Production of fungal volatile organic compounds in bedding materials Sanna Lappalainen University ofKuopio, Department ofEnvironmental Sciences, Kuopio, Finland. Current address: Uusimaa Regional Institute ofOccupational Health, Arinatie 3 A, FIN-00370 Helsinki, Finland, e-mail: sanna.lappalainen@occuphealth.fi Anna-Liisa Pasanen, Pertti Pasanen, Pentti Kalliokoski University ofKuopio, Department ofEnvironmental Sciences, PO Box 1627, FIN-70211 Kuopio, Finland The high relative humidity of the air and many potential growth media, such as bedding materials, hay and grains in the horse stable, for example, provide suitable conditions for fungal growth. Meta- bolic activity of four common agricultural fungi incubated in peat and wood shavings at 25°C and 4°C was characterized in this study using previously specified volatile metabolites of micro-organ- isms and C0 2 production as indicators. The volatile organic compounds were collected into Tenax resin and analysed by gas chromatography. Several microbial volatile organic compounds (MVOCs), e.g. 1-butanol, 2-hexanone, 2-heptanone, 3-octanone, l-octen-3-ol and 1-octanol were detected in laboratory experiments; however, these accounted for only 0.08-1.5% of total volatile organic com- pounds (TVOCs). Emission rates of MVOCs were 0,001-0.176 pg/kg of bedding materials per hour. Despite some limitations of the analytical method, certain individual MVOCs, 2-hexanone, 2-hep- tanone and 3-octanone, were also detected in concentrations of less than 4.6 pg/m 3 (0.07-0.31% of TVOC) in a horse stable where peat and shavings were used as bedding materials. MVOC emission rate was estimated to be 0.2-2.0 pg/kg x h 1 from bedding materials in the stable, being about ten times higher than the rates found in the laboratory experiments. Some compounds, e.g. 3-octanone and l-octen-3-01, can be assumed to originate mainly from microbial metabolisms. Key words', agricultural environment, microbial metabolites, microbial volatile organic compounds, peat, total volatile organic compounds, wood shavings Introduction Many reports on levels of volatile organic com- pounds in houses and offices have recently been published. Concentrations of total volatile organ- ic compounds (TVOCs) have ranged from 20 to 1900 pg/m 3 in houses and from 160 to 15 300 pg/m 3 in office buildings (Miller et al. 1988, Norbäck et al. 1993, Daisey et al. 1994, Ekberg 1994, Kostiainen 1995, Batterman and Peng 1995). Even though epidemiological data are not fully consistent, recent studies have shown an association between TVOC exposure and symp- © Agricultural and Food Science in Finland Manuscript received September 1996 219 Voi.6 (1997): 219-227. AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=q2mOfchjOXHattbG.GMgpViXZFdC0kYIp0pkNyQ.O_s28jffToqt2OnPKUj0ODujJxYp7gKeOT68v-GNrfAX201dNdGEHLe05Uwxiq5o5T-TmLXycKL77qDA_t4jdOyhRP2N5ZkDT_KYkcq0Qco37TvZMaLojNC21RxtwC0ufR1ei_gSglKxBOQWf2KzhEsfuk-vglGjlSpbmZIMlclxsOXbPPB5DqUE9orv4xOFO31dV5yO6zeU4BshNDN1EpM5gaS23g-piZXgdtaa-BBJbl-Lyoes79c0CMooOC6U3iGjh49aq104v_DumLdsRvQNfs5UYtCH_o3zmLGilK4FUnsKVlPqh98_MotDKppECGCsP_HzqHjXRIXvCNEA8w Lappalainen, S. el al. Production offungal volatile organic compounds toms. TVOC levels were 5-50 fold higher in “sick houses” than in normal houses (Kostiai- nen 1995),and significantly higher (67-8300 |ig/ m 3) in the dwellings of asthmatics than in those of subjects without such symptoms (Norbäck et al. 1993). Even low concentrations ofTVOC may cause irritative symptoms (in the eyes, nose and throat) or headache. Toxic effects may appear when the TVOC concentration exceeds 25 mg/m3 (Mplhave 1990). Laboratory experiments have shown that mi- cro-organisms, e.g., fungi, produce volatile me- tabolites, such as 2-methylfuran, 2-methyl-1-pro- panol, 3-methyl-1-butanol (Börjesson et al. 1989, 1993), l-octen-3-01, terpenes (Börjesson et al. 1989, 1990), ethylhexanol (Bjurman and Kris- tensson 1992, Ezeonu et al. 1994), 1-octanol (Kaminski et al. 1974) and 3-octanone (Kamin- ski et al. 1974, Börjesson 1993). Production of microbial volatile organic compounds (MVOCs) has been observed to depend on microbial fun- gal and bacterial species and growth conditions (Kaminski et al. 1974, Börjesson et al. 1989, 1993). MVOCs have also been found in the hu- man environment: Miller et al. (1988) detected several MVOCs, including 2-heptanone, octane, hexanone, 3-methyl-1-butanol and decanol, in the air of “sick buildings” in Canada. Ström et al. (1993) observed that the concentration of MVOCs in houses with microbial problems was significantly higher than that in unaffected houses and outdoor air. To our knowledge, no reports on TVOC or MVOC levels in the agricultural environment have yet been published so far. The high relative humidity of the air and the many different kinds of substrate such as bedding materials, hay and grains in horse stable, for example, provide good conditions for fungal growth. Airborne fungal spore levels are therefore usually quite high in the agricultural work environment, especially during the handling of hay and grains, when cul- turable spore concentrations typically range from 10s to 106 cfu/m3 in cow sheds (Kotimaa et al. 1984, Pasanen et al. 1989, Hanhela et al. 1995). We investigated here, the production of MVOCs and TVOCs in two bedding materials in labora- tory experiments and determined MVOC and TVOC emissions in a horse stable. Material and methods Laboratory experiments Incubation offungi in bedding materials The fungi chosen for this experiment were Fusarium poae, Paecilomyces variotii, Penicil- lium sp. and Wallemia sebi, all of which occur in abundance in bedding materials (Hanhela et al. 1995). The fungal strains were isolated from the bedding materials, hay and grain used in a horse stable that also served as a sampling site for MVOCs/VOCs in our study. F. poae, P. var- iotii and Penicillium sp. were cultured on 2% malt extract agar (MALT) and W. sebi was cul- tured on dichloral-glycerol agar (DG 18). Four samples of peat and wood shavings, 2 g of each, were weighed for each experiment. The samples were sterilized at 120°C for 30 minutes and sta- bilized in air-tight chambers (2L) at relative hu- midity of air (RH) 98% for 2 weeks. After stabi- lization, the spores of each fungal strain were suspended in sterile water and inoculated on one sample of peat and one of shavings (inoculation strength c. 106 cfu/g). Sterilized peat and shav- ings were used as controls. In addition, clean (not used) and dirty (used as bedding material in a horse stable), non-sterilized peat and shavings were incubated without inoculation of fungi. The samples were prepared for two experiments. The first experiment was carried out at 25°C for 6 days, which represented favourable growth con- ditions for the fungi and the second one at 4°C for 22 days, which simulates the winter condi- tions in stables. Sampling of volatile compounds Volatile organic compounds were collected into Tenax TA resin (150 mg per tube) from the cham- bers at an airflow rate of 100 ml/min for 10min. The clean air was led to the chambers through 220 AGRICULTURAL AND FOOD SCIENCE IN FINLAND activated carbon simultaneously with the collec- tion of volatile compounds. In the first experi- ment (at 25°C), TVOC samples were taken on the fourth day of the incubation, and MVOC sam- ples were collected on the sixth day of the incu- bation. In the second experiment (at 4°C), only MVOC samples were taken on the 14th and 22nd days of the incubation. Analysis of volatile compounds TVOCs and MVOCs were analysed by gas chro- matography (Hewlett Packard 5890) with a mass selective detector (GC-MS, Hewlett Packard 5970) fitted with a Thermal Desorption Cold Trap (TCT) injector. A capillary column (50 m x 0.33 mm, BP 10 SEG) was used with helium as the carrier gas. TVOCs were analysed in SCANNING (SCAN, ions 40-260 amu) mode and MVOCs in Selected lon Monitoring (SIM) mode. The temperature program of the SCAN- mode was: 40-200°C, 2 min at the initial tem- perature, at a rate of 5°C/min; that of the SIM mode was: 40-160°C, 2 min at the initial tem- perature, at a rate of 5°C/min and 160-200°C, 20°C/min. The compounds were identified us- ing the library of mass spectra (Hewlett Packard 599738). The SIM mode analysed seven com- pounds in the first experiment (2-hexanone, 2- heptanone, 3-octanone, l-octen-3-01, 3-octanol, 2-octanol and nonanal) and 13 compounds in the second experiment (2-hexanone, 2-heptanone, 3- octanone, 1-butanol, l-octen-3-01, 1-octanol, 2- ethyl-1-hexanol, nonanal, decanal, alfapinene, camphene, betapinene and limonene) all of which are considered to originate from the me- tabolism of most fungi (Kaminski et al. 1974, Börjesson et al. 1989, 1990, 1993). Deteilas of the sampling and analysis of MVOC have been given in elsewhere (Pasanen et al. 1996). Con- centrations ofTVOC and MVOC in the air (pg/m3 ) and the rate of MVOC emissions (pg/kg x h 1 from bedding materials) were calculated. Measurements of CO 2 The metabolic activity of the fungi was observed by C02 measurements (ADC analyser, the ana- lytical development, UK) every day in the first experiment and on the Ist, 7th, 14th, 21st and 22nd days of incubation in the second experi- ment. C0 2 emission rates (pg/kg x h 1 from bed- ding materials) were calculated. Field measurements Description ofhorse stable MVOCs and TVOCs were also measured in a stable containing 38 horse stalls and three stalls for horse washing. The floor area was 442 m 2 and the volume 1348 m 3. The floor area of each stall was 6.25-7.5 m 3, and the bedding material was about 10 cm thick when spread out smooth- ly. Thus, the mass of the bedding materials used in the whole stable was estimated to be about 3570 kg. The stable was equipped with mechan- ical ventilation, and the average air flow was approximately 6000 m3 /h. The stable was divid- ed into two equal parts separated by a partial wall. During the measurements peat served as the bedding materials in the first part of stable, and wooden shavings in the second part; normal- ly, shavings were the only bedding material. Two workers fed the horses dry hay and oats three times a day and cleaned the stalls once a day. Sampling of volatile compounds The samples (n=24) were collected at an airflow rate of 100 mL/min for 60 min into Tenax resin at a height of 1.5 m from four horse stalls where peat or shavings were used as bedding materi- als. The samples were collectedbefore the work- ers fed the horses (background) and while they cleaned the stalls. The sampling was carried out in winter and repeated three times (2 weeks be- tween each sampling). In addition, threeMVOC/ TVOC samples were collected outside the sta- ble and three unused Tenax resin tubes served as controls during the sampling. The samples were analysed by gas chromatography with a mass selective detector as described previously. MVOCs in the first and second measurements were analysed using the same SIM mode as in 221 Vol. 6(1997): 219-227. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Lappalainen, S. et al. Production offungal volatile organic compounds the first laboratory experiment and in the third measurement with the same SIM mode as in the second laboratory experiment. Results Laboratory experiments The potential MVOCs detected in the laborato- ry experiments are presented in Tables 1 and 2. The sum ofMVOC content includes 2-hexanone, 2- 3-octanone, 1-butanol, l-octen-3- ol and l-octanol, all considered to originate from microbes. Note, however, that the results for MVOC in the first and second experiments are not fully comparable, because there were more MVOCs in the SIM mode in the second than in the first experiment. On the other hand, low MVOC concentrations were also detected at the end of experiments in sterilized material sam- ples, indicating slight microbial activity in bed- ding materials despite sterilization (the concen- trations of 2-hexanone, 2-heptanone, 1-octen- -- and l-octanol varied from <0.4 pg/m3 to 33.5 pg/m 3 ). Only 3-octanone and 1-butanolwere not detected in any sterilized material samples. In general, the production of MVOC was 1-100 fold higher in shavings than in peat in both experiments. The rates of MVOC and C0 2 emissions are presented in Table 3. Penicillium sp. grew poor- ly in peat: in both experiments the C02 emission rate was at the same level as in sterilized sam- ples. The other fungi grew well in both peat and shavings. C0 2 production was 104-10 6 times higher than the amount of MVOCs released. The rates of MVOC emission from bedding materials ranged from 0.001 pg/kg x h 1 to 0.176 pg/ kg x h '. The concentrations of TVOCs varied from 510 to 3500 pg/m 3 in the inoculated bedding material samples (Table 1 and 2). The highest TVOC concentration was detected in the non- sterilized, used shavings. TVOC concentrations were 1300 pg/m 3 in the sterilized peat sample and 3500 pg/m3 in the sterilized shavings. Thus, sterilization of bedding materials did not have a major effect on TVOC level. More TVOCs were released from shavings than from peat due to the higher emissions of terpenes from the shavings. However, the level of MVOC was relatively low, comprising about 0.08-1.5% of the TVOC con- centration. Field measurements Low concentrations of MVOCs, such as 2-hex- anone (