Citric acid is ubiquitous in nature and exists as an intermediate in the citric acid cycle when carbohydrates are oxidized to carbon dioxide. It is solid at room temperature, melts at 153ºC and decomposes at higher temperatures into other products. It is responsible for the tart taste of various fruits in which it occurs, i.e. lemons, limes, figs, oranges, pineapples, pears and goose-berries. Because of its high solubility, palatability and low toxicity, it can be used in food, biochemical and pharmaceutical industries. These uses have placed greater stress on increased citric acid production and search for more efficient fermentation process. The worldwide demand of citric acid is about 6.0 ´ 10⁵ tons per year and it is bound to increase day by day. Many microorganisms such as fungi and bacteria can produce citric acid. The various fungi, which have been found to accumulate citric acid in their culture media, include strains of Aspergillus niger, A. awamori, Penicillium restrictum, Trichoderma viride, Mucor piriformis and Yarrowia lipolytica. But Aspergillus niger remained the organism of choice for the production of citric acid. The mutant strains might show several fold increase in citrate production as compared to wild-type cultures.

Although the surface culture process is still being used, most of the newly built plants have adopted the submerged fermentation process. Molasses is a desirable raw material for citric acid fermentation because of its availability and relatively low price. Incubation temperature plays an important role in the production of citric acid. Temperature between 25-30ºC was usually employed for culturing of Aspergillus nigerbut temperature above 35ºC was inhibitory to citric acid formation because of the increased the production of by-product acids and also inhibition of culture development. The maximum production of citric acid (6.5%) was obtained at pH 5.4 in molasses medium. The appropriate pH is important for the progress and successful termination of fermentation. The trace metals such as iron, zinc, copper, manganese present a critical problem in submerged fermentation. The organisms need major elements such as carbon, nitrogen, phosphorus and sulphur in addition to various trace elements for growth and citric acid production. The present work is concerned with the optimisation of cultural conditions for enhanced production of citric acid by mutant strain of Aspergillus nigerGCMC-7in stirred fermentor.

The mutant strain of Aspergillus niger GCMC-7 was used for citric acid fermentation. It was obtained from the culture collection of Biotechnology Research Labs., Government College Lahore, Pakistan. Hundred ml of molasses medium (Sugar 15%, pH 6.0) containing silica gel chips (0.75 mm dia) in 1.0 L cotton wool plugged Erlenmeyer flask, was sterilized. Small amount of conidia from the slant was aseptically transferred with the help of inoculating needle. The flask was incubated at 30ºC in a rotary incubator shaker (Gallenkamp, UK) at 300 rpm for 24 h. A laboratory scale stainless steel stirred fermentor (0012 M1E, New Brunswic, USA) of 15 L capacity with working volume of 9 L was used for citric acid fermentation. The fermentation medium consisting; clarified cane molasses 300.0 (sugar 15%), K₄Fe(CN)₆ 200 ppm at an appropriate initial pH. The vegetative inoculum was transferred to the production medium at a rate of 5% (v/v) based on total working volume of the fermentation medium. The incubation temperature was varied during different batches. Air supply was maintained at different levels from an aeration pump (MLK5, Pak. Made). Necessary agitation intensity was also maintained. Sterilized silicone oil (Antifoam AE-11, Merck, Germany) was used to control the foaming during fermentation. All the experiments were run parallel in triplicates. Sugar was estimated spectrophotometrically by DNS method (Tasun et al. 1970). A UV/VIS scanning spectrophotometer (Cecil-700 series, UK) was used for measuring colour intensity at required wavelength. Anhydrous citric acid was estimated using pyridine-acetic anhydride method.

A successful fermentation process depends both on an appropriate strain and optimisation of fermentation parameters. In the present work, cultural conditions such as sugar concentration, time profile of citric acid synthesis, incubation temperature, initial pH, agitation intensity and air supply were optimised by a mutant strain of Aspergillus niger GCMC-7 in a laboratory scale stirred fermentor. Cultural conditions for citric acid production by fungi vary from strain to strain and also depend on the type of process. The optimisation of cultural conditions is the key for high and consistent yields of metabolites like citric acid. In the present study, the mutant strain of Aspergillus niger GCMC-7 supported maximum production of citric acid (106.65 g/l) without supplements which is substantial. The addition of nitrogen sources and minerals like calcium and phosphate may further increase the production of citric acid, as required for an industrial process.

TASUN, K.; GHOSE, P. and GHEN, K. Sugar determination of DNS method. Biotechnology and Bioengineering, April 1970, vol. 12, p. 921.

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