CN101967498A - Method for producing biological organic acid by using dynamic expanding adsorption reactor system - Google Patents
Method for producing biological organic acid by using dynamic expanding adsorption reactor system Download PDFInfo
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Abstract
The invention belongs to the technical field of fermentation engineering, and particularly relates to a method for producing biological organic acid by using a dynamic expanding adsorption reactor system. In a microbial cell fermentation process, a biological organic acid target product is secreted in extracellular fermentation solution together with the growth of microbial cells, an enzyme system participating in the microbial cell growth metabolism is subjected to feedback suppression of the biological organic acid target product at the moment so that the metabolism velocity of the cells is reduced, and the fermentation solution with the microbial cells is directly pumped into an expanded bed chromatographic column filled with a separation medium in a dynamic expanding adsorption reactor so as to realize on-line adsorption of the biological organic acid target product through certain adsorption and separation processes and avoid plugging the chromatographic column; and the fermentation solution from which the biological organic acid target product is removed can be circularly refluxed to a fermentation tank. The suppression on the growth of the microbial cells is relieved, the growth vitality of the microbial cells is recovered, the biomass of the microbial cells is improved, and the yield of the biological organic acid product is increased.
Description
Technical field
The invention belongs to the fermentation engineering field, particularly use the method that dynamic expanding formula adsorptive reactor system produces biogenic organic acid.
Background technology
A feedback inhibition that a difficult problem is a products of cellular metabolism that runs into during with the various product of microorganism cells fermentative production, its phenomenon is: in the microorganism cells fermenting process, thalline is to nourish and grow breeding time in cell or the cell exocrine meta-bolites with specific substrate, after the concentration of some products of cellular metabolism reaches certain limit, target product no longer increases, impurity then rolls up, and causes fermentation production rate to improve, the separation that simultaneously a large amount of impurity also can interfere with subsequent.In time from fermentor tank, remove if adopt the process engineering means will feed back material, just might remove feedback inhibition, increase fermentation production rate, create huge economic benefit.Existing studies show that, remove feedback inhibition after, the productive rate of microorganism cells fermentation can increase substantially, output even can increase more than 50% even more.
Solve the feedback inhibition phenomenon from project angle, need research novel microorganism cell fermentation process.At present the microorganism cells fermenting process is divided into three kinds of modes: batch fermentation, semicontinuous fermentation, continuously ferment.Batch fermentation modes that adopt generally all are disposable feeding intake more in the production, disposable results product.Wherein must there be feedback inhibition to exist.Though the someone carried out the research of continuously fermenting,, add and continuously ferment contaminatedly easily that production concentration is low, wastage of material is many, is difficult to be accepted by fermentation industry so continuously ferment because that biological respinse is nothing like some chemical reactions is rapid.
Compare the mode of continuously fermenting, adopt the semicontinuous fermentation mode to have more prospect.It is not constant charging constantly and discharging as chemical reaction, but just carries out the charging and the discharging of fermentor tank (being bio-reactor) after the microorganism cells fermentation arrives certain phase.At present the semicontinuous fermentation great majority just batch fermentation after for some time stream add some raw materials and precursor, and do not remove product.This " semicontinuous " fermentation is called the fermentation of feed supplement formula again or stream adds the formula fermentation, has been widely used in the large scale fermentation process of secondary meta-bolitess such as microbiotic, has obtained very significant success, and the process that has improves output and reaches more than several times.
People have also explored at stream and have added on the basis of raw material, remove some products, and to reach real semicontinuous fermentation, this is the most effective to reducing feedback inhibition, and flow feeding usually is in order to control the metabolism in the fermentor tank.Although do a lot of work, the trial of removing product does not have stream, and to add raw material successful like that.The problem that exists is that the operation of removing product certainly leads to the influence to microorganism cells and enzymic activity, also must use separating medium when removing product, and separating medium itself also may produce the influence to the microorganism cells fermentation; Also have, the process of separated product usually comprises violent physicochemical change, remain on the mild conditions such as the normal temperature and pressure of fermentation, and gentle pH realizes that separated product is difficult down.
Based on above reason, to react and to separate the focus that the novel microorganism cell fermentation technology that is coupled just becomes domestic and international research.In recent years both at home and abroad after deliberation processes such as the fermentation of extractive fermentation, membrane reactor, ion-exchange absorption fermentation.For example, the scholar of China and France has studied respectively and has removed propionic acid with ultrafiltration membrane reactor and extraction process and suppress, and has realized separating of propionic acid and fermented liquid.But adding secondary amine and sodium hydroxide makes cost increase again as extraction agent and strippant.Although the result is encouraging for the ultrafiltration membrane reactor method, it is a headachy difficult problem that the pollution of film descends filtering rate rapidly.
Expanded Bed Adsorption (Expanded Bed Adsorption, EBA) technology is the very promising separation means that grows up the beginning of the nineties in last century, in the expanding bed chromatography column, be filled with separating medium, feed liquid is behind separating medium, the separating medium appropriateness expands, have big space between the separating medium, solid granule can directly pass through bed and the separated capture medium of target product in the feed liquid; Custom-designed separating medium can form and stablize the fractionated bed structure simultaneously, makes separating effect to work as with the fixed bed phase separation.Expanded Bed Adsorption collects solid-liquid separation, concentrate and the initial stage purifying among an operating unit, can directly from the fermented liquid, cell culture fluid or the homogenate that contain solid particulate, catch target product, thereby reduce operation steps, can reduce cost, shorten the operating time, improve yield.
The technology that dynamic expanding formula absorption and biological fermentation are coupled of the invention is applied to the biogenic organic acid production process, comprises the process integration etc. of on-line adsorption separation, acetic acid fermentation, citric acid fermentation, lactic fermentation, fumaric acid fermentation, tartaric acid fermentation, Succinic Acid fermentation and the long-chain biatomic acid fermentation of propionic acid in deoxyadenosyl cobalamin (VB12) fermenting process.These several products all are important biochemical industry products, it also is the representative that China has the leavened prod of certain market advantage in the world, but compared with developed countries, on fermentation technique and isolation technique, also has no small gap, quality product and cost face the constantly progressive threat that causes of developed country's technology, and being badly in need of development has independent intellectual property right, high yield, high yield, low cost and free of contamination fermentation engineering.
Summary of the invention
The feedback inhibition phenomenon that occurs when the objective of the invention is at using microbe cells produce leavened prod, from the integrated angle of bioprocess, expanding bed chromatography column and fermentor tank in the dynamic expanding formula adsorptive reactor directly are coupled, the creationary technology that dynamic expanding formula absorption and biological fermentation are coupled is applied to biogenic organic acid production, thereby a kind of method that dynamic expanding formula adsorptive reactor system produces biogenic organic acid of using is provided.
The present invention can overcome at present in utilizing microorganism cells or directly carrying out process that catalyzed reaction turns out a produce with enzyme system, usually be attended by metabolite when running up to finite concentration, the feedback inhibition phenomenon occurs, and causes the problem that product yield is low, raw material availability is low, production efficiency descends.The present invention is in the microorganism cells fermenting process, follow the growth meeting of microorganism cells in the fermented liquid of extracellular, to secrete the target product biogenic organic acid, the enzyme that participates in the microorganism cells growth metabolism this moment is that the feedback inhibition that is subjected to the target product biogenic organic acid makes when cellular metabolism speed slows down, directly pump in the expanding bed chromatography column that separating medium is housed in the dynamic expanding formula adsorptive reactor by the fermented liquid that will have microorganism cells, through certain adsorption separation process, realize on-line adsorption target product biogenic organic acid and do not cause the obstruction of chromatography column; Remove the fermented liquid fermentor tank that is back to capable of circulation of target product biogenic organic acid.The microorganism cells suffered inhibition of growing is removed, and microorganism cells recovers growth vigor, and the microorganism cells biomass improves, and the biogenic organic acid product production increases.
The method that application dynamic expanding formula adsorptive reactor of the present invention system produces biogenic organic acid may further comprise the steps:
(1) adopt ordinary method culturing micro-organisms cell seed, it is that the C source is used to prepare the microorganism cells seed culture medium 115 ℃ of independent down sterilizations after 30 minutes; Need cultured microorganism cell seed is inoculated in the microorganism cells seed culture medium in the seeding tank after sterilizing, is to adopt aerobic under 20-40 ℃ the normal pressure or the anaerobism mode was carried out elementary culturing micro-organisms cell seed 20-60 hour in temperature; Wherein: needing the volume ratio of cultured microorganism cell seed and microorganism cells seed culture medium is 5-10%; By percentage to the quality, the composition of microorganism cells seed culture medium: the C source 2-4% after the sterilization, N source 2-4%, inorganic salt 0.2-0.5%, somatomedin (inductor) 0.0005-0.01%, and water; And the pH that regulates substratum with mineral alkali is 6.5-7;
After preferably separating medium being soaked 12-24 hour with deionized water, use 1molL again
-1HCl solution soaking separating medium used the deionized water wash separating medium to neutral to remove organism and the metallic impurity in the separating medium in 4-6 hour then; Use 1molL
-1Separating medium 4-6 behind the NaOH washing by soaking hour, separating medium is converted into the free alkali type; Wash the separating medium that is converted into the free alkali type repeatedly to neutral with deionized water at last, wash-out goes out to be loaded on behind the unnecessary NaOH in the expanding bed chromatography column that the fermentor tank with dynamic expanding formula adsorptive reactor directly is coupled;
Described seeding tank is to be 121 ℃ in temperature to sterilize 30 minutes down;
Described separating medium is that the chemical ingredients of self is a macromolecular compound, its character is weakly-basic anion exchange separating medium, structure is made up of skeleton and functional group two portions, skeleton is a kind of in acrylic acid series, polystyrene, the epoxy resin, and functional group is selected from least a in the group that primary amine, secondary amine, tertiary amine, quaternary amine form.
(2) the C source is used to prepare fermention medium 115 ℃ of independent down sterilizations after 30 minutes; The cultured microorganism cells seed of step (1) is inoculated in the fermented liquid that obtains having microorganism cells in the fermention medium in the sterilized fermentor tank, is to adopt aerobic or anaerobism mode to carry out the fermentation culture of microorganism cells under 20-40 ℃ the normal pressure in temperature; Wherein: the volume ratio of cultured microorganism cells seed and fermention medium is 5-10%; By percentage to the quality, the composition of fermention medium: the C source 2-6% after the sterilization, N source 2-4%, inorganic salt 0.2-0.5%, somatomedin (inductor) 0.0005-0.01%, and water; And the pH that regulates fermention medium with mineral alkali is 6.5-7; Follow the growth and breeding of microorganism cells, the microorganism cells biomass is accumulated, the target product biogenic organic acid is constantly synthetic, when participating in the microorganism cells growth metabolism, the system of the enzyme in the microorganism cells (follows the growth and breeding of microorganism cells in the fermenting process, have in the microorganism cells in the process that corresponding enzyme ties up to microorganism cells growth and Product Metabolism and play specific function) time be subjected to the feedback inhibition of target product biogenic organic acid (mass concentration reaches 1-10%), make the pH of the fermented liquid that has microorganism cells (nutrient solution) in the fermentor tank be reduced to the 5-6 scope by 6.5-7, and the microorganism cells accretion rate that causes having in the fermented liquid of microorganism cells slows down, and opens the magnetic valve and the liquor pump of dynamic expanding formula adsorptive reactor;
Described fermentor tank is to be 121 ℃ in temperature to sterilize 30 minutes down;
(3) be that the fermented liquid that has microorganism cells of 5-6 directly enters in the chromatography column bottom of the expanding bed chromatography column from the dynamic expanding formula adsorptive reactor of step (1) with pH in step (2) fermentor tank, separating medium in the chromatography column is expanded, the height of the separating medium after the expansion and the aspect ratio of former separating medium are 2: 1-3: 1, there is big space between the separating medium, have in the fermented liquid of microorganism cells and contain certain density biogenic organic acid, when the fermented liquid that has microorganism cells is passed by the space between the separating medium in the expanding bed chromatography column, the separated medium of target product biogenic organic acid directly adsorbs, the fermented liquid that contains microorganism cells that passes by the space between the separating medium, be back to the fermentor tank from expanding bed chromatography column top, when the pH of the fermented liquid that has microorganism cells in the fermentor tank is back to 6.5-7, close the magnetic valve and the liquor pump of dynamic expanding formula adsorptive reactor; In the expanding bed chromatography column, can realize on-line adsorption target product biogenic organic acid and do not cause the obstruction of chromatography column, make the target product biogenic organic acid concentration in the fermentor tank reduce, the microorganism cells suffered inhibition of growing is removed, microorganism cells recovers growth vigor, the microorganism cells biomass continues to rise, and the pH that has the fermented liquid of microorganism cells is back to 6.5-7;
When the C source concentration in the fermention medium in the fermentor tank is lower than mass percentage concentration and is 1-2%, by the concentration ratio in C source in C source feed supplement liquid and the original fermentative medium formula is that 100: 1 feed supplement strategy carries out the feed supplement of C source, making C source concentration in the fermention medium return to mass percent is 2-6%, thereby make microorganism cells under sufficient nutritional status, keep vigorous continuous growth state, drop to up to the microorganism cells growth vigor and cell decline to occur or infect other microorganism cellss and (when decline appears in the growth of target microorganism cell, will infect corresponding phage, promptly infect other microorganism cellss) time stop fermentation, really realize the fermentation of semicontinuous microorganism cells;
The feed rate of C source feed supplement liquid is 0.1-0.5h
-1, the feed supplement mode is flow feeding or intermittent feed supplement;
(4) the separating medium water that is adsorbed with biogenic organic acid that step (3) is obtained carries out drip washing, carries out wash-out (separating medium after drip washing, wash-out, regeneration, the balance can reuse) with elutriant then; Collection desorption liquid, concentrated desorption liquid obtain biogenic organic acid.
Described elutriant is sodium hydroxide, sodium-chlor or their mixture.
The biogenic organic acid that application dynamic expanding formula adsorptive reactor of the present invention system produces is selected from a kind of in propionic acid, acetate, citric acid, lactic acid, fumaric acid, tartrate, Succinic Acid, the long-chain biatomic acid etc.
The magnetic valve of described unlatching dynamic expanding formula adsorptive reactor and liquor pump are that the pH according to the fermented liquid that has microorganism cells (nutrient solution) in step (2) fermentor tank is reduced to the 5-6 scope; When being back to 6.5-7, the pH of the fermented liquid that has microorganism cells (nutrient solution) in the fermentor tank closes the magnetic valve and the liquor pump of dynamic expanding formula adsorptive reactor, to carry out batch operation.
Described microorganism cells seed is a kind of, Succinic Acid actinobacillus, recombination bacillus coli or Succinic Acid anaerobism spirillum in a kind of, the lactobacillus in the propiono-bacterium etc.
Described C source is selected from a kind of in glucose, sucrose or the Semen Maydis powder etc.
Described N source is selected from a kind of in corn steep liquor, yeast extract paste or the peptone etc.
Described inorganic salt are selected from the group that dipotassium hydrogen phosphate, magnesium chloride, potassium primary phosphate, ammonium sulfate etc. are formed at least a.
Described somatomedin (inductor) is a cobalt chloride etc.
Described mineral alkali is sodium hydroxide, yellow soda ash, magnesiumcarbonate or ammoniacal liquor etc.
The present invention compared with prior art has following advantage:
1. the method that application dynamic expanding formula adsorptive reactor of the present invention system produces biogenic organic acid is separated coupled reactor with existing fermentation and is compared, application dynamic expanding formula adsorptive reactor of the present invention system separates the coupling reaction apparatus with existing fermentation to be had significantly different, the invention expanding bed chromatography column in the dynamic expanding formula adsorptive reactor is passed through respective valves, pipeline, liquor pump directly is coupled with fermentor tank, can be according to the particular organisms and physics and chemistry rule (the biological metabolism stream rule of particular studies object, fermented liquid physical chemistry rule etc.) set working parameter, can carry out Remote and analogue simulation by computer system, make chromatography column and directly being coupled of fermentor tank that to realize originally become possibility; And dynamic expanding formula adsorptive reactor of the present invention system is different with the fermentor tank of existing industrial application, has limit fermentation, limit separated product and removes the ability of inhibition, can realize the semicontinuity of fermentation mode, raises the efficiency, and reduces cost, and increases benefit.
2. the present invention adopts expanding bed chromatography column and fermentor tank directly to be coupled, and adsorbs certain meta-bolites selectively, rather than indiscriminate fermented liquid is taken away, is a new fermentation and separate the coupled process; And existing fixed bed chromatography column can't be realized this process, for utilizing existing fixed bed chromatography column to obtain fermented liquid, must a membrane separation apparatus be set before fixed bed chromatography post and separate microorganism cells and fermented liquid.The expanding bed that adopts in the inventive method allows to have the solid material and directly enters the expanding bed chromatography column, avoid fixed bed can not directly handle the absorption and the fermentor tank that contain solid materials and directly be coupled the blockage problem that is caused, omit extra additional membrane separation apparatus between fermentor tank and the expanding bed chromatography column, realized directly being coupled and the on-line adsorption product of expanding bed chromatography column and fermentor tank.
3. the present invention uses novelty, produce the feedback inhibition phenomenon ubiquity of various Product Metabolism products with microbial fermentation, the present invention not only can be applied in propionic acid/VB12 combined ferment process, but also can extend to other biogenic organic acid product, for example acetate, citric acid, lactic acid, fumaric acid, tartrate, Succinic Acid and long-chain biatomic acid etc.The present invention can reduce manufacturing cost, improves the quality of product, has important use and is worth.
Description of drawings
Fig. 1. application dynamic expanding formula adsorptive reactor of the present invention system produces a kind of dynamic expanding formula adsorptive reactor system schematic in the biogenic organic acid method.
Reference numeral
1. fermentor tank 2,22. pipelines 3. elutriant storage tanks
4. leacheate storage tank 5. regenerated liquid storage tanks 6. balance liquid storage tanks
7,9,10,11,12,13,14,15,23. magnetic valves
8. liquor pump 16,17,18. chromatography columns 19. product storage tanks
20. waste tank 21. detectors 24. conversion tank
Embodiment
Be example now, adopt batch fermentation and semicontinuous fermentation dual mode to illustrate that employing application dynamic expanding formula adsorptive reactor of the present invention system produces the advance of the technical system production propionic acid/VB12 of biogenic organic acid method with anaerobism production propionic acid/VB12 combined ferment and propionic acid ON-LINE SEPARATION.
Comparative Examples.
Utilize fermentor tank to carry out a batch fermentation
(1) preparation of seed
Adopt ordinary method culturing micro-organisms cell seed, it is that glucose is used to prepare propionibacterium freudenreichii (propionibacterium freudenreichii) seed culture medium 115 ℃ of independent down sterilizations after 30 minutes; The propionibacterium freudenreichii seed that need are cultivated is inoculated in the propionibacterium freudenreichii seed culture medium in the seeding tank after sterilizing, in temperature is to adopt the anaerobism mode to carry out elementary cultivation propionibacterium freudenreichii seed 40 hours under 30 ℃ the normal pressure; Wherein: needing the volume ratio of cultured microorganism cell seed and microorganism cells seed culture medium is 5-10%; By percentage to the quality, the composition of propionibacterium freudenreichii seed culture medium: the glucose 4% after the sterilization, corn steep liquor 4%, ammonium sulfate 0.5%, potassium primary phosphate 0.4%, lime carbonate 0.4%, cobalt chloride 0.0005%, and deionized water; And the pH that regulates substratum with NaOH is 6.5-7.0.
Seeding tank is to be 121 ℃ in temperature to sterilize 30 minutes down.
(2) fermentation stage
Glucose is used to prepare fermention medium 115 ℃ of independent down sterilizations after 30 minutes; The cultured propionibacterium freudenreichii seed of step (1) is inoculated in the fermented liquid that obtains having microorganism cells in the fermention medium in the sterilized fermentor tank, is to adopt the anaerobism mode to carry out the fermentation culture 90 hours of microorganism cells under 30 ℃ the normal pressure in temperature; Use ammoniacal liquor control fermented liquid pH to be 6.5-7.0 in the fermenting process, when growing to the logarithm later stage (50-60 hour), in fermented liquid, adds microorganism cells precursor 5,6-dimethylbenzimidazole (DMB) makes propionibacterium freudenreichii cell synthesizing deoxyadenosine cobalami (VB12), and whole fermentation process is regularly followed the tracks of detection of biological amount (OD value) and pH.Wherein, by percentage to the quality, the composition of propionibacterium freudenreichii fermention medium: the glucose 6% after the sterilization, corn steep liquor 4%, potassium primary phosphate 0.46, cobalt chloride 0.00127, and tap water; And the pH that regulates fermention medium with NaOH is 6.5-7.0.
Fermentor tank is to be 121 ℃ in temperature to sterilize 30 minutes down.
(3) owing to the chromatography column device in the dynamic expanding formula adsorptive reactor (separation link) is not applied in the fermenting process in the Comparative Examples, the original position on-line adsorption that promptly can't realize the propionic acid product reclaims, so high performance liquid chromatography (HPLC) testing product includes only VB12 after the fermentation ends.Experimental result sees Table 1.
See also Fig. 1.Used dynamic expanding formula adsorptive reactor system comprises the fermentor tank 1 that has whipping appts, pipeline 2, elutriant storage tank 3, leacheate storage tank 4, regenerated liquid storage tank 5, balance liquid storage tank 6, magnetic valve 7,9,10,11,12,13,14,15 and 23, liquor pump 8, chromatography column 16,17 and 18, product storage tank 19, waste tank 20, detector 21, pipeline 22, conversion tank 24 etc.
The bottom of the expanding bed chromatography column 16,17,18 of three parallel connections all has the circulation fluid import, and the top of expanding bed chromatography column all has the circulation fluid outlet.
One constitutes the circulation fluid main pipe rail 22 in circulation fluid loop, has 12,13 and 14 and 4 four way solenoid valves 9,10,11 and 15 of 3 three-way solenoid valves on this main pipe rail.
Wherein, the outlet of the circulation fluid of expanding bed chromatography column 16,17,18 is connected with three- way solenoid valve 12,13 and 14 by pipeline respectively; 3 three magnetic valves 12,13 and 14 on the circulation fluid main pipe rail at place, expanding bed chromatography column top are connected by the pipeline polyphone respectively, and a port of the three-way solenoid valve 14 on the circulation fluid export pipeline of first expanding bed chromatography column 16 in parallel is connected by the port of pipeline with the four way solenoid valve 15 of communication with detection device 21, and a port of the three-way solenoid valve 12 on the circulation fluid export pipeline of the 3rd expanding bed chromatography column 18 in parallel is connected by the port of pipeline with the magnetic valve 23 that is communicated with conversion tank 24; One port of four way solenoid valve 15 is connected with fermentor tank 1 by pipeline.
Four way solenoid valve 9,10,11 on the circulation fluid main pipe rail is connected by the circulation fluid import of pipeline with expanding bed chromatography column 16,17,18 respectively; 3 four magnetic valves 9,10 and 11 are connected by the pipeline polyphone respectively.
The outlet of liquor pump 8 is connected by the circulation fluid import of pipeline with four way solenoid valve 9,10,11 respectively; The import of liquor pump 8 is connected with a port of three-way solenoid valve 7 by pipeline; Two ports in addition of three-way solenoid valve 7 are connected with fermentor tank 1 by pipeline 2 respectively, and are connected with the elutriant storage tank 3 that respectively carries valve, leacheate storage tank 4, regenerated liquid storage tank 5 and balance liquid storage tank 6 by pipeline.
Use the above-mentioned dynamic expanding formula adsorptive reactor production propionic acid/VB12 of system
(1) adopt ordinary method culturing micro-organisms cell seed, it is that glucose is used to prepare propionibacterium freudenreichii (propionibacterium freudenreichii) seed culture medium 115 ℃ of independent down sterilizations after 30 minutes; The propionibacterium freudenreichii seed that need are cultivated is inoculated in the propionibacterium freudenreichii seed culture medium in the seeding tank after sterilizing, in temperature is to adopt the anaerobism mode to carry out elementary cultivation propionibacterium freudenreichii seed 40 hours under 30 ℃ the normal pressure; Wherein: needing the volume ratio of cultured microorganism cell seed and microorganism cells seed culture medium is 5-10%; By percentage to the quality, the composition of propionibacterium freudenreichii seed culture medium: glucose 4%, corn steep liquor 4%, ammonium sulfate 0.5%, potassium primary phosphate 0.4%, lime carbonate 0.4%, cobalt chloride 0.0005%, and deionized water; And the pH that regulates fermention medium with NaOH is 6.5-7.0.
Seeding tank is to be 121 ℃ in temperature to sterilize 30 minutes down.
Skeleton is that Resins, epoxy, functional group are primary, after the second month in a season, uncle, the weakly-basic anion in season exchange separating medium soak 12-24 hour with deionized water, uses 1molL
-1Organism and metallic impurity that the HCl solution soaking was removed in the separating medium in 4-6 hour are washed till neutrality with deionized water again; Use 1molL then
-1NaOH soaked 4-6 hour, and separating medium is converted into the free alkali type; Wash repeatedly to neutrality with deionized water at last, wash-out goes out unnecessary NaOH, is loaded on then in the expanding bed chromatography column 16,17,18 that the fermentor tank with dynamic expanding formula adsorptive reactor directly is coupled.
(2) glucose is used to prepare fermention medium 115 ℃ of independent down sterilizations after 30 minutes; The cultured propionibacterium freudenreichii seed of step (1) is inoculated in the fermented liquid that obtains having microorganism cells in the fermention medium in the sterilized fermentor tank 1, is to adopt the anaerobism mode to carry out the fermentation culture 120 hours of microorganism cells under 30 ℃ the normal pressure in temperature; Wherein: the volume ratio of propionibacterium freudenreichii seed and fermention medium is 10%; By percentage to the quality, the composition of fermention medium: the glucose 6% after the sterilization, corn steep liquor 4%, potassium primary phosphate 0.46, cobalt chloride 0.00127, and tap water; And the pH that regulates fermention medium with NaOH is 6.5-7.0.Follow the growth and breeding of propionibacterium freudenreichii cell, the microorganism cells biomass is accumulated, the target product propionic acid is constantly synthetic, when participating in the microorganism cells growth metabolism, the intracellular enzyme of propionibacterium freudenreichii system (follows the growth and breeding of microorganism cells in the fermenting process, have in the microorganism cells in the process that corresponding enzyme ties up to microorganism cells growth and Product Metabolism and play specific function) time be subjected to the feedback inhibition of propionic acid (mass concentration reaches 1.0%), make the pH of the cell culture fluid (fermented liquid) that has in the fermentor tank be reduced at 5.5 o'clock, the microorganism cells accretion rate that causes having in the fermented liquid of propionibacterium freudenreichii cell slows down, and opens the magnetic valve 7 and the liquor pump 8 of dynamic expanding formula adsorptive reactor.
Fermentor tank is to be 121 ℃ in temperature to sterilize 30 minutes down.
(3) with the pH in step (2) fermentor tank 1 be 5.5 fermented liquids that have a propionibacterium freudenreichii cell, 2 direct expanding bed chromatography columns 16 from the dynamic expanding formula adsorptive reactor of step (1) by the road, 17,18 bottom enters in the chromatography column, make chromatography column 16,17, separating medium in 18 expands, the height of the separating medium after the expansion and the aspect ratio of former separating medium are 2.5: 1, there is big space between the separating medium, have in the fermented liquid of propionibacterium freudenreichii cell and contain certain density propionic acid, when the fermented liquid that has microorganism cells by expanding bed chromatography column 16,17, when the space between the separating medium in 18 was passed, the target product propionic acid was with the negatively charged ion (OH of anionic form (propionate form) with the same nature on the good separating medium of regeneration
-) carrying out ion-exchange, propionate is adsorbed on the separating medium with anionic form, OH
-Is back to the fermentor tank 1 from expanding bed chromatography column top by replace, realizes the on-line adsorption propionic acid and do not cause the obstruction of chromatography column 16,17,18, make propionic acid concentration reduction in the fermentor tank 1 with fermented liquid.The fermented liquid that has a propionibacterium freudenreichii cell from magnetic valve 12,13,14,15 pump around circuits on expanding bed chromatography column 16,17,18 tops to fermentor tank 1, owing to contain OH in the backflow fermented liquid
-, when the pH of fermented liquid gos up to the suitableeest growing environment 6.5-7.0 of cell, close the magnetic valve 7 and the liquor pump 8 of dynamic expanding formula adsorptive reactor.The suffered inhibition of growing of the propionibacterium freudenreichii cell fermentation liquid of removing propionic acid is removed, and propionibacterium freudenreichii cellular-restoring growth vigor, cell concentration continue to rise.
When the glucose concn in the fermention medium in the fermentor tank 1 is lower than mass percentage concentration and is 2%, be that 60% glucose solution (feed supplement liquid) carries out feed supplement with mass concentration, feed rate is 0.1h
-1, the feed supplement mode is a flow feeding, making glucose concn in the fermention medium return to mass percent is 2-6%, thereby makes propionibacterium freudenreichii cell continuous growth under sufficient nutritional status, makes cell keep vigorous growth conditions.Drop to up to propionibacterium freudenreichii cell growth vigor and cell decline to occur or infect other microorganism cellss and (when decline appears in the growth of target microorganism cell, will infect corresponding phage, promptly infect other microorganisms) time stop fermentation, really realize the fermentation of semicontinuous microorganism cells.
(4) after primary sorption finishes, start corresponding solenoid valve 7,9,10,11,12,13,14 and 15 and liquor pump 8 respectively, open the valve of leacheate storage tank, the separating medium that is adsorbed with propionic acid that step (3) is obtained carries out drip washing with leacheate (deionized water), close the leacheate storage tank valve after drip washing is finished, leacheate enters waste tank 20.Open elutriant storage tank 3 valves then, (NaCl of 1mol/L) carries out wash-out with elutriant 3; Desorption liquid is collected desorption liquid in product storage tank 19 by the road by detector 21, concentrates desorption liquid behind the taking-up product and obtains propionic acid.Separating medium after drip washing, wash-out, regeneration, the balance can reuse.
When (5) the propionibacterium freudenreichii cell fermentation liquid in getting back to fermentor tank 1 ferments to cell growth 50-60 hour logarithm later stage (being that the continuous growth state no longer appears in the microorganism cells biomass), opens solenoid valve 7,10,11,23 and liquor pump 8, by the road 2,22, take out the part fermented liquid and be collected in a conversion tank 24, in conversion tank 24, add precursor 5,6-dimethylbenzimidazole (DMB) makes microorganism cells synthesizing deoxyadenosine cobalami (VB12), according to the volume of taking fermented liquid away, in fermentor tank 1, add and take the isopyknic substratum of fermented liquid away, carry out new seed cell inoculation once by 10% volume ratio, realize the continuity of fermenting process.
(6) product analysis method
The mensuration of VB12
To be made into mass concentration in 4000 rev/mins of centrifugal collecting cells be 2.5% bacteria suspension to the propionibacterium freudenreichii cell fermentation liquid of results from conversion tank 24,30 minutes smudge cellses of boiling water bath, discharge product VB12 in the propionibacterium freudenreichii cell, 10000 rev/mins of centrifugal collection supernatant liquors, supernatant liquor filters with the 0.2um cellulose filter membrane.In order to prevent its photodissociation, need lucifuge to carry out in the entire operation process.
Get and filter the test sample of good supernatant liquor as high performance liquid chromatography (HPLC), and chromatographic column: BeckmanC18 (5um, 4.6mm * 25cm); Chromatogram testing conditions: moving phase I is an acetonitrile, and moving phase II is sodium-acetate buffer (the pH value is 3.5): 25 ℃ of column temperatures; Detect wavelength 254nm; Flow velocity 1.0mLmin
-1Sample size 10ul.Elution requirement: 0-5 minute, mass concentration 15% acetonitrile constant speed wash-out; 5-15 minute, mass concentration 15-30% acetonitrile gradient wash-out.Measure VB12 output and adopt HPLC rapid analysis method [Zhang Yuming, food and fermentation industries, 2005,31 (1): 124-127].The results are shown in Table 1.
The mensuration of propionic acid
High performance liquid chromatography (HPLC): propionic acid is filtered test sample as HPLC through the 0.2um cellulose filter membrane, chromatographic column: BeckmanC18 (5um, 4.6mm * 25cm); Chromatographic condition: moving phase I is an acetonitrile, and moving phase II is 0.02molL
-1K
2HPO
4Damping fluid (it is 2.8 that strong phosphoric acid is regulated the pH value), V I: V II=8: 92; 30 ℃ of column temperatures; Detect wavelength: 215nm; Flow velocity: 1mLmin
-1Sample size: 10 μ L.The results are shown in Table 1.
In above-mentioned steps 1, the used bacterial classification propionibacterium freudenreichii (Propionibacterium freudenreichii) that ferments, CICC:10019 is available from Chinese industrial microbial strains preservation center.
Table 1 display application dynamic expanding formula adsorptive reactor system contrasts with the experimental result that tradition adds the fermentation system technical system of alkali control pH, as shown in Table 1, adopt dynamic expanding formula adsorptive reactor systems technology system to carry out the propionibacterium anaerobically fermenting and produce propionic acid/VB12, not only recyclable propionic acid product, microorganism cells biomass and VB12 output all are significantly improved simultaneously.
Table 1 is used dynamic expanding formula adsorptive reactor system and is carried out propionibacterium anaerobically fermenting production propionic acid/VB12 experimental result
Utilize dynamic expanding formula adsorptive reactor to produce the production technique that lactic acid adopted, and employed application dynamic expanding formula adsorptive reactor system is substantially the same manner as Example 1, different is:
(1) the microorganism cells seed is: plumule breast (acid) bacillus (Lactobacillus plantarum).
(2) the N source is changed to peptone by corn steep liquor in seed and the fermention medium, and the peptone mass concentration changes 2% in the seed culture medium, and the peptone mass concentration changes 2% in the fermention medium.
(3) fermenting process changes aerobic mode into.
Therefore (4) because present embodiment has only a kind of target product lactic acid, enter fermenting process, save microorganism cells during the fermentation and grow into the link that the logarithm later stage adds inductor.
Experimental result:
Table 2 is used dynamic expanding formula adsorptive reactor system and is carried out the experimental result that plumule breast (acid) bacillus aerobic fermentation is produced lactic acid
Embodiment 3. utilizes dynamic expanding formula adsorptive reactor system to produce Succinic Acid.
Utilize dynamic expanding formula adsorptive reactor to produce the production technique that Succinic Acid adopted, and employed application dynamic expanding formula adsorptive reactor system is substantially the same manner as Example 1, different is:
(1) the microorganism cells seed is: the Succinic Acid actinobacillus.
(2) the N source is changed to yeast powder by corn steep liquor in seed and the fermention medium, and the yeast powder mass concentration changes 2% in the seed culture medium, and the yeast powder mass concentration changes 2% in the fermention medium.
Therefore (3) because present embodiment has only a kind of target product Succinic Acid, enter fermenting process, save microorganism cells during the fermentation and grow into the link that the logarithm later stage adds inductor.
Experimental result:
Table 3 is used dynamic expanding formula adsorptive reactor system and is carried out the experimental result that Succinic Acid actinobacillus anaerobically fermenting is produced Succinic Acid
Claims (8)
1. use the method that dynamic expanding formula adsorptive reactor system produces biogenic organic acid for one kind, it is characterized in that this method may further comprise the steps:
(1) separating medium is loaded in the expanding bed chromatography column that the fermentor tank with dynamic expanding formula adsorptive reactor directly is coupled;
The structure of described separating medium is made up of skeleton and functional group two portions, and skeleton is a kind of in vinylformic acid, vinylbenzene, the Resins, epoxy, and functional group is selected from least a in the group that primary amine, secondary amine, tertiary amine, quaternary amine form;
(2) cultured microorganism cells seed is inoculated in the fermented liquid that obtains having microorganism cells in the fermention medium in the sterilized fermentor tank, adopts aerobic or the anaerobism mode is carried out the microorganism cells fermentation culture; Follow the growth and breeding of microorganism cells, the microorganism cells biomass is accumulated, the target product biogenic organic acid is constantly synthetic, when participating in the microorganism cells growth metabolism, the system of the enzyme in the microorganism cells is subjected to the feedback inhibition of target product biogenic organic acid, make the pH of the fermented liquid that has microorganism cells in the fermentor tank be reduced to the 5-6 scope by 6.5-7, and the microorganism cells accretion rate that causes having in the fermented liquid of microorganism cells slows down, and opens the magnetic valve and the liquor pump of dynamic expanding formula adsorptive reactor;
(3) be that the fermented liquid that has microorganism cells of 5-6 directly enters in the chromatography column bottom of the expanding bed chromatography column from the dynamic expanding formula adsorptive reactor of step (1) with pH in step (2) fermentor tank, separating medium in the chromatography column is expanded, the height of the separating medium after the expansion and the aspect ratio of former separating medium are 2: 1-3: 1, there is the space between the separating medium, have in the fermented liquid of microorganism cells and contain biogenic organic acid, when the fermented liquid that has microorganism cells is passed by the space between the separating medium in the expanding bed chromatography column, the separated medium of target product biogenic organic acid directly adsorbs, the fermented liquid that contains microorganism cells that passes by the space between the separating medium, be back to the fermentor tank from expanding bed chromatography column top, when the pH of the fermented liquid that has microorganism cells in the fermentor tank is back to 6.5-7, close the magnetic valve and the liquor pump of dynamic expanding formula adsorptive reactor;
When the C source concentration in the fermention medium in the fermentor tank is lower than mass percentage concentration and is 1-2%, by the concentration ratio in C source in C source feed supplement liquid and the original fermentative medium formula is that 100: 1 feed supplement strategy carries out the feed supplement of C source, and making C source concentration in the fermention medium return to mass percent is 2-6%;
(4) the separating medium water that is adsorbed with biogenic organic acid that step (3) is obtained carries out drip washing, carries out wash-out with elutriant then; Collection desorption liquid, concentrated desorption liquid obtain biogenic organic acid.
2. method according to claim 1 is characterized in that: described elutriant is sodium hydroxide, sodium-chlor or their mixture.
3. method according to claim 1 is characterized in that: described microorganism cells seed is selected from a kind of in propiono-bacterium, lactobacillus, Succinic Acid actinobacillus, recombination bacillus coli or the Succinic Acid anaerobism spirillum.
4. method according to claim 1 is characterized in that: the feed rate of described C source feed supplement liquid is 0.1-0.5h
-1The feed supplement mode is flow feeding or intermittent feed supplement.
5. according to claim 1 or 4 described methods, it is characterized in that: described C source is selected from a kind of in glucose, sucrose or the Semen Maydis powder.
6. method according to claim 1 is characterized in that: when described enzyme system in microorganism cells was subjected to the feedback inhibition of target product biogenic organic acid when participating in the microorganism cells growth metabolism, the mass concentration of target product biogenic organic acid was 1-10%.
7. according to claim 1 or 6 described methods, it is characterized in that: described biogenic organic acid is selected from a kind of in propionic acid, acetate, citric acid, lactic acid, fumaric acid, tartrate, Succinic Acid, the long-chain biatomic acid.
8. method according to claim 1 is characterized in that: described separating medium is after soaking with deionized water earlier, to use 1molL again
-1HCl solution soaking separating medium uses the deionized water wash separating medium to neutral to remove organism and the metallic impurity in the separating medium then; Use 1molL
-1Separating medium behind the NaOH washing by soaking is converted into the free alkali type with separating medium; Wash the separating medium that is converted into the free alkali type repeatedly to neutral with deionized water at last, wash-out goes out to be loaded on behind the unnecessary NaOH in the expanding bed chromatography column that the fermentor tank with dynamic expanding formula adsorptive reactor directly is coupled.
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| CN102391339A (en) * | 2011-09-14 | 2012-03-28 | 河北华荣制药有限公司 | Method for extracting cobamamide from aerobic fermentation liquor |
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| CN106906122A (en) * | 2017-05-09 | 2017-06-30 | 中国科学院过程工程研究所 | A kind of system and method for coproduction propionic acid and its salt and succinic acid and its salt |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102391339A (en) * | 2011-09-14 | 2012-03-28 | 河北华荣制药有限公司 | Method for extracting cobamamide from aerobic fermentation liquor |
| CN105755062A (en) * | 2016-03-31 | 2016-07-13 | 中国科学院过程工程研究所 | Method for producing long-chain dicarboxylic acid by regulating and controlling fermentation process through redox potential |
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| CN106906122A (en) * | 2017-05-09 | 2017-06-30 | 中国科学院过程工程研究所 | A kind of system and method for coproduction propionic acid and its salt and succinic acid and its salt |
| CN106916729A (en) * | 2017-05-09 | 2017-07-04 | 中国科学院过程工程研究所 | A kind of semicontinuous fermentation produces propionic acid with joint production vitamin B12Device and method |
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| CN106906122B (en) * | 2017-05-09 | 2023-11-21 | 中国科学院过程工程研究所 | System and method for co-producing propionic acid and salt thereof and succinic acid and salt thereof |
| CN112513246A (en) * | 2018-06-18 | 2021-03-16 | S&P 配料研发有限公司 | Isolation of microbial cell lines for the production of organic acids |
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