Jotrm~ oF Bxoscmrcc~.ANDBmE~rOmEErU~G
Vol. 91, No. 4, 382-389. 2001
Effect of Consumed Carbon to Nitrogen Ratio on Mycelial
Morphology and Arachidonic Acid Production in
Cultures of Mortierella alpina
YASUHISA KOIKE, l H O N G JIE CAI, 1 K E N I C H I HIGASHIYAMA, 2 SHIGEAKI FUJIKAWA, 2
AND E N O C H Y. PARK 1.
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka
University, 836 Ohya, Shizuoka 422-85291 and Institute for Fundamental Research, Suntory Ltd., 5-2-5
Yamazaki, Shimamot o-cho, Mishima-gun, Osaka 618-0001, 2 Japan
Received 30 October 2000/Accepted 12 January 2001
The influence of the consumed carbon to nitrogen (C/N) ratio on arachidonic acid (AA) production and
mycelial morphology was investigated in cultures of Mortierella alpina using shake flasks and a fermentor. The
consumed C / N ratio was varied from 5 to 32 under the condition that the total initial amount of carbon and
nitrogen sources was 50 g/l. Cellular yield increased markedly at C / N ratios below 7; carbon utilization was
switched from cellular growth to lipid biosynthesis in the C / N ratio range of 7-15; lipid biosynthesis was most
active when the C / N ratio was in the range of 15-32. However, for C / N ratios higher than 15, the mycelial
concentration decreased due to nitrogen limitation but the lipid yield still increased. In the presence of excess
nitrogen, the biomass concentration depended on the amount of the nitrogen source, but the AA yield was
inversely related to this. On the other hand, in the presence of excess carbon, the fatty acid concentration
increased with carbon source concentration but the A A concentration remained constant. From the viewpoint of
A A production, the optimum C / N ratio was in the range of 15 to 20 with a balance between the amounts of
carbon and nitrogen sources. When an enriched medium was used at a fixed C / N ratio of 20, the cellular and
AA concentrations were shown to be proportional to the total concentrations of carbon and nitrogen sources
in both flasks and the fermentor. The whole pellet size and width of pellet annular regions did not change with
increasing C / N ratio for C / N ratios below 20 in the flask cultures. However, when the C / N ratio was higher
than 20, these sizes increased in proportion to the C / N ratio.
[Key words:
Mortierellaalpina, arachidonic acid, mycelial morphology, C/N ratio]
natural nitrogen source for cell growth during a long
period of cultivation. Therefore, the kinetic models of
the consumed C / N ratio described previously (4, 13-15)
are not very appropriate to industrial cultivation.
The other consideration is the morphology of filamentous microorganisms, which usually varies between
"pellet" and "filamentous", depending on the culture conditions and the genotype of the strains. In the case of
Mortierella ramanniana, producing ~'-linolenic acid, a
higher amount of lipid was accumulated when fungus
exhibited the pellet morphology than when it exhibited the
filamentous morphology (16). To evaluate mycelial morphology quantitatively, Paul and Thomas (17) reviewed
some of the image analysis techniques used to quantify
the morphology of microorganisms from fermentation
samples, and describe some of the parameters used for
characterizing morphology, such as size, shape, roughness, and cell volume. Using image analysis morphology
can be characterized automatically (18). We have also
investigated the effects of shear stress on morphological
change in Streptomyces fradiae culture and relationship
between mycelial morphology and tyrosin productivity
(19).
In the case of Mortierella alpina cultures the morphology was affected by the dissolved oxygen concentration
(20), mineral addition (21), and the natural nitrogen
source (22). When the dissolved oxygen concentration
was maintained at 20-50 ppm the morphology changed
from filamentous to pellet (20). With the addition of
potassium dihydrogen phosphate the morphology was
Polyunsaturated fatty acids (PUFAs) are abundant in
humans and have an important role in the structure
and function of biological membranes (1). One of the
PUFAs, arachidonic acid (5, 8, 11, 14-cis-eicosatetraenoic
acid, AA), a biogenic precursor of prostaglandins and
leukotrienes, has been the subject of intensive medical
research (2, 3). Polyunsaturated fatty acids such as AA
have been reported to accumulate in ceils, algae, yeast
and fungi (4, 5). Mortierella fungi are known to accumulate AA to approximately 70% of the total fatty acids
(6). In general, it is assumed that PUFAs are accumulated due to a metabolic shift in the Krebs cycle caused by
activation of ATP-dependent citrate lyase. Therefore, a
very important factor affecting lipid over-production is
the metabolic shift caused by nitrogen limitation (7, 8).
Several researchers have reported on the influence of
various culture conditions on lipid production, i.e. composition of the culture medium (9), effect of nutrient
limitation (8), oxygen availability (10), pH and temperature (11). Among these parameters, nitrogen limitation
had a significant effect on lipid content and fatty acid
composition under oxygen-non-limited culture conditions
(12). However, these results were obtained from experiments in while a soluble nitrogen source was used. The
nitrogen sources used in industrial production are natural, and are usually insoluble in liquid media. Microorganisms use the nitrogen leached out from the insoluble
* Corresponding author, e-mail: yspark@agr.shizuoka.ac.jp
phone/fax: +81-(0)54-238-4887
382
VoL 91, 2001
EFFECT OF C/N RATIO ON ARACHIDONIC ACID PRODUCTION AND MYCELIAL MORPHOLOGY
filamentous, but in the case of the addition of sodium,
calcium, and magnesium ions the predominant morphology was of large pellets with diameters of 2-3 m m (21).
We have found that natural nitrogen sources such as
yeast extract, gluten meal, or corn steep liquor resulted
in the formation of circular pellets, but Pharmamedia,
fishmeal, or soybean meal resulted in the formation of
radial filamentous mycelia from a central pellet core
(22). However, despite the strong influence of the consumed C / N ratio on productivity in mycelial culture,
little is known about the effect of the consumed C / N
ratio on the morphology of fungi that accumulate lipids
within their mycelia.
Here, we describe the results of an investigation on
the morphology and AA productivity of the fungus, M.
alpina. The effects of consumed C / N ratio are discussed
with regard to morphological changes and A A productivity. The microscopic observations of the morphology are
described based on the whole pellet size, the pellet core
size, the ratio of the pellet annular region to the whole
pellet size, and the width of the pellet annular region
using image analysis for the characterization.
MATERIALS AND METHODS
M. alpina CBS 754.68
Microorganism and media
was used throughout this study. For the seed culture, the
medium used contained (g/0: glucose, 20; and yeast
extract (Oriental Yeast Co. Ltd., Tokyo), 10. For the
production culture, the medium used contained (g/0:
glucose, 40; soybean oil, 2; soybean meal (SM) (Ajinomoto
Co., Tokyo), 10; KH2PO4, 3; Na2SO4, 1; C a C I 2 . 2 H 2 0 ,
0.5; and MgCIz.6H20, 0.5. To investigate the effects of
consumed carbon to nitrogen (C/N) ratio on the mycelial
morphology and production of lipids and AA, the consumed C / N ratio was varied from 5 to 32 with the constraint that the total initial concentration of glucose and
the SM in batch culture was kept at 50 g/! as shown in
Table I. When the glucose concentration was fixed at
40 g/l, the consumed C / N ratio was varied from 10 to
24. To investigate the effect of glucose, the C / N ratio
was varied from 13.4 to 38.6 while also changing the initial SM concentration. These media conditions are listed
in Table 1. The pH of the media used was adjusted to
6.0 before sterilization.
Culture conditions
A spore suspension of M. alpina
CBS 754.68 maintained on agar slants at 103spores/
ml was inoculated into a test tube containing 10ml of
seed medium. The spores were grown at 28°C on a
reciprocal shaker at 120 spm (stroke per minute) for 30
h. For A A production, 5 ml of the seed culture was inoculated into a 500-ml Erlenmeyer flask containing 45 ml
of the production medium. The culture was carried out
at 28°C on the reciprocal shaker until the glucose was
exhausted. All cultures were carried out in triplicate and
the results were represented as an average value.
In the case of jar fermentor culture, 50 ml of the seed
culture was inoculated into a 3-1 jar fermentor (MDL300, Marubishi Co. Ltd., Tokyo) containing 1.5 1 of the
production medium. The culture was carried out at 28°C
until the glucose was exhausted. To avoid the effect of
shear stress on the mycelial morphology the agitation
and aeration rates were kept at 350rpm and 1 w m ,
respectively, throughout the experiments. To maintain
the dissolved oxygen concentration at a level higher than
30%, oxygen-enriched air (NewLife, AlrSep Co., Bur-
TABLE 1.
Experimental conditions for varying the consumed
C/N ratio
Initial concentration
(g//)
Glucose Soybean meal
5.0
10.0
15.0
20.0
30.0
35.0
37.5
40.0
42.5
47.5
383
45.0
40.0
35.0
30.0
20.0
15.0
12.5
10.0
7.5
2.5
Residual concentration
(g//)
Glucose
Nitrogen
0.3
0.0
0.1
0.1
0.5
0.9
0.3
5.7
4.2
32.6
0.8
0.5
0.5
0.6
0.3
0.1
0.1
0.0
0.0
0.0
Consumed
C/N ratio
(-)
4.9
5.6
6.7
8.6
12.0
14.9
18.9
20.4
23.7
31.7
Initial glucose concentration was 40 g/l, but SM concentration was
varied from 5 to 40 g/l.
40.0
40.0
40.0
40.0
5.0
20.0
30.0
40.0
18.3
0.0
0.1
0.2
0.0
0.2
0.4
0.6
24.1
14.9
11.7
10.0
Initial SM concentration was 10 g/l, but glucose concentration was
varied from 20 to 120 g/I.
20.0
40.0
60.0
80.0
100.0
120.0
10.0
10.0
10.0
10.0
10.0
10.0
0.0
5.7
19.5
31.9
36.9
48.2
0.0
0.0
0.0
0.0
0.0
0.0
13.4
20.4
23.4
27.1
34.4
38.6
falo, NY, USA) was used.
Image analysis
A binocular microscope (BX-60,
Olympus Co., Tokyo) or a stereoscopic microscope
(SZH-10, Olympus Co.) equipped with a monochrome
CCD camera (XC-77CE, Sony, Tokyo) was used for the
image analysis of mycelia. Captured images were fed
into a computer (Macintosh 8500/150) and analyzed
using image analysis software (IPLab Spectrum, Signal
Analytics Co., Virginia, USA). The captured images
were thresholded to obtain binary images. Opening was
then applied to the binary images for improvement of
their quality. Repeated opening cycles were performed
on each pellet until only the pellet core remained. The
subtraction of the pellet core from the whole pellet gave
the annular region. Throughout these experiments, the
annular regions were regarded as filamentous mycelia.
For each sample, images of at least 50 elements (defined
as either pellets or clumps of hyphae) were used for
image processing and determination of morphological
parameters. Average values were used to analyze the morphological parameters.
Equivalent diameters of the projected area of the
whole pellet (Din) and pellet core (Dpc) were calculated
by assuming a circular morphology for both (22). The
width of the pellet annular region (Lf) was also regarded
to be the thickness of the filamentous mycelia around
the pellet core. The L f was calculated using the whole
pellet and pellet core diameters as follows:
Lf---- D m - - D p c
2
Analytical methods
The fungal mycelia were harvested by filtration and washed with 50 ml of distilled
water. The filtered mycelia were dried at 105°C for 2 h
384
KOIKE ET AL.
J. BloSci. BIOENO.,
and weighed to obtain the dry cell weight.
Intracellular fatty acids (FA) and A A were extracted
from 20 mg of dried mycelia, trans-methylated in methanolic HCI and quantified by gas chromatography using
a flame-ionized detector (Shimadzu GC-14B, Shimadzu,
Tokyo). A glass column (3 m m × 2 m) was packed with
5% Advance DS on 80/100 mesh Chromosorb W
(Shimadzu, Kyoto). The temperatures of the column,
injection port, and detection chamber were 190, 240, and
250°C, respectively. The gas pressures of nitrogen, hydrogen, and air were 4, 0.6, and 0.5 kg/cm 2, respectively.
The residual glucose concentration was measured using
the DNS method (23).
The consumed C / N ratio was determined as follows,
= a ( G l c o - Glc) + 1~Smo
N
~'SMo- N
where a and/9 indicate the ratios of carbon contained in
the glucose and SM, respectively. The value for a was
0.4 and that for the/~ was 0.303. The ? value indicated
the ratio of nitrogen in SM and was 0.0847 as detrermined from analytical data. Glc and N denote the residual concentrations of glucose and nitrogen, respectively.
The subscript 0 indicates an initial concentration. The
residual nitrogen concentration in the culture medium
was measured by estimation of the ammonia formed
using Nessler's reagent after micro-Kjeldhal digestion of
cell-free medium samples.
I
II
III
25
--O-x
i
"
15
i,°
0
4
B
!,
3
1
o
i
~~
1.0
.,
I
~
0.8
3o 0.6
~. 0.4
~
AA/FA
RESULTS
0.2
Effect of C / N ratio on arachidonic acid production in
flask culture under a fixed initial a m o u n t of glucose and
0.0
SM
Under the condition of a fixed initial amount of
carbon and nitrogen sources of 50 g/1 over all, the C / N
ratio was varied from 5 to 32. Mycelial growth and fatty
acid production from M. alpina cells are divided into
three phases as shown in Fig. I. In phase I (a consumed
C / N ratio of less than 7) the carbon source is limited;
in phase II (a consumed C / N ratio of 7 to 15) utilization
of the carbon source switches from cell growth to lipid
biosynthesis; in phase III (a consumed C / N ratio of 15
to 32) lipid biosynthesis is most active and the nitrogen
source is limited.
During phase I, the mycelia concentrations (X) and its
concentration without fatty acids (X-FA) remained constant (Fig. 1A) and the ( X - F A ) / X ratio was higher than
0.9. The concentrations of F A and A A produced were
very low (Fig. 1B), and the A A / F A ratio was only 0.25.
Both the F A yield from consumed glucose (YFA/S) and
the A A yield from consumed glucose (YAA/S) were also
low (Fig. 1D). This indicates that the glucose was utilized only for mycelial growth and not for lipid biosynthesis below a consumed C / N ratio of 7.
However, during phase II, the mycelial concentration
increased; and the rate of lipid biosynthesis increased
rapidly (Fig. 1B). Therefore, YFA/S and YAms increased
markedly (Fig. 1D). In this phase, the carbon source was
utilized not for cell growth, but for lipid biosynthesis.
During phase III the mycelial concentration decreased
linearly with the increasing consumed C / N ratio (Fig.
IA). The YeA/s and YAA/S were high (Fig. 1D), indicating that mycelial growth was complete and lipid biosynthesis remained at the highest level. The ( X - F A ) / X ratio
decreased from 0.8 to 0.6 (Fig. 1A), because of the limitation of the nitrogen source.
C
:
,
t
0.12
iI
~
(X-FA)/X
I
I
o.o9
D
"~ 0.06
e~
"'~ 0.03
0
~V',
:.
,
+
YaWs
t
0
10
20
30
C/N ratio
FIG. 1. Effect of consumed C/N ratio on mycelial growth and
lipid production in flask culture of M. alpina. The sum of the initial
concentrations of glucose and SM was 50 g/1. A shows the mycelia
concentrations (X) and mycelial weight except for fatty acids (X-FA);
B, concentrations of arachidonic acid (AA) and total fatty acid (FA);
C, ratios of (X-FA)/Xand AA/FA; D, AA yield (YAms)and FA yield
(YFA/S)from consumed glucose. Cellular growth and Hpidbiosynthesis
are classified into phases I, II, and III.
Effect o f C / N ratio on A A production in the presence
of an excess concentration o f either glucose or SM
To investigate the effect of the consumed C / N ratio on
AA production a fixed concentration of either glucose or
SM, two separate experiments were carried out. In one,
the SM concentration was varied from 5 to 40 g/l while
the initial glucose concentration was fixed at 40 g/1 in
batch culture (a consumed C / N ratio of 10 to 24). In the
other, the glucose concentration was varied from 20 to
120g/l, but the initial SM concentration was fixed at
1 0 g / / ( a consumed C / N ratio of 13 to 39). The results
VOL 91, 2001
I
EFFECT OF C/N RATIO ON ARACHIDONIC ACID PRODUCTION AND MYCELIAL MORPHOLOGY
-o-
x - - I ~ X.FA
II
,-,
DlO • 1 5
I
•
X
30
~ o
~
~AA
0
.--.I~FA
--o-AA --o- ~-A
~#'-"'~B
"~4
~
l0
~ ,
r-.., r ~
o
3
II
1
'
0
•
'
"
'
n
~
YaW~
i°o:i
5
10
• 20
[ ] 27
I
,[]ln
[ ] 39
60
50
I
0.09
•23
5O
- I ~ X.FA
,o
~5
385
- o - YaWs
C
~" 0.10
40
o.0,
,
n
,
|
15 20
C/N ratio
.
i
25
0,00 . . . . . . . . .
"~ ~"
10 15 20 25 30 35 40
C/N ratio
FIG. 2. Effect of consumed C/N ratio on myceliai growth and
lipid production in flask culture of M. alpina. The initial glucose
concentration was 40 g/I and the SM concentration was varied (I); the
initial SM concentration was 10 g/I and the glucose concentration was
varied (II). A shows concentrations ofXand X-FA; B, concentrations
of AA and FA; C, YA~S.
are shown in Fig. 2.
W i t h an increase in the SM concentration at a fixed
initial glucose concentration (I in Fig. 2), when the consumed C / N ratio was close to 10, the concentration o f
mycelia was highest and then decreased with increasing
consumed C / N ratio. On the other hand, the FA concentration was independent o f the C / N ratio, but the
A A concentration decreased slightly, and YAA/S also decreased.
However, with an increase in the glucose concentration at a fixed initial SM concentration o f 10 g/! (II in
Fig. 2), mycelial concentrations remained constant despite increases in the consumed C / N ratio. With an increase in the consumed C / N ratio, the A A concentration
decreased, but the FA concentration increased linearly.
The yield o f A A f r o m consumed glucose decreased
linearly with increasing consumed C / N ratio.
The fatty acid compositions in these cultures are
shown in Fig. 3. In the case o f a fixed initial glucose concentration o f 40 g/l (Fig. 3I), when the consumed C / N
ratio increased, i.e. when the nitrogen source was limiting, the p r o p o r t i o n o f saturated fatty acids increased
and the p r o p o r t i o n o f the p o l y u n s a t u r a t e d fatty acid,
A A ( 2 0 : 4 ) increased. In the case o f a fixed initial SM
concentration o f 1 0 g / / (Fig. 3II), when the consumed
C / N ratio increased a n d the carbon source was in excess, the p r o p o r t i o n o f the saturated fatty acids increased, and the A A concentration decreased to half the
a m o u n t observed for consumed C / N ratios o f 20 and
39. These results indicate that for a fixed a m o u n t o f the
c a r b o n source, the A A concentration increased with an
increase in the a m o u n t o f the nitrogen source; for a
fixed a m o u n t o f the nitrogen source the A A concentration increased with a decrease in the a m o u n t o f the
carbon source.
Effect o f high-concentration carbon and nitrogen
~"
20
10
0
FIG. 3. Effect of the consumed C/N ratio on fatty acid composition in flask culture ofM. a l p i n a . Data in I and II were obtained from
the cultures the results for which are shown in Fig. 2I and 2II,
respectively. DGLA denotes dihomo-?-linolenic acid.
sources with a constant C / N ratio on A A production in
flasks and in a fermentor
Under a constant C / N
ratio o f 20, cultures were carried out in flasks a n d in a
fermentor, using m e d i a enriched 1- to 4-fold in b o t h SM
and glucose. The results for the fermentor culture are
shown in Fig. 4. Glucose was exhausted at 4 d, 7 d, 9 d,
a n d 10d when the 1- to 4-fold enriched m e d i a were
used, respectively. In all cultures, the nitrogen source
was consumed completely by 6 d or before. Since glucose
and SM were consumed completely there was no change
in the consumed C / N ratio o f 20. Final mycelial concentrations increased with increases in the degree o f m e d i u m
enrichment. The A A concentration depended on the
degree o f m e d i u m enrichment up to a 3-fold increase,
but decreased in the 4-fold enriched m e d i u m (Fig. 4C).
In the case o f 3- and 4-fold enriched media, m a n y
mycelial clumps were f o r m e d and attached to the glass
walls and impeller o f the fermentor. Moreover, it was
impossible to mix the culture b r o t h due to high viscosity
in the case o f the 4-fold enriched medium.
The yield coefficients, b o t h for the flasks a n d fermentor cultures are shown in Table 2. Cellular yields for
glucose (Yx/s) decreased with an increase in the degree
o f m e d i u m enrichment. The A A yield for glucose (YAA/S)
in the 4-fold enriched m e d i u m also decreased to half
to that in the control, while the FA yield for glucose
(YFA/S) increased gradually. The ratio o f A A to FA
decreased from 0.44 to 0.30 in the f e r m e n t o r culture
and was similar to that o f the flask cultures. This result
indicates that even though the consumed C / N ratio was
in an o p t i m a l range, when the nutrient concentration
was high, the mycelia metabolized the c a r b o n source
386
KOIKE ET AL.
J.
100
a
TABLE
=.I
•
~.o =
60
7!,020
So ( g / / ) a
SM0 (g//)b
....
a
A
50
AA/FA
30
.2[
40
10
80
20
120
30
160
40
17.96 (14.21) 27.40(27.63) 37.34(41.20) 43.40(49.85)
3.04 (2.78)
7.20 (6.79) 11.45 (12.97) 9.15 (14.63)
1.35 (1.27)
2.49 (3.17)
3.42 (3.75)
2.75 (3.62)
Fatty acid ratio ( - )
40
~
Yield coefficients for the same C/N ratio in the flask and
fermentor cultures
Concentration (gl/)
X
FA
AA
•~
2.
BIOSCI. BIOENG.,
0.44 (0.45)
0.35 (0.49)
0.30 (0.29)
0.30 (0.25)
Yield from glucose ( - )
21)
lO
O
o
i
B
i
i
i
i
t
i
i
i
i
3
1
I
Yx/s
YAA:S
YFA/S
I
2
I
I
I
4
6
Time
8
I
I
10
1
FIG. 4. Concentrations of residual glucose (A), mycelia (B), and
arachidonic acid (C) in fermentor culture of M. alpina. Initial concentrations (g//) of glucose and SM were 40 and 10 (O); 80 and 20 ( • );
120 and 30 (n); and 160 and 40 (m), respectively. In the cases of
concentrations of 120 and 30, and 160 and 40, sampling commenced
after 5 d of cultivation, because insoluble SM remained in the culture.
The final mycelial weight and AA concentration increased because
mycelia growing on the glass wall inside the ferrnentor were included
in the calculation.
and tended to produce F A , but not A A . In the 4-fold
enriched medium, concentrations o f mycelia and lipids
were lower than those o f the flask cultures.
The difference in yield coefficients between flask and
fermentor cultures might be due to shear stresses affecting the mycelial m o r p h o l o g y due to mechanical agitation
in the fermentor.
Effect of the consumed C / N ratio on mycelial mor.
phology in flask culture
P h o t o g r a p h s depicting the
m o r p h o l o g y o f mycelia cultured with different C / N
ratios are shown in Fig. 5. The m o r p h o l o g i c a l changes
occurring in relation to the consumed C / N ratio were
similar despite the use o f three different culture media.
With increasing consumed C / N ratio the whole pellet size
increased; smooth or hairy pellets were formed in all cases
(Fig. 5A). In the presence o f excess nitrogen, mycelia
formed long and narrow pellets (Fig. 5B). On the contrary, in the presence o f excess carbon, mycelia f o r m e d
hairy pellets (Fig. 5C). W h e n the carbon source concentration was high but the nitrogen source concentration
was low, with the consumed C / N ratio higher than 30,
the pellets grew larger than those observed at low C / N
ratios, and were hairy.
When the consumed C / N ratio was varied, the morphology was characterized and significant features are
shown in Fig. 6. The whole pellet size (A in Fig. 6) did
0.45 (0.39)
0.35 (0.39)
0.31 (0.38) 0.27(0.33)
3.37(3.50)×10-2 3.55(3.45)×10 2 2.85(3.46)×10-2 1.72(2.37)×10-2
0.76(0.77)×10-1 1.03(0.71)×10-l 0-95(1.20)X10 I 0.57(0,96)X10-I
Data are from the fermentor, but data in parentheses are from the
batch culture. The consumed C/N ratio in both cultures was 20.
a Initial glucose concentration.
b Initial SM concentration.
not change until a consumed C / N ratio o f 20 was
reached, but then increased gradually with an increase in
the consumed C / N ratio. However, the ratio o f the size
o f the pellet annular region to the whole pellet size was
0.8 (data not shown), which was independent o f the consumed C / N ratio or the culture media. The Lf remained
at 0.3-0.4 on average until a consumed C / N ratio o f 20
was reached, and then increased linearly with increasing
C / N ratio (Fig. 6B).
Effect of the consumed C / N ratio on mycefial morpholMorphological changes occurring
ogy in the fermentor
at a consumed C / N ratio o f 20 using the enriched media
in the fermentor are shown in Fig. 7. W h e n the initial
concentrations o f glucose and SM were 40 and 10 g/l,
respectively, the whole pellet sizes were larger than 1
m m 2 (Fig. 7A), which was similar to the size o f those
shown in Figs. 5 and 6. However, as the glucose concentration was increased to 120 a n d 160 g/l, the whole pellet
size and Lf decreased. This suggests that when the medium
was enriched the pellet size was smaller than in the control, but the mycelial concentration was higher (see Fig.
4). The m o r p h o l o g y o f mycelia in the fermentor was
similar to that o f mycelia in the flask culture but the size
was smaller (data not shown). A l t h o u g h the width o f
pellet annular regions was 0.45 m m on average at the
beginning o f the culture, the width was dependent on
the degree o f m e d i u m enrichment, decreasing linearly.
This means that the filaments a r o u n d the pellet core
might be shaved-off by shear stress and might form new
smaller entities.
DISCUSSION
The key to lipid accumulation is to allow the a m o u n t
o f nitrogen supplied to the culture to become exhausted
during cultivation. To investigate the effect o f limiting
the carbon or nitrogen source, we varied the consumed
C / N ratio under the condition that the sum o f the initial
concentrations o f the carbon and nitrogen sources was
fixed at 5 0 g / l . As indicated by m a n y researchers, the
fatty acid yield increases with increasing consumed C / N
ratio when nitrogen is limiting (Fig. 1).
To confirm this, the consumed C / N ratio was varied
VOL. 91, 2001
EFFECT OF C/N RATIO ON ARACHIDONIC ACID PRODUCTION AND MYCELIAL MORPHOLOGY
A
B
387
C
• Z ,,,
............~........~!ii~¸
FIG. 5. Photographs of mycelia cultured at various C/N ratios in flasks. Numbers in photographs denote the consumed C/N ratios. Photograph A was of mycelia grown in experiments the results of which are shown in Fig. 1; and photographs B and C, of mycelia grown in
experiments the results of which are shown in I and II in Fig. 2, respectively.
at a fixed initial concentration of the carbon source. In
this case the mycelial concentration depended on the
nitrogen source concentration. When nitrogen was in
excess, the proportion of polyunsaturated fatty acids
increased, but that of saturated fatty acids decreased. On
the other hand, when the consumed C / N ratio was increased at a fixed initial concentration of the nitrogen
source, the fatty acid concentration depended on the
initial concentration of the carbon source, but the concentration of A A was independent of this. When carbon
was in excess, the proportion of unsaturated fatty acids
decreased, but that of saturated fatty acids increased.
Increasing the already high C / N ratio led to a decrease
in the A A concentration to 50% (Fig. 3). When the C / N
ratio is high the rate of the desaturation reaction becomes
low, and the accumulated saturated fatty acids are not
converted to polyunsaturated fatty acids. From these
results, it was clear that the concentrations of carbon
and nitrogen sources should be balanced and that a suitable C / N ratio for A A production was around 20. These
results are similar to those obtained with other Mortierella sp. (15, 24). The optimum C / N ratio for lipid biosynthesis by Mortierella was 20 to 23. However, in the case
of the oleaginous yeasts, Rhodotorula glutinis (13, 25)
and Apiotrichum curvature (14, 26), the optimum C / N
ratio was higher than 40, a ratio at which the nitrogen is
probably suitably limited. Granger et al. (13) reported
that the cell growth and lipid production of R. glutinis
were independent of the C / N ratio below a consumed
C / N ratio of 10. When the consumed C / N ratio was
higher than 25 the lipid concentration was dependent on
the ratio. However, for C / N ratios higher than 40, lipid
synthesis declined. This indicates that the fungus required more nitrogen for lipid biosynthesis than does the
yeast. The difference in the optimum C / N ratio between
the yeast and fungus indicates the degree of nitrogen
limitation.
Using the optimum C / N ratio, when the initial concentrations of both carbon and nitrogen sources increased,
cellular growth and lipid production were proportional
to the increasing concentration until the carbon and
nitrogen sources were 120 and 40g/l, respectively.
However, at concentrations higher than this the A A concentration decreased despite an increase in the biomass
and FA concentration. In the culture containing initial
carbon and nitrogen concentrations of 160 and 40g/l,
respectively, it was very difficult to mix completely the
culture broth because of its high viscosity. Moreover,
many mycelia were attached to the fermentor wall and
to the impeller and its shaft. This probably resulted in
local deficiencies of dissolved oxygen in the culture due to
insufficient mixing in the reactor. Unsaturated fatty acids
are synthesized by an aerobic mechanism which involves
composed NAD(P)H-cytochrome b5 reductase, cytochrome bs, and a terminal cyanide-sensitive desaturase in
the cytoplasm (27, 28). Higashiyama et al. (20) reported
that the oxygen requirement for desaturation was higher
than for cell growth and FA production. They showed
that the optimum dissolved oxygen concentration for
A A production was 10-15 ppm, but at 3 ppm the FA
and X-FA yields did not decrease, but the A A yield was
lower than that obtained at a high dissolved oxygen concentration. Therefore, if the viscosity of the culture
broth is sufficiently low or the filament are converted to
388
KOIKE ET AL.
J. BIOSCI.BIOENG.,
l0
2
•
0
1.o
o.4
N~
0.2
0.0
0
20
C/N ratio
i0
30
40
FIG. 6. Effect of the consumed C/N ratio on morphological
parameters of M. alpina. A and B show the whole pellet size (Am)and
width of the pellet annular region (Lf), respectively. The morphological parameters pertain to mycelia grown in experiments the results
of which are shown in Fig. 1 ( • ); and in Fig. 2I (/x); experiments, in
Fig. 2II ( [] ).
small pellets, dissolved oxygen might not be limited in
the culture and then the A A production would be correlated with the nutrient concentration.
The most interesting results are that the whole pellet
size and the width o f the pellet annular region did not
change until the consumed C / N ratio was less than 20,
2.0
A
but then increased gradually with increasing consumed
C / N ratio. W h e n the m e d i u m was enriched at a fixed
C / N ratio o f 20, the whole pellet size and the width o f
the pellet annular region decreased with increasing o f
glucose and SM concentration.
In the case o f the fermentor, the whole pellet sizes
were smaller than those in the flask cultures and decreased over the course o f cultivation. The width o f the
pellet annular region remained constant during cultivation. These results indicate that the filaments were
b r o k e n by shear stress due to the mechanical agitation.
The resulting segmented mycelia m a y have grown to
form small mycelia in the presence o f nutrients.
H i g a s h i y a m a et al. (29) reported that hairy mycelia o f
M. alpina on the pellet surface were shaved off during
cultivations, a n d those filaments aggregated or became
entangled and increased in size. This result indicates that
the m o r p h o l o g y observed in flask culture cannot be observed in the fermentor culture due to differences in the
shear conditions and it was thought that the M. alpina
mycelium used in this study is very sensitive to shear
stress. In this study however, since the agitation rate was
fixed at 350 r p m throughout, there m a y not have been a
difference in shear stress conditions between the two
types o f cultures. The difference in m o r p h o l o g i c a l size
between cultures was due to the C / N ratios.
In this study, to produce A A f r o m a culture o f M.
alpina it was important to adjust the optimal C / N ratio in
the m e d i u m to a r o u n d 15-20, by balancing the carbon
and nitrogen concentrations. W h e n the consumed C / N
ratio was kept constant at 20, the a m o u n t o f A A
p r o d u c e d was p r o p o r t i o n a l to the carbon and nitrogen
concentrations. This result will be useful for A A production in a fed-batch operation. The mycelial m o r p h o l o g y
and the consumed C / N ratio were correlated in this
study. The whole pellet size and the width o f the pellet
annular region (layer o f filamentous mycelia a r o u n d the
pellet core) were independent o f the consumed C / N
ratio below 20 in both flask and fermentor cultures, but
increased gradually with increasing C / N ratio.
o4
~O . .
~
1.0
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