Process Biochemistry 40 (2005) 16051610

www.elsevier.com/locate/procbio

Submerged production of oxalic acid from glucose

by immobilized Aspergillus niger
Sushil K. Mandal, Pataki C. Banerjee*
Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India
Received 6 February 2004; received in revised form 7 April 2004; accepted 9 June 2004

Abstract

Oxalic acid is now considered as a useful chemical in various hydrometallurgical processes. For its massive applications in hydro-
metallurgy, cost-effective production of the acid is necessary. In this direction, fermentative production of the acid from glucose appears
attractive. Aspergillus niger, the main oxalic acid producing organism, in general oxidizes glucose rapidly to gluconic acid, and conversion of
this sugar to oxalic and citric acid is always sufficiently less. In this article, we have shown that immobilized mycelia (bioparticles) of A. niger
NCIM 548 strain secreted more oxalic acid from glucose than the amounts produced from either sucrose, lactose or glucose under submerged
condition. Germination of immobilized spores in polyurethane foam yielded bioparticles which produced more (average: 3-fold on 7th day
and 3.7-fold on 9th day) oxalic acid from glucose compared to free mycelium cells, mainly during the 2nd and 6th cycle. Recycled bioparticles
maintained average activity of acid production up to six cycles.
# 2004 Elsevier Ltd. All rights reserved.

Keywords: Aspergillus niger; Oxalic acid; Polyurethane foam; Spore immobilization

1. Introduction cesses, production of oxalic acid from a cheap carbon source

Oxalic acid, produced by many saprophytic and phyto-
pathogenic fungi, causes leaching and transformation of
insoluble inorganic metal compounds in nature through
acidification and via its chelating property [14]. These
activities have initiated studies directed towards utilization
of this acid in hydrometallurgy for improvement of clays,
kaolins, quartz and silica sands through dissolution of iron
mineral impurities [511]. Remediation of metal-contami-
nated soils with oxalic acid producing fungi has also been
contemplated [12].
Oxalic acid can be produced both by chemical and
fermentative processes, the first one being more common
nowadays. The usual chemical methods use sodium formate
(heating followed by H2SO4 treatment) or carbohydrates
(oxidation with HNO3) [13]. Due to increasing demand in
hydrometallurgy and for wider applications in other pro-
* Corresponding author. Tel.: +91-3324733491/0492/6793;
fax: +91-3324733967/24730284.
E-mail address: pcbanerjee@iicb.res.in (P.C. Banerjee).
by fermentation appears more attractive as an eco-friendly,
non-hazardous microbial process yielding this acid at lower
cost. Accordingly, fermentative production of this acid
has been studied with renewed interest in the last decade
[1417].
Among the variety of fungi producing oxalic acid [18],
Aspergillus niger is the organism of choice for its efficient
productivity [14,19]. This species produces mainly citric
and/or oxalic acid from sucrose and lactose depending on the
fermentation conditions, and glucose is usually converted
rapidly to gluconic acid [20,21]. Fermentative production of
oxalic acid for use in industrial hydrometallurgy will be
more cost-effective if a higher yield is obtained using
glucose as the carbon source rather than sucrose, which is
the preferred carbon source for oxalic acid production [14].
We reported previously that glucose-grown A. niger NCIM
548 culture filtrate leached more iron from china clay in
comparison with similar culture filtrates of other fungal
strains used and oxalic acid was found to be the compound
causing iron dissolution [10]. Although sucrose (as well as
lactose) is the preferred C-source for oxalic acid production

0032-9592/$ see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.06.013
1606 S.K. Mandal, P.C. Banerjee / Process Biochemistry 40 (2005) 16051610
by A. niger [1417], this observation indicated that the
NCIM 548 strain produced sufficient oxalic acid from
glucose, a cheap C-source, which is mostly converted to
gluconic acid under aerobic fermentation by other A. niger
strains. Therefore, studies on fermentative production of this
acid by the A. niger strain was undertaken. In this article, we
report that immobilized mycelium cells (in polyurethane
foam) of this strain excrete 33.7-fold more oxalic acid from
glucose compared to the free cells (mycelia) under sub-
merged condition. For standardization of fermentation con-
ditions, we initially studied oxalic acid production by free
cells of this strain. It may be mentioned that though the
production of gluconic [22,23] and citric [24] acids by
immobilized A. niger have been studied, this is the first
report of oxalic acid production by the immobilized mycelia
of this species.
2. Materials and methods
2.1. Microorganism
Aspergilus niger NCIM 548 strain used in this study was
kindly provided by Prof. A.K. Guha, Indian Association for
the Cultivation of Science, Kolkata, India.
2.2. Medium composition
The medium contained the following components in
(g/l): glucose (105.5) or sucrose (100) or lactose (102.6)
plus glucose (2.51)all with the same carbon content;
NaNO3 (1.5); KH2PO4 (0.5); MgSO4
7H2O (0.025); KCl
(0.025); and yeast extract (1.6) [14]. Medium pH was
adjusted to ca. 6 with 4 M NaOH before sterilization.
Twenty ml of universal pH indicator solution (Merck,
India) was added per litre medium for observing its pH
which was maintained within 6 to 7 by adding alkali during
fermentation.
2.3. Spore suspensions
Spores from 7-day stationary culture at 30 8C in potato-
dextrose broth were suspended in 0.001% (v/v) Triton X-100
solution, and were counted under a light microscope in a
Neubauer chamber.
2.4. Immobilization of A. niger spores in
polyurethane foam
Immobilization was done using spore suspension of
A. niger and polyurethane foam (PUF) cubes (ca. 2.2 mm,
density 40 kg/m3) by the adsorption technique described by
Sanroman et al. [24]. In brief, 1 g PUF cubes were mixed
with 50 ml medium containing 2.5  107 spores in a 250-ml
Erlenmeyer flask. The flask was shaken at 215 rpm for 3
days at 30 8C to produce bioparticles which were separated
by filtration and suspended in fresh medium.
2.5. Free cell fermentation
Fifty milliliter culture medium taken in a 250-ml
Erlenmeyer flask was inoculated with spores of A. niger
(range: 5  103 to 5  106 per ml medium), and was
incubated at 30 8C on an orbital shaker at 215 rpm for 7
days or for the number of days mentioned. Culture filtrate
from each triplicate set was analyzed for oxalic acid
content. During fermentation, the pH of the medium
was maintained at 6.5  0.5 with 4 M NaOH. Growth
and oxalic acid yield were monitored during the course of
fermentation.
2.6. Fermentation with bioparticles
In order to determine the oxalic acid producing activity
and stability of the bioparticles, several cycles of fermenta-
tion were carried out for either 7 or 9 days at 30 8C on an
orbital shaker at 215 rpm with the same PUF bioparticle
beads. After each fermentation the bio-particles were
washed with water, and a fresh medium of the same com-
position was added.
2.7. Analytical methods
Broths after fermentation were filtered through a 0.2-mm
membrane. Oxalate in the filtrate was estimated by titration
with KMnO4 after precipitating with CaCl2 [25,26].
Sugars were estimated colorimetrically using orcinol
[27]. Briefly, 1.5 ml of cold orcinol reagent (200 mg orcinol
dissolved in 100 ml of 70% (v/v) H2SO4 in water) was added
to 0.5 ml of diluted sample in a test tube, heated in a boiling
water bath for 20 min, and then cooled. Absorbance of the
samples was measured at 490 nm within an hour by a
spectrophotometer (Shimadzu UV1201). The amount of
sugar in the samples was determined from the standard
curve obtained using a solution of the respective sugar
(525 mg).
Biomass of A. niger grown under different conditions was
measured gravimetrically after separating the mycelia from
fermented broth by filtration, washing with water, and
drying at 80 8C to constant weight.
3. Results
3.1. Effect of inoculum concentration on oxalic
acid and biomass production
In the present study inoculum size was allowed to vary
from 5  103 to 5  106 spores per ml to determine the
optimum spore concentration for oxalic acid production
from glucose.
 S.K. Mandal, P.C. Banerjee / Process Biochemistry 40 (2005) 16051610
Fig. 1. Effect of inoculum size on oxalic acid production (&, mM; *, mM/
g biomass) from glucose. Fermentation was done for 7 days without
adjusting the medium pH. Average of two replicate experiments are
presented.
The results presented in Fig. 1 show that below 5 
103 spores/ml oxalic acid production was very low; the
amount of oxalic acid produced was a maximum at the
highest inoculum concentration (5  106 spores per ml)
used. Further increase in inoculum size (>5  106 spores/
ml) was not possible as the viscosity of the culture became
very high with the production of large amount of dense
biomass so that the liquid portion of the culture could not be
separated from mycelia. Therefore, an inoculum size of 5 
106 spores/ml was used in the shake-flask experiments.
Oxalic acid production from glucose per gram biomass also
increased with increase in the inoculum spore concentration
(Fig. 1).
3.2. Evaluation of carbon sources for oxalate
production by free cells
C-sources were used at concentrations equivalent to 42 g
of total C per litre of the medium [14]. Sucrose and lactose
were much better substrates than glucose for oxalic acid
production by this strain of A. niger. After 7 days of
fermentation, 124.8 mM (11.32 g/l), 84.8 mM (7.63 g/l)
Fig. 2. Effect of the C-source on oxalic acid (A) and biomass (B) production after 7 days.
1607
and 49.8 mM (4.48 g/l) oxalic acid was produced an average
from lactose, sucrose and glucose, respectively. However,
the yield of biomass was almost 1.5-fold higher on glucose
and sucrose than on lactose (Fig. 2).
The amount of oxalic acid (in g) produced per 100 g of
sugar utilised was 26.62 for lactose, while the same for
sucrose and glucose was only 8.13 and 5.75, respectively.
Similar trend was observed with respect to oxalate yield
(in g) per gram mycelium dry weight; the value for lactose,
sucrose and glucose was 0.92, 0.41 and 0.24, respectively.
Sucrose was metabolized more efficiently by this strain
than glucose, while lactose was slowly utilized. It was
noted that only 40% of lactose was consumed in 7 days,
while sucrose and glucose were consumed to the extent of
more than 88 and 73%, respectively, during the same
period.
3.3. Oxalic acid production by immobilized cells in
polyurethane foam (PUF)
The adsorption technique of immobilization [24] pro-
duces finely feathered mycelia of the fungus forming
spheres of bioparticles around the foam. Fig. 3 represents
oxalic acid production from glucose up to 9 days of fer-
mentation by bioparticles of immobilized cells. Although
the organism still produced oxalic acid, fermentation was
discontinued after the 9th day due to rapid increase of free
mycelia and turbidity of the culture. In Table 1, sugar
consumption of glucose by free and immobilized cells of
the strain is presented. It is evident from this table that on
average the immobilized cells used up more than 80% of
glucose within 7 days while free cells utilised sufficiently
smaller amount of the sugar within this period.
Fig. 4 shows oxalic acid production by bioparticles in
batch fermentation up to 7th cycle when a declining ten-
dency in oxalic acid production was noted. Oxalic acid
production increased from 77.7 mM (7.0 g/l) and 117 mM
(10.53 g/l) in the first cycle to 146 mM (13.14 g/l) and
178 mM (16.02 g/l) in the sixth cycle on the 7th and 9th
day of fermentation, respectively. The maximum amount
(230 mM or 20.6 g/l) of the acid was produced on the 9th
day in the second cycle.
1608 S.K. Mandal, P.C. Banerjee / Process Biochemistry 40 (2005) 16051610

Fig. 3. Production of oxalic acid from glucose by bioparticles of immo-

Fig. 4. Oxalic acid production from glucose by bio-particles on 7th (&) and
bilized cells.
9th (&) day in batch fermentations up to seventh cycle; pH of the medium
was kept at 67.

Table 1
Glucose utilization by free and immobilized cells of A. niger NCIM 548 after 7-day fermentation
Day Free cells Immobolized cells (cycle)
1 2 3 4 5 6 7
3 33.6  2.7 47.9  3.4
5 47.9  3.3 57.3  3.7
7 52.6  3.2 62.1  3.2 88.2  4.3 92.9  4.2 83.4  3.7 78.7  2.9 73.9  3.1 69.2  3.0
9 73.2  5.2 88.2  4.3 83.4  4.9 88.2  3.6 88.2  4.3 78.7  3.9 83.4  2.7 88.2  4.1

4. Discussion
A. niger strains, in general, secrete different carboxylic
acids including oxalic acid depending on the carbon source
and fermentative conditions [28]. However, in comparison to
other metabolites, fermentative production of oxalic acid has
never been deeply studied, and the acid has always been
considered as an unwanted by-product in citric acid fermen-
tation. Thus, instead of optimizing the process parameters
for large-scale production of the acid, efforts were directed
towards finding conditions under which secretion of this
metabolite could be avoided [21,29]. But due to the prob-
ability of increasing applications of oxalic acid in hydro-
metallurgy, some recent studies have been conducted on its
fermentative production by A. niger [1417]. Strasser et al.
[14] standardized the conditions for high-yield production of
oxalic acid by an A. niger strain in 1 M MES-buffered (pH
6.0) media because this is the major secreted acid product at
pH > 5 [30,31]. In sucrose and lactose permeate medium,
the strain produced almost equal amounts (175 mM versus
183 mM) of oxalic acid but converted glucose almost com-
pletely to gluconic acid (247 mM) in shake-flask experi-
ments after 7 days [14]. The same medium composition was
used here except that the use of MES, a costly buffer, which
was avoided by maintaining the broth pH with occasional
addition of alkali whenever the pH dropped to ca. 6.
The optimum inoculum size for this strain (Fig. 1) was
determined and found to be the same (5  106 spores/ml) as
suggested by Strasser et al. [14] for another A. niger strain
for oxalic acid production. Taking the cue from this experi-
ment, we used the same spore concentration of A. niger
NCIM 548 for immobilization as suggested before [24] for
citric acid production by immobilized A. niger in polyur-
ethane foam was used.
Lactose is not readily utilized by A. niger as a growth
substrate [32]. Initial studies in this laboratory and elsewhere
[23] also confirmed that lactose is slowly utilized by A. niger
NCIM 548 and biomass production (i.e. mycelial yield) was
sufficiently low even after 3 days fermentation. In presence
of glucose, the strain grew rapidly [23]. In this study,
however, a very small amount of glucose (0.1% of total
carbon) was added for initiation of growth which in turn
helped lactose utilization through induced synthesis of b-
galactosidase and other proteins in the presence of lactose
[33,34].
Medium pH strongly influences oxalic acid production by
A. niger because the enzyme oxaloacetate hydrolase is
induced at pH  4; further, maintenance of broth pH above
6 is necessary for accumulation of oxalic acid by A. niger
[14,2931,35]. In tune with these facts, it was also observed
that oxalic acid production was better if the initial medium
pH was 47 rather than 2, 3 or 8 when fermentation was
 S.K. Mandal, P.C. Banerjee / Process Biochemistry 40 (2005) 16051610
carried out for 7 days without adjusting the medium pH (data
not shown).
Although sucrose is the preferred substrate for fermen-
tative production of oxalic acid, nearly equal yields of oxalic
acid in lactose permeate and sucrose medium was reported
earlier [14]. Lactose was a better substrate than sucrose (Fig.
2) with the present A. niger strain, which produced much less
oxalic acid from glucose like other A. niger strains.
Immobilized cells of the A. niger NCIM 548 strain,
however, produced 33.7-fold more oxalic acid from glu-
cose compared to that by the free cells under identical shake-
flask conditions (Fig. 2A and Fig. 4). The amounts of acid
produced by free cells from sucrose and lactose (Fig. 2A)
[14] were also less compared to that produced by the
immobilized cells in the second to sixth cycles from glucose
(Fig. 4). Cameselle et al. [15] reported better (302 mM)
production of the acid from sucrose after 9-day fermenta-
tion; in this case, however, 1.5-fold amount (150 g/l) of
sucrose was used as substrate. Thus, it appears that immo-
bilized mycelia of the strain produced comparable, if not
higher, amounts of the acid from glucose than from sucrose
or lactose by free cells [14,30]. Since, the bioparticles could
be used several times and glucose is a low-cost C-source, it
may be contemplated that supply of oxalic acid at a cheaper
rate for hydrometallurgical operations might be achieved. It
is to be noted that this is the first report of oxalic acid
production by immobilized cells of A. niger.
Acknowledgements
We thank Prof. A.K. Guha of the Indian Association for
the Cultivation of Science, Kolkata for his valuable sugges-
tions. Authors thank Dr. B. Achari for revising the manu-
script. S.K. Mandal received a Senior Research Fellowship
from the Council of Scientific and Industrial Research, New
Delhi, India.
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