CHAPTER 22
MICROBIAL PRODUCTION OF ORGANIC ACIDS
22.1 CITRIC ACID
Citric acid is a tricarboxylic acid with the molecular formula:
CH2COOH
HO – CCOOH
CH2COOH
It was first isolated by Scheele from lemon juice in 1784. Microbial production of
citric acid started in USA in 1923. In 2000, the annual production of citric acid
worldwide was 736,000 MT.
22.1.1 PRODUCTION METHODS
Citric acid can be produced using molds, yeasts, or bacteria. Except for patents and
few research articles, literature details on yeast- and bacterial processes are still
scarce. Candida, Pichia, etc., are the main organisms studied in the yeast process. In
the bacterial process, mutated strains of Corynebacterium species have been reportedly
used. The mold process is by far the most important from trade fermentation point
of view. Today, improved strains of Aspergillus niger (mold) are universally used for
citric acid production.
22.1.1.1 The mold (fungal) process
Based on the fermentation differences, there are three main types of mold process
for citric acid production: (i) solid substrate fermentation (koji process), (ii) surface
culture, and (iii) submerged culture. Aspergillus niger strains used for the commercial
processes are very efficient, producing above 80 g citric acid per 100 g glucose. The
organism in general exhibits marked sensitivity to iron and zinc, particularly to iron,
in the medium. These elements promote growth of the organism but at the cost of
citric acid.
1. The koji process
This method is widely used in Japan. The method accounts for 1/5th of total citric
acid produced in Japan. The fermentation is carried out in moist wheat bran. Since
wheat bran is rich in minerals, only special, iron-tolerant strains of Aspergillus can be
used. Although wheat bran contains ~ 66% carbohydrate, it is probably
supplemented with suitable amounts of sugar for the fermentation. Ferrocyanides or
copper may also be supplemented. After sterilization of the substrate (which is in the
paste form with ~ 70% moisture content), the fermentation is carried out in batches,
in shallow trays or rotolouver-type drum fermenters, at a pH of 5.5. Temperature,
humidity, and air supply can be maintained in a manner similar to that for other koji
processes. Inoculation is done with pure mold spores. Mold spores are usually
suspended in 0.1% Tween 80 (a wetting agent) before mixing with the substrate. The
concentration of the spore is, of course, of prime importance, and is typically
maintained at 107 spores per ml of suspension. Fermentation is carried out at around
30-32°C until the pH of the bran extract falls to 1.8-2.0, which corresponds to about
6 days. Aeration rate is optimally maintained at 0.8 vol/vol/min. After fermentation
is over, the extract is recovered by maceration in water followed by filtration. The
liquor is purified by precipitation with lime followed by regeneration with H2SO4.
The final liquor is treated with activated carbon, concentrated, and crystallized. The
yield is low because of the difficulty in controlling trace metals and process
parameters.
2. Surface culture method
Production
The medium can be either synthetic (refined sugars) or complex (cane molasses).
When cane molasses is used, the excess minerals must be removed prior to
fermentation. Several treatment options are available for the same, for example,
deionizing, use of sequestrants, etc. In trade fermentation, the deleterious effect of
iron is counteracted by dosing alkali ferrocyanide (e.g., K4Fe[CN]6) as iron-chelating
agent. This compound becomes toxic if present in the medium in excess. It is
therefore customary to keep the free K4Fe[CN]6 below 20 μg/ml in the medium.
Molasses medium should be adjusted to a pH of 5-6 (usually with H2SO4) although
this is not the optimum pH for the mold. The main reason behind this is the
presence of acetic acid in molasses. Unionized acetate (which occurs at lower pH
values) represses spore germination. The pH is therefore brought to 5-7 to ionize
acetic acid and make germination of spores favorable. This is not a problem when
synthetic media are used. A pH as low as 2.5-3.0 can be used in the case of refined
media. The sugar concentration of the medium is maintained at 15%. This level
represents a good compromise. Sugar contents above 15% lead to increased residual
sugar and accumulation of oxalic acid in the beer while sugar contents below 15%
result in reduced yield.
Additional nitrogen requirement for the fermentation can be met by supplying
(NH4)2SO4. After normal sterilization procedure the medium is inoculated with
spores of Aspergillus niger grown separately. The concentration of spore is typically
105-107 per ml of medium.
The spores of the culture are prepared in a stepwise manner from lyophil vials. The
first step is to grow the organism in special sporulation agar. Thereafter it is
transferred to other suitable medium for either mycelium build up or spore
production. Both the forms (spores and mycelia) can be used for the main
fermentation. When spores are to be used, they are usually suspended in suitable
wetting agents, such as Tween 80, as the carrier.
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Fermentation is carried out in very high-purity shallow aluminum trays. The ratio of
medium volume to surface is maintained 1.22 ml/cm2 for maximum performance.
The organism is highly aerobic and hence must by supplied with adequate amount of
oxygen. This is met by supplying air at the rate of 0.5-1.5 vol/vol/min. The
inoculated trays are stacked in racks kept in rooms or compartment with provision
for ventilation and continuous aeration. The temperature is maintained at around
30°C and the duration of fermentation ranges from 7 to 14 days. The final broth
contains about 7% citric acid.
The fermentation must be continuously controlled. Laboratory analysis must be
periodically carried out for citric acid and residual sugar contents.
Recovery
The first step in the recovery is filtration of mycelia in rotary vacuum filter (Fig.
17.10a and 17.10b). The mycelia must be repeatedly washed with water but without
bringing about excess dilution. The citric acid in the liquor is precipitated out as
calcium citrate followed by regeneration with H2SO4. For detail of recovery, see
submerged fermentation described shortly.
3. Submerged culture method
Production
The preparation of medium is the same as that for surface culture. The inoculum is
normally in the mycelial form.
Fermentation is carried out in very high-grade stainless steel fermenters. The
fermenters can be of two main types, (i) stirred, aerated, baffled tank, and (ii) aerated tower
tank. The cultural condition is also similar to that of surface culture. Aeration is done
at a rate of 310-6 gram mole O2/ml/min. Aeration is very critical during the final
stage of fermentation: lack of O2 leads to remetabolism of citric acid. The temperature
is maintained at 30°C and the fermentation usually lasts for 4-5 days.
Stimulants are universally added in submerged fermentation. One of the most
important stimulants used in citric acid fermentation is methanol. It is added at the
rate of 3-4 % during fermentation. Methanol has been assumed to delay spore
formation and alter cell wall permeability in Aspergillus niger, thereby increasing the
yield. Antifoams (such as silicone oil, octadecanol, etc.) are also added during the
fermentation.
There are some variations in the submerged culture process. In one variation, the
fermentation is carried out in two stages, viz., growth stage and production stage in
separate vessels. In another variation, the mycelium is reused up to 3 times: this
eliminates the costly inoculum build-up stage.
Recovery
The beer is filtered in rotary vacuum filter to remove mycelia. The liquor can be
treated by any of the two different methods, viz., (i) classical method and (ii) solvent
extraction method, for refining citric acid. There are many other patented methods also.
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The classical method, which entails precipitation of citric acid with lime and
regeneration with H2SO4, is probably the most widely used method. The solvent
extraction method uses combination of solvents for extraction of citric acid and
back-extraction in alkaline aqueous phase as calcium citrate. The most commonly
used solvents are mixtures of tributyl phosphate plus kerosene, and tributyl phosphate plus
butyl acetate (100: 5-30).
The classical method of recovery
The filtered liquor is first treated with lime to selectively precipitate out oxalate. As a
rule, lime is added to the liquor in calculated amount to allow spontaneous rise in
temperature to 80-90°C. The precipitated calcium oxalate is removed by filtration and
the liquor treated again with hydrated lime. Hydrated lime is added to the liquor at
controlled rate (1 part hydrated lime per 2 parts liquor) over a period of 1 hr until the
temperature of the liquor reaches ~ 95°C. This event precipitates out citric acid as
calcium citrate. The insoluble calcium citrate is separated from the liquid portion by
filtration and the precipitate treated with equivalent amount of H2SO4 to regenerate
citric acid. The gypsum (calcium sulfate) resulting from this treatment is thrown away.
The final steps of purification consist of decolorization, crystallization, drying, and
packing. See Fig. 22.1 for an outline of recovery by classical process.
Filtered broth Lime
Decolorizing
Oxalate precipitation
Concentration
Filtration Hydrated lime
Ca-oxalate Crystallization Mother liquor
Ca-citrate precipitation
Centrifugation
Filtration H2SO4
Waste liquid Drying
Regeneration
Gypsum Sieving
Citric acid
Packing
Fig. 22.1 Recovery of citric acid by precipitation-regeneration method
At 40°C, citric acid crystallizes out as the anhydrous acid, and below 36.5°C as the
monohydrate.
Yield of citric acid
Under practical condition, the yield of citric acid is 70-90 g per 100 g of glucose. The
theoretical yields from sucrose and glucose anhydrous are 123% and 117%
respectively.
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22.1.2 BIOSYNTHESIS OF CITRIC ACID
Citric acid produced by commercial strains of Aspergillus niger is typically an overflow
product due to faulty operation of citric acid cycle. The major route of formation is
the condensation of acetyl-ScoA and oxaloacetate. Aspergillus niger uses 78% of the
sugar via EMP (Embden-Meyerhof-Parna) pathway. Oxaloacetate, which is
constantly needed in the synthetic step, is regenerated by carboxylation of triose
compounds or by glyoxylate cycle. It has been found that the enzymes leading to the
synthesis of cis aconitate and isocitrate is rather inefficient in citric acid producing
strains. This could be one of the reasons for overproduction of citric acid.
22.1.3 USES OF CITRIC ACID
Citric acid is used in food, confectionery and beverages, in pharmaceuticals and in
industrial fields. Its use depends on three properties: acidity, flavor, and salt
formation. A summary of the uses of citric acid is given in Table 22.1.
Citric acid forms a wide range of metallic salts including complexes with copper,
iron, manganese, magnesium, and calcium. These salts are used as sequestering
agents in industrial processes and as anticoagulant blood preservative. Citric acid also
exhibits antioxidant properties in fats and oils where it reduces metal-catalyzed
oxidation by clelating traces of metals such as iron. There are two components to its
use as a flavoring: the first is due its acidity, which has little aftertaste; the second to
its ability to enhance other flavors.
Table 22.1 Application of citric acid
Industry Property Market share
Food About 75%
Beverages Acidulant
Jellies, jams, etc. Flavoring
Fats and oils Antioxidant
Frozen foods Antioxidant
Pharmaceuticals About 10%
Effervescent Acid
Vitamins Antioxidant
Anticoagulants Sequestering
Iron preparations Salt formation
Cosmetics Buffering
Industrial About 15%
Cleaning (metals) Sequestering
Detergents Buffering
Photographic Buffering
Primer binding Sequestering
Polymerizations Sequestering
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A process to remove sulfur dioxide from flue gases has been developed where citric
acid is used as a scrubber, forming a complex ion which then reacts with H2S to give
elemental sulfur, regenerating citrate. This may become more important with
increased environmental pressures.
Citric acid esters of a range of alcohols are known: the triethyl-, butyl- and
acetyltributyl- esters are used as plasticizers in plastic films and monostyryl citrate is
used instead of citric acid as an antioxidant in oils and fats.
22.2 FUMARIC ACID
Fumaric acid was formerly produced by fermentation, but now it is produced by
hydrocarbon oxidation. The microbial process has fallen out of favor. The molecular
structure of the acid is:
H C COOH
HOOC C H
Fumaric acid is crystalline in nature. It is sparingly soluble in water (0.7 g/100 ml).
Sodium- and potassium salts are readily soluble in water. The acid finds use in
acidification of beverages, manufacture of aspartic acid, aspartame (a dipeptide
artificial sweetener composed of aspartic acid and phenylalanine), and polyester
fabrics. Trade fermentation utilizes improved strains of Rhizopus nigricans.
The acid is biosynthesized in the TCA cycle but can also be formed via glyoxylate
cycle. Since ethanol can also serve as a carbon source, the pathway appears to be
more than one.
22.2.1 MICROBIAL PRODUCTION
22.2.1.1 Cultural condition
Hexose sugars are the raw materials of choice. Molasses can also be used but the
invertase activity is not possessed by all strains. The most common nitrogen source
is ammonia or urea. Minerals play a very important role in fumaric acid
fermentation. Zinc in particular should be kept at suboptimal level. Excess zinc
allows the formation of acids other than fumaric acid. Methanol is generally used as
a stimulant but the effect of methanol is quite different from that in citric acid
production: the methanol molecule gets incorporated in the fumaric acid molecule.
22.2.1.2 Fermentation
No details are available for the fermentative production of fumarate. The inoculum
is probably prepared as in the case of citric acid. The fermentation is carried out
either as submerged- or surface culture, at 28-33°C. The pH is maintained at around
5-6 with constant addition of Na2CO3 or K2CO3. Continuous neutralization is
essential because the organism is very sensitive to acidity resulting from fumaric acid
accumulation. The acid being sparingly soluble in water tends to crystallize out in
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