1 Growth preferences on substrate, construction, and room location for indoor moulds and Actinomycetes Maria Nunez, Mari S. Sivertsen, Johan Mattsson Mycoteam as, Postboks 5 Blindern, N-0313 Oslo, Norway *Corresponding SUMMARY We describe the growth preferences of Actinomycetes and 13 common mould genera in moisture damaged buildings based on sampling and microscopic analyses of collections in Norway during the years Sampling has systematically been carried out on nine types of building materials, six room locations, and six construction types. Our results indicate that Ascotricha and Mycotrichum are commonly found in insulated outer walls in basements, Hyalodendron grows almost exclusively on wood in cold attics, and Chrysosporium in cold basements and crawl spaces. Aspergillus and Penicillium have a wider ecology as genera, and studies must be carried out at species level. KEYWORDS Mould damage, Actinomycetes, ecological preferences, indoor microbial ecology. 1 INTRODUCTION The ecological preferences of microbial growth indoors can help to address the source, extent and chronology of moisture damage in damp buildings. This is important when potential health risks and economic losses caused by moulds have to be addressed by legal instances, as these cases often end in litigation. Moulds are filamentous, mainly asexual fungi which grow rapidly on humid surfaces, and can release a high number of spores as well as other compounds to the air. Inhalation of mould products has been discussed as one of the major factors affecting indoor air quality (Bornehag et al. 2001). Several of the genera traditionally included in moulds belong taxonomically in Ascomycetes (i.e. Ascotricha, Chaetomium, Eurotium, Myxotrichum) or Basidiomycetes (Hormographiella, Hyalodendron). Between 200 and 600 mould species have been reported from buildings (Sedlbauer, 2001). Filamentous Actinomycetes, mainly the genera Streptomyces and Nocardiopsis, have also been related to moisture damage indoors (Andersson et al. 1998). Around 20-50% of dwellings in the northern Hemisphere sustain microbial growth (Nevalainen et al. 1998). Because the majority of indoor microbial studies focus on exposure assessments, air sampling and molecular techniques are often used to describe indoor microbial contamination (Yang, 2004). Many researchers use indirect means to characterize microbes in their natural habitats, arguing that morphological characters are difficult to use. Unfortunately, indirect tools cannot distinguish between viable microbial growth and fragments on settled dust. Therefore, the ecological knowledge of indoor microorganisms in situ is presently limited (Grant et al. 1989; Yang, 2007).
2 The prerequisites for microbial growth are present in damp buildings. However, growth requirements are often described for laboratory conditions at optimal RH and temperature on sterile culture media (Samson et al. 2002). These conditions are rarely met in the building environment, where fluctuations in temperature, humidity and nutrient availability occur (Grant et al. 1989; Viitanen, 1997), as well as competition with other microbes, and even predation (Mullen and O Connor, 2009). Predictions of microbial growth indoors after moisture damage would facilitate effective remediation, assessment of potential health exposure to residents, and selection of building materials and strategies to minimize microbial damage in the future. 2 MATERIALS/METHODS We have sampled a total of collections of Actinomycetes and mould genera in damp buildings in Norway from Samples include both bulk materials and tape lifts, collected by inspection, or sent to us by customers. Samples were identified to genus by optical microscopy. Filamentous Actinomycetes as such were not further identified. In this work we have analysed a subset of records comprising 14 taxa (Fig. 1) for which we know the room location, construction type, and substrate that support microbial growth (Table 1). About 1/3 of the collections were taken on tape lifts where substrate is not specified. These are not included in further substrate analysis. Fig. 1. All taxa, distribution in the data. Cladosporium Penicillium Aspergillus Actinomycetes Chaetomium Stachybotrys Acremonium Ulocladium Ascotricha Eurotium Myxotrichum Chrysosporium Trichoderma Hyalodendron % 7% 7% 6% 9% 1 15% 15% 22% Sampling has been carried out in every construction type for each room location, as specified in our results. We have not made any distinction among house types in our study. According to Lee and Jo (2006), this is not significant in microbial distribution indoors. For common water damages and risk constructions in Norwegian homes, see Holme et al. (2008). Analyses are performed with Windows Excel and JMP 9 ( SAS Institute 2010). Results are given in percentages. Combinations of room location and construction where taxa frequencies are under 5% are grouped into a category termed other locations. We have made two diagrams for each taxon that show location (room and construction type), and substrate preferences in percentages, as shown in Fig. 2 for Aspergillus and Fig. 3. for Hyalodendron.
3 Table 1. Substrate, room location and construction analysed, with data percentages. Substrates % Room location % Construction % Unknown 32,7 Warm room 44,1 Outer wall 54,2 Wood 27 Basement 27,6 Ceiling 19,2 Gypsum board 10,6 Bathroom 10,6 Wall against wet room 12 Wind barrier 6,8 Attic 7,3 Floor 11,5 Wall paper 6,5 Kitchen 6,0 Window sill 2,7 Concrete 5,8 Crawl space 4,4 Wall against warm room 0,3 Vinyl/Linoleum 3,8 Chipboard 3,7 Fibreboard 2,4 Paint 0,7 Fig. 2. Preferences for type location and substrate for Aspergillus. Fig. 2. Preferences for type location and substrate for Hyalodendron. 3 RESULTS The substrate preference of Actinomycetes is concrete (39,5%) and wood (23,7%). Out of 57% of all Actinomycetes grow in basements, and 46,6% of these grow on outer walls. 59% of those in basements grow on concrete. Acremonium grows mainly on outer walls (92,2%), mainly in warm rooms (62,7%) and in basements (29,5 %). They grow mainly on wood (16,5%) and wind barriers (15,4%). Ascotricha grows mainly inside insulated outer walls in the basement (83,5%), on gypsum boards (41,2%) and on wood (30,5%).
4 59% of Aspergillus grows on outer walls, 37% in warm rooms, and 22% in basements. It also grows on moist floors in warm rooms (1). Favourites substrates are wood (35%) and wall paper (14,8%), but Aspergillus species can grow on all moist porous substrates indoors. Chaetomium grows mainly in walls against wet rooms in bathrooms and kitchens (3), that is, walls containing water pipes, but it also grows on outer walls (27%) and floors (26%). Preferred substrates are chipboard (30,6%) and wood (30%), but also gypsum boards (17%). Chrysosporium grows mainly on ceilings in cold places (64%), such as basements (29%) and crawl spaces (14%), besides outer walls in basements (29%). The favourite substrates are wood (34%) and wind barrier (27%). Cladosporium grows on outer walls (57%), mainly in warm rooms (36%), but also in basements (2), and ceilings in cold attics (16%). The favourite substrates are wood (53%), especially in attics (89%), and concrete in basements (37,7%). Cladosporium is also the dominant species on window sills. Eurotium grows on all types of substrates, but mainly on wood (44%). It prefers outer walls in warm rooms (3), but also moist concrete floors covered with vinyl/linoleum (22%). Hyalodendron grows mainly on wood (93%), especially on ceilings in cold attics (89%). Myxotrichum grow mainly in isolated outer walls in basements (36%), outer walls in warm rooms (18%), and ceilings in crawl spaces (18%). 6 of the collections are made in basements and crawl spaces. Favourite substrates are wind barriers (58%) and wood (30%). Penicillium grows on different substrates, but mainly wood (48%). The highest percentage is on outer walls in warm rooms (40%). It also common in attic ceilings (12%) and outer walls in basements (1). Stachybotrys grows mainly in water damaged walls and floors in bathrooms and kitchens (39%), and outer walls in warm rooms (26%) and basements (17%). It grows mainly on gypsum boards in bathrooms (64,7%), and on wood (17,6%). Trichoderma: 58% grows in outer walls and floors in warm rooms, 5 grow in wood, and 23% on fibreboard and chipboard. Ulocladium grows on outer walls (74%), mainly on gypsum board (32%) and wall paper (26%). 60% of all collections grow on outer walls in warm rooms. 4 DISCUSSION Our results show that Cladosporium, Acremonium, Ulocladium and Actinomycetes follow the same growth pattern as to room location and construction, but they differ in substrate preference (wood for Cladosporium, wind barriers for Acremonium, gypsum boards for Ulocladium, and concrete for Actinomycetes). As to hydrothermal data from literature, Cladosporium is mesophyllic (80% RH), while Acremonium and Ulocladium are hydrophyllic (89-90% RH) and Actinomycetes grow over 95% RH. These differences point to a time succession following the amount of moisture on the wall. Alkaline ph conditions favour growth of Actinomycetes versus moulds. Acremonium, Cladosporium and Ulocladium grow on the phylloplane in nature, and are adapted to tolerate seasonal desiccation (Nielsen 2002),
5 as is the case for Actinomycetes (Andersson et al. 1999). The closest to phylloplane conditions in damp buildings are condensation surfaces subject to seasonal desiccation, that is, cold outer walls, where these microorganisms are most abundant. They can thus be used as indicators for condensation problems in houses. Ascotricha and Mycotrichum are commonly found inside isolated outer walls in basements and crawl spaces. Although we do not have hydrothermal data for these genera, we believe they grow after water penetration due to drainage problems. The main ecological difference between Ascotricha and Myxotrichum is substrate preference: gypsum boards for Ascotricha, and wood and wind barriers for Myxotrichum. Both genera are cellulolytic. Chaetomium, Stachybotrys and Trichoderma grow on wet constructions. These genera are cellulolytic. Again, there is a difference in substrate preference according to our results. Chaetomium prefers chipboards, Stachybotrys gypsum boards, and Trichoderma wood. As to hygrothermal data, the three genera are hydrophyllic, growing over 95%RH, that is, mainly after leakages. Although it is common to find Stachybotrys and Chaetomium growing in the same wet wall, Trichoderma is surprisingly missing. Whether this is because the genus is less competitive than Chaetomium and Stachybotrys remains to be addressed. The fact that both Chaetomium and Stachybotrys are also found on outer walls can be explained by leakages from windows and doors, but also when condensation is so extensive that liquid water is available. The presence of Chaetomium and Stachybotrys on condensation surfaces indicates that the moisture damage has been going on for a long time, and that the construction does not dry up during warmer periods. Hyalodendron is a specialist in cold attic ceilings. It grows exclusively on wood, chipboards and fiberboards, but never together with wood-rotting fungi (pers. obs.). This indicates that Hyalodendron grows after moisture problems leading to condensation. We have not found hydrothermal data for Hyalodendron in the literature, but the fungus is probably mesophyllic rather than hydrophyllic. Chrysosporium grows almost exclusively in cold places (crawling spaces and basements), on cellulose-rich substrates as wood and wind barrier, as the genus is cellulolytic. Chrysosporium seems to prefer stable humidity conditions, with relatively low RH (60-70%). Eurotium is also xerophilic (64-85%RH) and grows mainly on wood, but prefers warmer rooms. This fungus is a specialist on moist concrete floors covered with a damp barrier (f. ex. vinyl/linoleum). Aspergillus and Penicillium include too many species to make general conclusions on their ecology. Our experience is that different species occur on different substrates, constructions and room locations. Common for both genera is that they are the first successional genera after moisture damage, as they grow between 79-80% RH (Grant et al. 1989). Our results indicate that Aspergillus prefers warmer conditions than Penicillium, but both genera can grow together when RH rises over 75%. Our study shows firstly that optical microscopic is an easy, quick and cheap method to identify microbial growth in damp buildings. Secondly, that moisture source (liquid water, water vapour or condensation) and type of building material often determine the location, extent, and type of microorganisms growing indoors. Prediction of microbial growth in hidden constructions after moisture damage helps to address potential health exposure to residents, facilitates effective remediation, and continuously evaluates material properties and building strategies in order to minimize microbial growth in risk constructions.
6 ACKNOWLEDGEMENTS We thank all our Mycoteam colleagues for collections and identification of microorganisms, for discussions and always jovial working atmosphere. 6 REFERENCES Andersson M.A, Mikkola R., Kroppenstedt R.M., Rainey F.A., Peltola J., Helin J., Sivonen K. and Salkinoja-Salonen M.S The mitochondrial toxin produced by Streptomyces griseus strains isolated from an indoor environment is Valinomycin. Applied and Environmental Microbiology, 64(12), Andersson M.A., Tsitko I., Vuorio R. and Salkinoja-Salonen M.S Mycobacteria and related genera are major colonizers of a wall in a children's day care center. In: Bioaerosols, Fungi and Mycotoxins: Health Effects, Assessment, Prevention and Control, pp Bornehag C.G., Blomquist G., Gyntelberg F., Järvholm B., Malmberg P., Nordvall L., Nielsen A., Pershagen G. and Sundell J Dampness in buildings and health, Nordic interdisciplinary review of the scientific evidence on associations between exposure to "dampness" in buildings and health effects (NORDDAMP). Indoor Air, 11, Grant C., Hunter C.A., Flannigan B. and Bravery A.F The moisture requirements of moulds isolated from domestic dwellings. International Biodeterioration, 25, Holme J., Geving S. and Jenssen J.A Moisture and mould damage in Norwegian houses. In: Proceedings of the 8th Symposium on Building Physics in the Nordic Countries, Lyngby, Vol. 3, pp Lee J.H. and Jo W.K Characteristics of indoor and outdoor bioaerosols at Korean highrise apartment buildings. Environmental Research, 101, Li D.W and Yang C.S Fungal contamination as a major contributor to Sick Building Syndrome. Advances in Applied Microbiology, 55, Mullen G.R. and O Connor B.M Mites. In: Medical and Veterinary Entomology (2nd ed.), San Francisco, pp Nevalainen A., Partanen P., Jääskeläinen E., Hyvärinen A., Koskinen O., Meklin T., Vahteristo M., Koivisto J. and Husman T Prevalence of moisture problems in Finnish houses. Indoor Air, 4, Nielsen K.F Mould growth on building materials. Secondary metabolites, mycotoxins and biomarkers. Ph.D. Thesis, Biocentrum-DTU (Denmark), 106 pages. Samson R.A., Hoekstra E.S., Frisvad J.C. and Filtenborg O Introduction to food- and airborne fungi. Utretcht: Centraalbureau voor Schimmelcultures. Sedlbauer K Prediction of mould fungus formation on the surface of and inside building components. Ph.D. Thesis, University of Stuttgart (Germany), 246 pages. Viitanen H.A Modelling the time factor in the development of mould fungi. The effect of critical humidity and temperature conditions on pine and spruce sapwood, Holzforschung - International Journal of the Biology, Chemistry, Physics and Technology of Wood 51, Yang C.S Assessment of fungal contamination in buildings. EMLabP&K, 10 pages. Yang C.S A retrospective and forensic approach to assessment of fungal growth in the indoor environment. In: Sampling and analysis of indoor microorganisms. New Jersey, pp
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