CN1908176A - Method of realizing microorganism in situ separation and fermentation employing inorganic film - Google Patents
Method of realizing microorganism in situ separation and fermentation employing inorganic film Download PDFInfo
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- CN1908176A CN1908176A CN 200610015378 CN200610015378A CN1908176A CN 1908176 A CN1908176 A CN 1908176A CN 200610015378 CN200610015378 CN 200610015378 CN 200610015378 A CN200610015378 A CN 200610015378A CN 1908176 A CN1908176 A CN 1908176A
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Abstract
the invention discloses a realizing method of microbe original separating fermenting method through inorganic film in the high-density fermenting domain, which comprises the following parts: ferment tank, inorganic film component, chromatographic column, hollow fiber film component or extracting device, compensating tank, liquid reserving tank and peristaltic pump. the invention can separate object product or side-product from fermenting system, which inhibits microbe seriously.
Description
Technical field
The present invention is a kind of novel original position separate fermentation technology, belongs to microorganism high density fermentation field; Be particularly related to the method that adopts mineral membrane to realize microorganism in situ separation and fermentation.
Background technology
Traditional fermenting process is the batch culture process, thalli growth just stops breeding to certain degree, and the growth-inhibiting in the batch culture process is owing to following two aspects cause: (1) matrix suppresses phenomenon: the matrix of high density pair cell sometimes produces restraining effect.The biomass of high density fermentation can reach 60～150g/l, needs to drop into 2～5 times to the matrix of biomass, yet high density nutrition is unfavorable to the most of microbe growth.Press the Michaelis-Menton kinetics curve, when nutrient concentration is increased to when a certain amount of, growth shows the saturation type kinetic curve, further increase concentration of substrate, a kind of matrix inhibitory area just may take place, show as the prolongation of lag phase, specific growth rate reduces, degradation under the cell yield.(2) product suppresses: if the meta-bolites pair cell of cell has restraining effect, along with the accumulation of meta-bolites, the specific growth rate of cell will descend gradually.
At the drawback of traditional zymotic, what factory generally adopted at present is fed-batch fermentation, and this method can be regulated nutrient concentrations in the fermenting process on the one hand, makes its suitable thalli growth, prolongs the logarithmic phase of thalli growth, to improve product output; On the other hand, can also alleviate owing to the toxic action that by product that glycolysis produces brings thalli growth takes place initial sugared excessive concentration, so this training method is with in the high-density culture that is widely used in multiple microorganism.But fed-batch fermentation be owing to can not in time separate the target product or the metabolic by-prods that suppress thalli growth, thereby is difficult to reach very high cell concentration and target product output by fed-batch fermentation concerning thalline has the bacterial classification of severe inhibition effect for those fermentating metabolism products.
Adopt that membrane separation technique can be separated with solvent two-pack or multi-component solute, classification, concentrate, purify and purify, be used at present the separation and Extraction of fermentation ends after product, to separate, improve target product output be the task of top priority and in time will suppress the target product of thalli growth or metabolic by-prods original position in microbial fermentation.
Summary of the invention
The technical problem that solves:
The object of the present invention is to provide a kind of mineral membrane isolation technique of utilizing to have the target product of severe inhibition effect or metabolic by-prods in time from fermentation system, to separate to microbial cells with what produce in the fermenting process, thereby effectively removed the restraining effect of thalli growth, made the quick growth of thalline reach the very high concentration and the original position separate fermentation method of raising target product output.
Technical scheme:
Separatory membrane and membrane module comprise two kinds of mineral membrane and organic membrane.The organic membrane non-refractory is unsuitable for sterilization, and mineral membrane is compared with organic membrane, and it has high temperature high voltage resistant, physical strength height, and resistance to acids and bases is strong, advantages such as easy cleaning.These advantages are in time separated from fermentation system and are realized that the original position separate fermentation lays a good foundation suppressing the target product of thalli growth or metabolic by-prods for itself and fermentor tank are coupled.
The present invention provides a kind of novel fermentation device and fermentation process that adopts the original position isolation technique to realize the original position separate fermentation.
Adopting mineral membrane to realize the method for microorganism in situ separation and fermentation, is that mineral membrane isolation technique and microbial fermentation are coupled, by detecting, the concentration of the restriction thalline is grown fast in the control fermentor tank meta-bolites, carrying out the original position separate fermentation; Specific practice is to introduce the mineral membrane tripping device during the fermentation, make up original position separate fermentation reactor, logarithmic growth mid-term or later stage when the meta-bolites of thalline in the fermentor tank reaches the concentration that suppresses thalli growth, open this tripping device, fermented liquid is fed inorganic membrane assembly 4, after meta-bolites in the fermented liquid separated, obtain spissated fermented liquid, get back to fermentor tank 3 through the outlet of membrane module again, to be filtered solution flow to liquid storage through the Jacket outlet of membrane module to the substratum that contains meta-bolites after the filtration irritates 5 and squeeze into chromatographic column by peristaltic pump 2, hollow fiber film assembly or extraction plant 7, the target meta-bolites is removed, remaining filtered solution carries out degerming through second group of inorganic membrane assembly 8 again, return fermentor tank at last, continue fermentation; Treat that fermentation closes the mineral membrane tripping device when normally carrying out, when the meta-bolites that suppresses thalline in the fermentor tank not when regeneration, not regrowth of thalline, fermentation ends.
We design and have used original position separate fermentation reactor in order to implement the inventive method, it is mainly by fermentor tank 3, inorganic membrane assembly 4 and 8, chromatographic column, hollow fiber film assembly or extraction plant 7, feed supplement jar 1, container for storing liquid 5 and 6, peristaltic pump 2 is formed, and wherein, inorganic membrane assembly comprises two groups, first group of inorganic membrane assembly, be that inorganic membrane assembly 4 is used for filtering fermentating liquid to the disadvantageous meta-bolites of thalli growth, second group of inorganic membrane assembly, promptly inorganic membrane assembly 8, be used for degerming, they all are made up of film pipe and chuck; The mode of connection of whole reactor is, adopt pipeline with feed supplement jar 1 successively with peristaltic pump 2, fermentor tank 3, peristaltic pump 2, inorganic membrane assembly 4, container for storing liquid 5, peristaltic pump 2, chromatographic column (hollow fiber film assembly or extraction plant) 7, container for storing liquid 6, peristaltic pump 2 links to each other with inorganic membrane assembly 8.
Need to prove:
In the original position separate fermentation reactor, the inorganic membrane assembly 4 of filtration and degerming and 8 adopts inorganic ceramic film or stainless steel membranes, and its membrane pore size is less than 0.2 μ m.
In the original position separate fermentation reactor, select corresponding chromatographic column (or hollow fiber film assembly or extraction plant) according to the kind that will remove meta-bolites
The technological process that adopts mineral membrane isolation technique of the present invention and microbial fermentation to be coupled the realization high density fermentation is:
(1) the seed culture process is: after bacterial classification is activated, receives and carry out seed culture in the seed culture medium.
(2) the fermentation culture process is: after the fermention medium for preparing is cooled off through sterilizing, seed liquor is inoculated in the fermentor tank that fermention medium is housed according to suitable inoculum size, whole culturing process is taken a sample at set intervals, measure cell concentration, carbon source concentration, the concentration that suppresses the meta-bolites of thalli growth, the output of purpose product.
(3) the batch fermentation phase: parameters such as control leavening temperature and air flow, the fermentation initial stage, thalline grew into logarithmic phase fast because carbon, nitrogenous source, component of inorganic salts is abundant and the meta-bolites of a spot of inhibition thalline.
(4) the original position separate fermentation phase: logarithmic growth mid-term, the thalli growth metabolism is vigorous, produced the metabolic product of a large amount of inhibition thalli growths, start peristaltic pump fermented liquid is fed inorganic membrane assembly this moment, fermented liquid after the filtration returns fermentor tank, filtered solution feeds second group of inorganic membrane assembly again after the meta-bolites that chromatographic column (or hollow fiber film assembly or extraction plant) will suppress thalline is removed, turn back in the fermentor tank after the degerming.
(5) when not regrowth of thalline, enter the stage of stable development after, until fermentation ends.
Beneficial effect:
The invention provides a kind of mineral membrane isolation technique of utilizing has the target product of severe inhibition effect or metabolic by-prods in time to separate from fermentation system what produce in the fermenting process to microbial cells, thereby effectively removed the restraining effect of thalli growth, made the quick growth of thalline reach the very high concentration and the original position separate fermentation method of raising target product output.
Adopt the present invention to improve the growth velocity of thalline, and improved cell concentration, reach the purpose that improves target product output.Realize high-density culture, improved output.
Description of drawings
Fig. 1: be the synoptic diagram of original position separate fermentation reactor of the present invention.
Embodiment:
Embodiment 1:P.shermanii produces vitamins B
12The original position separate fermentation
Main primary metabolite propionic acid in the P.shermanii submerged fermentation process has serious restraining effect to thalli growth, and vitamins B
12Be secondary metabolite in the born of the same parents of P.shermanii, thereby timely propionic acid removed from fermentation system, remove the restraining effect of propionic acid, realize that high-density culture can improve vitamins B greatly thalline
12Output.
Original position separate fermentation reactor, mainly by fermentor tank 3, inorganic membrane assembly 4 and 8, chromatographic column 7, feed supplement jar 1, container for storing liquid 5 and 6, peristaltic pump 2 is formed, and wherein, inorganic membrane assembly comprises two groups, first group of inorganic membrane assembly, be that inorganic membrane assembly 4 is used for filtering fermentating liquid to the disadvantageous meta-bolites of thalli growth, second group of inorganic membrane assembly, promptly inorganic membrane assembly 8, be used for degerming, they all are made up of film pipe and chuck; The mode of connection of whole reactor is, adopt pipeline with feed supplement jar 1 successively with peristaltic pump 2, fermentor tank 3, peristaltic pump 2, inorganic membrane assembly 4, container for storing liquid 5, peristaltic pump 2, chromatographic column 7, container for storing liquid 6, peristaltic pump 2 links to each other with inorganic membrane assembly 8.
Logarithmic phase mid-term when the meta-bolites propionic acid of thalline in the fermentor tank reaches the concentration that suppresses thalli growth, open this tripping device, fermented liquid is fed inorganic membrane assembly (4), after meta-bolites in the fermented liquid separated, obtain spissated fermented liquid, get back to fermentor tank (3) through the outlet of membrane module again, to be filtered solution flow to liquid storage through the Jacket outlet of membrane module to the substratum that contains the meta-bolites propionic acid after the filtration irritates (5) and squeeze into chromatographic column (7) by peristaltic pump (2), the target meta-bolites is removed, remaining filtered solution carries out degerming through second group of inorganic membrane assembly (8) again, return fermentor tank at last, continue fermentation; Treat that fermentation closes the mineral membrane tripping device when normally carrying out, when the meta-bolites that suppresses thalline in the fermentor tank not when regeneration, not regrowth of thalline, fermentation ends.
Wherein 7L fermentor tank 3 has been adopted in experiment, and available from the high machine in Shanghai company, inorganic membrane assembly 4 and 8 adopts the inorganic ceramic membrane module, available from the outstanding film of Anhui generation Engineering Co., Ltd, aperture 0.2 μ m.Anionite-exchange resin 201x7 is available from Chemical Plant of Nankai Univ..
Specific practice is:
(1) seed culture medium: glucose 35g, corn steep liquor 20g (dry), ammonium sulfate 5g, potassium primary phosphate 4g, cobalt chloride 0.005g is dissolved in the 1000mL water, adds lime carbonate 2g behind the accent pH6.8-7.0.
Fermention medium: glucose 55g, corn steep liquor 35g (dry), ammonium sulfate 5g, potassium primary phosphate 8g, cobalt chloride 0.01g is dissolved in the 1000mL water, transfers pH6.8-7.0.In the fermentation, when residual sugar is reduced to 25g/L, add 0.01g/L precursor 5,6 dimethylbenzimidazoles.
(1) cultivates before the seed
Get the good solid seed pipe of growth, add 10mL physiological saline, scrape the puncture layer, insert in the ready liquid seed culture medium behind the preparation bacteria suspension, leave standstill in 30 ± 1 ℃ and cultivate 48h
(2) seed activation is cultivated
The seed of preceding cultivation is received in the fresh liquid seed culture medium with 10% inoculum size, left standstill in 30 ± 1 ℃ and cultivate 48h.
(4) prepare before the fermentation
At first fermentor tank and feed supplement jar are carried out slack tank sterilization, inorganic membrane assembly and the pipeline autoclaving that links to each other with fermentor tank thereof.The fresh fermention medium that will prepare the then fermentor tank of packing into after the real jar of sterilization connects pipeline.
(5) inoculation
Under the protection of flame circle, the inoculum size with 10% is inoculated in the 7L fermentor tank, and liquid amount is 5L, 30 ℃ of culture temperature.
(6) the batch fermentation stage
Earlier fermentation adopts static batch fermentation, controls 30 ℃ of leavening temperatures well, every the 4h sampling, measures the dry weight of thalline, remaining glucose content, and propionic acid content, and calculate the specific growth rate of thalline.
(7) the original position separate fermentation stage
Thalline is through ramp behind the lag phase, propionic acid content raises, when the propionic acid content in the fermented liquid reaches 10g/L, start peristaltic pump fermented liquid is fed first group of inorganic ceramic membrane module, filtered solution is after anion-exchange resin column is with the absorption of metabolism products such as propionic acid, feed second group of inorganic ceramic membrane module again, feed fermentor tank after the filtration sterilization; Measure propionic acid content, dry cell weight, remaining glucose content every 2h, and calculate the thalline specific growth rate; Propionic acid content in fermentor tank is lower than 5g/L, during the thalline ramp, stops peristaltic pump, carries out batch fermentation.Measure propionic acid content every 2h simultaneously.When the growth of thalline is suppressed once more, when promptly propionic acid content reaches 10g/L, the operation above repeating.When residual sugar is reduced to 25g/L in the fermented liquid, add 0.01g/L precursor 5,6 dimethylbenzimidazoles.
The result shows that comparing cell concentration with batch fermentation increases greatly, and dry cell weight is brought up to 35.99g/L by 13.5g/L, has improved 1.67 times, vitamins B
12Output be increased to 63.1mg/L by the 33.5mg/L of existing batch fermentation, improved 0.88 times, vitamins B
12Output obviously improve.
Embodiment 2:
The original position separation and Culture of Bacillus coagulans Bacillus cogulans TQ33
The lactic acid that produces in the Bacillus coagulans fermenting process has had strong inhibitory effects to the growth of thalline, thereby in time lactic acid is separated from fermented liquid, can improve the concentration of thalline and gemma greatly.The 7L fermentor tank that experiment is adopted is available from the high machine in Shanghai company, and the inorganic ceramic membrane module is available from the outstanding film of Anhui generation Engineering Co., Ltd, aperture 0.2 μ m.Anionite-exchange resin D354 purchases Zhengguang Resin Co., Ltd..
1: substratum
(1) seed culture medium (g/L)
Peptone 10.0, yeast extract paste 10.0, glucose 60.0, MgSO
47H
2O 0.5, CaCO
310.0.pH7.2-7.4。
(2) fermention medium (g/L)
Peptone 10.0, yeast extract paste 10.0, glucose 6.0, MgSO
47H
2O 1.0, K
2HPO
42.0, MnSO
450mg/L pH 7.0.
(3) GYP substratum (g/L)
2: culturing process
(1) 0～12h is lag phase, adopts batch culture.
(2) 12～18h are logarithmic growth early stage, and the lactic acid accumulation is less, adopt fed batch cultivation.Stream adds glucose and yeast extract paste mixed solution [m (glucose): m (yeast extract paste)=6: 1], and control glucose quality concentration is at 3.0～6.0g/L, and simultaneously, stream adds the NaOH solution of 2mol/L to keep pH about 7.0.
(3) 18～24h, be the logarithmic growth later stage, metabolism products such as lactic acid have accumulated more, lactic acid content in fermentor tank reaches 5g/L, start peristaltic pump fermented liquid is fed first group of inorganic ceramic membrane module this moment, filtered solution feeds second group of inorganic ceramic membrane module again after anionite-exchange resin is with the absorption of metabolism products such as lactic acid, feed fermentor tank after the filtration sterilization; Measure dlactic acid content, dry cell weight, remaining glucose content every 2h, and calculate the thalline specific growth rate; Lactic acid content in fermentor tank is lower than 5g/L, during the thalline ramp, stops peristaltic pump, carries out batch fermentation.Measure lactic acid content every 2h simultaneously.When the growth of thalline is suppressed once more, the operation above repeating.
(4) 24～48h are stationary phase, adopt batch culture.
3: the result
By the original position separation and Culture, cell concentration has reached 1.8
10The high-density of cfu/mL.Adopt batch culture behind the 24h, gemma forms rapidly.Because the cell density height, self-dissolving is more serious.During the 48h fermentation ends, total bacteria concentration is 1.6 * 10
10Cfu/mL, gemma concentration is 1.2 * 10
10Cfu/mL.
Other is with embodiment 1.
Embodiment 3:
Split-gill produces exocellular polysaccharide original position separate fermentation
The Split-gill exocellular polysaccharide is a kind of water miscible callose, and because of its active structure with uniqueness of the ramose β of good water-soluble and β-(1-6)-(1-3)-D glucose, in the adjusting immunologic function, antitumor, there is significant curative effect aspects such as radioprotective.About Split-gill exopolysaccharide molecule amount 40kD.The accumulation of Split-gill exocellular polysaccharide increases the viscosity of fermented liquid in the fermented liquid, is unfavorable for the growth of thalline, thereby in time separation of polysaccharides is gone out, and can help improving the output of exocellular polysaccharide.
The 7L fermentor tank that experiment is adopted is available from the high machine in Shanghai company, and inorganic stainless steel membrane assembly is available from Shanghai triumphant energy high-tech Engineering Co., Ltd, aperture 0.2 μ m.Macroporous adsorbent resin AB-8 is available from Chemical Plant of Nankai Univ..
1: substratum
(1) inclined-plane seed culture medium: PDA substratum (yeast extract powder content 5g/L).
(2) seed liquid nutrient medium: glucose 35g, yeast extract powder 3g, KH
2PO
40.5g, MgSO
47H
2O 0.5g, the tap water preparation is settled to 1L, and all the other are analytical pure except that the yeast extract powder, the pH nature.
(3) fermention medium: glucose 45g, yeast extract powder 3g, KH
2PO
40.5g, MgSO
47H
2O0.5g, the tap water preparation is settled to 1L, and all the other are analytical pure except that the yeast extract powder, the pH nature.
2: fermenting process
(1) 0-16h batch fermentation
Be inoculated into the 7L fermentor tank with 10% inoculum size, 27 ℃ of culture temperature, the pH nature, initial air flow 100L/h, mixing speed 200r/min, initial stage thalline thalline begin propagation, and polysaccharide begins secretion.
(2) 16-160h original position separate fermentation
Thalline is through ramp behind the lag phase, polysaccharide content increases sharply, polysaccharide causes fermentation broth viscosity to rise, when dissolved oxygen is lower than 20% in the fermented liquid, start peristaltic pump fermented liquid is fed first group of inorganic stainless steel membrane assembly, the macroporous adsorptive resins of filtered solution through the Split-gill exocellular polysaccharide there being special adsorption function feeds second group of inorganic stainless steel membrane assembly after polysaccharide is adsorbed again, feed fermentor tank after the filtration sterilization, and the nutritive ingredient of replenish loss.
(3) exocellular polysaccharide in the fermented liquid no longer increases, fermentation ends during not regrowth of thalline.
The yield of fermentation ends crude extracellular polysaccharide reaches 6.4g/L, is greatly improved than the 2.7g/L of batch fermentation.
Other is with embodiment 1.
Embodiment 4: Split-gill produces exocellular polysaccharide original position separate fermentation
The Split-gill exocellular polysaccharide is a kind of water miscible callose, and because of its active structure with uniqueness of the ramose β of good water-soluble and β-(1-6)-(1-3)-D glucose, in the adjusting immunologic function, antitumor, there is significant curative effect aspects such as radioprotective.About Split-gill exopolysaccharide molecule amount 40kD.The accumulation of Split-gill exocellular polysaccharide increases the viscosity of fermented liquid in the fermented liquid, has reduced dissolved oxygen of fermentation liquid, is unfavorable for the growth of thalline, thereby in time separation of polysaccharides is gone out, and can help improving the output of exocellular polysaccharide.The 7L fermentor tank that experiment is adopted is available from the high machine in Shanghai company, and inorganic stainless steel membrane assembly is available from Shanghai triumphant energy high-tech Engineering Co., Ltd, aperture 0.2 μ m.Hollow fiber film assembly 7 (molecular weight cut-off is less than 40kD) is available from film sky, Tianjin film Engineering Co., Ltd.
1: substratum
(1) inclined-plane seed culture medium: PDA substratum (yeast extract powder content 5g/L).
(2) seed liquid nutrient medium: glucose 35g, yeast extract powder 3g, KH
2PO
40.5g, MgSO
47H
2O 0.5g, the tap water preparation is settled to 1L, and all the other are analytical pure except that the yeast extract powder, the pH nature.
(3) fermention medium: glucose 45g, yeast extract powder 3g, KH
2PO
40.5g, MgSO
47H
2O0.5g,
The tap water preparation is settled to 1L, and all the other are analytical pure except that the yeast extract powder, the pH nature.
2: fermenting process
(1) 0-16h batch fermentation
Be inoculated into the 7L fermentor tank with 10% inoculum size, 27 ℃ of culture temperature, the pH nature, initial air flow 100L/h, mixing speed 200r/min, initial stage thalline thalline begin propagation, and polysaccharide begins secretion.
(2) 16-160h original position separate fermentation
Thalline is through ramp behind the lag phase, and polysaccharide content increases sharply, and polysaccharide causes fermentation broth viscosity to rise, and dissolved oxygen reduces, and when dissolved oxygen is lower than 20% in the fermented liquid, starts peristaltic pump.Fermented liquid is fed first group of inorganic stainless steel membrane assembly, and filtered solution is behind the hollow fiber film assembly of molecular weight cut-off less than 40kD, and concentrated solution is used for the separation of polysaccharide, and filtered solution feeds second group of inorganic stainless steel membrane assembly again, feeds fermentor tank after the filtration sterilization.
(3) exocellular polysaccharide in fermented liquid no longer increases, fermentation ends during not regrowth of thalline.
The yield of fermentation ends crude extracellular polysaccharide reaches 6.4g/L, is greatly improved than the 2.7g/L of batch fermentation.
Other is with embodiment 1.
Embodiment 5: butanols original position separate fermentation
In this year, petroleum resources are in short supply more and more obvious, and because the CO that the mass consumption of fossil oil is discharged in atmosphere
2Accumulation aggravation, so the recyclable organism energy source of biologic diesel oil becomes the research focus, and is poor but biofuel exists ignition characteristic, shortcomings such as black smoke generation are arranged, if add butanols therein, just can improve ignition characteristic and not produce black smoke, the significant performance that acts as a fuel of improving.From these viewpoints, examine closely as ancient industrial technology acetone butanol fermentation active day by day in recent years again in countries in the world.
It is extremely strong that the end product of acetone butanol fermentation suppresses level, when the concentration of butanols reaches 3g/L in the fermented liquid, restraining effect just occurred.If when butanol concentration surpassed this concentration, fermentation rate sharply descended, therefore need in time butanols to be separated from fermented liquid.The 7L fermentor tank that experiment is adopted is available from the high machine in Shanghai company, and inorganic stainless steel membrane assembly is available from Shanghai triumphant energy high-tech Engineering Co., Ltd, aperture 0.2 μ m.Concrete fermenting process:
(1) the batch fermentation stage, detects the concentration of butanols in the fermented liquid in real time.
(2) the original position separate fermentation stage, thalline is through ramp behind the lag phase, butanol content raises, when the concentration of butanols in the fermented liquid surpasses 3g/L, start peristaltic pump 2 fermented liquid is fed first group of inorganic stainless steel membrane assembly 4, filtered solution was through inhaling extraction plant 7, and biofuel wherein extracts away butanols, the nutrient solution of water feeds second group of inorganic stainless steel membrane assembly 8 again, feeds fermentor tank 3 after the filtration sterilization; Measure the content of butanols in the fermentation cylinder for fermentation liquid, the dry weight of thalline, remaining glucose content every 2h, and calculate the specific growth rate of thalline; Butanol content in fermentor tank is lower than 3g/L, during the thalline ramp, stops vacuum pump, carries out batch fermentation.When the concentration of butanols in the fermented liquid surpasses 3g/L, when thalli growth is suppressed once more, the operation above repeating.
(3) content of butanols no longer increases in fermented liquid, during not regrowth of thalline, and fermentation ends.
Fermentation ends, the content of butanols reaches 70g/L, is greatly improved than the 30g/L of batch fermentation.
Other is with embodiment 1.
Claims (4)
1. adopt mineral membrane to realize the method for microorganism in situ separation and fermentation, it is characterized in that mineral membrane isolation technique and microbial fermentation are coupled, by detecting, the concentration of the restriction thalline is grown fast in the control fermentor tank meta-bolites, carrying out the original position separate fermentation; Specific practice is to introduce the mineral membrane tripping device during the fermentation, make up original position separate fermentation reactor, logarithmic growth mid-term or later stage when the meta-bolites of thalline in the fermentor tank reaches the concentration that suppresses thalli growth, open this tripping device, fermented liquid is fed inorganic membrane assembly (4), after meta-bolites in the fermented liquid separated, obtain spissated fermented liquid, get back to fermentor tank (3) through the outlet of membrane module again, to be filtered solution flow to liquid storage through the Jacket outlet of membrane module to the substratum that contains meta-bolites after the filtration irritates (5) and squeeze into chromatographic column by peristaltic pump (2), hollow fiber film assembly or extraction plant (7), the target meta-bolites is removed, remaining filtered solution carries out filtration sterilization through second group of inorganic membrane assembly (8) again, return fermentor tank at last, continue fermentation; Treat that fermentation closes the mineral membrane tripping device when normally carrying out, when the meta-bolites that suppresses thalline in the fermentor tank not when regeneration, not regrowth of thalline, fermentation ends.
2. the original position separate fermentation reactor during claim 1 method is implemented, it is characterized in that mainly by fermentor tank (3), inorganic membrane assembly (4) and (8), chromatographic column, hollow fiber film assembly or extraction plant (7), feed supplement jar (1), container for storing liquid (5) and (6), peristaltic pump (2) is formed, and wherein, inorganic membrane assembly comprises two groups, first group of inorganic membrane assembly, be that inorganic membrane assembly (4) is used for filtering fermentating liquid to the disadvantageous meta-bolites of thalli growth, second group of inorganic membrane assembly, i.e. inorganic membrane assembly (8), be used for degerming, they all are made up of film pipe and chuck; The mode of connection of whole reactor is, adopt pipeline with feed supplement jar (1) successively with peristaltic pump (2), fermentor tank (3), peristaltic pump (2), inorganic membrane assembly (4), container for storing liquid (5), peristaltic pump (2), chromatographic column, hollow fiber film assembly or extraction plant (7), container for storing liquid (6), peristaltic pump (2) links to each other with inorganic membrane assembly (8).
3. original position separate fermentation reactor according to claim 1 and 2 is characterized in that filtering with inorganic membrane assembly (4) and (8) of degerming and adopts inorganic ceramic film or stainless steel membrane, and its membrane pore size is less than 0.2 μ m.
4. original position separate fermentation reactor according to claim 1 and 2 is characterized in that selecting corresponding chromatographic column, hollow fiber film assembly or extraction plant according to the kind that will remove meta-bolites.
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