2,697,062
United States PatentO
Patented Dec.’ 14, 1954
1
2,697,062
out
B‘ P‘
.,
0‘
Refraction
Speci?c
Index
Gravity
PROCESSING OF HYDROCARBONS
David L. Cramer, Long Beach, Calif., assignor to Texaco
Development Corporation, New York, N. Y., a corpo
ration of Delaware
'
No Drawing. Application March 30, 1951, ,
I Serial No. 218,546
'
to 205
205-215
215-225
225+
1. 4388
1. 4432
1. 4454
1. 4500
0. 8022
0.8118
0. 8155
0. 8217
178-261
1. 441
0.8118
10
Of the above cuts of kerosine, Cut D will usually give
the best yield and product as expressed in grams of mold
mycelia. Cut D will usually give a yield of over twice
_
10 Claims. (Cl. 195—3)
as much as Cut A, about 50% more than Cut B and
This invention relates to the production of biosyn 15 about 33% more than Cut C.
Commercial stove oil, having a B. P. range of 167° C.
thetics from hydrocarbons and more particularly to the
. to 277° C. can be similarly fractionated by ASTM dis
production of fat-like saponi?able'materials from hydro
tillation methods into the following four cuts:
carbons by the action of molds, yeasts, and other micro
organisms, the invention being an improvement over
the invention disclosed in the copending application of
out
R. G. Harris and R. J. Strawinski ?led Augustfl7, 1950
B" P’
o
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Refraction
Speci?c
Index‘
Gravity
bearing Serial No. 180,092 and entitledProcessing of
Hydrocarbons.
It has been known for some time that such micro
organisms have the ability to attack-ormetabolize some
materials such as carbohydrates. Since carbohydrates
to 221
221-235
235-252
1. 4541
1. 4583
1. 4631
0. 8279
0. 8351
0. 8415
252+
1. 4681
0. 8506
167-277
1. 4590
0. 8304
are relatively expensive raw materials and in most cases,
of greater value than the products produced therefrom,
Of the above, Cut I will usually give a yield of about
other raw materials have been sought. A considerable
three times the yield of Cut F measured in mold mycelia,
amount of work has been done with hydrocarbons but 30 of
about twice the yield of Cut G and about 1% times
to date it has not been generally successful and has not
the yield of Cut H.
been on a practical basis, apparently because of the
Relatively simple hydrocarbons such as isopentan'e,
failure to appreciate certain factors as hereinafter de
n-pentane, 2-2-dimethylbutane, 2,2,4-trimethylheptane,
scribed. Probably the most discouraging aspect of the
isoheptanes, isoheptenes, isooctanes, iso
hydrocarbon work done heretofore is the fact that the 35 n-heptane,
octenes, 2,3-dimethylpentane, cyclopentane, butylben
principal products have'been carbon dioxide and water.
zene, 2,2,5 - trimethylhexane, 2,3 - dimethylpentane,
Apparently, this has been due to a studied adherence to
Z-pentane, methylcyclopentane, and l-pentene can also
established methods of procedure and a failure to ap
be used as well as hexane.
preciate the possible effects of radical departures there
Microorganisms suitable for use include molds, yeasts
40
from.v
>
and the like. Typical examples are molds of the genus
Accordingly, it is an object of this invention to provide
Penicillium such as P. rocqueforti, P. glaucum, P. chryso
genum, P. patulum, and notatum, molds of the genus
Aspergillus such as A. fumigatus (strain TDC #946),
45 A. carbonarius, A. niger, A. ?avus and A. terreus. Typi
other products.
I
cal yeasts include M‘onilia murmanica, Monilia sitophile
Other objects and advantages of the invention will
and Saccharomyces.
I
appear from the following description taken in connec
The
majority
of
the
above
are
available
from
the
tion with the attached claims.
’
American Type Culture Collection. One such mold that
In brief, it has been discovered that under certain con
ditions, microorganisms such as molds, yeasts and the 50 igsssgitable for use is unidenti?ed except for, the number
like can be caused to grow quite rapidly upon relatively
One of the best microorganisms for use in the process
inexpensive substrates such as hydrocarbons in an emulsi
novel processes wherein hydrocarbons can be effectively
utilized in biosynthesis including the production of rela
tively high yields of fat-like saponi?able materials and
of this invention is Aspergillus fumigatus (strain TDC
?ed phase with the production of relatively large yields
of desirable products including fat-like saponi?able ma
terials, and other products. Such conditions include
No. 946) which has been identi?ed as follows:
Source of isolation.—A mixed sample composed of
soils taken from areas in which oils were undergoing de
such factors as the selection and maintenance of a proper
composition and water taken from sumps, tank bottoms,
and waters which had been in contact with re?nery
wastes; decomposing asphalt, Wax and used motor oil
pH range and temperature range, the selection of proper
nutrient salts, and the selection of a proper emulsifying
agent. In some cases, the selection of predetermined con
centrations of both salts and substrates is desirable. '
Broadly stated, the practice of the invention involves
60
the use of a nutrient salt solution such as a solution of
the mineral salts type with a pH in a selected range and
the use of a hydrocarbon, the use of an emulsifying
were included in the sample.
‘
Date of isolation.-October 1, 1947.
Location of isolatiom?Signal Hill Laboratory, The
Texas Company, Long Beach, California.
Media used for isolati0n.—A hydrocarbon-mineral
agent, preferably of a type capable of also functioning
salts medium composed of 5% Arabian crude oil
as a phosphorylating agent, the inoculation of 'the mix
ture with a selected microorganism or microorganisms,
Compound:
the maintenance of the mixture‘within predetermined
temperature and pH ranges for a time sufficient to effect
the desired biosynthesis, and the recovery of the desired 70
products.
Any type of hydrocarbon can be utilized in the proc
ess. Good results can be obtained with complex hydro
carbons such as crude oil, kerosine, parai?n wax, naphtha,
gasoline, gas oil, lubricating oils and bottom sediments
(topped) and the following medium at pH 4.5.
.
NaNOg ______________ __grams per liter__
KH2PO4
2.0
______________________ .._ 0____
1.0
MgSO4.7H2O __________________ __do____
‘KCl
'
do____
FeSO4.7H2O ___________________ __do____
0.5
0.5
0.01
Sea water
__________ _-_ _____ __milliliters__
10.0
Distilled water ________________ __do____ 990.0
The above is generally designated Tur?tt’s Yeast and
Mold Medium.
Conditions of isolation.-—Aerobic, at 40° C.
kerosine water white oil, sold by The Texas Company
Microscopic examination and a comparison of the es
under. the trade name “Crystalite” and having a B. P.
range of 178°= C.-261°> C. can be fractionated into ‘the 80 sential morphological features with those'of molds de
following cuts:
.
.
l
. _, scribed the literature indicatethat the mold belongs to
from oil tanks. Of the above, even better ‘results can
be secured with certain selected cuts. For instance, a
2,697,062
4
3
‘the j‘ztm'igatus group of the Aspergilli, the type species of
which vis Aspergillus fumigatus. A ‘complete description
stearate, etc., and R3 is the quaternary ammonium base
choline.
Other monoaminomonophospholipids such as cephalin
thereof can be found in “A Manual of the Aspergilli” by
Charles Thom, The William and Wilkins Company,
may be used as well as the diaminomonophospholipids of
Chapter X, pp. .148—151.
which the sphingomyelins are typical.
The proportion of emulsi?er or phospholipid used in
The character of the nutrient media or mineral salts
solution will vary to some extent in accordance with
the type of microorganism used and the type if hydro
carbon. Generally, it should contain N, P, K, S, Fe, Mg,
the general composition being well known in the art. A
typical medium is as‘ follows:
the process does not appear to be particularly critical.
Amounts varying from 0.1% by volume of the hydro
carbon to as much as 10 grams per 100 ml. hydrocar
10
The amount of emulsi?er used such as lecithin is so
small that it would not be used by the microorganism as a
Compound:
(NH4)2SO4 _____________ “grams/liter“
carbon source.
2.0
As an example of .a method embodying the present
invention, lecithin is added to kerosine in a proportion of
0.1 gram of lecithin to 100 ml. of vkerosine. The type
of hydrocarbon used is not critical nor is the above-in
dicated proportion of lecithin critical. Proportions as
high as l to ‘10 grams of lecithin to 100 ml. of kerosine
have been found to be equally effective.
A vsuitable sterile mineral salts or nutrient solution is
then prepared. Such asolution may vary widely in com
Ks‘HPOt ____________________ __‘do___a
2.0
‘NaI-I2PO4 ___________________ __do_..__
110
MgSO4 ______________________ __'do____
0.25
MnClz ______________________ __do____
001
CaClz ______________________ __do____
001
F6804 ______________________ __do_..__
0.01
KI _________________________ __do____ 0.00001
@1504 ______________________ __do____ 0.00001
Distilled water ___________ "milliliters"
1000
position and ‘proportion, the following being typical:
‘In some cases, the medium may be forti?ed or en
riched with accessory growth factors such as water ex
tract of yeast such as Saccharomyces cerevisiae.
It is to be understood that the character of ‘the me
dium may also be varied dependent upon the type of
product that it is desired to produce in the .most 30
abundance.
‘As regards the mechanical or contact aspect, the proc
ess may bepracticed in a number of different ways.
bon have been found to produce satisfactory results.
In
its simplest form, the growth may be accomplished ‘in a
static phase on the surface of the'liquidzmixture. Pre'r~~
erably it is practiced as a deep culture process by me
chanical agitation such as stirring or shakingin the case
of liquids. Inthe case of solid hydrocarbons, contact
may be established and:maintained by the method :de
Distilled water _____ ____ ___ _____________ __cc__
900
Sea water ________ __________ ____________ __cc__
100
K2HPO4 _____________________________ .._g___
(NH-Q2804 ___________________________ __g__
2.0
2.0
MgSO4 , _______________________________ __g__
0.25
MnClz
0.01
___.
_____
Q
CaClz _______________________________ __g__ 0.01
FeSO4 _______________________________ __g__ 0.01
211804 _______________________________ __g__ 0.005
KI __________________________________ __g__ 0.001
CuSOi _______________________________ __g__ 0.001
After sterilization, the pH ofsuch a solution is about
5
scribed in the copending application of Rhett G. Harris,
‘As already stated, the composition of the mineral salts
solution may be Widely variedprovidedthat the follow
Serial No. 12,896 ?led March.3, 1948 and entitled Rroc- *
esses Involving Action of‘ Microorganisms or Their
ing are supplied thereby:
'
Enzymes on Organic or InorganicSubstances whereinthe
Ammonium ionsuchas ammoniumsulfate or nitrate
hydrocarbon is adsorbed on a ?nely dividedzand relative~
Magnesium ion
ly inert adsorbent material such as. a clay. ‘Where the hy
Phosphate ion
45
drocarbon is in the gaseous phase, a systenrmay besetup
iron and Zinc ion (intraces)
gvherein the gas is recycled through .the inoculated-me
Manganese, calcium,vcopper and iodide ions which ap
pear to have a stimulatory effect
rum.
The pH with ‘molds may vary from 1 -,to .9. .With
yeasts, theipH vmay vary from 4 to9. its :selection :is
A suitable buffering system to absorb excess .aciditythat
may result from the growth process. .Good growths
governed by the particular microorganisms, ‘the (particu 50 havebeen secured :With an initial pH in the region of
lar hydrocarbon and the endproducts'desired.
4.5, ‘5.5 and 6.8.
'
The ‘same-is truefof temperature,-.the range beingifrom
20°"C. to '45 ° C. \Theactual time of'incubation,.;usually
.A mineral. salts solution, prepared -,_as described above,
from 7 to 12 days, is determined by the rate of growth
is addedto the keros‘ineflecithin. combinationin a pro
of the microorganisms. This
' is governed 1by-,the:ability i.‘ portion of nine,parts mineral salts solution to one part
of the enzymes, produced by the microorganisms to effect
kerosine-lecithin.
production of the desired products.
The mixture is then, stirred andan inoculum including
the mycelial :growth and-the .sporesofaseed culture of
The emulsifying agent may be one of the more common
and well-known agents for emulsifying hydrocarbon and
amold knowntoutilize thespeci?chydrocarbon as a
water but is preferably an agent which is preferentially ' L source of carbonandqenergyHis,added'in an amount of
soluble in'the hydrocarbon but not in'water. Thus, an
emulsion is obtained in Which Water is the dispersed
phase. This results in better contact of the'microor
ganism which is essentially hydrophilic and the hydro
from 10.1% .to 20% by'fvolume. The inoculum is
formedsas a thick suspepsion of theorganism in the
nutrientrmedium constituting about 10% by volume of
the totalcharge. ,The amount oftinoculum can be varied
phobic source of carbon. Stated otherwise, the resulting ’
mixture is preferably one in which water or‘ salt’ medium
widely since .such .variation ,is limiting only upon the
periodof .timesubsequently necessary for growth of the
globules are dispersed in hydrocarbon.
It is further desirable that the emulsifying agent be
characterized by apparent phosphorylating _. properties,
i. e., capable of entering into the mechanism of fat re
synthesis in the animal body.
Calcium stearateis .an example of one. of \the more
common emulsifying agents that might be used but
which do not‘ possess .phosphorylating. properties. Apre
ferred agent, capable. of functioning in. both .an. emulsify
ing and phosphorylating.capacity, is lecithin which may
be represented by .the.formula:
myceliato befully established in the desired submerged
culture.
While the method has been especially successful with
Aspergillus .fumigatus ‘(strain [TDC _No. .946), the
method {is t not restricted .to this particular organism.
Any-strain .of any species able-to. utilize the speci?c hy
drocarbon asla source of carbon and energy may be used.
The culture vessel is then held at' a substantially
constanttemperature in the range. of about .20“ C. to
45° ;'C., preferablyabout 30° :- 3°.C.,.and the. mixture
maintainedinan agitated condition ,by shakinglor stir
ring. ‘Sterile,airor.oxygen,.preferably tat superatmos
Pheric pressureuis introducedat .a vigorousrate into
80 the depths of the cu'ltureas through .a porous .bubbler.
,After .5.to 10 days’ operation under these_-conditions,
the moldmycelia?is harvested bya-mecharticral ,separa
tion such :as ?ltration vtor .centrifugingfrom the liquid
portion of the culture.
As compared with the same method but with the
where R1 ‘aridRz are fatty acid anions, such as oleate, 85
2,697,062
6
5
lecithin omitted, the yield of mold mycelia is increased
from 200% to 300%.
To recover the fat-like saponi?able material, the
mycelia can be dried and a crude extract prepared by
invention, as hereinbefore set forth, may be made
without departing from the spirit and scope thereof and,
therefore, only such limitations should be imposed as
are indicated in the appended claims.
I claim:
chloroform extraction. After removal of the solvent,
1. The method of synthesizing large yields of fat»
the residue is treated with approximately two-thirds its
like saponi?able materials from hydrocarbons which
weight of potassium hydroxide dissolved in alcohol and
comprises the steps of dispersing hydrocarbon with a
re?uxed to effect saponi?cation. Following saponi?ca
phospholipid in an aqueous nutrient medium for micro
tion about two-thirds of the solvent alcohol is removed
by heating. The residual soap solution is then extracted 10 organisms, inoculating the resultant mixture with a
microorganism selected from the group consisting of
with petroleum ether to remove the non-saponi?able
Penicillium molds, Aspergillus molds, Monilia yeasts
fraction. Following this step, the aqueous liquor is
and Saccharomyces yeasts, subjecting the inoculated mix
made strongly acid with a mineral acid and the free
ture to incubating conditions for a sustained period of
organic acids extracted with diethyl ether.
time, collecting the resultant solid material and separating
The saponi?able material thus recovered does not
synthesized fat-like materials therefrom.
consist exclusively of fatty acids of the usual aliphatic
2. The method of claim 1 wherein said phospholipid
character. Some products are found which react chemi
is lecithin.
,
cally very nearly the same, as the fatty acids but they
3. The method according to claim 1 in which said
are not the usual aliphatic fatty acids such as the
straight-chain acids containing an even number of car
bon atoms such as C10 acid (capric), C12 acid (lauric or
dodecenoic), etc., such acids being usually obtained from
the naturally-occurring fats.
phospholipid is cephalin.
4. The method according to claim 1 in which said
inoculated mixture is incubated at a temperature in
the range of 20 to 45° C. and at a pH of about 1 to 9.
5. The method according to claim 1 in which said
microorganism is Aspergillus fumigatus.
‘
6. The method according to claim 1 in which said
The mixed fatty acids obtained are in the nature of
pale yellow solids at room temperature with the charac
teristic appearance of a fat. A slightly, pleasant, fat
microorganism is Penicillium notatum.
like odor is present and the taste is similar to that of
7. The method according to claim 1 in which said
candle tallow or similar stearins. The product melts at
microorganism is Aspergillus ?avus.
about 29.5" C. and has a setting point of 17.5“ C. The
8. The method according to claim 1 in which said
mean molecular weight is 294, and the iodine number 30
microorganism is Monilia murmanica.
83.5. Of the common fats or fatty oils, the product
9. The method of increasing the yield of fat-like
most closely resembled the mixed fatty acids from olive
saponi?able materials obtained by the action of micro
oil.
organisms selected from the group consisting of the
In another method, lecithin is added in an amount of
0.01% the volume of kerosine. About ?fteen parts by
volume of nutrient medium, prepared as above described,
is added to two parts of the kerosine-lecithin mixture and
Penicillium molds, the Aspergillus molds, the Monilia
almost twice the yield of a control not containing lecithin.
If desired, the mycelia may be processed as de
phospholipid is lecithin.
yeasts and the Saccharomyces yeasts on the hydrocarbon
in aqueous nutrient medium which comprises the steps
of adding a phospholipid emulsifying agent to the mix
the mixture inoculated with Aspergillus fumigatus.
ture of hydrocarbon and nutrient medium.
After seven days’ incubation under agitation and aera
10. The method according to claim 9 in which said
tion, a yield, dry weight of mycelia is obtained which is 40
scribed in the afore-mentioned Harris and Strawinski
application to recover other products.
From the above it is believed apparent that the pres 45
ent invention provides a novel process for the synthesis
of relatively valuable fat-like saponi?able materials and
other products in relatively large yields from relatively
cheap raw materials, i. e., hydrocarbons.
The use of
an emulsifying agent, particularly an agent possessing
phosphorylating properties, is particularly advantageous
in submerged, stirred and aerated culture processes.
Obviously many modi?cations and variationsof the
References Cited in the ?le of this patent
UNITED STATES PATENTS
Number
1,753,641
1,835,998
Name
Date
Beckman ____________ __ Apr. 8, 1930
Giron _______________ __ Dec. 8, 1931
OTHER REFERENCES
Bushnell et al., I. Bact., 41, 653-73 (1941).
Zobell et al., Bull. Am. Assoc. Petroleum Geol., 27,
1175-93 (1943).