1987-Alterations of Respiratory Systems in Aspergillus niger under the Conditions of Citric Acid Fer




51 (5),




Alterations of Respiratory Systems in Aspergillus niger under the Conditions of Citric Acid Fermentation
Kohtaro Kirimura, Yuji Hirowatari and Shoji Usami
Department of Applied Chemistry, School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 160, Japan Received September 8, 1986 Under the conditions of citric acid fermentation, Aspergillus niger WU-2223L possessed at least two respiratory systems. Onesystem wassensitive to cyanide (CN) and the other was sensitive to salicylhydroxamic acid (SHAM). The respiration of mycelia was partially, inhibited by CNor SHAM,and was completely inhibited in the presence of both CNand SHAM.With culture time, the CN-sensitivity of respiration decreased and the SHAM-sensitivity increased. The respiratory rate itself decreased suddenly at 4 days and thereafter remained at a low level. The SHAM-sensitive respiration was localized in the mitochondria. The alterations of respiration in the mitochondria were similar to those observed in mycelial cells.

It has been shown that many higher plants and eucaryotic microorganismsrespire genetically or environmentally

the SHAM-sensitivity of respiration

by means of two

respiratory systems.1 ~3) The normal system is a cytochrome chain, that is sensitive to cyanide (CN) and antimycin A. The alternative one is chondria.

WU-2223L increased and the CN-sensitivity decreased during citric acid fermentation, probably resulting from changes in the capacities of these respiratory systems in mito-

in A. niger

insensitive to these inhibitors but sensitive to MATERIALS AND METHODS salicylhydroxamic acid (SHAM). The two respiratory systems are commonly accepted to compete for reducing equivalents on the Microorganism and culture conditions. Aspergillus niger WU-2223L,8) a good producer of citric acid, was used. oxygen side of dehydrogenases, forming a Conidia of the strain were suspended in 60ml of a branched electron transport system (see Fig. synthetic mediumat a concentration of 1 x 106/ml and then Zehentgruber et al.6) reported that the respiration of Aspergillus niger proceeds via a cytochrome chain and a SHAM-sensitive alflasks (500ml) at 30°C. The synthetic medium contained (per liter of distilled water): glucose, 120 g; (NH4)2SO4, 3 g; K2HPO4, 1 g; KH2PO4, 1 g; MgSO4-7H2O, 0.5g; MnSO4, 14mg; FeCl3-6H2O, lOmg; and 2% (v/v) methanol. The pH was initially adjusted to 3.0. Methanol promotes citric acid production and is not used as a nutrient.8) Isolation of mitochondria. The mitochondrial fraction (heavy particulate fraction) was prepared by the modified methods ofWatson and Smith9) and Uchiyama et al.10) On an indicated culture day, mycelia were harvested on a WhatmanGF/Afilter with suction and then washed twice with a 0.7 m KC1solution. Portions of the washed mycelia (lg wet weight) were suspended in 10ml of 50mMpotassium phosphate buffer, pH 6.0, containing 15mg of
chitinase (Godo Shusei Co., Tokyo), 80mg of hemicellulase (Miles Laboratories, Inc., Elkhart) and 0.7m KC1. Each mixture was incubated at 30°C in an

cultivated aerobically with shaking ( 120 rpm) in Sakaguchi

ternative system. Kubicek et al.n) reported that citric acid production by A. niger is strongly inhibited by SHAMduring the early-log-phase
whereas growth is not. These observations


suggest that participation
in citric acid


of SHAM-sensitive
by A.

Wehave studied citric acid fermentation by A. niger WU-2223L, and previously reported that mycelial respiration and acid productivity are influenced markedly by the culture medium constituents.8) This paper reports that


K. Kirimura, Y. Hirowatari and S. Usami

(60rpm) and then centrifuged at 2,500xg for 20min at 2°C. The mycelia after enzymic pretreatment were sustaining

Erlenmeyer flask (50ml) for 90min with gentle shaking

pended in 10 mMpotassium phosphate buffer, pH 7.2, con0.65m sorbitol and 0.5mM EDTA (SE buffer),

homogenized in a teflon/glass homogenizer (700 rpm) for 10min at 2°C, and then centrifuged at 3,000 x g for 10min at 2°C. The precipitate was discarded and the supernatant
was centrifuged at 20,000xg for 15min at 2°C. The

sedimented pellet was resuspended in SE buffer and then centrifuged at 20,000 xg for 15min at 2°C. The resulting pellet was used as the mitochondrial fraction. The latter was kept at 2°C and analyzed within 3hr. Measurement of respiration.
mycelia and isolated mitochondria in temperaturecontrolled reaction chambers was monitored with a Clarktype electrode (Yellow Springs Instruments, Cleveland, Ohio) by a modification of the method of Dutton.U) The respiration of mycelia was measured at 30°C in 3ml of a reaction mixture containing mycelia (1.0 mg dry weight/ ml) and 0.5m glucose in 10mM potassium phosphate buffer, pH7.2. The respiration of mitochondria was measured at 25°C in 2.5ml of a reaction mixture containing mitochondria (4mg protein/ml), 0.4m mannitol, 0.5 mM EDTA, 10mMKC1 and 2.5mg/ml of bovine serum al-

The respiration

of both

3 Xk. 2 C1 0 ? 120 ^ E80 o> en EIOOh ? ~ 80 h -2 60 C J 60 h ? 40 40 -I 20 20 °L o

8 Culture time (days)

40 ? O) 30~E .c> o 20 I >, k_ 10Q 10

Fig. 1. Time Course of Citric Aspergillus niger WU-2223L.


Symbols: citric pH (#).

acid (O), dry weight (A), glucose (??) and

bumin (fraction V, Sigma) in 10 mMpotassium phosphate
solution to give the final concentrations indicated

buffer, pH 7.2. Respiration measurements for the mito-

(A ) 0. 346 KCN s HA Mi

chondria were performed at State 3.n) Succinate, ADP, KCNand SHAM were added, injecting concentrated
in the

footnotes to figures.
Analytical methods. The amount of citric acid in the

0. 041 (B) \ S H A M o . 3 4 6 \ KC N I 0. 117 0 O. OI umo1 02/ mg dr y we i ght 2 m in
Fig. 2. Effects of CN and SHAMon the Mycelial Respiration of Aspergillus niger WU-2223L.

culture broth was determined by the method of Mollering and Gruber,12) and that of glucose by the Glucose-B-Test method (Wako Pure Chemical Ind., Tokyo). The weight of mycelia is expressed as that of the dried form. The protein content was determined by the method of Lowry et al.13)
with bovine serum albumin as a standard.


The time course of citric acjd fermentation by A. nigerWU-2223L is shown in Fig. 1. The

concentration ofcitric acid in the culture broth increased throughout the whole culture period (9 days). The dry weight of mycelia also
increased during the initial 6 days. In this

period, the growth of mycelia occurred in the culture broth even under low pH conditions (below 3.0). After 6 days, the dry weight of the
mycelia did not increase. Therefore the

amount of citric acid produced per dry weight

Mycelia at 5 days (at the late-log-phase) were used. At the points indicated by arrows, either KCNor SHAMwas added to the reaction mixture to give final concentrations of 2.5mMand 2.0mM,respectively. The values shown in the figure are the respiratory rates expressed in fimol O2/hr/mg (dry weight). The rate of inhibition by CN alone was 88.3% (A) and that by SHAMalone was 66.0% (B).

Respiratory Systems in A. niger


in this period was higher than that in the initial culture period. The effects of CNand SHAM the reson piration of the mycelia at 5 days are shown in Fig. 2. The respiration was partially inhibited

ratory system and the SHAM-sensitive one are correlated to each other. The respiratory rates and the sensitivities to CNand SHAM mycelia after various culof
ture periods are shown in Fig. 4. The res-

by CN or SHAM, and was completely inhibited in the presence of both CN and

piratory rate of mycelia decreased suddenly at
4 days and thereafter remained almost constant (Fig. 4(A)). The percent inhibition of

least two respiratory systems, one CNsensitive and the other SHAM-sensitive, are present in the mycelia of A. niger WU-2223L.

SHAM.These observations indicated that at

Additional experiments were performed using
antimycin A, a respiratory inhibitor of a cytochrome chain (data not shown). Respiration of the mycelia at 5 days was partially inhibited
by the addition sequent addition of antimycin A, and subof SHAM inhibited the res-

respiration decreased during the period between 5 days and 8 days (the late-log-phase

respiration by CN or SHAMis shown in Fig. 4(B). During citric acid fermentation, SHAMsensitive respiration wasobserved regardless of the culture period. The CN-sensitivity of the

piration completely. In a parallel experiment, the respiration was also partially inhibited by the addition of SHAM,and subsequent addition of antimycin A resulted in complete inhibition. These results indicated that the antimycin A-insensitive respiration was inhibited by SHAM, exactly as in the case of the
CN-insensitive respiration (Fig. 2). Therefore,

Fig. 1), and the SHAM-sensitivity increased, especially after 4 days (the late-log-phase). These observations indicated that the role of - 15 . ? f j >* , 1.0 p\ "5. ^ 0. 5 * 1 3 o (B) 10 0 90 ^ c 80 o f 70 c 60 50 (A )

and the beginning of the stationary phase, see

it was suggested that the CN- and antimycin A-sensitive respiratory system and the SHAMsensitive one form a branched electron transport system (Fig. 3), similar to those proposed for Neurospora crassa^ and higher plants.1 ~3) It should be noted that the extents of the inhibition by CN alone (Fig. 2(A)) and by SHAMalone (Fig. 2(B)) were not additive.
This suggested that the CN-sensitive SHAM NADH 02 / \ y Succinat e - > Fp Cyt. b - J> Cyt. c -> Cyt . aa3 Ant i m yc i n A Cya ni de ' \ respi-



A 6 8 Cu ltu re t im e (d ays )


Fig. 3. Branched Electron Transport System.
Xdenotes the antimycin A- and CN-insensitive oxidase system, and Y the hypothetical branch point, according to
Lambowitz et al.A) The sites of inhibition
Cyt, cytochrome; Fp, flavoprotein.

the Rates of Inhibition Mycelia of Aspergillus

Fig. 4. Time Courses of by CN or SHAM (B) (A) and the Respiratory Rate in the
niger WU-2223L.

In panel

(B), the rates

of inhibition

are shown as the

by drugs are

indicated by dashed lines.

sensitivities of the mycelial respiration to CN(A) and to SHAM(??). The final concentrations ofKCN and SHAM in the incubation mixture were the same as those in the footnote to Fig. 2.


K. Kirimura, Y. Hirowatari and S. Usami

(A succinate ax\vL0.01um gproteino.215N L_?\ A P ). o102/m H D?rv.;rt 2 m in 0. 26r (B) .0.21Succina te KCNo . 3SHAM ^ 29 \

than that at 1 day, whereas the CN-sensitivity at 9 days was lower than that at 1 day. The changes in respiration of mitochondria were similar to those observed in the case of mycelial cells, though the mitochondrial respi-

ADP 0 . U2 0 . 2 6 1 * K+ 0. 1 32 SHAM KCN Succin ate( C -^ A P ) D 0.132 o . u r \ KC N 0 . 2 4 ^ s H , A Ms u c c i n a t e ^ ^ ( D) J o . 1 1 1 ADP A. OU A \ ^ SHAM 0.111

ration was not completely blocked in the presence of both CN and SHAM.At present, it

remains unknownwhether or not mitochondria from A. niger WU-2223L contain CNand SHAM-insensitive respiratory system(s). However, these observations at least indicate that the capacity of the CN-sensitive respi-

ratory sytem decreased and that of the SHAMv

sensitive one increased fermentation.




o . u r O. 1 0 5

0 . 2 A6 N7 KCN a 124 0.10 4
Fig. 5. Effects ofCN and SHAMon the Respiration of Isolated Mitochondria of Aspergillus niger WU-2223L.

The nature of the SHAM-sensitive respiratory system remains poorly understood. There is, however, general agreement that it functions as a reoxidative system for NADH
without ATP formation,1~5'14'15) and the

The rates of State 3 respiration in mitochondria isolated at
1 day (A) (B), and at 9 days (C) (D) of culture were

branch point from the cytochrome chain has been reported to be at the level of ubiquinone
(see Fig. 3).14'15) In N. crassa*>5)

examined. Without inhibitors, the respiration at State 3 wasmaintained for at least 12minafter the addition of ADP. At the points indicated by arrows, drugs were added to the reaction mixtures. The final drug concentrations
were: succinate, 10him; ADP, 160fiu; KCN, 2.5mM;

the SHAM-

sensitive respiratory system is induced when
the cytochrome chain functions insufficiently due to mutation or inhibition of mitochondrial protein synthesis. In contrast, in Candida albicans16) or Histoplasma capsulatum,ll) the

SHAM, .0mM. The values shown in the figure are the 2 respiratory rates expressed in /imol O2/hr/mg protein. The rates of inhibition by CN were 56.9% (A) and 39.5% (C). The rates of inhibition by SHAMwere 10.9% (B) and
49.6% (D).

SHAM-sensitiverespiratory system is constitutive. Zehentgruber et al.6) reported that the SHAM-sensitive respiratory system in A. niger

the SHAM-sensitive respiration became more greater in the mycelia with culture time. Respiration of the mitochondria is shown in Fig. 5. The SHAM-sensitive respiration was found only in the mitochondrial fraction and no SHAM-sensitiverespiration was detected

appears to be constitutive since it was present at all stages of culture under varying nutrient

In the present study, it was found that the
SHAM-sensitivity of the respiration

and the CN-sensitivity decreased in both mycelial cells and mitochondria during citric acid


fermentation by A. niger WU-2223L. The SHAM-sensitive respiration in A. niger exists ess of mitochondria isolation. The respiratory similar to in the cases of rate of the mitochondria isolated at 9 days was in mitochondria, lower than that at 1 day. The respiration of other eucaryotic cells.1 ~5 14 15> The respiration

any other fractions produced during the procmitochondria was partially inhibited

by CN or of the isolated mitochondria exhibited higher sensitivity to SHAM 9 days than at 1 day, at ration of mitochondria at 9 days was higher whereas it exhibited higher sensitivity to CN at
SHAM. The SHAM-sensitivity of the respi-

Respiratory Systems in A. niger


1 day than at 9 days. The respiratory

rate of
1) M. F. HenryandE.
2) 3) (1975). J. M. (1976). Palmer,


the mitochondria isolated at 9 days was lower
than that at 1 day, similar to as observed for

J. Nyns, Sub. Cell.
Ann. Ann. Rev. Rev. Plant Plant

Physio!., Physiol, 27, 28,

4, 1
133 279

mycelial cells. These observations indicate that the low respiratory rate and the changes in the sensitivities of respiration to CNand SHAM in mycelial cells did not result from the regulation of respiration by some cytosolic fac-

T. Solomons, (1977).
108, 1087 (1971).

4) A. M. Lambowitz and C. W. Slayman, J. BacterioL,

tors but from changes in the capacities respiratory systems in mitochondria citric acid fermentation.

of the during

5) D. L. Edwards and F. Kwiecinski,
6) 610 (1973). O. Zehentgruber, C. P. Kubicek

J. BacterioL,


M. Rohr,

activities of enzymes in the tricarboxylic acid (TCA) cycle ofA. niger WU-2223L, and found that the specific activity of citrate synthase remained almost constant and those of such

Wepreviously examined the changes in the

FEMS Microbiol. Lett., 8, 71 (1980). 7) C. P. Kubicek, O. Zehentgruber, H. El-Kalak Rohr, Eur. J. Appl. Microbiol. Biotechnol.,

and M. 9, 101

S. Usami, Mem. School Science & Engineering Waseda
Univ., 42, 17 (1978). K. Watson and J. E. Smith,

enzymes as isocitrate dehydrogenase decreased during the period ofcitric acid accumulation.8*

/. BacterioL,

96, 1546

Similar results were obtained on solid fermentation with another A. niger strain.18) From these results, we have concluded that citric

H. Uchiyama, M. Ando, Y. Toyonaka and T. Tabuchi, Eur. J. Biochem., 125, 523 (1982). P. L. Dutton, Biochim. Biophys. Acta, 226, 63 (1971). H. Mollering and W. Gruber, Anal. Biochem., 17, 369

acid accumulation is caused by decreases in the activities of enzymes, such as isocitrate dehydrogenase, in the TCA cycle with culture time.8'18) It remains unclear whether or not the changes of respiratory systems are really cor-

O. H. Lowry, N. J. Rosebrough,A. L. Farr and R. J. Randall, /. Biol. Chem., 193, 265 (1951).

related with citric acid production. It is likely,

S. Huq and J. M. Palmer, "Plant Mitochondria," ed. by G. Ducet and C. Lance, Elsevier/North-Holland Biomedical Press, Amsterdam, 1978, p. 225. J. Doussiere, A. Sainsard-Chanet and P. V. Vignais,
Biochim. Biophys. Acta, 548, 236 (1979).

however, that the changes of respiratory systems are related to the unusual functioning of the TCAcycle during citric acid fermentation.
Acknowledgments. We wish to express our appre-

M. G. Sheperd, Arch. Microbiol.,

C. Moi-Chim and P. A. Sullivan, 116, 61 (1978).

ciation to Professor I. Yasumasu, Dr. A. Fujiwara and Dr. T. Hisahori (Department of Biology, School of Education, Waseda University) for their many valuable suggestions.

B. Maresca, A. M. Lambowitz, G. S. Kobayashi and G. Medoff, J. BacterioL, 138, 647 (1979). K. Kirimura, K. Kumagai, S. Morisada, S. Kawabe and S. Usami, Hakkokogaku, 62, 127 (1984).



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