Fatty Acid Toxicity and Methyl Ketone Production in Aspergillus niger

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1 JOURNAL OF BACTERIOLOGY, Jan. 197, p Vol. 11, No. 1 Copyright 197 American Society for Microbiology Printed in U.S.A. Fatty Acid Toxicity and Methyl Ketone Production in Aspergillus niger HAROLD L. LEWIS' AND DENNIS W. DARNALL2 Department of Biology, Texas Tech University, Lubbock, Texas 7949 Received for publication 18 October 1969 Vegetative hyphae of Aspergillus niger rapidly converted caproic acid into 2-pentanone. More caproic acid was required for maximal ketone production at alkaline as compared to acidic ph values. Further increases in caproate concentrations at each ph value tested (4.5, 5.5, 6.5, 7.5, and 8.5) resulted in inhibition of ketone production and 2 uptake. At alkaline ph values (8.5 and 7.5), oxygen uptake above the endogenous level and the production of 2-pentanone were parallel. This relationship did not hold at acidic ph values. At these ph values, ketone production continued (ph 6.5) or attained a maximum (ph 5.5 and 4.5) at caproate concentrations at which oxygen uptake was inhibited below endogenous levels. These data indicate that endogenous oxygen uptake was not inhibited by caproate at alkaline ph values at concentrations which did inhibit caproate oxidation and 2-pentanone production. Conversely, at acidic ph values, endogenous oxygen uptake was vigorously inhibited by caproate at concentrations at which exogenous fatty acid oxidation and 2-pentanone production were less affected. Simon-Beevers plots of these data showed that the undissociated acid was the permeant form of caproic acid. The fatty anion appeared to be the active or inhibitory form of caproate within the cell. Vegetative hyphae of A. niger were poorly buffered. Once the hyphae were washed and resuspended in phosphate buffer, they were well buffered towards inhibitory concentrations of caproic acid. These findings suggest that the primary mechanism(s) by which caproate inhibits oxygen uptake and ketone formation does not involve a change in the intracellular ph. As early as 1899, Biffen (2) reported the isolation of a fungus which grew on coconut meats with the production of a pleasant odor similar to amyl butyrate. Conversion of individual fatty acids to the corresponding methyl ketones by "pure cultures" of Penicillium glaucum was subsequently reported (6, 19, 2). In some cases, the expected ketones were obtained when "pure" triglycerides were provided as substrates. For example, Acklin (1) reported ketone yields as high as 48% of the theoretical value from tricaproin. Kiesel (13) studied the effects of fatty acids containing from one through six carbon atoms on Aspergillus niger. Caproic acid inhibited germination of spores, formation of mycelium, and production of conidia by this organism. No mention of ketone formation was made. An extensive study of the methyl ketone-forming ability of a large number of fungi was reported 1 Present address: Research Division, National Cotton Council of America, Memphis, Tenn Present address: Department of Chemistry, New Mexico State University, Las Cruces, N.M by Gehrig and Knight (9). They found that 9 of 11 Aspergillus species and 9 of 12 Penicillium species could produce 2-heptanone from caprylic acid. Inhibition by the acid was not discussed. Thaler and Geist (22) reported that acidic ph values were favorable to methyl ketone formation. Thaler and co-workers subsequently reported (21, 23) that P. glaucum produced the corresponding methyl ketones from beta-hydroxy acids and alpha, beta-unsaturated acids. These workers concluded that methyl ketone production proceeds according to the beta-oxidation pathway by decarboxylation of beta-keto-acids. Experiments with cell-free fungal enyme preparations support this hypothesis (8, 11, 25). Although much information has accumulated describing the inhibition of fungal metabolism by fatty acids and the production of methyl ketones from the acids by the same organisms, no studies of the relationship of the two processes have been reported. This paper constitutes an attempt to define the relationships and interactions of these phenomena in A. niger. 65 Downloaded from on June 21, 218 by guest

2 66 LEWIS AND DARNALL J. BACTERIOL. MATERIALS AND METHODS A. niger van Tieghem was isolated in our laboratory from old caproic acid solutions which possessed a strong odor of 2-pentanone. A single-spore isolate was obtained by dilution and pour plating on Capek Solution Agar (Difco) supplemented with.1% yeast extract. Stock cultures were maintained on the same medium. Spores of A. niger were grown on slants of glucose (4 g/liter)-yeast extract (5 g/liter)-neopeptone (1 g/liter)-agar (15 g/liter). Excellent sporulation was obtained on this medium after 4 to 5 days of incubation at 3 C. Spores were harvested by adding 1 ml of sterile.85% NaCl to the slant and rubbing gently with a stiff inoculating needle. The suspension was adjusted to contain 5 X 17 spores per ml, and 1. ml was added to each flask ofa solution containing, per liter: 4 g of glucose, 5 g of yeast extract, and 1 g of neopeptone (5 ml per 25-ml Erlenmeyer flask). All flasks were incubated on a rotary shaker (New Brunswick Scientific Co., New Brunswick, N. J.; Psychrotherm) operated at 18 rev/min at 3 C. After 24 hr of incubation, the vegetative hyphae were harvested, washed three times, and resuspended in.66 M Srensen phosphate buffer (Na2HPO4 plus KH2PO4) at the ph of the experiment. Harvesting and washing were done by centrifugation. Residual ungerminated spores were removed by washing through two layers ofcheesecloth. The cell volume was adjusted to.23 ml of cells per ml of cell suspension by either removal or addition of the phosphate buffer solution. Manometric measurements were done in a constant volume respirometer (Braun Instrument Co.). Each Warburg cup contained 1. ml of the above described cell suspension and 1. ml of.66 M Srensen phosphate buffer in the main compartment and 1. ml of fatty acid substrate at the proper concentration and ph in the side arms. Carbon dioxide was absorbed by.1 ml of 2% KOH in the center well. The gas phase was air. Oxygen uptake was followed for a period of 2 hr with readings taken every 3 min. Immediately after oxygen uptake experiments were complete, the contents of the Warburg flasks were acidified with.1 ml of concentrated HC1, and 2- pentanone was determined by gas-liquid chromatography. A sample of the acidified reaction mixture was injected onto a stainless-steel column [5 ft by j inch (154 by.3 cm)] packed with dimethyldichlorosilanetreated, acid-washed, 7/8 U.S. mesh Chromosorb W containing 2% phenyl-diethanolamine succinate as a stationary phase. The column was operated at 1 C with the injector block at 15 C. An Aerograph chromatograph (model A-6-D) equipped with a hydrogen flame ioniation detector was used for all analyses. The carrier gas was nitrogen flowing at 25 ml/min. The detector was operated at the same temperature as the column, since it was mounted in the column oven. Quantification was accomplished by peak height measurements and comparing these values to a standard curve obtained in the same way for pure 2-pentanone. Intracellular ph (phi) measurements were based on Kotyk's (14) demonstration that only the undissociated form of 2,4-dinitrophenol (DNP) penetrates the cell surface. The method was basically that described by Neal et al. (15). Cell suspensions were prepared in the same manner as used for manometric experiments. The reaction mixture was 7 ml of hyphal suspension, 7 ml of Srensen phosphate buffer at the desired ph, and 7 ml of either water or the fatty acid substrate at the desired concentration and ph value. The reaction mixture was equilibrated for 3 min on a rotary shaker (3 C, 18 rev/min) before the addition of DNP. The mixtures were equilibrated for 2 min after the addition of DNP and rapidly filtered through a membrane filter (Millipore Corp., Bedford, Mass.;.45 gm poresie). Intercellular space measurements were done by the gravimetric inulin space method (5). Optical density measurements were made with a Beckman model DB spectrophotometer. The following equation defines the distribution of the molecular species of caproic acid between the hyphae 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 concentration added to the system; [HA]. = undissociated caproic acid concentration in the liquid phase at equilibrium; [A-]. = caproate anion concentration in the liquid phase at equilibrium; [HA]i = undissociated caproic acid concentration in the cell phase at equilibrium; [A-]i = caproate ion concentration in the cell phase at equilibrium; Lf = liquid fraction of the system; and Cf = cell fraction of the system. The following two equations may be written concerning the ph values in the liquid fraction of the system (2) and the cell fraction of the system (3) at equilibrium: ph. = pka + log [A-]o/[HA]o (2) (3) phi = pka + log [A-]i/[HA]i Examination of these three equations reveals that [HA]tot, pho, phi, and pka (caproate) are known values. Similarly, [HA]I, [HA];, [A-]j, and [A-]. are unknown values. From Kotyk's work (14), it may be assumed that [HA]o = [HA]i. Therefore, we have three independent equations in three unknowns and may solve them simultaneously. This method was used for estimating [HA]i and [A-]i in vegetative hyphae of A. niger. Undissociated caproic acid concentrations for Simon-Beevers plots (18) were calculated by means of the well-known Henderson-Hasselbach equation. Since the degree of protolysis of weak acids is affected by changing ionic strengths, our calculations were corrected for this parameter by defining an apparent pka (pk'a; 7). A pka value of 4.85 at 25 C was taken for caproic acid (12). Titration curves for vegetative hyphae of A. niger were done on preparations of cells grown for 24 hr in the same manner as described above for manometric experiments. The hyphal preparations were titrated against.1 N KOH. Buffer values were calculated from the slope of the titration curve and represent relative values only, i.e., rate of change of ph per milliequivalent of hydroxyl ion added. Downloaded from on June 21, 218 by guest

3 VOL. 11, 197 FATTY ACID TOXICITY AND METHYL KETONE PRODUCTION RESULTS Based upon several reports of the inhibition of respiration in fungi by fatty acids (1,' 16, 17), experiments were designed to determine whether caproate inhibits respiration in vegetative hyphae of A. niger and to determine the relationship of the inhibition to 2-pentanone production. The response of vegetative hyphae to caproic acid was 'studied as a function of total fatty acid concentration and the ph of the external medium (pho). 2-Pentanone was never produced in the absence of caproate. At ph 8.5 (Fig. 1A), maximal 2 uptake and ketone production occurred at the same point, i.e.,.8% caproate. The same relationship held for inhibition. Ketone formation stopped when 2 consumption fell below the endogenous level. A similar relationship was found at ph 7.5. In this case, the total amount of caproate required for maximal stimulation or inhibition was less than at ph 8.5. At ph 6.5 (Fig. 1B), maximal 2 uptake and ketone production occurred at the same caproate concentration (.5%), and the hyphae seemed to continue 2-pentanone production after 2 uptake had decreased below the endogenous rate. This trend was even more pronounced at ph 5.5 (Fig. 1C), at which the parallel between 2 uptake and ketone production broke down completely. Maximal ketone production occurred at a caproate concentration (.3%) at which 2 uptake was inhibited below the en PER CENT CAPROIC ACID dogenous level. Furthermore, 2-pentanone continued to be formed at fatty acid concentrations at which 2 uptake was considerably lower than endogenous. At ph 4.5 (Fig. 1D), low concentrations of caproate inhibited 2 uptake below the endogenous level, whereas ketone production continued at a significant rate. Maximal 2-pentanone production occurred at a caproate concentration (.6%) approximately three times greater than that required to inhibit 2 uptake below the endogenous level. Simon-Beevers plots (18) for equi-effective concentrations of caproate towards vegetative hyphae of A. niger are shown in Fig. 2. Since the inhibition under investigation was a substrate inhibition, the acid' concentration which yielded a 5% reduction in maximal ketone production was selected for analysis. Similarly, the acid concentration required to effect a 5% reduction in maximal 2 uptake was taken for comparative purposes. The undissociated acid concentration required for 5% inhibition of ketone production increased only slightly (5.8 x 1-4 to 6.97 X 1-4 M) with an increase in external ph (pho) from 4.5 to 5.5. On the other hand, the undissociated acid concentration required to yield the same degree of inhibition between pho 5.5 and 8.5 decreased sharply (6.7 x 1-4 to 3. X 1-5 M). The same qualitative relationship was found for the inhibition of 2 uptake. However, 2 uptake was inhibited by 12 8, _2_l PER CENT CAPROIC ACID ENSOGEwOuS 67 Downloaded from on June 21, 218 by guest D9 D D6 PER CENT CAPROIC ACD PER CENT CAPROIC ACID FiG Pentanone production () and oxygen uptake () responses of vegetative hyphae of Aspergillus niger 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.

4 68 LEWIS AND DARNALL J. BACTERIOL. much lower concentrations of undissociated acid The variation in undissociated caproic acid at acidic pho values than was ketone production. concentration required for 5% inhibition of At higher pho values, they palralleled each other maximal 2 uptake or ketone production at quite closely. different ph. values suggested that the fatty acid altered the phi of the hyphae and that a fraction of the inhibition resulted from the altered phi (15). In all experiments, the hyphae were suspended in phosphate buffer before addition of 2. the fatty acid to maintain the desired pho. The phi varied almost directly as the ph of the phosphate buffer (pho) was changed (Fig. 3). How- -1. ever, the phi values of hyphae treated with stimulatory to inhibitory concentrations of w -2. caproate were not significantly different from the phi values obtained with phosphate buffer alone. This was true for all ph values tested x a- -3. (4.5, 5.5, 6.5, 7.5, and 8.5). The relationship of [A-]i and [HA]i to phi for 5% inhibition of.maximal 2 uptake and -4. ketone production is shown in Fig. 4. Increasing J EXTERNAL ph FIG. 2. Log caproic acid concentrations necessary to yield 5% inhibition of maximal 2 uptake and 2- pentanone production by vegetative hyphae of Aspergillus niger at various pho values. Symbols: *, log total caproic acid concentration to inhibit ketone production;, log total caproic acid concentration to inhibit 2 uptake; a, log undissociated caproic acid concentration to inhibit ketone production;, log undissociated caproic acid concentration to inhibit 2 uptake. I LUI a intracellular concentrations of anion were required to effect the same degree of inhibition as the phi increased. Changes in intracellular EXTERNAL ph caproic acid concentration to inhibit 2 uptake;, FiG. 3. Relcitionship of phi to pho of vegetative log caproate anion concentration to inhibit ketone hyphae of Asp;ergillus niger washed and resuspended production; *, log caproate anion concentration to in.22 M phlo)sphate buffer. inhibit 2 uptake. 1- (. U - 1. LLJ -2. ~ CL < -3. o -4. J -4C.- _ i Jo phi Ifi* s FIG. 4. Log intracellular caproic acid concentrations necessary to yield 5% inhibition of maximal 2 uptake and 2-pentanone production by vegetative hyphae of Aspergillus niger at various phi values Symbols:, log undissociated caproic acid concentra- --a I d' 11.1 tion to inhibit ketone production; O, log undissociated "It. Downloaded from on June 21, 218 by guest

5 VOL. 11, 197 FATTY ACID TOXICITY AND METHYL KETONE PRODUCTION 69 undissociated acid concentrations were the same as in the starting medium, which would be expected since the extracellular medium was well buffered. 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 endogenous Qo2 and phj; however, phi did not change as much at low ph. values (4.5 to 5.5) as at high ph. values (6.5 to 8.5), which appears to be reflected in the endogenous rates (Table 1). These data suggest that the hyphal contents are buffered to some extent at low ph values (4.5 to 5.5) but are more or less unbuffered at high ph values. A titration curve and buffer values for vegetative hyphal suspensions of A. niger are shown in Fig. 5. In all cases, the hyphal suspensions had a starting ph of approximately 3.5. At low ph values (4. to 5.), hyphal suspensions possessed a relatively high buffer value (.29 to.1). As the ph was increased the buffer value rapidly decreased to a minimum at ph 7.5 (.3) and then steadily increased to.17 at ph 1. Although the validity of such a titration curve may be questioned, the results obtained constitute a good fit for our physiological data and indicate that the hyphal contents are relatively well buffered at low ph values and rapidly approach the unbuffered condition at higher ph values. DISCUSSION Caproic acid behaved as a substrate inhibitor towards pregrown vegetative hyphae of A. niger. At alkaline pho values (Fig. 1A), 2 uptake over the endogenous level and 2-pentanone production followed each other almost mole per mole. These data support the theory (21) that methyl ketone production from fatty acid proceeds via the beta-oxidation pathway by decarboxylation of the beta-keto acid formed. Oxygen uptake above endogenous levels appeared to be solely a result of the oxidation of caproate to 2-pentanone. These results indicate that endogenous 2 uptake per se was not inhibited by caproate at alkaline pho values at concentrations which inhibited oxidation of exogenous caproate to 2-pentanone. At acidic pho values (Fig. IB, C, and D), this relationship failed to hold. Ketone production continued (ph, 6.5) and, in some cases (ph. 5.5 and 4.5), reached a maximum at which 2 uptake was inhibited well below endogenous levels. Thus, at acidic pho values, endogenous 2 uptake per se was vigorously inhibited by caproate concentrations at which exogenous fatty acid oxidation was not, at least not to as great an extent. Other workers (3, 4) have demonstrated that TABLE 1. Relationship of the rate of endogenous 2 uptake of vegetative hyphae oj A. niger to eaternal and intternial ph ph external ofextem Endogenous Q2' internal Apparent ph a Qo2 = microliters of 2 X hour-' X milli- grams of dry cell weight-'. exogenous acetate inhibits endogenous respiration 5 to 1% in P. chrysogenum and Neurospora crassa at a ph, value of 6.. In the present work, endogenous activity was most sensitive to caproate inhibition at acidic pho and ph i values near its optimum, and least sensitive at alkaline ph. and phi values where it was minimal (Table 1). These results show that the degree of inhibition of endogenous activity by exogenous substrates is not only a function of the past history of the organism, i.e., growth substrate, age, and conditions of culture, but also of the pho value of the manometric determination. The optimal pho value for 2-pentanone production was 6.5, which corresponded to a phi value of 6.7. This agrees with the report (8) that the optimal ph for the beta-keto acid decarboxylase from A. niger is 6.8. Neal et al. (15) proposed a mechanism for the toxicity of fatty acids toward yeast. They suggested that the undissociated acid enters the cell and dissociates, causing an acidification of the cell contents and a subsequent inhibition of respiratory and glycolytic enymes. Examination of the Simon-Beevers plots (Fig. 2) reveals that the undissociated acid is the permeant form of the molecule. The concept that intracellular acidification is the primary mechanism of inhibition is questionable, since the inhibition of either 2 uptake or ketone production by caproate in A. niger was independent of changes in phi at a given pho value. High concentrations of fatty acids, as used by Neal et al., can, undoubtedly, cause a decrease in phi value. Nevertheless, the inhibition could have been accomplished by lower concentrations of the acids which would not have altered the ph inside the cell. Thus, it seems necessary to distinguish between changes induced in cells by nonphysiological (high) concentrations of fatty acids as compared to physiological (low) concentrations, when one wishes to discuss the mechanism(s) by which these molecules inhibit cellular activities. Downloaded from on June 21, 218 by guest

6 7 LEWIS AND DARNALL J. BACTERIOL. Since the undissociated acid is the form of caproate which penetrates the cell, one might expect a given degree of inhibition with a given undissociated acid concentration regardless of the pho. That this is not the case is shown by the Simon-Beevers plots (Fig. 2). The most significant feature of these plots is that at high pho values less undissociated acid was required to cause the same degree of inhibition than at low ph. values. This relationship has been found to hold for many weak acids with respect to inhibition of yeast respiration (18). Furthermore, several theories, primarily involving cell permeability, have been advanced as explanations (24). However, if the increase in phi with increase in pho is taken into account (Fig. 3), a more direct explanation of the phenomenon may be obtained. If the fatty anion were the active form inside the cell and the undissociated acid the permeant form, then as the phi increased a smaller quantity of undissociated acid would be required in the external medium to effect a given concentration of anion inside the cell. Inspection of the log [A-]i-pHi plots (Fig. 4) shows that an increasing concentration of anion inside the cell was required to cause a 5% inhibition of either 2 UPtake or ketone production as the phi increased. This finding appears to be contradictory until one accounts for the binding of the anion to the enyme proteins involved. As the phi increased, the charge on all proteins within the cell should have become more negative and the affinity of these molecules for the fatty anion lowered. Therefore, a higher concentration of anion would be needed to cause a given degree of inhibition. Our experimental data are consistent with this concept and support the theory that the fatty anion is the active form of caproic acid with reference to inhibition of 2 uptake and 2-pentanone production in A. niger. Vegetative hyphae of A. niger appeared to approach the completely unbuffered state before washing and resuspension in phosphate buffer. Nevertheless, they were more highly buffered at low ph values (4.5 to 5.5) than at higher ones (5.5 to 8.5). This concept is supported by both the intracellular ph measurements (Fig. 3) and the titration curve (Fig. 5). After washing and resuspension in phosphate buffer, the hyphae appeared to possess good buffer strength, since increasing caproate concentrations caused no detectable change in the apparent phi. The degree of enyme inhibition produced by a compound such as caproic acid should depend on the buffer capacity of the cell. In general, two extreme cases may be discussed: completely buffered and completely unbuffered cells (24). If cells were completely buffered, the accumulation of fatty anion Co w -J -l 7.6 Q Q.I w.18 my.16 m.124 m JO r,.8 FM IGO ph FIG. 5. Titration curve and relative buffer values of vegetative hyphae of Aspergillus niger. Symbols: *, titration curve;, relative buffer values (i.e., rate of change ofph per milliequivalent ofhydroxyl ion added). would be much greater than in unbuffered cells. The greater the buffer capacity of a cell, the more undissociated acid that would penetrate and the greater the fatty anion or total inhibitor concentration in the cell. In reality, cells are neither unbuffered nor completely buffered, but fall somewhere between the two extremes. It would be very useful to know the true buffer capacity of cells, but no adequate quantitative work has yet been done. The data presented in this paper indicate that intracellular ph measurements in a defined physiological system can yield valuable information about the buffer capacity of cells. ACKNOWLEDGMENT This investigation was supported by grant D-175 from The Robert A. Welch Foundation, Houston, Tex. LITERATURE CITED 1. Acklin, Zur Biochemie des Penicillium glaucum. I. Ein Beitrag um Problem der Methylketonenbildung aus Triglyceriden bw. Fettsauren im Stoffwechsel des Schimmelpile. II. Die Bildung der Methylketone. Biochem. Z. 24: Biffen, R. H A fat destroying fungus. Ann. Bot. (London) 18: Blumenthal, H. J Endogenous metabolism of filamentous fungi. Ann. N.Y. Acad. Sci. 12: Blumenthal, H. J., H. Koffler, and E. C. Heath Biochemistry of filamentous fungi. V. Endogenous respiration during concurrent metabolism of exogenous substrates. J. Cell. Comp. Physiol. 5: Conway, E. J., and M. Downey An outer metabolic region of the yeast cell. Biochem. J. 47: Coppock, P. D., V. Subramaniam, and T. K. Walker The mechanism of the degradation of fatty acids by mould fungi. J. Chem. Soc. (London), p Edsall, J. T., and J. Wyman Biophysical chemistry. Academic Press Inc., New York. 8. Franke, W., A. Plateck, and G. Eichhorn Zur Kenntnis des Fettsaiureabbaus durch Schimmelpile. III. Uber eine Decarboxylase der mittleren beta-ketomonocarbon- Downloaded from on June 21, 218 by guest

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