of the present invention, there is provided a method of producing biodiesel from microalgae comprising the steps of: a) growing and culturing microalgae for a certain period of time; A second step of applying pressure stress to the microalgae for a certain period of time to convert the polar lipid bound to the cell membrane into the form of triglyceride; And a third step of converting biodiesel from triglyceride to biodiesel.

Hereinafter, the present invention will be described in detail.

Microalgae can convert solar energy to chemical energy and can convert carbon dioxide to biomass, such as carbohydrates, lipids, and proteins.

Microalgae cells are not composed of specific components and are similar to plants. In addition to substances such as pigments that are involved in photosynthesis, there are also substances with high digestible carbohydrates, enough protein to be used as food, and basic substances such as lipids suitable for foods (1 to 35%) or biofuels (20 to 80% (Table 1; chemical composition of microalgae biomass (% of dry mass)). Lipid components can be used as biodiesel (Figure 1).

The chemical composition of the microalgae biomass depends not only on the components of the medium but also on the environmental factors, the cell recovery process, and the cell drying process.

Hydrostatic pressure causes mortalities or near-lethal damage to bacteria and other microbial cells. Conventional studies have shown that high hydrostatic pressure on phospholipids and glycolipids attached to membranes and membranes present in bacterial cell envelope, such as proteins, known to be critical for normal functioning of the permeation barrier in bacterial cells Have been reported to have an effect. It is known that carbon dioxide pressure as high as 50 atm hinders microalgae from controlling the gas absorption and causes carbon dioxide to penetrate through the membrane, damaging the inside of the cell. However, the effect of a weak pressure of 5 to 15 atm on microalgae cells has not been studied yet.

The microalgae were able to store energy in the form of triglycerides (TAG) under stress conditions and, taking into account that pressure could affect the lipids bound to the membrane, the present inventors have found that stable storage Pressure properties in the translocation of intracellular lipids into TAG (more suitable for biodiesel production) in the form of TAG.

The present inventors have found that TAG accumulation in microalgae can be induced through stress exerted under mild pressure for a short time (for example, about 2 hours). In addition, the present inventors have found that, as a result of applying mild pressure stress to microalgae, the polar lipid bound to the cell membrane can be converted into the form of triglyceride.

On the other hand, microalgae culture is the most costly and takes a long time (eg, 7 to 14 days). Therefore, it is important to grow microalgae in a short period of time to produce large amounts of lipids.

The present invention is based on these discoveries, and distinguishes it from the process of proliferation and cultivation of microalgae for a certain period of time. The present invention provides a process for inducing triglyceride accumulation by applying mild pressure to a microalgae that does not damage microalgae for a short time . That is, the present invention is characterized in that the present invention includes a two-step cultivation which focuses on the growth of microalgae and the acquisition of biomass, and separately performs a triglyceride accumulation induction process through mild pressurization.

Mild pressurization (5 to 15 atm) induces stress on the cells of living microalgae, leading to rapid conversion of polar lipids bound to cell membranes to neutral lipids, particularly triglyceride (TAG) (Figure 4). A lipid class compositional study showed that glycolipids (membrane-associated lipid morphology) converted to neutral lipids (especially TAG) under pressure stress (FIG. 9). FIG. 10 shows an electron microscope photograph before and after the pressure treatment. It can be seen that the triglyceride (TAG) is increased by the pressure treatment.

The TAG was increased to a similar level at a microalgae cell concentration of 10-100 g cell / L in a pressurized reaction tank of 10-15 atm (Fig. 7), indicating that the present invention can be realized by a technique of a scale applicable in industry .

≪ Growth culture step of microalgae >

The step of proliferating microalgae according to the present invention may include cell growth as well as cell growth and material production.

Ideal microalgae have various requirements, but first, the productivity of intracellular metabolites should be high. High productivity can often be achieved through a two-stage process consisting of cell growth and material production.

Currently, the strains used for commercialization include Spirulina, Chlorella, Dunaliella, Haematococcsu, Nannochloropsis, and the like. In general, the composition of the medium for culturing microalgae differs depending on the species and culturing conditions.

Since large-scale processes for commercialization are generally difficult to maintain sterility and process control, it is important to select a strain that is stable and resistant to infection even in extreme environments such as extreme pH, high temperature, and high salinity.

Essential elements (carbon, nitrogen, phosphorus, sulfur) and trace elements (iron, magnesium, etc.) must be included in the culture medium for rapid growth of microalgae cells.

The temperature, pH, light intensity, nutrients (inorganic salts) and carbon dioxide concentration of the microalgae culture system can be tightly controlled to obtain maximum yield of biomass.

Microalgae can capture carbon dioxide from atmospheric or combustion flue-gases into biomass. In particular, Chlorococcum littorale shows high resistance to high concentrations of carbon dioxide up to 40% (v / v) and a CO2 fixation rate of more than 1 gcell / L / day. In addition, Scenedesmus obliquus and Spirulina show a high level of CO2 fixation in a three-stage tubular photobioreactor with a temperature of 30 ° C. Spirulina has a maximum growth rate of 0.44 / day at 6% carbon dioxide and a maximum productivity of 0.22 g / L / day at 6% carbon dioxide. Spirulina obliquus SJTU-3, Chlorella pyrenoidosa SJTU-2, and Botryococcus braunii SI-30 are high concentrations (30-50%) of carbon dioxide It is used for the accumulation of total lipids, polyunsaturated fatty acids and hydrocarbons. Flue-gas has high carbon dioxide concentration, which may cause inhibition of photosynthesis by photo-respiration, but it contains sulfur or nitrogen as another nutrient, which has the advantage of improving biomass productivity by about 30%.

Because microalgae are resistant to high concentration salts, they are used in various photobioreactors using fresh water, brackish water, highly saline water, marine water, Liquid culture is possible.

For example, the higher the intracellular oil content is, the higher the calorie of the biofuel produced by the microalgae becomes. The size of these biomass particles varies from a few μm (5-110 μm for Chlorella) to several hundred μm, similar to that of powdered coal or cellulose. Some microalgae, especially cyanobacteria ('blue algae'), can be cultivated without carbon or nitrogen nutrients and thus are efficient in terms of production costs.

The amount of light required for microalgae is smaller than that of higher plants but up to 400 mmol / m ² · s, metabolic activity increases as the amount of light increases. For example, the saturated light intensity of Chlorella and Scenedesmus is about 200 mmol / m ² · s. Chlorogleopsis species has an optimal light intensity of 36.9 mmol / m ² · s but is highly adaptable even at high light intensity (246.1 mmol / m ² · s) and low light intensity (36.9 mmol / m ² · s) It grows well. However, most microalgae that have undergone light independent nutrient growth at optimum light intensity are converted to heterotrophic growth when they are placed in low light conditions, and some strains exhibit mixed nutrient growth. However, at light intensity above a certain level, cell growth is lowered by light inhibition. The control of the optimal light intensity is more important in the indoor culture than in the outdoor culture, and the optimal value varies depending on the cell. Genetically, it can be changed by reducing the size of the chlorophyll antenna.

Temperature is a major factor controlling the physiological morphological response of microalgal cells. The higher the temperature, the higher the rate of cell growth. At optimal temperature, the microalgae enzymes exhibit the highest activity. The optimum temperature varies depending on the species of microalgae, but it is usually in the range of 25 to 35 ° C and varies depending on the environment such as light conditions.

Most microalgae have optimal neutral pH, but some microalgae species, such as Spirulina platensis and C. littorale, are best suited for alkaline (pH 9) or acidic (pH 4) conditions, respectively . Increasing the concentration of CO ₂ or CO ₃ ^2- in the bioreactor can have a profound effect on the pH control by changing the pH. NH ₃ and NH ₄ ^{+ in the} medium may also be oxidized to compete with water molecules to generate oxygen. Thus, in some cases the pH control of the photobioreactor is controlled by the concentration of CO ₂ and NH ₄ ⁺ .

Chlorella emersonii (63%), Chlorella minutissima (56%), Chlorella vulgaris (57.9%), Chlorella luteoviridis in the absence of nitrogen, (28.8%), Chlorella capsulata (11.4%), Chlorella pyrenoidosa (29.2%), Neochloris oleoabundans (35-54%), The lipid content of the fish increases. In particular, the proportion of triglyceride in the lipid of Neochloris is 80%, which is 2.2 times higher.

Most of the currently commercially produced microalgae (Chlorella, Spirulina, Dunaliella) grow in highly selective environments and thus are not contaminated by other species (algae, protozoa) in open cultures. In other words, Chlorella grows in a nutrient-rich medium, Spirulina grows in high pH and bicarbonate concentrations, and Dunaliella grows in very high salinity.

On the other hand, most other microalga species that do not have a selective benefit to the growing environment should be produced in a closed culture. Therefore, it has been shown that marine microalgae (Skeletonema, Chaetoceros, Thalassiosira, Tetraselmis, Isochrysis) used for aquacultural food and long - Large-scale cultivation techniques for most microalgae such as Crypthecodinium cohnii, which produces long-chain polyunsaturated fatty acids, have been developed specifically for each individual environment.

The accumulation of oxygen produced by photosynthesis of microalgae may reduce the productivity of microalgae, but carbon dioxide injection is more important and essential for microalgae culture. For this purpose, it is more appropriate to inject the industrial residual gas with a higher concentration than the low-concentration atmospheric gas into the bubbles. In order to reduce the energy cost of the injection, a strain capable of growing under the base condition is most suitable for increasing the mass transfer efficiency of the gas.

Among the microalgae culture technologies, the heterotrophic culture method is advantageous in terms of efficiency of capturing carbon dioxide and biomass but is costly. On the other hand, self-nutrient culture can lead to large-scale commercial processes if culture efficiency is increased through establishment of high concentration and mass culture technology. In addition, since the open type culture apparatus is easy to produce and operate, it is difficult to maintain culture conditions, is easily contaminated, and has a high cell harvesting cost. Therefore, a closed type incubator suitable for forming a selective culture environment such as temperature, pH, It is suitable for mass production. However, a closed culture system may include a specific type of optical incubator (vertical, flat plate, annular, plastic bags, air lifted glass, plastic tubular reactor, (eg, a plastic tubular reactor), optimization of internal processes such as energy-consuming pumping and sparging, and minimization of reactor material costs. At this time, for a strain such as Chlorococum, a semi-continuous culture technique with a dilution ratio of 20% (v / v) can be additionally used to smooth the capture of carbon dioxide. In general, closed photoreactors are suitable for the production of high value-added useful materials and open cultivation is suitable for producing biomass for biofuel production.

In general, the productivity of the optical incubator for continuous operation is obtained by multiplying the steady-state biomass concentration by the dilution rate, depending on the light intensity of the reactor surface (or interior), the hydrodynamic operating parameters, and so on. Among the various reactor types (helical, vertical, horizontal, etc.), tubular type reactors are the best in terms of solar absorption and shadow removal, carbon dioxide dispersion, temperature control, . In particular, if a conical helical tubular photobioreactor with optimized heat transfer is used, the optimal temperature of the fluid to be flowed into the interior is increased to produce more biomass by injecting less operating energy at various locations throughout the year It can be predicted according to season and time.

In addition, various microalgae such as Chlorella and Haematococcus pluvialis were cultured using a reactor and showed excellent productivity above the average.

The process of isolating and concentrating microalgae biomass should be an economical and efficient process. Methods such as extraction and separation using high energy such as high temperature and high pressure which have been developed in conventional chemical processes have not been effective in applying to biological process rather than chemical process. Biomass harvesting generally requires one or more solid-liquid separation steps and is an important process in the production of microalgae biomass. This collection process includes flocculation, filtration, flotation and centrifugal sedimentation, some of which are considerably energy consuming.

The biomass recovery process consists of two steps: bulk harvesting is the process of separating biomass from the bulk suspension. This process generally yields a total solid matter of 2-7% through a concentration factor of 100-800 times.

Subsequent thickening is a more energy intensive step that concentrates the slurry through techniques such as centrifugation, filtration, and ultrasonic aggregation.

On the cell membrane Combined  The polar lipid Triglyceride  A step of converting into a form>

The present invention relates to a method for producing triglyceride (TAG) in microalgae, which comprises applying pressure stress to a microalgae separately from the process for growing microalgae associated with an increase in biomass (in step 3 or 4 in FIG. 2) Characterized in that the step of converting the polar lipid bound to the form of triglyceride is carried out. In addition, the above step can induce triglyceride accumulation in microalgae (FIGS. 3 and 4).

Another characteristic of the present invention is that the pressure stress suitable for each microalgae converting the polar lipid bound to the cell membrane into the form of triglyceride can be selected through experiments.

In the step of converting the polar lipid bound to the cell membrane into the form of triglyceride, the pressure stress is preferably 5 to 15 atm, and the pressure stress is preferably 1 to 10 hours Lt; / RTI >

Pressure stress can be from 5 to 15 atm. Various gases such as nitrogen, helium, hydrogen, carbon dioxide, and methane can be used alone or in combination. It is also possible to utilize various industrial flue-gases containing CO ₂ , but caution is necessary since CO ₂ may reduce the pH of the microalgae culture medium and affect the growth and metabolism of microalgae. The use of air is desirable in terms of durability / price of the gas compressor or blower for gas price and gas supply.

In addition, along with pressure stress, TAG accumulation can be further increased if additional stresses, previously known to increase the TAG, are added. In addition to pressure stress, non-limiting examples of additional stress include fenpropimorph compound stress, osmotic stress, and light stress.

As shown in Figure 8, in addition to the pressure, other stresses such as compounds (fenpropimorph; 50 ppm), osmotic stress (sodium chloride; 1M) and strong light (200 μmol / m ² · s) , The TAG accumulation was further increased.

The present invention is characterized in that the first step is carried out for 1 to 14 days, preferably for 4 to 10 days, for 1 to 10 hours , Preferably from 1 hour to 6 hours.

The first and second steps may be carried out in separate reactors, i.e., photo-bioreactor and pressure reactor, respectively (Figure 5).

≪ Post step &

The microalgae can be recovered after the first step and / or after the second step.

Since microalgae are relatively small and single cells compared to other plant species, the cost of recovering the cells is high. Centrifugation is an expensive process, so filtration instead. Precipitation, floatation, etc. may be used. Or it is preferable to select a strain having high cohesiveness at a certain stage of growth.

The biomass drying process can be performed for high concentrations of biomass and final product. Typically, drying requires heat, so a methane drum dryer or other oven-type dryer can be used.

After drying, the microalgae may be crushed to extract the product. There are various cell crushing methods depending on the cell wall of the microalgae and the characteristics of the product. For low-cost cell disruption, strains smaller in size and larger in size than Nannochloropsis and larger in cell wall size and thinner in cell wall are suitable.

< From triglyceride Biodiesel  Transition phase>

Among the petroleum substitute fuels, biodiesel is a fatty acid alkyl ester obtained from triglyceride, which is the main component of vegetable oil or animal fat, by reaction with alcohol (mainly methanol) under various catalysts and reaction conditions.

Biodiesel is similar to existing petroleum diesel, so it can be used directly or in a certain ratio in diesel cars. Biodiesel is less toxic than petroleum diesel, has biodegradability, and emissions of automobiles using biodiesel as fuel are significantly lower than that of particulate matter (PM) or toxic gas.

The environmentally advantageous biodiesel is synthesized by the following reaction scheme 1, and various catalysts (for example, a basic catalyst such as KOH) and reaction conditions are studied in order to lower the production cost.

[Reaction Scheme 1]

On the other hand, biodiesel may have various kinds of saturated fatty acid groups and fatty acid compositions which constitute biodiesel as well as paraffin components.

In addition, biodiesel can be purified by column chromatography.

Since the present invention can accumulate large amounts of triglyceride-containing lipid droplets (LDs), it is possible to greatly reduce the oil production and extraction cost of the pre-stage for biodiesel production. Thus, it is possible to produce biodiesel from microalgae at a low cost and industrially applicable scale.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram for producing biodiesel from microalgae. FIG.
FIG. 2 is a diagram showing the steps of the biodiesel production process using microalgae as a raw material and the steps corresponding to each element technology.
Figure 3 shows a general lipid biosynthesis process.
FIG. 4 is a conceptual diagram of the present invention in which a polar lipid bound to a cell membrane is converted into a triglyceride form by applying mild pressure stress to microalgae for a short time to induce lipid translocation.
5 is a specific example of a pressure reactor in which pressure stress can be applied to the recovered microalgae according to the present invention.
FIG. 6 is a graph showing changes in TAG amount with pressure (1, 10 and 15 atm) and pressure application time.
Figure 7 is a graph showing the pressurization effect on TGA content at different biomass concentrations ((a) 10 g cell / L and (b) 100 g cell / L).
FIG. 8 shows a graph showing the relationship between (a) 50 ppm Fenpropimorph; (b) 1 M NaCl; (c) the combined pressurization effect on TGA content when high light is applied.
FIG. 9 is a Chlorella sp. 1 is a graph showing changes in lipid composition when KR-1 is pressurized. (a) total lipid content of cells; (b) Lipid content relative to different classes.
10 is a view showing an electron microscope photograph before and after the pressure treatment. (b) and (d) show enlarged photographs of (a) and (c), respectively. (L) lipid droplet; (S) starch; (C) chloroplast; (Red arrow) budding lipid droplet; (Blue arrow) membrane disintegration.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: strain and culture conditions

The native freshwater microalgae , Chlorella sp. KR-1 was isolated from a stream in Youngwol-gun, Korea. Microalgae cultivation was performed in a 7 L Pyrex bubble column photobioreactor (b-PBR, length 1180 mm; inner diameter 85 mm; working volume 6 L). At this time, air containing 10 (v / v)% carbon dioxide was supplied at a rate of 0.75 L / min and a 12 fluorescent lamp (intensity of light: ca. 80 μmol photons / m ² · s) were irradiated and cultured. The medium used was an improved photosynthetic N8 medium containing 3 mM potassium nitrate. The compound contained in 1 L of the enhanced N8 medium is as follows: 505.5 mg KNO ₃ , 740 mg KH ₂ PO ₄ , 259.8 mg Na ₂ HPO ₄ , 50 mg MgSO ₄ .7H ₂ O, 17.5 mg CaCl ₂ .2H ₂ O, 11.5 mg FeNaEDTA 3 H ₂ O, 3.2 mg ZnSO ₄ .7H ₂ O, 13 mg MnCl ₂ .4H ₂ O, 18.3 mg CuSO ₄ .5H ₂ O and 7 mg Al ₂ (SO ₄ ) ₃ .18H ₂ O The medium was sterilized by a 0.2 μm pore filter and the pH was adjusted to 6.5.

The b-PBR was continuously supplied with air containing 10 (v / v)% carbon dioxide from the bottom of the reactor and the supplied gas passed through a 0.2 μm PTFE ventilation filter (Minisart 2000, Satorius Stedium Biotech., Germany). The b-PBR was controlled by a mass-flow regulator (MKP, Korea) and a flow meter (Dwyer Instruments, USA) White fluorescent lamp, ~ 170 μmol / m ² · s).

Example  2: Pressure reactor and pressure experiment

A diagram of the pressure reactor is shown in FIG. The main components of the pressure reactor consist of a stainless steel reactor, a motorized agitator, a pressure gauge, a safety valve, a water jacket and a sample collection port on the bottom. Stainless steel tubing delivers pressurized gas from the cylinder to the reactor through a needle valve.

Cells were harvested by centrifugation (3800 x g , 10 min.) And prepared at the desired biomass concentration for pressure experiments, after 4 or 7 days of incubation according to Example 1.

The algae suspension having a known concentration and volume (ca. 100 mL) was transferred to a pressure cylinder and stirred at 100 rpm using a motorized stirrer. The temperature of the reactor was maintained at 30 캜 by using a water jacket connected to the circulating water bath. Air pressure of 5 to 15 atm was applied inside the reactor and cells grown under pressurized conditions were compared with cells grown under atmospheric pressure as a control.

Experimental Example  1: Analysis method and consideration

The biomass concentration was calculated multiplying OD ₆₆₀ by a factor of 0.2244. Here, the factor 0.2244 is a value calculated from a plot of OD ₆₆₀ value vs. dry-cell weight (DCW) variation. OD was measured with UV-Vis spectrophotometry (Optizen 2120UV, Mecasys Co., Korea). To measure the cell dry weight (DCW), fresh samples were filtered at different growth times using a weighted GF / C filter (Whatman, UK), washed twice with distilled water, Lt; 0 &gt; C for 2 hours to measure the weight. Cell dry weight (DCW) was calculated excluding the filter weight.

Example After a pressure test according to the second, the cells collected by centrifugation (3800x g, 10 min.) And by using a freeze-dryer (FD5512, IlShin BioBase Co., Korea) were freeze-dried for more than 4 days.

Total lipids extracted from about 10 mg of dry cells were mixed in 5 mL of chloroform / methanol (2: 1 v / v) and vigorously stirred for 6 hours. In vigorous stirring, 2 mL of distilled water was added for 5 minutes and centrifuged at 4000 x g . After centrifugation, the following organic layer was separated and filtered through a plastic syringe (Minisart SRP15, Satorius Stedium, Germany) with a 0.2 μm PTFE filter and dried with a rotary vacuum evaporator (EZ2 PLUS, Genevac Ltd., UK) .

To quantify the TAG, the lipid extract was redissolved in about 50 μL of chloroform and loaded onto a TLC plate (Silica gel Al foils, Fluka, USA). In order to separate the triglyceride from the TLC plate, it was developed with a hexane / diethyl ether / acetic acid mixed solvent (80/30/1, by volume). Acetone: Lipid spot was confirmed under UV by spraying 0.01% (w / v) primuline (Sigma) solution dissolved in distilled water (4: 1, v / v). The TAG band was collected from the TLC plates and quantitated. Quantitation was performed by densitometry and image analysis using ImageJ software (RSB, USA) or by gas chromatography (GC) analysis after transesterification.

The fatty acid content of TAG was measured by direct transesterification. The TAG band collected above was added to 2 mL of chloroform / methanol (2: 1 v / v) over 10 minutes and vigorously stirred. After stirring, 1 mL of chloroform, Heptadecanoic acid, 1 mL of methanol and 300 μL of sulfuric acid (95%) were added in this order, and the mixture was stirred well for 5 minutes and left at 100 ° C. for 10 minutes. An additional 1 mL of distilled water was added and mixed vigorously, then cooled to room temperature and centrifuged at 4000 x g . After centrifugation, the following organic layer was separated and filtered through a plastic syringe with a 0.2 μm PTFE filter.

FAME was analyzed using a gas chromatograph (GC) with a FIM detector (frame ionization detector) and an HP-INNOWax capillary column (0.32 mm ID x 30 m, Agilent Technologies, USA). Detailed GC analysis conditions are described in the prior art (Na et al., Biotechnology Letters , 2011, 33 (5), 957).

Fig. 6 is a graph showing the results of microalgae Chlorella sp. The relative TAG content of KR-1 over time is shown. Here, the TAG value at 0 hour of the control (1 atm) was regarded as 100%, and the relative TAG content was shown. In this case, the pressing time was about 2 hours, which was the most effective in terms of increasing the TAG content.

Figure 7 shows the microalgae Chlorella &lt; RTI ID = 0.0 &gt; sp . (TAG amount change) with respect to the FAME content of TGA in KR-1. The amount of TAG was also increased when treated with a high concentration of 100 g cell / L, which confirms that the present invention has a great advantage in the process aspect.

8 is Chlorella sp. With KR-1 pressure, (a) 50 ppm Fenpropimorph; (b) 1 M NaCl; (c) A graph showing the combined pressurizing effect on the FAME content of TGA when high light (200 μmol / m ² · s) was applied. In this case, the control group was subjected to pressure stress only at the pressures applied in FIGS. 8 (a), 8 (b) and 8 (c) for increasing the amount of TAG. The TAG was increased when the stress factors such as NaCl, Fen. (Chemical substance) and high light were added together, which is known to increase the TAG rather than the stress only.

Fig. 9 is a graph showing the results of the measurement of Chlorella sp. (A) the total lipid content of the cells; and (b) the lipid content relative to the different classes) when subjected to KR-1. Total lipid content did not change with the pressure factors. It can be seen from this that the change from Glycolipids (MGDG / DGDG - rich in DAG) to Neutral lipid (TAG) is induced from this because the variable amount is Neutral lipid (mainly TAG). That is, as shown in FIG. 4, the translocation of lipids was induced by the pressurization from the cell membrane of the cell and its organelle (chloroplast / mitochondria / endoplasmic reticulum).

Claims (11)

In the method for producing triglyceride (TAG) in microalgae,
And applying a pressure stress of 5 to 15 atm to the microalgae to convert the polar lipid bound to the cell membrane into the form of triglyceride in the microalgae.
In the method for producing triglyceride (TAG) in microalgae,
A first step of growing and culturing microalgae for a predetermined period of time; And
A second step of converting the polar lipid bound to the cell membrane into the form of triglyceride in the microalgae by applying a pressure stress of 5 to 15 atm to the microalgae for a certain period of time,
&Lt; / RTI &gt;
delete The method according to claim 1 or 2, wherein pressure stress is applied to the microalgae to induce triglyceride accumulation in the microalgae.
3. The method according to claim 1 or 2, further comprising the step of adding one or more stresses selected from the group consisting of fenpropimorph compound stress, osmotic stress, and light stress together with pressure stress to cause accumulation of triglyceride in the microalgae By weight of the triglyceride.
3. The method of claim 2, wherein the first step is performed for 1 to 14 days, &Lt; / RTI &gt; wherein the second step is performed during the second step.
[7] has been abandoned due to the registration fee. 3. The method of claim 2, wherein the first step and the second step are performed in the same reactor or in separate reactors, respectively.
3. The method of claim 2, further comprising the step of recovering the microalgae from the culture medium prior to the second step.
3. The method according to claim 1 or 2, wherein triglyceride accumulation in microalgae is induced under microbial cell concentration of 10 to 100 g cell / L and pressure of 10 to 15 atm.
3. The method according to claim 1 or 2, further comprising pulverizing or oil-extracting the microalgae subjected to pressure stress.
A method for producing biodiesel from microalgae,
A first step of growing and culturing microalgae for a predetermined period of time;
A second step of converting the polar lipid bound to the cell membrane into the form of triglyceride in the microalgae by applying pressure stress of 5 to 15 atm to the microalgae for a predetermined period of time; And
The third step of converting biodiesel from triglyceride
The method comprising the steps of:

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