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 applications. Table 1 lists the main acids that are produced commercially by chemical (C) or biotechnological (fermentation, F, or enzymatic, E) methods or extracted from winemaking residues (L). Citric, acetic, lactic, propionic, tartaric, fumaric, and malic acids are among the most versatile ingredients in the food and beverage industry because of their valuable properties, such as solubility, hygroscopicity, acidity, buffering capacity, and chelation (see Preservatives: Traditional Preservatives – Organic Acids). Citric acid accounts for around 80% of the food acidulant usage, whereas the use of phosphoric or acetic acids is limited, being almost exclusively utilized in cola soft drinks or in vinegar (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 the monohydrated form (molecular mass of 210.13 Da, relative density of 1.542 at 20 C, and heat of combustion of 1962 kJ mol1 at 25 C). It is a strong tricarboxylic acid (TCA; its dissociation constants being K1 ¼ 7.45 104, K2 ¼ 1.73 105, and K3 ¼ 4.02 107 at 25 C), highly soluble in water with pleasant acid taste. Citric acid was first isolated in 1784 by Scheele, who precipitated it as calcium citrate by adding calcium hydroxide (lime) to lemon juice. Before 1920, it was almost exclusively produced in Sicily by pressing lemons: The firm Arenella (Palermo, Italy) essentially established a monopoly until the advent of the citric acid fermentation technique in Table 1 Belgium (Societè des Produits Organiques de Tirlemont) in 1919 and in the United States (Chas. Pfizer & Co., New York) in 1923. About 10 years later, about 80% of the world’s citric acid was produced by the surface fermentation process. The submerged fermentation process began to be applied only after World War II. From 1950 to 1980, citric acid was mainly used in pharmaceutical or health products. In fact, in the early 1980s, its two largest manufacturers were Pfizer and Miles/Bayer, both suppliers of prescription drugs. Thereafter, as citric acid began to be used in the food and beverage sector in industrial and developing countries, its market size experienced significant growth and several new manufacturers were established in 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. In the early 1990s, a few manufacturers gave rise to the socalled citric acid cartel. The overcharges imposed on US buyers was estimated in the range of $116–309 million and, on January 29, 1997, Haarmann & Reimer Corp., a subsidiary of Bayer AG (D), pled guilty and paid a $50 million criminal fine. In March 1998, even Archer Daniels Midland Co. (ADM) agreed to pay $36 million to four citric acid customers that had opted out of the July 1997 civil class-action antitrust settlement. At that time, the global citric acid capacity was about 840 000 Mg (mega grams) per year with a growth rate of 5% per year. Afterward, the world citric industry became less concentrated and numerous new manufactures, especially in China, as well as Brazil, India, Indonesia, and Thailand, have entered the market, thus making the formation of cartels less probable. Moreover by the early 2000s, almost all citric acid manufacturing was globally integrated into the corn wet-milling 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) Lactic acid C2H4O2 C3H6O3 190 000 150 000 F 100% F 100% Propionic acid Fumaric acid Malic acid C3H6O2 C4H4O4 C4H6O5 130 000 12 000 10 000 Tartaric acid Itaconic acid Citric acid Gluconic acid C4H6O6 C5H6O4 C6H8O7 C6H12O7 28 000 15 000 1 800 000 87 000 C 100% C 100% C 70% E 30% L 100% C 100% F 100% F 100% Acetobacter aceti Lactobacillus spp. Rhizopus spp. Propionibacterium acidipropionici Rhizopus arrhizus – – Aspergillus terreus Aspergillus niger 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 Then. however. while it was about 1.96 kg1 depending on the amount ordered. and morphological changes. NRRL 599. thereafter. while in the fall 1990 it was as low as $1. the citric acid cartel accomplished its main goal of raising and keeping list price at $1. high dissolved oxygen (DO) concentration. Methods of Manufacture Citric acid production is mainly accomplished by the submerged fermentation process. unable to benefit from the economy of scale. the global citric acid production capacity reached almost 1. ATCC 9142. citric acid export from the mithocondria and cell.FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. Lactic. NRRL Y-1095) can be obtained from international culture collections. but citric acid–producing strains (A. respectively. low pH. but with limited success. lipolytica ATCC 20346. sucrose) with A.54 kg1 in the early 1997.8 kg1. The availability of the complete genome sequence of A.65 kg1. which results in a high adenosine triphosphate/ adenosine monophosphate ratio and.70–$0.e. Very low concentrations of Mn2þ (<10 mg m3) are critical..1 kg1 in June 2011. Improvement of strains for citric acid production traditionally has been carried out by mutagenesis and screening. Gluconic. and as much as 65–70% of global consumption. niger are induced by high sugar concentration. Low cytoplasmic levels of citrate may be responsible for reduced feedback inhibition of glycolysis. and manganese (Mnþ2) deficiency. and a-ketoglutarate dehydrogenase (KDH). and Propionic) industry either by acquisition (Pfizer and Miles/Bayer were bought by ADM and Tate & Lyle.80 kg1. citrate synthase (CS). to exit the business. The present economic crisis in Europe and the United States has newly reduced the market prices of (anhydrous) citric acid to US $0. are important. glucose carrier) or inactivation of genes encoding enzymes that produce allosteric inhibitors of hexokinase has been attempted. Improvement of yeast for citric acid production is directed to obtain strains with reduced isocitrate dehydrogenase and aconitase activities. in inactivation of nicotinamide adenine dinucleotide (NADþ)-specific isocitrate dehydrogenase (IDH). Key regulatory steps in the process include glucose transport and phosphorylation. niger are shown in Figure 1. thanks to the “citric acid conspiracy” in the years 1993–96. they steadily increased to reach a peak of $1. P. changes in membrane lipid composition and cell wall composition. in the additional production of citric acid. In the years 1987–89. trace metals). Y. NRRL Y-7576. It has been postulated that the activity of seven glycolytic enzymes needs to be increased to obtain increased production of citric acid. . while sugarcane or sugar beet molasses prevail in the Brazilian and Indian or European markets.76 kg1 in November 1994 to $1. Thereafter.39 kg1. The main anaplerotic reactions include the synthesis of OAA from pyruvate catalyzed by PC during production from glucose and the glyoxylate cycle during growth on n-alkanes. US list prices for citric acid anhydrous remained unchanged at $1.5 106 Mg in 2005.8 million metric tons (Mg) in 2010. probably because of the smaller contribution of investment and labor costs to its overall production costs.79 kg1. Other factors (i. the list price reduced to $1. S. in turn. as a direct result of the increase in the market prices for agricultural raw materials.87 kg1. or Mg) limitations coupled with a high rate of glucose utilization. NRRL 2270. pyruvate carboxylase (PC). In Europe. decreased activity of TCA cycle reactions that degrade citrate. China approximately accounted for more than 50% of global citric acid production capacity. phosphate and nitrogen concentrations. the severe competition resulted in selling prices of anhydrous citric acid decreasing to $0. Maize starch is mainly used in the United States and China. phosphofructokinase. Candida guillermondii). Citric acid overproduction in yeast is relatively insensitive to trace metals concentration and is triggered by nutrient (N. niger is likely to allow for the design of overproducing mutants. Overexpression of proteins critical to acidogenesis (hexokinase. Aspergillus niger. They result in decreased activity of the pentose phosphate pathway and increased glycolytic flux. Aspergillus wentii. respectively. Industrial strains are not freely available. The metabolic changes necessary for citric acid overproduction in A. the panorama of organic acid manufacturers has profoundly changed over the last decade. n-alkanes). Cellulosic materials are currently unused in citric acid production. all surface fermentation plants have been shut down during the past decade. and an anaplerotic reaction to replenish the oxaloacetate (OAA) used for the synthesis of citrate are all essential (see Metabolic Pathways: Release of Energy (Aerobic)).70$0. particularly corn. Accumulation of isocitrate (10–50% of the citrate produced) in excess with respect to the predicted equilibrium of aconitase probably is due to the high permeability of yeast mitochondria to isocitrate compared with citrate. ATCC 20390. From January 2008 to January 2009. Metabolic pathways involved in citric acid overproduction by A. Several substrates are used as fermentation substrates depending on the local availability. it lowered from $1. sucrose. In conclusion.7–$0. As a consequence.. thus forcing the smaller manufacturers. increased intracellular NHþ 4 pool and turnover of nucleic acids and proteins. even if there are projects to assess the technical feasibility of such feedstock materials in the citric acid industry. respectively) or by new process development (Cargill). while Europe and North America covered the 19 and 24%. By late 1989. see Aspergillus and Penicillium andTalaromyces: Introduction). but industrial processes have been developed only for the production of citric acid from sugars (glucose. 805 A high flux through the glycolysis. ATCC 11414. Then. and bacteria (Arthrobacter) produce citric acid from a variety of substrates (glucose. yeasts (Yarrowia lipolytica. The surface fermentation process currently accounts for only 5–10% of the world supply. niger. Smaller amounts of citric acid (<1%) are reported to be extracted from citrus fruits in Mexico and South America and to be produced by the solid-state process in Japan. niger and from sugars and n-alkanes with yeasts. Organisms and Metabolic Pathways Involved Several molds (Penicillium spp. In 2010. Trichoderma viride. export prices of Chinese (anhydrous) citric acid oscillated in the range of US $0. Gluconic.806 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. Lactic. and Propionic) . 1–0.or hydrocarbon-based media to overcome the main disadvantages of traditional mold fermentation (i. Low activity of NADPþ-specific IDH and KDH are a consequence of the effective removal of citrate. = Component Sucrose or glucose NH4NO3 (or other NHþ 4 salt) KH2PO4 MgSO47H2O Feþ2 Znþ2 Cuþ2 Mnþ2 Initial pH Range of concentrations Typical values Unit 125–225 0. tre. thus limiting the effective oxygen transfer rate with little or no citric Figure 1 Metabolic pathways for citric acid overproduction in Aspergillus niger. shorter fermentation times. PGI. alternative oxidase system. PT. sucCoA. pep. alternate oxidase system is used during acidogenesis to reoxidize the NADH produced during glycolysis. fru2. The most important steps in controlling glycolytic flux are glucose transport (simple diffusion is the main mechanism at high sugar concentrations. fru6P. mal. The fungus will develop different morphological forms (Figure 2). glucose.5 0. cis-aconitate. membrane bound invertase..and industrial-scale production of citric acid by A.5% w/v) have been claimed to enhance the citric acid yield. Manganese concentration has to be kept as low as possible (<10 mg m3). corn. especially molasses. Fermentation Process Inoculation is generally carried out by transferring aseptically the stock culture maintained on agar slants on other working slants. isocitrate. MDH. a-kg. CS activity in A. After w24 h incubation at 30 C. which initially is inhibited by trehalose-6phosphate. A salicylhydroxamic acid–sensitive. aco.2 kg m3 kg m3 mg m3 mg m3 mg m3 mg m3 – a To overcome the detrimental effects of iron and manganese on mycelium structure and restore proper morphology. which is produced in the anaplerotic reaction catalyzed by PC. pyruvate kinase. pyruvate transport system. Citric acid is produced from media containing high concentrations of simple sugars (molasses or glucose syrup) (see Fermentation (Industrial): Media for Industrial Fermentations).6-bisphosphate. dhp. but the inhibition is counteracted by the presence of high levels of NHþ 4 and by fructose-2. dihydroxiaceton phosphate. GC. CC.e. inhibitor. glycerol. Metals are removed by pretreatments of raw materials. PP. KDH. starch.5–3. putative citrate carrier. PK. a-ketoglutarate. PFK1.6 dP. and inhibits gluconeogenesis. citrate synthase.6 dP. The optimal iron concentration seems to depend on the fungal strain. 3–6% w/v. The inhibitory effect of Feþþ can be counterbalanced by the addition of copper and zinc salts during the inoculum development or during early mycelium growth in the production medium. which in turn is used as inoculum (5–10% v/v) for the industrial-scale production medium.0 2–1300 0–2900 1–10 200 0–46 2. Malate is produced from OAA by cytosolic MDH and acts as a counterion for citrate efflux from the mitochondrion. isocitrate dehydrogenase.5 180 1. phosphoglucose isomerase. PDH. pyr.2 mm in diameter). citrate. acCoA. IDH. pyruvate carboxylase. the conidia crop is inoculated in starch-rich seed-production media to yield up to 1011 spores cm3. The main advantages of these techniques are improved asepsis during fermentation. unbranched hyphae is to be avoided because this results in an enormous increase in the apparent viscosity of the culture broth. PFK2 activity is increased at high substrate concentration: its product. Their range of composition is given in Table 2. although A. The formation of a loose mycelium with long. niger as the fermenting organism. thus increasing carbon flux through glycolysis during acidogenesis. where it is oxidized back to OAA.5% w/v. oxaloacetate. ALD.025–0.6-bisphosphate. aldolase. Nitrogen is usually added as ammonium nitrate or sulfate. fructose-2. Lactic. HK. 6-phosphofructo-2-kinase. high sensitivity to trace metals and low production rates). pyruvate dehydrogenase.1–0.6-biphosphate (FBP). and greater product yields. may be responsible for acidogenesis. malate. niger Production Media Media sterilization is carried out batch wise at 121 C for 15–30 min at the laboratory or pilot scale or continuously using a plate–heat exchanger unit at the industrial scale. in which mycelium growth is restrained by nutrient (phosphorous. iron or zinc) limitation. counteracts citrate inhibition. phosphoenolpyruvate. phosphofructokinase. citrate transport system. Table 2 Composition for the production media used in the laboratory. products. malate dehydrogenase. Only relevant enzyme activities. and Propionic) 807 Both submerged. pyruvate. niger is regulated by the level of OAA. Malfunction of the normal respiratory chain is due to diminished activity of NADH ubiquinone reductase and other respiratory chain enzymes.5 0. PFK1 is feedback inhibited by citrate.1–2. fru1. no yeast-based process is currently known to be operating worldwide.and surface-culture fermentation processes use beet molasses or glucose syrup as the main raw material and use A. ica. but iron levels of 200 mg m3 were found to inhibit citrate production. ACT. AOX. which has a higher affinity than ACT. Substrates and products: glu. activator) are shown. automatic control of inoculation and fermentation procedures. aconitase.5–2 0. þ. FC: Fructose carrier. PC. low-affinity glucose carrier.FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. PFK2. peanut. oaa. niger has both low-affinity and high-affinity carriers for glucose) and hexokinase (HK) activity. 0. This culture may be directly transferred into 10–20 m3 seed fermenters to obtain a pellet-type inoculum consisting of 1–5 105 pellets dm3 (0. glucose-6-phosphate. succinyl-coenzyme A. TCC. cit.25 <200 200–1500a 200–1500a <2 2. Hexokinase. a-ketoglutarate dehydrogenase. gly. manganese. with cation-exchange resins or the addition of potassium hexacyanoferrate (HCF). FBP lowers the Michaelis–Menten constant (Km) of PFK1. acetyl-coenzyme A. which is insensitive to citrate inhibition. . and olive oils. Gluconic.5–6. Enzymes and transport systems: INV. glu6P. and effectors (. Submerged fermentation is carried out either in 150–200 m3 stirred-tank reactors or in 300–500 m3 (up to 1000 m3 as claimed by some manufacturers) bubble-column reactors.5 kg m3 kg m3 0. fructose-1. substrate. Some ingredients (methanol. A phosphorylated fragment of PFK1. fructose-6-phosphate. 0. proton pump. CS. In spite of the old and renewed interest in citric acid production by yeast grown submerged in sugar. trehalose. followed by restoration of the air supply.3–0. (b) stubby. Fermentation is exothermic and temperature has to be kept in the range 28–35 C.1–0. that are met by sparging 0. On the contrary.12–0. Mycelial clumps (whose structure is less compact than pellets) also may develop under high agitation speed. and the addition of alkali (NH3) is used to control pH at 2. Figure 3 shows the evolution of a typical batch citric acid fermentation in glucose-based media by A.3–0.5 kg m3 h1 result in microbial oxygen demand rates of 0. bulbous hyphae with frequent branching. Foaming is controlled by adding food-grade antifoam agents.5 kg O2 m3 h1.35 MPa to 0. during the second growth phase (idiophase). no acid production occurs. ranging from 0. niger in 400 m3 bubble-column fermenter. Low pH and high DO concentration are essential for citric acid production.6 once production of citric acid has started. acid production. . dense pellets (Figure 2(a)) with short stubby hyphae (Figure 2(b)) are generally regarded as the best morphological form for optimal citrate yields. the heat transfer surface required to keep the fermentation temperature constant would be w3. Initial decrease of pH is due to ammonium uptake. and Propionic) Figure 2 Pellet morphology of A. Temporary interruption to the air supply during fermentation does not seem to affect the performance of the culture on the condition that the DO level is greater than 20% of the saturation value. Gluconic.4 MPa and at the tank top. DO values of about 0 for as long as 85 min. such as HCF or zinc and copper sulfates. Extreme pH values (<1. Lactic. Frequent observation of pellet morphology during this early stage of the fermentation using a microscope allows hyphae proliferation to be controlled by the addition. and (c) degenerating pellet with pointed unbranched hyphae protruding from the pellet core (100). niger NRRL 2270 during citric production in a laboratory 2 dm3 stirred fermenter: (a) young pellet (100). acid production by almost nongrowing cells is observed. do not inhibit permanently mycelial growth and citrate production.4 volumes of air per medium volume per minute (vvm) at pressures at the sparger section of the fermenter not less than 0. of appropriate amounts of inhibiting compounds.15 MPa. respectively. niger NRRL 2270 in 2-dm3 stirred fermenter and by a mutant strain of A.6) limit productivity. in case of adverse development (Figure 2(c)). Two distinct phases are evident: during the primary growth phase (trophophase).808 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. The typical industrial-scale productivities of 1–1.2 m2 per m3 of fermentation medium. Assuming that the overall heat transfer coefficient and effective temperature difference between the fermenting medium and cooling water are of the order of 500 kJ m2 h1 and 5 C. depending on the (stirred or air-lift) fermenter type used. which are characteristic of citric acid production (400). but they do reduce the product yield coefficient up to 20%. small spherical.2–2.25–0. The precipitation process is used by the great majority of world citric acid manufacturers. B). Calitri. precipitation as calcium citrate tetrahydrate.. whereas the second term may be regarded as the nongrowth-associated product formation rate. the theoretical molar yield (z) of citric acid would be one or two if glucose or sucrose is used. The washed crystals and 98% w/w sulfuric acid are simultaneously..23) kg of citric acid monohydrate per kilogram of glucose (or sucrose) consumed. it was assumed that during the trophophase the microorganism (represented by a raw formula based on elemental analysis: CHnOpNq) replicates itself at the expanses of a generic carbon source in the presence of ammonia as the only nitrogen source. glucose (S: l. Liming temperature is critical.e. Italy).. Liquid extraction is used by Tate & Lyle Co. or other by-products as shown by the reaction in Eqn [2] (i. or liquid extraction (see Fermentation (Industrial): Recovery of Metabolites). it undergoes further growth while decreasing progressively its intracellular nitrogen content and excreting citric acid in a medium practically devoid of any nitrogen source (Figure 3). a subsidiary of Bayer Co. The industrial-scale trial was gently provided by Dr A. Trunfio c/o Palcitric SpA. In particular.6. and ammoniac nitrogen (N: D) versus time (t). but 10–25% of the expected product yield is needed as seed.17 (or 1. in greater filterability and washability because of its crystalline structure. A simplified process flowsheet of this method is shown in Figure 4. but separately. . In this way. niger NRRL 2270 in a 2 dm3 stirred fermenter (closed symbols) and by an industrial strain in a 400 m3 bubble-column fermenter (open symbols): Concentrations of mycelial biomass (X: A. before feeding a vacuum crystallizer operating at temperatures below (35 C) or above (62 C) the transition temperature (36. and Propionic) 809 A microscopic description of this fermentation using A. with the smaller figure generally being associated with the surface fermentation technique. Assuming that no carbon atom of sugar is converted into biomass. Citric acid may be recovered from the broths resulting from either the surface. both product formation (rP) and substrate consumption (rS) rates may be linearly related to cell growth rate (rx) and cell concentration (X). Citric acid is precipitated as calcium citrate by the addition of lime to the filtrate.5–0. and the laboratory-scale trial was performed by the authors at the University of Basilicata (Potenza. citric acid (P: n. This would be equivalent to 1. In both of the fermentation trials shown in Figure 3.or submerged-culture fermentations.) in the Dayton (OH. In practice. during the idiophase. USA) plants. such as sucrose syrups or crystals. including ADM in the United States. the well-known Luedeking– Piret kinetics for product formation has to be regarded as a special case: In fact. are used. The crystals are recovered using another continuous belt filter and then recycled back to the liming step.). >). No removal of oxalic acid is needed if the submerged-culture fermentation is used. Direct crystallization cannot be applied unless refined raw materials. Final refining of the filtrate is performed by decolorization on activated carbon and removal of residual calcium sulfate and iron and nickel salts on strong cation exchange and weak anion exchange (demineralization step). residual sugars and contaminants from the raw carbon source and soluble proteins from the autolysis of the fungus). In accordance with the Herbert–Pirt maintenance concept. Gluconic. carbon dioxide.8. fed to a mixer containing a 40% citric acid solution at pH 0. citric acid fermentation may be classified to be of the mixed-growth-associated product formation type. the industrial yields range from 57 to 81% of this theoretical value. The precipitate is washed to remove the impurities adsorbed onto it (i. using almost the same three methods – namely. Italy. Amorphous tricalcium citrate tetrahydrate generally is obtained at w70 C.6 C) between the monohydrate and anhydrous forms. Table 3 shows the simplified overall stoichiometric reactions occurring during the trophophase and idiophase of the fermentation examined.FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. when y and d are equal to 0). direct crystallization upon concentration of the filtered liquor. The citric acid fermentation may be mathematically described by means of the set of kinetic equations shown in Table 3. depending . Recovery and Purification Processes Figure 3 Time course of a typical batch citric acid fermentation from glucose-based media by A. USA) and Elkhart (IN. while crystalline dicalcium acid citrate is obtained at 90 C. The resulting solution (250–280 kg m3 of citric acid anhydrous) is concentrated using multiple-effect evaporators to about 700 kg m3. Mycelia and suspended particles are separated by continuous belt filters under vacuum. 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. with the oxygen penetration depth ranging from 110 to 300 mm in 2 mm pellets. to free the citric acid with the formation of a precipitate of calcium sulfate dihydrate (gypsum). Precipitation of dicalcium acid citrate results in one-third less consumption of lime and consequently of sulfuric acid for the subsequent regeneration of citric acid. Lactic. (formerly Haarmann & Reimer Co. while the filtrate has to be disposed.e. The residual citrate in the filtrate is precipitated as tricalcium citrate by further addition of lime to set the pH to 5. The low effective diffusivity of oxygen within the pellet limits mycelial activity to a peripherical spherical shell only. the first term in Eqn [10] may be described as the product formation rate in association with the mycelial growth rate. and YS/x. m. S. and instantaneous concentrations of mycelia. y. conversion rate of any reagent or product. P. Lactic. X. specific cell growth rate. specific rate of product formation (or substrate consumption) at zero cell growth rate. citric acid. YP/x. mycelium. XM. kinetic equations. YN/x. glucose. referred to the initial value. citrate concentration. sugar. and Propionic) Table 3 Citric acid fermentation: overall stoichiometric reactions. and substrate on unit cell biomass. z. mP (mS). stoichiometric coefficients. N.810 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. mM. critical concentration of nitrogen at the onset of citric acid production. b. tlim. Gluconic. mycelium concentration. overall duration of the citrate lag phase. 0. citrate. substrate concentration. referred to limiting concentration of the nitrogen source. overall duration of the cell lag phase. X. product. P. d. yield factors for ammoniac nitrogen. maximum mycelium concentration. nitrogen. S. and e. . Nlim. to. Subscripts: lim. cumulative nongrowth contribution to product formation. 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 glucose Idiophase reaction Kinetic equations C6 H12 O6 þ b O2 / y CHn Op Nq þ z C6 H8 O7 þ d CO2 þ e H2 O glucose rX ¼ dX ¼ mX X dt rN ¼ dN dX ¼ YN=X dt dt rN ¼ dN ¼ 0 dt ½3 ½6 for t tlim ½7 for t > tlim rP ¼ dP ¼ 0 for t tlim dt rP ¼ dP dX ¼ YP=X þ mP X dt dt ½8 ½9 for t > tlim ½10 dS dX þ mS X ¼ YS=X dt dt X ¼ X0 ½11 for t to X ¼ X0 e mM X ¼ ½5 for N < Nlim X m ¼ mM 1 XM rS ¼ ½4 for N Nlim for t to m ¼ mM ðt t0 Þ ½12 for t tlim XM XM 1þ 1 e mM ðt tlim Þ X0 ½13 for t > tlim ½14 for t < t0 N ¼ N0 ½2 citric acid mycelium m ¼ 0 Integral solutions of the differential kinetic equations ½1 mycelium N ¼ N0 YN=X ðX X0 Þ ½15 for t tlim ½16 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 ½20 N ¼ Nlim Aðt Þ ¼ for t tlim XM X h ln 1 M 1 e mM mM mM ðt tlim Þ i for t > tlim ½21 Nomenclature: A(t). ri. maximum specific cell growth rate. heat exchanger. cooling water. vacuum belt filter. holding tank. activated carbon adsorber. E. and Propionic) 811 Figure 4 Process flowsheet of a typical citric acid fermentation from glucose-based media by A. lime slurry. AF. BF. alkaline reagent. HT. production-bubble fermenter. centrifuge. DW. PE. Equipment and utility identification items: AE. plate–heat exchanger. CY. HS. seed-bubble fermenter. EV. D. SA. exhausted air. WE. water evaporated. cation exchanger. BD. low-pressure steam. cw. dicalcium citrate. nutrients and additives. Condensate. Se. . NA. SF. Lactic. EA. F. S. FI. antifoam agent. hot air. CE. M.FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. cyclone. sterile compressed air. HA. anion exchanger. sulfuric acid. mixer. grinder. CA. centrifugal pump. fluidized-bed drier. dicalcium citrate seed. holding tube. LS. demineralized water. evaporator. niger. C. dcc. c. tcc. vacuum crystallizer. tricalcium citrate. sterile pressure filter. Gluconic. PC. AL. CR. GR. that is straight away released in the medium. The environmental aspects of citric acid production have been assessed. Inc. Any improvement of strains of A. The conversion of glucose to gluconic acid is a simple oxidation process and may be carried out by a variety of processes – namely. Gluconic.. or the use of liquid membranes. electrochemical. Gluconic Acid D-Gluconic acid (2. namely trilaurylamine.g. niger usually were carried out by mutagenesis and selection. The replacement of molasses with raw or hydrolyzed starch. Pharmacopeia or Food Chemical Codex material. Although the effect of nitrogen deficiency on citric acid accumulation by A. would allow the citric acid to be separated in a single step and to be recovered without the formation of solid wastes for disposal. decolorized.e.or raw sucrosebased materials would simplify only the downstream processing. which presently is the only method of choice. equivalent to 2 mmol of intracellular NHþ 4 per gram of dry cell) was found to inhibit the morphological degeneration of pellets and postpone sporulation. are hazardous chemicals. whereas D-gluconog-lactone is made only in small quantities as a specialty chemical. food.. chemical.5.82). citric acid crystals do not need additional purification steps to meet specifications for U. In this way.. D-Glucono-d-lactone is used as a latent acid in baking powders for use in dry cake mixes. and sulfuric acid. decolorized fermentation broths by electrodialysis. the recovery of tricalcium (or trisodium) citrate from clarified. For instance. as well the adsorption of citric acid onto weakly basic anionic-exchange resins or zeolites using the simulated-moving bed chromatographic technology (Citrex Process. but they enter the cell to combine with glucose and form glucosamine.e. The extract is then heated and washed countercurrently with water. The calcium and iron gluconates are used in medicine to treat diseases of calcium and iron deficiency (such as osteoporosis and anemia). lime. presently is hampered in fungal processes by the deterioration of mycelial structure. or enzymatic catalysis. IL. and . In the liquid extraction process. Similarly. Feþ3). NHþ 4 ions simply are not deposited into the cell to form the so-called ammonium pool. and instant chemically leavened bread mixes. Future Developments Production of citric acid has not been much in the focus of modern molecular biology presumably because it is considered a mature area. the possibility of maintaining microbial cells active and controlling their growth and production processes for several weeks or months by immobilization within organic or inorganic matrices represents a further challenge to the technological modernization of this sector. This acid and its derivatives are used in the pharmaceutical. bottle washing. Several filamentous fungi of the genera Penicillium and Aspergillus. The replacement of the current batch fermentation with semicontinuous processes. in an aqueous solution.6-pentahydroxy pentane-1-carboxylic acid: C6H12O7) is an oxidation product of D-glucose. The effective relationship between the different compounds of the TCA cycle is to be studied further and controlled before the present batchproduction technology may be converted effectively into a prolonged fed-batch or continuous production process. and nontoxic food-grade solvent (i. a low level of ammonium ions (i.e.S. resulting in about 90% recovery yield and an aqueous citric acid concentrated solution. citric acid may be extracted from the fermentation broth using a highly selective. Pfizer & Co.3.4. the yeastlike fungus Aureobasidium pullulans.5-lactone (D-gluconod-lactone) and 1. meat processing. these processes appear to be either more expensive.. metal surface cleaning. and chemical industry because of their low toxicity and their ability to form water-soluble complexes with metallic ions (e. After the first isolation of calcium gluconate (1880) from glucose fermentation in the presence of CaCO3 by a strain of Mycoderma aceti. Despite the fact that most raw materials are of biological origin. USA) followed by desorption with water or dilute acidic solutions. Lactic. many ingredients. Several process alternatives have been suggested thus far to minimize the overall environmental impact of this process. which is passed through a granular activated–carbon column before undergoing the aforementioned evaporation and crystallization steps. and Propionic) on the form manufactured. the mechanism of which still is unknown.4-lactone (D-glucono-g-lactone). and fed back to liming. Further research at the U. UOP. Des Plaines.S. it was found that the environmental impact of citric acid production using whey was smaller than that using corn starch. low-price. especially in the presence of 5–10% of sodium hydroxide. C10 or C11 isoparaffin. water-insoluble amines. to increase volumetric productivity and reduce specific production costs. which. Caþ2. (New York. On the contrary. but metabolic and genetic engineering are likely to improve acidogenesis.15 kg of dried mycelium and 2 kg of gypsum per kilogram of citric acid anhydrous). the first one using hot air at 90 C and the second one using air conditioned at 20 C and relative humidity (RH) of 30–40% because of crystal hygroscopicity.812 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. 30 g m3.g. The mother liquor is partly (w20%) diluted with equipment-cleansing waters. feed. n-octanol. Department of Agriculture in cooperation with the Iowa State College led to the semicontinuous production of sodium gluconate from glucose using A. unstable. niger NRRL 67. tri-nbutyl phosphate. the remainder is in sequence decolorized and demineralized before being recycled to the crystallization unit. USA) started industrial-scale production of gluconic acid in 1923. The traditional recovery technology results in several problems because of disposal of liquid effluents (their chemical oxygen demand being about 20 kg m3) and solid by-products (i. Sodium gluconate is the main industrial product and it is used as a sequestering agent (e. Crystals are separated by centrifugation and dehydrated in a two-stage fluidized-bed dryer. such as ammonium nitrate... alkysulphoxides). the Chas. and rust removal) and to plasticize and retard the curing process of cement mixes. microbial fermentation. D-Gluconic acid is commercially available as 50% aqueous solution (density of 1230 kg m3 at 20 C and pH 1. or less efficient than the fermentation process. Currently. niger is well known. leads to a complex equilibrium between gluconic acid and its two lactones: 1. about 0. but a few gluconic acid–producing strains (A. Further crystallization at 30–70 C or at more than 70 C allows crystals of the d-lactone or g-lactone to be precipitated.4 120–350 0. is filtered.09 kg kg1) and gluconate productivities of 9–13 kg m3 h1. After the pH is adjusted at 4.0 kg m3 kg m3 kg m3 kg m3 kg m3 mg m3 mg m3 mg m3 mg m3 – 813 adjusted to 6–6. Such enzyme abstracts two hydrogen atoms from glucose.97–1 kg of gluconic acid per kilogram of glucose consumed (against a theoretical yield of 1. because of the slight toxicity of the D-() isomer. respectively. Gluconic.4–0. conidia are recovered from stock agar slants and are inoculated into vegetative seed–culture media (106 conidia cm3). Glucose syrups of 70 Brix strength are generally used as carbon source in the preparation of the fermentation medium. In the fed-batch operation.1–0. sodium lactate is used for carcass decontamination. niger Component Glucose NH4NO3 (or other NHþ 4 salt) KH2PO4 MgSO4$7H2O Agar Yeast extract Corn-steep liquor ZnSO4$7H2O CuSO4$5H2O FeCl3$6H2O MnSO4$7H2O Initial pH Vegetative seed –culture media Gluconic acid production media Unit 40 2. Table 4 lists the typical composition of the seed and production media used in laboratory. pullulans DSM 7085 recently has been developed.5 with sodium hydroxide. decolorized using a granular activated–carbon column. only the sodium process by batch-submerged fermentation from glucose syrups using A. The free acid is used as an acidulant/ preservative in several food products (cheese. which makes its recovery easier. meat. as well as many advantages over the traditional microbial fermentation processes. and a 2–5% v/v inoculum generally is used. niger is controlled by the enzyme glucose oxidase. Stepwise addition of glucose may be used to increase gluconate concentration to 580 kg m3. Industrial strains are not freely available. Lactic Acid Lactic acid (2-hydroxypropionic acid: C3H6O3) may be produced by chemical synthesis or fermentation. iron and manganese) are kept under control.5 with NaOH. pressure on the tank top (2–3 bar). beer) (see Preservatives: Traditional Preservatives – Organic Acids). and Propionic) bacteria (Acetobacter suboxidans. Penicillium spp.5 3. and controlled drug release. but industrial processes have been developed only for the production gluconic acid from glucose syrups with A.5 kg m3 kg m3 1. under vacuum-belt filtration or crossflow microfiltration and may be used as a source of glucose oxidase or may be disposed off via incineration. neutralized to pH 7.5 by addition of 30–50% NaOH solution to neutralize the gluconic acid formed). ammonium lactate is used as a source of nonprotein nitrogen in feeds.5 with sulfuric acid.FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. only the L-(þ) isomer is used by human metabolism and. At the end of fermentation. orthopedic implants. which to some extent hydrolyzes to gluconic acid. Although this novel fermentation process offers a new opportunity for commercial gluconic acid production. cooled at 33 C. jellies. all lactic acid manufacturing industries have switched to fermentation-based technologies (Table 1).5–6.and industrial-scale production of gluconic acid by A. which is converted into oxygen and water by the enzyme catalase. Lactic.e. The current economically viable industrial process for PLA production is via the dehydrated cyclic dilactate ester . but they have the advantage of excreting the glucose oxidase (an important by-product) into the medium. the medium is sterilized at 121 C for 15–30 min.1–0.. temperature (33 C). and then transferred into the fermentation vessel. Thus.3 0. NRRL 67) can be obtained from international culture collections. Pseudomonas ovalis. niger will be described in the following paragraphs. The formation of gluconic acid by A. pH (5. It is completed within w30 h with yield factors of 0. sodium and calcium stearoyl lactylates are used as emulsifiers and dough conditioners. L-(þ) and D-() lactic acid. pellet-like mycelia is obtained after incubation at 30 C for 15–24 h and is used to inoculate seed fermenters at a density of 20–50 pellets cm3. niger and Gluconobacter oxydans.) produce gluconic acid from glucose-based media. it is still confined to laboratory-scale applications. such as reabsorbable sutures. Of the two enantiomers. an omodimer containing two flavin adenine dinucleotide (FAD) moieties. Both glucose oxidase and catalase are constitutive endoenzymes in A. and then spray or drum dried. The pH is Table 4 Composition for the production media used in the laboratory. generally containing w300 kg m3 of sodium gluconate. the mycelium is removed using aseptic centrifugation.5 vvm). If 50% gluconic acid is required. the concentrated liquor may be passed through a cation exchanger to remove Naþ ions. niger. thus yielding the glucono-d-lactone. as well as several medical applications. and foam level.4 0 0 0 30 6. A highly productive process of gluconic acid using freegrowing cells of A. and disposable tableware.3 0 0 0. niger NRRL 3. The fermentation is carried out under continuous automatic control of sterile air sparging (1. it is preferred for food uses. concentrated under vacuum to 45–50% total solids. Its high conversion yields (90–98%) and rates (13–19 kg m3 h1) resulted in as high gluconate concentrations as 504 or 230–433 kg m3 in fed-batch or chemostat trials. compost. a polyester used for biodegradable plastics for food packaging. The large increase in lactic acid production is due to its use in the synthesis of polylactic acid (PLA).2–0. The FADH2 reacts with oxygen to form hydrogen peroxide. the mycelium may be reused up to five times without any loss in gluconate productivity provided that the levels of glucose oxidase activity and other microelements (i. generally produce less gluconate than Aspergillus.57 1 1 0 100 20 300 0 6. Because the chemical route yields a mixture of L-lactic acid and D-lactic acid and relies on costly raw materials. For inoculum development. and garbage bags. Gluconobacter spp.0–1. The clarified broth.5 0.and industrial-scale trials. In brief. and type of lactic acid isomer produced. although other nonfood uses are important (see Permitted Preservatives – Propionic Acid). coagulans and R. Batch fermentations result in high product concentration (120–150 kg m3) but in low productivity (2 kg m3 h1). Fermentation is carried out under anaerobic or microaerophilic conditions and lactic acid yield is usually between 85 and 98% with isomer purity as high as 99%. Undissociated lactic acid acts as a noncompetitive inhibitor for growth and lactic acid production by diffusing through the membrane and decreasing intracellular pH: pH control during fermentation reduces the inhibition. Lactic acid production is usually a growth-associated production process. to minimize competition for land and food. Because this may account for 30–35% of substrate costs. Homofermentative LAB ferment hexoses via the glycolytic pathway (see Metabolic Pathways: Release of Energy (Anaerobic)). stannous octoate at 0. Pyruvate is reduced to lactate by stereospecific lactate dehydrogenase(s) (L-LDH or D-LDH). Lactobacillus helveticus) tolerate higher concentrations of lactate and higher temperatures (48–52 C). because lactide is produced from the fermentation of renewable resources. These oligomers are then depolymerized by increasing the polycondensation temperature and lowering the pressure in the presence of transition metal–based catalysts (i. Lactobacillus amylophilus). but the maximum lactic acid concentration achievable is usually lower than 150 kg m3.5 by the automatic addition of NaOH. continuous fermentations with cell-recycle or immobilized cells give rise to higher productivities (20–80 kg m3 h1) and lower lactate concentrations (<50 kg m3). bulgaricus. ion-exchange resins. and molds (Rhizopus microsporus. They produce D-() or DL-lactic acid (some industrial strains that have been claimed to be L. delbrueckii produce L-(þ) lactic acid) and may be less suitable for food. facultatively anaerobic Bacillus species (B. acid tolerance. The pH is controlled at 5–6. Finally. Organisms and Metabolic Pathways Involved Lactic acid can be produced using homofermentative lactic acid bacteria (LAB). tensile strength >50 MPa). supplementation with milk protein hydrolysates (5–10 kg m3) and yeast extract (up to 20 kg m3) is required.. amylophilus) can hydrolyze starch. Nevertheless. and Streptococcus thermophilus). Other Organic Acids Produced by Fermentation Propionic Acid Propionic acid (C3H6O2) and its salts are used as mold inhibitors in bakery products. The choice of the species depends on several considerations.e. and thermophilic streptococci (Streptococcus thermophilus) have lower temperature optima or reduced acid tolerances.e. feed. nutritional needs. which produce optically pure L-(þ) or D-() lactic acid (see Lactobacillus: Introduction. even if smaller yields (as low as 70% for R. wood hydrolysates. Thermophilic lactobacilli (Lactobacillus delbrueckii subsp. End-product inhibition may be circumvented by using integrated fermentation processes. lactic acid is first polymerized into oligomers of PLA consisting of 30–70 lactyl units (–CHCH3– CO–O–). oryzae. casei. Alternative processes include the extraction by liquid membranes. lactic acid producing genetically modified strains of Escherichia coli and Saccharomyces cerevisiae have been developed. Recently. LDH is allosteric (activators: fructose-1. Recovery of lactate is made complicated by the high solubility of its salts. it is possible to obtain high molar mass polymers (100–300 kDa) with appropriate optical and mechanical properties (i.g. production of pure L-(þ) lactic acid.814 FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. crushed corncobs). thus involving higher productivity and yields and reduced contamination risks. usually in the form of yeast extract. Rhizopus oryzae). but nongrowthassociated production becomes significant when growth is limited by a lack of nutrients or high undissociated acid concentration. such as caprolactone. both being L(þ)-lactate producers. Conversely. can be incorporated to provide environmentally safe materials. Blair. electrodialysis. USA: 140 000 metric tons per year). It may be produced by fermentation by members of the genera . see Fermentation (Industrial): Media for Industrial Fermentations). Concomitant substrate and product inhibition has been reported for several species. Some species (Lb. Lactococci. the recent industrial use of electrodialysis with bipolar membranes in France resulted in the virtual elimination of gypsum waste production. starch and corn starch hydrolysates. and Propionic) (lactide) formation. Other comonomers. oryzae because of concomitant fumaric acid and ethanol production) and acid tolerance may offset the advantage of using lower amounts of supplements. hydrolysis of starch). but the pseudoplastic behavior of starchy substrates makes the pH control difficult.. Recently. Nebraska. such as corn starch (as in the industrial plant of Nature Works LLC. delbrueckii. Na2CO3. coagulans). genetic engineering has been used to produce L. helveticus and Lactobacillus plantarum strains. Gluconic. When whey permeate is used. or nanofiltration. beet and cane molasses. including the ability to use the type of sugars available in the substrate..05%) to distil the lactide.g. including electrodialysis. Lactic. they may be replaced by less demanding B. or biomedical applications. hydroxybenzoic acid. and ion exchange. in which lactic acid is removed from the culture broth by several techniques. growth temperature.. Conventional recovery by the precipitation method seems to be the most economical route. Lactococcus: Introduction. but they may be desirable for other reasons (e. delbrueckii subsp. and others. The traditional process involves precipitation of calcium lactate and regeneration of lactic acid by the addition of sulfuric acid followed by further purification steps (ion exchange and decolorization). by opening its ring. LAB are fastidious microorganisms and require supplementation of fermentation media with peptides and growth factors. or NH4OH or by the addition of CaCO3. L. mesophilic lactobacilli (Lactobacillus casei subsp. research studies have started to develop second-generation PLA products from lignocellulosic hydrolysates (e. Substrate Production and Recovery Lactic acid can be produced from a variety of raw substrates (whey and whey permeate. PLA appears to be a sustainable alternative to petroleum-based plastics. In particular.6-bisphosphate and Mnþ2) in lactococci and nonallosteric in homofermentative lactobacilli. Legisa. Fermentation (Industrial): Control of Fermentation Conditions. O. 2003. Gluconobacter. Process Biochemistry 44. Fukuda. acidipropionici). 2006.-T. Bread: Bread from Wheat Flour. Polymer Bulletin 63. For instance. 2009. Applied and Microbiology and Biotechnology 74.. Nampoothiri. Okano.). Citric acid production. Tanaka.G. John. The high microbial productivities (2–14 kg m3 h1) obtained in continuous fermentations using immobilized cells or membrane-recycle reactors. Organic acid production by filamentous fungi. Applied Microbiology and Biotechnology 75. M. Biotechnological production of enantiomeric pure lactic acid from renewable resources: recent achievements. See also: Arthrobacter. <1 kg m3 h1 in batch processes.P. freudenrichii.. Chapter 12. 307–340. and Selenomonas. Genetic Engineering. Kim. L. Branduardi. Ogino. and limits.. M.K. Applied Microbiology and Biotechnology 85. Mattey. Propionic acid production from glycerol by metabolically engineered Propionibacterium acidipropionici... L. Lactobacillus: Introduction. Veillonella. Fungi: Classification of the Deuteromycetes. might refocus industrial manufacturers toward the fermentation route. Advances in Fungal Biotechnology for Industry. R. 2000. 2007. I. use of a metabolically engineered strain of P. 1–17. Aspergillus. membrane transport and modeling. Microbial production of organic acids: expanding the markets.. Streptococcus thermophilus. Vinegar. J. M.M. Kumar. A.. Sauer. Yeasts: Production and Commercial Uses. Changes in primary metabolism leading to citric acid overflow in Aspergillus niger. Fermentation (Industrial): Recovery of Metabolites. Yang. A. Yoo.71 g per g of glycerol consumed and a propionic acid-to-acetic acid ratio of 22.. Bacillus: Introduction. 110–117. which invariably is produced from sugars and lactate). 2004. S... 2007. Advances in citric acid fermentation by Aspergillus niger: biochemical aspects.... Preservatives: Traditional Preservatives – Organic Acids. Agriculture.. 815 Further Reading Anastassiadis. Fermentation (Industrial): Media for Industrial Fermentations. and Propionic) Propionibacterium (P.. H. K. Clostridium. but currently it is produced by chemical synthesis because of the shortcomings of the fermentation route (low productivities. 2007. 413–423.S. and Medicine. Kulkarni. thoenii. Factors affecting the fermentative lactic acid production from renewable resources. C. 303–343. R. 2007. . Biotechnological production of gluconic acid: future implications.. A.. Singh.... Separation and Purification Technology 52. Magnuson. 2007. Gluconic. 1346–1351. 181–190. M. Yarrowia lipolytica (Candida Lipolytica). Kluwer Academic/Plenum Publishers.. Joshi. Biotechnology Annual Review 13.-K.. Joglekar. Propionibacterium.L. 713–722. (Eds.. Legisa.. J. Continuous gluconic acid production by isolated yeast-like mould strains of Aureobasidium pullulans. D. acidipropionici (ACK-Tet) resulted in a propionic acid concentration of 106 kg m3 with a product yield of 0... C.4. A. Fermentation (Industrial): Basic Considerations. In: Tkacz. Mattanovich. difficulty in product separation from acetic acid. 2009. Biotechnology Letters 29. Production of optically pure poly(lactic acid) from lactic acid. Fermentative production of lactic acid from biomass: an overview on process developments and future perspectives. like crude glycerol from the biodiesel industry. as well the possibility of obtaining pure propionic acid from alternative low-cost substrates. Porro.V. Fungi: The Fungal Hypha. Trends in Biotechnology 26 (2). Kondo. Zhang. A. Papagianni. pp.54–0. B. 244–263.. Babu. 2010.D. P. Permitted Preservatives – Propionic Acid. D. Rahman. Lactic.... Aivasidis.. Fungi: Classification of the Hemiascomycetes. 2007. Berovic. perspectives. D. New York.. M. Biotechnology Advances 25.. 524–534. K. Enzyme and Microbial Technology 26. K. 637–651. Lasure. S. Metabolic Pathways: Release of Energy (Aerobic). Fermented Foods: Fermentations of East and Southeast Asia. 100–108. Metabolic Pathways: Release of Energy (Anaerobic). low product concentrations. B. H.. M. Hofvendahl.FERMENTATION (INDUSTRIAL) j Production of Some Organic Acids (Citric. Lange. <50 kg m3.. Pandey.. Comparative assessment of downstream processing options for lactic acid. 87–107. D. Applied Microbiology and Biotechnology 61. T. S. Hahn–Hägerda. Wandrey. Escherichia coli: Escherichia coli.
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