Direct production of citric acid from raw starch by Aspergillus niger

Ikram-Ul Haq a,*, Sikander Ali a, Javed Iqbal b

a Biotechnology Research Laboratories, Department of Botany, Government College, Lahore, Pakistanb Department of Botany, University of the Punjab, Quaid-E-Azam Campus, Lahore 54590, Pakistan

Received 4 September 2001; received in revised form 25 April 2002; accepted 18 June 2002

Abstract

The present study deals with the direct production of citric acid from raw starch by Aspergillus niger . Shake flask and semi solid

culture methods were compared using A. niger GCB-47 (parental strain) and GCMC-7 (mutant strain). When cultivated in shaking

culture with 150 g/l soluble starch as a carbon source, the mutant strain GCMC-7 produced 69.5 g/l citric acid, which was, 1.48-fold

greater than the parental strain GCB-47. From a practical viewpoint, direct production of citric acid from corn and potato starch

was examined using semi-solid culture. On the basis of a comparison of kinetic parameters namely the volumetric substrate uptake

rate (Qs), the specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it

was observed that the mutant strain was a faster growing organism. The mutant strain GCMC-7 produced 71.4 and 92.9 g/l citric

acid, approximately 1.44 and 1.12 times as much as the parental strain GCB-47, from 200 g/l corn and potato starch, respectively.

The findings suggest that GCMC-7 possesses enhanced ability for sugar metabolism and citric acid production.

# 2002 Published by Elsevier Science Ltd.

Keywords: Citric acid; Direct production; Organic acids; Fermentation; Starch hydrolysis; Aspergillus niger ; Filamentous fungi; Mutants

1. Introduction

The entire worldwide demand for citric acid is met by

fermentative production mainly by the process involving

the filamentous fungus Aspergillus niger . Citric acid is a

commodity chemical, so, it is necessary to use inexpen-

sive and readily available raw materials in industrial

processes [1,2]. From this point of view, large amounts

of starchy materials are readily available since they are

cheap and renewable. In practice, starch hydrolysate is

generally used as a substrate, especially in submerged

fermentation [3]. Raw starch used without first being

hydrolysed generally gives a low yield of citric acid [4].

However, for direct production of citric acid from

starchy material, pretreatment processes could be

omitted and as a consequence the energy requirements

and cost of citric acid production could be reduced. The

present investigation deals with the direct production of

citric acid from corn and potato starch by a parental

strain of A. niger and a mutant strain GCMC-7 in semi-

solid culture. The yield of citric acid reached 47�/54%,

which is comparable to that from soluble starch under

shaking cultures.

2. Materials and methods

2.1. Microorganism and fermentation media

A. niger GCB-47, a hyper producer of citric acid in

semi-solid culture, was used as the parental strain. Strain

GCMC-7, a 2-deoxy-D-glucose-resistant mutant strain

induced from GCB-47 was also used. Synthetic mediumcontaining (%): NH4NO3 0.25, MgSO4 �/7H2O 0.025,

KH2PO4 0.1, glucose 15, initial pH 3.5 was used in

shaking culture. Since the solubility of corn and potato

starch in water is much lower than those of other

saccharides such as glucose or sucrose, these starches

were suspended in the medium and used only for semi-

solid culture. For semi-solid culture, 200 g/l of the

carbon source (corn or potato starch) was used. Allculture media were sterilized at 15 lb/in.2 pressure

(121 8C) for 15 min. Cultivations were carried out at

30 8C.* Corresponding author

E-mail address: ikrhaq@yahoo.com (I.-U. Haq).

Process Biochemistry 38 (2003) 921�/924

www.elsevier.com/locate/procbio

0032-9592/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.

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2.2. Fermentation techniques

Conidia were suspended in 60 ml synthetic medium

containing each carbon source. Orbital shaking wasmaintained at 160 rpm in 250 ml Erlenmeyer flasks for 7

days. After cultivation, the mycelia were removed by

filtration through Whatman filter No. 1 and the culture

filtrate used for analysis. Cultivation was carried out by

adding 1.0 ml of the conidial suspension (1.2�/106

conidia per ml) in 25 ml medium. For semi-solid culture,

3.2 g corn or potato starch in 15.0 ml basal synthetic

solution (pH 4.5), and 3.9 g sugar-cane bagasse wereadded in a Petri dish and sterilized. After cooling, 1.0 ml

of the conidial suspension was added to the semi-solid

medium and cultivation was initiated. Then 200 ml

distilled water was added in each sample at 60 8C. The

mixture was vigorously stirred for about 15 min and

filtered. The filtrate was then used as the semi-solid

culture extract for analysis.

2.3. Analysis

The concentration of citric acid in culture filtrate was

measured by titration with 0.1 N NaOH using phe-

nolphthalein as an indicator. After titration, analysis of

organic acids including citric acid was carried out

colorimetrically [5]. Growth was estimated by measuringthe mycelial dry weight of filtered mycelia [6]. In semi-

solid culture, it was impossible to separate the mycelia

from the bagasse, therefore, the mycelial dry weight was

not measured. Residual sugars were estimated by a DNS

method [7]. Kinetic relations were based on the proce-

dures of Pirt [8]. Duncan’s multiple range tests were

employed to analyze the data statistically [9].

3. Results and discussion

Corn and potato starch at concentrations of 10, 20,

50, 100, 200 and 250 g/l were examined for citric acid

production by GCB-47 using shake flask technique in250 ml Erlenmeyer flask (Table 1). The maximum

amount of citric acid produced with corn starch and

potato starch was 65.30 and 69.30 g/l, respectively.

Citric acid yield was highest at 200 g/l (m�/0.134 per h),

although there was little difference with 100 g/l starch.

In order to asses whether the mutant strain GCMC-7

would show enhanced production of citric acid from

starch, the mutant strain of A. niger GCMC-7 and the

parental strain GCB-47 were compared (Table 2), Both

cultures were cultivated in the medium containing 200 g/

l starch as a carbon source. The results demonstrated the

enhanced citrate potency from raw starch. Values of

citric acid obtained in the study are highly significant

(LSD 0.320 and 0.564 for parental and mutant strains,

respectively). Semi-solid culture method is suitable for

citric acid production from insoluble carbon sources

such as starch granules since stirring is unnecessary

during cultivation [10�/12].

A time course of citric acid production by the mutant

strain GCMC-7 with the medium containing 200 g/l

potato starch as the sole carbon source was undertaken

(Table 3). The maximum concentration of citric acid

produced after 168 h was 92.90 g/l (Yp/s�/0.874 g/g).

The amount produced by GCMC-7 was 1.48-fold higher

than that of GCB-47. These results suggest that the

enhanced starch hydrolytic activity related to the higher

citric acid production of GCMC-7 was due to increased

activity of glucoamylase in the fermented broth. More-

over, the rate of potato starch consumption by GCMC-

7 (Qp�/0.564 g/l per h) was faster than that of GCB-47,

which is highly significant. The mutant strain GCMC-7

possessed higher starch metabolizing and citric acid

producing abilities, which were probably related to some

difference in the property of catabolite repression

released in comparison with the parental strain. Taka-

tomi and Usami [13] reported that citric acid production

from the hydrolysate of sweet-potato starch gave a high

yield of 60% based on the amount of reducing sugar

supplied. Potato starch contains large amount of

amylopectin, a branched and water insoluble polysac-

charide as compared with the starch obtained from

corn, wheat or rice [4,14]. The culture filtrates of GCB-

47 and GCMC-7 cultivated with soluble starch in

Table 1

Citric acid production by A. niger GCB-47 in semi-solid culture

Starch concentration (g/l) Specific growth rate m (per h) Citric acid (g/l)

Corn starch Potato starch

10 0.118 3.25 3.06

20 0.121 3.35 5.27

50 0.123 8.10 19.50

100 0.123 48.60 50.40

200 0.134 65.30 69.30

250 0.126 54.52 61.62

Citric acid fermentation was carried out in SS-medium containing corn or potato starch at 30 8C for 3 days. Each value is an average of three

replicates. The values differ significantly at P B0.05. m (per h)� specific growth rate.

I.-U. Haq et al. / Process Biochemistry 38 (2003) 921�/924922

shaking culture efficiently saccharified potato starch.

Therefore, it was considered that direct production of

citric acid from potato starch might give a sufficient

yield. Citric acid productivity from potato starch was

higher than corn starch for both strains. No byproduct

organic acids other than from citric acid were detected in

the medium.

The data of Table 4 shows the comparison of kinetic

parameters for citric acid production from 200 g/l

potato starch as carbon source following growth of A.

niger and its mutant derivative. Maximum growth in

terms of specific growth rate (m) was only marginally

different during growth of the wild parent and the

mutant strain. However, when the cultures were mon-

itored for Yp/s, Qp and qs, there was a significant

enhancement (P B/0.05) in these variables in mutant

culture over the obtained for wild culture of A. niger . All

the other kinetic parameters [i.e. specific yield of citric

acid (Yp/x), specific productivity (qp) and volumetric

substrate utilization constant (Qs), etc.] of mutant strain

of A. niger GCMC-7 are several folds improved over the

parental strain GCB-47. These findings too suggest that

GCMC-7 possesses enhanced ability for sugar metabo-

lism and citric acid production.

3.1. Conclusion

For citric acid production from raw starch, prelimin-

ary liquefaction and saccharification of raw starch and

Table 2

Comparison of different carbon sources for the production of citric acid by using both GCB-47 (parent) and GCMC-7 (mutant) of A. niger

Carbon sources GCB-47 (parental) GCMC-7 (mutant)

CA (g/l) DCM (g/l) SU (g/l) CA(g/l) DCM (g/l) SU (g/l)

Soluble starch 45.1 18.5 72.0 69.5 16.5 80.5

Corn starch 44.2 21.0 125.5 71.4 19.5 116.0

Potato starch 85.3 19.5 132.0 92.9 16.0 125.0

LSD 0.320 0.112 0.567 0.564 0.124 0.583

HS HS HS HS HS HS

Incubation temperature�30 8C, soluble starch added�150 g/l, corn starch/potato starch added�200 g/l, fermentation period�7 days. Each

value is an average of three replicates. The values differ significantly at P 50.02. CA, citric acid; DCM, dry cell mass; SU, substrate utilized; HS,

highly significant; LSD, least significant difference.

Table 3

Comparison of kinetic parameters during time course study of citric acid production by mutant strain of A. niger GCMC-7 using potato raw starch

as carbon source (200 g/l)

Incubation period (h) Yp/s (g/g) Qp (g/l per h) Citric acid (g/l) Significance

24 0.329 0.082 4.09 �/

48 0.690 0.244 13.89 �/

72 0.668 0.292 26.45 �/

96 0.783 0.408 37.08 �/

120 0.891 0.512 49.98 �/

144 1.054 0.630 73.55 HS

168 0.874 0.564 92.90 HS

192 0.702 0.507 83.40 �/

HS denotes that the values are highly significant. The values differ significantly at P 50.20. Yp/s�g citric acid produced/g substrate consumed,

Qp�g citric acid produced/l per h.

Table 4

Kinetic parameters for production of citric acid from 200 g/l potato

starch as carbon source following growth of A. niger and its mutant

derivative

Kinetic parameters Parental strain GCB-47 Mutant strain GCMC-7

Citric acid formation parameters

Qp (g/l per h) 0.126 0 246

Yp/s (g/g) 0.174 0.507

Yp/x (g/g) 1.050 2.950

Qp (g/g cells per h) 0.017 0.020

Substrate consumption parameters

m (per h) 0.128 0.137

Yx/s (g cells/g) 0.164 0.192

Qs (g/l per h) 0.581 0.645

qs (g/g cell per h) 0.072 0.118

Each value is an average of three replicates. The values differ

significantly at P 50.05. m (per h)� specific growth rate, Yx/s�g

cells/g substrate utilized, Qs�g substrate consumed/l per h, qs�g

substrate consumed/g cells per h, Qp�g citric acid produced/l per h,

Yp/s�g citric acid produced/g substrate consumed, Yp/x�g citric

acid produced/g cells formed, qp�g citric acid produced/g cells per h.

I.-U. Haq et al. / Process Biochemistry 38 (2003) 921�/924 923

optimization of cultural conditions are normally neces-

sary. In the present study, direct production of citric

acid using semi-solid culture method and the mutant

strain GCMC-7 produced 92.90 g/l citric acid from 200g/l potato starch. The results obtained are highly

significant (Qp�/0.564 g/l per h). Fermentations were

completed after 168 h and yields based on the starch

supplied were comparable to those reported for citric

acid production from starch hydrolysate. The semi-solid

culture method with GCMC-7 might be useful in a

practical system for citric acid production from raw

starch without hydrolysis.

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