Method for culturing spore-forming bacteria, and method for producing useful substance

Abstract

An object of the present invention is to provide a novel culturing method by which spores can be efficiently produced. The present invention further provides a method for culturing sporulating bacteria, comprising adding a sporulation-inhibiting substance into a medium for culturing sporulating bacteria, wherein the carbon content in the medium is 9.1 g/L or more, and preferably further comprising a step of adding a sporulation-accelerating substance to the medium.

Claims

1. A method for culturing a sporulating bacterium, the method comprising: culturing a sporulating bacterium in a medium to which a sporulation-inhibiting substance is added, wherein the medium has a carbon content of 9.1 g/L or more, wherein the sporulation-inhibiting substance is lincomycin in an amount of 0.1 to 0.3 ppm wherein the sporulating bacterium is a Bacillus subtilis, wherein the culturing step produces at least 2.8×10.sup.9 spores per ml.

2. The method for culturing a sporulating bacterium according to claim 1, further comprising: adding a sporulation-accelerating substance to the medium.

3. The method for culturing a sporulating bacterium according to claim 2, wherein the sporulation-accelerating substance is added to the medium at a timing in a time period of from five hours to seventy hours after the start of culture.

4. The method for culturing a sporulating bacterium according to claim 2, wherein the sporulation-accelerating substance is a nucleobase analogue, an organic acid, an amino acid, an ammonium compound, a nitric acid compound, a nitrous acid compound or a mineral.

5. The method for culturing a sporulating bacterium according to claim 2, wherein the sporulation-accelerating substance is at least one substance selected from the group consisting of decoyinine, mizoribine, mycophenol, 6-azauracil, lactic acid and a salt thereof, acetic acid, an acetic acid salt, butyric acid, a butyric acid salt, manganese, ammonium, calcium, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, ammonium lactate, and ammonium acetate.

6. The method for culturing a sporulating bacterium according to claim 2, wherein the sporulation-accelerating substance is added at a concentration of 10 ppm to 10,000 ppm to the medium.

7. A method, comprising producing a substance by the culturing method of claim 1.

8. The method according to claim 7, wherein the substance is a spore of the sporulating bacterium.

9. The method according to claim 7, wherein the substance is a metabolite of the sporulating bacterium.

10. The method according to claim 9, wherein the metabolite is a cyclic lipopeptide.

11. The method according to claim 10, wherein the cyclic lipopeptide is at least one cyclic lipopeptide selected from the group consisting of iturin, surfactin, plipastatin, fengycin, bacillomycin, lichenysin, kurstakin, mycosubtilin, colistin, fusaricidin, paenibacterin, polymyxin, and pumilacidin.

12. A method of maintaining vegetative growth of a sporulating bacteria, culturing a sporulating bacterium in a medium to which a sporulation-inhibiting substance is added, wherein the medium has a carbon content of 9.1 g/L or more, and the sporulating bacterium produces one or more metabolites during vegetative growth, wherein the sporulation-inhibiting substance is lincomycin in an amount of 0.1 to 3 ppm wherein the concentration of one or more metabolites from the sporulating bacteria is increased compared to the sporulating bacteria cultured in a medium that has a carbon content of less than 9.1 g/L, and wherein the sporulating bacterium is a Bacillus subtilis, wherein the culturing step produces at least 2.8×10.sup.9 spores per ml.

13. The method according to claim 12, further comprising isolating the metabolites from the medium cultured.

14. A method for culturing a Bacillus subtilis, the method comprising: culturing a Bacillus subtilis in a medium to which lincomycin is added in an amount of 0.3 to 0.5 ppm, wherein the medium has a carbon content of 9.1 g/L or more, and then adding a sporulation-accelerating substance to the medium wherein the sporulation-accelerating substance is at least one substance selected from the group consisting of ammonium lactate, and ammonium acetate in an amount of 2000 to 4000 ppm.

Description

EXAMPLES

(1) The present invention will be described in detail below with reference to Examples, but is not limited to the following Examples.

Example 1

(2) <High-Concentration Growth (Bacillus subtilis) by Inhibition of Sporulation>

(3) Using a 500 ml Erlenmeyer flask (with baffles), each 100 ml of media listed in Table 1 was prepared and then autoclave sterilization was carried out. Glucose was separately sterilized and aseptically mixed in order to avoid Maillard reaction.

(4) The carbon contents of glucose, defatted soy flour, corn steep liquor, and a yeast extract among medium components were each calculated as 40% by weight of the total sugar amount and 50% by weight of the total protein amount. The total sugar amount was found by determining the reducing sugar concentration by Somogyi method after 2.5 hours of hydrolysis in acid at 100° C. The total protein amount was found by determining the total nitrogen amount by the Kjeldahl method, and then multiplying the amount by conversion factor, 6.25.

(5) TABLE-US-00001 TABLE 1 Medium Composition Concentration Component Manufacturer (g/L) Glucose Wako Pure Chemicals 12.500 Defatted soy flour Ajinomoto Healthy Supply 10.000 Corn steep liquor Roquette 2.500 Yeast extract Difco 2.000 MnCl.sub.2•4H.sub.2O Wako Pure Chemicals 0.090 NaCl Wako Pure Chemicals 0.500 KH.sub.2PO4 Wako Pure Chemicals 0.250 MgSO.sub.4•7H.sub.2O Wako Pure Chemicals 0.313 CaCl.sub.2 Wako Pure Chemicals 0.094 FeSO.sub.4 Wako Pure Chemicals 0.00019 Carbon content 9.1 Nitrogen content 1.1

(6) Test sections were set as shown in Table 2. According to the conditions of each test section, the filter-sterilized aqueous solution of each sporulation-inhibiting substance was added aseptically to each medium. One loopful of Bacillus subtilis MBI-600 was taken from a colony grown on a nutrient agar medium, inoculated, and then cultured with shaking at 30° C. and 150 rpm for 64 hours.

(7) Each of the thus obtained culture liquid was diluted 10-fold with sterile water, and then the bacterial cell concentration (vegetative cells and spores) and the sporulation rate (spore concentration/bacterial cell concentration) were measured using an optical microscope and a bacterial cell counter. The results are shown in Table 2.

(8) TABLE-US-00002 TABLE 2 Test Result Sporulation- Bacterial Spore inhibiting Concen- cell concen- concen- Test substance tration tration Sporulation tration No. added (ppm) (/mL) rate (/mL) 1 — — 3.2e+9 87% 2.8e+9 2 Lincomycin 0.1 4.7e+9 100%  4.7e+9 3 Lincomycin 1.0 7.0e+9  0% — 4 Erythromycin 0.1 3.5e+9 80% 2.8e+9 5 Erythromycin 1.0 6.2e+9  0% — 6 Streptomycin 12.5 4.3e+9 100%  4.3e+9 7 Streptomycin 15.0 5.5e+9 54% 3.0e+9

(9) The effect of inhibiting sporulation was not observed in the test section with the lincomycin concentration of 0.1 ppm, however, the effect of inhibiting sporulation was observed in the test section with the same of 1 ppm. Meanwhile, the bacterial cell concentrations tended to increase as the lincomycin concentrations increased.

(10) Similarly, the effect of inhibiting sporulation was not observed in the test section with the erythromycin concentration of 0.1 ppm, however, the effect of inhibiting sporulation was observed in the test section with the same of 1 ppm. Moreover, the bacterial cell concentrations tended to increase as the erythromycin concentrations increased.

(11) Furthermore, the effect of inhibiting sporulation was not observed in the test section with the streptomycin concentration of 12.5 ppm, however, the effect of inhibiting sporulation was observed in the test section with the same of 15.0 ppm. Meanwhile, the bacterial cell concentrations tended to increase as the streptomycin concentrations increased.

Example 2

(12) <High-Concentration Growth of Vegetative Cells by Inhibition of Sporulation and High-Level Production of Spores (Bacillus subtilis) by Induction of Sporulation>

(13) Using a 500 ml Erlenmeyer flask (with baffles), each 100 ml of media listed in Table 1 was prepared and then autoclave sterilization was carried out. Note that glucose was separately sterilized and aseptically mixed in order to avoid Maillard reaction.

(14) Test sections were set as described in Table 3. According to the conditions of each test section, the filter-sterilized aqueous solution of each sporulation-inhibiting substance was added aseptically to each medium. According to the conditions of each test section, one loopful of Bacillus subtilis MBI-600 was taken from a colony grown on a nutrient agar medium, inoculated, and then cultured with shaking at 30° C. and 150 rpm.

(15) Sixteen (16) hours after the start of culture, each sporulation-accelerating substance was added according to the conditions of each test section, culture was continued. Forty-five (45) hours after the start of culture, culture was stopped.

(16) Each of the thus obtained culture liquid was diluted 10-fold with sterile water, and then the bacterial cell concentration (vegetative cells and spores) and the sporulation rate (spore concentration/bacterial cell concentration) were measured using an optical microscope and a bacterial cell counter. The results are shown in Table 3.

(17) TABLE-US-00003 TABLE 3 Test Result Sporulation- Sporulation- Bacterial cell Spore Test inhibiting Concentration accelerating Concentration concentration Sporulation concentration No. substance added (ppm) substance added (ppm) (/mL) rate (/mL) 1 — — — — 3.2e+9 87% 2.8e+9 2 — — Ammonium 3,000 5.2e+9 91% 4.8e+9 lactate 3 — — Ammonium 3,000 4.6e+9 91% 4.2e+9 acetate 4 Lincomycin 0.3 — — 3.3e+9 80% 2.7e+9 5 Lincomycin 0.5 — — 4.4e+9  0% — 6 Lincomycin 1.0 — — 5.1e+9  0% — 7 Lincomycin 2.0 — — 5.9e+9  0% — 8 Lincomycin 0.3 Ammonium 3,000 5.6e+9 91% 5.1e+9 lactate 9 Lincomycin 0.5 Ammonium 3,000 7.2e+9 79% 5.7e+9 lactate 10 Lincomycin 0.5 Ammonium 2,000 6.4e+9 64% 4.1e+9 acetate 11 Lincomycin 0.5 Ammonium 3,000 7.6e+9 71% 5.4e+9 acetate 12 Lincomycin 0.5 Ammonium 4,000 8.4e+9 72% 6.1e+9 acetate 13 Chloramphenicol 1.4 — — 5.4e+9  0% — 14 Chloramphenicol 1.4 Decoyinine   200 5.4e+9 76% 4.1e+9

(18) The effect of inhibiting sporulation was not observed in the test section with the lincomycin concentration of 0.3 ppm, however, the effect of inhibiting sporulation was observed in the test section with the same of 0.5 ppm or more. Moreover, the bacterial cell concentrations tended to increase as the lincomycin concentrations increased.

(19) In the test sections in which ammonium lactate had been added as a sporulation-accelerating substance, improvement was observed in sporulation rate regardless of the lincomycin concentration, unlike test sections in which no such substance had been added. Particularly in the test sections with the lincomycin concentrations of 0.3 ppm and 0.5 ppm, in which ammonium lactate had been added, spore productivity was significantly higher than the same in the test sections in which no such substance had been added.

(20) In the test sections with the lincomycin concentration of 0.5 ppm, in which ammonium acetate had been added, a tendency was observed such that the bacterial cell concentration and the sporulation rate increased as the amount of ammonium acetate added increased. Spore productivity was also significantly higher in all of these test sections than those of the test sections in which no such substance had been added.

(21) In the test section with the chloramphenicol concentration of 1.4 ppm, the effect of inhibiting sporulation was observed. However, in the test section in which decoyinine had been added during culture in addition to chloramphenicol, improvement was observed in sporulation rate. Spore productivity was improved in both test sections more than that of the test sections in which no such substance had been added.

Example 3

(22) <High-Concentration Growth of Vegetative Cells by Inhibition of Sporulation and High-Level Production of Spores (Bacillus thuringiensis)>

(23) Using a 500 ml Erlenmeyer flask (with baffles), each 100 ml of media listed in Table 1 was prepared and then autoclave sterilization was carried out. Note that glucose was separately sterilized and aseptically mixed in order to avoid Maillard reaction.

(24) Test sections were set as shown in Table 4. According to the conditions of each test section, the filter-sterilized aqueous solution of each sporulation-inhibiting substance was added aseptically to each medium. According to the conditions of each test section, one loopful of Bacillus thuringiensis NBRC 101235 strain was taken from a colony grown on a nutrient agar medium, inoculated, and then cultured with shaking at 30° C. and 150 rpm for 40 hours.

(25) Each of the thus obtained culture liquid was diluted 10-fold with sterile water, and then the bacterial cell concentration (vegetative cells and spores) and the sporulation rate (spore concentration/bacterial cell concentration) were measured using an optical microscope and a bacterial cell counter. The results are shown in Table 4.

(26) TABLE-US-00004 TABLE 4 Test Result Sporulation- Bacterial Spore inhibiting Concen- cell concen- concen- Test substance tration tration Sporulation tration No. added (ppm) (/mL) rate (/mL) 1 — — 1.0e+9 54% .sup. 5e+8 2 Lincomycin 5.0 1.4e+9 86% 1.2e+9 3 Lincomycin 10.0 1.4e+9 71% 1.0e+9 4 Lincomycin 12.5 .sup. 9e+8 13% .sup. 1e+8 5 Chloramphenicol 2.0 1.6e+9 79% 1.3e+9 6 Chloramphenicol 6.0 .sup. 8e+6  0% — 7 Erythromycin 0.05 1.5e+9 73% 1.1e+9 8 Erythromycin 0.1 2.0e+9 16% .sup. 3e+8

(27) The effect of inhibiting sporulation was confirmed in the test section with the lincomycin concentration of 12.5 ppm. On the other hand, in the test sections with the lincomycin concentrations of 10.0 ppm or lower, the bacterial cell concentration and the spore concentration tended to increase.

(28) In the test section with the chloramphenicol concentration of 6.0 ppm, the growth and the effect of inhibiting sporulation were confirmed. On the other hand, in the test section with the same of 2.0 ppm, the bacterial cell concentration and the spore concentration tended to increase.

(29) In the test section with the erythromycin concentration of 0.1 ppm, the effect of inhibiting sporulation was confirmed. On the other hand, in the test section with the same of 0.05 ppm, the bacterial cell concentration and the spore concentration tended to increase.

(30) The bacterial cell concentration increased through the addition of a given amount of an antibiotic, and spore productivity improved through sufficient time (40 hours) of culturing without the addition of any sporulation-accelerating substance.

Example 4

(31) <High-Level Production of Surfactin (Bacillus subtilis) by Inhibition of Sporulation>

(32) Using a 500 ml Erlenmeyer flask (with baffles), each 100 ml of media listed in Table 5 was prepared and then autoclave sterilization was carried out. Note that glucose was separately sterilized and aseptically mixed in order to avoid Maillard reaction.

(33) Test sections were set as shown in Table 6. According to the conditions of each test section, the filter-sterilized aqueous solution of each sporulation-inhibiting substance was added aseptically to each medium. According to the conditions of each test section, one loopful of Bacillus subtilis MBI-600 strain was taken from a colony grown on a nutrient agar medium, inoculated, and then cultured with shaking at 30° C. and 150 rpm.

(34) TABLE-US-00005 TABLE 5 Medium Composition Concentration Component Manufacturer (g/L) Glucose Wako Pure Chemicals 25.000 Defatted soy flour Ajinomoto Healthy Supply 20.000 Com steep liquor Difco 5.000 Yeast extract Roquette 4.000 MnCl.sub.2•4H.sub.2O Difco 0.180 NaCl Wako Pure Chemicals 1.000 KH.sub.2PO4 Wako Pure Chemicals 0.500 MgSO.sub.4•7H.sub.2O Wako Pure Chemicals 0.625 CaCl.sub.2 Wako Pure Chemicals 0.188 FeSO.sub.4 Wako Pure Chemicals 0.00038 Carbon content 18.2 Nitrogen content 2.2

(35) Fifty (50) hours after the start of culture, culture was stopped.

(36) Each of the obtained cultured liquid was centrifuged using a refrigerated centrifuge (TOMY SEIKO Co., Ltd. MX-307) at 10,000 rpm and 20° C. for 30 minutes, thereby collecting a supernatant.

(37) To a solid-phase extraction column (Nihon Waters K.K. Oasis HLB 3 cc (400 mg) LP Extraction Cartridge), 3 ml of 0.1% TFA-containing acetonitrile was added and allowed to pass therethrough, and then 3 ml of 0.1% TFA-containing distilled water was added to pass through the column. Two ml of the thus collected centrifugal supernatant of each culture medium was added to pass through the column, 6 ml of 0.1% TFA-containing distilled water, and then 3 ml of 0.1% TFA-containing acetonitrile/distilled water (20:80, v/v) were allowed to pass through the column in sequence for washing. Next, 3 ml of 0.1% TFA-containing acetonitrile/distilled water (90:10, v/v) was allowed to pass through the column, thereby collecting an eluate. 0.1% TFA-containing acetonitrile/distilled water (90:10, v/v) was added, so that the amount of the collected solution was 4 ml, and then HPLC analysis was conducted under the following conditions.

(38) HPLC: Agilent Technologies, Inc. 1260 Infinity

(39) Column: Nihon Waters K.K. XBridge C18 5 μm 4.6×250 mm

(40) Mobile phase: A: 0.1% TFA-containing distilled water, B: 0.1% TFA-containing acetonitrile 0 to 5 minutes A 80%/B 20% 5 to 25 minutes A 80%/B 20%.fwdarw.B 100% 25 to 30 minutes B 100% 30 to 40 minutes A 80%/B 20% Flow rate: 1 ml/min Temperature: 40° C. Detection: UV205 nm Injection amount: 20 μL Authentic sample: Surfactin Sodium Salt (Wako Pure Chemicals) Concentration: 30 ppm, 120 ppm Solvent: 0.1% TFA-containing acetonitrile/distilled water (90:10, v/v)

(41) Each medium was compared with the authentic sample in terms of peak area detected at the elution time of 27.6 minutes and that of 28.4 minutes, thereby calculating the surfactin concentration in the culture medium.

(42) Results are shown in Table 6.

(43) TABLE-US-00006 TABLE 6 Test Result Sporulation- Bacterial cell Spore Surfactin Test inhibiting Concentration concentration Sporulation concentration concentration No. substance added (ppm) (/mL) rate (/mL) (ppm) 1 — — 5.8e+9 100% 5.8e+9 38.4 2 Erythromycin 0.1 8.1e+9  99% 8.0e+9 58.5 3 Erythromycin 0.2 6.9e+9 100% 6.9e+9 75.7

(44) In the test sections with the erythromycin concentrations of 0.1 ppm and 0.2 ppm, the spore concentration and the surfactin concentration tended to increase.

(45) Through addition of a given amount of an antibiotic, not only the productivity of spores but also the productivity of surfactin as a useful substance improved.