CN1130685A - Process for producing long-chain alpha, omega-dicarboxylic acid by synchronous fermentation of microbe - Google Patents

Abstract

A microbe synchronous fermentation process for producing long-chain alpha, omaga-dicarboxylic acid, especially dodecadiatomic acid (DC12) with high output features that after microbe strain (excellent saltant of Candida tropicalis) is inoculated into culture medium whose matrix is n-alkanes containing C11-C18, pH is controlled to 6.0-6.8 for thallus growth as priority while also production of diatomic acids by limited output, and when optical density of thallus growth (OD(X30,620nm)) reaches 0.6 and pH is controlled to 7.0-7.8, different diatomic acids with the same chain length as matrix is greatly produced. After inoculation for 40 hr, when acid output reaches 33.3 g/L, then output of acid is transferred to for fermentation as priority and can reach 145 g/l in 130 hr after inoculation.

Description

The method of long-chain production by simultaneous fermentation of microorganism alpha, omega-dicarboxylic acid

The present invention relates to the synchronous fermentation n-paraffins of microorganism and produce the long-chain alpha, omega-dibasic acid, n-dodecane (nC especially ferments ₁₂), high yield SL-AH (DC ₁₂) method.

Long-chain biatomic acid is the important source material of synthetic perfume, nylon engineering plastic, hot melt adhesive, resin, cold-resistant plasticizer etc.DC ₁₂Be synthetic senior nylon engineering plastic nylon 12, nylon 1212 and clothes are with the important source material of high-grade nylon hot-melt adhesive.Nylon 12 is that to develop carbochain at present in the world the longest, best in quality, and price is the highest, and annual production reaches 50,000 tons senior nylon engineering plastic.The Buddhist nun is to be gone into operation by German H ü Ls company and Switzerland Emser company in 1966 through 12, also all there were production in France, Japan, the U.S. afterwards, they adopt the pure chemistry methods more, are raw material with the divinyl, at high temperature, high pressure, under the catalyzer condition, produce through the reactions steps of seven complexity, step is many, and yield is low, the cost height, China can't produce at present.If with DC ₁₂Be raw material synthetic nylon 12, only need through four reactions steps, mild condition, step is few, the yield height, cost is low, be competitive power the strongest synthetic method.And DC ₁₂Occurring in nature does not exist, and is difficult to again on the chemical industry synthesize, and therefore, utilizes the special oxidation capacity of microorganism, transforms the n-dodecane in the oil at normal temperatures and pressures, produces DC ₁₂, open up DC ₁₂New source, for nylon 12, nylon 1212 and clothes provide inexpensive important source material with the industrial production of high-grade hot melt adhesive.

In patent documentation, have from the experimental example of n-dodecane fermentative production SL-AH, but all be laboratory level.In US 4339536, production DC is arranged ₁₂Experimental example, but only reach the 45g/L level, in CN1071951A, its experimental example 2 is asynchronous fermentative production DC ₁₂, DC in the automatic controlling tank of 3L ₁₂Reach 102g/L, transformation efficiency is 75%.

The used bacterial strain of the present invention is candida tropicalis (Candida tropicalis) UH-2-48, be to produce the candida tropicalis of mixed dibasic acid (referring to " microorganism journal " 20 (1): 88-93 with a strain oxidation normal alkane, 1980) be starting strain, by nitrous acid and ultraviolet repeatedly repeatedly mutagenesis screening cultivate, can be from C ₁₁-C ₁₇Various single normal alkane and mix normal alkane, especially n-dodecane, the diprotic acid of high production ground production respective chain length.Candida tropicalis UH-2-48 (hereinafter to be referred as UH-2-48) is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is: CGMCC NO.0239.

Seed culture medium of the present invention: the wort of (1) 10Be ' pol adds the solid inclined-plane that 2% agar is made; (2) malt juice liquid medium of 10Be ' pol; (3) the alkane seed culture medium comprises: KH ₂PO ₂6-12g/L, yeast extract paste 3-8g/L, sucrose 3-8g/L, corn steep liquor 3-8g/L urea 3-6g/L, heavy wax 40-70ml/L, tap water preparation, natural PH.

The process of cultivating seed is: get a transfering loop UH-2-48 yeast thalline, be coated on by ( 15 * 180 test tubes, every dress 6-7ml substratum is put into the inclined-plane after the sterilization) on the wort solid inclined-plane, cultivated 40 hours in 29-30 ℃.Getting an above-mentioned cultured UH-2-48 bacterial classification all scrapes in the 250mL triangular flask that 25ml alkane seed culture medium is housed, on 29-30 ℃ 220 rev/mins rotary shaker, cultivated 40-48 hour, as the shake flask fermentation seed or get two above-mentioned cultured UH-2-48 bacterial classifications and all scrape in the 5000ml triangular flask that the 500mL substratum is housed, in 200 rev/mins rotary shaker 29-30 ℃ of cultivations 4-48 hour, as the seed of first class seed pot.

The concrete grammar of producing long-chain biatomic acid, particularly SL-AH with UH-2-48 bacterial strain of the present invention is: through microscopy, the strain liquid of assorted bacterium does not insert PH5.5-9.0, the preferably C that contains 15-30% (V/V) of 6.-6.8 cultured ₁₁-C ₁₇In the mixed solution of normal alkane and 85-70 (V/V) fermention medium.Consisting of of fermention medium: alkali metal phosphate 6-14g/L is preferably 7-10g/L, sodium-chlor 0.5-2.0g/L, yeast extract paste 1-6g/L, be preferably 3-5g/L, corn steep liquor 0.5-2g/L, urea 0.5-2.5g/L, be preferably 1.0-2.0g/L, dimethyl sulfoxide (DMSO) 10-20ml/L, defoamer 400-1200PPm, heavy wax or sucrose 15-30g/L and some other known nutrition sources, under PH6.0-7.5 with said mixture at 25-34 ℃, be preferably in 27-31 ℃ of aerobic fermentation 72-170 hour.Within 40 or 48 hours, system PH is controlled at 6.0-6.8, based on thalli growth, produces the diprotic acid of some amount simultaneously, after 48 hours, between the system PH control 7.0-7.8, based on fermentation and acid.Since 72 hours, add a certain amount of normal alkane every day, make in the fermented liquid normal alkane concentration (V/V) all the time＞5%.Alkali metal phosphate can be from KH ₂P ₄, NaH ₂PO ₄, K ₂HPO ₄Or Na ₂HPO ₄In select a kind of.

After the fermentation ends, add an amount of water, add alkali to PH10-13, be heated to 80-95 ℃, carry out the breakdown of emulsion layering, the upper strata Oil residue recuperation is usefulness again, clear liquid in the middle of emitting, lower floor's thalline layer is handled once or press filtration again, merge clear liquid, add proper amount of active carbon 85-90 ℃ of decolouring 30 minutes, remove gac after, destainer is heated to 70-80 ℃, adds HCl or H ₂SO ₄Carry out acidizing crystal to PH4-5, be cooled to 30 ℃, press filtration, air blow drying, 60-70 ℃ of oven dry, white diprotic acid.

With UH-2-48 bacterial strain of the present invention and fermentation process, can produce C ₁₁-C ₁₇Various list-diprotic acid and mixed dibasic acid.Wherein in 3 tons of fermentor tanks, from nC ₁₂Fermentative production DC ₁₂The time, fermented 130 hours, produce the acid amount up to 145g/L, transformation efficiency reaches 90%, and the aftertreatment total recovery reaches more than 80%, DC ₁₂Purity reaches more than 95%.

Example 1.

(1) gets a transfering loop UH-2-48 bacterial classification, be coated on Ф 15 * 180 Boiling tube wort solid inclined-planes, cultivated two days for 30 ℃.

(2) get one of above-mentioned bacterial classification, insert and be equipped with in the 250mL triangular flask of 25ml alkane seed culture medium, on 200 rev/mins rotary shaker, cultivated 46 hours in 30 ℃.KH in the alkane seed culture medium ₂PO48g/L, yeast extract paste 3g/L, corn steep liquor 3g/L, sucrose 5g/L, urea 3g/L, heavy wax 50m/L, the tap water preparation, PH5.0 sterilized 30 minutes for 110 ℃.

(3) in the 500ml triangular flask of 15ml fermention medium is housed, insert the above-mentioned seed liquor of 3.5mL, 200 rev/mins rotary shaker top fermentations 4 days, transferred a PH to 7.5-8.0 with NaOH in per 24 hours.Contain KH in the fermention medium ₂PO ₄8g/L, yeast extract paste 2g/L, corn steep liquor 1g/L, Nacl 1g/L, urea 1.2g/L, heavy wax 30ml/L, nC ₁₂200mL/L, the tap water preparation, PH7.3 was 110 ℃ of sterilizations 30 minutes.After the fermentation ends, transfer PH to 3. ether extraction, remove ether, get white crystals,, calculate diprotic acid content with the titration of standard NaoH solution with 6moLHcl.DC as a result ₁₂Output is 61.6g/L, DC ₁₂Purity 97.83%.

Example 2.

According to the method for example 1, be normal alkane nC ₁₁DC as a result ₁₁Output is 46.5g/L, DC ₁₁Purity is 98.58%.

Example 3.

According to the method for example 1, be normal alkane nC ₁₄, do not increase the weight of wax, as a result DC ₁₄Output 72.3g/L, purity 96.88%.

Example 4.

According to the method for example 1, be normal alkane nC ₁₇, DC ₁₇Output is 54.1g/L, and purity is 97.27%.

Example 5.

(1) seed culture medium and cultural method and fermention medium are with example 1.

(2) 2000ml was cultivated two days, and OD (* 30, be 0.61 620nm), PH3.8, stalwartness, the UH-2-48 kind liquid that does not have assorted bacterium inserts the 350L seed culture medium is housed, in 40 minutes 500L seeding tank of 121 ℃ of sterilizations, 29 ℃ 350 rev/mins, tank pressure 0.8kg/cm ², air flow 1: 0.8 was cultivated 48 hours, as the seed of fermentation.

(3) cultivating in (2), stalwartness, the 350L kind liquid that does not have assorted bacterium inserts the 1800L fermention medium is housed, in 40 minutes the 3000L fermentor tanks of 121 ℃ of sterilizations, 30 ℃, 200 rev/mins, tank pressure 1kg/cm ², air flow 1: 0.7, during beginning, nC ₁₂240kg, in 40 hours, system PH is controlled at 6.0-6.8, and the thalline ramp also produces 33.3g/L DC ₁₂, hierarchy of control PH is 7.0-7.8 then, continues fermentation 90 hours, adds a certain amount of normal alkane every day, makes in the fermented liquid normal alkane concentration (V/V) all the time＞5%.Fermented DC 130 hours ₁₂Content is 145g/L, transformation efficiency 90%.After the fermentation ends, cumulative volume 2000L adds a certain amount of tap water, is heated to 80 ℃ and transfers PH10, put into standing demix jar layering one day, reclaim upper strata Residual oil 70kg, lower floor's bacterium layer is removed thalline by press filtration, cleaner liquid and middle level clear liquid merge, and add 0.7% gac, and 90 ℃ decoloured 20 minutes, gac is removed in press filtration, and the decolouring clear liquid is squeezed in the crystallizer, is heated to 70 ℃, add dense HCl and transfer PH to 4, be cooled to room temperature, filter press, air blow drying, solid substance get white DC 60 ℃ of oven dry ₁₂247kg, aftertreatment total recovery 85%, purity 97.1%.

Claims (2)

1. one kind is utilized microbial fermentation to produce α. the method for ω-12 carbon dicarboxylic acid, it is characterized in that with the n-dodecane being in the substratum of matrix, with candida tropicalis (Candida tropicalis) UH-2-48, i.e. formed diprotic acid is reclaimed in CGMCC NO.0239 fermentation then.

2. candida tropicalis (Candida Tropicalis) UH-2-48, i.e. CGMCCNO.0239 bacterial strain.