CN1041534C - Method and apparatus for controlling carbon source concentration in aerobic cultivation of microorganism - Google Patents
Method and apparatus for controlling carbon source concentration in aerobic cultivation of microorganism Download PDFInfo
- Publication number
- CN1041534C CN1041534C CN92101496A CN92101496A CN1041534C CN 1041534 C CN1041534 C CN 1041534C CN 92101496 A CN92101496 A CN 92101496A CN 92101496 A CN92101496 A CN 92101496A CN 1041534 C CN1041534 C CN 1041534C
- Authority
- CN
- China
- Prior art keywords
- carbon source
- culture
- feed rate
- phase
- signal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention relates to a method for controlling carbon source concentration in the aerobic cultivation of a microorganism. The substrate carbon source remains at a low level in a cultivation vessel during the culture feeding in aerobic fed-batch, continuous or cell-recycling continuous cultures. This is accomplished by monitoring the increase in pH or dissolved oxygen content in the culture medium and adding the feed solution intermittently into a cultivation vessel at a calculated feed rate using a feed control device controlled by a computer. The present invention further relates to a process for producing L-lysine by fermentation having the advantages over the prior methods, those being improved productivity, higher concentrations of accumulated product, and increased yields of L-lysine.
Description
The present invention relates to a kind of method of in aerobic cultivation of microorganism, controlling carbon source concentration.Specifically, the present invention relates to a kind of in ventilation fed batch cultivation, ventilation cultured continuously or cell recirculation culture of continuous cultivation.The matrix carbon source of controlling between charge period in the culture tank at culture makes it keep low-level method.Its way is that the increase of monitoring pH in culture medium or dissolved oxygen content utilizes by computer-controlled reinforced control device, adds feed liquid with the feed rate that calculates to the culture tank discontinuous.The device that is suitable for implementing a kind of like this method also is provided.
In order to utilize the various materials of Production by Microorganism Fermentation, for example each seed amino acid or nucleic acid perhaps in order to produce microorganism cells, as yeast cell, carry out aerated culture to microorganism.Industrial, the aerated culture of microorganism carries out with fed batch cultivation method, continuous culture method or cell recirculation culture method, and adopts such as carbon sources such as carbohydrates as main raw material.
In a kind of like this cultural method, must in the reinforced process of culture, make the carbon source (matrix) in the culture tank keep alap level as the concentration of carbohydrate.So just can reach many purposes, for example: prevent thick carbon source suppress matrix, by effective utilization cultivate that raw material reduces the loss of thick carbon source, contained residue carbon source contaminate environment in the waste liquid that stays after being easy to from final fermented liquid separated product and preventing separated product.
Under the situation of cultured continuously and cell recirculation cultured continuously, though in the reinforced process of culture also continuous blow-down nutrient solution and therefrom separated product.Must be in sepn process the flow control of carbon source such as carbohydrate at possible minimum level, so that carbon source is zero to the influence of sepn process substantially.Also must prevent the loss of raw material.In this class cultural method, also wish to avoid analyzing the manual operations of carbon source concentration, its way is to adopt automatic monitoring means to control the stability of carbon source concentration.
About in the reinforced process of culture, the concentration of carbon source such as carbohydrate being maintained low-level problem, once the someone had described certain methods in the past, these methods comprise with following index controls carbon source concentration as individual index, for example: oxygen-consumption, carbon dioxide gas quantity discharged, pH, by product growing amount, the add-on (amount of carbon source multiply by predetermined scale-up factor to ammonia residual and calculates) of ammonia when adding carbon source such as carbohydrate.According to these methods, can not very accurately measure the microorganism active in the culturing process, cause active in some cases when ANOMALOUS VARIATIONS takes place concentration can not obtain satisfied control.Owing to these reasons, in culturing process, can not effectively be controlled at low-level (for example being lower than 3 grams per liters) to carbon source concentration.
In addition.Also have another kind of currently known methods to disclose the consumption that only detects culture tank internal carbon source concentration in the culturing process, but detecting reliability is poor with dissolved oxygen concentration.If ventilation or agitation condition (mixing speed, air flow quantity) change, then dissolved oxygen concentration alters a great deal.In this case, transmitter detects the consumption of carbon source sometimes mistakenly, thereby the carbon source concentration in the culture tank can not be effectively controlled.Owing to these reasons, this method is also impracticable.
Therefore, very clear, need a kind of method and apparatus of in aerobic cultivation of microorganism, controlling carbon source concentration automatically at present.The invention provides a kind of like this method and apparatus to address the above problem.
Under the situation of batch treatment, the liquid nutrient medium that will contain carbon source and nitrogenous source places fermentor tank to carry out batch culture.Another kind method is to carry out adding continuously or off and on when culture feeds in raw material the substratum that only contains carbon source.
Under processed continuously situation, when cultivating substratum is infeeded in the fermentor tank continuously, and emit the nutrient solution of equal volume continuously, so that maintenance such as cell quantity or production concentration is constant.
An object of the present invention is to provide a kind of device of in aerobic cultivation of microorganism, controlling the method for carbon source concentration and being used to implement this method.
One embodiment of the invention relate to a kind of method of microorganism being carried out aerated culture with fed batch cultivation method, continuous culture method or cell recirculation continuous culture method, this method comprises, adds the carbon source feed liquid in the culture tank discontinuous by computer controlled automatic.
Another embodiment of the invention relates to a kind ofly carries out the device of aerated culture with fed batch cultivation method, continuous culture method or cell recirculation continuous culture method to microorganism, and this device comprises:
A culture tank, this culture tank are equipped with and are respectively applied for the pH that detects substratum in the culture tank and the transmitter of dissolved oxygen concentration, and one for receiving substratum ventilation and the whipping appts that feed liquid designs;
Be used to control the control device of feed liquid to the culture tank feed rate, this control device comprises: be used to receive respectively by the signal of pH transmitter and the output of dissolved oxygen concentration transmitter and export first signal converter of change over signal; Be used to receive change over signal, calculate feed rate and export the calculating device of feed rate signal with change over signal; Be used to the second signal transmodulator that receives the feed rate signal and export the feed rate change over signal; Be used to utilize the feed rate control device of feed rate change over signal control feed liquid to the culture tank feed rate.
From accompanying drawing and following detailed Description Of The Invention part, can obviously find out various other purposes of the present invention and advantage.
Fig. 1 has illustrated an embodiment of apparatus of the present invention.
Fig. 2 has illustrated the device that embodiment 1 is used.
Fig. 3 illustrates the used device of comparative example 1.
Fig. 4 represents the feeding culture condition of embodiment 1.The y axle is represented dissolved oxygen concentration, pH and the sugared concentration in the fermentor tank respectively in Fig. 4 (a), 4 (b) and 4 (c).The x axle is represented in Fig. 4 (a)-(c) and is increased progressively the time.
Fig. 5 illustrates among the embodiment 1 in for some time the situation that (hour, x axle) controls sugared concentration (y axle).
Fig. 6 illustrates the culture condition of comparative example 1.The y axle is represented sugared concentration, the x axle represent the time (hour).
The present invention partly relate to a kind of with fed-batch culture method, continuous culture method or cell again The method that the circulation continuous culture method is ventilated and cultivated microorganism, the advantage of the method is: Microorganism can be controlled at the suitable of carbon source feed liquid to the infiltration rate of carbon source such as carbohydrate in the reinforced process Work as charging rate; The consumption of carbon source can detect with good reliability and certainty; Carbon The detection that the source consumes can not ventilated or the impact of stirring condition. The method has also that other are excellent The point.
The present invention also provides the device of implementing said method.
Application artificially solves the problems referred to above of discussing in the background of invention part and has carried out extensively Research, found that, connect with fed-batch culture method, continuous culture method or cell recirculation Continuous cultivation is ventilated in the process of cultivating, if to the culture tank discontinuous add carbon source as Sugar, the carbon source feed liquid charging rate when then this time reinforced and follow-up each time is reinforced can be with pH's Increase or the increase of dissolved oxygen concentration is determined as index, this pH or dissolved oxygen concentration Increase is that the consumption by carbon source in the culture medium that fills in the culture tank causes. Along with first reinforced Finish from a rear charge period between the time interval different, according to pH in culture medium or dissolving The increase of oxygen is monitored and is controlled charging rate by a computer, has thereby solved earlier The difficult problem of technology.
Therefore, the present invention partly relates to a kind of microbial aerobic cultural method, wherein, and little Biological fed-batch ventilation is cultivated, is ventilated continuously and cultivate or continuously ventilation cultivation of cell recirculation In the process, be that feed liquid is added in the culture tank off and on when adding first the carbon source feed liquid, and with Predetermined charging rate and reinforced duration add feed liquid.
Stopping the material phase before certain charge period, the culture tank internal carbon source if COMPUTER DETECTION goes out (matrix) consumes and causes that pH increases or dissolved oxygen concentration increases, just begin for the second time reinforced or after Continuous reinforced, when reinforced, in culture tank, add feed liquid with the time of determining, charging rate is used tricks The calculation machine stops expecting that by this time of phase stops the charging rate meter of material feed liquid in the charge period before the phase with this Calculate, calculating principle is, if describedly stop expecting that the time of phase is long, then reduces charging rate, if The described time weak point that stops expecting the phase then strengthens charging rate.
The feature of the method is, in aerobic cultivation of microorganism, the matrix in the culture tank Concentration is controlled at constant low-level automatically.
For various materials of fermentative production or production cell itself, microorganism is carried out fed-batch aerated culture, continuous aerated culture or the continuous aerated culture of cell recirculation, (used here " continuously " speech is a generalized continuously in culture tank simultaneously, also comprise " off and on ") add the feed liquid that contains matrix carbon source (as carbohydrate), this is that people are known.Carry out fed-batch aerated culture of the present invention, continuously during the continuous aerated culture of aerated culture or cell recirculation, in feeding culture process described here, the charging process of feed liquid, also can make amendment to the known method of fed batch cultivation, cultured continuously or cell recirculation cultured continuously.
In general, if in main cultivation the before feeding culture, the concentration of substratum mesostroma carbon source (as carbohydrate) reaches a certain low-level, then begins the adding first of feed liquid in this feeding culture process at this moment.Predetermined feed liquid feed rate is determined by preliminary experiment in advance, generally equals main substrate consumption speed of cultivating when feeding culture begins (during reinforced first the beginning).The definite time length that feed liquid adds is a random time, and its range of choice should make the microbial consumption carbon source as the activity change of carbohydrate little (10 minutes to 24 hours scope in).
After reinforced first the finishing, if the matrix in the substratum is consumed, then the pH of substratum and dissolved oxygen concentration all will increase.When computer by pH transmitter and dissolved oxygen concentration sensor detecting during to this increases, computer just sends instruction to the device that the control feed liquid enters the culture tank flow velocity, thereby begins to add the second time feed liquid.
The increase of pH and the increase of dissolved oxygen concentration might not take place simultaneously.If between these two increases a time lag is arranged, then begin reinforced for the second time according to that increase that early detects.PH transmitter and dissolved oxygen concentration transmitter are damaged sometimes, therefore must often calibrate.So these two transmitters are not to use separately.Use two transmitters can improve the detecting reliability of substrate consumption greatly simultaneously.
Feed rate when for the second time adding feed liquid is the speed by calculating, and calculates principle to be, if it is long to stop the material phase, then reduces feed rate, lacks if stop the material phase, then strengthens feed rate.This just makes matrix feed rate and substrate consumption speed reach balance, thereby stops expecting the length of phase and reinforced first feed rate according between feeding in raw material the reinforced and second time first, the substrate concn of substratum in the culture tank is remained on desirable low-level.Computation process is undertaken by computer, but this computer program is easy to be designed by this area professional.An example of this program is shown in embodiment 1 described below.
The reinforced for the second time definite time length is according to determining with reinforced first similar principle of definite time.The for the second time reinforced time length might not be with reinforced identical first, only requires that this time selects in certain scope so that microbial consumption carbon source (matrix) is little as the activity change of carbohydrate.The selection of each reinforced time length should make the activity change of microbial consumption matrix in this time little, and can make that the substrate concn in the culture tank keeps low-level in the culturing process.
Each time adds the zero hour, the feed rate of feed liquid and determining of the time length of feeding in raw material after reaching for the third time, with reinforced similar for the second time.
Therefore, stopping the material phase of certain charge period, if COMPUTER DETECTION goes out owing to culture tank internal carbon source (matrix) consumption causes that pH increases or dissolved oxygen concentration increases, just begin for the second time reinforced and each time later on reinforced, reinforced time length and feed rate stop expecting that according to this time length of phase and preceding once reinforced feed rate calculate by computer, its calculating principle is, if it is long to stop the material phase, then reduce feed rate,, then strengthen feed rate if stop material phase weak point.
As mentioned above, might be when microorganism active be monitored in pointwise, feed rate and other reinforced conditions of regulation matrix carbon source (as carbohydrate) feed liquid.Also might be controlled at the substrate concn in the culture tank easily and be low to moderate below 5 grams per liters, further below 3 grams per liters.
The invention still further relates to the used device of aforesaid method, this device comprises: (ⅰ) culture tank, this culture tank is equipped with and is respectively applied for the pH that detects substratum in the culture tank and the transmitter of dissolved oxygen concentration, and for receiving ventilation and the whipping appts that feed liquid designs through current velocity controller; (ⅱ) computer of being furnished with two signal converters, wherein the mode of connection of (a) pH transmitter and dissolved oxygen concentration transmitter is, pH and dissolved oxygen concentration detection data that these two sensor detecting are gone out are imported computer respectively through same signal converter; (b) mode of connection of current velocity controller is, the feed liquid feed rate that computer calculates is gone out is imported current velocity controller through another signal converter.
Device of the present invention is the device with following function: detect the consumption of matrix carbon source such as carbohydrate; According to by the signal that is installed in pH transmitter (as pH electrode) on the culture tank and dissolved oxygen concentration transmitter (as dissolved oxygen electrode) input computer, by the feed rate of computer calculates carbon source feed liquid; The input of the feed rate that calculates is contained in the feed liquid current velocity controller on the carbon source filling tube of culture tank.
As known in the field, pH transmitter and dissolved oxygen sensor are often damaged easily, so should often calibrate.According to the present invention, each transmitter is not independent use, but is used in combination, thereby can improve the detecting reliability that carbon source such as carbohydrate consume greatly.When each transmitter uses separately, though reliability reduces but still produces effect.
Provide the following example and be in order to further specify the present invention, but should not think restrictive.
Embodiment
Device used among the embodiment 1-5 is shown in Fig. 2, [8] small-sized glass fermentor tank wherein, [9] pH electrode, [10] dissolved oxygen electrode, [11] Personal Computer, [12] analog/digital converter, [13] digital/analog converter, [14] take away pump, correspond respectively to [1] culture tank (fermentor tank) in the device shown in Figure 1, [2] pH transmitter: model F-635-D120-DH, make by ABLE Co. (Japan), [3] dissolved oxygen concentration transmitter: model S-1 is made by ABLE Co. (Japan), [4] computer, [5] signal converter (ⅰ) in this computer, [6] signal converter (ⅱ) in this computer, [7] flow rate control device.In Fig. 1-3, [15] comprise motor M corresponding to the ventilation and the whipping appts that are used for substratum is ventilated and stirred; [16] corresponding to the signal of stipulating feed rate; [17] corresponding to the feed liquid that flows into [14] take away pump or [7] flow rate control device.
With cell recirculation continuous culture method fermentation production of L-glutamic acid
At volume is the 30ml water-containing medium of packing in the bottle that shakes of 500ml, then in 115 ℃ of heat sterilizations 10 minutes.Contain in this substratum: 30 grams per liter glucose, 1 grams per liter KH
2PO
4, 0.4 grams per liter MgSO
47H
2O, 4 grams per liter ureas, 20 mg/litre FeSO
47H
2O, 20 mg/litre MnSO
44H
2O, 5 milliliters/rise soybean protein acid hydrolysate, 300 micrograms per litre vitamin Hs.After being cooled to room temperature, cultivated 24 hours down in 30 ℃ then inoculating lactic acid fermentation tyrothricin AJ 1511 (ATCC 13869) on substratum.
At the volume through sterilization in advance is that pack into seed culture medium and 270 milliliters of moisture main mediums contain 80 grams per liter cane molasses (as sugar), 1 grams per liter KH in 1 liter the small-sized glass fermentor tank in the main medium
2PO
4With 10 milliliters/rise the soybean protein acid hydrolysate.Then this mixture is placed down in 30 ℃.Speed with 300 ml/min feeds the filtration sterilization air, and begins to stir, and keeping pH with ammonia simultaneously is 7.5 (beginning main the cultivation).
As shown in Figure 2, small-sized glass fermentor tank links to each other with 16 Personal Computers.By being inserted in pH and the dissolved oxygen concentration simulating signal that pH electrode in the fermentor tank and dissolved oxygen concentration electrode detection go out, the analog/digital converter input Personal Computer in being contained in this Personal Computer.By moisture fed-batch medium (feed liquid) feed rate that this Personal Computer is stipulated and calculated, import take away pump through the digital/analog converter that is contained in this Personal Computer as simulating signal.
When beginning main the cultivation, on Personal Computer, set following condition: feed liquid initial charge speed (reinforced for the first time): 30 milliliters/hour; Increase detected carbohydrate matrix consumption data by pH: 7.7; Increase detected carbohydrate matrix consumption data by dissolved oxygen concentration: 20% (common level of dissolved oxygen concentration is 1-10%); Feed liquid is fed in raw material the time: 3 hours.
For thereby bacteria growing inhibiting produces L-glutamic acid, the concentration with 0.2% (weight) after main cultivation begins 5 hours adds the polyoxyethylene sorbitan monopalmitate.Proceed main the cultivation after 5 hours, Personal Computer is placed (running status automatically) under the controlled condition, and begin to add first feed liquid with 30 milliliters/hour predetermined feed rate.Be added to feed liquid in the fermentor tank and contain 180 grams per liter cane molasses (as sugar), 5 milliliters/rise soybean protein acid hydrolysate, 0.2% (weight) polyoxyethylene sorbitan monopalmitate.
Meanwhile, make substratum pass through an equatorial in advance through the millipore filtration of sterilization, make substratum be divided into 20 milliliters and contain cell solution and filtrate two portions with the flow of feed liquid feed rate+20 (milliliter/hour).To contain cell solution and send fermentor tank back to.From cell-free filtrate, collect L-L-glutamic acid with crystallization process.
After beginning added feed liquid first, computer just began automatic operation, and arrived the predetermined moment that adds feed liquid time (3 hours), and condition according to the rules stops to add feed liquid immediately automatically.Shown in the part of the A among Fig. 4, to interrupt along with reinforced, the sugared density loss in the fermentor tank becomes 0 grams per liter substantially.PH and dissolved oxygen concentration almost increase simultaneously this moment.
Arrive moment of substrate consumption detected value earlier at pH or dissolved oxygen concentration, computer calculates immediately and regulation adds the feed rate of feed liquid for the second time.Calculated value is transfused to take away pump.Added feed liquid once more 3 hours with the speed that calculates.After this repeat this step, very exactly the sugared concentration in the fermentor tank is controlled at the 0-2 grams per liter.
The feed rate of feed liquid during each for the second time reinforced later time is reinforced, stop the speed (υ) that material fed in raw material before the phase for the last time according to the time length that stops expecting the phase (τ) and this and stipulate that its principle is as follows:
If τ≤10 minute *, the feed rate of then stipulating fresh feed liquid is 1.1 υ.Here υ represents the feed rate of last charge period before this.
If * 10 minutes＜τ≤30 minute stipulate that then new feed rate is υ.
If * 30 minutes＜τ≤1 hour stipulates that then new feed rate is 0.9 υ.
If * 1 hour＜τ≤2 hour stipulate that then new feed rate is 0.8 υ.
Here, coefficient and the τ of υ become with culture condition sometimes, as cultivating type, matrix species, the character etc. of bacterium in one's power.The number of principle can suitably reduce or increase.As a reference, preliminary experiment is verified, does not satisfy the chance of τ＞2 hour in the culturing process of present embodiment.In addition, the reinforced time that at every turn adds feed liquid in the present embodiment all is defined as 3 hours, but as mentioned above, as long as bacterial activity changes not quite in the reinforced time, just there is no need to keep the reinforced time constant.
After the cultured continuously 80 hours, obtain 132 gram L-L-glutamic acid.As shown in Figure 5, automatically after the operation beginning, sugared concentration can fully automatically be stablized and is controlled at 2 grams per liters.
Comparative example 1 (prior art)
The feature of prior art is: use NH
3Control pH, sugaring (feed liquid) amount and NH
3Consumption is proportional.
At the volume through sterilization in advance is in 1 liter the small-sized glass fermentor tank, pack into the seed culture medium that makes by mode similar to Example 1 and 270 milliliters of main mediums identical with embodiment 1.This mixture is remained on 30 ℃, with the speed feeding filtration sterilization air of 300 ml/min.Begin to stir, keeping pH with ammonia simultaneously is 7.5 (beginning main the cultivation).
As shown in Figure 3, this small-sized glass fermentor tank links to each other with 16 Personal Computers.
The main cultivation began after 5 hours, and the concentration with 0.2% adds the polyoxyethylene sorbitan monopalmitate.After main cultivation is carried out 5 hours more continuously, Personal Computer is placed under the controllable state.
Simulating signal is imported Personal Computer from the mass flowmeter that links to each other with the ammonia pipe through being contained in the analog/digital converter in the Personal Computer.Calculated and NH every 1 hour
3The feed rate of the feed liquid of the proportional amount of add-on, the computer follow procedure is sent into take away pump as simulating signal through being contained in the digital/analog converter in the Personal Computer with feed rate then.
Fig. 6 A has illustrated experimental result, there is shown from cell and enters the L-L-glutamic acid culturing process of production phase NH vegetative period
3The ratio of feed rate and feed liquid feed rate, wherein the control of sugared concentration becomes very poor.Therefore scale-up factor changes after 13 hours.Even after this variation, this scale-up factor is also unstable, and it is so good to cause the control of sugared concentration in the fermentor tank not resemble Fig. 5.So the concentration of residue sugar (the sugared concentration in the fermentor tank) has been monitored 5 times.Because this unstable, can not only reduce to the concentration of residue sugar very low-level but also avoid sugared concentration in culturing process be 0 grams per liter.
After the cultured continuously 80 hours, obtain 120 gram L-L-glutamic acid, but this output is lower than the output of 132 grams among the embodiment 1.
Controlled concentration (grams per liter) the 5-20 0-2 feeding culture time of sugar in table 1 item compared comparative example 1 embodiment 1 (prior art) (invention) culturing process (hour) number of times (inferior) the 50 manual adjustment sugar consumption speed and the NH of the sugared concentration of 80 80 monitoring residues
3The number of times of feed rate ratio (inferior) 50 flows out sugar amount (gram) 28.5 0.1 glutamic acid yield (gram) 120 132 of system
Embodiment 2
With fed batch cultivation method fermentative production L-phenylalanine
In mode similar to Example 1, with Brevibacterium lactofermentus FERM BP-1071 fermentative production L-phenylalanine, different is to change cell recirculation cultured continuously into fed batch cultivation.
The method of the control method of substratum, culture tank, incubation time process and control feed liquid feed rate also is modified as follows.
(1) substratum
(a) seed culture medium sucrose 20 grams per liter phosphatase 11 grams per liter MgSO
4400 mg/litre FeSO
410 mg/litre MnSO
410 mg/litre ureas, 3 grams per liter ammonium acetates, 2 grams per liter tyrosinase 15 00 mg/litre KOH, 700 mg/litre protein hydrolysates (in total nitrogen content), 2 grams per liter vitamin Hs, 100 micrograms per litre vitamins Bs
120 ℃ of 100 micrograms per litre silicone foam killers, 20 micrograms per litre pH, 7.5 sterilizations, 20 minutes
(b) main medium glucose 150 grams per liter MgSO
4280 mg/litre MgSO
410 mg/litre phosphatase 79s, 00 mg/litre vitamin H, 500 micrograms per litre vitamins Bs
1120 ℃ of 20 micrograms per litre protein hydrolysates (in total nitrogen content), 8.7 grams per liter tyrosine, 1.0 grams per liter KOH, 700 mg/litre silicone foam killers, 2 micrograms per litre pH, 7.5 sterilizations, 20 minutes
(c) fed-batch medium
Identical with main medium except that using 250 grams per liter glucose.
(2) culture tank and cultural method:
(a) seed culture: in volume is 500 milliliters culturing bottle, pack into behind 30 milliliters of seed culture mediums, under 30 ℃, carry out shaking culture.
(b) the main cultivation: in volume is 1 liter small-sized glass fermentor tank, pack into behind 30 milliliters of seed culture fluids and the 270 milliliters of main mediums, under 30 ℃, carry out aerated culture (air flow quantity is 150 ml/min).Use NH
3Transfer pH to 7.5.
(3) incubation time:
(a) seed culture: 48 hours
(b) the main cultivation: 96 hours
(c) begin the reinforced time: the main cultivation began after 35 hours.
(4) method of control feed liquid feed rate:
Control feed rate by mode similar to Example 1, different is that initial charge speed is defined as 10 milliliters/hour.
Comparative example 2 (prior art)
Use NH
3Regulate pH, make control indexes feed liquid feed rate with pH.That is, when the increase of pH surpasses prescribed value, increase feed rate 5%; Do not see that pH increases, and then reduces feed rate 10% if there are 5 hours.
Embodiment 2 and comparative example 2 the results are summarized in table 2.
Controlled concentration (grams per liter) the 10-30 0-3 incubation time of sugar in table 2 item compared comparative example 2 embodiment 2 feeding culture (hour) 96 96 flow out sugar amount (gram) 10.5＜0.2 phenylalanine output (gram) 13 15.5 of systems
Produce yeast cell with the fed batch cultivation method
By mode similar to Example 1 cereuisiae fermentum CBS 1523 fermentative production yeast cell, different is to change cell recirculation cultured continuously into fed batch cultivation.
The method of substratum, culture tank, cultural method and control feed liquid feed rate also is modified as follows.
(a) seed culture medium glucose 30 grams per liter KH
2PO
41 grams per liter ammonium sulfate, 5 grams per liter MgSO
40.5 120 ℃ of grams per liter protein hydrolysate (in total nitrogen content) 1 grams per liter yeast extract 1 grams per liter pH 6.5 sterilizations, 15 minutes
(b) main medium glucose 50 grams per liter KH
2PO
41 grams per liter ammonium sulfate, 10 grams per liter MgSO
40.5 120 ℃ of grams per liter corn steep liquor 2 grams per liter pH 6.5 sterilizations, 15 minutes
(c) fed-batch medium
Identical with main medium except that using 500 grams per liter glucose and 50 grams per liter ammonium sulfate.
(2) culture tank and cultural method:
(a) seed culture: in volume is 500 milliliters culturing bottle, pack into behind 30 milliliters of seed culture mediums, under 30 ℃, carried out shaking culture 24 hours.
(b) the main cultivation: in volume is 1 liter small-sized glass fermentor tank, pack into behind 30 milliliters of seed culture fluids and the 270 milliliters of main mediums, under 30 ℃, carried out aerated culture 10 hours (air flow quantity is 150 ml/min).Use NH
3Transfer pH to 6.5.In addition, begin to add feed liquid (substratum) in main cultivation beginning after 10 hours.
(3) method of control feed liquid feed rate:
Control feed rate by mode similar to Example 1, different is that initial charge speed is defined as 5 milliliters/hour.
Comparative example 3 (prior art)
Make control indexes feed liquid feed rate (measuring the alcohol gas in the exhaust) with ethanol byproducts formation speed.That is, when the increase of ethanol formation speed surpasses prescribed value, increase feed rate; If ethanol formation speed is reduced to below the prescribed value, then increase feed rate.
Controlled concentration (grams per liter) the 5-20 0-3 incubation time of sugar in table 3 item compared comparative example 3 embodiment 3 feeding culture (hour) 50 50 ethanol formation amount (gram) 5-10 0-5 yeast cell dry weights (gram) 39 48
With fed batch cultivation method production of L-threonine by fermentation
By mode similar to Example 1 brevibacterium flavum FERM BP-2179 (CCTCCM89003) production of L-threonine by fermentation, different is to change cell recirculation cultured continuously into fed batch cultivation.
The method of the time course of substratum, culture tank, cultural method, cultivation, control feed liquid feed rate is modified as follows.
(1) substratum:
(a) seed culture medium glucose 20 grams per liter KH
2PO
41.0 grams per liter MgSO
4400 mg/litre FeSO
410 mg/litre MnSO
410 mg/litre urea 3.0 grams per liter ammonium acetates, 2.0 grams per liter protein hydrolysates (in total nitrogen content), 2 grams per liter vitamin Hs, 100 micrograms per litre vitamins Bs
1120 ℃ of 100 micrograms per litre silicone foam killers, 20 micrograms per litre pH, 7.5 sterilizations, 20 minutes
(b) main medium glucose 50 grams per liter MgSO
4400 mg/litre MnSO
410 mg/litre phosphoric acid, 2 grams per liter vitamin Hs, 50 micrograms per litre vitamins Bs
1120 ℃ of 5 mg/litre protein hydrolysates (in total nitrogen content), 1 grams per liter KOH, 1.5 grams per liter silicone foam killers, 2 micrograms per litre pH, 7.5 sterilizations, 20 minutes
(c) fed-batch medium
Use 500 grams per liter acetic acid aqueous solutions.
(2) culture tank and cultural method:
(a) seed culture: in volume is 500 milliliters culturing bottle, pack into behind 30 milliliters of seed culture mediums, under 30 ℃, carry out shaking culture.
(b) the main cultivation: in volume is 1 liter small-sized glass fermentor tank, pack into behind 30 milliliters of seed culture fluids and the 270 milliliters of main mediums, under 30 ℃, carry out aerated culture (air flow quantity is 150 ml/min).Use NH
3Transfer pH to 7.5.
(3) incubation time:
(a) seed culture: 40 hours
(b) the main cultivation: 100 hours
(c) begin time of feeding in raw material: mainly cultivate beginning after 20 hours.
(4) method of control feed liquid feed rate:
Control feed rate by mode similar to Example 1, different is that initial charge speed is defined as 1.5 milliliters/hour.
Comparative example 4 (prior art)
Make control indexes feed liquid feed rate with pH.That is, when the increase of pH surpasses prescribed value, with the feed rate adding feed liquid of a switched attenuator switched-mode timer with constant time and 0.5 ml/min.
Controlled concentration (grams per liter) the 10-30 0-3 incubation time of acetate in table 4 item compared comparative example 4 embodiment 4 feeding culture (hour) 100 100 flow out acetate amount (gram) 5.2＜0.2L-Threonine output (gram) 7.8 8.8 of systems
With fed batch cultivation method fermentative production guanosine
According to mode similar to Example 1 subtilis FERM BP-3601 fermentative production guanosine, different is to change cell recirculation cultured continuously into fed batch cultivation.
The method of substratum, culture tank, cultural method, incubation time process and control feed liquid feed rate is modified as follows:
(1) substratum:
(a) seed culture medium glucose 30 grams per liter phosphoric acid 0.4 grams per liter MgSO
4400 mg/litre FeSO
410 mg/litre MnSO
4120 ℃ of 10 mg/litre RNA, 5 grams per liter ammonium chlorides, 3 grams per liter yeast extract 500 mg/litre KOH, 700 mg/litre protein hydrolysates (in total nitrogen content), 2 grams per liter silicone foam killers, 200 mg/litre pH, 6.5 sterilizations, 20 minutes
(b) main medium glucose 200 grams per liter MgSO
4150 mg/litre MnSO
4120 ℃ of 10 mg/litre phosphatase 79s, 00 mg/litre ammonium chloride 5 grams per liter potassium chloride, 15 grams per liter protein hydrolysates (in total nitrogen content), 1.5 grams per liter RNA, 1.25 grams per liter DL-methionines, 500 mg/litre KOH, 600 mg/litre silicone defomaing agents, 2 micrograms per litre pH, 5 sterilizations, 20 minutes
(c) fed-batch medium
Use 500 grams per liter D/Ws.
(2) culture tank and cultural method:
(a) seed culture: in volume is 500 milliliters culturing bottle, pack into behind 30 milliliters of seed culture mediums, under 30 ℃, carry out shaking culture.
(b) the main cultivation: in volume is 1 liter small-sized glass fermentor tank, pack into behind 30 milliliters of seed culture fluids and the 270 milliliters of main mediums, under 30 ℃, carry out aerated culture (air flow quantity is 200 ml/min).Use NH
3Transfer pH to 6.5.
(3) incubation time:
(a) seed culture: 30 hours
(b) the main cultivation: 150 hours
(c) begin the reinforced time: the main cultivation began after 50 hours.
(4) method of control feed liquid feed rate:
Control feed rate by mode similar to Example 1, different is that initial charge speed is defined as 1 milliliter/hour.
Comparative example 5 (prior art)
Use NH
3Regulate pH, make control indexes feed liquid feed rate with pH.That is, when the increase of pH surpasses prescribed value, increase feed rate 5%; Do not see that pH increases, and then reduces feed rate 10% if there are 5 hours.
Controlled concentration (grams per liter) the 10-30 0-3 incubation time of sugar in table 5 item compared comparative example 5 embodiment 5 feeding culture (hour) 150 150 flow out sugar amount (gram) 15.0＜0.2 guanosine output (gram) 10.4 13.0 of systems
Claims (5)
1. method of in fed batch cultivation method, continuous culture method or cell recirculation continuous culture method, microorganism being carried out aerated culture, this method comprises:
Intermittent type adds the carbon source feed liquid, does not add the described carbon source phase so that each carbon source adding phase all is accompanied by one;
Wherein each described length that does not add the carbon source phase is to determine like this: the pH and the O that measure substratum
2Concentration is as described pH and O
2Concentration begin when increasing subsequently carbon source add the phase and
Wherein the adding speed of the described carbon source in carbon source adding phase (ⅰ) is to determine by time that does not add the carbon source phase (a) and connecting each other between the carbon source adding speed (b) of the adding carbon source that (a) is close to before that described carbon source adding phase (ⅰ) is close to before
So, if the time in described period (a) is long, then described carbon source add carbon source in the phase (ⅰ) add speed and for the carbon source that is close to previously adds the phase (ⅱ), descended and
So, if the time in described period (a) is short, then carbon source add speed and for the carbon source that is close to previously adds the phase (ⅱ), increased and
By above-mentioned control that described carbon source concentration remains on 0-5g/l in described substratum constant is low-level.
2. the process of claim 1 wherein that described carbon source is sugar.
3. one kind at fed-batch aerated culture, the continuous device of control matrix carbon source concentration in aerated culture or the continuous aerobic cultivation of cell recirculation, and this device comprises:
(ⅰ) culture tank, this culture tank are equipped with and are respectively applied for the pH that detects substratum in the culture tank and the transmitter of dissolved oxygen concentration, and one for receiving substratum ventilation and the whipping appts that feed liquid designs; With
(ⅱ) be used to control the control device of feed liquid to the culture tank feed rate, this control device comprises: be used to receive respectively by the signal of pH transmitter and the output of dissolved oxygen concentration transmitter and export first signal converter of change over signal; Be used to receive change over signal, calculate feed rate and export the calculating device of feed rate signal with change over signal; Be used to the second signal transmodulator that receives the feed rate signal and export the feed rate change over signal; Utilize the feed rate change over signal to control the feed rate control device of feed liquid with being used to the culture tank feed rate.
4. the device of claim 3, wherein said first signal converter comprises an analog/digital converter, described second signal transmodulator comprises a digital/analog converter.
5. the device of claim 3, wherein said feed rate control device comprises a feed rate controller and a take away pump.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12699291A JP3074781B2 (en) | 1991-03-12 | 1991-03-12 | Production method of L-lysine by fermentation method |
| JP126992/91 | 1991-03-12 | ||
| JP277938/91 | 1991-10-24 | ||
| JP27793891A JP2932791B2 (en) | 1990-11-30 | 1991-10-24 | Method and apparatus for controlling carbon source concentration in microbial aerobic culture |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN97112748A Division CN1117869C (en) | 1991-03-12 | 1997-06-10 | Process for producing L-lysine by fermentation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1064890A CN1064890A (en) | 1992-09-30 |
| CN1041534C true CN1041534C (en) | 1999-01-06 |
Family
ID=26463051
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN92101496A Expired - Lifetime CN1041534C (en) | 1991-03-12 | 1992-03-09 | Method and apparatus for controlling carbon source concentration in aerobic cultivation of microorganism |
| CN97112748A Expired - Lifetime CN1117869C (en) | 1991-03-12 | 1997-06-10 | Process for producing L-lysine by fermentation |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN97112748A Expired - Lifetime CN1117869C (en) | 1991-03-12 | 1997-06-10 | Process for producing L-lysine by fermentation |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN1041534C (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064404C (en) * | 1996-09-19 | 2001-04-11 | 中国科学院化工冶金研究所 | Cultivation method by controlling rhythm of somatic metabolism during the process of aerobe bacteria fermentation |
| US20170017891A1 (en) * | 2014-01-30 | 2017-01-19 | Valitacell Limited | A method of predicting relative fed batch production titer of a panel of clonally-derived producer cells |
| CN106222309A (en) * | 2016-07-28 | 2016-12-14 | 山东金朗生物科技有限公司 | A kind of fermentable produces the control of additive raw material method improving L alanine yield |
| CN108660168A (en) * | 2017-03-27 | 2018-10-16 | 上海医药工业研究院 | A kind of zymotechnique improving L-Trp yield |
| CN111099740B (en) * | 2018-10-26 | 2022-06-07 | 中国石油化工股份有限公司 | Feed supplement control method for chemoautotrophic microorganism culture process |
| CN116855370A (en) * | 2023-07-19 | 2023-10-10 | 安及义实业（上海）有限公司 | Automatic feeding device and method for bioreactor or fermentation tank |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3926738A (en) * | 1972-05-10 | 1975-12-16 | Wilson John D | Method and apparatus for control of biochemical processes |
| US4680267A (en) * | 1985-03-01 | 1987-07-14 | New Brunswick Scientific Company, Inc. | Fermentor control system |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2817172B2 (en) * | 1989-03-07 | 1998-10-27 | 味の素株式会社 | Production of L-lysine by fermentation |
-
1992
- 1992-03-09 CN CN92101496A patent/CN1041534C/en not_active Expired - Lifetime
-
1997
- 1997-06-10 CN CN97112748A patent/CN1117869C/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3926738A (en) * | 1972-05-10 | 1975-12-16 | Wilson John D | Method and apparatus for control of biochemical processes |
| US4680267A (en) * | 1985-03-01 | 1987-07-14 | New Brunswick Scientific Company, Inc. | Fermentor control system |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1183474A (en) | 1998-06-03 |
| CN1064890A (en) | 1992-09-30 |
| CN1117869C (en) | 2003-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5912113A (en) | Method and apparatus for controlling carbon source concentration in aerobic cultivation of a microorganism | |
| CN1121500C (en) | Method for production of dicarboxylic acids | |
| CN1038693C (en) | Process for fermentative preparation of amino acids | |
| CN1110316A (en) | A cultivation method and apparatus in aerobic culture of microorganism | |
| CN1820076A (en) | Method for producing organic acid | |
| CN102154160A (en) | Strain capable of producing L-arginine and method for producing L-arginine by same | |
| CN104487582A (en) | Method for preparing organic acid by batch-feeding carbon source substrate and base | |
| CN1041534C (en) | Method and apparatus for controlling carbon source concentration in aerobic cultivation of microorganism | |
| CN102604904B (en) | Production method of glucose dehydrogenase | |
| CN101967501B (en) | Method for producing lysine by feedback supplement based on pH | |
| US5139938A (en) | Production of substances with an acetic acid-producing and assimilating bacterium inhibited by acetic acid | |
| Pandey et al. | Microbial Production and Applications of L-lysine | |
| JP2932791B2 (en) | Method and apparatus for controlling carbon source concentration in microbial aerobic culture | |
| CN1029322C (en) | Method for producing L-sorbose and apparatus for culturing microorganisms | |
| CN117757859A (en) | Method for co-producing L-isoleucine and L-valine through microbial fermentation | |
| JP3074781B2 (en) | Production method of L-lysine by fermentation method | |
| CN1218043C (en) | Method for producing L-glutamic acid by continuous fermentation | |
| US5053328A (en) | Process for the fermentative preparation of L-amino acids from α-keto carboxylic acids | |
| JPS62289192A (en) | Continuous production of amino acid by fermentation | |
| Hong et al. | Semisolid state fermentation of Baker’s yeast in an Air-Fluidized bed fermentor | |
| CN112662713B (en) | Culture medium for producing L-lysine by high-density fermentation and method thereof | |
| CN117025496B (en) | Escherichia coli fermentation method of recombinant plasmid, culture medium system and application of culture medium system | |
| CN111411131B (en) | Glucose feed supplement control method for producing glutamic acid through fermentation | |
| Wu et al. | Glutamic acid production in an airlift reactor with net draft tube | |
| WO2002083836A2 (en) | Fermentor ammonium sulfate control |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C15 | Extension of patent right duration from 15 to 20 years for appl. with date before 31.12.1992 and still valid on 11.12.2001 (patent law change 1993) | ||
| OR01 | Other related matters | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Assignee: Sichuan ChuanHua Ajinomoto Co., Ltd. Assignor: Ajinomoto K. K. Contract record no.: Contract filing No. 025100030001 Denomination of invention: Method and apparatus for controlling carbon source concentration during microbial aeration culture Granted publication date: 19990106 License type: Common Record date: 20021118 |
|
| LIC | Patent licence contract for exploitation submitted for record |
Free format text: LICENCE Name of requester: SICHUAN CHUANHUA AJINOMOTO CO., LTD. Effective date: 20021118 |
|
| C17 | Cessation of patent right | ||
| CX01 | Expiry of patent term |
Expiration termination date: 20120309 Granted publication date: 19990106 |