FERMENTATION (INDUSTRIAL) Production of Some Organic Acids (Citric Gluconic LactiC and Propionic)

Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)M Moresi, Università della Tuscia, Viterbo, ItalyE Parente, Università della Basilicata, Potenza, Italy; and Istituto di Scienze dell’Alimentazione, Avellino, Italy

� 2014 Elsevier Ltd. All rights reserved.

Several organic acids are used in a variety of food and nonfoodapplications. Table 1 lists the main acids that are producedcommercially by chemical (C) or biotechnological (fermenta-tion, F, or enzymatic, E) methods or extracted from wine-making residues (L).

Citric, acetic, lactic, propionic, tartaric, fumaric, and malicacids are among the most versatile ingredients in the food andbeverage industry because of their valuable properties, such assolubility, hygroscopicity, acidity, buffering capacity, andchelation (see Preservatives: Traditional Preservatives –OrganicAcids).

Citric acid accounts for around 80% of the food acidulantusage, whereas the use of phosphoric or acetic acids is limited,being almost exclusively utilized in cola soft drinks or invinegar (see Vinegar), sauces, and condiments, respectively.

Citric Acid

Citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid: C6H8O7)is widely distributed in natural raw materials (such as lime,lemon, and raspberry) and is commercially available in themonohydrated form (molecular mass of 210.13Da, relativedensity of 1.542 at 20 �C, and heat of combustion of1962 kJmol�1 at 25 �C). It is a strong tricarboxylic acid(TCA; its dissociation constants being K1¼ 7.45� 10�4,K2¼ 1.73� 10�5, and K3¼ 4.02� 10�7 at 25 �C), highlysoluble in water with pleasant acid taste.

Citric acid was first isolated in 1784 by Scheele, whoprecipitated it as calcium citrate by adding calciumhydroxide (lime) to lemon juice. Before 1920, it was almostexclusively produced in Sicily by pressing lemons: The firmArenella (Palermo, Italy) essentially established a monopolyuntil the advent of the citric acid fermentation technique in

Table 1 Main organic acids: molecular formulas, world output, productio

Acidulant Chemical formula World output (metric ton

Acetic acid (vinegar) C2H4O2 190 000Lactic acid C3H6O3 150 000

Propionic acid C3H6O2 130 000Fumaric acid C4H4O4 12 000Malic acid C4H6O5 10 000

Tartaric acid C4H6O6 28 000Itaconic acid C5H6O4 15 000Citric acid C6H8O7 1 800 000Gluconic acid C6H12O7 87 000

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

804 Encyclopedia of Food Mic

Belgium (Societè des Produits Organiques de Tirlemont) in1919 and in the United States (Chas. Pfizer & Co., NewYork) in 1923.

About 10 years later, about 80% of the world’s citric acidwas produced by the surface fermentation process. Thesubmerged fermentation process began to be applied only afterWorld War II.

From 1950 to 1980, citric acid was mainly used in phar-maceutical or health products. In fact, in the early 1980s, itstwo largest manufacturers were Pfizer and Miles/Bayer, bothsuppliers of prescription drugs. Thereafter, as citric acid beganto be used in the food and beverage sector in industrial anddeveloping countries, its market size experienced significantgrowth and several new manufacturers were established inEurope and North America, as well as in China where severalsmall-scale fermentation units have produced citric acid fromsweet potatoes or cassava since the 1970s.

In the early 1990s, a few manufacturers gave rise to the so-called citric acid cartel. The overcharges imposed on US buyerswas estimated in the range of $116–309million and, onJanuary 29, 1997, Haarmann & Reimer Corp., a subsidiary ofBayer AG (D), pled guilty and paid a $50million criminal fine.In March 1998, even Archer Daniels Midland Co. (ADM)agreed to pay $36million to four citric acid customers that hadopted out of the July 1997 civil class-action antitrust settle-ment. At that time, the global citric acid capacity was about840 000Mg (mega grams) per year with a growth rate of 5%per year. Afterward, the world citric industry became lessconcentrated and numerous new manufactures, especially inChina, as well as Brazil, India, Indonesia, and Thailand, haveentered the market, thus making the formation of cartels lessprobable.

Moreover by the early 2000s, almost all citric acidmanufacturing was globally integrated into the corn wet-milling

n methods, and organisms

s) Production methods a,b Organism

F 100% Acetobacter aceti

F 100% Lactobacillus spp.Rhizopus spp.

C 100% Propionibacterium acidipropionici

C 100% Rhizopus arrhizus

C 70%E 30%

–

L 100% –

C 100% Aspergillus terreus

F 100% Aspergillus niger

F 100% Aspergillus niger

robiology, Volume 1 http://dx.doi.org/10.1016/B978-0-12-384730-0.00111-7

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 werebought by ADM and Tate & Lyle, respectively) or by new processdevelopment (Cargill).

In the years 1987–89, US list prices for citric acid anhydrousremained unchanged at $1.79 kg�1. By late 1989, the list pricereduced to $1.65 kg�1, while in the fall 1990 it was as low as$1.39 kg�1. Then, thanks to the “citric acid conspiracy” in theyears 1993–96, the citric acid cartel accomplished its main goalof raising and keeping list price at $1.87 kg�1. Then, it loweredfrom $1.76 kg�1 in November 1994 to $1.54 kg�1 in the early1997.

Thereafter, the severe competition resulted in selling pricesof anhydrous citric acid decreasing to $0.70–$0.80 kg�1, thusforcing the smaller manufacturers, unable to benefit from theeconomy of scale, to exit the business. As a consequence, thepanorama of organic acid manufacturers has profoundlychanged over the last decade. In 2010, China approximatelyaccounted for more than 50% of global citric acid productioncapacity, while Europe and North America covered the 19and 24%, respectively, and as much as 65–70% of globalconsumption.

Several substrates are used as fermentation substratesdepending on the local availability. Maize starch is mainly usedin the United States and China, while sugarcane or sugar beetmolasses prevail in the Brazilian and Indian or Europeanmarkets, respectively.

Cellulosic materials are currently unused in citric acidproduction, even if there are projects to assess the technicalfeasibility of such feedstock materials in the citric acidindustry.

From January 2008 to January 2009, export prices ofChinese (anhydrous) citric acid oscillated in the range of US$0.7–$0.8 kg�1; thereafter, they steadily increased to reacha peak of $1.1 kg�1 in June 2011, as a direct result of theincrease in the market prices for agricultural raw materials,particularly corn. The present economic crisis in Europe and theUnited States has newly reduced the market prices of (anhy-drous) citric acid to US $0.70�$0.96 kg�1 depending on theamount ordered.

In conclusion, the global citric acid production capacityreached almost 1.8 million metric tons (Mg) in 2010, whileit was about 1.5 � 106 Mg in 2005.

Organisms and Metabolic Pathways Involved

Several molds (Penicillium spp., Aspergillus niger, Aspergilluswentii, Trichoderma viride; see Aspergillus and Penicillium and-Talaromyces: Introduction), yeasts (Yarrowia lipolytica, Candidaguillermondii), and bacteria (Arthrobacter) produce citric acidfrom a variety of substrates (glucose, sucrose, n-alkanes), butindustrial processes have been developed only for theproduction of citric acid from sugars (glucose, sucrose) withA. niger and from sugars and n-alkanes with yeasts. Industrialstrains are not freely available, but citric acid–producingstrains (A. niger, NRRL 2270, NRRL 599, ATCC 11414, ATCC9142; Y. lipolytica ATCC 20346, ATCC 20390, NRRL Y-7576,NRRL Y-1095) can be obtained from international culturecollections.

Metabolic pathways involved in citric acid overproductionby A. niger are shown in Figure 1.

A high flux through the glycolysis, decreased activity of TCAcycle reactions that degrade citrate, and an anaplerotic reactionto replenish the oxaloacetate (OAA) used for the synthesis ofcitrate are all essential (see Metabolic Pathways: Release ofEnergy (Aerobic)). Key regulatory steps in the process includeglucose transport and phosphorylation, citric acid export fromthe mithocondria and cell, phosphofructokinase, pyruvatecarboxylase (PC), citrate synthase (CS), and a-ketoglutaratedehydrogenase (KDH).

The metabolic changes necessary for citric acid over-production in A. niger are induced by high sugar concentration,low pH, and manganese (Mnþ2) deficiency. Other factors (i.e.,phosphate and nitrogen concentrations, high dissolved oxygen(DO) concentration, trace metals), however, are important.Very low concentrations of Mn2þ (<10mgm�3) are critical.They result in decreased activity of the pentose phosphatepathway and increased glycolytic flux, increased intracellularNH4

þ pool and turnover of nucleic acids and proteins, changesin membrane lipid composition and cell wall composition,and morphological changes.

Improvement of strains for citric acid production tradi-tionally has been carried out by mutagenesis and screening.Overexpression of proteins critical to acidogenesis (hexokinase,glucose carrier) or inactivation of genes encoding enzymes thatproduce allosteric inhibitors of hexokinase has been attempted,but with limited success, in the additional production of citricacid. It has been postulated that the activity of seven glycolyticenzymes needs to be increased to obtain increased productionof citric acid. The availability of the complete genome sequenceof A. niger is likely to allow for the design of overproducingmutants.

Citric acid overproduction in yeast is relatively insensitive totrace metals concentration and is triggered by nutrient (N, S, P,or Mg) limitations coupled with a high rate of glucoseutilization, which results in a high adenosine triphosphate/adenosine monophosphate ratio and, in turn, in inactivation ofnicotinamide adenine dinucleotide (NADþ)-specific isocitratedehydrogenase (IDH). The main anaplerotic reactions includethe synthesis of OAA from pyruvate catalyzed by PC duringproduction from glucose and the glyoxylate cycle duringgrowth on n-alkanes. Accumulation of isocitrate (10–50% ofthe citrate produced) in excess with respect to the predictedequilibrium of aconitase probably is due to the high perme-ability of yeast mitochondria to isocitrate compared withcitrate. Low cytoplasmic levels of citrate may be responsible forreduced feedback inhibition of glycolysis. Improvement ofyeast for citric acid production is directed to obtain strains withreduced isocitrate dehydrogenase and aconitase activities.

Methods of Manufacture

Citric acid production is mainly accomplished by thesubmerged fermentation process, probably because of thesmaller contribution of investment and labor costs to its overallproduction costs. The surface fermentation process currentlyaccounts for only 5–10% of the world supply. In Europe, allsurface fermentation plants have been shut down during thepast decade. Smaller amounts of citric acid (<1%) are reportedto be extracted from citrus fruits in Mexico and South Americaand to be produced by the solid-state process in Japan.

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

Table 2 Composition for the production media used in thelaboratory- and industrial-scale production of citric acid by A. niger

Component

Range of

concentrations Typical values Unit

Sucrose or glucose 125–225 180 kg m�3

NH4NO3 (or otherNH4

þ salt)0.5–3.5 1.5 kg m�3

KH2PO4 0.5–2 0.5 kg m�3

MgSO47H2O 0.1–2.0 0.25 kg m�3

Feþ2 2–1300 <200 mgm�3

Znþ2 0–2900 200–1500a mgm�3

Cuþ2 1–10 200 200–1500a mgm�3

Mnþ2 0–46 <2 mgm�3

Initial pH 2.5–6.5 2.2 –

aTo overcome the detrimental effects of iron and manganese on mycelium structure

=


Both submerged- and surface-culture fermentation pro-cesses use beet molasses or glucose syrup as the main rawmaterial and use A. niger as the fermenting organism.

Submerged fermentation is carried out either in 150–200m3

stirred-tank reactors or in300–500m3 (up to1000m3 as claimedby some manufacturers) bubble-column reactors. The mainadvantages of these techniques are improved asepsis duringfermentation, automatic control of inoculation and fermentationprocedures, shorter fermentation times, and greater productyields.

In spite of the old and renewed interest in citric acidproduction by yeast grown submerged in sugar- or hydro-carbon-based media to overcome the main disadvantages oftraditional mold fermentation (i.e., high sensitivity to tracemetals and low production rates), no yeast-based process iscurrently known to be operating worldwide.

and restore proper morphology.

Production Media

Citric acid is produced from media containing high concen-trations of simple sugars (molasses or glucose syrup) (seeFermentation (Industrial): Media for Industrial Fermenta-tions), in which mycelium growth is restrained by nutrient(phosphorous, manganese, iron or zinc) limitation. Their rangeof composition is given in Table 2.

Nitrogen is usually added as ammonium nitrate or sulfate.Metals are removed by pretreatments of raw materials, espe-cially molasses, with cation-exchange resins or the addition ofpotassium hexacyanoferrate (HCF). The optimal iron concen-tration seems to depend on the fungal strain, but iron levels of200mgm�3 were found to inhibit citrate production. Theinhibitory effect of Feþþ can be counterbalanced by the addi-tion of copper and zinc salts during the inoculum developmentor during early mycelium growth in the production medium.Manganese concentration has to be kept as low as possible(<10mgm�3).

Some ingredients (methanol, 3–6%w/v; corn, peanut, andolive oils, 0.1–0.5%w/v; starch, 0.025–0.5%w/v) have beenclaimed to enhance the citric acid yield.

Figure 1 Metabolic pathways for citric acid overproduction in Aspergillus nig

(�, inhibitor; þ, activator) are shown. Enzymes and transport systems: INV,carrier; PP, proton pump; CC, putative citrate carrier; HK, Hexokinase; PGI, ph6-phosphofructo-2-kinase; ALD, aldolase; PK, pyruvate kinase; PC, pyruvate cTCC, citrate transport system; PDH, pyruvate dehydrogenase; CS, citrate synthdehydrogenase; AOX, alternative oxidase system. Substrates and products: glfru1,6 dP, fructose-1,6-bisphosphate; fru2,6 dP, fructose-2,6-bisphosphate; gpyr, pyruvate; oaa, oxaloacetate; mal, malate; cit, citrate; acCoA, acetyl-coenzysuccinyl-coenzyme A; tre, trehalose.

The most important steps in controlling glycolytic flux are glucose transpoalthough A. niger has both low-affinity and high-affinity carriers for glucose) aphosphate. PFK1 is feedback inhibited by citrate, but the inhibition is counterac(FBP). A phosphorylated fragment of PFK1, which is insensitive to citrate inhibitsubstrate concentration: its product, FBP lowers the Michaelis–Menten constanthus increasing carbon flux through glycolysis during acidogenesis. CS activianaplerotic reaction catalyzed by PC. Malate is produced from OAA by cytosoliwhere it is oxidized back to OAA. Low activity of NADPþ-specific IDH and KDHaffinity than ACT.

A salicylhydroxamic acid–sensitive, alternate oxidase system is used duringMalfunction of the normal respiratory chain is due to diminished activity of N

Media sterilization is carried out batch wise at 121 �C for15–30min at the laboratory or pilot scale or continuouslyusing a plate–heat exchanger unit at the industrial scale.

Fermentation Process

Inoculation is generally carried out by transferring asepticallythe stock culture maintained on agar slants on other workingslants. After w24 h incubation at 30 �C, the conidia crop isinoculated in starch-rich seed-production media to yield up to1011 spores cm�3. This culture may be directly transferred into10–20m3 seed fermenters to obtain a pellet-type inoculumconsisting of 1–5� 105 pellets dm�3 (0.1–0.2 mm in diam-eter), which in turn is used as inoculum (5–10% v/v) for theindustrial-scale production medium.

The fungus will develop different morphological forms(Figure 2).

The formation of a loose mycelium with long, unbranchedhyphae is to be avoided because this results in an enormousincrease in the apparent viscosity of the culture broth, thuslimiting the effective oxygen transfer rate with little or no citric

er. Only relevant enzyme activities, substrate, products, and effectorsmembrane bound invertase; GC, low-affinity glucose carrier; FC: Fructoseosphoglucose isomerase; PFK1, phosphofructokinase; PFK2,arboxylase; MDH, malate dehydrogenase; PT, pyruvate transport system;ase; ACT, aconitase; IDH, isocitrate dehydrogenase; KDH, a-ketoglutarateu, glucose; glu6P, glucose-6-phosphate; fru6P, fructose-6-phosphate;ly, glycerol; dhp, dihydroxiaceton phosphate; pep, phosphoenolpyruvate;me A; aco, cis-aconitate; ica, isocitrate; a-kg, a-ketoglutarate; sucCoA,

rt (simple diffusion is the main mechanism at high sugar concentrations,nd hexokinase (HK) activity, which initially is inhibited by trehalose-6-ted by the presence of high levels of NH4

þ and by fructose-2,6-biphosphateion, may be responsible for acidogenesis. PFK2 activity is increased at hight (Km) of PFK1, counteracts citrate inhibition, and inhibits gluconeogenesis,ty in A. niger is regulated by the level of OAA, which is produced in thec MDH and acts as a counterion for citrate efflux from the mitochondrion,are a consequence of the effective removal of citrate, which has a higher

acidogenesis to reoxidize the NADH produced during glycolysis.ADH ubiquinone reductase and other respiratory chain enzymes.

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 unbranchedhyphae protruding from the pellet core (100�).


acid production. On the contrary, small spherical, densepellets (Figure 2(a)) with short stubby hyphae (Figure 2(b))are generally regarded as the best morphological formfor optimal citrate yields. Frequent observation of pelletmorphology during this early stage of the fermentation usinga microscope allows hyphae proliferation to be controlled bythe addition, in case of adverse development (Figure 2(c)), ofappropriate amounts of inhibiting compounds, such as HCFor zinc and copper sulfates. Mycelial clumps (whose structureis less compact than pellets) also may develop under highagitation speed.

Fermentation is exothermic and temperature has to be keptin the range 28–35 �C. Assuming that the overall heat transfercoefficient and effective temperature difference between thefermenting medium and cooling water are of the order of500 kJ m�2 h�1 and 5 �C, respectively, the heat transfer surfacerequired to keep the fermentation temperature constant wouldbe w3.2m2 per m3 of fermentation medium.

Low pH and high DO concentration are essential for citricacid production. Initial decrease of pH is due to ammoniumuptake. Extreme pH values (<1.6) limit productivity, and theaddition of alkali (NH3) is used to control pH at 2.2–2.6 onceproduction of citric acid has started. The typical industrial-scale

productivities of 1–1.5 kgm�3 h�1 result in microbial oxygendemand rates of 0.3–0.5 kg O2 m�3 h�1, that are met bysparging 0.1–0.4 volumes of air per medium volume perminute (vvm) at pressures at the sparger section of thefermenter not less than 0.3–0.4MPa and at the tank top,ranging from 0.25–0.35MPa to 0.12–0.15MPa, depending onthe (stirred or air-lift) fermenter type used. Foaming iscontrolled by adding food-grade antifoam agents.

Temporary interruption to the air supply during fermenta-tion does not seem to affect the performance of the culture onthe condition that the DO level is greater than 20% of thesaturation value. DO values of about 0 for as long as 85min,followed by restoration of the air supply, do not inhibitpermanently mycelial growth and citrate production, but theydo reduce the product yield coefficient up to 20%.

Figure 3 shows the evolution of a typical batch citric acidfermentation in glucose-based media by A. niger NRRL 2270 in2-dm3 stirred fermenter and by a mutant strain of A. niger in400m3 bubble-column fermenter.

Two distinct phases are evident: during the primary growthphase (trophophase), no acid production occurs; during thesecond growth phase (idiophase), acid production by almostnongrowing cells is observed.

Figure 3 Time course of a typical batch citric acid fermentation fromglucose-based media by A. niger NRRL 2270 in a 2 dm3 stirred fermenter(closed symbols) and by an industrial strain in a 400 m3 bubble-columnfermenter (open symbols): Concentrations of mycelial biomass (X:A,>),glucose (S: l, B), citric acid (P: n, ,), and ammoniac nitrogen (N: D)versus time (t). The industrial-scale trial was gently provided by Dr A.Trunfio c/o Palcitric SpA, Calitri, Italy, and the laboratory-scale trial wasperformed by the authors at the University of Basilicata (Potenza, Italy).


Table 3 shows the simplified overall stoichiometric reac-tions occurring during the trophophase and idiophase of thefermentation examined. In particular, it was assumed thatduring the trophophase the microorganism (represented bya raw formula based on elemental analysis: CHnOpNq) repli-cates itself at the expanses of a generic carbon source in thepresence of ammonia as the only nitrogen source; during theidiophase, it undergoes further growth while decreasingprogressively its intracellular nitrogen content and excretingcitric acid in a medium practically devoid of any nitrogensource (Figure 3).

Assuming that no carbon atom of sugar is converted intobiomass, carbon dioxide, or other by-products as shown by thereaction in Eqn [2] (i.e., when y and d are equal to 0), thetheoretical molar yield (z) of citric acid would be one or two ifglucose or sucrose is used. This would be equivalent to 1.17 (or1.23) kg of citric acid monohydrate per kilogram of glucose(or sucrose) consumed. In practice, the industrial yields rangefrom 57 to 81% of this theoretical value, with the smallerfigure generally being associated with the surface fermentationtechnique.

The citric acid fermentation may be mathematicallydescribed by means of the set of kinetic equations shownin Table 3.

In accordance with the Herbert–Pirt maintenance concept,both product formation (rP) and substrate consumption (rS)rates may be linearly related to cell growth rate (rx) and cellconcentration (X). In this way, the well-known Luedeking–Piret kinetics for product formation has to be regarded asa special case: In fact, the first term in Eqn [10] may bedescribed as the product formation rate in association with themycelial growth rate, whereas the second termmay be regardedas the nongrowth-associated product formation rate. In both ofthe fermentation trials shown in Figure 3, citric acid fermen-tation may be classified to be of the mixed-growth-associatedproduct formation type.

A microscopic description of this fermentation usingA. niger pellets also has to account for oxygen diffusionphenomena from the bulk of the fermenting medium to thepellet surface and through the porous structure of the pelletitself. The low effective diffusivity of oxygen within the pelletlimits mycelial activity to a peripherical spherical shell only,with the oxygen penetration depth ranging from 110 to 300 mmin 2mm pellets.

Recovery and Purification Processes

Citric acid may be recovered from the broths resulting fromeither the surface- or submerged-culture fermentations, usingalmost the same three methods – namely, direct crystallizationupon concentration of the filtered liquor, precipitation ascalcium citrate tetrahydrate, or liquid extraction (see Fermenta-tion (Industrial): Recovery of Metabolites). Direct crystalliza-tion cannot be applied unless refined raw materials, such assucrose syrups or crystals, are used. Liquid extraction is used byTate & Lyle Co. (formerly Haarmann & Reimer Co., a subsidiaryof Bayer Co.) in the Dayton (OH, USA) and Elkhart (IN, USA)plants. The precipitation process is used by the great majority ofworld citric acid manufacturers, including ADM in the UnitedStates.

A simplified process flowsheet of this method is shownin Figure 4.

Mycelia and suspended particles are separated bycontinuous belt filters under vacuum. Citric acid is precipi-tated as calcium citrate by the addition of lime to the filtrate.Liming temperature is critical. Amorphous tricalcium citratetetrahydrate generally is obtained at w70 �C, while crystal-line dicalcium acid citrate is obtained at 90 �C. No removalof oxalic acid is needed if the submerged-culture fermenta-tion is used.

The residual citrate in the filtrate is precipitated as tricalciumcitrate by further addition of lime to set the pH to 5.8. Thecrystals are recovered using another continuous belt filter andthen recycled back to the liming step, while the filtrate has to bedisposed.

Precipitation of dicalcium acid citrate results in one-thirdless consumption of lime and consequently of sulfuric acid forthe subsequent regeneration of citric acid, in greater filterabilityand washability because of its crystalline structure, but 10–25%of the expected product yield is needed as seed. The precipitateis washed to remove the impurities adsorbed onto it (i.e.,residual sugars and contaminants from the raw carbon sourceand soluble proteins from the autolysis of the fungus). Thewashed crystals and 98%w/w sulfuric acid are simultaneously,but separately, fed to a mixer containing a 40% citric acidsolution at pH 0.5–0.6, to free the citric acid with the formationof a precipitate of calcium sulfate dihydrate (gypsum). Finalrefining of the filtrate is performed by decolorization on acti-vated carbon and removal of residual calcium sulfate and ironand nickel salts on strong cation exchange and weak anionexchange (demineralization step). The resulting solution(250–280 kgm�3 of citric acid anhydrous) is concentratedusing multiple-effect evaporators to about 700 kgm�3, beforefeeding a vacuum crystallizer operating at temperatures below(35 �C) or above (62 �C) the transition temperature (36.6 �C)between the monohydrate and anhydrous forms, depending

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

Equation or reaction

Trophophase reaction C6H12O6 þ a NH3 þ b O2glucose

/ y CHnOpNq þ d CO2 þ e H2Omycelium

½1�

Idiophase reaction C6H12O6 þ b O2glucose

/ y CHnOpNqmycelium

þ z C6H8O7 þ d CO2 þ e H2Ocitric acid

½2�

Kinetic equationsrX ¼ dX

dt¼ mX X ½3�

rN ¼ dNdt

¼ �YN=X

�dXdt

�for N � Nlim ½4�

rN ¼ dNdt

¼ 0 for N < Nlim ½5�

m ¼ 0 for t � to ½6�

m ¼ mM for t � tlim ½7�

m ¼ mM

�1� X

XM

�for t > tlim ½8�

rP ¼ dPdt

¼ 0 for t � tlim ½9�

rP ¼ dP

dt¼ YP=X

�dXdt

�þ mP X for t > tlim ½10�

rS ¼ �dSdt

¼ YS=X

�dXdt

�þ mS X ½11�

Integral solutions of thedifferential kinetic equations

X ¼ X0 for t � to ½12�

X ¼ X0 emM ðt�t0Þ for t � tlim ½13�

X ¼ XM

1þ�XMX0

� 1�e�mMðt�tlimÞ

for t > tlim ½14�

N ¼ N0 for t < t0 ½15�

N ¼ N0 � YN=X ðX � X0Þ for t � tlim ½16�

N ¼ Nlim for t > tlim ½17�

P ¼ P0 þ mP AðtÞ þ YP=X ðX � X0Þ ½18�

S ¼ S0 � ½mS Aðt Þ þ YS=X ðX � X0Þ� ½19�

AðtÞ ¼ 0 for t � tlim ½20�

Aðt Þ ¼ XMmM

ln�1� XM

mM

h1� emM ðt�tlimÞ

i�for t > tlim ½21�

Nomenclature: A(t), cumulative nongrowth contribution to product formation; b, y, z, d, and e, stoichiometric coefficients; mP (mS), specific rate ofproduct 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 cellgrowth rate; YN/x, YP/x, and YS/x, yield factors for ammoniac nitrogen, citrate, and substrate on unit cell biomass. Subscripts: lim, referred to limitingconcentration of the nitrogen source; N, nitrogen; P, citric acid; S, glucose; X, mycelium; 0, referred to the initial value.


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, dicalciumcitrate; 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;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.

FERMEN

TATIO

N(IN

DUSTR

IAL)j

Production

ofSom

eOrganic

Acids

(Citric,

Gluconic,

Lactic,and

Propionic)

811


on the form manufactured. Crystals are separated by centrifu-gation and dehydrated in a two-stage fluidized-bed dryer, thefirst one using hot air at 90 �C and the second one using airconditioned at 20 �C and relative humidity (RH) of 30–40%because of crystal hygroscopicity. The mother liquor is partly(w20%) diluted with equipment-cleansing waters, decolor-ized, and fed back to liming; the remainder is in sequencedecolorized and demineralized before being recycled to thecrystallization unit. In this way, citric acid crystals do not needadditional purification steps to meet specifications for U.S.Pharmacopeia or Food Chemical Codex material.

In the liquid extraction process, citric acid may be extractedfrom the fermentation broth using a highly selective, low-price,and nontoxic food-grade solvent (i.e., water-insoluble amines,namely trilaurylamine, n-octanol, C10 or C11 isoparaffin, tri-n-butyl phosphate, alkysulphoxides). The extract is then heatedand washed countercurrently with water, resulting in about90% recovery yield and an aqueous citric acid concentratedsolution, which is passed through a granular activated–carboncolumn before undergoing the aforementioned evaporationand crystallization steps.

Future Developments

Production of citric acid has not been much in the focus ofmodern molecular biology presumably because it is considereda mature area. Any improvement of strains of A. niger usuallywere carried out by mutagenesis and selection, but metabolicand genetic engineering are likely to improve acidogenesis.

The replacement of the current batch fermentation withsemicontinuous processes, to increase volumetric productivityand reduce specific production costs, presently is hampered infungal processes by the deterioration of mycelial structure, themechanism of which still is unknown. Although the effect ofnitrogen deficiency on citric acid accumulation by A. niger iswell known, a low level of ammonium ions (i.e., 30 gm�3,equivalent to 2mmol of intracellular NH4

þ per gram of dry cell)was found to inhibit the morphological degeneration of pelletsand postpone sporulation. NH4

þ ions simply are not depositedinto the cell to form the so-called ammonium pool, but theyenter the cell to combine with glucose and form glucosamine,that is straight away released in the medium. The effectiverelationship between the different compounds of the TCA cycleis to be studied further and controlled before the present batch-production technology may be converted effectively intoa prolonged fed-batch or continuous production process.

Similarly, the possibility of maintaining microbial cellsactive and controlling their growth and production processesfor several weeks or months by immobilization within organicor inorganic matrices represents a further challenge to thetechnological modernization of this sector.

The traditional recovery technology results in severalproblems because of disposal of liquid effluents (their chem-ical oxygen demand being about 20 kgm�3) and solidby-products (i.e., about 0.15 kg of dried mycelium and 2 kg ofgypsum per kilogram of citric acid anhydrous). Several processalternatives have been suggested thus far to minimize theoverall environmental impact of this process. The replacementof molasses with raw or hydrolyzed starch- or raw sucrose-based materials would simplify only the downstream

processing. On the contrary, the recovery of tricalcium (ortrisodium) citrate from clarified, decolorized fermentationbroths by electrodialysis, as well the adsorption of citric acidonto weakly basic anionic-exchange resins or zeolites using thesimulated-moving bed chromatographic technology (CitrexProcess, UOP, Des Plaines, IL, USA) followed by desorptionwith water or dilute acidic solutions, or the use of liquidmembranes, would allow the citric acid to be separated ina single step and to be recovered without the formation of solidwastes for disposal.

The environmental aspects of citric acid production havebeen assessed. Despite the fact that most raw materials are ofbiological origin, many ingredients, such as ammoniumnitrate, lime, and sulfuric acid, are hazardous chemicals. Forinstance, it was found that the environmental impact of citricacid production using whey was smaller than that using cornstarch.

Gluconic Acid

D-Gluconic acid (2,3,4,5,6-pentahydroxy pentane-1-carboxylicacid: C6H12O7) is an oxidation product of D-glucose, which, inan aqueous solution, leads to a complex equilibrium betweengluconic acid and its two lactones: 1,5-lactone (D-glucono-d-lactone) and 1,4-lactone (D-glucono-g-lactone).

D-Gluconic acid is commercially available as 50% aqueoussolution (density of 1230 kgm�3 at 20 �C and pH 1.82). Thisacid and its derivatives are used in the pharmaceutical, food,feed, and chemical industry because of their low toxicity andtheir ability to form water-soluble complexes with metallicions (e.g., Caþ2, Feþ3), especially in the presence of 5–10% ofsodium hydroxide. Sodium gluconate is the main industrialproduct and it is used as a sequestering agent (e.g., bottlewashing, metal surface cleaning, and rust removal) and toplasticize and retard the curing process of cement mixes. Thecalcium and iron gluconates are used in medicine to treatdiseases of calcium and iron deficiency (such as osteoporosisand anemia). D-Glucono-d-lactone is used as a latent acid inbaking powders for use in dry cake mixes, meat processing, andinstant chemically leavened bread mixes, whereas D-glucono-g-lactone is made only in small quantities as a specialtychemical.

The conversion of glucose to gluconic acid is a simpleoxidation process and may be carried out by a variety ofprocesses – namely, microbial fermentation, chemical, elec-trochemical, or enzymatic catalysis. Currently, these processesappear to be either more expensive, unstable, or less efficientthan the fermentation process, which presently is the onlymethod of choice.

After the first isolation of calcium gluconate (1880) fromglucose fermentation in the presence of CaCO3 by a strain ofMycoderma aceti, the Chas. Pfizer & Co., Inc. (New York, USA)started industrial-scale production of gluconic acid in 1923.Further research at the U.S. Department of Agriculture incooperation with the Iowa State College led to the semi-continuous production of sodium gluconate from glucoseusing A. niger NRRL 67.

Several filamentous fungi of the genera Penicillium andAspergillus, the yeastlike fungus Aureobasidium pullulans, and


bacteria (Acetobacter suboxidans, Pseudomonas ovalis, Glucono-bacter spp.) produce gluconic acid from glucose-based media,but industrial processes have been developed only for theproduction gluconic acid from glucose syrups with A. niger andGluconobacter oxydans. Industrial strains are not freely available,but a few gluconic acid–producing strains (A. niger NRRL 3,NRRL 67) can be obtained from international culture collec-tions. Penicillium spp. generally produce less gluconate thanAspergillus, but they have the advantage of excreting the glucoseoxidase (an important by-product) into the medium, whichmakes its recovery easier.

The formation of gluconic acid by A. niger is controlled bythe enzyme glucose oxidase, an omodimer containing twoflavin adenine dinucleotide (FAD) moieties. Such enzymeabstracts two hydrogen atoms from glucose, thus yielding theglucono-d-lactone, which to some extent hydrolyzes to glu-conic acid. The FADH2 reacts with oxygen to form hydrogenperoxide, which is converted into oxygen and water by theenzyme catalase. Both glucose oxidase and catalase areconstitutive endoenzymes in A. niger.

A highly productive process of gluconic acid using free-growing cells of A. pullulans DSM 7085 recently has beendeveloped. Its high conversion yields (90–98%) and rates(13–19 kgm�3 h�1) resulted in as high gluconate concentrationsas 504 or 230–433 kgm�3 in fed-batch or chemostat trials.Although this novel fermentation process offers a new opportu-nity for commercial gluconic acid production, as well as manyadvantages over the traditional microbial fermentation pro-cesses, it is still confined to laboratory-scale applications. Thus,only the sodium process by batch-submerged fermentation fromglucose syrups using A. niger will be described in the followingparagraphs.

Glucose syrups of 70�Brix strength are generally used ascarbon source in the preparation of the fermentation medium.Table 4 lists the typical composition of the seed andproduction media used in laboratory- and industrial-scaletrials.

After the pH is adjusted at 4.5 with sulfuric acid, themedium is sterilized at 121 �C for 15–30min, cooled at 33 �C,and then transferred into the fermentation vessel. The pH is

Table 4 Composition for the production media used in thelaboratory- and industrial-scale production of gluconic acid by A. niger

Component

Vegetative seed

–culture media

Gluconic acid

production media Unit

Glucose 40 120–350 kg m�3

NH4NO3 (or otherNH4

þ salt)2.4 0.4–0.5 kg m�3

KH2PO4 1.5 0.1–0.3 kg m�3

MgSO4$7H2O 3.57 0.1–0.3 kg m�3

Agar 1 0 kg m�3

Yeast extract 1 0 kg m�3

Corn-steep liquor 0 0.2–0.4 kg m�3

ZnSO4$7H2O 100 0 mgm�3

CuSO4$5H2O 20 0 mgm�3

FeCl3$6H2O 300 0 mgm�3

MnSO4$7H2O 0 30 mgm�3

Initial pH 6.5 6.0 –

adjusted to 6–6.5 with sodium hydroxide, and a 2–5% v/vinoculum generally is used. For inoculum development, con-idia are recovered from stock agar slants and are inoculated intovegetative seed–culture media (106 conidia cm�3); pellet-likemycelia is obtained after incubation at 30 �C for 15–24 h and isused to inoculate seed fermenters at a density of 20–50pellets cm�3.

The fermentation is carried out under continuous automaticcontrol of sterile air sparging (1.0–1.5 vvm), temperature(33 �C), pressure on the tank top (2–3 bar), pH (5.5–6.5 byaddition of 30–50% NaOH solution to neutralize the gluconicacid formed), and foam level. It is completed within w30 hwith yield factors of 0.97–1 kg of gluconic acid per kilogram ofglucose consumed (against a theoretical yield of 1.09 kg kg�1)and gluconate productivities of 9–13 kgm�3 h�1.

In the fed-batch operation, the mycelium may be reused upto five times without any loss in gluconate productivityprovided that the levels of glucose oxidase activity and othermicroelements (i.e., iron and manganese) are kept undercontrol. Stepwise addition of glucose may be used to increasegluconate concentration to 580 kgm�3.

At the end of fermentation, the mycelium is removed usingaseptic centrifugation, under vacuum-belt filtration or cross-flow microfiltration and may be used as a source of glucoseoxidase or may be disposed off via incineration. The clarifiedbroth, generally containingw300 kgm�3 of sodium gluconate,is filtered, decolorized using a granular activated–carboncolumn, concentrated under vacuum to 45–50% total solids,neutralized to pH 7.5 with NaOH, and then spray or drumdried. If 50% gluconic acid is required, the concentrated liquormay be passed through a cation exchanger to remove Naþ ions.Further crystallization at 30–70 �C or at more than 70 �Callows crystals of the d-lactone or g-lactone to be precipitated,respectively.

Lactic Acid

Lactic acid (2-hydroxypropionic acid: C3H6O3) may beproduced by chemical synthesis or fermentation. Of the twoenantiomers, L-(þ) and D-(�) lactic acid, only the L-(þ) isomeris used by human metabolism and, because of the slighttoxicity of the D-(�) isomer, it is preferred for food uses.Because the chemical route yields a mixture of L-lactic acid andD-lactic acid and relies on costly raw materials, all lactic acidmanufacturing industries have switched to fermentation-basedtechnologies (Table 1). The free acid is used as an acidulant/preservative in several food products (cheese, meat, jellies,beer) (see Preservatives: Traditional Preservatives – OrganicAcids); sodium lactate is used for carcass decontamination;ammonium lactate is used as a source of nonprotein nitrogenin feeds; sodium and calcium stearoyl lactylates are used asemulsifiers and dough conditioners. The large increase in lacticacid production is due to its use in the synthesis of polylacticacid (PLA), a polyester used for biodegradable plastics for foodpackaging, compost, and garbage bags, and disposable table-ware, as well as several medical applications, such as reab-sorbable sutures, orthopedic implants, and controlled drugrelease. The current economically viable industrial process forPLA production is via the dehydrated cyclic dilactate ester


(lactide) formation. In brief, lactic acid is first polymerized intooligomers of PLA consisting of 30–70 lactyl units (–CHCH3–

CO–O–). These oligomers are then depolymerized byincreasing the polycondensation temperature and lowering thepressure in the presence of transition metal–based catalysts(i.e., stannous octoate at 0.05%) to distil the lactide. Finally, byopening its ring, it is possible to obtain high molar masspolymers (100–300 kDa) with appropriate optical andmechanical properties (i.e., tensile strength >50MPa). Othercomonomers, such as caprolactone, hydroxybenzoic acid, andothers, can be incorporated to provide environmentally safematerials. PLA appears to be a sustainable alternative topetroleum-based plastics, because lactide is produced from thefermentation of renewable resources, such as corn starch (as inthe industrial plant of Nature Works LLC, Blair, Nebraska, USA:140 000metric tons per year). Nevertheless, to minimizecompetition for land and food, research studies have started todevelop second-generation PLA products from lignocellulosichydrolysates (e.g., crushed corncobs).

Organisms and Metabolic Pathways Involved

Lactic acid can be produced using homofermentative lacticacid bacteria (LAB), facultatively anaerobic Bacillus species(B. coagulans), and molds (Rhizopus microsporus, Rhizopusoryzae). Recently, lactic acid producing genetically modifiedstrains of Escherichia coli and Saccharomyces cerevisiae havebeen developed. The choice of the species depends on severalconsiderations, including the ability to use the type of sugarsavailable in the substrate, growth temperature, nutritionalneeds, acid tolerance, and type of lactic acid isomerproduced.

Thermophilic lactobacilli (Lactobacillus delbrueckii subsp.delbrueckii, L. delbrueckii subsp. bulgaricus, Lactobacillus helveticus)tolerate higher concentrations of lactate and higher temperatures(48–52 �C), thus involving higher productivity and yieldsand reduced contamination risks. They produce D-(�) or DL-lacticacid (some industrial strains that have been claimed to beL. delbrueckii produce L-(þ) lactic acid) and may be less suitablefor food, feed, or biomedical applications. Lactococci,mesophiliclactobacilli (Lactobacillus casei subsp. casei, Lactobacillus amylophi-lus), and thermophilic streptococci (Streptococcus thermophilus)have lower temperature optima or reduced acid tolerances, butthey may be desirable for other reasons (e.g., production of pureL-(þ) lactic acid, hydrolysis of starch). Recently, genetic engi-neering has been used to produce L. helveticus and Lactobacillusplantarum strains, which produce optically pure L-(þ) or D-(�)lactic acid (see Lactobacillus: Introduction; Lactococcus: Introduc-tion; and Streptococcus thermophilus).

Homofermentative LAB ferment hexoses via the glycolyticpathway (see Metabolic Pathways: Release of Energy (Anaer-obic)). Pyruvate is reduced to lactate by stereospecific lactatedehydrogenase(s) (L-LDH or D-LDH). LDH is allosteric (acti-vators: fructose-1,6-bisphosphate and Mnþ2) in lactococci andnonallosteric in homofermentative lactobacilli. Undissociatedlactic acid acts as a noncompetitive inhibitor for growth andlactic acid production by diffusing through the membrane anddecreasing intracellular pH: pH control during fermentationreduces the inhibition, but the maximum lactic acid concen-tration achievable is usually lower than 150 kgm�3.

Concomitant substrate and product inhibition has beenreported for several species.

LAB are fastidious microorganisms and require supple-mentation of fermentation media with peptides and growthfactors, usually in the form of yeast extract. Because this mayaccount for 30–35% of substrate costs, they may be replaced byless demanding B. coagulans and R. oryzae, both beingL(þ)-lactate producers, even if smaller yields (as low as 70%for R. oryzae because of concomitant fumaric acid and ethanolproduction) and acid tolerance may offset the advantage ofusing lower amounts of supplements.

Substrate Production and Recovery

Lactic acid can be produced from a variety of raw substrates(whey and whey permeate, beet and cane molasses, starch andcorn starch hydrolysates, wood hydrolysates; see Fermentation(Industrial): Media for Industrial Fermentations). Some species(Lb. amylophilus) can hydrolyze starch, but the pseudoplasticbehavior of starchy substrates makes the pH control difficult.When whey permeate is used, supplementation with milkprotein hydrolysates (5–10 kgm�3) and yeast extract (up to20 kgm�3) is required. Lactic acid production is usuallya growth-associated production process, but nongrowth-associated production becomes significant when growth islimited by a lack of nutrients or high undissociated acidconcentration. The pH is controlled at 5–6.5 by the automaticaddition of NaOH, Na2CO3, or NH4OH or by the addition ofCaCO3. Fermentation is carried out under anaerobic or micro-aerophilic conditions and lactic acid yield is usually between 85and 98% with isomer purity as high as 99%. Batch fermenta-tions result in high product concentration (120–150 kgm�3)but in low productivity (2 kgm�3 h�1). Conversely, continuousfermentations with cell-recycle or immobilized cells give rise tohigher productivities (20–80 kgm�3 h�1) and lower lactateconcentrations (<50 kgm�3). End-product inhibition may becircumvented by using integrated fermentation processes, inwhich lactic acid is removed from the culture broth by severaltechniques, including electrodialysis, ion-exchange resins, ornanofiltration.

Recovery of lactate is made complicated by the high solu-bility of its salts. The traditional process involves precipitationof calcium lactate and regeneration of lactic acid by the addi-tion of sulfuric acid followed by further purification steps (ionexchange and decolorization). Alternative processes include theextraction by liquid membranes, electrodialysis, and ionexchange. In particular, the recent industrial use of electrodi-alysis with bipolar membranes in France resulted in the virtualelimination of gypsum waste production. Conventionalrecovery by the precipitation method seems to be the mosteconomical route.

Other Organic Acids Produced by Fermentation

Propionic Acid

Propionic acid (C3H6O2) and its salts are used as mold inhib-itors in bakery products, although other nonfood uses areimportant (see Permitted Preservatives – Propionic Acid). It maybe produced by fermentation by members of the genera


Propionibacterium (P. freudenrichii, thoenii, acidipropionici), Veillo-nella, Clostridium, and Selenomonas, but currently it is producedby chemical synthesis because of the shortcomings of thefermentation route (low productivities, <1 kgm�3 h�1 in batchprocesses; low product concentrations,<50 kgm�3; difficulty inproduct separation from acetic acid, which invariably isproduced from sugars and lactate). The high microbialproductivities (2–14 kgm�3 h�1) obtained in continuousfermentations using immobilized cells or membrane-recyclereactors, as well the possibility of obtaining pure propionic acidfrom alternative low-cost substrates, like crude glycerol from thebiodiesel industry, might refocus industrial manufacturerstoward the fermentation route. For instance, use of a metaboli-cally engineered strain of P. acidipropionici (ACK-Tet) resulted ina propionic acid concentration of 106 kgm�3 with a productyield of 0.54–0.71 g per g of glycerol consumed and a propionicacid-to-acetic acid ratio of 22.4.

See also: Arthrobacter; Aspergillus; Bacillus: Introduction; Bread:Bread from Wheat Flour; Yarrowia lipolytica (Candida Lipolytica);Escherichia coli: Escherichia coli; Fermentation (Industrial):Basic Considerations; Fermentation (Industrial): Media forIndustrial Fermentations; Fermentation (Industrial): Control ofFermentation Conditions; Fermentation (Industrial): Recovery ofMetabolites; Fermented Foods: Fermentations of East andSoutheast Asia; Fungi: The Fungal Hypha; Fungi: Classificationof the Hemiascomycetes; Fungi: Classification of theDeuteromycetes; Genetic Engineering; Gluconobacter;Lactobacillus: Introduction; Metabolic Pathways: Release ofEnergy (Aerobic); Metabolic Pathways: Release of Energy(Anaerobic); Preservatives: Traditional Preservatives – OrganicAcids; Permitted Preservatives – Propionic Acid;Propionibacterium; Streptococcus thermophilus; Vinegar; Yeasts:Production and Commercial Uses.

Further Reading

Anastassiadis, S., Aivasidis, A., Wandrey, C., 2003. Continuous gluconic acidproduction by isolated yeast-like mould strains of Aureobasidium pullulans. AppliedMicrobiology and Biotechnology 61, 110–117.

Berovic, M., Legi�sa, M., 2007. Citric acid production. Biotechnology Annual Review 13,303–343.

Hofvendahl, K., Hahn–Hägerda, B., 2000. Factors affecting the fermentative lactic acidproduction from renewable resources. Enzyme and Microbial Technology 26,87–107.

Joglekar, H.G., Rahman, I., Babu, S., Kulkarni, B.D., Joshi, A., 2006. Comparativeassessment of downstream processing options for lactic acid. Separation andPurification Technology 52, 1–17.

John, R.P., Nampoothiri, K.M., Pandey, A., 2007. Fermentative production of lacticacid from biomass: an overview on process developments and future perspectives.Applied and Microbiology and Biotechnology 74, 524–534.

Legi�sa, M., Mattey, M., 2007. Changes in primary metabolism leading to citric acidoverflow in Aspergillus niger. Biotechnology Letters 29, 181–190.

Magnuson, J.K., Lasure, L.L., 2004. Organic acid production by filamentous fungi.Chapter 12. In: Tkacz, J.S., Lange, L. (Eds.), Advances in Fungal Biotechnology forIndustry, Agriculture, and Medicine. Kluwer Academic/Plenum Publishers, NewYork, pp. 307–340.

Okano, K., Tanaka, T., Ogino, C., Fukuda, H., Kondo, A., 2010. Biotechnological productionof enantiomeric pure lactic acid from renewable resources: recent achievements,perspectives, and limits. Applied Microbiology and Biotechnology 85, 413–423.

Papagianni, M., 2007. Advances in citric acid fermentation byAspergillus niger: biochemicalaspects, membrane transport and modeling. Biotechnology Advances 25, 244–263.

Sauer, M., Porro, D., Mattanovich, D., Branduardi, P., 2007. Microbial production oforganic acids: expanding the markets. Trends in Biotechnology 26 (2), 100–108.

Singh, O.V., Kumar, R., 2007. Biotechnological production of gluconic acid: futureimplications. Applied Microbiology and Biotechnology 75, 713–722.

Yoo, D.-K., Kim, D., 2009. Production of optically pure poly(lactic acid) from lactic acid.Polymer Bulletin 63, 637–651.

Zhang, A., Yang, S.-T., 2009. Propionic acid production from glycerol by meta-bolically engineered Propionibacterium acidipropionici. Process Biochemistry 44,1346–1351.

Documents

FERMENTATION (INDUSTRIAL) Production of Some Organic Acids (Citric Gluconic LactiC and Propionic)