Financial support: Pakistan Science Foundation under the project No supported the work P-GC/BIO/RES-283.

Keywords: agitator bioreactor, Aspergillus niger, citric acid, MnSO₄, recycling fed-batch cultivation system, TCA cycle.

Citric acid is one of the most commonly used acids in the food and pharmaceutical industries on account of its high solubility, palatability and low toxicity. Although it can be extracted from citrus fruits but submerged fungal fermentation processes - notably using filamentous fungi Aspergillus niger, principally produce it. However, in the cultures of these fungi, the growth of interlocking hyphae may result in high viscosity fermentation broths and consequent mass transfer and operational problems. For efficient citric acid production, growth of the Aspergillus in pellet form is desirable and this can be achieved by process optimisation. A number of carbon sources may be used for citric acid fermentation. For commercial reasons the use of molasses, sucrose or glucose syrups are favoured. The use of molasses in particular is desirable because of its availability and low cost. Considerable research effort has been expended in developing the citric acid production protocol (Torres and Kubicek, 1999). However, because it is a low volume, high value product, commercial profitability is dependent on keeping technical and scientific advances proprietary. Consequently, industrial developments in the citric acid production techniques are closely guarded by confidentiality agreements. Factors which affect Aspergillus growth and citric acid yields are many and may include, substrate and nitrogen concentrations, initial pH, dissolved oxygen and cation (especially Fe⁺², Mn⁺² and Cu⁺²) levels of the medium.

Citric acid is produced during trophophase as a metabolic product of the tricarboxylic acid cycle and the carbon entering the cycle is generally converted into biomass, metabolic energy and CO₂. Only a few small amounts would accumulate as citric acid under balanced growth. The main mechanism of accumulation is the manipulation of medium constituents and growth conditions. Several efforts were made to integrate the core of this knowledge in highly structured dynamical models. However, no recent attempts are reported about to gain insight trough macroscopic and energetic strategies, despite of some early precedents (Rohr, 1998). The availability of kinetic information under representative physiological conditions is usually scarce particularly in the case of pelletized fungus as Aspergillus. Recently, we have developed protocols for citric acid production as a future bioprocessing laboratory process. For this, five different industrial strains of Aspergillus niger were compared for citric acid production, biomass formation and substrate utilization on the basis of kinetic relation in fed-batch bioreactor. Addition of Mn⁺² ions in citrate broth of fed-batch culture not only remarkably enhanced citric acid production but also reduced the incubation period, thus making the process highly economical. High and consistent yield of citric acid and its kinetic status in fed-batch agitator bioreactor by adding MnSO₄ in the culture medium devised it as a novel technique on commercial basis citric acid productivity.     

Stainless steel fermentor (New Brunswick, USA) of 15 L capacity with working volume of 9 L was employed for citric acid fermentation. The fermentation medium consisting of (g/l); clarified cane molasses 300.0 (sugar 15%), K₄Fe(CN)₆ 200 ppm at pH 6.0 was used for fermentation. The vegetative inoculum was transferred to the production medium at a rate of 4% (v/v) based on total working volume of the fermentation medium. The incubation temperature was kept at 30±1°C throughout the fermentation period of 144 hours. Agitation speed of the stirred was kept at 200 rpm and aeration rate was maintained at 1.0 l/l/min. Sterilized silicone oil (antifoam AE-II) was used to control the foaming problem during fermentation.  'Mycelial dry weight' and 'Sugar' were estimated spectrophotometrically. A UV/VIS scanning spectrophotometer was used for measuring colour intensity. 'Anhydrous citric acid' was estimated using pyridine-acetic anhydride method.

Owing to the steadily increasing demand of citric acid for industrial purpose, its manufacture from molasses has proved to be of great importance to the sugar industry. Stability of the citric acid market and price range make it one of the most stable commodities that can be produced from molasses. The present investigation constitutes an attempt to design optimisation strategies of citric acid production rate in A. niger by integrating in a framework of relevant aspects of the A. niger physiology. In this regard, the most noticeable finding was the addition of 2.0 ´ 10^-6 M MnSO₄ in fed-batch bioreactor and remarkably highest yield of citric acid, ever recorded in the history of fermentation technology. The value of product yield coefficient (i.e. 1.661±0.02a g/g) of Aspergillus niger NG^GCB101 was highly significant (LSD > 0.0340).

TORRES, N.V. and KUBICEK, C.P. Glucose transport by Aspergillus niger during growth on high glucose concentrations. Applied Microbiology and Biotechnology, March 1999, vol. 47, p. 792-797.

ROHR, M. A century of citric acid fermentation and research. Food Technology and Biotechnology, March 1998, vol. 36, p. 163-171.

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