Utilization of Grape Pomace for Citric Acid

Production by Solid State Fermentation

Y. D. HANG ~ and E. E. W O O D A M S 2
A solid state fermentation method has been developed and is described for citric acid production from grape pomace by Aspergillus niger NRRL 567. The yields of citric acid varied with the pomace varieties and were dependent on the amount of methanol present in the pomace and the fermentation time and temperature. Under optimum fermentation conditions, this method reduced the pomace dry matter by about 43% and yielded more than
90 g of citric acid per kg of grape pomace fermented. The yield was more than 60% based on the amount of fermentable sugar consumed. These results suggest that the use of grape pomace for fungal production of citric
acid could represent an efficient method of minimizing the pomace disposal problems and concomitantly
producing a commercially valuable organic acid.

Grape pomace is the residue left after juice extraction

and constitutes about 16% of the gross grape tonnage (6).
In 1984, approximately 4.9 million metric tons of grapes
were produced in the United States (5). Grape pomace is
rich in carbohydrates, but its nitrogen and phosphorus
contents are low (6). At present, most pomace is dumped
on land despite the increasing disposal problems and the
efforts for by-product utilization (2).
Citric acid is widely used in the food and beverage
industries, pharmaceutical industry, and elsewhere. In
general, citric acid is commercially produced by microbial
submerged fermentation of molasses (3). Because of its
physical nature, grape pomace is not readily amenable to
submerged fermentation. For submerged fermentation to
occur, it was necessary to make a five parts dilution of the
pomace. The yield of citric acid from the dilute pomace
mash was too low to recover economically.
Solid state' fermentation refers to any fermentation
that occurs on solid or semi-solid substrate; such fermentations lead to the production of a variety of fermented
foods, such as cheese, meitauza, miso, soy sauce, sufu, and
tempeh (4). The objective of this study was to develop a
solid state fermentation method for citric acid production
from grape pomace.

sterilized at i21C for 15 minutes. Each flask was inoculated with an optimum quantity of spore inoculum (about
2 x 106 viable spores per flask) and incubated at 30C for
four days. Methanol was added to the flasks at a concentration of 3% (v/w) before fermentation. At the completion of the fermentation, the fermented materials were
extracted with water and the extracts were analyzed for
residual sugar and citric acid.
The citric acid content of grape pomace was determined by the microcolorimetric method of Taussky (7).
In this method, citric acid was oxidized with potassium
permanganate in the presence of bromine to pentabromoacetone, which gave a yellow color with sodium sulfide. The yellow color was measured with a Bausch and
Lomb Spectronic 20 at 420 nm against the reagent blank.
The amount of citric acid contributed by unfermented
pomace has been subtracted from that of the reported
data.
The residual sugar was analyzed as glucose by the
phenol-sulfuric acid method of Dubois et al. (1). In this
procedure, sugars reacted with phenol in the presence of
sulfuric acid to give a stable yellow-orange color with an
absorption maximum at 490 nm.

Materials and Methods

Figure 1 shows the enhanced effect of methanol on

citric acid production from grape pomace by A. niger
N R R L 567. Increasing the concentration of methanol
resulted in a marked increase in the production of citric
acid. The mold produced the greatest amount of citric
acid from grape pomace in the presence of methanol at a
concentration of 3% (v/w). The influence of methanol in
increasing citric acid production appears to be a general
phenomenon with strains of A. niger, and the use of
methanol has become a common practice (3). Methanol is
not assimilated by A. niger, and its exact role in stimulating the production of citric acid is still not clear. It is
likely that methanol affects the permeability properties
and enables greater excretion of citric acid (3).
Figure 2 demonstrates the time course of citric acid
production by A. niger N R R L 567 grown on grape
pomace in the presence of methanol at a concentration of
3% (v/w). The production of citric acid paralleled the
consumption of sugar. Citric acid production increased
rapidly between two and three days and reached the
maximum level on the fourth day. The sugar was reduced
to a minimum level of about 2% on the fourth day,

Results and Discussion

Pomace samples were obtained from our pilot-plant

juice processing unit and stored at - 2 3 C until needed.
Before its use, the pomace was ground through the 0.635
cm screen of a Fitz Mill, Model D (W. J. Fitzpatrick Co.,
Chicago, IL).
Aspergillus niger N R R L 567 was provided by C. W.
Hesseltine, Northern Regional Research Center, US Department of Agriculture, Peoria, Illinois. The culture was
grown on a potato dextrose agar slant at 30 to 33C for
seven days. A spore inoculum was prepared by adding 3
mL of sterile distilled water to the slant and shaking
vigorously for one minute.
Standard solid state fermentation experiments were
conducted as follows: Portions of 40 g of grape pomace
were introduced into 500-mL Erlenmeyer flasks and
1Associate Professor and 2Research Support Specialist, Department of Food Science and
Technology, Cornell University, Geneva, NY 14456.
Presented at the 36th Annual Meeting of the American Society for Enology and Viticulture,
Reno, Nevada (June 1985).
Manuscript submitted for publication 16 July 1985.
Copyright 1986 by the American Society for Enology and Viticulture. All rights reserved.

141
Am. J. Enol. Vitic., Vol. 37, No. 2, 1986

142 m GRAPE POMACE FOR CITRIC ACID

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TIME (days)

Fig. 1. Effect of methanol on fungal production of citric acid from

grape pomace. Experimental conditions were the same as the
standard method except that different methanol concentrations
were used.

Fig. 2. Time course of fungal production of citric acid from grape

pomace. Experimental conditions were the same as the standard
method except that pomace samples were fermented for varying
periods of time.

representing a reduction of more than 88%. The pomace

dry matter was reduced by about 43%. Kapoor et al. (3)
have reported that the time required for maximum citric
acid production by submerged fermentation of molasses
was about seven to 10 days.

Table 2 shows the effect of pomace varieties on the

production of citric acid by the mold. The yields of citric
acid varied from 560 to 600 g per kg of sugar fermented,
depending on the pomace varieties. This variation is
probably attributed to the intrinsic difference in the
chemical composition of pomace varieties.

Table 1. Effect of temperature on fungal production of citric acid

from grape pomace (initial sugar content, 17.3%) a.
Temperature
(C)

Residual
sugar

Yield of
citric acid

25
30
35

14.8
2.5
2.0

14
61
49

(%)

(%)b

Experimental conditions were the same as the standard method

except that pomace samples were fermented at different temperatures.

b Based on sugar consumed.

Temperature has a profound influence on the fungal

production of citric acid from grape pomace (Table 1).
The optimum temperature for citric acid production by
A. niger NRRL 567 grown on grape pomace in the
presence of 3% methanol was found to be 30C. The mold
produced only a small amount of citric acid at 25C in
four days. Sporulation, however, was more marked at
35C than at lower temperatures.
Table 2. Effect of pomace variety on citric acid production by A.
niger NRRL 567 a.
Pomace
variety

pH

Concord
Riesling
Concord/Riesling

3.4
3.8
3.6

Moisture
(%)
53.5
65.4
59.5

Sugar(%)
Initial Residual
10.5
17.3
13.8

1.6
2.0
1.7

Yield of
citric acid

(%)b
56
60
59

(1.1)
a

Experimental conditions were the same as the standard method

except that different pomace varieties were used.

b Based on sugar consumed.

Conclusions
A solid state fermentation method has been developed for the production of citric acid from grape pomace
by A. niger NRRL 567. This method reduced the pomace
dry matter by about 43% and yielded more than 90 g of citric acid per kg of grape pomace in the presence of 3%
methanol at 30C in four days. The yield was more than
60% based on the amount of fermentable sugar consumed. The results of this study indicate that the use of
grape pomace for fungal production of citric acid might
represent an efficient method of minimizing the pomace
disposal problems and concomitantly producing commercially valuable organic acid.

Literature C i t e d
1. Dubois, M., K. A. Giles, J. K. Hamilton, D. A. Roberts, and F.
Smith. Colorimetric methods of determination of sugars and related
substances. Anal. Chem. 28:350-6 (1956).
2. Famuyiwa, O., and C. S. Ough. Grape pomace: possibilities
as animal feed. Am. J. Enol. Vitic. 33:44-6 (1982).
3. Kapoor, K. K., K. Chandhary, and P. Tauro. Citric acid. In:
Prescott and Dunn's Industrial Microbiology, 4th Edition. G. Reed
(Ed.). pp 709-49. AVI Publishing Co., Westport, CT (1982).
4. Kronenberg, J.J., and Y. D. Hang. Biochemical changes
during meitauza fermentation. Nutr. Repts. Intl. 30:439-43 (1984).
5. New York Crop Reporting Service. Fruit Release No. 6-84.
Department of Agriculture and Markets, Albany, NY (1984).
6. Rice, A. C. Solid waste generation and by-product recovery
potential from winery residues. Am. J. Enol. Vitic. 27:21-6 (1976).
7. Taussky, H. H. A microcolorimetric method for the determination of citric acid. J. Biol. Chem. 181:195-8 (1949).

A m . J. E n o l . Vitic., V o l . 37, No. 2, 1 9 8 6