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Production of citric acid from corncobs by Aspergillus niger
Bioresource Technology 65 (1998) 251-253 © 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-8524/98 $19.00 ELSEVIER
PII:S0960-8524(98)00015-7
Short Communication containing corncobs were sterlized at 121°C for 15 min, cooled, inoculated (about 2 x 106 conidia/ flask), and incubated at 30°C for 72h in the presence of 3% (v/w) methanol. After fermentation, corncobs were extracted with distilled water in a Waring blender (Fisher Scientific Co., Pittsburgh, PA). After filtration, the filtrates were analyzed for citric acid and total sugar. Effect of methanol concentration on fungal production of citric acid from corncobs was carried out in the same manner as the standard method except that different methanol concentrations (0, 1, 2, 3, 4% v/w) were used. Effect of temperature (23, 30, 37°C) on fungal production of citric acid from corncobs was conducted in the same manner as the standard method. Effect of fermentation time (0, 24, 48, 72, 96, 120, 144 h) on fungal production of citric acid from corncobs was conducted in the same manner as the standard method. Total sugar was measured as glucose by the phenol-sulfuric acid method of Dubois et al. (1956). Citric acid was determined by HPLC under the following conditions: column, Bio-Rad HPX-87H (300×7.8mm); temperature, 65°C; mobile phase, 0-004 M H2SO4; flOW rate, 0"6 ml/min; and detector, Rainin refractive index detector, Model RI-1. Citric acid standard was obtained from Sigma Chemical Co., St Louis, MO. The statistical computer package program used to analyze the experimental data was Statistica General Manova (StatSoft, Inc., Tulsa, OK). The values that have no common superscript are significantly different (p<0-05) according Duncan's multiple range test.
Production of Citric Acid from Corncobs by Aspergillus niger Abstract Corncobs could serve as a substrate for citric acid production by Aspergillus nigeJ: Methanol had a significant effect on fungal production of citric acid from corncobs. Of the four cultures examined, .4. niger N R R L 2001 was found to produce the highest amount of citric acid (250 g/kg dry matter of corncobs) after 72 h of growth at 30°C in the presence of 3% methanol. The yield of citric acid was over 50% based on the amount of sugar consumed. © 1998 Elsevier Science Ltd./tll rights reserved.
INTRODUCTION Corncobs are an important by-product of the sweet corn processing industry that have been either used as animal feed or are returned to the harvested field (Inglett, 1970). In recent years, interest in the microbial conversion of food processing wastes into highvalue products has increased (Hang and Woodams, 1986, 1993; Hang et al., 1987; Shih and Hang, 1996; Tran and Mitchell, 1995). Citric acid is a commercially important product that has been produced by submerged fermentation of glucose or sucrose (Kapoor et al., 1982). The objective of the present study was to evaluate the feasibility of using corncobs as a substrate for citric acid production by A. niger under solid state fermentation conditions. METHODS
Corncobs were obtained from a commercial sweet corn processing plant and stored in a freezer until needed. Prior to use, they were chopped into small pieces in a Hobart chopper. Four citric acid-producing strains of A. niger, N R R L 2001, N R R L 2270, NRRL 328, and NRRL 599 (ARS culture collection, USDA, Nothern Regional Research Laboratory, Peoria, IL), were screened for their ability to produce citric acid on corncobs. Standard experiments were conducted in 500-ml Erlenmeyer flasks, each containing 50 g of chopped corncobs with a moisture content of 25%. All flasks
RESULTS AND DISCUSSION
Table 1 compares the production of citric acid by four citric acid-producing strains of `4. niger after 72 h of growth on corncobs at 30°C in the presence of 0-4% methanol (v/w). The molds produced only a small amount of citric acid (less than 60 g/kg dry weight) from corncobs in the absence of methanol. Addition of methanol resulted in a significant increase in citric acid production from corncobs by the four cultures examined. As the methanol 251
Short communication
252
Table 1. Effect of methanol on citric acid production by four strains of Aspergillus niger after 72 h of growth at 30°C
Citric acid yield (g/kg dry weight)
Methanol cone. (%, v/w) 0 1 2 3 4
NRRL 2001
NRRL 2270
NRRL 328
NRRL 599
53 + 8.29k 168 + 3.69f 227 ± 2.060 254__+4.92a 227 + 9.79cd
30 _ 1.50m 43 + 2.581 67 _ 3.56j 203 ___+2.89e 242 + 4.24b
58 + 4.24k 119 __+1.63h
6.5 +_0.52" 4.7 __+0.40n 28 q-5.86m 82___+4.16i 4.0 + 1.49"
168 q- 6.50 g
234 ___+7.04c 259 + 2.70a
The experimental values (n =4) that have no common superscript are significantly different (p<0.05) according to Duncan's multiple range test.
concentration was increased from 0 to 1, 2, and 3%, the yield of citric acid by A. niger N R R L 2001, for example, was increased from 53 to 168, 227, and 254g/kg dry weight of corncobs, respectively. Methanol at a level of 4% was inhibitory, as it gave significantly lower yields of citric acid by A. niger N R R L 2001 and A. niger N R R L 599. However, A. niger N R R L 2270 and A. niger N R R L 328 produced significantly more citric acid at 4% methanol than at lower methanol concentrations. Methanol has previously been reported to enhance fungal production of citric acid from apple, grape, kiwi and pineapple processing wastes (Hang and Woodams, 1986Hang and Woodams, 1993; Hang et al., 1987; Tran and Mitchell, 1995). Methanol is not assimilated by A. niger and its exact role in stimulating the production of citric acid by A. niger is still not clear. It is likely that methanol affects the permeability properties of the cell membrane and enables greater excretion of citric acid (Kapoor et al., 1982). Because of the results in Table 1, A. niger N R R L 2001 was used in all further experiments. As shown in Table 2, temperature was found to have a profound influence on fungal production of citric acid from corncobs with 3% methanol. A. niger N R R L 2001 produced the highest amount of citric acid (243 g/kg dry weight of corncobs) at 30°C after 72 h of growth on corncobs. Yields of citric acid at 23 and 37°C, for example, were only 86 and 167 g/kg dry weight of corncobs, respectively. Table 3 shows the time course of citric acid production at 30°C by A. niger N R R L 2001 from corncobs with 3% methanol. Citric acid production Table 2. Effect of temperature on citric acid production by A. niger NRRL 2001 after 72 h of growth on corncobs with 3% methanol
Temperature (°C) 23 30 37
Yield of citric acid (g/kg dry weight) 86.3 + 55.30c 243 _ 2.98a 167 + 2.96b
The experimental values (n = 4) that have no common superscript are significantly different (p<0.05) according to Duncan's multiple range test.
Table 3. Time course of citric acid production by A. niger NRRL 2001 from corncobs with 3% methanol at 30*C
Time (h) 0 24 48 72 96 120 144
Yield of citric acid (g/kg dry weight) 5.97 + 0.42f 8.28 + 0.55f 160+3.89 d 243 + 1.43a 228 + 3.45b 192_5.32 c 150 + 1.10c
The experimental values (n = 3) that have no common superscript are significantly different (p <0.05) according to Duncan's multiple range test.
increased rapidly between 24 and 48 h and reached the maximal level after 72h. Total sugar was reduced from an initial concentration of over 460 to about 50 g/kg of dry weight of corncobs after 72 h of fermentation. The yield of citric acid was over 50% based on the amount of sugar consumed. Extending the fermentation beyond 72 h resulted in oxidation of citric acid upon exhaustion of the fermentable sugars. For this reason mainly, the industrial citric acid process is always stopped short of complete utilization of the sugars (Foster, 1949). In this work, the average yield of citric acid was 254 g/kg dry weight of corncobs. Yields of citric acid from pineapple waste, apple pomace, kiwi peel, and grape pomace have been previously reported to be 161, 324, 234, and 267 g/kg dry weight, respectively (Hang and Woodams, 1984, 1986; Hang et al., 1987; Tran and Mitchell, 1995). It is concluded from the results of the present investigation that corncobs could serve as a potential source of raw material for the production of citric acid by A. niger N R R L 2001.
ACKNOWLEDGEMENTS This research was supported in part by Hatch funds (NYG233499). We extend thanks to Mr L. C. Wu for assistance in statistical analyses of the experimental data.
Short communication REFERENCES Dubois, M., Giles, K. A., Hamilton, J. K., Roberts, D. A. & Smith, F. (1956). Colorimetric determination of sugars and related substances. Analytical Chemistry, 28, 350-356. Foster, J. W (1949). ChemicalActivity of Fungi. Academic Press, New York. Hang, Y. D. & Woodams, E. E. (1984). Apple pomace: a potential substrate for citric acid production by Aspergillus niger. Biotechnology Letters, 6, 763-764. Hang, Y. D. & Woodams, E. E. (1986). Utilization of grape pomace for citric acid production by solid state fermentation. American Journal of Enology and Viticulture, 37, 141-142. Hang, Y. D. & Woodams, E. E. (1993). Production of diacetyl reductase by Geotrichum candidum from sauerkraut brine. Bioresource Technology, 43, 181-183. Hang, Y. D., Luh, B. S. & Woodams, E. E. (1987). Microbial production of citric acid from kiwi peel. Journal of Food Science, 52, 226-227. Inglett, G. E. (1970). Corn: Culture, Processing and
253
Products. The AVI Publishing Co., Westport, CT. Kapoor, K. K., Chaudhary, K. and Tauro, P. (1982) Citric acid. In Precott and Dunn's Industrial Microbiology, 4th edn, ed. G. Reed. The AVI Publishing Co., Westport, CT. Shih, C. T. & Hang, Y. D. (1996). Production of carotenoids by Rhodotorula rubra from sauerkraut brine. Food Science and Technology, 29, 570-572. Tran, C. T. & Mitchell, D. A. (1995). Pineapple waste: a novel substrate for citric acid production by solid state fermentation. Biotechnology Letters, 17, 1107-1110.
Y. D. Hang* & E. E. Woodams Department of Food Science and Technology, Cornell University, Geneva, NY 14456, USA (Received 24 April 1997; revised version received 7 January 1998; accepted 8 January 1998) *Author to whom correspondence should be addressed.
PII:S0960-8524(98)00015-7
Short Communication containing corncobs were sterlized at 121°C for 15 min, cooled, inoculated (about 2 x 106 conidia/ flask), and incubated at 30°C for 72h in the presence of 3% (v/w) methanol. After fermentation, corncobs were extracted with distilled water in a Waring blender (Fisher Scientific Co., Pittsburgh, PA). After filtration, the filtrates were analyzed for citric acid and total sugar. Effect of methanol concentration on fungal production of citric acid from corncobs was carried out in the same manner as the standard method except that different methanol concentrations (0, 1, 2, 3, 4% v/w) were used. Effect of temperature (23, 30, 37°C) on fungal production of citric acid from corncobs was conducted in the same manner as the standard method. Effect of fermentation time (0, 24, 48, 72, 96, 120, 144 h) on fungal production of citric acid from corncobs was conducted in the same manner as the standard method. Total sugar was measured as glucose by the phenol-sulfuric acid method of Dubois et al. (1956). Citric acid was determined by HPLC under the following conditions: column, Bio-Rad HPX-87H (300×7.8mm); temperature, 65°C; mobile phase, 0-004 M H2SO4; flOW rate, 0"6 ml/min; and detector, Rainin refractive index detector, Model RI-1. Citric acid standard was obtained from Sigma Chemical Co., St Louis, MO. The statistical computer package program used to analyze the experimental data was Statistica General Manova (StatSoft, Inc., Tulsa, OK). The values that have no common superscript are significantly different (p<0-05) according Duncan's multiple range test.
Production of Citric Acid from Corncobs by Aspergillus niger Abstract Corncobs could serve as a substrate for citric acid production by Aspergillus nigeJ: Methanol had a significant effect on fungal production of citric acid from corncobs. Of the four cultures examined, .4. niger N R R L 2001 was found to produce the highest amount of citric acid (250 g/kg dry matter of corncobs) after 72 h of growth at 30°C in the presence of 3% methanol. The yield of citric acid was over 50% based on the amount of sugar consumed. © 1998 Elsevier Science Ltd./tll rights reserved.
INTRODUCTION Corncobs are an important by-product of the sweet corn processing industry that have been either used as animal feed or are returned to the harvested field (Inglett, 1970). In recent years, interest in the microbial conversion of food processing wastes into highvalue products has increased (Hang and Woodams, 1986, 1993; Hang et al., 1987; Shih and Hang, 1996; Tran and Mitchell, 1995). Citric acid is a commercially important product that has been produced by submerged fermentation of glucose or sucrose (Kapoor et al., 1982). The objective of the present study was to evaluate the feasibility of using corncobs as a substrate for citric acid production by A. niger under solid state fermentation conditions. METHODS
Corncobs were obtained from a commercial sweet corn processing plant and stored in a freezer until needed. Prior to use, they were chopped into small pieces in a Hobart chopper. Four citric acid-producing strains of A. niger, N R R L 2001, N R R L 2270, NRRL 328, and NRRL 599 (ARS culture collection, USDA, Nothern Regional Research Laboratory, Peoria, IL), were screened for their ability to produce citric acid on corncobs. Standard experiments were conducted in 500-ml Erlenmeyer flasks, each containing 50 g of chopped corncobs with a moisture content of 25%. All flasks
RESULTS AND DISCUSSION
Table 1 compares the production of citric acid by four citric acid-producing strains of `4. niger after 72 h of growth on corncobs at 30°C in the presence of 0-4% methanol (v/w). The molds produced only a small amount of citric acid (less than 60 g/kg dry weight) from corncobs in the absence of methanol. Addition of methanol resulted in a significant increase in citric acid production from corncobs by the four cultures examined. As the methanol 251
Short communication
252
Table 1. Effect of methanol on citric acid production by four strains of Aspergillus niger after 72 h of growth at 30°C
Citric acid yield (g/kg dry weight)
Methanol cone. (%, v/w) 0 1 2 3 4
NRRL 2001
NRRL 2270
NRRL 328
NRRL 599
53 + 8.29k 168 + 3.69f 227 ± 2.060 254__+4.92a 227 + 9.79cd
30 _ 1.50m 43 + 2.581 67 _ 3.56j 203 ___+2.89e 242 + 4.24b
58 + 4.24k 119 __+1.63h
6.5 +_0.52" 4.7 __+0.40n 28 q-5.86m 82___+4.16i 4.0 + 1.49"
168 q- 6.50 g
234 ___+7.04c 259 + 2.70a
The experimental values (n =4) that have no common superscript are significantly different (p<0.05) according to Duncan's multiple range test.
concentration was increased from 0 to 1, 2, and 3%, the yield of citric acid by A. niger N R R L 2001, for example, was increased from 53 to 168, 227, and 254g/kg dry weight of corncobs, respectively. Methanol at a level of 4% was inhibitory, as it gave significantly lower yields of citric acid by A. niger N R R L 2001 and A. niger N R R L 599. However, A. niger N R R L 2270 and A. niger N R R L 328 produced significantly more citric acid at 4% methanol than at lower methanol concentrations. Methanol has previously been reported to enhance fungal production of citric acid from apple, grape, kiwi and pineapple processing wastes (Hang and Woodams, 1986Hang and Woodams, 1993; Hang et al., 1987; Tran and Mitchell, 1995). Methanol is not assimilated by A. niger and its exact role in stimulating the production of citric acid by A. niger is still not clear. It is likely that methanol affects the permeability properties of the cell membrane and enables greater excretion of citric acid (Kapoor et al., 1982). Because of the results in Table 1, A. niger N R R L 2001 was used in all further experiments. As shown in Table 2, temperature was found to have a profound influence on fungal production of citric acid from corncobs with 3% methanol. A. niger N R R L 2001 produced the highest amount of citric acid (243 g/kg dry weight of corncobs) at 30°C after 72 h of growth on corncobs. Yields of citric acid at 23 and 37°C, for example, were only 86 and 167 g/kg dry weight of corncobs, respectively. Table 3 shows the time course of citric acid production at 30°C by A. niger N R R L 2001 from corncobs with 3% methanol. Citric acid production Table 2. Effect of temperature on citric acid production by A. niger NRRL 2001 after 72 h of growth on corncobs with 3% methanol
Temperature (°C) 23 30 37
Yield of citric acid (g/kg dry weight) 86.3 + 55.30c 243 _ 2.98a 167 + 2.96b
The experimental values (n = 4) that have no common superscript are significantly different (p<0.05) according to Duncan's multiple range test.
Table 3. Time course of citric acid production by A. niger NRRL 2001 from corncobs with 3% methanol at 30*C
Time (h) 0 24 48 72 96 120 144
Yield of citric acid (g/kg dry weight) 5.97 + 0.42f 8.28 + 0.55f 160+3.89 d 243 + 1.43a 228 + 3.45b 192_5.32 c 150 + 1.10c
The experimental values (n = 3) that have no common superscript are significantly different (p <0.05) according to Duncan's multiple range test.
increased rapidly between 24 and 48 h and reached the maximal level after 72h. Total sugar was reduced from an initial concentration of over 460 to about 50 g/kg of dry weight of corncobs after 72 h of fermentation. The yield of citric acid was over 50% based on the amount of sugar consumed. Extending the fermentation beyond 72 h resulted in oxidation of citric acid upon exhaustion of the fermentable sugars. For this reason mainly, the industrial citric acid process is always stopped short of complete utilization of the sugars (Foster, 1949). In this work, the average yield of citric acid was 254 g/kg dry weight of corncobs. Yields of citric acid from pineapple waste, apple pomace, kiwi peel, and grape pomace have been previously reported to be 161, 324, 234, and 267 g/kg dry weight, respectively (Hang and Woodams, 1984, 1986; Hang et al., 1987; Tran and Mitchell, 1995). It is concluded from the results of the present investigation that corncobs could serve as a potential source of raw material for the production of citric acid by A. niger N R R L 2001.
ACKNOWLEDGEMENTS This research was supported in part by Hatch funds (NYG233499). We extend thanks to Mr L. C. Wu for assistance in statistical analyses of the experimental data.
Short communication REFERENCES Dubois, M., Giles, K. A., Hamilton, J. K., Roberts, D. A. & Smith, F. (1956). Colorimetric determination of sugars and related substances. Analytical Chemistry, 28, 350-356. Foster, J. W (1949). ChemicalActivity of Fungi. Academic Press, New York. Hang, Y. D. & Woodams, E. E. (1984). Apple pomace: a potential substrate for citric acid production by Aspergillus niger. Biotechnology Letters, 6, 763-764. Hang, Y. D. & Woodams, E. E. (1986). Utilization of grape pomace for citric acid production by solid state fermentation. American Journal of Enology and Viticulture, 37, 141-142. Hang, Y. D. & Woodams, E. E. (1993). Production of diacetyl reductase by Geotrichum candidum from sauerkraut brine. Bioresource Technology, 43, 181-183. Hang, Y. D., Luh, B. S. & Woodams, E. E. (1987). Microbial production of citric acid from kiwi peel. Journal of Food Science, 52, 226-227. Inglett, G. E. (1970). Corn: Culture, Processing and
253
Products. The AVI Publishing Co., Westport, CT. Kapoor, K. K., Chaudhary, K. and Tauro, P. (1982) Citric acid. In Precott and Dunn's Industrial Microbiology, 4th edn, ed. G. Reed. The AVI Publishing Co., Westport, CT. Shih, C. T. & Hang, Y. D. (1996). Production of carotenoids by Rhodotorula rubra from sauerkraut brine. Food Science and Technology, 29, 570-572. Tran, C. T. & Mitchell, D. A. (1995). Pineapple waste: a novel substrate for citric acid production by solid state fermentation. Biotechnology Letters, 17, 1107-1110.
Y. D. Hang* & E. E. Woodams Department of Food Science and Technology, Cornell University, Geneva, NY 14456, USA (Received 24 April 1997; revised version received 7 January 1998; accepted 8 January 1998) *Author to whom correspondence should be addressed.