CN115948622A - Microbial fermentation control method, device, system, equipment and medium - Google Patents
Microbial fermentation control method, device, system, equipment and medium Download PDFInfo
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- CN115948622A CN115948622A CN202211160593.9A CN202211160593A CN115948622A CN 115948622 A CN115948622 A CN 115948622A CN 202211160593 A CN202211160593 A CN 202211160593A CN 115948622 A CN115948622 A CN 115948622A
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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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
-
- 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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
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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
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P90/00—Enabling technologies with a potential contribution to greenhouse gas [GHG] emissions mitigation
- Y02P90/02—Total factory control, e.g. smart factories, flexible manufacturing systems [FMS] or integrated manufacturing systems [IMS]
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Abstract
The invention relates to the field of microbial fermentation control, and particularly provides a microbial fermentation control method, a device, a system, equipment and a medium, wherein the microbial fermentation control method comprises the following steps: collecting tail gas monitoring data of a fermentation process of microorganisms; inputting the tail gas monitoring data to a quantitative relation model for data analysis, and outputting a substrate consumption rate by the quantitative relation model; determining a feed rate command according to the substrate consumption rate; the feeding speed instruction is used for indicating feeding to the fermentation process according to the feeding speed. According to the invention, the feeding is accurately and quantitatively controlled, so that the real-time monitoring and accurate control of the whole fermentation process of the polyhydroxyalkanoate are realized, and the production intensity and the fermentation stability of PHA are effectively improved.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a microbial fermentation control method, a device, a system, equipment and a medium.
Background
Polyhydroxyalkanoate (PHA) is produced by fermentation mainly using sugars or lipids as a carbon source, in order to ensure a high PHA yield, the whole fermentation process generally needs to be regulated, and most of the common fermentation process control processes use parameters such as ph, dissolved oxygen, temperature, and pressure as the basis or means of a control strategy, however, most of the parameters can only reflect the physicochemical characteristics of a reaction system, and when the fermentation process is controlled by the parameters, the fermentation production capacity can be reduced due to the instability of the control process, and the fermentation yield and the fermentation quality are finally affected.
Disclosure of Invention
The invention provides a microbial fermentation control method, a device, a system, equipment and a medium, which are used for solving the technical defect of low fermentation production capacity in the existing microbial fermentation technology.
In a first aspect, the present invention provides a method for controlling microbial fermentation, comprising:
collecting tail gas monitoring data of a fermentation process of microorganisms;
inputting the tail gas monitoring data to a quantitative relation model for data analysis, and outputting a substrate consumption rate by the quantitative relation model;
a feed rate command determined from the substrate consumption rate;
the feeding rate instruction is used for indicating feeding to the fermentation process according to the feeding rate.
According to the microbial fermentation control method provided by the invention, the tail gas monitoring data comprise oxygen consumption rate, carbon dioxide generation rate, PHA synthesis oxygen consumption rate and PHA synthesis CO 2 Release rate, oxygen uptake rate by cellular respiration, cellular respiration CO 2 The rate of release.
According to the microbial fermentation control method provided by the invention, the quantitative relation model is used for calculating the substrate consumption rate based on the quantitative relation established among the oxygen consumption rate, the carbon dioxide generation rate and the substrate conversion rate;
the quantitative relationship model specifically performs the following steps:
determining a carbon dioxide generation component according to the carbon dioxide generation rate and a first coefficient;
determining a consumption difference value according to the oxygen consumption rate and the carbon dioxide generation component;
determining a consumption component according to the consumption difference and a second coefficient;
determining a substrate consumption rate according to the consumption component and the substrate conversion rate;
the first coefficient is formed by the oxygen consumption rate of cell respiration and the CO of cell respiration 2 Calculating the release rate; the second coefficient is composed of the first coefficient, the PHA synthesis oxygen consumption rate, the PHA synthesis oxygen consumption amount and the PHA synthesis CO 2 The release rate is calculated.
According to the invention, the microbial fermentation control method further comprises the following steps:
inputting the oxygen consumption rate to a fermentation stage confirmation model, confirming a current target fermentation stage by the fermentation stage confirmation model, and confirming the start and the end of a feeding program based on the current target fermentation stage;
confirming a material feeding control interval corresponding to the target fermentation stage based on the current target fermentation stage; regulating the feed rate based on the value of the feed control interval in combination with the substrate consumption rate.
According to the microbial fermentation control method provided by the invention, the target fermentation stage comprises a fermentation initial stage, a fermentation growth stage, a fermentation stabilization stage and a fermentation decline stage, and the corresponding feeding control intervals are respectively as follows: presetting a feeding speed, a first feeding control interval, a second feeding control interval and a third feeding control interval;
and the numerical values of the preset feeding speed, the first feeding control interval, the second feeding control interval and the third feeding control interval are preset feeding speeds.
According to the present invention, there is provided a method for controlling fermentation of a microorganism capable of accumulating polyhydroxyalkanoate in a cell, comprising the following genera: aeromonas, alcaligenes, azotobacter, bacillus, clostridium, halobacterium, nocardia, rhodospirillum, pseudomonas, ralstonia, and Acinetobacter.
In a second aspect, there is also provided a microbial fermentation control apparatus, comprising:
a collecting unit: the system is used for collecting tail gas monitoring data of a fermentation process of microorganisms;
an analysis unit: the system is used for inputting the tail gas monitoring data to a quantitative relation model for data analysis; outputting, by the quantitative relational model, a substrate consumption rate;
a determination unit: for determining a feed rate command based on the substrate consumption rate;
the feeding speed instruction is used for indicating feeding to the fermentation process according to the feeding speed.
According to the microbial fermentation control device of the present invention, the analysis unit further includes: for inputting the oxygen consumption rate to a fermentation stage validation model, validating a current target fermentation stage by the fermentation stage validation model, and validating the start and end of a feeding procedure based on the current target fermentation stage; the feeding control interval is used for confirming a feeding control interval corresponding to the target fermentation stage based on the current target fermentation stage; regulating the feed rate based on the value of the feed control interval in combination with the substrate consumption rate.
In a third aspect, a microbial fermentation control system is further provided, which includes the microbial fermentation control device, and is used for controlling the fermentation process of microorganisms according to the tail gas monitoring data;
further comprising:
a fermentation tank for providing a fermentation environment for the microorganisms;
the tail gas sampling pipeline is used for collecting tail gas from the fermentation tank;
a flow distributor for flow regulation;
the tail gas mass spectrum analysis device is used for analyzing the component information of the tail gas;
the tail gas state monitoring unit is used for acquiring tail gas monitoring data;
the microorganism is a microorganism capable of accumulating polyhydroxyalkanoate in a cell.
In a fourth aspect, an electronic device is further provided, which includes a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor implements the method for controlling microbial fermentation when executing the computer program.
In a fifth aspect, there is also provided a non-transitory computer readable storage medium having stored thereon a computer program which, when executed by a processor, implements the microbial fermentation control method.
The invention provides a microbial fermentation control method, a device, a system, equipment and a medium, which are characterized in that tail gas monitoring data are acquired in real time in the fermentation process of polyhydroxyalkanoate, the tail gas monitoring data are input to a quantitative relation model in real time, then the substrate consumption rate is determined, and the current required material supplementing speed is obtained through real-time accurate analysis; in addition, the feeding speed instruction can be further adjusted in a feeding control interval corresponding to the real-time fermentation stage of the polyhydroxyalkanoate according to the substrate consumption rate, and feeding control of all stages of processes including a new polyhydroxyalkanoate fermentation process and the like can be realized.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed for the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 is a schematic flow diagram of a method for controlling microbial fermentation provided by the present invention;
FIG. 2 is a schematic flow diagram of a fermentation stage validation model provided by the present invention;
FIG. 3 is a schematic flow chart of the present invention for calculating the consumption rate of a substrate based on a quantitative relationship;
FIG. 4 is a schematic structural diagram of a microbial fermentation control system provided by the present invention;
FIG. 5 is a view showing effects of a method for controlling fermentation using microorganisms according to the present invention;
FIG. 6 is a schematic structural view of a microbial fermentation control apparatus provided in the present invention;
fig. 7 is a schematic structural diagram of an electronic device provided by the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The microorganism referred to in the present invention includes microorganisms capable of accumulating polyhydroxyalkanoate in a cell, and specifically includes microorganisms of the following genera: aeromonas, alcaligenes, azotobacter, bacillus, clostridium, halobacterium, nocardia, rhodospirillum, pseudomonas, ralstonia, and Motorula, in order to describe more precisely the embodiments of the present invention, the present invention takes the microorganism producing Polyhydroxyalkanoate (PHA) as an example, and all the fermentation control methods in the following examples are described by the microorganism producing Polyhydroxyalkanoate (PHA), but this should not be construed as the present invention only being able to control the fermentation of Polyhydroxyalkanoate (PHA), and will not be described herein.
Polyhydroxyalkanoate (PHA) is a biopolymer polyester compound synthesized by microorganisms, exists in the form of granular inclusion in microbial cells, has good biocompatibility, degradability, and plasticity, and has great potential as an excellent biodegradable material in the fields of textile industry, agriculture, food, medical health, and the like. At present, the fermentation process using lipid substances as carbon sources has the problems of difficult substrate monitoring, overhigh foam, unstable process and the like, such as: in order to solve the above technical problems and effectively improve the production intensity and fermentation stability of PHA, the invention provides a fermentation control method for preparing polyhydroxyalkanoate, which monitors and accurately controls the whole fermentation process in real time in an efficient, accurate and timely manner, and fig. 1 is a schematic flow diagram of the microbial fermentation control method provided by the invention, and comprises the following steps:
collecting tail gas monitoring data of a fermentation process of microorganisms;
inputting the tail gas monitoring data to a quantitative relation model for data analysis; outputting, by the quantitative relational model, a substrate consumption rate;
determining a feed rate command according to the substrate consumption rate;
the feeding speed instruction is used for indicating feeding to the fermentation process according to the feeding speed.
In step 101, collecting tail gas monitoring data of fermentation process for preparing polyhydroxyalkanoate, taking PHA production strain as starting strain, performing slant screening, strain activation, primary seed culture, secondary seed culture to obtain high-activity seed solution, transferring the high-activity seed solution into a fermentation tank filled with PHA fermentation medium, controlling fermentation culture conditions, and subjecting the fermentation medium to fermentationAnd monitoring multidimensional parameters in the process in real time to obtain real-time tail gas monitoring data in the fermentation process of monitoring the polyhydroxyalkanoate. The exhaust monitoring data includes, but is not limited to: oxygen consumption rate, carbon dioxide production rate, PHA synthesis oxygen consumption rate, PHA synthesis CO 2 Release rate, oxygen uptake rate by cellular respiration, cellular respiration CO 2 The rate of release.
Alternatively, the PHA-producing strain is a microorganism capable of accumulating PHA intracellularly, including but not limited to Aeromonas, alcaligenes, azotobacter, bacillus, clostridium, halobacterium, nocardia, rhodospirillum, pseudomonas, ralstonia, moghania, and the like. Alternatively, the microorganism may be Alcaligenes autolyticus (Alcaligenes lipolytica), alcaligenes latus (Alcaligenes latus), eutropha rolfsii (Ralstonia eutropha), pseudomonas aeruginosa (Pseudomonas aeruginosa), rhodococcus rhodochrous (Rhodococcus opacus), bacillus subtilis (Bacillus subtilis), and the like.
Optionally, in the obtaining process of the high-activity seed solution, the secondary seed culture time is 8 hours to 14 hours, the dissolved oxygen 600 before transferring to the fermentation medium is 3 to 7, and the inoculation amount is 1-10%.
Alternatively, the polyhydroxyalkanoates PHA expressed in the present invention include, but are not limited to, poly- β -hydroxybutyrate PHB, copolymer PHBV of 3-hydroxybutyrate and 3-hydroxyvalerate, copolyester PHBHHx of 3-hydroxybutyrate with 3-hydroxyhexanoate, poly-3-hydroxybutyrate-4-hydroxybutyrate P34HB.
Optionally, the fermentation medium consists of: 10-20 g/L of grease, 1-5 g/L of disodium hydrogen phosphate, 0.5-2 g/L of monopotassium phosphate, 3-5 g/L of ammonium sulfate and 0.1-0.5 g/L of magnesium sulfate heptahydrate.
Optionally, the fermentation culture conditions at least comprise controlling the temperature between 28 ℃ and 34 ℃, controlling the pH value between 6.3 and 7.2, controlling the rotation speed between 200rpm and 1200rpm, and controlling the pressure between 0.02MPa and 0.1 MPa.
Optionally, the tail gas monitoring data includes an oxygen consumption rate and a carbon dioxide generation rate, and those skilled in the art understand that parameters such as a volume percentage of oxygen in air, a volume percentage of carbon dioxide in air, a volume percentage of nitrogen in air, a volume percentage of oxygen in tail gas, a volume percentage of carbon dioxide in tail gas, a volume percentage of nitrogen in tail gas, an air flow, a gas molar volume, and a fermentation liquid volume are acquired in real time monitoring of multidimensional parameters in a fermentation process, and the oxygen consumption rate and the carbon dioxide generation rate are determined by calculating according to the specific parameters, specifically, the oxygen consumption rate is determined by the following formula:
in formula (1), OUR is the oxygen consumption rate, O 2 (Reference) is the volume percentage of oxygen in air, O 2 Is the volume percentage of oxygen in the tail gas, N 2 (Reference) is the volume percentage of nitrogen in air, N 2 Is the volume percentage of nitrogen in the tail gas, F m Is the air flow rate, V m Is the gas molar volume, and V is the volume of the fermentation broth.
Accordingly, the carbon dioxide generation rate is determined by the following equation:
in the formula (2), CER is the rate of carbon dioxide generation, CO 2 (Reference) is the percentage by volume of carbon dioxide in air, CO 2 Is the volume percentage of carbon dioxide in the tail gas, N 2 (Reference) is the volume percentage of nitrogen in air, N 2 Is the volume percentage of nitrogen in the tail gas, F m Is the air flow rate, V m Is the gas molar volume and V is the volume of the fermentation broth.
In step 102, the exhaust gas monitoring data is input to a quantitative relationship model, and the substrate consumption rate output by the quantitative relationship model is obtained.
The quantitative relation model can reflect the quantitative relation between the input tail gas monitoring data and the substrate consumption rate, namely the corresponding substrate consumption rate can be determined according to the oxygen consumption rate and the carbon dioxide generation rate.
The specific substrate consumption rate can be determined according to the above formula (1) or formula (2), and the following formula can be referred to:
in the formula (3), V s For substrate consumption rate, α is the second coefficient, OUR is the oxygen consumption rate, β is the first coefficient, CER is the carbon dioxide generation rate, and Yeild is the substrate to product conversion. The first coefficient beta is formed by the oxygen consumption rate of cell respiration and the CO of cell respiration 2 Calculating the release rate; the second coefficient alpha is composed of the first coefficient beta, the PHA synthesis oxygen consumption rate, the PHA synthesis oxygen consumption amount and the PHA synthesis CO 2 The release rate is calculated.
In step 103, determining a feeding speed instruction according to the substrate consumption rate; wherein the feeding speed instruction is used for instructing feeding to the fermentation process according to the feeding speed. According to the method, the quantitative relation model of the tail gas monitoring data and the substrate consumption rate is established by taking the tail gas monitoring data as a main basis, the feeding flow acceleration is accurately regulated and controlled according to the model feedback result, the accurate monitoring and control of the state of the fermentation process are realized, and the efficient and stable operation of the fermentation is ensured.
Optionally, the method for controlling microbial fermentation provided by the invention further comprises: inputting the oxygen consumption rate to a fermentation stage confirmation model, confirming the current target fermentation stage by the fermentation stage confirmation model, and confirming the start and the end of a feeding program based on the current target fermentation stage. Confirming a material feeding control interval corresponding to the target fermentation stage based on the current target fermentation stage; regulating the feed rate based on the value of the feed control interval in combination with the substrate consumption rate.
Optionally, the fermentation stage validation model is configured to determine a target fermentation stage from a correspondence between all fermentation stages and oxygen consumption rates according to the oxygen consumption rate. And determining a target fermentation stage from the corresponding relation between all fermentation stages and the oxygen consumption rate according to the oxygen consumption rate, wherein all the fermentation stages are different fermentation stages according to the change of the polyhydroxyalkanoate PHA along with the time, optionally, all the fermentation stages comprise a fermentation initial stage, a fermentation growth stage, a fermentation stabilization stage and a fermentation decline stage, and the oxygen consumption rates in different fermentation stages are obviously different, so that the different fermentation stages of the polyhydroxyalkanoate PHA can be determined according to the oxygen consumption rate. When the fermentation is determined to be in the initial stage of fermentation, a feeding speed instruction is not issued, namely feeding is not needed, and a feeding program is not started; when the fermentation growth stage is determined, a feeding instruction is issued, namely a feeding program is started to be executed; and when the fermentation is at the end of the fermentation decline period, ending the fermentation process, confirming that the material supplementing speed instruction is not issued any more, namely ending the material supplementing program.
In step 103, controlling and adjusting the feeding instruction according to the target fermentation stage, and determining the feeding speed in the feeding instruction according to the substrate consumption rate, wherein each fermentation stage has a feeding control interval corresponding to the fermentation stage, and the feeding control interval is used for controlling the feeding speed.
Optionally, the target fermentation stage includes a fermentation initial stage, a fermentation growth stage, a fermentation stabilization stage, and a fermentation decline stage, and the corresponding feeding control intervals are respectively: presetting a feeding speed, a first feeding control interval, a second feeding control interval and a third feeding control interval.
The materials supplemented in the invention are oils and fats, including but not limited to edible vegetable oils and animal oils, wherein the edible vegetable oils include but not limited to soybean oil, palm oil, peanut oil, corn oil, rapeseed oil, peanut oil, sesame oil, canola oil, refined coconut oil, rice bran crude oil, cottonseed crude oil, rice oil, refined safflower oil, linseed oil, zanthoxylum oil; the animal oils include, but are not limited to, fish oil, chicken oil, beef tallow, mutton tallow, and lard.
The numerical values of the preset feeding speed, the first feeding control interval, the second feeding control interval and the third feeding control interval are preset feeding speeds.
Optionally, the preset feeding speed adopted in the invention comprises the step of determining the feeding speed within the feeding control interval range in a target fermentation stage corresponding to the time of the substrate consumption rate based on the substrate consumption rate in the historical data, and the feeding speed of feeding the oil is adjusted by taking the real-time substrate consumption rate as a parameter index in the fermentation process according to the historical sample data, for example, for a newly developed or new fermentation process, the fermentation is carried out according to the preset value of the feeding control interval in the fermentation process, and the fermentation is carried out while the real-time substrate consumption rate obtained by the invention is adjusted, so that the production intensity of PHA can be improved to the maximum extent in the whole fermentation process, the PHA fermentation efficiency is improved, and the technical problems of production rate reduction caused by excessive oil and fat accumulation due to excessive oil feeding speed in the fermentation substrate and possible high fermentation broth foam due to insufficient oil feeding number are solved.
The method can effectively improve the stability and the production intensity of the PHA fermentation process, and aiming at the growth characteristic difference of different fermentation stages, a quantitative relation model of tail gas detection parameters and substrate consumption rate is established by monitoring the change of the tail gas detection parameters in the fermentation process in real time, so that the consumption rate of the substrate is monitored in real time, the feeding rate of the fed-batch materials is accurately controlled, the real-time monitoring and the accurate control of the fermentation process are realized, and the method is suitable for large-scale industrial production.
The invention provides a microbial fermentation control method, a device, a system, equipment and a medium, which are used for acquiring input tail gas monitoring data in real time in the fermentation process of polyhydroxyalkanoate, inputting the tail gas monitoring data to a quantitative relation model in real time, determining the substrate consumption rate, and regulating and controlling the material supplementing speed instruction in a material supplementing control interval corresponding to the real-time fermentation stage of the polyhydroxyalkanoate according to the substrate consumption rate, so that the material supplementing control of all stages of the fermentation of the polyhydroxyalkanoate is realized.
Optionally, before determining the target fermentation stage from the correspondence between all fermentation stages and the oxygen consumption rate according to the oxygen consumption rate, the method includes:
under the condition that the oxygen consumption rate is 0 or less than a first preset threshold value, determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in the initial fermentation stage;
determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in a fermentation growth stage under the condition that the oxygen consumption rate is greater than or equal to a first preset threshold and is less than a second preset threshold;
determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in a fermentation stabilization stage under the condition that the oxygen consumption rate is greater than or equal to a second preset threshold and is less than a third preset threshold;
determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in a fermentation decay stage under the condition that the oxygen consumption rate shows a sign of beginning to decrease and is smaller than a third preset threshold;
and constructing the corresponding relation between all the fermentation stages and the oxygen consumption rate according to the corresponding relation between each of the fermentation initial stage, the fermentation growth stage, the fermentation stabilization stage and the fermentation decline stage and the oxygen consumption rate.
When the oxygen consumption rate is greater than or equal to a first preset threshold and less than a second preset threshold, determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in the initial fermentation stage, wherein the oxygen consumption rate is optionally 0mmol/L/h, and the first preset threshold is optionally 60mmol/L/h, that is, when the oxygen consumption rate is greater than or equal to 0mmol/L/h and less than 60mmol/L/h, determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in the initial fermentation stage.
And under the condition that the oxygen consumption rate is greater than or equal to a first preset threshold and less than a second preset threshold, determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in a fermentation growth stage, wherein the second preset threshold is optionally 120mmol/L/h, that is, under the condition that the oxygen consumption rate is greater than or equal to 60mmol/L/h and less than 120mmol/L/h, determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in the fermentation growth stage.
And under the condition that the oxygen consumption rate is greater than or equal to a second preset threshold and less than a third preset threshold, determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in a fermentation stable stage, wherein the third preset threshold is optionally 160mmol/L/h, that is, under the condition that the oxygen consumption rate is greater than or equal to 120mmol/L/h and less than 160mmol/L/h, determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in the fermentation stable stage.
And determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in a fermentation and death stage when the oxygen consumption rate begins to decrease and is smaller than a third preset threshold, namely determining that the polyhydroxyalkanoate at the moment corresponding to the oxygen consumption rate is in the fermentation and death stage when the oxygen consumption rate begins to be smaller than 160mmol/L/h, optionally in a range of 120mmol/L/h to 140 mmol/L/h.
In such embodiments, the oxygen consumption rate is monitored in real time, and the stage of fermentation of the polyhydroxyalkanoate is determined based on the oxygen consumption rate, specifically, the cells are grown under the initial bottom oil condition, i.e., the initial stage of fermentation, and the feeding rate is 0; after the cells enter an exponential growth phase, namely a fermentation growth phase, the OUR is between 60 and 120mmol/L/h, and the feeding speed is adjusted according to the consumption rate of the substrate; when the cells enter a growth stable phase, namely a fermentation stable stage, the OUR is between 120 and 160mmol/L/h, and the feeding speed is adjusted according to the consumption rate of the substrate; the cells gradually enter a decline period, namely a fermentation decline stage, the OUR is between 120 and 140mmol/L/h, and the feeding speed is adjusted according to the substrate consumption rate.
The corresponding relation between all the fermentation stages and the oxygen consumption rate is constructed according to the corresponding relation between each fermentation stage and the oxygen consumption rate in the initial fermentation stage, the growth fermentation stage, the stable fermentation stage and the decline fermentation stage.
Fig. 2 is a schematic flow diagram of a fermentation stage validation model provided by the present invention, the fermentation stage validation model further configured to:
confirming a fermentation initial stage and a corresponding relation with a preset feeding speed based on the current oxygen consumption rate;
confirming a fermentation growth stage and a corresponding relation with a first feeding control interval based on the current oxygen consumption rate;
confirming a fermentation stabilization stage and a corresponding relation with a second feeding control interval based on the current oxygen consumption rate;
confirming a corresponding relation between a fermentation decline stage and a third feeding control interval based on the current oxygen consumption rate;
and the numerical values of the preset feeding speed, the first feeding control interval, the second feeding control interval and the third feeding control interval are preset feeding speeds.
Optionally, confirming a feeding control interval corresponding to the target fermentation stage based on the current target fermentation stage; regulating said feed rate command based on the value of said feed control interval in combination with said substrate consumption rate, comprising:
in case the target fermentation stage is the initial stage of fermentation, no feed is required. In an alternative embodiment, the preset feeding rate is 0, that is, no feeding of the polyhydroxyalkanoate being fermented is needed, but a small amount of oil may be fed according to the preset feeding rate, that is, a feeding rate command is generated according to the preset feeding rate. As understood by those skilled in the art, the oil supplementing time point is the time point when the oil added into the culture medium is just started and the feeding is started after the oil added into the culture medium is consumed for a period of time, and the OD value is mainly used for determining whether the feeding is started or not, wherein the OD is a comprehensive index of the concentration, the chromaticity, the bacterial amount and the bacterial elongation and expansion of the fermentation culture medium. In general, feeding can be initiated when the OD is greater than or equal to 30.
Under the condition that the target fermentation stage is a fermentation growth stage, a fermentation stabilization stage or a fermentation decline stage, oil is supplemented according to a preset material supplementing speed, and then a real-time substrate consumption rate V is combined s And regulating and controlling the oil supplementing rate as the oil supplementing rate required at the current moment.
In step 201, the corresponding relationship between the initial fermentation stage and the preset feeding speed is determined, and the preset feeding speed is determined when the fermentation of the polyhydroxyalkanoate is in the initial fermentation stage, and the preset feeding speed is optionally 0, that is, the polyhydroxyalkanoate being fermented does not need to be fed in the whole fermentation stage of the polyhydroxyalkanoate.
In step 202, the corresponding relationship between the fermentation growth phase and a first feeding control interval is determined, in the case that the fermentation of polyhydroxyalkanoate is in the fermentation growth phase, the numerical value of the first feeding control interval, including the substrate consumption rate from the beginning of feeding in the fermentation growth phase to the substrate consumption rate before the beginning of the fermentation stabilization phase, is optionally 3g/L/h to 5g/L/h, that is, in the fermentation growth phase, the feeding speed is controlled according to the rate of 3g/L/h to 5 g/L/h.
In step 203, the corresponding relationship between the fermentation stabilization phase and a second feeding control interval is determined, and in the case that the fermentation of polyhydroxyalkanoate is in the fermentation stabilization phase, the numerical value of the second feeding control interval, including the substrate consumption rate from the start of feeding in the fermentation stabilization phase to the substrate consumption rate before the start of the fermentation decline phase, is optionally 5g/L/h to 10g/L/h, that is, in the fermentation stabilization phase, the feeding speed is controlled according to the rate from 5g/L/h to 10 g/L/h.
In step 204, the corresponding relationship between the fermentation decay phase and a third feeding control interval is determined, and under the condition that the fermentation of the polyhydroxyalkanoate is in the fermentation decay phase, the numerical value of the third feeding control interval comprises the substrate consumption rate from the beginning of feeding in the fermentation decay phase to the end of the fermentation, and the third feeding control interval is optionally 3g/L/h to 6g/L/h, namely, under the fermentation decay phase, the feeding speed is controlled according to the rate of 3g/L/h to 6 g/L/h.
In another alternative embodiment, a linear fit relationship may also be established based on the substrate consumption rate and the feeding rate for each feeding control interval, or an exponential fit relationship may be established to determine different feeding rates within each feeding control interval based on different substrate consumption rates.
FIG. 3 is a schematic flow chart of the present invention for calculating a substrate consumption rate from a quantitative relationship model for calculating the substrate consumption rate based on an established quantitative relationship between an oxygen consumption rate, a carbon dioxide generation rate, and a substrate conversion rate;
the quantitative relationship model specifically comprises the following execution steps:
determining a carbon dioxide generation component according to the carbon dioxide generation rate and a first coefficient;
determining a consumption difference value according to the oxygen consumption rate and the carbon dioxide generation component;
determining a consumption component according to the consumption difference and a second coefficient;
and determining the substrate consumption rate according to the consumption component and the substrate conversion rate.
Specifically, the establishment process of the quantitative relation model between the exhaust gas monitoring data and the substrate consumption rate is as follows:
OUR＝OUR1+OUR2 (4)
CER＝CER1+CER2 (5)
in the formulas (4) and (5), OUR is oxygen consumption rate, CER is carbon dioxide generation rate, OUR1 is PHA synthesis oxygen consumption rate, OUR2 is cell respiration oxygen consumption rate, and CER1 is PHA synthesis CO 2 Release rate, CER2 is cellular respiration CO 2 The release rate.
Further, the oxygen consumption rate and CO of PHA synthesis 2 Release rate ratio k 1 Comprises the following steps:
further, cellular respiration oxygen consumption rate and CO 2 The release rate ratio β is:
further, there are
Consumption of k according to the synthesis of 1g PHA 2 Oxygen-gramGas, then PHA synthesis rate V p Comprises the following steps:
assuming that the conversion of oil to PHA is Yield, the substrate consumption rate V s Comprises the following steps:
order to
Then it is got>
Namely, the formula (3).
In step 301, a carbon dioxide generation component is determined according to a product of the carbon dioxide generation rate and a first coefficient β, where a value range of the first coefficient is selectable between 1 and 3.
In step 302, a consumption difference is determined based on a difference between the oxygen consumption rate and the carbon dioxide production component, specifically by subtracting the carbon dioxide production component from the oxygen consumption rate.
In step 303, a consumption component is determined according to a product of the consumption difference and a second coefficient, and a value range of the second coefficient α is selectable between 0.5 and 0.8.
In step 304, determining the substrate consumption rate according to the quotient of the consumption component and the substrate-to-product conversion rate, specifically, refer to the above formula (3), which is not repeated herein, and optionally, the value of the substrate-to-product conversion rate ranges from 0.65 to 0.9.
FIG. 4 is a schematic structural diagram of a microbial fermentation control system provided by the present invention, which discloses a microbial fermentation control system comprising a microbial fermentation control device 6 for controlling a microbial fermentation process according to the off-gas monitoring data;
further comprising: a fermentation tank 1 for providing a fermentation environment for microorganisms;
the tail gas sampling pipeline 2 is used for collecting tail gas from the fermentation tank;
a flow distributor 3 for flow regulation;
the tail gas mass spectrum analysis device 4 is used for analyzing the component information of the tail gas;
the tail gas state monitoring unit 5 is used for acquiring tail gas monitoring data;
the microorganism is a microorganism capable of accumulating polyhydroxyalkanoate in a cell.
In the invention, multidimensional parameters in the fermentation process are realized through a tail gas mass spectrometer 4, a tail gas state monitoring unit 5 is used for acquiring tail gas monitoring data, fermentation tail gas is subjected to flow regulation from a fermentation tank 1 through a tail gas sampling pipeline 2 through a flow distributor 3 and then enters the tail gas mass spectrometer 4, the volume flow entering the tail gas mass spectrometer 4 is 0-2L/min, the tail gas mass spectrometer 4 can effectively detect the change of a series of component concentrations such as oxygen concentration, carbon dioxide concentration and nitrogen concentration in the tail gas, the tail gas mass spectrometer 4 transmits the detected result information to the tail gas state monitoring unit 5, the concentration content information of each component in the tail gas can be monitored in real time, and finally, based on a quantitative relation model between the tail gas monitoring parameters and substrate consumption rate, a microbial fermentation control device 6 is adjusted in real time to adjust the feeding speed. Specifically, the process for establishing the quantitative relation model between the exhaust monitoring data and the substrate consumption rate refers to the above process, and is not described herein again.
The present invention also includes a memory and a program or instructions stored on the memory and executable on the microbial fermentation control device 6, the program or instructions, when executed by the microbial fermentation control device 6, performing the fermentation control method for producing polyhydroxyalkanoate, the method comprising: collecting tail gas monitoring data of a fermentation process of microorganisms; respectively inputting the tail gas monitoring data to a quantitative relation model for data analysis; outputting, by the quantitative relational model, a substrate consumption rate; determining a feed rate command according to the substrate consumption rate; the feeding speed instruction is used for indicating feeding to the fermentation process according to the feeding speed.
In order to verify that the invention can play the roles of real-time monitoring and accurate control on the whole fermentation process, and can improve the PHA yield, the PHA production strength and the conversion rate from a substrate to a product, the invention is explained by combining the following experimental examples:
as comparative experiment example 1 of the present invention, the conventional procedure feeding was performed without using the control method of the present invention, that is, feeding was performed in the PHA production process according to the empirically set numerical value range:
seed culture: the PHBHHx is fermented by taking the eumycete roxburghii as a chassis strain, firstly, primary activation culture is carried out under the conditions of 30 ℃ and 200rpm, the culture is about 10, then, 1 percent (v/v) of the PHBHHx is inoculated into a seed culture medium, the culture is carried out for 10 hours under the conditions of 30 ℃ and 200rpm, and the seed culture medium comprises 10g/L of peptone, 3g/L of yeast powder and 3g/L of ammonium sulfate.
Fermentation culture: inoculating 10% of the strain into 35L of sterilized fermentation medium, wherein the fermentation conditions comprise temperature controlled at 30 ℃, pH controlled at 6.5, ventilation rate controlled at 1vvm, rotation speed controlled at 200rpm, pressure controlled at 0.04MPa, the fermentation medium is palm oil 10g/L, disodium hydrogen phosphate 1g/L, potassium dihydrogen phosphate 2g/L, ammonium sulfate 3g/L and magnesium sulfate heptahydrate 0.2g/L.
Procedure set empirically:
fermenting for 0-10 h, controlling the stirring speed and the dissolved oxygen coupling at 30%, and controlling the highest rotation speed at 300rpm;
fermenting for 10-30 h, and feeding the oil supplement at a constant speed at an oil supplement rate of 3 g/L/h;
fermenting for 30-50 h, and adjusting the oil supplementing rate to 6g/L/h;
fermenting for 50-56 h, adjusting the oil supplementing rate to 4g/L/h, maintaining the oil supplementing rate to a lower tank, and finally discharging the oil from the lower tank to obtain the PHA output of 8.40kg, the PHA production intensity of 3g/L/h and the conversion rate from a substrate to a product of 80%.
Experimental example 1: compared with comparative experiment example 1, the control method provided by the invention is adopted to feed materials in the PHA fermentation production process:
seed culture: as in comparative example 1, no further description is given here.
Fermentation culture: as in comparative example 1, no further description is given here.
Constructing a tail gas quantitative relation model: and (3) establishing a tail gas monitoring parameter according to the fermentation control condition, realizing monitoring of the tail gas parameter indexes OUR and CER in tail gas state monitoring software, and establishing a quantitative relational expression of tail gas monitoring data and substrate consumption rate.
The fermentation control based on the present invention results in the following process:
fermenting for 0-10 h, wherein the coupling of the stirring rotating speed and dissolved oxygen is controlled at 30%, and the highest rotating speed is 300rpm;
fermenting for 10-30 h, starting feeding and supplementing materials, and based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time, wherein the oil supplementing rate is controlled to be between 3.3 and 4.3 g/L/h;
fermenting for 30-50 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 4.3-6.4 g/L/h;
fermenting for 50-56 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 4.6-5.0 g/L/h; when the fermentation is finally discharged from the tank, compared with the feeding fermentation procedure of the first comparative example, the PHA yield in the feeding mode of the quantitative model is increased by 23.3 percent and reaches 10.36kg, the PHA production intensity is increased by 16.3 percent and reaches 3.49g/L/h, and the conversion rate from the substrate to the product is increased from 80 percent to 85 percent.
As comparative experiment example 2 of the present invention, the conventional programmed feeding method was adopted without using the control method of the present invention, that is, feeding was performed in the PHA production process by fermentation according to the empirically set numerical range:
seed culture: as in comparative example 1, no further description is given here.
Fermentation culture: the fermentation medium was 10g/L soybean oil, and the rest of the fermentation medium was identical to that in comparative example 1 and will not be described herein.
Procedure set empirically:
fermenting for 0-10 h, controlling the coupling of stirring speed and dissolved oxygen at 30% and the highest rotation speed of 300rpm;
fermenting for 10-30 h, and feeding the oil supplement at a constant speed at an oil supplement rate of 3 g/L/h;
fermenting for 30-50 h, and adjusting the oil supplementing rate to 5g/L/h;
the fermentation time is 50-56 h, the oil-supplementing rate is adjusted to 3g/L/h, the fermentation is maintained until the oil is discharged into the lower tank, and finally, the fermentation result is shown in the table 1, the PHA yield is 6.75kg, the PHA production intensity is 2.41g/L/h, and the conversion rate from the substrate to the product is 75%.
Experimental example 2: compared with comparative experiment example 2, the control method provided by the invention is adopted to supplement materials in the PHA fermentation production process:
seed culture: as in comparative Experimental example 1, no further description is given here.
Fermentation culture: the fermentation medium was 10g/L soybean oil, and the rest of the medium was identical to that in comparative example 1 and will not be described herein.
Constructing a tail gas quantitative relation model: and (3) establishing a tail gas monitoring parameter according to the fermentation control condition, realizing monitoring of the OUR and CER parameters in tail gas state monitoring software, and establishing a quantitative relational expression of the tail gas monitoring parameter and the substrate consumption rate.
The fermentation control based on the invention results in the following process:
fermenting for 0-10 h, wherein the stirring rotation speed and the dissolved oxygen coupling are controlled at 30%, and the maximum rotation speed is 300rpm;
fermenting for 10-30 h, starting feeding and supplementing materials, and based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time, wherein the oil supplementing rate is controlled to be between 3 and 4g/L/h;
fermenting for 30-50 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 3.5-5.5 g/L/h;
fermenting for 50-56 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 4.0-5.0 g/L/h; when the fermentation is finally discharged from the tank, compared with the programmed feeding fermentation, the fermentation result is increased by 15.6 percent and reaches 7.8kg by adopting the PHA yield under the quantitative model feeding mode, the PHA production intensity is increased by 15.8 percent and reaches 2.79g/L/h, and the fermentation is finished at the bottomThe conversion of material to product increased from 75% to 78%.
As comparative experiment example 3 of the present invention, the control method of the present invention was not used, and the conventional procedure feeding was used, that is, feeding was performed in the PHA production process according to the empirically set numerical range:
seed culture: PHB was fermented using Eumycota rolfsii as a chassis strain, and other elements were not described in detail in comparative example 1.
Fermentation culture: as in comparative example 1, no further description is given here.
Procedure set empirically:
fermenting for 0-10 h, controlling the coupling of stirring speed and dissolved oxygen at 30% and the highest rotation speed of 300rpm;
fermenting for 10-30 h, and feeding the oil supplement at a constant speed at an oil supplement rate of 3 g/L/h;
fermenting for 30-50 h, and adjusting the oil supplementing rate to 4.5g/L/h;
fermenting for 50-56 h, adjusting the oil supplementing rate to 3.5g/L/h, maintaining the oil supplementing rate to a lower tank, and finally discharging the oil from the lower tank, wherein the PHA yield is 6.65kg, the PHA production intensity is 2.38g/L/h, and the conversion rate from a substrate to a product is 76%.
Experimental example 3: compared with comparative experiment example 3, the control method provided by the invention is adopted to supplement materials in the PHA fermentation production process:
seed culture: PHB was fermented using a strain of Eutropha rolfsii as a chassis strain, otherwise, as in comparative example 1, and the details thereof are not repeated.
Fermentation culture: as in comparative example 1, no further description is given here.
Constructing a tail gas quantitative relation model: and (3) establishing a tail gas monitoring parameter according to the fermentation control condition, realizing monitoring of the OUR and CER parameters in tail gas state monitoring software, and establishing a quantitative relational expression of the tail gas monitoring parameter and the substrate consumption rate.
The fermentation control based on the invention results in the following process:
fermenting for 0-10 h, wherein the coupling of the stirring rotating speed and dissolved oxygen is controlled at 30%, and the highest rotating speed is 300rpm;
fermenting for 10-30 h, starting feeding materials in a flowing manner, and based on real timeRate of substrate consumption V s Adjusting the oil supplementing rate in real time, wherein the oil supplementing rate is controlled to be between 3 and 4g/L/h;
fermenting for 30-50 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 3-4.5 g/L/h;
fermenting for 50-56 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 3-3.5 g/L/h; upon final tank drop, the third example increased PHA production by 5.6% using the quantitative model feeding mode to 7.02kg, increased PHA production intensity by 5.5% to 2.51g/L/h, and increased substrate to product conversion from 76% to 78% compared to the third comparative example.
In order to further verify whether the fermentation control process is suitable for different fermentation conditions, the invention also makes experimental examples under the conditions of different active strains, different culture media, different rotating speeds and the like:
experimental example 4: adopting high-activity seeds and utilizing the control method of the invention to supplement materials in the process of producing PHA by fermentation:
seed culture: 3% (v/v) of the seed culture medium was inoculated in the same manner as in comparative experiment example 1, and the details thereof are omitted.
Fermentation culture: as in comparative example 1, no further description is given here.
Constructing a tail gas quantitative relation model: and (3) establishing a tail gas monitoring parameter according to the fermentation control condition, realizing monitoring of the tail gas parameter indexes OUR and CER in tail gas state monitoring software, and establishing a quantitative relational expression of the tail gas monitoring parameter and the substrate consumption rate.
The fermentation control based on the invention results in the following process:
fermenting for 0-10 h, wherein the stirring rotation speed and the dissolved oxygen coupling are controlled at 30%, and the maximum rotation speed is 300rpm;
fermenting for 10-30 h, starting feeding and supplementing materials, and based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time, wherein the oil supplementing rate is controlled to be between 4 and 5g/L/h;
fermenting for 30-50 h based on the real-time substrate consumption rate V s Real-time adjustment of oil supplyThe speed and the oil supplementing speed are 6 to 8g/L/h;
fermenting for 50-56 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 4-5 g/L/h; when the seeds are finally discharged from the tank, the PHA yield is further improved to 12.18kg under the modes of high-activity seeds and quantitative model feeding, the PHA production intensity reaches 3.95g/L/h, and the conversion rate from the substrate to the product is 82%.
Experimental example 5: adopting different fermentation culture media, and supplementing materials in the process of producing PHA by fermentation by using the control method of the invention:
seed culture: as in comparative Experimental example 1, no further description is given here.
Fermentation culture: the fermentation medium is 20g/L palm oil, 1g/L disodium hydrogen phosphate, 2g/L potassium dihydrogen phosphate, 3g/L ammonium sulfate and 0.1g/L magnesium sulfate heptahydrate, and other parts are the same as those in comparative experiment example 1 and are not described again.
Constructing a tail gas quantitative relation model: and (3) establishing a tail gas monitoring parameter according to the fermentation control condition, realizing monitoring of the tail gas parameter indexes OUR and CER in tail gas state monitoring software, and establishing a quantitative relational expression of the tail gas monitoring parameter and the substrate consumption rate.
The fermentation control based on the invention results in the following process:
fermenting for 0-10 h, wherein the coupling of the stirring rotating speed and dissolved oxygen is controlled at 30%, and the highest rotating speed is 300rpm;
fermenting for 10-30 h, starting feeding and supplementing materials, and based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time, wherein the oil supplementing rate is controlled to be between 3.5 and 4.5g/L/h;
fermenting for 30-50 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 5-7 g/L/h;
fermenting for 50-56 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 4.5-6.5 g/L/h; when the PHA is finally put into a tank, under different culture media and a quantitative model feeding mode, the PHA yield reaches 10.61kg, the PHA production intensity reaches 3.64g/L/h, and the conversion rate from the substrate to the product is 85 percent.
Experimental example 6: feeding materials in the process of producing PHA by fermentation by using different rotating speeds and the control method of the invention:
seed culture: as in comparative example 1, no further description is given here.
Fermentation culture: the fermentation conditions were 30 ℃ for temperature, 6.5 for pH, 1vvm for ventilation, 300rpm for rotation speed and 0.04MPa for pressure, and the same as in comparative example 1 are omitted here for brevity.
Constructing a tail gas quantitative relation model: and (3) establishing a tail gas monitoring parameter according to the fermentation control condition, realizing monitoring of the OUR and CER parameters in tail gas state monitoring software, and establishing a quantitative relational expression of the tail gas monitoring parameter and the substrate consumption rate.
The fermentation control based on the invention results in the following process:
fermenting for 0-10 h, wherein the stirring rotation speed and the dissolved oxygen coupling are controlled at 30%, and the maximum rotation speed is 300rpm;
fermenting for 10-30 h, starting feeding and supplementing materials, and based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time, wherein the oil supplementing rate is controlled to be between 3.3 and 4.3 g/L/h;
fermenting for 30-50 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 5.5-8.5 g/L/h;
fermenting for 50-56 h based on the real-time substrate consumption rate V s Adjusting the oil supplementing rate in real time to 6.5-4.5 g/L/h; when the final tank is taken out, under the conditions of increasing the rotating speed and feeding the quantitative model, the PHA yield reaches 10.79kg, the PHA production intensity reaches 3.71g/L/h, and the conversion rate of the substrate to the product is 83 percent.
In order to facilitate the examination of the correspondence between various influencing factors and the PHA production, the PHA production intensity and the conversion of the substrate into the product, the present invention summarizes the respective parameters and the test results of the above comparative examples 1-3 and examples 1-6, as shown in Table 1 below.
TABLE 1
Wherein, the oil supplement amount represents the total oil added mass at the end of fermentation, the PHA yield represents the product of PHA concentration and fermentation volume at the end of fermentation, the PHA production intensity represents the average PHA synthesis speed in the fermentation process, and the substrate-to-product conversion rate represents the ratio of the product PHA to the substrate oil mass.
As shown in FIG. 5, FIG. 5 is a graph showing the effect of the method for controlling fermentation using microorganisms according to the present invention, in which the PHA production strength and the substrate-to-product conversion rate (substrate conversion rate) of comparative examples 1-3 and examples 1-6 are further compared, and it can be seen that, in comparison between the production strength and the substrate conversion rate, examples 1-3 are respectively better than comparative examples 1-3; moreover, even under different fermentation conditions, the production intensity and the substrate conversion rate of the experimental examples 4-6 are higher than those of the comparative experimental example 1, and meanwhile, the stability of the control method provided by the invention is higher by combining the experimental example 1.
The invention provides a microbial fermentation control method, a device, a system, equipment and a medium, which take a fermentation process of polyhydroxyalkanoate as an example, input tail gas monitoring data is obtained in real time in the fermentation process of polyhydroxyalkanoate, the tail gas monitoring data is input into a quantitative relation model in real time to determine a substrate consumption rate, and a feeding speed instruction is regulated and controlled in a feeding control interval corresponding to a real-time fermentation stage of polyhydroxyalkanoate according to the substrate consumption rate in combination with the feeding control interval corresponding to the real-time fermentation stage of polyhydroxyalkanoate, so that feeding control of all stages of polyhydroxyalkanoate fermentation is realized.
Fig. 6 is a schematic structural diagram of a microbial fermentation control device provided by the present invention, and taking a fermentation control process of polyhydroxyalkanoate as an example, the present invention provides a fermentation control device for preparing polyhydroxyalkanoate, comprising:
the acquisition unit 51: the system is used for collecting tail gas monitoring data of a fermentation process of microorganisms;
the analyzing unit 52: the system is used for inputting the tail gas monitoring data to a quantitative relation model for data analysis; outputting, by the quantitative relational model, a substrate consumption rate;
the determination unit 53: for determining a feed rate command based on the substrate consumption rate;
the feeding rate instruction is used for indicating feeding to the fermentation process according to the feeding rate.
In the microbial fermentation control device according to the present invention, the analysis unit further includes: the system is used for inputting the oxygen consumption rate to a fermentation stage confirmation model, confirming a current target fermentation stage by the fermentation stage confirmation model, and confirming the start and the end of a material supplementing program based on the current target fermentation stage; the feeding control interval is used for confirming a feeding control interval corresponding to the target fermentation stage based on the current target fermentation stage; regulating the feed rate based on the value of the feed control interval in combination with the substrate consumption rate.
Fig. 7 is a schematic structural diagram of an electronic device provided by the present invention. As shown in fig. 7, the electronic device may include: a processor (processor) 610, a communication Interface (Communications Interface) 620, a memory (memory) 630 and a communication bus 640, wherein the processor 610, the communication Interface 620 and the memory 630 communicate with each other via the communication bus 640. The processor 610 may invoke logic instructions in the memory 630 to perform a microbial fermentation control method comprising: collecting tail gas monitoring data of a fermentation process of microorganisms; respectively inputting the tail gas monitoring data to a fermentation stage confirmation model and a quantitative relation model for data analysis; outputting a target fermentation stage by the fermentation stage confirmation model, and outputting a substrate consumption rate by the quantitative relation model; regulating and controlling a feeding speed instruction according to the substrate consumption rate and a feeding control interval of a target fermentation stage; the feeding rate instruction is used for indicating feeding to the fermentation process according to the feeding rate.
In addition, the logic instructions in the memory 630 may be implemented in software functional units and stored in a computer readable storage medium when the logic instructions are sold or used as independent products. Based on such understanding, the technical solution of the present invention may be embodied in the form of a software product, which is stored in a storage medium and includes instructions for causing a computer device (which may be a personal computer, a server, or a network device) to execute all or part of the steps of the method according to the embodiments of the present invention. And the aforementioned storage medium includes: a U-disk, a removable hard disk, a Read-Only Memory (ROM), a Random Access Memory (RAM), a magnetic disk or an optical disk, and other various media capable of storing program codes.
In another aspect, the present invention also provides a computer program product, the computer program product comprising a computer program, the computer program being stored on a non-transitory computer readable storage medium, wherein when the computer program is executed by a processor, the computer is capable of executing a method for controlling microbial fermentation provided by the above methods, the method comprising: collecting tail gas monitoring data of a fermentation process of microorganisms; respectively inputting the tail gas monitoring data to a fermentation stage confirmation model and a quantitative relation model for data analysis; outputting a target fermentation stage by the fermentation stage confirmation model, and outputting a substrate consumption rate by the quantitative relation model; determining or regulating a feeding speed instruction according to the substrate consumption rate and a feeding control interval of a target fermentation stage; the feeding speed instruction is used for indicating feeding to the fermentation process according to the feeding speed.
In yet another aspect, the present invention also provides a non-transitory computer readable storage medium having stored thereon a computer program that when executed by a processor is operative to perform the methods described above to provide a method for controlling microbial fermentation, the method comprising: collecting tail gas monitoring data of a fermentation process of microorganisms; respectively inputting the tail gas monitoring data to a fermentation stage confirmation model and a quantitative relation model for data analysis; outputting a target fermentation stage by the fermentation stage confirmation model, and outputting a substrate consumption rate by the quantitative relation model; determining or regulating a feeding speed instruction according to the substrate consumption rate and a feeding control interval of a target fermentation stage; the feeding speed instruction is used for indicating feeding to the fermentation process according to the feeding speed.
The above-described embodiments of the apparatus are merely illustrative, and the units described as separate parts may or may not be physically separate, and parts displayed as units may or may not be physical units, may be located in one place, or may be distributed on a plurality of network units. Some or all of the modules may be selected according to actual needs to achieve the purpose of the solution of the present embodiment. One of ordinary skill in the art can understand and implement it without inventive effort.
Through the above description of the embodiments, those skilled in the art will clearly understand that each embodiment may be implemented by software plus a necessary general hardware platform, and may also be implemented by hardware. With this understanding in mind, the above-described technical solutions may be embodied in the form of a software product, which can be stored in a computer-readable storage medium such as ROM/RAM, magnetic disk, optical disk, etc., and includes instructions for causing a computer device (which may be a personal computer, a server, or a network device, etc.) to execute the methods described in the embodiments or some parts of the embodiments.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for controlling microbial fermentation, comprising:
collecting tail gas monitoring data of a fermentation process of microorganisms;
inputting the tail gas monitoring data to a quantitative relation model for data analysis, and outputting a substrate consumption rate by the quantitative relation model;
determining a feed rate command according to the substrate consumption rate;
the feeding speed instruction is used for indicating feeding to the fermentation process according to the feeding speed.
2. The microbial fermentation control method of claim 1, wherein the tail gas monitoring data comprises oxygen consumption rate, carbon dioxide generation rate, PHA synthesis oxygen consumption rate, PHA synthesis CO 2 Release rate, oxygen uptake rate by cellular respiration, cellular respiration CO 2 The release rate;
the quantitative relation model is used for calculating the substrate consumption rate based on a quantitative relation established among the oxygen consumption rate, the carbon dioxide generation rate and the substrate conversion rate;
the quantitative relationship model specifically performs the following steps:
determining a carbon dioxide generation component according to the carbon dioxide generation rate and a first coefficient;
determining a consumption difference value according to the oxygen consumption rate and the carbon dioxide generation component;
determining a consumption component according to the consumption difference and a second coefficient;
determining a substrate consumption rate according to the consumption component and the substrate conversion rate;
the first coefficient is formed by the oxygen consumption rate of cell respiration and the CO of cell respiration 2 Calculating the release rate; the second coefficient is composed of the first coefficient, PHA synthesis oxygen consumption rate, PHA synthesis oxygen consumption and PHA synthesis CO 2 The release rate is calculated.
3. The method of claim 2, further comprising:
inputting the oxygen consumption rate to a fermentation stage confirmation model, confirming a current target fermentation stage by the fermentation stage confirmation model, and confirming the start and the end of a feeding program based on the current target fermentation stage;
confirming a material feeding control interval corresponding to the target fermentation stage based on the current target fermentation stage; regulating the feed rate based on the value of the feed control interval in combination with the substrate consumption rate.
4. The method of controlling fermentation of a microorganism according to claim 3,
the target fermentation stage comprises a fermentation initial stage, a fermentation growth stage, a fermentation stabilization stage and a fermentation decline stage, and the corresponding feeding control intervals are respectively as follows: presetting a feeding speed, a first feeding control interval, a second feeding control interval and a third feeding control interval;
and the numerical values of the preset feeding speed, the first feeding control interval, the second feeding control interval and the third feeding control interval are preset feeding speeds.
5. The method of controlling fermentation of a microorganism according to any one of claims 1 to 4, wherein said microorganism is a microorganism capable of intracellular accumulation of polyhydroxyalkanoate, and comprises a microorganism belonging to the genus: aeromonas, alcaligenes, azotobacter, bacillus, clostridium, halobacterium, nocardia, rhodospirillum, pseudomonas, ralstonia, and Acinetobacter.
6. A microbial fermentation control apparatus, comprising:
a collecting unit: the system is used for collecting tail gas monitoring data of a fermentation process of microorganisms;
an analysis unit: the system is used for inputting the tail gas monitoring data to a quantitative relation model for data analysis; outputting, by the quantitative relational model, a substrate consumption rate;
a determination unit: for determining a feed rate command based on the substrate consumption rate;
the feeding rate instruction is used for indicating feeding to the fermentation process according to the feeding rate.
7. The microbial fermentation control apparatus of claim 6, comprising:
the analysis unit further comprises: the system is used for inputting the oxygen consumption rate to a fermentation stage confirmation model, confirming the current target fermentation stage by the fermentation stage confirmation model, and confirming the start and the end of a feeding program based on the current target fermentation stage; the feeding control interval is used for confirming a feeding control interval corresponding to the target fermentation stage based on the current target fermentation stage; regulating the feed rate based on the value of the feed control interval in combination with the substrate consumption rate.
8. A microbial fermentation control system, comprising the microbial fermentation control device of claim 6 or 7, for controlling the fermentation process of microorganisms according to the off-gas monitoring data;
further comprising:
a fermentation tank for providing a fermentation environment for the microorganisms;
the tail gas sampling pipeline is used for collecting tail gas from the fermentation tank;
a flow distributor for flow regulation;
the tail gas mass spectrum analysis device is used for analyzing the component information of the tail gas;
the tail gas state monitoring unit is used for acquiring tail gas monitoring data;
the microorganism is a microorganism capable of accumulating polyhydroxyalkanoate in a cell.
9. An electronic device comprising a memory, a processor and a computer program stored on the memory and executable on the processor, wherein the processor implements the method of controlling microbial fermentation according to any one of claims 1 to 5 when executing the computer program.
10. A non-transitory computer-readable storage medium having stored thereon a computer program, wherein the computer program, when executed by a processor, implements the microbial fermentation control method of any one of claims 1 to 5.
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Address after: Building 1, No. 351 Yuexiu Road, Hongkou District, Shanghai 200072 Patentee after: Shanghai Blue Crystal Microbial Technology Co.,Ltd. Country or region after: China Address before: 102206 1st floor, building 4, courtyard 20, shengshengyuan Road, Life Science Park, Changping District, Beijing Patentee before: BLUEPHA Co.,Ltd. Country or region before: China |