KR101808429B1 - Device for microorganism isolation culture and re-use method of waste microbial culture medium - Google Patents

Abstract

The present invention relates to a microbial separation culture apparatus and a method for reusing a microbial culture waste medium, and more particularly, to a method for reusing waste microbes after cultivation of microbes, The present invention relates to a method for reusing a microbial culture waste medium and a microbial separation culture apparatus which can be used therefor.

Description

[0001] The present invention relates to a microbial separation culture apparatus and a method for reusing a microbial culture microbial culture medium,

The present invention relates to a microbial separation culture apparatus and a method for reusing a microbial culture waste medium, and more particularly, to a method for reusing waste microbes after cultivation of microbes, The present invention relates to a method for reusing a microbial culture waste medium and a microbial separation culture apparatus which can be used therefor.

Global warming is progressing rapidly due to the increasing concentration of greenhouse gases in the atmosphere since the industrialization era. Therefore, research and development for reducing carbon dioxide, which is the most important factor of global warming, is very urgent. To date, efforts have been made to solve the carbon dioxide capture and storage (CCS) by various methods. One of the most commonly used technologies is the chemical capture method, which is the core of carbon capture, but it has a disadvantage in that it consumes a lot of energy for regeneration of the rapidly saturated capture agent after converting other chemicals by reacting with carbon dioxide. There is also a physical absorption method, one of which is an ionic liquid, which is a liquid composed of large organic cations and small inorganic anions, making it an environmentally friendly process with little vapor pressure. The absorption heat for the carbon dioxide gas ionic liquid is about 11 kJ / mol, which can be considered physical sorption rather than chemical bonding. The ionic liquid has a high viscosity and low solvent loss, but has a disadvantage that the mass transfer is slow.

However, the biological CO2 reduction method proceeds through photosynthesis and is environmentally friendly as it has been based on the ecosystem of the earth until now. Particularly, the method of reducing the carbon dioxide using photosynthetic bacteria is more efficient than the plant having a long growth period because the process is easy to develop, the cultivation process condition is easy, It has an advantage that it can be easily cultured compared to algae in culture. It is also advantageous to use various molecular biological techniques.

More particularly, the present invention relates to a multi-photobioreactor and a method for cultivating a photosynthetic microorganism using the same. More particularly, the present invention relates to a multi-photobioreactor comprising at least one microbial growth A culture medium for culture; And a culturing zone provided in contact with the culturing area for cell growth and capable of producing useful products and at least one useful product producing area capable of containing a culture solution.

On the other hand, the waste medium that is discarded after the microorganisms have been cultured is classified as infectious waste and should be stored in the infectious waste collection container. Since the container containing the infectious waste is directly collected and handled by the authorized infectious waste disposal company, The treatment procedure is complicated, the treatment cost is high, and there is a problem of environmental pollution due to the carbon dioxide produced in the cultivation.

DISCLOSURE Technical Problem The present invention has been devised in order to solve the above problems, and it is an object of the present invention to provide a process for treating environmentally-friendly and troublesome waste medium as well as reduction of carbon dioxide through re- The present invention provides a method for reusing a microbial culture waste medium and a microbial separation culture apparatus that can be used therefor.

The present invention provides a method for reusing a microbial culture waste medium containing microbial cultivation of a photosynthetic bacterium in a pulmonary medium to produce carbon dioxide and biologically active substances and organic acids contained in the pulmonary medium.

According to a preferred embodiment of the present invention, the microorganism may include at least one member selected from the group consisting of Escherichia coli and Saccharomyces cerevisiae.

According to another preferred embodiment of the present invention, the medium is selected from the group consisting of YPD (Yeast extract 0.5-2 wt%, Peptone 1-3 wt% and Dextrose 1-3 wt%), LB (Luria-Bertani Yeast extract 0.1 0.1 to 1% by weight of yeast nigrogen base without amino acid, 0.1 to 1% by weight of casamino acid and 0.1 to 1% by weight of glucose (including 1 to 1% by weight, Tryptone 0.5 to 2% by weight and NaCl 0.5 to 2% ), Or M9 (0.5 to 5 g / l Na ₂ HPO ₄揃 7H ₂ O, 5 to 15 g / l KH ₂ PO ₄ , 1 to 10 g / l NaCl and 0.5 to 5 g / l NH ₄ Cl ).

According to another preferred embodiment of the present invention, the photosynthetic bacterium is selected from the group consisting of Rhodobacter sphaeroi mutant strain (deposit institution: National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, Accession No .: KACC91572P, deposit date: Aug. 12, 2010) And Rhodobacter sphaeroide. ≪ / RTI >

The present invention also relates to a microorganism incubator comprising a microorganism producing carbon dioxide; Bacterial incubators containing photosynthetic bacteria; And a connection culture for connecting between the microorganism incubator and the microbial culture incubator and allowing gas flow in the microbial incubator and the bacterial incubator; The present invention also provides an apparatus for separating and culturing microorganisms using microorganisms.

According to a preferred embodiment of the present invention, in the connection culture, carbon dioxide in the microorganism culture apparatus can be supplied to the bacterial incubator.

Further, the present invention relates to a method for producing an alcohol-producing strain, comprising the steps of: inoculating photosynthetic bacteria into a lung culture medium in which an alcohol-producing strain has been cultured, And further culturing an alcohol-producing strain in a cultivated waste medium to produce an alcohol; And a method of reusing the microbial culture waste medium.

According to a preferred embodiment of the present invention, the alcohol-producing bacterium may include at least one member selected from the group consisting of Escherichia coli and Saccharomyces cerevisiae.

According to another preferred embodiment of the present invention, the photosynthetic bacteria are selected from the group consisting of Rhodobacter sphaeroi mutant strains (deposited at National Academy of Agricultural Science, National Institute of Agricultural Science and Technology, Accession No .: KACC91572P, deposit date: Aug. 12, 2010) Bacterium, and bacterium spiroodide.

According to another preferred embodiment of the present invention, the cultivation, cultivation and further culture may be carried out at 25 to 35 ° C, 150 to 200 rpm and 10 to 150 hours under light conditions.

Hereinafter, terms of the present invention will be described.

The term "medium" of the present invention can be prepared to include an energy source, a carbon source, a nitrogen source, an inorganic salt, a growth factor and the like suitable for culturing microorganisms. Energy sources, carbon sources, nitrogen sources, inorganic salts, and growth factors suitable for culturing these microorganisms are known in the art. Specifically, Korean Patent Application No. 19950031490, Korean Patent Application No. 1019910018012, Korean Patent Reference can be made to those disclosed in Korean Patent Application No. 1019940001029, Korean Patent Application No. 019990050363, Korean Patent Application No. 1020060049936, and Jae-Beom Kim (Cultivation of High-Concentration Cell Oil of High Nucleic Acid Yeast Using Industrial Medium, 2001, Master Degree Thesis) . Such a medium may be a natural medium using natural materials such as milk, potato, blood, tomato crushed liquid, rice bran powder, soybean milk, molasses, or the like, or may be a synthetic medium artificially formed of nutrients, and may be a solid medium or a liquid medium.

The term "photosynthetic bacteria " of the present invention generally refers to bacteria that perform carbon assimilation using light energy, such as higher plants.

Further, the term "spent medium " of the present invention means a microorganism culture, and is a comprehensive meaning including not only the culture stock solution itself but also a liquid concentrate (i.e., concentrate) or a powder concentrate obtained by concentrating the culture stock solution.

In addition, the term "minimal medium " of the present invention means a medium in which at least one nutrient required by a bacterium is deficient and has a minimum necessary composition as a synthetic medium in which wild type cells of a certain organism can be proliferated .

The present invention can produce various useful physiologically active substances as well as reduction of carbon dioxide through reuse of discarded waste medium after cultivation of microorganisms. Therefore, the present invention can provide a highly economical microorganism culture which is eco- There is an effect of providing a method of reusing waste media and a microorganism separation culture apparatus which can be used therefor.

In addition, the present invention can provide a process system capable of immobilizing carbon dioxide produced in a microorganism in real time through separate culture of microorganisms, thereby obtaining a desired product from the microorganism, and at the same time, And a useful organic compound produced thereby can be obtained. Since this continuous organic process can be utilized as a carbon source or a nutrient source of Escherichia coli or Saccharomyces cerevisiae, the effect of reusing the waste medium is excellent .

1 is a cross-sectional view of a microbial separation culture apparatus according to a preferred embodiment of the present invention.
FIG. 2 is a graph showing the results of gas chromatography analysis of carbon dioxide through separate culture of saccharomyces cerevisiae and rhodobacter sphaero (RS: separation of rhodobacter speraceum and saccharomyces cerevisiae Culture, sis-S: Saccharomyces cerevisiae culture (control group), R-SD: Rhodobacter sphaeroide culture only (control group)).
FIG. 3 is a graph showing the results of gas chromatography analysis of carbon dioxide through separate culture of Escherichia coli and Rhodobacter sphaero (RE: separate culture of Rhodobacter sphaeroide and Escherichia coli, sis-E: E. coli only culture Control group), R-M9: rhodobacter sphaeroide only culture (control group)).
FIG. 4 is a graph showing the results of dry weight of separated cultures of rhodobacter sphaeroide and saccharomyces cerevisiae in terms of dry weight of cells obtained by culturing by the method of FIG. 2 (RS => R: R-SD => R: dry weight of Rhodobacter sphaeroide when Rhodobacter sphaeroid alone was cultured under the same conditions. , RS =>: dry weight of saccharomyces cerevisiae in a separate culture of rhodobacter sphaeroide and saccharomyces cerevisiae, sis-S => S: when only saccharomyces cerevisiae was cultured under the same conditions Dry weight of saccharose.
FIG. 5 is a graph showing the dry weight of the cells obtained by culturing in accordance with the method of FIG. 4, on the dry weight of the isolated cultures of Rhodobacter sphaeroides and Escherichia coli (RE => R: Rhodobacter sphaeroides And R: M9 => R: dry weight of Rhodobacter sphaeroid only when Rhodobacter sphaeroide was cultured under the same conditions, RE => E: Rhodobacter sphaeroide Eis = E: dry weight of Escherichia coli when Escherichia coli alone cultured under the same conditions)
FIG. 6 is a graph showing the results on the content of bacteriophyllophyll through separation cultivation of Saccharomyces cerevisiae or E. coli and Rhodobacter sphaeroide. (RE: Rhodobacter sphaeroide and Escherichia coli isolated culture, R-M9: , RS: separate culture of Rhodobacter sphaeroide and Saccharomyces cerevisiae, R-SD: cultivation of Rhodobacter sphaeroid alone under the same conditions)
FIG. 7 is a graph showing the results on the content of carotenoids through separate culture of Saccharomyces cerevisiae or E. coli and Rhodobacter sphaeroide (RE: Separation of Rhodobacter sphaeroides and E. coli, R-M9: , RS: separate culture of Rhodobacter sphaeroide and Saccharomyces cerevisiae, R-SD: cultivation of Rhodobacter sphaeroid alone under the same conditions)
FIG. 8 shows the results of analysis of glucose consumption rate according to the culture medium and strain of Saccharomyces cerevisiae in Experimental Example 2-1.
9 shows the results of analysis of the dry weight of saccharomyces cerevisiae according to the culture medium and strain of Saccharomyces cerevisiae in Experimental Example 2-2.
10 shows the results of analysis of ethanol production of Saccharomyces cerevisiae according to the culture medium and strain of Saccharomyces cerevisiae in Experimental Example 2-3.
Fig. 11 is a photograph showing the cultured state of Rhodobacter sphaeroide in the reused medium (used YPD: saccharomyces cerevisiae waste medium, used LB: coliform lungy medium).
FIG. 12 is a transmission electron microscope (TEM) image of Rhodobacter sprooid grown in the reused medium of FIG. 11 (used YPD: saccharomyces cerevisiae spent medium, used LB: coliform lung medium).
13 is a graph showing the results of analysis of the carbon dioxide abatement efficiency of Rhodobacter sphaeroide grown on a reused medium through gas chromatography.
14 is a graph showing the results for the concentration of malic acid according to Rhodobacter sphaeroi cultured on a reused YPD medium.
FIG. 15 is a graph showing the results for the concentration of lactic acid according to Rhodobacter sphaeroide cultured on reused LB medium. FIG.
16 is a graph showing the results of cell growth (dry weight) when SD medium and M9 medium as the minimum medium of Saccharomyces cerevisiae or Escherichia coli were re-used for culturing Rhodobacter sphaeroide (sist: Rhodobacter As a result of culturing in sistrom's media medium, which is a medium of speroid, used M9: medium in which Escherichia coli was cultured, used SD: medium in which Saccharomyces cerevisiae was cultured).
FIG. 17 is a graph showing the results of the content of the bacteriophilic chlorophyll according to FIG. 16 (sist: the medium which was used in sistrom's media medium used as Rhodobacter sphaeroide and used M9: Escherichia coli, used SD: The badge that cultivated Roman Ises Serve Bizet).
FIG. 18 is a graph showing the results of the content of carotenoids according to FIG. 16 (sist: a medium used for culturing Escherichia coli in a sistrom's media medium, Rhodobacter sphaeroide, used SD: A medium in which mycese serebesis was cultured).
19 is a graph showing the relationship between the concentration of glucose added, the concentration of produced ethanol and the concentration of ethanol produced when the saccharomyces cerevisiae was reused up to the last two times by reusing the waste medium after culturing in SD medium, A graph showing the results for efficiency.
FIG. 20 is a diagram illustrating the experimental procedure of FIG. 22; FIG.
FIG. 21 is a diagram illustrating the experimental procedure of FIG. 19.
Fig. 22 shows the results obtained by culturing Saccharomyces cerevisiae in an SD medium, using the SD medium used for culturing Rhodobacter sphaeroide, and then adding the glucose again to the medium to reuse the medium. A graph showing the results of the added glucose concentration, ethanol concentration, and ethanol production efficiency when reused.

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

As described above, the waste medium that is discarded after the microorganisms have been cultured is classified as infectious waste and should be stored in the infectious waste collection container. Since the container containing the infectious waste is directly collected and handled by the authorized infectious waste treatment company The treatment procedure of the waste medium is difficult, the treatment cost is high, and there is a problem of environmental pollution due to the carbon dioxide produced during the cultivation.

Accordingly, the present invention provides a method for reusing a microbial culture waste medium containing the step of inoculating photosynthetic bacteria into a pulmonary medium cultured with a microorganism to produce carbon dioxide reduction, a physiologically active substance and an organic acid contained in the pulmonary medium, Respectively. As a result, it is possible to produce various useful physiologically active substances as well as to reduce carbon dioxide by reusing the waste medium that is discarded after culturing the microorganisms. Therefore, it is possible to produce a very economical microorganism culture There is an effect of providing a method of reusing waste media and a microorganism separation culture apparatus which can be used therefor.

The method for reusing the waste medium is a method of reducing carbon dioxide by Rhodobacter sphaeroide in the waste medium by using carbon dioxide produced by culturing a microorganism containing Rhodobacter sphaeroid, which is a photosynthetic bacterium, in a waste medium as a carbon source, The production of rhodobacter sphaeroide-derived physiologically active substances and organic acids can be improved.

The medium of the present invention is not particularly limited as long as it includes a substance that can be added to a microorganism culture medium, but preferably includes yeast extract, tryptone, and peptone. YPD (containing 0.5 to 2% by weight of yeast extract, 1 to 3% by weight of peptone and 1 to 3% by weight of Dextrose), LB (Luria-Bertani; Yeast extract 0.1 to 1 wt% of yeast nigrogen base without amino acid, 0.1 to 1 wt% of casamino acid, and 0.1 to 1 wt% of SD (including 0.1 to 1 wt%, Tryptone 0.5 to 2 wt% and NaCl 0.5 to 2 wt% including glucose) or _{M9 (Na 2 HPO 4 · 7H} 2 O 0.5 ~ 5 g / l, KH 2 PO 4 5 ~ 15 g / l, NaCl 1 ~ 10 g / l , and _{NH 4 Cl 0.5 ~ 5 g /} l ). ≪ / RTI >

In addition, liquid media may be used, but are not limited thereto.

The microorganism that can be cultured by the medium is not particularly limited as long as it is a prokaryotic cell and / or a yeast, but preferably it may include a microorganism belonging to the genus Escherichia and / or yeast, more preferably a microorganism belonging to the genus Escherichia coli And may include Saccharomyces cerevisiae as an Escherichia coli and / or yeast microorganism.

The microorganisms Escherichia coli and yeast in the Escherichia coli are Saccharomyces cerevisiae, which is the most widely used industrial strains currently used in medicine, food, chemistry, energy, etc. Because it is widely used, and if it has similar culture and growth pattern based on it, it is considered that it can be fully utilized. Therefore, two most representative microorganisms are used in the present invention.

Furthermore, since the microorganisms produce carbon dioxide through growth, it is easier to analyze the reduction efficiency of the produced carbon dioxide and the production of the organic matter. Therefore, since the culture conditions are both anaerobic and aerobic, .

The photosynthetic bacterium is not particularly limited as long as it is an ordinary bacterium that carries out carbon assimilation using light energy such as higher plants. Preferably, the photosynthetic bacterium is Chlorobium sp., Chromatium sp. It comprises at least one of the group consisting of Rhodospirillum sp., Rhodopsendomonas sp., Rhodobacter sp. And Rhodopseudomonas sp. And more preferably a strain belonging to the group consisting of Rhodobacter sphaeroide mutant strain (Deposit Agency: National Institute of Agricultural Science, National Institute of Agricultural Science and Technology, Accession No .: KACC91572P, deposit date: Aug. 12, 2010) Or more species.

The above-mentioned Rhodobacter sphaeroide mutant strain (deposited by National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, Accession No .: KACC91572P, deposit date: August 12, 2010) was deposited with Rhodobacter sphaeroide (Depository: (N-methyl-N-nitro-N-nitrosoguanidine) as a parental strain by treating a strain of the present invention with a mutant strain (KCTC 1434, Compared to Rhodobacter sphaeroide, the growth promoting ability of biological and animal cells is improved, and it has remarkably excellent microbial physiological activity performance. The present invention is superior to other types of Rhodobacter sphaeroids in producing various organic materials using carbon dioxide, and thus exhibits an excellent effect in reusing waste media.

As can be seen from Experimental Examples 4 to 5, the Rhodobacter sphaeroide mutant strain (deposited at National Academy of Agricultural Science, National Institute of Agricultural Science and Technology, Accession No .: KACC91572P, deposit date: Aug. 12, 2010) It is preferable to reduce the amount of carbon dioxide contained in the waste medium by inoculating the waste in the culture medium, thereby improving the production amount of the biosurfactant and the organic acid, thereby reusing the waste medium.

The reduction of carbon dioxide in the waste medium is an effect of immobilizing carbon dioxide by using carbon dioxide generated from the culture of microorganisms containing Rhodobacter sphaerois in the waste medium as a carbon source. This is because the cell dry weight of Rhodobacter sphaeroide , It can be confirmed whether or not carbon dioxide produced during culturing the microorganism contained in the waste medium is used as a carbon source (see Experimental Example 4 and FIG. 13).

In addition, the production of physiologically active substances derived from Rhodobacter sphaeroides and organic acids can be improved based on reduction of carbon dioxide by Rhodobacter sphaeroides in the waste medium (see Experimental Example 5, Figs. 14 to 15).

The physiologically active substance is a physiologically active substance derived from Rhodobacter sphaeroides, and may preferably contain bacteriophilic chlorophyll and / or carotenoid.

If the organic acid is Rhodobacter sphaeroide-derived organic acid, the kind thereof is not particularly limited, but may be malic acid or lactic acid.

In addition, a microorganism incubator comprising a microorganism producing carbon dioxide; Bacterial incubators containing photosynthetic bacteria; And a connection culture for connecting between the microorganism incubator and the microbial culture incubator and allowing gas flow in the microbial incubator and the bacterial incubator; The present invention also provides an apparatus for separating and culturing microorganisms using microorganisms.

By directly immobilizing the carbon dioxide produced in the cultivation of the microorganism contained in the medium, the carbon dioxide in the waste medium is effectively reduced, and the carbon dioxide is supplied as a carbon source of the photosynthetic bacteria, thereby increasing the production amount of the microorganism-derived physiologically active substance and the organic acid .

FIG. 6 is a cross-sectional view of a microorganism separating and culturing apparatus according to Embodiment 1 of the present invention, which comprises a microorganism incubator 10 including a microorganism producing carbon dioxide; A bacterial incubator (20) comprising photosynthetic bacteria; And a connection culture path 30 connecting between the microbial culture device 10 and the bacterial culture device 20 and allowing the gas flow of the microbial culture device 10 and the bacterial culture device 20.

First, the microorganism incubator 10 cultivates a microorganism that produces carbon dioxide. The microorganism is not particularly limited as long as it is a microorganism that produces carbon dioxide. Preferably, the microorganism may include E. coli or saccharomyces cerevisiae.

The volume of the microorganism incubator is not limited, but may be preferably for a large-scale culture, and a sterilizable heat-resistant glass bottle or an Erlenmeyer flask may be used, but is not limited thereto.

In addition, the microorganism incubator 10 may further include an opening / closing means 21 for discharging carbon dioxide generated from the microorganisms to the flow path, thereby controlling the outflow of the carbon dioxide. The opening / closing means is not limited to the opening and closing means that normally controls the gas, but may preferably include a valve or a lever.

Next, the bacteria incubator 20 may further include an opening / closing unit 22 for introducing carbon dioxide generated from the microorganisms through a flow path. The opening / closing unit 21 is not limited to the opening / But may preferably include a valve or a lever.

Although the volume of the bacterial incubator is not limited, it may preferably be for a large-scale culture, and a sterilizable heat-resistant glass bottle or an Erlenmeyer flask may be used, but is not limited thereto.

Bacteria contained in the bacterial incubator are not particularly limited as long as they are ordinary photosynthetic bacteria that perform carbon assimilation using light energy, such as higher plants. Preferably, the bacteria include chlorobium sp., Chromatin 1 of the group consisting of Rhodospirillum sp., Rhodopsendomonas sp., Rhodobacter sp., and Rhodopseudomonas sp.), Rhodobacter sp. (Depository: National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, Accession No .: KACC91572P, deposit date: Aug. 12, 2010) and Rhodobacter sphaeroides ≪ RTI ID = 0.0 > and / or < / RTI >

Next, a connection culture furnace 30 is shown which connects between the microbial incubator and the bacterial incubator, and enables the gas flow of the microbial incubator and the bacterial incubator.

The connection culture furnace 30 is a passage for connecting the microorganism culture device and the bacterial culture device to the microbial culture device and the microbial culture device so as to allow the gas flow to flow therebetween. The length, the diameter and the material of the microbial culture device are not limited, Preferably a glass tube or a silicon tube.

The microorganism separating and culturing apparatus can directly reduce the carbon dioxide produced in the cultivation of the microorganism contained in the waste medium, thereby reducing the carbon dioxide in the waste medium. Using the carbon dioxide as the carbon source, the production amount of the physiologically active substance and the organic acid derived from Rhodobacter sphaeroide .

In addition, the microorganism separation culture apparatus can improve the culture of microorganisms and the culture of photosynthetic bacteria by introducing carbon dioxide produced by the microorganism culture into a bacterial culture apparatus and using the same for culturing photosynthetic bacteria.

Further comprising the steps of: culturing the microorganism producing carbon dioxide in a microorganism incubator; Culturing the photosynthetic bacteria in a bacterial incubator; And supplying carbon dioxide produced in the microorganism incubator to a bacterial incubator through a connection culture furnace. The present invention also provides a method for reusing microorganism culture waste media using the microorganism separation culture apparatus.

Thus, it is possible to provide a process system capable of immobilizing carbon dioxide produced in Escherichia coli or Saccharomyces cerevisiae in real time, and the two strains Escherichia coli or Saccharomyces cerevisiae Separate cultivation of Saccharomyces cerevisiae may allow continuous processing. Therefore, it is possible to obtain a desired product in E. coli or Saccharomyces cerevisiae, and at the same time, it is possible to obtain a useful organic compound which is produced by reduction of carbon dioxide in Rhodobacter sphaeroide, and also to recover such useful organic compound again from Escherichia coli or Saccharomyces A continuous process that can be utilized as a carbon source or a nutrient source of serebese is possible.

In the separation culture method, the carbon dioxide produced in culturing the microorganisms contained in the waste medium moves through a channel connected to the culture vessel in which rhodobacter sphaero is cultured, and the carbon dioxide is simultaneously immobilized by Rhodobacter spolide, It is the height. The immobilization of the carbon dioxide produced in culturing the microorganisms contained in the waste medium improves the production of the physiologically active substance derived from Rhodobacter sphaeroide and the organic acid, so that the waste medium can be reused (see FIG. 1).

Specifically, as shown in Example 1, the control group for the substantial reduction of carbon dioxide was inoculated with rosemary sphaeroide in the case of the medium in which the saccharomyces cerevisiae or the E. coli were not inoculated under the separate culture conditions, (See Figs. 2 to 3). As a result, when the Saccharomyces cerevisiae or Escherichia coli and Rhodobacter sphaeroide were separately cultured, it was confirmed that the carbon dioxide was decreased in comparison with the control group, and it was confirmed that the carbon dioxide produced by Saccharomyces cerevisiae or Escherichia coli This is the result of reduction by Rhodobacter sphaeroides.

On the other hand, in order to confirm whether or not the carbon of carbon dioxide was used as carbon source in immobilizing carbon dioxide produced in cultivation of Saccharomyces cerevisiae and Escherichia coli cultured in substantially Rhodobacter sphaeroideum, It was confirmed that the dry weight of the cells was further increased in the case where Lloyd and Saccharomyces cerevisiae or Escherichia coli were separately cultured together (see FIGS. 4 to 5). Therefore, the reason why the weight of Rhodobacter sphaeroide grown in the same medium is greater than the weight of Rhodobacter sphaeroid grown in the same medium can be judged as an increase in the dry weight due to the immobilization of more carbon dioxide because the difference is only the carbon dioxide concentration under the same conditions.

For the theoretical reasons, the CO2 fixation efficiency for reducing the carbon dioxide contained in the waste medium was higher than the CO2 fixation efficiency by the microbial separation culture by inoculating the photosynthetic bacteria into the waste microbial culture medium of Example 2, In fact, the efficiency of CO2 fixation by microbial separation cultures was higher.

Specifically, in the method for separating microorganisms and the method for reusing waste media as described in Example 2, carbon dioxide produced by culturing a microorganism contained in a waste medium is converted into carbon dioxide by Rhodobacter sphaero as a carbon source to increase carbon dioxide abatement efficiency by immobilizing carbon dioxide, Thereby increasing the production of physiologically active substances derived from Rhodobacter sphaeroide, thereby enabling the reuse of the waste medium. However, unlike the case of the waste medium re-use method of Example 2, the microorganism isolated culture method can be used by discharging the carbon dioxide produced during culturing the microorganisms contained in the waste medium to a culture container in which Rhodobacter sphaeroide is cultured through a communicated flow path And it is expected that it is possible to exclude the effect of interference between microorganisms because it is separated and cultured in each culture container. Therefore, the apparatus for separating and culturing microorganisms for reusing the waste medium and the method for separating the same using the same are very effective for reusing the waste medium.

Further, the separation culture of the microorganisms can be carried out by obtaining the desired product from microorganisms cultured together such as Escherichia coli or Saccharomyces cerevisiae, and simultaneously carrying out the carbon dioxide immobilization carried out through Rhodobacter sphaeroide and the resulting useful organic compound again It is advantageous as a system capable of continuous processing by being used as a carbon source or nutrient of a microorganism to be cultured.

In addition, the present invention relates to a method for culturing an alcohol-producing strain, comprising the step of inoculating photosynthetic bacteria into a lung culture medium in which alcohol-producing strains have been cultured, And further culturing an alcohol-producing strain in a cultivated waste medium to produce an alcohol; And a method of reusing the microbial culture waste medium.

First, photosynthetic bacteria are inoculated into the lung culture medium in which alcohol-producing strains have been cultured, and the lung culture medium is re-cultured.

The alcohol-producing microorganism is not particularly limited as long as it is a microorganism producing carbon dioxide. Preferably, the microorganism may include at least one selected from the group consisting of Escherichia coli and Saccharomyces cerevisiae.

The cultivation amount is not particularly limited as long as it is a condition for culturing photosynthetic bacteria, but it is preferably cultivated at 25 to 35 ° C and 100 to 250 rpm for 5 to 300 hours, more preferably 27 to 33 ° C and 150 To 200 rpm for 10 to 150 hours.

If the culture is carried out at a temperature lower than 25 ° C and / or higher than 35 ° C, the growth rate of the alcohol-producing microorganism may be inhibited or the production of the product may be inhibited.

If the culture is carried out at less than 100 rpm, the growth rate of the alcohol-producing bacteria may be inhibited or the production of the product may be inhibited. If the culture is performed in excess of 250 rpm, the growth rate of the alcohol- May be inhibited or excessive stirring may cause excessive bubbles to be generated.

If the culture is performed for less than 10 hours, there may be a problem that the growth of the alcohol-producing bacteria is insufficient or the production of the product is insufficient. If the culture is carried out for over 150 hours, the cells may be killed or the production of the product may decrease, A problem may arise.

The photosynthetic bacterium is not particularly limited as long as it is an ordinary bacterium that carries out carbon assimilation using light energy such as higher plants. Preferably, the photosynthetic bacterium is Chlorobium sp., Chromatium sp. It comprises at least one of the group consisting of Rhodospirillum sp., Rhodopsendomonas sp., Rhodobacter sp. And Rhodopseudomonas sp. And more preferably a strain belonging to the group consisting of Rhodobacter sphaeroide mutant strain (Deposit Agency: National Institute of Agricultural Science, National Institute of Agricultural Science and Technology, Accession No .: KACC91572P, deposit date: Aug. 12, 2010) Or more species.

According to another embodiment of the present invention, after the regeneration, the cells are removed, the pH is adjusted and filtered, and glucose is added to re-inoculate saccharomyces cerevisiae and cultured under the same conditions. As a result, the saccharomyces cerevisiae The higher yield of ethanol was obtained when the saccharomyces cerevisiae were cultured for the first time after cultivation of Rhodobacter sphaeroide in a medium which was reused to produce ethanol against the amount of glucose to be added (see FIG. 22).

In particular, in comparison with the control (Fig. 19), the yield of ethanol produced was higher than the value of the glucose concentration added, indicating that rooderabacter sphaeroide was produced in the production of ethanol by saccharomyces cerevisiae It seems that the organic matter was added to the medium and the saccharomyces cerevise acted as a carbon source when producing ethanol. In other words, by increasing the nutrient content of the SD medium by reusing the SD medium, which is the minimum medium in which the saccharomyces cerevisiae were cultured, to cultivate Rhodobacter sphaeroide, it is possible to further improve the efficiency of producing saccharomyces cerevisiae ethanol Can be obtained.

The higher ethanol production and yield were obtained by cultivating rhodobacter sphaeroide in the spent medium of saccharomyces cerevisiae cultured in a minimal medium other than the nutrient medium to prove that the method of reusing the waste medium of the present invention is effective will be.

As a result, the discarded waste medium is reused by using Rhodobacter sphaeroide, and it is more efficient in producing ethanol by the saccharomyces cerevisiae, and there is an eco-friendly effect in which the waste medium can be reused.

Next, an alcohol-producing strain is further cultured in a cultivated waste medium to produce an alcohol.

The alcohol-producing microorganism is not particularly limited as long as it is a microorganism producing carbon dioxide. Preferably, the microorganism may include at least one selected from the group consisting of Escherichia coli and Saccharomyces cerevisiae.

The alcohol may be a C ₁ to C ₅ alcohol, more preferably ethanol.

The additional culture is not particularly limited as long as it is a condition for culturing the microorganism, but it may be cultured at 25 to 40 ° C and 100 to 250 rpm for 5 to 150 hours, more preferably at 27 to 37 ° C and 150 To 200 rpm for 10 to 100 hours.

If the culture is carried out at a temperature lower than 25 ° C. and / or higher than 40 ° C., the growth rate of the alcohol-producing bacteria may be inhibited or the production of the product may be inhibited.

If the culture is carried out at less than 100 rpm, the growth rate of the alcohol-producing bacteria may be inhibited or the production of the product may be inhibited. If the culture is performed in excess of 250 rpm, the growth rate of the alcohol- May be inhibited or excessive stirring may cause excessive bubbles to be generated.

If the culture is performed for less than 5 hours, there may be a problem that the growth of the alcohol-producing bacteria is insufficient or the production of the product is insufficient. If the culture is carried out for more than 150 hours, the cells are killed or the product is reduced, A problem may arise.

According to one embodiment of the present invention, an alcohol producing strain produces ethanol through a fermentation process, and the ethanol can use a yeast (Saccharomyces cerevisiae) in the absence of oxygen. In addition, the yeast may produce ethanol as one of byproducts produced in the process of digesting glucose (C ₆ H ₁₂ O ₆ ) (metabolism), and may produce carbon dioxide (CO ₂ ) at the same time as the ethanol is produced .

The glucose is a central compound of carbohydrate metabolism, its utilization path is very complicated, and the path of decomposition as an energy source is particularly important. Glucose is first converted to glucose-6-phosphate by the action of hexokinase, which is then degraded to pyruvic acid. In aerobic conditions, it is decomposed into carbon dioxide and water via the TCA circuit. Glucose is broken down through this cell respiration to produce energy, and its energy is stored in the form of ATP. According to another embodiment of the present invention, since the energy is used for fermentation, respiration and the like, the step of adding glucose as an energy source for producing ethanol when the saccharomyces cerevisiae is regenerated in a waste medium May be included.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

[Example]

Example 1: Saccharomyces Serebee or E. coli and Rhodobacter Spearoid Immobilization of Carbon Dioxide through Separation Culture

Saccharomyces cerevisiae or E. coli produces carbon dioxide while growing. Therefore, a separate culture system connected with Rhodobacter sphaero was constructed so that these microorganisms can directly immobilize the carbon dioxide that they produce while growing.

1-1. Separation culture system

First, 60 ml of the sistrom's medium shown in the following Table 1 was placed in a 100 ml culture container, purged with argon gas for 7 minutes, and then purged with a mixed gas (nitrogen 95%, carbon dioxide 5%) for 20 seconds.

The culture broth was inoculated with 5% Rhodobacter sphaeroide, cultured at 30 ° C and 180 rpm for 24 hours, and Saccharomyces cerevisiae was cultured in SD (0.67% Yeast nigrogen base without amino acid, 0.5% casamino acid, 0.5% glucose), and a 25-mm silicone tube was connected to each of the culture vessels. Carbon dioxide produced upon cultivation of Saccharomyces cerevisiae was immediately transferred to Rhodobacter sphaeroide and immobilized And cultured at 30 ° C and 180 rpm under light conditions. In addition, these experiments were conducted by preparing three samples for accurate results.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a cross-sectional view showing an apparatus for separate culture of Rhodobacter sphaeroide and Saccharomyces cerevisiae or Escherichia coli.

KH ₂ PO ₄ 2.72 g (NH4) ₂ SO ₄ 0.5 g Succinic acid 4 g L-glutamic acid 0.1 g L-Aspartic acid 0.04 g NaCl 0.5 g Nitrilotriacetic acid 0.2 g MgSO ₄ .7H ₂ O 0.3 g CaCl ₂ .2H ₂ O 0.033 g FeSO ₄ .7H ₂ O 0.0002 g (NH ₄ ) ₆ Mo ₇ O ₂₄ (1% solution) 0.2 ml Trace element solution / 100 ml EDTA 1.765 g 0.1 ml ZnSO ₄ .7H ₂ O 10.95 g FeSO ₄ .7H ₂ O 5 g MnSO ₄ .H ₂ O 1.54 g CuSO ₄ · 5H ₂ O 0.392 g Co (NO 3) ₂ .6H ₂ O 0.248 g H ₃ BO ₃ 0.114 g Vitamin solution / 100 ml Nicotinic acid 1 g 0.1 ml Thiamine HCl 0.5 g Biotin 0.01 g Casamino acid 2 g Final volume 1 ℓ

1-2. Carbon dioxide fixation by separate culture system

The concentrations of carbon dioxide in the culture vessel were analyzed by gas chromatography by separately culturing the Rhodobacter sphaeroide and Saccharomyces cerevisiae or Escherichia coli for 12, 24, 48 and 72 hours.

The amount of carbon dioxide contained in the initial culture vessel was measured by GC (gas (gas)) method using a 60/80 carbonxen-1000 column (Supelco, Bellefonte, PA, USA) (DS 6200, Donam Instrument INC.), and the amount of carbon dioxide decreased with the incubation time.

Escherichia coli was also isolated from Rhodobacter sphaeroide in the same manner. The medium used for Escherichia coli was the M9 medium shown in Table 2 below. The control group for the reduction of the substantial carbon dioxide was compared with the case where the medium was not inoculated with Saccharomyces cerevisiae or Escherichia coli and the case where the medium was not inoculated with Rhodobacter sphaeroide under the conditions of the separate culture . The results are shown in Fig. 2 and Fig.

As a result, it was confirmed that carbon dioxide produced by Saccharomyces cerevisiae or Escherichia coli was reduced by Rhodobacter sphaeroides.

1M MgSO ₄ (sterile) 2 ml 20% glucose (sterile) 20 ml 1M CaCl ₂ (sterile) 0.1 ml M9 salts Na ₂ HPO ₄ .7H ₂ O 1.765 g 200 ml KH ₂ PO ₄ 10.95 g NaCl 5 g NH ₄ Cl 1.54 g Final volume 1 ℓ Sterilized Final volume 1 ℓ

1-3. Confirmation of carbon dioxide immobilization by separate culture system

On the other hand, the dry weight of the cells was measured in order to confirm whether roodobacter sphaerois used carbon of carbon dioxide as a carbon source for immobilizing carbon dioxide produced by saccharomyces cerevisiae and coliform culture.

The dry weight of each sample was taken at 10 ml from the culture medium and centrifuged at 7000 rpm at 4 ° C for 20 minutes. After collecting only the cells, the cells were washed twice with sterilized water, Lt; / RTI > for 48 hours. The results are shown in Figs.

As shown in Figs. 4 to 5, it was confirmed that the dry weight of cells was further increased when Rhodobacter sphaeroide and Saccharomyces cerevisiae or Escherichia coli were separately cultured together. Therefore, the reason why the weight of Rhodobacter sphaeroide grown in the same medium is greater than the weight of Rhodobacter sphaeroid grown in the same medium can be judged as an increase in the dry weight due to the immobilization of more carbon dioxide because the difference is only the carbon dioxide concentration under the same conditions.

Experimental Example 1: Saccharomyces Serebee or E. coli and Rhodobacter Spearoid Analysis of Bacteriophilic Chlorophyll and Carotenoids by Separation Culture

Bacterioclorophyll and carotenoid, which are physiologically active substances derived from Rhodobacter sprooid, were measured through co-cultivation of Rhodobacterium sp. Cultured in Example 1 through more carbon dioxide immobilization.

1-1. Identification of Bacteriophilus Chlorophyll

In order to measure the bacteriophilus, 10 ml of the culture was centrifuged at 3000 rpm for 10 minutes to recover only the cells. The cells were collected in a volume ratio of acetone: methanol: medium = 7: 2: The absorbance was measured at 772 ㎚ by UV spectrophotometer. The results are shown in Fig.

As can be seen in FIG. 6, higher concentrations of bacteriocin chlorophyll could be determined under conditions of rovovter spleoid isolated cultured with Saccharomyces cerevisiae or E. coli.

1-2. Identification of carotenoids

To measure carotenoid content, the culture was centrifuged at 10,000 rpm for 20 minutes, and the cells were washed twice with sterile water and lyophilized at -50 ° C for 48 hours. 3 M HCl was added to the dried cells, reacted at 28 ° C for 30 minutes at 100 rpm, and the supernatant was removed by centrifugation at 10000 rpm for 20 minutes. Acetone was added thereto, reacted at 28 ° C at 100 rpm for 30 minutes, centrifuged at 10000 rpm for 20 minutes, and the supernatant was recovered. The absorbance at 480 nm was measured by a UV spectrophotometer, and the content of carotenoid was confirmed using the following equation (1). The results are shown in FIG.

As can be seen in FIG. 7, higher concentrations of carotenoid could be determined under conditions of rovovter spleod isolated cultured with Saccharomyces cerevisiae or E. coli.

Experimental Example 2: Saccharomyces Serebee and Rhodobacter Spanish effect analysis of the strain difference Lloyd Mai access to the medium and the saccharide used in the connection of the separate culture

The medium contained in the SD medium, which is the minimal medium of saccharomyces cerevisiae used in the isolated culture of the cultivated Rhodobacter sphaeroi and saccharomyces cerevisiae cultured in Example 1, was 0.67 wt% yeast having no amino acid A medium containing 0.5 wt% casamino acid (BactoTM) and 0.5 wt% glucose was used as the culture medium.

Since casamino acid does not contain uracil and is a nutrient that can reinforce amino acids, nutritional requirement mutations in uracil are affected by growth even when casein hydrolyzate is added to the minimum medium .

The Saccharomyces cerevisiae [Matα pep4 :: HIS3 prb1 can1 his 3 ura 3-52] is an auxotrophic mutation for uracil and is affected by growth when there is no uracil in the medium. Therefore, we analyzed the effects of ethanol production, glucose consumption and carbon dioxide production on isolated cultures in which the saccharomyces cerevisiae was combined with rhodobacter sphaeroide. And to analyze the results obtained when pYES2 vector was transfected into Saccharomyces cerevisiae and Uracil strains, respectively.

2-1. Sakaromayses Analysis of Glucose Consumption by Serebee's Media Type and Strain

In SD medium, which is the minimal medium of saccharomyces cerevisiae used in the segregating culture of Saccharomyces cerevisiae and rhodobacter sphaeroide, the auxotrophic mutants for uracil, 0.67 wt% amino acid-free yeast nitrogen base (DifcoTM ) And 0.5% by weight glucose (using the same apparatus as in Fig. 1), and yeast nitrogen-free (DifcoTM) without 0.67 wt% amino acid, 0.5 wt% glucose and 0.5 wt% (Chung, BH, Seo, DJ & Nam, SW) High-level secretory production of recombinant human lipocortin- I by Saccharomyces cerevisiae. Proc Biochem 35: 97-101 (1999)) or pYES2 vector (Cat No. V825-20, Invitrogen Corporation, USA) The transformed Saccharomyces cerevisiae were ligated to each other.

The culture conditions and the method were the same as in Example 1, and the incubation time was 72 hours. After the final culture, the concentration of glucose in the cultured Saccharomyces cerevisiae was measured by HPLC (High-performance liguid chromatograph; Waters, MA, USA), and the glucose concentration And compared for the concentration of glucose consumed. The result is shown in Fig.

As shown in Fig. 8, in the case of saccharomyces cerevisiae (RS) which was cultured in connection with rhodobacter sphaeroide, regardless of the presence or absence of casein hydrolyzate of the medium, the type of saccharomyces cerevisiae And pYES2 transformation), it was confirmed that 100% of glucose was consumed.

However, in the case of wild-type Saccharomyces cerevisiae, in the case of not being cultured in combination with Rhodobacter sphaeroide (MS), the consumption rate of glucose was 78% in case of the medium containing no casein hydrolyzate and the casein hydrolyzate And 84% of the glucose consumption rate in the medium containing them.

On the other hand, in the case of Saccharomyces cerevisiae transformed into pYES2, the glucose consumption rate of 100% was confirmed even when the culture was not effected with rhodobacter sphaeroide because it was not affected by uracil.

Therefore, the wild type, a mutation in auxotrophic mutation of uracil in the cross-culture of Rhodobacter sphaeroide and Saccharomyces cerevisiae, had glucose consumption of less than 100% in the absence of uracil, It was judged that the connection cultivation of Cesserezevi had an effect of increasing the consumption rate of glucose.

As a result, the gas produced in the culture of rhodobacter sphaeroide is considered to be a factor affecting the growth of saccharomyces cerevisiae.

2-2. Sakaromayses Saccharomyces according to the type of medium and strain of Serebee Analysis of dry weight of serebese

After the final culture of Saccharomyces cerevisiae obtained in Experimental Example 2-1, 10 ml of the culture solution was centrifuged, and the cells were washed twice with distilled water collected only through the cells, and then the cells were dried through a freeze dryer and weighed Respectively. The results are shown in Fig.

As can be seen in FIG. 9, a higher dry weight was observed in the case of the cross-culture of Rhodobacter sphaeroide and Saccharomyces cerevisiae than in the case of no connection culture.

In particular, in the case of Rhodobacter sphaeroide not linked to saccharomyces cerevisiae cultured in a medium lacking casein hydrolyzate, the dry weight of saccharomyces cerevisiae was 0.14 g / l but the culture medium containing casein hydrolyzate In the case of Rhodobacter sphaeroide, which was not cultured with Saccharomyces cerevisiae cultured in the medium, the dry weight of Saccharomyces cerevisiae was 0.20 g / l.

As a result, it was judged by the effect of casein hydrolyzate. When the saccharomyces cerevisiae and rhodobacter sphaeroide were cross-cultured, the difference in dry weight of saccharomyces cerevisiae according to the presence or absence of casein hydrolyzate , Which is considered to be influenced by the difference in consumed glucose.

Further, in the case of transformed Saccharomyces cerevisiae, a higher dry weight was confirmed when cultured in combination with Rhodobacter sphaeroide. Therefore, a higher dry weight was confirmed when Rhodobacter sphaeroide and Saccharomyces cerevisiae were cross-cultured.

2-3. Sakaromayses Saccharomyces according to the type of medium and strain of Serebee Analysis of ethanol production of serebiza

After the final culture of Saccharomyces cerevisiae obtained in Experimental Example 2-1, the concentration of the culture solution in which the saccharomyces cerevisiae was cultured was measured by HPLC and the result was shown in FIG.

As can be seen in FIG. 10, in the case of saccharomyces cerevisiae cultured in connection with rhodobacter sphaeroide, higher ethanol could be confirmed than in the case of saccharomyces cerevisiae not linked to Rhodobacter sphaeroides. In particular, higher ethanol production was observed when cultured in a medium containing casein hydrolyzate than when cultured in a medium containing no casein hydrolyzate.

On the other hand, in the case of Saccharomyces cerevisiae transformed with pYES2, there was no significant effect on the presence or absence of the cultivation with Rhodobacter sphaeroide, since the amount of consumed glucose was the same, . However, in the case of saccharomyces cerevisiae, the connection culture of rhodobacter sphaeroide and saccharomyces cerevisiae affects not only the glucose consumption and dry weight but also the yield of ethanol production .

Therefore, it is considered that the saccharomyces cerevisiae cultivated in connection with Rhodobacter sphaeroide supplement or substitute necessary nutrients for growth inhibition due to lack of nutrients.

Example 2: Culture of rhodobacter sphaeroide mutants in re-used medium

Saccharomyces cerevisiae were cultured in YPD (Yeast extract 1%, Peptone-2%, Dextrose-2%) medium at 30 ° C and 180 rpm for 48 hours, and E. coli was treated with LB (Luria-Bertani; Yeast extract-0.5% , Tryptone-1%, NaCl-1%) at 37 ° C and 180 rpm for 24 hours.

After the cells were removed, the cells were filtered and sterilized. 30 ml of the cells were placed in a 50 ml culture container, and the mixture was purged with argon for 7 minutes. Then, a mixed gas (95% nitrogen, 5 carbon dioxide %) For 20 seconds.

A donor strain (donor agency: National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology, Accession No .: KACC91572P, deposit date: Aug. 12, 2010) or Rhodobacter sphaeroide : Microorganism Resource Center, Accession No .: KCTC 1434, deposit date: 1984) was inoculated at 5% and cultured at 30 ° C, 180 rpm and light condition for 36 hours, 84 hours or 132 hours. A cultured state of Rhodobacter sphaeroide mutant strain is shown in Fig.

As shown in Fig. 11, it was confirmed that the concentration of the Rhodobacter sphaeroi mutant strain cultured in the culture medium of Saccharomyces cerevisiae and Escherichia coli cultured in the culture medium was increased over time.

Experimental Example 3: Rhodobacter grown on reusable medium Speroid Transmission electron microscopy of mutant strains

The intracellular appearance of the Rhodobacter sphaeroi mutant strain cultured in Example 2 over time was examined. Specifically, a sample for a transmission electron microscope observation is produced as follows.

1) Saccharomyces cerevisiae and Escherichia coli cultured 10 ml of the culture broth was incubated for 36 hours, 84 hours, or 132 hours in the pulmonary culture medium, respectively, at 7000 rpm and 20 minutes at 4 캜 for 20 minutes Respectively.

2) The cells were washed twice with 2.5% glutaraldehyde, suspended in 2.5% glutaraldehyde, and reacted at 4 ° C for 2 hours to immobilize Rhodobacter sphaeroide mutant cells.

3) Centrifugation was performed and 2% agar solution was added to the collected cells, so that the cells and the agar solution were mixed and hardened. Thereafter, the cells were reacted with phosphate buffer (PBS, 1 M, pH 7.4) at 4 ° C for 10 minutes, washed twice, and reacted with 1% osmium tetroxide (OsO ₄ ) at 4 ° C for 1 hour and 30 minutes .

4) The reaction was repeated with phosphate buffer solution at 4 ° C for 10 minutes. After washing twice, 50%, 70%, 80% and 90% ethanol solutions were reacted for 15 minutes each, and 100% ethanol solution 15 minutes, three times.

5) Propylene oxide solution was reacted twice for 10 minutes, and then reacted for 1 hour 30 minutes in a mixture of propylene oxide: epon mixture at a volume ratio of 1: 1. Lt; RTI ID = 0.0 >% < / RTI > by volume.

6) Epon mixture solution for 2 hours, and then embedded. The mixture was incubated at 37 ° C for 24 hours, 45 ° C for 24 hours and 60 ° C for 48 hours.

7) Embedding was followed by microfabrication at a thickness of 60-100 nm using an ultramicrotome (Leica ultracut uct, Leica, Germany), followed by primary staining with uranyl acetate for 30 minutes , Washed, and then stained with lead citrate for 26 minutes, washed again, and observed with a transmission electron microscope (TEM: H-7650, Hitachi, Japan). The results are shown in Fig.

As shown in FIG. 12, the shape of PHB (polyhydroxybutyrate) having a round shape inside the cell was confirmed.

Experimental Example 4: Rhodobacter grown on reused medium Speroid Carbon dioxide reduction analysis of mutant strains

The effect of reducing the carbon dioxide by growth of the Rhodobacter sphaeroi strain cultured in Example 2 was examined.

Specifically, the amount of carbon dioxide contained in the first culture vessel due to the purging of the mixed gas (95% of nitrogen and 5% of carbon dioxide) was measured by using GC (gas) equipped with a 60/80 carbonxen-1000 column (Supelco, Bellefonte, (DS 6200, Donam Instrument INC.), and the amount of carbon dioxide reduced with the incubation time was measured.

As shown in FIG. 13, it was confirmed that carbon dioxide was reduced in both the LB medium and the YPD medium reused. As a result, it was confirmed that the carbon dioxide produced during cultivation of Saccharomyces cerevisiae and Escherichia coli was reduced by Rhodobacter sphaeroide mutant strains.

Experimental Example 5: Rhodobacter grown on reused medium The organic increase due to spheroid

It was confirmed that the nutrient content of the waste medium was increased by Rhodobacter sphaeroide (Depository: Microorganism Resource Center, Accession No .: KCTC 1434, Deposit date: 1984) grown in the reusable medium in Example 2 above.

Specifically, each of the re-used YPD medium and LB medium was subjected to HPLC (High-performance liguid chromatograph; Waters, Mailford, Japan) equipped with a column of Rspack KC-811 (diameter x height = 8 mm x 300 mm, Showa Denko, , MA, USA). The results are shown in Figs. 14 to 15. Fig.

As shown in Fig. 14, in the case of the YPD medium reused after cultivation of saccharomyces cerevisiae, the amount of malic acid in the medium was increased while Rhodobacter sphaeroide grew.

As shown in FIG. 15, it was confirmed that the amount of lactic acid was increased in the LB medium to be reused after culturing E. coli.

Example 3: Saccharomyces The minimum medium of Serebee or E. coli was applied to Rhodobacter 'S growth and its effect analysis when re-cultivation of the page Lloyd

After using LB and YPD media with relatively rich nutrients, it was possible to cultivate them when Rhodobacter sphaeroide was cultivated again in the waste medium. Therefore, evaluation of the possibility of sufficient culture of Rhodobacter sphaeroide and confirmation of the increase of nutrient content in the medium were confirmed even in the minimum medium which is less nutrient medium.

3-1. Growth of Rhodobacter sphaeroides in minimal media

The medium cultured in SD medium and M9 medium, which were the minimum medium of E. coli, and Saccharomyces cerevisiae in Example 1 was reused to cultivate Rhodobacter sphaeroide.

Specifically, the SD medium and the M9 medium used for the culture after the cultivation of Example 1 were centrifuged to remove the cells, and pure water was obtained, followed by filtering to remove foreign matter. Then, the pH of the culture medium was adjusted to 7.0 with KOH to adjust the pH of the culture medium. Then, 60 ml of the culture medium was placed in a 100 ml culture container, and the mixture was purged with argon gas for 7 minutes. Then, the mixture gas (95% nitrogen, 5% Lt; / RTI > for 20 seconds. Rhodobacter sphaeroide culture solution was inoculated at 5% in the medium purged with the above mixed gas, and cultured at 30 DEG C and 180 rpm in light condition. The culture was performed in the same manner using sistrom's medium as a control.

After the culture of the re-used medium and the control, the dry weight was confirmed by cell collection through centrifugation. The results are shown in Fig.

As shown in FIG. 16, when the dry weight of the cells was confirmed, Rhodobacter sphaeroide did not grow well in the reused M9 medium, but Rhodobacter sphaeroide was found to grow in the reused SD medium.

3-2. Analysis of Bacteriocin Chlorophyll and Carotenoids

The content of bacteriophilic chlorophyll and caroteroid according to the growth of Rhodobacter spolide in the minimum medium was measured in the same manner as in Experimental Example 1, and the measurement results are shown in Figs.

As shown in Figs. 17 to 18, the result was similar to that of Example 3-1 in terms of cell dry weight, and the content of bacteriorochlorophyll and carotenoid by Rhodobacter sphaeroide in the reused SD medium was also markedly increased I could confirm. However, in the case of the reused M9 medium, it is considered that the result is that the nutritional content is too low and the Rhodobacter sphaeroide is not sufficiently grown.

Example 4: Culture system

Saccharomyces cerevisiae is a representative ethanol producing strain. In this Example, SD medium, which is the minimum medium in which Saccharomyces cerevisiae was cultivated in Example 3, was reused for culturing Rhodobacter sphaeroide to increase the nutrient content of the SD medium, and again, saccharomyces cerevisiae And to obtain higher efficiency in the production of ethanol.

Specifically, the same SD medium as that of the SD medium of Example 1 and the saccharomyces cerevisiae were inoculated in a 100 ml culture container at 5%, and then cultured at 30 DEG C and 180 rpm for 48 hours. After the cells were cultured, the cells were removed by centrifugation. The pH was adjusted to 7.0 using potassium hydroxide, filtered, and then purged with argon gas for 7 minutes to culture Rhodobacter speroid. %, Carbon dioxide 5%) for 20 seconds, then inoculated with Rhodobacter sphaeroi culture 5%, and cultured at 30 ° C and 180 rpm for 48 hours. The cells were then removed in the same manner as described above, and the pH was adjusted using potassium hydroxide and then filtered.

Thereafter, 0.5% glucose was added, and the culture was inoculated with saccharomyces cerevisiae at a concentration of 5%, followed by cultivation at 30 ° C and 180 rpm for 48 hours. The experiment was conducted so as to be reused without discarding the medium 20).

As a control, Saccharomyces cerevisiae was cultured under the same conditions. After cultivation of Saccharomyces cerevisiae, the same conditions as those described above were used without culturing Rhodobacter sphaeroide, and then saccharomyces cerevisiae The non-yeast was cultured and the same procedure was repeated three times (see Fig. 19).

In order to confirm the re-use result of the reuse, the culture medium component analysis was analyzed using HPLC in the same manner as in Experimental Example 5 above. The results are shown in Fig. 20 and Fig.

As can be seen in Figures 19 and 22, saccharomyces cerevisiae was first cultured in a medium reusing to produce ethanol relative to the amount of glucose added, followed by saccharomyces cerevisiae for the first time The ethanol yield was higher than that when cultured.

Particularly, the yield of ethanol produced is higher than the value obtained by measuring the concentration of added glucose, so that the organic substance produced by Rhodobacter sphaeroide in the production of ethanol is added to the medium to produce saccharomyces cerevisiae, It is believed that cesium sorbic acid acts as a carbon source in the production of ethanol.

However, when the culture was re-cultured, the ethanol yield of the re-used medium was not increased due to the increase of the ethanol concentration. However, since the discarded medium is reused using Rhodobacter sphaeroide, and the ethanol production of the saccharomyces cerevisiae is further assisted, it is considered to be a more efficient and reusable environmentally friendly system.

Claims (3)

A microorganism incubator comprising a microorganism producing at least one carbon dioxide selected from the group consisting of Escherichia coli and Saccharomyces cerevisiae ;
Chlorobium sp., Chromatium sp., Rhodospirillum sp., Rhodopsendomonas sp., Rhodobacter sp., Rhodobacter sp., And Rhodobacter sp. And Rhodopseudomonas sp .; and a microbial cell culture system comprising a microbial cell culture system comprising at least one photosynthetic microorganism selected from the group consisting of Rhodopseudomonas sp. And
A connection culture which connects between the microorganism incubator and the microbial incubator and enables the gas flow of the microorganism incubator and the bacterial incubator;
Wherein the microorganism is cultured in a culture medium. The method of claim 1, wherein carbon dioxide in the microorganism culture apparatus is supplied to the microorganism culture apparatus. The method according to claim 1, wherein the bacterium belonging to the genus Rhodobacter is selected from the group consisting of a Rhodobacter sphaeroi mutant strain (deposited at National Academy of Agricultural Science, National Institute of Agricultural Science and Technology, Accession No .: KACC91572P, deposit date: Aug. 12, 2010) Wherein the microorganism is cultured in a culture medium.

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