CN102703523A - Method for producing butanol by mixed fermentation of bagasse and molasses serving as raw materials - Google Patents
Method for producing butanol by mixed fermentation of bagasse and molasses serving as raw materials Download PDFInfo
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- CN102703523A CN102703523A CN2012102282959A CN201210228295A CN102703523A CN 102703523 A CN102703523 A CN 102703523A CN 2012102282959 A CN2012102282959 A CN 2012102282959A CN 201210228295 A CN201210228295 A CN 201210228295A CN 102703523 A CN102703523 A CN 102703523A
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Abstract
The invention discloses a method for producing butanol by mixed fermentation of bagasse and molasses serving as raw materials. The method comprises the following steps of: hydrolyzing the crushed bagasse by using dilute acid, directly mixing the obtained hydrolysate and the molasses to form the fermentation raw materials, and fermenting the fermentation raw materials by using Clostridium beijerinckii gxas. 28CCTCC M201454 to produce the butanol. The yield of the butanol can reach 11.07 g/L. By adopting the method, the sugar making byproduct of sugarcane can be effectively used for producing the butanol through fermentation, the process is simple, the raw material cost for producing the butanol by microbial fermentation is reduced, and the method has social significance and application value.
Description
Technical field
The present invention relates to the working method of acetone, butanols, specifically, is that bagasse and the molasses with cheapness are raw material, through Bai Shi clostridium gxas.28 pentose and hexose is changed into the process of acetone and butanols, belongs to technical field of bioengineering.
Background technology
Butanols is a kind of important C4 hardware and software platform compound, is mainly used in the synthetic of n-butyl phthalate, butyl phosphate, Bing Xisuandingzhi and N-BUTYL ACETATE etc.; Also can be used as the extraction solvent of grease, medicine and spices and the fields such as additive of Synolac.Along with the development in pluralism of the biomass-making energy, because butanols has the energy density height, wetting ability is low, and steam forces down, characteristics such as octane value height, and being considered to has one of novel liquid fuel of development potentiality most.
Butylic fermentation once was second large leavened prod that the whole world is only second to ethanol fermentation.China has set up the enterprise of a plurality of ten thousand tons of production of butanol, mainly is to be raw material with corn, potato class, utilizes clostridium acetobutylicum fermentative prodn butanols.Because the raw materials cost of biological butanol accounts for 75 % of total cost, cost is too high to have retrained promoting the use of that biological butanol acts as a fuel.Limited since China in 2006 and to have utilized grain to produce the thing energy, the biomass energy of the non-grain of encourage growth next life.Utilizing raw material production butanols such as molasses, cassava, agricultural wastes and Mierocrystalline cellulose is a kind of trend, but since immature, the fermentation strain of raw material pretreatment technology to produce total solvent lower, butanols becomes the mass-produced bottleneck of biological butanol than problem such as not high.Fast development along with genomics and proteomics; The genome sequencing of clostridium acetobutylicum (Clostridium acetobutylicumATCC824) bacterial strain is accomplished (N lling J in calendar year 2001; Breton G; Omelchenko M V, et al. Genomesequence and comparative analysis of the solvent producing bacterium Clostridium acetobutylicum. Bacteriol, 2001; 183 (6): 4823-4838), genetic operating system successfully makes up; The genome sequencing of Bai Shi clostridium (Clostridium beijerinckii NCIMB8052) bacterial strain was accomplished in 2007; Molecular genetic os makes up (Copeland A; Lucas S; Lapidus A, et al. Clostridium beijerinckii NCIMB8052, complete genome.
Http:// www.ncbi.nlm.nih.gov/entrez/viewer.fcgi db=nucleotide& Val=CP000721)The transformation of domestic main research clostridium acetobutylicum, less to the research of Bai Shi clostridial.A lot of researchs show that Bai Shi clostridial raw material is composed and appropriate pH is wider, on the butylic fermentation industry, have bigger application potential.
Bagasse and molasses all are the main sub products of sugar industry, cheaply have that quantity is big, the place of production concentrates, and its composition is single not to need that sorting is handled, advantage such as quality controllable.Compare with the agricultural-forestry biomass resource of other types, bagasse and molasses all are the raw materials of good biomass refining industry, are important renewable biomass resources.China is the 3rd cane planting big country that is only second to Brazil and India, about more than 2,000 ten thousand tons of the output of annual bagasse, and molasses output reaches more than 3,000 ten thousand tons.Because the trans-utilization technique means falls behind, traditionally bagasse often go out of use need not or majority only be used as fuel, its utilization ratio is very low, has not only caused the waste of resource, but also has brought the pollution of environment.Bagasse and molasses with cheapness are that fermenting raw materials is produced the cost that butanols can reduce biological butanol, have the market competitiveness.
The application of cellulase degradation bagasse technology in industrial production prematurity still at present; But can obtain the mixing sugar of lower concentration through the dilute sulphuric acid hydrolysis; Staple is a wood sugar, and having in its hydrolysate manyly has inhibiting material to mikrobe: mainly contain degraded product, salt of aldehydes, organic acid, furans, lignin etc.Because the concentration of sugar is lower, generally need to concentrate and through detoxification as the butylic fermentation wine with dregs.It is the method that fermenting raw materials prepares butanols with the lignocellulose-like biomass that patent 201019026109.1 discloses a kind of, and wherein hydrolyzed solution need be used membrane separation technique and adsorption separation technology detoxification.Patent 200810141418.9 provides a kind of producing xylose with stalks cogeneration of propanone, butanols and alcoholic acid method utilized.It is the method for raw material production ethanol and butanols with the lignocellulose that patent 201111304843.7 discloses a kind of.The Bai Shi clostridium (Clostridium beijerinckii260) of nz David Jones professor seed selection can produce butanols (Qureshi N by simultaneous saccharification and fermentation wood fibre hydrolysis liquid; Saha B C; Hectora R E; Et a1.Butanol production from wheat straw by simultaneous saccharification and fermentation using Clostridium beijennckii:Part I:batch fermentation. Biomass and Bioenergy; 2008,32 (2): 168-175).
Summary of the invention
The purpose of this invention is to provide a kind of cane sugar manufacture by product with cheapness is the method that fermenting raw materials is produced butanols, replaces corn and potato starch raw material with non-grain biomass.The cane sugar manufacture by product raw material of cheapness according to the invention is bagasse and molasses.
The present invention achieves the above object through following technical scheme: the cane sugar manufacture by product with cheapness is the method that fermenting raw materials is produced butanols, comprises the steps:
1, pulverized 40～80 mesh sieves after the bagasse oven dry, with the dilute sulphuric acid hydrolysis of 0.25～3 w/v %, the solid-liquid ratio of bagasse and dilute sulphuric acid is 5～10 w/w %; Hydrolysis temperature is 110～130 ℃; Hydrolysis time is 30～90min, adds the alkali neutralization after the hydrolysis, filtered through gauze.
2, the gained hydrolyzed solution does not need detoxification treatment, in the bagasse hydrolyzed solution, directly adds the molasses of different ratios, and total reducing sugar quality percentage composition is 3～7%.
3, fermention medium: the carbon source in the substratum is bagasse hydrolyzed solution and molasses mixture; Total reducing sugar quality percentage composition is 3～7%; Nitrogenous source is 0.1～0.3% ammonium sulfate for the quality percentage composition, and the quality percentage composition of yeast powder is 0.1～0.5%, and inorganic salt are: any one or a few in calcium salt, sodium salt, phosphoric acid salt, sylvite, magnesium salts and the molysite; Transfer pH6.0～6.5,115 ℃ sterilization 15min.
4, with Bai Shi clostridium (Clostridium beijerinckii) gxas.28 seed liquor by volume percentage composition be that 5～10% inoculum size is inoculated in the fermention medium, in 32～39 ℃ of static fermentation 40～60h, obtain butanols, acetone and ethanol fermentation liquid.Utilize gas chromatograph check and analysis tunning.
Said Bai Shi clostridium (Clostridium beijerinckii) gxas.28 is deposited in Luojiashan, Wuchang, Wuhan City, Hubei Province China typical culture collection center, deposit number CCTCC M 2011454 on December 9th, 2011.
Outstanding advantage of the present invention is:
The present invention utilizes Bai Shi clostridium (Clostridium beijerinckii) gxas.28 can effectively transform five-carbon sugar and hexose generates acetone, butanols and ethanol; And obtained higher output and transformation efficiency, reduced the raw materials cost of microbial method fermentative prodn butanols.
Can effectively utilize sugar industry by product bagasse and molasses mixed fermentation to produce butanols, can reduce the raw materials cost of microbial method fermentative prodn butanols, have the market competitiveness.
Description of drawings
Fig. 1 for of the present invention be the process flow sheet of raw materials mix fermentative prodn butanols with bagasse and molasses.
Embodiment
Below further describe technical scheme of the present invention through specific embodiment.Method in following examples if no special instructions, is ordinary method.
Embodiment 1
Of the present invention is the method for raw materials mix fermentative prodn butanols with bagasse and molasses, comprises the steps:
The preparation of step 1, bagasse hydrolyzed solution
40～80 mesh sieves were pulverized in 60 ℃ of oven dry of bagasse.With the dilute sulphuric acid hydrolysis of 0.25～3 w/v %, wherein the solid-liquid ratio of bagasse and dilute sulphuric acid is 8 w/w %, and hydrolysis temperature is 121 ℃, and hydrolysis time is 60min, and hydrolysis is intact with the sodium hydroxide neutralization, crosses then and filters polysaccharide hydrolysis liquid.The DNS method is surveyed total reducing sugar, the content of wood sugar and glucose in the liquid chromatographic detection hydrolyzed solution, and results of hydrolysis is seen table 1.
Different dilute acid concentration 121 ℃ of hydrolysis 1h gained hydrolysis solution compositions of the bagasse of table 1 8% (material-water ratio) and percent hydrolysis
Step 2, be raw material with the bagasse hydrolyzed solution, with Bai Shi clostridium (Clostridium beijerinckii) gxas.28 fermentative prodn butanols.
Plate culture medium (TYA substratum): glucose 40g, Carnis Bovis seu Bubali cream 2g, yeast powder 2g, peptone 6g, ammonium acetate 3g, KH
2PO
40.5g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.01 g, agar 20 g, zero(ppm) water is settled to 1000mL, 6.5,121 ℃ of autoclaving 20 min of pH.
Seed culture medium: glucose 40g, Carnis Bovis seu Bubali cream 2g, yeast powder 2g, peptone 6g, ammonium acetate 3g, KH
2PO
40.5g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.01g, zero(ppm) water is settled to 1000mL, 6.5,121 ℃ of autoclaving 20 min of pH.
Fermention medium: yeast powder 2.5 g, lime carbonate 1.5 g, superphosphate of lime 0.35 g, ammonium sulfate 1.5 g distinguish constant volume to 1000 mL with the bagasse hydrolyzed solution of different dilute acid concentrations preparations, and pH 6.2～6.5.Above composition is mixed, be sub-packed in the triangular flask of 250 mL, the bottled 150mL of each triangle, plug sealing, 115 ℃ of autoclaving 15min.
Bai Shi clostridium (Clostridium beijerinckii) the gxas.28 coating TYA plate culture medium of getting the glycerine preservation places 37 ℃ of anaerobism incubators to cultivate 20h, chooses single bacterium colony in the TYA liquid nutrient medium, 37 ℃ of static OD that are cultured to
600Be about 1.0; 80 ℃ of heat shock 10min kill the nourishing body cell, are that 5～10% inoculum size is inoculated in the triangular flask of dress 150mL fermention medium 37 ℃ of static fermentation 60h according to volumn concentration; The gained fermented liquid is used the gas chromatographic detection analysis, and the fermentation result sees table 2.
Table 2 Bai Shi clostridium (Clostridium beijerinckii) gxas.28 strain fermentation bagasse hydrolyzed solution is produced acetone, ethanol and butanols
Table 2 is the result show: Bai Shi clostridium (Clostridium beijerinckii) gxas.28 is with the bagasse hydrolyzed solution fermentative prodn butanols of 0.5% volume dilute sulphuric acid preparation, and butanols output can reach 5.48 g/L.Along with the increase of hydrolyzed solution dilute sulphuric acid concentration, butanols productive rate and transformation efficiency begin to descend.
The pre-treatment cost, toxic substance of taking all factors into consideration bagasse are to the influence of bacterial strain and the factor of tunning transformation efficiency, and embodiment selects for use the dilute sulphuric acid hydrolysis bagasse of 0.5% volume to prepare hydrolyzed solution.
Embodiment 2
Step 1 is identical with embodiment 1.Plate culture medium and seed culture medium are with embodiment 1.
Step 2, Bai Shi clostridium (Clostridium beijerinckii) gxas.28 are raw materials mix fermentative prodn butanols with bagasse hydrolyzed solution and molasses
The fermention medium I: 80 sag molasses 50g, yeast powder 5g, lime carbonate 3.0g, superphosphate of lime 0.7g, ammonium sulfate 3.0g, the bagasse hydrolyzed solution of 0.5% volume dilute sulphuric acid preparation is settled to 1000mL, and pH 6.2～6.5.Above composition is mixed, be sub-packed in the triangular flask of 250 mL, the bottled 200mL of each triangle, plug sealing, 115 ℃ of autoclaving 15min.
The fermention medium II: 80 sag molasses 60g, yeast powder 5g, lime carbonate 3.0g, superphosphate of lime 0.7g, ammonium sulfate 3.0g, the bagasse hydrolyzed solution of 0.5% volume dilute sulphuric acid preparation is settled to 1000mL, and pH 6.2～6.5.Above composition is mixed, be sub-packed in the triangular flask of 250 mL, the bottled 200mL of each triangle, plug sealing, 115 ℃ of autoclaving 15min.
The fermention medium III: 80 sag molasses 70g, yeast powder 5g, lime carbonate 3.0g, superphosphate of lime 0.7g, ammonium sulfate 3.0g, the bagasse hydrolyzed solution of 0.5% volume dilute sulphuric acid preparation is settled to 1000mL, and pH 6.2～6.5.Above composition is mixed, be sub-packed in the triangular flask of 250 mL, the bottled 200mL of each triangle, plug sealing, 115 ℃ of autoclaving 15min.
The Bai Shi clostridium gxas.28 coating TYA plate culture medium of getting the glycerine preservation places 37 ℃ of anaerobism incubators to cultivate 20h, chooses single bacterium colony in the TYA liquid nutrient medium, 37 ℃ of static OD that are cultured to
600Be about 1.0; 80 ℃ of heat shock 10min kill the nourishing body cell, are that 5～10% inoculum size is inoculated in the triangular flask of dress 150mL fermention medium 37 ℃ of static fermentation 60h according to volumn concentration; The gained fermented liquid is used the gas chromatographic detection analysis, and the fermentation result sees table 3.
Table 3 Bai Shi clostridium (Clostridium beijerinckii) gxas.28 produces acetone, ethanol and butanols with bagasse hydrolyzed solution and molasses mixed fermentation
Table 3 is the result show: in the bagasse hydrolyzed solution of 0.5% volume dilute sulphuric acid preparation, add the molasses of different ratios, what transformation efficiency and butanols output were the highest is ferment substratum II, and butanols output is up to 11.07 g/L.
Embodiment 3
Plate culture medium and seed culture medium are with embodiment 1.
Bai Shi clostridium (Clostridium beijerinckii) gxas.28 is with glucose, wood sugar and be mixed into fermenting raw materials production butanols.
Fermention medium I: glucose 60g, Carnis Bovis seu Bubali cream 2g, yeast powder 2g, peptone 6g, ammonium acetate 3g, KH
2PO
40.5g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.01 g, agar 20 g, zero(ppm) water is settled to 1000mL.Above composition is mixed, be sub-packed in the triangular flask of 250 mL, the bottled 200mL of each triangle, plug sealing, 115 ℃ of autoclaving 15min.
Fermention medium II: wood sugar 60g, Carnis Bovis seu Bubali cream 2g, yeast powder 2g, peptone 6g, ammonium acetate 3g, KH
2PO
40.5g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.01 g, agar 20 g, zero(ppm) water is settled to 1000mL.Above composition is mixed, be sub-packed in the triangular flask of 250 mL, the bottled 200mL of each triangle, plug sealing, 115 ℃ of autoclaving 15min.
Fermention medium III: glucose 30g, wood sugar 30g, Carnis Bovis seu Bubali cream 2g, yeast powder 2g, peptone 6g, ammonium acetate 3g, KH
2PO
40.5g, MgSO
47H
2O 0.2g, FeSO
47H
2O 0.01 g, agar 20 g, zero(ppm) water is settled to 1000mL.Above composition is mixed, be sub-packed in the triangular flask of 250 mL, the bottled 200mL of each triangle, plug sealing, 115 ℃ of autoclaving 15min.
Bai Shi clostridium (Clostridium beijerinckii) the gxas.28 coating TYA plate culture medium of getting the glycerine preservation places 37 ℃ of anaerobism incubators to cultivate 20h, chooses single bacterium colony in the TYA liquid nutrient medium, 37 ℃ of static OD that are cultured to
600Be about 1.0; 80 ℃ of heat shock 10min kill the nourishing body cell, are that 5～10% inoculum size is inoculated in the triangular flask of dress 150mL fermention medium 37 ℃ of static fermentation 60h according to volumn concentration; The gained fermented liquid is used the gas chromatographic detection analysis, and the fermentation result sees table 4.
Table 4 Bai Shi clostridium (Clostridium beijerinckii) gxas.28 produces acetone, ethanol and butanols with glucose, wood sugar and mixed fermentation
Fermentation is the result show: Bai Shi clostridium (Clostridium beijerinckii) gxas.28 is effectively transforming glucose, xylose production acetone-butanol not only, and the performance that two kinds of sugar is mixed into fermenting raw materials production butanols is also fine.
Claims (7)
1. one kind is the method for raw materials mix fermentative prodn butanols with bagasse and molasses, it is characterized in that, this method may further comprise the steps:
A) utilize dilute sulphuric acid hydrolysis bagasse powder to prepare polysaccharide hydrolysis liquid,
B) the gained hydrolyzed solution directly mixes the carbon source as fermention medium with molasses, adds nitrogenous source and inorganic salt, regulates the pH value, sterilization,
C) with Bai Shi clostridium (Clostridium beijerinckii) gxas.28 fermentative prodn butanols.
2. according to claim 1 is the method for raw materials mix fermentative prodn butanols with bagasse and molasses, it is characterized in that: said bagasse powder is that bagasse was pulverized 40～80 mesh sieves after drying.
3. according to claim 1 is the method for raw materials mix fermentative prodn butanols with bagasse and molasses; It is characterized in that: the said dilute sulphuric acid hydrolysis bagasse powder that utilizes, described dilute sulphuric acid is the sulphuric acid soln of 0.25～3 w/v %, the solid-liquid ratio of bagasse and dilute sulphuric acid is 5～10 w/w %; Hydrolysis temperature is 110～130 ℃; Hydrolysis time is 30～90min, adds the alkali neutralization, filtered through gauze after hydrolysis is intact.
4. according to claim 1 is the method for raw materials mix fermentative prodn butanols with bagasse and molasses; It is characterized in that: the bagasse hydrolyzed solution directly mixes with molasses; Hydrolyzed solution does not need detoxification treatment; Directly in the bagasse hydrolyzed solution, add the molasses of different ratios, total reducing sugar quality percentage composition is 3～7%.
5. according to claim 1 is the method for raw materials mix fermentative prodn butanols with bagasse and molasses; It is characterized in that: the carbon source of said fermention medium is the mixture of bagasse hydrolyzed solution and molasses; Total reducing sugar quality percentage composition is 3～7%; Nitrogenous source is 0.1～0.3% ammonium sulfate for the quality percentage composition, and the quality percentage composition of yeast powder is 0.1～0.5%, and inorganic salt are: any one or a few in calcium salt, sodium salt, phosphoric acid salt, sylvite, magnesium salts and the molysite; Transfer pH6.0～6.5,115 ℃ sterilization 15min.
6. according to claim 1 is the method for raw materials mix fermentative prodn butanols with bagasse and molasses, it is characterized in that: said bacterial strain is Bai Shi clostridium (Clostridium beijerinckii) gxas.28 CCTCC M 2011454.
7. according to claim 1 is the method for raw materials mix fermentative prodn butanols with bagasse and molasses; It is characterized in that: said fermentative prodn butanols be with Bai Shi clostridium (Clostridium beijerinckii) gxas.28 by volume percentage composition be that 5～10% inoculum size is inoculated in the fermention medium; In 32～39 ℃ of static fermentation 40～60h, obtain butanols, acetone and ethanol fermentation liquid.
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| CN103667147A (en) * | 2013-12-11 | 2014-03-26 | 南京工业大学 | High ferulic acid stress resistance clostridium beijerinckii and application thereof |
| CN103898010A (en) * | 2014-03-10 | 2014-07-02 | 广州甘蔗糖业研究所 | High-tolerance clostridium beijerinckii and application thereof |
| CN108504616A (en) * | 2018-04-03 | 2018-09-07 | 广西科学院 | A kind of recombination Clostridium beijerinckii of high-efficiency fermenting sucrose and the method for improving Clostridium beijerinckii sucrose fermenting property |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667147A (en) * | 2013-12-11 | 2014-03-26 | 南京工业大学 | High ferulic acid stress resistance clostridium beijerinckii and application thereof |
| CN103667147B (en) * | 2013-12-11 | 2015-07-01 | 南京工业大学 | High ferulic acid stress resistance clostridium beijerinckii and application thereof |
| CN103898010A (en) * | 2014-03-10 | 2014-07-02 | 广州甘蔗糖业研究所 | High-tolerance clostridium beijerinckii and application thereof |
| WO2015135302A1 (en) * | 2014-03-10 | 2015-09-17 | 广州甘蔗糖业研究所 | Strain of clostridium beijerinckii with high tolerance and application thereof |
| CN103898010B (en) * | 2014-03-10 | 2016-04-20 | 广州甘蔗糖业研究所 | One plant height tolerance Bai Shi clostridium and application thereof |
| CN108504616A (en) * | 2018-04-03 | 2018-09-07 | 广西科学院 | A kind of recombination Clostridium beijerinckii of high-efficiency fermenting sucrose and the method for improving Clostridium beijerinckii sucrose fermenting property |
| CN108504616B (en) * | 2018-04-03 | 2020-05-29 | 广西科学院 | Recombinant clostridium beijerinckii for efficiently fermenting cane sugar and method for improving fermentation performance of clostridium beijerinckii cane sugar |
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