Archiv ffir Mikrobiologie 45, 111 -- 114 ( 1963 )

Istituto di Microbiologia Agraria e Tecnica dell' Universith di Perugia (Italia)

Yeasts in Cavern Environments By

ALESSANDRO )IARTINI

(Received December 15, 1962)

Very little da ta are avai lable on the presence of yeasts in calcareous soils of caverns. The researches on the ecology of yeasts in na tu re have neglected, up to present, the cavern environments , though the chemical and physical characteristics of cavern softs are very different from those of surface ones. However, the work conducted by the au thor of the present paper in two caverns a round Pe rug ia / I t a ly (MAt~TI~I 1962a, 1962b) has shown the occurrence of Blastomycetes on them.

The work reported in this paper was carried out following techniques a l ready used by the authors who studied the ecology of yeasts in na ture (CAP~IOTTI 1959, 1962; CASTELLI 1957). The results obta ined permit a comparat ive s tudy of the diffusion of yeasts in different habi ta ts and of the relations between species and envi ronment .

Methods Four soil samples were taken from the 'T ian delle Rotte" cavern (80U/PG)

situated near Campello sul Clitunno (Perugia/Italy). The first sample (A) was col- lected in the wood surrounding the entry of the cavern, the second one (B) on the bottom of the entrance pit, the third (C) along the wall of an internal room and the fourth (D) in the last cavity of the cavern.

Three other samples (E-F-G) always collected with the above mentioned pro- cedure, were coming from the cavern of "Parrano" (12U/Tt~). The sample indicated as H was taken from a deposit of bat guano of the Parrano cavern.

500 g of each soil were brought to the laboratory in sterile bottles and the en- richment cultures were prepared as follows: 100 ml of grape-must (pH 3.5 -- sugar 20~ in 150 ml Erlenmeyer flasks were inoculated with 2 g of each sample of soil and incubated at 25~ Microscopic examination of the growth were carried out every 24th hour while isolations were made at the end of a 10 day period by streaking on malt agar in plates.

Totally 61 pure cultures of yeasts were isolated. Their identification was made up following the procedure of LODDE~ and K~EGEI~ VAZr RI5 (1952) excepting the method of CAPgIOTTT (1955a) used for the carbon assimilation tests.

Results I n Tables 1 and 2 the results of identif icat ion work are referred. As

regards to any one isolated species, the number of strains isolated from each sample and their f requency on the cavern soils are reported. As can

Arch. :~ikrobiol., ]~d. 45 8

112 ALESSANDRO ~RTIEI :

be seen f r o m these t ab les t h e r e is a lways p r e s e n t in a l l t h e s amples a

y e a s t f lora p r e v a i l i n g l y c o m p o s e d b y spo rogenous species. T h e m o s t

w i d e l y d i s t r i b u t e d species in P l a n del le R o t t e c a v e r n is Saccharomyces

Table 1. Species isolated on the samples coming /rom the "Plan delle rotte" cavern

Species isolated

Sporogenous 1. Sacch. italicus Castelli 2. Sacch. cerevisiae Hansen 3. Sacch. capensis van der Walt 4. Sacch. ]ructuum Lodder et K. van Rij 5. Sacch. uvarum Beijerinck 6. Hansenula subpelliculosa Bedford 7. Sacch. microellipsodes Osterwalder 8. Sacch. smittii CapriotM 9. Debaryomyces, species I

10. Sacch. veronae Lodder et K. van Rij 11. Sacch. carlsbcrgensis t tansen 12. Sacch. ellipsoideus Hansen

. Asporogenous 13. K{oeckera magna de' Rossi

Total

4 4

n ~ of strains isolated from the samples

B C

i i 13 5 4 3 2

- 1

-- 2 - - 3

1 - - 1

- - 2

11 13

9

2

1 2

1

3 3

12

To~ally isolated strains

37 9 7 2 1 2 3 3 1

40

Table 2. Species isolated on the samples coming ]rom the "Parrano" cavern

Species isp]ated

I. Sacch. smittli Capriotti 7 2. Sacch. italicus Castelli 2 3. Sacch. oviformis Osterwa]der 4. Sacch. microellipsodes Osterwalder ~-- 5. Debaryomyces, spec ie s / /

n ~ of strains isolated from the samples

- - 2

- - 2

4

4 4

Totally isolated strains H

- - 9

- - 4

- - 4

3 3 1 1

4 21

The sample indicated as " H " comes from a bat guano deposit of the same cavern.

italicus Caste l l i w i t h 9 s t ra ins , whi le in t h e P a r r a n o c a v e r n is Saccharo- myces smittii C a p r i o t t i a lways w i t h 9 s t ra ins . I n t he f o r m e r c a v e r n

Saccharomyces cerevisiae H a n s e n is also p r e s e n t w i t h 7 s t ra ins whi le t h e

r e m a i n i n g species do n o t r each t h e s a m e f r e q u e n c y on t h e t o t a l o f i so l a t ed s t ra ins . M o r e o v e r we can p o i n t o u t t h e f ac t t h a t o n l y 3 s t ra ins o u t o f 40

Yeasts in cavern environments 113

were asporogenous and that 10 species out of 13 were belonging to the genus Saccharomyces.

In the latter cavern all the isolated species were sporogenous while 4 of them out of 5 could also be referred to the genus Saccharomyces.

The two Debaryomyces indicated as species I a n d / / , because of the fermentation and assimilation characters, could not be identified with any previously described species. For this reason we believe it necessary, before describing them as new species, to make a more detailed study which will be referred to in a following paper.

Discussion At the end of the above mentioned paper (MAnT~I 1962b) on the

microflora of Monte Cucco cavern we concluded that the presence of yeasts on cavern soils has to be ascribed to the transport action of bats and insects and that we cannot speak of as indigenous flora. In the present work also, the differences between the samples coming from the external soils and those collected inside the cavern are not so great as to change the mentioned conclusions. Very interesting is the marked prevalence of sporogenous species on the asporogenous ones: out of 61 isolated only 3 species were reported to the asporogenous genus Kloeckera.

A short comparison with the results obtained by CA~gIOT~I (1955b) on the yeasts found in some soils of Central I ta ly permits us to point out the fact that species as Saccharomyces ovi/ormis Osterwalder, Saccharo- myces microellipsodes Osterwalder and Saccharomyces ellipsoideus Hansen are present in both habitats. The other species also, Saccharomyces italicus Castelli, Saccharomyces uvarum Beijerinck, Hansenula subpelliculosa Bedford and Kloeckera magna De' Rossi, are normally present in the grape- musts coming from the vineyards of the same zone.

At length we can conclude that the presence of yeasts in habitats characterized by a low temperature (5~ all over the year) and by a content of only proteic substances is particularly interesting for the study of their ecology in nature. In this connection we have in mind of making a detailed study on soils of caverns completely isolated from the external environment.

Summary l~esearehes on the yeast flora of 8 soil samples collected from 2 caverns

of Umbria (Italy) show the presence of 15 different species of yeasts, 14 sporogenous and 1 asporogenous. Comparisons are made between yeasts flora of caverns and that of surface soils.

A c k n o w 1 e d g e m e n t. The author wishes to express his appreciation to the Speleo- logical Group of the Institute of Geology of the University of Perugia, Italy, directed by Professor CESARE LIP1)I BOlgCASIBI who made possible the collection of samples.

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114 ALESSANDRO I~ARTINI: Yeasts in cavern environments

References CAt'RIOTTI, A. : In torno al saggio di assimilazione degli zuccheri da par te dei lieviti.

Biochim. appl. (Parma) 4, 31 (1955a). - - I lieviti di alcuni terreni dell ' I tal ia Centrale. Riv. Biol. 47, 209 (1955b). - - The yeasts of certain soils from Sweden. Kungl. Lantbruksh6gskolans Annaler 25,

185 (1959). - - The yeasts of the Miami, Florida, area. Arch. Mikrobiol. 41, 142--146 (1962). CAST]~LLI, T. : Climate and agents of wine fermentation. Amer. J. Enol. 8, 149 (1957). :LODDER, J., and N. J. W. KaEGEa VAN ~IJ: The yeasts -- A taxonomic study.

Amsterdam: North Holland Publishing Co. 1952. MARTINI, A. : Alcune indagini preliminari sulla microflora delle caverne. ]:~ass.

Speleologica I ta l iana 16, 3 (1962a). - - La microflora della caverna di Monte Cuceo -- I a Nota: I blastomiceti. Ann. Fac.

Agraria Perugia 16 (1962b).

Dr. A. MARTINI, I s t i tu to di Microbiologia Agraria e Tecnica dell' UniversitA di Perugia, I tal ia