JP2008199914A - Liquid culture medium and method for proliferating and culturing photosynthetic bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a separated liquid obtained by subjecting a stock solution of Sake lees to solid and liquid separation as a liquid culture medium for culturing and to provide a method for mass-proliferation and mass-culturing of photosynthetic bacteria difficult to selectively proliferate in a short period of time. <P>SOLUTION: The separated liquid obtained by mixing the diluted photosynthetic bacteria with the stock solution of the Sake lees, stirring the resultant mixture, aggregating and separating the mixture and carrying out at least one treatment of regulating the pH of the separated liquid after the solid and liquid separation with the photosynthetic bacteria, reducing the BOD, conducting a mixing treatment, centrifuging, filtering or ultraviolet irradiation is used as the liquid culture medium for culturing. Thereby, the method for proliferating and culturing the remaining photosynthetic bacteria while irradiating the liquid culture medium with rays of light mainly containing wavelengths of 400-700 nm is provided. A method for proliferating and culturing the added photosynthetic bacteria or symbiotic strains is provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a liquid medium obtained by solid-liquid separation of alcoholic beverages and a method for growing and culturing photosynthetic bacteria using them.

JP-A-8-103273 and JP-A-11-32755 (hereinafter referred to as Patent Document 1) states that “(Production of self-proliferating immobilized cells) has an acetate residue of about 30% and a degree of polymerization of about 10 % -Polyvinyl alcohol aqueous solution 2 liters, 20% potassium silicate aqueous solution 0.53 liters, 0.7 liters of a medium in which the following medium components are dissolved, Rhodoshoot Monas capsulatorus culture solution 0.3 The liters were mixed and stirred at the same time, and an ungel solution having a final pH of 8.3 was filled into a transparent plastic container having a depth of 5 cm and a diameter of 10 cm.
The medium is 5 g of sodium propionate, 1 g of ammonium chloride, 0.8 g of potassium phosphate, 0.2 g of magnesium chloride, 0.1 g of sodium chloride, 0.05 g of calcium chloride, 0.5 g of sodium bicarbonate, and yeast extract per liter. 0.2 g.
Soon after, the ungel solution gelled, and self-proliferating immobilized cells of gel-like microorganisms were obtained. When cultured under illumination for 5 days, self-proliferating immobilized cells of microorganisms that grew brightly on the medium in the gel were obtained. When the number of viable bacteria was measured, 10 ⁸ order self-proliferating immobilized cells were obtained. (Examples of Patent Document 1: paragraphs 0011 to 0013) ".

Japanese Patent Laid-Open No. 9-238681 (hereinafter referred to as Patent Document 2) states that “10 g of succinic acid, 10 g of malic acid, 5 g of ammonium sulfate, 8 g of monopotassium phosphate, 2 g of magnesium sulfate, 1 g of sodium chloride, 0.5 g of calcium chloride and 2 g of yeast extract. In a nutrient substrate (medium) dissolved in 1 l of water, Rhodopseudomonas capsulata (Chemical Laboratories No. 879) and Chromatium vinosum (Chromatium vinosum: No. 890) In addition to inoculating the body, 10 g of Oya stone grains having a diameter of about 57 mm having fine pores having a diameter of 1 to 3 mm and 10 g of leech stone grains having a diameter of about 5 mm having fine pores having a diameter of 1 to 3 mm are added, A liquid A was prepared.
A solution B having a pH of 1.1 was prepared by adding 0.8 l of a nutrient substrate having the same composition as that of the nutrient substrate (medium), 0.7 l of water and 15.2 g of acetic acid to 2 l of a 10% aqueous polyvinyl alcohol solution.
A solution C having a pH of 11.9 was prepared by adding 400 g of 10% potassium aluminum sulfate, 4 g of sodium hydrogen carbonate and 4 g of sodium thiosulfate to 3 l of water.
In a 200 ml transparent container, 40 ml of liquid A, 80 ml of liquid B and 80 ml of liquid C are added simultaneously with stirring and mixing. Further, 5 g of cellulose fiber filler is added, stirred, placed and placed under light illumination. -It culture | cultivated at 25 degreeC for 7 days, and obtained the gel-like fixed microbial cell which displayed the crimson color. (Patent Document 2, paragraphs 0020 to 0023) ".

Japanese Patent Laid-Open No. 11-243946 (hereinafter referred to as Patent Document 3) shows a specific example in which photosynthetic bacteria are cultured according to the present invention according to the present invention. Sodium propionate is placed in a 10-t culture tank. 0.1%, monopotassium phosphate 0.5%, dipotassium phosphate 0.06%, ammonium sulfate 0.1%, magnesium sulfate heptahydrate 0.02%, sodium chloride 0.02%, calcium chloride・ Dihydrate 0.05%, Bacto yeast extract (Pifico) 0.01%, vitamin solution (thiamine hydrochloride 50 mg, niacin 50 mg, paraaminobenzoic acid 30 mg, pyridoxy hydrochloride 10 mg, and pithione 5 mg were dissolved in 100 ml of distilled water. 1 ml / liter trace element solution (EDTA-2Na 1000 g, FeCl26H2O 2000 mg, ZnCl2 100 mg, MnCl2 · 4H2O 100 mg, H3BO3 100 mg, CoCl2 · 2H2O 100 mg, Na2MoO4 · 2H2O 20 mg, CuCl2 · 2H2O 10 mg, NiCl2 · 6H2O1 liter in Na2SeO5 solution 9000 liters of the medium containing the medium was added, and sterilization was repeated three times for 3 hours at 100 ° C. After the medium was cooled to 30 ° C., it was cultured in a separate culture tank (Rhobacter capsule) 3 g wet weight / liter Aseptically inoculated 900 liters of the inoculum, stirred at 100 rpm for 10 minutes, stopped stirring and cultured, stirred for 10 minutes a day, cultured for 10 days, 34 A bacterial cell having a wet weight of kg was obtained. (Example of Patent Document 3 paragraph 0017) "
“The sterilized liquid medium is inoculated with the inoculum. The amount of the inoculated bacteria is preferably in the range of 1 to 20% of the liquid medium, especially about 10%. After the inoculation, the stirring blade 6 is intermittently rotated. The agitation can be performed for about 10 minutes a day at a rotation speed of 50 to 100 rpm, and frequent agitation is carried out by photosynthesis with dissolved oxygen in the contents of the container (mixture of photosynthetic bacteria and liquid medium). It is preferable to prevent the growth of bacteria other than bacteria, and depending on the type of photosynthetic bacteria, nitrogen is aerated from the shaft 6a.
The culture period is about 5 to 10 days. During this culture period, the liquid medium is sterilized as described above, so that the photosynthetic bacteria in the container contents preferentially grow. (Patent Document 3, paragraphs 0015 to 0016) ".

  Japanese Patent Application Laid-Open No. 2000-287675 (hereinafter referred to as Patent Document 4) states that “If there is sunlight, it can be produced in a double cycle (summer 15th) ...”, “Culture ratio for 10 liters of water-soluble culture base” Culture-added water Tap water containing chlorine 0.1PPm to 0.3PPm per day Open air sterilization for 24 hours (exclusion of airborne bacteria) ammonium chloride 10g yeast extrax 1g sodium hydrogen carbonate 10g peptone 1g sodium acetate (anhydrous) 10g Sodium propionate 2 g Sodium chloride 10 g Ammonium sulfate 2.5 g Dipotassium hydrogen phosphate 2 g Urea 2.5 g Magnesium sulfate 2 g Enzyme 2 gdL-malic acid 2.5 g (Example) (Example of Patent Document 4) ”

JP 2002-171963 (hereinafter referred to as Patent Document 5) states that “the present invention is a lactic acid bacterium, a Bacillus bacterium, a yeast, or the like that is compatible with a photosynthetic bacterium, or any one or more types of fermenting bacteria that ferment sugars. This is a method of inoculating sharpening juice and pre-fermenting it for several hours in advance, then inoculating photosynthetic bacteria into this sharpening juice and culturing for several days while irradiating and stirring with low light. What other nutrients are added This is an innovative technique for growing photosynthetic bacteria at a high concentration without adding a small amount of colloidal montmorillonite clay mineral, which enables long-term storage of photosynthetic bacteria (Patent Document 5). Paragraph 0023) ",
[[Example 1] After 1000 l of tap water was poured into 5 g of rice sharpening powder and sterilized by a known method, the following fermentation bacteria were inoculated.
1. 1. Lactic acid bacteria (Lactobacillus biidos), 2. Bacillus subtilis (Bacillus subtilis), Yeast (Sahalomyces cerevisiae)
After fermenting at 27 ° C for 24 hours, the culture solution in which three kinds of photosynthetic bacteria, Rhodobacter capsiyulet, Rhodocycles gelatinos, and ectothiorhospirilum were separately inoculated into this fermented broth was irradiated with an incandescent lamp (illuminance 1000 lux). The culture was carried out. After 5 days, the presence or absence of bacterial growth was measured by the concentration of red color generated for 3 days. (Example of Patent Document 5: paragraphs 0026 to 0027) ".

In JP-A-8-192180 (hereinafter referred to as Patent Document 6), “360 ppm propionic acid: 2,100 ppm butyric acid: less than 10 ppm valeric acid: 110 ppm caproic acid: less than 10 ppm BOD: 4,010 ppm TOC: 2,250 ppm
94 mL of a medium solution containing a medium component consisting of the above a was placed in a 500 mL Erlenmeyer flask, and the Lactobacillus acidophilus [ATCC 111975], a Saccharomyces cerevisiae [IFO 0309] and a photosynthetic bacterium were added to the medium liquid. Various species of Rhodopseudomonas sphaeroides [ATCC 33575] (10 ⁴ to 10 ⁵ cells / mL) were inoculated, and anaerobic mixed culture was performed at 30 ° C. for 7 days. The number of viable bacteria in the culture solution is 5 × 10 ⁵ lactic acid bacteria / mL, 8 × 10 ⁵ yeast / mL yeast, 9 × 10 ⁵ photosynthetic bacteria / mL, 2 × 10 ⁶ total viable bacteria / mL, total The bacterial cell concentration was 1.0 g / L. This mixed culture solution is added to 3.9 L / 5 L of activated sludge water consisting of the above b, room temperature, aeration time 2 hours / day (dissolved oxygen concentration at the time of aeration 2-3 ppm), light irradiation (incandescent lamp 1500 lux) Acclimatize for 30 days with a light-dark cycle every 12 hours. (Example of paragraph 0018 of Patent Document 1) ".

Patent No. 2511325 (hereinafter referred to as Patent Document 7) states that “the water filling volume of each photoreactor is 2.5 liters, but the light and dark culture tanks of green algae are 1.25 liters each. Naturally, the dark culture tank was not supplied with light energy, and the two hydrogen generation tanks (both water-filled volumes of 2.5 liters) were only supplied with light energy and stirred by a directional impeller. The residence time of each tank with respect to the raw water was set to 5 hours (the green tank culture tank was a total of 5 hours for both light and dark tanks). Is 4,000 to 5,000 mg / l, and microalgae is 8,000 to 10,000 mg / l, respectively.
Light energy is supplied by a xenon lamp (only visible light) in order to obtain quantitative data, and the light energy supply speed is 10 KW / m ³ · hr per tank volume and 50 W / m per light emitting area of the optical fiber. ^It was fixed at 2hr. The continuous treatment experiment on artificial sewage in Table 1 is continued for one month after the bacterial cell concentration is maintained at a predetermined concentration in each tank and reaches a so-called steady state, and the supply rate of raw water during this period is constant at 12 liters / day. It was. In this experiment, Rhodobacter sp. And Chromatium sp. Were used as photosynthetic bacteria, Oscillatoria sp. Was used as the cyanobacterium, and Chlamydomonas sp. Was used as the green algae. (Patent Document 7, paragraphs 0027 to 0028)
Is described.

  Japanese Patent No. 3184970 (hereinafter referred to as Patent Document 8) states that “this anaerobic granular sludge is (i) an ordinary non-particulate non-particulate consisting of acid-fermenting microorganisms and / or methane-fermenting microorganisms and (ii) photosynthetic bacteria. The anaerobic sludge can be obtained by anaerobic culture in an upward flow manner and self-aggregation (granulation) In the anaerobic granular sludge used in the present invention, the average particle size is in the liquid in the digester. The concentration in the digestion bath is 30 to 60% by weight, preferably 40 to 50% by weight.

As described above, “The digester was kept at 35 ° C., and from the third day of culture, light was continuously irradiated with an incandescent lamp at an intensity of 100 μE / m ² / s (intensity on the liquid surface). On the 14th, 20th and 21st days, the digester was kept in dark conditions for 24 hours, and after the culture, a solution containing organic acid, ammonia, phosphoric acid and the like was added to the digester. The composition of the supplied solution is shown in Table 1. The absorption spectra of the digestive juice under dark conditions and light irradiation conditions were measured, and in the case of light irradiation conditions, growth was induced from granular sludge. An absorption spectrum peculiar to bacteriochlorophyll of a photosynthetic bacterium was observed (paragraph 0016 of Patent Document 8) ".

In Japanese Examined Patent Publication No. 45-28234 (hereinafter referred to as Patent Document 9), “COD4800 ppm BOD7000 ppm ammonia 5800 ppm black wool wash waste liquid 1.2 ton is transferred to an aerobic culture tank (b), and air is aerated at 20-100 L / min. Then, aerobic bacteria contained in the waste liquid were grown and cultured for 48 hours at 37 ° C., thereby obtaining a treatment liquid that decomposed the waste liquid and exhibited a yellow color rich in organic acids, with a COD of 4800 ppm, The BOD was 7000 ppm and the ammonia was 5800 ppm, and then the treatment waste solution was transferred to the anaerobic culture tank (c), and a culture solution of red sulfur-free bacteria (using Rhodopseudomonas genus Capsulatas) was added to the treatment solution at about 1%. The upper part of an anaerobic culture tank after being used for 96 hours under the condition of degree C, 2000-10000 lux lighting As a result, the COD of the treatment waste liquid was lowered to 260 ppm, BOD 230 ppm, ammonia 190 ppm, and the liquid state was transparent. 0.9 kg of bacterial cells was obtained (Patent Document 9, page 6, line 12, line 1).
Since the pH of the aerobic culture tank (b) hardly changes until the end of the culture because of metal ions, amino compounds, etc. in the waste liquid, pH adjustment is unnecessary. (Patent Document 9, page 5, line 10, 34).

  Patent No. 3699987 (hereinafter referred to as Patent Document 10) states that “alkaline-resistant photosynthetic bacterium Rhodopseudomona as used in the present invention is a photosynthesis non-sulfur and is a species described in Bergey's Manual of Detergent Bacteriology Vol. 3, 1661-1682. These are alkaline and proliferate well and have bacteria chlorophyll a, neurosporene, lycopene, and the alkali-tolerant orchid-colored bacterium Synechococcus referred to in the present invention is Cyanobacteria, Bergey's Manual of Detergent Bacteriology, Vol. It is a kind of the species described in 1. It is alkaline and grows well, chlorophyll a, and β carotene Alkali resistance as used in the present invention mainly means a maximum growth rate at pH 8 to 10. Organic substance decomposition as used in the present invention mainly means domestic organic wastewater and livestock manure (patents). Reference 10 (paragraph 0007) ”.

According to Kazuo Yamamoto et al. (Hereinafter referred to as Non-Patent Document 1), “It is known that red non-sulfur bacteria are photosynthetic bacteria and can be used for the treatment of various high-concentration organic wastewater. However, it is difficult to selectively proliferate this non-sulfur bacterium at the actual treatment site to be a mixed culture system, and although it has been attracting attention for a while, unfortunately it has not yet spread.
In our laboratory, using an infrared filter, we can selectively propagate red non-sulfur bacteria in the treatment tank, and the grown cells can be used as a valuable resource such as fish food. We are developing. If this process is applied to wastewater treatment in food production plants, etc., it becomes a wastewater treatment process that consumes less energy, is low in cost, and is friendly to the environment. "Is written on the Internet."

JP-A-8-103273, JP-A-11-32755 Japanese Patent Laid-Open No. 9-238681 JP-A-11-243946 JP 2000-287675 A JP 2002-171963 A JP-A-8-192180 Japanese Patent No. 2511325 Japanese Patent No. 3184970 Japanese Patent Publication No. 45-28234 Japanese Patent No. 3699987 Kazuo Yamamoto et al., “Environmental Safety Research Center“ Development of simultaneous water treatment system for treatment of highly concentrated organic wastewater and production of valuable resources using photosynthetic bacteria ”, Internet.

However, each of these prior art search documents is a wonderful invention, making full use of useful technologies, but each uses a special medium composed of synthesized compounds, and the pH can be reduced by adding inorganic compounds. In consideration of preserving photosynthetic bacteria by various bacteria such as adjusting to alkalinity, gelled products and porous granular materials are used. Incubate under lighting for 5 days to 1 month.
In particular, there is no idea or description of growing and culturing using an alcoholic beverage having a pH of 3-4.

  The invention of the present application is to mix the alcoholic liquor stock solution with the diluted photosynthetic bacterium, stir and agglomerate and separate, adjust the pH of the separated liquid-solid separated solution with the photosynthetic bacterium, reduce BOD, mixing treatment, A method for cultivating a large amount of cultured liquid medium for culture mainly consisting of a separated liquid obtained by centrifugation, filtration or ultraviolet irradiation, and photosynthetic bacteria that are difficult to selectively grow in a short period of time The purpose is to provide.

    As a result of diligent research, the inventor of the present application mixes an alcoholic liquor stock solution with diluted photosynthetic bacteria, agitates and separates the mixture, and adjusts the pH of the separated solid-liquid separated solution with photosynthetic bacteria, thereby reducing BOD. The separation liquid obtained by performing at least one of mixing treatment, centrifugal separation / filtration or ultraviolet irradiation is used as a liquid medium for culturing and irradiated with light mainly containing a wavelength of 400 to 700 nm. However, the present invention was completed by a method of growing and culturing the remaining photosynthetic bacteria and a method of growing and culturing the added photosynthetic bacteria or symbiotic strains, thereby solving the above problems. That is,

  The invention of the present application uses a solid-liquid separation device to mix, stir, agglomerate and separate the alcoholic liquor stock solution in a dilute aqueous solution containing photosynthetic bacteria, in particular, red photosynthetic bacteria and / or synchrococcus. The agglomerated and separated agglomerated separation liquid is made into a solid content and a separation liquid, and further, the obtained separation liquid is aerated and mixed at a high speed without being aerated. Irradiation reduces the BOD of the separated solution, and further adds gelatin peptone and / or inorganic salts to create a culture medium for culture. Using these culture mediums, anaerobic A method for growing and cultivating the remaining photosynthetic bacteria in a short period of time while irradiating light mainly containing a wavelength of 400 to 700 nm in a natural atmosphere, Pressurized photosynthetic bacteria or symbiotic strains is obtained by implementing the method of growing culture in a short period of time.

The photosynthetic bacteria referred to in the present invention, Rodosupiriramu rubrum (Rhodospirillum rubrum) Rodosupiriramu genus such as (Rhodospirillum), Rhodopseudomonas Biridisu (Rhodopseudomonas viridis), Rhodopseudomonas Gerachinosa (Rhodopseudomonas gelatinos), Rhodopseudomonas plus tris (Rhodopseudomonas palustris), Rhodopseudomonas Rhodopseudomonas sulfidophila, Rhodopseudomonas CAPSULATAS, Rhodopseudomonas Spheroides, Rhodo pseudomona Rhodopseudomonas and Rhodobum um, and Rhodobum stagoids, such as Rhodacter smuter, and Rhodobacter smuter. .

The red photosynthetic bacteria (proteobacteria) referred to in the present invention are red non-sulfur bacteria, red sulfur bacteria, orchid bacteria, and the red non-sulfur bacteria are Rhodospiriraceae among the bacteria belonging to the photosynthetic bacteria. For example, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodomicrobium, and Rhodophilus are particularly limited to Rhodoculus. It can be used without Specific examples of such bacteria, Rhodopseudomonas Parusuchirusu (Rhodopseudomonas palstris), Rhodobacter Kapusuratasu (Rhodobacter capsulatus), Rhodobacter Sufaeroidesu (Rhodobacter sphaeroides), Rodoshikurusu Gerachinosasu (Rhodocyclus gelatinosus), Rodosupiriumu Rabamu (Rhodospirillum rubrum), Rhodopseudomonas Shufueroi Examples include bacteria such as death (Rhodopseudomonas shreroides), and alkali-resistant photosynthetic bacteria.

The alkali-resistant photosynthetic bacterium referred to in the present invention is an alkali-resistant photosynthetic bacterium (belonging to Rhodopseudomonas) and alkali-resistant orchid-colored bacterium (belonging to Synechococcus) described in Japanese Patent No. 3639905. Rhodopseudomos A strain with bacteria chlorophyll a, neurosporene, lycopene, alkali-resistant bacterial chlorophyll a, photosynthetic bacteria with neurosporene (Rhodopseudomos) B strain, alkali-resistant chlorophyll a and β-carotene (Synechococcus) refers to a group of microorganisms containing at least one selected from C strains.

As symbiotic strains referred to in the present invention, lactic acid bacteria, alcohol yeast, beer yeast, baker's yeast, liquor yeast, sake yeast, Bacillus, Streptococcus, Staphylococcus, Cryptococcus, Devariomyces (Debaryomyces), Endomycopsis (Endomycopsis), Hansenula (Hensenula), Kloekerosis (Pichia), Rhodotorulas, Saccharomyces (soccharomyces) Pseudomonas (Pse omonas, Aspergillus, Rhisopus, Kluyveromyces, Pleurotus, Kuehneromyces, Plumulinaceto, Vector. ), Nocardia.

  The liquor lees referred to in the present invention is a residue generated after alcoholic beverages are obtained during the production of alcoholic beverages. Here, as sake, sake, beer, liqueur, miscellaneous sake, shochu and the like can be mentioned. Examples of shochu include shochu shochu, wheat shochu, rice shochu, sesame shochu, hie shochu, corn shochu, and brown sugar shochu.

The liquid medium for culturing photosynthetic bacteria of the present invention is prepared by diluting photosynthetic bacteria (10 4 to 10 13 cells / mL) with water and diluting the photosynthetic bacteria treated water (10 2 to 10 12 12 liters / mL) is mixed with liquor lees 75 vol% or less and 1 vol% or more to produce agglomeration separation while stirring to obtain an agglomeration separation liquid, and this aggregation separation liquid is solidified using a solid-liquid separation device. It is obtained by solid-liquid separation into a product and a separated liquid.
The liquid medium for culturing photosynthetic bacteria according to the present invention is a solution obtained by diluting photosynthetic bacteria treated water (10 2 to 10 12 / mL), and the above separation liquid is 75 vol% or less and 1 vol% or more. Using a stirrer with a rotational speed of 1000 to 5000 rpm, the mixture is vigorously stirred while taking in air, and mixed until the pH does not drop below 6.0 or lower.
The liquid medium for culturing photosynthetic bacteria of the present invention can be obtained by solid-liquid separation of the mixing liquid obtained by the above mixing treatment into a solid and a separated liquid using a solid-liquid separation device.
The liquid medium for culturing photosynthetic bacteria of the present invention can be obtained by filtering and / or irradiating any of the above-mentioned separation liquids.
The culture medium of the microorganism of the present invention contains 0.5 to 75 g / liter of gelatin peptone and / or 0 to 75 g / liter of inorganic salts with respect to the culture medium for culture.
The microorganism culture medium of the present invention includes those characterized in that the medium is a gelled medium.

The method of growing and culturing according to the present invention is a method of growing and cultivating the remaining photosynthetic bacteria while irradiating the above-mentioned liquid culture medium for culturing under an anaerobic and / or aerobic atmosphere with light mainly containing a wavelength of 400 to 700 nm It is. Further, a method is also included in which a predetermined amount of a photosynthetic bacterium for proliferation is added to the liquid medium, and the cultivated photosynthetic bacterium is proliferated and cultured while irradiating light mainly containing a wavelength of 400 to 700 nm.
The method of the present invention is a method of growing and culturing photosynthetic bacteria in which the photosynthetic bacteria are red photosynthetic bacteria and / or orchid-colored bacteria (Synechococcus), and the red photosynthetic bacteria are grown and cultured in photosynthetic bacteria that are red non-sulfur bacteria. It is also a method.
Furthermore, the present invention can also be applied to a method of growing and cultivating photosynthetic bacteria, wherein a mixed bacterium in which symbiotic strains are mixed in an amount of 50% or less is used for the photosynthetic bacteria. The symbiotic strain is Bacillus, lactic acid bacteria, alcohol yeast, beer yeast, baker's yeast, liquor yeast, sake yeast, Streptococcus, Staphylococcus, Cryptococcus, Hansenula ), Saccharomyces, Shizosaccharomyces, and Toluropsis, a mixture of Bacillus and a photosynthetic bacterium characterized in that it is grown. A method is also included.
The method of the present invention also includes a method of separating and culturing the bacterial cells obtained by any one of the methods described above in the gel medium.

As described above, the present invention is effective as a liquid medium for cultivating microorganisms, particularly as a liquid medium for culturing photosynthetic bacteria, instead of a conventional specially formulated medium. Can be used.
In addition, photosynthetic bacteria that are difficult to selectively grow can be grown and cultured in a short period of time.
For example, by irradiating a red light-synthesizing bacterium (10 2 to 10 4 cells / mL) in an anaerobic atmosphere with a halogen lamp in 3 days (10 7 to 10 8 cells / mL) ) Can be grown and cultured. Cultivation using a specially formulated medium requires 12-14 days and can be processed in a very short period of time.
Furthermore, by gelling the liquid culture medium for culture of the present invention, the photosynthetic bacteria can be proliferated and cultured under anaerobic conditions, and can be used as a medium for differential culture.

As an example of an embodiment of the present invention, first, a basic concept of a liquor mash solid-liquid separation process is shown.
Cultured bacteria storage tank for storing cultured photosynthetic bacteria (10 4 to 10 13 cells / mL), and water until a predetermined number of cells (10 2 to 10 12 cells / mL) The treated water storage tank for storing the photosynthetic bacteria treated water obtained by diluting with the liquor, the liquor cake stock tank for storing the liquor cake stock solution transported from the liquor factory, and the liquor cake stock solution for the photosynthetic bacteria treated water are predetermined. A mixed stirring and aggregating and separating apparatus that conveys, mixes, agitates and agglomerates and separates the mixture so as to achieve a mixing ratio, and solid-liquid separation that separates the obtained agglomerated separated liquid into a separated liquid and a solid Device.
When simultaneous mixing and stirring deviates from the temperature range of 20 to 35 ° C. where the activity of photosynthetic bacteria is high, the aggregation and separation are completed within 10 minutes after the temperature range is reached. Aggregation and separation are performed even at a low concentration of 10 2 cells / mL. Therefore, when other photosynthetic bacteria and / or symbiotic strains are grown and cultured, they are agglutinated and separated at the lowest possible concentration.
The separated liquid subjected to the solid-liquid separation treatment contains organic acid, soluble protein, fine particles (such as fibrin), photosynthetic bacteria, and the like, and BOD is 4000 to 10000 ppm.
Organic acids, soluble proteins, and minute particles (such as fibrin) are eutrophic substances of photosynthetic bacteria.

As the solid-liquid separator, a commercially available solid-liquid separator such as a centrifugal separator, a centrifugal dehydrator, a twin cloth dehydrator, a filter or the like can be used alone or in combination.
Furthermore, the obtained separation liquid is put into a mixing apparatus, and a continuous centrifugal separation apparatus that performs solid-liquid separation processing on the separated separation liquid is connected. By this mixing treatment, solid-liquid separation can be promoted, pH can be adjusted to 6 or more, and BOD can also be reduced. The decrease in BOD is also evidence that photosynthetic bacteria are growing.

  As an example of the method of mixing treatment, the above-mentioned solid-liquid is mixed with high-speed stirring (1000 to 5000 rpm) while aerating the photosynthesis bacteria treated water obtained by dilution or high-speed stirring (1000 to 5000 rpm) while entraining air. The separation liquid separated into solid and liquid by the separation apparatus is continuously or intermittently added until PH becomes 6.0, preferably 6.5. At this time, the irradiation is performed while irradiating light mainly including a wavelength of 400 to 700 nm. This operation is referred to as a mixing process, and the components that constitute the operation are referred to as a mixing device.

This agitated separation liquid is called a mixing liquid, and includes a smoky and floating solid and a precipitated solid. When these solid particles are observed with a microscope, they are very small, 10 to 100 μm. This mixing liquid is subjected to solid-liquid separation using a continuous centrifugal separator. The obtained separation liquid is also referred to as a mixing separation liquid. Even when this mixing separation liquid was left standing for a whole day and night and the pH was measured, the value did not change. The BOD of the separation liquid is 1000 ppm or less, whereas it is 5000 ppm or more.
The pH of the separation liquid that has been subjected to the mixing step decreases with time. When this is mixed again, the pH value is restored.

If necessary, an ultraviolet irradiation device that sterilizes germs other than photosynthetic bacteria in the obtained separation liquid, since the germs are larger than photosynthetic bacteria, a filter with a fine filter of 300 mesh or more, and a continuous centrifuge To reduce or eliminate germs.
The separated liquid including the mixed separated liquid is treated in the next step and / or sterilizes bacteria other than photosynthetic bacteria by an ultraviolet irradiation device. This has the effect of eliminating bacteria other than symbiotic bacteria. Put the sterilized sterilized separation solution into the growth culture tank.
When processing the liquor bottle, it is returned to the treated water storage tank and / or the culture cell storage tank, and the photosynthetic bacteria are reused.

These separation liquids (including coagulation separation liquids, separation liquids, mixing liquids, and liquids that have been sterilized and sterilized) include organic acids, soluble proteins, minute particles (such as fibrin), and photosynthetic bacteria. The BOD is 4000 to 300 ppm, and organic acids, soluble proteins, and minute particles (such as fibrin) are eutrophic substances of photosynthetic bacteria. Therefore, these separated liquids are used as one composition of the liquid medium or the liquid medium itself.
In order to obtain a culture medium according to the properties of the target growth microorganism, a liquid medium having a composition containing 0.5 to 75 g / liter of gelatin peptone and / or 0 to 75 g / liter of inorganic salts with respect to the liquid medium. It is also preferable to do.
Furthermore, as a means for anaerobic, agar, gelatin, carrageenan, pectin or the like may be added to form a gel.

As one of the growth culture methods, the case of photosynthetic bacteria is shown.
In the case of anaerobic photosynthetic bacteria, the above-mentioned liquid medium is put into a growth culture tank that can be irradiated with light, and if it is the same photosynthetic bacterium as the liquor koji treatment, it is irradiated with light mainly containing a temperature of 20 to 45 ° C. and a wavelength of 400 to 700 nm. Incubate while It is allowed to stand without stirring, and after 24 hours or more, it turns red and it is visually observed that it is grown and cultured.
If it is another anaerobic photosynthetic bacterium, it is preferable to use a sterilized liquid medium, which is put into a growth culture tank that can be irradiated with light, inoculated with the desired anaerobic photosynthetic bacterium, and then stirred appropriately. It is allowed to stand and cultured while irradiating with light mainly containing a temperature of 20 to 40 ° C. and a wavelength of 400 to 700 nm. When 24 hours or more have elapsed, the color becomes red and it is visually observed that the cells are grown and cultured.
Since the photosynthetic bacteria actively move around in the liquid, they may be left standing, but an inert gas such as nitrogen gas may be introduced and gently stirred.
On the other hand, in the case of an aerobic photosynthetic bacterium, a light beam mainly containing a temperature of 20 to 45 degrees C and a wavelength of 400 to 700 nm by introducing sterilized air such as compressed air or oxygen gas into the growth culture tank instead of the above method. Incubate with irradiation.

Obtain 250L of the stock solution of shochu shochu from OG shochu. 100 g was collected from this and dried in a dryer 90-110 degrees. The weight was immediately measured using a 250 g scale, and a result of 4.54 g was obtained. The solid content of the shochu is 4.5 wt%.
Connect the metering pump P1 to the stock solution tank of shochu, the metering pump P2 to the culture Sato-treated water tank, connect the mixing pipe that can be stirred by the water wheel immediately after joining the pipes, and connect the outlet to the rotating drum of the rotary filter Plug in. A residence pipe was provided at the outlet, and this residence time was set within 2 minutes. Since the water wheel blades were rotated by the water pressure and stirred, the rotation speed was 200 rpm or less. The mixing ratio of 50-fold diluted cultured Sato bacterium treated water (a solution obtained by diluting 20 liters of cultured Sato bacterium into 1 ton of water) and shochu was set to 5: 1.

The rotary filter was charged at a flow rate of 8.5 L / min.
The filtration conditions of the rotary filter are a combination of a rotational speed of 42 rpm, a combination of 150 and a 120-mesh stainless steel filter, a tilt of the rotation axis θ = 4.2, a height of the spiral cutting plate 119 of 50 mm, and a spiral pitch of 150 mm. The operation was continued until the stock solution of shochu disappeared.
The separation filter drum is rotated while the flocculated separation liquid is filtered through the separation filter. The liquid is raised along the spiral plate. Within 2 minutes, store the semi-solid in a semi-solid state that does not completely filter moisture in the semi-solid receiving tank. Ten minutes later, the semi-solid was charged into a charging hopper of a screw press type pressing device with a semi-solid pump. Driving intermittently for 20 minutes. The operating conditions of the screw press type pressing device are a screw rotation speed of 90 rpm and a pore diameter of the pressing screen of 120 μm.

The squeezed liquid separated into solid and liquid was stored in a squeezed liquid receiving tank. The rotary filter A was recharged with a squeeze liquid charging pump. The pH of the separation liquid was 4.53. The BOD of the separated liquid was 2700 ppm.

The obtained separation liquid was processed by a mixing process.
[Test 1]
3 L of 40-fold diluted cultured Sato-treated water was placed in a 30 L plastic tank and stirred using a stirrer with a rotation speed of 1700 rpm while entraining air. The separation liquid was continuously added thereto at a flow rate of 82.5 mL / min, and the water temperature and pH were measured at 10 minute intervals. It was set so that the liquor cake and the 40-fold diluted cultured Sato-treated water might be 1: 6. The results are shown in Table 1. The change in pH treatment time is shown in FIG. Sampling was performed for BOD measurement at intervals of 20 minutes after 20 minutes. The measurement results are shown in Table 1. A change in the processing time of the BOD is shown in FIG.

The mixing separation liquid was sampled into two 500 mL PET bottles at intervals of 10 minutes. One was filtered as it was, the other was filtered using a paper filter used as a coffee filter, and the filtrate was again placed in a 500 mL PET bottle. These were arranged in a horizontal row and irradiated from 80 to 100 cm using a halogen lamp (500 W). Photographs were taken every 24 hours to observe the color of the liquid. Table 2 shows the change in color of green → brown → no change (colorless and transparent) → red → red color by numerical values of −4 → −2 → 0 → 3 → 6. The two types of numerical values are different in color between liquid and sediment. “Inherent” is expressed as “F” or “filter” when there is a solid substance, that is, as it is as the mixing separation liquid, or when the paper filter is filtered.

Photographs taken are shown in FIGS. FIG. 3 shows the change of the sample that was irradiated with the mixing separation solution “Eri-ri” for 10 to 80 minutes when 24, 48, 72, and 96 hours passed from the top.
FIG. 4 is a graph showing changes in what was irradiated and cultured for 10 to 80 minutes with the mixing separation solution “filter” in the order of 24, 48, 72, and 96 hours from the top.
FIG. 5 is a graph showing changes in 80-190 minutes of the mixed separation solution “Eri-ri” irradiated after 24, 48, 72, 96 hours in order from the top.
FIG. 6 shows the changes of 80-190 minutes of the mixed separation solution “filter” irradiated and cultured after 24, 48, 72, 96 hours in order from the top.

The separation liquid was added for 190 minutes and then mixed for 4 hours at the same rotational speed. The liquid temperature was 33.8 ° C. and the pH recovered to 8.05. As a result of measuring the BOD of this mixing separation liquid, it was 340 ppm.
This mixing separation liquid was filtered with a 325 mesh drum. The filtrate was put in a PET bottle, and the culture solution irradiated with a halogen lamp for 6 days turned dark green. FIG. 7 shows a photograph taken together with “irradiance” and “filter” irradiated for 6 days. Two of the medium that appear dark in the center are cultures that have turned green. It was confirmed that green photosynthetic bacteria were also grown and cultured.

[Comparative Example 1] (Method of culturing photosynthetic bacteria (Sato))
Mikawa Environmental Microorganisms Use a commercially available photosynthetic bacterial solution (original fungus) from Sato Laboratories. This is a photosynthetic bacterium isolated from the soil on the surface of the paddy field and expanded. It is crimson and smells like a dragonfly, has a pH of around 8.5, and has the following properties. 1. Detoxification of decomposition products such as hydrogen sulfide in soil. 2. Utilization of bacterial products such as vitamin B12, carotene, and nucleic acids. 3. Promotion of growth of actinomycetes in soil and compost "competition with fusarium". 4). Promotion of disappearance of enteric bacteria such as Escherichia coli and Salmonella from nature. 5. Denitrification of soil, sewage, compost, etc.
Therefore, in the expansion culture method of Motosato Sato marketed by the Sato Research Institute in Okazaki City, Aichi Prefecture, when protozoan bacteria aqueous solution (wine rose color, pH = 8.35) is observed with a microscope, A large number of various germs were confirmed. Protozoan aqueous solution (wine rosy color, pH = 8.35) was diluted 10 times and irradiated with ultraviolet rays to kill miscellaneous bacteria.
Water 40 L is poured into a 60 L water tank.
As a medium composition,
40g ammonium chloride
Sodium bicarbonate 40g
Sodium acetate 40g
Sodium chloride 40g
Dipotassium hydrogen phosphate 8g
Magnesium sulfate 8g
DL-malic acid 10g
Peptone 8g
Yeast extract 4g
And stir for 2 to 3 minutes to prepare medium water.

Prepare a culture pot that can block air and can be adjusted to a temperature of 25 to 40 degrees Celsius so as not to mix various germs, and can irradiate light.
40 L of medium water is put into this culture kettle, and 20 L of an aqueous solution containing only Sato genus bacteria is added and stirred. The temperature is adjusted at a temperature of about 32 degrees with a water temperature heater and cultured for 12 days in the sun. The colorless and transparent one turns red. pH = 8.5.
This is called cultured Sato bacteria.

[Comparative Example 2] (Cultivation method of Sato Bacillus)
Prepare commercially available heat-resistant Bacillus bacteria (agar-like, A and B) from Sato Laboratories in Okazaki City, Aichi Prefecture. Take half of the petri dish and put 1L of 40 ° C hot water in a 20L water tank and stir for 30 seconds. Add another 18 L of water (25 degrees).
Add 1 L of molasses. You may culture with honey instead of molasses. Stir for 1-2 min.
Temperature is adjusted at 32 degrees with a heater and aerated at 3 L / min with an air pump. It becomes dark black and pH = 3.6 at 24 hours. Store in a cool and dark place. This is called cultured Bacillus.
When cultured with honey, milky white, pH = 4.1.

[Comparative Example 3] (Cultivation method of alkali-resistant photosynthetic bacteria)
The cell purification material obtained by expanding and culturing the alkali-resistant photosynthetic bacteria obtained in Japanese Patent No. 3699987 was used. This fungus body purification material (commercially called eco-bacteria) is a mixture of red non-sulfur bacteria, orchid bacteria, and soil bacteria, and is used in the examples of Japanese Patent Application No. 2006-112336.
For example, 2 kg of eco-bacteria is mixed with 20 L of water, heated to 32 degrees, left to stand for 24 hours, and then filtered. The filtrate is irradiated with ultraviolet light (1200 μW, 40 seconds) to sterilize the soil fungus.
The obtained aqueous solution of alkali-resistant photosynthetic bacteria was carried out in the same manner as the culture method for cultivating Sato bacteria. However, it took 3 weeks to change to dark red. This which is pH = 8.3-8.7 is called culture sterilized eco-bacteria.

Obtain 200L of the stock solution of shochu shochu from OG shochu. 100 g was collected from this and dried in a dryer 90-110 degrees. The weight was immediately measured using a 250 g scale, and results of 3.90 g and 4.05 g were obtained. The solid content of the shochu is 3.98 wt%.
Experiments were conducted on coagulation separation and solid-liquid separation of shochu by simultaneous mixing and stirring. Connect the metering pump P1 to the stock solution tank of shochu, the metering pump P2 to the culture Sato-treated water tank, connect the mixing pipe that can be stirred by the water wheel immediately after joining the pipes, and connect the outlet to the rotating drum of the rotary filter Plug in. A residence pipe was provided at the outlet, and this residence time was set within 2 minutes. Since the water wheel blades were rotated by the water pressure and stirred, the rotation speed was 200 rpm or less. The mixing ratio of 50-fold diluted cultured Sato-treated water and shochu was set to 3: 1.
The rotary filter was charged at a flow rate of 8.5 L / min.
The filtration conditions of the rotary filter are a combination of a rotational speed of 42 rpm, a combination of 150 and a 120-mesh stainless steel filter, a tilt of the rotation axis θ = 4.2, a height of the spiral cutting plate 119 of 50 mm, and a spiral pitch of 150 mm. The operation was continued until the stock solution of shochu disappeared.
The separation filter drum is rotated while the flocculated separation liquid is filtered through the separation filter. The liquid is raised along the spiral plate. Within 2 minutes, store the semi-solid in a semi-solid state that does not completely filter moisture in the semi-solid receiving tank. Ten minutes later, the semi-solid was charged into a charging hopper of a screw press type pressing device with a semi-solid pump. Driving intermittently for 20 minutes. The operating conditions of the screw press type pressing device are a screw rotation speed of 90 rpm and a pore diameter of the pressing screen of 120 μm.

The solid was stored in a solid receiver. Immediately, the solid was weighed to give 3.172 Kg. Each 200 g of this sample was collected and dried with a dryer at 90 to 110 degrees. The dried solids were 54.72g and 55.18g. The solid content was 27.36 wt% and 27.59 wt%, respectively. The discharged solid from the screw press type pressing device was calculated to be 0.871 Kg. The residual solid weight collected from the rotary filter tank and the screw press type crusher cylinder was 8.669 kg. 300 g of this sample was collected and dried, and the solid weight was measured. The solid content was 3.14 wt% and 2.92 wt%, respectively. The solid matter was calculated to be 0.263 kg.
The squeezed liquid separated into solid and liquid was stored in a squeezed liquid receiving tank. The rotary filter A was recharged with a squeeze liquid charging pump. The pH of the separation liquid was 4.94. The precipitation height after 12 hours was 23 mm with respect to the separation liquid height of 260 mm. The BOD of the separated liquid was 3800 ppm.
By co-mixing and stirring, it was possible to agglomerate and separate within 5 minutes and then to separate into solid and liquid within a few minutes.

The obtained separation liquid was processed by a mixing process.
[Mixing test]
25 L of 50-fold diluted cultured Sato-treated water was placed in a 500 L plastic tank and stirred using a stirrer with a rotational speed of 3000 rpm while entraining air. To this, 688 g of the separated liquid was added at an interval of 10 minutes, and the water temperature and pH were measured. The pH recovered in 10 minutes, and even after stirring for 80 minutes and adding 5.5 L of the separation liquid, there was little decrease in pH. The BOD of this mixing separation liquid was 650 ppm.
This mixing separation liquid was subjected to solid-liquid separation using a centrifuge. The mixed separation liquid that has been mixed has a lower turbidity than the separation liquid, and is considerably transparent after centrifugation. The separation liquid has fine particles floating, has a high turbidity, and has a light brown color even after centrifugation. It can be seen that the amount of separated solids is also greatly reduced through the mixing process. The BOD of the centrifuged supernatant was 610 ppm. After adding 5.5 L of the separated liquid, stirring was continued at 3000 rpm for 180 minutes. BOD was 620 ppm.

Next, an experiment for aggregating separation was performed using a centrifuge. The centrifuge used is capable of setting eight centrifuge containers (referred to as capsules).
The residue of the separated liquid was stirred, 600 mL was collected, and the BOD of the liquid obtained by solid-liquid separation using a centrifuge was measured. It was 4500-4900 ppm. It was found that the centrifuge removed minute particles and lowered the BOD.

When the residue of the diluted liquid was measured using a halogen lamp (500 W) when the BOD was measured by an experiment of aggregating separation using these centrifuges, the color changed to red from the second day. It was confirmed that photosynthetic bacteria were growing.
It was found that these separated liquids, aggregating separated liquid, separated liquid, mixing liquid, and liquid obtained by sterilizing and sterilizing these, cultivate photosynthetic bacteria.

Agar (Shinano Agar Agricultural Cooperative) agar 15g is boiled in 1 liter of water and air-cooled.
5 mL and 50 mL of the separation liquid and mixing separation liquid of Example 1 were put into four 200 mL PET bottles, and the agar lysate was added and shaken. I put it in the refrigerator and left it all day and night. When it was taken out in a gel-like state and irradiated using a halogen lamp (500 W), it turned red from the second day. It was confirmed that photosynthetic bacteria were growing.
15 g of this agar contains 0.2 g of protein, 9.8 g of sodium, and 6.1 g of dietary fiber, and does not contain lipids or carbohydrates.
It can be seen that the cultured Sato bacteria contain anaerobic photosynthetic bacteria and are grown and cultured.

It is a figure which shows the process time change of pH of the influence which a mixing process has. It is a figure which shows the processing time change of BOD among the influences which a mixing process has. It is a photograph which shows the change of what irradiated the mixing isolation | separation liquid "inherent" from 10 minutes to 80 minutes. It is the photograph which shows what also irradiated the mixing separation liquid "filter" from 10 minutes to 80 minutes. It is a photograph which shows what irradiated and mixed the mixing separation liquid "inherent" from 80 minutes to 190 minutes. It is a photograph which shows what irradiated and mixed the mixing separation liquid "filter" from 80 minutes to 190 minutes. It is the photograph of the culture solution which irradiated the mixing isolation | separation liquid filtered with "Intrinsic", "Filter", and a 325 mesh drum with the halogen lamp for 6 days.

Claims (13)

  Diluted photosynthetic bacteria (10 4 to 10 13 cells / mL) with water and treated with photosynthetic bacteria treated water (10 2 to 10 12 cells / mL). It is obtained by mixing 75 vol% or less and 1 vol% or more, aggregating and separating with stirring to obtain an agglomerated separation liquid, and solid-liquid separation of the agglomerated separation liquid into a solid and a separated liquid using a solid-liquid separation device. Liquid medium for the cultivation of photosynthetic bacteria.   Using the stirrer at 1000 to 5000 rpm, the separation liquid of 75 vol% or less and 1 vol% or more of claim 1 with respect to the water treated with photosynthetic bacteria obtained by dilution (10 2 to 10 12 / mL). A liquid medium for culturing photosynthetic bacteria obtained by mixing with vigorous stirring while taking in air until the pH does not drop below 6.0.   A liquid medium for culturing photosynthetic bacteria obtained by solid-liquid separation of the mixing liquid obtained by the mixing treatment according to claim 2 into a solid and a separated liquid using a solid-liquid separation device.   A liquid medium for culturing photosynthetic bacteria obtained by filtering and / or irradiating with ultraviolet light the separation liquid according to any one of claims 1 to 3.   A microorganism medium containing 0.5 to 75 g / liter of gelatin peptone and / or 0 to 75 g / liter of inorganic salts with respect to the liquid medium according to any one of claims 1 to 4. 6. The microorganism culture medium according to claim 1, wherein the culture medium is a gelled medium.   A method for growing and cultivating the remaining photosynthetic bacteria while irradiating the liquid medium according to any one of claims 1 to 5 with light mainly containing a wavelength of 400 to 700 nm in an anaerobic and / or aerobic atmosphere.   A predetermined amount of a photosynthetic bacterium for proliferation is added to the liquid medium according to any one of claims 1 to 5, and irradiation with light mainly containing a wavelength of 400 to 700 nm is performed in an anaerobic and / or aerobic atmosphere. A method of growing and cultivating photosynthetic bacteria for propagation.   The method for growing and cultivating the photosynthetic bacterium according to any one of claims 1 to 8, wherein the photosynthetic bacterium is a red photosynthetic bacterium and / or a orchid bacterium (Synechococcus).   The method for growing and cultivating photosynthetic bacteria according to claim 9, wherein the red photosynthetic bacteria are red non-sulfur bacteria.   The method for growing and cultivating photosynthetic bacteria according to any one of claims 7 to 10, wherein a mixed bacterium in which 50% or less of symbiotic strains are mixed in the photosynthetic bacterium is used.   The symbiotic strain is Bacillus, lactic acid bacteria, alcohol yeast, beer yeast, baker's yeast, liquor yeast, sake yeast, Streptococcus, Staphylococcus, Cryptococcus, Hansenula ), Saccharomyces, Shizosaccharomyces, Toluropsis, or a mixture of Bacillus. A method for growing and cultivating photosynthetic bacteria.   A method for separating and culturing bacterial cells obtained by the method according to any one of claims 7 to 12 in the gel-like medium according to claim 6.

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