CN109868296A - A method of continuously ferment and produces long-chain biatomic acid - Google Patents
A method of continuously ferment and produces long-chain biatomic acid Download PDFInfo
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Abstract
The invention discloses a kind of methods continuously fermented and produce long-chain biatomic acid, after the 100-150h that ferments, continuous current adding substrate, while continuous flow adds the solution containing nutritive salt；And the dilution rate when fermentation is 0.001-0.006h‑1, preferably 0.0016-0.0054h‑1.Method of the invention guarantees thallus long period of activity on the basis of not increasing any equipment, to guarantee the continuous production of long-chain biatomic acid, so that long-chain biatomic acid yield increases considerably, yield also be increased.
Description
Technical field
The present invention relates to a kind of methods continuously fermented and produce long-chain biatomic acid.
Background technique
Long-chain biatomic acid (LDCA, HOOC (CH2) nCOOH, n >=7) application field is extensive, spy can be synthesized using it as raw material
Kind nylon (polyamide), fine perfumery, high-grade hot melt adhesive, cold resistant plasticizer, senior lubricant, advanced antirust agent, advanced paint
With coating etc..
In general, long-chain biatomic acid can produce to obtain with chemical synthesis or biological fermentation process.Chemical synthesis process
Synthetic route is long, and reaction condition is stringent, needs to carry out under high-temperature and high-pressure conditions, and harsher to catalyst requirement, because
This is of less types with the long-chain biatomic acid that chemical method synthesizes at industrial scale, only a small number of product such as 12 carbon long-chain biatomic acids
Kind.Biological fermentation process converts to obtain long-chain biatomic acid by microbial fermentation using long chain alkane as substrate；Its production process is normal
It is carried out under temperature, condition of normal pressure, and can be with large-scale production such as a variety of long-chain biatomic acids from C9 to C18.Therefore, long-chain binary
For the biological fermentation process of acid is produced compared to chemical synthesis, advantage is self-evident.
Currently, having had certain Research foundation for biological fermentation process production long-chain biatomic acid.For example, research initial stage,
The yield of long-chain biatomic acid is improved by carrying out mutagenesis to the bacterial strain for screening obtained production long-chain biatomic acid in oil field；Example again
Such as, by the way that the key enzyme in long-chain biatomic acid synthesis process, (such as P450 monooxygenase, FAO alcohol oxidase, FaldDH aldehyde are de-
Hydrogen enzyme etc.) research improve the yield of long-chain biatomic acid.Foreign countries have focused largely on to bacterium the research of long-chain biatomic acid bacterial strain
Strain carries out genetic modification, by blocking or weakening fatty acid beta oxidation related enzyme systems, strengthens fatty acid α-ω oxidative system to mention
The yield of high product.In terms of patent report, CN91109895.X, CN98121084.8, CN200310104982.0,
CN200410018255.7, CN200610029784.6 both provide the method for Production of Long Chain Dicarboxylic Acids Through Micro-Biological Fermen.Patent
CN201410065873.0 and CN201410067073.2, which is disclosed, carries out coupling fermentation production long-chain two using UF membrane thallus
The technology of first acid.There are also some reports to disclose the method etc. of string tank operation fermenting and producing long-chain biatomic acid.Patent
Although CN201510802343.4 discloses a kind of continuously ferment and produces the method and device of long-chain biatomic acid, but its fermentation liquid
It not only needs to increase film filter but also is coupled with seeding tank, continuously fermented by the seed liquor realization for periodically mending fresh
Process not only increases cost, and increases microbiological contamination risk.
Summary of the invention
The present invention provides a kind of method continuously fermented and produce long-chain biatomic acid, and method of the invention is not increasing any set
Guarantee thallus long period of activity on the basis of standby, to guarantee the continuous production of long-chain biatomic acid, so that long-chain biatomic acid yield is big
Amplitude increases, and yield also increased.
An object of the present invention, a method of continuously ferment and produces long-chain biatomic acid, after the 100-150h that ferments, with
0.001-0.006h-1The continuous current adding substrate of dilution rate, while continuous flow adds the solution containing nutritive salt.
One of the difficult point that long-chain biatomic acid continuously ferments is how to guarantee in prolonged fermentation process, keeps bacterium
Body activity and lasting production acid, and guarantee high binary acid acid production speed, yield and yield.Inventor passes through numerous studies, discovery
Using specific dilution rate, it can be realized good effect, thus obtain technical solution of the present invention.
Below to the further preferred technical solution of above-mentioned technical proposal, it is further described.
A preferred technical solution of the invention, the dilution rate are 0.0016-0.0054h-1。
A preferred technical solution of the invention, the substrate include the normal alkane of C9-C18, linear saturated fatty acids,
One of linear saturated fatty acids ester and salts of straight-chain saturated fatty acids are a variety of；Preferably include normal alkane, the straight chain of C11-C16
One of saturated fatty acid, linear saturated fatty acids ester and salts of straight-chain saturated fatty acids are a variety of；More preferably C11, C12,
In the normal alkane of C13, C15, C14 or C16, linear saturated fatty acids, linear saturated fatty acids ester and salts of straight-chain saturated fatty acids
One kind.
Further, the flow rate of substrate persistently produces microbial activity, binary acid acid production speed, yield and yield etc.
Acid effect, also play the role of it is positive, guarantee both reach certain balance, can be realized excellent effect.
A preferred technical solution of the invention, the flow rate V of the substrate(substrate)For 2-5mL/L/h.
A preferred technical solution of the invention, before the current adding substrate, controlling substrate content in the fermentation liquid is
2% (v/v) or more, the percentage are the percent by volume that the substrate accounts for fermentation liquid.
A preferred technical solution of the invention, the solution containing nutritive salt include following ingredient: glucose
0.0%-1.0% (w/v), corn pulp 0.05%-0.15% (w/v), yeast extract 0.0%-0.3% (w/v), potassium dihydrogen phosphate
0.005%-0.015% (w/v), urea 0.005%-0.015% (w/v) and ammonium sulfate 0.01%-0.15% (w/v), remaining
For water；The percentage is mass volume ratio, it may be assumed that each ingredient accounts for the mass volume ratio of solution, and fermentation arts technical staff knows
Road, the mass volume ratio are g/100mL.
Further, the flow rate of the solution containing nutritive salt to microbial activity, binary acid acid production speed, yield and obtains
Rate etc. persistently produce acid effect, also play the role of it is positive, guarantee both reach certain balance, can be realized excellent effect.
A preferred technical solution of the invention, the flow rate V of the solution containing nutritive salt(solution containing nutritive salt)For
0.5-3mL/L/h。
A preferred technical solution of the invention, bacterial strain used in the fermentation are candida tropicalis, such as can
With are as follows: Candida tropicalis (Candida tropicalis) bacterial strain CAT H1614, deposit number are CCTCC M
2015303。
A preferred technical solution of the invention, the long-chain biatomic acid include: chemical formula HOOC (CH2) nCOOH
Binary acid, wherein n >=7, including but not limited to: azelaic acid (HOOC (CH2)7COOH), decanedioic acid (HOOC (CH2)8COOH), ten
One carbon dicarboxylic acid (HOOC (CH2)9COOH, 1,9- nine carbon dicarboxylic acids or 1,11- eleven carbon diacids, the present invention in label for
" DC11 "), dodecanedicarboxylic acid (HOOC (CH2)10COOH, 1,10- ten carbon dicarboxylic acids or 1,12- dodecanedicarboxylic acid, the present invention
Middle label is DC12 "), tridecanyldicarboxylic acid (HOOC (CH2)11COOH, 1,11- ten one carbon dicarboxylic acids or 1,13 carbon two of 13-
First sour, label is DC13 "), tetradecane diacid (HOOC (CH in the present invention2)12COOH, 1,12- ten two carbon dicarboxylic acids or 1,
14- tetradecane diacid, the present invention in label be DC14 "), pentadecane binary acid (HOOC (CH2)13COOH, 1,13- ten three carbon
Label is DC15 "), 16-dicarboxylic acid (HOOC (CH in dicarboxylic acids or 1,15- pentadecane binary acid, the present invention2)14COOH,
1,14- ten four carbon dicarboxylic acids or 1,16- 16-dicarboxylic acid, label is DC16 " in the present invention), seventeen carbon diacids (HOOC
(CH2)15COOH, 1,15- pentadecane dicarboxylic acids or 1,17- seventeen carbon diacids, label is DC17 " in the present invention) and 18 carbon
Binary acid (HOOC (CH2)16COOH, 1,16- ten six carbon dicarboxylic acids or 1,18- octadecane diacid, in the present invention label for
" DC18 ") one of or it is a variety of.
A preferred technical solution of the invention, fermentating liquid volume is fermenter volume in the continuous ferment process
50-80%.Such as: when being continuously fermented using 30L fermentor, by substrate and the feed rate of the solution containing nutritive salt,
And discharge velocity, maintaining fermentation cylinder for fermentation liquid product is 15-22L.
The second object of the present invention, a kind of production method of long-chain biatomic acid, the production method include as described above
Continuously ferment the method for producing long-chain biatomic acid.
A preferred technical solution of the invention also carries out following behaviour before continuously fermenting and producing long-chain biatomic acid
Make:
Seed liquor is inoculated into fermentation medium, intermittent fed-batch fermentation is carried out, starts 10-100h in fermentation, is added
Substrate guarantees that substrate content is 2v/v% or more in fermentation liquid, and the percentage is the volume basis that the substrate accounts for fermentation liquid
Than.
Wherein, a preferred technical solution of the invention, the initial volume after the inoculation are fermenter volume
30%-50% (v/v), the percentage are that the volume of culture medium accounts for the percentage of fermenter volume.For example, when fermentor is
When the fermentor of 30L, initial volume 10-15L.
Wherein, a preferred technical solution of the invention, the inoculum concentration of the fermentation is 10%-30% (v/v), described
Percentage is the percent by volume relative to fermentation initial volume.
Wherein, a preferred technical solution of the invention, the cell concentration OD when fermentation620For 9-24 (dilution 30
Measured value again is 0.3-0.8).
Wherein, substrate can be added for batch in a preferred technical solution of the invention, the addition substrate.
Wherein, a preferred technical solution of the invention, the temperature of the fermentation are 28-32 DEG C.
Wherein, a preferred technical solution of the invention, the ventilation quantity of the fermentation are 0.3-0.7vvm.
Wherein, the tank pressure of a preferred technical solution of the invention, the fermentation is 0.05-0.14MPa, the pressure
For gauge pressure.
Wherein, a preferred technical solution of the invention, the substrate of fermentation starting addition 0-10% (v/v), institute
Stating percentage is the percent by volume relative to fermentation initial volume.
Wherein, a preferred technical solution of the invention, when fermentation, are stirred.
Wherein, a preferred technical solution of the invention, the dissolved oxygen amount of the fermentation are 10% or more.
Wherein, a preferred technical solution of the invention, the pH value of the fermentation are 7-8.5.
Of the invention continuously fermenting produces the production method of the method and the long-chain biatomic acid including it of long-chain biatomic acid,
Do not increase and guarantee thallus long period of activity on the basis of any equipment, to guarantee the continuous production of long-chain biatomic acid, so that long-chain
Binary acid yield increases considerably, and yield also increased.
Specific embodiment
Below by embodiment, the present invention is described in detail, so that the features and advantages of the present invention become apparent from.But it answers
This points out that for embodiment for understanding design of the invention, the scope of the present invention is not limited only to reality listed herein
Apply example.
In the present invention, the binary acid concentration in culture solution is measured, technology well known to those skilled in the art, example can be used
The measuring method as disclosed in Chinese patent ZL95117436.3.Specifically, the pH to 3.0 of fermentation liquid is adjusted with hydrochloric acid solution,
Then plus 100mL ether is for the binary acid in extractive fermentation liquid, then uses evaporation to remove ether, obtains binary acid powder；
In ethanol by the dissolution of obtained binary acid powder, and with the NaOH solution of 0.1mol/L it titrates, finally obtains in fermentation liquid
Binary acid titer.
In the present invention, unless otherwise indicated, " about " Lai Xiuzheng is used in the parameters such as component, reaction condition, concentration
Numerical value.Therefore numerical parameter in the specification and in the claims is an approximation, depends on the desired property of the present invention
Energy.At least, it is not considered as the limitation of doctrine of equivalents application protected to the claims in the present invention.At least, the number of each parameter
With rounding up, the digit of effective digital historically determines value.Although numerical value in a particular embodiment is as far as possible
It is accurate to accomplish, but since the systematic error of experimental test procedures necessarily causes any data that can all have certain error.
1 bacterial strain
Bacterial strain uses therefor of the present invention is one plant of Candida tropicalis (Candida tropicalis) bacterial strain CAT H1614,
Its deposit number is CCTCC M 2015303.
2 culture mediums
There are mainly three types of culture mediums used in of the invention.
Seed culture medium: sucrose 1%-3%, corn pulp 0.15%-1%, yeast extract 0.2%-1.5%, KH2PO40.4%-
1.5%, urea 0.05%-0.5%.
Fermentation medium: glucose 1%-5%, corn pulp 0.1%-0.9%, yeast extract 0.1%-0.5%, potassium nitrate
0.05%-1.2%, potassium dihydrogen phosphate 0.05%-1.0%, urea 0.05%-0.3%, ammonium sulfate 0.05%-0.3% and chlorine
Change sodium 0.05%-0.2%.
Supplemented medium: there are two types of supplemented mediums, and one kind is substrate；It is a kind of for the solution (glucose containing nutritive salt
0.0%-1.0%, corn pulp 0.05%-0.15%, yeast extract 0.0%-0.3%, potassium dihydrogen phosphate 0.005%-0.015%,
Urea 0.005%-0.015%, ammonium sulfate 0.01%-0.15%, remaining is water；The percentage is mass volume ratio, g/
100mL)。
The culture medium 20min that sterilizes at 121 DEG C is spare.
3 cultural methods
3.1 shake-flask seed culture process are that the glycerol tube strain of candida tropicalis is taken to be inoculated in equipped with seed culture medium
In 500mL triangular flask (liquid amount 50-100mL), initial pH is 6.0-6.5, at 28-32 DEG C, 200-250rpm shaking table culture 1-2
It.
3.2 seed tank culture techniques are to take shake-flask seed access equipped with (liquid amount 5- in the 10L seeding tank of seed culture medium
8L), at inoculum concentration 10%-30%, 28-32 DEG C, ventilation quantity 0.3-0.7vvm, tank presses 0.05-0.14MPa, keeps certain
Mixing speed controls the DO10% or more of seed culture process, cultivates 15-30h, cultivates the standard of mature seed as 30 times of dilution
OD afterwards620For 0.5-1.0.
4 fermentation process
Zymotechnique is that will be inoculated into the 30L fermentor containing fermentation medium in the resulting seed liquor of seed tank culture
In, initial volume is 10-15L, inoculum concentration 10%-30% (v/v, opposite initial volume of fermenting), fermentation starting addition after inoculation
The substrate (such as can be alkane) of 0-10% (v/v, opposite initial volume of fermenting), 28-32 DEG C of fermentation processes temperature are led to
Air quantity is about 0.3-0.7vvm, and tank pressure (gauge pressure) is about 0.05-0.14MPa, keeps certain mixing speed, controls dissolved oxygen 10%
More than, the pH value for adding the liquid alkaline control fermentation liquid of 10%-40% is 7-8.5；It is that bottom is added in 10-100h batch in fermentation period
Object controls substrate content in fermentation liquid and is not less than 2%.
After the 100-150h that ferments, start continuous current adding substrate, flow rate 2-5mL/L/h starts simultaneously at stream plus contains
The solution of nutritive salt, flow rate 0.5-3mL/L/h control the dense OD of bacterium620Between 0.3-0.8 (30 times of dilution), control out
Material rate makes fermentating liquid volume maintain 15-22L.
Embodiment 1
Seed culture medium: sucrose 3.0%, corn pulp 0.60%, yeast extract 1.0%, KH2PO41.2%, urea 0.30%.
Fermentation medium: glucose 3.0%, corn pulp 0.60%, yeast extract 0.30%, potassium nitrate 1.1%, biphosphate
Potassium 0.80%, urea 0.20%, ammonium sulfate 0.15% and sodium chloride 0.10%.
There are two types of supplemented mediums: one kind is carbon hendecane hydrocarbon；It is a kind of for solution (glucose 0.2%, ferment containing nutritive salt
Female cream 0.1%, corn pulp 0.15%, potassium dihydrogen phosphate 0.012%, urea 0.015%, remaining is water；The percentage is matter
Measure volume ratio, g/100mL).
Shake-flask seed culture process is that the glycerol tube strain of candida tropicalis is taken to be inoculated in equipped with seed culture medium
In 500mL triangular flask (liquid amount 50mL), initial pH is 220rpm shaking table culture 1.5 days at 6.3,29 DEG C.
Seed tank culture technique is that shake-flask seed access is taken to be equipped in the 10L seeding tank of seed culture medium (liquid amount 6L),
Inoculum concentration is 20%, and the liquid alkaline control pH for adding 25% is at 7.2,29 DEG C, and ventilation quantity 0.6vvm, tank presses 0.10MPa, keeps one
Fixed mixing speed controls the DO10% or more of seed culture process, cultivates 20h, cultivates the standard of mature seed as dilution 30
OD after times620It is 0.6.
Zymotechnique is that will be inoculated into the 30L fermentor containing fermentation medium in the resulting seed liquor of seed tank culture
In, initial volume is 15L after inoculation, inoculum concentration 25% (v/v, opposite ferment initial volume), fermentation starting addition 5% (v/v,
Opposite fermentation initial volume) alkane, 29 DEG C of fermentation processes temperature, ventilation quantity is about 0.5vvm, and tank pressure (gauge pressure) is about
0.10MPa keeps certain mixing speed, controls 10% or more dissolved oxygen, and the pH value for adding 30% liquid alkaline control fermentation liquid is
7-8.5；It is that 25h starts batch addition carbon hendecane hydrocarbon in fermentation period, controlling Determination of Alkane Content in fermentation liquid is 10% or more.
Start continuous flow in fermentation 125h and add carbon hendecane hydrocarbon, flow rate 1.6mL/L/h starts simultaneously at stream plus containing battalion
Support the solution of salt, flow rate 1.4mL/L/h, OD620Control is 0.55 (30 times of dilution), and control discharge velocity to ferment
Liquid product maintains 18L, dilution rate 0.003h-1。
The period continuously ferment as 300h, total fermentation period is 425h, and average rate of producing acid is 1.5g/L/h, and total yield is
93% (g/g).
Comparative example 1
Seed culture medium: sucrose 3.0%, corn pulp 0.60%, yeast extract 1.0%, KH2PO41.2%, urea 0.30%.
Fermentation medium: glucose 3.0%, corn pulp 0.60%, yeast extract 0.30%, potassium nitrate 1.1%, biphosphate
Potassium 0.80%, urea 0.20%, ammonium sulfate 0.15% and sodium chloride 0.10%.
Shake-flask seed culture process is that the glycerol tube strain of candida tropicalis is taken to be inoculated in equipped with seed culture medium
In 500mL triangular flask (liquid amount 50mL), initial pH is 220rpm shaking table culture 1.5 days at 6.3,29 DEG C.
Seed tank culture technique is that shake-flask seed access is taken to be equipped in the 10L seeding tank of seed culture medium (liquid amount 6L),
Inoculum concentration is 20%, and the liquid alkaline control pH for adding 25% is at 7.2,29 DEG C, and ventilation quantity 0.6vvm, tank presses 0.10MPa, keeps one
Fixed mixing speed controls the DO10% or more of seed culture process, cultivates 20h, cultivates the standard of mature seed as dilution 30
OD after times620It is 0.6.
Zymotechnique is that will be inoculated into the 30L fermentor containing fermentation medium in the resulting seed liquor of seed tank culture
In, initial volume is 15L after inoculation, inoculum concentration 25% (v/v, opposite ferment initial volume), fermentation starting addition 5% (v/v,
Opposite fermentation initial volume) alkane, 29 DEG C of fermentation processes temperature, ventilation quantity is about 0.5vvm, and tank pressure (gauge pressure) is about
0.10MPa keeps certain mixing speed, controls 10% or more dissolved oxygen, and the pH value for adding 30% liquid alkaline control fermentation liquid is
7-8.5；It is that 25h starts batch addition carbon hendecane hydrocarbon in fermentation period, controlling Determination of Alkane Content in fermentation liquid is 10% or more.
It fermenting after 160h, thallus vigor declines to a great extent, and stops tank when fermenting 165h, and average rate of producing acid is 1.2g/L/h,
Total yield is 89% (g/g).
From the comparison of embodiment 1 and comparative example 1: due to continuous flow plus alkane and nutritive salt, rate of producing acid is from 1.2g/
L/h is improved to 1.5g/L/h, improves 25%, total yield is improved from 89% to 93%.
Embodiment 2
Seed culture medium: sucrose 1%, corn pulp 0.30%, yeast extract 0.6%, KH2PO40.8%, urea 0.25%.
Fermentation medium: glucose 2.0%, corn pulp 0.20%, yeast extract 0.25%, potassium nitrate 0.05%, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.07%, urea 0.15%, ammonium sulfate 0.20% and sodium chloride 0.12%.
There are two types of supplemented mediums: one kind is carbon dodecane hydrocarbon；It is a kind of for solution (yeast extract 0.1%, sulphur containing nutritive salt
Sour ammonium 0.05%, corn pulp 0.05%, potassium dihydrogen phosphate 0.008%, urea 0.01%, remaining is water；The percentage is matter
Measure volume ratio, g/100mL).
Shake-flask seed culture process is that the glycerol tube strain of candida tropicalis is taken to be inoculated in equipped with seed culture medium
In 500mL triangular flask (liquid amount 60mL), initial pH is 250rpm shaking table culture 1 day at 6.5,32 DEG C.
Seed tank culture technique is that shake-flask seed access is taken to be equipped in the 10L seeding tank of seed culture medium (liquid amount 5L),
Inoculum concentration is 10%, and the liquid alkaline control pH for adding 40% is at 7.5,32 DEG C, and ventilation quantity 0.5vvm, tank presses 0.05MPa, keeps one
Fixed mixing speed controls the DO10% or more of seed culture process, cultivates 15h, cultivates the standard of mature seed as dilution 30
OD after times620It is 0.8.
Zymotechnique is that will be inoculated into the 30L fermentor containing fermentation medium in the resulting seed liquor of seed tank culture
In, initial volume is 10L after inoculation, inoculum concentration 30% (v/v, opposite ferment initial volume), fermentation starting addition 10% (v/v,
Opposite fermentation initial volume) alkane, 32 DEG C of fermentation processes temperature, ventilation quantity is about 0.7vvm, and tank pressure (gauge pressure) is about
0.05MPa keeps certain mixing speed, controls 10% or more dissolved oxygen, and the pH value for adding 40% liquid alkaline control fermentation liquid is
7-8.5；It is that 10h starts batch addition carbon dodecane hydrocarbon in fermentation period, controlling Determination of Alkane Content in fermentation liquid is 10% or more.
Start continuous flow in fermentation 100h and add carbon dodecane hydrocarbon, flow rate 2.5mL/L/h starts simultaneously at stream plus containing battalion
The solution of salt is supported, flow rate 0.5mL/L/h controls OD620For 0.4 (30 times of dilution), control discharge velocity makes fermentation liquid
Volume maintains 20L, dilution rate 0.003h-1。
The period continuously ferment as 400h, total fermentation period is 500h, and average rate of producing acid is 2.1g/L/h, and total yield is
100% (g/g).
Comparative example 2
Seed culture medium: sucrose 1%, corn pulp 0.30%, yeast extract 0.6%, KH2PO40.8%, urea 0.25%.
Fermentation medium: glucose 2.0%, corn pulp 0.20%, yeast extract 0.25%, potassium nitrate 0.05%, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.07%, urea 0.15%, ammonium sulfate 0.20% and sodium chloride 0.12%.
Shake-flask seed culture process is that the glycerol tube strain of candida tropicalis is taken to be inoculated in equipped with seed culture medium
In 500mL triangular flask (liquid amount 60mL), initial pH is 250rpm shaking table culture 1 day at 6.5,32 DEG C.
Seed tank culture technique is that shake-flask seed access is taken to be equipped in the 10L seeding tank of seed culture medium (liquid amount 5L),
Inoculum concentration is 10%, and the liquid alkaline control pH for adding 40% is at 7.5,32 DEG C, and ventilation quantity 0.5vvm, tank presses 0.05MPa, keeps one
Fixed mixing speed controls the DO10% or more of seed culture process, cultivates 15h, cultivates the standard of mature seed as dilution 30
OD after times620It is 0.8.
Zymotechnique is that will be inoculated into the 30L fermentor containing fermentation medium in the resulting seed liquor of seed tank culture
In, initial volume is 10L after inoculation, inoculum concentration 30% (v/v, opposite ferment initial volume), fermentation starting addition 10% (v/v,
Opposite fermentation initial volume) alkane, 32 DEG C of fermentation processes temperature, ventilation quantity is about 0.7vvm, and tank pressure (gauge pressure) is about
0.05MPa keeps certain mixing speed, controls 10% or more dissolved oxygen, and the pH value for adding 40% liquid alkaline control fermentation liquid is
7-8.5；It is that 10h starts batch addition carbon dodecane hydrocarbon in fermentation period, controlling Determination of Alkane Content in fermentation liquid is 10% or more.
It fermenting after 170h, thallus vigor declines to a great extent, and stops tank when fermenting 180h, and average rate of producing acid is 1.5g/L/h,
Total yield is 87% (g/g).
From the comparison of embodiment 2 and comparative example 2: due to continuous flow plus alkane and nutritive salt, rate of producing acid is from 1.5g/
L/h is improved to 2.1g/L/h, improves 40%, total yield is improved from 87% to 100%.
Embodiment 3
Seed culture medium: sucrose 3%, corn pulp 1%, yeast extract 0.2%, KH2PO40.9%, urea 0.5%.
Fermentation medium: glucose 5%, corn pulp 0.7%, yeast extract 0.3%, potassium nitrate 0.55%, potassium dihydrogen phosphate
0.60%, urea 0.20%, ammonium sulfate 0.20% and sodium chloride 0.15%.
There are two types of supplemented mediums: one kind is carbon tridecane hydrocarbon；It is a kind of for solution (glucose 0.5%, sulphur containing nutritive salt
Sour ammonium 0.15%, corn pulp 0.10%, potassium dihydrogen phosphate 0.008%, urea 0.007%, remaining is water；The percentage is matter
Measure volume ratio, g/100mL).
Shake-flask seed culture process is that the glycerol tube strain of candida tropicalis is taken to be inoculated in equipped with seed culture medium
In 500mL triangular flask (liquid amount 100mL), initial pH is 200rpm shaking table culture 2 days at 6.0,30 DEG C.
Seed tank culture technique is that shake-flask seed access is taken to be equipped in the 10L seeding tank of seed culture medium (liquid amount 8L),
Inoculum concentration is 30%, and the liquid alkaline control pH for adding 10% is at 7.0,30 DEG C, and ventilation quantity 0.3vvm, tank presses 0.14MPa, keeps one
Fixed mixing speed controls the DO10% or more of seed culture process, cultivates 30h, cultivates the standard of mature seed as dilution 30
OD after times620It is 1.0.
Zymotechnique is that will be inoculated into the 30L fermentor containing fermentation medium in the resulting seed liquor of seed tank culture
In, initial volume is 15L after inoculation, inoculum concentration 10% (v/v, opposite ferment initial volume), fermentation starting addition 3% (v/v,
Opposite fermentation initial volume) carbon tridecane hydrocarbon, 30 DEG C of fermentation processes temperature, ventilation quantity is about 0.30vvm, tank pressure (table
Pressure) it is about 0.14MPa, certain mixing speed is kept, 10% or more dissolved oxygen is controlled, adds 10% liquid alkaline control fermentation liquid
PH value is 7-8.5；It is that 80h starts batch addition alkane in fermentation period, controlling Determination of Alkane Content in fermentation liquid is 10% or more.
Start continuous flow in fermentation 150h and add carbon tridecane hydrocarbon, flow rate 1.36mL/L/h starts simultaneously at stream plus contains
The solution of nutritive salt, flow rate 1.36mL/L/h control thallus OD620It is 0.8, control discharge velocity makes fermented liquid
Product maintains 22L, dilution rate 0.0027h-1。
The period continuously ferment as 250h, total fermentation period is 400h, and average rate of producing acid is 1.6g/L/h, and total yield is
90% (g/g).
Comparative example 3
Seed culture medium: sucrose 3%, corn pulp 1%, yeast extract 0.2%, KH2PO40.9%, urea 0.5%.
Fermentation medium: glucose 5%, corn pulp 0.7%, yeast extract 0.3%, potassium nitrate 0.55%, potassium dihydrogen phosphate
0.60%, urea 0.20%, ammonium sulfate 0.20% and sodium chloride 0.15%.
Shake-flask seed culture process is that the glycerol tube strain of candida tropicalis is taken to be inoculated in equipped with seed culture medium
In 500mL triangular flask (liquid amount 100mL), initial pH is 200rpm shaking table culture 2 days at 6.0,30 DEG C.
Seed tank culture technique is that shake-flask seed access is taken to be equipped in the 10L seeding tank of seed culture medium (liquid amount 8L),
Inoculum concentration is 30%, and the liquid alkaline control pH for adding 10% is at 7.0,30 DEG C, and ventilation quantity 0.3vvm, tank presses 0.14MPa, keeps one
Fixed mixing speed controls the DO10% or more of seed culture process, cultivates 30h, cultivates the standard of mature seed as dilution 30
OD after times620It is 1.0.
Zymotechnique is that will be inoculated into the 30L fermentor containing fermentation medium in the resulting seed liquor of seed tank culture
In, initial volume is 15L after inoculation, inoculum concentration 10% (v/v, opposite ferment initial volume), fermentation starting addition 3% (v/v,
Opposite fermentation initial volume) carbon tridecane hydrocarbon, 30 DEG C of fermentation processes temperature, ventilation quantity is about 0.30vvm, tank pressure (table
Pressure) it is about 0.14MPa, certain mixing speed is kept, 10% or more dissolved oxygen is controlled, adds 10% liquid alkaline control fermentation liquid
PH value is 7-8.5；It is that 80h starts batch addition alkane in fermentation period, controlling Determination of Alkane Content in fermentation liquid is 10% or more.
It fermenting after 160h, thallus vigor declines to a great extent, and stops tank when fermenting 175h, and average rate of producing acid is 1.3g/L/h,
Total yield is 85% (g/g).
From the comparison of embodiment 3 and comparative example 3: due to continuous flow plus alkane and nutritive salt, rate of producing acid is from 1.3g/
L/h is improved to 1.6g/L/h, improves 23%, total yield is improved from 85% to 90%.
Embodiment 4
Seed culture medium: sucrose 1.3%, corn pulp 0.35%, yeast extract 0.45%, KH2PO41.1%, urea 0.5%.
Fermentation medium: glucose 4.5%, corn pulp 0.1%, yeast extract 0.15%, potassium nitrate 1.2%, biphosphate
Potassium 0.75%, urea 0.3%, ammonium sulfate 0.18% and sodium chloride 0.09%.
There are two types of supplemented mediums: one kind is carbon hexadecane hydrocarbon；It is a kind of for containing nutritive salt solution (ammonium sulfate 0.15%,
Corn pulp 0.15%, potassium dihydrogen phosphate 0.015%, urea 0.015%, remaining is water；Percentage is mass volume ratio, g/
100mL)。
Shake-flask seed culture process is that the glycerol tube strain of candida tropicalis is taken to be inoculated in equipped with seed culture medium
In 500mL triangular flask (liquid amount 50mL), initial pH is 240rpm shaking table culture 1.5 days at 6.3,28 DEG C.
Seed tank culture technique is that shake-flask seed access is taken to be equipped in the 10L seeding tank of seed culture medium (liquid amount 5L),
Inoculum concentration is 30%, and the liquid alkaline control pH for adding 15% is at 7.4,28 DEG C, and ventilation quantity 0.6vvm, tank presses 0.08MPa, keeps one
Fixed mixing speed controls the DO10% or more of seed culture process, cultivates 28h, cultivates the standard of mature seed as dilution 30
OD after times620It is 0.9.
Zymotechnique is that will be inoculated into the 30L fermentor containing fermentation medium in the resulting seed liquor of seed tank culture
In, initial volume is 13L after inoculation, inoculum concentration 30% (v/v, opposite ferment initial volume), fermentation starting addition 2% (v/v,
Opposite fermentation initial volume) alkane, 28 DEG C of fermentation processes temperature, ventilation quantity is about 0.6vvm, and tank pressure (gauge pressure) is about
0.08MPa keeps certain mixing speed, controls 10% or more dissolved oxygen, and the pH value for adding 15% liquid alkaline control fermentation liquid is
7-8.5；It is that 60h starts batch addition carbon hexadecane hydrocarbon in fermentation period, controlling Determination of Alkane Content in fermentation liquid is 10% or more.
Start continuous flow in fermentation 130h and add carbon hexadecane hydrocarbon, flow rate 1.35mL/L/h starts simultaneously at stream plus contains
The solution of nutritive salt, flow rate 1.47mL/L/h control thallus OD620For 0.65 (30 times of dilution), control discharge velocity makes
It obtains fermentating liquid volume and maintains 17L, dilution rate 0.0028h-1。
The period continuously ferment as 300h, total fermentation period is 430h, and average rate of producing acid is 1.7g/L/h, and total yield is
75% (g/g).
Comparative example 4
Seed culture medium: sucrose 1.3%, corn pulp 0.35%, yeast extract 0.45%, KH2PO41.1%, urea 0.5%.
Fermentation medium: glucose 4.5%, corn pulp 0.1%, yeast extract 0.15%, potassium nitrate 1.2%, biphosphate
Potassium 0.75%, urea 0.3%, ammonium sulfate 0.18% and sodium chloride 0.09%.
Shake-flask seed culture process is that the glycerol tube strain of candida tropicalis is taken to be inoculated in equipped with seed culture medium
In 500mL triangular flask (liquid amount 50mL), initial pH is 240rpm shaking table culture 1.5 days at 6.3,28 DEG C.
Seed tank culture technique is that shake-flask seed access is taken to be equipped in the 10L seeding tank of seed culture medium (liquid amount 5L),
Inoculum concentration is 30%, and the liquid alkaline control pH for adding 15% is at 7.4,28 DEG C, and ventilation quantity 0.6vvm, tank presses 0.08MPa, keeps one
Fixed mixing speed controls the DO10% or more of seed culture process, cultivates 28h, cultivates the standard of mature seed as dilution 30
OD after times620It is 0.9.
Zymotechnique is that will be inoculated into the 30L fermentor containing fermentation medium in the resulting seed liquor of seed tank culture
In, initial volume is 13L after inoculation, inoculum concentration 30% (v/v, opposite ferment initial volume), fermentation starting addition 2% (v/v,
Opposite fermentation initial volume) alkane, 28 DEG C of fermentation processes temperature, ventilation quantity is about 0.6vvm, and tank pressure (gauge pressure) is about
0.08MPa keeps certain mixing speed, controls 10% or more dissolved oxygen, and the pH value for adding 15% liquid alkaline control fermentation liquid is
7-8.5；It is that 60h starts batch addition carbon hexadecane hydrocarbon in fermentation period, controlling Determination of Alkane Content in fermentation liquid is 10% or more.
It fermenting after 150h, thallus vigor declines to a great extent, and stops tank when fermenting 165h, and average rate of producing acid is 1.3g/L/h,
Total yield is 68% (g/g).
From the comparison of embodiment 4 and comparative example 4: due to continuous flow plus alkane and nutritive salt, rate of producing acid is from 1.3g/
L/h is improved to 1.7g/L/h, improves 31%, total yield is improved from 68% to 75%.
Claims (10)
1. a kind of method for producing long-chain biatomic acid of continuously fermenting, it is characterised in that: after the 100-150h that ferments, with 0.001-
0.006h-1The continuous current adding substrate of dilution rate, while continuous flow adds the solution containing nutritive salt.
2. the method as described in claim 1, it is characterised in that: the dilution rate is 0.0016-0.0054h-1。
3. method according to claim 1 or 2, it is characterised in that: the substrate includes the normal alkane of C9-C18, linear saturation
One of fatty acid, linear saturated fatty acids ester and salts of straight-chain saturated fatty acids are a variety of；Preferably include the nalka of C11-C16
One of hydrocarbon, linear saturated fatty acids, linear saturated fatty acids ester and salts of straight-chain saturated fatty acids are a variety of；More preferably
Normal alkane, linear saturated fatty acids, linear saturated fatty acids ester and the linear saturation rouge of C11, C12, C13, C15, C14 or C16
One of fat hydrochlorate；
The flow rate V of the substrate(substrate)It is preferred that 2-5mL/L/h.
4. method as claimed in claim 3, it is characterised in that: before the current adding substrate, substrate content is in the fermentation liquid
2% (v/v) or more, the percentage are the percent by volume that the substrate accounts for fermentation liquid.
5. method according to any of claims 1-4, it is characterised in that: the solution containing nutritive salt include it is following at
Point: glucose 0.0%-1.0% (w/v), corn pulp 0.05%-0.15% (w/v), yeast extract 0.0%-0.3% (w/v), phosphorus
Acid dihydride potassium 0.005%-0.015% (w/v), urea 0.005%-0.015% (w/v) and ammonium sulfate 0.01%-0.15% (w/
V), remaining is water；The percentage is mass volume ratio, g/100mL；
The flow rate V of the solution containing nutritive salt(solution containing nutritive salt)It is preferred that 0.5-3mL/L/h.
6. method according to any of claims 1-4, it is characterised in that: the long-chain biatomic acid includes: chemical formula HOOC
(CH2) nCOOH binary acid, wherein n >=7, including but not limited to: azelaic acid, decanedioic acid, eleven carbon diacids, 12 carbon two
First acid, tridecanyldicarboxylic acid, tetradecane diacid, pentadecane binary acid, 16-dicarboxylic acid, seventeen carbon diacids and 18 carbon
One of binary acid is a variety of.
7. a kind of production method of long-chain biatomic acid, the production method includes as claimed in any one of claims 1 to 6 continuous
The method of fermenting and producing long-chain biatomic acid.
8. the method for claim 7, it is characterised in that: before continuously fermenting and producing long-chain biatomic acid, also carry out with
Lower operation:
Seed liquor is inoculated into fermentation medium, intermittent fed-batch fermentation is carried out, starts 10-100h in fermentation, substrate is added,
Guarantee that substrate content is 2% (v/v) or more in fermentation liquid, the percentage is the percent by volume that the substrate accounts for fermentation liquid.
9. method according to claim 8, it is characterised in that: the initial volume after the inoculation is fermenter volume
30%-50%, the percentage are that the volume of culture medium accounts for the percentage of fermenter volume；
And/or the inoculum concentration of the fermentation is 10%-30% (v/v), the percentage is the body relative to fermentation initial volume
Product percentage.
10. method as claimed in claim 8 or 9, it is characterised in that: the cell concentration OD when fermentation620For 9-24；
And/or the temperature of the fermentation is 28-32 DEG C；
And/or the ventilation quantity of the fermentation is 0.3-0.7vvm；
And/or the tank pressure of the fermentation is 0.05-0.14MPa, the pressure is gauge pressure；
And/or the dissolved oxygen amount of the fermentation is 10% or more；
And/or the pH value of the fermentation is 7-8.5.
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| CN112725400A (en) * | 2019-10-28 | 2021-04-30 | 中国石油化工股份有限公司 | Long-chain composition, long-chain composition kit, manufacturing method and application thereof |
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