Bioresource Technology 65 (1998) 251-253

1998 Elsevier Science Ltd. All rights reserved

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Short Communication
containing corncobs were sterlized at 121C for
15 min, cooled, inoculated (about 2 x 106 conidia/
flask), and incubated at 30C 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, 37C) 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
(3007.8mm); temperature, 65C; 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 30C 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 30C 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 30C

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 30C after
72 h of growth on corncobs. Yields of citric acid at
23 and 37C, 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 30C 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
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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.