CN106086087A - A kind of microbial co culture prepares the system of biomass monomer - Google Patents
A kind of microbial co culture prepares the system of biomass monomer Download PDFInfo
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Abstract
A kind of microbial co culture prepares the system of biomass monomer, contain with biomass as substrate, realize biomass degradation microorganism by special installation process adjustment and prepare subject monomers with subject monomers generation microbial co culture, it is characterized in that: use rumen microorganism and target product microbial co culture, the equipment of product can be separated in situ, it is achieved rumen microorganism and the symbiosis of target product microorganism and the preparation of high efficiency target product by regulation and control.
Description
Technical field
The present invention relates to biomass monomer preparation system, especially relate to utilize microbial co culture to prepare biomass list
The system of body.
Background technology
In recent years, occur multiple with non-grain biomass as raw material, utilize biological method prepare biomass monomer process and be
System.Ferment with yeast integration after some selection enzyme preparation saccharifying biomass, but enzyme preparation high cost.Some selection oxygen consumptions
Microorganism and yeast integration fermenting and preparing biological matter ethanol, owing to oxygen consumption fermentation production rate is relatively low.Existing fermentable system
Standby biomass monomer, needs also exist for biomass sterilizing, and energy consumption is huge, and batch fermentation is carried out, and fermentation can not be carried out continuously, antagonism
Effect is obvious, the problems such as fermentation efficiency is relatively low.
Summary of the invention
The purpose of invention is that providing a kind of realizes rumen microorganism by special installation process adjustment and produce material list
Body microbial co culture, the equipment that can be separated product by regulation and control in situ prepares biomass monomer, it is achieved low cost, high efficiency
The system preparing continuously biomass monomer.
A kind of microbial co culture of the present invention prepares the system of biomass monomer, contains with biomass as the end
Thing, realizes biomass degradation microorganism by special installation process adjustment and prepares target with subject monomers generation microbial co culture
Monomer, described use rumen microorganism and target product microbial co culture, can separate setting of product in situ by regulation and control
Prepare standby subject monomers.
Described, rumen microorganism carries out live body hot housing before gathering, and chooses ruminant, chooses the life preparing to utilize
Material is carbon source, administering transgenic animal, gradually decreases concentrated feed and adds, feeds preparation and co-culture subject monomers microorganism, mainly
Producing and ethanol microorganism, feeding laboratory animal January with.
Described, microorganism purification, use negative pressure stomach tube method to take ruminant tumor gastric content, anaerobism rolling is managed,
Purification rumen microorganism, builds small-sized continuous cultivation in anaerobic culture box and membrane separation device carries out Rumen Fungi and amplifies training
Support, after cultivating a period of time, culture medium gradually add producing and ethanol microbial metabolic products and carries out co-culturing direction domestication cultivation,
The rumen microorganism of purification includes cellulose-decomposing bacterium respectively Bacteroides succinogenes (Fibrobacter
Succinogenes), cud bacterium (Ruminococcus), fibrinolysin butanoic acid vibrio (Buutyrivibrio
Fibrisolvens), clostridium (Clostridium)；Cellulolytic fungi has Caecomyes single centre thalline, single flagellum trip
Zoospore, spherical, rhizoid system C.communis of branch, C.equi；Cyllamyces multicenter thalline, many flagellums are swum
Zoospore, spherical, the sporophore Cy.Aberensis of branch；Neocallimastix single centre thalline, many flagellums move about spore
Son, abundant silk strengthens rhizoid N.frontalis, N.patriciarum, N.hurleyensis, N.variabilis；
Piromyces single centre thalline, single flagellum zoospore thread rhizoid system P.communis, P.mae
P.dumbonicus P.rhizinflatus P.minutus P.spiralis Citronii ；Orpinomyces multicenter
Thalline, many flagellums zoospore, thread rhizoid system O.joyonii, O.intercalaris, O.bovis；
Anaeromyces multicenter thalline, single flagellum zoospore, silk strengthens rhizoid A.elegans, A.mucronatus, except routine
Technology obtains, and also can post acquisition at Chinese industrial Culture Collection.
Described target product strain includes producing and ethanol bacterium, butanol bacterium, produces butyric, produces anaerobism such as aminoacid bacterium and facultative
Anaerobe.
Described, the introducing of objective microbe the most exogenous producing and ethanol bacterium, selection can utilize pentose and six
The yeast first-selection candida mycoderma of carbon sugar, Ethanologic bacteria and available acetic acid mutant yeast bacterium, a kind of from the micro-life of Chinese industrial
Thing DSMZ obtains and can utilize the ethanol yeast of glucose and xylose simultaneously: numbering 1463 generic name:
Tropicalis generic name: Candida separates substrates: Chinese: candida tropicalis；1771 kinds of names of numbering:
Tannophilus generic name: Pachysolen separates substrates: Chinese: Pachysolen tannophilus；960 kinds of names of numbering:
Stipitis generic name: Pichia separates substrates: Chinese: pichia stipitis；Another kind is Ethanologic bacteria；The
Three kinds be can acetic acid utilize mutant yeast bacterium, the amplification culture later stage gradually add rumen microorganism cultivate metabolite to tumor
Stomach microorganism culturing condition carries out co-culturing direction domestication, mainly cultivation temperature 39 DEG C, mild alkaline conditions, low oxidoreduction electricity
Position, culture medium comprises the required trace element of rumen microorganism growth, and nitrogen source element, after starting, growth course nitrogen source, with straw
Protein in stalk straw, the protein that cell ablation produces, for main nitrogen, target product strain also includes producing butanol bacterium,
Produce butyric, produce anaerobism and the facultative anaerobes such as aminoacid bacterium.
Producing and ethanol bacterium addition cud continuous flow culture system after described domestication co-cultures, by training continuously
The system of supporting, it is ensured that product is separated by membrane separation device, does not produce antagonism, Rumen Fungi is along with growth generation in biomass
Thread rhizoid make biomass float to tank body top, rumen microorganism spore and be not fermented straw and stay tank body lower part.By
Characteristic in Rumen Fungi achieves continuous fermentation in a tank.
Described, producing and ethanol bacterium introducing aspect first-selection yeast, along with the research of cellulosic ethanol for many years, research worker is
Through taming the multiple strain that can utilize xylose and glucose simultaneously, what yeast strong adaptability was the most much studied tests
Card, can tame according to the condition of culture direction of Rumen Fungi.Existing yeast can't anaerobism be bred, but the product second of anaerobism
Alcohol antibacterial has anaerobism fertility, and ethanol production is the highest, and also can utilize xylose and glucose simultaneously.
Described, Rumen Fungi originally also can be with a large amount of competitive rumen bacteria symbiosis in cud, if but introducing second
Acid-utilising yeast, Rumen Fungi and this yeast-like fungi will belong to collaborative symbiosis, most useful for the realization co-cultured.Training altogether
In the microecological environment supported, microorganism is in addition to self adaptability, can ensure that elimination suppression is made by continuous film segregation apparatus
With, mainly cultivation temperature 39 DEG C, mild alkaline conditions, low oxidation-reduction potential-350mv.
Described, co-culture, co-culturing the cycle, need again to throw in producing and ethanol bacterium.The input cycle determines it is logical
Crossing the concentration rising decision measuring sugar, existing yeast can't anaerobism be bred, but the Ethanologic bacteria of anaerobism has anaerobism
Fertility, ethanol production is the highest, and also can utilize xylose and glucose simultaneously.
Described, the state of biomass, with to co-culture situation corresponding, co-culture with Rumen Fungi and yeast, chooses biology
Matter granule is relatively big at 1 ~ 10 millimeter, preferably 2 ~ 5 millimeters Rumen Fungis and and Ethanologic bacteria co-culture and also choose biological particles
Relatively big, during Rumen Fungi, rumen bacteria and producing and ethanol fungus co-culture, stalk particle is less at 1 to 3 millimeter, due to symbiosis
Suppressing other miscellaneous bacterias to produce, biomass need not sterilizing, it will substantially ethanol energy consumption is prepared in reduction.
Described, fermentation system uses 3 sets, and with alcohol yied as target, fermentation in parallel, through 24 hours by tank body
The straw fermented in top is got rid of outside tank, then feed supplement continuous fermentation, and 3 tank bodies start at times, when a tank body occurs
Problem can be introduced a fine variety by other tank bodies.
Described, biomass refer to corn straw, rice straw, wheat stalk, cotton stalk, waste of flax, preferably refer to
Corn straw stalks of rice, wheat, etc. leaf and skin ferment respectively, and the fermentation resultant product of stalks of rice, wheat, etc. leaf is as biological feedstuff raw material later use, and straw skin is sent out
Ferment residue is as biological fiber raw material later use.
The beneficial effect of the invention is: the present invention provides one to utilize microbial co culture fermenting and preparing biological matter monomer,
Microbial portion can pass on voluntarily, does not use enzyme preparation, uses continuous fermentation apparatus, separates product in situ, improves efficiency,
Microbial co culture, metabolite inhibits correspondence to co-culture the harmful bacteria of microorganism, it is not necessary to sterilizing pure culture, reduces energy
Source consumes.
Accompanying drawing explanation
The invention will be further described below in conjunction with the accompanying drawings:
A kind of microbial co culture of Fig. 1 prepares the system schematic of biomass monomer.
Detailed description of the invention:
In order to be more fully understood that the present invention, describe technical scheme in detail with instantiation below, but the present invention
It is not limited thereto.
Embodiment 1
Choosing ruminant goat, choosing the biomass preparing to utilize is that the stalks of rice, wheat, etc. leaf after straw separating treatment is carbon source, feeds
Laboratory animal, gradually decreases concentrated feed and adds, feed preparation and co-culture producing and ethanol microorganism, available pentose and hexose
Candida mycoderma, feeding laboratory animal January with.Negative pressure stomach tube method is used to take rumen content, anaerobism rolling pipe, purification mountain
Sheep rumen microorganism, cultivates in anaerobic culture box, after cultivating a period of time, gradually adds producing and ethanol microorganism generation in culture medium
Thank to product to carry out co-culturing direction domestication cultivation, send into small-sized continuous cultivation and membrane separation device carries out rumen microorganism and amplifies training
Supporting, the rumen microorganism cellulolytic fungi of purification has a Caecomyes single centre thalline, single flagellum zoospore, spherical, point
Rhizoid system C.communis propped up.Rumen microorganism puts into medium-sized continuous flow culture according to biomass weight 3 ~ 5% and film divides
Rumen microorganism amplification culture is carried out, after cultivating 8 hours from device.The introducing of exogenous producing and ethanol bacterium, selection can utilize
The yeast first-selection candida mycoderma of pentose and hexose, a kind of acquisition from China Research for Industrial Microbial Germ preservation center can be same
The ethanol yeast of Shi Liyong glucose and xylose: numbering 1463 generic name: tropicalis generic name: Candida un-mixing bases
Thing: Chinese: candida tropicalis, gradually adds rumen microorganism in the amplification culture later stage and cultivates metabolite to cud
Microorganism culturing condition carries out co-culturing direction domestication, mainly cultivation temperature 38 DEG C, mild alkaline conditions, low oxidoreduction electricity
Position.Producing and ethanol bacterium after domestication is co-cultured according in 6% addition cud continuous flow culture system of Biomass, cultivates
Base comprises the required trace element of rumen microorganism growth, and nitrogen source element, after starting, growth course nitrogen source, with straw straw
In protein, cell ablation produce protein, for main nitrogen, pass through continuous culture system, it is ensured that metabolite product
Ethanol etc. are separated by membrane separation device, do not produce antagonism, and different time sections collection is observed, and Rumen Fungi is along with at straw
The thread rhizoid growing generation on stalk makes straw float to tank body top, by tank body mechanism drain tank outside, rumen microorganism spore
Be not fermented straw and stay tank body lower part, be continuously added into solid-to-liquid ratio constant straw stalks of rice, wheat, etc. leaf and culture medium mixture, Biomass and
The solid-liquid ratio of culture medium is 1:15.Owing to the characteristic of Rumen Fungi achieves continuous fermentation in a tank.Co-culture,
Co-culturing the cycle, needing again to throw in producing and ethanol bacterium.The input cycle determines that the concentration by mensuration is sugared raises decision again,
This example 20 as a child after again add.Co-culture with Rumen Fungi and yeast, choose biological particles diameter at 3 ~ 5 millis
Rice, owing to symbiosis suppresses other miscellaneous bacterias to produce, biomass need not sterilizing, it will substantially ethanol energy consumption is prepared in reduction.Running
Above fermentation system parallel running is overlapped in middle employing 3, and with alcohol yied as target, fermentation in parallel, through 24 hours by tank body
The straw fermented in top is got rid of outside tank, then feed supplement continuous fermentation, and three tank bodies start at times, when a tank body occurs
Problem can be introduced a fine variety by other tank bodies.Using the fermentation resultant product of corn straw stalks of rice, wheat, etc. leaf as the follow-up profit of biological feedstuff raw material
With.
Embodiment 2
Choosing ruminant milch cow, choosing the biomass preparing to utilize is that the stalks of rice, wheat, etc. leaf after straw separating treatment is carbon source, feeds reality
Test animal, gradually decrease concentrated feed and add, feed interpolation preparation simultaneously and co-culture producing and ethanol microorganism, available pentose and six
The candida mycoderma of carbon sugar, more than feeding laboratory animal January.Using negative pressure stomach tube method to take rumen content, anaerobism is rolled
Pipe, purification cow rumen microorganism, cultivate in anaerobic culture box, after cultivating a period of time, culture medium gradually adds product second
Alcohol microbial metabolic products carries out co-culturing direction domestication and cultivates, send into small-sized continuous cultivation and membrane separation device to carry out cud micro-
Biomagnification is cultivated, rumen microorganism cud bacterium (Ruminococcus) of purification.Rumen microorganism according to biomass weight 3 ~
5% puts into medium-sized continuous flow culture and membrane separation device carries out rumen microorganism amplification culture, drawing of exogenous producing and ethanol bacterium
Enter, select to utilize the yeast first-selection candida mycoderma of pentose and hexose, a kind of from China Research for Industrial Microbial Germ guarantor
Center, Tibetan obtains and can utilize the ethanol yeast of glucose and xylose simultaneously: numbering 1463 generic name: tropicalis generic name:
Candida separates substrates: Chinese: candida tropicalis, gradually adds rumen microorganism in the amplification culture later stage and cultivates
Metabolite carries out co-culturing direction to rumen microorganism condition of culture and tames, mainly cultivation temperature 39 DEG C, and mild alkaline conditions is low
Oxidation-reduction potential.Producing and ethanol bacterium after domestication is carried out according in 5% addition cud continuous flow culture system of Biomass
Co-culturing, culture medium comprises the rumen microorganism required trace element of growth, nitrogen source element, after starting, and growth course nitrogen source,
With the protein in straw straw, the protein that cell ablation produces, for main nitrogen, pass through continuous culture system, it is ensured that generation
Thanking to product product ethanol etc. to be separated by membrane separation device, do not produce antagonism, different time sections collection is observed, yeast
Along with the thread rhizoid growing generation on straw makes straw float to tank body top, by tank body mechanism drain tank outside, cud is micro-
Biology is stayed not to be fermented and is stayed tank body lower part on straw stalks of rice, wheat, etc. leaf completely, is continuously added into solid-to-liquid ratio constant straw stalks of rice, wheat, etc. leaf and cultivation
Based mixtures, Biomass and culture medium ratio are 1:15.Owing to the characteristic of Rumen Fungi achieves continuous fermentation in a tank.
Existing yeast can't anaerobism breeding.Although Rumen Fungi originally also can with a large amount of competitive rumen bacterias altogether in cud
Raw, if but introducing acetic acid and utilize yeast, Rumen Fungi and this yeast-like fungi will belong to collaborative symbiosis, most useful for altogether
The realization cultivated.In Tiny ecosystem, microorganism is in addition to self adaptability, can ensure that elimination suppression by continuous film segregation apparatus
Effect.Co-culturing, existing yeast can't anaerobism be bred, and is co-culturing the cycle, is needing again to throw in producing and ethanol bacterium.Throw
The cycle of putting determine be by measure sugar concentration raise determine, this example 20 as a child after again add.The state of biomass
Corresponding with co-culturing situation, during rumen bacteria and producing and ethanol fungus co-culture, straw stalks of rice, wheat, etc. leaf granule is less, and particle diameter is at 1 ~ 3 milli
Rice, owing to symbiosis suppresses other miscellaneous bacterias to produce, biomass need not sterilizing, it will substantially ethanol energy consumption is prepared in reduction.Running
Above fermentation system parallel running is overlapped in middle employing 3, and with alcohol yied as target, fermentation in parallel, through 24 hours by tank body
The straw fermented in top is got rid of outside tank, then feed supplement continuous fermentation, and three tank bodies start at times, when a tank body occurs
Problem can be introduced a fine variety by other tank bodies.Corn straw stalks of rice, wheat, etc. leaf residues is made owing to melting albumen containing microorganism, can
Using as biological feedstuff or Fertilizer application.
Embodiment 3
Choosing ruminant cattle, choosing the biomass preparing to utilize is that the straw skin after straw separating treatment is carbon source, feeds reality
Test animal, gradually decrease concentrated feed and add, feed preparation and co-culture producing and ethanol microorganism, available pentose and the product of hexose
Ethanol bacterium, more than feeding laboratory animal January.Using negative pressure stomach tube method to take rumen content, anaerobism rolling pipe, purification is yellow
Bovine rumen microorganism, cultivates in anaerobic culture box, after cultivating a period of time, gradually adds producing and ethanol microorganism generation in culture medium
Thank to product to carry out co-culturing direction domestication cultivation, send into small-sized continuous cultivation and membrane separation device carries out rumen microorganism and amplifies training
Supporting, the rumen microorganism Neocallimastix single centre thalline of purification, many flagellums zoospore, rhizoid strengthened by abundant silk
N.frontalis, N.patriciarum, N.hurleyensis, N.variabilis therein one or more.Cud is micro-
Biology puts into medium-sized continuous flow culture according to biomass weight 3 ~ 5% and membrane separation device carries out rumen microorganism amplification culture,
The introducing of exogenous Ethanologic bacteria, selects to utilize the Ethanologic bacteria of pentose and hexose, and strain all can be from various places
DSMZ's mailing has the strain of correlation properties, gradually adds rumen microorganism in the amplification culture later stage and cultivates metabolism product
Thing carries out co-culturing direction to rumen microorganism condition of culture and tames, mainly cultivation temperature 36 ~ 39 DEG C, and mild alkaline conditions is low
Oxidation-reduction potential.Ethanologic bacteria after domestication is carried out according in 5% addition cud continuous flow culture system of Biomass
Co-culturing, culture medium comprises the rumen microorganism required trace element of growth, nitrogen source element, after starting, and growth course nitrogen source,
With the protein in straw straw, the protein that cell ablation produces, for main nitrogen, pass through continuous culture system, it is ensured that generation
Thanking to product product ethanol etc. to be separated by membrane separation device, do not produce antagonism, different time sections collection is observed, and cud is true
Bacterium makes straw float to tank body top along with the thread rhizoid growing generation on straw, by tank body mechanism drain tank outside, cud
Fungal spore and be not fermented straw and stay tank body lower part, is continuously added into the constant straw skin of solid-to-liquid ratio and culture medium mixture, raw
Thing amount and culture medium ratio are 1:15.Owing to the characteristic of Rumen Fungi achieves continuous fermentation in a tank.The product second of anaerobism
Alcohol antibacterial has anaerobism fertility, and ethanol production is the highest, and also can utilize xylose and glucose simultaneously.Rumen Fungi
Collaborative symbiosis will be belonged to, most useful for the realization co-cultured with this kind of Ethanologic bacteria.In Tiny ecosystem, microorganism is except certainly
Outside body adaptability, can ensure that elimination inhibitory action by continuous film segregation apparatus.Co-culture, co-culturing the cycle, no
Again throw in Ethanologic bacteria at needs or put into Ethanologic bacteria on a small quantity.The state of biomass with to co-culture situation corresponding,
Co-culture with Rumen Fungi and producing and ethanol bacterium, choose biological particles diameter at 3 ~ 5 millimeters, owing to symbiosis suppresses other miscellaneous bacterias to produce
Raw, biomass need not sterilizing, it will substantially ethanol energy consumption is prepared in reduction.Be in operation the employing 3 above fermentation system of set also
Through transport row, with alcohol yied as target, fermentation in parallel, the straw fermented on tank body top through 24 hours gets rid of tank
Outward, then feed supplement continuous fermentation, three tank bodies start at times, can be drawn by other tank bodies when a tank body goes wrong
Kind.Using maize straw skin residues as biological fiber raw material later use.
Embodiment 4
Choosing ruminant goat, choosing the biomass preparing to utilize is that the stalks of rice, wheat, etc. leaf after straw separating treatment is carbon source, feeds reality
Test animal, gradually decrease concentrated feed and add, feed preparation and co-culture producing and ethanol microorganism, produce for available rumen microorganism metabolism
The yeast of thing acetic acid, more than feeding laboratory animal January.Using negative pressure stomach tube method to take rumen content, anaerobism is rolled
Pipe, purified goat rumen microorganism, cultivate in anaerobic culture box, after cultivating a period of time, culture medium gradually adds product second
Alcohol microbial metabolic products carries out co-culturing direction domestication and cultivates, send into small-sized continuous cultivation and membrane separation device to carry out cud micro-
Biomagnification is cultivated, the rumen microorganism Anaeromyces multicenter thalline of purification, and single flagellum zoospore, rhizoid strengthened by silk
A.elegans, A.mucronatus.Therein one or more.Rumen microorganism is put into medium-sized according to biomass weight 3 ~ 5%
Continuous flow culture and membrane separation device carry out rumen microorganism amplification culture, the introducing of exogenous producing and ethanol bacterium, select permissible
Utilize acetic acid mutant yeast bacterium, the mutant yeast bacterium that acetic acid utilizes, gradually add rumen microorganism in the amplification culture later stage and cultivate
Metabolite carries out co-culturing direction to rumen microorganism condition of culture and tames, mainly cultivation temperature 39 DEG C, and mild alkaline conditions is low
Oxidation-reduction potential.The mutant yeast bacterium utilized by acetic acid after domestication is trained continuously according to 6% addition cud anaerobism of Biomass
Supporting in system and co-culture, culture medium comprises the required trace element of rumen microorganism growth, nitrogen source element, after starting,
Growth course nitrogen source, with the protein in straw straw, the protein that cell ablation produces, for main nitrogen, is trained by continuous
Support system, it is ensured that metabolite product ethanol etc. is separated by membrane separation device, does not produce antagonism, different time sections gather into
Row is observed, and Rumen Fungi makes straw float to tank body top, by tank body mechanism along with the thread rhizoid growing generation on straw
Outside drain tank, rumen microorganism spore and be not fermented straw and stay tank body lower part, it is continuously added into solid-to-liquid ratio constant straw stalks of rice, wheat, etc. leaf
It is 1:10 with culture medium mixture, Biomass and culture medium ratio.Connect in a tank owing to the characteristic of Rumen Fungi achieves
Supervention ferment.Introducing acetic acid utilizes yeast, Rumen Fungi and this yeast-like fungi will belong to collaborative symbiosis, most useful for common training
The realization supported.In Tiny ecosystem, microorganism is in addition to self adaptability, can ensure that elimination suppression is made by continuous film segregation apparatus
With.Co-culture, co-culturing the cycle, need again to throw in producing and ethanol bacterium.The input cycle determines by measuring acetic acid
Concentration raises and determines, existing yeast can't anaerobism breeding, the state of biomass is with to co-culture situation corresponding, with Rumen Fungi
During co-culturing with producing and ethanol fungus, straw stalks of rice, wheat, etc. leaf granule is less at 1 ~ 3 millimeter, owing to symbiosis suppresses other miscellaneous bacterias to produce, raw
Material need not sterilizing, it will substantially ethanol energy consumption is prepared in reduction.Be in operation the employing 3 above fermentation system of set through transport
OK, with alcohol yied as target, fermentation in parallel, the straw fermented on tank body top through 24 hours is got rid of outside tank, so
Rear feed supplement continuous fermentation, three tank bodies start at times, can be introduced a fine variety by other tank bodies when a tank body goes wrong.By jade
The fermentation resultant product of rice straw stalks of rice, wheat, etc. leaf is as biological feedstuff raw material later use.
Embodiment 5
Choosing ruminant goat, choosing the biomass preparing to utilize is that the stalks of rice, wheat, etc. leaf after straw separating treatment is carbon source, feeds reality
Test animal, gradually decrease concentrated feed and add, feed preparation and co-culture product product butyric, for available rumen microorganism metabolite
The product butyric of acetic acid, more than feeding laboratory animal January.Using negative pressure stomach tube method to take rumen content, anaerobism is rolled
Pipe, purified goat rumen microorganism, cultivate in anaerobic culture box, after cultivating a period of time, culture medium gradually adds product fourth
Acid bacteria microorganism metabolite carries out co-culturing direction domestication and cultivates, and sends into small-sized continuous cultivation and membrane separation device carries out cud
Microorganism amplification culture, the rumen microorganism Anaeromyces multicenter thalline of purification, single flagellum zoospore, rhizoid strengthened by silk
A.elegans, A.mucronatus.Therein one or more.Rumen microorganism is put into medium-sized according to biomass weight 3 ~ 5%
Continuous flow culture and membrane separation device carry out rumen microorganism amplification culture, and exogenous product produces the introducing of butyric, select to produce
Butyric, gradually adds rumen microorganism cultivation metabolite in the amplification culture later stage and carries out altogether to rumen microorganism condition of culture
Pedagogic objective is tamed, mainly cultivation temperature 39 DEG C, mild alkaline conditions, low oxidation-reduction potential.By the product butyric after domestication
Co-culturing according in 6% addition cud continuous flow culture system of Biomass, culture medium comprises rumen microorganism and grows institute
Needing trace element, nitrogen source element, after starting, growth course nitrogen source, with the protein in straw straw, cell ablation produces
Protein, for main nitrogen, pass through continuous culture system, it is ensured that metabolite product produces butanoic acid etc. and divided by membrane separation device
From, not producing antagonism, different time sections collection is observed, and Rumen Fungi is along with the thread vacation growing generation on straw
Root makes straw float to tank body top, by tank body mechanism drain tank outside, rumen microorganism spore and be not fermented straw and stay tank
Body bottom, is continuously added into solid-to-liquid ratio constant straw stalks of rice, wheat, etc. leaf and culture medium mixture, Biomass and culture medium ratio are 1:10.Due to
The characteristic of Rumen Fungi achieves continuous fermentation in a tank.Introducing and produce butyric, strain is that kind, tumor are registered in preservation center
Stomach fungus and product butyric will belong to collaborative symbiosis, most useful for the realization co-cultured.In Tiny ecosystem, microorganism is except certainly
Outside body adaptability, can ensure that elimination inhibitory action by continuous film segregation apparatus.Co-culture, co-culturing the cycle, need
Again to throw in product butyric.The input cycle determine be by measure produce butanoic acid concentration raise determine, the state of biomass with
Co-culturing situation corresponding, during co-culturing with Rumen Fungi and product butyric, straw stalks of rice, wheat, etc. leaf granule is less at 1 ~ 3 millimeter, due to
Symbiosis suppresses other miscellaneous bacterias to produce, and biomass need not sterilizing, it will substantially reduces preparation and produces butanoic acid energy consumption.Be in operation employing
3 overlap above fermentation system parallel runnings, to produce butanoic acid productivity as target, and fermentation in parallel, through 24 hours, tank body top is sent out
Straw that ferment completes is got rid of outside tank, then feed supplement continuous fermentation, and three tank bodies start at times, when a tank body goes wrong can
To be introduced a fine variety by other tank bodies.Using the fermentation resultant product of corn straw stalks of rice, wheat, etc. leaf as biological feedstuff raw material later use.
Embodiment 6
Choosing ruminant goat, choosing the biomass preparing to utilize is that the stalks of rice, wheat, etc. leaf after straw separating treatment is carbon source, feeds reality
Testing animal, gradually decrease concentrated feed and add, feed preparation and co-culture product butanol bacterium, butanol bacterium is that preservation center preserves kind, feeds
More than food laboratory animal January.Negative pressure stomach tube method is used to take rumen content, anaerobism rolling pipe, the micro-life of purified goat cud
Thing, cultivates in anaerobic culture box, after cultivating a period of time, gradually adds butanol bacteria microorganism metabolite and carry out in culture medium
Co-culture direction domestication to cultivate, send into small-sized continuous cultivation and membrane separation device carries out rumen microorganism amplification culture, purification
Rumen microorganism Anaeromyces multicenter thalline, single flagellum zoospore, silk strengthens rhizoid A.elegans,
A.mucronatus.Therein one or more.Rumen microorganism is put into medium-sized anaerobism according to biomass weight 3 ~ 5% and trains continuously
Support and membrane separation device carry out rumen microorganism amplification culture, the introducing of exogenous product butanol bacterium, the amplification culture later stage gradually
Add rumen microorganism cultivation metabolite to carry out co-culturing direction domestication to rumen microorganism condition of culture, mainly cultivate temperature
Spend 39 DEG C, mild alkaline conditions, low oxidation-reduction potential.Butanol bacterium after domestication is connected according to 6% addition cud anaerobism of Biomass
Co-culturing in continuous culture systems, culture medium comprises the required trace element of rumen microorganism growth, and nitrogen source element, for starting
After, growth course nitrogen source, with the protein in straw straw, the protein that cell ablation produces, for main nitrogen, by continuously
Culture systems, it is ensured that metabolite product butanol etc. is separated by membrane separation device, does not produce antagonism, different time sections collection
Observing, Rumen Fungi makes straw float to tank body top, by tank body machine along with the thread rhizoid growing generation on straw
Outside structure drain tank, rumen microorganism spore and be not fermented straw and stay tank body lower part, it is continuously added into the solid-to-liquid ratio constant straw stalks of rice, wheat, etc.
Leaf and culture medium mixture, Biomass and culture medium ratio are 1:10.Owing to the characteristic of Rumen Fungi achieves in a tank
Continuous fermentation.Introduce butanol bacterium, Rumen Fungi and this yeast-like fungi and will belong to collaborative symbiosis, most useful for the reality co-cultured
Existing.In Tiny ecosystem, microorganism is in addition to self adaptability, can ensure that elimination inhibitory action by continuous film segregation apparatus.Carry out
Co-culture, co-culturing the cycle, need again to throw in product butanol bacterium.The input cycle determines it is by measuring the rising of the concentration of butanol
Determine, existing butanol bacterium can't anaerobism breeding, the state of biomass is with to co-culture situation corresponding, with Rumen Fungi and butanol bacterium
During co-culturing, straw stalks of rice, wheat, etc. leaf granule is less at 1 ~ 3 millimeter, and owing to symbiosis suppresses other miscellaneous bacterias to produce, biomass need not go out
Bacterium, it will substantially butanol energy consumption is prepared in reduction.Be in operation the employing 3 above fermentation system parallel running of set, with alcohol yied
For target, fermentation in parallel, outside straw eliminating tank ferment on tank body top for 24 hours, then feed supplement is sent out continuously
Ferment, three tank bodies start at times, can be introduced a fine variety by other tank bodies when a tank body goes wrong.By corn straw stalks of rice, wheat, etc. leaf
Fermentation resultant product is as biological feedstuff raw material later use.
Embodiment 7
Choosing ruminant goat, choosing the biomass preparing to utilize is that the stalks of rice, wheat, etc. leaf after straw separating treatment is carbon source, feeds reality
Test animal, gradually decrease concentrated feed and add, feed preparation and co-culture aminoacid bacterium, be more than feeding laboratory animal January.Use negative
Pressure stomach tube method takes rumen content, anaerobism rolling pipe, purified goat rumen microorganism, cultivates, training in anaerobic culture box
After supporting a period of time, culture medium gradually adds producing and ethanol microbial metabolic products and carries out co-culturing direction domestication cultivation, send into
Small-sized continuous cultivation and membrane separation device carry out rumen microorganism amplification culture, and the rumen microorganism Anaeromyces of purification is many
Center thalline, single flagellum zoospore, silk strengthens rhizoid A.elegans, A.mucronatus.Therein one or more.Tumor
Stomach microorganism puts into medium-sized continuous flow culture according to biomass weight 3 ~ 5% and membrane separation device carries out rumen microorganism amplification
Cultivate, exogenous produce produce aminoacid bacterium introducing, the amplification culture later stage gradually add rumen microorganism cultivate metabolite to
Rumen microorganism condition of culture carries out co-culturing direction domestication, mainly cultivation temperature 39 DEG C, mild alkaline conditions, low oxidoreduction
Current potential.The aminoacid bacterium that produces after domestication is co-cultured according in 6% addition cud continuous flow culture system of Biomass,
Culture medium comprises the required trace element of rumen microorganism growth, and nitrogen source element, after starting, growth course nitrogen source, with straw
Protein in straw, the protein that cell ablation produces, for main nitrogen, passes through continuous culture system, it is ensured that metabolite
Product ethanol etc. are separated by membrane separation device, do not produce antagonism, and different time sections collection is observed, Rumen Fungi along with
The thread rhizoid growing generation on straw makes straw float to tank body top, by tank body mechanism drain tank outside, rumen microorganism
Spore and be not fermented straw and stay tank body lower part, is continuously added into solid-to-liquid ratio constant straw stalks of rice, wheat, etc. leaf and culture medium mixture, biological
Amount and culture medium ratio are 1:10.Owing to the characteristic of Rumen Fungi achieves continuous fermentation in a tank.Introduce and produce aminoacid
Bacterium, Rumen Fungi and product aminoacid bacterium will belong to collaborative symbiosis, most useful for the realization co-cultured.Microorganism in Tiny ecosystem
In addition to self adaptability, can ensure that elimination inhibitory action by continuous film segregation apparatus.Co-culture, co-culturing week
Phase, need again to throw in product and produce aminoacid bacterium.The input cycle determines it is by measuring product amino acid whose concentration rising decision, existing
Produce aminoacid bacterium can't anaerobism breeding, the state of biomass is with to co-culture situation corresponding, with Rumen Fungi and product aminoacid bacterium
During co-culturing, straw stalks of rice, wheat, etc. leaf granule is less at 1 ~ 3 millimeter, and owing to symbiosis suppresses other miscellaneous bacterias to produce, biomass need not go out
Bacterium, it will substantially ethanol energy consumption is prepared in reduction.Be in operation the employing 3 above fermentation system parallel running of set, to produce aminoacid
Productivity is target, fermentation in parallel, and the straw fermented on tank body top through 24 hours is got rid of outside tank, and then feed supplement is continuous
Fermentation, three tank bodies start at times, can be introduced a fine variety by other tank bodies when a tank body goes wrong.By corn straw stalks of rice, wheat, etc. leaf
Fermentation resultant product as biological feedstuff raw material later use.
Claims (10)
1. microbial co culture prepares a system for biomass monomer, contains with biomass as substrate, passes through special installation
Process adjustment realizes biomass degradation microorganism and prepares subject monomers with subject monomers generation microbial co culture, and its feature exists
In: use rumen microorganism and target product microbial co culture, the equipment of product can be separated in situ by regulation and control, it is achieved tumor
The symbiosis of stomach microorganism and target product microorganism and the preparation of high efficiency target product.
2. the system preparing biomass monomer according to microbial co culture a kind of described in claim 1 is characterized in that: at cud
Carrying out live body hot housing before microorganism collection, choose ruminant, choosing the biomass preparing to utilize is carbon source, administering transgenic
Animal, gradually decreases concentrated feed and adds, and feeds preparation and co-cultures the microorganism preparing monomer, more than feeding laboratory animal January.
3. according to claim 1, a kind of microbial co culture described in 2 is prepared the system of biomass monomer and be it is characterized in that: use
Negative pressure stomach tube method takes rumen content, anaerobism rolling pipe, purification rumen microorganism, builds small-sized company in anaerobic culture box
Continuous cultivation and membrane separation device carry out Rumen Fungi amplification culture, after cultivating a period of time, gradually add target and produce in culture medium
Thing microbial metabolic products carries out co-culturing direction domestication and cultivates, and the rumen microorganism of purification includes cellulose-decomposing bacterium respectively
There are Bacteroides succinogenes (Fibrobacter succinogenes), cud bacterium (Ruminococcus), fibrinolysin butanoic acid
Vibrio (Buutyrivibrio fibrisolvens), clostridium (Clostridium)；Cellulolytic fungi has Caecomyes
Single centre thalline, single flagellum zoospore, spherical, rhizoid system C.communis of branch, C.equi；Cyllamyces
Multicenter thalline, many flagellums zoospore, spherical, the Sporangium Cy.Aberensis of branch；During Neocallimastix is mono-
Heart thalline, many flagellums zoospore, abundant silk strengthens rhizoid N.frontalis, N.patriciarum,
N.hurleyensis, N.variabilis；Piromyces single centre thalline, single flagellum zoospore thread rhizoid system
P.communis, P.mae P.dumbonicus P.rhizinflatus P.minutus P.spiralis Citronii；
Orpinomyces multicenter thalline, many flagellums zoospore, thread rhizoid system O.joyonii, O.intercalaris,
O.bovis ；Anaeromyces multicenter thalline, single flagellum zoospore, silk strengthens rhizoid A.elegans,
A.mucronatus；Co-culturing the cud biology of middle use is isolated independent a kind of Rumen Fungi or it is micro-with other cuds
Biological combination.
4. the system preparing biomass monomer according to microbial co culture a kind of described in claim 1 is characterized in that: exogenous
The introducing of target product bacterium, selects to utilize the yeast first-selection candida mycoderma of pentose and hexose, Ethanologic bacteria and
Available acetic acid mutant yeast bacterium, a kind of obtain from China Research for Industrial Microbial Germ preservation center can utilize simultaneously glucose and
The ethanol yeast of xylose: numbering 1463 generic name: tropicalis generic name: Candida separates substrates: Chinese: heat
Band candida mycoderma；1771 kinds of names of numbering: tannophilus generic name: Pachysolen separates substrates: Chinese: addicted to
Tan pipe capsule yeast；960 kinds of names of numbering: stipitis generic name: Pichia separates substrates: Chinese: trunk finishes red ferment
Female；Another kind is Ethanologic bacteria；The third be can acetic acid utilize mutant yeast bacterium, gradually add tumor in the amplification culture later stage
Stomach microorganism culturing metabolite carries out co-culturing direction domestication, mainly cultivation temperature 39 to rumen microorganism condition of culture
DEG C, mild alkaline conditions, low oxidation-reduction potential, culture medium comprises the rumen microorganism required trace element of growth, and nitrogen source element is right
After starting, growth course nitrogen source, with the protein in straw straw, the protein that cell ablation produces, for main nitrogen；Mesh
Mark product strain also includes producing butanol bacterium, produces butyric, produces anaerobism and the facultative anaerobes such as aminoacid bacterium.
5. the system preparing biomass monomer according to microbial co culture a kind of described in claim 1 is characterized in that: will domestication
After target product bacterium add in cud continuous flow culture system and co-culture, pass through continuous culture system, it is ensured that cud
Microbial metabolic products is separated by membrane separation device with objective microbe product, does not produce antagonism, it is achieved rumen microorganism
The high efficiency degrading straw of exoenzyme, equipment batch mixing discharge exoenzyme degradation biological matter produce the most not by rumen microorganism
Utilize monosaccharide or double pool for target product microorganism utilize, Rumen Fungi along with the thread rhizoid growing generation on straw
Making biomass float to tank body top, the mechanism's removal system being fermented in equipment, rumen microorganism spore supplements not with new
It is fermented straw and stays tank body lower part, owing to the characteristic of Rumen Fungi achieves continuous fermentation in a tank, symbiosis culture.
6. the system preparing biomass monomer according to microbial co culture a kind of described in claim 1 is characterized in that: in target
Product equal bacterium introducing aspect first-selection yeast, along with the research of cellulosic ethanol for many years, research worker has been tamed multiple permissible
Utilize the strain of xylose and glucose simultaneously, the checking that yeast strong adaptability is the most much studied, can be according to cud
The condition of culture direction domestication of fungus；Secondly existing yeast can't anaerobism be bred, but the Ethanologic bacteria of anaerobism has
Anaerobism fertility, ethanol production is the highest, and also can utilize xylose and glucose simultaneously.
7. the system preparing biomass monomer according to microbial co culture a kind of described in claim 1 is characterized in that: although tumor
Stomach fungus originally also can be with a large amount of competitive rumen bacteria symbiosis in cud, if but introducing acetic acid and utilize yeast, cud
Fungus and this yeast-like fungi will belong to collaborative symbiosis, most useful for the realization co-cultured；Microorganism carries except early stage domestication
Outside self adaptability high, can ensure that elimination inhibitory action by continuous film segregation apparatus；Co-culture, co-culturing week
Phase, rumen microorganism can need again to throw in producing and ethanol bacterium with self-reproduction；The input cycle determines it is the concentration by measuring sugar
Raising and determine, existing yeast can't anaerobism be bred, but the Ethanologic bacteria of anaerobism has anaerobism fertility, and ethanol produces
Measure the highest, and also can utilize xylose and glucose simultaneously.
8. the system preparing biomass monomer according to microbial co culture a kind of described in claim 1 is characterized in that: biomass
State with to co-culture situation corresponding, co-culture with Rumen Fungi and yeast, choose biological particles relatively big, 2 ~ 10 millimeters,
Rumen Fungi has the mycelia entering biomass, and biomass need not consume mass energy and clay into power；Owing to symbiosis suppresses other
Miscellaneous bacteria produces, and biomass need not sterilizing, substantially reduction will prepare ethanol energy consumption equally.
9. the system preparing biomass monomer according to microbial co culture a kind of described in claim 1 is characterized in that: uses 3
Set fermentation system, with target product as target, fermentation in parallel, the straw fermented on tank body top through 24 hours is got rid of
Outside tank, then feed supplement continuous fermentation, three tank bodies start at times, can be drawn by other tank bodies when a tank body goes wrong
Kind.
10. the system preparing biomass monomer according to microbial co culture a kind of described in claim 1 is characterized in that: used
Biomass be various agricultural residue, i.e. corn straw, rice straw, wheat stalk, cotton stalk, waste of flax, mainly
For the stalks of rice, wheat, etc. leaf after corn straw stalks of rice, wheat, etc. leaf is separated with skin, ferment respectively.
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| PCT/CN2017/098071 WO2018033146A1 (en) | 2016-08-18 | 2017-08-18 | System for preparing biomass monomer by means of microbial coculture |
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