KR101694711B1 - Culturing method of microalgae for increasing lipid productivity - Google Patents

Abstract

The present invention relates to a method for culturing microalgae producing lipid, which is a raw material of biodiesel, and a method for culturing microalgae capable of increasing the growth rate of microalgae cells and the lipid content in microalgae cells.
According to the present invention as described above, it is possible to optimize the growth rate of the microalgae cells and the lipid content in the microalgae, such as the conditions of high concentration of nitrogen, the culture method under the above conditions, By providing culture conditions and a culture method, it is possible to increase the lipid productivity of microalgae, which is a raw material of biodiesel, and to mass-produce biodiesel using microalgae.

Description

TECHNICAL FIELD [0001] The present invention relates to a method of culturing microalgae that increases lipid productivity,

TECHNICAL FIELD The present invention relates to a method for culturing microalgae producing lipid, which is a raw material of biodiesel, and more particularly, to a microalgae culture method for enhancing microalgae growth rate and microalgae cell lipid content, And a method for culturing algae.

As the danger of oil depletion grows and as the importance of alternative energy becomes more and more important, we are making a global effort to develop alternative energy around the world. Among them, renewable energy that can replace fossil fuels is attracting attention as biofuel. Biofuels are fuels derived from biomass using plant and animal wastes, biological wastes, and bio-diesel and bio-ethanol that can replace gasoline and gasoline.

Biodiesel is an alternative to petroleum-based diesel fuel, which is produced based on renewable resources such as vegetable oil and animal fats. Especially microalgae, which are photosynthetic microorganisms, have the highest oil productivity among all biodiesel production crops, As a raw material.

More specifically, there are more than 100,000 kinds of microalgae. It is a photosynthetic organism that can supply water, sunlight and carbon dioxide only if it supplies photosynthesis and can grow unlimitedly. It can mass-produce in a short time in various environments and can fix carbon dioxide Also, because of high protein and lipid content, it can be used as biomass for food, feed or fuel production.

In particular, the lipid component contained in microalgae can be used as a raw material for the production of biodiesel used as a liquid fuel. That is, neutral lipids such as triglyceride among the lipid components of microalgae are extracted from cells and then converted into biodiesel alkyl fatty acid ester by a trans-esterification reaction with a low-molecular alcohol such as methanol.

Lipid productivity is calculated as (microalgae cell growth rate (g / L / d)) x (lipid content per cell dry weight (%)), microalgae biomass typically contains 15-30% lipid , And 50% or more in some special species. However, microalgae containing high quality lipids usually do not have high productivity of lipid production for biodiesel because of low growth rate. Therefore, it is advantageous to use microalgae with high growth rate even if lipid content is somewhat lower than 15 ~ 30%.

In addition, one of the methods for enhancing the lipid production of microalgae is to give a stressful condition instead of a normal growth condition. In many kinds of freshwater microalgae, the lipid content On the other hand, when there is a certain environmental stress on the culture condition, the growth is slowed, and the lipid synthesis is accelerated and the lipid content is often increased.

That is, some freshwater microalgae, including some chlorella species, tend to slow their growth and increase lipid yield when the nutrients, Nitrogen and Phosphorus, that are essential nutrients in culture are insufficient. In addition, other species have been reported to increase lipid production when iron, magnesium, and silicon are insufficient. In addition, when the temperature, pH, illumination, etc. are abnormally high or low, the amount of lipid production may increase. As a result, cultivation under stress conditions in which growth factors are deficient is utilized as a method for enhancing lipid production of microalgae.

However, the increase in the lipid content in the deficient state of the major growth factors of microalgae such as nitrogen is effective only in some freshwater species. In other species, the increase in the lipid content even in the stressed state in which the main growth factor is deficient In some cases, it does not occur or the lipid content decreases.

Especially, in marine microalga species growing at high salinity, cell growth rate and lipid content are often decreased in nitrogen deficiency state. In addition, since nitrogen is an essential factor for the growth of microalgae, even if the lipid content is increased in the nitrogen deficiency state, there is a problem that the final lipid production and productivity are lowered due to the decrease of the microalgae growth rate. Therefore, in order to improve the lipid productivity, a culture condition and culture method which can increase both the cell growth rate of microalgae and the lipid content in microalgae are needed.

Korean Patent Publication No. 10-2014-0121149 (method for producing biodiesel using simultaneous lipid extraction and transesterification of microalgae), Korean Patent No. 10-1403464 (microalgae lipid and reducing sugar And a microalgae culture system for increasing the content).

An object of the present invention is to provide a method for culturing microalgae for a predetermined period of time until the cell dry weight and the intracellular lipid content of a microalgae are both increased in a culture medium in which the concentration of nitrogen is maintained at a high concentration and the optimum concentration of organic carbon is added To thereby provide a microalgae culture method capable of increasing the production amount and productivity of lipid used as a raw material for biodiesel.

In order to accomplish the above object, the present invention provides a method for producing microalgae, which comprises culturing microalgae in a medium containing nitrogen at a high concentration of 300 to 5000 mg / L until a cell dry weight and a cell lipid content of the microalgae are both increased And a method for culturing microalgae that increases lipid productivity.

Further, the present invention is characterized in that the medium further comprises organic carbon.

The organic carbon is characterized by being glucose, glycerol, or acetate.

And the concentration of the organic carbon is 0.1 to 10% (w / v).

The nitrogen is characterized by being nitrate nitrogen (NO ₃ -N) or ammonia nitrogen (NH ₄ -N).

The nitrate nitrogen is at least one selected from the group consisting of sodium nitrate (NaNO ₃ ), potassium nitrate (KNO ₃ ) and ammonium nitrate (NH ₄ NO ₃ ).

The ammonia nitrogen is ammonium chloride (NH ₄ Cl), ammonium nitrate (NH ₄ NO _3), ammonium bicarbonate (NH ₄ HCO _3), ammonium acetate (CH ₃ COONH _4), urea _{(CO (NH 2) 2)} , Glycine (NH ₂ CH ₂ COOH) and yeast extract.

And nitrogen is continuously supplied at a time interval of 12 hours or more in order to maintain the concentration of nitrogen.

The culture medium of the microalgae salinity of 30 to 35 psu, temperature 10 to 40 ℃, light intensity of 20 to 500 μmol / m ² / s and 0.03 to 15% (v / v) feed rate of the air containing CO ₂ in 0.01 To 0.5 vvm. ≪ / RTI >

The microalgae may be selected from the group consisting of Tetraselmis , Dunaliella , Nannochloropsis , Isochrysis , Porphyridium , Coccomyxa , (Phaeodactylum), the eponymous kit Leeum (Schizochytrium), chlorella (chlorella), Chlamydomonas (Chlamydomonas), euglena (Euglena), Chitose in Karos (Chaetoceros), hematoxylin nose Syracuse (Haematococcus), creep te coordination nium (Crypthecodinium ), Neochloris , Navicula , and Arthrospira . In the present invention,

Also, the method for culturing the microalgae of the present invention comprises: (a) primary culturing microalgae in a medium having a nitrogen concentration of 75 mg / L; And (b) secondary culturing the primary cultured microalgae in a medium having a nitrogen concentration adjusted to a high concentration of 300 to 5000 mg / L.

According to the present invention as described above, it is possible to provide a method for culturing a microalgae for a predetermined period of time until the cell dry weight and the intracellular lipid content of the microalgae are both increased in a culture medium containing a high concentration of nitrogen, The growth rate and lipid content of the algae are both increased, thereby increasing the production and productivity of the raw material of biodiesel.

FIG. 1 (a) is a graph showing cell dry weight of microalgae as a result of culturing according to Example 1, and FIG. 1 (b) is a graph showing intracellular lipid content of microalgae as a result of culturing according to Example 1.
FIG. 2 (a) is a graph showing the cell dry weight of microalgae as a result of the culture according to Example 2, and FIG. 2 (b) is a graph showing the intracellular lipid content of microalgae as a result of the culture according to Example 2.

Hereinafter, the present invention will be described in detail.

Microalgae refers to creatures that have photosynthesis with photosynthetic pigments. It refers to aquatic organisms that have the size of a micro-unit that grows by fixing carbon dioxide with light as an energy source, such as sunlight, and by performing photosynthesis.

In the case of freshwater microalgae, if the concentration of nitrogen or phosphorus is insufficient, the genome can not be replicated properly, and cell division is inhibited. As a result, organic matter accumulated in photosynthesis is converted into fat and accumulated in cells, It is known. However, microalgae are very diverse in their kinds, and due to differences in lipid content, lipid characteristics, growth rate, and growth environment conditions for optimal growth depending on the species, The method of increasing the content is often not applied. Depending on the species, the excessive amount of certain components may act as a stress. Such excessive stress conditions do not affect the cell growth but affect only the carbon metabolism related to lipid production . Therefore, a method for increasing the lipid productivity for such microalgae species is required. Accordingly, in the present invention, the growth rate of microalgae and the lipid content in the microalgae are both increased to increase the lipid productivity by microalgae as a raw material of biodiesel The present invention relates to a method for culturing microalgae that can be cultivated in the soil.

Specifically, in the present invention, the microalgae are cultured in a medium containing nitrogen adjusted at a high concentration of 300 to 5000 mg / L, preferably 300 to 3500 mg / L, more preferably 350 to 1500 mg / L, The present invention also provides a method for culturing microalgae that increases lipid productivity, which comprises culturing the microalgae for a predetermined period of time until both the cell dry weight and the intracellular lipid content are increased.

Nitrogen is a major growth factor essential for microalgae cultivation. As the concentration of nitrogen in the culture process affects microalgae growth and lipid production, the present inventors have found that the growth rate of microalgae Numerous repeated experiments and analyzes were carried out under various conditions in order to find the high concentration of nitrogen condition which can increase the cell dry weight and the intracellular lipid content and ultimately increase the lipid productivity.

As a result, it was confirmed that the nitrogen concentration of 300 to 5000 mg / L was the optimum nitrogen concentration to increase the lipid productivity of the microalgae. When the nitrogen concentration in the culture medium is less than 300 mg / L, the amount of nitrogen supplied to basic metabolism such as protein and enzyme synthesis and photosynthesis of microalgae cells is insufficient, and carbon metabolism such as cell growth and lipid synthesis is inhibited, In contrast, when the nitrogen concentration in the culture medium exceeds 5000 mg / L, the activity of the enzyme reversibly reversed at an appropriate nitrogen concentration, and at high concentrations of nitrogen exceeding the appropriate range, the activity of the cell is inhibited And the concentration of the salt constituting the nitrogen compound is excessively increased, which may adversely affect the cell activity. Therefore, it is preferable that the concentration of nitrogen is 300 to 5000 mg / L in order to increase both the cell dry weight and the intracellular lipid content and ultimately increase the lipid productivity.

According to the microalgae culturing method of the present invention, the microalgae grow at a sufficient growth rate by nitrogen, the nitrogen consumption by the photosynthesis proceeds to a certain level, and the synthesis of the protein involved in lipid production and the related metabolism It is assumed that nitrogen is used in the promoted direction.

The nitrogen is characterized by being nitrate nitrogen (NO ₃ -N) or ammonia nitrogen (NH ₄ -N). The nitrate nitrogen is at least one selected from the group consisting of sodium nitrate (NaNO ₃ ), potassium nitrate (KNO ₃ ) and ammonium nitrate (NH ₄ NO ₃ ) .

The ammonium nitrogen is a nitrogen is present in an ammonia or an ammonium salt of various compounds of nitrogen, aqueous ammonium chloride (NH ₄ Cl), ammonium nitrate (NH ₄ NO _3), ammonium bicarbonate (NH ₄ HCO _3), ammonium acetate (CH ₃ COONH ₄ ), urea (CO (NH ₂ ) ₂ ), glycine (NH ₂ CH ₂ COOH) and yeast extract.

In addition, nitrogen is continuously supplied at a time interval of 12 hours or more in order to maintain the concentration of nitrogen. In order to monitor the concentration of nitrogen from time to time, ion chromatography, ultraviolet absorbance method, ion electrode method And the concentration is measured using the above.

The present invention is further characterized in that the culture medium further contains organic carbon. The organic carbon is preferably glucose, glycerol, or acetate, and the concentration thereof is preferably 0.1 to 10% (w / v).

In addition to the autotrophic carbon metabolism by carbon dioxide fixation, the microalgae, when supplied with the appropriate organic carbon as described above, are accompanied by a mixotrophic or hetotrophic carbon metabolism induced by organic fatty acid synthesis and triglyceride synthesis As shown in Fig.

In addition to the organic carbon, various organic substances contained in the wastewater can be used as a nutrient source for microalgae growth.

In addition, the CO ₂ According to the culture method of the micro-algae of the present invention the medium salinity of 30 to 35 psu, temperature 10 to 40 ℃, light intensity of 20 to 500 μmol / m ² / s and 0.03 to 15% (v / v) And a supply rate of air containing 0.01 to 0.5 vvm.

The microalgae species to which the microalgae culturing method for increasing the lipid productivity provided by the present invention can be applied include both freshwater and marine species, but it is more preferable to apply them to marine species microalgae.

When artificial cultivation of marine species microalgae is carried out, artificial seawater and artificial seawater medium supplemented with some nutrients for growth promotion are used to directly use seawater or to reduce the hassle of drinking water. The average salinity concentration of seawater is 30 to 35 g / L (30 to 35 ‰ (permile) or 30 to 35 psu (practical salinity unit)), and the saline solution mainly uses NaCl to control salinity. Marine species microalgae grow most normally in the normal salinity range of 30 to 35 psu because this concentration of salinity plays a role in balancing the intracellular osmotic balance. Therefore, it is preferable that the salinity of the medium is adjusted to 30 to 35 psu when the marine microalgae according to the present invention are cultured.

More specifically, microalgae to which the culture method of the present invention can be applied include microalgae such as Tetraselmis , Dunaliella , Nannochloropsis , Isochrysis , Porphyridium), nose Comics Inc. (Coccomyxa), L ohdak tea room (Phaeodactylum), the eponymous kit Leeum (Schizochytrium), chlorella (chlorella), Chlamydomonas (Chlamydomonas), euglena (Euglena), Chitose in Karos (Chaetoceros), H. And is characterized in that it is at least one selected from the group consisting of Haematococcus , Crypthecodinium , Neochloris , Navicula and Arthrospira .

Also, the method for culturing the microalgae of the present invention comprises: (a) primary culturing microalgae in a medium having a nitrogen concentration of 75 mg / L; And (b) secondary culturing the primary cultured microalgae in a medium having a nitrogen concentration adjusted to a high concentration of 300 to 5000 mg / L.

According to the method for culturing microalgae according to the present invention, it is possible to increase the growth rate and lipid content of microalgae even when cultured under high nitrogen concentration of 300 to 5000 mg / L from the beginning. However, In the case of the species whose content is increased but the growth rate is decreased, the cells are rapidly grown in the medium containing the nitrogen concentration of 75 mg / L, which is the standard nitrogen concentration of the artificial seawater culture medium (f / 2 medium) It is also possible to carry out two-step cultivation in which lipid is produced by converting to a high-concentration nitrogen condition and culturing.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1: Confirmation of increase in growth rate and lipid content of microalgae according to the microalgae culture method of the present invention (according to the change of nitrogen concentration)

In this example, the experiment was carried out in two separate steps to confirm the effect of growing microalgae cells moving from the standard concentration condition (75 mg / L) to the high concentration nitrogen condition.

First, CO ₂ was contained at a temperature of 25 ° C, a luminous intensity of 100 to 110 μmol / m ² / s, and 2% (v / v) in a normal artificial sea water medium (f / 2 medium) having a salt salinity of 35 psu ( Tetraselmis ) was inoculated at a concentration of 0.5 g / L on a dry weight basis, maintaining a concentration of sodium nitrate (NaNO ₃ ) of 75 mg / L.

At this time, NaNO ₃ was continuously supplied at intervals of 24 hours to maintain the NaNO ₃ concentration.

Subsequently, the cultures containing tetracellis grown at an appropriate concentration were divided and cultured at different concentrations (0, 75, 375, 750 and 1500 mg / L). Other conditions except nitrogen concentration were kept same as primary culture. At this time, NaNO ₃ was continuously supplied at intervals of 24 hours to maintain the NaNO ₃ concentration.

As a result, as shown in FIG. 1, at a concentration of 375, 750 and 1500 mg / L of nitrogen (NaNO ₃ ) higher than the standard nitrogen (NaNO ₃ ) concentration of 75 mg / L in the artificial seawater culture medium, The higher the nitrogen concentration, the higher the cell dry weight of tetracellmis during the same incubation time, and the higher the nitrogen concentration, the higher the growth rate.

In addition, the intracellular lipid content of Tetracellis was increased from 20% to 30% at a high concentration of 375, 750 and 1500 mg / L of nitrogen (NaNO ₃ ) for 60 hours. As a result, It can be confirmed that the increase of the lipid content is larger than that of the nitrogen concentration condition.

It can be seen from FIG. 1 that the culture time of about 60 hours is required until both the cell dry weight and the intracellular lipid content are maximally increased in the case of Tetracellis.

As described above, according to the microalgae culture method of the present invention, since the growth rate and the lipid content of the cells are simultaneously increased during culturing of microalgae, the lipid productivity (microalgae cell growth rate) x (lipid content per cell dry weight) Is increased.

Example 2: Confirmation of increase in growth rate and lipid content of microalgae according to the microalgae culture method of the present invention (with addition of organic carbon)

Air containing CO ₂ at a temperature of 25 ° C, a luminous intensity of 100 to 110 μmol / m ² / s and 2% (v / v) in a normal artificial sea water medium (f / 2 medium) having a salinity of 35 psu (practical salinity unit) And the concentration of NaNO ₃ was maintained at 75 mg / L. Tetracellis was inoculated at a concentration of 0.5 g / L in dry weight, and then primary cultured. At this time, NaNO ₃ was continuously supplied at intervals of 24 hours to maintain the NaNO ₃ concentration.

Thereafter, the culture solution containing the tetracellis grown at an appropriate concentration was divided into two different concentrations of nitrogen (75, 750, mg / L) and glycerol addition conditions (glycerol concentration 0, 0.5, 1% secondary and cultured., but the other conditions except for the nitrogen concentrations are maintained in the same manner as primary cultures, and every 12 hours to a concentration maintenance of NaNO ₃ NaNO ₃ for continuously supplying gave a filtration in order to stir the air (0.038% (v / v) CO ₂ ). In this experiment, the effect of using glycerol as a carbon source was minimized, so that the supply of CO ₂ was minimized and air was supplied for agitation of the culture solution.

As a result, as shown in Figure 2, the standard nitrogen in the artificial seawater medium (NaNO ₃₎ concentration of 75 mg / in case of L than 10 times the nitrogen of higher 750 mg / L (NaNO ₃₎ when cultured in concentrations By confirming that the cell dry weight of tetracellmis is higher for the same period of time, it can be seen that the growth rate increases at a high nitrogen concentration of 750 mg / L. In addition, the lipid content in the tetracellular mucosa was increased from 20% to 25% for 3 days in the high concentration of 750 mg / L of nitrogen (NaNO ₃ ) It can be seen that there is no change in intracellular lipid content when cultured under a nitrogen concentration condition.

In addition, when 5, 10 g / L of glycerol was added to the culture medium as organic carbon, the growth rate of the cells was further accelerated, and the cell concentration was greatly increased from 1.7 g / L to 3.7 g / L over 10 days there was.

As described above, according to the culture method and culture conditions including the nitrogen condition at the concentration provided by the present invention and the addition of the organic carbon at an appropriate concentration, the growth rate of the microalgae cells and the lipid content in the cell are simultaneously increased, It is possible to increase the lipid productivity.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

The method comprising adjusting the concentration of the salts of nitrogen to 75 mg / L 1 subculture microalgae tetrahydro cell Miss (Tetraselmis) in artificial sea water medium 35psu (practical salinity unit); And
Primary cultured microalgae were cultured in artificial seawater medium having a salinity of 35 psu (practical salinity unit) containing nitrogen and 0.1 to 10% (w / v) of organic carbon adjusted to a high concentration of 300 to 5000 mg / Step for tea cultivation
( Tetraselmis ). ≪ Desc / Clms Page number 20 >
delete The method according to claim 1,
Wherein the organic carbon is glucose, glycerol, or acetate. 2. The method of claim 1, wherein the organic carbon is glucose, glycerol, or acetate.
delete The method according to claim 1,
Wherein the nitrogen is nitrate nitrogen (NO ₃ -N) or ammonia nitrogen (NH ₄ -N).
6. The method of claim 5,
Wherein the nitrate nitrogen is at least one selected from the group consisting of sodium nitrate (NaNO ₃ ), potassium nitrate (KNO ₃ ) and ammonium nitrate (NH ₄ NO ₃ ). .
6. The method of claim 5,
The ammonia nitrogen is ammonium chloride (NH ₄ Cl), ammonium nitrate (NH ₄ NO _3), ammonium bicarbonate (NH ₄ HCO _3), ammonium acetate (CH ₃ COONH _4), urea _{(CO (NH 2) 2)} , Wherein the microorganism is at least one selected from the group consisting of glycine (NH ₂ CH ₂ COOH) and yeast extract.
The method according to claim 1,
Wherein the nitrogen is continuously supplied at a time interval of 12 hours or more in order to maintain the concentration of nitrogen.
The method according to claim 1,
The culture conditions further include a condition of an air supply rate of 0.01 to 0.5 vvm containing CO ₂ at a temperature of 10 to 40 ° C, an intensity of 20 to 500 μmol / m ² / s, and 0.03 to 15% (v / v) Wherein the method comprises the steps of:

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