An Overview of Citric acid Abstract: Citric acid ,a colourless organic compound belonging to the family of carboxylic acids present

practically in all plants and in many animal tissues and fluids. It is one of a series of compounds involved in the physiological oxidation of fats ,proteins, and carbohydrates to carbondioxide and water. Citric acid is manufactured by fermentation of cane sugar and molasses in the pressence of fungus ASPERGILLUS NIGER.It is used in confections and soft drinks(as a flavouring agent)in metal cleaning compositions and in improving the stability of foods and organic substances(by suppressing the deleterious action of dissolved metal salts).Current process techniques (fermentation--solid-state fermentation,submerged

fermentation)and current recovery methods (precipitation, filtration, solvent etraction, Adsorption, Absorption). This paper reviews recent developments on citric acid production by presenting a brief summary of the subject, describing micro-organisms, production techniques, and substrates, etc.

Key words: citric acid, Aspergillus niger, fermentation , solid-state fermentation, submerged fermentation, precipitation, filtration , solvent extraction, adsorption, absorption.

INTRODUCTION Citric acid is a weak organic acid with the formula C6H8O7. It is a natural preservative /conservative and is also used to add an acidic or sour taste to foods and drinks. In biochemistry, the conjugate base of citric acid(E-330), citrate, is important as an intermediate in the citric acid cycle, which occurs in the metabolism of all aerobic organisms. It consists of 3 carboxyl (RCOOH) groups.

Chemical structure Global production (www.niir.org There is a great worldwide demand for citric acid consumption due to its low toxicity when compared with other acidulants used mainly in the pharmaceutical and food industries. Other applications of citric acid can be found in detergents and cleaning products, cosmetics and toiletries, and other. Global production has now reached 1.4 million tonnes . The Indian demand is around 37000tonnes. The present import of citric acid is around 35000 tonnes. The projected demand for citric acid according to analyst is around 44000tonnes by the end of 2013 to 2014. The global production is around 1.75 million metric tonnes per annum and there is annual growth of 3.5-4.0 % in demand/consumption of citric acid.Taking into account the present demand supply scenario for domestic and global, price trends, new capacities can be created by entrepreneurs.Citric acid is a commodity chemical, and more than a million tonnes are produced every year by fermentation. It is used mainly as an acidifier, as a flavoring, and as a chelating agent.At room temperature, citric acid is a white crystalline powder. It can exist either in an anhydrous (water-free) form or as a monohydrate. The anhydrous form crystallizes from hot water, while the monohydrate forms when citric acid is crystallized from cold water. The monohydrate can be converted to the anhydrous form by heating above 78 C.

Citric acid also dissolves in absolute (anhydrous) ethanol (76 parts of citric acid per 100 parts of ethanol) at 15 C.In chemical structure, citric acid shares the properties of other carboxylic acids. When heated above 175 C, it decomposes through the loss of carbon dioxide and water (see decarboxylation).Citric acid is a slightly stronger acid than typical carboxylic acids because the anion can be stabilized by intramolecular hydrogen-bonding from other protic groups on citric acid. Natural Citric Acid - Sources & Benefits : Citric acid is a weak organic acid. Since it is a natural preservative, it used in preparing food, as well as adding taste to the food. Apart from being used in edible food stuffs, this organic acid also works well as a cleaning agent. It is also used in the production of dyes. For humans, citric acid plays an important role in the regulating the size of calcium crystals in the bone. It also keeps a check on the rate of glycolysis. Therefore is important that we include the right amount of this acid in our diet to keep our bones strong. Check out some of the natural citric acid sources: Fruits: Limes, lemon and oranges contain the highest amount of natural citric acid. This gives a tangy and sour taste to these fruits. The juices of these fruits are used as natural preservatives, thus not causing any side-effects to the body. Berries: Almost all types of berries such as cranberry, strawberry, raspberry, gooseberries, cherries etc. contain slight amounts of natural citric acid within them. Other Fruits: Tamarind and pineapples are other fruits that consist of this organic acid. Consuming pineapple juice helps in reducing mucus in the respiratory system. Vegetables: Lettuce and tomatoes are a rich source of natural citric acid. These are some of the natural citric acid sources. Let's have a look at the benefits of this acid on our health. Citric acid is helpful in naturally breaking down kidney stones and balance the acid content in urine.During illness, consumption of juices that contain this acid, helps in controlling bowel movements as well as preventing constipation.

One form of the acid, i.e. citrated calcium carbamide is sometimes used to treat alcoholic patients.Potassium citrate, another form of this acid is injected in the body to reduce the heart's contractions, thus reducing the difficulty of performing an open-heart surgery. Note: Citric acid may have adverse effects on people suffering from heartburn. Micro Organisms used for Citric Acid Production: ( Aboud-Zeid, A., and Ashy, M. A. (1984) Production of citric acid: A review. Agric. Wastes, 9, 51-7) Micro organisms Fungi Aspergillus niger References Hang & Woodams 1984, 1985, 1987, Roukas 1991, Garg & Hang 1995, Lu et al. 1997, Pintado et al. 1998, Vandenberghe et al., 1999b,c El Dein & Emaish, 1979 Grewal & Kalra, 1995 El Dein & Emaish, 1979 Karow & Waksman, 1947 Chen, 1994; Tran et al., 1998 Grewal & Kalra, 1995 Ikeno et al., 1975; Maddox et al., 1985; Kautola et al., 1992 Wojtatowicz et al., 1993; Rane & Sims, 1993 Kapelli et al., 1978 Ishi et al., 1972 Miall & Parker, 1975; Gutierrez et al., 1993 Omar & Parker, 1980 Uchio et al., 1975 Oh et al., 1973 Sardinas, 1972 Kroya fermentation Industry, 1970 Fukuda et al., 1970

A. aculeatus A. awamori A. Carbonarius A. wintii A.foetidus Penicillium janthinelum Yeasts Saccahromicopsis Lipolytica Candida tropicalis C. oleophila C. guilliermondii C. parapsilosis C. citroformans Hansenula anamola Bacteria Bacillus Arthrobacter paraffinens Corynebacterium sp.

Different strains: Aspergillus foetidus ACM 3996 (=FRR 3558) Three strains of Aspergillus niger ACM 4992 (=ATCC 9142), ACM 4993 (=ATCC 10577),

ACM 4994 (=ATCC 12846) were used for the production of citric acid.

A. niger ACM 4992 produced the highest amount of citric acid, with a yield of 19.4g of citric acid per 100g of dry fermented pineapple waste under optimum conditions, representing a yield of 0.74g citric acid/g sugar consumed. Optimal conditions were 65% (w/w) initial moisture content, 3% (v/w) methanol, 30C, an unadjusted initial pH of 3.4, a particle size of 2mm and 5ppm Fe2+. Citric acid production was best in flasks, with lower yields being obtained in tray and rotating drum bioreactors.

Aspergillus niger A. niger is a fungus and one of the most common species of the genus Aspergillus. It causes a disease called black mold on certain fruits and vegetables such as grapes, onions, and peanuts, and is a common contaminant of food. It is ubiquitous in soil and is commonly reported from indoor environments, where its black colonies can be confused with those of Stachybotrys (species of which have also been called "black mould").Some strains of A. niger have been reported to produce potent mycotoxins called ochratoxins other sources disagree, claiming this report is based upon misidentification of the fungal species. Recent evidence suggests some true A. niger strains do produce ochratoxin A. It also produces the isoflavone orobol. Abarca M, Bragulat M, Castell G, Cabaes F (1994). Ochratoxin A production by strains of Aspergillus niger var. niger. Appl Environ Microbiol 60 (7):2650 2. PMC 201698. PMID 8074536. Corynebacterium Nonpathogenic species of Corynebacterium are used for very important industrial applications, such as the production of amino acids, nucleotides, and other nutritional factors (Martn, 1989); bioconversion of steroids; degradation of hydrocarbons cheese aging and production of enzymes (Khurana et al., 2000). Some species produce metabolites similar to antibiotics:bacteriocins of the corynecin-linocin type antitumor agents etc. One of the most studied species is C. glutamicum, whose name refers to its capacity to produce glutamic acid in

aerobic conditions. It is used in the foods industry as monosodium glutamate (MSG) in the production of soy sauce and yogurt. Collins MD, Hoyles L, Foster G, Falsen E (May 2004). "Corynebacterium caspium sp. nov., from a Caspian seal (Phoca caspica)". Int. J. Syst. Evol. Microbiol. 54 (Pt 3): 925 8.doi:10.1099/ijs.0.02950-0. PMID 15143043 Lactobacillus Lactobacillus delbrueckii subsp. bulgaricus is commonly used alongside Streptococcus thermophilus[1] as a starter for making yogurt. The two species work in synergy, with L.d. bulgaricus producing amino acids from milk proteins, which are then used by S. thermophilus.[1] Both species produce lactic acid, which gives yogurt its tart flavor and acts as a preservative. The resulting decrease in pH also partially coagulates the milk proteins, such as casein, resulting in yogurt's thickness. While fermenting milk, L.d. bulgaricus produces acetaldehyde, one of the main yogurt aroma components.[3]Some strains of L.d. bulgaricus also produce bacteriocins, which have been shown to kill undesired bacteria in vitro.It is often helpful to sufferers of lactose intolerance,whose digestive systems lack the enzymes to break down lactose to simpler sugars. Lactobacillus delbrueckii subsp. bulgaricus (until 1984 known as Lactobacillus bulgaricus) It is one of several bacteria used for the production ofyogurt. It is also found in other naturally fermented products. First identified in 1905 by the Bulgarian doctor Stamen Grigorov, the bacterium feeds onlactose to produce lactic acid, which is used to preserve milk. It is a Grampositive rod that may appear long and filamentous. It is non-motile and does not form spores. It is regarded as aciduric or acidophilic, since it requires a low pH (around 5.44.6) to grow effectively. The bacterium has complex nutritional requirements. Courtin, P.; Rul, F. O. (2003). "Interactions between microorganisms in a simple ecosystem: yogurt bacteria as a study model

Production methods FERMENTATION:

Solid State Fermentation Submerged Fermentation Fermentation is a metabolic process that converts sugar to acids, gases and/or alcohol. It occurs in yeast and bacteria, but also in oxygen-starved muscle cells, as in the case of lactic acid fermentation. Fermentation is also used more broadly to refer to the bulk growth of microorganisms on a growth. French microbiologist Louis Pasteur is often remembered for his insights into fermentation and its microbial causes. The science of fermentation is known as zymology.Fermentation takes place in the absence of oxygen (when the electron transport chain is unusable) and becomes the cells primary means of ATP(energy) production.[1] It turns NADH and pyruvate produced in the glycolysis step into NAD+ and various small

molecules (see examples below). In the presence of O2, NADH and pyruvate are used in respiration; this is oxidative phosphorylation, it generates a lot more ATP in addition to that created by glycolysis, and for that reason cells generally benefit from avoiding fermentation when oxygen is available. Exceptions include obligate anaerobes, which cannot tolerate oxygen. Solid state fermentation: It is a biomolecule manufacturing process used in the food, pharmaceutical, cosmetic, fuel and textile industries. These biomolecules are

mostly metabolitesgenerated by microorganisms grown on a solid support selected for this purpose. This technology for the culture of microorganisms is an alternative to liquid or submerged fermentation, used predominantly for industrial purposes. [Pandey, A. (March 2003). "Solid-state fermentation". Biochem. Eng. J. 13 (23): 8184]

History Solid state fermentation has existed for several centuries. In Asia and Japan it is referred to as "Koji" fermentation.

Processes This process consists of depositing a solid culture substrate, such as rice or wheat bran, on flatbeds after seeding it with microorganisms; the substrate is then left in a temperaturecontrolled room for several days.Liquid state fermentation is performed in tanks, which can reach 1,001 to 2,500 square metres (10,770 to 26,910 sq ft) at an industrial scale. Liquid culture is ideal for the growing of unicellular organisms such as bacteria or yeasts.To achieve liquid aerobic fermentation, it is necessary to constantly supply the microorganism with oxygen, which is generally done via stirring the fermentation media. Accurately managing the synthesis of the desired metabolites requires regulating temperature, soluble oxygen, ionic strength and pH and control nutrients.Applying this growing technique to filamentous fungi leads to difficulties. The fungus develops in its vegetative form, generating hyphae or multicellular ramous filaments, while a septum separates the cells. As this mycelium develops in a liquid environment, it generates abundant viscosity in the growing medium, reducing oxygen solubility, while stirring disrupts the cell network increasing cell mortality. In nature, filamentous fungi grow on the ground, decomposing vegetal compounds under naturally ventilated conditions. Therefore, solid state fermentation enables the optimal development of filamentous fungi, allowing the mycelium to spread on the surface of solid compounds among which air can flow.Solid state fermentation uses culture substrates with low water levels (reduced water activity), which is particularly appropriate for mould. The methods used to grow filamentous fungi using solid state fermentation allow the best reproduction of their natural environment. The medium is saturated with water but little of it is free-flowing. The solid medium comprises both the substrate and the solid support on which the fermentation takes place. The substrate used is generally composed of vegetal byproducts such as beet pulp or wheat bran. At the beginning of the growth process, the substrates and solid culture compounds are non-soluble compounds composed of very large, biochemically complex molecules that the fungus will cut off to get essential C and N nutrients. To develop its natural substrate, the fungal organism sets forth its entire genetic potential to produce the metabolites necessary for its growth. The composition of the growth medium guides the microorganism's metabolism towards the production of enzymes that release bio-available single molecules such as sugars or amino acids by carving out macromolecules. Therefore, when selecting the components of the growth medium it is possible to guide the cells towards the production of the desired metabolite(s),

mainly enzymes that transform polymers (cellulose, hemicellulose, pectins, proteins) into single moieties in a very efficient and cost-effective manner. Compared to submerged fermentation processes, solid state fermentation is more costeffective: smaller vessels, lower water consumption, reduced wastewater treatment costs and lower energy consumption (no need to heat up water, poor mechanical energy input due to smooth stirring). Cultivating on heterogeneous substrates requires expertise to maintain optimal growth conditions. Air flow monitoring is key because it impacts temperature, oxygen supply and moisture. In order to maintain sufficient moisture content for the growth of filamentous fungus, waterlogged air is used and may require further addition of water. In most cases, solid state fermentation does not require a completely sterile environment as the initial sterilization of the fermentation substrate associated with the rapid colonization of the substrate by the fungous microorganism limits the development of the autochthonous flora. Submerged Fermentation: Submerged fermentation is the cultivation of microorganisms in liquid nutrient broth. Industrialenzymes can be produced using this process. This involves growing carefully selected microorganisms (bacteria and fungi) in closed vessels containing a rich broth of nutrients (thefermentation medium) and a high concentration of oxygen. As the microorganisms break down the nutrients, they release the desired enzymes into solution. Due to the development of large-scale fermentation technologies, the production of microbial enzymes accounts for a significant proportion of the biotechnology industrys total output. Fermentation takes place in large vessels(fermenter) with volumes of up to 1,000 cubic metres. The fermentation media sterilizes nutrients based on renewable raw materials like maize, sugars and soya. Most industrial enzymes are secreted by microorganisms into the fermentation medium in order to break down the carbon and nitrogen sources. Batch-fed and continuous fermentation processes are common. In the batch-fed process, sterilised nutrients are added to the fermenter during the growth of the biomass. In the continuous process, sterilised liquid nutrients are fed into the fermenter at thesame flow rate as the fermentation broth leaving the system. This will achieve a steadystate production. Parameters like temperature, pH, oxygen consumption and carbon dioxide formationare measured and controlled to optimise the fermentation process. Firstly, in harvesting enzymes from the fermentation medium one must remove insoluble products,

e.g. microbial cells. This is normally done by centrifugation. As most industrial enzymes are extracellular (secreted by cells into the external environment), they remain in the fermented broth after the biomass has been removed. The biomass can be recycled as a fertiliser, but first it must be treated with lime to inactivate the microorganisms and stabilise it during storage. The enzymes in the remaining broth are then concentrated by evaporation, membrane filtration or crystallization depending on their intended application. If pure enzyme preparations are required, they are usually isolated by gel or ion exchange chromatography. Certain applications require solid enzyme products, so the crude powder enzymes are made into granules to make them more convenient to use. Sometimes liquid formulations are preferred because they are easier to handle and dose along with other liquidingredients.( SC Prescott, CG Dunn - Industrial microbiology, 1949 - cabdirect.org) Factors Affecting Citric Acid Production (Vandenberghe, L. P. S., Soccol, C. R. Pandey, A. and Lebeault, J.-M. (1999c) Solidstate fermentation for synthesis of citric acid by Aspergillus niger. Biores. Technol. (in press) Medium and its components Carbon source: Citric acid accumulation is strongly affected by the nature of the carbon source. The presence of easily metabolized carbohydrates has been found essential for good production of citric acid. Hossain et al. (1984) showed that sucrose was the most favourable carbon source followed by glucose, fructose and galactose. Galactose contributed to a very low growth of fungi and did not favour citric acid accumulation.Other sources of carbon such as sorbose, ethanol, cellulose, manitol, lactic, malic and a -acetoglutaric acid, allow a limited growth and low production. Starch, pentoses (xyloses and arabinoses), sorbitol and pyruvic acid slow down growth, though the production is minimal (Yokoya, 1992).According to Kovats (1960), initial sugar concentration was critical for citric acid production and other organic acids produced by A. niger. Xu et al. (1989) reported that A.niger strains needed an initial sugar concentration of 1014% as optimal; no citric acid was produced at sugar concentration of less than 2.5%. Honecker et al. (1989) showed that immobilized cells of A.niger needed lower concentrations of sucrose

than free cells culture, in order to obtain high yields (200 g of citric acid/L for free cells culture, and 120 g/L for immobilized cells). Maddox et al. (1985) reported the influence of different sources of carbon on citric acid production by A. niger andSaccharomycopsis lipolytica. Glucose, maltose, galactose, xylose and arabinose were tested. Fermentation was carried out in 8 and 4 days, respectively, at 30C and 180 rpm. Better results were found for A. niger with 0.45 g of citric acid/ g of glucose corresponding to 27 g/L. S. lipolytica produced 0.41 g/g of glucose or 9 g/L which was not so bad.As presented previously, several raw materials can be employed successfully for citric acid production. There are some critical factors (costs, need of pretreatment), which should be considered for substrate determination. One another aspect is the presence of trace elements, which can act as inhibitors or stimulants. Consequently, sometimes it is necessary to conduce a pre-treatment, e.g.; precipitation of trace metals of molasses by potassium ferrocyanide. Nitrogen source: Citric acid production is directly influenced by the nitrogen source. Physiologically, ammonium salts are preferred, e.g. urea, ammonium sulfate, ammonium chlorure, peptone, malt extract, etc. Nitrogen consumption leads to pH decrease, which is very important point in citric acid fermentation (Rohr et al., 1983, Kubicek and Rohr, 1986). However, it is necessary to maintain pH values in the first day of fermentation prior to a certain quantity biomass production. Urea has a tampon effect, which assures pH control (Raimbault, 1980). The concentration of nitrogen source required for citric acid fermentation is 0.1 to 0.4 N /liter. A high nitrogen concentration increases fungal growth and the consumption of sugars, but decreases the amount of citric acid produced (Hang et al., 1977). Phosphorous source: Presence of phosphate in the medium has a great effect on the yield of citric acid. Potassium dihydrogen phosphate has been reported to be the most suitable phosphorous source. Shu and Johnson (1948) reported that phosphorous at concentration of 0.5 to 5.0 g/L was required by the fungus in a chemically defined medium for maximum production of citric acid. Phosphate is known to be essential for the growth and metabolism of A. niger (Shankaranand and Lonsane, 1994). Low levels of phosphate favour citric acid production, however, the presence of excess of phosphate was shown to lead to the formation of certain sugar acids, a decrease in the fixation of CO2, and the stimulation of growth. Phosphates acts at the level of enzyme activity and not at the level of gene expression (Kubicek et al., 1979). It is

interesting to note that different strains require distinct nitrogen and phosphorous concentrations in the medium. In fact, nitrogen and phosphorous limitation is a crucial factor in citric acid production as there is an interaction between them. Consequently, the study of their combined effect is necessary (Pintado et al., 1993; Chen, 1994). Pintado et al. (1998) reported how the culturing modality conditions the behavior of the micro-organisms referring to the tendencies of production as a function of the levels of N and P. The author used as first order an empirical model based on rotatable design to study the effect of both nutrients. As expected, for the two studied strains, a similar behavior was noticed, showing an improvement towards low levels of N and P in submerged culture, and toward high levels in solid state culture, and with superior productions for the last one. Shankaranand and Lonsane (1994) affirmed that the specificity of solid state culture is largely due to a lower diffusion rate of nutrients and metabolites, which occurs in low water activity conditions. Consequently, strains with large requirements of N and P seems to be disfavored, due to the restriction of accessibility to the nutrients in the medium. Trace elements: Trace element nutrition is probably the main factor influencing the yield of citric acid. A number of divalent metals such as zinc, manganese, iron, copper and magnesium have been found to affect citric acid production by A. niger. However, it is crucial to take into account the interdependence of medium constituents in SmF and, probably, in SSF. Zinc favoured the production of citric acid if added with KH2PO4. On the other hand, the presence of manganese ions and iron and zinc (in high concentrations) could cause the reduction of citric acid yields only in phosphate free medium. Shankaranand and Lonsane (1994) noticed that there were few differences in the response of A. niger to metal ions and minerals in SSF and in SmF systems. SSF systems were able to overcome the adverse effects of the high concentrations of these components in the medium. As a consequence of this, the addition of chelating agents such as potassium ferrocyanide to the medium proved to be of no use. Copper was found to complement the ability of iron at optimum level, to enhance the biosynthesis of citric acid. Manganese deficiency resulted in the repression of the anaerobic and TCA cycle enzymes with the exception of citrate synthetase. This led to overflow of citric acid as an end product of glycolysis (Kubicek and Rohr, 1978). A low level of manganese (ppm) was capable to reduce the yield of citric acid by 10%. Citric acid accumulation decreased by the addition of iron, which also had some effect on mycelial growth. Benuzzi and Segovia (1996)

reported that the presence of different copper concentrations in the pellet formation medium was very important in order to enhance a suitable structure, related to cellular physiology, for citric acid production. The optimal initial CuSO4.5H2O concentration was 78 mg/L.Magnesium is required both for growth as well as for citric acid production. Optimal concentration of magnesium sulfate was found in the range of 0.02-0.025% (Kapoor et al., 1983). Lower alcohols: Addition of lower alcohols enhances citric acid production from commercial glucose and other crude carbohydrate. Appropriate alcohols are methanol, ethanol, iso-propanol or methyl acetate. The optimal amount of methanol/ethanol depends upon the strain and the composition of the medium, generally optimum range being 1-3%. The effect of methanol or ethanol have been extensively studied by many authors (Hamissa, 1978; Mannomani and Sreekantiah, 1988; Georgieva et al., 1992; Dasgupta et al., 1994).Mannomani and Sreekantiah (1987) reported that addition of ethanol resulted in two-fold increase in citrate synthetase activity and 75% decrease in aconitase activity. Whereas the activities of other TCA cycle enzymes increased slightly. They also found that coconut oil influenced citric acid production in a sucrose medium when added at 3% (v/w). Alcohols have been shown to principally act on membrane permeability in micro-organisms by affecting phospholipid composition on the cytoplasmatic membrane (Orthofer et al., 1979). However Meixner et al. (1985) argued against a role of membrane permeability in citric acid accumulation. Ingram and Buttke (1984) found that alcohols stimulate citric acid production by affecting growth and sporulation through the action not only on the cell permeability but also the spatial organization of the membrane, or changes in lipid composition of the cell wall. Miscellaneous: Some compounds which are inhibitors of metabolism such as calcium fluoride, sodium fluoride and potassium fluoride have been found to accelerate the citric acid production, while, potassium ferrocyanide has been found to decrease the yield. There are many compounds, which act in many ways to favour citric acid accumulation. Some of them are capable to impair the action of metal ions and other toxic compounds influence growth during the initial phase. Some of these are: 4-Methyl-umbelliferone, 3-hydroxi-2-naphtoic, benzoic acid, 2-naphtoic acid, iron cyanide, quaternary ammonium compounds, amine oximes, starch, EDTA, vermiculite, etc. Process parameters

pH: The pH of a culture may change in response to microbial metabolic activities. The most obvious reason is the secretion of organic acids such as citric, acetic or lactic acids, which will cause the pH to decrease. Changes in pH kinetics depend highly also on the micro-organism. With Aspergillus sp., Penicillium sp. and Rhizopus sp., pH can drop very quickly until less than 3.0. For other groups of fungi such as Trichoderma, Sporotrichum, Pleurotussp., pH is more stable (between 4 and 5). Besides, the nature of the substrate also influences pH kinetics (Raimbault et al., 1997).Generally, a pH below 2.0 is required for optimum production of citric acid. A low initial pH has the advantage of checking contamination and inhibiting oxalic acid formation. A pH of 2.2 was reported to be optimum for the growth of the mould as well as for the production of citric acid (Srivastava and De, 1980) whereas, a higher pH i.e. 5.4 and 6.0-6.5 has been found optimum for citric acid production in molasses medium (Roukosu and Anenih, 1980). Aeration: Aeration has been shown to have a determinant effect on citric acid fermentation (Rohr et al., 1983; Dawson et al., 1986). Increased aeration rates led to enhanced yields and reduced fermentation time (Grewal and Kalra, 1995).The influence of dissolved oxygen concentration on citric acid formation has been examined. It is important to maintain the oxygen concentration above 25% saturation and interruptions in oxygen supply may be quite harmful (Kubicek et al., 1980). The high demand of oxygen is fulfilled by constructing appropriate aeration devices, which is also dependent on the viscosity of the fermentation broth. This is an additional reason why small compact pellets are the preferred mycelial forms of A. niger during fermentation (Kubicek and Rohr, 1986). When the organism turns into filamentous developments, e.g. due to metal contamination, the dissolved oxygen tension rapidly falls to less than 50% of its previous value, even if the dry weight has not increased by more than 5%. Aeration is performed during the whole fermentation with the same intensity through the medium at a rate of 0.5 to 1.5 vvm. However, because of economic reasons, it's usually preferred to start with a low aeration rate (0.1 to 0.4 vvm). High aeration rates lead to high amounts of foam, especially during the growth phase. Therefore, the addition of antifoaming agents and the construction of mechanical "defoamers" are required to tackle this problem.

Product Recovery: The recovery of citric acid from liquid fermentation is generally accomplished by three basic procedures, precipitation, extraction, and adsorption and absorption (mainly using ion exchange resins). Citric acid extraction has been described by the Food and Drug Administration (1975) of the United States and by Colin (1960,1962). Citric acid extracted by this method has been recommended suitable for use in food and drugs. Precipitation is the classical method and it is performed by the addition of calcium oxide hydrate (milk of lime) to form the slightly soluble tri-calcium citrate tetrahydrate. The precipitated tri-calcium citrate is removed by filtration and washed several times with water. It is then treated with sulphuric acid forming calcium sulphate, which is filtered off. Mother liquor containing citric acid is treated with active carbon and passed through cation and anion exchangers. Several anion-exchange resins are commercially available. Finally, the liquor is concentrated in vacuum crystallizers at 20-25C, forming citric acid monohydrate. Crystalization at temperatures higher to this is used to prepare anhydrous citric acid.

Recovery Processes: Precipitation Filtration Solvent extraction Adsorption Absorption Gas chromatography High pressure Liquid Chromatography Thin layer chromatography

Precipitation is the formation of a solid in a solution or inside another solid during a chemical reaction or by diffusion in a solid. When the reaction occurs in a liquid solution, the solid formed is called the precipitate. The chemical that causes the solid to form is called the precipitant. Without sufficient force of gravity (settling) to bring the solid particles together, the precipitate remains in suspension. After sedimentation, especially when using a centrifuge to press it into a compact mass, the precipitate may be referred to as a pellet. The precipitate-free liquid remaining above the solid is called the supernate or supernatant. Powders derived from precipitation have also historically been known as flowers.Precipitation may occur if the concentration of a compound exceeds its solubility (such as when mixing solvents or changing their temperature). Precipitation may occur rapidly from a supersaturated solution.In solids, precipitation occurs if the concentration of one solid is above the solubility limit in the host solid, due to e.g. rapid quenching or ion implantation, and the temperature is high enough that diffusion can lead to segregation into precipitates. Precipitation in solids is routinely used to synthesize nanoclusters.[1]

An important stage of the precipitation process is the onset of nucleation. The creation of a hypothetical solid particle includes the formation of aninterface, which requires

some energy based on the relative surface energy of the solid and the solution. If this energy is not available, and no suitable nucleation surface is available, supersaturation occurs.Precipitation reactions can be used for making pigments, removing salts from water in water treatment, and in classical qualitative inorganic analysis.Precipitation is also useful to isolate the products of a reaction during workup. Ideally, the product of the reaction is insoluble in the reaction solvent. Thus, it precipitates as it is formed, preferably forming pure crystals. An example of this would be the synthesis of porphyrins in refluxing propionic acid. By cooling the reaction mixture to room temperature, crystals of the porphyrin precipitate, and are collected by filtration:[2]

Precipitation may also occur when an antisolvent (a solvent in which the product is insoluble) is added, drastically reducing the solubility of the desired product. Thereafter, the precipitate may easily be separated by filtration, decanting, or centrifugation). An example would be the synthesis of chromic tetraphenylporphyrin chloride: water is added to the DMF reaction solution, and the product precipitates.[3] Precipitation is also useful in purifying products: crude bmim-Cl is taken up in acetonitrile, and dropped into ethyl acetate, where it precipitates.[4] Another important application of an antisolvent is in ethanol

precipitation of DNA. (Zumdahl, Steven S. (2005). Chemical Principles (5th ed.))

Filtration : (Article on "Water treatment solution: Filtration", retrieved on the 15th October 2013 from http://www.lenntech.com/chemistry/filtration.htm) It is commonly the mechanical or physical operation which is used for the separation of solids from fluids (liquids or gases) by interposing a medium through which only the fluid can pass. The fluid that pass through is called a filtrate. Oversize solids in the fluid are retained, but the separation is not complete; solids will be contaminated with some fluid and filtrate will contain fine particles (depending on the pore size and filter thickness). Filtration is also used to describe some biological processes, especially in water treatment and sewage treatment in which undesirable constituents are removed by absorption into a biological film grown on or in the filter medium as in slow sand filtration.

Methods There are many different methods of filtration; all aim to attain the separation of substances. Separation is achieved by some form of interaction between the substance or objects to be removed and the filter. The substance that is to pass through the filter must be a fluid, i.e. a liquid or gas. Methods of filtration vary depending on the location of the targeted material, i.e. whether it is dissolved in the fluid phase or suspended as a solid. Filter media Two main types of filter media are employed in any chemical laboratory surface filter, a solid sieve which traps the solid particles, with or without the aid of filter paper (e.g. Bchner funnel, Belt filter, Rotary vacuum-drum filter, Cross-flow filters, Screen filter), and a depth filter, a bed of granular material which retains the solid particles as it passes (e.g. sand filter). The first type allows the solid particles, i.e. the residue, to be collected intact; the second type does not permit this. However, the second type is less prone to clogging due to the greater surface area where the particles can be trapped. Also, when the solid particles are very fine, it is often cheaper and easier to discard the contaminated granules than to clean the solid sieve.

Filter media can be cleaned by rinsing with solvents or detergents. Alternatively, in engineering applications, such as swimming pool water treatment plants, they may be cleaned by backwashing. Self-cleaning screen filters utilize point-of-suction backwashing to clean the screen without interrupting system flow. Achieving flow through the filter Fluids flow through a filter due to a difference in pressure fluid flows from the high pressure side to the low pressure side of the filter, leaving some material behind. The simplest method to achieve this is by gravity and can be seen in the coffeemaker example. In the laboratory, pressure in the form of compressed air on the feed side (or vacuum on the filtrate side) may be applied to make the filtration process faster, though this may lead to clogging or the passage of fine particles. Alternatively, the liquid may flow through the filter by the force exerted by a pump, a method commonly used in industry when a reduced filtration time is important. In this case, the filter need not be mounted vertically. Filter aid Certain filter aids may be used to aid filtration. These are often

incompressible diatomaceous earth, or kieselguhr, which is composed primarily of silica. Also used are wood cellulose and other inert porous solids such as the cheaper and safer perlite.These filter aids can be used in two different ways. They can be used as a precoat before the slurry is filtered. This will prevent gelatinous-type solids from plugging the filter medium and also give a clearer filtrate. They can also be added to the slurry before filtration. This increases the porosity of the cake and reduces resistance of the cake during filtration. In a rotary filter, the filter aid may be applied as a precoat; subsequently, thin slices of this layer are sliced off with the cake.The use of filter aids is usually limited to cases where the cake is discarded or where the precipitate can be chemically separated from the filter.

Alternatives Filtration is a more efficient method for the separation of mixtures than decantation, but is much more time consuming. If very small amounts of solution are involved, most of the solution may be soaked up by the filter medium.An alternative to filtration is centrifugation instead of filtering the mixture of solid and liquid particles, the mixture is centrifuged to force the (usually) denser solid to the bottom, where it often forms a firm cake. The liquid above can then be decanted. This method is especially useful for separating solids which do not filter well, such as gelatinous or fine particles. These solids can clog or pass through the filter, respectively.

Product polishing It describes the final processing steps which end with packaging of the product in a form that is stable, easily transportable and convenient. Crystallization, desiccation,lyophilization and spray drying are typical unit operations. Depending on the product and its intended use, polishing may also include operations to sterilize the product and remove or deactivate trace contaminants which might compromise product safety. Such operations might include the removal of viruses or depyrogenation.For example, expanded bed adsorption (Vennapusa et al. 2008)

accomplishes removal of insolubles and product isolation in a single step. Affinity chromatography often isolates and purifies in a single step.

SOLVENT/Liquid- liquid extraction ( http://ull.chemistry.uakron.edu/chemsep/extraction/) It consists in transferring one (or more) solute(s) contained in a feed solution to another immiscible liquid (solvent). The solvent that is enriched in solute(s) is calledextract. The feed solution that is depleted in solute(s) is called raffinate.

Solvent extraction

Liquidliquid extraction also known as solvent extraction and partitioning, is a method to separate compounds based on their relative solubilities in two different immiscible liquids, usually water and an organic solvent. It is an extraction of a substance from one liquid into another liquid phase. Liquidliquid extraction is a basic technique in chemical laboratories, where it is performed using a separatory funnel. This type of process is commonly performed after a chemical reaction as part of the work-up.The term partitioning is commonly used to refer to the underlying chemical and physical processes involved in liquidliquid extraction but may be fully synonymous. The term solvent extraction can also refer to the separation of a substance from a mixture by preferentially dissolving that substance in a suitable solvent. In that case, a soluble compound is separated from an insoluble compound or a complex matrix. Solvent extraction is used in nuclear reprocessing, ore processing, the production of fine organic compounds, the processing of perfumes, the production of vegetable oils and biodiesel, and other industries.Liquidliquid extraction is possible in non-aqueous systems: In a system consisting of a molten metal in contact with molten salts, metals can be extracted from one phase to the other. This is related to a mercury electrode where a metal can be reduced, the metal will often then dissolve in the mercury to form an amalgam that modifies its electrochemistry greatly. For example, it is possible for sodium cations to be reduced at a mercury cathode to form sodium amalgam, while at an inert electrode (such as platinum) the sodium cations are not reduced. Instead, water is reduced to hydrogen. A detergent or fine solid can be used to stabilize an emulsion, or third phase..

Adsorption : ( absorption (chemistry). Memidex (WordNet) Dictionary/Thesaurus. Retrieved 2010-11-02) It is the adhesion of atoms, ions, or molecules from a gas, liquid, or dissolved solid to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. This process differs from absorption, in which a fluid (the absorbate) permeates or is dissolved by a liquid or solid (the absorbent). Adsorption is a surface-based process while absorption involves the whole volume of the material. The term sorption encompasses both processes, while desorption is the reverse of it. Adsorption is a surface phenomenon. Absorption: [(December 4, 2010) Absorption (Chemistry). Obtained on December 1, 2012 fromhttp://en.citizendium.org/wiki/Absorption_(chemistry)] It is a physical or chemical phenomenon or a process in which atoms, molecules, or ions enter some bulk phase gas,liquid, or solid material. This is a different process from adsorption, since molecules undergoing absorption are taken up by the volume, not by the surface (as in the case for adsorption). A more general term is sorption, which covers absorption, adsorption, and ion exchange. Absorption is a condition in which something takes in another substance.

Gas chromatography (GC): (Gas Chromatography. Linde AG. Retrieved 11 March 2012) It is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can bevaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture. In gas chromatography, the mobile phase (or "moving phase") is a carrier gas, usually an inert gas such as helium or an unreactive gas such asnitrogen.

The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column (an homage to the fractionating column used in distillation). The instrument used to perform gas chromatography is called a gas chromatograph (or aerograph, gas separator). The gaseous compounds being analyzed interact with the walls of the column, which is coated with a stationary phase. This causes each compound to elute at a different time, known as the retention time of the compound. The comparison of retention times is what gives GC its analytical usefulness.High-performance liquid chromatography (formerly referred to as highpressure liquid chromatography), (Lindsay, S. ; Kealey, D. (1987). High performance liquid chromatography)HPLC, is a technique in analytic chemistry used to separate the components in a mixture, to identify each component, and to quantify each component. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column. Thin-layer chromatography: (F. Geiss (1987): Fundamentals of thin layer chromatography planar chromatography, Heidelberg, Hthig) (TLC) is a chromatography technique used to separate non-volatile mixtures.[1] Thinlayer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the stationary phase.After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Specific examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers

in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents

APPLICATIONS: Industry Beverages Applications Provides tartness and complements fruits and berries flavors. Increases the effectiveness of antimicrobial preservatives. Used in pH adjustment to provide uniform acidity. Provides tartness, Minimizes sucrose inversion, Produces dark color in hard candies. Acts as acidulant. Lowers pH to inactive 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. Provides rapid dissolution of active ingredients. Acidulant in mild astringent formulation. Anticoagulant.

Jellies, Jams and Preserves Candy Frozen fruit Dairy products Fats and oils Pharmaceuticals

Cosmetics and toiletries pH adjustment, antioxidant as a metallic-ion chelator, buffering agent. Industrial applications Metal cleaning Sequestrant of metal ions, neutralizant, buffer agent Removes metal oxides from surface of ferrous and nonferrous metals, for preoperational and non operational cleaning of iron and copper oxides In electroplating, copper plating, metal cleaning, leather tanning, printing inks, bottle washing compounds, floor cement, textiles, photographic reagents, concrete, plaster, refractories and moulds, adhesives, paper, polymers, tobacco, waste treatment etc.

Others

Conclusion:

Critical analysis of current literature is focused on citric acid. Discussing the new citric acid producing microorganisms, methodologies, optimization of media composition, purification strategies, applications used in specific field. Citric acid is becoming increasingly important in high-value applications in industrial sector in the production of diary products, beverages, pharmaceuticals .Simultaneously, advances are being made in bioreactor and reaction technologies for effectively using the citric acid. The need of some pre-treatment of raw materials may enhance the fermentation efficiency. One area, which needs attention is the development of continuous culture techniques which have been attempted but only at the laboratory scale. Another area is the strain improvement with improved substrate utilization efficiency.

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