Note: Descriptions are shown in the official language in which they were submitted.
654
Process ~or the production of citric acid
~or a long time whey has been used for animal feed. More recently owing
to, among other things, the development of methods for ultracentri~uga-
tion of whey, protein has been separated from whey which ~eana that ~ ~ -
large amounts of whey permeate are obtained. This permeate has a very~
low protein content whlle the concentrations of lactose ànd salts are
:
about the same as in whey. Whey permeate,with its low protein contents,
~; is considered today as waste and as it has a great biological oxygen
consumption BS (BOD7) it is a waste being expensive to treat.
'
~o~ surprisingly, it has been found that the high lactose contents in
whey permeate can be used in a new process for the production of citric
, . . .
acid.
.
- : '
.
~ : :
- -- ..... .. .
5~
- ; This i~nventlon relates to pyruvie aeid and
eitrie aeid produetion and micro-organi.srs for use therein.
The invention in partieular provides a mierobiological
proeess using a laetose carbon source for produeing pyruvie
aeid and a further microbiological process (using a pyruvie
aeid-containing cultivation medium) for producing citrie
acid. Accordingly the invention can provide an overall
scheme for the production of eitrie acid from a laetose
~earbon` souree.
For a long time whey has been used for animal feed.
Reeently, as a result of, among other things, the develop-
ment of methods for ultrafiltration of whey, protein has
been separated from whey whieh means that large amounts of
whey permeate (i.e. the liquid left when protein has been
separated by ultrafiltration from whey~ are obtained. This
permeate has a very low protein eontent, but the eoneen-
trations of laetose and salts are about the same as in whey.
~hey permeate, with its low protein eontent, has hitherto
been regarded as waste. Sinee during treatment of it as
waste it has a high biologieal oxygen eonsumption, such
treatment is expensive.
Now, surprisingly, it has been found that whey perm-
eate, with its high lactose content, can be used in the
production of pyruvie aeid and/or eitrie acid by microbio-
logical means.
~` :
654- `
Citric acid, the final product o~ the present overall
scheme, is used to a lar~e extent in the FGod
industry as a flavouring substance and it can also be used
to a certain extent in the pharmaceutical industry and in
other chemical-based industries. Thus, for example, citric
acid can be used as a complexing agent in cleaning com~ounds
and for stack gas clèaning.
The presently utilised methods for producing citric
acid are quite old. ~he most common method is still the
surface fermentation of As~ergillus Niger in open vessels
using mGlasses as a carbon'source. Commonly molasses from
sugar-beet are utilised. However, there are also modern,
submerged methods (with or without continuous cultivation),
but these have only been put to commercial use in a few
cases.
Nearly all methods of manufacturin~ citric acid used
commercially today are based on the use of saccharose as a
carbon source, mostly in the form of molasses. ~owever, on
a smaller scale cellulose hydrolysates and starch have been
used as carbon sources. Also in U.S. Pa~ent Specification
No. 3,717,549 a method for the production of citric acid is
described in which a citric acid accumulative fermentation
strain is fermented under aerobic conditions. ~s suitable
carbon sources, starch, molasses, saccharose, glucose,
maltose, dextrin, fructose or galactose are suggested.
'~tL~ ~ '
,
; ! . ~
,
'
"', ' '
r~
.
Lactose is a form of sugar which most~v occurs in ~ilk.
~owever there are only a few micro-organisms which can use
lactose as a carbon source. These micro-organisms usually
occur in milk and they have ability to form ~-galactosidase..
. 5 It will be appreciated that there would be substantial
advantages in being able to utilise lactose in whey or whev
permeate as a carbon source in the manufacture o~ citric
acid (or pvruvic acid) as compared with the use of other
types of sugar or hydrolvsates of cellulose or starch as
carbon sources, in view of the fact that whey permeate is
now regarded only as a waste product.
As alreadv indicated, in the use of lactose as a
carbon source in the production of c~tric acid, the lactose
has to be used initially in the production of pyruvic acid
which can then be used to produce citric acid. One specific
problem with the~icrobiolo~ical process is the achieve.~ent
- o~ an accumulation of pyruvic acid in the culture medium in
such a way that it can be used to produce citric acid by a
subsequent microbiological process. Thus, the transform-
ation of pyruvic acid to C02 is preferably avoided as muchas possible.
According to one aspect of this invention there is
provided a process for the production of pyruvic acid which
comprises cultivatiny on a lactose-containing cultivation
medium a strain of Escherichia Coli ~hich is capable of
I
- '
'
3659L
forming pyruvic acid from a lactose carbon source and
accumulatlng said acid~ cultivation bein~ effected for a
sufficient time to form and accumulate pyruvic acid and, at
leas for a first part of that time, under aerobic conditions.
P~ruvic acid produced by the above process is one
possible source of pyruvic acid for use in another aspect
of the invention, which aspect is a process for the E~r~duc-
tion of citric acid which comprises cultivating on a
pyruvic acid-containing cultivation medium a strain of
Hansenula Wickerhamii which is capable of forming citric
acid from pyruvic acid, cultivation being effected under
conditions inhibiting metabolism of pyruvic acid to carbon
dioxide, and thereafter recovering citric acid from the
resulting cultivation broth. The cultivation broth
resulting from the process which is the first aspect of the
invention may be used as the cultivation medium for H.
Wickerhamii in the aforesaid second aspect.
This invention also naturally includes the products of
such processes.
In the first aspect of the invention it is highly
preferred that the strain used is E. Coli KG 93, F or a
mutant or variant thereof. A description of this new
organism is given hereinafter.
In the second aspect of the invention it is highly
~5 preferred that the strain used is H. I~ickerhamii CBS 4308
or a mutant or variant thereof~ ~ description of this new
--5
:
.
.
is~
.~ ~
organism is als~ ~lven hereinafter.
Preferred features of the present invention are illus-
- trated by a process for the production of citric acid in
which E. Coli KG 93, ~ is cultivated, in a first step, for
from 15 to 24 hours at a tem~erature of from 20-37C and at
a pH of 5.0 - 7.5 on a substrate consisting of whey permeate
to which has been added phosphates in an amount of from 0.8-
1.6 g/l and/or nitrates in an amount of from 0.8-1.2 g/l
and/or urea, and in which H. Wickerhamii CBS 4308, in a
second step, is cultivated for from 20-26 hours at a
temperature of f-~, 15-35C and at a pH- of from 4.5 - 6.5
on the cultivation broth resulting from the first step,
citric acid subsequently being isolated from the final
cultivation broth, for example, in a manner known per se.
The amounts quoted herein, in concentration terms, for
nitrate, phosphate~cyanide or other additional ingredients
which may be added to the cultivation media refer to weights
of compounds per unit volume in the resulting media.
A suitable substrate which can be used for the original
cultivation is a whey permeate having the average compos-
ition given below:
Per cent by weiqht -
fat 0.10 - 0.30
protein 0.01 - 0.1
lactose 4.5 - 5.5
--6--
,: . . - -,
,
65~
salts 0.2 - 0.8
lactic acid 0.05 - 0.15
and the remainder consisting of water.
Since there is often a deficiency, mostly of nitro-
genous compounds, in such a whey permeate substrate, it is
preferred to complement the substrate with nitrate or urea.
A single nitrate or a mixture of two or more nitrates can
be used for this purpose. For example, potassium nitrate
is a suitable compound. Furthermore, phosphorus~is prefer-
ably added to the substrate, most suita~ly in the form ofa phosphate such as KH2P04, K2HP04 or NaNH4HP0~. However,
other phosphates and phosphorus compounds might be used.
The substrate is preferably complemented so that the con-
centration of phosphate will be from 0.8 - 1.6 g/l,
preferably 1.2 g/l, and the concentration of nitrate from
0.8 - 1.2 g/l, preferably 1.0 g~l. Urea can be added in-
stead of nitrate.
~he preferred strain of micro-organism (Escherichia
Coli KG 93, ~ as denosited with the Microbiology Department
of the Universitv of UMEA, Sweden) used in the first step
of the two ste~ process described above is selected for its
abilitv to form pyruvic acid while the preferred strain of
micro-organism (Hansenula l~ickerhamii CBS 4308) used in the
second step is selected for its ability to form citric acid.
Suitably each separate cultivation is carried out
~...,~, I
... . . .
: '
~ ;y~ s~
continuousIy. ~hen transferring the cultivation liquid
from the first step to the second step, it is not necessary,
as a rule, to destroy the E. Coli organism. Thus, often
there is a difference in temperature and pH bet~een the two
steps, which has the effect that transferred cells of E.
Coli cannot com~ete with H. Wickerhamii. Howe~er, if
- desired, E. Coli can be destroyed after the first step.
It is especially advantageous to carrv out the E. Coli
cultivation at a temperature of about 37C and at a pH of
about 7. Conveniently, the- E. Coli cultivation is efEected
under aerobic conditions for about the first two-thirds of
the cultivation time, cultivation then being effected under
anaerobic conditions or without supply of further oxvgen.
Thus, air may be supplied to the E. Coli cultivation for
about the first 2/3 of the cultivation time, the supply of
air to the cultivation then being terminated. Generally,
the E. Coli cultivation step has a duration of about 18
hours.
It is preferable to have a temperature of about 30C
and a pH of about 5 for the H. I~ick`erhamii cultivationO
The pH during the two cultivations can be regulated in
various ways, e.g. ammonia gas can be used.
The transformation of pyruvic acid into C02 in the H.
~ickerhamii cultivation can be prevented by inter~ering
with the aerobic metabolism of the micro-organism. This
~1
-
s~ ~
can be achieved, for example, by adding cyanides to poison
the breathing of the micro-organisms during the H. Wicker-
hamii cultivation, by decreasing the amount of oxygen or by
adding aneromycin. Thus, advantageously an addition of,
for example, 0.1 - 0.5 g/l, of cyanide, for example in the
form of ferrocyanide or cyanoacetic acid, may be made to
the H. ~ickerhamii cultivation. However, as indicated above
aneromycin can be used instead of cyanides.
Citric acid can be recovered from the H. T~ickerhamii
cultivation in any convenient way. Thus, for example,
precipitation with calcium carbonate or calcium hydroxide
and su~se~uent filtration can be used.
The invention will now be exp'ained in more detail bv
means of specific Examples which relate to two stage
processes. Nonetheless, process information is given for
the separate cultivations.
Example 1
A whey permeate having ~he following content was used:
per cent by weight
fat 0.20
protein 0.05
lactose 5.0
lactic acid 0.1
salts 0.6
and the remainder consisting of water.
~J
-,
.' . :
54
The above 0.6 per cent by wei~ht of salts was made up
in the following way: ~
per cent by weight
Calcium Salts 0.06
Phosphorus Salts 0.06
Potassium Salts 0.16
Nitro~enous Salts 0.03
Sodium Chloride 0.3
The permeate was complemented with 1.2 g/l of phos-
lo phates and l.o 9/1 of nitrates.
- The complemented whey permeate was pumped into a
continuous fermentor (more particularly, a fermentor sold
under the registered Trade Mark CHEr~P~ which was provided
with a turbine agitator. The solution was inoculated with
15 - Escherichia Coli KG 93, F (as deposited with the Micro-
biologv Department of the Universitv of UMEA, Sweden). The
resultant cultivation was maintained at a temperature of
37C and at pH of about 7. The pH was kept at a stable
value by means of ammonia qas. Air was supplied from a
compressor to the cultivation.
After approximately 12 hours of cultivation, the air -
supply was stopped completely to achieve concentration of
pyruvic acid. After approximately another 6 hours of
cultivation without air supply, the liquid from the fermen-
tor was pumped continuously to another fermentor of the
--10--
:. .
6~
.
.. same type and thexe it wa5 inoculated with Hansenula
Wi'ckerhamii CBS 4308. This second cultivation was effected
at a temperature of 30C and at a pH of abou* 5. The
second cultivation was effected for about 24 hours and the
pH kept at a stable value by means of ammonia gas. In
addition, 0.3 g/l of ferro-cyanide was.added to the second
cultivation. .'
The yield of pyruvic acid from the first cultivation
was 32 g/l. The yield of citric acid after the whole
process was 43 g/l~
Example 2
The process according to Example 1 was repeated with
some modiications. Thus, the first cultivation (with
E. Coli KG 93, F ) was effected at a temperature of 32 C
and at a pH of 6.5. The first cultivation was carried out
for 16 hours with an air supply and then for 8 hours
without an air supply. The second cultivation (with H.
Wickerhamii CBS 4308) was carried out for 23 hours at a
temperature of 25C and at a pH of 5.5. The yield of citric
acid was 32 g/l.
Example 3
The process accordin~ to Example 1 was repeated with
some modifications. Thus, the first cultivation was effec-
ted at a tem~erature of 37C and a pH of 7.5. The cultivation
was carried out ~or 14 hours with an air supvly and then for
. ' , : . ~
: ' ` . . ~ , ' ,''
- . . -, ~ ~
- . . ~ : :
.
65~
7 hours without an air supply. The second cultlvation step
was carried out for 22 hours at ~ temperature of 30C and
a pH of 5.5. No addition of ferrocyanide was made. The
yield of citric acid was 42 g/l.
Example 4
The process according to Example 1 was repeated with
some ~odifications. Thus, the first cultivation was effec-
ted at a temperature of 37C and a pH of 6.5. The cultiv-
ation was carried out for 16 hours with an air supply and
then for 8 hours without an air supply. The second culti-
vation was carried out for 24 hours at a temperature of
25C and a pH of 5Ø The yield of citric acid was 50 g/l.
Example 5
The process according to Example 1 was repeated with
some modifications. Thus, the first cultivation was
effected at a temperature of 32C and a pH of 6.5. The
cultivation was carried out for 16 hours with an air supply
and then for 8 hours without an air sup~ly. Furthermore,
the second cultivation was carried out for 24 hours at a
temperature of 25C and a pH of 6.5. The yield of citric
acid ~as 1.6 ~/1.
The ne~ micro-or~anism Escherichia Coli KG 93, F as
referred to herein has also been de~osited at Delft University
of Technology, Laboratory of Microbiology, Delft, Holland
under the deposit number LMD 7853. The same micro-organism
-12~
. -
- . ~ . '
- ~ ~ ,.
' . ~ .
5~
.
,. has also been deposited wlth the Ger~an Collection of Micro-
organisms (DSM~ under the number DSM 1392 and is therefore
.also known (from this source) as E.` Coli DS~ 1392. Hansen-
ula Wickerhamii CBS 4308 has also been deposited with DSM
1380 and can therefore also be referred to as H. ~ickerhamii
DSM 1380.
The invention includes a micro-organism of the species
- E. Coli capable of forming pyruvic acid from a lactose
carbon source and accumulating said acid and having the
morphological and biochemical and physiological properties
described hereinafter. The invention further includes a
composition suitable as a cultivation medium for carrying
out a process for the production of citric acid using H.
Wickerhamii as referred to above and comprising a cul~iva-
tion broth produced by cutlivating E.' Coli as referred to inthe preceding paragraph in a cultivation ~edium containing
a lactose carbon source.
The E.''Coli.micro-organism of the invention has the
following properties:- ,-
I Morpholo~lcal properties
A. ' Surface icolonies- on solid me'dia
1) Shape : circular
2) Size : 2-3 ~m diameter
3) Colour : yellow
4) Opacity : opaque ..
5) Elevation : flat
6) Surface : smooth
7) Edge : entire
-13-
...~ I
J
.
:,
'
P65;4
.
- . 8) Emulsifiability : forms unifprmly turbid
-suspensions
9) Odour.: absent
B. Broth Cultu're
1)- Amount of yrowth : profuse
2) Turbidity : uniform
C. ' Characters
1) Gram reaction : negative
2) Shape : straight rods
3) Motility : no
II 'Biochemical'and physi-ol'ogi'ca'l`'Droperties
.
Resistant to streptomycin A
The invention also includes a micro-organism of the
species H.' Wick'erh'amii capable of formin~ citric acid from
a pyruvic acid carbon source and having the morphological .
and biochemical and physiological properties described
hereinbefore.
The'H. Wickerh'amii micro-organism of the invention ~.
has the following properties:-
orphol'oqica'1' properties
. .
` A. Surfa'ce''colo'nies on''solid me'dia
1) Shape : circular
2~ Size : 2-3 mm diameter
~1
`: `~ ~ ; `
`
;
.
3) Colour : white'
4) Opacity : opaque
5) Elevation : flat
' 6) Surface : smooth
7) Edge : entire
8) Emulsifiability : forms uniformly turbid
suspensions
9) Odour : ahsent
.. . .
, B. - Broth'~ulture
1) A~ount of growth : moderate
2) Turbidity : uniform
II Biochemcial and physiologica'l'properties'
1) Cannot ferment lactose
2~ Can use pyruvate as carbon source
1~ 3)' Can ferment glucose
r~ I
