KR20200058862A - Cultivating method of microalgae using titanium dioxide nano particles - Google Patents

Abstract

The present invention relates to a method for culturing microalgae by using titanium dioxide (TiO_2) nanoparticles and culture medium additives for culturing microalgae. More particularly, it has been confirmed that Synechocystis 6803 cells of microalgae, which are cultured in a culture medium treated with titanium dioxide (TiO_2) nanoparticles, have an effect of increasing an amount of producing phycobiliproteins and thylakoid membrane lipids without a change in cell growth compared to a control group so the method for treating titanium dioxide (TiO_2) nanoparticles may be provided as a method for culturing microalgae, which can easily mass-produce phycobiliproteins at a relatively low cost.

Description

Culture method of microalgae using titanium dioxide nanoparticles {Cultivating method of microalgae using titanium dioxide nanoparticles}

The present invention relates to a method for culturing microalgae using titanium dioxide (Titanium dioxide; TiO ₂ ) nanoparticles and a medium additive for culturing microalgae.

Microalgae are producers of marine ecosystems and are single-celled organisms that photosynthesize themselves, reaching hundreds of thousands of species and producing various bioactive substances according to their growth environment. In particular, microalgae that grow in extreme environments, such as deep seabeds or lava fields, have found new functional substances that are not found on land.

These microalgae can produce stable biomass due to high photosynthetic efficiency that can fix up to 10% of the incoming solar energy per unit area, and produce about 14 million tons per year due to their availability as food and biofuels worldwide. It is becoming a trend that the possibility of utilization as a material of high value-added useful substances such as functional foods, medicinal substances, and feed as well as biofuels is gradually increasing.

In particular, efforts to develop into health foods, cosmetics, medical materials and medicines utilizing natural bioactive substances such as antioxidant enzymes, carotenoids, picobili proteins, chlorophyll, lutein, and astaxanthin are being actively conducted worldwide.

Picobiliproteins (phycobiliproteins) are light collecting proteins with the structure of open ring tetrapyrrole, produced by cyanobacteria, red algae and silver flagella. It is present in the intracellular thylakoid membrane, and is a water-soluble protein and is classified into phycocyanin (C-PC), phycoerythrin (PE), and allophycocyanin (APC) according to absorbance.

Picobili protein has a rich color and is a water-soluble protein, so it can be used as a natural colorant in food, cosmetics, and medicine. Among them, phycocyanin is used as a natural pigment and fluorescent marker in ice cream and cosmetics, and research on antioxidant, anti-inflammatory and anti-cancer effects has been reported.

However, the cultivation of picobili protein is difficult and the protein content in the cell is not high, so the production cost is high and industrialization is difficult. In order to solve these problems, research and development is needed to increase the content of picobili proteins in cells through strain improvement and development of a culture process.

Korean Patent Publication No. 10-2016-0045168 (published on April 27, 2016)

The present invention is to provide a method for culturing microalgae in a medium containing titanium dioxide (TiO ₂ ) nanoparticles and a medium additive for culturing microalgae in order to increase the content of picobili proteins in microalgal cells. .

The present invention provides a method for culturing microalgae comprising culturing microalgae bacterial cells in a medium containing titanium dioxide (TiO ₂ ) nanoparticles.

In addition, the present invention provides a medium additive for culturing microalgae containing titanium dioxide (Titanium dioxide; TiO ₂ ) nanoparticles as an active ingredient.

According to the present invention, microalgae synocytis 6803 cells cultured in a medium treated with titanium dioxide (TiO ₂ ) nanoparticles have an effect of increasing the lipid production of the picobiliprotein and thylakoid membranes without changing cell growth compared to the control group. As confirmed, the titanium dioxide (TiO ₂ ) nanoparticle treatment method may be provided as a microalgal culture method that can easily mass-produce picobili proteins at a relatively low price.

1 is a result of confirming the growth of Synocysis 6803 cultured in the control and TiO2 treatment conditions.

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

The present inventors are studying a method for increasing the production of a low content of picobili protein in microalgal cells. The microalgal culture method through the treatment of titanium dioxide (TiO ₂ ) nanoparticles is easily available at a low price. The present invention was completed by confirming that it exhibits an effect of improving the production amount.

The present invention can provide a method for culturing microalgae comprising culturing microalgae cells in a medium containing titanium dioxide (TiO ₂ ) nanoparticles.

The titanium dioxide (TiO ₂ ) nanoparticles may be included in 10 ^-5 to 10 ^-1 parts by weight based on 100 parts by weight of the medium.

The microalgae cells may be cultured for 1 to 20 days in a medium containing titanium dioxide (TiO ₂ ) nanoparticles.

The microalgae microbial cell may be selected from the group consisting of the genera Synechocystis sp., Chlorella sp., Dunaliella sp. And Chlamydomonas sp. .

More preferably, it may be Sinechocystis PCC 6803 (Synechocystis sp.PCC 6803).

The microalgae culture method can increase the production of phycobiliproteins, chloroplasts and lipids in microalgae cells.

The picobili protein may be selected from the group consisting of phycocyanin (PC), phycoerythrin (PE), and allophycocyanin (APC).

The lipid may be selected from the group consisting of phosphatidylglycerols (PGs), monogalactosyldiacylglycerols (MGDGs) and sulfoquinovosyldiacylglycerols (SQDGs).

According to an embodiment of the present invention, picobiliproteins (PC, APC, and PE) in the Synocysis 6803 culture on the 8th and 16th days after treatment with TiO ₂ nanoparticles (25 mg / L) in the Synocysis 6803 cells As a result of confirming the production amount, as shown in Table 1, in the experimental group on the 8th day after treatment with TiO ₂ nanoparticles, the contents of phycocyanin (PC) and allophycocyanin (allophycocyanin, APC) in the picobili protein were 33.8% and 55.0%, respectively. Significantly increased, it was confirmed that the APC content increased by 22.4% on the 16th day after the TiO ₂ nanoparticle treatment. In addition, the phycoerythrin (PE) content increased slightly on the 8th and 16th days after treatment with TiO ₂ nanoparticles.

PC and PE of the present invention is located in the picobilisome rod, producing both and transferring energy to APC, APC having alpha and beta subunits collects energy as the subunit and transfers it to chlorophyll in the thylakoid membrane As a very important central component of the picobilisome that plays a role, it was confirmed that the TiO ₂ nanoparticle treatment according to the present invention greatly influences the process of transferring energy to chlorophyll and increases APC content. PE is a highly flexible picobili protein, a result of organ adaptation to environmental changes.

In addition, according to another embodiment of the present invention, as a result of comprehensive quantification of metabolites of Synecoctistis 6803 cells cultured by treating TiO ₂ nanoparticles and control Synecosistis 6803 cells by GC-MS analysis, the table A total of 34 lipid species were identified as 3, especially MGDGs on day 8 (18: 2/18: 3, 18: 2/18: 2, 18: 1/18: 2), PG (16: 0/16: 1) ) And SQDGs (16: 0/16: 1, 16: 0: 18: 4).

The MGDG, SQDG and PG of the present invention are known to be the main components of the thylakoid membrane in which photo-dependent photosynthesis is achieved in cyanobacteria and higher plants.

In addition, the present invention can provide a medium additive for culturing microalgae containing titanium dioxide (Titanium dioxide; TiO ₂ ) nanoparticles as an active ingredient.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example  1> culture conditions and TiO ₂ NPs  process

Synechocystis sp.PCC 6803 was obtained from the Pasteur Culture Collection, Pasteur Institute (Paris, France).

The strain was cultured at 30 ± 1 ° C. at 120 rpm using a shake incubator (NEX220SRL, Nexus Technologies, Seoul, Republic of Korea), and during the incubation period, 40 μmol photon m ^-2 s ^-1 fluorescent light was lowered for 16: 8 hours. / Investigate at night cycle.

Metal salt mixture A5 (trace metal mix A5) with cobalt 1 mL / L, ammonium iron citrate 6 mg / L, Na ₂ CO ₃ 20 mg / L and K ₂ HPO ₄ 30.5 mg / L Cells were cultured in blue-green medium (BG11, Sigma-Aldrich, St. Louis, MO, USA) and subcultured with fresh medium every week in the usual way to transfer 500 μL of stock culture into a 200 mL Erlenmeyer flask in a 200 mL Erlenmeyer flask Was inoculated.

To process TiO ₂ nanoparticles (NPs), 500 μL of stock culture was inoculated into 2.5 mL of TiO ₂ nanoparticle stock solution in a 250 mL Erlenmeyer flask and 100 mL of medium containing the same amount of distilled water as a control.

The TiO ₂ nanoparticles were obtained from Sigma-Aldrich (CAS No. 1317-70-0, particle size: <25 nm, anatase), and 1 g / L of a TiO ₂ nanoparticle stock solution was added to distilled water and ultrasonicator. (JAC-2010, Kodo, Hwaseong, Korea) was used to sonicate for 1 hour to prepare a nanoparticle dispersion.

Experiment before (day 0) was a nano-TiO ₂ particles are added to 25 mg / L concentration of the nanoparticle dispersion (2.5 mL) to TiO ₂ nanoparticles treated experimental group, control group was added to an equal volume of distilled water.

Each treatment group was repeatedly performed three times, and after treatment with TiO ₂ nanoparticles, Synocysis 6803 cells were collected on Day 0 (control), Day 8, and Day 16.

The collected cells were dried using a freeze dryer (FDU-1200, EYELA, Miyagi, Japan) and stored at -80 ° C for further analysis.

< Experimental Example  2> Confirm cell growth

To confirm the effect of TiO ₂ nanoparticles through the growth analysis of Synocysis 6803 cells, 200 μL of cell culture was transferred to a 96 well plate (Corning Inc., Corning, NY, USA) and a microplate spectrophotometer (xMarkTM, BIO) -RAD, California, USA) to confirm optical densities (OD) at 730 nm.

OD was measured three times daily, and calculated based on the average of biological and analytical replicates.

< Experimental Example  3> Check chlorophyll content

According to the previously reported method (18), the chlorophyll content in the Synecesistis 6803 culture was confirmed.

Briefly, 1 mL of the cell suspension of each sample was centrifuged at 4 ° C. and 10,000 × g for 10 minutes to remove the supernatant and to obtain a pellet.

After adding 0.9 mL of methanol to the pellet, cooled at 4 ° C. for 1 hour, centrifuged at 4 ° C. and 10,000 × g for 10 minutes, and the chlorophyll content was calculated using the absorbance of the methanol extract at 665 nm and the following formula.

< Experimental Example  4> Picobili protein ( Phycobiliproteins ) Check the content

According to the previously reported method (Hong, S. J. et al., Biotechnol. Bioprocess Eng. 2008), the picobiliprotein content in the Synocysis 6803 culture (1 mL cell suspension) was confirmed. The contents of phycocyanin (PC), allopicocyanin (APC) and phycoerythrin (PE) were calculated using the following equations 2, 3 and 4.

< Experimental Example  5> GC -Comprehensive metabolite identification using MS

According to the previously reported method (Kim, JY et al., PLoS One 2017), the freeze-dried Synocysis 6803 culture was weighed into a sample, and the total metabolism of each 10 mg sample was measured using GC-MS. Material analysis was performed.

< Experimental Example  6> DI -Confirmation of lipoma using MS

According to the previously reported method (Kim, SH et al., Food Chem. 2016), freeze-dried Synocysis 6803 cultures were weighed into samples and pure lipomas of 5 mg of samples each using DI-MS. Was analyzed.

< Experimental Example  7> Trace element identification using ICP-MS

An inductively coupled plasma mass spectrometry (ICP-MS) was measured using a NexION 350D ICP-mass spectrometer equipped with an argon plasma operating at 6000K to determine the content of Fe, Mg, Ti, Mn, and Zn in Synecesis 6803 cells. Confirmed.

For each analysis, 10 mg of freeze-dried Synocysis 6803 cells were added to 8 mL of nitric acid to digest for 30 minutes and adjusted to a final weight of the pretreated sample of 15 g.

Finally, before ICP-MS analysis, all samples were diluted 100-fold with respect to Fe and Mg, and Ti, Mn and Zn were diluted 10-fold to minimize the influence of the matrix and prevent salt formation.

Each sample was measured twice and the data values are expressed as mean ± standard deviation (SD).

< Example  1> TiO ₂  Confirm the effect of cell growth and chlorophyll content increase by nanoparticle treatment

In Synocystis 6803 cell growth, changes were checked daily for 16 days to confirm the effect of TiO ₂ nanoparticle treatment.

As a result, as shown in FIG. 1, there was no difference in growth characteristics between the treated cell group treated with TiO ₂ nanoparticles and the untreated control cell.

TiO ₂ nanoparticles exposed from the resulting cine Cauchy seutiseu was that it does not have a significant impact on the 6803 cell growth, which affect the TiO ₂ nanoparticles exposed in the study of the TiO ₂ nanoparticles exposure of 10 kinds of North American birds algae growth It is consistent with the research results that do not give.

In addition, it was confirmed in the change in the chlorophyll content of Synocysistis 6803 according to the TiO ₂ nanoparticle treatment.

As a result, as shown in Table 1, it was confirmed that there was no change in chlorophyll content of Synocysistis 6803 for the first 8 days after TiO ₂ nanoparticle treatment, while it was confirmed that chlorophyll content increased 62.2% after 16 days of treatment.

Since microalgae cells are trapped in large aggregates of TiO ₂ nanoparticles, their availability is limited and as a result, algal cell growth can be inhibited.

However, it was confirmed that the cultivation of Synocysis 6803 through the TiO ₂ nanoparticle treatment according to the present invention plays an important role in improving the function of chloroplast activity, thereby increasing the chloroplast content of Synocysis 6803.

< Example  2> TiO ₂  By nanoparticle treatment Picobili protein  Check the effect of increasing content

In order to confirm the effect of TiO ₂ nanoparticles on the Picobili protein content of Synocystis 6803, after treatment with TiO ₂ nanoparticles on Synocystis 6803 cells, picobilis in the Synocystis 6803 culture on days 8 and 16 Protein (PC, APC and PE) production was confirmed.

As a result, as shown in Table 1, compared to the control group, the amount of picobiliprotein production increased in the experimental group treated with TiO ₂ nanoparticles. In more detail, in the experimental group on the 8th day after the treatment with TiO ₂ nanoparticles, the contents of phycocyanin (PC) and allophycocyanin (allophycocyanin, APC) among the picobili proteins were significantly increased by 33.8% and 55.0%, respectively. On the 16th day after the TiO ₂ nanoparticle treatment, it was confirmed that the APC content increased by 22.4%. The phycoerythrin (PE) content was found to increase slightly, although not significantly, on the 8th and 16th days after the TiO ₂ nanoparticle treatment.

There are several possible explanations for the method in which the TiO ₂ nanoparticles have increased the content of picobili protein in the culture of microalgae.

First, as a protective reaction against oxidative stress by TiO ₂ nanoparticles, it may be to increase the content of picobiliprotein, an antioxidant, in the Synocysis 6803 cells.

Picobili protein is an important photosynthetic pigment protein that is present in microalgae and cyanobacteria and is known for its function as an antioxidant.

As TiO ₂ nanoparticles induce antioxidant stress in microalgal cells by generating reactive oxygen species, microalgae prevent cell damage by synthesizing antioxidant components such as pigments, glutathione, ascorbic acid, and phenolic compounds.

Second, as a compensation effect for photosynthesis inhibition according to the light blocking effect of the TiO ₂ nanoparticles, the content of picobili protein in the Synocystis 6803 cell may be increased.

Internalization of nanoparticles or cell surface binding may decrease the light utilization rate for photosynthesis, while increasing the photosynthetic pigment content that absorbs light to increase the light utilization rate.

Thus, the modulating action of TiO ₂ nanoparticles can promote protein utilization and increase the production of picobili proteins in the Synocystis 6803 cells.

< Example  3> TiO ₂  By nanoparticle treatment Synecesis  6803 Comprehensive metabolites and intact cells Geological species  Profile Check

Metabolic products of Synocystis 6803 cells cultured by treating TiO ₂ nanoparticles and control Synecesistis 6803 cells were comprehensively quantified by performing GC-MS analysis.

As a result, as shown in Table 2, 27 metabolites were identified and classified into different categories such as alcohol, amino acid, fatty acid, organic acid, and sugar.

Between the control group and the sample of the TiO ₂ nanoparticle treatment group, pyrrolidone carboxylic acid, threonine, linoleic acid, linolenic acid, pentadecanoic acid, lactic acid Eight metabolites, such as (lactic acid), succinic acid, and melibiose, showed a relative level difference, and in the Synocystis 6803 cell culture, each metabolite group was treated with TiO ₂ nanoparticles No change was seen.

In addition, through the DI-MS analysis, diversity of the intact lipid species in the Synocysis 6803 cells was confirmed in response to treatment with TiO ₂ nanoparticles.

As a result, referring to Table 3, a total of 34 lipid species were identified as follows: 5 phosphatidylglycerols (PGs), 1 phosphatidylinositol (PI), 7 monogalactosyldiacylglycerols (MGDGs), 3 digalactosyldiacylglycerols (DGDGs), 16 sulfoquinovosyldiacylglycerols (SQDGs) and 2 phytyl derivatives.

It was confirmed that the relative level of most pure lipid species was higher than that of the control group and the experimental group on the 8th and 16th days after the TiO ₂ nanoparticle treatment.

In particular, MGDGs the TiO ₂ nanoparticle treatment day 8 (18: 2/18: 3, 18 : 2/18: 2, 18: 1/18: 2), PG (16: 0/16: 1) and SQDGs ( The relative levels of 16: 0/16: 1 and 16: 0: 18: 4) were significantly increased.

In cyanobacteria and higher plants, MGDG, DGDG, SQDG and PG are known to be the main components of the thylakoid membrane, which undergoes photo-dependent photosynthesis.

It has been reported that MGDG constitutes the photosynthetic organ of Arabidopsis thaliana, and SQDG contributes to activity of photosystem II than photosystem I of Chlamydomonas reinhardtii, a green algae, and plays a role for chromosomal DNA replication in Synocysis 6803. It is known to perform.

In addition, PG is known to be an essential lipid species for the main photosynthetic activity of Synocysis, and a deficiency of PG has been reported to reduce chlorophyll content and inhibit oxygen evolution photosystem II in Synocysis PCC 7942 / ΔcdsA mutants41 , 42.

Similarly, increasing the chlorophyll content of a cine Cauchy seutiseu 6803 significantly in the experimental results of the invention TiO ₂ nanoparticles treated 16 days after it was confirmed that by increasing PG by the TiO ₂ nanoparticle treatment.

In addition, it was confirmed that the relative level increase of MGDG, PG and SQDG on the 8th day of culture was also related to an increase in the content of picobili proteins.

Photosynthesis is due to a complex interaction between a set of picobilisomes, acidic fats and coenzymes in a thylakoid membrane, and TiO ₂ nanoparticle treatment increases picobiliproteins (APC, PC) and acidic fats (MGDG, SQDG, and PG) By doing so, it was confirmed that it plays an important role in enhancing photosynthesis in the Synocysis 6803 culture.

The results are the first to confirm the correlation between the lipid species and the picobiliprotein and the change of the pure lipid species by treatment with TiO ₂ nanoparticles in Synecesistis 6803.

< Example  4> TiO ₂  Induced by nanoparticles Ti's Translocation  And Fe, Mg, Mn and Zn concentration changes

The concentrations of Ti, Fe, Mg, Mn, and Zn cells on the 8th and 16th day were checked using ICP-MS to confirm the effect of TiO ₂ nanoparticle treatment.

As a result, as shown in Table 4, it was confirmed that the Ti level in the cells was significantly increased on the 8th and 16th days after the TiO ₂ nanoparticles were treated in the medium.

From the above results, it was confirmed that Ti was translocated to Synocysis 6803 cells by treatment with TiO ₂ nanoparticles.

In a recent study, aggregation of TiO ₂ nanoparticles in microalgae and dislocation into cells were reported. In the present invention, the concentrations of Fe, Mg, Mn, and Zn were significantly reduced after 8 days of TiO ₂ nanoparticle treatment, and Fe, Mn And Zn was also reduced after 16 days.

Fe has been known to be an important factor in maintaining cell growth and picobili protein synthesis in microalgae.

Kuchmina's study reported iron binding enzyme as an essential enzyme in heme biosynthesis of picobili proteins, and Mg was also reported to be an important factor in increasing fatty acids in microalgae and biosynthesis of chlorophyll and MGDG. It has been confirmed that it plays an important role in the photosynthesis system of superalgae.

TiO ₂ nanoparticle treatment has been found to promote the use of Fe, Mg, Mn, and Zn to increase chlorophyll, picobiliprotein, and pure lipid species (mainly MGDG) levels and light system activity of Synocytistis 6803 cells. .

Since the specific parts of the present invention have been described in detail above, for those skilled in the art, it is clear that such specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

Method for culturing microalgae comprising culturing microalgae cells in a medium containing titanium dioxide (TiO ₂ ) nanoparticles. The method according to claim 1, wherein the titanium dioxide (TiO ₂ ) nanoparticles, microalgae culture method, characterized in that contained in 10 to ⁵ to 10 ^-1 parts by weight based on 100 parts by weight of the medium. The method according to claim 1, wherein the microalgae cells are cultured microalgae, characterized in that cultured for 1 to 20 days in a medium containing titanium dioxide (TiO ₂ ) nanoparticles. The method according to claim 1, wherein the microalgae is a group consisting of the genus Synechocytistis (Synechocystis sp.), Chlorella sp., Dunaliella sp. And Chlamydomonas sp. Microalgae culture method, characterized in that selected from. The method according to claim 1, wherein the method of culturing the microalgae is a method of cultivating microalgae, characterized by increasing the production of phycobiliproteins and lipids in microalgae cells. The method according to claim 5, wherein the picobili protein is selected from the group consisting of phycocyanin (phycocyanin, PC), phycoerythrin (PE), and allopicocyanin (allophycocyanin, APC) microalgae culture. Way. The method according to claim 5, wherein the lipid is selected from the group consisting of phosphatidylglycerols (phosphatidylglycerols; PGs), monogalactosyldiacylglycerols (MGDGs) and sulfoquinovosyldiacylglycerols (SQDGs). Microalgae culture method. Titanium dioxide (Titanium dioxide; TiO ₂ ) medium additive for culturing microalgae containing nanoparticles as an active ingredient.

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