Process for the microbiological oxidation of steroids

The improvement in a process for the production of 17-hydroxyandrosta-1,4-dien-3-one and androsta-1,4-diene-3,17-dione by the microbiological oxidation of a sterol, its 4-en-3-one derivative or its 1,4-dien-3-one derivative, which comprises adding to the culture medium at least one glyceride-containing substance selected from the group consisting of glycerides, fats, oil seeds and oil fruits in an amount sufficient that the culture medium contains at least 0.3 percent by weight of glycerides.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to an improved process for the production of 
17-hydroxyandrosta-1,4-dien-3-one and androsta-1,4-diene-3,17-dione by the 
microbiological oxidation of a sterol, its 4-en-3-one derivative or its 
1,4-dien-3-one derivative. 
2. Description of the Prior Art 
17-Hydroxyandrosta-1,4-dien-3-one and androsta-1,4-diene-3,17-dione are 
commercially significant intermediates for the synthesis of valuable 
steroidal hormones. 
It has long been known that 17-hydroxyandrosta-1,4-dien-3-one 
(1-dehydrotestosterone) (hereinafter referred to as DHT) and 
androsta-1,4-diene-3,17-dione (hereinafter referred to as ADD) are formed 
by the microbiological oxidation of sterols. However, this process suffers 
from the disadvantage that the formed DHT and ADD are subject to further 
oxidation which results in poor yields of DHT and ADD. U.S. Pat. No. 
3,388,042 to Arima et al., issued June 11, 1968, discloses the 
microbiological oxidation of sterols to ADD through the use of a chelating 
agent capable of forming chelate compounds with iron and copper ions which 
are present in the culture medium and which participate in the 
microbiological oxidation of ADD. Although this process permits the 
avoidance of the further oxidation of ADD to give excellent results, it 
has not achieved commercially attractive yields and conversions. 
Therefore, improvement in the process was required to improve such yields 
and conversions. 
It is well known that media containing fats and oils are employed in the 
microbiological oxidation of steroids. U.S. Pat. No. 2,756,179 to Josef 
Fried et al., issued July 24, 1956, describes in Example 1 the use of a 
medium containing about 0.22 percent by weight of soybean oil and about 
1.5 percent by weight of soybean meal in the microbiological oxidation of 
progesterone to ADD and DHT. U.S. Pat. No. 2,842,566 to Jean P. Rosselet 
et al., issued July 8, 1958, describes in Example 1 the use of a medium 
containing about 0.1 percent by weight of lard oil in the microbiological 
oxidation of 6.alpha. -methyl-11-ketoprogesterone to 1-dehydro-6.alpha. 
-methyladrenosterone and 1-dehydro-6.alpha. -methyl-11-ketotestosterone. 
U.S. Pat. No. 2,981,659 to Gunther S. Fonken et al., issued Apr. 25, 1961, 
described in Example 4A the use of a medium containing about 1.5 percent 
by weight of soybean meal and about 0.25 percent by weight of soybean oil 
in the microbiological oxidation of progesterone 20-ethylene ketal to 
1-dehydroprogesterone 20-ethylene ketal. It also describes in Example 5A 
the use of a medium containing about 0.22 percent by weight of soybean oil 
or about 0.2 percent by weight of lard oil in the microbiological 
oxidation of progesterone 20-ethylene ketal to 1-dehydroprogesterone 
20-ethylene ketal. 
U. S. Pat. No. 3,010,876 to Dan J. Badia et al., issued Nov. 28, 1961, 
describes in Example III the use of a medium containing about 0.27 percent 
by weight (10 ml./gal.) of soybean oil in the microbiological oxidation of 
Compound S to Compound F. 
U.S. Pat. No. 3,047,469 to Charles John Sih et al., issued July 31, 1962 
discloses the use of a medium containing about 0.22 percent by weight of 
soybean oil in dehydrogenating the A ring of steroids having A rings which 
are fully or partially saturated in that ring. 
U.S. Pat. No. 3,536,586 to Bong Kuk Lee et al., issued Oct. 27, 1970, 
discloses the use of a medium containing about 0.22 percent by weight of 
soybean oil and about 1.5 to 2.0 percent by weight of soybean meal in 
1-dehydrogenating and 16-hydroxylating a steroid which is saturated in the 
1,2-position and which has a replaceable hydrogen atom in the 16-position. 
Japanese Pat. published 16147/1971 describes in Example 4 the use of a 
medium containing about 0.1 percent by weight of soybean oil in the 
microbiological oxidation of cholesterol to ADD and 
androst-4-ene-3,17-dione. 
Japanese Pat. published 29193/1971 describes in Example 1 the use of a 
medium containing 0.2 percent by weight of soybean oil in the 
microbiological oxidation of lithocholic acid to ADD and 
androst-4-ene-3,17-dione. 
Biotechnology and Bioengineering 11 1255-1270 (1969) discloses the use of a 
medium containing about 0.22 percent by weight of soybean oil and about 
1.5 to 2.0 percent by weight of soybean oil meal in the microbiological 
oxidation of 
9.alpha.-fluoro-11.beta.,17,21-trihydroxy-pregn-4-ene-3,20-dione-21-acetat 
e to 
9.alpha.-fluoro-11.beta.,16.alpha.,17,21-tetrahydroxypregna-1,4-diene-3,20 
-dione. 
However, the addition of such a small amount of the fat to the culture 
medium in the microbiological oxidation of sterols results in only a 
slight increase in the yields of DHT and ADD. 
SUMMARY OF THE INVENTION 
It has now been discovered that when at least one glyceride-containing 
substance selected from the group consisting of glycerides, fats, oil 
seeds and oil fruits is added to the culture medium in an amount 
sufficient that the culture medium contains at least 0.3 percent by weight 
of glycerides, DHT and ADD are obtained at high yields and concentrations.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As stated above, this invention is directed to the microbiological 
oxidation of a sterol, its 4-en-3-one derivative or its 1,4-dien-3-one 
derivative to DHT and ADD. 
Although the process whereby the microbiological oxidation of sterols takes 
place is not fully understood, it is believed that the microbiological 
oxidation of sterols proceeds as follows: (see U.S. Pat. No. 3,388,042) 
##STR1## 
In the above formulas, R represents a side chain of 8 to 10 carbon atoms. 
The steroid substrates for the process of this invention are sterols, their 
4-en-3-one derivatives and their 1,4-dien-3-one derivatives. 
Sterols possess a hydroxy group at C-3, a double bond at C-5, a side chain 
of 8 to 10 carbon atoms at C-17, and in some cases, a double bond at C-7 
of the perhydrocyclopentanophenanthrene nucleus. 
Examples of such sterols are cholesterol, stigmasterol, campesterol, 
sitosterol, ergosterol, brassicasterol and fucosterol. Especially 
preferred are cholesterol, stigmasterol, campesterol and sitosterol. 
It goes without saying that the 4-en-3-one derivatives and 1,4-dien-3-one 
derivatives of the sterols which are the intermediate oxidation products 
proposed in the abovedescribed oxidation path of the sterols can also be 
employed as the starting materials of this invention. 
The concentration of the steroid substrate in the culture medium is not 
critical, and is generally in the range of from about 0.05 to about 5 
weight percent, preferably in the range of from about 0.1 to about 3 
weight percent. 
In accordance with the process of this invention, there is added to the 
culture medium at least one glyceride-containing substance selected from 
the group consisting of glycerides, fats, oil seeds and oil fruits. 
The glycerides which are added to the culture medium include 
monoglycerides, diglycerides and triglycerides. Likewise, single 
glycerides containing identical fatty acid residues, and mixed glycerides 
containing two or three different fatty acid residues can also be 
employed. The fatty acid residues include unsaturated fatty acid residues 
and saturated fatty acid residues. 
From the hydrophilic viewpoint, it is preferred that the fatty acid residue 
contains up to 26 carbon atoms. 
Examples of suitable single glycerides are monoglycerides such as 
.alpha.-monoacetin, .beta.-monoacetin, .alpha.-monopalmitin, 
.beta.-monopalmitin, .alpha.-monostearin, .beta.-monostearin, 
.alpha.-monoolein, .beta.-monoolein and the like; diglycerides such as 
.alpha.,.alpha.'-diacetin, .alpha.,.beta.-diacetin, 
.alpha.,.alpha.'-dipalmitin, .alpha.,.beta.-dipalmitin, 
.alpha.,.alpha.'-distearin, .alpha.,.beta.-distearin, 
.alpha.,.alpha.'-diolein, .alpha.,.beta.-diolein and the like; and 
triglycerides such as triacetin, trilaurin, trimyristin, tripalmitin, 
tristearin, triolein and the like. 
Examples of suitable mixed glycerides are 1-aceto-2,3-dipalmitin, 
1-palmito-2,3-dicaprin, 1-lauro-2-myristi-2-palmitin, 
2-oleo-1,3-dipalmitin and 2-stearo-1,3-diolein. In the process of this 
invention, fats can be used in place of the glycerides. 
As used hereinabove, and as will be used hereinafter and in the claims, the 
term "fats" is intended to include vegetable fats and oils, and animal 
fats and oils regardless of their physical state. 
Examples of suitable fats of plant origin are linseed oil, perilla oil, 
tung oil, sesame oil, corn oil, rapeseed oil, cottonseed oil, safflower 
oil, soybean oil, soya lecithin, camellia oil, rice bran oil, olive oil, 
castor oil, peanut oil, coconut oil, palm oil and palm kernel oil. 
Cottonseed oil, soybean oil, rapeseed oil, palm oil and palm kernel oil are 
especially preferred due to their stable supply. 
Examples of suitable fats of animal origin are fish oil, whale oil, beef 
tallow, lard, mutton tallow, beef foot oil and liver oil. 
Especially preferred are lard, fish oil and whale oil. Likewise, 
glyceride-containing oil seeds or oil fruits can also be used in place of 
the glycerides. 
As used hereinabove, and as will be used hereinafter and in the claims, the 
term "oil seeds and oil fruits" is intended to include fat-bearing seeds 
and fruits. 
Examples of suitable oil seeds and oil fruits are linseed, olive, sesame, 
rapeseed, cottonseed, soybean, peanut and rice bran. It is preferred that 
the oil seed and oil fruit be ground to a degree of fineness such that 
they are well assimilated. The glyceride-containing substance which is 
selected from the group consisting of glycerides, fats, oil seeds and oil 
fruits may be used individually, or if desired a mixture of two or more 
individual glyceride-containing substances may be employed. 
The glyceride-containing substance is added to the culture medium in such 
an amount that the culture medium contains from 0.3 to 4.0 percent of 
glycerides, preferably from 0.5 to 3.5 percent of glycerides, more 
preferably from 0.7 to 3.0 percent of glycerides, each based on the weight 
of the culture medium. 
The amount of the fats to be added will be calculated on the basis of the 
glyceride content of the fats. However, the main component of fats is 
glycerides, and accordingly, the weight of the fats is nearly equal to 
that of the glycerides. 
Likewise, the amount of the glyceride-containing oil seeds or oil fruits to 
be added will be calculated on the basis of the glyceride content of the 
oil seeds or oil fruits. For reference, the glyceride contents of typical 
oil seeds and oil fruits are shown in the following table. 
______________________________________ 
Oil Seed or Oil Fruit 
Glyceride Content (weight percent) 
______________________________________ 
Olive (fruit) 40 - 70 
Soybean 15 - 23 
Cottonseed 16 - 35 
Corn 4 - 6 
Sesame 35 - 56 
Rapeseed 22 - 50 
Peanut 29 - 39 
Camellia 35 
Palm 51 - 67 
Coconut 65 - 75 
______________________________________ 
The addition of vegetable oil meals along with the glyceride-containing 
substance to the culture medium has an especially beneficial effect on the 
production of DHT and ADD, and increases the yields of DHT and ADD 
considerably. 
As used hereinabove, and as will be used hereinafter and in the claims, the 
term "vegetable oil meals" is intended to include refuses of vegetable 
fats and oils, which are the crushed residue from the extraction of 
oil-bearing seeds or fruits. Depending upon the extractive process, 
varying percentages of protein and fats will remain in the meals. However, 
any vegetable oil meals may be employed. In general, commercially 
available vegetable oil meals are preferred. 
Examples of suitable vegetable oil meals are soybean oil meal, linseed oil 
meal, rapeseed oil meal, cottonseed meal, sesame oil meal, peanut oil meal 
and safflower oil meal. 
The amount of the vegetable oil meal to be added varies widely with the 
amount of the glyceride-containing substance to be used, and is generally 
in the range of from about 0.1 to about 50 times the weight of the 
glyceride-containing substance, preferably from about 0.3 to about 30 
times, and more preferably from about 0.6 to about 20 times the weight of 
the glyceride-containing substance. However, the concentration of the 
vegetable oil meal in the culture medium is normally in the range of about 
0.3 to about 15 percent, preferably from about 0.6 to about 10 percent, 
and more preferably from about 1 to about 8 percent, each based on the 
weight of the culture medium. The addition of a larger amount of the 
glyceride-containing substance requires the selection of suitable 
microorganisms capable of assimilating glycerides well. 
In fact, the use of a microorganism which assimilates glycerides to a lower 
extent results in almost no occurence of the microbiological oxidation, 
because glycerides are not well assimilated and, at the same time, the 
glycerides become massive. Accordingly, the microorganism which can be 
used in the process of this invention is required to be capable of 
assimilating sterols and glycerides to a higher extent. 
Examples of such microorganisms are those belonging to Arthrobacter, 
Nocardia, Fusarium, Microbacterium, Mycobacterium, Protaminobacter, 
Brevibacterium, Corynebacterium, Bacillus, Serratia, Azotobacter 
Streptomyces, Alkaligenes and Pseudomonas. 
Representative of the above microorganisms are 
Arthrobacter simplex (IAM 1660), 
brevibacterium lipolyticum (IAM 1398), 
mycobacterium smegmatis (IFO 3083), 
protaminobacter alboflavus (ATCC 8458), 
nocardia erythropolis (ATCC 4277), 
corynebacterium equi (IAM 1038) and 
Mycobacterium phlei (IFO 3158). 
Especially, preferred are Arthrobacter simplex and Brevibacterium 
lipolyticum. 
As used hereinabove, and as will be used hereinafter, the term "IAM" refers 
to Institute of Applied Microbiology, Tokyo University, Tokyo, Japan, and 
the term "IFO" refers to Institute for Fermentation, Osaka, Osaka, Japan. 
The process of this invention is not limited to the abovelisted 
microorganisms. 
A mixture of two or more of the above microorganisms may also be employed. 
In addition to the glyceride-containing substance and the vegetable oil 
meal, carbon sources, nitrogen sources and inorganic substances are 
incorporated in the culture medium. 
Examples of such carbon sources are hydrocarbon such as .eta.-paraffins, 
.alpha.-olefins, xylene and the like; alcohols such as methanol, ethanol, 
glycerol, higher alcohols and the like; organic acids such as succinic 
acid, acetic acid, higher fatty acids and the like, and the salts thereof; 
and saccharides such as starch, maltose, sucrose, glucose, rhamnose and 
the like. 
Natural nutrient sources containing carbon sources, nitrogen sources and 
other nutrient substances may be incorporated in the culture medium. 
Examples of such natural nutrient sources are molasses including hightest 
molasses, refinery molasses and xylose molasses; bagasse, corn cob, 
alfalfa, corn steep liquor, distillers' solubles, mieki (an aqueous 
solution of amino acids mixture prepared by the hydrolysis of soybean oil 
meal with HCl), fish meal, yeast, bran, meat extract, yeast extract, 
potato extract, malt extract, gluten, peptone, glutamates, asparagine, 
glycine, casein, casein hydrolysate and skimmed milk. 
Examples of the suitable inorganic substances which are incorporated in the 
culture medium are nitrogen sources such as ammonium sulfate, ammonium 
chloride and the like; potassium and phosphorus sources such as 
dipotassium hydrogenphosphate; and salts of iron, copper, magnesium, 
cobalt, zinc, calcium and the like. 
Other components, e.g., vitamins, can be present in the culture medium if 
they do not impede the function of the main components. 
The composition of the culture medium depends on the microorganism which is 
used. Carbon sources, nitrogen sources, potassium, phosphorous and 
magnesium are critical as components in the culture medium. 
An anti-foaming agent, e.g., polyoxyalkylene glycol, may be incorporated in 
the culture medium, if necessary. However, it need not always be added, 
since the glyceride-containing substance acts as an anti-foaming agent. 
The culture medium can contain a surface active agent. This is not 
required, but does normally render the culture medium more conducive to 
manipulation. 
Examples of the suitable surface active agents are polyoxyethylene sorbitan 
monostearate, sorbitan monopalmitate and polyethylene glycol monostearate. 
In order to obtain DHT and ADD in high yields, it is necessary to add to 
the culture medium an inhibitor for the oxidation of DHT and ADD. 
Examples of such inhibitors are nickel and cobalt salts, and chelating 
agents capable of forming chelate compounds with iron and copper ions. 
Representative of such chelating agents are 1,10-phenanthroline, 
2,2'-bipyridyl, 8-hydroxyquinoline, cupferron, isonicotinic acid 
hydrazide, o-phenylenediamine and sodium N,N'-diethyl-dithiocarbamate. 
Especially preferred are 1,10-phenanthroline, 2,2'-bipyridyl and 
8-hydroxyquinoline. 
The concentration of the chelating agent in the culture medium varies 
widely with the nature of the chelating agent and the composition of the 
culture medium. In general, it is in the range of from about 
1.times.10.sup.-5 to about 1.times.10.sup.-2 M. 
The time at which the chelating agent is added to the culture medium varies 
with the microorganism employed and the composition of the culture medium. 
In general, it is added at the end of a period of from 10 to 60 hours from 
the time of initiating the incubation. 
The incubation temperature is normally in the range of from about 
20.degree. C to about 37.degree. C. 
When a microorganism belonging to the genus Arthrobacter or the genus 
Brevibacterium is employed, the preferred incubation temperature is about 
30.degree. C. When a microorganism belonging to the genus Mycobacterium is 
employed, the preferred incubation temperature is about 35.degree. C. 
It is desirable that the initial pH of the culture medium be between pH 5 
and pH 8, and preferably about pH 7. 
The steroid substrate, after sterilization by dry heat or wet heat, is 
added in any suitable manner, such as in the form of a powder or a 
solution in a suitable solvent, e.g., dimethylformamide, or in the form of 
a suspension prepared by ultrasonically dispersing it. 
It is preferred that the steroid substrate and the surface active agent be 
simultaneously added because of the increased dispersion of the steroid 
substrate. 
The time at which the steroid substrate is added to the culture medium 
varies with the microorganism used and the nature of the steroid 
substrate. 
In general, the steroid substrate is added within 30 hours from the time of 
initiating the incubation. When the microorganism does not grow well, the 
steroid substrate may be added after 30 hours from the time of initiating 
the incubation. It is also possible to add the steroid substrate in 
portions, depending upon the growth of the microorganism. 
Because the glyceride-containing substance and the vegetable oil meal which 
are added to the culture medium in accordance with the process of this 
invention are effective in stimulating the growth of the microorganism at 
all stages in the microbiological oxidation, they are generally added at 
the time of initiating the incubation. 
It is believed that the glyceride-containing substance and the vegetable 
oil meal are especially effective in the oxidation of a side chain at C-17 
of the steroid. Accordingly, the addition of the glyceride-containing 
substance and the vegetable oil meal at the time of the addition of the 
steroid substrate or within about 50 hours from the time of the addition 
of the steroid substrate increases the yields of DHT and ADD considerably. 
It is also preferred to add the glyceride-containing substance in portions 
during the course of the fermentation to give the final concentration 
within the range described above, because it acts as an anti-foaming agent 
as stated above. The term "the final concentration" as used herein is 
defined as the concentration of the total glycerides contained in the 
added glyceride-containing substance in the culture medium, although the 
glycerides decrease during the fermentation as a result of assimilation. 
According to the process of this invention, DHT and ADD are obtained. In 
view of the proposed mechanism for the oxidation of the sterols as set 
forth herein above, it is believed that ADD is formed by the oxidation of 
the hydroxy group at C-17 of DHT. In fact, as shown in the attached 
drawing, the concentration of DHT which is higher at an early stage in the 
incubation decreases as the oxidation of the steroid substrate proceeds 
and, at the same time, the concentration of ADD increases. It follows from 
the above that, in order to attain a higher concentration of DHT, the 
incubation should be terminated at an early stage in the oxidation, 
whereas, in order to attain a higher concentration of ADD, the incubation 
should be continued for a long period of time. 
The incubation time required for obtaining DHT or ADD at a higher 
concentration varies widely with the microorganism, the incubation 
temperature and the composition of the culture medium. In order to attain 
a higher concentration of DHT, it is preferred to terminate the incubation 
at the end of a period of from about 15 to about 35 hours from the time of 
the addition of the chelating agent, whereas, in order to attain a higher 
concentration of ADD, it is preferred to terminate the incubation at the 
end of a period of from about 35 to about 90 hours from the time of the 
addition of the chelating agent. 
Upon completion of the fermentation, the resulting DHT and ADD are 
recovered from the fermentation broth by conventional methods. An 
especially advantageous method of recovering the products involves 
extracting the fermentation broth with a water-immischible organic 
solvent, such as methylene chloride, chloroform, ether, benzene, toluene, 
ethyl acetate or the like. 
After evaporation of the combined organic extracts, the resultant products 
can be purified by recrystallization from a suitable solvent, e.g., 
cyclohexane or by reprecipitation from a suitable combination of solvents, 
e.g., xylene-hexane. 
Alternatively, the products can be chromatographed over a column packed 
with silica or alumina to obtain the separated individual products in 
substantially pure form. Eluents such as petroleum ether, benzene, 
chloroform, ether, methanol and the like can be employed. The separated 
products as obtained from the chromatographic procedure may be further 
purified by recrystallization from a suitable solvent. 
Having generally described this invention, a more complete understanding 
can be obtained by reference to certain examples which are provided herein 
for purposes of illustration only are not intended to be limiting in any 
manner. In the following examples, analysis of the products is made by gas 
chromatography and the percentages are expressed in terms of area. Unless 
otherwise stated, the percentages in the following examples are by weight. 
EXAMPLE 1 
This Example illustrates the effect of the amount of cottonseed oil to be 
added on the yield of ADD. 
A seed medium having the following composition is prepared: 
1.0 percent of yeast extract, 
1.0 percent of meat extract, 
1.0 percent of peptone, and 
remainder --water 
The pH of the seed medium was adjusted to 7.0 with NaOH. To a 500 ml shaker 
flask is added 100 ml of the seed medium. The flask and its contents are 
sterilized by autoclaving for a period of 15 minutes at a temperature of 
120.degree. C, and then cooled. The medium is inoculated with a loopful of 
Arthrobacter simplex (IAM 1660) and the inoculated medium is incubated for 
a period of 48 hours at a temperature of 28.degree. C on a reciprocal 
shaker having a 7-cm stroke at 130 strokes per minute. 
A main fermentation medium having the same composition as that of the seed 
medium with the exception that the required amount of cottonseed oil shown 
in Table 1 is added to the medium to achieve the listed concentration. The 
pH of the main fermentation medium is adjusted to 7.0 with NaOH. To a 500 
ml shaker flask is added 50 ml of the main fermentation medium. The flask 
and its contents are sterilized by authoclaving for a period of 20 minutes 
at a temperature of 120.degree. C, and then cooled. The flask is 
inoculated with 2 ml of the seed culture broth obtained above and then 
incubated at a temperature of 30.degree. C on a reciprocal shaker having a 
7-cm stroke at 130 strokes per minute. 
At the end of 20 hours from the time of initiating the incubation, 150 mg 
of cholesterol suspended in 2 ml of water is added. At the end of 28 hours 
from the time of initiating the incubation, 4.0 mg of 2,2'-bipyridyl is 
added. At the end of 85 hours from the time of the addition of 
2,2'-bipyridyl, the incubation is stopped. The fermentation broth is 
extracted with 150 ml of ethyl acetate. The extract is concentrated, 
chromatographed on silica gel and eluted with benzene-acetone (1:1), 
thereby separating ADD from intermediate oxidation products and the 
steroid substrate. The yield of ADD is shown in Table 1. 
Employing a mixture of .beta.-sitosterol and campesterol (2:1), 
stigmasterol, cholest-4-en-3-one or cholesta-1,4-dien-3-one in place of 
cholesterol, the above-described incubation is repeated. 
In addition, the above-described incubation is repeated except that 
cottonseed oil is not added. 
The results are shown together in Table 1. 
TABLE 1 
______________________________________ 
Concen- 
tration Yield Re- 
of Cottonseed 
of maining 
Run oil (Weight) 
ADD Substrate 
No. Substrate Percent) (mg) (mg) 
______________________________________ 
1 Cholesterol 0 4.7 53.2 
2 " 0.15 5.2 45.3 
3 " 0.3 12.6 25.3 
4 " 0.5 15.7 18.7 
5 " 0.7 18.8 7.5 
6 " 1.0 25.8 0 
7 " 1.5 27.2 0 
8 " 2.5 20.8 2.2 
9 " 3.0 16.4 10.2 
10 " 3.5 13.5 12.8 
11 " 4.0 10.0 13.6 
12 Sitosterol + 0 2.0 75.0 
Campesterol (2:1) 
13 " 0.3 6.8 50.0 
14 " 0.5 6.9 48.8 
15 " 1.5 9.7 35.8 
16 Stigmasterol 0 1.0 88.8 
17 " 1.5 4.2 50.3 
18 Cholest-4-en-3-one 
0 8.4 17.5 
19 " 1.5 21.3 8.5 
20 Cholesta-1,4-dien-3-one 
0 6.9 12.6 
21 " 1.5 15.5 10.8 
______________________________________ 
EXAMPLE 2 
This Example illustrates the increased yield of ADD by the addition of the 
fat to the culture medium. 
A medium having the following composition is prepared: 
2.0 percent of sucrose, 
1.0 percent of NaNO.sub.3, 
1.0 percent of yeast extract, 
0.25 percent of K.sub.2 HPO.sub.4, 
0.03 percent of MgSO.sub.4.7H.sub.2 O, 
an amount of the fat to effect the concentration indicated in Table 2, and 
remainder-water 
The pH of the medium is adjusted to 7.0 with NaOH. To a 500 ml shaker flask 
is added 50 ml of the medium. The flask and its contents are sterilized by 
autoclaving for a period of 10 minutes at a temperature of 120.degree. C, 
and then cooled. The flask is inoculated with 1 ml of the seed culture 
broth which is obtained by the same procedure as in Example 1 with the 
exception that Brevibacterium lipolyticum (IAM 1398) is used instead of 
Arthrobacter simplex (IAM 1660). The inoculated medium is incubated at a 
temperature of 30.degree. C on a reciprocal shaker the same as that 
described in Example 1. At the end of 15 hours from the time of initiating 
the incubation, 200 mg of cholesterol is added to the medium. At the end 
of 28 hours from the time of initiating the incubation, 1.0 mg of 
1,10-phenanthroline is added. The incubation is continued for an 
additional 70 hours. At the end of this period, the products are extracted 
with ethyl acetate and separated in the same manner as in Example 1. The 
yield of ADD is shown in Table 2. 
TABLE 2 
______________________________________ 
Run Concentration 
Yield of ADD 
No. Fat (weight percent) 
(mg) 
______________________________________ 
1 None 0 12 
2 Soybean oil 0.15 15 
3 " 0.3 20 
4 " 0.5 37 
5 " 1.0 39 
6 " 2.0 41 
7 " 3.0 25 
8 " 4.0 15 
9 Soya lecithin 1.0 38 
10 " 2.0 38 
11 " 3.0 30 
12 " 4.0 18 
13 Peanut oil 2.0 40 
14 Sesame oil 2.0 30 
15 Olive oil 2.0 32 
16 Corn oil 2.0 33 
17 Rapeseed oil 2.0 38 
18 Rice bran oil 2.0 35 
19 Palm oil 2.0 35 
20 Coconut oil 2.0 36 
21 Cottonseed oil 
2.0 40 
22 Crude soybean oil 
2.0 32 
23 Lard 2.0 29 
24 Whale oil 2.0 32 
25 Castor oil 2.0 38 
26 Soybean oil plus 
2.0 43 
Castor oil 0.3 
______________________________________ 
EXAMPLE 3 
This Example illustrates an increase in the yield of ADD by the addition of 
the glyceride to the culture medium. Example 2 is repeated except that 3.0 
mg of 8-hydroxyquinoline is used in place of 1,10-phenanthroline, and that 
in place of the fat, an amount of the glyceride is added to effect the 
concentration shown in Table 3, and that the incubation is stopped at the 
end of 28 hours from the time of the addition of 8-hydroxyquinoline at the 
end of 56 hours from the time of initiating the incubation. When the 
incubation is stopped at this early stage, DHT is formed along with ADD. 
Upon completion of the incubation, the fermentation broth is treated in 
the same manner as in Example 1. 
The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Yield 
Run Concentration 
ADD DHT 
No. Glyceride (weight percent) 
(mg) (mg) 
______________________________________ 
1 None 0 14 2 
2 Tristearin 0.5 19 14 
3 " 1.0 25 18 
4 " 1.5 27 16 
5 " 2.0 23 16 
6 " 3.0 19 16 
7 " 4.0 15 5 
8 Triolein 1.5 21 25 
9 .alpha.-Monostearin 
1.5 27 18 
10 .alpha.,.alpha.'-Distearin 
1.5 20 14 
11 Tripalmitin 1.5 26 16 
______________________________________ 
The attached drawing illustrates the relationship between the incubation 
time, and the concentrations of cholesterol and the oxidation products 
thereof which are formed in Run 4 of this Example. 
EXAMPLE 4 
This Example illustrates the increased yield of ADD by the addition of 
rapeseed oil to the culture medium. 
A seed medium having the following composition is prepared: 
2.0 percent of .eta.-paraffin (main component C.sub.14), 
0.5 percent of ammonium sulfate, 
0.25 percent of K.sub.2 HPO.sub.4, 
0.1 percent of MgSO.sub.4.7H.sub.2 O, 
0.5 percent of corn steep liquor, and remainder-water 
The pH of the seed medium is adjusted to 7.5 with NaOH. 
To a 500 ml shaker flask is added 100 ml of the seed medium. The flask and 
its contents are sterilized by autoclaving for a period of 15 minutes at a 
temperature of 120.degree. C, and then cooled. The seed medium is 
inoculated with a loopful of Nocardia erythropolis (ATCC 4277) and the 
inoculated medium is incubated for a period of 35 hours at a temperature 
of 30.degree. C on a reciprocal shaker similar to that described in 
Example 1. 
A main fermentation medium having the following composition is prepared: 
1.0 percent of glucose 
0.5 percent of yeast extract 
0.5 percent of malt extract 
0.5 percent of meat extract 
0.1 percent of K.sub.2 HPO.sub.4 
0.02 percent of MgSO.sub.4.7H.sub.2 O 
1.0 percent of rapeseed oil 
The pH of the medium is adjusted to 7.0 with NaOH. 
To a 500 ml shaker flask is added 50 ml of the main fermentation medium. 
The flask and its contents are sterilized by autoclaving for a period of 
20 minutes at a temperature of 120.degree. C and then cooled. The flask is 
inoculated with 2 ml of the seed culture broth obtained above, and 
incubated at a temperature of 30.degree. C on a reciprocal shaker similar 
to that described in Example 1. At the end of 20 hours from the time of 
initiating the main fermentation, 150 mg of cholesterol is added. At the 
end of 28 hours from the time of initiating the main fermentation, 4.7 mg 
of 2,2'-bipyridyl is added. At the end of 70 hours from the time of the 
addition of 2,2'-bipyridyl, the incubation is stopped. Upon completion of 
the incubation, the same treatment as in Example 1 gives 12 mg of ADD. 
When the procedure described above is repeated with the exception that 
rapeseed oil is not added to the culture medium, 8 mg of ADD is obtained. 
EXAMPLE 5 
This Example illustrates the effect of the addition of rapeseed oil and 
soybean oil meal on the yield of ADD. 
A seed medium having the following composition is prepared: 
1.0 percent of glucose, 
0.3 percent of meat extract, 
1.0 percent of peptone, 
0.5 percent of NaCl, and remainder -- water 
The pH of the seed medium is adjusted to 7.2 with NaOH. 
To a 500 ml shaker flask is added 100 ml of the seed medium. The flask and 
its contents are sterilized by autoclaving for a period of 20 minutes at a 
temperature of 120.degree. C and then cooled. The flask is inoculated with 
a loopful of Mycobacterium phlei (IFO 3158) and incubated for a period of 
70 hours at a temperature of 35.degree. C on a reciprocal shaker the same 
as that described in Example 1. 
A main fermentation medium having the following composition is prepared: 
2.0 percent of corn steep liquor, 
2.0 percent of glucose, 
0.4 percent of sodium glutamate, 
0.2 percent of asparagine, 
0.2 percent of KH.sub.2 PO.sub.4, 
1.0 percent of rapeseed oil, and remainder -- water 
The pH of the medium is adjusted to 7.0 with NaOH. To a 500 ml shaker flask 
is added 50 ml of the main fermentation medium. The flask and its contents 
are sterilized by autoclaving for a period of 20 minutes at a temperature 
of 120.degree. C and then cooled. The flask is inoculated with 2 ml of the 
seed culture broth obtained above, and incubated at a temperature of 
35.degree. C on a reciprocal shaker the same as that described in Example 
1. 
At the end of 40 hours from the time of initiating the main fermentation, 
150 mg of cholesterol is added to the culture medium. At the end of 48 
hours from the time of initiating the main fermentation, 6.2 mg of 
2,2'-bipyridyl is added to the culture medium. At the end of 96 hours from 
the time of the addition of 2,2'-bipyridyl, the fermentation is stopped. 
Upon completion of the fermentation, the same treatment as in Example 1 
gives 25 mg of ADD. 
When the procedure described above is repeated with the exception that 4.0 
percent of soybean oil meal is present in the culture medium, 38 mg of ADD 
is obtained. 
When the procedure described above is repeated with the exception that 
rapeseed oil is not added to the culture medium, 20 mg of ADD is obtained. 
EXAMPLE 6 
This Example illustrates an increase in yield of ADD by the addition of the 
fat and the vegetable oil meal, and by the addition of the ground oil 
seed. 
Example 2 is repeated except that substances which are added to the culture 
medium are varied as indicated in Table 4, and that in place of 
1,10-phenanthroline, 2,2'-bipyridyl is added in the amount listed in Table 
4. 
The oil seeds which are used in this Example are ground in Waring Blender. 
TABLE 4 
______________________________________ 
2,2'- Yield 
Run Concentration 
bipyridyl 
of ADD 
No. Added Substance 
(weight percent) 
(mg) (mg) 
______________________________________ 
1 None 0 3.9 16 
2 Rapeseed oil 1.0 3.9 35 
3 Rapeseed oil 1.0 5.5 50 
Rapeseed oil meal 
2.0 
4 Ground Rapeseed 
2.5 5.5 45 
5 Linseed oil 1.0 3.9 33 
6 Linseed oil 1.0 5.5 42 
Linseed oil meal 
1.0 
7 Ground Linseed 
2.5 5.5 45 
8 Soybean oil 1.0 3.9 33 
9 Soybean oil 1.0 5.5 49 
Soybean oil meal 
1.0 
10 Ground soybean 
5.0 5.5 48 
11 Sesame oil 1.0 3.9 37 
12 Sesame oil 1.0 5.5 45 
Sesame oil meal 
2.0 
13 Ground sesame 
2.5 5.5 51 
______________________________________ 
EXAMPLE 7 
This Example illustrates the effect of the time of the addition of rapeseed 
oil on the yield of ADD. 
A medium having the following composition is prepared: 
2.0 percent of sucrose, 
1.0 percent of NaNO.sub.3, 
1.0 percent of yeast extract, 
0.25 percent of K.sub.2 HPO.sub.4, 
0.03 percent of MgSO.sub.4.7H.sub.2 O, 
3.0 percent of rapeseed oil, and remainder -- water 
The pH of the medium is adjusted to 7.0 with NaOH. To a 500 ml shaker flask 
is added 50 ml of the medium. The flask and its contents are sterilized by 
autoclaving for a period of 10 minutes at a temperature of 120.degree. C 
and then cooled. The flask is inoculated with 1 ml of the seed culture 
broth which is obtained in the same manner as in Example 2. The inoculated 
medium is incubated at a temperature of 30.degree. C on a reciprocal 
shaker similar to that described in Example 1. At the end of 25 hours from 
the time of initiating the incubation, 250 mg of cholesterol is added. At 
the end of 30 hours from the time of initiating the incubation, 5.5 mg of 
2,2'-bipyridyl is added. At the end of a period of time from the time of 
initiating the incubation as indicated in Table 5, 1.0 g of rapeseed oil 
is added to the culture medium. At the end of 110 hours from the time of 
initiating the incubation, the incubation is stopped. The formed ADD is 
separated in the same manner as in Example 1. 
The procedure described above is repeated with the exception that the time 
of the addition of rapeseed oil is varied as indicated in Table 5. 
TABLE 5 
______________________________________ 
Time of Addition of 
Rapeseed oil Yield of ADD 
(hours) (mg) 
______________________________________ 
0 47 
20 52 
30 48 
42 45 
70 23 
90 22 
no addition 22 
______________________________________ 
EXAMPLE 8 
This Example illustrates the increased yield of ADD by the addition of the 
vegetable oil meal along with the fat to the culture medium. A seed medium 
having the following composition is prepared: 
1.0 percent of yeast extract, 
1.0 percent of meat extract, 
1.0 percent of peptone, and remainder -- water 
The pH of the seed medium is adjusted to 7.0 with NaOH. To a 500 ml shaker 
flask is added 100 ml of the seed medium. The flask and its contents are 
sterilized by autoclaving for a period of 15 minutes at a temperature of 
120.degree. C, and then cooled. The medium is inoculated with a loopful of 
Arthrobacter simplex (IAM 1660) and the inoculated medium is incubated for 
a period of about 48 hours at a temperature of 28.degree. C on a 
reciprocal shaker similar to that described in Example 1. 
A main fermentation medium having the same composition as that of the seed 
medium with the exception that the required amounts of the fat and the 
vegetable oil meal shown in Table 6 are added to the medium to achieve the 
listed concentrations. 
The pH of the main fermentation medium is adjusted to 7.0 with NaOH. To a 
500 ml shaker flask is added 50 ml of the main fermentation medium. The 
flask and its contents are sterilized by autoclaving for a period of 20 
minutes at a temperature of 120.degree. C, and then cooled. The flask is 
inoculated with 2 ml of the seed culture broth obtained above and then 
placed on a reciprocal shaker having a 7-cm stroke. The main fermentation 
is initiated at a temperature of 30.degree. C. At the end of 20 hours from 
the time of initiating the incubation, 150 mg of cholesterol suspended in 
2 ml of water is added. At the end of 28 hours from the time of initiating 
the incubation, 6.2 mg of 2,2'-bipyridyl is added. At the end of 85 hours 
from the time of the addition of 2,2'-bipyridyl, the incubation is 
stopped. The fermentation broth is extracted with 150 ml of ethyl acetate. 
The extract is concentrated, chromatographed on silica gel and eluted with 
benzene-acetone (1:1), thereby separating ADD from intermediate oxidation 
products and the steroid substrate. The yield of ADD is shown in Table 6. 
Employing a mixture of .beta.-sitosterol and campesterol (2:1), 
stigmasterol, cholest-4-en-3-one or cholesta-1,4-dien-3-one in place of 
cholesterol, the above-described incubation is repeated. 
In addition, the above-described incubation is repeated except that the fat 
and the vegetable oil meal are not added to the culture medium. 
The results are shown together in Table 6. 
TABLE 6 
__________________________________________________________________________ 
Remaining 
Run Concentration 
Yield of ADD 
Substrate 
No. 
Substrate Fats and Vegetable oil meals 
(weight percent) 
(mg) (mg) 
__________________________________________________________________________ 
1 Cholesterol 
None 0 3 53 
2 " Linseed oil 1.0 26 0 
3 " Linseed oil meal 
2.0 18 0 
4 " Linseed oil 1.0 55 0 
Linseed oil meal 
2.0 
5 " Sesame oil 1.0 29 0 
6 " Sesame oil meal 2.0 20 0 
7 " Sesame oil 1.0 52 0 
Sesame oil meal 2.0 
8 Sitosterol- 
None 0 2 70 
Campesterol (2:1) 
9 " Linseed oil 1.0 10 40 
10 " Linseed oil meal 
2.0 7 58 
11 " Linseed oil 1.0 20 22 
Linseed oil meal 
2.0 
12 Stigmasterol 
None 0 1 86 
13 " Linseed oil 1.0 4 50 
14 " Linseed oil meal 
2.0 4 50 
15 " Linseed oil 1.0 9 38 
Linseed oil meal 
2.0 
16 Cholest-4-en-3-one 
None 0 5 18 
17 " Linseed oil 1.0 23 4 
18 " Linseed oil 1.0 55 0 
Linseed oil meal 
2.0 
19 Cholesta-1,4-dien- 
None 0 5 13 
3-one 0 
20 " Linseed oil 1.0 22 0 
21 " Linseed oil 1.0 53 0 
Linseed oil meal 
2.0 
__________________________________________________________________________ 
EXAMPLE 9 
This Example illustrates the effect of the amounts of soybean oil and 
soybean oil meal to be added on the yield of ADD. 
The procedure of Example 8 is repeated with following changes in amounts of 
the substances which are added to the culture medium: 
1. 200 mg of cholesterol is used as a substrate. 
2. Soybean oil and soybean oil meal are added in amounts required to give 
the concentrations as indicated in Table 7. 
3. When more than 8 percent of soybean oil meal is present in the culture 
medium, 7.8 mg of 2,2'-bipyridyl is added. 
The results are shown in Table 7. 
TABLE 7 
______________________________________ 
Run Soybean oil Soybean oil meal 
Yield of ADD 
No. (weight percent) 
(weight percent) 
(mg) 
______________________________________ 
1 0 0 3 
2 0 2.5 15 
3 0.15 2.5 15 
4 0.25 1.5 13 
5 0.25 2.5 16 
6 0.30 2.5 29 
7 0.50 2.5 41 
8 0.50 5.0 48 
9 0.50 8.0 51 
10 0.50 10.0 43 
11 0.50 15.0 38 
12 0.50 20.0 22 
13 1.0 0 36 
14 1.0 0.3 45 
15 1.0 0.6 51 
16 1.0 1.0 54 
17 1.0 2.5 53 
18 1.0 5.0 54 
19 1.0 8.0 52 
20 1.0 10.0 47 
21 1.0 15.0 36 
22 1.5 2.5 50 
23 3.0 0 25 
24 3.0 0.1 26 
25 3.0 0.3 36 
26 3.0 0.9 45 
27 3.0 1.8 47 
28 3.0 2.5 48 
29 3.5 2.5 45 
30 4.0 2.5 20 
31 6.0 2.5 8 
______________________________________ 
EXAMPLE 10 
This Example illustrates the effect of the addition of the fat and the 
vegetable oil meal on the yield of ADD. A medium having the following 
composition is prepared: 
2.0 percent of sucrose, 
1.0 percent of NaNO.sub.3, 
1.0 percent of yeast extract, 
0.25 percent of K.sub.2 HPO.sub.4, 
0.03 percent of MgSO.sub.4.7H.sub.2 O, 
amounts of the fat and the vegetable oil meal to give the concentration 
indicated in Table 8, and remainder-water 
The pH of the medium is adjusted to 7.0 with NaOH. To a 500 ml shaker flask 
is added 50 ml of the medium. The flask and its contents are sterilized by 
autoclaving for a period of 20 minutes at a temperature of 120.degree. C 
and then cooled. The flask is inoculated with 1 ml of the seed culture 
broth which is obtained in the same manner as in Example 8 with the 
exception that Brevibacterium Lipolyticum (IAM 1398) is employed in place 
of Arthrobaceter simplex (IAM 1660). The inoculated medium is incubated at 
a temperature of 30.degree. C on a reciprocal shaker the same as that 
described in Example 1. At the end of 15 hours from the time of initiating 
the incubation, 200 mg of cholesterol is added. At the end of 28 hours 
from the time of initiating the incubation, the amount of 2,2'-bipyridyl 
shown in Table 8 is added and then the fermentation is continued for an 
additional 70 hours. At the end of this period, the products are extracted 
with ethyl acetate and separated in the same manner as in Example 8. The 
yield of ADD is shown in Table 8. 
TABLE 8 
______________________________________ 
2,2'- Yield 
Run Fats and Vege- 
Concentration 
Bipyridyl 
of ADD 
No. table oil meals 
(weight percent) 
(mg) (mg) 
______________________________________ 
1 None 0 3.9 16 
2 Rapeseed oil 1.0 3.9 35 
3 Rapeseed oil 1.0 5.5 50 
Rapeseed oil meal 
2.0 
4 Linseed oil 1.0 3.9 33 
5 LInseed oil 1.0 5.5 42 
Linseed oil meal 
2.0 
6 Soybean oil 1.0 3.9 35 
7 Soybean oil 1.0 5.5 49 
Soybean oil meal 
2.0 
8 Sesame oil 1.0 3.9 37 
9 Sesame oil 1.0 5.5 45 
Sesame oil meal 
2.0 
10 Cottonseed oil 
1.0 3.9 32 
11 Cottonseed oil 
1.0 5.5 40 
Cottonseed meal 
2.0 
12 Peanut oil 1.0 3.9 37 
13 Peanut oil 1.0 5.5 50 
Peanut oil meal 
2.0 
______________________________________ 
EXAMPLE 11 
This Example illustrates the effect of the addition of the 
glyceride-containing substance and the vegetable oil meal on the yields of 
DHT and ADD. 
Example 10 is repeated except that the substances which are added to the 
culture medium are varied as indicated in Table 9, and that the amount of 
1,10-phenanthroline indicated in Table 9 is used in place of 
2,2'-bipyridyl, and that the incubation is stopped at the end of 28 hours 
from the time of addition of 1,10-phenanthrobine (at the end of 56 hours 
from the time of initiating the incubation). 
When the incubation is stopped at this early stage, DHT is formed along 
with ADD. The yields of DHT and ADD are shown in Table 9. 
TABLE 9 
______________________________________ 
Concen- 
tration 
1,10-Phen- 
Yield 
Run Added (weight anthroline 
ADD DHT 
No. Substances percent) (mg) (mg) (mg) 
______________________________________ 
1 None 0 1.3 7 4 
2 Triolein 1.0 1.3 24 25 
Linseed oil meal 
2.0 
3 Triolein 1.0 1.3 22 25 
Soybean oil meal 
2.0 
4 Triolein 1.0 1.3 25 23 
Sesame oil meal 
2.0 
5 Triolein 1.0 1.3 21 28 
Rapeseed oil meal 
2.0 
6 .alpha.-Monopalmitin 
1.0 1.3 23 21 
Sesame oil meal 
2.0 
7 .alpha.-Monopalmitin 
1.0 1.3 21 17 
Peanut oil meal 
2.0 
8 .alpha.-Monopalmitin 
1.0 1.3 19 21 
Cottonseed meal 
2.0 
9 .alpha.-Monopalmitin 
1.0 1.3 19 23 
Soybean oil meal 
2.0 
10 Palm oil 0.25 1.3 7 7 
Rapeseed oil meal 
2.0 
11 Palm oil 0.3 1.3 13 10 
Rapeseed oil meal 
2.0 
12 Palm oil 0.5 1.3 16 13 
Rapeseed oil meal 
2.0 
13 Palm oil 1.0 1.3 18 10 
Rapeseed oil meal 
0.3 
14 Palm oil 1.0 1.3 18 14 
Rapeseed oil meal 
0.6 
15 Palm oil 1.0 1.3 20 20 
Rapeseed oil meal 
1.0 
16 Palm oil 1.0 1.3 22 24 
Rapeseed oil meal 
2.0 
17 Palm oil 1.0 1.3 22 26 
Rapeseed oil meal 
5.0 
18 Palm oil 1.0 1.6 21 20 
Rapeseed oil meal 
10.0 
19 Palm oil 1.0 1.6 18 17 
Rapeseed oil meal 
15.0 
20 Palm oil 1.0 1.6 15 12 
Rapeseed oil meal 
20.0 
21 Lard 1.0 1.3 20 28 
Sesame oil meal 
2.0 
22 Lard 10.0 1.3 22 23 
Cottonseed meal 
2.0 
______________________________________ 
EXAMPLE 12 
This Example illustrates the effect of the simultaneous addition of the fat 
and the vegetable oil meal on the yield of ADD. 
A medium having the following composition is prepared: 
2.0 percent of corn steep liquor, 
2.0 percent of glucose, 
0.4 percent of sodium glutamate, 
0.2 percent of asparagine, 
0.2 percent of KH.sub.2 PO.sub.4, 
amounts of the fat and the vegetable oil meal to achieve the concentration 
listed in Table 10, and remainder - water 
The pH of the medium is adjusted to 7.0 by the addition of NaOH. To a 500 
ml shaker flask is added 50 ml of the medium. The flask and its contents 
are sterilized by autoclaving for a period of 20 minutes at a temperature 
of 120.degree. C. The flask is inoculated with 2 ml of the seed culture 
broth which is obtained in the same manner as in Example 5. The inoculated 
medium is incubated at a temperature of 35.degree. C on a reciprocal 
shaker similar to that described in Example 1. At the end of 40 hours from 
the time of initiating the incubation, 150 mg of cholesterol is added to 
the medium. 
At the end of 48 hours from the time of initiating the incubation, 4.2 mg 
of 8-hydroxyquinoline is added to the medium. At the end of 60 hours from 
the time of the addition of 8-hydroxyquinoline, the fermentation is 
terminated. Upon completion of the fermentation, the formed ADD is 
recovered from the fermentation broth by the same procedure as described 
in Example 1. The yield of ADD is shown in Table 10. 
TABLE 10 
______________________________________ 
Run Fat and Concentration 
Yield of ADD 
No. Vegetable oil meal 
(weight percent) 
(mg) 
______________________________________ 
1 None 0 12 
2 Linseed oil 0.5 26 
Linseed oil meal 
2.0 
3 Rapeseed oil 0.5 33 
Rapeseed oil meal 
2.0 
4 Cottonseed oil 
0.5 27 
Cottonseed meal 
2.0 
5 Cottonseed oil 
0.5 32 
Sesame oil meal 
2.0 
6 Cottonseed oil 
0.5 24 
Peanut oil meal 
2.0 
7 Cottonseed oil 
0.5 37 
Soybean oil meal 
2.0 
8 Soybean oil 0.5 22 
Soybean oil meal 
1.0 
9 Soybean oil 0.5 35 
Soybean oil meal 
2.0 
10 Soybean oil 0.5 38 
Soybean oil meal 
5.0 
11 Soybean oil 0.5 26 
Soybean oil meal 
8.0 
12 Soybean oil 0.5 19 
Soybean oil meal 
10.0 
______________________________________ 
EXAMPLE 13 
This Example also illustrates the effect of the addition of the fat and the 
vegetable oil meal on the yield of ADD. A medium having the following 
composition is prepared: 
2.0 percent of corn steep liquor, 
2.0 percent of glucose, 
0.4 percent of sodium glutamate, 
0.2 percent of asparagine, 
0.2 percent of KH.sub.2 PO.sub.4, 
amounts of the fat and the vegetable oil meal to effect the concentration 
listed in Table 11, and remainder -- water 
The pH of the medium is adjusted to 7.0 by the addition of NaOH. To a 500 
ml shaker flask is added 50 ml of the medium. The flask and its contents 
are sterilized by autoclaving for 20 minutes at 120.degree. C. The flask 
is inoculated with 2 ml of the seed culture broth which is obtained by the 
same procedure as in Example 5 with the exception that Protaminobacter 
alboflavus (ATCC 8458) is employed as a microorganism, and that the seed 
incubation is carried out for 50 hours at 30.degree. C. The inoculated 
medium is incubated at 30.degree. C on the same shaker as in Example 1. At 
the end of 25 hours from the time of initiating the incubation, 200 mg of 
cholesterol is added. At the end of 33 hours from the time of initiating 
the incubation, 6.2 mg of 2,2'-bipyridyl is added. At the end of 95 hours 
from the time of initiating the incubation, the incubation is stopped. 
Upon completion of the incubation, the formed ADD is recovered from the 
fermentation broth by the same procedure as described in Example 1. The 
yield of ADD is shown in Table 11. 
TABLE 11 
______________________________________ 
Run Fat and Concentration 
Yield of ADD 
No. Vegetable oil meal 
(weight percent) 
(mg) 
______________________________________ 
1 None 0 4 
2 Soybean oil 0.5 6 
3 Rapeseed oil 0.5 6 
4 Soybean oil meal 
3.0 9 
5 Rapeseed oil meal 
3.0 9 
6 Soybean oil 0.5 12 
Soybean oil meal 
3.0 
7 Rapeseed oil 0.5 13 
Rapeseed oil meal 
30 
______________________________________ 
Having now fully described the invention, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the invention as set 
forth herein.