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Патент USA US3083154

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March 26, 1963
M. w. sHEPARD
3,083,144
METHOD oF PRoDucING CITRIC ACID BY FERMENTATION
Filed April 4, 1960
EFFECT OF NITROGEN ADDITIONS
UPON RATE 0F PRODUCT/ON OF
C [77E/C` AC/D
MEHR/CK
BY
`
nge-PARO
'ì
l
/NVENTOR
ATTORNEY
United States Patent 0”
C@
3,083,144
Patented Mar. 26, 1963
l
2
this point can be readily ascertained by analysing samples
3,083,144
from a control run of citric acid production in which no
METHOD GF PRODUCING CITRIC ACID BY
FERMENTATION
nitrogen is added after the fermentation begins. By deter
_
Merrick W. Shepard, Elkhart, Ind., assignor_ to Miles
Laboratories, Inc., Elkhart, Ind., a corporation of In
diana
mining the rate of citric acid production at various times
during the fermentation cycle, and plotting this rate against
the age of the fermentation it is possible to determine the
point of maximum rate of citric acid production and
thereby the point at which the nitrogen nutrient should
be added. It should be pointed out, however, that while
'Ihis invention relates to the production of citric acid 10 this procedure is desirable it has been found that enhanced
Filed Apr. 4, 1960, Ser. No. 19,881
7 Claims. (Cl. 195-36)
from carbohydrate containing materials by submerged
production of citric acid is accomplished by nitrogen
vegetative fermentation methods. More specifically, this
additions throughout the course of fermentation starting
at any time after the tirst stage of growth of the citric acid
mycelia. For example, additions from the third through
the ninth day of the fermentation cycle have proved effec
tive to produce greatly improved yields of citric acid and
considerably more satisfactory utilization of the carbo
hydrate feed. In particular, additions of nitrogen nutrient
from about the third day of fermentation to about the
invention relates to a method for improving the yield of
citric acid in a submerged fermentation process. In one
of its more particular aspects this invention relates to the
nutrition of citric acid producing fungi.
Citric acid -has been produced in commercial quantities
by the fermentation of carbohydrate materials using vari
ous strains of citric acid producing fungi. Certain strains
of Aspergillus niger have proved to be particularly useful 20 seventh day have proved eminently satisfactory for this
purpose.
In addition, various strains of other
It will be appreciated that the nitrogen nutrient selected
species of fungi such as Aspergillus clavrztus, Aspergillus
for addition to the fermentation medium at this time is in
wentii, Aspergillus luclzuensis, Penicillìzlm citrinum, Pení
addition to any nitrogen which may have been originally
cìllium luteum, Mucor píriformis and others have been
used with varying degrees of success.
25 charged to the fermentation medium. Such initial charge,
which is present before development of citric acid pro
In such citric acid fermentations it has been found essen
ducing mycelia, has been found to be generally effective
tial to provide the citric acid producing organism with
to promote a certain amount of growth of the mycelia.
certain nutrients such as nitrogen, phosphorus, potassium
for this purpose,
and magnesium, which are required for growth of the
However, it is now known that this initial charge is not
organism and for stimulation of production of citric acid. 30 effective to cause production of optimum yields of citric
acid, yet the mere increasing of the concentration of nitro
In general a suitable fermentation medium may be
prepared by addition to a fermenter of the carbohydrate v gen originally charged does not have the desired effect
either. It is only by following the practice of the present
to be fermented together with the various nutrient mate
invention, namely by feeding to the fermentation medium
rials. The fermentation medium or broth is then inocu
an effective quantity of nitrogen at a stage during the
lated with spores of the organism being used. Fermenta
tion is allowed to proceed under carefully controlled con
ditions of temperature and pH for a period of time suffi
cient to obtain useful yields of citric acid. In this process
35 fermentation at which utilization of the increased con
centration of nitrogen is possible that optimum yields of
citric acid and corresponding utilization of the carbohy
it is generally found that the rate of production of citric
acid increases steadily with passage of time until the rate
drate medium are obtained.
~
a time subsequent to the beginning of fermentation. More «
of fermentation so that an amount of nutrient is used
of citric acid producing mycelia. -
normally would fall olf. The addition of an amount of a
The nitrogen nutrients used for the purpose of this in
vention are compounds which are capable of furnishing
reaches a maximum after which the rate of production of
ammonium ions to the fermenting broth. For example,
citric acid begins to fall off. In a few days the rate has
ammonia, both in gaseous and aqueous form, ammonium
fallen to a point at which production is no longer
hydroxide and ammonium carbonate may be used as the
economical.
It is a major object of this invention to prolong the 45 nitrogen nutrient. The commercial grade of ammonium
carbonate, which is satisfactory for use for this purpose,
length of time during which citric acid production proceeds
is actually a mixture of ammonium bicarbonate and
at a useful and economical rate.
Another object of this invention is to provide a process f ammonium carbamate. Other compounds which serve as
a source of the ammonium ion may also be used in the
for the production of citric acid which is capable of pro
process of this invention. Such compounds include, for
ducing increased yields of citric acid.
50 example, other ammonium salts such as ammonium nitrate,
A further object of this invention is to provide a process
ammonium sulfate and ammonium chloride, which are also
for production of citric acid which utilizes a larger propor
capable of furnishing ammonium ions. Ammonia is pre
tion of the carbohydrate material used for fermentation.
ferred Vfor this purpose because of the ease of handling
Other objects and advantages of this invention will be
come apparent in the course of the following detailed 55 and convenience of use of the various forms of ammonia.
The concentration of nitrogen nutrient added during the
disclosure and description.
Y
fermentation may be varied in accordance with the com
l"This invention consists broadly in the addition of Va
position of the fermentation medium and the conditions
nitrogen nutrient to the citric acid fermentation broth at
particularly, this invention concerns nitrogen addition at 60 Vwhich is suflicient to maintain the maximum rate of pro
a point in the fermentation cycle after the development - duction of citric acid beyond the point at which the rate
v»nitrogen nutrient sufficient to give a concentration in the
fermentation broth of from about 50 parts per million of
tion it is generally possible to increase the yield of citric .
nitrogen to about 5,000 parts per million of nitrogen is
acid by the addition of a suitable nitrogen source at any
VV65 generally found adequate to insure stabilization of citric
time subsequent to the development of citric acid pro
-acid production at a high rate for an extended period of
ducing mycelia. It is desirable to add the nitrogen nutrient _
time. By virtue of this maintenance of high production
ata point in the fermentation cycle near where the rate
of productionv of citric acid begins to drop ott from theV `i rates it becomes possible to shorten the over-all times of
and still realize economical yields of citric
maximum. This point may Vary from batch to batch and , fermentation
acid.
is, of course, dependent upon the make-up of the fermenta
While it is not desired to postulate a mechanism which
tion medium and the fermentation conditions. However,
may be responsible for this observed increase in yield
In’accordance with the improved process of this inven
3,0133, 144
EXAMPLE II
of critic acid, it is suggested that the additional source
of nutrient nitrogen may result in metabolic changes in
the citric acid producing organism so as to enhance its
citric acid producing ability. It is known, for example,
The fermentation broths were made up according to
the procedure of Example I except that the range of
that there is a pronounced change in the morphorolgy 5
sugar used was from 22,400
lb.
~
_ to 25,300 lb. (16.3
of citric acid producing strains of Aspergillus niger after
18-4‘70) Per batch» POÍaSSlUm amd Phosphate Was Present
about 16 hours to about 20 hours of fermentation.
in a concentration of 140 parts per million and magne
sium sulfate was used at the 1,000 parts per million level.
No ammonia was added after the start of fermentation.
The drawing illustrates the effect of ’nitrogen additions
Odngim fmuwe .mnuhd .MüLhe anh.,c 1 .deho.mofwWumOCwemdœ.d ren.IW .m m_ e
u_mœeüdinemdHS tnfe1VPm0.Oeouftmwn eîmdov CilXHWr Afbähß UECPC
n..
h
.,e
m...ma
lto ece
Od
V.
al
.1c0b
mikmbEo
defence 10 The rates of citric acid production (grams of citric acid
mdIn
produced per 100 ml. per day) for ten runs were av
t
V.
CaÍ
,m
mb aeI
ePXth. W.nüwsm
eraged and the averages were plotted against the age of
the fermentation in days. The results are shown in Table
min. n.D
15
I and as curve A of the FIGURE.
Also shown in Table I are data for an average of ten
runs in which 20 lb. (145 p.p.m.) of ammonia was added
on the fourth day after the start of fermentation, which
The fermentation medium was made up in accordance
with the teachings of United States Patent No. 21,’492 ’ 667 20
are plotted as curve B of the ñgure.
D ata for an average of ten runs in
. which
.
145 ppm.
'to Raymond L' sônfn an? tîl‘eoëargNB'dSchä/àgï’ând
of ammonia was added on the ñfth day after the start
Patent No' 2’492’ ’3 to
of fermentation are also shown in Table I and plotted
o u
'
O0 War
an
l ay'
mond L. Snell. The medium was prepared from Cuban
as curve C of the figure.
high-test molasses which was defecated,v decationized,
Table I
ñltered and made up to Volume. The initial charge to 25V
the fermenter was as follows:
Rate oi Citrie Acid Production (g.ll00 zul/day) on
Volume ___-___'________`__V__. 16,500 gal.
Sugar -____________________, 22,0S01b. (16.08%).
Iron _____-_______'___„_____. 0.40 ppm.
Potassium acid phosphate ___-. 18.75 lb. (137 p.p.m.).
Ca.m.1um cm O.nd e
_ _ _ _ _ _ _ _
_
_
_ _
_ _
_
Selected Days after Start of Fermentation
Day
30
Ammonia
Added
3rd.
7th
6th
5th
4th
8th
10th
9th
11th
11 2 .lb. ( 8 7 p P m. l
35
3 4 one
t
,
From these data and from a comparison of the curves
After the nutrient elements Were added to the deionized '
plotted in ,the ñgure it can be Seen that addition of am.
medium in the fermenter, the pI-I was adjusted from pI-IV
1.4--1.6 by addition of ammonia to a pH within the range
of pH 2.5-2.8. This required from about 400 parts per 40
million to 700 parts per million of nutrient nitrogen.
1
monia on the fourth or the fifth day not only prolongs
the rapid rate of citricV acid production andïthereby de
creases the length ‘of the fermentation cycle but also
The vfermenter was then inoculated with spores of Asper-
dition.
leads to a higher rate than occurs Without ammonia ad
The maximum rate is 'seen to Occur about two
gillus‘ niger Strain No. 139 according to standard pracdays after ammonia addition'
tice, and the fermentation
was allowed _to proceed for
h
d 45
The following example illustrates the improved rates
ñve days' on t e ñf?f day of fermrentauon
‘Pounns
of production of citric'acid achieved through the use of
0f anhydrous 'ammoma (800 Parts Per m11 lion) was
ammonium carbonate in the process of this invention.
added. The fermentation'medium Washarvestedl after
12 days and .the yield of citric acid was found to be
Y EXAMPLE In
85.2%.
50
VIt may be seen from this example that a yield ofV over
The fermentation mediawere made up according to the
85% was obtained using the process of this invention.
procedure of Example ÁI except that the media contained
17.63% reducing sugars, 0.25 parts per million iron, 0.1
Prior Vto use of the improvement of this invention, yields
of from about 65% to 75% were considered satisfactory.
parts per vmillion ZnJf“L and 200 parts per million of nitro
The following example illustrates'the Veffect of nitro- 55 gen as Vammonium carbonate at pH 2.72. The data ob~
tained are shown in Table II..
gen'addition upon the »rate of citric acid production.
Tabla '11.
a
vm
..
1
a
..
nA
m
m
.
l
oa
Aa.
r.
tr.it
Um
U.
.55R70 La3lœ0.-o2%M3m4502.l7I5952 neLQ4w.I„Üm13842dD50792 dLwÜ5J.dmmw73a4M1h5PH2/„200.395,wLQ6„IuLmÜo7cN342am5_2n007--D5 m0QL7'I.Ü.W8042a39m1h .M019UL 25x7) nm.0„Lnm..085769n10
0.2,
0
....
.
..t
.i
7i20.
75577.
0%22725
755.72
8,083,144
5
6
These data show that the rate of citric acid production
monium salts as a nitrogen containing nutrient effective
is markedly increased by the addition of ammonium car
to stabilize the production of citric acid beyond the point
bonate according to the process of this invention. The
at which said production normally tends to decrease, said
data for -the run in which 4,000 parts per million of
addition being made at a time which is no earlier than
nitrogen as ammonium carbonate was added to the orig- 5 about one day from the point of maximum rate of citric
inal fermentation medium and no nitrogen nutrient was
acid production in the absence of said added nutrient.
added after the beginning of fermentation show that the
2. In a process for the production of citric acid which
addition of even twice as much nitrogen nutrient prior
comprises yfermcnting a carbohydrate medium with a
to the beginning of fermentation does not result in the
citric acid producing strain of a fungus, said carbohy
improved rates of citric acid production which are ob- 10 drate medium containing nutrients including a nitrogen
served for additions in accordance with the teachings of
nutrient for said fungus, the improvement which com
this invention.
'
prises adding to said fermenting medium a quantity of a
The following example illustrates the use of ammonium
member selected from the group consisting of ammonia
hydroxide in the process of this invention and the remarkand ammonium salts as a nitrogen containing nutrient
able results achieved thereby.
15 suflficient to furnish nitrogen in a concentration of from
about 50 parts per million to about 5,000 parts per mil
EXAMPLE IV
lion, at a time from about the beginning of the third
The fermentation media were made up according to
day of fermentation to about the end of the seventh day
the process of Example I, except that the media contained
thereof.
17.06% initial sugar, 0.32 parts per million iron, 0.1 part 20
3. In a process for the production of citric acid by
per milliOIl Cll++, 0~1 Part Per million Zn++, and 540
submerged fermentation of a carbohydrate medium with
parts per million of nitrogen as ammonium hydroxide.
a citric acid producing strain of Aspergillus niger, said
The data obtained are shown in Table III.
medium containing nutrients, including a nitrogen nu
Table III
Ammonium
Rate of Citric Acid Production (g. /100 ml. lday)
Hydroxide Added
on Selected Days after Start of Fermentation
Days
p.p.m
3rd
4th
5th 0th
l 7th
8th
9th
10th 11th
0
0
00
540
270
270
1.20
1.42
1.40
0.72
0. 77
1.07
1.45
1.45
1.55
0.75
0.87
1.30
1.35
1.00
1.07
0.00
1. 00
1.42
1.32
1.37
1.50
0. 87
1.17
1.50
1.27
1.32
1.52
1. 22
1.45
1.52
1.17
1.30
1.05
1.12
1.27
1. 50
1.17
1.17
1.57
1.00
1. 25
1.42
0.82
0.77
0.22
1.12
1.25
0.45
540
0.95
1.17
1.32
1.47
1.07
1.07
1.37
0.07 ____ __
270
540
270
540
270
540
1.25
1.40
1.20
1.20
1.32
1.30
1.45
1.52
1.42
1.32
1.47
1.52
1.45 1.52
1.45 1. 00
1.35 1.30
1.37 1.32
1.47 .1.45
1. 20 1.52
1.55
1.70
1.15
1.20
1.20
1.37
1.55
1.77
1. 50
1. 57
1.22
1. 52
1.35
1.40
1.20
1.77
1.00
0.95
0.50
0.42
0.77
0.87
1.15
0.05
These data ShOW Clearly that the rate of citric «acid
Production iS 110t Only Stabilized at an increased rate for
an @Xteflded Period 0f time by the addiüm? 0f ammoni‘fm
0.70
0. 50
0.22
0. 87
0.70
0.07
0.27
0.15
0.22
0.30
0.45
0.20
trient, for said strain of Aspergillus niger, the improve
ment which comprises adding to the fermenting medium
an amount of a member selected from the group consist
hYdfPXld? in accordance with the teaclîmgs 0f, 'd_lîs 11?' 45 ing of ammonia and ammonium salts as a nitrogen con
Vennon’ but~a1s0 that the Tate of Pfoducilç’n of mmc acid
taining nutrient effective to furnish to said fermenting
îlîlatîlilâîta higher level through thls addmon of mtrogen
medium a concentration of from about 50 parts per mil
In summary, this invention is directed to the improved
îîâlî i0 359m 5’000 parts Ber inúhon of mtmgen’ Salld
process for the production of citric acid by the submerged 50 n . no? ‘31mg made at a point m the feimentatwnpyc e
vegetative fermentation of carbohydrates with citric acid
Whlch ls‘whlthuÄ about om? day of the Pomt of mfaxlmum
producing Strains of fungi’ Such as Aspergillus niger’
rate .of c1tr1c acid producuon 1n the absence of sa1d added
wherein a nitrogen nutrient is added to the fermentation
nument-
_
_
medium during fermentation »at a time at which the addi4- The process of claim 1 wherein the mtrogen _c011
tion of nitrogen is effective to increase `and maintain the 55 taining nutrient is an ammonium ion furnishing nutrient.
rate of citn'c acid production. As a result, yields obtained
5. The prÓCeSS 0f Claim 1 Whêrein the nitrogen c011
are greatly improved over yields previously attainable by
the use of known submerged fermentation processes and
fermentation times can be markedly reduced.
taining nutrient isv ammonia.
6~ The process of claim 3 wherein the nitrogen con
taining nutrient is ammonium hydroxide.
What iS Claimed iSï
60
7. The process of claim 1 wherein the nitrogen con
1. ÄIn a process for the production of citric acid which
taining nutrient is ammonium œrbonatg
comprises subjecting a carbohydrate to fermentation with
a citric acid producing strain of a fungus in the presence
of nutrients for said fungus, said nutrients including a
References Cited in the file of this patent
.
nitrogen nutrient, the improvement which comprises add- 65
ing to the fermentation broth a quantity of a member selected from the group consisting of ammonia and am-
UNITED STATES PATENTS
2,394,031
2,910,409
Waksmall et al --------- -- Feb» 5, 1946
Fried et al ------------ -- OCÍ- 27, 1959
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