Species-specific production of microbial volatile organic compounds (MVOC) by airborne fungi from a compost facility

Chemosphere,Vol. 39, No. 5, pp. 795-810. 1999 © 1999ElsevierScienceLtd.All rightsreserved 0045-6535/99/$ - see frontmatter

Pergamon

PII: S0045-6535(99)00015-6

SPECIES-SPECIFIC PRODUCTION OF MICROBIAL VOLATILE ORGANIC COMPOUNDS (MVOC)

BY A I R B O R N E F U N G I F R O M A C O M P O S T F A C I L I T Y

GUIDOFISCHER,REGINASCHWALBE,MANFREDM6LLEI~RENEOSTROWSKI, ANDWOLFGANGDOTT

Institute for Hygiene and Environmental Medicine, University Hospital, Technical University of Aachen, D-52074 Aachen, Germany (Receivedin Germany2 September1998;accepted4 December1998)

ABSTRACT

Thirteen airborne fungal species frequently isolated in composting plants were screened for microbial volatile organic compounds (MVOC), i.e., Aspergillus candidus, A. fumigatus, A. versicolor, Emericella

nidulans, Paecilomyces variotii, Penicillium brevicompactum, Penicillium clavigerum, Penicillium crustosum, Penicillium cyclopium, Penicillium expansum, Penicillium glabrum, Penicillium verruculosum, and Tritirachium oryzae. Air samples from pure cultures were sorbed on Tenax GR and analyzed by thermal desorption in combination with GC/MS. Various hydrocarbons of different chemical groups and a large number of terpenes were identified. Some compounds such as 3-methyl-l-butanol and 1-octen-3-ol were produced by a number of species, whereas some volatiles were specific for single species. An inventory of microbial metabolites will allow identification of potential health hazards due to an exposure to fungal propagules and metabolites in the workplace. Moreover, species-specific volatiles may serve as marker compounds for the selective detection of fungal species in indoor domestic and working environments. ©1999 Elsevier ScienceLtd. All rights reserved KEVWO~S: Airborne fungi, moulds, MVOC, microbial volatile organic compounds, chemotaxonomy, odour compounds, compost facility.

795

796 INTRODUCTION

Airborne fungal contaminants in compost facilities are becoming more important in regard to health hazards for workers and emission of spores and microbial metabolites into the environment. Apart from pathogenicity, the potential impact of bioaerosols on health is being widely discussed from both allergenic and toxicological points of view. To reliably estimate health risks for workers, it is not sufficient to measure total spore counts, which do not give any information about the toxic impact of fungal metabolites. Apart from fungal toxins (mycotoxins), microbial volatile organic compounds are discussed to have effects on human health, such as lethargy, headache, and irritation of the eyes and mucous membranes of the nose and throat [1]. In recent years, analysis of MVOC has been used as an indicator of fungal growth in both stored cereals and food [2 - 5]. The production of MVOC by fungi has been discussed, especially from the viewpoint of indoor pollution with microorganisms [6 - 8]. There is some evidence that indoor microbial contamination is indicated by the presence of certain MVOCs, that can be synthesized by a number of species [8]. Although the composition of microorganisms colonizing the building material was investigated in these studies, no information was given about the species-specific production of volatiles by certain fungi. In these investigations only single strains of species were tested for their physiological capacities. The relevance of fungal metabolites in working environments has not been sufficiently studied. Exposure to microbial volatile organic compounds is expected to be much higher in waste handling facilities than in other indoor environments, because of the extremely high microbial activity in the substrates. The odor compounds as well as non-pungent compounds must therefore be considered possible health hazards, and the identification of substances will help to elucidate the connection between the level of microbial volatiles in working places and health effects on humans. A clear distinction of the volatiles derived from plant debris and those produced by fungi in the biowaste is needed. Thus, the capacities of relevant fungal species to produce specific metabolites must be investigated. To get an indication of which compounds can be expected in situ, frequently occurring fungal species isolated from the air in compost facilities were screened for the production of microbial volatile organic compounds in pure culture. The investigation was performed as a screening to find a wide spectrum of fungal volatiles possibly occurring as air contaminants in compost plants. The objectives of our investigation were to study if the production of volatiles is consistent within different isolates of the same species, and if there are species-specific volatiles that may be suitable as marker compounds for the detection of certain fungal species,

797 MATERIAL AND METHODS

Fungal strains, growth conditions and culture The fungal strains tested had been recently isolated during preliminary investigations as air-borne fungi in a compost facility. All species were found to be among the most frequent ones in the air. Air samples were taken inside the housed parts of the facility using Sartorius MD-8 air sampler (GOttingen, Germany) and the samples were plated by dilution on diehlorane glycerol 18% agar (Oxoid). The strains were tested as fresh isolates within one to three weeks after isolation. Two isolates per species were screened for the production of MVOC. It was assured that the two isolates of one species were collected from distinct parts of the facility (loading area, compost pile hail, storage area) or from spatially separated piles within one area. The fungi were identified according to current taxonomic concepts [9 - 13]. If necessary, identification was confirmed by chemotaxonomic investigations on the mycotoxins using high performance liquid chromatography described by Frisvad and Thrane [14]. This was particularly helpful for the identification of terverticillate penicillia. All isolates tested in this study were initially subcultured on 2% malt extract agar slants and deposited in the Culture collection at the Institute for Hygiene and Environmental Medicine. Strains are referred to by their respective IHUA numbers. In the experiments, cultures were grown on yeast extract sucrose agar (YES). YES agar had proved to provide a wider spectrum of metabolites than current identification media such as malt extract agar or Czapek agar [12]. Strains of the following 13 species were tested: Aspergillus candidus 127.97, 132.97; Aspergillus fumigatus 164.97, 185.97, Aspergillus versicolor 278.97, 589.97 Emericella nidulans 166.97, 167.97; Paecilomyces variotii 592.97, 593.97; Penicillium

brevicompactum 594.97, 595.97; Penicillium clavigerum 598.97, 599.97, Penicillium cruslosum 41.97, 46.97; Penicillium cyclopium 245.97, 247.97; Penicillium expansum 602.97, 603.97; Penicillium glabrum 458.97, 463.97; Penicillium verruculosum 12.97, 14.97; Tritirachium oryzae 5.97, 125.97. The cultures were incubated at 22°C (room temperature) in daylight.

Assay for sampling of MVOC The assays were performed as long-time passive sampling on Tenax GR mesh 60/80 during a period of 10 days. The fungi were pre-incubated in YES-filled glass Petri dishes for about 4 to 6 days, until the agar surface was nearly two-thirds covered by the fungal colonies. The medium was nine-point inoculated to ensure a characteristic colony growth and to achieve a quick colonization of the agar surface. Spore suspensions were regarded as non-suitable for inoculation, since where there is dense colony growth on the plate, growth is inhibited and sporulation may be delayed, which can influence fungal physiology. With the inoculation technique used, the proportions of growing sterile mycelium and sporulating parts of the colony were regarded as best suited to imitate natural growth and combine different physiological states of the developing fungus in one experimental setup. After the pre-incubation time the lids of the Petri dishes were removed and replaced by glass funnels placed upside down containing Tenax GR tubes in the tube of the funnel (Fig. 1). The funnels were not connected gas-tight to the Petri dishes to ensure diffusion of oxygen

798 and carbon dioxide during the period of growth. Contamination by volatiles from outside the cultures was considered by measuring agar medium blanks.

Analysis of MVOC The analysis was done by thermal desorption and cold-trap injection on a Perkin Elmer ATD 400 in combination with a Perkin Elmer 4000 gas chromatograph and mass spectrometric detection (Finnigan ITD 800). ATD 400: primary desorption at 250°C for 3 min, valve temperature 180°C, cryo-focusing at -30°C, trap temperature 270°C for 5 min, input splittless, outlet 10 ml per min, transfer line temperature 200°C, desorption flow 100 ml per min, inlet flow 60 ml per min, column pressure 165 kPa. The temperature program of the gas chromatograph was set up as follows: initial temperature 37°C for 8 min, 5°C per min to 90°C, 90°C for 0.1 rain, 10°C per min to 330°C, 330°C for 7.3 min Column: Restek RTX 5:60 m, 0.25 mm ID, 2.5 ~zn df 5% polyphenylsilicone, polymer stable up to 330°C, minimal blood at 240°C. The volatile compounds were identified by comparison to libraries (Wiley 6, NBS and NIST) using calculated probability and comparison to the retention times of reference compounds. During mass-spectrometric analysis, scan intervals below M 49 were omitted to avoid disturbances from oxygen, carbon dioxide and water. This resulted in lower fit values for some substances given in Table 1 than would have been the case if the whole m/z interval had been scanned. In Table 1 only substances are reported when mass spectra coincided well with the library spectra and when the gas-chromatographic retention times were probable for the compounds suggested or could be identified with reference substances. Substances that did not coincide well with the library spectra in regard to single masses were labelled 'like'.

Referencecompounds The reference compounds used for the identification were the following: Itydrnearbons. The hydrocarbons included: hexene (Merck 820639, >96%), heptene (Merck 820622, >96%), 1nonene (Fluka 74320, -97%), 1-undecene (Fluka 94140, >95%). Alcohols. The alcohols included: 2-methyl-Ibutanol (Merck 806031, >98%), 1-octen-3-ol (Merck 814543, >97%), 3-octanol (Merck 821859, >97%), 2-pentanol (Merck 807501, >98%). Aldehydes. The aldehydes included: butyraldehyde (Merck 80155, >99%), hexanal (Merck 802672, >98%), isobutyraldehyde (Merck 801556, >95%), decanal (Merck 803211, >97%), octanal (Merck 806901, >98%), pelargonaldehyde (Merck 807166, >98%), propionaldehyde (Merck 822133, >98%), valeraldehyde (Merck, 808504, >98%). Ketnnes. The ketones included: 2-heptanone (Merck 818711, >98%), 3-heptanone (Merck 820621, >98%), 2-hexanone (Merck 804398, >98%), 3-hexanone (Merck 804533, >96%), 2-octanone (Merck 820926, >98%), 3-octanone (Merck 821860, >96%). Ethers. The ethers included: 2-methylfuran (Merck 820798, >99%). Esters. The esters included: butylacetate (Merck 101974, >99%), butylpropionate (Merck 822134, >98%), ethylbutyrate (Merck 800500, >98%), ethylisobutyrate (Merck 800501, >98%), ethylisovalerat (Merck 808541, >98%), hexylacetate (Merck 820555, >98%), isobutylacetate (Merck 820557, >98%), isobutylpropionate (Merck 822135, >98%), isovaleraldehyde (ICN 155131), propylacetate (Merck 803183, >98%). Terpenes and terpene derivatives. The terpenes included: (+)-calarene (Fluka 21025, >99%), (+)-camphene (Merck 820254, 95%), (IS)-(-) camphor (Aldrich 27,967-6, 99%), 5-(+)-3-carene (Merck 21986, >99%), (-)-trans-caryophyllene (Sigma C-9653), (R)-(+)limonene (Merck 818407, >96%), cymene (Aldrich), geosmin (Sigma G-5908, 98%), (R)-(-)-5-isopropyl-2-methyl-

799 1,3-cyclo-hexadiene (Merck 818569, >80%), myrcene (Aldrich 814593, ~75%), (1R)-(+)-ct-pinene (Merck 818632, >97%), (1S)-(-)-pinene (Merck 818405, >97%), ¢t-terpinene (Fluka 86473, -97%), ~'-terpinene (Fluka 86476, ~99%). The terpene derivatives included: cineol (Merck 159621), linalool (Merck 159645), nerolidol (Merck 818553, -95%, cis 40%, trans ~55%). Others. Hexanoie acid (Merck 800198, >98%).

RESULTS

A wide range of MVOCs (alkanes, alcohols, ketones, aldehydes, esters, ethers, terpenes and terpene derivatives) was found to be produced by the fungi in pure culture (Table 1). The MVOC spectra resulted in characteristic fingerprints for most species tested. Some volatiles were found in more than one species. 2Methyl-l-propanol (isobutanol), 2-methyl-l-butanol and 3-methyl-l-butanol (isopentanol) were found in high quantities for nearly all species tested. Penicillium crustosum did not produce 3-methyl-l-butanol, and for Penicillium cyclopium neither 2-methyl-l-butanol nor 3-methyl-l-butanol was found. Limonene was produced by all fungi except A. candidus and E. nidulans. 1-Octen-3-ol was found in four species, i.e., A. ¢andidus, A. versicolor, E. nidulans and Penicillium brevicompactum. In addition to these, many volatiles

were only found in single species. The two isolates of A. candidus showed nearly identical GC fingerprints (Fig. 2) and produced a few characteristic compounds that could be identified with less probability (Table 1). 1-Octen-3-ol was produced in relatively high quantities, but was also found in three other species: A. versicolor, E. nidulans and P. brevicompactum. 3-Cycloheptene-l-one and 3-octanone were found to be specific for A. candidus.

Besides these compounds, four other characteristic hydrocarbons were identified, of which 1,3,6-octatriene had the best fit (85%). The GC fingerprints ofA. fumigatus isolates 164.97 and 185.97 were highly similar (Fig. 3). Both isolates produced trans-13-famesene in high quantities. Moreover, camphene, ct-pinene and a 13-phellandrene-like compound were found exclusively for this species and therefore seemed to be characteristic for it. Three terpenes that could not be identified with the libraries used seemed to be characteristic as well. Moreover, limonene, 2-methyl-l-butanol, 3-methyl-l-butanol (isopentanol) and 2-methyl-l-propanol (isobutanol) were produced. A. versicolor showed a weak production of volatiles, with the exception of a series of terpenes with higher

molecular weight (Fig. 4). Only two of these compounds were identified, one being Z-curcumene and the other ct-muurolene. The combination of this series of terpenes was characteristic for the two strains of A. versicolor, but X-curcumene and ct-muurolene were also found in other species (Table 1). 1-(3-

Methylphenyl)-ethanone and 6-methyl-2-heptanone were found to be exclusively produced by A. versicolor. Moreover, the species produced 1-oeten-3-ol and limonene, as well as 2-methyl-l-butanol and 3-methyl-lbutanol (isopentanol) in trace amounts.

800

A -tic

Tenax TA Glasswool (silanized) Teflon sleeve

66

°I

46

Tenax GR (sampling) i 47

B

Spore filter

-TIC

?L_ T

Fungal colonies

" /' YES medium Fig. 1: Experimental setup for cultivation and sampling.

Fig. 3: GC chromatograms of Aspergillus fumigatus 164.97 (A) and 185.97 (B). The GC fingerprints show a high similarity in regard to the quality of compounds, fi-Farnesene (47) was specific for the species. Numbers identify the compounds as listed in Table 1.

A -11¢

A

9O

-TIC 23

22

96

6

21

16

~.L

I~llNt 11)3

~

u+wm

II~

.....

I

56

l i l t ,,

6

,

i

B ,

,

B -TIC

I

m

,

m

I

mu

Fig. 4: GC fingerprints of Aspergillus versicolor 278.97 (A) Fig. 2: GC chromatograms of Aspergillus candidus isolate 127.97 (A) and 132.97 (B). Numbers above peaks idantify the compounds as listed in Table 1. Cycinhepteu-l-one (23) was characteristic for the species within the two strains tested.

and 589.97 03). A number of terpenes of higher molecular weight were produced in relatively large amounts. The production of these terpenes seemed to be correlated with the intanse production of exudates on YES medium. 1-(3Me~hylphmayl)-ethanune (20) and 6-methyl-2-beptanone (21) were exclusively produced by this species. Numbers identify the compounds as listed in Table 1.

801 TABLE 1: Production of volatiles from Aspergillus candidus (A. can.), A. fumigatus (A. fum.), Aspergillus versicolor (A. ver.) Emericella nidulans (E. nid.), Paecilomyces variotii (Pae. var.), Penicillium brevicompactum (P. bre.), P. crustosum (P. cru.), P. clavigerum (P. cla.), P. cyclopium (P.cyc.), P. expansum (P.exp.) and P. glabrum (P. gla.) on YES agar. The fit values for the compounds listed were mostly above 75% and were given as characteristic compounds for a species when above 85%. If the fit values of substances were below 75%, these were listed only if the spectra of the compound corresponded well to the library spectra within the scanned region and the gaschromatographic retention times were probable for the suggested compound. No.

Compound

Production by: A. can.

Esters: 2-Methyl-butanoic acid methyl ester 2,3-Dimethyl-butanoi¢ acid methyl ester 4,4-Dimethyl-pentenoic acid methyl ester Hexanoie acid ethyl ester p-Mentha-6,8-dien-2-ol acetate 1-Oeten-3-ol ethyl ester Ethers: 7 2-Ethylfuran 8 275-Dimethylfaran 9 2,5 -Dimethylfuran-like 10 2*Ethyl-5-methyl-furan I1 lsopropylfuran 12 4-Methyl-2-(3-methyl-2 -butenyl)-furan ]3 2,3,5-Trimethylfuran 14 Furaneole 15 2-Acetyl-5-methylfuran 16 Methoxybenzene 17 1,2-Dimethoxybcozene 18 1-Methoxy-3 -methylbcnzene Aldehydes: 19 Decanal-like Ketones: 20 l -(3-Methylphenyl)--ethanone 21 6-Methyl-2-heptanone 22 3-Octanone 23 3-Cyelohepten-l-one 24 3-Cyclohepten- 1-one isomer 25 3-Hexanone 26 . 4-Methyl-3-hexanone-like 27 Bicyclo-(3.2.1 )-oeten-2-one Terpenes and terpene-like compounds: Aromadendrene 28 29 2-Methylenebomane 2-Methyl-2-bomene 30 2-Methyl-2-bomene isomer 31 32 x-Cadinene Camphene 33 34 8-4-Carene 2-1sopropenyl-2-camne-like 35 36 Carene-like 3,4-Dihydro-ionone-like 37 38 ~x-Chamigmne-like bcta-Caryophyllene 39 40 Elemol 41 X.-Curcumcne ! Dihydroedulan I 42 Bicycloelemene-like 43 44 Bicycloelemene 4 5 I ~-Elemene 46 cc-Farnescne 47 iTrans-l~-Famesene Gca'mac~ne A 48 I Germacrene B-like 49 50 Germacrene B 51 6-Guaiene-likc 52 I + ct-Longipmene 53 ct-Hmuleue-like 54 (x-lonol-like ltalicene 55

A. rum.

A. ver.

E. nid.

Pae. var.

P. bre.

P. cru.

P. eli.

P. e~e.

P. exp.

P. ~la.

+ + +

Fit

(%1 72 76 93 85 63 69 88 79

+ + + +

+

+ 1 2

+

2 2

79 67 63 R 74 72 59 59 95 81 79 74 R R 67 + +

1 + + +

2

2 +

+ + + +

+

2

59 65 R 83 91 74 79 88 63 79 94 cJ3 95 91 53 79 69 55 86

802 T a b l e 1 {continued) No.

Production by:

Compound

A. can. 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109

Limoncnc Mcgutigma-4,6(E),8(Z)-tricne Mcgastigraa..4,6(Z),8(E) -Iriene-like ~-Mumolene Myreene Nco-allo-O~imcnc 3-Mcthyl-4-methylen-bicycloocb2-cne-like [3-Phellandrcne ct-Pinene ct- Phellandrcne [~-Phellmdmnc-likc a-Terpinene ct-Terpinolene a-Yhujone-like Unknown terpene from lime oil Unknown terpene from P. elavigerurn Unknown terpene from P. clavlgerum Unknown terpene from P. cyclopmm Unknown terpenc from P. cyclopium Unknown terpene from A. fumtsatus Unknown terpene from A.fumlgatus Unknown tcrpenc from A. fumtgatus Unknown terpcne from P. glabrum Unknown lerpene from g nidulans Unknown terpene from E. nMulans Unknown terpenc from E. nidulans Unkuown Igrpene from P. variotti Alknholes: 8-2-Dodeeanol 4-Ethylbut~a.-4.-olide [3-Fenchyl alcohol 3-Methyl-1 -butanol (isopentanol) 2-Methyl-I -butanol 2-Methyl-1 -propanol (isobulanol) Citronellol 1-Octen-3-ol 2-Mcthylphenol AIImnes, alkencs, cycloalkanes: Dodecane 1-Ethyl-2-methylbcnzene 3,5,7-Trimethyl-2E,4E,6E,SE-deca~U'aen¢ 4-Methylen-spiro[2.4]hept~ae-like 3-Methyl-I -heptene 2-Methyl-2,4-hexadiene 1,2,3-Trimethylcyclohexane-like 3,5,5-Ttimethylhexene-like 1,3-Octadiene-like Bicyelooctan-2-one 1-xq~4cthylcncydopropyl)-cyclopentanol-like 1,3+6-Octattienc 1,3,5-Cyclooctatriene-like 1 1,3,5-Cyclooctatriene-like 2 1,3,5-Cyclooctatriene-like 3 Cyclooctene Styrene Sulphur compounds: Dimeth~ldisulfide ~compounds

A. ram.

A. ver.

E. nid. +

Pae. var.

P. bre. +

+ +

P. cru. +

P. cla. +

P. c~'c.

P. exp.

P. •la. +

+

R 95 50 63

2

+ + + + +

+ + + +

+ + +

+

+ + +

Fit (%) R 88 54 94 R 85 68 94 R 91

+ +

+ + +

+ + +

+

63 63 58 R 50 R 88

+ 1

68 91 89 59 74 56 31 63 59 67 85

+ + + +

16

14

11

21

l

+

+

+

22

12

15

+ 15

13

+

+

12

12

68 79 93

Substances identified with the use o f reference c o m p o u n d s are m a r k e d with R. For substances w h o s e identities are indicated by the m a s s spectrometric library, the fit values are g i v e n as percentage. I f percentages o f c o m p o u n d s listed ranged under 80%, the m a s s spectra fit well w i t h that o f the library, or substances are labelled 'like'. E v e n for reference c o m p o u n d s the fit values were s o m e t i m e s under 80%, since scan intervals b e l o w M 49 w e r e omitted to a v o i d disturbances f r o m oxygen, carbon dioxide and water. For 1-Octen-3-ol the fit value r a n g e d near 6 5 % w h e n analyzed as reference compound. This m u s t partly be ascribed to differences in m a s s detection s y s t e m s and is considered in the computer-assisted libraries such as W i l e y 6 b y listing several m a s s spectra. +: the c o m p o u n d w a s produced by both isolates o f a species; 1: the c o m p o u n d w a s produced by the isolates with the lower strain number; 2: the c o m p o u n d w a s produced b y the isolate with the h i g h e r strain number.

803 Likewise, isolate 166.97 and 167.97 ofEmericella nidulans showed nearly identical GC fingerprints (Fig. 5). For both isolates three different methyl-esters, 2-methyl-bu'tanoic acid methyl ester, 2,3-dimethylbutanoic acid methyl ester, and 4,4-dimethyl-pentenoic acid methyl ester were characteristic. Five different terpenes including timonene were identified, of which ct-terpinolene seemed to be specific for the species. Three terpenes that were characteristic for E. nidulans could not be identified with the libraries used (see Table 1). The abundant production of terpenes was specific for Paecilomyces variotii (Table 1, Fig. 6). 6-4-Carene, megastigma-4,6(E),8(Z)-triene,

myrcene, neo-allo-ocimene, and 13-phellandrene were identified with high

probabilities. Except for myrcene, all of the terpenes mentioned were exclusively found for P. variotii. Penicillium brevicompactum showed only weak production of MVOC, and the spectrum of compounds was not consistent within the two isolates tested (Fig. 7). Both isolates produced limonene, 3-methyl-Ibutanol (isopentanol), and l-octen-3-ol, the last two in higher quantities. 1,3-Octadiene was exclusively found in P. brevicompactum and therefore seemed to be characteristic for the species. Some volatiles identified with lower fit values were found in only one of the two isoates (see Table 1). For P. crustosum the production of four furan derivatives was characteristic. The species produced 2ethylfuran, 2,5-dimethylfuran, 2-ethyl-5-methylfuran, isopropylfuran and 2,3,5-trimethylfuran. The last derivative was also found in A. candidus, Paecilomyces variotii and Penicillium cyclopium. The GC fingerprints showed differences between the two isolates of P. crustosum, which is probably due to the quantity of compounds rather than the quality of the spectrum of volatiles (Fig. 8). The two isolates of P. clavigerum showed weak production of volatiles (Fig. 9). Nevertheless, some terpenes like caryophyllene, limonene, myrcene, and ct-terpinene were identified with higher probabilities and 13-elemene and a 6-guaiene-like compound with lower probabilities. Limonen was frequently produced by other species and myrcene was also found in Paecilomyces variotii. Finally, caryophyllene and ct-terpinene proved to be characteristic for P. clavigerum. In comparison with other species, the two isolates of P. cyclopium showed a very weak spectrum of volatile metabolites, although some terpenes were produced in higher amounts (Fig. I0). Germacrene A, which is one of them, 2-methyl-2-bomene, and a 4-methyl-2-(3-methyl-2-butenyl)-furan-like compound were found exclusively for Penicillium cyclopium.

Additionally, 2,5-dimethylfuran and 2,3,5-

trimethylfuran were found for the species, which were not characteristic since both were also produced in P. crustosum. In P. expansum characteristic GC fingerprints were found for both isolates (Fig. 11). Aromadendrene, bicycloelemene, dihydroedulane, and germacrene B seemed to be characteristic, since these terpenes were only found in this species. Moreover, 1-methoxy-3-methylbenzene (3-methyl-anisole) and three isomers of a 1,3,6-cyclooctatriene-like compound were produced both in isolate 602.97 and 603.97. 2-Methyl-1propanol, 2-methyl-l-butanol, and 3-methyl-l-butanol were not produced by Penicillium expansum.

804 A

A

-TkC

90

1

--T~C

3 56 68

107

79

u:tt:$o

81

oo:~G~

om~m

B

B

-11C

-nc

-

-

i w

L

Itllm

Fig. 5: GC fingerprints of Emericella nidulans 166197 (A) and 167.97 (B). 2-Methyl-butanoic acid methyl ester (I), 2,3dimethyl-butanoic acid methyl ester (2) and 4,4-dimethylpentenoic acid methyl ester (3) were found to be specific for the species. Numbers identify the compounds as listed in Table I.

Fig. 7: GC fingerprints of Penicillium brevicompactum 594.97 (A) and 595.97 (B). A number of volatiles was only found in one of the two strains. A 1,3-octadiene-like compound (100) was exclusively found in P. brevicompactura. Numbers identify the compounds as listed in Table 1.

A -rio

A

63

57

-nc

108

97

Llolm

10865

S3

61

38

13

54 94 32

m~N

B

8

LO 11

56

~m~m

m~ J

m~

I

, I M

m

i

B

-TIC

--T~

mi'Lw

hL.

J

Fig. 6: GC fingerprints of Paecilomyces variotii 592.97 (A) and 593.97 (B). Differences between the two isolates were found for the production of 2-methyl-l-butanol and 3-methyl-l-butaaol, whereas the production of a series of terpenes is consistent. 5-4Carene (34), megastigma-4,6(E),8(Z)-~'iene (57), neo-alloocimene (61), and !3-phellandrene (63) turned out to be specific. Numbers identify the compounds as listed in Table 1.

~ltm

mH

+

Fig. 8: C~ fingerprints of Penicillium crustosum 41.97 (A) and 46.97 (B). Differences in the chromatograms are mainly due to quantitative aspects. The combination of 2-ethylfuran (7), 2-ethyl-5-methylfuran (10), and isopropylfuran (11) turned out to be specific. Numbers identify the compounds as listed in Table 1.

805 A -TIC

A -llC 108

104

56

L..l ....

itl0m

B

5o

li li

I )

k, *t/t~

tutti

u uJ

J uu~

i

n.ll*

B -TIC

-TIC

L

F /:

r~2~

15

/

,

will

RJ i m

i

ilooo

itll

Fig. 9: GC fingerprints of Penicillium clavigerum 598.97 (A) and 599.97 (B). The volatiles specific for this species were identified with lower fit values. Nevertheless, the fingerprints of the two strains showed a high similarity. Numbers identify the compounds as listed in Table 1.

A

-r~¢

13 ~I07

" I

I MCI~

, ''~

,

imc,tm:mt

et4mlmt

I l o'l e

r u-lu~l

B -Tic

am.i~

InUatN

Fig. 10: GC fingerprints ofPenicillium cyclopium 245.97 (A) and 247.97 (B). The production of volutiles is rather weak in this species. Germacrene A (48), 2-methyl-2-bomene (30), and a 4-methyl-2-(3-methyl-2-butenyl)-furen-like compound (12) were exclusively found for P. cyclopium. Numbers identify the compounds as listed in Table 1.

,

FA

j~y'

/i

i

/ i r~

11: Characteristic GC-fingerprints of Penicillium expansum 602.97 (A) and 603.97 (B). Aromadendrene (28), Fig.

bicycloelemene (44), germaerene B (50), 3-methyl-anisole (17), and a dihydroedulane-like compound (42) were found to be specific. Numbers identify the compounds as listed in Table 1.

806 In P. verruculosum and Tritirachium oryzae extremely weak production of metabolites was observed. Even non-specific volatiles such as 2-methyl-l-propanol, 2-methyl-l-butanol, and 3-methyl-l-butanol were not found. P. verruculosum and Tritirachium oryzae are therefore not included in Table 1. In Table 2 the production of compounds characteristic for single species is summarized.

TABLE 2: Volatiles exclusively produced by both test strains of one species tested.

Species

Compounds

Aspergillus candidus

Hexanoic acid ethyl ester, methoxybenzene, 3-cycloheptene-l-one, 1,3,6-octatriene

A, fumigatus

p-Mentha-6,8-diene-2-ol acetate, camphene, trans-~-famesene, ct-pinene, three unknown terpenes

A. versicolor

1-(3 -Methylphenyl)-ethanone, 6-methyl-2-heptanone

Emericella nidulans

2,3-Dimethyl-butanoic acid methyl ester, 4,4-dimethyl-pentenoic acid methyl ester, 2-methyl-butanoic acid

Paecilomyces variotii

cS-4-Carene, megastigma-4,6(E),8(Z)-triene, neo-allo-ocimene, 13-phellandrene

Penicillium crustosum

2-Ethylfuran, 2-ethyl-5-methylfuran, isopropylfuran

P. clavigerum

13-Caryophyllene

P. cyclopium

2-Methyl-2-bornene, germacrene A

P. expansum

I-Methoxy-3-methylbenzene (3-methyl-anisole), aromadendrene, bicycloelemene

methylester, u-humulene like, a-terpinolene, three unknown terpenes

Isolates of Penicilliumbrevicompactum and Penicilliumglabrum are not includedsince the productionof metaboliteswas inconsistentwithinthe two differentstrainsof the same species. DISCUSSION

General indicators

Within the number of 109 compounds identified, some were found in several species (Table 1). 2-Methyl1-butanol was produced by all species except Penicillium crustosum, P. cyclopium and P. expansum. The isomer 3-methyl-l-butanol (isopentanol) was produced by all species except P. cyclopium and P. expansum. 1-Octen-3-ol was produced by 4 out of 13 species tested: Aspergillus candidus, A. versicolor, Emericella nidulans and Penicillium brevicompactum. These findings indicate the relevance of these

three compounds from the viewpoint of indoor pollution with fungi. The species A. versicolor and P. brevicompactum are generally known as fungal contaminants in indoor environments and must obviously

be regarded as a source of these MVOC. Larsen and Frisvad [15] found that smaller alcohols like 2methyl-l-propanol, 3-methyl-l-butanol and 1-pentanol were produced by several species, and some Cs compounds such as 1-octen-3-ol, 3-octanol and 3-octanone were produced by a range of species from the genus Penicillium. Str6m et al. [8] reported that the occurrence of 1-octen-3-ol in indoor environments correlated with fungal growth and indoor environmental complaints in damp Swedish houses. The above volatiles were also reported in an investigation by Sunesson et al. [16]. Thus, 3-methyl-l-butanol (isopentanol) and 1-octen-3-ol may serve as an indicator for fungal growth in general, but cannot serve as

807 a specific indicator for the occurrence of distinct taxa of fungi. In addition to the above mentioned compounds, 2,3,5-trimethylfuran was found in a number of species within this study, and may therefore serve as additional indicator for fungal growth in general. Compounds such as limonene and styrene were also found for several species in other investigations [15], but are not suited as general indicators for fungal growth, because these compounds can be derived from plants or building material, respectively.

Species-specific compounds and intraspecific variation Taking the chromatograms as fingerprints, a high similarity was observed within the two different strains of one species. The present results match well with those obtained by Larsen and Frisvad [15], who reported 1,3-dimethoxy-benzene to be produced by A. versicolor and 3-methyl-anisole by P. expansum. In our study, both substances turned out to be species-specific within the number of species and isolates tested. The compounds specific for A. versicolor were not found by Sunesson et al. [16], which is obviously due to the use of different culture media. However, a series of ketones was listed by these authors and two ketones were found to be specific in our experiments (Table 2). It is likely that among the great variety of compounds produced by the microorganisms, several species-specific can be found. Supposing a production on different substrata, these might be used as marker compounds for the selective detection of certain species in various environments. The inconsistency in Penicillium crustosum isolates 41.97 and 46.97 must be ascribed to quantitative rather than to qualitative differences in the spectra of metabolites. These differences were not reflected in colony morphology or in chemotaxonomic data from previous mycotoxin analysis (unpublished). Isolates of P. brevicompactum and P. glabrum gave inconsistent results. In many cases fresh isolates of

Penicillium brevicompactum differing in colony morphology had to be classified as P. brevicompactum series. Thus, the inconsistency in the production of volatiles may be an indication for the variability of the taxon. Likewise, in P. glabrum, species identification and delimitation from P. spinulosum is often unclear. Several isolates determined in our own investigations could not clearly be classified in one of the two species. Difficulties concerning the identification of naturally occurring Penicillia, even if sophisticated techniques are used, were described by Pitt et al. [17]. Although the two strains tested in this investigation are not sufficient for taxonomic evidence, this may be taken as an indication that the concept of P. brevicompactum and P. glabrum needs further taxonomic reevaluation.

Characteristic spectra of compounds In addition to single volatiles specific for certain species, the production of distinct chemical groups of compounds also seemed to be characteristic. For Emericella nidulans a combination of three methyl esters was found and P. crustosum produced a series of furan derivatives. Paecilomyces variotii was characterized by the production of a variety of terpenes, and for Penicillium cyclopium a combination of bomene derivatives was found. Apart from 2,3,5-trimethylfuran, the other furan derivatives were exclusively found in closely related Penicillium species, i.e., P. crustosum and P. cyclopium. The

808 spectrum of furan compounds may therefore serve as an indicator for species belonging to the P. aurantiogriseum complex. A certain combination of compounds may be suitable for describing a community of fungi. Stahl and Parkin [ 18] already showed that spectra of volatile metabolites can provide information on the nature of the soil microbial community. Likewise, it may be possible that the fungal community colonizing the biowaste can be described by the analysis of species-specific fungal volatiles. Exposure assessment and health impacts For compounds either indicating fungal growth in general or being species-specific, possible risk assessment and investigation of potential exposure in natural environments need further investigation. The relative amount of spore counts in the air does not necessarily reflect the relative abundance of species in the biowaste. Species not frequently found in the air may equally contribute to an emission of volatiles in working environments and, thus, more species than those presented here will have to be investigated for their physiological properties. In the present experiments, a synthetic medium with a high sugar content was used. The medium was used in several taxonomic studies on Penicillium systematics [13, 15, 19, 20, 21] and was recommended by Samson et al. [12] for the analysis of fungal metabolites. Larsen and Frisvad [15] showed that a range of volatile organic compounds could be produced on both synthetic and natural substrates. However, when volatiles are to serve as specific indicators for the occurrence of certain species in contaminated environments, it must be certain that these compounds can also be produced on natural substrates. Consequently, experiments are necessary to investigate the physiological properties of different species on natural substrates in vitro, such as compost or compostcontaining media. To reliably estimate health risks for workers, the amount of fungus and species-specific metabolites must be qualified and quantified. Because a great species diversity of fungi can be found in the air of compost plants and also in biowaste, a wide range of metabolites must be assumed to be produced by the fungi in natural habitats [22]. Non-volatile metabolites known as characteristic mycotoxins of.4. fumigatus were found by Fischer et. al. [23] in native bioaerosols from a compost facility. The spectrum of volatile organic compounds (VOC) in compost plants is manifold [24]. A wide variety of VOC, especially terpenes, can also originate from the plant material; others derive from bacteria. Therefore, the significance of fungal MVOC must be evaluated in comparison to the VOC deriving from plant debris. Since there have not been enough investigations on either species-specific MVOCs or the quantities of these compounds in the air, it is difficult to find a clear correlation between health effects and exposure to MVOCs. However, the influence of fungal populations colonizing the biowaste on the spectrum of MVOC must be taken into account when health hazards due to fungi are discussed. Further research In most species a number of compounds, mostly those produced in minor quantities, remained unidentified (Table 1). Many of these were found to be characteristic for certain species and, thus,

809 contributed basically to the similarity of the GC fingerprints of different strains within one species. The extended use of reference compounds will enlarge the number of compounds that can be identified. However, the results of the present investigation indicate that among the fungal volatiles several speciesspecific compounds exist, which may be used as specific markers for the presence of certain fungi in natural environments. The chemotaxonomic value of secondary metabolites has been demonstrated in several investigations [19, 20, 21, 25]. The present results have to be evaluated further by testing a greater number of species and isolates of one species on both synthetic and natural substrata. It is generally known that fungal strains can alter their physiological properties and metabolism when kept in culture collections for long periods. Therefore, with regard to a reliable exposure assessment in situ, it is necessary to investigate freshly isolated strains for their physiological properties.

ACKNOWLEDGEMENTS The corresponding author is subsidized by a dissertation grant (Az 6000/256) provided by the "Deutsche Bundesstiftung Umwelt" (DBU).

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