CN104263680B - Thermoanaerobacter ethanolicus and method for producing ethanol by using same - Google Patents
Thermoanaerobacter ethanolicus and method for producing ethanol by using same Download PDFInfo
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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
Technical field
A kind of bacterial strain producing ethanol and the acclimation method of its high concentration sugar tolerance raising, and produce second using this bacterial strain
The method of alcohol.The present invention relates to glucose, xylose as primary raw material, using thermophilic anaerobic bacillus
The method of (thermoanaerobacterium aotearoense scut27/ δ ldh) mutant fermentative production of ethanol.
Background technology
Ethanol is a kind of Novel clean-fuel, is the important directions that regenerative resource develops.Developing bio-ethanol is
Trend of the times.Second filial generation bio-ethanol utilizes Plant fiber to pass through biology again through pretreatment, mineral acid or cellulase hydrolysiss
Fermentation mode produces.And Hydrogen Energy because its cleaning, energy density is high, preparation method is various the features such as, attract the pass of researchers
Note.
High cost is with the major obstacle that cellulosic class material produces ethanol for fermenting raw materials.Using thermophilc anaerobe
(thermoanaerobacterium) fermentation can preferably solve this difficulty.Had with it for fermentation strain production of chemicals
Following several respects advantage (o-thong, s.et al., international journal of hydrogen energy,
2008.33 (4): 1204-1214): (1) reduces with the cellulase addition during cellulosic class fermenting raw materials.This be because
For such bacterial strain be in itself one class hemicellulose decompose microorganism, its can at high temperature hydrolyzed xylan fermentation produce multiple
Chemicals, thus decrease the demand of cellulase.(2) high temperature continuous fermentation production process, is conducive to the separation of ethanol pure
Change.(3) ethanol can be produced with raw material fermentation.Because industrial scale fermentation produces in ethanol process, pure-blood ferment is for equipment
Sterility requirements are higher, are therefore added significantly to Financial cost.But adopt Thermophilic Bacteria fermenting and producing, due to the micro- life of most room temperature
Thing is more difficult to be produced at high temperature, therefore not high to the sterility requirements of sweat, thus reduce further production cost.(4)
Can be fermentation raw material using high temperature organic wastewater, on the one hand this reduce production cost, on the other hand also mitigates environment pressure
Power.
One plant of thermophilic anaerobic bacillus (li, s., et al., bioresource is screened in applicant's early-stage Study
Technology, 2010,101 (22): 8718-8724, cai, y., et al., enzyme and microbial
Technology, 2011.48 (2): p.155-161), this bacterial strain being capable of the accumulation of the fermenting substrate such as effectively utilizes glucose, xylose
Ethanol, hydrogen etc..By it is carried out with genetic modification, knock out lactate dehydrogenase gene, after metabolic pathway transformation, mutant h2With
Ethanol production has been respectively increased 2.7 and 2.4 times.But this bacterial strain is in the case of high concentration sugar substrate, by high sugar osmotic stress
Impact, leads to the growthing lag phase long, leads to fermentation period too long, production cost increases considerably.And high concentration sugar substrate is resistance to
It is to realize cellulose series biomass simultaneous saccharification and fermentation to produce necessary to ethanol.
If it is possible to screen the thermophilic anaerobic bacillus bacterial strain of one plant of resistance to high sugar from the point of view of industrialization angle, improve thermophilic
Anaerobic bacillus(cillus anaerobicus) sugar concentration of substrate toleration, and character is stable, can be applicable to high concentration sugar fermenting substrate, shortens the fermentation lag phase,
Reduce technique amplification cost, so just can be more suitable for industrialized production.
Content of the invention
It is an object of the invention to provide a kind of bacterial strain producing ethanol and the method producing ethanol using this bacterial strain.
By starting strain thermophilic anaerobic bacillus (thermoanaerobacterium aotearoensescut27/ δ ldh)
P8g0 (Erythromycinresistant) is cultivated according to the method stepping up sugared concentration of substrate, repeat number generation, dense until obtaining here
Degree lower can after inoculating 6h thalline od600To 0.6 and character is stable for length.Take 1ml bacterium solution to be added in isolation medium, detesting
Cultivate 2-3 days in oxygen incubator, picking single bacterium colony, in the fermentation medium after culture 240h, ethanol final concentration reaches 28.11g/l,
Conversion ratio reaches 1.22mol/mol, and finally obtaining being capable of normal growth, the mutant p8g3#4 passing under high concentration substrate
(Erythromycinresistant).
Above-mentioned mutant is long white clouds thermophilic anaerobic bacillus (thermoanaerobacterium aotearoense)
P8g3#4, in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation (abbreviation cgmcc), deposit number
For cgmcc no.9000, preservation date is on April 3rd, 2014, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica.
Described p8g3#4 bacterial strain is: gram positive bacteria, elongated rod shape, and about 0.6-1.0 μm of diameter is about 2-15 μm, micro-
Microscopic observation its can tumbling motion.For strict anaerobess, optimum growth temperature is 55 DEG C, ph 6.3, and multiple sugar substrates can be utilized
Growth, such as glucose, xylose, mannose, Fructose, galactose, arabinose, trehalose, xylan, glucosan etc..This bacterial strain
Inherited character be: lactic acid metabolism Blocked-mutant, there is Erythromycinresistant, Erythromycinresistant concentration be 20-100ng/ μ l.
It is resistant to 120-150g/l sugar concentration of substrate, intracellular udp-gal (udp- α-d- galactose) and 1- (5- phosphoric acid-d- ribose)-
5- amino -4- imidazolyl carboxylic acid (cair) accumulation increases；The down-regulated expression of the mtgdp in phosphoglyceride metabolic pathway.
The method producing ethanol using bacterial strain described in claim 1: prepare the kind of thermophilic anaerobic bacillus cgmcc9000 first
Then thermophilic anaerobic bacillus cgmcc9000 seed liquor is transferred in fermentation medium by sub- liquid with 10-15%w/w inoculum concentration,
Stir culture under anaerobic condition, cultivation temperature 45-60 DEG C, incubation time is 50-250 hour, finally goes out second from separation of fermentative broth
Alcohol.
The total sugar concentration of described fermentation medium is 80-150g/l.
The initial ph of described fermentation medium is 5.5-6.5, and described speed of agitator is 120-200rpm.
The initial ph of described fermentation medium is 6.3.
Described fermentation medium components are: glucose 80-100, xylose 40-60, carbamide 1-10, yeast extract 1-5, chlorine
Change ammonium 0.5-3, unit g/l.
Described fermentation medium also includes following composition: potassium citrate salt 1-4, citric acid monohydrate 0.5-3, sodium sulfate
0.5-3, potassium dihydrogen phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate 0.5-3, four water ferrous chloride 0.05-0.5, two water chlorine
Change calcium 0.05-0.4, a water cysteine hydrochloric acid 0.5-3, two hydrochloric acid pyridoxamine 0-0.1, para-amino benzoic acid 0-0.01, d- give birth to
Thing element (d-biotin) 0-0.01, vitamin b12 0-0.01, vitamin b10-0.01, unit g/l.
The preparation of described seed liquor: thermophilic anaerobic bacillus cgmcc9000 are 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, xylose 2-4, and other components are identical with fermentation medium.Predominantly urinate
Plain 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/l.Preferably, also include following composition: potassium citrate salt 1-
4, citric acid monohydrate 0.5-3, sodium sulfate 0.5-3, potassium dihydrogen phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate 0.5-3, four
Water ferrous chloride 0.05-0.5, calcium chloride dihydrate 0.05-0.4, one water cysteine hydrochloric acid 0.5-3, two hydrochloric acid pyridoxamine 0-
0.1, para-amino benzoic acid 0-0.01, D-biotin (d-biotin) 0-0.01, vitamin b12 0-0.01, vitamin b10-
0.01, unit g/l.
Compared with prior art, the invention has the following beneficial effects:
(1) with this laboratory screening, build thermophilic anaerobic bacillus p8g0 obtaining as parental plant, carry out high sugar according to a conventional method
Substrate tolerance is tamed, and obtains mutant p8g3#4.This mutant has following inherited character: l- lactic dehydrogenase deficient,
Erythromycinresistant, sugared concentration of substrate tolerance range more than 120-150g/l, intracellular udp-gal (udp- α-d- galactose) and 1-
(5- phosphoric acid-d- ribose) -5- amino -4- imidazolyl carboxylic acid (cair) accumulation increases；The table of the mtgdp in phosphoglyceride metabolic pathway
Reach downward.
(2) ethanol is produced for substrate in 5l ferment tank with 120g/l glucose, xylose mixed sugar, the lag phase foreshortens to
About 1/4 originally, ethanol final concentration reaches 28g/l, and conversion ratio reaches 1.2mol/mol.The present invention provides the mutant of screening, can
Accumulate target product ethanol using high sugar substrate rapid fermentation, and the by-product producing is few, technique amplification is relatively easy to, and is suitable for
Industrialized production.
Brief description
Fig. 1 is the growth curve chart of the resistance to high sugar bacterial strain p8g3#4 and starting strain p8g0 after domestication.A) p8g0 is in difference
Growing state under carbon source concentration,-■ -30g/l,-● -81g/l,-▲ -120g/l；B) p8g3#4 is under different carbon source concentration
Growing state,--30g/l,-zero -81g/l,-△ -120g/l；Wherein sugared substrate is glucose and xylose (2:1, g:g).
Fig. 2 is that the cellular metabolism access function significance of p8g3#4 and p8g0 is enriched with schematic diagram.
A) abc transport protein system, right figure is Lactose in oligosaccharide and polyhydric alcohol movement system/l- arabinose transhipment egg
In vain, left figure is the branched-chain amino acid transport protein in phosphate and amino acid transporter system；B) double-component regulator control system (two-
Component system), glnl regulator control system；C) the lac family in phosphotransferase system (pts), right figure is Lactose
Transhipment, left figure is the transhipment of cellobiose；D) dna replicates related elements.Grey square frame represents the metabolite of accumulation.
Specific embodiment
Embodiment 1
The domestication of bacterial strain with separate
The resistance to high sugar thermophilic anaerobic bacillus bacterial strain of the present invention can so obtain:
In the present invention, the starting strain for screening is laboratory screening, the thermophilic anaerobic bacillus that are mutated and preserve
(thermoanaerobacterium aotearoense scut27/ δ ldh), is designated as p8g0 herein.
Screening culture medium: glucose: 80-100g l-1；Xylose: 40-60g l-1；(nh4)2so4: 1-5g l-1；
mgcl2·6h2O:1-5g l-1；kh2po4: 0.5-5g l-1；k2hpo4: 0.5-5g l-1；cacl·2h2O:0.1-2.0g
l-1；Na- β-glycerophosphate:2-10g l-1；feso4·7h2O:0.0001-0.001g l-1；Yeast extract:
2-10g·l-1；One water cysteine hydrochloric acid (c3h7no2s·hcl·h2O): 0.1-2.0g l-1；"diazoresorcinol": 0.001-
0.01g·l-1；Agar powder: 5-20g l-1；Erythromycin: 20-100ng/ μ l.
Starting strain p8g0 is to shake through 81g/l screening culture medium (glucose and xylose mass ratio is 2:1) anaerobism first
Bottle culture, treats strain growth light absorption value od600For 2.0 or more, new anaerobism Shake flask medium of transferring, repeat number generation, until
To at this concentration can inoculation 6h after thalline od600Long to 0.6, and the stable bacterial strain p8g1 of character；
As step (1) method, under the conditions of 102g/l glucose and xylose (2:1, g:g), obtain bacterial strain p8g2；
As step (1) method, under the conditions of 120g/l glucose and xylose (2:1, g:g), obtain bacterial strain p8g3；
Cultivated by flat board, 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, poor using transcription group method analysis p8g0 and p8g3#4 metabolic gene
Different, and differential gene is carried out with cluster analyses, identification leads to the gene table of two strain growth Difference of Metabolisms of p8g0 and p8g3#4
The reason reach in aspect.
By starting strain thermophilic anaerobic bacillus (thermoanaerobacterium aotearoensescut27/ δ ldh)
P8g0 and screening obtain to carry out transcript profile sequencing analysis in the mutant p8g3#4 of normal growth under high concentration substrate.Point
Difference expression gene in two samples of analysis, screening criteria is | logfc | >=1 (i.e. twice difference), in two samples of analysis
Difference expression gene, p8g0 and p8g3#4 has 69 genes and there is significant difference on gene expression abundance, with starting strain p8g0
Compare, have 35 and 24 genes, in bacterial strain logarithmic growth early stage, up-regulated and downward occur respectively, p8g3#4 is on transcript profile
With having differences property of p8g0.
(Fig. 2) is analyzed by kegg metabolic pathway, has 27 gene annotations in the middle of 20 signal paths, wherein annotate
To purine metabolism approach (purine metabolism), abc transport protein system (abc transporters), galactose generation
Thank approach (galactose metabolism), amino sugar and nucleotide sugar metabolic pathway (amino sugar and
Nucleotide sugar metabolism), nitrogen metabolic path-way (nitrogen metabolism), phosphotransferase system
Degradation pathway (the chloroalkane and of (phosphotransferase system), enpara and chloro-alkenes
Chloroalkene degradation), alanine, aspartic acid and glutamic acid metabolism approach (alanine, aspartate
And glutamate metabolism) and phosphoglyceride metabolic pathway (glycerophospholipid metabolism)
Gene more, respectively 5,5,4,4,4,4,3,3 and 2.
There is following discovery according to the expression analysis of differential gene: (1) is compared to the starting strain before domestication, mutant
The intracellular udp-gal of p8g3#4 (udp- α-d- galactose) and 1- (5- phosphoric acid-d- ribose) -5- amino -4- imidazolyl carboxylic acid
(cair) accumulation increases (Fig. 2 b)；(2) down-regulated expression (figure of the mtgdp in p8g3#4 intracellular phospholipid metabolism approach
2c), lead to intracellular glycerol content to increase (3) and abc transport protein rise (Fig. 2 a), lead to intracellular several small molecule solute,
As Lactose and branched-chain amino acid equal size increase；(4) dna replication capacity reduces (Fig. 2 d), phosphotransferase system (pts) lac
The movement system of the cellobiose in family is lowered.These differential gene expressions be raise or lower, its physiological foundation be in order to
The stress maintaining intraor extracellular osmotic balance and making, by improving intracellular water activity, makes cell volume and turgescence
Reach normal level, maintain the normal physiological metabolism of cell.
Embodiment 3
The growth differences of the resistance to high sugar bacterial strain after domestication and starting strain are compared in shake flask fermentation culture
The preparation of seed liquor:
Seed culture medium adopts mtc improved culture medium, and various combinations therein can be divided into a, b, c, d, e liquid, respectively high temperature
After sterilizing, then use in after the ratio mixing of 45:2:1:1:1.Specifically each liquid constituent is shown in Table 1.
Specifically, the compound method of seed culture medium is: prepare a, b, c, d liquid respectively and be loaded in serum bottle, evacuation, fills
Nitrogen, 115 DEG C of sterilizing 20min, save backup；E liquid filtration sterilization is injected directly into substitutes the bad for the good in nitrogen the serum bottle that sterilizes
Save backup；Take the serum bottle having been loaded with a liquid, above-mentioned b, c, d, e liquid preparing of respective volume is injected separately into using syringe
To working concentration, obtain final product seed culture medium.
The preparation of fermentation medium:
In fermentation medium constituent, in a liquid, carbon source concentration is respectively 30g/l, 81g/l or 120g/l, wherein Fructus Vitis viniferae
Sugar and xylose mass ratio are 2:1, and remaining constituent is consistent with seed culture based component.Its concrete process for preparation and seed culture
The operation processing methods such as basigamy system, sterilizing are consistent.
P8g0 and p8g3#4 of incubated overnight is seeded to ferment containing 50ml Fresh in 1:10 ratio respectively and trains
In the serum bottle of foster base.Fermentation liquid, in 55 DEG C of temperature, cultivates 84h in the shaking table of rotating speed 150rpm, sample every 6-12 hour and survey
Determine thalline od600, calculating somatic cells dry weight (Fig. 1):
Y=0.421x-0.006 (r2=0.99)
Wherein, y is dry cell weight (dcw), g/l；X is od600.
Can be seen that under normal culture medium concentration from Fig. 1 a, the dcw of starting strain p8g0 required for 0.5 when
Between be 8h, culture 24h after dcw can reach 1.5, reach peak 2.3 in 60h.When concentration of substrate is promoted to 81g/l, bacterium
Strain time lengthening required for 0.5 for the dcw, to 42h, equally needs longer time 82h under 120g/l.Under comparing, resistance to
High sugar bacterial strain p8g3#4 is in the growth (Fig. 1 b) under three concentration and starting strain growth tendency under normal culture medium concentration
Basically identical, indicate the shortcoming that resistance to high sugar bacterial strain p8g3#4 has overcome growthing lag phase length under high concentration substrate.
Embodiment 4
Fermentor cultivation compares Metabolite difference before and after domestication
Fermentation condition: by 10% inoculum concentration, seed liquor is accessed the nbs equipped with 3l culture medium (120g/l mixed sugar substrate)
The full-automatic reactor of company 5l.Before inoculation, reactor is first passed through nitrogen 30min, is passed through nitrogen 30min after inoculation again, to protect
Card yeasting anaerobic.
Metabolite ethanol, the detection of acetic acid, lactic acid and sugar consumption adopt waters 2695 high pressure liquid chromatography (hplc) to survey
Fixed.
1. the detection that glucose, xylose consume
Chromatographic column: aminex hpx-87p (biorad)
Mobile phase: ultra-pure water
Flow velocity: 0.6ml/min
Column temperature: 60 DEG C
Detector temperature: 40 DEG C
Sample preparation: 2ml fermentation liquid adds 1g caco3, vibrate 1min, under 16,000g, be centrifuged 5min, take supernatant to use
0.22 μm of membrane filtration.For detecting residual sugar.
Sample size: 10 μ l
Detector: Composition distribution
2. the detection of metabolite ethanol, acetic acid, lactic acid
Chromatographic column: aminex hpx-87h (biorad)
Mobile phase: 5mm h2so4
Flow velocity: 0.6ml/min
Column temperature: 60 DEG C
Detector temperature: 40 DEG C
Sample preparation: 1.9ml fermentation liquid adds 100 μ l 10%h2so4, it is centrifuged 5min under 16,000g, take supernatant to use
0.22 μm of membrane filtration.For detecting acid and ethanol.Sample size: 10 μ l
Detector: Composition distribution.
3rd, the lactic acid in mensure tunning, acetic acid and ethanol (table 1).Result shows, compared to starting strain p8g0, tames and dociles
The growthing lag phase of the bacterial strain p8g3#4 after change foreshortens to 24h from original 100h, and ferment end-stage cells dry weight also from
1.98g/l increases to 4.26g/l, improves about 2 times.And ethanol production and conversion ratio are also correspondingly improved, i.e. final second
Determining alcohol reaches 28.11g/l, and ethanol maximum conversion rate reaches 1.22mol/mol.
Table 1 domestication before and after bacterial strain with 120g/l mixed sugar as substrate on tank fermentation results compare
Claims (10)
1. a kind of thermophilic anaerobic bacillus are it is characterised in that this bacterial strain is long white clouds thermophilic anaerobic bacillus
(thermoanaerobacterium aotearoense) p8g3#4, general in China Committee for Culture Collection of Microorganisms
Logical microorganism center preservation, deposit number is cgmcc no.9000, and preservation date is on April 3rd, 2014.
2. bacterial strain described in a kind of utilization claim 1 produces the method for ethanol it is characterised in that preparing thermophilic anaerobic bacillus first
Then thermophilic anaerobic bacillus cgmcc9000 seed liquor is transferred to 10-15%w/w inoculum concentration and sends out by the seed liquor of cgmcc9000
In ferment culture medium, stir culture under anaerobic, cultivation temperature 45-60 DEG C, incubation time be 50-250 hour, finally from
Separation of fermentative broth goes out ethanol.
3. method according to claim 2 is it is characterised in that the total sugar concentration of described fermentation medium is 80-150g/l.
4. method according to claim 2 is it is characterised in that the initial ph of described fermentation medium is 5.5-6.5, described
Speed of agitator is 120-200rpm.
5. method according to claim 4 is it is characterised in that the initial ph of described fermentation medium is 6.3.
6. the method according to Claims 2 or 3 or 4 or 5 is it is characterised in that described fermentation medium components are: glucose
80-100, xylose 40-60, carbamide 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/l.
7. method according to claim 6 is it is characterised in that described fermentation medium also includes following composition: citric acid
Tripotassium salt 1-4, citric acid monohydrate 0.5-3, sodium sulfate 0.5-3, potassium dihydrogen phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate
0.5-3, four water ferrous chloride 0.05-0.5, calcium chloride dihydrate 0.05-0.4, one water cysteine hydrochloric acid 0.5-3, two hydrochloric acid pyrroles
Tremble amine 0-0.1, para-amino benzoic acid 0-0.01, D-biotin 0-0.01, vitamin b12 0-0.01, vitamin b10-0.01, single
Position g/l.
8. the method according to Claims 2 or 3 or 4 or 5 is it is characterised in that the preparation of described seed liquor: by 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 it is characterised in that described seed culture medium is: glucose 2-4, xylose 2-4,
Carbamide 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/l.
10. method according to claim 9 is it is characterised in that described seed culture medium also includes following composition: citric acid
Tripotassium salt 1-4, citric acid monohydrate 0.5-3, sodium sulfate 0.5-3, potassium dihydrogen phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate
0.5-3, four water ferrous chloride 0.05-0.5, calcium chloride dihydrate 0.05-0.4, one water cysteine hydrochloric acid 0.5-3, two hydrochloric acid pyrroles
Tremble amine 0-0.1, para-amino benzoic acid 0-0.01, D-biotin 0-0.01, vitamin b12 0-0.01, vitamin b10-0.01, single
Position g/l.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410453513.8A CN104263680B (en) | 2014-09-05 | 2014-09-05 | Thermoanaerobacter ethanolicus and method for producing ethanol by using same |
| SG11201701615RA SG11201701615RA (en) | 2014-09-05 | 2014-12-03 | A thermophilic anaerobic bacillus and a method for producing ethanol by using the same |
| PCT/CN2014/092862 WO2016033886A1 (en) | 2014-09-05 | 2014-12-03 | Thermoanaerobacterium and method for producing ethanol by using same |
Applications Claiming Priority (1)
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| 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 |
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| WO2016033886A1 (en) | 2016-03-10 |
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