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BiotechnologyAdvaacs,Vol. 13, No. 2, pp. 209--234, 1995 Copyright O 1995 Elsoviot 8 i ~ Ltd Printed in the US& All ri$hts reeerved 0734-9750/95 $29.00 + .00

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FUNGAL PRODUCTION OF CITRIC ACID H.S. G R E W A L and K.L KALRA Department of Microbiology, Punjab Agricultural University, Ludhiana-141 004, India ABSTRACT Citric acid is the principal organic acid found in citrus fruits. To meet increasing demands it is produced from carbohydrate feedstock by fermentation with the fungus Aspergillus niger and the yeasts of Candida spp. Effect of various fermentation conditions and the biochemistry of citric acid formation by A. niger have been discussed. Commercially citric acid is produced by surface, submerged and solid state fermentation techniques. Recovery of pure acid from fermentation broth is done primarily by precipitation with lime and also by solvent extraction. Key words: Aspergillus niger, citric acid, fermentation INTRODUCTION Citric acid (2-hydroxypropane-l,2,3-tricarboxylic acid) was first isolated and crystallized from lemon juice by Karl Wilhelm Scheele in 1784. This organic acid is found as a natural constituent of a variety of citrus fruits, pineapple, pear, peach and fig. Citrus fruits being a particularly rich source. Commercial production of citric acid commenced in England in 1860 from calcium citrate imported from

Italy. The discovery of formation of this acid by

fermentation can be traced back to 1893, when Whemer, the German Botanist, recognized it as a metabolite of the moulds Citromyces pfefferianis and C. glaber. His attempts to produce citric acid on commercial scale were unsuccessful. It was the work of Currie (25) that formed the basis of commercial citric acid production employing Aspergillus niger in a surface fermentation process. In 1950's an improved process employing submerged fermentation was developed in the United States. This process proved a turning point in the production of citric acid. Some of the major historical landmarks in the development of citric acid production have been reviewed (1, 121). Citric acid has a variety of uses in food, pharmaceuticals and industrial fields (36, Table 1). In view of the numerous applications of citric acid, its production by fermentation is increasing continually. About 400,000 tons of citric acid are produced annually by fermentation (94).
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H.S. OREWALandK. L. KALRA

Table 1. Applications of citric acid

Industry
Candies

Uses
Prevent crystallization of sucrose, produce dark colour in hard candies, inversion of sucrose. Acts as acidulant, adjusts pH to the range where pectin acts as gelling agent, provides the desired degree of tartness, tang and flavour. Adjusts pH to the desired levels and helps in setting of gelatin desserts. As acidulant in carbonated and sucrose based beverages, stimulates natural fruit flavour, incorporates tartness. Acts as stabilizer in commercially prepared juices of fruits and vegetables. Prevents turbidity of wines and ciders, prevents browning in some white wines, adjusts pH, inhibits oxidation. Neutralizes the residual lye, lowers pH to inactivate oxidative enzymes, protects ascorbic acid by inactivating trace metals. As emulsifier in ice creams and processed cheese, acidifying agent in many cheese products and as an antioxidant. Synergist for other antioxidants, as sequestrant. As effervescent in powders and tablets in combination with bicarbonates, solubilization action for cathartics, antioxidant in vitamin preparations, acidulant in mild astringent formulations, anticoagulant. For pH adjustment, antioxidant and buffering agent In electroplating, copper plating, metal cleaning, leather tanning, printing, inks, bottle washing compounds, floor cement, textiles, photographic reagents etc.

Jellies and jams

Gelatin desserts Soft drinks and syrups Fruits and vegetable juices Wines and ciders
Frozen fruits

Dairy products Animal fats and oils Pharmaceuticals

Cosmetics and toiletries Others

Citric acid production by A. niger is influenced by a number of culture parameters and, therefore, the aim of the present review is to discuss the fermentation conditions that govern citric acid production by this organism. The present review includes sections on micro-organisms, raw materials, fermentation conditions, biochemistry and product recovery. Several excellent reviews and articles on citric acid fermentation published earlier may also be consulted (1, 57, 74, 101, 121).

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MICRO-ORGANISMS
Over the years a large number of micro-organisms including fungi, yeasts and bacteria have been used for the production of citric acid. However, the fungus A. niger, first used by Currie (25), has remained the organism of choice for commercial production. The main advantages of using this organism are: (i) its ease of handling; (ii) its ability to ferment a wide variety of cheap raw materials; and (iii) high yields. A list of other micro-organisms capable of producing citric acid is given in Table 2.

Table 2. Microorganisms capable of producing citric acid

Fungi Aspergillus wentii (59)

Yeasts

A. carbona~ (111) A. aculeatus (111) A. awaraori Penicillium janthinellum

Saccharomycopsis lipo~tica (9, 88) (formerly Candida) Candida tropicalis (113) C. oleophila (53) C. guilliermondii (99) C. citroformans (144) Hansenula anamola (112)

Bacteria Bacillus licheniformis

(127)

Arthrobacter paraffinen~
(79)

Corynebaete~.an spp.
(34)

RAW MATERIALS A variety of raw materials such as molasses, starchy materials and hydrocarbons have been employed as substrates for commercial citric acid production. Hydrocarbons have served as substrate for citric acid production by yeasts. In view of availability and increasing price of hydrocarbons, the plants based on hydrocarbon substrates have switched over to carbohydrate feedstock. Molasses, a by-product of sugar industry, has remained a choice carbon source for citric acid production. Production from this raw material has been patented by many workers. Molasses has been acclaimed as a low cost raw material and it contains 40-55% of sugars in the form of sucrose, glucose and fructose. Both beet and cane molasses are used in the production of citric acid. The yield of citric acid produced in media containing beet molasses is higher than in media containing cane molasses because the latter has a high content of trace metals (calcium, magnesium, manganese, iron, zinc) which have a retarding effect on the synthesis of citric acid. As a result, cane

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molasses requires pretreatment for the reduction/removal of trace metals (18, 27, 35, 49, 77, 84, 85, 95, 126). Cane molasses also possess certain inhibitory substances which decrease the yield of citric acid (19, 27, 40, 41, 54, 55, 56, 111, 114, 124, 135). A wide variety of other raw materials have also been used for citric acid fermentation by a number of investigators (Table 3).

Table 3. Raw materials used for citric acid fermentation Raw material Cotton waste Turnip whey (enriched with molasses) Tubers ofAsphodelus aestivus Carob sugar Brewery waste Brewery waste Spent grain liquor Whey permeate Date syrup Wood hemicellulose Bagasse hydrolysates Cellulose hydrolysates Apple pomace Grape pomace Kiwi fruit peel Pineapple waste water Mandarin orange waste a - based on sugar consumed b - based on total sugars Type of fermentation Surface Surface Surface Surface Surface Submerged Submerged Submerged Submerged Submerged Submerged Solid state (SSF) SSF SSF SSF SSF SSF 88 a 60a 60a 50-60 b 55-65 b 60 a 40-60a 78.5 a 42-58a 63 a 19-33 a 60a 27-46a Yield (%) Reference 63 15 58 87 122, 123 42 38 51, 136 5 88 37 89 43, 44, 47 45 46 145 75

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CULTURE CONDITIONS There are considerable variations in culture conditions reported in the literature for citric acid production byA. niger. For attaining high productivity, it is essential that the medium contains major nutrients like carbon, nitrogen, phosphorous but also growth factors and trace metals. The fermentation process is influenced by aeration, temperature and pH. Effect of these factors on citric acid production is discussed below.

Carbon Source Production of citric acid from different carbon sources has been studied intensively and these sources have been shown to have marked effect on citric acid production byA. niger. Nature of sugars and their concentrations influence the yield of citric acid. A. niger can take up simple sugars such as glucose and fructose rapidly, although the usual carbon source used at industrial scale is sucrose which as such is not taken up by the fungus and the organism possesses an extracellular mycelial bound invertase which under acidic conditions of citric acid fermentation hydrolyses sucrose to its monomers (83). Hossain et

al. (52) reported that sucrose is the most favourable carbon source followed by glucose,
fructose and lactose. A. niger readily assimilates mannose, xylose and arabinose and produces citric acid from these sugars, although the yields were lower than from glucose (88). A mutant of A. niger yielded more citric acid form glucose followed by mannose, xylose and arabinose (116). Concentration of carbon source is critical to citric acid production, maximal production rate is usually achieved at 14-22% sugar concentration (132). Xu et al. (150) reported a sugar concentration of 10-14% as optimal; no citric acid was produced at sugar concentrations of less than 2.5% (151). A lower sugar concentration leads to lower yield of citric acid as well as accumulation of oxalic acid (65). Immobilized cells of A. niger need a lower initial sucrose concentration than free cells. High sucrose concentrations lead to a reduced yield and increased polyol formation (48).

Nitrogen Source
The nitrogen requirements for citric acid production by the fungus are generally met by the addition of a nitrogen source such as ammonium sulfate, ammonium nitrate, sodium nitrate, potassium nitrate, urea, etc. (30, 42, 55, 80, 89). The type and concentration of nitrogen source affect fungal growth and the synthesis of citric acid. Ammonium sulfate

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prolongs vegetative growth while ammonium nitrate favours a shorter period of vegetative growth. Ammonium nitrate (> 0.25%) leads to the accumulation of oxalic acid (39, 108). A high nitrogen concentration increases fungal growth and the consumption of sugars, but decreases the amount of citric acid produced (42, 116). Ammonium sulfate as nitrogen source results in increased production of citric acid without the formation of oxalic acid (146). Ammonium nitrate has been reported to be a better nitrogen source than ammonium carbonate (117, 118). A nitrogen limitation in continuous citric acid production has been reported (67). Phosphorous Source Besides carbon and nitrogen the presence of phosphate in the medium has a profound effect on the yield of citric acid. A higher concentration of phosphate promotes more growth and less acid production (60). Potassium dihydrogen phosphate (0.1%) has been reported to be the most suitable phosphorous source (55, 117). Addition of this salt (0.5 to 5.0 gL -1) had little effect on mycelial growth and the formation of citric acid from brewery waste (42). However, Shu and Johnson (133) reported that phosphorous concentration (0.5 to 5.0 gL -1) was required by the fungus in a chemically defined medium for maximum production of citric acid. The yield of citric acid produced byA.

niger is very

much retarded in a fermentation medium lacking potassium dihydrogen phosphate and dipotassium hydrogen phosphate (133). Influence of phosphate limitation on citric acid production by immobilized cells of A.

niger has been reported (48). It has been suggested

that phosphate ion might function in some manner other than as a simple nutrient and buffer (132). Phosphates act at the level of enzyme activity and not at the level of gene expression (70).

Fermentation pH
Maintenance of low pH is essential for maximum production of citric acid. The initial pH required depends on the carbon source used. Generally, pH below 2.0 is required for optimum production. A low initial pH has the advantage of checking contamination and inhibiting oxalic acid formation (130). At a pH of 4 or above, the formation of oxalic acid is accelerated due to high buffeting capacity of the medium (145). A pH of 2.2 has been reported to be optimum for the growth of the mould as well as for the production of citric acid (137) whereas, a higher pH i.e. 5.4 and 6.0-6.5 has been found optimum for citric acid

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production in molasses medium (55, 125). In a study of citric acid production from beet molasses in a continuous culture, Roukas and Harvey (124) found that at a pH value of 2.5, gluconic acid is the major product and citric acid being the predominant product at low pH. At higher pH, A. niger accumulates gluconic acid. This is brought about by extracellular partially mycelium bound glucose oxidase (102) which is inactivated below pH 2 (74).
Aeration

Citric acid fermentation is aerobic and the importance of oxygen has been emphasized by several studies (8, 21, 28, 29, 61, 72, 121, 129, 146). Increased aeration rates lead to enhanced yield and reduced fermentation time. Kovats and Gackowaska (66) reported that an interruption of aeration during batch fermentation adversely affected the process. A 20 minute interruption in aeration during idiophase did not reduce the viability of the organism but irreversibly destroyed its ability to produce and accumulate citric acid (72, 121). Critical dissolved oxygen tension (DOT) values of 9-10% of air saturation and 1213% of air saturation for growth and production phases, respectively, have been reported by Kubicek et aL (72) who also observed that production of citric acid steadily increased between DOT values of 20% and 75% saturation. Maximum citric acid yield and production rates were observed at higher DOT value (90% saturation) in fed batch culture (29). A considerable lowering of aeration demand occurs during diffused growth of citric acid producing A. niger in a submerged fermentation. The impeller speed affects considerably the morphology of the fungus and accumulation of citric acid (129). Trace Elements Trace element nutrition is probably the main factor influencing the yield of citric acid production. A number of divalent metals such as zinc, manganese, iron, copper and magnesium have been found to affect citric acid production by .4. niger (106, 123, 131, 132). When trace metals are growth limiting, citric acid accumulates in large quantities (20, 50, 126, 142, 143). Manganese strongly influence idiophase metabolism. In its presence, cell growth increases, sugar consumption diminishes and acidogenesis decreases drastically (88). Bowes and Mattey (12) reported that the presence of manganese in the medium inhibits citric acid accumulation. Manganese deficiency results in the repression of the

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H.S. GREWALmadK.L. KALRA

anaerobic and T C A cycle enzymes with the exception of citrate synthetase. This leads to an overflow of citric acid as an end product of glycolysis (68). Manganese at levels of 1 ppm reduced the yield of citric acid by 10% (20). Clark et al. (20) and Kisser et aL (64) proved the key regulatory role of manganese ions in citric acid fermentation. Citric acid accumulation decreased by the addition of iron which also had some effect on mycelial growth (73). Citric acid fermentation from molasses in the presence of ferrous ions (0.2 ppm) was affected and the addition of copper in the fermentation medium neutralized the deleterious effect of iron (32, 128). The effect of copper in counteracting the iron ions is due to its antagonistic effect on the uptake of manganese ions (138). Wold and Suzuki (148) reported that the concentration of zinc determines the course of citrate fermentation. At high zinc levels fungal cultures were maintained in growth phase and citric acid did not accumulate. At low zinc level growth became limited andA. niger passed into citric acid producing phase; addition of zinc to the acid-producing cultures resulted in their reversion to growth. Magnesium is required both for growth as well as for citric acid production. The optimum concentration of magnesium sulfate for maximum citric acid production varies from 0.02-0.025% (57). The effect of other trace elements as nickel, cobalt and molybdenum on citric acid biosynthesis by A. niger has been reported (147).

Lower Alcohols The use of alcohol in citric acid production was first reported by Moyer (104). He observed that methanol enhanced the formation of citric acid from commercial glucose and other crude carbohydrate sources. The optimum amount of methanol/ethanol depends upon the strain used and the composition of the medium; generally the optimum range being 1-3%. Stimulatory role of methanol or ethanol has been reported by a number of investigators (41, 42, 44, 46, 78, 89, 122). Manonmani and Sreekantiah (91) observed that addition of ethanol resulted in two fold increase in citrate synthetase activity and 75% decrease in the aconitase activity. The activities of other T C A cycle enzymes increased slightly. Increase in citric acid accumulation with the addition of ethanol might be due to slow degradation of citric acid consequent to reduction in aconitase activity (91). Bhat et aL (11) have stated that ethanol might be converted to acetyl-CoA which is required for citric acid formation. Ethanol also acts as carbon source which increases the inflow of carbon through the citric acid cycle.

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Methanol is not assimilated by A. niger and its exact role in stimulating the production of citric acid is not clear. Chaudhary et aL (19) reported that methanol helps in 'conditioning' of mycelia without impairing their metabolism. Presence of methanol might increase permeability of cell to citrate (57, 88).

Lipids
The supplementation of fermentation medium with lipid materials such as vegetable oils and fatty acids increases the citric acid production (76, 89, 99, 100). Mamonmani and Sreekantiah (90) reported that addition of vegetable oils influenced the citric acid yield with coconut oil (3%) being the best for maximum citric acid production. Fats and oils act as carbon sources as reported by Bhat et al. (11) and these are broken down to glycerol and fatty acids. The latter enters the TCA cycle via glycolysis, while the former enters directly by the formation of acetyl-CoA. Kumar and Ethiraj (76) reported that unsaturated oils like peanut oil have been observed to serve as alternate hydrogen acceptors in place of oxygen during fermentation. Mamonmani and Sreekantiah (90) suggested that coconut oil acts as additional source of acetyl-CoA, thus improving the yield.

Others

Addition of sodium monofluoroacetate to the fermentation medium increased the citric acid production (7, 89, 90). However, the results of Roukas and Kotzekidou (123) showed that addition of sodium monofluoroacetate to the brewery waste resulted in decreased yield of this acid. La Nauze (81) found that monofluoroacetate is toxic to the mould and does not stimulate citric acid yield. The effect of fluoroacetate in increasing citric acid accumulation has been observed to be an indirect effect with the specific inhibition of aconitase activity (103). Intu~oitors of metabolism such as calcium fluoride, sodium fluoride and potassium fluoride accelerate citric acid production (140), whereas, potassium ferrocyanide has been found to decrease the yield (7). Addition of mild oxidizing agents such as hydrogen peroxide, naphthaquinone and methylene blue to the fermentation medium increased the production of the acid (13). Kahlon (55) reported that organic metabolic inhibitors such as naphthol, resorcinol, cresol and benzaldehyde increase the citric acid production and suggested that these inhibitors might be affecting the activity of the enzymes aconitase and isocitrate dehydrogenase. Role of such compounds as sodium malonate, iodoacetate,

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H.S. GREWALa.udK. L. KALRA

sodium azidc, sodium arsenate, sodium fluoride and potassium ferricyanide in relation to citric acid production has also been reported (2). All et al. (4) found that addition of the insecticide 2,2-dichlorovinyl dimethyl phosphate (DDVP) delayed citric acid production and the utilization of inorganic phosphate by .4. niger. Addition of dithiocarbamates has been reported to enhance the production of citric acid under solid-substrate fermentation conditions (62).

INDUSTRIAL PROCESSES

The Surface Process Surface culture was the original production method employed for large scale manufacture of microbial citric acid. It was introduced in 1919 by Societe des Produits Organique in Belgium and in 1923 by Chas Pfizer and Co. in the United States. Despite the fact that more sophisticated fermentation methods (submerged process) have been developed, surface culture is still employed as it is simple to operate and install. The energy costs are lower than for the submerged process. The mycclium is grown as a surface mat in a large number of shallow trays/pans having a capacity of 50 to 100 L The trays are made of high purity aluminum or special grade steel and are stacked in stable racks in fermentation chambers. The chamber environment is almost aseptic. The carbohydrate sources which form fermentation media are refined or crude sucrose, high test cane syrup or beet molasses. Molasses, when used, is diluted to 15% sugars, the pH is adjusted to 5-7 and any required pretreatment is carried out. After the addition of the nutrients, the medium is sterilized, cooled and pumped into trays. Inoculation is done by introducing spores either by raising a spore suspension or by blowing spores over the surface of trays along with air. Spores germinate and form a mycelial mat. Generally a high number of spores is used for inoculation. The temperature is maintained at 28-300C and the relative humidity between 40-60%. During fermentation considerable heat is generated necessitating high aeration rates (74). Air provides oxygen to the organism and also controls fermentation temperature and relative humidity. Aeration requirement is considerably different in the germination and the production phases./~ novel two stage aeration system has been proposed to reduce aeration costs (98). As the fermentation progresses, the pH decreases to below 2.0. If

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during the fermentation pH rise to 3.0, oxalic acid and gluconic acid may be formed in considerable amounts. Fermentation period is 8-12 days. After completion of fermentation, the fermented liquor is poured out of the pans and separated from the mycelium for further processing. Fermentation yields are in the range of 70-75%.

The Submerged Fermentation Process This process in now popular for commercial production of citric acid. It requires less space, is less labour intensive and gives high production rate. Either the stirred tank reactor or the tower fermentors may be used. In view of the low pH level which develops during fermentation and the fact that citric acid is corrosive, the use of bioreactors made of acid resistant materials is desirable. Bioreactors used are, therefore, made of high grade steel. An other important consideration for bioreactors for citric acid production is the provision of an aeration system that can maintain a high dissolved oxygen level. With both types of bioreactors sterile air is sparged from the base, although additional inputs are often used with tower fermentors (92, 139). The influence of dissolved oxygen concentration on citric acid formation has been examined (72) and the dissolved oxygen levels are monitored by using electrodes. The high demand of oxygen hyA. niger is fulfilled by constructing appropriate aeration devices. The medium preparation in submerged fermentations involves appropriate dilution of molasses or other raw materials, pretreatment, addition of nutrients and sterilization in line or in the bioreactor. Inoculation is preformed either by adding a suspension of spores or of precultivated mycelia. When spores are used they need to be dispersed in the medium; therefore, addition of a surfactant is usually necessary. For precultivated mycelia the inoculum size is about 10% of the fresh medium. Air is sparged through the fermentation medium at a rate of 0.5 to 1.5 vvm throughout the fermentation; however, because of economic considerations, it is usually preferred to start with a low aeration rate (0.1 to 0.4 vvm). Considerable foaming may occur during fermentation. This is checked by mechanical foam breakers or chemical antifoam agents (10). Under optimal conditions fermentation is complete in 5 to 10 days depending upon the process specifics. Submerged fermentation can be performed by batch, continuous and fed batch modes, but generally it is carried out by the first mode. Process for continuous

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H.S. GREWALand K. L. KALRA

fermentation have been carried out at laboratory scale (67, 124) but commercially it has not been applied.

The Koji Process

The Koji process, developed in Japan, is the simplest process for production o f citric acid. This process is the solid state equivalent of surface fermentation. The raw materials used are sweet potato fibrous residues, rice or wheat bran and fruit wastes. The carbohydrate source is moistened with water to about 70% moisture, steamed for sterilization, placed in trays and inoculated by conidia of A. niger. The p H at the start of the fermentation is 5.5. The starch is hydrolysed by amylase produced by the fungus and subsequently converted to citric acid. The fermentation is complete in 4-5 days. The main problem of this process is the presence of trace elements which cannot be removed by standard methods. A number of reports on citric acid production from fruit pomace by solid state fermentation have been published (44, 45, 46, 75).

Production by Yeasts
Yeasts are also commercially employed for citric acid production from various carbon sources. A list of the yeasts capable of producing citric acid is given in Table 2. The main advantage of using yeasts in comparison with filamentous fungi are: (i) yeasts can tolerate high initial concentration of sugar; (ii) are insensitive to trace metals and can thus ferment crude carbon sources without any treatment; (iii) have a great potential for being used in continuous culture; and (iv) have a high fermentation rate. A number of patents and reports are available that describe the citric acid production from various carbohydrates with yeasts (9, 16, 17, 88, 99, 112). For commercial citric acid production by yeasts, tower fermentors having efficient cooling system are employed. Inoculum is prepared in a smaller fermentor and is subsequently transferred into the production fermentor. Temperature of the fermentation is maintained between 25-37C depending on the type of strain employed. The p H is generally >5.5, but can fall during fermentation. A continuous process for citric acid production using Candida cultured on cane molasses has been developed (99). A process for citric acid production from n-alkanes was first patented by Takeda Yakuhin Kogyo (141), but is no longer in operation because of the relatively expensive substrate.

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BIOCHEMISTRY OF CITRIC ACID PRODUCTION Lewis and Weinhouse (86) studied the formation of citric acid in A. niger using labelled acetate in the medium and suggested that citric acid is an overflow product formed by the faulty operation of the tricarboxylic acid (TEA) cycle. The biochemical reactions of the TCA cycle are well studied in fungi. The presence of TCA cycle enzymes in A. niger has been reported (14, 26, 69, 71, 96, 97, 105, 109, 110, 119). Two key enzymes that have been studied in detail in relation to citric acid fermentation are aconitase and isocitrate dehydrogenase. The activities of these enzymes have been shown to decrease to a very low level while the specific activity of the condensing enzymes increases during citric acid accumulation (81, 119, 120). Ramakrishnan and Martin (119) further indicated that isocitrate dehydrogenase was inhibited by citric acid and ferrocyanide. La Nauze (81) found that a 20-fold increase in the concentration of iron doubled the activity of aeonitase and citric acid accumulation decreased by 25%. Addition of potassium ferrocyanide to the broth directly inhibited aconitase and isocitrate dehydrogenase activities and stimulated citric acid accumulation. Nagashima and Usami (107) reported that aconitase activity was high in the lag phase and decreased rapidly in the stationary phase. The inhibition of aconitase by low levels of iron or by copper has been suggested as the reason for citrate accumulation (3, 109). While Kubicek and Rohr (73) reported that addition of Fe3+or Cu 2+ had no effect on the/n vivo activity of aconitase, these ions had a definite effect on citric acid formation. These workers concluded that inhibition of aconitase is not necessary for the citric acid fermentation. Effect of manganese on citric acid production has been discussed earlier in this review. The breakdown of isocitric acid is brought about by isocitrate dehydrogenase (TCA cycle) and isocitrate lyase (glyoxalate cycle). Isocitrate lyase is absent when citric acid is accumulating (68). Isocitrate dehydrogenase in A. niger is N A D H and NADPH specific and both the enzymes have been purified (14, 97). The NADH enzyme is cytoplasmic whereas, NADPH enzyme is located in the mitochondria and is inhibited by physiological concentrations of citrate (93). Mattey (93) reported that citric acid itself inhibits NADP specific mitochondrial isocitrate dehydrogenase of A. niger. The operation of this feed forward mechanism was considered to be responsible for citric acid accumulation. Although this explanation appears to be true, this mechanism cannot be responsible for the initiation of citric acid accumulation, as it would mean that all cultures of A. niger would accumulate high levels of citric acid. Also, the level of citrate in the early stages of

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H, s. GREWALand K. L. KALRA

growth are too low to achieve inhibition (97). However, this mechanism does not clearly explain the initiation of citric acid accumulation. The other mechanism which has been considered for citric acid formation is the regulation of a-ketogiutarate dehydrogenase. This enzyme has been characterized from

A. niger (96) and was identified as a regulatory point by enzyme activity measurements
(69); it is inhibited by physiological concentrations of oxaloacetate and NADH. It has been suggested that metabolism of glucose during initial stages of the process leads to an increase in cellular oxaloacetate levels (69) which decrease the catabolism of citrate at the ~t-ketoglutarate dehydrogenase and simultaneously increase the rate of citrate synthetase (31). The most probable mechanism which explains the initiation of citric acid formation has been proposed by Legisa and Mattey (82). On the basis of their observations they explained that accumulation of up to 1% glycerol by enlarged cells of A. niger is the cause of initial inhibition of NADP specific isocitrate dehydrogenase which leads to a build up of citrate via aconitase. Once the concentration of citrate reaches a critical value it brings about the feed-forward inhibition of NADPH specific isocitrate dehydrogenase despite the assimilation of glycerol from the medium. The condensing enzyme citrate synthetase brings about the biosynthesis of citric acid by the condensation of acetyl-CoA and oxaloacetic acid. This condensation of C 2 and C a compounds is the major route of citrate synthesis. Citrate synthetase has been shown to have little allosteric regulation (71). Oxaloacetic acid for citric acid formation is provided by the operation of citric acid cycle and by an anaplerotic reaction (22) for the high yield of citric acid. Condensation of carbon dioxide and pyruvate for the formation of oxaloacetie acid is carried out by pyruvate carboxylase (149); this enzyme has been isolated from A. niger (33). Once the level of citric acid in the cells is elevated, the acid has to be excreted. The excretion mechanism is not clear in A. niger. The overall success of citric acid production depends to a large extent on the regulation of the T C A cycle.

PRODUCT RECOVERY Recovery of citric acid from the fermented liquor directly by crystallization is not possible because it contains many unwanted materials which come from the raw material or from the autolysis of the microbial cells. For years, the precipitation method has been employed for the recovery of citric acid. In this method citric acid is precipitated as tricalcium citrate

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by the addition of lime to the fermented liquor. The precipitated tricalcium citrate is removed by filtration and washed several times with water. It is then treated with sulfuric acid in an acidualtor. The precipitate of calcium sulfate formed is filtered off. Mother liquor containing citric acid is treated with active carbon and passed through cation and anion exchangers. The liquor is concentrated by evaporation in vacuum at ca 40"C. Finally, crystallization is carried out in vacuum crystallizers at 20-25"C and crystals of citric acid monohydrate are formed. Solvent extraction is another method used for the recovery of citric acid. Use of 2butanol and tributyl phosphate for the extraction of citric acid have been reported (23, 24). The Food and Drug Administration of the United States recommended (6) a solvent extraction method using a mixture of n-octyl alcohol and tridodecylamine. The citric acid extracted by this method is suitable for use in food and drugs.

CONCLUSION AND FUTURE DEVELOPMENTS With a growing demand, the production of citric acid is expected to increase. The crude raw materials used in production, particularly cane molasses, require a pretreatment for removal of trace metals and this increases the cost of production. There is, thus, a need to develop microbial strains that can ferment untreated substrates. Presently, citric acid is produced by surface culture and submerged fermentation. Continuous culture techniques have been attempted but only at the laboratory scale. While the Aspergilli used in fermentations have been improved by mutation and selection, there is scope for further improvement using the recombinant DNA technology.

REFERENCES

A.Z.A. Abuzeid and M.A. Ashy, Production of citric acid: A review, Agr/c. Wastes, 9, 51-76 (1984). 2. P.IC Aggarwal, C.S. Bhat and L Vishwanathan, Effect of some metabolic inhibitors on citric acid production by Aspergillus niger, Enz. Microbial. TechnoL, 5, 369-372 (1983). 3. S.A. Ahmed, J.E. Smith and J.A. Anderson, Mitochondrial activity during citric acid production by Aspergillus niger, Trans. Br. MycoL Soc., 59, 51-61 (1972).

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4.

M.S. Ali, M. Rahmutullah, R. Jahan, H.K.M. Yusuf and A.,A_ Chaudhary, Effect of DDVP insecticide on citric acid fermentation in A. niger, Enz. Microbial.

Technol., 1, 127-128 (1979).

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