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Citric acid fermentation by gamma ray induced mutants of Aspergillus niger in different carbohydrate media
JOURNALOF FERMENTATIONAND BIOENGINEERING Vol. 70, No. 4, 286-288. 1990
Citric Acid Fermentation by Gamma Ray Induced Mutants of Aspergillus niger in Different Carbohydrate Media A N J U M A N A R A BEGUM, N A I Y Y U M C H O U D H U R Y , * AND M O H A M M A D S E R A J U L ISLAM
Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka, Bangladesh Received 18 November 1988/Accepted 19 July 1990 A natural isolate of Aspergillus niger, CA16, and two of its second step mutants, 136/40 and 277/30, grown on different sugar substrates gave maximum citric acid yields of 34, 70, and 126 mg/ml respectively in sucrose medium. Combination of two sugars in the medium at 50% of each improved the yields of citric acid for the sucrose: glucose, glucose: sorbitol, glucose: xylose, and xylose: sorbitol combinations with the mutant strains. Inclusion of galactose in combinations decreased the citric acid yield.
The majority of the published work on citric acid production in synthetic media refers to either glucose or sucrose (1) and little information is available on the use of lactose, galactose, mannose, or xylose by strains of Aspergillus niger. Glucose is a good sugar for citric acid production but sucrose is still better (2). Some sugars such as galactose and arabinose have been reported to inhibit citric acid production (3, 4). This investigation reports the effects of carbon sources, i.e. xylose, glucose, galactose, mannose, sorbitol, mannitol, sucrose, lactose, and polysaccharides such as starch, xylan, and CMC (carboxy methyl cellulose) on citric acid production by a natural isolate of A. niger and two of its gamma ray induced mutants. In addition some of the substrates with reasonable citric acid yields were combined in a 50 : 50 ratio to determine the effects of combinations of sugars on citric acid production. A natural strain of A. niger, CA16, and two of its second step mutants, 136/40 and 277/30, which gave higher citric acid yields from cane molasses, were selected for this study. Details of mutagenic improvement of the strain C A I 6 have been described elsewhere (5-7). Inocula consisted of spores suspended in sterile distilled water (1 × 106 107/ml) and fermentation was done in 100-ml Erlenmeyer flasks containing 25.0ml of medium, each with 14%0 m o n o (D-glucose, D-galactose, D-mannose, Dxylose, D-sorbitol, D-mannitol) or disaccharide (sucrose or lactose) or 1% polysaccharides (starch, xylan, or CMC) and Prescott salts (NH4NO3, 2.23 g/I; K2HPO4, 1.00 g/I; MgSO4-7H20, 0.23 g//) and pH adjusted to 4.0 with 1 N HC1. Total acidity was measured by titration with 0.1 N N a O H using phenolphthalein as the indicator. Citric acid and residual sugar in culture filtrates were measured spectrophotometrically by the method of Marier and Boulet (8) and Morse (9) respectively. Mycelial dry weight was measured by taking the weight after squeezing and drying the mycelial mat at 70°C for 48 h. The mutant strain 277/30 produced the highest amount of citric acid (126 mg/ml) from sucrose followed by that from glucose (42 mg/ml) compared to the parent, CAI6, and the other mutant, 136/40. No citric acid was produced by any of the strains from mannose, lactose, starch, xylan, or CMC. Some citric acid was formed by the mutants from sorbitol, xylose, fructose, and galactose. Maximum sugar
consumption ( 9 5 ~ ) and mycelial mat production (2.32 g/100 ml) for the strain 277/30 were found from glucose but in sucrose medium 93% sugar was consumed and the mycelial mat weight was 1.08g/100ml (Table 1). Similarly the strain 136/40 grew and produced citric acid on sucrose (70 mg/ml), glucose (20 mg/ml), sorbitol (20mg/ml), xylose (16 mg/ml), and galactose (8 mg/ml) and no citric acid was produced from mannose, lactose, or complex polysaccharides likes starch, xylan, and CMC by strain 136/40 (Table 1). Sugar consumption was maximum (94%o) in galactose medium in which the mycelial growth (dry weight basis) was the highest (2.80 gm/100 ml). The parental strain, CAI6, had its maximum citric acid yield (34 mg/ml) from sucrose and an insignificant amount from glucose (4mg/ml), galactose (6mg/ml), and sorbitol (2 mg/ml). From the rest of the sugars no citric acid was obtained (Table 1). Sugar consumption was the highest (917/00) with sorbitol, which was not reflected in citric acid production. The morphology of all three strains in starch, xylan, CMC, and lactose was changed to gelatinous mats. With combined carbon sources, citric acid yield by the high yielding mutant strain 277/30 was 66 mg/ml in the sucrose (7 ~ ) : glucose ( 7 ~ ) combination medium (Table 2) while in sucrose medium (149/00) alone the yield was 126 mg/ml (Table 1). Citric acid yield by this strain for combination of glucose: fructose, glucose: sorbitol glucose: xylose, and xylose: sorbitol combinations were 51,60, 40, and 52 m g / m l respectively (Table 2). Strain 136/40 had increased citric acid yield of 82 m g / m l on the sucrose: glucose ( 7 ~ : 7?/oo) combination (Table 2), which was higher than that obtained from the media containing sucrose (147/00) or glucose (14°/00) individually (Table 1). The glucose: galactose combination gave the lowest yield of citric acid, 8 and 10 mg/ml by strains 136/40 and 277/30 respectively, though the use of sugar was 94 and 727g, resulting in mycelial weights of 1.88 and 1.12g/100ml in these strains (Table 2). For the parent strain, the lowest citric acid yield was obtained with the xylose: galactose combination (2 mg/ml) on which sugar consumption was 509/ooand the mycelial mat production (dry weight basis) was 1.48 g/100 ml. The combination of glucose: fructose in the medium resulted in a citric acid yield higher than that obtained with glucose: galactose, glucose: sorbitol, glucose: xylan, and xylose: galactose combinations by the mutant strain, 136/40. Similarly for the mutant strain 277/30, citric acid yield for the glucose: fructose combination me-
+' Corresponding author. 286
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TABLE 1. Total titratable acidity (TTA), mycelial dry weight, citric acid production and sugar consumption of A. niger strain CA16, 136/40 and 277/30 grown for 11 d in media containing mono and dissaccharides (14°/00) and polymeric carbohydrates (1%) Carbon sources
Sucrose Glucose Fructose Galactose Uannose Sorbitol Mannitol Xylose lactose .Starch .Xylan -CMC
TABLE 2.
TTA (ml 0.1 N NaOH/ml medium)
Sugar consumed (%)
Mycelial dry weight (gm/100 ml)
Citric acid produced (mg/ml)
Citric acid in relation to sugar supplied (% w/w)
CAI6 136/40 277/30 5.0 13.2 17.6 2.8 6.4 10.8 2.2 1.2 1.6 1.0 2.2 3.6 1.4 2.2 2.4 1.2 5.4 5.2 1.6 2.0 1.20 1.2 2.8 4.4 0.8 3.8 1.6 0.8 1.0 2.0 1.25 2.2 0.8 2.8 0.8 1.4
CA16 136/40 277/30 65 80 93 73 73 95 51 61 64 27 94 71 68 42 77 91 91 93 7l 70 55 63 82 62 48 51 52 40 20 80 50 50 90 50 20 80
CAI6 136/40 277/30 1.04 2.4 1.08 1.92 1.51 2.32 2.28 1.76 1.80 0.84 2.80 1.93 1.80 2.80 1.24 1.80 1.32 1.40 0.88 0.94 1.08 1.64 1.52 1.05 0.40 0.40 0.40 0.12 0.08 0.08 0.08 0.12 0.12 0.08 0.04 0.08
CAI6 136/40 277/30 34 70 126 4 20 42 9 2.0 5 6 8 6 0 0 0 2 20 21 0 2 0 0 16 14 0 0 0 0 0 0 0 0 0 0 0 0
CAI6 136/40 277/30 24 50 90 2.8 14 30 6.1 1.4 3.5 4.2 5.7 4.3 0 0 0 1.4 14 15 0 2.1 0 0 11 l0 0 0 0 0 0 0 0 0 0 0 0 0
Total titratable acidity, mycelial dry weight, citric acid production and sugar consumption of A. niger, strains CAI6, 136/40 and 277/30 grown for 11 d in media containing combined sugar substrate (14%o) in 50 : 50 ratio
TTA on peak day of acid production Combination of sugar (ml 0.1 N NaOH/ml media) Sucrose: glucose Fructose: glucose Glucose: galactose Glucose: sorbitol Glucose: xylose Xylose: galactose Xylose: sorbitol
CA16 136/40 277/30 5.2 15.6 13.3 3.0 10.8 10.0 4.0 2.0 2.0 1.0 11.6 11.6 1.2 6.0 5.0 0.8 2.8 3.6 1.0 7.2 10.8
Sugar consumed (%)
Mycelial dry weight (gm/100 ml)
Citric acid produced (mg/ml)
Citric acid in relation to sugar supplied (% w/w)
CA16 136/40 277/30 78 91 92 76 84 78 57 94 72 58 87 96 60 85 87 50 84 77 50 91 95
CA16 136/40 277/30 1.98 1.0 2.44 1.50 2.08 2.41 1.40 1.88 1.12 0.90 1.57 1.12 1.54 1.84 1.92 1.48 1.33 1.67 1.16 0.68 1.09
CA16 136/40 277/30 24 82 66 21 64 51 8 8 10 6 48 60 34 25 40 2 14 20 0 78 52
CA16 136/40 277/30 17 58 47 15 45 36 5 5 7 4 34 42 24 17 28 1 10 14 0 20 37
d i u m was h i g h e r t h a n t h a t o b t a i n e d w i t h g l u c o s e : galacrose, g l u c o s e : x y l a n , a n d xylose: g a l a c t o s e c o m b i n a t i o n s ( T a b l e 2). H o w e v e r t h e yield was l o w e r t h a n t h a t o b s e r v e d in t h e s u c r o s e m e d i u m ( T a b l e 1). It c a n b e o b s e r v e d f r o m T a b l e 1 t h a t citric acid yield is very low in f r u c t o s e m e d i u m . T h e p a r e n t s t r a i n C A 1 6 h a d a yield o f 34 m g / m l in t h e glucose: xylose c o m b i n a t i o n , w h i c h was c o m p a r a b l e to t h a t o b t a i n e d f r o m s u c r o s e (14%0) a l o n e in t h e m e d i u m ( T a b l e 2). A. niger h a s b e e n r e p o r t e d t o a s s i m i l a t e m a n n o s e a n d xylose r e a d i l y a n d p r o d u c e citric acid a l t h o u g h t h e yield is low c o m p a r e d t o t h a t o b t a i n e d w i t h g l u c o s e (4). H o s s a i n et al. (10) r e p o r t e d little citric acid p r o d u c t i o n b y A . niger w i t h l a c t o s e a n d n o p r o d u c t i o n w i t h g a l a c t o s e as c a r b o n sources. Sucrose and glucose possibly suppress the activity of 2-oxoglutarate dehydrogenase, thereby causing accum u l a t i o n o f citric acid b y t h e s t r a i n s t e s t e d in this s t u d y . Thus the nature of the carbohydrate source has a marked effect o n citric acid p r o d u c t i o n a n d t h i s effect is a g a i n s t r a i n - d e p e n d e n t . A low yield o f citric acid was o b t a i n e d w h e n g a l a c t o s e was u s e d as t h e c a r b o n s o u r c e in c o m b i n a t i o n w i t h glucose w h e r e t h e yield in t h e c o m b i n a t i o n m e d i u m was less t h a n t h e t h e o r e t i c a l yield c a l c u l a t e d o n t h e b a s i s o f 50%o yield in m e d i u m c o n t a i n i n g h a l f t h e o r i g i n a l c o n c e n t r a t i o n o f t h e i n d i v i d u a l sugars. H o w e v e r , this effect o f galactose was n o t o b v i o u s w h e n u s e d in c o m b i n a t i o n w i t h t h e p e n t o s e s u g a r xylose. The mutant strains 136/40 and 277/30 showed higher citric acid yield w h e n s o r b i t o l was u s e d in s u g a r c o m b i n a t i o n s w i t h g l u c o s e a n d xylose. I n r e l a t i o n t o s o r b i t o l in t h e m e d i u m , t h e yield in t h e c o m b i n e d m e d i u m was c o m -
p a r a t i v e l y h i g h e r t h a n t h e t h e o r e t i c a l yield c a l c u l a t e d o n t h e b a s i s o f h a l f t h e c o n c e n t r a t i o n (7%o) o f i n d i v i d u a l s u g a r levels in t h e m e d i u m . T h u s it seems t h a t t h e p r e s e n c e o f s o r b i t o l in t h e m e d i u m c o n t a i n i n g g l u c o s e o r xylose f a v o r s citric acid a c c u m u l a t i o n b y all t h e t h r e e s t r a i n s . T h e n o t a b l e f e a t u r e o f this i n v e s t i g a t i o n was t h a t t h e p a r e n t s t r a i n C A 1 6 h a d a m a r k e d i n c r e a s e in citric acid yield ( 3 4 m g / m l ) in m e d i u m c o n t a i n i n g g l u c o s e ( 7 % ) + x y l o s e (7°/oo) in c o m b i n a t i o n . T h e s t r a i n h a d a yield o f 4 m g / m l in g l u c o s e m e d i u m a n d n o citric acid yield f r o m xylose a l o n g . T h e citric acid yield w i t h t h e glucose: xylose c o m b i n a t i o n was f o u n d to r e a c h t h e yield o b t a i n e d f r o m s u c r o s e a l o n e i n d i c a t i n g a l t e r e d m e t a b o l i c a c t i v i t y o f t h e p a r e n t s t r a i n in r e l a t i o n to citric acid p r o d u c t i v i t y in t h e p r e s e n c e o f glucose a n d xylose t o g e t h e r in t h e m e d i u m . REFERENCES 1. Kapoor, K. K., Chaudhury, K., and Tauro, P.: Citric acid, p. 709-747. In Gerald, R. (ed.), Industrial microbiology, 4th ed. AVI Publishing Co., Inc., Westport, Conn. (1982). 2. Srivastava, K. S. and Kamal, K.: Effect of some chemical factors on the growth and citric acid production by Aspergillus niger. J. Indian Botanical Soc., 58, 208-214 (1979). 3. Hossain, M., Brooks, J. D., and Maddox, I. S.: Galactose inhibition of citric acid production from glucose by Aspergillus niger. Appl. Microbial Biotechnol., 22(2), 98-102 (1985). 4. Maddox, I. S., Spencer, K., Greenwood, J. M., Dawson, M. W., and Brooks, J. D.: Production of citric acid from sugars present in wood hemicellulose using Aspergillus niger and Saccharomycopsis lipolytica. Biotechnol. Lett., 7, 815-818 (1985). 5. Hannan, M. A.: Variants of Aspergillus niger induced by gamma
288
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rays. Ind. J. Expt. Biol., 10, 370-381 (1972). 6. Hannan, M. A., Sarwar, M. G., Baten, A., and Choudhury, N.: Stepwise mutational improvement of Aspergillus niger for citric acid productivity in cane molasses. Folia Microbiol., 21,409-412 (1976). 7. Hannan, M. A., Rabbi, F., Rahmao, A. T. M., and Choudhury, N.: Analysis of some mutants of Aspergillus niger for citric acid production. J. Ferment. Technol., 51(8), 606-608 (1973).
J. FERMENT. BIOENG., 8. Marier, J. R. and Boulet, M.:Direct determination of citric acid in milk with improved pyridine--acetic anhydride method. J. Diary Sci., 41, 1683-1988 (1958). 9. Morse, E.E.: Anthrone in estimating low concentration of sucrose. Anal. Chem., 19, 1012-1013 (1947). 10. Hossain, M., Brooks, J. D., and Maddox, I. S.: The effect of the sugar source on citric acid production by Aspergillus niger. Appl. Microbiol. Biotechnol., 19, 393-397 (1984).
Citric Acid Fermentation by Gamma Ray Induced Mutants of Aspergillus niger in Different Carbohydrate Media A N J U M A N A R A BEGUM, N A I Y Y U M C H O U D H U R Y , * AND M O H A M M A D S E R A J U L ISLAM
Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka, Bangladesh Received 18 November 1988/Accepted 19 July 1990 A natural isolate of Aspergillus niger, CA16, and two of its second step mutants, 136/40 and 277/30, grown on different sugar substrates gave maximum citric acid yields of 34, 70, and 126 mg/ml respectively in sucrose medium. Combination of two sugars in the medium at 50% of each improved the yields of citric acid for the sucrose: glucose, glucose: sorbitol, glucose: xylose, and xylose: sorbitol combinations with the mutant strains. Inclusion of galactose in combinations decreased the citric acid yield.
The majority of the published work on citric acid production in synthetic media refers to either glucose or sucrose (1) and little information is available on the use of lactose, galactose, mannose, or xylose by strains of Aspergillus niger. Glucose is a good sugar for citric acid production but sucrose is still better (2). Some sugars such as galactose and arabinose have been reported to inhibit citric acid production (3, 4). This investigation reports the effects of carbon sources, i.e. xylose, glucose, galactose, mannose, sorbitol, mannitol, sucrose, lactose, and polysaccharides such as starch, xylan, and CMC (carboxy methyl cellulose) on citric acid production by a natural isolate of A. niger and two of its gamma ray induced mutants. In addition some of the substrates with reasonable citric acid yields were combined in a 50 : 50 ratio to determine the effects of combinations of sugars on citric acid production. A natural strain of A. niger, CA16, and two of its second step mutants, 136/40 and 277/30, which gave higher citric acid yields from cane molasses, were selected for this study. Details of mutagenic improvement of the strain C A I 6 have been described elsewhere (5-7). Inocula consisted of spores suspended in sterile distilled water (1 × 106 107/ml) and fermentation was done in 100-ml Erlenmeyer flasks containing 25.0ml of medium, each with 14%0 m o n o (D-glucose, D-galactose, D-mannose, Dxylose, D-sorbitol, D-mannitol) or disaccharide (sucrose or lactose) or 1% polysaccharides (starch, xylan, or CMC) and Prescott salts (NH4NO3, 2.23 g/I; K2HPO4, 1.00 g/I; MgSO4-7H20, 0.23 g//) and pH adjusted to 4.0 with 1 N HC1. Total acidity was measured by titration with 0.1 N N a O H using phenolphthalein as the indicator. Citric acid and residual sugar in culture filtrates were measured spectrophotometrically by the method of Marier and Boulet (8) and Morse (9) respectively. Mycelial dry weight was measured by taking the weight after squeezing and drying the mycelial mat at 70°C for 48 h. The mutant strain 277/30 produced the highest amount of citric acid (126 mg/ml) from sucrose followed by that from glucose (42 mg/ml) compared to the parent, CAI6, and the other mutant, 136/40. No citric acid was produced by any of the strains from mannose, lactose, starch, xylan, or CMC. Some citric acid was formed by the mutants from sorbitol, xylose, fructose, and galactose. Maximum sugar
consumption ( 9 5 ~ ) and mycelial mat production (2.32 g/100 ml) for the strain 277/30 were found from glucose but in sucrose medium 93% sugar was consumed and the mycelial mat weight was 1.08g/100ml (Table 1). Similarly the strain 136/40 grew and produced citric acid on sucrose (70 mg/ml), glucose (20 mg/ml), sorbitol (20mg/ml), xylose (16 mg/ml), and galactose (8 mg/ml) and no citric acid was produced from mannose, lactose, or complex polysaccharides likes starch, xylan, and CMC by strain 136/40 (Table 1). Sugar consumption was maximum (94%o) in galactose medium in which the mycelial growth (dry weight basis) was the highest (2.80 gm/100 ml). The parental strain, CAI6, had its maximum citric acid yield (34 mg/ml) from sucrose and an insignificant amount from glucose (4mg/ml), galactose (6mg/ml), and sorbitol (2 mg/ml). From the rest of the sugars no citric acid was obtained (Table 1). Sugar consumption was the highest (917/00) with sorbitol, which was not reflected in citric acid production. The morphology of all three strains in starch, xylan, CMC, and lactose was changed to gelatinous mats. With combined carbon sources, citric acid yield by the high yielding mutant strain 277/30 was 66 mg/ml in the sucrose (7 ~ ) : glucose ( 7 ~ ) combination medium (Table 2) while in sucrose medium (149/00) alone the yield was 126 mg/ml (Table 1). Citric acid yield by this strain for combination of glucose: fructose, glucose: sorbitol glucose: xylose, and xylose: sorbitol combinations were 51,60, 40, and 52 m g / m l respectively (Table 2). Strain 136/40 had increased citric acid yield of 82 m g / m l on the sucrose: glucose ( 7 ~ : 7?/oo) combination (Table 2), which was higher than that obtained from the media containing sucrose (147/00) or glucose (14°/00) individually (Table 1). The glucose: galactose combination gave the lowest yield of citric acid, 8 and 10 mg/ml by strains 136/40 and 277/30 respectively, though the use of sugar was 94 and 727g, resulting in mycelial weights of 1.88 and 1.12g/100ml in these strains (Table 2). For the parent strain, the lowest citric acid yield was obtained with the xylose: galactose combination (2 mg/ml) on which sugar consumption was 509/ooand the mycelial mat production (dry weight basis) was 1.48 g/100 ml. The combination of glucose: fructose in the medium resulted in a citric acid yield higher than that obtained with glucose: galactose, glucose: sorbitol, glucose: xylan, and xylose: galactose combinations by the mutant strain, 136/40. Similarly for the mutant strain 277/30, citric acid yield for the glucose: fructose combination me-
+' Corresponding author. 286
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NOTES
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TABLE 1. Total titratable acidity (TTA), mycelial dry weight, citric acid production and sugar consumption of A. niger strain CA16, 136/40 and 277/30 grown for 11 d in media containing mono and dissaccharides (14°/00) and polymeric carbohydrates (1%) Carbon sources
Sucrose Glucose Fructose Galactose Uannose Sorbitol Mannitol Xylose lactose .Starch .Xylan -CMC
TABLE 2.
TTA (ml 0.1 N NaOH/ml medium)
Sugar consumed (%)
Mycelial dry weight (gm/100 ml)
Citric acid produced (mg/ml)
Citric acid in relation to sugar supplied (% w/w)
CAI6 136/40 277/30 5.0 13.2 17.6 2.8 6.4 10.8 2.2 1.2 1.6 1.0 2.2 3.6 1.4 2.2 2.4 1.2 5.4 5.2 1.6 2.0 1.20 1.2 2.8 4.4 0.8 3.8 1.6 0.8 1.0 2.0 1.25 2.2 0.8 2.8 0.8 1.4
CA16 136/40 277/30 65 80 93 73 73 95 51 61 64 27 94 71 68 42 77 91 91 93 7l 70 55 63 82 62 48 51 52 40 20 80 50 50 90 50 20 80
CAI6 136/40 277/30 1.04 2.4 1.08 1.92 1.51 2.32 2.28 1.76 1.80 0.84 2.80 1.93 1.80 2.80 1.24 1.80 1.32 1.40 0.88 0.94 1.08 1.64 1.52 1.05 0.40 0.40 0.40 0.12 0.08 0.08 0.08 0.12 0.12 0.08 0.04 0.08
CAI6 136/40 277/30 34 70 126 4 20 42 9 2.0 5 6 8 6 0 0 0 2 20 21 0 2 0 0 16 14 0 0 0 0 0 0 0 0 0 0 0 0
CAI6 136/40 277/30 24 50 90 2.8 14 30 6.1 1.4 3.5 4.2 5.7 4.3 0 0 0 1.4 14 15 0 2.1 0 0 11 l0 0 0 0 0 0 0 0 0 0 0 0 0
Total titratable acidity, mycelial dry weight, citric acid production and sugar consumption of A. niger, strains CAI6, 136/40 and 277/30 grown for 11 d in media containing combined sugar substrate (14%o) in 50 : 50 ratio
TTA on peak day of acid production Combination of sugar (ml 0.1 N NaOH/ml media) Sucrose: glucose Fructose: glucose Glucose: galactose Glucose: sorbitol Glucose: xylose Xylose: galactose Xylose: sorbitol
CA16 136/40 277/30 5.2 15.6 13.3 3.0 10.8 10.0 4.0 2.0 2.0 1.0 11.6 11.6 1.2 6.0 5.0 0.8 2.8 3.6 1.0 7.2 10.8
Sugar consumed (%)
Mycelial dry weight (gm/100 ml)
Citric acid produced (mg/ml)
Citric acid in relation to sugar supplied (% w/w)
CA16 136/40 277/30 78 91 92 76 84 78 57 94 72 58 87 96 60 85 87 50 84 77 50 91 95
CA16 136/40 277/30 1.98 1.0 2.44 1.50 2.08 2.41 1.40 1.88 1.12 0.90 1.57 1.12 1.54 1.84 1.92 1.48 1.33 1.67 1.16 0.68 1.09
CA16 136/40 277/30 24 82 66 21 64 51 8 8 10 6 48 60 34 25 40 2 14 20 0 78 52
CA16 136/40 277/30 17 58 47 15 45 36 5 5 7 4 34 42 24 17 28 1 10 14 0 20 37
d i u m was h i g h e r t h a n t h a t o b t a i n e d w i t h g l u c o s e : galacrose, g l u c o s e : x y l a n , a n d xylose: g a l a c t o s e c o m b i n a t i o n s ( T a b l e 2). H o w e v e r t h e yield was l o w e r t h a n t h a t o b s e r v e d in t h e s u c r o s e m e d i u m ( T a b l e 1). It c a n b e o b s e r v e d f r o m T a b l e 1 t h a t citric acid yield is very low in f r u c t o s e m e d i u m . T h e p a r e n t s t r a i n C A 1 6 h a d a yield o f 34 m g / m l in t h e glucose: xylose c o m b i n a t i o n , w h i c h was c o m p a r a b l e to t h a t o b t a i n e d f r o m s u c r o s e (14%0) a l o n e in t h e m e d i u m ( T a b l e 2). A. niger h a s b e e n r e p o r t e d t o a s s i m i l a t e m a n n o s e a n d xylose r e a d i l y a n d p r o d u c e citric acid a l t h o u g h t h e yield is low c o m p a r e d t o t h a t o b t a i n e d w i t h g l u c o s e (4). H o s s a i n et al. (10) r e p o r t e d little citric acid p r o d u c t i o n b y A . niger w i t h l a c t o s e a n d n o p r o d u c t i o n w i t h g a l a c t o s e as c a r b o n sources. Sucrose and glucose possibly suppress the activity of 2-oxoglutarate dehydrogenase, thereby causing accum u l a t i o n o f citric acid b y t h e s t r a i n s t e s t e d in this s t u d y . Thus the nature of the carbohydrate source has a marked effect o n citric acid p r o d u c t i o n a n d t h i s effect is a g a i n s t r a i n - d e p e n d e n t . A low yield o f citric acid was o b t a i n e d w h e n g a l a c t o s e was u s e d as t h e c a r b o n s o u r c e in c o m b i n a t i o n w i t h glucose w h e r e t h e yield in t h e c o m b i n a t i o n m e d i u m was less t h a n t h e t h e o r e t i c a l yield c a l c u l a t e d o n t h e b a s i s o f 50%o yield in m e d i u m c o n t a i n i n g h a l f t h e o r i g i n a l c o n c e n t r a t i o n o f t h e i n d i v i d u a l sugars. H o w e v e r , this effect o f galactose was n o t o b v i o u s w h e n u s e d in c o m b i n a t i o n w i t h t h e p e n t o s e s u g a r xylose. The mutant strains 136/40 and 277/30 showed higher citric acid yield w h e n s o r b i t o l was u s e d in s u g a r c o m b i n a t i o n s w i t h g l u c o s e a n d xylose. I n r e l a t i o n t o s o r b i t o l in t h e m e d i u m , t h e yield in t h e c o m b i n e d m e d i u m was c o m -
p a r a t i v e l y h i g h e r t h a n t h e t h e o r e t i c a l yield c a l c u l a t e d o n t h e b a s i s o f h a l f t h e c o n c e n t r a t i o n (7%o) o f i n d i v i d u a l s u g a r levels in t h e m e d i u m . T h u s it seems t h a t t h e p r e s e n c e o f s o r b i t o l in t h e m e d i u m c o n t a i n i n g g l u c o s e o r xylose f a v o r s citric acid a c c u m u l a t i o n b y all t h e t h r e e s t r a i n s . T h e n o t a b l e f e a t u r e o f this i n v e s t i g a t i o n was t h a t t h e p a r e n t s t r a i n C A 1 6 h a d a m a r k e d i n c r e a s e in citric acid yield ( 3 4 m g / m l ) in m e d i u m c o n t a i n i n g g l u c o s e ( 7 % ) + x y l o s e (7°/oo) in c o m b i n a t i o n . T h e s t r a i n h a d a yield o f 4 m g / m l in g l u c o s e m e d i u m a n d n o citric acid yield f r o m xylose a l o n g . T h e citric acid yield w i t h t h e glucose: xylose c o m b i n a t i o n was f o u n d to r e a c h t h e yield o b t a i n e d f r o m s u c r o s e a l o n e i n d i c a t i n g a l t e r e d m e t a b o l i c a c t i v i t y o f t h e p a r e n t s t r a i n in r e l a t i o n to citric acid p r o d u c t i v i t y in t h e p r e s e n c e o f glucose a n d xylose t o g e t h e r in t h e m e d i u m . REFERENCES 1. Kapoor, K. K., Chaudhury, K., and Tauro, P.: Citric acid, p. 709-747. In Gerald, R. (ed.), Industrial microbiology, 4th ed. AVI Publishing Co., Inc., Westport, Conn. (1982). 2. Srivastava, K. S. and Kamal, K.: Effect of some chemical factors on the growth and citric acid production by Aspergillus niger. J. Indian Botanical Soc., 58, 208-214 (1979). 3. Hossain, M., Brooks, J. D., and Maddox, I. S.: Galactose inhibition of citric acid production from glucose by Aspergillus niger. Appl. Microbial Biotechnol., 22(2), 98-102 (1985). 4. Maddox, I. S., Spencer, K., Greenwood, J. M., Dawson, M. W., and Brooks, J. D.: Production of citric acid from sugars present in wood hemicellulose using Aspergillus niger and Saccharomycopsis lipolytica. Biotechnol. Lett., 7, 815-818 (1985). 5. Hannan, M. A.: Variants of Aspergillus niger induced by gamma
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J. FERMENT. BIOENG., 8. Marier, J. R. and Boulet, M.:Direct determination of citric acid in milk with improved pyridine--acetic anhydride method. J. Diary Sci., 41, 1683-1988 (1958). 9. Morse, E.E.: Anthrone in estimating low concentration of sucrose. Anal. Chem., 19, 1012-1013 (1947). 10. Hossain, M., Brooks, J. D., and Maddox, I. S.: The effect of the sugar source on citric acid production by Aspergillus niger. Appl. Microbiol. Biotechnol., 19, 393-397 (1984).