The selection of a suitable fungus strain is critical to the production of citric acid, because he fungus plays a central role in the process. A strain used in an industrial scale operation should have long term stability, high sporulation, good growth in the substrates, a short fermentation time, resistance to other microorganisms, and produce a high concentration of the acid (70.0-100.0 g/L) in different fermentation systems. Citric acid's production stability is difficult to maintain because of spontaneous mutation and autolysis of the strains during fermentation. These problems can be avoided by periodic reisolation from single spores, storage at low temperature (4.0-7.0°C), and avoidance of "foggy" patches of sterile mycelium (fungal mat lacking spores) when mass transfers are made (6).
Over the years, many microorganisms have been used for the production of citric acid. However, A. niger still remains the microorganism of choice in industrial scale production. Other microorganisms, such as yeasts and bacteria, produce potentially large amounts of citric acid (50.0-70.0 g/L), but until recently they have not been used for commercial production (3). There is general agreement that only selected strains of A. niger are useful citric acid producers because they can be handled easily, are inexpensive, and give high and consistent yields, thereby making the process economical. Other species of Aspergillus which have been found to accumulate citric acid include strains of A. awamori, A. fenicis, A. fonsecaeus, A. luchensis, A. fumaricus, A. wentii, A. saitoi, A. usami, A. phoenicus, A. lanosus, A. foetidus, and A. flavus as well as some strains of Penicillium such as P. janthinellum, P. simplicissimum, and P. restrictum (3,6,21-24). Fungi that produce citric acid include strains of Trichoderma viride, Mucor piriformis, Ustulina vulgaris, and species of Botrytis, Ascochyta, Absidia, Talaromyces, Acremonium, and Eupenicillium (3,6). The yields of citric acid obtained with these strains are lower than those obtained with strains of A. niger. It is generally recognized that A. niger consists of a large group of strains that differ from one another in their morphology and biochemical characteristics. The differences reported include: color of spores and mycelium, size and quantity of spores, mycelium size, substrate utilization, fermentation time, and ability to produce citric acid from different substrates (6,25-27). Industrial scale production by fungi has been carried out from chemically defined media or beet and cane molasses using surface or submerged fermentation. Today, almost all citric acid produced by fermentation is manufactured by strains of A. niger in submerged culture.
Although A. niger is the traditional producer of citric acid, in the past 30 years researchers have been attracted to the use of yeasts as citric acid producers (9). Yeasts have some advantages compared to A. niger strains. The fermentation time is short (half the time of A. niger fermentation) and thus, productivity is higher. Yeast strains are insensitive to molasses variations and can be used for developing a continuous process (3,5). Also, yeasts are not only more tolerant of contamination, but are capable of metabolizing high initial sugar or n-alkane concentrations (100.0-150.0 or 40.0-60.0 g/L, respectively), with rapid growth resulting in high productivity rates. Moreover, they have a greater tolerance for metal ions, thus allowing the use of less refined substrates. These capabilities can lead to significant reductions in substrate and waste treatment costs and in product recovery costs (10,11). The advantages of using n-alkanes over carbohydrates are potentially lower costs for substrates and higher citric acid concentrations (11). The major disadvantage of using yeasts is the production of isocitric acid during fermentation. The amount of isocitric acid produced depends on the yeast strains used, the chemical composition of the substrates, the fermentation system, and generally the conditions under which fermentation takes place. To overcome this problem, some methods have been developed to reduce the amount of isocitric acid produced. For instance, the utilization of mutants that are sensitive to fluoroacetate results in the production of very small amounts of isocitric acid (5).
The variety of yeasts that produce citric acid belong to the genus Candida, Saccharomycopsis, Hansenula, Pichia, Debaryomyces, Torulopsis, Kloeckera, Trichosporon, Torula, Rhodotorula, Sporobolomyces, Endomyces, Nocardia, Nematospora, Saccharomyces, and Zygosaccharomyces (3). Among these, strains of Candida are widely used for the production of citric acid. These strains include C. lipolytica, C. tropicalis, C. zeylanoides, C. fibrae, C. intermedia, C. parapsilosis, C. petrophilum, C. subtropicalis, C. oleophila, C. hitachinica, C. citrica, C. guilliermondii, and C. sucrosa (3,8-15,28-39). Recently, the production of citric acid by immobilized and mutant strains of C. lipolytica has been reported (40-51). The mechanism by which C. lipolytica produces a high concentration of citric acid is unclear. In general, unlimited growth of C. lipolytica in a rich medium results in low citric acid production. However, citric acid can be produced in increased amounts if yeast growth is properly restricted during the acid producing phase of fermentation (11). Production by yeasts, carried out in aerobic and agitated fermentation broth and at temperatures of 25-35°C, depended on the yeast strains and equipment used. The medium consisted of either glucose, molasses, n-alkanes, n-paraffins, methanol, butanol, ethanol, C12-16 alcohols, acetate, fatty acids, and natural oils, or fats supplemented with nitrogen sources such as (NH4)2SO4, NH4NO3, or NH4Cl, KH2PO4, MgSO4«7H2O, CaCO3, thiamin hydrochloride, vitamin B complex, and trace elements such as Fe2+, Zn2+, Mn2+, and Cu2+ (3,9-14,28-39). The medium was inoculated with 10-12% (v/v) of C. lipolytica (culture aged 48 h). Crolla and Kennedy (11) reported that excess inoculum concentration leads to high biomass concentration (20.0 g/L) and lower citric acid production (25.0 g/L), while low amounts of inoculum lead to long fermentation times.
Citric acid production by yeasts in the laboratory employs two phases: (1) a preliminary growth phase on a complete medium, followed by (2) a production phase without nitrogen sources. In some cases the medium is supplemented with limited amounts of nitrogen (1.0 g/L) in order to keep cellular activity at an acceptable level (10). In industrial scale production with Y. lipolytica, three steps have been identified: (1) the exponential growth phase, (2) the citrate production lag phase, and (3) the linear production phase. The production phase is connected to a preliminary reduction in intracellular nitrogen content (10). Fermentation runs from 3 to 6 days, with pH controlled at 4.5-6.5. Citric acid concentrations varied from 20 to -60 g/L,, depending on the strain used, the substrate (synthetic or byproduct), the fermentation system (surface culture or submerged fermentation), and the general conditions under which fermentation took place (initial sugar concentration, pH, and temperature). The yeast based process for citric acid production was maintained at neutral pH (5). Thus, the citrate produced was a sodium or calcium salt, which complicated recovery. In order to produce citric acid by yeast in industrial scale, further research is needed in selecting a high citrate producing strain and in optimizing its metabolic pathways as well as in understanding the best operating conditions for minimizing the production of isocitric acid (10).
Little information is available on the production of citrate by bacteria, as most of the references in the literature are to patents (3). Bacteria generally include Bacillus, Brevibacterium, Arthrobacter, Corynebacterium, Klebsiella, Aerobacter, Pseudomonas, and Micrococcus. Among these, B. subtilis, B. licheniformis, B. flavum, and A. paraffinens are the most promising (3,52). Kapoor et al. (3) reported that B. licheniformis grown in medium containing glucose, urea, calcium carbonate, and ammonium sulfate or glutamate (pH 7.0) produced 42.0 g/L of citric acid. Also, strains of A. paraffinens, Corynebacterium sp., and Bacillus sp. yielded maximum citric acid concentrations of 28-40 g/L when they were grown in media containing dodecane or a mixture of C12 -C14 paraffins (3). In all cases the fermentation was aerobic at 30-37°C for 2-5 days, depending on the strain and the composition of medium used. Generally, citric acid production by bacteria was 50-100% lower than that by fungi or yeasts. Nonetheless, these reports have opened a new avenue for the production of citric acid by bacteria.
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