CN107964506B - Fermentation feed supplement optimization control system and method - Google Patents
Fermentation feed supplement optimization control system and method Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 103
- 230000004151 fermentation Effects 0.000 title claims abstract description 103
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000005457 optimization Methods 0.000 title claims abstract description 18
- 239000006052 feed supplement Substances 0.000 title description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 28
- 239000008103 glucose Substances 0.000 claims abstract description 28
- 238000012544 monitoring process Methods 0.000 claims abstract description 17
- 230000003287 optical effect Effects 0.000 claims abstract description 15
- 239000001963 growth medium Substances 0.000 claims abstract description 14
- 241001052560 Thallis Species 0.000 claims abstract description 9
- 230000001502 supplementing effect Effects 0.000 claims abstract description 7
- 238000005086 pumping Methods 0.000 claims abstract description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 12
- 239000001301 oxygen Substances 0.000 claims description 12
- 229910052760 oxygen Inorganic materials 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 11
- 239000012526 feed medium Substances 0.000 claims description 10
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 8
- 230000006698 induction Effects 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 5
- 238000004868 gas analysis Methods 0.000 claims description 5
- 230000000241 respiratory effect Effects 0.000 claims description 5
- 239000001569 carbon dioxide Substances 0.000 claims description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 11
- 108090000623 proteins and genes Proteins 0.000 abstract description 11
- 239000000463 material Substances 0.000 abstract description 7
- 230000002349 favourable effect Effects 0.000 abstract 1
- 230000001276 controlling effect Effects 0.000 description 12
- 229940041514 candida albicans extract Drugs 0.000 description 10
- 239000012137 tryptone Substances 0.000 description 10
- 239000012138 yeast extract Substances 0.000 description 10
- 230000002572 peristaltic effect Effects 0.000 description 9
- 239000000843 powder Substances 0.000 description 8
- 235000013619 trace mineral Nutrition 0.000 description 8
- 239000011573 trace mineral Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 6
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000002518 antifoaming agent Substances 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000005070 sampling Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000013530 defoamer Substances 0.000 description 2
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
- 235000019799 monosodium phosphate Nutrition 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013178 mathematical model Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q3/00—Condition responsive control processes
Abstract
The invention discloses a fermentation feed optimization control system and a method, wherein the system comprises a fermentation tank, a feed bottle, a glucose electrode for monitoring the sugar concentration in the fermentation tank in real time, an optical density electrode for monitoring thallus OD in the fermentation tank in real time, an external feed pump for pumping a feed culture medium and an external computer control system, wherein the external feed pump and the external computer control system are connected with the glucose electrode, the optical density electrode and the feed pump, the running speed of the feed pump is controlled according to the sugar concentration, the thallus OD and a set specific growth rate, and the feed culture medium is fed from the feed bottle to the fermentation tank in a feed mode of controlling the specific growth rate and a continuous feeding mode. The system and the method can improve the automatic fermentation and material supplementing process, are favorable for improving the fermentation density of thalli and improving the expression of target protein.
Description
Technical Field
The invention relates to the technical field of fermentation, in particular to a fermentation feeding optimization control system and method.
Background
Fed-batch fermentation (Fed-batch fermentation), also known as semi-continuous fermentation, refers to a process in which fresh medium or certain nutrients are Fed intermittently or continuously during the batch fermentation. The application range is very wide at present and almost extends to the whole fermentation industry. The theoretical research of fed-batch fermentation is almost blank before the 70 th of the 20 th century, and in early industrial production, the feeding mode is very simple, the adopted mode is only limited to intermittent feeding and constant-speed feeding, and the control of fermentation is also based on experience. Until 1973, Yoshida et al, Japanese scholars, proposed the term "fed-batch fermentation" and theoretically established the first mathematical model, the study of fed-batch fermentation did not enter the theoretical stage of study. Since then, with the progress of research, fed-batch fermentation has made significant progress in three areas. In recent years, with the continuous deepening of theoretical research and application, the contents of fed-batch fermentation are greatly enriched.
At present, fed-batch fermentation is carried out by controlling the content of residual sugar or the specific growth rate in the fermentation process, and in the fermentation process, the thalli in a fermentation tank need to be sampled and detected for a period of time. In the material supplementing process, a peristaltic pump carried by an instrument is used, and the material is fed in an intermittent flow mode.
The defects of the prior art are shown in the following: (1) the feed supplementing speed needs to be fed back and regulated by continuously measuring OD and residual sugar in the fermentation process; (2) the requirement on manual operation in the fermentation process is high, and the time is long; (3) the contamination probability is increased in the fermentation process; (4) in the traditional fermentation liquor production process, a feeding system is a 'semi-starvation' feeding process, or all nutrient components of a culture medium are added at one time in the initial fermentation stage and are gradually consumed; or manual and intermittent feeding is carried out in the middle and later stages of fermentation, so that the overall ecological environment fluctuation of the culture medium is increased, and the loss of the fermentation yield is large.
Disclosure of Invention
The invention provides a fermentation feed supplement optimization control system and method, which can improve the fermentation feed supplement automation process and is beneficial to improving the thallus fermentation density and improving the target protein expression.
According to a first aspect of the present invention, the present invention provides a fermentation feeding optimization control system, which comprises a fermentation tank, a feeding bottle, a glucose electrode for monitoring the sugar concentration in the fermentation tank in real time, an optical density electrode for monitoring the bacterial body OD in the fermentation tank in real time, an external feeding pump for pumping in a feeding medium and an external computer control system, wherein the external feeding pump is connected with the glucose electrode, the optical density electrode and the feeding pump, the operation speed of the feeding pump is controlled according to the sugar concentration, the bacterial body OD and a set specific growth rate, and the feeding medium is fed from the feeding bottle to the fermentation tank in a feeding mode for controlling the specific growth rate and in a continuous feeding mode.
Further, the feeding speed of the feeding pump is obtained according to the following formula:
ωv＝(eμ·OD-OD)·V/c/V0；
wherein, ω isvRepresents the percentage of the feed pump's operating speed relative to its full speed; mu represents a set specific growth rate, i.e., the amount of bacteria increased per unit mass of bacteria per hour; v represents the fermentation volume; c represents the concentration of glucose in the feed medium; v0Represents the feed volume per unit time at which the feed pump was run at full speed.
Further, the system also includes a tail gas analysis mass spectrometer added at the tail gas end of the fermenter for measuring Oxygen Uptake Rate (OUR), carbon dioxide release rate (CER) and Respiratory Quotient (RQ).
Furthermore, the system also comprises a mobile terminal for monitoring the growth condition of the thalli in the fermentation tank in real time on line through a network.
According to a second aspect of the invention, the invention provides a fermentation feed optimization control method, comprising: monitoring the sugar concentration in the fermentation tank in real time by using a glucose electrode connected to the fermentation tank, and monitoring the thallus OD in the fermentation tank in real time by using an optical density electrode; and (3) controlling the running speed of the feed pump according to the sugar concentration, the thallus OD and the set specific growth rate by using an external computer control system connected with the glucose electrode, the optical density electrode and the feed pump, and supplementing a feed culture medium from a feed bottle to the fermentation tank in a feed mode of controlling the specific growth rate and a continuous feeding mode.
Further, the feeding speed of the feeding pump is obtained according to the following formula:
ωv＝(eμ·OD-OD)·V/c/V0；
wherein, ω isvRepresents the percentage of the feed pump's operating speed relative to its full speed; μ represents the set specific growth rate; v represents the fermentation volume; c represents the concentration of glucose in the feed medium; v0Represents the feed volume per unit time at which the feed pump was run at full speed.
Further, the method comprises adding a tail gas analysis mass spectrometer to the tail gas end of the fermentation tank for measuring Oxygen Uptake Rate (OUR), carbon dioxide release rate (CER) and Respiratory Quotient (RQ).
Further, the method further comprises: and (3) monitoring the growth condition of the thalli in the fermentation tank in real time on line by using a mobile terminal through a network.
Further, the method further comprises: the nutrient amount of the initial medium was controlled, and it was precisely after waiting until the nutrient was depleted that the cells needed to be fed and the OD reached the induction phase.
Further, the above method predicts the specific growth rate according to the standard of 1 OD increase of 1 liter of the bacterial solution per 1g of sugar to estimate the feeding rate of the next step, and automatically adjusts by computer.
The continuous addition of the feed medium is realized by adding a carbon source, a nitrogen source, inorganic salts and trace elements.
According to the fermentation feed optimization control system and method, an external feed pump is adopted, feed is fed in a feed feeding mode for controlling the specific growth rate and a continuous flow feeding mode, thalli can be controlled to grow and express protein at a certain speed in an induction period, normal folding of the protein is guaranteed, the protein expression level can be effectively improved, the protein expression level is improved by 30%, and the cell amount is improved by 35%. The system and the method of the invention are used for fermentation and material supplement, the operation process is simple and convenient, the sampling times are less, and the pollution probability is reduced.
Drawings
FIG. 1 is a schematic diagram of a fermentation feed optimization control system according to an embodiment of the present invention;
FIG. 2 is a graph showing the results of the protein yields of the supernatant and whole bacteria of the fermentation broth obtained by the fermentation method according to one embodiment of the present invention and one comparative example, wherein the supernatant 1 and the whole bacteria 1 represent the results of the comparative example, the fermentation tank is provided with a feeding pump, a semi-continuous feeding mode is adopted, the feeding strategy is to measure the residual sugar by sampling, and the semi-continuous feeding mode is adopted after the residual sugar is consumed; the supernatant 2 and the whole strain 2 represent the results of the examples, and the yield of the supernatant 1 and the whole strain 1 is improved by 30% compared with the comparative example.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
As shown in FIG. 1, the fermentation feed optimization control system according to an embodiment of the present invention comprises: the fermentation tank comprises a fermentation tank 1, a feeding bottle 2, a glucose electrode 3, an optical density electrode 4, an external feeding pump 5 and an external computer control system 6, wherein the glucose electrode 3 is used for monitoring the sugar concentration in the fermentation tank 1 in real time, the optical density electrode 4 is used for monitoring the thallus OD in the fermentation tank 1 in real time, the external feeding pump 5 is used for pumping a feeding culture medium, and the external computer control system 6 is connected with the glucose electrode 3, the optical density electrode 4 and the feeding pump 5 and is used for controlling the running speed of the feeding pump 5 according to the sugar concentration, the thallus OD and a set specific growth rate, so that the fermentation feeding optimization control system can feed the feeding culture medium from the feeding bottle 2 to the fermentation tank 1 in a feeding mode for controlling the specific growth rate and in a continuous flow feeding mode.
In one embodiment of the invention, the feed pump 5 is a peristaltic pump (e.g. lange peristaltic pump). The initial operating speed of the peristaltic pump is 1% of the full speed of the peristaltic pump.
In one embodiment of the present invention, the feed rate of the feed pump is given by the following formula:
ωv＝(eμ·OD-OD)·V/c/V0；
wherein, ω isvRepresents the percentage of the feed pump's operating speed relative to its full speed; μ represents the set specific growth rate; v represents the fermentation volume; c represents the concentration of glucose in the feed medium; v0Represents the feed volume per unit time at which the feed pump was run at full speed.
According to the technical scheme, a fermentation model is matched with a corresponding feeding mode, and automatic feedback control feeding fermentation is achieved by combining a computer; the fermentation tank is externally connected with a feed pump (such as a peristaltic pump), and a glucose electrode, an optical density electrode and an external computer control system are adopted; the feeding mode is to control the specific growth rate (the increased bacterial amount of the bacteria of unit mass per hour) and control the bacteria to grow and express protein at a certain speed in the induction period so as to ensure the normal folding of the protein; an external feeding pump is adopted, continuous feeding is carried out, and the stable growth of thalli in the fermentation process is ensured; the continuous addition of the feed medium is realized by adding carbon sources, nitrogen sources, inorganic salts and trace elements.
In addition, the technical scheme of the invention can also comprise the following technical means: adding a tail gas analysis mass spectrometer at the tail gas end of the fermentation tank for measuring OUR, CER and RQ and feeding back the growth condition of the thalli; controlling the nutrient amount of the initial medium, exactly after waiting until the nutrient is depleted, that the cells need to be fed and the OD reaches the induction phase; the specific growth rate is estimated according to the standard that 1 liter of bacterial liquid is increased by 1 OD per 1 gram of sugar so as to estimate the next feeding speed, and the feeding speed is automatically adjusted by a computer; and using a mobile terminal to monitor the growth condition of the thalli in the fermentation tank in real time on line through a network.
Example 1
Take fermentation of 30L Rosseta strain as an example.
(1) Primary culture medium: 10g/L of sodium chloride, 10g/L of tryptone and 5g/L of yeast extract powder.
(2) Secondary culture medium: tryptone 12g/L, yeast extract 24g/L, dipotassium phosphate trihydrate 16.45g/L, potassium dihydrogen phosphate 2.31g/L, and glycerol 5 g/L.
(3) The fermentation medium comprises: 48mmol/L disodium hydrogen phosphate, 20mmol/L sodium dihydrogen phosphate, 16mmol/L ammonium citrate, 9mmol/L magnesium sulfate heptahydrate, 24g/L glucose, 1.9g/L yeast extract powder, 3.8g/L tryptone, 0.1% trace elements, 0.09% antifoaming agent, and pH 7.2. Wherein magnesium sulfate heptahydrate, glucose and trace elements are sterilized separately.
(4) The feed medium comprises: 80mmol/L magnesium sulfate heptahydrate, 640g/L glucose, 38.8g/L yeast extract powder, 19.4g/L tryptone, 0.15 percent of trace elements and 0.09 percent of antifoaming agent. Wherein the yeast extract powder, tryptone and defoamer are sterilized separately.
(5) Single colonies on the plates were picked up in the primary medium, the primary seeds were cultured to OD 4.0, inoculated at 1% into the secondary medium, and inoculated into the fermentor at a rate of 3% volume ratio until OD reached 2.5.
(6) Setting the fermentation temperature at 37 ℃, not controlling the pH, correlating the dissolved oxygen and the rotating speed, controlling the dissolved oxygen to be more than 50 percent for culturing, feeding materials when the dissolved oxygen begins to rebound and about 7 hours, wherein the OD is about 20, and beginning to reduce the temperature to 30 ℃.
(7) The feed was carried out using a Lange peristaltic pump, at an initial feed rate of 1% of the full speed of the Lange peristaltic pump, at which time the pH was controlled at 6.5.
(8) Real-time monitoring is carried out by an optical density electrode and a glucose electrode, and according to the formula omega v ═ e (e/L) by sugar concentration, thallus OD, set specific growth rate of 0.11, fermentation volume, full-speed feeding speed (L/h) of a feeding pump, fed sugar concentration (640g/L) and the like0.11 OD-OD) 30/0.64/3240, calculating the feeding speed of the pump, controlling the content of residual sugar not to be lower than 0.5g/L, and if the concentration of residual sugar is higher than 0.5g/L, feeding the mixture at the speed of one hour. The pH control was manually increased by 0.03 per hour to a value of pH control starting at 6.5 and finally remaining unchanged after 7.0. The dissolved oxygen is controlled at 50% in relation to the rotational speed. The oxygen uptake rate and respiratory quotient of the thallus are observed in the process. The OD can reach 80 after 15 hours of induction.
Note: formula ω v ═ e0.11OD-OD) 30/0.64/3240, ω represents the percentage of the feed pump operating speed relative to full speed; 0.11 represents a controlled specific growth rate; 30 denotes a fermentation volume of 30L; 0.64 represents a glucose content of 0.64g in 1mL of feed medium; 3240 shows the feed volume at full feed pump speed of one hour at 3240 mL.
Comparative example 1
(1) Primary culture medium: 10g/L of sodium chloride, 10g/L of tryptone and 5g/L of yeast extract powder.
(2) Secondary culture medium: tryptone 12g/L, yeast extract 24g/L, dipotassium phosphate trihydrate 16.45g/L, potassium dihydrogen phosphate 2.31g/L, and glycerol 5 g/L.
(3) The fermentation medium comprises: 48mmol/L disodium hydrogen phosphate, 20mmol/L sodium dihydrogen phosphate, 16mmol/L ammonium citrate, 9mmol/L magnesium sulfate heptahydrate, 24g/L glucose, 1.9g/L yeast extract powder, 3.8g/L tryptone, 0.1% trace elements, 0.09% antifoaming agent, and pH 7.2. Wherein magnesium sulfate heptahydrate, glucose and trace elements are sterilized separately.
(4) The feed medium comprises: 80mmol/L magnesium sulfate heptahydrate, 640g/L glucose, 38.8g/L yeast extract powder, 19.4g/L tryptone, 0.15 percent of trace elements and 0.09 percent of antifoaming agent. Wherein the yeast extract powder, tryptone and defoamer are sterilized separately.
(5) Single colonies on the plates were picked up in the primary medium, the primary seeds were cultured to OD 4.0, inoculated at 1% into the secondary medium, and inoculated into the fermentor at a rate of 3% volume ratio until OD reached 2.5.
(6) The fermentation temperature is set to 37 ℃, the pH value is controlled to be 7.0, the dissolved oxygen and the rotating speed are related, the dissolved oxygen is controlled to be more than 50 percent for culture, and the OD is measured after the dissolved oxygen begins to rebound for about 7 hours. The feed was performed using the feed pump of the fermenter and was started to cool to 30 ℃.
(7) Feeding, taking off-line samples to measure OD, measuring residual sugar, and feeding at a constant speed when the amount of residual sugar is less than 1 g/L. The fermentation tank is provided with a feed pump. Feeding was carried out with 3 seconds feeding and 107 seconds stopping.
The results of the final fermentation feed process are shown in Table 1.
TABLE 1
The existing fermentation material supplementing method is to use a fermentation tank with a material supplementing pump and adopt a semi-continuous feeding mode. The feeding strategy is to measure the residual sugar by sampling and continuously feed glucose after the residual sugar is consumed. The protein yield electrophoresis results are shown as supernatant 1 and whole bacterium 1 in FIG. 2; according to the feeding method, an external feeding pump (peristaltic pump) is adopted, feeding is carried out in a feeding mode of controlling the specific growth rate and a continuous feeding mode, and the obtained protein yield electrophoresis results are shown as supernatant 2 and whole bacteria 2 in figure 2, and are improved by 30% compared with the yield of supernatant 1 and whole bacteria 1 obtained by the conventional fermentation feeding method.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (8)
1. A fermentation feed optimization control system is characterized by comprising a fermentation tank, a feed bottle, a glucose electrode for monitoring the sugar concentration in the fermentation tank in real time, an optical density electrode for monitoring thallus OD in the fermentation tank in real time, an external feed pump for pumping a feed culture medium and an external computer control system, wherein the external feed pump and the external computer control system are connected with the glucose electrode, the optical density electrode and the feed pump, the running speed of the feed pump is controlled according to the sugar concentration, the thallus OD and a set specific growth rate, and the feed culture medium is fed from the feed bottle to the fermentation tank in a feed mode of controlling the specific growth rate and a continuous feeding mode; the feeding speed of the feeding pump is obtained according to the following formula:
ωv=(eμ·OD-OD)·V/c/V0；
wherein, ω isvRepresents the percentage of the feed pump's operating speed relative to its full speed; mu represents a set specific growth rate, i.e., the amount of bacteria increased per unit mass of bacteria per hour; v represents the fermentation volume; c represents the concentration of glucose in the feed medium; v0Represents the feed volume per unit time at which the feed pump was run at full speed.
2. The fermentation feed optimization control system of claim 1, further comprising a tail gas analysis mass spectrometer added at the tail gas end of the fermentor for measuring oxygen uptake rate, carbon dioxide release rate and respiratory quotient.
3. The fermentation feeding optimization control system of claim 1, further comprising a mobile terminal for real-time online monitoring of thallus growth status in the fermentation tank through a network.
4. A fermentation feed optimization control method is characterized by comprising the following steps: monitoring the sugar concentration in the fermentation tank in real time by using a glucose electrode connected to the fermentation tank, and monitoring the thallus OD in the fermentation tank in real time by using an optical density electrode; controlling the running speed of the feed pump according to the sugar concentration, the thallus OD and the set specific growth rate by using an external computer control system connected with the glucose electrode, the optical density electrode and the feed pump, and supplementing a feed culture medium from a feed bottle to the fermentation tank in a feed mode of controlling the specific growth rate and a continuous feeding mode; the feeding speed of the feeding pump is obtained according to the following formula:
ωv=(eμ·OD-OD)·V/c/V0；
wherein, ω isvRepresents the percentage of the feed pump's operating speed relative to its full speed; μ represents the set specific growth rate; v represents the fermentation volume; c represents the concentration of glucose in the feed medium; v0Represents the feed volume per unit time at which the feed pump was run at full speed.
5. The fermentation feed optimization control method of claim 4, further comprising adding a tail gas analysis mass spectrometer at the tail gas end of the fermentor for measuring oxygen uptake rate, carbon dioxide release rate and respiratory quotient.
6. The fermentation feed optimization control method of claim 4, further comprising: and (3) monitoring the growth condition of the thalli in the fermentation tank in real time on line by using a mobile terminal through a network.
7. The fermentation feed optimization control method of claim 4, further comprising: the nutrient amount of the initial medium was controlled, and it was precisely after waiting until the nutrient was depleted that the cells needed to be fed and the OD reached the induction phase.
8. The optimized fermentation feeding control method as claimed in claim 4, wherein the method predicts the next feeding speed by predicting the specific growth rate according to the standard of 1 OD increase of 1L strain liquid per 1g sugar, and automatically adjusts by computer.
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