Bioresource Technology 98 (2007) 3464–3469

Citric acid production by a novel Aspergillus niger isolate:

I. Mutagenesis and cost reduction studies
¤
Walid A. Lotfy, Khaled M. Ghanem, Ehab R. El-Helow
Department of Botany, Faculty of Science, University of Alexandria, Alexandria 21526, Egypt

Received 10 September 2006; received in revised form 21 October 2006; accepted 4 November 2006
Available online 12 January 2007

Abstract

Ultraviolet-irradiation (UV), ethyl methane sulfonate (EMS) and acridine orange (AO) were used to induce citric acid overproduction
mutations in Aspergillus niger UMIP 2564. Among 15, eight of the mutant derivatives, were improved with respect to citric acid produc-
tion from sucrose in batch cultures. Maximum product yield (60.25%) was recorded by W5, a stable UV mutant, with approximately 3.2-
fold increase when compared to the parental wild type strain. In terms of the kinetic parameters for batch fermentation processes, the
mutation doubled the speciWc substrate uptake rate and achieved 4.5- and 7.5-fold improvements in citric acid productivity and speciWc
productivity, respectively. For reduction of the fermentation medium cost, corn steep liquor and calcium phosphate pre-treated beet
molasses were successfully used as substituents of nitrogen and carbon sources in the growth medium, respectively. These medium substi-
tutions resulted in a W5 citric acid fermentation culture with a product yield of 74.56%.
© 2006 Elsevier Ltd. All rights reserved.

Keywords: Aspergillus niger; Citric acid; Submerged fermentation; Mutation; Beet molasses

1. Introduction ent investigation, challenges were made for raising potent of

Citric acid fermentation is one of the largest biotechno-
logical industries as it is used in several other industrial
applications (Ali et al., 2001; Rohr, 1998). Improvement of
the microbial producer strain oVers the greatest opportu-
nity for cost reduction without signiWcant capital outlay
(Stanbury et al., 1995). This is achieved when a selected
strain can synthesize a higher proportion of the product
using the same amount of raw materials. Since citric acid is
an intermediate of energy metabolism, its concentration
can rise to appreciable amounts under conditions of meta-
bolic imbalances (Hutter, 1983).
Strains with superior characters, such as enhanced citric
acid production and increased rate of fermentation have been
previously selected after subjecting the genetic material to
physical or chemical mutagenic agents (Ikram-ul et al., 2003,
2004, 2005; Parekh et al., 2000; Rohr et al., 1983). In the pres-
Aspergillus niger UMIP 2564.04, a recently isolated fungal
strain, for citric acid production by random mutagenesis
using diVerent doses of UV-irradiation and chemical mut-
agenising agents. For further cost reduction, the ability of the
selected mutaginized strain to utilize agro-industrial by-prod-
ucts as carbon and nitrogen sources was also examined.
2. Methods
2.1. Microorganism
The wild type fungus used in this study is a new A. niger
strain isolated from an Egyptian soil sample and is depos-
ited in Pasteur Institute (Fungi Culture Collection) Paris,
France with the code UMIP 2564.04.
2.2. Media

*
Corresponding author. All media were sterilized by autoclaving at 121 °C for
E-mail address: elhelow@link.net (E.R. El-Helow). 15 min and the pH was adjusted before sterilization. The

0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.11.007
 W.A. Lotfy et al. / Bioresource Technology 98 (2007) 3464–3469
following culture media (g/l) were employed throughout
the work.
2.2.1. Sabouraud dextrose agar (SDA) for slant and plate
cultures
Mycological peptone, 10.0; glucose, 40.0; agar, 15.0 (pH
5.6 § 0.2).
2.2.1.1. Basal fermentation medium for citric acid production
(Khan et al., 1970). Sucrose, 160.0; NaNO3, 4.0; KH2PO4,
1.0; MgSO4 · 7H2O, 0.23; FeCl3, 0.02; ZnSO4, 0.0012;
MnCl2 · H2O, 0.0012 (pH 4 § 0.2).
For cost reduction of the fermentation medium, sucrose
was substituted by untreated and pre-treated beet molasses.
A physical treatment was carried out by repeated centrifu-
gation of onefold diluted crude solution of beet molasses at
4000 rpm until complete removal of muddy precipitates.
Chemical treatments included potassium ferrocyanide
(El-Abyad et al., 1992), Sulfuric acid (Mayilvahanan et al.,
1996), calcium phosphate (Mayilvahanan et al., 1996),
Sodium phytate (Wang, 1998) and methanol (El-Abyad
et al., 1992).
2.3. Preparation of spore suspensions
Spore suspensions of fungal strains were prepared by
washing 5-days old culture slants with sterilized saline solu-
tion (0.9% NaCl) with shaking vigorously for 1 min. Spores
were counted by a haemocytometer to adjust the count to
approximately 107 spores/ml.
2.4. Induction of mutation
A spore suspension was prepared from a 24 h-old slant
culture of A. niger (wild type) by suspending in sterile saline
water. A 30 W Perkin Elmer UV-lamp (Type G10T5-1/2L)
was used as the source of radiation. Ultraviolet mutagenesis
was applied as described by Ikram-ul et al. (2004) with
exposure times of 12, 18 and 24 min. Following the method
of Roy and Das (1977), chemical mutagenesis was carried
out by using ethyl methane sulfonate (EMS) and acridine
orange (AO), each dissolved in phosphate buVer, pH 7, with
a concentration of 50 g/ml. In all cases, mutant derivatives
were selected based on variations in colony morphology or
sporulation on SDA. PuriWed mutated colonies were
screened for citric acid production in liquid cultures.
2.5. Batch cultures
The fungus was allowed to grow in 100 ml aliquots of the
fermentation medium dispensed in 250 ml Erlenmeyer
Xasks. Each Xask was inoculated with 1% (v/v) inoculum
containing 107 spores/ml. Cultures were then incubated at
30 § 1 °C under shaken conditions at 300 rpm for the requi-
site time. Biomass was isolated by centrifugation at
5000 rpm for 10 min using swing-out rotor centrifuge and
supernatants were separated for necessary analyses. Each
3465
treatment was carried out in triplicate and the results
shown are the arithmetic means.
2.6. Analytical procedures
Elements in by-products were estimated by the method
described by Piper (1950), using Perkin Elmer model A
Analyst 100 Atomic Absorption. Biomass was determined
as described by Kirimura et al. (1988). Citric acid, glucose
and fructose were determined in culture Wltrates by a Gil-
son HPLC instrument equipped with a refractive index
detector (RID), Aminex HPX-87H column (7.8 £ 300 mm,
Bio-Rad, USA) for citric acid analysis and Aminex HPX-
87P column (7.8 £ 300 mm, Bio-Rad, USA) for glucose and
fructose analysis. The eluent used for analysis was 0.01 N
sulfuric acid solution. HPLC analyses were carried out
under the following operation conditions: pump Xow,
0.6 ml/min; column temperature, 40 °C and sample amount,
20 l. Concentrations were automatically calculated by Gil-
son Unipoint software. Sucrose concentration was calcu-
lated as the sum of glucose and fructose concentrations.
Yield of citric acid (%) D (grams of citric acid produced/
grams of original sugar) £ 100.
3. Results
3.1. Mutagenesis and screening for citric acid
hyperproduction
In a screening experiment, the parental culture of
A. niger and its mutant derivatives were grown on the basal
fermentation medium for 8 days and compared with respect
to citric acid yield and biomass production. As shown in
Fig. 1, most of the mutant derivatives, especially those
induced by UV, were improved with respect to acid produc-
tion. Mutants W1, W4, W5, W6, W8 and W9 showed
marked citric acid over-production records. Among those,
maximum product yield was achieved by the mutant W5
(60.25%) with approximately 3.2-fold increase when com-
pared to the wild type. Interestingly, the W5 culture resulted
in the lowest biomass yield. This mutant was obtained
through ultraviolet treatment, at a distance of 20 cm, for
18 min. Five successive cultures of this mutant on SDA
plates were further examined under the same fermentation
conditions. All cultures showed almost the same citric acid
concentration as well as biomass production records indi-
cating the stability of the mutaginized genotype.
3.2. Time course study of citric acid fermentation
Time course of growth, sugar utilization and citric acid
fermentation by the selected mutant W5 and its parental
wild type were investigated in batch cultures. Each culture
was separately terminated as complete sugar consumption
was accomplished. Conidial germination of W5 was
observed 9 h after inoculation. Swelling of the young
hyphal agglomerates was detected microscopically after the
3466 W.A. Lotfy et al. / Bioresource Technology 98 (2007) 3464–3469

70

Citric acid yield (%) & Biomass (g/l)

60
Citric acid yield Biomass
50

40

30

20

10

0
UV n

AO 1 4
15
E M 10
U 6

U 7

UV 8
UV 1

UV 2

UV 9

EM 11

AO 13
UV 3

UV 4

U 5
i

E M W1
W

W
W

W
ra

W
W

W
st

S
al
nt
re
Pa

Fig. 1. Citric acid production and biomass formation by A. niger parental strain, and genotypes (W1-W15) mutagenized by UV (ultraviolet radiation),
EMS (ethyl methane sulfonate) and AO (acridine orange).

Wrst 40 h of incubation. Eight hours later, the fungus was sugar consumption. This amount of citric acid represents
present in the form of spherical mycelial pellets which approximately 5.9-fold increase when compared to amount
improve the performance of the acid production process produced by the wild type culture within the same period of
(Haq et al., 2001; Pera and Callieri, 1997). In the case of the incubation. Total sucrose consumption by the parental
parental strain culture, fungal pellets were developed after strain required almost 9 days and the resulted biomass and
64 h. Longer incubation periods showed slight increasing in citric acid concentrations were 32.2 and 30.12 g/l, respec-
the diameters of the pellets. tively. Accordingly, the end mycelial dry weight of the
As shown in Fig. 2, biomass formation in the mutant mutant strain represented approximately 75% of the bio-
culture was relatively faster within the Wrst 4 days. How- mass harvested from the parental culture.
ever, the end mycelial dry weight of the mutant strain was In terms of the kinetic parameters for batch fermenta-
slightly less than the biomass harvested from the parental tion processes, determined after Pirt (1975) and presented
culture. The mutant started to produce citric acid within in Table 1, the mutation doubled the speciWc substrate
32 h while the parental strain started 16 h later. At the end uptake rate and markedly increased the speciWc citric acid
of the fermentation run of the mutant culture (152 h), the production per biomass. The selected A. niger mutant
recorded biomass and citric acid concentrations were achieved 4.5- and 7.5-fold improvements in citric acid pro-
approximately 24 and 96.3 g/l, respectively, with 100% ductivity and speciWc productivity, respectively.

100 180

90 160
Citric acid & Biomass (g/l)

80 140
70
Total sugar (g/l)

120
60
100
50
80
40
60
30

20 40

10 20

0 0
0 20 40 60 80 100 120 140
Fermentation time (hours)
Fig. 2. Time course of sugar consumption (䉭䉱), citric acid production (䊊䊉) and biomass formation (䊐䊏) by A. niger parental strain (open) and W5
(solid).
 W.A. Lotfy et al. / Bioresource Technology 98 (2007) 3464–3469 3467

Table 1 and 57%, respectively) which are comparable to the sucrose

Kinetic parameters and coeYcients of citric acid fermentation by A. niger culture result (60.52%).
W5 mutant and its parental strain
The eVects of some physical and chemical pre-treatments
Parameter Strain Fold increase on beet and cane molasses as substrates for citric acid pro-
Parental W5 duction were then investigated. Pre-treated beet molasses
Kinetics of substrate consumption showed citric acid concentrations ranged from 99.5 to
Substrate uptake rate 0.750 1.050 1.4 113.5 g/l while those of cane molasses recorded 92.5–98.08 g/
(g substrate/l/h) l (Table 2). Highest citric acid yield (70.94%) was achieved
SpeciWc substrate uptake rate 0.020 0.040 2.0
by beet molasses treated with 10 g/l calcium phosphate.
(g substrate/g cells/l/h)
Among all examined cultures, variations in biomass forma-
Product yield coeYcients tion were relatively slight.
Product yield coeYcient 0.190 0.600 3.2
(g product/g substrate)
DiVerent concentrations of calcium phosphate (5–20 g/l)
SpeciWc product yield 0.940 4.010 4.3 were examined for pre-treatment of beet molasses used for
coeYcient (g product/g cells) citric acid fermentation by W5. The records of product
Kinetics of citric acid formation yield ranged from 67.81% to 73.19% with the optimum
Productivity (g product/l/h) 0.140 0.630 4.5 result at calcium phosphate concentration of 15 g/l (Fig. 4).
SpeciWc productivity 0.004 0.030 7.5 On the other hand, formation of fungal biomass was almost
(g product/g cells/h) not aVected.
Cultures of parental strain and W5 were terminated after 212 and 152 h, Chemical analysis of untreated and calcium phosphate
respectively, as complete sugar consumption was accomplished. treated beet molasses showed that treatments resulted in
marked decreases in a group of heavy metals (Table 3). For
instance, the 10% calcium phosphate treatment removed
3.3. Utilization of agro-industrial by-products for citric acid approximately 65.8%, 55.9% and 53.8% of Cu, Zn and Si,
production by A. niger W5 respectively. Further removal of these metals was achieved
by increasing calcium phosphate concentration up to 15 g/l.
In an attempt to economize the fermentation compo- In addition to heavy metals, concentrations of other ele-
nents, sucrose in the basal medium was replaced by carbon ments such as K and Mg are also clearly reduced as a result
equivalent concentrations of crude beet molasses, cane of these treatments.
molasses, corn husks, corn cobs, lemon peels, rice husks, For further reduction of fermentation medium cost,
palm seeds, wheat straw and mango peels, one at a time. All sodium nitrate in the fermentation medium was also
cultures were shaken for 152 h. As shown in Fig. 3, beet and replaced by nitrogen equivalent concentrations of diVerent
cane molasses gave the highest citric acid yields (61.44% by-products namely; corn steep liquor, rice steep liquor and

70

Citric acid yield Biomass

Citric acid yield (%) & Biomass (g/l)

60

50

40

30

20

10

0
s

s
l

bs

s
s
s

w
tro

el

el
se

sk

sk
ed
se

ra
co

pe

pe
on

hu

hu
as

st
se
as

rn
C

on

go
ol

at
ol

ice
lm
Co
m

or

he
m

an

Pa

R
C

Le
e
et

W
M
an
Be

Fig. 3. The utilization of agro-industrial by-products as sucrose (control) substituents for citric acid production by A. niger W5.
3468 W.A. Lotfy et al. / Bioresource Technology 98 (2007) 3464–3469

Table 2
Citric acid production from diVerent treated beet and cane molasses
Treatment Beet molasses Cane molasses
Citric acid (g/l) Yield (%) Biomass (g/l) Citric acid (g/l) Yield (%) Biomass (g/l)
Control 98.33 61.46 23.70 91.25 57.03 24.41
Potassium ferrocyanide 107.20 67.00 23.60 95.40 59.63 24.17
Sodium phytate 105.70 66.06 23.63 94.60 59.13 24.24
Sulfuric acid 109.40 68.38 23.58 97.10 60.69 23.91
Centrifugation 102.30 63.94 23.66 93.30 58.31 24.31
Calcium phosphate 113.50 70.94 23.51 98.08 61.30 23.72
Methanol 99.50 62.19 23.69 92.50 57.81 24.38
Yield (%) D (grams of citric acid produced/grams of original sugar concentration) £ 100.

130 40

120
Citric acid (g/l) & yield (%)

110 30

Biomass (g/l)
100

90 20

80

70 10

60

50 0
0 5 10 15 20 25
Calcium phosphate concentration (g/l)
Fig. 4. The eVect of beet molasses treatment by diVerent calcium phosphate concentrations on citric acid production (䊏), citric acid yield (䊉) and biomass
formation (䉭) by A. niger W5.

Table 3
Chemical analyses of untreated and calcium phosphate treated beet molasses
Element Treatment
Untreated Calcium phosphate (10 g/l) Calcium phosphate (15 g/l)
Concentration (ppm) Concentration (ppm) Removal (%) Concentration (ppm) Removal (%)
K 9847.0 5400.5 45.2 5350.0 45.7
Na 2018.0 1680.6 16.7 1631.4 19.2
Mg 361.2 309.0 14.5 279.0 22.8
Zn 122.3 53.9 55.9 49.2 59.8
Ca 118.6 102.7 13.4 95.1 19.8
Fe 68.2 61.7 9.5 58.3 14.5
Si 25.9 12.0 53.8 11.8 54.5
Cu 23.1 7.9 65.8 6.2 73.2
Mn 21.3 20.2 5.2 19.2 9.9
Pb 10.2 9.6 5.9 9.5 6.9

cheese whey, one at a time. The citric acid yields obtained
by all cultures were very close (73.21–74.56%) with corn
steep liquor at the top. These results suggested that sodium
nitrate could be successfully replaced by any of the three
examined alternative nitrogen sources.
4. Discussion
Quantitative description of cellular processes is a vital
tool in the design of fermentation processes. The two most
important quantitative parameters, yield and productivity,
specify how the cells convert the substrate to the product
(Nielsen, 2004). Here, a stable A. niger UV mutant, W5,
achieved considerable fold increases in substrate uptake
rate, speciWc substrate uptake rate, product yield coeYcient,
speciWc product yield coeYcient, productivity as well as
speciWc productivity. Among all examined genotypes, this
mutant resulted in the lowest biomass yield and dramati-
cally shortened the period of substrate consumption.
Compared to some other wild type or mutated fungal cul-
tures, W5 showed improved records of speciWc product
yield coeYcient (4.010 g product/g cells) and citric acid
 W.A. Lotfy et al. / Bioresource Technology 98 (2007) 3464–3469
productivity (0.630 g product/l/h) (Ikram-ul et al., 2004;
Parvez et al., 1998; Kirimura et al., 1992; Bennet and Klich,
1992; Snedecor and Cochran, 1980).
Since citric acid is an intermediate of energy metabolism,
a mutaginized genetic background such as W5, may result
in metabolic imbalance that leads to citric acid accumula-
tion on account of biomass formation. A possible explana-
tion is the presence of lower aconitase activity levels in such
types of mutaginized cells (Shoukat et al., 1997) which leads
to abnormal lower rates of citrate consumption.
Fermentation economics are driven by the proWtability of
a marketed product. A key component of this value is based
on manufacturing cost per unit of product (Parekh, 1999).
For reduction of the fermentation medium cost, equivalent
concentrations of beet molasses and corn steep liquor
were successfully used as substituents of basal carbon and
nitrogen sources in the fermentation medium, respectively.
Pre-treatment of beet molasses with calcium phosphate dra-
matically reduced the concentrations of a number of heavy
metal ions that may retard the utilization of molasses or
aVect citric acid formation by fungal cells. These results are
possibly attributed to the presence of two phosphate groups
rich with negatively charged oxygen atoms in calcium phos-
phate that act as attractants for positively charged elements
present in crude beet molasses (Roukas and Kotzekidou,
1997; Mayilvahanan et al., 1996). In addition, untreated
corn steep liquor represents a favorable substrate for the
adjustment of trace metal balance in the fermentation broth
due to the presence of phytate which has 12 replaceable pro-
tons in the phytic molecule (Wang, 1998).
References
Ali, S., Haq, I., Qadeer, M.A., 2001. EVect of mineral nutrient on the pro-
duction of citric acid by Aspergillus niger. J. Biol. Sci. 32, 31–35.
Bennet, B.W., Klich, M.A., 1992. Aspergillus: Biology and Industrial Appli-
cations. Butterworth-Heinemann, USA. pp. 234–321.
El-Abyad, M., Hamissa, F., Gad, A., 1992. Treatment of beet molasses for
citric acid production by a potent strain of Aspergillus niger van Tieg-
hem. Bioresour. Technol. 39, 191–197.
Haq, I., Khurshid, S., Ali, S., Ashraf, H., Qadeer, M.A., Rajoka, M.I., 2001.
Mutation of Aspergillus niger for hyper-production of citric acid from
blackstrap molasses. World J. Microbiol. Biotechnol. 17, 35–37.
Hutter, R., 1983. Over production of microbial metabolites. In: Rehm,
H.J., Reed, J. (Eds.), Biotechnology, Microbial products, vol. 4. Verlag
Chemie, Weinheim, Germany, pp. 3–17.
Ikram-ul, H., Ali, S., Qadeer, M., Iqbal, J., 2003. Stimulatory eVect of alco-
hols on citric acid productivity by 2-deoxy D-glucose resistant culture
of Aspergillus niger GCB-47. Bioresour. Technol. 86, 227–233.
3469
Ikram-ul, H., Ali, S., Qadeer, M.A., Iqbal, J., 2004. Citric acid production
by selected mutants of Aspergillus niger from cane molasses. Bioresour.
Technol. 93, 125–130.
Ikram-ul, H., Ali, S., Qadeer, M.A., Iqbal, J., 2005. Optimization of nitrogen
for enhanced citric acid productivity by a 2-deoxy D-glucose resistant
culture of Aspergillus niger NGd-280. Bioresour. Technol. 96, 645–648.
Khan, M.A.A., Hassain, M.M., Khalique, M.A., Rahman, M.A., 1970.
Studies on methods of citric acid fermentation from molasses by
Aspergillus niger. Pak. J. Sci. Ind. Res. 13, 439–444.
Kirimura, K., Nakajima, I., Lee, S., Kawabe, S., Usami, S., 1988. Citric acid
production by the diploid strains of Aspergillus niger obtained by pro-
toplast fusion. Appl. Microbiol. Biotechnol. 27, 504–506.
Kirimura, K., Sarangbin, S., Rugsaseel, S., Usami, S., 1992. Citric acid pro-
duction by 2-deoxyglucose-resistant mutants of Aspergillus niger. Appl.
Microbiol. Biotechnol. 36, 573–577.
Mayilvahanan, D., Annadurai, G., Raju, V., Chellapandian, M., Krishnan,
M.R., Jayaraman, K., 1996. Citric acid production: strategies for reduc-
tion in cycle time for targeted yields. Bioprocess Eng. 15, 323–326.
Nielsen, J., 2004. Microbial process kinetics. In: Ratledge, C., Kristiansen,
B. (Eds.), Basic Biotechnology. Cambridge, University Press, UK, pp.
127–149.
Parekh, S., 1999. Strain improvement. In: Encyclopaedia of microbiology,
vol. 4. Academic Press, San Diego, USA, pp. 6170–6197.
Parekh, S., Vinci, V., Strobel, R., 2000. Improvement of microbial strains
and fermentation processes. Appl. Microbiol. Biotechnol. 54, 287–301.
Parvez, S., Rajoka, M.I., Ahmed, M.N., Latif, F., Shahid, R., Malik, K.A.,
1998. Citric acid production from sugar cane molasses by 2-deoxyglu-
cose-resistant mutant strain of Aspergillus niger. Folia Microbiol. 43,
59–62.
Pera, L.M., Callieri, D.A., 1997. InXuence of calcium on fungal growth,
hyphal morphology and citric acid production in Aspergillus niger.
Folia Microbiol. 42, 551–556.
Piper, C.S., 1950. Soil and Plant Analysis. Int. Sci. Publishers Inc., New
York.
Pirt, S.J., 1975. Principles of Microbes and Cell Cultivation. Black Wells
ScientiWc Corporation, London, UK. pp. 112–135.
Rohr, M., 1998. A century of citric acid fermentation and research. Food
Technol. Biotechnol. 36, 163–171.
Rohr, M., Kubicek, C., Kominex, J., 1983. Citric acid. In: Rehm, H.J.,
Reed, G. (Eds.), Biotechnology. Verlag Chemie, Weinheim, Germany,
pp. 419–454.
Roukas, T., Kotzekidou, P., 1997. Pretreatment of date syrup to increase
citric acid production. Enz. Microb. Technol. 21, 273–276.
Roy, P., Das, A., 1977. The mutagenic action of N, methyl N, nitro N, nitr-
osoguanidine on Aspergillus niger in relation to citric acid production.
Sci. Cult. 43, 451–463.
Shoukat, A., Ahmed, M.A., Akbar, G.R., 1997. Citric acid production by
Aspergillus niger by batch cultivation. J. Biotechnol. 7, 47–53.
Snedecor, G.W., Cochran, W.G., 1980. Statistical Methods, seventh ed.
Iowa State University, USA. pp. 32–43.
Stanbury, P., Whitaker, A., Hall, S., 1995. Fermentation economics. In:
Principles of Fermentation Technology, second ed. Pergamon Press,
Oxford, UK, pp. 331–341.
Wang, J., 1998. Improvement of citric acid production by Aspergillus niger
with addition of phytate to beet molasses. Bioresour. Technol. 65, 243–
245.