CN103805643B - A kind of method of producing long-chain biatomic acid - Google Patents
A kind of method of producing long-chain biatomic acid Download PDFInfo
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- CN103805643B CN103805643B CN201210439800.4A CN201210439800A CN103805643B CN 103805643 B CN103805643 B CN 103805643B CN 201210439800 A CN201210439800 A CN 201210439800A CN 103805643 B CN103805643 B CN 103805643B
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Abstract
A kind of method of producing long-chain biatomic acid, the method comprises following content: diprotic acid strain expanded culture is obtained seed liquor, and seed liquor carries out expansion fermentation, adds emulsification alkane in fermenting process, reaction terminates rear removing tropina, and crystallization obtains long-chain biatomic acid product.The inventive method improves the consistency in fermentation system between aqueous phase and oil phase, improves the transformation efficiency of alkane and the yield of dicarboxylic acid product in fermentation system.
Description
Technical field
The invention belongs to biological chemical field, relate generally to a kind of method utilizing fermentation mode to produce long-chain biatomic acid.
Background technology
Long-chain biatomic acid (Longchaindicarboxylicacids) refers to the aliphatic dicarboxylic acid (being called for short DCn) containing more than 10 carbon atoms in carbochain, comprising saturated and unsaturated dicarboxylic acid, is the fine chemical product that a class has important and extensive industrial use.Also be in chemical industry, synthesize the important source material such as fine perfumery, high performance nylon engineering plastics, high-grade nylon hot-melt adhesive, high-temperature electric medium, senior paint and coating, senior lubricant, cold-resistant plasticizer, resin, medicine and agricultural chemicals simultaneously.
Production of Long-chain Dicarboxylic Acids by Fermentation Methods is the effect utilizing the distinctive oxidation capacity of microorganism and extracellular microbial endoenzyme, at normal temperatures and pressures respectively by α, omega oxidation, the methyl oxidation at long-chain normal alkane two ends is become carboxyl, generates the various long-chain biatomic acids of respective chain length.The bacterium of petroleum hydrocarbon, mould and actinomycetic kind can be utilized a lot, and wherein the yeast of mycocandida (candida) is the high producing microbial of normal alkane fermentative production diprotic acid.Long-chain biatomic acid fermentation is typical gas phase (oxygen)-aqueous phase (fermented liquid)-oil phase (alkane)-solid phase (thalline) four phase system, in order to maintain the homogeneity of four phase system, usually adds emulsifying agent in the medium during the fermentation.
CN102115768A discloses the method that a kind of microbial synchronous fermentation n-hexadecane produces 16-dicarboxylic acid, adds tensio-active agent (polysorbate60) 0.1 ~ 2g/L to strengthen the emulsifying effectiveness of oil phase.Generally emulsifying agent is not composition needed for microbial metabolism, and therefore the mode of this additional emulsifying agent can have a negative impact to normal fermentable.
The present stage fermentation of long-chain biatomic acid generally adopts Batch fermentation, adds the form of alkane in fermenting process.CN102115767A discloses a kind of microbial synchronous fermentation n-undecane and produces 11 carbon dicarboxylic acid methods, by adding n-hexadecane at 60,90,120 hours, to make in fermented liquid normal alkane concentration all the time >=5%(V/V).In actual production process, in order to make the alkane added quick evenly mixed with water, generally adopt the method improving stirring velocity, but the raising of stirring velocity can make shearing force increase, thus affect the growth of fermentation thalli.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of method of producing long-chain biatomic acid.The method improve the consistency in fermentation system between aqueous phase and oil phase, improve the efficiency of fermentation.
A kind of method of producing long-chain biatomic acid, comprise following content: the diprotic acid strain expanded culture of preservation is obtained seed liquor, seed liquor carries out expansion fermentation, adds emulsification alkane in fermenting process, reaction terminates rear removing tropina, and crystallization obtains long-chain biatomic acid product.
In the inventive method, described diprotic acid bacterial classification is selected from the microorganism that normal alkane can be utilized to grow or the microorganism with complete α, omega oxidation approach, the one in preferred mycocandida, Cryptococcus, Endomyces, Hansenula anomala genus, pichia genus, Rhodotorula, torulopsis or Trichosporon etc.
In the inventive method, described enlarged culturing, carries out enlarged culturing according to 7 ~ 15 times, preferably 9 ~ 12 times of enlarged culturing.
In the inventive method, each component concentration of minimum medium is as follows: sucrose 20 ~ 50g/L, corn steep liquor 1 ~ 3g/L, yeast extract paste 1 ~ 2g/L, sodium-chlor 0.5 ~ 1.5g/L, potassium primary phosphate 3 ~ 8g/L, magnesium sulfate 1 ~ 2g/L, urea 1 ~ 5g/L.
In the inventive method, the volume of described expansion fermentation is 7 ~ 15 times of seed liquor volume, preferably 9 ~ 12 times.
In the inventive method, fermentation condition is: mixing speed is 100 ~ 700rpm, and air flow is 0.6 ~ 1.0VVM, and leavening temperature is 29 ~ 32 DEG C, and fermentation time is 138 ~ 144 hours.
In the inventive method, described fermenting process is regulated and controled by the mode of adjust pH step by step, and fermentation starts, hierarchy of control pH value is, after 4 ~ 5,24 hours, system pH is adjusted to 6.8 ~ 7, then improves a pH value at interval of 24 hours, each raising 0.1 ~ 0.5, until fermentation ends.
In the inventive method, add alkane concentration in emulsification alkane maintenance system during the fermentation and be not less than 4%, the volume at every turn adding emulsification alkane is 3% ~ 12% of fermentation cumulative volume.
In the inventive method, described emulsification alkane is that long chain alkane mixes with water, carries out that microwave treatment obtains.
Wherein long chain alkane is the alkane containing 10 ~ 16 carbon atoms in carbochain, and the volume ratio of long chain alkane and water is 3:1 ~ 10:1.
Wherein the power density of microwave treatment is the microwave power that 100 ~ 10kW/L(often rises oil-water mixture), microwave frequency is 300 ~ 3000MHz, and microwave treatment time is 10 ~ 30 minutes.
Compared with prior art, a kind of method tool producing long-chain biatomic acid of the present invention has the following advantages:
Microwave treatment is adopted alkane and water to be processed, microwave treatment can produce heat effect, utilize the feature of water molecules fast propagation microwave radiation energy, strengthen moving upward of water molecules, improve both mixed effects, facilitate the emulsification between oil phase and aqueous phase, improve the emulsifying effectiveness of alkane, improve the homogeneity of gas phase (oxygen)-aqueous phase (fermented liquid)-oil phase (alkane)-solid phase (thalline) four phase system, avoid the emulsifying agent of interpolation and high-speed stirring to the impact of fermenting process.By improve the transformation efficiency of alkane in fermentation system to the emulsification pre-treatment of alkane, and improve the yield of dicarboxylic acid product.
Embodiment
Below in conjunction with embodiment the detailed process of method is sent out to the present invention and effect is described, but be not limited to following examples.
Candida tropicalis (Candidatropicalis) mutant strain PF-UV-56 is selected to carry out the experiment of long chain alkane fermentative production long-chain biatomic acid as fermentation strain in the present embodiment, this mutant strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNO.0356.
Embodiment 1
The each component concentration of minimum medium is as follows: sucrose 30g/L, corn steep liquor 3g/L, yeast extract paste 2g/L, sodium-chlor 1g/L, potassium primary phosphate 5g/L, magnesium sulfate 1g/L, urea 4g/L.
(1) fermentation seed liquid is cultivated: slant preservation strain inoculation is carried out strain activation and culture in 3000mL shaking flask, and activation culture liquid amasss as 500mL, and culture temperature is 32 DEG C, and shaking table revolution is 120rpm, cultivates 48 hours.
(2) alkane pre-treatment: get 12 carbon alkane 3000mL and mix with 600mL water, microwave treatment 10 minutes, microwave frequency is 2450MHz, and power density is 4.2kW/L.
(3) diacid fermentation: the bacterial classification seed liquor 1000mL of activation is added in 6000mL fermention medium, then add ten two carbon alkanes of 600mL through microwave emulsification, pH value is 4.3, and agitation revolution is 400rpm, and ventilation is set to 7000mL/min; PH value was adjusted to 7.2 in 24 hours, and adds ten two carbon alkanes of 1200mL through microwave emulsification; Respectively pH value was raised 0.2 in 48 hours, 72 hours, and add ten two carbon alkanes of 600mL through microwave emulsification respectively, respectively pH value was raised 0.2 in 96 hours, 120 hours, until reaction in 144 hours terminates.In final fermentation system, SL-AH concentration is 150.1g/L, and the transformation efficiency of 12 carbon alkanes is 75.5%.
Embodiment 2
The each component concentration of minimum medium is as follows: sucrose 20g/L, corn steep liquor 1g/L, yeast extract paste 2g/L, sodium-chlor 1.5g/L, potassium primary phosphate 3g/L, magnesium sulfate 2g/L, urea 3g/L.
(1) fermentation seed liquid is cultivated: slant preservation strain inoculation is carried out strain activation and culture in 3000mL shaking flask, and activation culture liquid amasss as 500mL, and culture temperature is 32 DEG C, and shaking table revolution is 120rpm, cultivates 48 hours.
(2) alkane pre-treatment: get triclecane 3000mL and mix with 300mL water, microwave treatment 15 minutes, microwave frequency is 915MHz, and power density is 300W/L.
(3) diacid fermentation: the bacterial classification seed liquor 1000mL of activation is added in 6450mL fermention medium, and add the triclecane of 550mL through microwave emulsification, pH value is set to 4.5, and agitation revolution is 500rpm, and ventilation is set to 8000mL/min; PH value was adjusted to 7 in 24 hours, and adds the triclecane of 1100mL through microwave emulsification.Respectively pH value was raised 0.2 in 48 hours, 72 hours, and add the triclecane of 550mL through microwave emulsification respectively, respectively pH value was raised 0.2 in 96 hours, 120 hours, until reaction in 144 hours terminates.In final fermentation fermentation system, tridecanyldicarboxylic acid concentration is 138.6g/L, and the transformation efficiency of 12 carbon alkanes is 72.8%.
Embodiment 3
The each component concentration of minimum medium is as follows: sucrose 50g/L, corn steep liquor 2g/L, yeast extract paste 2g/L, sodium-chlor 1.5g/L, potassium primary phosphate 3g/L, magnesium sulfate 2g/L, urea 1g/L.
(1) fermentation seed liquid is cultivated: slant preservation strain inoculation is carried out strain activation and culture in 5000mL shaking flask, and activation culture liquid amasss as 900mL, and culture temperature is 32 DEG C, and shaking table revolution is 180rpm, cultivates 48 hours.
(2) alkane pre-treatment: get hexadecane hydrocarbon 3000mL and mix with 600mL water, microwave treatment 30 minutes, microwave frequency is 896MHz, and power density is 140W/L.
(3) diacid fermentation: the bacterial classification seed liquor 1000mL of activation is added in 5400mL fermention medium, and add the hexadecane hydrocarbon of 600mL through microwave emulsification, pH value is 4.3, and agitation revolution is 400rpm, and ventilation is set to 7000mL/min; PH value is littlely adjusted to 7 by 24 constantly, and adds the hexadecane hydrocarbon of 1200mL through microwave emulsification respectively.Respectively pH value was raised 0.2 in 48 hours, 72 hours, and add the hexadecane hydrocarbon of 600mL through microwave emulsification, respectively pH value was raised 0.2 in 96 hours, 120 hours, until reaction in 144 hours terminates.In final fermentation system, 16-dicarboxylic acid concentration is 123.1g/L, and the transformation efficiency of 12 carbon alkanes is 61.6%.
Comparative example 1
According to the experimental technique in embodiment 1, but the alkane added is without microwave emulsification.
(1) fermentation seed liquid is cultivated: slant preservation strain inoculation is carried out strain activation and culture in 3000mL shaking flask, and activation culture liquid amasss as 500mL, and culture temperature is 32 DEG C, and shaking table revolution is 120rpm, cultivates 48 hours.
(2) diacid fermentation: the bacterial classification seed liquor 1000mL of activation is added in 6000mL fermention medium (add polysorbate60 in substratum, concentration is 0.1g/L), and add 500mL 12 carbon alkane and 100mL water.PH value is set to 4.3, and agitation revolution is 700rpm, and ventilation is set to 7000mL/min.PH value was adjusted to 7.2 in 24 hours, and adds 1000mL 12 carbon alkane and 200mL water.Respectively pH value was raised 0.2 in 48 hours, 72 hours, and add 500mL 12 carbon alkane and 100mL water respectively, respectively pH value was raised 0.2 in 96 hours, 120 hours, until reaction in 144 hours terminates.In final fermentation system, SL-AH concentration is 125.7g/L, and the transformation efficiency of 12 carbon alkanes is 70.2%.
Claims (8)
1. produce the method for long-chain biatomic acid for one kind, it is characterized in that comprising following content: diprotic acid strain expanded culture is obtained seed liquor, seed liquor carries out expansion fermentation, adds emulsification alkane in fermenting process, reaction terminates rear removing tropina, and crystallization obtains long-chain biatomic acid product; Wherein said emulsification alkane is that long chain alkane mixes with water, carries out that microwave treatment obtains; Described long chain alkane is the alkane containing 10 ~ 16 carbon atoms in carbochain, and the volume ratio of long chain alkane and water is 3:1 ~ 10:1.
2. method according to claim 1, is characterized in that: described diprotic acid bacterial classification is selected from the microorganism that normal alkane can be utilized to grow or the microorganism with complete α, omega oxidation approach; Described diprotic acid bacterial classification is selected from the one in mycocandida, Cryptococcus, Endomyces, Hansenula anomala genus, pichia genus, Rhodotorula, torulopsis or Trichosporon.
3. method according to claim 1, is characterized in that: described diprotic acid bacterial classification is selected from the one in mycocandida, Cryptococcus, Endomyces, Hansenula anomala genus, pichia genus, Rhodotorula, torulopsis or Trichosporon.
4. method according to claim 1, is characterized in that: the volume of described expansion fermentation is 7 ~ 15 times of seed liquor volume.
5. method according to claim 1, is characterized in that: fermentation condition is: mixing speed is 100 ~ 700rpm, and air flow is 0.6 ~ 1VVM, and leavening temperature is 29 ~ 32 DEG C, and fermentation time is 138 ~ 144 hours.
6. method according to claim 1 or 5, it is characterized in that: described fermenting process is regulated and controled by the mode of adjust pH step by step, fermentation starts, hierarchy of control pH value is, after 4 ~ 5,15 ~ 24 hours, system pH is adjusted to 6.8 ~ 7.2, then at interval of 20 ~ 24 hours, improve a pH value, improve 0.1 ~ 0.5, until fermentation ends at every turn.
7. method according to claim 1, is characterized in that: the concentration of volume percent adding alkane in emulsification alkane maintenance system is during the fermentation not less than 4%, and the volume at every turn adding emulsification alkane is 3% ~ 12% of fermentation cumulative volume.
8. method according to claim 1, is characterized in that: the power density of microwave treatment is 100W/L ~ 10kW/L, and microwave frequency is 300MHz ~ 3000MHz, and microwave treatment time is 10 ~ 30 minutes.
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| CN111100884B (en) * | 2018-10-26 | 2022-03-08 | 中国石油化工股份有限公司 | Method for preparing long-chain dicarboxylic acid by fermentation |
| CN112725385B (en) * | 2019-10-28 | 2022-09-09 | 中国石油化工股份有限公司 | Method for preparing long-chain dicarboxylic acid by fermentation |
| CN112725400B (en) | 2019-10-28 | 2023-02-03 | 中国石油化工股份有限公司 | Long-chain composition, long-chain composition kit, manufacturing method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1071951A (en) * | 1991-10-29 | 1993-05-12 | 中国石油化工总公司抚顺石油化工研究院 | The method of asynchronous microbiological fermentative production long-chain alpha, omega-dibasic acid |
| CN1147016A (en) * | 1995-10-05 | 1997-04-09 | 中国石油化工总公司 | Method for treatment of alpha, omega dibasic acid fermentation liquor |
| CN1257126A (en) * | 1998-12-16 | 2000-06-21 | 中国石油化工集团公司 | Process for producing alpha, omega-long chain binary acid by using microorganism fermentation |
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Patent Citations (3)
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| CN1071951A (en) * | 1991-10-29 | 1993-05-12 | 中国石油化工总公司抚顺石油化工研究院 | The method of asynchronous microbiological fermentative production long-chain alpha, omega-dibasic acid |
| CN1147016A (en) * | 1995-10-05 | 1997-04-09 | 中国石油化工总公司 | Method for treatment of alpha, omega dibasic acid fermentation liquor |
| CN1257126A (en) * | 1998-12-16 | 2000-06-21 | 中国石油化工集团公司 | Process for producing alpha, omega-long chain binary acid by using microorganism fermentation |
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| 发酵法生产长链二元酸研究进展;徐成勇等;《生物工程进展》;20020425;第22卷(第2期);第66-69页 * |
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