CN104894174B - A kind of method that succinic acid is produced with bagasse fermenting raw materials - Google Patents
A kind of method that succinic acid is produced with bagasse fermenting raw materials Download PDFInfo
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Abstract
The invention discloses a kind of method that succinic acid is produced with bagasse fermenting raw materials, belong to technical field of bioengineering.The inventive method is to squeeze the Main By product bagasse of sugar industry as raw material, and by crushing, diluted alkaline pretreatment, and cellulase, zytase, the cohydrolysis of beta glucan enzyme and pectase obtains hydrolysis sugar liquid；Succinic acid is produced using Actinobacillus succinogenes fermentation hydrolysis liquid glucose, while the residue of bagasse hydrolysis is added in zymotic fluid so that fermentation can be run repeatedly, save seed culture, and the furfural in the pretreated raffinate of diluted alkaline can be extracted with organic solvent.The inventive method, the reasonable employment of bagasse hydrolytic residue is realized, reduce the seed culture link of normal fermentation, save fermentation costs, improve fermentation efficiency, low is required to Zymolysis Equipment simultaneously, it is not necessary to additional improvement is made to existing fermentation tank, is adapted to industrialized production.
Description
Technical field
The present invention relates to a kind of method that succinic acid is produced 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 that cane planting amount is the third-largest in the world
Country, south are the areas that China mainly grows cane, and annual sugarcane total output reaches more than 7,000 ten thousand tons, while can produce 700
Ten thousand tons of bagasse.
Key component is cellulose, hemicellulose and lignin in bagasse.Bagasse is mainly used for burning at present
Generate electricity (light science and technology, 2013, (11):11-12.), passed as feed stripped (Chinese Patent Application No. 94113455.5), replacement
The timber resources of system as papermaking and production high density material (Chinese Plastics, 2008, (02):57-61), the training as edible mushroom
Support base (edible mushroom, 1980, (02):12-16) and Development and Production high value added product：Such as Development and Production xylitol, (science and technology is logical
Report, 2004, (1):37-41), arabinose (amino acid and living resources, 2009, (1):8-12.), furfural (chemical science and technology city
, 2001, (11):12-15).Chinese patent CN 1060464C disclose prepares syringaldehyde with bagasse alkali-lignin catalysis oxidation
With the method for vanillic aldehyde.Chinese patent CN 103614435A disclose a kind of method that xylo-oligosaccharide is prepared from bagasse.
Succinic acid is a kind of industrial important C4 platform chemicals, in food, medicine, chemical industry and other field
Suffer from being widely applied.The culture medium carbon source of the fermentation production of succinic acid research institute of document report is mainly grape at present
Sugar, it is one of approach for reducing fermentation method production succinic acid cost to develop cheap carbon source.The reports such as Brazilian Borge are with dilute
Sulfuric acid solution bagasse, obtained liquid glucose is as carbon source, with the anaerobism shake flask fermentation fourths two of A.succinogenes CIP 106512
Sour 24h, the concentration for obtaining succinic acid are up to 22.5g.L-1(Industrial Microbiology and
Biotechnology,2011,38:1001-1011.).The dilute sulfuric acid acidolysis bagasse such as Xu Rong, then it is pre- with cellulose hydrolyzation
The residue of processing, bagasse hydrolyzate 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 fermenting and producings, yield is relatively low, the cost of subsequent extracted is added.The invention provides
A kind of method of bagasse effectively hydrolyzing and production high concentration succinic acid.
The content of the invention
It is an object of the invention to provide a kind of method that succinic acid is produced using cheap bagasse fermenting raw materials.
The method of the fermentation production of succinic acid, be bagasse is crushed, diluted alkaline pretreatment, the obtained hydrolysis sugar liquid of hydrolysis
As the carbon source of Actinobacillus succinogenes anaerobic fermentation succinic acid, 4~20g.L is added in zymotic fluid-1Bagasse hydrolysis after
Attachment carrier of the residue (dry weight) as somatic cells, repeated batch fermentation or Repeat fed-batch fermentation production succinic acid.
The bagasse crushes, diluted alkaline pretreatment, is to squeeze sugar industry discarded object in one embodiment of the invention
Bagasse is raw material, and dry bagasse is crushed, and crosses 20-40 mesh sieves, and 40-70 DEG C of drying preserves, and is 0.5-5% with mass concentration
NaOH solution, by solid-to-liquid ratio 1:1-1:15 (w/v), 121 DEG C of high temperature pre-process bagasse 2-4h, and bagasse is washed to obtain in filtering,
45-70 DEG C is dried for standby.
The bagasse crushes, diluted alkaline pretreatment, in one embodiment of the invention, after diluted alkaline pretreatment bagasse
Raffinate, with volume ratio be 1:0.5~1:1 chloroform extraction, obtains furfural crude product.
The hydrolysis, in one embodiment of the invention, be by the bagasse of pretreatment be made into mass concentration for 3~
10% solution, hydrolyzed under the synergy of cellulase, zytase, 1,4 beta-glucanase and pectase, 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, through centrifuging to obtain hydrolysis sugar liquid, solid
Residue after the hydrolysis of drying bagasse.
The hydrolysis, in one embodiment of the invention, cellulase, zytase, 1,4 beta-glucanase and pectin
Enzyme is respectively to the addition of every gram of substrate：10-35FPU.g-1、50-120U.g-1、10-80U.g-1And 5-30U.g-1。
Residue after the bagasse hydrolysis, in one embodiment of the invention, addition is 8~18g.L-1。
The repeated batch fermentation or Repeat fed-batch fermentation, in one embodiment of the invention, refer to use
The mode of batch fermentation or fed-batch fermentation produces succinic acid, static after each batch terminates, and treats sugarcane pulp water in zymotic fluid
After residue after solution sinks down into fermenter base, supernatant is shifted, then toward fresh fermentation medium is added in fermentation tank, is not required to
It is inoculated with, directly carries out next batch fermentation.
Methods described, in one embodiment of the invention, it is：(1) using bagasse hydrolysis sugar liquid as carbon source, hair is prepared
20~100g.L of bagasse hydrolyzate sugar concentration in ferment culture medium, wherein culture medium-1；(2) in the fermentation medium addition 4~
20g.L-1Bagasse hydrolysis after residue, then by 5~10% inoculum concentration be inoculated with, 35~40 DEG C of temperature, pH 5.8~
20~60h of batch fermentation or fed-batch fermentation under 6.5 anaerobic condition；(3) it is static after fermentation ends, treat sugarcane in zymotic fluid
After residue after pulp water solution sinks down into fermenter base, supernatant is shifted, then toward adding fresh fermentation medium in fermentation tank,
It need not be inoculated with, directly carry out next batch fermentation.
The fermentation medium of the step (1), in one embodiment of the invention, including：Bagasse hydrolyzate sugar
20~100g.L of concentration-1, 10~30g.L of corn steep liquor concentration-1, 0.05~0.5g.L of anhydrous calcium chloride concentration-1, dipotassium hydrogen phosphate
1~5g.L of concentration-1, 4~12g.L of phosphate dihydrogen sodium concentration-1, 0.05~0.5g.L of magnesium chloride hexahydrate concentration-1, vulcanize na concn
0.05~0.5g.L-1。
The fermentation medium of the step (1), in one embodiment of the invention, bagasse hydrolyzate sugar concentration
55g.L-1。
The fermentation of the step (2), in one embodiment of the invention, is passed through CO2/N2Gas controls fermentation process
For anaerobic condition, saturation NaHCO3Control fermentation pH 5.8~6.5.
The fed-batch fermentation of the step (2), it is that fermentation medium is initially sugared in one embodiment of the invention
Concentration is more than 50g/L fed-batch fermentation, when concentration of reduced sugar is less than 20g.L in zymotic fluid-1When, start to add bagasse hydrolysis
Liquid glucose is so that the sugared concentration in zymotic fluid is controlled in 10-20g.L-1, ferment 25-50h.
The step (2) is 3-5 batch of fermentation in one embodiment of the invention in batches.
The Actinobacillus succinogenes, in one embodiment of the invention, be A.ssuccinogenes F3-21 or
Actinobacillus succinogenes CGMCC NO.1593, CCTCC NO：M 2012036.
Fermentation ends in the step (3), in one embodiment of the invention, refer to zymotic fluid concentration of reduced sugar
Less than 10g.L-1Or less than 5g/L.
Methods described, in one embodiment of the invention, it is specifically：(1) bagasse pre-processes：Bagasse crushes
20-60 mesh sieves, 40-70 DEG C of drying, the NaOH solution for being 0.5-5% with mass concentration, according to solid-to-liquid ratio 1:1~1:20 (w/v),
121 DEG C of high temperature pre-process 1~4h of bagasse, and the bagasse that filtering washing is pre-processed, 45-70 DEG C is dried for standby；(2) sugarcane
Pulp water solution：The bagasse of pretreatment is made into the solution that mass concentration is 3~10%, and addition cellulase, zytase, β-Portugal gather
Carbohydrase and pectase, in 40~70 DEG C of temperature, pH is adjusted to 4.0~6.5, rotating speed 100-250r.min-1Under conditions of, hydrolysis 24~
72h；Its cellulase, zytase, 1,4 beta-glucanase and pectase are respectively to the addition of every gram of bagasse：10~
35FPU.g-1, 50~120U.g-1, 10~80U.g-1With 5~30U.g-1；Centrifugation, obtain bagasse hydrolysis sugar liquid and bagasse hydrolysis
Residue；(3) fermentation production of succinic acid：Bagasse hydrolysis sugar liquid concentration is adjusted, configures fermented and cultured, fermentation medium composition is：
Bagasse hydrolyzate sugar concentration 20-100g.L-1, corn steep liquor concentration 10-30g.L-1, anhydrous calcium chloride concentration 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, vulcanization
Na concn 0.05-0.5g.L-1；4~20g.L is added in the fermentation medium-1Bagasse hydrolysis after residue (with dry weight
Meter), then by 5~10% inoculum concentration be inoculated with seed liquor, 35~40 DEG C of temperature, pH 5.8~6.5 anaerobic condition under, hair
20~60h of ferment；It is static after fermentation ends, after the bagasse hydrolytic residue in zymotic fluid sinks down into fermenter base, in transfer
Clear liquid, then toward adding fresh fermentation medium in fermentation tank, it is not necessary to be inoculated with, directly carry out next batch fermentation.
The present invention is also claimed the succinic acid that is obtained according to methods described and its in food, chemistry and in terms of preparing medicine
Application.
Present invention also offers a kind of method for comprehensively utilizing bagasse, methods described includes：(1) bagasse through crush,
Carbon source of the hydrolysis sugar liquid that diluted alkaline pretreatment, hydrolysis obtain as Actinobacillus succinogenes anaerobic fermentation succinic acid, in zymotic fluid
4~20g.L of middle addition-1Bagasse hydrolysis after attachment carrier of the residue as somatic cells, repeated batch fermentation or repeatedly
Fed-batch fermentation produces succinic acid；(2) raffinate after diluted alkaline pretreatment bagasse, is 1 with volume ratio:0.5~1:1 chloroform
Extraction, obtains furfural crude product.The method that for details, reference can be made to above-mentioned fermentation production of succinic acid.
Beneficial effects of the present invention：
(1) reasonable employment of bagasse hydrolytic residue is realized, with 4~20g.L-1Bagasse hydrolytic residue as thalline
Adhere to carrier loop fermentation succinic acid, reduce the seed culture link of normal fermentation, saved fermentation costs, improved fermentation effect
Rate.(in normal fermentation, seed culture 12-24h, seed culture medium typically contains 10g.L-1Yeast extract above, seed account for raw material
The 2-5% of cost).Meanwhile Batch fermentation can be carried out repeatedly, the succinic acid concentration average of 3 batch cycle batch fermentations is
35g.L-1Left and right, it is 1.65g.L averagely to produce intensity-1.h-1, average saccharic acid conversion ratio is 0.81g.g-1, whole fermentation system fortune
Row 64h, without downward trend.
(2) technique of the invention, low is required to Zymolysis Equipment, it is not necessary to additional improvement is made to existing fermentation tank, fitted
Close industrialized production.
(3) it is fermentation raw material to employ and the cheap squeezing sugar industry discarded object bagasse of grain is not striven with people, multienzyme combination water
Bagasse is solved, has reached 90% total fiber element percent hydrolysis, compared with the acidic treatment of report, has been pre-processed with diluted alkaline, with reference to more
Enzyme synergetic hydrolysis, the hydrolysis sugared content of bagasse is improved, improve the concentration of hydrolyzate fermentation succinic acid-producing.Pass through benefit simultaneously
Expect batch fermentation, the concentration of succinic acid reaches 71g.L-1, succinic acid is 0.46g.g to the yield of bagasse-1。
(4) present invention is alleviated dependence of the succinic acid product to petrochemical material, promoted for the production cost of reduction succinic acid
The utilization of agricultural wastes resource, environmental protection, realize that sustainable recycling economy development has positive economic and social effect.
Brief description of the drawings
Fig. 1：Bagasse fermentation succinic acid process route；
Fig. 2：Multienzyme synergism hydrolysis pretreatment bagasse time graph；
Fig. 3：Conditional curve of the bagasse hydrolyzate as carbon source fed-batch fermentation succinic acid；
Fig. 4：Band slag 3L fermentation tank repeated batch fermentation succinic acid.
Embodiment
Residual sugar and organic acidity test：Pre-process various sugared concentration in bagasse hydrolyzate (glucose, xylose, cellobiose,
Arabinose, glucuronic acid etc.) measure and residual sugar in zymotic fluid and the concentration of organic acid surveyed using high performance liquid chromatography
It is fixed, refer to CN 101603059A patents.
Embodiment 1：Diluted alkaline pre-processes bagasse
The bagasse for taking 20g to be dried after crushing, mixed with mass concentration for 1% NaOH solution 300mL, in 121 DEG C of height
Temperature pretreatment bagasse 2h, filtering wash bagasse to neutrality, 65 DEG C of drying, obtain the bagasse of 13g pretreatments, weight damage
35% is lost, total fiber element content is 87.6%, its composition such as table 1.
Table 1 is the component that NREL methods measure oxygenation pretreatment bagasse
Embodiment 2：Bagasse after multienzyme synergism hydrolysis alkali process
The bagasse that wherein 3g oxygenation pretreatments are good is weighed, is transferred in 150mL triangular flask, adds cellulase 25FPU.g-1, zytase 85U.g-1, 1,4 beta-glucanase 40U.g-1, pectase 25U.g-1, pH5.0 citric acid buffer solution is added, it is fixed
Hold to 40mL, 40h is hydrolyzed on shaking bath at 50 DEG C, a sample is taken every 4h hours.Multienzyme synergism hydrolysis time figure
2.Hydrolysis 40h terminates, and concentration of reduced sugar is 65.6g.L in hydrolyzate-1, the total fiber element hydrolysis efficiency of bagasse is up to
90%, the concentration of glucose is 45.06g.L in bagasse hydrolyzate-1, xylose concentration 14.92g.L-1, glucuronic acid it is dense
Spend for 5.62g.L-1, the concentration ratio of glucose and xylose is close to 3:1.Multienzyme synergism hydrolysis pretreatment bagasse time graph is as schemed
Shown in 2.
Embodiment 3：Bagasse hydrolyzate fed-batch fermentation produces succinic acid
Bacterial strain：Actinobacillus succinogenes (Actinobacillu ssuccinogenes F3-21).Culture medium：Bagasse
Hydrolyzate sugar concentration 55g.L-1, corn steep liquor concentration 25g.L-1, anhydrous calcium chloride concentration 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, vulcanization na concn 0.1g.L-1, pH is adjusted to
6.0,121 DEG C of sterilizing 20min.
On 3L stirred fermentors, using fed-batch fermentation, the concentration of initial bagasse hydrolysis sugar liquid is 55g.L-1,
When remaining sugar concentration is reduced to 20g.L in zymotic fluid-1When, it is 200g.L to add up concentration of reduced sugar with peristaltic pump stream-1Concentration sugarcane
Slag hydrolysis sugar liquid, the sugared concentration in zymotic fluid is set to control in 10-20g.L-1.During fermentation 20-26h, production intensity may remain in
1.7g.L-1.h-1.Fermentation 50 hours, succinic acid-producing 71g.L-1, saccharic acid conversion ratio 0.82g.g-1, production intensity is 1.42g.L-1.h-1.As a result it is as shown in Figure 3.
Embodiment 4：Bagasse residue (SBR) is used as absorption carrier circulating fermentation succinic acid
Fermentation medium is with embodiment 3, and addition 10g.L-1SBR (amount of dry matter, constant weight being dried at 80 DEG C).Hair
121 DEG C of sterilizing 20min of ferment culture medium, access 5% (v/v) seed liquor, in 37 DEG C of fermentations, the rotating speed of fermentation tank is 150r.min-1,
With saturation NaHCO3It is 6.2 or so to control pH, when sugared concentration is reduced to below 5mg/L, terminates this wholesale ferment.Per Batch fermentation
Terminate, zymotic fluid is stood into 2-4h until bagasse natural subsidence is to the bottom of fermentation tank, with peristaltic pump by fermentation supernatant liquid pump
Out, then by fresh culture medium fermentation tank is pumped into, the fermentation system per batch is 1L, and iterative cycles operation is until fermentation knot
Beam.
3 batch cycle batch fermentations, as a result such as table 2 and Fig. 4.Succinic acid concentration average is 35g.L-1Left and right, succinic acid
It is 1.65g.L averagely to produce intensity-1.h-1, average saccharic acid conversion ratio is 0.81g.g-1, whole fermentation system operation 64h, do not have
Downward trend.
Result of the table 2 with slag circulation batch fermentation succinic acid
Embodiment 5：Furfural content in bagasse diluted alkaline pretreatment fluid
As described in Example 1, bagasse pretreatment is carried out, by obtained pretreatment fluid sulphur acid for adjusting pH to 3.0 left sides
It is right.Take 50mL pretreatment fluid and 50mL chloroform (volume ratio 1:1) carry out rocking at room temperature and mix 30min, stand 5h.Take
Machine phase, the content of furfural is determined at 276nm, is calculated in the treatment fluid of 100g bagasse, contains 2.6g furfurals.
Embodiment 6：Influence of bagasse residue (SBR) addition to circulating fermentation succinic acid
By the method for applying example 4, fermentation medium is configured, adds SBR respectively in 100mL triangular flasks, dosage is followed successively by
0.1g, 0.15g, 0.20g, 0.25g, 0.3g, 0.35g, 0.40g, 0.45g, 0.50g, the pH to 6.5 of zymotic fluid is adjusted, adds water
25mL is settled to, 121 DEG C of sterilizing 20min, 5% (v/v) seed liquor is accessed, 37 DEG C of anaerobism shake flask fermentations, stratification, pours out liquid
Body, survey the concentration of succinic acid in zymotic fluid, and add sterilized fermentation medium, repeated batch fermentation three times, as a result such as table
3。
The different SBR concentration of table 3 are to the succinic acid content (g.L that ferments-1) influence
Embodiment 7：Repeat fed-batch fermentation produces succinic acid
Fermentation production of succinic acid by the following method：
(1) sugarcane pre-processes；Bagasse crush 20-60 mesh sieves, 70 DEG C drying, with mass concentration be 5% NaOH solution,
According to solid-to-liquid ratio 1:1 (w/v), high temperature pretreatment bagasse 1h, the bagasse that filtering washing is pre-processed, 45 DEG C of drying are standby
With；Total fiber element content is 86.8% after pretreatment；
(2) bagasse hydrolyzes：The bagasse of pretreatment is made into the solution that mass concentration is 3%, and addition cellulase, wood are poly-
Carbohydrase, 1,4 beta-glucanase and pectase, in 40 DEG C of temperature, pH is adjusted to 6.5, rotating speed 100-250r.min-1Under conditions of, hydrolysis
72h；Its cellulase, zytase, 1,4 beta-glucanase and pectase are respectively to the addition of every gram of bagasse：
10FPU.g-1、50U.g-1、10U.g-1And 5U.g-1；Centrifugation, obtains bagasse hydrolysis sugar liquid and bagasse hydrolytic residue；In hydrolyzate
Concentration of reduced sugar is 60.6g.L-1。
(3) fermentation production of succinic acid：Bagasse hydrolysis sugar liquid concentration is adjusted, configures fermented and cultured, fermentation medium composition
For：Bagasse hydrolyzate sugar concentration 100g.L-1, corn steep liquor concentration 30g.L-1, anhydrous calcium chloride concentration 0.05g.L-1, phosphoric acid hydrogen
Two potassium concn 1g.L-1, phosphate dihydrogen sodium concentration 12g.L-1, magnesium chloride hexahydrate concentration 0.5g.L-1, vulcanization na concn 0.05g.L-1；
8g.L is added in the fermentation medium-1Bagasse hydrolysis after residue, then by 5% inoculum concentration be inoculated with butanedioic acid unwrapping wire bar
Bacterium CGMCC NO.1593 seed liquors, 40 DEG C of temperature, pH 6.5 anaerobic condition under ferment；When concentration of reduced sugar in zymotic fluid
Less than 20g.L-1When, start to add bagasse hydrolysis sugar liquid so that the sugared concentration in zymotic fluid is controlled in 10-20g.L-1, fermentation
40h；Static 1.5h after fermentation ends, the bagasse hydrolytic residue in zymotic fluid sink down into fermenter base, then shift supernatant
Liquid, then toward adding fresh fermentation medium in fermentation tank, it is not necessary to be inoculated with, directly carry out next batch fermentation.Repeatedly in batches
Fermentation 5 times.
As a result show, the succinic acid concentration average of repeated batch fermentation is 58g.L-1Left and right, the average production of succinic acid are strong
Spend for 1.61g.L-1.h-1, average saccharic acid conversion ratio is 0.80g.g-1, there is no in whole fermentation system running what is declined to become
Gesture.
Embodiment 8：Repeated batch fermentation produces succinic acid
Fermentation production of succinic acid by the following method：
(1) bagasse pre-processes；Bagasse crushes 20-60 mesh sieves, 40 DEG C of drying, with the NaOH that mass concentration is 0.5%
Solution, according to solid-to-liquid ratio 1:20 (w/v), 121 DEG C of high temperature pre-process bagasse 4h, and the bagasse pre-processed is washed in filtering,
70 DEG C are dried for standby；Total fiber element content is 84.6% after pretreatment；
(2) bagasse hydrolyzes：The bagasse of pretreatment is made into the solution that mass concentration is 10%, addition cellulase, wood
Dextranase, 1,4 beta-glucanase and pectase, in temperature 70 C, pH is adjusted to 4.0, rotating speed 100-250r.min-1Under conditions of, hydrolysis
24h；Its cellulase, zytase, 1,4 beta-glucanase and pectase are respectively to the addition of every gram of bagasse：
35FPU.g-1、120U.g-1、80U.g-1And 30U.g-1；Centrifugation, obtains bagasse hydrolysis sugar liquid and bagasse hydrolytic residue；Hydrolyzate
Middle concentration of reduced sugar is 62.8g.L-1, the total fiber element hydrolysis efficiency of bagasse is up to 89%, Portugal in bagasse hydrolyzate
The concentration of grape sugar is 42.86g.L-1, xylose concentration 14.32g.L-1, the concentration of glucuronic acid is 5.62g.L-1, glucose and
The concentration ratio of xylose is close to 3:1.
(3) fermentation production of succinic acid：Bagasse hydrolysis sugar liquid concentration is adjusted, configures fermented and cultured, fermentation medium composition
For：Bagasse hydrolyzate sugar concentration 20g.L-1, corn steep liquor concentration 10g.L-1, anhydrous calcium chloride concentration 0.5g.L-1, phosphoric acid hydrogen two
Potassium concn 5g.L-1, phosphate dihydrogen sodium concentration 4g.L-1, magnesium chloride hexahydrate concentration 0.05g.L-1, vulcanization na concn 0.5g.L-1；
4g.L is added in fermentation medium-1Bagasse hydrolysis after residue, then by 10% inoculum concentration be inoculated with butanedioic acid unwrapping wire bar
Bacterium CCTCC NO：The seed liquors of M 2012036,35 DEG C of temperature, pH 5.8 anaerobic condition under ferment, when remaining sugar concentration is less than
Fermentation ends during 5g/L, it is then static, after the bagasse hydrolytic residue in zymotic fluid sinks down into fermenter base, shift supernatant
Liquid, then toward adding fresh fermentation medium in fermentation tank, it is not necessary to be inoculated with, directly carry out next batch fermentation.Repeatedly in batches
Fermentation 4 times.
As a result show, 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 saccharic acid conversion ratio is 0.80g.g-1, entirely
There is no downward trend in fermentation system running.
Embodiment 9：Repeated batch fermentation produces succinic acid
Fermentation production of succinic acid by the following method：
(1) using bagasse hydrolysis sugar liquid as carbon source, fermentation medium is prepared, bagasse hydrolyzate sugar is dense wherein in culture medium
Spend 50g.L-1；(2) 12g.L is added in the fermentation medium-1Bagasse hydrolysis after residue, then connect by 8% inoculum concentration
Kind, 38 DEG C of temperature, pH 5.8~6.5 anaerobic condition under ferment 20h；(3) it is static after fermentation ends, treat sugarcane in zymotic fluid
After residue after pulp water solution sinks down into fermenter base, supernatant is shifted, then toward adding fresh fermentation medium in fermentation tank,
It need not be inoculated with, directly carry out next batch fermentation.
3 batch cycle batch fermentation results show that succinic acid concentration average is 32.6g.L-1Left and right, the average life of succinic acid
Production intensity is 1.64g.L-1.h-1, average saccharic acid conversion ratio is 0.82g.g-1, there is no what is declined during whole fermentation system fortune
Trend.
Embodiment 10：Repeated batch fermentation produces succinic acid
Fermentation production of succinic acid by the following method：
(1) using bagasse hydrolysis sugar liquid as carbon source, fermentation medium is prepared, bagasse hydrolyzate sugar is dense wherein in culture medium
Spend 80g.L-1；(2) 20g.L is added in the fermentation medium-1Bagasse hydrolysis after residue, then connect by 8% inoculum concentration
Kind, 38 DEG C of temperature, pH 5.8~6.5 anaerobic condition under ferment, when concentration of reduced sugar is less than 20g.L in zymotic fluid-1When, open
Begin to add bagasse hydrolysis sugar liquid so that the sugared concentration in zymotic fluid is controlled in 10-20g.L-1, ferment 60h；(3) after fermentation ends
It is static, after the residue after bagasse hydrolysis in zymotic fluid sinks down into fermenter base, supernatant is shifted, then toward in fermentation tank
Add fresh fermentation medium, it is not necessary to be inoculated with, directly carry out next batch fermentation.
5 batch cycle batch fermentation results show that the average production intensity of succinic acid is 1.58g.L-1.h-1, whole fermentation
The trend not being decreased obviously during system fortune.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (8)
- A kind of 1. method of fermentation production of succinic acid, it is characterised in that methods described be bagasse is crushed, diluted alkaline pretreatment, Hydrolyze the carbon source of obtained hydrolysis sugar liquid as Actinobacillus succinogenes anaerobic fermentation succinic acid, in zymotic fluid addition 4~ 20g·L-1Bagasse hydrolysis after attachment carrier of the residue as somatic cells, repeated batch fermentation or repeatedly fed-batch Fermentation production of succinic acid.
- 2. according to the method for claim 1, it is characterised in that the repeated batch fermentation or Repeat fed-batch fermentation, Refer to produce succinic acid by the way of batch fermentation or fed-batch fermentation, it is static after each batch terminates, treat in zymotic fluid After residue after bagasse hydrolysis sinks down into fermenter base, supernatant is shifted, then toward adding fresh culture in fermentation tank, It need not be inoculated with, directly carry out next batch fermentation.
- 3. according to the method for claim 1, it is characterised in that methods described is specifically：(1) using bagasse hydrolysis sugar liquid as Carbon source, prepares fermentation medium, 20~100gL of bagasse hydrolysis sugar liquid sugar concentration in culture medium-1；(2) in fermentation medium 4~20gL of middle addition-1Bagasse hydrolysis after residue, then by 5~10% inoculum concentration be inoculated with, in temperature 35~40 DEG C, 20~60h of batch fermentation or fed-batch fermentation under the anaerobic conditions of pH5.8~6.5；(3) it is static after fermentation ends, it is pending After residue in zymotic fluid after bagasse hydrolysis sinks down into fermenter base, supernatant is shifted, it is then fresh toward adding in fermentation tank Fermentation medium, it is not necessary to be inoculated with, directly carry out next batch fermentation.
- 4. according to the method for claim 3, it is characterised in that the fed-batch fermentation of the step (2) is fermented and cultured Base Initial sugar concentration is more than 50g/L fed-batch fermentation, when concentration of reduced sugar is less than 20gL in zymotic fluid-1When, start to add Bagasse hydrolysis sugar liquid is so that the sugared concentration in zymotic fluid is controlled in 10-20gL-1, ferment 25-50h.
- 5. according to the method for claim 3, it is characterised in that the fermentation medium is：Bagasse hydrolysis sugar liquid sugar is dense Spend 20~100gL-1, 10~30gL of corn steep liquor concentration-1, 0.05~0.5gL of anhydrous calcium chloride concentration-1, dipotassium hydrogen phosphate 1~5gL of concentration-1, 4~12gL of phosphate dihydrogen sodium concentration-1, 0.05~0.5gL of magnesium chloride hexahydrate concentration-1, vulcanized sodium is dense Spend 0.05~0.5gL-1。
- 6. according to the method for claim 1, it is characterised in that the system of the residue after hydrolysis sugar liquid and the bagasse hydrolysis Preparation Method is：Bagasse crushes 20-60 mesh sieves, 40-70 DEG C of drying, the NaOH solution for being 0.5-5% with mass concentration, according to solid Liquor ratio 1:1~1:20 (w/v), high temperature pretreatment 1~4h of bagasse, the bagasse that filtering washing is pre-processed, 45-70 DEG C of baking It is dry standby；Then by the bagasse of pretreatment be made into mass concentration be 3~10% solution, addition cellulase, zytase, 1,4 beta-glucanase and pectase, in 40~70 DEG C of temperature, pH is adjusted to 4.0~6.5, rotating speed 100-250rmin-1Under conditions of, Hydrolyze 24~72h；Its cellulase, zytase, 1,4 beta-glucanase and pectase are distinguished the addition of every gram of bagasse For：10~35FPUg-1, 50~120Ug-1, 10~80Ug-1With 5~30Ug-1；Centrifugation, obtains bagasse hydrolysis sugar liquid With bagasse hydrolytic residue.
- 7. according to the method for claim 1, it is characterised in that methods described is specifically：(1) bagasse pre-processes：Sugarcane The broken 20-60 mesh sieves of ground-slag, 40-70 DEG C of drying, the NaOH solution for being 0.5-5% with mass concentration, according to solid-to-liquid ratio 1:1~1:20 (w/v), high temperature pretreatment 1~4h of bagasse, the bagasse that filtering washing is pre-processed, 45-70 DEG C is dried for standby；(2) it is sweet Bagasse hydrolyzes：The bagasse of pretreatment is made into the solution that mass concentration is 3~10%, addition cellulase, zytase, β-Portugal Dextranase and pectase, in 40~70 DEG C of temperature, pH is adjusted to 4.0~6.5, rotating speed 100-250rmin-1Under conditions of, hydrolysis 24~72h；Its cellulase, zytase, 1,4 beta-glucanase and pectase are respectively to the addition of every gram of bagasse：10 ~35FPUg-1, 50~120Ug-1, 10~80Ug-1With 5~30Ug-1；Centrifugation, obtains bagasse hydrolysis sugar liquid and sugarcane Slag hydrolytic residue；(3) fermentation production of succinic acid：Bagasse hydrolysis sugar liquid concentration is adjusted, configures fermented and cultured, fermentation medium group Turn into：Bagasse hydrolysis sugar liquid sugar concentration 20-100gL-1, corn steep liquor concentration 10-30gL-1, anhydrous calcium chloride concentration 0.05-0.5g·L-1, dipotassium hydrogen phosphate concentration 1-5gL-1, phosphate dihydrogen sodium concentration 4-12gL-1, magnesium chloride hexahydrate concentration 0.05-0.5g·L-1, vulcanization na concn 0.05-0.5gL-1；4~20gL is added in the fermentation medium-1Sugarcane pulp water Residue after solution, then it is inoculated with seed liquor, the anaerobism bar in 35~40 DEG C of temperature, pH5.8~6.5 by 5~10% inoculum concentration Under part, 20~60h of batch fermentation or fed-batch fermentation；It is static after fermentation ends, treat the bagasse hydrolytic residue in zymotic fluid After sinking down into fermenter base, supernatant is shifted, then toward adding fresh fermentation medium in fermentation tank, it is not necessary to be inoculated with, directly Tap into the fermentation of row next batch.
- A kind of 8. method for comprehensively utilizing bagasse, it is characterised in that methods described includes：(1) bagasse is pre- through crushing, diluted alkaline Carbon source of the hydrolysis sugar liquid that processing, hydrolysis obtain as Actinobacillus succinogenes anaerobic fermentation succinic acid, adds 4 in zymotic fluid ~20gL-1Bagasse hydrolysis after attachment carrier of the residue as somatic cells, repeated batch fermentation or repeatedly feed supplement divide Criticize fermentation production of succinic acid；(2) raffinate after diluted alkaline pretreatment bagasse, is 1 with volume ratio:0.5~1:1 chloroform extraction, Obtain furfural crude product.
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| CN106811553B (en) * | 2017-02-09 | 2020-11-27 | 安徽农业大学 | Preparation method of prebiotics brown sugar |
| CN109013649A (en) * | 2018-07-24 | 2018-12-18 | 天峨县平昌生态农业有限公司 | A kind of sugarcane waste residue biodegrading process after hydrolysis |
| CN109097417B (en) * | 2018-08-17 | 2020-10-20 | 中国科学院青岛生物能源与过程研究所 | Whole-bacterium saccharification method for improving lignocellulose saccharification efficiency |
| CN112574925A (en) * | 2020-12-30 | 2021-03-30 | 广西科学院 | Furfural-tolerant actinobacillus succinogenes GXAS-137FM as well as 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 |
| CN117187309B (en) * | 2023-11-06 | 2024-03-08 | 山东小为生物科技有限公司 | Method for preparing succinic acid by bagasse fermentation |
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