The kinetic basis of the role of Ca++ ions for higher yield of citric acid in a repeated-

batch cultivation system

Ikram-ul-Haq1,*, Sikander Ali1, M.A. Qadeer2 and Javed Iqbal31Biotechnology Research Laboratories, Department of Botany, Government College University Lahore, Pakistan2National Centre of Excellence in Molecular Biology, Thokar Niaz Baig, C/B Road, University of the Punjab, Lahore,Pakistan3School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan*Author for correspondence: E-mails: ikrhaq@yahoo.com, hamad_ashraf@4u.net

Received 23 December 2002; accepted 23 May 2003

Keywords: Agitator bioreactor, Aspergillus niger, CaCl2, citric acid, repeated-batch culture, TCA cycle

Summary

The present investigation deals with role of Caþþ ions in increasing the yield of citric acid in a repeated-batchcultivation system (working volume 9-l) and its kinetic basis. Five different hyper-producing strains of Aspergillusniger were evaluated for citric acid production using clarified cane-molasses as basal substrate. Among the cultures,NGGCB101 (developed by u.v./chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/l, 6 days after mycelial inoculation. The addition of CaCl2 to the culture medium promoted the formation of smallrounded fluffy pellets (1.55 mm, diameter), which were desirable for citric acid productivity. CaCl2 at a level of2.0 lM, added during inoculation time, was optimized for commercial exploitation of molasses. During repeated-batch culturing, a yield of citric acid monohydrate of 128.68 g/l was obtained when the sampling vs. substratefeeding was maintained at 4-l (44.50% working volume). The incubation period was reduced from 6 to only 2 days.The values of kinetic parameters such as substrate consumption and product formation rates revealed thehyperproducibility of citric acid by the selected Aspergillus niger NGGCB101 (LSD ¼ 0.456a, HS). Case studies arehighly economical because of higher yield of product, lower energy consumption and the use of raw substratewithout any additional supplementation.

Introduction

Citric acid i.e., 2-hydroxy-1,2,3-propanetri-carboxylicacid (CH2COOH ÆCOH ÆCOOH ÆCH2COOH) is ubiq-uitous in nature and exists as an intermediate in theKrebs cycle (Kubicek 1998). It is solid at roomtemperature, melts at 153 �C and decomposes at highertemperature. It is responsible for the tart taste of variousfruits e.g., lemons, limes, oranges, pineapples, pears andgooseberries. It can also be produced by the fermenta-tion of glucose. It is a primary metabolite produced byAspergillus niger during the idiophase at which thegrowth drops and acid production becomes the maincellular activity (Berzi et al. 2001). The organisms needmajor elements such as nitrogen, phosphorus, calciumand sulphur in addition to carbon and various traceelements, for growth and subsequent citric acid produc-tion. The worldwide demand for citric acid is about6.0 · 105 tons per year (Karaffa & Kubicek 2003).For efficient citric acid production, growth of Asper-

gillus niger in pellet form is desirable and this can beachieved by process optimization (Wayman & Mattey2000). Factors which affect Aspergillus growth and citric

acid yield may include, incubation temperature, initialpH, substrate and nitrogen concentrations, dissolvedoxygen and cation level of the medium (Peskova et al.1996; Ali et al. 2001). In this manuscript, we report citricacid production as a bioprocessing laboratory process.For this, five different strains of Aspergillus niger werecompared for citric acid production, biomass formationand substrate utilization on a kinetic basis in a repeated-batch cultivation system. Addition of Caþþ ions in thebroth not only enhanced citric acid productivity but alsoreduced the incubation period, making the process moreeconomical.

Materials and methods

Organism

Five different cultures of Aspergillus niger were used inthe present study (Table 1) and were compared for citricacid productivity in an agitator bioreactor equippedwith cross baffles. These cultures were obtained from theculture collection of Biotechnology Research Labs,

World Journal of Microbiology & Biotechnology 19: 817–823, 2003. 817� 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Department of Botany, Government College UniversityLahore, Pakistan and maintained on sterilized potatodextrose agar medium (BDH, Germany), pH 5.6. Theculture slants were stored at 5 �C in the refrigerator. Allthe culture media were sterilized at 121 �C for 15 min,unless otherwise stated.

Vegetative inoculum and mutation

Hundred millilitre of molasses medium (sugar 15%, pH6.0) containing glass beads (2 mm, dia), in 1-l cottonwool plugged Erlenmeyer flask, was sterilized. A smallamount of conidia from the slant culture was asepticallytransferred with an inoculating needle. The flask wasincubated at 30 �C in an incubator shaker (Gallenkamp,UK) at 200 rev/min for 24 h. The conidia of Aspergillusniger were u.v.-irradiated following the method of Haqet al. (2001). The cell suspension was then treated withN-methyl N¢-nitro N-nitrosoguanidine (NG) accordingto the procedure of Roy & Das (1977).

Pre-treatment of molasses

Cane molasses obtained from Chunian Sugar Mills,Pakistan was clarified according to the method of Pandaet al. (1984). After pre-treatment, the sugar content wasmaintained at 15% (w/v) or desired levels and stored at15 �C in an amber coloured bottle.

Fermentation technique

A stainless steel fermentor of 15-l capacity (workingvolume 9-l) was employed for citric acid fermentation.

The fermentation medium consisting of (g/l); clarifiedcane molasses 300.0 (sugar 15%), K4Fe(CN)6 200 mg/lat pH 6.0 was used for fermentation. The vegetativeinoculum was transferred to the production medium at arate of 4.0% (v/v) based on total working volume of thefermentation medium. The incubation temperature was30 �C for 96–144 h. Agitation speed of the stirrer waskept at 200 rev/min and aeration rate was maintained at1.0 l/l/min. Sterilized silicone oil (antifoam AE-II) wasused to control foaming during fermentation.

Repeated-batch culturing

The sampling vs. repeated-batch was changed from 1.0to 5.0 l (11.50–55.50%). The samples were taken afterevery 24 h until citric acid production reached maximal.The same amount of sterilized medium was regularlyadded in the working vessel whenever the samples werewithdrawn. For each set of volumes, the procedure wasrepeated until acid production become low.

Assay methods

SugarSugar was estimated gravimetrically by the DNS method(Tasun et al. 1970). A double beam u.v./vis. scanningspectrophotometer (Model: Cecil-CE 7200-series, Agua-rius, UK) was used for measuring the % colour intensity.

Dry cell massDry cell mass was determined by filtering the culturemedium through weighed Whattman filter paper no. 44.The filtrate was used for further analysis. For the

Table 1. Comparison of kinetic parameters for the production of citric acid from cane-molasses following growth of different cultures of

Aspergillus niger.

Kinetic parameters Selected stock cultures of Aspergillus niger

NIAB-280 NGGCB101 NRRC-1243 Korea (ab-1) Zowang-12

Citric acid monohydrate 79.40 87.98 59.85 54.50 84.20

Substrate consumption parameters

l (h)1) 0.495 ± 0.02bc 0.593 ± 0.02a 0.4349 ± 0.01d 0.234 ± 0.02ef 0.548 ± 0.03ab

Yx/s (g cells/g) 0.194 ± 0.03ab 0.204 ± 0.03a 0.108 ± 0.02cd 0.098 ± 0.04d 0.148 ± 0.04c

Qs (g/l/h) 0.767 ± 0.02b 0.962 ± 0.02a 0.515 ± 0.02de 0.531 ± 0.03d 0.704 ± 0.04bc

qs (g/g cells/h) 0.063 ± 0.03bc 0.093 ± 0.03a 0.050 ± 0.01d 0.051 ± 0.02d 0.080 ± 0.03b

Qx (g cells/l/h) 0.073 ± 0.03b 0.078 ± 0.03a 0.061 ± 0.03d 0.063 ± 0.03d 0.074 ± 0.04b

Product formation parameters

Qp (g/l/h) 0.193 ± 0.03c 0.290 ± 0.03a 0.149 ± 0.03cd 0.236 ± 0.03b 0.248 ± 0.02b

Yp/s (g/g) 0.340 ± 0.02gh 0.940 ± 0.02a 0.872 ± 0.03b 0.442 ± 0.02ef 0.887 ± 0.03b

Yp/x (g/g cells) 3.600 ± 0.03ef 6.620 ± 0.03b 4.392 ± 0.04d 3.752 ± 0.02e 7.360 ± 0.04a

qp (g/g cells/h) 0.021 ± 0.02g 0.081 ± 0.02a 0.044 ± 0.02cd 0.072 ± 0.03b 0.043 ± 0.04cd

LSD 0.234bc 0.456a 0.098de 0.102d 0.248bc

Significance level (P) S HS – – S

Each value is an average of three parallel replicates. ± Indicates standard deviation among replicates. P indicates probability significance level.

The numbers differ significantly at P < 0.05. l (h)1) = growth rate constant, Yx/s = g cells/g substrate utilized, Qs = g substrate consumed/l/

h, qs = g substrate consumed/g cells/h, Qx = g cells formed/l/h, Qp = g citric acid produced/l/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/h. HS denotes that the values are highly significant

while S is for only significant values. LSD is for least significant difference.

818 Ikram-ul-Haq et al.

calculation of dry cell mass, mycelium was thoroughlywashed with tap water and dried at 105 �C for 2 h in anoven (Model: 1442A, Memmort, Germany) followingthe method of Haq & Daud (1995).

Total acidTotal acid was estimated by titrating 10.0 ml of dilutedculture filtrate against 0.1 M NaOH using phenolphtha-lein as an indicator. The total acid was reported in g/l.

% Total acid

¼ Titre� normality of alkali� equivalent weight of acid

Volume of sample� 1000

� 100

The equivalent weight of the acid is 70.

Citric acidCitric acid was estimated gravimetrically, using pyri-dine–acetic anhydride method as reported by Marrier &Boulet (1958). 1 ml of the diluted culture filtrate alongwith 1.30 ml of pyridine was added to a test tube andswirled briskly. Then 5.70 ml of acetic anhydride wasadded, and the test tube incubated at 32 ± 0.25 �C for30 min. The absorbance was measured at 405 nm andthe citric acid content of the sample was estimated withreference to a standard. The % of citric acid wasdetermined by using the formula.

% Citric acid ¼ Citric acid

Sugar used� 100

Ferrocyanide ionsFerrocyanide was estimated by the method of Marrier &Clark (1962). To 5.0 ml of culture filtrate 2 ml of 50%citric acid solution and 1.0 ml of ferric chloride reagentwere added. The contents were mixed thoroughly, setaside for 60–90 min at room temperature. A blank wasalso run parallel with 5.0 ml of distilled water in place offerrocyanide dilution, followed by the above procedure.The absorbance was measured at 690 nm. To correct forthe dark colour of the molasses, a duplicate aliquot wastreated similarly but with ferric chloride reagent re-placed by water. The absorbance of this sample wasused to correct the value of the test solution.

Kinetic study and statistical analysisThe kinetics of growth were measured (Pirt 1975) and thetreatment effects were compared after Snedecor & Coch-ran (1980). Significance has been presented as Duncanmultiple ranges in the form of probability (P) values.

Results and discussion

The data of Table 1 show the screening of the bestavailable stock culture of Aspergillus niger for citric acid

production in an agitator bioreactor. The concentrationof citric acid formed was in the range 54.50–87.98 g/l.The culture NGGCB101 (developed by u.v./Chemicalmutation in our labs) gave maximum citric acid pro-duction (87.98 g/l) followed by Zowang-12 (84.20 g/l)and NIAB-280 (79.40 g/l), respectively. Comparison ofkinetic relations (substrate consumption and productformation parameters) for citric acid fermentation wasalso made. The highest growth rate constant i.e.,l ¼ 0.593 ± 0.02a h)1 was obtained with Aspergillusniger strain NGGCB101. All the values of substrateconsumption rates (Yx/s, Qs, Qx and qs) and productformation coefficients (Qp, qp, Yp/s and Yp/x) forNGGCB101 were higher than the other strains. Thework is in accordance with that of with previousworkers (Roehr et al. 1987; Haq & Daud 1995). Delgado& Liao (1997) described that with the increase ofmycelial formation in the medium, there was a reductionin the yield of citric acid.The optimum time of incubation for maximal citric

acid production varies both with the organism andfermentation conditions. Figure 1 shows the time courseprofile for citric acid production by Aspergillus nigerNGGCB101 in a stirred fermentor. The fermentation wascarried out from 24 to 192 h. Maximum production ofcitric acid (119.6 g/l) was achieved at 96 h after incuba-tion. The sugar consumption, dry mycelial weight andfinal pH of the fermentation medium were 92.5, 13.5 g/land 2.1, respectively. Further increase in incubationperiod did not enhance citric acid production. Thismight be due to the decrease in amount of availablenitrogen in the fermentation medium, the age of thefungus, the presence of inhibitors produced by thefungus itself or the depletion of sugar contents. Similarwork has also been reported by Roehr (1998). Peskovaet al. (1996) reported the maximum yield of citric acid,8 days after the inoculation.

0

15

30

45

60

75

90

105

120

135

0 24 48 72 96 120 144 168 192Time course (h)

Dry

cel

l mas

s, S

ugar

use

d, C

itric

aci

d (g

/l)

0

1

2

3

4

5

6

Change in pH

Dry cell mass (g/l) Sugar used (g/l) Citric acid (g/l) Change in pH

Figure 1. Time course profile of citric acid production by mutant

strain of Aspergillus niger NGGCB101. Initial sugar concentration

150 g/l, initial pH 6.0, aeration rate 1.0 l/l/min, incubation tempera-

ture 30 �C. CaCl2 (2.0 lM) was added at the time of mycelial

inoculation.

Caþþ ion influence on citric acid production 819

The initial sugar concentration has been found todetermine the amount of citric acid and also the amountof other organic acids produced by Aspergillus niger.Figure 2 shows the effect of different sugar concentra-tions (60–210 g/l) of molasses medium for citric acidproduction by Aspergillus niger NGGCB101 in a stirredfermentor. The maximum amount of citric acid(118.95 g/l) was obtained in the medium containing150 g sugar/l. The consumption of sugar and drymycelial weight were 93.2 and 13.6 g/l, respectively. Inour finding, gradual reduction in citric acid formationwas observed when the sugar concentration of molassesmedium was further increased beyond 150 g/l. Thismight be due to overgrowth of the mycelium, resultingin increased viscosity of the medium and mass transferlimitation. Kubicek et al. (1994) described that with theincrease of mycelial formation in the medium, there wasreduction in the yield of citric acid. Normally, strains ofAspergillus niger need a fairly high concentration (150–180 g/l) of sugars in the medium. A lower concentrationof sugar leads to lower yields of citric acid as well as tothe accumulation of oxalic acid.In batchwise fermentation of citric acid, the produc-

tion started after a lag phase of 24 h and reachedmaximum at the onset of stationary or death phase. Theaddition of CaCl2 (2.0 lM) in the medium was found tobe economical, especially when the culture of Aspergillusniger NGGCB101 was used as the organism of choice(Table 2). This might be related to the higher yield ofcitric acid and reduction in incubation period (6–4 days). Small rounded pellets having 1.55 mm diameterwere desirable for maximal specific productivity (i.e.,0.928 g/l/h). The remaining of cultures did not showencouraging results with Caþþ ion addition to the broth.The Caþþ ions might have degraded cellular proteinsreleasing free NH4 ions. This decrease in intracellularproteins thus might cause more molasses carbon to gointo the TCA cycle to promote citric acid accumulation.However, Kisser et al. (1980) pointed out that Caþþ

0

20

40

60

80

100

120

140

160

180

60 90 120 150 180 210Initial sugar concentration (g/l)

Dry

cel

l mas

s, S

ugar

use

d, C

itric

aci

d (g

/l)

Dry cell mass (g/l) Sugar used (g/l) Citric acid (g/l)

Figure 2. Effect of initial sugar concentration on citric acid production

by mutant strain of Aspergillus niger NGGCB101. Fermentation period

96 h, initial pH 6.0, aeration rate 1.0 l/l/min, incubation temperature

30 �C. CaCl2 (2.0 lM) was added at the time of mycelial inoculation.Table

2.EffectofdifferentCaCl 2concentrationoncitric

acidferm

entationandcomparisonofdifferentculture

variables.

Parametricvariables

CaCl 2concentration(lM)

1.0

2.0

3.0

4.0

NIA

B-280

NG

GCB101

Zowang-12

NIA

B-280

NG

GCB101

Zowang-12

NIA

B-280

NG

GCB101

Zowang-12

NIA

B-280

NG

GCB101

Zowang-12

Totalacid(g/l)

90.20

95.44

92.64

82.85

125.04

94.60

75.42

105.45

82.30

69.96

90.02

71.50

Citricacid(g/l)

81.12

89.67

82.24

73.22

119.60

83.25

65.44

99.67

73.20

58.98

78.62

68.15

Dry

cellmass

(g/l)

17.50

14.55

17.50

15.50

13.50

16.50

12.00

14.75

14.00

8.50

12.50

12.65

Sugarused(g/l)

92.65

94.50

108.50

80.55

92.50

118.50

76.52

88.00

124.55

69.65

84.20

128.50

Sp.productivity(g/g/h)

0.875

0.898

0.852

0.567

0.928

0.872

0.459

0.878

0.812

0.426

0.760

0.782

Morphology

Pellets

Pellets

Pellets

Viscous

Pellets

Viscous

Viscous

Pellets

Viscous

Viscous

Pellets

Viscous

Pelletdiameter

(mm

2)

1.05

1.75

2.52

–1.55

––

1.40

––

1.25

–

Peakdayofproduct

form

ation

65

56

45

64

56

44

Initialsugarconcentration150g/l,initialpH

6.0,aerationrate

1.0

l/l/min,incubationtemperature

30�C

.Alltheresultsare

sum

meanofthreeparallelreplicates.CaCl 2concentrationswereadded

atthe

timeofmycelialinoculation.

820 Ikram-ul-Haq et al.

Table

3.EffectofCaCl 2concentrationontheproductionofcitric

acidbymutantstrain

ofAspergillusniger

NG

GCB101in

repeated-batchbioreactor.

Samplingvs.repeated-batch

CaCl 2conc.

(lM)

Peakdayfor

maxim

alproduct

form

ation

Totalacid(g/l)

Citricacid

monohydrate

(g/l)

Dry

cell

mass

(g/l)

FinalpH

Sugar(g/l)

Final

ferrocyanide

ionsconc.

(mg/l)

Mycelial

morphology

Litres

%Used

Residual

Control

––

693.34

88.55

14.55

2.3

94.5

55.5

50.58

Smallpellets

1.0

11.50

1.0

248.44

43.52

12.05

3.8

104.0

46.0

40.90

Largepellets

2.0

259.52

56.78

15.50

3.2

98.7

52.3

40.05

Mixed

mycelia

3.0

248.98

47.40

18.00

3.5

98.9

51.1

50.50

Finepellets

4.0

234.31

30.28

22.50

3.6

108.5

41.5

56.44

Finepellets

2.0

22.50

1.0

246.55

42.34

11.55

3.8

96.5

43.5

34.52

Viscous

2.0

268.95

65.55

14.06

3.1

102.0

48.0

36.50

Viscous

3.0

260.04

56.78

16.65

3.3

105.5

44.5

42.55

Finepellets

4.0

257.62

55.02

19.90

3.4

112.5

37.5

49.98

Finepellets

3.0

33.50

1.0

274.67

70.98

10.00

2.6

98.6

51.4

30.54

Mixed

mycelia

2.0

286.46

79.86

12.50

2.4

99.5

50.5

32.00

Mixed

mycelia

3.0

280.90

75.54

15.75

2.5

100.0

50.0

38.04

Gelatinous

4.0

278.74

74.24

18.90

2.5

108.5

41.5

39.50

Gummymass

4.0

44.50

1.0

298.66

92.80

9.50

2.1

94.6

45.4

22.45

Mixed

mycelia

2.0

2134.5

128.68

12.30

1.6

95.0

55.0

22.80

Fluffypellets

3.0

2106.6

98.42

13.50

1.9

103.5

46.5

24.55

Smallpellets

4.0

294.32

90.10

14.45

2.0

110.0

40.0

25.00

Smallpellets

5.0

55.50

1.0

379.34

78.25

8.00

2.4

120.5

29.5

20.80

Mixed

mycelia

2.0

395.90

91.20

9.50

2.2

115.0

35.0

21.34

Finepellets

3.0

392.22

87.52

10.50

2.5

104.5

45.5

24.05

Finepellets

4.0

389.56

85.40

12.40

2.6

100.5

49.5

24.52

Finepellets

Initialsugarconcentration150g/l,initialpH

6.0,aerationrate

1.0

l/l/min,incubationtemperature

30�C

,ferrocyanideionconcentration200mg/l.Totalvolumeoftheferm

entationmedium

waskept

constanti.e.,9-l(60%)throughoutforallthetreatm

ents.Onthebasisofsugarused,alltheresultsare

sum

meanofthreeparallel

replicates.

Caþþ ion influence on citric acid production 821

reduces the effect of Fe, as it is an antagonist of Mn ionuptake.The data of Table 3 reveal the effect of CaCl2

concentration on the production of citric acid by mutantstrain of Aspergillus niger NGGCB101 in a repeated-batch bioreactor. The control gave 88.55 g/l citric acidmonohydrate, 6 days after inoculation. Maximumamount of citric acid was achieved after 48 h ofincubation when sampling vs. repeated-batch was main-tained at 4.0 l (44.50%) with the addition of 2.0 lMCaCl2. Thus, adding same amount of CaCl2 butrecycling the medium and reusing of mycelia throughrepeated-batch culture in the agitator bioreactor re-duced the incubation period from 6 to 4 days with Caþþ

ions and then from 4 to only 2 days. Final pH of thefermented broth was recorded to be 1.6. The dry cellmass and sugar consumption was 12.30 and 95.0 g/l,respectively. Mycelial morphology was in the form ofsmall round fluffy pellets. Higher citric acid yield isdirectly related to pellet formation, which allows goodmixing and mass transfer. Conidia trap each other andform a core around which hyphae develop (Delgado &Liao 1997). Round uniform fluffy loose pellets(1.55 mm, diameter) and short stubby lateral hyphaewith swollen tips gave better citric acid production.The data indicate that in repeated-batch culture, citric

acid production is 1.68-fold higher than the control.Hence, the process is economically feasible for the yieldof citric acid monohydrate. Legisa &Mattey (1988) havepatented a continuous multistage process for citric acidproduction. In this process, culture medium was addedat a rate providing for its complete replacement in 24 h.The influence coefficient of the medium was kept at 4%.Therefore, our finding is not in agreement with the studyof these workers. The kinetic parameters of repeated-batch culture for citric acid production by Aspergillusniger NGGCB101 are given in Table 4. The maximumvalues of growth yield coefficients (Yp/s and Yp/x in g/g),volumetric rates (Qp and Qs in g/l/h) and specific rateconstants (qs and qx in g/g/l) were obtained whensampling vs. repeated-batch was maintained at 4.0-l,

supplemented with 2.0 lM CaCl2 during the time ofinoculation.

Conclusion

Stability of citric acid in market and price range makes itone of the most stable commodities that can beproduced from molasses. The present investigationconstitutes an attempt to design optimization strategiesfor the citric acid production rate in Aspergillus niger byintegrating in a framework of relevant aspects of thefungal physiology. In this regard, the noticeable findingwas that the addition of 2.0 lMCaCl2 in repeated-batchbioreactor led to a higher yield of citric acid. It ishypothesized that Caþþ ions make the mycelium fluffyand thus help in the easy excretion of citric acid fromvegetative cells. The repeated batch culture not onlyhelps in the optimal growth of organism but also helpsin the excretion of citric acid in the broth. The value ofproduct yield coefficient (i.e., 1.061 ± 0.02a g/g) byAspergillus niger NGGCB101 is highly significant(LSD < 0.0340).

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Table 4. Kinetic parameters of repeated-batch culture for enhanced citric acid production by Aspergillus niger NGGCB101.

Sampling vs.

repeated-batch

Growth yield coefficients (g/g) Volumetric rates (g/l/h) Specific rate constants (g/g/h) Significance

level P

Liters % Yp/s Yp/x Qp Qs qs qx

Control – 0.964 ± 0.03b 5.444 ± 0.01d 0.299 ± 0.02ef 0.922 ± 0.04d 0.075 ± 0.03e 0.041 ± 0.02f S

1.0 11.50 0.987 ± 0.02b 5.745 ± 0.01cd 0.678 ± 0.02bc 1.243 ± 0.02b 0.098 ± 0.02d 0.052 ± 0.02de –

2.0 22.50 0.710 ± 0.04d 6.081 ± 0.02c 0.601 ± 0.02c 0.930 ± 0.02d 0.128 ± 0.02bc 0.085 ± 0.03c –

3.0 33.50 0.878 ± 0.04c 7.223 ± 0.02b 0.817 ± 0.02b 1.009 ± 0.02bc 0.135 ± 0.02bc 0.108 ± 0.02ab S

4.0 44.50 1.061 ± 0.02a 8.607 ± 0.01a 0.943 ± 0.02a 1.799 ± 0.02a 0.238 ± 0.03a 0.113 ± 0.02a HS

5.0 55.50 0.303 ± 0.04ef 5.004 ± 0.02d 0.598 ± 0.03d 0.902 ± 0.02d 0.097 ± 0.05d 0.064 ± 0.04d –

LSD 0.0345 0.321 0.112 0.212 0.678 0.542

± Indicates standard deviation among replicates. P indicates probability significance level. HS denotes that the values are highly significant

while S is for only significant values. LSD is for least significant difference. The numbers differ significantly at P < 0.020. Yp/s = g citric acid

produced/g substrate consumed, Yp/x = g citric acid produced/g cells formed, Qp = g citric acid produced/l/h, Qs = g substrate consumed/l/

h, qp = g citric acid produced/g cells/h, qs = g substrate consumed/g cells/h.

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