Stimulatory effect of alcohols (methanol and ethanol) on citricacid productivity by a 2-deoxy DD-glucose resistant culture

of Aspergillus niger GCB-47

Ikram-Ul Haq a,*, Sikander Ali a, M.A. Qadeer b, Javed Iqbal c

a Biotechnology Research Labs, Department of Botany, Government College, Lahore, Pakistanb Centre of Excellence in Molecular Biology, Thokar Niaz Baig, University of the Punjab, Lahore, Pakistan

c Department of Botany, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan

Received 14 November 2001; received in revised form 18 July 2002; accepted 18 July 2002

Abstract

The present study describes citric acid fermentation by Aspergillus nigerGCB-47 in a 15-l stainless steel stirred fermentor. Among

the alcohols tested as stimulating agents, 1.0% (v/v) methanol was found to give maximum amount of anhydrous citric acid

(90:02� 2:2 g/l), 24 h after inoculation. This yield of citric acid was 1.96 fold higher than the control. Methanol has a direct effect on

mycelial morphology and it promotes pellet formation. It also increases the cell membrane permeability to provoke more citric acid

excretion from the mycelial cells. The sugar consumed and % citric acid was 108� 3:8 g/l and 80:39� 4:5%, respectively. The

desirable mycelial morphology was in the form of small round pellets having dry cell mass 14:5� 0:8 g/l. Addition of ethanol,

however, did not found to enhance citric acid production, significantly. The maximum value of Yp=x (i.e., 5:825� 0:25 g/g) was

observed when methanol was used as a stimulating agent. The best results of anhydrous citric acid were observed, 6 days after

inoculation when the initial pH of fermentation medium was kept at 6.0.

� 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Aspergillus niger; Citric acid; Stimulatory effect of alcohol; Methanol; Ethanol; Fermentation

1. Introduction

Citric acid has a wide range of applications and itsworldwide demand is increasing day by day. It is a pri-

mary product of Aspergillus niger metabolism (Pazouki

et al., 2000). A successful process depends both on ap-

propriate strain and optimisation of fermentation pa-

rameters. To hyper-produce the actual product desired,

the microorganisms must be grown under sub-optimal

conditions for biomass formation. The size and form of

mycelial pellets have a direct role on citric acid bio-synthesis in submerged fermentation (Saha et al., 1999).

The use of alcohols as a stimulant in citric acid produc-

tion may enhance the yield of citric acid (Wieczorek and

Braver, 1998). The stimulatory effect of methanol on

citric acid production can be explained in terms of my-

celial morphology as well as pellet shape and size (Sriv-

asta and Kamal, 1979). Citric acid production could be

increased by exploiting available resources and adding

the stimulatory agents to the fermentation medium (Pera

and Callieri, 1997). The problem is to determine the exactmorphology and size of mycelial pellets, desirable for

citric acid fermentation. A few research reports have

appeared in the literature to explain the effect of alcohols

on citric acid production. The purpose of the present

investigation was to determine the value of alcohols,

especially methanol (CH3OH), in stimulating the pro-

duction of citric acid by A. niger. For this a hyper-pro-

ducer culture ofA. nigerGCB-47 was selected. The crudecarbohydrate cane-molasses was used as substrate under

submerged fermentation conditions using a 15-l stirred

fermentor (working volume 9-l).

2. Methods

2.1. Organism

A hyper-producer strain of A. niger GCB-47, main-

tained on potato dextrose agar slants, was used in this

Bioresource Technology 86 (2003) 227–233

*Corresponding author.

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

0960-8524/03/$ - see front matter � 2002 Elsevier Science Ltd. All rights reserved.

PII: S0960-8524 (02 )00172-4

study. It was obtained from the Culture Collection ofBiotechnology Research Labs, Department of Botany,

Government College, Lahore (Haq et al., 2001). The

culture was previously stabilized by 0.026% 2-deoxy

DD-glucose addition and subjected to 0.015% N-methyl

N-nitro N-nitroso guanidine treatment for 15 min in an

ultraviolet chamber.

2.2. Pre-treatment of cane-molasses

Cane-molasses obtained from Chunian Sugar Mills,

Pakistan was used for present study. Sugar content of

molasses was about 60% and it was diluted to approxi-

mately a 25% sugar level. The molasses solution, afteradding 35 ml of 1 N H2SO4 per liter, was boiled for 30

min, cooled, neutralized with lime-water (CaO) and was

left over night for clarification (Srivasta and Kamal,

1979). The clear supernatant liquid was diluted to a 15%

sugar concentration.

2.3. Inoculum and fermentation conditions

Vegetative inoculum, prepared by taking 100 ml of

clarified cane-molasses (sugar 15% and initial pH 6.0)

with silica gel chips (15–20 in number and 1.25 mm dia-

meter each) in a 250 ml Erlenmeyer flask was seeded

with 1.0 ml of conidia (1:55� 106/ml). The flask wasincubated at 30 �C in rotary shaking incubator (Gal-

lenkamp UK, 200 rpm) for 24 h. A laboratory scale

stainless steel stirred fermentor, model GLSC-AF-199-

10 (15-l capacity) was employed for citric acid fermen-

tation. The working volume of the vessel was 9-l (60%

based on the total capacity of the vessel). The clarified

molasses contained (% w/v); total sugars 15.0, ash con-

tents 0.015, K4Fe(CN)6 concentration 200 ppm at initialpH 6.0. The medium was sterilized at 15 lbs/in.2 pressure

(121 �C) for 20 min. All fermentations were carried out

at 30 �C for 6 days (144 h) at a constant agitation in-

tensity (160 rpm). Aeration rate was maintained at 1.0 l/

l/min. Sterilized silicone oil (10%, antifoam AE-11) was

added to control the foaming during fermentation.

2.4. Assay techniques

Dry cell mass was determined by filtering mycelia

through weighed Whatman filter paper no. 44 and

drying at 110 �C for 2 h (Moreira et al., 1996). Citricacid was determined by titrating 1.0 ml of filtered broth

against 0.1 N NaOH, using phenolphthalein as an in-

dicator.

% citric acid

¼ titre� normality of alkali� eq: wt: of acid

volume of sample� 1000

Anhydrous citric acid was determined using the pyridineacetic anhydride method (Marrier and Boulet, 1958)

while residual sugars were estimated by the dinitro sal-

icylic acid method (Tasun et al., 1970).

2.5. Kinetic and statistical analyses

The kinetics analysis was based on the methods des-

cribed by Pirt (1975). All the results are sum means

of three parallel replicates. For statistical analysis

of values, Duncan�s multiple range tests were applied

(Snedecor and Cochran, 1980). The difference in values

was indicated in the form of probability (p < 0:05)values.

3. Results

3.1. Effect of different alcohols

The effect of different alcohols (CH3OH and

C2H5OH) on the production of citric acid was studied

(Table 1). The maximum amount of citric acid (72:55�2:5 g/l) was produced when methanol was added into the

molasses medium. Sugar consumption and dry cell masswere 110� 3:0 and 13:0� 1:0 g/l, respectively. The

maximum amount of citric acid on the basis of sugar

was 65:95� 2:6%. Mycelia were small (2–3 mm in di-

ameter) round pellets. Addition of ethanol, however, did

not enhance production and 56:25� 3:1 g/l citric acid

was obtained in the fermented broth. The control

(without alcohol addition) produced only 37:65� 2:0 g/l

citric acid. Therefore, methanol was found to be the beststimulant for citric acid production in the stirred fer-

mentor.

Table 1

Effect of different alcohols on the production of citric acid by mutant strain of A. niger GCB-47 in stirred fermentor

Different alcohols Total acid (g/l) Anhydrous citric acid (g/l) Dry cell mass (g/l) Sugar used (g/l) Mycelial morphology

Control 46.50� 2.5de 37.65� 2.0e 19.50� 1.2a 126� 4.5a Mixed mycelia

Methanol 81.62� 3.2a 72.55� 2.5a 13.00� 1.0c 110� 3.0c Small round pellets

Ethanol 62.55� 2.8cd 56.25� 3.1bc 14.50� 1.5b 118� 4.2bc Elongated mycelium

The fermentation was carried out using cane-molasses (150 g/l) as the basal fermentation medium (pH 6.0) at 30 �C for 6 days. The alcohol (1.0%)

was added just after (0 h) inoculation. � indicates the standard deviation between the three parallel replicates. The numbers given differ significantly

at p < 0:05 within each column.

228 I.-U. Haq et al. / Bioresource Technology 86 (2003) 227–233

3.2. Effect of methanol and ethanol concentration

It is clear from the data of Table 2 that the amount of

citric acid production at 0.5% concentration of metha-

nol was 50:70� 2:0 g/l. There was an increase in the

production of citric acid with the increase in methanol

concentration. The maximum amount of citric acid

(74:55� 1:8 g/l) was obtained when 1.0% methanol was

added to the medium. Sugar consumption was 100� 3:6g/l while dry cell mass was observed to be 12:5� 0:7 g/l

and exhibited small round pellets. Beyond 1.0%

methanol concentration, the production of citric acid

was gradually decreased. However, the production of

citric acid was not improved for all concentrations of

C2H5OH.

3.3. Effect of time of methanol addition

The effect of time of addition of methanol on pro-

duction of citric acid is shown in Table 3. The time in-terval was ranged from 0 to 48 h, after inoculation.

Production of citric acid gradually increased somewhat

when the time for addition of methanol was increased.

The highest yield of anhydrous citric acid (86:02� 2:2g/l) was obtained when 1.0% (v/v) methanol was added

to the production medium, 24 h after incubation.

Mycelia were present as small round pellets and had a

dry cell mass 14:5� 0:8 g/l. The consumption of sugar

and % citric acid on the basis of sugar used were 108�3:8 g/l and 80:39� 4:5%, respectively. Further increase

in the time of addition, did not enhance citric acid accu-

mulation.

3.4. Effect of partial replacement of ethanol with methanol

The data of Table 4 represent the effect of partial

replacement of ethanol to methanol ratio on citric acid

production by A. niger GCB-47 in a stainless stirred

fermentor. The maximum amount of citric acid

(62:76� 2:4 g/l) was achieved when C2H5OH to CH3OH

ratio was maintained at 0.4:0.6%, 24 h after inoculation.

The % citric acid on the basis of sugar used was

56:03� 2:5, having 112� 3:2 g/l sugar consumption.Mycelia exhibited fluffy pellets in their morphology and

their dry cell mass was 14:5� 0:9 g/l.

Table 2

Effect of different concentrations of methanol and ethanol on the production of citric acid by A. niger GCB-47 in stirred fermentor

Alcoholic concentration (%) Total acid (g/l) Anhydrous citric acid (g/l) Dry cell mass (g/l) Sugar used (g/l) Mycelial morphology

Methanol

0 45.16� 1.6e 39.55� 3.2e 15.0� 1.2b 123� 2.5a Large pellets

0.5 56.05� 2.8d 50.70� 2.0cd 12.5� 0.8d 98� 3.5cd Fine pellets

1.0 79.60� 2.5a 74.55� 1.8a 12.5� 0.7d 100� 3.6c Small round pellets

1.5 74.00� 1.7b 67.62� 1.5b 13.0� 1.0c 96� 4.2d Large pellets

2.0 66.50� 2.0c 58.55� 1.5c 14.7� 1.0bc 104� 4.5b Large pellets

2.5 60.02� 2.5cd 51.25� 2.2cd 16.5� 1.6a 118� 5.6a Gummy mass

Ethanol

0 45.78� 2.4e 41.65� 1.5b 16.5� 1.5a 119� 4.0c Large pellets

0.5 50.52� 1.5cd 42.50� 1.6b 13.5� 1.0bc 118� 5.5bc Viscous

1.0 63.10� 1.8a 56.85� 1.8a 14.0� 1.0b 121� 5.8b Intermediate pellets

1.5 57.14� 2.6b 47.05� 1.6b 14.0� 1.2b 122� 5.5ab Fluffy mass

2.0 52.62� 2.6c 44.65� 2.5b 14.5� 1.2ab 125� 5.5a Fluffy mass

2.5 49.55� 2.4d 40.52� 2.0bc 15.0� 0.9a 126� 6.2a Gelatinous

Sugar added 150 g/l, temperature 30 �C, initial pH 6.0, incubation period 6 days and agitation intensity 160 rpm. Alcohols were added just after (0 h)

incubation. The results are sum means of three parallel replicates. � indicates the standard deviation among the replicates. The values differ by letters

at p < 0:05 within each column.

Table 3

Effect of time of addition of methanol on the production of citric acid by A. niger GCB-47 in stirred fermentor

Time of addition of

methanol (h)

Total acid (g/l) Anhydrous citric acid

(g/l)

Dry cell mass (g/l) Sugar used (g/l) Mycelial morphology

0 81.70� 2.9bc 76.04� 2.0bc 14.0� 0.5b 110� 4.5a Fine round pellets

12 88.65� 2.6b 80.60� 2.2b 14.2� 0.5ab 105� 4.0bc Fine round pellets

24 92.14� 2.5a 86.02� 2.2a 14.5� 0.8a 108� 3.8b Small round pellets

36 86.56� 3.2b 78.00� 2.5b 14.0� 0.9b 112� 3.9a Intermediate pellets

48 78.60� 2.6c 72.55� 1.6c 13.7� 1.2c 109� 4.2ab Gelatinous

Sugar added 150 g/l, temperature 30 �C, initial pH 6.0, incubation period 6 days and agitation intensity 160 rpm. Methanol was added at a level of

1.0%. The values are sum means of three parallel replicates while � denotes the standard deviation among the replicates. The values differ

significantly at p < 0:05 within each column.

I.-U. Haq et al. / Bioresource Technology 86 (2003) 227–233 229

3.5. Initial pH and time profile of citric acid fermentation

The effect of different initial pH (5.0–7.0) on the

production of citric acid is shown in Fig. 1. Maximum

amount of citric acid (90.02 g/l) was achieved when

the initial pH of the fermentation medium was kept at

6.0 with methanol addition. The sugar consumption

was 112 g/l while dry mycelial weight was 14.5 g/l.When the pH was further increased from 6.0, the

production of citric acid decreased, gradually for

control as well as for the experimental. A time profile

of citric acid fermentation by A. niger was carried out

(Figs. 2–4). The fermentation was carried out for 1

through 9 days. Citric acid production of 7.5 g/l was

observed after 24 h and it reached a maximum of

89.62 g/l after 6 days of incubation with methanoladdition. The sugar consumption and dry mycelial

weight obtained after 6 days were 101 and 16 g/l,

respectively.

4. Discussion

One of the main problems for achieving bioreactor

performance under stable conditions with filamentous

fungi involves limiting hyphal growth as well as avoid-

ing diffusional restrictions. Different alcohols, particu-

larly methanol have a stimulatory effect on the

biosynthesis of citric acid in fermented broth. It mightbe because methanol increased the permeability of cell

membrane, resulting in better excretion of citric acid

from the mycelial cells. Citric acid fermentation is very

sensitive to the components of the medium, especially

iron, manganese and zinc (Zakowska and Joloka, 1984;

Sanjay and Sharma, 1994). The addition of slightly toxic

concentrations of low molecular weight alcohols to the

medium increases the tolerance level of trace metalsduring the fermentation. Methanol at 1.0% concen-

tration yielded maximal production of citric acid in the

present study. Methanol markedly depressed the syn-

Fig. 1. Effect of different initial pH on citric acid production by A. niger GCB-47 in stirred fermentor. Sugar added 150 g/l, temperature 30 �C, initialpH 6.0, aeration rate 1.0 l/l/min and fermentation period 6 days (144 h). The alcoholic addition was made after 24 h of incubation. The results are

sum mean of three parallel replicates. Y-error bars indicate the standard deviation among the replicates.

Table 4

Effect of partial replacement of ethanol to methanol concentration on citric acid production by A. niger GCB-47 in stirred fermentor

Alcohols (% v/v) Total acid (g/l) Anhydrous ci-

tric acid (g/l)

Dry cell mass

(g/l)

Sugar used (g/l) % citric acida Mycelial mor-

phologyEthanol Methanol

0.2 0.8 62.15� 2.5b 55.02� 2.0b 14.5� 0.9a 116� 4.1b 47.43� 2.0b Mixed pellets

0.4 0.6 66.52� 2.8a 62.76� 2.4a 14.5� 0.9a 112� 3.2bc 56.03� 2.5a Fluffy pellets

0.6 0.4 58.26� 3.5b 48.55� 2.0bc 13.5� 1.1b 127� 3.2a 38.23� 1.5c Gelatinous

0.8 0.2 51.04� 3.5bc 43.29� 1.9c 12.0� 1.2c 122� 2.8a 32.20� 2.2d Gelatinous

Sugar added 150 g/l, temperature 30 �C, initial pH 6.0, aeration rate 1.0 l/l/min and fermentation period 6 days (144 h). The alcoholic addition was

made after 24 h of incubation.aOn the basis of sugar used. The results are sum means of three parallel replicates. The numbers differ significantly by different letters at p < 0:05

within each column while � indicates the standard deviation among the replicates.

230 I.-U. Haq et al. / Bioresource Technology 86 (2003) 227–233

thesis of cell protein in the early stages of the cultivation

(Moyer, 1953) and also increased the metabolic activity

of the enzyme citrate synthase. From this, it can be

concluded that methanol-induced enzyme activities

could become suitable for citric acid production. When

the level of methanol concentration was increased fur-

ther, decreased production of citric acid occurred. This

might be due to the fact that the higher methanol con-centration in the medium disturbed the fungal metabo-

lism and mycelial morphology, which resulted in

decreased citric acid production. A similar type of work

has been carried out by Hang and Woodams (1986). In

the present study, addition of methanol after 24 h or

later was not found to be beneficial for the production

of citric acid. The data of Table 4 revealed that partial

replacement of ethanol to methanol concentration is not

economically feasible due to reduced yields of citric acid

and higher rate of energy consumption.

The improvement of citric acid production due to

methanol addition can be explained in terms of pellet

size controlled by means of air or oxygen. The proper

control of pellet size increased the active surface of

mycelia and allowed a better oxygen availability,which is a critical point in citrate synthesis during the

secondary metabolism of A. niger. Small round pellets

possessing diameters less than 3 mm have been found

to be highly desirable for maximal citric acid pro-

duction (Tauro, 1977). Methanol is responsible for

bed compaction and consequently, improving mass

transfer limitations and substrate conversion which

Fig. 2. Time profile of citric acid production by A. niger GCB-47 in stirred fermentor (same condition as in Fig. 1).

Fig. 3. Time profile of dry cell mass formation by A. niger GCB-47 in stirred fermentor (same condition as in Fig. 1).

I.-U. Haq et al. / Bioresource Technology 86 (2003) 227–233 231

increased production of citric acid. The comparison of

product and growth yield coefficients (Yp=x, Yp=s, Yx=s ing/g) for citric acid production by A. niger GCB-47 in

stirred fermentor was studied (Table 5). Maximum

value of Yp=x (i.e., 5:825� 0:25 g/g) was observed

when methanol (1%, v/v) was used as a stimulating

agent, 24 h after conidial inoculation. This value of

product yield coefficient is significantly higher com-pared with the findings of Pirt (1975). The value is

1.96 fold higher than the control (without alcohol).

Kinetic studies also revealed that ethanol enhanced

acid production (Yp=x ¼ 3:956� 0:20 g/g) but to a

lesser extent compared to methanol addition. The

ratio between citric acid production and substrate

utilization by methanol (0:883� 0:10 g/g) was much

improved with respect to ethanol and the control. The

low value of Yx=s (0:121� 0:02 g/g) in case of metha-

nol also justifies the better excretion of citric acid in

the broth culture.

The maintenance of a favourable pH is very essential

for the successful fermentation of citric acid. At lowinitial pH, the ferrocyanide ions may be more toxic for

the growth of mycelium in the medium. This finding is

an agreement with the observations of Pessoa et al.

(1982). A higher initial pH leads to the accumulation of

oxalic acid as reported by Shadafza et al. (1976). In

addition, low pH of cane-molasses has been found in-

hibitory for the growth of A. niger. The optimum time of

incubation for maximal citric acid production variesboth with the organism and fermentation conditions. In

batch-wise fermentation of citric acid, the production

started after a lag phase of one day and reached maxi-

mum at the onset of stationary phase or late exponential

phase. Further increase in incubation period did not

enhance citric acid production due to the age of fungi

and depletion of sugar contents in the culture broth.

Vergano et al. (1996) reported the maximum yield ofcitric acid i.e., 64.12 g/l, seven days after the inoculation.

So our finding may be of practical importance when

compared to previous workers because reduction of the

incubation period reduced the cost of citric acid pro-

duction.

5. Conclusion

Mycelial morphology has a profound effect on the

production of citric acid by A. niger. The addition ofmethanol not only influences pellet formation but also

increases the permeability of the cell membrane. The

culture of A. niger GCB-47 is DD-glc-resistant and has

Table 5

Comparison of product and growth yield coefficients (Yp=x, Yp=s and Yx=sin g/g) for citric acid production by A. niger GCB-47 in stirred fer-

mentor

Different alco-

hols (1.0%)Yield coefficients (g/g)

Yp=x Yp=s Yx=s

Control 3.254� 0.22bc 0.724� 0.12b 0.081� 0.01bc

Methanol 5.825� 0.25a 0.883� 0.10a 0.121� 0.02a

Ethanol 3.956� 0.20b 0.569� 0.10c 0.096� 0.02b

LSD 0.231 0.112 0.078

Probability

level hpiHS HS HS

Yp=x ðg=gÞ ¼ product (g/l)/cell mass formation (g/l), Yp=s ðg=gÞ ¼product (g/l)/substrate consumption (g/l), Yx=s ðg=gÞ ¼ cell mass for-

mation (g/l)/substrate consumption (g/l). � indicates the standard de-

viation among the three parallel replicates. The numbers differ

significantly at p < 0:05 within each column. HS denotes that the

values are highly significant and LSD abbreviates least significant

difference.

Fig. 4. Time profile of sugar utilization by A. niger GCB-47 in stirred fermentor (same condition as in Fig. 1).

232 I.-U. Haq et al. / Bioresource Technology 86 (2003) 227–233

high citrate synthase ability which allows high andconsistent yields of citric acid. Mycelial morphology of

filamentous fungi, in the form of small round pellets (<3

mm diameter) is proposed for maximal acid produc-

tion. The value of product yield coefficient i.e., Yp=x ¼5:825� 0:25 g/g in the present study is highly significant.

By optimising the role of Ca2þ ions and phytate addition

to the fermentation medium, the culture can be ex-

ploited on commercial-scale citric acid production.

Acknowledgement

Authors acknowledge Pakistan Science Foundation

for financial support.

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