ited States Patent Office 3,183,169

Patented May 1965

2
. 3,183,169 (5) Loeffler's blood serum: No growth
PREPARATION OFSALCYLIC ACID (6) Dorset egg medium: No growth -
André-R. Brilliaud, West Chester, Pa., assignor to Sun (7) Nitrate broth: Good growth, nitrite positive
Oil Company, Philadelphia, Pa., a corporation of New
Jersey (8) Kligler iron agar: Good growth, grey, smooth
colonies, no HS.
No Drawing. Fied July 22, 1963, Ser. No. 296,447 (9) Methyl Red-Voges Proskauer medium: Methyl Red
4 Caius (C. 95-28) negative; Voges Proskauer negative
This invention relates to the synthesis of salicylic acid. (10) Phenol Red lactose agar: Good growth, slight acid,
grey smooth colonies
More particularly, it relates to the production of salicylic 0 (11) Phenol Red mannitol agar: Good growth, tan
acid by the microbiological oxidation of naphthalene. smooth, circular colonies
Still more particularly, it relates to the production of
salicylic acid from naphthalene by fermentation of a (12) Phenol Red saccharose agar: Good growth, slight
acid, small, colorless, colonies
strain of microorganism of the Corynebacterium renale (13)
type. Glycerol agar: Small, grey, circular, Smooth co
lonies
Salicylic acid is economically a very valuable and use 5 (14) Phenol Red dextrose agar: Good growth, circular,
ful compound, particularly in the production of aspirin convex, smooth, colonies, acid
and the like. Thus, it is an object of this invention to (15) Nutrient agar: Small, cream colored, circular, con
provide a new source for this material. It is a further vex, entire, smooth, moist colonies
object to provide salicylic acid in high yield from a low 20 (16) Potato dextrose agar: No growth
cost starting material, naphthalene. (17) Litmus milk: Basic
It has now been found that high yields of salicylic (18) Sim's medium: Indole negative
acid may be obtained by the microbiological oxidation
of naphthalene in an appropriate mineral salts medium (19) Starch: No hydrolysis, circular, cream colored,
convex, entire, smooth, moist colonies
under aerobic conditions with a strain of microorganism (20) Catalase: Positive
selected from the genus Corynebacterium, and particul 25 (21) Gelatin: No liquefaction
larly with strains of a newly discovered microorganism
designated as Corynebacterium renale, a culture of which (22) Urea and Naph.: Circular, brown centers, tan,
convex colonies
has been deposited with the American Type Culture (23) Carbohydrate tests in Phenol Red broth using 0.5%
Collection in Washington, D.C., under the number 30 specific carbohydrate substrate:
ATCC 15,075.
The microorganism Corynebacterium renale ATCC (a) Mannitol-No acid, no gas
15,075 was isolated from soil in Marcus Hook, Penn (b) Levulose-No acid, no gas
sylvania. The organism was isolated in the following (c) Lactose-No acid, no gas
manner. A plot of ground was selected and fed regularly 35 (d) Sorbitol-No acid, no gas
with crystalline naphthalene and cultivated at 48-hour (e) Inositol-No acid, no gas
intervals. Calcium carbonate was added to the cultivated (f) Saccharose-No acid, no gas
soil periodically in order to keep environmental pH high. (g) Maltose-No acid, no gas
A sample of soil was than taken from the cultivated plot, Other naphthalene-oxidizing strains of salicylic acidi
dried, and pulverized. Approximately 40 of a gram of 40 producing microorganisms may similarly be selected
this material was sprinkled on the surface of agar con from soil samples or culture collections by conducting
taining Strawinski's medium and crystalline naphthalene the fermentation under the conditions described herein.
was added to an inverted. Petri dish cover. Incubation In general, it is desirable to select a specie producing at
was at 30-2 C. for 7 days at which time a salicylic acid least 1 gram of product per liter of whole broth.
colony was picked and purified. This organism was re 45 The nutrient medium for the culture of Corynebac
plated and at this time the colonial description was as fol terium renale organisms of this invention to produce
lows: yellowish brown, irregularly shaped, convex with salicylic acid should contain, in addition to naphthalene
erose margin. The culture was replated again with a as essentially the sole carbon and energy source, a source
slight change in colonial morphology being noted. It of available nitrogen and appropriate mineral salts. The
was now described as gray, circular, convex and en 50 naphthalene employed may be either in pure form or as
tire. Subsequent subcultures finally revealed stabiliza crude mixtures obtained from naphthalenic cuts of crude
tion of the colony as follows: brown with white center, oil. The quantity of naphthalene employed in this proc
circular, convex and entire. Maintenance of this cul ess may range from 0.5 to 10% of the total nutrient me
ture was on Strawinski's medium in the presence of dium and preferably should be from 1 to 3%. It is de
crystalline naphthalene. 55 sirable in carrying out this process that the naphthalene
The cultural and physiological characteristics whereby be introduced into the fermentation broth in small incre
Corynebacterium renale ATCC 15,075 is identified and ments so that the amount present at any one time is below
distinguished from other microorganisms are as follows: growth-limiting concentration. While the naphthalene
(1) Gram stain: Gram positive, club shaped, granules, may simply be added to the fermentation broth in pow
pleomorphic; size: varies from approximately 1.0 to 60 dered crystalline form, it has been found that the fermel
1.25 to approximately 1.0 to 2.0p. tation proceeds most effectively when the naphthalene is
(2) Methylene Blue stain: Pleomorphic, pairs with predispersed as an emulsion formed with a dilute aqueous
pointed ends, cocci, rods solution of a high molecular weight alcohol such as poiy
(3) Acid fast stain: Not acid fast vinyl alcohol.
(4) Phenol Red maltose agar: Good growth, very slight . 65 Examples of suitable sources of nitrogen are am
acid, tan, smooth, circular monium salts such as (NH4)2SO4 or, NHCl, or nitrate
salts such as NHNO3, NaNO3 or the like; urea, soybean
 3,183,169
3 4.
meal or other organic sources of available nitrogen may in an emulsion with a high molecular weight alcohol.
likewise be used. Care should be taken when using Thus, using the conditions described hereinabove, the
NaNO, however, to see that the pH does not rise much conversion of naphthalene to salicylic acid is uniformly
above 6.5 since degradation of salicylic acid tends to obtained at rates as high as approximately 90% of the
increase above this pH level. 5 naphthalene consumed.
The necessary mineral salts which must be present in The amount of salicylic acid in a particular fermenta
the medium may vary somewhat depending upon the tion broth may be determined not only by actual isola
organism. A suitable mineral salts composition contain tion of the acid in the manner described above but also
ing the necessary trace, elements is as follows: by the following chemical assay method:
Gms./liter 0 Anaphthalene shake flask which has produced salicylic
CaCl2 ------------------------------------- 0.02 acid is allowed to continue shaking until no more salicylic
FeSO4 ------------------------------------ 0.005 acid can be observed by FeCl3 spot test even after ex
KH2PO4----------------------------------- 0.4 tensive liquid-liquid extraction. To this fermentation
MgSO4 ------------------------------------ 0, 2 beer is added to weighed amount of salicylic acid and the
MnCl2 ------------------------------------ 0.002 pH adjusted to 6.5 whereupon the salicylic acid dis
NaHPO, ... was - a - - - as a m may wome m n as r or or her - me -m or r- a v me O.6
solves. A 0.5 ml. sample of this beer is then placed in
NaMoos ---------------------------------- 0.001 a test tube and a duplicate amount is placed in a similar
NH4NO3 ---------------------------------- 2.0 test tube as a reference blank. To the first test tube is
added 0.5 ml. of a 1% HNO solution while 1.0 ml. of a
While no other organic carbon source other than the 1% HNO3 solution is added to the blank tube. There
naphthalene need be present in the fermentation medium after, 0.5 ml. of a 5% Fe(NO3) reagent is added to the
for the successful conduct of this process, nevertheless, first tube. The color tube and the blank tube are then
it has been found that the rate of production of Salicylic shaken vigorously and 2.5 ml. of water is added to each
acid is increased slightly if such growth stimulating ma of the tubes. If the color in the tubes appears too dark
terials as yeast extract (e.g., purified Difico) at a concen to read in a spectrophotometer, the tubes should be
tration of 1 gm./liter is added. Other suitable growth diluted by adding water in multiples of 4.0 ml. The solu
stimulating materials which may also be used are such sub
stances as distillers solubles, corn steep liquor, or the tions are then filtered through cotton plugs and read on
a Beckman-DK2 spectrophotometer for absorbance at
like, 525 mp. After correction for the reading of a blank con
The process of this invention is desirably carried out 30 taining no salicylic acid, a straight line relationship is
at a temperature of from about 25 to 35° C., and pref found to exist between color absorbance and concentra
erably from 28° to 32° C. under aerobic conditions with tion of salicylic acid over a range of .05 to 1.0 mg. The
agitation. Although atmospheric concentration of oxy salicylic acid content of the unknown filtrate is calculated
gen is necessary for conversion of naphthalene to salicylic from a pre-prepared calibration curve plotting absorbance
acid, it has been found that increased oxygen concentra
tion does not increase the yield of salicylic acid. Thus, at 525 mp against mgs. per ml. of test material. The ac
sufficient aeration is generally obtained by mechanical tual amount of salicylic acid present is then determined
by the following relationship:
agitation during the fermentation period. The medium
should be maintained at a pH of from about 6.0 to 8.0, (1) Find the mg./test on the graph corresponding to the
and preferably at about pH 6.5 at which point maximum 40 (2)absorbance
mg/ml.=
at 525 mp.
salicylic acid accumulation results. For optimum yields
of salicylic acid, fermentation of the microorganism mg./testxdilution of colorx dilution of beer
Corynebacterium renale is conducted for about 24 to ml. beer added
96 hours, and preferably for about 48 hours, after which The following examples are given for purposes of
the salicylic acid is recovered.
The salicylic acid may be conveniently recovered from 45 illustration and not by way of limitation:
the fermentation broth by any of several methods knowr Example I
in the fermentation art. Thus, for example, Salicylic acid A series of fermentations were carried out in 300 ml.
may readily be recovered by filtering or centrifuging the sterilized dispo-plugged Erlenmeyer flasks, using a sterile
whole broth to remove the microorganism and other mineral salts medium of the following composition:
solids, then contacting the resulting liquor with activated
carbon to yield a product which is 99% pure based on re Grams/liter
agent grade salicylic acid. This recovery is most con CaCl2 ------------------------------------- 0.02
veniently achieved by adjusting the pH of the filtered or FeSO4 ------------------------------------ 0.005
centrifuged broth to 7, contacting this broth with 3 weight 55 KHPO4----------------------------------- 0.04
percent (based on salicylic acid content) of activated MgSO4 ------------------------------------ 0.20
carbon at 200' F., cooling and filtering the broth, and MnCl2 ------------------------------------ 0.002
acidifying it with a mineral acid such as hydrochloric to Na2HPO4---------------------------------- 0.6
a pH 1 to crystallize out 99% pure salicylic acid. This NaMoos ---------------------------------- 0.001
product may, if desired, be further purified by recrystal 60 NH4NO3 ---------------------------------- 2.00
lization from such solvents as acidic water. CaCO3 ------------------------------------ 1.00
Alternatively, salicylic acid may readily be recovered Naphthalene (2 gms.).
from filtered fermentation broth by neutralizing the The naphthalene was emulsified in 0.5% of polyvinyl
broth, contacting it with the chloride form of a weakly alcohol, sterilized, and added separately, as was the
basic anion exchange resin such as Amberlite IR-45, and sterilized CaCO3. The pH was adjusted to 6.5. Some
eluting the absorbed acid from the resin with acidified naphthalene was added initially (1 gram), and at 40 and
methanol. The salicylic acid is then conveniently recov 64 hours (0.5gm. each time).
ered from the methanol solution by evaporating it to One hundred ml. of the above medium was inoculated
dryness. with 1% by volume of a vegetative inoculum of the
In general, the present fermentation process results in culture Corynebacterium renale ATCC 15,075 and the
the fermentation of salicylic acid from naphthalene in 70 fermentation carried out on a gyratory shaker at ap
high yield; for example, yields of better than 6 grams per proximately 30° C.--2 for periods ranging from 24 to
liter of whole both are obtained when powdered crystal 96 hours, depending on the rate of production of salicylic
line naphthalene is used, while over 10 grams per liter acid. The pH was maintained at about 6.5 by periodic
of acid is recovered when the naphthalene is predispersed additions of CaCO3.
 3,183,169
5
During the fermentation period, samples of the fermen presence of varying nutrients.6 The mineral salts medium
tation broth were centrifuged to remove bacterial cells was the same as that used in Example 1. The naphthal
and residual CaCO3. The amount of salicylic acid in the ene was crystalline and added initially (1 gram). To
clear supernatant fluid was determined on a spectro each of sixflasks was added one of the following:
photometer. The following results were obtained: 5 (1) 0.1% soybean oil meal
Hours of Fermentation 1 --- (2) 0.1% yeast extract (Difco)
Fitrate No. (3) 0.1% yeast extract (S-50)
40 hr.2 64 hr. 88 hr. 9 hr. (4) 0.1% Pharmamedia (complex organic nutrient)
(5) 0.1% dextrose
0. (6) no nutrient
2, 3 2.5 5.0 5.5
2.2 3.0 7. 9 7. 9
0.84 4.5 6.9 7.4 The salicylic acid yields were determined by the sam
2.18
0.8
2.6
2.8
6.0
6.9
6.0
6.9
method as Example 1.
1.83 2.7 6.0 6.5
1.17 3, 6 7.5 8.7 5
0.04 2.9 8.6 9.4
1.5 3.2 6.0 6.75 Filtrate Time of Fermentationi
-- negative 2.2 6.9 7.3
--- negative 2.8 9.2 11.25
5, 61. 10 9.5 9.75 24hr. 48 hr, 72 hr. 96 hr,
4.88 1.5 9.9 10.1

1 Data indicate grams per liter of salicylic acid as determined spectro 1. Soy Bean.----- neg. neg. neg. neg.
photometrically. 2.
3,
Yeast Extrac
S-50---------
2.32
1, 12
4.13
1,94
4.75
2.33
5.72
3.20
20.5 grams of naphthalene added. 4. Pharmamedia- 1.21 1.76 .98 2.41
80.5 grams of naphthalene added. 5. Dextrose.-------- ---- neg. neg. neg, neg.
Example 2 6. Control--------------------------- .82 3.46 4.51 5.2
A series of fermentations were conducted following 25
the procedure of Example 1 using crystalline naphthalene 1 Data indicate grams per liter of salicylic acid as determined spectro
photometrically.
in place of the emulsified naphthalene. All of the naph I claim:
thalene. (1 gram) was added initially. The salicylic
acid yields were determined by the same procedure as 1. A process for preparing salicylic acid which com
Example 1. 30 prises subjecting naphthalene to the microorganism
Corynebacterium renale ATCC 15,075 in an aqueous nu
Time of Fermentation 1 trient medium under aerobic conditions.
Filtrate 2. The process according to claim 1 in which the naph
8 24 36 48 60 72 96 thalene is introduced into the fermentation medium as
hr. hr. hr. hr. hr. hr. hr. 35 an emulsion with a high molecular weight alcohol.
3. The process according to claim 2 in which the high
2.9l
5. 62
4.325. A0
6.23 7.78
6.2
9.10
molecular weight alcohol is polyvinyl alcohol.
.89 2.32 3, 45 5.23 4. The process which comprises subjecting naphthal
3.24
3.56
4.50 6.0
5,377.13
7.34
8.15 40
ene to the microorganism Corynebacterium renale ATCC
6.35 8.7. 9.31 - 7.23 15,075 in an aqueous nutrient medium under aerobic
5.32 6.
5.78 8.7 8.17
7.22 9.21
7.93
conditions at a pH of from about 6.0 to 7.0 and recover
ing the salicylic acid thus produced.
1 Data indicate grams per liter of Salicylic acid as determined spectro
photometrically. References Cited by the Examiner
45
Example 3 Klausmeier et al.: Journal of Bacteriology 73, 461
464, 1956.
A series of fermentations was conducted following
Example 1 to determine the salicylic acid yields in the A. LOUIS MONACELL, Primary Examiner.