Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species

f u n g a l b i o l o g y 1 1 6 ( 2 0 1 2 ) 9 4 1 e9 5 3

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Identification of volatile markers for indoor fungal growth and chemotaxonomic classification of Aspergillus species Viviana POLIZZIa,b, An ADAMSa, Svetlana V. MALYSHEVAb, Sarah DE SAEGERb, Carlos VAN PETEGHEMb, Antonio MORETTIc, Anna Maria PICCOd, Norbert DE KIMPEa,* a

Department of Sustainable Organic Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium b Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium c Institute of Sciences of Food Production, National Research Council, via Amendola 122/o, I-70126 Bari, Italy d Pavia University, Faculty of Sciences, Department of Territorial Ecology and Environment, via S. Epifanio 14, 27100 Pavia, Italy

article info

abstract

Article history:

Microbial volatile organic compounds (MVOCs) were collected in water-damaged buildings

Received 8 September 2011

to evaluate their use as possible indicators of indoor fungal growth. Fungal species isolated

Received in revised form

from contaminated buildings were screened for MVOC production on malt extract agar by

10 June 2012

means of headspace solid-phase microextraction followed by gas chromatography-mass

Accepted 13 June 2012

spectrometry (GCeMS) analysis. Some sesquiterpenes, specifically derived from fungal

Available online 30 June 2012

growth, were detected in the sampled environments and the corresponding fungal pro-

Corresponding Editor:

ducers were identified. Statistical analysis of the detected MVOC profiles allowed the iden-

Richard A. Humber

tification of species-specific MVOCs or MVOC patterns for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. In addition, Chaetomium spp. and Epicoccum

Keywords:

spp. were clearly differentiated by their volatile production from a group of 76 fungal

Aspergillus

strains belonging to different genera. These results are useful in the chemotaxonomic dis-

Chaetomium

crimination of fungal species, in aid to the classical morphological and molecular identifi-

Chemotaxonomy

cation techniques.

Indoor fungi

ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Microbial volatile organic compounds

Introduction Since the 1990s indoor air quality has received a lot of attention (Wallace 1991; Jones 1999) because of the increasing time people spend indoor. Indoor air pollution was identified as one of the top environmental risks by the U.S. Environmental Protection Agency (USEPA 1995). Along with particulate matter, gases such as ozone and nitrogen oxide, (microbial)

volatile organic compounds ((M)VOCs), cigarette smoke, building occupants’ activities, and outdoor ambient air are stated to be the most common sources of indoor air pollutants (Bernstein et al. 2008). MVOCs are byproducts of both the primary and secondary metabolism of fungi and bacteria, and they are detectable before any visible sign of microbial growth. For this reason, their use as early indicators of biocontamination has been proposed. Besides microbial metabolism, VOCs

* Corresponding author. Tel.: þ32 9 264 59 51; fax: þ32 9 264 62 43. E-mail addresses: [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected] 1878-6146/$ e see front matter ª 2012 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2012.06.001

942

also have other environmental sources including building materials such as wood or polyvinyl chloride, human activities, traffic, and smoking (Helmig et al. 1999; Schleibinger et al. 2002). Therefore, a volatile marker of indoor fungal growth has to be specifically produced only by these microorganisms. The identification of characteristic VOC patterns for dominant indoor moulds may provide very useful information for the development of methods for the detection and location of mould growth in buildings (Wilkins et al. 1997). The production of fungal volatiles has already been monitored on building materials or synthetic media using various methods (Pasanen et al. 1996; Fiedler et al. 2001; Claeson et al. 2002; Wilkins et al. 2003). The main MVOCs often reported as fungal growth indicators are 1-octen-3-ol, 3-octanone, geosmin (Sunesson et al. 1995; Fiedler et al. 2001), a-terpineol, and 2methylisoborneol (Nilsson et al. 1996; Larsen 1997). Hydrocarbon sesquiterpenes are among the most commonly identified microbial metabolites in laboratory experiments, yet they have not been reported indoors (Wilkins & Larsen 1995; Nilsson et al. 2004; Sunesson et al. 2006), probably because of analytical limitations. Due to the very low concentrations of MVOCs produced and due to the interferences with VOCs from other sources, it is still under discussion whether hidden indoor moulds can actually be revealed by the detection of MVOCs (Pasanen et al. 1998; Kim et al. 2007; Schleibinger et al. 2008). VOC emission by indoor fungi appears to be complex and are determined by several factors. In fact, their production is greatly affected not only by the fungal genus and species, but also by the growth phase and other conditions, such as nutrients, pH, humidity, and temperature (Korpi et al. 1998, 2009). Also, periods of high mould spore production may be preceded by increased MVOC production (Fedoruk et al. 2001). Moreover, some MVOCs are representative for the initial period of growth while others represent later stages, as recently shown by Matysik et al. (2008). For this reason, indoor air measurements by active sampling should ideally be performed in different time windows along 1 d and in different seasons. In addition, MVOC indoor levels are a balance between production rates, absorption to and desorption from building materials and furniture, and ventilation. Nutritional imbalances and disorders may often promote or trigger the production of several MVOCs (Turner 1971; Berry 1988; Bjurman 1999; Korpi et al. 2009). In fact, lack of primary carbon and nitrogen sources promotes terpene emissions while the availability of certain amino acids results in sulphur and/or nitrogen compounds (Korpi et al. 2009). In continuation of a previous report (Polizzi et al. 2009), 255 fungi and 133 (M)VOCs were collected from 11 water-damaged buildings. One of the main intentions was to analyze MVOCs as possible indicators of indoor fungal growth, by matching VOCs detected indoor with MVOC profile of isolated fungal strains. All isolated fungal species were screened in vitro for MVOC production to reveal potential species-specific MVOC patterns useful in chemotaxonomy. Chemotaxonomy, also called chemosystematics, is the attempt to classify and identify organisms according to demonstrable differences and similarities in their biochemical composition. This field is becoming more and more important for a polyphasic identification approach of microorganisms, i.e., the simultaneous use of

V. Polizzi et al.

morphological, molecular, and metabolic information to obtain a reliable fungal identification (Frisvad 2011). The possibility to highlight species- or genus-specific MVOCs or MVOC patterns has been proposed by some authors (Karlshøj & Larsen 2005; Zhang et al. 2010) and was investigated in this work within the fungal genus of Aspergillus together with a variety of other fungal genera, while Penicillium and Trichoderma genera are discussed elsewhere.

Materials and methods Reagents The following chemicals were used for MVOC and fungal sampling procedures: octane (98 %, SigmaeAldrich, Bornem, Belgium), nonane (99 %, Acros Organics, Geel, Belgium), decane (99 %, SigmaeAldrich), undecane (99 %, SigmaeAldrich), dodecane (99 %, SigmaeAldrich), tridecane (99 %, Acros Organics), tetradecane (99 %, SigmaeAldrich), pentadecane (99 %, SigmaeAldrich), hexadecane (99 %, Acros Organics), heptadecane (99 %, SigmaeAldrich), octadecane (99 %, Merck, Hohenbrunn, Germany), a-pinene (Acros Organics), b-pinene (Fluka, Bornem, Belgium), 2-phenylethanol (Acros Organics), eugenol (Fluka), 1-octen-3-ol (SigmaeAldrich), terpinolene (Fluka), absolute ethanol (SigmaeAldrich), Tween 80 (SigmaeAldrich), and NaCl. Distilled water was obtained using an AQUADEM Patrone system (model 22DF, Werner, Germany).

Materials Malt extract agar (MEA) was prepared as follows: 2 % malt extract (Oxoid, Basingstoke, UK), 0.1 % bacteriological peptone (Oxoid), 2 % bacteriological agar (Oxoid), and 2 % a-D-(þ)-glucose anhydrous (99 %, Acros Organics) were dissolved in distilled water. Dichloran glycerol 18 % (DG18) substrate (Oxoid) was prepared following the manufacturer’s instructions. Czapek Dox Agar and Potato Dextrose Agar (PDA) were purchased from Fluka. Sterile Petri dishes, inoculation needles, spatula, and pipettes (10 ml) were purchased from VWR International (Zaventem, Belgium). One-ml sterile pipettes were bought from Falcon (BD, Erembodegem, Belgium). For the sterilisations, a pressure cooker canner (Presto, VWR International) was used (121  C, 1.2 bar, 20 min).

Sampling procedures The sampling procedures were approved by the Ethical Committee of Ghent University Hospital (B6702006019, 17/07/2006). This study is a continuation of a previous investigation (Polizzi et al. 2009) in which the presence of mycotoxins, fungi, and (M)VOCs in seven water-damaged buildings located in Belgium was discussed (Table 1). Four additional waterdamaged buildings have been sampled. In all four cases, at least one of the inhabitants reported health problems, mainly respiratory problems starting after mould growth. Only one house was characterized by elevated relative humidity levels (63 %) without visible mould growth. It was decided to sample this house for the presence of some fungal speciesdAlternaria spp., Stachybotrys spp., Aspergillus fumigatus, and Aspergillus

Identification of fungal volatile markers and chemotaxonomic classification of Aspergillus species

943

Table 1 e Overview of the 12 buildings sampled. House n

Location

Date of sampling

Type of building

1

Huizingen

Sep. 2007

Private house

2

Boekentoren (Ghent) Zedelgem

Oct. 2007

Library

Oct. 2007

Private house

3

Health complaints

Degree of mould contamination

Airways irritation, cough Dizziness

>1 m2 but less than 5 m2

Heavy contaminationa

4

Ledeberg (Ghent)

Jan. 2008

Dormitory

Airways irritation, poor IAQb Airways irritation

5

Ghent

Jan. 2008

Office

Poor IAQ

6

Ghent

Jan. 2008

Office

7 8

Ghent Ghent

Jan. 2008 Nov. 2008

Storage room Private house

9 10

Ghent Wanzele

Mar. 2010 Jun. 2010

Private house Private house

11

Antwerpen

Jul. 2010

Library

12

Heusden

Jun. 2008

Control house

Allergic reaction upon entering the room Airways irritation Allergic reaction, poor IAQ Allergic reaction Smoke-, complaint-free, clean environment

Heavy contamination of books

Heavy contamination (visible fungal spores in condensation drops) Heavy fungal contamination hidden behind plasterboard Heavy fungal contamination hidden behind wallpaper Heavy contamination Heavy contamination Heavy contamination No visible mould growth Heavy contamination on books and surfaces Clean environment

a >5 m2. b IAQ ¼ indoor air quality.

versicolordbecause antibodies for these fungi had been found in blood analysis of the unhealthy inhabitant (data not shown). Also, one control house (i.e., a clean, smoke- and complaint-free environment) was sampled. An overview of the sampled buildings is given in Table 1. To sample visible fungal growth on surfaces, mycelium was sampled by ‘stick-to-it’ lift tape (Procare, Groningen, The Netherlands), composed of glasses with a square of glue of 2.5  2.5 cm that were gently pressed on the mycelium and pulled back. Alternatively, a sterile swab (VWR International) was pressed on the contaminated material surface and then immediately placed in a tube containing a sterile solution, forming in this way a spore suspension. When possible, mouldy building materials were cut away. To have an indication of the mycoflora present in the indoor air, MEA and DG18 Petri dishes (9 cm diameter) were exposed for 10 min to the air. MEA is a common medium for mould growth while DG 18 is more specific for isolation of xerophilic moulds, e.g., moulds that require low levels of free water for growth and can grow if air has more than 60 % relative humidity, like the genera Aspergillus and Penicillium. In fact, the medium formulation contains glycerol at 18 % (w/w) which lowers the water activity (aw) from 0.999 to 0.95. The medium also contains dichloran at 0.02 % which inhibits the spreading of other genera and allows unobscured growth of organisms that ordinarily form small colonies. The (M)VOCs in the houses were sampled by dynamic headspace absorption on Tenax and by Solid Phase Microextraction (SPME) followed by gas chromatography-mass spectrometry (GC–MS) analysis. The 50/30 mm divinylbenzene/ Carboxen/polydimethyl siloxane (DVB/Car/PDMS) SPME fibre (Supelco, Bornem, Belgium) was exposed to the mouldy

environment for 3.5 h. Moreover, depending on the possibility to leave the equipment in the buildings, SPME sampling was performed also for 30 min or 24 h. The same sampling times were applied to the dynamic headspace technique performed by means of a hand-made pump to which a Tenax adsorption tube (length 18 cm, i.d. 4 mm, o.d. 6 mm) was attached, filled with 250 mg of Tenax TA 60/80 mesh (Supelco, Bellefonte, PA, USA). Desorption and analysis were performed as described in Section 2.4.

Analytical procedures To collect fungal samples from dust, the procedure using the sterile swab was used (Section 2.3). The so-formed spore suspension was inoculated under sterile conditions on MEA and DG18 with 0.5 ml of undiluted and diluted suspension (102) (to avoid the masking effect of fast growing moulds on the slower growing ones). Colonies from both media were purified and compared to avoid duplications of the same fungal strain in these samples. When pieces of contaminated building materials were collected, extraction of the fungal strains from these pieces was performed by stirring them for 1 h in 50 ml of sterile physiological solution (0.85 % NaCl, 0.5 % Tween 80) and by inoculating 0.5 ml of this solution on the two media. This procedure permits the detection of fungal contaminations hidden on the inner side of the building material, not reachable by surface sampling. After incubation of the Petri dishes obtained by stick-to-it and swab samplings at 25  C, the growing colonies were purified and transferred on MEA, PDA or Czapek Agar substrates according to a previous genus identification of the growing colonies, and then morphologically identified at the species

944

level (Ellis 1971, 1976; Pitt 1979; Klich 2002). Colonies derived from different fungal sampling techniques (swab, exposure of the Petri dish to the indoor air, and extraction from contaminated building materials) were purified and always compared to avoid duplications of the same fungal strain in these samples. For all the other samples collected in houses 8, 9, 10, and 11 (Table 1), a first morphological identification at the genus level was carried out via a stereomicroscope (Bresser Advance ICD 10-160 X equipped with a Euromew illuminator EK-1, Germany). Stick-to-it samples taken in houses 1, 2, and 3 (Table 1), and strains derived from house samplings but isolated in the lab were sent for microscopic and molecular identification to Institute of Science and Food Production (ISPA, Bari, Italy). First, morphological identification was performed (Gams et al. 1980). For molecular identification, fungal strains were cultured for 48 h with shaking (125 rpm) at 25  C in 100 ml of Wickerham’s medium (4 % D-glucose, 0.5 % peptone, 0.3 % yeast extract, 0.3 % malt extract). The mycelium was vacuum-filtered on Whatman n 4 filter paper and washed with distilled water, frozen (20  C), and lyophilized. Lyophilized mycelium powder was used for genomic DNA isolation, according to the E.Z.N.A. Fungal DNA Miniprep Kit (Omega Bio-tek, Doraville, GA) instructions. Extracted DNA was dissolved in 100 ml of sterile water and stored at 20  C. DNA concentrations were estimated using a DNA ladder and electrophoresis. Sequence analyses were performed in order to confirm the identity of the strains established by morphological analysis. The species identification of the strains was obtained by sequencing a region of the b-tubulin gene by using primers Bt2a (Glass & Donaldson 1995) and T2 (O’Donnell & Cigelnik 1997). Glass & Donaldson (1995) and O’Donnell & Cigelnik (1997) developed a series of primers for amplifying b-tubulin from filamentous fungi, suitable for identification of a large spectrum of fungal genera. Polymerase chain reaction (PCR) was performed using the following conditions: denaturation at 94  C for 10 min; 35 cycles of denaturation at 94  C for 50 s, annealing at 53  C for 50 s, extension at 72  C for 1 min; final extension at 72  C for 7 min, followed by cooling at 4  C until recovery of the samples. Amplification of a 400 bp product was verified via gel electrophoresis on a 1.5 % agar gel. Once verified, PCR products were purified by filtration through Sephadex G-50 medium (Sigma) prior to DNA sequencing. Sequence analysis was performed with the Big Dye Terminator Cycle Sequencing Ready Reaction Kit for both strands (Applied Biosystems). The resulting sequences of all the isolates and reference strains were aligned by the Clustal method with DNAMAN (version 5.2.9, Lynnon Biosoft) or Bioedit (version 7.0.5, Ibis Therapeutics Carlsband, CA 92008) software. The b-tubulin homology sequences were searched performing a pairwise method in DDBJ/EMBL/GenBank International Nucleotide databases using Basic Local Alignments Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI) website. Eleven fungal sam
que de l’Universite  Cathoples were identified by the Mycothe lique de Louvain (MUCL, Belgium). Footnotes in Tables 3 and 4 give complete information on which identification procedure was used for each fungal sample. The fungal strains were cultivated as small surface cul€ lheim a/d Ruhr, Gertures in 20 ml SPME vials (Gerstel, Mu many) (Van Lancker et al. 2008). The vials were sterilized by

V. Polizzi et al.

autoclaving, filled with 3 ml of MEA, and inoculated by means of a sterile inoculation needle. The vials were closed with cotton, which was substituted for a magnetic crimp cap (20 mm, Gerstel) with a silicone/teflon septum (Gerstel) on the second day after inoculation. Blank substrates were treated and sampled as above except for the inoculation step. The fungal metabolites were extracted by headspace SPME during 30 min at 35  C. A 50/30 mm DVB/Car/PDMS fibre was used (Supelco, Bornem, Belgium). SPME extraction and desorption were performed automatically by means of a Multipurpose Sampler (MPS-2, Gerstel). The SPME desorption was performed for 2 min in the inlet of an Agilent 6890 Gas Chromatograph (GC) Plus (Agilent Technologies, Diegem, Belgium) coupled with a quadrupole mass spectrometer HP 5973 Mass Selective Detector (MSD), equipped with a programmed temperature va€ lheim a/d Ruhr, Gerporizer (CIS-4 PTV) injector (Gerstel, Mu many), and an HP5-MS capillary column (30 m  0.25 mm i.d.; coating thickness 0.25 mm). Working conditions were as follows: injector temperature 250  C, transfer line temperature 250  C, oven temperature: start 35  C, hold 5 min, programmed from 35 to 120  C at 5  C min1, from 120 to 280  C at 20  C min1, hold 3 min; carrier gas (He) 1.2 ml min1; splitless mode; Electron Impact (70 eV); acquisition parameters: scanned m/z: 40e200 (2e10 min), 40e300 (>10 min). Thermal desorption of the Tenax tubes and HSSE stir bar was carried out in a Thermodesorption System TDS2 from Gerstel coupled with the GCeMS system. The TDS2 was operated as follows: start 25  C and end at 260  C at a rate of 60  C s1, hold 7 min; purging gas helium; transfer line temperature to the injector 275  C. During thermal desorption the CIS-4 PTV inlet was held at 100  C by means of liquid N2 for 10 min to cryofocus the compounds. After complete desorption the inlet was heated to 260  C at 12  C s1. The GCeMS parameters were the same as for the SPME analysis. Fungal metabolites were identified by comparison of the obtained mass spectrum with mass spectral libraries (Nist 98; Wiley 6th; Adams 2007) and by comparison of the calculated linear retention index (RI) with literature values (Adams 2007; ElSayed 2011).

Statistical analysis Principal Component Analysis (PCA) was performed with Splus (version 8). The data were labelled as 0 or 1, depending on the absence or presence, respectively, of a specific MVOC for each fungal species. Then, the original variables (as separate compounds or as grouped volatiles) were transformed into a new set of variables, named principal components (PCs), in such a way that the first PCs account for the largest proportion of variation in the original data set and that the data can be visualized in a two-dimensional plot of PC1 vs. PC2.

Results and discussion Presence of fungi and (M)VOCs in water-damaged buildings The list of VOCs identified in the 12 sampled buildings (11 water-damaged and 1 control house, i.e., a clean, smokeand complaint-free environment) is presented in Table 2.

Identification of fungal volatile markers and chemotaxonomic classification of Aspergillus species

945

Table 2 e Identification of selected (M)VOCs in 12 building samplings. Compound

Toluenef Hexanal Octanef Furfural Ethylbenzene 1,3-Dimethylbenzene 1,2-Dimethylbenzene Styrenef Nonanef 2,5-Dimethyloctane a-Pinenef 5-Methyl-3-heptanone 1-Ethyl-2-methylbenzened Trimethylbenzene (1,2,3 or 1,2,5)d b-Pinenef 1,3,5-Trimethylbenzene Decanef Octanal d-3-Carene 1,4-Dichlorobenzene 2,6-Dimethylnonane or 4-methyldecaned p-Cymenef 1-Ethyl-2,4-dimethylbenzene Limonenef 2-Ethylhexanol Benzyl alcohold (E )-b-Ocimene 1-Methyl-2-propylbenzened 1-Ethyl-3,5-dimethylbenzened g-Terpinenef 2-Methyldecaned Acetophenone 3-Methyldecane Dihydromyrcenol 1,3-Dimethyl-2-ethylbenzene m-Cymenene p-Cymenene Undecanef Nonanal 2-Phenylethanolf 2-Ethylhexanoic acid Pentylcyclohexaned Camphor Benzyl acetate Menthol Octanoic acid 2-(2-Butoxyethoxy) ethanol a-Terpineolf Dodecanef Decanal Verbenonef 5-Hydroxymethyl2-furancarboxyaldehyde Carvone Geraniolf Nonanoic acid Bornyl acetatef 1(or 2)-Methylnaphthalene Tridecanef Undecanal 4-tert-Butylcyclohexyl acetated

RI expa

<800 800 800 830 863 867 893 1031 899 923 932 947 961 1002 975 993 999 1004 1009 1011 1022 1024 1025 1028 1037 1038 1048 1054 1059 1060 1063 1068 1070 1072 1086 1090 1091 1098 1104 1107 1127 1133 1151 1164 1173 1186 1189 1191 1199 1206 1213 1236 1246 1254 1277 1288 1293 1302 1305 1332

RI litb

House number 1

2

3

762e 800 800 831 858e 867e 892e 1031 900

þ þ

þ þ

939 944

þ

þ þ þ þ

þ þ þ þ

4

5

6

7

8

þ

þ

þ

þ

þ

þ

þ

9

10

11

12c

þ

þ þ

þ þ þ

þ þ

þ

þ þ þ þ þ

þ

þ

þ

þ

þ þ

þ

þ

þ þ

þ þ

þ þ

þ

þ þ

þ þ

þ þ þ þ þ

996e 980 994 1000 1004e 1008 1011

1026 1022e 1031 1032 1032 1050

þ þ

þ

þ

þ

þ

þ

þ þ

þ þ þ

þ

þ þ

þ

þ

þ þ þ

þ

þ

þ

þ

þ þ þ þ

þ

þ

þ

þ þ þ þ

þ þ

þ

þ

þ

þ þ

1243 1252 1280 1285 1298e 1300 1305

þ þ þ þ þ

þ þ

þ þ

þ

þ þ þ þ þ þ

þ þ

þ þ þ þ

þ

þ þ

þ þ þ þ

þ

þ

þ þ

1143e 1163 1171 1179e 1184e 1188 1200 1204 1205 1224e

þ

þ

1059

1129e

þ

þ

þ þ

1065 1073 1072 1085e 1085 1091 1100 1102

þ

þ þ

þ þ

þ

þ

þ þ þ

þ

þ

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þ

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þ

þ þ þ þ þ þ (continued on next page)

946

V. Polizzi et al.

Table 2 e (continued ) Compound

RI expa

RI litb

House number 1

a-Terpinyl acetate 2,4-Diisocyanato -1-methylbenzened Eugenolf a-Copaene Tetradecanef Diphenyl etherd Longifolenef Dodecanal Nopyl acetate (E )-a-Iononef 2-Methoxynaphtalene Geranyl acetone 2,6-Di-tert-butylp-benzoquinoned a-Isomethylionone 6-Methyl-g-ionone b-Iononef Valencenef Pentadecanef Butylated hydroxytoluene (BHT)f Eugenol acetate trans-Calamenene Lilial Isopentyl salicylate Pentyl salicylate 1-Hexadecenef 1-Methylethyl dodecanoated 4-Phenylundecane Dihydromethyl jasmonate Dioctyl ether 2,6-Diisopropylnaphthalened Hexyl salicylate Heptadecane 1,1,3-Trimethyl-3-phenyl-indaned 2-Hexyl-(E )-cinnamaldehyde Octadecane Isopropyl myristated Isopropyl tetradecanoate 5-Phenyltridecaned Other not identified benzene derivatives a b c d e f

1348 1353

1346

1356 1378 1397 1404 1407 1406 1426 1427 1449 1453 1468

1359 1376 1400

1481 1478 1489 1496 1498 1514

1478 1481 1487 1498 1500 1512

1517 1525 1528 1534 1573 1592 1625 1643 1658 1660 1677 1681 1697 1719 1752 1800 1809 1817 1820

1522 1522 1523 1535 1576 1593

1402 1408 1426 1428 1445 1453

2

3

4

5

6

7

8

9

10

þ þ þ þ

þ

þ þ þ

þ

þ

þ

þ

þ

þ þ þ

þ þ þ þ

þ

þ

þ

þ

þ þ þ þ þ

þ þ

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þ

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þ

þ

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þ

þ

þ

þ

þ

þ

þ þ þ

þ

þ þ þ þ þ

þ

1675 1700

12c þ þ

þ

1655

11

þ

þ

þ

þ þ þ

1748 1800

þ

1828

þ

þ þ

þ þ þ

þ

þ þ þ þ þ þ þ

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þ þ þ

þ

þ

þ

RI exp ¼ linear RI determined experimentally on HP5-MS stationary phase. RI lit ¼ linear RI value from literature (Adams 2007). Control house. Tentatively identified. El-Sayed (2011). In bold: typical fungal VOCs (cf. discussion Chapter 3). Compounds identified by comparison with reference standards.

The sampling technique yielding the highest (M)VOC concentrations was the dynamic headspace absorption on Tenax. SPMEeGCeMS showed a much lower intensity but confirmed the presence of almost all compounds detected by dynamic headspace. Concerning the analysis of indoor volatiles, some interesting results were gained in the four additional building investigations. In fact, the detection of some VOCs that could be specifically derived from moulds, namely 5-methyl-3-heptanone, longifolene, valencene, eugenol, and O-acetyl eugenol [(2-

methoxy-4-prop-2-enylphenyl) acetate], was revealed in one sampled place. To study the origin of these compounds, all fungal strains isolated from the contaminated building materials were screened in vitro for VOC production, and some fungal producers of valencene and eugenol were identified, in particular belonging to the genus of Penicillium for valencene and to Penicillium (results shown elsewhere), Aspergillus and Trichoderma for eugenol (results shown elsewhere). a-Copaene, geranyl acetone, trans-calamenene, and a-calacorene were detected in the house with predominant Aspergillus spp. These four compounds were

Identification of fungal volatile markers and chemotaxonomic classification of Aspergillus species

947

Table 3 e List of Aspergillus samples collected from damp buildings and analyzed for MVOC production by means of SPMEeGCeMS. Code

Aspergillus spp.

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 A25 A26 A27 A28

A. ustus Aspergillus spp. Aspergillus spp. A. flavusa Aspergillus spp. Aspergillus spp. A. ustus A. ustusa A. ochraceusa A. sydowiia A. sydowii Aspergillus spp. Aspergillus spp. A. ustusa A. flavusa A. sydowiia A. flavusa A. sydowiia Aspergillus spp. A. insuetus A. versicolora Epicoccum spp.a Aspergillus spp. Epicoccum spp.a A. ustusa A. versicolora Aspergillus spp. A. versicolora

Source

Code

Aspergillus spp.

Source

Wallpaper Air Dust Dust Dust Wall Wall Wall Dust Plint Wall Book Book Book Wallpaper Wall Silicon Wall Wallpaper Wallpaper Wallpaper Air Wood Air Pigeon feathers Air Air Air

A29 A30 A31 A32 A33 A34 A35 A36 A37 A38 A39 A40 A41 A42 A43 A44 A45 A46 A47 A48 A49 A50 A51 A52 A53 A54 A55 A56

Aspergillus spp. Aspergillus spp. A. versicolor Aspergillus spp. A. versicolora Aspergillus spp. Aspergillus spp. Aspergillus spp. Aspergillus spp. Aspergillus spp. Aspergillus spp. Eurotium herbarioruma Aspergillus spp. Aspergillus spp. Aspergillus spp. Aspergillus spp. Aspergillus spp. A. versicolora A. versicolor Aspergillus spp. A. ustus Eurotium amstelodamia Eurotium amstelodamia A. fumigatus A. ustus Epicoccum spp.a Epicoccum spp.a Epicoccum spp.a

Air Air Air Air Dust Pigeon feathers Dust Dust Pigeon feathers Dust Pigeon feathers Air Air Air Dust Curtain Dust Wallpaperb Glass window Dust Book Dust Dust Woodb Book Dust Air Air

a Morphologically identified by Prof. A.M. Picco (Pavia university, Italy). b Extracted from building material. All the mentioned fungal species have been molecularly identified by Dr A. Moretti (ISPA, Italy) as described in Section 2.4. Other samples were identified only at the genus level.

afterwards identified in the headspace of several Aspergillus ustus strains (cf. Section 3.2, Table 5). To the best of our knowledge, this is the first study in which fungal sesquiterpenes could be identified directly in a mouldy building since these MVOCs are generally identified only from cultures grown under laboratory conditions. In the present study, we present for the first time the list of all Aspergillus species (Table 3) and of fungi belonging to several other genera (Table 4) within the 255 fungal samples isolated from 11 mouldy environments. Penicillium and Trichoderma strains will be discussed elsewhere. Among 56 samples, seven different Aspergillus species were identified: A. ustus, Aspergillus flavus, Aspergillus ochraceus, Aspergillus sydowii, Aspergillus insuetus, Aspergillus versicolor, and Aspergillus fumigatus. For A. versicolor a widespread occurrence in indoor environments has been reported (Jussila et al. 2002; € ller et al. 2002; Nielsen 2003). Most of these strains are Mu also important fungal species in food mycology in terms of spoilage and production of secondary metabolites such as off-flavours and mycotoxins. Moreover, Aspergillus species normally have a tropical distribution but, in recent years, their increasing presence in indoor environments situated in temperate climates has been reported (Samson 2009). The predominant incidence of Cladosporium within the collected samples generally agrees with reports from cold and temperate regions (Nilsson et al. 2004). It should be noted that the

prevalent occurrence of Cladosporium is not reflected in Table 4 because for many Cladosporium strains a detailed MVOC analysis was not performed due to the repeatedly confirmed MVOC absence in this fungal genus. Some of the detected fungal generadChaetomium, Ulocladium, and Alternariadare often reported as important indicators of water damage (Yang & Heinsohn 2007).

In vitro MVOC profile of Aspergillus species To investigate the volatile profile of a large group of fungi, relevant for indoor environments, the in vitro production of fungal volatiles was investigated on MEA (Table 5). These experiments were designed to elucidate MVOC patterns typical for a certain fungal species, which would be useful for a fast and indicative fungal identification in vitro and/or as an additional identification tool for the separation of closely related fungi. This kind of study could also highlight compounds that are biosynthetically derived from the same fungal metabolic pathway, e.g., compounds that are emitted always simultaneously in different fungal cultures. Table 5 lists the profiles of volatile metabolites of the recovered Aspergillus strains. Despite the fact that in some cases the MVOC profile was not consistent within different strains of the same species, which has already been observed before

948

V. Polizzi et al.

Table 4 e List of fungal samples collected from damp buildings and analyzed for MVOC production by means of SPMEeGCeMS Code S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 S23 S24 S25 S26 S27 S28 S29 S30 S31 S32 S33 S34 S35 S36 S37 S38 S39 S40 S41 S42 S43 S44 S45 S46 S47 S48 S49 S50 S51 S52 S53 S54 S55 S56 S57 S58 S59 S60 S61 S62

Varia

Source

Acremonium strictuma Chaetomium globosuma Chaetomium murarum Chaetomium spp.a Chaetomium spp.a Dematioid anamorph Dematioid anamorph Unknown Unknown Epicoccum spp.a White mycelium Ulocladium chartarum Unknown Unknown Unknown White mycelium Dematioid anamorph Ulocladium botrytis Wallemia sebia Fusarium spp.a Dematioid anamorph White mycelium Dematioid anamorph White mycelium Dematioid anamorph Dematioid anamorph Dematioid anamorph Grey mycelium Rhizopus spp.a Botrytis cinereaa Rhizopus spp. Orange green mycelium Alternaria spp. Arthrinium spp. White mycelium Epicoccum spp.a Epicoccum spp.a White mycelium Alternaria spp. Unknown Epicoccum spp.a Arthrinium phaeospermuma Unknown White mycelium Monascus rubera Dematioid anamorph Botrytis cinereaa Dematioid anamorph Engyodontium album Isaria fumosorosea White mycelium Unknown Unknown Phoma spp.a Dematioid anamorph Dematioid anamorph Botrytis spp.a Cladosporium sphaerospermuma Dematioid anamorph C. sphaerospermuma Dematioid anamorph Dematioid anamorph

Wall Silicon Wall Collection Book Wallpaperb Dust Wood Dust Dust Dust Curtain Wall Dust Wallpaperb Dust Wallpaper Wallpaper Silicon Wall Wall Wood Wallpaper Wallpaper Wallpaper Wallpaper Wall Dust Wallpaperb Pigeon feathers Dust Wood Woodb Air Air Air Air Air Air Wood Wood Wood Air Air Woodb Air Air Air Wood Wood Wood Wood Wood Air Wood Wood Wood Glass window Air Glass window Leather chair Air

Table 4 e (continued ) Code

Varia

S63 S64 S65 S66 S67 S68 S69 S70 S71 S72a S72b S73 S74 S75 S76

a

C. herbarum C. sphaerospermuma C. sphaerospermuma Rhizopus spp.a Phoma spp.a Peacilomyces spp.a Unknown Epicoccum spp.a Periconia britannica Gilmaniella humicolaa Gonytrichum macrocladuma Colletotrichum asianum Rhizopus spp.a Epicoccum spp.a Epicoccum spp.a

Source Air Glass window Glass window Bookb Bookb Dust Book Air Air Wall Wall Dust Dust Air Air

a Morphologically identified by Prof. A.M. Picco (Pavia university, Italy). b Extracted from building material. All the mentioned fungal species have been molecularly identified by Dr A. Moretti (ISPA, Italy) as described in Section 2.4. Other samples were identified only at the genus level.

(Matysik et al. 2009), some valuable information can be concluded from Table 5. Firstly, 1,3-dimethoxybenzene can be proposed as a specific indicator of Aspergillus versicolor (group) growth within the Aspergillus genus. In fact, this MVOC was exclusively detected in A. versicolor cultures in previous studies (Wilkins & Larsen 1995; Fischer et al. 1999; Matysik et al. 2009). Also in this study, this compound was exclusively detected in the headspace of two Aspergillus sydowii strains (A. versicolor group) and one A. versicolor within the Aspergillus genus. In fact, 1,3dimethoxybenzene was detected also in the headspace of Alternaria, Paecilomyces, and various Penicillium species. Aspergillus ustus was characterized by a specific MVOC profile (Table 5). It was interesting to note that sample A53, initially unknown, could be tentatively identified as A. ustus based on the MVOC profile, and then confirmed by molecular identification. This finding proves the value of MVOCs in chemotaxonomy. Moreover, a-copaene, geranyl acetone, transcalamenene, and a-calacorene were detected in the indoor air of the interior from which some of these A. ustus samples were isolated. The same metabolite profile was detected also in some unknown samples, characterized by an orange-coloured mycelium, tentatively identified as Epicoccum spp. These strains are discussed together with the Aspergillus strains because of the absence of reproductive structures in Epicoccum spp., making the above-mentioned morphological identification uncertain, and because of the high similarity in volatile profile. Cadinenes, produced by Aspergillus flavus (A4), were previously reported only for aflatoxigenic strains of A. flavus (Zeringue et al. 1993). To the best of our knowledge, this is the first report on MVOC production by Aspergillus insuetus, A. ustus, A. sydowii, and Eurotium spp. 1,3-Dimethylbenzene, isoborneol derivatives, and 4-methoxystyrene were specific in our study for Eurotium amstelodami. This soil-borne fungus has been reported to evoke

Identification of fungal volatile markers and chemotaxonomic classification of Aspergillus species

949

Table 5 e MVOC profile of the identified Aspergillus and Chaetomium species (compounds mentioned in order of elution) grown on MEA. Fungal sample

N of strains or code

A. ustus

8

‘Epicoccum’a

5

A. flavus

A4 A15

A. sydowii

A10 A11

A. versicolor

A18 A33 A46 4

Eurotiumb

A40 A50-51

A. ochraceus A. insuetus A. fumigatus Chaetomium

A9 A20 A52 4

MVOC profile c

1-Octen-3-ol (1/8) , 2-phenylethanol (1/8), a-cubebene (7/8), eugenol (3/8), a-ylangene (4/8), a-copaene (6/8), b-bourbonene (6/8), b-cubebene (2/8), b-elemene (7/8), RI 1393 (1/8), cyperene (2/8), (E )-caryophyllene (1/8), b-copaene (5/8), a-trans-bergamotene (1/8), neryl acetone (3/8), RI 1447d (1/8), cis-muurola-3,5-diene (3/8), geranyl acetone (3/8), cis-cadina-1(6),4-diene (2/8), cis-muurola-4(14),5-diene (3/8), g-muurolene (4/8), ar-curcumene (1/8), germacrene D (6/8), trans-muurola-4 (14),5-diene (2/8), g-amorphene (1/8), a-selinene (1/8), a-muurolene (3/8), d-amorphene (1/8), g-cadinene (7/8), trans-calamenene (3/8), d-cadinene (7/8), trans-cadina-1,4-diene (4/8), a-cadinene (6/8), a-calacorene (6/8), (E )-nerolidol (1/8), (2Z,6Z )-farnesol (1/8), drimenol (2/8) 4-Methyl-3-hexanone (2/5), 4-methyl-3-hexanol (2/5), 5-ethyl-4-methyl-3heptanone (1/5), 3-(4-methyl-3-pentenyl)furan (1/5), ethyl benzoate (2/5), a-cubebene (3/5), a-ylangene (3/5), a-copaene (3/5), b-bourbonene (2/5), b-copaene (1/5), b-gurjunene (1/5), g-muurolene (4/5), germacrene D (2/5), a-muurolene (4/5), g-cadinene (5/5), trans-calamenene (3/5), d-cadinene (5/5), trans-cadina-1,4-diene (2/5), a-cadinene (2/5), a-calacorene (2/5) 2-Heptanone, g-cadinene, d-cadinene. A17 is a non-MVOC producer 1,3-Octadiene, 6-methyl-2-heptanone, 1-octen-3-ol, 2-phenylethanol, a-chamipinene, RI 1397, a-cedrene, b-cedrene, RI 1435, isobazzanene, b-acoradiene, ar-curcumene, g-curcumene, b-himachalene, a-chamigrene, cuparene 1,3-Octadiene, 1-octen-3-ol, 3-octanone, 1,3-dimethoxybenzene, b-cedrene 1-Octen-3-ol, 1,3-dimethoxybenzene, a-chamipinene, b-cedrene, b-himachalene, cuparene Camphene. A16 is a non-MVOC producer 1-Octen-3-ol, 3-octanone, a-pinene, b-pinene, camphene, terpinolene, 1,3-dimethoxybenzene. A21 is a non-MVOC producer 4-Methoxystyrene, d-deca-2,4-diene-lactone, 1,6,7-trimethylnaphthalene (identified by MS only) 1-Octen-3-ol (1/4), ethylbenzene (1/4), m-xylene (2/4), 2-methylisoborneol and derivatives (1/4), 1-heptanol (3/4), 4-methylanisole (1/4), (Z )-b-ocimene (1/4), terpinolene (1/4), daucene (1/4), a-chamipinene (2/4), RI 1397 (3/4), cyperene (1/4), b-cedrene (4/4), RI 1435 (3/4), isobazzanene (2/4), (E )-b-farnesene \(1/4), RI 1465 (3/4), b-acoradiene (3/4), RI 1477 (4/4), g-curcumene (1/4), a-zingiberene (1/4), a-himachalene (2/4), b-himachalene (1/4), isodaucene (1/4), a-chamigrene (2/4), cuparene (3/4), a-cuprenene (3/4), b-bisabolene (1/4), b-sesquiphellandrene (2/4), dauca-4(11),8-diene (1/4), trans-a-bisabolene (1/4) Non-MVOC producer 1,3-Dimethylbenzene, isoborneol derivatives, 2-phenylethanol, 4-methoxystyrene, RI1372, daucene, RI1405, RI1407, g-elemene, RI1458, RI1460, b-acoradiene, ar-curcumene, RI1489, isodaucene, cuparene, b-bisabolene, dauca-4(11),8-diene, RI1539, RI1541, 1,6,7-trimethylnaphthalene (identified by MS only) 2-Phenylethanol, RI 1914, RI 1933, RI 1952 Drimenol 3-Octanone, 3-octanol, 2-methyl-2-bornene, methyl benzoate, a-barbatene, diterpene H 1-Octen-3-ol, 3-octanone, 3-octanol, 2-phenylethanol, 3-octanyl acetate, RI1375, 3,5-dichloro-2,4-dimethyl-1-methoxybenzene

a Since the morphological identification as Epicoccum was not definitive, this group was included in the Aspergillus table based on MVOC production. b Eurotium is the perfect form of Aspergillus. c Numbers between brackets indicate the number of strains on the total for which this specific volatile was identified. d For unidentified compounds the linear RI is given, determined on an HP-5 MS stationary phase.

hypersensitivity pneumonitis, including the ‘farmer’s lung disease’ (Roussel et al. 2010), and for posing inhalation health risks to persons with a weak immune system. Indoors, this fungus has been isolated from floor, carpet and mattress dust, hospital air, cloth, and shoes. The concurrent presence of a-copaene, b-bourbonene, and a-calacorene on the one hand, and of b-acoradiene and

g-curcumene on the other hand, support the hypothesized common biosynthetic origin of these two groups from a germacradienyl cation and a bisabolyl cation, respectively, in the mevalonate pathway (Maia et al. 2000; Hong & Tantillo 2009; Garms et al. 2010). All the recovered fungal samples not belonging to Aspergillus, Penicillium, and Trichoderma were grouped together for the

950

evaluation of distinctive patterns of metabolites. The majority of these samples were characterized by a very weak production of secondary volatile metabolic products. There was a high consistency in the MVOC patterns of the four analyzed Chaetomium spp. which resulted in a clear differentiation of this genus from the others, especially by the unique presence of 3-octyl acetate. This compound is known to be a natural product emitted by melon that attracts fruit flies (Alagarmalai et al. 2009). Many of the detected MVOCs, such as 1-octen-3-ol, 2phenylethanol, b-elemene, and 1,3-dimethoxybenzene, were commonly produced by different fungal genera. The fact that a compound has been produced on MEA does not imply that it also will be produced on building materials. However, in a contemporaneous study where four selected fungal species were screened for VOC production on wallpaper and plasterboard (Polizzi et al. 2012) the authors noticed that, overall, the main MVOC profile detected on MEA was conserved also on the two building materials tested. Some of these compounds have also been reported in air samplings of mould-infested building environments (kitchen waste) and contaminated building materials (Wilkins & Larsen 1995). Although many studies investigating MVOC production by indoor moulds report often only this kind of compounds (Wilkins et al. 1997, 2000; Korpi et al. 1999; Matysik et al. 2009), such general metabolites are obviously not helpful in chemotaxonomy. However, they are suitable indicators of general indoor mould growth.

Statistical evaluation of the data by PCA Multivariate data analysis, or chemometrics, can reveal hidden patterns in complex data by reducing the information to a more comprehensive format. PCA is one specific kind of exploratory data techniques and was selected to get an insight in the variability of our data. Various classifications of MVOCs were examined to detect the best grouping variables for statistical discrimination of mould species within a genus, in particular the complete MVOC profile (model A), only terpene compounds (model B), terpenes classified by the number of rings in their chemical structure (model C) or classified by their cyclic skeleton (model D). The advantage of classifying compounds according to their chemical structure and/or (apparent) biosynthetic similarity in order to discuss volatile metabolite patterns has been underlined by Wilkins et al. (2000). In general, refining the model by deleting variables which showed low modelling power (nonterpene compounds found in many species) and by classifying the terpenes based on the number of rings in their chemical structure, resulted in a new model with PCs that accounted for a higher variance in the first PCs. The best classification for Aspergillus species based on their MVOC profile on MEA was achieved using model A, although only a small part of the variance was explained by PC1 and PC2 (cumulative proportion (c.p.) ¼ 36 %, i.e., variance represented by PC1 and PC2). This low value was not unexpected as a dimension reduction from 152 variables to two variables is achieved. The obtained classification was comparable to what was obtained with model C (and D), but model A enabled to distinguish an additional group of samples, as specified

V. Polizzi et al.

below. The biplot obtained with model A is shown in Fig. 1 while the group classification using model C and D is clarified in Table 6. The group situated on the lower right-hand side of the biplot (Fig. 1) included the two Eurotium species (A50 and A51) and was clearly separated from the others due to the high production of 2-methylisoborneol derivatives and an unidentified sesquiterpene. One strain of Aspergillus versicolor (A31) was distinguishable from the others because of its specific emission of monocyclic sesquiterpenes. A large group of samples in the middle included most of the Aspergillus ustus strains (A1, A7, A8, A14, and A25), three unknown fungi similar to Epicoccum spp. (A22, A24, and A55), and four unknown Aspergillus spp. (A12, A13, A32, and A49). The latter group of isolates showed morphological characteristics similar to A. ustus and might be referable to this fungal species, based on their MVOC profile. This large group of fungi in the middle of the biplot was characterized by a consistent production of bi and tricyclic sesquiterpenes. They were also classified together using model D (c.p. ¼ 93 %), i.e., by classifying sesquiterpenes based on their biosynthetic origin (Table 6). In fact, a number of MVOCs, all derived from the same biosynthetic pathway (Mann 1980) and having cadinane, bourbonene, copaene, and cubebane skeletons, were consistently produced by this group of fungi. A fourth group of samples in the upper right-hand side of the biplot appeared only using model A and was composed of two A. versicolor strains (A26, 28), one Aspergillus flavus (A15), and an unknown Aspergillus (A32). These fungal strains were the only producers of the sesquiterpenes with RI 1397, RI 1435, isobazzanene, b-acoradiene, and a-chamigrene (Fig. 1).

Fig. 1 e PCA biplot of Aspergillus spp. classified by model A (based on the complete MVOC profile; 152 volatiles not shown for reasons of clarity). Many of the fungal samples (codes in Table 3) overlap and are thus not differentiated by MVOC profile. Specific groups are circled and correspond to the groups specified in Table 6.

Identification of fungal volatile markers and chemotaxonomic classification of Aspergillus species

Table 6 e Classification of Aspergillus spp. by PCA using model C and D. Group

Code

Identification

1

A31

A. versicolor

2

A50 A51 A1 A7 A8 A12 A13 A14 A22 A24 A25 A32 A49 A55 A15 A26 A28 A32

Eurotium

3

4a

A. ustus A. ustus A. ustus Unknown Unknown A. ustus ‘Epicoccum spp.’ ‘Epicoccum spp.’ A. ustus Unknown Unknown ‘Epicoccum spp.’ A. flavus A. versicolor A. versicolor Aspergillus

Grouping variable Monocyclic sesquiterpenes 2-methylisoborneol derivatives Bicyclic sesquiterpenes Tricyclic sesquiterpenes Model D: cadinane, bourbonene, copaane, cubenane skeleton

RI 1397, RI 1435, isobazzanene, b-acoradiene, and a-chamigrene

a Group obtained only with model A.

Further experiments are planned to find an explanation why A. ustus strain A53 has a different volatile profile and is not grouped together with the other A. ustus strains. One explanation could be the different ability of the analyzed A. ustus strains to produce mycotoxins. The same can be hypothesized for the fungi classified in group 4 (Table 6) since both A. versicolor and A. flavus can produce the mycotoxin sterigmatocystin.

Fig. 2 e Classification of 74 fungal strains by PCA using model A (based on the complete MVOC profile). Many of the fungal samples (codes in Table 4) overlap and are thus not differentiated by MVOC profile. Specific groups are circled: S10, 36, 37, 41, 70: ‘Epicoccum’; S35, S38: unknown; S2-5: Chaetomium.

951

Such a relationship between mycotoxin production and volatile excretion has been shown before. In fact, an enhanced sesquiterpene production was found in trichothecene-producing Fusarium sambucinum strains compared to nontoxigenic strains of the same species (Jelen et al. 1995). In another study, a-gurjunene, (E )-caryophyllene, and cadinene were detected in aflatoxigenic strains of A. flavus but not in nonaflatoxigenic strains (Zeringue et al. 1993; Demyttenaere et al. 2003). The rest of the fungal samples not belonging to Aspergillus, Penicillium, and Trichoderma were statistically treated together using model A (Fig. 2). Even if the c.p. of PC 1 and 2 was quite low (26 %), the biplot is interesting because Chaetomium (S2eS5) and ‘Epicoccum’ spp. (S10, 32, 36, 37, 38, 41, and 70) were clearly separated from each other and from the others (76 samples in total). Chaetomium strains were differentiated from the others by the consistent production of 1-octen-3-ol, 3-octanone, 3-octanol, and the unique emission of 3-octyl acetate. It must be noted again that the morphological identification of the samples named as Epicoccum was not definitive due to the absence of reproductive structures.

Conclusions In the majority of indoor air samplings no mould-specific VOCs can be detected even in those environments where a strong mouldy odour is present. However, in two specific cases, volatile sesquiterpenes specifically derived from fungal metabolism were detected. In one of these environments there was no visible mould growth but surface and air sampling revealed an abnormal incidence of Aspergillus species. Therefore, the identification of specific target compounds for Aspergillus was possible, and this may provide an opportunity to increase the sensitivity of the analytical methodde.g., by using GCeSelected Ion Monitoring (SIM)eMSdand to detect MVOCs even when a low amount of mould is present indoors. Sesquiterpenes are generally not investigated or reported in research studies on MVOC presence in buildings, although our study clearly demonstrates their importance. Detection of sesquiterpenes in indoor environments may be the best approach to detect indicators of fungal growth. Concerning the chemotaxonomic field, this study confirms the possibility of using MVOC patterns in aid to the classical identification methodologies since species-specific MVOCs or MVOC patterns have been identified for Aspergillus versicolor group, Aspergillus ustus, and Eurotium amstelodami. These results were confirmed by statistical analysis. In this respect, it would be very useful for the scientific community to establish specific growth conditions (temperature, substrate) at which to perform screening of MVOC production, to enable a comparison between different studies, and to build a database for chemotaxonomic use.

Acknowledgements The authors are indebted to the Research Foundation e Flanders (Belgium) (FWO-Vlaanderen) for financial support and a Postdoctoral Fellowship to An Adams.

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