Biofilm formation by filamentous fungi recovered from a water system

Filamentous fungi have been constantly recovered from diverse aquatic environments including drinking water distribution systems. Althoughmost of the works are focused on the study of planktonic form, recent researches have shown that fungi develop biofilm within these systems. In this study, Aspergillus sp. (section Nigri), Aspergillus sp. (section Flavi), Alternaria sp., Botrytis sp., Cladosporium sp., and Penicillium sp. recovered from water biofilms were used to evaluate their capability to grow as biofilms under laboratorial conditions.Morphological and physiological characteristics were analysed using image analysis and biomass and cell activity estimation. All six isolates were able to form biofilm, though different patterns of development were observed. Only Alternaria sp. formed biofilm in water over 24h of analysis.MEB was shown to be the best culture media for biofilm formation. A direct correlation between biomass and cell activity was not observed, but biomass values and morphological parameters, that is, monolayer and EPS production, were directly correlated.Thus, the results present here highlight the capability of fungi to form biofilms and the emergent necessity to standardize methods for further research in this area

Biofilm formation by filamentous fungi recovered from a water system

Filamentous fungi have been consistently recovered from diverse aquatic environments including drinking water distribution systems. Although most of the work is focused on planktonic forms, recent research demostrates unequivocally that fungi develop biofilms within these systems. In this study, individual strains of Aspergillus (section Nigri), Aspergillus (section Flavi), Alternaria, Botrytis, Cladosporium and Penicillium, recovered from water biofilms, were used to evaluate their capability to grow as biofilms in vitro. Morphological and physiological characteristics were analysed using image analysis, and biomass and cell activity estimations. All six isolates were able to form biofilms, although different patterns of development were observed. Only Alternaria sp. formed biofilms in water within 24 h. Malt extract broth (MEB) was the optimal culture medium for biofilm formation. A direct correlation between biomass and cell activity was not observed, but the quantity of biomass and morphological parameters, i.e. monolayer and exopolysaccharides (EPS) production, were directly correlated. Thus, fungi are capable of forming biofilms and there remains a necessity to standardize methods for further research in this area

Quantification of fungal biomass in Penicillium brevicompactum biofilms by image analysis

Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/43719/200

Biofilm formation by filamentous fungi recovered from a water system

Filamentous fungi have been consistently recovered from diverse aquatic environments including drinking water distribution systems. Although most of the work is focused on planktonic forms, recent research demostrates unequivocally that fungi develop biofilms within these systems. In this study, individual strains of Aspergillus (section Nigri), Aspergillus (section Flavi), Alternaria, Botrytis, Cladosporium and Penicillium, recovered from water biofilms, were used to evaluate their capability to grow as biofilms in vitro. Morphological and physiological characteristics were analysed using image analysis, and biomass and cell activity estimations. All six isolates were able to form biofilms, although different patterns of development were observed. Only Alternaria sp. formed biofilms in water within 24 h. Malt extract broth (MEB) was the optimal culture medium for biofilm formation. A direct correlation between biomass and cell activity was not observed, but the quantity of biomass and morphological parameters, i.e. monolayer and exopolysaccharides (EPS) production, were directly correlated. Thus, fungi are capable of forming biofilms and there remains a necessity to standardize methods for further research in this area

Characterising filamentous fungal biofilm in drinking water distribution systems using microscopic and molecular techniques

Tese de doutoramento em Engenharia Química e BiológicaA qualidade microbiológica de água de consumo tem sido uma das grandes preocupações de fornecedores governamentais e privados. Sistemas de distribuição de água são complexos ambientes onde algas, bactérias, fungos e protozoários cohabitam. Muitos destes microrganismos são patogenicos ao homem. Uma vez no sistema de distribuição, microorganimos podem aderir às superfícies das tubulações e formar biofilmes. Biofilmes são comunidades microbianas nas quais seus constituintes apresentem características diferenciadas, por exemplo, maior resistência a fatores adversos como desidratação, altas temperaturas, condições oligotroficas e ação de biocidas. Fungos são conhecidos por habitarem diversos ambientes aquáticos. Sua presença em sistemas de distribuição de água está associada a problemas de bloqueio de tubulações, produção de metabólitos e mudanças em propriedades organolépticas como odor e turbidez, e consequentemente, os fungos estão ligados à perda da qualidade da água. Além disso, por conta da sua habilidade em aderir a superfícies, os fungos são capazes de crescer como biofilmes em diferentes materiais, inclindo as paredes das tubulações. O presente trabalho visou o estudo de biofilmes de fungos filamentosos em sistemas de distribuição de água. Para isto, técnicas de microscopia de fluorescência e fluorescent in situ hybridization (FISH) foram utilizadas pretendendo-se detectar, monitorar e caracterizar biofilmes de fungos filamentosos em condições reais e laboratoriais. Corantes e sondas fluorescentes específicos foram aplicados, i.e. Calcofluor White R2R, DAPI, FUN-1, e as sondas EUK516 FUN1429. Microspheres Adhesion Assay, coloração com Cristal Violeta e MTT foram também utilizados. Adicionalmente, foi projetado um amostrador para formação de biofilmes in situ. Como resultados, foi observado que biofilmes de fungos filamentosos, quando comparados com sua forma planquitonica, possuem um comportamento diferenciado com relação à resistência contra agentes desinfectantes e a nívels de hidrophobicidade celular. Biofilmes de fungos filamentosos foram detectados em tubulações, amostradores e sistemas laboratoriais. Fungus filamentosos isolados de sistemas de água são capazes de formar biofilmes, apresentando caracteristicas morfológicas e fisiológicas diferenciadas. Em conclusão, fungos filamentosos são capazes de formar biofilmes sob diversas condições reais e laboratoriais. É provável que os fungos desempenhem um papel importante nas interações nos biofilmes em sistemas de água e, conseqüentemente, também na qualidade microbiológica da água. Assim, os fungos devem ser incluídos como membros consistentes dos biofilmes. Os resultados relatados neste trabalho representam novos conceitos na pesquisa micológica, e mais estudos na área podem levar a novas descobertas sobre a biologia dos fungos.Microbial quality of drinking water has been one of the greatest concerns of governmental and privative water suppliers. Water distribution systems are complex environments where algae, bacterial, fungi and protozoa cohabit. Many of these microorganisms are pathogenic to human and are associated with loss of water quality. Once within a water distribution system, microorganisms can attach to pipe surface and form biofilms. Biofilms are microbial communities wherein theirs constituents present differentiate features when compared with their planktonic form, e.g. increased resistance against adverse factors such as dryness, high temperatures, oligotrophic conditions and biocides. Fungi are known to habit diverse aquatic environments. Their presence in water systems is associated with pipe blockage, produce of metabolites involved in organoleptic changes such as odour and turbidity, and consequently are linked with loss of water quality. Moreover, because of their ability to adhere to surfaces, fungi are able to grown as biofilms on different surfaces, including pipe walls. The present work aimed to study filamentous fungal biofilms in water distribution systems. For this, epifluorescence microscopy and fluorescent in situ hybridization (FISH) were applied intending to detect, monitor and characterise filamentous fungal biofilms in real and laboratorial water systems. Specific fluorescent dyes and probes were used, i.e. Calcofluor White R2R, DAPI, FUN-1, EUK516 and FUN1429 probes. Microspheres Adhesion Assay, Cristal Violet staining and MTT assay were also applied. Moreover, a device was designed for sampling fungal biofilms in situ. As results, was observed that filamentous fungal biofilms have differentiated behaviour concerning resistance against disinfectant and levels of cell hydrophobicity when compared with their planktonic form. Filamentous fungal biofilms were detected in replaced pipes, sampler devices and laboratorial water system. Moreover, filamentous fungi recovered from water systems were capable to form biofilms with specific morphological and physiological features. In conclusion, filamentous fungi are able to form biofilms under diverse real and laboratorial conditions. Fungi are likely to play an important role in microbial interactions within water biofilms and consequently in microbial water quality. Thus, fungi may be included as a consistent member of biofilms in drinking water systems. The results reported in this work represent new concepts in mycological research, and further studies in the area may lead to new insights in fungal biology

In situ detection of fungal biofilms in a water distribution system, Alto do Céu, Recife, Brazil

Recent investigations on water microbiology have shown that most of the biomass present in the drinking water network is located at the pipe walls as biofilms. Biofilms are organized in highly efficient and stable ecosystems and can be viewed as a survival mechanism; this way of life can provide protection from chemical, biological or physical stresses. Moreover, biofilms play a major role in the accumulation, protection and dissemination of pathogens through water distribution systems. Although filamentous fungi are especially adapted for growth on surfaces, fungal water biofilms have received less attention when compared with bacterial biofilms, thus remaining a lack of information in this field. The use of microscopy techniques associated with image analyses has become a valuable tool for in situ studies. Fluorescent in situ hybridization (FISH) and fluorescent dyes are non-invasive and nondestructive techniques which provide information on cell morphology, metabolism and phylogeny. In this work, we aimed to detect filamentous fungal biofilm in a water distribution system using FISH (EUK516 and FUN1429 probes) and Calcofluor staining in replaced pipes. We also presented a sampler developed to study in situ fungal biofilms formation in water distribution system. Calcofluor staining was a rapid and easy method to detect filamentous biofilms on pipes surfaces. Additionally, FISH provided phylogenetic information by the detection of eukaryotic and fungal cells. Ours results contributed to demonstrate the presence of fungi in water biofilms and emphasised that fungi play an important role in water biofilms and microbial water quality