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The relationship of some metal ions with citric acid production by Aspergillus niger using tamarind seed powder as raw material
[ j . Ferment. Technol., Vol. 64, No. 6, 561-565. 1986]
Note
The Relationship of Some Metal Ions with Citric Acid Production by Aspergillus niger Using Tamarind Seed Powder as Raw Material HEMANT
J.
PUROHIT a n d HATIM F. DAGINAWALA
Department of Biochemistryand Microbiology, Nagpur University,LIT Premises, Nagpur-440010, India Tamarind seed powder (TSP) was used as raw material for microbial production of citric acid. The maximum citric acid production was observed when the TSP-basal medium (TSP with inorganic nitrogen and phosphorus source) was supplemented with Mg a+, Fe *+, Cu ~+ and Zn s+. The use of methanol and ferrocyanide as a modulator for citric acid production was studied. O n methanol supplementation to the medium, ammonium nitrate was found to be better than ammonium carbonate as a source of inorganic nitrogen. Addition of ferrocyanide after sterilization to the medium containing methanol gave the maximum citrate production of 363 #g/ml.
Metal ions are the micronutrients which modulate the biochemical conversions, and direct the sequence of metabolic conversions to result in overproduction of desired seconda r y m e t a b o l i t e s . T h e s e l e c t e d s t r a i n s o f Aspergillus niger a c c u m u l a t e l a r g e a m o u n t s o f c i t r i c acid extracellularly when grown on a minimal s a l t s m e d i u m , 1) b u t a n o v e r a b u n d a n c e of the metal ions leads to excessive vegetative growth at the expense of citrate accumulation. ~ Traditionally, crude carbohydrate r a w m a t e r i a l is p r e - t r e a t e d t o r e m o v e u n d e sirable heavy metal ions. In the present studies, tamarind seed powder (TSP) was t r i e d as a r a w m a t e r i a l . It has a low ash content and approximately 62% alcoholinsoluble polysaccharide.s) I t is a l s o c h e a p e r t h a n o t h e r r a w m a t e r i a l s u s e d for c i t r i c a c i d p r o d u c t i o n s u c h as m o l a s s e s , w h e a t b r a n etc. The effect of ferrocyanide and methanol w a s also s t u d i e d as t h e s e also s t i m u l a t e t h e c i t r i c a c i d p r o d u c t i o n . 4,s~ M a t e r i a l s and M e t h o d s Strain Aspergilluz niger, a strain isolated from waste of a factory which produces TSP from tamarind seeds, was used.
Seed culture The sporulated culture on potato dextrose agar slant was scraped off, mixed with 5 ml of sterile distilled water, transferred to the surface of potato dextrose agar in a flask (25 ml of medium in a 250-mi borosil conical flask) and incubated at 30°C for 72 h. After complete sporulation of the culture, the growth was scraped off and mixed with 30 m] of sterile distilled water on a wrist shaker for I 0 min. Production medium The basal culture medium (TSP basal medium) contained 2% acid-treated TSP (5g TSP in 250 ml 9% HC1 was autoclaved at 115°C for 15 rain), 0.42g N2 equivalent per liter of (NH4)~CO3, and 0.20 g]100 ml KH~PO4, and had a pH of 3. The optimization of C : N : P ratio was done in earlier studies, e) The trace metal ion tamarind seed powder basal medium ( T M T B medium) contained TSP basal medium with 200 mg/l, MgSO4"THsO, 50 mg/l, FeSO4"7H20, 0.06 m M CuSO4.5H20 and 0.09 m M ZnSO4-7HzO. Culture conditions The fermentation was carried out statically. In the 250-mi conical flask, 50 ml of medium was sterilized at 15 lb pressure for 20 min. The sterilized medium was inoculated with 2% seed culture and incubated at 304-I°C with 60-80% relative humidity. The ferrocyanide was added before sterilization to TSP basal medium, and metal ions and methanol were added later. T h e effect of ferrocyanide after sterilization was studied by adding ferrocyanide solution to the sterile T M T B medium with methanol.
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Analysis of fermentation broth Reducing sugar in the fermented broth was estimated by the method of Nelson, v) and citric acid by the method of Marier and Boulet. 8) After the fermentation, i.e., 72 h of incubation, the broth was filtered through GF/C Whatman glass fiber paper under suction. The biomass retained on the paper was weighed after drying at 80°C for 18 h. The titrable acidity was expressed in terms of 0.01 N NaOH, using phenolphthalein as an indicator. Results and Discussion T h e m a x i m u m citric a c i d y i e l d was observed after 7 2 h o f i n c u b a t i o n . T h e o r g a n i s m utilizes t h e p o l y s a c c h a r i d e of T S P as substrate for citric a c i d p r o d u c t i o n , a n d therefore the c h a n g e in r e d u c i n g s u g a r level was also s t u d i e d for each p a r a m e t e r . Effect o f m e t a l i o n s Mg~+ a n d Fe2+ a r e essential m e t a l ions for m a x i m u m utilization o f sugars a n d for o v e r p r o d u c t i o n of citric a c i d b y A. niger.9) I n t h e p r e s e n t studies, w h e n M g 2+ was a d d e d a t 200 mg/l c o n c e n t r a t i o n as M g S O 4 " 7 H 2 0 , only 6 % increase in y i e l d was observed a n d only 4 % increase in yield was seen w i t h 50 mg/l o f F e S O , . 7 H 2 0 a d d i t i o n ( T a b l e 1). A decrease in biomass a n d increase in final p H w i t h
h i g h e r levels o f b o t h the m e t a l ions was observed. R e d u c i n g sugar decreased w i t h increase in M g *+ level a n d increased w i t h increasing c o n c e n t r a t i o n o f Fe*+, w h e n these m e t a l ions were a d d e d separately. I n t e r estingly, w i t h different c o m b i n a t i o n s of M g 2+ (100-200 mg/l) a n d F e S+ (25-75 mg//) a d d e d to the T S P b a s a l m e d i u m , the results differed d r a s t i c a l l y ( T a b l e 1). T h e simultaneous s u p p l e m e n t a t i o n o f 200 mg/l of M g 2+ a n d 50 mg/l o f F e 8+ increased the citric a c i d yield by 30%. T r a c e s o f o t h e r m e t a l ions such as Z n 2+ a n d C u 2+ also r e g u l a t e the physiology o f A. niger in favor o f citric a c i d production.9,10) T h e different cations were a d d e d a t 0.04 m M c o n c e n t r a t i o n after the sterilization o f T S P b a s a l m e d i u m a l o n g w i t h 200 mg/l of M g 2+ a n d 50 mg/l o f Fe *+. T h e s u p p l e m e n t a t i o n o f C u 2+ a n d Z n 2+ gave a n increase in citric a c i d yield only b y 5.0 a n d 2 . 0 % respectively ( T a b l e 2), b u t their s i m u l t a n e o u s a d d i t i o n further s t i m u l a t e d the citric a c i d p r o d u c t i o n . S i m i l a r findings h a v e b e e n r e p o r t e d earlier.9,x0) A t 0.02 m M Zn*+ a n d 0 . 0 6 m M Cu*+, a 2 2 % increase in citric a c i d y i e l d was observed, a n d a t 0.03 m M Zn*+ a n d 0.05 m M
Table 1. Optimization of Mg 2+ and Fe2+ relationship for citric acid production. Concn. of Mg 2+ (rag/l) 0
100
150
200
Concn. of Fez+ (rag/l)
Citric acid (#g/ml)
0 25 50 75 0 25 50 75 0 25 50 75 0 25 50 75
164 164 169 159 163 199 209 225 164 186 239 212 173 184 242 213
Reducing Biomass sugar (rag/100 ml) (rag/50 ml) 388 398 450 570 370 427 443 504 388 450 443 458 381 397 424 458
518 520 519 493 627 621 586 535 518 601 564 566 627 534 564 545
Final pH 2.30 4.00 4.30 4.90 3.50 3.80 3.95 3.80 3.60 3.85 3.75 3.90 3.60 3.85 3.90 3.75
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Table 2. Relationship between Zn a+ and Cu 2+ metal ion for citric acid production.* Concn. of Zn a+ (raM) Experiment 1 Control
Concn. of Cu ~+ (mM)
Citric acid (pg/ml)
Reducing Biomass sugar (mg/50 ml) (mg/100 ml)
Final pH
-0. 4 0. 6 ---
---0. 4 0. 6
242 248 240 252 267
424 217 191 116 106
564 388 481 431 431
3. 9 2. 8 3.0 2.8 2.9
0.010
0.05 0.06 0.07 0.05 0.06 0.07 0.05 0.06 0. 07
246 284 295 287 294 278 296 281 280
191 214 183 84 244 244 175 202 210
413 412 455 359 481 387 535 428 401
2.30 2.20 2.15 2.25 2.15 2.30 2.40 2.35 2.30
Experiment 2
0o 02
0.03
* Medium composition : TSP basal medium with 200 mg/l of Mg *+ and 50 mg/l of Fe~+. Cu*+ a 2 3 % increase was observed. T h e f o r m e r c o m b i n a t i o n was selected for f u r t h e r studies since m o r e r e d u c i n g s u g a r m i g h t b e a v a i l a b l e for further utilization. Citric a c i d f e r m e n t a t i o n involves t h e m a x i m u m c o n t r i b u t i o n o f t h e glycolytic p a t h w a y , w h i c h requires M g 2+ a n d Fe*+ as cofactors.g) T h e s u p p l e m e n t a t i o n o f Cu*+ has also b e e n shown to h e l p t h e c o n s u m p t i o n o f sugar, n i t r o g e n a n d phosphorus.11) Zinc plays an i m p o r t a n t role in m a i n t e n a n c e o f a specific physiological c o n d i t i o n f a v o r i n g m a x i m u m citric a c i d accumulation.12, "~
Effect o f m e t h a n o l a n d n i t r o g e n s o u r c e T h e a d d i t i o n o f m e t h a n o l to c r u d e c a r b o h y d r a t e r a w m a t e r i a l s induces citric a c i d p r o d u c t i o n . 14) I n e a r l i e r studies, 15) w e observed t h a t the isolated A. niger s t r a i n g r o w n on T S P p r e f e r r e d a m m o n i u m n i t r a t e as n i t r o g e n source in the presence o f m e t h a n o l . S i m i l a r findings h a v e b e e n r e p o r t e d b y o t h e r workers.") T h e effects o f 2 % a n d 3 % m e t h a n o l were c o m p a r e d a t various n i t r o g e n e q u i v a l e n t s (0.26 g N , - 0 . 7 0 g N , e q u i v a l e n t s p e r liter) o f a m m o n i u m n i t r a t e a n d a m m o n i u m c a r b o n a t e ( T a b l e 3).
T h e a d d i t i o n o f m e t h a n o l to T M T B m e d i u m w i t h a m m o n i u m c a r b o n a t e as nitrogen source i n h i b i t e d the citric a c i d p r o d u c t i o n . But citric a c i d y i e l d i n c r e a s e d b y 6 . 4 % w h e n a m m o n i u m c a r b o n a t e was r e p l a c e d b y a m m o n i u m n i t r a t e in T M T B w i t h 3 % m e t h a n o l . T h e r e d u c i n g s u g a r level increased w i t h increase in the level o f n i t r o g e n in the presence of 2% and 3% methanol when ammonium n i t r a t e was used as n i t r o g e n source. H o w ever, w h e n a m m o n i u m c a r b o n a t e was used as n i t r o g e n source, the r e d u c i n g sugar increase d w i t h i n c r e a s i n g n i t r o g e n level w i t h 2 % methanol, but decreased with 3% methanol. T h e biomass increased w i t h increasing n i t r o g e n level w h e n a m m o n i u m n i t r a t e was used as n i t r o g e n source, b u t showed the opposite trend when ammonium carbonate was used. Effect o f f e r r o c y a n l d e The addition o f f e r r o c y a n i d e ( 4 0 - 2 0 0 m g / / ) to T M T B m e d i u m w i t h 3 % (v/v) m e t h a n o l i n d u c e d citric a c i d p r o d u c t i o n ( T a b l e 4). Citric a c i d y i e l d increased b y 8 % w h e n 1 6 0 m g / l o f f e r r o c y a n i d e was a d d e d before sterilization. T h e r e d u c i n g s u g a r level a n d t i t r a b l e a c i d i t y
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Table 3. Effect of methanol on citric acid production by A. nigerin the presence of various levels of inorganic nitrogen source. Ammonium nitrate Cone. of Nitrogen Ammonium carbonate Methanol equivalents// Citric Reducing Biomass Titrable Citric Reducing Biomass Titrable (v/v) in g acid sugar (mg/50 ml) acidity acid sugar (rag/50 ml) acidity (/~g/ml) (rag/100 ml) (ml) (~g/ml) (mg-100 ml) (ml) 0o~ 0. 42 295 244 481 4. 0 287 218 368 4. 2 2o~
0.26 0.42 0.70
216 280 276
257 263 297
214 198 201
3.8 3.9 3.7
293 283 283
314 330 330
121 139 157
2.9 3.7 3.8
30/0
0. 26 0.42 0.70
273 297 198
287 273 253
136 93 90
4. 1 4.5 3.4
305 317 308
297 301 301
90 104 116
4. 3 4.3 4.7
decreased. A d d i t i o n o f f e r r o c y a n i d e after sterilization o f t h e m e d i u m gave 14% increase in citric a c i d yield w i t h only 120 mg/l c o n c e n t r a t i o n . T h e r e was a n increase in r e d u c i n g sugar level of 488 mg/100 ml, from 301 mg/100 m l o f the control. A s t e a d y increase in b i o m a s s w i t h ferrocya n i d e c o n c e n t r a t i o n was o b s e r v e d w h e n the f e r r o c y a n i d e was a d d e d before sterilization. Biomass p r o d u c t i o n increased 4.6-fold to 538 m g / 5 0 m l w h e n the o p t i m u m level o f f e r r o c y a n i d e was a d d e d before sterilization. H o w e v e r , o n l y a 1.6-fold increase in biomass was o b s e r v e d w h e n 120 mg/l of f e r r o c y a n i d e was a d d e d after sterilization, w h i c h is the o p t i m u m c o n c e n t r a t i o n for citric a c i d p r o d u c tion. G e n e r a l l y , f e r r o c y a n i d e a d d i t i o n before sterilization (heat t r e a t m e n t ) is effective for
r e m o v a l o f h e a v y m e t a l ions in molasses. 5) O n the o t h e r h a n d , H u s t e d e et al. h a v e shown t h a t the presence of f e r r o c y a n i d e in the e a r l y stages o f f e r m e n t a t i o n after p r e t r e a t m e n t ( r e m o v a l o f h e a v y m e t a l ion) o f molasses s t i m u l a t e d the citric a c i d p r o d u c t i o n , IT) suggesting its function as a m o d u l a t o r o f the T C A cycle. T h e s e findings a r e s u p p o r t e d b y the r e p o r t t h a t the a d d i t i o n o f f e r r o c y a n i d e after p r e t r e a t m e n t o f r a w m a t e r i a l in the presence o f m e t h a n o l also induces citric a c i d production.IS) F e r r o c y a n i d e a d d e d before sterilization m i g h t n o t show significant c h e l a t i n g effect in this s t u d y since t a m a r i n d seed p o w d e r has a low level o f h e a v y m e t a l ions.8) H o w e v e r , it acts as a m o d u l a t o r for the T C A cycle. T h e m a x i m u m yield of citric a c i d ( T a b l e 4, 363/~g/ml) is lower t h a n those in established
Table 4. Effect of ferrocyanide* on citric acid production by A. niger. Concentration of ferrocyanide (rag/l) 40 80 120 160 200
Experiment 1"* Citric acid (~g/ml) 255 263 279 341 329
Reducing Biomass sugar ml) (mg/50 ml) (rag/100 309 312 209 195 195
160 186 398 538 548
Experiment 2** Titrable acidity (ml) 2.9 2.4 2.3 2.3 1.9
Citric acid (~g/ml) 300 328 363 234 213
Reducing Biomass sugarml) (rag/50 ml) (rag/100 251 374 488 488 488
105 129 149 184 208
Titrable (ml) acidity 3. 1 3. 2 3.3 3. 0 2.9
* Control i. e. TMTB medium with 3% methanol, gave only 318/~g/ml citric acid with 4.7 ml of titrable acidity, 301 rag/100 ml of reducing sugar and 116 rag/50 ml of biomass. ** Experiment 1--Ferrocyanide added before sterilization. Experiment 2--Ferrocyanlde added after sterilization.
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Jour. (&i.) India, (1985) in press. 7) Nelson, N.: J. Biol. Chem., 153, 375 (1944). 8) Marier, J. R., Boulet, M. : J. Dairy Sci., 41, 1683 (1958). 9) Dasgupta, J., Nasim, S., Khan, A.W., Vora, V. C. : Indian J. Microbiol., 21, 288 (1981). 10) Kublcek, C.P., Rohr, M.: Eur. J. Appl. Microbiol., 4, 167 (1977). ll) Katarina, J., Cimmerman, A., Perldh, A.: Eur. J. Appl. Microbiol. Biotedmol., 33, 14 (1982). Acknowledgments 12) Williams, S. M. W., Suzuki, I." Can. J. Microbiol., One of the authors (HJP) is thankful to CSIR, 20, 1567 (1974). New Delhi for financial assistance. 13) Williams, S. M. W., Suzuki, I. : Can. J. Microbiol., 22, 1093 (1976)). References 14) Usami, S., Taketomi, N.: Kogyo Kogaku Zasshi, 1) Cielger, A., Raper, K.B.: Arch. Nlicrobiol., 37, 64, 2072 (1961). 188 (1960). 15) Purohit, H.J., Daginawala, H . F . : J. Soc. Pure 2) Trumpy, B.H., Millis, N.F.: J. Gen. Microbiol., Appl. Nat. Sci. (India), 1, 10 (1985). 30, 381 (1963). 16) Noguchi, Y., Arao, O.: J. Ferment. Technol., 38, 3) Srivastava, H. C., Singh, P.P.: CarbohydrateRes., 505 (1960). 4, 826 (1967). 17) Hustede, H., Hermann, R.: U.S. Patent, 3 4) Kumar, K., Ethiraj, S." Ind. Sugar. J., 78, 13 941656 (1976). (1976). 18) Nagashiraa, M., Usami, S.: Kogyo Kogaku Zasshi, 5) Clark, D.S., Ito, K., Horitsu, H.: Biotechnol. 72, 509 (1969). Bioeng., 8, 465 (1966). (Received March 3, 1986) 6) Purohit, H.J., Daginawala, H . F . : Nag. Univ.
procedures. Nevertheless, t h e r a w m a t e r i a l h e r e costs a b o u t o n e - t h i r d as m u c h as t h e c o n v e n t i o n a l r a w m a t e r i a l s . Also t h e usual p r e t r e a t m e n t , i.e., r e m o v a l o f m e t a l ions, is n o t r e q u i r e d w h e n this r a w m a t e r i a l is used. T h i s field still requires c o n s i d e r a b l e a t t e n t i o n , i n c l u d i n g d e v e l o p m e n t o f a m o r e efficient strain.
Note
The Relationship of Some Metal Ions with Citric Acid Production by Aspergillus niger Using Tamarind Seed Powder as Raw Material HEMANT
J.
PUROHIT a n d HATIM F. DAGINAWALA
Department of Biochemistryand Microbiology, Nagpur University,LIT Premises, Nagpur-440010, India Tamarind seed powder (TSP) was used as raw material for microbial production of citric acid. The maximum citric acid production was observed when the TSP-basal medium (TSP with inorganic nitrogen and phosphorus source) was supplemented with Mg a+, Fe *+, Cu ~+ and Zn s+. The use of methanol and ferrocyanide as a modulator for citric acid production was studied. O n methanol supplementation to the medium, ammonium nitrate was found to be better than ammonium carbonate as a source of inorganic nitrogen. Addition of ferrocyanide after sterilization to the medium containing methanol gave the maximum citrate production of 363 #g/ml.
Metal ions are the micronutrients which modulate the biochemical conversions, and direct the sequence of metabolic conversions to result in overproduction of desired seconda r y m e t a b o l i t e s . T h e s e l e c t e d s t r a i n s o f Aspergillus niger a c c u m u l a t e l a r g e a m o u n t s o f c i t r i c acid extracellularly when grown on a minimal s a l t s m e d i u m , 1) b u t a n o v e r a b u n d a n c e of the metal ions leads to excessive vegetative growth at the expense of citrate accumulation. ~ Traditionally, crude carbohydrate r a w m a t e r i a l is p r e - t r e a t e d t o r e m o v e u n d e sirable heavy metal ions. In the present studies, tamarind seed powder (TSP) was t r i e d as a r a w m a t e r i a l . It has a low ash content and approximately 62% alcoholinsoluble polysaccharide.s) I t is a l s o c h e a p e r t h a n o t h e r r a w m a t e r i a l s u s e d for c i t r i c a c i d p r o d u c t i o n s u c h as m o l a s s e s , w h e a t b r a n etc. The effect of ferrocyanide and methanol w a s also s t u d i e d as t h e s e also s t i m u l a t e t h e c i t r i c a c i d p r o d u c t i o n . 4,s~ M a t e r i a l s and M e t h o d s Strain Aspergilluz niger, a strain isolated from waste of a factory which produces TSP from tamarind seeds, was used.
Seed culture The sporulated culture on potato dextrose agar slant was scraped off, mixed with 5 ml of sterile distilled water, transferred to the surface of potato dextrose agar in a flask (25 ml of medium in a 250-mi borosil conical flask) and incubated at 30°C for 72 h. After complete sporulation of the culture, the growth was scraped off and mixed with 30 m] of sterile distilled water on a wrist shaker for I 0 min. Production medium The basal culture medium (TSP basal medium) contained 2% acid-treated TSP (5g TSP in 250 ml 9% HC1 was autoclaved at 115°C for 15 rain), 0.42g N2 equivalent per liter of (NH4)~CO3, and 0.20 g]100 ml KH~PO4, and had a pH of 3. The optimization of C : N : P ratio was done in earlier studies, e) The trace metal ion tamarind seed powder basal medium ( T M T B medium) contained TSP basal medium with 200 mg/l, MgSO4"THsO, 50 mg/l, FeSO4"7H20, 0.06 m M CuSO4.5H20 and 0.09 m M ZnSO4-7HzO. Culture conditions The fermentation was carried out statically. In the 250-mi conical flask, 50 ml of medium was sterilized at 15 lb pressure for 20 min. The sterilized medium was inoculated with 2% seed culture and incubated at 304-I°C with 60-80% relative humidity. The ferrocyanide was added before sterilization to TSP basal medium, and metal ions and methanol were added later. T h e effect of ferrocyanide after sterilization was studied by adding ferrocyanide solution to the sterile T M T B medium with methanol.
562
[J. Ferment. Technol.,
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Analysis of fermentation broth Reducing sugar in the fermented broth was estimated by the method of Nelson, v) and citric acid by the method of Marier and Boulet. 8) After the fermentation, i.e., 72 h of incubation, the broth was filtered through GF/C Whatman glass fiber paper under suction. The biomass retained on the paper was weighed after drying at 80°C for 18 h. The titrable acidity was expressed in terms of 0.01 N NaOH, using phenolphthalein as an indicator. Results and Discussion T h e m a x i m u m citric a c i d y i e l d was observed after 7 2 h o f i n c u b a t i o n . T h e o r g a n i s m utilizes t h e p o l y s a c c h a r i d e of T S P as substrate for citric a c i d p r o d u c t i o n , a n d therefore the c h a n g e in r e d u c i n g s u g a r level was also s t u d i e d for each p a r a m e t e r . Effect o f m e t a l i o n s Mg~+ a n d Fe2+ a r e essential m e t a l ions for m a x i m u m utilization o f sugars a n d for o v e r p r o d u c t i o n of citric a c i d b y A. niger.9) I n t h e p r e s e n t studies, w h e n M g 2+ was a d d e d a t 200 mg/l c o n c e n t r a t i o n as M g S O 4 " 7 H 2 0 , only 6 % increase in y i e l d was observed a n d only 4 % increase in yield was seen w i t h 50 mg/l o f F e S O , . 7 H 2 0 a d d i t i o n ( T a b l e 1). A decrease in biomass a n d increase in final p H w i t h
h i g h e r levels o f b o t h the m e t a l ions was observed. R e d u c i n g sugar decreased w i t h increase in M g *+ level a n d increased w i t h increasing c o n c e n t r a t i o n o f Fe*+, w h e n these m e t a l ions were a d d e d separately. I n t e r estingly, w i t h different c o m b i n a t i o n s of M g 2+ (100-200 mg/l) a n d F e S+ (25-75 mg//) a d d e d to the T S P b a s a l m e d i u m , the results differed d r a s t i c a l l y ( T a b l e 1). T h e simultaneous s u p p l e m e n t a t i o n o f 200 mg/l of M g 2+ a n d 50 mg/l o f F e 8+ increased the citric a c i d yield by 30%. T r a c e s o f o t h e r m e t a l ions such as Z n 2+ a n d C u 2+ also r e g u l a t e the physiology o f A. niger in favor o f citric a c i d production.9,10) T h e different cations were a d d e d a t 0.04 m M c o n c e n t r a t i o n after the sterilization o f T S P b a s a l m e d i u m a l o n g w i t h 200 mg/l of M g 2+ a n d 50 mg/l o f Fe *+. T h e s u p p l e m e n t a t i o n o f C u 2+ a n d Z n 2+ gave a n increase in citric a c i d yield only b y 5.0 a n d 2 . 0 % respectively ( T a b l e 2), b u t their s i m u l t a n e o u s a d d i t i o n further s t i m u l a t e d the citric a c i d p r o d u c t i o n . S i m i l a r findings h a v e b e e n r e p o r t e d earlier.9,x0) A t 0.02 m M Zn*+ a n d 0 . 0 6 m M Cu*+, a 2 2 % increase in citric a c i d y i e l d was observed, a n d a t 0.03 m M Zn*+ a n d 0.05 m M
Table 1. Optimization of Mg 2+ and Fe2+ relationship for citric acid production. Concn. of Mg 2+ (rag/l) 0
100
150
200
Concn. of Fez+ (rag/l)
Citric acid (#g/ml)
0 25 50 75 0 25 50 75 0 25 50 75 0 25 50 75
164 164 169 159 163 199 209 225 164 186 239 212 173 184 242 213
Reducing Biomass sugar (rag/100 ml) (rag/50 ml) 388 398 450 570 370 427 443 504 388 450 443 458 381 397 424 458
518 520 519 493 627 621 586 535 518 601 564 566 627 534 564 545
Final pH 2.30 4.00 4.30 4.90 3.50 3.80 3.95 3.80 3.60 3.85 3.75 3.90 3.60 3.85 3.90 3.75
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Table 2. Relationship between Zn a+ and Cu 2+ metal ion for citric acid production.* Concn. of Zn a+ (raM) Experiment 1 Control
Concn. of Cu ~+ (mM)
Citric acid (pg/ml)
Reducing Biomass sugar (mg/50 ml) (mg/100 ml)
Final pH
-0. 4 0. 6 ---
---0. 4 0. 6
242 248 240 252 267
424 217 191 116 106
564 388 481 431 431
3. 9 2. 8 3.0 2.8 2.9
0.010
0.05 0.06 0.07 0.05 0.06 0.07 0.05 0.06 0. 07
246 284 295 287 294 278 296 281 280
191 214 183 84 244 244 175 202 210
413 412 455 359 481 387 535 428 401
2.30 2.20 2.15 2.25 2.15 2.30 2.40 2.35 2.30
Experiment 2
0o 02
0.03
* Medium composition : TSP basal medium with 200 mg/l of Mg *+ and 50 mg/l of Fe~+. Cu*+ a 2 3 % increase was observed. T h e f o r m e r c o m b i n a t i o n was selected for f u r t h e r studies since m o r e r e d u c i n g s u g a r m i g h t b e a v a i l a b l e for further utilization. Citric a c i d f e r m e n t a t i o n involves t h e m a x i m u m c o n t r i b u t i o n o f t h e glycolytic p a t h w a y , w h i c h requires M g 2+ a n d Fe*+ as cofactors.g) T h e s u p p l e m e n t a t i o n o f Cu*+ has also b e e n shown to h e l p t h e c o n s u m p t i o n o f sugar, n i t r o g e n a n d phosphorus.11) Zinc plays an i m p o r t a n t role in m a i n t e n a n c e o f a specific physiological c o n d i t i o n f a v o r i n g m a x i m u m citric a c i d accumulation.12, "~
Effect o f m e t h a n o l a n d n i t r o g e n s o u r c e T h e a d d i t i o n o f m e t h a n o l to c r u d e c a r b o h y d r a t e r a w m a t e r i a l s induces citric a c i d p r o d u c t i o n . 14) I n e a r l i e r studies, 15) w e observed t h a t the isolated A. niger s t r a i n g r o w n on T S P p r e f e r r e d a m m o n i u m n i t r a t e as n i t r o g e n source in the presence o f m e t h a n o l . S i m i l a r findings h a v e b e e n r e p o r t e d b y o t h e r workers.") T h e effects o f 2 % a n d 3 % m e t h a n o l were c o m p a r e d a t various n i t r o g e n e q u i v a l e n t s (0.26 g N , - 0 . 7 0 g N , e q u i v a l e n t s p e r liter) o f a m m o n i u m n i t r a t e a n d a m m o n i u m c a r b o n a t e ( T a b l e 3).
T h e a d d i t i o n o f m e t h a n o l to T M T B m e d i u m w i t h a m m o n i u m c a r b o n a t e as nitrogen source i n h i b i t e d the citric a c i d p r o d u c t i o n . But citric a c i d y i e l d i n c r e a s e d b y 6 . 4 % w h e n a m m o n i u m c a r b o n a t e was r e p l a c e d b y a m m o n i u m n i t r a t e in T M T B w i t h 3 % m e t h a n o l . T h e r e d u c i n g s u g a r level increased w i t h increase in the level o f n i t r o g e n in the presence of 2% and 3% methanol when ammonium n i t r a t e was used as n i t r o g e n source. H o w ever, w h e n a m m o n i u m c a r b o n a t e was used as n i t r o g e n source, the r e d u c i n g sugar increase d w i t h i n c r e a s i n g n i t r o g e n level w i t h 2 % methanol, but decreased with 3% methanol. T h e biomass increased w i t h increasing n i t r o g e n level w h e n a m m o n i u m n i t r a t e was used as n i t r o g e n source, b u t showed the opposite trend when ammonium carbonate was used. Effect o f f e r r o c y a n l d e The addition o f f e r r o c y a n i d e ( 4 0 - 2 0 0 m g / / ) to T M T B m e d i u m w i t h 3 % (v/v) m e t h a n o l i n d u c e d citric a c i d p r o d u c t i o n ( T a b l e 4). Citric a c i d y i e l d increased b y 8 % w h e n 1 6 0 m g / l o f f e r r o c y a n i d e was a d d e d before sterilization. T h e r e d u c i n g s u g a r level a n d t i t r a b l e a c i d i t y
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Table 3. Effect of methanol on citric acid production by A. nigerin the presence of various levels of inorganic nitrogen source. Ammonium nitrate Cone. of Nitrogen Ammonium carbonate Methanol equivalents// Citric Reducing Biomass Titrable Citric Reducing Biomass Titrable (v/v) in g acid sugar (mg/50 ml) acidity acid sugar (rag/50 ml) acidity (/~g/ml) (rag/100 ml) (ml) (~g/ml) (mg-100 ml) (ml) 0o~ 0. 42 295 244 481 4. 0 287 218 368 4. 2 2o~
0.26 0.42 0.70
216 280 276
257 263 297
214 198 201
3.8 3.9 3.7
293 283 283
314 330 330
121 139 157
2.9 3.7 3.8
30/0
0. 26 0.42 0.70
273 297 198
287 273 253
136 93 90
4. 1 4.5 3.4
305 317 308
297 301 301
90 104 116
4. 3 4.3 4.7
decreased. A d d i t i o n o f f e r r o c y a n i d e after sterilization o f t h e m e d i u m gave 14% increase in citric a c i d yield w i t h only 120 mg/l c o n c e n t r a t i o n . T h e r e was a n increase in r e d u c i n g sugar level of 488 mg/100 ml, from 301 mg/100 m l o f the control. A s t e a d y increase in b i o m a s s w i t h ferrocya n i d e c o n c e n t r a t i o n was o b s e r v e d w h e n the f e r r o c y a n i d e was a d d e d before sterilization. Biomass p r o d u c t i o n increased 4.6-fold to 538 m g / 5 0 m l w h e n the o p t i m u m level o f f e r r o c y a n i d e was a d d e d before sterilization. H o w e v e r , o n l y a 1.6-fold increase in biomass was o b s e r v e d w h e n 120 mg/l of f e r r o c y a n i d e was a d d e d after sterilization, w h i c h is the o p t i m u m c o n c e n t r a t i o n for citric a c i d p r o d u c tion. G e n e r a l l y , f e r r o c y a n i d e a d d i t i o n before sterilization (heat t r e a t m e n t ) is effective for
r e m o v a l o f h e a v y m e t a l ions in molasses. 5) O n the o t h e r h a n d , H u s t e d e et al. h a v e shown t h a t the presence of f e r r o c y a n i d e in the e a r l y stages o f f e r m e n t a t i o n after p r e t r e a t m e n t ( r e m o v a l o f h e a v y m e t a l ion) o f molasses s t i m u l a t e d the citric a c i d p r o d u c t i o n , IT) suggesting its function as a m o d u l a t o r o f the T C A cycle. T h e s e findings a r e s u p p o r t e d b y the r e p o r t t h a t the a d d i t i o n o f f e r r o c y a n i d e after p r e t r e a t m e n t o f r a w m a t e r i a l in the presence o f m e t h a n o l also induces citric a c i d production.IS) F e r r o c y a n i d e a d d e d before sterilization m i g h t n o t show significant c h e l a t i n g effect in this s t u d y since t a m a r i n d seed p o w d e r has a low level o f h e a v y m e t a l ions.8) H o w e v e r , it acts as a m o d u l a t o r for the T C A cycle. T h e m a x i m u m yield of citric a c i d ( T a b l e 4, 363/~g/ml) is lower t h a n those in established
Table 4. Effect of ferrocyanide* on citric acid production by A. niger. Concentration of ferrocyanide (rag/l) 40 80 120 160 200
Experiment 1"* Citric acid (~g/ml) 255 263 279 341 329
Reducing Biomass sugar ml) (mg/50 ml) (rag/100 309 312 209 195 195
160 186 398 538 548
Experiment 2** Titrable acidity (ml) 2.9 2.4 2.3 2.3 1.9
Citric acid (~g/ml) 300 328 363 234 213
Reducing Biomass sugarml) (rag/50 ml) (rag/100 251 374 488 488 488
105 129 149 184 208
Titrable (ml) acidity 3. 1 3. 2 3.3 3. 0 2.9
* Control i. e. TMTB medium with 3% methanol, gave only 318/~g/ml citric acid with 4.7 ml of titrable acidity, 301 rag/100 ml of reducing sugar and 116 rag/50 ml of biomass. ** Experiment 1--Ferrocyanide added before sterilization. Experiment 2--Ferrocyanlde added after sterilization.
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Jour. (&i.) India, (1985) in press. 7) Nelson, N.: J. Biol. Chem., 153, 375 (1944). 8) Marier, J. R., Boulet, M. : J. Dairy Sci., 41, 1683 (1958). 9) Dasgupta, J., Nasim, S., Khan, A.W., Vora, V. C. : Indian J. Microbiol., 21, 288 (1981). 10) Kublcek, C.P., Rohr, M.: Eur. J. Appl. Microbiol., 4, 167 (1977). ll) Katarina, J., Cimmerman, A., Perldh, A.: Eur. J. Appl. Microbiol. Biotedmol., 33, 14 (1982). Acknowledgments 12) Williams, S. M. W., Suzuki, I." Can. J. Microbiol., One of the authors (HJP) is thankful to CSIR, 20, 1567 (1974). New Delhi for financial assistance. 13) Williams, S. M. W., Suzuki, I. : Can. J. Microbiol., 22, 1093 (1976)). References 14) Usami, S., Taketomi, N.: Kogyo Kogaku Zasshi, 1) Cielger, A., Raper, K.B.: Arch. Nlicrobiol., 37, 64, 2072 (1961). 188 (1960). 15) Purohit, H.J., Daginawala, H . F . : J. Soc. Pure 2) Trumpy, B.H., Millis, N.F.: J. Gen. Microbiol., Appl. Nat. Sci. (India), 1, 10 (1985). 30, 381 (1963). 16) Noguchi, Y., Arao, O.: J. Ferment. Technol., 38, 3) Srivastava, H. C., Singh, P.P.: CarbohydrateRes., 505 (1960). 4, 826 (1967). 17) Hustede, H., Hermann, R.: U.S. Patent, 3 4) Kumar, K., Ethiraj, S." Ind. Sugar. J., 78, 13 941656 (1976). (1976). 18) Nagashiraa, M., Usami, S.: Kogyo Kogaku Zasshi, 5) Clark, D.S., Ito, K., Horitsu, H.: Biotechnol. 72, 509 (1969). Bioeng., 8, 465 (1966). (Received March 3, 1986) 6) Purohit, H.J., Daginawala, H . F . : Nag. Univ.
procedures. Nevertheless, t h e r a w m a t e r i a l h e r e costs a b o u t o n e - t h i r d as m u c h as t h e c o n v e n t i o n a l r a w m a t e r i a l s . Also t h e usual p r e t r e a t m e n t , i.e., r e m o v a l o f m e t a l ions, is n o t r e q u i r e d w h e n this r a w m a t e r i a l is used. T h i s field still requires c o n s i d e r a b l e a t t e n t i o n , i n c l u d i n g d e v e l o p m e n t o f a m o r e efficient strain.