CN104894174A - Method for producing succinic acid by taking sugarcane bagasse as raw materials through fermentation - Google Patents

Method for producing succinic acid by taking sugarcane bagasse as raw materials through fermentation Download PDF

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CN104894174A
CN104894174A CN201510387997.5A CN201510387997A CN104894174A CN 104894174 A CN104894174 A CN 104894174A CN 201510387997 A CN201510387997 A CN 201510387997A CN 104894174 A CN104894174 A CN 104894174A
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bagasse
fermentation
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hydrolysis
succinic acid
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郑璞
淘声涛
查鑫华
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Shandong Xingqiang Chemical Industry Technology Research Institute Co Ltd
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Jiangnan University
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Abstract

The invention discloses a method for producing succinic acid by taking sugarcane bagasse as raw materials through fermentation and belongs to the technical field of bioengineering. The method is characterized by comprising the following steps of taking the sugarcane bagasse which is main byproducts in the sugar pressing industry as raw materials, and performing crushing, pretreatment with dilute alkali as well as common hydrolysis on cellulase, xylanase, beta-glucanase and pectinase to obtain a hydrolyzed sugar solution; fermenting the hydrolyzed sugar solution by using actinobacillus succinogenes to produce the succinic acid; meanwhile, residues left by hydrolyzing the sugarcane bagasse are added into fermented solution, so that the fermentation can be repeatedly operated, seed culture can be omitted and furfural in residual liquid obtained by extraction of organic solvent and pretreatment with the dilute alkali can be utilized. According to the method disclosed by the invention, reasonable utilization of hydrolyzed residues of the sugarcane bagasse is realized, and the conventional fermented seed culture link is reduced, so that the fermentation cost is reduced, and the fermentation efficiency is improved; besides, the requirement on fermentation equipment is low, extra improvement on an existing fermentation tank is not needed, and the method is suitable for industrial production.

Description

A kind of method of producing succinic acid with bagasse fermenting raw materials
Technical field
The present invention relates to a kind of method of producing succinic acid with bagasse fermenting raw materials, belong to technical field of bioengineering.
Background technology
Bagasse is a kind of important lignocellulose renewable raw materials, and China is the country that cane planting amount is the third-largest in the world, and south is the area that China mainly grows cane, and annual sugarcane ultimate production reaches more than 7,000 ten thousand tons, can produce the bagasse of 7,000,000 tons simultaneously.
In bagasse, main ingredient is Mierocrystalline cellulose, hemicellulose and xylogen.The main application of current bagasse is combustion power generation (light science and technology, 2013, (11): 11-12.), as cattle food (Chinese Patent Application No. 94113455.5), substitute traditional timber resources as papermaking and production high density material (Chinese Plastics, 2008, (02): 57-61), as the substratum (edible mushrooms of edible mushrooms, 1980, (02): 12-16), and Development and Production high value added product: as Development and Production Xylitol (science and technology circular, 2004, (1): 37-41), pectinose (amino acid and Biological resources, 2009, (1): 8-12.), furfural (chemical science and technology market, 2001, (11): 12-15).Chinese patent CN 1060464C discloses the method preparing syringic aldehyde and vanillin food grade,1000.000000ine mesh with bagasse alkali-lignin catalyzed oxidation.Chinese patent CN 103614435A discloses a kind of method preparing xylo-oligosaccharide from bagasse.
Succinic acid is industrial a kind of important C4 platform chemicals, all has a wide range of applications at food, medicine, chemical industry and other field.The culture medium carbon source mainly glucose of the fermentation production of succinic acid institute of current bibliographical information, developing cheap carbon source is one of approach reducing fermentative Production succinic acid cost.The report dilute sulphuric acid acidolysis bagasse such as Brazil Borge, the liquid glucose obtained is as carbon source, and with A.succinogenes CIP 106512 anaerobism shake flask fermentation succinic acid 24h, the concentration obtaining succinic acid is up to 22.5g.L -1(Industrial Microbiology and Biotechnology, 2011,38:1001-1011.).The dilute sulphuric acid acidolysis bagasse such as Xu Rong, the pretreated residue of reprocess fibre element enzymic hydrolysis, bagasse hydrolyzed solution fermentation succinic acid, obtains 20g.L -1succinic acid (chemical industry be in progress, 2013,04:874-877.).But with the succinic acid of these method fermentative production, output is lower, adds the cost of subsequent extracted.The invention provides a kind of method of bagasse effectively hydrolyzing and production high density succinic acid.
Summary of the invention
The object of this invention is to provide a kind of method utilizing cheap bagasse fermenting raw materials to produce succinic acid.
The method of described fermentation production of succinic acid, is using bagasse pulverizing, diluted alkaline pre-treatment, is hydrolyzed the hydrolysis sugar liquid that the obtains carbon source as Actinobacillus succinogenes anaerobically fermenting succinic acid, in fermented liquid, add 4 ~ 20g.L -1bagasse hydrolysis after residue (dry weight) as the attachment carrier of somatic cells, repeated batch fermentation or Repeat fed-batch fermentation produce succinic acid.
Described bagasse is pulverized, diluted alkaline pre-treatment, in one embodiment of the invention, be that to squeeze sugared trade waste bagasse be raw material, dry bagasse is pulverized, cross 20-40 mesh sieve, dry for 40-70 DEG C and preserve, be the NaOH solution of 0.5-5% by mass concentration, by solid-to-liquid ratio 1:1-1:15 (w/v), 121 DEG C of High Temperature Pre process bagasse 2-4h, bagasse is washed to obtain in filtration, 45-70 DEG C of dry for standby.
Described bagasse is pulverized, diluted alkaline pre-treatment, and in one embodiment of the invention, the raffinate after diluted alkaline pre-treatment bagasse is the chloroform extraction of 1:0.5 ~ 1:1 by volume ratio, obtains furfural crude product.
Described hydrolysis, in one embodiment of the invention, that pretreated bagasse is made into the solution that mass concentration is 3 ~ 10%, be hydrolyzed under the synergy of cellulase, zytase, beta-glucanase and polygalacturonase, hydrolysis temperature 40-70 DEG C, pH is adjusted to 4.0-6.5, rotating speed 100-250r.min -1, time 24-72h.After hydrolysis, obtain hydrolysis sugar liquid through centrifugal, the residue after bagasse hydrolysis is dried and obtained to solid.
Described hydrolysis, in one embodiment of the invention, cellulase, zytase, beta-glucanase and polygalacturonase to every gram of substrate addition be respectively: 10-35FPU.g -1, 50-120U.g -1, 10-80U.g -1and 5-30U.g -1.
Residue after described bagasse hydrolysis, in one embodiment of the invention, addition is 8 ~ 18g.L -1.
Described repeated batch fermentation or Repeat fed-batch fermentation, in one embodiment of the invention, refer to and adopt the mode of batch fermentation or fed-batch fermentation to produce succinic acid, after each batch terminates, static, after the residue after bagasse hydrolysis in fermented liquid sinks down into fermenter base, transfer supernatant liquor, then in fermentor tank, add fresh fermention medium, do not need inoculation, directly carry out next batch fermentation.
Described method, in one embodiment of the invention, is: (1) for carbon source, prepares fermention medium with bagasse hydrolysis sugar liquid, wherein bagasse hydrolyzed solution sugar concentration 20 ~ 100g.L in substratum -1; (2) 4 ~ 20g.L is added in the fermentation medium -1bagasse hydrolysis after residue, then by 5 ~ 10% inoculum size inoculation, batch fermentation or fed-batch fermentation 20 ~ 60h under the anaerobic condition of temperature 35 ~ 40 DEG C, pH 5.8 ~ 6.5; (3) static after fermentation ends, after the residue after bagasse hydrolysis in fermented liquid sinks down into fermenter base, transfer supernatant liquor, then adds fresh fermention medium in fermentor tank, does not need inoculation, directly carries out next batch fermentation.
The fermention medium of described step (1), in one embodiment of the invention, comprising: bagasse hydrolyzed solution sugar concentration 20 ~ 100g.L -1, corn steep liquor concentration 10 ~ 30g.L -1, anhydrous chlorides of rase calcium concn 0.05 ~ 0.5g.L -1, dipotassium hydrogen phosphate concentration 1 ~ 5g.L -1, phosphate dihydrogen sodium concentration 4 ~ 12g.L -1, magnesium chloride hexahydrate concentration 0.05 ~ 0.5g.L -1, sodium sulphite concentration 0.05 ~ 0.5g.L -1.
The fermention medium of described step (1), in one embodiment of the invention, bagasse hydrolyzed solution sugar concentration 55g.L -1.
The fermentation of described step (2), in one embodiment of the invention, passes into CO 2/ N 2it is anaerobic condition that gas controls fermenting process, saturated NaHCO 3control fermentation pH 5.8 ~ 6.5.
The fed-batch fermentation of described step (2), in one embodiment of the invention, the fed-batch fermentation of to be fermention medium Initial sugar concentration be more than 50g/L, when concentration of reduced sugar in fermented liquid is lower than 20g.L -1time, start to add bagasse hydrolysis sugar liquid and control at 10-20g.L to make the sugared concentration in fermented liquid -1, fermentation 25-50h.
Described step (2) in batches, in one embodiment of the invention, is fermentation 3-5 batch.
Described Actinobacillus succinogenes in one embodiment of the invention, is A.ssuccinogenes F3-21 or Actinobacillus succinogenes CGMCC NO.1593, CCTCC NO:M 2012036.
Fermentation ends in described step (3), in one embodiment of the invention, refers to that fermented liquid concentration of reduced sugar is less than 10g.L -1or be less than 5g/L.
Described method, in one embodiment of the invention, specifically: (1) bagasse pre-treatment: 20-60 mesh sieve pulverized by bagasse, 40-70 DEG C of oven dry, be the NaOH solution of 0.5-5% by mass concentration, according to solid-to-liquid ratio 1:1 ~ 1:20 (w/v), 121 DEG C of High Temperature Pre process bagasse 1 ~ 4h, filter washing and obtain pretreated bagasse, 45-70 DEG C of dry for standby; (2) bagasse hydrolysis: pretreated bagasse is made into the solution that mass concentration is 3 ~ 10%, add cellulase, zytase, beta-glucanase and polygalacturonase, temperature 40 ~ 70 DEG C, pH is adjusted to 4.0 ~ 6.5, rotating speed 100-250r.min -1condition under, hydrolysis 24 ~ 72h; Its cellulase, zytase, beta-glucanase and the polygalacturonase addition to every gram of bagasse is respectively: 10 ~ 35FPU.g -1, 50 ~ 120U.g -1, 10 ~ 80U.g -1with 5 ~ 30U.g -1; Centrifugal, obtain bagasse hydrolysis sugar liquid and bagasse hydrolytic residue; (3) fermentation production of succinic acid: regulate bagasse hydrolysis sugar liquid concentration, configuration fermentation culture, fermention medium consists of: bagasse hydrolyzed solution sugar concentration 20-100g.L -1, corn steep liquor concentration 10-30g.L -1, anhydrous chlorides of rase calcium concn 0.05-0.5g.L -1, dipotassium hydrogen phosphate concentration 1-5g.L -1, phosphate dihydrogen sodium concentration 4-12g.L -1, magnesium chloride hexahydrate concentration 0.05-0.5g.L -1, sodium sulphite concentration 0.05-0.5g.L -1; Add 4 ~ 20g.L in the fermentation medium -1bagasse hydrolysis after residue (with dry weight basis), then by 5 ~ 10% inoculum size inoculation seed liquor, under the anaerobic condition of temperature 35 ~ 40 DEG C, pH 5.8 ~ 6.5, fermentation 20 ~ 60h; Static after fermentation ends, after the bagasse hydrolytic residue in fermented liquid sinks down into fermenter base, transfer supernatant liquor, then adds fresh fermention medium in fermentor tank, does not need inoculation, directly carries out next batch fermentation.
The present invention's also claimed succinic acid of obtaining according to described method and in food, chemistry and the application prepared in medicine.
Present invention also offers a kind of method fully utilizing bagasse, described method comprises: (1) bagasse through pulverizing, diluted alkaline pre-treatment, be hydrolyzed the hydrolysis sugar liquid that the obtains carbon source as Actinobacillus succinogenes anaerobically fermenting succinic acid, in fermented liquid, add 4 ~ 20g.L -1bagasse hydrolysis after residue as the attachment carrier of somatic cells, repeated batch fermentation or Repeat fed-batch fermentation produce succinic acid; (2) raffinate after diluted alkaline pre-treatment bagasse is the chloroform extraction of 1:0.5 ~ 1:1 by volume ratio, obtains furfural crude product.Specifically can see the method for above-mentioned fermentation production of succinic acid.
Beneficial effect of the present invention:
(1) reasonable employment of bagasse hydrolytic residue is achieved, with 4 ~ 20g.L -1bagasse hydrolytic residue as thalline attachment carrier loop fermentation succinic acid, reduce the seed culture link of normal fermentation, saved fermentation costs, improve fermentation efficiency.(in normal fermentation, seed culture 12-24h, seed culture medium is generally containing 10g.L -1above yeast extract paste, seed accounts for the 2-5% of raw materials cost).Meanwhile, Batch fermentation can carry out repeatedly, and the succinic acid concentration average of 3 batch cycle batch fermentations is 35g.L -1left and right, average production intensity is 1.65g.L -1.h -1, average glucose acid invert ratio is 0.81g.g -1, whole fermentation system runs 64h, does not have downward trend.
(2) technique of the present invention, requires low to fermentation equipment, does not need to make additional improvement to existing fermentor tank, is applicable to suitability for industrialized production.
(3) the squeezing sugar trade waste bagasse that have employed the cheapness of not striving grain with people is fermentation raw material, multienzyme combined hydrolysis bagasse, reach the total fiber element percent hydrolysis of 90%, compared with the acidic treatment of report, with diluted alkaline pre-treatment, be hydrolyzed in conjunction with multienzyme synergism, improve the hydrolysis sugar content of bagasse, improve the concentration of hydrolyzed solution fermentation succinic acid-producing.Simultaneously by fed-batch fermentation, the concentration of succinic acid reaches 71g.L -1, succinic acid is 0.46g.g to the productive rate of bagasse -1.
(4) the present invention is for the production cost reducing succinic acid, and alleviate succinic acid product to the dependence of petrochemical material, promote the utilization of agricultural wastes resource, protection of the environment, realizing sustainable recycling economy development has positive economic and social effect.
Accompanying drawing explanation
Fig. 1: bagasse fermentation succinic acid operational path;
Fig. 2: multienzyme synergism hydrolysis pre-treatment bagasse time curve;
Fig. 3: bagasse hydrolyzed solution is as the conditional curve of carbon source fed-batch fermentation succinic acid;
Fig. 4: band slag 3L fermentor tank repeated batch fermentation succinic acid.
Embodiment
Residual sugar and organic acid measure: the residual sugar in pre-treatment bagasse hydrolyzed solution in the mensuration of various sugared concentration (glucose, wood sugar, cellobiose, pectinose, glucuronic acid etc.) and fermented liquid and organic acid concentration adopt high effective liquid chromatography for measuring, refer to CN 101603059A patent.
Embodiment 1: diluted alkaline pre-treatment bagasse
Get the bagasse that 20g pulverizes post-drying, the NaOH solution 300mL being 1% with mass concentration mixes, at 121 DEG C of High Temperature Pre process bagasse 2h, filtration is washed bagasse is extremely neutral, 65 DEG C of oven dry, obtain the pretreated bagasse of 13g, weight loss 35%, total fiber cellulose content is 87.6%, and its composition is as table 1.
Table 1 records the component of oxygenation pretreatment bagasse for NREL method
Embodiment 2: the bagasse after the process of multienzyme synergism hydrolysising alkali
Take the bagasse that wherein 3g oxygenation pretreatment is good, proceed in the triangular flask of 150mL, add cellulase 25FPU.g -1, zytase 85U.g -1, beta-glucanase 40U.g -1, polygalacturonase 25U.g -1, add the citric acid damping fluid of pH5.0, be settled to 40mL, at 50 DEG C, shaking bath is hydrolyzed 40h, gets a sample every 4h hour.Multienzyme synergism hydrolysis time Fig. 2.Hydrolysis 40h terminates, and in hydrolyzed solution, concentration of reduced sugar is 65.6g.L -1, the total fiber element hydrolysis efficiency of bagasse is up to 90%, and in bagasse hydrolyzed solution, the concentration of glucose is 45.06g.L -1, xylose concentration is 14.92g.L -1, the concentration of glucuronic acid is 5.62g.L -1, the concentration ratio of glucose and xylose is close to 3:1.Multienzyme synergism hydrolysis pre-treatment bagasse time curve as shown in Figure 2.
Embodiment 3: bagasse hydrolyzed solution fed-batch fermentation produces succinic acid
Bacterial strain: Actinobacillus succinogenes (Actinobacillu ssuccinogenes F3-21).Substratum: bagasse hydrolyzed solution sugar concentration 55g.L -1, corn steep liquor concentration 25g.L -1, anhydrous chlorides of rase calcium concn 0.2g.L -1, dipotassium hydrogen phosphate concentration 2g.L -1, phosphate dihydrogen sodium concentration 9.6g.L -1, magnesium chloride hexahydrate concentration 0.2g.L -1, sodium sulphite concentration 0.1g.L -1, pH is adjusted to 6.0,121 DEG C of sterilizing 20min.
On 3L stirred fermentor, adopt fed-batch fermentation, the concentration of initial bagasse hydrolysis sugar liquid is 55g.L -1, when in fermented liquid, remaining sugar concentration reduces to 20g.L -1time, adding up concentration of reduced sugar with peristaltic pump stream is 200g.L -1concentrated bagasse hydrolysis sugar liquid, the sugared concentration in fermented liquid is controlled at 10-20g.L -1.During fermentation 20-26h, production intensity can remain on 1.7g.L -1.h -1.Ferment 50 hours, succinic acid-producing 71g.L -1, glucose acid invert ratio is 0.82g.g -1, production intensity is 1.42g.L -1.h -1.Result as shown in Figure 3.
Embodiment 4: bagasse residue (SBR) is as absorption carrier circulating fermentation succinic acid
Fermention medium is with embodiment 3, and addition 10g.L -1sBR (amount of dry matter, 80 DEG C dry to constant weight).Fermention medium 121 DEG C of sterilizing 20min, access 5% (v/v) seed liquor, in 37 DEG C of fermentations, the rotating speed of fermentor tank is 150r.min -1, use saturated NaHCO 3control pH is about 6.2, when sugared concentration is reduced to below 5mg/L, terminates this batch fermentation.Every Batch fermentation terminates, fermented liquid is left standstill 2-4h until bagasse natural subsidence is to the bottom of fermentor tank, with peristaltic pump, fermented supernatant fluid is pumped, fresher substratum is pumped into fermentor tank, the fermentation system of every batch is 1L, and iterative cycles operation is until fermentation ends.
3 batch cycle batch fermentations, result is as table 2 and Fig. 4.Succinic acid concentration average is 35g.L -1left and right, the average production intensity of succinic acid is 1.65g.L -1.h -1, average glucose acid invert ratio is 0.81g.g -1, whole fermentation system runs 64h, does not have downward trend.
The result of table 2 with slag circulation batch fermentation succinic acid
Embodiment 5: furfural content in bagasse diluted alkaline pretreatment fluid
By the method for embodiment 1, carry out bagasse pre-treatment, by about the pretreatment fluid sulphur acid for adjusting pH to 3.0 that obtains.Carry out rocking under getting the pretreatment fluid of 50mL and chloroform (volume ratio 1:1) room temperature of 50mL and mix 30min, leave standstill 5h.Get organic phase, measure the content of furfural at 276nm place, calculate in the treatment solution of 100g bagasse, containing 2.6g furfural.
Embodiment 6: bagasse residue (SBR) addition is on the impact of circulating fermentation succinic acid
By the method executing example 4, configuration fermention medium, SBR is added respectively in 100mL triangular flask, consumption is followed successively by 0.1g, 0.15g, 0.20g, 0.25g, 0.3g, 0.35g, 0.40g, 0.45g, 0.50g, regulate the pH to 6.5 of fermented liquid, add water and be settled to 25mL, 121 DEG C of sterilizing 20min, access 5% (v/v) seed liquor, 37 DEG C of anaerobism shake flask fermentations, stratification, pouring liquids, surveys the concentration of succinic acid in fermented liquid, and adds sterilized fermention medium, repeated batch fermentation three times, result is as table 3.
The different SBR concentration of table 3 is to fermentation succinic acid content (g.L -1) impact
Embodiment 7: Repeat fed-batch fermentation produces succinic acid
Fermentation production of succinic acid by the following method:
(1) sugarcane pre-treatment; 20-60 mesh sieve pulverized by bagasse, and 70 DEG C of oven dry are the NaOH solution of 5% by mass concentration, and according to solid-to-liquid ratio 1:1 (w/v), High Temperature Pre process bagasse 1h, filters washing and obtain pretreated bagasse, 45 DEG C of dry for standby; After pre-treatment, total fiber cellulose content is 86.8%;
(2) bagasse hydrolysis: pretreated bagasse is made into the solution that mass concentration is 3%, add cellulase, zytase, beta-glucanase and polygalacturonase, temperature 40 DEG C, pH is adjusted to 6.5, rotating speed 100-250r.min -1condition under, hydrolysis 72h; Its cellulase, zytase, beta-glucanase and the polygalacturonase addition to every gram of bagasse is respectively: 10FPU.g -1, 50U.g -1, 10U.g -1and 5U.g -1; Centrifugal, obtain bagasse hydrolysis sugar liquid and bagasse hydrolytic residue; In hydrolyzed solution, concentration of reduced sugar is 60.6g.L -1.
(3) fermentation production of succinic acid: regulate bagasse hydrolysis sugar liquid concentration, configuration fermentation culture, fermention medium consists of: bagasse hydrolyzed solution sugar concentration 100g.L -1, corn steep liquor concentration 30g.L -1, anhydrous chlorides of rase calcium concn 0.05g.L -1, dipotassium hydrogen phosphate concentration 1g.L -1, phosphate dihydrogen sodium concentration 12g.L -1, magnesium chloride hexahydrate concentration 0.5g.L -1, sodium sulphite concentration 0.05g.L -1; Add 8g.L in the fermentation medium -1bagasse hydrolysis after residue, then by 5% inoculum size inoculation Actinobacillus succinogenes CGMCC NO.1593 seed liquor, at the anaerobic condition bottom fermentation of temperature 40 DEG C, pH 6.5; When concentration of reduced sugar in fermented liquid is lower than 20g.L -1time, start to add bagasse hydrolysis sugar liquid and control at 10-20g.L to make the sugared concentration in fermented liquid -1, fermentation 40h; Static 1.5h after fermentation ends, the bagasse hydrolytic residue in fermented liquid sinks down into fermenter base, then shifts supernatant liquor, then in fermentor tank, adds fresh fermention medium, does not need inoculation, directly carries out next batch fermentation.Repeated batch fermentation 5 times.
Result shows, and the succinic acid concentration average of repeated batch fermentation is 58g.L -1left and right, the average production intensity of succinic acid is 1.61g.L -1.h -1, average glucose acid invert ratio is 0.80g.g -1, in whole fermentation system operational process, there is no downward trend.
Embodiment 8: repeated batch fermentation produces succinic acid
Fermentation production of succinic acid by the following method:
(1) bagasse pre-treatment; 20-60 mesh sieve pulverized by bagasse, and 40 DEG C of oven dry are the NaOH solution of 0.5% by mass concentration, and according to solid-to-liquid ratio 1:20 (w/v), 121 DEG C of High Temperature Pre process bagasse 4h, filter washing and obtain pretreated bagasse, 70 DEG C of dry for standby; After pre-treatment, total fiber cellulose content is 84.6%;
(2) bagasse hydrolysis: pretreated bagasse is made into the solution that mass concentration is 10%, add cellulase, zytase, beta-glucanase and polygalacturonase, at temperature 70 C, pH is adjusted to 4.0, rotating speed 100-250r.min -1condition under, hydrolysis 24h; Its cellulase, zytase, beta-glucanase and the polygalacturonase addition to every gram of bagasse is respectively: 35FPU.g -1, 120U.g -1, 80U.g -1and 30U.g -1; Centrifugal, obtain bagasse hydrolysis sugar liquid and bagasse hydrolytic residue; In hydrolyzed solution, concentration of reduced sugar is 62.8g.L -1, the total fiber element hydrolysis efficiency of bagasse is up to 89%, and in bagasse hydrolyzed solution, the concentration of glucose is 42.86g.L -1, xylose concentration is 14.32g.L -1, the concentration of glucuronic acid is 5.62g.L -1, the concentration ratio of glucose and xylose is close to 3:1.
(3) fermentation production of succinic acid: regulate bagasse hydrolysis sugar liquid concentration, configuration fermentation culture, fermention medium consists of: bagasse hydrolyzed solution sugar concentration 20g.L -1, corn steep liquor concentration 10g.L -1, anhydrous chlorides of rase calcium concn 0.5g.L -1, dipotassium hydrogen phosphate concentration 5g.L -1, phosphate dihydrogen sodium concentration 4g.L -1, magnesium chloride hexahydrate concentration 0.05g.L -1, sodium sulphite concentration 0.5g.L -1; Add 4g.L in the fermentation medium -1bagasse hydrolysis after residue, then Actinobacillus succinogenes CCTCC NO:M 2012036 seed liquor is inoculated by the inoculum size of 10%, at the anaerobic condition bottom fermentation of temperature 35 DEG C, pH 5.8, when remaining sugar concentration is lower than fermentation ends during 5g/L, then static, after the bagasse hydrolytic residue in fermented liquid sinks down into fermenter base, transfer supernatant liquor, then in fermentor tank, add fresh fermention medium, do not need inoculation, directly carry out next batch fermentation.Repeated batch fermentation 4 times.
Result shows, repeated batch fermentation 3 times, succinic acid concentration average is respectively 35.2,33.2,30g.L -1, average out to 32.8g.L -1left and right, the average production intensity of succinic acid is 1.54g.L -1.h -1, average glucose acid invert ratio is 0.80g.g -1, in whole fermentation system operational process, there is no downward trend.
Embodiment 9: repeated batch fermentation produces succinic acid
Fermentation production of succinic acid by the following method:
(1) with bagasse hydrolysis sugar liquid for carbon source, prepare fermention medium, wherein in substratum bagasse hydrolyzed solution sugar concentration 50g.L -1; (2) 12g.L is added in the fermentation medium -1bagasse hydrolysis after residue, then by 8% inoculum size inoculation, at the anaerobic condition bottom fermentation 20h of temperature 38 DEG C, pH 5.8 ~ 6.5; (3) static after fermentation ends, after the residue after bagasse hydrolysis in fermented liquid sinks down into fermenter base, transfer supernatant liquor, then adds fresh fermention medium in fermentor tank, does not need inoculation, directly carries out next batch fermentation.
3 batch cycle batch fermentation result displays, succinic acid concentration average is 32.6g.L -1left and right, the average production intensity of succinic acid is 1.64g.L -1.h -1, average glucose acid invert ratio is 0.82g.g -1, in whole fermentation system fortune process, there is no downward trend.
Embodiment 10: repeated batch fermentation produces succinic acid
Fermentation production of succinic acid by the following method:
(1) with bagasse hydrolysis sugar liquid for carbon source, prepare fermention medium, wherein in substratum bagasse hydrolyzed solution sugar concentration 80g.L -1; (2) 20g.L is added in the fermentation medium -1bagasse hydrolysis after residue, then by 8% inoculum size inoculation, at the anaerobic condition bottom fermentation of temperature 38 DEG C, pH 5.8 ~ 6.5, when concentration of reduced sugar in fermented liquid is lower than 20g.L -1time, start to add bagasse hydrolysis sugar liquid and control at 10-20g.L to make the sugared concentration in fermented liquid -1, fermentation 60h; (3) static after fermentation ends, after the residue after bagasse hydrolysis in fermented liquid sinks down into fermenter base, transfer supernatant liquor, then adds fresh fermention medium in fermentor tank, does not need inoculation, directly carries out next batch fermentation.
5 batch cycle batch fermentation result displays, the average production intensity of succinic acid is 1.58g.L -1.h -1, in whole fermentation system fortune process, there is no obvious downward trend.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.

Claims (10)

1. a method for fermentation production of succinic acid, is characterized in that, described method is using bagasse pulverizing, diluted alkaline pre-treatment, is hydrolyzed the hydrolysis sugar liquid that the obtains carbon source as Actinobacillus succinogenes anaerobically fermenting succinic acid, in fermented liquid, add 4 ~ 20gL -1bagasse hydrolysis after residue as the attachment carrier of somatic cells, repeated batch fermentation or Repeat fed-batch fermentation produce succinic acid.
2. method according to claim 1, it is characterized in that, described repeated batch fermentation or Repeat fed-batch fermentation, refer to and adopt the mode of batch fermentation or fed-batch fermentation to produce succinic acid, after each batch terminates, static, after residue after bagasse hydrolysis in fermented liquid sinks down into fermenter base, transfer supernatant liquor, then adds fresh culture in fermentor tank, do not need inoculation, directly carry out next batch fermentation.
3. method according to claim 1, is characterized in that, described method specifically: (1) for carbon source, prepares fermention medium with bagasse hydrolysis sugar liquid, bagasse hydrolyzed solution sugar concentration 20 ~ 100gL in substratum -1; (2) 4 ~ 20gL is added in the fermentation medium -1bagasse hydrolysis after residue, then by 5 ~ 10% inoculum size inoculation, batch fermentation or fed-batch fermentation 20 ~ 60h under the anaerobic condition of temperature 35 ~ 40 DEG C, pH5.8 ~ 6.5; (3) static after fermentation ends, after the residue after bagasse hydrolysis in fermented liquid sinks down into fermenter base, transfer supernatant liquor, then adds fresh fermention medium in fermentor tank, does not need inoculation, directly carries out next batch fermentation.
4. method according to claim 3, is characterized in that, the fed-batch fermentation of the fed-batch fermentation of described step (2) to be fermention medium Initial sugar concentration be more than 50g/L, when concentration of reduced sugar in fermented liquid is lower than 20gL -1time, start to add bagasse hydrolysis sugar liquid and control at 10-20gL to make the sugared concentration in fermented liquid -1, fermentation 25-50h.
5. method according to claim 3, is characterized in that, described fermention medium is: bagasse hydrolyzed solution sugar concentration 20 ~ 100gL -1, corn steep liquor concentration 10 ~ 30gL -1, anhydrous chlorides of rase calcium concn 0.05 ~ 0.5gL -1, dipotassium hydrogen phosphate concentration 1 ~ 5gL -1, phosphate dihydrogen sodium concentration 4 ~ 12gL -1, magnesium chloride hexahydrate concentration 0.05 ~ 0.5gL -1, sodium sulphite concentration 0.05 ~ 0.5gL -1.
6. method according to claim 1, it is characterized in that, the preparation method of the residue after described hydrolysis sugar liquid and bagasse are hydrolyzed is: 20-60 mesh sieve pulverized by bagasse, 40-70 DEG C of oven dry, be the NaOH solution of 0.5-5% by mass concentration, according to solid-to-liquid ratio 1:1 ~ 1:20 (w/v), High Temperature Pre process bagasse 1 ~ 4h, filter washing and obtain pretreated bagasse, 45-70 DEG C of dry for standby; Then pretreated bagasse is made into the solution that mass concentration is 3 ~ 10%, add cellulase, zytase, beta-glucanase and polygalacturonase, temperature 40 ~ 70 DEG C, pH is adjusted to 4.0 ~ 6.5, rotating speed 100-250rmin -1condition under, hydrolysis 24 ~ 72h; Its cellulase, zytase, beta-glucanase and the polygalacturonase addition to every gram of bagasse is respectively: 10 ~ 35FPUg -1, 50 ~ 120Ug -1, 10 ~ 80Ug -1with 5 ~ 30Ug -1; Centrifugal, obtain bagasse hydrolysis sugar liquid and bagasse hydrolytic residue.
7. method according to claim 1, it is characterized in that, described method is specifically: (1) bagasse pre-treatment: 20-60 mesh sieve pulverized by bagasse, 40-70 DEG C of oven dry, be the NaOH solution of 0.5-5% by mass concentration, according to solid-to-liquid ratio 1:1 ~ 1:20 (w/v), High Temperature Pre process bagasse 1 ~ 4h, filter washing and obtain pretreated bagasse, 45-70 DEG C of dry for standby; (2) bagasse hydrolysis: pretreated bagasse is made into the solution that mass concentration is 3 ~ 10%, add cellulase, zytase, beta-glucanase and polygalacturonase, temperature 40 ~ 70 DEG C, pH is adjusted to 4.0 ~ 6.5, rotating speed 100-250rmin -1condition under, hydrolysis 24 ~ 72h; Its cellulase, zytase, beta-glucanase and the polygalacturonase addition to every gram of bagasse is respectively: 10 ~ 35FPUg -1, 50 ~ 120Ug -1, 10 ~ 80Ug -1with 5 ~ 30Ug -1; Centrifugal, obtain bagasse hydrolysis sugar liquid and bagasse hydrolytic residue; (3) fermentation production of succinic acid: regulate bagasse hydrolysis sugar liquid concentration, configuration fermentation culture, fermention medium consists of: bagasse hydrolyzed solution sugar concentration 20-100gL -1, corn steep liquor concentration 10-30gL -1, anhydrous chlorides of rase calcium concn 0.05-0.5gL -1, dipotassium hydrogen phosphate concentration 1-5gL -1, phosphate dihydrogen sodium concentration 4-12gL -1, magnesium chloride hexahydrate concentration 0.05-0.5gL -1, sodium sulphite concentration 0.05-0.5gL -1; Add 4 ~ 20gL in the fermentation medium -1bagasse hydrolysis after residue, then by 5 ~ 10% inoculum size inoculation seed liquor, under the anaerobic condition of temperature 35 ~ 40 DEG C, pH 5.8 ~ 6.5, batch fermentation or fed-batch fermentation 20 ~ 60h; Static after fermentation ends, after the bagasse hydrolytic residue in fermented liquid sinks down into fermenter base, transfer supernatant liquor, then adds fresh fermention medium in fermentor tank, does not need inoculation, directly carries out next batch fermentation.
8. according to the succinic acid that the arbitrary described method of claim 1-7 obtains.
9. succinic acid described in claim 8 is in food, chemistry and the application prepared in medicine.
10. one kind fully utilizes the method for bagasse, it is characterized in that, described method comprises: (1) bagasse through pulverizing, diluted alkaline pre-treatment, be hydrolyzed the hydrolysis sugar liquid that the obtains carbon source as Actinobacillus succinogenes anaerobically fermenting succinic acid, in fermented liquid, add 4 ~ 20gL -1bagasse hydrolysis after residue as the attachment carrier of somatic cells, repeated batch fermentation or Repeat fed-batch fermentation produce succinic acid; (2) raffinate after diluted alkaline pre-treatment bagasse is the chloroform extraction of 1:0.5 ~ 1:1 by volume ratio, obtains furfural crude product.
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CN113980868A (en) * 2021-12-02 2022-01-28 广西科学院 Actinobacillus succinogenes capable of tolerating pentamethyl furfural and breeding method and application thereof
CN113980868B (en) * 2021-12-02 2023-02-03 广西科学院 Actinobacillus succinogenes capable of tolerating pentamethyl furfural and breeding method and application thereof
CN114807250A (en) * 2022-06-08 2022-07-29 山东飞扬化工有限公司 Method for producing succinic acid by fermentation
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