CN111394398B - Method for preparing PHA (polyhydroxyalkanoate) by fermenting high-salt molasses serving as raw material - Google Patents
Method for preparing PHA (polyhydroxyalkanoate) by fermenting high-salt molasses serving as raw material Download PDFInfo
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- 235000013379 molasses Nutrition 0.000 title claims abstract description 100
- 238000000034 method Methods 0.000 title claims abstract description 45
- 239000002994 raw material Substances 0.000 title abstract description 18
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 title description 75
- 229920000903 polyhydroxyalkanoate Polymers 0.000 title description 75
- 238000000855 fermentation Methods 0.000 claims abstract description 254
- 230000004151 fermentation Effects 0.000 claims abstract description 253
- 235000000346 sugar Nutrition 0.000 claims abstract description 53
- 150000003839 salts Chemical class 0.000 claims abstract description 39
- 239000002609 medium Substances 0.000 claims description 71
- 235000015097 nutrients Nutrition 0.000 claims description 63
- 235000002639 sodium chloride Nutrition 0.000 claims description 56
- 239000000843 powder Substances 0.000 claims description 39
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 37
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 238000003756 stirring Methods 0.000 claims description 29
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 24
- 239000000126 substance Substances 0.000 claims description 23
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 17
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 14
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 14
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 12
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 12
- 230000001502 supplementing effect Effects 0.000 claims description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 8
- 239000001301 oxygen Substances 0.000 claims description 8
- 229910052760 oxygen Inorganic materials 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 239000013589 supplement Substances 0.000 claims description 6
- 241001653918 Halomonas sp. Species 0.000 claims description 4
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 claims description 4
- 235000015872 dietary supplement Nutrition 0.000 claims description 4
- 238000005273 aeration Methods 0.000 claims description 2
- 230000009469 supplementation Effects 0.000 claims description 2
- 241000144833 Halomonas salina Species 0.000 claims 1
- 241001466077 Salina Species 0.000 claims 1
- 241000588901 Zymomonas Species 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 32
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 52
- 239000000463 material Substances 0.000 description 43
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 30
- 229910052799 carbon Inorganic materials 0.000 description 30
- 229910052757 nitrogen Inorganic materials 0.000 description 28
- 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 21
- 239000008103 glucose Substances 0.000 description 21
- 241000206596 Halomonas Species 0.000 description 15
- 239000007788 liquid Substances 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 15
- 239000002028 Biomass Substances 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- 229930091371 Fructose Natural products 0.000 description 12
- 239000005715 Fructose Substances 0.000 description 12
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 12
- 229930006000 Sucrose Natural products 0.000 description 12
- 239000005720 sucrose Substances 0.000 description 12
- 230000004913 activation Effects 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 8
- 229910019142 PO4 Inorganic materials 0.000 description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 7
- 235000021317 phosphate Nutrition 0.000 description 7
- 159000000000 sodium salts Chemical class 0.000 description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical class OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 6
- 239000010452 phosphate Substances 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 5
- 159000000003 magnesium salts Chemical class 0.000 description 5
- 238000007670 refining Methods 0.000 description 5
- 238000011218 seed culture Methods 0.000 description 5
- 238000009423 ventilation Methods 0.000 description 5
- 240000000111 Saccharum officinarum Species 0.000 description 4
- 235000007201 Saccharum officinarum Nutrition 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 235000016068 Berberis vulgaris Nutrition 0.000 description 3
- 241000335053 Beta vulgaris Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- -1 glucose Chemical class 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 125000001477 organic nitrogen group Chemical group 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 101150026728 tesB gene Proteins 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000894556 Pharyngomonas kirbyi Species 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000013877 carbamide Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000002440 industrial waste Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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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
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
-
- 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the field of PHA fermentation, in particular to a method for preparing PHA by fermenting high-salt molasses serving as a raw material. The method comprises the following steps: inoculating PHA fermentation strains into a fermentation medium for fermentation under the condition of fermentation to produce PHA; wherein the fermentation medium contains high-salt molasses, the salt content is 8-11 weight percent and the sugar content is 40-70 weight percent based on the dry weight of the high-salt molasses. The method can effectively reduce the production cost of PHA, and the fermentation efficiency is also improved.
Description
Technical Field
The invention relates to the field of PHA fermentation, in particular to a method for preparing PHA by fermenting high-salt molasses serving as a raw material.
Background
The current society develops at a rapid pace, the environment is protected, the main melody of the contemporary chemical product is formed, and the prior fossil refining material is more and more difficult to meet the requirement of environmental protection. Under such a trend, the development and production of biodegradable materials are more urgent and necessary. PHA is a biodegradable, novel plastic material with similar material properties to traditional plastic products. PHA can be accumulated and stored in most microorganisms as an energy supply substance, and can be produced, extracted and purified on a large scale by utilizing a biological fermentation method.
In most PHA fermentation processes, a single sugar, primarily a monosaccharide, such as glucose, is often used as the primary carbon source for cultivation. PHA is accumulated mainly under unbalanced conditions of high carbon and low nitrogen, so that the use of glucose throughout the whole PHA fermentation process accounts for a large part of the overall fermentation cost. Therefore, finding an inexpensive carbon source which can replace glucose and has high fermentation performance can better save PHA fermentation cost.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a method for preparing PHA by fermenting high-salt molasses as a raw material, which can effectively reduce the production cost of PHA and improve the fermentation efficiency.
In some sugar processing plants, many industrial by-products are produced, molasses being a dark thick mass rich in sucrose, glucose, fructose. Some molasses are also enriched in salt by the processing process to form high salt molasses, which have a salt content of about 8% or more of the total composition. Although the high-salt molasses has high sugar content, the high-salt molasses has high salt concentration, high pigment content and high viscosity, so that the treatment difficulty of the high-salt molasses serving as a byproduct is increased. The inventor of the invention finds that the high-salt molasses is applied to the fermentation (halotolerant bacteria) production process of PHA to replace glucose for fermentation, on one hand, the use of the high-salt molasses reduces the addition of exogenous salt in the fermentation process, effectively improves the utilization rate of industrial byproducts, and reduces the cost; in the second aspect, the high-salt molasses is rich in various trace elements, N, P compounds, vitamins and the like which are necessary for the growth of microorganisms, can effectively promote the growth of thalli, can properly reduce the addition of corresponding raw materials in a culture medium or even directly replace conventional culture medium ingredients in the using process, reduces the cost of the culture medium, can still ensure the normal operation of PHA fermentation, and can improve the fermentation efficiency.
In order to achieve the above object, the present invention provides a method for producing PHA by fermentation, comprising inoculating a PHA fermenting strain into a fermentation medium for fermentation under conditions capable of producing PHA by fermentation;
wherein the fermentation medium contains high-salt molasses, the salt content is 8-11 weight percent and the sugar content is 40-70 weight percent based on the dry weight of the high-salt molasses.
Preferably, the fermentation medium contains high-salt molasses, yeast powder, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and sodium chloride; relative to 1L of fermentation medium, the content of high-salt molasses is 15-35ml, the content of yeast powder is 15-17g, the content of dipotassium hydrogen phosphate is 2-5g, the content of disodium hydrogen phosphate is 5-8g, the content of magnesium sulfate is 0.2-0.3g, and the content of sodium chloride is 45-55 g.
Preferably, the fermentation is carried out under the condition of stirring, the rotation speed of the stirring is 400-600rpm from 0h to 8-12 h;
from 8-12h to 16-20h, the stirring speed is 600-1000 rpm;
the rotation speed of the stirring is 400-600rpm from the fermentation time of 16-20h to the end of the fermentation.
Preferably, (1) the first nutrient is supplemented when the sugar content in the fermentation medium drops below 12g/L for the first time, preferably below 10g/L, more preferably below 5-8g/L, the first nutrient having a carbon to nitrogen ratio of 10-20: 1, the supplement amount of the first nutrient is 8-12 volume percent of the fermentation medium;
(2) when the first nutrient supplement is finished, supplementing a second nutrient, wherein the carbon-nitrogen ratio of the second nutrient is 30-50: 1, the supplementation amount of the second nutrient is 5-10 volume percent of the fermentation medium;
(3) and when the second nutrient supplement is finished, supplementing a third nutrient, wherein the third nutrient is high-salt molasses and the supplement amount of the third nutrient is 20-30 volume percent of the fermentation medium.
Through the technical scheme, the invention can obtain the following beneficial technical effects:
1) the method can fully utilize the advantages of high sugar content and high salt concentration in the high-salt molasses, provide sufficient carbon source supply and environmental requirements for fermentation of PHA fermentation strains, and bring a new way for utilization of the industrial waste;
2) the high-salt molasses has high salt content, and the salt addition can be reduced when a culture medium is prepared, so that the production cost is reduced;
3) the high-salt molasses has low price of about 800 yuan/ton, is more economical and practical than common glucose, and can reduce the cost of carbon sources by about 700 yuan/ton;
4) the high-salt molasses has high salt content, and the higher salt content can inhibit the growth of non-salt-tolerant bacteria, so that an effective bacteriostatic action is achieved, and the additional operation and economic cost brought by fermentation tank sterilization is reduced;
5) the high-salt molasses is rich in various trace elements, N, P compounds, vitamins and the like which are necessary for the growth of microorganisms, can effectively promote the growth of thalli, can properly reduce the addition of corresponding raw materials in a culture medium or even directly replace conventional culture medium ingredients in the use process, reduces the cost of the culture medium, and improves the fermentation efficiency;
6) the high salt molasses can also be applied to fermentation and feeding as a feeding carbon source for PHA production.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a method for preparing PHA through fermentation, which comprises the steps of inoculating PHA fermentation strains into a fermentation medium for fermentation under the condition of fermentation to produce PHA;
wherein the fermentation medium contains high-salt molasses, the salt content is 8-11 weight percent and the sugar content is 40-70 weight percent based on the dry weight of the high-salt molasses.
In the invention, the salt in the high-salt molasses refers to sodium chloride.
According to the invention, the high-salt molasses may be any molasses meeting the above requirements in terms of salt content and sugar content, for example, high-salt cane molasses and/or high-salt beet molasses.
According to a preferred embodiment of the invention, the high salt molasses is high salt cane molasses having a salt content of above 9 wt%, for example 9-11 wt%, and a sugar content of above 50 wt%, for example 50-70 wt%, on a dry weight basis, said sugars comprising glucose, sucrose and fructose, wherein the glucose content is 5-15 wt%, the sucrose content is 75-85 wt% and the fructose content is 5-12 wt%, based on total sugars.
According to the invention, the high-salt molasses can be crude high-salt molasses, also can be refined high-salt molasses, and also can be syrup obtained by decolorizing and diluting refined high-salt molasses.
According to the invention, the content of the high-salt molasses in the fermentation medium can be selected within a wide range, which can completely or partially replace the carbon source (glucose) in the fermentation medium. According to a preferred embodiment of the present invention, the high salt molasses completely replaces the carbon source (glucose) in the fermentation medium, and the content of the high salt molasses is 10-60mL, for example, 10mL, 15mL, 20mL, 25mL, 30mL, 35mL, 40mL, 45mL, 50mL, 55mL, 60mL, more preferably 15-35mL, relative to 1L of the fermentation medium.
According to the invention, the fermentation medium may also contain other components of the PHA fermentation process, such as nitrogen sources, phosphates, magnesium salts and sodium salts.
According to the present invention, the kind of the nitrogen source is not particularly limited as long as it can provide a nitrogen source required for the PHA fermentation broth, and may be an organic nitrogen source, for example, yeast powder, corn steep liquor powder, soybean meal powder, amino acids, etc., or an inorganic nitrogen source, for example, urea, ammonium sulfate, ammonium carbonate, etc. According to a preferred embodiment of the present invention, the nitrogen source is an organic nitrogen source, more preferably yeast powder.
The content of the nitrogen source can be selected from a wide range, and preferably, the content of the nitrogen source is 13 to 18g, for example, 13g, 14 g, 15g, 16g, 17g, 18g, and preferably 15 to 17g, based on 1L of the fermentation medium.
According to the invention, the phosphate may be a phosphate conventionally used in PHA fermentation processes, for example, sodium salts of phosphoric acid, potassium salts of phosphoric acid, preferably, the phosphate is dipotassium hydrogen phosphate and disodium hydrogen phosphate.
Wherein the content of the phosphate can be selected from a wide range, preferably, the content of the phosphate is 5-20g relative to 1L fermentation medium, for example, 5g, 7g, 9 g, 11 g, 13g, 15g, 17g, 19 g, 20 g; preferably 7-13 g.
According to a preferred embodiment of the invention, the phosphate is dipotassium hydrogen phosphate and disodium hydrogen phosphate, the content of dipotassium hydrogen phosphate being 2 to 5g, preferably 2.5 to 4g, and the content of disodium hydrogen phosphate being 5 to 8g, preferably 5.5 to 6.5g, relative to 1L of the fermentation medium.
According to the invention, the magnesium salt can be conventional soluble magnesium salts, but does not include phosphoric acid, preferably magnesium sulfate and/or magnesium chloride.
The amount of the magnesium salt is selected from a wide range, and preferably, the amount of the magnesium salt is 0.1 to 0.5g, for example, 0.1 g, 0.2g, 0.3g, 0.4 g, 0.5g, and preferably 0.2 to 0.3g, based on 1L of the fermentation medium.
According to the invention, the sodium salt may be any of the conventional sodium salts, but excluding the sodium salt of phosphoric acid, preferably the sodium salt is sodium chloride.
The content of the sodium salt can be selected from a wide range, and preferably, the content of the sodium salt is 40 to 70g, for example, 40g, 45g, 50g, 55g, 60g, 65g, 70g, and preferably 45 to 55g, based on 1L of the fermentation medium.
According to a preferred embodiment of the present invention, the fermentation medium contains high salt molasses, yeast powder, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and sodium chloride; relative to 1L of fermentation medium, the content of high-salt molasses is 15-35ml, the content of yeast powder is 15-17g, the content of dipotassium hydrogen phosphate is 2-5g, the content of disodium hydrogen phosphate is 5-8g, the content of magnesium sulfate is 0.2-0.3g, and the content of sodium chloride is 45-55 g.
The content of each substance as described above refers to the content of each substance in the medium, and does not refer to the total amount of each substance charged.
The temperature for fermentation of PHA according to the present invention may be the conventional fermentation temperature, and preferably, the temperature is 30 to 45 ℃ and may be, for example, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 37 ℃, 37.5 ℃, 38 ℃, 38.5 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃ or 45 ℃. More preferably, the temperature is 35-39 ℃.
The pH of fermentation of PHA according to the present invention may be at its conventional fermentation pH, preferably, the pH is in the range of 7-9, and may be, for example, 7, 7.5, 8, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 9. More preferably, the pH is 8.3 to 8.7. Wherein the pH can be adjusted using conventional bases, for example, 8 to 12mol/L sodium hydroxide solution.
The amount of dissolved oxygen in PHA fermentation according to the present invention may be the amount of dissolved oxygen in conventional fermentation, preferably 1-40%, for example, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%. More preferably, the dissolved oxygen amount is 1 to 30%.
According to the invention, the conditions for fermentation of the PHA also preferably comprise a control of the aeration, preferably in a quantity comprised between 0.5 and 1.5vvm, and may be, for example, 0.5vvm, 0.6vvm, 0.7vvm, 0.8vvm, 0.9vvm, 1vvm, 1.1vvm, 1.2vvm, 1.3vvm, 1.4vvm, 1.5vvm, preferably between 1 and 1.2 vvm.
According to the present invention, the fermentation conditions of PHA also preferably include agitation, wherein the rotation speed of the agitation can be selected within a wide range, preferably, the rotation speed of the agitation is 400-1000rpm, for example, 400rpm, 450rpm, 500rpm, 550rpm, 600rpm, 650rpm, 700rpm, 750rpm, 800rpm, 850rpm, 900rpm, 950rpm, 1000rpm, based on the 2-7L fermentor.
In the course of research, the inventors of the present invention found that the final fermentation effect can be further improved by controlling different stirring speeds in different fermentation stages. Preferably, the fermentation is carried out under stirring conditions, from 0h to 8-12h (for example, 0-8h, 0-9h, 0-10h, 0-11h, 0-12 h) preferably to 9-11h, and the stirring speed is 400-600 rpm;
from fermentation for 8-12h (preferably 9-11 h) to fermentation for 16-20h (for example, 8-16h, 9-17h, 10-18h, 11-19h, 12-20h, 9-20h, 10-20h, etc., preferably 17-19h, the specific starting time depends on the ending time of the previous stage), and the rotation speed of the stirring is 600-;
from 16-20h (preferably 17-19 h) to the end of fermentation (e.g., 16h to the end of fermentation, 17h to the end of fermentation, 18h to the end of fermentation, 19h to the end of fermentation, 20h to the end of fermentation, with the start time being determined by the end time of the previous stage), the rotation speed of the stirring is 400-600 rpm.
According to the invention, the fermentation of the PHA can be carried out without feed or with feed. According to a preferred embodiment of the invention, the fermentation of PHA is a fed-batch fermentation, i.e., the fermentation is supplemented with nutrients.
The timing of adding the nutrient can be determined according to the demand of the PHA fermentation strain for sugar, and preferably, when the sugar content in the fermentation medium is reduced to be less than 12g/L, preferably less than 10g/L, and more preferably to be less than 5-8g/L (for example, 5g/L, 6g/L, 7 g/L, and 8 g/L), the nutrient is started to be supplemented.
Wherein the amount of the nutrient is preferably such that the sugar content in the fermentation medium is controlled to be 5-20g/L, for example, 5g/L, 6g/L, 7 g/L, 8g/L, 9 g/L, 10g/L, 11 g/L, 12g/L, 13 g/L, 14 g/L, 15g/L, 16g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L.
Wherein the sugar content refers to the sugar content in the fermentation centrifugation supernatant measured by an SBA-90 biosensor analyzer.
Wherein, preferably, the sugar content in the fermentation system is monitored in real time during the fermentation process.
Wherein, when OD of the fermentation liquid is600The fermentation can be finished when the rate of increase is less than 5/h, and according to the fermentation experience of the inventor, when the fermentation is carried out for 36 to 48 hours, preferably 39 to 42 hours, the OD is600The speed increase of (2) can be reduced to the above level。
Wherein, OD600Refers to the absorbance of the fermentation broth in a spectrophotometer at a wavelength of 600 nm.
Wherein, preferably, OD in the fermentation system is monitored in real time during the fermentation process600。
According to the invention, the nutrient may be a feed in a conventional fermentation process, as long as the above requirements are met, for example, the nutrient may contain glucose. However, according to a preferred embodiment of the invention, the nutrient substance contains high salt molasses.
The inventor of the present invention further finds that the fermentation effect can be further improved by adding different nutrients at different stages of the fermentation, and preferably, the nutrient adding method comprises the following steps:
(1) supplementing a first nutrient when the sugar content in the fermentation medium drops below 12g/L for the first time, preferably below 12g/L, more preferably below 5-8g/L, the first nutrient having a carbon to nitrogen ratio of 10-20: 1, preferably 14-18:1, further preferably 15-17:1, the first nutrient being supplemented in an amount of 8-12 volume percent, further preferably 9-11 volume percent of the fermentation medium.
Preferably, the first nutrient contains high salt molasses and yeast powder.
Preferably, the content of the high salt molasses is 550-830ml and the content of the yeast powder is 80-120g relative to 1L of the first nutrient.
(2) And after the first nutrient substance is supplemented, supplementing a second nutrient substance, wherein the carbon-nitrogen ratio of the second nutrient substance is 30-50: 1, preferably 34 to 42:1, further preferably 38-42: 1; the second nutrient is supplemented in an amount of 5-10 volume percent, preferably 6-8 volume percent of the fermentation medium.
Preferably, the second nutrient contains high salt molasses and yeast powder.
Preferably, the content of the high salt molasses is 830-980ml and the content of the yeast powder is 40-80g relative to 1L of the second nutrient.
Wherein the second nutrient can be supplemented immediately after the first nutrient is supplemented, or at intervals such that the sugar content in the fermentation medium is 5-20g/L, preferably 8-15 g/L.
(3) And after the second nutrient supplement is finished, supplementing a third nutrient, wherein the third nutrient is high-salt molasses, and the supplement amount of the third nutrient is 20-30 volume percent, preferably 23-27 volume percent of the fermentation medium.
Wherein the third nutrient can be supplemented immediately after the second nutrient is supplemented, or at intervals such that the sugar content in the fermentation medium is 5-20g/L, preferably 8-15 g/L.
Preferably, the fermentation is terminated after the end of the addition of the third nutrient by continuing the fermentation for 1 to 3 hours.
According to the invention, the nutrient can be supplemented in an intermittent or fed-batch manner, and the person skilled in the art can determine the nutrient according to the actual situation.
According to the present invention, the fermentation method for producing PHA may be a continuous fermentation method or a batch fermentation method.
According to the invention, the PHA fermentation strain can be various conventional halophilic fermentation strains capable of producing PHA through fermentation, and preferably, the PHA fermentation strain is halophilic monad (a)Halomonas sp.) (ii) a More preferably, the PHA fermentation strain is halomonas (M.halophila)Halomonas sp.) TD01 with preservation number of CGMCC NO.4353 (CN 201010578858.8).
According to the present invention, the inoculum size of the fermentation strain may not be particularly limited, and preferably, the inoculum size of the fermentation strain is 9 to 15 volume percent with respect to 1L of the fermentation medium; for example, it may be 9 volume percent, 10 volume percent, 11 volume percent, 12 volume percent, 13 volume percent, 14 volume percent, 15 volume percent.
According to the invention, the fermentation strain inoculated into the fermentation medium is preferably an activated fermentation seed liquid, and the fermentation seedOD of liquid600The value is preferably 3 to 5.
The activation can be carried out by a conventional technique in the art, for example, inoculating a cryopreserved strain into a seed culture medium for activation culture. The seed culture medium can contain 5-10g/L yeast powder, 10-15g/L peptone and 50-60g/L sodium chloride, and is obtained by high-temperature and high-pressure sterilization.
Wherein, the conditions of the activation culture preferably comprise: culturing at 30-40 deg.C and rotation speed of 150-600To 3-5.
Among them, the activation culture is preferably a multi-stage activation culture, for example, 2 to 3 stages, so that a sufficiently activated seed solution is obtained.
According to the invention, the method also comprises the extraction of PHA from the fermentation broth resulting from the fermentation. The method of PHA extraction may be conventional in the art. For example, reference is made to the literature: chung A, Liu Q, Ouyang S P, et al, Microbial production of 3-hydroxydocanoic acid by phaoperon and dfatBA knock-out ofPseudomonas putidaKT2442 harboring tesB gene[J].Applied Microbiology&Biotechnology, 2009, 83(3):513-519.。
The present invention will be described in detail below by way of examples. In the following examples of the present invention,
the PHA fermentation strain is halomonas (Halomonas sp.) TD01 with preservation number of CGMCC NO.4353 from CN 201010578858.8;
the high-salt cane molasses 1 is a waste material produced in a sugar refining process of sugarcane, is in a thick liquid state, has the salt content of about 10 weight percent, contains glucose, sucrose and fructose in a dry-solid basis, has the sugar content of about 51 weight percent, and contains 11 weight percent of glucose, 81 weight percent of sucrose and 8 weight percent of fructose in total sugar;
the high-salt cane molasses 2 is a waste material produced in a sugar refining process of sugarcane, is in a thick liquid state, has the salt content of about 9 weight percent, and contains glucose, sucrose and fructose in a dry-solid basis, wherein the sugar content is about 60 weight percent, and the total sugar content comprises 15 weight percent of glucose, 75 weight percent of sucrose and 10 weight percent of fructose;
the high-salt cane molasses 3 is a waste material produced in a sugar refining process of sugarcane, is in a thick liquid state, has the salt content of about 11 weight percent, contains about 70 weight percent of glucose, sucrose and fructose in terms of dry solids, and contains 10 weight percent of glucose, 85 weight percent of sucrose and 5 weight percent of fructose in terms of total sugar;
the high-salt beet molasses 4 is waste material produced by the sugar refining and sugar making process of sugarcane, is in a thick liquid state, has the salt content of about 8 weight percent, and contains glucose, sucrose and fructose with the sugar content of about 40 weight percent in terms of dry solids, and has the glucose content of 5 weight percent, the sucrose content of 83 weight percent and the fructose content of 12 weight percent in terms of total sugar;
the sugar content is determined according to the SBA-90 biosensing analyzer method;
the determination method of the biomass of the halomonas in the fermentation broth comprises the steps of taking 25-45 ml of the fermentation broth, centrifuging (8000 rpm, 10 min), leaving precipitate, washing for 2 times by using sterile water, drying the washed precipitate by using a vacuum freeze dryer for 48 hours, and weighing;
reference is made to the following documents: chung A, Liu Q, Ouyang S P, et al, Microbial production of 3-hydroxydocanoic acid by Phaoperon and fadBA knock out of mutant ofPseudomonas putidaKT2442 harboring tesB gene[J]. Applied Microbiology&Biotechnology, 2009, 83(3): 513-;
the fermenter volume was 5L.
Preparation example
This preparation example is intended to illustrate the activation of the fermentation broth
Seed culture medium: contains 8g/L yeast powder, 12g/L peptone and 55g/L sodium chloride.
Inoculating Halomonas into seed culture medium, performing primary activation culture at 37 deg.C and 200rpm, and culturing to OD600Reaching about 4 to obtain first-grade seed liquid;
inoculating the primary seed liquid into a seed culture medium in an inoculation amount of 10 volume percentSecond activation culture was performed at 37 ℃ and 200rpm to OD600And obtaining a secondary seed liquid when the yield reaches about 4, and obtaining a fermented seed liquid.
Example 1
This example illustrates the method of fermentative preparation of PHA provided by the present invention
Fermentation medium: the content of high-salt molasses 1 was 25mL, the content of yeast powder was 17g, the content of dipotassium hydrogen phosphate was 3.5g, the content of disodium hydrogen phosphate was 6.2g, the content of magnesium sulfate was 0.25g, and the content of sodium chloride was 55g, relative to 1L of the fermentation medium. The pH was adjusted to 8.5.
Feeding: taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source; the carbon to nitrogen ratio was 16: 1. Wherein, relative to 1L of first nutrient substances, 1640 mL of high-salt molasses and 108g of yeast powder; the volume ratio of the first feeding material to the fermentation medium is 9: 100.
feeding II: taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source; the carbon to nitrogen ratio was 40: 1. Wherein, relative to 1L of second nutrient substances, the molasses is 900mL, and the yeast powder is 69.4 g; the volume ratio of the second supplementary material to the fermentation medium is 6: 100.
feeding materials: pure high-salt molasses 1 is used as a carbon source, and the volume ratio of the fed material III to the fermentation medium is 23: 100.
Inoculating the seed liquid prepared in the preparation example into a fermentation culture medium in an inoculation amount of 10 volume percent, and performing fermentation culture at 37 ℃ and a ventilation amount of 1vvm, wherein the pH value in the fermentation process is controlled to be about 8.5, and the dissolved oxygen amount is controlled to be 10-30 percent. In addition, the stirring speed is controlled in stages in the fermentation process, the fermentation is carried out for 0-10h, and the stirring speed is 400 rpm; fermenting for 10-18h, wherein the rotating speed of stirring is 800 rpm; fermenting for 18h until the fermentation is finished, wherein the rotating speed of the stirring is 400 rpm.
And in the fermentation process, the sugar content and the pH of the fermentation system are monitored in real time.
Feeding the first feeding material when the sugar content in the fermentation system is reduced to below 6g/L for the first time, feeding the second feeding material when the first feeding material is finished, and feeding the third feeding material when the second feeding material is finished.
The feeding flow rate of the feeding material enables the sugar content in the fermentation liquor to be maintained at about 10g/L, and after the feeding three-flow feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Example 2
This example illustrates the method of fermentative preparation of PHA provided by the present invention
Fermentation medium: the content of high-salt molasses 1 was 35ml, the content of yeast powder was 15g, the content of dipotassium hydrogen phosphate was 2.5g, the content of disodium hydrogen phosphate was 6.5g, the content of magnesium sulfate was 0.3, and the content of sodium chloride was 45g, relative to 1L of the fermentation medium. The pH was adjusted to 8.3.
Feeding: taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source; the carbon to nitrogen ratio was 15: 1. Wherein, relative to 1L of first nutrient substances, 1783 mL of high-salt molasses and 120g of yeast powder; the volume ratio of the first feeding material to the fermentation medium is 10: 100.
feeding II: taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source; the carbon to nitrogen ratio was 38: 1. Wherein, relative to 1L of second nutrient substances, 830mL of molasses and 80g of yeast powder; the volume ratio of the second supplementary material to the fermentation medium is 7: 100.
feeding materials: pure high-salt molasses 1 is used as a carbon source, and the volume ratio of the fed material III to the fermentation medium is 25: 100.
Inoculating the seed liquid prepared in the preparation example into a fermentation culture medium in an inoculation amount of 12 volume percent, performing fermentation culture at 35 ℃ under the condition of 1.1vvm ventilation rate, and simultaneously controlling the pH value to be about 8.3 and the dissolved oxygen amount to be 10-30 percent in the fermentation process. In addition, the stirring speed is controlled in stages in the fermentation process, the fermentation is carried out for 0-9h, and the stirring speed is 500 rpm; fermenting for 9-17h, wherein the rotating speed of stirring is 900 rpm; fermenting for 17h until the fermentation is finished, wherein the rotating speed of the stirring is 500 rpm.
And in the fermentation process, the sugar content and the pH of the fermentation system are monitored in real time.
Feeding the first material when the sugar content in the fermentation system is reduced to below 5g/L for the first time, feeding the second material when the first material feeding is finished, and feeding the third material when the second material feeding is finished.
The feeding flow rate of the feeding material enables the sugar content in the fermentation liquor to be maintained at about 8g/L, and after the feeding three-flow feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Example 3
This example illustrates the method of fermentative preparation of PHA provided by the present invention
Fermentation medium: the content of the high-salt molasses 1 was 15ml, the content of yeast powder was 16g, the content of dipotassium hydrogen phosphate was 4g, the content of disodium hydrogen phosphate was 5.5g, the content of magnesium sulfate was 0.2g, and the content of sodium chloride was 50g, relative to 1L of the fermentation medium. The pH was adjusted to 8.7.
Feeding: taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source; the carbon to nitrogen ratio was 17: 1. Wherein, relative to 1L of first nutrient substances, 1560 mL of high-salt molasses and 91g of yeast powder; the volume ratio of the first feeding material to the fermentation medium is 11: 100.
feeding II: taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source; the carbon to nitrogen ratio was 42: 1. Wherein, relative to 1L of second nutrient substances, 875 mL of molasses and 58g of yeast powder; the volume ratio of the second supplementary material to the fermentation medium is 8: 100.
feeding materials: pure high-salt molasses 1 is used as a carbon source, and the volume ratio of the fed material III to the fermentation medium is 27: 100.
Inoculating the seed liquid prepared in the preparation example into a fermentation culture medium in an inoculation amount of 15 volume percent, performing fermentation culture at 39 ℃ and under the condition of 1.2vvm ventilation rate, and simultaneously controlling the pH value in the fermentation process to be about 8.7 and the dissolved oxygen amount to be 10-30 percent. In addition, the stirring speed is controlled in stages in the fermentation process, the fermentation is carried out for 0-11h, and the stirring speed is 600 rpm; fermenting for 11-19h, wherein the rotating speed of stirring is 1000 rpm; fermenting for 19h until the fermentation is finished, wherein the rotating speed of the stirring is 600 rpm.
And in the fermentation process, the sugar content and the pH of the fermentation system are monitored in real time.
Feeding the first feeding material when the sugar content in the fermentation system is reduced to below 8g/L for the first time, feeding the second feeding material when the first feeding material is finished, and feeding the third feeding material when the second feeding material is finished.
The feeding flow rate of the feeding material enables the sugar content in the fermentation liquor to be maintained at about 15g/L, and after the feeding three-flow feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Example 4
This example illustrates the method of fermentative preparation of PHA provided by the present invention
Fermentation medium: the content of the high-salt molasses 1 was 40ml, the content of yeast powder was 10g, the content of dipotassium hydrogenphosphate was 2.0g, the content of disodium hydrogenphosphate was 5.0g, the content of magnesium sulfate was 0.15g, and the content of sodium chloride was 40g, relative to 1L of the fermentation medium. The pH was adjusted to 8.0.
Feeding: taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source; the carbon to nitrogen ratio was 18: 1. Wherein, relative to 1L of first nutrient substances, 1750 mL of high-salt molasses and 116 g of yeast powder; the volume ratio of the first feeding material to the fermentation medium is 8: 100.
feeding II: taking high-salt molasses 1 as a carbon source and yeast powder as a nitrogen source; the carbon to nitrogen ratio was 34: 1. Wherein, relative to 1L of second nutrient substances, the molasses is 900mL, and the yeast powder is 74 g; the volume ratio of the second supplementary material to the fermentation medium is 5: 100.
feeding materials: pure high-salt molasses 1 is used as a carbon source, and the volume ratio of the fed material III to the fermentation medium is 20: 100.
The seed solution prepared in the preparation example was inoculated into a fermentation medium at an inoculum size of 10 volume percent, and the fermentation was carried out at 30 ℃ and a ventilation of 1.5vvm while controlling the pH during the fermentation to about 8. In addition, the rotation speed was controlled to be about 800rpm during the fermentation.
And in the fermentation process, the sugar content and the pH of the fermentation system are monitored in real time.
Feeding the first material when the sugar content in the fermentation system is reduced to below 10g/L for the first time, feeding the second material when the first material feeding is finished, and feeding the third material when the second material feeding is finished.
The feeding flow rate of the feeding material enables the sugar content in the fermentation liquor to be maintained at about 5g/L, and after the feeding three-flow feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Example 5
This example illustrates the method of fermentative preparation of PHA provided by the present invention
Fermentation medium: the content of the high-salt molasses 1 was 15ml, the content of yeast powder was 20g, the content of dipotassium hydrogen phosphate was 5g, the content of disodium hydrogen phosphate was 8g, the content of magnesium sulfate was 0.5g, and the content of sodium chloride was 70g, relative to 1L of the fermentation medium. The pH was adjusted to 9.
Feeding: example 1 was used to feed one. The volume ratio of the fed material to the fermentation medium was 42: 100.
Inoculating the seed liquid prepared in the preparation example into a fermentation culture medium in an inoculation amount of 10 volume percent, performing fermentation culture at 45 ℃ and under the condition of 0.5vvm ventilation rate, and simultaneously controlling the pH value in the fermentation process to be about 9 and the dissolved oxygen amount to be 10-30 percent. In addition, the stirring speed is controlled in stages in the fermentation process, the fermentation is carried out for 0-12h, and the stirring speed is 400 rpm; fermenting for 12-20h, wherein the rotating speed of stirring is 800 rpm; fermenting for 20h until the fermentation is finished, wherein the rotating speed of the stirring is 400 rpm.
And in the fermentation process, the sugar content and the pH of the fermentation system are monitored in real time.
When the sugar content in the fermentation system drops below 12g/L for the first time, only one feed is fed.
The feeding speed of the feeding material keeps the sugar content in the fermentation liquor at about 20g/L, and after the feeding material feeding is finished, the fermentation is continued for 2 hours to finish the fermentation.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Example 6
This example illustrates the method of fermentative preparation of PHA provided by the present invention
PHA production was carried out according to the procedure of example 1, except that the high-salt molasses 1 was replaced with an equal amount of high-salt cane molasses 2.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Example 7
This example illustrates the method of fermentative preparation of PHA provided by the present invention
PHA production was carried out according to the method of example 1, except that the high-salt molasses 1 was replaced with an equal amount of high-salt cane molasses 3.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Example 8
This example illustrates the method of fermentative preparation of PHA provided by the present invention
PHA production was performed as in example 1, except that the high salt molasses 1 was replaced with an equal amount of high salt beet molasses 4.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Comparative example 1
This comparative example serves to illustrate a reference fermentative PHA production process
PHA production was carried out according to the procedure of example 4, except that the high-salt molasses 1 was replaced with an equal amount of low-salt molasses, and the sodium chloride content in the fermentation medium was adjusted so as to be the same as that in the fermentation medium of example 4.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Comparative example 2
This comparative example serves to illustrate a reference fermentative PHA production process
PHA production was carried out according to the procedure of example 4, except that the high-salt molasses 1 was replaced with a mixture of equal amounts of glucose, sucrose and fructose, and the content of sodium chloride in the fermentation medium was adjusted so as to be the same as that in the fermentation medium of example 4.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
Comparative example 3
This comparative example serves to illustrate a reference fermentative PHA production process
PHA production was carried out according to the procedure of example 4, except that the high-salt molasses 1 was replaced with an equal amount of glucose, and the sodium chloride content in the fermentation medium was adjusted to be the same as that in the fermentation medium of example 4.
The biomass of halomonas, PHA production and raw material cost per unit product in the fermentation broth are compared as shown in table 1.
TABLE 1
As can be seen from the results in Table 1, the embodiment 4 is compared with the comparative examples 1-2, the technical scheme of the invention can effectively reduce the production cost of PHA, and the embodiment 4 is compared with the comparative example 3, the technical scheme of the invention not only can effectively reduce the production cost of PHA, but also can improve the biomass of thalli in fermentation liquor and the yield of PHA. Comparing examples 1-3 with examples 4-5, it can be seen that controlling the fermentation process in the preferred mode of the present invention can further reduce the cost and increase the biomass of the cells and the yield of PHA in the fermentation broth. Comparing examples 1, 6 and 7 with example 8, it can be seen that selecting a specific high salt molasses and controlling the salt content and sugar content of the high salt molasses within the range of the present invention can further improve the fermentation effect.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (6)
1. A method for preparing PHA through fermentation is characterized in that PHA fermentation strains are inoculated into a fermentation medium for fermentation under the condition of fermentation to produce PHA;
wherein the fermentation medium contains high-salt molasses, the salt content is 8-11 weight percent and the sugar content is 40-70 weight percent based on the dry weight of the high-salt molasses;
wherein the fermentation culture medium consists of high-salt molasses, yeast powder, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate and sodium chloride; relative to 1L of fermentation medium, the content of high-salt molasses is 15-35ml, the content of yeast powder is 15-17g, the content of dipotassium hydrogen phosphate is 2-5g, the content of disodium hydrogen phosphate is 5-8g, the content of magnesium sulfate is 0.2-0.3g, and the content of sodium chloride is 45-55 g;
wherein the fermentation is carried out under the condition of stirring, the fermentation time is from 0h to 8-12h, and the stirring speed is 400-600 rpm;
from 8-12h to 16-20h, the stirring speed is 600-1000 rpm;
the rotation speed of the stirring is 400-600rpm from the fermentation time of 16-20h to the end of the fermentation;
wherein the method also comprises the step of supplementing nutrient substances in the fermentation process, wherein the supplement amount of the nutrient substances ensures that the sugar content in the fermentation medium is controlled to be 5-20 g/L;
the nutrient supplementing method comprises the following steps:
(1) supplementing a first nutrient when the sugar content in the fermentation medium is reduced to below 12g/L for the first time, wherein the carbon-nitrogen ratio of the first nutrient is 10-20: 1, the supplement amount of the first nutrient is 8-12 volume percent of the fermentation medium;
(2) and after the first nutrient substance is supplemented, supplementing a second nutrient substance, wherein the carbon-nitrogen ratio of the second nutrient substance is 30-50: 1, the supplementation amount of the second nutrient is 5-10 volume percent of the fermentation medium;
(3) and after the second nutrient supplement is finished, supplementing a third nutrient, wherein the third nutrient is high-salt molasses, and the supplement amount of the third nutrient is 20-30 volume percent of the fermentation medium.
2. The method of claim 1, wherein the conditions of the fermentation comprise: the temperature is 30-45 deg.C, pH is 7-9, and dissolved oxygen is 1-40%.
3. A process according to claim 1 or 2, wherein the fermentation is carried out under aeration in an amount of 0.5-1.5 vvm.
4. The method of claim 1, wherein the first nutrient comprises high salt molasses and yeast powder; and/or
The second nutrient substance contains high-salt molasses and yeast powder; and/or
The third nutrient is high salt molasses.
5. The method as claimed in claim 1, wherein the fermentation strain PHA is Halomonas salina (Zymomonas salina)Halomonas sp.)。
6. The method of claim 1, wherein the fermentation is a continuous fermentation or a batch fermentation.
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