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Attempts at improving citric acid fermentation by Aspergillus niger in beet-molasses medium
Bioresource Technology 84 (2002) 97–100
Short communication
Attempts at improving citric acid fermentation by Aspergillus niger in beet-molasses medium Nehad Z. Adham Chemistry of Nat. and Microb. Products Department, National Research Centre, Dokki, Cairo, Egypt Received 17 October 2001; received in revised form 29 October 2001; accepted 17 December 2001
Abstract Natural oils with high unsaturated fatty acids content when added at concentrations of 2% and 4% (v/v) to beet molasses (BM) medium caused a considerable increase in citric acid yield from Aspergillus niger. The fermentation capacities were also examined for production of citric acid using BM-oil media under different fermentation conditions. Maximum citric acid yield was achieved in surface culture in the presence of 4% olive oil after 12 days incubation. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Citric acid; Beet molasses; Oils; Aspergillus niger; Surface culture; Submerged culture
1. Introduction Citric acid (CA), an intermediate of the tricarboxylic acid cycle, is produced commercially almost exclusively by fermentation with Aspergillus niger, and widely used in the food beverage, chemical, pharmaceutical and other industries (Wang and Liu, 1996). The most widely used industrial substrate for CA production by A. niger is beet molasses (BM), a byproduct of beet sugar production (Clark and Lentz, 1963; Ogawa and Fazeli, 1976; Roukas and Alichanidis, 1988; Roukas and Harvey, 1988; Roukas, 1991; Hamissa et al., 1991; El-Abyad et al., 1992; Wang, 1998). Citric acid is produced commercially either by submerged fermentation (SmF) or liquid surface fermentation (LSF) processes employing BM as the substrate (Milson and Meers, 1985). The process organism A. niger is very sensitive to trace metal ions. Despite pretreatment with chelating agents such as sodium or potassium ferro-cyanide and ethylenediamine tetracetic acid (EDTA), molasses gives low yields of CA (Shankaranand and Lonsane, 1994). Many investigators have tried to improve the production of CA by various additives. Moyer (1953) found that methanol, ethanol and iso-propanol decreased growth but increased CA production from cane- and beet-molasses media. Since that time, a lot of work had been done on the effect of alcohol on citric acid fermentation (Hamissa, 1966; Szczodark and Ilczuk, 1975; Maddox et al., 1986; El-Batal et al., 1995; Roukas,
1999). Millis et al. (1963) studied the effect of various lipids on the yield of CA produced by A. niger and found that the addition of some natural oils with a high content of unsaturated fatty acids increased CA yield by about 20–50%. The present study was undertaken mainly to determine the effect of some oil supplements on CA fermentation of beet molasses by A. niger A20.
2. Methods 2.1. Microorganism A. niger A20 was kindly provided by the Centre of Culture Collection, National Research Centre (NRC), Cairo, Egypt. 2.2. Beet molasses The BM samples used in the present study were kindly supplied by the Beet Sugar Factory, Egypt. 2.3. Inoculum The slants of A. niger A20 were incubated on potato– dextrose agar medium (PDA) at 30 °C for 7 days. A spore suspension of about 105 –106 spores/ml was prepared in sterile distilled water containing about 0.01%
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 0 0 7 - X
98
N.Z. Adham / Bioresource Technology 84 (2002) 97–100
(v/v) tween-80. This solution was used as a source of inoculum. 2.4. Citric acid fermentation Unless otherwise stated, fermentation media were prepared by diluting BM with tap water to approximately 15% sugar concentration and centrifuging at 2500 rpm to remove suspended matter and the diluted BM was supplemented with 0.05 g% ðNH4 Þ2 SO4 , then dispensed in 50 ml portion into 250 ml Erlenmeyer flasks. Th pH was adjusted to 5.5–5.7. The flasks were autoclaved at 121 °C for 20 min, then inoculated with 1 ml spore suspension. The contents of the flasks were mixed thoroughly and incubated in a slanting position (surface fermentation condition) at 30 °C for 11 days. 2.5. Analytical techniques CA was determined by the colorimetric method of Lowenstein (1969). Total sugar content in the fermen-
tation liquor was determined after hydrolysis according to the modified method of Schoorl (1929). Each treatment was repeated three times and the results were reported as average of three repetitions. Estimation of mould growth. The amount of mould growth was estimated by determining the dry weight of mycelium formed. The contents of each flask were transferred to a weighed Whatman paper no. 541, washed three times with water and the mycelium was dried at 60 °C for 24 h. 3. Results and discussion 3.1. Effect of oil supplementation The data presented in Table 1 show that the production of CA was generally increased with the addition of tested oils. These oils increased CA yields effectively at 4% levels. Best CA yields were obtained with olive, sunflower and maize oils at 4% doses. Millis et al. (1963) suggested that the unsaturated lipids act as alternative
Table 1 Effect of addition of some oils on citric acid fermentation by A. niger A20 Tested oil (ml/100 ml)
Consumed sugar (g%)
CA (g/l)
CA yield (%)
Conversion (%)
Dry weight (g/flask)
Control (no addition)
10.8
29.7
20.3
27.4
2.60
Almond 2 4
14.6 14.19
25.3 47.47
17.3 32.5
17.3 33.4
3.76 3.80
Caster 2 4
14.34 14.54
45.7 52.91
31.2 36.2
31.8 36.3
3.75 4.42
Maize 2 4
14.28 14.6
50.89 58.9
34.8 40.3
35.6 40.3
4.07 4.51
Nigella 2 4
11.59 11.03
31.4 45.5
21.4 31.1
27.0 41.2
3.76 3.79
Olive 2 4
14.6 14.2
57.7 72.8
39.5 49.8
39.5 51.2
3.68 3.70
Peanut 2 4
12.5 12.4
40.6 47.4
27.8 32.4
32.3 38.2
3.56 4.13
Soybean 2 4
13.67 14.22
40.5 55.2
27.7 37.8
29.6 38.8
3.97 4.62
Sunflower 2 4
14.6 14.5
49.5 56.9
33.9 39.0
33.9 39.2
4.0 4.2
Initial sugar concentration ¼ 14.6%. The tested oils were separately supplemented at 2% and 4% (v/v) before autoclaving. CA producted CA yield% ¼ Original sugar
ðg=100 mlÞ ðg=100 mlÞ
100;
CA produced ðg=100 mlÞ 100: Conversion coefficient ðc:c:Þ ¼ Consumed sugar ðg=100 mlÞ
N.Z. Adham / Bioresource Technology 84 (2002) 97–100
Fig. 1. Effect of different oils on CA formation by A. niger A20 using surface and submerged culture techniques.
hydrogen acceptors to oxygen during the fermentation and thus improve the yield of citric acid. Relatively higher concentrations of sugars were assimilated and comparatively high conversion rates were recorded in the presence of the tested oils. Moreover, the fungal growth yields were considerably accelerated under these conditions. This experiment was followed by investigating CA accumulation after 5, 9 and 12 days in the presence of
99
the promising oil treatments, adopting both surface as well as submerged culture techniques (Fig. 1). Data shown in Table 2 showed that surface culture technique proved to be most suited for CA yield. This might have been due to differences in the response of A. niger to the metal ions and minerals upon using submerged or surface fermentation conditions. It has been extensively documented that Fe2þ stimulated aconitase activity which leads to a concurrent decrease in the rate of CA biosynthesis particularly in submerged fermentation (Peterson et al., 1983; La Nauze, 1966). The fermentation parameters obtained when the tested fungus was allowed to grow on BM-medium without oil supplementation (control treatment) showed that relatively higher CA outputs were maintained with the surface culture technique. Nevertheless, the submerged technique afforded higher biomass yield. Additionally, the superiority of the surface technique was traced in the presence of the favorite oil supplements (Table 2). Thus, under the latter cultivation conditions, the fermentation activities, in view of CA biosynthesis, were considerably enhanced. Better CA productivities were, however, achieved after 12 days incubation. The olive oil treatment supported maximum CA outputs followed by maize and then sunflower oil treatments. The submerged techniques afforded relatively lower fermentation efficiencies under all tested experimental conditions. No consistent relationship was recorded between growth and CA accumulation. Thus, while relatively higher growth values were recorded when the fungus was cultivated under submerged conditions, the CA formation proved to be adversely affected under the same conditions. These results may be correlated with the fact that, in citric acid fermentation two kinetic phases of sugar consumption occur. In the first phase, the most growth takes place, while in the second phase CA accumulates at the expense of the sugar consumption with only slight
Table 2 Effect of different oils on CA formation by A. niger A20 using surface and submerged culture techniques Different oils
Incubation period (days)
Surface culture Consumed sugar (g%)
CA (g/l)
Conversion (%)
Submerged culture
Control
5 9 12
7.9 9.1 10.84
7.2 27.2 29.7
9.1 29.8 27.4
Olive
5 9 12
3.6 11.7 14.2
13.2 56.3 72.9
Maize
5 9 12
7.2 12.0 14.6
Sunflower
5 9 12
7.9 12.7 14.5
Dry weight (g/flask)
Consumed sugar (g%)
CA (g/l)
Conversion (%)
Dry weight (g/flask)
1.9 2.7 2.6
8.76 13.4 14.6
8.6 5.1 5.5
9.8 3.8 3.7
3.5 4.88 3.96
36.3 48.1 51.3
2.75 3.34 3.7
8.3 13.9 14.6
7.00 7.2 5.3
8.4 5.2 3.5
5.36 6.88 5.78
15.6 53.2 58.9
21.7 44.1 4.0
3.43 5.17 4.51
8.4 11.7 14.4
6.1 6.9 6.0
7.3 5.9 4.2
4.62 5.72 3.72
13.9 54.8 56.9
17.6 43.2 39.2
2.8 4.1 4.2
8.8 11.5 14.5
6.0 7.0 5.8
6.8 6.1 4.0
4.58 5.7 3.9
100
N.Z. Adham / Bioresource Technology 84 (2002) 97–100
increase in growth (Gaden, 1955, 1959; Roukas and Kotzekidou, 1986; Roehr et al., 1987). El-Batal et al. (1995), using five different strains of A. niger, reported that relatively high CA productivities were achieved with only three cultures after 12 and 13 days adopting the surface culture technique. Similar results have been also recorded with Szczodark (1981). Summarizing the previously mentioned results, the supplementation of a surface culture with some oil makes a direct increase in CA yield possible in using molasses medium.
References Clark, D.S., Lentz, C.P., 1963. Submerged citric acid fermentation of beet molasses in Tank-Type fermentation. Biotechnol. Bioeng. 5, 193. El-Abyad, M.S., Hamissa, F.A., Gad, A.S., 1992. Treatment of beet molasses for citric acid production by a potent strain of Aspergillus niger van Teeghem. Bioresour. Technol. 39 (2), 191–197. El-Batal, A.I., Khalil, A.H., Mostafa, M.M., 1995. Citric acid production by gamma irradiated Aspergillus niger from treated beet molasses under different fermentation conditions. Egypt. J. Rad. Sci. Appl. 8 (2), 253–266. Gaden Jr., E.L., 1955. Chemical technology of fermentation. Chem. Eng. 63, 154–157. Gaden Jr., E.L., 1959. Fermentation process kinetics. J. Biochem. Microbiol. Technol. Eng. 8, 113–129. Hamissa, F.A., 1966. Chemical studies on citric acid fermentation. M.Sc. Thesis, Faculty of Science, Cairo University, Egypt. Hamissa, F.A., El-Abyad, M.S., Abdu, A., Gad, A.S., 1991. Raising potent UV mutants of Aspergillus niger van Tieghem for citric acid production from beet molasses. Bioresour. Technol. 39 (3), 209– 213. La Nauze, I.M., 1966. Aconitase and isocitirc dehydrogenase of Aspergillus niger in relation to citric acid production. J. Gen. Microbiol. 44, 73–81. Lowenstein, J.M., 1969. Chemical methods for citrate and aconitate. Methods Enzymol. 13, 513. Maddox, I.S., Hossain, M., Brooks, J.D., 1986. The effect of methanol on citric acid production from galactose by A. niger. Appl. Microbiol. Biotechnol. 23, 203–205. Milson, P.T., Meers, J.I., 1985. Citric acid. In: Blanch, H.W., Drew, S., Wang, D.I.C. (Eds.), Comprehensive Biotechnology. The Practice
of Biotechnology, Current Commodity Products, Vol. 3. Pergamon Press, Oxford, pp. 665–680. Millis, N.F., Trumpy, B.H., Palmer, B.M., 1963. The effect of lipids on citric acid production by an Aspergillus niger mutant. J. Gen. Microbiol. 30, 365–379. Moyer, A.J., 1953. Effect of alcohols on the mycological production of citric acid in surface and submerged culture. I. The nature of the alcohol effects. Appl. Microbiol. 11, 1–7. Ogawa, T., Fazeli, A., 1976. Additive effect of ferrocyanide treatment and step change of pH on citric acid production from Iranian beet molasses with Aspergillus niger. J. Ferment. Technol. 54, 66–67. Peterson, C.S., Johnson, V.A., Matlern, P.J., 1983. Evaluation of variation in mineral element concentrations in wheat flour and bran of different cultivars. Cereal Chem. 60, 450–455. Roehr, M., Kubicek, C.P., Zehentgruber, O., Orthofor, R., 1987. Accumulation and partial reconsumption of polyols during citric acid fermentation by A. niger. Appl. Microbiol. Biotechnol. 27, 235–239. Roukas, T., 1991. Production of citric acid from beet molasses by immobilized cells of Aspergillus niger. J. Food Sci. 56 (3), 878–880. Roukas, T., 1999. Citric acid production from carob pod by solid-state fermentation. Enzyme Microb. Technol. 24, 54–59. Roukas, T., Alichanidis, E., 1988. The effect of pH on the production of citric acid from beet molasses by surface fermentation. In: 8th International Biotechnol. Symposium, Paris, 218 Regie Publ. Roukas, T., Harvey, L., 1988. The effect of pH on production of citric and gluconic acids from beet molasses using continuous culture. Biotechnol. Lett. 10, 289–294. Roukas, T., Kotzekidou, P., 1986. Production of citric acid from brewery wastes by surface fermentation using Aspergillus niger. J. Food Sci. 51, 225–228. Schoorl, N., 1929. Sugar titration. Chem. Weekblad 26, 130–134. Shankaranand, V.S., Lonsane, B.K., 1994. Ability of Aspergillus niger to tolerate metal ions and minerals in a solid-state fermentation system for the production of citric acid. Process Biochem. 29, 29–37. Szczodark, J., 1981. Biosynthesis of citric acid in relation to the activity of selected enzymes of the Krebs cycle in Aspergillus niger mycelium. Eur. J. Appl. Microbiol. Biotechnol. 13, 107. Szczodark, J., Ilczuk, Z., 1975. Effect of alcohol on the synthesis of citric acid by the fungus Aspergillus niger. Przem. Ferment. Rolny 19 (6), 20–21. Wang, J., 1998. Improvement of citric acid production by Aspergillus niger with addition of phytate to beet molasses. Bioresour. Technol. 65, 243–245. Wang, J., Liu, P., 1996. Comparison of citric acid production by Aspergillus niger immobilized in gels and cryogels of polyacrylamide. J. Ind. Microbiol. 16, 351–353.
Short communication
Attempts at improving citric acid fermentation by Aspergillus niger in beet-molasses medium Nehad Z. Adham Chemistry of Nat. and Microb. Products Department, National Research Centre, Dokki, Cairo, Egypt Received 17 October 2001; received in revised form 29 October 2001; accepted 17 December 2001
Abstract Natural oils with high unsaturated fatty acids content when added at concentrations of 2% and 4% (v/v) to beet molasses (BM) medium caused a considerable increase in citric acid yield from Aspergillus niger. The fermentation capacities were also examined for production of citric acid using BM-oil media under different fermentation conditions. Maximum citric acid yield was achieved in surface culture in the presence of 4% olive oil after 12 days incubation. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Citric acid; Beet molasses; Oils; Aspergillus niger; Surface culture; Submerged culture
1. Introduction Citric acid (CA), an intermediate of the tricarboxylic acid cycle, is produced commercially almost exclusively by fermentation with Aspergillus niger, and widely used in the food beverage, chemical, pharmaceutical and other industries (Wang and Liu, 1996). The most widely used industrial substrate for CA production by A. niger is beet molasses (BM), a byproduct of beet sugar production (Clark and Lentz, 1963; Ogawa and Fazeli, 1976; Roukas and Alichanidis, 1988; Roukas and Harvey, 1988; Roukas, 1991; Hamissa et al., 1991; El-Abyad et al., 1992; Wang, 1998). Citric acid is produced commercially either by submerged fermentation (SmF) or liquid surface fermentation (LSF) processes employing BM as the substrate (Milson and Meers, 1985). The process organism A. niger is very sensitive to trace metal ions. Despite pretreatment with chelating agents such as sodium or potassium ferro-cyanide and ethylenediamine tetracetic acid (EDTA), molasses gives low yields of CA (Shankaranand and Lonsane, 1994). Many investigators have tried to improve the production of CA by various additives. Moyer (1953) found that methanol, ethanol and iso-propanol decreased growth but increased CA production from cane- and beet-molasses media. Since that time, a lot of work had been done on the effect of alcohol on citric acid fermentation (Hamissa, 1966; Szczodark and Ilczuk, 1975; Maddox et al., 1986; El-Batal et al., 1995; Roukas,
1999). Millis et al. (1963) studied the effect of various lipids on the yield of CA produced by A. niger and found that the addition of some natural oils with a high content of unsaturated fatty acids increased CA yield by about 20–50%. The present study was undertaken mainly to determine the effect of some oil supplements on CA fermentation of beet molasses by A. niger A20.
2. Methods 2.1. Microorganism A. niger A20 was kindly provided by the Centre of Culture Collection, National Research Centre (NRC), Cairo, Egypt. 2.2. Beet molasses The BM samples used in the present study were kindly supplied by the Beet Sugar Factory, Egypt. 2.3. Inoculum The slants of A. niger A20 were incubated on potato– dextrose agar medium (PDA) at 30 °C for 7 days. A spore suspension of about 105 –106 spores/ml was prepared in sterile distilled water containing about 0.01%
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 0 0 7 - X
98
N.Z. Adham / Bioresource Technology 84 (2002) 97–100
(v/v) tween-80. This solution was used as a source of inoculum. 2.4. Citric acid fermentation Unless otherwise stated, fermentation media were prepared by diluting BM with tap water to approximately 15% sugar concentration and centrifuging at 2500 rpm to remove suspended matter and the diluted BM was supplemented with 0.05 g% ðNH4 Þ2 SO4 , then dispensed in 50 ml portion into 250 ml Erlenmeyer flasks. Th pH was adjusted to 5.5–5.7. The flasks were autoclaved at 121 °C for 20 min, then inoculated with 1 ml spore suspension. The contents of the flasks were mixed thoroughly and incubated in a slanting position (surface fermentation condition) at 30 °C for 11 days. 2.5. Analytical techniques CA was determined by the colorimetric method of Lowenstein (1969). Total sugar content in the fermen-
tation liquor was determined after hydrolysis according to the modified method of Schoorl (1929). Each treatment was repeated three times and the results were reported as average of three repetitions. Estimation of mould growth. The amount of mould growth was estimated by determining the dry weight of mycelium formed. The contents of each flask were transferred to a weighed Whatman paper no. 541, washed three times with water and the mycelium was dried at 60 °C for 24 h. 3. Results and discussion 3.1. Effect of oil supplementation The data presented in Table 1 show that the production of CA was generally increased with the addition of tested oils. These oils increased CA yields effectively at 4% levels. Best CA yields were obtained with olive, sunflower and maize oils at 4% doses. Millis et al. (1963) suggested that the unsaturated lipids act as alternative
Table 1 Effect of addition of some oils on citric acid fermentation by A. niger A20 Tested oil (ml/100 ml)
Consumed sugar (g%)
CA (g/l)
CA yield (%)
Conversion (%)
Dry weight (g/flask)
Control (no addition)
10.8
29.7
20.3
27.4
2.60
Almond 2 4
14.6 14.19
25.3 47.47
17.3 32.5
17.3 33.4
3.76 3.80
Caster 2 4
14.34 14.54
45.7 52.91
31.2 36.2
31.8 36.3
3.75 4.42
Maize 2 4
14.28 14.6
50.89 58.9
34.8 40.3
35.6 40.3
4.07 4.51
Nigella 2 4
11.59 11.03
31.4 45.5
21.4 31.1
27.0 41.2
3.76 3.79
Olive 2 4
14.6 14.2
57.7 72.8
39.5 49.8
39.5 51.2
3.68 3.70
Peanut 2 4
12.5 12.4
40.6 47.4
27.8 32.4
32.3 38.2
3.56 4.13
Soybean 2 4
13.67 14.22
40.5 55.2
27.7 37.8
29.6 38.8
3.97 4.62
Sunflower 2 4
14.6 14.5
49.5 56.9
33.9 39.0
33.9 39.2
4.0 4.2
Initial sugar concentration ¼ 14.6%. The tested oils were separately supplemented at 2% and 4% (v/v) before autoclaving. CA producted CA yield% ¼ Original sugar
ðg=100 mlÞ ðg=100 mlÞ
100;
CA produced ðg=100 mlÞ 100: Conversion coefficient ðc:c:Þ ¼ Consumed sugar ðg=100 mlÞ
N.Z. Adham / Bioresource Technology 84 (2002) 97–100
Fig. 1. Effect of different oils on CA formation by A. niger A20 using surface and submerged culture techniques.
hydrogen acceptors to oxygen during the fermentation and thus improve the yield of citric acid. Relatively higher concentrations of sugars were assimilated and comparatively high conversion rates were recorded in the presence of the tested oils. Moreover, the fungal growth yields were considerably accelerated under these conditions. This experiment was followed by investigating CA accumulation after 5, 9 and 12 days in the presence of
99
the promising oil treatments, adopting both surface as well as submerged culture techniques (Fig. 1). Data shown in Table 2 showed that surface culture technique proved to be most suited for CA yield. This might have been due to differences in the response of A. niger to the metal ions and minerals upon using submerged or surface fermentation conditions. It has been extensively documented that Fe2þ stimulated aconitase activity which leads to a concurrent decrease in the rate of CA biosynthesis particularly in submerged fermentation (Peterson et al., 1983; La Nauze, 1966). The fermentation parameters obtained when the tested fungus was allowed to grow on BM-medium without oil supplementation (control treatment) showed that relatively higher CA outputs were maintained with the surface culture technique. Nevertheless, the submerged technique afforded higher biomass yield. Additionally, the superiority of the surface technique was traced in the presence of the favorite oil supplements (Table 2). Thus, under the latter cultivation conditions, the fermentation activities, in view of CA biosynthesis, were considerably enhanced. Better CA productivities were, however, achieved after 12 days incubation. The olive oil treatment supported maximum CA outputs followed by maize and then sunflower oil treatments. The submerged techniques afforded relatively lower fermentation efficiencies under all tested experimental conditions. No consistent relationship was recorded between growth and CA accumulation. Thus, while relatively higher growth values were recorded when the fungus was cultivated under submerged conditions, the CA formation proved to be adversely affected under the same conditions. These results may be correlated with the fact that, in citric acid fermentation two kinetic phases of sugar consumption occur. In the first phase, the most growth takes place, while in the second phase CA accumulates at the expense of the sugar consumption with only slight
Table 2 Effect of different oils on CA formation by A. niger A20 using surface and submerged culture techniques Different oils
Incubation period (days)
Surface culture Consumed sugar (g%)
CA (g/l)
Conversion (%)
Submerged culture
Control
5 9 12
7.9 9.1 10.84
7.2 27.2 29.7
9.1 29.8 27.4
Olive
5 9 12
3.6 11.7 14.2
13.2 56.3 72.9
Maize
5 9 12
7.2 12.0 14.6
Sunflower
5 9 12
7.9 12.7 14.5
Dry weight (g/flask)
Consumed sugar (g%)
CA (g/l)
Conversion (%)
Dry weight (g/flask)
1.9 2.7 2.6
8.76 13.4 14.6
8.6 5.1 5.5
9.8 3.8 3.7
3.5 4.88 3.96
36.3 48.1 51.3
2.75 3.34 3.7
8.3 13.9 14.6
7.00 7.2 5.3
8.4 5.2 3.5
5.36 6.88 5.78
15.6 53.2 58.9
21.7 44.1 4.0
3.43 5.17 4.51
8.4 11.7 14.4
6.1 6.9 6.0
7.3 5.9 4.2
4.62 5.72 3.72
13.9 54.8 56.9
17.6 43.2 39.2
2.8 4.1 4.2
8.8 11.5 14.5
6.0 7.0 5.8
6.8 6.1 4.0
4.58 5.7 3.9
100
N.Z. Adham / Bioresource Technology 84 (2002) 97–100
increase in growth (Gaden, 1955, 1959; Roukas and Kotzekidou, 1986; Roehr et al., 1987). El-Batal et al. (1995), using five different strains of A. niger, reported that relatively high CA productivities were achieved with only three cultures after 12 and 13 days adopting the surface culture technique. Similar results have been also recorded with Szczodark (1981). Summarizing the previously mentioned results, the supplementation of a surface culture with some oil makes a direct increase in CA yield possible in using molasses medium.
References Clark, D.S., Lentz, C.P., 1963. Submerged citric acid fermentation of beet molasses in Tank-Type fermentation. Biotechnol. Bioeng. 5, 193. El-Abyad, M.S., Hamissa, F.A., Gad, A.S., 1992. Treatment of beet molasses for citric acid production by a potent strain of Aspergillus niger van Teeghem. Bioresour. Technol. 39 (2), 191–197. El-Batal, A.I., Khalil, A.H., Mostafa, M.M., 1995. Citric acid production by gamma irradiated Aspergillus niger from treated beet molasses under different fermentation conditions. Egypt. J. Rad. Sci. Appl. 8 (2), 253–266. Gaden Jr., E.L., 1955. Chemical technology of fermentation. Chem. Eng. 63, 154–157. Gaden Jr., E.L., 1959. Fermentation process kinetics. J. Biochem. Microbiol. Technol. Eng. 8, 113–129. Hamissa, F.A., 1966. Chemical studies on citric acid fermentation. M.Sc. Thesis, Faculty of Science, Cairo University, Egypt. Hamissa, F.A., El-Abyad, M.S., Abdu, A., Gad, A.S., 1991. Raising potent UV mutants of Aspergillus niger van Tieghem for citric acid production from beet molasses. Bioresour. Technol. 39 (3), 209– 213. La Nauze, I.M., 1966. Aconitase and isocitirc dehydrogenase of Aspergillus niger in relation to citric acid production. J. Gen. Microbiol. 44, 73–81. Lowenstein, J.M., 1969. Chemical methods for citrate and aconitate. Methods Enzymol. 13, 513. Maddox, I.S., Hossain, M., Brooks, J.D., 1986. The effect of methanol on citric acid production from galactose by A. niger. Appl. Microbiol. Biotechnol. 23, 203–205. Milson, P.T., Meers, J.I., 1985. Citric acid. In: Blanch, H.W., Drew, S., Wang, D.I.C. (Eds.), Comprehensive Biotechnology. The Practice
of Biotechnology, Current Commodity Products, Vol. 3. Pergamon Press, Oxford, pp. 665–680. Millis, N.F., Trumpy, B.H., Palmer, B.M., 1963. The effect of lipids on citric acid production by an Aspergillus niger mutant. J. Gen. Microbiol. 30, 365–379. Moyer, A.J., 1953. Effect of alcohols on the mycological production of citric acid in surface and submerged culture. I. The nature of the alcohol effects. Appl. Microbiol. 11, 1–7. Ogawa, T., Fazeli, A., 1976. Additive effect of ferrocyanide treatment and step change of pH on citric acid production from Iranian beet molasses with Aspergillus niger. J. Ferment. Technol. 54, 66–67. Peterson, C.S., Johnson, V.A., Matlern, P.J., 1983. Evaluation of variation in mineral element concentrations in wheat flour and bran of different cultivars. Cereal Chem. 60, 450–455. Roehr, M., Kubicek, C.P., Zehentgruber, O., Orthofor, R., 1987. Accumulation and partial reconsumption of polyols during citric acid fermentation by A. niger. Appl. Microbiol. Biotechnol. 27, 235–239. Roukas, T., 1991. Production of citric acid from beet molasses by immobilized cells of Aspergillus niger. J. Food Sci. 56 (3), 878–880. Roukas, T., 1999. Citric acid production from carob pod by solid-state fermentation. Enzyme Microb. Technol. 24, 54–59. Roukas, T., Alichanidis, E., 1988. The effect of pH on the production of citric acid from beet molasses by surface fermentation. In: 8th International Biotechnol. Symposium, Paris, 218 Regie Publ. Roukas, T., Harvey, L., 1988. The effect of pH on production of citric and gluconic acids from beet molasses using continuous culture. Biotechnol. Lett. 10, 289–294. Roukas, T., Kotzekidou, P., 1986. Production of citric acid from brewery wastes by surface fermentation using Aspergillus niger. J. Food Sci. 51, 225–228. Schoorl, N., 1929. Sugar titration. Chem. Weekblad 26, 130–134. Shankaranand, V.S., Lonsane, B.K., 1994. Ability of Aspergillus niger to tolerate metal ions and minerals in a solid-state fermentation system for the production of citric acid. Process Biochem. 29, 29–37. Szczodark, J., 1981. Biosynthesis of citric acid in relation to the activity of selected enzymes of the Krebs cycle in Aspergillus niger mycelium. Eur. J. Appl. Microbiol. Biotechnol. 13, 107. Szczodark, J., Ilczuk, Z., 1975. Effect of alcohol on the synthesis of citric acid by the fungus Aspergillus niger. Przem. Ferment. Rolny 19 (6), 20–21. Wang, J., 1998. Improvement of citric acid production by Aspergillus niger with addition of phytate to beet molasses. Bioresour. Technol. 65, 243–245. Wang, J., Liu, P., 1996. Comparison of citric acid production by Aspergillus niger immobilized in gels and cryogels of polyacrylamide. J. Ind. Microbiol. 16, 351–353.