US20200199633A2 - Process for producing lactic acid or its salts from fermentation using thermotolerance bacillus bacteria - Google Patents
Process for producing lactic acid or its salts from fermentation using thermotolerance bacillus bacteria Download PDFInfo
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- US20200199633A2 US20200199633A2 US15/738,888 US201615738888A US2020199633A2 US 20200199633 A2 US20200199633 A2 US 20200199633A2 US 201615738888 A US201615738888 A US 201615738888A US 2020199633 A2 US2020199633 A2 US 2020199633A2
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- US
- United States
- Prior art keywords
- lactic acid
- producing lactic
- salts according
- carbon source
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 186
- 239000004310 lactic acid Substances 0.000 title claims abstract description 93
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 93
- 238000000034 method Methods 0.000 title claims abstract description 60
- 230000008569 process Effects 0.000 title claims abstract description 53
- 150000003839 salts Chemical class 0.000 title claims abstract description 51
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 16
- 238000000855 fermentation Methods 0.000 title description 29
- 230000004151 fermentation Effects 0.000 title description 29
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 57
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 57
- 241000894006 Bacteria Species 0.000 claims abstract description 53
- 238000011218 seed culture Methods 0.000 claims abstract description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 42
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 26
- 230000003287 optical effect Effects 0.000 claims description 17
- 235000000346 sugar Nutrition 0.000 claims description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 11
- 239000003637 basic solution Substances 0.000 claims description 10
- 241000283910 Bacillus acidiproducens Species 0.000 claims description 5
- 241000193749 Bacillus coagulans Species 0.000 claims description 5
- 239000000908 ammonium hydroxide Substances 0.000 claims description 5
- 229940054340 bacillus coagulans Drugs 0.000 claims description 5
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 claims description 4
- 150000002016 disaccharides Chemical class 0.000 claims description 4
- 150000002772 monosaccharides Chemical group 0.000 claims description 4
- 150000004043 trisaccharides Chemical class 0.000 claims description 4
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 3
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- ODEHMIGXGLNAKK-OESPXIITSA-N 6-kestotriose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 ODEHMIGXGLNAKK-OESPXIITSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 claims description 2
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 claims description 2
- 229930091371 Fructose Natural products 0.000 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims description 2
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 claims description 2
- DBTMGCOVALSLOR-VXXRBQRTSA-N alpha-D-Glcp-(1->3)-alpha-D-Glcp-(1->3)-D-Glcp Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](O)[C@@H](O[C@H]2[C@@H]([C@@H](CO)OC(O)[C@@H]2O)O)O[C@H](CO)[C@H]1O DBTMGCOVALSLOR-VXXRBQRTSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 2
- 229930182830 galactose Natural products 0.000 claims description 2
- DBTMGCOVALSLOR-AXAHEAMVSA-N galactotriose Natural products OC[C@@H]1O[C@@H](O[C@@H]2[C@@H](O)[C@H](CO)O[C@@H](O[C@H]3[C@@H](O)[C@H](O)O[C@@H](CO)[C@@H]3O)[C@@H]2O)[C@H](O)[C@H](O)[C@H]1O DBTMGCOVALSLOR-AXAHEAMVSA-N 0.000 claims description 2
- FBJQEBRMDXPWNX-FYHZSNTMSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H]2[C@H]([C@H](O)[C@@H](O)C(O)O2)O)O1 FBJQEBRMDXPWNX-FYHZSNTMSA-N 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 claims description 2
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical group O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims description 2
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 0.000 claims description 2
- 125000000185 sucrose group Chemical group 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 description 38
- 238000007792 addition Methods 0.000 description 13
- 230000000052 comparative effect Effects 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000000126 substance Substances 0.000 description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- 238000005273 aeration Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 3
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 239000008107 starch Substances 0.000 description 3
- 235000019698 starch Nutrition 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 2
- 239000001527 calcium lactate Substances 0.000 description 2
- 235000011086 calcium lactate Nutrition 0.000 description 2
- 229960002401 calcium lactate Drugs 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- AZUYLZMQTIKGSC-UHFFFAOYSA-N 1-[6-[4-(5-chloro-6-methyl-1H-indazol-4-yl)-5-methyl-3-(1-methylindazol-5-yl)pyrazol-1-yl]-2-azaspiro[3.3]heptan-2-yl]prop-2-en-1-one Chemical compound ClC=1C(=C2C=NNC2=CC=1C)C=1C(=NN(C=1C)C1CC2(CN(C2)C(C=C)=O)C1)C=1C=C2C=NN(C2=CC=1)C AZUYLZMQTIKGSC-UHFFFAOYSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- IUVCFHHAEHNCFT-INIZCTEOSA-N 2-[(1s)-1-[4-amino-3-(3-fluoro-4-propan-2-yloxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]ethyl]-6-fluoro-3-(3-fluorophenyl)chromen-4-one Chemical compound C1=C(F)C(OC(C)C)=CC=C1C(C1=C(N)N=CN=C11)=NN1[C@@H](C)C1=C(C=2C=C(F)C=CC=2)C(=O)C2=CC(F)=CC=C2O1 IUVCFHHAEHNCFT-INIZCTEOSA-N 0.000 description 1
- 241000209134 Arundinaria Species 0.000 description 1
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 1
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 1
- 229910052925 anhydrite Inorganic materials 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000002729 catgut Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000909 electrodialysis Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000010408 film Substances 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 235000021374 legumes Nutrition 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 235000019837 monoammonium phosphate Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- -1 starch or cellulose Chemical class 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000003039 volatile agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Definitions
- This invention relates to a process for producing lactic acid or its salts from fermentation using thermotolerance Bacillus bacteria, said process comprising the following steps:
- thermotolerance Bacillus bacteria cultivating thermotolerance Bacillus bacteria to obtain seed culture
- step (b) increasing cell number of bacteria by inoculating the seed culture obtained from step (a) into a fermenter containing an initial carbon source under an aerobic condition;
- step (c) fermenting the seed culture obtained from step (b) in the fermenter under a microaerobic condition to obtain lactic acid or its salts;
- step (b) comprising at least one addition of the carbon source under any one of the following conditions, which are independent to each other, to increase a concentration of the carbon source:
- This invention is in the field of biotechnology relating to the fermentation process and bacteria that can produce lactic acid.
- lactic acid is widely used in plastic industry, food and drug industry, and cosmetic industry.
- plastic industry lactic acid is widely used, especially for the production of polyester such as polylactic acid or poly(lactic-co-glycolic acid).
- Polymer produced from lactic acid has an advantage that it is biodegradable and biocompatible. Said polymer can be used in many applications such as fiber in textile, film, catgut packaging, and scaffold in medical.
- lactic acid such as chemical synthesis and biotechnology.
- the biotechnology has several advantages, including utilization of renewable resources for microbial fermentation such as cassava, corn, wheat, or cane.
- the microbial fermentation can produce lactic acid with high optical purity.
- lactic acid productions are the fermentation of sugar such as glucose, sucrose, maltose, or other carbohydrates such as starch or cellulose, wherein microorganisms that can produce lactic acid can be both bacteria and fungi.
- Bacteria in genus Lactobacillus, Leuconostoc , and Streptococcus are well known for the production of lactic acid from sugar under an anaerobic condition, leading to energy saving and providing higher yield than the one produced by fungi.
- said bacteria genus are fastidious bacteria, which need vitamins and essential amino acids for their growth.
- said bacteria genus cannot produce enzyme to convert starch into sugar, which needs the pretreatment step of converting starch to sugar prior to fermentation. This increases the production cost.
- One attempt to reduce the lactic acid production cost is the reduction of production step such as the use of thermotolerance Bacillus bacteria in lactic acid production.
- This is because normally the raw material of lactic acid production requires a pretreatment process prior to fermentation.
- pretreatment can be used, including mechanical treatment, heat treatment, chemical treatment, or enzyme treatment, depending on the physical, chemical, and nutritional property of such carbon sources.
- Said pretreatment processes are generally performed at high temperature in a range from about 50 to about 100° C. General bacteria cannot grow at said temperature. Therefore, an additional step is needed to reduce the temperature to a room temperature prior to using such carbon sources in lactic acid production. This results in complication of production process and higher production cost.
- lactic acid production cost is to eliminate the purifying step of lactic acid obtained from fermentation.
- the neutralizing agent is one of important factors. Generally, calcium carbonate (CaCO 3 ) that is used in the production of lactic acid generates calcium lactate as by product. Therefore, the acidification step is further needed to convert calcium lactate into lactic acid by using acid solution such as sulfuric acid (H 2 SO 4 ). This contributes to the complication of production process and high production cost. Moreover, said process causes calcium sulfate (CaSO 4 ) which is the undesired by product, leading to a problem to eliminate said substance, especially in the industrial scale production.
- U.S. Pat. No. 5,002,881 and WO2006124633 disclosed the use of ammonium hydroxide (NH 4 OH) as neutralizing agent during fermentation in lactic acid production.
- ammonium hydroxide is a volatile compound which could be harmful to operator's health.
- ammonium hydroxide is a weak basic; thus, it is needed to be used in high amount to control pH that results in a difficulty in separation and purification steps.
- this invention aims to improve the process for producing lactic acid or its salt from fermentation to provide high productivity and yield, wherein said process can be performed easily and reduce complicated steps.
- Any tools, equipment, methods, or chemicals mentioned here mean tools, equipment, methods, or chemicals commonly operated or used by those skilled in the art, unless explicated stated otherwise that they are tools, equipment, methods, or chemicals specifically used in this invention.
- Microaerobic condition means condition that air has been controlled to be limited without further adding of any gas, including air or inert gas to evacuate the existing air.
- “Concentration of carbon source” unless stated specifically, means the concentration of carbon source in such system such as in a fermenter at a specific time.
- This invention relates to the process for producing lactic acid or its salts from fermentation using thermotolerance Bacillus bacteria. Said process comprising the following steps:
- thermotolerance Bacillus bacteria (a) cultivating thermotolerance Bacillus bacteria to obtain a seed culture
- step (b) increasing cell number of bacteria by inoculating the seed culture obtained from step (a) into a fermenter containing an initial carbon source under an aerobic condition;
- step (c) fermenting the seed culture obtained from step (b) in the fermenter under a microaerobic condition to obtain lactic acid or its salts;
- step (b) comprising at least one addition of the carbon source under any one of the following conditions, which are independent to each other, to increase a concentration of carbon source:
- the addition of carbon source in step (b) in any above conditions increases the concentration of the carbon source in the fermenter to 75% or more comparing to the initial concentration.
- the addition of said carbon source includes the addition that results in the concentration larger than the initial concentration.
- the addition of carbon source is performed under the condition that the concentration of the carbon source in the fermenter reduces to 25% or less comparing to the initial concentration.
- the addition of the carbon source is performed under the step (b) is carried out for the time of at least one half.
- the addition of the carbon source is performed under the condition that the optical density (OD) of bacteria cell in the fermenter increases at least 10 to 50 times.
- OD optical density
- the concentration of the initial carbon source in step (b) may be in a range of 10 to 20 g/L, preferably about 15 g/L.
- said process for producing lactic acid or its salts may be carried out at the temperature in a range of about 45 to about 60° C., preferably at the temperature about 48 to 52° C.
- thermotolerance Bacillus genus in the step (a) may be selected from Bacillus acidiproducens, Bacillus coagulans , or a mixture of these bacteria.
- the initial optical density of bacteria cell in the step (a) is about 0.20 to 0.70, preferably 0.20 to 0.40.
- the step (a) may be carried out for about 2 to 5 hours, preferably about 3 to 4 hours.
- a bacteria cultivation media may contain nitrogen source that may be selected from yeast extract, peptone extract, beef extract and legume extract.
- the bacteria cultivation media and bacteria cell increasing media may contain inorganic nitrogen source that may be selected from ammonium chloride, ammonium nitrate, and ammonium dihydrogen phosphate.
- inorganic nitrogen source may be selected from ammonium chloride, ammonium nitrate, and ammonium dihydrogen phosphate.
- the step (b) may be carried out for about 2 to 5 hours, preferably about 3 to 4 hours, and most preferably about 4 hours.
- the step (c) further comprises at least one addition of the carbon source.
- the concentration of carbon source in step (c) may be in a range of about 100 to 200 g/L, preferably about 100 g/L.
- the step (c) is controlled to have pH of 6 to 7 with a use of basic solution, preferably have pH of 6.5 with a use of basic solution.
- the basic solution may be selected from sodium hydroxide (NaOH), ammonium hydroxide (NH 4 OH), potassium hydroxide (KOH), or a mixture thereof, preferably sodium hydroxide or potassium hydroxide, and most preferably sodium hydroxide.
- the carbon source used in the production of lactic acid or its salt is a fermentable sugar.
- the fermentable sugar is any sugar that can be found in nature or any sugar derived from a substance comprising sugar. Said sugar may be modified or unmodified.
- the fermentable sugar may be selected from, but not limited to monosaccharide, disaccharide, trisaccharide, or a mixture thereof.
- the monosaccharide may be selected from glucose, fructose, galactose, or a mixture thereof.
- the disaccharide may be selected from sucrose, lactose, maltose, cellobiose, or a mixture thereof.
- the trisaccharide may be selected from raffinose, isomaltotriose, maltotriose, nigerotriose, kestose, or a mixture thereof.
- the fermentable sugar is glucose
- the fermenter in step (b) and/or step (c) further comprises a mixer.
- the mixer has a speed in a range of about 150 to 450 rpm, preferably about 300 rpm.
- the change of fermenter condition from aerobic condition in step (b) to microaerobic condition in step (c) may be performed by stopping aeration in the fermenter in order that the remaining air from step (b) has been consumed, or adding nitrogen to replace air during fermentation in step (c).
- the fermentation in step (c) may be performed in batch, semi-batch, or continuous.
- said production of lactic acid or its salt may further comprises the step of separating and purifying the mixture obtained from step (c).
- the separation and purification may be selected from, but not limited to centrifugation, filtration, membrane filtration, flocculation, extraction, distillation, crystallization, filtration, ion exchange resin, or electrodialysis.
- Glucose and lactic acid are analyzed by high performance liquid chromatography using a Shimadzu equipped with Biorad, Aminex HPX-87H ion exclusion organic acid (300 mm ⁇ 7.8 mm) at a temperature around 45° C., and reflective index detector Shimadzu-RID-10A for detecting a signal comparing to a standard signal.
- Optical density (OD) of bacteria during cultivation and fermentation is analyzed by spectrophotometry at a wavelength 600 nm.
- Yield is calculated from a ratio of an amount of produced lactic acid to an amount of carbon source used during fermentation.
- Yield of produced lactic acid per bacteria cell (Y p/x ) represents the effectiveness of bacteria cell in the production of lactic acid, which is calculated from a ratio of an amount of produced lactic acid and a difference between the bacteria optical density after fermentation step and after increasing of bacteria cell step.
- media for culturing bacteria and increasing bacteria cell has composition (per liter) as following: 10 g glucose, 15 g yeast extract, 4 g ammonium chloride (NH 4 Cl), 5 g calcium hydroxide (Ca(OH) 2 ), and 20 mL saline solution.
- the cultivation of bacteria is carried out by adding thermotolerance Bacillus genus in a flask containing bacteria culturing media, wherein the initial concentration of bacteria is about 1% by volume and the initial optical density is about 0.30-0.40.
- the sample is centrifuged at about 250 rpm for 3 hours at a temperature of about 50° C. to obtain a seed culture.
- bacteria cell is increased by adding the seed culture in a 5 L fermenter containing about 2.5 L culture media with initial glucose concentration of about 15 g/L and calcium hydroxide (Ca(OH) 2 ) to control pH to be about 6.5.
- the step of increasing bacteria cell is performed at the temperature about 50° C. for about 3 hours under 1 vvm aeration and 300 rpm mixing.
- the aeration is stopped and about 1 L of 350 g/L glucose solution is added into the fermenter in order to achieve the initial glucose concentration of 100 g/L at the beginning of the fermentation step.
- the fermentation is operated under a microaerobic condition at the temperature of about 50° C. and the mixing speed of about 300 rpm by using various basic solutions in Table 1 to control pH to be about 6.5.
- the fermentation is performed until glucose is not detected.
- the products are centrifuged at about 10,000 rpm for about 5 minutes. The obtained products are analyzed for produced lactic acid and the optical density of bacteria during fermentation.
- thermotolerance Bacillus genus is added into a flask containing bacteria culturing media, wherein the initial concentration of bacteria is about 1% by volume and the initial optical density is about 0.30-0.40.
- the sample is centrifuged at about 250 rpm for 3 hours at a temperature of about 50° C. to obtain a seed culture.
- bacterial cell is increased by adding the seed culture in a 5 L fermenter containing about 2.5 L culture media with initial glucose concentration of about 15 g/L and calcium hydroxide (Ca(OH) 2 ) to control pH to be about 6.5.
- the step of increasing bacteria cell is performed at the temperature about 50° C. for about 2 hours under 1 vvm aeration and 300 rpm mixing.
- the step of increasing bacteria cell is operated for another 2 hours.
- the fermentation is performed under a microaerobic condition at the temperature of about 50° C. and the mixing speed of about 300 rpm by using various basic solutions in Table 1 to control pH to be about 6.5.
- the fermentation is performed until glucose is not detected.
- the products are centrifuged at about 10,000 rpm for about 5 minutes. The obtained products are analyzed for produced lactic acid and the optical density of bacteria during fermentation.
- examples A1, A2, A3 to comparative examples 1a, 2a, and 3a respectively which are the lactic acid production by using Bacillus acidiproducens strain
- the step of increasing bacteria cell comprising at least one addition of the carbon source results in the higher Y p/x , productivity, and yield.
- the lactic acid production according to the invention can use sodium hydroxide and potassium hydroxide solutions which are strong acids to control pH during fermentation and gives rise to high lactic acid yield.
- examples B1, B2, B3 to comparative examples 1b, 2b, and 3b, respectively which are the lactic acid production by using Bacillus coagulans strain it can be observed that at least one addition of the carbon source in the step of increasing bacteria cell results in greatly higher Y p/x . This indicates an increase of efficiency in utilizing carbon source.
- the lactic acid production according to the invention can enhance the efficiency of lactic acid production from thermotolerance Bacillus genus, which can be performed easily and can reduce complicated steps as indicated in the objective of this invention.
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Abstract
Description
- This invention relates to a process for producing lactic acid or its salts from fermentation using thermotolerance Bacillus bacteria, said process comprising the following steps:
- (a) cultivating thermotolerance Bacillus bacteria to obtain seed culture;
- (b) increasing cell number of bacteria by inoculating the seed culture obtained from step (a) into a fermenter containing an initial carbon source under an aerobic condition;
- (c) fermenting the seed culture obtained from step (b) in the fermenter under a microaerobic condition to obtain lactic acid or its salts;
- wherein the step (b) comprising at least one addition of the carbon source under any one of the following conditions, which are independent to each other, to increase a concentration of the carbon source:
-
- when the concentration of the carbon source in the fermenter reduces to 50% or less comparing to the initial concentration;
- when the step (b) is carried out for a time of at least one third;
- when an optical density (OD) of bacterial cell in the fermenter increases at least 10 times.
- This invention is in the field of biotechnology relating to the fermentation process and bacteria that can produce lactic acid.
- It is well known that lactic acid is widely used in plastic industry, food and drug industry, and cosmetic industry. For plastic industry, lactic acid is widely used, especially for the production of polyester such as polylactic acid or poly(lactic-co-glycolic acid). Polymer produced from lactic acid has an advantage that it is biodegradable and biocompatible. Said polymer can be used in many applications such as fiber in textile, film, catgut packaging, and scaffold in medical.
- At present, there are several production processes of lactic acid such as chemical synthesis and biotechnology. The biotechnology has several advantages, including utilization of renewable resources for microbial fermentation such as cassava, corn, wheat, or cane. Moreover, the microbial fermentation can produce lactic acid with high optical purity.
- Most of lactic acid productions are the fermentation of sugar such as glucose, sucrose, maltose, or other carbohydrates such as starch or cellulose, wherein microorganisms that can produce lactic acid can be both bacteria and fungi.
- Bacteria in genus Lactobacillus, Leuconostoc, and Streptococcus are well known for the production of lactic acid from sugar under an anaerobic condition, leading to energy saving and providing higher yield than the one produced by fungi. However, said bacteria genus are fastidious bacteria, which need vitamins and essential amino acids for their growth. Moreover, said bacteria genus cannot produce enzyme to convert starch into sugar, which needs the pretreatment step of converting starch to sugar prior to fermentation. This increases the production cost.
- One problem of polymer production from lactic acid is high production cost. It is necessary to develop the lactic acid production process with lower production cost, high yield and high productivity. In addition, there are several attempts to develop microorganisms and culturing steps in order to produce robust microorganisms that have ability to grow and proliferate with high rate.
- One attempt to reduce the lactic acid production cost is the reduction of production step such as the use of thermotolerance Bacillus bacteria in lactic acid production. This is because normally the raw material of lactic acid production requires a pretreatment process prior to fermentation. Several pretreatment can be used, including mechanical treatment, heat treatment, chemical treatment, or enzyme treatment, depending on the physical, chemical, and nutritional property of such carbon sources. Said pretreatment processes are generally performed at high temperature in a range from about 50 to about 100° C. General bacteria cannot grow at said temperature. Therefore, an additional step is needed to reduce the temperature to a room temperature prior to using such carbon sources in lactic acid production. This results in complication of production process and higher production cost.
- Therefore, there had been developments in production process of lactic acid using thermotolerance Bacillus bacteria such as Bacillus coagulans, Bacillus acidiproducens, Bacillus stearothermophilus, and Bacillus licheniformis as disclosed in Ouyang et al, (Appl. Biochem. Biotechnol., 2012, 168, 2387-2397), Wang et al, (Bioresour. Technol., 2011, 102, 8152-8158), and U.S. Pat. No. 8,119,376. However, it was found that the production of lactic acid as disclosed in said documents resulted in low productivity and yield, which was the limitation for the production of lactic acid in the industrial scale.
- One attempt to reduce lactic acid production cost is to eliminate the purifying step of lactic acid obtained from fermentation. The neutralizing agent is one of important factors. Generally, calcium carbonate (CaCO3) that is used in the production of lactic acid generates calcium lactate as by product. Therefore, the acidification step is further needed to convert calcium lactate into lactic acid by using acid solution such as sulfuric acid (H2SO4). This contributes to the complication of production process and high production cost. Moreover, said process causes calcium sulfate (CaSO4) which is the undesired by product, leading to a problem to eliminate said substance, especially in the industrial scale production.
- U.S. Pat. No. 5,002,881 and WO2006124633 disclosed the use of ammonium hydroxide (NH4OH) as neutralizing agent during fermentation in lactic acid production. However, ammonium hydroxide is a volatile compound which could be harmful to operator's health. Besides, ammonium hydroxide is a weak basic; thus, it is needed to be used in high amount to control pH that results in a difficulty in separation and purification steps.
- Ye et al, (Bioresour. Technol., 2013, 132, 38-44), Qin et al, (Bioresour. Technol., 2010, 101, 7570-7576) disclosed the use of strong basic such as sodium hydroxide (NaOH) and potassium hydroxide (KOH) during the fermentation of lactic acid production. However, it was found that the use of said basic caused the negative effect on the bacteria cell, resulting in low productivity and yield.
- From the reasons mentioned above, this invention aims to improve the process for producing lactic acid or its salt from fermentation to provide high productivity and yield, wherein said process can be performed easily and reduce complicated steps.
- Technical terms or scientific terms used herein have definitions as understood by those having an ordinary skill in the art, unless stated otherwise.
- Any tools, equipment, methods, or chemicals mentioned here mean tools, equipment, methods, or chemicals commonly operated or used by those skilled in the art, unless explicated stated otherwise that they are tools, equipment, methods, or chemicals specifically used in this invention.
- Use of singular noun or singular pronoun with “comprising” in the claims or the specification refers to “one” and also “one or more”, “at least one”, and “one or more than one”.
- Throughout this application, the term “about” is used to indicate that any value presented or showed herein may potentially vary or deviate. Such variation or deviation may result from errors of equipment, method, or from individual operator implementing equipment or method. These variations or deviations caused by the changes of physical properties.
- “Microaerobic condition” means condition that air has been controlled to be limited without further adding of any gas, including air or inert gas to evacuate the existing air.
- “Concentration of carbon source” unless stated specifically, means the concentration of carbon source in such system such as in a fermenter at a specific time.
- Hereafter, invention embodiments are shown without any purpose to limit the scope of the invention.
- This invention relates to the process for producing lactic acid or its salts from fermentation using thermotolerance Bacillus bacteria. Said process comprising the following steps:
- (a) cultivating thermotolerance Bacillus bacteria to obtain a seed culture;
- (b) increasing cell number of bacteria by inoculating the seed culture obtained from step (a) into a fermenter containing an initial carbon source under an aerobic condition;
- (c) fermenting the seed culture obtained from step (b) in the fermenter under a microaerobic condition to obtain lactic acid or its salts;
- wherein the step (b) comprising at least one addition of the carbon source under any one of the following conditions, which are independent to each other, to increase a concentration of carbon source:
-
- when the concentration of the carbon source in the fermenter reduces to 50% or less comparing to the initial concentration;
- when the step (b) is carried out for a time of at least one third;
- when an optical density (OD) of bacteria cell in the fermenter increases at least 10 times.
- In one embodiment, the addition of carbon source in step (b) in any above conditions increases the concentration of the carbon source in the fermenter to 75% or more comparing to the initial concentration. The addition of said carbon source includes the addition that results in the concentration larger than the initial concentration.
- Preferably, the addition of carbon source is performed under the condition that the concentration of the carbon source in the fermenter reduces to 25% or less comparing to the initial concentration.
- Preferably, the addition of the carbon source is performed under the step (b) is carried out for the time of at least one half.
- Preferably, the addition of the carbon source is performed under the condition that the optical density (OD) of bacteria cell in the fermenter increases at least 10 to 50 times.
- In one embodiment, the concentration of the initial carbon source in step (b) may be in a range of 10 to 20 g/L, preferably about 15 g/L.
- In one embodiment, said process for producing lactic acid or its salts may be carried out at the temperature in a range of about 45 to about 60° C., preferably at the temperature about 48 to 52° C.
- In one embodiment, the thermotolerance Bacillus genus in the step (a) may be selected from Bacillus acidiproducens, Bacillus coagulans, or a mixture of these bacteria.
- In one embodiment, the initial optical density of bacteria cell in the step (a) is about 0.20 to 0.70, preferably 0.20 to 0.40.
- In one embodiment, the step (a) may be carried out for about 2 to 5 hours, preferably about 3 to 4 hours.
- In one embodiment, a bacteria cultivation media may contain nitrogen source that may be selected from yeast extract, peptone extract, beef extract and legume extract.
- In one embodiment, the bacteria cultivation media and bacteria cell increasing media may contain inorganic nitrogen source that may be selected from ammonium chloride, ammonium nitrate, and ammonium dihydrogen phosphate. The use of said inorganic compounds for nitrogen source has benefit in the production of lactic acid in industrial scale because they are cheap, affordable, and can be quality controlled.
- In one embodiment, the step (b) may be carried out for about 2 to 5 hours, preferably about 3 to 4 hours, and most preferably about 4 hours.
- In one embodiment, the step (c) further comprises at least one addition of the carbon source.
- In one embodiment, the concentration of carbon source in step (c) may be in a range of about 100 to 200 g/L, preferably about 100 g/L.
- In one embodiment, the step (c) is controlled to have pH of 6 to 7 with a use of basic solution, preferably have pH of 6.5 with a use of basic solution.
- In one embodiment, the basic solution may be selected from sodium hydroxide (NaOH), ammonium hydroxide (NH4OH), potassium hydroxide (KOH), or a mixture thereof, preferably sodium hydroxide or potassium hydroxide, and most preferably sodium hydroxide.
- In one embodiment, the carbon source used in the production of lactic acid or its salt is a fermentable sugar.
- The fermentable sugar is any sugar that can be found in nature or any sugar derived from a substance comprising sugar. Said sugar may be modified or unmodified.
- The fermentable sugar may be selected from, but not limited to monosaccharide, disaccharide, trisaccharide, or a mixture thereof.
- In one embodiment, the monosaccharide may be selected from glucose, fructose, galactose, or a mixture thereof.
- In one embodiment, the disaccharide may be selected from sucrose, lactose, maltose, cellobiose, or a mixture thereof.
- In one embodiment, the trisaccharide may be selected from raffinose, isomaltotriose, maltotriose, nigerotriose, kestose, or a mixture thereof.
- Preferably, the fermentable sugar is glucose.
- In one embodiment, the fermenter in step (b) and/or step (c) further comprises a mixer.
- In one embodiment, the mixer has a speed in a range of about 150 to 450 rpm, preferably about 300 rpm.
- In one embodiment, the change of fermenter condition from aerobic condition in step (b) to microaerobic condition in step (c) may be performed by stopping aeration in the fermenter in order that the remaining air from step (b) has been consumed, or adding nitrogen to replace air during fermentation in step (c).
- In one embodiment, the fermentation in step (c) may be performed in batch, semi-batch, or continuous.
- In another embodiment, said production of lactic acid or its salt may further comprises the step of separating and purifying the mixture obtained from step (c).
- The separation and purification may be selected from, but not limited to centrifugation, filtration, membrane filtration, flocculation, extraction, distillation, crystallization, filtration, ion exchange resin, or electrodialysis.
- The following is the property testing according to the invention, wherein the methods and equipment used in the test are commonly used methods and are not intended to limit the scope of the invention.
- Glucose and lactic acid are analyzed by high performance liquid chromatography using a Shimadzu equipped with Biorad, Aminex HPX-87H ion exclusion organic acid (300 mm×7.8 mm) at a temperature around 45° C., and reflective index detector Shimadzu-RID-10A for detecting a signal comparing to a standard signal.
- Optical density (OD) of bacteria during cultivation and fermentation is analyzed by spectrophotometry at a wavelength 600 nm.
- Yield is calculated from a ratio of an amount of produced lactic acid to an amount of carbon source used during fermentation.
- Yield of produced lactic acid per bacteria cell (Yp/x) represents the effectiveness of bacteria cell in the production of lactic acid, which is calculated from a ratio of an amount of produced lactic acid and a difference between the bacteria optical density after fermentation step and after increasing of bacteria cell step.
- The following examples are presented to illustrate the present invention without limiting the scope of the invention.
- In the following examples, unless stated specifically, media for culturing bacteria and increasing bacteria cell has composition (per liter) as following: 10 g glucose, 15 g yeast extract, 4 g ammonium chloride (NH4Cl), 5 g calcium hydroxide (Ca(OH)2), and 20 mL saline solution.
- To study the effect of multiple additions of carbon source in the step of increasing bacteria cell on lactic acid or its salt production, a sample without adding carbon source in the step of increasing bacteria cell is used to compare with lactic acid production according to the invention.
- The cultivation of bacteria is carried out by adding thermotolerance Bacillus genus in a flask containing bacteria culturing media, wherein the initial concentration of bacteria is about 1% by volume and the initial optical density is about 0.30-0.40. The sample is centrifuged at about 250 rpm for 3 hours at a temperature of about 50° C. to obtain a seed culture. Then, bacteria cell is increased by adding the seed culture in a 5 L fermenter containing about 2.5 L culture media with initial glucose concentration of about 15 g/L and calcium hydroxide (Ca(OH)2) to control pH to be about 6.5. The step of increasing bacteria cell is performed at the temperature about 50° C. for about 3 hours under 1 vvm aeration and 300 rpm mixing. Then, the aeration is stopped and about 1 L of 350 g/L glucose solution is added into the fermenter in order to achieve the initial glucose concentration of 100 g/L at the beginning of the fermentation step. The fermentation is operated under a microaerobic condition at the temperature of about 50° C. and the mixing speed of about 300 rpm by using various basic solutions in Table 1 to control pH to be about 6.5. The fermentation is performed until glucose is not detected. Then, the products are centrifuged at about 10,000 rpm for about 5 minutes. The obtained products are analyzed for produced lactic acid and the optical density of bacteria during fermentation.
- The thermotolerance Bacillus genus is added into a flask containing bacteria culturing media, wherein the initial concentration of bacteria is about 1% by volume and the initial optical density is about 0.30-0.40. The sample is centrifuged at about 250 rpm for 3 hours at a temperature of about 50° C. to obtain a seed culture. Then, bacterial cell is increased by adding the seed culture in a 5 L fermenter containing about 2.5 L culture media with initial glucose concentration of about 15 g/L and calcium hydroxide (Ca(OH)2) to control pH to be about 6.5. The step of increasing bacteria cell is performed at the temperature about 50° C. for about 2 hours under 1 vvm aeration and 300 rpm mixing. Then, about 0.5 L of 90 g/L glucose solution was added. The step of increasing bacteria cell is operated for another 2 hours. Then, the fermentation is performed under a microaerobic condition at the temperature of about 50° C. and the mixing speed of about 300 rpm by using various basic solutions in Table 1 to control pH to be about 6.5. The fermentation is performed until glucose is not detected. Then, the products are centrifuged at about 10,000 rpm for about 5 minutes. The obtained products are analyzed for produced lactic acid and the optical density of bacteria during fermentation.
- From Table 1, when comparing examples A1, A2, A3 to comparative examples 1a, 2a, and 3a, respectively which are the lactic acid production by using Bacillus acidiproducens strain, it can be found that the step of increasing bacteria cell comprising at least one addition of the carbon source results in the higher Yp/x, productivity, and yield. Moreover, it can be found that the lactic acid production according to the invention can use sodium hydroxide and potassium hydroxide solutions which are strong acids to control pH during fermentation and gives rise to high lactic acid yield.
- Furthermore, when comparing examples B1, B2, B3 to comparative examples 1b, 2b, and 3b, respectively which are the lactic acid production by using Bacillus coagulans strain, it can be observed that at least one addition of the carbon source in the step of increasing bacteria cell results in greatly higher Yp/x. This indicates an increase of efficiency in utilizing carbon source.
- Therefore, regarding above results, it can be summarized that the lactic acid production according to the invention can enhance the efficiency of lactic acid production from thermotolerance Bacillus genus, which can be performed easily and can reduce complicated steps as indicated in the objective of this invention.
-
TABLE 1 The lactic acid bacteria production from thermotolerance Bacillus genus in various conditions Optical density Optical density Basic (OD) before (OD) after solution in Fermentation the step of the step of Optical density Lactic acid fermentation period increasing increasing (OD) after concentration Yield Productivity Example step (hr) bacteria cell bacteria cell fermentation (g/L) (g/g) (g/L/hr) Yp/x Bacillus acidiproducens strain Comparative 7M 17 0.32 7.7 20.7 87.3 0.87 5.1 6.7 Example 1a NH4OH Comparative 10M 19 0.32 7.0 22.0 99.9 0.94 5.6 6.6 Example 2a NaOH Comparative 10M 18 0.30 11.4 25.1 102.9 0.95 5.7 7.5 Example 3a KOH Example A1 7M 17 0.28 10.0 21.1 99.1 0.99 5.9 8.2 NH4OH Example A2 10M 18 0.35 11.3 22.6 108.9 1.04 6.1 8.8 NaOH Example A3 10M 19 0.31 12.5 23.7 102.0 0.92 5.5 9.1 KOH Bacillus coagulans strain Comparative 7M 18 0.31 9.3 21.9 93.5 0.90 5.2 7.4 Example 1b NH4OH Comparative 10M 12 0.32 10.4 20.4 94.4 0.94 7.9 9.4 Example 2b NaOH Comparative 10M 18 0.32 9.0 21.6 102.6 1.00 5.7 8.1 Example 3b KOH Example B1 7M 20 0.31 13.6 28.3 102.8 1.00 5.1 6.9 NH4OH Example B2 10M 20 0.38 10.9 17.2 108.0 1.00 5.4 17.5 NaOH Example B3 10M 20 0.26 9.5 16.5 102.9 0.97 5.2 14.7 KOH - Best mode of the invention is as disclosed in the detailed description.
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TH1501003766 | 2015-06-29 | ||
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