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Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)

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Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)

M Moresi, Università della Tuscia, Viterbo, Italy


E Parente, Università della Basilicata, Potenza, Italy; and Istituto di Scienze dell’Alimentazione, Avellino, Italy
Ó 2014 Elsevier Ltd. All rights reserved.

Several organic acids are used in a variety of food and nonfood Belgium (Societè des Produits Organiques de Tirlemont) in
applications. Table 1 lists the main acids that are produced 1919 and in the United States (Chas. Pfizer & Co., New
commercially by chemical (C) or biotechnological (fermenta- York) in 1923.
tion, F, or enzymatic, E) methods or extracted from wine- About 10 years later, about 80% of the world’s citric acid
making residues (L). was produced by the surface fermentation process. The
Citric, acetic, lactic, propionic, tartaric, fumaric, and malic submerged fermentation process began to be applied only after
acids are among the most versatile ingredients in the food and World War II.
beverage industry because of their valuable properties, such as From 1950 to 1980, citric acid was mainly used in phar-
solubility, hygroscopicity, acidity, buffering capacity, and maceutical or health products. In fact, in the early 1980s, its
chelation (see Preservatives: Traditional Preservatives – Organic two largest manufacturers were Pfizer and Miles/Bayer, both
Acids). suppliers of prescription drugs. Thereafter, as citric acid began
Citric acid accounts for around 80% of the food acidulant to be used in the food and beverage sector in industrial and
usage, whereas the use of phosphoric or acetic acids is limited, developing countries, its market size experienced significant
being almost exclusively utilized in cola soft drinks or in growth and several new manufacturers were established in
vinegar (see Vinegar), sauces, and condiments, respectively. Europe and North America, as well as in China where several
small-scale fermentation units have produced citric acid from
sweet potatoes or cassava since the 1970s.
Citric Acid In the early 1990s, a few manufacturers gave rise to the so-
called citric acid cartel. The overcharges imposed on US buyers
Citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid: C6H8O7) was estimated in the range of $116–309 million and, on
is widely distributed in natural raw materials (such as lime, January 29, 1997, Haarmann & Reimer Corp., a subsidiary of
lemon, and raspberry) and is commercially available in the Bayer AG (D), pled guilty and paid a $50 million criminal fine.
monohydrated form (molecular mass of 210.13 Da, relative In March 1998, even Archer Daniels Midland Co. (ADM)
density of 1.542 at 20  C, and heat of combustion of agreed to pay $36 million to four citric acid customers that had
1962 kJ mol1 at 25  C). It is a strong tricarboxylic acid opted out of the July 1997 civil class-action antitrust settle-
(TCA; its dissociation constants being K1 ¼ 7.45  104, ment. At that time, the global citric acid capacity was about
K2 ¼ 1.73  105, and K3 ¼ 4.02  107 at 25  C), highly 840 000 Mg (mega grams) per year with a growth rate of 5%
soluble in water with pleasant acid taste. per year. Afterward, the world citric industry became less
Citric acid was first isolated in 1784 by Scheele, who concentrated and numerous new manufactures, especially in
precipitated it as calcium citrate by adding calcium China, as well as Brazil, India, Indonesia, and Thailand, have
hydroxide (lime) to lemon juice. Before 1920, it was almost entered the market, thus making the formation of cartels less
exclusively produced in Sicily by pressing lemons: The firm probable.
Arenella (Palermo, Italy) essentially established a monopoly Moreover by the early 2000s, almost all citric acid
until the advent of the citric acid fermentation technique in manufacturing was globally integrated into the corn wet-milling

Table 1 Main organic acids: molecular formulas, world output, production methods, and organisms

Acidulant Chemical formula World output (metric tons) Production methods a,b Organism

Acetic acid (vinegar) C2H4O2 190 000 F 100% Acetobacter aceti


Lactic acid C3H6O3 150 000 F 100% Lactobacillus spp.
Rhizopus spp.
Propionic acid C3H6O2 130 000 C 100% Propionibacterium acidipropionici
Fumaric acid C4H4O4 12 000 C 100% Rhizopus arrhizus
Malic acid C4H6O5 10 000 C 70% –
E 30%
Tartaric acid C4H6O6 28 000 L 100% –
Itaconic acid C5H6O4 15 000 C 100% Aspergillus terreus
Citric acid C6H8O7 1 800 000 F 100% Aspergillus niger
Gluconic acid C6H12O7 87 000 F 100% Aspergillus niger

Note: Because of the lack of published data, the production figures are approximate.
a
Percentage of total production for food uses.
b
F, fermentation; C, chemical synthesis; E, enzymatic synthesis; L, leaching.

804 Encyclopedia of Food Microbiology, Volume 1 http://dx.doi.org/10.1016/B978-0-12-384730-0.00111-7


FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic) 805

industry either by acquisition (Pfizer and Miles/Bayer were A high flux through the glycolysis, decreased activity of TCA
bought by ADM and Tate & Lyle, respectively) or by new process cycle reactions that degrade citrate, and an anaplerotic reaction
development (Cargill). to replenish the oxaloacetate (OAA) used for the synthesis of
In the years 1987–89, US list prices for citric acid anhydrous citrate are all essential (see Metabolic Pathways: Release of
remained unchanged at $1.79 kg1. By late 1989, the list price Energy (Aerobic)). Key regulatory steps in the process include
reduced to $1.65 kg1, while in the fall 1990 it was as low as glucose transport and phosphorylation, citric acid export from
$1.39 kg1. Then, thanks to the “citric acid conspiracy” in the the mithocondria and cell, phosphofructokinase, pyruvate
years 1993–96, the citric acid cartel accomplished its main goal carboxylase (PC), citrate synthase (CS), and a-ketoglutarate
of raising and keeping list price at $1.87 kg1. Then, it lowered dehydrogenase (KDH).
from $1.76 kg1 in November 1994 to $1.54 kg1 in the early The metabolic changes necessary for citric acid over-
1997. production in A. niger are induced by high sugar concentration,
Thereafter, the severe competition resulted in selling prices low pH, and manganese (Mnþ2) deficiency. Other factors (i.e.,
of anhydrous citric acid decreasing to $0.70–$0.80 kg1, thus phosphate and nitrogen concentrations, high dissolved oxygen
forcing the smaller manufacturers, unable to benefit from the (DO) concentration, trace metals), however, are important.
economy of scale, to exit the business. As a consequence, the Very low concentrations of Mn2þ (<10 mg m3) are critical.
panorama of organic acid manufacturers has profoundly They result in decreased activity of the pentose phosphate
changed over the last decade. In 2010, China approximately pathway and increased glycolytic flux, increased intracellular
accounted for more than 50% of global citric acid production NHþ 4 pool and turnover of nucleic acids and proteins, changes
capacity, while Europe and North America covered the 19 in membrane lipid composition and cell wall composition,
and 24%, respectively, and as much as 65–70% of global and morphological changes.
consumption. Improvement of strains for citric acid production tradi-
Several substrates are used as fermentation substrates tionally has been carried out by mutagenesis and screening.
depending on the local availability. Maize starch is mainly used Overexpression of proteins critical to acidogenesis (hexokinase,
in the United States and China, while sugarcane or sugar beet glucose carrier) or inactivation of genes encoding enzymes that
molasses prevail in the Brazilian and Indian or European produce allosteric inhibitors of hexokinase has been attempted,
markets, respectively. but with limited success, in the additional production of citric
Cellulosic materials are currently unused in citric acid acid. It has been postulated that the activity of seven glycolytic
production, even if there are projects to assess the technical enzymes needs to be increased to obtain increased production
feasibility of such feedstock materials in the citric acid of citric acid. The availability of the complete genome sequence
industry. of A. niger is likely to allow for the design of overproducing
From January 2008 to January 2009, export prices of mutants.
Chinese (anhydrous) citric acid oscillated in the range of US Citric acid overproduction in yeast is relatively insensitive to
$0.7–$0.8 kg1; thereafter, they steadily increased to reach trace metals concentration and is triggered by nutrient (N, S, P,
a peak of $1.1 kg1 in June 2011, as a direct result of the or Mg) limitations coupled with a high rate of glucose
increase in the market prices for agricultural raw materials, utilization, which results in a high adenosine triphosphate/
particularly corn. The present economic crisis in Europe and the adenosine monophosphate ratio and, in turn, in inactivation of
United States has newly reduced the market prices of (anhy- nicotinamide adenine dinucleotide (NADþ)-specific isocitrate
drous) citric acid to US $0.70$0.96 kg1 depending on the dehydrogenase (IDH). The main anaplerotic reactions include
amount ordered. the synthesis of OAA from pyruvate catalyzed by PC during
In conclusion, the global citric acid production capacity production from glucose and the glyoxylate cycle during
reached almost 1.8 million metric tons (Mg) in 2010, while growth on n-alkanes. Accumulation of isocitrate (10–50% of
it was about 1.5  106 Mg in 2005. the citrate produced) in excess with respect to the predicted
equilibrium of aconitase probably is due to the high perme-
ability of yeast mitochondria to isocitrate compared with
Organisms and Metabolic Pathways Involved
citrate. Low cytoplasmic levels of citrate may be responsible for
Several molds (Penicillium spp., Aspergillus niger, Aspergillus reduced feedback inhibition of glycolysis. Improvement of
wentii, Trichoderma viride; see Aspergillus and Penicillium and- yeast for citric acid production is directed to obtain strains with
Talaromyces: Introduction), yeasts (Yarrowia lipolytica, Candida reduced isocitrate dehydrogenase and aconitase activities.
guillermondii), and bacteria (Arthrobacter) produce citric acid
from a variety of substrates (glucose, sucrose, n-alkanes), but
Methods of Manufacture
industrial processes have been developed only for the
production of citric acid from sugars (glucose, sucrose) with Citric acid production is mainly accomplished by the
A. niger and from sugars and n-alkanes with yeasts. Industrial submerged fermentation process, probably because of the
strains are not freely available, but citric acid–producing smaller contribution of investment and labor costs to its overall
strains (A. niger, NRRL 2270, NRRL 599, ATCC 11414, ATCC production costs. The surface fermentation process currently
9142; Y. lipolytica ATCC 20346, ATCC 20390, NRRL Y-7576, accounts for only 5–10% of the world supply. In Europe, all
NRRL Y-1095) can be obtained from international culture surface fermentation plants have been shut down during the
collections. past decade. Smaller amounts of citric acid (<1%) are reported
Metabolic pathways involved in citric acid overproduction to be extracted from citrus fruits in Mexico and South America
by A. niger are shown in Figure 1. and to be produced by the solid-state process in Japan.
806 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)
FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic) 807

Both submerged- and surface-culture fermentation pro- Table 2 Composition for the production media used in the
cesses use beet molasses or glucose syrup as the main raw laboratory- and industrial-scale production of citric acid by A. niger
material and use A. niger as the fermenting organism.
Range of
Submerged fermentation is carried out either in 150–200 m3
Component concentrations Typical values Unit
stirred-tank reactors or in 300–500 m3 (up to 1000 m3 as claimed
by some manufacturers) bubble-column reactors. The main Sucrose or glucose 125–225 180 kg m3
advantages of these techniques are improved asepsis during NH4NO3 (or other 0.5–3.5 1.5 kg m3
fermentation, automatic control of inoculation and fermentation NHþ 4 salt)
procedures, shorter fermentation times, and greater product KH2PO4 0.5–2 0.5 kg m3
yields. MgSO47H2O 0.1–2.0 0.25 kg m3
Feþ2 2–1300 <200 mg m3
In spite of the old and renewed interest in citric acid
Znþ2 0–2900 200–1500a mg m3
production by yeast grown submerged in sugar- or hydro-
Cuþ2 1–10 200 200–1500a mg m3
carbon-based media to overcome the main disadvantages of Mnþ2 0–46 <2 mg m3
traditional mold fermentation (i.e., high sensitivity to trace Initial pH 2.5–6.5 2.2 –
metals and low production rates), no yeast-based process is
a
currently known to be operating worldwide. To overcome the detrimental effects of iron and manganese on mycelium structure
and restore proper morphology.

Production Media
Media sterilization is carried out batch wise at 121  C for
Citric acid is produced from media containing high concen- 15–30 min at the laboratory or pilot scale or continuously
trations of simple sugars (molasses or glucose syrup) (see using a plate–heat exchanger unit at the industrial scale.
Fermentation (Industrial): Media for Industrial Fermenta-
tions), in which mycelium growth is restrained by nutrient
Fermentation Process
(phosphorous, manganese, iron or zinc) limitation. Their range
of composition is given in Table 2. Inoculation is generally carried out by transferring aseptically
Nitrogen is usually added as ammonium nitrate or sulfate. the stock culture maintained on agar slants on other working
Metals are removed by pretreatments of raw materials, espe- slants. After w24 h incubation at 30  C, the conidia crop is
cially molasses, with cation-exchange resins or the addition of inoculated in starch-rich seed-production media to yield up to
potassium hexacyanoferrate (HCF). The optimal iron concen- 1011 spores cm3. This culture may be directly transferred into
tration seems to depend on the fungal strain, but iron levels of 10–20 m3 seed fermenters to obtain a pellet-type inoculum
200 mg m3 were found to inhibit citrate production. The consisting of 1–5  105 pellets dm3 (0.1–0.2 mm in diam-
inhibitory effect of Feþþ can be counterbalanced by the addi- eter), which in turn is used as inoculum (5–10% v/v) for the
tion of copper and zinc salts during the inoculum development industrial-scale production medium.
or during early mycelium growth in the production medium. The fungus will develop different morphological forms
Manganese concentration has to be kept as low as possible (Figure 2).
(<10 mg m3). The formation of a loose mycelium with long, unbranched
Some ingredients (methanol, 3–6% w/v; corn, peanut, and hyphae is to be avoided because this results in an enormous
olive oils, 0.1–0.5% w/v; starch, 0.025–0.5% w/v) have been increase in the apparent viscosity of the culture broth, thus
claimed to enhance the citric acid yield. limiting the effective oxygen transfer rate with little or no citric

=
Figure 1 Metabolic pathways for citric acid overproduction in Aspergillus niger. Only relevant enzyme activities, substrate, products, and effectors
(, inhibitor; þ, activator) are shown. Enzymes and transport systems: INV, membrane bound invertase; GC, low-affinity glucose carrier; FC: Fructose
carrier; PP, proton pump; CC, putative citrate carrier; HK, Hexokinase; PGI, phosphoglucose isomerase; PFK1, phosphofructokinase; PFK2,
6-phosphofructo-2-kinase; ALD, aldolase; PK, pyruvate kinase; PC, pyruvate carboxylase; MDH, malate dehydrogenase; PT, pyruvate transport system;
TCC, citrate transport system; PDH, pyruvate dehydrogenase; CS, citrate synthase; ACT, aconitase; IDH, isocitrate dehydrogenase; KDH, a-ketoglutarate
dehydrogenase; AOX, alternative oxidase system. Substrates and products: glu, glucose; glu6P, glucose-6-phosphate; fru6P, fructose-6-phosphate;
fru1,6 dP, fructose-1,6-bisphosphate; fru2,6 dP, fructose-2,6-bisphosphate; gly, glycerol; dhp, dihydroxiaceton phosphate; pep, phosphoenolpyruvate;
pyr, pyruvate; oaa, oxaloacetate; mal, malate; cit, citrate; acCoA, acetyl-coenzyme A; aco, cis-aconitate; ica, isocitrate; a-kg, a-ketoglutarate; sucCoA,
succinyl-coenzyme A; tre, trehalose.
The most important steps in controlling glycolytic flux are glucose transport (simple diffusion is the main mechanism at high sugar concentrations,
although A. niger has both low-affinity and high-affinity carriers for glucose) and hexokinase (HK) activity, which initially is inhibited by trehalose-6-
phosphate. PFK1 is feedback inhibited by citrate, but the inhibition is counteracted by the presence of high levels of NHþ4 and by fructose-2,6-biphosphate
(FBP). A phosphorylated fragment of PFK1, which is insensitive to citrate inhibition, may be responsible for acidogenesis. PFK2 activity is increased at high
substrate concentration: its product, FBP lowers the Michaelis–Menten constant (Km) of PFK1, counteracts citrate inhibition, and inhibits gluconeogenesis,
thus increasing carbon flux through glycolysis during acidogenesis. CS activity in A. niger is regulated by the level of OAA, which is produced in the
anaplerotic reaction catalyzed by PC. Malate is produced from OAA by cytosolic MDH and acts as a counterion for citrate efflux from the mitochondrion,
where it is oxidized back to OAA. Low activity of NADPþ-specific IDH and KDH are a consequence of the effective removal of citrate, which has a higher
affinity than ACT.
A salicylhydroxamic acid–sensitive, alternate oxidase system is used during acidogenesis to reoxidize the NADH produced during glycolysis.
Malfunction of the normal respiratory chain is due to diminished activity of NADH ubiquinone reductase and other respiratory chain enzymes.
808 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)

Figure 2 Pellet morphology of A. niger NRRL 2270 during citric production in a laboratory 2 dm3 stirred fermenter: (a) young pellet (100); (b) stubby,
bulbous hyphae with frequent branching, which are characteristic of citric acid production (400); and (c) degenerating pellet with pointed unbranched
hyphae protruding from the pellet core (100).

acid production. On the contrary, small spherical, dense productivities of 1–1.5 kg m3 h1 result in microbial oxygen
pellets (Figure 2(a)) with short stubby hyphae (Figure 2(b)) demand rates of 0.3–0.5 kg O2 m3 h1, that are met by
are generally regarded as the best morphological form sparging 0.1–0.4 volumes of air per medium volume per
for optimal citrate yields. Frequent observation of pellet minute (vvm) at pressures at the sparger section of the
morphology during this early stage of the fermentation using fermenter not less than 0.3–0.4 MPa and at the tank top,
a microscope allows hyphae proliferation to be controlled by ranging from 0.25–0.35 MPa to 0.12–0.15 MPa, depending on
the addition, in case of adverse development (Figure 2(c)), of the (stirred or air-lift) fermenter type used. Foaming is
appropriate amounts of inhibiting compounds, such as HCF controlled by adding food-grade antifoam agents.
or zinc and copper sulfates. Mycelial clumps (whose structure Temporary interruption to the air supply during fermenta-
is less compact than pellets) also may develop under high tion does not seem to affect the performance of the culture on
agitation speed. the condition that the DO level is greater than 20% of the
Fermentation is exothermic and temperature has to be kept saturation value. DO values of about 0 for as long as 85 min,
in the range 28–35  C. Assuming that the overall heat transfer followed by restoration of the air supply, do not inhibit
coefficient and effective temperature difference between the permanently mycelial growth and citrate production, but they
fermenting medium and cooling water are of the order of do reduce the product yield coefficient up to 20%.
500 kJ m2 h1 and 5  C, respectively, the heat transfer surface Figure 3 shows the evolution of a typical batch citric acid
required to keep the fermentation temperature constant would fermentation in glucose-based media by A. niger NRRL 2270 in
be w3.2 m2 per m3 of fermentation medium. 2-dm3 stirred fermenter and by a mutant strain of A. niger in
Low pH and high DO concentration are essential for citric 400 m3 bubble-column fermenter.
acid production. Initial decrease of pH is due to ammonium Two distinct phases are evident: during the primary growth
uptake. Extreme pH values (<1.6) limit productivity, and the phase (trophophase), no acid production occurs; during the
addition of alkali (NH3) is used to control pH at 2.2–2.6 once second growth phase (idiophase), acid production by almost
production of citric acid has started. The typical industrial-scale nongrowing cells is observed.
FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic) 809

A microscopic description of this fermentation using


A. niger pellets also has to account for oxygen diffusion
phenomena from the bulk of the fermenting medium to the
pellet surface and through the porous structure of the pellet
itself. The low effective diffusivity of oxygen within the pellet
limits mycelial activity to a peripherical spherical shell only,
with the oxygen penetration depth ranging from 110 to 300 mm
in 2 mm pellets.

Recovery and Purification Processes


Citric acid may be recovered from the broths resulting from
either the surface- or submerged-culture fermentations, using
almost the same three methods – namely, direct crystallization
Figure 3 Time course of a typical batch citric acid fermentation from upon concentration of the filtered liquor, precipitation as
glucose-based media by A. niger NRRL 2270 in a 2 dm3 stirred fermenter calcium citrate tetrahydrate, or liquid extraction (see Fermenta-
(closed symbols) and by an industrial strain in a 400 m3 bubble-column tion (Industrial): Recovery of Metabolites). Direct crystalliza-
fermenter (open symbols): Concentrations of mycelial biomass (X: A, >), tion cannot be applied unless refined raw materials, such as
glucose (S: l, B), citric acid (P: n, ,), and ammoniac nitrogen (N: D) sucrose syrups or crystals, are used. Liquid extraction is used by
versus time (t). The industrial-scale trial was gently provided by Dr A. Tate & Lyle Co. (formerly Haarmann & Reimer Co., a subsidiary
Trunfio c/o Palcitric SpA, Calitri, Italy, and the laboratory-scale trial was
of Bayer Co.) in the Dayton (OH, USA) and Elkhart (IN, USA)
performed by the authors at the University of Basilicata (Potenza, Italy).
plants. The precipitation process is used by the great majority of
world citric acid manufacturers, including ADM in the United
States.
Table 3 shows the simplified overall stoichiometric reac- A simplified process flowsheet of this method is shown
tions occurring during the trophophase and idiophase of the in Figure 4.
fermentation examined. In particular, it was assumed that Mycelia and suspended particles are separated by
during the trophophase the microorganism (represented by continuous belt filters under vacuum. Citric acid is precipi-
a raw formula based on elemental analysis: CHnOpNq) repli- tated as calcium citrate by the addition of lime to the filtrate.
cates itself at the expanses of a generic carbon source in the Liming temperature is critical. Amorphous tricalcium citrate
presence of ammonia as the only nitrogen source; during the tetrahydrate generally is obtained at w70  C, while crystal-
idiophase, it undergoes further growth while decreasing line dicalcium acid citrate is obtained at 90  C. No removal
progressively its intracellular nitrogen content and excreting of oxalic acid is needed if the submerged-culture fermenta-
citric acid in a medium practically devoid of any nitrogen tion is used.
source (Figure 3). The residual citrate in the filtrate is precipitated as tricalcium
Assuming that no carbon atom of sugar is converted into citrate by further addition of lime to set the pH to 5.8. The
biomass, carbon dioxide, or other by-products as shown by the crystals are recovered using another continuous belt filter and
reaction in Eqn [2] (i.e., when y and d are equal to 0), the then recycled back to the liming step, while the filtrate has to be
theoretical molar yield (z) of citric acid would be one or two if disposed.
glucose or sucrose is used. This would be equivalent to 1.17 (or Precipitation of dicalcium acid citrate results in one-third
1.23) kg of citric acid monohydrate per kilogram of glucose less consumption of lime and consequently of sulfuric acid for
(or sucrose) consumed. In practice, the industrial yields range the subsequent regeneration of citric acid, in greater filterability
from 57 to 81% of this theoretical value, with the smaller and washability because of its crystalline structure, but 10–25%
figure generally being associated with the surface fermentation of the expected product yield is needed as seed. The precipitate
technique. is washed to remove the impurities adsorbed onto it (i.e.,
The citric acid fermentation may be mathematically residual sugars and contaminants from the raw carbon source
described by means of the set of kinetic equations shown and soluble proteins from the autolysis of the fungus). The
in Table 3. washed crystals and 98% w/w sulfuric acid are simultaneously,
In accordance with the Herbert–Pirt maintenance concept, but separately, fed to a mixer containing a 40% citric acid
both product formation (rP) and substrate consumption (rS) solution at pH 0.5–0.6, to free the citric acid with the formation
rates may be linearly related to cell growth rate (rx) and cell of a precipitate of calcium sulfate dihydrate (gypsum). Final
concentration (X). In this way, the well-known Luedeking– refining of the filtrate is performed by decolorization on acti-
Piret kinetics for product formation has to be regarded as vated carbon and removal of residual calcium sulfate and iron
a special case: In fact, the first term in Eqn [10] may be and nickel salts on strong cation exchange and weak anion
described as the product formation rate in association with the exchange (demineralization step). The resulting solution
mycelial growth rate, whereas the second term may be regarded (250–280 kg m3 of citric acid anhydrous) is concentrated
as the nongrowth-associated product formation rate. In both of using multiple-effect evaporators to about 700 kg m3, before
the fermentation trials shown in Figure 3, citric acid fermen- feeding a vacuum crystallizer operating at temperatures below
tation may be classified to be of the mixed-growth-associated (35  C) or above (62  C) the transition temperature (36.6  C)
product formation type. between the monohydrate and anhydrous forms, depending
810 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)

Table 3 Citric acid fermentation: overall stoichiometric reactions, kinetic equations, and instantaneous concentrations of
mycelia, product, sugar, and nitrogen sources

Equation or reaction

Trophophase reaction C6 H12 O6 þ a NH3 þ b O2 / y CHn Op Nq þ d CO2 þ e H2 O ½1


glucose mycelium

Idiophase reaction C6 H12 O6 þ b O2 / y CHn Op Nq þ z C6 H8 O7 þ d CO2 þ e H2 O ½2


glucose mycelium citric acid

Kinetic equations dX
rX ¼ ¼ mX X ½3
dt

 
dN dX
rN ¼ ¼ YN=X for N  Nlim ½4
dt dt

dN
rN ¼ ¼ 0 for N < Nlim ½5
dt

m ¼ 0 for t  to ½6

m ¼ mM for t  tlim ½7

 
X
m ¼ mM 1  for t > tlim ½8
XM

dP
rP ¼ ¼ 0 for t  tlim ½9
dt

 
dP dX
rP ¼ ¼ YP=X þ mP X for t > tlim ½10
dt dt

 
dS dX
rS ¼  ¼ YS=X þ mS X ½11
dt dt

Integral solutions of the X ¼ X0 for t  to ½12


differential kinetic equations
ðt t0 Þ
X ¼ X0 e mM for t  tlim ½13

XM
X ¼   for t > tlim ½14
XM
1þ  1 e mM ðt tlim Þ
X0

N ¼ N0 for t < t0 ½15

N ¼ N0  YN=X ðX  X0 Þ for t  tlim ½16

N ¼ Nlim for t > tlim ½17

P ¼ P0 þ mP Aðt Þ þ YP=X ðX  X0 Þ ½18

S ¼ S0  ½mS Aðt Þ þ YS=X ðX  X0 Þ ½19

Aðt Þ ¼ 0 for t  tlim ½20


 i
XM X h
Aðt Þ ¼ ln 1  M 1  e mM ðt tlim Þ
for t > tlim ½21
mM mM

Nomenclature: A(t), cumulative nongrowth contribution to product formation; b, y, z, d, and e, stoichiometric coefficients; mP (mS), specific rate of
product formation (or substrate consumption) at zero cell growth rate; Nlim, critical concentration of nitrogen at the onset of citric acid production; P,
citrate concentration; ri, conversion rate of any reagent or product; tlim, overall duration of the citrate lag phase; to, overall duration of the cell lag phase;
S, substrate concentration; X, mycelium concentration; XM, maximum mycelium concentration; m, specific cell growth rate; mM, maximum specific cell
growth rate; YN/x, YP/x, and YS/x, yield factors for ammoniac nitrogen, citrate, and substrate on unit cell biomass. Subscripts: lim, referred to limiting
concentration of the nitrogen source; N, nitrogen; P, citric acid; S, glucose; X, mycelium; 0, referred to the initial value.
FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)
Figure 4 Process flowsheet of a typical citric acid fermentation from glucose-based media by A. niger. Equipment and utility identification items: AE, anion exchanger; AF, antifoam agent; AL, alkaline reagent; BD,
fluidized-bed drier; BF, vacuum belt filter; C, centrifuge; c, Condensate; CA, activated carbon adsorber; CE, cation exchanger; CR, vacuum crystallizer; cw, cooling water; CY, cyclone; D, holding tank; dcc, dicalcium
citrate; DW, demineralized water; E, heat exchanger; EA, exhausted air; EV, evaporator; F, production-bubble fermenter; FI, sterile pressure filter; GR, grinder; HT, holding tube; LS, lime slurry; HA, hot air;

811
HS, sulfuric acid; M, mixer; NA, nutrients and additives; PC, centrifugal pump; PE, plate–heat exchanger; S, low-pressure steam; SA, sterile compressed air; Se, dicalcium citrate seed; SF, seed-bubble fermenter;
tcc, tricalcium citrate; WE, water evaporated.
812 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)

on the form manufactured. Crystals are separated by centrifu- processing. On the contrary, the recovery of tricalcium (or
gation and dehydrated in a two-stage fluidized-bed dryer, the trisodium) citrate from clarified, decolorized fermentation
first one using hot air at 90  C and the second one using air broths by electrodialysis, as well the adsorption of citric acid
conditioned at 20  C and relative humidity (RH) of 30–40% onto weakly basic anionic-exchange resins or zeolites using the
because of crystal hygroscopicity. The mother liquor is partly simulated-moving bed chromatographic technology (Citrex
(w20%) diluted with equipment-cleansing waters, decolor- Process, UOP, Des Plaines, IL, USA) followed by desorption
ized, and fed back to liming; the remainder is in sequence with water or dilute acidic solutions, or the use of liquid
decolorized and demineralized before being recycled to the membranes, would allow the citric acid to be separated in
crystallization unit. In this way, citric acid crystals do not need a single step and to be recovered without the formation of solid
additional purification steps to meet specifications for U.S. wastes for disposal.
Pharmacopeia or Food Chemical Codex material. The environmental aspects of citric acid production have
In the liquid extraction process, citric acid may be extracted been assessed. Despite the fact that most raw materials are of
from the fermentation broth using a highly selective, low-price, biological origin, many ingredients, such as ammonium
and nontoxic food-grade solvent (i.e., water-insoluble amines, nitrate, lime, and sulfuric acid, are hazardous chemicals. For
namely trilaurylamine, n-octanol, C10 or C11 isoparaffin, tri-n- instance, it was found that the environmental impact of citric
butyl phosphate, alkysulphoxides). The extract is then heated acid production using whey was smaller than that using corn
and washed countercurrently with water, resulting in about starch.
90% recovery yield and an aqueous citric acid concentrated
solution, which is passed through a granular activated–carbon
column before undergoing the aforementioned evaporation Gluconic Acid
and crystallization steps.
D-Gluconic acid (2,3,4,5,6-pentahydroxy pentane-1-carboxylic
acid: C6H12O7) is an oxidation product of D-glucose, which, in
Future Developments
an aqueous solution, leads to a complex equilibrium between
Production of citric acid has not been much in the focus of gluconic acid and its two lactones: 1,5-lactone (D-glucono-
modern molecular biology presumably because it is considered d-lactone) and 1,4-lactone (D-glucono-g-lactone).
a mature area. Any improvement of strains of A. niger usually D-Gluconic acid is commercially available as 50% aqueous
were carried out by mutagenesis and selection, but metabolic solution (density of 1230 kg m3 at 20  C and pH 1.82). This
and genetic engineering are likely to improve acidogenesis. acid and its derivatives are used in the pharmaceutical, food,
The replacement of the current batch fermentation with feed, and chemical industry because of their low toxicity and
semicontinuous processes, to increase volumetric productivity their ability to form water-soluble complexes with metallic
and reduce specific production costs, presently is hampered in ions (e.g., Caþ2, Feþ3), especially in the presence of 5–10% of
fungal processes by the deterioration of mycelial structure, the sodium hydroxide. Sodium gluconate is the main industrial
mechanism of which still is unknown. Although the effect of product and it is used as a sequestering agent (e.g., bottle
nitrogen deficiency on citric acid accumulation by A. niger is washing, metal surface cleaning, and rust removal) and to
well known, a low level of ammonium ions (i.e., 30 g m3, plasticize and retard the curing process of cement mixes. The
equivalent to 2 mmol of intracellular NHþ 4 per gram of dry cell) calcium and iron gluconates are used in medicine to treat
was found to inhibit the morphological degeneration of pellets diseases of calcium and iron deficiency (such as osteoporosis
and postpone sporulation. NHþ 4 ions simply are not deposited and anemia). D-Glucono-d-lactone is used as a latent acid in
into the cell to form the so-called ammonium pool, but they baking powders for use in dry cake mixes, meat processing, and
enter the cell to combine with glucose and form glucosamine, instant chemically leavened bread mixes, whereas D-glucono-
that is straight away released in the medium. The effective g-lactone is made only in small quantities as a specialty
relationship between the different compounds of the TCA cycle chemical.
is to be studied further and controlled before the present batch- The conversion of glucose to gluconic acid is a simple
production technology may be converted effectively into oxidation process and may be carried out by a variety of
a prolonged fed-batch or continuous production process. processes – namely, microbial fermentation, chemical, elec-
Similarly, the possibility of maintaining microbial cells trochemical, or enzymatic catalysis. Currently, these processes
active and controlling their growth and production processes appear to be either more expensive, unstable, or less efficient
for several weeks or months by immobilization within organic than the fermentation process, which presently is the only
or inorganic matrices represents a further challenge to the method of choice.
technological modernization of this sector. After the first isolation of calcium gluconate (1880) from
The traditional recovery technology results in several glucose fermentation in the presence of CaCO3 by a strain of
problems because of disposal of liquid effluents (their chem- Mycoderma aceti, the Chas. Pfizer & Co., Inc. (New York, USA)
ical oxygen demand being about 20 kg m3) and solid started industrial-scale production of gluconic acid in 1923.
by-products (i.e., about 0.15 kg of dried mycelium and 2 kg of Further research at the U.S. Department of Agriculture in
gypsum per kilogram of citric acid anhydrous). Several process cooperation with the Iowa State College led to the semi-
alternatives have been suggested thus far to minimize the continuous production of sodium gluconate from glucose
overall environmental impact of this process. The replacement using A. niger NRRL 67.
of molasses with raw or hydrolyzed starch- or raw sucrose- Several filamentous fungi of the genera Penicillium and
based materials would simplify only the downstream Aspergillus, the yeastlike fungus Aureobasidium pullulans, and
FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic) 813

bacteria (Acetobacter suboxidans, Pseudomonas ovalis, Glucono- adjusted to 6–6.5 with sodium hydroxide, and a 2–5% v/v
bacter spp.) produce gluconic acid from glucose-based media, inoculum generally is used. For inoculum development, con-
but industrial processes have been developed only for the idia are recovered from stock agar slants and are inoculated into
production gluconic acid from glucose syrups with A. niger and vegetative seed–culture media (106 conidia cm3); pellet-like
Gluconobacter oxydans. Industrial strains are not freely available, mycelia is obtained after incubation at 30  C for 15–24 h and is
but a few gluconic acid–producing strains (A. niger NRRL 3, used to inoculate seed fermenters at a density of 20–50
NRRL 67) can be obtained from international culture collec- pellets cm3.
tions. Penicillium spp. generally produce less gluconate than The fermentation is carried out under continuous automatic
Aspergillus, but they have the advantage of excreting the glucose control of sterile air sparging (1.0–1.5 vvm), temperature
oxidase (an important by-product) into the medium, which (33  C), pressure on the tank top (2–3 bar), pH (5.5–6.5 by
makes its recovery easier. addition of 30–50% NaOH solution to neutralize the gluconic
The formation of gluconic acid by A. niger is controlled by acid formed), and foam level. It is completed within w30 h
the enzyme glucose oxidase, an omodimer containing two with yield factors of 0.97–1 kg of gluconic acid per kilogram of
flavin adenine dinucleotide (FAD) moieties. Such enzyme glucose consumed (against a theoretical yield of 1.09 kg kg1)
abstracts two hydrogen atoms from glucose, thus yielding the and gluconate productivities of 9–13 kg m3 h1.
glucono-d-lactone, which to some extent hydrolyzes to glu- In the fed-batch operation, the mycelium may be reused up
conic acid. The FADH2 reacts with oxygen to form hydrogen to five times without any loss in gluconate productivity
peroxide, which is converted into oxygen and water by the provided that the levels of glucose oxidase activity and other
enzyme catalase. Both glucose oxidase and catalase are microelements (i.e., iron and manganese) are kept under
constitutive endoenzymes in A. niger. control. Stepwise addition of glucose may be used to increase
A highly productive process of gluconic acid using free- gluconate concentration to 580 kg m3.
growing cells of A. pullulans DSM 7085 recently has been At the end of fermentation, the mycelium is removed using
developed. Its high conversion yields (90–98%) and rates aseptic centrifugation, under vacuum-belt filtration or cross-
(13–19 kg m3 h1) resulted in as high gluconate concentrations flow microfiltration and may be used as a source of glucose
as 504 or 230–433 kg m3 in fed-batch or chemostat trials. oxidase or may be disposed off via incineration. The clarified
Although this novel fermentation process offers a new opportu- broth, generally containing w300 kg m3 of sodium gluconate,
nity for commercial gluconic acid production, as well as many is filtered, decolorized using a granular activated–carbon
advantages over the traditional microbial fermentation pro- column, concentrated under vacuum to 45–50% total solids,
cesses, it is still confined to laboratory-scale applications. Thus, neutralized to pH 7.5 with NaOH, and then spray or drum
only the sodium process by batch-submerged fermentation from dried. If 50% gluconic acid is required, the concentrated liquor
glucose syrups using A. niger will be described in the following may be passed through a cation exchanger to remove Naþ ions.
paragraphs. Further crystallization at 30–70  C or at more than 70  C
Glucose syrups of 70 Brix strength are generally used as allows crystals of the d-lactone or g-lactone to be precipitated,
carbon source in the preparation of the fermentation medium. respectively.
Table 4 lists the typical composition of the seed and
production media used in laboratory- and industrial-scale
trials. Lactic Acid
After the pH is adjusted at 4.5 with sulfuric acid, the
medium is sterilized at 121  C for 15–30 min, cooled at 33  C, Lactic acid (2-hydroxypropionic acid: C3H6O3) may be
and then transferred into the fermentation vessel. The pH is produced by chemical synthesis or fermentation. Of the two
enantiomers, L-(þ) and D-() lactic acid, only the L-(þ) isomer
is used by human metabolism and, because of the slight
Table 4 Composition for the production media used in the
toxicity of the D-() isomer, it is preferred for food uses.
laboratory- and industrial-scale production of gluconic acid by A. niger Because the chemical route yields a mixture of L-lactic acid and
D-lactic acid and relies on costly raw materials, all lactic acid
Vegetative seed Gluconic acid manufacturing industries have switched to fermentation-based
Component –culture media production media Unit technologies (Table 1). The free acid is used as an acidulant/
preservative in several food products (cheese, meat, jellies,
Glucose 40 120–350 kg m3
NH4NO3 (or other 2.4 0.4–0.5 kg m3 beer) (see Preservatives: Traditional Preservatives – Organic
NHþ 4 salt)
Acids); sodium lactate is used for carcass decontamination;
KH2PO4 1.5 0.1–0.3 kg m3 ammonium lactate is used as a source of nonprotein nitrogen
MgSO4$7H2O 3.57 0.1–0.3 kg m3 in feeds; sodium and calcium stearoyl lactylates are used as
Agar 1 0 kg m3 emulsifiers and dough conditioners. The large increase in lactic
Yeast extract 1 0 kg m3 acid production is due to its use in the synthesis of polylactic
Corn-steep liquor 0 0.2–0.4 kg m3 acid (PLA), a polyester used for biodegradable plastics for food
ZnSO4$7H2O 100 0 mg m3 packaging, compost, and garbage bags, and disposable table-
CuSO4$5H2O 20 0 mg m3
ware, as well as several medical applications, such as reab-
FeCl3$6H2O 300 0 mg m3
sorbable sutures, orthopedic implants, and controlled drug
MnSO4$7H2O 0 30 mg m3
Initial pH 6.5 6.0 – release. The current economically viable industrial process for
PLA production is via the dehydrated cyclic dilactate ester
814 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)

(lactide) formation. In brief, lactic acid is first polymerized into Concomitant substrate and product inhibition has been
oligomers of PLA consisting of 30–70 lactyl units (–CHCH3– reported for several species.
CO–O–). These oligomers are then depolymerized by LAB are fastidious microorganisms and require supple-
increasing the polycondensation temperature and lowering the mentation of fermentation media with peptides and growth
pressure in the presence of transition metal–based catalysts factors, usually in the form of yeast extract. Because this may
(i.e., stannous octoate at 0.05%) to distil the lactide. Finally, by account for 30–35% of substrate costs, they may be replaced by
opening its ring, it is possible to obtain high molar mass less demanding B. coagulans and R. oryzae, both being
polymers (100–300 kDa) with appropriate optical and L(þ)-lactate producers, even if smaller yields (as low as 70%
mechanical properties (i.e., tensile strength >50 MPa). Other for R. oryzae because of concomitant fumaric acid and ethanol
comonomers, such as caprolactone, hydroxybenzoic acid, and production) and acid tolerance may offset the advantage of
others, can be incorporated to provide environmentally safe using lower amounts of supplements.
materials. PLA appears to be a sustainable alternative to
petroleum-based plastics, because lactide is produced from the
Substrate Production and Recovery
fermentation of renewable resources, such as corn starch (as in
the industrial plant of Nature Works LLC, Blair, Nebraska, USA: Lactic acid can be produced from a variety of raw substrates
140 000 metric tons per year). Nevertheless, to minimize (whey and whey permeate, beet and cane molasses, starch and
competition for land and food, research studies have started to corn starch hydrolysates, wood hydrolysates; see Fermentation
develop second-generation PLA products from lignocellulosic (Industrial): Media for Industrial Fermentations). Some species
hydrolysates (e.g., crushed corncobs). (Lb. amylophilus) can hydrolyze starch, but the pseudoplastic
behavior of starchy substrates makes the pH control difficult.
When whey permeate is used, supplementation with milk
Organisms and Metabolic Pathways Involved
protein hydrolysates (5–10 kg m3) and yeast extract (up to
Lactic acid can be produced using homofermentative lactic 20 kg m3) is required. Lactic acid production is usually
acid bacteria (LAB), facultatively anaerobic Bacillus species a growth-associated production process, but nongrowth-
(B. coagulans), and molds (Rhizopus microsporus, Rhizopus associated production becomes significant when growth is
oryzae). Recently, lactic acid producing genetically modified limited by a lack of nutrients or high undissociated acid
strains of Escherichia coli and Saccharomyces cerevisiae have concentration. The pH is controlled at 5–6.5 by the automatic
been developed. The choice of the species depends on several addition of NaOH, Na2CO3, or NH4OH or by the addition of
considerations, including the ability to use the type of sugars CaCO3. Fermentation is carried out under anaerobic or micro-
available in the substrate, growth temperature, nutritional aerophilic conditions and lactic acid yield is usually between 85
needs, acid tolerance, and type of lactic acid isomer and 98% with isomer purity as high as 99%. Batch fermenta-
produced. tions result in high product concentration (120–150 kg m3)
Thermophilic lactobacilli (Lactobacillus delbrueckii subsp. but in low productivity (2 kg m3 h1). Conversely, continuous
delbrueckii, L. delbrueckii subsp. bulgaricus, Lactobacillus helveticus) fermentations with cell-recycle or immobilized cells give rise to
tolerate higher concentrations of lactate and higher temperatures higher productivities (20–80 kg m3 h1) and lower lactate
(48–52  C), thus involving higher productivity and yields concentrations (<50 kg m3). End-product inhibition may be
and reduced contamination risks. They produce D-() or DL-lactic circumvented by using integrated fermentation processes, in
acid (some industrial strains that have been claimed to be which lactic acid is removed from the culture broth by several
L. delbrueckii produce L-(þ) lactic acid) and may be less suitable techniques, including electrodialysis, ion-exchange resins, or
for food, feed, or biomedical applications. Lactococci, mesophilic nanofiltration.
lactobacilli (Lactobacillus casei subsp. casei, Lactobacillus amylophi- Recovery of lactate is made complicated by the high solu-
lus), and thermophilic streptococci (Streptococcus thermophilus) bility of its salts. The traditional process involves precipitation
have lower temperature optima or reduced acid tolerances, but of calcium lactate and regeneration of lactic acid by the addi-
they may be desirable for other reasons (e.g., production of pure tion of sulfuric acid followed by further purification steps (ion
L-(þ) lactic acid, hydrolysis of starch). Recently, genetic engi- exchange and decolorization). Alternative processes include the
neering has been used to produce L. helveticus and Lactobacillus extraction by liquid membranes, electrodialysis, and ion
plantarum strains, which produce optically pure L-(þ) or D-() exchange. In particular, the recent industrial use of electrodi-
lactic acid (see Lactobacillus: Introduction; Lactococcus: Introduc- alysis with bipolar membranes in France resulted in the virtual
tion; and Streptococcus thermophilus). elimination of gypsum waste production. Conventional
Homofermentative LAB ferment hexoses via the glycolytic recovery by the precipitation method seems to be the most
pathway (see Metabolic Pathways: Release of Energy (Anaer- economical route.
obic)). Pyruvate is reduced to lactate by stereospecific lactate
dehydrogenase(s) (L-LDH or D-LDH). LDH is allosteric (acti-
vators: fructose-1,6-bisphosphate and Mnþ2) in lactococci and Other Organic Acids Produced by Fermentation
nonallosteric in homofermentative lactobacilli. Undissociated
Propionic Acid
lactic acid acts as a noncompetitive inhibitor for growth and
lactic acid production by diffusing through the membrane and Propionic acid (C3H6O2) and its salts are used as mold inhib-
decreasing intracellular pH: pH control during fermentation itors in bakery products, although other nonfood uses are
reduces the inhibition, but the maximum lactic acid concen- important (see Permitted Preservatives – Propionic Acid). It may
tration achievable is usually lower than 150 kg m3. be produced by fermentation by members of the genera
FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic) 815

Propionibacterium (P. freudenrichii, thoenii, acidipropionici), Veillo- Further Reading


nella, Clostridium, and Selenomonas, but currently it is produced
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Berovic, M., Legisa, M., 2007. Citric acid production. Biotechnology Annual Review 13,
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productivities (2–14 kg m3 h1) obtained in continuous production from renewable resources. Enzyme and Microbial Technology 26,
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Joglekar, H.G., Rahman, I., Babu, S., Kulkarni, B.D., Joshi, A., 2006. Comparative
reactors, as well the possibility of obtaining pure propionic acid
assessment of downstream processing options for lactic acid. Separation and
from alternative low-cost substrates, like crude glycerol from the Purification Technology 52, 1–17.
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