CN103031348A - Method for preparing L-lactic acid by utilizing lignocellulose - Google Patents

Method for preparing L-lactic acid by utilizing lignocellulose Download PDF

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CN103031348A
CN103031348A CN 201110295619 CN201110295619A CN103031348A CN 103031348 A CN103031348 A CN 103031348A CN 201110295619 CN201110295619 CN 201110295619 CN 201110295619 A CN201110295619 A CN 201110295619A CN 103031348 A CN103031348 A CN 103031348A
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fermentation
bacterial strain
lactic acid
clostridium
gene
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崔球
刘亚君
洪伟
崔古贞
朱新术
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Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
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Abstract

The invention provides a method for preparing L-lactic acid by utilizing lignocellulose, relates to the technical field of microorganism fermentation, particularly relates to the technical field of producing organic acid through the microorganism fermentation, and particularly relates to a method for preparing the L-lactic acid of degrading the lignocellulose by utilizing a modified anaerobic clostridium bacterial strain through fermentation. With the adoption of the method, the L-lactic acid is produced through the condition that a genetic modified thermophilic anaerobic clostridium bacterial strain is used for a CBP (consolidated bioprocessing) technology and by taking the lignocellulose as a substrate, so that the production cost of the lactic acid is greatly reduced; and the method has good industrial application prospect.

Description

A kind of method of utilizing lignocellulose to prepare Pfansteihl
Technical field
The present invention relates to the microbial fermentation technology field, relate in particular to by microbial fermentation and produce the organic acid technical field, be specifically related to utilize the method for the anaerobism clostridium strains for degrading lignocellulose fermentation production of L-lactic acid that a strain transforms.
Background technology
Pfansteihl (L-lactic acid) is by the D-lactic acid that pyruvic acid is converted under the katalysis of LDH in biological fermentation process.Compare with D-ALPHA-Hydroxypropionic acid, because Pfansteihl can be participated in body metabolism, the good bio-intermiscibility such as without any side effects directly, it is widely used in the fields such as food, medicine, chemical industry.
Lignocellulose is renewable resources the abundantest on the earth, but only has only a few to be used effectively, and the overwhelming majority is used as waste disposal in environment, such as municipal wastes, causes on the contrary environmental pollution.China is large agricultural country, and approximately 1,500,000,000 tons of annual agriculture and forestry organic waste material total amounts are so utilize lignocellulose to have broad application prospects as starting material production different kind organism fuel and Chemicals.Lignocellulose three major polymers: cellulose, hemicellulose and xylogen three parts form, and account for respectively 35-50%, 20-35%, and 10-15%.Because its structure is extremely complicated, causes its degraded very difficult.
Integrate biological processing technology (Consolidated BioProcessing, CBP) utilize the lot of challenges that adopts unit operation and integrated optimization pattern to face in the technology for current lignocellulose, integrate by the reactions steps that the scripts such as the production of cellulase, cellulosic enzymic hydrolysis, pentose fermentation and zymohexose is discrete, greatly simplify production technique and effectively reduced cost, thereby improved the complex art economic target of the biological trans-utilization of Mierocrystalline cellulose.Based on these significant characteristics, CBP is considered to one of biological trans-utilization technological line of the most promising low-cost Mierocrystalline cellulose.
Clostridium thermocellum (Clostridium thermocellum) is to be considered at present be hopeful most one of microbe groups that satisfies the CBP requirement.Clostridium thermocellum can be at high temperature anaerobic condition bottom fermentation, and the cellulosome of its generation is present the most efficient crystalline cellulose hydrolyzation system.Clostridium thermocellum can be monose with cellulose conversion directly not only, and will to be hydrolyzed the cell-oligosaccharide that produces and glucose fermentation be the high value added product that lactic acid etc. is widely used in fields such as industry, grocery trades.Therefore, on the prospect of industrial applications, Clostridium thermocellum has more significant advantage than the non-cellulose degradation bacteria such as yeast, intestinal bacteria and zymomonas mobilis of using at present.Yet the lactic acid-producing ability of wild-type Clostridium thermocellum is lower, far can not reach industrialization production requirements.
Present method is to be based upon on the basis of the Clostridium thermocellum bacterial strain CT-ak/dh that a strain obtains by genetic modification.Keep wild strain can the cellulosic characteristics prerequisite of fast degradation under, this transformation bacterial strain has cut off the synthetic approach of its acetic acid when having strengthened the lactic acid route of synthesis, thereby will come from glucolytic most of metabolism conductance to lactic acid fermented direction, so that utilize the lignocellulose fermenting lactic acid to become possibility.Present method is utilized this transformation bacterial strain to set up and has been optimized the lignocellulose fermentation and prepares the method for Pfansteihl, thereby greatly reduces the lactic acid-producing cost, has good prospects for commercial application.
Summary of the invention
The present invention includes the anaerobism clostridium bacterial strain CT-ak/dh that sets up a strain genetic modification, and produce Pfansteihl two portions content with this strain fermentation degraded pretreated straw (lignocellulose).Technical problem to be solved can realize by following scheme:
1. the genetic modification of Clostridium thermocellum
The initial strain that the present invention uses is Clostridium thermocellum.
For knocking out goal gene, the plasmid of structure comprises that pHK01 (knocks out the plasmid skeleton, contains the replication orgin pno of the colibacillary replication orgin ColE1 of following original paper: a and Clostridium thermocellum; B Clostridium thermocellum identification copy in fact initial albumen repB; C paraxin (chloramphenicol) resistant gene cat; D macrolide antibiotics (macrolide-lincosamide-streptograminB) resistant gene mls), and make up on the pHK01 basis knock out plasmid pHK02, be used for knocking out the gene C the_1028 (E.C. 2.7.2.1) on the Clostridium thermocellum genome.Be expression alien gene, the encoding gene PpFbFP that at first adds the anaerobism fluorescin on the basis of pIMP1 or pHK01, the plasmid that makes up is defined as plasmid pEM01 or pEK01, adds gene order to be expressed, construction expression plasmid pEM02 or pEK02 on the two basis afterwards.
As shown in Figure 1, thus above-mentioned plasmid transformed to import to by electricity obtains corresponding purpose bacterial strain in the corresponding host strain.Gene knockout method among the present invention is based on that the principle of homologous recombination carries out, namely according to the nucleotide sequence of goal gene both sides on the Clostridium thermocellum genome, design respectively approximately the 1kb homology arm sequence of looking, and be structured on the corresponding knockout carrier, exchange restructuring by homology arm, so that the goal gene on the genome is replaced on the plasmid, screen accordingly the purpose that step reaches gene knockout thereby pass through again.The expression of foreign gene upward increases promotor and transcription terminator original paper by pEM02 or pEK02 to be controlled, and selected promotor sees Table 1.
Table 1. promotor kind of the present invention
Figure BSA00000585848200021
By a series of genetic modification, realized the knocking out of E.C. 2.7.2.1 encoding gene in the Clostridium thermocellum genome, and LDH (ldh) has been carried out heterogenous expression, thereby obtained bacterial strain CT-ak/ldh.The foreign gene source of selecting sees Table 2.
Carry out the foreign gene source of heterogenous expression among table 2. the present invention
Figure BSA00000585848200031
The mentioned gene that is used for heterogenous expression of the present invention also comprises the genes involved of listing through the table 2 of sequence modification transformation.
The culture medium prescription of transforming the bacterial strain screening use is: KH 2PO 41.5g, K 2HPO 42.9g, Urea 2.1g, MgCl 26H 2O1.0g, CaCl 22H 2O 150mg, FeSO 46H 2O 1.25mg, Cysteine hydrochloride 1.0g, Resazurin 2.0g, Cellobiose 5.0g, Yeast extract 6.0g, Morpholinopropane sulfonic acid (MOPS) 10.0g, Sodiumcitrate2H 2O 3.0g adds distilled water and is settled to 1L.Require to add thiamphenicol (Tm) 20mg or erythromycin (Ery) 20mg according to screening.
2. the pre-treatment of lignocellulose (stalk)
With behind the straw chopping and join and carry out attrition crushing in the mill, and add simultaneously the sodium hydroxide that concentration is 6%-8% (NaOH) solution, product insulation (60-150 ℃) 1 to 4 hour, processing is cooled to room temperature with the stalk fragment after finishing, and water flushing removes remaining alkali, to aqueous solution pH be 7.5-8.0.Then straw filtering is dewatered, dries and sterilize for subsequent usely, namely can be used as the carbon source substrate that Clostridium thermocellum (Clostridium thermocellum) is produced lactic acid.
3. Clostridium thermocellum high density fermentation
Clostridium thermocellum high density fermentation culture medium prescription:
KH 2PO 41.0-1.5g/L, K 2HPO 44.0-10.0g/L, nitrogenous source 3.0-7.0g/L, MgCl 26H 2O 1.0-5.0g/L, CaCl 22H 2O 50-250mg/L, FeSO 46H 2O 0.5-2.5mg/L, cysteine hydrochloride 1.0-5.0g/L, resazurin 1.0-10.0mg/L, carbon source 10.0-40.0g/L, morpholinopropane sulfonic acid (MOPS) 5.0-20.0g/L, yeast extract 5.0-20g/L, sodium citrate2H 2O 0.2-1.5g/L prepares fermention medium, and sterilization.Wherein, carbon source is cellulosic materials biomass (including but not limited to stalks, wooden chrysanthemum), and nitrogenous source is organic or inorganic nitrogenous source (including but not limited to urea, yeast extract, peptone, corn steep liquor, SODIUMNITRATE, Sodium Glutamate); Regulating pH before the sterilization is 7.0-8.5; If need to enlarge concentration of substrate, then must keep carbon-nitrogen ratio is 2: 1-10: 1.
The fermentation condition of Clostridium thermocellum is:
Temperature 50-65 ℃, anaerobism, tank pressure is 0.05-0.5MPa, mixing speed is 30-100rpm.Should control pH during high concentration of substrate (carbon source concentration is greater than 15g/L) fermentation constant in about 7.0-7.5, fermentation time is 24-48h.
4. the separation of Clostridium thermocellum fermented liquid and lactic acid content are measured
After the Clostridium thermocellum fermentation ends, fermented liquid separates with whizzer, discards solid collection liquid, utilizes lactic acid content in the nmr for the determination supernatant liquor.
5. according to above step, producing lactic acid by lignocellulose is that those skilled in the art can realize substantially.Based on method described in the invention, the direct conversion cellulosic materials biomass that can further develop are the technique of other products such as glucose, ethanol, acetic acid, have realized that cellulosic degraded and biological product transform the CBP circuit of accumulation.
Description of drawings
Fig. 1, plasmid and strain construction schematic diagram.
Embodiment
The present invention is further detailed explanation below in conjunction with specific examples.
Embodiment 1: Clostridium thermocellum is transformed the structure of bacterial strain CT-ak/ldh
1. make up pHK01.The cloning system that utilization does not rely on restriction enzyme site adds following original paper at plasmid, finally obtains the skeleton that the pHK01 conduct knocks out plasmid:
1) the replication orgin pno of colibacillary replication orgin ColE1 and Clostridium thermocellum
2) Clostridium thermocellum identification copies in fact initial albumen repB
3) paraxin (chloramphenicol) resistant gene catR
4) d macrolide antibiotics (macrolide-lincosamide-streptogramin B) resistant gene mls R)
2. make up on the basis of pHK01 and knock out plasmid pHK02.
Primers according to gene C the_1028 (ak) both sides on the Clostridium thermocellum genome, obtain respectively 5 ' end and 3 ' and hold approximately the 1kb homology arm sequence (arm) of looking, and utilization does not rely on the cloning system of restriction enzyme site, both sides arm is building up to respectively the both sides of the upper cat gene of pHK01, thereby obtains pHK02.
3. construction expression plasmid pEK01 or pEM01 on the basis of pHK01 or pIMP1.
Sequence (table 2) design primer according to goal gene PpFbFP, and add promoter region (table 1) at its 5 ' end respectively, add corresponding Transcription Termination subarea at 3 ' end, the cloning system that the PCR product utilization that obtains is not relied on restriction enzyme site is inserted on pHK01 or the pIMP1, finally obtains expression plasmid pEK01 or pEM01.
4. construction expression plasmid pEK02 or pEM02 on the basis of pEK01 or pEM01.
Sequence (table 2) design primer according to goal gene ldh, and after its 3 ' end terminator codon, add the RBS district respectively, the cloning system that the PCR product utilization that obtains is not relied on restriction enzyme site is inserted into the place ahead of the sequence of expression plasmid pEK01 or the upper PpFbFP of pEM01, the rear of promoter region finally obtains expression plasmid pEK02 or pEM02.
5. the culture medium prescription of Select to use is: KH 2PO 41.5g, K 2HPO 42.9g, Urea 2.1g, MgCl 26H 2O 1.0g, CaCl 22H 2O 150mg, FeSO 46H 2O 1.25mg, Cysteine hydrochloride 1.0g, Resazurin 2.0g, Cellobiose5.0g, Yeast extract 6.0g, Morpholinopropane sulfonic acid (MOPS) 10.0g, Sodium citrate 2H 2O3.0g adds distilled water and is settled to 1L.Require to add thiamphenicol (Tm) 20mg and erythromycin (Ery) 20mg. according to screening
6. the acquisition of gene knockout mutant strain.
1) gene Knockout involved in the present invention all carries out according to the homologous recombination principle.That is, the exchange restructuring by homology arm on corresponding knockout carrier and the genome so that the goal gene on the genome and the mutator gene on the plasmid replace, and operate by screening accordingly, finally obtains the purpose mutant strain.
2) acquisition of mutant strain CT-ak.
A) carry out electricity according to the method for patent 201110048349.9 and transform, plasmid pHK02 is imported in the host C.thermocellum cell, and utilize the Screening of Media positive transformant that adds thiamphenicol and erythromycin (Tm+Ery+).
B) transformant is transferred in the liquid nutrient medium of Tm+Ery-, and continuous passage is to lose plasmid.
C) again be transferred on the solid medium of Tm+Ery-, obtain bacterium colony, be inoculated into respectively in the liquid nutrient medium of Tm+Ery-and Tm+Ery+, in front a kind of substratum growth, the bacterium colony that rear a kind of substratum is not grown is mutant strain CT-ak.
D) by the ak gene in the pcr amplification mutant strain, further determine whether successfully to undergo mutation.
7. the acquisition of heterogenous expression bacterial strain.
1) gene heterogenous expression technology involved in the present invention all is foreign gene to be placed under the control of the promoter region that table 2 lists carry out.
2) acquisition of transformation bacterial strain CT-ak/ldh
A) carry out electricity according to the method for patent 201110048349.9 and transform, expression plasmid pEK02 or pEM02 are imported in the host CT-ak cell, and utilize the Screening of Media positive transformant of Tm+Ery+.
B) by ldh gene and PpFbFP gene in the pcr amplification bacterial strain, determine whether successfully to change over to foreign gene.
C) whether producing fluorescence by detect transforming bacterial strain, further determining whether successful expression of the ldh gene of external source and PpFbFP gene, finally obtain to transform bacterial strain CT-ak/ldh.
8. measure bacterial strain CT-ak/ldh and compare with wild strain, the LDH enzyme of its heterogenous expression is lived.
9. utilize nuclear magnetic resonance technique or LC-MS technology, measure the phenotype variation (acetic acid is synthetic, and ethanol is synthetic, and Pfansteihl is synthetic) that bacterial strain CT-ak/ldh compares with wild strain.
10. transform bacterial strain CT-ak/ldh acetic acid and ethanol production and be lower than detection line, Pfansteihl output reaches 3%.
Embodiment 2: the stalk Pretreatment Test
1. stalk is chopped into the 3-5cm fragment in advance, then puts into mill and push pulverizing, and the sodium hydroxide that adds simultaneously 6-8% in the machine carries out alkaline purification, at last with stalk fragment fibrillation.
2. the taking-up stalk powder is treated the temperature cool to room temperature, is filtered dry a pot suction filtration with crossing, and removes remaining alkali.Clean with large water gaging behind the suction filtration, abandon filtrate, until filtrate pH is 7.5-8.0.
3. with crossing the straw powder be filtered dry after pot suction filtration is processed, and 80 ℃ dry to constant weight, and detect its content of lignin.
4. the stalk after the method is processed, lignin removing rate can reach 80%.
5. the straw powder autoclave sterilization that takes by weighing after the processing is stand-by.
Embodiment 3:5L fermentor tank high density fermentation Clostridium thermocellum
1. by prescription " KH 2PO 41.0-1.5g/L, K 2HPO 44.0-10.0g/L, nitrogenous source 3.0-7.0g/L, MgCl 26H 2O 1.0-5.0g/L, CaCl 22H 2O 50-250mg/L, FeSO 46H 2O 0.5-2.5mg/L, cysteine hydrochloride 1.0-5.0g/L, resazurin 1.0-10.0mg/L, carbon source (pretreated straw or Mierocrystalline cellulose) 10.0-40.0g/L, morpholinopropane sulfonicacid (MOPS) 5.0-20.0g/L, yeast extract 5.0-20g/L, sodium citrate2H 2O 0.2-1.5g/L, thiamphenicol (Tm) 20mg/L " preparation fermention medium (adding after calcium, magnesium, the mother liquid of iron salt sterilization), 115 ℃, the 15min autoclave sterilization.
2. after sterilization finishes, constantly in fermentor tank, pass into nitrogen, to remove the oxygen in substratum and the fermentor tank.After substratum became the color of himself again by light blue or pink (color of resazurin), regulating medium pH constant was 7.0, and 55 ℃ of tank temperature access volume required CT-ak/ldh primary seed solution simultaneously, stopped ventilation, and 80rpm stirs fermentation.
Approximately behind the 48h carbon source all consumed, cell concentration reaches 12.0 (OD in the fermented liquid 600).
4. precipitate by centrifugal removal at the cultivation terminal point, collect fermented liquid, and measure the lactic acid content in the fermented liquid, its lactic acid production can reach 5%.
More than be sub to further describing that the present invention does in conjunction with specific embodiments.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in above-described embodiment and the specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.

Claims (13)

1. method of utilizing lignocellulose to prepare Pfansteihl, described method comprises: the genetic modification method of thermophilic anaerobic clostridium and fermentation optimization cultural method.
2. according to the method for claim 1, it is characterized in that described thermophilic anaerobic clostridium is the clostridium (Clostridium) that can utilize the cellulose raw lactic acid producing, comprise the derivative bacterial strain of Clostridium thermocellum (Clostridium thermocellum).
3. according to the method for claim 1-2, it is characterized in that described genetic modification method comprises the method for electric conversion, gene knockout and gene heterogenous expression.
4. according to the method for claim 3, the method that its described electric method that transforms is based on patent 201110048349.9 descriptions is carried out.
5. according to the method for claim 3, the method for its described gene knockout is based on that the principle of homologous recombination carries out.
6. according to the method for claim 1-5, the method for its described gene knockout is by making up pHK series plasmid, and is transformed into and carries out in the corresponding host cell.
7. according to the method for claim 6, its described pHK series plasmid comprises pHK01 and pHK02, and according to construction process described in the invention other plasmids take pHK01 and pHK02 as fundamental construction.
8. according to the method for claim 1-5, the method for its described genetic expression is by making up pEK series plasmid and pEM series plasmid, and is transformed into and carries out among the corresponding host.
9. according to the method for claim 8, its described pEK series plasmid comprises pEK01 and pEK02, and according to construction process described in the invention other plasmids take pEK01 and pEK02 as fundamental construction.
10. bacterial strain CT-ak/ldh that can utilize lignocellulose to prepare Pfansteihl is characterized in that any one method according to claim 1-9 makes up.
11. carry out the application of genetic modification according to the transformation bacterial strain of claim 10, it is characterized in that described method comprises:
A) selected Cthe_1028 (ak) is as knocking out object
B) selected ldh and PpFbFP are as the object of heterogenous expression
C) make up knockout carrier and expression vector
D) transform to wild strain importing knockout carrier by electricity, obtain mutant strain
E) transform to mutant strain importing expression vector by electricity, obtain to transform bacterial strain CT-ak/ldh.
12. the transformation bacterial strain according to claim 10-11 carries out fermentation culture, it is characterized in that described method comprises:
A) used high density fermentation culture medium prescription is " KH 2PO 41.0-1.5g/L, K 2HPO 44.0-10.0g/L, nitrogenous source 3.0-7.0g/L, MgCl 26H 2O 1.0-5.0g/L, CaCl 22H 2O 50-250mg/L, FeSO 46H 2O 0.5-2.5mg/L, cysteine hydrochloride 1.0-5.0g/L, resazurin 1.0-10.0mg/L, carbon source 10.0-40.0g/L, morpholinopropane sulfonic acid (MOPS) 5.0-20.0g/L, yeast extract 5.0-20g/L, sodiumcitrate2H 2O 0.2-1.5g/L ".Regulating pH before the sterilization is 7.0-8.5.
B) used carbon source is the cellulosic materials biomass, including but not limited to stalk, and wooden chrysanthemum.
C) used nitrogenous source is the organic or inorganic nitrogenous source, including but not limited to urea, and yeast extract, peptone, corn steep liquor, SODIUMNITRATE, Sodium Glutamate.
D) used fermentation condition is " temperature 50-65 ℃, anaerobism, tank pressure is 0.05-0.5MPa, mixing speed is 30-100rpm, pH is constant to be about 7.0-7.5, fermentation time is 24-48h ".
13. method according to claim 12 is characterized in that the mode batch fermentation of described fermentation, feed supplement-batch fermentation continuously ferments or semicontinuous fermentation.
CN 201110295619 2011-10-08 2011-10-08 Method for preparing L-lactic acid by utilizing lignocellulose Pending CN103031348A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106554961A (en) * 2015-09-25 2017-04-05 中国科学院青岛生物能源与过程研究所 A kind of anaerobism clostridium xylan inducible promoter and its application
CN106554960A (en) * 2015-09-25 2017-04-05 中国科学院青岛生物能源与过程研究所 A kind of anaerobism clostridium composing type high efficient expression starter and its application
CN108118020A (en) * 2018-01-16 2018-06-05 中国科学院青岛生物能源与过程研究所 Culture medium, preparation and its application of cellulose degradation microorganism

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106554961A (en) * 2015-09-25 2017-04-05 中国科学院青岛生物能源与过程研究所 A kind of anaerobism clostridium xylan inducible promoter and its application
CN106554960A (en) * 2015-09-25 2017-04-05 中国科学院青岛生物能源与过程研究所 A kind of anaerobism clostridium composing type high efficient expression starter and its application
CN106554960B (en) * 2015-09-25 2020-04-24 中国科学院青岛生物能源与过程研究所 Constitutive high-efficiency expression promoter of anaerobic clostridium and application thereof
CN106554961B (en) * 2015-09-25 2020-04-24 中国科学院青岛生物能源与过程研究所 Anaerobic clostridium xylan inducible promoter and application thereof
CN108118020A (en) * 2018-01-16 2018-06-05 中国科学院青岛生物能源与过程研究所 Culture medium, preparation and its application of cellulose degradation microorganism
CN108118020B (en) * 2018-01-16 2020-06-12 中国科学院青岛生物能源与过程研究所 Culture medium of cellulose degradation microorganism, preparation and application thereof

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Application publication date: 20130410