Citric Acid Fermentation by Fungi

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Volume 3, Issue 4 331 CITRIC ACID FERMENTATION

Authors: Christian P. Kubicek Max Rohr

Institute for Biochemical Technology Vienna Technical University

Vienna, Austria and Microbiology

Referee: H . 1. Rehm

Department of Microbiology Institute for Microbiology University of Westfallsche Wilhelrns Munscer. West Germany

I. INTRODUCTION

Citric acid (2-hydroxy-propane- 1,2,3-tricarboxyIic acid) was first discovered by Scheele in 1784 as a constituent of citrus fruits. However, since the discovery of the tricarboxylic acid cycle by Krebs, within which citric acid is a key intermediate, its ubiquitous occurrence in almost all living systems has been established. Due to its pleasant taste and also its low toxicity and ease of assimilation, interest in industrial production arose very early and was initially produced exclusively from citrus fruits. Soon after the first discovery of citric acid production by a fungus' in 1917, the first trials with Aspergillus niger were published. In the following years, several industries spread around the world and took over the microbial citric acid production; a survey on the history of this development was given recently and some milestones are cited in Table 1. By 1933, according to data quoted by Perquin,' the world production of citric acid amounted to 10,400 t, of which Italy contributed 1,800 t from lemons; the rest however was produced by fermentation. Today, the latest estimates, however, give values of more than 350,000 t/year which are exclusively produced by fermentation. Interestingly, A. niger is still almost exclusively the organism applied, although many investigations on screening for other microorganisms or strains have been carried out.

Recently, however, some more promising attempts have been made with n-alkane-assimi- lating yeasts, which have been utilized by a small number of factories.

Despite its great importance for the preparation of a bulk chemical with manifold appli- cations, citric acid fermentation by A. niger is still to be considered as a rather problematic fermentation process since it is influenced by a high number of variables which at present cannot always be accurately controlled and thus result in impaired yields. It is the object of the present review to put a special emphasis on the external and internal control of this fermentation process and the underlying biochemical mechanisms.

11. STRAINS AND STRAIN IMPROVEMENT

A detailed review on the strains reported to be useful for citric acid accumulation has been reported on previously3 and shall not be repeated here since many of the strains are not used on a commercial basis. On the other hand, several strains have been patented as industrial producer strains which do not essentially differ from others except for their acid- producing capacity and are protected solely for being exploited for commercial reasons.

Only a few classes or even genera of microorganisms have been reported to excrete substantial amounts of citric acid into the fermentation medium under certain conditions. This is not

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332 CRC Critical Reviews in Biotechnology Table 1

TIMETABLE OF DEVELOPMENTS IN CITRIC ACID FERMENTATION’

1893 1917 1919 1923 - 1938

About 1930 and later I 944 About 1947

and later 1968

Wehmer discovers fungal citric acid accumulation in Ci- rromyces (today identified as Penicillium s p . ) Systematic investigations of Currie on conditions for citric

acid production

First industrial plant in Belgium (Societi des Produits Organiques de Tirlemont)

Construction of further plants in England (Sturge), C.S.S.R.

(Kaznejov), U . S . (Pfizer). U.S.S.R. and Germany (Benckiser)

Bernhauers fundamental work on surface citric acid product ion

First submerged process (Sziisc)

Fundamental work on the submerged process by Johnson First patent on citric acid production from n-alkanes

Table 2

MICROORGANISMS CAPABLE OF CITRIC ACID PRODUCTION

Aspergillus niger Penicillium jan:hinellum4 P . restrictum4

A. wenti’

Trichoderma reeseib Arthrobacrer parafineus’

Saccharomycopsis lipolytica (formerly Candida)‘

S . oleophila’

Saccharomycopsis ~ p . ~

surprising in view of the fact that citric acid is a metabolite of energy metabolism, of which the concentration will only rise to appreciable amounts under conditions of drastic metabolic imbalances. Among those microorganisms for which an industrial process has been patented (see Table 2), only A . niger and certain yeast strains (mostly Saccharomycopsis sp.) are applied today; the latter being originally designed for use in the production of citric acid from n-paraffins.

Among the Aspergilli, the first producer strains were isolated from soil according to procedures, e.g., described by B e r n h a ~ e r . ~ Many of the successful strains from the first commercial attempts of citric acid production may be traced back to such early isolates.

Unfortunately, little of this work has been published, which is presumably, in addition to the general secrecy surrounding industrial citric acid fermentations, in part due to the very high yields given by existing strains under proper environmental conditions, and, on the other hand, to the fact that the fermentation conditions can influence a given strain to a high degree. Thus, far more research has been reported concerning medium optimization (see later).

As first pointed out by Wende1,‘O there are two principal methods of selecting varying portions of a certain population, namely, the “single-spore technique” and the ‘‘passage method”. In the single-spore technique, a suitable spore dilution is prepared and cultures from single spores are made and tested in separate assays. The rationale behind this is the heterogeneity of a given conidia population; the observed “productivity” of a certain in-

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Volume 3, Issue 4 333 oculum is thus a mean value obtained for the total conidia population; consequently, there must exist individuals with an even higher yield within this population. It is obvious that such a method requires a sensitive assay of the attempted product directly on the cultivation plate. Thus, James et a1.I’ have introduced the use of pH indicators as a means for rapidly detecting the highest acid-forming colonies. For obtaining only a limited diffusion of citric acid, they recommended the use of filter paper soaked with a kind of citric acid fermentation medium. Obviously, this bears the disadvantage that mineral acids, being formed by the consumption of cations of physiologically acid compounds from the nutrient medium or organic acids (gluconic acid, oxalic acid), will simulate the presence of citric acid. Thus, Rohr et al. l 2 have recently improved this method by incorporation of a specific stain for citric acid (paradimethylamino benzaldehyde) instead of the indicator. By this improvement, a semiquantitative means for the yield of citric acid can be deduced by simply calculating the ratio of the diameter of the citric acid zone over the diameter of the colony as shown in Figure 1. It has not yet been assessed however whether the calibration curve is applicable in a higher range of citric acid concentrations. However, a distinct advantage of this method is its applicability for reselection of “degenerated” strains, i.e., those which have lost their capacity to produce high amounts of citric acid after prolonged subcultivation. This phenomenon is most likely due to selective advantage of more vigorous back mutants accompanying successive transfers on agar slants. l 3 As shown in Figure 2, such degenerated cultures can effectively be reselected by application of the method cited above.

The “passage method” principally consists of plating a population on plates containing varying concentrations of a growth- or germination-inhibitory compound. This results in the selection of fractions from the population as a function of increasing concentrations of a selective agent. No literature is available on’the use of the “passage method” for improving citric acid-producing strains.

Surprisingly, little work has been undertaken on the improvement of citric acid-producing strains on a biochemical or genetic basis. Auxotrophic mutants ofA. niger have been isolated by some worker^,'^-'^ but the results failed to provide evidence for a stimulation of citric acid production by -certain auxotrophies. On the other hand, auxotrophic strains have been used as a foundation for genetic studies. Because of the lack of a sexual cycle in A. niger, the study of genetic factors in citric acid production must be carried out using parasexual cycle techniques. ^l9 Recent attempts h ~ w e v e P ’ ~ reported an unusual instability of the het- erozygous diploid nuclei formed, with a high rate of mitotic crossing over and haploidization occurring in the heterokaryotic hyphae prior to conidial formation.

Various procedures have been devised for isolating and testing mutant strains of several microorganisms that will be able to carry out citric acid accumulation from hydrocarbons;

this work is being carried out particularly in Japan. Although one of the earliest patents7 recommended the use of Arrhrobacter parafineus with approximately 100% yield, most of the following patents covered citric acid production by yeasts, especially of the genus Saccharomycopsis. Most of the mutants were primarily tested for growth on both n-paraffins and citric acid, and those considered as suitable were those which did not grow on the latter.

Since the byproduction of isocitric acid is one of the main problems associated with the n- paraffin process, many procedures for mutant isolation were devised to use inhibitors of aconitase (fluoroacetate or fluorocitrate) and to select those colonies which are especially sensitive to this compound. The rationale behind this is that mutant strains with low aconitase activity would exhibit a lower tolerance against this

Recently, several reports have dealt with basic genetic studies on potential citric acid- accumulating y e a ~ t s . ~ ~ - ~ ~ Up to now, however, these studies were restricted to the development of suitable genetic methodology.

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334 CRC Critical Reviews in Biotechnology

FIGURE I .

papers after drying and development of the acid zones.'LB

Photographs of (A) A . niger conidia growing on the nutrient soaked filter paper and (B) the Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Calgary on 06/01/12 For personal use only.

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Volume 3, Issue 4 335

O/O

t

j 1 . 2 2.4

I

^{1.2 2.4} ^,^{1.2 2.4} ^1.2 ^2.4

1.2 2.4 I

1.2 2.4

FIGURE 2.

tions of A . niger in the cqurse of selective transfers.

Distribution of citric acid productivity in conidia popula-

diameter of acid zone diameter of colony A . U . =

Values in brackets indicate the average A . U . value of the total conidial material used for single spore plating. (Data are taken from Rohr, M., Stadler, P. J., Salzbrunn, W. 0. J . . and Kubicek, C. P., Biotech. Lett.,

1, 28 I , 1979. With permission.)

111. CONDITIONS FOR FERMENTATION A. Citric Acid Production from Carbohydrates with A . niger

The accumulation of citric acid is strongly influenced by the composition of the nutrient medium. This effect is especially pronounced in the submerged process, and therefore, apart from first studies by Currie,'" systematic studies have been presented by Shu and J ~ h n s o n ~ " ~ ~ during their pioneering studies on submerged citric acid fermentation. Those medium constituents which have been found to exert an effect on citric acid fermentation are listed in Table 3, and will be discussed further in detail below.

1. Sugars

In general, only sugars which are rapidly taken up by the fungus are carbon sources

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336 CRC Critical Reviews in Biotechnology Table 3

GENERAL NUTRITIONAL REQUIREMENTS OF CITRIC ACID FERMENTATION BY ASPERGILLUS

NIGER

High sugar concentration (140 to 240 g/e) Ammonium salts (2 to 2.5 g/e)

pH (should be below 2 during the active phase of citric acid production) High oxygen tension in the medium (over 140 mbar)

Deficiency in trace metals (especially Mn”)

Balance between inorganic phosphate and Mn’+, Fe’+, and Zn’+

Table 4

CRUDE CARBOHYDRATES WHICH SERVED AS A SOURCE OF CARBON FOR

CITRIC ACID FERMENTATION BY ASPERGILLUS NIGER

Carbohydrate source Citric acid yield” Ref.

Date syrup 62 34

Cotton waste 60 33

Whey permeate 44-57 31. 32

Brewery waste 35

Carob sugar 4 0 6 0 36

Sweet potato pulp 31

Pineapple waste water 38

’ Citric acid yield is given as grams of citric acid per grams of initial sugar.

allowing a high final yield of citric acid (i.e., glucose, fructose, or sucrose). In most cases, sucrose, or its cheaper commercial source molasses, is used.

Although sucrose is not taken up by A . niger, precautions to achieve its hydrolysis prior to fermentation have proven unnecessary; this is in part due to the fact that a part of the sucrose is split during sterilization of the nutrient medium. Moreover, the fungus possesses an extracellular, mycelium-bound invertase, which is active under the acid conditions of citric acid fermentation and which is capable of rapidly hydrolyzing the sucrose supplied. 30.3”a

Polysaccharides, unless hydrolyzed, are generally not a useful raw material for citric acid fermentation since they are split too slowly (due to low activity of the respective hydrolases at the lower pH) to allow the high rate of sugar catabolism required for citric acid accumulation (see Section IV), although their use under exceptional conditions has been r e p ~ r t e d . ~ ’ - ~ ~ A representative selection of recent literature on the use of various sources of carbohydrates for citric acid accumulation is presented in Table 4. Several sources of crude carbohydrates have been used for citric acid production, e.g., beet and cane m o l a s ~ e s , ~ ~ . ~ ~ unrefined

~ u c r o s e , ~ ’ cane juice, citric rnola~ses,~’ and various hydr~lysates.~’ Most of these sources present the problem of heavy metal contamination (see later), and also in some cases the presence of growth inhibitory s u b ~ t a n c e s . ~ ~ . ~ ~ In any case, they have to be removed prior to fermentation. On the other hand, the presence of stimulatory compounds has also been r e p ~ r t e d , ~ ~ . ~ ’ although their chemical nature has not yet been elucidated.

*

* Concerning molasses, major differences can also be observed with respect to their origin (i.e., beet or cane molasses). In general. beet molasses are believed to be only applicable in surface fermentation, whereas cane molasses are preferred in submerged fermentation. Also, major differences have been observed in the use of molasses as carbon sources for citric acid fermentation with respect to their geographical origin, age, pretreatment in the sugar factory, and storage conditions.

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60 40 20

10 20 30

YoI W/VI SUGAR

FIGURE 3 . Influence of the initial sugar concentration on the final citric acid yield (grams citric acid per grams sugar) in citric acid fermentation by Aspergillus niger. U is yield per grams sugar consumed, O-C is yield per grams sugar added. (Data are taken from Shu, P. and Johnson, M. J . , Ind. Eng. Chern., 40, 1202, 1948. With permission.)

Not only the type but also the concentration of the carbon source is critical to citric acid fermentation: a maximal citric acid production rate is usually achieved at 14 to 22% of sugar in the nutrient medium (Figure 3).28 This is apparently related to stimulation of glycolytic sugar catabolism and C0,-fixation under these conditions (see below).

2 . Nitrogen

Nitrogen is usually supplied in the form of ammonium sulfate or ammonium nitrate.

Physiologically, acid ammonium compounds are preferred since their consumption concom- itantly lowers the pH of the medium to below 2, which is an additional prerequisite of citric acid fermentation.

The concentration of the nitrogen source required for citric acid fermentation is 0.1 to 0.4 g N per

t .

Furthermore, addition of NH,‘during fermentation has been recommended (Figure 4),48 provided it does not raise the pH of the medium. This effect is probably related to the stimulatory effect observed in vitro of ammonium ions on glycolysis in A . niger (see Section IV).

3 . Phosphate

Phosphate is a key nutrient of regulatory importance to microbial secondary metabolite production; its effect has also been demonstrated at an early stage with citric acid production by A . n i g e ~ - . ~ , ~ ~ In more recent investigations, however, it was established that -given that the metal ion limitation is correctly maintained (see below) - the effect of phosphate is not very p r o n ~ u n c e d . ~ ~ Actually, the concentrations of phosphate described as optimal for citric acid production by Shu and Johnsonz8 are sufficient for balanced growth. In cases of trace metal contamination, phosphate limitation can have a beneficial effect on citric acid productivity. At present, the biochemical basis of this “phosphate effect” is still not known,

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338 CRC Criiicat Reviews in Biotechnology

1.5

1 .o

0.5

3 9 d

FIGURE 4. lnfluence of addition of extra NH; on the volumetric productivity @I00 mP and per day) of citric acid fermentation by A. niger. ^0-C 1000 ppm NH;; 0-0 2000 ppm NH;; A-A 3000 PPm NH;; and n.z 4000 ppm NH,'.'O

but evidence has been reported that phosphate acts at the level of enzyme activity and not gene e x p r e s ~ i o n ; ~ ~ one of the first measurable changes accompanying a decrease in citric acid accumulation is a decrease in C0,-fixation. Additionally, Martin and Steels1 have observed an increased accumulation of sugar acids at the expense of citric acid in case of high phosphate concentrations.

4 . p H

The maintenance of a low pH (below 2) during citric acid fermentation is of extreme importance for a successful citric acid fermentation. The physiology of this effect is unknown at present, but it should be pointed out that the intracellular pH is only slightly influenced by this ~ o n d i t i o n . ~ ' " At a higher pH, A . niger accumulates gluconic acid, which is brought about by an extracellular partially mycelium-bound glucose o x i d a ~ e . ~ * This oxidase is induced by glucose as soon as the pH is raised above 4. The enzyme however is inactivated below pH 2 (Figure 5 ) , thus suggesting that the effect of pH on citric acid accumulation might in part be explained by elimination of reactions competing for sugar uptake. This assumption is further supported by comparing the rates of gluconic acid and citric acid production (20.0 and 0.7 g/t/hr, respectively).

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Volume 3 , Issue 4 339

1.5

2 3 4 5

PH

FIGURE 5 . Influence of the extracellular pH on the activity of extra- cellular glucose oxidase (0-0) and the glucose transport system

(A--n).S'.

5 . Trace Elements

Trace element nutrition is probably the main factor influencing the yield of citric acid fermentation. This is especially highlighted by the fact that an otherwise optimal nutrient medium for citric acid fermentation will not allow high production unless the trace element content is controlled carefully. On the other hand, if trace element nutrition is correct, other factors (sugar concentration, phosphate, and others) have only less pronounced effects. It

is well known that A . niger requires a certain amount of all trace elements for g r o ~ t h . ~ ~ - ~ ~ However, especially during submerged fermentation, a limitation in certain trace elements

is critical for citric acid production (Figure 6).29.55-@' Although certain effects were observed for zinc, iron, and manganese ions, with the latter as low as 3 p g / t , it drastically reduced the yield of citric acid under otherwise optimal c o n d i t i o n ~ . ~ ~ . ~ ~ . ~ ~ Investigations by Clark et a1.6' (Table 5) and Kisser et al.62 also confirm the key regulatory nature of manganese ions.

It is somewhat surprising that this effect of manganese has hitherto almost totally been neglected by the patent literature on citric acid production. Presumably this is related to the patent of S ~ h w e i g e r , ~ ~ who described the successful addition of copper ions as a counteractant of ferrous ion contamination in the nutrient medium. In these experiments, Schweiger added iron in quantities of up to 0.15 g / t Fe3+ (Figure 7); even assuming the highest available purity of the iron salt used, the contamination according to the manufacturers (0.005%, Merck per analysis) would indicate that the amount of iron introduces manganese ions in amounts critical for citric acid production (1 to 3 pg/e). The antagonistic action of copper ions has also been used as an argument in favor of iron being the key regulatory metal for citric acid accumulation. However, in the authors' laboratory it has been shown that copper ions can successfully counteract addition of manganese to citric acid fermentation media;63a these observations are further supported by recent findings that copper ions are inhibitors of cellular manganese uptake." The authors conclude, therefore, that manganese is the critical metal ion for citric acid fermentation.

The biochemistry of the manganese effect will be explained in more detail below. It should however be noted at this time that the addition of manganese ions to a citric acid fermentation will require active protein synthesis for inhibiting the further accumulation of citric acid,50

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340 CRC Critical Reviews in Biotechnology

5 ₁₀ 20 30 Mn

]

FIGURE 6. Effect of trace metals on citric acid production by A. niger. (A) Effect of Zn'+-ions on citric acid yield (grams citric acid per grams initial sugar, 0 ) and mycelium formation (A, MDW). (B) Effect of Fe3+ on citric acid yield (grams citric acid per grams initial sugar) at several defined concentrations of Zn'+ ( 0 , no Zn' +;

0 , 0.06 mg/t Zn"; 0 , 1.20 mgle Z n + + ; A , 0.48 mg/e Zn"). (C) Effect of Fe"' on citric acid yield (grams citric acid per grams initial sugar, A) and mycelium formation (0, MDW). (D) Effect of Mn" on citric acid yield (grams citric acid per grams initial sugar) in a high-phosphate ( 0 ) and low phosphate (a) nutrient medium. (Data were taken from Shu, P. and Johnson, M. J . , J . Bacferiol., 56, 577, 1948. With permission.)

thus providing evidence that manganese ions act at the level of protein synthesis and are not simply required as dissociable cofactors of enzymes involved in citrate breakdown.

6. Aeration

Aeration has been shown to have a critical effect on the submerged process of citric acid fermentation (Figure 8).65-68 It has even been shown that sparging with pure oxygen increases the yield of citric acid, but the expensiveness of the use of pure oxygen has precluded the application of this finding. Clark and Lentz,& however, have indicated that the gas phase of citric acid production can be recycled provided the CO, is trapped.

The requirement of citric acid fermentation for oxygen has the detrimental consequence that a short interruption of the oxygen supply, which has no effect on growth and viability, irreversibly affects the rate of citric acid production.68 The critical nature of such an interruption in the air supply is also dependent on the time and stage of fermentation. BattP9 has patented a procedure by which the detrimental effect of interruption of aeration can be prevented by raising the pH to 3 to 4 until the production of citric acid commences again.

Some aspects of the biochemistry of this "oxygen effect" will be discussed later.

7 . Other Factors of Influence

As we have shown above, the trace metal contamination of crude technically available

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Volume 3, Issue 4 341 Table 5

EFFECT OF METAL IONS ON CITRIC ACID FERMENTATION WITH ASPERGILLUS NIGER

Citric acid yield (g1100 g initial sugar) Metals added Amount (mgM‘) after 96 hr -

A13+

Ca’ ⁺ Co’ +

CU’ ⁺ Fe2+

Mg2+

NiZ+

Zn’ ⁺ Mn2 ⁺ Mixture of all Mixture of all except for Mn”

-

5.0 80.0 0. I 3.0 100.0

20.0 0. I 5.0 I .o

As above As above

8.8 7.3 9.0 9.0 8.6 7.5 8.8 8.6 7.5 1.5 I .4 1.4

Data are taken from Clark, D. S . , Ito, K., and Horitsu, H., Bio- rechnol. Bioeng., 8, 465, 1966. With permission.

75

s

0 50

-

v1 cy:

w

0

U

25

50 100 150 ppm Fe3+

FIGURE 7. Antagonization of Fe3+ inhibition of citric acid production by Cu2+. Conversion of sugar to citric acid is given in percent (grams citric acid per grams initial sugar). Cu2+ concentrations: V, 50 mg/e; A,

100 mgle; 0 , 200 mgle; 0 , 300 mde; ^{0 ,}⁵⁰⁰m g ~ e . ~ O Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of Calgary on 06/01/12 For personal use only.

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342 CRC Critical Reviews in Biotechnology w t-

CY

a

5 30

i = - u

.c

= E

n .

Ly \

20

n z

⁴

PEO 2

^=k

10

0 -

I - -

-

U

50 100 150

DOT I rnbarl

FIGURE 8.

acid fermentation medium on the citrate production rate.’”“

Influence of dissolved oxygen tension (DOT) of the citric

carbohydrates is one of the most critical factors in their use for citric acid fermentation.

Although their removal, by either cation exchange or complexing and precipitation with hexacyanoferrate (HCF), is the most appropriate method, this is not always possible. Several methods have thus been developed where the negative effect of trace metals could be abolished by the addition of certain additives, which will be discussed below.

Hexacyanoferrate - Treatment of crude carbohydrate sources with HCF is still the most frequently applied method for counteracting the inhibitory action of trace metal contamination by its chelating properties; an additional stirnulatory action on citric acid biosynthesis has been claimed but not yet proven.” It is nevertheless beneficial to the citric acid yield to always keep a slight excess of HCF in solution, probably to restrict fungal growth. It is impossible to predict the required amount of HCF, and therefore it must be determined by several trials at different scales. Usually the required amount is in the range of 10 to 200 mg HCF-ion per

C,

depending on the quality and brand of the raw material, and the trial is carried out while the solution is hot. It has been found that A . niger tolerates less HCF during submerged than during surface fermentation.

Lower alcohols - Originally found by Sakaguchi and Baba7* and later investigated in detail by M ~ y e r ’ ~ . ’ ~ is the antagonization of the trace metal contamination in crude carbohydrate sources by lower alcohols. These are probably the most important additives besides copper and HCF. Appropriate alcohols are methanol, ethanol, n-propanol, isopropanol, or methylacetate in concentrations from 1 to 5 % . When pure carbohydrates are used, however, the addition of alcohol is even detrimental indicating that its effect is the antagonization of trace metals. The biochemistry of the alcohol effect is still unexplained, but it is obviously related to the biochemical effects exerted by manganese (see later). Alcohols have been shown to principally influence membrane fluidity in microorganisms by affecting the phospholipid composition of the cytoplasmic membrane.75 Since manganese deficiency alters the phospholipid composition of the cytoplasma membrane of A . r ~ i g e r , ~ ~ an effect on membrane permeability would be very likely. However, recent investigation^^^ argue against a role of

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Volume 3, Issue 4 343 Table 6

LIPIDS INCREASING CITRIC ACID YIELD BY ASPERGILLUS NIGER"

Lipid added (2%, v/v) Natural oils

Vegetable

Citric acid (glC)

Almond Linseed Maize Olive Peanut Castor Tung Animal oils

Sperm Neatsfoot Lard Glycerides

Distearin Monooleate Unsaturated Oleic

I14 (79) 78 (66) 78 (46) 95 (65) 91 (62) 94 (70) 108 (87) 84 (70) 65 (50) 70 (50) 84 (73) 85 (73) 99 (68)

" Numbers in parentheses indicate the control experiment without lipid added.

The data were selected from Millis, N. F., Trumpy, B. H . , and Palmer, B. M., J . Gen.

Microbid., 30, 365, 1963. With permission.

membrane permeability in citric acid accumulation (see Section IV). This alcohol stimulation phenomenon clearly deserves further attention.

Fatty materials - Fatty materials are frequently used in the fermentation industry as antifoam agents. Millis et al.78 additionally found that certain natural oils, especially those containing a higher proportion of unsaturated fatty acids, can increase the final yield of citric acid without, however, affecting the final dry weight yield (Table 6). This procedure was later patented by Gold and Kieber.79 They found that a concentration of fatty acids of 0.05 to 0.3% had to be maintained during fermentation. Since the fatty acids are consumed, they have to be incrementally added during fermentation.

Other Compounds - Numerous other agents have been shown to increase the final yield of citric acid under defined conditions, as listed in Table 7. Most of them have been found empirically and thus no data on their presumptive physiological effects can be given. One compound, however, should be commented on. B r u c h m a n P found that H,O, stimulates the accumulation of citric acid, and this was interpreted to be due to its inhibitory effect on aconitase. It appears that H,O, is a rather unspecific inhibitor since it may act merely as an oxidant. Since aconitase is probably most active during citric acid fermentation (see later), the effect of H,O, is obviously related to other physiological events. A very likely explanation is that H,O, acts solely by providing an increased oxygen tension in the medium.

B. Citric Acid Production from n-Alkanes by Yeasts

About 10 years ago, hydrocarbon fermentations were euphorically welcomed since they enabled the conversion of not only a cheap, but an otherwise unwanted raw material into several useful products. Thus, in 1973, Gledhill et a1.** stated that hydrocarbon fermentation

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344 CRC Critical Reviews in Biotechnology Table 7

VARIOUS COMPOUNDS REPORTED TO INCREASE CITRIC ACID PRODUCTION

BY ASPERGILLUS NIGER

Ref.

4-Methyl-umbelliferone 3-Hydroxi-2-naphtoic acid Benzoic acid

2-Naphtoic acid Iron cyanide

Quaternary ammonium compounds Amine oximes

Starch EDTA

1,2-Diaminocyclohexane N,N‘ -tetraacetate Diethy lentriaminepentaacetate

Surface active agents H>Ol

Vermiculite

81

1

⁸²83, 84

85 86 87

Table 8 INTRACELLULAR

CONCENTRATIONS OF CITRATE AND ISOCITRATE IN ASPERGILLUS NIGER DURING CITRIC ACID FERMENTATION

Fermentation Citrate Isocitrate

condition (mM) (mM)

+

100 rng Cu2+ 4 . 1 0.36

+

^{50 mg}^Fez+ ^4.2 ^0.32

+

0.1 mg Mn2+ 2.1 0. I5

Nore: All data were obtained from 90-hr grown cultures. Concentrations are given as milli- molar, assuming average distribution within the mycelium.

From experiments in the authors’ laboratory. I n

of citrate production offers certain advantages over the traditional carbohydrate process, based on a comparison of substrate costs, product yield (140 to 150%, w/w, on hydrocarbons), production rates of 1.4 gltlhr and final broth concentrations of up to 225 gJt. Presently, ecological consciousness and political tensions with oil-producing countries such as Libya (the greatest n-paraffin reservoir in the world) have turned n-paraffins from an undesirable byproduct into a substrate in such short supply that it must be specially made. The other arguments in favor of the n-alkane process, however, still apply today. It is justified therefore to give some details on the current knowledge of this fermentation process.

An ideal medium composition for citric acid production from n-paraffins is given in Table 8: n-alkane fractions of C,, to C,, are generally preferred, the concentration used ranging between 3 to 10% (wlv). It must be borne in mind that they are water insoluble and one therefore has to deal with a four-phase system, thus making mass transfer (e.g., aeration) more difficult. Furukawa et aI.*’ have recently documented that C,,H,, and C,,H,, gave the highest yields of citric acid.

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Volume 3, Issue 4 345 The pH for n-alkane fermentation has to be held above 5.5, otherwise polyols (erythritol and mannitol) will accumulate at the expense of citric acid.” This presents some problems since both CaCO, as well as NH,OH, which can be used to neutralize the acid formed, are slightly inhibitory. Proposals to overcome this problem included addition of ion-exchange resins to the fermentation broth,” dialysis,’* or use of a semicontinuous cell recycle.88 Citric acid too inhibits its own production. To the best of our knowledge, however, these treatments have not been scaled up, and thus their effectiveness remains to be established.

A further peculiarity of the n-alkane fermentation process is the requirement of thiamin;

if it is omitted, a-keto acids will accumulate. This effect has not yet been analyzed on a biochemical basis, but is probably related to an increased requirement of thiamin pyro- phosphate as a coenzyme of oxidative decarboxylation during n-alkane degradation.

Inorganic phosphate has been reported to favor citric acid production when present only in limited amounts;89 in particular, the ratio of carbon to phosphorus should be high for a high yield.89 Other workers, however, have reported that the nitrogen concentration must be limited, and that the exhaustion of nitrogen from the medium (e.g., NH: salts) is the trigger for citrate a c c ~ m u l a t i o n . ~ ~ . ~ ~

The effect of aeration on citric acid production from n-paraffins is as yet controversial;

whereas Tabuchi and Hara90 reported a requirement for high aeration, with a concomitant decrease in acidogenesis when the aeration is lowered, Puklowski and R e h ~ n ~ ~ reported the opposite. An interpretation is complicated by the fact that the authors were using different species of Saccharomycopsis (i.e., lipolytica and parapsilosis).

Under these conditions, citric acid fermentation takes about 5 to 8 days; based on the high yield from n-paraffins (up to 140%), the final citric acid concentrations are considerable (over 20%), which facilitates recovery. However, the n-paraffin process bears the major disadvantage that considerable amounts of isocitric acid are accumulated as a byproduct. In certain cases, they can amount to equal the concentration of citrate. Separation during recovery is possible (due to the lower solubility of the tricalcium citrate). However, the prevention of isocitrate byproduction is preferred, but not completely possible. Improvements in the citrate to isocitrate ratio can however be achieved by several means: (1) omission of Fe-ions, constituents of the aconitase protein molecule, results in impaired aconitase activity and lower isocitrate levels;’6 (2) addition of monofluoroacetate, which is converted to mon- off uorocitrate, an inhibitor of aconitase, in the cell, in the presence of an uncoupling agent (e.g., 2,4-dinitrophenol);” (3) addition of certain alcohols (methanol, ethanol, lauryl-, stearyl-, and oleyl-al~ohol);~~ (4) alternatively, mutants low in aconitase activity have been selected based on their ability to grow on n-alkanes, but not on itr rate;^^ and ( 5 ) decrease the byproduction of isocitric acid by adding small amounts of Na,B,O,.IOH,O (0.1 mg/t).89 Higher amounts (1 mg/t) also decrease the amount of citrate produced without affecting growth.

IV. BIOCHEMISTRY AND METABOLIC REGULATION OF CITRIC ACID ACCUMULATION

A. Biochemistry of Citric Acid Production by A . niger

The biochemical pathways related to citric acid accumulation and their regulation have been the subject of numerous investigations; several metabolic schemes were proposed before the detection of the tricarboxylic acid cycle by Kornberg and Krebs,loo which are fully documented by Prescott and Dunn,’O’ but they are of mere historical interest here and thus beyond the scope of the present review. Figure 9 depicts the basic metabolic pathway of citric acid biosynthesis from glucose, which has been well established from investigations using radioactive labeling. ^lo*

Principally, the metabolic events related to citric acid accumulation can be divided into

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346 CRC Critical Reviews in Biotechnology

Glucose

Oxaloicetute Acetyl-C oA Citrate t

FIGURE 9. Generalized scheme of carbon flow to citrate in A. niger. Circled letters indicate metabolic sequences involved in citric acid accumulation: (a) glycolytic carbon breakdown; (b) oxidative decarboxylation and pyruvate carboxylation; and (c) tricarboxylic acid cycle.

three processes: (1) the breakdown of hexoses to pyruvate and acetyl-CoA by glycolysis;

(2) the “anaplerotic” formation of oxalacetate from pyruvate and CO,; and (3) the accumulation of citric acid within the tricarboxylic acid cycle. In the following, we will first describe the current knowledge about how these events contribute to citric acid accumulation and then will discuss how some of the important nutrient factors exert their influence.

1. Hexose Catabolism and Carbon Dioxide Fixation

A . niger possesses two pathways for the breakdown of hexoses, i.e., the hexosebisphos- phate (Embden Meyerhof Parnas) and the hexosemonophosphte (pentose phosphate) pathway, ^103-105but it is now well established that the hexosebisphopshate pathway contributes the major part, i.e., more than 8 O % . ’ O 6 . ’ O 7

This can also be calculated very easily from carbon balancing; according to the scheme in Figure 9, ideally no CO, should be lost during active citric acid production since CO, released during oxidative decarboxylation of pyruvate is used in the anaplerotic formation of oxalacetate. A further release of CO, in the pentose phosphate pathway, however, has no substrate for fixation and would thus be lost, which, however, is incompatible with the almost theoretical yields of citric acid obtained. A glycolytic carbon catabolism to the highest percentage possible is thus beneficial to citric acid accumulation. However, several workers have reported that during the early phase of citric acid fermentation more hexose is consumed than would correspond to the citric acid formed, and the converse was observed during the late phase of f e r m e n t a t i ~ n . ’ ~ ~ ~ ~ . ’ ~ ~ Most of these workers suspected the intermediate formation of nonreducing oligosaccharides, which are then converted to citric acid during later fermentation stages. In our laboratory, we have recently identified these “unknown” substances

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I r'

'-O.o

.r0

/ * . -

W O ' I

100 200

FIGURE 10. Accumulation of several polyols during citric acid accu- mulation by A . niger. 0 , Erythritol; 0 , glycerol; 0 , mannitol, A, arabitol.Iw

as a series of common polyhydric alcohols (mannitol, arabitol, erythritol, and glycerol) as shown in Figure 10. Their importance with regard to the physiology of citric acid fermentation remains as yet to be identified.log

As has been shown above, theoretically, one half of the catabolized hexose is converted to pyruvate, the other to acety1-CoA.lo2 The CO, formed during this reaction, however, is not lost, but is used by pyruvate carboxylase in the formation of oxalacetate. The importance of this "anaplerotic" reaction for the high yield of citric acid accumulation had been previously recognizedfo2 because it supplied the tricarboxylic acid cycle with an excess of oxalacetate. Citric acid production is tightly coupled to the rate of this C0,-fixation since it has recently been established that an almost stoichiometric relationship exists between C0,-fixation and citrate production.so The enzyme responsible for carrying out this reaction is pyruvate carboxylase (EC 6.4.1.1) which in Aspergilli is formed constitutively.'1° Recent studies by Hossain et al.l'I have further indicated that its activity increases with the concentration of glucose or sucrose in the nutrient medium. Unfortunately, the enzyme has not been completely characterized with respect to its kinetic and regulatory properties up to now.

Some purification and characterization has been carried out by Feir and Suzuki1I2 and Wongchai and Jefferson,'" but their results are not completely conclusive since recent results by Osmani and Scrutton113 with the enzyme of the closely related species A . nidulans revealed complex kinetic properties with respect to regulation by acetyl-CoA, aspartate, and a- ketoglutarate. Thus, previous findings on poor regulatory features of A . niger pyruvate carboxylase1'0.112 should be considered with caution. Woronick and Johnson'I4 have also investigated the presence of other enzyme activities .capable of carrying out C0,-fixation in crude extracts of a citric acid-producing A . niger. They reported the presence of a phos- phoenolpyruvate-carboxykinase (EC 4.1.1.32). However, the equilibrium of the enzymic reaction primarily favors the formation of phosphoenolpyruvate and CO,. An involvement of this enzyme in the anaplerotic fixation of CO, in A . niger is thus unlikely.

2 . The Role of the Tricarboxylic Acid Cycle

The importance of inhibiting certain reactions of the tricarboxylic acid cycle for a successful citric acid production has been subject of numerous arguments for the last 30 years, and it

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348 CRC Critical Reviews in Biorechnology Table 9

METABOLIC ALTERATIONS INDUCED BY MANGANESE DEFICIENCY IN ASPERGZLLUS NIGER DURING CITRIC ACID FERMENTATION

Ref.

Accumulation of amino acids and NH:

Elevated proteinase activity Elevated rate of protein degradation Increased monosome portion of ribosomes Decreased cellular phospholipid content

Plasma membrane phospholipid and fatty acid alterations Cell wall chitin elevation and decrease in @-I ,3-glucan Crippled, branched rnycelial development, single giant cells Increased glycolytic flux on the expense of the

pentose phosphate pathway

142 I 45 I45 I45 76 77 62 62 I07

appears that some general statements are necessary. It should be pointed out that the mere measurement of enzyme activities, as carried out by several workers,’o7.’ I 5 - l t 9 cannot provide information on the in vivo activity for the following reasons: (1) breakage of the fungal cell always results in a loss of compartmentation, thus vacuolar components (phenol derivatives, tannic acids, etc.) are released and may inactivate at least part of the enzymic activity;

moreover, in the case of citric acid fermentation, the extracellular citric acid may adsorb to the mycelium and thus be carried over into the extract and denaturate several enzymes by acidification unless the extraction buffer is readjusted in pH during extraction. (2) More important, however, is the fact that even if one assays the complete intracellular enzyme activity, this gives no correlation with the respective in vivo activity since intracellularly the enzyme is most probably not saturated with its substrate and cofactors, and its actual activity would thus be considerably less. For these two reasons, only papers dealing with the in vivo regulation of the tricarboxylic acid cycle shall be considered.

Pulse-labeling studies have up to now not completely resolved the question that for citric acid to accumulate the cycle needs to be inhibited. Shu et al. ^IZo concluded that approximately 40% of citric acid is accumulated from recycled dicarboxylic acids; this however would mean that yields of 80 to 100% as obtained in industrial practice are impossible. Cleland and Johnson,Io2 on the other hand, found evidence for an inhibition of the tricarboxylic cycle, but no data were offered to explain at which step the inhibition occurs. Within the metabolic sequence, aconitase catalyzes the breakdown of citrate; consequently an inhibition of aconitase by iron deficiency or copper addition has been claimed responsible for citrate accumulation.86-’2’.’22 Theoretically, however, there is no necessity for aconitase inhibition since the enzyme catalyzes at an equilibrium which is far on the side of citrate; thus an inhibition of isocitrate dehydrogenase could have a similar effect. Actually, measurement of intracellular metabolite concentrations have shown that aconitase is in equilibrium, even during citric acid accumulation (Table 9). It is thus unlikely that inhibition of aconitase is required for citric acid accumulation.

A further enzyme which has been implicated in the regulation of citric acid accumulation is isocitrate dehydrogenase.

matte^'^^

found that citric acid itself inhibits an NADP-specific isocitrate dehydrogenase, isolated from mitochondria of A. niger (K, of 0.15 mM). The operation of this “feedforward inhibition” was considered to be responsible for citric acid accumulation. This explanation, although convincing at first sight, has two interferences:

first, this would require a mechanism which increases the internal citrate pool to inhibitory levels; thus it cannot be the initial event of citrate accumulation. More important however is the fact that isocitrate breakdown is not solely catalyzed by NADP-specific isocitrate

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3 c d 2

= 1

aJ

L

t

U

.-

8

6.5 7.0 7.5

PH

FIGURE 11. Influence of pH on the inhibition of citrate on NADP- specific isocitrate dehydrogenase with either Mg2+ ( 0 ) or Mn" (A) as cofactor ions. Inhibition is expressed in terms of K, values (mM). The bars indicate intracellular citrate concentrations in A . niger under (A) not citric acid-producing and (B) citric acid-producing conditions.Iz9

dehydrogenase. A. niger contains, in addition, an NAD-linked enzyme'25 which is allosteric and activated by citrate, thus resembling the enzyme from other fungi. ^126.127The presence of the activity of this enzyme was doubtedIz4 as being caused by impurities, but we have recently been successful in demonstrating its activity in a citric acid-producing strain of A.

niger. 12" Moreover, it is established today that both isocitrate dehydrogenases can occur in the mitochondria, ¹²⁸and a detailed kinetic study would be required to decide whether citrate accumulation per se would affect the next flux through both isocitrate dehydrogenases. With respect to NADP-specific isocitrate dehydrogenase, we have recently carried out an extensive kinetic analysis of this enzyme from A . niger B 60Iz9 using a 200- to 300-fold purified preparation (obtained by salt fractionation, Reactive Blue agarose, and Mono Q ion-exchange fast liquid protein chromatography). The enzyme displays mere Michaelis-Menten-type ki- netics with isocitrate and NADP' , Mgz+, or Mn2+-isocitrate chelate being the physiological substrate, and is inhibited by citrate, a-ketoglutarate, NADPH, and glyoxalate plus oxalacetate. Although Bowes and Mattey130 had suggested that citrate inhibits the enzyme more strongly in the presence of Mgz+ ions as cofactor than in the presence of Mn2+ ions, this was not confirmed in the case of our strain B 60. 1 2 9 Actually, the inhibitory effect of citrate appeared to be comparable both in the presence of Mn2+ or Mgz+ (Figure 11). This was also investigated with respect to small changes in the cytoplasmic pH, which might occur during the accumulation of citric acid, but they were without significant effect. In summary, the results show that the K, of the NADP-specific isocitrate dehydrogenase for citrate (3 to 4 mM) is too high to explain citrate inhibition as a trigger of citrate accumulation. However, the citrate concentrations observed during citric acid fermentation (4 to 9 mM in the cell, averaged without considering c~mpartmentation)~~'.'~~ are high enough to inhibit isocitrate breakdown by NADP-IDH to a considerable extent.

Since this mechanism cannot satisfactorily explain the reason for citric acid accumulation,

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350 CRC Critical Reviews in Biotechnofogy

other mechanisms may be considered. We favor an explanation involving regulation of a- ketoglutarate dehydrogenase activity. 1 3 2 This enzyme step, catalyzing the conversion of a- ketoglutarate to succinyl-CoA, is a rate-limiting step in the tricarboxylic acid cycle and furthermore the only irreversible reaction of the cycle.’” Initial investigations involving enzyme activity determinations and metabolic measurements identified this step as a classical

“bottleneck” in citric acid breakdown.

’”

In view of other literature indicating the absence of this enzyme in filamentous fungi, an explanation was initially offered in terms of repression of this enzyme by medium components.’34.135 We have however recently succeeded in the isolation and characterization of this extremely labile enzyme from A . niger. 1 3 6 With respect to the mechanism of citric acid fermentation, it is most interesting that the enzyme is inhibited by physiological concentrations of oxalacetate and NADH. It was thus suggested that the increased supply of oxalacetate during citric acid fermentation, which leads to an increase in the cellular oxalacetate c~ncentration,’~’ decreases the catabolism of citrate at the ^a- ketoglutarate dehydrogenase step and, on the other hand, simultaneously increases the rate of citrate synthesis by citrate synthase. ^j3’ An examination of the regulatory properties of A . niger citrate s y n t h a ~ e ’ ~ ’ indicated that the enzyme is mainly regulated by the availability of oxalacetate. This metabolic situation would lead to a buildup of tricarboxylic acid cycle intermediates and of reduced niconicotamide nucleotide coenzymes (NADH, NADPH). The simultaneous accumulation of both a-ketoglutarate and NADH will decrease the activity of NAD (NADP)-isocitrate dehydrogenase, thereby stimulating a further increase in citrate up to a level where it can probably inhibit the enzyme itself (Figure 12).124.129

This explanation, although supported by a number of biochemical details, is of course still incomplete. In this connection, the most important unknown factor should not be concealed, i.e., how citrate transport out of the mitochondrium is regulated. Hitherto, this has not been studied with A . niger, although in yeasts the tricarboxylate carrier has been shown as being rate limiting to citrate accumulation.‘38 There is some possibility that this is linked to pyruvate carboxylase activity.

In A . niger, pyruvate carboxylase is a cytoplasmic enzyme.’38a A . niger has two malate d e h y d r ~ g e n a s e s ’ ~ ~ of which the cytoplasmic one exhibits a very high activity, thus ensuring that the oxalacetate formed is immediately converted to malate, which is a countersubstrate for the tricarboxylate carrier. ^I4OThus, the increased supply of oxalacetate could also increase the activity of the citrate transporter. Current investigations will clarify whether this hy- pothesis can stand.

3 . Regulation of Carbohydrare Breakdown

Once being transported out of the mitochondria, citrate must pass the cytoplasm before becoming secreted into the medium. Since the enzymes of carbohydrate breakdown (glycolysis, pentose phosphate pathway) are located in the cytoplasm, this leads to the next important point in citric acid accumulation, i.e., how the feedback inhibition of phosphofructokinase by citrate is overcome for allowing high concentrations of citrate to accumulate.

As has been noted above, carbohydrate breakdown by the glycolytic pathway is a prerequisite to citric acid fermentation. Phosphofructokinase from A . niger is inhibited by citrate, ^131.141 thus ruling out the simple possibility that citric acid is produced by organisms containing a citrate-insensitive phosphofructokinase. A kinetic investigation of the partially purified (af- finity chromatography on Blue Dextran@-Sepharose) enzyme from A . niger showed however that the inhibition by citrate can be counteracted by elevated concentrations of NH,‘ ions and inorganic phosphate. 1 3 ’ A study on the behavior of these intermediates further indicated that citric acid accumulation was accompanied by elevated intracellular concentrations of NH,‘ , but not of inorganic phosphate.141.’42 When the activity of the partially purified phosphofructokinase from A . niger was tested under conditions of in vivo concentrations of its substrates and effectors, it was ascertained that the concentrations of NH,‘ observed

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I

G L U C O S E I

NH;

Cytoplasm

I

i

OXALOACETATE PYRUVATE

WRUVATE

t

Acetyl-Co

I

A

1 c

OXALOACETATE

-c

FU MAR ATE SUCC I NATE

SUCCI NYL-COA

I &

_a-KETOGLUTAR ATE

L A ,

FIGURE 12.

indicates activation by the respective metabolite; ( - ) inhibition.

Metabolic scheme of interrelationships in citric acid accumulation; ( + )

during citric acid accumulation can actually antagonize the inhibition by citrate (Table 10).

Factors influencing the intracellular accumulation of NH,+ will be discussed in the next chapter.

Although application of the crossover theoremI3l indicated only a single regulatory point in glycolysis in A . niger, the probable existence of a second one at the pyruvate kinase step was checked.'43 It was found that pyruvate kinase from A. niger is principally a regulatory enzyme, but the enzyme is desensitized by fructose- 1,6-bisphosphate (FBP) against regulatory effects of other metabolites (i.e., ATP). The binding of FBP however is so tight (Kd,,*

= and the K, so small (below 0.1 @that it must be assumed that in vivo the I) enzyme is permanently unsusceptible to regulation. It is thus unlikely that a regulation at this step occurs in A. niger.

Most recently, however, an additional regulatory step was identified at the level of sugar transport by Mischak et al. who studied glucose transport into citric acid-producing mycelia of A . niger. Citrate strongly inhibits glucose transport (I,,5 15 M), which is not

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352 CRC Critical Reviews in Biotechnology Table 10

CONCENTRATIONS OF METABOLITES IN ASPERGILLUS NIGER MYCELIUM UNDER CITRIC

ACID-PRODUCING (MANGANESE DEFICIENT) AND

-