JPH06277078A - Method for producing parahydroxybenzaldehyde - Google Patents

Abstract

(57) [Abstract] [Purpose] A low-cost method for producing para-hydroxybenzaldehyde, which is useful as a raw material for pharmaceuticals and brighteners for plating, dyes, fragrance intermediates, agricultural chemicals, etc., by microbial oxidation with little energy required. Offer of. [Composition] Microbial Pseudomonas
putida) KS-0160, Pseudomonas putida (Ps
eudomonas putida) KS-0180 or Enterobacter cloacae IFO13
A method for producing parahydroxybenzaldehyde, which comprises converting paracresol into parahydroxybenzaldehyde using 535.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing parahydroxybenzaldehyde by converting paracresol using a microorganism. More specifically, Pseudomonas putida KS-0160, Pseudomonas
TECHNICAL FIELD The present invention relates to a method for producing parahydroxybenzaldehyde, which comprises culturing Putida KS-0180 or Enterobacter crocusae IFO13535, adding para-cresol to the culture solution, and collecting para-hydroxybenzaldehyde converted and produced in the culture solution. .

[0002]

2. Description of the Related Art Conventionally, the production of parahydroxybenzaldehyde has been carried out by subjecting phenol as a raw material to heating and reaction with chloroform, followed by steam distillation in the presence of acetic acid. However, the selectivity is by no means high, and the problem is that ortho-forms are mixed.

On the other hand, the production of parahydroxybenzaldehyde by microorganisms has been detected as a metabolic intermediate to protocatechuic acid in the paracresol metabolic pathway of Pseudomonas putida (S. Dagley and MD Patel; Bioch.
em.J., Vol.66, 227 (1970)), this research is
It has only been clarified that the metabolism of para-cresol proceeds via para-hydroxybenzaldehyde, and para-cresol is given as a raw material, which is converted to form para-hydroxybenzaldehyde, which is accumulated in the medium and collected. Does not suggest.

[0004]

An object of the present invention is to provide a novel method for producing parahydroxybenzaldehyde by utilizing an economical microbial conversion method which proceeds at room temperature and atmospheric pressure.

[0005]

[Means for Solving the Problems] The present inventors have focused on the substance conversion ability of microorganisms, which is more energy saving than the chemical synthesis method. That is, a co-oxidation (Co-oxidati) that gives a substrate to a microorganism to grow it, causes it to grow, and makes the culture solution coexist with para-cresol to oxidize and convert it.
The objective was to complete the method of producing para-hydroxybenzaldehyde by the method of (on) and accumulating it in a large amount without decomposing it. Therefore, as a result of searching for a microorganism having the ability to produce para-hydroxybenzaldehyde from para-cresol by co-oxidation, the present inventors have found that bacterial strains belonging to Pseudomonas and bacterial strains belonging to Enterobacter crocuse are A strain which converts cresol into parahydroxybenzaldehyde was found, and the present invention was completed.

That is, the present invention uses the microorganisms Pseudomonas putida KS-0160 strain, Pseudomonas putida KS-0180 strain, or Enterobacter crocusae IFO13535 strain to transform paracresol into parahydroxybenzaldehyde. A method for manufacturing the same is provided. Furthermore, the present invention provides a method for producing parahydroxybenzaldehyde, which comprises culturing the cells of the above-mentioned microorganism in a medium containing para-cresol.

Also provided is a method for producing parahydroxybenzaldehyde in which paracresol is converted into parahydroxybenzaldehyde by culturing and proliferating the cells of the above-mentioned microorganism, and then adding paracresol to continue the culture.

The bacterial strain used in the present invention is a strain identified as a bacterium belonging to the genus Pseudomonas and a bacterium belonging to the genus Enterobacter, which has the ability to convert para-cresol into para-hydroxybenzaldehyde. Good. In particular, bacteria belonging to Pseudomonas putida and Enterobacter crocusae are preferable, and Pseudomonas putida KS-016 is further preferable.
0, Pseudomonas putida KS-0180, Enterobacter crocusae IFO 13535 are preferred. Therefore, strains isolated from the soil should be used for the Vergis Manual of Systematic Bacteriology (Be
rgey's Mannual of Systematic Bacteriology), Vol.
Strains identified as strains of the genus Pseudomonas or the genus Enterobacter according to the criteria described in 1, 141-199 and 465-471 (Genus Enterobacter) (1984) may be used. Further, it may be obtained from an organization such as the Institute of Microscopy. Pseudomonas Pseudomonas putida KS-0160 strain was established on March 17, 1992 at the Institute of Microbial Technology, Ministry of International Trade and Industry.
It has been deposited / stored with a display for identifying it as nas putida KS-0160, and the deposit number is Microindustrial Research Institute No. 12879 (FERM P-12879). Pseudomonas putida KS-0180 strain
It was deposited and stored on the 17th of May at the Research Institute for Microbial Engineering, Ministry of International Trade and Industry, Institute of Industrial Science and Technology, with an identification label of Pseudomonas putida KS-0180, and the deposit number is Microbiology Research Institute No. 12880. (FERM P-128
80). Pseudomonas Petida KS-0160
Isolation and identification of the strain and Pseudomonas putida KS-0180 are described in Japanese Patent Application No. 4-145729 and Japanese Patent Application No.
As described in No. 145730.

The present inventors have found that strains belonging to the genus Pseudomonas and the genus Enterobacter, particularly Pseudomonas
Putida KS-0160 strain, Pseudomonas putida KS
-0180 strain and Enterobacter crocusae IF
As described above, the inventors have found that O13535 can convert para-cresol by a co-oxidation action to generate para-hydroxybenzaldehyde, and thus have reached the present invention.

The production method of the present invention comprises a strain belonging to the genus Pseudomonas and the genus Enterobacter, particularly Pseudomonas putida KS-0160, Pseudomonas putida KS-0180 and Enterobacter crocusae IFO13535. By utilizing the oxidation effect, the starting material is allowed to coexist in the culture solution of the microorganism, and the growth of the microorganism is carried out with a carbon source and a nitrogen source necessary for growth different from the starting material to be converted, and the grown microorganism Is to oxidize and convert the coexisting starting materials in the culture solution.

In the present invention, paracresol can be oxidized to parahydroxybenzaldehyde on an industrial scale by using a strain belonging to the genus Pseudomonas or a strain belonging to the genus Enterobacter. In particular, Pseudomonas putida KS-0160 strain, Pseudomonas putida KS-0180 strain and Enterobacter crocusae IFO13535 strain are used to oxidize para-cresol to para-hydroxybenzaldehyde.

The production method of the present invention includes a step of growing a microorganism and a step of converting para-cresol into para-hydroxybenzaldehyde using the microorganism. Further, the step of growing the microorganism (growth step) and the step of oxidizing paracresol to parahydroxybenzaldehyde using the microorganism (conversion step) may be separate steps or may be performed simultaneously. .

As a medium for growing the bacterial strain used in the method of the present invention (growth step), an ordinary bacterial medium may be used. Any medium can be used as long as it promotes The medium used at this time may contain appropriate carbon sources, nitrogen sources, inorganic salts and the like as medium components.

The medium used in the conversion step of the present invention may be the same medium as that for the growing bacterial cells, or may be a different medium, in order to grow the bacterial cells in the medium components. The medium may contain the same components as those contained in the medium.

As the carbon source, any carbon source which can be used by the strain of the present invention can be used. Such carbon sources as ribose, glucose, fructose, sugars such as mannose, soybean oil, lipids such as olive oil, methanol, ethanol, alcohols such as glycerol, succinic acid, malonic acid, acetic acid, propionic acid, organic acids such as butyric acid, waste Examples include agricultural product residues such as molasses. In the case of a medium for growing bacterial cells, the bacterial cells of the present invention as described above can be used 1
One or two or more carbon compounds can optionally be utilized as the carbon source. Further, in the case of the medium for conversion culture, all carbon sources used for growth can be used, but organic compounds and monosaccharides such as glucose and glycerin that are easy to use by the bacterium are preferable. The carbon source content varies depending on the type of carbon source, but is preferably 0.2% by weight or more in the medium. In particular, when Pseudomonas putida KS-0160 strain or Enterobacter crocusae IFO13535 strain is used, it is preferable to contain mannose in an amount of 0.2 to 10% by weight. Pseudomonas putida KS-018
When using 0 strain, fructose is 0.2 to 10% by weight.
It is preferably contained.

The nitrogen source is not particularly limited,
Organic nitrogen sources such as meat extract, peptone, yeast extract, corn steep liquor, casamino acid, urea and inorganic nitrogen sources such as ammonium nitrate and ammonium sulfate can be used. When an organic nitrogen source is used, it also contains carbon, and therefore it is not always necessary to add another carbon source in the case of the growth medium.

As the inorganic salts, various sulfates, phosphates, sodium salts, magnesium salts, calcium salts, potassium salts and the like can be used. Furthermore, a trace amount of heavy metal salts (eg iron salt, manganese salt, copper salt, zinc salt, molybdenum salt, cobalt salt, etc.) may be contained in the medium.

As the culture method, a shaking culture method, a deep aeration stirring culture method and the like can be used. The culture temperature is preferably 25 to 30 ° C., and the pH is preferably around neutral.
The number of culture days can be determined according to the progress of the reaction,
Usually, 3 days are suitable for cell growth in the main culture, and several hours to 1 day is suitable for conversion of the substrate. Furthermore, the yield of parahydroxybenzaldehyde is better when the substrate conversion is performed after 2 to 3 days than when the cell growth in the main culture is performed for less than 2 days. Further, if necessary, a defoaming agent such as fatty acid ester type or silicon type may be added.

The para-cresol used in the production method of the present invention may be added at the start of the cell growth culture, or may be added after the cell growth culture. Further, it may be added both during the expansion culture and after the expansion culture.

When cells are added after growth, it is desirable to add para-cresol when the cell concentration is 1.0 to 10.0 at an absorbance of 660 nm. The paracresol may be added at once or in small amounts, and may be added at the starting point of cell growth, or if added over time, one day after the growth. It may be added separately on the first or second day. Further, para-cresol may be continuously added so as to be 0.1 to 0.2% by weight.

The concentration of para-cresol to be added in the culture solution is preferably 3% by weight or less, particularly preferably 1% by weight or less, more preferably 0.1 to 0.5% by weight.
If it exceeds 3% by weight, the microorganisms do not sufficiently act, which is not preferable.

If para-cresol is added before or after the growth of the cells, the conversion rate of 2-3% is higher when the para-cresol is added after the growth of the cells.

Further, considering the growth step and the conversion step in terms of the combination of the times when paracresol is added, the following combinations are exemplified. 1) Paracresol is added at the starting point of the growth process and the growth and conversion processes are performed simultaneously. 2) After the microorganism is grown, paracresol is added to carry out a conversion reaction. Paracresol is added during the reaction process or both at the starting point and during the process. 3) Paracresol is added to the growth step and the conversion step at least once each time, and after the growth step, the cells are separated from the medium and transplanted into the medium for the conversion reaction.

The medium used in the above conversion step may be the same as the medium used in the growth step. In the method of 2) above, it is desirable to add para-cresol and continue the culture after the bacterial cells are grown.

After completion of the conversion reaction, the produced parahydroxybenzaldehyde is separated and purified from the culture solution by solvent extraction, column chromatography, neutralization, concentration, crystallization and the like, as in the case of the separation and purification of general organic compounds. Can be performed by appropriately combining means known to those skilled in the art. For example, a method in which solids such as bacterial cells are removed from the culture solution by centrifugation, the supernatant is concentrated, and then the concentrated solution is acidified to precipitate parahydroxybenzaldehyde for solid-liquid separation, or the supernatant is acidified. After that, there is a method of separating by extracting with an organic solvent such as diethyl ether, ethyl acetate, toluene, chloroform or a mixed solvent thereof. The composition organism thus obtained can be purified by various kinds of chromatography, recrystallization, or a combination thereof.

The parahydroxybenzaldehyde produced by the method of the present invention is useful as a raw material for pharmaceuticals, brighteners for plating, dyes, perfume intermediates, agricultural chemicals and the like.

The present invention will be described in more detail with reference to the following examples. Medium (I) and medium (II) used
It had the following composition. Composition of medium (I) disodium hydrogen phosphate 3.0 g 1 potassium hydrogen phosphate 2.0 g ammonium chloride 2.0 g magnesium sulfate heptahydrate 0.2 g sodium carbonate 0.1 g calcium chloride dihydrate 0.01 g Iron sulfate heptahydrate 0.005 g Mannose 2.0 g Yeast extract 1.0 g Ion-exchanged water 1.0 L pH 6.8 (after pH adjustment, sterilized at 120 ° C., 1.2 Kg / cm ² , 20 minutes)

Composition of medium (II) Disodium hydrogen phosphate 3.0 g 1 Potassium hydrogen phosphate 2.0 g Ammonium chloride 2.0 g Magnesium sulfate heptahydrate 0.2 g Sodium carbonate 0.1 g Calcium chloride dihydrate 0.01 g iron sulfate heptahydrate 0.005 g fructose 2.0 g yeast extract 1.0 g deionized water 1.0 L pH 6.8 (after pH adjustment, sterilized at 120 ° C., 1.2 Kg / cm ² for 20 minutes Used)

Example 1 This example illustrates the microbial conversion of para-cresol to para-hydroxybenzaldehyde by the method of the present invention. The above medium (I) 1
Pseudomonas putida KS-0160 strain strain 1 platinum loop was inoculated into 00 ml, pH 6.8, culture temperature 27 ° C,
Culture was carried out with shaking at 180 rpm overnight with shaking (preculture). 10 ml of the obtained culture solution was inoculated into a 300 ml flask containing 100 ml of the medium (I) to carry out shaking culture (main culture). The culture conditions were 27 ° C., pH 6.8, and shaking number 180 rpm. Two days and three days after the start of the main culture, 0.1 g each of para-cresol was added and the culture was performed for a total of 4 days. After completion of the culture, the cells were removed by centrifugation, concentrated to 20 ml under reduced pressure, and extracted with 20 ml of ethyl acetate three times. The organic layers were combined and concentrated to dryness to obtain crude crystals. The crude crystals were recrystallized with a 1: 1 mixed solvent of diisopropyl ether: isopropyl alcohol to obtain 20 mg (9%) of a purified product. The obtained substance was confirmed to be parahydroxybenzaldehyde by elemental analysis, IR and ¹ H-NMR measurement. Elemental analysis value (C ₈ H ₆ O ₂ ) calculated value: C 71.64%, H 4.50%, O 2
3.86% Found: C 70.55%, H 4.51%, O 2.
3.94% ¹ H-NMR (δppm, solvent DMSO, internal standard TM
S) 6.95 (d, 2H), 7.79 (d, 2H), 9.8
2 (s, 1H) IR (nujol): 3168, 1667, 1600,
1519, 1218, 1162, 835, 605 (cm
^-1 )

(Example 2) To the medium (I) having the same composition as that used in Example 1, 100 ml of the medium (III) added with 0.1% by weight of para-cresol was used.
1 Pseudomonas putida KS-0160 strain
A platinum loop was inoculated and cultured with shaking overnight. The obtained culture solution 10
30 ml prepared by adding 100 ml of the above medium (III)
Inoculate a 0 ml flask, culture temperature 27 ℃, pH
Shaking culture was carried out at 6.8 and a rotation speed of 180 rpm for 2 days. The amount of parahydroxybenzaldehyde produced in the culture broth after 2 days was 10 mg (9%).

For separation and purification of parahydroxybenzaldehyde from the culture solution, cells are removed by centrifugation, hydrochloric acid is added to the supernatant to adjust the pH to 4, and parahydroxybenzaldehyde is extracted and separated from this acidic solution with diethyl ether, followed by extraction. The liquid was concentrated under reduced pressure to obtain crude crystals. The crude crystals were recrystallized from a 1: 1 mixed solvent of diisopropyl ether: isopropyl alcohol in the same manner as in Example 1 to obtain 7 mg (6%) of white crystals.

Example 3 Six 300 ml Erlenmeyer flasks charged with 100 ml of the medium (I) having the same composition as that used in Example 1 were used, and Pseudomonas putida K was used.
Each flask was inoculated with 1 platinum loop of S-0160 strain, and cultured by shaking at 27 ° C., pH 6.8, 180 rpm for 24 hours. 600 ml of the obtained culture broth was combined and added to a 10 l jar fermenter containing 7 l of the medium (I) as a mother bacterium, and 15 g of para-cresol as a substrate was added at the same time for culturing. After 1 day, 12 g of para-cresol was further added to continue the culture, and the culture was continued for a total of 2 days. The culture conditions are a temperature of 27 ° C. and a pH of 6.
8, stirring was 300 rpm, and aeration was 0.5 volume / volume / minute. After the completion of the culture, the cells were removed by centrifugation at 10,000 × G for 20 minutes, adjusted to pH 4 with hydrochloric acid, and treated in the same manner as in Example 2 to obtain 3.05 g (10%) of parahydroxybenzaldehyde. .

(Example 4) 100 ml of the above medium (II)
Pseudomonas putida KS-0180 strain strain 1 platinum loop was inoculated and shake-cultured overnight at pH 6.8, a culture temperature of 27 ° C. and a shaking frequency of 180 rpm (preculture). 10 ml of the obtained culture broth was inoculated into a 300 ml flask containing 100 ml of the medium (II) to carry out shaking culture (main culture). Culture conditions are 27 ° C, pH 6.8, shaking number 1
It was 80 rpm. Two days and three days after the start of the main culture, 0.1 g each of paracresol was added to give a total of 4
Culture was carried out for one day. After completion of the culture, the cells were removed by centrifugation, concentrated to 20 ml under reduced pressure, and extracted with 20 ml of ethyl acetate three times. The organic layers were combined and concentrated to dryness to obtain crude crystals. The crude crystals are diisopropyl ether:
Recrystallization from a 1: 1 mixed solvent of isopropyl alcohol gave 25 mg (11%) of a purified product. The obtained substance was analyzed by elemental analysis, IR, and ¹ H-NMR measurement.
It was confirmed to be parahydroxybenzaldehyde.

Example 5 100 ml of the medium (IV) containing 0.1% by weight of para-cresol was added to the medium (II) having the same composition as that used in Example 4 in Example 1
1 Pseudomonas putida KS-0180 strain
A platinum loop was inoculated and cultured with shaking overnight. The obtained culture solution 10
300 ml prepared by adding 100 ml of the above medium (IV)
Inoculate a ml volumetric flask, culture temperature 27 ° C., pH 6.
8. Shaking culture was carried out at a rotation speed of 180 rpm for 2 days. Two
The amount of parahydroxybenzaldehyde produced in the culture solution after 12 days was 12 mg (10%).

For the separation and purification of parahydroxybenzaldehyde from the culture solution, the cells are removed by centrifugation, hydrochloric acid is added to the supernatant to adjust the pH to 4, and parahydroxybenzaldehyde is extracted and separated from this acidic solution with diethyl ether, followed by extraction. The liquid was concentrated under reduced pressure to obtain crude crystals. The crude crystals were recrystallized from a 1: 1 mixed solvent of diisopropyl ether: isopropyl alcohol in the same manner as in Example 1 to obtain 9 mg (7%) of white crystals.

(Example 6) 300 ml prepared by adding 100 ml of the medium (II) having the same composition as that used in Example 4
Using 6 Erlenmeyer flasks, 1 platinum loop of Pseudomonas putida strain KS-0180 was inoculated into each flask and shake-cultured at 27 ° C, pH 6.8, and 180 rpm for 24 hours. 600 ml of the obtained culture broth was combined and added to a 10 l jar fermenter containing 7 l of the medium (I) as a mother bacterium, and 15 g of para-cresol as a substrate was added at the same time for culturing. After 1 day, 12 g of para-cresol was further added to continue the culture, and the culture was continued for a total of 2 days. The culture conditions are a temperature of 27 ° C. and a pH of 6.
8, stirring was 300 rpm, and aeration was 0.5 volume / volume / minute. After completion of the culture, the cells were removed by centrifugation at 10,000 × G for 20 minutes, adjusted to pH 4 with hydrochloric acid, and treated in the same manner as in Example 2 to obtain 3.66 g (12%) of parahydroxybenzaldehyde. .

(Example 7) 100 ml of the above medium (I) was inoculated with 1 platinum loop of Enterobacter crocusae IFO 13535 strain, pH 6.8, culture temperature 27 ° C,
Culture was carried out with shaking at 180 rpm overnight with shaking (preculture). 10 ml of the obtained culture solution was inoculated into a 300 ml flask containing 100 ml of the medium (I) to carry out shaking culture (main culture). The culture conditions were 27 ° C., pH 6.8, and shaking number 180 rpm. Two days and three days after the start of the main culture, 0.1 g each of para-cresol was added, and the culture was performed for a total of 3 days. After completion of the culture, the cells were removed by centrifugation, concentrated to 20 ml under reduced pressure, and extracted with 20 ml of ethyl acetate three times. The organic layers were combined and concentrated to dryness to obtain crude crystals. The crude crystals were recrystallized from a 1: 1 mixed solvent of diisopropyl ether: isopropyl alcohol to obtain 19 mg (8.4%) of a purified product. The obtained substance was confirmed to be parahydroxybenzaldehyde by elemental analysis, IR and ¹ H-NMR measurement.

Example 8 100 ml of the medium (III) containing 0.1% by weight of para-cresol was added to the medium (I) having the same composition as that used in Example 1 in Example 1.
Like the Enterobacter crocusae IFO 135
One platinum loop of 35 strains was inoculated and cultured overnight with shaking. 10 ml of the obtained culture solution was added to 100 ml of the above medium (III).
Was inoculated into a 300-ml volume flask, and cultivated with shaking at a culture temperature of 27 ° C., a pH of 6.8, and a rotation speed of 180 rpm for 2 days. The amount of parahydroxybenzaldehyde produced in the culture broth after 2 days was 9.5 mg (7.6%).

For the separation and purification of parahydroxybenzaldehyde from the culture solution, the cells were removed by centrifugation, hydrochloric acid was added to the supernatant to adjust the pH to 4, and parahydroxybenzaldehyde was extracted and separated from this acidic solution with diethyl ether, followed by extraction. The liquid was concentrated under reduced pressure to obtain crude crystals. The crude crystals were recrystallized from a 1: 1 mixed solvent of diisopropyl ether: isopropyl alcohol in the same manner as in Example 1 to obtain 6 mg (4.8%) of white crystals.

(Example 9) Six 300 ml Erlenmeyer flasks charged with 100 ml of the medium (I) having the same composition as that used in Example 1 were used, and 1 strain of Enterobacter crocusae IFO 13535 strain was added to each flask. Inoculate with platinum loop, 24 at 180 ℃, 27 ℃, pH6.8
Culture was performed with shaking for an hour. 600 ml of the obtained culture broth were combined and added to a 10 l jar fermenter containing 7 l of the medium (I) as a mother bacterium, and 6 g of para-cresol as a substrate was added at the same time for culturing. One day later, 5 g of para-cresol was further added to continue the culture, and the culture was performed for a total of 2 days. Culture conditions are temperature 27 ℃, pH
6.8, stirring 300 rpm, aeration 0.5 volume / volume /
It was a minute. After completion of the culture, the cells were removed by centrifugation at 10,000 × G for 20 minutes, adjusted to pH 4 with hydrochloric acid, and treated in the same manner as in Example 2 to obtain 1.18 g (9.5%) of parahydroxybenzaldehyde. Obtained.

[0041]

EFFECTS OF THE INVENTION The method of the present invention requires almost no energy for parahydroxybenzaldehyde, which has been conventionally chemically synthesized by a high temperature reaction, and which is useful as a raw material for brighteners for medicines / plating, dyes, fragrance intermediates and agricultural chemicals. Without it, it could be manufactured inexpensively from para-cresol by microbial oxidation.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tokio Iizuka 1 Kawasaki-cho, Chuo-ku, Chiba-shi, Chiba Kawasaki Steel Corporation Technical Research Division (72) Inventor Kenichi Uehara Chuo-ku, Chiba Kawasaki Town No. 1 Kawasaki Steel Corporation Technical Research Division

Claims (5)

[Claims] 1. The microorganism Pseudomo
nas putida) KS-0160 is used to convert para-cresol into para-hydroxybenzaldehyde. 2. The microorganism Pseudomo.
nas putida) KS-0180 is used to convert para-cresol into para-hydroxybenzaldehyde. 3. The microorganism Enterobacter crocusae (En
terobacter cloacae) IFO13535 is used to convert para-cresol into para-hydroxybenzaldehyde. 4. The method for producing parahydroxybenzaldehyde according to claim 1, wherein the microorganism is cultured in a medium containing para-cresol. 5. After culturing and growing the microorganism, p-
A method for converting para-cresol into para-hydroxybenzaldehyde by adding cresol and continuing the culture.
4. The method for producing parahydroxybenzaldehyde according to any one of 3 above.