CN104911128A - Thermoanaerobacterium aotearoense and application thereof in lactic acid production - Google Patents
Thermoanaerobacterium aotearoense and application thereof in lactic acid production Download PDFInfo
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Abstract
The invention discloses a Thermoanaerobacterium aotearoense and an application thereof in lactic acid production. The strain is Thermoanaerobacterium aotearoense P9G4#16, and already collected in the China General Microbiological Culture Collection Centre, with a collection number of CGMCC NO. 10833 and a collection date of May 21, 2015. The strain P9G4#16 is capable of growing and being fermented under a high-concentration sugar substrate, and the yield of lactic acid is also increased. The strain disclosed by the invention is good in genetic stability and stable in yield traits; moreover, a fermentation lag phase is greatly shortened, thus saving energy consumption in a technique process, and greatly increasing the industrialization value.
Description
Technical field
The present invention relates to a kind of thermophilic anaerobic bacillus and producing the application in lactic acid.To the present invention relates to glucose, wood sugar or fructose as main raw material, utilize the method for thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoenseSCUT27/ Δ pta-ack) mutant strain fermenting lactic acid.
Background technology
Lactic acid is one of universally acknowledged three large organic acids, is widely used in the fields such as food, pharmacy, leather, makeup, chemical industry.85% of lactic acid total amount is applied to the relevant field of food and food, and remaining 15% for non-food industries.The lactic acid in the current world 90% is produced by fermentation using bacteria, uses this important industrial raw material of cheap biomass material fermenting lactic acid to have boundless prospect.
In biomass resource, except the glucose deriving from starchy material is used widely in fermentation industry, other renewable carbohydrate sources is not yet utilized effectively, and one of its reason is exactly that microorganism is poor to xylose utilization ability.The various chemical that utilization is cheap, the wood fibre resource fermentative production of rich reserves comprises lactic acid and substitute energy have very wide application prospect, be expected to the cost reducing current biological process synthesis of chemicals, thus promote its competitive power relative to chemical synthesis process.
High cost is with the major obstacle that cellulosic class material produces lactic acid for fermenting raw materials.Adopt thermophilc anaerobe (Thermoanaerobacterium) fermentation can solve this difficulty preferably.Research according to people such as Sommer P. shows (Sommer P.et al., Biochemical Society Transactions, 2004,32 (Pt 2): 283-289), with this bacterial strain production of chemicals, there is many advantages: (1) high temperature continuously ferments, and productive rate is high down in production, and inhibit the infection of miscellaneous bacteria, lactic acid can be produced by raw material fermentation.Higher to the sterility requirements of pure-blood ferment in industrial production, significantly add Financial cost.But when adopting thermophile bacteria fermentative production, the most of microbial growth of its high growth temperature surround inhibition, therefore can raw material fermentation, reduces production cost.(2) energy degraded cellulose hemicellulose biomass, are hydrolyzed into glucose and xylose and utilize, generating chemical and/or biofuel, reduce the addition of cellulase; (3) main metabolic product is few and simple, and carbon source has the high mole rate of recovery; (4) the hydrolyzed solution direct fermentation of farm crop and castoff can be utilized, alleviate environmental stress and reduce production cost.
A strain thermophilic anaerobic bacillus (Li is screened in contriver's early-stage Study, S., et al., Bioresource Technology, 2010,101 (22): 8718-8724), this bacterial strain can effectively utilize the fermenting substrate such as glucose, wood sugar to accumulate ethanol, lactic acid, hydrogen etc.By carrying out genetic modification to it, knock out phosphotransacetylase gene and Acetokinase gene, after pathways metabolism transformation, in fermentor tank under the fermentation of 50g/L glucose substrate, mutant strain lactic acid production improves 1.21 times, reaches 47.17g/L, produce acetic acid hardly, simultaneously ethanol production obviously declines (Yang et al.Biotechnology for Biofuels 2013,6:124, Li Shuan; Wang Jufang; Yang Xiaofeng; Wang little Ning; Utilize the engineering bacteria of wood-sugar fermentation production optical pure L-lactic acid in flat scholar is a kind of and builds .201110453684.7).But this bacterial strain is in high concentration sugar substrate situation, be subject to the impact of high sugared osmotic stress, cause bacterium almost can not grow, fermentation period is excessively of a specified duration, is not suitable for being applied in industrial production.And the tolerance of high concentration sugar substrate can lay the foundation for realizing cellulose series biomass simultaneous saccharification and fermentation production lactic acid.
From industrialization angle, if a strain can be screened can tolerate high concentration sugar substrate, and the thermophilic anaerobic bacillus strain that proterties is stable, namely can be applicable to high concentration sugar fermenting substrate, shorten the fermentation lag phase, reduce energy consumption, its application in suitability for industrialized production could be promoted like this.
Summary of the invention
The object of the present invention is to provide a kind of thermophilic anaerobic bacillus and producing the application in lactic acid.
In the present invention, the starting strain for screening be laboratory screening, sudden change and preserve thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoenseSCUT27/ Δ pta-ack) P9G0 (kalamycin resistance).
High concentration substrate tolerance acclimation method provided by the present invention, comprises the steps:
(1) cultivate thermophilic anaerobic bacillus P9G0, obtain seed liquor;
(2) seed liquor of above-mentioned steps (1) is transferred in the substratum of low concentration substrate with 10-15%w/w inoculum size, filter out at this concentration can inoculation 24h after thalline OD
600grow to 1.5 and the stable thermophilic anaerobic bacillus of proterties;
(3) seed liquor of above-mentioned steps (2) is transferred in the substratum of higher concentration substrate with 10-15%w/w inoculum size, filter out at this concentration can inoculation 24h after thalline OD
600grow to 1.5 and the stable thermophilic anaerobic bacillus of proterties;
(4) repeating step (3), progressively improve concentration of substrate until reach 120g/L, obtain under this culture medium condition inoculation 24h after thalline OD
600grow to 1.5 and the stable thermophilic anaerobic bacillus P9G4#16 (kalamycin resistance) of proterties.
Described P9G4#16 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: acetate metabolism Blocked-mutant, has kalamycin resistance, and kalamycin resistance concentration is 20-100ng/ μ l.80-150g/L sugar concentration of substrate can be tolerated.
P9G4#16 bacterial strain is producing the application in lactic acid, first the seed liquor of thermophilic anaerobic bacillus CGMC10833 is prepared, then 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 70-200 hour, finally goes out lactic acid 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 100-200rpm.Preferably, 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 0-0.01, vitamin B12 0-0.01, VITMAIN B1 0-0.01, unit g/L.
The preparation of described seed liquor: thermophilic anaerobic bacillus CGMC10833 is placed in seed culture medium, at 45-60 DEG C, rotating speed is 100-200rpm, cultivates 8-24h.
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.
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, VITMAIN B1 0-0.01, unit g/L.
Compared with prior art, the present invention has following beneficial effect:
(1) with this laboratory screening, build the thermophilic anaerobic bacillus P9G0 that obtains for parental plant, carry out high sugared substrate tolerance domestication by the method progressively improving concentration of substrate, obtain mutant strain P9G4#16.This mutant strain has following hereditary property: phosphotransacetylase and E.C. 2.7.2.1 defective type, kalamycin resistance, and sugared concentration of substrate tolerance range is more than 80-150g/L.
(2) the present invention filters out the thermophilic anaerobic bacillus strain of enduring high-concentration sugar substrate.Compared with the bacterial strain before domestication, solve the defect that it can not grow under high concentration sugar substrate, shorten fermentation period, improve fermentation capacity, and it is good to have genetic stability, yield traits is stablized, and the fermentation lag phase shortens greatly, save the power consumption in technological process, greatly improve its industrial value.
(3) with 100g/L glucose, fructose mixing sugar for substrate 5L ferment tank produce lactic acid, the lag phase is only 12h, and lactic acid final concentration reaches 77.7g/L, and transformation efficiency reaches 0.83g/g.The invention provides the mutant of screening, can utilize high sugared substrate quick fermentation accumulation target product lactic acid, and the by product produced is few, technique is amplified comparatively easy, is suitable for suitability for industrialized production.
The mutant strain that the present invention screens is Thermoanaerobacterium aotearoenseP9G4#16, in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (being called for short CGMCC), deposit number is CGMCC NO.10833, and preservation date is on May 21st, 2015.Preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Accompanying drawing explanation
Fig. 1 is the growth curve chart of resistance to height sugared bacterial strain P9G4 and starting strain P9G0 under 120g/L high concentration sugar substrate after domestication.-■-P9G4，-●-P9G0。Wherein sugared substrate is glucose and xylose (2:1, g:g).
Fig. 2 is that P9G4#16 utilizes food waste thing hydrolyzed solution to grow and fermentation situation.This hydrolyzed solution main component is glucose and fructose (9:1, g:g).-■-glucose ,-●-fructose ,-△-lactic acid ,-◇-ethanol ,-☆-dry cell weight.
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 aotearoenseSCUT27/ Δ pta-ack) P9G0 (kalamycin resistance).
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.
First starting strain P9G0 is through 60g/L substratum (glucose and xylose mass ratio is 2:1) anaerobism shake-flask culture, treats strain growth light absorption value OD
600be 1.5 or more, the anaerobism Shake flask medium of transferring new, repeat number generation, until obtain at this concentration can after inoculation 24h thalline OD
600grow to 1.5, and the bacterial strain P9G1 that proterties is stable;
The same with step (1) method, under 81g/L glucose and xylose (2:1, g:g) condition, obtain bacterial strain P9G2;
The same with step (1) method, under 102g/L glucose and xylose (2:1, g:g) condition, obtain bacterial strain P9G3;
The same with step (1) method, go down to posterity under 120g/L glucose and xylose (2:1, g:g) condition after 16 times, obtain the bacterial strain P9G4#16 that proterties is stable.
Embodiment 2
Ferment tank is cultivated and is compared the growth differences of P9G0 and P9G4#16 under high concentration sugar substrate
The preparation of fermention medium:
In fermention medium moiety, carbon source is the mixing sugar of 120g/L, and wherein glucose and wood sugar mass ratio are 2:1; Nitrogenous source is 5g/L Yeast extract; All the other B, D, E liquid 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.
Fermentation condition: by 10% inoculum size, is equipped with the full-automatic reactor of NBS company 5L of 2L fermention medium by seed liquor access.Before inoculation, reactor first passes into nitrogen 30min, passes into nitrogen 30min again after inoculation, to ensure yeasting anaerobic.Fermented liquid is temperature 55 DEG C, and cultured continuously 100h when rotating speed 150rpm, every 6-12 hour sampling and measuring thalline OD
600.
As can be seen from Figure 1, starting strain P9G0 almost can not grow in the mixing sugar substratum of 120g/L, lag phase indefinite extension; And the growthing lag phase of bacterial strain P9G4#16 after domestication is only 12h, be in the dry cell weight of plateau also up to 1.82g/L.Through domestication, P9G4#16 has obviously adapted to the environment of high concentration sugar substrate, substrate can be utilized rapidly to carry out metabolism, almost do not have the lag phase, in industrial production, reduce energy consumption.
Embodiment 3
Ferment tank is cultivated and is compared the growth differences of P9G0 and P9G4#16 under high concentration sugar substrate and Difference of Metabolism
The preparation of fermention medium:
In fermention medium moiety, carbon source is the mixing sugar of 100g/L, and wherein glucose and fructose mass ratio are 9:1; Nitrogenous source is 5g/L Yeast extract; Add PB buffer to 20mM as buffer environment.The operation processing methods such as its concrete process for preparation is prepared with seed culture medium, sterilizing are consistent.
Fermentation condition: by 10% inoculum size, is equipped with the full-automatic reactor of NBS company 5L of 2L fermention medium by seed liquor access.Before inoculation, reactor first passes into nitrogen 30min, passes into nitrogen 30min again after inoculation, to ensure yeasting anaerobic.Fermented liquid is temperature 55 DEG C, and cultured continuously when rotating speed 150rpm, every 6-12 hour sampling and measuring thalline OD
600and measure other metabolites.
Pass through OD
600the formula calculating somatic cells dry weight is
Y＝0.421X-0.006(R
2＝0.99)
Wherein, Y is dry cell weight (DCW), g/L; X is OD
600.
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, starting strain P9G0 almost can not grow in the mixing sugar substratum of 100g/L, lag phase indefinite extension; And the growthing lag phase of bacterial strain P9G4#16 after domestication is only 12h, and fermentation end-stage cells dry weight is also up to 3.34g/L.The output of lactic acid and transformation efficiency also have corresponding raising, and namely final lactic acid concn reaches 77.7g/L, and rotational rate of lactic acid reaches 0.83g/g.Almost can't detect acetic acid in tunning, by product is simple.
Table 1 tames front and back bacterial strain with 100g/L mixing sugar for tank fermentation results on substrate compares
Embodiment 4
P9G4#16 utilizes food waste thing hydrolyzed solution fermenting lactic acid
Using unsterilised for the hydrolyzed solution of certain food waste thing directly as fermention medium.After measured, wherein glucose content is 83.6 ± 5.9g/L, and fructose content is 9.5 ± 0.8g/L.Working concentration is 5g/L Angel Yeast extract (Angel yeast extract) is nitrogenous source; Add PB buffer to 20mM as buffer environment.
Fermentation condition: by 10% inoculum size, is equipped with the full-automatic reactor of NBS company 5L of 2L fermention medium by seed liquor access.Before inoculation, reactor first passes into nitrogen 30min, passes into nitrogen 30min again after inoculation, to ensure yeasting anaerobic.Fermented liquid is temperature 55 DEG C, and cultured continuously when rotating speed 150rpm, every 6-12 hour sampling and measuring thalline OD
600and measure other metabolites.
Fermentation results shows (Fig. 2), and the bacterial strain P9G4#16 after domestication when substratum is unsterilised, can utilize the hydrolyzed solution direct fermentation of food waste thing well, and the growthing lag phase is only 12h.The output of lactic acid is 78.35g/L, and transformation efficiency is 0.85g/g, and fermentation carbon balance in latter stage reaches 97.0 ± 6.This shows that this naturalized strain has overcome the shortcoming that the lag phase is longer under high concentration sugar substrate, and can utilize substratum raw material fermentation, has broad prospects in the industrial production.
Claims (10)
1. a thermophilic anaerobic bacillus, it is characterized in that, this bacterial strain is Thermoanaerobacterium aotearoenseP9G4#16, in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC NO.10833, and preservation date is on May 21st, 2015.
2. bacterial strain described in claim 1 is producing the application in lactic acid.
3. application according to claim 1, it is characterized in that, first the seed liquor of thermophilic anaerobic bacillus CGMC10833 is prepared, then 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 70-200 hour, finally goes out lactic acid from separation of fermentative broth.
4. application according to claim 3, is characterized in that, the total sugar concentration of described fermention medium is 80-150g/L.
5. application according to claim 3, is characterized in that, the initial pH of described fermention medium is 5.5-6.5, and described mixing speed is 100-200rpm.
6. the application according to claim 3 or 4 or 5, it 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. application 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 application according to claim 3 or 4 or 5, is characterized in that, the preparation of described seed liquor: thermophilic anaerobic bacillus CGMC10833 is placed in seed culture medium, and at 45-60 DEG C, rotating speed is 100-200rpm, cultivates 8-24h.
9. application 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. application 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.
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| CN114181859B (en) * | 2021-12-10 | 2023-09-01 | 中国科学院青岛生物能源与过程研究所 | Geobacillus stearothermophilus and method for producing lactic acid by using lignocellulose |
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