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CITRIC ACID, PROCESSES 651

Table 1. Citric Acid Contents of Various Fruits andVegetables

Fruit/vegetable Citric acid (wt %)

Lemon 4.08.0Grape 1.22.1Tangerine 0.91.2Orange 0.61.0Currant

Black 1.53.0Red 0.71.3

Raspberry 1.01.3Strawberry 0.60.8Apple 0.008Tomato 0.25Potato 0.350.5Asparagus 0.080.2Turnip 0.051.1Pea 0.05Corn 0.02Lettuce 0.16Eggplant 0.01

CITRIC ACID, PROCESSES

M.R.V. KRISHNANAnna UniversityChennai, India

KEY WORDS

Aspergillus nigerEnzyme CoAFermentationMolassesMoldpHSolid substrate fermentationSubmerged cultureSurface cultureYeast

OUTLINE

IntroductionMicrobial ProductionStorage of Citric AcidProduct Applications

Foods and BeveragesPharmaceuticalsCosmeticsMetallurgyIndustrial UsesOther Applications

The Technology of Citric Acid ProductionCitrus Fruits and PineapplesCane Molasses and Beet MolassesMolasses MediumSubmerged FermentationSolid-Substrate FermentationRelative Merits between Submerged and SurfaceFermentation

Biochemistry of Citric Acid ProductionExternal FactorsBiochemical FactorsCitric Acid Production by BacteriaRecovery of Citric Acid

ConclusionsBibliographyAdditional Reading

INTRODUCTION

Citric acid (C6H8O7) is a white or translucent solid with amolecular weight of 192.12. It occurs as a natural constit-uent in citron, lemon, lime, pineapple, pear, peach, andsimilar fruits (Table 1). It is also found in animal tissues.The popular forms of citric acid are the anhydrous forms,the monohydrate and sodium salts of the acid.

Citric acid was first isolated by Scheele in 1784 whenhe crystallized it from lemon juice. It was later synthesized(via symmetrical dicholoroacetone) from glycerol by Gri-mocex and Adam in 1880. It was first made in crystallizedform from lemon juice. Calcium citrate was precipitatedthrough the reaction of hot lemon juice with calcium car-bonate, followed by decomposition of the product with sul-phuric acid. Two hundred years later, this process is stillbeing used. However, raw material is the principal limitingfactor for production on a large scale. Thirty tons of lemonare needed to produce 1 ton of citric acid. The first com-mercial citric acid was prepared in 1860 in England fromcalcium citrate imported from Italy and Sicily. By 1880,France, Germany, and the United States had begun manu-facturing citric acid using similar methods. In 1913, recov-ery of citric acid from calcium citrate began in Italy, andby 1922, Italy produced 90% of the worlds supply.

MICROBIAL PRODUCTION

In 1893, Wehmer recognized citric acid as a microbial me-tabolite (1) (Table 2). Although his attempt to develop acommercial fermentation process with a species of Penicil-lium was unsuccessful, he established the basis for thesubsequent complete reorganization of the citric acid in-dustry. It was later confirmed that the use of Aspergillusniger (Fig. 1) favors sucrose fermentation to citric acid.

In 1923, a plant was started in New York to make citricacid by a fungal fermentation technique developed by Cur-rie (2). This production effectively broke the power of theItalian cartel. In this process, A. niger was grown on thesurface of a shallow pan of sugar medium. This surfacefermentation process was subsequently used in England,The Netherlands, Belgium, Germany, Switzerland, Argen-tina, and the former Soviet Union.

A new fungal fermentation process was introduced suc-cessfully in the United States in 1952. In this process,

652 CITRIC ACID, PROCESSES

Table 2. Chronology of Events in Citric Acid Production

Authors (ref.) Investigation/remarks

Wehmer 1893 (cited in 1) Demonstrated citric acid to be a metabolic product of certain molds.Currie 1917 (2) First chose Aspergillus niger for deriving citric acid.Doelger & Prescott 1934 (3) Fermentation above 30 C decreased citric acid yield and increased accumulation of oxalic

acid.Cahn 1935 (4) First to describe solid-state fermentation for production of citric acid.Mazzadroli 1938 (5) Potassium ferrocyanide could be added to eliminate excess Fe2.Szucs 1944 (6) Citric acid synthesis took place only after total assimilation of phosphates.Moyer 1953 (7) Suggested use of methanol as stimulant for increasing acid yield.Usami & Takatomi 1958 (1) Spores produced under submerged fermentation were poor, and spores from surface

culture were better acid formers.Usami et al. 1960 (9) Increase in aeration resulted in higher yields and reduction in cycle time.Kovats 1960 (10) Demonstrated lower concentration of sugar leads to lower yields of citric acid and

accumulation of oxalic acid.Schweiger 1961 (11) Iron concentration above 0.2 ppm affected yield of acid. However, addition of copper 0.1 to

500 ppm at the time of inoculation or during the first 50 h of fermentation counteredthe deleterious effect of iron.

Naguchi & Bando 1960 (12) Concentration of ammonium nitrate greater than 0.25% lead to accumulation of oxalicacid.

Sanchez-Marroquin et al. 1963 (13) Higher yields of citric acid occurred in a simple medium rather than a complex medium.Clark 1962 (14) Ferrocyanide addition on the acid yield 10200 lg/mL was tolerated during growth. But

less than 20 lg/mL helped acid production.Millis et al. 1963 (15) Addition of vegetable oils, fatty acids, etc., increased acid yield.Czech. Acad. Sci. 1964 (16) Mycelial digests from A. terreus or A. Niger stimulated acid production by 6070% when

added to the medium.Leopold 1965 (17) Addition of pressed bakers yeasts to culture medium increased acid yield from 61.5 to

72.1%.Bruchmann 1966 (18) Addition of mild oxidizing agents stimulated acid production.Sussman & Halvorson 1966 (19) Spore viability varies with age.Wendel 1967 (20) In the initial stages, citric acid diffused into the medium, which leads to a stratification

on the mycelium. This stratification inhibited fungal metabolism, leading to decreasedacid yields. This stratification can be eliminated by stirring.

Tabuchi et al. 1969 (21) Production of citric acid from n-paraffin using yeast, (Candida) medium composition.Hydrocarbon, 4060 gs; NH4Cl2, 2 gs; KH2PO4, 0.5 g; MgSO4, 0.5 g; cornsteep liquor,1 g; CaCo3, 30 g/L.

Khan et al. 1970 (22) Phosphates promoted more growth at less acid yields.Fedoseev et al. 1970 (23) Copper sulfate at 4.7 mg/100 g molasses resulted in better conversion of sugar to citric

acid.Kyowa Fermentation Industry 1970 (24) Citric acid production from dodecane (or) C12C14. Using arthrobacterium in aqueous

medium, inoculum 5% air at 3 vvma at 28 C, yielded 28 mg/mL.Sanchez-Marroquin et al. 1970 (25) The use of ion-exchange resin for reduction of metal content was better than chemical

treatment.Fukuda et al. 1970 (26) Use of Corynebacterium on n-paraffins; yield 41.4 mg/mL; culture period 64 h, 32 C.Leopold 1971 (17) Glycerol addition increased acid yield by 30%.Dhankar et al. 1972 (27) Acid yield improved by addition of peanut oil to molasses medium.Kumamoto & Okamura 1972 (28) Mn2, Ba2, Al3 had an effect on fungal morphology.Sardinas 1972 (29) Patented a process for citric acid production involving B. licheniformis.Chaudhary et al. 1972 (30) A low pH in molasses medium was inhibitory to growth.Ohmori & Ikeno 1973 (31) Bacterial production of citric acid from media containing isocitric acid.Dhankar et al. 1974 (32) Sodium nitrate at a concentration of 0.4% was superior to ammonium nitrate.Zhuravskii, 1974 (33) Patented continuous multistage process for citric acid production.Halama 1974 (34) Chemicals such as pentachloralphenolate, semicarbozone, tetracycline, etc., used for

control of bacterial growth.Ohtsuka et al. 1975 (35) Addition of malic hydrazide increased yield from 30 to 70%.Wold & Suzuki 1973 (36) The rate of acid production increased when AMP was added to the culture medium.Wold & Suzuki 1976 (37) Zn2 regulated growth and production.Brezhnoi et al. 1976 (38) Continuous production of citric acid from molasses.Choudhary 1978 (39) A 3-day-old slant culture was as good as 7- to 8-day-old spore.Krishnan & Natarajan 1987 (40) Liquidliquid extraction of citric acid.Krishnan & Annadurai 1992 (41) Precipitation of citric acid.Krishnan et al. 1996 (42) Reduction in cycle time for citric acid production.

a vvm, volume air per volume medium per minute.


Spore chain

Vesicle

Phialide

Conidiophore

Figure 1. Aspergillus niger.

Table 4. World Consumption Pattern of Citric Acid

Uses Consumption (%)

Food and beverages 60Pharmaceuticals 12Detergents 12Metal cleaning 6Textile dyes 5Cosmetics 3Others 2

Table 3. Global Production Pattern of Citric Acid

Place Production (%)

Western Europe 41North America 28South and Central America 11Far East, Australia, and New Zealand 11Other 9

A. niger was allowed to grow in the entire volume of theculture solution in large deep tanks. This process was in-troduced in Mexico in 1959 and in Israel in 1961.

The earliest attempt to produce citric acid by the sub-merged growth technique was that by Amelung, who aer-ated the culture by bubbling air through the solution. Nu-merous studies, subsequent to the work of Amelung,resulted in the development of two approaches to the con-trol of citric acid production in submerged cultures. In thefirst approach, Perquin produced citric acid by inducing theestablishment of a deficiency of one of the major nutrients,such as phosphate. In another approach, Johnson et al.studied the effect of manganese and iron and related theconcentration of these to the amount of inhibition of citricacid accumulation.

Further development of methods for the control of ironand manganese levels in the culture solution led to thepopularity of the submerged fermentation process, nowused in the United States, Mexico, and Israel. Excess ironmay be precipitated by ferrocyanide. The removal of ironand the presence of specific enzyme inhibitors that controlthe destruction of citric acid form the basis for the suc-cessful submerged fermentation process. Other mineralnutrients required for the submerged fermentation are thesame as those for surface fermentation.

The most interesting change in citric acid manufacturewas done by Miles Laboratories. They changed the rawmaterials from molasses to glucose, doubling the produc-tion. The company patented a process in which starchy ma-terial is first converted to sugar by enzymatic action andthen used as the raw material for citric acid production.They also claim that with the addition of various phenolsand other compounds to check the growth of A. niger, it ispossible to use less highly purified starting material. Thisis similar to the use of the lower alcohols, which has beenknown for many years and is being investigated now as anadditive to blackstrap molasses.

STORAGE OF CITRIC ACID

The popular forms of citric acid are the anhydrous forms,the monohydrate form and sodium salts of the acid. Bothcitric acid and citric monohydrate are available in varietyof sieve sizes. They are conventionally available as gran-ular, fine granular, and powder forms.

Crystalline anhydrous citric acid can be stored in dryform without difficulty, although highly humid conditionsand elevated temperatures should be avoided to preventcaking. The product should be stored in tight containers toprevent exposure to moist air. Several granulations arecommercially available with larger particle sizes havingless tendency toward caking. Materials packed with des-iccants are also available. Solutions of citric acid are cor-rosive to normal concrete, aluminium, carbon steel, copperalloys and should not be used with nylon, polycarbonates,polyamides, polyimides, or acrylics. Recommended mate-rials of construction for pipes, tanks, and pumps handlingcitric acid solutions are 316 stainless steel, fiberglass-reinforced polyester, polyethylene, polypropylene, andpolyvinyl chloride. Sodium and potassium citrates are not

as corrosive as citric acid but they should be handled inthe same type of equipment as citric acid.

PRODUCT APPLICATIONS

The food and pharmaceutical industries use citric acid ex-tensively because of its high solubility, pleasant sour taste,very low toxicity, and ready assimilability (Table 3). Citricacid also finds application in some cosmetic preparations,metal cleaning, electropickling, copper plating, secondaryoil recovery, and other industrial uses (Table 4).

Foods and Beverages

Citric acid is widely used in foods, beverages, and jams andjellies owing to its ability


Table 5. Some Leading Manufacturers and Suppliers ofCitric Acid

Manufacturers

Miles Laboratories Inc., Elkhart, IndianaJoh. A. Benckshiser Gmbh, Ludwingshafen/Rhein, GermanyRhone-Poulenc S.A., FranceJohn & E. Sturge Ltd., Birmingham, EnglandCargill Inc., Minneapolis, MinnesotaSan Fu Chemical Co., Ltd., ChinaBoehringer Ingelheim KG, Rhein, GermanyPfizer Inc., New JerseyFermenta Products, Quimicos, SA, BrazilShowa Chemical Co., Pvt Ltd., Osaka, Japan

Suppliers

Lurgi AG. Frankfurt am Main, GermanySnamprogetty SPA, San Donato, Milan, ItalyMannesman Anlagenbau, Dusseldorf, GermanyBoehringer Ingelheim KG, Burgerstrasse, GermanyVogelbusch GmbH, Austria

1. To maintain desired pH levels2. To impart refreshing and tingling taste3. To act as a flavor-enhancing agent4. To act as a color stabilizer

In candy manufacture, citric acid helps to enhance theflavor and taste of berries and other ingredients. As a verydependable agent for maintenance of pH, citric acid findswide application in the production of jams, jellies, pre-serves, soft drinks, syrup, and soft drink tablets. For pHcontrol and color stabilization, citric acid is used in fruitand vegetable juices. The pH prevents juice spoilage, andnatural taste and flavor are greatly enhanced. Lowering ofpH inactivates certain oxidative enzymes, thus improvingthe keeping qualities of frozen fruits, peaches, apricots,plums, pears, and cherries against flavor or color spoilage.Citric acid also combines with trace metals, helping avoidthe undesirable oxidation changes.

Pharmaceuticals

Citric acids wide application in the manufacture of drugsand pharmaceuticals is for enhancing the taste and flavor.It is used as an anticoagulant for blood. Powders and tab-lets owe their effervescent property to citric acid. The freeacid and its sodium and potassium salts are used as mildacidulants in astringent preparations. Several salts of cit-ric acid are used in the manufacture of antianemic tonics,vitamins, liver tonics, antipyretic agents, cough syrups,antidysentery, and antidiarrheal agents. It is also used ininducing fertility.

Cosmetics

Hair rinses and hair setting agents derive benefit from cit-ric acid. It is also used in astringent lotions, bleaching lo-tions, and others.

Metallurgy

Citric acid finds extensive use in metallurgical applicationand as a sequestering agent for such metals as iron, copper,zinc, nickel, cobalt, chromium, and manganese. The acidand ammonium salts are added for scale removal in boil-ers; cleaning of reactors could be done very well using theacid. Citric acid is widely used in electropickling of copperand its alloys and in copper coating. Any iron plugging inthe oil-flow line could be removed by the acid. Tanning li-quors, bottle washing compounds, and diazo printing pa-per all contain citric acid.

Industrial Uses

It is used in the manufacture of several esters, linoleum,inks, silvering compounds, and fabric dyes (Table 5). Citricacid enjoys a superior status above phosphates in themanufacturing of detergents.

Other Applications

Some of the algicides, pesticides, fish preservation, chem-icals, and insecticides are based on citric acid. Preservation

of latex from certain natural plants involves the use of cit-ric acid.

THE TECHNOLOGY OF CITRIC ACID PRODUCTION

Citric acid production centers around one strain, Aspergil-lus niger, which is still extensively used. In the late 1970s,processes involving Candida yeast became commercial.Candida guillermondi has unique benefits over A. niger inthat trace metal removal is not essential. It can also tol-erate higher pHs (3.5 to 5) and sugar concentrations foracid production. The rate of acid production is also faster.As in the case of A. niger, nitrogen limitation triggers acidproduction. Certain bacteria also produce citric acid on avariety of substrates.

Citric acid can be manufactured commercially by threemethods:

1. Surface culture technique

2. Submerged culture technique

3. Solid substrate technique

Commercially used substrates are citrus fruits (notadapted presently because the fruits are costly and sea-sonal), sucrose, glucose, molasses cane juice, and certainpetroleum fractions.

Shallow pan reactors, stirred tank fermenters, and air-lift fermenters are commonly used depending on the pro-cess requirements. Fermenters work in a semibatch modeor a continuous mode. It will be of interest to state herethat yields of product vary with the pretreatment to whichthe media are subjected. For example, yields for pretreat-ment with suitable exchange resins is 98% in the case ofsucrose and 75% for molasses; ferrocyanide-treated molas-ses yields 68% as contrasted to the untreated molasses, forwhich the yield is 62%.


Table 6. Analyses of Cane and Sugar Beet Molasses

Component Beet (%) Cane (%)

Water 20.0 16.5Sugar 62.0 53.0Nonsugars 10.0 19.0Ash 8.0 11.5

Citrus Fruits and Pineapples

Lemon peels and the white layer are removed and sepa-rated for the recovery of oil and pectin, respectively. Theremaining portion is pulped and filtered. The filtrate islikely to contain pectin and albumin, which are removedby spontaneous fermentation. In the case of pineapples,the waste and low quality fruits are crushed and filtered.The lemon juice and the pineapple juice, recovered sepa-rately, could contain 4% and 0.75% citric acid, respectively.Calcium carbonate or hydroxide is added to the individualjuice to the required amount, and the temperature is main-tained between 55 and 95 C. Any oxalate present is fil-tered preferentially before citric acid. The calcium citratecake obtained is washed and reacted with a small amountof sulfuric acid. The resultant slurry is filtered to recovercitric acid solution from the precipitated calcium sulfate.The clear filtrate is subjected to decolorization before re-covering the citric acid crystals by evaporation. This pro-cess is on the decline, as stated earlier.

Cane Molasses and Beet Molasses

Blackstrap molasses, about 40 Be with a sugar content of52 to 57% sugar, or beet molasses, 41 Be containing 48 to52% sugar, could be used for citric acid production (Table6). However, cane molasses should be purified before sub-jecting it to fermentation. Alternate methods for molassespurification are discussed below:

1. Sulfuric acid treatment. The pH of the molasses (10%total reducing sugar) is adjusted to 3.0 by adding 0.1N sulphuric acid. This is allowed to stand for 1.5 hand then centrifuged at 3,000 rpm for 15 min. Thesupernatant is collected and used.

2. Potassium ferrocyanide treatment. The molasses(10% TRS) is heated to 85 C for 30 minutes and cen-trifuged at 3,000 rpm for 15 min; 100 mL of the su-pernatant is collected, and 0.5 mL of K4 Fe(CN)6(10% solution) is added. The pH is adjusted to 6.5.

3. Tricalcium phosphate treatment. The pH of the mo-lasses (10% TRS) was adjusted to 7.0 by the additionof 0.1 N NaOH and treated with 2% (w/v) tricalciumphosphate followed by heating at 105 C for 5 min-utes. The mixture was cooled and centrifuged at3,000 rpm for 15 minutes. The supernatant wasused.

4. Tricalcium phosphate with hydrochloric acid treat-ment. The TCP-treated liquor was adjusted to pH 2.0by the addition of 0.1 N Hcl followed by vigorousshaking. The mixture was allowed to stand for 6 h.

The supernatant was used after centrifugation at3,000 rpm for 20 min.

5. Bentonite treatment. The pH of molasses (10% TRS)was adjusted to 7.0. Bentonite 2% (w/v) was addedand kept in a boiling water bath for 30 min. The so-lution was then centrifuged at 3,000 rpm for 15 min.The supernatant was collected and used.

Molasses Medium

Beet molasses is the most widely used raw material in theUnited States and Europe. Latin American and Caribbeanplants use sugar as raw material (3 tons of sucrose per tonof citric acid). Smaller plants in the Caribbean use citricwastes from citrus fruits. The production rates are Argen-tina, 2,000 tons per annum, Mexico, 1,000 tons per annum;and Uruguay, 500 tons per annum.

The beet molasses or pretreated cane molasses is takenin a mixing vessel where dilute sulphuric acid is added toregister a pH of 5.5 of 6.5. Phosphorous, potassium, andnitrogen are added as nutrients to the required amount forgrowth and citric acid production. This mixture is steril-ized with live steam. After this, a requisite amount of ster-ile water is added to give a sugar percentage of 15 to 20%.The feed is admitted into shallow aluminium pans ar-ranged in convenient stacks in a sterile room. Each tray isshallow (about 75-mm deep) but big enough to hold solu-tions up to 500 L. The temperature (28 to 32 C) and hu-midity (40 to 60%) are controlled for maximum yield. Thissugar medium is inoculated with the spores of a selectedstrain of A. niger. In about 8 to 10 days, the fermentationprocess is over (pH being about 2). The acid might be con-taminated with oxalic acid and gluconic acid. The tray con-tents are sent out for recovery of the acid, and the traysand the chamber are sterilized with steam. Dilute formicacid or sulfur dioxide could also be used for positive ster-ilization.

The recovery consists of adding hydrated lime (1 part oflime to every 2 parts of liquor) to the fermenter broth keptat 95 C. The precipitated calcium citrate could be reactedwith enough dilute sulfuric acid when citric acid remainsin solution, leaving behind calcium sulfate. Based on thesugar content of the raw materials, the yield could be 35to 65% by weight of sugar. In this process, pH maintenanceis difficult.

Submerged Fermentation

The majority of industries have adopted this process. Con-ventionally, the growth phase and the production phaseare two distinct regions. Two different media are used, onefor growth and the other for production (Table 7). After 3to 4 days of lush growth, the mycelia are separated fromthe growth medium and added to the production fermenterunder stirred conditions. In the production fermenter, thepH and temperature are maintained around 3 and 30 C,respectively. Air is sparged without interruption into thesolution at about 1 to 1.2 volume air per volume mediumper minute (vvm). In about 4 to 5 days, the production be-comes nearly complete with about 65 to 70% of sugar beingconverted to the final product.

The submerged process is admirably suited for a flexibleoperation. It is possible to introduce certain process accel-


Table 7. Medium for Citric Acid

ComponentSporulation

(g/L)Production

(g/L)

Sucrose 140 140Bactoagar 20 0.0Ammonium nitrate 2.5 2.5Potassium hydrogen phosphate 1.0 2.5Magnesium sulphate heptahydrate 0.25 0.25Copper ion 0.0048 0.00006Zinc ion 0.0038 0.00025Ferrous ion 0.0022 0.0013Manganous ion 0.001 0.001

HSCH2CH2NCCH2CH2NCCHCCH2OPOPOCH2

OH

Carbohydrates Lipids Proteins

Acetyl CoA

Pyruvic acid Fatty acid Amino acid

H HO O O

O

CH3

CH3

NH2

O

O

O

PO O

O

OH

OH

N

N N

N

AdenineRibose-3-phosphate

Pantothenic acidThio-ethyl amine

Figure 2. Enzyme CoA.

erators or nutrients at desired and convenient time inter-vals; activated carbon or ascorbic acid could be used as theaccelerator. The flexibility of such a fermenter is so goodthat sugar concentration and pH could be maintained atdesired values. The acid recovery is through the calciumcitrate process. For every ton of citric acid produced, oneneeds about 3,650 kg of molasses, 5 to 14 kgs of nutrient,650 kg of sulfuric acid, and 450 kg of lime. Although onemight use several organisms (A. niger is still the mostsought after), the yield depends mainly on the nutrientused, presence of trace metals, and above all, the pH andair supply.

In the new process by Miles Laboratories mentionedpreviously, sucrose has been replaced with glucose for acidproduction. Starch is converted to sugar through enzy-matic reaction, and the resulting sugar is the raw materialfor acid production. Specific chemicals control the growth

of the mold at the expense of citrate production. An inter-esting point is that their starting material is not of highpurity. They use very large fermentation vats into whichsucrose solution is pumped, and fungal spores are addedto help the growth and production cycles. After the cycle isover, the vats are charged with fresh medium.

Solid-Substrate Fermentation

In the solid-substrate fermentation process, the mold A.niger ferments molasses or sucrose adsorbed on beet orcane pulp. Carriers such as beet or cane pulp offer largeareas for microbial contact with the medium and ensuregood aeration. Bagasse is preferred over beet pulp becauseof its larger surface area and lower cost. In addition, ba-gasse can be used several times over. Bagasse is brokendown into fine pieces, 1 to 2 mm long, sterilized, andsoaked in molasses or sucrose solution before being inoc-ulated with the selected strain. The citric acid yields areabout 45% of the sugar content of molasses and 55% ofsucrose. This process is labor intensive and needs morefloor space, plus there is the likelihood of contamination.It is difficult to maintain pH. The process requires goodaeration with controlled humidities.

Relative Merits between Submerged and SurfaceFermentation

The starting sugar cane concentration and the yield per-centage are more or less the same for submerged and sur-face fermentation. However, the surface process is highlylabor intensive and not easily amenable for pH or tem-


Oxalacetic acidC4H4O6

Malic acidC6H6O5

Fumaric acidC4H4O4

NADHH

NADPHH

NADP

NAD

NADHH NAD

Succinic acidC4H6O4

Succinyl CoA

FADH2

FAD

Malic dehydrogenase

Succinic dehydrogenase

Succinyl CoAsynthetase

Mg

Fumarase

H2O

Citric acidC6H8O7

cis-Aconitic acidC6H6O6

Iso-citric acidC6H8O7

Oxalosuccinic acidC6H6O7

-Ketoglutaric acidC5H6O5

Aconitase

Iso-citric aciddehydrogenase

Mn

Aconitase

H2O

H2O

CO2

Condensing enzyme

Lipoic acidthymine

PyrophosphateCoA

Acetyl CoA

CoACoA

Pyruvic acid

CO2

NADNADH

Figure 3. Tricarboxylic acid-cycle.

perature control. Flexibility of operation is higher in thesubmerged process because addition or removal of chemi-cals is easier. The risk of contamination is less in the sub-merged process. The surface process needs a larger work-ing area, and the chance of pollution (caused by spores) isalso high. Oxalic acid formation is higher in surface fer-mentation.

One advantage of the surface process is that it is lessenergy intensive. Although automation of the submerged

process may be easier, in the event of contamination, thelosses suffered will be enormous.

BIOCHEMISTRY OF CITRIC ACID PRODUCTION

Successful production of citric acid depends on several fac-tors. External factors can be controlled directly, and bio-chemical factors, the various biological reactions that go


CO COA NADP ADP Pi

CH3

CH2

COOH

COOH

COOH

CH3COCOA CO2 NADH ATP

C6H8O7 SCOA

C6H8O7 C6H6O6 H2O

C6H6O6 H2O

C6H6O7 H2O NADPH2 ATP

succinyl SCOA CO2 NADH2 H2O

CH3COSCOA CO H2O

Pyr. dehydrogenasePyr. decarboxylase

Isocit. dehydrogenasedecarboxylase

Oxalo succinicdecarboxylase

a-ket. glut. dehydrodecarboxylase

Succinyl thiokinase

Succinicdehydrogenase

condensingenzyme

Aconitase

C6H8O7

C6H6O7

C4H6O5

C4H6O4 ATP SCOA

C4H4O4 FADH2 H2O ATP

C4H4O4 H2O

C5H6O5 NAD ADP Pi H2O SCOA

C5H6O5 CO2 H2O

C6H8O7 NADP ADP Pi

Aconitase

Fumarase

succinyl SCOA H2O ADP Pi

C4H6O4 FAD ADP Pi

Malicdehydrogenase

C4H4O5 H2O NADH2 ATPC4H6O5 NAD Pi ADP

The Krebs cycle

on inside the cell, can be controlled indirectly by controllingthe external factors.

External Factors

The type of sugar used and sugar levels The pH in the milieu The temperature of production The presence or absence of trace metals such as Fe,

Cu, Zn, Mn The presence or absence of phosphates and nitrates

in the medium The oxygen concentration in the environment

Biochemical Factors

Breakdown of sugars to pyruvic acid and acetyl CoA(Fig. 2)

Formation of oxalacetic acid from pyruvic acid andcarbon dioxide

Promotion of the enzymatic reaction necessary forcitric acid accumulation

Suppression of the enzymatic reaction that repressescitric acid production

Oxygen concentration in the medium and cell (citricacid production needs a copious supply of oxygen)

Favoring citric acid accumulation at the expense ofcell growth

Based on the external and biochemical factors, onecould draw the following conclusions:

1. Glycolytic and pyruvate enzymes should be activatedfor the citrate synthesis by maintenance of highsugar levels.

2. Repression of enzymes in the tricarboxylic acid(TCA) cycle would inhibit citrate production.

3. Manganese deficiency should be ensured for 2NH4generation, which would counter the inhibition ofphosphofructokinase by citrate.

4. A copious oxygen supply will overcome possible cellstarvation, even under emergent conditions.

5. Ideal pH maintenance encourages citrate productionrather than oxalic or gluconic acid formation.


In most cells, catabolism of simple sugar is the majorsource of energy. It starts with the glycolytic pathway. Glu-cose is broken down into two molecules of three-carboncompounds called pyruvic acid. The cell gains two ATPmolecules for each molecule of glucose entering the glyco-lytic pathway. The ATP molecules are storehouses of en-ergy, releasing energy necessary for cell work. The cata-bolic reaction also gives rise to NADH, which is useful forseveral anabolic reactions.

Depending on the type of cell and availability of oxygen,the pyruvic acid produced earlier can undergo two differenttypes of reactions. When anaerobic conditions prevail, py-ruvic acid is converted to lactic acid or ethyl alcohol. Underaerobic conditions, the pyruvic acid is converted to carbondioxide and acetyl CoA (Fig. 2). This acetyl CoA is furtherdegraded in the (Fig. 3) TCA cycle or election transportsystem to produce energy for cell work. Several other sub-strates exist that can give rise to acetyl CoA. For example,proteins and lipids can be degraded to provide acetyl CoA.Thus, cells can degrade several chemical compounds toacetyl CoA and subject them to the TCA cycle and electrontransport chain for obtaining energy.

The TCA cycle together with the electron transport sys-tem constitutes the cells primary metabolic furnace. Thesetwo combine to burn acetyl CoA in oxygen to liberate car-bon dioxide, water, and useful energy. This energy from theTCA cycle and electron transport chain is captured andtaken up in ATP for storage of energy.

During oxidation of one molecule of acetyl CoA (by 0theTCA cycle occurring in the mitochondrial matrix), one mol-ecule of flavoprotein (FP or FAB) and three molecules ofnicotinamide adenine dinucleotide (NAD) are reduced.These reduced coenzymes are oxidized by molecular oxy-gen by way of a system of enzymes and coenzymes calledthe respiratory chain or electron transport system, occur-ring in the inner mitochondrial membrane. During this ox-idation process, enormous amounts of energy are released,some of which is utilized by the inner membrane subunits.

The TCA cycle occurs in the following ways:

The pyruvic acid is decarboxylated; the acetyl groupcombines with CoA to give acetyl CoA. The H andNAD combine to give NADH. Energy transfer occurs,yielding ATP.

Active acetyl combines with oxalacetic acid to formcitric acid. The released SCoA can now combine withmore CH3CO.

Citric acid looses water to give rise to aconitic acid. Isocitric acid results as cis-aconitic acid gets rehy-

drated. Isocitric acid loses hydrogen to yield oxalosuccinic

acid. The H combines with NADP to form NADPH.Energy transport takes place, yielding ATP. Loss ofCO2 also occurs.

Decarboxylation of oxalosuccinic acid takes place toyield -ketoglutaric acid. Hydrogen reacts with NADto form NADH2. Energy is released to convert ADPto ATP.

SCoA combines with the succinyl group of -ketoglu-taric acid and gets converted to succinic acid, yieldingSCoA and ATP.

Dehydrogenation of succinic acid leads to fumaricacid. Hydrogen joins FAD to form FADH2 and energy.This leads to formation of ATP.

Malic acid is formed as fumaric acid gets hydrated. Malic acid is dehydrogenated to form oxalacetic acid.

Oxalacetic acid combines with active acetyl to formcitric acid and the cycle continues. The released hy-drogen forms NADH2 from NAD and energy releasedgives rise to ATP.

Several yeasts and bacteria can produce citric acid fromspecific substrates:

Citric Acid Production by YeastsCandida lipolyticaCandida tropicalisCandida zylenoidesCandida fibraeCandida intermediaCandida parpsilosisCandida petrophylumCandida subtropicalisCandida oleophilaCandida hitachinicaCandida citraCandida guillermondiCandida sucrosa

A variety of substrates can be utilized by the Candidaspecies. They could utilize glucose, acetic acid, calcium ac-etate, hydrocarbons, molasses, alcohols, fatty acids, andnatural oils (such as coconut oil). For example, Candidacan utilize glucose or molasses as indicated in the list. C.lipolytica can use n-paraffins, n-alkanes, and alkenes.

From Glucose From MolassesGlucose, 1018%NH4ClKH2PO4MgSO47H2O2230 C, 36

days

Sugar cane/beet molasses, 5250 g/L(NH4)2 SO4, NH4Cl, NH4NO30.31.5 VVM, 6 days5.56.50 pH for growth2.84 pH for production3,154 kg sugar r 1,119 kg

monocitrate

Citric Acid Production by Bacteria

Certain bacteria are preferred for producing citric acid be-cause of their low doubling time. Examples include:

Bacillus licheniformisBacillus subtilisBrevibacterium flavum }

Arthrobacterparaffinens can utilizedodecane (C12C14) at28 C over a period of72 h; yield 28 mg/mL

They utilize glucose orisocitric acid orhydrocarbons

Bacillus licheniformis canutilize glucose medium:3037 C 36120 h; pH 7;nitrogen salts act asnutrients; yield 42 g/L


14a 11a

16

17

15

14 13 11 109 8

7

65

5a

12

2b

2a

4

2

1

3

1 Glucose solution2 Demineralizer (trace metal control)2a Flash pasteurization2b Syrup cooler3 Inoculum4 Fermenter5 Filter6 Filter7 Acidulator8 Filter9 Demineralizer

10 Carbon column11 Evaporator11a Crystallizer12 Centrifuge13 Remelter14 Evaporator14a Crystallizer15 Centrifuge16 Drier17 Packaging

Air

CaCO3

H2SO4

Figure 4. Citric acid production and separation flowsheet.

Recovery of Citric Acid

Two important routes are followed. One is the precipitationas calcium citrate (Fig. 4). The second is the liquidliquidextraction technique as discussed by Krishnan and Nata-rajan (40). Solvents such as esters, ketones, amines, andalcohols have been successfully used for the recovery, asoutlined by Rieger and Kioustelidis (43).

CONCLUSIONS

Citric acid enjoys a unique place in several important ev-eryday applications. The capabilities of A. niger have beenalmost totally exploited by using several strains of its cul-ture and their mutants. The application of A. niger for cit-ric acid production has been stretched nearly to the limit.It is the wishful thinking of the author that some morework could be undertaken in producing such stains thatcould utilize unclarified raw materials in shorter periods.No effort should be spared in developing newer andcheaper chemicals (promoters) that could accelerate theproduction rate.

Care has been taken to cover the entire world literatureon citric acid production. However, some references mighthave been missed more because of specific constraints

rather than because of oversight or willful omissions. Ishould like to end by saying that some more work on theapplication of bacterial culture would be rewarding. It maynot be right to say that the last word has been spoken oncitric acid production.

Dissolve away difficulties,Filter out gloomAnd from the mother liquor of lifeGather crystals of (citric acid) joy.

Adapted from Krishnan.

BIBLIOGRAPHY

1. S.C. Prescott and C.G. Dunn, Industrial Microbiology,McGraw-Hill, New York, 1959.

2. J.N. Currie, J. Biol. Chem. 31, 1537 (1917).3. W.P. Doelger and S.C. Prescott, Ind. Eng. Chem. 26, 1142

(1934).

4. F.J. Cahn, Ind. Eng. Chem. 27, 201203 (1935).5. Fr. Pat. 833,631 (Oct. 26, 1938), G. Mazzadroli.

6. U.S. Pat. 2,353,771 (July 18, 1944) J. Szucs.

7. A.J. Moyer, Appl. Microbiol. 17 (1953).

citric acid-pages from encyclopedia of bioprocess tech

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