CN110982750A - High-density fermentation method for rhodopseudomonas palustris and application of high-density fermentation method - Google Patents
High-density fermentation method for rhodopseudomonas palustris and application of high-density fermentation method Download PDFInfo
- Publication number
- CN110982750A CN110982750A CN201911371787.1A CN201911371787A CN110982750A CN 110982750 A CN110982750 A CN 110982750A CN 201911371787 A CN201911371787 A CN 201911371787A CN 110982750 A CN110982750 A CN 110982750A
- Authority
- CN
- China
- Prior art keywords
- rhodopseudomonas palustris
- fermentation
- liquid
- density
- metabolizable
- 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.)
- Pending
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 79
- 230000004151 fermentation Effects 0.000 title claims abstract description 78
- 241000190950 Rhodopseudomonas palustris Species 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 27
- 230000000813 microbial effect Effects 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 10
- 239000003337 fertilizer Substances 0.000 claims abstract description 7
- 239000012629 purifying agent Substances 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000003674 animal food additive Substances 0.000 claims abstract description 6
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 73
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 50
- 239000001963 growth medium Substances 0.000 claims description 36
- 229910052757 nitrogen Inorganic materials 0.000 claims description 25
- 238000011218 seed culture Methods 0.000 claims description 25
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 15
- 229910052799 carbon Inorganic materials 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- 150000003839 salts Chemical class 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 14
- 238000009423 ventilation Methods 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 9
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 9
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 9
- 238000011081 inoculation Methods 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 6
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 4
- 244000144972 livestock Species 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 claims description 3
- 239000004254 Ammonium phosphate Substances 0.000 claims description 3
- 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 3
- 241001465754 Metazoa Species 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 3
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims description 3
- 235000019289 ammonium phosphates Nutrition 0.000 claims description 3
- 235000019730 animal feed additive Nutrition 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims 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 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 235000013877 carbamide Nutrition 0.000 claims description 3
- 235000013339 cereals Nutrition 0.000 claims description 3
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 3
- 235000013399 edible fruits Nutrition 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000001630 malic acid Substances 0.000 claims description 3
- 235000011090 malic acid Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 3
- 244000144977 poultry Species 0.000 claims description 3
- 235000019260 propionic acid Nutrition 0.000 claims description 3
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 239000001384 succinic acid Substances 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 235000013311 vegetables Nutrition 0.000 claims description 3
- 239000002351 wastewater Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 3
- 238000005286 illumination Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 abstract 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 4
- 239000001632 sodium acetate Substances 0.000 description 4
- 235000017281 sodium acetate Nutrition 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 235000017471 coenzyme Q10 Nutrition 0.000 description 1
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Polymers & Plastics (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of microorganisms, and relates to a rhodopseudomonas palustris high-density fermentation method and application thereof. The rhodopseudomonas palustris strain is derived from China industrial microorganism strain preservation management center, and has the preservation number: CICC 23812. The high-density fermentation method of rhodopseudomonas palustris sequentially adopts shake flasks to activate seeds, the activated shake flask seeds are inoculated into a primary fermentation tank to perform propagation to prepare primary seeds, the primary seeds are transferred into a secondary fermentation tank to perform fermentation production, fed-batch materials are adopted in the fermentation process to improve the strain density of fermentation liquor, and the strain density can reach at least 500 hundred million CFU/ml after 5 days of fermentation. The invention solves the problems of illumination requirement, long period, high rate of mixed bacteria, difficult control of product quality and the like in the prior art. Has the advantages of short fermentation period, stable product quality, low production cost and the like. The rhodopseudomonas palustris fermentation liquor obtained by the method can be further processed into a microbial feed additive, a microbial fertilizer and a microbial water purifying agent.
Description
Technical Field
The invention relates to a rhodopseudomonas palustris high-density fermentation method and application thereof, belonging to the technical field of microorganisms.
Background
Rhodopseudomonas palustris (Rhodopseudomonas palustris) is a photosynthetic bacterium which is widely researched and applied, belongs to Rhodopseudomonas palustris of the family Exoseriaceae, has various metabolic pathways such as nitrogen fixation, hydrogen absorption, sulfide utilization and the like, and can secrete various beneficial substances such as B vitamins, coenzyme Q, carotenoid and the like.
Currently, the artificial culture of rhodopseudomonas palustris is concentrated on light fermentation culture, namely, rhodopseudomonas palustris seeds are inoculated into a liquid culture medium in a fully transparent plastic barrel or a film bag and are subjected to anaerobic fermentation for 4-5 days, and finally rhodopseudomonas palustris fermentation liquor is obtained. However, the method can not ensure that the product is not polluted by mixed bacteria, the production is influenced under the conditions of cloudy days or low temperature, the density of the thalli of the fermentation liquor is not higher than 50 hundred million/mL, and the quality of the final product can not be guaranteed.
Disclosure of Invention
The invention aims to solve the defects of the problems and provides a rhodopseudomonas palustris high-density fermentation method and application thereof.
The invention is realized by adopting the following technical scheme:
the invention cultures activated strain through liquid seed culture medium, transfers to the first-stage fermentation tank to culture and obtain first-stage liquid, transfers to the fermentation tank to obtain high-density fermentation liquor through parameter control and fed-batch culture.
The technical scheme adopted by the invention is as follows:
a rhodopseudomonas palustris high-density fermentation method comprises the following steps:
a. inoculating the rhodopseudomonas palustris production seeds with the preservation number of CICC 23812 into 600-2000mL sterile liquid seed culture medium by the inoculation amount of 1-10 per mill, and culturing for 1-3 days on a constant temperature shaking bed with the temperature of 25-33 ℃ and the humidity of 40-80% at the rotating speed of 80-150rpm to obtain activated liquid;
b. b, inoculating the activated seed liquid of the rhodopseudomonas palustris in the step a into a primary fermentation tank filled with a sterile liquid seed culture medium, and culturing for 1-3 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation volume is 1:0.1-1:1 to obtain primary liquid;
c. and c, inoculating the activated primary liquid of the rhodopseudomonas palustris in the step b into a fermentation tank filled with a sterile liquid fermentation culture medium in an inoculation amount of 2-10%, culturing for 2-5 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation rate is 1:0.1-1:1, and increasing the bacterial density by adopting a fed-batch culture medium mode in the fermentation process to obtain a bacterial liquid with the bacterial density of not less than 500 hundred million CFU/ml, namely the high-density rhodopseudomonas palustris.
Preferably, the sterile liquid seed culture medium in the step a and the step b is: comprises metabolizable carbon source 10-50g/L, metabolizable protein nitrogen source 5-30g/L, dipotassium hydrogen phosphate 0.5-2.0g/L, magnesium sulfate heptahydrate 0.1-1.0g/L, adjusting pH to 5.0-8.0, and sterilizing at 121 deg.C for 30 min.
Preferably, the sterile liquid fermentation medium in step c is: comprises 10-50g/L of metabolizable carbon source, 5-30g/L of metabolizable protein nitrogen source, 5-30g/L of metabolizable inorganic nitrogen source, 0.5-2.0g/L of dipotassium phosphate, 0.1-1.0g/L of magnesium sulfate heptahydrate, 0.1-0.4g/L of ferrous sulfate heptahydrate, pH value is adjusted to be 5.0-8.0, and sterilization is carried out for 30 minutes at 121 ℃.
Preferably, the fed-batch fermentation medium in step c is: comprises 500g/L of metabolizable carbon source, 50-300g/L of metabolizable protein nitrogen source and 50-300g/L of metabolizable inorganic nitrogen source, the pH value is adjusted to 5.0-8.0, and the mixture is sterilized for 30 minutes at 121 ℃.
Preferably, the metabolizable carbon source is one or more of glucose, acetic acid and salts thereof, propionic acid and salts thereof, citric acid and salts thereof, malic acid and salts thereof, and succinic acid and salts thereof;
the metabolizable protein nitrogen source is one or a compound of yeast powder, peptone, beef extract, bean cake powder, corn steep liquor and corn protein powder;
the metabolizable inorganic nitrogen source is one or a compound of ammonium sulfate, ammonium chloride, ammonium phosphate and urea.
Preferably, the pH of the liquid seed culture medium or the liquid fermentation medium is 7.0 to 8.0.
Preferably, in the step a, the rhodopseudomonas palustris is inoculated into 1000mL of liquid seed culture medium in an inoculation amount of 1 per thousand, and cultured for 3 days on a constant temperature shaking bed with the temperature of 28 ℃ and the humidity of 60% at the rotation speed of 110-.
Preferably, in the step b, the activated rhodopseudomonas palustris liquid obtained in the step a is inoculated into a primary fermentation tank containing a sterile liquid seed culture medium for culture, and the primary rhodopseudomonas palustris liquid is obtained after the culture is cultured for 2 days under the conditions that the temperature is 28 ℃, the stirring speed is 100rpm, and the ventilation quantity is 1: 0.5.
Preferably, in the step c, the rhodopseudomonas palustris primary liquid obtained in the step b is inoculated into a fermentation tank containing a sterile liquid fermentation medium for culture, the culture is carried out for 5 days under the conditions that the temperature is 28 ℃, the stirring speed is 100rpm, and the ventilation quantity is 1:0.5, a metabolizable carbon source and a metabolizable nitrogen source are provided in a feeding and supplementing manner during the fermentation, and the rhodopseudomonas palustris fermentation liquid is obtained, and the density of bacteria is up to 560 hundred million CFU/ml.
The application of the high-density rhodopseudomonas palustris as a feed additive belongs to the field of feed additives of poultry, livestock, aquatic animals and special animal feed additives; or the microbial fertilizer is applied to vegetables, fruits and grain crops in the field of microbial fertilizer application; or the microbial water purifying agent can be used as a water purifying agent in the application fields of wastewater, riverways and ponds in various industries.
Compared with the prior art, the invention has the remarkable advantages that:
(1) the method can avoid contamination of bacteria, ensure smooth production, and ensure product quality.
(2) The method can avoid the dependence of equipment and process on illumination condition, and can be used for production in the absence of light.
(3) The method can obtain the bacterial density of more than 500 hundred million CFU/ml, and is the highest level in China at present.
(3) The method has low cost and low investment, and is suitable for mass production.
(4) The invention not only provides an industrialized high-density fermentation method for rhodopseudomonas palustris, but also enables the strain to have wide application prospect in the fields of livestock breeding, aquaculture, planting industry, wastewater treatment, river treatment and the like, and has higher social benefit.
Drawings
FIG. 1 is a diagram of activated liquid of Rhodopseudomonas palustris provided by the present invention.
FIG. 2 is a primary liquid diagram of Rhodopseudomonas palustris provided by the present invention.
FIG. 3 is a diagram of a high-density fermentation broth of Rhodopseudomonas palustris provided by the present invention.
Detailed Description
The following describes in detail a specific embodiment of the present invention with reference to the drawings.
See fig. 1-3.
The technical solutions of the present invention are further described below, but not limited thereto, and modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
The invention relates to a rhodopseudomonas palustris high-density fermentation method, which comprises the following steps:
a. inoculating the rhodopseudomonas palustris production seeds with the preservation number of CICC 23812 into 600-2000mL sterile liquid seed culture medium by the inoculation amount of 1-10 per mill, and culturing for 1-3 days on a constant temperature shaking bed with the temperature of 25-33 ℃ and the humidity of 40-80% at the rotating speed of 80-150rpm to obtain activated liquid;
b. b, inoculating the activated seed liquid of the rhodopseudomonas palustris in the step a into a primary fermentation tank filled with a sterile liquid seed culture medium, and culturing for 1-3 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation volume is 1:0.1-1:1 to obtain primary liquid;
c. and c, inoculating the activated primary liquid of the rhodopseudomonas palustris in the step b into a fermentation tank filled with a sterile liquid fermentation culture medium in an inoculation amount of 2-10%, culturing for 2-5 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation rate is 1:0.1-1:1, and increasing the bacterial density by adopting a fed-batch culture medium mode in the fermentation process to obtain a bacterial liquid with the bacterial density of not less than 500 hundred million CFU/ml, namely the high-density rhodopseudomonas palustris.
Preferably, the sterile liquid seed culture medium in the step a and the step b is: comprises metabolizable carbon source 10-50g/L, metabolizable protein nitrogen source 5-30g/L, dipotassium hydrogen phosphate 0.5-2.0g/L, magnesium sulfate heptahydrate 0.1-1.0g/L, adjusting pH to 5.0-8.0, and sterilizing at 121 deg.C for 30 min.
Preferably, the sterile liquid fermentation medium in step c is: comprises 10-50g/L of metabolizable carbon source, 5-30g/L of metabolizable protein nitrogen source, 5-30g/L of metabolizable inorganic nitrogen source, 0.5-2.0g/L of dipotassium phosphate, 0.1-1.0g/L of magnesium sulfate heptahydrate, 0.1-0.4g/L of ferrous sulfate heptahydrate, pH value is adjusted to be 5.0-8.0, and sterilization is carried out for 30 minutes at 121 ℃.
Preferably, the fed-batch fermentation medium in step c is: comprises 500g/L of metabolizable carbon source, 50-300g/L of metabolizable protein nitrogen source and 50-300g/L of metabolizable inorganic nitrogen source, the pH value is adjusted to 5.0-8.0, and the mixture is sterilized for 30 minutes at 121 ℃.
Preferably, the metabolizable carbon source is one or more of glucose, acetic acid and salts thereof, propionic acid and salts thereof, citric acid and salts thereof, malic acid and salts thereof, and succinic acid and salts thereof;
the metabolizable protein nitrogen source is one or a compound of yeast powder, peptone, beef extract, bean cake powder, corn steep liquor and corn protein powder;
the metabolizable inorganic nitrogen source is one or a compound of ammonium sulfate, ammonium chloride, ammonium phosphate and urea.
Preferably, the pH of the liquid seed culture medium or the liquid fermentation medium is 7.0 to 8.0.
Preferably, in the step a, the rhodopseudomonas palustris is inoculated into 1000mL of liquid seed culture medium in an inoculation amount of 1 per thousand, and cultured for 3 days on a constant temperature shaking bed with the temperature of 28 ℃ and the humidity of 60% at the rotation speed of 110-.
Preferably, in the step b, the activated rhodopseudomonas palustris liquid obtained in the step a is inoculated into a primary fermentation tank containing a sterile liquid seed culture medium for culture, and the primary rhodopseudomonas palustris liquid is obtained after the culture is cultured for 2 days under the conditions that the temperature is 28 ℃, the stirring speed is 100rpm, and the ventilation quantity is 1: 0.5.
Preferably, in the step c, the rhodopseudomonas palustris primary liquid obtained in the step b is inoculated into a fermentation tank containing a sterile liquid fermentation medium for culture, the culture is carried out for 5 days under the conditions that the temperature is 28 ℃, the stirring speed is 100rpm, and the ventilation quantity is 1:0.5, a metabolizable carbon source and a metabolizable nitrogen source are provided in a feeding and supplementing manner during the fermentation, and the rhodopseudomonas palustris fermentation liquid is obtained, and the density of bacteria is up to 560 hundred million CFU/ml.
The application of the high-density rhodopseudomonas palustris as a feed additive belongs to the field of feed additives of poultry, livestock, aquatic animals and special animal feed additives; or the microbial fertilizer is applied to vegetables, fruits and grain crops in the field of microbial fertilizer application; or the microbial water purifying agent can be used as a water purifying agent in the application fields of wastewater, riverways and ponds in various industries.
Example 1 preparation of activating solution
1) Preparing a liquid seed culture medium. The liquid seed culture medium of claim, wherein the liquid seed culture medium comprises the following components, sodium acetate 10g/L, yeast powder 10g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate heptahydrate 0.5g/L, pH 7.0-8.0, and sterilizing at 121 ℃ for 30 minutes.
2) Activating the liquid. Adding 1% of the production seeds into the culture medium, and culturing in a constant temperature shaking table at 28 deg.C and humidity of 60%, and rotating speed of 120 rpm. After 3 days of culture, activated seed solution was obtained as shown in FIG. 1.
Example 2 preparation of a first grade liquor
1) The liquid seed culture medium of claim, wherein the liquid seed culture medium comprises the following components, sodium acetate 10g/L, yeast powder 10g/L, dipotassium hydrogen phosphate 1.0g/L, magnesium sulfate heptahydrate 0.5g/L, pH 7.0-8.0, and sterilizing at 121 ℃ for 30 minutes.
2) Inoculating the activated seed solution into a primary fermentation tank filled with a liquid seed culture medium, and carrying out constant temperature treatment at 28 ℃, stirring at 120rpm, and ventilation volume of 1: culturing for 3 days under 0.5 condition to obtain the primary liquid of Rhodopseudomonas palustris, as shown in FIG. 2.
Example 3 high Density fermentation
1) The liquid fermentation medium of claim is prepared by weighing each component, sodium acetate 30g/L, yeast powder 10g/L, ammonium sulfate 10g/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate heptahydrate 1.0g/L, ferrous sulfate heptahydrate 0.4g/L, adjusting pH to 7.0-8.0, and sterilizing at 121 deg.C for 30 min.
2) Accurately weighing the components, 300g/L sodium acetate, 100g/L yeast powder and 100g/L ammonium sulfate according to the mixture ratio of the components in the liquid feed culture medium, adjusting the pH value to 5.0-8.0, and sterilizing at 121 ℃ for 30 minutes.
3) Inoculating all the first-stage liquid into a fermentation tank filled with a liquid fermentation culture medium, and carrying out constant temperature of 28 ℃, stirring at 120rpm and ventilation volume of 1: culturing for 5 days under the condition of 0.5, and feeding the supplemented culture medium timely according to the dissolved oxygen condition in the fermentation process to finally obtain the high-density fermentation liquor of the rhodopseudomonas palustris.
The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (10)
1. A rhodopseudomonas palustris high-density fermentation method is characterized by comprising the following steps:
a. inoculating the rhodopseudomonas palustris production seeds with the preservation number of CICC 23812 into 600-2000mL sterile liquid seed culture medium by the inoculation amount of 1-10 per mill, and culturing for 1-3 days on a constant temperature shaking bed with the temperature of 25-33 ℃ and the humidity of 40-80% at the rotating speed of 80-150rpm to obtain activated liquid;
b. b, inoculating the activated seed liquid of the rhodopseudomonas palustris in the step a into a primary fermentation tank filled with a sterile liquid seed culture medium, and culturing for 1-3 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation volume is 1:0.1-1:1 to obtain primary liquid;
c. and c, inoculating the activated primary liquid of the rhodopseudomonas palustris in the step b into a fermentation tank filled with a sterile liquid fermentation culture medium in an inoculation amount of 2-10%, culturing for 2-5 days under the conditions that the temperature is 25-33 ℃, the stirring speed is 80-200rpm, and the ventilation rate is 1:0.1-1:1, and increasing the bacterial density by adopting a fed-batch culture medium mode in the fermentation process to obtain a bacterial liquid with the bacterial density of not less than 500 hundred million CFU/ml, namely the high-density rhodopseudomonas palustris.
2. The rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein the sterile liquid seed culture medium in the step a and the step b is: comprises metabolizable carbon source 10-50g/L, metabolizable protein nitrogen source 5-30g/L, dipotassium hydrogen phosphate 0.5-2.0g/L, magnesium sulfate heptahydrate 0.1-1.0g/L, and adjusting pH to 5.0-8.0, sterilizing at 121 deg.C for 30 min.
3. The rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein the sterile liquid fermentation medium in the step c is: comprises 10-50g/L of metabolizable carbon source, 5-30g/L of metabolizable protein nitrogen source, 5-30g/L of metabolizable inorganic nitrogen source, 0.5-2.0g/L of dipotassium phosphate, 0.1-1.0g/L of magnesium sulfate heptahydrate, 0.1-0.4g/L of ferrous sulfate heptahydrate, pH value is adjusted to be 5.0-8.0, and sterilization is carried out for 30 minutes at 121 ℃.
4. The rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein the feed fermentation medium in the step c is: comprises 500g/L of metabolizable carbon source, 50-300g/L of metabolizable protein nitrogen source and 50-300g/L of metabolizable inorganic nitrogen source, the pH value is adjusted to 5.0-8.0, and the mixture is sterilized for 30 minutes at 121 ℃.
5. The rhodopseudomonas palustris high-density fermentation method according to one of the claims 2 or 3, wherein the metabolizable carbon source is one or more of glucose, acetic acid and salts thereof, propionic acid and salts thereof, citric acid and salts thereof, malic acid and salts thereof, and succinic acid and salts thereof;
the metabolizable protein nitrogen source is one or a compound of yeast powder, peptone, beef extract, bean cake powder, corn steep liquor and corn protein powder;
the metabolizable inorganic nitrogen source is one or a compound of ammonium sulfate, ammonium chloride, ammonium phosphate and urea.
6. The Rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein the pH of the liquid seed culture medium or liquid fermentation medium is 7.0-8.0.
7. The method for high-density fermentation of Rhodopseudomonas palustris as claimed in claim 1, wherein in step a, Rhodopseudomonas palustris is inoculated into 1000mL of liquid seed culture medium in an inoculum size of 1 ‰, and cultured on a constant temperature shaking bed with a temperature of 28 ℃ and a humidity of 60% for 3 days at a rotation speed of 110-.
8. The rhodopseudomonas palustris high-density fermentation method and the application thereof as claimed in claim 1, wherein in the step b, the rhodopseudomonas palustris activated liquid obtained in the step a is inoculated into a primary fermentation tank containing a sterile liquid seed culture medium for culture, and the primary rhodopseudomonas palustris liquid is obtained after 2 days of culture under the conditions of the temperature of 28 ℃, the stirring speed of 100rpm and the ventilation amount of 1: 0.5.
9. The rhodopseudomonas palustris high-density fermentation method according to claim 1, wherein in the step c, the rhodopseudomonas palustris primary liquid obtained in the step b is inoculated into a fermentation tank containing a sterile liquid fermentation medium for culture, the culture is carried out for 5 days under the conditions of 28 ℃ of temperature, 100rpm of stirring speed and 1:0.5 of ventilation, and a metabolizable carbon source and a metabolizable nitrogen source are provided by feeding and supplementing in the fermentation process to obtain the rhodopseudomonas palustris fermentation liquid with the bacterial density of 560 hundred million CFU/ml.
10. The use of rhodopseudomonas palustris of claim 1 as a feed additive in the field of poultry, livestock, aquatic animals, specialty animal feed additives; or the microbial fertilizer is applied to vegetables, fruits and grain crops in the field of microbial fertilizer application; or the microbial water purifying agent can be used as a water purifying agent in the application fields of wastewater, riverways and ponds in various industries.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911371787.1A CN110982750A (en) | 2019-12-26 | 2019-12-26 | High-density fermentation method for rhodopseudomonas palustris and application of high-density fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911371787.1A CN110982750A (en) | 2019-12-26 | 2019-12-26 | High-density fermentation method for rhodopseudomonas palustris and application of high-density fermentation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110982750A true CN110982750A (en) | 2020-04-10 |
Family
ID=70077631
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911371787.1A Pending CN110982750A (en) | 2019-12-26 | 2019-12-26 | High-density fermentation method for rhodopseudomonas palustris and application of high-density fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110982750A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112625956A (en) * | 2020-12-25 | 2021-04-09 | 江苏苏港和顺生物科技有限公司 | Method for culturing photosynthetic bacteria by utilizing membrane concentrated biogas slurry |
| CN113151012A (en) * | 2021-05-11 | 2021-07-23 | 河北大河生物科技有限公司 | High-density fermentation method of antrodia camphorata |
| CN114891682A (en) * | 2022-05-18 | 2022-08-12 | 北京蓝晶微生物科技有限公司 | A kind of rapid culture method and culture medium of Rhodopseudomonas swamps |
| CN116254196A (en) * | 2022-12-30 | 2023-06-13 | 厦门昶科生物工程有限公司 | Rhodopseudomonas palustris, culture medium, fermentation liquor and fermentation method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050446A (en) * | 2007-03-21 | 2007-10-10 | 珠海市农业科学研究中心 | Swamp Rhodopseudomonas of using nitrite nitrogen in high effect, and application |
| CN101407774A (en) * | 2008-11-20 | 2009-04-15 | 中国科学院广州地球化学研究所 | Preparation technique of photosynthetic bacteria preparation |
| CN102363793A (en) * | 2011-10-25 | 2012-02-29 | 杭州江南科学研究院有限公司 | Preparation method of photosynthetic bacteria flocculant |
| CN102747021A (en) * | 2012-07-23 | 2012-10-24 | 通威股份有限公司 | Seed production method of primary photosynthetic bacterium strain with high viable count |
| CN106135285A (en) * | 2016-05-19 | 2016-11-23 | 湖南省植物保护研究所 | The application in prevention and control rice blast of the Rhodopseudomonas palustris biocontrol agent |
-
2019
- 2019-12-26 CN CN201911371787.1A patent/CN110982750A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050446A (en) * | 2007-03-21 | 2007-10-10 | 珠海市农业科学研究中心 | Swamp Rhodopseudomonas of using nitrite nitrogen in high effect, and application |
| CN101407774A (en) * | 2008-11-20 | 2009-04-15 | 中国科学院广州地球化学研究所 | Preparation technique of photosynthetic bacteria preparation |
| CN102363793A (en) * | 2011-10-25 | 2012-02-29 | 杭州江南科学研究院有限公司 | Preparation method of photosynthetic bacteria flocculant |
| CN102747021A (en) * | 2012-07-23 | 2012-10-24 | 通威股份有限公司 | Seed production method of primary photosynthetic bacterium strain with high viable count |
| CN106135285A (en) * | 2016-05-19 | 2016-11-23 | 湖南省植物保护研究所 | The application in prevention and control rice blast of the Rhodopseudomonas palustris biocontrol agent |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112625956A (en) * | 2020-12-25 | 2021-04-09 | 江苏苏港和顺生物科技有限公司 | Method for culturing photosynthetic bacteria by utilizing membrane concentrated biogas slurry |
| CN113151012A (en) * | 2021-05-11 | 2021-07-23 | 河北大河生物科技有限公司 | High-density fermentation method of antrodia camphorata |
| CN114891682A (en) * | 2022-05-18 | 2022-08-12 | 北京蓝晶微生物科技有限公司 | A kind of rapid culture method and culture medium of Rhodopseudomonas swamps |
| CN116254196A (en) * | 2022-12-30 | 2023-06-13 | 厦门昶科生物工程有限公司 | Rhodopseudomonas palustris, culture medium, fermentation liquor and fermentation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109251874B (en) | Probiotic preparation and preparation method and application thereof | |
| CN105347511B (en) | A kind of complex microorganism preparations used for aquiculture and method | |
| CN100588624C (en) | Microorganism renovation agent of water environment and preparation method thereof | |
| CN110982750A (en) | High-density fermentation method for rhodopseudomonas palustris and application of high-density fermentation method | |
| CN101595943B (en) | Production method of additive of bait for photosynthetic bacteria aquaculture | |
| CN101591631B (en) | EM original dew and production method thereof | |
| CN111235071B (en) | Rhodopseudomonas palustris culture medium and application thereof | |
| CN101386827A (en) | Method for producing inocula for livestock and poultry by multi-thalli mixed liquid | |
| CN107176691A (en) | It is a kind of for spacetabs type microorganism improver of water quality of aquaculture and preparation method thereof | |
| CN108587975A (en) | A kind of Rhodopseudomonas palustris culture medium and cultural method | |
| CN104593303B (en) | A kind of liquid complex micro organism fungicide and its production method | |
| CN108660090A (en) | A kind of complex microorganism preparations used for aquiculture and method | |
| CN103184174B (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN107034165B (en) | High-density fermentation medium for enterococcus faecalis and fermentation process thereof | |
| CN108865935A (en) | A kind of probiotics of water quality improvement and preparation method thereof | |
| CN109548962B (en) | A kind of compound microbial inoculum containing Lactobacillus plantarum and application thereof | |
| CN101624234A (en) | Microorganism water treatment agent in culturing water and preparation technique thereof | |
| CN103641229A (en) | Method for regulating and controlling of water quality of Litopenaeus Vannamei culture pond | |
| CN102864104A (en) | Preparation method and application of composite bacteria preparation for livestock and poultry fermented feed | |
| CN114437975A (en) | Lactobacillus coagulans strain for producing lactic acid and application thereof | |
| CN109055264A (en) | A kind of microbial bacterial agent and preparation method thereof for holothruian cultures | |
| CN111100830B (en) | A kind of composite microecological preparation for aquaculture water purification and its application | |
| CN104894028A (en) | Fishery ocean microbial ecological preparation and preparation method thereof | |
| CN102919611A (en) | Manufacturing method of fermented bait for larval sea cucumber | |
| CN106244489A (en) | A kind of chrysophyceae and the method for photosynthetic bacteria mixed fermentation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200410 |