CN109097416A - Lignocellulosic one kettle way bioconversion method - Google Patents
Lignocellulosic one kettle way bioconversion method Download PDFInfo
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Abstract
The present invention provides the methods of lignocellulosic one kettle way bioconversion, comprising the following steps: (1) mechanical treatment: lignocellulosic material is crushed, and cleaning launches into closed reactor, adds water, is impregnated.(2) it is chemically treated: ammonium sulfite being added into reactor, after sulfonation is disposed, flows out black liquor；(3) material cleans: water being added into reactor, is uniformly mixed；It is passed through high pressure nitrogen, flows out black liquor；(4) it is saccharified: saccharification culture medium, autoclave sterilization being added into container；Inoculation produces cellulosome bacterial strain seed liquor, and saccharification obtains the liquid glucose containing glucose.(5) it ferments: accessing the aerobic heterotrophic microorganism seed liquor of activation into the fermentation medium of acquisition, ferment.Method of the present invention, simplifies processing step and the requirement to equipment, and reduces carbon source cost and use enzyme cost, while solving the problem of complex utilization of agriculture and forestry organic waste material, with huge environmental benefit and social benefit.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of bioconversion method of lignocellulosic, and in particular to a kind of
Full bacterium method for saccharifying for lignocellulosic.
Background technique
In recent years, the reproducible energy, material or chemicals are obtained from biomass obtains the common concern of countries in the world.
It is that raw material produces second generation biologically based fuels or chemicals using lignocellulose-like biomass, can become substitute stone on a large scale
The key of oil-based products.Lignocellulose-like biomass is present on earth renewable biomass resources the most abundant, by it
The energy, chemicals or material are converted into huge application and development prospect.Particularly, China is large agricultural country, annual
Agricultural crop straw yield is more than 900,000,000 tons.On the other hand, environmental pollution caused by crop straw burning and the problems such as traffic accidents,
Also promote country to release planning, scheme and the measure of a series of stalk comprehensive utilizations, agriculture waste biomass comprehensive is pushed to utilize
Industrialized development.
The conversion of lignocellulosic to fuel or chemicals is usually required by three pretreatment, saccharification, fermentation steps
Unit, the usual technical conditions of different step unit have very big difference, and whole operation process is complicated, energy consumption is high, generates simultaneously
A large amount of waste water.Therefore, occur the relevant technologies of saccharification and fermenting step integration, including fractional hydrolysis zymotechnique in the world
(SHF), simultaneous saccharification and fermentation technique (SSF) and three kinds of biological processing technology (CBP) is integrated.Wherein, SHF and SSF are required
First in independent reactor carry out originated from fungus free cellulose enzyme system production, then to raw material after pretreatment into
The enzyme hydrolysis and fermentation of row cellulose.CBP is by each relatively independent sport technique segment such as the production of cellulase and enzymatic hydrolysis and fermentation
It is integrated into same step, (Curr Opin Biotechnol 2005,16:577-583) is carried out in same reactor, has
Simple flow reduces the advantages such as equipment requirement.
In addition, researcher has also carried out the research of preconditioning technique Yu downstream process integration technology.The human hairs such as Kataeva
Existing, Thermophilic Bacterium Caldicellulosiruptor bescii can be hydrolyzed without pretreated substrate (Energy&
Environmental Science.2013.6:2186-2195.Application for a patent for invention 201010149132.2 discloses " a kind of
The method of cellulosic material saccharification ".This method is the cellulosome based on a kind of secretion of high temperature thermophilc anaerobe, and the cellulose is small
Body is capable of the avicel cellulose of effective degrading texture complexity.This application can make pretreated without method physically or chemically
Cellulosic material directly carries out fermentation or enzymatic treatment, and effectively hydrolyzing is reduced sugar.But to there is efficiency too low for the two, and
The problem of final product complicated component, does not have the value of application still.Application for a patent for invention 201610202657.5, which discloses, " to be utilized
The method that biomass ' one kettle way ' prepares fermentable sugars or bio-ethanol "；Method includes the following steps: (1) is by mechanical treatment
And stirring is added into low temperature sodium hydroxide/urea liquid system to room temperature in the biomass after being sieved；(2) acid for adjusting pH is added,
Enzyme hydrolysis is added；Sugared content in enzymolysis liquid is measured, enzymatic hydrolysis conversion ratio sugared in biomass material is calculated；(3) 24~48h of enzyme hydrolysis
Afterwards, strain is inoculated with into enzymolysis liquid, fermenting and preparing biological ethyl alcohol measures the yield of target product.This application will pretreatment, enzyme water
Solution, fermentation one kettle way are completed, and it is convenient to operate, time-consuming short, the loss of lignocellulosic substrate and a large amount of waste water during reducing
Generation.However, this method needs to be added the neutralization that acid carries out lye, chemicals usage is big, and the yield of final product is lower.
In addition, this application enzymolysis process uses free cellulose enzyme preparation, since existing enzyme preparation technology is by company of developed country ridge
It is disconnected therefore generally with high costs using the production technology of cellulase preparation, do not have economy.
Sulfonation method is industrially to use sulphite or bisulfites will using the method for more removing lignin
Sulphonation of Lignin, the lignin after sulfonation are easier to remove due to hydrophilic increase.Lv Jiaqing etc. is located in advance using ammonium sulfite
Wheat straw stock waste residue (Chinese papermaking, 2015,34,1-6) is managed, is pre-processed at 165 DEG C with 16% ammonium sulfite, through PFI defibrination
Obtained substrate, enzyme hydrolysis total reducing sugar conversion ratio is up to 67%.Yu et al. research discovery (Bioresource Technology,
2016,199,188-193), with 5.21% magnesium sulfite, 60min, about 90% lignin and 80% are handled at 170 DEG C
Hemicellulose can remove, the cellulose conversion ratio after enzyme hydrolysis is up to 90%.However, the pretreated wooden fibre of sulfonation method
It ties up in plain raw material rich in free lignin component, that is, forms black liquor, and pH value is high, there is significant inhibition to enzyme preparation,
It is generally necessary to be cleaned repeatedly after blanking with special washing device, next step enzymatic saccharification just can be carried out；And individually make
With sulfonation preprocessing lignocellulose raw material, what is obtained after enzyme hydrolysis is also mixed sugar, and hemicellulose utilization efficiency is lower.Invention
Patent application 201610133959.1 discloses " a method of pretreatment separation lignocellulose-like biomass ", the side
Then method is presoaked the following steps are included: 1) first by lignocellulose-like biomass Mechanical Crushing to suitable particle size
Stain；2) solid obtained in step 1) is mixed with water or vapor and carries out hydrothermal pretreatment；3) it will be passed through obtained in step 2)
Solid material after hydrothermal pretreatment and cleaning is mixed with sulfonation pretreatment medical fluid carries out sulfonation pretreatment.This application is pre- by hydro-thermal
Processing and sulfonation pretreated group altogether, both can effectively remove hemicellulose, can also effectively remove lignin, to make bottom
Fibres cellulose content significantly improves, and substantially improves its enzyme hydrolysis efficiency.
Compared with dependent on the technique of enzyme preparation, the biology saccharification strategy based on the full bacterium catalysis of cellulosome, which has, simplifies stream
Journey reduces the advantages such as equipment requirement, is the process route for being most suitable for cellulose series biomass bioconversion and utilizing.Cellulosome is
A kind of extracellular multienzyme complex with labyrinth and component is known most efficient cellulose degradation system in nature
One of.Currently, the full bacterium catalysis saccharification of lignocellulosic mainly utilizes the high temperature anaerobic based on this production cellulosome of Clostridium thermocellum
Bacterium is as full bacterium catalyst.So far, the report still without full bacterium catalysis saccharification " one kettle way " technology of lignocellulosic.
Summary of the invention
For lignocellulosic in the prior art utilize in the presence of the step of it is cumbersome, technology matching is poor, it is with high costs,
The problems such as seriously polluted, the present invention provides the methods of lignocellulosic one kettle way bioconversion, at low cost, process is simple
Etc. advantages, and reduce discharge of wastewater, industrially application prospect is good.
The method of lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: lignocellulosic material is mechanically pulverized, and is washed with water, then from closed reactor
The feeding port of side is launched into closed reactor, is added water according to solid and liquid weight volume ratio 1:2-1:6, is uniformly mixed, impregnates.
Wherein, the fragment partial size of lignocellulosic material is no more than 2cm, soaking time 2-4h after the mechanical crushing.
The lignocellulosic material is one of corn stover, wheat straw, brush wood branch, wood chip, corncob, straw and waste paper or more
The combination of kind.
(2) be chemically treated: appropriate ammonium sulfite is added into closed reactor, under the conditions of 160-190 DEG C of temperature into
Row sulfonation handles 1-4h.After completion of the reaction, the discharge port for opening closed reactor bottom, makes black liquor via provisioned in discharge port
Filter device flows out in storage tank；The storage tank is connected by pipeline with closed reactor.When in closed reactor
It when pressure is down to atmospheric pressure, is vacuumized using the vacuum pump at the top of storage tank, makes to form negative pressure in storage tank, black liquor further flows
Out；Until the water content of material reaches 30%-50% in closed reactor, discharge port is closed.
Wherein, the final concentration of 9-15% (w/v) of the closed reactor sulfite ammonium.
(3) material cleans: water being added into closed reactor, is uniformly mixed；High pressure nitrogen is passed through into closed reactor,
So that reactor pressure is reached 10-12 standard atmospheric pressure, then open closed reactor bottom discharge port, make black liquor via
Filter device provisioned in discharge port flows out in storage tank.When the pressure in closed reactor is down to atmospheric pressure, storage is utilized
The vacuum pump of hiding tank top vacuumizes, and makes to form negative pressure in storage tank, black liquor further flows out；The material into closed reactor
Water content reaches 30%-50%, closes discharge port.The amount that water is added is the 1-2 of volume of material after step (2) processing
Times.
Wherein, material cleaning step can repeat 1-4 times, until the pH of material is not higher than 7.5.It is removed by filtering and washing
Black liquor not only eliminates the addition of acid, reduces the use of chemicals；And black liquor can prepare organic fertilizer also by modification
Process economy is improved in field, reduces discharge of wastewater.In addition, the pretreated material of sulfonation complies fully with after filtering and washing
The requirement of saccharification step, this is the committed step for realizing " one kettle way ".
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:2-1:25, saccharification culture medium, high temperature being added into closed container
High pressure sterilization；Inoculation produces cellulosome bacterial strain seed liquor, using batch type mode, in 34-65 DEG C of temperature condition and
Saccharification reaction is carried out under the pH of 5.8-6.5, obtains the liquid glucose containing glucose.Using the blending manner of intermittent stirring, guaranteeing
Under the premise of mass-transfer efficiency, the energy consumption in saccharification stage and the loss to equipment can be greatly reduced, improve saccharification efficiency.
Wherein, the batch type are as follows: every 1-6 hours stirring 5-10 minutes, the revolving speed of the batch type was
0.5-20rpm.In saccharifying, make pH control in 5.8-6.5 by flowing adding sodium hydroxide.
The production cellulosome bacterial strain is Clostridium thermocellum, the molten fine clostridium of yellow, thermophilic fiber clostridium, solution fiber clostridium, defibering
Tie up vinegar vibrios, molten fiber vacation bacteroid, Ruminococcus albus or yellow Ruminococcus；Inoculum concentration is 2-10% (v/v).
The saccharification culture medium is that dipotassium hydrogen phosphate 2.9g/L, potassium dihydrogen phosphate 1.5g/L, urea are added in every liter of water
0.8g/L, calcium chloride 0.1g/L, magnesium chloride 1.8g/L, ferrous sulfate 0.0005g/L, vulcanized sodium 2g/L, corn pulp 4g/L, lemon
Sour trisodium 2g/L, pH 6.5-7.5.
(5) it ferments: when concentration of glucose reaches 25-180g/L in closed reactor, being passed through filtrated air, addition culture
Base component obtains fermentation medium；Then the aerobic heterotrophic microorganism seed liquor for accessing activation, stirs evenly, at 15-38 DEG C
Temperature condition is mended and is fermented 1.5-7 days under nitrogen, pH6.5-9.0, is obtained aerobic heterotrophic microorganism fermentation liquid, is measured biomass.
Wherein, the oleaginous microorganism is schizochytrium limacinum, micro- quasi- ball algae, Mortierella alpina, chlorella, phaffiafhodozyma
Or Candida, inoculum concentration are 5-10% (v/v).Revolving speed 100-200rpm, the ventilatory capacity 0.2-2vvm of the stirring.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Lignocellulosic one kettle way bioconversion device, including closed reactor 5 and the storage being connected with vacuum plant 7
Tank 8；The top of the closed reactor 5 is equipped with air inlet 2 and feed inlet 3, and the inside of the closed reactor 5 is equipped with heating dress
It sets, temperature regulating device and the agitating paddle 4 being connected with motor 1, the bottom of the closed reactor 5 are equipped with discharge port 6；The discharging
Mouth 6 is connected with the entrance of storage tank 8；Filter device is set on the discharge port 6, and the bottom of the storage tank 8 is provided
Mouthful.Using the device, the one kettle way bioconversion of lignocellulosic may be implemented, overcome and lack in the prior art and one kettle way
The problem of matched device.
Beneficial effects of the present invention:
(1) method of the one kettle way bioconversion of lignocellulosic of the present invention by pretreatment, is saccharified and fermented
Journey series connection, and carried out in the same reactor, greatly simplify processing step and the requirement to equipment.
(2) present invention is exempted from using the saccharification for being realized lignocellulosic based on the cellulase preparation for producing cellulosome bacterium
In addition to the addition for enzyme preparation of dissociating, significantly reduce the saccharification stage uses enzyme cost.
(3) present invention using lignocellulose biomass be raw material combine oil fermentation produce, on the one hand reduce carbon source at
This, on the other hand also solves the problem of complex utilization of agriculture and forestry organic waste material, has huge environmental benefit and social benefit.
Detailed description of the invention
Attached drawing 1 is the structural schematic diagram of lignocellulosic one kettle way bioconversion device of the present invention.
Specific embodiment
The present invention will be further explained with reference to the examples below.
Embodiment 1: lignocellulosic one kettle way biotransformation method
The method of lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: corn stover is mechanically pulverized, until fragment partial size is no more than 2cm；It washes with water, so
It launches afterwards from the feeding port above closed reactor into closed reactor, adds water, mixing according to solid and liquid weight volume ratio 1:2
Uniformly, 4h is impregnated.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 11% (w/v), at 175 DEG C
Temperature under the conditions of carry out sulfonation handle 3.5h.After completion of the reaction, open closed reactor bottom discharge port, make black liquor via
Filter device provisioned in discharge port flows out in storage tank；The storage tank is connected by pipeline with closed reactor.When
It when pressure in closed reactor is down to atmospheric pressure, is vacuumized using the vacuum pump at the top of storage tank, makes to be formed in storage tank negative
Pressure, black liquor further flow out；Until the water content of material reaches 30%-50% in closed reactor, discharge port is closed.
(3) material cleans: the water of 1 times of volume of material being added into closed reactor；High pressure is passed through into closed reactor
Nitrogen makes reactor pressure reach 12 standard atmospheric pressures, then opens the discharge port of closed reactor bottom, passes through black liquor
The filter device as provisioned in discharge port flows out in storage tank.When the pressure in closed reactor is down to atmospheric pressure, utilize
Vacuum pump at the top of storage tank vacuumizes, and makes to form negative pressure in storage tank, black liquor further flows out；The material into closed reactor
Water content reach 30%-50%, close discharge port.Material cleaning step is repeated 2 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:6, saccharification culture medium being added into closed container, high temperature and pressure is gone out
Bacterium；It is inoculated with Clostridium thermocellum seed liquor, inoculum concentration is 5% (v/v)；Using batch type mode, in 60 DEG C of temperature conditions and
Saccharification reaction is carried out under the pH of 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 5 minutes for every 1 hour, the revolving speed of the batch type is 5rpm.Saccharifying
In, make pH control in 5.8-6.5 by flowing adding sodium hydroxide.
The saccharification culture medium is that dipotassium hydrogen phosphate 2.9g/L, potassium dihydrogen phosphate 1.5g/L, urea are added in every liter of water
0.8g/L, calcium chloride 0.1g/L, magnesium chloride 1.8g/L, ferrous sulfate 0.0005g/L, vulcanized sodium 2g/L, corn pulp 4g/L, lemon
Sour trisodium 2g/L, pH 6.5-7.5.
(5) it ferments: when concentration of glucose reaches 70-80g/L in closed reactor, being passed through filtrated air, addition culture
Base component obtains fermentation medium.Then the schizochytrium limacinum seed liquor of activation is accessed, inoculum concentration is 5% (v/v), is stirred evenly；
It ferments 3 days at revolving speed 100rpm, ventilatory capacity 0.2vvm, 25 DEG C of temperature condition, benefit nitrogen, pH6.5, it is micro- to obtain aerobic heterotrophism
Bio-fermented liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 2: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: wheat straw is mechanically pulverized, until fragment partial size is no more than 2cm；Wash with water, then from
Feeding port above closed reactor is launched into closed reactor, adds water according to solid and liquid weight volume ratio 1:3, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 12% (w/v), at 180 DEG C
Temperature under the conditions of carry out sulfonation handle 4h.After completion of the reaction, flow out to black liquor in storage tank, until object in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 1 times of volume of material being added into closed reactor；High pressure is passed through into closed reactor
Nitrogen makes reactor pressure reach 12 standard atmospheric pressures, then opens discharge port, flow out to black liquor in storage tank, directly
Into closed reactor, the water content of material reaches 30%-50%, closes discharge port.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:3.5, saccharification culture medium, high temperature and pressure being added into closed container
Sterilizing；It is inoculated with Clostridium thermocellum seed liquor, inoculum concentration is 6% (v/v)；Using batch type mode, in 60 DEG C of temperature condition
With saccharification reaction is carried out under the pH of 5.8-6.5, obtain the liquid glucose containing glucose.
The batch type are as follows: stir 5 minutes for every 2 hours, the revolving speed of the batch type is 2rpm.Saccharifying
In, make pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 140-150g/L in closed reactor, being passed through filtrated air, addition training
Base component is supported, fermentation medium is obtained.Then micro- quasi- ball algae seed liquor of activation is accessed, inoculum concentration is 8% (v/v), and stirring is equal
It is even；It ferments 6 days, obtains aerobic different at revolving speed 150rpm, ventilatory capacity 1.0vvm, 30 DEG C of temperature condition, benefit nitrogen, pH8.0
Microbial fermentation solution is supported, biomass is measured.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 3: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: brush wood branch is mechanically pulverized, until fragment partial size is no more than 2cm；It washes with water, so
It launches afterwards from the feeding port above closed reactor into closed reactor, adds water, mixing according to solid and liquid weight volume ratio 1:4
Uniformly, 2-4h is impregnated.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 13% (w/v), at 185 DEG C
Temperature under the conditions of carry out sulfonation handle 1h.After completion of the reaction, flow out to black liquor in storage tank, until object in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 1.5 times of volume of material being added into closed reactor；Height is passed through into closed reactor
Nitrogen is pressed, so that reactor pressure is reached 12 standard atmospheric pressures, then opens discharge port, flow out to black liquor in storage tank,
Until the water content of material reaches 30%-50% in closed reactor, discharge port is closed.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:5, saccharification culture medium being added into closed container, high temperature and pressure is gone out
Bacterium；It is inoculated with Clostridium thermocellum seed liquor, inoculum concentration is 8% (v/v)；Using batch type mode, in 60 DEG C of temperature conditions and
Saccharification reaction is carried out under the pH of 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 10 minutes for every 3 hours, the revolving speed of the batch type is 0.5rpm.Saccharification
In the process, make pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 90-110g/L in closed reactor, being passed through filtrated air, addition culture
Base component obtains fermentation medium.Then the Mortierella alpina seed liquor of activation is accessed, inoculum concentration is 10% (v/v), and stirring is equal
It is even；It ferments 6.5 days, obtains aerobic at revolving speed 200rpm, ventilatory capacity 2.0vvm, 28 DEG C of temperature condition, benefit nitrogen, pH7.5
Heterotrophic microorganism fermentation liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 4: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: wood chip is mechanically pulverized, until fragment partial size is no more than 2cm；Wash with water, then from
Feeding port above closed reactor is launched into closed reactor, adds water according to solid and liquid weight volume ratio 1:5, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 14% (w/v), at 190 DEG C
Temperature under the conditions of carry out sulfonation handle 1.5h.After completion of the reaction, flow out to black liquor in storage tank, until in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 1.5 times of volume of material being added into closed reactor；Height is passed through into closed reactor
Nitrogen is pressed, so that reactor pressure is reached 10 standard atmospheric pressures, then opens discharge port, flow out to black liquor in storage tank,
Until the water content of material reaches 30%-50% in closed reactor, discharge port is closed.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:8, saccharification culture medium being added into closed container, high temperature and pressure is gone out
Bacterium；It is inoculated with Clostridium thermocellum seed liquor, inoculum concentration is 10% (v/v)；Using batch type mode, in 60 DEG C of temperature conditions and
Saccharification reaction is carried out under the pH of 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 10 minutes for every 6 hours, the revolving speed of the batch type is 20rpm.It was saccharified
Cheng Zhong makes pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 60-70g/L in closed reactor, being passed through filtrated air, addition culture
Base component obtains fermentation medium.Then the chlorella seed liquor of activation is accessed, inoculum concentration is 6% (v/v), is stirred evenly；?
Revolving speed 150rpm, it ventilatory capacity 0.5vvm, 15 DEG C of temperature condition, mends and ferments 2.5 days under nitrogen, pH7.0, it is micro- to obtain aerobic heterotrophism
Bio-fermented liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 5: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: straw is mechanically pulverized, until fragment partial size is no more than 2cm；Wash with water, then from
Feeding port above closed reactor is launched into closed reactor, adds water according to solid and liquid weight volume ratio 1:6, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 15% (w/v), at 160 DEG C
Temperature under the conditions of carry out sulfonation handle 2h.After completion of the reaction, flow out to black liquor in storage tank, until object in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 2 times of volume of material being added into closed reactor；High pressure is passed through into closed reactor
Nitrogen makes reactor pressure reach 10 standard atmospheric pressures, then opens discharge port, flow out to black liquor in storage tank, directly
Into closed reactor, the water content of material reaches 30%-50%, closes discharge port.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:10, saccharification culture medium, high temperature and pressure being added into closed container
Sterilizing；It is inoculated with Clostridium thermocellum seed liquor, inoculum concentration is 2% (v/v)；Using batch type mode, in 60 DEG C of temperature condition
With saccharification reaction is carried out under the pH of 5.8-6.5, obtain the liquid glucose containing glucose.
The batch type are as follows: stir 5 minutes for every 4 hours, the revolving speed of the batch type is 10rpm.It was saccharified
Cheng Zhong makes pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 50-60g/L in closed reactor, being passed through filtrated air, addition culture
Base component obtains fermentation medium.Then the Candida seed liquor of activation is accessed, inoculum concentration is 8% (v/v), is stirred evenly；
It ferments 5 days at revolving speed 150rpm, ventilatory capacity 1.2vvm, 38 DEG C of temperature condition, benefit nitrogen, pH9.0, it is micro- to obtain aerobic heterotrophism
Bio-fermented liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 6: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: waste paper is mechanically pulverized, until fragment partial size is no more than 2cm；Wash with water, then from
Feeding port above closed reactor is launched into closed reactor, adds water according to solid and liquid weight volume ratio 1:2, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 9% (w/v), at 165 DEG C
Sulfonation is carried out under the conditions of temperature handles 2.5h.After completion of the reaction, flow out to black liquor in storage tank, until object in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 2 times of volume of material being added into closed reactor；High pressure is passed through into closed reactor
Nitrogen makes reactor pressure reach 11 standard atmospheric pressures, then opens discharge port, flow out to black liquor in storage tank, directly
Into closed reactor, the water content of material reaches 30%-50%, closes discharge port.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:2, saccharification culture medium being added into closed container, high temperature and pressure is gone out
Bacterium；It is inoculated with the molten fine clostridium seed liquor of yellow, inoculum concentration is 6% (v/v)；Using batch type mode, in 65 DEG C of temperature strip
Saccharification reaction is carried out under the pH of part and 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 10 minutes for every 5 hours, the revolving speed of the batch type is 15rpm.It was saccharified
Cheng Zhong makes pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 160-180g/L in closed reactor, being passed through filtrated air, addition training
Base component is supported, fermentation medium is obtained.Then the phaffiafhodozyma seed liquor of activation is accessed, inoculum concentration is 10% (v/v), stirring
Uniformly；It ferments 7 days, obtains aerobic at revolving speed 150rpm, ventilatory capacity 2.0vvm, 18 DEG C of temperature condition, benefit nitrogen, pH7.5
Heterotrophic microorganism fermentation liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 7: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: wheat straw is mechanically pulverized, until fragment partial size is no more than 2cm；Wash with water, then from
Feeding port above closed reactor is launched into closed reactor, adds water according to solid and liquid weight volume ratio 1:3, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 10% (w/v), at 170 DEG C
Temperature under the conditions of carry out sulfonation handle 3h.After completion of the reaction, flow out to black liquor in storage tank, until object in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 1.2 times of volume of material being added into closed reactor；Height is passed through into closed reactor
Nitrogen is pressed, so that reactor pressure is reached 11 standard atmospheric pressures, then opens discharge port, flow out to black liquor in storage tank,
Until the water content of material reaches 30%-50% in closed reactor, discharge port is closed.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:15, saccharification culture medium, high temperature and pressure being added into closed container
Sterilizing；It is inoculated with thermophilic fiber clostridium seed liquor, inoculum concentration is 6% (v/v)；Using batch type mode, in 37 DEG C of temperature strip
Saccharification reaction is carried out under the pH of part and 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 8 minutes for every 3 hours, the revolving speed of the batch type is 12rpm.It was saccharified
Cheng Zhong makes pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 30-50g/L in closed reactor, being passed through filtrated air, addition culture
Base component obtains fermentation medium.Then the schizochytrium limacinum seed liquor of activation is accessed, inoculum concentration is 5% (v/v), is stirred evenly；
It ferments 3 days at revolving speed 100rpm, ventilatory capacity 0.8vvm, 28 DEG C of temperature condition, benefit nitrogen, pH7.0, it is micro- to obtain aerobic heterotrophism
Bio-fermented liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 8: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: wheat straw is mechanically pulverized, until fragment partial size is no more than 2cm；Wash with water, then from
Feeding port above closed reactor is launched into closed reactor, adds water according to solid and liquid weight volume ratio 1:4, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 11% (w/v), at 175 DEG C
Temperature under the conditions of carry out sulfonation handle 3.5h.After completion of the reaction, flow out to black liquor in storage tank, until in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 1.2 times of volume of material being added into closed reactor；Height is passed through into closed reactor
Nitrogen is pressed, so that reactor pressure is reached 11 standard atmospheric pressures, then opens discharge port, flow out to black liquor in storage tank,
Until the water content of material reaches 30%-50% in closed reactor, discharge port is closed.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:10, saccharification culture medium, high temperature and pressure being added into closed container
Sterilizing；Inoculation solution fiber clostridium seed liquor, inoculum concentration are 6% (v/v)；Using batch type mode, in 34 DEG C of temperature strip
Saccharification reaction is carried out under the pH of part and 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 10 minutes for every 3 hours, the revolving speed of the batch type is 8rpm.It was saccharified
Cheng Zhong makes pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 40-60g/L in closed reactor, being passed through filtrated air, addition culture
Base component obtains fermentation medium.Then the schizochytrium limacinum seed liquor of activation is accessed, inoculum concentration is 8% (v/v), is stirred evenly；
It ferments 5.5 days at revolving speed 100rpm, ventilatory capacity 1.5vvm, 35 DEG C of temperature condition, benefit nitrogen, pH7.0, obtains aerobic heterotrophism
Microbial fermentation solution measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 9: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: wheat straw is mechanically pulverized, until fragment partial size is no more than 2cm；Wash with water, then from
Feeding port above closed reactor is launched into closed reactor, adds water according to solid and liquid weight volume ratio 1:5, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 12% (w/v), at 180 DEG C
Temperature under the conditions of carry out sulfonation handle 4h.After completion of the reaction, flow out to black liquor in storage tank, until object in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 1.8 times of volume of material being added into closed reactor；Height is passed through into closed reactor
Nitrogen is pressed, so that reactor pressure is reached 10.5 standard atmospheric pressures, then opens discharge port, black liquor is made to flow out to storage tank
In, until the water content of material reaches 30%-50% in closed reactor, close discharge port.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:4.5, saccharification culture medium, high temperature and pressure being added into closed container
Sterilizing；It is inoculated with Acetivibrio cellulolyticus seed liquor, inoculum concentration is 8% (v/v)；Using batch type mode, in 37 DEG C of temperature
Saccharification reaction is carried out under the pH of condition and 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 8 minutes for every 4 hours, the revolving speed of the batch type is 6rpm.Saccharifying
In, make pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 110-130g/L in closed reactor, being passed through filtrated air, addition training
Base component is supported, fermentation medium is obtained.Then the schizochytrium limacinum seed liquor of activation is accessed, inoculum concentration is 8% (v/v), and stirring is equal
It is even；It ferments 3.5 days, obtains aerobic at revolving speed 100rpm, ventilatory capacity 1.8vvm, 25 DEG C of temperature condition, benefit nitrogen, pH7.0
Heterotrophic microorganism fermentation liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 10: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: corncob is mechanically pulverized, until fragment partial size is no more than 2cm；It washes with water, then
It launches from the feeding port above closed reactor into closed reactor, adds water according to solid and liquid weight volume ratio 1:6, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 13% (w/v), at 185 DEG C
Temperature under the conditions of carry out sulfonation handle 1h.After completion of the reaction, flow out to black liquor in storage tank, until object in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 1.8 times of volume of material being added into closed reactor；Height is passed through into closed reactor
Nitrogen is pressed, so that reactor pressure is reached 10.5 standard atmospheric pressures, then opens discharge port, black liquor is made to flow out to storage tank
In, until the water content of material reaches 30%-50% in closed reactor, close discharge port.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:5, saccharification culture medium being added into closed container, high temperature and pressure is gone out
Bacterium；It is inoculated with molten fiber vacation bacteroid seed liquor, inoculum concentration is 8% (v/v)；Using batch type mode, in 42 DEG C of temperature
Saccharification reaction is carried out under the pH of condition and 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 10 minutes for every 6 hours, the revolving speed of the batch type is 15rpm.It was saccharified
Cheng Zhong makes pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 60-80g/L in closed reactor, being passed through filtrated air, addition culture
Base component obtains fermentation medium.Then the Mortierella alpina seed liquor of activation is accessed, inoculum concentration is 10% (v/v), and stirring is equal
It is even；It ferments 3.5 days, obtains aerobic at revolving speed 200rpm, ventilatory capacity 1.5vvm, 26 DEG C of temperature condition, benefit nitrogen, pH7.0
Heterotrophic microorganism fermentation liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 11: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: waste paper is mechanically pulverized, until fragment partial size is no more than 2cm；Wash with water, then from
Feeding port above closed reactor is launched into closed reactor, adds water according to solid and liquid weight volume ratio 1:3, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 14% (w/v), at 190 DEG C
Temperature under the conditions of carry out sulfonation handle 1.5h.After completion of the reaction, flow out to black liquor in storage tank, until in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 2 times of volume of material being added into closed reactor；High pressure is passed through into closed reactor
Nitrogen makes reactor pressure reach 11.5 standard atmospheric pressures, then opens discharge port, flow out to black liquor in storage tank,
Until the water content of material reaches 30%-50% in closed reactor, discharge port is closed.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:25, saccharification culture medium, high temperature and pressure being added into closed container
Sterilizing；It is inoculated with Ruminococcus albus seed liquor, inoculum concentration is 8% (v/v)；Using batch type mode, in 37 DEG C of temperature
Saccharification reaction is carried out under the pH of condition and 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 6 minutes for every 1 hour, the revolving speed of the batch type is 5rpm.Saccharifying
In, make pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 25-35g/L in closed reactor, being passed through filtrated air, addition culture
Base component obtains fermentation medium.Then the Mortierella alpina seed liquor of activation is accessed, inoculum concentration is 6% (v/v), and stirring is equal
It is even；It ferments 1.5 days, obtains aerobic at revolving speed 200rpm, ventilatory capacity 1.5vvm, 38 DEG C of temperature condition, benefit nitrogen, pH6.5
Heterotrophic microorganism fermentation liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 12: lignocellulosic one kettle way biotransformation method
Unlike the first embodiment, the method for lignocellulosic one kettle way bioconversion, comprising the following steps:
(1) mechanical treatment: corncob is mechanically pulverized, until fragment partial size is no more than 2cm；It washes with water, then
It launches from the feeding port above closed reactor into closed reactor, adds water according to solid and liquid weight volume ratio 1:4, mixing is equal
It is even, impregnate 2-4h.
(2) it is chemically treated: ammonium sulfite being added into closed reactor, until its final concentration of 15% (w/v), at 160 DEG C
Temperature under the conditions of carry out sulfonation handle 2h.After completion of the reaction, flow out to black liquor in storage tank, until object in closed reactor
The water content of material reaches 30%-50%, closes discharge port.
(3) material cleans: the water of 2 times of volume of material being added into closed reactor；High pressure is passed through into closed reactor
Nitrogen makes reactor pressure reach 11.5 standard atmospheric pressures, then opens discharge port, flow out to black liquor in storage tank,
Until the water content of material reaches 30%-50% in closed reactor, discharge port is closed.
Above-mentioned material cleaning step is repeated 4 times, until the pH of material is not higher than 7.5.
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:3, saccharification culture medium being added into closed container, high temperature and pressure is gone out
Bacterium；It is inoculated with yellow Ruminococcus seed liquor, inoculum concentration is 8% (v/v)；Using batch type mode, in 37 DEG C of temperature strip
Saccharification reaction is carried out under the pH of part and 5.8-6.5, obtains the liquid glucose containing glucose.
The batch type are as follows: stir 6 minutes for every 1 hour, the revolving speed of the batch type is 2rpm.Saccharifying
In, make pH control in 5.8-6.5 by flowing adding sodium hydroxide.
(5) it ferments: when concentration of glucose reaches 100-120g/L in closed reactor, being passed through filtrated air, addition training
Base component is supported, fermentation medium is obtained.Then the Mortierella alpina seed liquor of activation is accessed, inoculum concentration is 8% (v/v), stirring
Uniformly；It ferments 7 days, obtains aerobic at revolving speed 200rpm, ventilatory capacity 2.0vvm, 15 DEG C of temperature condition, benefit nitrogen, pH7.5
Heterotrophic microorganism fermentation liquid measures biomass.
The nutrient media components are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L,
Potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6
3-200 mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
Embodiment 13: lignocellulosic one kettle way bioconversion device
Lignocellulosic one kettle way bioconversion device, including closed reactor 5 and the storage being connected with vacuum plant 7
Tank 8；The top of the closed reactor 5 is equipped with air inlet 2 and feed inlet 3, and the inside of the closed reactor 5 is equipped with heating dress
It sets, temperature regulating device and the agitating paddle 4 being connected with motor 1, the bottom of the closed reactor 5 are equipped with discharge port 6；The discharging
Mouth 6 is connected with the entrance of storage tank 8；Filter device is set on the discharge port 6, and outlet is arranged in the bottom of the storage tank 8.
Using the device, the one kettle way bioconversion of lignocellulosic may be implemented, overcome and lack in the prior art and one kettle way
The problem for the device matched.
The result of 1 embodiment 1-12 lignocellulosic one kettle way bioconversion of table
As shown in Table 1, using lignocellulosic one kettle way biotransformation method of the present invention, cellulose sugar rate is
80.9-90.8%, using the liquid glucose culture schizochytrium limacinum, micro- quasi- ball algae, Mortierella alpina, chlorella, phaffiafhodozyma and
Candida, biomass obtained can achieve or the level of the technology higher than the progress of existing substep.The conversion ratio of glucose,
The conversion ratio of glucose to biomass i.e. in liquid glucose is 31.2-51.2%, reaches the higher level of existing fermentation technique.
Lignocellulosic one kettle way biotransformation method of the present invention, pretreatment, saccharification and fermentation process are connected on
It carries out in the same reactor, under the premise of biomass and inversion rate of glucose reach the higher level of the prior art, simplifies
Processing step and the requirement to equipment, reduce production cost, have great importance for industrialization.Moreover, of the invention
The addition for eliminating free enzyme preparation, significantly reduce the saccharification stage uses enzyme cost.
Claims (10)
1. lignocellulosic one kettle way biotransformation method, it is characterised in that: the following steps are included:
(1) mechanical treatment: lignocellulosic material is mechanically pulverized, and cleaning is thrown in closed reactor, according to solid-liquid
W/v 1:2-1:6 adds water, is uniformly mixed, and impregnates；
(2) it is chemically treated: appropriate ammonium sulfite being added into closed reactor, carries out sulphur under the conditions of 160-190 DEG C of temperature
Change processing 1-4h；After completion of the reaction, the discharge port for opening closed reactor bottom, flow to black liquor in storage tank；When closed anti-
It when the pressure in device being answered to be down to atmospheric pressure, is vacuumized using the vacuum pump at the top of storage tank, makes to form negative pressure in storage tank, black liquor
It further flows out；Until the water content of material reaches 30%-50% in closed reactor, discharge port is closed；
(3) material cleans: water being added into closed reactor, is uniformly mixed；It is passed through high pressure nitrogen into closed reactor, makes anti-
It answers pressure in device to reach 10-12 standard atmospheric pressure, then opens the discharge port of closed reactor bottom, black liquor is made to flow to storage
In tank；When the pressure in closed reactor is down to atmospheric pressure, is vacuumized, made in storage tank using the vacuum pump at the top of storage tank
Negative pressure is formed, black liquor further flows out；Into closed reactor, the water content of material reaches 30%-50%, closes discharge port；
(4) it is saccharified: according to the solid and liquid weight volume ratio of 1:2-1:25, saccharification culture medium being added into closed reactor, high temperature is high
Pressure sterilizing；Inoculation produces cellulosome bacterial strain seed liquor, using batch type mode, in 34-65 DEG C of temperature condition and 5.8-
Saccharification reaction is carried out under 6.5 pH, obtains the liquid glucose containing glucose；
(5) it ferments: when concentration of glucose reaches 25-180g/L in closed reactor, being passed through filtrated air, add culture medium group
Point, obtain fermentation medium；Then the aerobic heterotrophic microorganism seed liquor for accessing activation, stirs evenly, in 15-38 DEG C of temperature
Condition is mended and is fermented 1.5-7 days under nitrogen, pH6.5-9.0, is obtained aerobic heterotrophic microorganism fermentation liquid, is measured biomass.
2. lignocellulosic one kettle way biotransformation method according to claim 1, it is characterised in that: described in step (3)
The amount that water is added is 1-2 times of volume of material after step (2) processing；The step (3) repeats 1-4 times, until the pH of material
Not higher than 7.5.
3. lignocellulosic one kettle way biotransformation method according to claim 2, it is characterised in that: step (1) described machine
The fragment partial size of lignocellulosic material is no more than 2cm, soaking time 2-4h after tool crushes；The lignocellulosic material is
One of corn stover, wheat straw, brush wood branch, wood chip, corncob, straw and waste paper or a variety of combinations.
4. lignocellulosic one kettle way biotransformation method according to claim 2, it is characterised in that: step (2) is described close
Close the final concentration of 9-15% (w/v) of reactor sulfite ammonium.
5. lignocellulosic one kettle way biotransformation method according to claim 2, it is characterised in that: described in step (4)
Batch type are as follows: every 1-6 hours stirring 5-10 minutes, the revolving speed of the batch type was 0.5-20rpm.
6. lignocellulosic one kettle way biotransformation method according to claim 2, it is characterised in that: described in step (4)
Production cellulosome bacterial strain is Clostridium thermocellum, the molten fine clostridium of yellow, thermophilic fiber clostridium, solution fiber clostridium, Acetivibrio cellulolyticus, molten fibre
Tie up false bacteroid, Ruminococcus albus or yellow Ruminococcus；Inoculum concentration is 2-10% (v/v).
7. lignocellulosic one kettle way biotransformation method described in any one of -6 according to claim 1, it is characterised in that: institute
The saccharification culture medium stated specifically: dipotassium hydrogen phosphate 2.9g/L, potassium dihydrogen phosphate 1.5g/L, urea 0.8g/ are added in every liter of water
L, calcium chloride 0.1g/L, magnesium chloride 1.8g/L, ferrous sulfate 0.0005g/L, vulcanized sodium 2g/L, corn pulp 4g/L, citric acid three
Sodium 2g/L, pH 6.5-7.5.
8. lignocellulosic one kettle way biotransformation method described in any one of -6 according to claim 1, it is characterised in that: step
Suddenly aerobic heterotrophic microorganism described in (5) is schizochytrium limacinum, micro- quasi- ball algae, Mortierella alpina, chlorella, phaffiafhodozyma or vacation
Silk yeast, inoculum concentration are 5-10% (v/v)；Revolving speed 100-200rpm, the ventilatory capacity 0.2-2vvm of the stirring.
9. lignocellulosic one kettle way biotransformation method described in any one of -6 according to claim 1, it is characterised in that: step
Suddenly nutrient media components described in (5) are as follows: yeast extract 0.2-30g/L, peptone 0.2-20g/L, corn pulp 6-30g/L, phosphorus
Acid dihydride potassium 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 0-30g/L, vitamin B1 3-200mg/L, vitamin B6 3-
200mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
10. lignocellulosic one kettle way bioconversion device, it is characterised in that: including closed reactor 5 and with 7 phase of vacuum plant
The storage tank 8 of connection；The top of the closed reactor 5 is equipped with air inlet 2 and feed inlet 3, the inside of the closed reactor 5
Equipped with heating device, temperature regulating device and the agitating paddle 4 being connected with motor 1, the bottom of the closed reactor 5 is equipped with discharge port
6；The discharge port 6 is connected with the entrance of storage tank 8；Filter device, the bottom of the storage tank 8 are set on the discharge port 6
Portion setting outlet.
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| CN114807269A (en) * | 2022-06-08 | 2022-07-29 | 中国科学院青岛生物能源与过程研究所 | Lignocellulose whole-cell saccharification technology adopting oxygen treatment method |
| CN115029399A (en) * | 2022-06-08 | 2022-09-09 | 中国科学院青岛生物能源与过程研究所 | Raw material treatment method for lignocellulose whole-cell saccharification |
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