CN113088456A - Method for controlling growth form of filamentous fungi - Google Patents
Method for controlling growth form of filamentous fungi Download PDFInfo
- Publication number
- CN113088456A CN113088456A CN202110346541.XA CN202110346541A CN113088456A CN 113088456 A CN113088456 A CN 113088456A CN 202110346541 A CN202110346541 A CN 202110346541A CN 113088456 A CN113088456 A CN 113088456A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- culture
- filamentous fungi
- culture medium
- medium
- 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.)
- Granted
Links
- 241000233866 Fungi Species 0.000 title claims abstract description 48
- 238000000034 method Methods 0.000 title claims abstract description 33
- 230000004151 fermentation Effects 0.000 claims abstract description 58
- 238000000855 fermentation Methods 0.000 claims abstract description 57
- 239000001963 growth medium Substances 0.000 claims abstract description 41
- 238000011218 seed culture Methods 0.000 claims abstract description 39
- -1 alcohol amine Chemical class 0.000 claims abstract description 14
- 239000000126 substance Substances 0.000 claims abstract description 14
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 16
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 claims description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 241000228212 Aspergillus Species 0.000 claims description 2
- 241000235527 Rhizopus Species 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 238000012258 culturing Methods 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 5
- 239000002253 acid Substances 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 239000012798 spherical particle Substances 0.000 abstract 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 16
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 15
- 230000001276 controlling effect Effects 0.000 description 14
- 240000005384 Rhizopus oryzae Species 0.000 description 11
- 241000228245 Aspergillus niger Species 0.000 description 10
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 9
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 9
- 235000011090 malic acid Nutrition 0.000 description 9
- 239000001530 fumaric acid Substances 0.000 description 8
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 241001052560 Thallis Species 0.000 description 7
- 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 description 6
- 239000007836 KH2PO4 Substances 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 229910052564 epsomite Inorganic materials 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 229940116298 l- malic acid Drugs 0.000 description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 6
- 235000013619 trace mineral Nutrition 0.000 description 5
- 239000011573 trace mineral Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 229910052603 melanterite Inorganic materials 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 108010023063 Bacto-peptone Proteins 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 238000012136 culture method Methods 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 230000002349 favourable effect Effects 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 229940099690 malic acid Drugs 0.000 description 3
- 239000001630 malic acid Substances 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 description 3
- 239000011686 zinc sulphate Substances 0.000 description 3
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 229940079919 digestives enzyme preparation Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 241000479753 Aspergillus niger ATCC 1015 Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 229960004543 anhydrous citric acid Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960004106 citric acid Drugs 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12N1/14—Fungi; Culture media therefor
-
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for controlling growth form of filamentous fungi, which mainly comprises the steps of adding alcohol amine substances into a culture medium in the process of culturing filamentous fungi seeds and then culturing. According to the invention, the alcohol amine substance with a certain concentration is added into the filamentous fungus seed culture medium, so that the seed form is controlled to be spherical particles beneficial to the later fermentation period. The method can obtain the spherical filamentous fungus mycelium with smooth surface, has no toxic or side effect, is safe and efficient, can be used for industrial acid production, has low cost, is simple, convenient, easy, convenient, strong in operability, obvious in effect, good in repeatability and wide in operating condition range, can effectively reduce the requirement on equipment, and has industrial competitiveness compared with other technical means.
Description
Technical Field
The invention belongs to bioengineering, and particularly relates to a method for controlling growth form of filamentous fungi.
Background
Filamentous fungi have wide application in industrial production, and can be used for producing industrial enzyme preparations, organic acids, antibiotics and the like. In industrial production, filamentous fungi in submerged fermentation processes typically exist in three forms: clumpy, flocculent, and spherical. The filamentous fungi with different forms can directly influence physical parameters, matrix transmission and the like in a fermentation system, so the growth form is important for the generation of fermentation products and the control of the fermentation process. Different target products have different requirements on the shape of the thalli because the growth shape of the thalli has obvious influence on cell metabolic pathways, product accumulation and separation and extraction, for example, when aspergillus niger is used for producing enzyme preparations, the growth shape of the thalli is often required to be controlled to be in a dispersed flocculent mycelium shape, and when aspergillus niger is used for fermenting chitosan, the spherical shape of the thalli is more beneficial to improving the product yield. Therefore, the control of the growth morphology of filamentous fungi is the focus of research in industrial fermentation processes.
The lumpy fungus can be wound on a stirrer or attached to a wall in the fermentation process of the stirring type fermentation tank, so that the limitation of mass transfer and oxygen transfer is caused, and finally, a target product cannot be obtained or the yield is low. The spherical fungi can improve the insufficient conditions, improve the rheological property of fermentation liquor, effectively improve the mass transfer and oxygen transfer performance and reduce energy consumption, but the spherical fungi also need to be controlled in a regular and proper size, and the regular and proper spherical fungi are beneficial to the production of products such as organic acid and the like. It is worth emphasizing that the morphology of the filamentous fungi is influenced by many factors in the fermentation process, including the influence of strains, the influence of growth nutrition environment, the influence of fermentation operation conditions and the like, so that the problems of difficult control of mycelium morphology, poor parallelism and repeatability and the like in the experimental process occur. Therefore, it is necessary to find a convenient and efficient morphology control technique to solve this problem. In the process of producing an organic acid by using a filamentous fungus, a spherical shape is recognized as an optimal form. The existing various technical schemes related to the control of the morphology of the filamentous fungi have characteristics and disadvantages of the existing technical schemes.
The existing patents for controlling the morphology of filamentous fungi are mainly: a device for mechanically controlling the size of a mycelial fungus pellet in a fermentation process; a method for constructing a mutant strain for directionally improving the metabolic yield of filamentous fungi by morphological optimization. In the aspect of regulating and controlling production equipment, the problems of high cost investment, high precision requirement and the like exist. In the aspect of strain breeding, the characteristics of strains are changed through strain breeding or metabolism transformation is carried out, and finally the obtained strains can become strains under various culture conditions, but the strain breeding and the mutation of the strains in metabolic engineering have randomness, so that the screening work can only be carried out aiming at certain or a plurality of specific characters, the screening period has no time limit, and the strain degeneration phenomenon exists.
In conclusion, the existing technology related to the shape control of the filamentous fungi has the problems of complex operation method, harsh process conditions, low controllability and the like. Therefore, the design of an efficient and convenient technical scheme for controlling the fermentation growth form of the filamentous fungi is a necessary requirement.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a method for controlling the growth form of filamentous fungi, which can efficiently and conveniently control the growth form of the filamentous fungi in the fermentation process, improve the fermentation yield of a target product and solve the defects of harsh control conditions of the balling of the filamentous fungi, poor repeatability, uneven distribution of the diameters of bacteria balls and the like in the prior art.
The technical scheme is as follows: in order to achieve the above object, the present invention provides a method for controlling growth morphology of filamentous fungi, comprising the steps of: in the process of culturing filamentous fungi seeds, alcohol amine substances are added into a culture medium for seed culture so as to control the growth of hyphae into a form favorable for later-stage fermentation, and then fermentation culture is carried out.
Wherein the filamentous fungus is Aspergillus or Rhizopus.
Wherein, the alcohol amine substance comprises ethanolamine, diethanolamine and triethanolamine.
Wherein the culture medium is a culture medium containing a carbon source, a nitrogen source and inorganic salts.
The filamentous fungal seed culture and fermentation medium of the present invention is understood to be the medium for growth (seed culture) and fermentation of filamentous fungi of the prior art. For example, a conventional fermentation medium for producing fumaric acid or malic acid by fermenting rhizopus oryzae, a conventional fermentation medium for producing citric acid or malic acid by fermenting aspergillus niger, and the like. The nutrient substances necessary for the growth of the filamentous fungi comprise a carbon source, a nitrogen source, inorganic salt and water, and are prepared into a solution with a certain concentration, sterilized and cooled.
Preferably, the addition amount of the alcohol amine substances is 0.1-2.0% of the volume of the alcohol amine substances in the culture medium. After the seed culture is finished, transferring the seed culture solution to a fermentation culture medium by the inoculation amount of 5-25% of the volume ratio.
Preferably, in the mycelium growth stage, i.e., the seed culture stage, the alcohol amine substances are added into the seed culture medium for seed culture to obtain a certain amount of spherical filamentous fungus mycelium, and then the filamentous fungus mycelium is transferred into a fermentation culture medium for fermentation culture.
Wherein, the seed culture medium is added with alcohol amine substances for seed culture, and then is transferred into a fermentation culture medium for fermentation culture according to the volume ratio of 10-20%.
Furthermore, alcohol amine substances with proper concentration are added into a filamentous fungus seed culture medium to control the thalli to be round granules with uniform shapes and sizes, and then a filamentous fungus seed culture solution is added into a fermentation culture medium according to a certain proportion for liquid fermentation culture, so that the filamentous fungus with biological catalytic activity is kept to be spherical in the whole fermentation process.
Wherein the seed culture condition is that the seed is cultured for 18-24 hours at 28-35 ℃ and the rotating speed is 200 rpm; the fermentation culture condition is that the culture is carried out for 60-120 hours at 28-35 ℃ and the rotating speed is 200 rpm.
Wherein the population of biocatalytically active filamentous fungi is used as a seed for fermentative production of a pre-cultured or product for extraction of the desired biomass.
The invention utilizes alcamines to control thalli to form a spherical shape with a smooth surface, the size of the thalli is consistent, and the shape control is important for the production of related products of filamentous fungus fermentation.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) compared with the prior art for controlling the shape of the fungus balls of the filamentous fungi, the method has the advantages of low cost, simplicity, convenience, practicability, strong operability, obvious effect and better repeatability.
(2) By the method for controlling the form, the yield of fermentation products such as fumaric acid, malic acid and the like can be effectively increased. Compared with the prior art, the method increases the output of fermentation products, improves the income of enterprises producing the products, and can effectively reduce the requirement on equipment precision during production by using the alcohol amine substances to control the shape of the bacteria balls, thereby reducing the cost of production equipment and ensuring that the method has competitive advantage in industrial application.
(3) Adding alcamines substances with proper concentration into a filamentous fungus seed culture medium, culturing to obtain spherical filamentous fungi, controlling the shape of the filamentous fungi, and inoculating a seed culture solution into a fermentation culture medium in a proper proportion to perform fermentation to produce acid. The invention has good repeatability, the diameter distribution of the obtained bacteria balls is uniform, the surface is smooth, the fermented product has no toxic or side effect, and the invention is safe and efficient.
Drawings
FIG. 1 shows the morphology of Rhizopus oryzae under different culture conditions;
FIG. 2 shows the fumaric acid production by Rhizopus oryzae;
FIG. 3 shows the Aspergillus niger morphology under different culture conditions;
FIG. 4 shows the yield of L-malic acid produced by Aspergillus niger.
Detailed Description
The invention will be better understood from the following examples. It is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating the present invention and should not be construed as limiting the present invention as detailed in the claims. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.
The strains used in the present invention are the typical representative filamentous fungi Rhizopus oryzae ATCC 20344 and Aspergillus niger ATCC 1015 which are commonly used in industrial production.
Example 1
Method for controlling rhizopus oryzae bacterium ball shape by adding triethanolamine
(1) Preparation of a culture medium: the seed culture medium comprises 20g/L glucose, 1g/L urea and KH2PO4 0.3g/L，MgSO4·7H2O 0.25g/L，ZnSO4·7H2O 0.066g/L，FeSO4·7H2O0.01 g/L, sterilizing at 115 deg.C for 30 min.
(2) Seed culture: adding 1.5% triethanolamine based on the volume of the culture medium in the step (1) while inoculating the medium at a concentration of 1X 107The rhizopus oryzae spore suspension of each/mL is 1mL, and the seed solution is obtained after culturing for 24 hours at 35 ℃ and 200 rpm.
Simultaneously adopting the same culture method except that the triethanolamine is not added in the step (2) and the diluted H is used instead of adding the triethanolamine in the step (2) of adopting the same culture method2SO4Culturing the seedsAdjusting the pH value of the medium to 3.0, inoculating two groups of seed culture solutions for culture as controls 1 and 2;
the results are shown in FIG. 1, in which the Rhizopus oryzae mycelia of control 1 are flocculent (A) and diluted H2SO4The mycelium adjusted to pH 3.0 showed a spherical shape with a rough surface (B), while the mycelium in the medium added with 1.5% triethanolamine showed a spherical shape with a smooth surface and a uniform diameter distribution (C). The result shows that the addition of the alcamines with proper dosage in the seed culture stage can control the filamentous fungi rhizopus oryzae to form uniform spherical shape which is favorable for the later fermentation stage, and the repeatability is good; rhizopus oryzae cannot be used for producing acid.
Example 2
Fumaric acid fermentation production by utilizing triethanolamine to control growth form of rhizopus oryzae bacteria balls
(1) Preparation of a culture medium: the seed culture medium comprises 20g/L glucose, 1g/L urea and KH2PO4 0.3g/L，MgSO4·7H2O 0.25g/L，ZnSO4·7H2O 0.066g/L，FeSO4·7H2O 0.01g/L。
The fermentation medium comprises 80g/L glucose, 0.2g/L urea and KH2PO4 0.15g/L，ZnSO4·7H2O0.05g/L，FeSO4·7H2O 0.068g/L，MgSO4·7H2O 0.1g/L，CaCO360 g/L. The culture medium was sterilized at 115 ℃ for 30 min.
(2) Seed culture: adding 1.0% triethanolamine of the culture medium volume into the seed culture medium of the step (1), and inoculating at the same time the concentration is 1X 1071mL of rhizopus oryzae spore suspension per mL is cultured at 35 ℃ and 200rpm for 24 hours to obtain seed liquid;
meanwhile, dilute H is used instead of triethanolamine in the step (2)2SO4The pH of the seed medium was adjusted to 3.0 and the seed solution was inoculated for culture as a control.
(3) Fermentation culture: inoculating the seed culture solutions of the experimental group and the control group into a fermentation culture medium respectively in an inoculation amount of 10% by volume, wherein the fermentation time of the two groups is 72 hours, 35 ℃ and 200rpm, and obtaining the fumaric acid fermentation liquor.
(4) As shown in FIG. 2, the final fumaric acid concentration in the fermentation broth was measured by High Performance Liquid Chromatography (HPLC) ultraviolet detection using a Berkele Aminex HPX-87H column with a mobile phase of 5mM dilute sulfuric acid, a column temperature of 30 ℃, a mobile phase flow rate of 0.6mL/min, and a wavelength of 210 nm. The experimental group is 28.6g/L, the control group is 22.5g/L, and after triethanolamine is added, the final concentration of the target product fumaric acid in the experimental group is improved by 27.1 percent compared with the control group. The data show that the improvement effect on the yield of related products is obvious after the triethanolamine is added to control the good form.
Example 3
Method for controlling aspergillus niger bacteria ball shape by adding ethanolamine
(1) Preparation of a culture medium: the seed culture medium comprises 40g/L glucose, 6g/L bactopeptone and KH2PO4 0.75g/L，K2HPO4 0.75g/L，MgSO4·7H2O 0.1g/L，CaCl2·7H20.1g/L of O and trace elements. Wherein the trace elements comprise 0.005g/L NaCl and 0.005g/L FeSO4·7H2O and 0.001 citric acid anhydrous, sterilizing at 115 deg.C for 30 min.
(2) Seed culture: adding 0.1% ethanolamine by volume of the culture medium to the culture medium of the step (1) while inoculating at a concentration of 1X 108Culturing 1mL of Aspergillus niger spore suspension at 28 ℃ and 200rpm for 19 hours to obtain seed liquid;
meanwhile, the same culture method is adopted, only the seed culture solution in the step (2) without adding ethanolamine is used as a control;
the results are shown in FIG. 3, which shows that the mycelia without any treatment (without ethanolamine) are irregularly spherical (A) and the mycelia with smooth surface and uniform diameter distribution (B) in the culture medium with 0.1% ethanolamine. The result shows that the addition of the appropriate dose of the alcamines in the seed culture stage can control the filamentous fungus Aspergillus niger to form a uniform spherical shape which is favorable for the later fermentation stage, and the repeatability is good.
Example 4
L-malic acid fermentation production by using method of controlling growth form of aspergillus niger bacteria balls by triethanolamine
(1) Preparation of a culture medium: the seed culture medium comprises 40g/L glucose, 6g/L bactopeptone and KH2PO4 0.75g/L，K2HPO4 0.75g/L，MgSO4·7H2O 0.1g/L，CaCl2·7H20.1g/L of O and trace elements.
The fermentation medium comprises 100g/L glucose, 6g/L bactopeptone and KH2PO4 0.15g/L，K2HPO4 0.15g/L，MgSO4·7H2O 0.1g/L，CaCl2·7H20.1g/L of O and trace elements. Wherein the trace elements comprise 0.005g/L NaCl and 0.005g/L FeSO4·7H2O and 0.001g/L of anhydrous citric acid, and the culture medium is sterilized at 115 ℃ for 30 min.
(2) Seed culture: adding 0.2% triethanolamine of the culture medium volume into the seed culture medium of the step (1), and inoculating at the same time the concentration is 1 multiplied by 108Culturing 1mL of Aspergillus niger spore suspension at 28 ℃ and 200rpm for 19 hours to obtain seed liquid;
meanwhile, the seed culture solution which is prepared by the same method except that the triethanolamine is not added is used as a control.
(3) Fermentation culture: inoculating the seed culture solutions of the experimental group and the control group into a fermentation culture medium respectively in an inoculation amount of 20% by volume, wherein the fermentation time of the two groups is 96 hours; obtaining L-malic acid fermentation liquor at 28 ℃ and 200 rpm.
(4) As shown in FIG. 4, the final concentration of the target product L-malic acid in the fermentation broth was measured by High Performance Liquid Chromatography (HPLC) using a Berkele Aminex HPX-87H column with a mobile phase of 5mM dilute sulfuric acid, a column temperature of 30 ℃ and a mobile phase flow rate of 0.6mL/min at a wavelength of 210 nm. The experimental group is 60.2g/L, the control group is 48.5g/L, and the final concentration of the triethanolamine target product L-malic acid added in the experimental group is improved by 24.1 percent compared with the control group.
Example 5
Example 5 the same procedure as in example 1, except that: the alcamines used in the step (2) are diethanolamine, and the addition amount of the alcamines is 2.0% of the volume of the culture medium; cultured at 35 ℃ and 200rpm for 18 hours.
Example 6
Example 6 is the same as example 3, except that: the alcamines used in the step (2) are diethanolamine, and the addition amount of the alcamines is that the volume of the alcamines accounts for 0.5% of the volume of the culture medium; incubated at 28 ℃ and 150rpm for 24 hours.
Example 7
Example 7 is the same as example 2, except that: and (4) fermenting for 120 hours at 35 ℃ and 150rpm to obtain fumaric acid fermentation liquor.
Example 8
Example 8 is the same as example 4 except that: and (4) fermenting for 60 hours at 28 ℃ and 200rpm to obtain the L-malic acid fermentation liquor in the step (3).
Claims (8)
1. A method for controlling the growth morphology of filamentous fungi, said method comprising adding an alcohol amine to the culture medium during the cultivation of the filamentous fungi.
2. The method according to claim 1, wherein the filamentous fungus is Aspergillus or Rhizopus.
3. The method according to claim 1, wherein the alcohol amine comprises one or more of ethanolamine, diethanolamine, and triethanolamine.
4. A method for controlling the growth morphology of filamentous fungi of claim 1, wherein the medium is a medium comprising a carbon source, a nitrogen source and inorganic salts.
5. The method according to claim 1, wherein the amount of the alcamines is preferably 0.1-2.0% by volume of the culture medium.
6. The method according to claim 1, wherein the step of adding the alcohol amine substance to the seed culture medium for seed culture is performed during the mycelium growth stage, and then the seed culture is transferred to the fermentation medium for fermentation culture.
7. The method according to claim 6, wherein the seed culture medium is supplemented with an alcohol amine substance for seed culture, and then transferred to a fermentation medium at a volume ratio of 10-20% for fermentation culture.
8. The method for controlling the growth morphology of filamentous fungi of claim 6, wherein the seed culture conditions are culture at 28-35 ℃ for 18-24 hours at a rotation speed of 150-200 rpm; the fermentation culture conditions are that the culture is carried out for 60-120 hours at 28-35 ℃ and the rotating speed is 150-200 rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110346541.XA CN113088456B (en) | 2021-03-31 | 2021-03-31 | Method for controlling growth form of filamentous fungi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110346541.XA CN113088456B (en) | 2021-03-31 | 2021-03-31 | Method for controlling growth form of filamentous fungi |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113088456A true CN113088456A (en) | 2021-07-09 |
| CN113088456B CN113088456B (en) | 2023-03-21 |
Family
ID=76671948
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110346541.XA Active CN113088456B (en) | 2021-03-31 | 2021-03-31 | Method for controlling growth form of filamentous fungi |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113088456B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635823A (en) * | 2016-10-19 | 2017-05-10 | 中国科学院过程工程研究所 | Method for controlling growth morphology of mycelia |
-
2021
- 2021-03-31 CN CN202110346541.XA patent/CN113088456B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635823A (en) * | 2016-10-19 | 2017-05-10 | 中国科学院过程工程研究所 | Method for controlling growth morphology of mycelia |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113088456B (en) | 2023-03-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tay et al. | Production of L (+)‐lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor | |
| Xu et al. | Key technologies for the industrial production of fumaric acid by fermentation | |
| Liao et al. | Co-production of fumaric acid and chitin from a nitrogen-rich lignocellulosic material–dairy manure–using a pelletized filamentous fungus Rhizopus oryzae ATCC 20344 | |
| Zhou et al. | Production of fumaric acid from biodiesel-derived crude glycerol by Rhizopus arrhizus | |
| WO2020042481A1 (en) | Method for continuously culturing aspergillus niger seeds and producing citric acid using same | |
| CN110499345B (en) | Fermentation method of vitamin k2(MK-7 type) | |
| Fu et al. | High-efficiency l-lactic acid production by Rhizopus oryzae using a novel modified one-step fermentation strategy | |
| Reddy et al. | Homofermentative production of optically pure l-lactic acid from sucrose and mixed sugars by batch fermentation of Enterococcus faecalis RKY1 | |
| CN101497901A (en) | Novel technological process for producing high optical purity L-lactic acid by semi-continuous high-density fermentation of Rhizopus oryzae | |
| CN102304480B (en) | Lactobacillus rhamnose strain for producing L-lactic acid efficiently and method for producing L-lactic acid by fermenting cassava and sugarcane molasses | |
| Yu et al. | Enhanced production of L (+)-lactic acid by floc-form culture of Rhizopus oryzae | |
| CN103597087B (en) | The preparation method of butyric acid, butanol and butyl ester | |
| US20140106421A1 (en) | Methods Of Producing Carboxylic Acids And/Or Alcohols | |
| CN112831437B (en) | Bacillus subtilis and fermentation method for high yield of indoleacetic acid and high spore formation rate | |
| CN109652469A (en) | A method of Pfansteihl is prepared using lactobacillus paracasei fermentation | |
| Liu et al. | Optimization of L-(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL 395 | |
| CN102093990B (en) | Method for producing low temperature amylases through microbial fermentation | |
| CN104561140B (en) | A kind of method of preparation of citric acid by fermentation | |
| CN113088456B (en) | Method for controlling growth form of filamentous fungi | |
| Crognale et al. | Fed-batch gluconic acid production from Penicillium variabile P16 under different feeding strategies | |
| CN102212484B (en) | Method for controlling growthform of filamentous fungi during fermentation process | |
| CN106957882B (en) | Method for preparing citric acid by fermentation | |
| CN105154360B (en) | A kind of cultural method of Comamonas testosteroni HY-08D | |
| CN101463370B (en) | Method for preparing L-lactic acid by fermenting potato starch by Rhizopus oryzae | |
| CN104561139A (en) | Method for increasing final cell density of microorganisms and shortening culture time |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |