CN109055459A - Full bacterium method for saccharifying for lignocellulosic - Google Patents
Full bacterium method for saccharifying for lignocellulosic Download PDFInfo
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Abstract
The present invention provides the full bacterium method for saccharifying for lignocellulosic, comprising the following steps: (1) seed liquor optimizes: under anaerobic, will produce cellulosome bacterial strain using glucose as the culture medium of sole carbon source, carries out passage and attenuation；It is then seeded into the dextrose culture-medium for being added to pretreated lignocellulosic material and carries out inducing and acclimating.(2) raw material pre-preg: pretreated lignocellulosic material and culture medium solution are uniformly mixed.(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the reaction system after the pre-preg that step (2) obtains, saccharification reaction is carried out, obtains the liquid glucose containing glucose.This method not only substantially reduces the period of seed growth phase, reduces the cost of seed liquor, the period of full bacterium saccharification is also shortened, to lay a good foundation for industrialized process.
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 on a large scale
The key of petroleum-based products.
Lignocellulose-like biomass is present on earth renewable biomass resources the most abundant, is translated into energy
Source, chemicals or material have huge application and development prospect.Particularly, China is large agricultural country, annual crops straw
Stalk 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 promotes state
Family releases planning, scheme and the measure of a series of stalk comprehensive utilizations.So agriculture waste biomass comprehensive is pushed to utilize
The Significance of Sustainable Development of industrialized development, foundation and society to China's green circulation economy is great.
However, the used enzyme preparation technology of biomass saccharification is at present by company monopolizing of developed country, therefrom caused by enzyme
High cost become restrict lignocellulosic industrial applications critical issue.In addition, technical difficulty also counteract it is wooden
The development and application of cellulose conversion technology.Therefore, developing low-cost, efficient saccharification technology, to developing country
It is particularly necessary.
Compared with dependent on the Mashing process of enzyme preparation, the biology of " one kettle way " full bacterium catalysis of biological processing technology is integrated
Saccharification strategy has simple flow, reduces the advantages such as equipment requirement, is most suitable for cellulose series biomass bioconversion and utilizes
Process route.The core of " one kettle way " technology is using efficient full bacterium catalyst, i.e., a kind of to have lignocellulosic substrate
Cellulosic substrate hydrolysis can be efficiently the microorganism of fermentable sugars by degradation capability.Currently, the full bacterium of lignocellulosic
Catalysis saccharification is main using the high temperature anaerobic bacterium based on Clostridium thermocellum this production cellulosome as full bacterium catalyst.Fiber is small
Body is a kind of extracellular multienzyme complex with labyrinth and component, is known most efficient cellulose drop in nature
One of enzymatic hydrolysis system.The full bacterium catalysis saccharification of lignocellulosic includes full bacterium catalyst preculture (seed growth phase) and full bacterium sugar
The two step semi-successive cultivation modes changed.In seed growth phase, usually there are two types of methods at present: (1) with commercially available cellobiose or
Person's cellulose is cultivated as carbon source, and seed growth is very fast, but expensive；(2) using preprocessing biomass raw material as carbon source
Seed culture is carried out, although this method can reduce cost, the seed culture period is longer.
Glucose is the common microculture carbon source of fermentation industry, and price is lower, and is the product of saccharification of cellulose.
However, currently, glucose can't be applied to the preculture of full bacterium catalyst.This is because, when using glucose as sole carbon source
When cultivating Clostridium thermocellum, tens hours growth lag phases are not only had, and the yield that will lead to cellulosome is aobvious
Writing reduces, long so as to cause the production cycle, in addition, the cellulose degradation vigor of full bacterium catalyst obtained is lower.Yoav etc.
People by Clostridium thermocellum bacterial strain using glucose as the culture medium of sole carbon source in tamed (Yoav, S., Barak, Y.,
Shamshoum, M., et al., Biotechnology for biofuels 2017,10:222), solve glucose conduct
Caused by single carbon source the problem of growth lag phase.However, this report is intended merely to obtain thallus faster and fiber is small
Body, there is no solving, cellulosome yield is significantly reduced and the cellulose degradation vigor of full bacterium catalyst obtained is lower
The problem of.In addition, being saccharified the stage in full bacterium, seed liquor is inoculated into high solids content preprocessing lignocellulose substrate
After extensive saccharification system, generally requires 2-5 days phase buffers and just start enzymatic saccharification process, it is long to also result in the production cycle,
To increase artificial, the energy consumption cost of production process.
Therefore, the technical problem that in the full bacterium catalysis saccharification of lignocellulosic prepared by the seed liquor of seed growth phase, and
Period length, problem at high cost present in full bacterium saccharification step are the lignocellulosic biomass saccharification skills based on the catalysis of full bacterium
Art is difficult to large-scale application in one of industrial main bottleneck.
Summary of the invention
It is asked for the period is long, seed liquor preparation cost is high in the presence of the full bacterium saccharification of lignocellulosic in the prior art
Topic, the present invention provides the full bacterium method for saccharifying for lignocellulosic, this method not only substantially reduces seed growth phase
Period, reduce the cost of seed liquor, also shorten the period of full bacterium saccharification, to establish base for industrialized process
Plinth.
Technical solution of the present invention:
Full bacterium method for saccharifying for lignocellulosic, comprising the following steps:
(1) seed liquor optimizes: under anaerobic, will produce cellulosome bacterial strain using glucose as the culture of sole carbon source
Base that is, in dextrose culture-medium, carries out passage and attenuation；It is then seeded into and is added to pretreated lignocellulosic material
Inducing and acclimating is carried out in dextrose culture-medium.To obtain Seedling height speed, high microsteping corpusculum yield, high microsteping corpusculum than living
The full bacterium saccharification seed liquor of power.
The concentration that the dextrose culture-medium contains glucose is 1-20g/L；Described passage and attenuation progress 2-5 times, often
Secondary passage is to be seeded in fresh culture to train according to 0.2-2% (volume fraction) after producing cellulosome strain culturing
It supports.The additive amount of the lignocellulosic material is 0.1-2.0g/L；The temperature condition of the inducing and acclimating is 34-65 DEG C；Institute
The time for stating inducing and acclimating is 12-48 hours.Pretreated lignocellulosic material is added into dextrose culture-medium, i.e.,
Substrate is pre-processed, is in order to which the cellulosome of Induced matching synthesizes.
The Rate activity and cellulose of the cellulosome of present invention seed liquor obtained are the cellulosome one of carbon source production
It causes, the fiber higher than the cellulosome produced using cellobiose as carbon source, cellulosome yield and cellulose for carbon source production
Corpusculum is consistent, and seed growth speed is consistent as the seed of carbon source for growth with using cellobiose, and being higher than cellulose is carbon source for growth
Seed, much higher than preprocessing lignocellulose be carbon source for growth seed.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:3-1:50, by pretreated lignocellulosic material
It is uniformly mixed under the speed conditions of 0.5-100r/min in a reservoir with culture medium solution and continues 1-48h；The pretreatment
Lignocellulosic material afterwards is identical with step (1), it is therefore an objective to it is abundant to realize that substrate and culture medium carry out before saccharification starts
Mixing, be conducive to growth and the vigor of full bacterium catalyst, thus realize faster saccharification.The container is with stirring
The anaerobic fermentation equipment or anaerobism of paddle rotate vortex mixer；The anaerobic fermentation equipment with agitating paddle is realized by stirring
It is uniformly mixed, and the whole rolling flip realization that anaerobism rotation vortex mixer then passes through container is uniformly mixed.
(3) full bacterium saccharification: after the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the pre-preg that step (2) obtains
Reaction system in, and carry out saccharification reaction under the speed conditions of 0.5-100r/min, obtain the liquid glucose containing glucose.
The volume fraction that the inoculum concentration of the full bacterium saccharification seed liquor is 0.1-10%.Stream plus hydroxide can be passed through in saccharifying
The mode of sodium makes pH control in 5.8-6.2.
Wherein, the production cellulosome bacterial strain is Clostridium thermocellum, the molten fine clostridium of yellow, thermophilic fiber clostridium, solution fiber shuttle
Bacterium, Acetivibrio cellulolyticus, molten fiber vacation bacteroid, Ruminococcus albus or yellow Ruminococcus.The pretreatment is alkali
One of method, acid system, hydro-thermal method, steam explosion method and sulfonation method preconditioning technique or a variety of combinations.The lignocellulosic is former
Material is one of corn stover, wheat straw, brush wood branch, wood chip, corncob, straw and waste paper or a variety of combinations.
Wherein, dextrose culture-medium described in step (1) is that dipotassium hydrogen phosphate 2.9g/L, biphosphate are added in every liter of water
Potassium 1.5g/L, urea 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, trisodium citrate 2g/L, glucose 1-20g/L, pH 6.5-7.5.
Beneficial effects of the present invention:
(1) the full bacterium method for saccharifying of the present invention for lignocellulosic is low with relatively inexpensive glucose addition
The preprocessing lignocellulose biomass of concentration is carbon source, cellulosome bacterial strain is produced for cultivating Clostridium thermocellum etc., as full bacterium
The seed liquor of saccharification；Compared with prior art, the seed liquor of the full bacterium saccharification realizes the effect of cost reduction and cycle time
Fruit overcomes the problem that you can't have both at the same time in the prior art.
(2) the full bacterium method for saccharifying of lignocellulosic of the present invention, it is significant to shorten using the technology of raw material pre-preg
The adaptation phase buffer of full bacterium saccharification improves the efficiency of full bacterium saccharification, to reduce period and the cost of entire saccharifying.
Specific embodiment
The present invention will be further explained with reference to the examples below.
Embodiment 1:
Full bacterium method for saccharifying for lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by Clostridium thermocellum using the glucose of 5g/L as the culture medium of sole carbon source, i.e., glucose is trained
It supports in base, according to the inoculative proportion of 1% volume fraction, carries out 3 secondary cultures；It is then seeded into addition 0.1g/L dry weight
In the dextrose culture-medium for pre-processing corn stover, inducing and acclimating 48h under the conditions of 55 DEG C of temperature, to obtain Seedling height
Speed, high microsteping corpusculum yield, the full bacterium saccharification seed liquor of high microsteping corpusculum Rate activity.The dextrose culture-medium, which removes, to be contained
Have outside 5g/L glucose, also there is dipotassium hydrogen phosphate 2.9g/L, potassium dihydrogen phosphate 1.5g/L, urea 0.8g/L, chlorine in every liter of water
Change calcium 0.1g/L, magnesium chloride 1.8g/L, ferrous sulfate 0.0005g/L, vulcanized sodium 2g/L, corn pulp 4g/L, trisodium citrate
2g/L,pH 6.5-7.5.Pretreated lignocellulosic material is added into dextrose culture-medium, i.e. pretreatment substrate, be
In order to which the cellulosome of Induced matching synthesizes.The preprocess method is to adopt in document (Chinese papermaking, 2015,34,1-6)
Sulfonation method pretreatment.
After step (1), cellulosome Rate activity, yield and the cellular biomass of seed liquor are detected.Wherein: (1)
The detection of cellulosome yield are as follows: the cellulosome in seed liquor is extracted using cellulose absorption method, with the progress of Bradford method
Quantification of protein, to obtain cellulosome yield (the milligram number of cellulosome in per unit volume seed liquor).(2) fiber
The detection of corpusculum Rate activity are as follows: to pre-process corn stover as substrate, the hydrolysis vigor of the cellulosome of Detection and Extraction.Hydrolysis
Experiment continues 24 hours under the conditions of 55 DEG C, then with the concentration of the reduced sugar of DNS method detection hydrolysis release.According to reduced sugar
With cellulosome Production rate cellulosome Rate activity (U/mg), wherein enzyme-activity unit (U) is defined as under determination condition, often
Hour hydrocellulose substrate generates protein content (mg) required for 1mg reduced sugar.(3) detection of cellular biomass are as follows: will
Seed liquor centrifugation after precipitating be hydrolyzed with sodium hydroxide, then using Bradford method detection precipitating in cell protein it is dense
It spends (the milligram number of protein precipitation in per unit volume seed liquor), is grown according to growth curve and protein precipitation concentration analysis
The time required to mid-log phase (hour), the speed of growth is judged.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:6.5, by the pretreated lignocellulosic material of 1kg
It is uniformly mixed under the speed conditions of 50r/min with the culture medium solution of 6.5L, and continues 4h；It is described pretreated wooden
Cellulosic material is identical with step (1), it is therefore an objective to realize that substrate is adequately mixed with culture medium before saccharification starts,
Be conducive to growth and the vigor of full bacterium catalyst, to realize faster saccharification.It is described to be uniformly mixed as by being stirred in container
The stirring for mixing device, which is realized, to be uniformly mixed.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated with according to the inoculum concentration of 10% volume fraction
In reaction system after the pre-preg obtained to step (2), and continue to carry out saccharification reaction under the speed conditions of 50r/min,
Obtain the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate bottom
Conversion ratio of the cellulose to soluble sugar in object.
Wherein, lag phase of the conversion ratio lower than the hydrolysis that the timing definition of 10% (mass ratio) is lignocellulosic substrate
(day).The timing definition that conversion ratio reaches 80% (mass ratio) is the hydrolysis period (day) of lignocellulosic substrate.
Embodiment 2:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by Clostridium thermocellum, pre-processing corn stover using 5g/L, as the culture medium of carbon source, (glucose is trained
The ingredient of feeding base in addition to glucose) in cultivate 48h.After step (1), seed liquor is detected using the method for embodiment 1
Cellulosome Rate activity, yield and cellular biomass.
(2) full bacterium saccharification: according to the inoculum concentration of 10% volume fraction, seed liquor is directly inoculated into and is pre-processed containing 1kg
Full bacterium saccharification, pretreated feedstock and the saccharification of full bacterium are carried out in the reaction system of the culture medium solution of corn stover and 6.5L afterwards
Condition it is same as Example 1.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate
Conversion ratio of the middle cellulose to soluble sugar.
Embodiment 3:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by Clostridium thermocellum using the glucose of 10g/L as the culture medium of sole carbon source, i.e., glucose is trained
It supports in base, according to the inoculative proportion of 1% volume fraction, carries out 3 secondary cultures；It is then seeded into addition 0.5g/L dry weight
In the dextrose culture-medium for pre-processing wheat stalk, inducing and acclimating 36h under the conditions of 58 DEG C of temperature, to obtain Seedling height
Speed, high microsteping corpusculum yield, the full bacterium saccharification seed liquor of high microsteping corpusculum Rate activity.The preprocess method is
The hydro-thermal and sulfonation method combined pretreatment method used in CN201610133959.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:5, by the pretreated lignocellulosic material of 1kg and
The culture medium solution of 5L is uniformly mixed under the speed conditions of 25r/min, and continues 36h；The pretreated wood fibre
Plain raw material is identical with step (1).
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the inoculum concentration of 10% volume fraction
In reaction system after the pre-preg that step (2) obtains, and continue to carry out saccharification reaction under the speed conditions of 25r/min, obtain
To the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate
Conversion ratio of the middle cellulose to soluble sugar.
Embodiment 4:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by Clostridium thermocellum using the glucose of 20g/L as the culture medium of sole carbon source, i.e., glucose is trained
It supports in base, according to the inoculative proportion of 1% volume fraction, carries out 2 secondary cultures；It is then seeded into the pre- of addition 2g/L dry weight
Handle shrub branch dextrose culture-medium in, under the conditions of 60 DEG C of temperature inducing and acclimating for 24 hours, thus obtain Seedling height speed
The full bacterium saccharification seed liquor of degree, high microsteping corpusculum yield, high microsteping corpusculum Rate activity.The preprocess method is document
(Bin Li,et al.Recent progress on the pretreatment and fractionation of
lignocelluloses for Biorefinery at QIBEBT.Journal of Bioresources and
Bioproducts, 2017,2 (1), 4-9) in alkaline process preconditioning technique.After step (1), using the side of embodiment 1
Cellulosome Rate activity, yield and the cellular biomass of method detection seed liquor.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:8, by the pretreated lignocellulosic material of 1kg and
The culture medium solution of 8L is uniformly mixed under the speed conditions of 45r/min, and continues 28h；The pretreated wood fibre
Plain raw material is identical with step (1).(3) full bacterium saccharification: by the full bacterium saccharification seed liquor of step (1) optimization with 6% volume point
Several inoculum concentrations is inoculated into the reaction system after the pre-preg that step (2) obtains, and is continued under the speed conditions of 45r/min
Saccharification reaction is carried out, the liquid glucose containing glucose is obtained.In the process, reduced sugar in every 12 hours sample detection fermentation liquids
Content.
Embodiment 5:
As different from Example 4, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by Clostridium thermocellum using 20g/L microcrystalline cellulose as culture medium (the glucose culture of carbon source
The ingredient of base in addition to glucose) in culture for 24 hours.After step (1), seed liquor is detected using the method for embodiment 1
Cellulosome Rate activity, yield and cellular biomass.
(2) full bacterium saccharification: according to the inoculum concentration of 8% volume fraction, seed liquor is directly inoculated into containing after 1kg pretreatment
Shrub branch and 8L culture medium solution reaction system in carry out full bacterium saccharification, the condition of pretreated feedstock and the saccharification of full bacterium
It is same as Example 4.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate in fiber
Element arrives the conversion ratio of soluble sugar.
Embodiment 6:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by Clostridium thermocellum using the glucose of 5g/L as the culture medium of sole carbon source, i.e. glucose culture
In base, according to the inoculative proportion of 0.1% volume fraction, 5 secondary cultures are carried out；It is then seeded into addition 0.5g/L dry weight
In the dextrose culture-medium of pre-treatment of chips, under the conditions of 60 DEG C of temperature inducing and acclimating for 24 hours, thus obtain Seedling height speed,
High microsteping corpusculum yield, the full bacterium saccharification seed liquor of high microsteping corpusculum Rate activity.The preprocess method is document
The preconditioning technique that alkaline process in (Biotechnology for Biofuels, 2014,7:116) is combined with hydro-thermal.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:8, by the pretreated lignocellulosic material of 100kg
It is uniformly mixed, and continues for 24 hours under the speed conditions of 1.5r/min with the culture medium solution of 800L；The pretreated wood
Matter cellulosic material is identical with step (1).It is described to be uniformly mixed to realize that mixing is equal by the whole rolling flip of container
It is even.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the inoculum concentration of 8% volume fraction
In reaction system after the pre-preg that step (2) obtains, and continue to carry out saccharification reaction under the speed conditions of 1.5r/min,
Obtain the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate bottom
Conversion ratio of the cellulose to soluble sugar in object.
Embodiment 7:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by Clostridium thermocellum using the glucose of 5g/L as the culture medium of sole carbon source, i.e. glucose culture
In base, according to the inoculative proportion of 2% volume fraction, 2 secondary cultures are carried out；It is then seeded into the pre- place of addition 1g/L dry weight
In the dextrose culture-medium for managing straw, inducing and acclimating 48h under the conditions of 60 DEG C of temperature, to obtain Seedling height speed, high fibre
Tie up corpusculum yield, the full bacterium saccharification seed liquor of high microsteping corpusculum Rate activity.The preprocess method is document (cellulose section
Learn and technology, 2002,3,47-52) in steam explosion preconditioning technique.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:11, the pretreated lignocellulosic of 100kg is former
The culture medium solution of material and 1100L are uniformly mixed under the speed conditions of 0.5r/min, and continue 48h；It is described pretreated
Lignocellulosic material is identical with step (1).It is described to be uniformly mixed to realize that mixing is equal by the whole rolling flip of container
It is even.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the inoculum concentration of 2% volume fraction
In reaction system after the pre-preg that step (2) obtains, and saccharification reaction is carried out under the speed conditions of 1.6r/min, obtain
Liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate in
Conversion ratio of the cellulose to soluble sugar.
Embodiment 8:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by Clostridium thermocellum using the glucose of 5g/L as the culture medium of sole carbon source, i.e. glucose culture
In base, according to the inoculative proportion of 0.5% volume fraction, 4 secondary cultures are carried out；It is then seeded into the pre- of addition 2g/L dry weight
In the dextrose culture-medium for handling waste paper, inducing and acclimating 12h under the conditions of 60 DEG C of temperature, to obtain Seedling height speed, height
Cellulosome yield, the full bacterium saccharification seed liquor of high microsteping corpusculum Rate activity.The preprocess method is document
Hydrothermal pretreatment technology in (Bioresource Technology, 2004,91,93-100).After step (1), adopt
With cellulosome Rate activity, yield and the cellular biomass of the method detection seed liquor of embodiment 1.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:10, the pretreated lignocellulosic of 0.2kg is former
The culture medium solution of material and 2L are uniformly mixed under the speed conditions of 5.0r/min, and continue 40h；The pretreated wood
Matter cellulosic material is identical with step (1).It is described to be uniformly mixed to realize that mixing is equal by the whole rolling flip of container
It is even.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the inoculum concentration of 4% volume fraction
In reaction system after the pre-preg that step (2) obtains, and continue to carry out saccharification reaction under the speed conditions of 5.0r/min,
Obtain the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate bottom
Conversion ratio of the cellulose to soluble sugar in object.
Embodiment 9:
As different from Example 8, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by Clostridium thermocellum (dextrose culture-medium removes using 5g/L cellobiose as the culture medium of carbon source
Ingredient other than glucose) in cultivate 12h.After step (1), using the fiber of the method detection seed liquor of embodiment 1
Corpusculum Rate activity, yield and cellular biomass.
(2) full bacterium saccharification: according to the inoculum concentration of 4% volume fraction, seed liquor is directly inoculated into and is pre-processed containing 0.2kg
The condition of full bacterium saccharification, pretreated feedstock and the saccharification of full bacterium is carried out in the reaction system of the culture medium solution of waste paper and 2L afterwards
It is same as Example 8.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate in fiber
Element arrives the conversion ratio of soluble sugar.
Embodiment 10:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by the molten fine clostridium of yellow using the glucose of 10g/L as the culture medium of sole carbon source, i.e. glucose
In culture medium, according to the inoculative proportion of 1% volume fraction, 3 secondary cultures are carried out；It is then seeded into addition 0.5g/L dry weight
Pretreatment wheat straw dextrose culture-medium in, under the conditions of 65 DEG C of temperature inducing and acclimating for 24 hours, thus obtain Seedling height speed
The full bacterium saccharification seed liquor of degree, high microsteping corpusculum yield, high microsteping corpusculum Rate activity.The preprocess method is the same as embodiment 1
In sulfonation preconditioning technique.After step (1), lived using the cellulosome ratio of the method detection seed liquor of embodiment 1
Power, yield and cellular biomass.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:15, the pretreated lignocellulosic of 0.2kg is former
The culture medium solution of material and 3L are uniformly mixed under the speed conditions of 20r/min, and continue 12h；It is described pretreated wooden
Cellulosic material is identical with step (1).It is described to be uniformly mixed to realize that mixing is equal by the stirring of agitating device in container
It is even.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the inoculum concentration of 5% volume fraction
In reaction system after the pre-preg that step (2) obtains, and continue to carry out saccharification reaction under the speed conditions of 20r/min, obtain
To the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate
Conversion ratio of the middle cellulose to soluble sugar.
Embodiment 11:
As different from Example 10, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by the molten fine clostridium of yellow using 10g/L microcrystalline cellulose as the culture medium (glucose of carbon source
The ingredient of culture medium in addition to glucose) in culture for 24 hours.After step (1), seed is detected using the method for embodiment 1
Cellulosome Rate activity, yield and the cellular biomass of liquid.
(2) full bacterium saccharification: according to the inoculum concentration of 5% volume fraction, seed liquor is directly inoculated into and is pre-processed containing 0.2kg
The condition of full bacterium saccharification, pretreated feedstock and the saccharification of full bacterium is carried out in the reaction system of the culture medium solution of wheat straw and 3L afterwards
It is same as in Example 10.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate fine in substrate
Dimension element arrives the conversion ratio of soluble sugar.
Embodiment 12:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by thermophilic fiber clostridium using the glucose of 10g/L as the culture medium of sole carbon source, i.e., glucose is trained
It supports in base, according to the inoculative proportion of 1% volume fraction, carries out 3 secondary cultures；It is then seeded into addition 0.5g/L dry weight
Pre-process wheat straw dextrose culture-medium in, inducing and acclimating 36h under the conditions of 37 DEG C of temperature, thus obtain Seedling height speed,
High microsteping corpusculum yield, the full bacterium saccharification seed liquor of high microsteping corpusculum Rate activity.The preprocess method is the same as in embodiment 1
Sulfonation preconditioning technique.After step (1), lived using the cellulosome ratio of the method detection seed liquor of embodiment 1
Power, yield and cellular biomass.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:20, the pretreated lignocellulosic of 0.2kg is former
The culture medium solution of material and 4L are uniformly mixed under the speed conditions of 20r/min, and continue 10h；It is described pretreated wooden
Cellulosic material is identical with step (1).It is described to be uniformly mixed to realize that mixing is equal by the stirring of agitating device in container
It is even.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the inoculum concentration of 1% volume fraction
In reaction system after the pre-preg that step (2) obtains, and continue to carry out saccharification reaction under the speed conditions of 20r/min, obtain
To the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate
Conversion ratio of the middle cellulose to soluble sugar.
Embodiment 13:
As different from Example 12, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by thermophilic fiber clostridium using 10g/L microcrystalline cellulose as culture medium (the glucose training of carbon source
The ingredient of feeding base in addition to glucose) in cultivate 36h.After step (1), seed liquor is detected using the method for embodiment 1
Cellulosome Rate activity, yield and cellular biomass.
(2) full bacterium saccharification: according to the inoculum concentration of 1% volume fraction, seed liquor is directly inoculated into and is pre-processed containing 0.2kg
The condition of full bacterium saccharification, pretreated feedstock and the saccharification of full bacterium is carried out in the reaction system of the culture medium solution of wheat straw and 4L afterwards
It is identical as embodiment 12.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate fine in substrate
Dimension element arrives the conversion ratio of soluble sugar.
Embodiment 14:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by solution fiber clostridium using the glucose of 10g/L as the culture medium of sole carbon source, i.e., glucose is trained
It supports in base, according to the inoculative proportion of 1% volume fraction, carries out 3 secondary cultures；It is then seeded into addition 0.5g/L dry weight
Pre-process wheat straw dextrose culture-medium in, inducing and acclimating 36h under the conditions of 34 DEG C of temperature, thus obtain Seedling height speed,
High microsteping corpusculum yield, the full bacterium saccharification seed liquor of high microsteping corpusculum Rate activity.The preprocess method is the same as in embodiment 2
Preconditioning technique.After step (1), using the cellulosome Rate activity of the method detection seed liquor of embodiment 1, produce
Amount and cellular biomass.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:40, the pretreated lignocellulosic of 0.1kg is former
The culture medium solution of material and 4L are uniformly mixed under the speed conditions of 75r/min, and continue 5h；It is described pretreated wooden
Cellulosic material is identical with step (1).It is described to be uniformly mixed to realize that mixing is equal by the stirring of agitating device in container
It is even.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated with the inoculum concentration of 0.5% volume fraction
In reaction system after the pre-preg obtained to step (2), and continue to carry out saccharification reaction under the speed conditions of 75r/min,
Obtain the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate bottom
Conversion ratio of the cellulose to soluble sugar in object.
Embodiment 15:
As different from Example 12, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by solution fiber clostridium using 10g/L cellobiose as culture medium (the glucose culture of carbon source
The ingredient of base in addition to glucose) in cultivate 36h.After step (1), seed liquor is detected using the method for embodiment 1
Cellulosome Rate activity, yield and cellular biomass.
(2) full bacterium saccharification: according to the inoculum concentration of 0.5% volume fraction, seed liquor is directly inoculated into and is located in advance containing 0.1kg
The item of full bacterium saccharification, pretreated feedstock and the saccharification of full bacterium is carried out in the reaction system of the culture medium solution of wheat straw and 4L after reason
Part is identical as embodiment 14.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate in
Conversion ratio of the cellulose to soluble sugar.
Embodiment 16:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by Acetivibrio cellulolyticus using the glucose of 10g/L as the culture medium of sole carbon source, i.e. glucose
In culture medium, according to the inoculative proportion of 1% volume fraction, 3 secondary cultures are carried out；It is then seeded into addition 0.5g/L dry weight
Pretreatment corncob dextrose culture-medium in, inducing and acclimating 36h under the conditions of 37 DEG C of temperature, to obtain Seedling height
Speed, high microsteping corpusculum yield, the full bacterium saccharification seed liquor of high microsteping corpusculum Rate activity.The preprocess method is the same as implementation
Steam explosion method preconditioning technique in example 5.After step (1), the fiber using the method detection seed liquor of embodiment 1 is small
Body Rate activity, yield and cellular biomass.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:50, the pretreated lignocellulosic of 0.1kg is former
The culture medium solution of material and 5L are uniformly mixed under the speed conditions of 100r/min, and continue 1h；It is described pretreated wooden
Cellulosic material is identical with step (1).It is described to be uniformly mixed to realize that mixing is equal by the stirring of agitating device in container
It is even.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated with the inoculum concentration of 0.1% volume fraction
In reaction system after the pre-preg obtained to step (2), and it is anti-to continue to carry out being saccharified under the speed conditions of 100r/min
It answers, obtains the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate
Conversion ratio of the cellulose to soluble sugar in substrate.
Embodiment 17:
As different from Example 16, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by Acetivibrio cellulolyticus using 10g/L cellobiose as culture medium (the glucose training of carbon source
The ingredient of feeding base in addition to glucose) in cultivate 36h.After step (1), seed liquor is detected using the method for embodiment 1
Cellulosome Rate activity, yield and cellular biomass.
(2) full bacterium saccharification: according to the inoculum concentration of 0.1% volume fraction, seed liquor is directly inoculated into and is located in advance containing 0.1kg
Full bacterium saccharification is carried out in the reaction system of the culture medium solution of corncob and 5L after reason, what pretreated feedstock and full bacterium were saccharified
Condition is identical as embodiment 16.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate
Conversion ratio of the middle cellulose to soluble sugar.
Embodiment 18:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by molten fiber vacation bacteroid using the glucose of 5g/L as the culture medium of sole carbon source, i.e. grape
In sugar culture-medium, according to the inoculative proportion of 2% volume fraction, 2 secondary cultures are carried out；It is then seeded into addition 1g/L dry weight
Pretreatment waste paper dextrose culture-medium in, inducing and acclimating 40h under the conditions of 42 DEG C of temperature, thus obtain Seedling height speed
The full bacterium saccharification seed liquor of degree, high microsteping corpusculum yield, high microsteping corpusculum Rate activity.The preprocess method is the same as embodiment 5
In steam explosion method preconditioning technique.After step (1), using the cellulosome ratio of the method detection seed liquor of embodiment 1
Vigor, yield and cellular biomass.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:25, the pretreated lignocellulosic of 0.2kg is former
The culture medium solution of material and 5L are uniformly mixed under the speed conditions of 1.6r/min, and continue 12h；The pretreated wood
Matter cellulosic material is identical with step (1).It is described to be uniformly mixed to realize that mixing is equal by the whole rolling flip of container
It is even.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the inoculum concentration of 5% volume fraction
In reaction system after the pre-preg that step (2) obtains, and continue to carry out saccharification reaction under the speed conditions of 1.6r/min,
Obtain the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate bottom
Conversion ratio of the cellulose to soluble sugar in object.
Embodiment 19:
As different from Example 18, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by molten fiber vacation bacteroid using 10g/L pretreatment waste paper as the culture medium (grape of carbon source
The ingredient of sugar culture-medium in addition to glucose) in cultivate 40h.After step (1), kind is detected using the method for embodiment 1
Cellulosome Rate activity, yield and the cellular biomass of sub- liquid.
(2) full bacterium saccharification: according to the inoculum concentration of 5% volume fraction, seed liquor is directly inoculated into and is pre-processed containing 0.2kg
Full bacterium saccharification, pretreated feedstock and full bacterium are carried out in the reaction system of the culture medium solution of lignocellulosic material and 5L afterwards
The condition of saccharification is identical as embodiment 18.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, meter
Conversion ratio of the cellulose to soluble sugar in calculation substrate.
Embodiment 20:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by Ruminococcus albus using the glucose of 20g/L as the culture medium of sole carbon source, i.e. glucose
In culture medium, according to the inoculative proportion of 1% volume fraction, 2 secondary cultures are carried out；It is then seeded into addition 2g/L dry weight
In the dextrose culture-medium for pre-processing corncob, inducing and acclimating 40h under the conditions of 37 DEG C of temperature, to obtain Seedling height speed
The full bacterium saccharification seed liquor of degree, high microsteping corpusculum yield, high microsteping corpusculum Rate activity.The preprocess method is the same as embodiment 5
In steam explosion method preconditioning technique.After step (1), using the cellulosome ratio of the method detection seed liquor of embodiment 1
Vigor, yield and cellular biomass.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:3, by the pretreated lignocellulosic material of 0.2kg
It is uniformly mixed under the speed conditions of 2r/min with the culture medium solution of 0.6L, and continues 48h；It is described pretreated wooden
Cellulosic material is identical with step (1).It is described to be uniformly mixed to be realized and being uniformly mixed by the whole rolling flip of container.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the inoculum concentration of 10% volume fraction
In reaction system after the pre-preg that step (2) obtains, and continue to carry out saccharification reaction under the speed conditions of 2r/min, obtain
To the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate
Conversion ratio of the middle cellulose to soluble sugar.
Embodiment 21:
As different from Example 20, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by Ruminococcus albus using 20g/L pretreatment corncob as the culture medium (grape of carbon source
The ingredient of sugar culture-medium in addition to glucose) in cultivate 40h.After step (1), kind is detected using the method for embodiment 1
Cellulosome Rate activity, yield and the cellular biomass of sub- liquid.
(2) full bacterium saccharification: according to the inoculum concentration of 10% volume fraction, seed liquor is directly inoculated into and is located in advance containing 0.2kg
Carry out full bacterium saccharification in the reaction system of the culture medium solution of lignocellulosic material and 0.6L after reason, pretreated feedstock and
The condition of full bacterium saccharification is identical as embodiment 20.In the process, reduced sugar contains in every 12 hours sample detection fermentation liquids
It measures, conversion ratio of the cellulose to soluble sugar in calculating substrate.
Embodiment 22:
Unlike the first embodiment, for the full bacterium method for saccharifying of lignocellulosic, comprising the following steps:
(1) seed liquor optimizes:
Under anaerobic, by yellow Ruminococcus using the glucose of 20g/L as the culture medium of sole carbon source, i.e. glucose
In culture medium, according to the inoculative proportion of 1% volume fraction, 2 secondary cultures are carried out；It is then seeded into addition 2g/L dry weight
In the dextrose culture-medium for pre-processing corncob, inducing and acclimating 48h under the conditions of 40 DEG C of temperature, to obtain Seedling height speed
The full bacterium saccharification seed liquor of degree, high microsteping corpusculum yield, high microsteping corpusculum Rate activity.The preprocess method is that document is (raw
Object process, 2010,3,66-72) the dilute acid hydrolysis preconditioning technique in.After step (1), using embodiment 1
Cellulosome Rate activity, yield and the cellular biomass of method detection seed liquor.
(2) raw material pre-preg: according to the solid and liquid weight volume ratio of 1:5, by the pretreated lignocellulosic material of 0.4kg
It is uniformly mixed under the speed conditions of 10r/min with the culture medium solution of 2L, and continues 36h；The pretreated wooden fibre
It is identical with step (1) to tie up plain raw material.It is described to be uniformly mixed to be realized and being uniformly mixed by the whole rolling flip of container.
(3) full bacterium saccharification: the full bacterium saccharification seed liquor of step (1) optimization is inoculated into the inoculum concentration of 10% volume fraction
In reaction system after the pre-preg that step (2) obtains, and continue to carry out saccharification reaction under the speed conditions of 10r/min, obtain
To the liquid glucose containing glucose.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content, calculate substrate
Conversion ratio of the middle cellulose to soluble sugar.
Embodiment 23:
As different from Example 22, for the full bacterium method for saccharifying of lignocellulosic, without domestication and pre-soak step,
The following steps are included:
(1) seed liquor culture: by yellow Ruminococcus using 20g/L pretreatment corncob as the culture medium (grape of carbon source
The ingredient of sugar culture-medium in addition to glucose) in cultivate 48h.After step (1), kind is detected using the method for embodiment 1
Cellulosome Rate activity, yield and the cellular biomass of sub- liquid.
(2) full bacterium saccharification: according to the inoculum concentration of 10% volume fraction, seed liquor is directly inoculated into and is located in advance containing 0.4kg
Carry out full bacterium saccharification in the reaction system of the culture medium solution of lignocellulosic material and 2L after reason, pretreated feedstock and complete
The condition of bacterium saccharification is identical as embodiment 22.In the process, in every 12 hours sample detection fermentation liquids reduced sugar content,
Conversion ratio of the cellulose to soluble sugar in calculating substrate.
Index parameter in the full bacterium saccharifying of 1. embodiment 1-22 of table
Note:
1. the speed of growth refers to grow into mid-log phase needed for the time.
2. lag phase refers to that the conversion ratio of cellulose to soluble sugar in substrate in full bacterium saccharifying is lower than 10% (quality
Than) time.
3. the hydrolysis period refers to that the conversion ratio of cellulose to soluble sugar in substrate in full bacterium saccharifying reaches 80% (matter
Measure ratio) time.
As shown in Table 1,
(1) according to the comparison of four groups of embodiments 8-9,14-15 and 16-17 it is found that using described herein for wooden
The full bacterium method for saccharifying of cellulose, the Rate activity of the cellulosome of obtained seed liquor are 4.6-17.8U/mg；Cellulosome
Yield be 4.6-6.3mg/mL；Seed growth speed is 10-36 hours.The yield of glucose is 10.6-54.8g/ in liquid glucose
L；Lag phase is 0.5-1.0 days；Saccharification period is 6.5-14 days.
Use cellobiose for carbon source in the case where, the Rate activity of cellulosome is 1.3-4.1U/mg；The production of cellulosome
Amount is 1.4-2.1mg/mL；Seed growth speed is 12-36 hours.The yield of glucose is 10.7-50.3g/L in liquid glucose；Prolong
Demurrage is 2-3 days；Saccharification period is 10-17.5 days.
Illustrate, compared with using the case where cellobiose is carbon source, using described herein for lignocellulosic
The Rate activity of cellulosome and the yield of cellulosome of full bacterium method for saccharifying seed liquor obtained greatly improve；Retardation
Phase and saccharification period greatly shorten；The yield of glucose almost maintains an equal level in seed growth speed and liquid glucose.
(2) according to the comparison of four groups of embodiments 4-5,10-11 and 12-13 it is found that being used for wooden fibre with described herein
The full bacterium method for saccharifying of element is tieed up, the Rate activity of the cellulosome of obtained seed liquor is 4.1-17.7U/mg；Cellulosome
Yield is 13.9-25.5mg/mL；Seed growth speed is 16-32 hours.The yield of glucose is 26.6-62.5g/ in liquid glucose
L；Lag phase is 0.5-1.5 days；Saccharification period is 7.5-13.5 days.
Use microcrystalline cellulose for carbon source in the case where, the Rate activity of cellulosome is 3.3-17.1U/mg；Cellulosome
Yield be 15.0-21.3mg/mL；Seed growth speed is 24-40 hours.The yield of glucose is 23.9- in liquid glucose
63.1g/L；Lag phase is 2.5-4.5 days；Saccharification period is 12-18 days.
Illustrate, compared with using the case where microcrystalline cellulose is carbon source, is used for lignocellulosic using described herein
The Rate activity of cellulosome of full bacterium method for saccharifying seed liquor obtained, glucose in the yield of cellulosome and liquid glucose
Yield almost maintain an equal level；And seed growth speed greatly improves, lag phase and saccharification period greatly shorten.
(3) according to the comparison of four groups of embodiments 1-2,18-19,20-21 and 22-23 it is found that using use described herein
In the full bacterium method for saccharifying of lignocellulosic, the Rate activity of the cellulosome of obtained seed liquor is 4.1-14.8U/mg；It is fine
The yield for tieing up corpusculum is 7.9-15.2mg/mL；Seed growth speed is 40-48 hours.The yield of glucose is in liquid glucose
27.1-112.5 g/L；Lag phase is 0.5-1.5 days；Saccharification period is 9.5-18.5 days.
Use lignocellulosic for carbon source in the case where, the Rate activity of cellulosome is 5.1-15.3U/mg；Cellulosome
Yield be 8.1-14.8mg/mL；Seed growth speed is 66-72 hours.The yield of glucose is 24.7- in liquid glucose
103.1g/L；Lag phase is 2.5-5.0 days；Saccharification period is 15-26.5 days.
Illustrate, compared with using the case where lignocellulosic is carbon source, is used for lignocellulosic using described herein
The Rate activity of cellulosome of full bacterium method for saccharifying seed liquor obtained, glucose in the yield of cellulosome and liquid glucose
Yield almost maintain an equal level；And seed growth speed greatly improves, lag phase and saccharification period greatly shorten.
In summary, the Rate activity of the cellulosome of (1) present invention seed liquor obtained and microcrystalline cellulose are carbon source
The cellulosome of production is consistent, higher than the cellulosome produced using cellobiose as carbon source；(2) present invention seed obtained
The cellulosome yield of liquid is consistent with the cellulosome that cellulose is carbon source production；Higher than what is produced using cellobiose as carbon source
Cellulosome；(3) the seed growth speed of present invention seed liquor obtained with using cellobiose as the seed one of carbon source for growth
It causes, higher than the seed that cellulose is carbon source for growth, much higher than the seed that preprocessing lignocellulose is carbon source for growth；(4) this hair
Lag phase and saccharification period in bright with using cellobiose/microcrystalline cellulose/preprocessing lignocellulose as carbon source compared with,
It greatly shortens.
This explanation, the full bacterium method for saccharifying of the present invention for lignocellulosic are added with relatively inexpensive glucose
The preprocessing lignocellulose biomass for adding low concentration is carbon source, produces cellulosome bacterial strain for cultivating Clostridium thermocellum etc., as
The seed liquor of full bacterium saccharification；Compared with prior art, the seed liquor of the full bacterium saccharification realizes cost reduction and cycle time
Effect, overcome the problem that you can't have both at the same time in the prior art.In addition, the full bacterium of lignocellulosic of the present invention
Method for saccharifying significantly shortens the adaptation phase buffer (lag phase) of full bacterium saccharification, especially using the technology of raw material pre-preg
It is the efficiency of full bacterium saccharification to be improved, to reduce period and the cost of entire saccharifying under conditions of high solids content.
Claims (10)
1. being used for the full bacterium method for saccharifying of lignocellulosic, it is characterised in that: the following steps are included:
(1) seed liquor optimizes: under anaerobic, will produce cellulosome bacterial strain and carries out passage and attenuation in dextrose culture-medium；
It is then seeded into the dextrose culture-medium for being added to pretreated lignocellulosic material and carries out inducing and acclimating, to obtain
Full bacterium saccharification seed liquor after optimization；The dextrose culture-medium is the culture medium using glucose as sole carbon source；
(2) raw material pre-preg: according to certain solid and liquid weight volume ratio, by pretreated lignocellulosic material and culture medium
Solution is uniformly mixed in a reservoir, and certain time；The pretreated lignocellulosic material and phase in step (1)
Together；
(3) full bacterium saccharification: anti-after the full bacterium saccharification seed liquor of step (1) optimization to be inoculated into the pre-preg that step (2) obtains
It answers in system, and carries out saccharification reaction under the speed conditions of 0.5-100r/min, obtain the liquid glucose containing glucose.
2. the full bacterium method for saccharifying according to claim 1 for lignocellulosic, it is characterised in that: step (1) is described
The concentration of glucose is 1-20g/L in dextrose culture-medium；The passage and attenuation carries out 2-5 times；The lignocellulosic material
Additive amount be 0.1-2.0g/L.
3. the full bacterium method for saccharifying according to claim 1 for lignocellulosic, it is characterised in that: the inducing and acclimating
Temperature condition be 34-65 DEG C；The time of the inducing and acclimating is 12-48 hours.
4. the full bacterium method for saccharifying according to claim 1 for lignocellulosic, it is characterised in that: step (2) is described
Lignocellulosic material and culture medium solution solid and liquid weight volume ratio be 1:3-1:50；The lignocellulosic material and
Culture medium solution is uniformly mixed under the speed conditions of 0.5-100r/min and continues 1-48h.
5. the full bacterium method for saccharifying according to claim 4 for lignocellulosic, it is characterised in that: the container is band
There are the anaerobic fermentation equipment or anaerobism rotation vortex mixer of agitating paddle.
6. the full bacterium method for saccharifying according to claim 1 for lignocellulosic, it is characterised in that: step (3) is described
Full bacterium saccharification seed liquor inoculum concentration be 0.1-10% volume fraction.
7. the full bacterium method for saccharifying of lignocellulosic is used for described in any one of -6 according to claim 1, it is characterised in that:
Wherein, the production cellulosome bacterial strain is Clostridium thermocellum, the molten fine clostridium of yellow, thermophilic fiber clostridium, solution fiber clostridium, solution fiber
Vinegar vibrios, molten fiber vacation bacteroid, Ruminococcus albus or yellow Ruminococcus.
8. the full bacterium method for saccharifying of lignocellulosic is used for described in any one of -6 according to claim 1, it is characterised in that:
The pretreatment is one of alkaline process, diluted acid method, hydro-thermal method, steam explosion method and sulfonation method preconditioning technique or a variety of groups
It closes.
9. the full bacterium method for saccharifying of lignocellulosic is used for described in any one of -6 according to claim 1, it is characterised in that:
The lignocellulosic material be corn stover, wheat straw, brush wood branch, wood chip, one of corncob, straw and waste paper or
A variety of combinations.
10. being used for the full bacterium method for saccharifying of lignocellulosic described in any one of -6 according to claim 1, feature exists
In: dextrose culture-medium described in step (1) be added in every liter of water dipotassium hydrogen phosphate 2.9g/L, potassium dihydrogen phosphate 1.5g/L,
Urea 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,
Trisodium citrate 2g/L, glucose 1-20g/L, pH 6.5-7.5.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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