CN101215583B - Method for preparing succinic acid by coupling fermentation and film separation unit - Google Patents
Method for preparing succinic acid by coupling fermentation and film separation unit Download PDFInfo
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- CN101215583B CN101215583B CN2008100192877A CN200810019287A CN101215583B CN 101215583 B CN101215583 B CN 101215583B CN 2008100192877 A CN2008100192877 A CN 2008100192877A CN 200810019287 A CN200810019287 A CN 200810019287A CN 101215583 B CN101215583 B CN 101215583B
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Abstract
The invention discloses a process for preparing succinic acid through coupling fermentation and membrane separation units, the process for preparation comprises following steps: firstly, inoculating fermentation seeds of bacteria strains which produce the succinic acid into a fermentation tank to culture, then, coupling microfiltration or ultrafiltration membrane separation units with the fermentation tank to realize semi-continuously or continuously preparing the succinic acid with fermentation, separating out part of fermentation liquor, releasing from the control of metabolites, returning somatic cells and components which are cut off by the membrane separation units back into the fermentation tank and continuingly fermenting, continuingly using substrate which is not consumed in the fermentation liquor which is nanofiltrated, reclaimed, and separated to ferment, and simultaneously adding the fermentation culture medium to keep the volume of the fermentation liquor in the fermentation tank constant. The invention not only can release from the control of the metabolites and increase the production strength of the succinic acid, but also uses reducing sugar which is not consumed in the liquor which is nanofiltrated, reclaimed, and separated, simultaneously concentration is available, the technical condition is moderate, environmental pollution is little, which is easy to control automatically and has wide social and economic benefits.
Description
Technical field
The invention belongs to technical field of bioengineering, relate in particular to and utilize fermentation and film separation unit to be coupled to prepare the method for Succinic Acid.
Background technology
Succinic Acid (Succinic Acid, Butanedioic Acid) claims succsinic acid again, it is a kind of important binary organic carboxyl acid, can be used as the intermediate of synthetic complex organic compound, be widely used in pharmacy, food-processing, synthetic materials, tensio-active agent, protective coating, dyestuff and other industry.Yet chemosynthesis Succinic Acid traditionally makes Succinic Acid be restricted as the widespread use of basic chemical raw materials because of reasons such as cost and environmental pollutions.It is raw material with lower-cost reproducible biomass and greenhouse gases carbonic acid gas that fermentation method prepares Succinic Acid, pollutes and lacks, and energy consumption is low, is therefore just in depth carrying out in the U.S., Japan and other countries with the research and development of Production by Microorganism Fermentation Succinic Acid.The patent of utilizing the microbial fermentation preparation to extract Succinic Acid has: US5034105 (1991), US5168055 (1992), US5143834 (1992), US5958744 (1999) etc., its patent protection content relates generally to fermentation bacterial classification, zymotechnique and extraction process etc.
Utilize at present microbial fermentation to prepare the traditional batch fermentation technology of the many employings of Succinic Acid both at home and abroad, during the fermentation, a large amount of accumulation along with by products such as product Succinic Acid and formic acid, acetate, seriously suppressed Succinic Acid produce bacterium growth and and then influence the resultant velocity of Succinic Acid, cause its fermentative production level low, therefore in the Succinic Acid fermenting process, often need to add a large amount of calcium magnesium salts and neutralize a large amount of organic acids that produce to guarantee normally carrying out of fermentation.In above-mentioned disclosed patent,, adopt these technologies to have following problem through putting into practice and discovering:
Calcium magnesium salts neutralization has brought a large amount of impurity for the Succinic Acid fermentation system, has increased the difficulty of later stage separation and Extraction, as product loss in the calcium sulfate filtration procedure up to more than 30%; High ionic strength causes ion exchange resin seriously polluted, and working capacity descends rapidly, the resin regeneration difficulty; The calcium ions and magnesium ions of high density also can cause the solvent extraction inefficiency or pollute electrodialytic membranes causing energy consumption to increase sharply.
Adopt calcium magnesium salts neutral batch fermentation to prepare Succinic Acid in a word, the separating technology step is many, and yield is low, soda acid/solvent-oil ratio is big, and production equipment is easily contaminated, the liquid and waste slag produced serious environment pollution of generation, the running cost height has finally limited the market competitiveness that fermentation method prepares Succinic Acid.Therefore how removing the meta-bolites inhibition in the Succinic Acid fermenting process, the production intensity that improves Succinic Acid and yield is that biological process prepares the key that Succinic Acid is realized industrialization.
At present film separation unit and bio-reactor are coupled, can realize preferably that somatic cells received much attention with separating of meta-bolites, successfully report is all arranged in the research of alcohol prepared by fermenting, acetone-butanols, lactic acid, propionic acid and cell high-density culture.But do not see the patent report that film separation unit and fermentation coupling is carried out continuous or semicontinuous preparation Succinic Acid at present.
But the reaction-separation coupled fermentation based on membrane separation technique prepares in the organic acid research at present, the micro-filtrations that adopt based on the screening principle more, ultra-filtration membrane is as single separation assembly, this type of design can only realize separating of cell and fermented liquid, be difficult to realize separating of product and substrate, cause the loss that does not consume substrate and other nutritive elements in the fermented liquid inevitably, in order to address this problem, forefathers are at micro-filtration, coupled ion exchange in the ultra-filtration membrane circulating biological reactor, chemical extraction, means such as electrodialysis reclaim the substrate that does not consume, but these means maybe need consume a large amount of acid-alkali regeneration resins, or easily cause dissolvent residual unfavorable to fermenting, maybe need consume a large amount of electric energy.And nanofiltration membrane (molecular weight cut-off is 100-1000 dalton) between reverse osmosis and ultrafiltration, its separating mechanism is relevant with the Coulomb repulsion of molecular weight screening and membrane sepn laminar surface, biological micromolecule and high valence ion to molecular weight≤1000 have separating effect preferably, separate to concentrate at microbiotic, the aspect is existing uses for amino acid separation and purification etc., does not see that efficient membrane sepn means such as using nanofiltration reclaim the patent report of substrate at present from the Succinic Acid fermented liquid.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of utilizing the fermentation and the coupling of film separation unit to prepare Succinic Acid, suppress with the meta-bolites of removing in the Succinic Acid fermenting process, improve Succinic Acid fermentative production level, recovery and the utilization that does not consume substrate in realization response-separation coupling process simultaneously, the utilization ratio of raising substrate.
The present invention is for solving the problems of the technologies described above, and the technical scheme that is adopted is as follows:
A kind of fermentation is coupled with film separation unit and prepares the method for Succinic Acid, comprises the steps:
1, the ferment-seeded of preparation succinic acid-producing bacterial strain;
2, preparation fermention medium;
3, fermention medium is packed into fermentor tank inserts fermentor tank with ferment-seeded again;
4, early stage, anaerobically fermenting was produced Succinic Acid;
5, make the part fermentation broth stream go out fermentor tank, stream adds fermention medium to keep in the fermentor tank fermentating liquid volume constant simultaneously;
6, the fermented liquid in the fermentor tank continues fermentation production of succinic acid; The fermented liquid that flows out fermentor tank simultaneously is earlier through micro-filtration or ultra filtration unit, pass through nano-filtration unit again, returned in the fermentor tank by somatic cells and component that micro-filtration or ultra filtration unit are held back, the substrate that does not consume in the fermented liquid of being held back by nano-filtration unit also returns fermentor tank, collects the nanofiltration membrane penetrating fluid.
Wherein, the succinic acid-producing bacterial strain described in the step 1 is for producing succsinic acid actinobacillus Actinobacillussuccinogenes.
Wherein, the method for preparing ferment-seeded described in the step 1 is made up of following steps:
The preparation of a, seed culture medium: seed culture medium is the conventional liq substratum that carbon source, nitrogenous source and inorganic salt can be provided that contains carbohydrate of pH 6.0～7.0; During slant culture, add agar in the seed culture medium again.Wherein carbohydrate is one or more in the carbon sources such as glucose, pectinose, wood sugar, seminose; Nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or more in ammonium acetate, the ammonium chloride, and nitrogen-containing organic compound is one or more in peptone, yeast extract paste, corn steep liquor, the extractum carnis; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, manganese salt, the molysite.
B. slant culture: the succinic acid-producing bacterial strain carried out plate streaking in slant medium after, cultivate in the anaerobism incubator, incubator feeds and contains N
2, CO
2And H
2Mixed gas, N
2, CO
2And H
2Volume ratio be 8: 1: 1, temperature is 30～40 ℃, cultivates 24～48h, is used for seed culture medium inoculation and bacterial strain preservation.
C. ferment-seeded is cultivated: the consumption of seed culture medium is 20～80mL in every 100mL serum bottle, feeds to contain N
2, CO
2Mixed gas, N
2, CO
2Volume ratio be 4: 1,115～121 ℃ of sterilization 15～30min, the succinic acid-producing bacterial classification is inserted in cooling back, culture temperature is 30～40 ℃, rotating speed 100～200r/min, fermentation time is 10～14h, as ferment-seeded.
Wherein, the described fermention medium of step 2 is the conventional liq substratum that carbon source, nitrogenous source and inorganic salt can be provided that contains carbohydrate of pH 6.0～7.0, and adds VITAMIN.Wherein carbohydrate is one or more in the carbon sources such as glucose, pectinose, wood sugar, seminose, biomass (as maize peel, corn cob, straw) hydrolysis sugar liquid; Nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or more in ammonium acetate, the ammonium chloride, and nitrogen-containing organic compound is one or more in peptone, yeast extract paste, corn steep liquor, the extractum carnis; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, manganese salt, the molysite; VITAMIN is folic acid, vitamin H, vitamins B
12, Thioctic Acid, nicotinic acid, pantothenic acid, VitB1 or vitamins B
6In one or more.
Wherein, in the step 3, every 5L fermentation cylinder for fermentation culture volume is 2～4L, and the inoculation volume of Succinic Acid ferment-seeded accounts for 3～7% of culture volume.
Wherein, the method that the described anaerobically fermenting of step 4 is produced Succinic Acid is: 35～40 ℃ of temperature, fermentor tank feeds CO
2Gas, air flow are 0.75～1.5L/min, mixing speed 100～300rpm, and fermenting process adopts yellow soda ash, ammoniacal liquor, sodium hydroxide, sulfuric acid or hydrochloric acid, and the pH of the hierarchy of control carries out anaerobically fermenting 10～15h in early stage 6.0～7.5.
Wherein, step 5 can adopt semicontinuous mode (claiming semicontinuous fermentation again), promptly every 4～6h, 40～67% fermented liquids is isolated fermentor tank, and fermentating liquid volume is constant in the fermentor tank of afterfermentation substratum in fermentor tank, and maintenance simultaneously.Also can adopt continuous mode (claiming again to continuously ferment), promptly with 0.1～0.8h
-1Dilution rate (dilution rate is meant that stream in the unit time adds to the ratio of liquor capacity and initial fermentating liquid volume in the fermentor tank among the present invention) stream add fermention medium, discharge fermented liquid simultaneously, constant to keep in the fermentor tank fermentating liquid volume.
Wherein, in step 6 micro-filtration or the ultra filtration unit, employed microfiltration membrane aperture is 0.1～0.2 μ m, and the molecular weight cut-off of ultra-filtration membrane is 8000～100000 dalton, its membrane module form is tubular fibre formula or tubular membrane, and its membrane material is organic membrane or inorganic ceramic film; For organic membrane, can adopt 1～4% sodium hydroxide solution to carry out chemical disinfection before using; To inorganic ceramic film, can adopt 110～120 ℃ the damp and hot sterilization of steam before using or carry out chemical disinfection with 1～4% sodium hydroxide solution.In the nano-filtration unit, the molecular weight cut-off of nanofiltration membrane is 100～200 dalton, adopts the rolling nanofiltration membrane component, needs to disinfect through 1～4% sodium hydroxide solution before using.
Wherein, in the step 6, fermented liquid is after flowing out micro-filtration or ultra filtration unit, earlier with sulfuric acid or salt acid for adjusting pH value to 3.0～4.5; Enter nano-filtration unit then, working pressure is 7～20bar, and temperature is 10～30 ℃; At this moment, nanofiltration membrane can be held back glucose, wood sugar, pectinose, divalent metal salt and organic nitrogen source, and hold back partial pigment in the feed liquid (reducing the activated carbon dosage that colourity can reduce downstream process), and inhibition meta-bolitess such as Succinic Acid, formic acid and acetate can see through nanofiltration membrane; It is 30～60g/L that sugar in fermented liquid is concentrated into sugared concentration by nanofiltration, and formic acid, acetic acid concentration all is lower than 2g/L, when Succinic Acid concentration is lower than 5g/L, stops nanofiltration; The nanofiltration concentrated solution of holding back is regulated pH value to 6.0～6.5 with sodium hydroxide, after molecular weight cut-off is 10000 daltonian ultrafiltration membrance filters, directly be back in the fermentor tank again.
The nanofiltration penetrating fluid of collecting can be proceeded the downstream separation operation of Succinic Acid.
The method flow diagram of fermentation of the present invention and film separation unit coupling preparation Succinic Acid is seen Fig. 1.
Beneficial effect: the present invention adopts film separation unit and fermenting process coupling to realize the semicontinuous or preparation Succinic Acid that continuously ferments, not only can remove in the fermenting process meta-bolites to the inhibition of thalli growth, significantly improve the production intensity of Succinic Acid, and do not need to add in process of production any calcium, magnesium salts neutralization; Utilize nanofiltration to reclaim when not consuming reducing sugar in the parting liquid, realized concentrating and substrate and product separation of substrate, do not need to use any organic solvent and ion exchange resin.Method provided by the invention is when realizing the Succinic Acid high production intensity, with cell separate with product, substrate reclaims with technology and fermenting process close-coupled such as concentrate, the complex steps of prior art existence, the defective that the soda acid consumption is big, pollution is heavy, energy consumption is high and productive labor intensity is big have been overcome, the production process mild condition, be easy to automatization control and industrialization, have significant social and economic benefit.
Description of drawings
Fig. 1 is the method flow diagram of fermentation with film separation unit coupling preparation Succinic Acid.
Embodiment
The following examples elaborate to the present invention, but to the present invention without limits.
Embodiment 1:
(1) seed culture medium: glucose 10g/L, yeast extract paste 5g/L, peptone 5g/L, NaCl 1g/L, NaHCO
310g/L, Na
2HPO
49～15g/L, KH
2PO
44～12g/L, feeds N at (adding agar 20g/L during slant culture)
2: CO
2=4: 1 gas mixture 2min, 121 ℃ of sterilization 20min, cooling;
(2) fermention medium: glucose 40g/L, yeast extract paste 10～50g/L, peptone 10～50g/L, MnCl
20.05g/L, FeCl
20.05g/L, CaCl
20.05g/L, K
2HPO
42.0g/L VITAMIN is added folic acid 5mg/L, vitamin H 5mg/L, vitamins B
125mg/L, Thioctic Acid 5mg/L, nicotinic acid 5mg/L, pantothenic acid 5mg/L, VitB1 5mg/L, vitamins B
65mg/L, pH 7.0;
(3) be the product succsinic acid actinobacillus bacterial strain of CGMCC No.1716 carries out plate streaking in the seed slant medium after, to cultivate in the anaerobism incubator deposit number, the gas mixture ratio is in the control incubator: N
2: CO
2: H
2=8: 1: 1, the bacterial strain behind the cultivation 36h encircled in the 100ml serum bottle that contains 50ml seed liquid nutrient medium with inoculation articulating one, rotating speed 150r/min, and 37 ℃ of cultivations are used for above-mentioned fermention medium inoculation after 12 hours.The substratum liquid amount is 3L when adopting the 5L fermentor cultivation, and inoculum size is 5% (v/v), and mixing speed is at 200rpm, and 37 ℃, pH is 7.0 with the regulation and control of 250g/L yellow soda ash, and the air flow of fermentor tank carbonic acid gas is 0.75L/min.Behind the fermentation 12h, the Succinic Acid production intensity has reached 2.0g/Lh, and thalli growth enters logarithmic phase latter stage, and the total concn of Succinic Acid that accumulates in the system and formic acid, acetate has reached 30g/L, glucose concn is 10g/L in the fermented liquid, prepares the coupling microfiltration membrane this moment and carries out semicontinuous fermentation;
(4) adopt the organic microfiltration membrane of tubular fibre formula of 0.1 μ m, soak 6h with 1% sodium hydroxide solution before using and carry out disinfection, the back is washed to water outlet pH8.5 with sterilized water and is sealed standby.Microfiltration membrane is connected with fermentor tank, under the pressure of 0.5ba, carries out micro-filtration, leach fermented liquid, cell and hold back part and return a jar relaying supervention ferment, filtrate stops to separate during to 2.0L (be equivalent to jar interior original volume 67%), and stream adds the fermention medium that the 1.5L glucose concn is 60g/L; The filtrate of collecting is adjusted to pH4.0 with concentrated hydrochloric acid, selecting molecular weight cut-off for use is that 100 daltonian rolling nanofiltration membrane are carried out nanofiltration separation, and working pressure is 15bar, and feed temperature is controlled at 30 ℃, the glucose rejection reaches 90% in the nanofiltration separation process, the rejection of Succinic Acid, formic acid, acetate all＜8% is collected concentrated solution 0.5L, and glucose concn reaches 40g/L, transfer to pH6.0 with sodium hydroxide, stream adds in the fermentor tank behind ultrafiltration membrance filter, keeps jar internal volume constant, continues fermentation.
After this separate and the feed supplement operation every 5h, be divided into from feed supplement 6 times, continue 30h, the Succinic Acid production intensity reaches 3.0g/Lh.The Succinic Acid production intensity is than identical glucose concn and add 40g/L magnesiumcarbonate and carry out the neutral batch fermentation and increased by 50%, than improving 84% without calcium magnesium salts neutral batch fermentation.
Embodiment 2:
(1) seed culture medium, fermention medium are all identical with embodiment 1.
(2) be that the product succsinic acid actinobacillus bacterial strain of CGMCC No.1716 prepares ferment-seeded with deposit number, method is with embodiment 1.The substratum liquid amount is 3L when adopting the 5L fermentor cultivation, and inoculum size is 4% (v/v), and mixing speed is at 300rpm, 37 ℃, to regulate and control to pH 6.8 with 280g/L yellow soda ash, and the air flow of fermentor tank carbonic acid gas is 1.0L/min.Behind the fermentation 11h, the Succinic Acid production intensity has reached 2.2g/Lh, and thalli growth logarithmic phase latter stage, the total concn of Succinic Acid that accumulates in the system and formic acid, acetate has reached 29g/L, glucose concn is 11g/L in the fermented liquid at this moment, prepares the coupling ultra-filtration membrane this moment and carries out semicontinuous fermentation;
(3) adopting molecular weight cut-off is 100000 daltonian tubular type ceramic super-filtering films, at 121 ℃ of sterilization 20min, standby before using.Ultra-filtration membrane is connected with fermentor tank, under the pressure of 2ba, carries out ultrafiltration, leach fermented liquid, cell and hold back part and return a jar relaying supervention ferment, filtrate is during to 1.5L (be equivalent to jar interior original volume 50%), stops to separate, and stream adds the fermention medium that the 1L glucose concn is 70g/L.
The 1.5L filtrate that ultra-filtration and separation is obtained is adjusted to pH4.0 with concentrated hydrochloric acid, selecting molecular weight cut-off for use is that 150 daltonian rolling nanofiltration membrane are carried out nanofiltration separation, working pressure is 10bar, feed temperature is controlled at 15 ℃, filtrate is concentrated into 0.5L, and the glucose rejection reaches 88% in the nanofiltration separation process, and the rejection of Succinic Acid, formic acid, acetate all＜5%, glucose reaches 31g/L in the concentrated solution, formic acid 1g/L, acetate 0.5g/L, Succinic Acid 2g/L.Nanofiltration simultaneously reaches 80% to the percent of decolourization that sees through liquid.
Carry out identical lock out operation every 6h, at fed-batch medium simultaneously, the glucose concentrated solution that nanofiltration is reclaimed transfers to pH6.0 with sodium hydroxide, directly returns after uf processing in the fermentor tank, keeps jar internal volume constant.Whole semicontinuous fermentation process continues 50h, separates feed supplement 5 times, is total to consumption of glucose 470g in the fermentation, succinic acid-producing 325g, and the Succinic Acid production intensity is 2.17g/Lh.Under the condition of initial glucose 40g/L, Succinic Acid mass yield 0.69g/g glucose, the Succinic Acid production intensity is than identical glucose concn and add 40g/L magnesiumcarbonate and carry out the neutral batch fermentation and increased by 8.5%, than improving 33% without the U.S. salt neutral of calcium batch fermentation.
Embodiment 3:
With deposit number is that the bacterial strain of CGMCC No.1716 adopts and to prepare the fermentor tank seed with embodiment 1 identical method, the substratum liquid amount is 3L during the 5L fermentor cultivation, glucose concn is 25g/L in the fermention medium, all the other components are with embodiment 1, adopt 10N sodium hydroxide to regulate pH 6.80, feed CO
2Air flow is 0.75L/min, the anaerobic environment that keeps fermentation system, inoculum size is 5% (v/v), rotating speed 250r/min, cultivate 37 ℃ of fermentation culture 10.5h after, the Succinic Acid production intensity has reached 1.7g/Lh, the total concn of Succinic Acid that accumulates in the system and formic acid, acetate has reached 23g/L, and glucose concn is 2g/L only, prepares the coupling microfiltration membrane and continuously ferments.
The tubular type ceramic microfiltration membrane of the 0.14 μ m that employing is disinfected through 4% sodium hydroxide, microfiltration membrane is connected with fermentor tank, separate fermentation liquid, cell and hold back the part return in the jar, fed-batch medium (glucose concn is 30g/L) guarantees that jar internal volume is constant simultaneously, and dilution rate is respectively 0.1～0.8h
-1, stream adds glucose 600g respectively, the results are shown in Table 1.
The different dilution rates of table 1 are to the influence of Succinic Acid production intensity
| Dilution rate h -1 | 0.1 | 0.2 | 0.5 | 0.6 | 0.8 |
| Succinic Acid production intensity (g/Lh) | 3.4 | 4.2 | 8.5 | 10.7 | 13.0 |
| Succinic Acid mass yield (g/g glucose) | 0.82 | 0.78 | 0.67 | 0.62 | 0.55 |
Compare with semicontinuous fermentation, the Succinic Acid production intensity reaches as high as 13.0g/Lh in the process of continuously fermenting.At 0.1h
-1Dilution rate under, the Succinic Acid mass yield can reach 0.82g/g glucose.
Embodiment 4:
Fermented liquid (the glucose 9g/L that ultra-filtration and separation in the semicontinuous fermentation process is obtained, Succinic Acid 20～24g/L, formic acid 4～5g/L, acetate 6～7g/L) is adjusted to pH4.0 as the nanofiltration separation raw material with hydrochloric acid, selecting molecular weight cut-off for use is that 150 daltonian nanofiltration membrane are separated under the working pressure of 7～20bar respectively, separated volume is 1.5L, and the nanofiltration membrane penetrating fluid is collected in 18 ℃ of service temperature controls in the nanofiltration process, trapped fluid returns in the feeding liquid, the results are shown in Table 2.
Table 2 different pressures reclaims the influence of glucose to nanofiltration separation
| Pressure (ba) | Glucose rejection (%) | Succinic Acid rejection (%) | Formic acid rejection (%) | Acetate rejection (%) | Membrane permeation flux (L/m 2h) | Percent of decolourization (%) |
| 7 | 83.3 | 3.5 | 4.3 | 0.9 | 20.8 | 76 |
| 10 | 87.2 | 4.2 | 4.4 | 1.6 | 36.5 | 81 |
| 12 | 89.5 | 4.8 | 4.8 | 1.8 | 38.2 | 83 |
| 15 | 91.6 | 4.6 | 5.0 | 2.1 | 42.3 | 86 |
| 18 | 92.2 | 4.1 | 4.7 | 2.4 | 39.6 | 88 |
| 20 | 90.3 | 3.7 | 4.7 | 2.6 | 37.0 | 89 |
When as seen working pressure was 15～20ba, nanofiltration membrane can reach 90% to the rejection of glucose.Formic acid in the trapped fluid, acetic acid concentration all≤2g/L, Succinic Acid concentration≤5g/L, percent of decolourization is all 〉=85%.
Embodiment 5:
With deposit number is that the bacterial strain of CGMCC No.1716 adopts and to prepare the fermentor tank seed with embodiment 1 identical method, the substratum liquid amount is 3L during the 5L fermentor cultivation, total reducing sugars concentration is that 50g/L is (being carbon source with the cornhusk hydrolysate in the fermention medium, wherein glucose 20g/L, wood sugar 18g/L, pectinose 12g/L), all the other components are with embodiment 1, adopt 2M yellow soda ash to regulate pH 6.70, feed CO
2Air flow is 0.75L/min, keeps the anaerobic environment of fermentation system, and inoculum size is 5% (v/v), rotating speed 250r/min, after cultivating 37 ℃ of fermentation culture 15h, bacterial strain succinic acid-producing speed has been stabilized in 2.5g/Lh, and the total concn of Succinic Acid that accumulates in the system and formic acid, acetate has reached 30g/L, this moment, residual reducing sugar total concn was 12g/L (glucose 2.5g/L, wood sugar 3g/L, pectinose 6.5g/L), prepare the coupling microfiltration membrane and continuously ferment.
The hollow fiber microfiltration membrane of the 0.1 μ m that employing is disinfected through 4% sodium hydroxide, microfiltration membrane is connected with fermentor tank, beginning separate fermentation liquid, cell and hold back the part return in the jar, separate the fermented liquid that obtains in the process of will continuously fermenting and be adjusted to pH3.0～4.5 with hydrochloric acid respectively, selecting molecular weight cut-off for use is that 100 daltonian nanofiltration membrane are separated nanofiltration separation respectively under the working pressure of 15bar, 20 ℃ of service temperature controls, concentrate 3 times, the nanofiltration separation effect sees Table 3.
As seen pH≤4.0 o'clock, nanofiltration membrane can reach about 90% the rejection of reducing sugar.Inhibition meta-bolites concentration such as formic acid, acetate, Succinic Acid all≤5%, percent of decolourization is all 〉=85%.Total reducing sugars concentration is about 50g/L in the concentrated solution, transfers to pH6.0.
The dilution rate of fed-batch medium in fermentor tank (with the cornhusk hydrolysate is carbon source, and total reducing sugars concentration is 60g/L, wherein glucose 25g/L, wood sugar 22g/L, pectinose 13g/L) is 0.1h
-1, the reducing sugar concentrated solution that nanofiltration is reclaimed directly returns after uf processing in the fermentor tank simultaneously, and the control dilution rate is 0.1h
-1, keep the fermentor tank internal volume constant.The whole process of continuously fermenting continues 40h, and the Succinic Acid production intensity is 2.55g/Lh, compares raising 59% with adding calcium magnesium salts neutral batch fermentation, and the Succinic Acid mass yield reaches the 0.71g/g reducing sugar, has improved 9.2%.
The different pH of table 3 reclaim the influence of glucose to nanofiltration separation
| pH | Total reducing sugar rejection (%) | Succinic Acid rejection (%) | Formic acid rejection (%) | Acetate rejection (%) | Membrane permeation flux (L/m 2h) | Percent of decolourization (%) |
| 3.0 | 93.5 | 2.5 | 2.3 | 0.5 | 30.3 | 88 |
| 3.5 | 93.2 | 3.7 | 4.4 | 1.0 | 26.7 | 85 |
| 4.0 | 91.6 | 4.4 | 4.8 | 3.8 | 15.8 | 86 |
| 4.5 | 83.9 | 9.1 | 6.3 | 4.4 | 10.2 | 83 |
Claims (6)
1. the coupling of fermentation and film separation unit prepares the method for Succinic Acid, it is characterized in that this method comprises the steps:
(1) ferment-seeded of preparation succinic acid-producing bacterial strain;
(2) preparation fermention medium;
(3) fermention medium is packed into fermentor tank inserts fermentor tank with ferment-seeded again;
(4) early stage, anaerobically fermenting was produced Succinic Acid;
(5) make the part fermentation broth stream go out fermentor tank, stream adds fermention medium to keep in the fermentor tank fermentating liquid volume constant simultaneously;
(6) fermented liquid in the fermentor tank continues fermentation production of succinic acid; The fermented liquid that flows out fermentor tank simultaneously is earlier through micro-filtration or ultra filtration unit, pass through nano-filtration unit again, returned in the fermentor tank by somatic cells and component that micro-filtration or ultra filtration unit are held back, the substrate that does not consume in the fermented liquid of being held back by nano-filtration unit also returns fermentor tank, collects the nanofiltration membrane penetrating fluid;
Wherein, step (5) adopts semicontinuous mode or continuous mode; When step (5) adopts semicontinuous mode, every 4～6h, 40～67% fermented liquid is isolated fermentor tank, afterfermentation substratum in fermentor tank keeps the interior fermentating liquid volume of fermentor tank constant simultaneously; When step (5) adopts continuous mode, with 0.1～0.8h
-1Dilution rate stream add fermention medium, discharge fermented liquid simultaneously, keep in the fermentor tank fermentating liquid volume constant;
Wherein, in step (6) micro-filtration or the ultra filtration unit, employed microfiltration membrane aperture is 0.1～0.2 μ m, and the molecular weight cut-off of ultra-filtration membrane is 8000～100000 dalton, its membrane module form is tubular fibre formula or tubular membrane, and its membrane material is organic membrane or inorganic ceramic film; In the nano-filtration unit, the molecular weight cut-off of nanofiltration membrane is 100～200 dalton, adopts the rolling nanofiltration membrane component;
Wherein, in the step (6), fermented liquid is after flowing out micro-filtration or ultra filtration unit, earlier with sulfuric acid or salt acid for adjusting pH value to 3.0～4.5; Enter nano-filtration unit then, working pressure is 7～20bar, and temperature is 10～30 ℃; It is 30～60g/L that sugar in fermented liquid is concentrated into sugared concentration by nanofiltration, and formic acid, acetic acid concentration all is lower than 2g/L, when Succinic Acid concentration is lower than 5g/L, stops nanofiltration; The nanofiltration concentrated solution of holding back is regulated pH value to 6.0～6.5 with sodium hydroxide, after molecular weight cut-off is 10000 daltonian ultrafiltration membrance filters, directly be back in the fermentor tank again.
2. fermentation according to claim 1 and film separation unit coupling prepare the method for Succinic Acid, it is characterized in that the succinic acid-producing bacterial strain described in the step (1) is for producing succsinic acid actinobacillus (Actinobacillus succinogenes).
3. the coupling of fermentation according to claim 1 and film separation unit prepares the method for Succinic Acid, it is characterized in that the method for preparing ferment-seeded described in the step (1) is made up of following steps:
The preparation of a, seed culture medium: seed culture medium is the conventional liq substratum that carbon source, nitrogenous source and inorganic salt can be provided that contains carbohydrate of pH 6.0～7.0; During slant culture, add agar in the seed culture medium again; Wherein carbohydrate is one or more in glucose, pectinose, wood sugar or the seminose; Nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or more in ammonium acetate or the ammonium chloride, and nitrogen-containing organic compound is one or more in peptone, yeast extract paste, corn steep liquor or the extractum carnis; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, manganese salt or the molysite;
B. slant culture: the succinic acid-producing bacterial strain carried out plate streaking in slant medium after, cultivate in the anaerobism incubator, incubator feeds and contains N
2, CO
2And H
2Mixed gas, N
2, CO
2And H
2Volume ratio be 8: 1: 1, temperature is 30～40 ℃, cultivates 24～48h, is used for seed culture medium inoculation and bacterial strain preservation;
C. ferment-seeded is cultivated: the consumption of seed culture medium is 20～80mL in every 100mL serum bottle, feeds to contain N
2, CO
2Mixed gas, N
2, CO
2Volume ratio be 4: 1,115～121 ℃ of sterilization 15～30min, the succinic acid-producing bacterial classification is inserted in cooling back, culture temperature is 30～40 ℃, rotating speed 100～200r/min, fermentation time is 10～14h, as ferment-seeded.
4. fermentation according to claim 1 and film separation unit coupling prepare the method for Succinic Acid, it is characterized in that the conventional liq substratum that carbon source, nitrogenous source and inorganic salt can be provided that contain carbohydrate of the described fermention medium of step (2), and add VITAMIN for pH 6.0～7.0; Wherein carbohydrate is one or more in glucose, pectinose, wood sugar, seminose or the biomass by hydrolyzation liquid glucose; Nitrogenous source is the organic or inorganic nitrogenous compound, and wherein inorganic nitrogen-containing compound is one or more in ammonium acetate or the ammonium chloride, and nitrogen-containing organic compound is one or more in peptone, yeast extract paste, corn steep liquor or the extractum carnis; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, phosphoric acid salt, manganese salt or the molysite; VITAMIN is folic acid, vitamin H, vitamins B
12, Thioctic Acid, nicotinic acid, pantothenic acid, VitB1 or vitamins B
6In one or more.
5. fermentation according to claim 1 and film separation unit coupling prepare the method for Succinic Acid, it is characterized in that in the step (3) that every 5L fermentation cylinder for fermentation culture volume is 2～4L, and the inoculation volume of Succinic Acid ferment-seeded accounts for 3～7% of culture volume.
6. fermentation according to claim 1 and film separation unit coupling prepare the method for Succinic Acid, and it is characterized in that the method for the described anaerobically fermenting production of step (4) Succinic Acid is: 35～40 ℃ of temperature, fermentor tank feeds CO
2Gas, air flow are 0.75～1.5L/min, mixing speed 100～300rpm, and fermenting process adopts yellow soda ash, ammoniacal liquor, sodium hydroxide, sulfuric acid or hydrochloric acid, and the pH of the hierarchy of control carries out anaerobically fermenting 10～15h in early stage 6.0～7.5.
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