WO1995021145A2

WO1995021145A2 - FERMENTATION PRODUCTION OF LONG CHAIN α,φ-DICARBOXYLIC ACIDS FROM ALKANES BY USE OF A MICROORGANISM

Info

Publication number: WO1995021145A2
Application number: PCT/IB1995/000093
Authority: WO
Inventors: Yuan Tong Chen; Yue Chuan Pang; Ting Liu; Xiu Zhen Hao
Original assignee: Institute Of Microbiology, Chinese Academy Of Sciences; Technology Marketing & Management, L.L.C.; W & K International, Inc.
Priority date: 1994-01-28
Filing date: 1995-01-27
Publication date: 1995-08-10
Also published as: CN1030146C; WO1995021145A3; AU1544695A; CN1092108A

Abstract

Provided is a method for producing long chain α,φ-dicarboxylic acids by fermentation from long chain alkanes and recovering the produced dicarboxylic acids. Also provided is a novel microorganism useful for carrying out the method of the invention.

Description

FERMENTATION PRODUCTION OF LONG CHAIN , ω-DICARBOXYLIC ACIDS FROM ALKANES

BY USE OF A MICROORGANISM

This invention involves the fermentative methods of producing α, ω- dicarboxylic acids, especially pentadecanedioic acid, from corresponding alkanes. Long chain dicarboxylic acids are important raw materials used in manufacturing perfume, nylon, engineering plastics, hot melting adhesives, resins and cold resistant plasticizers. Dicaiboxylic acids containing more than twelve carbon atoms do not exist in nature, and there are no economical and practical chemical synthetic methods. Therefore, the method of transforming normal alkanes in petroleum to long chain dicarboxylic acid under common temperature and pressure has long been of interest and subject of investigation by scientists.

Pentadecanedioic acid is an important raw material for synthesis of cyclopentadecanone and muskone (3-methylcyclopentadecanone). The latter is the leading effective component responsible for the physiological activity of natural musk and can take the place of natural musk to compound traditional

Chinese medicines. Clinical tests indicated that the curative effect of man- made muskone was as good as that of natural musk.

As normal pentadecane is an alkane that can be assimilated easily by microorganisms, it is quite difficult to produce and obtain a high yield of pen¬ tadecanedioic acid. Although it is known in the patent literature that long chain dicarboxylic acids can be produced by microbial transformation of alkanes, no report has been found to deal with pentadecanedioic acid (DC 15) from corresponding normal alkane, pentadecane, (nCis), especially the report of high yield of DC15. Some reports on production of dicarboxylic acids through microbial fermentation technology refer to U.S. Patent 4,339,536, which deals mainly with the production of DC12 and DC13, with yield of acids of only about 45 g/L.

In U. S. Patent 4,624,920, which deals mainly with experiments with DC14 and

DC16 using 50ml and 500ml flasks, the yield of acid was extremely low, only about 3 mg/L. In Chinese Patents 87105445 and 89102548, which deal mainly with methods of producing DCiβ and DCπ, the yields of acids in a 16-liter bioreactor were 120g/L and 133g/L, respectively.

The yield of DC is reported by Japanese scientist Namio Uemura was

90g/L using a 3-liter bioreactor; a mutant strain M2030 of Candida tropicalis was used in his experiment. Another example of DCis production was reported by Shen Yong Qiang, Institute of Shanghai Plant Physiology, Shanghai, China.

A strain of Candida tropicalis N-15 in the state of concentrated static cells with cell concentration of 15 x 10/ml was used to transform normal pentadecane to pentadecanedioic acid with the yield of 109g/L. See Acta Phytophysiologica Sinica, No 1, Vol 6, 1980. The yield of DCis was only 77.2g/L after an eight- day fermentation in a laboratory experiment. As for other reports about the production of DCι , the yield of DCis was only about 40 to 50g/L. Therefore, it is still a problem to be solved to produce a high yield of DCis by microbial fermentation. The object of this invention is to develop a method of producing a single long chain dicarboxylic acid in the range from Cn to Cn and mixed dicarboxylic acids, especially a method of producing high yield of DCis.

A strain of Candida tropicalis which can produce mixed dicarboxylic acid from normal alkane was used as a starting strain in the process of repeated treatments under NO2 and UV light. An improved mutant strain, Candida tropicalis NP-6-126, was developed and was used in the invention. See Acta

Microbiologica Sinica 20(l):88-93, 1980. The strain can produce high yields of mixed dicarboxylic acids and a single dicarboxylic acid in the range from Cn to Cn, from corresponding normal alkanes, especially high yields of DCis and nCis. The strain of Candida tropicalis NP-6-126, known as NP-6-126, is now deposited in the center of General Microbiology of China Committee for

Culture Collection of Microorganisms. The deposit number is CGMCC NO.

0206.

PHYSIOLOGICAL CHARACTERISTICS OF NP-6-126 ARE AS FOLLOWS:

1. Fermentation of carbohydrates :

Glucose +, Glactose +, Sucrose +, Maltose +, Lactose -,

2. Assimilation: Glucose +, Galactose +, Sorbose -, Sucrose +, Maltose +, Cellobiose +,

Trehalose +, Lactose -, Melibiose -, Raffinose -, Melezitose +, Inulin -,

Soluble starch +, Xylose +, L-Arabinose +, D-Arabinose -, Ribose -,

Rhamnose -, -Methylglucoside +, Glycerine +, Ethanol +, Erythπtol -,

Mannitol +, Inositol -, Ribitol +, Galactitol -, Glucitol +, Sodium citrate -, Sodium dibutyrate +, Calcium lactate -, 3. Requirements for growth factors:

Biotin ++, Vitamin Bi ++, Vitamin B2 +, Vitamin Bβ +, Vitamin B12 +,

Folic acid +, Nicotinic acid +, Pantothenic acid +, Inositol +,

P-aminobenzoic acid +, 4. Others:

Nitrate -, Peptonized milk -, Decomposition of arbute seed acid -,

Solidified milk -, Lipase -,

MORPHOLOGICAL CHARACTERISTICS: Cream white color, folded type, the morphology of colony is cake shaped and crisp cake shaped.

CULTURE CHARACTERISTICS :

There are many long pseudomycelium in the liquid medium of maltose juice. There are some short pseudomycelium in the alkane medium used for seed culture. There are many single oval cells in the feπnentation medium.

(1) Slant culture of 10°Be maltose juice and 2% agar powder.

(2) Liquid medium of 10°Be maltose juice.

(3) Seed medium of alkane: KH2PO4 6- 12g/L, Corn extract 3-8g/L, Yeast extract 3-8g/L,

Sucrose 3-8g/L, Urea 3-6g/L, Heavy wax or normal pentadecane

40-70 ml/L, dissolved with tap water, natural pH. The process of seed culture:

Take one loop of mycelium of Candida tropicalis NP-6-126, smear it on the slant culture of maltose juice (15 x 180 tubes, each slant culture contains 6- 7 ml medium). Culture at 28-30°C for 40 hours, take one of the above tubes, scrape mycelium on it completely and transfer mycelium into a 250 ml flask containing 25 ml seed culture of alkane culture at 28-30°C on a rotating shaker with 180 rpm for 44 to 48 hours, the culture is prepared for grade one seed fermentor.

The method of the invention, used for producing long chain dicarboxylic acid, especially pentadecanedioic acid by using NP-6-126, is shown as follows:

Transfer feπnentation seed into feπnentation liquid mixed with 15 to

45% (v/v) normal alkane ranging from Cn to C19 and 85 to 55% (v/v) fermentation medium at pH 5.5 to 9.0, preferably within the pH range of 6.5 to

7.5. The composition of the fermentation medium is as follows: Alkaline metal phosphate 6-14g/L, preferably within 7-10 g/L, sodium chloride 0.5-2.0 g/L, yeast extract 1-6 g/L, preferably within 1.0-2.0 g/L, nitrate 5-15 g/L, preferably within 6-12 g/L, dimethyl-sulphoxide 10-20 ml, anti-foam compound 400-1200 ppm and some other conventional nutrients. Culture at 25-35°C, preferably within 27-31°C, with air input for 48 to 170 hours, add NaOH solution to adjust pH of fermentation broth every 6-8 hours so that pH can be controlled within 7.5 to 8.5. Finally, extract the produced dicarboxylic acid from the fermentation broth. At the beginning of feπnentation, the content of normal alkane is about 10-20% (v/v), from then on, it is supplemented at certain intervals to keep the concentration of normal alkane in the fermentation medium higher than 5% (v/v). Alkaline metal phosphate used may be selected from KH2PO4, NaH2Pθ4, K2HPO4 and Na2HP04; the nitrate used may be potas¬ sium or sodium nitrate.

At the end of fermentation, some water is added to the broth, NaOH so¬ lution is added to adjust the pH within a pH range of 10 to 12, heat the broth until the temperature is about 85 to 90°C, after de-emulsifying, recover the remaining alkane on the upper layer to give a clear liquid, extract the mycelium layer at the bottom by filtration under pressure or centrifugation, collect total clear liquid. Some active carbon is added into the liquid to decolour it at 85 to

90°C for 30 minutes. After removing the active carbon, the decolourized liquid is heated to 60 to 70°C. Acidify the liquid with HCI or H2SO4 to form a crystalline product at pH 4-5. When the temperature of liquid reaches 30°C, filter out the crystals under pressure, drying the crystals with air and drying at

60°C to obtain white crystals of pentadecanedioic acid.

Every kind of single or mixed dicarboxylic acid product which ranges from Cn to C19 can be produced by the strain NP-6-126 and the methods of the invention. Among them, the yield of DCis from nCis reaches 170-180g/L after a fermentation of 6 days using a 1/5 ton fermentor. The final extract rate is about 80%, the purity is about 96%.

EXAMPLE 1

1) Take one loop of mycelium of NP_6-126, smear it on the slant culture of maltose juice (15 x 180 tubes), culture at 30°C for two days.

2) Take one of the above tubes, scrape the mycelium off it completely, and transfer it into a 250-ml flask containing 25 ml of alkane seed medium, culture at 30°C on a rotating shaker with 220 rpm for 48 hours.

The composition of seed medium of alkane is as follows: KH2PO4 8g/L, yeast extract 5 g/L, com extract 3 g/L, sucrose 5 g/L, urea 3 g/L, heavy wax

50 ml L, dissolved with tap water, pH 5.0. 3) Inoculate 3.5 ml seed medium of alkane into a 500-ml flask containing 15 ml fermentation medium, culture at 30°C on a rotating shaker with 200 rpm for 4 days, add NaOH solution to adjust pH within 7.5-8.0 every 24 hours. ^'"•

The composition of feπnentation medium is as follows:

KH2PO4 8g/L, com extract 1 g/L, NaCI 1.5 g/L, urea 1 g/L, normal pentade- cane 200 m/L, anti-foam agent 500 ppm, KNO3 6 g/L, dissolved with tap water, pH 7.5, autoclave at 110°C for 30 minutes. At the end of fermentation, adjust pH to 3, extrate the broth with ether, after evaporating ether, white crystals of acid product separates out, add some of neutral hot alcohol to dissolve the crystal, titrate the solution with standard NaOH solution and calculate the content of acid product. The yield of DCn is about 53.6 g/L, the purity of DCn is 98.11% by analysis using gas chromatography.

EXAMPLE 2 The method is the same as Example 1, except NCis is used as normal alkane, the yield of DCis is 73.5 g/L, purity is 96.31%. EXAMPLE 3

The method is the same as Example 1, except nCπ is used as normal alkane, the yield of DCn is 53.9 g/L, purity is 98.2%.

EXAMPLE 4

Seed medium and culture medium are the same as Example 1, fermen¬ tation medium is as follows:

KH2PO4 8g/L, NaOH 1 g/L, yeast extract 2 g/L, com extract 1 g/L, KNO3 9 g/L, dimethyl sulphoxide 6 ml/L, anti-foam agent 600 ppm, urea 1/2 g/L, normal pentadecane 200 ml L, dissolved with tap water, pH 7.5, add 8-liter fermentation medium into a 16-liter automatic control fermentor, autoclave at

121°C for 30 minutes, after cooling to 32°C, 1500 ml of liquid seed medium is i used to inoculate fermentor. Culture period of the seed is 48 hours.

Contaminating microorganisms should be absent in seed medium as confirmed by microscopic examination. Fermentation starts at 29°C, agitation rate is 800 rpm, air input is 1: 1, fermentor pressure is 1 Kg/cm, pH is controlled at 7.5 after a 24-hour fermentation, the yield of DCis reaches 130.1 g/L after 165 hours' fermentation.

EXAMPLE 5

Seed medium and culture method are the same as Example 1, fermentation medium is the same as Example 2.

Add 1500 liters of fermentation medium into a 2500-liter fermentor, autoclave at 121°C for 30 minutes, after cooling to 32°C, 400 liters of liquid seed medium of NP-6-126 is inoculated into fermentor, contaminating microorganisms should be absent in the seed medium as shown by microscopic examination. Fermentation starts at 29°C, agitation rate is 200 rpm, air input is 1:0.8, fermentor pressure is 0.8 Kg/cm^. At the beginning of fermentation, the volume of nCis is 300 liter, NaOH solution is added into the fermentor to adjust pH within 7.5 every 8-9 hours after 24 hours of fermentation, 120 liters of nCis is supplemented daily after 3 days of fermentation and at least 3 times. The content of DCis in clear fermentation liquid is 178 g/L after 6 days of fermenta¬ tion. When fermentation is over, the same volume of water is added into the fermentor, and heated to 90°C. NaOH solution is added into the fermentor to adjust pH to 10, after cooling to 50°C, the broth is kept in a separating tank for one day, then, 60 liters of remaining alkane on the upper layer is removed, mycelium layer at the bottom is subjected to filter under pressure, the mycelium is removed, filtrate and clear liquid from the middle of the broth are merged together, then, add 0.7% active carbon to decolour the solution at 90°C for 15 minutes. After removing active carbon by filtration under pressure, decolourized liquid is put into the acidifying tank, add water till the concentration of DCis is about 4%, heat the solution to 70°C, and acidify it with concentrated HCI to pH 4, cooling until temperature is about 30°C. Filter the liquid by use of pressure, drying the crystal with air and stoving at 60°C to obtain 229 Kg white DCis. Final extract rate is about 85.76%, purity is 96.5%.

Claims

What is Claimed is:

1. A method for producing a long chain α,ω -dicarboxylic acid by fermentation using the microorganism Candida tropicalis NP-6-126, CGMCC NO. 0206 in a medium having a long chain alkane and recovering the long chain , ω- dicarboxylic acid formed.

2. A method of Claim 1 wherein the long chain alkane used in the fermentation medium has 11 to 17 carbon atoms..

3. A method of Claim 1 wherein the long chain alkane of the fermentation medium is n-pentadecane and the product formed is α,-ω -pentadecanedioic acid.

4. The microorganism Candida tropicalis NP-6-126, CGMCC NO. 0206 strain mutants and variants thereof which are capable of producing a long chain dicarboxylic acid in accordance with the method of Claim 1.