Professional Documents
Culture Documents
1917 The Citric Acid Fermentation of A. Niger
1917 The Citric Acid Fermentation of A. Niger
1917 The Citric Acid Fermentation of A. Niger
ACID
FERMENTATION
NIGER.*
BY
(From
the Research
JAMES
Laboratories,
Dairy
of Agriculture,
PLATES
(Received
OF ASPERGILLUS
N. CURRIE.
Division,
Washington.)
1 AND
for publication,
United
States Departmeut
2.
April
20, 1917.)
INTRODUCTION.
THE
i6
6 Zahorski,
J. N. Currie
17
described in detail, although general conclusions from such preliminary work may be related.
Results were sometimes obtained
which could not be duplicated.
Such results have not been
included without comment to this effect.
Methods Employed.
18
Mineral
Requirements of Fungi.
carbon-dioxide-free air through the flasks containing the growing mold and absorbing the carbon dioxide produced in caustic
potash.
Diligent search was made for other organic acids, especially
malic and tartaric.
These were never found and in all probability
are never formed at all. The total acidity is nearly exactly accounted for by the sum of the oxalic and citric acids regardless
of the proportion in which they occur.
Both citric and oxalic acids have repeatedly been isolated and
identified.
The oxalic acid can be recovered directly from the
fermented
liquors by evaporation
and crystallization.
Citric
acid because of its very great solubility has been recovered only
through the calcium salt. Several pounds of calcium citrate
have been prepared which by analysis corresponds to the formula
Caa(CaHs0J24H~0 and in no wise differs from calcium citrate
prepared from the citrus fruits.
The citric acid prepared from
this calcium citrate by decomposition with sulfuric acid has sometimes crystallized out anhydrous and sometimes with one molecule of water.
There is some discussion in the literature on the isomerism of
citric acid. Witter* claimed that an anhydrous isomeric citric
.acid was obtained by recrystallizing a sample of ordinary citric
acid which had been dehydrated at 130C. His conclusion was
based on chemical data. On the other hand, Meyer9 concluded
from physicochemical data that the acids were identical.
The
anhydrous acid was the more stable form at high temperatures
and the hydrated acid the more stable form at low temperatures.
The conclusions of Meyer are in all likelihood correct.
J. N, Currie
gm.
..........
..........
.........
.........
..
.
.........
.........
.........
.........
.........
........
........
........
........
........
. ... ..,.
..
. .
,.
.. .
.. .
..
...
..
..
..
..
1,500 cc.
70
4
4
0.6
0.6
0.4
0.25
0.07
0.07
0.07
Distilled water.. . . . .
Cane sugar.. . . . . . . .
Tartaric acid.. . . . . .
Ammonium tartrate..
Ammonium phosphate.
Potassium carbonate.
Magnesium carbonate.
Ammonium sulfate..
Zinc sulfate..
. . ... ..
Ferrous sulfate.. . . . . . . .
Potassium silicate.. . . . .
20
gm.
Distilled water.. . . .
Cane sugar.. . . .
Ammonium dihydrogen
phosphate.. . . .
Potassium chloride......................................
Magnesium sulfate......................................
..
.
. . .
1,000 cc.
30
2
0.2
0.2
The water used in making up the medium and for recrystallizing the salts was doubly distilled and received in flasks lined
All the salts were recrystalwith a pure high melting paraffin.
lized three times in porcelain dishes and finally twice in a platinum
dish. The cane sugar was crystallized five times from redistilled
alcohol and acetone, and finally twice from alcohol in a platinum
dish. To what extent this method has been used to purify saccharose is not known to the writer.
Ethyl alcohol of 90 per cent
strength is saturated at the boiling point with saccharose. When
the solution has cooled, acetone is added until an abundant precipitate of saccharose falls out.
The medium was sterilized in a platinum dish covered with
tin foil and, when cool, poured into sterile paraffined flasks.
With all of these precautions to assure the absence of iron,
Aspergillus niger spored just as abundantly as on a medium of
the same composition to which iron salts were added. Transfers
to this iron-free medium from cultures already grown upon it
likewise showed no impairment
of ability to produce spores
(Fig. 1).
The same cultures are seen in Fig. 2 grown upon a medium
containing iron, but failing in all but two cases to form more than
a few scattered patches of spores. This medium had the following
composition per 1,000 cc.
J. N. Currie
. .
. . ..
... ... ...
... ... ...
... ... ...
. . .
.. . . . .
.. ... ... .. .
.. . . ... .... ..
.... ... .... ... ..
... .... ... ... ...
..
.
. . .
.. ..
....
150
. .
2.5
. .
1.0
...
0.2
. ..
0.01
. . . 19 CC. N/5
of Metabolism
acid+oxalic
of Aspergillus
of Aspergillus
acid+carbon
niger.
niger may
dioxide-tmycelium.
A., Bull.
Torrey
Bot.
Club,
1904,
xxxi,
291.
Saccharose...............................................
NHaNOa..
. .
.
KH2POb..
. . .. . . . . .
..
MgSOa.7HzO.
.. . . . .
FeSOd.7H20.
. . .. . . . . . . . . .
HCl..
. .... .... .... ... ...
21
22
TABLE
Composition
oj Media
NO.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Sacchal-X0.
NaNOa
I
I
100
150
150
50
50
50
50
50
50
50
50
50
50
50
50
50
50
NH&O3
I
NHnH*POd
Asparagine.
I
NaN03
I
1NH,NOs
Gm.
per
Nitrogen.
--
50
50
50
in
I,
KHaPOa
--
1.5
2.0
3.0
3.0
2.5
2.5
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.5
2.5
1.2
1.2
1.0
1.0
1.0
1.0
1.0
1.0
1,000
KC1
0.5
0.5
0:2
0.2
0.2
0.2
1.0
1.0
2.0
2.0
0.2
0.2
1.0
1.0
1.0
1.0
Cc.
f$.
0.2
0.2
0.5
0.5
0.25
0.25
0.4
0.4
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
--
2%3
0.01
0.01
0.01
0.01
0.01
0.01
0.01
We commonly think of respiration as an oxidation which proceeds with the consumption of oxygen and the elimination of
carbon dioxide. Throughout
the plant kingdom there are
numerous instances where this respiratory process stops short of
carbon dioxide, and results in the formation
of the common
organic acids, tartar& oxalic, citric, malic, and succinic. All of
J. N. Currie
23
TABLE
Products
No.
--
of Metabolism
of
culture.
-_
II.
Age.
Carbon
dioxide.
in
niaer upon
!l%ble i.
-
h3lic
o.f Media
-
ah 1. t3tric
--
--
60 Cc.
ach 1. ABycelium.
-_
-_
dagys
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
* Not
142
142
142
142
28.7
28.7
142
69.4
28.7
28.7
28.7
28.7
28.7
28.7
28.7
28.7
69.4
69.4
69.4
69.4
1.797
2.126
2.268
3.468
6.632
5.926
1.686
1.707
1.353
1.530
1.558
1.582
1.482
1.410
*
*
8
9
8
8
8
8
8
8
7
7
7
7
7
7
7
7
8
8
7
7
2.378
2.094
2.053
2.029
-
1.254;
I.3638
1.5514
1.9757
1.2061
1.4356
0.7809
0.6348
0.6345
0.6743
0.7288
0.7368
0.7835
0.7905
0.5254
0.6785
1.7715
1.3169
1.2719
0.9605
0.4914
0.6048
0.6401
0.4133
0.0945
0.0158
0.5353
0.2041
0.0869
0.1310
Trace.
I
I
0.1739
0.0756
*
*
0.1220
0.0439
0.0000
0.5812
3.5000
3.2906
0.2907
0.5801
0.3139
0.5012
0.4221
0.4475
0.3342
0.3106
0.4116
0.7028
*
*
0.2167
0.1083
0.3038
0.2828
-
0.372
0.520
0.641
0.775
0.969
0.985
0.525
0.481
0.417
0.380
0.507
0.498
0.475
0.477
*
*
0.679
0.597,
0.636
0.629.
determined.
only the major or more obvious reactions with which they are
concerned. Many of the students of the chemical activities of
Aspergillus niger have taken account of only one or at most two
of the products of metabolism.
The stimulating effect of various
substances has been repeatedly studied by determining only the
weight of the mycelium.
In, this study an effort has been made
J. N. Currie
25
Lime juice
Lime
juice
Lemonjuice
Lemon
juice
yeasts
yeasts
Vinegar
3
Wine.s
BbuLgaricus
4
Beers
B.coli
Streptococci
Water
8
TEXT-Fb.
1. pH of biological fluids.
*HCl
AskiTer
AskiTer
26
m.
Water...................................................
1,OOOcc.
Saccharose..............................................
50
NaNO *...........................................
.. ... ..
2
KHaPO ~.......................,........................
1
MgS04.7HzO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.2
Acid to designated pH.
Clark and Lubsl5 give the hydrogen ion concentration of a culture of Aspergillus niger they examined as 2 X 10-Z N (pH 1.7).
They did not attempt to determine a limit.
This result together
with the fact that the cultures readily produce a solution containing 10 per cent of citric acid shows that the limiting hydrogen ion
concentration is very high in comparison with most organisms.
It is of the same order of magnitude as that of lime and lemon
juice (Text-Fig. 1).
Ten cultures of Aspergillus niger and one culture resembling
Wehmers Citromyces p.fefferianus were inoculated into media
made up to definite hydrogen ion concentrations with three acids,
citric, oxalic, and hydrochloric.
The hydrogen ion concentration
was determined by the calorimetric scheme of Clark and Lubs.16
The media employed had the following composition :
J. N. Currie
27
TABLE
Inhibiting
Effect
ofHydrogen
Hydrochlcric
Ion
III.
Concenlralion
acid.
on Aspcrgillus
Odie
niger.
acid.
Citric
--
acid.
96 .
+
+
? +t fS
+
+ t+++
+
++
++
+t
74 . . ++
+
+
+ +t
+ t+++
+
+
++
+
+
I
+
?
49 . . ++
+
+
+
+
++
+t
+
+ t+++
0
?
0 t+
50 . . . ++
+ +
0 +
+
?
+
+
? 0 + +
?
0
57 .
+ +
+
+
++
+
?
I
?
?
0
++y
? + +
?
69.4..
++
++
+
0
0
0
47 . . ++
++?
0 +
+
0
+
?
I
0
0
0
111 . . ++
++
0
0 +
+
+
+
+
C
?
?
?
0
142 .
++?
? +
0
++
+
?
+
Penicillium
45
+ ++ tt
++
++
++
+
+ t+ ++ ++
+
- + indicates positive growth; ? indicates germination;
0 indicates no development.
_
In& and
Eng. Chem.,
0.0
-
1.4
+
+
?
?
?
?
?
0
?
0
+
-._
had previously been selected as the best one for the citric acid
fermentation.
A glance at the columns representing the pII and the per cent
of acid present shows clearly the importance of considering the
hydrogen ion concentration.
The titratable acidity of the medium
containing the citric acid at pH 1.4 is 62 times that of the medium
containing the hydrochloric acid and having the same pH and the
same inhibiting
effect. In this connection the ,writer cannot
resist the temptation to point out the possible bearing of these
facts on the problem of preserving the juice of the citrus fruits.18
28
I9 Clark
and
2o Hallerbach,
25.
Lubs,
W.,
J. &act.,
1917, ii, 219.
Die Citronenstiure
and
ihre
Derivate,
Berlin,
1911,
Wehmer2 states that one of the chief reasons for the failure of
his process was the difliculty encountered in preventing his media
from becoming contaminated
with organisms which interfered
with the citric acid fermentation.
A glance at the tables given
by Clark and Lubslg will show that few bacteria will grow below
a pH of about 3.5. It would require at least 10 per cent of citric
acid to lower the pH from this point to the region where the
hydrogen ion concentration would begin to interfere with growth.
It is therefore clear that the fermentation can be started off at a
hydrogen ion concentration that will greatly reduce the chances
of infection.
This is the best argument against combining the
acid in the form of calcium citrate as the fermentation proceeds.
Wehme? states that the yield of citric acid was increased by adding calcium carbonate for some of the citric acid was thereby removed from the field of action and could not be consumed by the
growing mold. This observation is not in accord with the experience of the writer.
Higher yields have been obtained and in a
shorter time in the absence of calcium carbonate.
When the
fermentation
proceeds properly there is little consumption of
citric acid as long as sugar is present.
Even if calcium carbonate
were present the substrate in immediate contact with the mold
mycelium would contain free citric acid or calcium citrate in solution and the chance for the consumption of the products of fermentation would not be materially lessened.
The element of time should also be taken into consideration.
Hallerbach20 states that Wehmers process failed because it required too long a time on a technical scale. The fermentation
proceeds much more rapidly in an acid medium than in one to
which calcium carbonate has been added. This is probably partly
due to the reaction and partly to the disturbance of the equilibrium of salts in solution in the medium.
The magnesium and
phosphate radicles, both of which are essential to the growth of
the mold, would be at least partially precipitated.
There is also a possibility of recovering the citric acid directly
from the fermented liquor, without going through the expensive
process of separating and decomposing the calcium citrate.
This
J. N. Currie
. ...
. .,.
. ...
. ..
...
. ..
.
. ..
.
.
.
.
.. .
.. .
. .
... .
.
.
.
.
.
.
.
.
... .
.. .
. .
.. .
.
.
.
.
1,OOOcc.
50
2
1
0.5
0.5
0.01
........
.........
.........
.........
........
........
........
........
.........
.........
.........
. . . . . . . . ..t
.
.,..........
.,..........
. Varying
1,OKL
50
1
0.25
.0.25
0.01
quantity.
30
of Va@ng
Quantitiies
1.42. Results
niger
I kidity
of Nitrogen
Expressed
by titration.
Oxalic
IV.
of Asperof Medium.
acid.
W&high
Citric
acid.
yy-
_-
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
166.9
169.3
162.2
161 .O
152.4
158.2
158.0
152.9
149.2
114.8
-
108.1
130.6
137.5
141 .o
143.5
150.9
152.5
154.9
153.2
124.1
58.3
39.7
27.2
24.4
14.5
14.1
5.7
6.5
7.8
060
gm.
0.398
0.500
0.548
0.634
0.606
0.616
0.643
0.637
0.644
0.695
.....................
.Varying
gm.
1,000 cc.
..
50
.
2.0
.
0.2
quantity.
Effect
gillus
gm.
Water...................................................
Saccharose.............................................
NaNOa..
_. . . . . . . . . . . . . . .
KHePOh..
.... ... ... ... ... .
KC1 . . . . . . . . . . . . .
.. . .
FeS04.7Hz0.
... ... ... . . .
MgS01.7HzO
.. .. . . . . . . . . . . .. . .
...
. .
. .
.
... .
. ...
.. ...
. ..
Expressed
Acidity
by
titration.
Fermentation
of As60 Cc. of Midium.
on ths Acid
in ~/lo
Cc. per
Oxalic
--
acid.
Citric
acid.
--
--
gm.
Wm.
0.2
0.4
0.6
115.2
136.4
127.4
52.8
64.6
55.2
45.6
55.7
52.0
0.335
0.457
0.382
69.4
0.2
0.4
0.6
110.4
118.8
97.6
0.0
0.0
14.4
94.3
101.5
63.0
0.535
0.520
0.494
28.7
0.2
0.4
0.6
143.2
120.4
119.2
10.0
6.0
6.8
117.1
98.2
93.8
0.631
0.543
0.522
74
0.2
0.4
0.6
159.4
124.8
151.2
17.6
22.8
23.8
133.8
81.6
lO9.2
0.498
0.572
0.492
142
The cultures were examined when 5 days old. .From the results it appears that the most favorable quantity of MgS04.7Hz0
for a medium of the above composition lies. between 0.1 and 0.2
gm. per liter (Table VI).
-
TABLE
Effect
of Varying
Aspergillus
Quantities
niger
14.8.
Results
of
M&O4
titra-
VI.
of MgSO4.7H20
Expressed
Medium.
Oxalic
acid.
on the Acid
E&mentation
in ~/lo
Cc. per 60 Cc.
Citric
gm.
0.01
0.05
0.10
0.15
0.20
0.25
0.30
0.40
acid.
Weid&;p1y-
am.
31.3
72.0
103.5
112.8
93.3
94.3
89.0
82.5
31.0
75.0
109.8
103.8
83.3
86.8
78.0
69.5
31
Trace.
2.1
10.8
9.4
16.5
8.8
8.6
0.123
0.394
0.521
0.551
0.547
0.460
0.456
0.436
of
culture.
--
V.
of Potassium
Quantities
Results
. . . . . . . . . . . . . . . .Varying quantity.
TABLE
Effect
of Varying
pergillus
niger.
1,OOOcc.
50
2
1
0.25
0.01
32
..
. .
MgS04.7HzO.. .
KC1 .
..
. .. . .. . .
FeS0,.7Hz0. .. . .
.. .
.
.
.. . . .
.. . . ... ... ..
.
. .. .. ... .. ..
50
1
3
0.25
0.25
. . . . . . . . . . . . . . . . . . . . . . Varying quantity.
The cultures were examined on the 7th day. No determinations were made other than total acidity.
The results are shown
below (50 cc. of medium were employed).
1,000 cc.
50
.
.
. .. ... . .
2
KH2PO,................................................
1
MgSOa.7HzO..
.
. . . . . . . , _. . . . . . . . . . . . . . .
0.5
salts.
+ 0.5 KCl.
+
t-o.5
+ 0.,5 NaCl.
0.01
FeS04.7Hz0.
Saccharose.............................................
KHzPO4................................................
J. N. Currie
33
The results in Table VII show that the duplication of the potassium radicle and the introduction of chlorides and iron are not
only unnecessary but in most cases even unfavorable to the acid
fermentation of Aspergillus niger.
Expressed
No. of
medium.
Culture.
--
in
( hlic
Acidity.
--
VII.
Cc. ~/lo
acid
?r 60 Cc.
Citric
-Of
acid
--
Medium.
Weight of
Oxalic and I
mycelium.
citric.
_
_
_
148.8
142.8
152.0
137.6
91.0
103.6
118.6
88.6
61.8
44.0
40.8
60.4
152.8
147.6
157.4
149.0
om.
0.506
0.469
0.495
0.486
135.6
111.4
79.6
103.0
41.4
38.0
42.4
42.0
94.8
83.5
42.9
75.5
136.2
121.5
85.3
117.5
0.462
0.472
0.454
0.461
140.0
82.8
78.6
92.8
67.0
43.2
32.6
37.0
66.1
35.7
47.9
51.9
133.1
78.9
80.5
88.9
0.511
0.521
0.570
0.532
74
115.4
39.2
45:4
44.4
72.0
27.0
32.0
34.8
47.3
10.4
19.0
12.4
119.3
37.4
41.0
37.2
0.438
0.458
0.493
0.438
96
91.2
78.8
71.2
81.4
44.4
34.0
34.4
36.8
45.9
44.3
42.1
43.0
90.1
78.3
76.5
79.8
0.597
0.549
0.617
0.591
142
69.4
28.7
.2
3
4
TABLE
Results
34
125 -150
.
. . 2.0 -2.5
~ . . . . . . . . . 0.75-1.0
. . . . . . . 0.20-O. 25
21 Wehmer,
C.,
U. S. Patent
No.
515,033,
Feb.
20, 1894.
The medium proposed by Wehme+ contained a small quantity of mineral salts (NHdNOs, KH,POI, and MgS04) . Zahorski5
used small*amounts of nutrient salts such as ammonium nitrate,
potassium phosphate, and magnesium sulfate.
The addition of hydrochloric acid to the mixture of inorganic
salts proposed above has several advantages.
It raises the hydrogen ion concentration to a point that makes complete sterilization possible at a single heating in steam at atmospheric pressure
for 30 minutes.
As previously pointed out, it greatly reduces
the dangers of infection of the liquors with organisms which
might interfere with the citric acid fermentation without inhibiting
the growth of the mold with which the liquors are inoculated.
Many fermentations have been conducted in Erlenmeyer flasks
on this medium.
The general course of the fermentation is quite
similar. There is little development of acid during the first 2
or 3 days. When a vigorous mycelial felt has developed the rise
in acidity is very rapid, about 2 per cent in 24 hours, until the 7th
or 8th day. After remaining nearly constant for 2 or 3 days the
acidity begins to decline. When the. fermentation
proceeds
properly the mold does not spore but remains white as shown in
Figs. 3 and 4.
The course of the fermentation
under varying conditions is
shown by the curves in Text-fig. 2. Curve I shows the course of
the development of acid in a 12.5 per cent cane sugar solution.
100 cc. of the medium were contained in a 300 cc. Erlenmeyer
flask. 5 cc. were removed at intervals and the acidity was determined by titration.
Curve II shows the development of acid
in a 15 per cent cane sugar solution.
Curve III shows the course
of the fermentation in a shallow pan (Fig. 5) containing 1 liter
of a I5 per cent cane sugar solution.
The mycelium had been
developed in a previous fermentation and the rise in acidity began
at once.
J. N. Currie
35
14
12
246
16
18
20
22
.2
cdntend with, aside from the infection of his liquors with undesirable organisms, was the varying fermentative
power of his cultures (Variabilitdt des Garverm6gens). Throughout this study it
has been emphasized that a complex biological reaction is involved
which results in a number of products and which cannot be expressed by a simple equation, representing the oxidation of a
sugar to citric acid. All of the coriditions that majr influence
the course of the fermetitation are not within the control of the
experimenter.
Good results can be had only by the adoption of
conditions that prove successful and can be duplicated with a
high degree of uniformity.
per cent
1
2
3
11.54
11.51
ll.ocl
Elm.
3.8789
3.8209
3.7673
per cent
per cent
15.52
15.28
15.07
10.35
10.19
10.05
per cent
3.95
4.69
5.93
OF
PLATES.
1.
FIG.
1. Ten strains of Aspergillus niger. Showing sporification
on a
medium coritaining no iron.
FIG. 2. Ten strains of Aspergillus niger.
Showing failure to spore on a
medium containing iron.
ff Ehrlich,
While most of the experiments have been conducted in Erlenmeyer flasks of various sizes, the fermentation
can be conducted
with equal success in shallow pans. 1 liter of a 15 per cent saccharose solution was fermented in each of three pans (Fig. 4).
The fermentation
was continued for 8 days. About 800 cc. of
liquor could be recovered from each pan by pressing out the mycelium in a hand filter press. Analytical data on the liquid from
each pan are shown in the table below:
J. N. Currie
PLATE
37
2.
FIG. 1.
Fermentation.)
Acid
Citric
(Currie:
1.
PLATE
XXXI.
VOL
CHEMISTRY,
OF BIOLOGICAL
JOURNAL
THE
THE
JOURNAL
OF BIOLOGICAL
CHEMISTRY,
3.
FIG.
4.
FIG.
5.
XXXI.
PLATE
2.
FIQ.
VOL.
Khnrie:
James N. Currie
J. Biol. Chem. 1917, 31:15-37.
ARTICLE:
THE CITRIC ACID FERMENTATION
OF ASPERGILLUS NIGER