EFFECT OF ADDITIONAL MINERAL IONS ON CITRIC ACID PRODUCTIVITY BY ASPERGILLUS NIGER NG-110
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1 BIOLOGIA 21, 47 (1&2), PP ISSN EFFECT OF ADDITIONAL MINERAL IONS ON CITRIC ACID PRODUCTIVITY BY ASPERGILLUS NIGER NG-11 SIKANDER ALI 1, IKRAM-UL-HAQ 1 AND JAVED IQBAL 2 1 Biotechnology Research Labs, Department of Botany, Govt. College, Lahore, Pakistan 2 School of Biological Sciences, University of the Punjab, Lahore, Pakistan ABSTRACT: The mutant strain of Aspergillus niger NG-11 was evaluated for the present investigation of citric acid production. The cultural conditions optimized during the course of study were ph and K 4Fe(CN) 6, K 2HPO 4 and CaCl 2 concentrations. Cane-molasses was utilized as the basal fermentation medium. The mutant NG-11 produces maximal amount of citric acid when the ph of the medium was 6. When the concentrations of K 4Fe(CN) 6, K 2HPO 4 and CaCl 2 were 2 ppm,.2% w/v and 2.%, the maximum citric acid production was 69.3±.8 g/l, 81.21±.2 g/l and 8.76±.2 g/l, respectively. Keywords: Aspergillus niger, cane-molasses, fermentation, potassium ferrocyanide. INTRODUCTION Citric acid is one of the tricarboxylic acids produced as intermediates in Krebs cycle. It can be obtained from oranges, lemons etc. It can also be produced from various microorganisms such as bacteria, filamentous fungi and yeast by applying various fermentation techniques (Roskosu & Anenil, 198). Because of its high solubility, palatability and low toxicity, citric acid has now become one of the most commonly used acids. Approximately, 75% of this compound is used as food acidulate and 12% in pharmaceutical industry (Haq et al., 2). Various fungi have been evaluated for citric acid production but one of the prerequisites for abundant citric acid production is Aspergillus niger (Maddox & Brooks, 1998), and has dominated others both in laboratory and industrial scale. The present study is concerned with the effect of ph and some nutritional parameters such as concentrations of K 4 Fe(CN 6 ), K 2 HPO 4 and CaCl 2 on citric acid bioproduction and their kinetic analysis. Clarified cane-molasses was employed as the basal fermentation medium throughout the investigation. MATERIALS AND METHODS Organism and Inoculum Preparation The mutant strain of Aspergillus niger NG-11 was obtained from the cultural stock of Biotechnology Research Laboratory, GC Univ; Lahore, Pakistan. The culture was maintained on potato dextrose agar slants and preserved at 4 C. Conidial inoculum was used in the shake flask technique. Conidia from 3-5 days old slant culture were used for inoculation. The conidial suspension was prepared in sterilized.5% Monoxal O.T. (Dioctyle ester of sodium sulfosuccinic acid). Ten millilitre of Monoxal O.T. was added to each slant having profuse
2 BIOLOGIA SIKANDER ALI, ET AL 6 conidial growth on its surface. Inoculating needle was used for breaking the clumps of conidia and test tube was shaken vigorously to obtain homogenous mixture of conidial suspension. One millilitre of the suspension contained conidia. The count was made on a haemocytometer. Fermentation Technique Twenty-five millilitre of clarified cane molasses with 15% sugar level (initial ph 6.) was taken in each of the 25. ml Erlenmeyer flasks. The flasks were cotton plugged and autoclaved at 121ºC for 15 minutes. After cooling at room temperature, the flasks were inoculated with 1. ml of conidial suspension. One millilitre of 2 ppm sterilized potassium ferrocyanide, K 4 Fe(CN) 6 was added at the time of inoculation when the medium was hot. The flasks were then incubated at a rotary incubator shaker (Gallenkamp PLC, UK) at 3ºC for 7 days. The shaking speed was kept at 16 rpm. After incubation, fermented broth was filtered and filtrate was used for the estimation of citric acid and residual sugar contents. Analytical Techniques Residual sugar was estimated by DNS method (Tasun et al., 1958). Dry cell mass was determined by filtering the culture medium through pre-weighed Whatmann filter paper No. 44. The filtrate was used for further analysis while for the calculation of dry cell mass, mycelium was thoroughly washed with tap water and dried at 15ºC for 2 h (Haq & Daud, 1995). Citric acid anhydrous was estimated spectrophotometrically using pyridineacetic anhydride method. RESULTS Effect of Initial ph Effect of changing initial ph of the basal medium on the production of citric acid, sugar consumption and mycelial dry weight by mould culture was investigated. Fermentation medium with initial ph 6. resulted in maximum citric acid production (65.2±.2 g/l). The sugar consumption and mycelial dry weights were 93.5±2. and 16.±.3 g/l, respectively. The percentage yield of citric acid on the basis of sugar used was 69.73%. Fig., 1 shows the comparison of specific growth rate (, h -1 ) for citric acid fermentation. Among all the initial ph, the value of 4. was comparatively significant than others. Effect of Different Concentrations of Potassium Ferrocyanide Effect of different concentrations of potassium ferrocyanide (5-3 ppm) on citric acid fermentation by a strain of A. niger NG-11 was investigated in shake flask. Potassium ferrocyanide was added to the sterilized fermentation medium just before inoculation when the medium was hot. The fermentation medium containing 2 ppm potassium ferrocyanide showed the maximum citric acid production (69.3±.8 g/l). The sugar consumption and mycelial dry weight were 83.5±4. and 25.3±.4 g/l, respectively. The percentage yield of citric acid on the basis of sugar consumed in the medium containing K 4 Fe(CN) 6 (2 ppm) was 8.99%. A decrease in citric acid production was observed, when the concentration of potassium ferrocyanide was increased or decreased from 2 ppm. The level of K 4 Fe(CN) 6 2 ppm concentration was found to be optimum for citric acid production. Product and growth yield coefficients as kinetic
3 Vol. 47(1&2) CITRIC ACID PRODUCTION BY A. NIGER. 61 parameters were also studied for citric acid using different concentration of potassium ferrocyanide (Fig. 2). The values for Yp/s and Yp/x at 5ppm K 4 Fe(CN) 6 were found to be more significant than the others. Effect of Different Concentrations of Dipotassium Hydrogen Phosphate The effect of K 2 HPO 4 in different concentrations ranging from.15% -.3% w/v, on citric acid fermentation by mutant strain of A. niger NG-11 in shake flask was observed. The addition of K 2 HPO 4 in the molasses medium resulted in maximum citric acid production (81.21±.2 g/l) in concentration.2% w/v. Sugar consumption and mycelial dry weight were 92.2±3.5 and 2.4±.2 g/l respectively. The percentage yield on the basis of sugar used was 88.11%. The mould growth was in the form of small round pellets, observed in the fermented broth. Product and growth yield coefficients as kinetic parameters were also studied for citric acid using different sources of phosphate and their concentrations (Fig., 3). The values for Yp/s and Yp/x (g/g) at 1. % KH 2 PO 4 were found to be significant. Effect of Different Concentrations of Calcium chloride The concentration of calcium chloride varied from 1.-2.%. The production of citric acid was increased with the increase in the concentration of CaCl 2 and found to be optimum when 2.% CaCl 2 was added to the fermentation medium (8.76±.2 g/l). The percent yield of citric acid was 82.4% with sugar consumption 98.±2. g/l while the dry weight of mycelia was 2.92±.2 g/l. Further increase in the concentration of CaCl 2 reduced the secretion of citric acid from mycelia. Product and growth yield coefficients as kinetic parameters were also studied for citric acid using different sources of phosphate and their concentrations (Fig., 4). The values for Yp/s and Yp/x (g/g) at 1. % KH 2 PO 4 were found to be significant. DISCUSSION The present study describes the effect of different conditions such as ph, effect of different concentrations of potassium ferrocyanide, dipotassium hydrogen phosphate and Calcium chloride on citric acid production by a mutant strains of A. niger NG-11 obtained from Government College University Biotechnology Lab. Initial ph of basal medium is very essential for the successful fermentation of citric acid. Effect of different initial ph of molasses medium of citric acid production was studied and maximum yield (65.2±.2 g/l) was obtained when initial ph of the fermentation medium was 6.. Any increase or decrease in the ph, greatly reduced citric acid biosynthesis. It might be due to that the ferrocyanide was more toxic at lower ph for growth of mycelium in molasses medium. This had been reported by Pessoa et al. (1984). A higher ph caused accumulation of oxalic acid. The effect of different concentrations of K 4 Fe(CN) 6 on citric acid fermentation by a mutant strain of A. niger NG-11 was carried out in shake flask. The addition of K 4 Fe(CN) 6 (2 ppm) at the time of inoculation did not increase the citric acid production. The insoluble complexes of ferrocyanide with heavy metals acted as metal buffers in the fermentation medium, which made the metal ions available at concentration suitable for citric acid production (Akbar et al., 1967). It was also due to the fact that it checked the mycelial growth and also inhibited the activity of
4 BIOLOGIA SIKANDER ALI, ET AL 62 enzyme aconitase (Shankaranand & Lonsane, 1993). Calcium also played an important role in citric acid fermentation with its effect on fungal growth, hyphal morphology and citric acid production. The addition of CaCl 2 to the fermentation medium lowered the final biomass and increased the uptake of phosphate, sucrose and citric acid production (Pera & Callieri, 1997). Calcium chloride also reduced the biomass concentration and increased the volumetric productivity as well as specific production rate (Rajoka et al., 1998; Pera & Callieri, 1997). The production rate, however, was dependant on the concentration and the type of organism. In the present study the 1.5% level of CaCl 2 was found to be the best for citric acid production by A. niger. The kinetic parameters such as growth yield coefficients (Y p/s, Y p/x, Y x/s in g/g), volumetric rates (Q p, Q s, Q x in g/l/h) and specific substrate rates (q p, q s in g/g cells/h) were also undertaken. The mutant strain of A. niger NG-11 showed improved values for Y p/s, Y p/x, and Y x/s. Similar kind of work had also been reported by Pirt (1975). Maximum growth in terms of specific growth rate ( in h -1 ) was only marginally different during growth of mutant A. niger GCB- 47 on 15 g/l carbohydrates in molasses at 3 C (than 32 C or 165 g/l sugar). However, when the culture was monitored for Y x/s, Q s and q s, there was a significant enhancement in these variables at optimal nutritional conditions, i.e., incubation temperature 3 C, initial sugar concentration 15 g/l, methanol 1%, NH 4 NO 3.15%, CaCl 2 2.% K 2 HPO 4.2% and an incubation period of 168 h (7 days). This indicated that the mutant strain used in the current studies is a faster growing organism and have the ability to hyper produce citric acid without additional replacements. The study was directly substantiated with the findings of Rajoka et al. (1998). Maximum values for Y p/s, Q p and q p were several folds improved over the previous studies. REFERENCES Akbar, M., Ahmad, F. & Qadeer, M.A., Chelation of metal ions in citric acid fermentation by Aspergillus niger. Pak. J. Sci. Ind. Res., 1: Haq, I., Rehman, A. & Ali S., 2. Citric acid fermentation by Aspergillus niger. Pak. J. Biol. Sci., 4: Haq, P.B. & Daud, D.A., Process of mycelial dry weight calculation for citric acid. J. Biotechnol., 9: Maddox, I.S. & Brooks, J.D., Fermentation, merits and future perspectives. Proc. Biochem., 33: Marrier, J.R. & Boulet, M., Direct determination of citric acid in milk with an improved, pyridine acetic anhydride method. J. Dairy Sci., 41: Pera, L.M. & Callieri, D.A., Influence of calcium on fungal growth, hyphal morphology and citric acid production in Aspergillus niger. J. Technol., 42: Pessoa, D.F., Castro, A.C.D. & Leite, S.G.F., Citric acid fermentation with Aspergillus niger. Rev. Microbiol., 15: Pirt, S.J Principles of Microbes & Cell Cultivation. Black Wells Scientific, London, UK, pp:
5 Specific growth rate/h Vol. 47(1&2) CITRIC ACID PRODUCTION BY A. NIGER. 63 Rajoka, M.I., Ahmad, M.N., Shahid, R., Latif, F. & Parvez, S., Citric acid production from sugar cane molasses by cultures of Aspergillus niger. Biologia, 44: Roskosu, A.A. & Anenil, C.A., 198. Effect of various conditions on the production of citric acid during fermentation of molasses by Aspergillus niger. J. Microbial Technol., 2: Shankaranand, V.S. & Lonsane, B.K., The use of sugarcane as substrate for the production of citric acid by solid-state fermentation. World J. Microbiol. Biotechnol., 9: Tasun, K., Chose, P. & Glien, K., 197. Sugar determination by DNS method. Biotechnol. Bioeng., 12: Initial ph Figure 1: Comparison of specific growth rate for citric acid fermentation. Kinetic parameters: Specific growth rate, μ (h -1 ) = g cell mass produced /l/h values differ significantly at p.5
6 Product and growth yield coefficient (Yp/x, Yp/s in g/g) Product and growth yield coefficients (Yp/s, Yp/x in g/g) BIOLOGIA SIKANDER ALI, ET AL Control Time course study at 75 ppm of Potassium ferrocyanide Yp/s Yp/x Figure 2: Comparison of product and growth yield coefficients for citric acid fermentation. Kinetic parameters: Yp/s= g citric acid produced/g substrate consumed, p/x= g citric acid produced/ g cells formed values differ significantly at p Control Different phosphate sources and their concentrations (%) Yp/s (Di potassium hydrogen phosphate) Yp/x (Di potassium hydrogen phosphate) Figure 3: Comparison of product and growth yield coefficients for citric acid fermentation. Kinetic parameters: Yp/s= g citric acid produced/ g substrate consumed, Yp/x= g citric acid produced/ g cells formed values differ significantly at p.5.
7 Product and growth yield coefficient (Yp/x, Yp/s in g/g) Vol. 47(1&2) CITRIC ACID PRODUCTION BY A. NIGER Different calcium cloride concentrations (% ) Yp/s Yp/x Figure 4: Comparison of product and growth yield coefficients for citric acid fermentation. Kinetic parameters: Yp/s= g citric acid produced/ g substrate consumed, Yp/x= g citric acid produced/ g cells formed values differ significantly at p.5.
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