World Journal of Microbiology & Biotechnology 14, 399±404

Selection of a strain of Aspergillus for the

production of citric acid from pineapple waste
in solid-state fermentation

C.T. Tran,* L.I. Sly and D.A. Mitchell

Aspergillus foetidus ACM 3996 (ˆFRR 3558) and three strains of Aspergillus niger ACM 4992 (ˆATCC 9142), ACM
4993 (ˆATCC 10577), ACM 4994 (ˆATCC 12846) were compared for the production of citric acid from pineapple
peel in solid-state fermentation. A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4 g
of citric acid per 100 g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74 g
citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol,
30 °C, an unadjusted initial pH of 3.4, a particle size of 2 mm and 5 ppm Fe2+. Citric acid production was best in
¯asks, with lower yields being obtained in tray and rotating drum bioreactors.

Key words: Aspergillus niger, citric acid, pineapple waste, solid-state fermentation.

Citric acid is widely used in the food, beverage, phar-
maceutical and cosmetic industries and ®nds applica-
tions in a range of other industries, from textiles to
electroplating (Bodie et al. 1994). World-wide demand for
citric acid far exceeds its production (Rohr et al. 1983).
Developing countries could reduce the burden of citric
acid imports on their foreign exchange if they could
produce citric acid locally. This could be achieved by the
solid-state fermentation (SSF) of wastes, since SSF pro-
cesses can often perform economically at relatively low
capacity. In addition, production of citric acid could
offset the disposal costs of the wastes.
Various waste materials have been evaluated for citric
acid production by SSF, including sugar cane bagasse,
grape pomace, apple pomace, wheat bran, sugarcane-
pressmud, coffee husk, kiwifruit peel, kumara, taro and
cassava ®brous waste (Lakshminarayana et al. 1975;
Hang & Woodams 1984, 1985, 1986a, b, 1987; Hang et al.
1987; Shankaranand & Lonsane, 1992, 1993, 1994a; Lu
et al. 1995).
Pineapple peel is a byproduct of pineapple juice ex-
traction and its disposal can pose serious environmental
problems. We have previously shown that pineapple
waste has potential as a substrate for the production of
citric acid by SSF (Tran & Mitchell, 1995). In the present
study we compare several citric acid producing strains,
and optimize citric acid production by the most prom-
ising strain, Aspergillus niger ACM 4992.
Materials and Methods
Micro-organism
A. niger ACM 4992 (ˆATCC 9142), ACM 4993 (ˆATCC 10577),
ACM 4994 (ˆATCC 12846) and Aspergillus foetidus ACM 3996
(ˆFRR 3558) (obtained from the Australian Collection of Mic-
roorganisms, Department of Microbiology, The University of
Queensland) were incubated on PDA slants at 30 °C for 7 days
to allow sporulation and were then stored at 4 °C. Slants were
shaken vigorously for 1 min with 3 ml of sterile water to prepare
spore suspensions containing about 1 ´ 107 spores/ml.

Solid Substrate Preparation and Inoculation

C.T. Tran was and D.A. Mitchell is with the Department of Chemical Engineering
and L.I. Sly is with the Department of Microbiology, The University of Queens-
land, Brisbane, Queensland, 4072, Australia; fax 61 7 33654199. C.T. Tran is
now with the Food Industries Research Institute, Km 8 Nguyen Trai Rd, Dong
Da, Hanoi, Vietnam; fax 84 4 8584554. *Corresponding author.
Wet pineapple waste (Golden Circle Cannery, Brisbane, Aus-
tralia) was dried at 52 °C for 60 h to 7% (w/w) moisture and
stored at room temperature. Prior to use, the dried pineapple
waste was ground with a hammer mill and screened to obtain
the desired particle size. Except for the experiment investigating

ã 1998 Rapid Science Publishers

World Journal of Microbiology & Biotechnology, Vol 14, 1998 399

C.T. Tran et al.
particle size, a 2 mm particle size was used. For solid-state fer-
mentations the dried pineapple waste was adjusted to 65% (w/
w) moisture with distilled water (unless otherwise indicated),
autoclaved (121 °C for 15 min) and allowed to cool. When
methanol was added it was ®lter-sterilized and added prior to
inoculation. Inoculation was at a rate of 2 ml of spore suspen-
sion per 10 g of dried pineapple waste. All cultures were incu-
bated at 30 °C, except for the experiment testing the effect of
temperature.
Flask Experiments
A 10 g quantity of dried pineapple waste was used in each
300 ml Erlenmeyer ¯ask. Treatments varied as noted later. All
experiments were carried out in duplicate ¯asks.
Surface and Submerged Culture
A 100 ml volume of distilled water was added to 5 g portions of
dried pineapple waste in each 300 ml Erlenmeyer ¯ask. Flasks
were autoclaved (121 °C for 15 min), cooled, and inoculated
with 0.2 ml of spore suspension. For submerged culture, ¯asks
were incubated on a rotary shaker at 210 rev/min; for surface
culture the ¯asks remained static.
Bioreactor Experiments
The rotating drum bioreactor was a glass tube (0.18 m diameter
by 1.2 m long) ®tted with end caps and placed on horizontal
rollers in a chamber controlled at 30 °C. Filtered, humidi®ed air
at 30 °C was passed through the bioreactor headspace at 3 l/
min. The bioreactor contained 600 g of dried pineapple waste
processed as described above, and was rotated at 7 rev/min.
When methanol was added the ®nal concentration was 3%
(v/w) (i.e. 3 ml/100 g moist substrate). The trays were 40 ´
28 ´ 8 cm high and contained 500 g of dried pineapple waste
processed as described above; 3% (v/w) methanol was added.
Samples were removed daily for assay of citric acid and residual
sugar.
Results and Discussion
Comparison of Strains for Production of Citric Acid
from Pineapple Waste by SSF
Because the optimum conditions for citric acid produc-
tion vary with the strain, the comparison was made with
a range of moisture contents (Figure 1). For A. foetidus
ACM 3996 and A. niger ACM 4992 and 4994 a moisture
content of 65% (w/w) was optimal. A. niger ACM 4993
gave its highest citric acid content at 60% moisture. The
highest citric acid content of 16.4 g/100 g dry fermented
pineapple waste (DFPW), obtained with A. niger ACM
4992, corresponds to a yield of 0.58 g citric acid/g initial
sugar (0.65 g citric acid/g sugar consumed).
These yields are lower than the 30 g/100 g of apple
pomace reported by Hang & Woodams (1987) for citric
acid production by A. niger NRRL 567 at moisture con-
tents between 65 and 75% and supplemented with 3% of
methanol. Optimal moisture contents from 55% to 75%
have been reported for various strains of A. niger (Hang
& Woodams 1987). The effects of methanol concentra-
tions from 0 to 4% (v/w) on citric acid yields from the
four strains were compared at the optimal initial mois-
ture contents (Figure 2). For A. niger ACM 4992 and
A. niger 4994 the effect of methanol was relatively small.
As noted by Tran & Mitchell (1995), methanol improved
citric acid production by A. foetidus ACM 3996 with op-
timal citric acid contents of around 16 g/100 g DFPW
obtained with 3% (v/w) methanol.

Extraction of Citric Acid

A 10 ml volume of deionized water was added per g of fer-
mented material. Flasks were agitated for 60 min on a rotary
shaker and the slurry was ®ltered through Whatman No.1 ®lter
paper. The solids were washed with deionized water several
times, and the ®ltrate and washings were made up to 500 ml
with deionized water and ®ltered through a 0.2 lm ®lter
membrane.

Analytical Techniques
For moisture contents, samples were dried to a constant weight
at 105 °C. Citric acid was measured with a high-pressure liq-
uid chromatography (HPLC) system consisting of a Water As-
sociates M.45 pump connected to a Perkin Eluent series 200
refractive index detector, and a Waters 740 data module
integrator. The column was a cation exchange, Biorad HPX 87
analytical column in the (H+) form, with an Aminex H+
Microguard cartridge. The column was eluted with 4 mM sul-
phuric acid at 0.6 ml/min. Sucrose, fructose, and glucose were
estimated in a HPLC system comprising a Water Associates QA-
I analyser connected to a Model R 401 refractive Index Detector,
a Water Associates 740 data module integrator, and a cation Figure 1. Effect of moisture content on citric acid production from
exchange±shodex S-801/S column (7.8 mm ´ 250 mm) in the pineapple waste by Aspergillus foetidus ACM 3996 (s) and Asper-
sodium (Na+) form. The column was eluted with water at gillus niger strains ACM 4992 (,), ACM 4993 (h) and ACM 4994 (e),
0.5 ml/min. after 3 days, incubation at 30 °C.

400 World Journal of Microbiology & Biotechnology, Vol 14, 1998

 Citric acid from pineapple waste in SSF

of 65% (w/w). Maximum citric acid contents at 25 and

30 °C were very similar, namely 17 g/100 g DFPW after
4 days, but there was no growth at 35 °C. This may be a
problem in SSF since overheating is a common problem,
and could potentially decrease citric acid yields. In con-
trast, Hang & Woodams (1986b) noted growth of A. niger
at 35 °C, although sporulation was greater than at lower
temperatures.
For the effect of pH, pineapple waste with an initial
pH of 3.4 was compared against pineapple wastes ad-
justed to initial pH values of 2 and 5. Cultures contained
3% (v/w) methanol. The citric acid content was only 4 g/
100 g after 4 days with an initial pH of 2, but as high as
19 g/100 g DFPW after 4 days, with initial pH values of
either 3.4 or 5. The pH was not adjusted in further ex-
periments.

Effect of Culture Method on Citric Acid Production

by Aspergillus niger ACM 4992
Citric acid production, expressed as g citric acid/100 g
Figure 2. Effect of methanol on the production of citric acid from initial dry pineapple waste (IDPW) to enable comparison
pineapple waste by Aspergillus foetidus ACM 3996 (s) and Asper- between the culture methods, was best in SSF compared
gillus niger ACM 4992 (,), 4993 (h) and 4994 (e), after 4 days, with submerged and surface culture (Figure 3). In the
incubation at 30 °C and an initial moisture content of 65% (w/w).
absence of methanol, citric acid levels were relatively low
A. niger ACM 4993 (j) was also incubated with an initial moisture
content of 60% (w/w). in submerged and surface culture, and signi®cant oxalic
acid was produced. Oxalic acid contents at 3 days were
10.9 g/100 g IDPW for submerged culture, 2.5 g/100 g
Because A. niger ACM 4992 produced high citric acid
IDPW for surface culture and only 0.7 g/100 g IDPW for
yields over a range of moisture contents (Figure 1) and
both in the presence and absence of methanol (Figure 2),
it was chosen for further study. Since it can be dif®cult to
control conditions in SSF, it is advantageous to have an
organism with high product yields over a range of con-
ditions. The citric acid content of 17.8 g/100 g DFPW
dried pineapple waste obtained with A. niger ACM 4992
in the presence of 3% methanol represents a yield of
0.64 g citric acid/g initial sugar (0.67 g citric acid/g
sugar consumed).
The improvement of citric acid yields by methanol is a
general phenomenon with strains of A. niger and its ad-
dition has become a common practice in citric acid pro-
duction (Kapoor et al. 1982). Methanol is not assimilated
by A. niger and its role in stimulating the production of
citric acid is to increase cell permeability, enabling
greater excretion of citric acid (Kapoor et al. 1982).
However, it should be noted that the optimal methanol
concentration varies among strains and it can also be
inhibitory, as was observed for strain ACM 4993 above
2% methanol (Figure 2).

Effect of pH and Temperature on Citric Acid Production

by Aspergillus niger ACM 4992
Citric acid production by A. niger ACM 4992 was com-
pared at 25, 30 and 35 °C in the presence of 3% (v/w)
methanol and using the optimal initial moisture content
Figure 3. Comparison of citric acid production from pineapple waste
by Aspergillus niger ACM 4992 in submerged (h, j), surface (e, r)
and solid-state fermentation (s, d). Fermentations were carried out
both in the absence (hollow symbols) and presence of 3% (v/w)
methanol (solid symbols).

World Journal of Microbiology & Biotechnology, Vol 14, 1998 401

C.T. Tran et al.

SSF. In the presence of methanol, oxalic acid production Table 1. Effect of trace elements on citric acid production from
was suppressed for all culture modes: at 3 days the pineapple waste by Aspergillus niger ACM 4992.
contents were 1.0, 0.7 and 0.0 g/100 g IDPW for sub-
merged, surface and solid-state culture, respectively. Citric acid (g/100 g DFPW)
Because good yields and negligible oxalic acid were Level (p.p.m.) Fe2+ Cu2+ Zn2+ Mn2+ Mg2+
produced in SSF, both in the presence and absence of
0 (control) 13.2 17.5 18.1 18.2 17.6
methanol, SSF is the preferred method for citric acid
0.2 13.0 16.5 15.3 18.1
production from pineapple waste. 0.4 13.4 16.5 18.1
0.6 17.2
Effect of Particle Size on Citric Acid Production 0.8 14.0 12.8
by Aspergillus niger ACM 4992 1.0 15.5 12.1 16.9 17.5
In SSF microorganisms grow on the particle surface and 2 15.0 12.2 15.4
therefore initially attack the exposed substrate. The par- 5 16.6 16.8 16.5
10 16.1 13.2 16.8 18.3 16.7
ticle size of the substrate affects the surface area to vol-
20 14.1 15.9 16.6 17.4
ume ratio of the material and the packing density within 100 12.3 4.2 13.6 18.1 18.6
the substrate mass. The surface area to volume ratio in- 200 18.0
creases as the particle size decreases. If the particles are
too small they pack together too tightly, and the inter- Mg2+ had no effect from 0 to 200 p.p.m. Citric acid con-
particle diffusion of oxygen becomes dif®cult. However, tent decreased slightly with increasing Zn2+ content.
if the particles are too big then much of the substrate will Cu2+ inhibited citric acid production, with 100 p.p.m. of
remain inaccessible to the microorganism (Mitchell et al. Cu2+ reducing yields to less than 25% of the control.
1992). Based on these results, pineapple waste was supple-
A. niger ACM 4992 produced 17.4, 17.7, 18.8 and mented with 5 p.p.m. Fe2+ in the further experiments.
18.2 g citric acid/100 g DFPW for particle sizes of 0.75, 1, Citric acid production in submerged liquid culture is
2 and 3 mm, respectively. These are not signi®cantly quite sensitive to the levels of Fe2+, Zn2+ and Mn2+. When
different. However, ®ltering during the recovery process impure carbohydrate sources are used, growth rather
was easiest with the 2 mm particles, so this size was used than citric acid production is favoured at Fe2+ levels as
for further investigations. low as 0.2 p.p.m. (Noyes 1969) and Zn2+ levels as low as
Effect of Ferrocyanide on Citric Acid Production 1 p.p.m. (Shu & Johnson 1948). Likewise levels of Mn2+
by Aspergillus niger ACM 4992 as low as 0.4 p.p.b. reduce citric acid yields (Clark et al.
The production of citric acid by fungi is affected by metal 1966). In the present study, citric acid production was
ions in the medium (Kapoor et al. 1982). Ferrocyanide is unaffected by these trace metals at 20 p.p.m. con®rming
sometimes added to chelate trace metals, such as Fe2+ that the effects of high trace metal contents are overcome
and Zn2+, during autoclaving, to reduce their availability in SSF (Shankaranand & Lonsane 1994b). This may occur
to the microorganism and thereby promote citric acid owing to the distributed nature of the substrate. Growth
production (Purohit & Daginawala 1986). Therefore, the at the surface of particles causes concentration gradients,
effect of ferrocyanide was investigated, but citric acid which might allow metal ions to become limiting at the
production was not stimulated over the range 0± surface, even though they may be present at higher
150 p.p.m., with citric acid levels obtained after 4 days concentrations in the particle interior.
decreasing from 18.2 to 16.0 g/100 g DFPW. The citric The effect of metal ions on citric acid production in
acid content was only 5.2 g/100 g DFPW with 200 p.p.m. SSF varies with the substrate and microorganism, since
ferrocyanide, possibly because all the trace metals were Shankaranand & Lonsane (1994a,b) found that for SSF of
chelated. Ferrocyanide was not added in the following wheat bran by A. niger CFTRI 30, citric acid yields were
experiments. increased 1.4-fold with the addition of 0.1 p.p.m. Zn2+,
1.5-fold with the addition of 0.05 p.p.m. Mn2+ and 1.9-
Effect of Metal Ions on Citric Acid production fold with the addition of 20 p.p.m. Mg2+.
by Aspergillus niger ACM 4992 Copper stimulates A. niger to produce citric acid in-
The dried pineapple waste was supplemented with stead of biomass (Tomilsom et al. 1951). The addition of
various concentrations of FeSO4.7H2O, CuSO4.H2O, 10 p.p.m. Cu2+ to ferrocyanide-treated carob sugars
ZnSO4.7H2O, MnSO4.H2O or MgSO4.7H2O (Table 1). during submerged liquid fermentation by A. niger NRC
Only supplementation with Fe2+ between 1 and A-1-233 considerably increased the yield (Roukas &
10 p.p.m. signi®cantly improved citric acid production Kotzekidou 1987) whereas in SSF of wheat bran by
(P ˆ 0.05), increasing citric acid content by 22% over the A. niger CFTRI 30 addition of 0.2 p.p.m. of Cu2+ increased
control. Mn2+ had no effect from 0 to 100 p.p.m. and citric acid production 1.9-fold (Shankaranand & Lonsane

402 World Journal of Microbiology & Biotechnology, Vol 14, 1998


1994b). However, for the pineapple waste in the present
study, addition of Cu2+ decreased the citric acid contents.
Time course for citric acid production
by Aspergillus niger ACM 4992
The time course for citric acid production by A. niger
ACM 4992 was followed using pineapple waste supple-
mented with 5 p.p.m. Fe2+. On day three the citric acid
content was 18.0 g/100 g DFPW. The maximal citric acid
content of 19.4 g/100 g DFPW was reached on the day 4,
representing a yield of 0.74 g citric acid/g sugar con-
sumed. The citric acid content then decreased slowly to
reach 16.0 g/100 g DFPW on day 7.
Citric Acid Production in Larger-scale Bioreactors in SSF
Citric acid production from pineapple waste in tray and
rotating drum bioreactors was compared with produc-
tion in ¯asks. Citric acid production was best in ¯asks,
with a maximum citric acid content of 14.0 g/100 g
DFPW obtained after 3 days (Figure 4). The citric acid
content was lower than in previous experiments because
pineapple waste originating from a different region was
used. In the tray bioreactor and the rotating drum bio-
reactor operated with and without 3% (v/w) methanol,
the citric acid content peaked at 2 days at less than 8 g/
100 g DFPW and rapidly fell to zero.
For SSF at laboratory scale, citric acid is commonly
produced in Erlenmeyer ¯asks (Hang & Woodams, 1984,
1985; Hang et al. 1987; Shankaranand & Lonsane 1992,
1993, 1994a, b; Lu et al. 1995). Flasks are suitable for in-
Figure 4. Citric acid production from pineapple waste by A. niger
ACM 4992 in ¯ask (h), tray (,), and rotating drum bioreactors (s), in
the presence of 3% (v/w) methanol. The rotating drum bioreactor was
also operated in the absence of methanol (d).
Citric acid from pineapple waste in SSF
vestigation in the laboratory because they are easy to
handle. If a large number of separate ¯asks are used, then
all ¯asks should be representative and individual ¯asks
can be removed daily without disrupting other samples.
In a larger bioreactor, the removal of samples can disrupt
fungal mycelia which may adversely affect growth.
Bioreactor design and operation for citric acid pro-
duction by SSF requires much more attention. Trays are
most often used for large-scale citric acid production
(Lakshminarayana et al. 1975). The tray technique is
simple, but requires a large area and is dif®cult to au-
tomate and is therefore labour intensive; they also allow
relatively little control over the cultural conditions. Ro-
tating drum bioreactors offer more potential for control,
but they can damage fungal mycelia, and heat removal
may still be a problem (Lonsane et al. 1985). Another
bioreactor with potential is the packed bed, which has
been used for citric acid production from apple pomace
(Hang 1988). This bioreactor deserves further attention
because it allows some control over cultural conditions
without damaging fungal mycelia.
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