CN104263680A - Thermoanaerobacter ethanolicus and method for producing ethanol by using same - Google Patents

Thermoanaerobacter ethanolicus and method for producing ethanol by using same Download PDF

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CN104263680A
CN104263680A CN201410453513.8A CN201410453513A CN104263680A CN 104263680 A CN104263680 A CN 104263680A CN 201410453513 A CN201410453513 A CN 201410453513A CN 104263680 A CN104263680 A CN 104263680A
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ethanol
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fermentation
thermophilic anaerobic
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CN104263680B (en
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李爽
赖志城
王菊芳
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South China University of Technology SCUT
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/205—Bacterial isolates
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Abstract

The invention relates to the field of microbial strains, particularly a Thermoanaerobacter ethanolicus and a method for producing ethanol by using the same. The strain is Thermoanaerobacterium aotearoense P8G3#4, and is collected by China General Microbiological Culture Collection Center; and the collection number is CGMCC No.9000, and the collection date is April 3rd, 2014. The method for producing ethanol by using the strain comprises the following steps: preparing a Thermoanaerobacter ethanolicus CGMCC 9000 seed solution, inoculating the Thermoanaerobacter ethanolicus CGMCC 9000 into a fermentation culture medium at the inoculum size of 10-15%w/w, stirring and culturing under anaerobic conditions, and finally, separating ethanol from the fermentation liquid. The strain has the advantages of favorable hereditary stability and stable yield character, shortens the fermentation lag period to 1/4 of the original fermentation lag period, and saves the energy consumption.

Description

A kind of thermophilic anaerobic bacillus and utilize it to produce the method for ethanol
Technical field
Produce the bacterial strain of ethanol and an acclimation method for its high concentration sugar tolerance raising, and utilize this bacterial strain to produce the method for ethanol.To the present invention relates to glucose, wood sugar as main raw material, utilize the method for thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoense SCUT27/ Δ ldh) mutant strain fermentative production of ethanol.
Background technology
Ethanol is a kind of Novel clean-fuel, is the important directions that renewable energy source develops.Development bio-ethanol is trend of the times.S-generation bio-ethanol utilizes vegetable fibre to be produced by biological fermentation mode through pre-treatment, mineral acid or cellulase hydrolysis again.And Hydrogen Energy is because of features such as it are clean, energy density is high, preparation method is various, attract the concern of investigators.
High cost is with the major obstacle that cellulosic class material produces ethanol for fermenting raw materials.Adopt thermophilc anaerobe (Thermoanaerobacterium) fermentation can solve this difficulty preferably.With it for fermentation strain production of chemicals has the advantage (O-Thong of following several respects, S.et al., International Journal of Hydrogen Energy, 2008.33 (4): 1204-1214): (1) reduces with the cellulase addition in cellulosic class fermenting raw materials process.This is because namely such bacterial strain itself is a class hemicellulose decompose microorganism, it at high temperature hydrolyzed xylan fermentation can produce multi-chemical, thus decreases the demand of cellulase.(2) high temperature continuously ferments production process, is conducive to the separation and purification of ethanol.(3) ethanol can be produced by raw material fermentation.Due in industrial scale fermentation production ethanol process, pure-blood ferment is higher for the sterility requirements of equipment, therefore significantly adds Financial cost.But adopt thermophile bacteria fermentative production, at high temperature produce because most normal temperature microorganism is more difficult, therefore not high to the sterility requirements of fermenting process, thus reduce further production cost.(4) high temperature organic waste water can be utilized for fermentation raw material, this reduces production cost on the one hand, also mitigates environmental stress on the other hand.
A strain thermophilic anaerobic bacillus (Li is screened in applicant's early-stage Study, S., et al., Bioresource Technology, 2010,101 (22): 8718-8724, Cai, Y., et al., Enzyme and Microbial Technology, 2011.48 (2): p.155-161), this bacterial strain can effectively utilize the fermenting substrate such as glucose, wood sugar to accumulate ethanol, hydrogen etc.By carrying out genetic modification to it, knock out lactate dehydrogenase gene, after pathways metabolism transformation, mutant H 2improve 2.7 and 2.4 times respectively with ethanol production.But this bacterial strain is in high concentration sugar substrate situation, be subject to the impact of high sugared osmotic stress, cause the growthing lag phase long, cause fermentation period excessively of a specified duration, production cost increases considerably.And high concentration sugar substrate tolerance be realize cellulose series biomass simultaneous saccharification and fermentation produce ethanol necessary.
From industrialization angle, if the thermophilic anaerobic bacillus strain of the resistance to high sugar of a strain can be screened, improve thermophilic anaerobic bacillus sugar concentration of substrate tolerance, and proterties is stablized, can be applicable to high concentration sugar fermenting substrate, shorten the fermentation lag phase, reduce technique and amplify cost, so just can be more suitable for suitability for industrialized production.
Summary of the invention
The object of the present invention is to provide a kind of produce ethanol bacterial strain and utilize this bacterial strain to produce the method for ethanol.
Starting strain thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoenseSCUT27/ Δ ldh) P8G0 (Erythromycinresistant) is cultivated according to the method progressively improving sugared concentration of substrate, repeat number generation, until obtain at this concentration can inoculation 6h after thalline OD 600grow to 0.6 and also proterties stablize.Getting 1mL bacterium liquid is added in isolation medium, 2-3 days is cultivated in anaerobic culture box, picking list bacterium colony, after cultivating 240h in the fermentation medium, ethanol final concentration reaches 28.11g/L, transformation efficiency reaches 1.22mol/mol, and finally obtaining can normal growth, the mutant strain P8G3#4 (Erythromycinresistant) that goes down to posterity under high concentration substrate.
Said mutation strain is long white clouds thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoense) P8G3#4, in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (being called for short CGMCC), deposit number is CGMCC NO.9000, preservation date is on April 3rd, 2014, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Described P8G3#4 bacterial strain is: gram-positive microorganism, elongated rod shape, and diameter is about 0.6-1.0 μm, is about 2-15 μm, basis of microscopic observation its can tumbling motion.For strictly anaerobic bacterium, optimum growth temperature is 55 DEG C, pH 6.3, can utilize multiple sugared substrate growth, as glucose, wood sugar, seminose, fructose, semi-lactosi, pectinose, trehalose, xylan, dextran etc.The hereditary property of this bacterial strain is: lactic acid metabolism Blocked-mutant, has Erythromycinresistant, and Erythromycinresistant concentration is 20-100ng/ μ l.Can tolerate 120-150g/L sugar concentration of substrate, in cell, UDP-Gal (UDP-α-D-semi-lactosi) and 1-(5-phosphoric acid-D-ribose)-5-amino-4-imidazolyl carboxylic acid (CAIR) accumulates increase; The down-regulated expression of the mtGDP in glyceryl phosphatide pathways metabolism.
Bacterial strain described in claim 1 is utilized to produce the method for ethanol: the seed liquor first preparing thermophilic anaerobic bacillus CGMCC9000, then thermophilic anaerobic bacillus CGMCC9000 seed liquor is transferred in fermention medium with 10-15%w/w inoculum size, under anaerobic stir culture, culture temperature 45-60 DEG C, incubation time is 50-250 hour, finally goes out ethanol from separation of fermentative broth.
The total sugar concentration of described fermention medium is 80-150g/L.
The initial pH of described fermention medium is 5.5-6.5, and described mixing speed is 120-200rpm.
The initial pH of described fermention medium is 6.3.
Described fermentation medium components is: glucose 80-100, wood sugar 40-60, urea 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/L.
Described fermention medium also comprises following composition: citric acid tri potassium salt 1-4, Citric acid monohydrate Food grade 0.5-3, sodium sulfate 0.5-3, potassium primary phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate 0.5-3, four water iron protochloride 0.05-0.5, Calcium dichloride dihydrate 0.05-0.4, a water halfcystine hydrochloric acid 0.5-3, two hydrochloric acid Pyridoxylamine 0-0.1, para-amino benzoic acid 0-0.01, Bio (D-biotin) 0-0.01, vitamin B12 0-0.01, vitaminB10-0.01, unit g/L.
The preparation of described seed liquor: thermophilic anaerobic bacillus CGMCC9000 is placed in seed culture medium, at 45-60 DEG C, rotating speed is 100-250rpm, cultivates 8-24h.
Described seed culture medium is: glucose 2-4, wood sugar 2-4, and other component is identical with fermention medium.Be mainly urea 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/L.Preferably, also comprise following composition: citric acid tri potassium salt 1-4, Citric acid monohydrate Food grade 0.5-3, sodium sulfate 0.5-3, potassium primary phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate 0.5-3, four water iron protochloride 0.05-0.5, Calcium dichloride dihydrate 0.05-0.4, one water halfcystine hydrochloric acid 0.5-3, two hydrochloric acid Pyridoxylamine 0-0.1, para-amino benzoic acid 0-0.01, Bio (D-biotin) 0-0.01, vitamin B12 0-0.01, vitaminB10-0.01, unit g/L.
Compared with prior art, the present invention has following beneficial effect:
(1) with this laboratory screening, build the thermophilic anaerobic bacillus P8G0 that obtains for parental plant, carry out high sugared substrate tolerance domestication according to a conventional method, obtain mutant strain P8G3#4.This mutant strain has following hereditary property: LDH defective type, Erythromycinresistant, sugar concentration of substrate tolerance range is more than 120-150g/L, and in cell, UDP-Gal (UDP-α-D-semi-lactosi) and 1-(5-phosphoric acid-D-ribose)-5-amino-4-imidazolyl carboxylic acid (CAIR) accumulates increase; The down-regulated expression of the mtGDP in glyceryl phosphatide pathways metabolism.
(2) produce ethanol at 5L ferment tank with 120g/L glucose, wood sugar mixing sugar for substrate, the lag phase foreshortens to original about 1/4, and ethanol final concentration reaches 28g/L, and transformation efficiency reaches 1.2mol/mol.The invention provides the mutant of screening, can utilize high sugared substrate quick fermentation accumulation target product ethanol, and the by product produced is few, technique is amplified comparatively easy, is suitable for suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is the growth curve chart of the resistance to height sugared bacterial strain P8G3#4 and starting strain P8G0 after domestication.A) growing state of P8G0 under different carbon source concentration ,-■-30g/L ,-●-81g/L ,-▲-120g/L; B) growing state of P8G3#4 under different carbon source concentration ,--30g/L ,-zero-81g/L ,-△-120g/L; Wherein sugared substrate is glucose and xylose (2:1, g:g).
Fig. 2 is the cellular metabolism access function significance enrichment schematic diagram of P8G3#4 and P8G0.
A) abc transport protein system, right figure is lactose/L-arabinose translocator in oligose and polyvalent alcohol movement system, and left figure is the branched-chain amino acid translocator in phosphoric acid salt and amino acid transporter system; B) two component regulator control system (Two-component system), GlnL regulator control system; C) the Lac family in phosphotransferase system (PTS), right figure is the transhipment of lactose, and left figure is the transhipment of cellobiose; D) DNA replication dna related elements.The metabolite of grey box indicating accumulation.
Embodiment
Embodiment 1
The domestication of bacterial strain be separated
The sugared thermophilic anaerobic bacillus strain of resistance to height of the present invention can obtain like this:
In the present invention, the starting strain for screening be laboratory screening, sudden change and preserve thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoense SCUT27/ Δ ldh), be designated as P8G0 herein.
Screening culture medium: glucose: 80-100gL -1; Wood sugar: 40-60gL -1; (NH 4) 2sO 4: 1-5gL -1; MgCl 26H 2o:1-5gL -1; KH 2pO 4: 0.5-5gL -1; K 2hPO 4: 0.5-5gL -1; CaCl2H 2o:0.1-2.0gL -1; Na-β-glycerophosphate:2-10gL -1; FeSO 47H 2o:0.0001-0.001gL -1; Yeast extract: 2-10gL -1; One water halfcystine hydrochloric acid (C 3h 7nO 2sHClH 2o): 0.1-2.0gL -1; Resazurin: 0.001-0.01gL -1; Agar powder: 5-20gL -1; Erythromycin: 20-100ng/ μ l.
First starting strain P8G0 is through 81g/L screening culture medium (glucose and xylose mass ratio is 2:1) anaerobism shake-flask culture, treats strain growth light absorption value OD 600be 2.0 or more, the anaerobism Shake flask medium of transferring new, repeat number generation, until obtain at this concentration can after inoculation 6h thalline OD 600grow to 0.6, and the bacterial strain P8G1 that proterties is stable;
The same with step (1) method, under 102g/L glucose and xylose (2:1, g:g) condition, obtain bacterial strain P8G2;
The same with step (1) method, under 120g/L glucose and xylose (2:1, g:g) condition, obtain bacterial strain P8G3;
By slat chain conveyor, obtain single bacterium colony P8G3#4.
Embodiment 2
Identification of strains after P8G3#4 domestication
In order to identify the difference of bacterial strain before and after domestication, adopt transcription group methods analyst P8G0 and P8G3#4 metabolic gene difference, and cluster analysis is carried out to differential gene, qualification causes the reason in the genetic expression aspect of P8G0 and P8G3#4 two strain growth Difference of Metabolisms.
Starting strain thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoenseSCUT27/ Δ ldh) P8G0 and screening are obtained at the mutant strain P8G3#4 of normal growth under high concentration substrate, to carry out transcript profile sequencing analysis.Analyze the difference expression gene in two samples, screening criteria is | logFC|>=1 (i.e. twice difference), analyze the difference expression gene in two samples, P8G0 and P8G3#4 has 69 genes and there is significant difference on gene expression abundance, compared with starting strain P8G0, have respectively 35 and 24 genes bacterial strain logarithmic growth early stage there is up-regulated and downward, P8G3#4 on transcript profile with the P8G0 property of there are differences.
Analyze (Fig. 2) by KEGG metabolic pathway, have in the middle of 27 gene annotations to 20 signal paths, wherein annotation is to purine metabolism approach (Purine metabolism), abc transport protein system (ABC transporters), galactose metabolism approach (Galactose metabolism), aminosugar and nucleotide sugar pathways metabolism (Amino sugar and nucleotide sugar metabolism), nitrogen metabolic path-way (Nitrogen metabolism), phosphotransferase system (Phosphotransferase system), the degradation pathway (Chloroalkane and chloroalkene degradation) of enpara and chloro-alkenes, L-Ala, aspartic acid and glutamic acid metabolism approach (Alanine, aspartate and glutamate metabolism) and the gene of glyceryl phosphatide pathways metabolism (Glycerophospholipid metabolism) more, be respectively 5, 5, 4, 4, 4, 4, 3, article 3 and 2.
Following discovery is had: (1), compared to the starting strain before domestication, in mutant strain P8G3#4 cell, UDP-Gal (UDP-α-D-semi-lactosi) and 1-(5-phosphoric acid-D-ribose)-5-amino-4-imidazolyl carboxylic acid (CAIR) accumulates increases (Fig. 2 B) according to the expression analysis of differential gene; (2) down-regulated expression (Fig. 2 C) of the mtGDP in P8G3#4 intracellular phospholipid metabolism approach, intracellular glycerol content is caused to increase (3) and abc transport protein upregulation (Fig. 2 A), cause several small molecules solute in born of the same parents, as lactose and branched-chain amino acid equal size increase; (4) DNA replication dna ability reduces (Fig. 2 D), and the movement system of the cellobiose in phosphotransferase system (PTS) Lac family is lowered.These differential gene expressions raise or lower, and its physiological foundation is the stress reaction of making to maintain intraor extracellular osmotic balance, by improving water activity in cell, makes cell volume and turgescence reach normal level, maintains the normal physiological metabolism of cell.
Embodiment 3
Shake flask fermentation cultivates the growth differences of the sugared bacterial strain of resistance to height after comparing domestication and starting strain
The preparation of seed liquor:
Seed culture medium adopts MTC improved culture medium, and wherein each kind combination can be divided into A, B, C, D, E liquid, respectively after high-temperature sterilization, then uses in after the ratio mixing of 45:2:1:1:1.Concrete each liquid moiety is in table 1.
Concrete, the compound method of seed culture medium is: prepare A, B, C, D liquid respectively and be loaded in serum bottle, vacuumize, inflated with nitrogen, and 115 DEG C of sterilizing 20min, save backup; The filtration sterilization of E liquid is injected directly into the nitrogen saving backup in the serum bottle of sterilizing of substituting the bad for the good; Get the serum bottle that A liquid is housed, adopt syringe to inject above-mentioned B, C, D, E liquid prepared of respective volume respectively to working concentration, obtain seed culture medium.
The preparation of fermention medium:
In fermention medium moiety, in A liquid, carbon source concentration is respectively 30g/L, 81g/L or 120g/L, and wherein glucose and wood sugar mass ratio are 2:1, and all the other moietys are consistent with seed culture based component.The operation processing methods such as its concrete process for preparation is prepared with seed culture medium, sterilizing are consistent.
P8G0 and P8G3#4 of incubated overnight is seeded in the serum bottle containing 50mL Fresh fermention medium in 1:10 ratio respectively.Fermented liquid, temperature 55 DEG C, cultivated 84h in the shaking table of rotating speed 150rpm, every 6-12 hour sampling and measuring thalline OD 600, calculate somatic cells dry weight (Fig. 1):
Y＝0.421X-0.006(R 2＝0.99)
Wherein, Y is dry cell weight (DCW), g/L; X is OD 600.
As can be seen from Fig. 1 a, under normal substratum concentration, the DCW of starting strain P8G0 is 8h to the time required for 0.5, and after cultivating 24h, DCW can reach 1.5, reaches maximum 2.3 at 60h.When concentration of substrate is promoted to 81g/L, the time lengthening required for bacterial strain DCW to 0.5, to 42h, needs the time 82h more grown equally under 120g/L.Under comparing, the growth of resistance to height sugared bacterial strain P8G3#4 under three concentration (Fig. 1 b) is basically identical with starting strain growth tendency under normal substratum concentration, indicates the sugared bacterial strain P8G3#4 of resistance to height and has overcome the shortcoming that the growthing lag phase is long under high concentration substrate.
Embodiment 4
Fermentor cultivation compares Metabolite difference before and after domestication
Fermentation condition: by 10% inoculum size, is equipped with the full-automatic reactor of NBS company 5L of 3L substratum (120g/L mixing sugar substrate) by seed liquor access.Before inoculation, reactor first passes into nitrogen 30min, passes into nitrogen 30min again after inoculation, to ensure yeasting anaerobic.
The detection of meta-bolites ethanol, acetic acid, lactic acid and sugar consumption adopts Waters 2695 high pressure liquid chromatography (HPLC) to measure.
1. the detection of glucose, wood sugar consumption
Chromatographic column: Aminex HPX-87P (Biorad)
Moving phase: ultrapure water
Flow velocity: 0.6ml/min
Column temperature: 60 DEG C
Detector temperature: 40 DEG C
Sample preparation: 2ml fermented liquid adds 1g CaCO 3, vibration 1min, centrifugal 5min under 16,000g, get supernatant 0.22 μm of membrane filtration.For detecting residual sugar.
Sample size: 10 μ l
Detector: Composition distribution
2. the detection of meta-bolites ethanol, acetic acid, lactic acid
Chromatographic column: Aminex HPX-87H (Biorad)
Moving phase: 5mM H 2sO 4
Flow velocity: 0.6ml/min
Column temperature: 60 DEG C
Detector temperature: 40 DEG C
Sample preparation: 1.9ml fermented liquid adds 100 μ l 10%H 2sO 4, centrifugal 5min under 16,000g, gets supernatant 0.22 μm of membrane filtration.For detecting acid and ethanol.Sample size: 10 μ l
Detector: Composition distribution.
3, lactic acid, acetic acid and the ethanol (table 1) in tunning is measured.Result shows, compared to starting strain P8G0, the growthing lag phase of the bacterial strain P8G3#4 after domestication foreshortens to 24h from original 100h, and fermentation end-stage cells dry weight is also increased to 4.26g/L from 1.98g/L, improves about 2 times.And ethanol production and transformation efficiency also have corresponding raising, namely final alcohol concn reaches 28.11g/L, and ethanol maximum conversion rate reaches 1.22mol/mol.
Table 1 tames front and back bacterial strain with 120g/L mixing sugar for tank fermentation results on substrate compares

Claims (10)

1. a thermophilic anaerobic bacillus, it is characterized in that, this bacterial strain is long white clouds thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoense) P8G3#4, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC NO.9000, and preservation date is on April 3rd, 2014.
2. the method utilizing bacterial strain described in claim 1 to produce ethanol, it is characterized in that, first the seed liquor of thermophilic anaerobic bacillus CGMCC9000 is prepared, then thermophilic anaerobic bacillus CGMCC9000 seed liquor is transferred in fermention medium with 10-15%w/w inoculum size, under anaerobic stir culture, culture temperature 45-60 DEG C, incubation time is 50-250 hour, finally goes out ethanol from separation of fermentative broth.
3. method according to claim 2, is characterized in that, the total sugar concentration of described fermention medium is 80-150g/L.
4. method according to claim 2, is characterized in that, the initial pH of described fermention medium is 5.5-6.5, and described mixing speed is 120-200rpm.
5. method according to claim 4, is characterized in that, the initial pH of described fermention medium is 6.3.
6. the method according to Claims 2 or 3 or 4 or 5, is characterized in that, described fermentation medium components is: glucose 80-100, wood sugar 40-60, urea 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/L.
7. method according to claim 6, it is characterized in that, described fermention medium also comprises following composition: citric acid tri potassium salt 1-4, Citric acid monohydrate Food grade 0.5-3, sodium sulfate 0.5-3, potassium primary phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate 0.5-3, four water iron protochloride 0.05-0.5, Calcium dichloride dihydrate 0.05-0.4, a water halfcystine hydrochloric acid 0.5-3, two hydrochloric acid Pyridoxylamine 0-0.1, para-amino benzoic acid 0-0.01, Bio 0-0.01, vitamin B12 0-0.01, vitaminB10-0.01, unit g/L.
8. the method according to Claims 2 or 3 or 4 or 5, is characterized in that, the preparation of described seed liquor: thermophilic anaerobic bacillus CGMCC9000 is placed in seed culture medium, and at 45-60 DEG C, rotating speed is 100-250rpm, cultivates 8-24h.
9. method according to claim 8, is characterized in that, described seed culture medium is: glucose 2-4, wood sugar 2-4, urea 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/L.
10. method according to claim 9, it is characterized in that, described seed culture medium also comprises following composition: citric acid tri potassium salt 1-4, Citric acid monohydrate Food grade 0.5-3, sodium sulfate 0.5-3, potassium primary phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate 0.5-3, four water iron protochloride 0.05-0.5, Calcium dichloride dihydrate 0.05-0.4, a water halfcystine hydrochloric acid 0.5-3, two hydrochloric acid Pyridoxylamine 0-0.1, para-amino benzoic acid 0-0.01, Bio 0-0.01, vitamin B12 0-0.01, vitaminB10-0.01, unit g/L.
CN201410453513.8A 2014-09-05 2014-09-05 Thermoanaerobacter ethanolicus and method for producing ethanol by using same Expired - Fee Related CN104263680B (en)

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CN104911128A (en) * 2015-06-10 2015-09-16 华南理工大学 Thermoanaerobacterium aotearoense and application thereof in lactic acid production
CN104911128B (en) * 2015-06-10 2019-01-29 华南理工大学 A kind of thermophilic anaerobic bacillus and its application in production lactic acid
CN107164567A (en) * 2017-04-28 2017-09-15 昆明理工大学 A kind of method that high temperature anaerobic bacterium controlled based on pH value produces ethanol
CN111041039A (en) * 2019-12-04 2020-04-21 江苏大学 Thermophilic anaerobic ethanol bacillus genome editing vector and application thereof
CN111041039B (en) * 2019-12-04 2023-01-17 江苏大学 Thermophilic anaerobic ethanol bacillus genome editing vector and application thereof

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