Mixed function oxidase inhibitors affect production of the off-flavor microbial metabolite geosmin

Mixed function oxidase inhibitors affect production of the off-flavor microbial metabolite geosmin

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 38, 7680 (19%) Mixed Function Oxidase Inhibitors Affect Production Off-Flavor Microbial Metabolite Geosmin’...

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PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

38, 7680 (19%)

Mixed Function Oxidase Inhibitors Affect Production Off-Flavor Microbial Metabolite Geosmin’

of the

CHRISTOPHERP. DIONIGI,DAPHNE A. GREENE,DAVID F. MILLIE, AND PETERB. JOHNSEN United States Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, Food Flavor Quality Laboratory, P.O. Box 19687, New Orleans, Louisiana 70179 Received April 10, 1990; accepted June 15, 1990 Geosmin (l , lO-trans-dimethyl-trans-(9)-decalol) is produced by the tilamentous bacteria Streptomyces tendae Ettlinger and imparts an intense muddy “off-flavor” which degrades the quality of drinking water and pond-raised fish. The effects of the cytochrome Pd5,, mixed-function oxidase inhibitors piperonyl butoxide [a-[2-(2-butoxyethyoxy)ethoxy]-4,5-(methylenedioxy)-2-propyltoluene], and MGK-264 [N-octyl bicycloheptene dicarboximide] on dry matter, pigment, and geosmin production were determined in S. tendae. Neither piperonyl butoxide nor MGK-264 affected dry matter production, but both compounds decreased pigment synthesis compared to untreated controls. Exposure to MGK-264 and piperonyl butoxide produced differing effects on geosmin production. Piperonyl butoxide (300 llM) induced approximately a 400% increase in geosmin levels, while MGK-264 (300 @I) reduced geosmin levels approximately 40% compared to untreated controls. 8 1990 Academic press, hc.

INTRODUCTION

cursor, possibly farnesyl pyrophosphate (3, 9). Geosmin contains a hydroxyl group, indicating that at some point in the pathway a hydroxylation reaction occurs. Hydroxylation reactions involving 1,Ccineole, a naturally occurring monoterpenoid, have been reported in several strains of Streptomyces griseus Krainsky and S. punipalus Waksman (10). Cytochrome Pd5a mixed function oxidases (MFO) catalyze hydroxylation reactions in a wide variety of organisms, including bacteria (11). Piperonyl butoxide [ol-[2-(2-butoxyethoxyl-4,5-(methylenedioxy)-2-propyltoluene] and MGK-264 [Noctyl bicycloheptene dicarboximide] inhibit hydroxylation of certain pesticides and other xenobiotics in insect and weed species (11). However, the effects of these compounds on organisms that produce offflavor metabolites have not been reported. The objectives of this research were to determine the effects of piperonyl butoxide and MGK-264 on S. tendae Ettlinger, a geosmin-producing filamentous bacterium.

Geosmin (1, lo-trans-dimethyl-trans(9)-decalol) (Fig. 1) is produced by several species of algae (1, 2) and bacteria (3-5). Concentrations as low as 10 rig/liter geosmin can impart a musty “off-flavor” and odor to drinking water (6) and can seriously degrade the flavor quality of pondraised fish (7). The use of pesticides to control blooms of geosmin-producing organisms can cause massive die-offs of algal populations, resulting in a depletion of dissolved oxygen and a release of nutrients that can stimulate additional blooms (8). Currently, there are no known compounds which inhibit off-flavor metabolite biosynthesis and are not toxic to the microbial weeds that produce off-flavors. Although the biochemical pathway of geosmin synthesis has not been determined, evidence exists for a terpenoid prer Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

MATERIALSANDMETHODS Chemical treatments. Stock solutions 76

0048-3575190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any fomt reserved.

of

OXIDASE

INHIBITORS

w3

I cy> I CH3

bH

FIG. 1. The structure trans-dimethyl- tram-(9)-decaloi).

of geosmin

(l,lO-

technical grade piperonyl butoxide and MGK-264 (both from McLaughlin, Gormley and King Co., Minneapolis, MN) were prepared by mixing sufficient amounts of these compounds with 100% ethanol so that when 40 ~1 of stock solution was added to 100 ml of media the resultant concentration was either 0, 50, 100, 200, or 300 pB. Microbial

culture.

Streptomyces

tendae

[ATCC 31160; obtained from P. Engel, U.S. Department of Agriculture, New Orleans, LA] was grown in 100 X 15mm polystyrene petri dishes containing 20 ml of Hickey and Tresner media (12) solidified with 1.2% (w/v) bacteriological grade agar. Liquid cultures were grown unshaken in 250-ml glass flasks containing 50 ml of Hickey and Tresner media with no agar. Inoculum was prepared by briefly sonicating spores scraped from one petri dish in 1 ml of Mill&Q reagent grade water. Each dish or flask received 0.10 ml of inoculum and was incubated in the dark at 28°C. Calorimetry. Lightness (L) values, as determined by a Hunter D25-PC2 colorimeter, are measurements of reflected light intensity, and can be used to quantify pigments in solid samples (13). L is equal to 100(Y/Y#z, where: Y is a tristimulus value, and Y,,is the tristimulus value for a perfect diffuser for the illuminant used. Sample L values are related to a white tristimulus standard tile (L = lOO),and a black calibration standard tile (L = 0). Therefore, the greater the L, the lesspigmented the sample. Each treatment dish was placed over the measurement port (19 mm diameter) of the calorimeter, with a white backing tile

AND GEOSMIN

77

placed on top of the treatment dish. L values were recorded at four locations on each treatment dish, and the mean of the four L values were used as an index of pigment production. Chromatography. Methods for off-flavor metabolite extraction from a liquid medium (6) were modified for use with a solid medium. A known sample weight of solid medium (2-3 g) was obtained from each petri dish and placed in a lOO-ml glass beaker. The sample was covered with 20 ml of spectro-grade methylene chloride solvent. Then 50 p,l of 25 ppm 2-undecanone in 100% ethanol was added as an internal standard prior to finely chopping the solid medium with a stainless steel spatula. The solvent was dehydrated through approximately 5 g of anhydrous sodium sulfate and collected in a 50-ml conical glass centrifuge vial. Sample concentration and preparation were performed as described by Johnsen and Kuan (6). Gas chromatography was performed on a Hewlett-Packard Model 5890 equipped with a flame ionization detector, a HewlettPackard Ultra 2 crosslinked capillary column (50 m x 0.2 mm i.d.) coated internally with a 0.33-pm film of 5% phenylmethylsilicone, and a Hewlett-Packard Model 3392A integrator. Injections (1 l.~l) were performed by a Hewlett-Packard 7673A automatic sampler. The injector port temperature was 200°C the flame ionization detector was heated to 27O”C, and the inlet split ratio was 3.25: 1. The oven temperature was held at 100°Cfor 5 min, then programmed at S”C/min to 2OO”C, then at 10”Clmin to 275°C and held at 275°Cfor 30 min. Helium served as the carrier gas (0.26 ml/mm at 1OO’C)and the make-up gas (34.2 mYmin). Gas flow rates for the flame ionization detector were 30.2 mYmin for hydrogen and 378 ml/min for air. Compounds of interest were identified by comparing retention times with those of authentic standards. In addition, flame ionization peaks corresponding to geosmin and the internal standard were verified by mass spectrometry.

78

DIONIGI

Growth determinations. When grown on solid media, S. tendae produces a mass of mycelium which can form spores as the cultures mature. This prevents an accurate quantification of dry matter. In liquid media, S. tendae does not sporulate, and the cells can be easily separated from the media. Therefore, to quantify dry matter accumulation, cells were vacuum-filtered from the liquid medium through a preweighed, glass microfiber filter (Whatman GF/C), and oven-dried (1 hr at IOO’C). Cell dry weight was determined gravimetrically. Statistical analysis. Dosage effects of MGK-264 and piperonyl butoxide were determined in separate complete randomized blocks experiments. Each dosage was replicated three times per experiment. Each experiment was then replicated in time. Original data from experiments concerning either MGK-264 or piperonyl butoxide were combined and analyzed according to McIntosh (14) before percentage of control values were calculated. RESULTS

Pigment levels. Initially, the solid medium appeared colorless, but as the cultures of S. tendae grew the media became brownish yellow. Exposure to both piperonyl butoxide and MGK-264 inhibited pigment production which resulted in L values that were significantly greater (P 2 0.0001 for both piperonyl butoxide and MGK-264) than those of the untreated controls (Figs. 2 and 3). Geosmin levels. Except for the solidifying agent (agar), the ingredients of the solid and liquid media were the same. However, geosmin could not be detected by olfaction or gas chromatography and spore formation was not observed in the liquid medium, whereas both spores and geosmin were observed in solid medium. In solidified medium, piperonyl butoxide (300 tcM) increased (P z 0.0001) the concentration of geosmin by approximately 400% over untreated controls (Fig. 4). However, exposure to MGK-264 (300 ~J,M)

ET AL.

3,LLd 0

50

--_-.

100

Piperonyl

200

Butoxide

300

(PM)

FIG. 2. Effect of piperonyl butoxide on the colorimeter lightness (L) value of Streptomyces tendae cultures grown on solid medium. The greater the L value the less pigmented the sample. Data are expressed 22 (SE) of a dosage mean (n = 12).

reduced (P 3 0.0001) geosmin concentrations by approximately 40% compared to untreated controls (Fig. 5). Growth effects. Following inoculation, the liquid media became turbid and untreated controls in the piperonyl butoxide and MGK-264 experiments contained 0.042 and 0.030 g of dry matter, respectively. However, neither piperonyl butoxide nor MGK-264 significantly decreased (P 2 0.38 and P 5 0.65, respectively) cell dry weight compared to untreated controls. DISCUSSION

Although S. tendae produced a measurable amount of dry matter, neither piperonyl butoxide nor MGK-264 inhibited the growth of this taxon. However, greater dosages and other culture conditions were not investigated and may affect the response of this organism to these com43,

,

0

50

100

MGK-264

200

300

(@A)

FIG. 3. Efhect of MGK-264 on the calorimeter lightness (L) value of Streptomyces tendae cultures grown on solid medium. The greater the L value the less pigmented the sample. Data are expressed *I (SE) of a dosage mean (n = 12).

OXIDASE

e -F 6

450i

: b” .E

3001

INHIBITORS

t-

,.

375. /

A---

2251

//-.I ~~- 0’ I

i

i :

,+

E 15oi G

75 ci.

i’ 0

_I -~--50 100

Piperonyl

;

200 Butoxide

300 &CLM)

FIG. 4. Effect of piperonyl butoxide on geosmin concentration in Streptomyces tendae cultures. Data are expressed 21 (SE) of a dosage mean (n = 12). Untreated controls contained 256 ppb (w/w) geosmin.

pounds. Geosmin production has been associated with sporulation (3). In this investigation, when S. ten&e were grown in liquid medium, neither spores nor geosmin were produced. When S. tendae was grown on a solid form of the same medium, both spores and geosmin were produced, indicating that the consistency of the culture media may affect geosmin synthesis. Although the physiological role of geosmin is unknown, geosmin may be involved in propagation. Inhibition of pigment production by both compounds and geosmin production by MGK-264 indicates the possible involvement of MFO in these processes. Although the chemical identities of the pigments produced by S. tendae are unknown, they may be derived from the terpenoid pathway below famesyl pyrophosphate as in higher plants (15, 16) and in the bacterium, Bacillus megaterium (QM Bl551) (17). Increases

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GEOSMIN

79

in geosmin concentrations could be due to an inhibition of pigment synthesis by piperonyl butoxide. An “overflow” of carbon into geosmin synthesis was observed when the geosmin-producing cyanobacterium, Oscillatoria brevis Kutz., was exposed to herbicides that inhibit terpenoid pigment synthesis below the proposed famesyl pyrophosphate to geosmin branch point (9). However, MGK-264 also inhibited pigment production with no increase in geosmin synthesis which does not support this overflow hypothesis. In many strains of Streptomycetes, the production of odorous substances tends to be genetically unstable and may be associated with transposons and amplifiable chromosomal and extrachromosomal elements (18). The stimulation of geosmin production by piperonyl butoxide may be due to increased expression of geosmin production rather than an overflow of carbon into geosmin synthesis. Although piperonyl butoxide is usually considered an enzyme inhibitor, repeated exposure may induce the activity of MFO (D-21). Cell growth on media containing piperonyl butoxide may have induced the activity of MFO involved in geosmin production. Regardless of the mechanism of the stimulation of geosmin synthesis, the production of geosmin in S. tendae is not inhibited by piperonyl butoxide. Although little information concerning M6K-264 exists in the literature [see (22)], this compound has been reported to competitively inhibit MFO (11). Conceivably, M6K-264 may chemically resemble intermediates in the terepene pathway and thus competitively inhibit both geosmin and pigment synthesis in S. tendae. ACKNOWLEDGMENTS

FIG. 5. Effect of MGK-264 on geosmin concentration in Streptomyces tendae cultures. Data are expressed +I (SE) of a dosage mean (n = 12). Untreated controls contained 1820 ppb (w/w) geosmin.

The authors thank S. W. Lloyd for his help with the gas chromatography and mass spectrometry, L. L. Munchausen for her creative input, J. R. Coats, M. A. Klich, B. A. Moore, B. G. Montalbano, and W. J. Connick for their help in the preparation of this manuscript. P. Engel, C. M. Vides, Jr., and P. Y. Harris for their help with microbial cultures, and S. E. Rogosh-

DIONIGI

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ET AL.

eske and the McLaughlin Gormley and King Co. for their technical assistance. REFERENCES 1. G. Izaguirre, C. J. Hwang, S. W. Kranswer, and M. J. McGuire, Geosmin and 2-methylisoborneol from cyanobacteria in three water supply systems, Appl. Environ. Microbial. 43, 708 (1982). 2. H. Naes, H. C. Utkilen, and A. F. Post, Geosmin production in the cyanobacterium Oscillatoria brevis, Arch. Microbial. 151, 407 (1989). 3. R. Bentley and R. Meganathan, Geosmin and methylisobomeol biosynthesis in Streptomyces, evidence for a isoprenoid pathway and its absence in nondiIferentiating isolates, FEBS Lett. 3, 220 (1982). 4. N. N. Gerber, Volatile substances from actinomycetes: Their role in the odor pollution of water, Water Sci. Techol. 15, 115 (1983). 5. 0. Yagi, N. Sugiura, and R. Sudo, Chemical and physical factors in the production of musty odor by Streptomyces spp. isolated from Lake Kasumigaura, Agric. Biol. Chem. 51, 2081 (1988). 6. P. B. Johnsen and J. W. Kuan, Simplified method to quantify geosmin and 2-methylisobomeol concentrations in water and microbiological cultures, J. Chromatography, 409, 337 (1987). 7. R. T. Lovell, I. Y. Lelana, C. E. Boyd, and M. S. Armstrong, Geosmin and musty-muddy flavors in pond-raised channel catfish, Trans. Amer. Fish. Sot. 115, 485 (1986). 8. C. S. Tucker, Water quality, in “Channel Catfish Culture,” (C. S. Tucker, Ed.), pp. 135, Elsevier, New York, 1985. 9. H. Naes, H. C. Utkilen, and A. F. Post, Factors influencing geosmin production by the cyanobacterium Oscillatoria brevis, Water Sci. Techol. 20, 125 (1988). 10. J. P. N. Rosazza, J. J. Steffens, F. S. Sariaslani, A. Goswami, J. M. Beale, S. Reeg, and R. Chapman, Microbial hydroxylation of 1,4cineole, Appl. Environ. Microbial. 53, 2482 (1987). 11. E. Hodgson and L. G. Tate, Cytochrome P-450

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