Abstract Under surface culture condition, a number of cheap indigenous raw materials like cane molasses and jackfruits were selected for citric acid fermentation and gamma ray induced mutant strains of 14/20 and 79/20 of Aspergillus niger were used. The rate of citric acid production was also influenced by biomass production and residual sugar used. In both cases on the day 13 without presence of Prescott salt for the mutant strain of Aspergillus niger 14/20 the citric acid production was highest. The biomass production was found different in various fermentation medium. It was found to increase with the increase of fermentation period and maximum biomass was found on day 13. It is also found that biomass production was higher in mutant strain of A. niger 14/20 than 79/20. In addition to this, wet-weight biomass was higher than dry weight biomass. Without presence of Prescott salt, wet weight of mycelial body of the fungus Aspergillus niger 14/20 was found highest on day 13 in mixed substrates which was 160.15. Mixed substrate prepared with molasses and jackfruit media was proved to be the best and potential for biomass production. Sugar utilization rate was also different in various media during citric acid production by A. niger 14/20 and 79/20. Prescott salt was also found to have remarkable effect on sugar utilization for citric acid production except on days 0, 3 and 5. In presence of Prescott salt, sugar utilization was lower than without presence of Prescott salt in the molasses, jackfruit and mixed fermentation media during production of citric acid. With the increase of fermentation period, sugar concentration in the medium was reduced and maximum reduction was found on day 13. Without presence of Prescott salt and in mixed fermentation medium the highest production of citric acid was found by further mutated Aspergillus niger 14/20 which was about 16.16mg/ml.

1. Introduction

One of the most important fungi used in industrial microbiology, Aspergillus niger (A. niger) has been employed for many years for the commercial production of citric acid ^[1]. However, the worldwide demand for citric acid is increasing faster than its production and more economical processes are required ^[2].

A. niger is most commonly used for citric acid production ^{[3, 4, 5]}. This is because of the fact that this organism has capacity to utilize varieties of substrates due to its well-developed enzymatic system ^[6]. A. niger is normally a haploid fungus producing white septate hypha which is profusely branched. It produces black mass of conidia, which are found in chain arising from the secondary sterigmata. Citric acid is mainly produced by a fungus A. niger by utilizing starchy and sugar substrates ^{[7, 8]}. Deolger and Prescott (1934) found a medium (Sucrose 140g/l, NH₄NO_3, 2.23g/l; K₂HPO_4, 1.00g/l and MgSO_4.7H₂O, 0.23g/l) to be most satisfactory for A. niger for giving high yields of citric acid which is termed as Prescott salt. If more than 0.25% of ammonium nitrite, 0.15% of potassium monohydrozen phosphate and 0.3% of magnesium sulfate were used oxalic acid formation increased and the yield of citric acid decreased ^[9]. In spite of being developing country, Bangladesh is trying to develop in the field of food and pharmaceutical industry. Any increase in citric acid production would be of potential interest and hence there is an obvious need to consider all possible ways in which this might be achieved. The present investigation was therefore, undertaken with a view to determine the feasibility of using raw and cheap materials such as molasses and jackfruit for citric acid production and optimization under fermentation condition on these substrates.

2. Materials and Methods

Citric acid producing strains of A. niger designated as 14/20 and 79/20 (Hannan et al., 1973) were used in this present study. 14/20 and 79/20 are second step mutants derived from the strain HB3 which is the first step mutant from the wild type strain CA16 (Hannan et al., 1973 and Begum et al., 1988). The culture was maintained on agar slants containing 1% malt extract, 1% yeast extract, 1.5% dextrose and 2.5% bacto agar ^[9].

Cane molasses containing 20% water, 62% sugar, non-sugar contents 10% and 8% inorganic salts (ash contents), making a blackish homogenous liquid with high viscosity. Ash contents include ions such as Mg, Mn, Al, Fe and Zn in variable ratio (Prescott and Dunn’s, 1987). The other substrate bright yellow colored jackfruit was used in the experiments for the comparative study ^[10].

Molasses was clarified by appropriate dilution with water and boiled the solution for half an hour. The clarified molasses was then kept overnight for sedimentation of suspended particles. In order to remove the coarse particle in the solution it was filtered through absorbent cotton and sediment was discarded ^[10].

Jackfruit was washed with tap water for several times. Thereafter jackfruit was sliced thinly and dried in dryer at 50°C. The substrate was powdered by using a grinding machine. Dried powder of jackfruit was hydrolyzed separately in 300ml solution of 0.05N HCl and autoclaved at 121°C temperature, 15 Ibs pressure for 20 minutes. The hydrolyzed materials were then filtered through thin cloth ^[10].

Equal amount of jackfruit and molasses were hydrolyzed in 300ml solution of 0.05N HCl and autoclaved at 121°C temperature, 15 lbs pressure for 20 minutes. The hydrolyzed materials were then filtered through thin cloth. The media was then kept overnight for sedimentation of suspended particles resides in molasses ^[10].

Experiments carried out for the following substrates: (i) Molasses in presence of Prescott salt (ii) Molasses without presence of Prescott salt (iii) Jackfruit in presence of Prescott salt (iv) Jackfruit without presence of Prescott salt (v) Mixed in presence of Prescott salt and (vi) Mixed without presence of Prescott salt.

Initially 14% sugar, pH 5 and incubation temperature (30ºC) were optimized in case of all used media and the given strains for citric acid fermentation. 14% sucrose solution was found better moisture level for citric acid fermentation ^[11]. A concentration higher than 14 to 18 % however, leads to greater amount of residual sugar making the process uneconomical and when the concentration of the media is more than needed it become inhibitory for citric acid production. And as a result citric acid production also decreases. In the present study, the molasses, jackfruit and mixed substrate media was prepared with calculating the 14% sugar concentration first and thus high yield of citric acid was produced^[12] investigated that in respect to citric acid production all the strains (318, 79/20, 14/20 and CA16 wild) gave more citric acid at pH 5 than the other pH values (3.5, 4.0, 4.5). In the present study, pH 5 in molasses, jackfruit and mixed substrate media was maintained for optimum production of citric acid. The optimum temperature range of 26-30°C has been proposed for good citric acid yields and rapid rate of accumulation (Doelger and Prescott, 1934). A temperature of 30ºC was found to be the best for citric acid fermentation (Qp = 0.667 ± 0.02a g/l/h) ^[13]. The following parameters were selected to find out which one was better for citric acid fermentation: Sugar with Prescott salt and Sugar without Prescott salt (NH₄NO_3, 2.23g/l; K₂HPO_4, 1.00g/l and MgSO_4.7H₂O, 0.23g/l). Fermentation was carried out in 100ml conical flasks containing 25ml medium. Titration was done from the day 7 of incubation. Total titrable acid values were determined by freshly prepared 0.1N NaOH. Citric acid and residual sugar were estimated between 3-13 days of culture broth by the Marrier and Boulet method (1958) and Anthrone sulphuric acid method (Morse, 1947) respectively.

3. Results and Discussion

Increased biomass of A. niger strains was found with the increased period of time. This increase was mainly for the production of mycelial body of the fungus and their sporulation. Without presence of Prescott salt biomass weight was highest for fermented product of A. niger 14/20.

Without presence of Prescott salt, in the molasses fermentation medium the wet weight of mycelial body of the fungus A. niger 14/20 was found comparatively higher than in the presence of Prescott salt on days 3, 5, 7, 9, 11 and 13 (Table 1). Without presence of Prescott salt, in the same fermentation medium and on the same respective day the wet weight of mycelial body of the fungus A. niger 79/20 was found comparatively higher than in the presence of Prescott salt (Table 2). On the other hand, higher amount of dry weight of mycelial body of the fungus A. niger 14/20 was found 30.05 mg/ml on day 13 without presence of Prescott salt. It is reported that, significantly higher amount of dry weight of mycelial body of the fungus A. niger 14/20 was found 29.76mg/ml on day 13 with the absence of Prescott salt ^[14]. In presence of Prescott salt dry weight was found lower for each day measurement. Without presence of Prescott salt biomass weight was high for fermented product of A. niger 14/20 in molasses medium. Without presence of Prescott salt biomass weight of A. niger 79/20 fermented product was respectively lower than A. niger 14/20 strain.

In presence of Prescott salt, Wet-weight for both strains of fungus A. niger 14/20 and 79/20 were found comparatively lower in the jackfruit fermentation medium than without presence of Prescott salt on days 3, 5, 7, 9, 11 and 13. On the other hand, higher amount of dry weight of mycelial body of the fungus A. niger 79/20 was found 29.20mg/ml on day 13 without presence of Prescott salt. It is reported that in the pumpkin fermentation medium, significantly higher amount of dry weight of mycelial body of the fungus A. niger 79/20 was found 27.89mg/ml on day 13 with the absence of Prescott salt ^[14]. In presence of Prescott salt dry weight was lower for each day measurement. Without presence of Prescott salt biomass weight was higher for fermented product of A. niger 14/20 in jackfruit medium than A. niger 79/20.

In presence of Prescott salt, wet weight of mycelial body of the fungus A. niger 14/20 was found 21.20, 36.10, 47.05, 82.70, 115.00 and 134.00mg/ml respectively in the mixed fermentation media on day 3, 5, 7, 9, 11 and 13. Without presence of Prescott salt, wet weight was found 30.17, 43.20, 57.70, 128.50, 144.40 and 160.15mg/ml on the respective day. On the other hand, higher amount of wet weight of mycelial body of the fungus A. niger 79/20 was found 155.70mg/ml on day 13 with the absence of Prescott salt. It is reported that in the molasses and pumpkin mixed fermentation medium, significantly higher amount of wet weight of mycelial body of the fungus A. niger 79/20 was found 155.29mg/ml on day 13 with the absence of Prescott salt. In presence of Prescott salt dry weight was lower for each day measurement ^[14].

In presence of Prescott salt, dry weight of mycelial body of the fungus A. niger 14/20 was found comparatively lower in the mixed fermentation medium than without presence of Prescott salt on days 3, 5, 7, 9, 11 and 13. In presence of Prescott salt, dry weight of mycelial body of the fungus A. niger 79/20 was found comparatively lower in the mixed fermentation medium than without presence of Prescott salt on the same respective days. Without presence of Prescott salt biomass weight was higher for fermented product of A. niger 14/20 than that of A. niger 79/20 on the same mixed medium.

Through this result we observe that the biomass production was not same in all fermentation medium. Without presence of Prescott salt biomass weight was higher for fermented product of A. niger 14/20 than A. niger 79/20 when compared.

Table 1. Weight of biomass at different days of fermentation by Aspergillus niger 14/20

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Table 2. Weight of biomass at different days of fermentation by Aspergillus niger 79/20

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During citric acid production by A. niger 14/20 and 79/20, sugar utilization rate was different in various medium. Prescott salt was also found to have significant effect on sugar utilization for citric acid production except on days 0 and 3. With the increase of fermentation period sugar concentration in the medium was reduced and maximum reduction was found on day 13.

Without presence of Prescott salt, highest amount of sugar was utilized by A.niger 14/20 on the day 13 on molasses fermentation medium (Figure 1).

For the A. niger 79/20 in the presence of Prescott salt, residual sugar was found 145, 140, 130, 125, 110, 102 and 98.05mg/ml respectively in the fermentation medium on day 0, 3, 5, 7, 9, 11 and 13. Lowest residual sugar was 61.10 mg/ml on 13 day without the presence of Prescott salt means highest sugar utilization (Figure 2). Maximum amount of sugar was utilized by A. niger 14/20 without presence of Prescott salt on the day 13 on molasses fermentation medium than A. niger 79/20. It is reported that, the values of sugar utilization on days 0 and 3 are not statistically significant but the following days sugar utilization in presence of Prescott salt and without salt both are significant both the strains A. niger 14/20 and 79/20 ^[14].

In presence of Prescott salt, utilization of residual sugar of the fungus A. niger 14/20 was found 146, 139, 138, 130, 110, 103 and 97mg/ml respectively in the jackfruit fermentation medium on day 0, 3, 5, 7, 9, 11 and 13. On the other hand, the residual sugar 142, 135, 129, 115, 98, 74 and 65.57mg/ml was found respectively without the presence of Prescott salt on the same respective days. On the pumpkin fermentation medium it is reported that, the residual sugar 143, 137.9, 129.69, 116.37, 100.25, 75.21 and 63.51mg/ml was found respectively without the presence of Prescott salt on the same respective day ^[14]. Highest amount of sugar was utilized by A. niger 14/20 without presence of Prescott salt on the day 13 on the jackfruit fermentation medium (Figure 1).

Figure 1. Sugar utilization at different days of fermentation during citric acid production by Aspergillus niger 14/20. Collections of bars with different alphabets are significantly different at p<0.05. Data was analyzed by repeated t-test with respect to zero (0) day data. Mol, Jack and Mix indicate Molasses, Jackfruit and Mixed respectively. Plus sign (+) stand for with Prescott salt and minus sign (-) stands for without Prescott salt.

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Figure 2. Sugar utilization at different days of fermentation during citric acid production by Aspergillus niger 79/20. Collections of bars with different alphabets are significantly different at p<0.05. Data was analyzed by repeated t-test with respect to zero (0) day data. Mol, Jack and Mix indicate Molasses, Jackfruit and Mixed respectively. Plus sign (+) stand for with Prescott salt and minus sign (-) stands for without Prescott salt.

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In presence of Prescott salt, residual sugar was found 148, 139, 135, 130,115, 105 and 99.99 mg/ml respectively in the fermentation medium by Aspergillus niger 14/20 on day 0, 3, 5, 7, 9, 11 and 13. On the other hand, the residual sugar without the presence of Prescott salt in mixed fermentation medium was 142, 136, 135, 118, 100, 73 and 60.89mg/ml respectively on the same respective days. Utilization of residual sugar without the presence of Prescott salt in mixed fermentation medium was higher than with the presence of Prescott salt except 0, 3 and 5 days (Figure 1). On the molasses and pumpkin mixed fermentation medium it is also reported that, Utilization of residual sugar without the presence of Prescott salt in mixed fermentation medium was higher than with the presence of Prescott salt except 0, 3 and 5 days ^[14].

Utilization of residual sugar without the presence of Prescott salt in mixed fermentation medium was higher than with the presence of Prescott salt by A. niger 14/20. And A. niger 14/20 utilized more sugar than by A. niger 79/20 utilized from the same fermentation medium.

Citric acid production was also different with various fermentation media by A. niger 14/20 and 79/20 strains. It was found to significantly increase with the increase of fermentation period and maximum citric acid production was found on day 13. In the presence of Prescott salt citric acid production was found significantly lower than without presence of Prescott salt.

In the presence of Prescott salt, the citric acid production by A. niger 14/20 was shown on Figure 3 for day 7, 9, 11 and 13. On the day 13, citric acid production was 6.38mg/ml. Again, citric acid production on day 7, 9, 11 and 13 without the presence of Prescott salt was 5.5, 6.45, 7.20 and 9.22mg/ml respectively (Figure 4). Citric acid production by A. niger 79/20 on day 7, 9, 11 and 13 in the presence of Prescott salt was shown in Figure 5. On the other hand, Citric acid production in the same respective day without presence of Prescott salt was 5.0, 6.50, 7.0 and 9.08mg/ml respectively (Figure 6). It is also reported that, significantly highest amount of citric acid was produced by A. niger 14/20 without presence of Prescott salt on the day 13 on the molasses fermentation medium ^[14].

Figure 3. Citric acid production in different days of fermentation in various substrates (with prescott salt) by Aspergillus niger 14/20. Mol, Jack and Mix indicate Molasses, Jackfruit and Mixed respectively.

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Figure 4. Citric acid production in different days of fermentation in various substrates (without prescott salt) by Aspergillus niger 14/20. Mol, Jack and Mix indicate Molasses, Jackfruit and Mixed respectively.

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Figure 5. Citric acid production in different days of fermentation in various substrates (with prescott salt) by Aspergillus niger 79/20. Mol, Jack and Mix indicate Molasses, Jackfruit and Mixed respectively.

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Figure 6. Citric acid production in different days of fermentation in various substrates (without prescott salt) by Aspergillus niger 79/20. Mol, Jack and Mix indicate Molasses, Jackfruit and Mixed respectively.

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In presence of Prescott salt, citric acid production by the fungus A. niger 14/20 was significantly lower than without presence of Prescott salt in the jackfruit fermentation media on day 7, 9, 11, 13. On the other hand, the highest citric acid production on day 13 without the presence of Prescott salt was 11.85mg/ml. It is reported that, on the pumpkin fermentation medium the highest citric acid production on day 13 without the presence of Prescott salt was 10.35mg/ml ^[14]. In presence of Prescott salt, production of citric acid of the fungus A. niger 79/20 found in the jackfruit fermentation media on day 7, 9, 11 and 13 was 2.60, 3.45, 3.90 and 4.20mg/ml respectively. On the other hand, the citric acid production on day 7, 9, 11 and 13 without the presence of Prescott salt was 6.50, 7.80, 8.60 and 11.75mg/ml respectively. Highest amount of citric acid was produced by A. niger 14/20 than A. niger 79/20 without presence of Prescott salt on the day 13 in the jackfruit fermentation medium.

In presence of Prescott salt, citric acid production in mixed fermentation medium by A. niger 14/20 on day 7, 9, 11 and 13 was found 3.0, 4.60, 5.50 and 6.27mg/ml respectively. On the other hand, citric acid production on day 7, 9, 11 and 13 without the presence of Prescott salt in mixed fermentation medium was 9.50, 12.0, 13.80 and 16.16mg/ml respectively. On the molasses and pumpkin fermentation medium it is reported that, citric acid production on day 3, 5, 7, 9, 11 and 13 without the presence of Prescott salt in mixed fermentation medium was 2.37, 4.55, 8.55, 10.95, 12.55 and 14.86 ^[14]. In presence of Prescott salt citric acid production in the fermentation medium by A. niger 79/20 was lower than without the presence of Prescott salt. Citric acid production on 13 day was 15.94mg/ml in mixed fermentation medium. Production of citric acid without the presence of Prescott salt in mixed fermentation media is higher than with the presence of Prescott salt.

4. Conclusion

The production of citric acid was not same in all fermentation medium. Without the presence of Prescott salt highest citric acid production was found in mixed fermentation medium throughout the fermentation period and lowest production of citric acid was found in molasses media. Whereas with the presence of Prescott salt lowest production of citric acid was found in jackfruit medium and highest amount of citric acid was produced in molasses medium throughout the fermentation period.

And in both cases A. niger 79/20 produces lower amount of citric acid than A. niger 14/20.

From the experiments it can be opined that both molasses and jackfruit could be used for the production of citric acid but in case of mixed substrate the citric acid production is much economically feasible and was proved more potential for citric acid production. It is apparent from the results that the maximum yield of citric acid was also dependent on sugar level and biomass.

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