CN108841912A - A kind of technique preparing and separate biological butanol and bio-ethanol - Google Patents
A kind of technique preparing and separate biological butanol and bio-ethanol Download PDFInfo
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Abstract
The invention belongs to fermentation technical fields, disclose a kind of technique for preparing and separating biological butanol and bio-ethanol comprising following steps：Step 1）Handle stalk, step 2）Prepare fermentation medium, step 3）Produce alcohol, step 4）It separates and pure and mild prepares albumen powder.Present invention process can obtain biological butanol, bio-ethanol and mycoprotein powder simultaneously, improve added value.
Description
Technical field
The invention belongs to fermentation technical fields, and in particular to a kind of work for preparing and separating biological butanol and bio-ethanol
Skill.
Background technique
Butanol is colourless liquid, there is vinosity, with ethyl alcohol, ether and other a variety of immiscible organic solvents, steam and air shape
At explosive mixture, explosion limit 1.45-11.25（Volume）.Mainly for the manufacture of phthalic acid, aliphatic dibasic acid and
The N-butyl class plasticizer of phosphoric acid, they are widely used in various plastics and rubber product and producing butyladehyde, fourth in organic synthesis
The raw material of acid, butylamine and butyl lactate etc..Biological butanol is bio-fuel similar with bio-ethanol.Its raw material and production technology
It is similar to bio-ethanol, but the steam pressure of biological butanol is low, it is big to the tolerance of water impurity when being mixed with gasoline, and corrode
Property is smaller, compared with existing bio-fuel, higher mixing ratio can be reached with gasoline, without being transformed to vehicle.
Butanol can be used fermentation process similar with ethyl alcohol and produce.But, compared with ethyl alcohol, the cost of production of butanol wants high
Much, that is to say, that production butanol need to be higher with facilities, the investment cost such as biggish evaporation, heating, cooling.Therefore, life is realized
The commercialized key of object butanol is to improve Raw material processing into the conversion ratio of butanol, accelerates conversion process.This depends on high-performance bio
The exploitation of catalyst and the optimization of process design.The same with ethyl alcohol, biological butanol conventional production methods can also consume largely
Agricultural product, for this purpose, just solving the problems, such as to strive grain with people in the new technology of a variety of biology base waste material production butanol of research and utilization.It passes
The preparation method of the biological butanol of system is to be prepared using grains such as corn, wheat, soybean as raw material by fermentation.But
Producing bio-fuel as raw material using grain cannot not only satisfy social needs, but also can jeopardize grain security.There is researcher to refer to
Out, even if all corns of U.S.'s plantation and soybean are all used to produce bioenergy, American society's gasoline can only also be met respectively
The 6% of 12% and diesel oil demand of demand.And corn and soybean first have to meet grain, feed and other economic needs, it can not
It can all be used to produce bio-fuel.Expert indicates, produces the direction that biological butanol is future development by raw material of non-grain crop,
Future, energy industry was expected using plant fiber element, such as grain straw next life generation butanol.Currently, biological butanol industry is also located
In the primary stage.In recent years, since oil price is constantly soaring, the enterprise of not rare strategic perspective takes part in grinding for biological butanol
Study carefully.
Bio-ethanol, which refers to, converts fuel alcohol for various biomass by the fermentation of microorganism.It can individually or with
Ethanol petrol is made as motor vehicle fuel in gasoline mixture.The alcohol fuel overwhelming majority of current industrialized production is made with grain
Object is raw material, has size limit and unsustainable property in the long run.Using lignocellulosic as the second generation of raw material biology
Alcohol fuel is the key that determine the following extensive substitution petroleum.
Chinese patent " CN102796692A " discloses a kind of pair of clostridium acetobutylicum and carries out genetic modification, can be more efficient
Ground utilizes xylose and arabinose, butanol, ethyl alcohol and the acetone equal solvent of higher concentration.Chinese patent " CN103409470A "
It discloses and Yeasts, the method that mixed fermentation produces butanol, ethyl alcohol and acetone is followed by using first inoculation clostridium acetobutylicum.
Corn stover belongs to agriculture waste, is arbitrarily abandoned or is burned mostly, but burns and will cause environmental pollution,
Through being prohibited by country, currently, utilization method of many enterprises in research corn stover.Due to fiber in former corn stover
Element, the fine and close complicated structure of hemicellulose and lignin, cellulose it is more difficult by enzyme hydrolysis at monosaccharide, how corn stover is carried out
It handles so that strain fermentation production ethyl alcohol is the direction that we study.It is had become using the biomass ferments butanol such as lignocellulosic
For a hot spot of current research.Selection production alcohol bacterium improves and compatibility, is a breach to improve alcohol yield.Applicant
Before research " using corn stover be main fermenting raw materials produce biological butanol and bio-ethanol method ", be using stalk
Primary raw material fermentation preparation obtains mixed alcohol, and on this basis, applicant has made further research.
Summary of the invention
On the basis of preamble work, the present invention provides a kind of techniques for preparing and separating biological butanol and bio-ethanol.
The present invention is achieved by the following technical solution：
A kind of technique preparing and separate biological butanol and bio-ethanol comprising following steps：Step 1）Handle stalk, step
2）Prepare fermentation medium, step 3）Produce alcohol, step 4）It separates and pure and mild prepares albumen powder.
Further, described method includes following steps：
Step 1）Handle stalk：Corn stalk powder is broken within 5cm first, is placed in pressure 1.5-2MPa, residence time 10-
Steam blasting pretreatment is carried out under conditions of 15min, then carries out explosion；50 will be crossed by the corn stover of Steam explosion treatment
Mesh collects screenings, is then added to screenings in the water of 1.5-2 times of weight, is heated to 60 DEG C, under heat-retaining condition
100rpm stirs 90min, is then heated to 121 DEG C, keeps the temperature 10min, and cooled to room temperature obtains culture solution；
Step 2）Prepare fermentation medium：Koning trichoderma seed liquor and aspergillus niger seed liquor are mixed, then connecing according to 6-8%
Kind of amount is inoculated into culture solution, cultivates 96h, and control mixing speed is 100rpm when culture, and cultivation temperature is 32 DEG C, by stream plus
The pH that ammonium hydroxide controls incubation is 4-5；After the completion of culture, ultrasonic treatment, then adjusting temperature is 55 DEG C, adds lysozyme, protects
Under the conditions of temperature, digest 12 hours；Then 121 DEG C of enzyme deactivation 5min, last cooled to room temperature is to get fermentation medium；
Step 3）Produce alcohol：Clostridium acetobutylicum seed liquor is linked into obtain in anaerobic fermentation tank containing fermentation medium and is trained
It supports, temperature is 30-32 DEG C, incubation time 24-36h, is then inoculated with Neuraspora crassa seed liquor, continues to cultivate 48-72h, training
After supporting, fermentation liquid is taken, 10min is centrifuged with 8000rpm, collects supernatant liquid and bacterial sediment；
Step 4）It separates and pure and mild prepares albumen powder：Supernatant liquid is obtained into crude product by vacuum distillation, then passes through rectifying and takes off
Water obtains biological butanol and bio-ethanol；Bacterial sediment is washed, then as drying and processing under the conditions of 80 DEG C, is finally crushed
Up to albumen powder.
Preferably,
The density of the koning trichoderma seed liquor is 1 × 108cfu/mL。
Preferably,
The density of the aspergillus niger seed liquor is 1 × 108cfu/mL。
Preferably,
The processing time of the ultrasound is 40min, and ultrasonic frequency is 30KHz.
Preferably,
The additive amount of the lysozyme is 20,000 U：1L solution.
Preferably,
The inoculum concentration of the clostridium acetobutylicum seed liquor is 5-7%.
Preferably,
The inoculum concentration of the Neuraspora crassa seed liquor is 8-10%.
Preferably,
The koning trichoderma seed liquor and aspergillus niger seed liquor are according to 2-3:The volume ratio of 1-2 mixes.
The specific bacterial strain that embodiment of the present invention is selected is koning trichoderma ATCC18649, aspergillus niger ATCC1015, acetone fourth
Alcohol clostridium ATCC824, Neuraspora crassa ATCC44317；The seed liquor of each bacterial strain can be recorded often according to textbook or document
Training method is advised to obtain.
Compared with prior art, the beneficial effect that the present invention obtains mainly includes but is not limited to several aspects：
The mixed solvent that fermentation obtains has been carried out distillation separating treatment by the present invention, and mycoprotein is processed, and is mentioned
The high added value of industry；
The present invention carries out broken wall treatment, broken wall to thallus in the technique for preparing fermentation medium, using ultrasonic wave added lysozyme
Effect is good, avoids impacting subsequent strain growth using organic solvent；
Steam blasting pretreatment makes hemicellulose and lignin portion degrade, and destroys lignin and hemicellulose to cellulose
Package action, destroy the crystal region of cellulose, the porosity and internal surface area of raw material increase, thus after being more advantageous to
The enzyme hydrolysis of continuous cellulose, which acts on, to be carried out.
The present invention has carried out crushing and explosion treatment to agricultural wastes straw, can provide for koning trichoderma and aspergillus niger
Normal growth conditions, raw material relative moderate, can reduce entreprise cost；Corn stalk is handled using koning trichoderma and aspergillus niger
Then stalk digests thallus, obtain the culture medium containing reduced sugar and mycoprotein, produces alcohol for subsequent bacterial strain anaerobism and uses, mentions
High fermentation efficiency, and agricultural wastes are utilized, it reduces costs.The present invention utilizes the cavitation of ultrasonic wave, generates
Partial high pressure high temperature, impacts somatic cells, leads to cytomorphosis and rupture, assists carrying out broken wall dissolution using lysozyme
Mycoprotein is obtained, the mycoprotein liquid concentration of preparation is high, is suitble to anaerobic fermentation, and reduce costs.
Koning trichoderma fermentation production endo-glucanase enzyme activity is higher, but generally existing β-glucose in produced cellulase
The low defect of glycosides enzyme activity, and Aspergillus Niger beta-glucosidase ability is higher, produces inscribe and exoglucanase ability is lower.
The two is subjected to cooperative fermentation stalk, Filter paperlyase and the active of beta-glucosidase greatly improve, the reduced sugar in fermentation liquid
Yield also correspondinglys increase.Cellulose generates the hexose based on glucose after hydrolysis, and obtains after hydrolysis of hemicellulose
Pentose based on xylose, but pentose is relatively difficult to can use glucose using alcohol, present invention selection is produced by microorganism
Clostridium acetobutylicum be used to produce butanol and ethyl alcohol, and Neuraspora crassa is used to utilize xylose producing and ethanol；It is inoculated with acetone fourth first
Alcohol clostridium can use glucose fermentation and produce alcohol, but cannot utilize xylose, when glucose is reduced to a certain concentration, at this time third
Ketone Clostridium acetobutylicum has been dominant microflora, access Neuraspora crassa, can use the xylose producing and ethanol of high concentration, without with
Clostridium acetobutylicum competitive utilization glucose, the two can be common to generate butanol and ethyl alcohol with cooperative fermentation.
Detailed description of the invention
Fig. 1：The inulinase-producing activity of different strains；
Fig. 2：Influence of the different strains fermentation method to alcohol amount is produced；
Fig. 3：Influence of the mixed fermentation time to alcohol amount is produced.
Specific embodiment
Those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that
All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair
It is bright.Product and method of the invention is described by preferred embodiment, and related personnel can obviously not depart from this hair
Product as described herein and method are modified in bright content, spirit and scope or appropriate changes and combinations, to realize and answer
Use the technology of the present invention.For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.
Embodiment 1
A kind of technique preparing and separate biological butanol and bio-ethanol comprising following steps：
Corn stalk powder is broken within 5cm first, is subsequently placed under conditions of pressure 1.5MPa, residence time 15min and carries out
Steam blasting pretreatment, then carries out explosion；50 meshes will be crossed by the corn stover of Steam explosion treatment, collect screenings,
Then screenings is added in the water of 2 times of weight, is heated to 60 DEG C, 100rpm stirs 90min under heat-retaining condition, then heats
To 121 DEG C, 10min is kept the temperature, cooled to room temperature obtains culture solution；
By koning trichoderma seed liquor（Density is 1 × 108cfu/mL）With aspergillus niger seed liquor（Density is 1 × 108cfu/mL）It presses
According to 2:1 volume ratio mixing, is then inoculated into culture solution according to the inoculum concentration of 6-8%, cultivates 96h, control stirring speed when culture
Degree is 100rpm, and it is 5 by the pH that Feeding ammonia water controls incubation that cultivation temperature, which is 32 DEG C,；After the completion of culture, ultrasonic treatment
40min, supersonic frequency 30KHz, then adjusting temperature is 55 DEG C, adds lysozyme, the additive amount of lysozyme is 20,000 U：1L is molten
Liquid under heat-retaining condition, digests 12 hours；Then 121 DEG C of enzyme deactivation 5min, last cooled to room temperature is to get fermentation medium；
By clostridium acetobutylicum seed liquor（Density is 3 × 108cfu/mL）According to 7%（Volume ratio）Inoculum concentration be linked into containing
It is cultivated in the anaerobic fermentation tank of fermentation medium, temperature is 32 DEG C, then incubation time 36h is inoculated with Neuraspora crassa
Seed liquor（Density is 3 × 108cfu/mL）, inoculum concentration is 10%（Volume ratio）；Continue culture 72h and takes hair after fermentation
Zymotic fluid is centrifuged 10min with 8000rpm, collects supernatant liquid and bacterial sediment, then butanol in gas chromatographic detection supernatant liquid
With the content of ethyl alcohol；Crude product is obtained by vacuum distillation, then by rectifying and dewatering process, obtains biological butanol and biological second
Alcohol；Bacterial sediment is washed, then as drying and processing under the conditions of 80 DEG C, is finally crushed up to albumen powder.
Embodiment 2
It is the method that main fermenting raw materials produce biological butanol and bio-ethanol using corn stover comprising following steps：
Corn stalk powder is broken within 5cm first, is subsequently placed under conditions of pressure 2MPa, residence time 10min and is steamed
The broken pretreatment of steam explosion, then carries out explosion；50 meshes will be crossed by the corn stover of Steam explosion treatment, collects screenings, so
Screenings is added to afterwards in the water of 1.5 times of weight, is heated to 60 DEG C, 100rpm stirs 90min under heat-retaining condition, then heats
To 121 DEG C, 10min is kept the temperature, cooled to room temperature obtains culture solution；
By koning trichoderma seed liquor（Density is 1 × 108cfu/mL）With aspergillus niger seed liquor（Density is 1 × 108cfu/mL）It presses
According to 3:2 volume ratio mixing, is then inoculated into culture solution according to the inoculum concentration of 6-8%, cultivates 96h, control stirring speed when culture
Degree is 100rpm, and it is 4.5 by the pH that Feeding ammonia water controls incubation that cultivation temperature, which is 32 DEG C,；After the completion of culture, at ultrasound
40min, supersonic frequency 30KHz are managed, then adjusting temperature is 55 DEG C, add lysozyme, the additive amount of lysozyme is 20,000 U：1L is molten
Liquid under heat-retaining condition, digests 12 hours；Then 121 DEG C of enzyme deactivation 5min, last cooled to room temperature is to get fermentation medium；
By clostridium acetobutylicum seed liquor（Density is 2 × 108cfu/mL）According to 7%（Volume ratio）Inoculum concentration be linked into containing
It is cultivated in the anaerobic fermentation tank of fermentation medium, temperature is 30 DEG C, and incubation time is for 24 hours, to be then inoculated with Neuraspora crassa
Seed liquor（Density is 2 × 108cfu/mL）, inoculum concentration is 10%（Volume ratio）；Continue culture 72h and takes hair after fermentation
Zymotic fluid is centrifuged 10min with 8000rpm, collects supernatant liquid and bacterial sediment, then butanol in gas chromatographic detection supernatant liquid
With the content of ethyl alcohol；Crude product is obtained by vacuum distillation, then by rectifying and dewatering process, obtains biological butanol and biological second
Alcohol；Bacterial sediment is washed, then as drying and processing under the conditions of 80 DEG C, is finally crushed to get albumen powder.
Embodiment 3
Variation of the explosion to corn stover main component, is specifically shown in Table 1：
Table 1
| Index | Before explosion | After explosion |
| Hemicellulose and cellulose % | 61.2 | 48.9 |
| Lignin % | 22.7 | 15.6 |
| Xylo-oligosaccharide % | 0 | 6.9 |
| Cellooligosaccharide | 0 | 3.1 |
Conclusion：Explosion causes stalk cell wall to destroy, part hemicellulose and cellulose degradation and dissolve out, be conducive to subsequent fiber
Enzymatic hydrolysis of the plain enzyme to cellulose；Cellulose crystallity and degree of polymerization decline in explosion preprocessing process, hemicellulose is by from water
Solution effect is degraded into xylose etc., can be used as the carbon source of bacterial strain.
Embodiment 4
By taking embodiment 1 as an example, influence of the mode of independent bacterial strain and compound strain processing stalk to inulinase-producing activity is had detected, specifically
Inulinase-producing activity is as shown in Figure 1, koning trichoderma fermentation production filter paper enzyme activity is higher, but there are β-glucose in produced cellulase
The low defect of glycosides enzyme activity, and Aspergillus Niger beta-glucosidase ability is higher, production Filter paperlyase ability is lower, and the two is assisted
Same fermented stalk, Filter paperlyase and beta-glucosidase activity greatly improve, enzyme activity respectively reach 357U/ml and
395U/ml。
The main component of fermentation medium of the present invention is as shown in table 2：
Table 2
| Component | Six sugared content g/l | Pentasaccharides content g/l | Protein content g/l |
| Koning trichoderma | 51.8 | 33.4 | 17.1 |
| Aspergillus niger | 39.2 | 26.8 | 13.9 |
| Mixed fermentation | 73.6 | 45.7 | 21.4 |
As shown in table 2, six sugar and five pool rich contents in fermentation medium of the present invention, protein content is more than 20g/L, be can be used as
The carbon source and carbon source of clostridium acetobutylicum and Neuraspora crassa anaerobic fermentation come using.
Embodiment 5
Different strains fermentation produces influence of the alcohol mode to alcohol amount is produced：
Using embodiment 1 as experimental group, control group is set, wherein control group 1：Only with clostridium acetobutylicum, fermentation time is
108h；Control group 2：Only with Neuraspora crassa, fermentation time 108h；Control group 3：Inoculating two kinds bacterial strain simultaneously, when fermentation
Between be 108h.As shown in Fig. 2, the butanol content highest of experimental group, the ethanol content highest of control group 3, but butanol yield compared with
It is low, it may be possible to because Neuraspora crassa is easy to generate clostridium acetobutylicum the competition of six sugar carbon sources, to be unfavorable for producing the acetone of butanol
Clostridium acetobutylicum；The ethanol production of experimental group and the gap of control group 3 are not obvious, the fermentation production rate of comprehensive butanol and ethyl alcohol, real
The production alcohol performance for testing group is best.As shown in figure 3, butanol and ethyl alcohol obviously increase, for 24 hours with the increase of mixed fermentation time
Afterwards, butanol amplification is substantially reduced, and ethyl alcohol amplification is obvious, and fermentation time selects 48-72h best to alcohol performance is produced.
Finally it should be noted that：The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although reference
Invention is explained in detail for previous embodiment, those skilled in the art should understand that：It still can be right
Technical solution documented by previous embodiment is modified or equivalent replacement of some of the technical features；And these
It modifies or replaces, the spirit and scope for technical solution of the embodiment of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (9)
1. a kind of technique for preparing and separating biological butanol and bio-ethanol comprising following steps：Step 1）Handle stalk, step
Rapid 2）Prepare fermentation medium, step 3）Produce alcohol, step 4）It separates and pure and mild prepares albumen powder.
2. the method according to claim 1, wherein described method includes following steps：
Step 1）Handle stalk：Corn stalk powder is broken within 5cm first, is placed in pressure 1.5-2MPa, residence time 10-
Steam blasting pretreatment is carried out under conditions of 15min, then carries out explosion；50 will be crossed by the corn stover of Steam explosion treatment
Mesh collects screenings, is then added to screenings in the water of 1.5-2 times of weight, is heated to 60 DEG C, under heat-retaining condition
100rpm stirs 90min, is then heated to 121 DEG C, keeps the temperature 10min, and cooled to room temperature obtains culture solution；
Step 2）Prepare fermentation medium：Koning trichoderma seed liquor and aspergillus niger seed liquor are mixed, then connecing according to 6-8%
Kind of amount is inoculated into culture solution, cultivates 96h, and control mixing speed is 100rpm when culture, and cultivation temperature is 32 DEG C, by stream plus
The pH that ammonium hydroxide controls incubation is 4-5；After the completion of culture, ultrasonic treatment, then adjusting temperature is 55 DEG C, adds lysozyme, protects
Under the conditions of temperature, digest 12 hours；Then 121 DEG C of enzyme deactivation 5min, last cooled to room temperature is to get fermentation medium；
Step 3）Produce alcohol：Clostridium acetobutylicum seed liquor is linked into obtain in anaerobic fermentation tank containing fermentation medium and is trained
It supports, temperature is 30-32 DEG C, incubation time 24-36h, is then inoculated with Neuraspora crassa seed liquor, continues to cultivate 48-72h, training
After supporting, fermentation liquid is taken, 10min is centrifuged with 8000rpm, collects supernatant liquid and bacterial sediment；
Step 4）It separates and pure and mild prepares albumen powder：Supernatant liquid is obtained into crude product by vacuum distillation, then passes through rectifying and takes off
Water obtains biological butanol and bio-ethanol；Bacterial sediment is washed, then as drying and processing under the conditions of 80 DEG C, is finally crushed
Up to albumen powder.
3. according to the method described in claim 2, it is characterized in that, the density of the koning trichoderma seed liquor is 1 × 108cfu/
mL。
4. according to the method described in claim 2, it is characterized in that, the density of the aspergillus niger seed liquor is 1 × 108cfu/mL。
5. according to the method described in claim 2, it is characterized in that, the processing time of the ultrasound is 40min, ultrasonic frequency
For 30KHz.
6. according to the method described in claim 2, it is characterized in that, the additive amount of the lysozyme is 20,000 U：1L solution.
7. according to the method described in claim 2, it is characterized in that, the inoculum concentration of the clostridium acetobutylicum seed liquor is 5-
7%.
8. according to the method described in claim 2, it is characterized in that, the inoculum concentration of the Neuraspora crassa seed liquor is 8-
10%.
9. according to the method described in claim 2, it is characterized in that, the koning trichoderma seed liquor and aspergillus niger seed liquor according to
2-3:The volume ratio of 1-2 mixes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810632494.3A CN108841912B (en) | 2018-06-20 | 2018-06-20 | Process for preparing and separating bio-butanol and bio-ethanol |
Applications Claiming Priority (1)
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| CN101492700A (en) * | 2009-03-05 | 2009-07-29 | 王建设 | Intensive processing method for stalk articles or agricultural castoff |
| CN103409470A (en) * | 2013-05-15 | 2013-11-27 | 中国科学院广州能源研究所 | Method for producing ethanol, butanol and acetone by utilizing segmented and mixed fermentation of mixed sugar containing pentose and hexose |
| US20160289661A1 (en) * | 2013-11-22 | 2016-10-06 | IFP Energies Nouvelles | Endoglucanase variants having improved activity, and uses of same |
| CN106834361A (en) * | 2017-03-24 | 2017-06-13 | 黑龙江中丹建业生物能源有限公司 | The method that stalk produces cellulosic ethanol |
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| CN101492700A (en) * | 2009-03-05 | 2009-07-29 | 王建设 | Intensive processing method for stalk articles or agricultural castoff |
| CN103409470A (en) * | 2013-05-15 | 2013-11-27 | 中国科学院广州能源研究所 | Method for producing ethanol, butanol and acetone by utilizing segmented and mixed fermentation of mixed sugar containing pentose and hexose |
| US20160289661A1 (en) * | 2013-11-22 | 2016-10-06 | IFP Energies Nouvelles | Endoglucanase variants having improved activity, and uses of same |
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