CN105200094B - A method of utilizing microbial fermentation lignocellulosic material producing and ethanol - Google Patents
A method of utilizing microbial fermentation lignocellulosic material producing and ethanol Download PDFInfo
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Abstract
The present invention provides a kind of method using microbial fermentation lignocellulosic material producing and ethanol, including digesting the lignocellulosic material to obtain lignocellulosic material enzymolysis liquid, the microorganism being expanded and is cultivated, it obtains scale-up medium, the scale-up medium is seeded in the lignocellulosic material enzymolysis liquid, the step of fermenting is adjusted by intermittent oxygen supply.The method of the invention can greatly improve the yield of ethyl alcohol.
Description
Technical field
The invention belongs to field of microbial fermentation, and in particular to a kind of to produce second using microbial fermentation lignocellulosic material
The method of alcohol.
Background technique
The non-renewable fossil energy of many such as petroleum is increasingly depleted so that renewable energy especially bio-fuel by
To more and more concerns, and bring huge business opportunity and social effect.
According to the related definition of International Energy Agency (IEA), standard biologic fuel includes that glycosyl and starch base ethyl alcohol, oil plant are made
Object base biodiesel, the vegetable oil that can directly utilize and the biogas being made by anaerobic digestion；Advanced bio-fuel technology packet
Include the transformation technology referred to also in research and development, pilot scale or demonstration phase, commonly referred to as second-generation technology or third-generation technology.It is this kind of
It does not include the hydrogenated vegetable oil (HVO) refined with animal tallow and vegetable oil, and produced with lignocellulose-containing biomass
Bio-fuel, such as ethyl alcohol, fischer-tropsch liquids and synthetic natural gas.
Ethyl alcohol is clean regeneratable liquors fuel, and many countries have begun using the vapour for being added to certain proportion ethyl alcohol
Oil-gasohol, to replace the consumption of gasoline.This New-type fuel can alleviate the wear rate of petroleum and reduce automobile
Tail gas pollution has greatly application and development potentiality.China since 2001 promote the use of gasohol, at present gasohol
About the 20% of gasoline-like fuel total flow is accounted for, and in the impetus increased year by year.
Primary raw material used in production ethyl alcohol is the cereal crops such as corn both at home and abroad at present.It is continuous with world population
Increase, grain is increasingly in short supply, therefore in the long run, and cereal crops are not the desirable feedstocks for producing ethyl alcohol.Meanwhile having every year big
The improper processing of mode of burning etc. of the agriculture and forestry waste wood fibrous material (such as corn stover) of amount, not only causes raw material
Waste, and polluted environment.Lignocellulosic is mainly made of cellulose, hemicellulose, lignin, in agriculture waste wood
The content of three is about in matter fibrous material: cellulose accounts for 30~40%, and hemicellulose accounts for 20~30%, and lignin accounts for 10~
25%.Wherein glucose is the main component units of cellulose, and xylose is the main component units of hemicellulose.In plant fiber
Xylose accounts for 30% or so in material hydrolyzate, is the most abundant sugar of nature after glucose, thus utilizes glucose wood
Sugared common fermentation produces ethyl alcohol abundant raw materials, has a vast market foreground.
In general, belonging to advanced bio-fuel technology with the production that the biomass of lignocellulose-containing carries out bio-fuel
Scope.Generally use the technique of biochemical transformation using lignocellulose-containing raw material production ethyl alcohol, including pretreatment, hydrolysis,
Fermentation and recycling and etc..By pretreatment, the accessibility of enzyme and cellulosic material is increased, so that improves raw material can
Usability；After hydrolysis (including enzymatic hydrolysis), hemicellulose main decomposition is C5 sugar, and cellulose main decomposition is C6 sugar.Due to
The undefined structure of hemicellulose is easier to be hydrolyzed to C5 sugar.In pretreatment stage and enzymatic hydrolysis stage, Partial fermentation can be obtained
Mortifier, such as formic acid, acetic acid, furfural etc..
But the predicament accordingly faced is, the microorganism that alcohol can be efficiently produced in nature is typically only capable to utilize glucose etc.
Hexose produces ethyl alcohol, cannot utilize xylose；And a small number of microorganisms that alcohol can be produced using xylose, alcohol inferior capabilities are produced, this limitation
Effective use to lignocellulosic in nature.Yeast is transformed by metabolic engineering, and utilizes its metabolic conversion
C5 sugar mainly passes through following reaction: Xylose reductase relies on NADPH and xylose is reduced to xylitol, and xylitol is de- in xylitol again
Xylulose is converted under hydrogen enzyme effect；X 5P is formed through Xylulokinase phosphorylation again, subsequently into phosphopentose
Approach (PPP).The intermediate product glucose 6-phosphate and glyceraldehyde 3-phosphate of PPP approach form pyruvic acid by diphosphate pathway,
Pyruvic acid or ethyl alcohol is reduced to through pyruvate decarboxylase, alcohol dehydrogenase decarboxylation.
Under the promotion of technique for gene engineering, researcher, which obtains a batch, can use the weight of xylose metabolism production ethyl alcohol
Group fermentative microorganism.However, especially extensive industrialization stage, above-mentioned microorganism is expanded in actual industrialized production
Big culture growth rate is slower, it is difficult to realize large-scale application, and due to the wooden fibre of microbial metabolism sugar containing C5 and C6 sugar
The consumption otherness demand supplied during plain raw material enzymolysis liquid to fermentation is tieed up, the conversion ratio mistake in actual production is always resulted in
It is low, also become one of the difficult point for limiting the development of this technology.
Summary of the invention
Therefore, it is an object of the invention to overcome microbial metabolism in the prior art containing the lignocellulosic of C5 and C6 sugar original
Expect the consumption otherness demand supplied during enzymolysis liquid to fermentation, the too low defect of conversion ratio in caused actual production, from
And a kind of method for efficiently utilizing microbial fermentation lignocellulosic material producing and ethanol is provided, to improve the conversion ratio of ethyl alcohol.
For this purpose, the present invention provides a kind of method using microbial fermentation lignocellulosic material producing and ethanol, including as follows
Step:
1), the lignocellulosic material is digested, obtains lignocellulosic material enzymolysis liquid；
2), the microorganism is expanded and is cultivated, scale-up medium is obtained；
3), the scale-up medium is seeded in the lignocellulosic material enzymolysis liquid by 5-20%, control reaction
25-38 DEG C of temperature, pH5.0-7.0 carry out fermenting and producing, control and carry out intermittent oxygen supply, ventilatory capacity in the fermentation process
0.01-0.5mL/L, duration of ventilation 0.5h, interval time 5-6h, fermentation obtain fermentation liquid；
4) it, separates the fermentation liquid and obtains ethyl alcohol.
In the step 3), air ventilatory capacity 0.1mL/L-0.2mL/L in fermentation process is controlled.
The lignocellulosic material be selected from corn stover, corncob, hardwood, cork, shuck, grass, paper, barley-straw,
One or more of wheat straw, leaf, cottonseed wadding, withy, oat shell.
Structurally, lignocellulosic mainly includes cellulose, hemicellulose and lignin.Preferably, the lignin
Cellulosic material includes at least 30wt%, preferably at least 50wt%, more preferably at least 70wt%, even more desirably at least 90wt%
Lignocellulosic.It should be understood that can also include other components in lignocellulose-containing raw material, such as protein material forms sediment
Powder, sugar, such as fermentable sugared and/or not fermentable sugar.
The enzyme is selected from cellulase, hemicellulase, amylase, protease, glucoamylase, lipase.The fibre
Tieing up plain enzyme includes but is not limited to cellobiohydrolase (cellobiohydrolase I and cellobiohydrolase II) and inscribe Portugal
Glycan and β-glucosyl enzym.
Also contain soybean cake powder, corn-dodger powder, corn pulp, fish meal or yeast extract in the lignocellulosic material enzymolysis liquid
One or more of be used as nitrogen source, contain disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphoric acid hydrogen
One or more of diammonium is used as inorganic salts, and selective contains urea and/or inhibitor.
The carbon source contained in the fermentation medium, by weight ratio, glucose 5-20%, xylose 3-8%；Make
For in one of fermentation medium preferred content, glucose 6-12%, xylose 3-6%.
Also contain one or both of the protein as nitrogen source, free amino acid in the fermentation medium.Wherein
The content of protein in the fermentation medium is 3-4g/L, is mainly used for nitrogen source needed for providing the microorganism growth；It is described
Free amino acid includes alanine, proline, phenylalanine, arginine, isoleucine, methionine, glutamic acid, threonine, Guang
One or more of propylhomoserin, leucine and valine.
The inorganic salts contained in the fermentation medium are selected from disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid
One or more of hydrogen dipotassium, diammonium hydrogen phosphate, and content of the inorganic salts in the fermentation medium is 0.5-4g/
L, preferably 1-2.5g/L.
Also contain one or more of vitamin, microelement in the fermentation medium.The vitamin includes B race
Vitamin, the B family vitamin include at least biotin, folic acid；The microelement include copper, calcium, iron, zinc, lead, silver, chromium,
One or more of Yan acid, pyridoxic acid, thiamines and calcium pantothenate.
Also containing the bacteriostatic agent for being used to inhibit varied bacteria growing in fermentation medium of the present invention, certainly, if fermentation
There is no miscellaneous bacteria in incubation, above-mentioned bacteriostatic agent can not also be added, the bacteriostatic agent is selected from penicillin, ampicillin, chain
One or more of mycin, chloramphenicol, terramycin, tetracycline, preferably penicillin and/or streptomysin, in fermentation medium
The working concentration of bacteriostatic agent is 10-50 unit/mL, preferably 15-25 unit/mL, more preferably 20 units/mL.
When the fermentation medium derives from the lignocellulosic material enzymolysis liquid, ratio by weight, grape
The content of sugar is 1-25%, and the content of xylose is 1-10%；Preferably, the content of glucose is 5-20%, and the content of xylose is
3-8%；It is furthermore preferred that the content of glucose is 6-12%, the content of xylose is 3-6%.
Meanwhile although it will be understood by those skilled in the art that certain density acetic acid may produce the microbial cell
Raw inhibiting effect, but still can permit that there are a small amount of acetic acid in lignocellulosic material enzymolysis liquid, the acetic acid is in institute
Stating the content in fermentation medium is preferably 0.1-1%, more preferably 0.1-0.8%.
The expansion culture expands incubation step by least one level extremely to expand in culture medium the microbial inoculant
The microorganism is expanded culture, it is dense by adjusting the oxygen supply expanded in incubation in the expansion incubation step
Degree realizes the expansion culture to the microorganism.
Expand incubation step using level-one to expand culture the microorganism, include the following steps:
The seed liquor is seeded to according to the inoculum concentration of 5-20% and is spread cultivation in tank containing the level-one for expanding culture medium, control
25-35 DEG C of temperature, pH4-6.5, revolving speed 180-220rpm, ventilatory capacity 0.08-0.5L/L.min expand culture 6-12h；Then adjust
Turn over speed 40-100rpm, ventilatory capacity 0.18-0.5L/L.min, expand culture to the microorganism concn reach (0.1-0.5) ×
109/mL。
Expand incubation step using second level to expand culture the microorganism, include the following steps:
1) seed liquor is seeded to according to the inoculum concentration of 5-20% and is spread cultivation tank containing the level-one for expanding culture medium
In, 25-35 DEG C of temperature, pH4-6.5, revolving speed 180-220rpm, ventilatory capacity 0.08-0.5L/L.min are controlled, is expanded culture
Reach (0.1-0.5) × 10 to the microorganism concn9/mL；
2) microorganism that will spread cultivation obtained in step 1) is seeded to according to the inoculum concentration of 5-20% to be cultivated containing the expansion
The second level of base spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-100rpm, ventilatory capacity 0.18-0.5L/L.min,
It expands culture to the microorganism concn and reaches (0.1-0.5) × 109/mL。
Expand incubation step using three-level to expand culture the microorganism, include the following steps:
1) seed liquor is seeded to according to the inoculum concentration of 5-20% and is spread cultivation tank containing the level-one for expanding culture medium
In, 25-35 DEG C of temperature, pH4-6.5, revolving speed 180-220rpm, ventilatory capacity 0.08-0.5L/L.min are controlled, is expanded culture
Reach (0.1-0.5) × 10 to the microorganism concn9/mL；
2) microorganism that will spread cultivation obtained in step 1) is seeded to according to the inoculum concentration of 5-20% to be cultivated containing the expansion
The second level of base spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-60rpm, ventilatory capacity 0.18-0.5L/L.min,
It expands culture to the microorganism concn and reaches (0.1-0.5) × 109/mL；
3) microorganism that will spread cultivation obtained in step 2) is seeded to according to the inoculum concentration of 5-20% to be cultivated containing the expansion
The three-level of base spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-60rpm, ventilatory capacity 0.01-0.2L/L.min,
It expands culture to the microorganism concn and reaches (0.1-0.5) × 109/mL。
Expand incubation step using level Four to expand culture the microorganism, include the following steps:
1) seed liquor is seeded to according to the inoculum concentration of 5-20% and is spread cultivation tank containing the level-one for expanding culture medium
In, 25-35 DEG C of temperature, pH4-6.5, revolving speed 180-220rpm, ventilatory capacity 0.08-0.5L/L.min are controlled, is expanded culture
Reach (0.1-0.5) × 10 to the microorganism concn9/mL；
2) microorganism that will spread cultivation obtained in step 1) is seeded to according to the inoculum concentration of 5-20% to be cultivated containing the expansion
The second level of base spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-60rpm, ventilatory capacity 0.18-0.5L/L.min,
It expands culture to the microorganism concn and reaches (0.1-0.5) × 109/mL；
3) microorganism that will spread cultivation obtained in step 2) is seeded to according to the inoculum concentration of 5-20% to be cultivated containing the expansion
The three-level of base spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-60rpm, ventilatory capacity 0.01-0.2L/L.min,
It expands culture to the microorganism concn and reaches (0.1-0.5) × 109/mL；
4) microorganism that will spread cultivation obtained in step 3) is seeded to according to the inoculum concentration of 5-20% to be cultivated containing the expansion
The level Four of base spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, ventilatory capacity 0.0005-0.02L/L.min, carries out expansion training
It supports to the microorganism concn and reaches (0.1-0.5) × 109/mL。
In the expansion incubation at different levels, the level-one tank that spreads cultivation is that 50L spreads cultivation tank, and the second level tank that spreads cultivation is that 500L spreads cultivation tank,
Three-level spreads cultivation tank as 5 cubes of tanks that spread cultivation, and level Four spreads cultivation tank as 50 cubes of tanks that spread cultivation.
During the expansion at different levels culture, the expansion culture medium it is independent of each other containing corn mash, molasses,
One or more of glucose, sweet sorghum stalk juice or lignocellulosic material enzymolysis liquid as carbon source, containing soybean cake powder,
One or more of corn-dodger powder, corn pulp, fish meal, yeast extract are as nitrogen source；And it is selective containing urea, it is inorganic
Salt, amino acid, microelement and/or inhibitor.
The expansion culture can generate ethanol Step for subsequent fermentation and provide enough micro organism quantities, wherein expanding
Culture medium is the source of nutrition that microorganism obtains existence, and activity and yield to microbial growth, enzyme have direct shadow
It rings.
The selecting property of scale-up medium base includes peptone, corn mash, molasses, glucose, sweet sorghum stalk juice
Or lignocellulosic material enzymolysis liquid.
As a preference, the expansion culture medium is peptone, corn mash, molasses, glucose, sweet sorghum stalk
One or more of juice or lignocellulosic material enzymolysis liquid, and the content for expanding glucose in culture medium is by weight
Degree is 2-10%, preferred content 4-6%.
As another preferred embodiment, the culture medium containing 4% weight percent glucose in the expansion culture medium, Huo Zhehan
Weight percent is the corn mash or molasses of 4% glucose.
Alternatively preferred embodiment, the expansion culture medium can also be that diluted concentration is 10- by weight percentage
75% lignocellulosic material enzymolysis liquid, wherein the lignocellulose raw material enzymolysis liquid contains by weight percentage when not being diluted
There are glucose 1-25%, xylose 1-10%；Preferably, glucose content 5-20%, Xylose Content are preferably 3-8%；It is more excellent
Choosing, glucose content 6-12%, Xylose Content 3-6%.Preferably, the lignocellulosic material enzymolysis liquid is as institute
When stating expansion culture medium, diluted concentration is 20-30% by weight percentage.
Required nitrogen source, microelement, vitamin etc. when preferably as another, to provide the microorganism expansion culture
Substance, it is described to expand the culture medium also soybean cake powder containing 5-10g/L, corn-dodger powder, the jade that dry matter content is 40-60wt%
The mixture of Rice & peanut milk, fish meal and/or yeast extract, preferably 8-10g/L；To provide for the microorganism including alanine, dried meat
Propylhomoserin, phenylalanine, arginine, isoleucine, methionine, glutamic acid, threonine, cystine, leucine and/or valine
A variety of amino acid including free amino acid, including copper, calcium, iron, zinc, lead, silver, chromium, Yan acid, pyridoxic acid, thiamines and/or pantothenic acid
Various trace elements including calcium and the various vitamins including B family vitamin.
As a further preference, urea and/or inorganic salts are also contained in the expansion culture medium, the urea is described
Expanding the content in culture medium is 2-5g/L, preferably 3-4g/L；The inorganic salts be selected from disodium hydrogen phosphate, sodium dihydrogen phosphate,
One or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate, preferably potassium dihydrogen phosphate and/or phosphoric acid hydrogen two
Ammonium, the content in the expansion culture medium is 0.5-4g/L, preferably 1-3g/L.It is furthermore preferred that containing in the expansion culture medium
There are the potassium dihydrogen phosphate of 1.5g/L and the diammonium hydrogen phosphate of 1.5g/L.
Under normal conditions, addition bacteriostatic agent is not needed if entered without miscellaneous bacteria in the expansion incubation, if there is
When miscellaneous bacteria enters, bacteriostatic agent preferably can be added in the expansion culture medium as further, the bacteriostatic agent is selected from blueness
One or more of mycin, ampicillin, streptomysin, chloramphenicol, terramycin, tetracycline, preferably penicillin or/strepto-
Element, it is described expansion liquid medium described in bacteriostatic agent working concentration be 10-50 unit/mL, preferably 15-25 unit/mL,
More preferably 20 units/mL.
It further include that the microorganism is cultivated into 12-24h in seed culture medium before expanding culture for the microorganism
Reach (0.1-0.5) × 10 to the microorganism concn9The step of/ml.
The microorganism is cultivated using seed culture medium, it is therefore an objective to microorganism described in rapid amplifying, to reach as early as possible
To the cell concentration cultivating the Institute of Micro-biology and needing is expanded, the seed culture medium can be using existing seed in the prior art
Culture medium, such as YEPD.
The YEPD refers to that medium component is yeast powder 10g/L, peptone 20g/L, glucose 20g/L, goes out through packing
It is mixed with to obtain after bacterium.
The microorganism is selected from Saccharomyces Cerevisiae in S accharomyces cerevisiae, pachysolen tannophilus Pachysolen
Tannophilus, pichia stipitis Pichia stipitis, shehatae candida Candida shehatae, aroma ferment
Female Brettanomyces naardenensis, very thin Candida Candida tenuis, candida tropicalis Candida
Tropicalis, match ditch Pichia pastoris Pichia segobiensis, Bacteroides polypragmatus Bacteroides
Polypragmatus, chrysanthemum Erwinia Erwinia chrysanthem, plant klebsiella spp Klebsiella
Planticola, thermophilic anaerobic ethyl alcohol bacterium Thermoanaerobacter ethanolicus, spherical spiral body Spirochaeta
Coccoides sp., plant fermentation clostridium Clostridium phytofermentas sp., Fusarium oxysporum Fusarium
Oxysporum, Neuraspora crassa Neurospora crassa, fusarium avenaceum Fusarium avenaceum, motion fermentation list
Born of the same parents bacterium Zymomonas mobilis, Escherichia coli Escherichia coli and recombinant Saccharomyces cerevisiae S.cerevisiae, again
Group zymomonas mobilis Zymomonas mobilis, recombination bacillus coli Escherichia coli and art technology
Other known bacterial strains of C5 and C6 co-fermentation producing and ethanol can be carried out known to personnel.
Mentioned microorganism selected by the method for the invention is isolated from nature, or is changed by genetic engineering
It is obtained after making, including bacterium, fungi and yeast wine brewing.
Wherein, the recombinant Saccharomyces cerevisiae S.cerevisiae be by the xylose transport enzyme of fungi in saccharomyces cerevisiae into
Row expression selects xylose isomerase (XI) approach or xylose reduction-xylitol oxidative pathway (XR-XDH) to carry out xylulose conversion,
And phosphopentose downstream metabolic path is modified to strengthen the ability of xylose metabolism ethanol conversion, available saccharomyces cerevisiae
Bacterium is documented in disclosed in patent WO9742307A, WO9513362A, US20110027847, CN1966694A, CN101205525A
Bacterial strain.
The transformation of the recombination zymomonas mobilis Zymomonas mobilis can (xylose be different by the xylA of E.coli
Structure enzyme gene), xylB (xylulokinase gene), talB (transketolase gene), tktA (transaldolase gene) imported into
In Z.mobilis, for example, patent US5843760, US5514583, WO200851348A, WO200958927A,
WO200944868A or WO200958938A.
The recombination bacillus coli Escherichia coli, referring to (will carry zymomonas mobilis containing PET operon
Bacterium pyruvic dehydrogenase and alcohol dehydrogenase gene) plasmid be transformed into E.coli bacterial strain can make recombinate E.coli sugar and
The central metabolites object that pentose metabolism is formed-pyruvic acid turns to ethyl alcohol production, such as the bacterium reported in patent CN101875912A
Strain.
Preferably, the microorganism is selected from Saccharomyces Cerevisiae in S accharomyces cerevisiae, zymomonas mobilis
Zymomonas mobilis and/or E. coli.
It is further preferred that the microorganism is Saccharomyces Cerevisiae in S accharomyces cerevisiae, motion fermentation list
Born of the same parents bacterium Zymomonas mobilis.
Separating alcohol can use conventional method known in the art, for example, by using distillation, this can be used from fermentation liquid
Conventional distil-lation equipment described in field, such as the destilling tower with double pass tray and cross-current trays.Because fermentation spirituosity produces
The suspended solids content of object is higher, and in general, double fluid sieve column plate or crossing current valve tower plate are preferred.In various preferred implementations
In scheme, the tower including the valve tower plate that flows over is preferably as often providing higher turndown ratio by the valve tower plate that flows over
With higher efficiency.
A kind of method using microbial fermentation lignocellulosic material producing and ethanol of the invention has the advantage that
1. the method for the invention using microbial fermentation lignocellulosic material producing and ethanol, by the way that lignocellulosic is former
Lignocellulosic material enzymolysis liquid is obtained after material enzymatic hydrolysis, the microbial inoculant after then will be enlarged by culture to above-mentioned lignocellulosic
In raw material enzymolysis liquid, the fermentation strategies of intermittent oxygen supply are taken during the fermentation, microorganism is sufficiently comprehensively utilized
The efficiency of glucose and xylose in enzymolysis liquid, producing and ethanol greatly improves.
2. the method for the invention using microbial fermentation lignocellulosic material producing and ethanol, used lignocellulosic
Raw material is selected from corn stover, corncob, hardwood, cork, shuck, grass, paper, barley-straw, wheat straw, leaf, cottonseed wadding, willow
One or more of branch, oat shell, thus can not only have largely using the organic waste for being difficult to rationally dispose in reality
Conducive to environment is improved, mitigate the pressure for handling above-mentioned organic waste, and the ethyl alcohol of high level can also be produced, as clear
Clean fuel uses, to mitigate the utilization of fossil fuel, slows down the speed of Fossil fuel consumption, and protect environment；On
Raw material sources abundance is stated, the producing and ethanol that ferments using grain is can avoid, has saved grain, reduced the production cost of ethyl alcohol.
4. the method for the invention using microbial fermentation lignocellulosic material producing and ethanol, above-mentioned lignocellulosic material
In enzymolysis liquid nitrogen can also be used as containing one or more of soybean cake powder, corn-dodger powder, corn pulp, fish meal or yeast extract
Source is made containing one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate
For inorganic salts, and it is selective containing urea and/or inhibitor etc., it can provide needed for mentioned microorganism during the fermentation
Various nutriments cooperate the fermentation strategies of above-mentioned intermittent oxygen supply to promote the fast breeding of microorganism, improve the quality of fermentation
And speed.
5. the method for the invention using microbial fermentation lignocellulosic material producing and ethanol, passes through the expansion of at least one level
Incubation step cultivates the microorganism, and in the expansion incubation step, adjusts for oxygen concentration, so that described
Microorganism is quickly rised in value, and fermentative activity enhances.
6. the method for the invention using microbial fermentation lignocellulosic material producing and ethanol, used microorganism be from
Nature is isolated, or obtains after genetically engineered, including bacterium, fungi and yeast wine brewing etc., above-mentioned micro- life
Object, can be efficiently former using lignocellulosic under the production method of different fermentations stage of the invention using varying environment condition
Expect that enzymolysis liquid fermentation generates ethyl alcohol.
Specific embodiment
Below with reference to embodiment to a kind of method using microbial fermentation lignocellulosic material producing and ethanol of the invention
Do more specifical description.
Embodiment 1
The present embodiment provides a kind of expansion cultural methods of common fermentation C5 and C6 production ethanol microbe, to recombinate wine brewing ferment
Experiment, the recombinant Saccharomyces cerevisiae are expanded culture for female S.cerevisiae424A (LNH-ST)
S.cerevisiae424A (LNH-ST) (such as Cellulosic ethanol production from AFEX-treated
corn stover using Saccharomyces cerevisiae424A(LNH-ST),M.W.Lau,B.E.Dale,Proc
Natl Acad Sci USA,106(2009),pp.1368-1373；Two-step SSCF to convert AFEX-
treated switchgrass to ethanol using commercial enzymes and Saccharomyces
cerevisiae424A(LNH-ST),M.Jin,M.W.Lau,V.Balan,B.E.Dale,Bioresour Technol,101
(2010), pp.8171-8178 etc. are reported), it is commercially available acquisition, the expansion cultural method expands incubation step, tool using level-one
Body includes:
A, seed liquor incubation step
Prepare YEPD culture medium, the above-mentioned thallus of preservation is seeded in YEPD culture medium, control 25 DEG C of temperature, pH6.0,
Revolving speed 200rpm, ventilatory capacity 0.2L/L.min cultivate 12h, until above-mentioned strain concentration is (0.1-0.5) × 109/ml；
The YEPD culture medium specifically contains yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and adjusts pH6.5.
B, expand incubation step
Seed liquor after culture is expanded into culture medium according to the level-one that contains that the inoculum concentration access volume of 5% (v/v) is 50L
Level-one spread cultivation in tank, 35 DEG C of temperature of control, pH4, revolving speed 180rpm are passed through air, and ventilatory capacity 0.5L/L.min cultivates 12h；
Then adjustment revolving speed be 40rpm, ventilatory capacity 0.18L/L.min, expand culture 12h, until strain concentration reach (0.1-0.5) ×
109/ml；
It includes lignocellulosic material enzymolysis liquid, the corn pulp for being diluted with water to 30wt% that the level-one, which expands culture medium,
(dry matter content 40%) 15g/L, KH2PO41.5g/L、(NH4)2HPO42.5g/L is configured molten using deionized water or softened water
Liquid, packing sterilizing, mixes, and 2g/L urea is added after cooling.
Embodiment 2
The present embodiment provides a kind of expansion cultural methods of common fermentation C5 and C6 production ethanol microbe, with tropical false silk ferment
Experiment is expanded culture for female Candida tropicalis, the candida tropicalis Candida tropicalis is
Commercially available gained, the expansion cultural method expand incubation step using second level, specifically include:
A, seed liquor incubation step
Prepare YEPD culture medium, the above-mentioned thallus of preservation is seeded in YEPD culture medium, control 25 DEG C of temperature, pH6.0,
Revolving speed 200rpm, ventilatory capacity 0.2L/L.min, culture to above-mentioned strain concentration are (0.1-0.5) × 109/ml；
The YEPD culture medium specifically contains yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and adjusts pH6.0.
B, expand incubation step
It 1) is that expanding containing level-one for 50L is trained according to the inoculum concentration access volume of 20% (v/v) by the seed liquor after culture
The level-one of feeding base spreads cultivation in tank, and 25 DEG C of temperature of control, pH6.5, revolving speed 220rpm are passed through air, ventilatory capacity 0.08L/L.min, training
16h is supported, until strain concentration reaches (0.1-0.5) × 109/ml；
2) level-one level-one that step the obtains liquid that spreads cultivation that spreads cultivation is seeded to volume and is according to the inoculum concentration of 20% (v/v)
The second level for expanding culture medium containing second level of 500L spreads cultivation in tank, and 25 DEG C of temperature of control, pH6.5, revolving speed 100rpm are passed through air,
Ventilatory capacity 0.18L/L.min cultivates 12h, until strain concentration reaches (0.1-0.5) × 109/ml；
The level-one expands culture medium and second level expansion culture medium is identical, is the glucose for being 4% containing mass percent
Expand culture medium, and contains corn pulp (dry matter content 40%) 15g/L, KH2PO41.5g/L、(NH4)2HPO41.5g/L；Go from
Sub- water or softened water configure solution, and packing sterilizing mixes, and 3g/L urea is added after cooling.
Embodiment 3
The present embodiment provides a kind of expansion cultural methods of common fermentation C5 and C6 production ethanol microbe, to recombinate wine brewing ferment
It is expanded culture experiment (with embodiment 1) for female S.cerevisiae424A (LNH-ST), the expansion cultural method is adopted
Expand incubation step with three-level, specifically include:
A, seed liquor incubation step
Prepare YEPD culture medium, the above-mentioned thallus of preservation is seeded in YEPD culture medium, control 30 DEG C of temperature, pH6.0,
Ventilatory capacity 0.2L/L.min, revolving speed 200rpm cultivate 14h, until above-mentioned strain concentration is (0.2-0.3) × 109/ml；
The YEPD culture medium specifically contains yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and adjusts pH6.5.
B, expand incubation step
It 1) is that expanding containing level-one for 50L is trained according to the inoculum concentration access volume of 10% (v/v) by the seed liquor after culture
The level-one of feeding base spreads cultivation in tank, and 30 DEG C of temperature of control, pH5.0, revolving speed 200rpm are passed through air, ventilatory capacity 0.5L/L.min, training
It supports 14h and reaches (0.2-0.3) × 10 to strain concentration9/ml；
2) level-one level-one that step the obtains liquid that spreads cultivation that spreads cultivation is seeded to volume and is according to the inoculum concentration of 15% (v/v)
The second level for expanding culture medium containing second level of 500L spreads cultivation in tank, and 25 DEG C of temperature of control, pH6.0, revolving speed 100rpm are passed through air,
Ventilatory capacity 0.5L/L.min, culture 16h to strain concentration reach (0.1-0.5) × 109/ml；
3) according to the inoculum concentration of 5% (v/v) by the second level spread cultivation the second level that step obtains spread cultivation liquid be seeded to volume be 5
The three-level for expanding culture medium containing three-level of cubic meter spreads cultivation in tank, and 33 DEG C of temperature of control, pH6.2, revolving speed 60rpm are passed through sky
Gas, ventilatory capacity 0.01L/L.min, culture 16h to strain concentration reach (0.2-0.3) × 109/ml；
The level-one expands culture medium and second level expansion culture medium is identical, are as follows: 4wt% containing corn mash, after sterilizing
3g/L urea is added；
The three-level expands culture medium are as follows: matter cellulose material enzymolysis liquid, the corn pulp for being diluted with water to 30wt% wood are (dry
Content of material 40%) 15g/L, KH2PO41.5g/L、(NH4)2HPO41.5g/L deionized water or softened water configure solution, packing
Sterilizing mixes after cooling, and 3g/L urea is added.
Embodiment 4
The present embodiment provides a kind of expansion cultural methods of common fermentation C5 and C6 production ethanol microbe, to recombinate wine brewing ferment
Fermenting experiment (with embodiment 1) is carried out for female S.cerevisiae424A (LNH-ST), the expansion cultural method uses four
Grade expands incubation step, specifically includes:
A, seed liquor incubation step
Prepare YEPD culture medium, the above-mentioned thallus of preservation is seeded in YEPD culture medium, control 30 DEG C of temperature, pH6.0,
Ventilatory capacity 0.2L/L.min, revolving speed 200rpm cultivate 20h, until above-mentioned strain concentration is (0.2-0.3) × 109/ml；
The YEPD culture medium specifically contains yeast powder 5g/L, peptone 15g/L, glucose 15g/L, and adjusts pH6.0.
B, expand incubation step
It 1) is that expanding containing level-one for 50L is trained according to the inoculum concentration access volume of 10% (v/v) by the seed liquor after culture
The level-one of feeding base spreads cultivation in tank, and 33 DEG C of temperature of control, pH4.8, revolving speed 200rpm are passed through air, ventilatory capacity 0.4L/L.min, training
It supports 14h and reaches (0.2-0.3) × 10 to strain concentration9/ml；
2) level-one level-one that step the obtains liquid that spreads cultivation that spreads cultivation is seeded to volume and is according to the inoculum concentration of 17% (v/v)
The second level for expanding culture medium containing second level of 500L spreads cultivation in tank, and 30 DEG C of temperature of control, pH5, revolving speed 60rpm are passed through air, leads to
Tolerance 0.3L/L.min, culture 14h to strain concentration reach (0.2-0.3) × 109/ml；
3) second level second level that step the obtains liquid that spreads cultivation that spreads cultivation is seeded to volume and is according to the inoculum concentration of 18% (v/v)
5 cubic metres of the three-level for expanding culture medium containing three-level spreads cultivation in tank, and 35 DEG C of temperature of control, pH6.5, revolving speed 60rpm are passed through sky
Gas, ventilatory capacity 0.18L/L.min, culture 16h to strain concentration reach (0.2-0.3) × 109/ml；
4) three-level three-level that step the obtains liquid that spreads cultivation that spreads cultivation is seeded to volume and is according to the inoculum concentration of 20% (v/v)
30 cubic metres of the level Four for expanding culture medium containing level Four spreads cultivation in tank, controls 33 DEG C of temperature, pH5.8, ventilatory capacity 0.01L/
L.min, culture 16h to strain concentration reach (0.2-0.3) × 109/ml。
The level-one expands culture medium and second level expands culture medium and expands culture medium and second level with the level-one in embodiment 3
It is identical to expand culture medium；
The three-level expands culture medium and level Four expansion culture medium is identical as the three-level expansion culture medium in embodiment 3.
In embodiment 1,3,4, the raw material of lignocellulosic material enzymolysis liquid is selected from corn stover, and preparation method is such as
Under:
Step (1): being broken for the particle that granularity is 10-100mm for corn stover, put the raw materials into scroll feeder, right
It carries out extrusion dehydration to dry 20%-50%, forms fine and close material plug, constantly enters process container in guarantee cellulosic material
While the steam that can also resist in container leak；
Step (2): the material plug formed in step 1 is mixed into its matter into cellulosic material after scroll feeder processing
The dilute sulphuric acid solution of amount 2% obtains acid cellulose raw material after mixing into sour blending tank；
Step (3): low-pressure steam of the acid cellulose raw material in step 2 in boiling process container, with 0.6MPa (G)
Contact is handled, and handles the time between 15-25min, and appearance then is discharged in the way of the discharge of certain frequency intermittent decompression
Device, this process can open the structure of cellulosic material, hemicellulose fraction made to degrade, and portion of cellulose is exposed on surface；
Step (4): biomass processes product is washed, and adjusting pH value is 5.0, after being heated to 50 DEG C, with every gram of product
Dry weight meter, the cellulase that 20-50 filter paper enzyme activity unit of force is added calculate, and squeezing into cellulase, (letter Co., Ltd, Novi mentions
For), and 72h is mixed in heat preservation at 50 DEG C, obtains the enzymolysis liquid.
The expansion culture strain liquid that will be cultivated in embodiment 1-4, is connected to by 10% inoculum concentration containing fermentation medium
Fermentor in, control in fermentation process 30 DEG C of temperature, pH6.0, revolving speed 200rpm are passed through air, ventilatory capacity 0.2L/L.min,
Ferment 16h, detects tunning.
The fermentation medium are as follows: glucose 8wt%, xylose 4.5wt%, KH2PO40.5g/L、(NH4)2HPO42g/L；It goes
Ionized water or softened water configure solution, and packing sterilizing mixes after cooling.
| Spread cultivation series | Originate sugared concentration (g/L) | Terminal concentration of alcohol (g/L) | Total used time (spread cultivation+ferment (h) | |
| Embodiment 1 | 1 | 77.00+43.42 | 30.96 | 28 |
| Embodiment 2 | 2 | 77.00+43.42 | 41.30 | 44 |
| Embodiment 3 | 3 | 77.00+43.42 | 45.78 | 62 |
| Embodiment 4 | 4 | 77.00+43.42 | 50.35 | 76 |
Embodiment 5
The present embodiment provides a kind of methods using microbial fermentation lignocellulosic material producing and ethanol, to recombinate wine brewing ferment
Fermenting experiment, the recombinant Saccharomyces cerevisiae S.cerevisiae424A are carried out for female S.cerevisiae424A (LNH-ST)
(LNH-ST) (such as Cellulosic ethanol production from AFEX-treated corn stover using
Saccharomyces cerevisiae424A (LNH-ST), M.W.Lau, B.E.Dale, Proc Natl Acad Sci USA,
106 (2009), pp.1368-1373；Two-step SSCF to convert AFEX-treated switchgrass to
Ethanol using commercial enzymes and Saccharomyces cerevisiae424A (LNH-ST),
M.Jin, M.W.Lau, V.Balan, B.E.Dale, Bioresour Technol, 101 (2010), the equal report of pp.8171-8178
Road), it is commercially available acquisition, the method using microbial fermentation lignocellulosic material producing and ethanol specifically includes: by wooden fibre
The step of tieing up the step of plain raw material digests the step of lignocellulosic material enzymolysis liquid is made, seed culture, strain expansion culture,
And the step of fermentation producing and ethanol, it specifically includes:
A, the step of lignocellulosic material enzymolysis liquid is made in enzymatic hydrolysis lignocellulosic material
The raw material of the lignocellulosic material enzymolysis liquid is selected from corn stover, and preparation method is as follows:
Step 1): corn stover is broken for the particle that granularity is 10-100mm, scroll feeder is putted the raw materials into, to it
Extrusion dehydration is carried out to dry 20%-50%, fine and close material plug is formed, constantly enters process container in guarantee cellulosic material
The steam that can also be resisted in container simultaneously leaks；
Step 2): the material plug formed in step 1) is mixed into its matter into cellulosic material after scroll feeder processing
The dilute sulphuric acid solution of amount 2% obtains acid cellulose raw material after mixing into sour blending tank；
Step 3): low-pressure steam of the acid cellulose raw material in step 2) in boiling process container, with 0.6MPa (G)
Contact is handled, and handles the time between 15-25min, and appearance then is discharged in the way of the discharge of certain frequency intermittent decompression
Device, this process can open the structure of cellulosic material, hemicellulose fraction made to degrade, and portion of cellulose is exposed on surface；
Step 4): biomass processes product is washed, and adjusting pH value is 5.0, after being heated to 50 DEG C, with the dry of every gram of product
Restatement, the cellulase that 20-50 filter paper enzyme activity unit of force is added calculate, and squeeze into cellulase (Novi believes that Co., Ltd provides),
And 72h is mixed in heat preservation at 50 DEG C, obtains the enzymolysis liquid.
B, seed liquor incubation step
Prepare YEPD culture medium, the above-mentioned thallus of preservation is seeded in YEPD culture medium, control 30 DEG C of temperature, pH6.0,
Revolving speed 200rpm, ventilatory capacity 0.2L/L.min cultivate 20h, until above-mentioned strain concentration is (0.2-0.3) × 109/ml；
The YEPD culture medium specifically contains yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and adjusts pH6.0.
C, expand incubation step
It 1) is that expanding containing level-one for 50L is cultivated according to the inoculum concentration access volume of 5% (v/v) by the seed liquor after culture
The level-one of base spreads cultivation in tank, and 30 DEG C of temperature of control, pH6.0, revolving speed 200rpm are passed through air, ventilatory capacity 0.1L/L.min, culture
16h reaches (0.2-0.3) × 10 to strain concentration9/ml；
2) level-one level-one that step the obtains liquid that spreads cultivation that spreads cultivation is seeded to volume and is according to the inoculum concentration of 5% (v/v)
The second level for expanding culture medium containing second level of 500L spreads cultivation in tank, and 30 DEG C of temperature of control, pH6.0, revolving speed 50rpm are passed through air,
Ventilatory capacity 0.2L/L.min, culture 16h to strain concentration reach (0.1-0.5) × 109/ml；
The level-one expands culture medium and second level expansion culture medium is identical, is the glucose for being 4% containing mass percent
Expand culture medium, and contains corn pulp (dry matter content 40%) 15g/L, KH2PO41.5g/L、(NH4)2HPO41.5g/L；Go from
Sub- water or softened water configure solution, and packing sterilizing mixes, and 3g/L urea is added after cooling.
D, fermentation producing and ethanol step
Second level after taking the second level to expand culture spreads cultivation liquid, is seeded to according to the inoculum concentration of 10% (v/v) containing upper
Lignocellulosic material enzymolysis liquid is stated as in the fermentor of fermentation medium, controls 28 DEG C of temperature, pH6.0, ventilatory capacity
0.01mL/L, interval are ventilated, duration of ventilation 0.5h, interval time 6h, and ferment 72h, obtain tunning.
Embodiment 6
The present embodiment provides a kind of methods using microbial fermentation lignocellulosic material producing and ethanol, to recombinate wine brewing ferment
Fermenting experiment (with embodiment 5) is carried out for female S.cerevisiae424A (LNH-ST), the method tool of the fermentation producing and ethanol
The step of the step of body includes: lignocellulosic material enzymolysis liquid that lignocellulosic material enzymatic hydrolysis is obtained, seed culture, strain
The step of expanding culture, and the step of fermentation producing and ethanol, specifically include:
A, the step of lignocellulosic material enzymolysis liquid is made in enzymatic hydrolysis lignocellulosic material
With embodiment 5, difference is, used lignocellulosic material is corncob and grass.
B, seed liquor incubation step
Prepare YEPD culture medium, the above-mentioned thallus of preservation is seeded in YEPD culture medium, control 25 DEG C of temperature, pH6.0,
Revolving speed 200rpm, ventilatory capacity 0.2L/L.min cultivate 12h, until above-mentioned strain concentration is (0.1-0.5) × 109/ml；
The YEPD culture medium specifically contains yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and adjusts pH6.5.
C, expand incubation step
Seed liquor after culture is expanded into culture according to the level-one that contains that the inoculum concentration access volume of 10% (v/v) is 50L
The level-one of base spreads cultivation in tank, and 30 DEG C of temperature of control, pH6.0, revolving speed 200rpm are passed through air, ventilatory capacity 0.05L/L.min, culture
14h reaches (0.2-0.3) × 10 to strain concentration9/ml；
In the inoculum concentration according to 10%, the liquid that spreads cultivation of the level-one after taking described spread cultivation is seeded to the second level of 500L and spreads cultivation in tank,
30 DEG C of temperature of control, pH6.0, revolving speed 40rpm are passed through air, ventilatory capacity 0.3L/L.min, and culture 14h to strain concentration reaches
(0.2-0.3)×109/ml；
It is that the lignocellulosic material enzymolysis liquid for being diluted with water to 30wt%, corn pulp are (dry that the level-one, which expands culture medium,
Content of material 40%) 15g/L, KH2PO41.5g/L、(NH4)2HPO41.5g/L deionized water or softened water configure solution, packing
Sterilizing mixes after cooling, and 2g/L urea is added.
D, fermentation producing and ethanol step
The liquid that spreads cultivation after above-mentioned expansion culture is taken, is seeded to according to the inoculum concentration of 10% (v/v) containing fermented and cultured
In the fermentor of base, 25 DEG C of temperature, pH5, ventilatory capacity 0.5mL/L are controlled, interval is ventilated, duration of ventilation 0.5h, interval time
For 6h, ferment 72h；Obtain tunning；
The fermentation medium are as follows: above-mentioned lignocellulosic material enzymolysis liquid, the corn pulp for being diluted with water to 30wt% are (dry
Content of material 45wt%) 7.5g/L, KH2PO40.5g/L, (NH4)2HPO42g/L, fermentation adjusts pH before starting be 5.0.
Embodiment 7
The present embodiment provides a kind of methods using microbial fermentation lignocellulosic material producing and ethanol, to recombinate wine brewing ferment
Fermenting experiment (with embodiment 5) is carried out for female S.cerevisiae424A (LNH-ST), the method tool of the fermentation producing and ethanol
The step of the step of body includes: lignocellulosic material enzymolysis liquid that lignocellulosic material enzymatic hydrolysis is obtained, seed culture, strain
The step of expanding culture, and the step of fermentation producing and ethanol, specifically include:
A, the step of lignocellulosic material enzymolysis liquid is made in enzymatic hydrolysis lignocellulosic material
With embodiment 5, difference is, used lignocellulosic material is barley-straw and wheat-straw.
B, seed liquor incubation step
Prepare YEPD culture medium, the above-mentioned thallus of preservation is seeded in YEPD culture medium, control 30 DEG C of temperature, pH6.0,
Revolving speed 200rpm, ventilatory capacity 0.2L/L.min cultivate 14h, until above-mentioned strain concentration is (0.2-0.3) × 109/ml；
The YEPD culture medium specifically contains yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and adjusts pH6.0-
6.5。
C, expand incubation step
Level-one culture: the level-one of the present embodiment expands culture medium and expands culture medium using 4% glucose: wherein containing glucose
4wt%, corn pulp (dry matter content 40%) 15g/L, KH2PO41.5g/L, (NH4) 2HPO41.5g/L, deionized water or soft
Change water and configure solution, packing sterilizing mixes after cooling, 5g/L urea is added；The method and parameter of culture is same as Example 6；
Second level culture: same as Example 6.
D, fermentation producing and ethanol step
Expand the liquid that spreads cultivation after culture, is seeded to volume according to the inoculum concentration of 10% (v/v) and is 30 cubic metres and contain
Have in the fermentor of fermentation medium, control 30 DEG C of temperature, pH6.5, ventilatory capacity 0.1mL/L, interval is ventilated, and duration of ventilation is
0.5h, interval time 5h, ferment 72h, obtains tunning；
It is supplied oxygen in fermentation process using micro- oxygen equipment.
The fermentation medium are as follows: be diluted with water to lignocellulosic material enzymolysis liquid, the corn pulp (dry matter of 30wt%
Content 40wt%) 7.5g/L, KH2PO40.5g/L, (NH4)2HPO42g/L, fermentation adjusts pH before starting be 6.0.
Embodiment 8
The present embodiment provides a kind of methods using microbial fermentation lignocellulosic material producing and ethanol, to recombinate wine brewing ferment
Fermenting experiment (with embodiment 5) is carried out for female S.cerevisiae424A (LNH-ST), the method tool of the fermentation producing and ethanol
The step of body includes: the step of lignocellulosic material enzymolysis liquid is made in enzymatic hydrolysis lignocellulosic material, seed culture, strain expand
It the step of big culture, and the step of fermentation producing and ethanol, specifically includes:
A, the step of lignocellulosic material enzymolysis liquid is made in enzymatic hydrolysis lignocellulosic material
With embodiment 5, difference is, lignocellulosic material used in the present embodiment is paper, leaf and oat shell.
B, seed liquor incubation step
YEPD culture medium is prepared, the above-mentioned Candida tropicalis body of preservation is seeded in YEPD culture medium, control temperature
Spend 30 DEG C, pH6.0, revolving speed 200rpm, ventilatory capacity 0.2L/L.min, cultivate 20h, to above-mentioned strain concentration for (0.2-0.3) ×
109/ml；
The YEPD culture medium specifically contains yeast powder 10g/L, peptone 20g/L, glucose 20g/L, and adjusts pH6.0-
6.5。
C, expand incubation step
With embodiment 6；
Difference is: the level-one of the present embodiment expands culture medium and uses corn mash: 4wt% containing corn mash goes out
3g/L urea is added after bacterium.
D, fermentation producing and ethanol step
The liquid that spreads cultivation expanded after cultivating is taken, 5 containing fermentation medium are seeded to according to the inoculum concentration of 8% (v/v)
In the fermentor of cubic meter, 37 DEG C of temperature, pH4.8, ventilatory capacity 0.2mL/L are controlled, interval is ventilated, duration of ventilation 0.5h,
It is 6h every the time；
It is supplied oxygen in fermentation process using micro- oxygen equipment；Used fermentation medium is same as Example 7.
Embodiment 9
The seed liquor culture of strain described in the present embodiment, the method for expanding culture one grade fermemtation culture and parameter and seed
Liquid culture medium, the selection of ferment liquid culture medium and the liquid culture medium that spreads cultivation are same as Example 7, and difference is only that, are fermenting
During culture, using the condition for culture of continuously fermenting, 28 DEG C of fermentation temperature, pH6.0 are controlled, keeps ventilatory capacity 0.5mL/L
Fermentation 72h is carried out, tunning is detected.
The tunning content for detecting above-described embodiment 5-9, see the table below.
Obviously, above-described embodiment is only intended to clearly illustrate example, and does not limit the embodiments.For
For those of ordinary skill in the art, other various forms of variations or change can also be made on the basis of the above description
It is dynamic.There is no necessity and possibility to exhaust all the enbodiments.And obvious variation extended from this or change
It moves still within the protection scope of the invention.
Claims (14)
1. a kind of method using microbial fermentation lignocellulosic material producing and ethanol, which comprises the steps of:
1), the lignocellulosic material is digested, obtains lignocellulosic material enzymolysis liquid, and former in the lignocellulosic
Expect that nitrogen source, inorganic salts are added in enzymolysis liquid, urea and/or bacteriostatic agent is simultaneously selectively added；
2), microorganism is expanded and is cultivated, scale-up medium is obtained；The microorganism is that can carry out C5 and C6 co-fermentation to produce second
The bacterial strain of alcohol；The expansion incubation step is that the microbial inoculant is expanded by least one level and trained to expanding in culture medium
Step is supported to expand culture the microorganism；
3) scale-up medium is seeded in the lignocellulosic material enzymolysis liquid by 5-20%, controls reaction temperature
25-38 DEG C, pH5.0-7.0 progress fermenting and producing, control and carry out intermittent oxygen supply in the fermentation process, ventilatory capacity 0.01-
0.5mL/L.min, duration of ventilation 10-30min, interval time 5-6h, fermentation obtain fermentation liquid；
4) it, separates the fermentation liquid and obtains ethyl alcohol.
2. utilizing the method for microbial fermentation lignocellulosic material producing and ethanol according to claim 1, which is characterized in that institute
It states in step 3), controls air ventilatory capacity 0.1-0.2mL/L.min in fermentation process.
3. the method according to claim 1 or claim 2 using microbial fermentation lignocellulosic material producing and ethanol, feature exist
In the lignocellulosic material is selected from corn stover, corncob, hardwood, cork, shuck, grass, paper, barley-straw, wheat
One or more of bar, leaf, cottonseed wadding, withy, oat shell.
4. utilizing the method for microbial fermentation lignocellulosic material producing and ethanol according to claim 3, which is characterized in that institute
It states lignocellulosic material and digests used enzyme selected from cellulase, hemicellulase, amylase, protease, glucose starch
Enzyme, lipase.
5. the method for utilizing microbial fermentation lignocellulosic material producing and ethanol according to claim 1 or described in any one of 2,
It is characterized in that, the nitrogen source is one or more of soybean cake powder, corn-dodger powder, corn pulp, fish meal or yeast extract；The nothing
Machine salt is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate.
6. the method for utilizing microbial fermentation lignocellulosic material producing and ethanol according to claim 1 or described in any one of 2,
It is characterized in that, in the expansion incubation step, expands realizing for oxygen concentration to micro- life in incubation by adjusting
The expansion culture of object.
7. utilizing the method for microbial fermentation lignocellulosic material producing and ethanol according to claim 6, which is characterized in that make
Expand incubation step with level-one to expand culture the microorganism, include the following steps:
The seed liquor of the microorganism is seeded to according to the inoculum concentration of 5-20% and is spread cultivation in tank containing the level-one for expanding culture medium,
25-35 DEG C of temperature, pH4-6.5, revolving speed 180-220rpm, ventilatory capacity 0.08-0.5L/L.min are controlled, culture 6-12h is expanded；With
Revolving speed 40-100rpm, ventilatory capacity 0.18-0.5L/L.min are adjusted afterwards, are expanded culture to the microorganism concn and are reached (0.1-
0.5)×109/mL。
8. utilizing the method for microbial fermentation lignocellulosic material producing and ethanol according to claim 6, which is characterized in that make
Expand incubation step with second level to expand culture the microorganism, include the following steps:
1) seed liquor of the microorganism is seeded to according to the inoculum concentration of 5-20% and is expanded containing the level-one for expanding culture medium
It trains in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 180-220rpm, ventilatory capacity 0.08-0.5L/L.min, expanded
Culture to the microorganism concn reaches (0.1-0.5) × 109/mL；
2) microorganism that will spread cultivation obtained in step 1) is seeded to according to the inoculum concentration of 5-20% containing the expansion culture medium
Second level spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-100rpm, ventilatory capacity 0.18-0.5L/L.min, carries out
Expand culture to the microorganism concn and reaches (0.1-0.5) × 109/mL。
9. utilizing the method for microbial fermentation lignocellulosic material producing and ethanol according to claim 6, which is characterized in that make
Expand incubation step with three-level to expand culture the microorganism, include the following steps:
1) seed liquor of the microorganism is seeded to according to the inoculum concentration of 5-20% and is expanded containing the level-one for expanding culture medium
It trains in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 180-220rpm, ventilatory capacity 0.08-0.5L/L.min, expanded
Culture to the microorganism concn reaches (0.1-0.5) × 109/mL；
2) microorganism that will spread cultivation obtained in step 1) is seeded to according to the inoculum concentration of 5-20% containing the expansion culture medium
Second level spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-60rpm, ventilatory capacity 0.18-0.5L/L.min, carries out
Expand culture to the microorganism concn and reaches (0.1-0.5) × 109/mL；
3) microorganism that will spread cultivation obtained in step 2) is seeded to according to the inoculum concentration of 5-20% containing the expansion culture medium
Three-level spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-60rpm, ventilatory capacity 0.01-0.2L/L.min, carries out
Expand culture to the microorganism concn and reaches (0.1-0.5) × 109/mL。
10. utilizing the method for microbial fermentation lignocellulosic material producing and ethanol according to claim 6, which is characterized in that
Expand incubation step using level Four to expand culture the microorganism, include the following steps:
1) seed liquor of the microorganism is seeded to according to the inoculum concentration of 5-20% and is expanded containing the level-one for expanding culture medium
It trains in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 180-220rpm, ventilatory capacity 0.08-0.5L/L.min, expanded
Culture to the microorganism concn reaches (0.1-0.5) × 109/mL；
2) microorganism that will spread cultivation obtained in step 1) is seeded to according to the inoculum concentration of 5-20% containing the expansion culture medium
Second level spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-60rpm, ventilatory capacity 0.18-0.5L/L.min, carries out
Expand culture to the microorganism concn and reaches (0.1-0.5) × 109/mL；
3) microorganism that will spread cultivation obtained in step 2) is seeded to according to the inoculum concentration of 5-20% containing the expansion culture medium
Three-level spreads cultivation in tank, controls 25-35 DEG C of temperature, pH4-6.5, revolving speed 40-60rpm, ventilatory capacity 0.01-0.2L/L.min, carries out
Expand culture to the microorganism concn and reaches (0.1-0.5) × 109/mL；
4) microorganism that will spread cultivation obtained in step 3) is seeded to according to the inoculum concentration of 5-20% containing the expansion culture medium
Level Four spreads cultivation in tank, control 25-35 DEG C of temperature, pH4-6.5, ventilatory capacity 0.0005-0.02L/L.min, expand culture to
The microorganism concn reaches (0.1-0.5) × 109/mL。
11. the method that microbial fermentation lignocellulosic material producing and ethanol is utilized according to any one of claim 7-10,
It is characterized in that, in the expansion incubation at different levels, the level-one tank that spreads cultivation is that 50L spreads cultivation tank, and the second level tank that spreads cultivation spreads cultivation for 500L
Tank, three-level spread cultivation tank as 5 cubes of tanks that spread cultivation, and level Four spreads cultivation tank as 50 cubes of tanks that spread cultivation.
12. according to any one of the claim 7-10 method using microbial fermentation lignocellulosic material producing and ethanol,
It is characterized in that, during the expansion cultures at different levels, the expansion culture medium is independent of each other to contain corn mash, sugar
One or more of honey, glucose, sweet sorghum stalk juice or lignocellulosic material enzymolysis liquid contain soyabean cake as carbon source
One or more of powder, corn-dodger powder, corn pulp, fish meal, yeast extract are as nitrogen source；And selective contains urea, nothing
Machine salt, amino acid, microelement and/or bacteriostatic agent.
13. the method that microbial fermentation lignocellulosic material producing and ethanol is utilized according to any one of claim 7-10,
It is characterized in that, further including that the microorganism is cultivated 12-24h in seed culture medium before expanding the culture microorganism
Reach (0.1-0.5) × 10 to the microorganism concn9The step of/ml.
14. utilizing the method for microbial fermentation lignocellulosic material producing and ethanol according to claim 1, which is characterized in that
The microorganism is selected from Saccharomyces Cerevisiae in S accharomyces cerevisiae, pachysolen tannophilus Pachysolen
Tannophilus, pichia stipitis Pichia stipitis, shehatae candida Candida shehatae, aroma ferment
Female Brettanomyces naardenensis, very thin Candida Candida tenuis, candida tropicalis Candida
Tropicalis, match ditch Pichia pastoris Pichia segobiensis, Bacteroides polypragmatus Bacteroides
Polypragmatus, chrysanthemum Erwinia Erwinia chrysanthem, plant klebsiella spp Klebsiella
Planticola, thermophilic anaerobic ethyl alcohol bacterium Thermoanaerobacter ethanolicus, spherical spiral body Spirochaeta
Coccoides sp., plant fermentation clostridium Clostridium phytofermentas sp., Fusarium oxysporum Fusarium
Oxysporum, Neuraspora crassa Neurospora crassa, fusarium avenaceum Fusarium avenaceum, motion fermentation list
Born of the same parents bacterium Zymomonas mobilis, Escherichia coli Escherichia coli and recombinant Saccharomyces cerevisiae S.cerevisiae, again
Group zymomonas mobilis Zymomonas mobilis, recombination bacillus coli Escherichia coli.
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| GB201620658D0 (en) * | 2016-12-05 | 2017-01-18 | Univ Stellenbosch | Recombinant yeast and use thereof |
| CN106906150A (en) * | 2017-03-24 | 2017-06-30 | 黑龙江中丹建业生物能源有限公司 | The expansion method of the hexose co-fermentation saccharomycete of new metabolism five |
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| CN113025506B (en) * | 2019-12-24 | 2022-12-09 | 中国科学院天津工业生物技术研究所 | Recombinant filamentous fungus for producing ethanol and construction and application thereof |
| CN111849811B (en) * | 2020-07-16 | 2022-08-26 | 山佳经典(北京)科技有限公司 | Compound bacterium system for efficiently decomposing lignocellulose materials and culture method thereof |
| CN113881710B (en) * | 2021-10-29 | 2023-09-22 | 安徽工程大学 | Method for co-production of biological hydrogen and microalgae grease by lignocellulose fermentation |
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