KR20140022212A - Culturing method of microalgae for producing biodiesel by regulating salinity in wastewater - Google Patents

Abstract

The present invention relates to a method for mass-culturing freshwater microalgae containing lipoid by culturing strains of microalgae tolerant toward wastewater and salinity in wastewater of which the salinity is controlled. The microalgae culturing method according to the present invention obtains a growth rate of microalgae more excellent than a microalgae culturing method using normal wastewater by a salinity condition and enables the production of a high-quality fatty acid. Additionally, the present invention enables precise wastewater treatment as a rate of removing nitrogen and phosphorous is excellent by culturing microalgae in wastewater of which the salinity is controlled. Furthermore, the present invention is environment-friendly and obtains excellent economic efficiency in an aspect of purifying and recycling resources by utilizing wastewater and food wastewater as a salinity controlling element.

Description

Technical Field [0001] The present invention relates to a method for mass production of freshwater microalgae for the production of biodiesel by controlling the salinity in wastewater,

The present invention relates to a method for mass-culturing freshwater microalgae for biodiesel production, and more particularly, to a method for mass culturing freshwater microalgae for biodiesel production, and more particularly, to a method for mass culture of freshwater microalgae for biodiesel production, To a method for mass-culturing microalgae.

The microalgae have high efficiency in purification of wastewater water pollution by removing nitrogen and phosphorus. Microalgae biomass produced by this activity is bioethanol, lipid (fatty acid) It is the source of renewable energy to produce bio-butanol and biodiesel [Park, Jae-il, Woo-hee, Lee, Jae-hwa, 2008. Bio-energy production from marine algae: status and outlook. Journal of Chemical Engineering. 46 (5): 833-844).

Most of the algae studied so far are marine algae living in the ocean, and recent studies on biodiesel production of microalgae cultivated in wastewater and lakes have been conducted. Light, carbon dioxide, nitrogen, phosphorus and salinity are known to be important growth factors of freshwater microalgae. However, no studies on the growth and biodiesel production due to the effect of salinity on various microalgae have been made.

Recently, studies on the origin of organic carbon source, nitrogen and phosphorus for the growth of freshwater microalgae in livestock wastewater and wastewater have been carried out recently. However, studies on mass production and biodiesel optimization by salinity concentration are insufficient .

Accordingly, a problem to be solved by the present invention is to cultivate microalgae, cultivate microalgae capable of solving the energy problem by mass-producing biodiesel by establishing saltiness conditions in the medium capable of producing optimal microalgae growth and lipid production Method.

Also, it is intended to provide a method for cultivation of microalgae containing high-quality algae and a method for cultivation of microalgae that can treat wastewater at a high altitude and purify resources and utilize wastewater to regulate salinity.

In order to solve the above problems,

The present invention relates to a method for culturing freshwater microalgae containing lipid by culturing a freshwater microalgae culture medium in a microalgae culture medium having a controlled salinity,

Wherein the microalgae culture medium is adjusted to a salt concentration of 25-200 mM NaCl (2-12 ‰). The present invention also provides a method for culturing freshwater microalgae for biodiesel production.

According to one embodiment of the present invention, the microalgae culture medium may be at least one selected from wastewater, animal wastewater, agricultural wastewater, industrial wastewater, sewage sludge, water sludge, sewage treatment plant influent water and sewage treatment plant effluent.

According to an embodiment of the present invention, the salinity can be controlled by mixing the salt in the seawater or the desalination filtrate into the microalgae culture medium.

According to one embodiment of the present invention, the culture medium containing the freshwater microalgae strains is placed in a fluorescent stirrer and cultured at a culture temperature of 25 to 30 ° C, pH of 7.2 to 7.6, light intensity of 40 to 60 μmol / -200 rpm.

According to one embodiment of the present invention, the method of culturing freshwater microalgae can remove nitrogen, phosphorus, organic carbon, and inorganic carbon in the microalgae culture medium while producing fresh water microalgae containing lipid.

According to one embodiment of the present invention, the freshwater microalgae strains are selected from the group consisting of Chlamydomonas mexicana), the boat may be at least one selected from Lactococcus Rio beurawooniyi (Botryococcus braunii), species Chlorella (Chlorella. sp.), and three or four des mousse species (Scenedesmus. sp.).

According to the microalgae culturing method according to the present invention, the microalgae growth rate is higher than that in general wastewater through saltiness conditions, and high quality fatty acid production is possible. In addition, it can cultivate microalgae in the salinity-controlled wastewater, and it is possible to treat the wastewater with a high degree of removal such as nitrogen and phosphorus, and it is possible to utilize the wastewater and the wastewater as the salinity control factor. It is environment-friendly and economical.

FIGS. 1A to 1B are graphs showing the growth rates of microalgae cultured in different microalgae culture broth and wastewater according to salinity conditions. FIG.
FIGS. 2A and 2B are graphs showing the biomass dry weight and lipid content increase rate in microalgae culturing liquid culture medium and wastewater under different saltiness conditions, respectively.
FIG. 3A is a graph showing the composition of fatty acids in a microalgae culture broth with different salinity conditions, and FIG. 3B is a graph showing fatty acid content increase rates in microalgae culturing under different salinity conditions in wastewater (C 18 : 3n6, C18: 3n3, C18: 2n6c C18: 1n9c, C18: 0, C17: 1, C16: 1, C16: 0).
FIG. 4 is a graph showing reduction efficiencies of nitrogen, phosphorus, and inorganic carbon in wastewater when microalgae culture is performed under different salinity conditions.

Hereinafter, the present invention will be described in more detail.

The present invention separates and identifies freshwater microalgae species having resistance to wastewater and salinity, and cultivates high-grade microalgae according to the control of salinity after inoculation in a wastewater or microalgae culture, an economy in which high-quality biodiesel production and wastewater treatment are performed The present invention relates to a method for cultivating microalgae. Particularly, it is characterized in that microbial growth is maximized at a specific salinity concentration, and high-quality biodiesel can be produced due to a low gelation point as the amount of unsaturated fatty acids increases upon microalgae growth.

The present invention relates to a method for culturing freshwater microalgae containing high quality by culturing a microalgae culture medium of Chlamydomonas resistant to wastewater and saltiness in a microalgae culture medium, And a salinity of 25-200 mM NaCl (2-12 ‰).

In the case of microalgae, glycerol is produced in response to the osmotic phenomenon due to a defense mechanism against high salinity of the concentration. It accumulates in the body with triacylglycerols (TAG) together with other inorganic carbon and nutrients, , And high quality.

Freshwater microalgae strains are classified as Chlamydomonas mexicana , Botryococcus may be braunii), species Chlorella (Chlorella. sp.), and three or four des mousse species (Scenedesmus. sp.) can be at least one selected from, preferably from Chlamydomonas Mexicana (Chlamydomonas mexicana).

In addition, the microalgae culture medium contained BBM medium (KH ₂ PO ₄ , CaCl ₂ .2H ₂ O, MgSO ₄ .7H ₂ O, NaNO ₃ , K ₂ HPO ₄ , NaCl, H ₃ BO ₃ And a trace element, and the trace element may be ZnSO ₄ .7H ₂ O, MnCl ₂ .4H ₂ O, MoO ₃ , CuSO ₄ .5H ₂ O, Co (NO ₃ ) ₂ .6H ₂ O, Na ₂ EDTA, KOH, FeSO ₄ And H ₂ SO ₄ .

In particular, it is more preferable to cultivate microalgae in a large amount by salinity condition in wastewater. The wastewater may be at least one selected from livestock wastewater, agricultural wastewater, industrial wastewater, sewage sludge, water sludge, inflow water from sewage treatment plant, have.

In addition, when controlling the salt concentration in the culture medium, in addition to the chemical agent of NaCl, seawater, the number of harmful substances in the power generating facility, and saline in the desalination filtrate can be used.

The present invention is characterized by producing fresh water microalgae containing lipids and removing nitrogen, phosphorus, organic carbon and inorganic carbon from the microalgae culture medium.

The present invention also encompasses a method for separating lipids from wastewater and salinity tolerant strains or cultures thereof and producing high quality biodiesel using the lipids.

Generally, a method of producing biodiesel using lipids includes transesterification and D-glycerin production. The present invention can also produce high-quality biodiesel using the above-described conventional biodiesel production method. have. The lipid can be separated by the Sulfophosphovanillin method, the Kunkel method, the Brugdon method or the like, and the lipid is separated from the culture solution of the present invention by this method.

As a result of analysis of fatty acids in the lipids separated from the culture broth cultured by the culture method according to the present invention, the unsaturated fatty acid was increased from 49% to 62% in the optimum growth condition as described in the following examples .

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It will be clear to those who have knowledge.

<Examples>

Example 1. Culture of freshwater microalgae according to salinity conditions in a culture medium

Using a liquid medium (BBM) having the physicochemical composition shown in the following [Table 1], microalgae Clamidomonas Mexica or YSL07 ( Chlamydomonas mexicana YSL07) were cultured.

5 ml of the above strain was added to a 250 ml Erlenmeyer flask at 25-30 ° C under pH 7.2-7.6 for incubation for about 20 days in a fluorescent stirrer. The stirring was maintained at 150 rpm and the illuminance was kept at 50 μmol / m 2 -sec.

In addition, the salinity of the liquid medium was varied in the range of 0-100 mM (0-6 ‰) as shown in Table 2 below.

ingredient Content (per liter) KH ₂ PO ₄ 175 mg CaCl ₂ .2H ₂ O 25 mg MgSO ₄ .7H ₂ O 75 mg NaNO ₃ 250 mg K ₂ HPO ₄ 75 mg NaCl 25 mg H ₃ BO ₃ 11.42 mg Trace element ZnSO ₄ .7H ₂ O 8.82 g MnCl ₂ .4H ₂ O 1.44 g MoO ₃ 0.71 g CuSO ₄ · 5H ₂ O 1.57 g Co (NO ₃ ) ₂ 6H ₂ O 0.49 g Solution 1 Na ₂ EDTA 50 g KOH 3.1 g Solution 2 FeSO ₄ 4.98 g H ₂ SO ₄ 1 mL

Classification (liquid medium) NaCl dose (mM) 0 0.43 10 25 50 100 Total salinity (‰) 0.3 0.4 1.2 1.9 3.5 6.2 Conductivity (mS cm ^-1 ) 0.7 0.9 2.0 3.7 6.5 11.1

Example 2. Culture of freshwater microalgae according to saltiness conditions in wastewater

Using the wastewater having the physicochemical composition shown in the following [Table 3], the microalgae Clamidomonas Mexica and YSL07 ( Chlamydomonas mexicana YSL07) were cultured.

5 ml of the above strain was added to a 250 ml Erlenmeyer flask at 25-30 ° C under pH 7.2-7.6 for incubation for about 20 days in a fluorescent stirrer. The stirring was maintained at 150 rpm and the illuminance was kept at 50 μmol / m 2 -sec.

In addition, the salinity of the wastewater was varied in the range of 0-400 mM (0-25 ‰) as shown in Table 4 below.

Parameters Influent pH 8.0 ± 0.1 Total nitrogen (TN) (mg L ^-1 ) 18 ± 0.3 Total phosphorus (TP) (mg L ^-1 ) 1.4 ± 0.1 Total inorganic carbon (TIC) (mg L ^-1 ) 33.4 ± 0.3 Chemical oxygen demand (COD) (mg L ^-1 ) 63 ± 0.9 Sulfate (mg L ^-1 ) 30 ± 1 Nitrate (mg L ^-1 ) 32 ± 0.1 Chloride (mg L ^-1 ) 68 ± 0.2 Calcium (mg L ^-1 ) 29 ± 0.3 Sodium (mg L ^-1 ) 23 ± 0.2 Potassium (mg L ^-1 ) 8.8 ± 0.1 Magnesium (mg L ^-1 ) 4.0 ± 0.1 Boron (mg L ^-1 ) 0.4 ± 0.1 Iron (mg L ^-1 ) 0.1 Manganese (mg L ^-1 ) 0.1 Barium (mg L ^-1 ) 0.1 Zinc (mg L ^-1 ) 0.04 Aluminum (mg L ^-1 ) 0.04 Copper (mg L ^-1 ) 0.02

Classification (Wastewater) NaCl dose (mM) 0 50 100 200 300 400 Total salinity (‰) 0.2 3.2 6.1 11.8 19.7 25.1 Conductivity (mS cm ^-1 ) 0.7 0.9 2.0 3.7 6.5 11.1

As in Example 1, the microbial culture culture medium for 20 days showed a growth rate of microalgae up to a concentration of 0-25 mM, but decreased from 50 mM (Fig. 1A). The maximum growth rate was measured at 0.8 g / L at 25 mM salinity, and the dry weight was increased 6-7 times higher than that of liquid medium (BBM) without salinity.

Also, as in the case of Example 2, the growth rate was increased from 0 to 200 mM for 10 days according to the saltiness condition in the wastewater, but decreased from 300 mM (Fig. 1B). The maximum growth rate was measured as 0.8 g / L at 100-200 mM salinity and the dry weight increased up to 3 times more than the wastewater condition without salinity condition.

As described above, microalgae grow in response to the osmotic phenomenon due to a defense mechanism against a high concentration of salinity, and the growth rate is reduced due to inhibition of light-induced photosynthesis by the salt concentration above the growth limit. On the other hand, the Na ⁺ ion enhances the uptake of bicarbonate ions, which are important for photosynthesis.

Experimental Example 1. Determination of lipid content and fatty acid composition according to salinity concentration

Fatty acid content and composition were determined by Lepage and Roy [Lepage, G., C.C. Roy (1984) Improved recovery of fatty acid through direct transesterification without prior extraction or purification, Journal of Lipid Research, 25, 1391-1396.

Mix RM3, Mix RM5, GLC50, GLC70 (Supelco, USA), heptadecanoic acid, gamma-linolenic acid (Supelco, USA] was used. A microalgae lipid sample, which had been mass-measured, was injected into a glass tube (11 mL, DH.GL28020, Daihan Scientific, Korea) with a teflon stopper, and 2 mL of chloroform-methanol (2: 1, vol / vol) Were mixed with a vortex mixer (Vorex Genius 3. Ika, Italy). 1 mL (500 μg / L) of chloroform containing an internal standard substance nonadecanoic acid (Sigma Co., USA), 1 mL of methanol, and 300 μL of sulfuric acid were sequentially added to a glass tube, Mixed. The tube was placed in a constant-temperature water bath and reacted at 100 ° C for 10 minutes. The tubes were cooled to room temperature, and 1 mL of distilled water was added. The mixture was vigorously mixed with a mixer for 5 minutes, followed by centrifugation at 4,000 rpm for 10 minutes to separate the layers. The lower layer (organic phase) was sampled with a disposable PP syringe (Norm-ject, Germany) and subjected to gas chromatography with an automatic injector after filtration with a disposable 0.22 μm PVDF syringe filter (Millex-Gv, Millipore, USA) Agilent, USA).

As a result of the analysis, when cultured for 20 days according to the salinity condition in the liquid medium for microalgae cultivation as in Example 1, the lipophilic production was increased as well as the growth rate up to the concentration of 0-25 mM (FIGS. The maximum lipid content per g was 37% at 25 mM salinity, and the lipid content was about 2.5 times higher than that of BBM without salinity.

In case of fatty acid composition, the unsaturated fatty acid suitable for biodiesel is distributed at a relatively high level at 25 mM, so that it can be used as a constituent element of biodiesel (FIG. 3A). In addition, it can be seen that oleic acid (C 18: 1 n 9c) is highly distributed in fatty acids having relatively higher oxidation stability than unsaturated fatty acids under the condition of 10 mM salinity under the condition of 25 mM, so that high quality biodiesel production is possible.

Strain Medium Cultivation (day) NaCl dose (mM) Lipid content (%) C. mexicana GU732420 Liquid medium (BBM) 20 0 15 10 24 25 37 50 25 100 19

In addition, as shown in Example 2, the growth rate of the wastewater was increased by 10-200 days according to the salinity condition of the wastewater, and the lipid production was also increased (Fig. 3B and Table 6). The maximum lipid content per g was 38% under the salinity condition of 400 mM and the lipid content was increased about twice as much as that of the wastewater without the salinity condition. However, lipid production, the value of microalgae growth and lipid content, is the highest at 200 mM salinity.

In the case of fatty acid composition, about 70% or more of unsaturated fatty acids suitable for biodiesel are relatively high (palmitic acid (27-31%), heptadecenoic acid (13-18%), oleic acid 7%), linolelaidic acid (17-20%), and linolenic acid (28-32%). On the other hand, the change in the composition of fatty acids is less than the result of microalgae culture (BBM).

Strains NaCl dose (mM) Medium Lipids (%) Chla . mexicana
(GU732420) 0 Wastewater 17 50 27 100 28 200 29 300 33 400 38

Thus, the microalgae produce glycerol in response to the osmotic phenomenon due to the defense mechanism at high concentration of salinity and accumulate in the body with triacylglycerols (TAG) together with other inorganic carbon and nutrients, .

Experimental Example 2. Confirmation of Nitrogen and Phosphorus Removal in Wastewater and Salinity Condition

Algae in different salinity conditions were seeded to examine nitrogen and phosphorus removal from algae obtained by the above Examples 1 and 2 into wastewater after MF (microfiltration). Each of the reaction vessels was maintained at room temperature of 25 to 27 DEG C and the pH was maintained at 7.8 to 8 in a natural state and proceeded to a batch experiment. It took about 10 days for the initial adaptation of the post - breeding birds, and subsequent removal of nitrogen and phosphorus occurred irrespective of the initial concentration of wastewater. In Fig. 4, nitrogen, phosphorus and inorganic carbon were removed at 100, 80 and 68%, respectively, at a salinity condition of 200 mM 10 days after the algae reaction proceeded.

Claims (6)

The present invention relates to a method for culturing freshwater microalgae containing lipid by culturing a freshwater microalgae culture medium in a microalgae culture medium having a controlled salinity,
The microalgal culture medium is a method of culturing freshwater microalgae for biodiesel production, characterized in that the salinity is adjusted to 25-200 mM NaCl (2-12 ‰). The method of claim 1,
The microalgal culture medium is a wastewater, cultivation of freshwater microalgae for biodiesel production, characterized in that at least one selected from livestock wastewater, agricultural wastewater, industrial wastewater, sewage sludge, purified water sludge, sewage treatment plant influent and sewage treatment plant effluent. Way. The method of claim 1,
Wherein the control of the salinity is carried out by mixing salt in the seawater or the desalination filtrate into the microalgae culture medium to regulate the salt content of the microalgae. The method of claim 1,
The culture medium containing the freshwater microalgae strains is placed in a fluorescent stirrer and cultured at a culture temperature of 25-30 ° C, pH 7.2-7.6, light intensity 40-60 μmol / m 2 -sec and stirring speed 100-200 rpm Wherein the method comprises the steps of: (a) cultivating freshwater microalgae for biodiesel production. The method of claim 1,
The method for culturing freshwater microalgae is characterized by producing fresh water microalgae containing lipids and removing nitrogen, phosphorus, organic carbon and inorganic carbon from the microalgae culture medium. Lt; / RTI &gt; The method of claim 1,
The freshwater microalgae strains include Chlamydomonas mexicana and Botryococcus brownies. braunii ), Chlorella species ( Chlorella . sp.) and Sendesmus species ( Scedesmus . sp.) is a culture method of freshwater microalgae for biodiesel production, characterized in that at least one selected from.

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