CN1177007A - Cultivation method by controlling rhythm of somatic metabolism during the process of aerobe bacteria fermentation - Google Patents
Cultivation method by controlling rhythm of somatic metabolism during the process of aerobe bacteria fermentation Download PDFInfo
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- CN1177007A CN1177007A CN 96119825 CN96119825A CN1177007A CN 1177007 A CN1177007 A CN 1177007A CN 96119825 CN96119825 CN 96119825 CN 96119825 A CN96119825 A CN 96119825A CN 1177007 A CN1177007 A CN 1177007A
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Abstract
The present method makes the two non-linear time-varied biochemical variables (CO2 and pH) which represent the metabolic condition of bacteria during fermentation, connect into a correlation of synchronized reverse phase, and through the control system, such as computer system, changes the different operating conditions of process so as to cause the bacteria proceed enzymatic biochemical reaction under designed oscillation rhythm. The invention can raise the conversion rate of fermentation and shorten the fermentation period.
Description
The present invention relates to a kind of aerobic cultural method, this method is that the Fed-Batch process in the fermentor tank is regarded as a space-time chaos system, and on-line Control non-linear variable is wherein finished the enzymatic biochemical reaction of living organisms under rhythm and pace of moving things state.
In typical yeast culture program in the biochemical industry is (the about 2M of volume in a seed fermentation jar of elder generation
3) carry out cultivating seeds, seed culture medium is transported in the seeding tank behind the scalding, the microorganism cells of crossing through sterile culture (bacterial strain) is tapped into jar allows its grow, is just to be transported to Primary Fermentation equipment when being in logarithmic phase to begin fermentation culture and obtain product through certain hour (about 16 hours) when cell number shows microorganism, and prior art all is at the fermentation biological process in the Primary Fermentation equipment about monitoring and control.
The Primary Fermentation process generally is divided into three phases, and earlier fermentation is the long bacterium stage, and ferment middle and later stage are product accumulation and the drainage stage metabolic processes of thalline just.In aerobic culturing process, attemperation, pH, ventilation and stream add nitrogenous source timely and accurately, and carbon source can be controlled thalline breeding, base consumption speed, improves product and forms speed.Most of up to now fermentation industries still adopt every several hrs to take out sample in fermentor tank, and off-line carries out biochemical analysis, measure cell concentration, carbon source concentration and production concentration, so that take corresponding operating to carry out process control.
The prior art that European patent EP 0661380A2 " aerobic microorganism cultured method and equipment " is introduced comprises method and apparatus, its method be exploitation in a kind of on-line measurement fermented liquid cell concentration, carbon source concentration and production concentration and the method for ammonium ion concentration, go to calculate the stream rate of acceleration according to these observed values.Quantitatively the carbon source concentration and the ammonium ion concentration of control fermented liquid make aerobic microorganism keep the suitable growth of microbial bacteria in the Fed-Batch culturing process.The equipment of implementing this method comprises a near infrared spectrometer and the computer that joins of instrument and comprise that with function unit that computer joins temperature, pH, stream add carbon source, tank pressure and ventilation therewith.Computer and function unit thereof are formed an on-line system.Automatically got one time fermentation liquid, and carried out on-line analysis in per 10 minutes by near infrared spectrometer.The method of on-line analysis is that the rule of thumb data that provides according to calibration curve is calculated: carbon source concentration=32.36+0.900 * 1295.2 * (F[2270]-1019.0 * F
[2180]-165.7 * F[1982]+848.3 * F[2100]) aminoglutaric acid concentration=-4.064+1.079 * (383.2 * F[2190]+1233.0 * F[2139]
+ 122.9 * F[1982]-998.5 * F[2100]) cell concentration=-1.570+0.321 * (100.1 * F[2348]+71.18 * F
[2208]+and 53.83 * F[1445] ammonium ion concentration=-2.817+0.856 * (846.5 * F[1818]+878.6 * F
[1778]-15.8 * F[2100]) determine that according to the result of on-line analysis stream adds carbon source speed: 1. as analytical results＞SV+0.2 stream rate of acceleration=0.8 * C source wear rate 2.SV+0.2 〉=analytical results＞SV+0.1 stream rate of acceleration=0.9 * C3. analytical results=SV stream rate of acceleration=1 * C4.SV-0.1＞analytical results 〉=SV-0.2 stream rate of acceleration=1.1 * C5.SV-0.2＞analytical results stream rate of acceleration=1.2 * C
The analytical results input computer of near infrared spectrometer, computer calculates the stream rate of acceleration according to the method described above, wherein SV represents roughly to consume in per 10 minutes how many carbon sources (this is the artificial set(ting)value of determining), carbon concentration difference when carbon source wear rate C was meant before ten minutes with ten minutes and the ratio of time, five kinds of stream rates of acceleration are determined a kind ofly by computer, carry out stream by controller and add.
The cell concentration that calculates according to above-mentioned formula is used for determining the thalli growth state, carries out temperature control:
Initial temperature heats up for the first time and heats up for the second time
31.5 ℃ when cell concentration 〉=5.6g/l as cell concentration 〉=8.0g/l
36 ℃ of 39 ℃ of prior aries remain on certain value substantially to pH, tank pressure and air flow, do not regulate.
Prior art has following weak point:
1. the cultural method that proposes of prior art only is to replace hand sampling and be transported near infrared spectrometer carrying out analytical calculation with computer, does not have advanced new technology aspect the cell concentration in the measurement fermented liquid and the method for other concentration.In fact domesticly adopt farinfrared spectrophotometer 721 to measure cell concentrations and carbon source concentration already and with enzyme membrane sensing technology mensuration production concentration, these all are classic methods.
2. the online test method of prior art proposition is the linear systematic quantification method of a kind of static state, and the specific absorption of sample under the illumination of various wavelength according to experimental formula and typical curve, calculated cell concentration, carbon source concentration and production concentration.In fact, the culturing process of microorganism shows at random and non-linear behavior, usually not that a pile rule of thumb data can be lived by frame, for example with prior art detect cell concentration reflected is the thalline number that comprises dead thalline, the situation of truly growing that can not reflect thalline is so the evaluation method of this classics can only be as a reference.
3. to have ignored one of most important condition that aerobic microorganism cultivates be the supply of oxygen to prior art, and in fact the aerobic situation of thalline is that breeding along with bacterium is with metabolic different steps and different.Prior art thinks that the cell concentration that pH, tank pressure and ventilation maintain certain value substantially and do not pass through to monitor changes pH and air flow, illustrates that the method for prior art is unreasonable.
4. the emphasis of the cultural method of prior art proposition is the carbon source concentration according to online detection, estimates the carbon source wear rate, goes to determine the stream rate of acceleration of carbon source.Here there are two kinds of errors: the one, near infrared spectrometer is to the different measuring error that produce of the raw-material transmittance of different carbon sources; The 2nd, the progressive error that the carbon source wear rate calculates, because being the carbon concentration with current mensuration, the wear rate of carbon source deducts the carbon concentration last time measured divided by the time, so once the stream rate of acceleration of Ji Suaning is inaccurate, will cause back 10 minutes stream rates of acceleration inaccurate, later on per 10 minutes and the like, progressive error directly influences the exactness that stream adds carbon source.
5. the purpose with the online detection ammonium ion of near infrared spectrometer is to add nitrogenous source for stream in existing patent, and existing patent also is to add nitrogenous source for stream with its purpose of pH electrode detection pH value, these two is the foundation that stream adds nitrogenous source with which kind of detection, because of two kinds of tests do not have association, and do not have between ammonium ion concentration and the amino yet related, so control ammonium ion concentration does not have new Practical significance.
The present invention proposes the new cultural method of a cover, is the picked-up of excitation microbial bacteria to carbon, nitrogen, oxygen, promotes thalli growth to grow and metabolic activity, finally improves the method for output.The key character of the inventive method is in aerobic cultivation, controlling microbial in nutrient solution pH and the carbonic acid gas in the exhaust, make them present anti-phase rhythm and pace of moving things state.So-called rhythm and pace of moving things state, the pulsation or the oscillation behavior that have some cycles and strong and weak changes in amplitude exactly.Anti-phase rhythm and pace of moving things state is meant CO
2Identical with the period of change of pH, but CO worked as
2During rising, pH descends, and CO
2During decline, pH rises.CO
2When peaking, pH just is in low ebb, as shown in Figure 1.The method that produces metabolic rhythm is to use computer monitoring CO
2With pH, and by the synergy of computer run control algolithm with the realization operations.The technological line of the inventive method is as follows:
1. determine CO according to the different steps of thalline in culturing process
2With the oscillation amplitude (set(ting)value) of pH, work as CO
2When not reaching set(ting)value, change temperature, reduce air quantity, replenish vitamin H etc., make CO
2Rise; Work as CO
2When reaching set(ting)value, raising air quantity, stream add nitrogenous source, stream adds carbon source etc., makes CO
2In the following periodical change of set(ting)value.
2. according to the pH regulator scope in each stage in the culturing process, add nitrogenous source with stream and control period of oscillation.Various operations in the culturing process comprise that changing temperature, pH, oxygen-supplying amount and stream adds nitrogenous source, carbon source etc., all are to finish with step or ON/OFF mode.
In a kind of like this cultural method, detected a CO every 1 minute
2And pH, and, judge the consumption situation quantitatively supply in time of nitrogenous source and carbon source through computer with their dynamic trend of graphical representation that shows in real time.With the fundamental difference of prior art be that the inventive method has been carried out dynamic optimal control to culturing process because at any time can be according to CO
2Adjust the operating and setting value with the graphics track of pH, so various operations all can be simplified complicated technology in the culturing process by quantification, all weak points of prior art all can be avoided in the background material.
The specific practice of the control metabolic rhythm that relates in the aerobic microorganism cultural method that the present invention proposes is to set up 4 groups of control loops, as shown in Figure 2, and wherein with pH and CO
2The pass be core, by multivariable controller they and stream add nitrogenous source, change ventilation, stream adds vitamin H, stream adds carbon source and combines and carry out Collaborative Control.These four groups of loops are described below respectively:
Control loop (1): stream adds nitrogenous source as input variable, pH and CO
2Be output variable, by many
The given pH set(ting)value of variable controller, control nitrogenous source stream adds.
Control loop (2): change the input variable of ventilation, pH and CO as process
2For output becomes
The amount, by multivariable controller CO
2Variation feed back to input, by changeable
The given CO of amount controller
2Set(ting)value, control air quantity.
Control loop (3): it is input variable that stream adds vitamin H, pH and CO
2Be output variable, by many
Variable controller is CO
2Variation feed back to input, by the multivariable controller control
The stream dosage of system vitamin H perhaps changes CO
2Set(ting)value.
Control loop (4): it is input variable that stream adds carbon source, pH and CO
2Be output variable, by changeable
Amount controller is CO
2Variation feed back to input, regulate by multivariable controller
The intermittent flow dosage of carbon source, and regulate CO
2The oscillation amplitude set(ting)value.
In above-mentioned four groups of control loops, the input of loop (1) must adopt close/open valve to automatically perform, its cocircuit (2)～(4) can be automatically also can manual operation (control loop (2) is used manual operation usually).
Multivariable controller is the programmodule that is stored in the computer microprocessor, is used for order to carry out the expectant control algorithm.The control algolithm of multivariable controller comprises following two aspect contents in the methods of the invention:
1. the setting of period of oscillation:
The size of period of oscillation is directly proportional with the degree of regulation of pH.Degree of regulation is big, and period of oscillation is big; Regulate
Spend for a short time, period of oscillation is little.
PH span of control=pH set(ting)value ± pH regulator degree
PH regulator degree=pH regulator rate % * (range under the last range-pH of pH)
N=per unit pH value descends the required time in period of oscillation=n * pH regulator degree formula
2. the adjusting of oscillation amplitude:
Artificial intelligence logic evaluation algorithm is adopted in the adjusting of oscillation amplitude, for example: earlier fermentation about 0～6
Between hour, improve air flow and make CO
2Reach maximum value; Between about 6～24 hours of the ferment middle
Improve air flow once more, stream adds nitrogenous source and makes CO simultaneously
2Be controlled at the median size amount, the ferment middle master
To usually regulate CO with improving air flow and additional biology
2Amplitude.During this period approximately from 16 hours
Beginning determines that with reference to the concentration of substrate of off-line analysis stream adds carbon source, keeps CO
2Amplitude.In fermentation
About 24 hours～30 hours of later stage is to reduce air flow control CO
2At certain value.The whole cultivation
Journey CO
2Amplitude be controlled at large, medium and small three kinds of values.Be to realize the method that the present invention proposes, in used device and the device each integral part as shown in Figure 3, the structure of aerobic fermentation process metabolic rhythm control device comprises transmitter and instrument, controlling box, computer system, driving and topworks's four parts.
1. transmitter and instrument: be used for temperature in the online detection fermentation culture, pH and exhaust CO
2
2. controlling box: form by the multiple interfaces card, be used for connecting instrument and computer, carry out data gathering
With the generation control signal.
3. computer system: the control that is used for exercising supervision, carry out the multivariable control algorithm, process is carried out
Intelligent decision-making, comprising graphic presentation and warning, data storage and figure figurate number
According to I/O.
4. drive and topworks, be used for finishing the operations under the optimum rhythm and pace of moving things control.Four integral parts of said apparatus are mainly finished detection, process supervision and decision-making, three kinds of functions of control.The connection of each components interior and detailed effect are described below:
1. detect: the main detection limit of the present invention is temperature, pH and CO
2, wherein the transmitter of temperature and pH is installed in respectively on the fermentor tank tank body, is inserted in jar interior substratum, and sensor output signal is transported to the interior signal conditioning circuit of controlling box, CO through transmitter
2Detection be that the pipeline of drawing from the fermentor tank ventage is through moisture eliminator and strainer input CO
2Measurer, the signal conditioning circuit in the electrical signal of the measuring input controlling box.
2. process supervision and decision-making: these functions are finished by controlling box and computer.
(1) controlling box: by power card, communication card, signal condition card and modulus, digital-to-analog conversion, number go into/count card release to form, respectively the effect that blocks is as follows: power card is used to provide ± 12, ± the 15V D.C. regulated power supply.Communication card is the expansion of computer bus in controlling box, and computer is connected with controlling box, transmits the signal that control signal and reception come from controlling box.The signal condition clamping through isolation, amplification, impedance matching, flows to mould/number conversion card after receiving and coming from the signal of measuring instrument, finishes data gathering by computer.
(2) computer: comprise little processing counter, batch count off certificate and figure output printer, indicating meter, four major partss of storer, the effect of each parts is as follows: little processing counter carries out process monitoring and warning on the one hand, carry controlling box the data acquisition signal of coming to use graphic presentation on display screen, be convenient to operator and monitor culturing process, when culturing process occurs when unusual from employing acousto-optic hint, be convenient to manual intervention, eliminate unusual; Rely on microprocessor to automatically perform control algolithm (artificial intelligence logic evaluation algorithm as previously mentioned) on the other hand, determine the set(ting)value of each control loop, send operational order simultaneously and go into/count card release by the number of controlling box and go to drive topworks.Criticize centre or final technical information that newspaper and figure output printer are used for exporting every batch of cultivation.Indicating meter is used for showing all figures and data.Storer is used for the storing technology profile.
3. control: the control of fermenting process is finished by driving mechanism and topworks by computer and controlling box, and main functional component comprises driving circuit and topworks.Wherein driving circuit accepts to come from the control signal of controlling box, drives topworks with rly..Topworks comprises that stream adds two batch total measuring device and the valves that nitrogenous source and stream add carbon source, and valve is directly installed on the delivery port of fermentation tank deck, and gauger is installed in and is used for the quantitative flow dosage in the pipeline.
The method of the control bacterial metabolism rhythm and pace of moving things that the present invention proposes is not subjected to the different restriction of microbial bacteria kind, is suitable for any aerobic culturing process.Be used to produce each seed amino acid, nucleic acid and Gu Long acid for example are used in the culturing process of producing L-L-glutamic acid, and is effective equally to T6-13 bacterium or F9114 bacterium, has excitation thalli growth and metabolic activity equally, promotes the effect of thalline excretory function.No matter be batch fermentation technology (output once feeds intake) or feed supplement-batch fermentation technology (Fed-Batch) process, present method can obtain to improve the productive rate and the effect in the cycle of shortening equally.Present method nationality helps the dynamic trajectory that computer shows microbial respiratory and consumes nitrogenous source, directly carry out relevant automatically control, dependency to the cell concentration of off-line sampling analysis, carbon source concentration, production concentration is more much smaller than any other cultural method, fermentor tank used in the present invention in addition can be any specification and Any shape, for example: the stirred-tank fermenter of various tonnages or airlift fermentor.Also having, without any restriction, for example can be urea, gas ammonia or liquefied ammonia for the kind of used nitrogenous source.These illustrate that all the inventive method is particularly suitable for plant-scale fermenting process.
Description of drawings 1.CO
2And nonlinear correspondence relation Fig. 2 between the pH. with CO
2With pH be structure general diagram Fig. 4 of the thick block diagram 3. aerobic fermentation metabolic rhythm control device of rhythm and pace of moving things control method of master loop. produce 150M at L-glutamic acid
3Application example 1. on the fermentor tank and example 2. Fig. 5. nonlinear Control real-time graph Fig. 6 of application example 1. and example 2.. the fermentation state off-line analysis of application example 1. and example 2. is Fig. 7 as a result. do not adopt the 150M of the utility model and rhythm and pace of moving things control method
3Equipment comparative example Fig. 8 of fermentor tank. on-line Control figure Fig. 9 of comparative example. the fermentation state off-line analysis result of comparative example
Application example: the industry spot matching equipment synoptic diagram of usefulness control bacterial metabolism rhythm and pace of moving things method fermentative production L-glutamic acid is 1. examples one as shown in Figure 4: industrial glutamic acid fermentation, 150 tons of scale microbial bacteria SF119 tyrothricin fermentor tank 150M
3Stirred pot
Constituent concentration culture medium prescription amylum hydrolysate of the sugar 16.07%
1.1～1.2％
Sal epsom 0.06～0.07%
Corn steep liquor 0.43%
Molasses 0.04%
Three grades 35 ℃～37 ℃～39 ℃ of Repone K 0.1% control condition temperature
Tank pressure 0.7MPa
Air quantity is set three grades and is carried wind 700 → 1100 → 1500 → 1800
Three grades are fallen wind → 1600 → 1500 → 1300
PH sets 7.4 → 7.3 → 7.2 → 7.1 → 7.0
150 rev/mins of stirring velocitys
Stop fermentation condition residual sugar＜0.6%
Fermentation period 30 hours
Stream sugaring amount 3.53% cultural method: substratum was through sterilization (140 ℃) 40 minutes in the main fermentation tank, and the cooling back is from seed
Jar inserts SF119 bacterium liquid to begin to cultivate, and Figure 4 shows that 150M
3Adorn on the fermentor tank
The fermentating metabolism rhythm and pace of moving things control device that is equipped with, wherein online detection limit be temperature, pH,
CO
2, the offline inspection signal is cell concentration, remaining sugar concentration and aminoglutaric acid concentration.The setting of the rhythm and pace of moving things
The earlier fermentation ferment middle fermentation later stage
Period of oscillation (minute) 7.5 7.5 7.5
Oscillation amplitude (CO
2%) 8～13 6～8 0～6 control loops (1):
CO
2Loop set(ting)value with pH
PH CO
2Regularly (hour)
7.4 8％＜CO
2＜13％ 0＜t＜12
7.2 8％＜CO
2＜10％ 12＜t＜24
7.1 5％＜CO
2＜8％ 24＜t＜28
7.0 0％＜CO
2＜5％ 28＜t＜30
6.9 30＜t＜32 control loops (2)
Ventilation and CO
2Between relation (using the manual shift valve)
Ventilation (rice
3/ hour) CO
2The content timing (hour)
700 CO
2＝0 t＝0
1100 CO
2＞4％ t＜8
1500 4％＜CO
2＜12％ t＜8
1800 CO
2≥12％ t＜8
1600 9％≤CO
2＜12％ t＞24
1500 5％＜CO
2＜19％ t＞24
1300 2%CO
2＜5% t＞24 control loops (3)
This batch fermentation does not add vitamin H, because the thalline turbidity of off-line analysis 6 hours
Have a net increase of through reaching＞0.6g/l.While CO
2Amplitude limit reaches 12%, and the thalline number is described
Good with developmental state, need not add vitamin H again.Control loop (4)
Stream sugaring control residual sugar content CO
2Content
5.5 tons of S of 24.4% liquid glucose≤3.6% 5%＜CO
2＜8%
(test tank work output) 4 tons of S≤2.6% 5%＜CO
2＜8%
3 tons of S≤1.6% 5%＜CO
2＜8% batch fermentation acid production rate 10%, transformation efficiency 60.8%, the fermentation period 30 hours control rhythm and pace of moving things as Fig. 5 (on) shown in, three kinds of off-line state analytical resultss of glutamic acid fermentation process as Fig. 6 (on) shown in.2. example two: industrial glutamic acid fermentation, 150 tons of scale microbial bacteria SF119 tyrothricin fermentor tank 150M
3Stirred pot
Constituent concentration culture medium prescription amylum hydrolysate of the sugar 16.2%
1.1～1.2%
Sal epsom 0.06～0.07%
Corn steep liquor 0.45%
Molasses 0.042%
Three grades 35 ℃～37 ℃～39 ℃ of Repone K 0.1% control condition temperature
Tank pressure 0.7MPa
Air quantity is set three grades and is carried wind 700 → 1100 → 1500 → 1800
Three grades are fallen wind → 1600 → 1500 → 1300
PH sets 7.4 → 7.3 → 7.2 → 7.1 → 7.0
150 rev/mins of stirring velocitys
Stop fermentation condition residual sugar＜0.6%
Fermentation period 32 hours
Stream sugaring amount 3.1% cultural method: in the main fermentation tank substratum through sterilization (140 ℃) 40 minutes, the cooling back from
Seeding tank inserts SF119 bacterium liquid to begin to cultivate, and Figure 4 shows that 150M
3Send out
The fermentating metabolism rhythm and pace of moving things control device of equipping on the ferment jar, wherein online detection limit
Be temperature, pH, CO
2, the offline inspection signal is a cell concentration, residual sugar
Concentration and aminoglutaric acid concentration.Rhythm and pace of moving things set(ting)value
The earlier fermentation ferment middle fermentation later stage
Period of oscillation (minute) 15 15 10.5
Oscillation amplitude (CO
2%) 8～13 6～8 0～6 control loops (1):
CO
2Loop set(ting)value with pH
PH CO
2Regularly (hour)
7.4 8％＜CO
2＜13％ 0＜t＜12
7.2 8％＜CO
2＜10％ 12＜t＜24
7.1 5％＜CO
2＜8％ 24＜t＜28
7.0 0％＜CO
2＜5％ 28＜t＜30
6.9 30＜t＜32 control loops (2)
Ventilation and CO
2Between relation (using the manual shift valve)
Ventilation (rice
3/ hour) CO
2The content timing (hour)
700 CO
2＝0 t＝0
1100 CO
2＞4％ t＜8
1500 4％＜CO
2＜12％?t＜8
1800 CO
2≥12％ t＜8
1600 9％≤CO
2＜12％?t＞24
1500 5％＜CO
2＜19％?t＞24
1300 2%CO
2＜5% t＞24 control loops (3)
This batch fermentation does not add vitamin H, because the thalline turbidity of off-line analysis 6 hours
Reached and had a net increase of＞0.6kg/l.While CO
2Amplitude limit reaches 12%, explanation
Thalline number and developmental state are good, need not add vitamin H again.Control loop (4)
Stream sugaring control residual sugar content CO
2Content
5.5 tons of S of 24.4% liquid glucose≤3.6% 5%＜CO
2＜8
％
(test tank work output) 4 tons of S≤2.6% 5%＜CO
2＜8
％
3 tons of S≤1.6% 5%＜CO
2＜8
The % result of fermenting: acid production rate 9.21%, transformation efficiency 58%, fermentation period 32 hours.The control rhythm and pace of moving things is shown in Fig. 5 (descending), and the state analysis result is shown in Fig. 6 (descending).Example 2 has been grown 2 hours than the cycle of example 1, and acid production rate and transformation efficiency corresponding points are all hanged down, and illustrates that the metabolism control cycle of this bacterial strain is smaller more suitable.3. comparative example: industrial L-glutamic acid, the equipment situation of 150 tons (not adopting 150 tons of stirred pots of rhythm and pace of moving things control method) is microbial bacteria SF119 tyrothricin fermentor tank 150M as shown in Figure 7
3Stirred pot
Constituent concentration culture medium prescription amylum hydrolysate of the sugar 16.07%
1.1～1.2%
Sal epsom 0.06～0.07%
Corn steep liquor 0.43%
Molasses 0.04%
Three grades 35 ℃～37 ℃～39 ℃ of Repone K 0.1% control condition temperature
Tank pressure 0.7MPa
Air quantity is set three grades and is carried wind 700 → 1100 → 1500 → 1800
Three grades are fallen wind → 1600 → 1500 → 1300
PH sets 7.4 → 7.3 → 7.2 → 7.1 → 7.0
150 rev/mins of stirring velocitys
Stop fermentation condition residual sugar＜0.6%
Fermentation period 30 hours
Stream sugaring amount 3.53% cultural method: in the main fermentation tank substratum through sterilization (140 ℃) 40 minutes, the cooling back from
Seeding tank inserts SF119 bacterium liquid to begin to cultivate, and Figure 4 shows that 150M
3Fermentation
The fermentating metabolism rhythm and pace of moving things control device of equipment on the jar, wherein online detection limit are temperature
Degree, pH, CO
2, the offline inspection signal be cell concentration, remaining sugar concentration and
Aminoglutaric acid concentration.Loop (1)
According to the pH value, personnel selection wage adjustment liquefied ammonia+gas ammonia
The pH timing (hour)
7.3 0＜t＜6
7.2 6＜t＜16
7.1 16＜t＜24
7.0 24＜t＜28
6.9 28＜t＜32 loops (2)
Ventilation and CO
2Between relation (using the manual shift valve)
Ventilation (rice
3/ hour) CO
2The content timing (hour)
700 CO
2＝0 t＝0
1100 CO
2＞4％ t＜8
1500 4％＜CO
2＜12％ t＜8
1800 CO
2≥12％ t＜8
1600 9％≤CO
2＜12％ t＞24
1500 5％＜CO
2＜19％ t＞24
1300 2%CO
2＜5% t＞this batch fermentation of 24 loops (3) not stream adds vitamin H.
Loop (4)
Stream sugaring control residual sugar content CO
2Content
5.5 tons of S of 24.4% liquid glucose≤3.6% 5%＜CO
2＜8%
(test tank work output) 3.4 tons of S≤2.6% 5%＜CO
2＜8%
2～3 tons of S≤1.6% 5%＜CO
2＜8%
All various conditions of this example and example 1. examples 2. are similar, and different is not carry out the metabolism vibration rhythm and pace of moving things
Control, but according to CO
2Variation tendency regulate the speed that vitamin H, stream sugaring and stream add liquefied ammonia
PH is at appropriate value in control, and the figure of its on-line Control is shown in 8.The fermentation result as shown in Figure 9.
Acid production rate 8.6%, transformation efficiency 57%, 32 hours cycles.Economic benefit: adopt the control of the microbial metabolism rhythm and pace of moving things to carry out fermenting process and can obtain following benefit: 1. the ferment strength 0.1～0.2 that improves the unit volume fermentor tank; 2. make acid production rate improve 5～10% relatively, transformation efficiency improves 5% relatively; 3. can shorten fermentation period, save material and save electricity, water, energy and be converted into average every batch and reduce cost more than 8%.
Claims (18)
1. an aerobic microorganism cultural method is characterized in that the metabolic rule of on-line Control microbial cells in fermentation process, makes it be in rhythm and pace of moving things state.
2. according to the described aerobic fermentation culture method of claim 1, wherein artificial the or automatic operation of all of microbial cultivation process all adopts step or ON/OFF mode to carry out so that the metabolism of thalline is in the oscillatory regime of some cycles and amplitude.
3. according to the described aerobic fermentation culture method of claim 2, wherein computer control is adopted in operation automatically.
4. according to the described aerobic fermentation culture method of claim 2, the automatic monitored control system formed by computer system, transmitter and instrument, driving and topworks's four parts of automatic operation system wherein.
5. according to the described aerobic fermentation culture method of claim 4, wherein the control method of the bacterial metabolism rhythm and pace of moving things is with CO
2Related with PH, become the nonlinear dependence loop when setting up.
6. according to the described aerobic fermentation culture method of claim 4, with control PH and CO
2For core is set up following four groups of control loops: control loop (1): stream adds nitrogenous source as input variable, pH and CO
2Be output variable, pass through multivariate
The given pH set(ting)value of controller, control nitrogenous source stream adds.Control loop (2): change the input variable of ventilation, pH and CO as process
2Be output variable, logical
Cross multivariable controller CO
2Variation feed back to input, given by multivariable controller
CO
3Set(ting)value, control air quantity.Control loop (3): it is input variable that stream adds vitamin H, pH and CO
2Be output variable, pass through multivariate
Controller is CO
2Variation feed back to input, by multivariable controller control vitamin H
The stream dosage perhaps changes CO
2Set(ting)value.Control loop (4): it is input variable that stream adds carbon source, pH and CO
2Be output variable, by the multivariate control
The system device is CO
2Variation feed back to input, regulate the intermittence of carbon source by multivariable controller
The stream dosage, and regulate CO
2The oscillation amplitude set(ting)value.
7. according to the described aerobic fermentation culture method of claim 6, wherein the input of multivariable controller comprises that adjustment air flow, stream add vitamin H, stream adds carbon source and stream adds nitrogenous source; The output of multivariable controller is CO
2
8. use infrared spectrum analyser to detect CO according to the described aerobic fermentation culture method of claim 4
2Concentration.
9. use the pH value of pH electrode and transmitters sense fermented liquid according to the described aerobic fermentation culture method of claim 4.
10. according to the described aerobic fermentation culture method of claim 4, wherein metabolic rhythm monitoring rule is to determine by the metabolism track that real-time graph shows.
11. according to the described aerobic fermentation culture method of claim 2, wherein to add the mode of nitrogenous source be on/off by the computer control magnetic valve to stream.
12. according to the described aerobic fermentation culture method of claim 2, wherein the metabolism oscillation amplitude of thalline be add vitamin H by changing air flow, stream, stream adds carbon source and stream adds nitrogenous source, adjusts CO
2Concentration.
13. according to the described aerobic fermentation culture method of claim 12, wherein the metabolism of thalline is following definite period of oscillation:
PH span of control=pH set(ting)value ± pH regulator degree
PH regulator degree=pH regulator rate % * (range under the last range-pH of pH)
N descends required for the pH of the unit of making value in period of oscillation=n * pH regulator degree formula
Unit time.
14. according to the described aerobic fermentation culture method of claim 1, wherein tunning is an amino acid.
15. according to the described aerobic fermentation culture method of claim 1, wherein tunning is a nucleic acid.
16. according to the described aerobic fermentation culture method of claim 1, wherein tunning is ancient dragon acid.
17. according to the described aerobic fermentation culture method of claim 1, wherein fermentor tank comprises the airlift fermentor and the stirred-tank fermenter of various volume scales.
18. according to the described aerobic fermentation culture method of claim 1, fermentation wherein can be batch fermentation or feed supplement-batch fermentation.
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| CN96119825A CN1064404C (en) | 1996-09-19 | 1996-09-19 | Cultivation method by controlling rhythm of somatic metabolism during the process of aerobe bacteria fermentation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009121211A1 (en) * | 2008-04-03 | 2009-10-08 | 西门子公司 | System and method for fermentation control |
| CN103614289A (en) * | 2013-10-30 | 2014-03-05 | 浙江科技学院 | Shaking table used for microorganism culture and bacterial strain screening |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH078231B2 (en) * | 1985-03-25 | 1995-02-01 | 株式会社日立製作所 | Culture control method and culture control device |
| DD297444A5 (en) * | 1990-07-09 | 1992-01-09 | Jenapharm Gmbh,De | METHOD AND DEVICE FOR FOLLOWING CONTROL OF AEROBIC BIOPROCESSES |
| CN1041534C (en) * | 1991-03-12 | 1999-01-06 | 味之素株式会社 | Method and apparatus for controlling carbon source concentration in aerobic cultivation of microorganism |
| JPH07184634A (en) * | 1993-12-28 | 1995-07-25 | Ajinomoto Co Inc | Culture method for aerobic culture of microorganism and apparatus therefor |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009121211A1 (en) * | 2008-04-03 | 2009-10-08 | 西门子公司 | System and method for fermentation control |
| CN103614289A (en) * | 2013-10-30 | 2014-03-05 | 浙江科技学院 | Shaking table used for microorganism culture and bacterial strain screening |
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