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Available online 13 October 2022, Bacteria and filamentous fungi (ff) are commonly encountered in biofilms developed in drinking water (DW) distribution systems (DWDS). Despite their intimate ecological relationships, researchers tend to study bacteria and ff separately. This work assesses the impact of bacteria-ff association in biofilm formation and tolerance to chlorination. One strain of Acinetobacter calcoaceticus isolated from DW was used as a model bacterium. Penicillium brevicompactum and P. expansum isolated from DW were the ff selected. Single species and inter-kingdom adhesion and biofilm formation occurred under two shear stress () conditions (0.05 and 1.6 Pa). The sessile structures were further characterized in terms of biomass production, respiratory activity and structure. The results showed that 1.6 Pa of shear stress and A. calcoaceticus-ff association favoured biofilm production. Inter-kingdom biofilms produced more biomass than A. calcoaceticus single species and reduced A. calcoaceticus susceptibility to disinfection, particularly to high sodium hypochlorite (SHC) concentrations. In addition, P. brevicompactum formed single species biofilms highly resistant to removal and inactivation by SHC. The presence of P. brevicompactum or P. expansum in inter-kingdom biofilms significantly decreased SHC removal and inactivation effects in comparison to the bacterial biofilms alone, proposing that using bacteria to form biofilms representative of DWDS can provide inaccurate conclusions, particularly in terms of biofilm production and susceptibility to disinfection., This work was financially supported by LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE) - funded by national funds through the FCT/MCTES (PIDDAC); Projects Germirrad - POCI-01-0247-FEDER-072237; PRESAGE - Aquatic/0007/2020, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalizaçao ˜ (POCI) and by national funds (PIDDAC) through FCT/MCTES; Project “HealthyWaters – Identification, Elimination, Social Awareness and Education of Water Chemical and Biological Micropollutants with Health and Environmental Implications”, with reference NORTE-01-0145-FEDER-000069, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This study was further supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020., info:eu-repo/semantics/publishedVersion

The presence of biofilms in plumbing systems is often related to organoleptic alterations in drinking water, the acceleration of pipe corrosion, and the proliferation of opportunistic pathogens. As a result of these adverse effects, there is not completely effective strategy to control biofilms and a biofilm-free plumbing system is utopic. It is known that the materials used in plumbing construction play a key role in biofilm formation, microbial diversity, structure, and pathogen proliferation. However, there is no consensus on which type of material has the major impact on biofilm prevention. The discrepancies between different published reports may be due to the high complexity of plumbing systems and the multitude of variables that change among studies. In this chapter, the influence of different metallic and polymeric materials on biofilm formation in plumbing systems is reviewed. Metallic materials are highly associated with biofilm development, corrosion susceptibility, and disinfectant decay. However, polymeric materials are prone to biofilm development, mainly due to the leaching of organic compounds into drinking water. Moreover, the use of different materials simultaneously in the same plumbing system may also favor material corrosion and biofilm development. Finally, this chapter presents a summary on the most commonly used techniques to assess and evaluate biofilm formation on different surface materials., This work was financially supported by Base Funding—UIDB/00511/2020 of LEPABE funded by national funds through the FCT/MCTES (PIDDAC); Project Biocide_for_Biofilm— PTDC/BII-BTI/30219/2017—POCI-01-0145-FEDER-030219; Germirrad—POCI-01-0247- FEDER-072237; pBio4.0—POCI-01-0247-FEDER-033298, funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES. This study was further supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

A sobrevivência dos mais aptos é um princípio biológico aplicável a todos os seres vivos e, embora organismos diferentes tenham desenvolvido mecanismos próprios para maximizar as suas hipóteses de sobrevivência, todos possuem um fator comum — a sobrevivência está diretamente relacionada com a capacidade de adaptação às variações das características do meio ambiente. As bactérias são particularmente adaptáveis a tais variações, em virtude da sua taxa de reprodução extremamente elevada, o que lhes permite a transferência de características de sobrevivência para gerações futuras em períodos de tempo muito curtos. Adicionalmente, muitas espécies bacterianas têm uma tendência natural para se fixar a superfícies, multiplicar e incorporar numa matriz viscosa produzida pelos microrganismos colonizadores, formando biofilmes1 Este trabalho foi financiado por: Financiamento Base — UIDB/00511/2020 da Unidade de Investigação — Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia — LEPABE — financiado por fundos nacionais através da FCT/MCTES (PIDDAC); Financiamento Base — UIDB/04469/2020 da Unidade de Investigação Centro de Engenharia Biológica — financiado por fundos nacionais através da FCT/MCTES (PIDDAC); financiamento CIMO (UIDB/00690/2020) através de fundos nacionais FCT/MCTES; Financiamento Base — UIDB/04033/2020 da Unidade de Investigação — Centro de Investigação e Tecnologias Agroambientais e Biológicas — CITAB — financiado por fundos nacionais através da FCT/MCTES (PIDDAC); BioTecNorte (NORTE-01-0145-FEDER-000004) no âmbito do Programa Operacional Regional do Norte — Norte2020; Projetos: Biocide_for_Biofilm — PTDC/BII-BTI/30219/2017 — POCI-01-0145-FEDER-030219; AlgaValor — POCI-01-0247-FEDER-035234; LISBOA-01-0247-FEDER-035234; ALG-01-0247-FEDER-035234; pBio4.0 — POCI-01-0247-FEDER-033298; Germirrad — POCI-01-0247-FEDER-072237, financiados pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) e com o apoio financeiro da FCT/MCTES através de fundos nacionais (PIDDAC) e ANI. info:eu-repo/semantics/publishedVersion

LegionellaDB is the first database on Legionella outbreaks; it is based on a metadata analysis of peer-reviewed manuscripts from PubMed and SCOPUS. LegionellaDB is dynamic and extensible, allowing users to search for specific outbreaks, suggest additional information to be included after curation, visualize statistical representations on specific outbreaks, and download selected data. The database is maintained online., This work was financially supported by Base Funding – UIDB/00511/2020 of LEPABE and UIDB/00081/2020 of CIQUP funded by national funds through the FCT/MCTES (PIDDAC); Project Biocide_for_Biofilm – PTDC/ BII-BTI/30219/2017 – POCI-01-0145-FEDER-030219, ABFISH – PTDC/ASP-PES/28397/2017 – POCI-01- 0145-FEDER-028397 and ALGAVALOR – POCI-01- 0247-FEDER-035234, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte; Applied Molecular Biosciences Unit – UCIBIO, which is financed by national funds from FCT (UIDB/04378/2020). H.S.F. acknowledges FCT for his PhD grant SFRH/BD/115396/2016., info:eu-repo/semantics/publishedVersion

Legionella pneumophila is an intracellular pathogen, ubiquitous in the environment and considered opportunistic. It is the leading cause of legionellosis, which can be present in its nonpneumonic form (Pontiac fever) and acute pneumonic form (Legionnaires` disease – LD). In the soil and aquatic systems, L. pneumophila can invade and survive intracellularly in various protozoans. The ability to proliferate within biofilms provides additional protection from environmental stresses, such as disinfection. Human infection by L. pneumophila occurs after the inhalation or aspiration of aerosols containing the pathogen. Upon infection, alveolar macrophages can be invaded and used by L. pneumophila for replication, resembling the infection of protozoan hosts in the environment. The ability of L. pneumophila to overcome the killing mechanisms of phagocytes depends on the Dot/Icm type IV secretion system – a specialized protein translocation system vital for the intracellular survival of the pathogen and for establishing a replicative niche known as the Legionella-containing vacuole. Following host cell lysis, the released bacteria infect other host cells, beginning a new cycle of infection., This work was financially supported by Base Funding – UIDB/00511/2020 of LEPABE and funded by national funds through the FCT/MCTES (PIDDAC); Project Biocide_for_Biofilm – PTDC/BII-BTI/30219/2017 – POCI-01-0145-FEDER-030219, pBio4.0 – POCI-01-0247-FEDER-033298, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE 01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Os biofilmes são o resultado da tendência natural dos microrganismos se fixarem a superfícies (bióticas ou abióticas), multiplicarem e incorporarem numa matriz viscosa de substâncias poliméricas extracelulares (EPS). À medida que o biofilme se desenvolve, o descolamento e os processos de crescimento entram em equilíbrio, levando a uma quantidade constante de biomassa na superfície e à libertação de células do biofilme que permitem a colonização de superfícies vizinhas e a formação de novas comunidades sésseis. EPS dos biofilmes são responsáveis pela ligação entre as células e com outros materiais particulados. A adaptação bem sucedida das bactérias às mudanças das condições naturais requer também que o microrganismo possa sentir e responder ao seu ambiente externo e, em consequência, modular a expressão dos seus genes. Mesmo que os biofilmes sejam provavelmente a primeira forma de vida comunitária na Terra, a incorporação de aspetos científicos da ciência e tecnologia dos biofilmes nos conteúdos de ensino pré ou pós graduado é uma tarefa desafiante, uma vez que esta área de investigação está em constante evolução., Este trabalho foi financiado por: Financiamento Base - UIDB/00511/2020 da Unidade de Investigação - Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia – LEPABE; financiamento CIMO (UIDB/00690/2020); financiamento CITAB UIDB/04033/2020 e UIDP/04033/2020; financiamento CEB UIDB/04469/2020 - por fundos nacionais através da FCT/MCTES (PIDDAC) e PT2020. - Projeto PTDC/BII-BTI/30219/2017 - POCI-01-0145-FEDER-030219 e POCI 01-0247-FEDER-072237, financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER), através do COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) e com o apoio financeiro da FCT/MCTES através de fundos nacionais (PIDDAC)., info:eu-repo/semantics/publishedVersion

The selection of materials for plumbing application has potential implications on the chemical and microbiological quality of the delivered water. This work aims to evaluate the action of materials with different copper content (0, 57, 96 and 100%) on biofilm formation and control by chlorination and mechanical stress. A strain of Stenotrophomonas maltophilia isolated from drinking water was used as model microorganism and biofilms were developed in a rotating cylinder reactor (RCR) using realism-based shear stress conditions. Biofilms were characterized phenotypically and exposed to three control strategies: 10 mg/l of free chlorine for 10 min; an increased shear stress (equivalent to 1.5 m/s of fluid velocity); and the combination of both treatments. Biofilms formed on the copper materials had lower wet mass and produced significantly lower amounts of extracellular proteins than those formed on stainless steel (0% of copper content). Although, the effects of copper materials on biofilm cell density was not significant, these materials had important impact on the efficacy of chemical and/or mechanical treatments. Biofilms formed on 96 or 100% copper materials had lower content of culturable bacteria than that observed on stainless steel after exposure to chlorine or shear stress. The mechanical treatment used had no relevant effects in biofilm control. The combination of chemical and mechanical treatments only caused higher culturability reduction than chlorine in biofilms formed on 57% copper alloy. The number of viable cells present in bulk water after biofilm treatment with chlorine was lower when biofilms were formed on any of the copper surface. The overall results are of potential importance on the selection of materials for drinking water distribution systems, particularly for house and hospital plumbing systems to overcome the effects from chlorine decay. Copper alloys may have a positive public health impact by reducing the number of viable cells in the delivered water after chlorine exposure and improving the disinfection of DW systems. Moreover, the results demonstrate that residual chlorine and mechanical stress, two strategies conventionally used for disinfection of drinking water distribution systems, failed in S. maltophilia biofilm control., This work was the result of the projects: UIDB/00511/2020 of the Laboratory for ProcessEngineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds throughthe FCT/MCTES(PIDDAC); PTDC/BII-BTI/30219/2017-POCI-01-0145-FEDER-030219;POCI-01-0145-FEDER-006939, funded by FEDER funds through COMPETE2020 – ProgramaOperacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) throughFCT/MCTES; NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, supported by NortePortugal Regional Operational Programme (NORTE 2020), under PORTUGAL 2020 PartnershipAgreement, through the European Regional Development Fund (ERDF), info:eu-repo/semantics/publishedVersion

The chemical disinfectant chlorine, as chlorine gas (Cl2) and sodium hypochlorite (NaOCl), has been commonly used for drinking water treatment.1,2 Although the recommended residual concentration of free chlorine allows to some extent the control of microbial growth in the bulk water, the occurrence of biofilms in chlorinated drinking water distribution systems (DWDS) has been frequently reported.3,4 Therefore, the main goal of this study was the study of alternative biocides to control biofilm development in DWDS. The effects of sodium dichloroisocyanurate (NaDCC), trichloroisocyanuric acid (TCCA), and pentapotassium bis(peroxymonosulphate) bis(sulphate) (OXONE®) were analysed against two emerging pathogens isolated from drinking water, Acinetobacter calcoaceticus and Stenotrophomonas maltophilia. The determination of the minimum bactericidal concentrations (MBC) of the selected biocides were based on the European Standard EN 1276, with MBC between 1.56 to 6.25 mg/L for NaDCC, 2.5 to 3.75 mg/L for TCCA, and 172 to 688 mg/L for OXONE®. Inactivation curves were developed and fitted to microbial survival models. The effects of biocides on cytoplasmic membrane integrity were assessed by propidium iodide uptake. The action on biofilm control was analysed against 48 h old biofilms developed on polyvinyl chloride (PVC) and stainless steel (SS) coupons using a 24-wells microtiter plate assay. The bacteria culturability and removal assessment were determined by colony forming units (CFU) enumeration on R2A agar, and by 4,6-diamidino-2-phenylindole (DAPI) staining, respectively. This study reinforces biofilms as chronic contaminants of DWDS and highlights that the understanding of antimicrobial susceptibility of microorganisms to biocides is an important step in the design of effective biofilm control strategies in order to provide to consumers drinking water of adequate microbiological quality., This work was financially supported by: Base Funding - UIDB/00511/2020 of the Laboratory forProcess Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national fundsthrough the FCT/MCTES (PIDDAC); PTDC/BII-BTI/30219/2017 - POCI-01-0145-FEDER-030219;POCI-01-0145-FEDER-006939, funded by FEDER funds through COMPETE2020 – ProgramaOperacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; and PhD grant awarded to Isabel Maria Oliveira (SFRH/BD/138117/2018)., info:eu-repo/semantics/publishedVersion

The presence of biofilms in drinking water distribution system (DWDS) constitutes one of the currently recognized hazards affecting the microbiological quality of drinking water (DW). Also, biofilms can alter the taste, odor, and the visual appearance of water, which is an indication of poor DW quality and may lead to a number of unwanted effects on the quality of the distributed water. Very few reports on filamentous fungi (ff) biofilms can be found in the literature mainly because these fungi do not conform completely to the biofilm definitions that are usually proposed for bacteria. Nevertheless, filamentous fungi are microorganisms that due to their (1) absorptive nutrition mode, (2) secretion of extracellular enzymes to digest complex molecules, and (3) apical hyphal growth that form lattices, which are excellent candidates for biofilm formation. However, this aspect is still poorly understood. In several environments, bacteria and ff cohabit and interact with each other, which has a wide range of applications and influences both organisms, sometimes creating an increase in resistance to antibiotics and antifungal agents. This chapter provides an overview on the presence of ff in DWDS, including their interaction with bacteria and potential biofilm formation., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020dPrograma Operacional Regional do Norte. Further support was obtained from the European Investment Funds by FEDER/COMPETE/POCI Operational Competitiveness and Internationalization Programme, under Projects: POCI-01-0145-FEDER-030219; POCI-01-0247-FEDER-035234; POCI-01-0247-FEDER033298; POCI-01-0145-FEDER-028397; and POCI-01-0145-FEDER-006958 and National Funds by FCTdPortuguese Foundation for Science and Technology., info:eu-repo/semantics/publishedVersion

[Excerpt] Biofilms constitute one of the major microbial problems in drinking water distribution systems (DWDS) that most contribute to the deterioration of water quality. Knowledge of drinking water biofilms has been mainly obtained from studies on bacterial biofilms even though, under natural conditions, they are usually viewed as complex communities where different organisms are present. including filamentous fungi. The ecology of a biofilm is a complex function of different factors, including the presence of microbial metabolites and molecules (cell-cell signalling communication) excreted by the microbial inhabitants of the biofilm. Quorum sensing (QS) controls different population density- dependent processes, including biofilm formation. Eukaryotes have. however, the ability to interfere with bacterial communication by producing molecular signals that interact with bacterial QS. The aim of this study was to assess the effect of patulin, a fungal secondary metabolite, and N-(3-Oxododecanoyl)- L-homoserine lactone (3-Oxo-C12-HSL), a QS signalling molecule, on interkingdom biofilm formation between bacteria and fungi isolated from a DWDS. [....]

info:eu-repo/semantics/publishedVersion

The use of chemical biocides in medical, industrial and domestic environments is a first line defense in the prevention and control of microbial growth. Although biocide treatments eliminate most surface contamination, some microorganisms may survive and give rise to substantial problems, and numerous reports have highlighted the survival of microorganisms after cleaning and disinfection in different environments. Microorganisms may have intrinsic resistance to biocides which is commonly associated with cellular impermeability. However, the continuous exposure to biocides may increase microbial resistance by cellular mutations or acquisition genetic elements. The increasing use of biocides is also a concern related to their putative environmental toxicity. Therefore, new biocidal solutions are needed to combat effectively the evolution of microbes developing resistance while having a low or no environmental toxicity impact. Phytochemicals are an attractive source of eco-friendly, relatively inexpensive and widely available new broad-spectrum antimicrobials with low levels of cutaneous cytotoxicity and environmental toxicity. This article will highlight the cleaning and disinfection processes in microbial growth control as well as the properties of biocides commonly used, the processes involved in their antimicrobial action, the factors influencing their efficacy, and the mechanisms of cellular and biofilm resistance. In addition, it will cover the main classes of phytochemicals with antimicrobial properties and their mode of action., This work was the result of the projects: POCI-01-0145-FEDER-030219; POCI-01-0145-FEDER-028397; POCI-01–0145-FEDER006939 (Laboratory for Process Engineering, Environment, Biotechnology and Energy – UID/EQU/00511/2013) funded by the European Regional Development Fund (ERDF), through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds, through FCT – Fundação para a Ciência e a Tecnologia, and the project NORTE‐01-0145‐FEDER‐000005 – LEPABE-2-ECO-INNOVATION, supported by North Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF)., info:eu-repo/semantics/publishedVersion

The main focus so far in the study of biofilm formation in drinking water has been bacteria. Studies on biofilm formation involving filamentous fungi are, therefore, scarce. This study aimed to assess and characterize the ability of these microorganisms to interact with bacteria whilst forming inter-kingdom biofilms. Biofilms were analysed in terms of total biomass, metabolic activity, bacterial colony forming units and morphology by epifluorescence microscopy. The quantitative methods revealed that biofilm mass increased over time for both single and inter-kingdom biofilms, while specific metabolic activity decreased, in general, along the time points evaluated. Microscopic data visually confirmed the biofilm mass increase over time. This study shows that fungal stage development is important in the first 24 h of biofilm formation. Inter-kingdom biofilm formation is microorganism dependent and inter-kingdom biofilms may provide an advantage to the opportunistic bacterium Acinetobacter calcoaceticus to replicate and proliferate when compared with Methylobacterium oryzae.

Biofilms are heterogeneous and complex structures of microorganisms, typically adhered on a surface and presenting sophisticated singular and collective behaviours. This protected mode of growth allows the microorganisms to survive in hostile environments. Research on biofilms has progressed rapidly in the last decade. Due to the fact that biofilms have required the development of new analytical tools, many recent advances have resulted from collaborations between biologists, engineers and mathematicians. The scientific community has come to understand many things about the particular biology of microbial biofilms through a variety of microscopic, physical, chemical, and molecular techniques of study. "Recent trends in biofilm science and technology" provides a remarkable amount of knowledge on the processes that regulate biofilm formation; on the methods used for their formation, monitoring, characterization and mathematical modelling; on the problems/advantages caused by their presence in the food industry, environment and medical fields; and describes the current and emergent strategies for their control. The information in "Recent trends in biofilm science and technology" is designed to be of use to researchers and professionals working on fundamental aspects of biofilm formation and control and to be helpful in conducting biofilm studies and in the consistent interpretation of results., This work was financially supported by Base Funding UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE)dfunded by national funds through the FCT/MCTES (PIDDAC); projects PTDC/BII-BTI/30219/2017 - POCI-01-0145-FEDER-030219, POCI-01e0145-FEDER-028397, POCI-01e0145-FEDER-033298, and POCI 01e0145-FEDER-035234 funded by FEDER funds through COMPETE2020 Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through the FCT/MCTES. FCT under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Sabbatical grant SFRH/BSAB/150379/2019., info:eu-repo/semantics/publishedVersion

Dissertação de mestrado em Biotecnologia, Drinking Water Distribution Systems (DWDS) is essential for the delivery of high-quality and safe drinking water (DW). However, DWDS allows the establishment of a dynamic microbiological ecosystem, where microorganisms are present in both planktonic and biofilm states. Microorganisms adhered to the surfaces of pipes are dominant. A biofilm can be defined as a sessile community of microorganisms irreversibly attached to a surface or substratum and each other, which are embedded in an extracellular polymeric substances (EPS) matrix that they produce and excrete. The biofilms formed in a DWDS are an interkingdom complex community since under natural conditions is rare the formation of monospecies biofilms. This diversity leads to a multiplicity of complex relationships involving intraspecies and interspecies interactions. In addition, biofilm formation in DWDS can be affected by a variety of biotic and abiotic factors, namely: environmental factors (temperature, pH), residual concentration of disinfectant, type and availability of nutrients, hydrodynamic conditions, design of the network, pipe material and sediment accumulation. The control of biofilm formation in DWDS is essential to make sure that the water delivered to the consumer is microbiologically safe. In this work, six different fungi were first evaluated in terms of growth kinetics and biofilm formation. The six fungi were: P. expansum, P. brevicompactum, F. oxysporum, A. versicolor, Alternaria sp, and Mucor sp. A stock of spore suspension of each fungus was made, and the biofilm assay was executed. For biofilm formation, 200 µL of spore suspension R2B was added into each well and spectrophotometric-based methods (crystal violet method for biomass quantification and resazurin method for metabolic activity quantification) were used to monitor the biofilm formation over time. Macro and Microscopic characterization were also performed for each fungus. In the end, the fungus which presented higher biomass formation and metabolic activity was Alternaria sp., and the values were 20.070 ± 3.825 and 3695.625 ± 802.910, respectively. This fungus was chosen for further studies to understand its behavior under different process conditions. The conditions chosen were the hydrodynamics, the nutrient concentration, the presence of chlorine, and an interkingdom association. Relative to the hydrodynamics, four conditions were evaluated (static, 30, 150, and 200 rpm). After three days of incubation, some significant differences between conditions were observed. The use of 200 rpm caused a significant difference (ρ, Os Sistemas de Distribuição de Água Potável (DWDS) são essenciais para o fornecimento de água potável de alta qualidade e segura (DW). Contudo, os DWDS permitem o estabelecimento de um ecossistema microbiológico dinâmico, onde os microrganismos estão presentes tanto em estados planctónicos como de biofilme. Os microrganismos aderidos nas superfícies das tubagens são dominantes. Um biofilme pode ser definido como uma comunidade séssil de microrganismos irreversivelmente ligados a uma superfície ou substrato e uns aos outros, que estão embutidos numa matriz extracelular de substâncias poliméricas (EPS) que produzem e excretam. Os biofilmes formados numa DWDS são uma comunidade complexa do interreino, uma vez que em condições naturais é rara a formação de biofilmes monoespécie. Esta diversidade conduz a uma multiplicidade de relações complexas envolvendo interações intraespécies e interespécies. Além disso, a formação de biofilmes no DWDS pode ser afetada por uma variedade de fatores bióticos e abióticos, nomeadamente: fatores ambientais (temperatura, pH), concentração residual de desinfetante, tipo e disponibilidade de nutrientes, condições hidrodinâmicas, conceção de rede, material de tubagem e acumulação de sedimentos. O controlo da formação do biofilme no DWDS é essencial para garantir que a água entregue ao consumidor é microbiologicamente segura. Neste trabalho, seis fungos diferentes foram primeiramente avaliados em termos de cinética de crescimento e formação de biofilme. Os seis fungos foram: P. expansum, P. brevicompactum, F. oxysporum, A. versicolor, Alternaria sp, e Mucor sp. Foi feito um stock de suspensão de esporos de cada fungo, e o ensaio do biofilme foi executado. Para a formação do biofilme, foram adicionados 200 µL de suspensão de esporos e caldo R2A em cada poço e foram utilizados métodos espectrofotométricos (método violeta cristal para quantificação da biomassa e método de resazurina para quantificação da atividade metabólica) para monitorizar a formação do biofilme ao longo do tempo. Foi também realizada a caracterização macroscópica e microscópica de cada fungo. No final, o fungo que apresentou maior formação de biomassa e atividade metabólica foi a Alternaria sp., e os valores foram de 20,070 ± 3,825 e 3695,625 ± 802,910, respetivamente. Este fungo foi escolhido para mais estudos a fim de compreender o seu comportamento sob diferentes condições de processo. As condições escolhidas foram a hidrodinâmica, a concentração de nutrientes, a presença de cloro, e uma associação interrelacionada. Em relação à hidrodinâmica, foram avaliadas quatro condições (estática, 30, 150, e 200 rpm). Após três dias de incubação, foram observadas algumas diferenças significativas entre as condições. A utilização de 200 rpm causou uma diferença significativa (ρ