CN106801026A - Bacterial strain and its production method used by a kind of utilization xylose mother liquid fermentation succinic acid-producing - Google Patents
Bacterial strain and its production method used by a kind of utilization xylose mother liquid fermentation succinic acid-producing Download PDFInfo
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- CN106801026A CN106801026A CN201710076734.1A CN201710076734A CN106801026A CN 106801026 A CN106801026 A CN 106801026A CN 201710076734 A CN201710076734 A CN 201710076734A CN 106801026 A CN106801026 A CN 106801026A
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- Prior art keywords
- mother liquid
- succinic acid
- xylose mother
- fermentation
- concentration
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- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 title claims abstract description 170
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title claims abstract description 154
- 239000007788 liquid Substances 0.000 title claims abstract description 108
- 238000000855 fermentation Methods 0.000 title claims abstract description 102
- 230000004151 fermentation Effects 0.000 title claims abstract description 100
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 title claims abstract description 86
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 title claims abstract description 86
- 239000001384 succinic acid Substances 0.000 title claims abstract description 65
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 38
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 19
- 241000948980 Actinobacillus succinogenes Species 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 33
- 229960005137 succinic acid Drugs 0.000 claims description 62
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 56
- 239000002609 medium Substances 0.000 claims description 31
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 28
- 229910052757 nitrogen Inorganic materials 0.000 claims description 28
- 241000894006 Bacteria Species 0.000 claims description 27
- 240000008042 Zea mays Species 0.000 claims description 23
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 23
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 22
- 235000005822 corn Nutrition 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 22
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 22
- 239000001963 growth medium Substances 0.000 claims description 21
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims description 20
- 239000002054 inoculum Substances 0.000 claims description 20
- 239000012530 fluid Substances 0.000 claims description 19
- 238000011218 seed culture Methods 0.000 claims description 18
- 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 16
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 16
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 15
- 239000001110 calcium chloride Substances 0.000 claims description 15
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 15
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 12
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 10
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 claims description 9
- 239000001095 magnesium carbonate Substances 0.000 claims description 9
- 229910000021 magnesium carbonate Inorganic materials 0.000 claims description 9
- 230000033228 biological regulation Effects 0.000 claims description 8
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 8
- 239000012467 final product Substances 0.000 claims description 8
- 235000012054 meals Nutrition 0.000 claims description 8
- 239000006174 pH buffer Substances 0.000 claims description 8
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 8
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 5
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 claims description 5
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 claims description 5
- 239000000347 magnesium hydroxide Substances 0.000 claims description 5
- 229910001862 magnesium hydroxide Inorganic materials 0.000 claims description 5
- 239000011591 potassium Substances 0.000 claims description 5
- 229910052700 potassium Inorganic materials 0.000 claims description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
- 235000003276 Apios tuberosa Nutrition 0.000 claims description 3
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 3
- 235000010744 Arachis villosulicarpa Nutrition 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 239000000047 product Substances 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 241000193830 Bacillus <bacterium> Species 0.000 claims 1
- 244000133018 Panax trifolius Species 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000029219 regulation of pH Effects 0.000 claims 1
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 238000005457 optimization Methods 0.000 abstract description 2
- 238000004064 recycling Methods 0.000 abstract description 2
- 235000011044 succinic acid Nutrition 0.000 description 54
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 11
- 239000002994 raw material Substances 0.000 description 11
- 235000013339 cereals Nutrition 0.000 description 9
- 239000003002 pH adjusting agent Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 239000012452 mother liquor Substances 0.000 description 6
- 241000606750 Actinobacillus Species 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000417230 Actinobacillus succinogenes 130Z Species 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 244000105624 Arachis hypogaea Species 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 210000002429 large intestine Anatomy 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- -1 poly butylene succinate Polymers 0.000 description 2
- 239000004631 polybutylene succinate Substances 0.000 description 2
- 229920002961 polybutylene succinate Polymers 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- OXHNLMTVIGZXSG-UHFFFAOYSA-N 1-Methylpyrrole Chemical class CN1C=CC=C1 OXHNLMTVIGZXSG-UHFFFAOYSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 description 1
- 241000609240 Ambelania acida Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 244000205754 Colocasia esculenta Species 0.000 description 1
- 235000006481 Colocasia esculenta Nutrition 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- WWUVLNPEWUTCEI-KRWDZBQOSA-N Flowerone Chemical compound C1=C(O)C(CC=C(C)C)=CC([C@H]2OC3=C(O)C(O)=CC(O)=C3C(=O)C2)=C1 WWUVLNPEWUTCEI-KRWDZBQOSA-N 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- 244000207740 Lemna minor Species 0.000 description 1
- 235000006439 Lemna minor Nutrition 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 241000606860 Pasteurella Species 0.000 description 1
- 241000606752 Pasteurellaceae Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 235000001855 Portulaca oleracea Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000001361 adipic acid Substances 0.000 description 1
- 235000011037 adipic acid Nutrition 0.000 description 1
- 239000002154 agricultural waste Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- 239000010905 bagasse Substances 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001555 benzenes Chemical class 0.000 description 1
- 239000004621 biodegradable polymer Substances 0.000 description 1
- 229920002988 biodegradable polymer Polymers 0.000 description 1
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 1
- OVYQSRKFHNKIBM-UHFFFAOYSA-N butanedioic acid Chemical compound OC(=O)CCC(O)=O.OC(=O)CCC(O)=O OVYQSRKFHNKIBM-UHFFFAOYSA-N 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 210000004027 cell Anatomy 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
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 229940099112 cornstarch Drugs 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- YVBUWVMMORNHHZ-UHFFFAOYSA-N flowerone Natural products CC(=C)CCc1cc(ccc1O)C1CC(=O)c2c(O)cc(O)c(O)c2O1 YVBUWVMMORNHHZ-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229940031958 magnesium carbonate hydroxide Drugs 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 150000003742 xyloses Chemical class 0.000 description 1
Classifications
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- 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
-
- 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
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- 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
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses the bacterial strain and its production method used by a kind of utilization xylose mother liquid fermentation succinic acid-producing, and the bacterial strain is Actinobacillus succinogenes（Actinobacillus succinogenes）PZ, deposit number is：CCTCC NO:M2016396.Actinobacillus succinogenes of the invention can utilize the xylose mother liquid fermentation production of succinic acid of high concentration, and succinic acid yield reaches as high as 95g/L, and succinic acid yield is up to 85%.Method of the present invention low cost, yield are high, process is simple, realize to xylose mother liquid high-efficiency resource recycling, and optimization and the environmental protection aspect to resource have great importance.
Description
Technical field
The invention belongs to industrial microorganism fermentation technical field, and in particular to one kind is using xylose mother liquid fermentation succinic acid-producing
Bacterial strain used and its production method.
Background technology
Succinic acid (Succinicacid), is a kind of common natural have also known as butanedioic acid (butanedioic acid)
Machine acid, discovery is separated by Georgius in initial 1546 from amber, thus is gained the name.The molecular formula of succinic acid is C4H6O4, molecule
It is 118.09 to measure, sour, and relative density is 1.572, and fusing point is 188 DEG C, 235 DEG C of boiling point (decomposition temperature).
A kind of important C4 platform chemicals of succinic acid, are BDO, tetrahydrofuran, gamma-butyrolacton, N- methyl pyrroles
The base stock of the important bulk chemical such as pyrrolidone, adipic acid and specialty chemicals, be widely used in food, plastics, medicine,
The industry such as spices, can synthesize more than 250 kinds of chemical products with substituted benzene.Moulded for synthesis in wherein most promising field
Material, it is synthesis poly butylene succinate (PBS), polyethylene glycol succinate (PES), polypropylene glycol succinate (PPS)
Deng the primary raw material of biodegradable polymer.
At present, the main production process of succinic acid is synthesized by chemically of oil, not only needs consumption a large amount of not
Reproducible petrochemical material, production cost is higher, and environmental pollution is serious.
Compared with chemical synthesis, microbe fermentation method has advantages below：(1) raw material is cheap, reproducible
Biomass resource；(2) a large amount of CO are can absorb in fermentation process2, environmental protection；(3) fermentation condition is gentle.Therefore, microorganism hair
Ferment method gets most of the attention in recent years, the study hotspot as recent domestic.
At present, about fermentation production of succinic acid raw material used in addition to glucose, predominantly grain raw material, such as U.S.
Argonne laboratories have carried out the research of Maize Production succinic acid, Japanese aginomoto and Mitsubishi Chemical Ind mutual association carry out with
Cornstarch is the research that fermenting raw materials produce succinic acid.But due to flourishing for the biological industry of grain raw material in recent years,
There is shortage in grain, and substantially, depending on grain unduly in addition can also influence the grain security of country to rise in price trend, so using
Cheap agricultural wastes are one of keys of reduction succinic acid production cost for raw material carries out succinic acid fermentation.Non- grain forms sediment
Powder raw material (cassava, banana dasheen, duckweed etc.), cane molasses, whey and cellulose etc. are used to produce succinic acid.At present, with change
Learn synthetic method to compare, the cost of producing succinic acid by microbial fermentation is still higher, it is necessary to further explore more cheap raw material.
Xylose mother liquid is to extract the accessory substance produced during xylose, price with cellulosic materials such as corncob, bagasse
It is very cheap.Under usual condition, often producing 1 ton of crystalline xylose can produce 1.5 tons of xylose mother liquid.Containing up in xylose mother liquid
60% or so mixed sugar, mainly xylose, arabinose and a small amount of glucose, with stronger development and application potentiality.In
State patent CN104789606A discloses a kind of method of utilization xylose mother liquid fermentation production of succinic acid, and it utilizes and recombinates large intestine bar
The method that bacterium (E.coli) xylose-fermenting mother liquor for fermentation produces succinic acid, needs to carry out detoxification to xylose mother liquid pre- in fermentation process
Treatment, not only increases production cost, and preprocessing process can cause carbohydrate to lose.Additionally, the strain for being used is large intestine
Oxydans genetic engineering bacterial strain, the shortcomings of there is bacterial strain unstability and fermentation process and produce endotoxin, limits its range of application.
Chinese patent 201010310881.0 discloses a kind of method that utilization fermenting crops straws produce succinic acid, is digested with stalk
The mixed liquor of liquid and xylose mother liquid produces succinic acid as carbon source through fermentation, is needed by soda boiling because prepared by straw enzymolysis, divided
From steps such as, enzymolysis, complex process, enzymolysis time is long, high cost, and the amount that xylose mother liquid is used is few, and concentration is low, and xylose is female
The utilization rate of liquid is not high.Chinese patent 200910089501.0 discloses a kind of method that utilization corncob produces succinic acid, profit
One or more in the mother liquor of isolated the first liquid glucose, the second liquid glucose or the first liquid glucose is hydrolyzed with corncob, using fourth
Diacid production bacterial strain ferment obtaining succinic acid.When being fermented using the mother liquor of the first liquid glucose, the yield of succinic acid is only
It is 5.46%, yield is extremely low, it is impossible to reach the requirement of industrialized production.
The content of the invention
The purpose of the present invention be directed to raw material high cost in existing succinic acid fermenting and producing, yield poorly, complex process is asked
Topic, there is provided bacterial strain and its production method used by a kind of utilization xylose mother liquid fermentation succinic acid-producing, method of the present invention low cost,
Yield is high, process is simple, and succinic acid yield reaches as high as 95g/L, and succinic acid yield is up to 85%.
The present invention is achieved through the following technical solutions：
A kind of bacterial strain used by utilization xylose mother liquid fermentation succinic acid-producing, the bacterial strain is Actinobacillus succinogenes
(Actinobacillus succinogenes) PZ, deposit number is：CCTCC NO:M2016396.
Actinobacillus succinogenes (Actinobacillus succinogenes) PZ of the present invention, preservation date is 2016
On July 19, in, depositary institution's full name is China typical culture collection center, referred to as CCTCC, depositary institution address：China
Wuhan Wuhan University, deposit number is CCTCC NO:M2016396.
The present invention also provides a kind of method that utilization xylose mother liquid ferments succinic acid-producing, including application deposit number is
CCTCC NO:The Actinobacillus succinogenes fermentation production of succinic acid of M2016396.
The Actinobacillus succinogenes obtained with the present invention, by the method for xylose mother liquid fermentation production of succinic acid, including
Following steps：
During Actinobacillus succinogenes PZ single bacterium colonies on flat board are accessed into seed culture medium, in anaerobic culture box or common
36-38 DEG C of culture 16-20h in incubator, bacterium number carries out two grades and expands numerous culture up to more than 300,000,000, according still further to 5% (V/V) inoculum concentration
8h, bacterium number obtains liquid seed up to more than 300,000,000；Liquid seed is inoculated into fermented and cultured by the inoculum concentration of 5-15% (V/V)
In base, 6.5-7.0 is maintained using pH buffer regulation zymotic fluid pH, full of N2Or CO2Environment in, rotating speed 100-200r/
Min, temperature is 36-38 DEG C, and batch fermentation or fed batch fermentation 30-72h obtain final product succinic acid.
Further illustrated as of the invention, the composition and concentration of the seed culture medium are：Glucose 8g/L, dusty yeast
12g/L, corn pulp 4g/L, NaHCO34g/L, NaH2PO49.6g/L, K2HPO41.55g/L。
Further illustrated as of the invention, the composition and concentration of the fermentation medium are during batch fermentation：Xylose is female
Liquid total reducing sugar 30-100g/L, nitrogen source 5-20g/L, potassium dihydrogen phosphate 2-10g/L, sodium acid carbonate 2-10g/L, calcium chloride 0.3-1.0g/
L, magnesium chloride 0.3-1.0g/L, the total sugar concentration of the xylose mother liquid is 50-80% (W:V).
Further illustrated as of the invention, the composition and concentration of the fermentation medium are during fed batch fermentation：Wood
Sugared mother liquor initial total sugar 30-40g/L, nitrogen source 5-20g/L, potassium dihydrogen phosphate 2-10g/L, sodium acid carbonate 2-10g/L, calcium chloride
0.3-1.0g/L, magnesium chloride 0.3-1.0g/L, intermittent flow adds the xylose mother liquid that total sugar concentration is 200-300g/L in fermentation process,
The total reducing sugar of zymotic fluid is controlled for 30-35g/L, the total sugar concentration of the xylose mother liquid is 50-80% (W:V).
Further illustrated as of the invention, the nitrogen source of the fermentation medium is corn pulp, dusty yeast, soy meal, flower
One or more in raw cake powder are mixed with arbitrary proportion.
Further illustrated as of the invention, the pH adjusting agent is basic magnesium carbonate, sodium carbonate, magnesium hydroxide, hydrogen-oxygen
One or more in change sodium are mixed with arbitrary proportion, and total concentration is 20-80g/L.
Further illustrated as of the invention, the composition and concentration of the fermentation medium are during batch fermentation：Xylose is female
Liquid total reducing sugar 40g/L, corn pulp 12g/L, potassium dihydrogen phosphate 6g/L, sodium acid carbonate 6g/L, calcium chloride 0.6g/L, magnesium chloride 0.7g/
L, the total sugar concentration of the xylose mother liquid is 60% (W:V).
Further illustrated as of the invention, the composition and concentration of the fermentation medium are during fed batch fermentation：Wood
Sugared mother liquor initial total sugar 40g/L, corn pulp 20g/L, potassium dihydrogen phosphate 10g/L, sodium acid carbonate 10g/L, calcium chloride 1.0g/L, chlorine
Change magnesium 1.0g/L, intermittent flow adds total sugar concentration to be the xylose mother liquid of 300g/L in fermentation process, and the total reducing sugar for controlling zymotic fluid is
35g/L, the total sugar concentration of the xylose mother liquid is 80% (W:V).
Compared with prior art, advantages of the present invention and have the beneficial effect that：
1st, the present invention replaces the glucose of costliness or cereal crops to be sent out as carbon source by the use of trade waste xylose mother liquid
Ferment produces succinic acid, has on the one hand saved substantial amounts of grain, ensures China's grain security, reduces production cost, on the other hand real
Xylose mother liquid high-efficiency resource recycling is showed, optimization and the environmental protection aspect to resource have great importance.
2nd, the present invention produces succinic acid by fermenting raw materials of xylose mother liquid, it is not necessary to carry out detoxification treatment, it is not required that match somebody with somebody
Other carbon source co-fermentations are closed, production method is simple, efficient, and succinic acid yield reaches as high as 95g/L, and succinic acid yield is up to
85%.
Brief description of the drawings
Fig. 1 is the Yield mapping of different nitrogen sources fermentation succinic acid-producing and its accessory substance in fermentation medium.
Specific embodiment
The following is Actinobacillus succinogenes CCTCC NO:The seed selection of M2016396, identification, life of being fermented using xylose mother liquid
The specific embodiment of succinic acid-producing.
Embodiment 1
The seed selection and identification of Actinobacillus succinogenes
1st, the method for the orthogenesis seed selection of Actinobacillus succinogenes is：Seed liquor is replaced first with the glycerine of 10g/L
Glucose be carbon source as the culture medium that carries out of orientation, anaerobic culture box is positioned over after inoculation, 37 DEG C of culture 12-18h are carried out
Passage, glycerol concentration increases to 20g/L after cultivating 7-10 days, the like, after about taming 90 days, the glycerol concentration of culture medium
Can improve to 80g/L, seed selection obtains one plant of Actinobacillus succinogenes PZ, the bacterial strain can be cultivated in glycerol concentration 80g/L and concentrated
Well-grown.Find that Actinobacillus succinogenes (Actinobacillus succinogenes) PZ can be in 100g/L simultaneously
Xylose mother liquid in growth it is preferable, it is possible to high concentration succinic acid is produced in fermentation.
2nd, the Actinobacillus succinogenes PZ for obtaining above-mentioned seed selection carries out 16S rDNA identified for genes, and according to《Primary
Outstanding formula Bacteria Identification handbook》Physiology and biochemistry identification is carried out, qualification result is：Actinobacillus succinogenes PZ produces butanedioic acid with bacterial strain
Actinobacillus GXAS137 similarities are 100%, are 99.5% with bacterial strain Actinobacillus succinogenes 130Z similarities.By shape
State and physiology, biochemical identification：Actinobacillus succinogenes PZ belongs to Gram-negative bacteria, amphimicrobian, and cell is in rod-short, nothing
Flagellum, without gemma, does not move, and does not also produce spore, can utilize common most of carbon source succinic acid-producings.
By above qualification result, it can be assumed that Actinobacillus succinogenes PZ belongs to Pasteurella section
(Pasteurellaceae) Actinobacillus succinogenes (Actinobacillus of Actinobacillus (Actinobacillus)
succinogenes).It has been preserved in China typical culture collection center, referred to as CCTCC, depositary institution address：China
Wuhan Wuhan University, deposit number is CCTCC NO:M2016396, preservation date is on July 19th, 2016.
Embodiment 2
A kind of method of utilization xylose mother liquid fermentation production of succinic acid, comprises the following steps：
By on flat board Actinobacillus succinogenes PZ single bacterium colonies access seed culture medium in, 37 DEG C in anaerobic culture box
Culture 16h, bacterium number carries out two grades and expands numerous culture 8h up to more than 300,000,000, according still further to 5% (V/V) inoculum concentration, bacterium number up to more than 300,000,000, i.e.,
Obtain liquid seed；Liquid seed is inoculated into the 250mL anaerobism bottle equipped with fermentation medium by the inoculum concentration of 5% (V/V),
Liquid amount is 150mL, 6.5-7.0 is maintained using pH buffer regulation zymotic fluid pH, full of N2Environment in, rotating speed
100r/min, temperature is 36 DEG C, and batch fermentation 30h obtains final product succinic acid.
The composition and concentration of the seed culture medium be：Glucose 8g/L, dusty yeast 12g/L, corn pulp 4g/L,
NaHCO34g/L, NaH2PO49.6g/L, K2HPO41.55g/L。
The composition and concentration of the fermentation medium be：Xylose mother liquid total reducing sugar 30g/L, nitrogen source 5g/L, potassium dihydrogen phosphate 2g/
L, sodium acid carbonate 2g/L, calcium chloride 0.3g/L, magnesium chloride 0.3g/L, the total sugar concentration of the xylose mother liquid is 50% (W:V).
The nitrogen source of the fermentation medium is corn pulp, dusty yeast, soy meal, groundnut meal with arbitrary proportion mixing.
The pH adjusting agent is that basic magnesium carbonate and sodium carbonate are mixed with arbitrary proportion, and total concentration is 20g/L.
The yield of the present embodiment succinic acid is 22.73g/L, and yield is 75.77%.
Succinic acid yield (%) is defined as：Often consume the grams of succinic acid produced by 1g xyloses.
Analysis method of the present invention：
Sample treatment：Zymotic fluid 12000r/min at room temperature, is centrifuged 10min, takes supernatant, is then 0.22 μm with aperture
Sterilised membrane filter filtering, with high performance liquid chromatography (HPLC) detect zymotic fluid succinic acid and residual concentration of reduced sugar.
Organic acidity test：HPLC methods, wear peace Utimat3000, automatic sampler, chromatographic column：Rezex ROA-organic
Acid300 × 7.8mm, mobile phase 2.5mmol/L H2SO4, pH2.5,45 DEG C of column temperature, sample size 10uL, flow velocity 0.6mL/min,
UV-detector wavelength 210nm.
Biomass estimation：Using spectrophotometer (DU 800UV/VIS Spectrophotometer, Beckman, USA)
It is measured in 660nm, sample is first with 0.2M HCl treatment, the contained MgCO of dissolving3, 12000r/min centrifugation 10min,
Again with distillation washing three times, to remove contained pigment and impurity.
Embodiment 3
A kind of method of utilization xylose mother liquid fermentation production of succinic acid, comprises the following steps：
By on flat board Actinobacillus succinogenes PZ single bacterium colonies access seed culture medium in, 38 DEG C in standard incubator
Culture 20h, bacterium number carries out two grades and expands numerous culture 8h up to more than 300,000,000, according still further to 5% (V/V) inoculum concentration, bacterium number up to more than 300,000,000, i.e.,
Obtain liquid seed；Liquid seed is inoculated into the 250mL anaerobism bottle equipped with fermentation medium by the inoculum concentration of 15% (V/V)
In, liquid amount is 150mL, 6.5-7.0 is maintained using pH buffer regulation zymotic fluid pH, full of CO2Environment in, rotating speed
200r/min, temperature is 38 DEG C, and batch fermentation 72h obtains final product succinic acid.
The composition and concentration of the seed culture medium be：Glucose 8g/L, dusty yeast 12g/L, corn pulp 4g/L,
NaHCO34g/L, NaH2PO49.6g/L, K2HPO41.55g/L。
The composition and concentration of the fermentation medium be：Xylose mother liquid total reducing sugar 100g/L, nitrogen source 20g/L, potassium dihydrogen phosphate
10g/L, sodium acid carbonate 10g/L, calcium chloride 1.0g/L, magnesium chloride 1.0g/L, the total sugar concentration of the xylose mother liquid is 80% (W:
V)。
The nitrogen source of the fermentation medium is that corn pulp and dusty yeast are mixed with arbitrary proportion.
The pH adjusting agent is basic magnesium carbonate, sodium carbonate, magnesium hydroxide, NaOH are mixed with arbitrary proportion, total dense
It is 80g/L to spend.
The yield of the present embodiment succinic acid is 33.65g/L, and yield is 33.65%.
Embodiment 4
A kind of method of utilization xylose mother liquid fermentation production of succinic acid, comprises the following steps：
By on flat board Actinobacillus succinogenes PZ single bacterium colonies access seed culture medium in, 36 DEG C in standard incubator
Culture 18h, bacterium number carries out two grades and expands numerous culture 8h up to more than 300,000,000, according still further to 5% (V/V) inoculum concentration, bacterium number up to more than 300,000,000, i.e.,
Obtain liquid seed；Liquid seed is inoculated into the 250mL anaerobism bottle equipped with fermentation medium by the inoculum concentration of 10% (V/V)
In, liquid amount is 150mL, 6.5-7.0 is maintained using pH buffer regulation zymotic fluid pH, full of CO2Environment in, rotating speed
200r/min, temperature is 37 DEG C, and batch fermentation 40h obtains final product succinic acid.
The composition and concentration of the seed culture medium be：Glucose 8g/L, dusty yeast 12g/L, corn pulp 4g/L,
NaHCO34g/L, NaH2PO49.6g/L, K2HPO41.55g/L。
The composition and concentration of the fermentation medium be：Xylose mother liquid total reducing sugar 40g/L, nitrogen source 12g/L, potassium dihydrogen phosphate
6g/L, sodium acid carbonate 6g/L, calcium chloride 0.6g/L, magnesium chloride 0.7g/L, the total sugar concentration of the xylose mother liquid is 60% (W:
V)。
The nitrogen source of the fermentation medium is corn pulp.
The pH adjusting agent is basic magnesium carbonate, and total concentration is 50g/L.
The yield of the present embodiment succinic acid is 33.72g/L, and yield is 84.30%.
Embodiment 5
A kind of method of utilization xylose mother liquid fermentation production of succinic acid, comprises the following steps：
By on flat board Actinobacillus succinogenes PZ single bacterium colonies access seed culture medium in, 37 DEG C in anaerobic culture box
Culture 16h, bacterium number carries out two grades and expands numerous culture 8h up to more than 300,000,000, according still further to 5% (V/V) inoculum concentration, bacterium number up to more than 300,000,000, i.e.,
Obtain liquid seed；Liquid seed is inoculated into fermentation medium by the inoculum concentration of 5% (V/V), using 2L ferment tanks,
Liquid amount is 1200mL, 6.5-7.0 is maintained using pH buffer regulation zymotic fluid pH, full of N2Environment in, rotating speed
100r/min, temperature is 36 DEG C, and fed batch fermentation 30h obtains final product succinic acid.
The composition and concentration of the seed culture medium be：Glucose 8g/L, dusty yeast 12g/L, corn pulp 4g/L,
NaHCO34g/L, NaH2PO49.6g/L, K2HPO41.55g/L。
The composition and concentration of the fermentation medium be：Xylose mother liquid initial total sugar is 30g/L, nitrogen source 5g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 2g/L, sodium acid carbonate 2g/L, calcium chloride 0.3g/L, magnesium chloride 0.3g/L, intermittent flow adds the total sugar concentration to be in fermentation process
The xylose mother liquid of 200g/L, controls the total reducing sugar of zymotic fluid for 30g/L, and the total sugar concentration of the xylose mother liquid is 50% (W:V).
The nitrogen source of the fermentation medium is that corn pulp and soy meal are mixed with arbitrary proportion.
The pH adjusting agent is that basic magnesium carbonate and magnesium hydroxide are mixed with arbitrary proportion, and total concentration is 20g/L.
The yield of the present embodiment succinic acid is 23.01g/L, and yield is 76.70%.
Embodiment 6
A kind of method of utilization xylose mother liquid fermentation production of succinic acid, comprises the following steps：
By on flat board Actinobacillus succinogenes PZ single bacterium colonies access seed culture medium in, 38 DEG C in standard incubator
Culture 20h, bacterium number carries out two grades and expands numerous culture 8h up to more than 300,000,000, according still further to 5% (V/V) inoculum concentration, bacterium number up to more than 300,000,000, i.e.,
Obtain liquid seed；Liquid seed is inoculated into fermentation medium by the inoculum concentration of 15% (V/V), is sent out using 2L fermentation tanks
Ferment, liquid amount is 1200mL, 6.5-7.0 is maintained using pH buffer regulation zymotic fluid pH, full of CO2Environment in, turn
Fast 200r/min, temperature is 37 DEG C, and fed batch fermentation 72h obtains final product succinic acid.
The composition and concentration of the seed culture medium be：Glucose 8g/L, dusty yeast 12g/L, corn pulp 4g/L,
NaHCO34g/L, NaH2PO49.6g/L, K2HPO41.55g/L。
The composition and concentration of the fermentation medium be：Xylose mother liquid initial total sugar is 40g/L, nitrogen source 20g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 10g/L, sodium acid carbonate 10g/L, calcium chloride 1.0g/L, magnesium chloride 1.0g/L, intermittent flow adds total sugar concentration in fermentation process
It is the xylose mother liquid of 300g/L, controls the total reducing sugar of zymotic fluid for 35g/L, the total sugar concentration of final zymotic fluid reaches 120g/L, institute
The total sugar concentration for stating xylose mother liquid is 80% (W:V).
The nitrogen source of the fermentation medium is that corn pulp, dusty yeast, soy meal and groundnut meal are mixed with arbitrary proportion.
The pH adjusting agent is basic magnesium carbonate, sodium carbonate, magnesium hydroxide, NaOH are mixed with arbitrary proportion, total dense
It is 80g/L to spend.
The yield of the present embodiment succinic acid is 94.34g/L, and yield is 78.62%.
Embodiment 7
A kind of method of utilization xylose mother liquid fermentation production of succinic acid, comprises the following steps：
By on flat board Actinobacillus succinogenes PZ single bacterium colonies access seed culture medium in, 36 DEG C in standard incubator
Culture 18h, bacterium number carries out two grades and expands numerous culture 8h up to more than 300,000,000, according still further to 5% (V/V) inoculum concentration, bacterium number up to more than 300,000,000, i.e.,
Obtain liquid seed；Liquid seed is inoculated into fermentation medium by the inoculum concentration of 10% (V/V), is sent out using 2L fermentation tanks
Ferment, liquid amount is 1200mL, 6.5-7.0 is maintained using pH buffer regulation zymotic fluid pH, full of CO2Environment in, turn
Fast 200r/min, temperature is 37 DEG C, and fed batch fermentation 70h obtains final product succinic acid.
The composition and concentration of the seed culture medium be：Glucose 8g/L, dusty yeast 12g/L, corn pulp 4g/L,
NaHCO34g/L, NaH2PO49.6g/L, K2HPO41.55g/L。
The composition and concentration of the fermentation medium be：Xylose mother liquid initial total sugar is 35g/L, nitrogen source 12g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 6g/L, sodium acid carbonate 6g/L, calcium chloride 0.7g/L, magnesium chloride 0.8g/L, intermittent flow adds the total sugar concentration to be in fermentation process
The xylose mother liquid of 250g/L, controls the total reducing sugar of zymotic fluid for 35g/L, and the total sugar concentration of final zymotic fluid reaches 100g/L, described
The total sugar concentration of xylose mother liquid is 65% (W:V).
The nitrogen source of the fermentation medium be corn pulp,.
The pH adjusting agent is basic magnesium carbonate, and concentration is 60g/L.
The yield of the present embodiment succinic acid is 79.56g/L, and yield is 79.56%.
Embodiment 8
Actinobacillus succinogenes CCTCC NO:M2016396 xylose mother liquid tolerance tests
By in the Actinobacillus succinogenes bacterial strain access seed culture medium of different numberings, trained for 37 DEG C in anaerobic culture box
16h is supported, in the seed culture medium of the xylose mother liquid for containing various concentrations according still further to the access of 5% (V/V) inoculum concentration, after culture 24h, is seen
The upgrowth situation of different numbering Actinobacillus succinogenes is examined, as a result such as table 1.
The different Actinobacillus succinogenes bacterial strain xylose mother liquid tolerance tests of table 1
| Total reducing sugar (g/L) | A.succinogenes PZ | A.succinogenes GXAS 137 | A.succinogenes 130Z |
| 30 | +++ | +++ | +++ |
| 40 | +++ | +++ | +++ |
| 50 | +++ | +++ | ++ |
| 60 | +++ | ++ | + |
| 70 | +++ | + | - |
| 80 | +++ | - | - |
| 90 | ++ | - | - |
| 100 | ++ | - | - |
Wherein：+++ well-grown is represented, ++ represent that growth is preferable ,+expression slightly grows ,-represent and cannot grow.
As can be seen from Table 1：Actinobacillus succinogenes (Actinobacillus succinogenes) PZ is female to xylose
The tolerance of liquid preferably, when xylose mother liquid initial total sugar is 100g/L, still grows preferably, and strains A .succinogenes
GXAS 137 and A.succinogenes 130Z cannot grow.
Embodiment 9
Most suitable xylose mother liquid initial total sugar concentration experiment
By in Actinobacillus succinogenes PZ bacterial strains access seed culture medium, 16h is cultivated for 37 DEG C in anaerobic culture box, then
Two grades are carried out according to 5% (V/V) inoculum concentration and expand numerous culture 8h, that is, obtain liquid seed.Dress is accessed by the inoculum concentration of 8% (V/V)
Have in the 250mL anaerobism bottle of fermentation medium, liquid amount is 150mL, and the xylose mother liquid of different total sugar concentrations is added respectively, then is added
Plus dusty yeast 14g/L, potassium dihydrogen phosphate 6g/L, sodium acid carbonate 6g/L, calcium chloride 0.7g/L, magnesium chloride 0.8g/L, add 50g/L
Basic magnesium carbonate adjust pH in 6.5-7.0, be placed in 37 DEG C of batch fermentation 60h in incubator.Experimental result such as table 2.
Influence of the xylose mother liquid of the different total sugar concentrations of table 2 to succinic acid yield
As can be seen from Table 2：The xylose mother liquid of different total sugar concentrations is larger to batch fermentation production succinic acid yield effect,
When initial total sugar concentration is 70g/L, the yield highest of succinic acid reaches 48.06g/L.
Embodiment 10
Different nitrogen sources fermentation test
According to the method for Actinobacillus succinogenes PZ xylose-fermenting mother liquor succinic acid-producings in embodiment 9, initial total sugar is dense
It is 70g/L to spend, and adds different types of nitrogen source, and total nitrogen addition is 15g/L, is compareed without any nitrogen source, and inoculum concentration is 8%
(V/V), it is inoculated into the 250mL anaerobism bottle equipped with fermentation medium, liquid amount is 150mL, is placed in incubator 37 DEG C in batches
Fermentation 60h, succinic acid and main byproducts content, are as a result shown in Fig. 1.
As seen from Figure 1：When xylose mother liquid carries out succinic acid fermentation without nitrogen source, succinic acid yield only has 2.36g/
L, most suitable nitrogen source is dusty yeast and corn pulp, and succinic acid yield is respectively 46.68g/L, 45.37g/L, is secondly peptone yield
For 29.98g/L, beef extract yield are 22.08g/L, effect is poor when urea is nitrogen source, and succinic acid yield only has 2.57g/L.Examine
Consider the factors such as succinic acid production cost and industrialization production, selection corn pulp is nitrogen source, compared with dusty yeast is for nitrogen source, can be shown
Write the production cost for reducing succinic acid.
Claims (10)
1. the bacterial strain used by a kind of utilization xylose mother liquid fermentation succinic acid-producing, it is characterised in that the bacterial strain is to produce butanedioic acid unwrapping wire
Bacillus（Actinobacillus succinogenes）PZ, deposit number is： CCTCC NO: M2016396.
2. a kind of utilization xylose mother liquid ferments the method for succinic acid-producing, it is characterised in that using the product described in claim 1
Actinobacillus succinogenes fermentation production of succinic acid.
3. a kind of method of utilization xylose mother liquid fermentation production of succinic acid as claimed in claim 2, it is characterised in that including following
Step：During Actinobacillus succinogenes PZ single bacterium colonies on flat board are accessed into seed culture medium, in anaerobic culture box or common training
Support case in 36-38 DEG C culture 16-20h, bacterium number up to more than 300,000,000, according still further to 5%（V/V）Inoculum concentration carries out two grades and expands numerous culture 8h,
Bacterium number obtains liquid seed up to more than 300,000,000；Liquid seed is inoculated into fermentation medium by the inoculum concentration of 5-15% (V/V)
In, 6.5-7.0 is maintained using pH buffer regulation zymotic fluid pH, full of N2Or CO2Environment in, rotating speed 100-200r/
Min, temperature is 36-38 DEG C, and batch fermentation or fed batch fermentation 30-72h obtain final product succinic acid.
4. according to claim 3 using the method for xylose mother liquid fermentation production of succinic acid, it is characterised in that the seed training
Support base composition and concentration be：Glucose 8g/L, dusty yeast 12g/L, corn pulp 4g/L, NaHCO34g/L, NaH2PO49.6g/L,
K2HPO41.55g/L。
5. according to claim 3 using the method for xylose mother liquid fermentation production of succinic acid, it is characterised in that during batch fermentation
The composition and concentration of the fermentation medium be：Xylose mother liquid total reducing sugar 30-100g/L, nitrogen source 5-20g/L, potassium dihydrogen phosphate 2-
10g/L, sodium acid carbonate 2-10g/L, calcium chloride 0.3-1.0g/L, magnesium chloride 0.3-1.0g/L, the total reducing sugar of the xylose mother liquid are dense
It is 50-80% to spend（W:V）.
6. according to claim 3 using the method for xylose mother liquid fermentation production of succinic acid, it is characterised in that batch feeding is sent out
The composition and concentration of the fermentation medium are during ferment：Xylose mother liquid initial total sugar 30-40 g/L, nitrogen source 5-20g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 2-10g/L, sodium acid carbonate 2-10g/L, calcium chloride 0.3-1.0g/L, magnesium chloride 0.3-1.0g/L, interval in fermentation process
Stream plus total sugar concentration are the xylose mother liquid of 200-300g/L, control the total reducing sugar of zymotic fluid for 30-35 g/L, the xylose mother liquid
Total sugar concentration is 50-80%（W:V）.
7. using the method for xylose mother liquid fermentation production of succinic acid according to claim 5 or 6, it is characterised in that the hair
The nitrogen source of ferment culture medium is mixed for one or more in corn pulp, dusty yeast, soy meal, groundnut meal with arbitrary proportion.
8. according to claim 3 using the method for xylose mother liquid fermentation production of succinic acid, it is characterised in that the pH regulations
Agent is mixed for one or more in basic magnesium carbonate, sodium carbonate, magnesium hydroxide, NaOH with arbitrary proportion, and total concentration is
20-80g/L。
9. according to claim 7 using the method for xylose mother liquid fermentation production of succinic acid, it is characterised in that during batch fermentation
The composition and concentration of the fermentation medium be：Xylose mother liquid total reducing sugar 40g/L, corn pulp 12g/L, potassium dihydrogen phosphate 6g/L, carbon
Sour hydrogen sodium 6g/L, calcium chloride 0.6g/L, magnesium chloride 0.7g/L, the total sugar concentration of the xylose mother liquid is 60%（W:V）.
10. according to claim 7 using the method for xylose mother liquid fermentation production of succinic acid, it is characterised in that batch feeding
The composition and concentration of the fermentation medium are during fermentation：Xylose mother liquid initial total sugar 40 g/L, corn pulp 20g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium 10g/L, sodium acid carbonate 10g/L, calcium chloride 1.0g/L, magnesium chloride 1.0g/L, intermittent flow adds total sugar concentration in fermentation process
It is the xylose mother liquid of 300g/L, controls the total reducing sugar of zymotic fluid for 35 g/L, the total sugar concentration of the xylose mother liquid is 80%（W:V）.
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| CN106995794A (en) * | 2017-04-19 | 2017-08-01 | 广西科学院 | A kind of Actinobacillus succinogenes engineered strain and its construction method and purposes for improving succinic acid yield |
| CN109536565A (en) * | 2018-12-20 | 2019-03-29 | 南京工业大学 | A method of succinic acid is produced using the sugared high temperature anaerobic bacterium of pyrolysis and Actinobacillus succinogenes mixed fungus fermentation |
| CN111676252A (en) * | 2020-07-15 | 2020-09-18 | 广西民族大学 | Method for reducing generation of acetic acid serving as succinic acid fermentation by-product |
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| CN101250561A (en) * | 2008-04-15 | 2008-08-27 | 中国科学院微生物研究所 | Method for producing butanol and butanedioic acid by fermentation |
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| CN106995794A (en) * | 2017-04-19 | 2017-08-01 | 广西科学院 | A kind of Actinobacillus succinogenes engineered strain and its construction method and purposes for improving succinic acid yield |
| CN109536565A (en) * | 2018-12-20 | 2019-03-29 | 南京工业大学 | A method of succinic acid is produced using the sugared high temperature anaerobic bacterium of pyrolysis and Actinobacillus succinogenes mixed fungus fermentation |
| CN111676252A (en) * | 2020-07-15 | 2020-09-18 | 广西民族大学 | Method for reducing generation of acetic acid serving as succinic acid fermentation by-product |
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