CN104789606A - Method for fermentation production of succinic acid by use of xylose mother liquid - Google Patents
Method for fermentation production of succinic acid by use of xylose mother liquid Download PDFInfo
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Abstract
The invention discloses a method for fermentation production of a succinic acid by use of a xylose mother liquid. The xylose mother liquid is subjected to inhibitor removal treatment, and the method mainly comprises three methods, namely a lime method, an evaporation method and an ion-exchange resin method. The method comprises the following steps: by virtue of escherichia coli WH121 built in a laboratory, under the anaerobic condition containing CO2 or N2, adding water to the xylose mother liquid which is subjected to inhibitor removal treatment, adjusting to 20-100g/L, and carrying out fed-batch fermentation to produce the succinic acid, wherein the concentration of the succinic acid in the obtained fermented product can reach 30-100g/L; the yield is 0.6-1.2g of succinic acid for each gram of total sugar; and the productivity is 0.6-3.5g/L-h. The method has the outstanding advantages that expensive glucose is replaced with a xylose mother liquid material as a carbon source for fermentation production of the succinic acid; the method is friendly to environment; a lot of grains are saved; the grain safety in China is ensured; meanwhile, the amount of a nitrogen source in fermentation can be reduced; the production cost of the succinic acid is reduced; on the other hand, the sugar price can be adjusted to ensure the rights and interests of farmers; by virtue of renewable biological resources, the problem of petrochemical resource shortage for chemical synthesis of the succinic acid can be relieved; and sustainable development is facilitated.
Description
One, technical field
The invention belongs to technical field of microbial fermentation, being specifically related to a kind of method utilizing xylose mother liquid fermentation production of succinic acid, relating to recombination bacillus coli WH121 prepares the method for succinic acid application when taking xylose mother liquid as carbon source (comprising directly as carbon source and limestone-based process (Overliming) pre-treatment, method of evaporation, ion exchange method) simultaneously.
Two, background technology
Succinic acid (succinic acid), is also called succsinic acid, is extensively present in the tissue of various plants and humans and animals, is one of mesostate of tricarboxylic acid cycle (TCA).Because being isolated name the earliest from amber.The molecular formula of succinic acid is C
4h
6o
4, molecular weight is 118.09, its structural formula as:
Succinic acid is a kind of di-carboxylic acid, and containing two carboxyls, two carboxylic acids in succinic acid can dewater generation succinyl oxide, and can be dissociated into amber acid radical negatively charged ion and proton in water, the pH value of the 0.1mol/L aqueous solution is 2.7.Succsinic acid chemical property is enlivened, and can participate in a lot of chemical reaction, be mainly reduction reaction, oxidizing reaction, esterification, halogenating reaction etc.
Succinic acid is ataractic moiety.In pharmaceutical industries, hemostatic agent, toxinicide and diuretic(s) can be prepared with succinic acid, anticorrosion, to regulate pH and hydrotropy effect can be played.In addition, one of mesostate that succinic acid circulates as tricarboxylic acid (TCA), has the status of forming a connecting link, is therefore widely used as pharmaceutical intermediate in the substance metabolism and energy metabolism of organism.In agricultural, succinic acid, through processing the percentage of germination that can be used for improving seed, promotes crop growth, also can be used as the additive except herbal medicine and the large smut of prevention and therapy; Succinic acid can be used as monogastric animal such as the fodder additives of chicken, pig etc. and provides energy for it; Cellulosic succsinic acid acidylate can improve the receptivity of plant to water, can make thickening material etc. after starch succsinic acid acidylate.
Along with the application of succinic acid is more and more wider, also get more and more to the research of the various production method of succinic acid, just current research, succinic acid mainly can be obtained by chemical synthesis, biotransformation method, this three productions method of microbe fermentation method.The market value of current succinic acid is about 2 ~ 30,000 tons every year, industrial primarily of toxilic acid or the synthesis of maleic anhydride hydrogenation catalyst.Although the market of succinic acid is little at present, if succinic acid can be competed mutually with the maleic anhydride deriving from oil in price, then it can substitute maleic anhydride as platform chemicals, has great market demand.Bioenergy ethanol, butanols or large platform chemicals etc. are produced as a kind of reproducible by fermentable sugar source or ligno-cellulose hydrolysate; the biology of petroleum replacing refining manufactures the mode of production; contribute to promoting recycling economy development; alleviate current energy source crisis, be subjected to worldwide extensive concern.
The market demand cumulative year after year of succinic acid.Chemically produce succinic acid, not only can consume petrochemical material non-renewable in a large number, and can cause severe contamination to environment, production cost affects very large by oil price in addition.Utilize xylose mother liquid fermentation production of succinic acid; by the by product xylose mother liquid resource utilization of Xylitol industry, not only can avoid consuming petrochemical material, and production process is pollution-free; energy stabilizing carbon dioxide, should have positive meaning for alleviation Greenhouse effect and protection of the environment.
Along with the application of succinic acid is more and more wider, also get more and more to the research of the various production method of succinic acid, just current research, succinic acid mainly can be obtained by chemical synthesis, biotransformation method, this three productions method of microbe fermentation method.
(1) chemical synthesis process
The primary synthetic methods of current industrial succinic acid is chemical synthesis, raw material mainly oil or the liquefied petroleum gas (LPG) of use.Chemical synthesis mainly comprises toxilic acid electrochemical reduction method, toxilic acid shortening method, paraffin oxidation style and methyl chloroacetate cyaniding hydrolysis method etc.At present, industrial main use shortening method carrys out synthesizing succinic acid, and toxilic acid shortening method adopts butane to be that substrate generates maleic anhydride, and then carries out hydrogenation reaction under the effect of catalyzer, produces succinic acid.The method productive rate is high and without obvious side reaction, but operational condition requires high, and the catalyzer of use is expensive, and complex process, seriously polluted.
(2) biotransformation method synthesizing succinic acid
Bio-transformation is using fumaric acid as substrate, rely on the fumaric reductase that thalline produces, fumaric acid one step is converted into succsinic acid 1993, petroleum chemistry company of the Mitsubishi researchists such as the IKUTAMIKI of Japan have changed into succinic acid fumaric acid, and they carry out transforming with the fumaric reductase that Brevibacterium flavum MJ-233 fermentation produces.The Ryu of Korea S etc. filter out a strain excrement intestines club Enterococcus faeclis RKY1 in calendar year 2001, and this bacterial strain can transform fumaric acid and generate succsinic acid.This bacterial strain, when utilizing glycerine or glucose, can produce fumaric reductase, thus makes its richness change succsinic acid into, and the transformation efficiency of succinic acid is respectively 95% and 99%.Biotransformation method is that the production of succsinic acid provides a kind of new thinking, but needs a large amount of use substrate fumaric acid, because fumaric acid is also a kind of important raw materials for production, in fact biotransformation method is not a kind of production ways of economy yet.
(3) producing succinic acid by microbial fermentation
Succinic acid is the intermediate product of Tricarboxylic Acid Metabolism in organism, and many strictly anaerobic bacteriums and facultative anaerobe are all using succinate as the important intermediate of its metabolism.Therefore the microorganism of natural succinic acid-producing can be screened from occurring in nature.By gene engineering method, the genetic engineering bacterium of succinic acid-producing can be obtained.These genetic engineering bacteriums mainly contain succinic acid-producing actinobacillus Actinobacillus succinogenes, succsinic acid bacterium Mannheimia succiniciproducens, Anaerobiospirillum succinoproducens Anaerobiospirillum succiniciproducens and recombination bacillus coli recombinant Escherichia coli are produced in mannheim.Early stage research is strictly anaerobic spirillum A.succiniciproducens ATCC29305 (being registered as ATCC 53488 afterwards) (IntJ Syst Bacteriol.1999 comparatively successfully; 49:207-216), be separated, Gram-negative from the oral cavity of sleuth, glucose etc. can be utilized to produce succinic acid, lactic acid, formic acid and acetic acid etc., Korea S Lee etc. have studied respectively affects its growth and the principal element of producing acid, points out high CO
2succinic acid can be accumulated, low CO in level and pH 6.2 environment
2level and pH 7.2 accumulate lactic acid; Add a certain amount of vitamin H, increase consumption sugar and cell concn, the output of succinic acid can be improved.In fact, the ratio (4: 1) between succinic acid and acetic acid is the impact not being subject to glucose concn.A kind of novel film-bioreactor-electrodialysis integrated systems of research such as France Soucaille, are coupled by electrodialysis unit and cell cycle reactor and remove succinate and acetate.This system produces succinate 83g/L, average succinate productive rate 1.35mol/mol, succsinic acid volume production efficiency 10.4g/L-h, provide succinic acid production method (the Japanese Patent of a kind of high product concentration and high efficiency, 5068576,1993).But the defect one of A.succiniciproducens is fermenting process needs absolute anaerobism, two is have the possibility causing mankind's septicemia, although it is lower to infect probability, potential toxicity still can not be ignored.
Wood sugar is the low heat value new function sweeting agent prepared by agricultural wastes corn cob, bagasse etc., is widely used in the industries such as food, medicine, chemical industry.The crystalline xylose of usual production 1 ton is by the xylose mother liquid of generation 1.5 tons, and these xylose mother liquids often can only be sold with the form of byproduct, and price is very cheap, and its utility value is low especially.At present, discarded xylose mother liquid major part is directly discharged, and serious environment pollution, only a small amount of for producing caramel colorant etc., utilization ratio is very low, has a strong impact on the economic benefit of xylose production industry.Therefore, the treatment process of research xylose mother liquid, turns waste into wealth, protection of the environment, and improving xylose production industrial economy benefit, is very important problem.
But China is the country of a populous grain relative shortage, a large amount of by external food import every year.Be that the biological fermentation process of raw material is produced succinic acid and can be consumed a large amount of grain with grain, and utilize xylose mother liquid to produce succinic acid for fermenting raw materials, accomplished not strive with people grain, be conducive to alleviating crisis in food.The raw material of current fermentation production of succinic acid has glucose, lignocellulose, stalk, corn cob, vinasse etc., utilizes xylose mother liquid to produce succinic acid for fermenting raw materials, extends the raw material sources of succinic acid fermentation, for the production of succinic acid provides new approaches.Similar is CN 10395481 A as publication No., a kind of method utilizing sugar cane juice fermentation production of succinic acid by name, and publication No. is CN 101948879 A, the method utilizing banana dasheen fermentation production of succinic acid by name, being that fermenting raw materials produces succinic acid with sugar cane juice and banana dasheen respectively, is produce the effective ways of succinic acid by agricultural wastes.At present, be that the research of raw material production succinic acid has no report with xylose mother liquid.
Summary of the invention
The object of the invention is for prior art Problems existing, thering is provided a kind of xylose mother liquid that can utilize to produce the method for succinic acid for fermenting raw materials, providing the application of xylose mother liquid preparing in succinic acid and recombination bacillus coli engineering bacteria WH121 preparing the application in the method for succinic acid for carbon source with pretreated xylose mother liquid simultaneously.
The following technical scheme of the present invention achieves the above object: a kind of xylose mother liquid that utilizes, for the method for fermenting raw materials production succinic acid, comprises the following steps:
Utilize xylose mother liquid for a method for fermenting raw materials production succinic acid, it is characterized in that, comprise the following steps:
(1) xylose mother liquid inhibition process, mainly comprises and removes inhibition in xylose mother liquid by limestone-based process, method of evaporation, ion-exchange-resin process three kinds of methods;
(2) xylose mother liquid through the process of derepression thing is added water adjustment sugar to 20 ~ 125g/L, add nitrogenous source, inorganic salt, pH buffer reagent be configured to succinic acid fermention medium;
(3) accessing seed culture medium by flat board producing intestinal bacteria WH121, cultivating 16 ~ 20h at anaerobic culture box;
(4) according to inoculum size be 1 ~ 10% ratio seed is added the fermention medium after optimization, Anaerobic culturel 24 ~ 72h, in gained tunning, succinic acid concentration is 30 ~ 100g/L, and yield is 0.6 ~ 1.2g succinic acid/g total reducing sugar, and productive rate is 0.6 ~ 3.5g/L-h.
Described intestinal bacteria WH121, this laboratory is preserved.E.coli WH121 obtains for introducing plasmid pTrc-cppc process LAN phosphoric acid enol pyruvic acid carboxylase (ppc) gene in E.coli WH1515, and this gene is from anabena sp.7120.WH1515 [pflB::Cam ldhA::Tn10ptsG400::Kan in W1485] is built voluntarily by this laboratory.
The component protein peptone 10g/L of described seed culture medium, yeast extract paste extract 5g/L, NaCl 5g/L, glucose sugar starting point concentration is 0 ~ 15g/L two kinds.
Described fermention medium is that what comprise that experiment of single factor optimization and response phase method optimize take xylose mother liquid as the fermention medium of carbon source.Screen remarkable factor by Plackett-Burman experimental design, Steepest Ascent steepest climbing experiment, Box-Behnken contrived experiment is to xylose mother liquid medium optimization.Its composition is peptone 5 ~ 20g/L, yeast extract 5 ~ 20g/L, MgSO
47H
2o 0.1 ~ 1g/L, Na
2hPO
412H
2o 0.2 ~ 1g/L, NaH
2pO
42H
2o 2 ~ 20g/L, (NH
4)
2sO
47H
2o1 ~ 20g/L, CaCl
20.1 ~ 5g/L, Initial sugar concentration is 20 ~ 100g/L.
Described pH buffer reagent is carbonate, ammoniacal liquor, NaOH, and described carbonate is magnesium basic carbonate or sodium carbonate, and described fermentation condition is aerobic or passes into N
2or CO
2.Priority scheme is that magnesium basic carbonate regulates fermentation pH, passes into CO
2; As adopted the alkalescence such as ammoniacal liquor, NaOH to regulate, CO must be passed into
2.
Described nitrogenous source comprises organic nitrogen source and inorganic nitrogen-sourced; Described organic nitrogen source comprises yeast powder, peptone, pancreatic extract; Described inorganic nitrogen-sourcedly comprise urea, ammonium sulfate.
Described fermentation mode can adopt batch fermentation, fed-batch fermentation or continuously ferment.
Described pretreatment process comprises:
(1) limestone-based process pre-treatment: the Ca (OH) adopting 10 ~ 50% (w/v)
2or the NaOH of 5 ~ 30% (w/v), regulate pH to 10, filter after leaving standstill 1 ~ 5h, filtrate H
2sO
4regulate pH to 3.5 ~ 5.5; Then use sulfiting, add Na
2sO
3, concentration is 1 ~ 5%, boils 15 ~ 30min, filters after 1 ~ 5h, uses H
2sO
4be adjusted to pH 5.5 to refilter;
(2) evaporate: decompression rotary evaporation, is concentrated into original volume 60 ~ 90% respectively by each group of sample, adds water to original volume afterwards, regulates pH to 5.5 (NaOH 5 ~ 30%w/v);
(3), under the condition of anionresin: pH 6 ~ 10, do to spend the night exchanging process with anionite-exchange resin XAD-8.
DNS method is adopted to measure the concentration of total reducing sugars in xylose mother liquid.Getting 182g Seignette salt is dissolved in 500mL hot water, adds the 2mol/L NaOH solution of 262mL, 6.3g 3,5-dinitrosalicylic acid, 5.0g phenol, 5.0g sodium sulphite anhydrous 99.3 successively, be stirred to reagent and dissolve completely in hot solution.After solution cooling, be transferred in 1000mL volumetric flask, be settled to scale with distilled water, be stored in brown reagent bottle, use after keeping in Dark Place one week.Draw glucose standard curve.In colorimetric cylinder, add DNS reagent and the 2mL reaction solution sample of 1.5mL, in boiling water, heat 5min.Room temperature is cooled to after having reacted.With JASCO-530 type spectrophotometer, the absorbancy of working sample under 540nm wavelength.
The 723N type visible spectrophotometer that biomass adopts Shanghai precision scientific instrument company to produce measures, and use wavelength is 600nm.Absorbancy (OD600) obtains by the following method with the corresponding relation of dry cell weight: the fermented liquid getting 10mL, it is centrifugal and collect thalline, re-use the HCl of 0.5mol/L and deionized water and wash this thalline respectively three times and centrifugal, finally dry to constant weight under 60 DEG C of conditions, obtaining dry cell weight corresponding to 1OD600 is 0.38g.
Various sugars (glucose, wood sugar, semi-lactosi, pectinose etc.) in fermented liquid and organic acid concentration are detected by chromatographic analyzer of liquid phase, detector is UV-detector (Agilent Technologies, and differential refraction detector (Agilent Technologies G1315D), G1362A), analytical column is HPX-87H column (300mm × 4.6mm, Bio-Rad).Column temperature is 25 DEG C, and moving phase is pH 2.5,5mmol/LH
2sO
4solution, flow velocity is 0.6mL/min, and sample size is 20 μ L.
The colibacillary culture temperature of the present invention's research is 37 DEG C, cultivates in LB substratum or the substratum after optimizing.According to the needs of different situations, add ammonia benzyl microbiotic and IPTG inductor.
Outstanding advantages of the present invention and beneficial effect:
1. be that fermenting raw materials produces succinic acid with xylose mother liquid, in gained tunning, succinic acid concentration is 30 ~ 100g/L, and yield is 0.6 ~ 1.2g succinic acid/g total reducing sugar, and productivity is 0.6 ~ 3.5g/L-h.
2. utilize the glucose of xylose mother liquid raw material substitution costliness or food crop to produce succinic acid for carbon source through fermentation, can a large amount of grain be saved on the one hand, ensure the grain security of China, nitrogenous source usage quantity in fermentation can be reduced simultaneously, reduce the production cost of succinic acid.Sugared valency can be regulated on the other hand, ensure peasant's rights and interests.
3. utilize renewable biomass resource to produce succinic acid, the problem of the fossil resources anxiety of chemosynthesis succinic acid can be alleviated, be conducive to social sustainable development.
Be below the specific embodiment utilizing the method for xylose mother liquid fermentation production of succinic acid of the present invention, but the present invention is not limited.
Accompanying drawing explanation
Fig. 1 recombination bacillus coli fermenting xylose mother liquor shake flask fermentation succinic acid-producing, substratum is the substratum after response phase method is optimized.
Embodiment 1
Different pretreatments method is on the impact of xylose mother liquid succinic acid-producing
Respectively by limestone-based process, method of evaporation, ion-exchange-resin process three kinds of method pre-treatment:
(1) limestone-based process: the Ca (OH) adopting 20% (w/v)
2or the NaOH of 10% (w/v), regulate pH to 10, filter after leaving standstill 1h, filtrate H
2sO
4regulate pH to 5.5; Then use sulfiting, add Na
2sO
3, concentration is 1%, boils 15min, filters after 1h, uses H
2sO
4be adjusted to pH 5.5 to refilter;
(2) evaporate: decompression rotary evaporation, is concentrated into original volume 90% respectively by each group of sample, adds water to original volume afterwards, regulates pH to 5.5 (NaOH 10%w/v);
(3), under the condition of anionresin: pH 10, do to spend the night exchanging process with anionite-exchange resin XAD-8.
Pretreated xylose mother liquid is used as fermentation substrate, and fermentation medium components is peptone 20g/L, yeast extract 10g/L, MgSO
47H
2o 0.3g/L, Na
2hPO
412H
2o 0.45g/L, NaH
2pO
42H
2o 6g/L, (NH
4)
2sO
47H
2o 3g/L, CaCl
20.2g/L, Initial sugar concentration is 40g/L.Fermentation net result is as shown in table 1.
Table 1 different pretreatments method is on the impact of intestinal bacteria succinic acid-producing
Embodiment 2
Take xylose mother liquid as carbon source, the succinic acid in the substratum after response phase method optimization is produced
First xylose mother liquid is adopted limestone-based process process: the Ca (OH) namely adopting 20% (w/v)
2or the NaOH of 10% (w/v), regulate pH to 10, filter after leaving standstill 1h, filtrate H
2sO
4regulate pH to 5.5; Then use sulfiting, add Na
2sO
3, concentration is 1%, boils 15min, filters after 1h, uses H
2sO
4be adjusted to pH 5.5 to refilter;
Access seed culture medium by flat board producing intestinal bacteria WH121, the composition of seed culture medium is peptone 10g/L, yeast extract paste extract 5g/L, NaCl 5g/L, and glucose sugar starting point concentration is 5g/L.According to inoculum size be 8% ratio seed is added response phase method optimize after fermention medium, Anaerobic culturel.
Take xylose mother liquid as carbon source, the optimal medium of the fermentation of response phase method optimization is: 82.62g/L initial total sugar, 42.27g/LMgCO
3, and 17.84g/L yeast extract paste, 20g/L peptone, 0.3g/LMgSO
47H
2o, 0.6g/LNa
2hPO
412H
2o, 4g/L NaH
2pO2H
2o, 2g/L (NH
4)
2sO
47H
2o, 0.4g/L CaCl
2; Optimal conditions of fermentation is: pH 7.0; Leavening temperature is 37 DEG C.
Final recombination bacillus coli WH121 consumes 37.01g/L wood sugar respectively in 84h, 13.86g/L pectinose, 12.00g/L glucose, other reducing sugars of 8.70g/L semi-lactosi and some trace.Succinic acid maximum production is 52.09 ± 0.21g/L, and yield is 0.63 ± 0.03g/g total reducing sugar.Wherein succinic acid output is close to predictor (53.18g/L).
Embodiment 3
Be carbon source with xylose mother liquid, the succinic acid in fermentor tank is produced
After method of evaporation pre-treatment, namely reduce pressure rotary evaporation, each group of sample is concentrated into original volume 85% respectively, adds water to original volume afterwards, regulates pH to 5.5 (NaOH 20%w/v).
Access seed culture medium by flat board producing intestinal bacteria WH121, the composition of seed culture medium is peptone 10g/L, yeast extract paste extract 5g/L, NaCl 5g/L, and glucose sugar starting point concentration is 2g/L.According to inoculum size be 5% ratio seed is added response phase method optimize after fermention medium, Anaerobic culturel.
Be carbon source with xylose mother liquid, the fermention medium in fermentor tank is: 80g/L initial total sugar, 40g/L MgCO
3, and 15g/L yeast extract paste, 20g/L peptone, 0.3g/L MgSO
47H
2o, 0.6g/L Na
2hPO
412H
2o, 4g/LNaH
2pO2H
2o, 2g/L (NH
4)
2sO
47H
2o, 0.4g/LCaCl
2; Optimal conditions of fermentation is: pH 7.0; Leavening temperature is 37 DEG C.In two-stage, oxygen-supply quantity is 4vvm, by NaOH and H
2s0
4regulation and control pH maintains 7.0, and temperature maintains 37 DEG C, and agitator speed is 400RPM.After 4h, stop logical oxygen, and add IPTG inductor, logical CO
2amount is 2vvm, MgCO
3as alkaline neutraliser control pH 6.8, agitator speed is 180RPM.
In gained tunning, succinic acid concentration is 65g/L, and yield is 1.1g succinic acid/g total reducing sugar, and productivity is 1.55g/L-h.
Embodiment 4
Take xylose mother liquid as carbon source, the succinic acid under fed-batch fermentation mode is produced
Adopt anion exchange method pre-treatment to remove inhibition in xylose mother liquid, be i.e., under the condition of pH 10, do to spend the night exchanging process with anionite-exchange resin XAD-8.
Access seed culture medium by flat board producing intestinal bacteria WH121, the composition of seed culture medium is peptone 10g/L, yeast extract paste extract 5g/L, NaCl 5g/L, and glucose sugar starting point concentration is 10g/L.According to inoculum size be 8% ratio seed is added response phase method optimize after fermention medium, Anaerobic culturel.
Be carbon source with xylose mother liquid, the fermention medium in fermentor tank is: 80g/L initial total sugar, 40g/L MgCO
3, and 15g/L yeast extract paste, 20g/L peptone, 0.3g/L MgSO
47H
2o, 0.6g/L Na
2hPO
412H
2o, 4g/LNaH
2pO2H
2o, 2g/L (NH
4)
2sO
47H
2o, 0.4g/L CaCl
2; Optimal conditions of fermentation is: pH 7.0; Leavening temperature is 37 DEG C.In two-stage, oxygen-supply quantity is 4vvm, by NaOH and H
2sO
4regulation and control pH maintains 7.0, and temperature maintains 37 DEG C, and agitator speed is 400RPM.After 4h, stop logical oxygen, and add IPTG inductor, logical CO
2amount is 2vvm, MgCO
3as alkaline neutraliser control pH 6.8, agitator speed is 180RPM.When reducing sugar content in fermented liquid is lower than 10g/L, the xylose mother liquid liquid containing 200g/L reducing sugar is added in fermented liquid with suitable speed, makes sugared stable content at 10g/L.Experimental result is shown in Table 2.As can be seen from Table 2: be 45g/L in initial xylose mother liquid total concn, through fed-batch fermentation 48h, succinic acid concentration reaches 79.44g/L, and fermentation residual sugar is 2.18g/L, and succinic acid yield is 1.17g/g.Production intensity is 1.66g/L-h.
Table 2 3L fermentor tank batch feeding test-results
Claims (6)
1. utilize xylose mother liquid for a method for fermenting raw materials production succinic acid, it is characterized in that, comprise the following steps:
(1) xylose mother liquid inhibition process, mainly comprises and removes inhibition in xylose mother liquid by Wingdale (Overliming) method, method of evaporation, ion-exchange-resin process three kinds of methods;
(2) xylose mother liquid through the process of derepression thing is added water adjustment sugar to 75 ~ 125g/L, add nitrogenous source, inorganic salt, pH buffer reagent be configured to succinic acid fermention medium;
(3) accessing seed culture medium by flat board producing intestinal bacteria WH121, cultivating 16-20h at anaerobic culture box; The component protein peptone 10g/L of described seed culture medium, yeast extract paste extract 5g/L, NaCl 5g/L, glucose 0 ~ 15g/L.
(4) according to inoculum size be 1 ~ 10% ratio seed is added the fermention medium after optimization, Anaerobic culturel 24 ~ 72h, gained fermentation in primary product be succinic acid.
Described fermentation medium components and concentration thereof for its composition be peptone 5 ~ 20g/L, yeast extract 5 ~ 20g/L, MgSO
47H
2o 0.1 ~ 1g/L, Na
2hPO
412H
2o 0.2 ~ 1g/L, NaH
2pO
42H
2o 2 ~ 20g/L, (NH4)
2sO
47H
2o1 ~ 20g/L, CaCl
20.1 ~ 5g/L, Initial sugar concentration is 20 ~ 100g/L.
2. the method utilizing xylose mother liquid fermentation production of succinic acid according to claims 1, it is characterized in that, described pH buffer reagent is carbonate, ammoniacal liquor, NaOH, and described carbonate is magnesium basic carbonate or sodium carbonate, and described fermentation condition is aerobic or passes into N
2or CO
2.Priority scheme is that magnesium basic carbonate regulates fermentation pH, passes into CO
2; As adopted the alkalescence such as ammoniacal liquor, NaOH to regulate, CO must be passed into
2.
3. the nitrogenous source according to claims 1 comprises organic nitrogen source and inorganic nitrogen-sourced; Described organic nitrogen source comprises yeast powder, peptone, pancreatic extract; Described inorganic nitrogen-sourcedly comprise urea, ammonium sulfate.
4. the method utilizing xylose mother liquid fermentation production of succinic acid according to claims 1, is characterized in that, batch fermentation, fed-batch fermentation that described fermentation mode adopts or continuously ferment.
5. the pretreated three kinds of methods of xylose mother liquid according to claim 1, comprise Wingdale (Overliming) method, method of evaporation, ion-exchange-resin process.
6. other method of the xylose mother liquid fermentation production of succinic acid derivative described in 5 is wanted according to right.
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| CN106801026A (en) * | 2017-02-13 | 2017-06-06 | 广西科学院 | Bacterial strain and its production method used by a kind of utilization xylose mother liquid fermentation succinic acid-producing |
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| CN104293817A (en) * | 2013-07-15 | 2015-01-21 | 重庆大学 | Construction methods and uses of recombinant plasmid for producing succinic acid, and genetic engineering bacterium |
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| CN104293817A (en) * | 2013-07-15 | 2015-01-21 | 重庆大学 | Construction methods and uses of recombinant plasmid for producing succinic acid, and genetic engineering bacterium |
Non-Patent Citations (3)
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| DAN WANG ET AL.: "High cell density fermentation via a metabolically engineered Escherichia coli for the enhanced production of succinic acid.", 《J CHEM TECHNOL BIOTECHNOL》 * |
| HONGHUI WANG ET AL.: "Succinic acid production from xylose mother liquor by recombinant Escherichia coli strain.", 《BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT》 * |
| SIMONA LARSSON ET AL.: "Comparison of Different Methods for the Detoxification of Lignocellulose Hydrolyzates of Spruce.", 《APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106801026A (en) * | 2017-02-13 | 2017-06-06 | 广西科学院 | Bacterial strain and its production method used by a kind of utilization xylose mother liquid fermentation succinic acid-producing |
| CN106801026B (en) * | 2017-02-13 | 2020-05-12 | 广西科学院 | Bacterial strain for producing succinic acid by fermentation of xylose mother liquor and production method thereof |
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