JOURNAL OF BACTERIOLOGY, Jan. 1970, p. 65-71 Vol. 101, No. 1Copyright 0 1970 American Society for Microbiology Printed in U.S.A.

Fatty Acid Toxicity and Methyl KetoneProduction in Aspergillus niger

HAROLD L. LEWIS' AND DENNIS W. DARNALL2Department of Biology, Texas Tech University, Lubbock, Texas 79409

Received for publication 18 October 1969

Vegetative hyphae of Aspergillus niger rapidly converted caproic acid into 2-penta-none. More caproic acid was required for maximal ketone production at alkaline ascompared to acidic pH values. Further increases in caproate concentrations at eachpH value tested (4.5, 5.5, 6.5, 7.5, and 8.5) resulted in inhibition of ketone produc-tion and 02 uptake. At alkaline pH values (8.5 and 7.5), oxygen uptake above theendogenous level and the production of 2-pentanone were parallel. This relation-ship did not hold at acidic pH values. At these pH values, ketone production con-tinued (pH 6.5) or attained a maximum (pH 5.5 and 4.5) at caproate concentrationsat which oxygen uptake was inhibited below endogenous levels. These data indicatethat endogenous oxygen uptake was not inhibited by caproate at alkaline pH valuesat concentrations which did inhibit caproate oxidation and 2-pentanone production.Conversely, at acidicpH values, endogenous oxygen uptake was vigorously inhibitedby caproate at concentrations at which exogenous fatty acid oxidation and 2-penta-none production were less affected. Simon-Beevers plots of these data showed thatthe undissociated acid was the permeant form of caproic acid. The fatty anion ap-peared to be the active or inhibitory form of caproate within the cell. Vegetativehyphae of A. niger were poorly buffered. Once the hyphae were washed and re-suspended in phosphate buffer, they were well buffered towards inhibitory concen-trations of caproic acid. These findings suggest that the primary mechanism(s) bywhich caproate inhibits oxygen uptake and ketone formation does not involve achange in the intracellular pH.

As early as 1899, Biffen (2) reported theisolation of a fungus which grew on coconutmeats with the production of a pleasant odorsimilar to amyl butyrate. Conversion of individualfatty acids to the corresponding methyl ketonesby "pure cultures" of Penicillium glaucum wassubsequently reported (6, 19, 20). In some cases,the expected ketones were obtained when "pure"triglycerides were provided as substrates. Forexample, Acklin (1) reported ketone yields ashigh as 48% of the theoretical value from tri-caproin.

Kiesel (13) studied the effects of fatty acidscontaining from one through six carbon atomson Aspergillus niger. Caproic acid inhibitedgermination of spores, formation of mycelium,and production of conidia by this organism. Nomention of ketone formation was made. Anextensive study of the methyl ketone-formingability of a large number of fungi was reported

1 Present address: Research Division, National Cotton Councilof America, Memphis, Tenn. 38112.

2 Present address: Department of Chemistry, New MexicoState University, Las Cruces, N.M. 88001.

by Gehrig and Knight (9). They found that 9 of11 Aspergillus species and 9 of 12 Penicilliumspecies could produce 2-heptanone from caprylicacid. Inhibition by the acid was not discussed.

Thaler and Geist (22) reported that acidic pHvalues were favorable to methyl ketone forma-tion. Thaler and co-workers subsequently re-ported (21, 23) that P. glaucum produced thecorresponding methyl ketones from beta-hydroxyacids and alpha, beta-unsaturated acids. Theseworkers concluded that methyl ketone productionproceeds according to the beta-oxidation pathwayby decarboxylation of beta-keto-acids. Experi-ments with cell-free fungal enzyme preparationssupport this hypothesis (8, 11, 25).Although much information has accumulated

describing the inhibition of fungal metabolism byfatty acids and the production of methyl ketonesfrom the acids by the same organisms, no studiesof the relationship of the two processes have beenreported. This paper constitutes an attempt todefine the relationships and interactions of thesephenomena in A. niger.

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MATERIALS AND METHODS

A. niger van Tieghem was isolated in our laboratoryfrom old caproic acid solutions which possessed astrong odor of 2-pentanone. A single-spore isolate wasobtained by dilution and pour plating on CzapekSolution Agar (Difco) supplemented with 0.1% yeastextract. Stock cultures were maintained on the samemedium.

Spores of A. niger were grown on slants of glucose(40 g/liter)-yeast extract (5 g/liter)-neopeptone (10g/liter)-agar (15 g/liter). Excellent sporulation was ob-tained on this medium after 4 to 5 days of incubationat 30 C. Spores were harvested by adding 10 ml ofsterile 0.85% NaCl to the slant and rubbing gentlywith a stiff inoculating needle. The suspension wasadjusted to contain 5 X 107 spores per ml, and 1.0ml was added to each flask ofa solution containing, perliter: 40 g of glucose, 5 g of yeast extract, and 10 g ofneopeptone (50 ml per 250-ml Erlenmeyer flask). Allflasks were incubated on a rotary shaker (New Bruns-wick Scientific Co., New Brunswick, N. J.; Psychro-therm) operated at 180 rev/min at 30 C. After 24 hrof incubation, the vegetative hyphae were harvested,washed three times, and resuspended in 0.066 M S0ren-sen phosphate buffer (Na2HPO4 plus KH2PO4) at thepH of the experiment. Harvesting and washing weredone by centrifugation. Residual ungerminated sporeswere removed by washing through two layers ofcheese-cloth. The cell volume was adjusted to 0.230 ml ofcells per ml of cell suspension by either removal oraddition of the phosphate buffer solution.

Manometric measurements were done in a constantvolume respirometer (Braun Instrument Co.). EachWarburg cup contained 1.0 ml of the above describedcell suspension and 1.0 ml of 0.066 M S0rensen phos-phate buffer in the main compartment and 1.0 ml offatty acid substrate at the proper concentration andpH in the side arms. Carbon dioxide was absorbed by0.1 ml of 20% KOH in the center well. The gas phasewas air. Oxygen uptake was followed for a period of2 hr with readings taken every 30 min.

Immediately after oxygen uptake experiments werecomplete, the contents of the Warburg flasks wereacidified with 0.1 ml of concentrated HC1, and 2-pentanone was determined by gas-liquid chromatog-raphy. A sample of the acidified reaction mixture wasinjected onto a stainless-steel column [5 ft by j inch(154 by 0.3 cm)] packed with dimethyldichlorosilane-treated, acid-washed, 70/80 U.S. mesh ChromosorbW containing 20% phenyl-diethanolamine succinateas a stationary phase. The column was operated at100 C with the injector block at 150 C. An Aerographchromatograph (model A-600-D) equipped with ahydrogen flame ionization detector was used for allanalyses. The carrier gas was nitrogen flowing at 25ml/min. The detector was operated at the same tem-perature as the column, since it was mounted in thecolumn oven. Quantification was accomplished bypeak height measurements and comparing these valuesto a standard curve obtained in the same way forpure 2-pentanone.

Intracellular pH (pHi) measurements were basedon Kotyk's (14) demonstration that only the undis-

sociated form of 2,4-dinitrophenol (DNP) pene-trates the cell surface. The method was basically thatdescribed by Neal et al. (15). Cell suspensions wereprepared in the same manner as used for manometricexperiments. The reaction mixture was 7 ml of hyphalsuspension, 7 ml of S0rensen phosphate buffer at thedesired pH, and 7 ml of either water or the fatty acidsubstrate at the desired concentration and pH value.The reaction mixture was equilibrated for 30 min on arotary shaker (30 C, 180 rev/min) before the additionof DNP. The mixtures were equilibrated for 20 minafter the addition ofDNP and rapidly filtered througha membrane filter (Millipore Corp., Bedford, Mass.;0.45 gm poresize). Intercellular space measurementswere done by the gravimetric inulin space method (5).Optical density measurements were made with a Beck-man model DB spectrophotometer.The following equation defines the distribution of

the molecular species of caproic acid between thehyphae and the suspending medium at equilibrium:

[HA]t.t = [HA]o(Lf) + [HA]i (Cf) + [A-]. (Lf)+ [A-]i(Cf) (1)

where [HA]tot = total caproic acid concentrationadded to the system; [HA]. = undissociated caproicacid concentration in the liquid phase at equilibrium;[A-]. = caproate anion concentration in the liquidphase at equilibrium; [HA]i = undissociated caproicacid concentration in the cell phase at equilibrium;[A-]i = caproate ion concentration in the cell phaseat equilibrium; Lf = liquid fraction of the system;and Cf = cell fraction of the system. The followingtwo equations may be written concerning the pHvalues in the liquid fraction of the system (2) and thecell fraction of the system (3) at equilibrium:

pH. = pKa + log [A-]o/[HA]opHi = pKa + log [A-]i/[HA]i

(2)(3)

Examination of these three equations reveals that[HA]tot, pHo, pHi, and pKa (caproate) are knownvalues. Similarly, [HA]I, [HA];, [A-]j, and [A-]. areunknown values. From Kotyk's work (14), it may beassumed that [HA]o = [HA]i. Therefore, we havethree independent equations in three unknowns andmay solve them simultaneously. This method was usedfor estimating [HA]i and [A-]i in vegetative hyphaeof A. niger.

Undissociated caproic acid concentrations forSimon-Beevers plots (18) were calculated by meansof the well-known Henderson-Hasselbach equation.Since the degree of protolysis of weak acids is affectedby changing ionic strengths, our calculations werecorrected for this parameter by defining an apparentpKa (pK'a; 7). A pKa value of 4.85 at 25 C wastaken for caproic acid (12).

Titration curves for vegetative hyphae of A. nigerwere done on preparations of cells grown for 24 hr inthe same manner as described above for manometricexperiments. The hyphal preparations were titratedagainst 0.01 N KOH. Buffer values were calculatedfrom the slope of the titration curve and representrelative values only, i.e., rate of change of pH permilliequivalent of hydroxyl ion added.

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RESULTSBased upon several reports of the inhibition of

respiration in fungi by fatty acids (10,' 16, 17),experiments were designed to determine whethercaproate inhibits respiration in vegetative hyphaeof A. niger and to determine the relationship ofthe inhibition to 2-pentanone production. Theresponse of vegetative hyphae to caproic acidwas 'studied as a function of total fatty acidconcentration and the pH of the external medium(pHO). 2-Pentanone was never produced in theabsence of caproate.At pH 8.5 (Fig. 1A), maximal 02 uptake and

ketone production occurred at the same point,i.e., 0.80% caproate. The same relationship heldfor inhibition. Ketone formation stopped when02 consumption fell below the endogenous level.A similar relationship was found at pH 7.5. Inthis case, the total amount of caproate requiredfor maximal stimulation or inhibition was lessthan at pH 8.5.At pH 6.5 (Fig. 1B), maximal 02 uptake and

ketone production occurred at the same caproateconcentration (0.05%), and the hyphae seemedto continue 2-pentanone production after 02uptake had decreased below the endogenous rate.This trend was even more pronounced at pH 5.5(Fig. 1C), at which the parallel between 02uptake and ketone production broke downcompletely. Maximal ketone production oc-curred at a caproate concentration (0.03%) atwhich 02 uptake was inhibited below the en-

dogenous level. Furthermore, 2-pentanone con-tinued to be formed at fatty acid concentrationsat which 02 uptake was considerably lower thanendogenous.At pH 4.5 (Fig. 1D), low concentrations of

caproate inhibited 02 uptake below the endoge-nous level, whereas ketone production continuedat a significant rate. Maximal 2-pentanone pro-duction occurred at a caproate concentration(0.006%) approximately three times greater thanthat required to inhibit 02 uptake below the en-dogenous level.

Simon-Beevers plots (18) for equi-effectiveconcentrations of caproate towards vegetativehyphae of A. niger are shown in Fig. 2. Sincethe inhibition under investigation was a sub-strate inhibition, the acid' concentration whichyielded a 50% reduction in maximal ketoneproduction was selected for analysis. Similarly,the acid concentration required to effect a 50%reduction in maximal 02 uptake was taken forcomparative purposes. The undissociated acidconcentration required for 50% inhibition ofketone production increased only slightly (5.08 x10-4 to 6.97 X 10-4 M) with an increase inexternal pH (pHo) from 4.5 to 5.5. On theother hand, the undissociated acid concentrationrequired to yield the same degree of inhibitionbetween pHo 5.5 and 8.5 decreased sharply(6.7 x 10-4 to 3.00 X 10-5 M). The same quali-tative relationship was found for the inhibition of02 uptake. However, 02 uptake was inhibited by

12

8,1086

1420

-2

2 4 6 8 10 12 14 16 182022242628PER CENT CAPROIC ACID

.1 .2 3 4 5PER CENT CAPROIC ACID

4

32

-2-3

ENSOGEwOuS

.01 .02 .03 .04 .05 002 .004 006 008 D09 D12 .04.- D06PER CENT CAPROIC ACD PER CENT CAPROIC ACID

FiG. 1. 2-Pentanone production (0) and oxygen uptake (0) responses of vegetative hyphae of Aspergillusniger in the presence ofstimulatory to inhibitory concentrations ofcaproic acid. A, pH 8.5; B, pH 6.5; C, pH 5.5;D,pH 4.S.

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LEWIS AND DARNALL

much lower concentrations ofat acidic pHo values than wasAt higher pHo values, they paquite closely.

2 0.000z -1.0000

w -2.00

0x -3.00a-0

-4.000J

undissociated acid The variation in undissociated caproic acidketone production. concentration required for 50% inhibition ofLralleled each other maximal 02 uptake or ketone production at

different pH. values suggested that the fatty acidaltered the pHi of the hyphae and that a fractionof the inhibition resulted from the altered pHi(15). In all experiments, the hyphae were sus-pended in phosphate buffer before addition ofthe fatty acid to maintain the desired pHo. ThepHi varied almost directly as the pH of the phos-phate buffer (pHo) was changed (Fig. 3). How-ever, the pHi values of hyphae treated withstimulatory to inhibitory concentrations ofcaproate were not significantly different from thepHi values obtained with phosphate bufferalone. This was true for all pH0 values tested(4.5, 5.5, 6.5, 7.5, and 8.5).The relationship of [A-]i and [HA]i to pHi

for 50% inhibition of .maximal 02 uptake andketone production is shown in Fig. 4. Increasingintracellular concentrations of anion were re-quired to effect the same degree of inhibition asthe pHi increased. Changes in intracellular

45 5.5 6.5 75 8.5EXTERNAL pH

FIG. 2. Log caproic acid concentrations necessaryto yield 50% inhibition of maximal 02 uptake and 2-pentanone production by vegetative hyphae of Asper-gillus niger at various pHo values. Symbols: *, logtotal caproic acid concentration to inhibit ketone pro-duction; 0, log total caproic acid concentration toinhibit 02 uptake; a, log undissociated caproic acidconcentration to inhibit ketone production; 0, logundissociated caproic acid concentration to inhibit 02uptake.

10.0

I

zLUI

z

z

a-.

9.00

8.00

700

6.00

5.00

FiG. 3. Relchyphae of Aspin 0.022 M phlo

1-

( 0.00z0U

- 1.00LLJ

-2.000~CL< -3.00

o -4.00J

4.5 5.5 6.5 75 8.5pHi

FIG. 4. Log intracellular caproic acid concentra-tions necessary to yield 50% inhibition of maximal 02

Ifi*| s | uptake and 2-pentanone production by vegetativehyphae of Aspergillus niger at various pHi values.

4.5 5.5 65 75 8.5 Symbols: 0, log undissociated caproic acid concentra-EXTERNAL pH tion to inhibit ketone production; O, log undissociatedEXTERNALpH caproic acid concentration to inhibit 02 uptake; 0,

itionship of pHi to pHo of vegetative log caproate anion concentration to inhibit ketone;ergillus niger washed and resuspended production; *, log caproate anion concentration to)sphate buffer. inhibit 02 uptake.

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VOL. 101, 1970 FATTY ACID TOXICITY AND METHYL KETONE PRODUCTION

undissociated acid concentrations were the sameas in the starting medium, which would be ex-pected since the extracellular medium was wellbuffered.The endogenous Qo, was lowest at alkaline

pH. values, increased to a maximum at pH. 5.5,and decreased at pHo 4.5 (Table 1). In general,this same relationship was found for endogenousQo2 and pHj; however, pHi did not change asmuch at low pH. values (4.5 to 5.5) as at highpH. values (6.5 to 8.5), which appears to be re-flected in the endogenous rates (Table 1). Thesedata suggest that the hyphal contents are bufferedto some extent at low pH values (4.5 to 5.5) butare more or less unbuffered at high pH values.A titration curve and buffer values for vegetativehyphal suspensions of A. niger are shown in Fig.5. In all cases, the hyphal suspensions had astarting pH of approximately 3.5. At low pHvalues (4.0 to 5.0), hyphal suspensions possesseda relatively high buffer value (0.29 to 0.10). Asthe pH was increased the buffer value rapidly de-creased to a minimum at pH 7.5 (0.03) and thensteadily increased to 0.17 at pH 10. Although thevalidity of such a titration curve may be ques-tioned, the results obtained constitute a good fitfor our physiological data and indicate that thehyphal contents are relatively well buffered atlow pH values and rapidly approach the un-buffered condition at higher pH values.

DISCUSSION

Caproic acid behaved as a substrate inhibi-tor towards pregrown vegetative hyphae of A.niger. At alkaline pHo values (Fig. 1A), 02uptake over the endogenous level and 2-penta-none production followed each other almostmole per mole. These data support the theory(21) that methyl ketone production from fattyacid proceeds via the beta-oxidation pathway bydecarboxylation of the beta-keto acid formed.Oxygen uptake above endogenous levels appearedto be solely a result of the oxidation of caproateto 2-pentanone. These results indicate that endog-enous 02 uptake per se was not inhibited bycaproate at alkaline pHo values at concentrationswhich inhibited oxidation of exogenous caproateto 2-pentanone. At acidic pHo values (Fig. IB,C, and D), this relationship failed to hold. Ketoneproduction continued (pH, 6.5) and, in somecases (pH. 5.5 and 4.5), reached a maximum atwhich 02 uptake was inhibited well below endog-enous levels. Thus, at acidic pHo values, endog-enous 02 uptake per se was vigorously inhibitedby caproate concentrations at which exogenousfatty acid oxidation was not, at least not to asgreat an extent.

Other workers (3, 4) have demonstrated that

TABLE 1. Relationship of the rate of endogenous 02uptake of vegetative hyphae oJ A. niger to

eAternal and intternial pH

pH of external Endogenous Q02' Apparentofextem internal pH

4.50 36.3 5.415.50 51.1 5.926.50 22.5 6.707.50 13.3 7.618.50 15.2 8.59

a Qo2 = microliters of 02 Xgrams of dry cell weight-'.

hour-' X milli-

exogenous acetate inhibits endogenous respira-tion 50 to 100% in P. chrysogenum and Neuro-spora crassa at a pH, value of 6.0. In the presentwork, endogenous activity was most sensitive tocaproate inhibition at acidic pHo and pH i valuesnear its optimum, and least sensitive at alkalinepH. and pHi values where it was minimal (Ta-ble 1). These results show that the degree of in-hibition of endogenous activity by exogenous sub-strates is not only a function of the past historyof the organism, i.e., growth substrate, age, andconditions of culture, but also of the pHo valueof the manometric determination.The optimal pHo value for 2-pentanone pro-

duction was 6.5, which corresponded to a pHivalue of 6.7. This agrees with the report (8) thatthe optimal pH for the beta-keto acid decarboxyl-ase from A. niger is 6.8.

Neal et al. (15) proposed a mechanism for thetoxicity of fatty acids toward yeast. They sug-gested that the undissociated acid enters the celland dissociates, causing an acidification of thecell contents and a subsequent inhibition of res-piratory and glycolytic enzymes. Examinationof the Simon-Beevers plots (Fig. 2) reveals thatthe undissociated acid is the permeant form ofthe molecule. The concept that intracellular acidi-fication is the primary mechanism of inhibition isquestionable, since the inhibition of either 02uptake or ketone production by caproate in A.niger was independent of changes in pHi at agiven pHo value. High concentrations of fattyacids, as used by Neal et al., can, undoubtedly,cause a decrease in pHi value. Nevertheless, theinhibition could have been accomplished bylower concentrations of the acids which wouldnot have altered the pH inside the cell. Thus, itseems necessary to distinguish between changesinduced in cells by nonphysiological (high) con-centrations of fatty acids as compared to physio-logical (low) concentrations, when one wishes todiscuss the mechanism(s) by which these mole-cules inhibit cellular activities.

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Since the undissociated acid is the form ofcaproate which penetrates the cell, one might ex-pect a given degree of inhibition with a given un-dissociated acid concentration regardless of thepHo. That this is not the case is shown by theSimon-Beevers plots (Fig. 2). The most signifi-cant feature of these plots is that at high pHovalues less undissociated acid was required tocause the same degree of inhibition than at lowpH. values. This relationship has been found tohold for many weak acids with respect to inhibi-tion of yeast respiration (18). Furthermore,several theories, primarily involving cell perme-ability, have been advanced as explanations (24).However, if the increase in pHi with increase inpHo is taken into account (Fig. 3), a more directexplanation of the phenomenon may be obtained.If the fatty anion were the active form inside thecell and the undissociated acid the permeantform, then as the pHi increased a smaller quan-tity of undissociated acid would be required in theexternal medium to effect a given concentrationof anion inside the cell. Inspection of the log[A-]i-pHi plots (Fig. 4) shows that an increasingconcentration of anion inside the cell was re-quired to cause a 50% inhibition of either 02 UP-take or ketone production as the pHi increased.This finding appears to be contradictory until oneaccounts for the binding of the anion to the en-zyme proteins involved. As the pHi increased, thecharge on all proteins within the cell should havebecome more negative and the affinity of thesemolecules for the fatty anion lowered. Therefore,a higher concentration of anion would be neededto cause a given degree of inhibition. Our experi-mental data are consistent with this concept andsupport the theory that the fatty anion is the ac-tive form of caproic acid with reference to inhibi-tion of 02 uptake and 2-pentanone production inA. niger.

Vegetative hyphae of A. niger appeared to ap-proach the completely unbuffered state beforewashing and resuspension in phosphate buffer.Nevertheless, they were more highly buffered atlow pH values (4.5 to 5.5) than at higher ones(5.5 to 8.5). This concept is supported by boththe intracellular pH measurements (Fig. 3) andthe titration curve (Fig. 5). After washing andresuspension in phosphate buffer, the hyphae ap-peared to possess good buffer strength, since in-creasing caproate concentrations caused no de-tectable change in the apparent pHi. The degreeof enzyme inhibition produced by a compoundsuch as caproic acid should depend on the buffercapacity of the cell. In general, two extreme casesmay be discussed: completely buffered and com-pletely unbuffered cells (24). If cells were com-pletely buffered, the accumulation of fatty anion

0Coz

w

-J-l

070

0.60

Q50

040

030

020

Q.I

0

3028.262422.20 w.18 mY.16 m.124 m

JO r,.08 FM0604020

3.5 4.5 5.5 65 75 8.5 9.5 IGOpH

FIG. 5. Titration curve and relative buffer values ofvegetative hyphae of Aspergillus niger. Symbols: *,titration curve; 0, relative buffer values (i.e., rate ofchange ofpHper milliequivalent ofhydroxyl ion added).

would be much greater than in unbuffered cells.The greater the buffer capacity of a cell, the moreundissociated acid that would penetrate and thegreater the fatty anion or total inhibitor concen-tration in the cell. In reality, cells are neither un-buffered nor completely buffered, but fall some-where between the two extremes. It would be veryuseful to know the true buffer capacity of cells,but no adequate quantitative work has yet beendone. The data presented in this paper indicatethat intracellular pH measurements in a definedphysiological system can yield valuable informa-tion about the buffer capacity of cells.

ACKNOWLEDGMENT

This investigation was supported by grant D-175 from TheRobert A. Welch Foundation, Houston, Tex.

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4. Blumenthal, H. J., H. Koffler, and E. C. Heath. 1957. Biochem-istry of filamentous fungi. V. Endogenous respirationduring concurrent metabolism of exogenous substrates.J. Cell. Comp. Physiol. 50:471-497.

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6. Coppock, P. D., V. Subramaniam, and T. K. Walker. 1928.The mechanism of the degradation of fatty acids by mouldfungi. J. Chem. Soc. (London), p. 1422-1427.

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8. Franke, W., A. Platzeck, and G. Eichhorn. 1961. Zur Kennt-nis des Fettsaiureabbaus durch Schimmelpilze. III. Ubereine Decarboxylase der mittleren beta-Ketomonocarbon-

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14. Kotyk, A. 1962. Uptake of 2,4-dinitrophenol by the yeast cell.Folia Microbiol. 7:109-114.

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biological activities of weak acids and bases. 1. The mostusual relationship between pH and activity. New Phytol.51:163-190.

19. Stairkle, M. 1924. Die Methylketone im oxidativen Abbaueiniger Triglyceride (bzw. Fettsauren) durch Schimmelpilzeunter Berucksichtigung der besondern Ranziditait desKokosfettes. I. Die Bedeutung der Methylketone im derButterranziditat. II. Ober die Entstehung und Bedsutungder Methylketone als Aromastoffe im Roquefortkase.Biochem. Z. 151:371-415.

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22. Thaler, H., and G. Geist. 1939. Zur Chemie der Ketonran-zigkeit. 1. Ober den Abbau gesittigter Fettsauren durchPeni.illium glaucum. Biochem. Z. 302:121-136.

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