Appl Microbiol Biotechnol (1992) 36:518-524
Applied
A comparative study on the formation of citric acid
and polyols and on morphological changes of three strains
of free and immobilized Asperyillus niyer
Sanaa Hamdy Omar*, Siegfried Honecker, and Hans-Jiirgen Rehm
Institut for Mikrobiologie der Westf~ilischen Wilhelms-Universit~t, Corrensstrasse 3, W-4400 Miinster, Federal Republic of Germany
Received 31 July 1991/Accepted 6 September 1991
Summary. The formation of citric acid, oxalic acid, ery-
thritol and glycerol by three strains of Asperoillus niger
immobilized in calcium alginate was investigated and
compared with that of free cells when cultivated in
shake flasks under phosphate limitation. Morphologi-
cal changes were followed using an electron micro-
scope. The production of acids and polyols, the con-
sumption of glucose and fructose, and also the morpho-
logical changes were strain-dependent. The results also
reflected the influence of long storage of a strain on
productivity, morphological behaviour and phosphate
consumption.
Introduction
In the past decade various investigations dealing with
citric acid production by free and immobilized cells of
different strains of Aspergillus niger have been pub-
lished (Eikmeier and Rehm 1984, 1987a, b; Horitsu et
al. 1985; Vaija and Linko 1986; Xu et al. 1989a, b;
Honecker et al. 1989; Bisping et al. 1990b).
It is a well-established fact that rapid accumulation
of citric acid in a submerged fermentation is associated
with certain fungal morphologies. Changes in cellular
m o r p h o l o g y are characterized by short, stubby, forked,
and bent mycelia with numerous swollen spherical ves-
icles and bulbous hyphae (Kisser et al. 1980; Eikmeier
and Rehm 1984; Tsay and To 1987; Honecker et al.
1989; Legisa and Mattey 1986a, b, 1988).
In the present investigation the production of citric
acid, oxalic acid and polyols, consumption of sugars,
and the morphological changes in free and immobilized
spores of three strains of A. niger were studied. Besides
this the influence of storage on these factors and on
phosphate consumption were investigated.
* Present address: Botany Department, Faculty of Science, Mo-
harram Bey, Alexandria University, Alexandria, Egypt
Offprint requests to: H.-J. Rehm
Microbiology
Biotechnology
© Springer-Verlag 1992
Materials and methods
Microorganisms. Three strains of A. niger: strain 180 derived from
ATCC 11414, Wisconsin 72-4 K Shu (CMI 75353, NRRL 2270,
known for production of citric acid, aconitic hydrase, and lipid
biosynthesis); strain 836 obtained from Sturge (Selby, North
Yorkshire, UK); and strain 1120 (isolated from soil, MOnster,
FRG, June 1988) were used in this comparative investigation.
Spore suspensions for inoculation were obtained from cultures on
malt-yeast-agar slants after 7 days cultivation at 30°C using a
0.9% NaC1/0.1% Tween 80 solution and placed in a sonifier for
5 min to separate the clumped spores. Strain 836 (old) had been
stored in a refrigerator for about 1 year.
Immobilization. Spore suspensions were entrapped in 3% calcium
alginate. The beads were prepared using a motor-driven syringe
press as described by Eikmeier and Rehm (1987a). Sodium algi-
nate (commercial name "Manugel DJX") was purchased from AI-
ginate Industries (Hamburg, FRG).
To reach maximal citric acid production different phosphate
concentrations are needed by free and immobilized cells. Also, for
the formation of the same specific supply of biomass by free and
immobilized cells, the ratio of the other ionic ingredients (supple-
mentations) are kept proportional to each other. The following
media were therefore prepared separately for each case.
Media. The medium for free cells contained (per litre): sucrose,
160g; KHzPO4, 400mg; NHnNO3, 1600mg; MgSO4.7H20,
1200 mg; FeSOa.7H20, 0.26 mg; ZnSO4.7 H20, 0.67 mg. The me-
dium for immobilized cells contained (per litre): sucrose, 160 g;
KHzPO4, 150mg; NH4NO3, 600mg; MgSO4.7H20, 450mg;
FeSO4.7 HzO, 0.1 mg; ZnSO4.7 H20, 0.25 mg. Sucrose was purif-
ied by passing it through a cation exchange column packed with
Lewatit S 100.
Cultivation and Sampling. Shake cultures (at 200 rpm) of free and
immobilized spore suspension were produced in Fernbaeh flasks
with a capacity of 450 ml and a working volume of 150 ml at
30° C. Sampling (in triplicate) was done by taking 1 ml of medium
with cells (free or in beads) from each flask with the aid of an
inverted sterilized 5-ml capacity pipette at various intervals rang-
ing from 17 h to 7 days.
Analyses. Concentrations of citric acid, oxalic acid, glucose, fruc-
tose, erythritol, and glycerol were determined by HPLC equipped
with an RI detector ERC-7512 (Erma, Tokyo, Japan) and a
sampler following Pfeiffer and Radler (1985) with some modifica-
tion. As the mobile phase, 6.5 mM H2SO4 was used with a flow
rate 0.6 ml/min and at a column temperature of 70° C. For further
 519

details see Honecker et al. (1989) and Bisping et al. (1990a). Phos-
phate was determined spectrophotometrically at 870 nm using the
procedure of Fiske and Subbarow (1925) after slight modifica-
tion.
Scanning electron microscopy (SEM). For SEM the cells (free and
immobilized) were dehydrated in an ascending ethanol series fol-
lowed by isopropyl alcohol. After critical-point drying using a
Balzers critical-point dryer type CPD 010 and sputtering with
gold using a Balzers Sputtering device, micrographs were taken
with a Philips (Eindhoven, The Netherlands) 500 X scanning elec-
tron microscope.
Chemicals. The chemicals used throughout this investigation were
of analytical grade.
Results
Polyol and acid formation by A. niger 180
The results presented in Fig. 1A-F show that this im-
mobilized strain produced 36-40 g 1-1 citric acid within
7 days, while the free cells formed much less citric acid
(24.5 g 1-1) in the same period. This species did not
produce either oxalic acid or polyols when immobil-
ized. Free cells, on the other hand, produced about
1 g 1-1 polyols.
Both immobilized and free cells preferred glucose to
fructose, but immobilized cells used more of the glu-
cose than the free cells.
Polyol and acid formation by A. niger 1120
The results presented in Fig. 2A-F show that this strain
produced smaller quantities of citric acid than the other
two strains, namely 180 and 836 (young cultures) when
immobilized. The production of citric acid was about
30 g 1-1. The acid production of free cells was nearly
the same as that of the immobilized cells (approx.
28 g 1-1). This strain was able to accumulate significant
amounts of oxalic acid when immobilized. Free ceils
did not form oxalic acid.
Free cells produced more polyols than the immobil-
ized culture (Fig. 2E and F). The quantities of both su-
gars used were less than those consumed by A. niger
180 with both free and immobilized cells and therefore
the citric acid yield was higher.
Polyol and acid formation by A. niger 836
Old stored cultures. The results in Fig. 3A-F demon-
strate that very low quantities of citric acid were pro-
duced by immobilized (approx. 15 g 1-1) and free cells
(ca. 5 g 1-1) of a culture stored in a refrigerator at 4 ° C
for approx. 1 year (Fig. 3A and B). Oxalic acid was not
produced. This strain preferred fructose to glucose, and
the immobilized cells consumed nearly the double
quantity of fructose compared to free cells (Fig. 3C and

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Fig. 1. Fermentation profiles of free (A, C, E) and immobilized Fig. 2. Fermentation profiles of free (A, C, E) and immobilized
(B, D, F) cells of Aspergillus niger strain 180: Q, citric acid; (3, (B, D, F) cells of A. niger strain 1120: Q, citric acid; V, oxalic
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