CN109943598A - A kind of method of fermenting and producing long-chain biatomic acid - Google Patents
A kind of method of fermenting and producing long-chain biatomic acid Download PDFInfo
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- CN109943598A CN109943598A CN201711379606.0A CN201711379606A CN109943598A CN 109943598 A CN109943598 A CN 109943598A CN 201711379606 A CN201711379606 A CN 201711379606A CN 109943598 A CN109943598 A CN 109943598A
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Abstract
The invention discloses a kind of method of fermenting and producing long-chain biatomic acid, cell concentration is in the fermentation process with optical density OD620Meter, OD620It is 10~30.Method of the invention can significantly improve the fermentation conversion rate of long-chain biatomic acid, and low in cost, easy to operate, be suitble to large-scale industrial production.
Description
Technical field
The present invention relates to fermentation arts, and in particular to a kind of method of fermenting and producing long-chain biatomic acid.
Background technique
Long-chain biatomic acid has very extensive purposes, can synthesize extraordinary polyamide, height by raw material of long-chain biatomic acid
Grade fragrance, high-grade hot melt adhesive, cold resistant plasticizer, senior lubricant, advanced antirust agent, advanced paint and coating etc..Long-chain binary
Acid can usually be synthesized with chemical method or bioanalysis.Chemical method synthetic route is long, and reaction needs high temperature and pressure, wants to catalyst
Ask comparison harsh, therefore long-chain biatomic acid at industrial scale is of less types, only a small number of product such as 12 carbon long-chain biatomic acids
Kind.And bioanalysis is to be obtained using long chain alkane as substrate by microorganism conversion, utilizes the special function of microorganism diterminal oxidation
Long-chain biatomic acid can be prepared by fermenting substrate of n-alkane.The mechanism study of long-chain biatomic acid is generated by microbiological oxidation alkane
Known to: its main reaction is α, and omega oxidation, side reaction is alpha-oxidation decarboxylation and beta oxidation, makes the product long-chain biatomic acid quilt generated
Further oxidative degradation.Beta oxidation, which is mainly grown to microorganism, provides energy, maintains the conversion activity of microbial cell, this just leads
Cause substrate alkane that cannot be completely converted into product binary acid, and the conversion ratio of alkane is to Guan Chong for the production cost of binary acid
It wants.
Chinese patent CN102808004A and CN1259424C disclose a kind of side for improving fermentation yield of long-chain dicarboxylic acid
Method.This method improves fermentation conversion rate by adding alpha-oxidation decarboxylation or beta oxidation inhibitor during the fermentation, uses
Inhibitor is the reagents such as chlorpromazine hydrochloride, halogenated aliphatic acid, and such reagent price is expensive, increases the extraction and purification process of binary acid
And cost, and conversion ratio highest is only increased to 89% (w/w).Chinese patent CN105755062A discloses a kind of utilize and aoxidizes also
The method that former current potential regulates and controls fermentation process production long-chain biatomic acid.This method combines oxygen by adjusting ventilatory capacity, speed of agitator
Agent and reducing agent realize that two stages ORP of long-chain biatomic acid control fermentation, but do not refer to binary acid yield.Chinese patent
CN103074325A discloses a kind of method of mutagenesis of the Candida tropicalis of long-chain biatomic acid, and this method utilizes induction mutation of bacterium
Superior strain is screened, but does not refer to binary acid conversion ratio.The document of foreign study long-chain biatomic acid is to carry out base to bacterial strain mostly
Because of transformation, by blocking or weakening the related enzyme systems of fatty acid beta oxidation, the enzyme system for strengthening fatty acid α-ω oxidation produces to improve
The yield of product.
In conclusion the technique of Production of Long-chain Dicarboxylic Acids by Fermentation Methods in the prior art, fermentation conversion rate is not generally high, and each
Kind improves the method higher cost of long-chain biatomic acid fermentation conversion rate, and effect is not very significant, does not have still in the prior art
One kind can efficiently improve long-chain biatomic acid fermentation conversion rate, and method easy to operate, at low cost.
Summary of the invention
It is not high in order to solve long-chain biatomic acid fermentation conversion rate existing in the prior art, in order to improve fermentation conversion rate,
It needs to increase great amount of cost and effect and inapparent defect, a kind of method of fermenting and producing long-chain biatomic acid is provided, the present invention
Method can significantly improve the fermentation conversion rate of long-chain biatomic acid, and it is low in cost, it is easy to operate, be suitble to heavy industrialization
Production.
A kind of method of fermenting and producing long-chain biatomic acid, the fermentation is using alkane as substrate, thallus in the fermentation process
Concentration is with optical density OD620Meter, OD620It is 10~30, preferably 12~21.
In the prior art, there are many researchs to the method for improving long-chain biatomic acid fermentation conversion rate, but there has been no pass through control
Fermentation process cell concentration processed is (with optical density OD620) just improve the report of fermentation conversion rate.Inventor passes through to long-chain binary
Many researchs of acid fermentation technique, discovery: cell concentration has a great impact to the fermentation conversion rate of long-chain biatomic acid.Pass through
In a specific range by cell concentration control, it can effectively improve the fermentation conversion rate of long-chain biatomic acid；On this basis, it sends out
Bright people deep structure research phenomenon in terms of mechanism, discovery: why cell density has a significant impact fermentation conversion rate, be
Because cell density has an impact to generation of the conversion of substrate, intermediate product, by-product etc., only in certain equilibrium condition
Under, just it is able to achieve the fermentation conversion rate of high long-chain biatomic acid；One side cell concentration is excessively high, and substrate conversion rate is too fast, auxiliary
Enzyme NADPH is insufficient, and beta-oxidation enhancing causes the substrate transformation rate low, and the accumulation of intermediate product hydroxycarboxylic acid excessively leads to product
Quality decline；Another aspect cell concentration is too low, and substrate conversion rate is excessively slow, and consumption substrate excessively maintains energy to cause to convert
Rate reduces, and fermentation period extension causes to produce strength reduction, increases production cost.Therefore the reasonable cell concentration of control can be effective
Improve the fermentation conversion rate of long-chain biatomic acid.
More than, the preferred technical solution of scheme, is described further in view of the above technology.
The present invention to the control method of cell concentration without limitation, it is only necessary to control cell concentration in particular range i.e.
It can；In general, the method for conventional control cell concentration is suitable for the present invention.
A preferred technical solution of the invention, in the fermentation process, in the early period of the fermentation, the thallus is dense
Degree reaches OD620It is 15~21.
A preferred technical solution of the invention, in the fermentation process, the later period of the fermentation, the cell concentration
Reach OD620It is 12~18.
The cell concentration of a preferred technical solution of the invention, the earlier fermentation is more than or equal to the fermentation later period
Cell concentration.
Those skilled in the art are according to common sense in the field, it can be determined that the stage of fermentation.In general, before the fermentation
Phase is that fermentation starts to 30~60h of fermentation.The fermentation later period is that fermentation starts 30-60h to fermentation ends.
In general, the time of the fermentation is 140~180h.
A preferred technical solution of the invention, the culture medium of the fermentation include: carbon source, nitrogen source, phosphorus source, micro gold
Belong to element source and growth factor.
Wherein, the carbon source includes either one of glucose, sucrose, maltose, molasses, methanol, ethyl alcohol or more
Kind；It is highly preferred that the carbon source include either glucose, sucrose it is one or more.The carbon source concentration preferably 20~
40g/L.Other technological parameters and condition are cooperated by control carbon source kind and its concentration in earlier fermentation, control OD620In spy
Determine in range.
Wherein, the nitrogen source includes either organic nitrogen and/or inorganic nitrogen, and organic nitrogen includes but is not limited to yeast extract, egg
One of white peptone, corn pulp are a variety of, and inorganic nitrogen includes but is not limited to one of urea, ammonium sulfate, potassium nitrate or a variety of；
Preferably, the nitrogen source includes one or two kinds of mixing of either ammonium sulfate, potassium nitrate.The concentration of the nitrogen source is preferred
0.5~6g/L.Preferably, the concentration of the ammonium sulfate is 0.5~3g/L.Preferably, the concentration of the potassium nitrate be 0.5~
3g/L.Other technological parameters and condition are cooperated by control nitrogen source type and its concentration in fermentation middle and later periods (i.e. transition phase),
Control OD620In particular range.
Wherein, phosphorus source includes either one of phosphate, dibasic alkaliine and dihydric phosphate or a variety of；It is excellent
Selection of land, the phosphate include either one of potassium phosphate, sodium ascorbyl phosphate, ammonium phosphate salt or a variety of；It is highly preferred that institute
It states phosphate and includes either potassium dihydrogen phosphate.The concentration of phosphorus source preferably 1~3g/L.In the fermentation middle and later periods, (that is: substrate turns
The change phase), by control phosphorus source type and its concentration, cooperate other technological parameters and condition, controls OD620In particular range.
Wherein, the minor metallic element source includes potassium, calcium, magnesium, iron, copper, zinc, the sulfate of manganese, hydrochloride and nitric acid
One of salt is a variety of.The concentration preferably 0.1~50ppm in the minor metallic element source.
Wherein, the growth factor includes one of amino acid, citric acid and vitamin or a variety of；It is highly preferred that institute
State the mixing that growth factor includes either one or both of citric acid, biotin.The growth factor concentration is preferred
0.01~1ppm.
A preferred technical solution of the invention, the strain of the fermentation include candida tropicalis (Candida
) or candida sake (Candidasake) Tropicalis.Such as: it can be candida tropicalis (Candida
Tropicalis) (deposit number is CCTCC M203052) or candida tropicalis (Candida Tropicalis)
CATN145 (deposit number is CCTCC M 2011192) or candida sake (Candidasake) CATH4013 (deposit number
For CCTCC M2011486) or candida sake (Candidasake) CATH4014 (deposit number CCTCC
M2011487) perhaps candida sake (Candidasake) CATH4012 (deposit number be CCTCC M2011485) or
Candida sake (Candidasake) CATH4016 (deposit number is CCTCC M2011488) or candida sake
(Candidasake) CATH430 (deposit number is CCTCC M2011489).
A preferred technical solution of the invention, the substrate include the normal alkane of C9~C18, linear saturation fat
One of acid, linear saturated fatty acids ester and salts of straight-chain saturated fatty acids are a variety of；Preferably include C11~C16 normal alkane,
One of linear saturated fatty acids, linear saturated fatty acids ester and salts of straight-chain saturated fatty acids are a variety of；More preferably C11,
Normal alkane, linear saturated fatty acids, linear saturated fatty acids ester and the linear saturated fatty acids of C12, C13, C15, C14 or C16
One of salt.
A preferred technical solution of the invention, the tank temperature when fermentation are 28~32 DEG C.
A preferred technical solution of the invention, the ventilation quantity when fermentation are 0.3~0.7vvm.
A preferred technical solution of the invention, the tank pressure when fermentation is 0.05~0.14Mpa (gauge pressure).
A preferred technical solution of the invention, the ferment control pH are 5.0~8.5.
Those skilled in the art are according to common sense in the field, it can be determined that the stage of fermentation.In general, before the fermentation
Phase is that fermentation starts to 30~60h of fermentation.The fermentation later period is that fermentation starts 30~60h to fermentation ends.
A preferred technical solution of the invention, the fermentation period are 100~180h.
The method of fermenting and producing long-chain biatomic acid of the invention can significantly improve the fermentation conversion rate of long-chain biatomic acid, and
It is low in cost, it is easy to operate, it is suitble to large-scale industrial production.
Specific embodiment
The present invention provides a kind of side that long-chain biatomic acid fermentation conversion rate is improved by control fermentation process cell concentration
Method.Wherein, cell concentration is with the optical density OD of fermentation liquid620Characterization.Those skilled in the art understand: OD620Measurement it is general
Measured value is measured to obtain after prepare liquid is diluted, then measured value is obtained into OD multiplied by extension rate620Absolute value.The present invention passes through
Carbon source concentration or nitrogen concentration or phosphorus source concentration in fermentation medium are controlled, other technological parameters and condition are cooperated, to control hair
Ferment process cell concentration is in a suitable range, to improve fermentation conversion rate.
The method of fermenting and producing long-chain biatomic acid, comprising the following steps:
A) actication of culture；
B seed liquor) is prepared in seeding tank using seed culture medium；
C) seed liquor is inoculated into the fermentor containing fermentation medium, fermentation process adds substrate；In fermentation process
Cell concentration is with optical density OD620Meter, OD620It is 10~30, preferably 12~21；It is further preferred that cell concentration early period of fermentation reaches
OD620It is 15~21, the later period cell concentration of fermentation reaches OD620It is 12~18；It is furthermore preferred that the cell concentration of earlier fermentation is big
In the cell concentration for being equal to the fermentation later period.
In one embodiment of the present of invention, step A) actication of culture is the following steps are included: take candida tropicalis
The glycerol stock of (Candida Tropicalis) or candida sake (Candidasake) shakes in the shaking flask of YPD culture medium
Bed culture, 200~250rpm of revolving speed, cultivate 24~48h by 28~30 DEG C of cultivation temperature；
Step A) in, YPD culture medium includes: peptone 10g/kg, yeast extract 5g/kg, and glucose 10g/kg, pH are natural.
In one embodiment of the present of invention, step B) in the parameter of seeding tank be: 28~30 DEG C of cultivation temperature, ventilation quantity
0.3~0.6vvm, tank pressure are 0.08~0.1MPa (gauge pressure), and 12~36h of incubation time, seed maturity index is OD620For 15~
30；
Step B) in seed culture medium be water-soluble liquid culture medium, preferably include following component: 10~30g/L of sucrose, corn
Starch 1.5~10g/L, 1~10g/L of yeast extract, 4~12g/L of potassium dihydrogen phosphate, 0.5~5g/L of urea, 0~30ml/ of alkane
L, water-soluble liquid culture medium is prepared with water, and is sterilized.
In one embodiment of the present of invention, step C) in, 28~32 DEG C of fermentation processes temperature, ventilation quantity is 0.3~
0.7vvm, tank pressure are 0.05~0.14MPa (gauge pressure), keep certain mixing speed to control 10% or more dissolved oxygen；It adds
The pH value of the NaOH solution control fermentation liquid of 10%~40% (w/v)；The early period (that is: thalli growth phase) of fermentation, control pH are
3.5~6.5；And/or middle and later periods (that is: substrate transition phase) the control pH of the fermentation is 5.0~8.5；Fermentation process adds bottom
Object, total fermentation period are 100~180 hours.
In one embodiment of the present of invention, step C) in, 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 the normal alkane, straight of C11~C16
One of chain saturated fatty acid, linear saturated fatty acids ester and salts of straight-chain saturated fatty acids are a variety of；More preferably C11,
Normal alkane, linear saturated fatty acids, linear saturated fatty acids ester and the linear saturated fatty acids of C12, C13, C15, C14 or C16
One of salt；
In one embodiment of the present of invention, step C) in fermentation medium include: carbon source, nitrogen source, phosphorus source, trace meter member
Plain source, growth factor；
Carbon source includes either one of glucose, sucrose, maltose, molasses, methanol, ethyl alcohol or a variety of；More preferably
Ground, carbon source include one or two kinds of mixing of either glucose, sucrose；Carbon source concentration is 20~40g/L.
Nitrogen source can be organic nitrogen and/or inorganic nitrogen, and organic nitrogen includes but is not limited to yeast extract, peptone, in corn pulp
It is one or more, inorganic nitrogen includes but is not limited to one of urea, ammonium sulfate, potassium nitrate or a variety of；Preferably, nitrogen source packet
Include one or two kinds of mixing of either ammonium sulfate, potassium nitrate；The concentration of nitrogen source is 0.5~3g/L.
Phosphorus source includes either one of phosphoric acid normal salt, dibasic alkaliine and dihydric phosphate or a variety of, preferably phosphorus
Hydrochlorate includes either one of potassium phosphate, sodium ascorbyl phosphate, ammonium phosphate salt or a variety of；It is highly preferred that phosphate include or
Person is potassium dihydrogen phosphate.Phosphorus source concentration is 1~3g/L.
Minor metallic element source includes one of potassium, calcium, magnesium, iron, copper, zinc, the sulfate of manganese, hydrochloride and nitrate
Or it is a variety of.The concentration in microelement source is 0.1~50ppm.
Growth factor includes one of amino acid, citric acid and vitamin or a variety of；Preferably include either citric acid,
The mixing of one or both of biotin.Growth factor concentration is 0.01~1ppm.
In fermentation process, in the early period of fermentation, cell concentration reaches OD620It is 15~21；The later period of fermentation, cell concentration
Reach OD620It is 12~18.
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.
It is such as not particularly illustrated, concentration of the present invention is mass percent concentration.
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 ZL 95117436.3.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.
Embodiment 1
1) actication of culture:
Taking candida tropicalis (Candida Tropicalis) CATN145, (deposit number is CCTCC M
2011192) it (includes: peptone 10g/kg, ferment that glycerol tube strain, which is inoculated in equipped with 30ml YPD seed culture medium,
Female cream 5g/kg, glucose 10g/kg) seed bottle in, pH is naturally, at 29 DEG C, 220rpm shaking table culture 1 day；
2) seed liquor is prepared:
Taking shake-flask seed access that 5L seed culture medium is housed (includes: sucrose 20g/L, corn pulp 3g/L, yeast extract
5g/L, potassium dihydrogen phosphate 7g/L, urea 2g/L) seeding tank in, inoculum concentration is 10% (v/v, opposite ferment initial volume), is connect
The initial ph value of system is at 6.0,29 DEG C after kind, and ventilation quantity 0.4vvm, tank presses 0.08MPa, cultivates 18h, pH in incubation
Naturally 3, OD are dropped to620It grows to 15 or more；
3) it ferments:
Seed is inoculated into containing fermentation medium (glucose 30g/L, potassium nitrate 3g/L, potassium dihydrogen phosphate 2.4g/L, sulphur
Sour ammonium 3g/L, calcium chloride 40ppm, citric acid 0.1ppm) fermentor in, 30 DEG C of fermentation processes temperature, ventilation quantity is about
0.4vvm, tank pressure (gauge pressure) is about 0.12MPa, and control dissolved oxygen is not less than 20%；Start when fermentation period is 10~20 hours
Substrate n-dodecane hydrocarbon is added portionwise, controls Determination of Alkane Content in fermentation liquid and is no more than 10% (v/v).Fermentation process adds 30%
Liquid alkaline controls the pH value 5.0-8.5 of fermentation liquid, until fermentation ends；Earlier fermentation (thalli growth phase) OD620It is 18~21, hair
Ferment later period (substrate (alkane) transition phase) OD620Be 15~18, total fermentation period is 145 hours, whens fermentation ends residual hydrocarbon content base
This is 0.
The production acid concentration 150mg/g of LCDA, alkane mass transitions rate 92%.
Embodiment 2
1) actication of culture:
Taking candida tropicalis (Candida Tropicalis) CATN145, (deposit number is CCTCC M
2011192) it (includes: peptone 10g/kg, ferment that glycerol tube strain, which is inoculated in equipped with 30ml YPD seed culture medium,
Female cream 5g/kg, glucose 10g/kg) seed bottle in, pH is naturally, at 29 DEG C, 220rpm shaking table culture 1 day；
2) seed liquor is prepared:
Taking shake-flask seed access that seed culture medium is housed (includes: sucrose 30g/L, corn pulp 4g/L, yeast extract 5g/
L, potassium dihydrogen phosphate 8g/L, urea 3g/L) seeding tank in, inoculum concentration is 10% (v/v, opposite ferment initial volume), inoculation
The initial ph value of system is at 6.0,29 DEG C afterwards, and ventilation quantity 0.4vvm, tank presses 0.08MPa, cultivates 18h, in incubation pH from
So drop to 3, OD620It grows to 15 or more；
3) it ferments:
Seed is inoculated into containing fermentation medium (glucose 30g/L, potassium nitrate 2g/L, potassium dihydrogen phosphate 2.5g/L, sulphur
Sour ammonium 1.5g/L, magnesium sulfate 50ppm, citric acid 1ppm) fermentor in, 30 DEG C of fermentation processes temperature, ventilation quantity is about
0.4vvm, tank pressure (gauge pressure) is about 0.12MPa, and control dissolved oxygen is not less than 20%；Start when fermentation period is 10~20 hours
Substrate n-dodecane hydrocarbon is added portionwise, controls Determination of Alkane Content in fermentation liquid and is no more than 10% (v/v).Fermentation process adds 30%
Liquid alkaline controls the pH value 5.0-8.5 of fermentation liquid, until fermentation ends；Earlier fermentation (thalli growth phase) OD620It is 18~21, hair
Ferment later period (substrate (alkane) transition phase) OD620Be 12~15, total fermentation period is 160 hours, whens fermentation ends residual hydrocarbon content base
This is 0.
The production acid concentration 160mg/g of LCDA, alkane mass transitions rate 94%.
Embodiment 3
1) actication of culture:
Take the glycerol tube strain of candida tropicalis (Candida Tropicalis) (deposit number is CCTCC M203052)
It is inoculated in the kind equipped with 30ml YPD seed culture medium (including: peptone 10g/kg, yeast extract 5g/kg, glucose 10g/kg)
In sub- bottle, pH is naturally, at 29 DEG C, and 220rpm shaking table culture 1 day；
2) seed liquor is prepared:
Taking shake-flask seed access that 5L seed culture medium is housed (includes: sucrose 2g/L, corn pulp 4g/L, yeast extract 4g/
L, potassium dihydrogen phosphate 5g/L, urea 3g/L) seeding tank in, inoculum concentration is 10% (v/v, opposite ferment initial volume), inoculation
The initial ph value of system is at 6.0,29 DEG C afterwards, and ventilation quantity 0.4vvm, tank presses 0.08MPa, cultivates 18h, in incubation pH from
So drop to 3, OD620It grows to 15 or more；
3) it ferments:
Seed is inoculated into containing fermentation medium (glucose 25g/L, potassium nitrate 2g/L, potassium dihydrogen phosphate 2.5g/L, sulphur
Sour ammonium 1.5g/L, magnesium sulfate 50ppm, citric acid 1ppm) 30 DEG C of fermentation cylinder for fermentation process control temp, ventilation quantity is about
0.4vvm, tank pressure (gauge pressure) is about 0.12MPa, and control dissolved oxygen is not less than 20%；Start when fermentation period is 10~20 hours
Substrate n-dodecane hydrocarbon is added portionwise, controls Determination of Alkane Content in fermentation liquid and is no more than 10% (v/v).Fermentation process adds 30%
Liquid alkaline controls the pH value 5.0-8.5 of fermentation liquid, until fermentation ends；Earlier fermentation (thalli growth phase) OD620It is 15~18, hair
Ferment later period (substrate (alkane) transition phase) OD620Be 12~15, total fermentation period is 170 hours, whens fermentation ends residual hydrocarbon content base
This is 0.
The production acid concentration 170mg/g of LCDA, alkane mass transitions rate 96%.
Embodiment 4
1) actication of culture:
Take the glycerol tube bacterium of candida sake (Candidasake) CATH4012 (deposit number is CCTCC M2011485)
Kind is inoculated in equipped with 30ml YPD seed culture medium (including: peptone 10g/kg, yeast extract 5g/kg, glucose 10g/kg)
In seed bottle, pH is naturally, at 29 DEG C, and 220rpm shaking table culture 1 day；
2) seed liquor is prepared:
Taking shake-flask seed access that 5L seed culture medium is housed (includes: sucrose 30g/L, corn pulp 4g/L, yeast extract
5g/L, potassium dihydrogen phosphate 6g/L, urea 4g/L) seeding tank in, inoculum concentration is 10% (v/v, opposite ferment initial volume), is connect
The initial ph value of system is at 6.0,29 DEG C after kind, and ventilation quantity 0.4vvm, tank presses 0.08MPa, cultivates 18h, pH in incubation
Naturally 3, OD are dropped to620It grows to 15 or more；
3) it ferments:
Seed is inoculated into containing fermentation medium (glucose 25g/L, potassium nitrate 2g/L, potassium dihydrogen phosphate 2.5g/L, sulphur
Sour ammonium 1.5g/L, magnesium sulfate 50ppm, citric acid 1ppm) fermentor in, 30 DEG C of fermentation processes temperature, ventilation quantity is about
0.4vvm, tank pressure (gauge pressure) is about 0.12MPa, and control dissolved oxygen is not less than 20%.Start when fermentation period is 10~20 hours
Substrate n-tridecane hydrocarbon is added portionwise, controls Determination of Alkane Content in fermentation liquid and is no more than 10% (v/v)；Fermentation process adds 30%
Liquid alkaline controls the pH value 5.0-8.5 of fermentation liquid, until fermentation ends；Earlier fermentation (thalli growth phase) OD620It is 15~18, hair
Ferment later period (substrate (alkane) transition phase) OD620Be 12~15, total fermentation period is 170 hours, whens fermentation ends residual hydrocarbon content base
This is 0.
The production acid concentration of LCDA produces acid 160mg/g, alkane mass transitions rate 93%.
Embodiment 5
1) actication of culture:
Take the glycerol tube bacterium of candida sake (Candidasake) CATH4012 (deposit number is CCTCC M2011485)
Kind is inoculated in equipped with 30ml YPD seed culture medium (including: peptone 10g/kg, yeast extract 5g/kg, glucose 10g/kg)
In seed bottle, pH is naturally, at 29 DEG C, and 220rpm shaking table culture 1 day；
2) seed liquor is prepared:
Taking shake-flask seed access that 5L seed culture medium is housed (includes: sucrose 20g/L, corn pulp 5g/L, yeast extract
3g/L, potassium dihydrogen phosphate 7g/L, urea 3g/L) seeding tank in, inoculum concentration is 10% (v/v, opposite ferment initial volume), is connect
The initial ph value of system is at 6.0,29 DEG C after kind, and ventilation quantity 0.4vvm, tank presses 0.08MPa, cultivates 18h, pH in incubation
Naturally 3, OD are dropped to620It grows to 15 or more；
3) it ferments:
Seed is inoculated into containing fermentation medium (glucose 25g/L, potassium nitrate 2g/L, potassium dihydrogen phosphate 2.5g/L, sulphur
Sour ammonium 1.5g/L, magnesium sulfate 50ppm, citric acid 1ppm) fermentor in, 30 DEG C of fermentation processes temperature, ventilation quantity is about
0.6vvm, tank pressure (gauge pressure) is about 0.1MPa, and control dissolved oxygen is not less than 20%.Start point when fermentation period is 10~20 hours
It criticizes and substrate hexadecane hydrocarbon is added, control Determination of Alkane Content in fermentation liquid and be no more than 10% (v/v)；Fermentation process adds 30%
Liquid alkaline controls the pH value 5.0-8.5 of fermentation liquid, until fermentation ends；Earlier fermentation (thalli growth phase) OD620It is 18~21, hair
Ferment later period (substrate (alkane) transition phase) OD620Be 15~18, total fermentation period is 150 hours, whens fermentation ends residual hydrocarbon content base
This is 0.
The production acid concentration of LCDA produces acid 120mg/g, alkane mass transitions rate 75%.
Comparative example 1
1) actication of culture:
Take the glycerol tube strain of candida tropicalis (Candida Tropicalis) (deposit number is CCTCC M203052)
It is inoculated in the seed equipped with 30mlYPD seed culture medium (including: peptone 10g/kg, yeast extract 5g/kg, glucose 10g/kg)
In bottle, pH is naturally, at 29 DEG C, and 220rpm shaking table culture 1 day；
2) seed liquor is prepared:
Taking shake-flask seed access that 5L seed culture medium is housed (includes: sucrose 30g/L, corn pulp 5g/L, yeast extract
5g/L, potassium dihydrogen phosphate 8g/L, urea 3g/L) seeding tank in, inoculum concentration is 10% (v/v, opposite ferment initial volume), is connect
The initial ph value of system is at 6.0,29 DEG C after kind, and ventilation quantity 0.4vvm, tank presses 0.08MPa, cultivates 18h, pH in incubation
Naturally 3, OD are dropped to620It grows to 15 or more；
3) it ferments:
Seed is inoculated into containing fermentation medium (glucose 50g/L, potassium nitrate 5g/L, potassium dihydrogen phosphate 5g/L, sulfuric acid
Ammonium 5g/L, magnesium sulfate 50ppm, citric acid 1ppm) fermentor in, 30 DEG C of fermentation processes temperature, ventilation quantity is about
0.4vvm, tank pressure (gauge pressure) is about 0.12MPa, and control dissolved oxygen is not less than 20%；Start when fermentation period is 10~20 hours
Substrate n-dodecane hydrocarbon is added, controls Determination of Alkane Content in fermentation liquid and is no more than 10% (v/v)；Fermentation process adds 30% liquid alkaline
The pH value 5.0-8.5 of fermentation liquid is controlled, until fermentation ends；Earlier fermentation (thalli growth phase) OD620It is 31~36, after fermentation
Phase (substrate (alkane) transition phase) OD620It is 29~33, total fermentation period is 130 hours, and whens fermentation ends, residual hydrocarbon content was essentially
0。
The production acid concentration 130mg/g of LCDA, alkane mass transitions rate 84%.
Comparative example 2
1) actication of culture:
Taking candida tropicalis (Candida Tropicalis) CATN145, (deposit number is CCTCC M
2011192) it (includes: peptone 10g/kg, ferment that glycerol tube strain, which is inoculated in equipped with 30ml YPD seed culture medium,
Female cream 5g/kg, glucose 10g/kg) seed bottle in, pH is naturally, at 29 DEG C, 220rpm shaking table culture 1 day；
2) seed liquor is prepared:
Taking shake-flask seed access that seed culture medium is housed (includes: sucrose 20g/L, corn pulp 3g/L, yeast extract 5g/
L, potassium dihydrogen phosphate 8g/L, urea 4g/L) seeding tank in, inoculum concentration is 10% (v/v, opposite ferment initial volume), inoculation
The initial ph value of system is at 6.0,29 DEG C afterwards, and ventilation quantity 0.4vvm, tank presses 0.08MPa, cultivates 18h, in incubation pH from
So drop to 3, OD620It grows to 15 or more；
3) it ferments:
Seed is inoculated into containing fermentation medium (glucose 10g/L, potassium nitrate 4g/L, potassium dihydrogen phosphate 5g/L, sulfuric acid
Ammonium 4g/L, magnesium sulfate 5ppm, citric acid 1ppm)) fermentor in, 30 DEG C of fermentation processes temperature, ventilation quantity is about
0.4vvm, tank pressure (gauge pressure) is about 0.12MPa, and control dissolved oxygen is not less than 20%；Start when fermentation period is 10~20 hours
Substrate n-dodecane hydrocarbon is added portionwise, controls Determination of Alkane Content in fermentation liquid and is no more than 10% (v/v).Fermentation process adds 30%
Liquid alkaline controls the pH value 5.0-8.5 of fermentation liquid, until fermentation ends；Earlier fermentation (thalli growth phase) OD620It is 9~12, fermentation
Later period (substrate (alkane) transition phase) OD620It is 6~9, total fermentation period is 220 hours, and whens fermentation ends, residual hydrocarbon content was essentially
0。
The production acid concentration 135mg/g of LCDA, alkane mass transitions rate 80%.
Claims (10)
1. a kind of method of fermenting and producing long-chain biatomic acid, it is characterised in that: cell concentration is in the fermentation process with optical density
OD620Meter, OD620It is 10~30, preferably 12~21.
2. the method as described in claim 1, it is characterised in that: in the fermentation process, in the early period of the fermentation, the bacterium
Bulk concentration reaches OD620It is 15~21；The early period of the fermentation is that fermentation starts to 30~60h of fermentation.
3. the method as described in claim 1, it is characterised in that: in the fermentation process, the later period of the fermentation, the thallus
Concentration reaches OD620It is 12~18；The fermentation later period is that fermentation starts 30-60h to fermentation ends.
4. the method according to claim 1, it is characterised in that: the culture medium of the fermentation includes: carbon source, nitrogen
Source, phosphorus source, minor metallic element source and growth factor.
5. method as claimed in claim 4, it is characterised in that: the carbon source includes glucose, sucrose, maltose, molasses, first
One of alcohol, ethyl alcohol are a variety of；It is highly preferred that the carbon source include glucose, sucrose it is one or more；The carbon source is dense
Spend preferably 20~40g/L.
6. method as claimed in claim 4, it is characterised in that: the nitrogen source includes organic nitrogen and/or inorganic nitrogen, organic nitrogen packet
One of yeast extract, peptone, corn pulp or a variety of are included but are not limited to, inorganic nitrogen includes but is not limited to urea, ammonium sulfate, nitre
One of sour potassium is a variety of；Preferably, the nitrogen source includes one or two kinds of mixing of ammonium sulfate, potassium nitrate；The nitrogen
The concentration in source preferably 0.5~3g/L.
7. method as claimed in claim 4, it is characterised in that: phosphorus source includes phosphate, dibasic alkaliine and di(2-ethylhexyl)phosphate
One of hydrogen salt is a variety of；Preferably, the phosphate include one of potassium phosphate, sodium ascorbyl phosphate, ammonium phosphate salt or
It is a variety of；It is highly preferred that the phosphate includes potassium dihydrogen phosphate；The concentration of phosphorus source preferably 1~3g/L.
8. the method according to claim 1 to 7, it is characterised in that: the strain of the fermentation includes candida tropicalis
(Candida Tropicalis) or candida sake (Candidasake), preferably candida tropicalis (Candida
Tropicalis) (deposit number is CCTCC M203052) or candida tropicalis (Candida Tropicalis)
CATN145 (deposit number is CCTCC M 2011192) or candida sake (Candidasake) CATH4013 (deposit number
For CCTCC M2011486) or candida sake (Candidasake) CATH4014 (deposit number CCTCC
M2011487) perhaps candida sake (Candidasake) CATH4012 (deposit number be CCTCC M2011485) or
Candida sake (Candidasake) CATH4016 (deposit number is CCTCC M2011488) or candida sake
(Candidasake) CATH430 (deposit number is CCTCC M2011489).
9. the method according to claim 1 to 7, it is characterised in that: the substrate of the fermentation include C9~C18 just
One of alkane, linear saturated fatty acids, linear saturated fatty acids ester and salts of straight-chain saturated fatty acids are a variety of；It preferably includes
One of normal alkane, linear saturated fatty acids, linear saturated fatty acids ester and salts of straight-chain saturated fatty acids of C11~C16 or
It is a variety of；The more preferably normal alkane of C11, C12, C13, C15, C14 or C16, linear saturated fatty acids, linear saturated fatty acids ester
One of with salts of straight-chain saturated fatty acids.
10. such as the described in any item methods of claim 1-9, it is characterised in that: the tank temperature when fermentation is 28~32 DEG C；
And/or the ventilation quantity when fermentation is 0.3~0.7vvm；
And/or the tank pressure when fermentation is 0.05~0.14Mpa, the pressure is gauge pressure；
And/or the pH when fermentation is 5.0~8.5；
And/or the fermentation period is 100~180h.
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| WO2021073011A1 (en) * | 2019-10-18 | 2021-04-22 | 上海凯赛生物技术股份有限公司 | Strain for producing long-chain dicarboxylic acids and fermentation method therefor |
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