APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1976, p. 408414Copyright ©) 1976 American Society for Microbiology

Vol. 31, No. 3Printed in U.S.A.

Morphology and Ultrastructure of a Penicillium sp. Grown on

n-Hexadecane or Peptone1A. M. CUNDELL,2* W. C. MUELLER, AND R. W. TRAXLER

Department of Plant Pathology-Entomology, University of Rhode Island, Kingston, Rhode Island 02881

Received for publication 22 September 1975

The morphology and ultrastructure of a Penicillium sp. grown on n-hexade-cane or on peptone in shake-culture were compared using transmission andscanning electron microscopy. The fungus grew as hollow mycelial balls sur-rounding individual hydrocarbon droplets on n-hexadecane and as solid mycelialballs on peptone. A dense layer of fungal mycelium that showed irregular forms,fusion, and increase in hyphal size formed at the hydrocarbon-water interface.Inclusions were present in the hexadecane-grown fungus that were absent whenthe Penicillium sp. was grown on peptone. Problems of fixation made it difficultto differentiate detailed changes in the cytoplasm when the fungus was exam-ined with the transmission electron microscope.

The importance of fungi in the biodegrada-tion ofpetroleum hydrocarbons in the estuarineenvironment has been recognized (1, 4). Somedata have been published on the ultrastructureof hydrocarbon-grown bacteria (2, 7) and yeasts(8, 13), but photomicrographs of hydrocarbon-grown fungi appear to be absent from the liter-ature. When the bacteria and yeasts weregrown on aliphatic hydrocarbons in shake-cul-ture, inclusions formed within the cells thatwere absent from cells grown on water-solublenutrients. Osumi et al. (13) have also reportedthe presence of microbodies in yeast grown inhydrocarbon that were absent in nutrient-grown cells.The present study was undertaken to com-

pare the morphology and ultrastructure of aPenicillium sp. when grown in liquid cultureon n-hexadecane or peptone and to establishwhether inclusions were formed within the hy-drocarbon-grown fungus in a manner found forbacteria and yeast. Members of the genus Peni-cillium have been previously shown to grow atthe expense of aliphatic hydrocarbons (1, 4, 9).

MATERIALS AND METHODS

Isolation. Sediment and water samples were col-lected from a polluted salt marsh at the mouth ofBuckeye Brook, Mill Cove, Narragansett Bay,which was heavily contaminated with residual fuel

' This paper is contribution no. 1637 of the Rhode IslandAgricultural Experiment Station.

2Present address: Engineering Science Laboratory, Har-vard University, Cambridge, Mass. 02138.

oil from the grounding of the Liberian registeredtanker Pennant on April 1, 1973 (14). The dominantgrass in the salt marsh was Spartina alterniflora,Loisel. Fungal isolates obtained by the inoculationof Sabouraud maltose agar, Cooke rose bengal agar,and Emerson agar plates with suitable diluents ofthe samples were screened for their ability to growat the expense of hydrocarbons on no. 1 fuel oil inliquid culture. A Penicillium sp. that was capable ofgrowth on n-hexadecane, 1-octadecene, and pristanewas selected for further study, because it grew rap-idly with high mycelial yields. The mode of sporula-tion and the presence of sclerotia when the funguswas cultivated on solid media suggested that theorganism belonged to the Raistrickii series.

Cultivation. The fungus was grown at room tem-perature in liquid culture on a mineral salts me-dium (NH4Cl, 1 g; Na2HPO4, 6 g; KH2PO4, 3 g; NaCl,5 g; MgSO4 7H20, 0.1 g; distilled water, 1,000 ml; pH6.8) containing 0.5% (vol/vol) n-hexadecane or 0.5%(wt/vol) neopeptone (Difco). The inocula were sporesuspensions of the fungus, and the incubation timewas from 3 to 5 days in shake-culture.

Fixation and staining procedure. Considerabledifficulty was encountered in fixing the fungal my-celium because the cell wall apparently preventedadequate penetration of the fixative. As a result, thefungal cytoplasm presented a dense and amorphousappearance, making the differentiation of organ-elles difficult. Other workers, experiencing similardifficulties, have achieved good fixation by usingextended fixation times (13) or by enzymaticallyremoving the cell wall (10). We achieved fixationthat was adequate to reveal differences in internalstructures of the peptone and hydrocarbon-grownfungi by permanganate treatment or by fixing withosmium tetroxide after a long prefixation in parafor-maldehyde-glutaraldehyde (6).

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PENICILLIUM GROWTH ON n-HEXADECANE OR PEPTONE 409

The mycelium was fixed with paraformaldehyde-glutaraldehyde (6) in 0.1 M phosphate buffer, pH6.8, for 90 min, washed in buffer, and then postfixedwith 0.5% unbuffered aqueous potassium perman-ganate for 30 min. Alternatively, mycelium wasfixed for 8 to 12 h in paraformaldehyde-glutaralde-hyde, washed, and then postfixed with 1% osmiumtetroxide for 2 h. Increasing the prefixation timewith the permanganate treatment did not seem toimprove the fixation. The fixed material waswashed in buffer and dehydrated in a graded seriesof acetone-water mixtures.

Transmission electron microscopy. The dehy-drated fungal material was embedded in a low-vis-cosity embedding medium (Spurr medium) accord-ing to procedures standard in this laboratory (11).Thin sections stained with uranyl acetate and leadcitrate were examined with Hitachi HS-9 electronmicroscope operating at 75 kV.

Scanning electron microscopy. The dehydratedfungal material was dried by the critical pointmethod (15), mounted on specimen stubs, platedwith palladium-gold alloy in a vacuum evaporator,and inspected with a Cambridge S-4 scanning elec-tron microscope (3, 5).

RESULTS AND DISCUSSIONThe fungus grew as mycelial balls in shake-

culture. The morphological response of the hy-drocarbon-utilizingPenicillium sp. to substratewas apparent from the scanning and transmis-sion electron micrographs.The mycelial balls formed by the fungus

growing on peptone were found not to be hol-low. Scanning electron photomicrographsshowed a uniform mycelial mass (Fig. la) withindividual hyphae coated with a dense depositof what was apparently the substrate (Fig. 2a).Transmission electron photomicrographs of os-mium-fixed and permanganate-fixed materialshowed a typical fungal ultrastructure (Fig. 3).Cytoplasm in the hyphae was very dense, butmitochondria, nuclei, vacuoles, and lipid bodieswere prominent. Permanganate fixationcaused considerable deformation of mitochon-dria, the condensation of vacuoles, and forma-tion of small electron-lucent areas that mayhave contained glycogen (8, 16) around the pe-riphery of the fungal cells. The deposition ofmedium about the fungal mycelium was appar-ent in the transmission photomicrographs.Similar deposition has been reported by Mur-phy et al. (12) when the corn pathogen Diplodiamaydis was grown on a peptone-yeast extractbroth.Examination of wet mounts of the hexade-

cane-grown culture with a light microscope re-vealed the accumulation of germinating fungalspores at the hydrocarbon-water interface of

the hexadecane droplets during the first days ofincubation in shake-culture. Mycelium fromthe germinating spores enveloped the hydrocar-bon droplets, forming an inner rind of densefungal hyphae that penetrated the surface ofthe hydrocarbon and less dense hyphae grow-ing away from the hydrocarbon.The mycelial balls, grown on the hexadecane

in shake-culture, which contained a centralcore of hydrocarbon, were found to be emptyafter fixation, dehydration, and critical-pointdrying (Fig. lb). These balls could be crackedopen like an egg shell with a glass needle, andthe inner and outer surfaces ofthe fungal myce-lium could be examined with the scanning elec-tron microscope. The hyphae from the innerregion were densely packed, irregular in form,enlarged, and fused (Fig. 2c and d), whereas thehyphae on the outer margin of the mycelial ballwere less densely packed, regular in form, andoccasionally bore conidia (Fig. 2b).Transmission electron photomicrographs

taken of thin sections cut from the inner andouter regions of the mycelial ball illustrated adifference in the ultrastructure of the hydrocar-bon-grown fungus within the two regions. Themycelium of the inner layer, consisting of adense mass of fungal hyphae, contained anabundance of inclusions (presumed to be hydro-carbon or a hydrocarbon oxidation product)(Fig. 4). In contrast, photomicrographs of thematerial from the outer margins of the hydro-carbon-grown mycelial balls showed an ultra-structure that resembled that of the fungusgrown on peptone in shake-culture (similar topreparations shown in Fig. 3).Because of the difficulties with fixation, it is

not feasible to make a detailed comparison ofthe ultrastructure of the fungus when grown onhexadecane and peptone. However, inclusionspresent in the hydrocarbon-grown myceliumwere absent in the peptone-grown fungus.These inclusions that presumably are derivedfrom the hydrocarbon in the medium appearsimilar to those described in bacteria and yeast(2, 7, 8). These findings support the generalitythat hydrocarbons are sequestered in bacteria,yeast, and fungi. The pooling of the hydrocar-bons within inclusions implies the active trans-port of the hydrocarbons against a concentra-tion gradient (7). However, in the absence ofsupporting biochemical data, only the similar-ity of the inclusions of the hydrocarbon-grownfungus compared with that of bacteria andyeast can be stressed. No evidence was found ofthe microbodies described by Osumi et al. (13)in hydrocarbon-grown yeast.

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410 CUNDELL, MUELLER, AND TRAXLER

D %- ;_.4#FIG. 1. Scanning electron photomicrographs ofa Penicillium sp. grown in shake-culture. Marker indicates

100 ,im. (a) Mycelial ball grown on 0.5% (wtlvol) neopeptone. ( x100) (b) Bisected hollow mycelial ball grownon 0.5% (vollvol) n-hexadecane. Fungus has formed a dense rind at the water-hydrocarbon interface. (xlOO)

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c d_FIG. 2. Scanning electron photomicrographs ofa Penicillium sp. grown in shake-culture. (a) Surface ofthe

mycelial ball grown on neopeptone. Note the particulate matter on the hyphal surfaces. Marker indicates 20Am. (x500) (b) Outer margin of the mycelial ball grown on n-hexadecane. Conidia indicated by arrows.Marker indicates 50 ,um. ( x200) (c) Fungal rind at the hydrocarbon-water interface ofthe mycelial ball grownon n-hexadecane. Marker indicates 50 tLm. (x200) (d) Inner surface of the fungal rind showing deformationand fusion of individual hyphal strands. Marker indicates 5 ,im. (x2,000)

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FIG. 3. Transmission electron photomicrographs of a Penicillium sp. grown on neopeptone in shakeculture. (V) Vacuoles, (M) mitochondria, (N) nuclei, (L) lipid bodies, and (0) polysaccharide granules. (a)Longitudinal section ofan aldehyde-0s04-fixed hypha. Marker indicates 0.66 lim (x15,000) (b) Longitudi-nal section ofa permanganate-fixed hypha. Marker indicates 0.45 jim. (x15,000) (c) Transverse section ofanaldehyde-0s04-fixed hypha. Marker indicates 0.5 lim. (x15.,000) (d) Transverse section of a permanganate-fixed hypha. Marker indicates 0.5 g~m. (x15,000)

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FIG. 4. 7'ransmission electron photomicrographs of the mycelial layer at the hydrocarbon-water interfaceofa Penicillium sp. grown on hexadecane in shake-culture. Inclusions indicated by arrows. Markers indicate0.5 tim. (a) Aldehyde-OsO4 fixation. (x20,00) (b) Permanganate fixation. (x20,OOO)

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414 CUNDELL, MUELLER, AND TRAXLER

ACKNOWLEDGMENTSWe wish to thank R. D. Goos, Botany Department, Uni-

versity of Rhode Island, for identifying the fungus, J. McN.Sieburth, Paul Johnson, and Donald Scales, GraduateSchool of Oceanography, University of Rhode Island, forassistance with the scanning electron microscopy, and R. R.Young for the isolation of hydrocarbon-degrading fungi.

This investigation was supported by Office of Naval Re-search contract N00014-6A-02515-0013.

LITERATURE CITED

1. Ahearn, D. G., and S. P. Meyers. 1972. The role of fungiin the decomposition of hydrocarbons in the marineenvironment, p. 12-18. In A. W. Walters and E. H.Hueck-van der Plas (ed.), Biodeterioration of ma-terials. Applied Science Publishers, London.

2. Atlas, R. M., and C. E. Heintz. 1973. Ultrastructure oftwo species of oil-degrading marine bacteria. Can. J.Microbiol. 19:43-45.

3. Brooks, R. D., R. D. Goos, J. M. Sieburth. 1972. Fungalinfestation of the surface and interior vessels offreshly collected driftwood. Mar. Biol. 16:274-278.

4. Cerniglia, C. E., and J. J. Perry. 1973. Crude oil degra-dation by microorganisms isolated from the marineenvironment. Z. Allg. Mikrobiol. 13:299-306.

5. Cundell, A. M., and A. P. Mulcock, 1975. The biodegra-dation of vulcanized rubber. Dev. Ind. Microbiol.16:88-90.

6. Karnovsky, M. J. 1965. A formaldehyde-glutaraldehydefixation of high osmolality for use in electron micros-copy. J. Cell Biol. 27:137A.

7. Kennedy, R. S., W. R. Finnerty, K. Saudarsanan, andR. A. Young. 1975. Microbial assimilation of hydro-carbon. I. The fine-structure of a hydrocarbon-oxidiz-ing Acinetobacter sp. Arch. Microbiol. 102:75-83.

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8. Ludvik, J., V. Munk, and M. Dostalek. 1968. Ultra-structural changes in the yeast Candida lipolyticacaused by penetration of hydrocabons into the cell.Experientia 24:1066-1069.

9. Markovetz, A. J., J. Cazin, and J. E. Allen. 1968. As-similation of alkanes and alkenes by fungi. Appl.Microbiol. 16:487-489.

10. May, R. 1974. Verbesserung der Glutaraldehyde -°OS4- Fixierung von Hefen durch Vorfixierung mit For-maldehyd und enzymatischen Zellwandabbau. Z.Allg. Mikrobiol. 14:161-166.

11. Mueller, W. C., and C. H. Beckman. 1974. Ultrastruc-ture of the phenol-storing cells in the roots of banana.Physiol. Plant Pathol. 4:187-190.

12. Murphy, J. A., L. L. Campbell, and A. J. Pappelis.1974. Morphological observation of Diplodia maydison synthetic and natural substrates as revealed byscanning electron microscopy. Appl. Microbiol.27:232-250.

13. Osumi, M., N. Miwa, Y. Teranishi, A. Tanaka, and S.Fukui. 1967. Ultrastructure of Candida yeasts grownon n-alkanes. Arch. Microbiol. 99:181-201.

14. Pierce, J. H., A. M. Cundell, and R. W. Traxler. 1975.The biodegradation and persistence of spilled resid-ual fuel oil on an estuarine beach. Appl. Microbiol.29:646-652.

15. Porter, K. R., D. Kelley, and P. M. Andrews. 1972. Thepreparation of cultured cells and soft tissues for scan-ning electron microscopy, p. 1-19. In Proceedings, 5thAnnual Steroscan Colloquium. Kent Cambridge Sci-entific Inc., Chicago.

16. Wergin, W. P. 1972. Ultrastructural comparison of mi-crobodies in pathogenic and saprophytic hyphae ofFusarium oxysporum. f. sp. lycopersici. Phytopathol-ogy 62:1045-1051.

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