The production of organic acids.

Critical Reviews in Biotechnology, 12(1/2):87-132 (1992)

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The Production of Organic Acids Michael Mattey, Ph.0. The University of Strathclyde in Glasgow, Department of Bioscience and Biotechnology, 31 Taylor Street, Glasgow G4 ONR, Scotland

ABSTRACT: The production of organic acids covers two aspects: first, the metabolic pathways involved in the biosynthesis, and, second, the industrial process strategy adopted. The review seeks to show the underlying biochemical similarities in the biosynthesis of organic acids and the resulting similarities in the commercial processes. Two groups of acids are defined, those with a “long” biosynthetic path from glucose, involving much of the glycolytic pathway and the tricarboxylic acid cycle, and those acids with a “short pathway”, essentially a biotransfonnation of glucose. The regulation of the pathways and the future developments in metabolic control theory and genetic manipulations relating to them are considered. The organisms used industrially are also limited, Aspergillus sp. and Candida yeasts; again the underlying metabolic similarities lead to similar strategies for all the acids discussed.

KEY WORDS: organic acids; microbial, hydroxy acids; itaconic.

1. INTRODUCTION A. Scope Organic acids contain one or more carboxylic acid groups and are widely distributed in nature, occurring in animal, plant, and microbial sources. The carboxyl group may be covalently linked in amides, esters, peptides, etc. and thus is often masked. The production of organic acids on an industrial scale is largely confined to acids of microbial origin; this is the group of compounds in which biological production has an economic advantage over chemical synthesis. Without considering acids of animal and plant origin, there is still a formidable list of acidic compounds that have been isolated from microbial sources. Over 130 such acids have been described.’ They range from various simple, unsubstituted acids such as formic2 to complex, glycosylated acids such as pyolipic acid.3 The monocarboxylic acids range from formic, acetic,2,4-6and p r o p i o n i ~to ~,steariq6 ~ with branch chain acids such as isovaleric4 also occurring.

More than a dozen di- and tricarboxylic acids have been described, while over 50 hydroxy- (or keto-) acids of microbial origin are known, including a number of commercially important acids such as c i t r i ~ , ~i,t~a,c*~ n i cand , ~ lac ti^.^^^ Among the many sugar-derived acids described, g l u c ~ n i c ~ ~and ~ ~ascorbicI2 ~ ~ ’ ~ ~ are ’ ’ the ones produced in bulk. Acids containing nitrogen and sulfur are also known, but, with the exception of the nutritionally important amino a c i d ~ ’ ~and ,’~ some antibiotics,” they are of laboratory interest only. This review concentrates largely on the production of hydroxy-acids of microbial origin that are produced in industrial quantities. Itaconic acid, due to its metabolic relationship to citric acid, is also included. General reviews of this area have been written by Lockwood,’6 Milsom,” and Ilczuk. ** The majority of the organic acids produced by microorganisms can be readily placed into two main gropus, depending on their metabolic origin: (1) acids from the main metabolic sequence of aerobic organisms, that is, the tricarboxylic acid cycle and glycolysis, and (2) acids arising

0738-855lf92/$.50 0 1992 by CRC Press, Inc.

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from the direct oxidation of glucose. This second group, produced by only one or two enzymatic steps from glucose, are in this respect akin to biotransformations. Recent developments have used the concept of biotransformation to produce organic acids, using other acids as starting materials. In general the biosynthetic routes to group 1 organic acids can use a variety of starting materials. Citric, itaconic, lactic, and malic acids fall into the first group, while gluconic and 2-0x0gluconic acids are in the second. Acetic acid should be considered a biotransformation of ethanol, but could be regarded as a group I compound as ethanol is itself produced by a group 1 process. Two aspects of the production of organic acids will be considered - the biochemical and the industrial. In both areas there are many similarities between the various acids in their biochemical pathways and production techniques. The strategies used both by the organisms and by the manufacturers have shown parallel development.

B. Uses and Properties 1. Citric Acid In terms of bulk production, citric acid is one of the most important of the organic acids. It is estimated that some 400,000 tons per year are now produced largely by processes involving Aspergillus niger, although an increasing amount is produced by Yarrowia lipolytica (asexual form Candida syn. Saccharomycopsis) -based process. Such processes are commonly referred to as fermentations, although P a s t e ~ r would '~ not have considered such highly aerobic systems as such! The uses and properties of citric acid have been reviewed.20 Citric acid was first described in 1784 by Scheele,21who isolated it from lemon juice. This remained the major source until 1919, when the first industrial process using A. niger began in Belgium. Commercial production from Italian lemons commenced about 1826 in England by John and Edmund Sturge, but the Italians established a virtual monopoly of production in the next 100 years, and the product remained ex-

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pensive as a result. Two strategies were examined to find alternative sources of citric acid - the chemical and the microbiological routes. Citric acid was synthesized from glycerol in 1880;22several other routes using different starting materials have been published since then. These have proven unsuitable mainly on cost grounds as the starting materials were often worth more than the product, although poor yields due to the number of reaction steps in the syntheses and precautions due to the hazardous compounds involved contributed to the failure to find a chemical solution to the problem. The first indication of a microbiological solution was the observation by WehmeP in 1893 that certain ' 'Citromyces" (now Penicillium) accumulated citric acid. Although this did not lead directly to a commercial process, the subsequent search for other organisms capable of this synthesis did. CurrieZ4in 1917 found a strain of A. niger capable of significant accumulation of citric acid from a sugar medium. This work formed the basis of the citric acid plant established by Chas. Pfizer & Co. Inc. in the U.S. in 1923. Other processes were established in Europe, all using variants of the surface culture method, with sucrose as raw materiaLZ5Processes using the cheaper beet molasses were soon introduced. Submerged culture processes were developed about 1940. They have not yet displaced the surface culture processes entirely, although it is unlikely new surface plants would be economical in a developed country. From about 1965, methods using yeasts were developed, first from carbohydrates and then from n-alkanes.26At this time hydrocarbons were relatively cheap, and plants were built to use the method. The economics have altered since then; plants built to utilize both yeast technologies have apparently switched back to carbohydrate feedstocks. Citric acid is used in food, confectionary, and beverages; in pharmaceuticals; and in industrial fields. Its uses depend on three properties: acidity, flavor, and salt formation. Chemically, citric acid is 2-hydroxy-l,2,3propane tricarboxylic acid (77-92-9). It has three pK, values at pH 3.1, 4.7, and 6.4.27As these three values are relatively close together, the second H+is appreciably dissociated before the first


is completed, and the third is similar. Due to this overlapping, the solution is well buffered throughout the titration curve, and there are no breaks from about pH 2 (the approximate pH of a 0.2 M solution) to pH 7. Citric acid forms a wide range of metallic salts, including complexes with copper, iron, manganese, magnesium, and calcium. These salts are the reason for its use as a sequestering agent in industrial processes and as an anticoagulent blood preservative. The salts also are the basis of its antioxidant properties in fats and oils in which it reduces metal-catalyzed oxidation by chelating traces of metals such as iron. There are two components to the use of citric acid as a flavoring: its acidity, which leaves little aftertaste, and its ability to enhance other flavors. A process to remove sulfur dioxide from flue gases has been developed2' in which citric acid is used as a scrubber, forming a complex ion (citrate2-HS0,2-) that then reacts with H2S to give elemental sulfur, regenerating citrate. This may become more important with increased environmental pressures. The applications of citric acid are summarized in Table 1 .

citric acid production processes, a number of yeasts have been discovered to accumulate ita~~Rhodotor~la~~ conic acid. Both C a ~ z d i d aand strains have been described and patented. The chemical route to itaconate production has also been examined, particularly the destructive distillation of citric acid. Although improvements have been made, the economic balance remains with the microbially produced material. Other chemical syntheses have been proposed, but none has achieved commercial success. Itaconic acid (mol wt 131) is soluble in water, from which it can be easily crystallized. The melting point is indeterminate, the acid decomposes above 162", and the quoted range is from 162" to 168". It is slightly soluble in organic solvents and is not particularly toxic, despite its reactive methylene group.45The diesters of itaconic acid are readily prepared; the dimethyl and dibutyl esters are used as copolymers and are produced in commercial quantities. The uses of itaconic acid have been reviewed by B i l l i n g t ~ n ~ ~ and L u ~ k i n . ~ ~ The ability of the methylene group to take part in polymerization reactions, either as homopolymers or, more usefully, as a heteropolymers, is the key property that makes itaconic 2. ltaconic Acid acid a commercially useful product. The resulting polymers have many free carboxyl groups with Itaconic acid (methylene succinic acid 97the acid, or ester groups when the diesters are 65-4) was first isolated as a product of the deused. This confers hydrophilic properties on the structive distillation of citric acid.29It was idenpolymers and also improves the binding of many tified as a microbial product by K i n ~ s h i t a , ~ ~ , ~dyes. ' S tyrene-butadiene-itaconic acid polymers are used in carpet backing and paper coatings. who isolated it from the growth medium of the A second significant reaction of itaconic acid osmophilic A . itmanicus. Patents on the industrial use of this strain were obtained in 196132by is the formation of N-substituted pyrrolidinones using untreated molasses as growth medium, and with amines. Such pyrrolidinones are used in defurther studies by Kobayashi and T a b u ~ h i ~ ~ -tergents, ~~ shampoos, and other products in which extended the range of media. their surface activity is exploited.48 The preferred industrial process, however, uses the closely related A . terreus, some strains of which were first identified as producers of 3. Gluconic Acid itaconate by Calam et al.36in 1939. Further strain Gluconic acid is derived from D-glucose and development was carried out at the Northern Rehas the same stereochemical configuration. It is gion Research Laboratory of the U.S. Departproduced by an oxidative reaction from glucose ment of Agriculture, first isolating NRRL 26537 to give pentahydroxycaproic acid (526-95-4), and NRRL 1960,3842which have formed the bawhich has a melting point of 13 1 and a specific sis of still further mutation and strain selection rotation of *Od = - 6.7" (H20).49Gluconic acid work. is readily soluble in water and is usually supplied in a manner parallel to the development of O

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TABLE 1 Applications of Citric Acid Pronertv utilized Market share


Food Industry Beverages Jellies Jam and other preserves Fats and oils

Acidulent;

flavoring

Flavoring;

acidulent

Antioxidant

(metal complex)

Frozen foods

Antioxidant

About 75%

Pharmaceutical Effervescent products Vitamin preparations Anticoagulents Iron preparations Cosmetics

Acid;

flavor

Antioxidant About 10% Sequestering: Salt formation Buffering;

buffering antioxidant

Industrial Cleaning (metals) Detergents Photographic reagents Primer Binder Polymerizations

Sequestering Buffering; Buffering

About 15% Sequestering Sequestering

as a 50% solution. In solution it is in equilibrium with D-glucono-delta-lactone (90-80-2) and Dglucono-gamma-lactone ( 1 198-69-2), and it dissociates into a gluconate ion and a proton to give a complex system. The apparent dissociation constant for gluconic acid should be corrected for lactone formation. Calculations suggest a pK, of 3.749or 3.62,50making it somewhat weaker than citric acid. The lactones and gluconic acid can be purified from aqueous solutions by crystallization at defined temperatures, seeding a solution where one product predominates with the appropriate crystals. Below 30" gluconic acid can be isolated; between 36 and 5705' the b-lactone is produced and above 70°, the y-la~tone.~' Because the various equilibria change slowly, up to 12 h may be required to obtain the y-lactone. Apart

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Sequestering

from a 50% solution, the main commercial product is the b-lactone. Gluconic acid is nontoxic and is approved for food use. The &lactone is used in baking powders for instant products such as cake and bread mixes. The other main use of gluconic acid is in the form of its salts for pharmaceutical and medical products. Many salts have been described, and this area has been reviewed by Sawyer.53 The calcium and iron salts are used medically to treat deficiency states, such as various anemias and the brittle bone syndrome. The advantage of gluconate salts, in addition to their lack of toxicity, is their stability, particularly to sterilization, and their solubility, resulting in a high concentration of the desired ion. These properties


are exploited in other pharmacological preparations such as chlorhexidine gluconate, a lowirritancy phenolic antiseptic. Industrial uses for gluconic acid are largely based on its chelating properties, particularly for calcium and iron. In dishwashing powders it is used to prevent the formation of calcium and magnesium hydroxides in hard water districts. Such washing powders are strongly alkaline; commercial glass-washing preparations may contain 5% NaOH. The resulting scum of mixed hydroxides is not easily rinsed away, but the ability of gluconates to sequester these metals prevents the p r ~ b l e m .The ~ ~ .ability ~ ~ of gluconates to form chelates in the presence of NaOH is attributed to the ionization of hydroxyl groups to form chelate rings.52 4. Lactic Acid Lactic acid (2-hydroxypropanoic acid) is one of the oldest known acids56and is widely distributed in nature. From its formula it can be seen to contain an asymmetric carbon atom and as a result exists in three forms. The racemic version (589-82-3) is normally the final product obtained by carbohydrate fermentation with various Lactobacilli, although some strains of Lactobacillus caseis' and Rhizopus oryzue6 produce the L( +) isomer (79-33-4) (S-).The D( -) isomer (1032641-7) (R - ) can also be produced by a fermentation route.58The use of racemic or D- forms of lactic acid limits the amount that can be consumed, as human lactate dehydrogenase only can metabolize L-( + ) lactate. The accumulation of the D - isomer can result in renal acidosis. The World Health Organization (WHO) limits consumption of the D-isomer to 100 mg/kg/d as a result. Lactic acid has been made by fermentation on a commercial scale since 1881, although a competing chemical synthesis now supplies a significant fraction of the market .59,60 Many applications use lactic acid solutions; technical grades (50 to 88%) are purified mainly by filtration and decolorization; higher grades can be produced by solvent extraction, vacuum distillation, or esterification.6' The degree of purification determines

the price and the use to which it is put; technical grade is the least pure, followed in order of increasing purity by edible, plastics, and pharmaceutical lactic acid. The price difference is about 1:4 overall. Lactic acid is used largely in the food industry, although some is used in the manufacture of plastics. Calcium stearyl 2-lactylate is a bread additive, and calcium lactate is an ingredient in baking powders. It is also used as an acidulent in many foods and beverages because of its pleasant acidic flavor and its preserving qualities. Cheese production, seasoning, and fruit and fish canning are further examples of the uses of lactic acid in the food industry. In an industrial context it is used as a mordant for textile printing and dying. In tanning it is employed for dehairing and tanning hides. L( + ) actic acid is used in plastics. It can be polymerized to a polylactide that is used in the manufacture of plastic foils. Production of lactic acid is estimated to be 35,000 to 40,000 tons per year, of which about one third is synthetically produced.

5. Malic Acid L-( - )Malic acid (hydroxybutanedioic acid 6915-15-7 is produced by microbiological means using L . brevis with a yield of 100% (on weight basis) from glucose, according to Yarnada.@' This yield does not seem to have been achieved by synthesis from glucose or an equivalent carbon source under industrial conditions, but may be achieved by biotransformation of fumaric acid (transcystallization). Malic acid has a melting point of 100". It decomposes at 140", and a D = - 2.3" in water. Its main use is as an acidulent in food and beverages, where it competes with citric acid. Organoleptic assessments suggest that it might be preferable to citric acid in its aftertaste, but this is not sufficiently pronounced to offset the cost difference between the two. Most malic acid is chemically synthesized as a racemic mixture. As well as a microbiological process based on Lactobacillus, a method employing Yarrowiu (Cundida) yeasts has been described.62

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6. Tartaric Acid Tartaric acid (2,3-dihydroxybutanedioicacid) having has four optical isomers: D- (147-71-7), the configuration S-[R* ,I?*]; DL- ( 1 33-37-9) [R*,R*]; L- (87-69-34)R-[R*,R*]; and meso(147-73-9) R* ,S*. The naturally occurring D-( ) isomer has an optical rotation of 15" (20% in water) and a melting point of 150".It is widely distributed in nature, particularly in fruit. The main source is the residue from wine manufacture, which contains large quantities of potassium tartarate. Like many of the organic acids, its primary use is in the food industry. The estimated market size is about 50,000 tons per year.


+

7. 2-Oxogluconic Acid This acid is used as the raw material for the production of D-araboascorbic acid6' (erythorbic acid), one of the antioxidants used in the food industry.

II. BIOCHEMISTRY A. Group 1 Acids The efforts to understand the mechanisms underlying the accumulation of large quantities of organic acids by microorganisms date back almost as far as the observations of the acids themselves. Citric acid has been by far the most extensively studied of the acids; little biochemical or physiological work has been done on the others. The majority of the studies on citric acid have been carried out on Aspergillus niger. The acids themselves are related metabolically, and there are also similarities in the organisms used in the processes. The biochemistry and physiology of citric acid production in both A. niger and Cundidu have been extensively reviewed recently by Kubicek and ROhr.64The salient points are reviewed here to provide a framework to understand the possible mechanisms in other, less well studied, systems. Three experimental approaches have been employed: first, the manipulation of culture conditions; second, the study of metabolic se-

92

quences; and third, enzyme studies. Much of the early work has been reviewed repeatedly, for example, Foster (1949y Prescott and Dunn (1959),66 and Johnson (1954).67 The accumulation of high concentration of acids by microorganisms is not a normal phenomenon, although, for example, A . niger strains all excrete some acids, even if only to a small extent. High yields are brought about by manipulation of the growth medium and growth conditions of the organism, and by some elements of strain selection and mutation. Because the industrial process depends on the enhancement of a naturally occurring mechanism, many of the studies have concentrated on the nutritional requirements and the effects of various constraints on enzyme synthesis. Some radioactive tracer studies have been carried out and some studies of mutants, but a significant, and often noted,w omission is the lack of any significant genetic work. Particularly since the advent of genetic engineering techniques, this is a puzzling situation. It may be that this lack is only apparent, as much industrial research in this area is, of necessity, secret. There is little patent protection to be obtained on such well-established techniques; therefore, confidentiality is important. Academic studies on the genetics of filamentous fungi have tended to be concentrated on A . n i d u l ~ n s , ~ ~ . ~ ~ which is not in the same group as A . niger. A second modern methodology that has not yet been applied to this area is the metabolic control theory. 7 ~ 7 4 Many of the reports that have been published over the years appear contradictory, or are presented as such. There are a few obvious reasons for this. First, it is clear that different strains of the same organism may adopt different strategies to achieve the same end. For example, A. niger strain B6075has a phosphofructokinase that shows citrate inhibition, like the analogous enzyme in many other organisms. However, strain 83276does not exhibit citrate inhibition of phosphofructokinase and also maintains a low cytoplasmic citrate concentration, even during citric acid accumulation. Second, the yields reported in many studies are so low that the organism is not being studied under the condition of the industrial process. An example is the study of Wold and Suzuki7' in


which yields were 4 to 8 mM after 72 h from a sucrose concentration of 8 g/l , only a fraction of the 600 mM (final concentration) obtained in practice. Under such conditions, side reactions (as far as acid accumulation is concerned) can distort the interpretation. Third, the reasons for the initiation acid accumulation and the physiological requirements are not yet known in detail. It is therefore difficult to be sure a given observation is central to acid accumulation or whether it is secondary or even coincidental. The first systematic study on the influence of the growth medium on citric acid production was carried out by Shu and J o h n ~ o n .They ~~,~~ used the Wisconsin 72-4 strain of A. niger and examined the effects of macro- and micronutrients. It is clear that in order to accumulate, citric acid growth must be restricted, but it is not clear whether phosphate or nitrogen is the necessary factor for limitation. Kubicek and RohPO showed that citric acid accumulation occurred in their strain whenever phosphate was limiting, even when nitrogen was not, which is in agreement Kriswith the earlier report of S z u c ~ .However, ~l tiansen and Sinclair,82using a continuous culture system, found a sharp maximum of citric acid production at 0.8 kg mP3ammonium nitrate and concluded that nitrogen limitation was essential for citric acid production. In an industrial situation, where molasses is used as a carbon source, quantities of nitrogenous materials such as betaine are not utilized, so that phosphate limitation is more likely than total nitrogen limitation. Studies reported by Mattey and LOW^^^ indicate that exhaustion of ammonium ions is more significant than total nitrogen depletion for citric acid accumulation, but the possibility is that either phosphate or nitrogen may be effective, depending on the strain and conditions. Normally in defined media, nitrogen is supplied as ammonium sulfate or nitrate. The advantage of ammonium salts is that they decrease the pH as they are consumed, which is also a requirement for citric acid accumulation. Ammonium ions are rapidly removed from the medium with the excretion of a stoichiometric amount of protons, with a resultant lowering of the pH. This occurs within 24 h of spore germination in a suitable medium.83

Phosphate does not have to be limiting, as can be seen from the data of Shu and Johns~n,’~ but when trace metal levels are not limiting, additional phosphate results in side reactions and increased g r ~ w t h .The ~ ~ reason , ~ ~ for the effect of phosphate on growth is metabolic rather than genetic86and may be related to the role of phosphate in the regulation of a number of enzyme systems or even in the overall energetic^.^' The pH of the medium is vital to a good yield of citric acid. The pH should fall below 2 within a few hours of the initiation of the process; otherwise, yields are reduced. There are three major reasons for this: first, the competition for substrate by glucose oxidase; second, the accumulation of citric acid in the medium; and third, of lesser importance under asceptic conditions, a reduction in the likelihood of contamination. Glucose oxidase in induced in or on A. niger mycelium (it is a periplasmic enzyme; see the section on gluconic acid) when the pH is above 4, but the enzyme is inactivated below pH 2.The production rates for citric and gluconic acids are typically 0.7 and 20 g/l/h, so that the role of pH in inactivating glucose oxidase is important to the final yield. The pH of the medium will affect the ionization of citric acid. At pH values around 2 most of the citrate will be present as &rateo or citrate’- , whereas at the internal pH of about 7 the citrate will be mainly in the form of citrate2-. It has been suggested that only citrate2ions can be transported out of the mycelium.88 The carbon source for the citric acid process has been the focus of much study, frequently with a view to the utilization of polysaccharide sources. A. niger can take up simple sugars such as glucose and fructose rapidly, although the usual industrial source is sucrose, which does not seem to be taken up as such by the fungus. A. niger has a potent extracellular invertase bound to the mycelium,89and this rapidly hydrolyzes sucrose; a further degree of hydrolysis of the sucrose results from the preparatory sterilizationprocedures. Beet and cane molasses serve as the industrial sucrose source. They are variable products with seasonal and production differencesg0and a complex composition. Cane molasses does not give good yields in surface processes but is used in the submerged process. All molasses and other

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gested as being required in limiting amounts for crude carbon sources may need pretreatment to a successful process.111These include Fe2+,Cu2+, lower heavy metal levels (see later). Using a purZn2+, Mn2+, and Mg2+ Polysaccharides do not give good yields of ified glucose medium with the Wisconsin 74-4 citric acid without pretreatment. Their rate of strain, Shu and Johnson found optimal levels of hydrolysis is too slow to allow the high levels of 0.3 ppm Zn and 1.3 ppm Fe. Any addition of sugars to accumulate that are required so that Mn resulted in reduced yields. Indeed, Mn levels citric acid is produced rather than biomass. Many in spores were sufficient to reduce yields.79Other sources of complex carbohydrates have been investigations confirmed the general findings and tested, including various w a ~ t e s ~and l - ~unusual ~ the importance of Mn levels to the A . niger prosources such as date syrup,96 and sweet c ~ s s . "One ~ problem with all studies on trace potatos.'* In addition to the problem of metal metals is that of purification. For example, most contamination previously mentioned, these sources are reported to contain both inhibitoryw.'OO studies on Fe levels have used high-purity Fe salts, but even these contain, among other trace and stimulatory101.102 substances. impurities, about 0.005% Mn, enough to lead to As well as the nature of the sugars, their Mn inhibition of citric acid accumulation when concentation influences the rate of production and levels of added Fe are about 0.1%. the yield. The influence of sugar concentration The effect of Cu in reducing the effects of was demonstrated in 1948 by Shu and Johnson,'* Fe could equally well be due to its antagonistic who studied the effect of the initial concentration effect on the uptake of Mn ions.114The imporof sugar on the final citric acid yield and found tance of reducing Mn and possibly other trace a maximum at about 15% sugar, beyond which metal levels has led to a number of treatments, the yield per gram sugar fell, but not the overall among them the addition of ferrocyanide ions1I5 amount of citric acid, although this fell above to the molasses as a pretreatment, or to the me25% sugar. studied the effect of changdium itself.lI6 Clark et a1.113 found that as little ing the sucrose concentration in a disc fermenterlM as 1 ppb of Mn reduced the yield of citric acid during citric acid production (Figure 1) at 4 d by 10%. A second effect of ferrocyanide seems growth, when acid production was maximal. A to be as a direct growth inhibitor that leads to concentration of more than 6%sucrose was necincreased citric acid yields by limiting the amount essary to maintain citric acid production. A. niger requires certain essential trace metof carbon diverted into biomass.1'7.118On the it seems that the limitation of als for g r ~ ~ t h .It ~is widely ~ - ~ observed ~ ~ ~ ~ available ~ - ~ evidence ~ ~ Mn is of primary importance; the levels of Fe that trace metal and other metabolic requirements and Zn are probably related to the diversion of show a two-stage growth response, with increascarbon between biomass and citric acid, while ing growth at low levels and inhibition of growth above a certain optimum level. Although this is Cu (and ferrocyanide) also acts on the growth mechanisms rather than those leading to citric true for A . niger growth, the production of citric acid. It should be noted that Kubicek et al." have acid requires a degree of growth limitation. The demonstrated that the role of Mn is also indirect role and identification of the trace metal requirein the sense that protein synthesis is required ment of A . niger illustrate an important concept before Mn exerts its effect. in understanding the biochemistry and physiolAlthough the A. niger citric acid process is ogy of such a process. Many factors will affect clearly aerobic, the importance of oxygen has the final yield, but some are related to the primary been emphasized by several studies. 119--122An inmetabolic ''lesion" that enables the accumulacrease in the oxygen concentration results in an tion of citric acid, and the majority relate to secincrease in citric acid yields, and an interruption ondary strategies that optimize the yield. It is in the aeration has a damaging effect,'22 so that often difficult to identify which is which, but if a transient interruption will redirect metabolism some strains do not require a certain factor then toward biomass production. This effect can be it is part of the secondary strategy, not a primary ameliorated by raising the pH to about 4 until one. aeration recommences. I Z 2 A number of divalent metals have been sug-


. 7 8 , 7 y 7 1 1 ' * 1 1 2

94

0.5


0 -4

0.3

0.2

0.1

rn V

A 0

,

I

3

I

4

I

I

5

I

6

Days

FIGURE 1. Effects of sucrose concentrations on rates of citric acid production in disc fermenter with change of sucrose from 14%. 2% sucrose; V-V 6% sucrose; F V 10% sucrose; 0-0 14% sucrose; 0-• 18% sucrose; H20% sucrose. (From Divers, SM., Ph.D. thesis, University of Strathclyde, Glasgow, 1978. With permission.)

+-+

The metabolic pathways leading to citric acid have been the subject of much discussion. Several reaction sequences were proposed before the central role of the tricarboxylic acid cycle was elucidated by Krebs et al. 123-126These hypotheses have been reviewed by Prescott and Dunn and ~ t h e r s ~ but . ' ~ are ' not now considered likely. The basic metabolic sequences related to citrate metabolism are well established, and the principle relationships are shown in Figure 2. Two radioactive tracer studies have yielded information on the pathway. The first such study concluded that 40% of the citric acid was produced from the reaction of a pair of two-carbon compounds and 60% from the reaction of a one-carbon fragment with a three-carbon compound.128Shu et al.129 concluded that about 40%of the citric acid resulted from recycled four-carbon acids. This

would mean that the sort of yield commonly obtained industrially of greater than 80% would be impossible. Other studies showed little such recycling;IZ8-l3O however, in these experiments the yield was higher than that achieved by Shu et al., lZ9 which may explain the difference. Cleland and used 3,4-14C glucose as a pulse label in a system, giving a good yield of citric acid. Quantitative recovery of the label was obtained from carbon 4 of the citric acid, whereas carbon 6 had about 15% of the original label. No other carbons were significantly labeled. This led to the conclusion that there was no recycling of the citric acid and that it was formed by a series of reactions involving two three-carbon units, one of which was decarboxylated, the other of which was carboxylated, the resulting two-carbon and four-carbon units reacting to produce citric acid.

95

Sucrose

n-Alkane


I1

/ \

F r 11 c t 0 s e

Alkan-1-01

Glucose

Alkan-1-oic acid

Glucose-6-P cc k'rurtose-6--P

I 12

co2

a - h x o q 1 u t a r a te

Malate

FIGURE 2. The Metabolic Relationships of citrate metabolism from carbohydrates or n-alkanes. (1) Alkane monooxygenase; alkane, reduced rubridoxin:oxygen 1-oxidoreductase. 1.14.15.3; (2) Alcohol dehydrogenase; alcohol: NAD+ oxidoreductase. 1.1.1.I ; (3) Aldehyde dehydrogenase; aldehyde: NAD' oxidoreductase. 1.2.1.3; (4) Hexokinase; ATP:D-hexose 6-phosphotransferase. 2.7.1.1; (5) Fructokinase; ATP:Dfructose-6-phosphotransferase. 2.7.1.4; (6) @-oxidation;(7) Embden-Myerhof-Parnas pathway; (8) Pyruvate carboxylase; pyruvate:carbon dioxide ligase(ADP). 6.4.1.1; (9) Pyruvate dehydrogenase; pyruvate:lipoate oxidoreductase (acceptor-acylating) 1.2.4.1; (10) Citrate synthase; citrate oxaloacetate-lyase (CoA-acylating). 4.1.3.7; (11 ) Aconitase; citrate(is0citrate)hydro-lyase. 4.2.1.3; (12) lsocitrate dehydrogenase; threo-Dsisocitrate:NAD+ oxidoreductase (decarboxylating). 1.1.1.41; isocitrate dehydrogenase;threo-Ds-isocitrate:NADP+oxidoreductase (decarboxylating). 1.1.1 -42;(13) Isocitrate lyase; threo-Ds-isocitrate glyoxylate-lyase. 4.1.3.1; (14) Malate synthase; 1 Malate glyoxylate-lyase(CoA-acetylating). 4.1.3.2; (15) Oxaloacetate hydrolase; oxaloacetate acetylhydrolase. 3.7.1 . l .

-

96


Some evidence to support this comes from the mass balance studies of Verhoff and Spradlin,I3' whose analysis suggested two schemes, one identical to Cleland and Johnson's; the other, which they favored, involving oxalate production from oxaloacetate by a hydrolase; reduction of the oxalate to glyoxylate; condensation of the glyoxylate with succinate by isocitrate lyase, operating in the reverse of the direction associated with the glyoxylate cycle; and then metabolism of the isocitrate through the tricarboxylic acid cycle. In support of this scheme, they quote the work of Joshi and Ramakri~hnan'~~ to suggest that oxaloacetic hydrolase is present in A. niger. In fact, that study showed that the oxaloacetic hydrolase was not found when citric acid was accumulating, a result supported by other studies.133This enzyme may behave like glucose oxidase, responding to the external pH, although the mechanism for such a response is unknown. The enzymes of the tricarboxylic acid cycle have been assayed in several studies, and some of them have been purified. The presence of tricarboxylic acid cycle enzymes has been d e m o n ~ t r a t e d ~in~ A. ~ - 'niger ~ under a variety of conditions. Clearly some aspect of the tricarboxylic acid cycle must be operating at a rate less than the carbon flux entering the cycle for citric acid to accumulate. Two enzymes - aconitase and isocitrate dehydrogenase -have formed the focus of many older studies. The observation of Ramakrishnan and Martin'34suggested that aconitase activity and isocitrate dehydrogenase activity disappeared during citric acid accumulation. They also indicated that isocitrate dehydrogenase was inhibited by citric acid and by ferrocyanide, an observation that was ignored at the time. Other worker^'^^,'^^ have shown the presence of these enzymes under citric acid-accumulating conditions, and isocitrate dehydrogenase has been purified. The apparent discrepancy between these observations and others described later is probably due to the chelating ability of citric acid and ferrocyanide. It is difficult to remove citric acid from mycelium that is actively producing it. Although the presence of aconitase has been established, the inhibition of aconitase by low levels of Fe or by Cu additions has been suggested as the reason for citrate accumulation. 138,139,147 The equilibrium catalyzed by acon-

itase is in favor of itr rate,'^^,'^^ some 95% being citric acid. The actual metabolite concentrations measured in m i t o c h ~ n d r i a ' ~show ~ * ~that ~ ~ the *~ enzyme does bring about an equilibrium between citrate and isocitrate in the mycelium, so that the inhibition by low Fe concentrations is not important in vivo. The situation with respect to isocitrate dehydrogenase, although complex, is now clear. Two pathways for the breakdown of isocitrate are possible, either through isocitrate dehydrogenase (tricarboxylic acid cycle) or by isocitrate lyase (glyoxylate cycle). Although isocitrate lyase is present under some conditions, it is absent when citric acid is accumulating.80Isocitrate dehydrogenase activity in A. niger is brought about either by a NAD- or the NADP-catalyzed enzyme. Both have been isolated and purified. 140~141 The major mitochondrial enzyme seems to be the NADP-dependent e n z ~ m e , ~which ~,'~ has ~ an apThe suggesparent K , of 0.15 mM for tion was made that this "feed forward" inhibition might be responsible for the accumulation of citric acid. Although that appears to be true, this mechanism obviously cannot be responsible for the initiation of citric acid accumulation, as that would imply that all cultures of A. niger would accumulate high levels of citric acid. Further, the levels of citrate in the early stages of growth are too low to achieve i n h i b i t i ~ n . ~The ~ ' , kinetics ~ noted by Bowes and Mattey151.152 in mitochondrial extracts were complex, indicating that it was not a single isocitrate dehydrogenaseenzyme that was present. The situation in the strain used by Kubicek (B60) is different in that only a single enzyme was found, with a K,of 3 to 4 mM, and in that the identical enzyme was found in both the mitochondrial and the cytoplasmic compartments," which is a most unusual situation. Radioactive trace studies indicate clearly that during the period when citric acid is accumulating in the medium, the restriction in the tricarboxylic acid cycle is at isocitrate dehydrogenase.149The exchange of radiolabeled CO, with citric acid cycle intermediates was measured in vivo conditions, and no exchange at the position removed during the isocitrate dehydrogenase step was observed. Other labeling patterns were as predicted for isocitrate dehydrogenase restriction. Aconitase was active and brought about the expected

97


equilibrium in vivo. The ability of COz to enter the mitochondrial compartment is shown by the presence of pyruvate carboxylase in the mito~ h o n d r i o n , " ~although .'~~ it appears to be present in the cytoplasmic compartment as well. The probable explanation for this restriction is that the substrate for isocitrate dehydrogenase is either Mg or Mn isocitrate, and that citrate, by virtue of its ability to chelate these metals, competes with isocitrate and reduces the effective concentration of the metal isocitrate to levels at which the rate of the isocitrate dehydrogenase reaction is reduced. This effect would be enhanced by ATP competition for the Mgz+. Calculations of the expected levels of intermediate^'^^ support this suggestion. Such chelation would affect both NAD and NADP enzymes. It is worth noting that the cytoplasmic levels of citrate are much lower than the mitochondrial levels,76so that isocitrate leaving the mitochondrial compartment may be decarboxylated by the cytoplasmic isocitrate dehydrogenase, allowing some recycling of citrate. The chelation of divalent metals by citrate also explains other observations, including that of Bowes and Mattey,15'j in which strains of A. niger showed citrate inhibition of isocitrate dehydrogenase in proportion to their citric acid yields. An unresolved question is the mechanism by which citrate accumulation is initiated. Two hypotheses have been put forward: one evokes the regulation of a-ketoglutarate dehydrogenase a ~ t i v i t y ;the ' ~ ~other suggests a role for the early production of polyols. 157 a-Ketoglutarate dehydrogenase has been purified from A. niger.lM It was identified as a regulatory point by enzyme activity measurem e n t ~ , ' ~and * the suggestion was made that this enzyme was repressed during citric acid product i ~ n . The ' ~ ~purified enzyme is inhibited by oxaloacetate and NADH at levels consistent with the measured concentrations. It was suggested that the breakdown of glucose during the initial stages of the process leads to an increase in the oxaloacetate concentration, which in turn leads to inhibition of a-ketoglutarate dehydrogenase and the stimulation of citrate synthetase, which has been shown to have little allosteric ~egulation'~' and is mainly responsive to changes in oxaloacetate levels. The level of oxaloacetate

98

will depend on the activity of the carbohydrate breakdown pathways. The enzymes of the glycolytic pathway have long been known to be present in A. niger,'59v'M) and the pentose phosphate pathway has also been shown to be present and functional, at least in Except the early stages of the process. 161~162~157 for the early stages of the process, the glycolytic sequence is the major pathway,L63having more than 80%of the carbon flux. With the glycolytic pathway, no net CO, production should occur; the release of C 0 2at the pyruvate dehydrogenase step is balanced by the anaplerotic reaction at pyruvate carboxylase, leading to oxaloacetate . Carbon balances for citric acid production indicate that this is so. This is not the situation during the first few hours after spore germination, however. The consumption in the early stages of more hexose than could be accounted for as biomass or citric acid, with the converse being true in the latter stages, has been reported by several studies.78,89,'aThis was shown to be due to the formation of polyols, principally glycerol and erythritol in the first instance,'65 but with arabitol and mannitol as minor components. Phosphofructokinase in many organisms is subject to citrate regulation, whereby citrate potentiates ATP i n h i b i t i ~ n . ' ~The ~ . ' enzyme ~~ in A. niger is similar in some ~ t u d i e sThe . ~ ~enzyme ~~~ in this case was released from inhibition by ammonium ions, which are elevated in citric acidfound that accumulating conditions. '" the enzyme from a low-yielding strain (627) was also inhibited strongly by 5.5 mM citrate, but the effect of ammonium ions was not apparent in vivo. A high-yielding strain (832) showed slight activation of phosphofructokinase under the same conditions. It is worth noting that the cytoplasmic levels of citrate are low, however (Table 2), so that the significance of these effects is not clear. The situation is complicated by the inability to measure the cytoplasmic concentration of ATP, or indeed the cytoplasmic concentrations of ammonium ions. Aldolase has been shown to require Zn for activity,"j9 which suggests that Zn deficiency, rather than causing citric acid accumulation, ' 0 9 would limit the flux through glycolysis. Another possible regulatory enzyme, by analogy with other organisms, is pyruvate carboxylase.

TABLE 2 Distribution of Citric Acid in A. niger Mycelium


Strain

832 51.433 50.565 31.824 627 27.809

Mycelium MitoCytoplasmic medium Yield concn. chondrial (mM) (mM) (mM) PH (W)

4.66 3.4 3.0 2.4 1.6 0.8

21 .o 15.0 13.0 10.8 6.8 2.8

0.58 0.44 0.42 0.35 0.33 0.3

1.9 2.2 2.4 2.1 2.1 2.2

74.0 58.6 44.0 30.4 5.6 2.9

Data compiled from B o w ~ sand ' ~ unpublished data.

This enzyme has been studied by several workers. It has been i ~ o l a t e d ' ~ ~and * ' ~shown ' to be inhibited by aspartate, but, unlike the analogous enzyme in the closely related A . n i d u l ~ n s , 'it~ ~ did not appear to be regulated by acetyl CoA or a-ketoglutarate. However, its activity did seem to depend on some function of the glucose or sucrose concentration in the medium. 173 The metabolism of the fungus following spore germination has been studied with respect to glycerol production. A characteristic series of morphological changes O C C U ~ S " ~in which after germination, enlarged, bulbous cells form, from which filamentous hyphae emerge. At the same time as the morphological changes, a change in the main flux of carbon from the pentose phosphate pathway to the glycolytic pathways occurs. The highest concentration of glycerol was found about the same time as the change in morphology. Glycerol, which may function as an o~moregulator,'~~ is formed in the cytoplasmic compartment by a NADP-dependent glycerol dehydrogenase. 177,178 It is not retained within the mycelium but diffuses into the medium and into the mitochondrial compartment, where other glycerol dehydrogenase enzymes are found. 179 It reaches a concentration of 175 mM in the medium,Is0 which is more than enough to inhibit the NADP-dependent isocitrate dehydrogenase (K, 8 The levels of glycerol produced are dependent on the sucrose concentration initially present.'" Above 5% glucose gives a glycerol concentration that would potentially inhibit isocitrate dehydrogenase. This is the same order

as that of the sucrose concentrations that were observed by to support the continuation of citric acid production. Both the regulation of a-ketogluterate dehydrogenase and the glycerol inhibition mechaisms are plausible and are supported by the evidence available at present. It is not possible to decide which is the more important. Indeed, it may be that both are necessary, for the presence of glycerol in the early stages provides an additional source of carbon that is not restricted in its entry by a transport system. In this respect the observation of InayaP9 that the glycerol dehydrogenase present in the first 24 h after spore germination has a rate constant that strongly favors the synthesis of glycerol from dihydroxyacetone, but that this is replaced by an enzyme that favors glycerol degradation at the same time the pentose phosphate/glycolysis switch occ u r ~ ,may ' ~ ~be significant. Once citrate accumulation has been initiated and the mitochondria1 levels have been elevated, the citrate has to be exported. The transport mechanisms involved in this and in sugar uptake are not understood in A. niger. The kinetics of glucose transport have been measured , I 8 ' and some hints of the influence of pH on citrate exodus have been obtained.88 The existence of an ammonium uptake system has been shown, and the excretion of protons in stoichiometric amounts has been d e m o n ~ t r a t e dbut , ~ ~the existence of the equivalent of the mitochondrial tricarboxylic acid carrier present, for example in yeasts18*has been only assumed; no citrate carriers have been iso-

99


lated from the cell membrane. It is not known whether the efflux of citrate from the cytoplasmic compartment is an active process or the result of the diffusion of one of the ionized forms of citrate down a concentration gradient, maintained as a result of the pH difference between the cytoplasm (about pH 7) and the external environment (below pH 2). The internal pH has been suggested as an important factor, in that many enzymes are sensitive to changes in the pH 6 to 7 range. The hypothesis advanced by Punekar et al. I E 3 suggests that the Mn effect is mediated by glutamine synthetase, with the change in internal pH acting as a switch between the Mn2+ and Mg2+ forms. Legisa and KidricIE0have made the suggestion that NADP-specific isocitrate dehydrogenase may also be affected. Using fluorescent probe^"^ and P71-NMR,179 changes in average cellular pH from pH 7.1 to pH 6.5 have been observed. These pH changes are significant if they represent changes in the cytoplasmic compartment, but the distribution of pH between the cytoplasm and, for example, the large vacuoles found in the mycelium is not known. The effects of Mn have been catalogued, particulary by Kubicek and co-workers. Effects on amino acids and ammonium,16nproteinase activity,IE4phospholipid and fatty acid and cell walls187have been noted. It appears that the Mn effect cannot at present be simply related to any particular enzyme activity.64The effect on cell walls and the resulting morphological changes are consequences of the Mn2+deficiency but are not related to citric acid production. IE7 Mutants that retain their filamentous form but produce citric acid are known’8Eand are used in batch and continuous processes. The effect of Mn2+ deficiency on the cell membrane may influence transport activitie~,”~ but no clear evidence has so far been produced. Indeed, some evidence to the contrary exists as far as the glucose transport system is concerned.IE6In this system no significant changes in the kinetics of glucose transport were found when Mn deficiency was imposed. The other clear effect of Mn deficiency is on ammonium levels,168although the ammoniuml proton exchange reaction is not affected by Mn deficiency. IE9 The elevated ammonium levels could be related to increased protein breakdown,

100

and one effect of the elevated levels could be to relieve citrate inhibition of phosphofructokinase activity.’” One problem with high internal ammonium levels is the futile cycle that will occur when ammonia, which is in equilibrium with ammonium ions, diffuses across the cell membrane down the concentration gradient that will exist between the nearly neutral pH of the cytoplasm and the pH around 2 in the medium (Figure 3). The biochemistry of citric acid production by yeasts will depend on the carbon source -either n-alkanes or glucose. 1 9 1 There are some general differences from the A. niger process: first, the lack of effect of Mn deficiency; second, the higher pH required (around pH 5 instead of pH 2); third, the requirement for nitrogen limitation to initiate citrate synthesis; and fourth, a requirement for thiamin. The requirement for a higher pH is related to the production of polyols, mainly erythritol and mannitol, instead of citric acid192at pH Values below 5.5. Citric acid at this pH inhibits its further accumulation, which may reflect on the transport process for citrate. If thiamin is not added, oxoacids accumulate, presumably due to limitations in the oxidative decarboxylation steps in n-alkane degradation. Like A. niger, the yeasts respond to phosphate l i m i t a t i ~ n , ’but ~ ~ the main stimulus for citrate production is nitrogen limitation. 194~195 The exhaustion of nitrogen is thought to alter the adenine nucleotide balance so that a decrease in AMP results. This is a known allosteric regulator of the mitochondria1 NAD-dependent isocitrate dehydrogenaseIY6and has been shown in Cundidu lipolyticu as well. 197-199 This has some parallels with the situation in A . niger, in which the NADPdependent isocitrate dehydrogenase shows inhibition by citrate, probably due to chelation.2m The accumulation of citrate and isocitrate will depend on the activity of aconitaseZo1and the activity of the tricarboxylic acid carrier, which has a high affiity for citrate in similar yeasts,202.203 a K , of 2.2 mM. The metabolism of n-alkanes by yeasts is known in ~ u t l i n e , ~although ~ * ~ * ~the complexity of the compartmentation involved means that the details remain to be elucidated. The uptake of nalkanes is dependent on their lack of water solubility, which makes diffusion through the lipid

(PH 7.0)

INSIDE L

NH4+ k

p

-

3

t

H+

I I I I

Glucose


I I

1

I I I I I I

R .COOH

It

I

I I I I I I I

R.COO-

t

H+

t

I ...I I ADP t Pi

ATP ,-PADP

7.m-

t

... ...

Pi

I

I

I

I I

I I

--

I

I I I I

1

I

t

m4Cl

NH3

NH4+

t

H+

H+

t

c1-

OUTSIDE FIGURE 3. Futile cycle for ammonia. monium uptake and proton excretion).

------------

(PH 2.0) Diffusion through membrane;

membrane easy but increases the difficulty of mass transfer between the alkane substrate, through the aqueous phase, to the cells. The analysis of the influence of process parameters such as aeration in such a system is also of increased difficulty. The optimum chain length for alkanes is 14 to 15.193The influence of aeration is not clear; high aeration rates have been reported to enhance citric acid p r ~ d u c t i o n ,but ' ~ ~the opposite has also been reported. l y 5 Although different Suc-

+active transport (am-

churornycopsis species were used in the two studies, it seems doubtful that such diametrically opposed effects can be explained away as species differences. Once taken up, n-alkanes are hydroxylated by a microsomal enzyme system, and the resulting alcohols are oxidized to aldehydes, then to fatty acids, in a process involving microsoma1 and peroxisomal enzymes. The co-enzyme A esters of the fatty acids are oxidized by a peroxisomal @-oxidativepathway to give acetyl CoA.

101


The further metabolism of acetate is via the glytion of mutants with a low aconitase activoxylate cycle, to give succinate and malate. One ity.2'5,216 These were selected by their ability to of these products is converted into oxalacetate, grow on n-alkanes but not citric acid. Limitation which is transferred to the mitochondrial comof the production of isocitrate from citrate will partment. Acetyl CoA is transferred via the carresult in increased yields of citric acid and denitine acyl transferase system to the mitochoncreased biomass; further limitations could result drion, and citrate is produced by citrate synthase. in insufficient isocitrate to provide the substrate The level of isocitrate lyase206-209is the main for isocitrate lyase and isocitrate dehydrogenase, factor in the level of citrate production, and its at which point the yields would drop, assuming importance is emphasized by the study of temthe organism was still viable. perature-dependent mutants.210 The biochemistry of itaconic acid synthesis The role of the glyoxylate cycle as an anais less well understood than that of citric acid, plerotic sequence is a major difference between with some contradictory findings reported. But the alkane process and the glucose process, rather on balance the most likely scheme is closely rethan a difference between A . niger and the yeasts. lated to the biosynthesis of citric acid by A. niger. Under conditions in which the substrate is a twoThree schemes have been proposed: (1) formacarbon compound, the yeast uses the glyoxylate tion of itaconate from ~is-aconitate,~'( 2 ) forcycle in the classic manner; from glucose, pymation from citramalic and (3) formation ruvate carboxylase provides the synthesis of fourThe metabolic from l,2,3-tricarboxypropane.**E carbon metabolites. relationships of itaconic acid and its metabolites An early problem with the production of citare shown in Figure 4.30*219 ric acid from n-alkanes was the presence of isoThe scheme suggested by Kinoshita30 procitrate in the product. The levels are greater than posed that in A . terreus cis-aconitate was decarthose anticipated from the aconitase equilibrium, boxylated to itaconate. Itaconate itself can be often being as high as 40% rather than the 7% metabolized by A. terreus,z20giving rise to itaor less expected.Iz6This has been explained2" as tartaric acid (2-hydroxy-2-hydroxymethylbutaaccumulation of isocitrate in the cytoplasm to nedioic acid) and citramalic acid (2-hydroxy-2high levels as a result of the absence of aconitase methylbutanedioic acid). The conversion of itafrom that compartment and high rates of transport conate to itatartarate has been ascribed to the from the mitochondrial compartment. This is difpresence of itaconate oxidase,221but this enzyme ficult to imagine when isocitrate is being used as was not found by Jakubowska and Metodiewa,220 a substrate for the glyoxylate cycle. It is perhaps who suggested on this basis that itatartarate was significant that isocitrate lyase-defective an intermediate in itaconate synthesis from citramutants"* show a higher isocitrate-to-citrate ratio malate. The citramalate would arise from pyruthan the parent strain, suggesting that regulation vata and acetyl CoA. Studies by Nowakowskaat the level of isocitrate lyase may be the cause on mitochondria, both inWaszczuk et al.222.223 of the isocitrate accumulations. The strategies tact and disrupted, isolated from A . terreus, found used to mitigate this effect, although apparently that they would not oxidize citric acid cycle invaried, probably have a common effect, that is, termediates. The researchers concluded from this the partial inhibition of aconitase to limit the forthat citric acid cycle enzymes were not present mation of isocitrate in the first place. These stratand could not be important in itaconate synthesis. egies are (1) the use of an Fe-free The scheme they supported218suggested the conwhich results in impairment of aconitase activity; version of pyruvate to acetyl phosphate and the (2) the addition of monofluoroacetic acid214to progressive condensation of three molecules of the grown medium, which must result in the forthis to give 1,2,3-tricarboxypropane,which was mation of monofluorocitrate, a classical inhibitor converted to aconitate, then decarboxylated to of aconitase, and possibly also a toxic final proditaconate. However, the failure to oxidize citric uct; ( 3 ) the addition of sodium t e t r a b ~ r a t e , ' ~ ~ acid cycle intermediates was observed whether which probably complexes Fe; and (4) the selecthe mitochondria were isolated from itaconate-

102


co2

-b

Citrate

10

cis-Aeonitate

Citramalate

-Citramalyl

4

Itaconate

CoA

Itaconyl CoA

Itatartarate

FIGURE 4. The metabolic relationships of itaconate metabolism. (1) Pyruvate carboxylase; pyruvate:carbondioxide ligase(ADP).6.4.1.1; (2) Pyruvatedehydrogenase; pyruvate:lipoate oxidoreductase (acceptor-acylating) 1.2.4.1; (3) Citrate synthase; citrate oxaloacetate-lyase (CoA-acylating). 4.1.3.7; (4) Aconitase; citrate(is0citrate)hydro-lyase.4.2.1.3; (5) Aconitate decarboxylase; cis-aconitate carboxy-lyase. 4.1.1.6; (6) “ltaconate Oxidase”; (7) Succinic thiokinase; succinate:CoA ligase(GDP-forming).6.2.1.4; (8) ltaconyl CoA hydratase; citrarnalyl CoA hydro-lyase. 4.2.1.56; (9) Citramalate CoA transferase; succinyl CoA:Citramalate CoA transferase. 2.8.3.7; (10) Citrarnalate CoA lyase; citrarnalyl-CoA-pyruvate-lyase. 4.1.3.25.

103


producing cultures or not, which, while still difficult to explain, suggests the observation is not connected to itaconate biosynthesis. The pathway proposed by Kinoshita has the same final step as the previous scheme, that is, the decarboxylation of cis-aconitate. It differs in that the source of aconitate is citrate. A. terreus has been shown to convert citric acid into itaconic acid both in intact mycelium224and in but in the latter case aconitate was a more effective substrate. Evidence for the biosynthesis of itaconic acid from citric acid, via dehydration to aconitic acid and the decarboxylation of this acid, was produced by Bentley and Thiessen in a series of three papers .22G228 Aconitase and cis-aconitate decarboxylase were shown to be present in extracts from surface-grown A. terreus. The aconitase reaction mechanism was, as expected, from previous studies, while the decarboxylase was shown to remove Cs of aconitate, which is the expected reaction with a P,y-unsaturated acid. The radioactivity from 1-I4C-acetate was found to be incorporated into the two carboxyls of itaconate equally while 60% of the radioactivity was found in C5 (the methylene carbon) when 2-I4Cacetate was used. The carbons 1, 2, 3, and 4 of itaconic acid were shown to be derived from SUCcinic acid without cleavage of the carbon skeleton. The decarboxylation of cis-aconitate was shown to take place at the primary carboxyl, which was followed by an allylic rearrangement.228This mechanism has been confirmed by ,H NMR.229 By carrying out the reactions in deuterated water, Bentley and Thiessen228were able to show the reaction mechanism involved the addition of a proton to form a carbonium ion intermediate. The involvement of citric acid as an intermediate is confirmed by the results of W i n ~ k i l l . ~ ~ ~ The radioactive tracer studies might also be consistent with the scheme involving 1,2,3-tricarboxypropane as an intermediate, but mass balance calculations show that the scheme involving this intermediate is not consistent with the pubIf the lished yields of 60 to 70% by overall stoichiometry is assumed to be one itaconate from one hexose, with no allowance for biomass or CO, loss, then the theoretical yield is 72.2%. As one CO, is lost for each of the three pyruvate molecules converted to acetyl phos-

-

104

phate in the 1,2,3-tricarboxypropanescheme, the stoichiometry becomes 1 :1.5; the theoretical yield is then just over 48%, unless the assumption of considerable recycling of COz is made. This recycling would not be consistent with the observed pattern of radioactivity. The biochemistry of itaconate synthesis appears generally rather similar to that of citric acid. The general path from hexose to tricarboxylic acid intermediate is the same, and a severe flux limitation appears to occur in the same place. The production of polyols shows parallels, as do such factors as some trace metal deficiency requirements, pH conditions, nitrogen and carbon sources, and phosphate effects. A model of A. niger with the addition of cis-aconitate decarboxylase might not be inappropriate. Malic acid is produced microbiologically from fumaric acid, and although fumaric acid can be produced from glucose by Rhizopus spp. ,232,233 it is produced chemically on an industrial scale.2" The conversion of fumarate to malate appears to utilize fumarase, which catalyzes a reversible hydration-dehydration. No cofactors are required and the reaction is stereospe~ific.~~~ Not only have microorganisms been but also isolated ~ ~even fumarase from Lactobaccillus b r e v i ~ ,and pig heart238has been employed. A process using Candida yeasts with n-alkanes as a substrate has been described.239Other strains of Candid0 in the same produced 2-oxoglutaric, SUCcinic, and fumaric acids. Similar strains of Candida also produce citric and isocitric acids from n-alkanes, so that it seems likely that the basic metabolic sequences employed for all these tricarboxylic acid cycle acids are the same, with oxidation of the alkane to acetate, an anaplerotic role for the glyoxylate cycle, and the synthesis of one or more of a variety of tricarboxylic cycle acids, depending on the particular regulatory status, fluxes, and concentrations of intermediates present under particular growth conditions. The acid that is eventually excreted will depend, among other things, on the relative activities of the TCA-cycle enzymes and the rate of export from the mitochondria and the cell membrane. The biochemical pathway for the microbiological synthesis of lactic acid from glucose by homolactic fermentation is simply a variant of

the glycolytic pathway. The efficiency of the process is such that yields of 96% of D( -)-lactic acid are normal.240*24


6. The Group 2 Acids The conversion of glucose into gluconic acid is a two-step process involving the oxidation of beta-D-glucose into the D-glucono-S-lactone and the subsequent hydrolysis of the lactone to gluconic acid or gluconate. Two industrial processes are used: the primary process for gluconates uses A. niger, whereas the alternative process using Gluconobacter suboxydans (Acetobacter)can also give 2-oxogluconic acid, 5-oxogluconic acid, and tartaric acid. The metabolic relationships are shown in Figure 5. The initial step in the A. niger process is carried out by glucose oxidase (beta-D-glucose:oxygen oxidoreductase, EC1.1.3.4), which removes two hydrogen atoms from beta-D-glucopyranose to give the lactone and hydrogen peroxide. The hydrogen peroxide is cleaved by catalase, while the lactone is hydrolyzed either spontaneously or by glucono-6-lactonase.242-244 Glucose oxidase was first described by Miiller,245 and its properties were ~ t u d i e d .The ~ ~enzyme ~~~* is a flavoprotein that is itself reduced during the reaction, then reoxidized by oxygen, giving hydrogen peroxide as one Both glucose oxidase and catalase were claimed to be located in peroxisomes,250 thus preventing peroxide toxicity, although this location is disputed.251The substrate for the oxidase is the beta form of glucose, which is about 150 times more effective than the alpha isomer, although the presence of a mutarotase means the the alpha form is also effectively utilized. The regulation of the synthesis of glucose oxidase and gluconic acid has not been studied in detail. It is assumed that there is a direct relationship between enzyme activity and gluconic acid accumulation. No regulatory activity has been described, except for the report of the studies of Mishak et a1.,251.64 which described the relationship between external pH and glucose oxidase activity. The enzyme was partially mycelium bound and also extracellular, not peroxisomal. It was induced by glucose at pH

values above 4, while being inactivated below pH 2. This is consistent with the observation of Tessi et al.252that gluconate is the predominant product at higher pH levels. Accumulation of the lactone product reduces the rate of glucose oxidation so that its hydrolysis is important for the productivity of the process. The rate of spontaneous hydrolysis is high at neutral pH values, but at acidic pH values the enzymic hydrolysis is more important. Gluconic acid can also be synthesized in A . niger by a pathway involving hexokinase, glucose-6-phosphate dehydrogenase, and a phosphomonoe~terase,~~~ but this seems not to be significant under the conditions used in the industrial processes. The ability to form gluconic acid is widespread in the Pseudomonadaceae and in most Acetobacter species, in particular Acetobacter (Gluconobacter) suboxydans. This organism has two glucose dehydrogenase enzymes,254 one of which, a soluble, NADP-specific enzyme, may be induced by high glucose levels. The other enzyme, which is particulate, is not very active, while the glucose-6-phosphate pathway is apparently repressed at high glucose concentrations. Many observations of “induction” or “repression” in the field of fungal metabolism are based on observations of enzyme activity alone and may therefore have other explanations. A feature of the Gluconobacter process is that further oxidation of gluconate can, and often does, occur. The formation of 2-oxogluconate from gluconate is carried out by a specific enzyme. ~-gluconate:(acceptor)2-oxidoreductase, which is most active when oxidations are carried out at neutral pH values. Some strains of GZuconobacteF are able to oxidize gluconate to the 5-0x0 derivative at both neutral and acidic pH levels; this can be further oxidized to tartaric although little is known of the enzymology (Figure 5). The 2-0x0 derivative does not form tartaric acid. Formation of the 2,5 dioxogluconate also occurs in some strains of Gluconobacter. Under normal process conditions, A. niger accumulates gluconic acid, but at least some strains can be adapted to grow on gluconate. 257-259

105

1


a-D-glucose

1 p-D-

Glucose

I

Glucose-6-P

D-Gluconic acid

5-Oxogluconate

6

2-Oxogluconate

12

2.5-Dioxogluconate

) -

6-Phosphogluconate

2-OX0-3-deO~

gluconate

2-0x0-3-deoxy-

1

7

EMP

phosphogluconate

pyruvate + glyceraldehyde

PP

It

ED

FIGURE 5. The metabolic relationships in gluconate metabolism. (1) Aldose mutarotase; aldose 1-epimerase. 5.1.3.3; (2) Glucose oxidase; D-g1ucose:oxygen oxidoreductase. 1.I .3.4; (3) Glucose dehydrogenase; D-glucose:NAD 1-oxidoreductase. 1.1.1.118; (4) Glucose dehydrogenase; D-g1ucose:NADP I -oxidoreductase. 1.1.1.1 19; (5) o-GluConO-6-laCtOnaSe (or spontaneous); D-glucono-&lactone hydro-lyase. 3.1.1.17; (6) Gluconokinase; ATP:Dgluconate 6-phosphotransferase. 2.7.1.1 2; (7) 2-gluconate dehydrogenase; ~-gluconate:(acceptor)2oxidoreductase. 1.1.99.3; (8) 5-Gluconate dehydrogenase; D-gluconate:(acceptor)5-oxidoreductase;(9) Ketogluconokinase; 2-ketO-D-glUCOnate 6-phosphotransferase. 2.7.1 .13; (10) 2-ketogluconate dehydrogenase; 2-ketO-Dgluconate:(acceptor)5-oxidoreductase. 1.1.99.4; (11) Hexokinase; ATP:D-hexose 6-phosphotransferase. 2.7.1.1 ; (12) Glucose-6-phosphate dehydrogenase; o-gIucose-6-phosphate:NADP+1-0xidoreductase. 1.1.I .49; (13)Phosphogluconate dehydratase; 6-phospho-~-gluconatate hydro-lyase. 4.2.1.12; EMP = Embden-Myerhof-Parnas pathway; ED = Entner-Doudoroff pathway; PP = Pentose phosphate pathway. +

106

+

111. INDUSTRIAL PROCESSES


A. Citric Acid Both A . niger and Yarrowia lipolytica are used in industrial processes. The methods can be subdivided into surface or submerged systems, and either carbohydrate of n-alkanes are used as feedstock. 1. A. niger Media

Several carbohydrate sources are employed. The critical factor in determining which is used is cost, both of the raw material and of any pretreatment. Beet molasses, cane molasses, glucose syrup, or even sucrose are used. Both beet and cane molasses are variable in quality, depending on seasonal factors such as rainfall, sunshine hours, etc. as well as differences in refinery techniques. The composition of molasses is complex,9oand the reasons for the variations are not understood, despite many studies. The only reliable method known to determine quality is empirical performance tests, which can also determine the best pretreatment for a particular sample. Considerable effort is devoted to this testing. The main problem with all carbohydrate sources is metal levels; Zn, Fe, Mn, and Mg have been mentioned in this context. In molasses this control of metal levels is achieved by the addition of sodium or potassium ferrocyanide.260In the patents, potassium ferrocyanide or other equivalent ferri- or ferrocyanide salt was added either before inoculation or 8 h after inoculation. The ability of ferro- or ferricyanides to form complex anions has been known for many years. The nature of the anions was revealed by X-ray analysis.261 A stirnulatory effect on growth and citric acid production, in addition to the trace metal chelating effect of ferrocyanide, has been claimed.262 Optimum treatment levels are usuaIly sIightIy in excess of those needed to chelate the metals, probably a result in growth inhibition and thus restricted biomass production. A similar fungicidal effect is produced by the addition of Cu salts, which are used in some industrial treat-

ments when simple glucose or sucrose media are employed. The amounts of added ferrocyanide are in the range of 0.5 to 3 g/l of fermentation medium, normally in the 1.5- to 2-g range. Other nutrients are supplied to molasses media if required, but most of the organic salts needed are already present. The other method used for reducing the trace metal ion levels is ion exchange,263but this is applicable only when a relatively simple carbohydrate source such as glucose syrup is used, otherwise the ion-exchange capacity of the resin is rapidly exhausted by other ions. Where relatively pure glucose or sucrose is used as a carbon source, nitrogen, phosphates, and other nutrients are added.79 The nitrogen requirement is usually supplied as ammonium salts, either sulfate or nitrate. The range of concentration varies from 1 to 3 g/l. Addition of further ammonium salts during the process has been suggested2&but is probably not economical in industrial practice. Phosphates are usually added to give a final concentration of between 1 and 2.5 g/l, which is probably optimal for balanced growth. The general requirements are summarized in Table 3.

2. The Surface Process This is the original method employed on a iarge scale and was introduced around 1920. Despite the more sophisticated methods that have been developed, the process is still employed as the energy costs are lower than those for the submerged process, but the labor costs are higher. Although it is still employed in regions with high labor costs, only existing plants, where the capital costs have been written down, are economical; it is doubtful whether new plants of this type would be economical under present conditions. The details of this process are not well documented, despite its long history, due to the restriction of information about process details and performance by the manufacturers. The mycelium is grown as a surface mat in a large number of shallow trays with a capacity of 50 to 100 1. The surface area of each tray is about 5 m2, and the depth of medium is between 5 and 20 cm. The trays, which are manufactured from high-

107


TABLE 3 General Requirements for Citric Acid Production By A. niger Parameter

Component

Carbon source

Sucrose, molasses, etc. Ammonium salts Urea Potassium phosphate Manganese Zinc Iron Air

Nitrogen source

Phosphate source Trace metals Oxygen PH

purity aluminum or stainless steel, are stacked in racks in a chamber constructed so that the process can be carried out under conditions approaching the aseptic. The material used for the trays depends on the handling methods employed. Molasses is diluted to the required concentration, about 15%, and the pH is adjusted to between 5 and 7. This initial pH is used because spores of A. niger will not germinate at lower values in molasses. This has been identified as an effect of acetic a ~ i d ;the ~ un-ionized ~ ~ . ~ ~ acid ~ prevents germination, but the mechanism by which this occurs is not known. Any required pretreatment such as ferrocyanide or nutrient additions is carried out, and the medium is sterilized usually by boiling. After cooling, the medium is pumped into the trays within the ventilated chamber. Inoculation is directly from spores, either added as a suspension or introduced into the air blown over the trays. Industrial practice seems to use a high number of spores for inoculation, which presumably decreases the length of the unproductive phases of growth and may give a filamentous growth rather than pellets.267 Aeration is of particular importance in this process as it fulfills two functions -oxygenation and heat removal - in addition to its role in spore inoculation. The requirement varies with the stage of growth. Initially, sterile air at low rates is required to prevent contamination during 108

Level

Range (911)

High

60-1 40-240

Low

2-2.5-3.0

Low

1-2.5-3.0

Deficiency Low Low High Low

Metabolic engineering of carbon and redox flow in the production of small organic acids.

The organic synthesis of unsaturated fatty acids.

Discovery and Engineering of Pathways for Production of α-Branched Organic Acids.

Effect of host diet on production of organic acids and methane by cockroach gut bacteria.

Microbial production of specialty organic acids from renewable and waste materials.

Initial pH of medium affects organic acids production but do not affect phosphate solubilization.

Direct production of organic acids from starch by cell surface-engineered Corynebacterium glutamicum in anaerobic conditions.

The influence of the buffering capacity on the production of organic acids and alcohols from wastewater in anaerobic reactor.

Volatile fatty acids production from sewage organic matter by combined bioflocculation and anaerobic fermentation.

Amino Acids Hydrolyzed from Animal Carcasses Are a Good Additive for the Production of Bio-organic Fertilizer.

The Roles of Organic Acids in C4 Photosynthesis.

Gastric emptying of organic acids in the dog.

Thin-layer chromatographic method for the identification of organic acids.

Organic acids in urine from preterm infants.

Organic acids tunably catalyze carbonic acid decomposition.

Extraction of medium chain fatty acids from organic municipal waste and subsequent production of bio-based fuels.

Stimulation of monokine production by lipoteichoic acids.

The production of extracellular amino acids by rumen bacteria.

New developments in the chemo-enzymatic production of amino acids.

Control of Meloidogyne incognita Using Mixtures of Organic Acids.

Analysis of kinetic labeling of amino acids and organic acids by GC-MS.

Bacterial production of short-chain organic acids and trehalose from levulinic acid: a potential cellulose-derived building block as a feedstock for microbial production.

Quantitation of organic acids in amniotic fluid by gas chromatography.

Organic acids and bases: review of toxicological studies.