CN104911128B - A kind of thermophilic anaerobic bacillus and its application in production lactic acid - Google Patents
A kind of thermophilic anaerobic bacillus and its application in production lactic acid Download PDFInfo
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- C12P7/56—Lactic acid
Abstract
The invention discloses a kind of thermophilic anaerobic bacillus and its applications in production lactic acid, the bacterial strain is Thermoanaerobacterium aotearoenseP9G4#16, in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number is CGMCC NO.10833, and the deposit date is on Mays 21st, 2015.Bacterial strain P9G4#16 can grow and ferment under high concentration sugar substrate, and lactic acid production is also improved.Bacterial strain of the present invention has genetic stability good, and yield traits are stablized, and the lag phase of fermenting greatly shortens, and saves the energy consumption in technical process, greatly improves its industrial value.
Description
Technical field
The present invention relates to a kind of thermophilic anaerobic bacillus and its applications in production lactic acid.The present invention relates to glucose,
Xylose or fructose are primary raw material, utilize thermophilic anaerobic bacillus (Thermoanaerobacterium
AotearoenseSCUT27/ Δ pta-ack) mutant strain fermenting lactic acid method.
Background technique
Lactic acid is universally acknowledged one of three big organic acids, is widely used in food, pharmacy, leather, cosmetics, chemical industry etc.
Field.The 85% of lactic acid total amount is applied to food and the relevant field of food, and remaining 15% is used for non-food industries.At present
The lactic acid in the world 90% is generated by bacterial fermentation, uses this important raw material of industry of cheap biomass material fermenting lactic acid
With boundless prospect.
In biomass resource, in addition to the glucose from starchy material is used widely in fermentation industry,
Effective use is not yet received in other renewable carbohydrate sources, and one of reason is exactly microorganism to xylose utilization ability
It is poor.Utilize various chemicals and substitution energy of cheap, rich reserves the wood fibre resource fermenting and producing including lactic acid
Source have very wide application prospect, be expected to reduce the cost of current bioanalysis synthesis of chemicals, thus promoted its relative to
The competitiveness of chemical synthesis process.
It is that cost is excessively high using the major obstacle that cellulosic substance produces lactic acid as fermenting raw materials.Using thermophilc anaerobe
(Thermoanaerobacterium) fermentation can preferably solve the difficulty.According to Sommer P.'s et al. research shows that
(Sommer P.et al., Biochemical Society Transactions, 2004,32 (Pt 2): 283-289), with this
Bacterial strain production of chemicals has many advantages: (1) high temperature, which continuously ferments, produces lower yield height, and inhibits the infection of miscellaneous bacteria, can
Lactic acid is produced with raw material fermentation.It is higher to the sterility requirements of pure-blood ferment in industrial production, it is added significantly to economic cost.But it adopts
When with Thermophilic Bacteria fermenting and producing, high growth temperature environment inhibits the growth of most of microorganism, therefore can be dropped with raw material fermentation
Low production cost.(2) energy degraded cellulose hemicellulose biomass, is hydrolyzed into glucose and xylose and utilizes, and generates chemistry
Product and/or bio-fuel reduce the additive amount of cellulase；(3) main metabolic product is few and simple, and carbon source has high mole
The rate of recovery；(4) the hydrolyzate direct fermentation that can utilize crops and castoff, alleviates environmental pressure and reduces life
Produce cost.
One plant of thermophilic anaerobic bacillus (Li, S., et al., Bioresource are screened in inventor's early-stage study
Technology, 2010,101 (22): 8718-8724), which can efficiently use the fermenting substrates such as glucose, xylose product
Tired ethyl alcohol, lactic acid, hydrogen etc..By carrying out genetic modification to it, phosphotransacetylase enzyme gene and Acetokinase gene, generation are knocked out
After thanking to approach transformation, in fermentor under the fermentation of 50g/L glucose substrate, mutant strain lactic acid production improves 1.21 times, reaches
47.17g/L, hardly production acetic acid, while ethanol production is decreased obviously (Yang et al.Biotechnology for
Biofuels 2013,6:124, Li Shuan；Wang Jufang；Yang Xiaofeng；Wang little Ning；In flat scholar one kind light can be produced using wood-sugar fermentation
Learn the engineering bacteria and its building .201110453684.7 of pure Pfansteihl).But the bacterial strain is in high concentration sugar substrate, by
The influence of high sugar osmotic stress, causes bacterium that can hardly grow, fermentation period too long, is not suitable for being applied in industrial production.
And the tolerance of high concentration sugar substrate can lay the foundation to realize that cellulose series biomass simultaneous saccharification and fermentation produces lactic acid.
From the point of view of industrializing angle, if it is possible to screen one plant and be resistant to high concentration sugar substrate, and stable thermophilic of character
Hot anaerobic bacillus(cillus anaerobicus) bacterial strain can be applied to high concentration sugar fermenting substrate, shorten the fermentation lag phase, reduces energy consumption, could promote in this way
Into its application in industrialized production.
Summary of the invention
The purpose of the present invention is to provide a kind of thermophilic anaerobic bacillus and its applications in production lactic acid.
In the present invention, the starting strain for screening is laboratory screening, mutation and the thermophilic anaerobic bacillus saved
(Thermoanaerobacterium aotearoenseSCUT27/ Δ pta-ack) P9G0 (kalamycin resistance).
High concentration substrate tolerance acclimation method provided by the present invention, includes the following steps:
(1) thermophilic anaerobic bacillus P9G0 is cultivated, seed liquor is obtained；
(2) seed liquor of above-mentioned steps (1) is transferred to the culture medium of low concentration substrate with 10-15%w/w inoculum concentration
In, rear thallus OD for 24 hours can be inoculated at this concentration by filtering out600It is long to 1.5 and the stable thermophilic anaerobic bacillus of character；
(3) seed liquor of above-mentioned steps (2) is transferred to the culture medium of higher concentration substrate with 10-15%w/w inoculum concentration
In, rear thallus OD for 24 hours can be inoculated at this concentration by filtering out600It is long to 1.5 and the stable thermophilic anaerobic bacillus of character；
(4) step (3) are repeated, step up concentration of substrate until reaching 120g/L, obtain under the culture medium condition
Thallus OD after inoculation for 24 hours600It is long to 1.5 and the stable thermophilic anaerobic bacillus P9G4#16 (kalamycin resistance) of character.
The P9G4#16 bacterial strain are as follows: gram-positive bacteria, elongated rod shape, are about 2-15 μm by about 0.6-1.0 μm of diameter, micro-
It can tumbling motion under the microscope.For strict anaerobes, optimum growth temperature is 55 DEG C, pH 6.3, using a variety of sugared substrates
Growth, such as glucose, xylose, mannose, fructose, galactolipin, arabinose, trehalose, xylan, glucan.The bacterial strain
Hereditary capacity are as follows: acetate metabolism Blocked-mutant, have kalamycin resistance, kalamycin resistance concentration be 20-
100ng/μl.It is resistant to 80-150g/L sugar concentration of substrate.
Application of the P9G4#16 bacterial strain in production lactic acid, prepares the seed liquor of thermophilic anaerobic bacillus CGMC10833 first,
Then seed liquor is transferred in fermentation medium with 10-15%w/w inoculum concentration, under anaerobic stir culture, culture temperature
45-60 DEG C of degree, incubation time are 70-200 hours, finally go out lactic acid from separation of fermentative broth.
The total sugar concentration of the fermentation medium is 80-150g/L.
The initial pH of the fermentation medium is 5.5-6.5, and the speed of agitator is 100-200rpm.Preferably, described
The initial pH of fermentation medium is 6.3.
The fermentation medium components are as follows: glucose 80-100, xylose 40-60, urea 1-10, yeast extract 1-5, chlorine
Change ammonium 0.5-3, unit g/L.
The fermentation medium further includes following ingredient: citric acid tri potassium salt 1-4, Citric Acid Mono 0.5-3, sodium sulphate
0.5-3, potassium dihydrogen phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate 0.5-3, four water frerrous chloride 0.05-0.5, two water chlorine
Change calcium 0.05-0.4, water cysteine hydrochloric acid a 0.5-3, two hydrochloric acid pyridoxamine 0-0.1, p-aminobenzoic acid 0-0.01, D- is raw
Object element 0-0.01, vitamin B12 0-0.01, vitamin B1 0-0.01, unit g/L.
The preparation of the seed liquor: thermophilic anaerobic bacillus CGMC10833 is placed in seed culture medium, at 45-60 DEG C, is turned
Speed is 100-200rpm, cultivates 8-24h.
The seed culture medium are as follows: glucose 2-4, xylose 2-4, urea 1-10, yeast extract 1-5, ammonium chloride 0.5-
3, unit g/L.
The seed culture medium further includes following ingredient: citric acid tri potassium salt 1-4, Citric Acid Mono 0.5-3, sodium sulphate
0.5-3, potassium dihydrogen phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate 0.5-3, four water frerrous chloride 0.05-0.5, two water chlorine
Change calcium 0.05-0.4, water cysteine hydrochloric acid a 0.5-3, two hydrochloric acid pyridoxamine 0-0.1, p-aminobenzoic acid 0-0.01, D- is raw
Object element 0-0.01, vitamin B12 0-0.01, vitamin B1 0-0.01, unit g/L.
Compared with prior art, the invention has the following beneficial effects:
(1) the thermophilic anaerobic bacillus P9G0 obtained using this laboratory screening, building is dense by stepping up substrate as parental plant
The method of degree carries out high sugared substrate tolerance domestication, obtains mutant strain P9G4#16.The mutant strain has following hereditary capacity: phosphorus
Sour transacetylase and acetokinase deficiency, kalamycin resistance, sugared concentration of substrate tolerance range are more than 80-150g/L.
(2) present invention filters out the thermophilic anaerobic bacillus strain of enduring high-concentration sugar substrate.Compared with the bacterial strain before domestication, solution
It has determined the defect that it cannot grow under high concentration sugar substrate, has shortened fermentation period, improve fermentability, and there is heredity
Stability is good, and yield traits are stablized, and the lag phase of fermenting greatly shortens, and saves the energy consumption in technical process, greatly improves it
Industrial value.
It (3) is that substrate in 5L ferment tank produces lactic acid using 100g/L glucose, fructose mixed sugar, the lag phase is only
12h, lactic acid final concentration reach 77.7g/L, and conversion ratio reaches 0.83g/g.The present invention provides the mutant of screening, can utilize high sugared bottom
Object Rapid Fermentation accumulates target product lactic acid, and the by-product generated is few, and technique amplification is relatively simple, is suitable for industrial metaplasia
It produces.
The mutant strain that the present invention screens is Thermoanaerobacterium aotearoenseP9G4#16, in
State's Microbiological Culture Collection administration committee common micro-organisms center preservation (abbreviation CGMCC), deposit number CGMCC
NO.10833, the deposit date is on Mays 21st, 2015.Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China
Institute of microbiology of the academy of sciences.
Detailed description of the invention
Fig. 1 is the resistance to height sugar bacterial strain P9G4 and growth of the starting strain P9G0 under 120g/L high concentration sugar substrate after domestication
Curve graph.- ■-P9G4 ,-●-P9G0.Wherein sugared substrate is glucose and xylose (2:1, g:g).
Fig. 2 is that P9G4#16 utilizes the growth of food waste object hydrolyzate and situation of fermenting.The hydrolyzate main component is grape
Sugar and fructose (9:1, g:g).- ■-glucose ,-●-fructose ,-△-lactic acid ,-◇-ethyl alcohol ,-☆-dry cell weight.
Specific embodiment
Embodiment 1
The domestication of bacterial strain with separate
Resistance to high sugared thermophilic anaerobic bacillus bacterial strain of the invention can obtain in this way:
In the present invention, the starting strain for screening is laboratory screening, mutation and the thermophilic anaerobic bacillus saved
(Thermoanaerobacterium aotearoenseSCUT27/ Δ pta-ack) P9G0 (kalamycin resistance).
The preparation of seed liquor:
Seed culture medium uses MTC improved culture medium, and one of the various combinations can be divided into A, B, C, D, E liquid, respectively high temperature
It is used after sterilizing, then after being mixed in the ratio of 45:2:1:1:1.Specific each liquid constituent is shown in Table 1.
Specifically, the preparation method of seed culture medium are as follows: prepare A, B, C, D liquid respectively loaded in serum bottle, vacuumize, fill
Nitrogen, 115 DEG C of sterilizing 20min, saves backup；E liquid filtration sterilization is injected directly into substituted the bad for the good nitrogen and the serum bottle of sterilizing
It saves backup；The serum bottle for having been loaded with A liquid is taken, above-mentioned prepared B, C, D, E liquid of respective volume is injected separately into using syringe
To working concentration to get seed culture medium.
Starting strain P9G0 is by 60g/L culture medium (glucose and xylose mass ratio is 2:1) anaerobism shaking flask training first
It supports, to strain growth light absorption value OD600It is 1.5 or more, the anaerobism Shake flask medium for transferring new, repeat number generation, until obtaining
Rear thallus OD for 24 hours can be inoculated under this concentration600It is long to 1.5, and the bacterial strain P9G1 that character is stable；
As step (1) method, bacterial strain P9G2 is obtained under the conditions of 81g/L glucose and xylose (2:1, g:g)；
As step (1) method, bacterial strain P9G3 is obtained under the conditions of 102g/L glucose and xylose (2:1, g:g)；
As step (1) method, after being passed on 16 times under the conditions of 120g/L glucose and xylose (2:1, g:g), obtain
The bacterial strain P9G4#16 stable to character.
Embodiment 2
The growth differences of P9G0 and P9G4#16 under high concentration sugar substrate are compared in ferment tank culture
The preparation of fermentation medium:
In fermentation medium constituent, carbon source is that the mixed sugar of 120g/L, wherein glucose and xylose mass ratio are 2:
1；Nitrogen source is 5g/L Yeast extract；Remaining B, D, E liquid constituent is consistent with seed culture based component.It is specifically prepared
The operation processing methods such as process and seed culture medium prepare, sterilizing are consistent.
Fermentation condition: pressing 10% inoculum concentration, and seed liquor access is equipped with to the NBS company 5L Full-automatic reverse of 2L fermentation medium
Answer kettle.Before inoculation, reaction kettle is first passed through nitrogen 30min, is passed through nitrogen 30min after inoculation again, to guarantee yeasting anaerobic.
Fermentation liquid continuously cultivated 100h in the case where 55 DEG C of revolving speed 150rpm of temperature, every 6-12 hours sampling and measuring thallus OD600。
It will be seen from figure 1 that starting strain P9G0 can hardly be grown in the mixing sugar culture-medium of 120g/L, the lag phase
Indefinite extension；And the growthing lag phase of the bacterial strain P9G4#16 after taming is only 12h, the dry cell weight in plateau is also up to
1.82g/L.By domestication, P9G4#16 has obviously adapted to the environment of high concentration sugar substrate, can carry out generation using substrate rapidly
It thanks, almost without the lag phase, energy consumption is reduced in industrial production.
Embodiment 3
The growth differences and Difference of Metabolism of P9G0 and P9G4#16 under high concentration sugar substrate are compared in ferment tank culture
The preparation of fermentation medium:
In fermentation medium constituent, carbon source is that the mixed sugar of 100g/L, wherein glucose and fructose mass ratio are 9:
1；Nitrogen source is 5g/L Yeast extract；PB buffer to 20mM is added as buffer environment.Its specific process for preparation and kind
The operation processing methods such as sub- culture medium preparation, sterilizing are consistent.
Fermentation condition: pressing 10% inoculum concentration, and seed liquor access is equipped with to the NBS company 5L Full-automatic reverse of 2L fermentation medium
Answer kettle.Before inoculation, reaction kettle is first passed through nitrogen 30min, is passed through nitrogen 30min after inoculation again, to guarantee yeasting anaerobic.
Fermentation liquid was continuously cultivated in the case where 55 DEG C of revolving speed 150rpm of temperature, every 6-12 hours sampling and measuring thallus OD600And
Measure other metabolins.
Pass through OD600Calculate somatic cells dry weight formula be
Y=0.421X-0.006 (R2=0.99)
Wherein, Y is dry cell weight (DCW), g/L；X is OD600。
The detection that metabolite ethyl alcohol, acetic acid, lactic acid and sugar consume is surveyed using 2695 high pressure liquid chromatography of Waters (HPLC)
It is fixed.
1. the detection of glucose, xylose consumption
Chromatographic column: Aminex HPX-87P (Biorad)
Mobile phase: ultrapure water
Flow velocity: 0.6ml/min
Column temperature: 60 DEG C
Detector temperature: 40 DEG C
Sample preparation: 1g CaCO is added in 2ml fermentation liquid3, 1min is vibrated, 5min is centrifuged at 16,000g, supernatant is taken to use
0.22 μm of film filtering.For detecting residual sugar.
Sample volume: 10 μ l
Detector: Composition distribution
2. the detection of metabolite ethyl alcohol, 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: 100 μ l 10%H are added in 1.9ml fermentation liquid2SO4, it is centrifuged 5min at 16,000g, supernatant is taken to use
0.22 μm of film filtering.For detecting acid and ethyl alcohol.Sample volume: 10 μ l
Detector: Composition distribution.
3, lactic acid, acetic acid and the ethyl alcohol (table 1) in tunning are measured.The result shows that starting strain P9G0 is in 100g/L
Mixing sugar culture-medium in can hardly grow, lag phase indefinite extension；And the growthing lag phase of the bacterial strain P9G4#16 after taming
Only 12h, and end-stage cells dry weight of fermenting also is up to 3.34g/L.The yield and conversion ratio of lactic acid are also correspondingly improved, i.e.,
Final lactic acid concn reaches 77.7g/L, and rotational rate of lactic acid reaches 0.83g/g.Acetic acid, by-product are nearly no detectable in tunning
Object is simple.
The domestication of table 1 front and back bacterial strain compares by tank fermentation results on substrate of 100g/L mixed sugar
Embodiment 4
P9G4#16 utilizes food waste object hydrolyzate fermenting lactic acid
The hydrolyzate of certain food waste object is unsterilised directly as fermentation medium.After measured, wherein glucose contains
Amount is 83.6 ± 5.9g/L, and fructose content is 9.5 ± 0.8g/L.It the use of concentration is 5g/L Angel Yeast extract (Angel
Yeast extract) it is nitrogen source；PB buffer to 20mM is added as buffer environment.
Fermentation condition: pressing 10% inoculum concentration, and seed liquor access is equipped with to the NBS company 5L Full-automatic reverse of 2L fermentation medium
Answer kettle.Before inoculation, reaction kettle is first passed through nitrogen 30min, is passed through nitrogen 30min after inoculation again, to guarantee yeasting anaerobic.
Fermentation liquid was continuously cultivated in the case where 55 DEG C of revolving speed 150rpm of temperature, every 6-12 hours sampling and measuring thallus OD600And
Measure other metabolins.
Fermentation results show (Fig. 2), and the bacterial strain P9G4#16 after domestication can be in the case where culture medium be unsterilised, well
Ground utilizes the hydrolyzate direct fermentation of food waste object, and the growthing lag phase is only 12h.The yield of lactic acid is 78.35g/L, is turned
Rate is 0.85g/g, and fermentation latter stage Carbon balance reaches 97.0 ± 6.This shows that the naturalized strain has overcome in high concentration sugar
Substrate lower lag phase longer disadvantage, and can use culture medium raw material fermentation, it has broad prospects in the industrial production.
Claims (10)
1. a kind of thermophilic anaerobic bacillus, which is characterized in that the bacterial strain is Thermoanaerobacterium
AotearoenseP9G4#16, in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preservation is compiled
Number be CGMCC NO.10833, the deposit date is on Mays 21st, 2015.
2. application of the bacterial strain described in claim 1 in production lactic acid.
3. application according to claim 1, which is characterized in that preparation thermophilic anaerobic bacillus CGMCC NO.10833's first
Then seed liquor is transferred in fermentation medium, under anaerobic stir culture by seed liquor with 10-15%w/w inoculum concentration,
45-60 DEG C of cultivation temperature, incubation time is 70-200 hours, finally goes out lactic acid from separation of fermentative broth.
4. application according to claim 3, which is characterized in that the total sugar concentration of the fermentation medium is 80-150g/L.
5. application according to claim 3, which is characterized in that the initial pH of the fermentation medium is 5.5-6.5, described
Speed of agitator is 100-200rpm.
6. according to application described in claim 3 or 4 or 5, which is characterized in that the fermentation medium components are as follows: glucose 80-
100, xylose 40-60, urea 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/L.
7. application according to claim 6, which is characterized in that the fermentation medium further includes following ingredient: citric acid
Tripotassium salt 1-4, Citric Acid Mono 0.5-3, sodium sulphate 0.5-3, potassium dihydrogen phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate
0.5-3, four water frerrous chloride 0.05-0.5, calcium chloride dihydrate 0.05-0.4, a water cysteine hydrochloric acid 0.5-3, two hydrochloric acid pyrroles
Tremble amine 0-0.1, p-aminobenzoic acid 0-0.01, D-Biotin 0-0.01, vitamin B12 0-0.01, vitamin B1 0-0.01,
Unit g/L.
8. according to application described in claim 3 or 4 or 5, which is characterized in that the preparation of the seed liquor: by thermophilic anaerobic bar
Bacterium CGMCC NO.10833 is placed in seed culture medium, at 45-60 DEG C, revolving speed 100-200rpm, cultivates 8-24h.
9. application according to claim 8, which is characterized in that the seed culture medium are as follows: glucose 2-4, xylose 2-4,
Urea 1-10, yeast extract 1-5, ammonium chloride 0.5-3, unit g/L.
10. application according to claim 9, which is characterized in that the seed culture medium further includes following ingredient: citric acid
Tripotassium salt 1-4, Citric Acid Mono 0.5-3, sodium sulphate 0.5-3, potassium dihydrogen phosphate 0.5-3, sodium bicarbonate 1-4, magnesium chloride hexahydrate
0.5-3, four water frerrous chloride 0.05-0.5, calcium chloride dihydrate 0.05-0.4, a water cysteine hydrochloric acid 0.5-3, two hydrochloric acid pyrroles
Tremble amine 0-0.1, p-aminobenzoic acid 0-0.01, D-Biotin 0-0.01, vitamin B12 0-0.01, vitamin B1 0-0.01,
Unit g/L.
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| High efficiency hydrogen production from glucose/xylose by the ldh-deleted Thermoanaerobacterium strain;Shuang Li，et al;《Bioresource Technology》;20100715;第22卷(第101期);全文 * |
| 嗜热厌氧杆菌利用甘蔗渣发酵产氢及其高糖耐受菌株的转录特征研究;赖志城;《中国优秀硕士学位论文全文数据库》;20150315(第3期);第16-17页、第39-40页、第47-48页 * |
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