CN106467920A - A kind of method that starch simultaneous saccharification and fermentation produces butanol - Google Patents
A kind of method that starch simultaneous saccharification and fermentation produces butanol Download PDFInfo
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Abstract
The invention discloses a kind of method that starch simultaneous saccharification and fermentation produces butanol, including（1）The activation of seed：Butanol fermentation bacterium is accessed activation preparation seed liquor in RCM culture medium；（2）The liquefaction of starch：It is 5wt%-10wt% that starch is adjusted to dry substance concentration, and regulation pH value is 6.0-6.5, adds starch liquefacation enzyme, is heated to 60-95 DEG C and is liquefied；（3）Preparation P2 culture medium：P2 culture medium is prepared for stock solution with liquefying starch；（4）Simultaneous saccharification and fermentation：After the starch P2 culture medium deoxygenation after sterilizing, access step（1）The butanol fermentation bacterium seed liquor of activation, is simultaneously introduced diastase, carries out anaerobic fermentation and produces butanol.The inventive method not only avoid the inhibitory action that glucose accumulation produces to saccharifying enzyme, decreases enzyme dosage, reduces production cost；And avoid the inhibitory action to fermented bacterium primary growth for the enzymolysis solution middle and high concentration glucose, shorten fermentation period, improve total solvent yield.
Description
Technical field
The invention belongs to technical field of biological fermentation and in particular to a kind of starch simultaneous saccharification and fermentation produce butanol method.
Background technology
Butanol is a kind of important industrial chemicals, is widely used in various plastics and the manufacture of rubber and the synthesis of the chemicals such as butyraldehyde, butanoic acid, butylamine and butyl lactate.In recent years, with the development of downstream industry, the market demand ramps, and the import volume of China's butanol in 2013 is at 400,000 tons about；Butanol is also the novel biological fuel of another great potential after alcohol fuel, its calorific value, octane number are suitable with gasoline, and steam forces down, miscible with gasoline arbitrarily ratio, usage safety performance is high, and it is discarded not produce environmental pollution produced by the conventional fossil fuel such as SOx and NOx.In addition, butanol will not corrosion pipeline, not hygroscopic, rely on existing gasoline feed pipe road and distribution channel just can realize remotely conveying, based on above good characteristic, biological butanol is classified as second filial generation bio-fuel by the United Nations's International Energy Agency.
Fermentable produces butanol as the potentiality of a kind of great development of important conversion technology and industrialization, for the biomass material of fermentable, mainly has three classes according to traditional division：Sugar, starch and lignocellulose.Currently, with starch-based bio matter, such as Semen Maydiss, Semen Tritici aestivi, Maninot esculenta crantz. etc., the radix as fermentable raw material is the most ripe, market share also highest.Thus starch is widely used as a kind of raw material of high-quality, but production cost is higher, and the productivity ratio of butanol is somewhat limited.
The saccharifying of starch and biomass can carry out step by step with fermentation it is also possible to synchronous carried out.Substep diastatic fermentation refers to the first liquefying-saccharifying in the presence of amylase and saccharifying enzyme by starch and biomass, is then fermented using this sugar liquid.Thang etc.（Thang V H et al.
ApplBiochemBiotechnol,2010,161:157-170）UsingClostridium
SaccharoperbutylacetonicumN1-4 substep diastatic fermentation corn starch, total solvent yield is 20.7g/L.Although this method can make saccharifying and sweat carry out respectively under conditions of optimum, the high concentration glucose after Starch Hydrolysis can suppress the activity of saccharifying enzyme thus reducing enzymolysis efficiency, and then affects efficiency of pcr product.
Simultaneous saccharification and fermentation refers to that starchy material saccharifying and sweat are synchronous in same reactor to be carried out, and saves the inhibitory action reducing Fructus Vitis viniferae sugar accumulation while equipment investment to saccharifying enzyme and fermented bacterium primary growth.Wang Xian etc.（The process optimization of sweet potato dregs simultaneous saccharification and fermentation production ethanol, Transactions of the Chinese Society of Agricultural Engineering, 2012,28:
256-261）Use simultaneous saccharification and fermentation technique in the process optimization of Rhizoma Dioscoreae esculentae fermenting and producing ethanol, mixed after adding Thermostable α-Amylase, be placed in the liquefaction of water bath with thermostatic control shaking table, it is cooled to 30 DEG C after liquefaction, adjusts pH value, add ammonium sulfate, saccharifying enzyme, the yeast after activation, standing for fermentation.It is 4 that the method needs the pH adjusting simultaneous saccharification and fermentation system in advance, then starts follow-up saccharifying and fermentation.Different with the optimal conditionss of fermentation yet with saccharifying, and the characteristic due to bacterial strain itself, the method is only applicable to yeast fermentation ethanol, during for butanol fermentation, effect on driving birds is not good.
CN103898165 A discloses a kind of method that starch-based bio matter simultaneous saccharification and fermentation produces ethanol, including：Using α-amylase and saccharifying enzyme, saccharifying is carried out to starch-based bio matter in saccharifying kettle；Collect the saccharified liquid after enzymolysis；And using Yeast culture, saccharified liquid is carried out fermenting in fermentation cauldron and obtain the fermentation liquid containing ethanol；It is characterized in that：Collect part saccharified liquid from saccharifying kettle while saccharifying and be delivered to defecator, solid phase after filtering is back to saccharifying kettle, and liquid phase after filtering is delivered to fermentation cauldron, the fermentation liquid circulation of same volume is back to saccharifying kettle simultaneously.The method simply achieves saccharifying and fermentation synchronization in time, not carries out in same reactor.
Content of the invention
For the deficiencies in the prior art, the present invention provides a kind of method that starch simultaneous saccharification and fermentation produces butanol.The inventive method not only avoid the inhibitory action that glucose accumulation produces to saccharifying enzyme, decreases enzyme dosage, reduces production cost；And avoid the inhibitory action to fermented bacterium primary growth for the enzymolysis solution middle and high concentration glucose, shorten fermentation period, improve total solvent yield.
The method that starch simultaneous saccharification and fermentation of the present invention produces butanol, including following content：
（1）The activation of seed：Butanol fermentation bacterium is accessed activation preparation seed liquor in RCM culture medium；
（2）The liquefaction of starch：It is 5wt%-10wt% that starch is adjusted to dry substance concentration, and regulation pH value is 6.0-6.5, adds starch liquefacation enzyme, is heated to 60-95 DEG C and is liquefied；
（3）Preparation P2 culture medium：P2 culture medium is prepared for stock solution with liquefying starch；
（4）Simultaneous saccharification and fermentation：After the starch P2 culture medium deoxygenation after sterilizing, access step（1）The butanol fermentation bacterium seed liquor of activation, is simultaneously introduced diastase, carries out anaerobic fermentation and produces butanol.
Step of the present invention（1）Described butanol fermentation bacterium is clostridium acetobutylicum（Clostridium
acetobutylicum) or Clostridium beijerinckii（Clostridium beijerinckii）, such as can using clostridium acetobutylicum (Clostridium
acetobutylicum) ATCC 824, it is purchased from American Type Culture Collection center；Or using Clostridium beijerinckii CM20 described in CN201410731297.9, its Classification And Nomenclature is Clostridium beijerinckii（Clostridium
beijerinckii）, it was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 17th, 2014, deposit number is CGMCC No.9354.Present invention preferably uses Clostridium beijerinckii CM20, this bacterial strain due to the specificity of itself, toleration and better adaptability in this fermentation system, butanol yield is higher.
Step of the present invention（1）Described RCM culture medium prescription is commonly used in the art, Ju Ti Pei Fang（In terms of g/L）For：Peptone 10, beef powder 10, yeast powder 3.0, glucose 5, soluble starch 1.0, sodium chloride 5.0, Sodium Acetate Trihydrate 3.0, L-cysteine hydrochloride 0.5, agar 0.5, with pure water configuration, 115 DEG C of sterilizing 20min.
Step of the present invention（1）Described seed activation is to access the spore liquid of butanol fermentation bacterium in RCM culture medium, and at 36-38 DEG C, Anaerobic culturel 16-20h obtains seed liquor.
Step of the present invention（2）Described starch can be corn starch, wheaten starch, sweet potato starch etc., preferably corn starch.Starch is adjusted to by dry substance concentration using pure water（The quality of contained dried starch in the mixed system of 100g starch and water）For 6wt%-8wt%, it is 6.0-6.5 using alkali liquor regulation system pH value, add starch liquefacation enzyme after adjusting pH value, be heated to 70-90 DEG C, constant temperature stirs 2-5h.Starch liquefacation enzyme is high-temperature resistant alpha-amylase, and addition is 6 × 10-4-
3×10-3G/g starch.
Step of the present invention（3）P2 culture medium is prepared for stock solution with liquefying starch, the formula of P2 culture medium is well-known to those skilled in the art.Ju Ti Pei Fang（In terms of g/L）For：Yeast powder 1, CH3COONH4
2.2, KH2PO40.5, K2HPO4
0.5, MnSO40.01, NaCl 0.01, MgSO4·7H2O
0.2, FeSO40.01, para-amino benzoic acid 0.001, vitamin B1 0.001, biotin 0.001e-3,115 DEG C of sterilizing 20min.
Step of the present invention（4）Purge first with high pure nitrogen, then add the mode deoxygenation of oxygen scavenger, oxygen scavenger is 1g sodium hydrosulfite and 0.6g sodium carbonate is dissolved in 10mL pure water and is obtained.Then according to volume ratio 5%-10% accesses step（1）The seed liquor of activation, is simultaneously introduced diastase, and addition is 0.4 × 10-3-2.0×10-2G/g starch.
Step of the present invention（4）The temperature of fermenting and producing butanol is 35-38 DEG C, and rotating speed is 50-200rpm, and fermentation time is 72-120h.
Compared with prior art, the present invention obtains following beneficial effect：
1st, adopt simultaneous saccharification and fermentation butanol system of the present invention, it is possible to achieve starch saccharification and strain fermentation are synchronously carried out, it is to avoid the inhibitory action to saccharifying enzyme generation for the glucose accumulation, decrease enzyme dosage, reduce production cost；And avoid the inhibitory action to fermented bacterium primary growth for the hydrolyzed solution middle and high concentration glucose during substep diastatic fermentation, shorten fermentation period, improve total solvent yield.
2nd, simultaneous saccharification and fermentation method of the present invention need not filter to saccharified liquid, band slag fermentation, and corn starch saccharifying and fermentation carry out it is not necessary to regulate and control pH in same reactor, and technological operation is simple, reduces production intensity, improves the productivity ratio of solvent.
Biomaterial preservation explanation
The Clostridium beijerinckii that the present invention provides（Clostridium
beijerinckii）CM20 bacterial strain, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica；Deposit number：CGMCC No. 9354；Preservation date：On 06 17th, 2014.
Specific embodiment
Below by embodiment, the invention will be further described.Embodiment is only limitted to the present invention is described rather than limits the scope of the present invention.In the present invention, wt% is mass fraction.
Embodiment 1
With clostridium acetobutylicum (Clostridium
acetobutylicum) ATCC 824 is the strain of fermenting and producing butanol, starch adopts corn starch.
Preparation RCM culture medium, is calculated as with g/L：Peptone 10, beef powder 10, yeast powder 3.0, glucose 5.0, soluble starch 1.0, sodium chloride 5.0, Sodium Acetate Trihydrate 3.0, L-cysteine hydrochloride 0.5, agar 0.5, with pure water configuration, 115 DEG C of sterilizing 20min.
（1）The activation of seed
The spore liquid of ATCC 824 bacterial strain is accessed in RCM culture medium, Anaerobic culturel 20h at 37 DEG C, the seed liquor of activation is obtained.
（2）The liquefaction of starch
Using pure water, corn starch being adjusted to dry substance concentration is 8wt%, using certain density NaOH solution regulation system pH to 6.0, adds starch liquefacation enzyme Liquozyme
Supra（Commercially available）, addition is 6 × 10-4G/g corn starch, is heated to 90 DEG C under constant agitation, and constant temperature stirs 3h.
（3）Preparation P2 culture medium
Fermentation medium is prepared for stock solution with liquefied corn starch, adds following material to be configured to P2 culture medium in terms of g/L：Yeast powder 1, CH3COONH4
2.2, KH2PO40.5, K2HPO4
0.5, MnSO40.01, NaCl 0.01, MgSO4·7H2O
0.2, FeSO40.01, para-amino benzoic acid 0.001, vitamin B1 0.001, biotin 0.001e-3,115 DEG C of sterilizing 20min.
（4）Simultaneous saccharification and fermentation
Deoxygenation is carried out to the corn starch P2 culture medium after sterilizing, first with high pure nitrogen purging, then adds oxygen scavenger, oxygen scavenger is 1g sodium hydrosulfite and 0.6g sodium carbonate is dissolved in 10mL pure water and is obtained.Then according to the step that volume ratio accesses 10%（1）The seed liquor of activation, is simultaneously introduced 0.6 × 10-3The diastase SuHong GA of g/g corn starch（Commercially available）, 100rpm, anaerobic fermentation 120h under the conditions of 37 DEG C.
It is 11.738g/L using butanol content in liquid chromatogram measuring fermentation liquid, using content of acetone 5.122g/L in gas Chromatographic Determination fermentation liquid, be 5g/L using ethanol content in bio-sensing analysis-e/or determining fermentation liquid, total solvent content is 21.86g/L.
Embodiment 2
With clostridium acetobutylicum (Clostridium
acetobutylicum) ATCC 824 is the strain of fermenting and producing butanol, starch adopts corn starch.
（1）The activation of seed
The spore liquid of ATCC 824 bacterial strain is accessed in RCM culture medium, Anaerobic culturel 18h at 38 DEG C, the seed liquor of activation is obtained.
（2）The liquefaction of starch
Using pure water, corn starch being adjusted to dry substance concentration is 6wt%, using certain density NaOH solution regulation system pH to 6.5, adds starch liquefacation enzyme Liquozyme
Supra（Commercially available）, addition is 3 × 10-3G/g corn starch, is heated to 75 DEG C under constant agitation, and constant temperature stirs 5h.
（3）Preparation P2 culture medium
Fermentation medium is prepared for stock solution with liquefied corn starch, adds following material to be configured to P2 culture medium in terms of g/L：Yeast powder 1, CH3COONH4
2.2, KH2PO40.5, K2HPO4
0.5, MnSO40.01, NaCl 0.01, MgSO4·7H2O
0.2, FeSO40.01, para-amino benzoic acid 0.001, vitamin B1 0.001, biotin 0.001e-3,115 DEG C of sterilizing 20min.
（4）Simultaneous saccharification and fermentation
Deoxygenation is carried out to the corn starch P2 culture medium after sterilizing, first with high pure nitrogen purging, then adds oxygen scavenger, oxygen scavenger is 1g sodium hydrosulfite and 0.6g sodium carbonate is dissolved in 10mL pure water and is obtained.Then according to the step that volume ratio accesses 5%（1）The seed liquor of activation, is simultaneously introduced 1.2 × 10-3Anaerobic fermentation 120h under the conditions of diastase SuHong GA, 100rpm, 37 DEG C of g/g corn starch.
It is 11.126g/L using butanol content in liquid chromatogram measuring fermentation liquid, using content of acetone 4.658g/L in gas Chromatographic Determination fermentation liquid, be 4g/L using ethanol content in bio-sensing analysis-e/or determining fermentation liquid, total solvent content is 19.784g/L.
Embodiment 3
, with embodiment 1, difference is for handling process and operating condition：Using the Clostridium beijerinckii described in CN201410731297.9（Clostridium
beijerinckii）CM20 is zymocyte.Fermentation 80h, in fermentation liquid, the yield of butanol, acetone and ethanol is respectively 12.639g/L, 6.015g/L and 5.6g/L after testing, and solvent total output is 24.254g/L.As can be seen here, in fermentation system of the present invention, using Clostridium beijerinckii（Clostridium
beijerinckii）CM20 can significantly improve solvent total output.
Comparative example 1
（1）The activation of seed
The spore liquid of ATCC 824 bacterial strain is accessed in RCM culture medium, Anaerobic culturel 20h at 37 DEG C, the seed liquor of activation is obtained.
（2）The liquefaction of starch
Using pure water, corn starch being adjusted to dry substance concentration is 8wt%, using certain density NaOH solution regulation system pH to 6.0, adds starch liquefacation enzyme Liquozyme
Supra（Commercially available）, addition is 6 × 10-4G/g corn starch, is heated to 90 DEG C under constant agitation, and constant temperature stirs 3h.It is cooled to 60 DEG C, use H2SO4Solution regulation system pH, to 4.5, adds 0.6 × 10-3The diastase SuHong GA of g/g corn starch, constant temperature stirs 72h, makes corn starch saccharifying liquid.
（3）Preparation P2 culture medium
Fermentation medium is prepared for stock solution with corn starch saccharifying liquid, adds following material to be configured to P2 culture medium in terms of g/L：Yeast powder 1, CH3COONH4
2.2, KH2PO40.5, K2HPO4
0.5, MnSO40.01, NaCl 0.01, MgSO4·7H2O
0.2, FeSO40.01, para-amino benzoic acid 0.001, vitamin B1 0.001, biotin 0.001e-3,115 DEG C of sterilizing 20min.
（4）Simultaneous saccharification and fermentation
Deoxygenation is carried out to the corn starch P2 culture medium after sterilizing, first with high pure nitrogen purging, then adds oxygen scavenger, oxygen scavenger is 1g sodium hydrosulfite and 0.6g sodium carbonate is dissolved in 10mL pure water and is obtained.Then according to the step that volume ratio accesses 10%（1）The seed liquor of activation, 100rpm, anaerobic fermentation 120h under the conditions of 37 DEG C.
After fermentation ends, in fermentation liquid, the yield of butanol, acetone and ethanol is respectively 10.714g/L, 4.918g/L and 1.7g/L after testing, and solvent total output is 17.332g/L.
Comparative example 2
, with comparative example 1, difference is for handling process and operating condition：Using the Clostridium beijerinckii described in CN201410731297.9（Clostridium
beijerinckii）CM20 is zymocyte.After fermentation 96h terminates, in fermentation liquid, the yield of butanol, acetone and ethanol is respectively 11.358g/L, 5.297g/L and 2.1g/L after testing, and solvent total output is 18.755g/L.
Comparative example 3
Handling process and the same document of operating condition " sweet potato dregs simultaneous saccharification and fermentation produces the process optimization of ethanol " are described.Difference is to adopt corn starch, mixes after adding Thermostable α-Amylase, is placed in the liquefaction of water bath with thermostatic control shaking table, is cooled to 30 DEG C after liquefaction, and adjusting pH value is 4, and culture medium adopts the formula of P2 culture medium, adds saccharifying enzyme and butanol fermentation bacterium（Clostridium
Acetobutylicum 824 or Clostridium beijerinckii CM20）, standing for fermentation.It is saccharifying appropriate pH that this system needs to adjust pH value, yet with the difference of strain nature and product, after the 120h that ferments, Clostridium
Acetobutylicum 824 or Clostridium beijerinckii CM20 bacterial strain almost do not grow, and in fermentation liquid, solvent total output is almost nil.As can be seen here it is adaptable to the simultaneous saccharification and fermentation system of yeast fermentation ethanol is not particularly suited for butanol fermentation bacterium.
Claims (11)
1. a kind of starch simultaneous saccharification and fermentation produces the method for butanol it is characterised in that including following content：
（1）The activation of seed：Butanol fermentation bacterium is accessed activation preparation seed liquor in RCM culture medium；
（2）The liquefaction of starch：It is 5wt%-10wt% that starch is adjusted to dry substance concentration, and regulation pH value is 6.0-6.5, adds starch liquefacation enzyme, is heated to 60-95 DEG C and is liquefied；
（3）Preparation P2 culture medium：P2 culture medium is prepared for stock solution with liquefying starch；
（4）Simultaneous saccharification and fermentation：After the starch P2 culture medium deoxygenation after sterilizing, access step（1）The butanol fermentation bacterium seed liquor of activation, is simultaneously introduced diastase, carries out anaerobic fermentation and produces butanol.
2. method according to claim 1 it is characterised in that：Step（1）Described butanol fermentation bacterium is clostridium acetobutylicum（Clostridium acetobutylicum) or Clostridium beijerinckii（Clostridium beijerinckii）.
3. method according to claim 2 it is characterised in that：Step（1）Described butanol fermentation bacterium adopt clostridium acetobutylicum (Clostridium acetobutylicum) ATCC 824, it is purchased from American Type Culture Collection center.
4. method according to claim 2 it is characterised in that：Step（1）Described butanol fermentation bacterium adopts Clostridium beijerinckii CM20 described in CN201410731297.9, and its Classification And Nomenclature is Clostridium beijerinckii（Clostridium beijerinckii）, it was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 17th, 2014, deposit number is CGMCC No.9354.
5. method according to claim 1 it is characterised in that：Step（1）Described RCM culture medium Ju Ti Pei Fang（In terms of g/L）For：Peptone 10, beef powder 10, yeast powder 3.0, glucose 5, soluble starch 1.0, sodium chloride 5.0, Sodium Acetate Trihydrate 3.0, L-cysteine hydrochloride 0.5, agar 0.5, with pure water configuration, 115 DEG C of sterilizing 20min.
6. method according to claim 1 it is characterised in that：Step（1）Described seed activation is to access the spore liquid of butanol fermentation bacterium in RCM culture medium, and at 36-38 DEG C, Anaerobic culturel 16-20h obtains seed liquor.
7. method according to claim 1 it is characterised in that：Step（2）Described starch is corn starch, wheaten starch or sweet potato starch；Using pure water, starch being adjusted to dry substance concentration is 6wt%-8wt%, is 6.0-6.5 using alkali liquor regulation system pH value, adds starch liquefacation enzyme, be heated to 70-90 DEG C, constant temperature stirs 2-5h after adjusting pH value；Starch liquefacation enzyme is high-temperature resistant alpha-amylase, and addition is 6 × 10-4- 3×10-3G/g starch.
8. method according to claim 1 it is characterised in that：Step（3）P2 culture medium, the formula of P2 culture medium are prepared for stock solution with liquefying starch（In terms of g/L）For：Yeast powder 1, CH3COONH42.2, KH2PO40.5, K2HPO40.5, MnSO40.01, NaCl 0.01, MgSO4·7H2O 0.2, FeSO40.01, para-amino benzoic acid 0.001, vitamin B1 0.001, biotin 0.001e-3,115 DEG C of sterilizing 20min.
9. method according to claim 1 it is characterised in that：Step（4）Purge first with high pure nitrogen, then add the mode deoxygenation of oxygen scavenger, oxygen scavenger is 1g sodium hydrosulfite and 0.6g sodium carbonate is dissolved in 10mL pure water and is obtained.
10. method according to claim 1 it is characterised in that：Step（4）Access step according to volume ratio 5%-10%（1）The seed liquor of activation, is simultaneously introduced diastase, and addition is 0.4 × 10-3-2.0×10-2G/g starch.
11. methods according to claim 1 it is characterised in that：Step（4）The temperature of fermenting and producing butanol is 35-38 DEG C, and rotating speed is 50-200rpm, and fermentation time is 72-120h.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559778A (en) * | 2012-02-14 | 2012-07-11 | 南京工业大学 | Fermentation medium and method for producing butanol by fermentation of same |
| CN105713851A (en) * | 2014-12-05 | 2016-06-29 | 中国石油化工股份有限公司 | Clostridium beijerinckii strain and applications thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559778A (en) * | 2012-02-14 | 2012-07-11 | 南京工业大学 | Fermentation medium and method for producing butanol by fermentation of same |
| CN105713851A (en) * | 2014-12-05 | 2016-06-29 | 中国石油化工股份有限公司 | Clostridium beijerinckii strain and applications thereof |
Non-Patent Citations (3)
| Title |
|---|
| PHILIPPIDIS GP, SMITH TK, WYMAN CE: "Study of the enzymatic hydrolysis of cellulose for production of fuel ethanol by the simultaneous saccharification and fermentation process", 《BIOTECHNOL BIOENG》 * |
| 于鹏: "降解木糖渣生产丁醇的初步研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
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