Organic and Fatty Acid Production, Microbial

I Goldberg and J S Rokem, The Hebrew University of Jerusalem, Jerusalem, Israel

ª 2009 Elsevier Inc. All rights reserved.

Defining Statement Fatty Acids

Introduction Conclusions
Organic Acids Further Reading

Glossary the major pathway for the complete oxidation of

eukaryotic organisms Organisms having one or more
cells, with well-defined membrane-bound nuclei.
filamentous fungi Fungi that grow as long,
multicellular strands.
genetic engineering Technique used to modify
genetic information of microorganisms, reprogramming
for a desired purpose (e.g., to produce a substance it
would not naturally produce).
global transcription machinery
engineering Approach for reprogramming gene
transcription to elicit cellular phenotypes important for
technological applications.
metabolic engineering Improvement of cellular
activities by manipulation of enzymatic, transport, and
regulatory functions of the cell with the use of
recombinant DNA technology.
metabolomics Metabolite profiling, measuring the real
outcome of the potential changes suggested by
genomics or proteomics. It investigates regulation and
metabolic fluxes in individual microorganisms.
molar yield Gram dry weight of cells or product per
mole of substrate (e.g., carbon source) utilized.
oxidative TCA cycle The tricarboxylic acid (TCA) cycle,
also known as the Krebs cycle or the citric acid cycle, is
acetyl-CoA to CO2 (catabolism) and the generation of
energy in the form of ATP. Reactions of the TCA cycle
also function in metabolic processes (anabolism) other
than energy generation.
primary metabolites Metabolites produced during and
required for growth of the microorganism.
prokaryotes Bacteria and blue green algae
(cyanobacteria) with nuclear material not limited by
membrane and DNA not organized in chromosomes.
reductive TCA cycle Biochemical pathway including
the carboxylation of pyruvic acid to oxaloacetic acid,
which can be further converted to L-malic acid, fumaric
acid, and/or succinic acid, dependent on the organism
in use. The reactions are in the reverse direction of the
oxidative TCA cycle and no energy is generated.
submerged fermentation Production of different
substances by growing producer microorganisms in
depth in liquid culture.
systems biology Systematic study of interactions in
the organism using modeling, global analysis (or ome
analysis), mapping of interactions between cellular
components, and quantification of dynamic responses
in living cells resulting in the quantitative description of
the biological system under study.

Abbreviations GRAS generally recognized as safe

2-KGA 2-keto-D-gluconic acid PHA Polyhydroxyalkonate
5-KGA 5-keto-D-gluconic acid PHB 3-hydroxybutyric acid
ARA arachidonic acid PUFA polyunsaturated fatty acids
DHA docosahexaenoic acid SCFA short chain fatty acids
EFA essential fatty acids SCO single-cell oil
ESA L-trans-2,3-epoxysuccinic acid TCA tricarboxylic acid
GLA -lineolenic acid

421
422 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial

Defining Statement
Organic acids are used as food acidulants and building
blocks for other useful chemicals. Fatty acids were
recently recognized for their beneficial health properties
and a prospective energy source. The potential of various
microorganisms to synthesize various organic and fatty
acids will be highlighted with emphasis on the existing
industrial large-scale production processes.
Introduction
(i.e., the use of natural L-malic acid in foods instead of the
chemically produced DL-malic acid), and recent require-
ments for a specific isomer of the acid for biodegradable
and natural polymer production (i.e., L- or D-lactic acid)
may result in much improved economics for the imple-
mentation of the biological route for the industrial
production of organic acids (Table 1).
This article describes the potential of various micro-
organisms to synthesize high amounts of organic and fatty
acids. It emphasizes those acids that are produced in the
industry by large-scale fermentation processes.

Organic and fatty acids are broadly distributed in nature
and were used by humans in their natural forms since early
ages. Presently, organic acids are used mostly as food
acidulants and as building blocks for other useful chemicals
of low and high molecular weight (polymers). Although
high concentrations of acids are produced by various
microorganisms, only for a few acids the microbiological
production process is important and an economic alterna-
tive to chemical synthesis. Within the world’s fermentation
market, organic and fatty acids constitute a significant
portion, especially with the production of citric acid,
L-lactic acid (see ‘Lactic acid, microbially produced’),
acetic acid (see ‘Acetic acid production’), gluconic acid,
and itaconic acid (Table 1). Recent developments in the
biotechnology field, environmental pressures, increased
recognition of the positive contribution of specific fatty
acids to health and prevention of chronic disease, steady
increase in price of petrochemicals, integration of fermen-
tation and agricultural products (e.g., corn), consumer
awareness for natural instead of chemical food constituents
Organic Acids
Organic acids are intermediates or end products of cellular
metabolism. Accumulation of various organic acids is the
basis for a varied and extensive industry, often based on
biological production methods. The majority of the organic
acids discussed in this article are produced and excreted to
the medium at high concentrations by filamentous
fungi such as Aspergillus sp. (citric, L-malic, gluconic, itaco-
nic, L-trans-2,3-epoxysuccinic, gallic, and kojic acid) and
Rhizopus sp. (fumaric, L-lactic, and gallic acid). Most of
these acids are intermediate metabolites in the primary
biosynthetic metabolism and, therefore, do not accumulate
under normal growth conditions (primary metabolites).
Stress appears to be a common requirement for the unu-
sually high production and accumulation of these acids,
since under stress conditions most of the carbon source is
used for acids production (metabolite) rather than increase
in biomass production (growth). Other acids (2-ketogluco-
nic, 5-ketogluconic, acrylic, and adipic acid) are produced

Table 1 Production of selected organic acids by chemical and biological routesa

Acid Annual production (tonnes) Chemical process Biological process Year

Food acids
Citric acid 1 600 000  þ 2007
Acetic acid 10 000 000 þ þ 2005
Lactic acid 240 000  þ 2006
D-Gluconic 87 000b  þ 2004
L-Tartaric 35 000  þ 2003
Malic acid 60 000 þ þ 2006
Ascorbic acid 110 000 þ (þ) 2001
Fumaric acid 90 000 þ  2008
Building blocks
Polyhydroxyalkonates 50 000  þ 2006
Itaconic 30 000  þ 2006
Succinic acid 15 000 þ þ 2003
Acrylic 3 400 000 þ  2005
Adipic 2 760 000 þ  2004
Propionic acid 176 000c þ 2003
a
The estimated annual world production of the various acids is based on information in the published literature. The order of the acids is based on the
volume produced by biological routes.
b
The worldwide consumption of D-gluconic acid based on major industrial applications.
c
Estimated consumption in the United States.
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial 423

by bacteria under aerobic conditions, while succinic acid is
produced by bacteria under anaerobic conditions. The
formation of the latter acid is usually the way by which
these bacteria regenerate NAD from the accumulated
NADH, and thus the accumulation of succinic acid strictly
parallels and is required for growth.
Food Acids
Citric acid
Citric acid (2-hydroxy-1,2,3 propanetricarboxylic acid,
C6H8O7) (Figure 1) is a white or colorless, odorless,
crystalline solid. It is highly soluble in water, freely solu-
ble in ethanol, and slightly soluble in ether. Citric acid has
three carboxyl groups and, therefore, is a good buffer for
pH control.
O OH
O O
HO OH
OH
Figure 1 Citric acid.
The discovery of citric acid has been credited to the
eighth century alchemist Jabir Ibn Hayyan. The acid
was first isolated in pure form in 1784 by Carl Wilhelm
Scheele, who crystallized it from lemon juice.
Citric acid is found in almost all plants and in many
microorganisms and animal tissues and fluids. It is a key
element in the physiological oxidation of fats, proteins,
and carbohydrates to carbon dioxide and water.
Citric acid, with very low toxicity, is mainly used as a
flavoring agent and preservative in food and beverages,
bestowing tartness to fruit and soft drinks. Citrate salts of
different metals are used to deliver these metals, in a
biologically available form, in food supplements. Citric
acid is used in household cleaning chemicals and phar-
maceuticals, for storage of blood, in tablets and ointments,
and also in cosmetic preparations. It acts as a bacterial
inhabitant and as an antioxidant.
The biosynthesis of citric acid has been well studied;
however, all the reactions that lead to citric acid are still
not fully understood. Citric acid is a primary metabolite
formed in the tricarboxylic acid (TCA) cycle, discovered
in 1937 by Hans Adolf Krebs (Figure 2). It has been
known for a long time that there is a condensation of a

O NAD + NADH O
–
CH3 C COO CH3 C SCoA
Pyruvate AcetylCoA
CoASH CO2 CoASH
DH complex
– –
COO CH2COO
NADH –
C O HO C COO
– Citrate –
CH2COO synthase CH2COO
NAD + Oxaloacetate Citrate
Malate Aconitase
DH
– –
COO CH2COO
–
H C OH H C COO
– –
CH2COO HO CHCOO
Malate Isocitrate
Kreb’s
NAD +
Fumerase TCA cycle Isocitrate
H2O NADH DH
– –
H COO CO2 CH2COO
C
H C H
C –
C COO
–OOC H
Fumerate O 2-oxo glutarate
NAD +
Succinate GDP NADH
DH GTP + Pi CoASH
–
FADH2 – CH2COO DH complex
CH2COO
– CH2 CO2
FAD CH2COO
C SCoA
Succinate CoASH O Succinyl CoA
Succinyl CoA synthetase
Figure 2 The tricarboxylic acid (TCA) cycle (also Krebs cycle and citric acid cycle). With permission from Dr. R. Paltel, Humboldt State
University, CA, USA.
424 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
C4 moiety with a C2 moiety, both metabolites of pyruvic
acid, to form the C6 molecule of citric acid. In high-
glucose media the high yields obtained for citric acid
are explained by two different reactions occurring with
pyruvic acid. One of the two pyruvic acid molecules (C3),
formed by the glycolytic pathway from glucose (C6), is
converted to acetyl CoA (C2). The other molecule is
carboxylated by a reaction catalyzed by pyruvate carbox-
ylase, which is a part of the reductive direction of the
TCA cycle and found to occur in the cytosol in Aspergillus
niger (Figure 3). The product of this reaction is oxaloa-
cetic acid (C4). This acid is first reduced to malic acid
(C4), and enters the mitochondria via a malate–citrate
transporter, converted to malic acid, which together
with acetyl CoA will form the citric acid. The enzyme
performing this reaction, citrate synthase, is only present
in the mitochondria.
The current world production of citric acid is esti-
mated to be 1.6 million tonnes per year (Table 1),
whereof 99% is produced by microbial fermentation.
Several microbial species are able to accumulate citric
acid during primary metabolism; both fungi, such as
A. niger, Aspergillus wentii, Penicillium luteum, and
Trichoderma viride, yeast like Candida guilliermondii, and
Saccharomycopsis lipolytica, and bacteria such as
Glucose
CO2
Pyruvate Pyruvate
PYC PDH
CO2
Oxaloacetate
Oxaloacetate
MDH
MDH
L-malate
L-malate
FUM FUM
Fumarate
Fumarate
FRD
SDH
Succinate
Cytosol
Figure 3 The reductive reactions of the TCA cycle. The abbreviations of the enzymes are ACO, aconitase; CIT, citrate synthase; FUM,
fumarase; FRD, fumarate reductase; IDH, isocitrate dehydrogenase; KGD-, ketoglutarate dehydrogenase; MDH, malate
dehydrogenase; PDH, pyruvate dehydrogenase; PYC, pyruvate carboxylase.
Arthrobacter paraffineus and Corynebacterium sp. For produc-
tion, A. niger is used (Figure 4).
Modern fermentation technologies yield 95 kg of citric
acid from 100 kg of sugar supplied, with final concentra-
tions of over 200 g l1. The modifications that have led to
the high producing industrial strains are well-kept secrets;
however, mutant isolations by academic laboratories
include high sugar tolerance and growth and acid produc-
tion at low pH. This is an example where fermentation of
sugar(s) is converted to an end product (citric acid) at
very high efficiency. The medium for optimal production
of citric acid should contain a sugar concentration of
between 120 and 250 g l1, a nitrogen source (ammonium
salts) over 2 g l1, a phosphate concentration between 0.2
and 1.0 g l1, Mn < 108 mol l1, Zn < 106 mol l1 and
Fe < 104 mol l1. The dissolved oxygen concentration
should be > 150 mbar, and the pH is 1.6–2.2.
As can be seen for other acids of the TCA cycle
(fumaric and L-malic acid, see below), the high molar
yield values obtained by the different filamentous fungi
are due to the presence of a compartmentalized (in this
case cytosolic) activity of a carboxylation enzymatic reac-
tion, resulting in the fixation of carbon dioxide by the
unique pyruvate carboxylase (in most eukaryotic cells
located in the mitochondria) (Figure 3).
AcetylCoA Mitochondrion
Citrate
CIT
ACO
Isocitrate
IDH
α -Ketoglutarate
KGD
Succinate
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial 425

Figure 4 Aspergillus niger. (Left panel) Slide culture, 400. With permission of H. Mirhendi, Tehran University of Medical Sciences,
Tehran, Iran. (Right panel) Scanning electron micrograph of asexual reproductive apparatus. Partially freeze-dried. Reproduced from
Read ND (1991) Low temperature scanning electron microscopy of fungi and fungus-plant interactions. In: Mendgen K and Leseman D-
E (eds.) Electron Microscopy of Plant Pathogens, pp. 17–29. Berlin: Springer Verlag, with permission from Springer Science and
Business Media.

Two methods are used by industry to manufacture
citric acid, the submerged fermentation process
(Figure 5) and the surface method. Small amounts are
made by solid-state fermentations, mainly in East Asia.
The main industrial organism in use today is A. niger.
Before the 1973 oil crises, use of yeast grown on n-alkanes
under submerged conditions was also in use. The yeast
most in use was Yarrowia lipolytica grown on straight chain
paraffins (see also ‘Isocitric acid’).
The surface method is still in use, being less labor- and
energy-intensive and with much lower susceptibility to
trace metal ions. The fermentation is carried out in alu-
minum trays, where spores are dispersed on the surface of
the nutrient medium. The fungus forms a mat and yields

Nutrients

Steam

Molasses Molasses Medium

tank dilution preparation

Filter Water
Medium Inoculum
sterilization fermenter

Air
filter Main
fermenter

Air composer

Waste Ca(OH)2
mycelium H2SO4

Broth
storage

CaSO4

To
effluent To crystallisation
treatment

Figure 5 Manufacturing of citric acid by a submerged process. Reproduced from Meers JL and Milsom PE (1987) Organic acids and
amino acids. In: Bu’lock J and Kristiansen B (eds.) Basic Biotechnology, 1st edn., pp. 359–383. London, UK: Academic Press, with
permission from Elsevier.
426 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
reported are between 0.7 and 0.9 g g1 of sugar after 1–2
weeks.
The submerged fermentation has higher efficiency and
is easier to automate. The carbon source, usually from
renewable resources, needs to be analyzed for trace
metals, and if present, these should be removed, to avoid
inhibition of citric acid accumulation. Both aerated
towers, where the air enters at the bottom of the vessel,
and stirred tank fermentors are in use by industry. The
required growth mode is the formation of pellets made of
the fungal mycelium, with a diameter of 0.2–0.5 mm, that
sediment swiftly at harvest. The low pH of the process is
critical for obtaining a high yield of citric acid and to
avoid the potential accumulation of oxalic acid. The
initial inoculum for vegetative growth is, in most cases,
spores that require a pH above 5.0 in order to germinate.
For production, the pH should be <2, which also reduces
the risk for contamination.
The purification of citric acid from the fermentation
broth starts with filtration with the help of filter aids to
obtain good separation of the citric acid containing broth
and the mycelium (Figure 5). To avoid oxalic acid in the
final product, lime (calcium hydroxide) is added at low
pH and calcium oxalate precipitates and is separated
from the liquid. Citric acid is then precipitated by
increasing lime concentration and raising the temperature
to 70–90  C and the pH to 7.2, followed by collection of
calcium citrate using rotating filters. To obtain higher
purity of citric acid, sulfuric acid is added, reprecipitating
calcium sulfate. The citric acid is further treated with
activated carbon, cation exchangers, and anion exchan-
gers and finally crystallized either as citric acid or as citric
acid monohydrate. The use of solvent extraction (alipha-
tic alcohols and ketones, amines and phosphines with
hydrocarbons are mainly used) for citric acid purification
has been introduced more recently and is also used by
industry.
The recent sequencing of an A. niger strain has shed
more light on the potential biochemical mechanisms
involved in acid accumulation; however, it should be
noted that the sequenced strain is not known to be a
major producer of citric acid. Genes coding for several
of the enzymes involved in the crucial formation of oxa-
OH OH O
HO
OH
OH OH
Figure 6 Gluconic acid.
good chelator at high pH, with better activity than com-
monly used chelators.
Gluconic acid was discovered in 1870 by Hlasiwetz
and Habermann, when glucose was oxidized with chlor-
ine. In 1922 it was isolated from a strain of A. niger. Later,
other filamentous fungi, such as Penicillium, Scopulariopsis,
Gonatobotrys, and Gliocladium, and also oxidative bacteria,
such as strains of Pseudomonas, Gluconobacter (Acetobacter),
Moraxella, Micrococcus, Enterobacter, and Zymomonas were
found to produce gluconic acid. Already in the 1940s it
was possible to obtain good yields of gluconic acid using
A. niger by fermentation, neutralizing the accumulating
acid with calcium carbonate.
The physiological functions of gluconic acid accumu-
lation for these organisms are not clear; one possibility is
its contribution to the competitiveness of the organism,
removing glucose from the close environment. In the case
of P. expansum (a phytopathogenic fungus), it was demon-
strated that secreted gluconic acid contributed to the
colonization and disease development of apple tissues
by this fungus.
Gluconic acid is used in the manufacture of metal,
leather, and food. It has been accredited with the cap-
ability of inhibiting bitterness in foods. Sodium gluconate
is permitted in food and it has GRAS (generally recog-
nized as safe) status. This salt is also utilized as a
sequestering agent in many detergents, and added to
cement to improve the hardening process.
The formation of gluconic acid is different from most
other organic acids, since it is formed outside the cyto-
plasmic membrane, by the enzyme glucose oxidase. This
enzyme has been shown to be localized in the cell wall,
at least for fungi known to accumulate gluconic acid.
Glucose in the medium is oxidized in a two-step
reaction to gluconic acid; first glucose oxidase oxidizes
loacetic acid were found. The identification of the various
activities comprising citric acid metabolism and the
implicated transporter genes provides the basis for a
more complete understanding of the efficient accumula-
tion of citric acid by A. niger.
Gluconic acid
Gluconic acid (2,3,4,5,6-pentahydroxy caproic acid,
C6H12O7) (Figure 6) is a noncorrosive, nontoxic, mild
organic acid with a brown clear appearance. It is very
soluble in water and has a mild and refreshing taste. It is a
-D-glucopyranose to D-glucono-1,5 lactone with the for-
mation of hydrogen peroxide, acted upon by catalase to
form water and oxygen (Figure 7). The hydrolysis of the
lactone is spontaneous in aqueous solutions, but occurs six
times faster with the enzyme gluconolactonase, resulting
in gluconic acid (Figure 7).
The main route of gluconic acid production has been
by fermentation, mainly using A. niger in submerged fer-
mentations. The two most important parameters of the
fermentation is a high concentration of dissolved oxygen,
used directly in the biosynthesis, and keeping pH 4.5–6.5,
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
β -D-glucose
FAD
Glucose
oxidase FADH2
Glucono-δ-lactone
Lactonase/
spontaneous H2O
Gluconic acid
Glucose + ½O2
Figure 7 Biosynthesis of gluconic acid. Reproduced from Ramachandran S, Fontanille P, Pandey A, and Larroche C (2006) Gluconic
acid: Properties, applications and microbial production. Food Technology and Biotechnology 44: 185–195.
traditionally achieved by addition of calcium carbonate as
the neutralizing agent. Best results are obtained with a
high glucose concentration (110–250 g l1), low concen-
trations of nitrogen and phosphorus (<20 mmol l1), and
low concentrations of metal ions. Fermentations with
almost quantitative yield (>90% on a molar basis) are
completed in less than 24 h. The fact that the oxidation
reactions occur outside of the cells allows for reuse of
the mycelium up to 14 times. In the current industrial
method, neutralization with NaOH allows for the use of
higher initial sugar concentrations (up to 350 g l1) and
the pH is then maintained close to 6.5. Recently, enzy-
matic processes have been introduced by conversion of
glucose syrups with less formation of byproducts and
resulting in fewer problems in the downstream process.
There are alternative ways of gluconic acid production
by chemical, electrochemical, and bioelectrochemical
routes, but with lower yields than the fermentation pro-
cesses. Bacteria, mainly Gluconobacter oxydans, are reported
to be used by industry. Recently, a process based on
Acetobacter methanolicus was developed. The process uti-
lizes methanol as the carbon source for growth, with the
addition of glucose for its conversion to gluconic acid.
L(þ)-Tartaric acid
L(þ)-Tartaric acid (L-2R,3R-dihydrobutanedioic acid,
C4H6O6) (Figure 8) derives its name from the medieval,
alchemical term tartarus. It is a white, crystalline powder,
odorless, and with an acidic taste. It is a strong organic
acid, widely distributed in nature, and classified as a fruit
acid (it is the most expensive fruit acid). The acid has two
stereogenic atoms and it exists in three stereoisomeric
forms – L(þ), D(), and the DL-racemic tartaric acid,
which is distinct from the meso-tartaric acid. Although
the dextrorotatory D()-isomer is the ‘unnatural’ form
of the acid, its occurrence in small amounts in nature
has been demonstrated. L-Tartaric acid is present in its
427
H2O2
Catalase
½O2
½O2
Gluconic acid
free form or combined with potassium, calcium, and
magnesium. It is highly soluble in water, methanol, etha-
nol, and glycerol but is insoluble in chloroform.
Although tartaric acid was first isolated by the alche-
mist Jabir Ibn Hayyan, Carl Wilhelm Scheele is credited
for its discovery in 1769.
L-Tartaric acid is present in the juices of various fruits,
particularly in tamarinds, unripe grapes, and is one of the
main acids in wine.
L-Tartaric acid is an extremely versatile acid and it is
utilized in a wide range of industries. The largest single
application for tartaric acid is as a raw material for
the manufacturing of emulsifiers used for bread improve-
ment. An important salt of tartaric acid, potassium
hydrogen tartarate (or cream of tartar), has applications
as an acidulant for baking powder and sugar confection-
ery. Tartaric acid is used in the food industry (in
particular, in jams, fruit juices, pickles, soft drinks, etc.),
in the production of various construction materials, and in
certain medicines as an inert bulk substance, since it is not
metabolized by the human body. The potassium sodium
salt, called Rochelle salt, was the first compound used as a
piezoelectric crystal. The acid is a useful raw material for
the synthesis of other chiral molecules (e.g., L(þ)-, D()-
tartaric acids are used as chiral auxiliary reagents in the
oxidation of alkenes to enantiomerically pure epoxides).
Tartaric acid can be produced by natural and synthetic
routes. The natural route involves the recovery of the
reddish precipitated salt, potassium bitartarate, from
argol, the sediment in wine vats. The synthetic chemical
route involves the production of the racemic mixture of
tartaric acid from maleic anhydride.
Since the availability of L(þ)-tartaric acid from wine is
dependent on the climate, and because the chemical pro-
cess results in the racemic mixture of the acid, attempts
have been made to explore alternative biological ways of
producing the high-cost tartaric acid. Several patents and
428 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
OH O temperature (180–220  C) and high pressure (14–18 bar),
HO
OH
O OH
Figure 8 L-Tartaric acid.
reports suggest that various bacteria (such as
Corynebacterium sp., Rhodococcus sp., Alcaligenes levotartaricus,
Acinetobacter tartaricus, and Pseudomonas agrobacterium)
show a high enzymatic activity of the stereo-specific
cis-epoxysuccinic acid hydrolase to form L(þ)-tartaric
acid. Industrial processes based on these organisms are
not known.
Malic acid
Malic acid (2-hydroxybutanedioic acid, C4H6O5)
(Figure 9) is a white, odorless, crystalline solid. In con-
trast to other fruit acids, it is very hygroscopic and has a
tendency to lump. Malic acid is a dicarboxylic acid and
has an asymmetric carbon and occurs as L(the natural)-
and D-isomers.
Malic acid was first described by Sheele who, in 1785,
isolated this acid from unripe apples. The name malic is
from the Latin for apple, malum.
Malic acid is found in other fruits such as grapes, water-
melons, cherries, and in vegetables such as carrots and
broccoli. This acid is mainly used in food applications
including candy and beverages. It gives a tart taste, lowers
the pH, has antimicrobial effects, and confers special
blending and flavor-fixing properties. There are also non-
food applications such as use for metal cleaning and
finishing, textile finishing, electroless plating, pharmaceu-
ticals, infusions, and paints.
The biosynthesis of L-malic acid from carbohydrates
(glucose) is similar to citric and fumaric acid, where the
carboxylation of pyruvic acid leads to oxaloacetic acid
that is reduced to L-malic acid (Figure 3). The carbox-
ylation reaction that leads to L-malic acid accumulation
allows for a high acid molar yield (based on the glucose
utilized).
World production of malic acid is estimated at around
60 000 tonnes per year (Table 1), most of this is made by
chemical hydration of maleic or fumaric acid, using high
O HO
HO
OH
O OH
Figure 9 L-Malic acid.
yielding the racemic mixture of DL-malic acid.
Biological processes result in the L-isomer of the acid. In
one process, species of Brevibacterium flavum or
B. ammoniagenes with high fumarase (fumarate hydratase)
activity are immobilized, in either polyacrylamide or
-carrageenan, and are used to convert fumarate to
L-malate. In another efficient process, the yeast
Saccharomyces cerevisiae was amplified for the enzyme fumar-
ase by cloning the single homologous nuclear gene
downstream of a strong promoter. The overproducing strain
can be used as free cells or can be immobilized (e.g., in
agarose beads or microspheres); it converted fumarate to L-
malate at a maximal rate of 65 mmol l1 g1 h1, with a yield
of approximately 85%.
After optimization of the fermentation conditions, fila-
mentous fungi, such as Aspergillus flavus using glucose,
accumulate over 110 g l1 of L-malate. Other species
(e.g., Aspergillus oryzae and A. niger) have also been found
to accumulate L-malic acid from glucose. Since these
latter fungi are GRAS organisms, these are suitable for
food applications. The one-step fermentation of sugars to
L-malic acid is not practiced by industry as yet.
L-Ascorbic acid (vitamin C)
L-Ascorbic acid (3-keto-L-gulofuranolactone, vitamin C,
C6H8O6) (Figure 10) is a colorless crystal, white or very
pale yellow crystalline powder. It is sensitive and
will decompose at prolonged exposure to light and air.
Vitamin C is an essential nutrient for higher primates and
its deficiency will cause scurvy, probably due to the
stimulation, by vitamin C, of the oxidation enzymes (oxi-
dases) involved in the formation of collagen. The gene
encoding l-gulono-1,4-lactone oxidase is absent in these
animals, and therefore an external source of vitamin C is
necessary. L-Ascorbic acid is a strong antioxidant and it
protects toward oxidative stress and has been implicated
to protect tissues and hinder cardiovascular disease and
cancer.
In 1928, Albert Szent Györgi discovered vitamin C
(the third vitamin found, and given the third letter in
the alphabet) and solved a long debate over the active
ingredient with antiscurvy activity. The name ascorbic
acid is derived from a scorbuticus meaning against scurvy.
Vitamin C was the first vitamin to be artificially synthe-
sized in 1935.
HO
O O
HO OH
Figure 10 L-Ascorbic acid.
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial 429

L-Ascorbic acid is found in citrus fruits such as lemon,
lime, and orange and also in vegetables such as cabbage
and processed cabbage (sauerkraut). Long before the iso-
lation of L-ascorbic acid, it was known that including
lemons and oranges in the diet prevented scurvy.
L-Ascorbic acid is used on a large scale (110 000 tonnes
in 2001, see Table 1) as an antioxidant in food, animal
feed, beverages, pharmaceutical formulations, and cos-
metic applications.
The manufacture of L-ascorbic acid is by the
Reichstein–Grüssner synthetic pathway introduced in
1934 (Figure 11). It is a chemical process with one step
carried out by biocatalysis; conversion of D-sorbitol to
L-sorbose by the bacterium Gluconobacter suboxydans (or
by the formerly used Acetobacter xylinum). The yield of
ascorbic acid from the starting material glucose is esti-
mated to be around 50%. The process is energy
consuming with high temperatures and pressures neces-
sary for some steps of the process. Economic factors have
given rise to tremendous interest for use of alternative
processes.
There are several alternatives based on biological
processes with 2-keto-L-gulonic acid (2-KLG) as the
key intermediate. Potential use of the genera
Gluconobacter (Acetobacter), Ketogulonicigenium, Pseudomonas,
Erwinia, and Corynebacterium has been looked into
furthermore. In the so-called two-stage process, the che-
mical reactions of the Reichstein–Grüssner process to
produce di-acetone-ketogulonic acid are replaced by a
second fermentation step, which results in the formation
of 2-KLG (Figure 12).
Another biological route, the ‘one organism route’, has
great potential to become commercialized. Here glucose is
converted to 2-KLG in one-step fermentation process,
using Erwinia sp. (able to convert glucose efficiently to
2,5-diketo-D-gluconic acid) engineered with a gene
encoding 2,5diketo-D-gluconic acid reductase from
Corynebacterium glutamicum, resulting in more than 120 g l1
of 2-KLG formed in 120 h of fermentation (Figure 13).
Although biotechnological processes are being exploited
for the production of ascorbic acid and Reichstein–
Grüssner intermediates, the future goal should be the total
replacement of the chemical route by efficient
biocatalytical systems for production, using cost-saving
starting materials.
Fumaric acid
Fumaric acid (2-butenedioic acid trans, C4H4O4)
(Figure 14) derives its name from the fact that the acid
is found in plants that belong to the genus Fumaria, a
common European herb. Fumaric acid is the trans-isomer
of symmetric, unsaturated dicarboxylic acid; the cis-iso-
mer is maleic acid. It is produced as a colorless, crystalline
powder with a fruit-like taste (a fruit acid), and it is a weak
acid which forms diesters, has low solubility in water, and
it undergoes additions across the double bond.

CH2OH CH2OH CH2OH CH2OH

HO–/KMnO4 or
HO HO O O NaOCl/nickel
catalyst or
HO H2/cat HO Acetobacter HO O O air oxidation
OH OH Suboxydans OH H+ O
HO HO HO O
CHO CH2OH CH2OH H2C–O

D-glucose D-sorbitol L-sorbose Diacetone-L-sorbose

H3O+, Δ

COOH COOH COOMe CO

O O O HO

O H3O+ HO MeOH HO MeO– HO

O OH H+ OH O
O HO HO HO
H2C–O CH2OH CH2OH CH2OH

Diacetone-2-keto 2-keto-L-gulonic Methyl 2-keto- Ascorbic acid

L-gulonic acid acid (2-KLG) L-gulonate

Figure 11 The Reichstein–Grüssner process for the synthesis of L-ascorbic acid. Reproduced from Anderson S, Berman Marks C,
Lazarus R, et al. (1985) Production of 2-keto-L-gulonate, an intermediate in L-ascorbate synthesis, by a genetically modified Erwinia
herbicola. Science 230: 144–149, with permission from AAAS.
430 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial

Bacteria and in vitro

(1) in vitro by Reichstein-Grüssner’s method
Diacetonesorbose Diacetone-2KLGA
(2) Sugisawa/ Hoshino
PQQ-SLDH FAD-SDH
D-Sorbitol L-Sorbose L-Sorbosone
Gluconobacter Gluconobacter
Gluconobacter
PQQ-SNDH
Acetobacter
NAD(P)-SNDH
Pseudomonas
+H2/Catalyst L-Gulonate

PQQ-SLDH
5-Keto-D-gluconate
Gluconobacter
PQQ- (4) Gray Esterification
GDH Gray’s method Gray’s method 2-Keto-L- Lactonisation
D-Glucose D-Gluconate D-ldonate L-Ascorbic acid
Gluconobacter gulonate in vitro
Erwinia FAD- (3) Sonoyama CH2HO
Pantoea G-2-DH H OH O
Pseudomonas 2-Keto-D-gluconate
Gluconobacter O
Erwinia
FAD- H
Pantoea
2-KGADH
Pseudomonas
2,5-Diketo- 2,5-DKGR OH OH
D-gluconate Corynebacterium

Figure 12 Alternative processes for L-ascorbic acid production. Reproduced from Bremus C, Herrman U, Bringer-Meyer S, and
Sahm H (2006) L-Ascorbic acid production. Journal of Biotechnology 124: 196–205, with permission from Elsevier.

140
Fumaric and maleic acids were discovered in 1817 by
Braconnet and independently by Vauquelin during dry
120 distillation of malic acid.
100 Fumaric acid is widely used in the food industry as an
2-KLG Conc. (g/L)

acidulant because it is nontoxic and is the least expensive

80
of the food-grade acids. Fumaric acid solubility in water is
60 low (0.6 g per 100 g at 25  C), and, therefore, in order to
increase its application in various foods a cold water-
40
soluble (CWS) fumaric acid, which contains a wetting
20 agent, for example 0.3% w/w dioctyl sodium sulfosucci-
0
nate, is used. Fumaric acid is used for the industrial
0 20 40 60 80 100 120 preparation of L-malic acid catalyzed by the enzyme
Time (h)
fumarase (see ‘Malic acid’) and L-aspartic acid, a compo-
Figure 13 Formation of 2-KLG from glucose in a single nent of aspartame, by the enzyme aspartase. Other
production host. Reproduced from Chotani G, Dodge T, Hsu A, industrial uses of fumaric acid are in jet printing inks,
et al. (2000) Commercial production of chemicals using pathway
engineering. Biochimica et Biophysica Acta 1543: 434–455, with
plastics surface coating and paper sizing, and as an inter-
permission from Elsevier. mediate in the preparation of unsaturated polyester and
alkyd resins. Fumaric acid is used by the pharmaceutical
industry to produce alexipharmic sodium dimercaptosuc-
cinate and ferrous fumarate, as an optical bleaching agent,
in formulations for alternative medicine or as fumaric acid
O
esters monoethylfumarate and dimethylfumarate to treat
OH
psoriasis.
Fumaric acid does not accumulate under normal
O growth conditions of microorganisms and its synthesis
OH is carried out through succinic acid by the mitochondrial
Figure 14 Fumaric acid. oxidative TCA cycle (Figure 2). Various species of the
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
filamentous fungus Rhizopus (such as Rhizopus nigricans
and Rhizopus oryzae) produce high concentrations of this
acid, which is excreted to the medium. In Rhizopus, most
of the fumaric acid is formed from pyruvic acid via a
carboxylation reaction, yielding oxaloacetic acid, which
is converted directly to L-malic acid and then to fumaric
acid. These reactions, which are catalyzed by pyruvate
carboxylase, malate dehydrogenase, and fumarase,
respectively, are localized in the cytosol and are part of
the reductive TCA cycle (Figure 3), carried out under
aerobic conditions. The production of fumaric acid from
glucose through the reductive reactions of the TCA
cycle does not provide net energy and is balanced for
carbon, oxygen, and hydrogen. Therefore, part of the
pyruvic acid must be utilized through the oxidative
TCA cycle to provide energy, mainly for maintenance
requirements. It should be noted that, in general, pyru-
vate carboxylase in eukaryotic organisms is localized in
mitochondria, while in certain acid producing filamen-
tous fungi (e.g., Rhizopus and Aspergillus), the enzyme is
situated exclusively in the cytosol and in some cases in
both compartments. The cytosolic localization of this
enzyme appears to be important for the ability of these
organisms to accumulate high concentrations of organic
acids.
In the 1940s, fumaric acid was made by fermentation
on a commercial scale (about 4000 tonnes per year) using
a strain of the fungus Rhizopus arrhizus (later named R.
oryzae). It was the first submerged fermentation process
with molds and served as a model for implementing and
scaling up the techniques used for submerged fermenta-
tions. The biological production of fumaric acid was
terminated when the chemical synthesis became
economically more attractive. Fumaric acid is a symme-
trical molecule having no isomers and, therefore, the
biological process offers no specific advantage over the
chemical process. Fumaric acid is presently produced by a
chemical process through the isomerization of maleic acid
(or maleic anhydride), obtained from a catalytic vapor-
phase oxidation of benzene or C4 hydrocarbons.
The accumulation and excretion of fumaric acid from
glucose by R. oryzae (about 100 g l1 of fumaric acid)
occurs under aerobic conditions in a high-glucose (an
initial concentration of 120 g l1) medium containing a
limited amount of nitrogen and a neutralizing agent
(CaCO3). Fumaric acid (molar yield of approximately
100%; moles acid produced per mole of glucose utilized
 100), L-malic acid (15 mol%), and succinic acid
(5 mol%) are the major acids formed during fermentation.
C4 acid (fumaric, L-malic, and succinic acid) molar yields
of 120–145% are obtained after 4–5 days. The high
fumaric acid molar yield and C4 acid molar yield confirm
that these acids are produced via a carboxylation reaction
of pyruvic acid.
431
Isocitric acid
Isocitric acid ((1R,2S)-1-hydroxypropane-1,2,3-tricarbox-
ylate, C6H8O7) (Figure 15) is a TCA intermediate and an
isomer of citric acid. It exists in four isomers and is a chiral
molecule, and thus has two enantiomers of each of the
isomers.
Isocitric acid (the ()-D-threo-isomer) is synthesized
by all cells as it is an intermediate of the TCA cycle
(Figure 2). Isocitric acid is synthesized from citric acid
via the intermediate cis-aconitic acid by the enzyme aco-
nitase (aconitate hydratase). Isocitric acid is a minor
organic acid found in most fruit juices, especially in black-
berries, youngberries, and boyberries, and in vegetables,
especially in carrot. The determination of D-isocitric acid
has become of importance in the analysis of fruit juices for
the detection of illegal additives (adulteration). Since the
quantities of citric (see ‘Citric acid’) and isocitric acids are
correlated in fruit juices, a too high ratio of citric to
isocitric acid indicates the addition of citric acid.
Isocitric acid is mostly used in the food industry (food
additive) as a food acidulant, and because of being a chiral
molecule, isocitric acid is used in the pharmaceutical
industry and as a potential raw material for synthesis of
expensive chemicals.
Racemic isocitric acid was first prepared by Fittig and
Miller in 1889 by the condensation of chloral with sodium
succinate in the presence of acetic anhydride and by the
subsequent hydrolysis of the resulting trichloromethylpar-
aconic acid to isocitric acid; this is isolated as the lactone.
Formation of citric and isocitric acid by yeasts using
various carbon sources has been the focus of investigations
for the past four decades. Most of the research was direc-
ted for production of citric acid while isocitric acid was
considered as an undesirable by-product. Y. (Saccharomyces
or Candida) lipolytica, several Candida species (such as
Candida oleophila, Candida parapsilosis, Candida brumptii,
C. guilliermondii, Candida zeylanoides, Candida pulcherrima,
and Candida chalmersi), Pichia polymorpha, and Hansenula
pelliculosa are examples of yeasts that are capable of produ-
cing different citric acid/isocitric acid ratios and quantities
using various carbon sources, such as hexadecane, tetra-
decane, glucose, n-paraffins, calcium acetate, calcium
butyrate, soybean oil, fish oil, canola oil, ethanol, and
glycerol. Fermentation conditions, type of carbon source,
COOH
1
H C OH
HOOC
2
C H
3
CH2
COOH
Figure 15 Isocitric acid.
432 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
and the organism utilized determine the amount of iso-
citric acid formed. It was reported that with Y. lipolytica
grown on canola oil, up to about 75 g l1 of isocitric acid
and 110 g l1 of citric acid were produced. It can be
expected that an increase in isocitric acid synthesis, on
the expense of citric acid, can be obtained in a mutant of Y.
lipolytica having an amplified activity of aconitase. This
strain could then be used more economically than the
wild-type strain, for the commercial production of isoci-
tric acid.
The complete genome sequence of Y. lipolytica has
been determined and is opening up the potential for use
of this yeast to produce specific acids (i.e., isocitric) at
high productivity.
In the early 1970s, polyhydroxybutyrate was manufac-
tured by the use of Alcaligenes sp., accumulating up to 70%
of cell dry weight as polymer. However, this process had
its problems such as that the product showed brittleness
and poor mechanical characteristics. A copolymer of
3-hydroxybutyrate and 3-hydroxyvalerate, BIOPOL,
was developed and is used for medical devices such as
bioengineered hearts, surgical sutures, meshes, and ortho-
pedic fixtures. Another copolymer of 3-hydroxybutyrate
and 3-hydroxyhexanoate is used in the production of
nonwovens, binders, flexible packaging, synthetic paper,
and medical devices. A recombinant Escherichia coli, able
to produce a mixture of 3-hydroxybutyrate and 3-hydro-
xyoctanoate to 90% of its cell dry weight in 24 h, has been
developed.
The biosynthesis of PHA consists of three enzymes,
Building Blocks
Polyhydroxyalkonates
Polyhydroxyalkonates (PHA) is a large group of polymers
based on linear polyesters of 3, 4, 5 and 6-hydroxyacids
(Figure 16).
The molecular weight of PHA can be up to 2 million
Daltons. All the monomers are enantiomerically pure and
in the R-configuration. The PHA exhibit thermoplastic
and elastomeric properties and are easily degraded to
carbon dioxide and water. They are considered as good
replacements for petroleum-derived polymers, in terms
of physical-chemical characteristics and biodegradability.
In 1923, Lemoigne found that spore-forming bacilli
make 3-hydroxybutyric acid. In 1927, after using chloro-
form extraction, he showed that a Bacillus sp. accumulated
the polymer, which consists of the monomer of 3-hydro-
xybutyric acid (PHB), where PHB is a specific case of
PHA. There are a multitude of possible monomers, of
either 3–5 carbon atoms designed as short chain-length
PHA or 6–14 carbon atoms, called medium chain-length
PHA. A wide variety of bacteria, both Gram negative and
Gram positive, have the capability to synthesize PHA.
Among these are species of Pseudomonas, Bacillus, Ralstonia,
Aeromonas, Rhodobacter, and even certain Archae, such as
Haloferax sulfurifontis. The main species in use by industry
was initially designated as Alcaligenes sp., and today it is
called Cupriavidus sp. The PHA function as energy sto-
rage compounds for the bacteria and are found as discrete
cytoplasmic inclusions in the cells.
PHA synthase,  kethothiolase, and  reductase, which
are all found in producing microorganisms. Supply of
monomers and polymerization are the two main steps in
biosynthesis, and PHA formed is dependent on the carbon
source used as well as on the different pathways (three)
that are present in different microorganisms; pathway I
where the carbon source (sugar) is the precursor, through
the formation of acetyl-coA, pathway II where fatty acid
degradation delivers the precursors, and pathway III
where the polymer is formed through the route of fatty
acid biosynthesis.
Itaconic acid
Itaconic acid (methylenesuccinic acid, C5H6O4)
(Figure 17) is a white colorless crystalline, hygroscopic
powder soluble in water, ethanol, and acetone. It is an
unsaturated diprotic acid, which derives its unique che-
mical properties from the conjugation of one of its two
carboxylic acid groups with its methylene group.
Itaconic acid was discovered by Baup in 1837 as a
product of pyrolytic distillation of citric acid. The name
itaconic was devised as an anagram of aconitic.
Itaconic acid is formed in fermentation of some sugars.
In 1929, Kinoshita first showed the acid to be a metabolic
product of Aspergillus itaconicus. A derivative of itaconic
acid (trans-phenylitaconic acid) was isolated from another
natural source (Artemisia argyi).
The biosynthetic pathway of itaconic acid from glu-
cose is similar to that of citric acid, which occurs via
the glycolytic pathway and anaplerotic formation of

R1 O R2 O

CH C CH C
O (CH2)x O (CH2)x O n

Figure 16 General structure of polyhydroxyalkonates. Reproduced from Philip S, Keshavarz T, and Roy I (2007)
Polyhydroxyalkanoates: Biodegradable polymers with a range of applications. Journal of Chemical Technology and Biotechnology 82:
233–247, with permission from Society of Chemical Industry, granted by John Wiley & Sons Ltd on behalf of SCI.
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial 433

O about 80 g l1 during a cultivation at 39–42  C for 8–10

OH
HO
O
Figure 17 Itaconic acid.
oxaloacetate by CO2 fixation and via the TCA cycle
(Figure 2). Itaconic acid is formed by the cytosolic
enzyme aconitate decarboxylase from cis-aconitic acid.
Another biosynthetic pathway from pyruvate through
citramalic acid, citraconic acid, and itartaric acid also
results in itaconic acid (Figure 18).
In contrast to several other organic acids (e.g., citric,
isocitric, lactic, fumaric, and L-malic acid) itaconic acid is
used exclusively in nonfood applications, especially in the
polymer industry. Itaconic acid derivatives are used in
medicine, cosmetics, lubricants, thickeners, and herbi-
cides (e.g., substituted itaconic acid anilides).
Itaconic acid is produced solely by batch submerged
fungal fermentation. Aspergillus terreus has been used from
the 1940s in the fermentation process, which is similar to
that of citric acid (see ‘Citric acid’), that is, it requires an
excess of readily metabolizable sugar (glucose syrup,
crude starch hydrolysates, and decationized molasses –
up to 200 g l1 sugar), continuous aeration, a low initial
pH (between 3 and 5), sufficient nitrogen, high magnesium
sulfate concentration (0.5%), low phosphate to limit bio-
mass production, and a limitation in metal ions (zinc,
copper, and iron). However, there exists one significant
difference in that the sensitivity of this fungus to the
formed acid, in contrast to A. niger, necessitates maintain-
ing of the pH at 2.8–3.1 throughout the fermentation, in
order to obtain high amounts of the acid. At present, the
published production yield of itaconic acid is about 85%
of theoretical, accompanied by product concentrations of
days. Recovery of itaconic acid is accomplished by first
separating the fungal biomass by filtration followed by
evaporation, treatment with active carbon, and crystalliza-
tion and recrystallization. Actual markets for itaconic acid
are currently limited because the fungal fermentation is
carried out at a relatively high cost. New biotechnological
approaches, such as published immobilization techniques,
screening programs for other producing organisms (such
as yeast), and genetic engineering of A. terreus (the anno-
tated genome sequence of A terreus strain NIH 2624 has
been publicly released), or of A. niger, could lead to higher
production of itaconic acid. Also, the use of alternative
substrates may reduce costs and thus open the market for
new and expanded applications of this acid.
Kojic acid
Kojic acid (5-hydroxy-2-(hydroxymethyl)-4-pyrone,
C6H6O4) (Figure 19) is a chelating organic acid freely
soluble in water, ethanol, and acetone.
Kojic acid is a fungal metabolic product produced by a
few species of Aspergillus, especially by A. oryzae, which has
the Japanese common name koji. This acid is a by-
product in the fermentation process of malting rice, for
use in the manufacturing of sake, the Japanese rice wine.
Kojic acid was first isolated in 1907 by Saito from
mycelia of A. oryzae grown on steamed rice. In 1912
Yabuta gave it the name kojic acid, and only in 1924 he
deciphered the correct structure of the molecule of this
acid.
Kojic acid is an inhibitor of growth of bacteria, fungi,
and multiplication of viruses. It inhibits the catecholase
activity of tyrosinase, which is rate-limiting, and an essen-
tial enzyme in the biosynthesis of the skin pigment melanin.
Due to its properties as antibacterial, antioxidative, and

(a) Via the tricarboxylic acid cycle

° H
CH3 · CO 2
Acetic acid ° H
CH2 · CO ° H
CH2 · CO
2 2 ° CH2
+ CO2
CO · CO2H C(OH)CO2H C · CO2H C · CO2H

CH2 · CO2H CH2 · CO2H CH · CO2H CH2 · CO2H

Oxaloacetic acid Citric acid Aconitic acid Itaconic acid
(b) Via citramalic acid
CH3 CH3 CH2

CO · CO2H C(OH)CO2H C · CO2H

Pyruvic acid
+ ° H
CH2 · CO ° H
CH2 · CO
° H 2 2
CH3 · CO 2
Acetic acid Citramalic acid Itaconic acid
Figure 18 Biosynthetic routes to itaconic acid. Reproduced from Winskill N (1983) Tricarboxylic acid cycle activity in relation to
Itaconic acid biosynthesis by Aspergillus terreus. Journal of General Microbiology 129: 2877–2883, with permission from Society for
General Microbiology.
434 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
O Succinic acid (from Latin succinum, amber) was discov-
OH
HO
O world especially, in amber (3–8% by weight) and plant
Figure 19 Kojic acid.
color-protectant, kojic acid is used in the cosmetic industry,
and in the food industry as a precursor for flavor enhancers
(maltol and ethyl maltol), on cut fruits to prevent oxidative
browning, antistaling of fruits and vegetables, and in sea
food and meat to preserve pink and red colors. Kojic acid is
consumed widely in the Japanese diet with the belief that it
is of benefit to health (a functional food).
Kojic acid is produced chemically via pyranoid 3,2-
enolones, or biologically by the direct fermentation of
glucose by A. oryzae and A. flavus, where yield values of
70–90% of the acid are obtained, based on carbon source.
When immobilized A. oryzae cells, entrapped in alginate
gel beads, were used with glucose as the carbon source,
kojic acid began to crystallize upon reaching a concentra-
tion of about 83 g l1. The carbon source can also be
pyruvic acid, acetic acid, glycerol, and/or ethanol. The
production using glucose by these filamentous fungi is
very similar to that of citric acid and itaconic acid pro-
cesses (i.e., at high sugar concentration and low phosphate
concentration). By changing the fermentation parameters,
such as stirring or aeration rate, some strains can be
converted from citric acid or itaconic acid production to
kojic acid production. This can be explained by the direct
conversion of glucose, without splitting of the carbon
chain, to kojic acid, as well as to citric and itaconic
acids. By this proposed pathway, glucose is converted
to D-gluconic acid by glucose dehydrogenase and
D-gluconic acid is converted to kojic acid by gluconate
dehydrogenase. However, direct evidence for the invol-
vement of these enzymes in kojic acid biosynthesis has not
been shown.
Succinic acid
Succinic acid (butanedioic acid, C4H6O4) (Figure 20) is a
symmetric, four-carbon, dicarboxylic acid, which forms
colorless, odorless crystals with characteristic acid taste.
The acid is soluble in water and slightly soluble in etha-
nol, ether, acetone, and glycerol.
O
HO
OH
O anaerobic bacteria, such as E. coli, Enterococcus flavescens,
Figure 20 Succinic acid.
ered in 1546 by Agricola by dry distillation (heating in
vacuum) of amber.
Succinic acid is distributed widely through the natural
and animal tissues, and in microorganisms. It plays a
significant role in intermediary metabolism (i.e., mainly
in the TCA cycle (Figure 2) and the glyoxylate pathway
(Figure 21)).
Succinic acid is used for production of polymers,
clothing fibers, plasticizers, solvents, paints, inks, food
and feed additives, pharmaceuticals, perfumes, and an
array of industrial and consumer products.
Succinic acid is synthesized as an intermediate of the
TCA cycle (Figure 2), operating under aerobic condi-
tions, from -ketoglutaric acid by -ketoglutarate
dehydrogenase. Succinic acid is an intermediate in the
glyoxylate bypass (Figure 21). Succinic acid is produced
as an end product by the reductive TCA cycle (Figure 3),
under anaerobic conditions. While the biosynthesis of the
acid through the first two aerobic pathways converts only
four of the six carbons from glucose into the four-carbon
succinic acid product, the anaerobic reductive TCA path-
way produces two four-carbon acids for every glucose
molecule utilized, due to the carboxylation reaction of
phosphoenolpyruvate carboxylase. Therefore, anaerobic
fermentations are preferred to aerobic fermentations for
efficient and economical succinic acid production.
Succinic acid is produced mainly by the chemical
catalytic hydrogenation of maleic or fumaric acids
(about 15 000 tonnes per year – see Table 1). Food-
grade acid is probably produced through a fermentation
and separation process. The market potential for products
based on succinic acid (e.g., 1,4-butanediol, tetrahydro-
furan, and adipic acid) is large, and, therefore, there is
intensive ongoing research to develop an efficient biolo-
gical route for its production.
Several biological routes to produce succinic acid are
known but can as yet not compete with the chemical
processes, mainly because the acid is a symmetrical mole-
cule, similar to the fumaric acid production processes (see
‘Fumaric acid’). The first route is the bioconversion from
fumarate to succinate, which is carried out by Aerobacter
aerogenes strains, or by E. coli strains amplified for fumarate
reductase. With the last bacterium, the bioconversion was
shown to occur in the presence of glucose and under
anaerobic conditions. A complete and rapid conversion
of fumarate into succinate required high cell densities. It
was found that the succinate molar yield (calculated from
the utilized fumarate) was higher than 100%, suggesting
that in addition to fumarate, glucose is converted
to succinate. Production of succinic acid by fermentation
from glucose was shown to occur by various
Acinetobacter succinogenes, Anaerobiospirillum succiniciproducens,
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial 435

Glyoxylate cycle
O
CH3 C SCoA
AcetylCoA
CoASH

CH2COO–
COO–
HO C COO–
C O Citrate
CH2COO– synthase CH2COO–
Oxaloacetate Citrate
Aconitase
NADH Malate CH2COO–
DH
NAD+ H C COO–
HO CHCOO–
COO–
Isocitrate
H C OH
CH2COO– H2O Isocitrate
Malate O lyase
HCCOO–
O Malate
Glyoxalate
CH3 C SCoA synthase
AcetylCoA CoASH

CH2COO–

CH2COO–
Succinate
Figure 21 The glyoxylate pathway. With permission from Dr. R. Paltel, Humboldt State University: CA, USA.

Mannheimia succiniciproducens, and by the yeast S. cerevisiae. COOH

Succinic acid can be produced under aerobic conditions
(see above) by Fusarium sp., Aspergillus sp., Penicillium sim- HCOH
plicissimum, and R. oryzae, which excrete the acid but
in rather low amounts and low yields. With A. succinogenes HOCH
anaerobic fermentation, the succinic acid produced
amounts to 110 g l1, with a yield value of 83–87% from HCOH
glucose. Recent work that includes genetic engineering
and immobilization techniques may improve the biologi- CO
cal process to become competitive with the chemical
CH2OH
process.
Figure 22 5-Keto-D-gluconic acid (5-KGA).
5-Keto-D-gluconic acid
5-Keto-D-gluconic acid (5-KGA, C6H10O7) (Figure 22) COOH
was first obtained in 1940 by Stubbs and his colleagues,
via a fermentation process. CO
The production from glucose of 5-KGA, and subse-
quently its conversion to 2-ketogluconic acid (2-KGA) HOCH
(see ‘Ascorbic acid’) (Figure 23), is carried out by strains
of the bacterium G. oxydans. Three different consecutive HCOH
and membrane-bound periplasmic enzymes catalyze
this conversion. The first enzyme is pyrroloquinoline HOCH
quinine (PQQ)-dependent glucose dehydrogenase,
which catalyzes the oxidation of glucose to D-gluconic CH2OH
acid. The second enzyme is a PQQ-dependent Figure 23 2-Keto-D-gluconic acid (2-KGA).
436 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
gluconate-5-dehydrogenase, which converts D-gluconic
acid to 5-KGA. The third enzyme is a flavin-dependent
gluconate-2-dehydrogenase, which converts 5-KGA to
2-KGA. This bacterium contains another set of
these enzymes for the oxidation of glucose to the keto-
gluconic acids, which are soluble and NADPþ-dependent
enzymes. Since 2-KGA is an undesirable by-product in
this fermentation, a mutant of G. oxydans was obtained in
which the membrane-bound gluconate-2-dehydrogenase
was inactivated. This mutant converted the available glu-
cose almost completely (84%) into 5-KGA.
5-KGA can be converted effectively into L(þ)-tartaric
acid (see ‘L(þ)-Tartaric acid’). The recent success in
deciphering the genome of G. oxydans made it possible
to develop a recombinant strain producing 5-KGA effi-
ciently from glucose. This will enable the development of
an economical biological process for tartaric acid, as
compared to the existing process from argol, and thus
increase its availability.
2-Keto-D-gluconic acid
2-Keto-D-gluconic acid (2-KGA, C6H10O7) (Figure 23)
is used commercially as an intermediate in the production
In 1843 Ferdinand Redtenbacher oxidized acrolein
with aqueous silver oxide and isolated acrylic acid.
Acrylates are used in formulating paints and disper-
sions for paints, inks, and adhesives. Acrylates are used in
manufacturing cleaning products, antioxidant agents,
amphoteric surfactants, aqueous resins, and dispensions
for textiles and papers. Methyl acrylate is used in making
vitamin B1. Acrylate polymers have been successfully
used in the manufacturing of hygienic products, deter-
gents, and waste water treatment chemicals.
Acrylic acid is produced from propylene, a gaseous
product of oil refineries, by a two-step gas-phase oxida-
tion via carolein. This process has almost wholly replaced
alternative technologies (i.e., acrylonitrile hydrolysis and
the Reppe process). In the past decade, efforts have been
made to use propane, which is more widely available and
even lower priced than propylene, to produce acrylic
acid.
A combination of a sugar-based fermentation to pro-
duce an intermediate compound, which will then be
converted by chemical catalysis to acrylic acid, provides
multiple routes that may be feasible for the economical
production of this acid (Figure 25). Only one of the
of D-isoascorbic acid (a cheap isomer of L-ascorbic acid,
which is absorbed in the body and has limited antiscorbu-
tic activity). Its accumulation by different strains of
G. oxydans, Acetobacter pasteurianus, A. aceti, A. cerinus,
Enterobacter intermedium, and Klebsiella aerogenes has been
described. 2-KGA can be produced at high concentrations
with strains of the genus Pseudomonas and by Serratia
marcescens. With the latter bacterium, a fermentation pro-
cess was described that resulted in 95–100% of the
theoretical yield of 2-KGA from glucose.
Acrylic acid
Acrylic acid (2-propanoic acid, C3H4O2), (Figure 24) is
the simplest unsaturated carboxylic acid. It has both a
double bond and a carboxyl group linked to its C3, and
these two functional groups enable its polymerization. In
its pure form, acrylic acid is a clear, colorless liquid with a
characteristic acrid odor. It is a major bulk chemical,
being produced at over 3.4 million tonnes per year
(2005, Table 1). It is miscible with water, alcohols, ethers,
and chloroform. The esters and the salts of acrylic acid are
collectively known as acrylates. Acrylates readily com-
bine with themselves or other monomers (e.g., amides,
acrylonitrile, vinyl, sterene, and butadiene) by reacting
at their double bond, forming homopolymers (e.g., poly-
acrylic acid) or copolymers.
O colleagues in 1884 by oxidation of castor oil with nitric
HO
Figure 24 Acrylic acid.
intermediates (e.g., -alanine, methylcitric acid, methyl-
malonyl CoA, lactic, acid and 3-hydroxypropionic acid),
lactic acid, is commercially available today (see ‘Lactic
acid, microbially produced’).
Dehydration of lactic acid leads directly to acrylic
acid. However, when chemical catalysts were used large
amounts of side-products were formed. A biological con-
version of lactic acid to acrylic acid was studied in
Clostridium propionicum, but this process has not been
implemented in industry. A possible pathway of interest
is shown in Figure 26, where 3-hydroxypropionic acid is
produced at a theoretical yield of 100% from glucose and
is then chemically converted in a cost-effective process to
acrylic acid.
Another possibility is to use the enzyme nitrilase
(produced by bacteria such as Rhodococcus rhodochrous,
Alcaligenes faecalis, and Klebsiella ozaenae) to convert acet-
onitrile to acrylic acid. The highest accumulation of
acrylic acid, using a periodic substrate feeding strategy,
was 390 g l1, with almost 100% molar conversion yield.
Adipic acid
Adipic acid (butane-1,4-dicarboxylic acid, C6H10O4)
(Figure 27) is a white crystalline powder of C6-straight
chain dicarboxylic acid, and is one of the most used
chemicals in the world today (Table 1). It is slightly
soluble in water and soluble in alcohol and acetone.
Adipic acid was first obtained by Dieterle and his
acid.
Adipic acid, despite its name (in Latin adipis is fat), is a
product of the oxidative rancidity of fats (lipid peroxidation).
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial 437

Biomass (sugars) Fermentation intermediates Chemical product

O

OH OH
OH O
HO O
HO Lactic acid OH
HO OH O O
Glucose HO OH HO H Acrylic acid
3-Hydroxypropionic acid or aldehyde
Figure 25 Routes to acrylic acid from renewable carbon sources. With permission from Dr. J. Holladay. Pacific Northwest National
Laboratory, WA, USA.

Glucose
NH2

NADH O O O O

ATP
H2N OH OH OH
1 2 3 Malonate
Pyruvate L-α-alanine β-Alanine
semialdehyde

Glutamate α-Ketoglutarate α-Ketoglutarate Glutamate

4
NADH

1 – Pyruvate/alanine aminotransferase
2 – Alanine 2,3-aminomutase
3 – β-Alanine aminotransferase 3-HP
4 – 3-HP dehydrogenase HO O

OH

Figure 26 Biosynthesis of 3-hydroxy propionic acid from glucose. With permission from Dr. D. Cameron, Koshla Ventures, CA, USA.

O The 14-kb gene cluster for cyclohexanol oxidation in

OH
HO
O ygenase, a hydrolase, and a transcriptional regulator.
Figure 27 Adipic acid.
Adipic acid consumption is linked almost 90% to
nylon (nylon-6.6) production by the polycondensation
with 1,6-hexamethylenediamine. Adipic acid is used in
manufacturing plasticizers, lubricant components, and
polyester polyols for polyurethane systems. The acid
and its derivatives are also used in the food industry (as
gelling aid, acidulant, flavoring, leavening, and buffering
reagents), and for the preparation of pesticides, dyes,
textile treatment agents, fungicides, and pharmaceuticals
(e.g., cephalosporin intermediates).
Presently, almost all of the commercial adipic acid is
produced from cyclohexane, obtained by the hydrogena-
tion of benzene, through two sequential oxidation
processes.
A plausible biological alternative can be the oxidation
of cyclohexanol or cyclohexanone to adipic acid by
strains of Acinetobacter, Pseudomonas, and Xanthobacter.
Acinetobacter sp. contains genes encoding two alcohol
dehydrogenases, an aldehyde dehydrogenase, a monoox-
A recombinant E. coli containing the 14-kb gene cluster
was able to convert cyclohexanol to adipic acid.
Theoretically, adipic acid can be produced from
hexanoic acid (maybe by a Pseudomonas sp.), which is a
product of anaerobic glucose fermentation by
Megasphaera elsdenii. This bacterium metabolizes glucose
or maltose into acetic, butyric, and hexanoic acids by
serial condensations of C2 units produced from pyruvic
acid. The yields of hexanoic acid are 5–8 g l1 from
40 g l1 glucose in a stirred batch fermentation. Other
possibilities for the biological production of adipic acid
include the production of minute amounts of the acid
from aliphatic amines or diamines (e.g., dodecamethyle-
nediamine) by Nocardia sp., and the conversion of
straight chain monocarboxylic acids (e.g., myristic acid)
into dicarboxylic acids including adipic acid by Pichia
carboniferous. Both these biological processes are very
inefficient and require substantial optimization to be
competitive with the chemical process.
438 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
L-trans-2,3-Epoxysuccinic acid
L-trans-2,3-Epoxysuccinic acid (ESA, C4H4O5)
(Figure 28) is a rare natural acid and it was first isolated
by Sakaguchi and his colleagues in 1939, from the culture
broths of Paecilomyces variotii and Talaromyces flavus. Few
other fungi (such as A. fumigatus, A. clavatus, Monilia for-
mosa, and Penicillium viniferum) produce this compound.
ESA is hydrated to meso-tartaric acid by the enzyme
fumarase from pig heart and by a hydrolase obtained from
Pseudomonas putida. ESA can be used as a good starting
material for the synthesis of optically active compounds
such as specific single beta-lactam antibiotics. This epox-
ydicarboxylic acid can be converted into dialkyltin
epoxysuccinates, which are important plasticizer stabili-
zers for polyvinyl chlorides as well as for cross-linkable
epoxy-containing film-forming polyamides.
This acid may be regarded as an oxygenation product
of fumaric acid (Figure 14), whereby the oxygen mole-
cule is incorporated to the double bond of fumaric acid to
form the epoxide carbons. Optimization of the fermenta-
tion of P. variotii on glucose (12%, w/v) resulted in a 41%
weight yield of ESA (or 61% of the theoretical 100%
molar yield).
Gallic acid
Gallic acid (3,4,5-trihydroxybenzoic acid, C7H6O5)
(Figure 29) forms white, yellowish-white, or pale fawn-
colored crystals of organic acid. It is soluble in alcohol and
ether but its solubility in water is low. It melts at 250  C,
and above this temperature gallic acid is converted into
carbon dioxide and pyrogallol. The gallic acid molecule
contains two functional groups, hydroxyl groups and a
carboxylic acid group, and therefore two molecules of the
acid can interreact to yield numerous esters and salts,
including digallic acid.
Gallic acid was discovered by Carl Wilhelm Scheele in
1786.
Gallic acid is found in the leaves of bearberry, in
pomegranate root bark, gallnuts, witch hazel, sumac, tea
HOOC H
C Short Chain Fatty Acids
O
C SCFA, also called volatile acids, are any of a large group
H COOH
Figure 28 L-trans-2,3-Epoxysuccinic acid.
O OH
HO OH
OH
Figure 29 Gallic acid.
leaves, oak bark, and many other plants, both in its free
state and as part of the tannin molecule.
Gallic acid is utilized in the ink industry, dye industry,
food industry (antioxidants and preservatives), and, most
importantly, in the pharmaceutical industry (as a raw
material for the production of the hallucinogenic alkaloid,
mescaline, and trimethoprim, a broad-spectrum antibio-
tic). Upon heating, gallic acid is converted to pyrogallol or
1,2,3-trihydroxybenzene, which are used in laboratories
for absorbing oxygen, and in the production of azo dyes
and photographic developers.
Gallic acid is produced chemically by the hydrolysis of
tannic acid by sulfuric acid at 110–120  C.
Using the free and the immobilized filamentous fungi
R. oryzae and Aspergillus foetidus, solid-state or submerged
fermentation processes for the production of tannase (an
inducible extracellular tannin acyl hydrolase) and the
hydrolysis of tannic acid extracted from tara powder (a
low-priced raw material), or tannin-rich substrates, were
developed. Following the extraction of gallic acid (e.g., by
diethyl ether), these processes resulted in good yield of
gallic acid (up to about 95% of available tannic acid), with
a minimum energy input and probably more economical,
as compared to the conventional industrial acid hydro-
lysis process.
Fatty Acids
Fatty acids are monocarboxylic acids with a long hydro-
carbon chain (having the general formula CnH2nþ1COOH),
usually linear, saturated or unsaturated acids, with an even
number of carbon atoms. Most of the fatty acids in nature
are combined, usually with glycerol, as triglyceride in fats
and, thus, these are important components of lipids in
plants, animals, and microorganisms. In this article, we will
focus on two classes of fatty acids: short chain fatty acids
(SCFA) and polyunsaturated fatty acids (PUFA), since
these are, or may be, produced by microorganisms on an
industrial scale.
of monobasic acids, especially those found in animal and
vegetable fats and oils, with a chain length of 2–6 carbon
atoms. The major SCFA are acetic (see ‘Acetic acid pro-
duction’), propionic, butyric, and valeric acids. These
acids are the major end products normally produced by
the bacterial fermentation of dietary carbohydrates and
fiber polysaccharides in the colon. These acids are con-
sidered as improved dietary components that may have a
significant role in protecting against diseases. Acetic, pro-
pionic, and n-butyric acids account for 83% of the SCFA
produced. These acids can be used in fuel production, in
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial 439

addition to specific other uses for each of these acids
(described below). The SCFA can be produced by an
active and stable mixed culture of anaerobic bacteria
(e.g., initially obtained from sewage sludge digestor efflu-
ent), which utilized H2 and CO2 (the major products from
the gasification of a wide spectrum of fossil and renewable
resources).
Butyric acid
Butyric acid (n-butanoic acid, C4H8O2) (Figure 30) is a
carboxylic acid, which forms an oily colorless liquid that
solidifies at 8  C and boils at 164  C. It is soluble in water,
ethanol, and ether. Isobutyric acid (2-methylpropanoic
acid) is an isomer of butyric acid and has similar chemical
properties but different physical properties (i.e., it boils at
155  C).
O from acetyl CoA, obtained from the degradation of fatty
OH
Figure 30 Butyric acid.
Butyric acid was discovered in 1869 by Lieben and
Rossi. In Latin, butyric acid means the acid of butter, as it
was first discovered in rancid butter (butyric acid is
hydrolyzed from the glyceride and causes a very unplea-
sant odor).
Butyric acid is a short chain saturated fatty acid found
in the form of esters in animal fats and plant oils.
Butyric acid is utilized in the production of various
butyrate esters (e.g., methyl butyrate), which have pleasant
aromas and tastes and are used as additives in foods,
perfumes, flavorings, varnishes, pharmaceuticals, and dis-
infectants. It is also used for the production of plastics,
plasticizers, surfactants, and textile auxiliaries. Butyric
acid and its derivatives (e.g., tributyrin) are also being
considered as potential anticancer agents; the acid induces
cytodifferentiation of a wide variety of neoplastic cells.
Butyric acid is synthesized under anaerobic conditions
acids, or from pyruvic acid via a unique reaction catalyzed
by pyruvate-ferredoxin oxireductase, which forms acetyl
CoA, CO2, and H2 (Figure 31). Acetyl CoA is converted

Glucose

Glucose-6-P

Fructose-6-P

Embden-meyerhof pathway

Pyruvate
~P CO2 NADH
CO2 + H2 + CO2 + H2 +
2 Acetyl-CoA Acetyl-CoA Oxaloacetate Lactate
NADH

Acetoacetyl-CoA P Fumarate Acrylyl-CoA

H2
β -OH Butyryl-CoA Acetyl-P Succinate
~P

Crotonyl-CoA ~P Methylmalonyl-CoA Propionyl-CoA

Propionyl-CoA

Butyrate Acetate Propionate

Figure 31 Biosynthesis of acetic, butyric, and propionic acids from glucose.

440 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
to butyryl CoA, which is then converted to butyric acid.
Three molecules of ATP are produced per each molecule
of utilized glucose during this anaerobic fermentation, as
compared to 2 moles of ATP produced during the pro-
duction of lactic acid and/or ethanol, and 4 moles of ATP
produced during the anaerobic production of acetic acid.
Butyric acid can be prepared by the chemical oxida-
tion of butyraldehyde which has been obtained from
propylene by oxosynthesis. However, the butyric acid
obtained this way cannot be regarded as a natural product
and, therefore, is not used in the food industry. The acid
can be extracted from butter (containing 2–4% of acid)
but this method is too expensive. Butyric acid is produced
as end product of fermentation of sugar by obligate anae-
robic bacteria, first discovered in 1861 by Pasteur;
Clostridium butyricum being the most prominent and main
bacterium, but other organisms such as C. kluyveri, C.
beijerinckii, C. barkeri, C. acetobutylicum, C. thermobutyricum,
C. thermopalmarium, C. pasteurianum, Butyribacterium sp.,
Sarcina sp., Megasphera sp., Fusobacterium nucleatum,
Peptococcus asacelarolyticus, Butyrivibrio fibrisolvens,
Pseudobutyrivibrio ruminis, and Eubacterium limosum are
also used. Optimal cultivation conditions are 35–37  C,
an atmosphere of pure CO2, N2, and a pH range of 4.5–
7.0. The Clostridia can utilize hexoses, several pentoses,
and polysaccharides. Butyrate production is favorable in a
fed-batch, glucose-limited, and slow-growing culture. It is
also stimulated under phosphate and nitrogen limitation.
Batch, fed-batch, or continuous processes combined with
cell recycle or immobilization are applicable for these
anaerobic fermentations. Inhibition of the fermentation
caused by the butyric acid formed can be avoided by the
on-line removal of the acid by various methods (e.g.,
distillation and evaporation, permeate electrodialysis,
and adsorption). Butyric acid can also be removed by
extraction, or by extraction combined with simultaneous
stripping of the organic phase (liquid membrane) into the
second aqueous phase (pertraction).
Propionic acid
Propionic acid (propanoic acid, C3H6O2) (Figure 32) is a
naturally occurring three-carbon carboxylic acid. In the
pure state, it is a colorless, water soluble, and corrosive
liquid with a sharp, somewhat unpleasant odor.
Propionic acid was first described in 1844 by Johann
Gottlieb, who found it among the degradation products of
sugar. Its name is derived from the Latin words protos and
pion, which mean first and fat, respectively.
.... O
O may be conceivable that the fermentation route could
CH3 CH2 C ..
..OH OH
Figure 32 Propionic acid.
Propionic acid is used in the manufacture of herbi-
cides, fine chemical intermediates, rubber chemicals,
emulsions, and environmentally friendly solvents for
coating formulations, artificial fruit flavors, pharmaceuti-
cals, and modified synthetic cellulose fibers (e.g., cellulose
acetate propionate). Propionic acid inhibits growth of
mold and various bacteria and is used as a preservative
for food (especially bread and other baked goods as its
sodium or calcium salts), animal feed (directly or as its
ammonium salt), and grains.
Propionic acid is biosynthesized as propionyl CoA,
which is a product of the catabolism of fatty acids having
odd numbers of carbon atoms, and of the degradation
of some amino acids. Propionic acid bacteria (e.g.,
Propionibacterium shermanii) produce this acid as a product
of their anaerobic metabolism of sugars. The initial cata-
bolism of glucose to pyruvic acid follows glycolysis, and
the NADH formed is oxidized via a pathway by which
propionic acid is produced. Pyruvic acid accepts a
carboxyl group from methylmalonyl CoA by a transcar-
boxylase reaction, leading to the formation of oxaloacetic
acid and propionic acid (Figure 31). Propionibacterium sp.
(and other bacteria such as C. propionicum, M. elsdenii, and
Prevotella ruminicola) also ferments lactic acid with the
production of propionic acid, acetic acid, and CO2
(Figure 31).
Commercial production of propionic acid is mainly
carried out via chemical synthesis from petroleum feed-
stocks by three process routes: ethylene hydroformylation/
oxidation, carboxylation of ethylene with carbon monoxide
and water, and direct oxidation of hydrocarbons. Propionic
acid is also produced, in minor quantities, as a by-product
of acetic acid manufacturing.
Fermentation is an attractive route to produce this acid
from renewable sugars (such as glucose, xylose, maltose,
sucrose, and lactose) by propionic acid bacteria (e.g.,
P. shermanii and P. acidipropionici). These bacteria play
important roles in the dairy industry in the development
of the characteristic flavors and holes (or eyes) production
(by CO2 evolution) in Swiss-type cheese. However, this
fermentation process is inefficient and only barely com-
petes with the chemical processes for the production of
propionic acid. The fermentation parameters that affect
the cost of the fermentation process are the low rates and
the maximal amounts of the product, and the formation of
undesirable by-products in addition to propionic acid.
Thus, only small amounts of this acid are produced by
fermentation of whey and are used as a natural product in
foods to adhere to the labeling rules of natural foods.
Since the genome sequence of Propionibacterium freudenrei-
chii shermanii ATCC9614 is being almost completed, it
become economically valuable. Propionic acid can also
be obtained by oxidation of propanol with G. oxydans, in a
fed-batch fermentation process, resulting in 56 g l1 of the
 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
sodium propionate (the yields of the acid obtained are
near the theoretical values) after 70 h. Since the bacterium
cannot grow on propanol as a sole carbon source, but is
able to convert it to propionic acid, glycerol was used as
an assimilatable cosubstrate.
Valeric acid
Valeric acid (pentanoic acid, C5H10O2) (Figure 33) is a
straight, saturated chain, alkyl carboxylic acid. It is a
colorless, oily liquid with a very unpleasant odor of stale
-Linolenic acid (GLA).
cheese. The acid exists in four isomeric forms, one of
which contains an asymmetric carbon atom and conse-
quently occurs in two optically active modifications and
one optically inactive modification.
Valeric acid is found naturally as free or as esters in the
vegetable and animal kingdoms.
Valeric acid, and its esters, is mainly used in perfumes
and cosmetics, as food additives because of the fruity flavor
of the esters, and as plasticizers and pharmaceuticals.
Valeric acid can be extracted by boiling water or soda
from the roots of Angelica archangelica and Valeriana offici-
nalis (from which valeric acid gets the name). The acid
can also be obtained by oxidizing fermentation amyl
alcohol with chromic acid. The acid can be formed by
microorganisms during the anaerobic fermentation of
CO2 and H2 (discussed above) and by the fermentation
of other carbon sources, such as wastewater solids.
Polyunsaturated Fatty Acids
PUFA are fatty acids in which more than one double bond
(generally separated by a single methylene group) exists
within the representative molecule. The term PUFA is
used only for acids with three up to six double bonds as
those found in fish oil or brain tissue. These acids (also
called essential fatty acids (EFAs)) are necessary fats that
humans cannot synthesize, and must be obtained through
their diet. There are two families of EFAs: omega-3 and
omega-6 fatty acids, in which the position of the first
double bond, from the terminal methyl group of the
molecule, is indicated by the number following ‘omega’.
There is a great variety of polyunsaturated fatty acids,
and here we will discuss those that are of commercial
interest and possible to produce by microorganisms,
namely, arachidonic acid, C20H32O2 (ARA) (Figure 34);
-lineolenic acid, C18H30O2 (GLA) (Figure 35); and
docosahexaenoic acid, C22H32O2 (DHA) (Figure 36).
-Linolenic acid is now obtained primarily from
Microbial lipid production is of interest when the
desired fatty acids are difficult to obtain from plant or
O renewed commercially successful production from 2000.
OH
Figure 33 Valeric acid.
441
O ω
6 1
HO 1 5 8 11 14
Figure 34 Arachidonic acid (ARA).
O ω
6 1
HO
1 6 9 12
Figure 35
O ω
3 1
HO
1 4 7 10 13 16 19
Figure 36 Docosahexaenoic acid (DHA).
animal oils. The formation of fatty acids for commercial
purposes is called single-cell oil (SCO). Prokaryotes are
known to form a variety of fatty acid structures including,
branched chains, cyclopropane rings, and trans-double
bonds, whereas eukaryotes only form straight chains
with cis-double bonds. The fatty acids of interest (ARA,
GLA, and DHA) are all straight chains containing solely
cis-double bonds and with methylene interruption
(CH¼CH-CH2-CH¼CH). Most microorganisms do not
accumulate triacylglycerol lipids and their main lipid is in
the form of phospholipids. Microorganisms that do
accumulate more than 20% of their dry weight as tria-
cylglycerol lipids are called oleaginous and are of
commercial interest. Fatty acids are important for both
structure and storage, and also as precursors for a range of
cell signaling molecules, involved in inflammatory
responses, blood coagulation, and reproductive function.
ARA and DHA are of particular interest, since they are
common in neural tissues both of the eye and of the brain.
They are both present in human breast milk and are
added to infant formula in more than 60 countries, with
beneficial effects on visual and intellectual development.
In 1985, the first commercial process growing the
fungus Mucor circinelloides for SCO was started in Japan.
The fungus was grown in stirred-tank fermentors fol-
lowed by extraction of the oil and final refinement and
purification. The product was rich in GLA (18–20% of
the oil) and was produced for 6 years when the introduc-
tion of plant-derived ‘borage oil’ became a cheaper source
of GLA.
the seed of the evening primrose plant.
In case the dietary requirement is for a single acid,
this is best met by microbial oil, which has lead to
Three organisms are in use for DHA production,
Crypthecodinium cohnii, Schizochyturium sp., and Ulkenia sp.,
all grown by submerged fermentations. ARA was shown
442 Applied Microbiology: Industrial | Organic and Fatty Acid Production, Microbial
to be a major component of the oil from Mortierella alpine.
Various strains of M. alpina are used in processes in
Europe, China, and possibly also in Japan.
PUFA materials are introduced as dietary supplements
for adults especially as DHA-rich oils, to help prevent
coronary heart problems. There are also suggestions that
DHA may be useful as a dietary supplement to prevent
the onset of degenerative disorders such as Alzheimer’s
disease.
Conclusions
As is evident from this article, there is a strong competi-
tion, based mainly on economical considerations, between
the chemical and the fermentation industries to produce
organic acids. At the present time, chemical processes,
being more economical than the fermentation processes,
are the existing industrial manufacturing processes not
only for symmetrical molecules (e.g., fumaric acid and
succinic acid) but also for asymmetrical molecules (e.g.,
malic acid). Unlocking the biochemical networks and the
regulatory mechanisms of the producer strains, and
implementing modern technologies of genetic and meta-
bolic engineering (e.g., global transcription machinery
engineering, systems biology) may provide an opportu-
nity for their manipulation by amplifying or deleting
critical steps in metabolism to improve acid production
or to overproduce novel acids. In addition, the newly
emerging information will allow the efficient production
of acids (e.g., L-lactic and succinic acids) by a better
suitable and specifically tailored microorganism (e.g., A.
niger) under different production conditions (e.g., micro-
aerophilic or aerobic instead of anaerobic conditions).
These approaches may be a major breakthrough in the
scientific methodology that can improve considerably the
economy of industrial processes employing biological
systems for the production of acids.
See also: Acetic Acid Production; Lactic Acid, Microbially
Produced; Vitamins and Vitamin-like Compounds:
Microbial Production
Further Reading
Berovic M and Legisa M (2007) Citric acid production. Biotechnology
Annual Reviews 13: 303–343.
Engel CAR, Straathof AJJ, Zijlmans TW, Van Gulik WM, and Van der
Vielen LAM (2008) Fumeric acid production by fermentation. Applied
Microbiology and Biotechnology 78: 379–389.
Goldberg I, Rokem JS, and Pines O (2006) Organic acids: Old
metabolites, new themes. Journal of Chemical Technology and
Biotechnology 81: 1601–1611.
Kubicek CP and Karaffa L (2006) Organic acids. In: Ratledge C and
Kristiansen B (eds.) Basic Biotechnology, pp. 359–380. Cambridge:
Cambridge University Press.
Magnuson J and Lasure LL (2004) Organic acid production by
filamentous fungi. In: Lang J and Land L (eds.) Advances in Fungal
Biotechnology for Industry, Agriculture, and Medicine, pp. 307–340.
New York: Kluwer Academic/Plenum Publishers.
Papagianni M (2007) Advances in citric acid fermentation by Aspergillus
niger: Biochemical aspects, membrane transport and modeling.
Biotechnology Advances 25: 244–263.
Roehr M and Kubicek CP (1996) Further organic acids. In: Rehm HJ and
Reed G (eds.) Biotechnology, vol. 6. pp. 363–380. Weinheim:
Whiley-VCH.
Roehr M, Kubicek CP, and Kominek J (1996) Citric acid. In: Rehm HJ
and Reed G (eds.) Biotechnology, vol. 6. pp. 307–346. Weinheim:
Whiley-VCH.
Russel NJ and Nichols DS (1999) Polyunsaturated fatty acids in marine
bacteria – a dogma rewritten. Microbiology 145: 767–779.
Steinbuchel A (1996) PHB and other polyhydroxyalkanoic acids.
In: Rehm HJ and Reed G (eds.) Biotechnology, vol. 6 pp. 403–464.
Weinheim: Whiley-VCH.
Wynn JP and Anderson AJ (2006) Microbial polysaccharides and single
cell oils. In: Ratledge C and Kristiansen B (eds.) Basic Biotechnology,
pp. 381–401. Cambridge: Cambridge University Press.