A high performance fermentation process for citric acid production by Aspergillusniger NGGCB-101, using vermiculite as an additive in a stirred bioreactor

Ikram-ul-Haq1, Sikander Ali1,*,�, M.A. Qadeer2 and Javed Iqbal31Department of Botany, Biotechnology Research Labs, G.C. University, Lahore, Pakistan (E-mail: ikrhaq@yahoo.com)2National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan3The School of Biological Sciences, University of the Punjab, Lahore, Pakistan*Author for correspondence: Tel.: +92-42-921-1634, Fax: +92-42-724-3198, E-mail: alisbiotech@yahoo.com�Present address: Department of Plant Soil Science and General Agriculture, Southern Illinois University atCarbondale, Carbondale, IL 62901, USA. Tel.: +1-618-457-0516, Fax: +1-618-453-7457, E-mail: alis@siu.edu

Received 27 January 2003; accepted 11 November 2003

Keywords: Aspergillus niger, cane-molasses, citric acid, fermentation, fermentor, vermiculite

Summary

The present study describes the use of vermiculite for enhanced citric acid productivity by a mutant strain ofAspergillus niger NGGCB-101 in a stirred bioreactor of 15.0 l capacity. The maximum amount of citric acid (96.10 g/l) was obtained with the control 144 h after mycelial inoculation. To enhance citric acid production, varying levelsof vermiculite were added as an additive into the fermentation medium. The best results were observed when0.20 lg/l vermiculite was added into the medium 24 h after inoculation resulting in the production of 146.88 g citricacid monohydrate/l. The dry cell mass and residual sugar were 11.75 and 55.90 g/l, respectively. Mixed mycelialpellets (1.08–1.28 mm, dia) were observed in the fermented culture broth. When the culture grown at differentvermiculite levels was monitored for Qp, Qs and qp, there was a significant enhancement (P £ 0.05) in these variablesover the control (vermiculite-free). Based on these results, it is concluded that vermiculite might affect mycelialmorphology and subsequent TCA cycle performance to improve carbon source utilization by the mould, basicparameters for high performance citric acid fermentation.

Introduction

There is a growing trend to enhance microbial citric acidproduction, due to its wide range of uses in the food andpharmaceutical industries (Haq et al. 2001). The rawmaterials like beet or cane-molasses, starch hydrolysateand glycerol residues are abundantly available forexploitation by microbial fermentations. They containvarious levels of available sugars, ranging from 40–60%with additional ionic salts. The production of metabo-lites is strongly dependent on the fungal strain andfermentation medium used (Kubicek & Rohr 1985;Maria & Wladyslaw 1989). A potent strain and a cheapcarbon source may also raise the status of the fermen-tation process; either directly or indirectly, and so affectthe economics of citric acid fermentation (Majolli &Aguirre 1999).Trace metals present in blackstrap molasses such as

iron, zinc, copper and manganese pose problems duringsubmerged citric acid fermentation, therefore concentra-tion of these heavy metals should be maintained foroptimal mycelial growth (Neilsen 1992; Pera & Callieri1997). The addition of vermiculite (0.02–0.10 lg/l) into

the medium at the time of inoculation or during the first40 h of fermentation counteracts the deleterious effect ofcertain trace metals like zinc and copper (Fedoseev et al.1970). The use of soil type vermiculite in fungalfermentations is increasing due to enhancement inproteins and metabolites (Tvuawa & Chandn 1986).The present studies highlight that vermiculite additionmay influence pellet formation, facilitate enzyme secre-tion and subsequently increase the citric acid production.

Materials and methods

Organism

Aspergillus niger strain NGGCB-101 was obtained fromavailable stock culture of Biotechnology Research Labs,G.C. University Lahore, Pakistan and maintained on thepotato dextrose agar slants (pH 4.8).

Molasses pre-treatment

Blackstrap molasses obtained from Kamalia SugarMills, Pakistan was pre-treated according to Panda

World Journal of Microbiology & Biotechnology 20: 463–467, 2004. 463� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

et al. (1984). The sugar content of molasses was typi-cally about 45.0%. Thirty-five millilitres of 0.05 MH2SO4 were added to per l molasses medium and placedin a water bath at 90 ± 2 �C for about 1 h. Aftercooling at room temperature, the medium was neutral-ized with lime (CaO) and left overnight for clarification.Two layers were formed, the upper shiny black and thelower yellowish brown, due to the presence of tracemetal hydroxides. The clear supernatant was decantedand diluted to 15.0% sugar.

Inoculum preparation

First 45.0 ml Vogel’s medium (0.5% KH2PO4, 0.2%NH4NO3, 0.4% (NH4)2SO4, 0.02% MgSO4, 0.1%peptone, 0.2% yeast extract at pH 5.5) were added toa 250 ml conical flask. Chromic acid-washed marblechips (12–15 in number) were added to break themycelial pellets. As a carbon source, 2.0 ml glucose(50.0% w/v) was then added into the Vogel’s medium.After autoclaving at 121 �C for 15 min and cooling atroom temperature, the flask was seeded with a loopful ofA. niger conidia aseptically. The inoculum was allowedto grow at 30 �C in a rotary incubator shaker (Gal-lenkamp, UK, 160 rev/min) for 24 h. Next the cells wereharvested, centrifuged at 8331 · g for 15 min, washedtwice with saline water (NaCl 0.85%, yeast extract0.50%) and resuspended in distilled water. Opticaldensity was measured (610 nm wavelength) in a doublebeam UV/VIS scanning spectrophotometer and main-tained at 0.5 · 10)2 dilution.

Fermentation technique

A stainless steel stirred bioreactor (GLSC-AF-199-10)of 9.0 l working volume (total capacity 15.0 l) wasemployed for the microbial cultivations. The fermenta-tion medium consisted of clarified blackstrap cane-molasses containing: initial sugar 15.0, NH4NO3 0.025,ash contents 0.45, trace metals like iron, zinc, aluminium(0.035% w/v) and K4Fe(CN)6 200 mg/l (initial pH 6.0)was sterilized at 121 �C (15 lb/in.2 pressure) for 20 min.Vermiculite (Analytical grade, Sigma, USA) concentra-tion varied from 0.10 to 0.40 lg/l added 8–48 h afterinoculation. Mycelial inoculum was added at a level of4.0% (v/v). Incubation temperature was kept at 30 �Cfor 144 h. The aeration rate was maintained at 1.0 l/l/min with a 35.0% dissolved O2 level (found optimal).Agitation intensity was 200 rev/min in all experiments.Sterilized silicone oil (10.0% antifoam AE-II) was usedto control foaming during fermentation. Samples weretaken every 24 h.

Assay methods

Dry cell mass was determined gravimetrically using thedry weight method after Pirt (1975). Citric acid andsugars in the fermented culture broth were determinedby the DNS method (Ghose 1969). Kinetic parameters

and statistical tests for batch fermentations were basedon Pirt (1975).

Results and discussion

The rates of citric acid production by the mutant strainAspergillus niger NGGCB-101 in a stirred bioreactor withcontrol (vermiculite-free) and experimental batches(0.20 lg/l vermiculite added at the time of inoculation)were compared (Figures 1 and 2). The maximumamount of citric acid obtained with the control 144 hafter mycelial inoculation was recorded at 96.10 g/l. Thedecreased citric acid production after 144 h might bedue to exhaustion of nutrients in the fermentationmedium and the age of the fungus, resulting in thedecreased metabolic rates. Citric acid excretion normallydecreases at the onset of stationary phase (Panda et al.1984). The dry cell mass and residual sugar were 14.20and 62.08 g/l, respectively. Small round fungal pellets(1.25–1.40 mm, dia) were found at the optimal citricacid production level. However, experimental batchcultivation gave 123.78 g/l citric acid productivity only96 h after incubation. Thus reduction in the incubationtime period for citric acid production took place. Inaddition, net citric acid productivity was 1.29-foldhigher than the control.To enhance citric acid productivity, the effect of the

addition of various vermiculite levels was investigated(Table 1). In the present study, vermiculite concentra-tion ranged from 0.10 to 0.40 lg/l added at the start offermentation. However, citric acid biosynthesis wasobserved to be maximal (124.62 g/l) when the concen-tration of vermiculite was maintained at 0.20 lg/l added96 h after the start of the production phase. Theenhancement in citric acid biosynthesis is thus sub-stantial. The dry cell mass and residual sugar were 12.00and 54.45 g/l, respectively (small round fungal pelletshaving 1.12 mm, dia). However, production of citricacid monohydrate was markedly decreased at higherlevels of vermiculite, due to a change of mycelialmorphology from pellets to a gelatinous form, substan-tiating the findings of Gulkhera (1997). The residualsugar and dry cell mass were also reduced.The effect of vermiculite addition time on the citric

acid production by Aspergillus niger was also examined(Table 2). Samples were taken every 24 h during thefermentation. As far as citric acid production is con-cerned, the best results (146.88 g/l citric acid monohy-drate) were obtained when 0.20 lg vermiculite/l wasadded 24 h after mycelial inoculation. The dry cell massand residual sugar were slightly lower than the previousexperimental batch i.e., 11.75 and 94.10 g/l, respectively(Figure 2). Mixed fungal pellets (0.08–1.28 mm, dia)overtook small round pellets, which revealed the effectof vermiculite on mycelial cells and subsequent higherproduct formation (Gulkhera 1997). In the presentstudy, the vermiculite might have an effect on theperformance of the TCA cycle to enhance its efficiency.

464 I. Haq et al.

The mould capacity to utilize carbon source was alsoincreased, resulting in higher product formation asreported by Fedoseev et al. (1970).A comparison of kinetic parameters for vermiculite

addition on citric acid production in the stirred biore-actor is highlighted in Table 3. All values at differentconcentrations of vermiculite were significantly im-proved for Yx/s, Yp/s and Yp/x over the control (withoutvermiculite addition). The maximum growth in terms of

volumetric rate for mycelial formation (Qx) 96 h afterincubation was only marginally different during fermen-tation at 0.20 and 0.25 lg/l, respectively. Similar find-ings have also been reported by Tvuawa & Chandn(1986) and Bruchmann (1961), but our report on citricacid productivity is many fold (<2.8–3.5) higher thanthese workers. In addition, when the culture wasmonitored for Qp and qs, there was a significantenhancement (P £ 0.05) in these variables at 0.20 lg/l

0

15

30

45

60

75

90

105

120

135

150

0 24 48 72 96 120 144 168 192 216 240Rate of citric acid fermentation with control (h)

Dry

cel

l mas

s, R

esid

ual s

ugar

, Citr

ic a

cid

(g/l)

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

Figure 1. The rate of citric acid fermentation by mutant strain of Aspergillus niger NGGCB-101 in stirred bioreactor (Incubation temperature

30 �C, initial pH 6.0, aeration rate 1.0 l/l/min, initial sugar concentration 150 g/l. Control batch i.e., without vermiculite addition).

0

15

30

45

60

75

90

105

120

135

150

0 24 48 72 96 120 144 168 192 216 240

Rate of citric acid fermentation with experimental (h)

Dry

cel

l mas

s, R

esid

ual s

ugar

, Citr

ic a

cid (g

/l)

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

Figure 2. The rate of citric acid fermentation by mutant strain of Aspergillus niger NGGCB-101 in stirred bioreactor (Incubation temperature

30 �C, initial pH 6.0, aeration rate 1.0 l/l/min, initial sugar concentration 150 g/l. Experimental batch i.e., with 0.20 lg/l vermiculite addition at

the time of inoculation).

A high performance fermentation process for citric acid production 465

vermiculite over other levels 96 h after the incubation.In particular, value for qp (i.e., specific productivity rate)at 0.20 lg/l is highly significant 96 h after incubation.The maximum Yp/s, Yp/x, Qp and qp were several foldimproved over those from some other Aspergillus orCellulomonas spp. (Schweiger 1961; Maria & Wladyslaw1989; Haq et al. 2001). In the present study, the ratecoefficient of citric acid production proportional togrowth rate was almost constant regardless of oxygentension (Unpublished data).

Conclusion

The improvement in citric acid fermentation technologymay be attributed to the impact of additives on mycelialmorphology for enhanced yields. In the present inves-tigation, most notable are the reduction in incubationperiod (144 to only 96 h) and the commercially accept-able citric acid productivity level (146.88 g/l) achievedby adding 0.20 lg vermiculite/l into the fermentationmedium 24 h after mycelial inoculation. We concludethat vermiculite addition somehow enhances the TCAcycle efficiency of mycelial cells for A. niger, resulting inhigher levels of product formation.

Table 1. The effect of vermiculite addition on citric acid fermentation of Aspergillus niger NGGCB-101 in stirred bioreactor*.

Vermiculite

concentration

(lg/l)

Concentration (g/l) Peak hour

for maximal

citric acid

production

Mycelial

morphology

Pellet diameter

(mm)

Dry cell mass Residual sugar Citric acid

monohydrate

0.10 12.80 51.50 104.46 120 Mixed pellets 1.35–1.60

0.15 12.15 53.20 109.50 116 Small pellets 1.20

0.20 12.00 53.45 124.62 96 Small pellets 1.12

0.25 11.30 55.90 116.10 96 Fine pellets 0.65

0.30 11.10 56.20 102.55 92 Fluffy mass –

0.35 10.20 61.85 89.26 84 Gelatinous –

0.40 9.55 67.60 81.86 80 Gelatinous –

* Incubation temperature 30 �C, initial pH 6.0, aeration rate 1.0 l/l/min, initial sugar concentration 150 g/l. Vermiculite was added at the time

of inoculation. Samples were taken every 24 h of fermentation.

Table 2. The effect of time of vermiculite addition on citric acid fermentation by Aspergillus niger NGGCB-101 in stirred bioreactor*.

Vermiculite

addition time (h)

Concentration (g/l) Mycelial morphology Pellet diameter (mm)

Dry cell mass Residual sugar Citric acid

monohydrate

8 12.65 51.10 126.10 Small pellets 1.16

16 12.10 55.35 131.64 Small pellets 1.12

24 11.75 55.90 146.88 Mixed pellets 1.08–1.28

32 11.60 56.20 122.05 Mixed pellets 1.15–1.55

40 11.50 59.40 121.16 Elongated mycelia –

48 11.20 62.55 114.42 Dumpy mass –

* Incubation temperature 30 �C, initial pH 6.0, aeration rate 1.0 l/l/min, initial sugar concentration 150 g/l, vermiculite concentration 0.20 lg/l.

Table 3. The comparison of kinetic parameters for vermiculite

addition on citric acid fermentation by Aspergillus niger NG-

GCB101 in stirred bioreactor 96 h after the incubation.

Kinetic parameters Vermiculite conc. (lg/l)

0.20 0.25

Specific growth rate

l (h)1) 0.725 40.509

Citric acid formation parameters

Qp (g/l/h) 1.325 0.599

Yp/s (g/g) 0.956 0.662

Yp/x (g/g) 8.293 6.755

qp (g/g cells/h) 0.082 0.026

Substrate consumption parameters

Yx/s (g cells/g) 0.010 0.009

Qs (g/l/h) 0.267 0.180

qs (g/g cells/h) 0.048 0.061

Qx (g cells/l/h) 0.024 0.026

Least significant difference 0.566 0.312

Significance level hP i HS S

Kinetic parameters: l(h)1) = specific growth rate, 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, 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.

HS is for the ‘highly significant’ while S for ‘significant’ values.

466 I. Haq et al.

Acknowledgements

We are thankful to the officials of PSF, Islamabad,Pakistan who partially funded this project. The com-ments and English proof reading by Dr David A.Lightfoot, Professor of Molecular Biology and Genom-ics, SIU at Carbondale, IL, USA are greatly appreci-ated.

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A high performance fermentation process for citric acid production 467